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THE AMERICAN JOURNAL 


CHARLES R. BARDEEN 
University of Wisconsin 


Henry H. DonaLpson 
The Wistar Institute 


Simon H. GAGE 
Cornell University 


OF 


EDITORIAL BOARD 


G. Cart HUBER 
University of Michigan 


GEORGE S. HuNTINGTON 
Columbia University 


Henry McE. KNower, 
SECRETARY 
University of Cincinnati 


VOLUME 17 
1914-1915 


ANATOMY 


FRANKLIN P. MALL 
Johns Hopkins University 


J. Puayratr McMourricu 
University of Toronto 


GrorGeE A. PIERSOL 
University of Pennsylvania 


THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY 


PHILADELPHIA, PA. 


CONTENTS 


1914-1915 


No.1. NOVEMBER 


E. V. Cowpry. The comparative distribution of mitochondria in spinal 
ganglion cells of vertebrates. Fourteen figures (three plates)......... 


F. W. Toyne. The anatomy of a 17.8 mm. human embryo. Eight figures 


Rosert BENNETT Bean. The stature and the eruption of the permanent 
teeth of American, German-American and Filippino children. Deduc- 
tions from the measurements and examination of 1445 public school chil- 
dren in Ann Arbor, Michigan, and 776 in Manila, P. I. Five diagrams 


No. 2. JANUARY 


Otto FREDERIC KAMPMEIER. On the origin of lymphatics in Bufo. Thirty- 
END). TEI CUR ESE 5 te a iehay Brak ea Pes Rane CEP eh me Rie oN ort 


Henry K. Davis. Astatistical study of the thoracic ductinman. Thirty- 
Osa OAT Cama PANN fea ff ot arch EM calc ch RE alas La an hebae 6h Meuhatte Stee 


No. 3. MARCH 


_Ricwarp E. Scammon. The histogenesis of the selachian liver. Forty-five 
FS MTONE (SEEN: PIAUER) Pale cs a Ge teres Shel ee iG aula cc Aree, Bia Bek 


J. A. BADERTSCHER. The development of the thymus in the pig. I. Mor- 
phogenesis. Twelve figures (two plates).................0ec000- 


MarGARET REED LEWIS AND WARREN Harmon Lewis. Mitochondria (and 
other cytoplasmic structures) in tissue cultures. Twenty-six figures. . 


No. 4. MAY 


RanpDourx West. The origin and early development of the posterior lymph 
heartvmetnevenck. Mourteen: figures... 0.0... vee sels we se ee eens oe ke 


J. A. BaprrtscHer. Development of the thymus in the pig. II. Histo- 
genesis. -Pimpernlates: (eight figures) ..2.05 550.5000. ease esas sc eee ees 


L. Bork. On the premature obliteration of sutures in the human skull.... 


113 


161 


211 


245 


317 


339 


403 


495 


; wee" 


eesheus, bys ok 


THE COMPARATIVE DISTRIBUTION OF MITO- 
CHONDRIA IN SPINAL GANGLION CELLS _ 
OF VERTEBRATES! 


E. V. COWDRY 
Department of Anatomy, Johns Hopkins University 


FOURTEEN FIGURES (THREE PLATES) 


CONTENTS 
TET BI RONG HOMER IIICO HN ae era es cen ey rts te RE a Sc ude c a AER aN 1 
TE AHR SIEM HUTS oo ee a Ree ene TERRA Oe RNG SRRTCIE ch cece NCR Bitrate ot Re eee 2 
nee tie ea TEC RIMeCEMOMS 1 aches Gio gail, tA MARAE Bick es sheune clste ee wahoo, «th iin Jae ene 
PSST AUTO TAS ee ree eee a NL rr ane Tu Ae ALPINE ps (UP OMECY Woe) cy onlay ea 11 
TDS OTS Vana e be Oe a VN RY 8 a ee, DACA Seta Vani RT NA ce RR Te OEE ne 16 
TRESIIILTER, 5 beste thc MMe ay ok es Ue gi pe ERM CORE Toe MIU <i BRAM er Senne AR MIEN SRS oP 21 
INTRODUCTION 


This paper constitutes part of a program of research, outlined 
some years ago, the object of which is to lay a sound foundation 
for the study of functional changes in nerve cells. In the first 
place, I confined my attention to a single type of nerve cell and 
brought the most refined methods of general cytology to bear 
upon an analysis of its cytoplasmic constituents (12); I then 
began a study of the same cytoplasmic constituents in devel- 
opment, paying particular attention to the histogenesis of neuro- 
fibrils (14a); and I now wish to present some observations, 
based upon a study of the comparative distribution of nerve 
cell granulations, which I believe throw some light on the 
structural relations and physiological significance of both the 
mitochondria and the Nissl substance. They indicate that 
mitochondria are fundamental constituents of spinal ganglion 
cells, which probably play a part in metabolism; and that the 
Nissl substance occurs in living spinal ganglion cells as a dil- 


1 Aided by the Carnegie Institution. 
1 


THB AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 
NOVEMBER, 1914 


2 E. V. COWDRY 


fuse, continuous deposit instead of in the form of granules and 
flakes of various size and shape. I have received valuable help 
from my father in this work, for which I am very thankful. 


LITERATURE 


I have prepared the following summary so that what we know 
of the comparative distribution of mitochondria in vertebrate 
nerve cells may be seen at a glance. In it, the forms in which 
mitochondria are described by me for the first time, are given 
in italics, the others being recorded in plain type: 


Mammalia: Primates Homo? 
Macacus rhesus 
Rodentia Guinea-pig (Nageotte ’09a and 710; Laignel- 


Lavastine and Jonnesco ’11) 

Lepus (Levi ’96; Held ’97 a; Nageotte ’09 b; 
Schirokogoroff 713) 

Mus norvegicus albinus 


Carnivora Felis (Altmann ’90; Lobenhoffer ’06) Canis (Lob- 
enhoffer ’06) 
Aves: Gallinae Gallus (Duesberg 710) 
Columba (Cowdry ’12) 
Reptilia: Ophidia Eutaenia sirtalis 
Chelonia Testudo graeca (Busana 712) 
Pseudemys hieroglyphica 
Amphibia: Anura Rana (Altmann ’90); Tadpole (Duesberg 712) 
Rana palustris 
Urodela Necturus maculatus 
Pisces: Teleostei Salmo (Furst ’02; Lobenhoffer ’06) 


Cyclostomata: Petromyzontes Petromyzon marinus and Ammocoetes branchi- 
alis (Mawas 710) 


The evidence which these investigators have presented will 


now be considered. 
Guinea-pig 


Nageotte (’09 a, p. 472) demonstrated certain bacilli-like bodies 
in peripheral nerve fibers fixed in Tellyesniczky’s bichromate 
acetic fluid and stained with Altmann’s anilin fuchsin. He found 
them also after preliminary fixation in 10 per cent formalin fol- 
lowed by Tellyesniczky’s fluid and staining in the same way. 


2 Collin (’13, p. 1123) has recorded the occurrence of mitochondria in the 
neuroglia cells of the spinal cord of man. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 3 


He claimed that they were mitochondria on account of their 
morphology. He did not specify either the tissue or the animal 
selected, but I gather from a second paper (710, p. 41) that it 
was the sciatic nerve of a guinea pig which he used. In this 
second paper he employed an osmic bichromate mixture (pre- 
sumably that of Altmann) and succeeded in staining bodies of 
somewhat different form and cytoplasmic distribution which, 
to my mind, resemble mitochondria much more closely. 

Laignel-Lavastine and Victor Jonnesco (11, p. 699) observed 
granules, rods and rows of granules in the Purkinje cells of the 
cerebellum. The technique consisted of fixation in 12 per cent 
formalin, followed by treatment with Weigert’s neuroglia mor- 
dant, staining with hematoxylin and by the methods of Altmann 
and Benda. They employed, in addition, Regaud’s formol-bi- 
chromate-hematoxylin technique and concluded that the struc- 
tures thus revealed were mitochondria. 


Lepus 


Levi (96, p. 3) studied granules which he called ‘fucsinofili 
(rossi)’ in spinal ganglion cells by means of Galleotti’s modifica- 
tion of Altmann’s method, which consists of using methyl green 
as a differentiator in place of picric acid. He observed rod- 
like bodies which took the anilin fuchsin deeply and could be 
distinguished with ease from the green colored Nissl bodies. 
These structures occurred in the axone hillock where the Nissl 
substance is absent. Their staining reactions, form and dis- 
tribution are sufficient basis on which to conclude that they 
are mitochondria. 

Held (97a, p. 293) observed bodies, which he called neuro- 
somes, in spinal ganglion cells (plate 11, figs. 1 and 2) and in 
Purkinje cells (plate 11, figs. 7-9). In a second contribution 
C97b, p. 307) he records the observation of similar bodies in 
the anterior horn cells of the lumbar region of the spinal cord 
treated by an iron hematoxylin method devised by himself (plate 
12, fig. 7). I have already shown (’12, p. 497) that these bodies 
are mitochondria. 


4 E. V. COWDRY 


Nageotte (09 b, p. 825) applied the Altmann method, the 
Benda method and iron hematoxylin as advocated by Meves 
to the nervous system. By means of all three he demonstrated 
bodies in anterior horn cells, Purkinje cells, neuroglia cells and 
ependymal cells which he believed to be mitochondria on the 
basis of their characteristic morphology and distribution. 

Schirokogoroff (18, p. 522) observed structures which he con- 
cluded to be mitochondria on account of their filamentous and 
rod-like shape, cytoplasmic distribution and independence of the 
Nissl bodies, in spinal ganglion cells, in the cells of the spinal 
cord, medulla, brain and retina by fixation in Regaud’s fluid and 
staining according to the directions of Altmann, Heidenhain 
and Benda. 

I have confirmed these observations by making preparations 
of mitochondria in Purkinje cells by Bensley’s anilin fuchsin 
methyl green method. 


Felis 


Altmann (90, p. 52) described and figured granules (bioblasts) 
in nerve cells by means of his well-known method of technique. 
An analysis of his descriptions and figures shows that some of 
these bodies are mitochondria. In plate 11, figure 3 they are 
illustrated, stained brilliantly with anilin fuchsin after fixation 
in his osmic acid mixture, in the Purkinje cells of the cerebellum 
(p. 52). They occur in the form of granules which tend to be 
arranged in rows and they are present in the axone as well as 
in the dendrites, which excludes the possibility of confusion with 
the Nissl substance. His figure (plate 13, fig. 1) and his de- 
scription (p. 538) of granules in the cells of the granule layer of 
the cerebellum are not sufficiently precise to justify the identifi- 
cation of the bodies as mitochondria; but his illustrations (plate 
14, figs. 1-2) of rod-like and filamentous structures in the wall 
of the cerebral vesicle and neural tube of a cat embryo undoubt- 
edly relate to mitochondria. 

Lobenhoffer (06, p. 491) found granules and rods, similar .to 
those described by Altmann, in the cells of the spinal cord, brain 
and retina by Schridde’s modification of Altmann’s technique. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 5 


He clearly showed their independence of the Nissl substance by 
counterstaining with toluidin blue, so that in the same cell the 
granules of Altmann were colored red and the Nissl substance 
blue. The fixation and staining reactions of these Altmann 
granules, together with their rod-like form and eytoplasmie dis- 
tribution, show that they are in truth mitochondria. 


Canis 


Lobenhoffer (’06, p. 491) also found bodies, which he likewise 
termed Altmann’s granules, in the cells of the spinal cord, brain 
and retina of the dog. I have no hesitation whatever in calling 
these granules mitochondria since they correspond very closely 
with mitochondria which I have observed in the cells of the 
Gasserian ganglion of the dog by Bensley’s anilin fuchsin methyl 
green technique. 


Gallus 


Duesberg (10, p. 612) recorded chondriosomes (mitochondria) 
in adult ganglion cells (spinal?) by the Bendamethod. He 
states, however, on the same page, that no elements stainable 
by the Benda method occur in the adult nerve fiber. 

I have found, by the application of Bensley’s anilin fuchsin 
methyl green method, that mitochondria are very abundant in 
the cell bodies and medullated processes of adult spinal ganglion 
cells of the fowl. | 


Columba 


I have already demonstrated, in a previous paper (712, p. 497), 
that mitochondria occur in adult spinal ganglion cells of the 
pigeon. 

Testudo graeca 


Busana (12, p. 621) studied granules and rods, which he 
styled mitochondria, in the cells of the spinal ganglia, cord, 
medulla, optic lobes and cerebellum by means of the Regaud 
method, as modified by Luna, and the Benda method. They 
presented different characteristics in the large and in the small 


6 E. V. COWDRY 


cells. In the former he observed them in the form of granules, 
and, in the latter, as granules and very tiny rods. This differ- 
entiation apparently (p. 620) applies to the large and small 
cells of the spinal ganglion as well as to those found in other 
parts of the brain. There can be no question that the bodies 
which he described as mitochondria are mitochondria. 


Rana 


Altmann (’90) also observed and figured (plate 11, fig. 2) 
structures in the spinal ganglion cells of the frog which may be 
identified with mitochondria for reasons similar to those already 
given in detail in the case of the cat. 

Duesberg (712, p. 809) made some observations on plastosomes 
(mitochondria) in the ganglion cells of tadpoles, but is unwilling 
to arrive at any conclusion regarding them. 


Petromyzon marinus Lin. and Ammocoetes branchialis Bloch 


Mawas (710, p. 126) investigated the structure of the spinal 
ganglion cells of these two forms by the Regaud method. His 
description apparently applied to the former. He found that 
in the adult the nerve cells may be divided into two groups, the 
large ones and the small ones. In the small cells he found gran- 
ules and filaments, distributed throughout the cytoplasm, elec- 
tively stained in black with the hematoxylin. The larger cells 
differ in that the cytoplasm is less intensely stained and that 
there are present in addition a number of vesicles which are 
considerably larger than the granules and filaments. These 
vesicles seem to be more numerous in the region of the nucleus 
but they extend into the dendritic processes as well. Where 
they are absent the granules and filaments take their place. 
He concluded that the granules and filaments of the small cells 
are mitochondria and that the vesicles are allied to them. 

It is doubtful whether the descriptions of Furst (02), Motta- 
Coco and Lombardo (’03) and Motta-Coco (’04) relate to 
mitochondria. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 7 

Furst (’02, p. 389) described peculiar rings, threads and knots 
in the ganglion cells of salmon embryos, but he studied, in addi- 
tion (p. 391), carp, Trutta fario, Coregonus laveretus, etc. He 
states that in the first place he only used Perenyi’s fluid, but 
that later he made use of sublimate acetic and other mixtures 
without obtaining such good preparations as he did with Perenyi’s 
fluid. He says (p. 389) that these structures occurred in the 
cytoplasm of cranial and spinal ganglion cells, but not in the 
ganglion cells of the brain and spinal cord, which is rather ambigu- 
ous. There is considerable difference of opmion among investi- 
gators with regard to the nature of these bodies. Van der Stricht 
(09, p. 21) thinks that they are mitochondria, while Duesberg 
(12, p. 806) states his conviction that they do not belong to 
the category of plastosomes (mitochondria). Although I ac- 
knowledge that mitochondria sometimes occur in the form of 
rings, threads and knots, I consider that since the fixatives which: 
he employed are not adapted for the demonstration of mito- 
chondria, the absence of these strange structures in the ganglion 
cells of the brain and spinal cord, together with a careful scrutiny 
of his figures are insurmountable obstacles against the conclu- 
sion that he was dealing with mitochondria. 

Motta-Coco and Lombardo (’08, p. 637) described certain 
bodies which they called ‘granulazioni fucsinofili’ in the spinal 
ganglion cells of the rabbit and frog. They state (p. 640) that 
the spinal ganglion cells contain in their cytoplasm and nuclei 
a certain number of fuchsinophile granules of variable dimen- 
sions, which are situated in the interfibrillar spaces and in the 
achromatic network of the nucleus. They employed Flemming’s 
fluid, Muller’s fluid and a solution of chromic acid and formalin, 
as fixatives. They stained with methylene blue, safranin and 
eosin and by Levi’s method of coloring with fuchsin and differ- 
entiating in alcoholic picric acid. One gains the impression that 
they believe their ‘granulazioni fucsinofili’ to be the same struc- 
tures as those described by Levi (’96, p. 3). Motta-Coco ex- 
tended the work in a second paper (’04). Duesberg (12, p. 809) 
asserts that the plastochondrial (mitochondrial) nature of these 
bodies is very doubtful, and I am in accord. with him, particu- 


8 EK. V. COWDRY 


larly so on account of Motta-Coco and Lombardo’s statement 
that the granulazioni fucsinofili occur within the nuclei. Never- 
theless, the bodies which they find in the cytoplasm may be 
mitochondria; those described by Levi certainly are. 


It may well be asked why the observations dealing with mito- 
chondria in nerve cells are so scattered and so few? The reasons 
are technical, psychological and theoretical. 

The fixatives in general use for the study of the nervous sys- 
tem exercise a destructive action on mitochondria. Mitochon- 
dria are completely dissolved, for instance, by the acetic acid 
in Carnoy’s 6:3:1 fluid. It is on account of this property that 
the acetic acid in the mitochondrial fixatives devised by Benda, 
Meves, Bensley and others is reduced to a maximum of a few 
drops only. Formalin generally destroys them unless its action 
is modified by the addition of some other ingredient. Regaud 
combines potassium bichromate with it for this purpose. Alcohol 
and corrosive sublimate are also bad fixatives for mitochondria. 
And conversely the chemicals best adapted for the preservation 
of mitochondria, like osmic acid and potassium bichromate, are 
but little used in neurological technique because of their poor 
penetration. The concentration of attention upon the nucleus 
and nuclear changes made this condition of affairs worse because 
the very chemicals which destroy mitochondria give the clearest 
nuclear detail. 

The observations which first laid the basis for our present 
conception of the significance of mitochondria were those of 
Benda (’99) on sex cells. The unfortunate lack of correlation 
between neurologists and psychiatrists, on the one hand, and 
general cytologists, on the other, has resulted in the former 
ignoring the significance of these observations of Benda, followed 
as they were by the important work of Meves, Duesberg and 
others, for more than a decade. The position assumed by hema- 
tologists is quite analogous, because we are only now, largely 
through the efforts of cytologists, beginning to hear something 
of mitochondria in blood cells. 

Meves (08, p. 845) advanced a theory which has exercised a 
dominating influence upon the trend of mitochondrial investi- 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 9 


gation. It was that with the specialization of the embryo into 
different organs and tissues, primitively similar cells assume 
special functions which find expression in characteristic struc- 
tures or differentiations. All these products, no matter how 
heterogeneous they may be, arise through the metamorphosis 
of one and the same elementary plasma constituent, the chon- 
driosomes (mitochondria). Thus the neurofibrils are, accord- 
ing to his conception, to be classified as products of mitochondria. 
Hoven’s (’10, p. 475) work on the transformation of mitochon- 
dria into neurofibrils in the developing nerve cell has generally 
been accepted (Firket 711, p. 545; Arnold 712, p. 289, and others) 
as a ratification of Meves’ hypothesis, although Duesberg (’12, 
p. 745), under whose direction Hoven did his work, does not 
claim that he demonstrated his point conclusively. Since it 
was supposed that the mitochondria became transformed into 
neurofibrils, it was natural to believe that mitochondria are 
absent in the adult nerve cell after neurofibril formation has 
ceased. Thus we find that Meves (’10, p. 655), Duesberg (10, 
p. 612) and Hoven (10, p. 478) have expressed their opinion 
that there are no mitochondria in fully developed nerve cells. 
Consequently investigators have looked for them and failed to 
find them. I repeated Hoven’s work and found that the facts 
do not justify his conclusion that mitochondria become trans- 
formed into neurofibrils (Cowdry ’14a, p. 414). Now that a 
reaction is taking place against Meves’ hypothesis (vide Gure- 
witsch 713, p. 126; Levi, ’13, p. 550; Cowdry ’14 a, p. 409, et al.) 
we may look forward to the study of mitochondria in nerve cells 
receiving the attention which it certainly merits. 


MATERIAL AND METHODS 


This investigation has been limited to spinal ganglion cells. 
Spinal ganglia were selected because they are easy to obtain, 
fluids penetrate them rapidly and because I believe that they 
constitute suitable material for experimental studies. All the 
animals, with one exception (Homo), were adults. The sex of 
the animals and the dates of the observations are recorded. 
Sizes were estimated by inserting, in the ocular, a micrometer 


10 . E. V. COWDRY 


dise, each space on which had been estimated, with the com- 
bination used (Zeiss apochromatic objective 1.5 mm. and com- 
pensating ocular 4), to be equivalent to 1.5y. Cells, the greatest 
diameter of which measured 30u or less, were classified as small, 
the others being referred to as large. 

In the study of fresh tissues the greatest care was taken to 
obtain isotonic media. Where possible the cells were observed 
in their native tissue juices without the addition of any foreign 
fluid. Occasionally, however, it was found necessary to employ 
some sodium chloride, Ringer’s or Locke’s solution. I used janus 
ereen (M. L. B.) and diethylsafranin, which I made myself 
(14b) from diethylsafraninazodimethylanilin, extensively as 
vital dyes for mitochondria; and nilblau B extra (B. A. 58. F.), 
methylene blue medicinale (M. L. B.) and new methylene blues 
GB, N, NSS, NSSF, NX, R and RRR (Cassella), which Dr. 
H. M. Evans kindly gave to me, for the lipoid globules and 
Nissl substance. 

The standard mitochondrial methods of Bensley (711, p. 309), 
Altmann (’90, p. 27), Benda (’02, p. 752), Meves (08, p. 832) 
and Regaud (’11, p. 3) were employed in the study of fixed 
tissues. The technique as originally outlined by these investi- 
gators was adhered to with as little deviation as possible. The 
temperature and time were not kept constant. Bergamot oil 
was often substituted for xylol as a clearing agent, but only after 
experimentation had shown that doing so did not alter the 
- specificity of the stain. The duration of impregnation in paraf- 
fin at 60°C.+ varied from one-half to two hours according to 
the size of the piece of tissue. Solubilities were tested by fix- 
ation in fluids containing different amounts of acetic acid, alco- 
hol, corrosive sublimate and formalin, because it is well known 
that these substances exercise a destructive influence on mito- 
chondria. This was done by fixation in Zenker’s fluid with and 
without acetic acid, Carnoy’s fluid, absolute alcohol and formalin. 

The observations were carefully controlled by comparing the 
unstained cells with vitally stained ones and permanent prep- 
arations. Where possible a number of animals of the same 
species were studied in order to eliminate individual variations. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 11 
OBSERVATIONS 


The evidence which I have gathered together for the identifi- 
cation of mitochondria in the spinal! ganglion cells of vertebrates’ 
is based on a consideration of their morphology, distribution, 
staining reactions and solubilities. It is set forth in table 1. 
The spinal ganglion cells of man, for example, contain rod-lke 
bodies; which occur in the axone and axone hillock, as well as 


TABLE 1 
Lh - wane : Se 

Seale aia ns EN eng 

3 Scie en rite aoiee < S| 2 | 

Melee zeal EiS/S) (8isis| =! 

ANIMAL ee eae st es 2 O a) S | S| MITOCHONDRIA 

Erle tealteee rected ake | elie Wiesel ete sells city NR PE | 

elalaleiel2is/e2l/ebl/e@lale|/s/B/e 

7 Aap) en ie s/Aa]Al] 4 Ol elmzaliioullion| 

sl eelele Slalelalsjalalelals 

ei/2/S/alalalalsiala/s/8]s <| 5 
Homo: 2.4.4 +/+) +| ng asic) valk elle ao alae) present, fig. 1, m 
IMACHCUS eae —itplecsl. 8) aioe} se] | + | eal) alee present, fig. 2, m 
Guinea=pig. sos) FE), ep ee] +) 4,4)? })+)-) -— —| present, fig. 3, m 
IMEDISES cerca ove S68) SS SS) SE Se Sell Sel) Sr =|] a} i) Present, she. 4 cam 
Pigeon: .):.. +/+) +) of +e +i t+ + Sl ta al al —| present, fig. 5, m 
Eutaenia...) +) +) +) +) +) +| ? + | | | present, fig. 6, m 
Pseudemys | --) --) -F) -| -F) -F) -Fl +} + —|?)—| — —| present, fig. 7, m 
AN Assis oes +) +) +) 4} +] +) H+) +) H+ eae! | —' present, fig. 8,m 
Neetumns. ii) seb eke) erly]? |? | | | present, fig. 9, m 

ee tsh | ont a | Pop Al deol 


in the cell body; which stain characteristically by the mito- 
chondrial methods of Bensley, Altmann and others; which are 
fixed by the action of 2 per cent osmic acid and are destroyed 
by the acetic acid in Zenker’s fluid. They may, therefore, be 
termed mitochondria and are represented in figure 1, m. In 
the table ‘+’ signifies a positive reaction, ‘—’ a negative one. 
‘?’ a doubtful one and, where there is no record, it is to be under- 
stood that the test has not been applied. 


3 Mitochondria also occur in the nerve cells of invertebrates. I have dem- 
onstrated them with both janus green and the anilin fuchsin methyl green method 
in the nerve cells of Callinectes hastatus, Cancer borealis, Limulus polyphemus, 
Fulgur canaliculatus and Nereis virens; and with janus green alone in Eshna, 
Loligo pealii, Homarus americanus, Venus mercenaria and Mytilus edulis. They 
are rather more variable in number, size and staining reactions in invertebrate 
than in vertebrate nerve cells. 


1 E. V. COWDRY 


Mitochondria evidently occur in the spinal ganglion cells of 
all the forms which I have studied. Their appearance, in speci- 
mens prepared by Bensley’s method, is illustrated by the figures 
on plates 1 and 2. The figures show the form relations very 
well, but the color values could not be reproduced on account 
of the expense. The following description applies only to those 
parts of the nerve cell found within the spinal ganglian. The 
peripheral and central processes have, of necessity, been ignored. 

The morphology of the mitochondria is remarkably constant. 
They vary, in all the animals investigated, from granules (0.25— 
0.754, measured in Bensley preparations) to rods (1-2y) and 
filaments (2-4). Sometimes the granules are arranged in rows. 
The rods may be dumb-bell shaped or pear-shaped and the 
filaments occasionally exhibit varicosities. I am unable to dis- 
tinguish between the spinal ganglion cells of any of these animals 
on the basis of their mitochondrial content alone. Morpho- 
logically the mitochondria in the spinal ganglion cells of man 
are identical with those of the monkey, guinea-pig, white rat, 
etc. 

The arrangement of mitochondria within the cell is subject 
to but slight variation. They are generally distributed evenly 
throughout the cell body. Occasionally they are more numer- 
ous in the region bordering on the axone hillock. This was 
observed in the human cell illustrated in figure 1. In the large 
cells the mitochondria are generally found between the flakes 
of Nissl substance, while in the small cells they are imbedded in 
it (fgs. 10a, 12 and 13). In some of the small cells the mito- 
chondria are confined to the central cytoplasm (fig. 13 b). This 
condition is rare. They tend to be oriented parallel to the cell 
wall so that they are placed more or less concentrically about 
the nucleus. The guinea-pig cell shown in figure 3 illustrates 
this very nicely. In the medullated processes they are always 
arranged with their long axes parallel to the length of the process. 
I have not studied their relations in non-medullated fibers. 

The microchemical reactions of mitochondria are likewise very 
constant. Janus green and diethylsafranin gave very disap- 
pointirg results, as compared with their action on blood cells, 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS tS 


for instance. I find that they stain the mitochondria in embry- 
onie nerve cells of vertebrates (chick), and in fully differentiated 
nerve cells of invertebrates (edible crab, Callinectes hastatus) 
much more briliantly than the mitochondria in the spinal gan- 
glion cells of the adult vertebrates which I have examined. 

The relative amount of mitochondria is apparently constant 
in the nerve cells of the different animals although there is a 
certain amount of variation among the spinal ganglion cells of 
the same animal, which could not be accurately determined on 
account of the difficulty in enumerating them. The amount 
of mitochondria illustrated in the figures would be more uniform 
than it is were it not for this individual variation. Where the 
mitochondria are few in number lipoid granules are abundant 
and vice versa. 

The relation of mitochondria to lipoid globules was studied in 
detail in the guinea-pig, but a reciprocal relationship between 
the relative amounts of the two was observed in all the animals 
investigated. It is easy enough to distinguish typical mitochon- 
dria (fig. 2, m) from typical lipoid (J) but it is impossible to 
determine whether the intermediate stages (7) are true mito- 
chondria or true lipoid. Coincident with the change in shape 
from mitochondria to lipoid there is a progressive increase in 
the resistance to acetic acid. The mitochondrial methods show 
no difference between the fixation and staining properties of 
mitochondria ‘and lipoid, but the lipoid globules are not so 
readily destroyed by fixation in Carnoy’s fluid and Zenker. By 
varying the concentration of the acetic acid in Zenker’s fluid 
a series of gradations may be obtained in spinal ganglion cells 
of the pigeon between lipoid and no mitochondria, on the one 
hand, and lipoid plus the normal amount of mitochondria, on 
the other. Both mitochondria and lipoid stain with nilblau B 
extra in the cells of all the animals, but the actual transforma- 
tion of the one into the other could not be followed. Probably 


‘{ have applied the term ‘lipoid’ (derived from the Greek, Xizos, fat) to these 
globules, loosely, without meaning to convey any exact knowlege of their 
composition. It is possible that their composition may differ in the various 
animals which I have studied. 


14 E. V. COWDRY 


this is due to the difficulty of keeping the nerve cell under obser- 
vation, in approximately normal environment, for a sufficient 
length of time. 

The lipoid globules are of about the same size in the different 
animals. They are always spherical and vary from 1 to 5y in 
diameter (measurements made on unstained cells in approxi- 
mately isotonic media). Their arrangement in the cell is per- 
fectly typical. They occur in clumps in any part of the cyto- 
plasm. They sometimes extend into the processes, but rarely 
attain a size of more than 1 when thus situated. They vary 
in amount in different cells of the same animal, sometimes being 
absent and sometimes present in abundance. The large and the 
small cells show them in equal number. Their presence is not 
accompanied by any sign of pathological change. 

The Nissl substance, also, occurs in the spinal ganglion cells 
of man, monkey, guinea-pig, white rat, pigeon, snake, turtle, 
frog and necturus. Its morphology is more variable than that 
of either the mitochondria or the lipoid. It presents constant 
differences in the large and in the small cells (30u or less, meas- 
ured in fixed tissues) of man, monkey, guinea-pig and white 
rat. I am not yet prepared to make definite statements about 
the other animals. In the cells, which have been fixed, it occurs 
in irregular aggregates of variable size and shape, which are 
absent in the axone hillock and are larger about the periphery 
than in the more central parts of the cell; but in small cells it 
is generally (though not invariably) present as a diffuse, con- 
tinuous, amorphous deposit. All gradations exist between these 
extremes. Figure 10 shows some cells occurring in a single 
section of a guinea pig’s spinal ganglion prepared by Bensley’s 
method. At the top the diffuse condition is seen, at the bottom, 
well formed Nissl bodies. It is not due to the distance of*the 
cells from the surface, and resultant variations in the rate of 
penetration of the fixative, because the cell shown in figure 12 
is in actual contact with the one illustrated in figure 10d. They 
both typify the condition mentioned although the fixative acted 
on them both in the same way and at the same time. Fixation 
in 2 per cent osmic acid and in Altmann’s osmic bichromate 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 15 


mixture gives similar results. When sublimate acetic, picro- 
sulphuric or Carnoy’s fluid are used, in place of the acetic osmic 
bichromate mixture of Bensley, the Nissl substance occurs in 
the form of discrete, well formed masses in both types of cells. 
This is shown in figure 11, drawn from a spinal ganglion cell of 
a guinea-pig fixed in Carnoy’s 6:3:1 fluid and stained with anilin 
fuchsin methyl green (compare figs. 10 and 11). Attempts to 
devise a fixative which would give a uniform Nissl substance in 
the large cells failed. The diffuse Nissl substance in the small 
cells stains with variable intensity with basic dyes (methyl green, 
fig. 13, a and 6). 

All my attempts to see formed Nissl bodies in unstained cells 
were unavailing. The least toxic methylene blues (methylene 
blue medicinale and the new methylene blues GB, N, NSS, 
NSSF, NX, R, and RRR), applied to spinal ganglion cells of 
the frog (chosen because with a cold-blooded animal the warm 
stage may be dispensed with), give first a diffuse coloration of 
the cytoplasm followed by the appearance of irregular masses 
of stained material, which look something like the Nissl bodies 
of fixed tissues. JI was unable to determine whether these blue 
stained bodies represent pre-existent structures in the cytoplasm 
which are invisible in the unstained cells by virtue of their low 
refractive index. 

Pigment was seen only in the spinal ganglion cells of Necturus 
and Rana. It has a bright orange color in Necturus and occurs 
in a variety of forms. Sometimes as highly colored masses 
(0.5-1u measured in fresh, unstained cells) which tend to fuse 
together in a conglomerate way (1-6). It may occur as spher- 
ules of variable size (0.5-10u) and intensity of coloration; sickle- 
shaped bodies (4.54 long by 0.5u wide), threads of variable 
length and diffuse masses may also be made out. It varies in 
amount in different cells of the same animal and in different © 
animals. The pigment in Rana is bright orange in color. It 
occurs in the shape of globules and angular masses of consider- 
able range in size (0.5-1.5u) either distributed evenly through- 
out the cell or else gathered together in clumps. It is absent 


16 E. V. COWDRY 


in the processes. No sickle-shaped bodies were seen. The pig- 
ment is Just as abundant in the small cells as in the large ones. 
No relationship was observed between the pigment and mito- 
chondria, although I would not deny that such may exist. 


DISCUSSION 


The analysis of the literature and the observations which I 
have recorded show that mitochondria occur in the nerve cells 
of representative examples df the chief vertebrate groups. Their 
properties are so constant that the spinal ganglion cells of man 
cannot be distinguished from those of any of the other forms, 
which I have studied, on the basis of their mitochondrial content. 

A similar condition prevails in the developing nerve cell, in 
which I have found mitochondria in stages from chick embryos, 
before the differentiation of any somites, to adult fowls. My 
series consists, in addition to the embryos already described 
(Cowdry ’14a, p. 397), of preparations of four-day embryos, 
of chicks just hatched and adult fowls. Unfortunately the later 
stages are not very close together, but the series, as a whole, 
is sufficiently complete to show that the relative amount, micro- 
chemical properties and arrangement of mitochondria are approxi- 
mately constant, although their morphology changes progres- 
sively. In the spinal ganglion cells of a 35-somite chick, for 
example, their average length is from 3 to 5u, but in the spinal 
ganglion cells of an adult fowl they are seldom more than ly 
in length. These measurements were made on fixed and stained 
material. 

I believe that this constancy of mitochondria in the phylogeny 
and ontogeny of the nerve cell is significant, for it may serve 
as a clue to their function. Their presence, the constancy of their 
microchemical properties, relative amount and distribution within 
the cell in the different stages of evolution and development may, 
perhaps, be explained on the supposition that their function is 
common to these same stages. It must be, therefore, a funda- 
mental and a basic function, inseparably connected with the 
life of the cell and subject, of course, to qualitative as well as 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS iL 


quantitative fluctuations. Mitochondria occur in all parts of 
the nerve cell, in the axone as well as in the dendrites, for these 
basic chemical reactions to which I refer are common to the 
whole protoplasm. Herein the mitochondria differ from the 
Nissl substance, which we must look upon as a more specialized 
cell organ. While I regret and deplore the absence of experi- 
mental evidence, I nevertheless feel myself justified in enter- 
taining, on these grounds, for the time being at least, the hy- 
pothesis that mitochondria are concerned with the metabolism 
of the nerve cell. 

This conception is supported by evidence from analogy con- 
cerning mitochondria in other than nerve cells. "They may almost 
(but not quite) be regarded as coexistent with protoplasm. They 
occur, with few exceptions, in the cells of plants (Guilliermond 
12. p. 412; Maximow 713, p. 242, and many others) as well 
as in those of animals. They are transmitted from one gen- 
eration to another through the medium of the egg, and, in 
some cases, of the sperm also (Meves 713, p. 225). On cell 
division, whether it be by mitosis or amitosis, they are dis- 
tributed in approximately equal amounts to the two daughter 
cells (Romeis 713, p. 17). Cells in which mitochondria do not 
occur are less numerous but no less instructive. All at- 
tempts to demonstrate them in bacteria (Guilliermond ’11, 
p. 200), in the most superficial cells of the epidermis (Firket 
11, p. 544) and in the circulating red blood cells of man (Cowdry 
’14b, p. 17) have failed. Moreover, Guilliermond (12, p. 379) 
states that he cannot demonstrate mitochondria in the later 
stages of the life cycle of barley, wheat, maize, bean and pea. 
Now bacteria are primitive organisms in which the occurrence 
of a nucleus is disputed, and the epidermal cells and blood cor- 
puscles are, like the cereals mentioned by Guilliermond, terminal 
stages in cytomorphosis. We may conclude, therefore, that 
mitochondria are present in the majority of actively functioning 
cells, that they decrease progressively with a diminution in cell 
activities and that they are absent in the most primitive organ- 
isms. In other words, that, so far as we know, the ground 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


18 E. V. COWDRY 


substance, alone, of the constituents of the cytoplasm is more 
fundamental. These considerations, coupled with the fact that 
the mitochondria, wherever they occur, show a certain uni- 
formity with respect to their morphology and microchemical 
properties, to my mind, support the view that they play a part 
in metabolism. 

We do not know in what way they may be associated with 
metabolic processes in nerve cells, but I believe that Fauré- 
Fremiet, Mayer and Schaeffer (710, p. 95) have furnished us with 
a clue. They found, by a detailed study of solubilities, fixatives 
and stains, that mitochondria are chemically a lipoid albumin 
complex. This is of course a vague statement, because there 
are many different sorts of lipoid and a multitude of albumins, 
but it is nevertheless of importance. The reciprocal relations 
which I have described between mitochondria and lipoid, the 
observations of Mawas referred to on page 6, those of M. R. 
Lewis and W. H. Lewis (14, p. 332) on tissue cultures, and, 
above all, the artificial imitation of mitochondria by Lo6wschin 
support this view. Lowschin (713, p. 203) made the so-called 
‘‘Myelinformen” of lecithin in water, different salt and albumin 
solutions (resulting in the formation of lecithalbumin) which 
showed masses with the same morphology as mitochondria. He 
observed granules, rods, threads and rows of granules. The 
granules divided directly and the threads longitudinally. He 
was able to influence their form by changing the physicochemical 
properties of their environment. They were soluble in acetic 
acid and could be fixed by formalin, osmic and chromic acid. 
In a later paper (14, p. 269) he discovered that particles of 
lecithin and cholesterin suspended in glycerin-gelatin, when fixed, 
stained like mitochondria by the various mitochondrial methods. 
Kingsbury (711, p. 316), also, has emphasized the similarity which 
obtains in the microchemical reactions of mitochondria and lipoid. 
I find, moreover, that janus green, in addition to being a vital 
dye for mitochondria, stains both lecithin and egg albumin 
(Kahlbaum), the latter more intensely. 

Notwithstanding the astonishing general similarity of mito- 
chondria in diverse types of cells slight, but perplexing, varia- 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 19 


tions do occur in their morphology, resistance to acetic acid and 
other’ fixatives and in their staining properties which require 
to be explained. It is here, I believe, that a knowledge of their 
constitution helps us, if we postulate slight variations in the 
relative amounts of ipoid and albumin. Not only the amount 
but also the properties of mitochondria vary with changes in 
metabolism. This is true in spermatogenesis where Regaud (’08, 
p. 661) has detected a progressive increase in the resistance of 
mitochondria to acetic acid. We may here be dealing with an 
increased consumption of albumin which would tend to increase 
the relative amount of lipoid in the mitochondria and in this 
way increase their resistance to acetic acid. The failure of 
Jordan (711, p. 59) and Wildman (713, p. 427) to observe mito- 
chondria in the early stages of spermatogenesis may thus be 
explained because they used fixatives with a constant percentage 
of acetic acid, which perhaps destroyed the mitochondria of low 
resistance and left the others. It is possible that the formation 
of lipoid in nerve cells is a process essentially similar but carried 
to an extreme. In any case the importance of being able to 
see In the living nerve cell and in many others deposits of the 
nature of mitochondria, by means of the vital dye janus green 
can searcely be overestimated. 

The Nissl substance is, in a sense, a more specialized con- 
stituent of nerve cells. Recent investigation has shown that 
material very closely allied to it occurs in many types of cells, 
muscle cells and gland cells for example. Substances of this 
sort are grouped together and called ‘chromidia.’ Bensley’s 
demonstration (11, p. 359) that the chromidial substance is 
present in the living acinus cells of the pancreas as a continuous 
deposit which would lead one to believe that Held and others 
(vide Barker ’99, p. 131) may be right in their assertions, which 
have been too frequently ignored, that the Nissl bodies, instead 
of being pre-formed elements in the living nerve cell, result from 
the coagulation (or precipitation) of a substance present in a 
diffuse, amorphous state. I have recorded some observations 
which apparently support this notion, so far as the spinal ganglion 
cells of vertebrates are concerned, my experience with other types 
of nerve cells being too limited to justify any assertions. 


20 E. V. COWDRY 


I have failed to observe Nissl bodies in unstained spinal gan- 
glion cells teased out in isotonic media, and the vital dyes which 
I have used (Methylene blue medicinale (M. L. B.) and New 
Methylene blue GB, N, NSS, NSSF, NX, R and RRR) give 
first a diffuse staining of the ground substance followed by the 
appearance of typically blue stained Nissl bodies, which look 
very much like coagula. I would be inclined to interpret the 
gradations which I have observed in fixed preparations between 
the diffuse Nissl substance in the small spinal ganglion cells of 
the guinea-pig and the well formed Nissl bodies in the large 
ones (fig. 10, a, b, ¢ and d) as due to a difference in the coagula- 
bility of a Nissl substance originally present in the diffuse state 
in all. The fact that by the use of other fixatives, which are 
perhaps more energetic coagulants or precipitants, formed Nissl 
bodies may also be seen in the small cells (fig. 11, a) supports 
this view. 

On this supposition the fact that the Nissl bodies are larger 
in the peripheral cytoplasm would easily be explained because 
the action of the fixative is more powerful there. The‘chromo- 
phile cells’ (Barker ’99, p. 123) may be interpreted as cells in 
which the coagulability of the diffuse Nissl substance is reduced. 
I do not desire to draw a sharp line of demarcation between the 
terms ‘coagulation’ and ‘precipitation.’ Researches on a differ- 
entiation between functional groups of nerve cells, on the basis 
of the appearance of their Nissl bodies as seen in fixed prepara- 
tions (Malone ’10, etc.) stand as genuine contributions, even 
though they be interpreted on a hypothetical difference in coag- 
ulability due to a quantitative or a qualitative change in the 
Nissl substance or both. For if the diffuse Nissl substance is 
present in different concentration in certain types of cells, the 
coagula resulting from similar fixation may be different. Or it 
may be that the Nissl substance actually differs in kind. The 
great mass of work which has been done on nerve cell physi- 
ology, with the Nissl bodies as indicators (Dolley 713, ete.), is 
best interpreted on the first supposition, of a quantitative change; 
for here, coincident with the reduction in the Nissl substance in 
fatigue, the Nissl bodies become smaller and present a more dif- 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS Zl 


fuse appearance (chromatolysis). In other words, with the dis- 
charge of function the concentration of the diffuse Nissl substance 
is lessened and its coagulability decreased so that the aggregates 
become smaller and smaller until finally no coagula result. The 
amount of chromatolysis would be inversely proportional to 
the concentration of the Nissl substance and its degree of 
coagulability. 


RESULTS 


1. Mitochondria occur in the spinal ganglion cells of man, 
monkey, guinea-pig, white rat, pigeon, snake, turtle, frog and 
necturus, in which they are characterized by the constancy of 
their morphology, distribution, relative amount and microchemi- 
cal properties. 

2. There is a reciprocal relation between the amount of mito- 
chondria and lipoid granules in the spinal ganglion cells of these 
vertebrates. 

3. The coagulability of the Nissl substance, on fixation, in- 
creases progressively in the gradation which exists between the 
small and the large spinal ganglion cells of man, monkey, guinea- 
pig and white rat. 


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EXPLANATION OF PLATES 


All the figures were drawn by me with Zeiss apochromatic objective 1.5 mm.., 
aperture 1.30, compensating ocular 4 and camera lucida. They were reduced 
one-fourth in reproduction so that their magnification as they now appear on 
the plates is 960 diameters. Only spinal ganglion cells, cut about 4y in thickness, 
are represented. I wish to acknowledge many helpful suggestions from Mr. 
James F. Didusch. 


ABBREVIATIONS 


c., canalicular apparatus 7., Intermediate between (m) and (lL) 
l., lipoid s., Sheath cells 
m., mitochondria n., Nissl substance 

PLATE 1 


EXPLANATION OF FIGURES 


lto4 These figures are intended to illustrate the generality of the occurrence 
of mitochondria in the spinal ganglion cells of different vertebrates. All the 
drawings have been made from specimens prepared by Bensley’s method, by 
which the mitochondria and lipoid are colored red and the Nissl substance green. 
Unfortunately the colors could not be represented. 

1 Homo, colored, female, 2 years (?). Anatomical diagnosis: miliary tuber- 
culosis, tuberculous pneumonia and terminal acute bronchopneumonia; Johns 
Hopkins Hospital autopsy no. 4095; fixed 4 hours after death; (this material was 
obtained through the courtesy of Dr. Whipple of the Johns Hopkins Hospital). 

2 Macacus rhesus, female, adult; chloroformed March 11, 1914. 

3 Guinea-pig, female, 332 grams; decapitated March 24, 1914. 

4 Mus norvegicus albinus, female, length (snout to root of tail) 19ems.; decap- 
itated February 27, 1914. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS PLATE 1 
E. V. COWDRY 


E. V. C. del. 


PLATE 2 


EXPLANATION OF FIGURES 


5 to 9 The preparations illustrated on this plate are also designed to show 
the comparative distribution of mitochondria in the spinal ganglion cells of verte- 
brates. All of them have been drawn from preparations made by Bensley’s 
method. Plates 1 and 2 should be compared. 

5 Pigeon, adult; decapitated, April 18, 1913. 

6 Eutaenia sirtalis (garter snake), male, total length 62 cms.; decapitated 
April 21, 1914. 

7 Rana palustris, adult; decapitated, January 22, 1914. 

8 Pseudemys hieroglyphica (terrapin), male, length 17 cms.; decapitated, 
March 7, 1914. 

9 Necturus maculatus, female, length (snout to anus) 23 ems.; decapitated, 
March 26, 1914. 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS PLATE 2 
a. V. COWDRY 


E. Y. C. del. 


PLATE 3 
EXPLANATION OF FIGURES 


10to 14 These figures illustrate the difference in the coagulability of the Nissl 
substance in the large and small spinal ganglion cells of the guinea-pig and mon- 
key. All, except figure 11, have been drawn from specimens prepared by Bensley’s 
method. Figure 11 is from a guinea-pig’s spinal ganglion, fixed in Carnoy’s 
6:3:1 fluid and stained with anilin fuchsin methyl green (p. 15). 

10 Four cells from a single section of a guinea-pig’s spinal ganglion, prepared 
by Bensley’s method, showing the graduation between the diffuse Nissl sub- 
stance (n.) in the small cells (a and 6) and the sharply defined Nissl bodies in the 
large ones (c and d). The mitochondria (m) are the same in both types of cells 
(p. 14). 

11 Two cells from the spinal ganglion of a guinea-pig fixed in Carnoy’s 6:3:1 
fluid and stained with anilin fuchsin methyl green. Here the Nissl substance (n) 
is present in the form of discrete granules in both the large and the small cells. 
The mitochondria are absent, having been destroyed by the fixative (p. 15). 

12 From a small spinal ganglion cell in direct contact with the large cell 
illustrated in figure 10d. The Nissl substance within it is present as a diffuse 
deposit (p. 14). 

13. Two small spinal ganglion cells of a monkey (same animal as fig. 2) to show 
that the diffuse Nissl substance often stains with variable intensity. It is much 
lighter in (a) than in (b) (p. 15). 

14 Spinal ganglion cell, from the same preparation, also showing the diffuse 
Nissl substance (7) axone (p. 15). 


PLATE 3 


MITOCHONDRIA IN VERTEBRATE NERVE CELLS 


E. V. COWDRY 


E. V. C. del. 


THE ANATOMY OF A 17.8 MM. HUMAN EMBRYO! 


fF. W. THYNG 
From the Department of Comparative Anatomy of Harvard Medical School 


EIGHT FIGURES 


CONTENTS 

littEO GIG DLONRE Re Hee Soh creer ee EOC Se ert ye ar mere Ec Ad) iio. a ER 33 
EE TMA Ise a bUTCSs ice eee ean ne ae ana cement AE. cin a Pein 33 
TD Wa SRL aINVEUTENASLAEN ANG gi ce alee acne Sons ceey cits ig bes ae ae ee RS tO 35 
<Oseral orate Bern eat ates cre to cas hug tore Baud ollie ctmats A eg eee ats Paneer ita 3 35 
Haase ect Mieco. Senn AO eRe ei Tee, Whe atm tee tame Oued cena ae 36 
Salient ane VANACLS ssn Sc et thase e aPsch seen o Mune AMER 2. a Ae 40 

@ eso pliaig Wistar ny ete eee ce ose he Pe er Ey nie ota J Yok Gs A ee ee 40 
SION al mts te ORF ren Sc tec eee Ne ee kt RRSP Monte vtec Ree meee ed 41 
TELAWEPSISU TITANS sive Goad aes aiken re ttre eee ok Ae 8 2 Oe OR oe ea aie Vee et eee ee a 42 
JCA SBR athe de ecacpr aks peatavetie ctr aR RNR eas Lo city Ace aU Raa PR a ORD Daten ce 43 

JAR AVOIO RIS a Laika nh achat Sea OF REEMA Calera SATE 5, SEU ee RR i OR ge Unt at 44 
JOS] OMAK ONEY 2 EV SUEIO ON Cook siocns ses chek moe Steak eM ee lett Ree ee ce BW Rn ma aes 45 
BTR eat We remenen rere Poked cae Ne, a Ns ob acres RARER AAS. vas, ohcSee he. tach ey set ee 45 

Mer ACh C aeeaey a eee Ie iepce se fre eeiecee ns Ses eee eR = RE EO ce ere to ane Meena 45 
JOAUUGYERS) = ces acu unatteaperaes acta a ERC N tee ERT CooL As ania Cone earrtae , AEENCES Bw es 45 
Pleualee anaes: hes ee Geet yt eS Ob erage nes oh beer teks ete, oc 45 
HOO eMC G GOIN Mme yo iia. attest, edhe | tye he ope a Csicete Utes odtoce face ayn tae Re 46 
Wiolttrarnenia cena uO Ciyigsts a coca cls cos Rae Teabags niceranic seeks hei Seas bi ane Cl 46 
VWROUU ERE GC NOG) ger a Shaken IBD ab. Rae CDC ay ect 1372 ie cance oP TGC WO Be OMe 46 

IN hiallereilarne lie Gaye estsce ea eh A coe aN tea he UR ees AUR ges 47 
Bill ere ee we sists Naas Poe Coo ect Bact! Mati Sere tae th Rowers. ia apne Ms 47 
Wirogennpallsimushaace camer 1A tee mee a RES ieee eta LEE PRE Pacts eee 47 
INE Came DEO SMart cco bee rot eee BAe hry ak eran eee anes eae en PRN ec ee a 48 
(OKCTRTUDNL CIB NERS Shae Ene cre ert Se SO EE pe I each re ie a A RR ae rE es 48 
ten essere mr Loe cc NT ie Cte a tk ate Wels hes iN, Sy Als oe date ee rhe 48 
SS (CIO RRARG TAU FEW Feta eset nr enue enlist em OO ca. Rye me Peet Nel Ce ean 48 
HOME Oi. 25.5 LS See ERE VOR EE LAL ie Gee TONE 50 

IN EIAVO TUS SRP SUSIE INE eek eet ec Aue no Raced ta Ra 9 Ne Fae Penh PP 50 
LENBEND > ould gua aR Og aT a toot ae SOO gd a 50 

BEA Grace melanie bre ey.n Teh Se keira, Ee wien iy LR hake yt ibaa eb duty: 50 

ID AUCTIONED Koa See ene Re enh, eRe a ge ne 51 

ule sernerars lniy! orn epee an eee Rae eke Oi Oy Mews ek GOP ae ee oe 51 
LSUGINETE 6 a5 57h 0 REN ee cet ee ee te 52 

IME femcerial meine Ne te Ae eo eet) oe De es 52 
Mivoler Ge ten ume meer Soe, on ks Nn on Ree ea, A eee oe a aas bh atlanaeee 53 


‘This work has been aided by a grant from the Elizabeth Thompson Science 
Fund. 
31 


32 


iS) S]LAI TGR CLO) 00 Dee ren RIM eia. Shad mio omiin Ours Ol Coren omit oleh orton on Soe ranels eros O C 54 
@ranral Werves s24 2 ciict ah eR ere oe eee Bo NAN eae ae Oke oc rare 54 
ININ:. OLLA GE OMILS Se Svea reset Boeri oe te tore, ets sce ar ica ear eae 54 
ING: “OD GHCUS ors Aero eecoe R eeLe S  a tgete 54 
IN|. OCUlOMOLOTIIS# seem rees er teat o Seeclens Fa stake eee eee eee 54 
N.S GE OCHIC PISO Aree sate wee eee perc ok ak ws ocatee cs RI Oe ro Se 54 
IN. . EEN @ON UMS pacha cela oses) Sen Sar oss a. ois wn eo cle tn genes Gree ES 55 
Ne abducenisa eect roe oes sake ects nce otitis er eee 56 
INE Pa Cia liste cerns epee tral peers Src Ayigtits s,s Reviaireye: uadesaa seo rok ae Aa 57 
ING SA CUS EL CUS eae eever terete tes oer Ghe vie a ws saat otcle Sie a Ri Opa ae 57 
Necclossophamymeecusten sot otc atiecae? aoe neo Gee hero ee 58 
IBN GI AUIS Giy Rerutp ou ataiog 6 dine eae Ree Rea ee naman PG TERS aa onO otns'o o - 59 
INGBAICCESS OTIS PRR Roe frie ass tachi AA.» GlrbIN ISR IAC COTE a re ae ee 63 
Neg VOPLOSSUB ts Bees tene © wel Ass so oe tbe wae ee ee oe en 64 

Spimallmenyes saree cere aera oe sees ose Sean te ae eee ee 65 

ODN) CSHENICE Isis a eho ead ro eee VaR one Reno: oa ties Say ERomp rs alka ov < 65 
EMSC LORE aise ee eee ee Ree ates wie 2 Sic va aveletd HSE SiG ere SS EO ve ee 68 

Hees Se Me a aa eae ee ae ead ee mR Mereee IN ee ee Weim oS 68 

| DEN iia a cas eats crete tiated ae ee eee ae a ee We Rl het dan ox 68 

IN oon Ae Racin c Sates ep eC eee een chs Ke tore ne GiGi one mad o0'0 de 69 

Vascularisiysthemisner cepcimecscs @. iat se Suck eke ooee Stare ger Rene ae ae 70 

15 [EER rR ASS o. Msves As choke okt el Ren ie tortoise at eee, etn SAU ola no 7G 
SIMUSMVEMOSUS eee cea ake eens kod ae ee re a 
Red aA EN UNO Gs, ees Ree eRmran eee Ares chon AonB con halen ia aa © 70 
IRCA HELO TOIKG! (Wy seo te GARNER ore a's, ie lo Rie o romb'aacut bp,on dion oath. ql 
NetGear trite ey cls.) vss,1cte ease Ae ACRE eo ee ee ee 71 
The hibpvemuniel Gerace cia ae = doves ah eo ee ae er orc ee al 

(AT LCTICS MOE TRE ORs ae i oe ae RO ORO ee 72 
Systemucqderivatives of the aorticsarehest-c.-% - 1. 5. eee eee 72 
Palmom ayn anvenies:\..\ 2.5 emcee tele sem eae moe cee bee ee eee 76 
Derivatives of the occipital and cervical segmental vessels.......... 76 
Parietaluarteries of the tramk.....)..21.042 ences ee ode oe 
VaASCeral@anvenleStmnciace cae Stan cae eee ee ee eee 79 
Were s AHETICS ea Meer ns acs vo clea ARIA Y Slord ete Bie ee ee 82 

\WGTO EES oan ested SSRIS Gk ae ee OE ME en EID VT eo os 83 
Antenlonand commonicardimal system sqse eee eee eee eee 83 
Rostenionicandimal’sy.stemiy...7.¢on otek c on. eee eee 87 
‘PRontalSsyis hemmee cra ae dees ou isaacon vend Haske sia. ease ee aa ROS on ones Pena 90 
Pulmonary veins (see left atrium). 

I Lina) of NEN ACIS eBags eecunnce Ra he to cee ore a REC CRTRAR e piea rao Arar BARA AC cal 3 Gd 91 
SEVCOUE) TUNATI RAI SONG, oS occ oy oueen bao b oe sdonoeeEsEebavoeuoLe 91 
SACCuUss UTM ATISNMe LOIN, Wet crce Cains sehen oe Oe rae lon Tee 91 
SAC CUSUMESEMUCE CU Stet: so ve iar et tai sPecuat aaa ee nea Ree oa 92 
@istern ac ly lites ee esse thew leks Ws Waneihe s clelis on SeA ESE CR ee 92 
Saccialyamphaticignostenlionesann cata iccs ae. Selec ae ee 92 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 33 


INTRODUCTION 


This work was undertaken at the suggestion of Prof. C. 8. 
Minot, and carried on for the most part in his laboratory during 
the years 1906 and 1907 when the author held an Austin Teach- 
ing Fellowship in Histology and Embryology at the Harvard 
Medical School. Its completion, unfortunately delayed by 
other work, has been accomplished in the Anatomical Depart- 
ment of the University and Bellevue Hospital Medical College. 
During the progress of the work many helpful suggestions have 
been received from Professors Minot and IF. T. Lewis of Harvard, 
and Prof. H. D. Senior of Bellevue, for which I am very grateful. 
I also desire to express my gratitude to Mr. W. T. Oliver of 
Lynn, Massachusetts, for the careful manner in which he has 
reproduced in finished form my original drawings. 

The reconstructions upon which this work is based, were made 
from transverse sections of Embryo 839 of the Harvard Embryo- 
igrical Collection, chiefly by the modified graphic reconstruction 
inethod of His. The shading usually has been inferred from a 
study of the sections, but in a few instances wax models were 
made of regions requiring a fuller interpretation. 

This embryo (extra-uterine) measured in formalin 17.8 mm., 
greatest length, with a neck breech of 16.7 mm. The greatest 
length in 80 per cent alcohol was 13.6 mm. In previous papers 
in which this embryo was referred to (Thyng ’08, and Lewis and 
Thyng ’08), the latter measurement was given. 


EXTERNAL FEATURES 


The external features of this embryo are seen in profile view 
in text figure 1, a reproduction of figure 104 in Minot’s (’10) 
“Laboratory text-book of embryology,” also in part in plates 3 
and 5. The neck-bend is approximately a right angle; the 
cephalic flexure is also very nearly a right angled bend, so that 
the oral aperture is in close proximity to the cardiac region. The 
dorsal flexure has disappeared almost entirely, only a. slight 
elevation persisting to mark its earlier position. Above this 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


34 F. W. THYNG 


Text fig. 1 Human embryo of 17.8 mm. (H. E. C. 839). X 5 diams. (after 
Minot).? 


elevation there is a shallow depression, said to disappear in the 
course of development. 

A distinct groove, extending transversely between the medial 
angles of the developing eyes, separates the forehead from the 
root of the nose. The maxillary process of either side has joined 
the adjacent lateral and median nasal processes, obliterating the 
naso-optic grooves. The nares are open, but separated by a 
rather low, broad septum. <A triangular space still intervenes 
between the globular processes so that the median region of the 
upper lip is not well differentiated. The median groove between 
the ventral ends of the mandibular arches has been obliterated, 
but differentiation between the chin and lip regions has not 


2A reproduction of figure 104, page 153 of ‘Laboratory Textbook of Embry- 
ology,’’ Charles Sedgwick Minot, edition of 1910, published by P. Blakiston’s 
Son and Company, Philadelphia. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 35 


occurred. The line of fusion between the first and second bran- 
chial arches is marked ventrally by a transverse groove, dorsal 
to which is seen the fossa conchae. The grooves between the 
other branchial arches have disappeared. 

The limb buds extend nearly perpendicularly to the longitu- 
dinal axis of the body. The upper project slightly beyond the 
ventral border of the body, and show a differentiation of arm, 
forearm, and clearly outlined digits. The latter protrude slightly 
beyond the border of the hand-plate. Upon the lower limb 
buds are slight indications of developing toes. 

Circular thickenings of the epidermis on the lateral body walls 
mark the developing mammae. In section these thickenings ap- 
pear slightly convex on the surface, and project into the under- 
lying mesenchyma. The umbilical cord as it leaves the body 
wall bends towards the right. 


DIGESTIVE SYSTEM 


Oral cavity. The primitive oral cavity appears in median 
sagittal section in plate 1, but is represented more fully in plate 
4, a portion of the tongue having been cut away. It is a short, 
dorso-ventrally compressed passage consisting of a roof and a floor, 
the epithelia of which meet laterally at the angle of the mouth. 
The external aperture of the primitive oral cavity, the rima oris, 
(R.or.) is indicated as seen from the exterior in plates 2, 3 and 
5; as seen in median sagittal section in plates 1 and 4. It isa 
narrow, horizontal expanded orifice, concave dorsally where it is 
bounded by the fused maxillary and median nasal processes, 
convex ventrally where it is formed by the united mandibular 
processes. 

Dorsally the oral cavity communicates with the pharynx, the 
division between the two being marked in the median line on the 
roof by the stalk of the hypophysis (Hyp.) While the roof, as 
thus bounded, is of considerable extent, the floor is very lim- 
ited, consisting of merely the anlage of the lower lip and teeth. 

The lip-grooves are just beginning to indent the oral epithe- 
lium. The lower lip-groove is seen in sagittal section in plate I. 


36 F. W. THYNG 


Into the anterior part of the roof of the primitive oral cavity 
open the large primitive choanae (Ch.pr.) of the olfactory vesicles, 
separated from one another by a primitive nasal septum. The 
roof also presents on either side of the median line, a prominent 
longitudinal ridge, the palate process (Pr.pl.). This process of 
the right maxillary arch is clearly shown in plate 4 where a por- 
tion of the tongue has been removed. It begins at the intermaxil- 
lary process (Pr.i.m.) and extends dorsally, lateral to the choana 
(Ch.pr.) along the primitive oral cavity and the cephalic part of 
the pharynx. Its free ventral border nearly reaches the floor of 
the pharynx in the region of the alveolo-lingual groove. The roof 
of the oral cavity between the palate processes thus forms a high 
arch which receives the dorsum of the developing tongue. It is 
evident that the primitive oral cavity now comprises a portion 
which will be cut off later by the union of the palate processes 
and nasal septum, to form in part the nasal cavities of the adult. 

The hypophysis (Hyp.), alluded to above, consists of a distal 
spade-like portion, connected to the oral epithelium in the median 
line by a slender stalk with reduced lumen. It is represented in 
side view in plate 4, in median sagittal section in plate 1. Its 
flattened body impinges upon the ventral surface of the infundib- 
ulum, on either side of which it projects dorsally as a short, 
blunt process. On its cephalic surface there is a distinct ridge 
or fold, continuous with the anterior surface of the stalk. 

Pharynx. <A left lateral view of the entodermal wall of the 
pharynx is seen in plates 2 and 5. The interior, as it appears 
in median sagittal section, is represented in plate 1. It isa 
broad, dorso-ventrally compressed canal which narrows rapidly 
in passing caudally to divide into trachea (7T’r.) and oesophagus 
(Oe.). The epithelium of the roof and floor of the pharynx meet 
to form an external ridge, which extends from near the angle 
of the mouth to the Jateral border of the oesophagus. Corre- 
sponding with the ridge there is an internal furrow. 

The tongue (plate 1) is a comparatively broad elevation of the 
floor of the pharynx, composed of a large cephalic part (t’) in- 
timately fused with a smaller caudo-lateral division (t’’), the root. 
A surface view of the dorsum of the tongue would show the line of 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 300 


fusion of these two parts to be a V-shaped groove with the apex of 
the V pointing caudad. In plate 1, the apex and right limb of this 
groove, sulcus terminalis, are represented, the apex marking the 
place of origin of the median thyreoid gland. Laterally the tongue 
is bounded by deep alveolo-lingual grooves which converge ceph- 
alad so as to separate its tip from the subjacent mandible. 

According to Kallius (01) the anterior anlage of the tongue is 
derived chiefly from the dorsum of the ventral ends of the man- 
dibular processes, the so-called lingual folds, the tuberculum 
impar of His contributing only a small part thereto. Hammar, 
however, believes (’01), that the tuberculum impar is a transitory 
structure, and that the tip and body of the tongue are formed by a 
considerable area of the floor of the oral (pharyngeal) cavity. In 
regard to the development of the root of the tongue there is some 
disagreement in that His derived it (’85) from the ventral ends 
of the second and third visceral arches, while Born (’83) and 
Hammar (’01) limit it to the second arch. 

Posterior to the root of the tongue, and fused with it, there 
is a broad, bilobate elevation (Hp.) which represents the epi- 
glottis. It is a derivative of the third visceral arch (Born ’83, 
and Hammar ’01). 

The first pharyngeal pouch (Ph.P.1, plate 5), the cavity of 
which ultimately, will form the tuba auditiva and cavum tym- 
pani, is seen at this stage to be an extensive, lateral, pointed, 
evagination of the pharyngeal wall, extending somewhat dorsally 
toward the depression of the primary meatus acusticus externus. 
It presents three surfaces, dorsal, cephalo-ventral, and caudo- 
ventral. The dorsal surface, which cannot be seen in the draw- 
ing, is triangular in outline; medially it passes over into the dorso- 
lateral wall of the pharynx. Dorsal to it is the cochlear division 
of the otocyst. The caudal boundary of this surface is the poste- 
rior tympanal ridge. The cephalic boundary is marked by a 
ridge overlying the tubo-tympanal sulcus of Moldenhauer (’77). 
This ridge extends from the tip of the first pharyngeal pouch in a 
cephalic and medial direction to the oral epithelium between the 
hypophysis and the angle of the mouth. The cephalo-ventral 
surface of the first pharyngeal pouch is concave. A dorso-ven- 


38 F. W. THYNG 


tral ridge, representing the entodermal part of the first closing- 
plate, separates it from the caudo-ventral surface. Upon the 
caudo-ventral surface near the tip of the pouch there is a slight 
groove. This, the ‘tensor groove’ of Hammar (’02), marks the 
place of formation of the tensor tendon. 

The second pharyngeal pouch (Ph.P.2, plate 2) appears on 
either side as a low evagination from the lateral pharyngeal wall. 
It is situated just caudal to the first pouch and projects towards 
the cephalic aspect of the glossopharyngeal nerve. According 
to Hammar (03) this evagination represents only the dorsal part 
of the primary pouch. A deeply staining cyst is present in this 
embryo on either side of the pharynx, which evidently belongs 
to either the ectodermal or entodermal part of the second bran- 
chial groove. The left cyst is situated just lateral to the left 
glossopharyngeal nerve, while the right is just cephalad of the right 
glossopharyngeal nerve and in close relation to the second pouch. 
Piersol (’88) found that in rabbit embryos there were formed, in 
the development of the second pharyngeal pouches, two epithelial 
tubes on either side, one from the entoderm and the other from 
the ectoderm, both of which subsequently atrophied. The 
former, however, persisted longer than the latter. Hammar 
(03) described and figured a structure in human embryos pro- 
truding above the margin of the tonsilar pouch. In early stages 
this was connected with the ectoderm, and hence he concluded 
that it was of ectodermal origin. Fox (’08) did not find any 
ectodermal remnant in this region of the pig embryo, but de- 
scribed and figured a long filiform process continuous with each 
of the second pharyngeal pouches. 

The third pharyngeal pouches (plates 2 and 6) have lost their 
connection with the pharynx. They are now represented each 
by a compact cylinder (Thy., plates 2 and 6) in the side of the 
neck, and which contains only a slight lumen. The cylinders 
converge caudally toward the median line and end approximately 
at the level of the aortic arch (Arc.ao.). The right and left eylin- 
ders become, eventually, the corresponding lobes of the thymus 
gland with the exception of the cephalic extremities which are 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 39 


compact epithelial masses (not marked off in the figures) differing 
in structure from the rest of the anlagen. The cephalic portion 
of each cylinder is closely applied to the lateral wall of the com- 
mon carotid artery, and is the part described by Katschenko 
(87) as the nodulus thymicus, and by Fox (’08) as the carotid 
gland. The recent work of Hammar (11) substantiates the view 
that this part eventually becomes separated from the thymic 
cylinder and (coming to lie at the caudo-dorsal border of the 
lateral part of the thyreoid gland) forms with its fellow of the 
other side the caudal pair of para-thyreoids. The cephalic ends 
of the thymic cylinders also show two hollow projections, a medial 
one, extending toward the pharynx and ending blindly, dorsal to 
the common carotid artery; and a more lateral process extending 
cephalad and ending blindly alongside the ventro-medial surface 
of the vagus nerve where the latter is crossed on its lateral side 
by the hypoglossal. The former or medial of these processes is 
evidently the remains of the thymo-pharyngeal duct. The lat- 
eral process seems to be the remains of the cervical sinus fused 
with the third pharyngeal pouch, as maintained for corresponding 
structures by Katschenko (87), Fox (08), and Hammar (11), 
and not an outgrowth from the thymus as conjectured by Pren- 
ant (94) and Bell (05). Katschenko from a study of this 
structure in the pig, maintained that it formed a considerable 
portion of the head of the thymus, a view since corroborated by 
Prenant (94) who, however, considers it of entodermal origin. 
Fox (08) found that in the cat it apparently atrophied early in 
development, and that in later stages of the rabbit it had largely, 
if not entirely disappeared. He is inclined to think that, when it 
does persist, it does not form an integral part of the thymus, but 
merely an associated structure. 

The fourth pharyngeal pouch of either side, exclusive of the 
so-called ultimobranchial (postbranchial) body (Greil ’05) is 
represented by a solid epithelial mass (P-thyr. IV) seen in plates 2 
and 6. These masses, which represent the cephalic pair of para- 
thyreoids, are situated dorsal to the lateral lobe of the thyreoid 
and are entirely separate from the pharynx. That on the right 


40) F. W. THYNG 


side is bilobate and somewhat removed from the proliferating 
entoderm of the thyreoid, but on the left the two are intimately 
connected. 

The thyreoid gland (plates 2 and 6) is distinctly U-shaped, with 
the concavity of the U directed cephalad. The level of the slender 
connecting bar of the median thyreoid (Thyr.m.) is seen in plates 
1 and 6. The arms of the U, derivatives in part of the ultimo- 
branchial bodies (which are still discernible although intimately 
connected with the median thyreoid) are widened considerably 
dorso-ventrally, and terminate at a level corresponding with the 
cephalic ends of the thymic cylinders. Numerous proliferating 
cords of cells make its surface somewhat irregular. Its connec- 
tion on the left with the parathyreoid anlage of the fourth pharyn- 
geal pouch has been referred to above. 

Salivary glands. The parotid and submaxillary glands are 
shown in plates 2 and 5. The parotid gland is represented by a 
small, solid cord of cells (Gl.p.) partly constricted off from the 
ridge leading caudad from the angle of the mouth and overlying 
the sulcus buccalis. The submaxillary gland is represented by a 
solid cord of cells (Gl.s.) but is larger than the parotid anlage. It 
projects from the caudal part of the floor of the alveolo-lingual 
groove into the underlying mesenchyma which for some space 
around the gland consists of closely packed cells for the develop- 
ment of a capsule. Its extremity is broader than the stalk, and 
shows slight indications of proliferating buds. The anlagen of 
the sublingual glands have not developed at this stage. That 
they are the latest of the salivary glands to develop, has been 
noted by His (’85), Chievitz (85), Hammar (01), Paulet (10) 
and others. 

Oesophagus. The oesophagus is comparatively long at this 
stage. The entodermal part of the tube only is figured in plates 
land 2. This consists of an epithelium, containing four or more 
layers of nuclei. Numerous irregular cavities, as seen by Schultze 
(97), and others, are found within the epithelium. They occur 
in scattered situations, and are separated from the surrounding 
mesenchyma usually by a single layer of columnar or cuboidal 
cells. Inno case were they found to connect with the lumen of the 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 41 


oesophagus. They are apparently vacuoles as maintained by 
Forssner (07), and Johnson (10). A granular coagulum was 
invariably found within them, but Kreuter (’05) who has studied 
these structures concludes that a degeneration of cells does not 
occur. For a general account of these structures see Lewis (’12). 


(aes : * “SS ck tae 
we oh as ~~ fox De 
| 


Vf D.pancv 


Pancd/ Panev= ~D. pained. 


“Dchol 


Text fig. 2. Wax-plate reconstruction of the stomach, the duodenum and the 
pancreas; the model is represented somewhat ventrally, from the right side. 
A. du., antrum duodenale; C., corpus gastri; D. chol., ductus choledochus; D. cyst., 
ductus cysticus; D. hep., ductus hepaticus; D. panc. d., ductus pancreatis dorsalis; 
D. pane. v., ductus pancreatis ventralis; F’., fundus zastri; Oe., oesophagus; Panc. 
d., pancreas dorsale; Panc. v., pancreas ventrale; P. py., pars pylorica gastri 
(H. E. C. 839). X 55 diams. 


Stomach. The stomach has assumed practically the adult po- 
sition. It is represented by the entodermal lining only, in plate 1 
and text figure 2 (Ga.,C). Its primitive dorsal border representing 
the greater curvature has revolved to the left, while its ventral 
border, now identified by the lesser curvature, faces toward the 
right. Its entodermal lining is an epithelium containing four or 
more layers of nuclei. It presents at the cardiac end a prominent, 
dorsally directed pouch, the fundus (F.), which according to 
Keith and Jones (’01) develops as a localized outgrowth. That 


42 ; F. W. THYNG 


this outgrowth is from the left side of the primitive stomach is 
evident, a relation evidenced in the adult by the reflection line 
of the lieno-gastric ligament. The body (C.) extends caudad 
and ventrally, passing into the attenuated pars pylorica (P.py.) 
which ends at a dilated portion of the duodenum (A.du.), the duo- 
denal antrum of Retzius (57). This, according to Lewis (’12) 
always marks the position of the pylorus. The external surface 
of the epithelium is for the most part smooth, but the internal 
surface is indented by slight grooves representing the beginning 
of the gastric pits. 

Intestine. Most of the small intestine and all of the large are 
shown in plates 1 and 2 (Duo., Int.t., Int.cr., Int.r.). The duode- 
nal division of the small intestine (Duo.) leading from the pyloric 
end of the stomach passes transversely across the median line 
from the left to right. Here it bends dorsally and receives the 
duct of the dorsal pancreas (D.panc.d.), and the bile duct (D.chol.) 
(text fig. 2). The small intestine (/nt.t.) then extends in a caudal 
and ventral direction, a little to the right of the median line, into 
the umbilical cord. In the umbilical cord it is bent twice in the 
sagittal plane at approximately 90°. On the left side of the 
second or cephalic bend (pl.2) it is continuous with the yolk-stalk 
(D.vit.). The portion of the small intestine beyond the second 
bend returns toward the body, cephalad of the part described, 
and terminates at the caecum. 

The caecum forms a considerable dilatation and ends in the 
vermiform process (Pr.ver.) which projects ventrally and to the 
left. 

The colon (Int.cr.) extends from the caecum, dorsally in the 
median plane, crossing to the left of the duodenum. Opposite the 
caudal extremity of the Wolffian body it turns caudally, and at an 
arbitrary point becomes the rectum (/nt.r.). It is evident that 
the primitive U-shaped loop of intestine has undergone in this 
embryo a rotation of approximately 180°. 

The epithelium of the duodenum a short distance caudad to the 
bile duct presents on its left side one prominent diverticulum, 
directed cephalad. Indications of similar outgrowths occur at 
twelve other places along the portion of the small intestine within 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 43 


the umbilical coelom. The lumen of the duodenum, beginning 
somewhat cephalad of the duct of the dorsal pancreas and ex- 
tending caudad to the duodenal diverticulum above mentioned, 
is subdivided into two or three parts by a proliferation of the 
epithelium in a manner similar to that already described by 
Tandler (00) in a human embryo of 14.5 mm. This observa- 
tion has been confirmed by Forssner (’07), Johnson (’10) and 
Lewis (12). Beyond this proliferation the lumen of the small 
intestine is either a small cylindrical passage or a slight slit. 

The yolk-stalk has a lumen for a short distance beyond its 
connection with the epithelium of the intestine. It then becomes 
a cord of degenerating cells, showing here and there traces of a 
lumen. Whether the yolk-stalk is still connected with a rudimen- 
tary yolk-sac could not be determined as these parts were cut 
away in the embryo. 

The lumen of the colon as it leaves the caecum, is a very small 
cylindrical passage, but as it nears the rectum, it becomes a 
transversely directed slit (corresponding to the shape of the 
intestine which is compressed dorso-ventrally). 

The cephalic portion of the rectum is a little larger in diameter 
than the colon, while the terminal part is more attenuated. The 
latter is circular in transverse section and the lumen is reduced to 
a very small cylindrical passage, which, however, does not connect 
with the shallow external depression (An.) between the protrud- 
ing genital folds. 

Inver. The liver (see plates 1, 2 and 4, Hepar) occupies the 
greater part of the cephalic and ventral regions of the abdominal 
cavity. The right lobe is much the larger, and extends from the 
cephalic end of the abdominal cavity on the right, caudad to a 
point on a level with the crossing of the duodenum by the colon. 
It is joined to the dorsal abdominal wall on the right of the 
dorsal mesogastrium, at the ventral region of the right supra- 
renal gland, by the plica venae cavae of Ravn (’89) (caval mes- 
entery). 

The caudate lobe is located between the caval mesentery and 
the lesser omentum. It projects somewhat toward the left 
into the bursa omentalis. The position of the quadrate lobe 


44 F. W. THYNG 


can be determined from plate 2. It is situated to the right of 
the vesica fellea (Ves.fel.), lying between this and the umbilical 
vein (V.um.s., plate 4). 

The hepatic duct (D.hep., plate 2, and text figure 2) takes 
origin from the hepatic trabeculae of the medial surface of the 
right lobe, ventral to the entrance of the portal vein (V.P.). It 
extends dorsally and to the left for a short distance and then cau- 
dad, uniting with the small cystic duct (D.cyst) to form the ductus 
choledochus (D.chol.). 

The cystic duct has a slender lumen, and leads in a nearly 
dorso-ventral direction from a small, distal dilatation, the gall- 
bladder (Ves.fel., plate 2). The latter is closely applied to the 
ventro-medial surface of the right lobe of the liver. 

The common bile duct is considerably larger in diameter than 
either the hepatic or cystic ducts. It has a well-defined lumen, 
and extends caudad through the lesser omentum in an S-shaped 
course to open into the duodenum. This it does upon its left 
side, a short distance beyond the pyloric end of the stomach. 
The epithelium of the gall-bladder and of the cystic and common 
bile ducts, is devoid of knob-like buds and diverticula, met with 
in these situations in other embryos. 

Pancreas. The pancreas of this embryo has been described, 
and pictured in a previous paper (Thyng ’08), but for the sake of 
completeness it will be described briefly in this connection. It 
consists of two parts, a dorsal and a ventral pancreas (Panc.d. 
and v., text figure 2). The dorsal pancreas is considerably the 
larger, and extends distally into the mesogastrium. It is essen- 
tially a long, irregular, hollow mass of epithelium with prolifer- 
ating branches of varying length which in turn often give off 
hollow buds. Its duct (D.panc.d.) is larger than that of the ven- 
tral pancreas, contains a well-defined lumen, and opens into 
the left side of the duodenum nearer the stomach than the bile 
duct. 

The ventral pancreas is in close relation with the proximal 
part of the dorsal anlage, the two having anastomosed ventral to 
the portal vein, and on the left of the common bile duct. Like the 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 45 


dorsal pancreas, the ventral also shows a branching condition of 
its epithelium. The duct of the ventral pancreas (D.panc.v.) is 
short, and opens into the bile duct (D.chol.) near its entrance into 
the duodenum. 


RESPIRATORY SYSTEM 


The olfactory pits are described with the sense organs. 

The entodermal lining of the larynx and trachea are represented 
as seen in ventral view in plate 6; from the left side in plate 2; 
and in the median sagittal section in plate 1. In plate 1 the 
anlage of the right lung is seen from the left side. 

Larynx. The larynx, which opens from the pharynx by a 
T-shaped aperture, is placed immediately caudal to the epiglottis. 
The pedicle of the T or interarytaenoid notch, extends dorso- 
ventrally between the arytaenoid protuberances, and is bounded 
laterally by the aryepiglottic folds. A median raphé (R.) ex- 
tends dorso-ventrally across the larynx, in a somewhat caudal 
direction, so as to close it temporarily. 

Trachea. The trachea passes caudally to bifurcate into the 
two bronchi. The root of the left bronchus is shown in section 
in plate 1 (Br.s.) but, the greater portion of this bronchus and 
the corresponding lung have been removed. 

Lungs. The entodermal outpocketings of the right lung and its 
pulmonary vessels (A.pul. and Vv.pul.d.) are represented in plate 
1, as seen through the mediastinum. An eparterial (tracheal) 
bronchus is present on the right. It is situated dorsal to the 
right pulmonary artery, and a branch of the latter passes ceph- 
alad of this bronchus. The oesophagus (Oe.) passes between the 
developing lungs. 

Pleural cavity. Each pleural cavity is closed off completely 
from the pericardial by the pleuro-pericardial membrane, but 
still communicates with the abdominal cavity. The aperture 
on the left is very small. 

A blind prolongation of the right pleural cavity begins medially 
and dorsally to the anlage of the root of the right lung. From 
here it extends caudally and somewhat ventrally along the right 


46 F. W. THYNG 


side of the oesophagus to the diaphragm. It then passes be- 
tween the oesophagus and diaphragm and ends at a situation 
approximately corresponding to the level of the most caudal 
extension of the right pleural cavity. This diverticulum is 
ventral to the main pleural cavity and evidently corresponds 
to the space, termed by Broman (’04) the infracardial bursa. 
The infracardial bursa in this case differs from that described 
by Broman in that the primitive connection with the pleural 
cavity has been retained. The retention of this connection is of 
interest in that such a recess of the right pleural cavity exists 
normally in animals possessing an infracardial lobe of the right 
lung, and may be expected to occur occasionally in man. 
UROGENITAL SYSTEM 

The parts of the urogenital system are shown in plate 2 in 
which the urogenital sinus, the left Wolffian body and duct, 
the left metanephros and ureter, the left genital ridge, and Miil- 
lerian duct are shown from the left side. 

Wolffian ridge and body. The Wolffian ridge extends almost 
the entire length of the abdominal cavity, its anterior three- 
fourths being occupied by the Wolffian body. The ridge tapers 
off bluntly at its cephalic end; caudad of the area of mesonephritic 
tubules (7.W.) it dwindles to a slight elevation upon the abdom- 
inal wall in which the Wolffian duct (D.W.) passes to the urogeni- 
tal sinus (S.u.-g.). Under the influence of the rapidly growing 
liver and suprarenal glands the ridge has moved laterally from 
its primitively dorso-medial position. 

Wolffian duct. The Wolffian duct (D.W.) passes through the 
ventro-lateral region of the Wolffian body receiving the tributary 
mesonephric tubules (7'.W.). From the caudal end of the 
Wolffian body it extends through the remainder of the Wolffian 
ridge to end by piercing the dorso-lateral wall of the urogenital 
sinus (S.u.-g.). Close to its entrance into the sinus there is a 
slight dorsal enlargement of the duct which suggests the first 
anlage of the seminal vesicle. It should be noted, however, that, 
according to the investigations of Pallin (01), the vesiculus 
seminalis first appears at a much later stage of development. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 47 


Miillerian duct. The Miillerian duct ().M.) les parallel with 
the Wolffian, being a little ventral to it. Its cephalic end opens 
freely into the abdominal cavity. The opening which occurs 
near the cephalic end of the Wolffian body, shows something of a 
fimbriated condition (fim.). In the caudal direction the Miil- 
lerian duct seems to terminate in a blind pointed end close to the 
Wolffian duct. The point of termination is about on a level with 
the junction of the middle and caudal thirds of the Wolffian body. 

Bladder. The ventral segment of the cloaca is divisible into 
two parts, a cephalic portion (B) which will ultimately form the 
bladder and a caudal portion (S.u.-g.) the urogenital sinus. 

The region of the bladder anlage adjoining the urogenital sinus 
is expanded on either side to produce a dorso-lateral ridge. Into 
each ridge at the caudal extremity of the bladder anlage opens the 
corresponding ureter. The portion of the bladder remote from 
the urogenital sinus is continued as the allantois into the caudal 
wall of the umbilical cord. The proximal portion of the allantois 
consists of a solid cord of cells, the urachus, but distally isolated 
portions of the original lumen are found. 

Urogenital sinus. The cephalic region of the urogenital smus 
receives the Wolffian ducts. The orifices of these are placed on 
either side, a short distance from the median line. The remainder 
of the sinus extends into the median caudal part of the genital 
tubercle (Pa.gen.) as a laterally compressed structure. In profile 
view this part of the sinus appears somewhat triangular in outline. 
Its dorsal region encloses a cavity, but its latero-ventral walls 
are approximated to form an incomplete raphé, the urogenital 
membrane (Mem.u.-g.). This membrane is broken down in its 
extreme caudal part so that the urogenital smus opens to the 
exterior. 

The lower part of the sinus becomes the vestibule in the female, 
while in the male it forms the main portion of the urethra. The 
female urethra and the proximal part of the male urethra being 
formed by a canal differentiated later between the bladder anlage 
and the urogenital sinus. 

Metanephros. The metanephros (Met., pl. 2; see also pl. 1) is 
situated dorsal to the middle part of the Wolffian body, extending 


48 F. W. THYNG 


approximately from a level of the twelfth thoracic to the second 
lumbar nerve, and appears externally as an oval, lobulated body. 
It consists of approximately eight branches arising from the ceph- 
alic extremity of the ureter, the renal pelvis. Each branch 
ends in a bilobated ampulla which is surrounded by condensed 
‘nephrogenic tissue.’ The ureter as it leaves the developing 
metanephros, extends at first caudally and medially in the ven- 
tral part of the dorsal body wall. It then passes ventrally in the 
lateral parieties and opens into the extreme lateral wall of the 
anlage of the bladder. The ureteral orifices are slightly cepha- 
lad of those by which the Wolffian ducts communicate with the 
urogenital sinus. 

Genital ridges. ‘The ovaries are two compact, longitudinal pro- 
tuberances projecting from the medial borders of the Wolffian 
bodies. They taper at either extremity, more gradually at the 
cephalic than the caudal. The cephalic end of the left ovary 
(G.R.) is partly hidden from view in plate 2 by the Wolffian body, 
but it does not extend quite to the cephalic pole of the latter. 
The caudal end of the ovary is on a level with that of the meso- 
nephros by which it is hidden in the drawing. The cephalic 
region of each ovary is deeply marked by infolding of the germ- 
inal epithelium. 


DUCTLESS GLANDS 


The right suprarenal is shown in plate 4, the spleen in plate 2. 
The thyreoid, thymus, and parathyreoid glands are described in 
connection with the pharynx (pp. 38-40) and shown in plates 2 
and 6. 

Suprarenal gland. The suprarenal glands (Gl.s.-r.) are some- 
what oval bodies of considerable size, developing in the ventral 
region of the dorsal body wall between the Wolffian body and the 
dorsal aorta. Their cephalic extremities lie at the level of the 
ninth thoracic nerves, i.e., a short distance cephalad of the 
caudal extension of the pleural cavities, of which they form, in 
part, the caudo-lateral wall. Their caudal extremities lie on a 
level with the twelfth thoracic nerves, i.e., slightly beyond the 
cephalic extremity of the Wolffian body. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 49 


Each gland has developed in the path of the cephalic part of the 
subeardinal vein of that side, and the vein has been subdivided 
to form the sinusoidal channels now found within it. Simusoids 
in the suprarenal glands of mammals were demonstrated histo- 
logically by Minot (00). 

The cells composing the cortex are arranged in peripheral 
layers, surrounding a loose reticular core. These comprise what 
is usually termed the interrenal part of the suprarenal glands, 
from their resemblance to the interrenal bodies of Selachians. 
These interrenal bodies are being invaded on their medial sur- 
faces by numerous scattered clumps of deeply staiming cells, 
derived from the sympathetic ganglia, often termed the sym- 
patho-chromaffine organs. 

A deeply staining, oval mass of cells is present on either side 
of the inferior mesenteric artery, ventro-lateral to the aorta. 
Caudally these bodies reach the level of the proximal part of the 
common iliac arteries, while cephalad they are continued by scat- 
tered, smaller groups of cells to the caudal extremities of the 
suprarenal glands. They have a rich blood supply, and are 
intimately related to the sympathetic system in their locality. 
They unquestionably correspond to the aortic bodies discovered 
by Zuckerkandl (01). 

It seems very probable to the author that each of these main 
groups of cells has been partially isolated from the suprarenal 
gland of its side by the development of the large dorso-ventral 
segment of the supra-ureteral venous channel (plate 4) which now 
intervenes between them. Further evidence of this interpreta- 
tion is the fact that a closer relation between the two exists on the 
left side in this embryo, where the vein in question is much smaller 
than its companion on the right. 

Spleen. The spleen (Lien.) is clearly recognizable as a small, 
protuberance of the mesogastrium, containing dense mesenchyma. 
The tissue directly ventral to this protuberance, is permeated by 
a vascular network, supplied by the splenic artery, and drained 
by the splenic vein (V.li., plate 4). 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


50 F. W. THYNG 
NERVOUS SYSTEM 
Brain 


The surface of the brain is represented from the left in plate 
2, and from the right in plate 3. The brain is shown in median 
sagittal section from the right side in plates 1 and 4. By the 
cephalic flexure (Flex.ceph.) which occurs in the mesencephalon 
(Mesen.) the fore-brain is bent at an acute angle to the hind- 
brain. The cervical flexure (Flex.cerv.) is nearly a right angle. 

Telencephalon. The telencephalon (Telen.) is sharply marked 
off dorso-laterally from the diencephalon (Dien.) by a deep ex- 
ternal groove and a corresponding internal ridge or fold, the 
velum transversum. This fold forms the caudal boundary of the 
interventricular foramen (/’o.int.). From this fold the line of 
demarcation extends ventrally just behind the optic evaginations 
to the postoptic recess (.po.-op.). The telencephalon thus 
bounded contains a median cavity, the anterior part of third 
ventricle. The latter communicates with the lateral ventricles 
by comparatively large crescentic openings, the interventricular 
foramina (Fo.int.). The median cavity is bounded anteriorly by 
the lamina terminalis. The large oval hemispheres represent 
the dorsal zones of the telencephalon. Each protrudes consider- 
ably beyond the lamina terminalis and presents orally two inter- 
nal depressions with corresponding external swellings. These are 
the developing anterior and posterior olfactory lobes (thin.). 
Caudal and dorsal to the olfactory area the wall of each hemi- 
sphere ismuch thickened to form the corpus striatum (C.str.) which 
appears as a prominent swelling on the ventral surface in front 
of the praeoptic recess (R.p.-op.). Externally the position of the 
corpus striatum is marked by a shallow depression, the develop- 
ing lateral fossa. The praeoptic recess is a slight groove passing 
transversely across the lamina terminalis into the optic stalk of 
either side. It represents the cephalic extension of the sulcus 
limitans, His (92), Johnston (09) and others. The ventral 
zones of the telencephalon comprise the area between the prae- 
optic and the postoptic recesses, which marks the place of later 
development of the optic chiasma. In regard to the area of 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 51 


evagination of the optic vesicles it is somewhat questionable 
whether this belongs primarily with the tel- or di-encephalon. 

Diencephalon. The boundary between the diencephalon and 
the mesencephalon (Mesen.) is a slight constriction extending 
nearly transversely across the brain toward the tuberculum 
posterius (T'ub.p.). An ill defined furrow, sulcus limitans, ex- 
tends cephalo-caudad along the internal surface of the dienceph- 
alon separating the dorsal zone above from the ventral zone or 
hypothalamus below. The roof of the diencephalon is thin; near 
its caudal limit there is a slight evagination which represents the 
first appearance of the epiphysis (Corp.pin.). 

The ventral part of the dorsal zone caudad of the interventricu- 
lar foramen is thickened, forming on the medial surface a low 
ridge, the developing optic thalamus. Dorsal to the ‘thicken- 
ing, the internal surface of each dorsal zone presents a prominent 
concavity. The lateral wall of the hypothalamus is thickened. 
Caudad of the recessus postopticus (R.po.-op.) the cavity of the 
diencephalon extends into a small median evagination from the 
floor-plate. The evagination is the anlage of the infundibular 
gland (Glinf.). The thickened knob-like termination of the 
gland is embraced by the forked distal end of the hypophysis 
(Hyp.). It becomes the neural lobe of the adult pituitary. 

Mesencephalon. The conventional boundary between the 
mesencephalon (Mesen.) and metencephalon (Meten.) is the con- 
stricted portion or isthmus (/sth.). As already stated a slight 
constriction extending nearly transversely across the brain 
toward the tuberculum posterius (J'ub.p.) divides it from the 
diencephalon. The roof-plate is comparatively thin, and bears 
no trace of the longitudinal ridge which has been described as 
occurring later. The floor-plate is considerably thicker than the 
roof-plate. The dorsal and ventral zones are distinct. The 
cavity of the mesencephalon (cerebral aqueduct) is more expanded 
in the caudal two-thirds of the mesencephalon, a large oval con- 
cavity appearing on the internal surface of each dorsal zone. 
The ventral zones are considerably thickened so that in the region 
of the oculo-motor nerve they project ventrally below the floor- 
plate. Externally the surface of the mesencephalon ;is smooth 


a2 F. W. THYNG 


except for slight dorso-lateral depressions on either side indicating 
the area of division into anterior and posterior colliculi. 

Isthmus. The isthmus ([sth.) usually is described as the con- 
stricted portion between the mesencephalon and the metenceph- 
alon. It was considered by His (’92), and given in the Basel 
nomenclature (’95) as a distinct segment of the rhombencephalon 
which formed a marked ring somewhat narrower dorsally than 
ventrally. 

In the corresponding area of the brain of this embryo there is 
on the internal surface of each ventro-lateral wall, a distinct 
transverse groove. The two grooves unite ventrally in a recess 
or sulcus (Sul.) caused by a depression of the floor-plate, per- 
ceptible on the external surface as a distinct elevation (H.7-p.) 
the eminentia interpenduncularis of His (’92). This suleus was 
noted by Burckhardt (’91) and considered by him to be of general 
occurrence. It was observed in the human embryos by His 
(92), and named by him the isthmus groove. Kupffer (’03—’05) 
named it the sulcus intraencephalicus posterior. The author 
believes that the transverse grooves mentioned above are the 
ventral continuations of the caudal mesencephalic neuromere; 
also that the adjacent brain wall should be regarded as the caudal 
part of the mesencephalon rather than a distinct division of the 
rhombencephalon. 

Metencephalon. The metencephalon (Meten.) is the division 
of the primitive rhombencephalon from which the pons and 
cerebellum are developed. Its separation from the myelenceph- 
alon (Myelen.) is indicated, in part, by an internal transverse 
ridge (Pl.ch.p.) and a corresponding external groove, representing 
the developing plica chorioidea posterior. 

The roof-plate is enormously expanded so that it forms not 
only the roof, but the greater part of the lateral wall of the meten- 
cephalon. The remaining or cephalic part of the lateral wall is 
formed on either side by a thickened band which is joined cau- 
dally to the ependymal roof-plate by a thinner intermediate layer 
known as the rhombic lip. This band on either side represents 
the corresponding dorsal zone (Z.dors.). It extends obliquely 
cephalad and medially to merge into the slightly thickened roof- 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 53 


plate, caudad of the isthmus. It will form the corresponding 
lateral portion of the cerebellum. The vermis is believed to be 
developed from the slightly thickened roof-plate intervening 
between these thickenings. 

The ventral zones (Z.vent.) of the metencephalon, which form 
the pons, are deep longitudinal bands separated by a thick median 
raphé which represents the floor-plate. They extend caudad 
to the angle made by the pontal flexure where they blend with 
the corresponding zones of the medulla. 

Myelencephalon. The myelencephalon (Myelen.) is the re- 
maining portion of the brain, which arches over the cervical 
flexure (Flex.cerv.) and joins the spinal cord. The roof-plate is 
exceedingly thin. It is widest in the region of the plica choroidea 
posterior where it forms the roof and the dorsal half of the lateral 
wall. Its caudal extension gradually tapers out to pass into the 
narrow roof-plate of the spinal cord. It becomes the caudal part 
of the posterior medullary velum. 

The dorsal zones are thick. In the region near the spinal cord 
they are nearly vertical, but, by becoming progressively oblique, 
their internal surfaces form the lateral region of the ventricular 
floor at the cephalic end of the myelencephalon. The ventral 
zones, as in the metencephalon, are thick longitudinal bands on 
either side of the median line. In the floor of the ventricle a 
median longitudinal groove extends between them. Ventral 
to the groove a thickened raphé, floor-plate, unites the ventral 
zones. As the raphé approaches the region of the spinal cord it 
gradually becomes thinner. 


Spinal cord 


The spinal cord (Md.sp.) is represented in median sagittal 
section in plates 1 and 4. The surface is shown, in part, in plates 
2 and 3. It has a narrow slit-like cavity, somewhat expanded 
dorsally. The lateral walls of the cord have clearly marked ven- 
tral and dorsal zones which are continued into the corresponding 
zones of the myelencephalon. 


54 F. W. THYNG 


Cranial nerves 


Bh 


The cranial nerves of the right side are displayed in plate 3, 
and those of the left are shown in plates 2 and 5. Plate 6 shows 
some of the cranial nerves on both sides. 

Nn. olfactorw. Numerous nerve fibers (Nn.olf., plate 1) ex- . 
tend from the dorsal and medial surfaces of the nasal epithelium, 
and from the vomero-nasal organ (Org.vom.-nas.) to the olfactory 
area (Rhin.) of the telencephalon. In plates 2 and 3 the trunk 
formed by these nerves is represented as a stump. Among the 
fibers are numerous groups of cells which are not represented 
in the reconstruction. These cells perhaps have migrated from 
the nasal epithelium along the nerve fibers. 

N. opticus. Fibers are present, extending from the retinal 
layer of the optic vesicle along the optic stalk to the correspond- 
ing ridge (optic) of the brain. 

N. oculomotorius. The oculomotor nerve (N.oc., plates 2, 3 
and 5) issues from the ventro-lateral wall of the mesencephalon 
(Mesen.) by numerous small rootlets. it extends ventrally and 
cephalad, passing lateral to the posterior cerebral artery (A.cer.p.), 
and medial to the cavernous sinus (S.cav.) and the ophthalmic 
nerve (N.oph.). From the ophthalmic nerve it acquires a small 
sensory branch. Caudad of the optic stalk it gives off a branch 
to the anlage of the superior rectus muscle, but its main trunk 
is continued to the partially differentiated anlage for the inferior 
and medial recti and inferior oblique muscles. 

N. trochlearts. The trochlear nerve (N.troch., plates 2 and 3) 
issues from the roof of the isthmus and extends ventrally in a 
sinuous course to the orbit. In its course it passes just cephalad 
of the superior cerebellar artery (A.cereb.s., plate 2) and lateral 
to the anterior cerebral vein. In the orbital region it passes 
dorsal to the anlage of the lateral rectus muscle, and medial to 
the frontal ramus (N.fr., pl. 3) of the ophthalmic nerve. It 
terminates in the anlage of the superior oblique muscle. The 
trochlearis receives a small sensory branch from the ophthalmic 
nerve (N.oph., pl. 3). 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO oo 


N. trigeminus. The trigeminal nerve (plates 2, 3, 4 and 5) is 
composed of sensory and motor components. The sensory fibers 
arise from the large semilunar ganglion (G.s-l.), which lies lateral 
to the cavernous sinus (S.cav., plates 4 and 5), and form a large 
trunk which enters the latero-ventral surface of the metencepha- 
lon (Meten.). The motor fibers issue from the metencephalon at 
a point slightly ventro-cephalad of the sensory root. They form 
a trunk of considerable size which crosses the medial surface of the 
semilunar ganglion to join the mandibular nerve (plate 4). The 
peripheral fibers leave the semilunar ganglion as three main 
branches, the ophthalmic (N.oph.), the maxillary (N.mz.), and 
the mandibular (N.md.). 

The ophthalmic nerve passes to the orbit. Dorsal to the optic 
stalk it gives a branch to both the oculomotor and trochlear’ 
nerves, then divides into naso-ciliary and frontal nerves. The 
frontal (V.fr.) passes dorsal to the superior rectus and superior 
oblique muscles, and breaks up into several branches of which 
the supraorbital may be recognized by its dorsal direction. The 
naso-ciliary (V.na.-cil.) passes ventral to these muscles, and can 
be followed into the cephalic part of the corresponding lateral 
nasal process. The maxillary nerve (N.mzx.) soon after leaving 
the semilunar ganglion becomes a bundle of loosely connected 
fibers extending into the maxillary process ventral to the optic 
vesicle (Ves.op.). 

The mandibular nerve (N.md.) receives, in addition to the sen- 
sory fibers from the semilunar ganglion, the motor part of the 
trigeminal nerve. It divides into a small cephalic and a large 
caudal trunk. The former or buccal nerve at first extends 
cephalad in company with the infra-orbital branch of the 
stapedial artery (A.stp., pl. 2). Soon leaving this the buccal 
nerve crosses the anlage of the parotid gland (Gl.p.), and passing 
cephalad, furnishes branches to the epithelium near the angle 
of the rima oris. The larger, caudal trunk of the mandibular 
as 1t crosses the tubo-tympanal ridge, divides into three branches, 
the auriculo-temporal, the inferior alveolar, and the lingual. The 
latter near its origin is joined by the chorda tympani branch 
(N .ch-tymp., plate 3; ch.-ty., plate 2) of the facial nerve. 


56 F. W. THYNG 


The auriculo-temporal has two roots of origin which em- 
brace a branch of the stapedial artery. It extends at first 
ventrally and laterally, and then bends dorsally, giving off 
twigs to the epithelium in the neighborhood of the tuberculum 
tragicum. 

The inferior alveolar (N.alv.inf.) crosses the lateral border of 
the pharynx, and follows the lateral side of Meckel’s cartilage 
beneath the pharyngeal floor. It soon gives off the mylohyoid 
branch which passes ventrally, lateral to Meckel’s cartilage, to 
the anlage of the mylohyoid muscle. The inferior alveolar then 
continues cephalad in the mandibular process of the mandibular 
arch where it divides into a dorsal and a ventral branch. The 
former supplies the oral epithelium of the corresponding side, 
the latter or mental nerve supplies the ectoderm on the Vventro- 
lateral surface of the mandibular process. 

The lingual nerve (NV .ling.) is formed by the union of mandibu- 
lar and chorda tympani fibers (N.ch.tymp.) medial to Meckel’s 
cartilage. It extends cephalad for a distance between the carti- 
lage and the alveolo-lingual ridge where the submaxillary gland 
(Gl.smx.) has developed. Here it passes into the anlage of the 
submaxillary ganglion (plate 3). From the ganglion it issues as 
several bundles which curve medially around the alveolo-lingual 
ridge into the lateral part of the tongue. In this situation 
branches extend cephalad between the ridge and the hypoglossal 
nerve and, after repeated subdivisions, are ultimately distributed 
to the epithelium of the tongue. 

N. abducens. The abducens (N.ab.) issues from the ventral 
wall of the metencephalon (Meten.) by several rootlets which are 
hidden in the drawings by the overlying auditory and facial 
nerves. A caudal aberrant root is present on either side which 
extends from the region of the glossopharyngeal and vagus 
nerves to join the abducens. Similar aberrant roots of the 
abducens have been observed by Elze (’07) and represented by 
Bremer (’08). The abducens extends ventrally and somewhat 
laterally towards the orbital region. It passes dorsal to the in- 
ternal carotid artery, obliquely across the medial side of the 
cavernous sinus and ophthalmic vein. The abducens then turns 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 57 


laterally between the vein and the oculomotor nerve to enter the 
anlage of the lateral rectus muscle. 

N. facialis. The motor root of the facial nerve (plate 3) issues 
from the ventro-lateral wall of the metencephalon (Meten.) and 
passes to the geniculate ganglion (G.gn.) by which its fibers are 
enveloped for a short distance. The sensory root (pars inter- 
media, N.int.) arises from the geniculate ganglion and enters the 
metencephalon immediately caudo-lateral to the motor root. 
The mixed facial trunk which emerges at the caudo-ventral 
border of the geniculate ganglion (G.gn.) represents the post- 
trematic ramus of the first pharyngeal pouch. It takes a caudal 
and lateral direction. Having given off the chorda tympani 
(N.ch.tymp., pl. 3, ch.ty., pl. 2) it ends in several small branches. 

The chorda tympani nerve leaves the facial trunk at an acute 
angle and extends cephalad, ventral to the auditory pouch, to 
join the lingual branch of the mandibular nerve as described 
above. From the geniculate ganglion a small nerve (N.pet.s.m.) 
extends at first ventrally, and then bends sharply cephalad 
medial to the mandibular nerve and the tubo-tympanal ridge. 
It is the great superficial petrosal nerve. As it bends cephalad 
it gives off caudally a short twig (anastomotic with the tympanic 
plexus) to meet the tympanic branch (N.tym.) of the glosso- 
pharyngeal nerve: The nerve arising from this junction (the 
anlage of the tympanic plexus) is the small superficial petrosal. 

N. acusticus. The ganglion acusticum and otic vesicle are 
shown in plate 3. In plates 2 and 5 the otic vesicle has been 
removed. The ganglion acusticum is partially differentiated into 
cochlear and vestibular divisions. The vestibular part (G.ves.) 
lies cephalad of the utriculo-saccular division of the otic vesicle 
and lateral to the cochlear part of the ganglion, so that the 
latter is mostly hidden from view in plate 3. Four nerve trunks 
proceed from the vestibular division, a cephalic which divides 
into two rami to supply the ampullae of the superior and lateral 
semicircular canals; a caudal which passes medial to the utriculo- 
saccular division of the otic vesicle to supply the posterior semi- 
circular canal; and two intermediate branches, the cephalic of 
which extends to the developing utricle, the caudal to the saccule. 


58 F. W. THYNG 


From the cochlear division short fibers extend ventrally to the 
cochlear duct (D.c.). The central fibers of the ganglion acus- 
ticum (N.acus.) enter the caudo-ventral wall of the metencephalon 
slightly dorsal and caudal to the sensory root of the facialis. 

N. glosso-pharyngeus. The motor fibers of the glosso-pharyn- 
geal nerve (plates 2, 3 and 6) issue from the myelencephalon just 
ventral to the entering sensory roots. The latter. are hidden 
partially from view in plate 3 by the ductus endolymphaticus 
(D.end.) and crus commune, but are exposed in plate 2. 

The small ganglion superius (G.sup.) lies at. the medial side of 
the ampulla of the posterior semicircular canal, separated from it 
by a narrow zone of the developing otic capsule. It extends 
slightly dorsal to the ampulla, and apparently involves only the 
more posterior and medial of the fibers of the nerve. 

The ganglion petrosum (G.petros.) is a large ganglion, the 
caudo-lateral surface of which is closely applied to the trunk and 
ganglion nodosum of the vagus which somewhat overlap it. The 
medial surface of this ganglion is in contact with the internal 
carotid artery and the accompanying sympathetic fibers. From 
the cephalic part of the ganglion arises the tympanic nerve 
(N.tym.) which extends cephalad lateral to the internal carotid 
artery and nearly parallel with it. It passes dorsal to the re- 
mains of the second pharyngeal pouch and to the auditory (first 
pharyngeal) pouch, and lateral to the stapedial artery close to 
its origin. A little cephalad of the stapedial artery it is joined 
by a slender branch from the great superficial petrosal as de- 
scribed above (p. 57). The small superficial petrosal, resulting 
from this communication, I have been able to trace as far as a 
point near the caudo-ventral border of the semilunar ganglion. * 

Beyond the ganglion petrosum the glossopharyngeal nerve 
skirts closely around the caudal aspect of the remains of the 
second pharyngeal pouch and reaches the ganglion nodosum. 
At the ventral border of the lateral pharyngeal wall it receives a 
branch (not shown in the figure) from the superior cervical sym- 
pathetic ganglion. This branch, extending around the medial 
side of the internal carotid artery, joins the dorsal aspect of the 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 59 


glossopharyngeal. The glossopharyngeal nerve, having passed on 
the cephalic aspect of the ganglion nodosum, leaves the vagus, 
but gives to it a communicating branch (also omitted from the 
reconstruction) which contributes fibers to the pharyngeal 
branch of the vagus. The glossopharyngeal then extends medi- 
ally, bifurcating into a lateral or pharyngeal branch and a medial 
or lingual branch. The pharyngeal branch (plate 6) sends a 
twig to the developing stylo-pharyngeus muscle, and continues 
cephalad, for a short distance, along the pharynx. The lingual 
branch which is somewhat larger than the pharyngeal, also gives 
off a small branch to the pharynx, and then passes in a cephalo- 
medial direction to the lateral.side of the caudal part of the 
tongue where it splits up into many branches. 

The glossopharyngeal trunk which extends peripherally from 
the ganglion nodosum, is usually considered as the posttrematic 
ramus of the second branchial cleft. 

N. vagus. The vagus (N.vag., plates 2, 3 and 6) is a large, 
mixed nerve, its main sensory component being derived from two 
ganglia, the jugular (G.jug.) and the nodosum (G.nodos.). The 
vagus acquires additional sensory fibers from an irregular series 
or chain of ganglionic masses (Gg.hyp.) situated caudad of the 
jugular ganglion and dorsal to the accessory nerve (N.acc.). 
These irregular clumps of cells without doubt represent hypo- 
glossal ganglia, but the exact number of ganglia formed by them 
is uncertain. Prentiss (’11) in dissected pig embryos frequently 
found fibers from two or three of such ganglia passing ventrally 
to join the corresponding motor roots of the hypoglossal nerve. 
Such fibers, however, were not found in thisembryo. The chain 
of ganglia (Gg.hyp.) on the left side (plate 2) is continuous with 
the first cervical ganglion which is very slender in its middle 
part. : 

Small motor rootlets issuing from the myelencephalon ventral 
to the entering sensory fibers, together with fibers from the ac- 
cessory, furnish the motor components of the vagus. The vagus, 
therefore, from its sensory as well as from its motor composition, 
is a compound nerve, as has been maintained by many investi- 
gators. 


60 F. W. THYNG 


Fach vagus nerve extends caudad upon the medial side of the 
internal jugular vein as far as the common cardinal vein. It then 
occupies the angle between the oesophagus and trachea until the 
latter bifurcates. From here each nerve, as it continues caudad, 
passes dorsally to the bronchus of its own side where it gives off 
pulmonary branches. More caudally the nerves are in close 
relation to the oesophagus, forming a coarse plexus superfcial 
to the anlage of the external muscular layer. From the oesopha- 
gus the vagi spread onto the stomach and give branches to the 
neighboring viscera. 

An auricular branch of the vagus has not been identified on 
either side of this embryo. It is possibly a nerve of late develop- 
ment. In rare instances its absence has been noted in the adult. 

A short, rather ill-defined pharyrgeal branch is present on 
either side (but not reconstructed). Each arises from the medial 
side of the cephalic part of the ganglion nodosum, and extends to 
the pharynx, passing ventral to the proximal part of the internal 
carotid artery. ‘To the formation of this nerve on either side the 
glossopharyngeal seems to contribute fibers as mentioned above. 
These branches of the vagus are apparently late in becoming 
formed into definite trunks. 

The superior laryngeal nerve (N.l.s., pl. 3; N.laryng.s., 
pl. 6) arises at the ganglion nodosum, slightly caudad of its 
middle. It is represented in plate 3 as a stump, but is shown in 
part in plate 6. It extends ventrally and medially, dorsal to 
the distal part of the common carotid artery, and caudad of the 
detached (from pharynx) end of the thymico-pharyngeal duct, 
to divide into two branches, internal and external. 

The internal branch passes medially over the cephalic border 
of the lateral lamina of the thyreoid cartilage, and divides into 
dorsal and ventral branches. The dorsal extends in the mesen- 
chyma between the entodermal portions of the oesophagus and 
larynx. The ventral extends caudad, medial to the lateral lamina 
of the thyreoid cartilage. The external branch of the superior 
laryngeal is smaller than the internal, and can be traced caudally 
along the lateral aspect of the thyreoid cartilage for a short 
distance. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 61 


Froriep (’85) has shown conclusively that the superior laryn- 
geal nerve is the posttrematic ramus of the third branchial 
cleft. 

Another branch (R.p-tr.) of the left vagus arises from the 
ganglion nodosum directly caudad of the place of origin of the 
superior laryngeal. It is a small strand which extends caudally, 
at first between the vagus and the lateral lobe of the thyreoid 
gland, and dorsal to the thymus and left common carotid artery. 
Having passed the caudal extremity of the thymus gland it is in 
relation dorsally with the vagus, medially with the aortic arch, 
and laterally with the internal jugular vein. The nerve finally 
winds around the fourth aortic arch, and extends cephalad lateral 
to the recurrent of that side. It passes dorsal to the parathy- 
reoid of the fourth pharyrgeal pouch and turning cephalad, 
appears at first dorsal and then dorso-medial to the lateral lobe 
of the thyreoid gland. It ultimately becomes exhausted by giv- 
ing off fibers which extend ventrally in the region of the termina- 
tion of the external ramus of the superior laryngeal nerve. 

The corresponding nerve on the right takes origin from the 
dorso-medial side of the right ganglion nodosum, where the hypo- 
glossal nerve crosses the vagus laterally. As it extends caudad 
it lies close to the medial side of the vagus, and it is conceivable 
that in later development the two nerves might be inclosed in a 
common connective tissue sheath. It finally winds around the 
right fourth aortic arch (A. subclavia) in the concavity of the 
larger recurrent nerve, and returns cephalad, passing lateral to 
the parathyreoid of the fourth pharyngeal pouch, but having the 
same relation to the lateral lobe of the thyreoid as the corre- 
sponding nerve on the right. It has a shorter course in the neck 
than that of the right side, the right fourth aortic being situated 
considerably more cephalad than the left. 

From their site of origin, and from their relation to the fourth 
aortic arches and to the parathyreoids of the fourth pouches, it 
can scarcely be doubted that these nerves represent the post- 
trematic rami of the fourth pharyngeal pouches. 

These nerves are obviously comparable to the branches of the 
vagus identified by Froriep (’85) in cow embryos (8.7 to 8.8 mm. 


62 .F. W. THYNG 


in length) as the posttrematic rami of the fourth pharyngeal 
pouches, and figured in Taf. I, figs. I and I1, II, I15. Froriep, ” 
however, could find this nerve only in the young stages. 

Lewis (06) found in the 12 mm. pig embryo a small nerve 
running beside the postbranchial body which he thought might 
be comparable to the nerve described by Froriep as the post- 
trematic ramus for the fourth pouch. 

Elze (07) identified this posttrematic branch of the vagus 
in a human embryo of about 7 mm., (p. 427, text figs. 7-8), but 
could not find the nerve in two older human embryos (II and ITT), 
measuring (greatest length) 9.5 and 11 mm. respectively. Hence, 
like Froriep, he concluded that in man the existence of this post- 
trematic ramus of the vagus is transitory. 

In this embryo it is seen that these posttrematic rami become 
closely associated with the recurrent, especially on the right 
side where the pulmonary aortic arch atrophies, and the author 
believes that they occasionally, at least, persist in the adult. 
The support for this conclusion is based not only upon their 
presence in an embryo of this stage of development (17.8 mm.), 
but upon the observations of Wrisberg. Wrisberg (Henle’s 
Anatomie des Menschen, Bd. 3, p. 441, 1868) observed in three 
cases a reduplication of the right recurrent nerve. The extra 
branch was much smaller than the normal, and it accompanied 
the latter upwards between oesophagus and trachea, in much the 
same way as occurs in this embryo. 

The recurrent nerves are displayed in plate 6. The right recur- 
rent nerve (N.rec.). arises from the vagus at the caudal border 
of the right fourth aortic arch (A. subeclavia), and passes ceph- 
alad in the neck, dorsal to the parathyreoid of the fourth pharyn- 
geal pouch (P.-thyr.IV). Here it gives off some oesophageal 
branches, and then continues to the medial side. of the right 
lamina of the thyreoid cartilage where it becomes exhausted by 
giving off branches which turn ventrally to the anlagen of the 
laryngeal muscles. 

The left recurrent nerve (N.rec.s.) arises from the vagus at 
a more caudal level than the right, viz., at the caudal border of 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 63 


the pulmonary arch (ductus arteriosus). Winding around this 
arch ventro-dorsally, it extends cephalad. In the neck it has 
relations similar to those of the corresponding nerve on the right, 
with the exception that it passes medial to the parathyreoid IV 
instead of dorsal. 

It has been suggested by Froriep (85) that the recurrent nerves 
may well be considered as trunks formed by the fusion of bran- 
chial nerves for clefts which fail to develop. This interpretation, 
again advanced by Lewis in 1906, gains support from the relation 
of the posttrematic rami IV to the recurrent nerves in this em- 
bryo, especially on the right side where the two nerves are brought 
close together, the caudal aortic arches having atrophied. 

One of the inferior cardiac branches of the right vagus is seen 
in plate 3. It (N.c.2.) arises from the dorso-medial wall of the 
vagus caudad of the place of origin of the recurrent nerve. It 
extends caudad in the angle between the oesophagus and the 
trachea, being dorsal to the vagus and medial to the internal jugu- 
lar vein. It eventually becomes in part incorporated in the deep 
cardiac plexus, a part rejoining the vagus. Slightly caudad of 
the above nerve another inferior cardiac ramus from the vagus 
extends medially to the anlage of the deep cardiac plexus. It 
is hidden in the reconstruction by the overlying vagus. An in- 
ferior cardiac nerve was found on the left side corresponding to 
the right nerve shown in plate 3, but it has been omitted in the 
reconstructions. It arises from the left vagus just after it has 
given off its recurrent ramus, hence at a considerably more 
caudal level than the right inferior cardiac. It extends caudad 
in the angle between the oesophagus and the trachea to the deep 
cardiac plexus. 

N. accessorius. The accessory nerve (N.acc., mate 2 and 3) 
is formed by a series of small rootlets which emerge from the 
lateral surface of the spinal cord and myelencephalon. The 
most caudally placed root of the series issues at the level of, and 
in close proximity to, the dorsal root of the second cervical nerve. 
In this embryo the place of emergence of the most caudally 
placed root is similar on the two sides, and has been noted in 


64 F. W. THYNG 


consequence of its variation in other human embryos. Streeter 
(04) places the level at the third or fourth cervical, but finds 
that sometimes it is more caudally placed. 

The main trunk of the nerve arches cephalad and ventrally 
under the hypoglossal ganglia (Gg.hyp.) to become incorporated 
with the vagus ventral to the jugular ganglion. , At the cephalo- 
ventral border of the ganglion nodosum of the vagus, the ramus 
externus (f.ev., plate 3) of the aecessory nerve curves laterally 
and dorsally around the lateral side of the internal jugular vein 
(plate 6) to the developirg sterno-cleido-mastoid muscle. 
From here it passes around the cephalic part of the adjacent 
lymph sac to reach the anlage of the trapezius muscle. In the 
angle between the internal jugular vein and the lymph sac 
(S.jug.) it communicates with the great auricular nerve. 

N. hypoglossus. Numerous, small, hypoglossal rootlets issue 
from the ventral wall of the myelencephalon on either side, and 
each group converges to form four trunks. These, after crossing 
the lateral side of the vertebral artery (A.vert.), further join to 
form the two main roots of the hypoglossal nerve (\.hyp., 
plates 2 and 3). 

On the right side, in addition to the four trunks seen on the 
left, a vestigial root joins the fourth trunk (plate 3). Such ves- 
tigial roots of the hypoglossal nerve have been found by Bremer 
(08) to occur frequently in embryos of man, pig, sheep and dog, 
and almost constantly in the turtle and chick. 

It will be noted that the caudal of the four hypoglossal roots 
on the left (plate 2) passes through an arterial fenestra of the 
verteLral artery. <A similar, but more extensive anastomosis of 
arterial branches with the vertebral, enclosing the hypoglossal 
roots has been shown by Elze (07), (Taf. 15, fig. 2). They show 
how, by means of island-formation, the vertebral artery in the 
adult may come to pass between the roots of the hypoglossal 
nerve or, in rare cases, extend lateral to the entire nerve. The 
hypoglossal ganglia have been considered with the description of 
the vagus. 

The hypoglossus receives a branch from the first cervical nerve 
(one from the second cervical in addition on the right), and ex- 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 65 


tends ventrally between the vagus and the internal jugular vein. 
At the ventral border of the vagus, a little caudad of the termina- 
tion of the linguo-facial vein, it gives off its ramus descendens. 
The latter extends caudad, ventral to the internal jugular vein, 
and joins the descendens cervicalis to form the ansa hypoglossi. 
The hypoglossus then continues cephalad, medial to the linguo- 
facial vein and the developing submaxillary gland (plate 5) to 
divide in the tongue into muscular branches. 


Spinal nerves 


Plates 1 and 4 show portions of the ventral divisions (rami 
anteriores) of the spinal nerves (Nn.sp.); plate 3 gives a lateral 
view of the right cervical nerves and plexus; and plates 2 and 5, 
a lateral view of the left cervical nerves and plexus. In plates 5 
and 6 the relations of these nerves to the jugular lymph sac 
(S.jug.) are shown. It will be noted that some of the ganglia 
are still connected by ganglionic bridges. 

Nn. cervicales. The cervical nerves divide just beyond the 
union of the dorsal and ventral roots into dorsal and ventral pri- 
mary divisions (rami posteriores and anteriores). The dorsal 
rami are shown as stumps with the exception of the great occipital 
branch (N.occ.m.) from the second, a piece of which has been 
added. The ventral primary divisions run ventrally and cau- 
dally, and the second to the sixth inclusive, extend laterally to 
the vertebral artery (A.vert.). 

The first cervical nerve has a long slender ganglion (plates 2 
and 3), the dorsal part of which overlies the accessory nerve 
(N.acc.). The ganglion shows signs of atrophy in its middle 
part, especially on the left side. As the nerve extends ventrally 
it is in close contact for a distance with the vertebral artery (plates 
1 and 2), which it crosses on the medial side. A short communi- 
eating branch connects the first cervical nerve with the ventral 
primary division of the second cervical. The fibers of this 
communicating branch join the hypoglossal nerve, and are thought 
to assist in forming the ramus descendens hypoglossi. 

The ventral primary division of the second cervical nerve be- 
haves differently on the two sides. On the left (plate 2) beyond 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. l 


66 F, W. THYNG 


the communicating branch with the first cervical nerve, it passes 
caudad to join a branch from the third. The junction occurs 
dorsal to the internal jugular vein and forms the descendens 
cervicalis which extends ventrally between the internal jugular 
vein and jugular lymph sac (plates 5 and 6). ‘The descendens 
cervicalis joins the ramus descendens hypoglossi to form the ansa 
(An.hyp.). 

On the right side (plate 3), beyond the communicating branch 
with the first cervical nerve, the ventral primary divisions of the 
second cervical bifureates. One branch joins the third cervical 
to form the descendens cervicalis, while the other passes ventrally 
between the vagus and internal jugular vein to join the hypo- 
glossus. The fibers passing direct to the hypoglossus, therefore, 
reach the ansa by way of the ramus descendens hypoglossi. 

The ventral primary division of the third cervical nerve also 
differs on the two sides. On the right (plate 3) distal to the 
communicating branch with the second cervical nerve, it divides 
into a cephalic and a caudal branch. The caudal branch fur- 
nishes medially the ramus descendens cervicalis; and laterally a 
branch which unites with one from the fourth cervical to form the 
supra-clavicular nerves (Vn. s-cl.). 

On the left side the ventral division of the third cervical 
(plate 2) also divides into two branches. The more cephalic of 
these first gives off medially a branch which passes between the 
vagus and jugular lymph sac to the ramus descendens cervicalis. 
Then, continuing ventrally between the dorsal portions of the 
internal jugular vein and adjacent lymph sac, it furnishes (plates 
5 and 6) the great auricular (N.aur.m.), small occipital, and 
cutaneous colli (N.c.c.) nerves. The great auricular nerve 
pierces the dorsal part of the cephalic segment of the saccus 
jugularis, and here communicates with the ramus externus of the 
accessory nerve. The small occipital and cutaneous colli nerves 
appear a little more caudally between the two cephalic segments 
of the lymph sac. At the place of origin of the small occipital 
and the cutaneous colli, the third cervical nerve has a communi- 
cating branch with the fourth cervical, probably to assist in the 
formation of the supraclavicular nerves (Nn.s-cl.) as on the 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 67 


right side. The more caudal branch of the ventral ramus of the 
third cervical nerve extends caudad, and gradually tapers out. 
Possibly this branch would have joined, eventually, the phrenic 
nerve (N.phr.). 

The fourth cervical nerve on either side (plates 2 and 3) con- 
tributes to the formation of the supraclavicular nerves (Vn.s-cl.), 
which extend ventrally along the cephalic border of the jugulo- 
cephalic vein (plate 5). It also sends a branch to the phrenicus 
(N.phr.), and to the ventral ramus of the fifth cervical. The root 
which the phrenic derives from this nerve (plate 2) passes be- 
tween the two terminal branches of the thyreo-cervical artery. 

The ventral primary division of the fifth cervical nerve gives 
off the upper root of the long thoracic nerve, and then joins with 
the sixth cervical to form the upper trunk of the brachial plexus 
(plate 3). Just before joining, however, the fifth cervical nerve 
gives off its phrenic root which, extending caudad, dorsal to the 
thyreo-cervical artery (plate 2), meets the branch from the fourth 
cervical nerve. Each phrenic nerve (N.phr.), thus formed, ac- 
companies the internal mammary artery (A.mam.z.) dorso- 
ventrally around the caudal border of the terminal portion of the 
subclavian vein (V.scl., plate 5). The nerve here leaves the 
artery, and at first extends in the somatopleure along the lateral 
side of the anterior cardinal vein, then between this vein and the 
pleural cavity, and finally in the pleuro-pericardial membrane, 
lateral to the common cardinal. The nerves ultimately reach 
the anlage of the diaphragm on either side of the common hepatic 
vein (the right phrenic being for some distance in close relation 
to the latter). 

The ventral primary division of the sixth cervical nerve, dorsal 
to where it is joined by that of the fifth, gives off the middle root 
of the long thoracic nerve. 

The ventral primary division of the seventh cervical nerve 
furnishes the lower or caudal root of the long thoracic nerve, and 
becomes the middle trunk of the brachial plexus. 

The ventral primary division of the eighth cervical and that 
of the first thoracic nerves unite to form the lower trunk of the 
brachial plexus. 


68 F. W. THYNG 
SENSE ORGANS 
Hye 


The eye-ball is distinctly outlined, and the anlagen of the 
eyelids form two prominent arches. The optic vesicle (Ves.op.), | 
its stalk (Op.s.), and the lens (L.) of the right eye are shown in 
plate 3; the same structures on the left in plate 5. 

The optic stalk is slender and the lens is now devoid of a cavity. 
Into the loose mesenchyma, occupying the vitreous chamber 
extend the hyaloid artery and its branches. Other blood vessels 
reach this chamber through the circular fissure at the margin of 
the lens. 


Kar 


The fossa conchae (text fig. 1) is a depression rather broad 
and shallow dorsally, but narrow and deep ventrally. The man- 
dibular border of the fossa is formed by a fold especially promi- 
nent in its ventral part where it presents a conspicuous projection. 
This, the tuberculum tragicum, extends laterally and somewhat 
toward the fossa. The ridge at the hyoid border of the fossa is 
more prominent than that on the mandibular border and is 
bounded caudally by the retroauricular groove. At the ventral 
end of the vidge is a well marked protuberance which overhangs the 
fossa directly opposite the tuberculum tragicum. It represents 
the tuberculum antitragicum. The portion of the ridge dorsal 
to this tubercle is the developing helix. In the central part of 
the floor of the fossa is the low elevation which represents the 
tuberculum membrane tympani of Hammar (’02). 

The lateral aspect of the right otic vesicle is represented in 
plate 3 and the medial in plate 4. The stumps of the nerves 
which innervate it are seen in plate 2. The utricular (Ut.), 
saccular (Sac.), and cochlear divisions (D.c.), are not sharply 
differentiated from one another, and the coalesced lamellae of the 
lateral canal have not been absorbed as yet. The ductus endo- 
lymphaticus widens into the saccus, and extends for a consider- 
able distance dorsally. A peculiar anomaly occurs in this oto- 
cyst; a short hollow diverticulum projecting from the utricle 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 69 


just dorsal to the ampulla of the lateral semicircular canal. 
This diverticulum joins the crus commune of the superior and 
posterior semicircular canals without, however, opening into it. 
This diverticulum. does not occur upon the left side, but a simi- 
lar condition in other human embryos of the Harvard Collection, 
has been observed by the author. 


Nose 


The nares are represented in plates 3 and 5, and the ectodermal 
lining of the olfactory vesicles in plates 1, 2 and 4. 

The nares (Na.) are open, but separated by a broad septum, 
From the naris each olfactory pit (Ves.olf.) extends dorso-cau- 
dally as a small tube, oval in transverse section. Each tube 
shortly expands to form the main nasal cavity which opens into 
the fore part of the roof of the mouth by a large primitive choana 
(Chipr.)s 

In lateral view (plate 2) the epithelial wall of the olfactory 
vesicle presents cephalad a large concavity partially subdivided 
by a slight external ridge (unfortunately not shown in the draw- 
ing) into a shallow dorsal depression, the nasoturbinal (agger 
fasi), and a more extensive ventral one (Maz-turb.), the maxillo- 
turbinal (concha inferior). The maxilloturbinal is limited ven- 
trally by a pronounced, somewhat dorsally curled fold of the 
vesicular wall, containing within the meatus nasi inferior. 

At the dorsal part of the choanal extremity of the olfactory 
vesicle there is seen in a lateral view a slight indentation (Hth.- 
turb.[), representing the developing ethmoturbinal I (concha 
media). The ethmoturbinal I, although it now forms in part the 
lateral wall of the olfactory vesicle, has been shown by Peter (’01) 
to be a derivative of the septal wall. The ridge appearing be- 
tween the ethmo- and maxillo-turbinal overlies the meatus nasi 
medius. The palatal process forms the lateral boundary of the 
choanal extremity of the olfactory vesicle. 

On the medial wall of the olfactory vesicle (plates 1 and 4) 
. 1s seen the small, tubular outgrowth (Org.vom.-nas.) for the 
vomero-nasal organ (Jacobson’s), rudimentary in man. 


70 F. W. THYNG 


The nasolacrimal duct (not shown in the reconstructions) 
begins at the medial angle of the eye in an expanded end, dis- 
connected from the ocular epithelium, and extends medially to 
end near the epithelium of the meatus nasi inferior. It is a solid, 
irregularly branching, cord of cells, entirely surrounded by mesen- 
chyma. Outgrowths from the ocular extremity for the superior 
and inferior lacrimal ducts have not developed. 


meVASCULAR SYSTEM 
Heart 


The heart in its relation to the surrounding organs is shown 
in plates 1 and 4. The section shown in plate 1 passes to the left 
of the atrial and ventricular septa, and therefore, opens the left 
atrium and ventricle. In plate 4 the section passes through the 
cavities of the right side. The ventricular trabeculae are repre- 
sented somewhat diagrammatically. 

The sinus venosus (S.v., pl. 4) receives the common hepatic 
(V.hep.com.) and the right and left common cardinal veins 
(Vv.card.c.d. and s.), and opens into the right atrium. 

Upon the dorsal wall of the right atriwm (At.d., plate 4) is the 
sagittally directed, sinu-atrial orifice. ‘The latter forms a nar- 
row slit, bounded laterally by the right and left sinus valves 
(V.v.s.). The cephalic ends of the valves converge and meetin a 
ridge. The continuation of this ridge, which can be traced for 
some distance along the cephalic part of the interior of the atrium, 
is the septum spurium. 

The caudal extremity of the left sinus valve meets the right 
side of the septum primum at the caudal part of the right atrium. 
Just where these two structures meet ventrally there is a ridge or 
tubercle, which probably represents the caudal end of the future 
septum secundum. Born (’89) goes into no detail with regard 
to the earlier stages in the development of the septum secundum, 
which is shown completely formed in his figure 29. From the 
relation which the tubercle bears to the septum primum and the 
left sinus valve it can scarcely be doubted that it would eventually , 
form part of the adult limbus fossae ovalis. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO rel 


A large portion of the right sinus valve subsequently disappears 
but its caudal part will persist to form the valvulae venae cavae 
inferioris and sinus coronarii. On the right of the atrio-ventric- 
ular orifice a portion of the developing tricuspid valve (V.t.) is 
seen. mAb 

The principal outlet for the right ventricle (Vent.d.) is now 
the truncus pulmonalis (7'r.pul.), although the presence of the 
small interventricular opening (F.int., pl. 1) still allows some 
blood to pass out by way of the aorta. The aortic septum (S.) is 
practically complete at this stage, and is seen in plate 4, separat- 
ing the root of the aorta from the conus arteriosus (Con.art.) 
of the right ventricle. 

The left atrium (At.s., pl. 1) is partially separated from its 
fellow on the right by the septum primum. A quadrilateral 
opening in the septum, the ostium secundum (0.s.), places the 
two atria in direct communication. Leading into the left atrium 
on its dorsal side two pulmonary veins are shown (Vv.pul.d.). 
Between the left atrium and ventricle there is a marked protu- 
berance of the fused endocardinal cushions, which represents in 
part the anlage of the bicuspid valve. The vessel (V.card.com.s.) 
seen in section immediately caudad of the left atrium is the part 
of the left common cardinal which will become the coronary 
sinus. 

The left ventricle is still in communication with the right 
by a small opening (F.int.) the foramen interventriculare 
which is directed obliquely dorso-ventrally from left to right. 
This eventually becomes closed towards the right ventricle by 
complete fusion between the aortic and interventricular septa. 
When this has occurred, the aorta (7’r.aor.) will become the only 
outlet of the left ventricle. The ventricular wall immediately 
ventral to the anlage of the tricuspid valve (V.t.) separates the 
left ventricle from the right atrium, and has been named by 
Hochstetter (’98) the septum atrio-ventriculare. This septum 
together with that formed by the fusion of the aortic (S.) and 
interventricular septa, form the septum membranaceum ventric- 

-ulorum of the adult heart. 


Vp F. W. THYNG 


Arteries 


The arteries of the right side are shown in plate 1 from the 
left side. The arteries of the left side of the head and neck are 
shown in plate 2. 

Systemic derivatives of the aortic arches. The truncus aorticus 
(Tr. aor., plate 1) leaves the left ventricle of the heart, extends 
in the dorsal mesocardium across the pericardial cavity, and 
immediately divides into two ventral aortae. The left ventral 
aorta gives off the short fourth left aortic arch which becomes 
part of the arcus aortae, and continues cephalad as the !eft 
common carotid. It is shown in this plate as a stump, but is 
displayed fully in plate 2. 

The left common carotid extends cephalad between the thymic 
and thyreoid anlagen to the region of the larynx, where it divides 
into a dorsal and a ventral branch, the internal and the external 
carotid arteries, respectively. 

The external carotid (A.car.ex.) represents the continuation 
of the ventral aorta. It is a short stem which terminates by 
dividing into five branches, viz.: superior thyreoid, occipital, 
lingual, external maxilary and posterior auricular. The superior 
thyreoid (A.thyr.s. and d., plates 1 and 6) runs medially skirting the 
cephalic extremity of the lateral lobe of the thyreoid gland, and 
at once breaks up into small branches. The occipital (A.occ., 
plates 2 and 6) takes a cephal‘c and dorsal direction around the 
lateral side of the internal jugular vein, and gives off the sterno- 
mastoid branch. The lingual (A.ling., plates 2 and 6) runs 
medially to the tongue, giving off laterally a branch to the sub- 
maxillary gland. The external maxillary (A.maz.ex., plates 2 
and 6) extends ventrally along the mandibular arch. The pos- 
terior auricular (A.aur.p., plates 2 and 6) is a large vessel which, 
having given off the stylo-mastoid branch curves laterally behind 
the primary external acoustic meatus and passes dorsally. The 
ascending pharyngeal and temporal arteries have not been iden- 
tified. The internal maxillary, as such, is still wanting in this 
embryo; it is foreshadowed by the stapedial branch of the in- 
ternal carotid to be described later. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 73 


The left internal carotid (A.car.2.) comprises the third aortic 
arch, the left dorso-lateral aorta cephalad of the third arch, 
and a terminal portion or branch from the first aortic arch. It 
curves at first dorsally and cephalad, and then extends directly 
cephalad, dorsal to the roof of the pharynx. It passes medial to 
the cranial nerves and bifurcates, lateral to the diencephalon 
(Dien.) into caudal and cephalic divisions. The former is the 
posterior communicating artery (A.com.p.). This extending 
caudad gives off the posterior chorioidal artery, and joins the left 
posterior cerebral (A.cer.p.). The other terminal division of the 
internal carotid artery divides into two branches, the anterior 
chorioidal (A.chr.a.), and a stem common to the middle cerebral 
(A.cer.m.) and the anterior cerebral (A.cer.a.) arteries. Besides 
the terminal branches two other branchesof the left internal carot- 
id are shown here, the ophthalmic and the stapedial. The oph- 
thalmie artery (A.oph.) arises from the internal carotid artery 
medial to the ophthalmic nerve (N.oph.). It extends cephalad 
and laterally, ventral to the optic stalk which it penetrates, be- 
coming the central artery of the retina. 

The stapedial artery (A.stp., pl. 2) arises from the internal 
carotid near the middle of the pharynx, and passing through the 
left stapedial cartilage, runs as shown in plate 2, towards the semi- 
lunar ganglion (G.s.-l.). A short distance from the ganglion it 
divides into two branches, dorsal and ventral. The dorsal or 
supraorbital branch passes lateral to the semilunar ganglion into 
the region of the orbit. The ventral division soon bifurcates, 
and the resulting branches pass to the medial and lateral sides, 
respectively, of the mandibular nerve. The medial branch, 
having communicated with the lateral by a branch passing ven- 
tral to the mandibular nerve, first accompanies the buccal branch 
of the mandibular and later the maxillary nerve, as the infra- 
orbital artery. The lateral branch passes between the roots of 
origin of the auriculo-temporal nerve and, having communicated 
with the medial branch, becomes the inferior alveolar which ac- 
companies the nerve of the same name (N.alv.inf.). 

The development and importance of the stapedial artery have 
been demonstrated admirably by Tandler (’02). He finds that 


74 F. W. THYNG 


this artery represents a persistent portion of the dorsal part of the 
second aortic arch, and an intermediate segment of the first arch; 
the two being united by a longitudinal anastomosis. In certain 
mammals the stapedial artery persists throughout life, but in man 
an anastomosis between the external carotid artery and the man- 
dibular ramus of the stapedial results in the formation of the adult 
internal maxillary artery to which the stapedial transfers its 
branches. In this embryo the anastomosis is yet to form; there 
can be no doubt, however, that it will join the lateral branch of the 
ventral division of the stapedial between the auriculo-temporal 
nerve and the branch of communication between the lateral and 
medial branches. As a result of such an anastomosis the lateral 
branch of the ventral division, together with the dorsal division 
(supraorbital of Tandler), would become the middle meningeal. 
The middle meningeal and inferior alveolar would then spring. 
from the internal maxillary. The original main trunk of the 
stapedial would persist in part as the carotico-tympanic branch 
of the internal carotid and the superior tympanic branch of the 
middle meningeal. It is extremely probable that the medial 
branch of the original ventral division of the stapedial is repre- 
sented in the adult by the accessory meningeal branch of the 
internal maxillary. 

The right ventral aorta (plate 1) takes a cephalic direction 
from the truncus aorticus. It soon gives off a short dorsal 
branch, the fourth aortic arch, which becomes part of the right 
subclavian. The portion of the right ventral aorta proximal to 
the fourth arch becomes the innominate artery (A.anon.), the 
smaller distal continuation represents the right common carotid. 
The relations of the latter are essentially the same as those just 
given for the corresponding artery on the left. A branch, how- 
ever, from the proximal part of the right internal carotid, accom- 
panies the hypoglossal nerve. This, the hypoglossus artery 
(A .hyp.d., plate 1) has been noted in the human embryo by Zim- 
merman (89), Tandler (’02) and Ingalls (’07), and in the 
rabbit by Hochstetter (90). The hypoglossus artery of the 
left side had disappeared in this embryo. The hypoglossus 
arteries represent the second pair of primitive intersegmental 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 4b 


arteries from the dorso-lateral aortae, later becoming temporary 
branches of the internal carotids. . 

The dorso-lateral aortae have ceased to exist as complete 
trunks, for their continuity has been interrupted in the segment 
of each intervening between the third and fourth aortic arches. 

The segments cephalad of the third pair of aortic arches con- 
sist of two symmetrical vessels which enter into the formation of 
the internal carotid arteries, as already stated. The segments 
caudad of the fourth pair of arches, consist of two very unsym- 
metrical vessels which converge and unite, opposite the second 
pair of thoracic nerves, to form the dorso-median aorta (Ao.d.m.). 

The caudal segment of the right dorso-lateral aorta is still 
large in the neighborhood of the fourth aortic arch, with which it 
participates in the formation of the right subclavian artery 
(A.scl.d.). The remainder of this segment of the right dorso- 
lateral aorta is reduced to a fibrous cord containing only a trace 
of a lumen. The caudal segment of the left dorso-lateral aorta 
is large and contributes to the formation of the permanent arcus 
aortae. 

Although the intermediate segments of the dorso-lateral aortae 
have lost all connection with the third pair of aortic arches, they 
still persist as a pair of vestigial arterial tubes (S.) projecting 
from the cephalic aspect of the fourth aortic arches. 

The fourth aortic arches extend from the place of origin of the 
common carotids, upon the right and left sides, to the vestigial 
tubes above mentioned. They are both short, wide trunks, of 
which the right occupies a more cephalic position. The right 
fourth aortic arch eventually forms the proximal part of the right 
subclavian artery. The left participates in the formation of the 
arcus aortae. 

Pulmonary arteries. The truncus pulmonalis (Tr.pul., plates 1, 
2 and 4) begins in the conus arteriosus (Con.art.) of the right 
ventricle which is almost completely separated from the truncus 
aorticus (vestibule). Of the pulmonary arches, only the left per- 
sists in its entirety. The truncus pulmonalis is now continued 
directly into the left pulmonary arch which opens into the left 
dorso-lateral aorta in common with the left fourth arch. 


76 F. W. THYNG 


The two small pulmonary arteries (Aa.pul., plate 1) extend 
caudad from the main pulmonary trunk, one to each developing 
lung. The right vessel is reconstructed more completely than 
the left, and represents not only the right pulmonary artery, 
but the proximal portion of the right pulmonary arch, as has been 
shown by Bremer (02). The left pulmonary artery is shown 
only asastump. It represents the left pulmonary artery proper. 
The segment of the pulmonary arch (D.a., plate 2) between the 
left pulmonary artery and the left dorsal aorta is the ductus 
arteriosus. 

Derivatives of the occipital and cervical segmental arteries. The 
right hypoglossus (intersegmental) artery (see p. 75) and the 
proximal parts of the cephalic six (five on the right side) pairs of 
cervical intersegmental arteries have disappeared. The distal 
parts of these intersegmentals have now become branches of 
the large vertebral arteries (A.vert.), formed from the series of 
post-costal anastomoses between them. The seventh inter- 
segmental artery (sixth on the right side) has retained its connec- 
tion with the dorso-lateral aorta of its own side to form a portion 
of the subclavian and the proximal part of the corresponding 
vertebral artery. The vertebral arteries extend cephalad medial 
to the cervical nerves, with the exception of the first pair which 
they accompany towards the spinal cord. The sudden change in 
direction of the vertebral arteries opposite the suboccipital pair 
of nerves arises from the fact that the portion of these arteries 
between the atlas and occiput develops from the first cervical 
intersegmental spinal rami. The vertebral arteries pass ventral 
to the myelencephalon and medial to the hypoglossal and vagus 
nerves. Opposite the glossopharyngeal the arteries of the two 
sides unite to form an enormous median vessel, the basilar (A .bas.). 
The basilar artery extends cephalad in the median line between the 
notochord and the rhombencephalon. Between the trochlear 
nerves (N.troch.) it divides into two lateral branches, the posterior 
cerebral arteries. Each posterior cerebral artery (A.cer.p.) 
communicates ventrally with a branch (posterior communicating) 
of the internal carotid artery (A.car.z.) to participate in the for- 
mation of the circulus arteriosus. It also dispenses several 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO dk 


branches to the lateral and dorsal surfaces of the di- and 
mesencephalon (Dien. and Mesen.), a considerable branch to the 
mesencephalon passing through the rootlets of the oculomotor 
nerve (N.oc., plate 2). The left superior cerebellar artery 
(A.cereb.s.) arising from the basilar is seen in plate 2, just 
caudad of the trochlear nerve. Its branches overlie the isthmus 
and the metencephalon. The other branches of the basilar 
are seen in part in plate 1, those on the left side having been cut 
away. 

The left subclavian artery (A.scl., plate 2) is a branch of the 
corresponding seventh cervical intersegmental artery. Beyond 
the origin of the vertebral artery (A.vert.), the thyreo-cervical 
(Tr.thyr.-cerv.) arises from the cephalic aspect of the subclavian; 
the costo-cervical (7'r.cost.-cerv.) from the dorsal; and the internal 
mammary (A.mam.i.) from the ventral aspect. The costo- 
cervical divides, as usual, into the deep cervical and superior 
intercostal; the others can be identified by their direction. The 
remaining portion of the subclavian trunk extends ventro-later- 
ally into the upper limb. 

On the right side (plate 1) there is a common anomaly, the 
sixth (not the seventh) cervical intersegmental being connected 
with the right dorso-lateral aorta. By means of the right dorso- 
lateral aorta and a short precostal anastomosis the sixth inter- 
segmental is connected with the seventh. 

Consequently the right subclavian comprises three elements 
(exclusive of the parts derived from the fourth aortic arch and 
dorso-lateral aorta). These are: (1) the root of the sixth cer- 
vical intersegmental artery; (2) a longitudinal precostal anasto- 
mosis connecting the sixth and seventh intersegmentals; and (3) 
a portion of the seventh intersegmental. From the first element 
arises the right vertebral artery (A.vert.d.), the root of which 
represents a part of the sixth cervical segmental. From the 
third element the right superior intercostal arises on the caudal 
‘aspect; the thyreo-cervical on the cephalic; the deep cervical on 
the dorsal, and the internal mammary on the medial aspect. 
The deep cervical is derived from the dorsal ramus of the seventh 
intersegmental artery. The internal mammary represents the 


78 | F. W. THYNG 


real continuation of the ventral ramus of the seventh cervical 
intersegmental. The continuation of the subclavian beyond the 
point of origin of the internal mammary appears to represent a 
lateral branch of the ventral ramus of the seventh intersegmental. 
By the anomaly found on the right side of this embryo, the deep 
cervical and superior intercostal arteries come to have separate 
origins from the subclavian as sometimes occur in the adult. 

Parietal arteries of the trunk. From the level of the second 
pair of thoracic nerves, where the dorso-lateral aortae unite, the 
single dorso-median aorta (Ao.d.m.) continues throughout the 
trunk to end as the middle sacral (A.s.m.). The dorsal inter- 
segmental arteries arise in regular pairs from the dorso-median 
aorta. Those of the right side are shown in plate 1, the corre- 
sponding vessels on the left having been omitted. 

The dorsal intersegmental arteries curve laterally and dorsally, 
passing lateral to the sympathetic, and either pass dorsally to 
become muscular branches or continue medially as spinal rami 
into the vertebral canal. In the latter situation the spinal 
rami pass laterally to the spinal cord to form the posterior spinal 
arteries or join to form a bilateral ventral longitudinal anasto- 
mosis which extends from the vertebral artery (directly caudad 
of the myelencephalon) along the spinal cord into the tail process. 
Some of the spinal rami bifurcate contributing thereby a branch 
to each anterior and posterior spinal artery of that side. 

That the embryonic right anterior spinal anastomosis eventually 
joins its fellow of the opposite side to form the single anterior 
spinal artery of the adult was first indicated by His (86). Re- 
cently the matter has been treated in greater detail by Sterzi 
(04), and Evans (09 and 712). The latter observers conclude 
that fusion between the two stems does not occur, but that the 
single adult artery represents the selected channel persisting 
from the alternative paths offered by the two longitudinal vessels 
and the plexiform connections between them. 

In the thorax and abdomen the ventral rami of the dorsal inter- , 
segmentals become the aortic intercostal and lumbar arteries, 
respectively. Some of the former are represented as short stumps 
in plate 1. The ventral rami of the first and second thoracic 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 79 


pairs of dorsal intersegmentals have lost their connection with the 
dorso-median aorta. They are now connected with the sub- 
clavian arteries by the precostal anastomosis which forms the 
adult superior intercostal. 

Visceral arteries. 'The ventral visceral arteries and the lateral 
visceral arteries of the right side are indicated in plate 1. 

In the thorax several small, ventral visceral arteries (Aa.oe.) 
extend from the aorta through the mediastinum to the oesophagus. 
According to Broman (’08) these oesophageal arteries are of 
secondary formation, the primary segmental arteries having early 
disappeared. 

The coeliac artery (A.coel.) leaves the aorta slightly caudad 
of the 11th thoracic pair of dorsal intersegmental arteries. It 
extends between the medial surfaces of the anlagen of the supra- 
renal glands, and entering the mesogastrium at once gives off the 
left gastric artery which turns cephalo-ventrally. The coeliac 
then continues in the mesogastrium (hidden in the reconstruction 
by the stomach, Ga.), and having given off the splenic artery it 
becomes the hepatic. The former artery extends in the meso- 
gastrium to the splenic anlage; the latter enters the lesser omen- 
tum and divides into the hepatic artery proper and the gastro- 
duodenal. The hepatic proper can be seen in the reconstruction, 
entering the liver just ventral to the portal vein (V.P.). It gives 
off the small cystic branch (not shown in the reconstruction) 
which accompanies the cystic duct. The gastro-duodenal artery 
can be traced distally between the left side of the duodenum and 
the proximal part of the dorsal pancreas. 

Tandler (’03) finds that the coeliac artery in a 17 mm. human 
embryo arises from the aorta slightly above the level of the 20th 
intersegmental (12th thoracic) artery. 

The superior mesenteric artery (A.mes.s.) takes origin from 
the aorta approximately on a level with the 12th thoracic pair 
of dorsal intersegmental arteries. It extends ventrally between 
the caudal ends of the suprarenal glands (plate 4) into the dorsal 
mesentery. It courses to the left of the duodenum and passes 
dorsal to the vena omphalomesenterica. In the dorsal mesen- 
tery it gives off numerous small branches, and crosses on the right 


80 F. W. THYNG 


of the large intestine. In the umbilical cord, after again crossing 
the gut (on the left side of the small intestine), it follows the 
yolk-stalk. In a 17 mm. human embryo studied by Tandler 
(03) the origin of this artery is opposite the 21st intersegmental 
(1st lumbar) artery. 

On a level with the 2nd lumbar pair of dorsal intersegmental 
arteries the inferior mesenteric (A.mes.i.) arises from the aorta. 
It is a small ventral visceral branch, extending ventrally and 
somewhat caudally between the aortic bodies (of Zuckerkandl) 
into the dorsal mesentery. In the dorsal mesentery it gives off 
several branches which ramify in the region of the colon. Tand- 
ler (03) found this artery in a 17 mm. human embryo arising 
from the aorta at the level of the 23d intersegmental (8d lumbar) 
artery. 

That the arteries of the gut migrate cephalo-caudad was 
demonstrated by Mall (’91 and ’97), but comparison of the origins 
of these arteries in this embryo with those described by Tandler 
(03) in an embryo of approximately the same length, indicates 
that the rate of migration is somewhat variable, or we may have 
to do merely with variations in the point of origin, such as have 
been demonstrated for the superior mesenteric artery. 

Just caudad of the pleural cavities and on a level with the 10th 
thoracic pair of dorsal intersegmental arteries, there appears a 
left lateral visceral artery. It extends from the aorta laterally 
and caudad first along the medial border of, and then through, 
the suprarenal gland to the Wolffian body. Apparently this 
vessel becomes the left inferior phrenic artery which furnishes a 
superior suprarenal branch in the adult. Another pair of lateral 
visceral (mesonephric) arteries (A.s.-r.) presumably the middle 
suprarenal of the adult leaves the aorta near the origin of the 
superior mesenteric artery. This pair of vessels extends laterally 
in a cephalo-dorsal direction to the anlagen of the suprarenal 
glands. 

Beginning just caudad of the place of origin of the superior 
mesenteric artery the left components of four successive pairs of 
lateral visceral (mesonephric) arteries are seen in the reconstruc- 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 81 


tion. The cephalic two have a common stem of origin from the 
aorta. 

The first passes through the caudal part of the suprarenal 
gland to the Wolffian body. It presumably becomes the inferior 
suprarenal branch of the left renal. 

The second skirts the caudal part of the suprarenal anlage, 
passing dorsal to the left suprarenal vein (left subcardinal), and 
ends in the region medial to the metanephros of that side. It 
may be concluded, therefore, that this artery (including the 
common trunk referred to above) becomes the left renal. 

The corresponding artery (renal) on the right arises from the 
aorta dorsal to the left renal vein (renal anastomosis) and ex- 
tends to the Wolffian body, passing dorsal to the subcardinal seg- 
ment of the inferior vena cava. 

Broman (’08) finds that the renal arteries take origin from 
the 21st or 22nd aortic segment. 

The third mesonephrie artery is small and, hence, of little 
moment. The corresponding one on the right passes in a strand 
of mesenchyma through the inferior vena cava (i.e., the supra- 
ureteral channel). 

The fourth (A.sp.7.) is of considerable caliber. It takes origin 
at the level of the 21st dorsal intersegmental arteries, dorsal to 
the termination of the left suprarenal vein, and extends caudad, 
lateral to the anlage of the corresponding spermatic vein, to the 
Wolffian body. In the Wolffian body it supplies several glomeruli, 
lying adjacent to the most prominent part of the genital ridge. 

The corresponding artery on the right arises from the aorta 
dorsal to the renal anastomosis. It extends ventral to the vena 
cava inferior (i.e., the supra-ureteral channel) and caudad along 
the lateral side of the anlage of the right spermatic vein to the 
Wolffian body. 

These arteries, from their relations, the author believes, are 
the embryonic representatives of the internal spermatic (ova- 
rian) arteries of the adult. 

Broman (’08) finds that usually the spermatic arteries arise 
from the 22nd aortic segment. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO.1 


82 F. W. THYNG 


Iliac arteries. Each common iliac artery (Aa.il.com.) arises 
from the lateral wall of the aorta directly opposite the third lum- 
bar pair of dorsal intersegmental arteries, and on a level with the 
2nd lumbar nerves. At first of small caliber the common iliacs 
soon increase notably in diameter. The right common iliac 
artery is the more fully displayed in the reconstruction (plate 1). 
It extends laterally and caudad, passing ventral to the caudal 
segment of the inferior vena cava, medial to the metanephros, 
and dorsal to the ureter and Wolffian body (compare plate 2). 
This relation of the common iliac artery, which is preserved in 
the adult, is probably that of a dorsal intersegmental artery. 

The right common iliac artery terminates in two branches, the 
external iliac (A.il.ext.), a medium sized artery which accom- 
panies the corresponding vein, and the arteria hypogastrica 
(A .hypogas.). 

The external iliac artery first extends laterally and gives off 
a branch directed cephalad, the inferior epigastric (not shown in 
the reconstruction). It then continues caudad in the proximal 
part of the posterior limb-bud to finally join the A. ischiadica. 

The hypogastric artery is only a short trunk which divides, 
lateral to the ureter, into dorsal and ventral branches (the so- 
called anterior and posterior divisions of adult anatomy). The 
dorsal branch appears in the drawing only as a short stump, but 
probably here continues as the superior gluteal artery. The ven- 
tral branch soon divides into the large umbilical artery (A.wm.d.) 
and a short trunk common to the sciatic and internal pudendal 
arteries (A.isch.d. and A.pud.i.). The umbilical artery extends 
ventrally in the lateral body wall across the lateral side of the 
anlage of the bladder into the caudal wall of the umbilical cord. 

The sciatic (inferior gluteal and A. comitans n. ischiadica) is 
at first dorso-lateral to the corresponding vein (V.isch., plate 4) 
with which it continues into the posterior limb-bud. The tap 
between it and the femoral has already occurred. 

The internal pudendal is at first ventral to the sciatic vein but 
soon bends ventrally into the cephalo-lateral part of the genital 
papilla where it becomes the artery of the clitoris or penis, accord- 
ing to sex. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 83 


Veins 


The general distribution of the veins of the right side of the 
body is shown in plate 4. Plate 5 is a left lateral, and plate 
6 a ventral view of part of the anterior cardinal system. 

Anterior and common cardinal system. Between the dorso- 
medial surfaces of the cerebral hemispheres (plate 4) appears the 
paired anlage of the superior sagittal sinus (S.sag.sup.). This 
vessel together with numerous other tributaries arising on the 
lateral surface of the tel-, di-, and mes-encephalon (Telen.,Dien., 
and Mesen.) contributes to form the anterior cerebral vein 
(V.cer.a.) which joins the cephalic end of the cavernous sinus 
(S.cav.). The ophthalmic vein (V.oph.) which is represented more 
fully in plate 5, joins the cavernous sinus ventral to the entrance 
of the anterior cerebral vein. 

Venules arising on the lateral surface of the metencephalon 
(Meten.) unite to form the middle cerebral vein (V.cer.m.) which 
passes around the caudal border of the semilunar ganglion 

*(G.s-l.) to enter the cavernous sinus. 

The cavernous sinus is medial to the semilunar ganglion, and 
represents a persistent portion of the primitive anterior cardinal 
vein. The original anterior cardinal from the cavernous sinus to 
the site of the future jugular foramen, has disappeared, and a 
temporary vein, the vena capitis lateralis (V.cap.lat.), has formed 
lateral to the otocyst and the adjacent cranial nerves. The vena 
capitis lateralis is joined at the caudal aspect of the otocyst by 
the posterior cerebral vein (V.cer.p.) which takes its origin from 
a capillary plexus overlying the caudo-lateral surface of the 
myelencephalon (Myelen.). 

From the point at which it receives the posterior cerebral vein 
the right anterior cardinal (internal jugular) takes a direct 
course towards the region of the heart. Dorsal to the heart it 
unites with the azygos vein (V.az.) to form the right common 
cardinal (V.card.c.d.). The right common cardinal, which be- 
comes the proximal part of the adult vena cava superior, empties 
through the sinus venosus (S.v.) into the right atrium (Ait.d.). 


84 F. W. THYNG 


The extra-cranial part of the right anterior cardinal is repre- 
sented in the adult by the right internal jugular and innominate 
veins, and by the part of the superior cava distal to the point of 
entrance of the vena azygos. 

The extra-cranial part of the left anterior cardinal (plate 5) at 
this time is symmetrical in size and position with its fellow of the 
right side. Just before entering the sinus venosus the left com- 
mon cardinal (V.card.c.s.) turns to the right and occupies the 
suleus coronarius on the diaphragmatic surface of the heart. 
It is shown in the latter situation in plates 1 and 4. 

The vertebral veins of which the right (V.ver.d.) is recon- 
structed in plate 4, take the major share in the drainage of the 
cervical intersegmental veins. They open on either side into the 
dorsal aspect of the common cardinal near the termination of the 
azygos and hemiazygos respectively. 

The above description of the extra-cranial portions of the 
anterior cardinal veins includes only the main venous channels 
draining the sinuses of the developing dura mater. In the 
opinion of the author the true ‘anterior cardinals’ are not repre- 
sented by these channels alone, but by the main trunks of the — 
vertebral veins as well. This opinion is supported by the occur- 
rence in a pig embryo of 7.8 mm. (Thyng ’11) of a series of fen- 
estrae in the dorsal portion of the anterior cardinal vein (fig. 2) 
which appears to foreshadow the segregation of the dorsally 
placed vertebral vein from the main ventral channel (corre- 
sponding to the anterior cardinal as described above). A ver- 
tebral vein, thus formed, would receive the cervical interseg- 
mental veins as does the vertebral in this embryo. 

The right and left linguo-facial veins (V.ling-fac.) (Grosser 
01, Lewis ’09, and others) are shown in plates 4 and 5, 
respectively, and the terminal parts of both are seen in plate 6. 
Each arises in tributaries from its own side of the tongue, man- 
dible and face. The trunk, thus formed, enters the ventral wall 
of the internal jugular vein of its own side, near the p'ace where 
the latter is crossed medially by the hypoglossal nerve (N.hyp.). 
Earlier in development each linguo-facial vein enters the ventral 
wall of the anterior cardinal at a more caudal level, i.e., immedi- 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 85 


ately cephalad of the pericardial cavity. Or it may open into 
the common or even posterior cardinal vein (Lewis ’09, p. 34). 

Immediately cephalad of the pericardial cavity there now opens 
into the ventral wall of each anterior cardinal a vein (V.thym.-thyr., 
plate 6) arising from a venous plexus of the thymic and thyreoid 
anlagen. For this vein the term, vena thymico-thyreoidea, does 
not seem inappropriate. 

The right and left thymico-thyreoid veins, it seems to the 
author, undoubtedly unite to form the transverse anastomosis 
which eventually becomes the left innominate vein. Szawlowski 
(91) arrived at a similar conclusion from a study of these veins 
in the human embryo, as did Anikew (’09) who investigated the 
question in pig embryos and in a human embryo of 17.5 mm. 
The vena anonyma sinistra when fully formed, would thus re- 
ceive tributaries from the caudal part of the thyreoid anlage, 
the vv. thyreoidez inferiores and ima of the adult. 

Dorsal to the vena thymico-thyreoidea the right anterior car- 
dinal receives a vein, arising in a plexus situated at the cephalic 
end of the thyreoid gland, and the caudo-dorsal wall of the 
pharynx. It is possible that this vessel may represent the 
middle thyreoid vein. 

The subclavian veins, of which the left is shown in plate 5, 
(V.scl.), are formed by the union of the thoraco-epigastric 
(V.th.-ep.) and brachial veins. The latter vein begins as the 
primitive ulnar (V.ul.pr.) which forms a venous loop at the cir- 
cumference of the hand-plate. Each subclavian (plate 6) joins 
the dorso-lateral aspect of the internal jugular vein of its own 
side somewhat caudal to the place of entrance of the vena thy- 
mico-thyreoidea. The plexiform termination of the subclavian 
(plate 5), the foramina of which transmit branches of the brachial 
plexus, would indicate that this vein is still migrating in the 
cephalic direction. 

The terminations of the external jugular veins (V.jug.ez.) 
are distinct in plate 6. The terminal part of the left external 
jugular vein (V.jug.ex.s., plate 5) enters the internal jugular on 
a level with the caudal extremity of the jugular lymph sac 
(Sac.jug.). It is also connected, more dorsally, by a considerable 


86 F. W. THYNG 


channel with the terminal part of the subclavian vein at about 
the same level. The connection with the internal jugular is 
(usually) temporary, in which case the communication with the 
subclavian represents the future permanent outlet of the adult 
vessel. Sometimes a reverse condition occurs. Distally each 
external jugular vein is connected through a capillary plexus, 
caudad of the fossa conchae (shown, in part, in plate 5) with the 
linguo-facial vein and its tributaries from the hyoid arch. 

The cephalic vein (V.ceph.) of which only a part is represented 
here, occupies the radial border of the arm, and passes super- 
ficial to the clavicle (which is just beginning to ossify) to joi 
the external jugular. A similar condition of this vein has been 
represented for a human embryo of 22.8 mm. by Lewis (’09, fig. 
4), and for a human embryo of 20 mm. by Evans (’12, fig. 478). 
The portion of the cephalic vein superficial to the clavicle (V.jug.- 
ceph.) has been named the jugulo-cephalic vein. It usually 
atrophies since the cephalic commonly acquires a new connection 
with the axillary. The jugulo-cephalic occasionally persists in 
the adult, in which case the cephalic remains partially or entirely 
tributary to the external jugular vein. 

The proximal end of the external jugular vein receives ven- 
trally the anterior jugular vein (platie 5), proceeding from a 
superficial venous plexus of the neck. A similar venous con- 
nection between the external and anterior jugular veins has been 
represented in a human embryo of 22.8 mm. by Lewis (09, fig. 
4). Since the anterior jugular vein is in the adult normally a 
tributary of the external, it may be supposed that that part of 
the external jugular vein which now opens into the internal 
jugular, finally becomes the terminal part of the anterior jugular. 

Posterior cardinal system. The primitive posterior cardinals 
> in great part have lost their identity since they have been reduced 
to sinusoidal channels by the developing Wolffian bodies. ‘These 
channels, some of which gain prominence have been reduced fur- 
_ ther or interrupted by the developing metanephros and supra- 

renal gland. 

Minot (’98) demonstrated that the posterior cardinal veins in 
pig embryos become subdivided by the mesonephric tubules into 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 87 


sinus-like channels, and in 1900 he introduced additional histo- 
logical evidence of the presence of these channels to which he 
applied the name ‘sinusoids.’ It is also noteworthy that Hoch- 
stetter (93) expressed doubt whether in man the azygos and 
hemiazygos veins could be considered in their entirety as rem- 
nants of the posterior cardinals. 

Only the right posterior cardinal derivatives have been recon- 
structed (plate 4). 

The middle sacral component (V.s.m.) of the right posterior 
cardinal begins in the tail-process and extends cephalad ventro- 
lateral to the arteria sacralis media (A.s.m., pl. 1), receiving as 
tributaries the right caudal intersegmental veins. 

At the level of the fourth and fifth lumbar intersegmental veins 
the right vena sacralis media is connected to its companion vein on 
the left by a relatively large, transverse anastomosis (a’), passing 
ventral to the middle sacral artery. Other anastomoses of the 
middle sacral veins exist more caudally. 

It is by virtue of this large transverse anastomosis (which 
becomes the terminal part of the left common iliac vein of the 
adult) that the left middle sacral and the veins of the left lower 
extremity, come to drain into the inferior vena cava. 

Two venous channels (sub- and supra-ureteral) extend cephalad 
from the right middle sacral, enclosing between them an area of 
mesenchyma through which passes the ureter (Ur.). The smaller, 
sub-ureteral channel having at first passed dorsal to the Wolfhan 
body, with the sinusoids of which it is intimately connected, 
bends medially to terminate in the caudal part of the persistent 
portion of the right subeardinal vein (V.scard.d.) described below. 
This channel from its relation to the ureter and right Wolfhan 
body, corresponds perfectly with Hochstetter’s ‘Urnierenvene.’ 
It is of importance as Hochstetter (’93) pointed out, in that from 
its cephalic portion and the tributary mesonephric sinusoids 
there probably arises the right spermatic vein. 

The channel extending from the vena sacralis media, dorso- 
medial to the ureter (supra-ureteral), is much larger than the 
sub-ureteral. It extends cephalad, lateral to the aorta, and 
receives as tributaries the last two thoracic and first four lumbar 


eo 


SS8 F. W. THYNG 


intersegmental veins. Opposite the Ist lumbar spinal nerve 
it bends ventrally, and is continued as a large dorso-ventral 
channel (compare Sabin ’09, fig. 11), which terminates in the 
caudal extremity of the part of the subcardinal which still per- 
sists. This dorso-ventral segment of the supra-ureteral channel, 
extends medial to the metanephros and Wolffian body from 
both of which it receives tributary veins. One (or more) of 
these tributaries undoubtedly forms the right renal vein (or 
veins). The supra-ureteral channel forms a considerable portion 
of the definitive inferior vena cava. 

The subcardinal veins are derived from Wolffian sinusoids 
in the region ventral to the mesonephric arteries (Lewis ’02). 

At this stage the right subcardinal (V.scard.d.) begins at the 
renal anastomosis (7), which is formed by the confluence of the 
sub- and supra-ureteral channels from both sides of the body. 
From the renal anastomosis the right subcardinal vein extends 
cephalad, ventral to the right suprarenal gland (Gl.s.-r.d.), as a 
component of the vena cava inferior. 

By the development of the suprarenal gland the cephalic part 
of the V. subcardinalis (which previously opened into the posterior 
cardinal vein on a level now represented by the cephalic extremity 
of the gland) has been reduced to sinusoidal channels. The 
cephalic end of the portion of the right subcardinal vein, now 
persisting, represents the anlage of the right suprarenal vein 
(V.s.-r.d.) which connects, as does the azygos, with the suprarenal 
sinusoids. 

Tt should be stated here that the presence of sinusoids in the 
suprarenal glands of mammals and other vertebrates, has been 
demonstrated histologically by Minot (’00). 

Also significant in this connection are the observations of 
Lewis (’02, p. 236) upon the veins in question in a rabbit embryo, 
11 mm. in length. He says ‘‘at the upper end of the veins, on 
either side, cardinal and subcardinal anastomose in condensed 
mesenchyma probably connected with the suprarenal anlage.” 

The large renal anastomosis (a), referred to above, unites the 
two subcardinals. It crosses the median line, ventral to the aorta, 
a little caudad of the superior mesenteric artery (A.mes.s.) of 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 89 


which a short piece has been added in plate 4. It persists as 
the terminal part of the left renal vein. 

Simultaneously with the transformation of the subcardinal 
veins that part of the right subcardinal, situated ventral to the 
developing suprarenal gland has tapped the hepatic sinusoids as 
described by Lewis (02). In this manner there has developed 
that segment of the vena cava inferior, intervening between the 
subeardinal and common hepatic (V.hep.com.) veins. 

The entire vena cava inferior, as shown in plate 4, is composed, 
therefore, of four parts. These are the supra-ureteral channel; 
a segment of the original right subcardinal (V.scard.d.) interme- 
diate between the renal anastomosis and the termination of the 
anlage of the right suprarenal vein (V.s.-r.d.); a large channel 
passing through the plica venae cavae formed by the tapping of 
the hepatic sinusoids by the right subcardinal, and the terminal 
part, the vena hepatica communis (V.hep.com.), which empties 
into the right atrium through the sinus venosus (S.v.). 

The course of the vena azygos (V.az.) along the lateral side of 
the aorta has been interrupted in the region of the suprarenal 
gland, so that it now receives the first ten thoracic intersegmental 
veins (the first three indirectly, for they unite to form a common 
trunk, the vena intercostalis suprema dextra). 

The vena azygos, a little cephalad of the termination of the 
* vena intercostalis suprema, also receives a small ventral trib- 
utary, the cephalic remnant of an earlier mesonephric sinu- 
soid, figured in pig embryos, by Lewis (’03), Davis (710), and 
Thyng (11). This vessel has been designated by Davis (10) 
the ventro-lateral vein of the mesonephros. 

At the level of the 10th thoracic intersegmental vein the vena 
azygos communicates with the hemiazygos by a transverse anas- 
tomosis, passing dorsal to the aorta. Slightly cephalad of this 
anastomosis tributaries from the suprarenal gland (referred to 
above) join the azygos. 

Although the left posterior cardinal system has not been recon- 
structed, the following observations are here recorded for the sake 
of completeness. 


90 F. W. THYNG 


The sub-ureteral channel (Urnierenvene of Hochstetter) has 
‘been obliterated caudally. The supra-ureteral channel is not 
only much smaller than its companion on the right, but is sub- 
divided caudally by numerous mesenchymal septa, indicative of 
commencing atrophy. 

The left subcardinal vein, caudad of the renal anastomosis 
has degenerated, but its cephalic extremity is connected with the 
suprarenal sinusoids as on the right. It will ultimately form the 
left suprarenal vein, a tributary to the left renal. 

The vena hemiazygos still opens into the left common cardinal. 
It is connected with the azygos by the anastomosis at the level 
of the 10th thoracic spinal nerve, mentioned above. 

Portal system. The left umbilical ven (V.wm.s.) is repre- 
sented in plate 4. In the liver it communicates with the hepatic 
sinusoids, its blood passing chiefly through an especially large 
sinusoid, the ductus venosus (D.v.) which joins the left side of the 
common hepatic vein (vena cava inferioris). 

The trunk (V.vit.) formed by the fused vitelline veins (Begg 12) 
passes through the coelom in a separate strand of mesentery 
(plate 2). In the dorsal mesentery of the duodenum it is joined 
by the superior mesenteric vein (V.mes.s.) of which only thestump 
is here shown. The trunk formed by this union, the vena 
omphalo-mesenterica (V.omp.-mes.), now bends cephalad, and 
passes dorsal to the anastomosis of the dorsal and ventral pan- ° 
creatic outgrowths. After receiving the henal vein (V.lv.) it 
becomes the vena portae (V.P.) which enters the liver where it 
joms the hepatic sinusoids, and, by means of a considerable 
channel, the right side of the ductus venosus. 

The hepatic sinusoids discharge into the vena cava inferior 
(common hepatic segment) by way of the hepatic veins (Vv.hep.). 


Lymphatics 


The jugular lymph saes (S.jug.), comparable to those dis- 
covered by Sabin (’02) are seen in plate 6. The left also appears 
in plate 5. They are large, nearly symmetrical sacs somewhat 
constricted into segments, and are situated one on either side, 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 91 


immediately lateral to the internal jugular vein. Through the 
sacs pass branches of the cervical plexus. 

Saccus jugularis sinister. The cephalic end of the left sac 
communicates with the internal jugular vein through a small 
channel (a). There is also a large caudal connection between 
the sac and vein which cannot be seen in the reconstruction. 
The latter opening is in the caudo-medial wall of the sac near 
the temporary opening of the external jugular vein (V.jug.ev.) 
into the internal jugular. Incomplete valves, dorsally and ven- 
trally placed, guard the opening. 

The branches of the cervical plexus which pass through this sac 
are seen in plate 5. Through the cephalic extremity extends the 
great auricular (N.aur.m.). A little more caudally there issues a 
branch of the third cervical which is joined lateral to the sac by 
a branch of the fourth cervical nerve. The trunk thus formed, 
immediately gives off the small occipital, and then bends ventrally 
across the sac as the N. cutaneous colli. 

The vasa lymphatica superficialia (Vas.lym.sup.), arising from 
the cephalo-lateral portion of the left lymph sac, are seen plainly 
in plate 6. They extend laterally and dorsally into the subcu- 
taneous tissue. A prolongation from the caudal extremity of 
the left sac overlies the lateral aspect of the terminal part of 
the subclavian vein. 

Saccus jugularis dexter. The cephalic end of the right lymph 
sac does not communicate with the internal jugular vein as 
does the left. Caudally at a level corresponding approximately 
with the large communication on the left, the right sac opens 
into the internal jugular. The opening in this case is a very 
small slit-like aperture between two valves, a lateral and a medial. 
The lateral valve is adjacent to the permanent termination of the 
external jugular vein, while the medial projects into the cavity 
of the internal jugular. 

A prolongation of the sac overlies the lateral surface of the 
proximal portion of the subclavian vein as occurred on the left. 

Saccus mesentericus. There is a plexus of vessels, situated 
immediately ventral to the aorta, which extends for the most part 
between the proximal parts of the superior and inferior mesen- 


9? F. W. THYNG 


teric arteries. The cephalic part of this plexus lies between the 
subcardinal segment of the inferior vena cava and the left supra- 
renal vein (left subcardinal), the caudal portion lies dorsal to the 
renal anastomosis. In places the diameter of the plexus is equal 
to, or even greater than, that of the adjacent aorta. Although 
the lumina of the vessels are closely packed with corpuscles, it is 
difficult to connect the vessels with any of the definitely formed 
veins. Nevertheless, it would be impossible at least without 
reconstructions to say that such connections do not exist. From 
the relations of the plexus, given above, there seems little doubt 
that it corresponds to the anlage of the mesenteric lymph sac, 
discovered in rabbit embryos by Lewis (02 and ’05). 

Baetjer (08) who has investigated the development of this sac 
in a series of pig embryos, concludes that it originates in a series 
of small veins which separate from the renal anastomosis. 

Cisterna chylt. A series of anastomosing venous channels is 
found on the right and left side, dorso-lateral to the aorta. The 
cephalic end of this plexus apparently connects with the azygos 
and hemiazygos veins respectively. Many of the channels open 
into the supra-ureteral venous channels, previously described. 
The intersegmental arteries in this locality extend dorsal to these 
channels, although offshoots of the latter frequently anastomose 
between the successive pairs of arteries, dorsal to the aorta.. 
These vessels may represent the anlage of the cisterna chyli, for 
they correspond in position to the anlage as described by Sabin 
(09). 

Sacct lymphatict posteriores. Definite posterior lymph sacs 
in relation to the sciatic veins have not been found, but numerous 
small tributaries, entering the proximal part of the veins may 
foreshadow them. Sabin (’09) states that they first appear in an 
embryo of 20 mm., as a plexus of small veins. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 93 


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Forssner, H. 1907 Die angeborenen Darm- und Osophagusatresien. Anat. 
Hefte, Heft 102, S. 1-163, Taf. 1-9. 

Fox, H. 1908 The pharyngeal pouches and their derivatives in the Mammalia. 
Am. Jour. Anat., vol. 8, pp. 187-250. 

Froriep, A. 1885 Ueber Anlagen von Sinnesorganen am Facialis, Glosso- 
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Abth., S. 1-55, Taf. 1-2. 


94 


GREIL, / 


GROSSER, 


F. W. THYNG 


1905 Uber die Anlage der Lungen, sowie der ultimobranchialen 
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1907 Die Elemente des Kopvenensystems der Wirbeltiere Verh. d. 
Anat. Ges. 21, S. 179-192. 


Hammar, J. A. 1901 Notiz iiber die Entwickelung der Zunge und der Mund- 


HENLE, J. 


His, 


We 


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1902 Studien tiber die Entwickelung des Vorderdarms und einiger 
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1903 Abt. II. Das Schicksal der zweiten Schlundspalte. Zur 
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1893 Beitrige zur Entwickelungsgeschichte des Venensystems der 
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1898 Ueber die Pars membranacea sépti. Vortrag gehalten in der 
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Incauus, N. W. 1907 Beschreibung eines menschlichen Embryos von 4.9 mm. 


Arch. fiir mikr. Anat., Bd. 70, S. 506-576, Taf. 29-31. 


Jounson, F. P. 1910 The development of the mucous membrane of the cesopha- 


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Jounston, J. B. 1909 The morphology of the forebrain vesicle in vertebrates. 


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15, S. 41-42. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 95 


KastscHENKo, N. 1887 Das Schicksal der embryonalen Schlundspalten bei 
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Lewis, F. T., ann Tuync, F. W. 1908 The regular occurrence of intestinal 
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Matt, F. P. 1891 A human embryo of twenty-six days old. Jour. Morph., 
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76, S. 658-681, Taf. 25-26. 


96 F. W. THYNG 


Perer, K. 1901 Die Entwickelung des Geruchsorgans, u.s.w. Hertwig’s 
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Tryne, F. W. 1908 Models of the pancreas in embryos of the pig, rabbit, cat 
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17-45. 

ZIMMERMAN, W. 1889 Rekonstruktion eines menschlichen Embryos von 7 mm. 
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naealraum des Menschen. Verh. d. Anat. Ges. 15, S. 95-107. 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 97 


ABBREVIATIONS 


All., allantois 

An.hyp.(Ans.hyp.), ansa hypoglossi 

An., anus 

Ao.dors.lat., aorta dorsalis lateralis 

Ao.d.m., Ao. dorsalis mediana 

Arc.ao., arcus aortae 

A.anon., arteria anonyma 

A.aur.p., A. auricularis posterior 

A.bas., A. basilaris 

A.car.c.d., A. carotis communis dextra 

A.car.c.s., A. carotis communis sin- 
istra 

A.car.ex., A. carotis externa 

A.car.i.d., A. carotis interna dextra 

A.car.i.s., A. carotis interna sinistra 

A.cer.a., A. cerebri anterior 

A.cer.m., A. cerebri media 

A.cer.p., A. cerebri posterior 

A.cereb.s., A. cerebelli superior 

A.chr.a., A. chorioidea anterior 

A.chr.p., A. chorioidea posterior 

A.coel., A. coeliaca 

Aa.com.p., Aa. communicantes poste- 
riores 

A.hyp.d., A. hypoglossa dextra 

Aa.il.com.d. and s., Aa. iliacae com- 
munes, dextra et sinistra 

A.il.ex., A. iliaca externa 


A.isch.d., A. ischiadica dextra (A. 
glutea inferior et a. comitans n. 
ischiadici) 


A.ling., A. lingualis 

A.mami., A. mammaria interna 
A.mazx.ex., A. maxillaris externa 
A.mes.s., A. mesenterica superior 
A.mes.i., A. mesenterica inferior 
A.occ., A. occipitalis 

Aa., Aa. oesophageae 

A.oph., A. ophthalmica 
A.pud.i., A. pudenda interna 
Aa.pul., Aa. pulmonales 

A.s.m., A. sacralis media 

A.scl., A. subclavia 

A.sp.i., A. spermatica interna 
A.stp., A. stapedia 

A.s-r., A. suprarenalis 


A.thyr.d., A. thyreoidea dextra 

A.thyr.s., A. thyreoidea sinistra 

A.um.d., A. umbilicalis dextra 

A.vert.d., A. vertebralis dextra 

A.vert.s., A. vertebralis sinistra 

At.d., atrium dextrum 

At.s., At. sinistrum 

B., vesicula urinaria 

Br.s., bronchus sinister 

B.phary., bursa pharyngea 

Ch.pr., choana primitiva 

Con.art., conus arteriosus 

Corp.pin., corpus pineale 

Corp.str., corpus striatum 

Dien., diencephalon 

D.a., ductus arteriosus 

D.c., D. cochlearis 

D.cyst., D. eysticus 

D.end., D. endolymphaticus 

D.hep., D. hepaticus 

D.p.d., D. pancreatis dorsalis 

D.v., D. venosus 

D.vit., D. vitellinus 

D.M., D. Mulleri 

D.W., D. Wolffii 

Duo., duodenum 

Em.i-p., eminentia interpeduncularis 

Ep., epiglottis 

Eth.-turb.I., ethmo-turbinale I 

Fim., fimbriae 

Flex.ceph., flexura cephalica 

Flex.cerv., flexura cervicalis 

Fl.pl., floor-plate 

Fo.ep., foramen epiploicum 

Fo.int., foramen interventriculare 
(Monroi) 

Fo.iv., foramen interventriculare 

G.gn., ganglion geniculatum 

Gg. hyp., Gg. hypoglossa 

G.jug., G. jugulare 

G.nodos., G. nodosum 

G.petros.(G.p.), G. petrosum 

G.s-l., G. semilunaris 

G.sup., G. superius 

G.ves., G. vestibularis 

Ga., gaster 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


98 F. W. THYNG 


G.R., genital ridge 

Gl.inf., glandula infundibularis 

Gl.p., Gl. parotidis 

Gl.sma.(Gl.s.), Gl. submaxillaris 

Gl. s-r.d., Gl. suprarenalis dextra 

Hyp., hypophysis 

Int.cr., intestinum crassum 

Int.r.(Int.rec.), intestinum rectum 

Int.t., intestinum tenue 

Isth., isthmus 

L., lens 

Md., mandible 

Maz.-turb., maxillo-turbinale 

Md.sp., medulla spinalis 

Mem. u.-g., membrana urogenitalis 

Mesen., mesencephalon 

Mesogas., mesogastrium 

Met., metencephalon 

Myelen., myelencephalon 

Na., naris 

N.a., aberrant nerve of myelencepha- 
lon 

N.abd.(N.ab.), nervus abducens 

N.acc., N. accessorius 

N.alv.inf., N. alveolaris inferior 

N.acus., N. acusticus 

N.aur.m., N. auricularis magnus 

N.c.i., N. cardiacus inferior 

N.ch.tymp.(Ch.ty.), N. chorda tympani 

N.c.c., N. cutaneous colli 

N.fac., N. facialis 

N.fr., N. frontalis 

N.glos., N. glossopharyngeus 

N.hyp., N. hypoglossus 

N.int., N. intermedius 

N.laryng.s.(N.l.s.), N. 
perior 

N.1.(l.), N. lingualis 

N.md., N. mandibularis 

N.mza., N. maxillaris 

N.na.-cil., N. nasociliaris 

N.occ.m., N. occipitalis major 

N.oc., N. oculomotorius 

Nn.olf., Nn. olfactorii 

N.oph., N. ophthalmicus 

N.pet.s.m., N. petrosus  superficialis 
major 

N.phr., N. phrenicus 


laryngeus su- 


N.rec.s., N. recurrens sinister 

Nn.sp., Nn. spinales 

Nn.s.-cl.(Nn.s-c.), Nn. supraclavicu- 
lares (common trunk) 

N.troch., N. trochlearis 

N.tym., N. tympanicus 

N.vag., N. vagus 

Oe., oesophagus 

Op.s., optic stalk 

Or.vom.-nas., organon vomero-nasale 

O.s., ostium secundum 

Panc.d.and v., pancreas dorsale et ven- 
trale 

Pa.gen., papilla genitalis 

P.-thyr.IV., parathyreoidea IV 

Ph.P.1, pharyngeal pouch 1 

Ph.P.2, pharyngeal pouch 2 
only) 

Ph., pharynx 

Pl.ch.p., plica chorioidea posterior 

Pr.i-m., processus intermaxillaris 

Pr.pl., processus palatinus 

Pr.ver., processus vermiformis 

R., raphé 

R.des., ramus descendens (hypoglossi) 

R.ex., ramus externus (accessorii) 

R.p-tr., ramus posttrematicus (of 
fourth pharyngeal pouch) 

R.po-op., recessus postopticus 

R.p-op., recessus preopticus 

Rhin., rhinencephalon 

R.or., rima oris 

Rf.pl., roof-plate 

Sac., sacculus 


(region 


Sac.jug.(S.jug.), saccus lymphaticus 
jugularis 

S., septum aorticum (aortico-pulmo- 
nale) 


S.u.-r., septum uro-rectale 

S.cav., sinus cavernosus 

S.sag.sup., S. sagittalis superior 

S.u.-g., S. uro-genitalis 

S.v., S. venosus 

S (plate 1) vestigial portions of dorso- 
lateral aortae 

Sul., sulcus (mesencephali?) 

T.1., ganglion thoracicale primum 

Telen., telencephalon 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 99 


Thy., thymus 

Thyr., glandula thyreoidea 

Thyr.m., gl. thyreoidea media 

t’ and t”, anterior and posterior parts of 
the tongue 

Tr., trachea 

Tr.aor., truncus aorticus 

Tr.cost.cerv., Tr. costocervicalis 

Tr.pul., Tr. pulmonalis 

Tr.thyr.-cerv., Tr. thyreocervicalis 

Tub.p., tauberculum posterius 

T.W., tubuli Wolffi 

Umb.c., umbilical cord 

Ur., ureter 

Ut., utriculus 

Vas.lymp.sup., vasa lymphatica super- 
ficialia 

Vv.v.s., valvulae sinus venosi 

V.b., valvula bicuspidalis 

V.t., V. tricuspidalis 

V.az., vena azygos 

V.cap.lat., V. capitis lateralis 

V.card.a., V. cardinalis anterior 

V.card.c.d., V. cardinalis communis 
dextra 

V.card.c.s.(V.card.com.s), V. cardinalis 
communis sinistra 

V.card.p., V. cardinalis posterior 

V.ceph., V. cephalica 

V.cer.a., V. cerebralis anterior 

V.cer.m., V. cerebralis media 

V.cer.p., V. cerebralis posterior 

V.fem., V. femoralis 

Vv.hep., Vv. hepaticae 

V.hep.com., V. hep. communis 


V.isch., V. ischiadica 

V.jug.-ceph., V. jugulo-cephalica 

V.jug.ex., V. jugularis externa 

V.jug.t., V. jugularis interna 

V.li., V. lienalis 

V.ling.-fac., V. linguo-facialis 

V.mes.s., V. mesenterica superior 

V.oph., V. ophthalmica 

V.omp.-mes., V. omphalo-mesenterica 

V.P., V. portae 

Vov.pul.d., Vv. pulmonales dextrae 

V.s.m., V. sacralis media 

V.scard.d., V. subcardinalis dextra 

V.scl., V. subclavia 

V.s-r.d., V. suprarenalis dextra 

V.th.-ep., V. thoracico-epigastrica 

V.thy.-thyr., V. thymico-thyreoidea 

V.ul.pr., V. ulnaris primitiva 

V.um.s., V. umbilicalis sinistra 

V.ver.d., V. vertebralis dextra 

V.v., Vv. vitellinae (fused) 

Vent.d., ventriculus dexter 

Ves.fel., vesica fellea 

Ves.op., vesicula optica 

x, renal anastomosis (V. renalis sin- 
istra) 

x’, transverse iliac anastomosis (V. 
iliaca communis sinistra) 

X, strand of mesentery containing the 
fused vitelline veins 

y, branch of right jugular lymph sae 

Z.dors., zona dorsalis 

Z.vent., zona ventralis 

1 to 8, ganglia cervicales 


Aca oy 
A.th yf. a Thyr-o 
med. 


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PLATE 1 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO 
F. W. THYNG 


Reconstruction to illustrate chiefly the interior of the brain and the spinal cord; the digestive 
system and its appendages; the arterial system; the left atrium and ventricle of the heart, and in 
part the urogenital system of a 17.8 mm. human embryo (H. E. C. 839). X 11.2 diams. 


101 


PLATE 2 
EXPLANATION OF FIGURE 


This plate consists of two reconstructions. The upper shows a left lateral 
view of the brain and cervical cord with the nerves in situ, the aortic arch, and 
other arteries of the left side of the head and neck. It also represents the oral, 
nasal and pharyngeal epithelia; the left thymic and thyreoid anlagen; and illus- 
trates in a measure the relation of the nerves and arteries to these epithelial 
structures. The lower reconstruction shows the pancreas and spleen within the 
mesogastrium (a portion of the stomach having been removed); the left genital 
ridge, and the left meso- and metanephros with their ducts opening independently 
into the urogenital division of the cloaca (H. E. C. 839). » 11.2 diams. 


103 


Nint.& Gan. Nacus. 


: fae s 


G orp. 
Bey Dien 


ea “ep: 
Achra. (e* /\ = Tadd CS 

x eLea Acer. a, a 4 ey 

NSS cAS ; 


Fo.ep. Dicyst 


Ves. fel. D.pd. Dhep Oe 


All. Pa.gen. 
104 


PLATE 2 


ANATOMY OE A 17.8 MM. HUMAN EMBRYO 
F. W. THYNG 


105 


Dien. 
\ SS ZN.tyTe ‘ , 
<\ (fac , 
= VI i & A Telen 
hk 7 Nmx 
“Sey . 
S 
C1. N.ch.tymp. ee oo 
Nn.s-cl. Anhyp. N.pet.s.m. Nn.olf. Nna.-cil. 


106 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO PLATE 3 
F. W. THYNG 


Reconstruction showing the right side of the brain and the cervical cord; the right cranial and cervi- 
ical nerves; the internal ear, and the optic vesicle of a 17.8 mm. human embryo (H. E. C. 839). x 11.9 
\diams. 


107 


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LL B 
Pri-m7 7 pe 


to47Z 
SG KG, Z 
S b 
Zi LEE S 
Za = ah 


Pa.gen. 


108 


PLATE 4 


ANATOMY OF A 17.8 MM. HUMAN EMBRYJ 
F. W. THYNG 


Reconstruction to show the right atrium and ventricle of the heart; the venous system of 
the right side; and the left umbilical vein of a 17.8 human embryo (H. E. C. 839). X 11.2 
diams. 

109 


nm Gyves. G.sup. Nvag. Vicerp. Nshyp. 


Noe. 
—ralnaNY 
, 2 ny 
EX ; 
Cc 


rt 
v. 


| CATA.C.S. 


110 


ANATOMY OF A 17.8 MM. HUMAN EMBRYO PLATE 5 
F. W. THYNG 


& 


Reconstruction to show the lateral aspect of the left cephalic and cervical veins, and the left 
jugular lymph sac of a 17.8 mm. human embryo (H. E. C. 839). X 11.9 diams. 


111 


MM. HUMAN EMBRYO 


W. THYNG 


ANATOMY OF A 17. 


PLATE 6 


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112 


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THE STATURE AND THE ERUPTION OF THE PERMA- 
NENT TEETH OF AMERICAN, GERMAN-AMERICAN 
AND FILIPINO CHILDREN. DEDUCTIONS FROM 
THE MEASUREMENTS AND EXAMINATION OF 1445 
PUBLIC SCHOOL CHILDREN IN ANN ARBOR, MICH- 
IGAN, AND 776 IN MANILA, P. I. 


ROBERT BENNETT BEAN 


From the Anatomical Laboratory, the Tulane University of Louisiana 


FIVE DIAGRAMS 


CONTENTS 
Introduction......... Pe ys «en Ee ey MM ONS 5p Sc coc oh cee UR ee eee 114 
IVilstte ict SMeeeresetn ean: Pasar tnt ere <8 oc SMa MD cate TE Etre s anchetelers 114 
WCAC ISS AS A RTC ae ae a eee sted DE ey 2s By oe ae cee eee GO eee ae oie 115 
‘TROVE ASLEPHRDUREX sesers Ore Manet YB oct NeMeR ea ae apeS eae Loto Oe epee Datei ak Micon Gracies erode: 117 
Ieetherstarnunrerom bhevroups Compared ssereanaae. «oi. ates ser cece. 117 
2 ikeviewormune literature-and GdisCuUssiONer .. /u2 > scenes ge shen pe ee 119 
3. Periods of acceleration and retardation in growth................. 128 
GENEVE VAGLN ECV are Saher eRe Oe a emits Ca ae oe a gee Te oe a ee 125 
it, Eheteruptiomio£ the permanenit beethy con cs foc 25 vaeig apne se ee si 125 
2. The alternation of growth in stature and the eruption of the teeth.. 126 
3. The eruption of the individual permanent teeth.................. 128 
4) Review ob she literature and Giscussiomne.:. ee oicn el. sac eciae ee 133 
whe law.of alternation.in development) .o.7..2¢.c daxjssed faves ete oe bs oe oie 137 
dibesmorpholoric tyme ana tne, teen. 62 ...45 Ghee ates ame oo Svis sees ote cess ober 146 


1. The morphologic type and the eruption of the permanent teeth... 147 
2. The morphologic type and the number of good sets of permanent 


LHSTE Hay 5 OUNETSTOUAN Bearer Se AEN eSy Geter aloe aetna SIO cae’) oC ORNS EO otra aE 147 

3. The morphologic type and the average number of decayed perma- 
METI ALC CUO NESCTHIUMP ET CrI ae hes ict SRR oe ORT reins Ole pee cies 148 

4. The relations of the morphologic type in thee several groups, races 
CHISTES CES As, -' 8 ANG Ie ape 9 Sane OC A ae 148 

The physiological standard of the teeth, by which may be determined 
the mental and physical development of the individual.............. 152 
sheyechoolsonaderandethenteeunme ame see b ieriee cone Sila enter Mee ciate = 153 
PES URLG ee on eee ern eRe OF aY Peee PME Mee 8. Be like Boe TY cabo aed ge eke 155 
1 By] ON IayiaPO ON os hve Soden odd ok OCR CS OAH Oo Dea OnE er ee eer arc 158 

113 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


114 ROBERT BENNETT BEAN 


Data: 2221 school children 
630 Filipino boys 
776 146 Filipino girls 


322 German boys 
628 306 German girls 


407 American boys 
817 410 American girls 


2221 “otal 
INTRODUCTION 


The records on which this study is based were made in 1906, 
1907 and 1908 and have lain fallow for more than five years, be- 
cause of excessive teaching duties and lack of library facilities. 
However, after an extensive survey of the literature I have come 
to the conclusion that no other study of its kind has ever been 
made, except that of Hrdli¢ka on the North American Indians 
(81), and it was through the initiative of Dr. Hrdlicka that this 
work was undertaken. Observations on the teeth of many people 
have been made from time to time but no extensive detailed ob- 
servations have ever before been published, not even of Euro- 
peans, and no absolute standards have been established as to the 
time of eruption or extent of decay in males and females and 
from the standpoint of race. This study is an attempt in that 
direction, but leaves much to be desired, especially as the num- 
ber of Filipinos examined at each age in the early years is but 
small. 

MATERIALS 


During the school term of 1906-07 I examined and measured 
every available child in the public schools of Ann Arbor. This 
was made possible through the codperation of Dr. MceMurrich, 
and the superintendent of schools and the teachers who showed 
commendable zeal in furthering the work. The teeth of the 
children were examined by Dr. Bunting who also assisted me in 
making the records. Two groups of children were segregated, 
one of American parentage solely, the other of German, or Ameri- 


e 


ERUPTION OF THE PERMANENT TEETH 105 


can and German parentage. The titles American and German 
will be used for the twq groups. When taken together the 
German and American children will be called European. Chil- 
dren were grouped as German if two grandparents or if either 
parent came from Germany, and whenever the name was Ger- 
man and the parentage was not given the child was included in 
the German group, although there were only a few records of 
this kind. There is a large colony of Germans both in the town 
of Ann Arbor and in the adjacent country and the majority of 
the Germans included in the records are of pure German ex- 
traction. A large number of children of the teaching staff at 
the University of Michigan are included in the American group 
and a large number of rural children are included in the German 
group. The whole lot of children may be called sub-urban. 

The Manila school children were examined by myself in 1907— 
08 at the Normal and Trade schools, where pupils from all parts 
of the Philippine Archipelago come to be educated as teachers 
and mechanics, carpenters, etc. The young children attend 
classes taught by the older ones at the Normal School and are 
necessarily from Manila and vicinity. The majority of the chil- 
dren are mestizos, mixed Spanish, Chinese and Filipino, and 
represent the littoral population of the archipelago fairly well. 
A few individuals are probably of pure Spanish extraction. No 
attempt was made to segregate the mestizos or those of Chinese 
or Spanish extraction from the presumably pure Filipinos be- 
cause any such attempt is necessarily imperfect and wili be so 
until we understand better than we do at present the workings 
of Mendelian heredity in man. 


METHODS 


The age of the children was obtained from each individual and 
verified as far as possible through the teachers. In Manila the 
school registers were also examined and the age verified in this 
way. Discrepancies occur in spite of all efforts to avoid them, 
but I believe nearly all the ages are exact, and the few that may 
not be are approximately correct. The year and the month of 


116 ROBERT BENNETT BEAN 


birth were obtained in all except a few instances and the child 
reckoned to be a certain age if it is anywhere within six months 
of that age. For instance, if a child was born in January 1902 
and the records were made in July 1908, the child was recorded 
as six years old, but if the records were made in July 1907 the 
child was recorded as five years old, and if the records were made 
in August, 1908, the child was recorded as seven years old. This 
method is practically the same as obtaining from the child the 
nearest birthday, although in giving that the child is apt to 
miscalculate. 

The stature was obtained by using a stationary vertical gradu- 
ated scale with sliding arm to come down on the child’s head. 
This was pressed firmly and several readings were made for each 
record, the child standing erect with its back against the scale. 
The children were measured without shoes when feasible, other- 
wise allowance was made for the height of the shoe heel. The 
average or ‘mean’ stature is used although the ‘median’ and the 
‘mode’ will be presented. 

Many other measurements were made, including sitting stature, 
weight, head and face dimensions, with observations of the hair 
and eyes, but these are reserved for future publication. The 
teeth were examined for eruption, decay, absence, or irregularity. 
For detailed observations see the table of records, which shows 
the condition of eruption and decay of each tooth in each indi- 
vidual, and will be published either separately or on file in The 
Wistar Institute of Anatomy in Philadelphia. 

The standard deviation, coefficient of correlation, probable 
error, etc., are not given, because the characteristics of the indi- 
vidual rather than the mass are desired. However, the average, 
the mean, the mode, and the extremes have been computed, and 
these with other factors are utilized in an attempt to determine 
individual characteristics. 

At the end the morphologic type, the sex, and the race are used 
to assist in explaining individual differences, such as why one 
child has a certain tooth erupted 5 years earlier than another 
child has the same kind of tooth erupted, which occurs with great 
frequency. 


ERUPTION OF THE PERMANENT TEETH 117 
THE STATURE 
1. Stature of the groups compared 


It is to be noted that all the boys and girls of each group have 
about the same stature at the age of seven years, and the stature 
varies more both before and after this age. At five years the 
Igorot boys are 97 cin. tall, the Filipino boys 105.8 cm. tall, 
the German boys are 107.5 cm. tall, the German girls 110.8 em. 
tall, and the American girls and boys each 112.5 em. tall. The 
difference between the smallest and tallest group at five years is 
15.5 cm., whereas the difference at seven years is only 4 cm. 
There are so few individuals at the age of five years in each 
group, the greatest number in any group is five individuals, that 
the differences given are of slight importance. 

After the age of seven years the differences become significant. 
The Igorots are still much below the average of any other group, 
and the Filipino boys are also low in stature, but between 12 and 
15 years very nearly equal to the German boys. There is a 
constant decrease in the increment of growth in the Filipino 
girls, which decrease from the age of seven years onward is greater 
than that of any other group. 

The growth of the Filipino girls almost ceases between 14 and 
15 years, and there is apparently no growth after the age of 18 
years. ‘The stature of the Filipino boys is less than that of the 
Filipino girls until the age of 12 years is reached at which time 
the boys pass the girls in a rapid growth that decreases suddenly 
at the age of 17, although there is a slight increment of growth 
in the Filipino boys even after the age of 20 is passed. 

The stature of the German girls is less than that of the German 
boys before the age of 11, greater between the ages of 12 and 14, 
after which it is again less, but very slightly so. There is a sud- 
den check in the growth of the German girls at 14 but after 15 
the growth again accelerates. The growth of the German boys 
is fairly uniform throughout and there is no evidence of cessa- 
tion at the age of 16. The growth of the German boys is, how- 
ever, slightly more rapid after the age of 12 than before. There 
is also a slight acceleration of growth in the German boys up 


118 ROBERT BENNETT BEAN 


to the age of 8 and a slight retardation between the ages of 8 
and 12. 

The stature of the American boys and girls crosses and recrosses 
up to the age of 10 years, that of the girls in general is less be- 
fore this age. Between the ages of 10 and 11 the stature of the 
girls becomes greater than that of the boys until the age of 14 is 
reached when the growth of the girls ceases, to continue slowly 
after the age of 15, at which time the stature of the girls becomes 
less than that of the boys, and it remains so thereafter. The 
growth of the American girls from 15 to 17 years of age is re- 
tarded at a slightly slower rate than that of the Filipino boys, 
and there is a fairly uniform parallel growth of the two except for 
the sudden cessation in growth of the American girls from 14 to 
15 years, the American girls at all ages being taller than the 
Filipino boys. The growth of the American and German boys 
is fairly parallel, the American boys slightly taller at each age 
except 8 when the German boys are taller. The American boys’ 
growth is retarded from 5 to 7, accelerated from 7 to 10, again 
retarded from 10 to 13. 

The stature of the American and German girls is fairly parallel 
until the age of 15, the German less than the American, but after 
the age of 15 the stature of the German girls exceeds that of the 
American girls. 

In general the stature of the Filipino is less than that of the 
German and the stature of the German less than that of the 
American. The growth of the Filipino girls and boys and of the 
American girls has its final retardation earlier than that of the 
German girls and boys and American boys, the last three giving 
no evidence of this when the records cease at 18 years of age. 

The stature of the Filipinos beyond the age of 20 is greater 
by 5.2 em. than that of the adult Filipinos of Taytay (3), a village 
near Manila. The boys belong to the well-to-do class. The Tay- 
tayans are poor which may account for this difference. The Tay- 
tayans are also less mixed with the Spanish and Chinese than the 
school boys which is another factor in accounting for the differ- 
ence in stature. 


ERUPTION OF THE PERMANENT TEETH 119 


The stature of the Filipino boys is 11 cm. greater than that 
of the girls beyond the age of 20, and this is in excess of the 
usual sexual difference, which is often not more than 5 cm. 
This may be due to a greater inheritance of the father’s stature 
by the boys and of the mothers’ stature by the girls because the 
fathers of many of the children are Spanish and Chinese and the 
mothers are Filipinos, and the stature of the Spanish and Chinese 
is greater than that of the Filipinos. This may be a confirmation 
of Pearson’s findings that males inherit stature from the father 
and females from the mother. 

There are so few Filipinos below the age of 16 for each sex | 
that the average has only an approximate value. There is also 
greater variability in stature at the ages of 12, 13 and 14 among 
the Filipinos, than either before or after. For instance at the 
age of 10, the extremes are only 15 cm. apart, and at the age of 
15 only 25 cm. apart, whereas at 12, 13 and 14 years, they are 
30, 35 and 35 cm. apart respectively. It is noted that the Ger- 
man and American children exhibit a variability that is greater 
with advancing years. The reverse is true of the Filipinos. 


2. Review of literature and discussion 


When the stature of the Ann Arbor boys of American parent- 
age is compared with the stature of the Boston boys (13,691, 
Bowditch (11)) of American parentage it is found that the Ann 
Arbor boys are about 5 cm. taller at the ages of 5 and 6, and this 
difference decreases until the age of 15 is reached after which the 
boys of both places have about the same stature. This is true 
also of the Nebraska boys (5,476, Hastings (26)) when compared 
with the Ann Arbor boys, and the difference with them at the 
early ages is even more marked in favor of the Ann Arbor boys. 
It is evident that the boys of Ann Arbor attain their early stature 
quicker than the boys of Boston. This is probably due to the 
fact that the children of Ann Arbor have a more favorable envi- 
ronment than the children of Boston. Bowditch found that the 
boys of the private schools in Boston were taller than the boys 
of the public schools, and taller than the boys of the laboring 


120 ROBERT BENNETT BEAN 


classes in England. It is generally recognized that the children 
of the well-to-do grow more rapidly than the children of the 
poor, that is, early in life. There may be some influence due to 
the selected parentage and not alone due to environment. A 
great number of the boys examined at Ann Arbor were chil- 
dren of members of the Faculty of the University of Michigan, 
or children of students, and this might have some influence. 

The German boys of Ann Arbor (324, Bean) are also taller than 
the German boys of Boston (752, Bowditch), although this dif- 
ference is only about 2 or 3 cm. until the age of 15 is reached 
when both groups become equal in stature. This difference may 
be due to environment alone because the Germans of Boston and 
Ann Arbor probably come from a similar stock. 

The same difference that exists between the American boys of 
Ann Arbor and of Boston exists to a limited extent between the 
girls of the two places. The girls of Ann Arbor (409, Bean) are 
about 5 em. taller at the age of 5 and 6 years than the Boston 
girls (10,874, Bowditch) of the same age and this difference de- 
creases until the age of 15 is reached, at which time the stature 
is only about 2 cm. apart. The Ann Arbor girls pass the Ann 
Arbor boys in stature a half year earlier than this takes place 
in Boston (Ann Arbor, 10, Boston 103) and the boys do not again 
reach the stature of the girls in Ann Arbor until a half year later 
than in Boston (Ann Arbor 15 years, Boston 143 years). 

Peckhan (44) reports the results of measuring 5,136 girls, and 
5,117 boys of Milwaukee, in which the boys, both German boys 
and American, were taller than the Boston boys from 13 years 
onward (Bowditch 13,691 boys, 10,874 girls), with a rapid jn- 
crease of stature from 16 to 19 years. The girls at all ages both 
German and American were taller than the Boston girls. The 
Milwaukee children differ from the Ann Arbor children in that 
the former have a late rapid growth whereas the latter have an 
early rapid growth. Is the late rapid growth of the Milwaukee 
children due to the Norse (long head) stock and is the early 
rapid growth of the Ann Arbor children due to the large 
south German (broad head) element? Or is it a matter of 
environment? 


ERUPTION OF THE PERMANENT TEETH 124 


Schwerz (47) reports the results of measuring 960 boys and 818 
girls of Schaffhausen, Switzerland, in which the Swiss boys are 
4 cm. less in stature at all ages than the Ann Arbor American 
boys, and the increase in rate of growth is at about the same 
periods, 9, 14 and 17 years. The Ann Arbor German boys have 
1 or 2 cm. greater stature than the Swiss boys until the age 
of 14 years after which the German boys attain a stature which is 
4 cm. greater at 15 years and 7 cm. greater at 16 years, this, too, 
in spite of the fact that the Swiss boys are six months older at 
each period than the Ann Arbor German boys. The differences 
that exist between the Swiss boys and the Ann Arbor German 
boys also exist to about the same extent between Swiss girls and 
Ann Arbor girls. The periods of rapid growth are about the same 
in the two groups. 

The cephalic index, face index, etc. of the two groups are about 
the same thus signifying that the two groups, Swiss and Ann 
Arbor Germans, are of the same stock, the Alpine or middle 
Kuropean, rather than of the Nordic or Mediterranean stocks. 
Schwerz compares the Berlin German children with the Turin 
Italian children in both the poorer classes and the well-to-do 
classes, with the result that the Berlin children are taller at each 
age in both classes and both sexes. The well-to-do boys of 
Berlin have about the same stature at all ages as the American 
boys at Ann Arbor and the boys of the poor in Berlin are only 
slightly less tall at each age (1 to 2 em.) than the Ann Arbor 
German boys. The well-to-do girls of Berlin are slightly taller 
at each age than the American girls of Ann Arbor, and the poor 
girls of Berlin are only slightly less tall (1 to 2 em.) than the Ann 
Arbor German girls. The Turin boys and girls are in all groups 
and at all ages from 5 to 10 cm. less in stature than the Berlin 
children. Thus the three stocks of Europe, Nordic, Alpine and 
Mediterranean, are represented by the Berlin, Schaffhausen and 
Turin children respectively (?) and it would appear that the 
Milwaukee children are Nordic, the Ann Arbor American chil- 
dren are Nordic and Alpine, and the Ann Arbor German children 
are Alpine. The effect of nutrition or environment cannot be 


1 ay ROBERT BENNETT BEAN 


stated for the several groups but no doubt its influence accounts 
for some variation. 

When the Filipino boys measured by Bobbitt (10) are compared 
with those I measured, the difference in stature is in favor of 
the latter to the extent of about 2 cm., after the age of 11 years; 
at 10 and 11 the two groups are equal, and before that Bobbitt’s 
Filipino children are on the average 1 cm. taller down to and 
including the age of 6. 

I measured only 11 Filipino girls below the age of 12 years, 
therefore they need not be considered. From 12 years onward, 
however, the girls measured exceed those measured by Bobbitt 
from 1 cm. at 12 and 13 years, to 5 cm. at 20 years and over. 
Not only this but Bobbitt gives the age from one birthday to 
the next as of the preceding birthday, whereas I give the age as 
of the six months before and after the birthday. For instance, 
he gives all children from 10 to 11 years of age as 10 years of 
age, whereas I give all children 9 years 6 months to 10 years 5 
months as 10 years of age. His Filipino children are not only 
smaller but in each group are six months older than the same 
group given by me. 

The children measured by Bobbitt were of three sorts. The 
oldest were about 75 per cent from the provinces, the youngest 
were largely from Manila and the intermediates were about 
equally divided between the two places. The majority of the 
children I measured were from the provinces, except those below 
the age of 10 years, who were all from Manila. Bobbitt did not 
include children who had evidence of mestizo blood (Spanish 
and Chinese), whereas I included all the children as they became 
available, regardless of race or color. A few were crosses of the 
American white and Filipino: the differences in stature may be 
explained by the greater amount of foreign blood in the chil- 
dren I measured, although when the children from the provinces 
are in the majority the stature is taller than when the children 
from the city of Manila are in the majority. It appears that 
European stock and rural environment may both increase the 
stature of the Filipino. 


ERUPTION OF THE PERMANENT TEETH 123 


3. Periods of acceleration and retardation in growth 


Growth occurs in waves. A period of acceleration is followed 
by a period of retardation. The periods occur at different times 
in the different sexes and races. 

The first period of acceleration of which we have any record 
occurs between the ages of 6 and 10 years, after which there is a 
retardation followed by another acceleration about the age of 13 
to 15. These periods may be of interest in relation to the erup- 
tion and decay of the teeth. 

Disregarding the first and second periods of rapid growth, of 
which we have no record here, it may be seen that the third 
period of growth begins at about the age of 7 years and continues 
until about the age of 10, being most rapid from 7 to 8, and at 
the age of 10 years the third period of growth begins. 

The third and fourth periods of rapid growth are earlier for 
the girls than for the boys and the fourth period of rapid growth 
is shorter for the girls than for the boys. The result is that the 
girls become larger than the boys between the ages of 7 and 15, 
and remain so until the fourth acceleration sets in for the boys, 
when they outstrip the girls in stature and remain taller there- 
after. 

The stature of the Filipinos is less than that of the Germansand 
Americans and this becomes more evident after the age of 14 
years. 

The stature of the Igorot boys is less than that of the Filipino 
boys and girls except at the ages of 7 to 8 when it is greater than 
that of the Filipino boys, and at the age of 20 when it is greater 
than that of the Filipino girls. 

The Igorot boys attain their growth later than either the Fili- 
pino girls or the Filipino boys, but earlier than the German or 
American boys and girls. 

The four groups may be arranged in order of precocity of 
acceleration periods as follows: 1, Filipino; 2, Igorot; 3, German; 
4, American. The acceleration periods of the German begin 
earlier than those of the American but the ultimate stature is 


124 ROBERT BENNETT BEAN 


reached later in the German. This follows a law, general in 
nature, that the precocity or rapidity of development is inverse 
to the ultimate size. 

The precocity of the group is inverse to the ultimate stature; 
the Americans, the tallest, the Filipinos, the smallest, with the 
Igorots and Germans intermediate: the Germans taller than the 
Igorots. 

The Filipinos may have an early rapid development which is 
from the European standpoint premature, and a late maturity 
that is incomplete, at least it looks as if growth is continued 
up to a later age in the Filipinos than in the Europeans, but the 
extent of development is less. 7 

The Ann Arbor American children, the Milwaukee and the 
Berlin children are similar in stature and in periods of growth. 
The Ann Arbor German children, the Boston German children and 
the Swiss children are likewise similar in stature and in periods of 
growth. The Swiss children, however, show considerable retar- 
dation in growth from the age of 15 onwards, whereas the Ameri- 
ean German children show no retardation until the age of 17 is 
reached. All the six groups are different from the Turin chil- 
dren of South Europe. In the latter there is an early rapid growth 
followed by retardation, and a later rapid growth followed by 
retardation that is earlier than in the other groups. In this they 
resemble the Filipinos. 

The urban children of Boston are less rapid in their early de- 
velopment than the sub-urban children of Ann Arbor and the 
urban children of Milwaukee are more rapid in their late de- 
velopment that the sub-urban children of Ann Arbor. The 
early development of the Ann Arbor and Milwaukee children is 
about the same and the later development of the Ann Arbor and 
Boston children is about the same. 

Factors in race and climate or heredity and environment both 
probably cause. these differences. Neither can be excluded. 


Or 


ERUPTION OF THE PERMANENT TEETH 12 


THE TEETH 


The consideration of the teeth will be taken up under the 
following headings: 1. The eruption of the permanent teeth; 
2. The alternation of growth in stature and eruption of the per- 
manent teeth; 3. The eruption of the individual permanent 
teeth; 4. Review of the literature and discussion. 

The youngest children whose teeth I examined were five years 
old, and at that age all the temporary teeth have erupted, there- 
fore the time of the eruption of the temporary teeth cannot be 
given. The decay of the temporary teeth occurs partly after 
the age of five years, and this will be considered, in addition to 
the eruption and decay of the permanent teeth. 


1. The eruption of the permanent teeth 


This is determined by taking the average number of teeth 
erupted or erupting at each age, as well as the ‘median,’ the ‘mode’ 
and the ‘extremes,’ after which the periods of acceleration and 
retardation in the eruption of the permanent teeth will be given. 

The eruption of the permanent teeth begins about the age of 5 
years, slightly earlier among the girls and slightly later among 
the boys of the German and American groups, but considerably 
earlier than this among the Filipinos of both sexes. The Ger- 
mans and Americans of both sexes have almost exactly the same 
number of permanent teeth at the ages of 6, 7 and 8 years, after 
which the girls have a greater number of teeth than the boys until 
the age of 15 is reached, when the number of teeth in the two 
sexes is again about the same. 

The girls of each group are more precocious in the eruption of 
the permanent teeth than the boys, they have a greater number 
of permanent teeth at each age, and the difference is greatest 
between the ages of 8 and 15 years, for at the age of 11 years the 
American girls have an average of 21.3 permanent teeth present, 
and the American boys have an average of only 17.3. The differ- 
ence between the German boys and girls at this age is not so 
great, and they have only 16.7 and 19.5 teeth present for the boys 
and girls respectively. The difference between the Filipino boys 


126 ROBERT BENNETT BEAN 


and girls is still less, and they have a greater number of teeth 
present than either the German or Americans at the age of 11 
years, 24.3 and 27.0 for the boys and girls respectively. 

The Filipinos are much more precocious than either the Ger- 
mans or Americans in the eruption of the permanent teeth, and 
there is little difference between the boys and girls in the number 
of teeth present at each age. The Americans are a little more 
precocious than the Germans in the eruption of the permanent 
teeth, and the difference between the boys and girls is greater 
among the Americans than among the Germans and Filipinos. 

About the age of 15 years the children of both sexes in all 
the groups have nearly the same number of teeth, and after this 
age the German and American children maintain the same num- 
ber of teeth up to the time at which the records cease (18 years), 
but the Filipino children continue to gain in the number of their 
teeth to the age of 20 and beyond, although the majority of the 
Filipinos have acquired the complete set of 32 teeth at the age 
of 20 years. This is due to the early eruption of the third molars 
among the Filipinos (begins at 13) and their later eruption among 
the Germans and Americans (begins at or after 16). 

Sixty odd negro boys and girls, mostly mulattoes, were exam- 
ined at Ann Arbor, and it is found that the negro boys and the 
American boys are almost parallel in the time of the eruption of 
their teeth, and the negro girls are only slightly more precocious 
than the American girls. 


2. The alternation of growth in stature and the eruption of the 
permanent teeth 


The eruption of the permanent teeth does not occur with the 
same rapidity at each age, but there are waves of eruption of the 
teeth like the waves of growth, although the two are not syn- 
chronous. The periods of acceleration in the eruption of the 
permanent teeth occur about the ages of 7 and 11 years, and the 
periods of retardation follow those of acceleration. It will be 
seen that the periods of acceleration in the eruption of the per- 
manent teeth initiate or precede the periods of rapid increase in 


ERUPTION OF THE PERMANENT TEETH 127 


stature. The eruption of the teeth through the gums is but 
evidence of their previous rapid development, and there is 
doubtless a period of rest throughout the body between the 
periods of rapid development of the teeth and the periods of 
rapid growth in stature. 

Others have repeatedly demonstrated that the six months 
following birth is a period of rapid increase in length of the 
infant, which is followed by the eruption of the temporary teeth, 
all of which are through the gums by the age of three years. 
After this there is a period of rest which is followed by a second 
period of growth in stature about the age of five years, suc- 
ceeding which the permanent teeth begin to develop, and this 
development is most rapid about the age of 7 years. This is 
followed by the third period of rapid growth in stature about the 
age of 8 years after which comes a period of rest and then the 
second acceleration in the eruption of the permanent teeth occurs 
between 10 and 11 years. The final rapid growth in stature 
comes after this at about 12 years of age, and immediately pre- 
cedes puberty. Following puberty the increase in stature and 
the eruption of the teeth are delayed, especially in the girls. 
However, the third period of the acceleration in the eruption of 
the teeth (second period of acceleration in the eruption of the 
permanent teeth), the girls are about one year earlier than the 
boys, and the period of growth following this is about two years 
earlier in the girls than in the boys; (see The law of alternation 
in development, p. 137). 

Résumé. After this brief consideration of the average or 
‘mean,’ time of eruption of the permanent teeth, the ‘median,’ 
the ‘mode’ and the ‘extremes,’ as well as the periods of accelera- 
tion and retardation in the eruption, it is to be noted that the 
second teeth begin to erupt about 5 years of age, slightly earlier 
among the girls than among the boys and earlier among the 
Filipinos than among the Germans and the Americans, and 
latest of all among the Germans. 

The girls are more precocious in the periods of acceleration of 
the eruption of the teeth than the boys, as well as in the average 
number of teeth, the ‘median’ and ‘extremes’ at any age. The 


128 ROBERT BENNETT BEAN 


Filipinos are likewise more precocious than the Americans, who 
are slightly more precocious than the Germans. 

There is an alternation in the periods of acceleration and retar- 
dation in the eruption of the permanent teeth, and there is also an 
alternation in the periods of acceleration of the growth in stature 
and the periods of acceleration in the eruption of the permanent 
teeth. 


3. The tume of eruption of each individual tooth 


This is to be determined for each sex and race by means of the 
beginning of eruption, the end of eruption, and the average per 
cent of the teeth erupted at specified ages during which time the 
teeth are erupting in all the groups. The beginning of eruption 
may be utilized: that is the time at which the tooth first erupts 
in any individual in a race-sex group; the end of eruption, that 
is the time at which the tooth last erupts in any race-sex group; 
the ‘median,’ the time half way between the beginning of erup- 
tion and the end of eruption, may be taken as the time of erup- 
tion; or the time when 50 per cent of the teeth of any type have 
erupted in a sex-race group may be taken as the time of eruption. 
For purposes of comparing the groups with each other the 
average number of teeth erupted at the ages in which all the 
comparable groups have the teeth erupting at the same time may 
be utilized. This may be called the average per cent. As, for 
instance, the right upper second molar is erupting in all the groups 
during the ages of 12, 13 and 14 years, and the average per cent 
erupted at these ages is as follows: 

per cent per cent per cent 


Filipino boys...... 85 American boys......53 German boys......51 
Filipino girls..:... 94 American girls...... 68 German girls......58 


From this we may gather that the Filipinos are more preco- 
cious than the other two groups, the Americans are more pre- 
cocious than the Germans, and the girls are more precocious than 
the boys in the eruption of the right upper second molar teeth. 

The median incisors. The median incisors begin to erupt in 
some individuals before the age of five years, and the teeth are 


ERUPTION OF THE PERMANENT TEETH 129 


nearly if not all through the gums at the age of 10 years. There 
is practically no difference in the time of eruption on the two 
sides, but the lower median incisors erupt about a year earlier 
than the upper, a little less than a year on the left side and a 
little more than a year on the right side. 

The median incisors erupt in the following order: (1) lower 
right; (2) lower left; (3) upper left; (4) upper right. The left 
upper median incisors erupt at approximately the same time in 
the Germans and Americans, the left lower erupt earlier in the 
Americans than in the Germans, and the right upper erupt earlier 
in the Americans than in the Germans, although the German 
girls may be slightly more precocious than the American girls. 
The median incisors erupt earlier in the Filipinos than in either 
the Germans or Americans. The median incisors erupt earlier 
in the German and American girls than in the German and 
American boys. 

The lateral incisors. The lateral incisors begin to erupt in 
some individuals as early as the age of 6 years, and the teeth 
are nearly if not all through the gums at the age of 13 years. 
There is practically no difference in the time of eruption on the 
two sides, but the lower lateral incisors erupt about a year 
earlier than the upper, a little less than a year on the left side 
and a little more than a year on the right side. 

The lateral incisors erupt in the following order: (1) lower 
right; (2) lower left; (3) upper left; (4) upper right. The lateral 
incisors erupt earlier in the girls than in the boys. The lateral 
incisors erupt earlier in the Filipinos than in the Germans and 
Americans, although there is very little difference in time for the 
eruption of the lower teeth of this type. 

There is so little difference in time of eruption between the 
Germans and Americans that they may be considered as having 
the lateral incisors erupted at the same time, but the slight 
difference indicates that the Germans are more backward than 
the Americans. 

The canines. The canines begin to erupt in some individuals 
as early as the age of 8 years, and the teeth are nearly all, if not 
all, through the gums at the age of 15 years. There is practically 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


130 ROBERT BENNETT BEAN 


no difference in the time of eruption of the canine teeth on the 
two sides of the mouth but the lower canines erupt earlier than 
the upper. The difference in time between the eruption of the 
lower and upper canines is about one year in the American boys, 
less than a year in the German boys, and more than a year in 
the German and American girls. There is no order of eruption 
of the canines except that the lower erupt earlier than the upper. 

The canines erupt earlier in the girls than in the boys, except 
that the upper canines erupt earlier in the Filipino boys than in 
the Filipino girls. The sexual differences amount to at least 
two years for the Filipimos, between one and two years for the 
Germans, and about one year for the Americans. 

The upper canines erupt at about the same time in the Filipino, 
German and American girls, but the lower canines erupt at least 
three years earlier in the Filipino girls than in the German and 
American girls. There is almost no difference between the Ger- 
man and American girls in this. The canines erupt anywhere up 
to four years earlier in the Filipino boys than in the American or 
German boys, and the American boys are slightly more precocious 
than the German boys. The Filipinos of both sexes are preco- 
cious in the eruption of the canine teeth, the Germans are back- 
ward and the Americans are intermediate. 

The median premolars. The median premolars begin to erupt 
in some individuals as early as the age of 8 years and the teeth 
are nearly if not all through the gums at the age of 15 years. 

There is practically no difference in the time of eruption on the 
two sides of the mouth, but the upper median premolars erupi 
slightly earlier than the lower, although this difference is con- 
siderably less than one year. 

The median premolars erupt in the following order: (1) left 
upper; (2) right upper; (3) right lower; (4) left lower. The 
median premolars erupt first in the Filipinos, second in the Ameri- 
cans and third in the Germans, although the differences are not 
great between the last two. The median premolars erupt earlier 
in the girls than in the boys, except that apparently the upper 
premolars erupt slightly earlier in the Filipino boys than in the 
Filipino girls, but this is questionable. 


ERUPTION OF THE PERMANENT TEETH 131 


The lateral premolars. The lateral premolars begin to erupt 
in some individuals at the age of 8 years, and they are all through 
the gums in all individuals at the age of 16 years. There is very 
little difference in time of eruption on the two sides of the mouth, 
especially of the lower lateral premolars, although the left upper 
erupts earlier than the right upper. The order of eruption is as 
follows: (1) left upper; (2) right upper; (8) right lower, and (4) 
left lower. The upper lateral premolars erupt less than a year 
earlier than the lower. 

The lateral premolars erupt first in the Filipinos, and second 
in the Germans and Americans, at almost the same time for each 
of the latter. The lateral premolars erupt earlier in the girls 
than in the boys. 

The first molars. The first molars begin to erupt in some 
individuals before the age of five years, and nearly all, if not all, 
the first molars are erupted in all individuals at the age of 12 
years. 

There is practically no difference in the time of eruption of 
the first molars on the two sides of the mouth, but the lower first 
molars erupt slightly earlier than the upper, therefore the order 
of eruption is (1) and (2), lower first molars, (3) and (4), 
upper first molars. The lower first molars erupt earlier than the 
upper, the interval being considerably less than a year. The 
first molars erupt later in the Filipinos than in the Germans or 
Americans who are almost exactly alike in the time of eruption of 
these teeth. 

The first molars erupt about one year earlier in the girls of 
each group than in the boys. 

The second molars. The second molars begin to erupt at about 
the age of 10 years—slightly earlier in the Filipinos—and their 
eruption is completed in all, or nearly all, the individuals at the 
age of 16. 

There is practically no difference in the time of eruption of 
the second molars on the two sides of the mouth, but the lower 
second molars erupt about a year earlier than the upper, therefore 
the order of eruption is (1) and (2) lower second molars, and (3) 
and (4) upper second molars. The second molars erupt earlier 


132 ROBERT BENNETT BEAN 


in the Filipios than in the Germans and Americans, and earlier 
in the Americans than in the Germans. 

The second molars erupt more than a year earlier in the girls 
than in the boys, except in the Filipinos, where the boys are 
slightly more precocious than the girls, although the lower second 
molars erupt earlier in the Filipino girls than in the Filipino 
boys. 

The third molars. The third molars begin to appear in the 
Filipinos at the age of 13, and from 61 per cent to 83 per cent of 
the third molars have erupted in the Filipinos at the age of 20 
and over. 

The third molars have appeared in none of the German and 
American boys up to the age of 18 years, but they begin to appear 
in the American girls at the age of 16 years, and the left lower 
third molar has begun to erupt in the German girls also at the 
age of 16 years. The girls therefore appear to be more precocious 
in the eruption of the third molars than the boys, and the American 
girls more precocious than the German girls. The latter is not 
true among the Filipinos at the beginning of eruption, which 
occurs first among the Filipino boys, but as age advances up to 
20 the Filipino girls become more advanced than the Filipino 
boys, and at the age of 20 and thereafter the girls have a greater 
average number of third molar teeth erupted than the boys. 

There is practically no difference in the time of eruption of 
the third molar teeth on the two sides of the mouth, but the 
lower third molar erupt slightly earlier than the upper. 

Résumé. On pages 128 to 132 we have considered the eruption 
of the teeth from the standpoint of the beginning of eruption, 
the end of eruption, and the average per cent. The results areas 
follows: The order of eruption in groups is (1) Filipino girls, 
(2) Filipino boys, (3) American girls, (4) German girls, (5) 
American boys, (6) German boys. 

The order of the eruption of the individual teeth of the German 
and American group is (1) lower median incisors, (2) lower first 
molars, (3) upper first molars, (4) upper median incisors, (5) 
lower lateral incisors, (6) upper lateral incisors, (7, 8) upper 
median premolars and lower canines, (9) lower median premolars, 


ERUPTION OF THE PERMANENT TEETH 133 


(10, 11) upper lateral premolars and upper canines, (12) lower 
lateral premolars, (13) lower second molars, (14) upper second 
molars, (15) lower third molars, (16) upper third molars. 

The order of eruption of the individual teeth in the Filipinos 
is the same as the above except that the lower first molars and 
the lower median incisors erupt at the same time, the upper 
median incisors and the upper first molars change places, the 
lower canines erupt before any of the premolars, and the upper 
canines erupt at the same time'as the upper median premolars. 

If these results are compared with those obtained from the time 
at which 50 per cent of the teeth are erupted little difference 
will be found. The order of eruption in the sex-race groups is 
the same, and the order of eruption of the types of teeth isthe 
same, but the order of eruption of a few of the individual teeth 
is slightly different. This is manifest chiefly in the relative time 
of eruption of the first molars and median incisors, and of the 
canines and premolars. The meaning of this is that the first 
molars and median incisors are intimately related in time of 
development, if they are not synchronous, and the same is true 
of the canines and premolars. In either case the canines are 
more precocious in the Filipinos than in the Germans and 
Americans. 


4. Review of the literature and discussion 


We may arrange the teeth in the order found in their eruption 
among the German and American children of Ann Arbor taking 
the time at which 50 per cent have erupted and with this compare 
records for the French by Magitot-Broca (12, 13, 36, 37, 38, 39), 
Mayet (40), and Cherot (14), for the German by Welcker (51), 
and for the English by Livy (35). 

It may be said that the time of the eruption of the teeth as 
given by Cherot for the French is almost the same as that of the 
Ann Arbor German and American children, or only a little earlier, 
whereas those of Magitot-Broca, and Mayet for the French are 
earlier, and those of Welcker for the Germans are later than those 
for the Ann Arbor children. The records of Welcker are only 
slightly later than those for the Ann Arbor German boys 


134 ROBERT BENNETT BEAN 


and those of Magitot-Broca are about the same as for the 
Ann Arbor American boys. The Americans and the French 
appear to be more precocious than the Germans. The English 
are precocious in the eruption of the lateral premolars, and back- 
ward in the eruption of the lateral incisors, but otherwise they 
conform fairly well in the eruption of the teeth to the Germans. 

It is generally recognized that girls are more precocious than 
boys in the eruption of the teeth, and that the lower teeth erupt 
earlier than the upper, except the premolars, but Livy found 
among 4,000 children of the workers of Bolton, England, that 
the upper canines invariably precede the lower in the girls, whereas 
the lower canines invariably precede the upper in the boys. The 
upper canines erupt a year later than the lower among the Ger- 
man, American and Filipino girls that I examined, and less than a 
year later among the boys of the three groups. 

Spokes (48) found among British children that the eruption 
of the canines occurs between the time of eruption of the median 
and lateral premolars, which is a verification of what I found 
among the German and American children of Ann Arbor, but 
among the Filipinos the canines erupt before the premolars. 1 
found that the lower canines erupt between the time of eruption 
of the upper and lower median premolars, and the upper canines 
erupt between the time of eruption of the upper and lower lat- 
eral premolars. ‘This may explain the contention of Owen (51) of 
England on the one side, who contends that the canines erupt 
before the premolars (canines, 7 to 9 years, premolars, 8 to 10 
years), and Welcker, Sommering (51), Hyrtl (51), Henle (54), and 
Blumenbach (51) on the other, who contend that the median 
premolars erupt before the canines. It may be, however, that 
the canines erupt earlier among the British than among the Ger- 
mans, just as the canines erupt earlier among the Filipinos than 
among the children of Ann Arbor, or at least this may be true for 
some parts of the British Empire. Livy did not find it so for the 
children of the laboring classes of Bolton, England. 

The work of Cherot is of value because it is of recent date, and 
because he procured data from 20 to 30 children of each sex at 
each age. The time of the beginning of eruption and of the 


ERUPTION OF THE PERMANENT TEETH 135 


end of eruption as he found them in the French may be com- 
pared with my records of the Ann Arbor children. 

It is to be seen that the French children are earlier than the 
children of Ann Arbor both in the beginning of eruption and in 
the end of the eruption of the teeth, except in the beginning of 
eruption of the lower median incisors and upper canines, where- 
fore it is evident that the French at any age are more mature in 
the eruption of the teeth than the children of Ann Arbor. 

Cherot believes there are four periods of acceleration in the 
eruption of the permanent teeth, from six to eight and a half years 
when some teeth are erupting all the time; from 10 to 11 years; 
at 12 years; and about 20.5 years during the eruption of the third 
molars. The periods of 10 to 11 and at 12 should be combined 
because they run together, thus leaving three periods, which have 
been associated by others with the eruption of the three series 
of molar teeth. The periods of dentition given by Cherot would 
then correspond to the periods of acceleration given on page 127. 

Hrdli¢ka (30, 31) has published the only detailed data of the 
examination of children’s teeth that I have been able to find in the 
literature. He examined the teeth of the Apache and Pima 
Indians of North America, and from his records he concludes 
that the teeth of the Indians appear at about the same ages as 
the teeth of the whites, with the exception of the canines and 
second molars which apparently erupt earlier in the Indians than 
in the whites. I have calculated the beginning of eruption and 
the end of eruption of the teeth in the Indians, which is based 
on the stature rather than the age, because the age of the Indians 
was not obtained by Hrdli¢ka. The age is calculated from the 
stature by Hrdlicka, and he remarks that it is doubtless imperfect. 

The order of eruption is similar to that of the Europeans or 
white peoples, and different from that of the Filipinos, because in 
the latter the canines erupt before the premolars, whereas in the 
others the canines erupt at the time of the eruption of the pre- 
molars, or as noted above: the lower canines erupt between the 
eruption of the upper and lower median premolars and the upper 
canines erupt between the eruption of the upper and lower 
lateral premolars. 


136 ROBERT BENNETT BEAN 


The eruption of the teeth begins later in the Indians than in 
the whites, and ends earlier. The teeth of the Indians erupt more 
promptly than those of the whites and the canines, second and 
third molars erupt earlier in the Indians than in the whites. 

To test the relative precocity of the Indians, I have calculated 
the average age of all the Indian, Filipino, German and American 
children and the average number of teeth at all ages in all the 
groups. The ratio of the age to the number of teeth erupted 
in the order of precocity, is shown below: 


Age: teeth Age: teeth 
(@) milinchianyain| Seana LOO iGo (5) (Germanvorrlst eee. 100: 167.0 
()) Jambioriov) gangs ooo od soe 100: 175.0 (G@) Mindiantboysse ene 100: 153.6 
(3) Filipino boys.......... 100: 170.3 (7) American boys........ 100: 153.0 
(4) American girlsaey ono 100: 170.0 (8) uGenmaniboyss-eeneee 100: 150.0 


The Indian girls are the most precocious girls, and the German 
boys are the most backward boys. The Filipino boys are the 
most precocious boys, and the German girls are the most back- 
ward girls. The German boys and girls are the most backward, 
the American boys and girls are next to the most precocious. 
The girls are more precocious than the boys, but the difference 
is not great among the Filipinos and is greatest among the 
Americans. 

The ratio of the groups when both sexes are combined is: 


Age: teeth Age: teeth 
(1), Bulipimos ty. ecient 100: 172.6 (2) Indians..........:......- 1002 165205 
(8) RAI eT Can Serer ieee LON) Sel G13 (4) Germans. at.5. ee OORMaSe5 


The Filipinos are the most precocious, the Germans are the 
most backward, and the Americans and Indians are between the 
other two, the Indians more like the Filipinos and the Americans 
more like the Germans. The Indian women have a greater 
precocity than the Filipinos, and the Indian boys are almost 
exactly like the German and American boys. 

Boas (6, 7, 8) and Boas and Wissler (9) have made reports as to . 
the eruption of the permanent teeth in American whites but their 
work has been inaccessible to me, and what I give is based on 


ERUPTION OF THE PERMANENT TEETH ileyve 


abstracts. Boas published the following results recently with 
the remark that they ‘‘are not very accurate:” 


AGE IN YEARS 


ies | Boys Girls 
| Boas | Bean Boas Rean 
Inner permanent incisors.... . | 7.5 | 7.0 7.0 | 6.65 
Outer permanent incisors..... | 9.5 | 8.4 8.9 | 8.00 
icnapideteeee (itt itn Gude OS 11.1 9.0 |) 40:6 
CLITA Ieee FAG eee ae gg | 11.2 eG. | AS 10.5 
NEcondemolarceesem nae a eee 13.2 12.6 12.8 12.0 


I do not know how the calculations were made by Boas but 
when compared with my results of the time when approximately 
50 per cent of the teeth are erupted, his records show a retarded 
eruption of all the teeth except the premolars, which are preco- 
cious and erupt more than a year earlier than the premolars of 
the Germans and Americans of Ann Arbor. The canines in his 
records erupt about two years later than the ‘bicuspids’ (pre- 
molars?), which is very unusual. Boas and Wissler also place 
the end of eruption of the permanent teeth as follows: First 
molars, 9 years; median incisors, 12 years; bicuspids, 6 to 12 
years; canines, 6 to 15 years; lateral incisors, 15 years; and 
second molars, 7 to 15 years. So far as I am aware, they are the 
only records that place the eruption of the canines after the bicus- 
pids and the lateral incisors after both. 


THE LAW OF ALTERNATION IN DEVELOPMENT 


Donaldson (17-22), Jackson (32-34), Hatai (27, 28) and others 
have demonstrated the alternate periods of development of the 
parts of the body without stating a law that would apply to this 
phenomenon, and I have simply added to their work the results 
of my observations and from the combined data I have deduced 
a law which may be formulated tentatively somewhat as follows: 

There are one or more periods of acceleration alternating with 
periods of retardation in the development of each structural unit 
or organ of the body. The periods of acceleration in the development 


138 ROBERT BENNETT BEAN 


of one structure may be synchronous with the periods of retardation 
in the development of another, and the two may be called comple- 
mentary structures. Hach organ or structure has a critical period 
when it is developing most rapidly, and when tt is probably most 
susceptible to its environment. 

Jackson determined the growth in volume of the parts of the 
body in relation to the whole from observations on 43 human 
embryos and fetuses, and other similar material. The time at 
which the structure attains its greatest relative size is given, 
but this may or may not be the time of greatest relative accel- 


Trunk Head 
Heart Brain 


Mo. ! 2 


Liver Kidneys 


[cial Spleen thymus and thyroid a 
8 5 6 7 8 


Diagram 1 Prenatal growthinman. The curved lines represent the month 
at which the organs named above grow most rapidly. The diagonal lines repre- 
sent the approximate relative rate of growth of the organs named thereon. 
The number of the months is written below. 


Adrenals 


eration in growth, and it certainly is not its greatest absolute 
extent of growth, all of which should be considered in any study 
of growth. 

The growth of any part of the body should be determined in at 
least three ways. First, the amount of growth in relation to the 
size of the part previous to the period of growth; second, the 
amount of growth in relation to the adult size of the part; and 
third, the amount of growth of the part in relation to the total 
growth of the individual. The first is important because it 
would give the real activity of the part at different stages of its 
development, yet this method is not usually adopted. Diagrams 
1 and 2 will serve, however, to illustrate the relative growth of 
the body parts. 


ERUPTION OF THE PERMANENT TEETH 139 


From these diagrams it will be seen that there are three general 
_types of growth in the white rat as demonstrated by Hatai and 
Donaldson: (1) represented by the brain, which is ‘‘characterized 
by a very rapid growth in weight at an early period and after this 
period the rate of growth is much reduced;” (2) represented by the 
extremities, spleen, thymus and thyroid which are characterized 
by a relatively rapid rise at an early period followed by a straight 
line at an angle from the base line always much greater than that 
of type 1; (3) represented by the sex glands, which have an 
irregular growth, first slow, then rapid, then slow again. 

In any consideration of development it must be remembered 
that general laws should apply to all forms of mammals, at least, 
yet inasmuch as certain parts may be pathological (the brain of 
man is a pathological organ in relation to other forms and to evo- 
lution) any laws made for one form may not apply to another. 

The first part of the white rat to develop in prenatal growth 
is the trunk, including the heart, spinal cord and somites, which 
form 65 per cent of the total body weight in the first month of 
intrauterine life. Next the head develops, including the brain, 
skull, eyeballs and face, which forms 45 per cent of the total body 
volume at 2 months. The extremities maintain a uniform de- 
velopment throughout the prenatal period after their initial 
rise in the second month, and so do the spleen, thymus and thy- 
roid, although the relative growth of the last three is small com- 
pared to the relative growth of the extremities. The individual 
organs have a maximum relative volume during the prenatal 
period of growth as follows: heart, first month; brain, second 
month; liver, third month; lungs, fourth month; and kidneys, 
seventh month. 

The head in man is reciprocal in its growth to the trunk, and 
the trunk and the parts of the extremities are reciprocal to each 
other and to the extremities, as demonstrated by Godin (23, 24) 
and Pfitzner (45, 46), therefore these parts may be called com- 
plementary structures. The development of the heart is coin- 
cident with the development of the trunk, and the development 
of the head is coincident with the development of the brain, 
therefore the heart and brain may be called complementary 


140 ROBERT BENNETT BEAN 


BRAIN, CORD, EYEBALLS, | THYMUS, HEART, | STOMACH, 2 
LUNGS, SPLEEN | KIDNEYS, ADRENALS | INTESTINE, LIVER | Se uae 
E (ea rers Slee, = | E yee tees YI ea 
Rat Ist period (7 days) 2 (20 days) | 3 (6 wks.) 4 (puberty) 


| 


Man 1-2 yrs. | 3-5 yrs. | 7-10 yrs. puberty 


Diagram 2 Postnatal growth in the white rat 


structures. The development of the heart is also reciprocal to 
that of the lungs. The heart develops in time before the de- 
velopment of the brain and the lungs, but it is complementary 
to both. Likewise the liver may be reciprocal to the lungs, 
heart and brain in its development, and so each may be recip- 
rocal to the other, but if we examine adjacent organs in their 
development, such as the head and trunk, the heart and lungs, 
and the liver and intestine, as well also as the upper and lower 
teeth, their reciprocal development makes them logical comple- 
ments of each other. 


1 I 2 TT? }) apes Meme oT la a Oe | ay; 
STATURE TEETH |STATURE| TEETH |STATURE, TEETH } STATURE SEX | TEETH 
} | | 
|. 


1-6 mos. | 1? syns. \5 yrs. |7 yrs.| 8 yrs. | LL \agst | 12 yrs. "puberty 20 yrs. 


—|——_—— ———— 


Diagram 3 Periods of growth in stature and eruption of the teeth . 


The postnatal development of the structures of the white rat as 
given by Jackson may be grouped into four periods, and I have 
roughly approximated these periods for man. The brain and 
lungs develop most rapidly soon after birth, the heart and kid- 
neys a little later, followed by the development of the stomach, 
intestine and liver. The sex glands develop irregularly but their 
most rapid period of development immediately precedes puberty. 

I have roughly approximated the periods of most rapid growth 
in stature and the most rapid development of the teeth after 
birth, and have placed them in diagram 3. This is a tentative 
scheme and awaits further observations for confirmation. 

The first period of postnatal growth is the most rapid of all, 
and is associated with the development of the trunk and extremi- 
ties. This is followed by the eruption of the temporary teeth, 
associated with the rapid development of the brain. This re- 


ERUPTION OF THE PERMANENT TEETH 141 


minds us of the development of the trunk in the first month of 
prenatal life, followed by the rapid development of the head 
(brain). Between the arrival of the temporary teeth and the 
second acceleration of growth in stature (2 to 5 years), the heart 
and kidneys are apparently developing most rapidly. The 
eruption of the first permanent teeth, followed by the third 
acceleration of growth in stature, is related to the most rapid 
postnatal development of the liver, stomach and intestine. The 
third period of rapid tooth development followed by the fourth 
period of rapid growth in stature succeeds this and precedes 
puberty, after which the growth of the boys is retarded and the 
growth of girls almost ceases. There is need of more detailed 


Diagram 4 The order of eruption of the permanent teeth in Europeans. 
One side only of the mouth is represented, the left side as viewed from in front. 
The dots represent the individual teeth, whose symbols are placed between them. 
The order of eruption of the teeth is represented by numbers and by the lines 
connecting the dots. 7, incisors; c, canines; p, premolars; m, molars. 


data relating to the postnatal periods of growth in stature, in 
the teeth, and in the other structures of the human body. Until 
this is forthcoming there can be no final statement as to the 
exact relations of these periods, but enough is known of both 
prenatal and postnatal development to justify the law of alterna- 
tion in development. 

This law is applicable to the eruption of the individual teeth. 
No two adjacent teeth erupt at the same time except the median 
incisors, and the lower canines and median premolars. The 
median line represents a barrier, therefore the places filled by the 
median incisors are almost like remote areas. Diagram 4 rep- 
resents the order of eruption of the teeth on one side of the mouth 
in the Germans and Americans. 


142 ROBERT BENNETT BEAN 


The order of eruption is represented by serial numbers and 
followed by the line with barbs, beginning with the lower first 
molar and terminating with the upper third molar. The line 
skips from maxilla to mandible or mandible to maxilla nine 
times, and five times it goes from one tooth to another in the 
same Jaw. 

There are two waves of growth, one from the median line later- 
ally, which includes the incisors, canines and premolars, 20 teeth 
in all, that erupt more rapidly (from 6 to 11 years) than the 
other wave, which includes the first, second and third molars, 
only 12 teeth in all, that erupt more slowly (from 6 to 20 years 
or later). Four molars erupt every 6 years, the first molars at 
6 years of age, the second molars at 12 years of age, and the 
third molars at about 18 years of age. We may illustrate the 


WASEV NT VENN 


Diagram 5 The eruption of the teeth of the Filipinos and the Europeans 
in two series. A, American: F, Filipino. Each tooth is represented by a dot. 
Under the dots are symbols for the tooth; 7, incisor; c, canines; p, premolars; 
m, molars. The order of eruption is represented by the lines. 


two waves of growth by diagram 5, showing the relative time 
of development of each tooth in the two waves. 

When the permanent teeth begin to erupt the lower teeth erupt 
before the upper in the first period of acceleration in eruption 
(the first molars, incisors and canines); the upper teeth erupt 
before the lower in the second period of acceleration in eruption 
(the premolars) ; and the lower teeth again erupt before the upper 
in the final stages of eruption (the second and third molars). 

The alternation in development seems to apply to abnormal 
as well as normal development, although insufficient data are as 
yet available to demonstrate this conclusively. If one structure 
is unusually precocious in the periods of acceleration in develop- 
ment its complementary structure will be backward in the 
periods of acceleration, and vice versa. The eruption of the teeth 


ERUPTION OF THE PERMANENT TEETH 143 


of the Filipino boys may illustrate this. Take the lower teeth, 
for instance. In the median series the median incisors are 
precocious, hence the lateral incisors are relatively backward, 
hence the canines are relatively precocious, hence the median 
premolars are relatively backward, and hence the lateral pre- 
molars are relatively precocious. In the lateral series the first 
molars are relatively precocious, hence the second molars are rela- 
tively backward, and hence the third molars are relatively pre- 
cocious. The alternation is also true for the lateral series of the 
upper teeth where the first molars are backward, the second molars 
are precocious and the third molars are backward, which is not 
only an alternation of adjacent teeth, but also of the similar 
teeth of opposite jaws, because the lower first molars are pre- 
cocious, and the upper first molars are backward, the lower second 
molars are backward and the upper second molars are precocious, 
and the lower third molars are precocious and the upper third 
molars are backward. 

Racial differences in growth also fall within the law of alterna- 
tion in development. It is evident from this and other studies 
already published or at present under way that the Filipinos 
mature later than the Europeans in morphologic form, especially 
of the face, head, nose, etc., or never reach the state of the mature 
European, yet the Filipinos mature earlier than the Europeans 
(Ann Arbor Germans and Americans) in stature and in the erup- 
tion of the teeth. Does the early development of the teeth and 
stature in the Filipinos cause the late maturity of the head and 
face, and the early development of the head and face of the EKuro- 
pean cause a late development of the teeth and stature, or are the 
differences incidental, or caused by other factors? 

A presentation of the supposed causes of alternation in develop- 
ment would be incomplete without suggesting the influence of 
mechanical factors and the internal secretions. The alternation 
in development of the teeth may be produced by a more rapid 
growth of one tooth than another due either to an initial stimulus, 
or to a better blood supply. The adjacent tooth may be crowded 
back by the precocity of the one that develops first and the 
substances used in the building of the latter delay the growth of 


144 ROBERT BENNETT BEAN 


the adjacent tooth. The position or size of the blood vessels 
may determine the precocity of the tooth, just as the greater 
quantity of pure blood going to the head in the fetus may ac- 
count for the early precocity of that part. There may be blood 
vessels to the first molars and median incisors that are larger than 
those to the other teeth at first, or there may be an initial stimu- 
lus to these teeth that is greater than to the other teeth. Biting 
~ and chewing in the region of the median incisors and first molars 
is apt to be greater at first than in the region of the other teeth. 
In the same way the rapid growth of the lungs soon after birth 
and the closure of the ductus arteriosus and foramen ovale may be 
explained by the shunting of the blood stream from its fetal 
course through the foramen ovale and ductus arteriosis to its 
postnatal course through the right ventricle and pulmonary 
artery. The activity of the lungs immediately after birth sucks 
the blood through the pulmonary artery and right ventricle that 
formerly went through the foramen ovale and the ductus arterio- 
sus to the body, and thus allows the closure of the last two 
channels. | 

The ductless glands with their internal secretions poured into 
the blood stream play a part in development that is little under- 
stood, but if the normal effect of the secretions may be inferred 
from their abnormal effect, we may know more than we think, or 
understand. I cannot enter here into a review of the literature 
of the internal secretions, which is enormous, but I wish to pre- 
sent a few facts that may be relevant. We may infer from 
recent work that the hypophysis influences the growth of the 
bones, and there is some indication of the antagonistic action of 
the sex glands and the hypophysis, which may account for the 
retardation of the growth of the bones (stature) after puberty. 

The growth of the sex glands is irregular after birth, according 
to Jackson and Hatai, and if this irregularity in their development 
bears any relation to the periods of acceleration in stature, we 
may have a causal relationship. 

The thyroid influences growth, because hypothyroidism pro- 
duces cretinism and hyperthyroidism produces rapid differen- 
tiation with irritability of the nervous system, when either occurs 


ERUPTION OF THE PERMANENT TEETH 145 


during development. <A person of 13 to 14 may appear to be 20 
to 25 years of age. If later in life, hypothyroidism causes simple 
goiter, and hyperthyroidism causes exophthalmic goiter. Ex- 
ophthalmic goiter, or hyperthyroidism occurs invariably in hyper- 
ontomorphs, and eretins are hypodntomorphs, therefore the 
hyperactivity of the thyroid gland may have something to do 
with the hypermorphism of Europeans, and the hypoactivity of 
the thyroid gland may have something to do with the hypomor- 
phism of the Filipinos and other peoples (see p. 148). Climatic 
conditions, habits, food, water, and animal parasites may ac- 
count in part for the differences. 

The sexual activity of a people may have a profound effect upon 
their bodily form and mental condition through the interactivi- 
ties of the secretions of the sexual glands and the other glands of 
internal secretions. A great deal more has yet to be done on the 
effect of the internal secretions before definite results can be 
assured, but the indications are that the secret of multiple activi- 
ties resides in them and the explanation of many phenomena of 
development may be there, as well. Factors of selection, of 
evolution and involution, of progression or regression, of pro- 
gressive metamorphosis or of retrograde metamorphosis should 
be mentioned here. The third molars and canines are retro- 
grading, more in Americans than Filipinos, more in Germans 
than Indians. 

The process of selection in evolution cannot be passed over as a 
factor in the eruption of the teeth. Biting and chewing, or gnaw- 
ing and grinding, are the essentials in man, rather than holding 
and tearing, therefore the incisors and first molars develop early 
and are larger, and the canines develop late and are small. It is 
to be presumed that in prehistoric times those individuals in 
whom the incisors and molars developed early would be better 
fitted in the struggle for existence, and their kind would be 
propagated to a greater extent than those who had the incisors 
and molars developed late. The third molars are at present 
undergoing retrograde metamorphosis among Europeans; they 
appear late, and in some eases fail to appear. It is probable 
that the second molars erupt in man later than they once did. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 1 


146 ROBERT BENNETT BEAN 


The canines are also undergoing retrograde metamorphosis as 
indicated by their size in prehistoric times and today. The Fili- 
pinos and Indians, in whom the canines erupt early, are more 
like the prehistoric men than are the Germans and Americans, 
in whom the canines erupt late. The same is also true of the 
second and third molars, which also erupt earlier among the 
Filipinos and Indians than among the Germans and Americans. 


THE MORPHOLOGIC TYPE AND THE TEETH 


In a former study of Filipino students (2) I called attention 
to the bad teeth of what I then called the Iberian type, but 
have since named the hyper-onto-morph. This type is usually 
small, slender, narrow nosed, and has a long slender face with 
pointed chin, and a long narrow head with projecting occipital 
region, although at times the type may be fat, the face may be 
short and not slender, the nose may be stubby, and the head may 
be rounded; but at all times the type has characteristic ears, 
with everted anthelix, tragus and antitragus, and rolled back 
helix. The type is identical with the Mediterranean race of 
Sergi. In studies subsequent to those on the Filipino students 
I made observations in Manila (4), in Taytay (8), a town near 
Manila, and in New Orleans (5). I have demonstrated that this 
type is what I call an epitheliopath, a type which is very suscept- 
ible to diseases of the structures derived from epithelium, such as 
the lungs, alimentary canal and central nervous system. The 
hyper-onto-morph is quite distinct from the round-headed, round- 
faced type with more or less infantile nose and bowl-shaped ears. 
However, no observations were made of the ear form of the school 
children represented in this study, hence only the cephalic and 
facial indices, and the relative size of the occipital and parietal 
regions of the head can be utilized here. This will not effect a 
complete segregation of the hyper-onto-morph and the hypo-onto- 
morph, but it will indicate that a complete segregation such as 
might be accomplished by the use of the ear form and other fac- 
tors would give a more marked contrast between the two types 


ERUPTION OF THE PERMANENT TEETH 147 


in the eruption of the teeth, in the number of good sets present, 
and in the average number of bad teeth. As it is, the segregation 
by cephalic and facial indices gives considerable difference in all 
three of these conditions. 

The two types are segregated by putting those with a cephalic 
index below 80 and a facial index below 120 in one group, and those 
with a cephalic index of 80 and over and a facial index of 120 and 
over in another, and the remainder, or those with a cephalic index 
of 80 and over and a facial index of 120 and less, or those with a 
cephalic index of 80 and less and a facial index of 120 and over, 
ina third group. The three will be called the hyper-onto-morph, 
hypo-onto-morph and remainder. Below the age of 11 years the 
dividing figure of the cephalic index is 81 instead of 80. 


1. The average number of teeth 


The Filipinos from 5 to 10 years of age may be neglected be- 
cause of the few individuals who come under observation. The 
average number of teeth erupted among the German and American 
boys from 5 to 10 years of age is 


Hyper 9.87 Hypo 9.14 Remainder 9.45 


The average number of teeth erupted in all the groups from 11 
to 16 years of age, including both sexes, is 


Hyper 26.6 Hypo 24.1 Remainder 25.4 


The hyper- is more precocious than the hypo- from 10 to 16 
years of age. It is therefore evident that the hyper-, at all ages, 
is more precocious in the eruption of the permanent teeth than 
the hypo-, and this difference amounts to one or more teeth. 


2. The percentage of good sets of teeth 


The percentage of good sets of teeth among the German and 
American boys and girls from 5 to 10 years of age is 


Hyper 80.33 Hypo 80.98 Remainder 82.8 


148 ROBERT BENNETT BEAN 


The percentage of good sets of teeth among the boys and girls 
of all the groups from 11 to 16 years is 


Hyper 18.3 Hypo 46.8 Remainder 34.4 
Among the Filipinos alone from the age of 17 to 30, both sexes, 
it is 
Hyper 9.3 Hypo 34.3 Remainder 24.2 


It is evident that the hyper- has fewer good sets of teeth at 
all ages than the hypo-. 


3. The average number of bad teeth 


The Filipinos below the age of 10 years may be disregarded. 
The average number of bad teeth below the age of 10 years among 
the German and American boys and girls is 


Hyper 0.468 Hypo 0.36 temainder 0.30 


The average number of decayed teeth in all the groups from 
11 to 16 years of age is 


Hyper 3.35 Hypo 1.79 Remainder 2.09 


The average number of decayed teeth among the Filipinos 
alone from 17 to 30 years of age is 


Hyper 3.33 Hypo 2.53 Remainder 3.08 


It is evident from a consideration of the average number of bad 
permanent teeth that the hyper-s have the worst teeth and the 
hypo-s have the best. 


4. Racial and sexual differences 


Racial differences may be found by calculating the relative 
number of hyper- and hypo- individuals in each group. Be- 
tween the ages of 5 and 16 the Filipinos have 1 hyper- to 15.2 
hypo-s, the Germans have 1 hyper- to 5.3 hypo-s, and the Amer- 
icans have 1 hyper- to 1 hypo-. From 17 to 30 years the Fili- 
pinos have 1 hyper- to 3.8 hypo-s. 


ERUPTION OF THE PERMANENT TEETH 149 


Sexual differences may be determined in the same manner. The 
Filipino girls have 1 hyper- to 28 hypo-s; and the Filipino boys 
have 1 hyper- to 4.6 hypo-s; the German girls have | hyper- to 
7.1 hypo-s, and the German boys have 1 hyper- to 4.3 hypo-s; 
and the American girls have | hyper- to 1.2 hypo-s, and the Amer- 
ican boys have 1 hyper- to 0.84 hypo-. * 

It is also noticed that the relative number of hyper-s increases 
with age. This is especially noticeable with the American boys 
for whom the ratio is 1 hyper- to 3 hypo-s at 8 years of age, and 
12 hyper-s to 1 hypo- at 16 years of age. 

Hyper-morphism is a condition, of greater age, of development 
in a certain direction, a male condition, and a condition charac- 
teristic of the American white. Hypo-morphism is a condition of 
less age, of development in another direction, a female condition, 
and a condition characteristic of the Filipino. 

A number of other conditions besides the shape of the head and 
face characterize the hyper- and the hypo- types, such as the shape 
of the nose, the relative length of the extremities and of their 
parts, the ear form, the size of the regions of the head, etc., but 
it will suffice to give only one of these here, that is, the relative 
size of the occipital and parietal regions of the head. 

The circumferences of the forehead, frontal, parietal and occipi- 
tal regions of the head of the school children were taken but they 
will be reserved for future studies of head form that are at present 
under way, only the relative size of the occipital and parietal 
regions of the head to each other will be given here. 

The points selected from which to measure the circumferences 
of the regions of the head are the dorsal extremities of the middle 
roots of the zygoma on each side of the head immediately ven- 
tral to the external ear, a place that is easily accessible and dis- 
tinctly felt. The tape was passed from the point on the right 
side to the point on the left side around the maximum protuber- 
ance of the parietal and occipital regions of the head, and this 
distance recorded as the circumference of the part. An index is 
obtained that is called the occipito-parietal index, by multiply- 
ing the occipital circumference by 100 and dividing by the pa- 
rietal circumference. This gives the occipital circumference in 


150 ROBERT BENNETT BEAN 


terms of the parietal, the latter always being 100. A high in- 
dex denotes a large occipital region, a low index the reverse. 

The occipito-parietal index in the German and American chil- 
dren from 5 to 10 years of age is 


Hyper 80.9 Hypo 79.9 
and from 11 to 16 years of age is 
. Hyper 82.2 Hypo 79.4 


The occipito-parietal index in the Filipino boys is 


5 to 10 years Hyper 88.4 (1) Hypo 78.3 
11 to 16 years Hyper 81.9 Hypo 76.7 
17 to 30 years Hyper 83.4 Hypo 79.9 


The index of the Filipinos is less than that of the Germans and 
Americans at the same age, and the index of the hypo- in the Fili- 
pino from 17 to 30 years of age has reached that of the Germans 
and Americans at the age of 5 to 10 years, but the index of the 
hyper- among the Filipinos at 17 to 30 years of age has passed 
that of the Germans and Americans at the age of 11 to 16. The 
index increases with age except that the hypo-s among the Ger- 
mans and Americans do not change. 

The hypo- has a relatively smaller occipital region in the Fili- 
pino than in the German, and a relatively larger occipital region 
in the American than in the German. The same is not true for 
the hyper-, for in them the occipital region is about the same 
for the American and Filipino, but relatively smaller for the 
German. 

The sexual differences indicate that the male has a relatively 
larger occipital region than the female, and the female a relatively 
larger parietal region than the male although the difference is not 
great. 

No other differences will be presented here, although differences 
in stature, weight, nose form, ear form, facial angle, cranio-facial 
index, etc., will be given in subsequent publications, but enough 
has been done to establish the hyper-onto-morph and the hypo- 
onto-morph as entities and to indicate their identity. 


ERUPTION OF THE PERMANENT TEETH P51 


Magitot (37) called attention to the differences in the decay of 
the teeth in Normandy and Brittany, and attributed the difference 
to the type of people in the two compartments of France. In 
Brittany are the Celts or Gauls, who are small, with dark hair 
and eyes, broad head, and with good teeth; in Normandy are the 
Belgians or Kymries, who are tall, with fair hair and eyes, long 
head, and with bad teeth. The color of hair and eyes seems to be 
incidental, because in those I examined with bad teeth and of 
hyper-onto-morph form, the color of hair and eyes was of all 
shades. In southern Europe the long head has dark hair and 
eyes and in northern Europe fair hair and eyes, and in central 
Europe the broad head has dark hair and eyes and in eastern 
Europe the same form has fair hair and eyes. The morphologic 
type is independent of pigmentation and I believe the time of 
eruption of the permanent teeth and the extent of their decay 
are due to inherent differences in the morphologic type of the 
individual. 

The teeth of the hyper- form are undergoing retrograde meta- 
morphosis because they appear early and decay early. It is 
believed that the third molar in man is undergoing retrograde 
metamorphosis more rapidly than the other teeth and there are 
indications that the canines are also undergoing rapid retrograde 
metamorphosis, because the canines appear late and the third 
molars sometimes do not appear at all. It is strange to say that 
the teeth of the hyper- are undergoing retrograde metamorphosis 
because they appear early and decay early and the canines and 
molars are undergoing retrograde metamorphosis because they 
appear late or not at all, yet such is the inference. In the case of 
the hyper- there is rapid differentiation with early decay and in 
the canines and premolars retardation or failure to appear 
through backwardness. Retrograde metamorphosis, as in the 
human ear, seems to come about with precocity; involution prob- 
ably occurring earlier and earlier with each succeeding generation, 
therefore the hyper- is a very much involved form. Involution 
in the teeth may occur in the same way until crowding causes 
the third molars and canines to appear late and finally to fail to 
appear. There may also be more than one method of evolution. 


152 ROBERT BENNETT BEAN 


The hypo- and hyper- are both undergoing retrograde meta- 
morphosis, the hyper- in America, the hypo- in Asia, and the 
Meso form carries the evolution for the future. 

It is noted that the Filipinos are more precocious than the 
Americans in the eruption of the permanent teeth, although the 
Filipinos are more hypo- than the Americans, and the hypo- is 
more backward than the hyper- in this respect. Therefore the 
precocity of the Filipinos must be attributed to something other 
than hypermorphism. Whether it is race, climate, food, water, 
animal parasites, or something else, is problematic. 


PHYSIOLOGICAL STANDARD 


Each tooth may be considered from the standpoint of the time 
at which it first appears in any individual, and the time at which it 
last appears in any individual, as well as from the time at which 
50 per cent of the individuals have the tooth erupted, and the time 
during which the greatest number of individuals have the tooth 
erupting. Suppose we take the left upper second molar tooth 
in the American girls. This tooth has not erupted in any Ameri- 
can girl at 9 years of age, but 2 per cent appear at 10 years, and 
the latest age at which any erupt is 15 years, all are erupted at 
16 years. Approximately 50 per cent have the tooth erupted at 
12 years, therefore there is a shorter period for the eruption 
of the first 50 per cent (10 to 12 years), than for the eruption of 
the second 50 per cent (12 to 15 years). The greatest number 
of teeth erupt between 11 and 12 years (41 per cent) although there 
is a second period of rapid eruption between 13 and 14 years. 
This may be a segregation of the precocious and backward Ameri- 
can girls. 

In like manner we may obtain the same data for all the other 
teeth of the American girls, and of the teeth of all the other boys 
and girls. The upper teeth of the same type on the two sides, as 
the upper median incisors, erupt at so nearly the same time that 
they may be put together. The periods of most rapid eruption 
almost invariably coincides with the time at which 50 per cent of 
the teeth have erupted therefore the limits of the first mentioned 
period will cover the second. Table 1 (p. 155) has been constructed 
showing the results, and this table will be the physiological stand- 


ERUPTION OF THE PERMANENT TEETH 153 


ard for each group of individuals in relation to the eruption of 
each tooth. The ages given are the ages at which the teeth nor- 
mally appear. If the teeth appear earlier in any individual, that 
individual is precocious, and if later, backward. 

The physiological standard may be utilized to greater advan- 
tage with increasing age, because the racial and sexual differences 
increase with age. A word as to one way in which such a table 
may be of use. Take a German girl of 10 years, and all the in- 
cisor teeth, the first molars, premolars and canines may be 
erupted, whereas a German boy may not have all these teeth 
erupted until 11 years of age, yet at 11 years he may be at the 
same physiological standard as the girl at 10 years. Should 
either one have all the teeth mentioned erupted at the age speci- 
fied there would be evident precocity. In like manner evident 
backwardness may be determined. 

The teeth are more convenient and more exact as a means of 
determining the physiological standard than stature, or weight, or 
the growth of the bones, or secondary sexual characters, ete., and 
they may be of greater value than any other means that can be 
utilized. The teeth can be seen, recognized and counted by 
almost anyone after a little experience, and they are either pres- 
ent or absent, therefore very definite. 

Precocity in the eruption of the permanent teeth is a sign of 
hyper-morphism, and hyper-onto-morphs are epitheliopaths, who 
are especially susceptible to diseases of the alimentary canal, the 
lungs and the central nervous system, therefore precocious chil- 
dren should be shielded from injuries to the susceptible organs. 

Backwardness in the eruption of the permanent teeth is a sign 
of hypo-morphism, and hypomorphs should be shielded from 
injuries to their susceptible organs. 


THE SCHOOL GRADE AND THE TEETH 


The modal grade, or the grade that has the greatest number of 
individuals at each age, varies little with sex but the Germans are 
about one year behind the Americans. This corresponds with the 
backwardness of the Germans in the eruption of the permanent 
teeth, and indicates a correlation of mental and dental develop- 


154 ROBERT BENNETT BEAN 


ment. This is true not only of the modal grade, but also of the 
grades above and below the mode. At each age, from 7 to 14 
inclusive, the children who are below the modal grade have an 
average of 0.9 of a tooth less than those in the modal grade, and 
the children who are above the modal grade have an average of 
0.8 of a tooth more than those in the modal grade. The differ- 
ence is greatest at 10 and 11 years, when the second period of the 
second dentition is at its height, and the difference at these twa 
years amounts to 1.5 fewer and 1.8 more teeth for those below and 
above the mode respectively. . 

It will be recalled that there is a period of one year during 
which the greater number of teeth of any form erupt, and during 
this year in nearly every case, 50 per cent of that form of tooth 
has erupted. From this a physiological standard, or age of 
eruption of each tooth, was determined. Evidence is therefore 
produced to indicate that the eruption of the teeth is a better 
criterion than age as a standard of both physical and mental 
development. 

This evidence is not conclusive for each individual, but only 
as an average or modal factor, because some individuals with fewer 
teeth than normal are above the modal grade, and some individu- 
als with more teeth than normal are below the modal grade. 
Other factors therefore play a part and must not be overlooked. : 
For instance, one child who was advanced in school grade beyond 
her age, and who had less than the normal number of teeth 
present, was the child of a teacher, and had evidently been pushed 
in school work. However, the condition of the teeth, both as to 
eruption and decay, may be utilized and may be of value in de- 
termining the relative development of the individual. 

Maturity in the Filipinos is different from maturity in the 
Europeans, at least the face, body form, extremities, ete., of the 
Filipinos differ from the same parts in the Europeans, and the 
time of definite maturity of the parts is different. Each people 
is probably an expression of different conditions, and each repreé- 
sents development in a different direction, at a different rate, 
and to a different extent. The adult Filipino resembles the infant 
Kuropean in morphologic type more than it does the adult 


ERUPTION OF THE PERMANENT TEETH 155 


European. The condition of the Filipino in stature and in the 
eruption of the permanent teeth seems to be an early precocity 
superseded by an early retardation in development, but in mor- 
phologic type the Filipino is retarded throughout the period of 
development. 


RESUME 


1. The eruption of the teeth in relation to the development of the 
individual: the physiological standard 


The time of most rapid eruption of the teeth and the time 
at which 50 per cent have erupted is the same, and may be called 
the physiological standard of the teeth. This is shown in table 
1, for the Americans, Germans, and Filipinos. 


TABLE 1 
GIRLS, BOYS, 

AMERICAN AMERICAN FILIPINOS, 

AND GERMAN, | AND GERMAN, 


FIRST MOLARS 6 YEARS 6.5 YEARS | 5? YEARS 
MVM CU ATT RITTCTS OTS) 9: iN teas. 20Gd ater as cued ie 2 6.5 | 7.0 | 5? 
Hea tenaleincisonsa nse setae cies 8.0 | 8.5 6? 
MEAT NeMIOLATS tanec os checuevs Wel = oe | 10.0 | 11.0 | 8.0 
Canintesmere ns See sn oenie biti. SES Mla ks. | 10.5 Ut 25 7.0 
Matenale prem Glare sy: ic. Ce See cs 12S | 10.0 | Me) 9.0 

| 5 10.5 


SeconGdumolars tere eer as sek lee Rigerictaee s | 5 | 1D. 


2. The eruption of the teeth in relation to stature: periods of rapid 


development 
TABLE 2 

1 PERIOD | 2 PERIOD | 3 PERIOD 4 PERIOD 

ue 
| 

IBOySastabure:...).5 acts 1-6 mos. | 3-5 years | 7-9 years 12-14 years 
IBoysmteet ins. eeiyaee aes 6 mos.-2 yrs. | 6-9 years | 10-18 years 
Ginlsestatunethinc. sactescee nee 1-6 mos. | 2-4 years | 6.5-7.5 years | 10-14 years 


Ginls’-teeth. ..57 35. ; ey, oer 6 mos.-2 yrs. | 6-8 years | 9-11 years 


156 ROBERT BENNETT BEAN 


3. The eruption of the teeth in relation to race 


The American white is taken as the standard. Order of erup- 
tion: (1) Filipino, (2) French, (8) American Indian, (4) American 
white, (5) German. The time at which 50 per cent of the teeth 
have erupted: French 6 months to 1 year earlier than American 
white. German 6 months to 2 years later than American white. 
American Indian is from 1 to 6 months earlier than the Ameri- 
can white. Filipino | to 4 years earlier than American white. 


4. The eruption of the teeth in relation to sex 


American girls are from 0.2 years for the incisors, to 1.4 years 
for the canines, earlier than the American boys. German girls 
are from 0.3 years for the incisors to 1.1 years for the canines, 
earlier than the German boys. Filipino girls and boys are more 
nearly alike in the time of eruption. 


5. The eruption of the teeth in relation to school grade 


The modal grade is the grade which has the greatest number of 
individuals at each age. For instance at 7 years of age the 
second grade is the modal grade. At ages 7 to 14 inclusive, 
children above the modal grade have 0.8 more teeth, and children 
below the modal grade have 0.9 fewer teeth, than children at the 
modal grade. The difference is greatest at 10 and 11 years, 1.e., 
during the most rapid eruption of the second part of the period of 
second dentition, when children above the modal grade have 1.8 
more teeth, and children below the modal grade have 1.5 fewer 
teeth, than children at the modal grade. 


6. The eruption and decay of the teeth in relation to morphologic 
form 


Two types, extremes: (1) Hypo- equals infantile, round head, 
broad face and nose, large parietal and small occipital region of 
head. (2) Hyper- equals long head, face, nose, small parietal, 
large occipital region of the head (table 3): 


ERUPTION OF THE PERMANENT TEETH 157 


TABLE 3 
Teeth erupted: 
Below 10 years Hyper, 9.87 Hypo, 9.14 | German and 
Above 10 years Hyper, 26.6 Hypo, 24.1 J American 
Good sets: 
Below 10 years Hyper, 80.3 % Hypo, 81 % \ German and 
Above 10 years Hyper, 18.3 % Hypo, 46.8% { American 
17-30 years Hyper, 9.3 Hypo, 34.3 Filipino 
Average number of decayed teeth 
Below 10 years Hyper, 0.47 Hypo, 0.36 \ German and 
Above 10 years Hyper, 3.35 Hypo, 1.8 J American 
17-30 years Hyper, 3.33 Hypo, 2.53 Filipino 
Characteristics of the individuals in each group: § 
5-16 years 17-30 years Gils Boys 
Hyper, Hypo Hyper, Hypo Hyper, Hypo Hyper Hypo 
Filipinos 1 15.2 1 3.8 1 28.0 1 4.6 
Germans 1 5.3 1 el: 1 4.3 
Americans 1 1.0 1 1.2 1 0.84 


American boys have 1 hyper- to 3 hypo-s at 8 years, and 12 
hyper-s to 1 hypo- at 16 years. 


7. The law of alternation of development 


There are one or more periods of acceleration alternating with 
periods of retardation in the development of the structures of the 
body. Each organ or structure has a critical period when it is 
developing most rapidly, and when it is probably most susceptible 
to its environment. The periods of acceleration in the develop- 
ment of one structure are synchronous with the periods of retar- 
dation in the development of another, and the two may be called 
complementary structures. 

The relative number of hyper-onto-morphs is greatest among 
the Americans, least among the Filipinos, and nearly as great 
among the Germans as among the Americans. Hypo-morphism 
decreases with age, and hyper-morphism increases, so that whereas 
among the Filipinos there are 15.2 hypo-s to 1 hyper- between the 
ages of 5 and 16, there are only 3.8 hypo-s to 1 hyper-= from 16 to 
30 years of age. Hypo-morphism is a condition of less maturity 
than hyper-morphism. Apparently the Filipinos mature more 
slowly than the Americans and Germans in morphologic form, 


158 ROBERT BENNETT BEAN 


although they mature earlier in stature and in the eruption of their 
permanent teeth, which, again, may be only another expression 
of compensation in the law of alternation in development. 

The teeth are useful ‘as a physiological standard for deter- 
mining the relative development of the individual, physically 
and mentally. 


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ERUPTION OF THE PERMANENT TEETH 159 


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ON THE ORIGIN OF LYMPHATICS IN BUFO 


OTTO FREDERIC KAMPMEIER 
From the Anatomical Laboratories, School of Medicine, University of Pittsburgh 


THIRTY-FIVE FIGURES! 


The question of the origin of lymphatics continues to incite 
the most animated controversy in the field of anatomy in America 
at the present time. Consequently all observations bearing 
directly upon this problem are eagerly awaited and are received 
with enthusiasm by investigators specially interested in it. For 
the last two years the author has focused his attention upon the 
development of the lymphatic vessels in Amphibia with the hope 
of being able to discover an evidence to the solution of that 
problem which could not be controverted by any criticism, and 
incidentally of adding to our as yet fragmentary knowledge of 
the phylogenesis of the lymphatic system. Although such a 
hope may have been too sanguine, inasmuch as the evidence 
offered does not entirely reveal how much fact and how much 
fiction there is in the various hypotheses propounded hitherto, 
the observations reported here differ so decidedly from observa- 
tions made in the past and at the same time fit in so readily with 
them that we trust much has been accomplished towards a speedy 
termination of the dispute. An extensive paper dealing with the 
genesis of the lymph hearts, ducts and sinuses in Amphibia is in 
the process of preparation. Some months, however, will inevi- 
tably elapse before this work is in its final form. It has seemed 
expedient, therefore, to publish immediately for the benefit of 
the writer’s co-workers in lymphatic research a partial account 
embodying the central point of his investigations. 

Early embryos of the American and the European common 
toads constitute the chief material of this inquiry. The first 
were collected near Princeton, New Jersey, the second were 


‘Cost of illustrations in part borne by the Laboratories. 
161 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 2 
JANUARY, 1915 


162 OTTO FREDERIC KAMPMEIER 


gathered in the marshes of the Isar near Munich and prepared 
in Professor Rtickert’s laboratory at the Anatomical Institute. 
The specific descriptions in the following pages are based entirely 
upon the specimens secured in Europe and, strictly speaking, 
pertain only to them. The serial sections of the native toad were 
not used extensively because they were prepared first, that is, 
at a time when the writer’s practice in making perfect series of 
yolk-laden Amphibian larvae was still in its trial and error phase. 
Such experimentation, particularly in regard to fixation, embed- 
ding and sectioning, was conducive of better results with the 
second batch of specimens, which were procured last spring? 
while the writer was engaged in study abroad. Mention of the 
methods of technic as well as a review of the work of other in- 
vestigators relating to the formation and growth of lymphatics 
in Amphibia will be reserved for the later article. Bufo em- 
bryos were chosen in preference to frog embryos because their 
mesenchyme appears less scanty and less loose in texture, a 
condition which it was supposed might prove helpful in distin- 
guishing between lymphatic anlagen and tissue spaces. Never- 
theless, it will become evident presently that such a precaution 
was needless. 

At the beginhing of the inquiry the gaining of exact special 
information was beset with what at first seemed a very discon- 
certing obstacle, namely, the enormous number of yolk spherules 
which are closely massed in all tissues of the young larvae and 
cover and obscure everything except darkly staining cell nuclei. 
The effort required to follow the development of such material 
with some degree of accuracy exceeds the ingenuity and patience 
expended in the preparation of satisfactory serial sections. Many 
details of the earliest genetic changes cannot -:be followed with 
certainty until much of the yolk has vanished. But in this 
respect a comparison of young and somewhat older stages will 
yield the important fact that the yolk does not disappear uni- 
formly from the embryonic body. In other words, the period 
in which the yolk substance is lost is different for the various 


* April and May, 1913. The manuscript was completed in the spring of 1914, 


ORIGIN OF LYMPHATICS IN BUFO 163 


tissues. Of course this does not necessarily imply that certain 
tissues are more active than others and use up the stored nutri- 
tive supply more rapidly. On the contrary, some may be more 
richly supplied at the outset. Thus organs derived directly 
from entoderm retain traces of yolk much longer than others. 
These considerations suddenly led to the inspiration that the 
presence of yolk might after all prove to be an advantage since 
it might point out the relationship of lymphatic endothelium. 
Such a natural and simple factor, if found and capable of being 
utilized as an instrument of proof, would carry far more weight 
than any possible evidence obtained by the use of special stain- 
ing processes, injections, or other artificial procedure. A. critical 
study of the behavior of the several tissues in this respect has 
convinced the writer of the soundness of this principle. Refer- 
ring especially to the head region, which in vertebrate embryos is 
ever in advance of other parts of the organism in degree of devel- 
opment, it was observed that the mesenchymal cells dispose of 
their yolk content much earlier than do blood corpuscles and 
vascular endothelia, both haemal and lymphatic. To be more 
specific, in 6 and 7 mm. embryos of the European common toad 
the cephalic mesenchyme is virtually destitute of yolk, while 
the lining of the lymphatic anlagen contains large globules even 
in specimens measuring 9 mm. in length. To the author’s 
mind these facts indicate fundamental differences between the 
two tissues and show that in Amphibia, at least, the lym- 
phatic intima arises not by direct differentiation of mesenchymal 
elements. The idea of similarity of origin but diversity due 
to function cannot be urged against this argument, for at the 
time when the yolk-filled rudiments of the large cranial lymph 
channels appear the embryonic connective tissue of the same 
region has lost most of its yolk granules. 

Before entering into an interpretation of the appended figures 
illustrating the source of certain lymph vessels, a brief descrip- 
tion of a later embryonic phase, when an effective system of lym- 
phatics is already in existence, is essential to the proper under- 
standing of the narrative. This can best be done by referring 
the reader to Hoyer’s figure 417 in the seventh edition of Wieder- 


164 OTTO FREDERIC KAMPMEIER 


scheim’s ‘‘Vergleichende Anatomie der Wirbeltiere,” or to his 
original article which depicts such a condition in a frog tadpole. 
An enormous lymph sinus, probably tantamount to the several 
cephalic subcutaneous sacs of the adult, is seen to occupy almost 
entirely the ventral and lateral territory of the head. On each 
side this reservoir passes backward as a short slender duct which 
opens into the anterior lymph heart, situated in the region of the 
fore-limb bud. A second set of vessels, draining the posterior 
portions of the body, unite cranially to form a single trunk, which 
communicates with the duct coming from the head at its point 
of entrance into the lymph heart. Besides the chief systemic 
vessels, numerous smaller subsidiary channels and_ plexuses 
spring from them; but these will not be considered in the present 
paper and may therefore be disregarded. 

The inception of the large ventral cephalic lymph sinus will 
be discussed fully, for in the writer’s opinion it offers a very 
clear case of the derivation of lymphatic endothelium from the 
lining of blood vascular channels. This lymph vessel is an 
especially favorable object for study not only on account of its 
size, but because it originates in the immediate vicinity of veins 
located in a broad uninterrupted expanse of mesenchyme which 
is loosely woven yet sufficiently abundant in number of cells 
to facilitate an examination and comparison of the rédle played 
by these structures during the formation of it. Figure 1, inserted 
for the purpose of orientation, represents a cross-section of the 
head of an 11 mm. embryo and illustrates the situation of this 
resevoir (l.) relative to other organs. The position also of the 
external jugulars (j.d. and j.s.)* should be carefully noted, for 
the five succeeding plates portray events that take place proxi- 
mately around these veins. Beginning with the stage of the 
almost completed lymph sinus, the territory ventral to the mouth 
cavity (m.c.) In consecutively younger embryos was scrutinized 
with the oil immersion lens. The important revelations are set 
forth in the camera lucida sketches reproduced on plates 1 to 6 
inclusive. To duplicate as nearly as is feasible the course by 
which the data were obtained, the descriptions commence with 


8 Goette called the external jugular (Gruby and Ecker) the inferior jugular. 


ORIGIN OF LYMPHATICS IN BUFO 165 


that of a fairly definitive stage in the organization of the sinus 
and then treat of progressively earlier conditions, which may 
subsequently be summarized in a more logical manner. ; 

In figure 1 the ventral cephalic lymph sinus (l.) of an 11 mm. 
embryo is shown in section as a wide clear chamber, the periphery 
of which is sharply defined and the cavity is not interrupted by 
partitions. Disregarding the disparity in magnifications, with 
it should be compared the one represented in figure 13, from a 
9 mm. embryo, where it (/.) is seen to be less broad dorso-ven- 
trally and to be crossed wholly or part way by tissue strands (. 
and s.). Imagined in its entirety, these trabeculae are very 
numerous and exhibit several variations. Some are proportion- 
ately thick (¢., figs. 13 and 14) and are composed of a core of 
mesenchyme (m.) covered with endothelium; others are more 
tenuous, barely stretching across the lumen; and still others 
exist as spurs (s.) of varying lengths which project into it from 
the surface. Furthermore, there are scattered freely through- 
out the cavity small clumps of lightly staining débris (d.) whose 
appearance would suggest their being cellular shreds or frag- 
ments of former trabeculae or partitions, which had perchance 
become separated from the walls of the sinus during its formation. 
Such an explanation presents itself as the most credible one. 
Pictured in figure 14 individual yolk spherules are conspicuous 
here and there in the endothelial cytoplasm generally in the 
neighborhood of a nucleus. At this stage the lining cells also 
possess all of the intrinsic qualities of typical endothelia and 
accordingly it is an easy matter to distinguish them from the 
stellate mesenchymal cells. 

Figure 12 delineates a little more than the right half of a section 
through the sinus (/.) in an 8 mm. embryo and plainly indicates 
its plexiform character at this period. This is confirmed by an 
enlarged graphic reconstruction! which shows it to be a network 
of interanastomosing vessels arranged one layer deep in a slightly 
curved plane, and which, viewed from the ventral surface, brings 
to mind a coarsely and irregularly meshed sieve. Followed 

4 Wax models and graphic reconstructions of crucial stages in the development 


of the lymph hearts, ducts and sinuses of the toad will be pictured in the later 
contribution. 


166 OTTO FREDERIC KAMPMEIER 


throughout its total extent, the sinus is found to be nowhere in 
connection with the veins, although it closely approaches the 
external jugular of each side at its extreme anterior limit; nor has 
it as yet established junction with the lymph hearts. 

Plate 4 is representative of conditions observed in a 7 mm. 
embryo. The upper sketch, figure 10, drawn from a section of 
the region eventually occupied by the completed sinus, shows 
this vessel as a double or bilateral rudiment (/.) which does not 
communicate from side to side, but each half is isolated from the 
other and consists of several individual anlagen which are de- 
veloped ventro-laterally of the external jugulars (j.d. and j.s.). 
In the lower picture, figure 11, is sketched a highly magnified 
area on the left including the vein (j.d.) and three lymphatic 
anlagen (/., J.) in cross section. One of these is solid, being 
laden with yolk globules, and the larger two are hollow but 
possess walls which are dense and firm. With reference to yolk 
content, the drawing, a faithful copy of the actual state of affairs, 
impresses the distinction between mesenchyme and lymphatics 
so forcibly that further words to the same effect are superfluous. 
Indeed, there are occasional mesenchymal elements which do 
contain yolk, but then it is usually in the form of minute granules 
and is distributed thinly in the protoplasm. Other distinguish- 
ing marks are not so apparent. In all probability the lymphatic 
endothelial nuclei are on the whole somewhat smaller and more 
compact than those of the connective tissue at this relatively 
late stage, but manifold exceptions are encountered among them 
and consequently the observer would hesitate to emphasize such 
a difference unduly. 

Turning to the next previous stages, 6 and 5 mm. embryos, 
more interesting phenomena were witnessed, which are in part 
reproduced on plates 3 and 2. Figure 7 was drawn from a, trans- 
verse section of a 6 mm. specimen to exhibit the features in the 
environment of the left .external jugular (j.s.). At this level 
the larger one of the two sinus anlagen (l.) contains a small 
slit-like lumen, the confines of which are thick and are packed 
with yolk spherules. This lymphatic followed from end to end 
was discovered to be applied to the lining of the vein at its ante- 


ORIGIN OF LYMPHATICS IN BUFO 167 


rior extremity and then to accompany the vein back a consider- 
able distance, sometimes lying hard against it, at other times 
slightly aloof from it, as displayed in the sketch, and finally 
becoming a solid attenuated cell cord that ends freely in the 
mesenchyme. The smaller sinus anlage, shown in the same sketch 
(fig. 7), stands in similar relations to the vein, but it is less ex- 
tensive than the other, less wide in diameter and discloses fewer 
vacuoles. The two anlagen are strictly independent of one 
another. Beyond their terminations several shorter anlagen 
are met with which are essentially like those described. 

A circumscribed area on the right side of another 6 mm. larvae 
is portrayed in the second drawing, figure 8. Medially a sinus 
rudiment (1.1.) is inseparably attached to the intima of the 
external jugular (7.d.); at least no visible boundary line can be de- 
tected between the adjoining walls of the two structures. More- 
over, the lumen of the lymphatic is bisected by a thin cytoplas- 
mic filament which passes from the inner combined venous and 
lymphatic wall to the free outer wall. After extending through 
a number of sections in this manner, the anlage becomes de- 
tached from the vein and pursues its way parallel to it alone 
through the mesenchyme until it bends downward and comes in 
touch with another lymphatic undergoing development on the 
ventral side of the vein. This is clearly set forth in figure 9, 
where the anlage labelled /.3 is approximated by anlage 1.1, 
which is identical with or, more correctly stated, a continuation 
of the one (/.1) situated on the dextral wall of the blood channel 
in figure 8. Eight sections, each 6 micra in thickness, intervene 
between the two levels. The reader perceives that the lymphatic 
rudiment (/.1) not only severs connection with the vein in this 
short stretch, but it becomes broader and at the hinder level 
(fig. 9) lacks a lumen in consequence of the large yolk corpuscles 
that crowd every available nook and corner in it. This condition 
is true of the greater part of its course. The rudiment 1.3 (fig. 
9) is of brief length, appears behind the level represented in 
figure 8 and possesses a large cavity proportionately, which, 
separated from that of the bloodvessel (j.d.) merely by a thin 
partition, resembles strikingly the extra-intimal spaces existing 


168 OTTO FREDERIC KAMPMEIER 


in mammalian embryos of certain ages. Another anlage (1.2, 
fig. 8) begins anteriorly as a distinct thickening of the lining of 
the external jugular (j.d.), but after two or three sections it 
breaks loose from the vein and proceeds posteriorly as a compact 
yolk-stuffed endothelial column (J.2, fig. 9) surrounded by mesen- 
chymal cells. Shghtly farther back the same anlage has acquired 
a lumen. After a variable course, in which it lies occasionally 
against the venous intima and now and then buds collateral 
sprouts, it terminates near the posterior niveau of the thyroid 
anlage, just in front of the heart. 

The earliest initial stages in the genesis of the ventral cephalic 
lymph sinus occur in 5 mm. embryos. The sketches on plate 2 
illustrating its inception are representative of the phases observed. 
Figures 2, 3 and 4 must be considered together since they treat 
of the same anlage the origin and character of which they plainly 
depict. In figure 2° attention should first be directed to the 
endothelium of the external jugular (j.s.) which is very undulat- 
ing and nodular, each node or protuberance consisting of a cell 
and a cluster of yolk corpuscles. Then, one cannot but be 
impressed by the knoblike appendage (/.) which protrudes from 
the ventral venous wall into the open tissue reticulum. This 
endothelial projection is the anterior end of a developing lymphatic 
channel and, as the figure suggests, arises indisputably by pro- 
liferation from the lining of the bloodvessel. Its nucleus in the 
section (fig. 2) does not differ materially from mesenchymal 
nuclei except for the depressions in its contour which are caused 
by the crowding of the yolk bodies against it. A large typical 
stellate tissue cell is shown in the lower left corner of the picture 
(fig. 2). Like a number of such cells, its protoplasm at this 
stage incloses a few scattered yolk granules. Figure 3 represents 
the next successive section in which the lymphatic anlage (l.) 
is joined to the vein (j.s.) solely by delicate cytoplasmic threads. 

5 Near the left hand margin of figure 2, the lumen of the external jugular ap- 
pears to be crossed by an endothelial partition. But this is not the fact. The 
sketch is of a section taken immediately behind the level in which a medial branch 
leaves the vein at a very acute angle. Hence the strand of endothelium repre- 


sents the point at which the walls of trunk and tributary meet, and the two cavities 
are the lumina of these vessels respectively. 


ORIGIN OF LYMPHATICS IN BUFO 169 


Three sections back, the diameter of the external jugular (/.s., 
fig. 4) has markedly contracted while that of the incipient lymph 
channel has increased. This rudiment is solid from end to end, 
being stuffed with yolk, which attribute sharply demarcates it 
from the embryonic connective tissue. To repeat what has 
been said but a moment ago, the visible differences between 
lymphatic endothelial nuclei and mesenchymal nuclei are too 
trivial at this early genetic period to warrant our emphasizing 
them as positive differential characteristics. 

One of the earliest sinus anlagen observed by the writer is 
shown in section in figures 5 and 6. It is of very brief extent, 
being only as long as the total thickness of four or five sections. 
In the first (fig. 5) of these two sketches it appears as a compact 
protuberance (/.) on the intima of the right external jugular 
(j.d.). The structure labelled b.c. in the same drawing and which 
at first glance might be mistaken for another initial lymphatic 
is a blood cell closely pressed against the lining of the vein. In 
very young toad larvae blood cells like vascular walls are abund- 
antly supplied with yolk. Overlooking one section we come to 
figure 6. Here two features should be noted in particular: 
firstly, the blind slit-like space within the lymphatic anlage, 
and secondly, the appearance of a boundary between anlage 
and vein which however is still imperfect since the line of division 
extends only partway. The cavity of the anlage is not in con- 
nection with that of the vein. 

From the data so clearly displayed in the camera lucida sketches 
reinforced by much similar evidence at the writer’s command, 
the following generalization or coherent account of the genesis 
of the ventral cephalic lymph sinus can be constructed. In 5mm. 
embryos, a stage in which the mesenchyme and the vascular 
endothelia of the head already differ to a noteworthy degree in 
the fact that the former is more meagerly furnished with yolk, 
knot-like thickenings occur at unequal intervals on the lining of 
the external jugular veins in the direction of their long axis. 
These thickenings, potentially lymphatics, are few in number and 
arise unquestionably as proliferations of the venous intimal cells 
and like them are provided with many yolk spherules which are 


120 OTTO FREDERIC KAMPMEIER 


generally large and lie closely crowded in the cytoplasm. De- 
pendent apparently on the degree of development, these cell 
aggregations present somewhat different characteristics Some 
have the form of a bulging compact local swelling of the venous 
wall and can be pursued through a few sections only. Others 
- are quite as short in extent but manifest one or several small 
crevice-like cavities which it appears never open into the lumen 
of the parent vein. Still others, older doubtlessly, exist as 
comparatively long yolk-stuffed sprouts, one end of which adheres 
to the bloodvessel and the other lies some distance aloof among 
the thinly scattered large mesenchymal cells. Such growths, 
at this time, are solid cell cords for the greater part of their course, 
but between the masses of yolk ill-defined and irregular vacuities 
now and then occur which intimate an intracellular origin. 
Solely superficial differences, if any, can be detected between the 
nuclei of the lymphatic endothelial buds and those of the mesen- 
chyme. The former seem to be denser in chromatic substance, 
although this is by no means diagnostic, for in this regard much 
variation has been found among nuclei according to the mitotic 
phase active at the time of fixation. <A difference which may be 
more pronounced, yet a difference which after all is neither causal 
nor specific but is secondary, is the more frequent occurrence 
in endothelial nuclei of an indented periphery due evidently to 
the pressure of the yolk spheroids upon the nuclear membrane. 
As has been and will be emphasized again, the character which 
visibly differentiates the endothelial from the mesenchymal 
cell in the early genetic stages of the cephalic region is the pres- 
ence of much yolk. From this it is obvious that were both tissues 
devoid of yolk it would scarcely be possible to follow the incipient 
lymphatics through the meshwork of mesenchyme; and even 
though all the details of their formation were known the seasoned 
observer would suffer considerable perplexity in localizing and 
tracing them. On the contrary, with a large quantity of yolk 
present in one of these two tissues and absent in the other, the 
lymphatic anlagen, after they have been once recognized as such, 
can be pursued from section to section with exceeding ease. In 
the sections stained with hematoxylin and orange G the contrast 


ORIGIN OF LYMPHATICS IN BUFO LAE 


between the brilliant brownish yellow color of the yolk-rich 
tissue and the blue of the yolk-poor is very striking, and in the 
colored sketches an attempt has been made to express the dis- 
tinction as strongly. 

In succeeding stages, 6 and 7 mm. embryos, the anlagen of 
the lymph sinus have in the main received a lumen. Their 
confines are still thick and heavy and the contour of the cavity 
is irregular on account of the many large protruding yolk spheres 
packed in the cytoplasm of the endothelium. Frequently 
protoplasmic bridges or trabeculae traverse the cavity from side 
to side and nuclei have been observed to be included in them. 
Such a condition, coupled with the fact that yolk globules oc- 
casionally lie freely in the lumen of an anlage, would point to 
the derivation of that lumen from intracellular vacuoles by their 
enlargement and coalescence. By this time all of the initial 
lymphatics have grown much in length and have also expanded 
proportionately in caliber. Some of them still retain their 
original junction with the intima of the vein; others have lost 
this connection and exist in the mesenchyme as longer or shorter 
discrete endothelial and yolk-lined fusiform spaces. Other such 
spaces cling to the parent vein almost throughout their entire 
extent and hence bear an extraordinary resemblance to the extra- 
venous spaces of mammalian embryos. Blood corpuscles have 
not been seen in the cavities of these structures; yet this finding 
“is not as significant as it might seem, for as the sketches show, 
even in the external jugular veins of the specimens studied there 
is a paucity of them as a result apparently of fixation currents 
and contractions. 

The transformation of the discontinuous bilateral lymphatic 
rudiments into one broad continuous Sinus is a very rapid one. 
During the stages previous to older 7 mm. embryos the anlagen 
had developed chiefly in a longitudinal direction, that is, along 
the cranial portion of the external jugulars, but now they begin 
to germinate collateral sprouts in an oblique direction towards 
the mid-ventral line, especially at the level of the thyroid anlage. 
These sprouts again proliferate buds, the tips of which grow out, 
soon collide and fuse with one another, and their cavities become 


172 OTTO FREDERIC KAMPMEIER 


confluent. Others continue to grow medially until they meet 
those coming from the opposite side. In this manner a very 
complicated plexus of interanastomosing lymph vessels is estab- 
lished in the ventral territory of the head. This plexus, as 
reconstructions of it show, is remarkable for its regularity and 
symmetry, roughly suggesting the form and extent of the finished 
lymph resevoir. Such a phase has been reached in the younger 
8 mm. embryos. The channels constantly distend and in effect 
the walls of adjacent ones are rapidly approximated so that the 
mesenchyme filling the meshes of the lymphatic network is 
diminished in amount. In older 8 mm. embryos the sides of 
contiguous vessels have met and begin to break down and dis- 
appear at the place of coincidence; in this way the plexiform 
character of the developing sinus is progressively obliterated. 
Transverse sections at this stage show many mesenchymal and 
endothelial strands and partitions crossing the cavity of the 
sinus dorso-ventrally and giving to it a multi-locular appearance, 
which compares favorably with a similar transient phase in the 
embryonic history of the thoracic duct in the pig. Such trabec- 
ulae are still conspicuous in 9 mm. specimens, but later these 
last vestiges of boundaries between originally independent chan- 
nels vanish and leave the sinus a vast uninterrupted lymph 
chamber. 

During these genetic processes, the yolk content of the lym- 
phatic anlagen has suffered marked diminution, and coexistent 
with this change the intimal cell has passed through a gradual 
metamorphosis from the stage of an undifferentiated generalized 
cell to that of a specialized endothelial-like cell. In 10 mm. 
larvae a casual yolk spherule may still be found in the confines 
of the sinus, and the lining*cells have assumed the features typical 
of all well-formed endothelia. From now on the prime alter- 
ation which the lymph sinus undergoes is the establishment of 
cont nuity between it and the other components of the lymphatic 
channel system; other minor changes are chiefly in the nature of 
growth. But such considerations are beyond the scope and pur- 
pose of this paper, and a discussion of them will be postponed 
until a later date. 


ORIGIN OF LYMPHATICS IN BUFO 173 


To forestall the criticism of insufficient data, a few typical and 
decisive stages in the genesis of two other lymphatic vessels will 
be briefly detailed. These stages shall primarily show the occur- 
rence of discontinuity in a developing lymph duct just as the 
developing sinus has shown in the first instance the origin of its 
endothelium from venous intima. The channels to be considered 
are the lymph ducts situated laterally, a pair on each side, and 
extended through the entire length of tail and trunk to open into 
the anterior lymph hearts (cf. Hoyer’s fig. 417, 7th edition, 
Wiederscheim’s ‘‘Vergleichende Anatomie der Wirbeltiere ’). 
For a more precise conception of their place in the anatomy 
of the embryo the reader is directed to figure 15, which pictures 
a little more than the sinistro-dorsal quarter of a section through 
the mid-trunk region of a 9 mm. embryo. The lymph ducts, 
l.s. and 1.2., are seen to be located between epidermis and myo- 
tome (m.s.), the first (/.s.) near the superior or dorsal border of 
this structure, and the second (l.7.) near the inferior or ventral 
border. To make more comprehensive the succeeding sketches, 
the relative position and direction of two or three neighboring 
organs should be pointed out. The Wolffian or pronephric 
duct (w.) is intercalated between the lower margin of the muscle 
segment and the dorsal peritoneum or roof of the coelom. Along 
its free walls run two veins which in reality are the medial and 
lateral divisions of but one, the posteardinal. Regarded in their 
longitudinal aspect, the postcardinal divisions are bound _ to- 
gether by numerous cross-anastomoses, which pass over and 
under the pronephric duct at unequal but frequent intervals and 
hence produce a cylindrical vascular network which closely in- 
vests this duct. The postcardinal vein of each side also give 
off in regular sequence the intersegmental veins (7.v.), every one 
of which supplies chiefly a myotome. 


® According to Goette (’75) the postcardinal veins in Anura lie medial to the 
pronephric ducts. But the writer can not subscribe to this statement uncon- 
ditionally, for it was observed in young toad embryos that the postcardinals 
resemble more nearly those of Urodeles (Hochstetter) where they surround the 
pronephric ducts as vascular sheaths. It is true, the portion of the cylindrical 
postcardinal network lying medially in Bufo is on the whole larger and doubtlessly 
constitutes the main channel. But this channel and the portion of the posteardinal 


174 OTTO FREDERIC KAMPMEIER 


In 6 and 7 mm. larvae, the collateral tributaries of consecutive 
intersegmental veins tend to anastomose with one another and 
give rise to a longitudinal channel near the upper border of the 
muscle somites. It is along this vessel that the dorsal or supe- 
rior lateral lymph duct originates. It is formed by the fusion 
of several discontinuous anlagen which evidently are engendered 
by the attendant vein. Figures 16 to 25 inclusive, drawn from 
sections of a 6 mm. embryo, reveal in detail the character of such 
an anlage situated in the posterior half of the trunk, thus far 
removed from the anterior lymph heart. A comparison of these 
sketches with figure 15 will show that topographically this rudi- 
ment lies in the pathway ultimately occupied by the completed 
duct. It begins blindly and it ends blindly. It is relatively 
long, extending through forty-six sections. It is closely applied 
to the venous wall throughout by far the major part of its course. 
In figure 16 its anterior tip (/.) is indicated some distance dorsad 
of the intersegmental vein (7.v.), but seven sections distally the 
vein has approached the lymphatic (l., fig. 17) by bending up- 
ward and backward. After two additional sections the vessels 
are in contact (fig. 18), and it is impossible to discern a boundary 
line between their adjoining walls. During the remainder of 
its course the lymphatic anlage remains attached to the venous 
intima. Four sections back of the level represented in figure 18, 
it (1., fig. 19) appears on the side of the vein (7.v.) as a lump 
solidly packed with yolk spheres, and as such it continues for 
five or six sections. In figure 20 the lumen of the anlage ((.) 
is bisected by a broad protoplasmic partition containing a nucleus 
and a yolk corpuscle. In the ten following sections, one of which 
is illustrated in figure 21, a similar condition prevails. Passing 
over fourteen further sections, we meet with a sprout (fig. 22) 
which is given off dorsally by the lymphatic anlage (l.), but 


complex situated laterally are not straight uniform longitudinal channels through- 
out their course, since either one at times bends around the pronephric duct, 
fuses with the other on the opposite side for a short distance, then again becomes 
independent and resumes its former position. Thus the separation of the post- 
cardinal vein into a medial and a lateral division is a more or less arbitrary one, 
here instituted for the sake of convenience and clearness in the descriptions. 


ORIGIN OF LYMPHATICS IN BUFO 175 


strangely enough it squeezes between vein (7.v.) and myotome 
(m.s.). Three sections caudad an endothelial nucleus curiously 
protrudes into the lumen of the lymphatic (fig. 23). Seven 
sections beyond this level the anlage has become compressed and 
in transverse section appears as a crescent shaped cavity (l., 
fig. 24) clinging to the venous lining. Three additional sections 
bring us just beyond its end (fig. 25); in fact, it terminates at the 
large endothelial nucleus on the ventral venous wall. In this 
(fig. 25) and the following levels there is no indication of the lym- 
phatic anlage. Between it and the anterior lymph sac one 
hundred and sixty sections intervene, but in this interval two 
similar anlagen occur which, however, are of much shorter length. 
In another younger 6 mm. embryo the longitudinal venous 
channel of the same locality, in which the anlage just discussed 
has its being, pursues its way alone and without a dependent 
structure adhering to its walls, save for a distance of three sec- 
tions where a small lymphatic rudiment (/.) is intimately associ- 
ated with it (7.v.), as indicated in the sketch, figure 26. Its 
appearance brings to mind some of the initial rudiments of the 
cephalic lymph sinus, for instance, one (1.3) shown in figure 9. 
In the foregoing description of two genetic stages of the supe- 
rior lateral lymph duct no mention was made of the mesenchyme. 
It requires but cursory notice, for in the youngest toad larvae 
it is extremely sparse between trunk myotomes and epidermis; 
only after the larvae have attained the length of 8 mm. does the 
territory between these two structures expand and the mesen- 
chyme invade it more plentifully. On the other hand, in the 
area in which the formation of the inferior lateral lymph duct 
takes place, that is lateral to the Wolffian duct and the post- 
cardinal, the mesenchyme is considerable even in 6 and 7 mm. 
embryos. Moreover, it loses its yolk content much later than 
does that in the head; yolk bodies may be found in its cells long 
after the rudiments of the lymphatic duct had their inception. 
Thus it is evident that, though the trunk mesenchyme pro- 
portionately contains less yolk than do the endothelia, the pres- 
ence or absence of this substance can not strictly be used as a 
differential character in the development of the lymph duct 


176 OTTO FREDERIC KAMPMEIER 


rudiments in the trunk region. It is clear also that were the 
cephalic mesenchyme more amply furnished with yolk from the 
start and would lose it more tardily, the discovery of the funda- 
mental moment in lymphatic development in toad embryos 
would have been by far more difficult and tedious and the evidence 
less striking. Yet the judicious reader cannot contend for the 
direct mesenchymal origin of the lateral lymph ducts of the 
trunk after he has carefully inspected the figures illustrating their 
anlagen; nor can he, though this remark be irrelevant at this 
’ point, conscientiously claim for them continuity in development. 

The genesis of the inferior lateral lymph duct accompanying 
the postcardinal vein (cf. fig. 15) evinces the actuality of dis- 
continuity in a large developing lymphatic vessel probably more 
forcibly than does the genesis of the two vessels already considered. 
Figure 27 was drawn from a section of a 6 mm. embryo through 
the posterior portion of the anterior lymph heart. From the 
ventro-lateral aspect of the heart (U.h.) a vessel (l.), the foremost 
anlage of the lymph duct, is given off which at first turns down- 
ward between pronephric duct (w.) and epidermis (ep.) and then 
backward. After continuing in this direction for eight sections, 
it ends blindly, the termination being shown in figure 28 (l.). 
Between this and the anterior limit of the next succeeding anlage 
there is an interval of twenty-five sections in which lymphatic 
rudiments or anything resembling them are absent, at least are 
not in evidence. The territory between pronephric duct, lateral 
posteardinal division and epidermis is in possession of apparently 
only mesenchymal cells as pictured in the sketched section, 
figure 29, which typifies the character of this region. After such 
an interval the blunt tip of a discrete endothelial-lined space 
suddenly springs into view (l., fig. 30). The next section (fig. 31) 
reveals plainly other salient features of this rudiment (/.). It 
composes a well-defined and closed cavity, the confines of which 
are strong and firm, and like those of the vein (p./.) contain many 
yolk globules; in brief, there is no feature, except perhaps its 
large rotund nuclei, which could cause it to be confused with the 
mesenchyme. But the nuclei do not offer a serious hazard in 
the matter, for we have seen that the nuclei of the initial lymph 


ORIGIN OF LYMPHATICS IN BUFO 177 


sinus anlagen, as well, resemble mesenchymal nuclei indistin- 
guishably. What lends interest to the case is that the anlage is 
exceedingly short, being limited to six sections, though its caliber 
is proportionately broad. It ends as suddenly as it begins 
(compare figs. 32 and 33 with 30). In its abrupt course it lies 
adjacent to the vein (p.l.), and at its posterior end (J., figs. 32 
and 33) the two vessels are in contact but their lumina do not 
communicate. Proceeding distally from this level, nothing is 
encountered which might suggest a lymphatic anlage until 
twenty-eight sections have been passed over, where another 
similar endothelial-lined space exists in a similar position. 

Figures 34 and 35 illustrate a rudiment of the inferior lateral 
lymph duct on the right side of another 6 mm. embryo. More 
than fifty sections intervene between the anterior lymph heart 
and the blind anlage (/.) pictured here. In all notable qualities 
it is like the anlage described and figured last, except that it is 
longer, extending through eighteen sections, and is larger in 
circumference (fig. 34). Near its posterior limit it becomes 
much compressed and flattened out against the venous intima 
to which it is apparently firmly adherent (/., fig. 35). The 
terminal portion, however, is not attached to the vein (p./.) 
but lies slightly removed in the mesenchyme. 

The discontinuous anlagen described above are not isolated 
cases; several such anlagen may be found in all 6 and 7 mm. 
toad larvae, which are the critical genetic stages of the lateral 
lymph ducts. In older 7 mm. larvae these discrete lymph 
vessel rudiments have elongated and by further increment and 
by coalescence are creating continuous channels. Yet it is ex- 
ceedingly interesting to note that the tips or ends of consecutive 
anlagen do not always strike each other squarely; the writer 
has frequently observed the posterior tip of one and the anterior 
of another a considerable distance apart in the same section. In 
other words, the anlagen in their growth and elongation had 
shoved past one another without immediately meeting. Eventu- 
ally they become confluent by the gemmation of lateral sprouts 
or the dilatation of their lumina. This doubtlessly explains the 
irregularity and sinuosity of their course at an early period after 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 2 


178 OTTO FREDERIC KAMPMEIER 


the establishment of continuity. The writer would show figures 
portraying these phases, but the number of illustrations already 
far exceeds his intentions; moreover, such conditions are con- 
cerned more especially with their later development and will 
receive consideration in the subsequent contribution. At that 
time the profuse plexus or network will also be considered which 
in 8 and 9 mm. embryos is developed between the two lateral 
lymph ducts and from which ultimately the definitive lateral 
subcutaneous lymph vessels of the trunk are, so to speak, 
crystallized. 

The history of science, in fact the history of lymphatic re- 
search alone, has so often shown the fallacy of theorizing from 
insufficient or problematical data and premises, that the writer 
feels little inclination to base upon a simple finding an hypothesis 
that shall attempt to harmonize or properly valuate the work 
of other investigators at variance with his own. Only after 
observation has been corroborated repeatedly or from several 
viewpoints and the demonstration of fact is final can a law be 
formulated which is sound, comprehensive and stripped of all 
opinions and prejudices. Suggestions thrown out, however, to 
give direction to inquiry are ever seasonable and little hesitation 
is felt in expressing poignant ones. The purpose, then, of the 
theoretical considerations in the following paragraphs is neither 
to defend nor to refute any one view of lymphatic development; 
nor is it the aim to effect a compromise between conflicting views 
or to promulgate a new one. Consistent with the conditions in 
Bufo, such considerations are offered as plausible possibilities, 
the truth or error of which subsequent researches on other verte- 
brate embryos will determine. 

If the interpretation of the structures described in this treatise 
is the only possible one as the author believes it to be; if the 
lymphatic system of Amphibia is homologous to that of other 
vertebrate animals as we expect it to be; and if the morphological 
dogma; like structure, like origin, is infallible, as all biologists 
tacitly assume it to be, then the view of the direct mesenchymal 
origin of lymphatics seems to be untenable But not only is this 
theory affected; the opposing one also must certainly be radically 


ORIGIN OF LYMPHATICS IN BUFO 179 


modified. If we are to have agreement in our accepted belief 
of the mode of lymphatic genesis, the two chief antagonistic 
schools of investigators are compelled to re-examine their material 
with a new unbiased attitude, both those who maintain that 
lymph ducts are formed in situ from mesenchyme and discontinu- 
ously, and those who contend for the view of the venous origin 
of such vessels, of their continuity in development and of their 
centrifugal growth and spreading from a few definite foci in the 
body. The idea that strikes the writer primarily is that most 
workers have been deceived as to the time when lymphatic anlagen 
first make their appearance; it is conceivable how structures 
which have been described as the incipient anlagen of a lymph 
duct may already represent a much later phase. The char- 
acteristics which the lymphatic rudiments, discussed in this 
paper, manifest, such as solidity or imperfect vacuolation and 
discontinuity, though venous in origin, irrefutably show that the 
injection method would have been utterly incapable of revealing 
them at this time. Might not the same contention apply to 
the study of lymphatics in other vertebrate embryos, and might 
not anlagen exist long before injections could attest their existence. 
Is most of the other work on lymphatic development exempt 
from similar criticism? In their suppositions investigators have 
been more or less led astray by appearances, and in no other 
field of inquiry perhaps do appearances intimate so little of the 
truth. In a previous paper on the development of the thoracic 
duct in the pig embryo, the writer has described and figured very 
definite spaces which lie in the vicinity of the cardinal veins or 
their tributaries and in the path of the future definitive duct, 
and which he believed to be lined by cells mesenchymal in de- 
rivation. This conclusion was reached because the intimal 
cells of the thoracic duct anlagen at their inception visibly re- 
sembled the embryonic connective tissue elements very closely 
in certain seemingly important qualities. In finding that the 
character which marks incipient endothelium from mesenchyme 
particularly in the head region of young toad larvae is its abund- 
ance of yolk while other cell attributes appear identical, the 
present inquiry has convinced him of other potent possibilities. 


180 OTTO FREDERIC KAMPMEIER 


Are the discontinuous thoracic duct rudiments, like those of the 
ventral cephalic sinus in Amphibia, derived from endothelial 
proliferations which have severed their connections with the 
parent veins and have acquired lumina, subsequently to meet 
and become confluent with one another to create a continuous 
channel? Is the relation of these isolated cavities to some of the 
confused group of Mayer-Lewis anlagen a much more intimate 
one than has been supposed by most investigators? 

Recently the discussion has centered largely around an in- 
jected and sectioned pig embryo, series no. 23a, of the Johns 
Hopkins University Embryological Collection. Professor Sabin 
originally held’ that the developing thoracic duct in this specimen 
was completely filled with the injecta. Some time later the writer 
was given the privilege of examining and describing this particular 
embryo. He pointed out’ the existence of a long, blind, dilated 
space which follows closely upon the injected portion of the duct 
and is sharply demarcated from the neighboring veins as well as 
from the indefinite connective tissue interstices, a contrast 
admirably brought out in the photographs published at that time. 
Sabin now argues’ that the injection in this case was not a per- 
fect one. She accepts the space as a part of the thoracic duct 
anlage, but assumes, firstly, that it is united with the injected 
vessel by a very frail connection, which, if at all possible, must be 
extremely difficult to distinguish from the surrounding tissue 
reticulum, and secondly, that the pressure of the injection was 
entirely inadequate to force the fluid through the narrow passage. 
Leaving aside a consideration of the contradictory evidence which 
has been explicitly expressed in previous papers,!® the writer 
would ask Professor Sabin, whether her explanation of discon- 
tinuities in an inchoate lymphatic duct is the most likely one, in 
view of the observed conditions in Amphibia. The view of the 


7 In a report before the American Association of Anatomists, Ithaca, 1910. 

8’ Kampmeier; Anat. Rec., vol. 6, no. 5, June, 1912. 

* Sabin; Anat. Rec., vol. 6, no. 7, August, 1912, and Johns Hopkins Hospital 
Reports, new series, no. 5. 

° Kampmeier; Am. Jour. Anat., vol. 18, no. 4, September, 1912, and Anat 
Rec., vol. 6, no, 5. 1912. 


ORIGIN OF LYMPHATICS IN BUFO 1IS1 


origin of lymphatic channels from a few very definite centers in 
the organism has been too dogmatically asserted, and those 
investigators who have strongly adhered to it now find it difficult 
to consider exceptions, which, there is a probability, may prove 
the rule. In an analysis of the researches on the genesis of the 
lympathics we do not find a single really valid objection, based 
on correct interpretation of observations, to discredit the view 
of the origin of a lymph duct from a number of points, that is, 
from mutliple anlagen, which in toad embryos are _ proliferated 
from the intima of the vein which the definitive duct accompanies. 
The present findings, in Bufo, then, will permit of a partial 
acceptance of both the ‘‘centrifugal growth theory” and the 
“discontinuous, im situ mesenchymal origin” of lymph ducts. 
That the lymphatic endothelium, here considered, arises from 
venous endothelium has been shown beyond the shadow of a 
doubt, and the writer consistently, though tentatively, abandons 
the hypothesis of its derivation in other vertebrates from mesen- 
chymal cells, unless their lympathic system is shown to behave 
differently, which is scarcely conceivable. But the other tenets 
of the ‘outgrowth theory,’ such as continuity and centralization 
in lymphatic development, he can not accept, for the present 
observations reinforce strongly the diametrically opposite doc- 
trines of discontinuity and multiple origin. The ventral cephalic 
Iyvmph sinus is a product of the walls of the external jugular 
veins; its anlagen arise not at one point but individually at 
intervals along the entire extent of the cranial division of these 
veins. Even after the lymph sinus has become a single chamber 
by the coalescence of its rudiments, it remains as a blind reservoir 
until a relatively late stage when it joins the lymph hearts. The 
lateral lymphatic ducts of the trunk, too, do not spring from one 
center but are formed and acquire continuity by the fusion of a 
number of anlagen, which originate along the posteardinals and 
their dorsal tributaries, the intersegmental veins. To satisfy 
the ‘outgrowth theory,’ the lymphatic vessels mentioned, the 
sinus and at least the anterior half of the lateral ducts, should 
develop as continuous growths from the anterior lymph hearts, 


182 OTTO FREDERIC KAMPMEIER 


which correspond, as the writer will show in the later paper, to 
the two anterior centers, the jugular lymph sacs, in Mammalia. 

Finally, a certain peculiarity in the development of lymphatic 
channels in Bufo has suggested a possible homology between the 
incipient anlagen here observed and _ peri-venous lymphatic 
spaces first discovered and described by Huntington and McClure 
in cat embryos." Opponents of this view of lymphatic formation 
maintain that such spaces are artificial, due to shrinkage, though 
adequate proof to uphold this contention is not forthcoming. 
As has been shown in the figures, there occur in certain stages of 
the toad larvae hollow lymphatic anlagen which hug the vein 
closely and consequently resemble extra-intimal spaces, but which 
have been shown to be really intra-intimal in nature since their 
lumen arises as a vacuolation of the endothelial proliferation. 
Might not a similar condition be found to prevail in mammalian 
embryos were the genesis of extra-intimal spaces followed back 
far enough? 

In the writer’s judgment, it is only by carrying out extensive 
and minute cytological studies in all classes of vertebrate embryos, 
to determine specific cell character and behavior of both mesen- 
chyme and endothelium during the very early genetic stages, 
that we can arrive most surely and quickly at a uniform and com- 
prehensive conception of lymphatic development and can mea- 
sure the amount of truth and the number of fallacies inherent 
in the several respective theories hitherto advanced. This may 
prove to be an arduous task inasmuch as a natural and peculiar 
diagnostic trait, like that of yolk content, to distinguish incipient 
lymphatic intima from mesenchyme, probably does not exist in 
embryos other than Amphibian, but by the invention and appli- 
cation of different staining methods the results would perhaps 
be quite as surprising. 


1 Huntington and McClure; Am. Jour. Anat., vol. 6, 1907, Anat. Rec., no. 3. 


ORIGIN OF LYMPHATICS IN BUFO 183 
APPENDIX 


At the time when the foregoing observations had already been 
made, a reprint of a preliminary report by 8. Fedorowiez on the 
development of the lymph vessels in the larvae of Anura fell into 
the writer’s hands. (Untersuchungen tber die Entwicklung der 
Lymphgefisse bei Anurenlarven. Vorliufige Mitteilung. Ex- 
trait du Bulletin de |’Academie des Sciences de Cracovie, June, 
1918. SS. Fedorowiez). Although his observations pertain to the 
development of the posterior lymph hearts in Bufo, he expresses 
a view essentially similar to that advanced in this treatise. The 
fact that two investigators have arrived at the same conclusions 
independently of each other gives additional weight to their 
work. Since Fedorowicz has the priority, the writer gladly 
submits this contribution in corroboration of his work. 


PLATES 


All of the sections from which the following illustrations were made are stained 
with hematoxylin and orange G and are six microns in thickness. Figures 2 to 9, 
11, and 16 to 26 inclusive are camera lucida sketches drawn with a 2 mm. oil immer- 
sion objective and a no. 8 ocular. Figures 1, 10, 12 to 15, and 27 to 35 were pro- 
duced with the aid of the Edinger Projection Apparatus. All of the figures were 
drawn as nearly as was possible at one plane of focus or optical section. 


PLATH 1 
EXPLANATION OF FIGURE 


1 Transverse section of a 11 mm. toad embryo through the head at the level 
of the eye. Series 35, slide 1, row 9, section 5. > 150. Reduced to X 75. 


REFERENCES 


l., ventral cephalic lymph sinus j.d., j.8., right and left external jugular 
1.l., U.l., lateral extensions or wings of veins 
the sinus which are joined to it fur- — m.c., mouth cavity 


ther forward 


184 


ORIGIN OF LYMPHATICS IN BUFO PLATE 1 
OTTO FREDERIC KAMPMEIER 


Sees Oe 


iy nets 


S 


Veiuts 


1 es 


< 


zs 


es 


RG Das ~<A 


bales 


Car a) 
“2 


PLATH 2 
EXPLANATION OF FIGURES 


2to06 Five transverse sections of a5 mm. toad embryo through a small portion 
of the ventral region of the head to show the initial anlagen of the lymph sinus. 
Series 25. > 1100. 


REFERENCES 


l., l., sinus anlagen t.j7., jugular tributary (see footnote 5, 
j.d., j.s., right and left external jugular page 168) 
veins b.c., blood corpuscle 


2and3 Successive sections from the left side. Slide 1, row 4, sections 19 and 
20. 

4 From the same side, three sections caudad. Slide 1, row 4, section 25. 
Compare the lymphatic rudiment (1) with the mesenchyme in regard to yolk, 
and note its solidity. 

5 and 6 Two alternate sections from the right side. Slide 1, row 4, sections 
18 and 20. Observe the cavity of the lymphatic (1) in the second sketch. 


186 


ORIGIN OF LYMPHATICS IN BUFO PLATE 2 
OTTO FREDERIC KAMPMEIER 


187 


PLATE 3 
EXPLANATION OF FIGURES 


7 Transverse section of a 6 mm. toad embryo through the sinistro-ventral 
region of the head. Series 54, slide 1, row 4, section 8. X 1100. 
REFERENCES 
/., sinus anlagen 
j.s., left external jugular vein 


Sand9 Transverse sections of another 6 mm. toad embryo through the dextro- 
ventral region of the head. Series 53, slide 1, row 5, section 13, and row 6, section 
4.- 1100. Eight sections intervene between these two levels. 


REFERENCES 


(1.1, 1.2, 1.3, sinus anlagen 
j.d., right external jugular vein 
t.j7., wall of a tributary cut tangentially 


ORIGIN OF LYMPHATICS IN BUFO PLATE 3 
OTTO FREDERIC KAMPMEIER 


PLATE 4 


EXPLANATION OF FIGURES 


10 Transverse section of a 7 mm. toad embryo through the ventral region of 
the head. Series 52, slide 1, row 4, section 5. 300. 

11 A small area near the lower left corner of the same section (fig. 10). 
x 1100. 


REFERENCES 


j.d., j.s., right and left external jugular —_(l., /., simus anlagen 
veins m.c., mouth cavity 


190 


4 


PLATE 


ORIGIN OF LYMPHATICS IN BUFO 


OTTO FREDERIC KAMPMEIER 


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191 


PLATE 5 
EXPLANATION OF FIGURES 


12 Transverse section of an 8 mm. toad embryo through a little more than the 
right half of the territory ventral to the mouth cavity (cf. fig. 1). Series 27, slide 
1, row 5, section 8. > 500. 


REFERENCES 


l. to l., plexiform anlage of sinus e.c., right external carotid artery 
j.d., right external jugular vein m.a., muscle anlagen 


13 Transverse section of a 9 mm. toad embryo through the ventral region of 
the head. Series 2, slide 1, row 8, section 5. X 300. 


REFERENCES 
l., multilocular anlage of sinus j.d., j.8., right and left external jugular 
t., s., tissue strands and trabeculae, veins 


remnants of walls between originally — m.c., mouth cavity 
independent channels 


192 


ORIGIN OF LYMPHATICS IN BUFO PLATE 53 
OTTO FREDERIC KAMPMEIER 


2. ti or ae tee 
Zia Gee a. 0 —e 
<A ey : Sp 982 
~ i Si <0: is gis 
SIC 


Vis. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 2 


PLATE 6 
EXPLANATION OF FIGURE 


14 A small portion of the section shown in figure 13. > 1100. 


REFERENCES 
/., lumen of sinus d., cellular débris, probably vestiges 
t., s., tissue spurs and trabeculae of former partitions 
m., mesenchymal cell 


b 


194 


ORIGIN OF LYMPHATICS IN BUFO 
OTTO FREDERIC KAMPMUIER 


195 


PLATE 6 


14 


PLATE 7 
EXPLANATION OF FIGURES 


15 Transverse section of a 9 mm. toad embryo through the middle of the 
trunk. Series 2, slide 3, row 7, section 2. X 150. 


REFERENCES 


l.s., l.d., superior and inferior lateral  p.m., p.l., medial and lateral divisions 


lymphatic ducts of the postcardinal vein 
i.v., intersegmental vein w., Wolffian or pronephric duct 


m.s., mauscle segment 


16 Asmall area of a section of a7 mm. toad embryo through the left mid trunk 
region. Series 52, slide 3, row 4, section 16. X 1100. 


REFERENCES 


l., anterior tip of a blind anlage of the 7.v., intersegmental vein 
developing superior lateral lymph — ep., epidermis 
duct m.s., myotome 


PLATE 7 


ORIGIN OF LYMPHATICS IN BUFO 


OTTO FREDERIC KAMPMEIER 


15 


197 


PLATE 8 
EXPLANATION OF FIGURES 


17, 18, 19, 20, 21 and 22 Transverse sections to show different levels of the 
lymphatic anlage the tip of which is pictured in figure 16. Series 52, slide 3, row 4, 
.section 24; row 5, sections 2, 6, 9, 11; row 6, section 1. X 1100. 


REFERENCES 


l., anlage of the superior lateral lymph  7.v., intersegmental vein 
duct ep., epidermis 
m.s., myotome 


198 


ORIGIN OF LYMPHATICS IN BUFO . PLATE 8 
OTTO FREDERIC KAMPMEIDR 


199 


PLATE 9 
EXPLANATION OF FIGURES 


23, 24 and 25. Transverse sections to show three additional levels of the lym- 
phatic rudiment illustrated in the sketches on plate 8. Series 52, slide 3, row 
6, sections 4, 11, 18. X 1100. 

26 Transverse section of a 6 mm. toad embryo through approximately the 
same region (cf. figs. 17-25) to show an earlier condition, evidently, of the superior 
lateral lymph duct. Series 53, slide 4, row 5, section 17. X 1100. 


REFERENCES 


l., anlage of superior lateral lymph duct — ep., epidermis 
7.v., intersegmental vein m.s., muscle segment 


27 Transverse section of a6 mm. toad embryo at the level of the left anterior 
lymph heart. Series 53, slide 3, row 3, section 138. X 1100. 


REFERENCES 


l.h., posterior portion of the anterior p.m., p.l., medial and lateral divisions 


lymph heart of the postcardinal 
l., anterior extent of the developing  b.c., blood corpuscles 

inferior lateral lymph duct c.c., coelomic cavity 
7.v., intersegmental vein ep., epidermis 


m.s., muscle segment 


200 


9 


4 


PLATE 


ORIGIN OF LYMPHATICS IN BUFO 


OTTO FREDERIC KAMPMEIER 


201 


PLATE 10 
EXPLANATION OF FIGURES 


28 Transverse section a short distance behind the left lymph heart indicated 
in figure 27. Series 53, slide 3, row 4, section 3. X 1100. 


REFERENCES 


l., extreme posterior tip of the anterior p.a., dorsal anastomosis connecting 


anlage of the inferior lateral lymph the two postcardinal divisions 
duct, shown in figure 27 as a ventral b.c., blood corpuscles 
sprout of the lymph heart ep., epidermis 


p.l., lateral division of the postcardinal —w., pronephric duct 
REFERENCES 


29 HKighteen sections caudad of the level represented by figure 28. X 1100. 
Series 53, slide 3, row 5, section 4. X 1100. 


m., mesenchymal cells; no indication p.a., ventral postcardinal anastomosis; 
of a lymphatic anlage other explanations the same as above 


202 


ORIGIN OF LYMPHATICS IN BUFO PLATE 10 
OTTO FREDERIC KAMPMEIER 


PLATE 11 


EXPLANATION OF FIGURES 


30 and 31 Successive sections after an interval of nine sections behind that 
pictured in figure 29. Series 53, slide 3, row 5, section 12. X 1100. 


REFERENCES 


/., discontinuous anlage of the inferior p.a., dorsal anastomosis between post- 


lateral lymph duct; figure 30 shows cardinal divisions 
the anterior tip w., pronephrie duct 
p-m., p.l., medial and lateral post-  ep., epidermis 


-ardinal divisions 


204 


ORIGIN OF LYMPHATICS IN BUFO PLATE 11 
OTTO FREDERIC KAMPMEIER 


205 


PLATE 12 


EXPLANATION OF FIGURES 


32 and 33 Consecutive sections after an interval of four sections posterior to 
those represented on plate 12. Series 53, slide 3, row 5, section 16; row 6, section 1. 
1100: 


REFERENCES 


l., discontinuous anlage of the inferior w., pronephric duct 

lateral lymph duct. Figure 33 shows — ep., epidermis 

the extreme posterior tip p.a., dorsal anastomosis between post- 
p.l., lateral posteardinal division cardinal divisions 
c.c., coelomic cavity 


206 


ORIGIN OF LYMPHATICS IN BUFO PLATE 12 
OTTO FREDERIC KAMPMEIER 


PLATE 13 
EXPLANATION OF FIGURES 


34and 35 Transverse sections of another 6 mm. toad embryo through the right 
side of the mid trunk region. Series 54, slide 3, row 3, sections 4and10. X 1100. 


REFERENCES 


l., discontinuous anlage of the right  p.a., dorsal postcardinal anastomosis 
inferior lateral lymph duct. The  b.c., blood cells 
last figure shows the anlage clinging 
to the venous wall 

p.m., p.l., medial and lateral post- 
cardinal divisions 


ep., epidermis 
w., pronephric duct 


ORIGIN OF LYMPHATICS IN BUFO PLATE 13 
OTTO FREDERIC KAMPMEIER 


209 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 2 


AS STAI SLiCAte SUD Y Ob THE THORAGKE 
DUCH IN MAN! 


HENRY K. DAVIS 


From the Anatomical Laboratory of the Cornell University Medical College, 
Ithaca, New York 


THIRTY-TWO-FIGURES 
INTRODUCTION 


It has long been known that the thoracic duct in man presents 
a certain amount of variation and in addition to the usually 
deseribed ducts various marked anomalous conditions have 
been noted. This investigation was undertaken to determine the 
percentage of occurrence of the different variations of the thoracic 
duct. An attempt has also been made to explain these vari- 
ations from an embryological standpoint. The various types 
of duct that might develop from the primitive embryological 
network have been indicated and the ducts here described to- 
gether with those described by other investigators have been 
divided into corresponding groups. 


MATERIAL AND METHODS 


This paper is based upon the records of the dissection of 22 
cadavers in the Anatomical Laboratory of the Cornell Univer- 
sity Medical College, Ithaca, New York. Forty-two cadavers, 
on which autopsies had been performed, were examined, but 
many of them had to be discarded on account of injury to the 
duct at the post mortem. In 11 of these however, the duct 
was found complete and records were taken of the ducts in these 
bodies. The other 11 records were taken from bodies which 
were dissected by the medical students. During the course of 


‘From a thesis presented to the faculty of the Graduate School of Cornell 
University for the degree of Master of Arts, June, 1914. 


211 


le, HENRY K. DAVIS 


the dissection of the abdomen, thorax, and base of the neck, I 
supervised the students’ work so that no injury would occur 
to the duct and made the dissection of the duct myself. 

The thoracic duct in each case was injected with a carmine 
gelatin mass (Lee ’05). At first, I attempted to make the in- 
jection from the cephalic portion of the duct but could not make 
the injection mass flow caudad on account of the valves. By 
experimenting, I found that by exposing the duct Just cephalic 
to where it pierces the diaphragm and making the injection 
from this point, that the injecting mass flowed freely caudad as 
well as cephalad. This seems to indicate that the valves are 
much more efficient in the cephalic than in the caudal portion 
of the duct. Before the injection, the innominate, vertebral, 
subclavian, and internal jugular veins were clamped. This 
insured a good filling of the duct. After the injecting mass had 
been allowed to cool and gelatinize, a careful dissection and a 
natural sized drawing of the duet was made. 


EMBRYOLOGY 


According to Sabin (’09) the thoracic duct in human embryos 
begins in the abdominal cavity at the cisterna chyli as two ducts. 
These pass cephalad through the thorax, the right duct crossing, 
at about the level of the 4th thoracic vertebra, dorsal to the 
aorta to join its fellow of the opposite side. There is thus formed 
on the left side a single trunk that connects with the jugular 
portion of the thoracic duct. The jugular portion of the thoracic 
duct is a caudal outgrowth of the jugular lymph sac on the left 
side. Sabin, however, has not been able to find the jugular 
portion, that is, the caudal outgrowth of the right jugular lymph 
sac, connecting with the thoracic duct. She was able to trace 
it to the root of the lung but could find no connection of it with 
the thoracic duct in this region. For this reason the duct as 
described by Sabin is not a complete bilaterally symmetrical 
duct. In the embryo there is a distinct right and left duct. 
These two ducts are connected by numerous cross anastomoses 
thus forming a plexus of lymphatic vessels along the course 


THORACIC DUCT IN MAN 2S 


of the aorta. Sabin (09) reports the first appearance of the 
cisterna chyli in 23 mm. human embryos. Here it is a definite 
sac opposite the 3rd and 4th lumbar vertebrae. The thoracic 
duct is first found in human embryos of 24 mm. In human 
embryos of 30 mm. the thoracic duct is complete. 

Sabin (02) in her study of the thoracic duct in pig embryos 
found it to be essentially the same as she found in human embryos. 

Lewis (06) found the thoracic duct in the rabbit embryo to 
be practically the same as Sabin found in the human and pig 
embryos. 

Huntington (11) in his study of the lymphatic vessels of the cat 
states that the thoracic duct is ‘“‘potentially bilaterally symmetri- 
cal’ and he pictures a bilaterally symmetrical duct in figure 29, 
plate 22. 

It is interesting also to note that Sala (99-00) and Pensa 
(O8—’09) picture bilaterally symmetrical thoracic ducts in birds 


DIVISION OF THE THORACIC DUCTS INTO GROUPS 


Assuming that the embryonic thoracic duct is bilaterally 
symmetrical and that the duct in the adult is produced by the 
persistence and growth of a part of the embryonic duct and the 
disappearance of other parts, one might expect to find variations 
in the adult thoracic duct. These variations depend then upon 
which portions of the embryonic thoracic ducts atrophy and dis- 
appear and which continue to develop. These possible varieties 
of the thoracic duct may be divided into the following types. 

Type 1. To this type of thoracic duct belong those ducts 
which would retain more or less the early embryological conditions 
and would consist of a completely bilaterally symmetrical duct 
connected by numerous cross anastomoses (fig. 1). 

Type 2. In this type of thoracie duct we would have caudad 
the persistence of the original double thoracic duct of the embryo. 
There would be a right and left duct, which starting in the ab- 
dominal cavity would pass cephalad through the thorax and at 
about the level of the 4th thoracic vertebra, the right duct would 
cross by persistence of one of the embryonic cross anastomosing 


214 HENRY K. DAVIS 


branches to join the duct of the left side. A single trunk would 
be thus formed which would empty into the venous system of 
the left side. The cephalic portion of the right duct would fail 
to connect with the thoracic duct and would remain as the right 
lymphatic duct (fig. 2). 

Type 3. In this type of thoracic duct we would have caudad 
the persistence of the original double thoracic duct of the embryo. 
There would be a right and a left duct, which starting in the 
abdominal cavity, would pass cephalad through the thorax and 
the left duet would cross by persistence of one of the embryonic 
cross anastomosing branches to join the duct of the right side. 
A single trunk would thus be formed which would empty into 
the venous system of the right side. The cephalic portion of the 
left duct would fail to connect with the thoracic duct and would 
remain as a left lymphatic duct which would be comparable to 
the usual right lymphatic duct (fig. 3). 

Type 4. In this type of thoracic duct, we would have cephalad 
the persistence of the original double thoracic duct of the embryo. 
There would be complete atrophy of the caudal portion of the 
left duet and the cephalic portion of the left duct would join 
the right duct through the persistence of one of the embryonic 
cross anastomosing branches (fig. 4). 

Type 5. In this type of thoracic duct, we would have cephalad 
the persistence of the original double thoracic duct of the embryo. 
The caudal portion of the right duct would be completely atro- 
phied and the cephalic portion of the right duct would join the 
left duct through the persistence of one of the embryonic cross 
anastomosing branches (fig. 5). 

Type 6. In this type of thoracic duct, we would have the 
persistence of the cephalic portion of the left duct and the caudal 
portion of the right duct. These two segments would be joined 
together by the persistence of one of the embryonic cross anas- 
tomosing branches. The caudal portion of the left duct would 
be completely atrophied and the cephalic portion of the right 
duct would persist as the right lymphatic duct (fig. 6). 

Type 7. In this type of thoracic duct, we would have the 
persistence of the cephalic portion of the right duct and the 


THORACIC 


Right Duct ( Left Duct Right Duct ||/) 


ON 


is 


C/ 
Si) 


Right Duct Left Duct 


Right Duct Left Duct 


DUCT IN MAN 


2|Left Duct 


Right Duct) | 


Fig. A Schematic representation of the embryonic lymph channels. 

Fig. 1 Type 1; schematic representation of the embryonic lymph channels 
which might persist. 

Fig. 2 Type 2 


which might persist. 


Fig. 3 Type 3; schematic representation of the embryonic lymph channels 


which might persist. 


Fig. 4 Type 4; schematic representation of the embryonic lymph channels 


which might persist. 


; schematic representation of the embryonic lymph channels 


DAG HENRY K. DAVIS 


caudal portion of the left duct. These two segments would 
be joined together through the persistence of one of the embryonic 
cross anastomosing branches. The caudal portion of the right 
duct would be completely atrophied and the cephalic portion 
of the left duet would not connect with the thoracic duct and it 
would persist as a left lymphatic duct (fig. 7). 

Type 8. In this type of thoracic duct, we would have the 
complete persistence of the right embryonic duct. The caudal 
portion of the left duct would be completely atrophied and the 
cephalic portion of the left duct would not be connected with the 
thoracic duct and would persist only as the left lymphatic duct 
which would be comparable to the usual right lymphatic duct 
(fig. 8). 

Type 9. In this type of thoracic duct, we would have the 
complete persistence of the left embryonic duct. The caudal 
portion of the right duct would be completely atrophied and the 
cephalic portion of the right duct would not connect with the 
thoracic duct and would persist only as the right lymphatic duct 
(fig. 9). 

It should be noted that Types 2 and 3, 4 and 5, 6 and 7, 8 and 
9 are respectively the reverse of one another in that those channels 
which persist in one atrophy in the other and vice versa. 


Group I 


Winslow (66), Cruickshank (790), Sémmering (’92), and 
Hommel (’37) deseribe bilaterally symmetrical thoracic ducts. 
The thoracic ducts start in the abdominal cavity as two ducts 
which pass cephalad through the thorax, one opening into the 
venous system of the left side and the other into the venous system 
of the right side. The right duct lies to the right of the aorta and 
the left duct on the left side of the aorta. These two ducts are 
joined together by numerous cross anastomoses. I found no 
ducts of this type. It is clearly evident that the thoracic ducts 
described by the above investigators belong to Type 1 (fig. 1). 


Vig 


which 


THORACIC 


\( MW Left Duct 


X 
i 
OS 
Ve A \(\| 
1. 5 C IN 
If) D 
2 5 Right Duct J K| 
y \V 
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LY VA 


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= 


aX 


Ss 


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Right Duct 


8 


.5 Type 5; schematic 
might persist. 


Fig.6 Type 6; schematic 
which might persist. 

Fig. 7 Type 7; schematic 
which might persist. 

Fig. 8 Type 8; schematic 
which might persist. 

Fig.9 Type 9; schematic 
which might persist. 


esa" 
} 


representation 
representation 
representation 
representation 


representation 


DUCT IN MAN 


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S 


of the embryoni 


of the embryonic 


of the embryonic 


g 


» lymph 
» lymph 


lymph 


of the embryonic lymph 


of the embryonic 


» lymph 


Left Duct 


Left Duct 


channels 


channels 


channels 


channels 


channels 


218 HENRY K. DAVIS 
Group II 


The thoracic ducts of this group (cases 1-6, figs. 10-15) begin 
in the abdominal cavity as two ducts which extend cephalad 
through the thorax. The right duct lies to the right of the 
aorta and the left duct to the left of the aorta. The right duct 
crosses in the thorax at the level of the 4th thoracic vertebra 
dorsal to the aorta to join the left duct forming a single trunk 
which empties into the venous system on the left side at the base 
of the neck. The two ducts are connected by cross anastomosing 
channels. This type of duct occurred in 6 cases out of 22, or 
in 27.27 per cent. This form of duct corresponds to the thoracic 
duet represented in Type 2. In proportion to the completeness 
of the persistence of embryonic conditions these ducts have been 
divided into three divisions, A, B and C. Lower (’80) and 
Nuhn (’49) describe a similar thoracic duct. 

Division A. The thoracic duct of this division (fig. 10) begins 
in the abdominal cavity as two ducts which pass cephalad through 
the thorax. The right duct at the upper level of the 5th thoracic 
vertebra begins to incline to the left and passing dorsal to the 
aorta reaches the left side and at the level of the lower third of 
the body of the 2nd thoracic vertebra joins the left duct forming 
a single trunk which continues cephalad to open into the left 
subclavian vein a short distance from its junction with the in- 
ternal jugular vein. The right duct lies to the right of the 
aorta and is situated between the aorta and the vena azygos 
major. The left duct lies to the left of the aorta. The two ducts 
are of equal calibre and are connected by numerous cross anas- 
tomoses. Three of these are especially well developed (1) a 
cephalic one which connects the cephalic end of the right duct 
with the left; (2) one which is situated opposite the body of the 
9th thoracic vertebra nearly transverse in direction; (3) and 
one starting in the abdominal cavity on the right side and passing 
cephalad and to the left to join the left duct opposite the body 
of the 11th thoracic vertebra. There is no cisterna chyli. It is 
represented in this case by a plexus of lymphatic vessels. This 
type of duct occurred in 1 case out of 22, or in 4.545 per cent. 


THORACIC DUCT IN MAN 219 


Internal Jugular 


Left Internal Jugular Vein ey Subclavian Internal 
~ —, 


fol Trunk Jugular Trunk~_ Subclavian 


Left Internal Jugular pe |) Left 


Vertebral 
Left\, 1st Thoracic Vertebra ZEN 


ak 


‘Subclavian 2a 
Z : Vein Left 7 eS 
C= Subclavian” —\\ ae 
i=» \ Left Vein FS pi int —s Thoracic 
Innominate tere Duct 
“~~ Vein 
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Vein 
Sys 
me 
ZO 
77 Thoracic 
WA 
a = Duct 
\Wa 
iS 
i) 3 laterals 
4 \ 
NS )Collaterals 
mir. 
TEs _ 


Intestinal 


Trunk SV Ya \ Ny 


4 | oe ‘ Lumbar beth 
Y mphatics 
Right Left ars el 
Lumbar 


Lumbar ANN 4 
Lymphatics sil S Lymphatics 
Mt == << $__— /f 
lO 


Fig. 10 Type 1; thoracic duct in a male white subject, age 35. Note the double 
duct and the abundant cross anastomoses. 

Fig. 11 Type 1; thoracic duct in a male white subject, age 51. Note the 
double duct and the reduction in size of the left duct. 


220 HENRY K. DAVIS 


Division B. The thoracic duct of this division (fig. 11) begins 
in the abdominal cavity as two ducts which pass cephalad through 
the thorax. The right duct at the upper level of the 5th thoracic 
vertebra begins to incline to the left and passing dorsal to the 
aorta reaches the left side and joins the left duct at the level of 
the lower third of the body of the 2nd thoracic vertebra forming 
a single trunk which passes cephalad. This trunk at the lower 
level of the 7th cervical vertebra divides into three branches which 
do not become united again before opening into the venous 
system. The most cephalic branch opens into the left subclavian 
vein a short distance distal to its junction with the left internal 
jugular vein. The intermediate branch opens into the angulus 
venosus formed by the junction of the left internal jugular and 
left subclavian veins and the most caudal branch opens into the 
left vertebral vein a short distance medial to its Junction with 
the left innominate vein. The right duct hes to the right of the 
aorta and is placed between the aorta and the vena azygos major. 
The left duct lies to the left of the aorta. The two ducts are of 
unequal size, the right duct being of much greater caliber than 
the left duct. The cross anastomosing channels in this case 
are not as numerous as in case | (fig. 10). The chief anastomosis 
is the cephalic one which joins the cephalic end of the right duct 
with the left duct. The reduction in caliber of the left duct 
and the decrease in the number of cross anastomoses point to a 
stage in the atrophy of the left duct. There is no cisterna chyli 
present. It is represented by a plexus of lymphatic vessels. 
This type of duct occurred in 1 case out of 22, or in 4.545 per 
cent (fig. 2). 

Division C. In cases 3 and 4 (figs. 12-13), in cases 5 and 
6 (figs. 14-15) there is a partial doubling of the caudal portion 
of the thoracic duct. In each case the caudal portion of the right 
duct is complete but the left duct has partially atrophied. 

In case 3 (fig. 12) the thoracic duct begins in the abdominal 
cavity as two ducts which pass cephalad into the thorax. The 
right duct lies to the right of the aorta and is placed between 
the aorta and vena azygos major. It begins to incline to the 
left opposite the inferior level of the body of the 4th thoracic 


Internal 


Left Internal Jugular Vein /Jugular Trunk 
, . Internal 
\Subclavian Jugular Trunk Subclavian 
WA Trunk Trunk ~ 
So Left Interna! Jugular Vein WN 
Rett e 
(7 Ait Cee J 
Ist Thoracic Vertebra a | WV. ia Us 
\YSubclavian Left a a ip 
Subclavian Ve 
(8 V3 Vein 
= ( “5 innominate leita < > 
eS » _S P Innominate: _ 
Lymph Glands Fi HG V 
ee Sax ein 
Ye Ze) S by, 
ge 


~— Thoracic 


Thoracic 
Duct 
ee 4 
Collaterals TO ser 
i = 
Lumbar [ Lumbar 


esa | 
Lymphatics ed 


\ Lymphatics 
(WH WS 
IN: 


vty 


Intestina 
Trunk 


eas 
Fig. 12 Type 1; thoracic duct in a male white subject, age 72. Note the 
incomplete duct of the left side. 
Fig. 13 Type 1; thoracic duct in a female white subject, age 69. Note the 
incomplete duct of the left side and the abundant cross anastomoses between it 
and the duct on the right side. 


y) 


9 9 1 


aa 


Dee, HENRY K. DAVIS 


vertebra and crossing dorsal to the aorta reaches the left side 
where it continues its course cephalad to open into the angulus 
venosus formed by the junction of the left internal jugular and 
left subclavian veins. The right duct lies to the right of the 
aorta. It passes up into the thorax from the abdominal cavity 
and ends at the lower level of the 7th thoracic vertebra. The 
portion cephalad of this has atrophied. The two ducts are 
joined together by numerous cross anastomoses. There is no 
cisterna chyli present. It is represented by a plexus of lymphatic 
vessels. 

In figures 13, 14 and 15, the right duct corresponds to the 
right duct described in case 3 (fig. 12) with the exception that it 
empties into the left subclavian vein instead of the angulus 
venosus. The left ducts are essentially the same as in case 3 
(fig. 12). In case 4 (fig. 13) there is no cisterna chyli but there 
is a lymphatic plexus. n case 5 (fig. 14) there are two cysternae 
chyli. The right duct is a direct continuation of the right cis- 
terna chyli. The left cisterna chyli is connected to both the 
right and left ducts. The left duct, however, is not a direct 
continuation from the left cisterna chyli. In case 6 (fig. 15) 
there is a single cisterna chyli. The right duct is a direct con- 
tinuation of this cisterna. The left duct is alsc connected with 
it. In this case there is a division of the right duct into two 
branches. This bifurcation takes place at the lower level of the 
body of the 6th thoracic vertebra and the two branches unite 
again to form a single trunk at the upper level of the body of 
the 5th thoracic vertebra. In these 4 cases the lymphatics on 
the left side of the aorta including the left duct, drain in a caudal 
direction. Lymph glands are associated with the thoracic duct 
in cases 3, 4 and 5 (figs. 12-14). A further account of these will 
be given in dealing with the variations. 

The arrangement of the two ducts in these 4 cases points 
to an originally double thoracic duct, as in case 2 (fig. 11). There 
is represented in these 4 cases another more advanced stage 
in the atrophy of the left duct. In case 2 there was a reduction 
in the size of the left duct, while in these 4 cases there is a reduction 
in size and a complete atrophy of a portion of the left duct. This 
type of duct occurred in 4 cases out of the 22, or in 18.18 per cent. 


THORACIC DUCT IN MAN Dae 


Internal 
Left Internal Jugular NA asesies Trunk internal 
3 poe Trunk Jugular Trunk Subeiavinniicanic 
Ist ie a Left Interna! Jugular Vein 
Left \ Ist Thoracic Vertebsa = alt 
Subclavian Left z ; 
“LR Aaa & vel Subclavian— 1 > 
3) ane Vein Sa ald 
; ii. Left ei { < 
Ad BZA Innominate Lett : ir | Nog 
P ras 7} i ( \\ \s 
Lymph = ‘ee : 1K ts Vein Innominateg, alll a If git: 
Glands “ Pitas } 2 Vein & i fy Ry) fi =o Z 
i| i} 
Wy = 
“Thoracic Duct 
Collateral 
Collaterals 
ollaterals 
$ nf : ly i volleterals 
1 eas Hi $ 
ia 
Cisterna {\/) Ef “l) \ | 
! i AN) 
BS Chyli >] 
sa OES [7 \\\ : 
Cisterna gekes y VN) = & 


hyli Nop 
Evil . Lymph ony 
7 f, 


Intestinal _ (4 Glands 


Trunk fk N V Cisterna Chyli Intestinal Lymph 
Cofi PoPe Trunk ¥ s/ Gland 
eyZ LIN 
oe CY x 
VA = A y) | 
Sh Sexes) f D Rignt 4. my i Left 
\ |-—/ Lumbar Trunk f 1} 3) 
Right aaa j 7 De eer Ds Gi ; i umbar Trunk 
Lumbar. i Yt NS Lumbar = ve 
Lymphatios Qf ie 


His toll i 


Fig. 14 Type 1; thoracic duct in male white subject, age 56. Note the two 
cisterna chyli and the incomplete duct on the left side. 

Fig. 15 Type 1; thoracic duct in a male white subject, age 67. Note the 
incomplete duct on the left side. 


224 HENRY K. DAVIS 


Group IIT 


Breschet (736) describes a thoracic duct as seen by Haller 
which was double in its caudal portion. The thoracic duct in 
this case start in the abdominal cavity as two ducts, which 
pass cephalad into the thorax, one lying on each side of the 
aorta. In the cephalic portion of the thorax, the left duct crosses 
over to the right side and the right and left ducts both open 
into the left angulus venosus. This form of duct belongs to 
Type 3 (fig. 3). I found no ducts of this type ainong my own 
eases and could find no other cases described in the literature. 


Group IV 


Butler (03), Lauth (35), Patruban (’44), Diemerbroeck (’85), 
Cousin (98), and Walther (described by Haller ’46), describe a 
thoracic duct which starts in the abdominal cavity as a single 
trunk and passing cephalad into the thorax on the right side of 
the aorta divides into two branches at about the level of the 4th 
thoracic vertebra. The right branch opens into the angulus 
venosus on the right side and the left branch passing dorsal to 
the aorta opens into the angulus venosus of the left side. The 
type of duct described by these investigators belongs clearly 
to Type 4 (fig. 4). I found no ducts of this type among my 
own cases. 


Group V 


The thoracic duct in this type starts in the abdominal cavity 
as a single duct and passing cephalad into the thorax on the left 
side of the aorta divides into two branches. The right branch 
opens into the right angulus venosus and the left branch into the 
left angulus venosus. I have been unable to find ducts of this 
type described in literature and there were none among my own 
cases. 


THORACIC DUCT IN MAN 225 
Group VI 


In 14 instances in my series (figs. 16-29) the thoracic duct 
begins in the abdominal cavity as a single trunk which passes 
cephalad into the thorax and at the level of the 5th to the 
3rd thoracic vertebra begins to incline to the left and finally 
passes to the left of the median line of the bodies of the thoracic 
vertebrae. The duct continues cephalad and at the level of the 
2nd thoracic to the 6th cervical vertebra changes its course 
passing cephalad, ventrad, and to the left, and then caudad and 
slightly ventrad to open into the venous system at the base of 
the neck. 

fneSisinstances (figs. 16, Li, -20i21 0 23,. 26, 27, 29), the 
thoracic duct begins to incline to the left opposite the body of 
the 5th thoracic vertebra; opposite the body of the 6th thoracic 
vertebra in 1 instance (fig. 28); opposite the body of the 4th 
thoracic vertebra in 4 instances (figs. 18, 19, 24, 25); and 
opposite the body of the 3rd thoracic vertebra in 1 instance 
(fig. 22). The terminal portion of the thoracic duct changes its 
course opposite the 2nd thoracic vertebra in 1 instance (fig. 20); 
opposite the 1st thoracic vertebra in 6 instances (figs. 16, 17, 
19, 21, 24, 26); opposite the 7th cervical vertebra in 6 instances 
(figs. 18, 23, 25, 27, 28, 29); and opposite the 6th cervical verte- 
bra in | instance (fig. 22). 

The mode of termination is somewhat variable. In 5 in- 
stances (figs. 16, 20, 22, 27, 28) the thoracic duct terminates 
by a single opening into the left subclavian vein; into the left 
subclavian vein by 2 branches in 1 instance (fig. 17); into 
the left angulus venosus by a single branch in 5 instances 
(figs. 18, 19, 21, 24, 25); inte the left angulus venosus by 2 branches 
in 1 instance (fig. 29;) into the left internal jugular by a single 
branch in 1 instance (fig. 26); and into the posterior wall of the 
left innominate vein by a single branch in 1 instance (fig. 23). 

The thoracic ducts begin in the abdominal cavity by the 
confluence of the lumbar lymphatics and sometimes the intestinal 
trunk or by a lymphatic plexus in which the lumbar trunks are 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 2 


226 HENRY K. DAVIS 


indistinct. Cases 14 and 18 represent such a plexus. In all 
the other cases of this type, the right and left lumbar trunks are 
distinct. The intestinal trunk joins the caudal extremity of 
the thoracic duct in 6 instances (figs. 16, 17, 19, 20, 24, 28); 
the right lumbar lymphatics in 3 instances (figs. 18, 25, 29); 
the left lumbar lymphatics in 4 instances (figs. 22, 28, 26, 27); 
and in both the right and left lumbar lymphatics in 1 instance 
(hie. 21. 

The caudal extremity of the thoracic duct in 9 instances 
(figs. 16, 17, 18, 20, 22, 24, 26, 28, 29) presents an ampulliform 
dilatation, the cisterna chyli. This is absent in 5 instances, 
and in its place in 4 instances (figs. 21, 23, 25, 27) there is a 
lymphatic plexus. In 1 instance (fig. 19), there is neither a 
cisterna chyli nor lymphatic plexus. 

In addition to the right and left lumber and intestinal branches, 
the thoracic duct may receive the following branches: (1) col- 
laterals which drain the intercostal spaces (present in all cases) ; 
(2) efferent vessels which drain the posterior mediastinal lymph 
nodes; (3) the left internal jugular trunk in all cases; and (4) 
the left subclavian trunk in eases 14,15 and 16. The collaterals 
mentioned above drain, as a rule, more than one intercostal 
space. There is not, however, a collateral for each intercostal 
space. The trunks draining the posterior mediastinal nodes 
had been destroyed in most of the cases. 

Lymph glands are associated with the thoracic duct in 11 
instances (figs. 16, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29). In 
9 instances (figs. 17, 18, 19, 21, 24, 25, 26, 28, 29) the duct 
divides into two branches which unite again after a short distance 
to form a single trunk. These were called ‘insulae’ by Haller 
(75). In case 20 (fig. 29) there is a bifurcation of the terminal 
portion of the duct and each branch presents an ampulliform 
dilatation, similar to a cisterna chyli. 

In the abdominal portion, the thoracic ducts of this group lie 
ventral to the bodies of the first 2 lumbar and 12th thoracic 
vertebrae and between the crura of the diaphragm or under cover 
of the right crus. Ventrad they are in relation with the right 
side of the abdominal aorta. 


‘THORACIC DUCT IN MAN 227 


ie ' Internal 
nterna 
: Jugular Trunk Jugular Trunk Subclavian Trunk 
Left Internal Jugular Vein eae seinstiscueie 
Ist va > UL ZSubclavian = i a -Insula 
Thoracic!” Se -— _ Trunk |st Thoracic Vertebra GE NST ‘a 
Vertebra=— Ny A Deft Left 4 = 
Jp \ Subelavian Subclavian— == 
ii \, = Vein Vein 


Saat « ‘Left 
“eau a») 5 Innominate 
Pa (EE eee Vein 
a in 


Collaterals 


Thoracic 


BN 


= Sd (ee: 
oy Poo ) 

Dit a a 

i ii 3= 


=e SN 
h Hy) 


MyM 3 Cisterna 
VAS Chyli 


AYN — 
Righ cy, NY ailaft 


Lymph 
Glands 


Yi 
1 Yip 


Left ~\ 

<P v i 
Innominate= Ee 
Vein 


= ay 
Heer Collaterals 


Collaterals ollaterals 


Wen) Joy) & = 
Thoracic Duct 


) Lumbar Trunk 


Fig. 16 Type 6; thoracic duct in a male white subject, age 62. Note the 
position of the cisterna chyli opposite the body of the 11th vertebra. 

Fig. 17 Type 6; thoracic duct in a male white subject, age 22. Note the insula 
at the terminal portion of the duct and the opening of the duct by two branches 


into the left subclavian vein. 


Internal 


puduler Trunk 
ie lar Vein. 
eft Internal Jugular Sin oa Snel Internal 
A Jugular Trunk 
a att Subclavian 


= pia __. Left Internal 
=v). a Subclavian aaa IE y, Trunk 
TG -Vein Ist Thoracic Vertebra\|'|’ hyp 
: \/) ¥ 4) ) Seay OS * we = a A ¢ 
NIN he Lett Left a LY, oxy = 
— OE 5 eee Innominate Subclavian Ce 
Dy Aa “S Vein Vein = == Thoracic 
2 > Du 
Thoracic Y= Left : 
Duct Innominatess 


Vein 


Kr 


) 


Collaterals a 


me 


ah We 
ue lies 
i 


\ yl: ~My 
Collaterals SY) ay 


Fig. 18 Type 6; thoracic duct in a female white subject, age 39. Note the 
insula at the terminal portion of the duct. 
Fig. 19 Type 6; thoracic duct in a female white subject, age 50. Note the 
caudal portion of the duct; there is no cisterna chyli or lymphatic plexus. 
228 


Internal 
Left Internal Jugular Vein 


om LING 


Ist \ =a (x ? ‘NN ya E—~ 
Thoracic y= . oy ip pel 
Vertebra — ASS Left 

> KS Subclavian 
—Ah7~—__ \ Vein 
—© \Left 
Thoracic 

Duct 
Lymph 
Glands 
Cisterna : 

Chyli S| == 

i nl — 
UIA \\ ea 7 Intestinal Trunk 
(= 

Right PAM. 16 NON Left 

Lumbar Trunk¥(- >) Lumbar Trunk 


iM 
iN 
») )) 


area 

" Dery 
ep ht 
‘ Uhh 


Mihi 2 Vi 


a] 


20 


terminal portion. 


Jugular Trunk 


= eli Subelavian Trunk Left Internal Jugular Vein— 


1st Thoracic Vertebra — ch er) 


Internal 


Jugular Trunk Subclavian Trunk 


CRRA i 


Subclavian > ve oh 
Vein RO | 

F : Ir=5 
Vein fos Va 


“Collaterals 


Thoracic — 
Duct: = 
= (Nine } 
Ee OM z 
ollaterals 
a 
Lymph 
Gland 
: ntestinal 
Trunk 
: \ 1 Ny os ~ 
Right: , i ul Left 


4) wih 
Lumbar Trunk<f— = =e) Lumbar Trunk 


ee 
TD i// 
i sf 


—_—— 


<= — i 
Ni ‘hi 


pi yn 3 


- Uh 


2| 


Fig. 20 Type 6; thoracic duct in a male white subject, age 57. Note the large 
lymph gland associated with the thoracic portion of the duct. 

Fig. 21 Type 6; thoracic duct in a female white subject, age 66. Note the 
plexiform arrangement of the caudal portion of the duct and the insula at its 


230 HENRY K. DAVIS 


Internal 
Jugular Trun 
: Subclavian. 
: Internal Left Internal Jugular Vein j 
Left Internal Jugular Vein “jy aca aiak <A V, Trunk 
\ elt Ist Thoracic Vertebray! i” Fe — 
ia ae | ays 
__\ Subélavian Trunk Left 3 
SN Ne Subclavia 7 fs 
am =Ist\Thoracic Vertebra \,_. Thoracic 
B® oN “yi os fee Vein &e Duct 
‘ io = as x e 
cer ENE aE ail = ,\ Subclavian Left 
SEs i: Vein Innominate? 
4 Re Ne w/o NS Vain 
SP XZ Ly y S Left 
\ Innominate 
Vein 
lhl t> Collateral 
TTY -—> Collaterals 3 
is 


Trunk 


Left 


Fig. 22 Type 6; thoracic duct in a female white subject, age 42. Note the 
abrupt manner in which the right duct crosses over to the left side. 

Fig. 23 Type 6; thoracic duct in a male white subject, age 43. Note the 
plexiform arrangement of the caudal portion of the duct and its termination into 
the posterior wall of the innominate vein. 


ic = am 7 


taternal Internal 
Left Internal Jugular Vein _Augular Trunk Jugular Trunk. Subclavian 
2 ANNA ___ Left Internal Jugular Vein 
Ist ARN —~ Subclavian ~~ ¢ 
Z < G (yi Ry \ 

“Trunk Ist Thoracic Verteprase), Bey ( 
= UN ere ese 

S Subclavian aie = ee 
‘ ubclavian Vein ee ie 


Left _~.M 
c Innominates —“2 
Thoracic | = Innominate Vein S = 
Duct “"v-» Vein Ce 1 
Se pas 
pe eiNe Wie 
Thoracic —3— 


Duct ’ a : "i 


NE Collaterals 


Lymph 
Glands \. 
= Collateral 
Cs 
Cisterna 
Chyli Intestinal > —Lymph 
Trunk \/ Gland 
Lumbar 7s Lumbar 
Trunk (€ Trunk 
‘ iN Ri TH Left 
Why ght > e 
Lumbar , Je) 1 Lumar 
Lymphatics Wii a8 fh Lymphatics 
Mitineee es fe 


25 


Fig. 24 Type 6; thoracic duct in an adult male white subject. Note the 
lymph glands along the thoracic portion of the duct and position of the cisterna 
chyli. 

Fig. 25 Type 6; thoracic duct in a female white subject, age 51. Note the 
plexiform arrangement of the caudal portion of the duct and the insula associated 
with its thoracic portion. 


231 


252 HENRY K. DAVIS 


Pea Internal 
Left Internal Jugular Vein Sei Tuek Left Internal Jugular Vein 4U9H!@r Trunk 
\ 
, J YS Subelavian 
Subclavian Trunk Left Se MI oN 
Ist Subclavian23— : =" (hen ates 
Thoracics< Vein 4 = —_ 
Vertebra “= , : “ — DY 
(a N Left gS A\ hs > ; 1st 
eb vila re Se iftisy_b Thoracic 
en NEF I h(_2 Verteb 
— So )) . vertebra 
@ NC | pc Ge: 
Innominate - a cae V2 
3 . : a 
Thoracic 
Duct 
Thoracic a, (WSS 
wa me—= ‘Collaterals 


Collaterals Collateral <—Vk a 


= ise J) 


Y 


Me Intestinal 
Intestinal Trunk Fg i 

= Lymph ae ff i \ JE 

ae) Glands SSP A) 
Cisterna Chyliai™ Sj yy yy 
Right oI Tai Right icon /) 
Lumbar WA as aebee 
Lymphatics YG f fl to lY femipheties 

eA 


26 


Fig. 26 Type 6; thor 
cisterna chyli and the termination of the du 
Fig. 27 Type 6; thoracic duct in a ma 
lymph glands associated with the caudal portion of the duct an 


into the left subclavian vein. 


acie duct in a male white subject, age 21. Note the 
et into the left internal jugular vein. 
le white subject, age 70. Note the 
d its termination 


THORACIC DUCT IN MAN Dae 


Left Internal Jugular Vein 


Subclavian Internal Jugular Trunk \ 4 Subclavian 
=A hy) eee 
2 JZ Munk Left Keay Ss aN) Trunk 
ee he ai Subclavian — Sf NY —— 
4 -_ aS z~Left Vein oy | == Ze 
Vv aN meme Cok ubclavian Left Sh Ss PO 
Gaga Bx. /f i Vein | nnominate. S) Thoracic 
: ‘mera pe Vein ——— | Vertebra 
Thoracic < | = pS 


\\ 


f ; 
Peat: 
Sams ae: 
‘ ie a 
Png (1) >| | SG 
liqmuy At \ 
Q, 5 2). 


IN 


, age 60. Note the 


Fig. 28 Type 6; thoracic duct in a male white subject 
cisterna chyli and the two insulae. 

Fig. 29 Type 6; thoracic duct in a male white subject 
cisterna chyli and the two ampulliform dilations at the termin 


, age 61. Note the 
al bend of the duct. 


234 HENRY K. DAVIS 


In their thoracic portion, they lie at first within the posterior 
mediastinum, but cephalad they enter the superior mediastinum. 
«In the posterior mediastinum, they lie ventrad to the bodies of 
the 11th to the 5th thoracic vertebrae, and have ventral to 
them the pericardium, the oesophagus, and the arch of the 
aorta. The thoracic aorta lies to the left and to the right are the 
right pleura and the greater azygos vein. The caudal right 
intercostal arteries pass between them and the bodies of the 
vertebrae, as does also the terminal portion of the hemiazygos 
vein. In the superior mediastinum, they rest upon the caudal 
part of the longus colli muscle, being separated by it from the 
bodies of the three cephalic thoracic vertebrae. Ventrad they 
are in relation with the origin of the left subclavian artery and 
with the vertebral vein; to the left is the pleura and to the right 
are the oesophagus and the left recurrent laryngeal nerve. 

The arch of these is in relation caudad with the apex of the 
left lung and with the left subclavian artery. Dorsad and to the 
left is the vertebral vein and to the right and ventrad are the 
left common carotid artery, the left internal jugular vein, and 
the left vagus nerve. 

This type of duct belongs clearly to Type 6 of my classification. 
It occurred in 14 cases out of 22, or in 63.63 per cent. This 
type of duct is described as normal by all anatomists. 


Group VII 


The thoracic duct of this type begins in the abdominal cavity 
as a single trunk and passes cephalad into the thorax lying on the 
left side of the aorta. In the thorax it crosses over to the right 
side and opens into the right angulus venosus. I was unable 
to find any ducts of this type described in the literature, nor did 
I find any among my own cases. It seems strange that ducts 
of this type and also of Type 5 have not been reported, inasmuch 
as ducts of all the other types have been found. 


THORACIC DUCT IN MAN Day 


Group VIII 


In case 21 (fig. 30) the thoracic duct begins in the abdominal 
cavity from a plexus of lymphatic vessels and passes cephalad into 
the thorax. In its course through the thorax, it lies to the right 
of the aorta, placed between it and the vena azygos major. At 
the level of the lower third of the 1st thoracic vertebra, the duct 
divides into two branches which do not become reunited before 
emptying into the venous system of the right side. The cephalic 
branch ascends to the 6th cervical vertebra opposite the body of 
which it begins to incline to the right and divides into two branches 
which become united again after a course of 20 mm. to form a 
single trunk which opens into the right internal jugular trunk 
which opens into the right internal jugular vein a short distance 
cephalad of its junction with the right subclavian vein. Another 
branch is given off from this cephalic branch just after it bifur- 
cates and which opens into the right internal jugular vein cephalad 
to the opening of the branch just described. The more caudal 
branch of the thoracic duct passes cephalo-laterad, then iatero- 
caudad and ventrad to open into the posterior aspect of the right 
internal jugular vein. 

The thoracic duct receives the lumbar and intestinal lymphatics 
in its abdominal portion, collaterals in its abdominal portion, 
collaterals draining the intercostal spaces in its thoracic portion, 
and the right internal jugular lymphatic trunk in its cervical 
portion. 

There are no vascular peculiarities associated with the duct 
in this case and there is a left lymphatic duct comparable to the 
usual right lymphatic duct. 

Watson (’72), Todd (39), Haller (’75), Cruickshank (’90), 
and Fleischmann (715) describe cases similar to this, in which 
the thoracic duct runs its entire course on the right side and 
opens into the venous system at the base of the neck on the 
right side. This type of thoracic duct belongs clearly to Type 
8 of my classification. It occurred in 1 case out of 22 in my 
series, or in 4.545 per cent. 


236 HENRY K. DAVIS 


Group IX 


In case 22 (fig. 31) the thoracic duct begins at the upper level 
of the 9th thoracic vertebra by the confluence of a plexus of 
lymphatic vessels. It lies on the left side of the aorta and in 
this position continues its direction cephalad and opposite the 
body of the 1st thoracic vertebra begins to incline to the left and 
then caudad dividing into two branches which open into the left 
subclavian vein. The caudal branch terminates singly but the 
cephalic branch divides into three branches just before its termi- 
nation and opens into the left subclavian vein by three branches. 
This duct in its abdominal portion receives the lumbar and 
intestinal lymphatics, in its thoracic portion the collaterals 
which drain the intercostal spaces and in its cervical portion the 
left internal jugular and subclavian trunks. 

Cameron (’02) describes a similar case in which the thoracic 
duct runs its entire course on the left side of the aorta. This 
type of duct belongs clearly to Type 9 of my classification. It 
occurred in 1 case out of 22 in my series, or in 4.545 per cent. 


VARIATIONS - 


In 11 cases out of 22, or in 50 per cent, there is a cisterna chy _ 
present (figs. 14-18, 20, 22, 24, 26, 28, 29). In one of these 
cases (fig. 14) there is a double cisterna chyli. There is also a 
double cisterna chyli in case 23 (fig. 832). This case differs from 
the previous one in that a cisterna chyli is placed on each lumbar 
trunk and the two lumbar trunks unite to form a single thoracic 
duct. This case has not been considered among my series be- 
cause nearly the entire thoracic portion of the duct had been 
destroyed at the post mortem. Jossifow (’06) reports a similar 
case in which there was a cisterna chyli on each of the lumbar 
trunks. Instead of the caudal portion of the duct being dilated 
as a cisterna chyli it may be represented by a plexus of lymph 
channels. This condition was found in 10 cases out of the 22, 
or in 45:45 per cent (figs. 10-13, 21; 23, ‘25, 27; 30; a1).. Ing 
case out of the 22, or in 4.545 per cent, the thoracic duct is formed 
by the confluence of the right and left lumbar and intestinal 


THORACIC DUCT IN MAN Dai 


Internal Jug la r Trunk 


\h.. Right Internal Jugular Vein 
> ; Ee 
ie GreG TS? mom nema 
Tall = So Subclavian Jugular Trunk Subclavian 
ZT pe Vein Left Internal Jugular Vein 


vA 


aN | 


eupelevien Se \ a= 


aN =F A cama ah 

HSS ight Ist Thoracic Vertebra —N\) \-’-)) “9 

ii o> “Innominate = 4 
Po ~ Vein Left =z. 


HH) = 


y “hi Y 


ollaterals 


. big 3 ” 
pi 


Pe Wea LAN 


Wwe, 


—— 
| 


Lymphatics (are 3 


f} 


30 


Fig. 30 Type 8; thoracic duct in a male white subject, age 55. Note the 
plexiform arrangement of the caudal portion of the duct and its termination into 
the right internal jugular vein by three branches. 

Fig. 31 Type 9; thoracic duct in a male white subject, age 71. Note the 
position of the duct on the left side, and its quadruple termination into the left 
subclavian vein. 


238 HENRY K. DAVIS 


lymphatics without the formation of a cisterna chyli and there 
is no lymphatic plexus (fig. 19). 

The cisterna chyli is placed opposite the body of the 11th 
thoracic vertebra in 2 cases out of 22, or in 9.09 per cent (figs. 16 
and 22); opposite the bodies of the 11th and 12th thoracic verte- 
brae in 2 cases out of the 22, or in 9.09 per cent (figs. 15 and 18); 
opposite the bodies of the 12th thoracic and 1st lumbar vertebrae 
in 4 cases out of 22, or 18.18 per cent (figs. 14, 17, 20, 29) ; opposite 
the bodies of the 1st and 2nd lumbar vertebrae in 3 cases out of 


eal \) Cisterna 
2 =a —Chyli 


Right De IK ©) 


TY Lumbar Trunk 
oe 


ee 


NF | 

/} Het »' | 
Ay Mis tah 
BP ea / 


Fig. 32 Thoracic duct in an adult male white subject. Note the cisterna 
chyli associated with each lumbar trunk. 


the 22, or in 13.635 per cent (figs. 24, 26, 28); and opposite the 
bodies of the 2nd and 8rd lumbar vertebrae in 1 case out of 22 or 
in 4.545 per cent (fig. 14). 

The intestinal trunk empties into the left lumbar trunk in 7 
cases out of 22, or in 31.815 per cent (figs. 11, 12, 16, 22, 23, 26, 
29); into the right lumbar trunk in 5 cases out of 22, or in 22.725 
per cent (figs. 18, 15, 18, 25, 29); into both the right and left 
lumbar trunks in 1 case out of 22, or in 4.545 per cent (fig. 21); 
into the cisterna chyli in 5 cases out of the 22, or in 22.725 per 
cent (figs. 14, 17, 20, 24, 28); into a lymph plexus in 2 cases out 


THORACIC DUCT IN MAN 239 


of 22, or in 9.09 per cent (figs. 30, 31); and into the thoracic duct 
in 2 cases out of the 22, or in 9.09 per cent (figs. 10, 19). 

The point at which the thoracic duct lying on the right side of 
the aorta begins to incline to the left is subject to some variation. 
The inclination begins opposite the body of the 3rd thoracic 
vertebra in 1 case out of 22, or in 4.545 per cent (fig. 22); opposite 
the body of the 4th thoracic vertebra in 5 cases out of 22, or in 
22.725 per cent (figs. 14, 18, 19, 24, 25); opposite the body of the 
5th thoracic vertebra in 12 cases out of 22, or in 54.54 per cent 
(figs. 10-13, 16, 17, 20, 21, 23, 26, 27, 29); and opposite the body 
of the 6th thoracic vertebra in 2 cases out of 22, or in 9.09 per cent 
(figs. 15, 18). In 1 case out of 22, or in 4.545 per cent the duct 
lying on the right side of the aorta did not cross over to the left 
side (fig. 30) and in 1 case out of 22, or in 4.545 per cent there was 
no duct on the right side of the aorta (fig. 31). 

There is a divisiqn of the thoracic duct into two branches which 
unite again to form a single trunk. This has been termed an 
‘insula’ by Haller. One or more insulae occurred in 13 cases 
out of the 22, or in 59.085 per cent (figs. 10, 12, 18, 15, 17, 18, 20, 
2120, 26; 28, 29,30). 

In 10 cases out of the 22, or in 45.45 per cent (figs. 10, 12, 13, 
14,19, 20, 23, 24, 28, 29) lymph glandsaresituated along the thoracic 
portion of the thoracic duct. According to Sabin (12) lymph 
glands develop from a lymphatic plexus and as Pensa (’08-’09) 
remarks, lymph glands may occur anywhere along the course 
of the thoracic duct. This one may expect, I think, if he recall 
the early embryonic plexiform arrangement of the thoracic duct. 

I have observed among my cases the forms of terminations 
shown in table 1. Terminations of the thoracic duct similar 
to those described in my cases have been reported by Parsons 
and Sargent (’09), Wendel (’98), and Verneuil (’53), who cites 
Boullard’s cases (tables 2 and 3). 

Table 4 is a comparison of the percentages of the different 
modes of termination of the thoracic duct as given in tables 1 
to 3. From table 4, it will be seen that my results agree quite 
closely with those of Boullard and with those of Parsons and 
Sargent. 


240 HENRY K. DAVIS 


SUMMARY 


The thoracic duct may be double throughout its entire extent, 
one channel lying on each side of the aorta and opening into the 
venous system of the corresponding side. This type of duct is 
similar to the diagram, figure 1. Ducts of this type have been 
described by four authors. 

The thoracic duct may be partially doubled and open into the 
venous system of the left side (figs. 10-15). This type of duct 


TABLE 1 
Termination 
MODE WHERE CASES | PER CENT FIGURES 
Singlets A ene | Left angulus venosus 5 | 22.725 | 12, 18, 21, 24, 
25 
Singles ee eee | Left subclavian 10 | 45.450 | 10, 13-16, 19, 
| , 20, 22, 27, 28 
Single...........| Left internal jugular } 1 | 4.845 26 
Sumele...tke ele | Left innominate i! 4.545 23 
Woublen cain. | Left subclavian 1 | 4.545 Li 
Doublepee2e. a2: | 1 branch in I. int. jug. | . 
| 1 branch in ang. ven. 1), 445 ee 29 
riple: peer cse | 2 branches in 1. int. jug. 
| 1 branch in 1. vertebral 1 | 4.545 11 
ripple nee ewes « R. internal jug. 12 Ao545 30° 
Quadruple.......) Left subclavian 1) 49545 31 
TABLE 2 
Parsons and Sargent’s cases 
Termination 
MODE WHERE | NO. OF CASES | PER CENT 
Single. foi). 244. | Left internal jugular | 28 | 70.00 
Ns 80/83 |< aero oe | Left angulus venosus | 3 | 7.50 
Double..........| Left internal jugular | 4 | 10.25 
Double..........| Left internal jugular and some | 
other vein | 2 5.00 
Double sae: cece i-br: in 1: int. jug: 
| 1 br. in 1. subclavian 1 2.50 
Quadruple.......; Left int. jug. 1 2.50 
Quadruple.......) 1 br. in left int. jug. 


| 3 br. in left subclavian il 2.50 


THORACIC DUCT IN MAN 241 


occurred in 6 instances in my series of 22, or in 27.27 per cent. 
This type of thoracic duct has been described by two authors. 

The thoracic duct may be partially doubled and open into 
the venous system of the right side. This type of duct is similar 
to the diagram, figure 3. One author has described a duct of 
this type. 

The thoracic duct may pass cephalad into the thorax on the 
right side of the aorta as a single duct and divide into two branches, 
one branch connecting with the venous system of the left side 
and the other branch with the venous system of the right side. 
This type of duct is similar to the duct in the diagram, figure 4. 
Thoracic ducts of this kind have been described by six authors. 


TABLE 3 
Wendel’s cases 


Termination 


MODE NO. OF CASES PER CENT 
SUNG, 3 gd Gab Seed eleetree ERNE Ret a Gace ee aT ec Crt cutee 9 52.941 
IDOI DS ie: a coed Moo oc ORS Nees ENC OTTER ee 3 17.647 
‘Wie ONG 5 a5 3516.5 6 eo ss oR Ue ODO clo re en Ren ed Gone 1 5.882 
IIo epee ere, race erat ey east 0. '.0.4 02 ana 4 23 .528 


Boullard’s cases, reported by Verneuil 


Termination 
SHUG 5 cod 5 her etour Stee a orale tes Peer ach aie ae ROMP raee ae eR er 18 74.98 
ID Yael aless, sis: oe eR NC ec etc Canela a et ee ee Rn Leer 3 12.49 
lini CMP ccm rai Ne Coney crepe cs ala Baia eee: 2 8.33 
4.16 


Sib Ol Cage rete are ree a Re me da A! ae) Rene ana 1 


TABLE 4 


PARSONS 


MODE OF TERMINATION ANDSARGENT’S BOULESED, 5 WEND EE = DAVIS’ CASES: 

SEE RIEIC CASES: CASES: 

CASES: 

Per cent Per cent Per cent Per cent 
‘Slval( ta (Cee el a ee OD 74.98 52.941 77.265 
ID OWI oa ee eee Res eye rpe ed ox 17.50 12.49 17.647 9.09 
‘Titel Ae eee oon ee 8.33 5.882 9.09 
imap e. «4. ed stecky vive oe 5.00 4.545 
MRO CAT PO Gaye, ss wisps! oe cuceeecrs edna 23 .528 


SHESIOMG| oes eet ee eee roe 4.16 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 2 


242 HENRY K. DAVIS 


The thoracic duct may be single and pass cephalad into the 
thorax on the right side of the aorta and at about the level of 
the 5th thoracic vertebra cross over to the left side and open 
into the venous system of the left side. This type of duct 
occurred in 14 instances in my series of 22, or in 63.63 per cent 
(figs. 16-29). This is the most predominant type of thoracic duct 
and is described as normal by all anatomists. 

The thoracie duct may lie to the right of the aorta in its entire 
extent and open into the venous system of the right side. This 
type of duct occurred in 1 instance in my series of 22, or in 
4.545 per cent (fig. 30). Ducts of this type have been described 
by five authors. 

The thoracic duct may le to the left of the aorta in its entire 
extent and open into the venous system of the left side. This 
type of duct occurred in 1 instance in my series of 22, or in 
4.545 per cent (fig. 31). A similar thoracic duct has been de- 
scribed by one author. 

Assuming that the thoracic duct is developmentally bilaterally 
symmetrical, one might expect to find in the adult some cases 
in which a single duct was situated on the left side of the aorta 
and divided in the thorax into two branches, one of which would 
open into the venous system of the left side and the other into 
the venous system of the right side. This type of duct would 
be similar to the diagram, figure 5. I found no duct of this type 
in my own series, nor could I find any described in the literature. 

Again, assuming that the thoracic duct develops with bilater- 
al symmetry, it may start in the abdominal cavity as a single 
duct and pass cephalad into the thorax on the left side of the 
aorta and at about the level :f the 5th thoracic vertebra cross 
over to the right side and open into the venous system of the 
right side. This type of thoracic duct would be similar to the 
diagram, figure 7. I found no ducts of this type among my own 
cases nor could I find any described in the literature. It seems 
strange that no ducts of these last two types have been reported 
inasmuch as ducts of all the other types have been found and 
reported. 


THORACIC DUCT IN MAN 243 


A cisterna chyli is present in 50 per cent of my cases (figs. 
14-18, 20, 22, 24, 26, 28, 29) from which results it is evident that 
a cisterna chyli is not present as often as one would suspect 
from the descriptions of the thoracic duct in modern anatomical 
textbooks. 

In 59.085 per cent of my cases, there is an insula associated 
with the thoracic duct (figs. 10, 12, 13, 15, 17, 18, 20, 21, 25, 26, 
28, 29, 30). Haller (75) considered this the normal condition. 

The thoracic duct terminates singly in 77.265 per cent of my 
cases (figs. 10-16, 18-28); doubly, in 9.09 per cent of the cases 
(figs. 17, 29); triply, in 9.09 per cent of the cases (figs. 11, 30; 
and quadruply, in 4.545 per cent of the cases (fig. 31). 

The thoracic duct terminated in the left subclavian vein in 
59.085 per cent of my cases (figs. 10, 13-17, 19, 20, 22, 27, 28, 31); 
in the left innominate in 4.545 per cent of the cases (fig. 28); 
in the left angulus venosus in 22.725 per cent of the cases (figs. 
12, 18, 21, 24, 25); in the left internal jugular in 4.545 per cent 
of the cases (fig. 26); in the right internal jugular in 4.545 per 
cent of the cases (fig. 30); in the left internal jugular and left 
angulus venosus in 4.545 per cent of the cases (fig. 29); and in 
the left internal jugular and left vertebral vein in 4.545 per cent 
of the cases (fig. 11). 


In conclusion, it is a pleasure to thank Dr. Abram T. Kerr for 
many valuable suggestions and continued interest throughout 
the course of this work. 


BIBLIOGRAPHY 


BreESCHET, G. 18386 Le systeme lymphatique. 

Buruer, C.8. 1903 Anabnormal thoracic duct. Journal of Medical Research, 
Boston, vol. 10, p. 154. 

CaMERON, 8S. 1903 Case of right aortic arch with abnormal disposition of the 
left innominate vein and thoracic duct. Lancet, London, vol. 2, p. 670. 

Cousin, G. 1898 Anomalies du canal thoracique. Bull. Soc. Anat., Paris, 
tome 73, p. 334. 

CRUICKSHANK, W. 1790 Anatomy of the absorbing vessels. 

DIEMERBROECK, T. 1685 Opera omnia anatomica et medica. 

EKustacuius, B. 1564 Opuscula anatomica, Venetiis. 

FLEISCHMANN, G. 1815 Leichen6ffnungen. 


244 HENRY K. DAVIS 


dardint 


Hauer, A. 1775 Elementa physiologiae corporis humani. Tome 8. 
1746 Disputationes anatomicae selectae. Tome 1. 

Homer, D. 1737 Litterarium ad rei commercium medicae et scientiae. Norim- 
berg, p. 162. 

Huntinaton, G. 8. 1911 The anatomy and development of the systemic lym- 
phatic vessels in the domestic cat. 

Josstrow, G. M. 1906 Der Anfang des Ductus Thoracicus und dessen Erweite- 
rung. Archiv fiir Anat. und Physiol., Anat. Abt., Tafl. 1. 

Lautu, E. A. 1885 Nouveau manuel de l’anatomiste. 

Ler, A. B. 1905 The microtomists vade-mecum. 

Lewis, F. T. 1906 The ties of the lymphatic system in rabbits. Am. 
Jour. Anat., vol. 5, p. 95. 

Lower, R. 1680 We. ae 

Nuun,, A. 1849 Untersuchungen und Beobachtungen a. d. der Gebiete der 
Anatomie. Heidelberg. 

Parsons, F. C., and Sarcent, P. W.G. 1909 Termination of the thoracic duct. 
Lancet, London, vol. 1, p. 1178 

von PaTRUBAN, C. E. 1844 Medicinische Jahrbiicher des Kais. Koénigl. Oester- 
reichischen Stattes. Bd. 39, Seite 24. 

Precqurt, J. 1661 Experimenta nova anatomica. Amsterdam. 

Pensa, A. 1908-09 Studio sulla morphologia e sulla topographia della eysterna 
chili e del ductus thoracicus nell’ uomo ed in altri Mammifera. Ricerche 
fatte nel Laboratoria di Anatomia Normale della R. Universita di 
Roma, vol. 14, p. 1. 

Sasin, F. R. 1901-02 On the origin of the lymphatic system from the veins 
and the development of the lymph hearts and the thoracic duct in the 
pig. Am. Jour. Anat., vol. 1, p. 367. 
1909 The lymphatic system in human embryos with a consideration 
of the morphology of the system as a whole. Am. Jour. Anat., vol. 9, 
p. 41. 
1912. The development of the lymphatic system. Keibel and Mall’s 
human embryology, vol. 2, p. 734. 

Saua, L. 1899-00 Sullo sviluppo dei cuori limfatici e dotti toracici nell’ embrione 
di pollo. Ricerche fatte nel Laboratoria di Anatomia Normale della 
R. Universita di Roma. Table 14, fig. 16. 

Topp, R. B. 18389 Cyclopedia of anatomy and physiology, vol. 3, p. 232. 

VERNEUIL, A. G. 1853 Le systeme veineux. Paris. 

Watson, M. 1872 Note on the termination of the thoracic duct on the right 
side. Jour. Anat. and Physiol., vol. 6, p. 427. 

WENDEL, W. 1898 Ueber die Verletzung des Ductus Thoracicus am Halse und 
ihre Heilungs-méglichkeit. Deutsche Zeitschrift fiir Chirurgie, Leip- 
zig. Bd. 48, p. 4387. 

Winstow, M. 1866 Exposition anatomique de la structure du corps humain, 
tome 3. 


THE HISTOGENESIS OF THE SELACHIAN LIVER 


RICHARD E. SCAMMON 


Institute of Anatomy, University of Minnesota 


FORTY-FIVE FIGURES (SEVEN PLATES) 


CONTENTS 

ee OULU GION eer eta a Ks Sei atl eee Biro Re rcrah eh Hd og aces o'n! Sha taelehine a old 245 
Wevelapment or the hepatic Gylimgens.: .0\-q. gees: a2. esa es ets k cede wn dete 246 
ley LUTE REE Toe aa a Oe Boe Sire a PO coat hale At J Secs a a a 246 
Pe Mablyed eve lOpme»mites cnt: luce ty stata ae eRe SNeE aks foie ahi cases) Gat eyarepeies 249 
Som NCRPLOCESSUOL ANASTOMOSIS Hs. 4a eae nen eet cere = 8 }spatcs (a sehen e ceepauetere 253 
dhs LDP ysTeroats FENIeMG) 0} 60% = 0) Ney ve ts Pe ko em SUEUR SS 2. 5c cr ne 258 
Ae Grow bh Ofeiheyhepaticen et woke rerio 26 ces ss nee ce epee 258 

b. Changes in size and structure of the hepatic cylinders........ 261 
5. Development of the hepatic tubules in different forms of selachians.. 266 
Development of the minor rami of the hepatic ducts........................ 269 

evelopment of the hepatic mesenchyma. .../o20 82 28 .. ki kes eee sees ee Bae 27 
MistenveOrghe mepatic SIMUSHIOS. ho... s'.\\ cea. as a hoa knee «Ante nue 282 
Summary... “fe ee AU TS a0 SOU rnc 5 Se ct AP 294 
[SYS INSpa ea yo) SRE eR 8 lh on ie Ro) OT ae Ae oe 299 

INTRODUCTION 


The structure of the adult selachian liver is far removed from 
that type which is generally considered characteristic of the 
organ in ertebrates. This variance from the common verte- 
brate type is seen in the great accumulation of fat in the hepatic 
cells, in the comparatively slight development of the bile duct 
system and in the absence of lobulation of the kind generally 
found in higher vertebrates... However, these pecularities which 


1 The histology of the adult elasmobranch liver was first briefly described by 
Leydig (’51) from observations on Chimaera. He published a more complete 
account dealing with several forms of selachians in 1853. Later descriptions of 
the general histology of the adult liver have been given by Shore and Jones (89), 
Pilliet (90), and Holm (’97). Deflandre (’05) has investigated the fat content 
of the hepatic cells, and Monti (’98) has studied the bile capillaries by the Golgi 
method. 

245 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 3 
MARCH, 1915 


246 RICHARD E. SCAMMON 


distinguish the selachian liver are not manifested until a com- 
paratively late stage in the development of the organ. In 
earlier stages characters common to the liver in all vertebrates, 
but which are often masked or modified in higher forms, are 
shown with unusual clearness. It is chiefly with these more 
fundamental characters such as the formation and anastomoses 


of the hepatic cylinders, the differentiation of the minor bile | 


ducts and the relation of the parenchymatous and vascular 
structures in the liver, that this paper has to do. The specific 
characters of the selachian liver, which have been mentioned, 
have been considered only incidentally. 

The main material employed in this study consisted of embryos 
of Squalus acanthias, but specimens of Raia batis, Torpedo 
ocellata, Mustelus canis, Mustelus laevis and Squatina angelus 
have been used for supplemental and comparative work. For 
a large part of the Acanthias material, and for the specimens 
of Mustelus laevis, I am particularly indebted to the late Dr. 
Charles S. Minot, who not only permitted the removal of 
numerous series from the Harvard Embryological Collection, 
but also provided special material for use in this work. 


DEVELOPMENT OF THE HEPATIC CYLINDERS 
1. Literature 


Our conception of the glandular structure of the vertebrate 
liver rests upon a large number of observations made mainly 
in the first half of the last century and culminating in the work of 
Eberth (66) and Herring (’72). Since that time it has been 
recognized that the liver is a compound gland with a more or 
less regularly branching system of ducts and with a terminal 
network of anastomosing end pieces, and our knowledge of the 
details of this network has been greatly extended by the Golgi 
method in the hands of Retzius (’92), Hendrickson (’96) and 
others. 

It is generally stated that the anastomotic type of liver is a 
modification of the compound branching gland type: Of this 
the best proof is the phylogenetic one, for in the lower cyclos- 


ee ee * ne ye 


HISTOGENESIS OF THE LIVER 247 


tomes, the liver is indeed a true compound branching gland, 
as has been shown by the work of Retzius (92), Holm (97), 
Cole (’13) and others. In Petromyzon, however, anastomoses 
occur occasionally in the embryo (Brachet ’97) and are very 
numerous in the adult (Renaut 797). On the side of embryology 
little evidence of this modification has been offered, although 
the statement that the liver arises in many vertebrate embryos 
as a branching gland, and that it takes on its adult reticular 
structure through the anastomoses of its end pieces, is common 
enough in texts. But an examination of the literature shows no 
more complete account of this early period of the histogenesis 
of the liver than that given by Remak (’55) excepting Hilton’s 
work (’03) on the pig which has hardly received the attention 
that it deserves. The study of the actual course of the anasto- 
mosis of the end pieces has also been neglected, and the only 
clear statement to be found regarding this process is not in 
connection with the liver at all but in Laguesse’s (’96) description 
of the histogenesis of the pancreas where he found numerous 
anastomoses at different stages. 

The literature of the histogenesis of the liver in selachians 
is scanty and has been to a large extent reviewed in the com- 
prehensive works of Oppel (’00) and Fiessinger (711). 

Balfour’s (’76) account was the first to appear and as it has 
formed the basis of all later descriptions, it is given here in full: 


By stage K the hepatic diverticula have begun to bud out a number 
of small hollow knobs. These rapidly increase in length and number 
and form the so-called hepatic cylinders. They anastomose and unite 
-together so that by stage L there is constructed a regular network. 
As the cylinders increase in length their lumen becomes very small] 
but appears never to vanish. 


Hammar (’93) in the course of a study devoted particularly 
to the development of the larger hepatic structures illustrates the 
development of the hepatic cylinders in two models of the liver 
of Torpedo embryos corresponding in development to Balfour’s 
stages L and M. He makes no comment upon them aside from 
stating that the trabeculae increase in number and decrease in 
caliber in the period intervening between the two stages. 


248 RICHARD E. SCAMMON 


Brachet (95) studying Torpedo embryos determined with 
accuracy the area of the hepatic diverticulum which gives rise 
to the hepatic tubules, and confirmed Balfour’s description of 
the structure of the hepatic cylinders in later stages. 

Holm (’97) figured and described very briefly sections of the 
liver of two Scyllium embryos of advanced stages. 

Most of our later information concerning the embryonic 
hepatic parenchyma comes from the studies of Braus (’96) on em- 
bryos of Acanthias, Spinax and Scyllium. Confirming Balfour’s 
observations in regard to early stages he noted a complete and 
regular anastomosis of the tubules in Acanthias embryos of 38 
mm. Here the tubules were of even size and consisted in cross 
section of seven cells surrounding a lumen of variable size but 
distinctly larger than that seen in the adult. The hepatic cells 
of this stage were free from fat. In older embryos of Spinax and 
Acanthias the cells were fat laden. Braus saw no side branches 
nor blind endings of any hepatic capillaries. 

Choronshitzky (’00), studying Torpedo, found several secondary 
hollow outgrowths from the hepatic pouch in his ‘Stadium II,” 
which corresponds approximately to Balfour’s stage K, and to 
the Normal-plate Nos. 22-24. In ‘Stadium III” which is 
represented by considerably older embryos, the liver pouches 
give rise to a number of small hollow buds, the cavities of which 
communicate with that of the pouch. ‘‘Die Leber macht im 
alleemeinen den Eindruck einer verzweigten Driise.” By “Sta- 
dium IV” the hollow buds have been transformed into much 
branched hepatic trabeculae which contain no traces of lumina. 
Choronshitzky’s opinion of the mechanical influence of the blood 
vessels on the formation of the hepatic tubules will be discussed 
in a later part of this paper. 

Minot (’00) in discussing the development of sinusoids, men- 
tions the presence of the first short hepatic cylinders in an Acan- 
thias embryo of 11.5 mm. and speaks of their anastomosis and 
growth in older stages. He noted the interesting retardment 
of development which is to be seen in later stages in the caudal 
tip of the liver as compared with the cardiac end. 


> 


HISTOGENESIS OF THE LIVER 249 


Debeyre (’09) made use of observations upon the development 
of the hepatic cylinders in Acanthias to lay the ghost of the 
theory of the mesodermal origin of the hepatic parenchyma, 
which had been again raised a short time before by Géraudel 
(07). He gives no complete or detailed history of the cylinders 
but notes, with illustrations, their general appearance in embryos 
16, 22, and 30 mm. in length, respectively. In the latter he 
recognized the beginnning of a period of pronounced increase 
in the diameter of the cylinders. Debeyre noted the presence 
of numerous granules in the apices of the hepatic cells and bases 
upon this the interesting suggestion that the liver may serve 
as an organ of internal secretion during a part of embryonic 
life. 


2. Early development of the hepatic tubules 


In this account the structures which have been variously 
termed hepatic cords, trabeculae, cylinders and tubules will 
be spoken of as tubules as long as they remain as portions of 
simple or branching unanastomosed glands. The term hepatic 
cylinders will be employed for the same structures after the 
process of anastomosis has taken place. 

The exact time when the anlagen of the hepatic tubules first 
make their appearance is somewhat variable. In general they 
are first to be seen in embryos from 7.5 to 9 mm. in length, being 
somewhat younger than Balfour’s stage K? and corresponding 
to numbers 22 and 23 of the Normal plate series. Such embryos 
have from fifty to sixty-five segments and four or five pairs of 
gill pouches of which the anterior three or four may open extern- 
ally. The spiral valve is in the process of formation, making 
at this time one or two complete turns of the intestine and the 
vitelline duct is reduced to a short wide canal. The form of 
liver anlage at this stage is represented somewhat diagrammati- 
cally in figure 1. The organ consists of a ventral median pouch 
from the foregut just anterior to the vitelline duct. The anterior 

2 In correlating embryos with stages of Balfour’s series only the general develop- 


ment of the embryo has been considered and not the state of development of the 
organ under discussion. 


250 RICHARD E. SCAMMON 


part of this pouch is full and rounded and may be termed the pars 
hepatica mediana. From the median pouch spring two large 
lateral pouches which form together the pars hepatica lateralis. 
‘In stages just preceding the appearance of the tubule anlagen the 
lateral pouches are smooth and globose and project outward almost 
at right angles to the median hepatic pouch. At the time when 
the tubules are formed, however, the lateral pouches are flattened : 
transversely and have entered upon a pronounced dorsal growth. 
Connected with the liver pouch above and in front and with the 
anterior wall of the yolk stalk behind is a small thick walled 
sac, the anlage of the gall bladder. The hepatic tubules take 


Fore gut 


Fig. 1 Lateral view of a semi-diagrammatic reconstruction of the foregut 
and liver of an Acanthias embryo 9 mm. long. The areas represented in stipple 
give rise to hepatic tubules. 


origin entirely from the pars hepatica mediana and the pars he- 
patica lateralis. These areas are indicated in stipple in figure 1. 

The tubule anlagen arise in two forms: as slight longitudinal 
ridges upon the lower part of the outer surface of the lateral 
pouches and as very small irregularities of the dorsal margins 
of the same structures. When first observed the longitudinal 
ridges are two to four in number on either side. They extend 
almost the entire length of the lateral pouches and are distinctly 
separated by shallow lateral furrows. Sometimes these ridges 
may be subdivided longitudinally at their ends. Figure 17 is a 
view from the left side and below of the liver of an embryo 7.5 


HISTOGENESIS OF THE LIVER 251 


mm. in length (S.C. 14). This specimen bears two ridges on the 
left hepatic pouch, but the right pouch is entirely smooth. The 
irregularities of the margin of the hepatic pouches, which also 
form tubules, are at first so ill defined as to be scarcely noticeable 
unless reconstructed. Then it is seen that the formerly straight 
dorsal margin has a wavy contour. 

The tubule ridges rapidly increase both in size and number. 
New ridges appear ventral and mesial to the earlier ones and 
extend to the base of the lateral pouches and upon the ventral 
surface of the pars hepatica medialis of the median liver pouch. 
No new ridges appear dorsal to the first ones and I thin that no 
new ridges arise between older ones. By the time the embryo 
reaches a length of 10 to 12 mm. each lateral pouch wall and 
lateral half of the pars hepatica medialis bears seven to ten 
tubule ridges. This is the total number formed on either side, 
and when tubules thereafter arise from the pouch directly, they 
do so as individual tubules and not in the form of tubule ridges. 
’ Figure 18 is a view from the left side and below of a reconstruction 
of the liver of an Acanthias embryo 10 mm. in length (S.C. 20) 
showing the later form of the tubule ridges and the beginning 
of the differentiation of tubules from them. 

Before all the tubule ridges are formed the ones which first 
develop are broken up by transverse or oblique furrows into 
rather irregular rows of low mound like elevations. These 
elevations are the anlagen of the individual hepatic tubules and 
may be seen in figure 18 referred to above. They are semi- 
circular or nearly so in cross section and nearly twice as long as 
broad, their greater length being always directed antero-poste- 
riorly. Almost immediately the tubules begin an active outward 
growth and each is differentiated into a distal extremity which 
often is large and pouch like, and a’ more slender proximal stalk 
which is connected with the hepatic pouch and which is circular 
in cross section. The further growth of the tubules takes place 
by the formation of tubules of the second order from the distal 


3 In designating embryos the following abbreviations will be employed: H.E.C., 
Harvard Embryological Collection; K.U.E.C., Embryological Collection, De- 
partment of Zoology, University of Kansas; S.C., author’s collection. 


252 RICHARD E. SCAMMON 


expansions of the primary ones. The secondary tubules are short 
conical projections rarely over one and one-half times as long as 
their greatest diameter. They arise from the sides of the distal 
expansion and almost always extend out at right angles to the 
axis of the primary tubule. Those primary tubules which arise 
from the sides of the hepatic pouches and are packed in among 
their fellows commonly assume a T-shaped form with the limbs 
of the T directed antero-posteriorly. ‘The primary tubules which 
arise from the margins of the hepatic pouches become somewhat 
larger than those formed from the tubule ridges and give rise to 
from three to seven secondary tubules from their expanded 
distal chambers. These secondary branches may again sub- 
divide into branches of the third order and upon these in turn 
there may occasionally be found nipple-like projections which 
represent the fourth order of tubules. In the large majority 
of cases, however, anastomosis takes place before tubules of the 
fourth or even the third order are formed. Figures 19 and 20 
are of wax reconstructions of tubules from the lateral hepatic — 
pouch of an embryo 13.3 mm. in length (8.°C. No. 18). In figure 
19 one sees the beginning of the outpouching of the distal ex- 
pansion into secondary tubules. These structures are well 
marked projections in the older tubule shown in figure 20. Figures 
22 and 23 are two views of a hepatic tubule from the dorsal 
margin of the left hepatic pouch of an embryo 15 mm. long 
(H.E.C. 227). The latter figure shows tubules of the fourth 
order. Figure 21 is a wax reconstruction of two young primary 
tubules from the ventral surface of the pars hepatica medialis 
of the same 15 mm. embryo. One of these shows the beginning 
of tubules of the second order. ~ 

When the tubule ridges first appear on the hepatic pouches 
they are due to the increased thickness of the epithelium in these 
places and not to an evagination of the pouch wall. In cross 
sections of the ridges (see fig. 28, a cross section of the lateral 
wall of the left hepatic pouch of an embryo of 8 mm. K.U.E.C. 
542) it is noticeable that the nuclei which in other parts of the 
wall are arranged in two interlocking rows tend to be reduced 


HISTOGENESIS OF THE LIVER 253 


to a single row which lies near the external surface of the epithe- 
lium. ‘There seems to be but a single layer of very high columnar 
cells at this point but the cell boundaries are not very clear. 
There is always a broad clear zone of cytoplasm towards the 
lumen of the pouch opposite the tubule ridge. Mitotic figures 
do not occur in the ridges but are frequent in the zones of epithe- 
lium between them. Following the formation of the ridge there 
appears a very shallow trench on the internal surface of the 
epithelium. The lumina of the individual tubules appear as the 
tubules themselves are differentiated through the breaking up 
of the ridges into rounded anlagen. One then finds in each tubule 
anlage a narrow slit-like cleft passing between the cell walls 
at right angles to the pouch cavity. This is shown in figure 29. 
Thus at first the tubule cells do not lie at right angles to the tubule 
lumen but nearly parallel toit. Later the cells assume an oblique 
position (see again fig. 29) and finally come to lie in the typical 
radiating position in relation to the lumen (fig. 30). As the 
tubules develop the cells become shorter and their nuclei change 
from elongately ovoid to nearly spherical bodies. 


3. The anastomosis of the hepatic tubules 


The liver is transformed from a gland of the branching to one 
of the reticular type by the anastomosis of its end pieces. The 
process begins in Acanthias in embryos from 12 to 15 mm. in 
length. Such embryos have from 70 to 85 pairs of somites and 
correspond roughly with Balfour’s stages L and K and with Nos. 
25-26 of the Normal plate series. The tubules arising from the 
dorsal portions of the lateral liver pouches precede in their 
differentiation those of other regions and it is generally among 
these dorsally placed tubules that anastomoses are first found. 
Later the tubules of the lower parts of the lateral pouches and 
finally those of the pars hepatica medialis enter upon the process. 
Variants from this general plan of procedure are not uncommon. 
Figure 2, of a sagittal section of the left hepatic pouch of an 
embryo 14 mm. long (S.C. 30) shows how general anastomoses 
are when once they begin in a given region. 


254. RICHARD E. SCAMMON 


No particular degree of differentiation seems necessary before 
a*tubule takes part in an anastomosis, and when this process 
begins in any region both branched and simple tubules fuse 
indifferently. The commonest form of anastomosis is that 
established by the end to end fusion of tubules, but tubules may 


Fig. 2 Sagittal section of the anastomosing tubules of the left hepatic pouch 
of an Acanthias embryo 14 mm. long (8.C. 30.) X 100. 


also join end to side or side to side. The last type is much com- 
moner in later stages when more opportunities are offered for this 
form of contact. Anastomoses also seem to take place with 
equal frequency between tubules formerly quite separate or 
between minor branches of the same tubule when it is possible 
for such branches to come in contact. 


HISTOGENESIS OF THE LIVER 255 


Figure 24 is of a wax reconstruction of three anastomosing 
tubules from the left hepatic pouch of an embryo 15 mm. in 
length (H.E.C. 227). The pouch wall from which the tubules 
spring is cut away squarely around the base of each tubule. 
Tubules A and B are completely anastomosed end to end but 
there is still a distinct constriction at the plane where they have 
joined and the lumen within is considerably narrower than in 
the bodies of the tubules. Although not clearly shown in the 
figure both A and B are T-shaped and in each case the other 
tip of the tubule ends freely without anastomosis. Tubule C 
is a compound one and the subdivision which passes over to join 
with B is a branch of the third order. There is as yet no actual 
anastomosis between C’ and B, but the epithelial walls of the 
two are in direct contact. 

The lack of any account of the histology of anastomosis seems 
to warrant a rather full description of the process here. The 
process may be rather arbitrarily divided into three steps or 
stages. In the first step the tubules come in actual contact; 
in the second there occurs the fusion of their walls and a re- 
arrangement of the cells forming them, and in the third there is 
an establishment of a connecting channel between the two 
original lumina. 

These stages of anastomosis can be observed in the liver of 
Acanthias embryos of any length from 13 to 45 mm. They are 
more easily followed, however, in young specimens. The figures 
which are used here to illustrate the process have been taken 
from embryos under 20 mm. in length. In each case the sections 
have been followed through and the tubules involved have been 
reconstructed to make sure that the picture presented was not 
due to an oblique plane of section or a misinterpretation of a 
segment of a single complicated tubule. 

Early stages of contact of the anastomosing tubules are illus- 
trated by figure 30 which is a transverse section of the left hepatic - 
pouch of an embryo 13.3 mm. long (8.C. 18). Numerous tubules 
extend outward from the lateral wall of the hepatic pouch. Of 
these the larger number are still in the form of simple tubules 
which are expanded distally but a few have entered upon anasto- 


256 RICHARD E. SCAMMON 


mosis. The blood vessels pass among the tubules covering them 
in part with a thin film of endothelium which is interrupted at 
some places by the attachment of strands of mesenchyma and at 
others by the apposition of the distal surfaces of tubules with 
the insheathing layer of splanchnic mesothelium. Whether this 
endothelial covering forms an absolutely continuous partition 
without fenestra between the blood and the tubule epithelium 
cannot be definitely determined without injected specimens, 
but there seem to be places where the separation is not complete. 
Anastomosis is inaugurated by the contact of the involved tubules. 
At first a few mesenchymal or endothelial cells may separate the 
tubules but these are apparently pressed to either side so that the 
entodermal cells soon lie in actual contact. Often at first only 
four or five tubule cells actually meet, but shortly there is formed 
an area of contact which generally is not quite so large as the 
caliber of either tubule involved. Sometimes the cells of one 
tubule indent the wall of the consort but this is not the common 
rule. There are no basement membranes about the hepatic 
tubules, but the basal margins of the cells seem a little thickened 
so that for a time after contact the line between the cells of the 
two anastomosing tubules is still distinct. With the disappearance 
of this line the tubules may be considered as fairly fused. 

The connection between the two fused tubules is often drawn . 
out a little forming a short stalk or bridge between them. This 
will be termed here the connecting stalk. An indentation upon 
the external surface of this stalk indicates the line of fusion of the 
tubules. The connecting stalk, when present, is generally a little 
less in diameter than are either of the tubules and the boundaries 
-of the cells forming it are not clear. One can follow the cells, 
however, by the position of their nuclei. Although at first sight 
the nuclei appear scattered without order in the stalk or at the 
point of anastomosis, a little study enables one to determine with 
some accuracy which nuclei are contributed by each tubule. 
The two rows of nuclei approach one another and may interlock 
but their radial arrangement in regard to the lumina of the two 
original tubules is not lost at first. They then pass to one side 
or the other of the connecting stalk, leaving a clear cytoplasmic 


HISTOGENESIS OF THE LIVER 257 


core in which cell boundaries are faintly distinguishable. In so 
doing the axes of the nuclei rotate through an angle of 90° so 
that instead of being parallel to the long axis of the connecting 
stalk as at first, they are now at right angles toit. Such rotation 
can be determined, of course, only in young tubules where the 
nuclei are oval and not circular in section. Figure 26, drawn 
from an embryo 19 mm. in length (S.C. 3), shows an early step 
in these changes. Figure 3A is a graphic reconstruction of 
the tubules shown in cross section in figure 26. In this section 
the rows of nuclei belonging to the two tubules involved are 
distinguishable although the process of migration of the nuclei 
to the sides of the connecting piece is clearly under way. In 
figure 36 of a later stage from an embryo 14 mm. in length 
(S.C. 30), all the nuclei with the exception of one have passed to 
the sides of the connecting stalk. 

In following the course of the nuclei in tubule anastomosis 
one is but tracing the movements of the cells in which they are 
contained, for it is hardly to be considered that the nuclei shift 
their axes within the cells, and moreover the few faint cell bound- 
aries which may be made out show the same changes in position 
as do the nuclei. The hepatic cells of the connecting stalk have 
shifted through an are of about 90° and when a lumen is estab- 
lished through the center of the connecting stalk it is bounded at 
least in greater part by the same cell surfaces which were presented 
to the lumina in the original tubules. In other words, while 
the tubule cells involved in anastomosis have shifted in position, 
their surfaces and their axes will bear the same relation to the 
new lumen which they did to the former one. Their long axes 
will be at right angles to the lumen while the inner and outer 
surfaces of the cell remain constant in both the original position 
in the simple tubule and the later position in the anastomotic 
segment. The polarity of the cell, in the sense of the term as 
used by Rabl (’88, ’90), is not disturbed by anastomosis. 

The lumen of the anastomosis is formed by clefts which ex- 
tend out from the lumina of the formerly simple tubules. These 
clefts are at first small and irregular. They pass between the 
rather irregular borders of the radially arranged cells of the con- 


258 RICHARD E. SCAMMON 


necting stalk or plate, and, meeting, become confluent. It is 
not until some time after these clefts have joined that the lumen 
of the anastomosis acquires its full size and regularity. A late 
stage in the establishment of this connecting channel is shown 
in figure 27 from an embryo 19 mm. long (8.C. 3). Here the 
clefts are stil! separated by a single cell. Figure 3 B is a graphic 
reconstruction of the tubules involved in this anastomosis. 


Fig. 3 Graphic reconstructions of anastomosing hepatic tubules in Acanthias. 
Detailed drawings of the sections indicated with dotted lines will be found in plate 
2. A, tubules from an embryo 19 mm. long (S.C. 8) (see fig. 26). B, tubules from 
an embryo 19 mm. long (S.C. 2) (see fig. 27). > 100. 


4. Later history of the hepatic cylinders 


a. Growth of the hepatic network. Immediately after anasto- 
mosis has occurred the liver increases in size very rapidly. At 
first this growth is due almost entirely to the increase in number 
of the hepatic cylinders, but later the greater portion is brought 
about by the tremendous enlargement of the hepatic sinusoids. 
After anastomosis new cylinders are added to the existing net- 
work in three ways: by the formation of tubules from the remains 
of the hepatic pouches, by the formation of blind sprouts or 
buds from the sides of cylinders forming the network and by the 
production of new cylinders at the periphery of the network from 
the cylinders located there. These methods of addition to the 
hepatic network cease in the order given and the proportional 
amount contributed to the network by the several methods is 
in inverse order to that in which they are listed above. 


HISTOGENESIS OF THE LIVER 259 


The direct production of new hepatic cylinders from the 
hepatic pouches contributes but little to the bulk of the hepatic 
network and continues for only a short period after anastomoses 
are formed. As described in section 2, there are numerous 
tubules which arise at the bases of or between tubules which have 
previously sprung from the hepatic pouches. These younger 
tubules are at first small nipple shaped elevations arising singly 
and not from ridges. So long as the earlier tubules remain simple 
the later tubules follow them closely in their development, but 
when anastomoses become common among the older tubules 
and the sinusoidal circulation is well established, the younger 
tubules anastomose almost immediately after their formation 
either with one another or with cylinders of the already estab- 
lished network. In later stages hepatic tubules may sometimes 
arise as loops, the ends of which are attached to the wail of the 
hepatic pouch. By the time the embryo reaches a length of 
approximately 20mm. (Normal plate Nos. 27-28, Balfour’s stage 
N) the hepatic pouches are transformed into veritable hepatic 
ducts and thereafter no new hepatic tubules arise from them. 

Hepatic cylinders, as has been seen, give rise to secondary 
buds while still in the form of single tubules, and this budding 
process continues long after anastomosis. The process can be 
most clearly demonstrated by the means of thick sections of 
which figure 41 from an embryo 24 mm. in length is an example. 


4 The method of preparation of the thick sections used in this study was as 
follows: The embryo was infiltrated with celloidin and cemented to a piece of 
infiltrated spleen or liver which in turn was fastened to a fiber block. The speci- 
men was then cleared by Gilson’s method, which makes the block almost trang- 
parent. The block was then placed in the microtome clamp and a strong beam of 
light from a condenser directed upon the object. A Greenough binocular micro- 
scope equipped with low power lenses was set up over the object. With this 
arrangement it was possible to follow in detail the process of section cutting. 
Sections were then carefully cut away until the exact region desired was reached. 
By focusing with the binocular it was possible to determine the approximate 
thickness of the section needed to just include the desired structures, and this 
section was then removed with a single cut. Sections made in this way are often 
superior to reconstructions for the study of the form of very small structures. 
They are best stained in a very dilute carmin solution and cleared for a long 
period in.cedar oil, after which they can be observed with the binocular micro- 
scope, or better with the aid of an Abby binocular eyepiece. 


260 RICHARD E. SCAMMON 


In the area of approximately 1 mm. represented here there are 
five blind buds projecting from the cylinders into the sinusoids. 
This method of addition to the network continues for a con- 
siderable period. I have found no traces of new buds in the body 
of the liver after the great increase in size of the cylinders when 
the embryo reaches a length of about 40 mm. However, in the 
portions of the liver which are the last to be formed, 1.e., the dor- 
sal margins and the posterior tips of the lateral lobes, this method 
of cylinder formation continues until the embryo reaches a length 
of 50 to 60 mm. 

While in the earlier stages of the development of the hepatic 
network the increase comes perhaps equally from peripheral 
and interstitial growth, in later stages the latter method is by 
far the more important. The hepatic network terminates periph- 
erally in a large number of blind knobs which by their growth 
and division give rise to a large amount of hepatic tissue. The 
cells of these terminal knobs remain in a comparatively undiffenti- 
ated condition while those of the more central part of the network 
are undergoing rapid changes in structure. Figure 43 shows a 
small portion of the tip of the lateral lobe of an embryo 20 mm. 
long. The mesothelium covering the liver has been removed. 
Here the terminal knobs are seen projecting from the general 
network and are often attached to the mesothelial sheath by 
strands of mesenchyma. This specimen was prepared by cutting 
a thick celloidin section of the region desired by the method 
already described. The celloidin was then dissolved away and 
the mesothelial covering stripped from the fragment with fine 
forceps. ge 

The peripheral addition to the hepatic network takes place 
at first over the entire surface of the liver. But like the inter- 
stitial method of addition it is later limited to the tips of the 
lateral lobes and to the dorsal margins of both the body and 
lateral lobes of the liver which at a comparatively late stage 
grow rapidly upward between the stomach and the lateral body 
walls. In embryos 60 mm. in length these areas are much re- 
stricted and they cannot be seen in an embryo 80 mm. long. 


HISTOGENESIS OF THE LIVER 261 


In the rapidly growing parts of the liver the cylinders often 
terminate peripherally in expanded end-bulbs or vesicles which 
contain a lumen many times the diameter of that of the typical 
cylinder. Such vesicles are found in embryos from 25 to 40 mm. 
in length but not in older specimens. Their walls consist of a 
low columnar or cuboidal epithelium. Figure 4 is a cross section 
of one of these structures from the lateral lobe of an embryo 
36.6 mm. long (S.C. 10). The significance of these vesicles is 
“unknown to me. 


Fig. 4 Terminal vesicle of a hepatic cylinder from the lateral lobe of an Acan- 
thias embryo 36.6 mm. long (8.C. 10). * 400. JL, lumen of hepatic cylinder; 
V, terminal vesicle; X, side branch from central lumen of cylinder. 


b. Changes in the size and structure of the hepatic cylinders. 
When the hepatic tubules enter upon anastomoses they are 
irregular in form and of variable caliber. The lumina of the 
tubules are large and irregular and are generally surrounded in 
cross section by 14 or 15 eells, if one may judge from the number 
of nuclei present, for cell boundaries are often indistinct at this 
time. 

Table 1 shows some of the changes which take place in the 
‘course of the later development of the cylinders. The measure- 
ments and cell counts given in this table are in each case averages 
determined from twenty fair cross sections of cylinders of the 


THE AMBRICAN JOURNAL OF ANATOMY, VOL. 17, No.3 


262 é RICHARD E. SCAMMON 


posterior part of the median lobe of the liver. ‘The cross sections 
of cylinders were taken at random from this region, except 
that those lying in the rapidly growing peripheral zone were 
avoided in each case. 

In the first specimen of the series the number of cells bounding 
the lumen in cross section averages 12.5. The diameter of the 
tubules has become more uniform and averages 58 micra. Ex- 
amples of such tubules are illustrated in figure 31. At about this 
time the liver begins to increase greatly in size. This growth is — 
due in part to the actual increase in number of hepatic cells, as 


TABLE 1 


Measurements of the hepatic cylinders in Acanthias embryos 


LENGTH NP SEEANEH DA ISUITAE) AVERAGE DIAMETER 
PaeeT? «| promgasignemon’ (| Paauarias 

15.0 | 12.5 58.0 
20.6 10.0 50.0 
25.0 8.0 37.6 
28.0 7.0 35.6 
33.0 | 6.6 37.0 
37.0 | 6.0 38.5 
47.0 | 5.2 40.0 
50.0 4.6 46.0 
60.0 | 5.3 39.0 
80.0 | 5.0 43.0 

| 3.7 54.0 


95.0 


is indicated by the presence of numerous mitotic figures in the 
hepatic cylinders, but a much greater part of the increment is 
due to the establishment of the huge hepatic sinusoids. With 
this increase in size of the sinusoids the tubules are distinctly 
reduced in size, their average diameter dropping from 58 micra 
in an embryo 15 mm. in length to 37.6 micra in one 25 mm. and 
35.6, in one 28 mm. in length. This reduction in diameter may 
be due somewhat to the decrease in the size of the lumen, but is’ 
caused mainly by the actual decrease in the number of cells 
surrounding the lumen at any one plane. The process is a con- 
tinuous one after anastomosis is established, but is more rapid 


HISTOGENESIS OF THE LIVER 263 


at first, dropping from an average of 12.5 in an embryo of 15 mm. 
to 7in one of 28mm. I think that this rapid and early reduction 
is due to the stretching of the tubules caused by the dilatation 
of the sinusoids among them. The tubules are attached to each 
other and to the mesothelial wall of the liver by strands of mesen- 
chyma which may aid in the process by pulling upon the cylinders 
as the mesothelial wall is rapidly stretched in’ all directions. 
These mesenchymal strands can be seen in figure 43 of the liver 
of an embryo 20 mm. long. 

After the embryo reaches a length of from 28 to 30 mm. the 
hepatic cylinders again begin to increase in size. They rise 
rapidly in diameter from an average of 35.6 micra in an embryo 
28 mm. long to 46 micra in an embryo of 50 mm. and 54 micra 
in an embryo of 95 mm. This growth is still noticeable in an 
embryo of 220 mm. in which, however, the cells were too col- 
lapsed to admit of accurate measurement. While the diameter 
of the tubules increases, the size of the lumen and the number 
of cells surrounding it at any given plane as steadily decreases. 
Thus the average number of cells seen in cross section of a cylinder 
at 25 mm. is 8, at 50 mm. 4.6, and at 95 mm. 3.7. This indi- 
cates that the increase in size of the cylinder is due to the growth 
of the individual cells forming it and not to their multiplication, 
as is the early increase in size found in embryos from 13 to 16 mm. 
long. 

Almost all this growth is due to the deposition of fat in the 
hepatic cells, the nuclei of which remain stationary or actually 
decrease in size. While the cells begin to increase in size even 
when the cylinders are becoming reduced in caliber, due to factors 
already mentioned, this growth is not sufficient to make up for 
the reduction until the embryo reaches a length of more than 
30 mm. 

The thick sections illustrated in figures 40, 41 and 42 show 
graphically some of the changes just described. Figure 40 is 
a section of a liver in which the process of anastomosis is well 
under way. It shows the large size and irregular caliber of both 
the cylinders and their lumina. Figure 41 is from a specimen 
in which the hepatic sinusoids have nearly reached their highest 


264 RICHARD E. SCAMMON 


development and in which the cylinders are reduced to slender 
tubes. Figure 42 is from a somewhat older specimen in which 
the cylinders have again begun to increase in diameter. The 
changes in the number of cells surrounding the lumen of the 
cylinder in cross section are shown in the four figures forming 
plate 3. 

My remarks’ upon the changes in the finer structure of the 
hepatic cells can be regarded as little more than notes made in 
the course of the general study of the growth of the cylinders. 

During the development of the hepatic tubules and cylinders, 
the nuclei of. the cells forming them are modified in shape, size 
and structure. At the time when the tubule ridges first appear 
upon the hepatic pouches the hepatic nuclei are elongately oval 
in outline, their longer axes averaging from 14 to 15 micra and 
their lesser axes from one-third to one-half of this length (fig. 28).* 
With the definite outpouching of the hepatic tubules the nuclei 
become broader and shorter, but their volume remains practi- 
cally unchanged (figs. 29, 30). In an embryo 10 mm. in length 
(S.C. 20) from which tubules have been described and figured 
in the preceding part of this paper, the hepatic nuclei average 
8 micra in diameter and 11 micra in length. Anastomosis has 
apparently no effect upon either the size or structure of the nuclei. 
In embryos from 10 to 20 mm. long one can follow the change 
in shape of the majority of the nuclei from broadly oval to spheri- 
cal bodies. In a 20.6 mm. embryo (H.E.C. 1494, fig. 32) the 
great majority of the nuclei are spherical and have a diameter 
of 10 micra. Thereafter they gradually decrease in size even as 
the cells grow in size through an increase in fat content. In an 
embryo 47.3 mm. long (8.C. 11) the average diameter of the 
nuclei is 7.5 micra and in one of 95 mm. (H.E.C. 1882) it is little 
if any less. As the fat accumulates in the liver cells the nuclei 
may again change in shape, being in many cases pressed against 
the margin of the cell and assuming an oval or crescentic outline 
suggesting the form of the nuclei found in true fat cells. The 
nuclei which remain spherical have an average diameter of 7 
micra or a little less (in embryos 200-240 mm. long). 


6 All measurements given here were made from paraffin sections. 


HISTOGENESIS OF THE LIVER 265 


In early stages the nucleus ‘is always in the basal portion of the 
cell. After anastomosis they are found more centrally located. 
In later stages they are found scattered, sometimes in the center of 
the cell surrounded with a film of protoplasm from which threads 
extend to the cell periphery; often close to the lumen wall of the 
cell and sometimes near some other point in its periphery. 

Before the tubule anlagen appear the hepatic nuclei contain 
one, two or three large masses of chromatin which surround 
nucleoli and are generally applied closely to the nucleus wall. 
These chromatin masses are round or oval in shape and smooth 
in outline except for one or two small chromatin threads which 
may extend outward from each mass. The remaining space of 
the nucleus is filled with a clear nuclear sap through which run 
a few delicate and faintly staining fibrils. Such nuclei are 
characteristic of the young Acanthias embryo being found also 
in the cells of the mesenchyma, mesothelium, walls of the medul- 
lary canal and in the mesonephric tubules and duct. Those of 
the mesenchyma have been fully described by McGill (10) 
in a study of the development of the striated muscle of the 
oesophagus in the dogfish. The figures in Neal’s (14) recent 
work illustrate the similar structure of the nuclei in the nervous 
system. Figure 28 shows the structure of a number of these 
nuclei stained with iron-hematoxylin. As the hepatic cells are 
differentiated the chromatin masses above mentioned become 
more irregular and there are given off from them a number of 
chromatin strands which eventually form a coarse network. 
The chromatin masses are reduced in size and may become 
detached from the nuclear wall. They also may be somewhat 
broken up and present a granular appearance. On the other 
hand, the chromatin threads which have originated from them 
may fuse forming secondary and generally smaller karyosomes 
which do not surround nucleoli. These changes are shown in 
figures 31 to 34, 38 and 39. The adult hepatic nucleus is rich in 
chromatin which is arranged in a coarse network containing 
several karyosomes some of which are probably the remains of 
the original ones and some of which are the result of secondary 
aggregation of chromatin granules from the chromatin threads. 


266 RICHARD E. SCAMMON 


Most of these karyosomes are applied to the nuclear wall. The 
hepatic cell retains the typical embryonic nuclear structure 
much longer than do the cells of the mesenchyma, mesothelium, 
nervous system or urogenital system. This typical embryonic 
structure is lost first in the cells forming the gall bladder and 
major hepatic ducts, next in the minor hepatic ducts which are 
formed from cylinders already well started upon a development 
towards typical hepatic parenchyma, and finally from the hepatic 
cells proper. 

As has been remarked, fat droplets as indicated by vacuoles 
in the protoplasm of the hepatic cells appear, when the embryos 
obtain a length of about 25 mm. The use of special reagents 
would no doubt demonstrate the presence of fat prior to this 
stage. The droplets are found at first at the base of the cell, but 
later, in embryos 65 to 95 mm. long, droplets are found scattered 
through the entire cell body reducing the protoplasm to the 
network which has been described for the adult hepatic cell of 
selachians by Shore and Jones (’89) and Pilliet (’90). 

In Acanthias embryos about 30 mm. long the gall bladder 
begins to press against the hepatic tissue which lies on either 
side and above it. This process is probably brought about by 
the great growth of the internal yolk-sac which lies below the 
gall bladder. This pressure of the gall bladder causes some 
degeneration of the hepatic tissue immediately surrounding it. 
Toldt and Zuckerkandl (’78) have described a similar process in 
the human embryo. 


5. Development of the hepatic tubules in other forms of selachians 


The development of the hepatic tubules in Torpedo, Raia 
and Mustelus differs somewhat from that of Acanthias. In 
these forms the lateral hepatic pouches do not reach the great 
development found in Acanthias and tubules are formed from 
these structures at a comparatively earlier stage. The omphalo- 
mesenteric veins are somewhat larger than in Acanthias and at 
the time when the individual hepatic tubules develop veinous 
channels are found both medial and lateral to the hepatic pouches. 


HISTOGENESIS OF THE LIVER 267 


The tubules which are formed from the dorsal parts of the pouches 
and of the pars hepatica medialis arise singly or in small clusters 
as do those in the same situation in Acanthias, but in the forms 
under discussion the dorsal or marginal tubules form a much 
greater part of the whole number produced than is the case in 
Acanthias. In Torpedo and in Mustelus the tubules from the 
ventral portion of the lateral pouches arise as in Acanthias from 
tubule ridges. These ridges are but slightly elevated and the 
corresponding grooves on the internal surfaces of the pouches 
are wide and shallow. The individual tubules which form from 
these ridges do not remain as mound-like structures arranged in 
rows but grow out almost at once and like the dorsal tubulesappear 
as slender tubes extending outward between the veinous sprouts. 
In most cases the hepatic tubules which are first formed come in 
contact with the splanchnic mesothelium covering the liver. 

The omphalo-mesenteric veins in these forms increase in size 
very rapidly and before tubule formation has progressed very 
far the anterior, ventral and lateral surfaces of the liver pouch 
are practically surrounded by a venous lake. All tubules which 
arise after this period project into this sinus and in their growth 
they push its endothelial wall before them. This process is 
illustrated in figure 44, which is of a thick frontal section of the 
liver of an embryo of Mustelus canis approximately 12 mm. long. 
The number of tubules arising directly from the pouch walls is 
small in these forms as compared with Acanthias for the surface 
area of the pouches is much reduced from the first. The large 
majority of the later tubules is formed by the branching of the 
earlier ones. 

In Acanthias the hepatic tubules in the earlier period of their 
development are short, broad and pouch-like and the branches 
which arise from them are nipple-like projections. In the other 
forms studied both the primary and secondary tubules are slender 
elongated tubes which are early united in a complex branching 
and anastomosing network, the meshes of which are separated 
by sinusoids of large size. Figure 44 shows an anastomosis of a 
young hepatic tubule in Mustelus, and figure 45 of a similar 


268 RICHARD E. SCAMMON 


section of an embryo about 4 mm. longer of the same species 
shows the complete establishment of the hepatic network. 

While the early tubules of Raia, Mustelus and Torpedo are 
smaller in cross-section than are those of Acanthias, the lumina 
of these tubules are considerably larger. The cells lining the 
lumina are cubical or low columnar in outline as compared with 
the high columnar type found in Acanthias. The nuclear struc- 
ture is quite similar to that of the hepatic cells in Acanthias, the 
chromatin covered nucleoli described for that form being par- 
ticularly prominent in Torpedo and Mustelus. 

The later history of the cylinders is quite similar to that of 
Acanthias. The cylinders rapidly increase in diameter and the 
contained lumina become smaller. The increase in diameter 
of the cylinders is due, as in Acanthias, to the growth of the 
individual cells, and the number of cells about the lumen in 
any given section steadily decreases. The nuclear changes are 
similar to those found in Acanthias. In such specimens of Mus- 
telus and Squatina as J have examined the fat contained in hepatic 
cells remains in discrete droplets instead of forming one large 
mass as is generally the case in Acanthias. The same is true to 
some extent of Torpedo (fig. 35). 

Pilliet (90) has described areas of comparatively undifferenti- 
ated cells in the adult selachian liver. These cells form the 
portions of the cylinders which lie about the hepatic-portal veins. 
They are of comparatively small size and contain centrally 
placed nuclei which stain deeply with alum-carmin. The fat 
content of the cells is less than of those cells located elsewhere, 
and particularly of those lying in the neighborhood of the hepatic 
veins. Pilliet regards these smaller cells as reserves or nests of 
young cells which, from their position near the nourishing vessels, 
contribute to the growth of the organ. Apparently he did not 
find them in all the specimens which he examined. 

I have seen no evidence of a retardment of the differentiation 
of the cells near the hepatic-portal veins in Acanthias embryos, 
and if such occurs it must be at a late period in the development 
of the organ. ‘There are numerous small cells in the immediate 
neighborhood of the hepatic-portal vessels, but they all form 


’ 


HISTOGENESIS OF THE LIVER 269 


portions of the terminal bile ducts, the development of which 
will be described in a later part of this paper. Such embryos of 
Torpedo as I have examined agree with Acanthias in lack of 
any definite nests or reserves of undifferentiated cells about 
the hepatic-portal veins. Jn well advanced Mustelus embryos, 
however, the hepatic cells which surround the larger branches 
of the hepatic-portal veins do remain somewhat smaller than those 
of the remainder of the liver and are not so completely charged 
with fat. 

In summary it may be said that the chief differences between 
the two types of selachian liver, that represented by Acanthias 
and that by the several other forms mentioned, lies almost en- 
tirely in the earlier stages. These differences seem to be depend- 
ent on the difference in the size and arrangement of the omphalo- 
mesenteric veins at the time of the formation of the hepatic 
tubules. 


DEVELOPMENT OF THE MINOR RAMI OF THE HEPATIC DUCTS 


The formation of the minor rami of the hepatic ducts is closely 
associated with the history of the hepatic cylinders. In a pre- 
ceding publication (Scammon 713) it was stated that all of the 
major and some of the minor rami of the hepatic ducts arise from 
the constricted bases of certain fairly definitely placed clusters 
of hepatic tubules. In following the history of these tubule 
clusters it was found that they become separated from the 
hepatic pouch from which they arise by a broad and rather in- 
definite peduncle which is at first hardly more than an extension 
of the pouch wall. Later this peduncle becomes constricted and 
elongated, forming a small branch from the pouch which by this 
time is transformed into a segment of the hepatic duct. This 
development begins before tubule anastomosis gets fairly under 
way and continues at the time when anastomoses are taking 
place. No new major rami arise as outpouchings after anastomo- 
sis is established and aside from the actual lengthening of already 
established rami, which is not great, all further growth of these 
structures takes place by the transformation of pre-existent 
hepatic tubules into ducts. 


270 RICHARD E. SCAMMON 


This illustrates well the two methods of hepatic duct formation. 
The first and more primitive type is that suggested by Minot 
(93, p. 763) in which the duct is the result of a direct outpouch- 
ing of the wall of the hepatic diverticulum. In the second and 
specialized type the duct is produced by the transformation of 
portions of the network of hepatic cylinders. Selachians clearly 
stand near the bottom of the scale in this phase of development. 
Here the ductus choledochus, the hepatic ducts and their major 
rami arise as outpouchings and only the minor rami and the most 
distal portions of the major ones are of trabecular origin. In 
ganoids, amphibians, reptiles and birds the ductus choledochus 
and the proximal part, at least, of the hepatic ducts are formed by 
outpouching and the remainder of the duct system from tra- 
beculae. In the mammals apparently the ductus choledochus 
alone is the result of an outpouching, but further investigation 
may change this conception. 

The differentiation of hepatic cylinders into bile ducts is very 
closely associated with their relations to the blood vessels. Bile 
ducts are only formed from cylinders which are in contact with 
the main trunks of the hepatic-portal veins or their larger and 
more definite branches. Still more striking is the fact that the 
side of the cylinder towards the vessels precedes in a very marked 
degree the differentiation of the opposite side and in fact in the 
smallest ducts the cells of the opposite side of the cylinder may 
never be transformed into duct epithelium at all but complete 
the ordinary development of true hepatic cells. Such a terminal 
duct from the liver of an embryo 95 mm. in length is shown in 
figure 37. On the other hand, of the many cylinders which 
closely surround the vascular trunk only a very small percentage 
is transformed into ducts. The development of the minor 
ducts is extremely small in proportion to the amount of hepatic 
parenchyma, smaller, I think, than in any other group of verte- 
brates. There is absolutely no indication of any system of 
intercalated ducts. 

In the differentiation of a hepatic cylinder into a bile duct the 
former first approaches more nearly a perfect circle in cross 


HISTOGENESIS OF THE LIVER PFA 


section and the lumen distinctly enlarges. The cells on the 
vascular side of the lumen are reduced in actual height but be- 
come more columnar in form because of the still greater reduction 
of the size of their bases. At the same time the nuclei which 
in hepatic cylinders are round or broadly oval in section and lie 
near the center of the cells become elongately oval in outline and 
tend to retreat to the bases of the cells. The cells are so di- 
minished in size that the nuclei which increase little, if any, in 
bulk almost fill them. The nuclei lose their typical structure 
of a clear karyoplasm containing one or two large chromatin 
masses from which radiate chromatin threads and present in- 
stead a reticular chromatin network made up of evenly distrib- 
uted granules of about the same size. The protoplasm becomes 
homogeneous and colors darkly with plasma stains. An example 
of such a developing duct at an early stage is shown in figure 39. 
Approximately one-third of the duct which abuts upon a blood 
vessel shows considerable progress in differentiation, while the 
cells of the segment opposite it are true hepatic cells. Between 
the two are zones of transitional cells. To the side of this duct is 
a smaller one in still an earlier stage of differentiation. The out- 
line sketch shown in figure 5 illustrates the changes in shape of 
the hepatic cells at the time of duct formation. 

The cells of the large hepatic ducts which are formed from the 
hepatic pouches and their evaginations show much the same 
steps in cytomorphosis as do those just described. The nuclei 
of the larger ducts, however, are oval at the start and so undergo 
no changes in form, but the change in chromatin arrangernent 
is the same as in the minor ducts. In the gall bladder the same 
changes also take place but at a late stage (60-80 mm.) the 
nuclei again become circular in cross section and come to occupy 
the centers of the cells which are much elongated. 

It is sometimes stated that the bile duct epithelium is formed 
of cells of a more primitive type than those of the cylinders or 
trabeculae. In the forms under discussion, however, the bile 
duct cells have departed farther from the embryonic type than 
the parencyhmal cells, if we may judge by their nuclear structure. 


272 RICHARD E. SCAMMON 


Fig. 5 Minor hepatic duct with connecting hepatic cylinders from the median 
lobe of the liver of an Acanthias embryo 37 mm. long (H.E.C. 3538). > 400. 
Lumina of hepatic duct and cylinders solid black; hepatic cells in stippled outline; 
sinusoids, S, in unbroken outline. 


DEVELOPMENT OF .THE HEPATIC MESENCHYMA 


It is well known that the mesenchymal tissue of the selachian 
liver is extremely scanty in the embryo although fairly abund- 
ant around the blood vessels in the adult. No particular study 
has been made of its origin, but Minot (’01) in his study of 
sinusoids, and more recently Debeyre (’09) in following the de- 
velopment of the hepatic cylinders, have noted the occasional 
delamination of mesenchymal cells from the mesothelium cover- 
ing the liver and the presence of such elements along the walls 
of the hepatic sinuses. Debeyre also figures a cross section of a 
small portion of the periphery of the liver of an Acanthias embryo 
30 mm. long, which shows small spurs of mesenchymal tissue 
extending inward from the mesothelium between the hepatic 


HISTOGENESIS OF THE LIVER 2s 


cylinders. The origin of the hepatic mesenchyma can be followed 
with little difficulty in Acanthias. Here it appears that this 
material is probably derived completely from the mesothelium: 
but at two distinct periods and from two different regions, and 
that in both cases this proliferation is associated with distinct 
irregularities of splanchnic mesothelium. 

The first mesenchymal proliferation appears at a little later 
time than the formation of the stroma of that portion of the gut 
which lies posterior to the liver. In Acanthias embryos 5 mm. 
in length the liver pouch is but slightly differentiated and con- 
sists of two shallow diverticula which lie in the lateral walls of 
the archenteron and are fused anteriorly to form the median 
hepatic pouch. The archenteron of this region is clothed on 
either side by a layer of splanchnic mesoderm which is continuous 
above with the radix mesenterica and below with the splanchnic 
layer of the blastoderm. At the radix this layer is much thickened 
but it is reduced to a moderately thin layer over the sides of the 
archenteron. The irregular endothelial walls of the omphalo-- 
mesenteric veins intervene between the ventral part of the 
_archenteron and the mesothelium, but no mesenchyma is present. 
Irregular processes from the mesothelium, however, do extend 
inward and in places it appears as though cells were about to 
be delaminated. A short time later the hepatic pouch increases 
much in size and extends anteriorly far in front of the anterior 
intestinal portal. With this change the ventral parts of the 
investing layers of splanchnic mesothelium are brought in con- 
tact and eventually fuse, thus forming for some time a ventral 
mesentery. In connection with this process there appear two 
distinct sets of mesothelial irregularities. These consist of the 
mesothelial villi on the right side and of numerous irregular folds 
on the left. 

The mesothelial villi found in connection with the covering 
of the selachian liver were first described by Choronshitzky (’00), 
although they were observed in other forms long before that 
time. Hochstetter (00) has given their later history in Acanthias 
in connection with his study of the formation of the septum 
transversum. As Hochstetter has stated, these structures are 


274. RICHARD E. SCAMMON 


found only on the right side. They appear in embryos of 
45 to 30 segments as a thickened plate of the mesothelium 
overlying the right omphalo-mesenteric vein (fig. 6A). This 
plate is soon thrown into a series of pouch like irregularities, and 
the spaces thus formed are filled with a delicate network of 
protoplasmic processes from the mesothelial cells. The cores 
of the villi are thus at no time really empty, and are soon occupied 
by delaminated mesenchymal cells. From the bases of these 
villi mesenchymal cells are proliferated apparently both from the 
walls and the mesenchymal core, and soon come to form a small 
mass on the right side lying just below the omphalo-mesenteric 
vein. On the opposite side the irregularities are not villi but 
longitudinal folds. Unlike villi they do not arise from a thickened 
plate, the mesothelium at this point remaining as thin as else- 
where at the time of formation. Afterward the cells become 
more columnar and a mass of mesenchyma, larger but similar 
in other respects to that of the opposite side, is proliferated and 
underlies the left omphalo-mesenteric vein. Figure 7 shows 
the position and extent of these two mesenchymal proliferations 
in an embryo 7.5 mm. long (H.E.C. 1496). Soon after this 
stage the ventral mesentery breaks down and the ventral surface 
of the liver is free throughout its extent. With this process 
there is a delamination of mesenchymal cells along the median . 
ventral line which unites the lateral ones already described, and 
there is thus formed a general ventral bed of mesenchyma which, 
as growth proceeds, forms a coating about the gall bladder and 
constitutes a large loose-meshed mass which extends forward 
from the gall bladder to the anterior mesothelial wall of the 
liver. 

This constitutes the first and ventral contribution of mesen- 
chyma to the liver from its mesothelial envelope. In its entire 
extent it produced the mesenchyma which surrounds the gall 
bladder, and later the vessels adjacent to it, the covering of the 
cystic duct, and to an undeterminable degree the sparse mesen- 
chymal tissue of the lower part of the hepatic parenchyma. 
The irregularities of the mesothelium on the right side do not | 
continue for any great period. By the time the embryo reaches 


HISTOGENESIS OF THE LIVER Dae 


Fig. 6 <A series of transverse sections of liver anlagen of Acanthias embryos 
of different ages. ‘The mesothelium is represented in solid black, the mesenchyma 
in coarse stipple and the entodermal structures in outline; the blood-vessels are 
omitted. A, embryo 6.4 mm. long (S.C. 19); B, embryo 9 mm. long (H.E.C. 
1495); C, embryo 10 mm. long (C.S. 20); D, embryo 15 mm. long (H.E.C. 1494); 
E, embryo 20.6 mm. long (H.E.C..1494); all x 50. 


276 RICHARD E. SCAMMON 


a length of 15 mm. the mesothelium is smooth and much reduced 
in thickness. When the embryo reaches a length of 18 mm. the 
villi are reduced to small conical projections and are absent in 
embryos of 20 mm. Thereafter the mesothelium covering the 
ventral part of the liver on both sides becomes steadily reduced 
in thickness, and in embryos of 25 mm. and longer it is an ex- 
ceedingly thin layer of squamous epithelium. 

The second and dorsal mesenchymal proliferation begins at a 
later stage than does the ventral one. In an embryo 6.4 mm. in 
length (S.C. 19) (fig. 6 A) the dorsal portion of the mesothelium 
- is seen to be slightly thinner than that of the ventral zone. As 
the lateral pouches of the liver are pushed upward, the omphalo- 
mesenteric veins come to lie mainly dorsal to these structures 
and the mesothelium over them is gradually thickened. This 
thickening is most noticeable at the lateral margins of the dorsal 
- surface (fig 6B). Soon after, in embryos of 10 to 12 mm., the 
mesothelium of this region becomes distinctly thickened in places 
and is invaginated forming tubules the walls of which are continu- 
ous with the splanchnic mesothelium and the lumina with the coe- 
lomic cavity (fig. 6 C and fig. 7). Often these tubules first appear 
as long trenches on the coelomic surface of the mesothelium. 
The sides of these trenches coalesce, thus roofing over the de- 
pression and forming tubules which open into the coelom at both 
ends. Other tubules grow in and end blindly. Examples of 
these early tubules are shown in figure 8. Although fairly 
regular at the start, these structures soon become irregular in 
caliber and form, and very often anastomose. Their lumina 
may be occluded in places thus forming mesothelial cysts which 
are generally connected by solid stalks with the covering meso- 
thelium. At the time of their highest development the tubules 
form an anastomotic network which fills the dorsal fifth of the 
anterior part of the liver and they are then easily mistaken at 
first sight for true hepatic tubules (fig. 6D). More careful 
study shows a number of differences between the two structures. 
Aside from the nuclear differences to be mentioned later, the 
mesothelial tubules are more irregular than the hepatic cylinders, 
their walls are thinner both actually and relatively as compared 


HISTOGENESIS OF THE LIVER PA UTE 


to their lumina, the cells are more columnar and there is no 
definite external cuticula as there seems to be in the hepatic 
cells at this time. The cytoplasm of the cells is also more granu- 
lar. The mesothelial tubules may extend downward and come 


Fig. 7 Transverse section through the anterior part of the anlage of the 
liver of an Acanthias embryo 7.5 mm. long (H.E.C. 1496). x 400. B, tangential 
section of the mesothelial wall surrounding radicle of the left omphalo-mesenteric 
vein; M.hep.p., median hepatic pouch; M.v., mesothelial villus; V.l., left omphalo 
mesenteric vein; V.7., right omphalo-mesenteric vein; X, proliferated mesenchyma. 


in contact with the hepatic cylinders, but generally they are 
replaced in this region by cords of mesenchymal cells. Figure 9 
is a cross-section of a portion of the liver of an Acanthias embryo 
15 mm. in length (H.E.C. 227) showing these tubules (Mes.t.) in 
the highest state of development. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No.3 
MARCH, 1915 


278 RICHARD E. SCAMMON 


Fig. 8 Transverse section of the mesothelial covering of the dorso-lateral 
margin of the liver of an Acanthias embryo 13.3 mm. long (S.C. 18). X 400. 
M.t., mesothelial tubules. 

Fig. 9 Transverse section of the dorsal portion of the liver of an Acanthias 
embryo 15 mm. long (H.E.C. 227). x 400. H.t., hepatic tubules; M.t., mesothe- 
lial tubules. 


HISTOGENESIS OF THE LIVER 279 


The mesothelial tubules do not remain long as such. The 
lumina are soon obliterated and the solid cords thus formed 
break up into mesenchymal strands. This process begins at 
the inner end of the cords and can be seen in figure 9. By the 
time the embryo reaches a length of 23 to 25 mm. the cords are 
entirely replaced by such mesenchymal strands which run in 
among the tubules which have now invaded this region (fig. 38). 
At 28 to 30 mm. even these strands have entirely disappeared 
and mesenchymal cells can be found only occasionally. The 
formation of mesenchyma by tubular ingrowths of mesothelium 
does not preclude the occasional delamination of single cells 
from the splanchnic mesoderm direct, but this delamination 
contributes but little to the total of the hepatic mesenchyma. 

The nuclei of the splanchnic mesothelium in embryos 5 or 6 mm. 
in length, before mesenchymal delamination begins, are broadly 
oval in outline. They have the typical structure which is found 
in the nuclei of most of the tissues at this time. Each nucleus 
contains two or three large masses staining black with Heiden- 
hain’s hematoxylin. Each of these masses consists of a nucleolus 
surrounded by a thick layer of chromatin. The remainder of 
the nucleus is filled with a clear karyoplasm with a few faint 
strands of chromatin: These nuclei have been well described by 
MeGill (10) who studied the mesenchyma of the foregut region 
in the dogfish. As cells are delaminated from the mesothelium 
and as the mesothelial tubules are broken up into a mesenchymal 
syncytium, most of the nucleoli disappear and the chromatin 
is gradually scattered in a finely divided network. Similar 
changes take place in the nuclei of mesothelial cells but at a later 
period. A comparison of figures 7 and 38 will show the two 
extremes of this change. Mitotic figures are numerous in the 
mesothelium but scarce in the mesenchyma derived from it. 

The relation of the mesothelial tubules to the sinusoids is of 
interest in the light of Bremer’s (714) recent work on the earliest 
blood vessels in man. Bremer found mesothelial cords from the 
extra-embryonic coelome connected with the angiocysts and 
solid cords of the vascular net in the body stalk, and he suggests 
that the elements of the vascular net arise from these ingrowths. 


280 RICHARD E. SCAMMON 


I have not found that the cavities in fhe mesothelial tubules or 
cysts of the selachian liver connect with the blood spaces. As 
the walls of these structures break up into mesenchymal strands 
it is impossible with ordinary section and staining methods to 
distinguish between them and the endothelium surrounding the 
adjoining sinusoids. This is particularly true if one tries to follow 
the mesenchymal strands which penetrate between the hepatic 
cylinders in later stages such as the one shown in figure 38. 
The mesenchymal and endothelial cells form free anastomoses. 
McGill (10) apparently found the same condition in a much 
younger embryo, judging from figure 9 of her paper with which 
the section shown in my figure 7 is in agreement. 

The reduction in the thickness of the mesothelial covering of 
the liver and the disappearance of the mesothelial villi is no doubt 
due in part to the great growth of the contents of the former, 

‘particularly of the hepatic cylinders and sinusoids. This in- 
crease takes place rather suddenly in embryos from 18 to 20 mm. 
in length and the reduction in the thickness of the mesothelium 
is to some extent coincident with it. It is noticeable, however, 
that in many cases when a hepatic cylinder comes in contact 
with the covering mesothelium the latter layer is distinctly 
reduced in thickness, its constituent cells: change from a high 
columnar to a cuboidal form and the nuclei from oval to nearly 
spherical bodies. This change is probably not due to pressure 
for in places where two tubules come in contact with the meso- 
thelium and are separated by a distance hardly equal to their 
own diameter the mesothelium becomes thin at the points of 
contact and remains thickened over the small intervening portion. 
Were the effect of tubule contact only a matter of pressure, one 
might expect some flattening of this intervening place as well. 
Also the mesothelium is not pushed outward at these points of 
contact but remains on a level with the surrounding surface. 

One is tempted to suggest the homology of this dorsal mesen- 
chyma-producing mass with the ‘Vorleber’ of Kolliker and His. 
But. this similarity is only a superficial one as the mass just 
described does not appear until after the liver is well developed, 


HISTOGENESIS OF THE LIVER 281 


while in birds and mamfnals the ‘Vorleber’ is a precursor of 
that organ. Also the selachian mesenchymal mass lies in the 
dorsal and posterior part of the liver along its free upper surface, 
while the ‘Vorleber’ lies anteriorly and is broadly connected with 
the dorsal wall of the coelom. Ziegler (’88) showed long ago that 
the mesenchyma of the intestine in selachians arose from two 
longitudinal zones of proliferation from the splanchnic mesoderm. 
One of these zones lies above the archenteron and the other 


¢ 

° 
oe 
eo 
= 

- 
- 

= 

~ 


Fig. 10 A comparison of Ziegler’s figure: A, of the mesenchymal tissue in 
the trunk region of a selachian embryo with a semi-diagrammatic figure; B, show- 
ing the origin of the mesenchyma in the hepatic region of an Acanthias embryo. 


ventral to it. The method of development of the hepatic mesen- 
chyma indicates that these dorsal and ventral zones of pro- 
liferation extend forward into the liver region as well and remain 
distinct for a considerable period even after the liver has separated 
from the gut above it. A comparison of a semi-diagrammatic 
figure of a cross-section through the hepatic region with Ziegler’s 
diagram of a similar section through the middle of a trunk seg- 
ment shows the essential agreement in the origin of the splanchnic 
mesenchyma in the two regions (fig. 10). 


282 RICHARD E. SCAMMON 


HISTORY OF THE HEPATI@ SINUSOIDS 


The sinusoids of the selachian liver are most striking objects 
and at one time form decidedly more than half of the entire bulk 
of the organ. Minot (’00) has given the only description of them 
which is at all complete as a part of his exposition of the nature 
of the sinusoidal circulation. Brief notes are also to be found 
in the papers of Holm (97) and Debeyre (’09). The general 
development of the veins of the liver in these fishes has been 
studied by Rabl (’92), Hoffmann (’93) and Hochstetter (’93). 
The early development of the omphalo-mesenteric veins has 
been observed in detail by Mayer (’87) and by Ruckert (’88). 
Therefore I shall give only the briefest outline of the general 
history of the veins, and will begin with the conditions found in 
Acanthias embryos from 8 to 10 mm. in length. In such embryos 
there are paired omphalo-mesenteric veins extending forward 
to the sinus venosus on either side of the gut. The left vein is 
somewhat larger than the right. At first the right and left 
trunks are quite separate but soon they form two anastomoses, 
one posterior to the pancreas and the other, a little later, just 
posterior to the liver below the foregut. From the latter anasto- 
mosis the veins pass forward on either side of the foregut and 
medial to the lateral hepatic pouches until they reach the anterior 
end of the liver where they join and form the sinus venosus. 
It is important to note that in Acanthias in early stages only the 
medial surfaces of the lateral pouches are in contact with vascular 
channels. There are no vascular channels between the lateral 
surfaces of the hepatic pouches and the mesothelium covering 
them. 

Figure 11 A shows a reconstruction of a somewhat later stage 
in which important changes have taken place. Now only the 
left omphalo-mesenteric vein passes through the groove between 
foregut and lateral hepatic pouch to join the sinus venosus. ‘The 
right vessel ends blindly anteriorly and is connected with the left 
by the large anastomosis which lies behind the liver. In the 
meantime either a single vessel or several vascular sprigs have 
grown back on either side from the sinus venosus and passing 


HISTOGENESIS OF THE LIVER 283 


Fig. 11 Semi-diagrammatic graphic reconstructions (from frontal sections) 
of the liver veins, as seen from above, of three Acanthias embryos. The hepatic- 
portal veins are represented in solid black, the hepatic veins and sinus venosus 
in heavy stipple and the hepatic parenchyma in light stipple. In figure 12 A, 
the connection between the liver and the foregut and the upper parts of the lateral 
hepatic pouches are represented as cut away dorsally. A, embryo 15 mm. long 
(H.E.C. 230); B, embryo 20 mm. long (S.C. 31); C, embryo 41 mm. long (H.E.C. 
371). H.p., right lateral hepatic pouch; St., stalk connecting liver pouch with 
foregut above; S.v., sinus venosus; V.h.d., right hepatic vein; V.h.-p.d., right 
hepatic-portal vein. 


284 RICHARD E. SCAMMON 


along on the lateral surfaces of lateral hepatic pouches have 
formed a vascular plexus consisting of several large sinuses inter- 
rupted by small lacunae. In the drawing the omphalo-mesenteric 
veins, properly speaking, are represented in solid black and the 
sinus venosus and the veins developed from it in stipple. The 
posterior part of the omphalo-mesenteric veins may now be called 
‘hepatic-portal’ veins, this being the term commonly used to 
designate them in the adult, while the veins which are efferent 
in their drainage will be designated by the term hepatic veins, 
which is the name applied to them in works on adult anatomy. 

In the stage just described tubules have formed only on the 
lateral surfaces of the hepatic pouches. Soon thereafter tubule 
formation, takes place with great rapidity along the margins of 
the pouches and from the pars hepatic medialis as well. The 
latter process results in breaking up the clear passageway of the 
blood along the left omphalo-mesenteric vein to the sinus venosus 
and in place of this one discrete passage there are formed a number 
of small irregular venous channels which pass from the posterior 
portions of the omphalo-mesenteric veins (hepatic-portal veins) 
in among the tubules and join the hepatic veins. 

At this time there is a marked growth of the posterior lobes 
of the liver which brings about several changes in the position of 
the venous trunks. These may be seen by comparing figures 
11 B and 11C which are graphic reconstructions of embryos 
18 mm. and 41 mm. long respectively. The branches of the 
early hepatic-portal vein which extend backward along the inner 
surface of the posterior lobes assume more and more importance, 
becoming in the later stage the posterior continuations of the 
main trunks of the vein. The former main trunks which extended 
forward are relatively reduced in size and appear as branches of 
these posterior vessels. At the same time the sinus venosus 
becomes more completely separated from the liver as the septum 
transversum is formed and the lateral hepatic efferent trunks 
converge towards the middle line. In so doing they take up 
the two larger venous radicles which formerly lay medial to them 
and represented the remains of the extreme anterior ends of the 
former omphalo-mesenteric veins. Thus these vessels, as may 


HISTOGENESIS OF THE LIVER 285 


be seen by comparing B and C of figure 11, become branches of 
the hepatic veins instead of direct tributaries of the sinus venosus. 
In older embryos an increase in the caliber of the hepatic veins 
posterior to their entrance into the sinus venosus indicates the 
position of the hepatic sinuses which become so prominent in 
some selachians and which have been discussed in some detail 
by Neuville (01). 

In selachians as compared with the higher groups of vertebrates 
the primitive liver veins are retained in the adult almost in their 
entirety. The main vessels are established early before the 
sinusoidal complex is well developed and these trunks remain 
almost complete, although they may vary in size with changes in 
the hepatic parenchyma. In figure 14, for example, the main 
trunks of the hepatic vein can be seen in each section although 
the size of these trunks varies greatly. The only primitive 
hepatic vessel which is really completely broken up into sinusoids 
by the hepatic cylinders is the small segment of the left omphalo- 
mesenteric vein which passes over the pars hepatica medialis to 
join the sinus venosus and even the anterior and posterior ends 
of this vessel remain as branches of the left hepatic and the 
hepatic-portal veins respectively. The growth in length of the 
hepatic and hepatic-portal veins takes place at the ends of the 
lateral lobes of the liver. Here there exist venous sinuses which 


~ are joined by both veins and which are interrupted by only 


occasional hepatic cylinders sheathed with endothelium. From 
these sinuses the posterior ends of the afferent (hepatic-portal) 
and efferent (hepatic) veins are differentiated by the growth of 
a septum of hepatic cylinders which at first form a loose mesh- 
work and later become a compact wall through which only 
small capillary sinusoids pass from one vein to another. Some 
of the venous branches of the second order represent remains of 
original venous trunks as for example the two largest tributaries 
of the hepatic veins from the median lobe. The others are the 
representatives of the larger passageways which have pushed be- 
tween the fairly constant tubule clusters which arise from the 


hepaticpouches. Each of thelarger branches of the hepatic-portal 


vein in the median lobe of the liver accompanies for a short dis- 


286 RICHARD E. SCAMMON 


tance a secondary hepatic duct derived from one of these tubule 
clusters. As a rule the tubule ridges of the lateral surfaces of 
the hepatic pouches are completely formed and are beginning 
to break up into rows of individual anlagen before these blood 
vessels invade their vicinity. Different embryos, however, show 
some variation in this respect. The vessels pass backward 
between these rows of tubules and form a vascular plexus inter- 
rupted by a few small lacunae. After this process is quite well 
under way, the main vascular trunk of the left omphalo-mesenteric 
vein is interrupted by the growth of the pars hepatica medialis 
and as a result vascular sprouts extend out from the posterior 
part of this vessel and its fellow of the opposite side. These 
sprouts join with the plexuses of the hepatic veins and there is 
thus produced a set of sinusoidal vessels connecting the remains 
of the omphalo-mesenteric (hepatic-portal) venous system with 
the true hepatic veins. The extension of this network is gradual, 
and is not completely established until the tubules are well differ- 
entiated and anastomosis between the tubules begins to take 
place. The establishment of the complete sinuosidal circulation 
and of tubule anastomosis is practically simultaneous. In 
Acanthias, as in the pig according to Hilton (’03) and in reptiles 
(Hammer ’93), there is no reason to believe that the blood vessels 
have anything to do with the formation of the earliest hepatic 
tubules. Certainly there is no breaking up of a solid mass of 
liver cells as described in the frog by Shore (’93) or any indentation 
of the wall of the lateral liver pouches as has been seen in Torpedo 
by Choronshitzky (00). The vessels last of all penetrate among 
the tubules of the ventral surface of lateral hepatic pouches. 
Figure 12 is a frontal section through the tubules of this region 
of an embryo 15 mm. in length (H.E.C. 228). It will be seen 
that the tubules are separated only by scattered mesenchymal 
cells with the exception of a single vascular sprig. 

Minot (’00) has remarked that in the liver, and in the pro- 
nephros and mesenephros as well, the sinusoids which at first 
may be small, increase in size until they reach a certain maximum 
and then decrease. To secure some idea of the extent of the 
changes which take place in this process, the area in cross-section 


HISTOGENESIS OF THE LIVER 287 


of the blood spaces of selected sections have been measured 
from a series of livers of selachian embryos. Hight sections were 
selected for measurement from each liver. To secure sections 
representing about the same relative planes in each specimen 


HUW Po/YY Lr 
iy pb 
JSON gY_ 


® 
Oo 


WY oe We 


ie 
eo OL 
qty W) 2 
AMC & 
sd) 0 


Fig. 12 Frontal section through the tubules arising from the ventral surface 
of the pars hepatica medialis of an Acanthias embryo 15 mm. long (H.E.C. 230). 
< 150. Hepatic cell nuclei are shaded with vertical lines, nuclei of mesenchymal 
cells drawn in outline. A single vascular twig is seen in cross section above the 
largest hepatic tubule. 


the following method was employed. The liver was considered 
as divisible into three parts, as represented in figure 13. The 
first segment (A to B, fig. 13) consisted of that part of the liver 
anterior to the foremost part of the curved cystic duct. The 
second segment (B to C) extended from the cystic duct to the 


288 RICHARD E. SCAMMON 


plane where the anterior lobe of the liver joins with the two lateral 
and posterior ones. The two lateral lobes form the third seg- 
ment (C' to D). The first segment, A to B, was divided into 
three equal parts and the sections falling upon the planes separat- 
ing these parts (1 and 2) were used for measurement. Two 
sections were selected in the same manner from the second seg- 
ment B to C. The third segment C to D was divided into five 
equal parts and the four sections falling on the dividing planes 


AUaENE Hs 49 D 


i 
i 
i 


ee eS eee) 


! 
! 
r 
1 
1 
t 
1 
i 
1 
1 
1 
i 
' 
i 
j 
! 
i 
i 
q 
i) 
1 
1 


Jef See ee SSNS SSN 
a ee Se ee —----f 


----:|--~--~---~-~-_]-----q@ 
-=--)s—-=—-—~——..-— ]-_----g) 
ee 
ee Na a ee ar ee tee EST 


Fig. 13 Diagram of the liver of an Acanthias embryo showing method of 
selecting sections for measurements of the blood vessels. Bile ducts and gall 
bladder represented in black. 


_used for measurement. In this segment only the left lobe was 
measured in each case. The sections thus determined were 
drawn with the camera lucida or projection apparatus and the 
total area, the total area of the vascular bed in cross-section, and 
the area of the larger vascular trunks in cross-section were then 
determined by means of a planometer.6 From this data the 
figures given in table 2 were calculated. Such a method can lay 
no claim to great accuracy. The possibility of error is quite 
large and the method of determining the position of the sections 
has some objections, for all parts of the liver do not grow at the 


6 It is impossible to determine the exact area of the large trunks in the earlier 
stages because of their great irregularity and their many connections with the 
adjoining sinusoids. In embryos 20 to 21 mm. long the area of the main venous 
trunks forms about 20 per cent of the entire cross section area of the vascular 
spaces of the liver. This drops to about 15 per cent in embryos of 25 to 28 mm. 
and increases thereafter with the relative reduction of the sinusoids. In the last 
member of the series in table 2, the main trunks formed about 30 per cent of the 
total cross section area of the blood vessels. 


HISTOGENESIS OF THE LIVER 289 


same rate and the development of the vascular supply of the 
organ is closely related to its growth. Such a study, however, 
does give us some rough approximations which may be of value. 

A study of table 2 brings out clearly the three stages in the 
vascularization of the liver in Acanthias. The first stage found 
in an. embryo between 12 and 20 mm. in length is one in which 
the tubules are growing with great rapidity and the sinusoids 
are only cleft like endothelium lined spaces (figs. 14 A and B). 
It is only in the lateral lobes of the liver where the hepatic and 
the hepatic-portal veins unite in large sinuses that the vascular 
percentage of the organ is high. During the latter part of this 
period tubule anastomoses are: forming in large number. With 
anastomosis the vascular supply of the organ increases with the 
greatest rapidity and the tubules, taking on tbe form of slender 
cylinders, are separated by very large vascular spaces. This 
phase, which is inaugurated rather suddenly, continues while the 
embryo grows from a length of about 20 mm. to a length of 


TABLE 2 
Measurements of the hepatic vascular spaces in Acanthias embryos 


a = 
Be | B q LEVELS OF SECTION 
Of) «8 x, 
Mog | = = 
hte) AE al Ga | 
am) 2 B aire Oe)? Celene 4 5 6 pened Ve 
4 Sie a “as i | | i a %d Cale Ie 
. | | } F 
HEC T.al 0.238 0.321 0.314 0.314 | 0.029 0.019+ 
15.0 997 eevee 0.035 0.044 0.020 , 0.021 0.005 0.006— 
| V.%l 18.3 | 13.8 6.3 Ga | 26.3 30.0 
SC. | ay. 0.308 | 0.423 0.581 0.502 0.160 | 0.099 0.065 0.027 
20.5 | 5 V.a. 0.030 | 0.047 0.031 0.112 ORL 28 0).024 0.012 0.007 
| | V.% 9.8 nd 14.0 | 22.4 | 10.8 | 24.5 19.7 nated 
aot LS Ee ec cae 
HEC T.a. | 0.738 0.942 | 0.742 0.934 0.412 0.232 0.234 0.178 
20.6 | 1494 V.a..| 0.349 0:373 | 0.385 0.392 | 0.172 0.124 0.141 0.112 
| V.% | 47.3 39.7 | 52.0 | 42.0 | 41.8 53.4 60.3 68.5 
| | ‘ ) 
th ae | j Sei ] 7 Tali aeeens 
| S.C ean 1.120 1.440 | 1.210 1.190 0.357 | 0.324 | 0.3803 0.204 
28.0) ” 6 bc ulbe Vielen: |-@ 08698: 0.616 | OF D915 9} 0522 0.205 0.167 0.173 0.157 
eA 62.4 42.8 48.9 | 43.9 | 57.5 ; 51.5 | 57.0 77.0 
£5 3 pe | i | | J ue Eee Stee |) Bik: oo) le ths 
3.C eI as 2.060 | 2.120 2.340 2.270 0.669 | 0.595 0.590 0.357 
36.6 29. | V.a. | 0.403 0.578 | 0.559 0.644 | 0.152 | 0.108 | 0.084 | 0.051 
| V.% 19.6 27.3 | 23.9 28.4 | 22.8 | 18.3 | 14.4 | 14.4 
u } = — =—— 


1 T.a., total area of cross section in square mm.; V.a., area of vascular spaces in square mm.; V.%, 


percentage of total area occupied by vascular spaces. 


290 RICHARD E. SCAMMON 


about 30 mm. During this period roughly one-half of the bulk 
of the liver is made up of vascular spaces and of this half much 
the greater part is in the form of sinusoids. Figure 14C is a 
section through a plane corresponding to figure 14 B, and shows 
graphically the extent of the blood vessels at the time when they 
form the larger part of the liver. New tubules develop in great 
numbers during this time. Finally a period of reduction in the 
size of the sinusoids sets in. This reduction is brought about 
entirely by the increase in the size of tubules already formed. 
By this increase in the parenchyma the sinusoids are reduced to 
the ‘capillary sinusoids’ of Minot. This process is very notice- 
able in embryos between 35 and 45 mm. in length. It continues 
probably to the time of birth. Figure 14 E is of a cross section 
of the liver of an embryo 47.3 mm. long and shows the marked 
decrease in the size of the sinusoids at that time. 

The increase in size of the sinusoids is both actual and relative. 
The decrease in the total area (in cross-section) of the sinusoids 
is at first only a relative one, but later,for a short period at least, 
it is actual as well as relative. 

A point which table 2 does not bring out is that the size of the 
sinusoids is not determined by their position in relation to the 
larger vessels but is dependent upon the stage of development 
of the parenchyma with which they are interwoven. The 
sinusoids do not form a tapering system of vessels largest near 
the veins which receive them. This has been in a way pointed 
out in the section upon the growth of the hepatic cylinder net- 
work. In the lateral lobes for example, the growth of the tissue 
is at first entirely backward and during this period sinusoids of 
large size are generally distributed throughout the lobe (fig. 15 A). 
After the lobes have completed the greater part of their posterior 
growth there begins a great increase in the hepatic tissue along 
their dorsal margins and the lobes gradually push upward on 
either side of the intestine. With this change in the area of rapid 
growth there also occurs a change in the sinusoids which, as is 
shown in figure 15 B, continue to form practically half of the 
dorsal portion of the lobes while in the ventral portions they 
form less than 10 per cent of the total area in cross-section. 


Fig. 14 A series of transverse sections of the livers of Acanthias embryos of 
different ages showing the development of the sinusoids which are represented 
in solid black. All sections, with the exception of A, are taken at a plane equally 
distant from the anterior end of the liver and the anterior énd of the gall bladder; 
all X 35. A, embryo 13.3 mm. long (S.C. 18); B, embryo 20.5 mm. long (S.C. 5); 
C, embryo 28 mm. long (S.C. 6); D, embryo 47.3 mm. long (S.C. Wid). 


291 


292 RICHARD E. SCAMMON 


Similar changes are seen in earlier stages in the main median 
lobe of the liver and in the process of the cystic lobe which ex- 
tends into the yolk stalk coelome. 

In Mustelus and Torpedo the early development of the hepatic 
sinusoids is quite different from that seen in Acanthias. In 
these forms the omphalo-mesenteric veins become much enlarged 
at an early stage and lie both dorso-medial and ventro-lateral 
to the hepatic pouches instead of only medial to them in Acanthias. 
As a result the hepatic tubules are in contact with the walls of 
large vascular chambers from the time of their first appearance 
and as they grow they can only do so by pushing the endothelial 
walls of the vessels before them. The tubules, as has been re- 
marked, are slender and elongated and unite by anastomoses 
soon after their formation. Thus there is no stage in these forms 
corresponding to the early one in Acanthias where the sinusoids 
are small cleft-like spaces. The proportion of the volume of the 
liver formed by sinusoids is very high at first but is steadily 
reduced as development proceeds. Figure 16 of two transverse 
sections of Mustelus embryos 16.7 mm. and 22.5 mm. in length 
respectively illustrates these points, as do also the thick frontal 
sections shown in figures 44 and 45. The larger vasculartrunks of 
the liver in Mustelus and Torpedo are mapped out by the growth 
of the hepatic cylinder network between them. As in Acanthias 
the main liver veins are marked out early in the development 
of the organ, and are not formed by the fusion of smaller vascular 
spaces. 

In both the forms under discussion and in Acanthias the 
hepatic sinusoids are formed by the intercresence of the hepatic 
cylinders and the omphalo-mesenteric veins. In Mustelus and 
Torpedo this intercresence is due to the invasion of the space 
occupied by the vein. In Acanthias, in early stages, the inter- 
cresence is due to the penetration of the venous sprouts about 
the tubules which are already established. In later stages in 
Acanthias the cylinders increase by growing into venous spaces 
and the end result in either case is the same. Thus both the 
methods of sinusoid development postulated by Minot (’00) and 
by Lewis (’04) are found in these forms. 


HISTOGENESIS OF THE LIVER 293 


. 


Fig. 15 Two transverse sections through the middle of the left lateral lobe of 
Acanthias embryos; both < 75. A, embryo 28 mm. long (S.C. 6): B, embryo 
47.3 mm. long (S.C. 11): sinusoids represented in black. 

Fig. 16 Transverse sections through the middle of the median lobe of two 
embryos of Mustelus canis. A, embryo 16.7 mm. long (S.C. 32): B, embryo 22.5 
mm. long (S.C. 33); both x 35; sinusoids represented in black. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 3 


294 RICHARD E. SCAMMON 


In Acanthias the establishment of the sinusoidal circulation 
and the process of anastomosis of the hepatic tubules occur 
almost simultaneously. In Mustelus and Torpedo the hepatic 
tubules anastomose immediately after their intercresence with 
the omphalo-mesenteric veins. At first sight there would seem 
to be some relation between the sinusoidal type of circulation 
and the anastomosis of glandular end-pieces. This seems to be 
supported by the evidence that the pancreas of ganoids has free 
anastomoses with a sinusoidal circulation and these conditions 
are also found in the paraphysis of Necturus (Warren ’05) and 
in the islands of Langerhans in Mammalia (deWitt ’06). Sinus- 
oids also occur in other ductless glands which anastomose. 
On the other hand I have found no report of anastomoses between 
the tubules of the mesonephros, and a free sinousoidal circulation 
exists in this organ. The tubules of the embryonic mammalian 
pancreas are known to anastomose freely, and there is no record 
of a sinusoidal circulation in this organ. Similarly Braus (’00) 
finds a free anastomosis of the end pieces of the bulbo-urethral 
gland which has the capillary type of circulation and Bremer 
(11) has found the same conditions in the testis.’ It is evident 
that no general rule can be laid down regarding this relation, at 
least with our present knowledge of the subject. 


SUMMARY 
A. Development of the hepatic parenchyma 


1. Hepatic tubules are first represented by longitudinal ridges 
formed on the external surfaces of the pars hepatica lateralis 
and medialis, and by slight irregularities of the margins of the 
pouches forming the pars hepatica lateralis. 

2. The hepatic ridges are converted into irregular rows of 
tubule anlagen by transverse constructions. 

3. The individual tubule anlagen thus formed grow outward 
and are differentiated into expanded terminal chambers and 

7 Occasional anastomoses are found in a number of glands such as the gastric 
and uterine glands. It is to be expected that such occasional connections will 


be found in almost any gland which is studied carefully with the aid of recon- 
structions. 


HISTOGENESIS OF THE LIVER 295 


constricted proximal necks. Secondary tubules of the second, 
third or fourth order may be produced from the terminal expanded 
chamber before tubule anastomoses are formed. , 

4. In Acanthias the hepatic tubules may differentiate in regions 
into which blood bessels have not penetrated. 

5. Tubule anastomosis and the complete establishment of 
sinusoidal circulation are practically simultaneous. 

6. Anastomoses of tubules may take place end to end, side 
to end, or side to side. The frequency of the several types is 
in the order stated. 

7. The process of anastomosis involves the stages of (a) the 
contact and fusion of the tubules involved, (b) the rearrangement 
of cells in the area of confluence, and (c) the establishment of a 
new connecting lumen. 

8. While the cell position is shifted in anastomosis, the cell 
axis remains unchanged. 

9. After anastomosis the increase in the number of hepatic 
cylinders takes place in three ways, viz: (a) the formation of he- 
patie cylinders directly from the pouch wall; (b) the inter- 
stitial development of cylinders from blind sprouts from the net- 
work; (c) the peripheral growth of the terminal hepatic cylinders. 

10. These methods of increase cease in the order named and 
contribute to the network inversely to the order given above. 

11. Increase in the number of cylinders of the hepatic network 
first ceases in the body of the median lobe and last in the tips of 
the lateral lobes and along the dorsal margins of the lateral lobes 
and connecting portion. 

12. The number of cells surrounding the lumen of the hepatic 
cylinder in cross-section drops from an average of 12 + in the 
tubule at the time of anastomosis (14 to 15 mm.) to 3 + in an 
embryo 95 mm. long. 

13. The diameter of the hepatic cylinders increases up to and 
immediately after the time of anastomosis. With the enormous 
increase in the size of the sinusoids following a short time after 
anastomosis, the diameter for a time decreases. Thereafter 
the diameter of the cylinders undergoes a steady increase at least 
to the time of birth or hatching. 


296 RICHARD E. SCAMMON 


14. The early increase in size of the cylinders is due to a muiti- 
plication of cells. The later increase is due to a growth in the 
size of the individual cells. 

15 The hepatic nuclei which are originally large and elongately 
oval, become somewhat reduced in size and spherical in shape 
at the time when the tubules are formed from the tubule ridges. 
Thereafter they undergo a slow shrinkage in size up to the time of 
birth. In later stages they may again become oval or gibbus in 
outline, due to the pressure of the intracellular fat. The nuclei 
are at first basal in position and later become either central or 
peripheral. 

16. Hepatic nuclei first have the common embryonic type of 
structure which they retain longer than do the nuclei of most 
tissue. The embryonic type of structure is lost first in the 
nuclei of the gall bladder and major hepatic ducts, next in the 
minor hepatic ducts, and finally in the hepatic cylinders. 

17. Fat appears in the cylinders at an early stage and eventually 
fills almost the entire cell as in the typical fat cell. 

18. Selachians in which the omphalo-mesenteric veins are 
early developed to large proportions have slender hepatie tubules 
which indent the walls of the vessels and anastomose at a very 
early stage. 


B. Development of the minor ducts 


1. Two forms of bile duct formation exist in the selachian liver, 
that of evagination of the liver pouch, and that of transformation 
of pre-existing cylinders into bile ducts. The first and most 
primitive is more active in the selachians than in higher verte- 
brates and forms the major ducts and the proximal parts of the 
minor ones. The distal parts of the minor ducts are formed by 
cylinder transformation. 

2. Ducts are only differentiated from cylinders which le in 
contact with branches of the omphalo-mesenteric veins. The 
side of the cylinder lying towards the vein is always the first 
to differentiate and in the smaller ducts is the only part to be 
differentiated from the true hepatic cells. 


HISTOGENESIS OF THE LIVER 297 


3. The cells of the bile duct epithelium are structurally farther 
removed from the primitive hepatic cell type than are the cells 
of the hepatic cylinders. 


C. The hepatic mesenchyma 


1. The hepatic mesenchyma is derived from the splanchnic 
mesothelium from two zones and at two periods. In both cases 
the mesenchymal proliferation is associated with marked irregu- 
larities of the mesothelium. 

2. The first mesenchymal proliferation is ventral and is as- 
sociated with the formation of mesothelial villi on the right side 
and irregular mesothelial growths about the omphalo-mesenteric 
vein on the left side. This proliferation forms the mesenchymal 
tissue about the gall bladder, cystic duct, main trunk of the 
omphalo-mesenteric vein, and to an indeterminable degree the 
interstitial mesenchymal tissue of the ventral part of the liver. 

3. The second and dorsal proliferation is associated with the 
formation of mesothelial funnels and anastomosing mesothelial 
tubules which at one time occupy the dorsal fifth of the liver. 
These mesothelial invaginations break down into mesenchymal 
cords which in turn form the interstitial mesenchymal tissue of 
the dorsal part of the liver. 

4. These two zones of mesenchymal proliferation correspond 
with the two zones of splanchnic mesenchymal proliferation of 
the trunk region of selachian embryos as described by H. E. 
Ziegler. 

5. The mesothelial covering of the liver is at first made of 
columnar cells which later become squamous and _ thereafter 
apparently give rise to little or no mesenchymal tissue. The 
last areas from which the primitive form of mesothelium dis- 
appears are the tips of the lateral lobes and the dorsal margins 
of the lateral and median lobes. 

6. Generally the splanchnic mesothelium becomes reduced 
at once from a columnar to a squamous cell layer upon contact 
with a hepatic tubule or cylinder. 


298 RICHARD E. SCAMMON 


7. The primitive embryonic type of nuclear structure is lost 
in the mesenchymal cells as they are proliferated from the meso- 
thelium. No sharp line of demarcation could be drawn between 
endothelial and mesenchymal cells. 


D. Hepatic sinusoids 


1. Hepatic sinusoids arise in selachians by intercresence of 
the hepatic cylinders with the omphalo-mesenteric vein. This 
intercresence may be brought about either (a) by the growth of 
the rami of the omphalo-mesenteric vein about the cylinders as in 
Acanthias, or (b) by the invagination of the vessel wall by grow- 
ing cylinders as in Mustelus and Torpedo. 

2. In the first type there may be recognized three stages of 
sinusoid development: (a) The first in which the sinusoids are 
sparse and differ from capillaries only in their terminations at 
either side in veins; (b) The second stage in which the sinusoids 
are greatly enlarged constituting approximately 50 per cent of 
the liver; (c) The final stage in which the sinusoids take the form 
of the capillary sinusoids of Minot. In the second type of inter- 
cresence only the last two stages are present. 

3. The reduction of the sinusoids from stage (b) to stage (c) 
is due to the growth in size of the hepatic cylinders and not due 
to their increase in number, for the most active increase in num- 
ber of cylinders takes place prior to or early in stage (b). 


HISTOGENESIS OF THE LIVER 299 


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B to G. Journ. Anat. Phys., vol. 10. (Also: A monograph on the 
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Bracuet, A. 1896 Recherches sur le développement du pancréas et du foie 
(Sélaciens, Reptiles, Mammiféres). Journ. de |’Anat., t. 32. 
1897 a Sur le développement du foie et sur le pancréas de l’Ammo- 
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1897 b Die Entwickelung und Histogenese der Leber und des Pan- 
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Braus, H. 1896 Untersuchungen zur vergleichenden Histologie der Leber der 
Wirbelthiere. Semon. Zool. Forschungsreisen. 4 Lief. 


1900 Ueber den feineren Bau der Glandula bulbourthralis (Cowper- 
*schen Driise) des Menschen. Anat. Anz., Bd. 17. 


Bremer, J. L. 1911 The morphology of the tubules of the human testis and 
epididymis. Am. Jour. Anat., vol. 11. 


1914 The earliest blood-vessels in man. Anat. Rec., vol. 8. 


CuHorRONSHITZKY, B. 1900 Die Entstehung der Miltz, Leber, Gallenblase, Bauch- 
speicheldruse und des Pfortader-systems bei den verschiedenen Abtei- 
lungen der Wirbeltiere. Anat. Hefte, Bd. 13. 

Cots, F. J. 1913 A monograph on the general morphology of the myxinoid 


fishes based on a study of Myxine. Part V: The anatomy of the gut 
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Deseyre, A. 1909 Le foie est-il d’origine endodermique ou mesodermique? 
Biob. Anat., t. 19. 

DEFLANDRE, C. 1904 Le fonction adipogénique du foie dans la série animale. 
Journ. de l’Anat., t. 40. 


Esertu, C. J. 1867 Untersuchungen iiber die normale und _ pathologische 
Leber. Arch. f. path. Anat. u. Phys., Bd. 39. 


Fressincer, N. 1911 La cellule hepatique. Revue gén. d’histologie, t. 4, 
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GmhRAvUDEL, E. 1907 Le parenchyma hépatique et les voies biliaires. Journ. 
de l’Anat., t. 43. 


Hammar, J. A. 1893 Einige Plattenmodelle zur Beleuchtung der friiheren 
embryonalen Leberentwicklung. Arch. Anat. Ent. (Also: Nova Acta 
Reg. Soc. Sci. Upsala, Ser. 3). 

Henprickson, W. F. 1898 The development of the bile-capillaries as revealed 
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Herina E. 1866 Ueber den Bau der Wirbelthierleber. Sitzungsb. k. Akad. 
Wiss. Wein. Bd. 54. Abth. 1. 


300 RICHARD E. SCAMMON 


Herine, E. 1872 The liver. Stricker: Manual of human and comparative his- 
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Hitton, D. C. 1903 The early morphogenesis and histogenesis of the liver in 
Sus scrofa domesticus. Trans. Am. Mier. Soce., vol. 24. 


Hocusterter, F. 1893 Entwicklungsgeschichte des Gefiiss-systems. Ergeb. 
Gly Ames Wie IM. Wiel, 2 


1900 Ueber die Entstehung der Scheidenwand zwischen Pericardial- 
und Peritonealhohle und iiber die Bildung des Canalis pericardiaco- 
peritonealis bei Embryonen von Acanthias vulgaris. Morph. Jahrb., 
Bd. 29. 


Horrmann, C. K. 1893 Zur Entwicklungsgeschichte des Venensystems bei 
den Selachiern. Morph. Jahrb., Bd. 20. 


Hom, J. F. 1897 Ueber den feineren Bau der Leber bei den niederen Wirbel- 
tieren. Zool. Jahrb., Anat. Abt., Bd. 10. 


LacuessE, E. 1896 Recherches sur l’histogénie du pancréas chez le mouton. 
Journ, Ce WAIS, Ws os 


Lewis, F. T. 1904 The question of sinusoids. Anat. Anz., Bd. 25. 


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1852 Beitrage zur microskopischen Anatomie und Entwicklungs- 
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McGitu, C. 1910 The early histogenesis of striated muscle in the pig and the 
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Mayer, P. 1889 Ueber die Entwicklung des Herzens und der grossen Gefas- 
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Minot, C. S. 1892 Human embryology. New York. 
1900 On a hitherto unrecognized form of blood circulation without 


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1905 Genetic interpretations in the domain of anatomy. Am. Jour. 
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Monti, R. 1898 Ricerche anatomo-comparative sulla minuta innervazione 
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Nevvittz, H. 1901 Contribution a l’étude de la vascularization intestinale 
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OpreL, A. 1900 Lehrbuch der vergleichenden microscopischen Anatomie der 
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Pitiiet, A. H. 1890 Recherches sur la structure du foie des sélaciens. Compt. 
rend. Soc. Biol. Paris, ann. 42. 


HISTOGENESIS OF THE LIVER 301 


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Berlin 1850-55. 

Renavt, J. 1897 Traité d’histologie pratique, t. 2, Paris. 


Rtcxert, J. 1888 Ueber die Entstehung der endothelialen Anlagen des Herzens 
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ScammMon, R. E. 1911 Normal plates of the development of Squalus acanthias. 
Normentaf. d. Ent. d. Wirbeltiere, Jena. 


1913 The development of the elasmobranch liver. Am. Jour. Anat., 
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SHore, T. W., and Jones, H. L. 1889 On the structure of the vertebrate liver. 
Journ. Phys., vol. 10. 


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Selachiern. Arch. mikr. Anat., Bd. 32. 


PLATE 1 


EXPLANATION OF FIGURES 


17 Reconstruction of the hepatic anlage of an Acanthias embryo 7.5 mm. 
long (S.C. 14) showing longitudinal tubule ridges upon the wall of the lateral 
hepatic pouch. 

18 Reconstruction of the hepatic anlage of an Acanthias embryo 10 mm. long 
(S.C. 20) showing the formation of individual tubule anlagen from the tubule 
ridges. 

19 Reconstruction of a hepatic tubule of an Acanthias embryo 13.3 mm. 
long (S.C. 18) showing division into dorsal chamber and proximal neck and the 
secondary tubules arising from the former. 

20 Reconstruction of a more highly developed tubule; from the same speci- 
men as figure 19. 

21 Reconstruction of two simple tubules from the lateral hepatic pouch of an 
Acanthias embryo 15 mm. long (H.E.C. 227). 

22 and 23. Two views of a reconstruction of a highly developed hepatic tubule 
just prior to anastomosis; some of the secondary tubules are in contact; from the 
same specimen as figure 21. 

24 Reconstruction of three anastomosing tubules; from the same specimen as 
figure 21; tubules A and B are completely fused; tubules C and A are in an early 


stages of the process. 


HISTOGENESIS OF THE LIVER 


PLATE 1 
RICHARD E. SCAMMON 


303 


PLATE 2 


EXPLANATION OF FIGURES 


25 to 27 Sections of hepatic tubules of Acanthias illustrating the process of 
anastomosis; (fig. 36 represents a step between figs. 26 and 27). 

25 Early contact stage in anastomosis; from an embryo 13.3 mm. long (S.C. 
18); alum hematoxylin. X 400. 

26 Fusion of tubules; from an embryo 19 mm. long (S.C. 3); alum hematoxylin. 
x 400. ; 

27 ~Establishment of connecting lumen between two anastomosed tubules. 
From an embryo 19 mm. long (8.C. 2); alum hematoxylin. X 400. Graphic 
reconstructions of these tubules, shown in cross section in figures 26 and 27, are 
illustrated in figure 2, page 224. 

28 to 30 Sections of the external walls of lateral hepatic pouches of Acanthias 
embryos illustrating the formation of the hepatic tubules. 

28 Formation of tubule ridges; from an embryo 8 mm. long (K.U.E.C. 
545); iron hematoxylin. 600. 

29 Early outpouching of individual tubules. From an embryo 10 mm. 
long (S.C. 20); alum hematoxylin.  X 400. 

30 Completely formed tubules at the time when anastomoses first appear; 
from an embryo 13.3 mm. long (S.C. 18); alum hematoxylin. > 400. 


304 


PLATE 2 


HISTOGENESIS OF THE LIVER 


RICHARD E. SCAMMON 


28 


PLATE 3 


EXPLANATION OF FIGURES 


31 to 34 Four sections of livers of Acanthias embryos of different ages, illus- 
trating the changes in the hepatic cylinders; all * 400. 
31 Section of the liver of an embryo 16 mm. long (K.U.E.C. 547); iron 


hematoxylin. 
32 Section of the liver of an embryo 20.6 mm. long (H.E.C. 1494); iron 


hematoxylin. 

33 Section of the liver of an embryo 32 mm. long (H.E.C. 1652); iron 
hematoxylin. 

34 Section of the liver of an embryo (of Squalus suckli1) 95 mm. long (H.E.C. 
1882); alum cochineal. 


306 


PLATE 3 


HISTOGENESIS OF THE LIVER 
RICHARD E. SCAMMON 


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33 


PLATE 4 


EXPLANATION OF FIGURES 


35 Transverse section of the margin of the liver of an embryo of Torpedo 
ocellata 29 mm. long; alum hematoxylin. 350. 

36 Section of an anastomosis between two hepatic tubules of an Acanthias 
embryo 14 mm. long (S.C. 30); alum hematoxylin. »X 500. (This section shows 
a stage intermediate to those shown in figures 26 and 27). 

37 Transverse section of a terminal bile duct of an embryo 95 mm. long (H.E. 
C. 1882); alum cochineal. X 400. 

38 Hepatic tubules and mesenchymal ingrowths from a transverse section of 
the dorsal margin of the liver of an Acanthias embryo 24.7 mm. long (H.E.C. 
1492); iron hematoxylin. »X 400. M, mesenchymal ingrowths. H.t., hepatic 
tubules. 

39 Transverse section of a developing bile duct of an Acanthias embryo 24.7 
mm. long (H.E.C. 1492); iron hematoxylin. X 400. 


308 


PLATE 4 


HISTOGENESIS OF THE LIVER 


RICHARD E. SCAMMON 


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39 


309 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No.3 


PLATE 5 


EXPLANATION OF FIGURES 


40 to 42 Portions of thick sections of livers of Acanthias embryos of dif- 
ferent ages. The method of preparation is described in footnote 4, page 225. 
All approximately 200. 

40 Thick section of the liver of an embryo approximately 15 mm. long. 

41 Thick section of the liver of anembryo approximately 21 mm. long. 

42 Thick section of the liver of an embryo approximately 30 mm. long. 


3510 


PLATE 5 


HISTOGENESIS OF THE LIVER 
RICHARD E. SCAMMON 


311 


PLATE 6 
EXPLANATION OF FIGURES 


43 Preparation of the periphery of the posterior lobe of an Acanthias embryo 
approximately 20 mm. long showing the terminal buds of the hepatic network 
and the attachment of the network to the mesothelial covering by means of mesen- 
chymal strands. 

44 Thick frontal section of the liver of an embryo of Mustelus canis approxi- 
mately 12 mm. long. The large hepatic duct and the hepatic tubules lie in a 
vascular sinus which is represented in black. The vertical band seen on the right 
hand side is a section of the mesothelial covering of the liver. 


312 


HISTOGENESIS OF THE LIVER PLATE 6 
RICHARD E. SCAMMON 


313 


PEATE 4 


EXPLANATION OF FIGURE 


45 Thick frontal section of the liver of an embryo of Mustelus canis approxi- 
mately 16 mm. long. : 


314 


HISTOGENESIS OF THE LIVER 
RICHARD E. SCAMMON 


PLATE 7 


315 


i 
i 
{ 
i 


i: 


THE DEVELOPMENT OF THE THYMUS IN THE PIG 


I. MORPHOGENESIS 


J. A. BADERTSCHER 


From the Department of Histology and Embryology, Cornell University, 
Ithaca, N. Y. 


TWELVE FIGURES—TWO PLATES 


HISTORICAL 


It has been definitely established by many investigators that 
the thymus of all mammals is of epithelial origin. More recent 
investigations have shown, however, that the epithelial anlage 
of the thymus is not derived from the same germ layer in all 
mammals. Investigators are agreed on the point that the thy- 
mus of mammals, when it is of a purely entodermal origin, is 
a, derivative of the ventrally directed epithelial diverticulum of 
the third pharyngeal pouch. It has also been quite definitely 
settled that in some mammals (mouse, Roud ’00) the thymus 
is entirely of ectodermal origin. The mixed (ectodermal-ento- 
dermal) origin of the thymus in some mammals has not yet been 
generally accepted. In pig embryos it is the close topographi- 
eal relation that exists between the cervical vesicle and the third 
pharyngeal pouch that makes a mixed origin of the thymus 
possible. 

Among some of the workers on the early development of the 
thymus of the pig may be mentioned Fischelis, Kastschenko, 
Zotterman, Born, Bell, and Fox, the first three of whom attribute 
to the thymus an ectodermal-entodermal origin. Fischelis (’85) 
derived the thymus from the third pharyngeal pouch and the 
third branchial groove. According to this investigation these 
two fuse, and from their point of fusion each contributes about 
one-half to a ventrally directed downgrowth, the anlage of the 
thynrus. This conclusion is erroneous, for that portion of the 

317 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 3 


318 J. A. BADERTSCHER 


thymus which is derived from the third pharyngeal pouch is 
a comparatively long mid-ventrally directed epithelial tube before 
the cervical sinus is fused to it. He also makes no mention of 
the XII cranial nerve which plays an important part in modi- 
fying the topographical relations of the anterior portion of the 
thymus to surrounding structures. Basing his conclusions on 
inaccurate observations, his views in regard to a mixed origin 
of the thymus have now only an historical value. 

The first detailed study of the early development of the thymus 
in the pig was made by Kastschenko (’87). He describes the 
mesial portion of the sinus cervicalis, which he calls the ‘vesicula 
thymica,’ as fusing with the anterior end of the epithelial anlage 
of the thymus, which is derived from the third pharyngeal 
pouch. In the shiftings of some of the structures in the neck, 
that occur in young embryos during growth, the lateral portion 
of the cervical vesicle is separated mechanically from its mesial 
portion by the hypoglossal nerve. The free lateral portion 
of the cervical vesicle gives rise to the ‘thymus superficialis’ 
which is necessarily of ectodermal origin. He claims that 
the superficial thymus is not a constant structure, for, in a 
30 mm. embryo that he examined, it was not present. The 
anterior end of the thymus to which the mesial portion of sinus 
cervicalis has fused, plus the parathyroid, that lies close to it, 
he designates the thymus head; while the large remaining por- 
tion of the thymus which is of a purely entodermal origin, plus 
the thymus head, he calls the ‘thymus profunda.’ The largest 
embryo examined by him was 82 mm. in length. 

Zotterman (711) also made a detailed study of the morpho- 
_ genesis of the thymus of the pig. Her conclusions are in accord 

with those of Kastschenko with the exception that in about 
one-half of the specimens examined the superficial thymus was 
connected with the thymus head by a cord of cells that looped 
over the hypoglossal nerve. The superficial thymus was found 
in all the specimens examined and in the largest (105 mm.) 
investigated all the features common to the thymus (cortex, 
medulla, Hassall’s corpuscles, etc.) were present. 

Fox (’08) agrees with Kastschenko that the superficial thymus 


DEVELOPMENT OF THE THYMUS 319 


of the pig arises by a constriction of the fundus praecervicalis, 
but claims that in embryos up to 35mm. in length, the oldest 
stage he examined, its histological structure does not resemble 
that of the thymus more than any other branching epithelial 
mass. 

Born (’83) derived the thymus anlage in pig embryos from the 
third pharyngeal pouch, while Bell (’06) also is inclined to believe 
that the ectoderm takes no part in the formation of the thymus. 


MATERIAL AND METHODS 


For investigation of the early stages of the morphogenesis of 
the thymus, the excellent collection of pig embryo series, 3 to 
42 mm. in length, in the Department of Histology and Embry- 
ology of Cornell University, proved very helpful. In addition 
to these, five embryos ranging from 9 to 21.5 mm. in length, 
and the neck and upper thoracic region of eight embryos ranging 
from 32 to 95 mm. in length, were sectioned transversely. These 
sections, 10 microns in thickness, were stained with hematoxylin 
and eosin. From series of this group reconstructions of the 
pharyngeal region were made. Many dissections exposing the 
thymus were made of the neck and upper thoracic region of 
embryos from 100 to 280 mm. in length (full term). The thy- 
mus of a pig one day old was also examined. 


MORPHOGENESIS 


In the investigation on the morphogenesis of the thymus special 
_ attention was constantly directed toward the development of 
the superficial thymus because its existence is not yet generally 
accepted and, since the latest developmental stage in which it 
was investigated by Zotterman was only 105 mm. in length, its 
fate is not definitely known. 

An 11 mm. embryo was the developmental stage chosen as 
the starting point for the study of the morphogenesis of the 
thymus. At this stage the ectodermal and entodermal parts 
of the branchial grooves and pharyngeal pouches can still be 


320 J. A. BADERTSCHER 


distinguished from each other without difficulty. The sinus 
cervicalis, formed by the rapid growth in a caudal direction of 
the mandibular and hyoid arches and the more retarded growth 
of the branchial arches proper, is already well mapped out. As 
this stage shows well accepted relations and developmental steps 
it needs no further description. 

Embryo of 14.5 mm. (figures 1 and 2). This is the youngest 
developmental stage from which a reconstruction of one side 
of the neck was made. Since the determination of the real 
origin of the thymus was one of the crucial points in this inves- 
tigation only that part of the neck containing the anterior por- 
tion of the thymus.anlage and the vesicula cervicalis was modelled. 

The posterior edge of the hyoid arch has grown over the open- 
ing of the sinus cervicalis, shutting it off from the exterior. The 
cavity thus formed is the vesicula cervicalis (V.c.) or the ‘vesicula 
thymica’ of Kastschenko.! The vesicula cervicalis, now widely 
separated from the ectoderm (Kci.), is still connected with it 
by a heavy cord of cells, the ductus cervicalis (D.c.). Only in 
places through its entire extent are traces of a lumen left. To 
the outer end of the vesicula cervicalis is attached a cord of cells 
that runs in an antero-ventral and mesial direction and con- 
nects with the second pharyngeal pouch. This is the ductus 
branchialis (D.b.).. The anterior one-fifth of this structure pos- 
sesses a lumen which is continuous with that of the second pouch. 
At this stage it is impossible to determine the extent of the part 
that is of entodermal origin and the extent that is of ectodermal 
origin. The boundary line between the two has disappeared 
through the obliteration of the lumen. Fox (’08) was unable 
to find the ductus branchialis in pig embryos but demonstrated 
a long diverticulum—‘filiform process’—arising from the ventro- 
lateral angle of the second pouch and connected with the ecto- 


1 “According to H. Rabl (’09) the term ‘vesicula cervicalis’ is to be applied 
to the entire complex, including the two ductus branchiales; Hammar uses the 
term ‘vesicula praecervicalis’ only for the vesicular portion that is associated 
with the third pharyngeal pouch, this portion being approximately identical 
with the ‘fundus praecervicalis’ (cervicalis) of His and H. Rabl, as well as with 
the ‘vesicula thymica’ of Kastschenko and the ‘sinus vesicle’ of Zuckerhandl.’’ 
(Quoted from Keibel and Mall’s Human embryology, vol. 2, p. 456). 


DEVELOPMENT OF THE THYMUS 5 Pa! 


derm. This process was not present in any of the embryos I 
examined. 

The vesicula cervicalis (fig. 2, V.c.) lies lateral to the third 
pharyngeal pouch, between the cephalo-dorsal portion of the 
parathyroid (Pt. 3) and the caudo-ventral part of the ganglion 
nodosum (G.n.) and a short distance anterior to the hypoglossal 
nerve (N.XJI). Its general shape is fusiform, with its long 
axis almost perpendicular to the surface ectoderm. ‘The middle 
third is solid, but each extremity contains a cavity. The expan- 
sion of the central portion is due to a proliferation of the cells 
of its anterior wall which presses tightly against the dorsal sur- 
face of the parathyroid. The pressure against the parathyroid 
has apparently caused the obliteration of the cavity of the 
vesicula cervicalis in its central portion. Its inner third lies 
closely along the ventral side of the ganglion nodosum into which 
its curved end projects and with which it apparently is fused. 

The parathyroid (Pt. 3) is now a massive structure lying 
lateral to the third pharyngeal pouch (S.b.3), anterior to the 
hypoglossal nerve with which it is in contact, ventral to the 
ganglion nodosum and dorsal to the carotid artery (A.c.). Its 
general shape is that of a hemisphere with its flat side turned 
toward the vagus nerve and the vesicula cervicalis. A depres- 
sion in both the vesicula cervicalis and the parathyroid mark 
the points of most intimate contact between the two structures. 

The entodermal anlage of the thymus (7.e.) is now, through- 
out its greatest extent, a solid cord of cells, and still attached to 
the third pharyngeal pouch. Its anterior end lies closely against 
the parathyroid and the hypoglossal nerve. From its point of 
origin it extends caudally, and, with the exception of about one- 
fourth of the posterior portion, lies ventro-laterally to the carotid 
artery and the vagus nerve. ‘The caudal portion makes a rather 
sharp turn in a ventro-mesial direction and occupies the upper 
part of the pericardial region. The diameter of the caudal part 
is considerably greater than that of the remaining portion. This 
is due to the presence of a large lumen and thick walls in this 
region. In the central and anterior portions only a few slight 
traces of a lumen persist. The vesicula cervicalis and the an- 


322 J. A. BADERTSCHER 


terior portion of the entodermal thymus do not come in contact 
with each other in this developmental stage. 

Embryo of 17.5 mm. (figures 3 and 4). During the interval 
between this and the previous stage shiftings in the pharyngeal 
region have taken place that have changed the relation of some 
of the parts to each other. The ductus cervicalis (D.c.), now a 
solid cord of cells, is still connected with the ectoderm. The 
ductus branchialis on the left side, which was not modelled, has 
lost its connection with the outer end of the vesicula cervicalis. 
No traces of it in this region can be seen. It is, however, still 
connected with the second branchial pouch from which it extends 
for a short distance toward the point of its former attachment.? 
On the right side it is still a continuous solid cord of cells: ex- 
tending from the second pouch to the vesicula cervicalis. In a 
21 mm. pig embryo Zotterman (11) had demonstrated the ductus 
branchialis as a continuous cord of cells while the ductus cervi- 
calis as being broken. In the embryos which I examined the 
ductus branchialis was always the first to become discontinuous. 

The vesicula cervicalis (V.c.) no longer lies perpendicular to 
the ectoderm. The vesicula cervicalis medialis (V.c.m.)? extends 
from its point of attachment to the ganglion nodosum in an 
antero-lateral direction to the hypoglossal nerve (V.XIJ) around 
which it forms an acute angle. Frem the nerve the vesicula 
cervicalis lateralis (v.c.l.) extends for a short distance in a caudo- 
lateral direction. This is the same general direction taken by 
the ductus cervicalis which is connected to the vesicula cervi- 
calis lateralis and the ectoderm. ‘The vesicula cervicalis medialis 
is tightly wedged in between the vagus on its dorsal side and 
the parathyroid gland and a small portion of the thymus on its 


2 A reconstruction of this remnant was deemed unnecessary since it takes no 
part in the formation of the thymus and would have needlessly increased the 
size of the model. 

3 From the reconstruction as represented in figures 3 and 4 it will be seen that 
the vesicula cervicalis now loops over the hypoglossal nerve. For the sake of 
simplicity as well as for clearness, that portion of the vesicula cervicalis lying 
between the nerve and the pharynx will be termed the ‘vesicula cervicalis medialis’ 
while the part lying between the nerve and the surface ectoderm will be termed 
the ‘vesicula cervicalis lateralis.’ 


ok pe ee 


DEVELOPMENT OF THE THYMUS 32a 


ventral aspect. Its caudal portion is greatly flattened but as 
it approaches the nerve it gradually assumes a cylindrical form 
which also is the shape of the vesicula cervicalis lateralis. A 
part of its flattened caudal portion dips into the ganglion nodosum 
while a portion lies in close contact with the thymus. Fusion 
between the thymus and the vesicle has apparently not yet 
taken place, for the boundary of both can still be clearly deter- 
mined. The lumen of the vesicula cervicalis is for the most 
part obliterated. Only slight traces here and there in its course 
persist. It is largest in the portion that dips into the ganglion 
nodosum. Here the lumen is large and the wall of this portion 
of the vesicle is no thicker than that of earlier stages. Appar- 
ently no cell proliferation takes place in this region. The sur- 
face of the entire vesicula cervicalis is more or less irregular. 
An idea of its shape can best be obtained by referring to figure 
4 in which the hypoglossal nerve and a part of the ganglion 
nodosum were removed, thus almost entirely exposing it. 

The parathyroid (Pt. 3) is an elongated and very irregular 
mass of cells that is tightly packed in between the cervical 
vesicle and vagus nerve on its dorsal aspect, and the carotid 
artery on its mesial surface. Its caudo-mesial and caudo-lateral 
portions are in contact with the thymus while its anterior portion 
is on a lével with the arch of the vesicula cervicalis over the 
hypoglossal nerve. 

The thymus (7..e.) is considerably longer than in the preceding 
stage. Its cephalic and caudal ends have about the same relative 
position to the other structures as in the 14.5 mm. embryo. Its 
greater length at this stage is due to growth which has kept 
pace with the growth of the pharynx. It is still connected with 
the third pharyngeal pouch by a greatly attenuated cord of cells. 
Its anterior portion (figs. 3-4) is fused to the caudal aspect of 
the parathyroid from which it extends caudally. As in the pre- 
ceding stage, the caudal portion makes a sharp turn in a ventro- 
mesial direction and lies over the upper portion of the peri- 
cardium. The caudal portion of the right thymus extends across 
the mesial plane while the same region of the left thymus lies 
to the left of the mesial plane and extends farther caudally than 


324 J. A. BADERTSCHER 


the right one. The anterior and central portions are cylindrical 
in outline, having an almost uniform diameter. In the left 
thymus the lumen of the anterior and central regions has entirely 
disappeared, while in the right thymus only a trace of it persists 
in the central portion. The caudal portion in the pericardial 
region is greatly enlarged. The lumen in this region is broken 
but in places is quite large in diameter. The walls are very 
thick and irregular, no longer retaining their cylindrical shape. 

The anterior portion of the thymus also extends for a short 
distance along the ventro-lateral aspect of the parathyroid. It 
thus has two prongs between which lies the epithelial body. 
This condition is not present on the right side and was not 
observed in other specimens of about the same developmental 
stage. In stages earlier than this the parathyroids are anterior 
to the hypoglossal nerve. The anterior portion of the thymus 
is in close contact with both, as shown in figure 2. In the 
shiftings that occurred during the interval between this and the 
previous stage it appears that a portion of the thymus was 
carried along by the nerve and strung along the parathyroid 
thus bringing about the split condition of its anterior end. 

Embryo of 21.5 mm. (figure 5). In this stage the vesicula 
cervicalis has lost its connection with the ectoderm. The duc- 
tus cervicalis has entirely disappeared. The vesicula cervicalis 
lateralis (7.s.-V.c.l.) lies lateral to the hypoglossal nerve 
(N.XIT). It is a large fusiform shaped mass of cells containing 
in its anterior portion a narrow tortuous lumen. This structure 
is of a purely ectodermal origin and represents the ‘thymus 
superficialis’ of Kastschenko. It is connected to the vesicula 
cervicalis medialis by the pars intermedia or connecting band 
(P.2.) that loops over the hypoglossal nerve. This band was 
not observed by Kastschenko, hence he held that the superficial 
thymus remained free from the remaining portion of the thymus. 
The vesicula cervicalis medialis, also greatly expanded, has lost 
its connection with the ganglion nodosum, possesses no lumen, 
and lies along the antero-lateral side of the massive para- 
thyroid (Pi. 3) where it is fused with the anterior portion of 
the thymus. 


DEVELOPMENT OF THE THYMUS 325 


The anterior portion of the thymus (7'.e.) has lost its connec- 
tion with the pharynx and lies on the dorso-lateral side of the 
parathyroid and is fused with the vesicula cervicalis medialis. 
In the region of the fused portion it contains a cavity of con- 
siderable size while the remaining portion along the epithelial 
body is without a lumen. From the epithelial body the 
thymus extends in a caudal and a slightly medial and ven- 
tral direction as a solid cord of cells. Just anterior to its 
entrance into the thoracic cavity it is slightly enlarged. The 
extreme caudal portion which lies within the thoracic cavity 
turns abruptly in a ventral direction, is greatly flattened, and 
in contact with the pericardium. The thoracic segments of the 
right and left thymus at this stage lie closely together but are 
not fused. Bell, however, in a 20 mm. embryo, describes them 
as being fused. 

The hypoglossal nerve now forms an acute angle with that 
portion of the vagus lying immediately posterior to it. In the 
two preceding stages that were modelled, the corresponding 
angle formed by these two nerves was obtuse instead of acute. 
This change in the form of angle between the earlier and later 
stages apparently is due to shiftings—a consideration of which 
is to follow—that take place in the neck during the growth of 
young embryos by which a stress appears to be exerted on the 
hypoglossal nerve by the cervical vesicle. 

The thymus at this early stage (21.5 mm.) can be divided into 
seven regions, most of which in the later stages become very 
pronounced. They are: (1) The ‘superficial thymus’ which rep- 
resents the vesicula cervicalis lateralis and is of a purely ecto- 
dermal origin; (2) the ‘thymus head’ which represents the struc- 
ture formed by the fusion of the vesicula cervicalis medialis and 
the anterior portion of the entodermal anlage of the thymus; 
(3) the ‘connecting band’ which loops over the hypoglossal nerve 
and connects the superficial thymus with the thymus head and 
is of a purely ectodermal origin; (4) the ‘mid-cervical segment’ 
which is an enlargement of the thymus between the intermediary 
and cervico-thoracie cords; (5) the ‘intermediary cord’ which 
connects the thymus head with the mid-cervical segment; (6) 


326 J. A. BADERTSCHER 


the ‘thoracic segment’ which lies in the anterior portion of the 
thorax and is spread over a portion of the pericardium; and 
(7) the ‘cervico-thoracic cord’ which unites the mid-cervical 
segment to the thoracic segment. This system of nomenclature, 
for which we are indebted to Kastschenko, Zotterman, and Bell, 
will be used in the discussion of all the later developmental 
stages. 

The different regions of the thymus described in the 21.5 mm. 
embryo were examined microscopically in the following five 
stages. These will be briefly described in order to present a 
more complete developmental history up to a 95 mm. embryo, 
which was the oldest stage in which a part of the pharyngeal 
region was reconstructed. 

Embryo of 26 mm. The thymus as a whole is considerably 
larger than in the preceding stage. The surface of the super- 
ficial thymus, the thymus head, and the thoracic segment has 
become very irregular due to outgrowths of epithelial buds from 
the main stem. This budding represents the beginning of lobu- 
lation and had already started in the preceding stage. Lobula- 
tion of the mid-cervical segment has just begun. ‘The super- 
ficial thymus extends only slightly farther caudally from the 
hypoglossal nerve than in the preceding stage. The connecting 
band on the right side has disappeared but the superficial thymus 
has retained its usual topographical relation to the thymus head. 
From the parathyroid body the general direction of the thymus 
is in a caudo-mesia land ventral direction. The intermediary 
cord on the right side is only a very slender cord of cells while 
that of the left side has a considerably greater diameter. The 
cervico-thoraciec cords are short and have a uniform diameter 
of small dimension. The thoracic segments lie in contact with 
the anterior and ventral portion of the pericardium. The two 
segments lie close together and have fused in some places along 
their median sides. 

Embryo of 832 mm. The connecting band on the left side is 
broken. No traces of it can be seen in connection with the 
thymus head but a remnant of it is still attached to the super- 


DEVELOPMENT OF THE THYMUS 327 


ficial thymus and extends as a greatly attenuated cord of cells 
toward the hypoglossal nerve. The general features of the entire 
thymus at this stage so closely resemble those of the 26 mm. 
embryo that a detailed description is unnecessary. The only 
difference of importance is the greater size of the organ as a 
whole and of the epithelial buds from the main stem. 

Embryo of 40 mm. The connecting band is continuous around 
the hypoglossal nerve on both sides. The parathyroids are 
slightly elongated and lie along the dorso-mesial side of the 
central third of the thymus head. Many of the primary epi- 
thelial buds of the enlarged segments (the superficial thymus, 
thymus heads, mid-cervical and thoracic segments) have sent 
out processes, thus marking the beginning of secondary lobula- 
tion. The intermediary cords are greatly attenuated and show 
no signs of budding. The transition from the thymus head to 
the intermediary cords and from the latter to the mid-cervical 
segment is very abrupt. The cervico-thoracic cords are short 
and lie near each other a short distance ventral to the trachea. 
The thoracic segments of both the right and left thymus are 
now fused along the greater part of their median plane. They 
are a little larger than those in the previous stage and have the 
same general position over the anterior and ventral portion of 
the pericardium. 

Embryo of 52 mm. The connecting band on each side loops 
over the hypoglossal nerve and connects the thymus head with 
the superficial thymus. The one on the left side is a compara- 
tively large and irregularly modelled cord of differentiated thymic 
tissue while the one on the right side is a slender and greatly 
attenuated cord of epithelial cells. The intermediary cords are 
still greatly attenuated cords of epithelial cells but are now 
studded here and there with small epithelial buds. The left 
cervico-thoracic cord is still slender with a nearly uniform diam- 
eter while the right one is much larger and has undergone lobu- 
lation. Both are now of differentiated thymic structure. The 
enlarged segments of the thymus are appreciably larger than 
those in the 40 mm. embryo. They have undergone extended 


328 J. A. BADERTSCHER 


secondary lobulation the lobes of which on account of their 
large size lie in general more closely together than those in the 
previous stage. The parathyroid lies partly imbedded in the 
dorso-mesial aspect of the thymus head slightly anterior to its 
central portion. The superficial thymus lies closely along the 
antero-lateral aspect of the thymus head but is not fused to it. 

Embryo of 63 mm. The connecting band on both the right 
and left sides loops over the hypoglossal nerve. They are com- 
paratively large and have an irregular surface similar to the 
left connecting band in the preceding stage. Aside from their 
greater size the large segments of the thymus in this stage present 
no striking morphological changes from those of the 52 mm. 
embryo. 

Embryo of 95 mm. (figures 6 and 7). This is the oldest stage 
in which the anterior portion of the thymus was modelled. The 
left thymus was chosen for reconstruction although the right 
one would have done equally well. Figure 6 represents a lateral 
aspect of the thymus head (C.t.) and the superficial thymus 
(T.s.) The thymus head lies alongside the common carotid 
artery (A.c.), its anterior end lying near the bifurcation into the 
external and internal carotid arteries. The parathyroid (Pé. 3) 
lies about midway between the two ends of the thymus head 
along its dorsal border and is closely attached to it. The super- 
ficial thymus (7’.s.) lies Along the anterior half of the lateral 
aspect of the thymus head. Its dorsal and ventral borders are 
almost parallel to each other. The caudal border is rounding 
while its anterior part tapers irregularly into the slender connect- 
ing band (P.2.) which loops over the hypoglossal nerve (V.XJJ) 
-and is connected with the thymus head. Figure 7 represents a 
ventral aspect of the same structures as seen in figure 6. The 
superficial thymus (7’.s.) and the thymus head (C.t.) are flat- 
tened laterally. The anterior portion of the thymus head lies 
in contact with the dorsal border of the hypoglossal nerve. The 
superficial thymus les closely against the thymus head but is 
not fused with it. Its posterior border on the right side gradu- 
ally tapers down to a thin edge in contrast to the blunt posterior 
border of the left superficial thymus. The diameter of the inter- 


=—.¢ Sty = 


IES S Ces 5 


DEVELOPMENT OF THE THYMUS 329 


mediary cords is considerably greater than in the preceding 
stages. Lobules are now present along their entire extent. They 
are a little shorter in this stage than in the 63 mm. embryo while 
the mid-cervical segment is somewhat longer. The cervico-tho- 
racic cords are short and lie closely together. The thoracic seg- 
ments are thin and flat and spread out over the pericardium to 
the left of the median line. They extend only a short distance 
to the right beyond the median line. 

Embryos of 105, 140, 170 and 280 mm., and pig 1 day post 
partem (text figures A, B, C, D and EH, respectively). In these 
stages the structures covering the thymus were removed and 
the entire organ on the left side, undisturbed, was exposed to 
view. Diagrammatic drawings, representing accurately the out- 
line of the lateral aspect of the different regions of the thymus, 
were made. In all cases specimens were selected in which the 
connecting band on both sides looped over the hypoglossal nerve 
and connected the superficial thymus with the thymus head. 

By referring to the figures cited above it will be seen that the 
comparative size of the superficial thymus (7’.s.) and the thymus 
head (C.é.) vary somewhat in different developmental stages. 
In general, the proportional size of the former to the latter is 
greater in earlier than in later developmental stages. From 
numerous dissections that were made it was found that the 
comparative sizes of the two structures vary considerably in 
embryos of about the same developmental stage or even in those 
of the same litter. Figure A represents about the average com- 
parative size of the superficial thymus and the thymus head in 
embryos of about 105 mm. in length, while the size of the super- 
ficial thymus of an 140 mm. embryo as represented in figure B 
is considerably larger than it ordinarily occurs in corresponding 
developmental stages. Variations in size of the superficial thymi 
in the same embryo also occur; e.g., the right one in a 170 mm. 
embryo was an oblong flap that covered the anterior one-fourth 
of the lateral surface of the thymus head, while the left one is 
much smaller as represented in figure C. Jn all the embryos 
examined the superficial thymus was always closely associated 
with the thymus head but never fused with it. 


330 J. A. BADERTSCHER 


Text figs. A, B, C, D Outline drawings of the exposed left thymus of embryos 
respectively 105, 140, 170 and 280 mm. (full term) in length; natural size. C.ct., 
cervico-thoracic cord; C.i., intermediary cord; C.t., caput thymus = thymus 
head; N.XII, hypoglossal nerve; S.m., mid-cervical segment; S.th., thoracic seg- 
ment; 7’.s., thymus superficialis. 

Text fig. E Outline drawing of the exposed left thymus of a ‘runty’ pig, one 
day old and only 240 mm. in length; natural size. The thymus in this speci- 
men was a few millimeters shorter than that in the full-term embryo; this is 
perhaps due to the fact that the specimen was a ‘runt’. C.ct., cervico-thoracic 
cord; C.i., intermediary cord; C.t., caput thymus = thymus head; N.XIJJ, 
hypoglossal nerve; S.m., mid-cervical segment; S.th., thoracic segment; T.s., 
thymus superficialis. 


DEVELOPMENT OF THE THYMUS Sal 


From the thymus head the intermediary cord (C.7.) and the 
mid-cervical segment (S.m.) extend in a meso-ventral direction 
to the anterior aperture of the thorax ventral to the trachea. 
In comparatively early stages they are more or less tortuous as 
represented in figure A, while in later stages their course is 
nearly straight. The mid-cervical segment in early stages is 
short and lies immediately anterior to the thorax while the inter- 
mediary cord is comparatively long as represented in figure A. 
As development proceeds the mid-cervical segment gradually 
becomes longer while the intermediary fe becomes shorter as 
represented in the figures. 

The cervico-thoracic segment (C.ct.) in all stages is short and 
of a comparatively small diameter. It lies in the extreme ven- 
tral portion of the anterior aperture of the thorax. In early 
stages the cords of the right and left thymus lie closely together 
and in later stages they fuse with each other. 

The thoracic segment (S.th.) in later developmental stages is 
composed of the thoracic portions of both the right and left 
thymus which have fused in this region. In embryos about 
105 mm. in length, and later stages, it is spread over the antero- 
ventral surface of the left side of the pericardium. The swinging 
of the right segment toward the left side is already noticeable 
ina42mm.embryo. This segment is thickest along the median 
line (3.5 to 4 mm. in full term embryos) and gradually tapers 
down to a thin irregular edge. 

The connecting band was present on both sides in the majority 
of specimens examined. It may, however, be absent either on 
one or on both sides. Its rupture is apparently due to the growth 
in length of the thymus not keeping pace with the growth in 
length of the neck. The expanded caudal portion of the thymus 
being firmly anchored in the anterior portion of the thoracic cavity, 
on account of the unequal rate of growth between the neck and 
the thymus, will exert a pull on that portion of the organ in the 
neck and thus greatly attenuate or tear the connecting band. 
Also there is thus a stress exerted on the hypoglossal nerve which 
apparently tends slightly to change its direction, as stated in 
the description of the thymus in 21.5 mm. embryo (p. 325). 


aoe J. A. BADERTSCHER 


A microscopical examination was made of the superficial thy- 
mus in various developmental stages, including that of two full- 
term embryos. Jt was found that the histogenetic processes of 
this segment kept pace with those in the segments of the thymus 
which have a purely entodermal origin. 


CONCLUSIONS 


The thymus of the pig has an ectodermal-entodermal origin. 
The respective origin of each segment is as follows: 

1. The superficial thymus, which is a derivative of the cervical 
vesicle, has a purely ectodermal origin. It is a constant struc- 
ture and, therefore, forms an integral part of the organ. 

2. The connecting band is also a derivative of the cervical 
vesicle and has, therefore, a purely ectodermal origin. In the 
majority of embryos it persists to birth but may be absent 
either on one or on both sides. 

3. The thymus head, in which is lodged the parathyroid III, 
is formed by a fusion of a portion of the cervical vesicle to the 
anterior end of the epithelial diverticulum derived from the third 
pharyngeal pouch. It has, therefore, an ectodermal-entodermal 
origin. 

4. The intermediary and cervico-thoracic cords, and the mid- 
cervical and thoracic segments are derived wholly from the epi- 
thelial diverticulum of the third pharyngeal pouch and have, 
therefore, a purely entodermal origin. 


I wish to thank Dr. B. F. Kingsbury for the aid given me in 
this work. I am also indebted to Dr. David Marine, of the 
Western Reserve University, for sending me many formalin- 
preserved embryos of various sizes from which most of the draw- 
ings of the exposed thymus were made. 


DEVELOPMENT OF THE THYMUS aoe 


LITERATURE CITED 


Bett, E. T. 1906 The development of the thymus. Amer. Jour. Anat., vol. 5. 

Born, C. 1883 Uber die Derivate der embryonalen Schlundbogen und Schlund- 
spalten bei Siugetieren. Arch. f. mikr. Anat., Bd. 22. 

Fiscuenis, P. 1885 Beitrige zur Kenntnis der Entwickelungsgeschichte der Gl. 
thyreoidea und Gl. thymus. Arch. f. mikr. Anat., Bd. 25. 

Fox, H. 1908 The pharyngeal pouches and their derivatives in the Mammalia. 
Am. Jour. Anat., vol. 8. 

IXASTSCHENKO, N. 1887 Das Schicksal der embryonalen Schlundspalten bei 
Siugetieren. Arch. f. mikr. Anat., Bd. 30. 

Roup, A. 1900 Contribution a l'étude de l’origine et de |’évolution de la thy- 
roide laterale et due thymus chez le campagnol. Bull. Soc. vaudoise 
des Sc. natur., T. 36. 

ZOTTERMAN, A. 1911 Die Schweinthymus als eine Thymus ecto-entodermalis. 
Anat. Anz., Bd. 38. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 5 


ABBREVIATIONS 


A.c., carotid artery Pt.3, parathyroid derived from third 
C.t., caput thymus = thymus head pharyngeal pouch 

D.b., ductus branchialis S.b. 2, sacculus branchialis II = second 
D.c., ductus cervicalis pharyngeal pouch 

Ect., ectoderm S.b.3, sacculus branchialis III = third 
G.n., ganglion nodosum pharyngeal pouch 

G.s.c., superior cervical ganglion S.b.4, sacculus branchialis 1V = fourth 
N:X., vagus nerve , pharyngeal pouch 

N.XII., hypoglossal nerve T.e., entodermal thymus 

P., pharynx T.s., thymus superficialis 

Pi., connecting band V.c., vesicula cervicalis 


V.c.l., vesicula cervicalis lateralis 
V.c.m., vesicula cervicalis medialis 


PLATE 1 
EXPLANATION OF FIGURES 


Figures 1 to 7 were drawn by Miss Cora Whitman, from wax models which 
were made by the author. The text figures A to H were drawn by the author. 


1 Drawing of a reconstruction of the pharynx and derivatives of the second 
and third pharyngeal pouches of the right side, including portions of the struc- 
tures closely associated with the pharyngeal derivatives and a portion of the 
ectoderm. Ventral aspect; pig embryo 14.5 mm. in length. Model x 92, reduced 
one-half. 

2 Drawing of a reconstruction of a portion of the pharynx, the third pharyn- 
geal pouch, anterior portion of the thymus anlage, parathyroid 3, cervical vesicle, 
and associates of the above named structures. Caudo-ventral aspect. The 
model represented in this figure was made from the same side of the same em- 
bryo (14.5 mm.) from which the model represented in figure 1 was made. Model 
245, reduced one-half. 

3 Drawing of a reconstruction of the same structures as enumerated under 
figure 2. This model was made to show specially the relation of the cervical 
vesicle to the thymus anlage and the hypoglossal nerve after the shifting of the 
structures in the neck have become quite noticeable. Ventral aspect, left side; 
pig embryo 17.5 mm. in length. Model < 182, reduced one-half. 


334 


DEVELOPMENT OF THE THYMUS PLATE | 
J. A. BADERTSCHER 


PLATE 2 
EXPLANATION OF FIGURES 


4 Drawing of the same model as represented in figure 3, with the hypoglossal 
nerve and a portion of the ganglion nodosum removed to expose the cervical 
vesicle and more clearly to show its relation to the thymus anlage. Dorso-lateral 
aspect; reduced one-half. 

5 Drawing of a reconstruction showing the relation of the vesicula cervicalis 
lateralis (7'.s.) to the thymus head (C.t.) and their topographical relation to 
neighboring structures. In this stage the thymus and the cervical vesicle, which 
have fused, have lost their connection respectively with the entoderm and ecto- 
derm. Right side, lateral aspect; pig 21.5 mm. in length. Model x 182, reduced 
one-half. 

6 Drawing of a reconstruction showing the topographical relation of the 
superficial thymus to the thymus head. These two structures, in the specimen 
from which the reconstruction of the thymus was made, are connected with 
each other by the connecting band (P.7.) which loops over the hypoglossal nerve. 
Left thymus, lateral aspect. 30, reduced one-half. 

7 Drawing of model represented in figure 6; ventral aspect. 


336 


DEVELOPMENT OF THE THYMUS PLATE 2 
J. A. BADERTSCHER 


337 


MITOCHONDRIA (AND OTHER CYTOPLASMIC 
STRUCTURES) IN TISSUE CULTURES 


MARGARET REED LEWIS AND.WARREN HARMON LEWIS! 


From the Anatomical Laboratory, Johns Hopkins Medical School, and the Marine 
Biological Laboratory, Woods Hole, Mass. 


TWENTY-SIX FIGURES 


CONTENTS 
IEAM PROKOUDIOTANC Nat... ane ght Stchits Gren artnet ig Mika scale Preuss Gisin 0 Ace eee hee bh: 340 
WOGIMING, t.3 ore eRe acto eae Oh SOG ee ON ote feb rSlre: SOMO Ry a RE Dy oe Oe 341 
RUDE ANOVA os 25 crete ate Ot Re SELEY oct ES A oy Me 344 
WiitOochondriacese se athe otter coe ree eee sc ie Oe Del ae oe 347 
JEONG Ol MMNUTCENOMNOITA, -cooocodonobooonsedodcietovndodausnoedarasonsucss 348 
SHApeyOremitochOnaii Aas cance icy ce cde ene sy okete oA nea hn 352 
STAC LOteNGOCH ON GIN ee Mica AA yuan anes cle eM es heat ee ee oe 359 
NiumbersotemnbochoOndritiscpayc ests acticin noe ee os on ees 360 
Quantity of mitochondria... 2st ash > ieee aot: Sake etn ce Doth ewes 360 
Relation between position, size, number and quantity of mitochondria... 362 
DECE MERLE mT OCMOMORA wrcha.cchea So Scene mul a Ae cota lrs henna hi aaa 363 
INieoelacrielBiet Tah MANOS, dap ooooas eames & eceroeuie bide ase aoe Salminen one 365 
Miro chomdmicnnmnrchiter ete kaimd Skoicelll Saas een nn ene 372 
| Bisa graven ean TEM SHOT eR Ase A orp tage ne MCR Ra Ai Cet Ae Ree are eRe erm eels 373 
TRIBE EL AKOTA) EXCISE ane Dee ae ae OER RS Re Sikes, SR eke nL Se bat ee eta 373 
VCACuION SG Ona calTeSt eae ric ctaphin Orsitc ciongn 5 Aree ae ae etre, ot me aaa 374 
Readchonsronaylolarchlorotormrether 4 ssee see eee ee ee ae eee 374 
Reaction to hyper andthypotonic solutions:...J5.0. 0.5602 o> eens ene. 374 
TEM SEY CILANGTEUHUG)LOVELEIES cael Mtl 5 elles aera ne 8 neg a ke Bab 374 
WUTC AGREES 2 & ORM ey Ave 1 each ANA CU Ne Sg Me a 9 a OS 376 
AIGHOV TIS) ARSE OF 5 NSC RNS. i sai CENSOR OP Ren MN PNENE ig ee Reco ete Ne ined Cm a ARETE 376 
Nile blue B extra and brilliant cresyl blue 2b... 2.2.0 -...0.0..-25 0560s se: 377 
GVO SVE degaene AED EA LS). 3's hn ON he eo a Oe Ey IRS, ey a 379 
Certain other cell structures and their relation to the mitochondria........ 380 
CGAL Sie en meee rey 0 We Wo nh OE URN Cae Oi aS kun Wa 380 
NSC LOL CS heyaerce encom ste he pee MA hea A Ce yrs POR As ees 381 
Retr clab lester enn sida We eh ak eee bale e Nawete ad Maly 386 
Wait ea lecuele tar seins lee oe etl Aas a se in oes ce SE ens s See ree noe S os 390 
ANUAOLOIST ES} CEU aKOUTENG 5 71 (G2) KC Ne Cray Ge Re a a 391 
IDS ODISETVOIN SO Bis oS Sedan ie Say RE SY ys RO Rel ac 392 
Conc lusionts sae pene ty ee nen Se oa gia aehi os Ase e304 
EDO COED Lye TS EU NS 6G thik ie, ocsssle. sib os Aedes REG Oe ORR 397 


1 We are indebted to the Marine Biological Laboratory for the use of a room 


during the summer of 1914. 
339 


340 MARGARET R. LEWIS AND WARREN H. LEWIS 


INTRODUCTION 


Tissue cultures afford a new and somewhat different method 
from that usually employed for the study of many cell struc- 
tures. It enables one to compare the living with the fixed mate- 
rial. In fact, one can study the same cell while living, during 
the process of fixation, and later as a stained permanent prep- 
aration. It also enables one to follow the changes which take 
place in the living cell from minute to minute. Above all, tissue 
cultures afford a method by which we can experiment on the 
cells and mitochondria. And through such methods only do 
we believe a correct interpretation of the significance of mito- 
chondria is to be found. 

In spite of the new and different environment of the tissue, 
i.e., Its isolation from the rest of the embryo; the substitution 
of a simple Locke’s solution for normal plasma; the contact with 
the cover-slip; and the absence of a circulation, which continu- 
ally renews the food-supply and removes the waste, the cells 
of the tissue cultures are apparently quite normal during the 
first two or three days and exhibit no noticeable changes except 
the characteristic configuration of the growth. How greatly the 
new environment disturbs the normal metabolic processes of the 
cell is impossible to surmise. The cells are in such a thin layer 
that each cell is probably as well bathed by the Locke’s solu- 
tion as in the embryo it would have been bathed by plasma 
or lymph. 

In the older cultures the cells lose their normal appearance 
and show signs of degeneration. Migration, growth and mitosis 
cease, the cells become smaller and show both cytoplasmic and 
nuclear changes. This may be due to the fact that the medium 
lacks both the inorganic and organic substances necessary for 
the prolonged continuance of life, but when we consider that the 
same degeneration takes place when tissues are explanted into 
a plasma medium it seems more probable that the degener- 
ation is due to an excess of waste products accumulated around 
the cell. 


MITOCHONDRIA IN TISSUE CULTURES 341 


It is during the first two or three days then that we may 
compare the cells and their structures with those found in the 
embryo. The mitochondria have been studied during this early 
period when ‘their appearance and behavior can be considered 
normal. The close resemblance of the mitochondria found dur- 
ing this early period to those found in the chick by other ob- 
servers (Benda, Meves, Duesberg, Dubrueil, Cowdry, ete.) shows 
that they at least are not noticeably altered in the culture. We 
are justified, we believe in assuming that our findings concern- 
ing mitochondria apply as well to the normal cells within the 
embryo as they do to the cells of the tissue culture. 


TECHNIC 


The ordinary technic for the cultivation of tissues in Locke’s 
solution as described by Lewis and Lewis (’11, 12) was used. 
We found great variations in the amount, duration and character 
of growth in different solutions. This was apparently not due 
to the slight variation that occurs in the weighing out of the 
salts or sugar, which enter into the composition of the solution, 
since these can be varied considerably and good growth obtained. 
The trouble lies either in the distilled water, a contaminated 
container for the solution, poor chick material, or some manip- 
ulation during the process of explantation, which we vary un- 
knowingly. In repeating this work one should make several 
trials until a solution favorable for growth is obtained. When a 
favorable solution is once obtained it can be kept for months, 
provided the dextrose is not added’ to the stock solution. 

Chick embryos were taken out of the egg under aseptic condi- 
tions and put into 10 or 20 ce. of sterile Locke’s solution (NaCl 
0.9 per cent, CaCl,0.025 per cent, KCl 0.042 per cent, NaHCO; 
0.02 per cent, dextrose 0.25 per cent at 39°C. A piece a few milli- 
meters in diameter of the desired tissue was then cut out and 
placed in another dish which contained 10 or 20 ce. of sterile 
Locke’s solution at 39°C. This small piece was then cut up 
into numerous very small pieces. These were drawn up into 


342 MARGARET R. LEWIS AND WARREN H. LEWIS 


a fine pipette, usually one at a time, with some of the solution 
and placed each on a sterile cover-slip which was inverted onto 
a vaseline (melting point, 46 + °C.) ring on a hollow ground slide. 
All instruments and cover-slips were sterilized by passing them 
through the flame, and aseptic precautions were observed 
throughout. 

Great care should be taken to insure absolute cleanliness of 
the cover-slips. The migrating and dividing cells, as we have 
already stated, adhere to the cover-slip and utilize it as a means 
of support, and the presence of grease seems to prevent them 
from getting a foothold. The small drop should spread out 
evenly and thinly over the center of the cover-slip so that the 
surface tension keeps the explanted piece in contact with the 
cover-slip. The stereotropic cells can thus easily creep out from 
the piece to the cover-slip on which they migrate towards the 
periphery of the drop. In cases where the drop is too deep and 
the small explanted piece falls away from the cover-slip the con- 
vex surface of the drop may act as a support for growth. 

Growth began within ten to twenty hours and reached a 
maximum in extent and showed the greatest number of mitotic 
figures on the second or third day. The cultures were incu- 
bated at 39°C. to 40°C. in an electric incubator (with a glass 
in the door). The presence of the electric light which was placed 
in the same chamber with the cultures for the purpose of main- 
taining the temperature of the incubator did not seem to affect 
the growth. Cultures apparently grow as well in the light as 
in the dark. 

Around the piece of explanted tissue the new growth forms 
a more or less radiating reticulum, a syncytium, or a membrane- 
like sheet of cells with varying numbers of isolated cells. The 
growth may be several cells in thickness near the old piece, but 
toward the periphery there is usually only a single layer of 
flattened cells which are often scarcely 2 u in thickness (fig. 1). 
The entire contents of these peripheral cells can be observed 
with very little change in focus. The growth is so closely at- 
tached to the cover-slip that in many cases the explanted piece 
can be torn away without injury to the new growth. 


MITOCHONDRIA IN TISSUE CULTURES 343 


a. Pea a. Ay 
~ 2 bs “S Yo me, > 
a,“ ' 6 iV EX : 
‘ be ay = 
: "oo 2 
~ RD heh oL ye N, < 
; : Vantes? eb wy he 
x Ch a >: “- f * Py : . uf —_— 
, BP eae Sak y Ode 
ae * « ‘ 2 Tr 
ane —~—* oe id Led - 
ow My hla 
)* Aloe JE 
* me *» ss Fe PI a 
a 9 : pas Selng 
’ ar ~~ = A he : 
are ; 
h . * % 
N 
~ 


Fig. 1 Photograph of part of a 2-day culture of intestine from an 8-day chick. 
The black mass is the explanted piece, which is surrounded by the new growth 
of connective tissue and smooth muscle; there are 13 mitotic figures in this part 
of the culture; osmic vapor and iron hematoxylin. 


Although several different kinds of cells have been identified 
in the living cultures (Lewis and Lewis) as, for instance, the 
mesenchyme and connective tissue cells, the heart and smooth 
muscle syncytium, the endodermal membrane, the yolk mem- 
brane, the nerve cell, the kidney tubule cell, ete., nevertheless, 
the general cytoplasmic structure of the living cell, regardless 
of the kind of cell, is practically the same except in cases where 
the cells contain secretory granules. The cytoplasm appears 
as a homogenous substance within which are several types of 
granules, i.e., refractive fat globules, various shaped mitochon- 


344 MARGARET R. LEWIS AND WARREN H. LEWIS 


dria and other granules. The nucleus appears as a finely gran- 
ular body surrounded by a definite nuclear wall with one or 
more nucleoli. The nucleolus is never a round compact body, 
but instead is a coarsely granular ragged body, often large in 
proportion to the size of the nucleus. The nucleolus can read- 
ily be seen with the low power even when the outline of the 
nucleus cannot be distinguished. At one side of the nucleus 
there is usually present the central body (idiozome). 

When permanent preparations were desired the cover-slip was 
removed from the vaseline ring and the entire culture fixed to 
the cover-slip by means of osmic acid vapor. After fixation 
the explanted piece was often torn off from the cover-slip, leaving 
the new growth, in order to facilitate certain staining processes. 
Since the growth is very thin it was unnecessary to cut sections. 
The cover-slip with the fixed growth was treated as one would 
sections on a slide. ; 


FIXATION 


The entire process of fixation can be watched and studied 
upon any cell, as, for example, one that has been under obser- 
vation for some time. While the preparation is observed under 
the microscope some of the fixing solution can be introduced 
into the cavity of the shde through an opening made in the 
vaseline ring, which seals the coverslip to the slide. The speci- 
men can be fixed with either vapor or fluid. If a vapor is used, 
as of osmic acid, a small drop of a 2 per cent solution of osmic 
acid is introduced on the bottom of the cavity so that it does 
not come in contact with the hanging drop. If a solution is 
used, enough is introduced to fill the entire cavity and mix with 
the hanging drop. 

The vapor from a freshly made 2 per cent osmic acid solu- 
tion gave the best results, and when used with care resulted 
in a fixed cell, which more closely resembled the living cell than 
any other method we have used. The osmic acid vapour seems 
to cause a precipitation of the cell structures in the form of 
minute granules. Even after such fixed specimens are stained 
by means of iron hematoxylin the general character of the cyto- 


MITOCHONDRIA IN TISSUE CULTURES 345 


plasm and nucleus are not noticeably altered except for the 
staining. 

B. F. Kingsbury (12) states that according to Rawitz (’07), 
Kollarewsky (’87) and Eisen (’00) osmic acid does not preserve 
nuclear details. So far as can be seen from our material the 
living cell exhibits few nuclear details and even osmic acid vapor 
differentiates more clearly the nuclear structures than they can 
be distinguished in the living cell. 

The mitochondria are so well fixed by osmic acid vapor or 
by a fixing solution which contains osmic acid that it has been 
suggested that the mitochondria may be artifacts due to osmic 
fixation. Vapor from strong formalin which has been carefully 
neutralized (Mann ’02 and also Bensley °11 recommend that 
formalin be freed from acid by careful neutralization and redis- 
tillation) gave good results in regard to fixation not only of the 
mitochondria but of all cellular structures. Unfortunately, the 
mitochondria did not stain well after formalin fixation. Iodine 
vapor from a crystal of iodine often afforded good results in 
regard to the spindle fibers and also the mitochondria, especially 
so when followed by Bensley’s anilin fuchsin, methylene green 
stain, but 1odine was an uncertain fixative. Osmie acid solu- 
tions do not give as uniformly good results as the vapor. 

Any fixing solution which contained acid (acetic, hydrochloric, 
sulphuric, etc.) proved useless as a fixative for tissue cultures. 
The vapor from such acids coagulated the entire cell before 
the fluid touched the preparations. The mitochondria rapidly 
changed into small granular rings, which later were completely 
lost in the coagulated network of cytoplasm. The nucleus lost 
its homogenous finely granular structure and a coarse network 
appeared. The nucleolus became a small round body. This re- 
sembles closely the usual textbook figure of a cell, which by 
long association one has come to believe represents a cell but 
which actually resembles the living cell not at all. 

When a living cell (fig. 2a) was exposed to the action of 
vapor from 2 per cent glacial acetic this coagulation effect was 
soon apparent, as shown in figure 2b. The cytoplasmic and 
nuclear networks rapidly appeared while the mitochondria, which 


346 MARGARET R. LEWIS AND WARREN H. LEWIS 


>= —sS \ = loavele 


acme Oui ee 11.02r 11.03 11.04 am 12.54pm 12.55 pm 12.56 pm 
e 


TTL LLY fo FF 


1.30 pm 1.34pm 1.36 pm 
L 10 Het ate a eile ee ate E15) elon eG ael6r pm 


b d 
[eo soa on oe Se See See ee 
C- ca a 
11.16am 11.17 11.18 11.19 11.20 11.21 11.22 11.23 am 
Cc 
11 pm 
nee a bs Vas : 
1.8 pm 


Fig. 2. A, sketch of living cell with nucleus and mitochondria, the cytoplasm 
should be homogenous. b, the same cell after exposure to the vapor of 2 per 
cent acetic acid; both cytoplasm and nucleus show reticular structure due to 
coagulation; the mitochondria are destroyed except for a few granular remains. 

Fig.3 Changes in shape of mitochondria as observed in living cells; a, changes 
by bending during a period of 4 minutes; b, by shortening and elongating and 
shifting along the mitochondrium of the mitochondrial substance, time 6 minutes; 
c, by fusion of granules; in the course of 7 minutes the four granules a, 6, c, d 
became fused into two granules ab and cd; a, b, c from a 2-day culture of mesen- 
chyme from a 43-day chick; d, changes in shape and fusion of mitochondria to 
form network in the course of 6 minutes; e, changes in shape and fusion, 2 minutes; 
/, changes in form of a single mitochondrium during a period of 7 minutes; d, e 
and f from a 3-day culture of mesenchyme from a 4-day chick. 


MITOCHONDRIA IN TISSUE CULTURES 347 


were clearly seen in the living cell as long rods and threads 
rapidly disappeared. Brunn (’84) found that the Eberth bodies, 
which have since been shown to be mitochondria, are dissolved 
by acetic acid. Duesberg (’11) states the fact that they are 
destroyed by acetic acid to be one of the criteria for mitochon- 
dria. Prolonged fixation in osmic acid after the osmic vapor 
has already fixed the preparation did not cause any distortion 
of the nucleus or the cytoplasmic structures. The mitochondria 
became somewhat blackened and the fat globules were first 
yellow-brown and later became a dark brown. Even after a 
month no change appeared in the cytoplasm which in any way 
indicated the presence of a canilicular apparatus as was found 
in certain other cells by Kopsch (’02), Sjovall (06), and Cowdry 
(712). 

A careful study of the living cell together with a study of the 
effect of various fixatives shows that while the mitochondria 
are only successfully fixed by osmic acid, they are by no means 
artifacts due to osmie acid fixation. 


MITOCHONDRIA 


The mitochondria are always present in the living cells of 
the tissue cultures and after some experience can be easily recog- 
nized. They are never as conspicuous, however, as the fat 
globules or the nucleus. The mitochondria are slightly refract- 
ive bodies which vary greatly in shape, size, and position. 
In the living cell these bodies are never quiet, but are contin- 
ually changing in shape, size, and position. Often as many as 
fifteen or twenty shapes may be exhibited by a single mitochon- 
drium within as many minutes (fig. 3). This extreme plasticity 
of the mitochondria is a very important characteristic and was 
shown in every preparation examined. It is certainly a feature 
which must be reckoned with in any attempt to classify or to 
analyze their behavior from fixed material. 

The chaotic condition of the literature in respect to the ter- 
minology and criteria for mitochondria and other cytoplasmic 
bodies renders it difficult often to correlate our findings with 


348 MARGARET R. LEWIS AND WARREN H. LEWIS 


those of other observers. It is important then that we should 
as far as possible submit the bodies herein under consideration 
to already established criteria for mitochondria. 

While these bodies fulfil Benda’s (’99) original criterion for 
mitochondria in embryonic cells in that they stain blue with 
alizarine, we have made no effort to fulfil Montgomery’s (11) 
criterion that they must show an unbroken cycle from egg to 
somatic cell to anlage sex cell and back to the fertilized egg. 
They do, however, correspond with Duesberg’s (711) criterion 
for mitochondria in the adult cell, in that they are seen in the 
fresh preparation, dissolved by acetic acid, preserved by osmic 
acid, and stain by the same dyes as the mitochondria in em- 
bryonic cells, that is, green with Janus green in the fresh prep- 
arations (Michaelis ’99, Laguesse ’99, Bensley ’11, Cowdry ’12); 
stain blue with Benda’s stain (Benda ’03, Meves, ’08, Duesberg 
09); red with Bensley’s anilin fuchsin, methylen green stain 
(Bensley ’11, Cowdry 712), and black with Heidenhain’s iron 
hematoxylin. 

Janus green caused the death of the growth after a few hours, 
and frequently the mitochondria separated into granules (fig. 21). 
For this reason Janus green was used only to identify various 
granules as mitochondria but never for any observations upon 
the changes in shape, size or quantity of mitochondria. 

These bodies have been given various names—mitochondria 
and chondriomiten by Benda; chondrioconten, chondriosomen, 
chondrion and plastosomen by Meves; plasmafaden, plasma- 
k6ren by Retzius; paramiton or miton by Flemming; micro- 
somen by Van Beneden; granules and filament by Altman, ete. 


Position of mitochondria 


Great variation occurs in the arrangement of the mitochondria 
even in the same kind of cells in the same preparation, not 
only in the living but also in the fixed preparations. It is not 
uncommon for the mitochondria to be more or less evenly 
scattered throughout the cytoplasm and the various processes 
of the cell. They have been observed even in the extremely 


MITOCHONDRIA IN TISSUE CULTURES 349 


slender processes that are scarcely larger in diameter than a 
mitochondrium. This rather uniformly scattered arrangement 
usually occurs during mitosis and here likewise the cell processes 
may contain mitochondria. The spindle area is usually free 
from mitochondria (figs. 4, 15, 16, 17). Infrequently in the 
late anaphase the mitochondria may collect along the plane 
of division. In elongated cells the mitochondria are usually 
arranged at either end of the nucleus with their long axis more 
or less parallel to the long axis of the cell. However, in many 
of the cells the mitochondria are more numerous about the 
nucleus or about the central body than towards the periphery 
of the cell, where they may be scattered or entirely absent 
(figs. 4, 5, 12, 17). The central body is an extremely finely 
granular body at one side of the nucleus and has been so-called 
by us because the mitochondria frequently radiate around this 
body and because it is a non-committal term. Usually the idio- 
zome (or nebenkern, for discussion of correct terminology see 
Wilson 711) can be seen within this body and occasionally the 
centrasome can be made out within the idiozome. The central 
body is more clearly seen in the living cell than in the fixed 
cell, but in some cells this body cannot be distinguished and the 
mitochondria appear more or less radially arranged around the 
nucleus. At times the mitochondria may be confined to one 
side only of the nucleus, usually the side on which lies the cen- 
tral body. This radial arrangement about the central body 
has been described by Eberth (66), Vejdovsky (07), Meves (09), 
Veratti (09). In some preparations this arrangement is so 
marked that one cannot but wonder if there is not some definite 
relationship between the two, and it is not difficult to under- 
stand why Vejdovsky (’07) believed that the mitochondria were 
products of the activity of the centrasome. 

The mitochondria, however, are continually altering their posi- 
tion, not only in relation to the nucleus and central body but 
also in relation to each other. They seem to be continually 
emerging from the mass near the nucleus or near the central 
body and to migrate out towards the periphery. Also those 
towards the periphery often return to the central mass. There 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No.3 


350 MARGARET R. LEWIS AND WARREN H. LEWIS 


\¢ 


LING 3 Giees 
wv JOSME Noe 2 /-: 
Os ee er se 
SEA GGL ee, cay 
\’ Oe OOS AS AS Ae 
fall hee 
mat aca Wy f Oe SL 


fo € ae ai a \ 
Sh : BNNs 
/ = ie \ \ \ ili 1 
aS » \ / \ 


Fig. 4 A, b,c, d,e, cells from a 2-day culture of heart from a 5-day chick. 
a, cell with 69 mitochondria of granular type, somewhat radially arranged about 
the central body. 6, cell with 125 mitochondria of very different shapes and sizes, 
about the central body and scattered through the cytoplasm. c, cell with 37 
mitochondria about the central body, mostly granular in type; X 1080 diam. 
d, cell with about 90 mitochondrig about nucleus and central body, mostly rod- 
and thread-shaped; X 540 diam. e, group of six adjoining cells; as in a the cells 
with granular and short, rod-shaped mitochondria show the latter arranged about 
the nucleus and central body; one cell contains 40 and the other 54 mitochondria 
of the short and long rod- and thread-shaped types, which are arranged more 


MITOCHONDRIA IN TISSUE CULTURES 351 


about the nucleus and central body; the dividing cell has 118 mitochondria which 
are scattered more evenly through the cytoplasm than in the other cells; X 790 
diam.; osmic acid vapor and iron hematoxylin; f, cell from a 2-day culture of 
heart from a 6-day chick cell with 152 mitochondria, mostly rod- and thread- 
shaped, arranged about nucleus and central body; X 790 diam. g, cell froma 
2-day culture of heart from a 4-day chick. Only two cells in entire culture show 
these ring-formed mitochondria and in these two the cytoplasm was abnormal, 
perhaps dead. Bensley stain; X 790 diam. 

Fig. 5 A, 6, c,d, four adjoining cells from a 2-day culture of heart from a 
5-day chick; X 790 diam. The resting cells, a, b and c, have 47, 51 and 48 mito- 
chondria respectively, while cell d in early prophase has 89. These cells exhibit 
great variety in the shapes of the mitochondria; small and large granules, spin- 
dles, short rods, long rods and threads are present; the mitochondria are some- 
what more scattered than the cytoplasm in cell d. e, cell with 38 mitochondria 
which vary greatly in size and shape; from the same specimen; X 540; osmic 
acid vapor and iron hematoxylin. /, g, mesenchyme cells from 2-day cultures 
of intestine from a 7-day chick embryo; f, many of the mitochondria are united 
in networks; < 540 diam.; g, some ot the mitochondria branch and anastomose 
to form a complicated network which appears to extend from one nuclear area 
to another, where the cells form a syncytium; X 790 diam.; only a portion of the 
cells and network is shown; osmic acid vapor and iron hematoxylin. 


352 MARGARET R. LEWIS AND WARREN H. LEWIS 


is probably no relation between the movement of the cell and 
the movement of the mitochondria, for the cell processes change 
their position so slowly that there is often no noticeable change 
for several hours, while the mitochondria change position rapidly 
and continually. 

Occasionally in a syncytium of cells a mitochondrium may 
pass over the cytoplasmic bridge from one cell to another (fig. 
5g). In some cases a mitochondrial thread may pass over the 
bridge into another cell and later return. 

What is it that governs the arrangement of the mitochondria? 
Is it the shape of the cell, the influence of the central body or 
“of the nucleus, the internal structure of the cytoplasm, or do 
the metabolic activities of the cell govern the size, shape and 
arrangement of the mitochondria? 


Shape of mitochondria 


The mitochondria exhibit extraordinary diversity of form often 
in the same preparation, even in adjoining cells of the same 
type (figs. 4, 5). Not infrequently a single cell may contain 
mitochondria of diverse shapes (fig. 5e). These different mito- 
chondrial shapes may be more or less localized in different parts 
of the cytoplasm (fig. 5 e) or may be more or less mixed together 
(fig. 4b). The extraordinary diversity in form of the mito- 
chondria shown by cells of the same type lying side-by-side in 
the same preparation is sometimes very striking. Such differ- 
ences occur in the young growing cells after division, in older 
resting cells and even during the various stages of mitosis. 
Again, we may find in the same preparation groups of cells 
in one part of the growth, that have very similarly shaped 
mitochondria, while in another part practically all of the cells 
may have quite differently shaped mitochondria. In such prep- 
arations all gradations in shape and size, from minute granules 
to larger and larger ones, or from rods to threads of various 
lengths, or threads and networks, etc., can be seen in adjoining 
cells of the same type or even in the same cell. Just as the 
fixed preparations show such gradations, we find that all sorts 


MITOCHONDRIA IN TISSUE CULTURES abs 


of transformations from one shape into another can be watched 
in the living cell. 

Mitochondria of various shapes have been described by other 
observers, and so definite did some of the shapes appear to be 
that they were given various names, which today are without 
much significance. Nevertheless, it is convenient to classify 
mitochondria as follows (fig. 10). 


Small granules Threads 
Dumb-bell-shaped granules Loops 
Spindle-shaped granules Rings 
Large granules Network 
Rods 


The degenerate mitochondria also show more or less definite 
shapes (fig. 13). 

Mitochondria continually change shape as by bending in vari- 
ous directions (fig. 3a), or by shortening and thickening or 
elongating and thinning (fig. 3b); at times this thickening and 
thinning seems almost like a pulsation along the length of the 
mitochondrium. These various shapes of mitochondria are not 

fixed or constant in any cell. Rods or threads may change into 
- granules; threads may fuse or branch into networks (fig. 3 d, 6, 7); 
or granules may fuse to form larger granules (fig. 3c). Degen- 
erating mitochondria may separate into granules and _ vesicles 
(fig. 13). 

Ring-shaped mitochondria are seldom found in these prepa- 
rations. Occasionally a living cell may contain one or two large 
or small ring-shaped mitochondria which rapidly change into 
threads, rods or granules. A few fixed and stained preparations 
show one or two cells at the periphery of a large growth which 
contain ring-shaped mitochondria exclusively (fig. 4g). Kings- 
bury (11) has suggested the possibility that mitochondria which 
contain a large amount of lipoid are reduced by osmic acid only 
at the surface, and the central part later dissolves out, which 
produces the appearance of rings. These ring-shaped mito- 
chondria can hardly produce fat or lipoid droplets (Dubreuil 
11, 718) since they are seldom present in cells in which fat is 
being formed. 


354 MARGARET R. LEWIS AND WARREN H. LEWIS 


a Fe ee ee ~~ 4.00 P.M 


Fig. 6 From a living culture about 24 hours old, of a piece of heart from an 
6-day chick, showing branching, fusion and splitting of two or three mitochondria 
during a period of about 1 hour. 


MITOCHONDRIA IN TISSUE CULTURES 355 


ae 


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a Ne 
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b 


2.40 P.M 2.42 P.M. 2.43 P.M 2.45 P.M. 
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2.55 P.M. 2.56 P.M. 2.57 P.M. 2.58 P.M. 


3.00 P.M 3.02 P.M. 3.05 P.M 


Fig. 7 Changes in shape and anastomoses of a few mitochondria during a 
period of 25 minutes in a living mesenchyme cell. The changes were so rapid 
that it was not always possible to draw each mitochondrium; they can be fol- 
lowed by the lettering; 24-hour old culture from a 6-day chick embryo. 


The mitochondria are frequently arranged in the form of a 
network (fig. 5 f, g) which may involve many of the mitochondria 
or only a few of them. A study of the fixed preparations and 
especially a study of the living cells shows conclusively that 
Mislavsky (11) is correct in his contention that the mitochondria 
do fuse and branch to form networks. We have observed all 


356 MARGARET R. LEWIS AND WARREN H. LEWIS 


stages of the formation of network in the living cell. These 
networks continually change shape (figs. 6, 7). New branches 
appear, old ones change shape or position or break away, and 
at times the entire network may break down into loops, threads 
and granules without any apparent change on the part of the 
cell. From our observations it appears that the network is 


Fig. 8 Mitochondria from a 3-day culture of intestine from a 7-day chick; 
osmic vapor and iron hematoxylin; X 2250 diam.; various forms of mitochondria, 
which come from the breaking down of a network, into loops, rings, threads, etc. 


very unstable and rapidly breaks down into granules, loops and 
threads. Figure 8 shows such loops and rings in a fixed specimen. 

There has been some discussion as to which shape of mito- 
chondria is the more primitive. Meves (’08) claimed that in 
the twenty-hour chick embryo the mitochondria are present 


MITOCHONDRIA IN TISSUE CULTURES 307 


only as very thin threads, but that at forty-eight hours the 
threads are thicker and also some granules are present. These 
mitochondria have heavy stained edges with a clear mark 
substance. 

Duesberg (’08) finds the same for the chick, but in the rabbit 
he describes the mitochondria in the early fertilized egg as small 
granules, which increase in volume and become large granules 
at the end of the third day. The large granules have a clear 
central part and dark outer edge. They flow together and build 
rods and threads. Rubaschkin (’11) finds only granules in the 
early guinea-pig development. He claims that the granular 
form of mitochondria is the most primitive and indifferent form. 

So far as could be observed, there is no special difference in 
the shape of the mitochondria present in the cells of the growth 
from a piece of a three-day chick embryo from that present in 
the growth from a piece of ten-day chick embryo. Only those 
cells show exclusively the small granules, which contain many 
fat globules or vacuoles. We have observed the cells of a 51 
hours growth which contained only the granular type of mito- 
chondria to contain at 70 hours mostly thread types (fig. 9 a-f). 
The threads were formed by the stretching out of the granules 
rather than by fusion of granules although such fusion of granules 
does take place. When a preparation is studied from day to 
day it is clear that the shape of the mitochondria changes and 
that no one shape is constant for any one age. 

Brown (’13) finds that in the male germ cells of Notonerta 
the mitochondrial fibers and threads arise in part at least from 
spherical-shaped mitochondria. : 

Schaxel (’11) claims that the shape of the mitochondria varies 
with the method of fixing and staining inasmuch as by the 
Benda treatment the rod-like forms predominate while after 
the Altman treatment the granular type predominates. While 
there have been few observations made as to the effect of various 
technical methods upon the shape of the mitochondria they 
appear to be such malleable structures that it is quite probable 
that their shape could be altered by different methods. 


358 MARGARET R. LEWIS AND WARREN H. LEWIS 


Pi 


g 4.50. P.M. h 4.55 P.M i 500P.M j 5:04PM kK 5.08 P.M {5.15PM 


Fig. 9 Mitochondria in living cell from a culture of heart from a 43-day chick. 
a, camera drawing at 2.20 p.m., when culture was 51 hours old; b, same cell at 
3.20 P.M.; c, at 4.20 p.m.; d, at 4.55 p.M.; e, at 5.15 p.m.; at this time all the cells 
had a similar type of mitochondria; f, camera drawing at 9.20 a.m. of next day, 
culture 70 hours old. All the cells in the culture had the same type of mito- 
chondria asine. At4 p.m. most of the cells began to show signs of degeneration 
and a fresh drop of Locke’s solution was put on preparation; the thread-like 
mitochondria, as seen in f, began to fuse into large spindle-shaped masses near 
the nucleus and central body, as in g, where all the mitochondria now present 
at 4.50 p.m. are shown, and the changes which they underwent during the next 
25 minutes are in this particular cell shown in h, 7, j, k and l. 


MITOCHONDRIA IN TISSUE CULTURES 359 


Size of mitochondria 


The mitochondria vary so greatly in size (fig. 10) that were 
it not for prolonged study of them and the use of a specific 
vital stain such as Janus green it would be difficult to believe 
that they all belong in the same class of granules. Even in a 
single cell great variation occurs from very minute granules 
which are scarcely visible to relatively large masses (figs. 4, 5, 9). 


IO ONL | {Le roniters 


Fig. 10 Camera lucida drawings of-mitochondria of various sizes and shapes 
from different cells and specimens; osmic acid vapor and iron hematoxylin; X 
790 diam. 

Fig. 11 Endodermal cells from 3-day culture of allantois from a 7-day chick; 
Bensley’s aniline fuschsin methylene green stain; X 790 diam. 


Occasionally a cell is seen in which all the mitochondria appear 
to be swollen up and much larger than those in the surrounding 
cells (fig. 11). To what this is due is not known. 

A mitochondrium under observation frequently seems to change 
in size as well as shape, but so far no micrometer measurements 
have been made to determine this point. Definite increase in 
size has frequently been seen, due to fusion of two granules to 
form a larger granule or to fusion of rods into threads; and 
occasionally all the mitochondria in the cell may become col- 


360 MARGARET R. LEWIS AND WARREN H. LEWIS 


lected in several very large granules (fig. 9). In certain patho- 
logical conditions Barratt (13) has found that the mitochondria 
become abnormally large and stain clearly. 


Number of mitochondria 


The number of mitochondria varies greatly in cells of the 
same kind in the same preparation (fig. 12) and in different 
preparations (figs. 4, 5, 12). Numerous counts of the mito- 
chondria in the same kind of cells in the same preparation show 
that there is no one number of mitochondria peculiar to any 
one kind of cell or to any one stage in the development of the 
cell. The number of the mitochondria appear to decrease and 
to increase under various conditions. This may result from 
fusion or division of the mitochondria without much change in 
the quantity of mitochondrial substance; or this may be accom- 
panied by a corresponding increase or decrease in the amount 
of mitochondrial substance, independent of any fusion or divi- 
sion of the already existing mitochondria. This would indicate 
that some of the mitochondria may at times entirely disappear 
and that possibly new ones may arise de novo in the cytoplasm. 
Sometimes most of the cells in a growth undergo such changes. 
When observed on one day they may have rather few mito- 
chondria, while on the following day most of the cells may 
contain a marked increase in the number of mitochondria, or 
the opposite phenomenon may take place. This may or may 
not be accompanied by a corresponding change in the quantity 
of mitochondrial substance. Prolonged action of heat causes 
a decrease in the size and number of the mitochondria, and 
it is hoped that further experimental work will determine what 
conditions cause such changes in the ordinary cultures. 


Quantity of mitochondria 


By the quantity of mitochondria we mean the total mass 
of the mitochondrial substance within a cell. This can only 
be roughly estimated, as some cells with many very small mito- 
chondria have a smaller quantity of mitochondrial substance 
than others with fewer but larger mitochondria. However, in 


MITOCHONDRIA IN TISSUE CULTURES 361 


Fig. 12 A, mesenchyme cells from a 2-day culture of intestine from a 5-day 
chick, showing marked differences in shape, size and number of mitochondria. 
The four cells have 74, 8, 27 and 6 mitochondria; Bensley stain; X 790 diam,; 
b, two adjoining cells from a 2-day culture of heart from a 7-day chick; granular 
type of mitochondria, one cell has 38 and the other 111 mitochondria. Osmic 
acid and iron hematoxylin; < 540 diam.; c, two endodermal cells from a 2-day 
culture of allantois from a 4-day chick; the larger cell contains about 128 and the 
other 27 mitochondria; Bensley stain; x 790 diam. 


many cases of adjoining cells (figs. 11,12b) or of the same 
kind of cells in different parts of the preparation (fig. 12 ¢) 
there can be no doubt that the quantity of mitochondria is 
markedly increased or very much decreased. This increase in 
the quantity of mitochondria is most marked in a few scat- 
tered cells in the growth from a piece of allantois (fig. 11). 

Cells with few mitochondria do not necessarily have larger 
mitochondria and there seems to be no definite relation between 
size and number or number and quantity. The quantity in the 
cell differs so widely that it has so far been impossible to con- 
nect the quantity of mitochondria with any one factor. Possibly 
it is dependent upon the metabolism of the individual cell. 


362 MARGARET R. LEWIS AND WARREN H. LEWIS 


This is also true of cells undergoing division, for there seems 
to be no amount of mitochondria characteristic of any one 
phase of division. Also the variation in the quantity of mito- 
chondria present in any one phase of division is considerable, 
as can be seen (figs. 14, 15, 16). Daughter cells usually have 
a smaller quantity of mitochondria than the metaphase cell or 
than the resting cell (fig. 17). 

As to the question whether the amount of mitochondria in- 
creases during mitosis it is impossible to state. So far, we have 
only one definite observation that this is true. In this case the 
living culture was subjected to a temperature of 46°C. for two 
hours, during which period the mitochondria decreased decidedly 
in number and size. Two cells which were under observation 
suddenly began to pass into prophase and during this process 
the number of mitochondria in these two cells increased until 
they contained more than they had before the experiment was 
begun. Although several subsequent experiments with increased 
heat caused a decrease in the quantity of mitochondria no cell 
division was observed. 

No agent but heat has so far been observed which caused a 
change in the amount of the mitochondria without injury to 
the cell. However, it is evident that certain metabolic condi- 
tions must cause a change in the quantity of mitochondria. 


Relation between position, size, number and quantity of 
mitochondria 


No definite relation between the position, size, number and 
quantity of the mitochondria has been observed in the cells of 
the tissue cultures, still there is a more or less marked manner 
in which the mitochondria occur in the cells. Frequently the 
long threads or short rods are plentiful and scattered throughout 
the cytoplasm with or without a definite central body. When 
the mitochondria are in the form of large granules and thick rods 
they are fewer in number and are arranged more or less radially 
around a central body. When only a very few mitochondrial 
granules are present they are usually of the large granule type. 


MITOCHONDRIA IN TISSUE CULTURES 363 


All phases in the development of the cell, i.e., daughter cell, 
growing cell, resting cell and dividing cell can be found with 
any one of the above combinations of the mitochondria. How- 
ever, it must not be forgotten that many cells contain part of 
one kind of mitochondria and part of another and that any 
one shape of mitochondria may turn into another at any time 
during observation, and that no one shape of mitochondria 
remains as such for a very long interval of time, but changes 
into another. 


hd 
i ee 
7 ————. 
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b 
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a hae a te © hbe 
oes 345 Pm e 3247 em 


Fig. 13 A, cell from a 2-day culture of heart from a 7-day chick; practically 
all the mitochondria are degenerated, those in the region of the central body 
show most advanced stage of granular rings; X 790 diam.; b, degenerating mito- 
chondria from mesenchyme cell of a 2-day culture of intestine from a 4-day 
chick; X 920 diam.;c, cell from a 2-day culture of heart of a 5-day chick; all the 
mitochondria have degenerated into granular rings; osmic acid vapor and iron 
hematoxylin; X 790 diam.; d, process of degeneration in a single mitochondrium 
produced by the action of acetic acid vapor on a living cell; e, effect of CO, on 
another mitochondrium in 2 minutes. 


Degenerate mitochondria 


Degenerate mitochondria of various shapes are occasionally 
found in these preparations (fig. 13). A study of the cells of 
the older growths shows that all the mitochondria do not neces- 
‘sarily degenerate at the same time. Some cells are found which 
contain many normal mitochondria, some partly degenerate, 
and others entirely degenerate. 


364 MARGARET R. LEWIS AND WARREN H. LEWIS 


This degeneration appears first in the mitochondria around the 
central body and later in those scattered at the periphery 
(fig. 13a). 

The process of degeneration of the mitochondria can be most 
successfully observed when produced by some outside agency 
such as carbonic acid gas or vapor from a weak acid solution 
(fig. 18d, e). When the death of the cell is produced experi- 
mentally the mitochondria become first a series of granules which 
soon become slightly vesicular although at this stage they still 
stain in the characteristic manner. Then these vesicles sepa- 
rate and rapidly become small, finely granular rings or shadows. 
These no longer stain like mitochondria but more like the cyto- 
plasm, i.e., brownish green with Bensley’s anilin fuchsin, 
methylen green or pale gray with Heidenhain’s iron hemo- 
toxylin, and in the living cell the Janus green does not stain 
them green. It is apparent that some change has taken place 
which has completely changed not only the morphology but also 
the composition of the mitochondria. 

These degenerate mitochondria correspond in many ways to 
the ‘‘grains du segregation’? described by Dubreuil in the lymph 
cells, but are unquestionably degenerate mitochondria, and they 
can be produced in any cell of these growths by means of various 
agents such as carbonic acid gas, chloretone, acid vapor, hydro- 
gen peroxide and potassium permanganate. 

Meves (710) and Duesberg (710) simultaneously found that 
poorly fixed mitochondria show granulation and small bladder 
forms. Other observers have found that granulation is due to 
delay in fixation after death or to disease, as Mayer and Rathery 
(07) experimental polyuria; Takaki (07) polyuria or prolonged 
fast; Policard (’10) experimental poluria and after injection of 
phlorizin; Policard and Garnier (’07), Cesa Bianchi (’10), Heiden- 
hain (711) also obtained similar results. 

Beckton (’10) claims that in a certain tumor no mitochondria 
were present in the tumor cells. In view of the observations of 
Beckwith (14) it may be possible that certain cells can exist 
without mitochondria, but it seems more probable that the 
apparent lack of mitochondria in the tumor cell described by 


MITOCHONDRIA IN TISSUE CULTURES 365 


Beckton may have been caused by delay in fixing the material 
so that the mitochondria became degenerate, or the mitochondria 
may have been present only as degenerate structures which 
did not stain. 


Mitochondria in mitosis 


Naturally, the question at once arises: What is the rdéle of the 
mitochondria during division of the cell? Many observers be- 
lieve that the mitochondria form a palisade about the spindle 
during late anaphase and then divide and one-half of each mito- 


Fig. 14 Arrangement of mitochondria in the prophase stage; a, from a 3-day 
culture of intestine from an 8-day chick embryo; X 1080 diam.; b, from a 3-day 
culture of intestine from a 7-day chick; X 790 diam.; c, from a 2-day culture of 
heart from a 5-day chick embryo; X 540 diam. 


chondrium passes to each daughter cell (Benda, Duesberg, Meves, 
etc.). Meves (713) in his work on ascaris egg goes so far as to 
state that not only are the mitochondria present in the egg and 
spermatozoon, but also that the male mitochondria are carried 
into the egg by the spermatozoon and so each egg receives not 
only female but also male mitochondria and the granules result- 
ing from the fusion of the male and female mitochondria are 
distributed to each cell of the embryo. In view of the behavior 
of the mitochondria Meves suggests that they may play a part 
in inheritance. 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 3 


366 MARGARET R. LEWIS AND WARREN H. LEWIS 


A study of the fixed specimens seems to show that the mito- 
chondria retain somewhat their original character and shape 
during mitosis (figs. 14-17). They are, however, almost always 
shorter and more scattered through the cytoplasm than in the 
surrounding cells (figs. 4, 5, 14 a, 15 b, d, 17, 18). There are 
usually as many and often more mitochondria in the early stages 
of the dividing cell than in the neighboring cells (figs. 4, 5, 
14a, b, 15:b). 

There is no indication in the fixed specimens of any arrange- 
ment of the mitochondria about the spindle in such a manner 
that they would undergo division into two parts in the plane 
of cleavage of the dividing cell. On the other hand, all of our 
specimens seem to show that the mitochondria tend to become 
more evenly scattered through the cytoplasm during division, 
and those that happen to be on either side of the cleavage plane 
are carried into the respective daughter cells. 

Since each daughter cell contains only about one-half the 
number of mitochondria found in the mother cell at the time of 
division we must assume that there is an increase sometime 
during the life of the cell between one division and the next, 
otherwise the number would rapidly decrease during each suc- 
cessive division. Now the question is: When does this increase 
take place? Is it during the so-called resting period, or during 
mitosis? In some of the fixed preparations where mitotic figures, 
daughter cells and young growing cells are numerous, it is possi- 
ble to arrange cells in a series according to the stage of recon- 


Fig. 15 Arrangement of mitochondria during metaphase; a, b,-c, f, cells from 
2-day cultures of heart from 5-day chick embryos; d, e, cells from a 3-day culture 
of intestine from an 8-day chick embryo; X 540 diam. 

Fig. 16 Arrangement of mitochondria during anaphase and telephase and 
young daughter cells, a, b, c, from a 3-day culture of intestine from an 8-day 
chick; X 540 diam. Cell a, anaphase has 156 mitochondria, the two daughter 
cells, b, have 12? and 125 each, while the older daughter cell, c, has 151 mito- 
chondria; the neighboring adult cells in this region have been 70 to 160 mito- 
chondria; d, 3-day culture of intestine from a 7-day chick; the two daughter cells 
with the smaller dark nuclei have 92 and 49 mitochondria, while the adjoining 
resting cells have only 77 and 48 mitochondria each; * 790; e, daughter cell from 
a 2-day culture of heart from a 5-day chick, with very different type of mitochon- 
dria; X 540 diam.; osmic acid vapor and iron hematoxylin. 


MITOCHONDRIA IN TISSUE CULTURES 367 


368 MARGARET R. LEWIS AND WARREN H. LEWIS 


struction of the nucleus, as indicated in figures 17 and 18. The 
younger nuclei are smaller, darker and more compact and the 
cells are smaller. The older cells are larger and contain larger 
nuclei which are less and less deeply stained. In such a series 
(fig. 17) the number of mitochondria increases from about 40 
to 150. On the other hand, the old resting cell (k) with a very 
pale nucleus has only 32. In one series (fig. 181, j, k, 1) the 
number increases from 24 to 140. In figure 18 one of the two 
daughter cells (ce) has 37, the dividing cell (a) has 140, while the 
two neighboring resting cells (e) and (f) have 39 and 47. Again, 
in figure 18, the young daughter cells (d, d) have 37 each while 
older neighboring cells (g, h) have 56 and 58 each. On the other 
hand, another dividing cell (b) near this same group has but 
60 mitochondria. 

From such observations one might conclude that there is a 
gradual increase in the number and in the size of the mito- 
chondria during the growth period of the daughter cells. The 
greatest increase both in number and size seems to occur then 
during the so-called ‘resting’ period which is in reality a period 
of growth both for the mitochondria and for the nucleus. On 
the other hand, while we are unable to determine definitely 
whether the number of mitochondria actually increases during 
the early stage of mitosis there are frequently indications that 
such cells have more mitochondria than mature cells Cell d 
(fig. 5) early prophase has 89 while the resting cells a, b, ¢ have 
47, 51 and 48 mitochondria each. In figure 4e the dividing 
cell has 118 while the three neighboring cells have 102, 126 
and 62 mitochondria each. The two cells with the larger nuclei 
are probably older resting cells and each has about the same 
number of mitochondria as in the dividing cell. 

Numerous other specimens seem to show that the dividing 
cells often have more mitochondria than any of the fullgrown 
resting cells in the immediate neighborhood. Sometimes this is 
so marked that there is every indication that the number of the 
mitochondria in some instances may increase considerably dur- 
ing mitosis. It seems probable thereforee that mitochondria 
increase in number both during the resting period and during 


MITOCHONDRIA IN TISSUE CULTURES 369 


Fig. 17 Cells from a 2-day culture of heart from a 5-day chick embryo; X 540 
diam.; a, very young daughter cells with 42 mitochondria in each cell; 6, slightly 
older daughter cells with 42 and 35 mitochondria; c, older daughter cells with 48 
and 75 mitochondria; d, older daughter cells with 43 and 38 mitochondria; e, 
older cells with 66 mitochondria; f, older cell with 96; g, still older cell with 156; 
h, prophase with 197 (?); 7, metaphase with 117; 7, anaphase with 174; k, old 
resting cell with only 32 mitochondria. 


370 MARGARET R. LEWIS AND WARREN H. LEWIS 


mitosis; perhaps in some more during the resting period; in others 
more during mitosis and in still others during both periods or 
only during one. It is very unlikely that one can arrive at a 
satisfactory solution of such a problem from fixed material, 
since the bodies we are dealing with are subject to such great 
changes in number and size during life. The number of mito- 
chondria is not of much value as an indicator of the total quan- 
tity of mitochondrial substance. 


Fig. 18 Cells from a 2-day culture of heart from a 5-day chick; X 540 diam. 
Cell a in anaphase has about 140 mitochondria, while b has only about 60, the 
young daughter cell c has 37, the two daughter cells d, d have 36 each; the older 
cells e, f, g and h have 39, 47, 58 and 56 mitochondria respectively. In the series 
1, j, k, l, the young cell 7 has only 24, the older cells 7 and & have 41 and 42, while 
the mature cell / has 140 mitochondria. 


We have indicated that the daughter cells not only have 
about one-half the number of mitochrondria found in the mature 
cells, but that the mitochondria are sometimes smaller. 

Does the increase in number during the growth period come 
about through division of preéxisting mitochondria (a process 
which frequently takes place) or do mitochondria arise de novo? 
So far as our observations go, either or both processes may occur. 


MITOCHONDRIA IN TISSUE CULTURES Sat 


The only certain method to determine just how and when the 
mitochondria increase is to follow several living cells through 
complete cycles. Unfortunately, the cells of tissue culture often 
round up during late metaphase and anaphase (fig. 15 e, f) so 
that it is impossible, except in a few cases, to follow the indi- 
vidual mitochondrium throughout cell division. 

The process of mitosis is an exceedingly slow one compared 
with that described in other tissues: Prophase 10 to 20 minutes, 
metaphase and anaphase, 1 to 2 hours; while the period from 
anaphase including telophase to the daughter cells is an exceed- 
ingly short one, never more than five minutes from the time the 
chromosomes are arranged at the opposite poles of the spindle 
until the cytoplasm is divided, except for slender processes, and 
such stages are correspondingly few in number in the permanent 
preparations. 

We have not been able to follow the number of mitochondria 
through a ‘complete cycle of the cell in the living cultures. We 
have, however, been able to watch the behavior of the mito- 
chondria during mitosis in a few living cells. Usually the mito- 
chondria are scattered throughout the cytoplasm and remain so 
during cell division. About one-half of the mitochondria pass 
to each daughter cell, namely, those which happen to be on one 
side or the other of the cleavage plane. In two or three cells 
during late anaphase most of the mitochondria became arranged 
in rather of a broad zone around the spindle in the area through 
which the division plane later formed and one-half of the number 
of mitochondria passed into each daughter cell. There was no 
indication of any division of the mitochondrial granules;'in fact, 
in one cell it was clearly observed that several thread-shaped 
mitochondria passed over entire into one of the daughter cells. 
A division of the mitochondria such as observed by Meves (’08) 
and Duesberg (’10) was never observed. We find as did Buch- 
ner (09, 710, ’11) that this characteristic arrangement of the 
mitochondria during division of the cell is by no means a con- 
stant occurrence. 

We have already stated that we are uncertain whether there 
is an actual or only an apparent increase in the amount of 


372 MARGARET R. LEWIS AND WARREN H. LEWIS 


mitochondria during mitosis. So far we have only one direct 
experimental observation to offer, and in this particular case 
there was an actual increase in the number and possibly also 
in the quantity. In this experiment the temperature had been 
raised from 39 to 46°C. and was retained at 46°C. for two hours. 
There resulted a decided decrease in the amount of mitochondria 
within all the cells. Two cells began to divide. The nuclear 
wall disappeared, the nucleoli faded and the chromosomes ap- 
peared. These cells, which a few minutes before had contained 
only a very few mitochondria, now became full of short dumb- 
bell-shaped rods, while the resting cells did not undergo any 
change. So far as could be seen by most careful observation, 
this increase in quantity of mitochondria was not due to the 
division of the existing granules. 


Mitochondria in different kinds of cells 


Regardless of the fact that the mitochondria constantly change 
in shape, size and quantity in any one cell, there is a character- 
istic appearance of the mitochondria in certain kinds of cells, 
as, for instance, the short, rod and dumbbell shapes are most 
frequently found in the cells of the endodermal membrane; 
the long threads, rods and sometimes loops are found more fre- 
quently in the connective tissue; the small granules and short 
rods are frequent in nerve fibers and cells, and are often much 
smaller than those of the connective tissue cells over which the 
nerve fiber passes; a striated arrangement together with scat- 
tered granules is characteristic of the fibroblasts; and the large 
eranules are more frequently seen in the heart and smooth 
muscle syncitium than in any other kind of tissue. At times 
the growth from the explanted intestine or heart contains only 
cells with thread- and rod-shaped mitochondria. Again, a large 
proportion of such cells contain only large granules. These 
granules are frequently so large that they are clearly seen with 
the low power. They are collected about the central body and 
appear to be more refractive than other types of mitochondria. 
Occasionally these large granules fuse. That they are not a 


MITOCHONDRIA IN TISSUE CULTURES oun 


degenerate form of mitochondria is shown by the fact that such 
cells frequently divide. In case of mitosis the mitochondria 
spread around the nucleus, and the large granules become short 
rods or dumbbell-shaped rods. 

While these certain characteristic appearances of the mito- 
chondria are found as a rule in the different kinds of cells, never- 
theless the shape, position, size and quantity vary so much that 
it is not always possible to distinguish the kind of cell by the 
appearance of the mitochondria. 


[IE 3 
/ . i] 
sii ae ae, EE TIE 
6 6.00 P.M 6.20 P.M 
Ammonia vapor Glacial acetic 1% Cc 
eee e = —_— — ohioy 
—_—— e = Sa eo ee — CAs et | 
: 315 PM 4.00 P.M 
1.30 P.M. +135 P.M. 1.37-3.00 PM. 3.10 P.M. ‘ 2 
qd Normal Hypotonic Sol, Hypertonic Sol, | Hypotonic Sol. Hypertonic Sol. Hypotonic Sol. 
d 


Fig. 19 A, effect of 2 per cent glacial acetic acid vapor on the nucleus and 
adjoining mitochondria and upon a single thread-like mitochondria; b, effect of 
strong ammonia water vapor on another nucleus with adjoining mitochondria 
and on a single thread-like mitochondrium, the reaction in both cases was almost 
instantaneous; c, effect of ammonia vapor on a single mitochondrium followed 
after 20 minutes by the vapor of 1 per cent glacial zoo acid; d, effect of hypo- 
and hypertonic solutions on 4 mitochondria. 


EXPERIMENTAL WORK 


Mitochondria in the living cell react rapidly and definitely 
to certain stimuli and in many cases they react more rapidly 
than either the cell as a whole or any other structure of the cell. 
This reaction, to be sure, often resembles a disintegration of 
the mitochondria and results in the rapid formation of varicose 
mitochondria and then the separation of the varicose mito- 
chondria into a number of small, finely granular rings. 


Reaction to acids 


When the culture is subjected to the action of carbonic acid 
gas (fig. 13 e) or the vapor of acetic, sulphuric, hydrochloric, 
chromic and other acids (fig. 19 a, 13 d) the mitochondrial threads 
rapidly assume a varicose condition and soon separate into a 


374 MARGARET R. LEWIS AND WARREN H. LEWIS 


number of small granular rings of uniform size. Hydrogen per- 
oxide, potassium permanganate and chlorotone, each produce a 
similar result. 


Reaction to alkalies 


Alkalies, ammonia gas and sodium hydroxide, on the other 
hand, cause the mitochondria to swell without any sign of vari- 
cosity. The nucleus also becomes larger and more transparent 
(fig. 19 b). 

If the ammonia vapor is followed by vapor from acetic acid 
the acid will cause the mitochondria and also the nucleus to 
return to the normal condition. We have not succeeded in 
stopping the action of the acid at this point, however, and the 
mitochondria become degenerate rings (fig. 19 ¢). 


Reaction to xylol, chloroform, ether 


Xylol, chloroform, and ether simply remove the mitochondrial 
material, or possibly dissolve the mitochondria and leave shadow 
forms or slight traces of degenerate mitochondria. 


Reaction to hyper and hypotonic solutions 


Changes in osmotic pressure affect the mitochondria often 
before any change is seen in the cytoplasm. Hypertonic solu- 
tions shrink the mitochondria while hypotonic solutions cause 
them to become swollen. The effect of a hypertonic solution 
can be removed by a decrease in the osmotic pressure of the 
solution, and, vice versa, that of a hypotonic by an increase in 
the osmotic pressure (fig. 19 d). 


Reaction to heat 


Heat gives interesting results. With an increase in the tem- 
perature of the warm stage on which the preparation is studied 
from 40 to 48°C., the mitochondria become round granules within 
fifteen or twenty minutes, regardless of their previous shape 
(fig. 20). The size of these round granules is determined by 


MITOCHONDRIA IN TISSUE CULTURES 375 


the size of the mitochondrial thread or rod before the heat began 
to act. When the heat is applied the mitochondria do not divide 
into a number of granules, as is sometimes the case when Janus 
green is used, but each one rounds up as a whole and forms one 
round granule for each mitochondrium. With rapid cooling of 


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6.00 P.M 40°C 600+ PM 46°C 6.01 P.M 47°C. 6.01+ PM. 48°C, 6.02 P.M. 47°C. 
= 
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G02 FIM S756 6.03 PM 47°C 6.04PM 46°C 6.05 P.M, 46°C. 6.06 P.M. 45°C. 
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$ Cold water passed = = 1 or 2 migrated 
&S, through the coils = towards the central 
61SPM 46°C of the warm stage iam body; 3 or 4 fol- 
618 PM 30°C 6.20. P.M. 44°C lowed at 6.32 P.M 
= 
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6.25 P.M 44°C 630 PM 46°C 6.32 P.M. 45°C. 6.35 PM. 48°C. 637/EM 45°C 
Cold water passed ae > ae = 
through the coils : f = 
of the warm stage 645 PM 39°C 647PM 45°C, 7.00 P.M. 48°C. 


Fig. 20 Part of living cell, drawn with camera lucida, showing position” of 
4 mitochondria which were drawn at intervals, while the temperature was first 
increased and kept at 46 to 48°C. for 15 minutes, then cooled to 39° for 3 minutes 
and again increased to 44 to 46° for 20 minutes; again cooled to 39° for 8 min- 
utes, and finally increased to 48° again. 


a] 


the preparation, by passing cold water through the coils of the 
warm stage, the mitochondria return to their normal shape. 
Prolonged heat, such as 46°C. for over an hour, has in a few 
instances reduced the number and also the size of the mito- 
chondria in a given cell. 


376 MARGARET R. LEWIS AND WARREN H. LEWIS 


VITAL DYES 
Janus green 


Janus green (di-ethyl saffranin azo di-methyl] aniline) has been 
considered a more or less specific stain for mitochondria in the 
living cell, according to Laguesse (’99), Michaelis (99), Bensley 
(11), Cowdry (12-14). Unfortunately, in our preparations, 
while the dye stained the mitochondria a brilliant blue-green, 
it was also toxic to the cells, and even the weakest solution 
(1-200,000) which definitely stained the mitochondria caused 
the death of the cells within a few hours. Not only did the 
dye prevent further growth, but in most instances it also caused 
various amounts of distortion of the mitochondria. In a few 


é H ry ‘ 
a c 
Fig. 21 Changes exhibited by 4 different cells after application of Janus green; 


in a and 6b the mitochondria were all long threads before the Janus green was 
applied and had begun to split up before the drawings could be made. 


cases the mitochondria moved, changed shape and appeared quite 
normal, although distinctly stained, but usually the mitochon- 
drial threads or rods separated into granules (fig. 21), shortly 
after the stain was applied. This is an indication of a slight 
degree of degeneration on the part of the mitochondria (see 
experimental work) and possibly the cell is already injured, 
although not so greatly as to interfere immediately with the 
activities of the cell, as in many cases the cell continued to 
move after the stain had been applied and in one observation 
on a heart muscle cell in which the mitochondria granules were 
deeply stained with a (1—100,000) Janus green solution the cell 
continued to beat for one hour and forty minutes. At the end 
of this time the stain had faded out and the cell ceased to beat. 

The dye was dissolved in the Locke’s solution, which was 
used for that particular explantation, and after a drop of the 
solution containing the dye warmed to 39°C. had been dropped 


MITOCHONDRIA IN TISSUE CULTURES 377 


on the growth it was drawn off and the tissue again bathed in 
a fresh drop of the warm solution free from dye. The mito- 
chondria take up-the dye within a few minutes and remain 
stained from thirty minutes to two hours. So far as we have 
observed, the intensity with which the mitochondria stain does 
not depend upon the strength of the solution. A very weak 
solution (1—100,000) gives as intensely stained mitochondria as 
does a strong solution (1-5000). A weak solution, such as 
1-100,000 Janus green, stains only the mitochondria a blue- 
green, while the cytoplasm, nucleus, and nucleolus remain clear. 
A strong solution (1—-5000), however, stains the cytoplasm a pale 
green, the mitochondria a darker green, the nucleolus green, 
and the nucleus a more or less violet-green. 


Nile blue B extra and brilliant cresyl blue 2b 


Aside from Janus green, no dye used in these observations 
stained the mitochondria in the living cell. Both nile blue A 
concentrated or B extra and brilliant cresyl blue 2 B, however, 
did stain the mitochondria after the death of the cell, especially 
after fixation either with neutralized formalin vapor or osmic 
acid vapor. This is interesting in connection with the work 
of Lorrain Smith (’08) on differential stains for fats. He states 
as follows: 

It was observed that watery solutions of nile blue sulphate (A), a 
colour stuff of the oxazine series, stains the fat globules contained in 
tissue cells in various colours. In the majority of cases the fat globules 
are stained a brilliant red; occasionally globules are present which take 
a blue stain, and not infrequently the colour is due to a mixture of blue 
and) red.!° )/"% . We may express the reaction in the following 
way: The fatty acid combines with the oxazine base to form a blue 
soap, whereas both neutral fat and fatty acid merely dissolve the rela- 
tively weak oxazone base (red). 

He remarks in relation to tissues fixed with formalin that 
the globules stain readily either red or blue according to their 
composition: 

When a globule contains a small amount of fatty acid and a large 
amount of oxazone base is present in the solution of the dye, the globule 


becomes predominately red, whereas if the stain is relatively weak in 
oxazone the blue colour of the oxazine staining is more apparent. 


378 


MARGARET R. LEWIS AND WARREN H. LEWIS 


We found that not only do nile blue (A concentrated and B 
extra) and brilliant cresyl blue (2b) show the above changes 
of color found by Smith (’08) with fats but also that each dye 
changes from blue to pink in the presence of certain other sub- 
stances as shown in table 1. 

Both brilliant cresyl blue 2b and nile blue B extra are toxic 
to the cell, and a preparation never lived more than an hour 
after even the weakest (1—200,000) solution of the nile blue B 


extra. 


Brilliant cresyl blue 2 b is less toxic than nile blue and 


each of these stains is in a way antiseptic, for no infection took 
place after the stain was used although the dye was not steri- 
lized. The color reactions with these stains on the living and on 
the dead cells are shown in table 2. 


TABLE 1 
NILE BLUE | BRILLIANT CRESYL 
B EXTRA BLUE 2B 
Sodium carbonate...............; (in solution) | blue _ blue 
Sodium carbonate............... | (dry) red | bluish violet 
Lithium carbonate.............. (in solution) | pink | violet 
Potassium hydroxide............ | pink | pink 
Sodium hydroxide............... | pink | pink 
Ammonia Wateiy.c. oa. <3 «sisted 2 precipitate red dirty brown 
| solution 
TABLE 2 
i me a ch 
| 
CYTOPLASM] NUCLEUS |NUCLEOLUS) peoormrs | \(f10.22) | GRANULES | DRIA 
Living cell 
Nile blue clear clear clear __refrac- _ pink | blue clear 
B extra tive | 
Brilliant clear clear pale | refrac- | pink | purple | clear 
cresyl blue | blue __ tive | 
2b ae oe. Sale oe al ik a: 
Dead or fixed cell 
Nile blue | pale blue blue- blue clear | blue | blue 
B extra blue | violet | | 
Brilliant | pale _—blue- blue | blue clear purple  gray- 
cresyl blue | violet violet | | | | violet 
2b | | 


& 


MITOCHONDRIA IN TISSUE CULTURES 379 


The difference in the results obtained when these dyes are 
used upon dead cells and when used upon living cells shows 
clearly that the chemical conditions which exist in the living 
cell are quite different from those in the dead cell. What hap- 
pens in the living cell to prevent the mitochondria and fat glob- 
ules from taking on the pink or blue color which is assumed 
immediately upon the death of the cell? Was the dye itself 
oxidized and why did the vacuoles and certain other granules 
stain? The vacuole certainly does not take the pink color due 
to the presence of fat of any kind, for death of the cell would 
hardly remove the fat but would only change it possibly from 
neutral to acid fat and the vacuole should then change from 
pink to blue color instead of fading out entirely. If on the other 
hand the pink color is due to the alkaline nature of the vacuoles, 
why then does it not either remain pink or else become blue? 
Why does the nucleus remain unstained until death of the cell 
begins and then the nucleolus first take on the stain and later 
the nucleus? Is the pale blue color of the nucleus after brilliant 
cresyl blue 2 b in the living cell a delicate indicator that the cell 
is injured by the dye? These are but a few of the questions 
suggested by the different action of these dyes upon the dead 
and the living cell and which must be left for the physiological 
chemist to solve. 

This change is most readily seen when a cell has first been 
stained while it is living and then fixed under the microscope. 
As the preparation dies the pink vacuoles fade out and the 
nucleolus, the nucleus, cytoplasm, fat globules and mitochondria 
stain. This is not due to the direct action of the fixative upon 
the stain itself since a fixed preparation which has been well 
washed with Locke’s solution gives the same results with these 
dyes. 

Lodine 


It might be mentioned in this connection that while the vapor 
from a crystal of iodine did fix the mitochondria as reddish 
brown threads, rods and granules, there was no evidence of 
any port wine colored granules of glycogen attached to any 


380 MARGARET R. LEWIS AND WARREN H. LEWIS 


mitochondrium nor within the loop or ring shaped mitochondria. 
A few glycogen granules were occasionally present, however, 
as could be distinguished by the color reaction. The fat glob- 
ules stain first a pale port wine color which later becomes black- 
ened. If Guilliermond’s (’13) conclusion that the loop-shaped 
mitochondria give off glycogen granules is correct, one would 
certainly expect to find that iodine used in connection with un- 
fixed material would show this at least during the final stage 
in the formation of the glycogen when the granule lies free but 
still in the neighborhood of the mitochondrium from which it 
came. 


CERTAIN OTHER CELL STRUCTURES AND THEIR RELATION TO THE 
MITOCHONDRIA 


Granules 


Certain other granules were present in most of the cells of 
these growths, but so far, these granules have not been carefully 
studied. They can be differentiated from mitochondria of simi- 
lar shape by the greater rapidity with which the granules move 
through the cytoplasm. Certain of the vital dyes which color 
these granules leave the mitochondria unstained. Neutral red 
usually stains one or several granules near the central body. 
Nile blue B extra and brilliant cresyl blue 2 b also stain certain 
granules near the central body. In cells which contain the body 
we have termed vacuole (see below) one or more of these granules 
are present within the vacuoles and are stained blue within a 
pink vacuole (nile blue B extra) or purple within a pink vacuole 
(brilliant cresyl blue 2b). These granules are few in number in 
the normal cell but plentiful in cells which contain many vacuoles. 
Other vital dyes stain certain granules within the cell, but so 
far as our observations go they are the same as the neutral red 
granules or else as the nile blue, and brilliant cresyl blue granules. 

At times the granules within the vacuole take the Janus green 
color as a very pale green, but no other relation between these 
granules and the mitochondria has been found. 


MITOCHONDRIA IN TISSUE CULTURES 381 


Vacuoles 


There are two distinct types of degeneration of the cells of 
the tissue cultures. The cell either suspends activities, rounds 
up and dies, or else the cell continues its usual activities but the 
cytoplasm becomes filled up with vacuoles and the mitochondria 
become small granules (fig. 22). In a healthy cell a vacuole is 
often seen to come and go in the cytoplasm, but when several 
vacuoles remain in the cytoplasm degeneration has begun and 
the cell never again resumes its normal appearance, but con- 
tinues to accumulate vacuoles until most of the cytoplasm is 
used up and only a network which contains scattered granules 
remains. 


Fig. 22 Cell from a 3-day culture of intestine from a 7-day chick embryo; 
the cell has a number of vacuoles near the nucleus, most of the vacuoles contain 
one or more granules; X 1580 diam. 


In the fixed and stained preparations the vacuole appears 
either as a clear space often difficult to differentiate from the 
fat globule space, or it appears as a clear space within which is 
a faintly stained granular substance (gray with Heidenhain’s 
iron hematoxylin or red brown with Bensely’s anilin fuchsin, 
methylen green stain). 

In the living cell these vacuoles are distinctly different from 
the fat droplets. They appear to be fluid spaces not at all 
refractive, in fact, they resemble a hole in the cytoplasm. Small 
dancing granules which vary in number from one to many may 
be suspended in the fluid of the vacuole or closely attached to 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 3 


382 MARGARET R. LEWIS AND WARREN H. LEWIS 


the side. These granules usually stain a pale green with Janus 
green stain. | 

Nile blue B extra and brilliant cresyl blue 2b each act as a 
differential stain for these bodies. The vacuole stains pink and 
the granules blue (nile blue B extra) or purple (brilliant cresyl 
‘blue 2'b, fig. 23): 


a, ee eee 


10.46 A.M. 10.48 A.M. 


Ay. © © ©) .@554b .@) (Se soa | 
i Aye 11 A.M. 11.5 A.M. 11.12 A.M. 
EON 4 ©. wm @ Ge = aes 
a Ae ani sea 11.30 A.M. b 15pm,  18P.M. 


\X 11.8 A.M. 
Soe Q) X11.5 A.M. ®) ca 
Cc KR d a e . 

a 
oss —— @55 o---: oo ~ X12.30 P.M. 
X12 Noon 12.2 P.M. 


(8) vac.b. 11.20 A.M. 


vac.a Vac. b.C © © we ( t a 
© 2 11.20 A.M. 12 Noon 12.20 P.M. 
Oo 
\ [ 4 t ' fg fo 6. ie 
e) 12.45 P.M. 1.45 P.M. 
a 
e 
° Vac. c. @) ©) @ Cae e, f° a 

! 12.15 P.M. 12.27 P.M. 1P.M. 


Fig. 23 Observations on the behavior of vacuoles in the living cell with 
brilliant cresyl blue 2 B (see text). 


MITOCHONDRIA IN TISSUE CULTURES 383 


Brilliant cresyl blue 2b, which is much less toxic than nile 
blue B extra, shows a most interesting behavior on the part of 
these vacuoles and granules. A vacuole may appear in the cyto- 
plasm as a clear unstained space which at first contains no 
granules, but within five to ten minutes the granules appear as 
dancing bodies as though they were the result of some conden- 
sation and precipitation within the vacuole. This process con- 
tinues and the vacuole takes on first a pale violet color, but later 
a bright pink, and the granules condense into one or two purple 
granules. Then the vacuole exhibits various movements such as 
sending out long pink streamers or threads or becoming U-shaped. 
Such a vacuole may decrease in size until only the purple gran- 
ule can be seen. When a cell which contains many vacuoles 
is stained, all gradations between the pale non-granular vacuole 
to the single purple granule can be seen. 

One of the many observations made upon healthy cells in 
which vacuoles appear is given in full below (fig. 23). 


10.45 a.m. 1 gtt. (1-100,000) brilliant cresyl blue 2b in Locke’s 
solution + 0.25 per cent dextrose was placed on the preparation. 

10.50 a.m. The cytoplasm remains clear, several purple granules 
appear. The mitochondria are unstained, slightly refractive bodies, 
the fat globules are unstained and highly refractive, the nucleus is 
unstained, but the nucleolus is a pale blue (fig. 23 a). There is present 
one vacuole in the cell, which is stained a brilliant pink and contains 
a large purple granule. The vacuole sends off a long pink streamer 
quite as long as the thread-like mitochondria but not so thick (fig. 
23, vac.a.). The vacuole manifests great activity. The streamer at 
times becomes detached from the vacuole and fades out. Again it 
appears to be drawn into the vacuole or sent out from the vacuole. 
Fig. 23 b. 

11.00 a.m. The streamer no longer appears, and the vacuole itself 
begins to change shape (fig. 23 b) and continued until 11.30 when the 
vacuole began to grow smaller and a deeper pink. It then remained 
more or less quiet but grew much smaller in size. 

1.05 p.m. The vacuole again sent off a pink streamer which lasted 
only two or three minutes, after which the small vacuole with one 
large granule remained quiet. 

11.00 a.m. A small granule (x) at the other side of the nucleus moves 
rapidly to and fro between the nucleus and the periphery several times 
(fig. 23 c,d, e, f). A streamer of pink follows the purple granule until 
at 12 noon while the granule moved rapidly towards the nucleus the 
streamer broke off two pink granules which instantly faded out. 


« 
384 MARGARET R. LEWIS AND WARREN H. LEWIS 


12.30 a.m. The granule shows no sign of pink vacuole or streamer 
and remains quiet near the nucleus. 

11.20 a.m. A clear unstained vacuole (vac.b.) appeared in the cyto- 
plasm between two of the purple granules (fig. 23 g) and behaved as 
follows, (fig. 23 h). 

11.30 a.m. It became a pale violet vacuole with a few dancing un- 
stained granules. 

11.45 a.m. It was a violet vacuole with purple granules. 

11.50 a.m. Violet vacuole became pink with purple granules. 

12.00 Noon. The vacuole condensed into a small bright pink vacuole 
with only one purple granule. 

12.13 p.m. The vacuole entirely disappeared and only the purple 
granule remained. 

12.20 p.m. Purple granule sent out a pink streamer. 

12.45 p.m. The pink streamer osculates and is rapidly sent out and 
drawn in again. 

1.45 p.m. The granule became quiet and the streamer disappeared. 
The purple granules later moved as a rod from the periphery of the 
cell in towards the nucleus and back several times. It passed over 
and under the mitochondria without hindrance. Other purple gran- 
ules in the cell moved rapidly without streamers, some as double gran- 
ules, others as rods or as single round granules. 

12.10 p.m. A pale space appeared in an adjoining cell (fig. 23 1). 

12.15 p.m. This space became pale violet. 

12.20 p.m. Violet color changed to pink. 

12.25 p.m. Granules appeared in the vacuole. 

12.27 p.m. Granules became deep purple granules. 

The mitochondria and the fat globules remain unstained in all the 
cells of the growth. In some other cells of the growth many pink vacu- 
oles are present and also many purple granules. In such cells the 
mitochondria are mostly small granules and only a few rod- or thread- 
like ones remain. In the cell under observation the mitochondria did 
not change type although they were continually changing shape. 


There was no direct connection between the mitochondria 
and the formation of the vacuoles in the above observation, and 
yet in many cells there is often a coincident change in the shape 
of the mitochondria until in cells which contain many vacuoles 
within the cytoplasm the mitochondria are no longer in the 
shape of rods and threads but then appear as small granules. 

As stated above, the fixed and stained preparations (fig. 24 a) 
do seem to show all stages in the formation of the vacuoles 
from the mitochondria just as Dubreuil and Guilliermond have 
shown the formation of bodies from the mitochondria. How- 
ever, the fact that the vacuoles have been observed to arise 


pie 


MITOCHONDRIA IN TISSUE CULTURES 385 


independently of the mitochondria, although there is a coincident 
change in the shape of the mitochondria makes one exceedingly 
wary of accepting any evidence from the fixed and stained prep- 
arations in this regard without corroboration from observations 
upon the living cell. 


——=_- -—>_ —~. a ee 


d 


Fig. 24 One-day culture of heart from a 5-day chick embryo; a, cell with 
various shaped mitochondria similar to those figured by Dubreuil and Guillier- 
mond, which led them to conclude that mitochondria formed fat and other 
bodies. Observations on this cell while living gave no evidence for the forma- 
tion of such bodies from the mitochondria; 6 and ¢ show successive forms of 
two mitochondria from the above cell and d also shows changes exhibited by a 
single mitochondrium that Guilliermond might have interpreted as showing the 
formation of a droplet; e, the rod-shaped mitochondrium which is applied closely 
to the vacuole was observed, while the cell was living, to migrate from some little 
distance to the vacuole; it had no connection with the formation of the vacuole. 
If the specimen had been fixed to show the condition, as in e, one might have 
concluded that the mitochondrium had something to do with the formation of 
the vacuole or droplet. 


Certainly the mitochondria are intimately connected with any 
change in the cytoplasm, often as in the case of heat without 
manifestation of change by other bodies in the cytoplasm, and 
it is probable that any change which takes place in the cyto- 
plasm such as would cause the formation of vacuoles or other 
bodies would also have an influence upon the mitochondria of 
that cell. 


386 MARGARET R. LEWIS AND WARREN H. LEWIS 


Fat globules 


The connection between the mitochondria and the formation 
of fat is a very complex and much discussed subject. It has 
undoubtedly been shown that the mitochondria are bodies which 
contain lipoid (Fauré-Fremiet ’09; Regaud and Mawas ’09; 
Fauré-Fremiet, Mayer, Schaeffer, 710; Regaud 710; Mawas ’10; 
Mayer, Rathery, Schaeffer, °10; Duesberg 711; Dubreuil 713; 
Cowdry ’14). Our experimental work shows that the mito- 
chondria act in many ways like bodies which contain lipoid. 
They are soluble in xylol, chloroform or ether, are slightly black- 
ened by means of osmic acid, and in fixed preparations are stained 
blue by means of nile blue B extra and yellow by means of Sudan 
Ill. It seems probable that the bodies which contain lipoid 
should form the fat globules, and many observers have tried 
to establish this (Metzner ’90, Zoja ’91, Loyez ’09, Russo ’09, 
Dubreuil 713). Others have claimed that the mitochondria are 
indirectly connected with the formation of fat (Bluntschli ’04, 
Van der Stricht ’05, Van Durme ’07, Lams and Doorme ’08, 
Schoonjams ’09). 

The masterly papers of Dubreuil (11, °13) appear to show 
clearly and concisely each step in the formation of fat droplets 
from the mitochondria, and without doubt from the fixed mate- 
rial which Dubreuil had at hand it seemed to be the logical con- 
clusion that the fat is formed from the mitochondria. Guillier- 
mond (’13) in a set of observations equally clear uses many 
figures similar to those of Dubreuil, but reaches the conclusion 
that the mitochondria form the glycogen granules of certain 
cells. It is certainly evident from our observations that no defi- 
nite conclusions can be drawn from the morphology of the mito- 
chondria present in any one cell at any one time. Various 
chemical tests and continued observation of a given mitochon- 
drium are necessary to establish any morphological conclusion. 

In our fixed preparations (fig. 24 a) all the figures shown by 
Dubreuil as evidence that the mitochondria form the fat can 
be found, i.e., threads, loops, rings and fat droplets, but the 
study of any one such mitochondrium in the living cell has 


MITOCHONDRIA IN TISSUE CULTURES 387 


never shown that fat droplets arise from mitochondria (fig. 
24b, c, d). A thread may form a loop, but the loop changes 
back again into a thread instead of continuing into a ring. 
Various rings studied have never changed into globules during 
observation but have become rods or threads or granules. Such 
appearances as figure 24 e, were caused by the migration of a 
mitochondrium to the edge of a vacuole and not as both Dubreuil 
and Guilliermond might conclude, that the mitochondrium 
formed the vacuole. Certain granules or thick rods seen in the 
living cell have the appearance of hollow bodies in the perma- 
nent preparations and correspond to some of Dubreuil’s figures. 
This appearance may be due to fixation as Kingsbury (’11) 
suggests, i.e., that the osmic acid reduced more at the surface 
and later the more soluble interior is dissolved out. Both 
Meves (’08) and Duesberg (’11) describe the clear inner part of 
the mitochondrium, to quote Duesberg, the mitochondria were 
first present in the early rabbit embryo as small granules but 
these increase in volume and become large granules at the end 
of the third day. They have a clear central part with a dark 
outer edge. Such appearance was seldom seen in the living 
cell and it is possible that these as well as certain figures of 
Dubreuil and Guilliermond were formed by the method of fix- 
ation. Cells which contain both loop and ring shaped mito- 
chondria frequently show no sign of fat formation, while other 
cells which are accumulating fat show no mitochondria of the 
shape which Dubreuil leads us to suppose form the fat droplets. 
There are three distinct types of fat in these tissue culture 
growths. First, that in the cells which grow out from tissues 
that at the time of explantation of the piece of tissue, con- 
tained fat droplets as the yolk membrane or the migrating fat 
cells. There seems to be a predetermined ability on the part 
of these cells to form fat, as is clearly shown where the growth 
from the yolk membrane adjoins that from the connective tissue 
(fig. 25). Each new yolk membrane cell contains fat droplets 
similar to those of the explanted piece of the yolk membrane. 
In these cells the mitochondria are usually in the form of small 
granules and the fat droplet is surrounded by granules which 


388 MARGARET R. LEWIS AND WARREN H. LEWIS 


stain like mitochondria. In the migrating fat cells which contain 
few fat globules some of the mitochondria may be in the form 
of threads or short rods but there is a coincident change in the 
shape of the mitochondria with the accumulation of fat drop- 
lets so that a cell which is crowded full of fat droplets contains 


Fig. 25 Photograph of part of a 2-day culture of intestine from a 6-day chick. 
The explanted piece of intestine is from the region where the yolk-sac is attached, 
and the cells on the left ot the culture are similar to those from cultures of the 
yolk-sac; each endodermal cell has one or two large fat globules; on the right are 
mesenchyme cells free or almost free from fat. 


only small granule shaped mitochondria. The fat droplets are 
outlined by a row of granules which stain lke mitochondria 
(fig. 26 b). 

The second type of fat is one or two small round refractive 
granules found in almost all the cells of the growths. These 
fat globules have not been observed to increase markedly in 


MITOCHONDRIA IN TISSUE CULTURES 389 


size or to change their shape. During mitosis they remain 
stationary and all may pass over to one daughter cell or part 
to one and part to the other daughter cell. No relation between 
these fat globules and the mitochondria was observed. Cells 
which contain one or no fat globules often contain loop or ring 
shaped mitochondria, but prolonged observation of these has 
not shown any increase in the amount of fat. The third type 
of fat is that of an accumulation of fat droplets in many of the 


Fig. 26 A, b, cells from a 2-day culture of heart from a 10-day chick in which 
fat is accumulating; a was studied while living, after staining with nile blue 
B extra, after osmie acid vapor, after nile blue B extra again, Sudan III and 
Bensley’s aniline fuchsin methylene green. No relation could be found between 
mitochondria and the formation of fat; b, a typical wandering fat cell with many 
small granular mitochondria about the fat droplets; c, cell from a 2-day culture 
of heart from a 11-day chick, accumulating fat; prolonged fixation with osmic 
acid followed by iron hematoxylin; the fat appears as dark granules. 


cells of a preparation due to some unknown cause (fig. 26 a, ¢). 
These cells accumulate fat droplets from day to day, and some 
cells may become crowded full of fat droplets within forty-eight 
hours. Such cells should show the relation between the mito- 
chondria and the fat globules were such a relation present, but 
so far as our observations go none such could be established. 
These three types of droplets are undoubtedly fat. It is 
possible to treat the same cell with various fat stains in succes- 


390 MARGARET R. LEWIS AND WARREN H. LEWIS 


sion and to compare the results. The cell shown in figure 26 a 
was first studied and drawn while living. The clear, refractive 
fat globules were easily recognized. A drop of nile blue B extra 
(1-100,000) was added without any change in the appearance of 
either the fat globules or of the mitochondria. After a few 
minutes this was washed off and the preparation was fixed in 
osmic acid vapor for a few minutes. The same cell was then 
examined and the fat globules were stained a yellow brown, 
while the mitochondria remained clear. A drop of nile blue 
B extra was then added. The fat globules took a dark blue 
stain and the mitochondria a pale blue. A drop of Sudan III 
was then added and the fat became yellow while the mitochondria 
stained a trace of blue. The specimen was then dehydrated 
and stained with Bensley’s anilin fuchsin, methylene green. The 
fat droplets were dissolved and the mitochondria stained a bril- 
liant red. 

Early in the experimental work it was observed that the mito- 
chondria under certain conditions became granules around a 
vesicle. This vesicle stained pink with the nile blue B extra in 
the living cell, and at that time it was supposed that this indi- 
cated the formation of a fat by the mitochondria since Lorrain 
Smith (’08) had shown that nile blue stained neutral fat pink 
in tissue cells. Later it was observed that nile blue B extra 
only stains fat in the dead and not in the living cell, and there- 
fore there was no indication that the mitochondria are in any 
way connected with the formation of fat. 

So far as our observations go they show no direct relation 
between the mitochondria and the formation of fat, although 
in some cases there is a coincident change in shape of the mito- 
chondria with the accumulation of fat droplets. 


Canalicular system 


One of the interesting cytoplasmic structures, the canalicular 
system, found by other observers, has as yet not been observed 
in these living cells. 

Bensley (11) by means of neutral red observed the small 
canaliculi as clear spaces in the deeply stained pancreatic cell. 


MITOCHONDRIA IN TISSUE CULTURES 391 


In the cells of tissue cultures neutral red stains only a few gran- 
ules unless used in such strong solutions as to stain the entire 
cytoplasm. In such cases a few clear unstained spaces were seen, 
but a study of the living cell and of the same cell fixed after 
the neutral red stain by means of osmic vapor and stained with 
Bensley’s anilin fuchsin, methylen green stain demonstrated that 
the clear space seen in the cells stained with strong neutral red 
solution are only the unstained mitochondria. 

The description of the Binnennetz given by Perroncito (11) 
certainly resembles in many ways the behavior of the mito- 
chondria in the tissue culture cells. He finds a network which 
is like that sometimes seen in these cells, and the ‘corona’ of 
granules shown in some of his figures appears very much like 
the mitochondria granules radiating out around the central body. 
In some of our permanent preparations where vacuoles are 
present these spaces have all the appearance of the canalicular 
system. 

Prolonged fixation in osmic acid did not reveal the canalicular 
system, although the mitochondria became slightly blackened 
by the action of the osmic acid. However, none of the special 
stains for the canalicular system were used, as we desire to deal 
only with the structures seen in the living cell. 


Amitosis and giant cells 


Many cells of these growths contain two or more nuclei and 
the membrane within the nucleus, which Childs (’07) described 
as connected with amitosis, is occasionally seen in such cells, 
but no definite relation between such cells and the mitochondria 
has been observed. Certainly in some giant cells containing 
many nuclei, the number of mitochondria present is far greater 
than that present in a normal cell of the same growth, in fact, 
it is so much greater that it seems to be definitely related to the 
amount of nuclear material and to the extent of the cytoplasm. 
These cells show clearly that there is some other method of 
increase in the number of mitochondria than that of division 
at the time of mitosis, for these giant cells appear to be formed 


392 MARGARET R. LEWIS AND WARREN H. LEWIS 


by an amitotic division of the nucleus without a coincident 
division of the cytoplasm. 

In regard to the structures of the differentiated cell, such as 
muscle fibrillae, ete., we have no observations to offer. How- 
ever, from the behavior of the mitochondria in various shaped 
cells it is quite evident that any change which affected the 
morphology of the cell might also change the position of the 
mitochondria in such a way that they might appear to be con- 
nected with the formation of the differentiating structure. 


DISCUSSION 


We have made no attempt to formulate a theory from the 
above observations in regard to the origin or function of the 
mitochondria. A review of the literature shows that the mito- 
chondria have been found in almost every kind of cell. They 
are present in the oocyte and spermatocyte (Benda °97, Van 
der Stricht 00, Meves ’11, and others) and are carried over by 
the spermatozoon into the egg’ cell in fertilization (Benda ‘11, 
Meves 711); they are abundant in cells of the young embryo 
(Meves ’08, Rubaschkin 711); they occur in plant cells as well 
as in the cells of most animals, including certain of the Protozoa 
(Lams ’09, Duesberg ’10, Meves ’04, Guilliermond ’12). It is 
claimed that they form certain cytoplasmic structures such as 
the fibrillae of the connective tissue (Meves ’10), the neuro- 
fibrillae in the growing neuroblast (Hoven 710), the myofibrillae 
(Duesberg 710, Torraca 714) the fibrillae of the epithelial cell 
(Herxheimer ’89, Korotneff ’09, Fauré-Fremiet 710, Firket 711); 
that they play a part in the process of cornification (Firket ’11); 
that they form the secretory granules, directly or indirectly, in 
the salivary (Regaud and Mawas ’09, Bouin ’05), gastric 
(Schultze 11), mammary (Hoven ’11) and other glands (Schultze 
11). They are described in the rods of the urinary tubule 
cells (Schultze ’11, Regaud ’08), in the intestinal cells (Champy 
10), in the liver cell (Policard ’09). They may form the test 
of the foraminifera (Fauré-Fremiet ’13). They are described in 
connection with the formation of the retina cells (Leboueq ’09). 


MITOCHONDRIA IN TISSUE’ CULTURES 393 


Numerous observers have claimed that they form the fat directly 
(Altmann ’89-’95, Metzner ’90, Zoja 791; Arnold ’07, Russo 
07, Loyez ’09, Van der Stricht ’05, Policard ’09, Frissinger 
09, Regaud °10, Fauré-Fremiet ’10, Dubreuil 713); indirectly 
(Bluntschli 04, Van der Stricht 705, Van Durme ’07, Lams and 
Doorme ’08, Schoonjans ’08). It is claimed that they form the 
leucoplastids, chloroplastids and chromoplastids and possibly the 
glycogen (Guilliermond ’12—’13). 

The above theories seem impossible to correlate. It seems 
evident that the mitochondria are too universal in all kinds of 
cells to have the function of forming any one of the above struc- 
tures of differentiated tissue, and in the light of what cytological 
chemistry is known, it appears practically impossible for the 
mitochondria to form all the cell structures mentioned above. 
In view of the fact that the mitochondria are found not only 
in almost all animal cells but in plant cells as well it seems 
more probable that they play a role in the more general physi- 
ology of the cell. It may be possible that they are concerned 
with respiration. As suggested by Kingsbury (712), they may 
represent the structural expression of the reducing substances 
concerned in cellular respiration, which process Matthews (’05) 
has described in his theory of protoplasmic respiration. Accord- 
ing to Matthews, the activity of the cell causes reducing bodies 
to be formed in the cytoplasm for whose neutralization oxygen 
is necessary. The hpoid nature of the mitochondria makes it 
possible to consider them as reducing bodies and certainly the 
mitochondria exhibit activities which may be due to the fact 
that they are continually formed in the cytoplasm and con- 
tinually oxidized. On the other hand, the mitochondria may 
have to do with assimilation or they may even be stored-up 
food-stuff themselves, which are continually used up and restored 
again. Beckwith (’14) holds that the mitochondria are unneces- 
sary for the life of the cell or for the development of such a 
complicated structure as a Hydractinia ciliated planula. The 
fact that such a large group of observers should each have evi- 
dence to show that the mitochondria form some one structure 
of the differentiated cell shows that the mitochondria must be 


394 MARGARET R. LEWIS AND WARREN H. LEWIS 


intimately connected with all transformations of the cytoplasm. 
On the other hand, we must bear in mind the fact that many 
observers have neglected to identify the body which they had 
under observation in such a manner that one can be certain 
that they had the same body which another observer would 
term mitochondria. It is quite doubtful whether all the bodies 
called mitochondria are really the same. 

The criterion for mitochondria in the embryonic cell, as stated 
by Duesberg after Montgomery, is one which the observer would 
hesitate to carry out, but some criterion in the sex cell, in the 
embryonic cells and also in the adult cells should be established 
for the mitochondria, which all workers will endeavor to fulfil, 
in order that there may be some common ground for discussion 
of the results obtained by the numerous observers at work in 
this field. 


CONCLUSION 


1. Tissue cultures afford an excellent method for observations 
upon an undisturbed cell as it lives, divides and grows in a 
medium of known chemical constitution; for experimental work 
on a living cell; and for the study of the process of fixation. 

2. These living cells do not correspond to the usual concep- 
tion of a cell obtained from the study of fixed material. Both 
cytoplasm and nucleus are finely granular, almost homogenous 
in appearance. There is no sign of a reticular or of an alveolar 
structure of either the cytoplasm or nucleus. Osmic acid 
vapor is the best fixative for these cells. 

3. Mitochondria are present in all the cells of these growths 
as slightly refractive, large or small granules, rods and threads, 
similar to those of the chick embryo cell. The mitochondria 
can be followed and studied in the living unstained cell for 
hours. 

4. The mitochondria may be scattered throughout the cyto- 
plasm or they may be located around the nucleus or around 
the idiozome. Any one mitochondrium may change its position 
in regard to other mitochondria or in regard to the entire cell. 
Mitochondria located around the centra_some may later migrate 


MITOCHONDRIA IN TISSUE CULTURES 395 


out and become scattered through the cytoplasm, or those scat- 
tered throughout the cytoplasm may become located around the 
nucleus. During mitosis the mitochondria become more evenly 
scattered throughout the cytoplasm, except in the spindle area, 
where they are usually absent. 

5. Any and every shape granule from a minute to a large 
granule, from small short rods to long threads, loops, rings and 
networks of various shapes and sizes can be found. Any one 
type of mitochondria such as a granule, rod or thread may at 
times change into any other type or may fuse with another 
mitochondrium, or it may divide into one or several mitochon- 
dria. Every type of mitochondria is continually changing 
shape and may assume as many as fifteen or twenty shapes in — 
ten minutes. The shape of all the mitochondria in a cell can 
be changed by experimental means such as heat or hyper- or 
hypotonic solutions. 

6. The mitochondria vary greatly in size from minute granules 
to irregularly shaped, large granules, from short rods to long 
threads. The size of a single mitochondrium may change by 
the fusion of two or more granules or by the division of a single 
mitochondrium. ‘They also appear to increase or decrease without 
such fusion or division. 

7. The number of mitochondria in a single cell varies from two 
or three to over two hundred. The number of mitochondria 
is not constant for any one kind of cell or for any phase of any 
one kind of cell. Daughter cells contain about one-half the 
number of mitochondria present in the mother cell. The number 
of mitochondria increases from the daughter cell to the mature 
dividing cell, and apparently also at times during mitosis. 

8. The quantity of mitochondria is not constant for any one 
kind of cell. Some cells with many small granular mitochondria 
contain less mitochondrial substance than other cells with a few 
large granules. 

9. Degenerating mitochondria become first a series of gran- 
ules; later the granules become vesicles and then separate into a 
number of small finely granular rings which stain like the cyto- 
plasm rather than like mitochondria. 


396 MARGARET R. LEWIS AND WARREN H. LEWIS 


10. The mitochondria become more or less scattered through- 
out the cytoplasm in an indifferent manner and decrease in size 
during mitosis. About one-half the quantity of mitochondria 
is separated into each daughter cell by the plane of division. 
The individual mitochondria pass over entire into one or the 
other daughter cell and do not each divide into two halves, each 
going to one daughter cell, as usually described. 

11. There are some characteristic differences in the mito- 
chondria of different kinds of cells, but these are not constant 
enough to be sufficient to distinguish the kinds of cells. 

12. The mitochondria are extremely plastic bodies and often 
react more rapidly than any other cell structure. They are easily 
_ influenced in shape and quantity by varous agents, such as heat, 
carbon dioxide, acids, alkalies, fat solvents, and potassium per- 
manganate, or by changes in osmotic pressure of the surround- 
ing medium. 

13. The mitochondria are stained in these living cells by 
Janus green but not by nile blue B extra or brilliant cresyl blue 
2 b except in the dead cell. 

14. Other granules are present in the cells which are not 
related to mitochondria. 

15. Mitochondria show at times a coincident change in shape 
with the formation of fat droplets or vacuoles in the cytoplasm, 
but there is no evidence in these cells of a direct relation between 
the mitochondria and the formation of either the fat droplets 
or the vacuoles. 

16.. In giant cells the number and quantity of mitochondrial 
substances is greatly increased above that of the normal cells, 
somewhat in proportion to the increase in the amount of the 
cytoplasm and nuclear material. 

The mitochondria are extremely variable bodies, which are 
continually moving and changing shape in the cytoplasm. There 
are no definite types of mitochondria, as any one type may change 
into another. They appear to arise in the cytoplasm and to be 
used up by cellular activity. They are, in all probability, 
bodies connected with the metabolic activity of the cell. 


MITOCHONDRIA IN TISSUE CULTURES 397 


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Ai : 
: rips 


THE ORIGIN AND EARLY DEVELOPMENT OF THE 
POSTERIOR LYMPH HEART IN THE CHICK 


RANDOLPH WEST 


From the Laboratories of Comparative Anatomy, Princeton and Columbia 
Universities 


FOURTEEN FIGURES 
INTRODUCTION 


During the autumn of 1912 Professor McClure suggested 
to the writer the advisability of working out the early develop- 
ment of the posterior lymph heart in the chick, with especial 
reference to the source of its endothelium. Throughout the 
following winter the problem was carried on under Professor 
MeClure’s supervision at Princeton University, while during 
the past year it has been continued under the direction of Pro- 
fessor Huntington at Columbia University. 

Sala (1) in 1900 described the development of the posterior 
lymph heart of the bird, and gave a review of the literature to 
that date. In the caudal sections of an embryo of six days and 
eighteen hours incubation he finds that: 


In the mesenchyme which stands in the lateral relation to the caudal 
myotomes and corresponds to the lateral branches of the first five 
coccygeal veins, a progressive excavation occurs of little spaces or 
fissures which soon enter into communication with the lateral venous 
branches themselvyes—one would say in fact that these fissures are 
only simple dilatations and ramifications of the veins themselves. 


If the writer interprets him correctly, Sala states that the lymph 
hearts are formed by an addition of spaces to the veins, and then 
a few lines later intimates that these spaces might be considered 
as “ramifications of the veins themselves.’ He also states that 
the ‘fissures’ are at first few in number and are arranged in a 
linear series, parallel to the axis of the vertebral column, corre- 

403 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 
May, 1915 


404 RANDOLPH WEST 


sponding to the point of penetration of each venous branch of 
the intermuscular septum, and that afterward they gradually 
increase in number and come to lie near each other. He points 
out that at the end of the seventh day many of the little ‘fissures’ 
have fused to give rise to larger spaces, so that the spaces, sepa- 
rate at first, have finally established irregular communications 
between themselves, by breaking down their mesenchymal 
partitions. He goes on to show that by the end of the eighth 
day the ensemble of the cavities is transformed into a kind of 
a sac, still communicating with the first five coccygeal veins and 
later with the general lymphatic system, which develops inde- 
pendently by fusion of intercellular mesenchymal spaces at first 
appearing along the veins of the hypogastric plexus. The 
cavities at this stage often contain red blood cells and some- 
times appear quite full of them, and by a condensation of 
mesenchymal cells the wall of the lymph hearts are formed. 
The rest of this paper, which does not especially concern us, 
shows that the lymph hearts increase in volume up to the six- 
teenth day, that the first and fifth coccygeal veins lose their con- 
nections with the hearts during this period, and that the con- 
nection of the lymph hearts with the independently developed 
general lymphatic system occurs toward the end of the tenth 
day. During the remainder of embryonic life the lymph hearts 
persist, but shortly after the chick is hatched they commence 
to degenerate. Traces of the degenerating lymph hearts were 
found in a chicken thirty-five days after hatching. 

Mierzejewski, in 1909 (2), published an article on the origin 
of the lymphatic vessels in birds, which was presented by M. H. 
Hoyer before the Academy of Sciences of Cracow. Concerning 
the origin of the posterior lymph hearts he agrees with Sala, 
except that he holds that the first anlagen appear in the middle 
of the sixth day of incubation, and not, as Sala states, in the 
first hours of the seventh day. 

Stromsten (3) has published two papers in 1910 and 1911 on 
the development of the posterior lymph heart in turtles. He 
finds that their development is initiated in the logger-head turtle 
by the vacuolization of the post-iliaec mesenchymal tissue during 


ORIGIN OF THE POSTERIOR LYMPH HEART 405 


the latter part of the second week of development, and that 
the spongy tissue thus formed is invaded by capillaries from the 
dorso-lateral branches of the caudal portion of the postcardinal 
veins. The capillaries do not communicate primarily with 
the mesenchymal spaces. Near the close of the third week, 
parallel veno-lymphatic channels are formed in this spongy 
area by the confluence of mesenchymal spaces with one another 
and with the invading capillaries. These veno-lymphatics 
anastomose freely with each other and communicate by two or 
three openings with the veins running along their mesial bor- 
ders. Finally a condensation of mesenchyme and an invasion 
of muscle cells form the wall, while a confluence of the veno- 
lymphatic sinuses gives rise to the single sac-like cavity of the 
adult form of the lymph heart. 

The subject rested at this point until 1912 when E. L. Clark 
(4) cited observations, based on injections, to show that in the 
chick of five days and twenty hours, in the region later occupied 
by the posterior lymph heart, there exists a lymphatic plexus 
connected with the coceygeal veins, but not with the haemal 
capillaries which bear a superficial relation to the lvmphatic 
vessels. She also shows that the lymphatic plexus is of a differ- 
ent pattern than the blood capillary plexus and is filled with stag- 
nant blood, which she considers as backed up from the cocecyg- 
eal veins. This state of affairs undoubtedly exists in the chick 
of five days and twenty hours but the observation, aside from 
its morphological value, throws no light on the origin and mode 
of growth of the lymphatic plexus. 

EK. R. and E. L. Clark (5) in a paper in the same number of 
The Anatomical Record attempt to prove, by observing the 
first appearance and early growth of this blood filled lymphatic 
plexus in the living chick embryo of about five days, that it is 
formed by a purely centrifugal outgrowth from the coccygeal 
veins. ‘To quote from their article: 


The first lymphatics in the tail region of the chick arise as direct 
lateral buds from several of the main dorsal intersegmental coccygeal 
veins, and not by the transformation of a previously functioning 
blood vessel plexus. From now on the lymphatic endothelium is 


406 RANDOLPH WEST 


specific and spreads by a steady centrifugal extension 
The buds send out processes forming clusters. From the clusters, in 
turn, processes are sent out which anastomose with one another, form- 
ing a plexus. Simultaneously, processes grow toward the surface from 
the clusters, and give rise to the superficial plexus of peripheral lym- 
phaties of the posterior part of the body. There is no essential dif- 
ference between the manner of growth of the peripheral lymphatics 
and that of the plexus which is to form the lymph heart (p. 258). 


In June 1913 Miller (6) in a preliminary note on the develop- 
ment of the thoracic duct of the chick states that certain aggre- 
gations of mesenchymal cells mentioned by Sala (1) ‘‘com- 
prise developing blood cells which are differentiated in situ 
out of the indifferent mesenchymal syncytium, that these blood 
cells then gain access to the lymph channelsmaking up the devel- 
oping thoracic duct, and that finally the haemal cellular ele- 
ments in question, reach the blood stream via the thoracic duct 
and the jugular lymph sae.’ He clearly recognizes that lym- 
phatic channels may serve to transmit blood cells arising 77 situ 
in the mesenchyme to the haemal channels and distinguishes 
this function of the lymphatics by the term ‘haemorphic.’ He 
further states that ‘‘the lymphatics arise as isolated lacunae 
directly from mesenchymal intercellular spaces and are not in 
any sense derived from the veins, and subsequently coalesce 
to form the continuous channel of the thoracic duct.’ The 
possibility of venous origin of these lymphatics or of the back- 
ing up of their blood content from the veins is excluded by the 
total absence of the azygos system in the Sauropsida. In his 
completed paper of September 1913 Miller (6) gives his results 
in greater detail. He states that the lacunae in question are 
bounded at first by indifferent mesenchymal cells which become 
flattened to form cells which are morphologically equivalent to 
endothelial cells. 

Hoyer in June 1913 presented Fedorowiez’s ‘‘Untersuchung 
iiber die Entwickelung der Lymphgefiisse bei Anurenlarven”’ 
(7) before the Academy of Sciences of Cracow. Fedorowicz, 
working on Bufo vulgaris, Bufo viridis, Rana esculenta, and Rana 
temporaria found cell strands developing from the surface of 
the lymph heart. In these strands intercellular spaces and 


Low 


ORIGIN OF THE POSTERIOR LYMPH HEART 407 


finally lumina, which could not be injected from the lymph 
heart, appeared. The lumen of each strand he found to be 
lined with endothelial cells. By the continuation of the space 
formation lymphatic vessels developed which connected second- 
arily with the similarly acquired lumina of other cell strands 
which had appeared within the heart. It was not until this 
connection was established that it was possible to inject the 
lymphatic vessels from the heart. 

Allen (8) in a recent important publicatien on Polistotrema 
(Bdellostoma) describes the caudal lymph heart as arising from 
isolated mesenchymal spaces in the region of the anterior end 
of the two branches of the caudal vein, and the ultimate fusion 
of these spaces by the breaking down of their partitions. Inci- 
dent to this process certain cells in the interior of the system 
of spaces become spherical and are transformed into red blood 
corpuscles. Secondarily the cavity of the lymph heart estab- 
lishes connections with the caudal vein by the same process, 
that is a breaking down of mesenchymal partitions while periph- 
erally the cavity is enlarged by the new formation of isolated 
mesenchymal spaces and their ultimate annexation. Coinci- 
dentally the mesenchymal cells bordering the cavity of the 
lymph heart flatten to form its endothelium. Allen in con- 
clusion says that his 
* * * * studies thus far indicate that the most primitive form of 
lymphatic system are veins that function for both lymphatics and 
veins. Hence it would be expected that ontogeny would repeat the 
phylogeny of the lymphatics, and instead of having their origin directly 
from the veins, they would begin directly as the veins did, by the vacuo- 
lization of the original mesenchyme. 


These vessels Allen has designated ‘veno-lymphatics.’ The 
recognition of haemopoesis in the vicinity of developing lym- 
phatiecs and from their endothelium is of major morphological 
importance and the substantial agreement between the results 
of Allen and of Miller should go far to clear up some of the 
difficulties that have beset the study of the ontogeny of the 
lymphatic system. The term veno-lymphatic was used by 
Huntington and McClure (9) in their studies of the mammalian 


408 RANDOLPH WEST 


jugular lymph sac to designate constituents of the sac which 
were found at first to contain blood and later to be devoid of 
blood content. The term veno-lymphatic was simply meant to 
cover these two conditions of the vessels; for at the time of 
their studies the criterion of content seemed most available to 
discriminate between lymphatic and haemal channels. The 
work of Miller and of Allen demonstrating the 7m situ formation 
of blood cells and their carriage by lymphatics affords a com- 
plete and satisfactory explanation of these earlier observations, 
and Miller’s term haemophoric lymphatic satisfactorily de- 
scribes the actual conditions, and it is to be hoped in interest of 
clarity will replace veno-lymphatic. This question was fully 
considered by Huntington (10) at the Thirtieth Session of the 
American Association of Anatomists. 

The present investigation is concerned with the earliest appear- 
ance of the posterior lymph hearts in the chick. They are two 
in number and bilaterally symmetrical. Each one arises in 
the mesenchyme lateral to the caudal muscle plate and posterior 
to the hind limb bud. Before the lymph heart assumes the form 
of a single sac-like cavity there exists in this same area a plexus 
of lymphatic vessels which later coalesce to form the single 
cavity of the lymph heart. Both the completed lymphatic 
plexus and later the lymph heart are in connection with several 
of the most anterior coccygeal veins by means of their lateral 
branches which pierce the caudal muscle plate, drain the lym- 
phaties, and then pass outward in the younger embryos to drain 
a haemal capillary plexus, which bears a superficial relation to 
the lymphatic plexus. 

It is the purpose of this paper to show that the plexus of 
lymphatic vessels, which later enters into the formation of the 
posterior lymph heart arises by the confiuence of independent 
mesenchymal spaces which connect secondarily with the veins; 
that these spaces are bounded at first by mesenchymal cells which 
become flattened to form an endothelium, and that both in the 
endothelial lymphatic walls and in the adjacent mesenchyme an 
active haemopoesis is taking place. 


ORIGIN OF THE POSTERIOR LYMPH HEART 409 


MATERIAL 


Forty-one of the forty-five embryos used in this work were 
injected with India ink through the large vitelline blood vessels, 
the injection usually being pushed to the point of extravasation 
for the haemal capillaries. Of the four embryos not injected 


TABLE 1 


List of sectioned embryos 


Length in mm. after Age in days Hours Series 
fixation 
Gado 4 12 371 
if 4 16 382A 
8 4 16 31A 
8.5 4 18 5A 
8.5 4 18 33A 
8.5 4 20 21A 
9 4 18 29A 
9 4 20 Q27A 
9 4 20 23A 
9 4 21 22A 
9 4 21 4A 
9 5 1 7A 
9.5 f 18 30A 
9.5 4 18 28A 
9.5 4 20 244 
9.5 4 20 8A 
10 1 u 13A 
10 +f 21 9A 
10.5 4 20 26A 
10.5 4 20 25A 
11 4 21 20A 
Wi 5 0 12A 
11 5 1 ’ 6A 
11 5 13 2A 
11.5 5 a 18A 
LES 5 a 19A 
eS 5 3 14A 
11.5 5 1 10A 
12 5 6 34A 
WH) 5 10 326 
13.5 6 1 1A 
13.5 te 3A 
14 5 20 17A 
14.5 ? 11A 
15 5 20 15A 
15 5 20 16A 


410 RANDOLPH WEST 


through the vitelline vessels three were injected directly into 
the posterior lymph heart plexus and one (12 mm.) was not in- 
jected at all. All material was fixed in Zenker’s fluid. Thirty- 
six of the embryos were cut into 10 » and 7 u serial sections and 
stained on the slide with eosin and methyl! blue by Mann’s method. 
One or two series were stained with Delafield’s hemotoxylin 
and orange G, but this method gave a very poor differentiation 
of the blood cells. The nine embryos not sectioned were cleared 
by the Spateholz method and examined in folo under the binocu- 
lar microscope (table 1). 


OBSERVATIONS 


A. FORMATION OF BLOOD CELLS FROM THE MESENCHYME AND 
THEIR ENTRANCE INTO THE CIRCULATION VIA THE DEVEL- 
OPING HAEMAL CAPILLARIES, PRIOR TO THE FORMATION OF 
LYMPHATICS 


As the appearance of numerous blood cells in the mesenchyme 
and the extension of the haemal capillaries, previously referred 
to, is the first change which occurs in the mesenchyme lateral 
to the caudal muscle plate in the caudal region of the embryo, 
these processes will be considered first. When the lymphatic 
anlagen first appear, in the 10.5 mm. embryo, the haemal capil- 
lary plexus has reached a very high degree of complexity and 
from this time onward merely holds its own or develops compara- 
tively slowly. 

The youngest embryo examined was one of 6.75 mm. In 
this specimen the mesenchyme lateral to the muscle plate was 
uniformly loose, and very nearly indifferent. A few rather 
rounded eosinophile cells were observed in each section. Some of 
these cells contained one or two large eosinophile granules. Oc- 
casional venous branches pierced the muscle plate to drain the 
mesenchyme lateral to it. 

The same area in the 7 mm. embryo presents several changes. 
The mesenchyme is much more compact, being equal in den- 
sity to the mesenchyme which lies medial to the muscle plate. 
Groups of differentiating blood cells are much more abundant. 


ORIGIN OF THE POSTERIOR LYMPH HEART 411 


These cells are becoming rounded, with a diameter of 7 to 8 u. 
Their cytoplasm is neutrophile or eosinophile and contains 
several strongly eosinophile granules. The nucleus is slightly 
more basophile than the cytoplasm. Eosinophile granules were 
also observed in the cytoplasm of some of the mesenchyme cells. 
There is usually a free space of 2 to 3 » about each differentiating 
cell, which is not encroached upon by the surrounding mesen- 
chyme. Lateral branches of the coccygeal veins pierce the caudal 
muscle plate at regular intervals but the capillaries which they 
drain are few in number. 

The 8.5 mm. embryo presents a very similar state of affairs, 
except that the capillaries emptying into the lateral branches 
of the coceygeal veins are somewhat more numerous, and the 
differentiating blood cells also occur in greater numbers. As 
may be seen from figure 1, 5, the haemal capillaries are injected 
to the point of extravasation, but the differentiating eosinophile 
cells (7) are absolutely independent of them, nor are there any 
eosinophile cells medial to the caudal muscle plate. 

From this stage on until the embryo reaches the length of 
10.5 or 11 mm. (fig. 2), the capillary plexus steadily increases 
in richness and complexity, while the blood cells differentiating 
from the mesenchyme become scarcer. The capillary plexus 
has invaded the area formerly occupied by differentiating blood 
cells, and blood cells in the mesenchyme have decreased until 
only a small fraction of those present in the 8.5 mm. embryo 
remap. 

These blood cells have, then, either degenerated and dis- 
appeared, or have been drained off by the capillary plexus. 
The present investigation has not been of such a character as 
to warrant tracing the complete history of the blood cells which 
differentiate from the mesenchyme but representatives of both 
the red and white blood cell lines have been identified in the 
tissue spaces. 

That these cells are drained off by the extending capillaries 
is indicated by the fact that within five or six hours we find 
first a practically indifferent mesenchyme, a little later a very 
active haemopoesis taking place in it and finally a general vas- 


412 - RANDOLPH WEST 


cularization of the tissue accompanied by a marked decrease in 
the number of blood cells in the tissue spaces. It seems highly 
improbable that decided haemopoesis should take place only to 
let the cells formed disintegrate three or four hours later without 
having entered a vessel, and moreover none of the blood cells 
observed in the tissues appeared to be disintegrating. Mc- 
Whorter and Whipple (11) in their study of the chick blasto- 
derm in vitro have observed a to-and-fro movement of the 
blood cells in the tissue spaces synchronous with the heart beat, 
and have also observed the entrance of these cells into the 
general circulation following their rhythmical movement. This 
phenomenon might be regarded as a plasmatie pulse, which 
would eventually force any blood cells lying free in the tissue 
spaces into the general circulation. In addition those cells 
having the power of amoeboid movement could enter the vessels 
by diapedesis through the capillary walls. 


B. DEVELOPMENT OF THE LYMPHATIC PLEXUS AND ACCOM- 
PANYING HAEMOPOESIS 


The changes about to be described take place only in the 
mesenchyme lateral to the caudal muscle plates in the posterior 
region of the embryo, the mesenchyme lying medial to the 
muscle plates maintaining its compact indifferent character. 
For the sake of clearness we shall first consider the Histogenesis 
and then the Morphogenesis of the developing plexus of lymphatic 
vessels. 


Fig. 1 Chick 8.5 mm., Series 21, Slide 1, Row 3, Section 2. X 200. Photo- 
micrograph of transverse section of caudal end of the embryo. 


1, Notochord 5, Haemal capillaries 
2, Neural tube 6, Caudal muscle plate 
8, Coceygeal vein 7, Differentiating blood cells 


4, Coccygeal artery 


414 RANDOLPH WEST 


1. Histogenesis 


In the embryo of 10.5 mm. (about 4 days and 22 hours) we 
observe two new phenomena; the formation of spaces bounded 
by mesenchymal cells which eventually become flattened to form 
an endothelium, and the appearance of certain strands of flattened 
cells in the mesenchyme. Haemopoesis continues to take 
place in the mesenchyme and also from endothelial cells of the 
lymphatic walls as soon as these are formed. 

Throughout the younger stages until the embryo has reached 
the length of 10.5 mm. the mesenchyme lateral to the caudal 
muscle plate is of a uniform degree of compactness equal to 
that of the mesenchyme medial to the muscle plates. The 10.5 
mm. embryo, however, shows a slight, but distinct loosening 
of the mesenchyme just lateral to the muscle plate, between the 
points of penetration of the lateral branches of coccygeal veins 
and at certain points the loosening of the tissue is more marked, 
giving rise to small mesenchymal spaces. The spaces still 
bounded by mesenchyme are more numerous in the 11 mm. 
embryo (fig. 2, 8) and some differentiating blood cells have be- 
come included in them (fig. 5, 7). Certain of the spaces nearest 
the veins have acquired a venous connection at this stage and 
in the injected embryos appear as small knob like processes 
(fig. 5, 10) of a larger caliber than the veins with which they 
connect. filled with blood cells, and lined by endothelium. These 
knobs correspond in shape to the mesenchymal spaces men- 
tioned. It is to be expected that when a space connects witha 
vein and is subjected to the pressure and friction of the general 
circulation, that the cells bounding it will tend to become flat- 
tened. And the fact that in later stages, when the stiil isolated 
spaces become larger and are under a greater plasmatic pressure 


Fig. 2 Chick 11 mm., Series 20, Slide 1, Row 4, Section 3. X 200. Photo- 
micrograph of transverse section of the caudal end of the embryo. 


1, Notochord 5, Haemal capillaries 
2, Neural tube 6, Caudal muscle plate 
3, Coccygeal vein 8, Mesenchymal space 


4, Coecygeal artery 9, Lateral branch of coeeygeal vein 


a 
i 
Pi 


BES EUS 


ORIGIN OF THE POSTERIOR LYMPH HEART 


Dot hem +e 
Pence, 
> aren 
Wirt et 
10% 


2 


416 RANDOLPH WEST 


the bounding cells do flatten, renders it highly probable that a 
similar process takes place in the case of the smaller spaces which 
first acquire a venous connection. 

As was pointed out by E. R. Clark (12) at the Christmas meet- 
ing of the Anatomical Society in 1913, there are present in the 
mesenchyme lateral to the caudal muscle plate in the posterior 
region of the embryo certain strands of flattened cells which 
Clark holds to be outgrowths from the venous endothelium and 
to be always capable of being traced back to the veins. These» 
cells, he says, contain nuclei which may be distinguished from 
the mesenchyme nuclei by their morphological and staining 
characters. 

That strands of flattened cells, sometimes with continuous 
lumina, sometimes with an interrupted lumen or with no lumen 
at all occur in the chick as early as 9.5 mm. and more abun- 
dantly in the later stages, is true. But that they can be clearly 
distinguished from mesenchyme cells, and that they can always 
be traced back to a venous endothelium, are at least open 
questions. 

E. R. Clark (12) describes the endothelial nucleus as being 
rather pale and elongated with one or two definite reddish 
discoid nucleoli, while the mesenchymal nucleus he holds to be 
darker, and more chromatic with one or two irregular bluish 
nucleoh, not sharply differentiated from the surrounding chro- 
matin material. A careful examination, however, reveals a 
series of graduated stages between these two forms of nuclei. 
A shght change in the focus of the microscope will make a bluish 
nucleolus appear reddish, and vice versa, while a careful study 
of the tissue reveals great variance in the amount of chromatin 


Fig. 3 Chick 15 mm., Series 16, Slide 2, Row 4, Section 8. X 300. Photo- 
micrograph of transverse section of the caudal end of the embryo. 


1, Notochord 7, Differentiating blood cells 

2. Neural tube 8, Mesenchymal space 

3, Coceygeal vein 9, Lateral branch of coceygeal vein 
4, Coceygeal artery 10, Lymphatic connected with vein 
5, Haemal capillaries 11, Aorta 


6, Caudal muscle plate 


Rated: 


verge § 


418 RANDOLPH WEST 


in the various nuclei. That the typical nucleus of the fully 
differentiated endothelial cell may be distinguished. from that 
of the indifferent mesenchyme cell we do not deny, but that in- 
termediate stages between the two exist, in the case in question 
we likewise hold to be true. And unless it be cut parallel to its 
long axis, it is practically impossible to distinguish even the 
fully differentiated endothelial nucleus from the mesenchymal 
nucleus. 

As for the statement that these flattened rows of cells are al- 
ways connected with a preéxisting endothelium it must be re- 
membered that practically every cell in the embryo is, at this 
stage, in syncytial relation with every other cell, the blood cells 
excepted. So in a certain sense a protoplasmic connection be- 
tween flattened cells and preéxisting endothelium may be demon- 
strated by passing over the protoplasm of indifferent mesenchyme 
cells. To assume that because all endothelium in the embryo 
is in syncytial relationship it is therefore derived from some 
preéxisting endothelium, appears unwarranted. Can it not be 
said with equal truth that since the embryonic vascular endothe- 
lium is in syncytial relationship with the mesenchyme it is 
therefore derived from the mesenchyme? This being the case, 
we know that there are in the mesenchyme certain flattened 
cells which are not connected with any preéxisting endothelium 
otherwise than by means of the protoplasm of the mesenchymal 
syneytium. The isolation of these flattened cells from any 
other endothelium and the fact that all possible gradations 


Fig. 4 Chick 8.5 mm., Series 21, Slide 1, Row 3, Section 2. X 500. Fhoto- 
micrograph of transverse section of the caudal end of the embryo. 

Fig. 5 Chick 11 mm., Series 20, Slide 1, Row 4, Section 4. X 600. Photo- 
micrograph of transverse section of the caudal end of the embryo. 


Figure 4. Figure 5. 
3, Coceygeal vein 3, Coceygeal vein 
5, Haemal capillaries 5, Haemal capillaries 
6, Caudal muscle plate 6, Caudal muscle plate 
7, Differentiating blood cells 7, Differentiating blood cells 


8, Mesenchymal spaces 
10, Lymphatie connected with vein 


' 


ORIGIN OF THE POSTERIOR LYMPH HEART 


419 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 


420 RANDOLPH WEST 


exist between them and the typical mesenchymal cells shows 
clearly that an in situ differentiation of endothelial cells takes 
place (fig. 6, 13, and fig. 7, 19). The cells so formed may then 
bound isolated cysts filled with plasma (fig. 6, 72) which some- 
times enclose a differentiating blood cell. These plasmatocysts 
then proceed to grow together connecting up with one another 
and with the veins, and it is probable that they form in some 
instances a connecting link between the veins and the large 
lacunae in the mesenchyme. The early appearance of the 
blood-filled lymphatic plexus connected with the veins in the 
living chick, which E. R. and E. L. Clark (5) describe as follows, 
lends weight to such an interpretation of the facts: 


The first evidence of lymphatics in the tail region of the living chick 
is the appearance of separate knobs filled with stagnant blood just 
lateral to the coccygeal veins. Soon after these knobs appear similar 
ones develop about them which have fne connections with them. 
* * * * Their injection shows discreet tiny clusters, somewhat 
like bunches of grapes (p. 254). 


Figure 6, a section of the caudal region of an 11 mm. embryo, 
shows an isolated plasmatocyst (7/2). This section and the adja- 
cent sections were studied with the greatest care under the oil 
immersion lense, and the two elongated cells (73) with pale nuclei 
and distinet nucleoli bounding the cyst were not in connection 
with any other endothelium. 

Figure 7, a section of the caudal region of a 15 mm. embryo, 
shows a structure which some might describe as a venous sprout. 
The injection mass has entered the lumen for a short distance in 
large amounts. Then the lumen becomes somewhat constricted, 
and beyond that point only occasional ink granules can be found. 
Finally the lumen terminates and a long flat cell (73) follows in 
which two distinet nucleoli are seen, beyond which is a space (75) 
bounded by a delicate strand of cytoplasm on either side. This 
space contains a differentiating red blood cell (7). The adja- 
cent sections have also been examined with great care, and the 
one directly preceding shows one rather elongated flattened cell 
with a pale nucleus forming the floor and probably the end of the 
plasmatocyst containing the blood cell just described. Several 


ORIGIN OF THE POSTERIOR LYMPH HEART 421 


of the mesenchymal cells near by, in the direction in which this 
‘sprout’ would extend, show a tendency to become elongated (19), 
but they are separated from the endothelial cell by indifferent 
mesenchymal cells, and their nuclei are quite chromatic. They 
probably represent cells which are about to flatten and to limit 
a plasmatocyst. 

Since disconnected plasmatocysts have been found; since 
all gradations between an indifferent mesenchymal cell and a 
typical endothelial cell have been observed; and since, in the 
section just described, we find most distally an uninjected plas- 
matocyst, containing a differentiating blood cell, then a single 
endothelial cell enclosing no lumen, and finally a lumen con- 
nected with the veins into which the injection mass has entered, 
it does not seem justifiable to call this structure a venous sprout. 
It should rather be considered as a plasmatocyst which has 
differentiated in situ, and connected secondarily with the vein. 
Whether the endothelial cells between the plasmatocyst and 
the vein arise by an in situ differentiation, or by a mutual growth 
of the plasmatocyst and the vein toward each other, it is im- 
possible in this particular case to determine definitely by the 
study of sections or injections. ‘The latter interpretation would 
in no way invalidate the fundamental conception that endothelium 
arises in situ from mesenchyme. It merely implies that en- 
dothelial cells once formed are capable of proliferation, as cells 
in general are. It should be noted that discontinuity of the 
lumen of the ‘sprout’ present in figure 7 shows clearly the utter 
inadequacy of the injection method for demonstrating all of 
the endothelium in the embryo. 

As regards the further development of the blind spaces in 
the mesenchyme, we have seen that in the 10.5 and 11 mm. 
embryos there exist a number of spaces in the mesenchyme Just 
lateral to the caudal muscle plate, and that these spaces are 
bounded by mesenchymal cells. Some of these spaces are con- 
nected at this stage with the lateral branches of the coccygeal 
veins, and certain blood cells, differentiating from the mesen- 
chyme, have become included in some of the disconnected 
spaces. In the 12.5 mm. and 13.5 mm. embryos more and 


422 RANDOLPH WEST 


more spaces continue to connect with the veins, either directly 
or by means of the delicate hollowed ‘cell strands’ already de- 
scribed, and as the spaces acquire venous connection, they may 
become filled with blood backed up from the general circulation 
especially in injected embryos. The spaces which have not as 
vet attained a venous connection, increase in size, several smaller 
spaces coalescing by a breaking down of their cell boundaries 
to form a single larger space (fig. 9, 8; fig. 8, 8; fig. 3, 8). As 
the plasmatic pressure becomes greater, the indifferent mesen- 
chyme cells which bounded these spaces become flattened to 
form cells which are identical in appearance with endothelial 
cells (fig. 9, 8). The first spaces about which endothelial cells 
were detected were in a 13.5 mm. embryo, although the cells 
bounding the spaces were somewhat flattened in the 11.5 mm. 
and 12.5 mm. embryos. The fact that the cells about a single 
isolated space may be in part endothelial and in part mesen- 
chymal, with many intermediate stages between the two, indi- 
cates that an in situ differentiation of endothelium from mesen- 
chyme is taking place. 

The haemopoesis, which was described as taking place before 
the lymphatic anlagen appear, continues, but much less rapidly 
than formerly. We have seen that the mesenchyme lateral 
to the caudal muscle plate was first practically indifferent and 
non-vascular. Then came a wave of haemopoesis, followed 


Fig. 6 Chick 11 mm., Series 20, Slide 1, Row 3, Section 7. x 500. Photo- 
micrograph ot transverse section of caudal end of the embryo. 

Fig. 7 Chick 15 mm., Series 16, Slide 2, Row 4, Section 6. > 500. Photo- 
micrograph of transverse section of caudal end of the embryo. 


Figure 6 7, Differentiating blood cell 
8, Mesenchymal space 
9, Lateral branch of coceygeal vein 
' 13, Elongated cell with pale nucleus 
and distinct nucleoli 
14, Lumen continuous with vein 
15, Lumen not continuous with vein 
19, Isolated flattened cell, with pale 
3, Coceygeal vein nucleus and distinct nucleoli 
6, Caudal muscle plate 


6, Caudal muscle plate 

9, Lateral branch of coccygeal vein 

12, Isolated plasmatocyst 

13, Elongated cell with pale nucelus 
and distinct nucleoli 


Figure 7 


424 RANDOLPH WEST 


quickly by a vascularization of the tissue anda decrease in the 
number of blood cells in the tissue spaces. ‘This takes the em- 
bryo up to the 10.5 mm. stage, when the lymphatic anlagen 
first appear. From this time onward certain mesenchyme cells 
still seem to become rounded, break away from the surrounding 
syneytium, and acquire eosinophile granules. In other cells the 
cytoplasm becomes eosinophile more evenly, forming erythro- 
cytes. These cells, which le in the tissue spaces, for the most 
part become included in the lymphatic anlagen, and as these 
anlagen acquire a venous connection, reach the general circulation. 

For the first time in the 12.5 mm. embryo groups of rounded 
strongly basophile cells, may be observed to be differentiating 
from the endothelium near the junction of the lymphatics and 
veins. Small clumps of rounded cells, more strongly basophile 
than the mesenchyme or endothelial cells, are seen forming and 
apparently splitting off from the endothelium of the lymphatics 
(fig. 10, 76). In some of the older embryos the cytoplasm of these 
cells acquires an eosinophile tinge. These cells are identical with 
the erythroblasts described by Dantschakoff (13). Finally, in the 
13.5, 14.5 and 15mm. embryos large aggregations of slightly baso- 
phile cells with conspicuous eosinophile granules (fig. 11, 17) 
are seen differentiating and splitting off from the lymphatic 
endothelium. 

One final point must be noted, although it does not concern 
the endothelium of the lymphatic plexus. In the 14.5 mm. 
embryo strands of three or four myoblasts appear in the now 


Fig. 8 Chick 14 mm., Series 17, Slide 2, Row 1, Section 6. X 300. Photo- 
micrograph of transverse section of caudal end of the embryo. 

Fig. 9 Chick 15 mm., Series 16, Slide 2, Row 4, Section 6. X 600. Photo- 
micrograph of transverse section of caudal end of the embryo. 


Figure 8 Figure 9 
3, Coccygeal vein 3, Coceygeal vein 
§, Haemal capillaries 6, Caudal muscle plate 
6, Caudal muscle plate 8, Isolated space, bounding cells be- 
8, Mesenchymal space coming flattened 
10, Lymphatie connected with veins 9, Lateral branch of coceygeal vein 


10, Lymphatic connected with vein 


oe re en 


pve 
5 Oa 


Pag 


* 


426 RANDOLPH WEST 


vacuolated mesenchyme just lateral to the caudal muscle plate 
and parallel to the axis of the notochord, and occasional very 
small longitudinal spaces may be seen in the most lateral portion 
of the caudal muscle plate. In one or two sections, one end of 
the strand of myoblasts was seen to be in connection with the 
muscle plate. Whether these cells were splitting off from the 
muscle plate by delamination, or whether they were forming 
from the mesenchyme and being added to it by accretion, it 
was not possible to determine in the material available. 


2. Morphogenesis 


Up to this point we have considered the histogenetic changes 
which take place in the developing lymphatic plexus, and we 
shall now consider the morphogenesis of the plexus. For this 
purpose four wax reconstructions have been made by the method 
of Born, three of which are here reproduced. 

Chick of 11 mm. Reconstructions of vessels and isolated spaces 
of the caudal region. X 150. Figure 12: Arteries black, veins 
and capillaries white, isolated spaces yellow. The posteardinal 
vein and the aorta run a few sections above the upper level of 
this reconstruction, but the coccygeal branches of the aorta 
(fig. 12, 4) and a little more externally the coccygeal veins (3) 
which drain into the posteardinals, are seen running downward 
at right angles and dorsal to the axis of the vertebral column. All 
of these structures are medial to the caudal muscle plate, which 
has been omitted from this reconstruction for the sake of simplic- 
ity. This muscle plate extends in a plane, parallel to the ecto- 


Tig. 10 Chick 12 mm., Series 46, Slide 1, Row 4, Section 4. X 500. Photo-. 
micrograph of transverse section of caudal end of the embryo. Uninjected. 

Fig. 11 Chick 15 mm., Series 16, Slide 2, Row 4, Section 7. > 600. Photo- 
micrograph of transverse section of caudal end of the embryo. 


Figure 10 Figure 11 
3, Coceygeal vein 5, Haemal capillaries 
6, Caudal muscle plate 17, Blood cells differentiating from 
16, Blood cells differentiating from lymphatie walls 


endothelium 


*& ay > 


a ey 


428 RANDOLPH WEST 


derm, just lateral to the coccygeal veins. ‘Two or three lateral 
branches of each coccygeal vein (fig. 12, 9) pierce the muscle plate 
and proceeding directly outward terminate in a plexus of haemal 
capillaries which lie directly beneath the ectoderm. 

The lymphatic plexus, which later forms the lymph heart, 
develops in the mesenchyme between the caudal muscle plate 
and this superficial plexus of haemal capillaries. A number of 
isolated spaces, bounded by mesenchyme cells which are still 
practically unflattened, are seen (fig. 12, 8; fig. 5, 8) to occupy 
the position just alluded to. They have been studied very 
carefully with oil immersion lenses and are absolutely inde- 
pendent of any vascular connection, either with the lateral 
branches of the coceygeal veins or the haemal capillaries; they 
occur only caudal to the level of the hind limb bud and only 
lateral to the muscle plate. 

Chick of 14 mm. Reconstruction of the blood vessels of the 
caudal region, and the lymphatic plexus in so far as it forms a 
continuous channel connected with the veins. X< 150. Figure 13: 
Arteries black, veins and capillaries white, lymphatics connected 
with veins, green. The isolated spaces have been omitted from 
this reconstruction in order that the lymphatic plexus connected 
with the veins might be more clearly shown. The reconstruc- 
tion has been drawn from the side and somewhat from above 
and the aorta and posteardinals have been shown in the draw- 
ing as folded upward and outward. We have in this recon- 
struction practically the same arrangement of arteries, veins 
and haemal capillaries as was described for the 11 mm. embryo. 
The two posteardinal veins (fig. 13, 78) are seen above and some- 
what lateral to the aorta; they anastomose above that vessel, 
and receive the coceygeal veins both cranial and caudal to their 
anastomosis. The coccygeal veins (fig. 13, 3) as before, pass 
downward, at right angles to the axis of the vertebral colu nn, 
close to the caudal muscle plate, and give off lateral branches 
(fig. 13, 9 a, b) which pierce the muscle plate. It will be seen 
that a plexus of lymphatic vessels connected with the coccygeal 
veins has been established between the haemal capillaries and 
the muscle plate, which is characterized by the irregular size 


ORIGIN OF THE POSTERIOR LYMPH HEART 429 


of its vessels, prominent knob-like enlargements occurring where- 
ever a large independent space previously existed. This plexus, 
as has been noted, usually fills with stagnant blood, backed up 
from the venous circulation. There is no connection between 
the lymphatic plexus and the haemal circulation except at the 
point where the lateral branches of the coceygeal veins have 
just pierced the muscle plate. 

We now see that the lateral branches of the five or six most 
cranial cocecygeal veins pierce the muscle plate, drain the lym- 
phatic plexus and then pass outward to drain the haemal capil- 
lary plexus (fig. 13, 9a). Soon that portion of the lateral branches 
of the coccygeal veins distal to the lymphatic taps degenerates, 
thus severing the connection of these veins with the haemal 
capillaries, so that those lateral coceygeal branches which drain 
the lymphatic plexus, cease to function otherwise than for the 
lymphatic drainage (fig. 13, 9b). An examination of several 
injected embryos cleared by the method of Spateholz showed 
this point clearly; the haemal capillary plexus being drained in 
the 15 mm. embryo by the most dorsal portions of the coceygeal 
veins with only two of the lateral cocevgeal branches assisting 
them. although in the embryo of 11.5 mm. five or six lateral 
coccygeal branches drained the plexus of haemal capillaries. 
One 17.5 mm. embryo which was examined in cross sections, 
showed no connection between the lateral branches of the five 
or six coccygeal veins which drain the lymphatic plexus and the 
haemal capillaries. 

Chick of 15 mm. Reconstruction of the caudal vessels. & 150. 
Antero-lateral view. Figure 14: Arteries black, veins and capil- 
laries white, lymphatics connected with the veins green, isolated 
spaces yellow. In this reconstruction the coccygeal veins (3) are 
seen extending downward from the posteardinals (/8) and the 
coccygeal arteries (4) from the aorta (//). The coccygeal 
veins give off lateral branches (9) which pierce the caudal muscle 
plate—which has been omitted from this reconstruction—and 
then proceed laterally to drain the lymphatic plexus (green) 
and at the points where the lymphatics are not as yet formed 
to any extent, the haemal capillary plexus (white). The lym- 


430 RANDOLPH WEST 


phatic plexus may be clearly seen to occupy the area which in 
the reconstruction of the 11 mm. chick was filled only by iso- 
lated mesenchymal spaces. A great number of these isolated 
spaces (yellow, 8) still exist, not connected as yet with the lym- 
phatic plexus. They occur in greater numbers medial to the 
lymphatic plexus which is connected with the veins (green, /0) 
that is between it and the caudal muscle plate, than they do 
lateral to the lymphatic plexus, although quite a number, as 
may be seen from the figure, occupy the latter position. It is 
especially interesting to note that the isolated spaces le on all 
sides of the lymphatic plexus, seeming to precede it and form 
in an area which an hour or two later is occupied by the con- 
tinuous plexus of lymphatics, connected with the coccygeal 
veins. Such outlying isolated spaces are clearly shown at the 
cranial end of this reconstruction. 


Fig. 12 Reconstruction of caudal vessels of a chick of 11 mm., Series 20. 
x 150. Antro-lateral view; arteries in black; veins and capillaries in white; 
isolated spaces in yellow. 

Fig. 13 Reconstruction of caudal vessels of a chick of 14mm., Series 17. 
< 150. Antro-lateral view; arteries black; veins and capillaries white; lym- 
phatie plexus connected with veins, green. The disconnected mesenchymal 
spaces have been omitted from this reconstruction. 

Fig. 14 Reconstruction of caudal vessels of a chick of 15 mm. Series 16. 
< 150. Antro-lateral view; arteries black; veins and capillaries white; lym- 
phaties connected with veins, green; mesenchymal spaces yellow. 


Figure 12 9b, Lateral branches of coccygeal vein 
draining lymphatics only 

10, Lymphatic plexus connected with 
the veins (green) 

18, Posteardinal veins 


3, Coceygeal vein 
4, Coceygeal artery 
8, Mesenchymal spaces (green) 
9, Lateral branches coccygeal veins 
draining haemal capillaries 
Figure 14 


Higume ls 8, Coccygeal vein 


53, Coccygeal vein 4, Coceygeal artery 

4, Coceygeal artery 8, Disconnected mesenchymal space 

5, Haemal capillary plexus 9, Lateral branch of coccygeal vein 

9, Lateral branches of coccygeal vein 10, Lymphatic plexus connected with 
9a, Lateral branches of coccygeal veins veins 


draining haemal capillaries andlym- 11, Aorta 
phaties 18, Posteardinal vein 


ORIGIN OF THE POSTERIOR LYMPH HEART 431 


432 RANDOLPH WEST 


As this investigation has been concerned solely with the 
origin of the lymphatic plexus which later forms the lymph heart, 
the later history of the lymph heart has not been studied. <A 
cursory examination of a 16, 16.5, 17 and 18 mm. embryo would 
indicate that the conclusions of Sala are in the main correct, 
and that the plexus coalesces to form the single cavity of the 
lymph heart. The formation of the musculature, valves and 
the number of venous taps in the stages later than 15 mm. has 
not been studied. 


GENERAL DISCUSSION 


Of the previous investigators of the posterior lymph heart 
in the chick, Sala (1) and Mierzejewski (2) have not committed 
themselves as to the origin of the lymphatic endothelium, while 
EK. R. and E. L. Clark hold that the lymphatics are outgrowths 
from the veins and that the endothelium is specific. E. R. and 
K. L. Clark (5) have studied the growth of the lymphatic plexus 
in the living chick under the binocular microscope, using the 
stagnant blood backed up in the growing lymphatics from the 
veins as the index to lymphatic growth. To quote from their 
paper: 

Since stagnant blood in the interior of the lymphatics is the index 
on which these studies are based, it was important to determine whether 
the blood always fills the lymphatics to their tips. This was tested 
in two ways, by pressure over the part filled with blood to see if it 
could be forced farther; and by injection. As a result of numerous 
tests by both of these methods it was found that in these early stages, 
practically all of the lymphatics, save very fine connections are filled 
with blood. * * * * Hence, since the blood fills the successive 
extensions of the lymphatic as soon as formed, the use of the stagnant 
blood as an index for the study of lymphatic development is justifiable 
(p. 255), 


This method has overlooked even the possibility of the presence 
of disconnected mesenchymal spaces entering into the formation 
of this lymphatic plexus. How, extravasation excepted, could 
pressure over the blood-filled plexus or injections into it, reveal 
disconnected lymphatic anlagen in the form of blind spaces in 
the mesenchyme? It is obviously impossible to detect small 


ORIGIN OF THE POSTERIOR LYMPH HEART 433 


mesenchymal spaces filled with colorless lymph by examining a 
living chick under the binocular microscope. The tests just 
described would serve to show that the blood fills the lymphatics 
to their tips only in so far as they formed a continuous channel 
connected with the veins, and would utterly fail to reveal any dis- 
connected anlagen in the form of independent mesenchymal 
spaces. The appearance of a centrifugal outgrowth of the 
lymphatic plexus from the veins is s¢mulated if stagnant blood 
or any other form of injection be used as an index to the lymphatic 
development, for the mesenchymal spaces lying next to veins 
are the first to make the venous connection and fill with blood 
backed up from the general circulation. Then the spaces a 
little more distal join the spaces already connected, in turn are 
filled with blood, and so on until the entire blood-filled lymphatic 
plexus is formed. Thus, while the development is proceeding 
by the centripetal addition of disconnected anlagen, the stag- 
nant blood in the plexus is extending in a centrifugal direction. 
In discussing the blood-contents of the early lymphatic plexus, 
which later forms the posterior lymph heart, the active haemo- 
poesis in the surrounding mesenchyme is the only factor of mor- 
phological and genetic significance. The accidental or normal 
backing up of circulating blood into the lymphatic plexus, after 
it has secondarily established a connection with the veins, is of 
no significance as far as the genesis of the lymphatic structures 
is concerned. But the in situ origin of blood cells from the 
mesenchyme and their conveyance, via the lymphatics, into the 
general haemal circulation is of great importance, and at once 
places the posterior lymph hearts in the chick in the category 
of haemophoric lymphatics, such as are met with in the thoracic 
duct of the same form and in other vertebrates in various degrees 
of development, as has been brought out in Huntington’s paper 
of July 1914 (10). 

Until it can be absolutely proven by some other method than 
that of injection that all lymphatic development is centrifugal 
growth in continuity, with invariable continuity of lumen as 
well, such methods as this will seem to beg the question; for they 
can afford evidence only of the degree of the centrifugal exten- 


434 RANDOLPH WEST 


sion of the lymphatics and by no means serve as a test of the 
process by which this extension is effected once the question of 
annexation of mesenchymal spaces has been raised. They serve 
simply as a measure of the process and do not indicate its nature. 

It may be argued that the spaces here described are due to 
the action of fixing fluids. But if this were so they certainly 
would not appear only in the region of the embryo in which the 
lymphatics are developing, and only during the short period of 
embryonic history during which the lymphatic vessels are formed 
nor would the border cells of an artefact be flattened to form 
endothelium. That the spaces exist in the fixed and sectioned 
embryos, is clearly shown in the accompanying photomicro- 
graphs, and it is safe to conclude that they exist in the living 
embryo as well. 

But, is there any evidence that the venous endothelium does 
not invade this vacuolized tissue and grow out to line these 
independent spaces? There is: The spaces are in the younger 
embryos (10.5 mm.) bounded by mesenchyme cells, but as the 
embryo becomes larger and the spaces increase in size the bound- 
ing cells become flattened and gradations between mesenchymal 
cell and endothelial cell are found bounding the spaces (fig. 9, 
S). Nor is there ever found an endothelial tube within the 
flattened cells. The idea just discussed has been suggested by 
Knower (14) without, so far as the writer is aware, the slightest 
objective evidence in its support. The spaces form and acquire 
a venous connection so rapidly that in only a few cases are iso- 
lated spaces found bounded by fully developed endothelial 
cells, which are disconnected with any preéxisting endothelium. 
But many spaces are found surrounded more or less completely 
by endothelium and in the remainder of their periphery by cells 
ranging from unmodified mesenchyme to almost typical en- 
dothelial cells. 

Mesothelium has been produced experimentally from con- 
nective tissue, by introducing the factors of pressure and fric- 
tion, by W. G. Clark (15). He has used non-irritating solid 
and fluid foreign bodies; celloidin and paraffin, injected into 
the cornea and subcutaneous tissue, and mucus which was allowed 


ORIGIN OF THE POSTERIOR LYMPH HEART 435 


to flow through a fistula. He concludes that ‘“‘the fact that con- 
nective tissue cells are changed in form by physical agents 
into flat closely disposed cells, the outline of which may be de- 
fined by silver salts makes tenable the conclusion that the ex- 
posed connective tissue cells * * * may become flattened 
by pressure or friction or both.’ Therefore, mesothelium and 
endothelium, both being tissues of mesenchymal origin, owe 
their production to identical mechanical factors. 

To summarize: The evidence found from the study of in- 
jected embryos indicates that the lymphatic plexus which later 
enters into the formation of the posterior lymph heart, arises 
by the confluence of independent mesenchymal spaces which 
connect secondarily with the veins; that these spaces are bounded 
at first by mesenchymal cells which later become flattened to 
form an endothelium and that both in the endothelial lymphatic 
walls and the adjacent mesenchyme an active haemopoesis, 
the products of which reach the general circulation via the lym- 
phatic plexus, is taking place. 


In conclusion, | wish to thank Professor MeClure and Pro- 
fessor Huntington who bave directed this work for their constant 
guidance and eriticism; Professor Schulte and Professor Miller 
for many valuable suggestions; Dr. McWhorter for the care 
that he has expended on the microphotographs, and Mr. Peter- 
sen for his drawings of the very complex reconstructions. 


BIBLIOGRAPHY 


In the order in which the articles are mentioned in this paper. 

(1) Sana, L. 1900 Richerche fatta nel Lab. di Anat., Norm della R. Univ. di 
Roma, vol. 7, p. 263. 

(2) MirrzErmmwsk1, L. 1909 Beitrag zur Entwicklung des Lymphgefiss- 
systems der V6gel. Bulletin de 1’ Academie des Sciences de Cracovie, 
Juillet. 

(3) Srromsten, F. A. (1) 1910 A contribution to the anatomy and develop- 
ment of the posterior lymph hearts in turtles. Publication No. 132 
of the Carnegie Institution of Washington, pp. 77-87. (2) 1911 On 
the relation between the mesenchyme spaces and the development 
of the posterior lymph hearts of turtles. Anat. Rec., vol. 5, no. 4, 
April. 


(7) 


(8) 


(10) 


(13) 


(14) 


RANDOLPH WEST 


Cruark, E. L. 1912 General observations on early superficial lymphatics 
in living chick embryos. Anat. Rec., vol. 6, no. 6, June, p. 247. 
CuiarK, E. R., and Cuark, E. L. 1912 Observations on the development 
of the earliest lymphatics in the region of the posterior lymph heart 

in living chick embryos. Anat. Rec. vol. 6, June, p. 253. 

Miuier, A. M. 1913 (1) Haemorphic function of the thoracic duct 
in the chick. Science, new series, vol. 37, no. 962, June, p. 879. (2) 
1913 Histogenesis and morphogenesis of the thoracic duct in the 
chick: Development of the blood cells and their passage to the 
blood stream via the thoracic duct. Am. Jour. Anat., vol. 15, no. 2, 
September. 

Frpprowicz, 8S. 1913 Untersuchung uber die Entwickelung der Lymph- 
gefiisse bie Anurenlarven. Bulletin de 1’ Acad. des Sciences de Cra- 
covie, Series B, Juin, pp. ‘290-297. 

Auten. W.F. 1913 Studies on the development of the veno lymphatics 
in the tailregionot PolistotremaStouti: First communication. Quart. 
Jour. Microse. Science, vol. 59, Part 2, July. 

Huntineton, G. S., and McCuurn, C. F. W. 1910 The anatomy and 
development of the jugular lymph sacs in the domestic cat. Amer. 
Jour. Anat., vol. 10. 

Huntineton, G. 8S. (1) 1914 The genetic relations of lymphatic and 
haemal vascular channels in the embryos of amniotes. Proceedings 
Am. Assn. Anat., Thirtieth Session, Anat. Rec., vol. 8, no. 2, Febru- 
ary. (2) 1914 The development of the mammalian jugular lymph 
sac, of the tributary primitive ulnar lymphatic and of the thoracic 
ducts from the viewpoint of recent investigations of vertebrate lym- 
phatic ontogeny, together with a consideration of the genetic relations 
of lymphatic and haemal vascular channel in the embryos of amniotes. 
Am. Jour. Anat., vol. 16, no. 3, July. 

McWuorter, J. E., and Wuiprir, A. O. 1912 The development of 
the blastoderm chick in vitro. Anat. Rec., vol. 6, no. 3. 

Criark, E. R. 1914 On certain morphological and staining character- 
istics of the nuclei of lymphatic and blood vascular endothelium and 
of mesenchyme cells in chick embryos. Proceedings Am. Assn. 
Anat., Thirtieth Session. Anat. Rec., vol. 8, no. 2, February. 

DantTscHAkorr, WERA 1908 (1) Untersuchung iiber die Entwicklung 
des Blutes und Bindgewebes bei den Végeln. Anatomische Hefte, 
Beste, pe Adale 

Knowerr, H. McE. 1914 <A comparative study of the embryonic blood 
vessels and lymphatics in amphibia. Proceedings Am. Assn. Anat., 
Thirtieth Session, Anat. Rec., vol. 8, no. 2, February. 

Crark, W.G. 1914 Experimental mesothelium. Proceedings Am. Assn. 
Anat., Thirtieth Session, Anat. Record, February, pp. 95-96. 


THE DEVELOPMENT OF THE THYMUS IN THE PIG 


Il. HISTOGENESIS 


J. A. BADERTSCHER 


From the Department of Histology and Embryology, Cornell University, 
Ithaca, N. Y. 


THREE PLATES (EIGHT FIGURES) 


CONTENTS 

ra O CLIT ETO Teer eM eer eee checks Uae eee meet A a wai. saddle te aepeentae RS 437 
INMtaige rial sin clen € Ghd strraresteeciy chr Secteur pOILEe | sealer coches << <rara\ oo eueenetelerensrsieiars 438 
TEVTISRONE GY Lisa er Babes costes oto oN ie cate SERENE Oi eC cE ca eee 440 
TETSU ORIGIG STIS sees Bg Uiceel ates ys = CORNER cae 20 tn PRR 1 eC 443 
iene murely epithelial (pochiss sus fans chokes cee. ote Ela c een coteaeenee 444 

2. The epoch of lymphocyte infiltration and lymphocyte proliferation and 
HOS NOTE Olt TAY MENCMMING Ss oseauepEedboodomsabooddd obgon6 se 448 
3. The epoch of the formation of red blood-cells and granular leucocytes.. 465 
(COMG| WENO y en ela hake Ka BOC Oa toaae ae RRR a ee ne oe eee tate, Sco 484 
Jor 6) GPT OLA Se chopatcasen tus haneib Go idee Sicko OIC CIRLC CLA CRNRE APPEAR Sin ans Eternia ion <0 486 

INTRODUCTION 


There is perhaps no organ in the body whose mode of devel- 
opment has given rise to so bitterly contested and so widely 
divergent views as has that of the thymus. This is particularly 
true of its histogenesis. The source and nature of the small 
round cells that make up the greatest mass of the organ in its 
fully developed condition; the origin and nature of its intra- 
lobular supporting structure; the origin and significance of its 
granular cells and of the Hassall’s corpuscles; the extent to which 
red blood-cells and granular leucocytes are formed in it; have 
during the past thirty-five years attracted the attention of many 
investigators, and yet the only point upon which they unanimously 
agree is that the thymus is, in part at least, of epithelial origin. 
This disparity of views cannot be due to differences in the devel- 
opment of the thymus in different animal forms, for some workers 

437 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 


438 J. A. BADERTSCHER 


who have extended their investigations over a comparatively 
wide range of species and classes of animals have found that the 
developmental processes involved are practically the same for 
the different types of animals investigated. 

While the investigation presented in this paper deals with 
the histogenesis of the thymus as a whole, special consideration 
is given, (1) to the origin and nature of the small round cells, 
and (2) to the origin of free erythrocytes and eosinophile cells 
that are present in both the interlobular septa and the paren- 
chyma of the thymus in later developmental stages. Though 
comparatively little attention has been paid to the develop- 
ment of the reticulum and the thymic bodies, they nevertheless 
have received a consideration sufficient to determine their origin. 


MATERIAL AND METHODS 


The material used for the histogenesis of the thymus was — 
collected in great abundance at a packing house. Often the 
embryos still showed signs of life while they were being measured 
and prepared for the fixing fluid. The upper jaw, the cranium, 
and the posterior thoracic wall were removed from embryos 
from 10 to 20 mm. in length. The part containing the thymus 
was thus made comparatively small and fixed well. Embryos 
from 20 to 55 mm. in length were treated in a similar manner 
and in addition the sides were trimmed and the cervical vertebrae 
removed. in order to reduce the size of the piece. From embryos 
ranging from 60 to 165 mm. in length only portions of the thymus 
with some of the surrounding tissues were removed. From all 
these stages the entire superficial thymus and thymus head, 
and parts of the mid-cervical and thoracic portions were pro- 
cured. From embryos 180 to 289 mm. in length (full term) 
the superficial thymus and portions of the thymus head and mid- 
cervical segment were removed. The left thymus was usually -° 
selected in those stages from which only a portion of the organ 
was removed. The lengths in millimeters of the different 
developmental stages of which the thymus was prepared for a 
study of its histogenesis are as follows: 17, 20, 23, 25, 26, 27, 


DEVELOPMENT OF THE THYMUS 439 


28, 30, 33, 35, 36, 37, 40, 42, 45, 50, 50, 55, 60, 65, 68, 78, 85, 100, 
LOPS, 125, 135% 1405165; 180; 190; 210, 230, 270;-and, 280; 
These figures represent the length of the embryos while in a 
fresh condition. 

Helly’s fluid (Zenker-formol) was almost exclusively used 
for fixing the material. This fixer does not destroy the baso- 
philic character of the cytoplasm of the lymphocytes and appar- 
ently produces no appreciable alteration in the hemoglobin of 
the red blood-cells found in the thymus. A few embryos of 
different developmental stages were fixed in Zenker’s fluid, 
mainly to check up the results of the work done by investigators 
who employed this fixer for a histogenetic investigation of the 
thymus. It was found that Zenker’s fluid is not at all suitable 
for work of this nature since, to a large extent, it destroys the 
basophilic character of the lymphocytes, the preservation of 
which is of inestimable value in tracing out the origin of the first 
lymphocytes found in the thymus. The tissue was imbedded 
in paraffine and cut in sections 3 to 5 » in thickness. Only such 
sections as were desired were spread on slides. - These always 
included sections of the superficial thymus, the thymus head, 
and cervical and thoracic segments.!. For the preservation of 
cells with basophilic granules the material was fixed in 95 per 
cent alcohol. 

Hasting’s modification of Nocht’s Romanowsky blood stain 
proved to be of the greatest value for this work and was almost 
exclusively used in the investigation of the histogenesis of the 
thymus. In properly differentiated sections the cytoplasm 
of the lymphocytes, which has a distinctly basophilic character, 
stains a light blue, while the cytoplasm of the epithelial cells 
of the thymus and that of the mesenchymal cells stains a light 
red color. The lymphocytes can thus be distinguished from the 
epithelial nuclei with comparative ease. Erythrocytes and the 
granules of eosinophile cells are stained intensely red, while 
the granules of the cells with basophilic granules (fixed in 95 
per cent alcohol, are stained blue. In tissue fixed in Zenker’s 


' For a discussion of the different regions of the thymus in the pig reference 
should be made to Part I of this investigation (’14). 


440 J. A. BADERTSCHER 


fluid (used by Bell) the basophilic character of the cytoplasm 
of the lymphocytes is lost, thus rendering it difficult to distin- 
guish a large or medium sized lymphocyte from some of the 
smaller epithelial nuclei of the thymus. Mallory’s connective tis- 
sue stain was also used for staining the connective tissue fibers of 
the thymus. 

HISTORICAL 


Only a brief bistorical sketch will be given to outline, in a 
general way, the views regarding the histogenesis of the thymus. 
For a comprehensive review of the literature on this subject 
reference should be made to Hammer’s work of 1910. 

The investigations that have been made of the histogenesis 
of the thymus of various classes and species of animals have 
led to the formation of two general theories, viz., the pseudo- 
morphosis theory and the transformation theory, each of which 
has been more or less modified by the different investigators of 
this subject. As it is beyond the scope of this work to give a 
detailed discussion of each theory and its modifications they 
will be discussed only in a general way. The pseudomorphosis 
theory will be first considered. In its original setting this theory 
held that the epithelial anlage of the thymus is gradually in- 
vaded by mesenchymal and adenoid tissue. This process dis- 
places the epithelial cells and the only remnants of them in the 
fully developed thymus are the Hassall’s corpuscles. This view 
was held by Maurer for the thymus of teleosts (’86) and for the 
thymus of Urodela and Anura (’88). He gives no detailed 
description of the development of the reticulum, or of the 
invasion of the epithelial anlage of the thymus by the lympho- 
cytes. 

Von Ebner held a somewhat modified view of the pseudo- 
morphosis theory as set forth above. According to him, the 
reticulum and Hassall’s corpuscles of the medulla are derived 
directly from the cells of the original epithelial anlage, while 
the entire cortex with its reticulum, lymphocytes, and blood 
vessels, and also the lymphocytes of the medulla, are of mesen- 
chymal origin. 


DEVELOPMENT OF THE THYMUS 441 


The latest modification of the pseudomorphosis theory which 
is now generally accepted by investigators belonging to this 
school, was set forth by Hammer in his investigations of the 
thymus of human embryos (05). According to him, the retic- 
ulum of both the cortex and medulla and the Hassall’s corpus- 
cles are of epithelial origin. He then was in doubt as to the 
origin of the lymphocytes in the thymus, but called attention to 
the presence of ‘wanderzellen’ (lymphocytes) in the immediate 
vicinity of the organ before and some time after they were pres- 
ent in it. He also observed darkly stained cells in the thymus 
of early developmental stages which, with only moderately high 
magnification, could easily be mistaken for lymphocytes, but 
with very high magnification could readily be recognized as 
degenerating epithelial cells. He thus cast a doubt on the ori- 
gin of lymphocytes from the epithelial cells of the thymus, as 
is still held by some investigators, and pointed out as probable 
an infiltration of the thymus with extrathymic lymphocytes 
which have migrated into it from the surrounding mesenchyme. 
This last view he was unable to prove conclusively on account of 
a lack of sufficient range of developmental stages. His investi- 
gations on numerous developmental stages of the Teleostean 
thymus (’08) also led him to conclude that the fixed elements of the 
thymus are of epithelial origin. He fully believed, however, 
that the lymphocytes first present in the thymus of the Teleost 
migrate into it from the mesenchyme and there, through repeated 
division, give rise to the numerous lymphocytes found in it 
in its fully developed condition. In his latest work (11) on 
the development of the human thymus he makes no mention of 
a migration of lymphocytes into the thymus from the surround- 
ing mesenchyme. 

Maximow in his work (’09 b) on the developing thymus of 
mammals (rabbit, guinea-pig, cat, rat and mouse) and on the 
thymus of the Axolotol (12) also holds that the fixed elements 
of the thymus are of epithelial origin, while the lymphocytes 
first present in that organ have migrated into it from the mesen- 
chyme and, through repeated division, form the numerous 
small lymphocytes of the thymus in later developmental stages. 


ad 


449 J. A. BADERTSCHER 


His views of the developing mammalian thymus are, therefore, 
similar to Hammer’s views of the developing Teleostean thymus. 

The chief exponents of the transformation theory are Prenant 
(94), Maurer in his later work (’99), Bell (06), Stohr (06), 
and Dustin (11). The main point of this theory which they 
most strenuously defend is that the lymphocytes arise from trans- 
formed epithelial cells of the thymus. The epithelial cells 
proliferate rapidly. A part of the daughter cells transform into 
lymphocytes while the undifferentiated portions continue to 
proliferate and are the source of succeeding generations of epithe- 
lial cells and lymphocytes. All hold that the epithelial cells 
give rise to the reticulum and Hassall’s corpuscles, excepting 
Dustin (’11) who claims that the reticulum is of mesenchymal 
origin. These investigators regard the small round cells of the 
thymus as real lymphocytes. 

Sto6hr also derived the small round cells of the thymus from 
epithelial cells, but claimed tbat except for their similarity 
in structure to the small lymphocytes in the blood, they have 
nothing in common with them. They never enter the blood 
stream, they remain epithelial cells as long as they exist, and 
have the power to enlarge and change back to typical epithelial 
cells. In the medulla, according to Stohr, are found real 
lymphocytes that have entered from the blood. He, however, 
fails to explain how they can be distinguished from the small 
round cells (epithelial cells). when they lie side by side. He also 
derives the reticulum and Hassall’s corpuscles from the epithelial 
cells of the thymus. 

It is now generally accepted that the thymic bodies are derived 
from the epithelial cells of the thymus. Hammar and Bell 
have given a thorough description of their development. 

From this brief historical sketch it can be seen that the nature 
of the development of the thymus is by no means a settled ques- 
tion. I wish to state at this point that the results of my investi- 
gation of the histogenesis of the thymus agree with these of Ham- 
mar (’08) and Maximow. 

A brief historical sketch relative to the origin and develop- 
ment of the free erythrocytes and granular cells of the thymus 
will be given in connection with their consideration. 


DEVELOPMENT OF THE THYMUS 443 
HISTOGENESIS 


To determine the origin of the different cellular elements 
that are found in the fully developed thymus it is necessary to 
begin with stages in which the thymus is purely epithelial, and 
to use a differential stain by which one can definitely distinguish 
a lymphocyte from an epithelial cell. The latter fact was em- 
phasized by Maximow (’09 b) who accomplished this differen- 
tiation by fixing the tissue in Helly’s fluid and staining with 
eosin-azure. <A similar differential staining was accomplished 
by me by fixing the tissue, as stated above, in Helly’s fluid and 
staining with Hasting’s Nocht’s blood stain. The material 
prepared for the histogenesis of the thymus begins with an 
embryo 17 mm. in length. Of the many stages that were pre- 
pared and examined there are chosen for description only a few 
series of successively older stages, each of which is decidedly 
advanced in development over the previous stage and yet closely 
enough connected with it so that the developmental history 
will be continuous. 

The histogenesis of the thymus may conveniently be divided 
into epochs, each of which is characterized by more or less 
distinct developmental features. They are: (1) a purely epithelial 
epoch which extends from its earliest development as an out- 
pocketing from the third pharyngeal pouch and the forma- 
tion of the cervical vesicle to the appearance of the first lympho- 
cytes in the epithelial anlage of the organ; (2) The epoch of 
lymphocyte infiltration and lymphocyte proliferation, and the 
formation of the reticulum. This epoch begins with the appear- 
ance of the first lymphocytes in the thymus. The invasion 
of the thymus by lymphocytes from the surrounding mesenchyme 
continues probably up to stages 180 mm. in length while the 
proliferation of the lymphocytes in the thymus still continues 
in full term embryos and doubtless after birth. During this 
epoch the cortical and medullary portions of the lobules appear 
first in stages ranging from 65 to 75 mm. in length. The reticu- . 
lum, which according to the nature of its development is formed 
gradually, is fully developed in embryos 180 mm. in length; 


444 J. A. BADERTSCHER 


(3) the epoch of the formation of red blood-cells and the develop- 
ment of granular leucocytes. Although an occasional red blood- 
cell is formed in the thymus shortly after the appearance of 
the first lymphocytes, this epoch properly begins in embryos 
about 55 mm. in length for it is at this developmental stage that 
erythrocytes are beginning to be formed in comparatively large 
numbers. Granular cells appear first in appreciably large 
numbers in embryos 125 mm. in length. The formation of both 
erythrocytes and granular cells in the thymus still continued in 
the full term embryo. 


1. The purely epithelial epoch 


A 23 mm. embryo is the first stage in which the histological 
structure of the thymus will be described. The thymus at this 
stage is a purely epithelial structure. It has the form of a greatly 
elongated mass of protoplasm and is a syncytium. No cell 
walls are present. The cytoplasm of the superficial thymus, 
the thymus head, and the mid-cervical and thoracic segments 
contain many vacuoles which vary in size anywhere from the 
(apparent) size of a small pinhead to that of an epithelial nucleus 
when magnified 1300 diameters. The vacuoles in the inter- 
mediary and cervico-thoracic cords are comparatively few in 
number. Fine, branching, and rather deeply stained proto- 
plasmic threads give the syncytium a distinctly reticular appear- 
ance. These protoplasmic threads, however, must not be con- 
fused with the reticulum in later developmental stages. The 
outer surface of the enlarged portions of the thymus is already 
quite irregular, being studded over with blunt epithelial buds 
which are the beginnings of lobules. No basement membrane 
is present. 

The form and size of the epithelial nuclei vary considerably. 
While the majority are sligbtly ellipsoidal in shape, some are 
spherical and others slightly irregular in outline. They are 
quite regularly distributed through the different segments of the 
organ, lying farther apart in the more vacuolar regions than in 


7 pore 


DEVELOPMENT OF THE THYMUS 445 


the intermediary and cervico-thoracic cords where only a few 
vacuoles are present. Those lying near the surface are quite 
regularly arranged. The long axis of the oval ones is usually 
perpendicular to the surface. The more centrally located cells 
have no regular arrangement. A very distinct nuclear mem- 
brane is present. They possess a quite rich supply of chromatin 
which is distributed mostly in the form of fine threads, giving it 
a reticular structure. About one-half of the nuclei possess two 
nucleoli while the other half contains but one. Occasionally 
one can be found with three nucleoli. They are large and occupy 
no definite position in tbe nucleus. In the oval nuclei they may 
lie near the ends or near the center, while in the round nuclei 
they may occupy an eccentric position. The nuclei at this 
stage do not all stain with the same intensity. In the enlarged 
segments of the thymus some stain much more intensely than 
the majority and with only moderately high magnification 
could easily be mistaken for transforming stages leading to the 
development of lymphocytes, which, however, is not the case. 
A consideration of their real significance will be given in connec- 
tion with a discussion of the origin of the lymphocytes in the 
thymus. Nuclear division at this stage goes on rapidly in the 
enlarged segments of the thymus. Even with a magnification 
of 1300 diameters often three nuclei in mitotic division can be 
brought into a microscopic field. 

To determine whether or not cells migrate into the thymus 
it is necessary to make a study of the connective tissue, at least 
in the earlier stages, as painstaking as the study of the thymus 
itself. The mesenchymal cells are of the spindle or stellate 
type. Their protoplasmic processes often unite with those 
of neighboring cells. Many fine fibers are scattered in the 
meshes between the cells giving the appearance of a network. 
The mesenchyme so closely invests the thymus that in places 
the cytoplasmic processes of the mesenchymal cells appear to 
be fused with the cytoplasm of the epithelial cells. The nuclei 
are large and spherical or oval in shape, and contain about as 
much chromatin as the epithelial nuclei of the thymus. 


446 J. A. BADERTSCHER 


Large lymphocytes? are found scattered here and there through- 
out the mesenchyme of the neck and upper thoracic regions which 
were the only regions examined. They are characterized by 
a wide rim of basophilic, nongranular cytoplasm and a large 
nucleus containing a generous amount of chromatin. Their 
shape varies; some are nearly spherical while others have an 
irregular outline with one or more projecting pseudopodia. 
When treated with Hasting’s Nocht’s blood stain the cytoplasm 
takes on a distinct bluish hue the deepness of which may vary 
in different lymphocytes found in a single section, thus indi- 
cating that some are more basophilic than others. The rela- 
tion of faintly stained to the more deeply stained cells will be 
considered later on in this paper. The nucleus is sharply de- 
fined from the cytoplasm by a distinct nuclear membrane. The 
chromatin is in the form of irregular and deeply stained gran- 
ules which vary much in size. Some of the granules adhere 
to the nuclear membrane, while others are scattered in the less 
deeply stained nucleoplasm. In most nuclei only one nucleolus 
is present but some contain two. The shape of the nucleus 
often conforms to the shape of the cell body. In round lym- 
phocytes it usually is round while in the irregular shaped lym- 
phocytes it may also be irregular in shape. It is impossible 
to mistake an irregularly shaped lymphocyte with its blue 
stained cytoplasm and its nucleus rich in chromatin for a spindle 
or stellate shaped mesenchymal cell with slightly reticulated 
and lightly red stained cytoplasm and a nucleus containing 
considerably less chromatin. 

While the lymphocytes are scattered singly throughout the 
entire mesenchyme, local accumulations are also found. These 
are most pronounced in the upper thoracic region near the large 
blood vessels and the thoracic segments of the thymus. Most 
profound growth activity is apparent in the mesenchyme sur- 
rounding the thoracic segments and it is here that transition 
stages from mesenchymal cells to lymphocytes frequently 
occur. Large lymphocytes are quite numerous. Even with 


2 The ‘Wanderzellen’ of Maximow and other investigators are regarded as being 
identical to the large lymphocytes. 


DEVELOPMENT OF THE THYMUS 447 


a magnification of 1300 diameters five were found in a single 
microscopic field. Those with only a slightly basophilic cyto- . 
plasm are quite numerous. 

Other cellular elements such as giant cells, the megaloblasts 
of Maximow, normoblasts and definitive erythrocytes also 
occur. These elements and the granular cells found in the 
thymus in later developmental stages are discussed further on 
in this paper. 

The occurrence of lymphocytes in the mesenchyme before they 
are present in the thymus is of the greatest significance from a 
histogenetic view point, for Bell (06) in describing the thymus 
of a 45 mm. pig embryo says: ‘““There are no lymphocytes in 
the connective tissue around the thymus or in the blood at this 
stage,’’ and of a 70 mm. embryo he says: “There are a few lym- 
phocytes outside the thymus in the interlobular tissue in this 
region; * * * * JT have never seen lymphocytes outside 
the thymus where there were none inside it, but they appear 
outside shortly after they are formed here.’’ The results of 
my observations are contradictory to those of Bell. Ina17 mm. 
embryo an occasional lymphocyte can be found in the mesen- 
chyme while in the 23mm. embryo an occasional lymphocyte 
was found in the blood. His failure to detect lymphocytes in 
the mesenchyme in stages less than 70 mm. in length was per- 
haps due to the use of Zenker’s fixing fluid which, as stated 
above, destroys the basophilic character of the cytoplasm. 
Beard (’02), in his work on the smooth skate, positively asserts 
that the first lympbocytes in the body are found in the thymus. 
Embryo of 26 mm. In this stage the lobes of the superficial 
thymus, the thymus head, and the thoracic segments have greatly 
enlarged. Lobules are beginning to grow out from them. The 
cervical segment, which enters its period of rapid develop- 
ment a little later than the thymus head and the thoracic seg- 
ment, is now quite pronounced and is studded over with short, 
blunt epithelial buds. No lobules have yet started to grow out 
from the intermediary and cervico-thoracic cords. The sur- 
face of the thymus is quite definitely marked from the mesen- 
chyme but no basement membrane is present. The vacuoles 


448 J. A. BADERTSCHER 


in the syneytium are somewhat more numerous than in the pre- 
ceding stage. They vary greatly in size. Some are in contact 
with the epithelial nuclei while others have no connection with 
them. No consideration was given to the mode of their forma- 
tion. They will again be considered in a later developmental 
stage. 

Large lymphocytes, as in the previous stage, are found plenti- 
fully in the mesenchyme surrounding tbe thoracic segment of 
the thymus. They are more numerous in the region of the larger 
blood vessels of the thorax than in those parts of the connective 
tissue containing only smaller vessels where, however, they can 
be found without much searching. Around the superficial and 
head thymus local accumulations now occur. In general, they 
are more numerous than in the preceding stage. 


2. The epoch of lymphocyte infiltration and lymphocyte proliferation 
and the formation of the reticulum 


30 mm. embryo. The thymus of this stage is decidedly in 
advance of the 26 mm. stage just described. The lobules of 
the superficial and head thymus and the cervical and thoracic 
segments have greatly enlarged while those of the intermediary 


and cervico-thoracic cords have started to develop. The mesen- 


chyme occupies all the spaces between the lobules and is some- 
what denser than that surrounding the thymus. Blood vessels 
are numerous in the connective tissue septa but none are present 
in the lobules. At this stage the lumen of the blood vessels is 
comparatively large, and their walls are thin, being made up of 
large endothelial cells only. 

The structure of the epithelial nuclei of the thymus is the 
same as in the preceding stage. Mitoses (fig. 1, M.e.N., also 
fig. 2, 37 mm.) are quite frequent. The “large dark nuclei” 
and “small dark nuclei (lymphoblasts),” (figs. land 2, D.e.N.), 
according to Bell’s nomenclature, are present as in the 23 mm. 
embryo. These I regard as epithelial nuclei in the first stages 
of degeneration. A completely degenerated epithelial nucleus 
is also present (fig. 1, D.e.N’). Its chromatin has massed into 


po ip alc oepeengtetlalie 


DEVELOPMENT OF THE THYMUS 449 


deeply stained clumps which lie in a clear space that has almost 
the same size and shape as a normal nucleus. 

The cytoplasmic syncytium of the epithelial anlage has the 
same general structure as thatina23mm.embryo. The vacuoles 
(fig. 1, V) are, however, more numerous and the vacuolation of 
the cytoplasm bas reached its greatest height in this stage. No 
basement membrane is present but the anlage is quite sharply 
defined from the surrounding mesenchyme which closely invests 
the thymus. As in earlier stages some of the protoplasmic 
processes of the mesenchymal cells are apparently fused with 
the outer surface of the cytoplasmic syneytium of the epithelium. 

The point of greatest interest and importance in this devel- 
opmental stage is the presence of lymphocytes (fig. 1, L.L.) 
in the thymus anlage. They are present in small numbers 
in the superficial thymus, thymus head, and in the thoracic 
segment. None were found in the mid-cervical segment which 
in this and younger stages is not as far advanced as the head 
and thoracic segments. Lymphocytes, however, occur in the 
thymus before the 30 mm. developmental stage. In a 25 mm. 
embryo a single lymphocyte was found in the thymus head. 
None were seen in the thymus of a 26 mm. embryo. In a 27 
mm. embryo one lymphocyte was found in one of eleven sections 
prepared from the thoracic segment. In a 28 mm. embryo 
only a few could be demonstrated. In the stage being described 
they are present in small but appreciable numbers, hence, this 
stage was chosen for the discussion of the origin of the lympho- 
cytes inthe thymus. Their location in the lobules varies. Some 
are found in or near the center of the lobules while others lie 
near the periphery. All the lymphocytes that were found in 
the thymus in this and somewhat later stages are large lympho- 
cytes. No small lymphocytes such as make up the bulk of the 
organ in late developmental stages, are present. The large 
lymphocytes are characterized by a generous amount of non- 
granular cytoplasm which is distinctly basophilic in its character. 
This basophilic character of the cytoplasm enables one to dis- 
tinguish it unmistakably from the cytoplasm of the epithelial 
cells. On account of their power of undergoing amoeboid 


450 J. A. BADERTSCHER 


movement they take on various forms. Some are round with a 
layer of cytoplasm of uniform thickness around the nucleus. 
Some are irregularly oblong with the bulk of the cytoplasm massed 
at one or both poles of the nucleus. Others are very irregular 
in outline, sending out one or more pseudopods. The size of 
the large lymphocytes varies somewhat but-all possess a large 
amount of cytoplasm. In the smaller of the large lymphocytes 
the nucleus is proportionally smaller than in the larger ones. 

The nuclei of the lymphocytes are large, but on the whole 
a little smaller than the epithelial nuclei, i.e., the nuclei of the 
large lymphocytes are smaller than the large epithelial nuclei 
while the nuclei of the smaller lymphocytes are smaller than 
the small epithelial nuclei. A distinct nuclear membrane is 
present. The form of the nuclei may vary considerably. Some 
are spherical, some oval in outline, while others have a very 
irregular shape. The varied forms of nuclei are undoubtedly 
brought about through the motile activity of the lymphocytes. 
The chromatin is generally distributed in the form of larger or 
smaller irregular granules some of which are attached to the 
nuclear membrane. These are often united with each other 
by fine irregular chromatin threads. The nucleoli are large, 
and usually very irregular in outline, having an extremely jagged 
surface. On the whole the nucleus of a large lymphocyte con- 
tains a more generous supply of chromatin than does an epithelial 
nucleus. The amount of chromatin present and the manner of 
its distribution in the two kinds of nuclei is, however, not the 
main feature by which one can readily distinguish a large lym- 
phocyte from an epithelial nucleus. The basophilic character 
of the cytoplasm of the lymphocytes is the main distinguishing - 
feature between them. At no stage in the histogenesis of the 
thymus is the value of a differential stain more appreciable 
than at the stage marking the appearance of the first lympho- 
cytes, for, by the use of it, a lymphocyte can be distinguished 
unmistakably from an epithelial cell or nucleus. 

The most critical developmental stages in which the source of 
the lymphocytes in the thymus is to be determined, are those 
in which only a few lymphocytes are found. The evidence indi- 


DEVELOPMENT OF THE THYMUS 451 


cating a transformation of epithelial cells into lymphocytes, 
and the evidence indicating an infiltration of the epithelial 
thymus by lymphocytes from the mesenchyme surrounding it or 
from the blood, were carefully followed out. The transformation 
theory will be first considered. 

In describing the structure of the thymus of a 23 mm. embryo 
it was stated that not all of the epithelial nuclei stain with the 
same intensity. In the enlarged portions of the thymus—super- 
ficial thymus, thymus head and mid-cervical and thoracic seg- 
ments—are found normally shaped nuclei (spherical and ellip- 
soidal forms; fig. 1, D.e.N.), of both the larger and smaller 
types, the nucleoplasm of which stains much more intensely 
than it does in the great majority of epithelial nuclei present. 
The nucleoplasm, however, does not stain with the same degree 
of intensity in all of the darkly stained nuclei for, in some the 
deeply stained nucleoplasm almost completely masks the chro- 
matin fibrils and granules while in others the chromatin is seen 
some what more clearly. Thus the transition forms, observed 
by Bell, occur between the more usual clear type of nuclei (which 
I regard as the normal nuclei) and the more deeply stained ones. 
In the stage (80 mm.) being described they are less numerous in 
the intermediary and cervico-thoracic cords than in the en- 
larged segments of the thymus. In a single section through 
the thoracic segment nineteen were found. In a 23 mm. em- 
bryo only an occasionally one was found in the intermediary 
and cervico-thoracic cords, while in a section through the super- 
ficial thymus ten were counted, thus indicating that they appear 
first in those portions of the organ that develop. most rapidly. 
In a 17 mm. embryo none of the intensely deeply stained nuclei 
were present although a few were found that were stained some- 
what more deeply than the great majority of normal nuclei 
present. 

A consideration of these darkly stained nuclei is of the great- 
est importance, for Prenant (’94), Bell (’06), and others appar- 
ently have taken these cells to be the forerunners of the first 
formed lymphocytes in the thymus. <A close examination of 
them, therefore, is necessary in order to reveal their fate. Be- 


452 J. A. BADERTSCHER 


sides the normal shaped dark nuclei others are found that are 
very irregular in outline and greatly distorted. Varying degrees 
of deformed nuclei can be found between the more deeply stained 
normally shaped types and the greatly distorted ones. Others 
are again found that are, without doubt, undergoing degeneration. 
Figures 2 and 5 represent each a small portion of a section through 
the thymus head of a 37 and 36 mm. embryo respectively and 
were drawn specially to show the degenerating cells.* In some 
of the degenerated epithelial nuclei (D.e.N’.) represented in 
figure 5 the chromatin is massed together in one, two or three 
clumps that stain intensely deep blue or blue-black. These 
chromatin masses usually le in clear spaces around some of 
which the distorted nuclear membrane apparently still persists. 
Occasionally a slightly elongated nucleus can be found in one 
end of which is a clump of chromatin lying in a nuclear vacuole 
while in the opposite end the nuclear threads and the nucleo- 
plasm are stained as deeply as that in the nuclei described 
above. Again, here and there in the cytoplasm of epithelial 
cells may be found larger and smaller deeply stained parti- 
cles which are evidently the débris of degenerated epithelial 
nuclei (fig. 5, D.d.e.N.). To the succession of microscopic 
pictures—deeply stained and normal shaped nuclei, deeply 
stained and distorted nuclei, and nuclei that have fallen to 
pieces—described above and drawn in figures 1, 2, and 5, only 
one interpretation, it seems to me, can be given, namely, that 
the deeply stained normal shaped epithelial nuclei are on their 
way to degeneration and do not transform into lymphocytes. 
Some of the epithelial cells show signs of degeneration in com- 
paratively early stages as stated above. They are, however, 
most numerous and most pronounced in stages from about 
30 mm. to about 45 mm. in, length. They are also found in later 
stages and will be referred to again. 

With a proper fixer, a suitable stain, and a high magnification, 
such as was used for this work, the plump lymphocytes stand 

3 Judging from the greater number of lymphocytes present and the great 
number of completely degenerated epithelial cells, the thymus of the 36 mm. 


embryo is slightly farther advanced in its development than that of the 37 mm. 
stage. 


DEVELOPMENT OF THE THYMUS 453 


out in sharp contrast to the dark epithelial nuclei. Now, what 
structural features of the dark epithelial nuclei give them a 
semblance to small lymphocytes? As the amount of cytoplasm 
around the nucleus of a small lymphocyte is meager and often 
difficult to demonstrate, a comparison between the two must 
be confined largely to the structure of the nucleus. Prenant 
observed that the nucleoplasm of the small dark epithelial 
nuclei stained so intensely as to mask its internal structure. 
Bell makes no mention of the affinity of the nucleoplasm for 
basic stains. They apparently contain no greater amount of 
chromatin than do the normal epithelial nuclei. Their diffused 
dark color is due to the affinity of the nucleoplasm for a basic 
stain. This is not the case of the nuclei of the small lympho- 
eyces. Their dark color is due to the deeply stained chromatin 
granules which, in proportion to the size of the cells, is much 
greater in amount than that found in the epithelial nuclei. The 
nucleoplasm of the small lymphocytes, which is meager in amount, 
but with a high magnification easily demonstrable, is quite 
as clear as that ordinarily found in nuclei, for example in normal 
epithelial nuclei of the thymus anlage. This fact alone, namely, 
clear nucleoplasm and an abundance of chromatin in the nucleus 
of a small lymphocyte in contrast to the deeply stained nucleo- 
plasm and a smaller amount of chromatin in the small dark 
epithelial cells, is sufficient to cause one immediately to doubt 
the identity of the two kinds of cells. Prenant writes of degen- 
erated epithelial nuclei in the thymus of a 28 mm. and later 
stages of sheep embryos but apparently saw no connection be- 
tween them and the darkly stained nuclei. Bell does not men- 
tion degenerating nuclei. 

Most of the investigators who have made a detailed study of 
the histogenesis of the thymus and who adhere to the trans- 
formation theory of the formation of the lymphocytes lay a 
great dedl of stress on the vacuolation of the cytoplasmic syncy- 
tium as a factor in the histogenesis of the small lymphocytes. 
This is particularly true of Prenant and Bell. The latter calls 
the small dark epithelial nuclei, while still imbedded in the 
cytoplasm of the epithelial cells, lymphoblasts. During the 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, NO. 4 


454 + J. A. BADERTSCHER 


process of vacuolation some of the lymphoblasts become free 
and lie in vacuoles. They then are called lymphocytes. My 
observation on the histogenesis of the thymus in the numerous 
pig embryos examined warrants no distinction in the nomen- 
clature between the two. Referring again to figures 1, 2 and 5, 
it will be seen that some of the small dark epithelial nuclei lie 
entirely in the cytoplasm and some in contact with vacuoles. 
Occasionally one can be found that lies free in a vacuole (none 
of the latter happened to be present in the portions of the sec- 
tions from which the figures were drawn). The structure of the 
nucleus is the same whether they lie in the vacuoles or in the 
cytoplasm. This also is true of the large lymphocytes (l.L.). 
Some are entirely imbedded in the cytoplasm of the epithelial 
cells while others are in contact with or entirely in the vacuoles. 
The structure of all is the same and there is no reason why they 
should not all bear the same name. The significance of the 
vacuoles and their mode of formation in the thymus is unknown 
tome. They are already present in embryos of 17 mm. in length, 
and slightly increased in size and number in later stages (30 
to 40 mm.). 

In considering the genetic relationship of the small dark 
epithelial nuclei, the ‘lymphoblasts’ of Bell, to the large dark 
epithelial nuclei, Bell expresses a doubt by saying that ‘“‘the 
large dark nuclei probably divide by mitosis and form the lympho- 
blasts.” I was unable to find any of the dark epithelial nuclei 
in mitosis although I earnestly searched for them. To me it 
appears that the small and large dark nuclei are derived respec- 
tively from small and large normal epithelial cells, their degree 
of darkness in stained preparations depending on the extent of 
degeneration. Also it cannot be that the small dark nuclei are 
formed through a contraction of the large ones for the structure 
of both is the same. Prenant, in the developing thymus of the 
sheep, described and figured direct cell division. I wads unable 
to find amitotic cell division in the thymus of the pig. 

The true source of the lymphocytes first present in the thy- 
mus will now be considered. Reference has already been made 
to the presence of lymphocytes in the thymus anlage of a 30 mm. 


DEVELOPMENT OF THE THYMUS 455 


embryo. Some are represented in figure 1 (L.L.). In slightly 
later stages they have become more numerous as represented 
in figures 2 and 5 (Z.L.). All the lymphocytes present in the 
thymus of these early stages are large lymphocytes. No small 
lymphocytes are present. No transition forms from the normal 
epithelial nuclei to the large plump lymphocytes with a generous 
amount of basophilic cytoplasm can be seen. When first present 
in the thymus they are there in a fully developed condition. 
It is, therefore, evident that their source must not be sought in 
the thymus anlage. It was stated above that large lympho- 
cytes were present here and there in the mesenchyme of a 17 
mm. embryo. In successively older stages their numbers grad- 
ually increase until in stages ranging in length from 25 to 30 
mm. they can be found in all parts of the mesenchyme without 
much searching. In these later stages, however, they are most 
numerous in the neighborhood of the thymus and the large 
blood vessels in the anterior portion of the thorax. Figure 
1 represents a portion of a lobule of the thymus head and sur- 
rounding mesenchyme of a 30 mm. embryo. Three lympho- 
cytes (L.L.) can be seen in the mesenchyme, two of which have 
a structure identical to those in the thymus. In one (lower 
corner to the right) the cytoplasm has a distinctly lighter hue, 
1.e., less basophilic, than the other two. Only very seldom 
can this latter type be found in the thymus anlage (fig. 2, D.L.; 
lower border to the left). These will be considered farther on 
in the paper. One of the lymphocytes (fig. 1) is in contact 
with the surface of the thymus. The microscopic picture, which 
is reproduced in the figure, is suggestive. Since lymphocytes 
are found in the mesenchyme in the neighborhood of the thymus 
before they are found in it, and since there are no transition forms 
between epithelial cells and lymphocytes nor any blood vessels 
in the thymus anlage, only one conclusion can be drawn in re- 
gard to the source of the lymphocytes first present in the thymus, 
namely, that they have migrated into the thymus from the 
surrounding mesenchyme. The lymphocyte bordering on the 
surface of the thymus was apparently about to enter it when the 
material was fixed. Many similar conditions exist, indicating 


456 J. A. BADERTSCHER 
the entrance of lymphocytes into the thymus at the time of the 
fixation of the material. 

Usually there are no indications on the surface of the lobules 
to mark the place where lymphocytes have entered it. On 
account of the plasticity of the cytoplasmic syncytium we can 
assume that the gaps formed in it by the entrance of the lympho- 
cytes immediately close up. Not infrequently, however, places 
can be found where the surface of a lobule is dented in and a 
lymphocyte located in the thymus near the depression (fig. 2, 
L.L., lower border to the left). Also occasionally a lymphocyte 
can be found in a lobule some distance away from the periph- 
ery with a trail (fig. 2, 7.) leading to the surface of the lobule. 
This trail apparently marks the path that a very active lympho- 
cyte took in its migration from its place of entrance to the posi- 
tion it now occupies. To similar microscopic pictures as repre- 
sented above, Maximow has given a like interpretation. The 
first lymphocyte present in the thymus then must come from 
the mesenchyme and not from transformed epithelial nuclei. 

Another type of cells which are comparatively few in number 
and found only in the earlier developmental stages deserves 
mention before passing on to a later developmental stage. These 
cells (fig. 1, X.) are characterized by rather deeply stained nu- 
clei which resemble closely the degenerating epithelial nuclei 
discussed above. The cell wall, if present, is indistinct. Their 
cytoplasm can be distinguished from the cytoplasm of the epithe- 
lial cells only by its darker color. The majority of the cells are 
long and drawn out and usually lie near the surface of the thy- 
mus anlage with the long axis of the cell nearly perpendicular 
to the surface. These were most numerous in the 17 mm. 
stage and entirely absent from the 40 mm. and later develop- 
mental stages. This type of cells was also observed by Maxi- 
mow (’09 b) who derived them from epithelial cells which for 
a time assume such form then revert to the usual type of epithe- 
lial cells. Their origin and significance are unknown to me. 

Embryo 42 mm. (fig. 3). The lobes of the superficial thymus, 
the thymus head, and of the mid-cervical and thoracic segments 
have greatly increased in size. A few lymphocytes are now 


DEVELOPMENT OF THE THYMUS 457 


present in the intermediary and cervico-thoracic cords. No 
blood vessels are present in the thymus. The walls of the 
blood vessels of the interlobular connective tissue septa are made 
up of endothelium only. The mesenchyme around the super- 
ficial and head thymus and the thoracic segment is much looser 
in its structure than in the corresponding regions in previous 
stages. Around the intermediary and cervico-thoracice cords 
and the mid-cervical segment it has a somewhat denser struc- 
ture than around the above named regions of this stage. 

As in previous stages completely degenerated epithelial nuclei 
(fig. 3, D.e.N’.) are present. Epithelial nuclei (D.e.N.) in the 
first stages of degeneration are also present but they are not as 
numerous as in the 36 mm. embryo. Mitoses of epithelial 
nuclei (M.e.N.) are quite numerous. The vacuoles are not 
as numerous as in stages ranging from 25 to 37 mm. in length. 
The most striking feature of this stage, however, is the large 
number of lymphocytes that are present in the thymus. They 
no longer all belong to the type of large lymphocytes but now 
and then a small lymphocyte (S.LZ.) is found. These are char- 
acterized by a rather small nucleus which is richly laden with 
chromatin and surrounded by only a very thin layer of cyto- 
plasm which is often difficult to demonstrate. Intermediate 
stages between the large and small lymphocytes make up a 
relatively large proportion of all present. Some in mitotic 
division can be found without much searching. Mitosis of 
epithelial nuclei and large lymphocytes can be distinguished 
from each other without much difficulty. ‘The chromosomes of 
the lymphocytes are shorter, somewhat thicker, and more 
closely packed together than those of epithelial cells. The 
basophilic cytoplasm of the lymphocytes also is sharply out- 
lined in contrast to the cytoplasm of epithelial syncytium. 
The absence of blood vessels in the thymus at this stage, the 
absence of transition forms between epithelial cells and lym- 
phocytes, the unbroken’ series of intermediate stages between 
the large and small lymphocytes, and the frequent mitoses 
found among them all, are evidences that undoubtedly point 
to the conclusion that the large lymphocytes through repeated 


458 J. A. BADERTSCHER 


division give rise to the small lymphocytes. This view of the 
origin of the small lymphocytes in the thymus is in accord with 
that of Hammer for teleosts (08) and with that of Maximow 
for mammals (’09 b). 

The mesenchyme of the interlobular septa (S.z.) in the head 
and thoracic segments contains a large number of large and 
intermediate sized lymphocytes. A few small lymphocytes 
are also present. The mesenchyme surrounding the above 
named regions also contains a relatively greater number of 
lymphocytes than it does in corresponding regions of the pre- 
vious stage described. 

An occasional nucleated red blood-cell can be found lying free 
in the thymus. At this stage they are scarcely more numerous 
than in the previous stage. None were present in that part 
of the section from which the figure was drawn. LEosinophile 
cells also can be found occasionally in any part of the mesen- 
chyme. They were first found in embryos 35 mm. in length. 
None were seen in the thymus. 

Embryo of 65 mm. (fig. 4). The lobules are more numerous 
than in the previous stage along the entire extent of the organ. 
Those of the enlarged regions of the thymus are much more 
voluminous than those of the intermediary and cervico-thoracic 
cords. An almost interrupted layer of greatly attenuated mes- 
enchymal cells closely invests the outer surface of the lobes and 
apparently forms a limiting membrane (fig. 3, L.M.) for the 
outer surface of the thymus. This membrane is present in 
slightly earlier stages. Blood vessels (BI.V.) are numerous in 
the interlobular septa. No thick walled vessels are yet present. 
The walls of most of them are made up of endothelium only. 
A few small blood vessels of an essentially capillary nature can 
now be found in the center as well as in the periphery of the 
thymic lobules. 

The thymus now contains many lymphocytes. The small 
lymphocytes (S.L.) are more numerous than in the previous 
stage. The medium-sized lymphocytes have also greatly in- 
creased in number while the number of large lymphocytes has 
remained about the same. Mitoses of both the lymphocytes 
(M.L.) and the epithelial nuclei (1/.e.N.) are of frequent occur- 


DEVELOPMENT OF THE THYMUS 459 


rence. Only an occasional deeply stained epithelial cell can 
be found. Completely degenerated epithelial nuclei can be 
seen scattered here and there throughout an entire section. 
The connective tissue of the interlobular septa now contains 
numerous lymphocytes of all sizes. The deep portions of some 
of the septa are so completely gorged with them that it is diffi- 
cult to distinguish clearly where the septa end. An especially 
favorable place for lymphocytes to collect seems to be along 
the course of blood vessels of the septa. They can be found 
strung along in rows on one side of the vessels or the accumula- 
tion may extend entirely around it. The vacuoles of the syn- 
cytium are not as numerous as in the preceding stage. Most 
of them have become occupied with lymphocytes. In later 
stages they are altogether absent. 

An almost uninterrupted zone of epithelial syncytium (Z.pr.) 
extends around the periphery of the thymus. It is from one 
to three epithelial nuclei deep and, on account of the few lym- 
phocytes which it contains, appears quite clear in contrast to 
the deeper portion of the syncytium in which are found many 
lymphocytes. It is not pronounced along the interlobular 
septa. Mitoses of the epithelial nuclei are more numerous in 
this zone than they are in the deeper portions of the lobules, 
hence, Prenant called it the zone of proliferation. According to 
him both lymphocytes and reticulum cells are formed from this 
zone. ‘This, however, cannot be the case for the transition forms 
from epithelial nuclei to lymphocytes are not present. Con- 
sidering the fact that the epithelial zone is most pronounced 
only on the convex peripheral surface of the lobules, and that 
it is present only during the period of rapid growth of the thy- 
mus, it can rightly be regarded as a zone of proliferation for 
epithelial cells but not for lymphocytes. It is mainly from this 
zone that the reticulum of the peripheral margin of the cortex 
is formed while the thymus is rapidly growing in thickness. This 
interpretation of the significance of this zone is in accord with 
that of Maximow. 

This develomental stage marks the appearance of the medulla. 
Longitudinal sections through the thymus head show that the 
epithelial syneytium of almost the entire central stem has under- 


460 J. A. BADERTSCHER 


gone changes. The medulla of the lobes, in some of which at 
this stage it is but slightly developed, is continuous with that of 
the central stem. The deep portions of some of the interlobular 
septa are almost in contact with it while others are separated 
from it by a cortical layer of considerable thickness. The 
medulla is formed directly from the epithelial syncytium. In 
sections stained with Hasting’s Nocht’s stain it is easily dis- 
tinguished from the cytoplasmic syncytium of the cortex byits 
brighter red color. The initiative changes marking its appear- 
ance are apparently chemical in their nature as pointed out by 
Bell, for the syncytium in some parts of the central stem and 
in the center of some of the lobules is stained a bright red even 
before any morphological changes have set in. The morphologi- 
cal changes of the epithelial structure occur very soon after, or 
almost simultaneously with, the chemical changes. The epithe- 
lial cells hypertrophy. The nuclei become large and relatively 
clear when compared with those of the cortex. The cytoplasm 
of the syncytium also increases in amount. Its anastomosing 
processes are no longer thin and attenuated as they now appear 
in the cortex but have become more or less massive bands. 
Although the cortex and medulla are quite sharply defined the 
eytoplasmic processes of the epithelial cells. lying along the 
line of demarcation between these two structures are contin- 
uous with each other. 

Soon after the medulla has started to develop some of the 
epithelial nuclei contained in it greatly increase in size, grow- 
ing much larger than the majority of hypertrophied nuclei. 
These may be found singly or in groups of two or three and 
mark the beginning:of Hassall’s corpuscles. 

All the different types of lymphocytes (large, medium-sized, 
and small) found in the cortex are also found scattered in the 
meshes of the reticulum of the medulla where they are, how- 
ever, much less numerous than in the former place. According 
to Maximow (’09 b) the disappearance of the lymphocytes from 
the medulla, when it is first formed, is due to their migration 
into the cortex and to degeneration. His interpretation does not 
seem to explain similar conditions existing in the thymus of the 


ee ee ee ee ee 


a 
OT ied ted 


NT Sete en ot Se SI oS 


DEVELOPMENT OF THE THYMUS 461 


pig, for only very seldom can degenerated cells be found which 
may be degenerated epithelial nuclei, and as to whether they 
migrate into the cortex it is indeed difficult to establish in fixed 
material when no circumstantial evidence is present indicative 
of their migration. A more plausible interpretation seems to 
be that during the hypertrophy of the epithelial cells the medul- 
lary portion of the thymus greatly increases in volume through 
the enlarging of both the nuclei and anastomosing processes of 
the syncytium thus separating the lymphocytes farther apart. 
Just as many are present in the rapidly newly formed medulla 
as there were in the syncytium from which the medulla was 
formed only they are scattered over a larger area making them 
to appear less numerous. ‘This interpretation is made plausible 
when the great rapidity of its initial development is considered, 
e.g., in the thymus of a 60 mm. embryo no traces of the medulla 
were present while in a 65 mm. embryo it has reached a stage of 
development as described above. 

The reticulum of the cortex also is formed from the cyto- 
plasmic syncytium of the epithelial cells. Its development, 
unlike that of the medulla, is gradual. The change from the 
rather coarse syncytial network of younger stages to fine and 
greatly attentuated threads making up the reticulum in the 
fully developed thymus is due to the lymphocytes constantly 
increasing in numbers in its meshes thus gradually separating 
the cell bodies of the reticulum farther apart. In all of the 
developmental stages studied mitosis of the epithelial nuclei 
could be found, being, however, more numerous in younger 
than in later developmental stages. 

In this and slightly earlier stages (55 mm.) nucleated and non- 
nucleated red blood-cells lying free in the parenchyma of the 
thymus are of frequent occurrence. While some are scattered 
about singly they usually occur in groups. An _ occasional 
eosinophile cell can also be found. In the interlobular septa 
phagocytes can be found without much searching. 

The thymus head and superficial thymus were so oriented 
on the microtome that sections of both of these regions were 
made by a single stroke of the knife. This made a comparison 


462 J. A. BADERTSCHER 


of their histological structure easy as they lay side by side on 
the slide. No difference in structure could be distinguished 
between the two, thus indicating that the histogenetic processes 
of that portion of the thymus derived from the ectoderm keep 
pace with that portion derived from the entoderm. 

Embryos 85, 100, 125, 165, and 180 mm. in length. In these 
developmental stages all the structures found in the fully devel- 
oped thymus are laid down and will, therefore, be considered 
only briefly. The average size of the lobules and the thymus 
as a whole increases in the successively older stages. In the 
first four stages the thymic septa have still a very loose struc- 
ture while in the 180 mm. embryo they are quite narrow and 
correspondingly denser. Many of the septa are broadly ex- 
panded where the larger interlobular blood vessels are harbored. 
The thymic septa of the 85 and 100 mm. stages are characterized 
by the large number of all types of lymphocytes (large, medium- 
sized, and small) which they contain. The presence of so many 
lymphocytes in the septa is a feature that is most marked in 
developmental stages from 65 to about 115 mm. in length. The 
septa of the 125 and 165 mm. stages contain many lymphocytes 
but on the whole they are less numerous than in the earlier stages 
cited. In the 180 mm. embryo the lymphocytes are mostly 
confined to the deep expanded portions of the septa which are 
often gorged with them. The number of lymphocytes in the 
connective tissue immediately surrounding the thymus is rela- 
tively small when compared with the number present in the septa. 
Mitoses of all types of lymphocytes are of frequent occurrence. 

A marked feature of the cortex in these stages is the large 
number of small lymphocytes which it contains. Excepting 
the 85 mm. stage they make up the largest proportion of all the 
lymphocytes present. Mitoses of all types of lymphocytes are 
of frequent occurrence while mitoses of epithelial nuclei can occa- 
sionally be found. The epithelial (reticulum) nuclei are, in gen- 
eral, smaller than those found in younger stages but their struc- 
ture has remained unchanged. The reticulum composing the 
strands are greatly attenuated and only in very thin sections 
can it be satisfactorily demonstrated. Its meshes are filled 
with lymphocytes. Vacuoles are no longer present. 


DEVELOPMENT OF THE THYMUS 463 


The clear epithelial zone around the periphery of the thymus 
is present in all the stages excepting the 180 mm. embryo. Around 
the thymus head of the 165 mm. embryo it is at its highest de- 
velopment. This is contradictory to the observations of Bell 
who states that this zone has disappeared in a 140 mm. embryo. 
Mitoses of epithelial cells in this zone are quite numerous and in 
no stage is it entirely free from lymphocytes. The limiting 
membrane could no longer be distinguished around the thymic 
lobules in the 180 mm. stage. It apparently has become blended 
with the thin capsule that invests the thymus of this and later 
stages. 

Im all these stages the medulla contains a relatively much 
larger number of lymphocytes than in the 65 mm. embryo, which 
makes it appear less conspicuous. This is especially the case 
in the 180 mm. embryo, but even in that stage in suitably stained 
preparations it is still quite sharply defined from the cortex. 
Mitoses of all types of lymphocytes occur here as in the cortex.. 
In the 180 mm. embryo Hassall’s corpuscles are more numerous 
than in the earlier stages, while some are still in the process of 
formation. The reticulum on the whole is much coarser than 
in the cortex and hence more easily demonstrable. 

In the 180 mm. embryo deeply stained (degenerating) epithe- 
lial nuclei can be found only after prolonged searching while in 
the earlier stages they are of more frequent occurrence in both 
the cortex and medulla. Débris of degenerated cells, some of 
which in these stages is undoubtedly composed of nuclei ex- 
truded from normoblasts, also occurs. 

A discussion of the red blood-cells and granular leucocytes 
will be considered later. 

Embryo 270 mm. (full term). Since one of the main objects 
of the investigation of the developing thymus was to determine 
the origin and fate of the superficial thymus its histological 
structure will, therefore, be considered. The lobules are now 
closely packed together. The cortex has greatly increased in 
thickness over that of the 180 mm. embryo. The lymphocytes 
are no more closely packed together than in the previous stage, 
room having been made for the additional number by an increase 
in the volume of the organ. While the small lymphocytes are 


464 J. A. BADERTSCHER 


by far the most numerous, large ones are still plentiful in all 
parts of the cortex. Even with a magnification of 1300 diameters 
eleven were counted in a single microscopic field. All grada- 
tions between the large and small ones are present. Mitoses 
of all the different types occur. 

The medulla is still quite sharply defined from the cortex. 
It contains less lymphocytes than the cortex. In some places 
where the medulla of the lobules joins with that of the central 
stem it comes in contact with the deep portion of the interlobular 
septa. It contains all the different types of lymphocytes that 
are present in the cortex and mitoses among them can be demon- 
strated without much difficulty. Hassall’s corpuscles are more 
numerous than in the 180 mm. stage and an occasional one can 
still be found in the process of formation. 

In the medulla the strands of the reticulum are “oti wavy 
and in general are much coarser than those of the cortex. Also 
-the epithelial nuclei are on the whole larger and clearer, and 
surrounded by a more generous amount of cytoplasm than those 
in the cortex. Mitoses of epithelial nuclei in both the cortex 
and medulla can only very seldom be found. In sections treated 
with Mallory’s connective tissue stain fibrillae can be seen to 
come off from the interlobular septa and the capsule and extend 
a distance of from one to four cells deep into the cortex. In 
both the cortex and medulla the same condition prevails between 
the adventitia of the larger blood vessels (which are very few) 
and the reticulum. I was unable to determine whether the 
connective tissue fibers fuse with the reticulum. This intimate 
relation of the connective tissue of the septa and of the large 
blood vessels to the reticulum was observed by Mietens (’08) 
but denied by Maximow (’09 b). No connective tissue fibers 
aside from those mentioned above could be demonstrated in 
either the cortex or medulla. Bell, however, by using Jack- 
son’s modification of Mallory’s stain states that he was able to 
demonstrate them thinly scattered through both the cortex 
and medulla in all late developmental stages. 

The interlobular connective tissue septa are greatly reduced 
in thickness, being widest in those places which lodge the larger 


So I ee er nr aie eo 


a nor 


. 
DEVELOPMENT OF THE THYMUS 465 


blood vessels and at the points of intersection of two or more 
septa. In some places prolongations of the septa dip down into 
the cortex of the lobules. These secondary septa approach 
very nearly the medulla but seldom enter it and are usually 
expanded at their deeper ends where they may lodge larger 
blood vessels. The structure of the wider portions of the septa 
is usually looser than the thinner parts. Small blood vessels of 
a capillary nature are found through the septa and ean often 
be seen entering the cortex. Lymphocytes are present only in 
comparatively small numbers. In the more compact portions of 
the septa they may be entirely absent. 

A discussion of the red blood-cells and the granular leucocytes 
in full term embryos will follow. 


3. The epoch of the formation of the red blood-cells and the develop- 
ment. of granular leucocytes 


Investigators disagree as to the extent of the formation of 
red blood-cells in the thymus. Many have observed red blood- 
cells lying free in the parenchyma of the thymus during both 
its growth and involution but to my knowledge no extended 
investigation through a wide range of developmental stages 
has yet been made of their origin. Afanassiew (’77) apparently 
was the first to consider their origin. He held that during 
the development of the thymus a rearrangement of some of 
the blood vessels took place resulting in the formation of 
the concentric (Hassall’s) corpuscles. During this process 
some of the blood vessels are ruptured thus permitting erythro- 
cytes as well as leucocytes to escape into the parenchyma where 
they then may be found singly or in groups. In mammals the 
red blood-cells usually underwent degeneration. He regarded 
the thymus a hemolytic organ. 

Watney (’82) also observed erythrocytes, ‘hemoglobin masses,’ 
and cells containing fragments of hemoglobin in their cytoplasm 
in the thymus of mammals, birds, reptiles, and fishes. He does 
not state whether the erythrocytes and hemoglobin masses are 
derived from cells in the parenchyma or whether they have 


466 ' J. A. BADERTSCHER 


passed into it from blood vessels but regards the thymus as a 
source of some of the ‘‘colored blood corpuscles.” 

Prymac (02) holds that during involution of the teleostean 
thymus numerous red blood cells are formed from the small 
round cells. Erthrocytes also escape into the parenchyma 
from the blood vessels. All undergo degeneration. The prod- 
ucts of degeneration of the greater number of red cells are gran- 
ules which are taken up by indifferent thymus elements, while 
that of the smaller portion is in the form of pigment which 
accumulates in masses in the parenchyma. 

Schaffer (’93) in the thymus of the rabbit and cat found red 
blood-cells in various stages of development, and transition 
forms between the leucocytes and nucleated red blood-cells. 
He believes that the thymus has a hematopoietic function. 

Bell (06) in the thymus of a 240 mm. pig found numerous 
erythrocytes, lying singly and in groups, in the cortex while 
free erythrocytes were rarely to be found in the medulla. He 
does not consider their origin. 

Maximow (’09) was not able to recognize definite erythro- 
blasts or transition forms in the parenchyma of the thymus but 
thinks that lymphocytes have been confused with them. In 
the interlobular septa, however, he found collections of erythro- 
blasts and normoblasts, or briefly, all transition forms from large 
lymphocytes to erythrocytes. He does not believe that red 
blood-cells are formed in the thymus. 

Other investigators have observed free erythrocytes in the 
thymus in various stages of its development. Some make no 
mention of their origin while others suggest a possible. origin 
but do not trace out their cytomorphosis. 

Granular cells have been observed in the thymus by many 
investigators but the views regarding their origin and nature, 
which will be only briefly summarized, are conflicting. During 
the latter part of fetal life and the remaining period of growth 
and involution, Watney (’82) found in the medulla of the thymus 
of birds, reptiles, and mammals many granular cells. He divided 
them into*four classes which were connected with each other by 
intermediate forms. He found them especially numerous along 


7 
i 
‘ 
' 


DEVELOPMENT OF THE THYMUS 467 


the course of blood vessels to the outer tunic of which many 
were attached. He derived them all from connective tissue 
cells. 

Schaffer (91) apparently was the first investigator to observe 
eosinophile cells in the thymus. In various developmental 
stages of the human thymus he found large numbers of eosino- 
phile cells in the connective tissue surrounding the thymus, in 
the interlobular septa, and along the course of some of the cap- 
illaries in the medulla. A few also were found in the cortex. 
The size of the granules vary and the nucleus he described as 
being round. His investigations of the eosinophile cells in 
the thymus were then too incomplete to say anything definitely 
regarding their origin but he believed that they were not identi- 
eal with the ‘granular cells’ of Watney. In the medulla of 
the involuting thymus of the mouse he (’09) found many eosino- 
phile cells and numerous free granules that stained intensely 
with eosin. These granules he regarded as products of degen- 
erated epithelial cells. Within the lobules and in the inter- 
lobular septa of the involuting thymus many plasma cells were 
also present. These he derived from the small lymphocytes 
of the thymus. Many had the appearance of undergoing de- 
generation. 

Goodall (’05) is of the opinion that the pseudo-eosinophile 
cells in the region of Hassall’s corpuscles in the thymus of the 
guinea-pig, are derived from the blood. 

Maximow (’09) claims that different types of granular cells 
were found in the thymus in different species of mammals. In 
the more advanced stages of rabbit embryos an appreciable 
number of pseudo-eosinophile myelocytes were found in the 
interlobular septa; cortex and medulla. Only a few mast cells 
were found, most of which were in the septa. In guinea-pig 
embryos psuedo-eosinophile myelocytes were seldom found. 
In cat embryos 35 to 50 mm. in length special myelocytes and 
leucocytes were found in quite large numbers, while in embryos 
120 to 130 mm. long numerous mast cells were found in the deeper 
portions of the cortex and in the medulla. In the septa they 
were less numerous. Only an occasional eosinophile cell was 


468 J. A. BADERTSCHER 


found in the interlobular septa of the thymus in the above named 
animals. A few were found in the parenchyma of the thymus 
in a rat embryo 19 mm. in length. The different types of cells 
named above are derived from lymphocytes and the granules 
in all of them are products of the cell in which they are contained. 

In the description of the later developmental stages of the 
thymus mention was made of free nucleated and non-nucleated 
red blood-cells and eosinophile cells in both the parenchyma and 
interlobular septa of the thymus. Through the investigations 
of Maximow (’09 a), and others, who have traced the develop- 
ment of the blood from early developmental stages in which the 
cells of the blood islands were still undifferentiated to later stages 
in which all the different types of blood-cells were found in the 
circulating blood, the view of a common ancestor for all the 
different types of blood-cells has been quite generally accepted. 
This primitive or undifferentiated blood-cell is structurally very 
much hke a large lymphocyte and by some regarded identical with 
it. Also, Maximow and others have shown that erythrocytes, 
and granular cells develop from mesenchymal cells of the intra- 
embryonic mesenchyme.' 

Since erythrocytes were already present in the blood streams 
of the youngest embryos collected for this work, the blood islands 
and other hematopoietic regions were not investigated. In the 
mesenchyme, however, the development of the free erythrocytes 
and granular cells was traced apparently from their source. 
A consideration, therefore, of the source of the above named 
cells found in the interlobular septa (mesenchyme) of the thymus 
will be made first, for a knowledge of their origin will aid in 
determining the origin of free erythrocytes and granular cells 


4 For a detailed account of the development of free blood-cells in the mesen- 
chyme, reference should be made to Maximow’s work of 1906. With the methods 
of technic used for this work I was able to confirm most of his conclusions re- 
garding the origin of mesenchymal blood-cells. Hence, I have adopted ten- 
tatively the nomenclature employed by him. A detailed account of my observa- 
tions would unnecessarily lengthen this article. The descriptions and drawings, 
therefore, will be only sufficiently detailed to be within the limits of clearness 
and accuracy. The primitive blood-cells or ‘Wanderzellen’ have been termed 
‘large lymphocytes’ throughout this work. 


DEVELOPMENT OF THE THYMUS 469 


in the parenchyma of the thymus which, to anticipate, develop 
from the same type of undifferentiated cells as those in the 
mesenchyme. 

In early stages the development of the free blood-cells can be 
demonstrated in most any portion of the mesenchyme in the 
neck and upper thoracic region of young embryos. There are, 
however, localized regions where this process is carried on even 
in later developmental stages. The interlobular septa of the 
thymus are particularly favorable places to study the develop- 
ment of blood-cells in well advanced embryos. The thymus of 
an embryo 125 mm. in length was selected for the cytomorphosis 
of the erythrocytes, eosinophile cells, and phagocytes, although 
somewhat later stages could have been used. The connective 
tissue of the septa is loosely arranged and contains many transi- 
tional forms leading from the connective tissue cells to the above 
named elements. A few stellate-shaped connective tissue cells 
are still found but the spindle shaped type is most numerous. 
In figure 7 is a series of diagrams representing a suggested ‘cell 
lineage’ between connective tissue cells and their derivatives, 
1.e., erythrocytes (blood-plastids of Minot), phagocytes, and 
granular cells. Diagram a represents a connective tissue cell. 
The cytoplasm is finely granular and is only very slightly baso- 
philic. In some small vacuoles occur. No cell membrane 
could be demonstrated. The nucleus may be round or oval and 
has a distinct nuclear membrane. The chromatin is in the 
form of small irregularly shaped granules many of which are 
joined together by very fine chromatin threads. The nuclei 
vary in number from one to three. Diagram e represents a 
transformed mesenchymal cell. Its protoplasmic processes are 
retracted and now lie free in the septa. The cytoplasm has 
slightly but appreciably increased in amount and has become 
more basophilic and now is homogeneous. The nuclear changes 
are represented apparently by a slight massing of the chromatin, 
the granules becoming slightly coarser and less numerous. In 
some connective tissue cells the cytoplasm becomes more baso- 
philic and the nuclear changes occur even before its processes 
have been retracted (diagram 6). This type of connective 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 


470 J. A. BADERTSCHER 


tissue cells is interesting in that its transformation to the free 
cell can be easily followed. Now and then a transformed mesen- 
chymal cell, d, can be found, the cytoplasm of which is quite 
pale, being no more or only slightly more basophilic than that 
in the ordinary connective tissue cells. Its cytoplasm, how- 
ever, is homogeneous and its nuclear structure similar to that of 
ordinary large lymphocytes. This type of cells evidently has 
the power to wander about in the mesenchyme for occasionally 
(very seldom) ean one be seen in the epithelial anlage of the thy- 
mus (fig 2, lower border to the left). These were observed 
by Maximow who claims that their cytoplasm soon turns baso- 
philic after they are formed. Judging from their structure 
and the small number present his interpretation is correct and 
they must therefore be considered as belonging to the same 
type of cells as those in which the cytoplasm is more basophilic. 
In some transforming mesenchymal cells, c, the cytoplasm be- 
comes basophilic and the chromatin increases in amount while 
the protoplasmic processes are being retracted, i.e., the cyto- 
plasmic and nuclear changes take place simultaneously. This 
process results in the formation of a cell in which the cytoplasm 
is less basophilic and the nucleus contains less coarse granules 
than in a fully developed large lymphocyte. These cells (young 
lymphocytes) transform into the typical large lymphocytes as 
represented in diagram e. Since this type of cells is of more 
frequent occurrence than those represented in diagrams b and 
d it is assumed that this is the most usual manner by which 
a mesenchymal cell transforms into a lymphocyte. The type 
of cells under consideration and represented in diagrams 
c, d and e are the primitive mesamoeboids of Minot and the 
primary wandering cells of Maximow and others. With Max- 
imow and others, I agree that they are essentially identical 
with the large lymphocytes which term was given them in the 
account of the histogenesis of the thymus in the early stages 
of its development. 

The power of the lymphocytes to develop in different directions 
is clearly manifested in the interlobular septa of this stage in 
which many lymphocytes of all types are found. Judging 


Oe ee ee ee ee eee eee eee 2 


am a oe 


DEVELOPMENT OF THE THYMUS 471 


from the mitoses that some are undergoing, the large lympho- 
cytes through repeated divisions become smaller and form 
small lymphocytes, f. Whether or not the small lymphocytes 
have the power to grow and again form large lymphocytes, as 
claimed by some investigators, is difficult to demonstrate. The 
point of interest and importance is the development of erythro- 
cytes and granular cells from the lymphocytes. In some of 
the transition stages between the large and small lymphocytes, 
or for convenience, the large and medium-sized lymphocytes, 
changes occur in both their nucleus and cytoplasm. The latter 
stains a faint brick-red indicating the presence of hemoglobin 
while the nucleus becomes granular. These are the mega- 
loblasts of Maximow or erythroblasts, g. In some cells, h, the 
nucleus has the characteristic granular structure of the erythro- 
blasts while the cytoplasm still retains its basophilic character, 
or is dimmed only slightly by a faint trace of hemoglobin. These 
are the younger forms of erythroblasts and aid in tracing the 
source of the older ones. They may be found lying singly but 
usually occur in groups. Mitoses of erythroblasts can be found 
without much searching. Diagram 7 represents a normoblast. 
The cells of this type are on the whole a little smaller than the 
erythroblasts from which they are derived. Through the 
extrusion of their nuclei they are transformed into erythrocytes, 
j. That the nuclei are extruded is indicated by deeply stained 
degenerating nuclei or fragments of them lying free among the 
cells in a group made up of a mixture of both erythrocytes and 
normoblasts. Thus the free erythrocytes of the interlobular 
septa, as stated by Maximow, are derived from the lymphocytes, 
their ultimate source being from transformed mesenchymal cells. 
Whether or not they enter the circulation will be considered 
presently. While they are found in the septa in quite early 
stages they are most numerous in this region in embryos rang- 
ing from 115 to 165 mm. in length, the greatest number 
being present at about the 125 mm. stage. The superficial and 
head thymus of a 270 mm. (full term) fetus contained a few, 
singly and in groups, in the deeper and looser portions of the 
septa. 


472 J. A. BADERTSCHER 


With this brief review of the origin of the erythrocytes in the 
interlobular septa we are prepared to consider their source in the 
cortex and medulla of the thymus. In every stage from the 55 
mm. to the full term embryo that was examined, red blood- 
cells were found singly and in groups in the thymus. In the 
developmental stages approaching maturity a larger number of 
the lobules contain groups of red cells than in younger stages. 
Some lobules contain two or three groups some of which are 
quite large. Also red cells lying singly in the thymus are more 
numerous in the later than in the earlier stages. The super- 
ficial thymus and the thymus head were found to contain a 
relatively larger number than the mid-cervical segment. Un- 
fortunately, the thoracic segment of late developmental stages 
was not collected, so I was unable to make a comparison of their 
number with that of the other segments of the thymus. The 
superficial thymi and the thymus heads of two full term fetuses 
(270 and 280 mm in length) contained a comparatively larger 
number of red blood-cells than the corresponding segments of 
somewhat earlier stages. In the thymus of the 280 mm. fetus 
the red blood-cells were about equally distributed in the two 
segments while the red cells in the superficial thymus of the 270 
mm. embryo were much more numerous than in the thymus 
head. In full term embryos groups of red blood-cells are found 
in both the cortex and medulla of the thymus. In the younger 
stages no groups of red blood-cells were found in the medulla 
although they may be found lying singly in that region. 

An occasional nucleated red blood-cell can be found in the 
thymus of embryos 35 to 50 mm. in length. Erythrocytes in 
these stages are very seldom found. They do not come from 
the blood for blood capillaries have not yet penetrated the lobules 
at this stage. In an embryo 55 mm. in length, in which only a 
few capillaries are found in the lobules, they are much more 
numerous than in the preceding stages. Nucleated and non- 
nucleated red cells can be found singly among the lymphocytes 
which at this stage are already quite numerous but the striking 
feature is that they are present in groups (fig. 6). They vary 
somewhat in size as do those in the interlobular septa but the 


— =< 


DEVELOPMENT OF THE THYMUS 473 


majority in the thymus have a smaller average diameter than 
those in the latter place. Their contour is often very irregular 
which is due to their lying closely together when found in groups 
or wedged in between lymphocytes and epithelial cells when 
they occur singly. The nuclei of some have the characteristic 
coarsely granular structure of erythroblasts (Hrb.) and young 
normoblasts, while in others the nuclei are pyknotic. The red- 
dish hemoglobin-containing cytoplasm of the nucleated red cells 
varies in its amount in different cells but is easily recognized 
even with moderately high magnification in the larger and 
medium sized cells. Some of the nucleated red cells have two 
nucleoli (A. Hrb.) which stain intensely, are round, and of equal 
size. These apparently are undergoing amitotic division. Ery- 
throcytes (#rc.) are quite numerously scattered among the 
erythroblasts. Aside from the irregular outline of some of the 
nucleated red cells found in the thymus of this stage they com- 
pare favorably in all other respects with the free erythroblasts 
and normoblasts found in the septa. Also they have the same 
origin, namely, from the lymphocytes. 

The lymphocytes in the thymus in which the origin of the 
red blood-cells was just considered belong almost entirely to 
the large and medium-sized type. Only a very few small ones 
are present. It is, therefore, necessary to consider the origin 
of the numerous free erythrocytes in the thymus of late develop- 
mental stages in which the large majority of all the lymphocytes 
belong to the small type. The superficial thymus of a 270 
mm. (full term) fetus was selected for this purpose because the 
red blood-cells in the thymus of this stage are more numerous 
than in any other examined. The great majority lie in groups 
which, in a section, appear as smaller or larger bright red irregu- 
lar patches or as long tortuous streamers. The proportion of 
lymphocytes to the red cells varies in different groups. In 
some the lymphocytes are most numerous, in others they are 
about equal in number, while in still others the red cells greatly 
predominate. Also, in some groups the erythrocytes make 
up nearly the entire number of red blood-cells, only a few nu- 
cleated red cells being present. In some groups many nucleated 


474. J. A. BADERTSCHER 


red cells are found among the erythrocytes, while other groups 
are composed almost entirely of nucleated red cells. Both the 
erythrocytes and nucleated red blood-cells vary in size, but the 
small ones greatly predominate over the medium sized and larger 
ones. They are usually irregular in outline. The structure 
of the nuclei vary as those in the 55 mm. stage. An interesting 
and most helpful feature in tracing out the origin of erythrocytes 
in late developmental stages is the presence of lymphocytes 
with granular nuclei, the structure of which is the same as that 
of the erythroblasts and normoblasts found in the interlobular 
septa, the only difference being their smaller size. Only a few 
small lymphocytes with this type of nucleus were found in the 
thymus of the 55 mm. stage and in the interlobular septa of the 
125 mm. embryo. They stain intensely and when examined 
with lenses of low magnification appear as black dots in con- 
trast to the other small lymphocytes among which they lie. 
Maximow (’09 b) in writing of the erythropoetic function of 
the thymus, which he denies, makes mention of this type of 
lymphocytes but on the ground that they contained no hemo- 
globin he does not consider them to be normoblasts. It is true 
that in many lymphocytes with this type of nucleus, some- 
times entire groups, no traces of hemoglobin can be detected in 
their cytoplasm even when highly magnified (x 2000). But, 
many small cells can also be found with similarly granular 
nuclei and with a distinct reddish tinge which indicates the 
presence of hemoglobin in their cytoplasm. These are small 
erythroblasts that are derived from small lymphocytes and the 
small cells referred to by Maximow, and so plentifully found 
in the thymus of this developmental stage, are transition forms 
between the ordinary small lymphocytes and the small erythro- 
blasts. On account of the meagre amount of cytoplasm in these 
erythroblasts they appear much as if the nucleoplasm was 
stained slightly red. But that is not the ease for the red stained 
cytoplasm, of those in the late normoblast stage in which the 
nucleus has become shrunken and pyknotic, stands out sharply, 
although it is small in amount. Transition forms are often 
scattered along the border of groups of red cells containing many 


DEVELOPMENT OF THE THYMUS 475: 


erythrocytes, and in groups of nucleated red cells they are almost 
invariably found scattered among the erythroblasts and normo- 
blasts. Typical large erythroblasts and normoblasts such. as 
occur in the thymus of the 55 mm. stage are also present in the 
thymus of late developmental stages; but in these stages they: 
make up only a small proportion of the erythroblasts. This is: 
-accounted for by the fact that the small nucleated red cells are 
derived from the small lymphocytes while the large ones are 
derived from large and medium-sized lymphocytes which are 
comparatively few in number in late developmental stages. 

The débris of degenerated nuclei extruded from the normo- 
blasts can often be found scattered among erythrocytes. It 
is not uncommon for this débris to collect in heaps which appear 
in sections as deeply stained dark structureless patches in a group 
of erythrocytes. Why the degenerated nuclei have a tendency 
to flow together can only be conjectured; also why the red blood- 
cells are mostly formed in groups instead of uniformly through- 
out a lobule is unknown to me. 

The blood stream of a 55 mm. embryo contains nucleated 
red blood-cells and since capillaries are already found in the 
thymus in this developmental stage it might still be argued, 
_ as is held by some, that the free erythrocytes in the thymus are 
derived from the blood. This, however, cannot be the case for 
in a full term fetus in which many erythrocytes and nucleated 
red blood-cells are found in the parenchyma of the thymus no 
nucleated red cells were found in the blood stream. 

From the observations cited above I must conclude that the 
erythrocytes in the meshes of the reticulum of the thymus are 
derived from the lymphocytes. Furthermore, on this con- 
clusion are hinged three important features two of which strongly 
reflect on the nature of the small round cells of the thymus, 
while one furnishes additional proof for the pseudomorphosis 
theory of the histogenesis of the thymus. They are: (1) Since 
both the lymphocytes of the mesenchyme and the small round 
cells of the thymus have the power to transform into erythro- 
cytes they are potentially alike. The small thymic cells are, 
therefore, lymphocytes and not epithelial cells as held by StGhr 


476 J. A. BADERTSCHER 


(706); (2) The lymphocytes can be regarded as undifferentiated 
blood-cells and under certain conditions are, in some organs, 
potentially like the primitive blood-cells, and (3) The like poten- 
tiality of the lymphocytes in both the thymus and the mesen- 
chyme is additional evidence that the lymphocytes first present 
in the thymus have migrated into it from the mesenchyme. 

Whether or not any of the erythrocytes formed in the thymus 
or in the mesenchyme surrounding it enter the circulation is 
difficult to determine in fixed material. Some undoubtedly un- 
dergo degeneration. In the mesenchyme of early stages and 
in the interlobular septa of later developmental stages some 
erythrocytes are present the cytoplasm of which is granular in- 
stead of homogeneous. In some cells the granules are small, 
round, and of a quite uniform size while in others the granules 
vary greatly in size. Some are apparently about to break up 
into a small number of irregularly shaped fragments. I am 
confident that these erythrocytes are degenerating forms and 
are not artifacts, for in the same microscopic field may be found 
numerous other cellular elements (connective tissue cells, lym- 
phocytes, nucleated and non-nucleated red cells) all of which 
have the appearance of a good preservation. Also not infre- 
quently erythrocytes can be found that have completely fallen 
to pieces, the débris of degeneration being in the form of vary- 
ing sized globules and irregularly shaped fragments, or irregular 
groups or long drawn out rows of small deeply red stained (eosino- 
phile) granules. The latter may be derived directly from the 
disintegration of granular erythrocytes or from the further dis- 
integration of large fragments of them. Some of the red cells 
undergo degeneration while still in the normoblast stage. These 
are characterized by a pyknotic nucleus and more or less gran- 
ular cytoplasm. Except for their small size and the type of 
nucleus they contain, some could easily be mistaken for small 
eosinophile cells. 

Degeneration of some of the free erythrocytes in the lobules 
of the thymus takes place in a manner similar to that described 
above. Groups of free eosinophile granules can be found in 
the thymus of all developmental stages in which red blood-cells 


DEVELOPMENT OF THE THYMUS 477 


are formed in that organ. The free eosinophile granules are never 
very numerous but the thymus of late developmental stages in 
which the erythrocytes are comparatively numerous contains 
more than the thymus of early stages. Erythrocytes and normo- 
blasts with granular (degenerating) cytoplasm are also present. 
The red blood-cells in the thymus usually have an irregular 
outline. This is not a sign of degeneration but is brought about 
by purely mechanical factors as stated above. In late develop- 
mental stages phagocytes ingested with erythrocytes and other 
degenerated products can occasionally be found in the lobules of 
the thymus. These, however, appear first and are more numer- 
ous in the interlobular septa. In some groups of red cells in 
the superficial thymus of the 270 mm. fetus some of the erythro- 
cytes are apparently fused, forming as seen in section, irregularly 
and deeply red stained and quite homogeneous patches which 
contain only a few lymphocytes. Whether or not the fused 
erythrocytes undergo degeneration could not be determined with 
the material at hand. The thymus of post-natal pigs needs 
to be investigated to determine the fate of the numerous free 
erythrocytes present in that organ. It is evident, however, 
that not all, if any, enter the circulation. 

In all the developmental stages examined eosinophile cells® 
are, in general, more numerous in the connective tissue of the 
interlobular septa than in the lobules of the thymus. A con- 
sideration first of their origin in the former place will, therefore, 
aid in determining their origin in the lobules. Eosinophile 
cells are already present in the mesenchyme of quite young 
stages (20 to 25 mm.). In all these and in somewhat older 
stages (55 mm.) they are not found in localized areas but may be 
found in almost any part of the connective tissue. In the 55 
mm. embryo they are somewhat more numerous than in the 
younger stages but can be found only after considerable search- 
ing. From embryos more than 55 mm. in length only the 


5 The value of the distinction of eosinophile myelocytes (myeloid eosinophiles) 
and eosinophile leucocytes is not considered and cells containing eosinophile 
granules are therefore simply designated as eosinophile cells regardless of the 
shape of their nucleus. 


478 J. A. BADERTSCHER 


thymus was removed. In late developmental stages, there- 
fore, only the eosinophile cells in the interlobular septa will be 
considered. The septa of the thymus in embryos from 65 to 


85 mm. in length contain only a few eosinophile cells. In some 


sections none were found. In the 100 mm. stage they can be 
found without much searching while in the 110 mm. stage a 
single group was found in the sections prepared from the thymus 
head while those lying singly are more numerous than in the 
previous stage. In a 125 mm. embryo groups of eosinophile 
cells are of frequent occurrence and lie usually along the course 
of blood vessels but some are also present in the deep looser por- 
tions of the septa. They are also found lying singly in the 
septa. The greatest numbers occur in stages 165 and 180 mm. 
long. In the former stage they were more numerous in the 
superficial thymus than in the thymus head or cervical segment, 
and on the whole more numerous than in the latter stage in which 
their distribution was about equal in the different segments 
examined. In the full term fetus (270 mm.) they are much 
less numerous in the septa than in the 180 mm. stage. In the 
last three stages many eosinophile cells are found lying singly 
in the deep looser portions of the septa but the large majority 
are found in groups which almost without exception are found 
in the immediate vicinity of the larger blood vessels where the 
structure of the septa is comparatively loose. Some groups 
extend entirely around blood vessels (fig. 8, Ho.C.) while 
others lie only to one side of them. In some groups the eosino- 
phile cells lie closely together while in others they are more 
loosely arranged. Without exception a greater or less number 
of large and medium-sized lymphocytes are promiscuously 
scattered among the eosinophile cells. 

The eosinophile cells vary in size from very large to medium- 
sized lymphocytes. The outline of the greater number is spheri- 
cal but when they lie closely together or in close contact with 
other cellular elements they may have an irregular shape. The 
eosinophile granules are coarse, round, and of a nearly uniform 
size. Their number varies greatly in different cells. In some 
a few granules may be found in a group to one side of the nucleus, 


LS 


> ae ee ee ee a 


a 


DEVELOPMENT OF THE THYMUS 479 


in others they are thinly and quite evenly scattered throughout 
the basophilic cytoplasm, while still others are completely 
gorged with them. 
* A striking peculiarity is that the large majority are mono- 
nuclear. Only very seldom can one of the polymorphonuclear 
type be found. The nuclei are round, slightly indented, or 
crescentic in outline and are usually eccentrically located in the 
cell. In those cells that are gorged with granules the nuclei 
are crowded to one edge of the cell and stand out conspicuously 
among the eosinophile granules. The structure of the nuclei 
is identical with that of the nuclei in the large lymphocytes which 
have been described. | 

The thymus of a 125 mm. embryo was chosen to consider 
the origin of the eosinophile cells. In this stage the inter- 
lobular septa, loose in structure, contain numerous lympho- 
cytes, red blood-cells, and many eosinophile cells lying both 
singly and in groups. Also in a single group can be found 
eosinophile cells containing varying numbers of granules, as 
stated above. An interesting and instructive feature often 
to be observed is the presence of large lymphocytes containing 
only from one to three or four eosinophile granules which are of 
the same size and shape as those found in cells completely gorged 
with them. Often in very limited areas—covered by very 
slightly moving the slide under high magnification—can be 
found large lymphocytes and‘a series of eosinophile cells with 
gradually increasing numbers of granules (fig. 7, l.m.n.). Only 
one interpretation can be given to microscopic pictures of this 
kind, namely, that the eosinophile cells are derived from lympho- 
cytes. This conclusion also accounts for the large numbers of 
eosinophile cells along the course of blood vessels in late develop- 
mental stages, for it is along the blood vessels—in the loose por- 
tions of the septa—that the lymphocytes are most numerous. 

I believe that the groups of eosinophile cells in the septa are 
identical with the granular cells of Watney which he found in 
the interlobular septa of the thymus in various classes of ani- 
mals, although none of the cells were attached to the tunica 
externa of the vessels, as was observed by him. The ultimate 


480 J. A. BADERTSCHER 


source of the eosinophile cells in the interlobular septa of the 
thymus of the pig is the same as that of the granular cells of 
Watney, the only difference is that he derived them directly 
from connective tissue cells while in the pig thymus they are 
derived from transformed connective tissue cells, the large 
lymphocytes. 

Of course, in fixed material it is difficult to determine whether 
all the lymphocytes along the blood vessels are derived from 
the loose connective tissue in which the vessels lie or whether 
some come from the blood. Two features are in favor of the 
former view; (1) transition forms from connective tissue cells to 
lymphocytes are of frequent occurrence. The lymphocytes 
thus formed through division also increase in number; (2) 
diapedesis of the leucocytes is thought of as taking place only 
through thin walled blood vessels, but the lymphocytes and 
eosinophile cells are as numerous along the course of thick 
walled vessels as along those of a capillary nature. Another 
possible source of the lymphocytes in the septa is from the par- 
enchyma of the thymus. However, in late stages that por- 
tion of the thymus contains mostly small lymphocytes and 
judging from the small number of small lymphocytes present 
in the septa very few have migrated into them from the par- 
enchyma. Only a few eosinophile cells were found undergoing 
mitosis, so the number of this type of cells formed through their 
proliferation is almost neglible. 

The source and nature of all the granules in eosinophile cells 
is difficult to determine. There is, however, no evidence indi- 
cating that the granules are débris of degenerated epithelial 
cells, as held by Schaffer (’09), but ample evidence that 
not all are products of the protoplasmic activities of the cells 
containing them, which view is held by Maximow for the ori- 
gin of the granules of the myelocytes found in the thymus of 
various animals. Mention was made of free eosinophile gran- 
ules (fig.,8, Ho.G.) in the interlobular septa where free red blood- 
cells also occur. These can be traced directly to degenerated 
red blood-cells, but the free granules usually observed in the 
septa of any developmental stage do not seem to be numerous 


| 


DEVELOPMENT OF THE THYMUS 481 


enough to account for all of the granules in the numerous eosino- 
phile cells even though all should be ingested by lymphocytes. 
However, lymphocytes with only a few granules in their cyto- 
plasm and lying among free eosinophile granules suggests that 
some eosinophile cells are simply lymphocytes ingested with 
débris of degenerated erythrocytes. This view of the origin 
of the granules in eosinophile cells is held by Weidenreich (’08, 
08, mammals), and by Badertscher (718, amphibia) in a some- 
what modified form in that some of the granules are also formed 
from the débris of degenerated muscle tissue. Also circumstan- 
tial evidence indicating the formation of eosinophile granules 
from erythrocytes is not wanting and may be enumerated as 
follows: (1) The free red blood-cells appear in the interlobular 
septa in advance of eosinophile cells; (2) The red blood-cells 
appear in large numbers in earlier developmental stages than 
do large numbers of eosinophile cells, e.g., in the septa of the 
thymus of a 125 mm. embryo the red blood-cells are more numer- 
ous than in any other developmental stage while the largest 
number of eosinophile cells occur in the septa of the thymus 
of a 165 mm. fetus; (3) As the free red blood-cells in the septa 
of late stages begin to decrease in number the eosinophile cells 
decrease in number in correspondingly later stages, e.g., the 
red blood-cells in the thymic septa of 165 and 180 mm. fetuses 
are not as numerous as in the 125 mm. embryo but the eosino- 
phile cells in the 270 mm. embryo are less numerous than in 
the 165 and 180 mm. fetuses. These facts can be stated in a 
general way by saying that the height and decrease of erythro- 
cyte formation in the septa are followed respectively by the 
height and decrease of eosinophile cell formation in somewhat 
later stages. If the granules in eosinophile cells are products 
of degenerated erythrocytes this apparent relationship exist- 
ing between these two types of cells can be accounted for only 
on the assumption that the majority of free red cells in the septa 
undergo dissolution and the products of degeneration taken up 
by the lymphocytes, possibly in soluble form, and in them 
transformed into granules. 


482 J. A. BADERTSCHER 


Cells of a peculiar type (fig. 7, &). are quite frequently found 
among lymphocytes and eosinophile cells in the thymic septa. 
They are derived from large lymphocytes and are characterized 
by a part of or the entire superficial layer of the basophilic cyto- 
plasm staining a deep red similar to the erythrocytes or the 
granules in eosinophile cells. Their nuclei have the character- 
istic structure of those in the lymphocytes or eosinophile cells. 
They cannot, therefore, be erythroblasts which have granular 
nuclei but must be classed with the eosinophile cells. The 
cells of this type are never very numerous and the youngest 
stage in which they. were found was in the body mesenchyme 
of a25mm.embryo. They occur most frequently in the thymic 
septa of quite late developmental stages. 

The origin of the eosinophile cells in the lobules of the thymus 
can now be discussed briefly. Their structure is the same as 
of those in the interlobular septa. They belong to the mono- 
nuclear type. They were first found in the lobules of the thy- 
mus of a 42 mm. embryo. In this stage they are very rare and 
can be found only after prolonged searching. Their number 
increases in successively advanced developmental stages. In 
the 125 mm. embryo they are readily found in both the cortex 
and medulla. In the 180 mm. embryo a group of them was 
found in the medulla of the mid-cervical segment while those 
lying singly are more numerous than in younger stages. In 
the full term fetus they are present in appreciably greater num- 
bers than in the previous stage, groups of them being found 
in both the cortex and medulla and many can be found lying 
singly. Since the red blood-cells were considered particularly 
in the superficial thymus of a 270 mm. (full term) fetus the eosino- 
phile cells also in that region will be emphasized. Some groups 
of eosinophile cells are found in the immediate vicinity of blood 
vessels but as many are found that are not associated with the 
vessels. The groups occur most frequently along the border 
of or near the vicinity of groups of erythrocytes but some groups 
are isolated and as far as position is concerned their origin does 
not seem to bear any relation to erythrocytes. Here as in the 
interlobular septa the origin of some is, undoubtedly, from the 


eee eS Se oe 


DEVELOPMENT OF THE THYMUS 483 


large lymphocytes that have ingested eosinophile granules (débris 
of degenerated erythrocytes) which as was stated above can be 
found lying free in the meshes of the reticulum among the lym- 
phocytes. The free eosinophile granules do not seem to be 
numerous enough, as in the case of the septa, to account for all 
granules found in eosinophile cells. However, an apparent 
general relationship exists between the latter type of cells and 
the erythrocytes which indicates that at least some of the gran- 
ules of eosinophile leucocytes are derived from degenerated 
erythrocytes. The features indicating this relationship may be 
expressed as follows: (1) As in the thymic septa, the red blood- 
cells are present in advance of the eosinophile cells; (2) The 
eosinophile cells increase in numbers in successively advanced 
developmental stages as do also the red blood-cells; (8) They 
are most numerous in the thymus of a full term fetus in which 
developmental stage the red blood cells are also most numerous; 
(4) In the thymus of a 270 mm. embryo the eosinophile cells 
are more numerous in the superficial thymus than in the thymus 
head, the difference in the numbers corresponding favorably 
to the difference in the numbers of red blood-cells which are 
much more numerous in the former than in the latter segment. 
Here also it must be said that if all the granules of the eosino- 
phile leucocytes in the lobules are derived from degenerated 
erythrocytes it must also be assumed that their degenerated 
products are taken up in soluble form by the lymphocytes in 
which it is transformed into granules. 

Phagocytes (fig. 7, 0. and p.) are found in the interlobular 
septa of the thymus in a wide range of developmental stages. 
They are most numerous in those stages in which the septa 
have a loose structure and contain many lymphocytes. They 
possess a large amount of cytoplasm which in some cells is 
vacuolar. Some are gorged with ingested material which con- 
sists mainly of lymphocytes (apparently) in various stages 
of degeneration. Occasionally one can be found in which an 
entire erythrocyte or a part of one makes up a part of the in- 
gested material. They arise directly from connective tissue 
cells some of which contain ingested particles even before their 


484 J. A. BADERTSCHER 


protoplasmic processes have been withdrawn. The phagocytes - 


vary greatly in size. Some are from two to three times as large 
as the largest lymphocytes. Only a few were found in the lobules 
of the thymus of late developmental stages. 

Cysts were found in the thymus in embryos 55, 65, 110, 125, 
165 and 180 mm. in length. They vary in size and shape 
and all are lined with simple cuboidal or low columnar epithelium 
which is ciliated only in patches. The cilia are long and slen- 
der. No consideration was given to their origin. 


CONCLUSIONS 


1. The lymphocytes first present in the thymus are all large 
lymphocytes and have migrated into it from the mesenchyme. 

2. The numerous small round cells of the thymus are formed 
by the repeated division of the large lymphocytes which thus 
become small, and also by their own proliferation. 

3. Judging from the source and structure of the small round 
cells they are small lymphocytes and are identical with the small 
lymphocytes of the blood. The thymus, therefore, may well 
be considered as a source of some of the small lymphocytes found 
in the circulating blood. 

4. The reticulum of the thymus is of epithelial origin and is 
formed passively by its meshes becoming filled with lympho- 
cytes which separate the nodal nuclei farther apart and thus 
greatly attenuate the protoplasmic processes of the syncytium. 

5. The Hassall’s corpuscles are of epithelial origin. 

6. The free red blood-cells and eosinophbile cells found in both 
interlobular septa and the thymic lobules are derived from lym- 
phocytes in situ. 

7. Whether or not any of the erythrocytes formed in the 
thymus enter the circulating blood is difficult to determine in 
fixed material. Some of the free erythrocytes undoubtedly 
undergo degeneration and the products of disintegration of those 
existing in the form of eosinophile granules are taken up by the 
lymphocytes which thus become transformed into eosinophile 
leucocytes. 


DEVELOPMENT OF THE THYMUS 485 


8. It was impossible to trace the origin of all the eosinophile 
granules in the eosinophile cells directly to degenerated red 
blood-cells. However, the fact, that the height and decrease 
of the formation of red blood-cells in the septa is followed by the 
height and decrease of the formation of eosinophile cells, is cir- 
cumstantial evidence that a relationship exists between the 
disappearance of the free erythrocytes and the formation of 
free eosinophile cells. 

9. The histogenesis of the thymus may be divided into epochs 
each of which is characterized by more or less distinct develop- 
mental features. They are: 

(1) The purely epithelial epoch which extends from its origin 
as an outpocketing from the third pharyngeal pouch and the 
formation of the cervical vesicle to the appearance of the first 
lymphocytes in the thymus. 

(2) The epoch of lymphocyte infiltration and lymphocyte 
proliferation and the formation of the reticulum. The in- 
filtration of the thymus by extrathymic lymphocytes from the 
mesenchyme surrounding it begins in embryos from 25 to 30 
mm. in length and probably continues up to stages 180 mm. in 
length, while their proliferation in the thymus undoubtedly 
continues after birth. The reticulum, which according to the 
nature of its development is formed gradually, differentiates 
~ into the cortex and the medulla in developmental stages 65 to 
75 mm. in length, and is fully formed in embryos 180 mm. in 
length. 

(3) The epoch of the formation of red blood-cells and the 
development of granular cells. An occasional red blood-cell is 
found in the thymic lobules shortly after lymphocytes are found 
in them. They are, however, first present in appreciably large 
numbers in stages of about 55 mm. in length and are most numer- 
ous in the thymus of full term embryos. In the interlobular 
septa of the thymus the greatest number occurs in stages of 
about 125 mm. in length while only a few are found in embryos 
of 180 mm. in length to full term. 

EKosinophile cells were first found in the thymic lobules of a 
42 mm. embryo but occur first in appreciably large numbers 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 


486 J. A. BADERTSCHER 


in embryos of about 180 mm. in length and are most numerous 
in the parenchyma of the thymus of full term embryos. In 
the interlobular septa they are seldom found in embryos from 
65 to 85 mm. in length. They occur first in appreciably large 
numbers in the septa of embryos of about 125 mm. in length 
and are most numerous in embryos 165 to 185 mm. long but 
are still present in the septa in full term embryos. 


I wish to thank Prof. B. F. Kingsbury for the aid and encour- 
agement given me on this work, and Prof. 8. H. Gage for many 
suggestions. 


BIBLIOGRAPHY 


AFANASSIEW, B. 1877 Uber die concentrischen Kérper der Thymus. Arch. 
f. mikr. Anat., Bd. 14. 

BapDERTSCHER, J. A. 1913 Muscle degeneration and its relation to the forma- 
tion of eosinophile leucocytes in amphibia (Salamandra atra). Am. 
Jour. Anat., vol. 15. 
1915 The development of the thymus in the pig. Part I: Morpho- 
genesis. Am. Jour. Anat., vol. 17. 

Brearp, J. 1902 The origin and histogenesis of the thymus in Raja batis. 
Zool. Jahrb. Abt. f. Anat. u. Ontogen., Bd. 17. 

Betu, E. T. 1906 The development of the thymus. Amer. Jour. Anat., 
vol. 5. 

Dustin, A. P. 1911 Le thymus de |’Axolotl. Arch. de Biolog., vol. 26. 

von Esner, V. 1902 A. Koelliker’s Handbuch der Gewebelehre des Men- 
schen, Bd. 3, 6 Aufl, p. 328. 

Goopatu, A. 1905 The post natal changes in the thymus of guinea-pigs and 
the effect of castration on the thymus structure. Jour. Physiol., 
vol. 32. 

Hammar, J. A. 1905 Zur Histogenese und Involution der Thymusdriise. 
Anat. Anz., Bd. 27. 
1908 Zur Kenntnis der Teleostierthymus. Arch. f. mikr. Anat., 
Bd. 73. 
1910 Fiinfzig Jahre Thymusforschung. Kritische Ubersicht der 
normalen Morphologie. Ergeb. d. Anat. u. Entwickl., Bd. 19. 
1911 Zur groberen Morphologie und Morphogenie der Menschen- 
thymus. Anat. Hefte, vol. 43. 

Mavrer, F. 1886 Schilddriise und Thymus der Teleostier. Morph. Jahrb., 
Bde 115 
1888 Schilddriise, Thymus und Kiemenreste der Amphibien. Morph. 
Jahrb., Bd. 13. 
1899 Die Schilddriise, Thymus und andere Schlund-spaltenderivate 
bei der Eidechse. Morph. Jahrb. 


DEVELOPMENT OF THE THYMUS 487 


Maximow, A. 1906 Uber die Zellformen des lockeren Bindegewebes. Arch. f. 
mikr. Anat., Bd. 67. 

1909 a Der Lymphozyt als gemeinsame Stammzelle der verschiedenen 
Blutelemente in der embryonalen Entwickelung und im postfétalen 
Leben der Siugetiere. Folia haemat., Bd. 8. 

1909 b Untersuchungen iiber Blut und Bindegewebe. II. Uber die 
Histogenese der Thymus bei Siugetieren. Arch. f. mikr. Anat., Bd. 74. 
1912 Untersuchungen iiber Blut und Bindegewebe. IV. Uber die 
Histogenese der Thymus bei Amphibien. Arch. f. mikr. Anat., Bd. 79. 

Mirrens, H. 1908 Zur Kenntnis der Thymusreticulum und seiner Bezie- 
hungen zu dem der Lymphdriisen, nebst einigen Bemerkungen tiber 
die Winterschlafdriise. Jenaische Zeitschr., Bd. 44. 

PRENANT, A. 1894 Contribution A l’étude organique et histologique du thy- 
mus, de la glande thyroide et de la glande carotidienne. La Cellule, 
e10: 

Prymak, T. 1902 Beitrige zur Kenntnis des feineren Baues und der Involu- 
tion der Thymusdriise bei den Teleostiern. Anat. Anz., Bd. 21. 

ScHaFrrer, J. 1891 Uber das Vorkommen eosinophiler Zellen in der Mensch- 
lichen Thymus. Central. f. d. med. Wiss. 

1893 Uber den feineren Bau der Thymus und deren Beziehung zur 
Blutbildung. Sitzungsb. d. K. Akad. d.Wiss. Wein., Math.-nat. KI. 
Abt. 3., Bd. 102. 

ScHAFFER, J., UND Rast, H. 1909 Das thyreo- thymische System des Maul- 
wurfes und der Spitzmaus. I. Morphologie und Histologie. Sit- 
zungsber. d. Wiener Akad., Math. nat. Kl. Abt. 3., vol. 118. 

Sréur, Pu. 1906 Uber die Natur der Thymuselemente. Anat. Hefte, Bd. 31. 

Watney, H. 1882 The minute anatomy of the thymus. Phil. Transact. 
Roya Soc, vole li73;, ps 3: 

Weipenreicu, F. 1908 a Morphologische und experimentelle Untersuchungen 
uber Entstehungen und Bedeutung der eosinophile Leucocyten. Anat. 
Anz., Bd. 32. 

1908 b Beitrige zur Kenntniss der granulierten Leucocyten. Arch. 
i, immer, AMonghes. excl, 7%, 


REFERENCES ON PLATES 


A.Erc., amitosis of erythroblasts 

bl.v., blood vessel 

D.d.e.N., débris of degenerated epithe- 
lial nuclei 

D.e.N., degenerating epithelial nucelus 

D.e.N’., completely degenerated epi- 
thelial nucleus 

E.N., epithelial nucleus 

Eo.C., eosinophile cells 

Eo.G., free eosinophile granules 

Erb., erythroblast 

Erc., erythrocyte 

L.L., large lymphocyte 

L.M., limiting membrane 


M.C., mesenchymal cell 

M.e.N., mitosis of epithelial nuclei 

Me.L., medium sized lymphocyte 

M.L., mitosis of lymphocyte 

Nmb., normoblast 

Pc., phagocyte 

S.i., interlobular septa 

S.L., small lymphocyte 

tial 

V., vacuole 

X., cell of unknown origin and _ sig- 
nificance 

Z.pr., zone of rapid proliferation of 
epithelial cells 


JeIbANIE I) il 
EXPLANATION OF FIGURES 


1 Camera lucida drawing of a portion of a lobule of a section of the right thy- 
mus head in a 30 mm. embryo. The infiltration of the thymus by extrathymic 
lymphocytes from the surrounding mesenchyme has just begun. In order to 
reduce the size of the drawing the very large lymphocyte represented in the 
outer border of the mesenchyme was drawn a little nearer the thymus than it 
really is. 1.5 mm. Zeiss App. objective, X 12 Comp. ocular; reduced one-half. 

2 Camera lucida drawing of a portion of a lobule of a section of the left thor- 
acie segment of the thymus in a 37 mm. embryo. The thymus in this develop- 
mental stage is slightly more advanced in development than in the 30 mm. 
embryo. This drawing was made to show particularly the large number of 
epithelial nuclei that are in the first stages of degeneration and the trail that was 
apparently made by an active lymphocyte that migrated into the thymus from the 
mesenchyme surrounding it. 1.5mm. Zeiss App. objective, * 12 Comp. ocular; 
reduced one-half. 


488 


DEVELOPMENT OF THYMUS IN THE PIG PLATE 1 
J. A. BADERTSCHER 


PLATE 2 
EXPLANATION OF FIGURES 


3 Camera lucida drawing of portions of two lobules of the left thymus head 
in an embryo 42 mm. in length. In the thymus of this stage many lympho- 
cytes are present most of which are large and medium-sized. Only a few small 
lymphocytes are present. Mitoses cf both epithelial nuclei and lymphocytes 
occur. The lymphocytes in the interlobular septa are quite numerous. 1.5mm. 
App. objective, X 4 Comp. ocular; reduced one-fourth. 

4 Camera lucida drawing of portions of two lobules of the left thymus head 
ina 65mm.embryo. The thymus of this stage contains numerous lymphocytes 
most of which are small ones. Mitosesof lymphocytes are comparatively numer- 
ous. Many lymphocytes are found in the interlobular septum. 1.5 mm. App. 
objective, X 4 Comp. ocular; reduced one-fourth. 


490 


DEVELOPMENT OF THYMUS IN THE PIG PLATE 2 
J. A. BADERTSCHER 


; =3(¢ oS) gs . 3 
& e ae 
Pee 


i ¢ 
aah 


ey cl 
ND S55 8) 


pc 


PLATE 3 
EXPLANATION OF FIGURES 


5 Camera lucida drawing of a portion of a thymic lobule in a 36mm. embryo 
to show specially epithelial nuclei in various stages of degeneration. 1.5 mm. 
Zeiss App. objective, * 8 Comp. ocular; reduced one-half. 

6 Camera lucida drawing of a portion ot a lobule of the thymus in a 55 mm. 
embryo to show especially free nucleated and non-nucleated red blood-cells. 
In the portion drawn one erythroblast is in mitotic division while several are in 
amitotic division. 1.5 mm. Zeiss App. objective X 8 Comp. ocular; reduced 
one-half. 

7 Diagrams showing the different types of cells that are derived from mesen- 
chymal cells. The direction of the arrows shows the relation of the different types 
of cells to each other; a, mesenchymal cell; b, c and d, transforming mesenchymal 
cells; e, large lymphocyte; /, small lymphocyte; g and h, erythroblasts; 7, normo- 
blast; 7, erythrocyte; k, lymphocyte capped with a layer of hemoglobin; /, m and 
n show the formation of eosinophile leucocytes, and o and p, phagocytes; a, b, 
k and p are camera lucida drawings while the remainder are free-hand drawings 
from actual specimens. All were drawn from specimens in the interlobular 
septa of a 125 mm. embryo excepting o and p, which were drawn from specimens 
in an interlobular septum of a 110 mm. embryo. 1.5 mm. App. objective, xX 8 
Comp. ocular; reduced one-half. 

8 Camera lucida drawing of a portion of an interlobular septum of the thy- 
mus in a 165 mm. embryo showing specially eosinophile leucocytes and a few free 
eosinophile granules. 


492 


PLATE 3 


DEVELOPMENT OF THYMUS IN THE PIG 


J. A. BADERTSCHER 


493 


ON THE PREMATURE OBLITERATION OF SUTURES 
IN THE HUMAN SKULL 


L. BOLK 
From the Anatomical Institute, University of Amsterdam (Holland) 


INTRODUCTION 


In the developmental history of the human skull, there is a 
period in which the phenomena of development are as yet fairly 
unknown to us: viz., the phase of life included between the third 
year and the adult state. The reason of this is quite clear. It 
is impossible to acquire a perfect knowledge of an object of such 
intricate structure as the human skull, unless investigation is 
made of a very great number of infantile skulls. Now, the 
number of non-adult skulls, except those belonging to children 
of one and two years old, found in the anatomical institutes is 
generally quite restricted. This was the case with the anatomi- 
cal institute of the University of Amsterdam, until two years 
ago, when it became the possessor of about two thousand infan- 
tile human skulls. This collection may be utilized for inves- 
tigations of many totally different natures and my intention is 
to communicate occasionally in this journal the results of some 
investigations worked out by myself or by my pupils on the ma- 
terial of this splendid collection. 

The present paper will refer to the sutures of the cerebral 
part of the skull. 

It is a well known fact, that the bones of the human skull 
coalesce either during the developmental period or in a more 
advanced phase of life. In the first case, coalescence takes place 
for the greater part during the foetal period, in the second case 
at a date varying extraordinarily for each suture. Therefore 
in human life a phase exists during which skull-bones do not 
unite, beginning about the fourth year, when the metopic suture 

495 


496 ie BOlk 


has closed itself and the different parts of the occipital bone are 
united with each other. The sutures still existing at this date 
of life, are the so-called persisting sutures remaining for a shorter 
or longer time after the individual has become full grown. Re- 
garding the variability of age in which these sutures disappear 
there are already some extensive and carefully written com- 
munications, for instance by Fredericg and Nibbe. Yet it is a 
well-known fact that now and then one of these persisting sutures 
does close during childhood before the individual has reached 
the adult state. Of this fact, and its influence on the shape of 
the skull, the casuistic literature is already very abundant, 
but a systematical inquiry into this phenomenon is as yet 
wanting. In the present paper I wish to deal with the results 
of my examination of the premature concrescences of bones in 
the human skull, results acquired by the investigation of 1820 
skulls of non-adult individuals. I regarded the obliteration of 
the occipito-sphenoidal synchondrosis as a criterion of the 
adult condition of the skull. The youngest skulls at my disposal 
already possessed their complete milk-dentition, therefore in this 
communication skulls of the first two years are not mentioned. 

Before beginning my investigation I divided my collection 
into seven groups, in accordance with the developmental phase 
of the dentition. Of the different groups a brief description 
follows: 

Group I. This first group is composed of the very young 
skulls, with complete milk dentition, and therefore with the 
following dental formula: 

ie le Co Wi, TA. 
These skulls, 725 in number, are those of children who died 
between the third and sixth year. 

Group II. This group includes the skulls in which, besides 
the complete milk dentition, the first permanent molar tooth 
is also present. The dental formula of these skulls (of children 
aged 6-7 years) is as follows: 


i. 1. c. m. m. M. 


This second set comprises exactly 400 skulls. 


OBLITERATION OF SUTURES IN SKULL 497 


Group IIT. In this group the eruption of the permanent 
central incisor had taken place. There were 168 of such skulls 
belonging to children aged about 7 or 8 years. The dental 
formula is as follows: 


In tle @5 1m, ittle MME 


Group IV. This group contains 157 infant skulls in which 
the lateral permanent incisor has made its appearance corre- 
sponding with the age of 8 or 9 years. The dental formula of 
this series is to be written: 


Ih, Il, @s Wn, 107, IMI. 


Group V. In this set were included the 109 skulls in which 
the first milk-molar was lost, and the dental formula is the 
following: 

Ile Iho @> 1P5 Tens WH 


Such a set of teeth corresponds with an age of 9-11 years. 

Group VI. Includes 203 skulls between 11 and 13 years, in 
which the second premolar and the canine are present. The 
order of eruption of both of these teeth is not a constant one. 
Although in a considerable majority of skulls the second pre- 
molar precedes the canine, yet there were a certain number in 
which the eruption of the second premolar evidently succeeds 
the eruption of the canine. Therefore I have included all 
these skulls in one set. Its dental formula is as follows: 


Ialege PME 


Group VII. This last group contains all the skulls (58 in num- 
ber) with a complete set of teeth, save the third molar. These 
skulls, of which the dental formula is 


PASC. PP. MMe. 


come from individuals who died between the age of 13 years 
and the adult state. 

I have found in my whole collection but three skulls, not 
yet completely developed, in which the eruption of the third 
molar had already taken place. It must therefore be considered 
as a rule that the wisdom tooth makes its eruption after the 


498 1. 2B OLK 


termination of the development of the skull. Exceptions to 
this rule are very infrequent. 

Table 1 gives a brief résumé of the above described groups 
of my collection: 


TABLE 1 
GROUP DENTAL FORMULA AGE NUMBER 

Lee es ilo He Os fans Toa 3-6 725 

lea ee ee ee No Jka (5 Oly wats IE. 6-7 400 
Toit, Wyn Pee ee Ils th x 10M foot, IL 7-8 168 
1 TRY Geeta em Pee ee 8 [eleven Vie 8-9 157 
VE 2 i 2 ceca ees Ws We @5 IRs seals IMI. 9-11 109 
Valie Se Sak bo Cone ree ele ee 11-13 203 
AVAl Bl ean tre chamen bors Is Wy (Gh, IP. Tes aM, IME 13-20 58 


Soon after the beginning of my investigations, the fact struck 
me that the closing of so-called persisting sutures in skulls of 
non-adults occurs more frequently than I had supposed. How- 
ever, this is not the case with all cranial sutures in the same 
degree. In some a premature concrescence is an unusual rarity, 
but on the other hand there are some in which the concrescence 
occurs so often, that it can scarcely be considered as an anom- 
aly. Now in this communication, I will discuss first: those 
sutures which I found most frequently closed, and second: 
those in which coalescence appeared as a very exceptional 
variety. 


PREMATURE OBLITERATION OF THE MASTO-OCCIPITAL SUTURE 


The examination of this suture produced one of the most sur- 
prising results of my investigation. Fredericg, in his very ex- 
tensive and valuable paper ‘‘On the obliteration of the cranial 
suture,’! asserts that the coalescence of the occipital and the 
temporal bones does not occur before the thirty-first year, it 
being a very rare exception when it has already occurred in the 
twenty-first or twenty-fourth year (loc. cit., p. 441). On an- 
other page in the same work the author strongly points out the 


1 Zeitschrift fur Morphologie und Anthropologie, B. 9, 1906. 


OBLITERATION OF SUTURES IN SKULL 499 


fact that the masto-occipital suture belongs to those persisting 
the longest. 

Now it is important to note that this author had at his dis- 
posal only a small number of skulls of 20 to 30 years and that 
his investigation was made principally on adult skulls. If the 
investigator had extended his examination upon a sufficient 
number of non-adult skulls, his conclusion would, no doubt, 
have been quite different. For the coalescence of the occipital 
and petrosal bone in the skull of infants is not at all a rare 
event. On the contrary amongst my material there even was a 
considerable and unexpected number of skulls, showing complete 
or partial closure of the masto-occipital suture, either on one side 
or on both. Moreover not in all cases was the coalescence 
restricted to this one suture, but in a large number of skulls 
two or three or even four sutures were totally or partially obliter- 
ated. Here I wish to treat separately the cases in which only 
the masto-occipital suture was closed and in which the oblitera- 
tion was of a more extensive nature. I will begin with the 
first group. 

It is scarcely necessary to particularly mention that in case 
of closure of the masto-occipital suture the coalescence of the 
two bones can be a total or a partial one. In the second table 
this fact is taken into consideration. As a rule the coales- 
cence of the petrosal and occipital bones begins midway in the 
suture, passing in the majority of cases from this point towards 
the masto-parietal suture, in consequence of which, in a partial 
closure, it is most often the upper half which is obliterated. 
Table 2 shows the results of my examination on the masto- 
occipital suture. This table demonstrates at once the quite 
unexpected fact, that in the human non-adult skulls the masto- 
occipital suture is found closed so often, that one is inclined 
to consider this phenomenon no longer as an anomaly. Let us 
consider the frequency of this obliteration. It is not possible 
to recognize, with the aid of table 2 (p. 500), the absolute num- 
ber of skulls in which the suture showed obliteration, a certain 
number being twice mentioned, viz. the skulls entirely closed 
on the one side and partially on the other; the skulls in which 


500 L. BOLK 


TABLE 2 


CLOSURE OF MASTO-OCCIPITAL SUTURE 


GROUP NUMBER | 


Both sides Right Left 
entirely partial entirely partial entirely Partial 
Tee: Mert SOs Se eG led 12 20 9 21 
II 400 12 5 5 11 9 1 
| ET Be eee ee 168 10 2 YY 4 3 2 
I\ 157 7 1 2 4 2 1 
\ 109 6 0) 0 1 2 1 
Wiles 203 9 5 8 3 6 5 
Walaa ree er 58 3 1 4 1 3 3 
25 


1820 63 33 44 34 40 

on both sides the suture was partially or entirely closed are men- 
tioned once, and also those in which the suture on one side only 
was partially obliterated. Moreover an uncertain number re- 
mains in which the suture is totally closed on one side. Tak- 
ing this into consideration, I found amongst about 1820 skulls 
of non-adults at least the number of 63 + 25 + 44 + 40 = 172 
with closure of the masto-occipital suture either on both sides 
or on one side only. Reckoning the number of skulls with a 
total closure on one side to be 10, we can conclude that in 10 
per cent of our non-adults the said suture shows more or less 
signs of obliteration. Therefore Fredericg’s conclusion is not 
right, when he writes that the coalescence of the petrosal and 
occipital bone in the third decennium of life rarely occurs. Even 
before the twentieth year the coalescence is not exceptional. 
The preceding table shows yet another phenomenon of no less 
importance. At what age does this obliteration take place? 
Our table includes skulls from about three years up to the adult 
state. Now two possibilities must be considered. Either the 
coalescence may begin at each date of this period, or the com- 
mencement of the process is limited to a shorter or longer phase 
of it. In the first ease the number of synostotic skulls increases 
while the age advances; in the second case such a correlation is 
wanting. Now for the solution of this problem it is a happy 
coincidence that the number of skulls in Group I is considerable. 


OBLITERATION OF SUTURES IN SKULL 501 


This group includes 725 skulls with a complete milk-dentition. 
And, proceeding in the same manner as before, it appears that in 
the whole collection the number of skulls showing a closure 
of the masto-occipital suture in this group must be at least as 
follows: Complete obliteration on both sides 16 times, partial 
on both sides 11. A partial closure on the right side only 20 
times, and on the left side 21 times, amounting to 68 skulls 
out of 725. Resuming, we find the following remarkable re- 
sult. In 1820 skulls varying in age between 3 to 20 years, the 
masto-occipital suture is obliterated wholly or partially 172 
times, making about 10 per cent, and in 725 skulls of infants 
aged 3 to 6 years, the closure occurred 68 times, also coming 
to about 10 per cent. In this early stage of childhood, the ob- 
literation is found in the same proportion as in the total number 
of skulls including the whole developmental period. Hence 
the following conclusion is obvious: The number of infantile 
skulls with closure of the masto-occipital suture does not in- 
crease after the age of six or seven years; the premature ob- 
literation of the said suture is limited to a circumscribed period 
of infancy, beginning as a rule before the end of the sixth year. 

This fact deserves our full attention in reference to its etio- 
logical interpretation. For the question arises whether this 
premature obliteration is a pathological phenomenon, or one 
of a purely physiological nature. When working out my sta- 
tistical material I doubted at first the physiological nature. 
I took into consideration the possibility of this process being 
caused by some inflammation in the neighborhood of the suture, 
especially in the tympanic cavity. No doubt an otitis media 
will cause a hyperaemic state in the adjacent parts of the skull, 
and one can imagine that under the influence of the latter a 
coalescence of the occipital and petrosal bone may occur. The 
consideration, however, that certainly not 10 per cent of our 
children undergo an inflammation of the middle-ear is sufficient 
to reject the idea of this pathological cause for the obliteration. 
Moreover there was yet another circumstance pleading against 
such a supposition. As we will demonstrate in the following 
paragraph of this paper, the sagittal suture is also very often the 


THE AMERICAN JOURNAL OF ANATOMY, VOL. 17, No. 4 


502 L. BOLK 


seat of a premature obliteration, and it is almost improbable 
that this process is effected by an influence originating from the 
middle-ear. Therefore it is necessary to explain the great fre- 
quency of the closure of the masto-occipital suture in infantile 
skulls in a quite different manner. We will return to this ques- 
tion after having discussed the premature obliteration of the 
sagittal suture, which resembles in many points the masto- 
occipital. The number of all non-adult skulls, showing a closure 
of the masto-occipital sutures amounts to about 10 per cent, 
and we have found the same proportion in infants’ skulls aged 
3 to 6 years. The process is therefore limited to the period 
before the commencement of the dentition and is not extended 
over the whole period of growth of the individual. This fact 
is proved by another statement given in table 2. It appears 
that the number of partial coalescences diminishes when the 
age of the skulls advances and that on the contrary the num- 
ber of total coalescences increases with the age of the individuals. 
To prove this let us compare the first two and the last two 
groups with each other. In the first two groups are included 
the skulls of children from 3 to 7 years. The total number of 
these is 1125. In 28, or nearly 25 per cent, of these, a com- 
plete coalescence of the masto-occipital suture was seen on 
both sides. In the Groups VI and VII, including the skulls of 
12 years and more to the adult state, there were 261 skulls, and 
of these there were 12, or about 5 per cent, with complete closure 
on both sides. The difference appears still more considerable 
by comparing the unilateral coalescence. In the first two 
groups there are 12 + 5 + 9 + 9 = 35 completely closed sutures 
on one side and 20 + 11 + 21 +7 = 59 partially obliterated. 
Therefore, in the very young skulls (Groups I and II) the cases 
with partial obliteration greatly outnumber those with complete 
obliteration. After the twelfth year (Groups VI and VII) the 
correlation becomes reversed; total obliteration being then 
more common than partial, proved by the following addi- 
tion: totally closed 8 + 4+ 6+ 3 = 21 and partially closed 
3+14+5+4+3 = 12. 


OBLITERATION OF SUTURES IN SKULL 503 


Summarizing the preceding results our investigation leads 
us to the following conclusions with reference to the masto- 
occipital suture. In the infantile skull there is found a premature 
closure of the suture between the mastoid and occipital bone 
either on one or on both sides in about 10 per cent of the cases. 
This process is not pathological but ought to be considered as 
merely physiological. The beginning of the coalescence between 
the two bones is restricted to earlier stages. After the child 
has reached its seventh year it has but little chance to be sub- 
ject to the said premature synostosis. 

In the preceding pages we only considered the skulls in 
which exclusively the masto-occipital suture was closed and 
all others were intact. There were, however, in my collection 
of infants’ skulls a certain number showing a more complicated 
condition in which premature obliteration was seen in more than 
one suture. For the sake of brevity we will postpone the ex- 
amination of these cases until after the description of the skulls 
with a single obliteration. 


THE PREMATURE OBLITERATION OF THE SAGITTAL SUTURE 


In this suture too my investigation resulted in unexpected 
results, the frequency of premature closing being more consid- 
erable than I anticipated. 

The premature closure of this suture has attracted the atten- 
tion of many anatomists, more so than the masto-occipital 
suture. The frequency of the latter’s synostosis was till now an 
unknown fact in the anatomy of the skull. In general it was 
acknowledged. that a premature closure of the sagittal suture 
occurred occasionally, although investigations with statements 
are as yet wanting. The cause of this difference between two 
homologous phenomena: is near at hand. In case of closure 
of the sagittal suture, the possibility arises of a deformity of 
the skull, more considerable the sooner in life the process com- 
mences. This anomaly is known as scapho-cephaly (which 
name was introduced by von Baer), because when excessively 
deformed the skull becomes boat-shaped. A premature coales- 


504 “2, “BOLK 


cence of the occipital and mastoid bones on the contrary does 
not cause a striking deformation of the skull or the head. In 
some cases of a premature union of these two bones I met with a 
somewhat peculiar form of the occipital region of the skull. 
But this peculiarity can scarcely be observed during life because 
the greater part of this region of the skull is covered by the 
muscles of the neck. Now on the contrary, the deformity be- 
comes more visible when obliteration of the sagittal suture 
occurs in early life. The effect of this process is clearly visible 
and an extensive literature in all the principal languages has 
treated of this subject. We may distinguish two schools of 
method in this literature, the purely descriptive and the etio- 
logical. The investigations of the former simply reported the 
description of the scaphocephalic skulls, without referring to the 
origin of the deformity. 

The naturalists of the latter school did not limit their subject 
to a simple description, but they went more to the bottom 
of the problem and tried to point out the genetical cause of the 
deformity. The opinions of this group were directed in that 
way principally by a work of Virchow. In it the author dem- 
onstrates that the anatomical details characteristic of the 
scaphocephalic skull, were due to the coalescence of the two 
parietal bones in an early stage of development. Before Vir- 
chow this hypothesis had been defended by von Baer, but the 
correlation between cause and effect was clearly demonstrated 
for the first time by Virchow. 

However, though I intend to write about the genetical rela- 
tion between skull deformation and premature obliteration of 
sutures in a following paper, still I wish to lay stress here upon 
the justness of an observation already made by Huxley, and 
which was confirmed by my investigation. This famous natur- 
alist demonstrated infantile skulls, absolutely normal in shape 
and size, although the sagittal suture was entirely obliterated. 
One might observe that in such cases the individual died shortly 
after the synostosis of the suture and that the skull had there- 
fore no time to deform. To this supposition I will reply that 
the number of skulls with premature obliteration and without: 


OBLITERATION OF SUTURES IN SKULL 505 


any sign of scaphocephalic deformation in my collection is too 
large to accept this point of view. But as mentioned before I 
will return to this question later on. 

The number of skulls with premature closure of the sagittal 
suture was a fairly large one. After finding this fact the ques- 
tion arose whether this process should be considered either 
pathological or physiological. 

To justify the putting of this question some observations may 
precede upon the variability of the closure of this suture in the 
adult. The opinions of the writers diverge greatly as to the 
age in which the normal obliteration of the sagittal suture com- 
mences. According to Tapmord the process begins normally 
at the age of 40 to 45 years, a conclusion also accepted by Ribbe. 
In the text book of human anatomy the average age of the 
closure is given as about 50 years. Dwight, on the contrary, 
lays stress upon the fact that the obliteration commences be- 
tween the twentieth to thirtieth year, although the individual 
variability is considerable, while the process can occasionally 
be postponed till a fairly old age. In his admirable paper, 
already mentioned, Fredericg shows that in 22 out of 34 human 
skulls, varying between 20 to 30 years, the suture commences 
to disappear. In this connection the author cited an observation 
of Schwalbe, who always found the sagittal suture either 
partially or entirely coalesced after the fortieth year. 

The process of obliteration however can proceed very slowly, 
and it even happens that in skulls 80 years of age, the two 
parietal bones are not yet totally united. Based upon the re- 
sults of the investigations of Schwalbe and Fredericg, the 
following point of view presents itself. It is proved, and we 
need not doubt the reality of the fact, that the beginning of the 
obliteration of the sagittal suture is seen fairly often between 
the twentieth and thirtieth year. But this fact was found by 
merely examining skulls older than 20 years. Until the present 
time young skulls have not yet been investigated as to the 
occurrence of the closure of the sagittal suture. And if it 
becomes clear in the course of such an examination, that such 
a closure in infantile skulls is not an exception, then I must 


506 Li; ‘BOLK 


say a doubt ought to arise whether such cases have been rightly 
considered as pathological. It is true that it is premature, for 
the individual has not attained his adult stage, but why patho- 
logical? Could it not be possible that the normal variability 
is even more extensive and that the age at which the obliteration 
may begin, which as Fredericg truly found, reaches the thresh- 
old of manhood, may also include a restricted period of childhood? 

The problem will be thoroughly examined later on. 

The results of my researches upon the said suture are ar- 
ranged systematically in the following table 3. 


TABLE 3 


Obliteration of the sagittal suture 


OBLITERATED 
GROUP DENTAL FORMULA NUMBER 

partially entirely 
Pada’ aviehrntrdes See wae Mla Wy a ceele, sea 725 10 2 
1) Se Pees Stic side hy te (ean taal AY 400 8 4 
1) Gl May Net At Ae ie a JIS als Gy 10015 soa YE 168 4 2 
TLS eA esis Oe Bae Ty @mem:. M 157 3 3 
VES Aa a ane ee Tec eee Vi 109 2 2 
NARS Nir Peer eee at Al pete: ECE Ve es IML 203 il 6 
WALT ey tence sts anaes hicieron seca ee ea Vien iVie 58 0 0 
1820 28 19 


I wish once more to emphasize that in this table only those 
skulls. are referred to in which the process of obliteration was 
limited to the sagittal suture. 

This table shows that in 47 skulls out of 1820 there was a 
partial or total closure of the sagittal suture, making 2} per 
cent. I had not expected to find such a considerable number. 
The cases of partial obliteration outnumber those of entire 
closure, a condition which is in no way surprising. For the 
majority of the skulls are those of children, who died early in 
life, so that the process of uniting had scarcely time to be extended 
along the whole suture. 

In truth the fact that an entire obliteration was found in 19 
skulls, making 1 per cent, surprises us as highly as the large 


OBLITERATION OF SUTURES IN SKULL HOT 


frequency of the obliteration in general. For by the investiga- 
tion of Fredericg and Ribbe it is made clear that total oblitera- 
tion of the sagittal suture in the adult required a fairly long 
period. Taking this fact into consideration the large number of 
entirely closed sutures in infantile skulls.awakes a strong sus- 
picion that the obliteration, beginning in an early period of 
life, proceeds more quickly than those taking place in the more 
advanced phase of life. The increased intensity of all physio-+ 
logical and histological processes natural to youth, evidently 
influenced also the process of premature obliteration. 

Now we will enter into the problem whether the obliteration 
- of the infantile skull is pathological or not. It is clear that 
this problem is not solved by observing that the union of the 
two parietal bones, when occurring at an early date in life, causes 
deformity of the skull to a certain extent, for the effect of an 
intrinsically normal process may become under circumstances an 
abnormal one, while the proper nature of the process is not al- 
tered by it. One must distinguish formal and causal genesis. 

Moreover one may not conclude that the closure must be of 
a pathological nature only because it occurs before the full 
development of the body is reached. For (1) many sutures 
in the skull disappear during this stage of life and, (2) I call 
attention to the result of my investigation in which I showed, 
after examination of about 800 skulls of apes and monkeys,? 
that in a large number of genera of primates, and especially 
in anthropoids, synostosis of the sagittal suture happens before 
the individual is full-grown. Thus, in forms with which the 
human being stands in close phylogenetical connection, the 
premature synostosis of the two parietal bones appears to be 
normal. Here the process bears a purely physiological character. 
Why should we refuse then to consider it also physiological in 
man? These arguments however are purely theoretical and 
through them a decisive answer to the question proposed is 
not possible. Let us try to find it, by examining more closely 
the contents of table 3. It showed us that in infantile skulls 


2 Zeitschrift fur Morphologie und Anthropologie, B. 15, 1912. 


508 L. BOLK 


the obliteration appeared in 23 per cent. As I pointed out, 
there are two possibilities. The process is either confined to a 
definite period of development, or it can happen during its 
whole course. To determine which of the two possibilities 
really occurs, we have only to observe the frequency of pre- 
- mature obliteration appearing in the two groups of youngest 
skulls, containing those of children from 3 to 6 years. Their 
total number is 1125. Amongst these skulls there were 24 with 
partial or entire closure of the sagittal suture, amounting to 
2.1 per cent. The conclusion therefore is quite simple and lies 
close at hand. 

Amongst 1820 skulls of non-adult individuals (aged 3 to 20 
years) there are found 47, or 2.5 per cent, in which the parietal 
bones are united, and amongst 1125 skulls of children, less than 
7 years of age, I count 24, or 2.1 per cent, in which coalescence 
had taken place. Consequently the number of skulls with 
synostosis of the sagittal suture scarcely increases after the 
seventh year. 

The period during which the obliteration of this suture in in- 
fancy begins reaches a limit therefore in the seventh year. The 
tendency to premature closing is not extended over the whole 
period of growth, but practically stops after the seventh year. 
I recall the fact that exactly the same relation was found in 
the masto-occipital suture. 

Referring to the suture just mentioned, still another cir- 
cumstance presents itself, proving that the number of prematurely 
closed sutures do not augment after the seventh year, 7.e., the 
proportion between *the partially and totally closed sutures. 
The former diminish as the skulls reach a more advanced age. 
To demonstrate this I beg the reader to look at the last two 
rows on table 3. In Groups I, II and III (skulls up to 8 years 
of age) the partially closed sutures exceed in number those en- 
tirely closed; in the Groups IV and V (skulls up to 9 and 10 
years of age) an equal number of each is found, and finally in 
Groups VI and VII the entirely obliterated surpass the partially 
closed ones. I may conclude, therefore, that the process once 
commenced is of a progressive nature. 


OBLITERATION OF SUTURES IN SKULL 509 


ON THE GENETIC SIGNIFICANCE OF THE PREMATURE OBLITERA- 
TION OF THE SAGITTAL AND MASTO-OCCIPITAL SUTURES 


The facts, demonstrated in the foregoing paragraphs as to the 
sagittal and masto-occipital suture, exhibit so much resem- 
blance in some principal points, that it is desirable to treat these 
sutures together from a more general point of view. My reason 
for intercalating these considerations here and for not waiting 
till the description of the premature closure of all the sutures 
is given, is founded on the circumstance that in the other sutures 
premature obliteration is very seldom seen, and does not occur 
with the regularity which characterizes the two sutures above 
mentioned. The following points of resemblance between the 
two sutures may be mentioned. Firstly, the frequency of pre- 
mature obliteration. Especially in the masto-occipital suture 
this is so often found, that one may well question why this 
phenomenon has remained unknown: in literature until now. 
The synostosis of the masto-occipital suture is more frequent 
than that of the sagittal suture. On the other hand one should 
not forget that the former suture is paired and the chance of a 
premature closure therefore is doubled. Secondly, both sutures 
have the fact in common that the commencement of the process 
of closure is confined to a circumscribed phase of the develop- 
ment ending approximately in the seventh year. By this limi- 
tation in time the process attains a peculiar character. There 
is, aS one might say, in the development of man a stage, dur- 
ing which he exhibits an intensified tendency to obliteration 
of some sutures. Beyond this stage, this disposition seems to 
be lost. The weight of this tendency is not at all a small one, 
as is proved by the fact that a premature obliteration of the 
masto-occipital suture is seen in more than 10 per cent of the 
skulls, and of the sagittal suture in 2.5 per cent. This un- 
expected large number of cases with premature synostosis gives 
a predominant significance to the problem of the etiology 
of this anomaly. This question has already been mentioned, 
7.e., is this synostosis of skull bones a pathological phenomenon? 

In the literature on this subject generally the opinion is ad- 
vocated that premature synostosis of skull bones is a symptom 


510 ; Lie BOLK 


of some constitutional disease. And as a rule rhachitis or 
heredity syphilis are accused of being the primary causes of the 
anatomical anomaly. 

It is clear that the literature on this subject principally refers 
to the sagittal suture, because the deformity, which in some 
cases results from the premature closure of the latter, has long 
‘since attracted the attention of anatomists. Concerning this 
deformity, scaphocephaly as von Baer first called it, an extensive 
literature exists, in which the question is widely discussed whether 
scaphocephaly can be acquired after birth, or is in each case 
already present in the foetal skull. Although we shall not 
enter into this question, it seems necessary to state the fact 
that in all the skulls described in the preceding paragraphs, 
the synostosis of the skull-bones had undoubtedly taken place 
after birth and in the majority of the cases at the age between 
the third and seventh year. 

Still I cannot agree with the opinion of the investigators, who 
consider the premature obliteration as the result of rhachitical 
or syphilitical disposition of the individual and will give some 
arguments against this theory. 

My first objection is based on the large number of skulls with 
premature closure. If rhachitis or syphilis is the cause of it, 
one must not shrink from the conclusion that one or the other 
of these diseases has affected more than 15 per cent of the 
individuals. 

I admit this argument to be purely theoretical and therefore 
of a problematical value. Still there are other reasons why 
the pathological nature of the premature obliteration should be 
denied. In my collection of skulls there were, as need scarcely 
be mentioned, a certain number with evident symptoms of 
rhachitis: Hydrocephaly, flattened occipital region, defective 
development of the enamel of the teeth, ete. A special exami- 
nation has shown to me that the positive rhachitical skulls 
were characterized in no manner by an increased tendency to 
premature synostosis of the skull-bones. Amongst these rha- 
chitical skulls there were naturally a certain number with pre- 
mature closure of the sagittal or masto-occipital suture, but the 


OBLITERATION OF SUTURES IN SKULL Shi 


proportion in which this happened was not larger than in the 
wormal skulls. This fact is further strengthened by the cir- 
cumstance that in most cases of premature obliteration no other 
symptoms of rhachitis were visible, they possessed a normal 
structure of the bone tissue and of the tooth-enamel. 

Another argument pleading against the rhachitical character 
of the premature obliteration is the great regularity with which 
the process commences and proceeds. In all the skulls de- 
scribed in the foregoing paragraph, it was clear that the synos- 
tosis of the sagittal suture regularly commenced at the very 
point where in normal cases the obliteration begins, 7.e., in the 
obelion. Should the process be of a pathological nature the 
starting-point of the synostosis should be very inconstant. 

Finally, if the obliteration is really the effect of some general 
constitutional disease, how can we understand that the process 
confines itself to the whole length of one suture only? In the 
sagittal suture the synostosis is often complete, extending from 
the bregma to the lambda point. Why, one may ask, does 
not the process continue along the coronal and lambda sutures? 
Is such an anatomically strictly confined extension of the proc- 
ess in accordance with a supposed pathological origin? I 
must admit that these arguments prove nowhere decisively 
that the premature synostosis cannot be caused by some con- 
stitutional disease. But on the whole I think that they form a 
strong evidence against it. On the other hand, I will by no 
means absolutely deny all genetical correlation between anom- 
alies in the system of sutures of the infantile skulls and con- 
stitutional diseases. I willingly admit the possibility of such a 
relation, but I wish to reserve it for those cases in which an en- 
tire or partial closure of several sutures is seen in an often very 
irregular manner. ; 

Now the question arises as to the real significance of the pre- 
mature closure. If it is not, as I just made clear, the result 
of some pathological cause, from which point of view is the phe- 
nomenon to be explained? I believe I am able to give such an 
explanation, and I wish to give in the following pages a brief 
account of my opinion upon this subject. 


ore Lb. BOLK 


Some years ago I published an extensive investigation upon 
the normal obliteration of the sutures in Primates. The re 
sults of this inquiry were based upon the examination of more 
than 800 skulls of platyrrhine and catarrhine monkeys and a 
considerable number of skulls of anthropoid apes, all present 
in the anatomical museum of the University of Amsterdam. 
As to the problem interesting us in the present paper, we may 
limit ourselves to the conclusions relative to the anthropoid 
skulls. 

There are striking differences in the process of obliteration 
between man and apes. These differences concern the age in 
which normal obliteration takes place and the order of suc- 
cession in which the closure in the different sutures begins. 
In man, as a rule, the principal sutures persist for a longer or 
shorter time after the complete formation of the skull. The 
same happens in some genera of American monkeys; but in 
apes the sutures can close immediately after the skull is 
full grown. At this moment the general growth of the 
individual is not yet finished, and though it is, for reasons near 
at hand, impossible to know the age in which the obliteration 
begins, it is sure that the process commences, and perhaps in 
some sutures is even finished, before the animal has attained its 
adult state. 

The significance of this premature synostosis of the skull 
bones in apes may be found in the strong development of the 
muscles of mastication, arising from almost the whole surface 
of the braincase, and moreover in Gorilla and male Orangs from 
strong crests developing exactly in the line of union of the parietal 
and occipital bones. 

Now it is obvious that in the apes, as well as in man, there 
exists: a relation between the growth of the brain and the brain- 
case. In apes, as a rule, the different bones of the skull can- 
not unite together before the brain has attained its final volume. 
This is so clear and simple that it is altogether unnecessary to 
enlarge upon it. As it is, the conclusion lies close at hand that 
the sutures in the braincase of apes disappear immediately 
after their physiological function is finished. The physiological 


s 


OBLITERATION OF SUTURES IN SKULL 513 


function of the sutures is to produce new osseous tissue along 
the margin of the skull bones for the sake of the enlarging of the 
braincase. This function is continued as long as the brain- 
case needs enlarging, 7.e., as long as the brain increases in volume. 
Summarizing, I think it is clear, that in apes the sutures com- 
mence to obliterate as soon as the enlargement of the brain 
has ceased. And in this respect there is a remarkable differ- 
ence between man and apes. In the former the sutures often 
persist a long time after the brain has ceased growing. 

We can now return to our starting point and consider the 
question whether there is some relation between the normal 
progress of suture-obliteration in apes and premature oblitera- 
tion in man. There is no doubt about the fact that man stands 
in nearer phylogenetical relation to the anthropoids than to any 
other representative of the primate stem. Therefore, since, 
as a rule, the sutures begin to disappear in apes shortly after 
the brain is full-grown, which happens in youthful animals, 
we have the right to conclude that the condition in man is 
of a progressive nature. This condition, 7.e., the persistence 
of the sutures during a certain period of the adult state, must 
be considered as a peculiarity acquired by man during the 
earliest phase of his phylogenetic evolution. 

This conclusion gives rise to the following question. Should 
not the premature obliteration of the sutures in the braincase 
of man be considered an atavistic phenomenon? ‘This hy- 
pothesis deserves our full attention. If the statement is ac- 
cepted as true, that in human ancestors the sutures closed as 
those of the anthropoids of today, 7.e., at an early stage of life, 
then the occasional premature obliteration in recent man loses 
its non-proved pathological character and becomes more in- 
telligible. For we know that each quality newly acquired in 
the evolution of beings often requires a long space of time be- 
fore it becomes absolutely fixed. During this period the ante- 
cedent condition reappears individually now and then. For my 
part I think I may conclude that the premature closure of sutures 
in infant-skulls is such an atavistic phenomenon. 


514 BOLE 


ON THE OBLITERATION OF ONE OF THE OTHER SUTURES OF THE 
SKULL 


In considering the occurrence of premature obliteration a 
striking difference is observed between the sagittal and masto- 
occipital suture on the one hand and all the other sutures on 
the other. <A special discussion therefore upon the sutures just 
mentioned seems desirable in every respect. The frequency of 
premature closure in the other sutures being very small, there 
is no ground to describe each of these in a special paragraph. 
I will subsequently communicate the results of my investigation 
on each of these sutures. I wish to point out that for the present 
only those cases are being discussed in which the obliteration is 
limited to one single suture. 

I shall begin with the coronal suture. There is a notable 
difference between the coronal and sagittal suture concerning 
the starting point from which the obliteration begins. This 
point is always the same in the sagittal suture, it is the so- 
called obelion. I have found no cases in which the frontal half 
of this suture was closed, while the occipital was left open. In 
the coronal suture on the contrary this regularity does not 
exist at all and the synostosis between the parietal and frontal 
bones may commence at any point of the suture. Moreover the 
synostosis in most cases is asymmetrical and only proceeds more 
symmetrically when starting at the bregma-point. These differ- 
ences clearly show that the process in the coronal suture in some 
ways is of another character compared with the sagittal suture. 

In table 4 I gathered the cases in which the coronal suture 
was partially or totally obliterated. 

A comparison of the contents of this table and the former, 
referring to the’ sagittal and masto-occipital suture, shows 
immediately that here one has to reckon with a different cate- 
gory of phenomena. For a non-complicated obliteration of 
the coronal suture only appeared in 6 of the 1820 infantile 
skulls. Therefore one can surely consider a premature ob- 
literation of this suture to be exceptional. Once more I lay 
stress upon this fact, because it is of great importance for the 
general question concerning the etiological nature of the pre- 


OBLITERATION OF SUTURES IN SKULL 515 


TABLE 4 
Obliteration of the sagittal suture 


OBLITERATION © 
GROUP f | NUMBER 
entire partial 
lay Ghueis noo EIR eee 725 1 2 
Nia: Meda re 400 0 1 
TUE ees acc ia 168 0 1 
TNs ear ta sky 5! sxe tis | 157 0 0 
NO ee ne ae | 109 0 0 
WA Coed Seta Boe ee ae 203 0 1 
AVAL) ees See Serie ee an 58 0 0 
1820 1 5 


mature concrescence. In the preceding paragraph I dem- 
onstrated my view on the significance of the premature closure 
of the sagittal and masto-occipital suture, and in particular 
I objected there to the conception of a pathological process, 
result of a general constitutional disease, causing the obliteration 
of these sutures. For when in two sutures (which possess as 
to the development of the skull identical significance, as is the 
case with the sagittal and coronal sutures) a premature ob- 
literation appears in the former 47 times and in the latter only 6 
times, then one must conclude that other and more special in- 
fluences have to be regarded as causing the difference. If the 
obliteration was caused by a general and constitutional disease, 
one would expect the number of premature obliterations in both 
sutures to be almost the same. Here I repeat that I do not wish 
to deny that general diseases of the skeletal system can evoke 
an unfavorable influence on the sutures of the skull. Then still 
there is no reason why the osteogenesis, which can be dis- 
turbed in all other subdivisions of the skeleton by diseases of 
the bony tissue, should remain always normal in the skull. 
The abnormal process should present a character of generality 
and irregularity and the suture-system should show different 
signs of the disturbing influence. In the sagittal and masto- 
occipital suture the obliteration shows too clearly a sharply 
defined morphological character. 


516 L. BOLK 


In the coronal suture, however, it appears to be of a more 
irregular character, as follows from the fact that in cases of 
partial concrescence at one time a certain point of the suture is 
obliterated, at another time again a different point. In the 
five cases of partial concrescence I found the following con- 
ditions: once the right half was totally obliterated and of the 
left half the lower part; once only the right half was totally - 
obliterated; once the upper part of the right half, once the upper 
part of the left half and once the lower part of the left half. 
The contrast with the sagittal suture in which the synostosis 
regularly appears in the occipital half is indeed very mani- 
fest. 

Rarer still than the non-complicated synostosis of the coronal 
suture is that of the parieto-temporal (squamosal) sutures. 
On the whole I only encountered three infantile skulls of my col- 
lection in which this was the case: 7.e., two in Group I and one 
in Group II. Twice the middle part of this suture on the left 
side of the skull was obliterated, and once the hindmost part 
of the suture on both sides. These cases do not call for a 
special consideration. 

A premature synostosis of the fronto-sphenoidal suture I 
found four times in infantile skulls belonging to the first, second, 
fourth and sixth groups. It was a remarkable thing that the 
process appeared symmetrically, in all these cases the synos- 
tosis being noted on both sides. This does not seem to me to 
be of a special significance, for the suture between the sphenoidal 
and parietal bone I only found obliterated once unilaterally 
- (left side) in an infantile skull from Group III. I found, more- 
over, in this same group a skull in which a part of the left half 
of the lambdoid suture has disappeared. 

These are the cases in which a synostosis, total or partial, of 
only one single suture was seen. Before we pass to the exami- 
nation of the more complicated cases I will give a table (5) 
containing the facts heretofore stated. 

This table shows very clearly the typical place occupied more 
particularly by the masto-occipital suture but also by the 
sagittal suture with regard to the premature obliteration. 


OBLITERATION OF SUTURES IN SKULL Bi ey 


TABLE 5 


Premature synostosis in one suture only 
Number of skulls 1820 


SUTURE | NUMBER OF OBLITERATIONS 
Masto-occipital...... 180 
Samrital. 3. eee | 47 
Coronal. .24))...qge | 6 
Squamosals../..... . 3.0] 3 
Fronto-sphenoidal... . 4 
Spheno-parietal......| 1 


| 241 


THE PREMATURE OBLITERATION OF TWO SUTURES AND MORE 


We will begin with the discussion of the more simple cases in 
which only two sutures were prematurely closed. It is quite 
natural that amongst this group the coincidence of a synostosis 
in the sagittal and masto-occipital suture appears most fre- 
quently. One will remember that the obliteration of the masto- 
occipital suture, either unilateral or bilateral, has been found 
in no less than 10 per cent of the infantile skulls. The proba- 
bility therefore that such an instance can be complicated with 
an obliteration of the sagittal suture is not small. Now, the 
same possibilities may occur in these cases, as in the non-com- 
plicated synostosis of the masto-occipital suture. Together 
with the sagittal suture the masto-occipital can be obliterated 
bilaterally or unilaterally, totally or partially. It does not 
seem necessary for me to describe all these cases in detail, as 
no principle is involved. I will restrict the communication to 
those cases in which the premature obliteration appeared in 
both sutures. A summary of this is seen in table 6. 

As this table shows, we find among 1820 skulls 19 in which 
at the same time the sagittal and masto-occipital sutures were 
no longer intact, this making 1 per cent. The absolute numbers 
are too small to decide whether the frequency increases accord- 
ing to the age of the individuals. One can demonstrate how- 
ever that a predisposition of these two sutures toward a pre- 
mature closure is revealed by the relative large number of cases 


THE AMERICAN JOURNAL OF ANATOMY, VOl.. 17, No. 4 a 


518 L. BOLK 


TABLE 6 


Obliteration of the masto-occipital suture in skulls in which also the sagittal suture 
is totally or partially closed 


GROUP BILATERAL LEFT ONLY RIGHT ONLY TOTAL 
Pe eavepetehaney sto 2 1 2 5 
1 i) Celie neha 1 0 1 2 
We rece curiae 3 0 0 3 
DEVI Ha, Stree svar 2 0 0 2 
i a ee ese as 0 0 0 0 
Wis ais ee ee 4 1 0 5 
WaT 2.9 erate ieoade 1 0 1 2 
13 2 4 19 


in which this combination coincides. It can be proved in the 
following simple way. The frequency of the premature ob- 
-literation exclusively in the masto-occipital suture is 10 per 
cent, that of the sagittal suture 3 per cent. Therefore, when 
both phenomena were totally independent of each other a 
combination of both should then according to the rules of proba- 
bility never come to 1 per cent, as we have been able to de- 
termine. Thus this very frequent coincidence can only be 
explained on the assumption that the cause of the premature 
obliteration in one of the two sutures at the same time increases 
the predisposition to a simultaneous obliteration in the other 
suture. In one of the preceding paragraphs I have developed 
my view as to the cause of the obliteration. This too is sufficient 
to explain the relative large frequency of the simultaneous 
obliteration in the sagittal and masto-occipital sutures. 

The other cases in which together with the masto-occipital 
suture yet another was obliterated were the following. In 
three cases the masto-parietal and in one case the coronal suture 
was obliterated simultaneously with the masto-occipital suture. 
Finally I found a case in which the posterior half of the squamosal 
suture and the whole masto-parietal suture were obliterated. 

On the whole there were found in my collection 24 infantile 
skulls in which two sutures were coalesced. 

Finally there were amongst my material a small number of 
skulls in which the premature obliteration had assumed a more 


OBLITERATION OF SUTURES IN SKULL 519 


extensive character and showed a more irregular form. It is 
impossible to divide these cases according to a presumed point of 
view in groups. Therefore I will give only a simple description 
of them. 

Amongst the infantile skulls with milk-dentition (Group I) 
I found the following cases of complicated closure: 

1. On the right side: the posterior half of the squamosal 
suture, the masto-parietal and the masto-occipital suture; on 
the left side: the masto-parietal and masto-occipital suture. 

2. On the right side: the whole of the squamosal suture, the 
masto-parietal and the masto-occipital; on the left side only the 
masto-occipital suture. 

3. On the right side: the spheno-frontal and spheno-parietal 
suture, the lower half of the coronal suture, the posterior half 
of the squamosal suture, the masto-parietal and masto-occipital 
suture; on the left side the spheno-frontal, the spheno-parietal, 
the lower part of the coronal, the lower part of the lambdoid 
and the masto-occipital suture. 

4. The occipital half of the sagittal and the right half of the 
lambdoid sutures. 

5. On the right side: the lower half of the coronal, the hinder 
part of the squamosal, the masto-parietal and the masto-occipital 
suture. On the left side the coronal suture. 

6. The sutura sagittalis totally. On the right side: the 
spheno-parietal, the squamosal, the masto-parietal and masto- 
occipital and the lower half of the lambdoid suture. On the 
left side: the spheno-parietal and the masto-occipital sutures. 

7. The occipital part of the sutura sagittalis. On the right 
side: the spheno-parietal and masto-occipital suture. On the left 
side: the squamosal, masto-parietal and masto-occipital sutures. 

8. The sutura sagittalis. On the right and left side the 
occipital half of the squamosal, the masto-parietal and the 
masto-occipital suture. 

9. The sutura sagittalis. On the right and left side the mesial 
part of the coronal suture. 

Amongst the skulls of Group II the following cases were 
found: 


520 L. BOLK 


10. On the right side: the lower half of the coronal, the whole 
of the masto-occipital suture. On the left side: the coronal 
suture and partly the masto-occipital suture. 

11. On the right side: the masto-occipital and masto-parietal 
suture. On the left side: the hinder part of the squamosal, the 
masto-parietal and masto-occipital suture. 

12. The sutura sagittalis. On the right side: the masto- 
occipital suture; on the left side the lower half of the coronal, 
the masto-parietal and masto-occipital suture. 

Amongst the skulls of Group III, the following case was 
found: 

13. On the right side: the whole of the squamosal and the 
masto-occipital sutures. On the left side: the masto-occipital 
suture. 

Amongst the infantile skulls of Group IV, the following cases 
were found: 

14. On the right side: the hinder part of the squamosal and 
the masto-occipital suture; on the left side: the squamosal, 
masto-parietal and masto-occipital sutures. 

15. The sutura sagittalis. On the right and left side: the 
spheno-parietal, the masto-parietal and masto-occipital sutures. 

Amongst the skulls of Group V I found the following cases: 

16. The sutura sagittalis partially on the right and left side: 
a hinder part of the squamosal and the masto-parietal sutures. 

17. On the right side: the posterior half of the squamosal, 
the masto-parietal, the masto-occipital sutures. On the left — 
side as on the right, and moreover the lower half of the lambdoidal 
suture. 

Amongst the skulls of Group VII I found the following case: 

18. The sutura sagittalis partially. On the right side: the 
masto-occipital suture. On the left side: the posterior half of 
the squamosal, the masto-parietal and the masto-occipital 
suture. The number of non-adult skulls in which the premature 
obliteration of the sutures assumes an irregular character on a 
larger scale amounts to 1 per cent (18 skulls amongst 1820). 

When examining these cases more closely, and seeking to deter- 
mine the question in which suture the greatest amount of ob- 


OBLITERATION OF SUTURES IN SKULL 53] 


literation occurs in cases of more extensive premature closure, 
the very interesting fact presents itself that it is the squamosal 
suture. To demonstrate this fact, I recall that amongst 1820 
infantile skulls there were only three in which only the suture 
mentioned showed signs of obliteration, forming a striking 
difference with the sagittal suture, in which this appeared 47 
times. On the other hand I find amongst 18 skulls with more 
extensive and irregular premature obliteration not less than 
11 in which the suture squamosa was no longer intact, and 
only 9 in which the sutura sagittalis was partially or totally 
obliterated. I call attention to this fact because it speaks in 
favor of my opinion that the isolated obliteration of the sagittal 
suture is caused by a special cause. 

Finally I shall proceed to give a general view of the sutural 
obliteration in our collection of skulls considered as a whole. 
In this collection, consisting of 1820 skulls, I found a prema- 
ture obliteration either in a single suture or in more, in no less 
than 343 skulls. This amounts to 19 per cent. This result, 
due largely to the very frequent obliteration of the masto- 
occipital suture, I did not expect, neither should it have been 
expected by anybody. 

In the preceding paragraphs I pointed out how frequently 
only a single suture in a skull showed a more or less extensive 
obliteration, while all others remained intact. This, of course, 
did not give a real idea as to the number of times in which each 
of the sutures amongst the 1820 skulls really was obliterated, 
because amongst these, the cases in which more than one suture 
showed signs of obliteration, were not counted. In the table 
7 I give a short summary which shows how many times 
each suture was obliterated either totally or partially. If this 
occurred, as is possible in paired sutures, on both sides, the case 
is only once counted. 

The extraordinary frequency of the obliteration in the masto- 
occipital suture is very obvious, and no less that of the sagittal 
suture. To estimate the frequency correctly one has to com- 
pare, of course, the bilateral sutures with each other, and also 
the unpaired. Then the difference between the sutura masto- 


522 L. BOLK 


TABLE 7 


Absolute frequency of premature obliteration 
in 1820 skulls 


times 
Sut. masto-occipitalis......... 272 
Sut: Sagittaliguee:.:.. 2. = 20el) 71 
Sutsisquamosaseer. oss .c2). oe see Li 
Sut. parieto-mastoidea........ 16 
Dut. COLOMAMS EN... . s.ncnav en 12 
Sut. parieto-sphenoidalis...... 5 
Sut. fronto-sphenoidalis....... 5 
SUt MAMA oOIdea:.. ..:. + Suis See 5 


occipitalis with 272 cases or 15 per cent and the sutura squamosa 
with 17 cases, or 1 per cent is no less surprising than that of the 
sutura sagittalis with 71 cases or 4 per cent and of the coronal 
suture with 12 cases or 0.6 per cent. Once more it is demon- 
strated by these relations that a premature obliteration of 
the sagittal suture occurs more often than was formerly be- 
lieved, while that of the masto-occipital suture occurs so often 
that it can scarcely be considered an anomaly. 

From the annotations, collected during my investigation, I 
finally will communicate a very interesting observation. As 
generally known it may happen in the skull of man that the 
sutura frontalis persists. According to the communications of 
the authors, this should be the case in about 6 per cent. In 
Dutch skulls the persistence of this suture is found in not quite 
9 per cent. It has struck me, however, that I did not find 
in my collection of non-adult skulls the coincidence of a per- 
sisting sutura frontalis and premature obliteration in one of the 
other sutures. This seems not inconceivable in case of the 
sagittal suture, for this suture and the frontal can be con- 
sidered as two parts of one, extending from the nasion to the 
lambda. But also nearly the same was stated as to the masto- 
occipital suture. There were, as mentioned, 272 skulls with 
premature obliteration of this suture, but according to the 
general relation, one should expect to find amongst these skulls, 
9 per cent or 24 with a ‘persisting metopical suture. In reality 
I only found two cases. 


OBLITERATION OF SUTURES IN SKULL eA 


This fact however is not altogether inconceivable. When the 
metopical suture, obliterating normally between the second and 
third year, persists this fact points to a decreasing tendency of 
the sutures of the skull to coalesce. And that in such skulls a 
premature obliteration does not take place, seems to me a 
very natural phenomenon. This fact can be considered as a 
new proof of the justness of my opinion that premature ob- 
literation of the sutures is not caused by a general pathological 
influence, but that it is a phenomenon of which the origin has 
to be looked for in the sutures themselves and in the process 
of growth localized in them. 


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