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THE DEVELOPMENT OF THE FROG'S EGG

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THE DEVELOPMENT OF THE

FROG'S EGG

AN INTRODUCTIOX TO
EXPERIMENTAL EMBRYOLOGY

BY

THOMAS HUNT MORGAN, Ph.D.

PROFESSOR OF BIOLOGY, BKYN MAWR COLLEGE

THE MACMILLAN COMPANY

LONDON: MACMILLAN & CO., Ltd.

1897

All rights reserved

COPTEIGHT, 1S97,

By the MACMILLAN COMPANY.

NorfaooD Press

J. S. Cushing & Co. - Berwick & Smith
Norwood Mass. U.S.A.

PEEFACE

The development of the frog's egg was first made known
through the studies of Swammerdam, Spalhmzani, Rusconi,
and von Baer. Their work laid the basis for all later research.
More recently the experiments of Pfliiger and of Roux on this
egg have turned the attention of embryologists to the study
of development from an experimental standpoint. Owing to
the ease with which the frog's egg can be obtained, and its
tenacity of life in a confined space, as well as its suitability for
experimental work, it is an admirable subject with which to
begin the study of vertebrate development.

In the following pages an attempt is made to bring together
the most important results of studies of the development of
the frog's egg. I have attempted to give a continuous account
of the development, as far as that is possible, from the time
when the egg is forming to the moment when the young tad-
pole issues from the jelly-membranes. Especial weight has
been laid on the results of experimental work, in the belief
that the evidence from this source is the most instructive for
an interpretation of the development. The evidence from the
study of the normal development has, however, not been neg-
lected, and wherever it has been possible I have attempted to
combine the results of experiment and of observation, with the
hope of more full}' elucidating the changes that take place.
Occasionally departures have been made from the immediate
subject in hand in order to consider the results of other work
having a close bearing on the problem under discussion. I
have done this in the hope of pointing out more definite con-
clusions than could be drawn from the evidence of the frog's
egg alone.

In treating the general problems of development, I have tried
to keep as near to the evidence as possible. I have intention-

vi PREFACE

ally avoided at times the discussion of the more theoretical
problems arising from the experiment, for it seems to me that
such discussions are out of place in a volume of this sort. Only
the early stages of the development have been considered,
because almost all of the experimental work on the frog's egg
has been done on the early stages, and also because I am more
familiar with the development and with the experiments of this
period. Moreover, the later stages have been recently most
admirably described by Marshall in his Vertebrate Embryology.

A few words of personal explanation may be added. For
several years I have been collecting the material for the present
volume, but as the literature is so extensive and as I have had
other work to do first, I made but slow progress. In the
summer of 1893 I set seriously to work, and owe much to the
admirable facilities offered by the University of Berlin. I take
pleasure in acknowledging my indebtedness to Geheimrath
Professor Fr. E. Schulze for many privileges and kindnesses
extended to me in Berlin. The work was continued irregu-
larly during the winter of 1893-1894 while enjoying the oppor-
tunities of the Stazione Zoologica in Naples. During the
winter of 1894-1895 the material was brousfht together and in
the summer of 1896 at Zurich the manuscript was almost com-
pleted. I gladly take this opportunity to thank Professor
Arnold Lang for many courtesies extended to me during two
visits to Ziirich. Dr. Driesch has most kindly looked over
some of the chapters, and has made many valuable sugges-
tions. Dr. H. H. Field has also examined a part of the
manuscript and helped me in several directions. To Professor
E. B. Wilson I am under heavy obligations, and owe much
to his valuable suo-o-estions and corrections. To Dr. H.
Randolph I owe a debt of gratitude for kindly advice and
criticism. I am also greatly indebted to Professor Joseph W.
Warren and to Professor E. A. Andrews for advice in con-
nection with the revision of the proof.

CONTENTS

PACK

Introduction ^i

CHAPTER I

The Formation of the Sex-cells 1

Sperinatogenesis.

" Direct " Division of the Germ-cells.

Oogenesis.

Comparison of Spermatogenesis with Oogenesis.

CHAPTER n

Polar Bodies and Fertilization 15

Extrusion of the First Polar Body and Egg-laying.
The Jelly of the Egg, and the Second Polar Body.
Entrance of Spermatozoon and Copulation of Pronuclei.

CHAPTER m

Experiments in Cross-fertilization 26

Experiments of Pfliiger and of Born on Frogs' Eggs.
Experiments on Other Forms.
Experiments of Rauber and of Boveri.

CHAPTER IV

Cleavage of the Egg 32

Normal Cleavage.

Correspondence of the First Cleavage-plane and the Median-plane

of the Embryo.
Roux's Experiments with Oil-drops.
Historical Account of the Cleavage of the Frog's Egg.

vii

28910

viii CONTENTS

CHAPTER V

PAGE

Early Development of the Embryo 50

The Blastopore.

External Changes after the Closure of the Blastopore.

CHAPTER YI

Formation of the Germ-layers 63

His's Experiments with Elastic Plates.
The Formation of the Embryo by Concrescence.
The Formation of the Archenteron.
The OvergTowth of the Blastoporic Rim.
The Origin of the Mesoderm.

Different Accounts of the Origin of the Archenteron and Meso-
derm.
Later Development of the Mesoderm and Origin of the Notochord.

CHAPTER VII
The Production of Abnormal Embryos with Spina Bifida . 75

CHAPTER VIII

Pfluger's Experiments on the Frog's Egg 81

The Effect of Gravity on the Direction of the Cleavage.

The Relation of the Planes of Cleavage to the Axes of the

Embryo.
Conclusions from the Experiments.

CHAPTER IX

Experiments of Born and of Roux 90

Changes that take Place in the Interior of the Egg after Rotation.
The Cleavage of the Egg in a Centrifugal Machine.

CHAPTER X

Modification of Cleavage by Compression of the Egg . . 95

Effect of Compressing the Segmenting Egg between Parallel Plates.

Conclusions from the Experiments.

The Distribution of the Nuclei in the Compressed Egg.

CONTENTS ix

CHAPTER XI

PAGE

The Effect of Injuring One of the First Two Blastomeres 106
Roux's Experiment of " Killing " One of the First Two Blasto-
meres.
Further Experiments by Others (Hertwig, Entires and "Walter,
Schultze, Wetzel, Morgan).

CHAPTER Xn

Interpretations of the Experiments; and Conclusions . . 123
Roux's Mosaic Theory of Development.
Theory of Driesch and of Hertwig of the Equivalency of the Early

Blastomeres.
Roux's Subsidiary Hypothesis.
Experiments on Other Forms.
General Conclusions.

CHAPTER XIII

Organs from the Endoderm 137

The Closure of the Blastopore, and the Formation of the Neuren-

teric Canal.
The Digestive Tract and the Gill-slits.

CHAPTER XIV

Organs from the Mesoderm 146

The Mesodermic Somites.
The Heart and Blood-vessels.
The Pronephros.

CHAPTER XV

Organs from the Ectoderm 159

The Central Nervous System.

The Eyes.

The Ears.

The Nerves.

The Appearance of Cilia on the Surface of the Embryo.

^ CONTENTS

CHAPTER XVI

PAGE

. 168

Effects of Temperature and of Light on Development

APPENDIX ^"^^

LITERATURE . 173

INDEX ^^'^

INTRODUCTION

The eggs of most of our species of frogs are laid in the
spring. In some cases they are set free ahuost immediately on
the emergence of the frogs from their winter sleep ; in other
cases the eggs are not laid until some weeks or even months
after the frogs have awakened. In almost every instance the
eggs are deposited in water and usually in quiet pools or ponds,
or in protected bays along streams where the water has backed
up and has come to rest. Sometimes the bunches of eggs are
stuck to sticks, grass, submerged sedge, or even to stones ; in
other cases the bunches are not fastened.

The copulation precedes and lasts through the laying-period ;
a single male fertilizing all the eggs laid by one female. The
sperm pours out of the cloaca of the male at the moment when
the eggs pass out of the female.

Both the male and the female sexual products, the eggs and
spermatozoa, are ripened during the summer and autumn of
the year preceding the deposition of the eggs, — at least this
is the more usual process. The origin of these sexual cells
must first be studied in order to more fully understand their
relation to each other, and the part they play in the subsequent
development.

XI

/

DEYELOPxAIENT OF THE FEOG'S EGG

-oo>»<oo-

CHAPTER I

THE FORMATION OF THE SEX-CELLS

The development of the sex-cells is generally divided into
three periods : 1) a multiplication-period, during which the
primitive germ-cells pass through a large number of divisions ;
2) a growth-period, in which the primitive germ-cells, that
have become reduced in size through repeated division, grow
larger ; 3) a maturation-period, when only two divisions take
place, between which the nucleus does not pass into a resting-
stage. At the end of this last division the male germ-cells
undergo histological changes by which they become trans-
formed into spermatozoa. 1

Spermatogenesis

The changes that take place in the testes of the frog have
not been so fully worked out as in some other animals ; we
may therefore follow, first, the method of development of the

1 This is a modification of the terminology of v. la Valette St. George, whose
nomenclature of spermatogenesis is still often used. La Valette's classification
is as follows : —

The primordial germ-cells give rise to spermatogonia, which cease to divide
after a time and increase in size. Each spermatogonium is thus converted into
a primary spermatocyte. Each primary spermatocyte divides into two cells, the
spermatocytes of the second order, and each of these divides once more, with-
out a resting-period, to form two spermatids. In this way four spermatids are
formed from each primary spermatocyte. Each spermatid is then changed
directly into a spermatozoon.

B 1

DEVELOPMENT OF THE FROG'S EGG

[Ch. I

spermatozoon in two forms in which tlie process is better
known, and then consider the special case of the frog.

The development of the spermatozoa of Gryllotafpa, the
mole-cricket, has been described by vom Rath ('92, '95). As
the process of spermatogenesis is relatively simple in this form,
and as it is, according to vom Rath, much like the process that
takes place in the frog, we may therefore first briefly consider
the changes in Gryllotalpa.

First Period. A cell in the resting-stage at this time shows
a large nucleus with a distinct membrane enclosing a network
of fine chromatin (Fig. 1, A). The beginning of the cleavage
is indicated by the withdrawal of the chromatin from the
nuclear membrane and the thickening of the fibres of the

A B c D

Fig. 1. — Division of sperm-mother-cells in Gryllotalpa. (After vom Rath.)

chromatic network. The tangled mass of threads, or net-
work, then takes a somewhat excentric position. This thread
seems to consist of linin, on which chromatin-granules are
arranged. Sometimes the thread can be seen to be split along
its length into two parts. The halves of the thread remain,
however, closely sticking to each other. The double thread
then breaks up by cross-division into twelve equal segments,
or chromosomes (Fig. 1, B). The chromosomes next become
shorter, and finally spherical, and come to lie in an equa-
torial plate (Fig. 1, C). When the chromatin is still in the
skein-stage, two minute bodies are seen in the protoplasm just
outside of the nuclear membrane (Fig. 1, B). These are the
two centrosomes, which separate more and more from each
other, and finally come to lie on opposite sides of the nucleus.
A protoplasmic spindle develops between the two centrosomes
(Fig. 1, C) and the fibres of the spindle become fixed to the

Ch. I]

FORMATIOX OF THE SEX-CELLS

chromatin-granules of the equatorial plate. Each of the twelve
chromatin-granules divides into two equal parts and the halves
migrate toward one or the other of the centrosomes (Fig. 1, D).
The cell-protoplasm next divides into two parts, so that two
new cells are formed. Each cell contains twelve chromosomes.
In this way the primitive sperm-cells continue to increase in
number by a series of cell-divisions, all like that just described.

Fig. 2. — The formation of spermatozoa in Gryllotalpa. The two maturation-divi-
sions. (After vom Rath.)

Second Period. A period of rest then follows, during which
the cells grow larger. During this time the chromatin is again
arranged in a fine network.

Third Period. Two successive and most peculiar cell-
divisions now take place. The chromatin-network becomes
thicker, and forms a tangled skein of threads (Fig. 2, A, B).

4 DEVELOPMENT OF THE FROG'S EGG [Ch. I

Each thread is split longitudinally into two parts. Two cen-
trosomes again appear. The chromatin-thread next breaks up
into six bent rods or chromosomes (Fig. 2, C). There is some
doubt as to the way in which the next change is brought
about. The account of vom Rath, which we follow here, seems
to be in harmony with the process that is known to take place
in some other forms during this period of development of the
germ-cells. It appears that the halves of each of the six bent
rods begin to separate from each other except at the ends
of the rods, where the halves remain united. Each rod is
in this way converted into a ring (Fig. 2, D). These rings
are often so bent on themselves that they form a loop. The
six chromatin-rings lie close to the periphery of the nucleus.
The rings contract and become smaller and thicker (Fig. 2, E),
This stage lasts but a short time and is succeeded by a stage
shown in Fig. 2, F, G. Out of each ring four star-like granules
are formed, the tetrad or " Vierer-gruppe." The four granules
of each set are closely held together by clear linin threads. If
each granule be counted as a distinct chromosome, then there
are present at this time six groups of four chromosomes each, or
twenty-four chromosomes. These twenty-four chromosomes be-
come attached to the fibres of the achromatic spindle (Fig. 2, H)
and arrange themselves into an equatorial double plate. Then
twelve of these granules united in pairs wander toward one pole
of the cell and twelve toward the other pole, and the division
of the cell takes place (Fig. 2, I). This process is spoken of as
the first maturation-division. Without passing into a resting-
stage, a second division of each cell follows (Fig. 2, J). A new
karyokinetic spindle is formed and the twelve chromosomes
are separated into two plates of six chromosomes each, which
go to their respective poles. Each of the two new cells con-
tains therefore only six chromosomes (Fig. 2, K). The number
of chromosomes is now reduced to half the normal 7iumber
present in the other cells of the body of the animal. Each of
the four cells formed by these two consecutive divisions of the
sperm-mother-cell then differentiates into a spermatozoon (Fig.
2, L). Each spermatozoon consists of three parts, — a head, a
middle piece, and a tail. The head is formed almost entirely
out of the nucleus of the j)arent-cell of the spermatozoon, as

Ch. I] FORMATION OF THE SEX-CELLS 5

seen in Fig. 7, A, B, C. It is probable that a very thin Liyer
of cytophism covers the outer surface of the head. The chro-
matin is densely packed into the head-piece, and cannot be
resolved into its component chromosomes. The middle piece
lies just back of the head.^ In some animals this middle piece
is known to enter the egg with the spermatozoon and a part of
it becomes the centrosome, which then divides into two centro-
somes and around these arise the achromatic rays of the dividing
egg. The tail of the spermatozoon is generally described as
coming from the cytoplasm of the cell.

The development of the spermatozoon in the salamander
has been carefully studied by Flemming ('87), vom Rath
('93), Meves (*96), and others. There are certain remark-
able processes that take place in the spermatogenesis of
these Amphibia that seem to occur also in the frog, but as
they have not been as carefully worked out in the latter form
we may examine first the changes that take place in the sala-
mander. Each year after the male has lost its supply of
sperm, new spermatozoa begin to develop. The epithelial
cells linino- the cavities of the testes divide at first after a
type of cleavage called, by Flemming, homoeotypic. This first
period of activity produces the first generation of spermato-
cytes, which divide according to another type, the heterotypic.

The cells of the second generation of spermatocytes also di-
vide in the same way, but with an occasional homoeotypic cleav-
age. Finally, in the third generation of spermatocytes, both
types of cleavage occur. The products of the third generation
transform directly into spermatozoa. In the heterotypic division
the process is as follows. The chromatin is at first arranged in
a thick thread, having a definite arrangement. The skein-stage
follows, and a longitudinal splitting of the chromatin-thread is
apparent. A thickening of the thread then takes place, and
it breaks up into twelve chromosomes (only half the number
present in other cells of the body) (Fig. 3, A, B). At the free
ends of the bent chromosomes, each of which is split longitudi-
nally, the halves fuse together (Fig. 3, B), but elsewhere the

1 Its origin in the frog has not been definitely made out. It is probably
cytoplasmic in origin (Fig. 7, A, B, C).

DEVELOPMENT OF THE FROG'S EGG

[Ch. 1

halves of the chromosomes separate from each other along the
longitudinal line of division. The process is similar to the ring-
formation of Gryllotalpa. In this way twelve loops are formed
from the twelve chromosomes. The bent ends of the new loops
or rings correspond to the middle portions of the earlier rods or
chromosomes (see the + and - signs in Fig. 3, A, C). ]\Iean-
while the achromatic spindle between the centrosomes has devel-
oped, and the loops of chromatin are arranged on the threads of
the spindle, as seen in Fig. 3, B. At the next stage each loop

B

E F

Fig. 3. — Heterotypic type of nuclear division in Salamandra. (After Flemming.)

breaks at the equator, i.e. at the point where the ends of the
rods fused at an earlier period, and begins to migrate toward
its centrosome (Fig 3, D). While this migration of the twelve
bent chromosomes is taking place, each chromosome may be
seen again to split longitudinally, although the two halves
remain in contact (Fig. 3, E). The cell then passes into a
restino'-stage.

In the Iwmoeotijinc divnion the first phase, the spireme, is simi-

Cii. 1]

FORMATION OF THE SEX-CELLS

lar to the last, i.e. it is a skein (Fig. 4, A) with longitudinally
split thread. Twelve bent rods appear and become shorter than
the bent rods of the heterotypic type. These rods then arrange
themselves about the middle of the achromatic spindle (Fig.
4, B). The twelve bent rods divide each into two by separa-
tion along a longitudinal line, and t\vent3'-four rods are present.
Immediately twelve of these migrate toward one pole, and
twelve toward the other, and the cell-division follows (Fig. 4,
C, D, E, F). The cells then come to rest.

B

D E - F

Fig. 4. — Homceotypic type of nuclear division in Salamandra. (After Flemming.)

The end result in the two types of cleavage is the same, but
the details are, as described, different. It is important to note
that the number of chromosomes is half that of the number of
chromosomes in the other cells of the bod}-.

Vom Rath maintains that a fourth generation of cells appears
in the development of the spermatozoa of the salamander. Flem-
ming supposed that at the end of the third generation of cells,
described above, the differentiation into spermatozoa began, but

8

DEVELOPMENT OF THE FROG'S EGG

[Ch. I

vom Rath has found that at the end of the third generation
large cells appear with huge nuclei (Fig. 5, B), in which there
are twelve groups of chromosomes. Each group or tetrad is
composed of four granules. There are, therefore, present forty-
eight spherical chromosomes united in groups of four. These
tetrads arose from a heterotypic spindle, and in the following-
way. As the twelve loops (which are now double, making
twenty-four loops) passed toward one pole they became much
thicker (Fig. 5, A). The middle point of union of each of the
twenty-four loops broke (Fig. 5, B), and the portions rounded
up, so that there were present forty-eight chromosomes arranged
in twelve groups of four chromosomes each (Fig. 5, B). Imme-
diately after the formation of the tetrads the groups of chromo-
somes arranged themselves along the rays of the achromatic

A B C

Fig. 5. — Formation of tetrads in testis of Salamandra. (After vom Rath.)

spindle (Fig. 5, C). The tetrads next passed toward the
equator of the spindle, and there they divided, so that two of
each of the four chromosomes passed toward one pole of the cell
(as in Gryllotalpa). In this way two new cells are formed
with twenty-four chromosomes each. A second division suc-
ceeds without an intervening resting-stage, and the number of
chromosomes is reduced, so that each cell has twelve chromo-
somes. The cells resulting from the last division, having each
twelve chromosomes, differentiate each into a spermatozoon.

The second division, according to some workers (Boveri,
Hertwig, and Brauer), is the result of a second longitudinal
division. But vom Rath holds that this second division in the
Amphibia and in Gryllotalpa is the result of a cross-division of

Ch. I] FORMATIOX OF THE SEX-CELLS 9

the threads. According to Boveri, the meaning of the forma-
tion of the tetrad is only the precocious separation of the chro-
matin-threads for two rapidly succeeding divisions (without an
intermediate resting-stage). The doubling of the chromosomes
previous to division has, he thinks, no further significance than
the preparation for two quickly succeeding divisions. It is not
obvious, however, in the development of the spermatozoon why
this rapid division should take place at tliis time and at no
other in the life of the cell.

Meves ('96) has most recently reexamined the development
of the spermatozoon in the salamander. His results differ in
several respects from the earlier results of Flemming, and in
one essential respect from the work of vom Rath, According
to Meves, the germ-cells undergo many divisions in the upper
part of the testis. The chromatic figure is that of the usual
type of division ; and ttventy-four chromosomes are present. As
a result of the division, the cells become smaller, and each cell
becomes surrounded by a layer of connective tissue. Each of
these cells then divides many times according to the usual type
of division, so that clusters of cells are produced surrounded by
a common wall of connective tissue. Then follows the resting-
period, in which the cells enlarge. After this the maturation-
divisions take place. Meves thinks that most probably each
cell divides only twice during this period, as in other forms.
The first division is heterot^'pic, and now for the first time the
number of cltromosomes is reduced to twelve. Without a resting-
period each cell again divides, the twelve chromosomes splitting
longitudinally. This second division is homceotypic. Each
cell, containing twelve chromosomes, then transforms directly
into a spermatozoon.

Meves shows therefore that Flemming was mistaken in regard
to the number of cell-generations that are present in the sper-
matogenesis of the salamander, and further that Flemming
failed to make out the real sequence of the generations and
the number of chromosomes present in each. ]\Iore important
is Meves' statement that, normally, there is not a formation of
tetrads as vom Rath had affirmed. At present it is impossible
to decide between the divergent accounts of ^Nleves and vom

10

DEVELOPMENT OF THE FROG'S EGG

[Cii. I

Rath, and we must suspend judgment until further work throws
more light upon the question.

The spermatogenesis of the frog has not been worked out in
the same detail as that of the salamander, yet vom Rath ('95)
has made certain important statements in regard to it. The
prophase of the mitoses, before the ripening period, has in the
frog a close resemblance to the skein-stage of the heterotypic
and homoeotyj^ic variations in the salamander. But, on the
other hand, in the metakinetic stage the peculiarities of the
honiffiotypic and heterotypic forms, as described by Flemming,

B

Fig. (5. — Stages in the last maturation-division of sperm-cells of Frog.
(After vom Rath.) (Figs. C, D, F slightly modified.)

are absent. The formation of the chromatin-rings and tetrad-
groups in the frog (Fig. 6, A, B. C) differs from that of the sala-
mander and is much more like that of Gryllotalpa. The rings,
owing to the strong contraction of the segments, are relatively
small in the frog, but proportionately thick (Fig. (3, B). From
each ring arise the four spherical chromosomes of each tetrad-
group (Fig. 6, C). The ring-stage lasts quite long in Rana,
judging from the frequency of its presence. The rings lie at
the periphery of the nucleus.

Ch. I]

FORMATION OF THE SEX-CELLS

11

The shape of the spermatozoon is very different in different
species of frogs. In some species the head of the spermatozoon
is drawn out into a fine point (Fig. 7, A, E) ; in other forms it
ends bluntly (Fig. 7, D). The middle piece is easily found in
some spermatozoa, but in others only by the application of
special reagents. In the European toad (Fig. 7, A) the tail of

V

M

B

/

Fig. 7. — Spermatozoa. A. Bufo cinereus. B-C. Two stages in development of sper-
matozoon; D. Fully formed spermatozoon of Hyla arborea. E. Spermatozoon
of Raua esculenta (the tail is too short). (After v. la Valette St. George.)

the spermatozoon is formed by a flat membrane with a thick-
ened border. In Hyla arborea and Rana esculenta (Fig. 7, D, E)
the tail of the spermatozoon is like a long lash or thread. In
Rana esculenta the head measures .015-.021 mm. in length and
the tail .04 mm. in length.

"Direct" Division of the Germ-cells

In the testes of the frog and of other Amphibia are often
found germ-cells whose nuclei have very irregular outlines.

12

DEVELOPMENT OF THE FROG'S EGG

[Ch. I

Centrosomes can generally be demonstrated in these resting-cells.
Other cells have the nuclei broken up into a number of smaller
spheres. Still other nuclei may have a deep depression on one
side, as though the nucleus were dividing into two by constric-
tion. These nuclei have often been described as dividing by
amitotic or direct division, i.e. without the characteristic mitotic
division. Meves ('91) has stated that in the testes of the sala-
mander amitotic division occurs regularly, and he believes that
such cells will later form spermatozoa. Other authors (Bellonci
and vom Rath), admitting that such a division may take place,
affirm that such cells are in process of degeneration, and never
subsequently form spermatozoa. Vom Rath declares that cells
that have once divided by amitosis can never again divide by
karyokinesis (mitosis), and that such cells degenerate later, and
do not ever develop into sex-cells.

Oogenesis

The origin of the egg in the ovary of the frog has been
studied by Schultze (*87, e), but many important details are

still unknown. The egg
derived from one of the
cells of the outer layer of
the ovary is surrounded
by a large number of fol-
licle-cells. The nucleus
of the Qgg consists of fine
chromatic threads, of a
nuclear sap, and of scat-
tered nucleoli. As the
egg enlarges the nucleus
also enlarges, and the
chromatin stains more
faintly and appears in the
form of scattered threads.
The nucleoli stain well
and become larger and more numerous as the egg enlarges,
and are found generally around the periphery of the nucleus.
Certain portions of the protoplasm now begin to stain dif-

FiG. 8. — Ovarian egg of Raua.

Ch. I] FORMATION OF THE SEX-CELLS 13

fereiitly from the rest, and are spoken of as the yolk-nuclei.
They seem, in some way not fully understood, to be con-
nected with the development of the yolk-granules.^ The
yolk-granules, at first small and scattered, grow larger and
become more numerous (Fig. 8). Before the egg leaves the
ovary, the nucleus wanders toward the periphery and places
itself under the black pole of the egg
(Fig. 9). When the surface of the
egg is examined, it shows a lighter
area, owing to the displacement of the
pigment-granules in the region occu-
pied by the nucleus. The nucleoli at
this time migrate toward tlie centre of
the nucleus,^ there disintegrate, and
finally disappear. The chromatin-mate-
rial draws together at this time into Fig. 9. — Section of ripe ova-

, , , . , 1 1 ii rian egg. (After Hertwig.)

threads which stain more deeply, the

nuclear membrane disappears, an achromatic spindle develops,

and the egg is ready to extrude the first polar body.

Comparison of Spermatogenesis with Oogenesis

The method of extrusion of the polar bodies is described in
the next chapter, but we may anticipate this account in order
to consider here a remarkable parallel that has been discovered
between the formation of the polar bodies and the formation of
the spermatozoa. In the latter, as we have seen, two successive
divisions follow each other during the maturation-period tvith-
out an mtervening re sting -stage. The tetrad-groups are present
at the beginning of the process. After the two maturation-
divisions the number of chromosomes is reduced to half the
number characteristic for the species.^ The same phenomena
appear when the polar bodies are extruded from the egg. After
the extrusion of the first polar body, the spindle for the second

1 Will ('84) describes the yolk-nuclei as arising from constrictions of the
nucleus set free with their nucleoli into the protoplasm.

2 Schultze aifirms that the later chromatin comes from these nucleoli, but
Born has corrected this statement.

3 The reduction in number of the chromosomes seems in some forms to take
place before the tetrad-period.

14 DEVELOPMENT OF THE FROG'S EGG [Ch. T

polar body forms immedi?ite\j tvithout a reatincj-period. Further,
it is found, after the extrusion of the second polar body, that the
number of chromosomes in the Q^g is reduced to half the num-
ber characteristic for the somatic cells. Tetrads have also been
described as occurring just before the extrusion of the polar
bodies. In many cases the first polar body divides into two,
so that three polar bodies are present. These three polar bodies
and the egg seem to correspond to the four spermatozoa from
each spermatocyte. All four spermatozoa are functional, but
onlj^the egg (and not its three polar bodies) is capable of devel-
opment. Weismann has utilized the discovery of the reduction of
the number of chromosomes to build up an elaborate and highly
speculative theoiy of heredity. The reduction division is, ac-
cording to Weismann, not simply a quantitative division of the
chromatic thread, but is at one stage at least a qualitative
division. The reduction of the chromosomes to half the number
pi-esent in the other cells of the body seems, according to Weis-
mann and others, to be a preparation for fertilization. Since
the spermatozoon brings into the Qgg only half the number of
chromosomes found in the somatic cells of the animal, and
since the egg-nucleus supplies the other half, the number of
chromosomes will thus remain constant for the species from
generation to generation.

CHAPTER II

POLAR BODIES AND FERTILIZATION

Whether the egg leaves the ovary by means of its own
activity, or by some other mechanism, we do not know. That
the egg itself takes some part in the process seems possible
from the fact that it is set free only at a particular moment
of its maturation, i.e. at a time when certain processes have
taken place in its interior. This same process takes place
' simultaneously in all the eggs in the ovary. The separation of
the Qgg from the ovary is not dependent upon the act of copu-
lation, for several cases are on record in which isolated females
were found to have eggs in the body-cavity and oviducts.

The egg set free in the ccelomic cavity is covered by an ex-
tremely thin membrane, the egg-membrane or vitelline mem-
brane. The egg itself is very soft and easily broken if handled.
Later, when in the oviduct, the protoplasm seems to become
more firm.

The egg shows a white and a dark hemisphere. The relative
distribution of superficial pigment in the egg determines the
extent of the white and dark surfaces. The outer layer of pig-
ment in the black hemisphere seems to be in close contact with,
or fixed to, the vitelline membrane, but the pigment lying in
the protoplasm beneath the outer layer is free to move with any
movement of the protoplasm (Figs. 8, 9). The relative ex-
tent of surface of the egg that is black or white is variable in
different species, and even in different females of the same
species ; but all the eggs from one female show approximately
the same distribution of pigment.

Extrusion of the First Polar Body axd Egg-laying

Just prior to the extrusion of the egg into the body-cavity of
the frog, the nucleus undergoes a remarkable change, so that in

15

16 DEVELOPMENT OF THE FROG'S EGG [Cii. n

place of a large watery nucleus only a small mass of chromatic
substance, lying in the j)rotoplasm, is present. An achromatic
spindle appears, and the chromatin in the form of granules is
arranged at the equator of the spindle. The spindle lies at the
surface of the egg near the centre of the black hemisphere
(Fig. 11, A). It lies also in the centre of the fovea, which is
found on the surface of the egg. The fovea marks the former
position of the large ovarian nucleus, and although tlie nuclear
membrane of the original nucleus lias disappeared, and its
watery cavity has been encroached upon by the surrounding
protoplasm, yet the pigment has not penetrated very deeply
into this region. The eggs pass in this condition from the
body-cavity into the oviducts. Newport ('51) believed that,
owing to the close attachment of the oviducts at their inner
openings to the walls of the pericardium, at each contraction of
the heart the slit-like openings of the oviducts would gape open,
and any eggs in the vicinity might be forced into the mouths
of the tubes. Also, he thought that owing to the muscu-
lar movements of the body, and the resulting shifting of the
internal organs, the eggs sooner or later pass near the openings
of the oviduct, and are then carried into the tube. At any
rate, there seems to be not much ground for the older state-
ment that the mouths of the oviducts actually grasp the eggs
by a muscular movement like that of swallowing. According
to Nussbaum ("95), the eggs, wlien set free from the ovary into
the body-cavity of the frog, are carried into tlie open mouths
of the oviducts by the motion of the cilia of the ccelomic epi-
thelium. These cilia drive anteriorly any bodies lying free
in the body-cavity. If, for instance, eggs taken from one frog
be placed in the vicinity of the openings of the oviducts in
the body-cavity of another frog, they will be carried into the
open mouths of the oviducts by the action of the cilia in that
region.

The cilia do not cover the entire surface of the coelomic
epithelium, and there are certain recesses in the body-cavity
destitute of cilia. The eggs that accumulate in these recesses
will be sooner or later forced out into the general cavity as a
result of the alternate contractions and expansions of the ven-
tral musculature of the body-wall, as well as l^y the changes

Ch. II] POLAR BODIES AXD FERTILIZATION 17

produced by the filling and emptying of the lungs, and by the
movements of the heart.

Swammerdam's account in 1737 describes the passage of the
egg from the ovaries to the oviducts by way of the coelomic
space. Spallanzani in 1785 observed that the females of Bufo
igneus, isolated before union with the male, could still la}' their
eggs. One of the tree-frogs has its eggs in the uterus before
it unites with the male. On the other hand, Spallanzani stated
that females of the stinking toad if isolated while the eggs are
still in the ovaries will retain their eggs, but if separated after
having paired will then deposit their eggs. According to the
evidence of several authors, Rana temporaria when isolated will,
in certain cases at least, set free its eggs.

It has been suggested that the embrace of the male is me-
chanically necessary in order that the eggs may pass from the
ovary into the oviducts,

but this is certainl}' not „ '" ~^

always the case, and if
not necessary in one form , ^

is probably not necessary *!•*?

in others. The sexual
excitement set up by the
tight embrace of the male
may however be neces-
sary in some species for
the successful perform-
ance of egg-laying. The \^
eggs pass one by one n,
down the length of the
oviducts, ultimately to Fig. lO.— Egg iu jelly. (Aiter Schultze.)
reacli the lower portion

of the tube, the so-called uterus, A^'here the eggs accumulate.
If a frog is killed at the height of the breeding season, free
eggs are often found in the body-cavity, and a series of eggs
passing individually down the ovarian tubes, as well as an
accumulation of eggs in the uteri. In their passage through
the oviducts the eggs undergo certain internal changes and re-
ceive also their egg-coats. In the tubes of the oviducts the nu-
clear spindle divides, so that half of the original chromatin goes
c

18

DEVEi^OPMENT OF THE FROG'S EGG

[Cn. II

to one pole of the spindle, and half to the other. The spindle
has assumed, during this time, a radial position with respect to
the egg, so that we may speak of a distal and of a proximal or
central end (Fig. 11, A). The distal end pushes out into a
protrusion of protoplasm that has simultaneously formed at this

B

i

1 ^-.-

r

D

o

F

Fig. 11. — Extrusion of first polar body and fertilization of egg of Toad. (From
preparations made by Helen D. King.) A. First polar spindle. B. First polar
body extruded; second jjolar spindle present. C. Entrance of spermatozoon.
D. Male and female pronuclei. E. Apposition of two pronuclei. F. First seg-
mentation-spindle.

point of the surface of the Qgg. This protrusion of protoplasm
with its enclosed half of the nucleus gradually pinches off from
the surface of the egg, and there is thus formed the first polar
body (Fig. 11, B). The egg gets a thin layer of gelatinous

Ch. II] POLAR BODIES AXD FERTILIZATION 19

substance around it soon after entering the oviduct, i.e. before
it has reached the first part of the convoluted portion. Tliis is
the so-called chorion, — a thin investino- membrane which ad-
heres closely to the vitelline layer around the egg. During the
remainder of the passage through the oviducal tube the egg gets
two otlier distinct gelatinous layers (Fig. 10). The middle
layer of the three is, according to Newport, a watery layer of
considerable thickness. The outer gelatinous covering is also
thick and serves to stick the eggs together in a bunch, and even
to stick the bunches of eggs, when laid, to surrounding objects.

The si)awning of certain species of frogs takes place very rap-
idly, and by a single effort. Newport says that the process
takes place in a few seconds or less than a minute, and that all
the eggs that have accumulated in the uteri are laid at once.
When laid, the egg-cluster forms a rounded mass which is, at
first, scarcely as large as a walnut. The eggs then seem to con-
sist almost entirely of dark-colored "yelks" with thin gelatinous
envelopes. "Up to about this period the ova remain undisturbed
in the water in a mass as they are expelled, and lie indiscrimi-
nately, some with the dark and some with the white portion of
the yelk uppermost or horizontal. But during the time that
has passed since the ova have been in contact with the water,
the envelopes have imbibed fluid and expanded until these in-
vestments of the yelk have a thickness equal to about two-
thirds of the diameter of the yelk itself."

"The yelks, that have remained up to this time with their
white surface uppermost, now change their position spontane-
ously by a partial rotation of the whole mass oi each on its
axis, within the vitelline membrane, until the dark surface of
the whole is placed uppermost. Whether this change of posi-
tion is merely the result of expansion of the vitelline membrane
at this period, or whether it be also connected, as we may
fairly believe, with changes going on in the interior of the
yelk, I am not prepared to decide."

The Jelly of the Egg, and the Second Polar Body

The jelly around the frog's egg serves, no doubt, as a pro-
tection to the egg. The soft eggs are kept in spherical shape

20 DEVELOPMENT OF THE FROG'S EGG [Cn. II

and protected from injury from without. The slime protects
them from water-snails that will eat the eggs if they are shelled
out from the jelly. The jelly may also protect them against
water-birds. The eggs and young tadpoles seem, however, in
themselves to be distasteful to certain Crustacea (Bernard and
Bratuschek, '91).

This jelly has the physical peculiarity of allowing the sun's
rays to pass through, but hinders reflection of the rays from
the interior to the outside. The result is that in the sunlight
the mass of eggs is at a higher temperature than the surround-
ing water, and as the eggs of many frogs are laid in the early
spring, when the water is quite cool, this property of the jelly
helps to hasten their development.

Hertwig ('77) thought that a change takes place in the inte-
rior of the egg after fertilization, so that a difference in the
specific gravity of different parts of the egg is brought about.
Schultze ('87), however, pointed out that at this period the
egg contracts slightly from its vitelline membrane, and between
the egg and its membrane a fluid collects, that is probably
squeezed out of the egg itself. The egg, freed from its inner-
most coat which held it in place, then rapidly orients itself wnth
respect to gravity. Unfertilized eggs will also, after a time,
slowly rotate, and in these it can be seen that the separation
of the egg from its membrane is less perfect than in fertilized
eggs. "At the moment when the ovum is expelled from the
body, the envelope is merely a thin gelatinous layer, its entire
diameter being equal only to about one-sixth of the diameter
of the yelk. After it has been one minute in water, and begun
to imbibe and expand, it is then equal to about one-fourth of
the diameter of the yelk. At the end of ttvo minutes it is en-
larged to one-third, and in three minutes, to one-half the diame-
ter of this body. In four minutes, it exceeds three-fifths, and
in six minutes, two-thirds, and it continues to imbibe flnid and
expand at the same rate, until, at from ten to fifteen minutes,
it very nearly equals in thickness the whole diameter of the
yelk ; and at half an hour it is one-fourth greater than this.
At the end of three hours the membranes have acquired nearly
their full size."

'•The expansion of the envelope is greatly retarded at the

Ch. II] POLAR BODIES AND FERTILIZATION 21

end of the third or fourth hour, until after cleavage of the yelk
has taken place, when it again proceeds, but much more slowly
than at first." ^

In Rana fusca the extrusion of the second polar body takes
place one half-hour after fertilization, and the process can be
seen under a low magnifying glass or even with the naked
eye. A whitish speck appears in the black hemisphere near
the point at which the first polar body was extruded. It is
necessary, however, to make sections of the Qgg to discover the
further changes that are taking place. Schultze ('87) has
given a careful description of the process. The nucleus that
remains in the egg after the extrusion of the first polar body
assumes once more a horizontal position, but does not go into a
restiny-stage (Fig. 11, li), i.e. the chromatic loops or threads
do not re-fuse into a network nor does a nuclear membrane
form. The chromatin arranges itself on a new spindle. The
latter then assumes a more or less radial position, and the
second polar body is extruded half an hour after the egg is
laid. It is probable that the second polar body is not ex-
truded under normal conditions until after a spermatozoon has
entered the egg.

One and a half hours after the egg is laid, another change
may be seen taking place. Near to or at the apex of the black
pole the egg is seen to flatten, and an accumulation of fluid
is found here between the egg and its vitelline membrane
(Fig. 10). At or near the centre of this flattened portion one
may see the fovea, and near or in it the polar bodies appear on
the flattened disc. This chamber formed between the flattened
egg and the inner membrane was seen by Newport and called
the "respiratory chamber." It may ultimately be as large as
one-sixth the diameter of the whole egg. Schultze points out
that it lies somewhat excentrically witli respect to the egg-axis
(Fig. 10). The clear fluid in this chamber has been supposed
to be the watery contents of the original large nucleus of the
Qggi which has been squeezed out of the egg. Very little
evidence has as yet been given to support this view. Some
of the older embryologists thought that this fluid represented

1 Newport ('51), p. 193.

22 DEVELOPMENT OF THE FROG'S EGG [Cn. 11

the original egg-nucleus itself, which was squeezed out of tlie
egg at this time. Now, however, since we know the com^^leto
history of the nucleus during this period, the suggestion of its
entire loss by the egg does not call for serious criticism.

Entrance of Spermatozoon and Copulation of

Pronuclei

The sperm of the male is poured out into the water, and
probably over the eggs themselves at the moment wlien they
are laid, and the spermatozoa begin at once to bore into the
jelly of the egg-mass (Fig. 10).

Kupffer has described the entrance of the spermatozoon into
the eggs of Bufo variabilis. When the head of a spermatozoon
touches the egg-membrane, the protoplasm of the egg draws
back slightly at the point of contact, but C[uickly returns again
to its first position. The period of penetration of the sperma-
tozoon from the moment of contact of the sperm-head until the
spermatozoon disappears into the egg, lasts in some cases from
one to one and a half minutes, in other cases only three-fourths
of a minute. Several spermatozoa were observed by Kupffer
to enter each egg.

Other spermatozoa reach the egg-membrane, but do not seem
to be able to enter the egg. In the regions where these sper-
matozoa lie, the surface of the egg rises up in small protuber-
ances. This process occurs about fifteen minutes after the first
spermatozoa have entered, and lasts about one or two minutes,
after which the protuberances sink back into the egg. The
spermatozoa in the regions of the j)rotuberances are left outside
the egg-membrane. This peculiar phenomenon is described by
Kupffer as a counter demonstration of the egg against those
spermatozoa that have not been able to enter. Eggs that have
been artificially fertilized show, when cut into sections, that one
hour after fertilization a dark pigmented streak is formed,
reaching from the pigmented coating of the egg into the yolk-
mass. The process takes place in the upper or dark hemi-
sphere, and regularly at one side of the centre of the dark field
near to the edge of the white border. The streak takes a
somewhat oblique course toward the centre of the egg. At

Ch. II] POLAR BODIES AND FERTILIZATION 23

the central end the dark streak is rounded, and encloses a clear
spot.

In this clear region one sees a distinct pronucleus about nine
microns (/z) in diameter. Eggs one and a half hours after
fertilization show that the pigmented streak has penetrated
deeper into the egg, and in the frog the male pronucleus has
enlarged to 32 by 22 fi (Fig. 11, D, for the toad).

At this stage another nucleus is present in the frog's egg,
and this lies not far from the end of the pigmented streak
(Fig. 11, D). This measures 22 /x, and has the same structure
as the male nucleus. These two nuclei are undoubtedly the
male and female pronuclei. We now know that the female
pronucleus has come directly from the original egg-nucleus,
which has, after extruding its two polar bodies, i^enetrated
once more deeper into the egg. The complete history has not
been traced in the frog, but there can be no reasonable doubt
as to what takes place. In the newt (and in the toad) the
history has been followed, and it is found that the female pro-
nucleus arises from the egg-nucleus after the extrusion of the
polar bodies.

In the next half-hour Hertwig has found that the nuclei
approach more nearly to each other, and the pigment-streak
penetrates deeper into the egg, the swollen end enlarges,
and the two large oval male and female pronuclei are then
found together in the swollen end of the streak (Fig. 11, E).
In a preparation of an older stage both nuclei have increased
in volume to 35 /x, and have flattened against each other. They
then fuse into one nucleus which measures 44 fx, (Fig. 11, F, toad).
The resulting nucleus, the segmentation-nucleus, is surrounded
b}' clear protoplasm and then by a pigment-coat. From the
segmentation-nucleus a streak of pigment extends to the dark
surface of the egg and marks the path of entrance of the
spermatozoon. All preparations after two and a half hours
showed the union of the two j)ronuclei.

If the jelly be examined after the eggs have been laid,
several or many spermatozoa can be seen boring their way
through the jelly toward the egg. Some will have reached
the inner layers, and still others lie in the outer coats (Fig.
10). It is probable that after one spermatozoon has succeeded

24 DEVELOPMENT OF THE FROG'S EGG [Ch. II

in forcing its Avay throngh the inner coat and into tlie Qgg,
changes then take place in the egg that prevent or make
difftcult the further entrance of other spermatozoa. The con-
traction of the Qggi noted above, may possibly have something
to do with the process. If, however, two or more sper-
matozoa should reach the surface of the egg at about the
same moment, it is not improbable that more than one might
enter. ^ Both may then pass toward the female pronucleus, but
in the frog it is probable that after one male pronucleus has
fused with the female pronucleus, the further progress of other
male pronuclei that happen to get into the egg is stopped.

It is sometimes said that the female pronucleus attracts t\\e
male pronucleus, but the approach of the two may be due to
changes in the protoplasm ; for the migration of the pronuclei
through the egg is probably in most cases brought about by
the protoplasm of the egg under the influence of the j^ronuclei,
and the pronuclei themselves are merely passively carried
along.

In the newt (Jordan, '93) it seems to be usual for more than
one spermatozoon to enter, but only one of these fuses with the
female pronucleus. The others subsequently degenerate and
go to pieces. In the eggs of other animals, as the starfish,
polyspermy, or the entrance of more than one spermatozoon
into the egg^ brings about disastrous results, causing irregular
division of the nucleus and subsequent irregularities in the
segmentation of the egg. In these eggs the field of action is
small, and the male pronuclei or their centrosomes mutually
influence one another and the female pronucleus. In large
eggs with much yolk, such as those of the Amphibia and of tlie
Sauropsida, the spermatozoa may be too far apart to affect one
another or the segmentation-nucleus, and after the fusion of
one male pronucleus with the female the movement of the
other male pronuclei towards the female pronucleus seems to
stop.

The head of the spermatozoon enters the egg to become the
male pronucleus. The tail of the spermatozoon is left at the

lit is probable that Kupffer's ('82) account does not apply to eggs under
normal conditions.

Ch. II] POLAR BODIES AND FERTILIZATION 25

surface of the egg, or if a part enter the egg it takes no share
in the subsequent changes. The middle piece of the sperma-
tozoon is now known to contain a body that phiys a most con-
spicuous part in many animals in the division or cleavage of
the egg. The middle piece enters with the head of the sper-
matozoon. It contains the centrosome, which divides, and
around each centre an elaborate sj^stem of rays develops. The
two centrosomes migrate to opposite sides of the segmen-
tation-nucleus, and between the two appears the spindle of
the first cleavage. In the frog the history of the middle
piece and centrosome, and the origin of the segmentation-
spindle have not yet been worked out.

CHAPTER III

EXPERIMENTS IN CROSS-FERTILIZATION

A NUMBER of attempts have been made to fertilize the eggs
of one species of frog with the spermatozoa of another species.
Rusconi experimented in 1840 with the toad {$ ) and the green
water-frog, Rana esculenta (9 ). Lataste in 1878 attempted to
cross-fertilize the eggs of different species of urodeles with
Felobates fuscus and P. cultripes.

Experiments of Pfluger and of Born

The most extensive and important work is that of Pfliiger ('82)
and of Born ('83). These investigators have made a large
number of experiments in crossing different races and species
of Anura. When the sperm of Rana fusca was placed with
the eggs of Bufo vulgaris, the eggs segmented and developed
as far as the " morula " stage, and then without exception died.^
Conversely, when the sperm from B. vulgaris was used with the
eggs of R. fusca, no result followed, not even the segmentation
of the egg (except in one experiment where two eggs out of one
hundred divided irregularly). Eggs of R. fusca placed in a
water-extract of the testes of R. esculenta remained unfertil-
ized. But eggs of R. esculenta placed with the sperm of R.
fusca developed regularly, with few exceptions, as far as the
blastula stage, and then died. Crossing various species of Tri-
tons gave no results. But eggs of Rana fusca were acted upon
by the sperm of Triton alpestris and Triton tpeniatus, inasmuch
as they began to show irregular cleavage-lines. Later they
died. The reverse cross gave no result.

Rana fusca and Rana arvalis are very similar in appearance,
but are apparently separate species. Cross-fertilization was

1 Pfluger ('82).
26

Ch. Ill] EXPERIMENTS IN CROSS-FERTILIZATION 27

here possible (R. fusca, S , R. arvalis, ? ). Tadpoles developed
from the crossed eggs, and some of these ultimately transformed
into frogs. Pfliiger got similar results with the same species, and
also found that the reverse cross (R. fusca, 9 , and R. arvalis, $ )
gave no result. Born found that the eggs of Bufo cinereus
could readily be fertilized with the sperm of Bufo variabilis.
All the eggs segmented regularly, the larvae left the jelly, and
developed into frogs.

In respect to the closeness of the relation between the species,
Born says that we can be quite certain that the two species of
Rana arvalis and R. fusca are much more nearly related than the
two species of Bufo. The success of cross-fertilizing depends
apparently less on the degree of relationship, as shown by the
similarity of color and habits, than on the similarity of the male
sexual products (Pfliiger). Although R. fusca and R. arvalis
seem to be very closely allied species, they have very different
spermatozoa ; in fact, the spermatozoa are as different as the
spermatozoa of R. fusca and R. esculenta.^ The two species of
toads (Bufo) have very similar spermatozoa, which differ only
in size, but this difference is so slight that, were the two kinds
mixed together, one could scarcely distinguish between them.
It is apparently owing to the difference in form of the sperma-
tozoa of the R. fusca and R. arvalis, and to the similarity of the
spermatozoa of B. cinereus and B. variabilis that the results are
due.

Pfliiger has made a large number of reciprocal crosses between
different races of R. fusca. ''The different races are as fertile
inter se as are individuals of the same race." Pfliiger concluded,
after comparing the results of all of his experiments on cross-
fertilization, that in general tJiose spermatozoa are most successful
for purposes of cross-fertilization that have the thinnest and most
pointed heads. That in general those eggs are most easily fer-
tilized that belong to species having spermatozoa with thick
heads. The results, then, he thought, depend largely upon me-
chanical conditions ; for where the head is small and pointed, the
spermatozoon can bore its way more successfully into the eggs

1 R. arvalis and R. esculenta have similar sperm. Born and Pfliiger found
tliat the crossed eggs segmented irregularly, and that later the embryos all died.

28 DEVELOPMENT OF THE FROG'S EGG [Cii. Ill

of its own and of other species. If the head is large, the sper-
matozoon can force its way only into those eggs that are adapted
to spermatozoa with large heads. For instance, the sperma-
tozoa of R. fusca have thinner heads than any others, and
the head is, moreover, very pointed. These spermatozoa can
fertilize eggs of nearly all other species (R. arvalis, R. escii-
lenta, B. communis). Conversely, the thick-headed spermatozoa
of R. arvalis and the blunt-headed spermatozoa of R. esculenta
cannot get into the eggs of R. fusca.

The spermatozoon of B. communis, which has a very pointed
but sometvhat larger head than that of R. fusca, appears never-
theless to be able at times to penetrate the eggs of R. fusca and
to fertilize them. That the spermatozoon of Triton can enter
the eggs of R. fusca is explained very easily when we remember
that the sharp thin head of the Triton spermatozoon is best
adapted of all species to penetrate any egg. We see, too, that
the thick-headed spermatozoon Avith a blunt anterior end, such
as those of R. arvalis and R. esculenta, cannot fertilize the eggs
of any other species. And finally, to confirm the conclusion,
we find that these two species, R. arvalis and R. esculenta,
which have large-headed spermatozoa, are alone capable of
reciprocal crossing. Pfluger believed that the eggs have the
greatest capacity for cross-fertilization at the height of the
breedinaf season, and the same statement holds, but in a much
less degree, for the spermatozoa.

Experiments on Other Forms

Hertwig has objected to Pfliiger's conclusions on the ground
that the eggs of the sea-urchin are much more capable of cross-
fertilization after they have begun to suffer change either from
being kept some time in sea-water, or from the application of
drugs. He thought that the frogs kept by Pfiiiger had been
also under artificial conditions. Further, Hertwig concluded,
from his results on sea-urchins, that the possibility of crossing
does not depend entirely upon the external conditions, but to
a large extent upon some unknown property of the egg. Eggs
in good condition are able to prevent the entrance of foreign
spermatozoa, but as soon as they begin to lose their irritability,
they can no longer resist the entrance.

Ch. Ill] EXPERniENTS IX CROSS-FERTILIZATIOX 29

Born obtained some interesting results as to the relations
existing between the number of spermatozoa in a fluid-extract
of the testis and the power of the fluid-extract to fertilize eggs.
He insists that in some cases there is a necessary connection
between the two. It is far from clear how this is possible, and
the result may depend on other causes which are introduced
along with the solutions employed. ^Moreover, the further
question of polyspermy of such eggs complicates the results.
Born believes that many cases of irregular segmentation of
crossed eggs are due to the entrance of several or many sper-
matozoa into the egg, which act as centres for protoplasmic
accumulations. Such a segmentation he calls "barock" seg-
mentation. On the other hand, Pfliiger suggests that the
irregular cleavage of certain of the crossed eggs is the result
of the disintegration of the male pronuclei, so that the chro-
matin is scattered, and then acts on the protoplasm, producing
an irregular division.

Recent results have shown that polyspermy is a normal oc-
currence in some amphibian eggs, and, despite the presence
of several spermatozoa, normal cleavage and normal embryos
result. The changes that take place within the cross-fertilized
eggs must be more carefully studied before a final decision can
be reached in regard to the meaning of some of the experiments
described above.

We must not confuse two factors that enter into the problem
of cross-fertilization. On the one hand, the spermatozoon may
not be able to push through the gelatinous coatings of the egg,
or it may not be able to bore through the outer surface of the
egg itself, or it might be unable to enter the protoplasm if the
latter were entirely free from its coats. ^ On the other hand,
even if the spermatozoon could successfully enter and combine
with the female pronucleus, it does not follow that the egg
would deVelop. We now know that so many factors enter
into the problem of fertilization of the egg that it is not sur-
prising when we find that two pronuclei that have ever so
slight differences are not able to carry out the complicated
machinery of cell-division and development.

1 As in the case of naked pieces of protoplasm of the egg of species of sea-urchins.

30 DEVELOPMENT OF THE FROG'S EGG [Cii. Ill

The eggs of the starfish can be fertilized by the spermatozoa
of the sea-urchin, — ^ forms much more different than any two
species, genera, or even families of frogs, and the early stages
of segmentation, and the formation of a swimming blastula and
gastrula may be passed through ; but the later embryonic devel-
opment is not carried out, and after a time the gastrulas die.^

Hertwig's experiments ('77) on polyspermy in the eggs of
echinoderms show that when several spermatozoa enter the
same egg a karyokinetic spindle is formed around each of the
resulting male pronuclei and many or all of the pronuclei
divide. Often the spindles are so near together that they
mutually influence one another and most complicated karyo-
kinetic figures result. Subsequently the protoplasm breaks up
around the pronuclei in a most irregular way, and generally
such eggs do not give rise to even the earliest stages of devel-
opment. The phenomenon is so similar to the '• barock " seg-
mentation of the frog's egg that it seems possible that in the
latter the result is brought about in the same way as in the
echinoderms.

Experiments of Rauber and of Boveri

Rauber, in 1886, tried to carry out the following interesting
experiment. The segmentation-nucleus of a frog's egg, one
hour after fertilization, was removed by means of a fine pipette.
The same "process was carried out with a toad's egg. The
nucleus of the toad's egg was then placed in the frog's egg
that had had its nucleus removed, and the nucleus of the
frog's egg was placed in the toad's egg. Unfortunately,
neither egg developed. The results of such an experiment
would be of the greatest importance if the experiment could
be successfully carried out ; for in this way we should hope to
discover whether the characters of the embryo come from the
nucleus or from the protoplasm of the egg.

Boveri, in 1889, made somewhat similar experiments with
the egg of the sea-urchin. When the eggs are shaken in a
small tube, they are broken into fragments, some with nuclei
and others without. When a sufficiently large non-nucleated

1 Morgan, '93, Anat. Anzeiger.

Ch. Ill] EXPERIMENTS IN CROSS-FERTILIZATION 31

fragment is penetrated by one spermatozoon, the fragment
develops. Such a fragment contains only half the number of
chromosomes of the normal fertilized egg.'^ Boveri isolated
some of these fragments, and said that they give rise to small
embryos normal in structure. Boveri stated, further, that if a
non-nucleated fragment of the egg of one species of sea-urchin
is entered by one spermatozoon of another species, the result-
ing larva is like the larva of the father (i.e. it is like the larva
of the individual from which the spermatozoon comes). If
this result should prove true,^ it would show that the nucleus
and not the protoplasm determines the character of the larva.

1 Morgan, '95, Anat. Anzeigcr.

2 Seeliger ('95) and myself ('95) have repeated Boveri's experiment and have
tried to sliow that the evidence on which Boveri based his conclusion in regard
to the paternal character of the crossed larva is insufficient.

CHAPTER IV

CLEAVAGE OF THE EGG

"When the eeof comes to rest in its membranes after fertiliza-
tion has taken place, it will be found that the egg-axis assumes
an oblique position with respect to the vertical. The degree of
obliquity may be different for the eggs of different species of
frogs, but in some species it is carried so far that, when the
egg is looked at from above, a crescent of the white hemisphere
can be seen on one side of the egg. Roux has stated that the
declination of the egg-axis takes place only after the entrance
of the spermatozoon, and toward that side into which the sper-
matozoon has penetrated. 1 He was able to determine this by
artificially fertilizing the egg at definite points. By means of
a small pipette, water containing spermatozoa was brought in
contact with the jelly somewhere near the upper hemisphere of
an egg. Presumably the spermatozoon will then take the short-
est path to the egg. Roux found that the egg after a time gen-
erally rotated on its axis toward the point at which the artificial
fertilization was supposed to have taken place.

Normal Cleavage

The first furrow appears on the egg about two and one half
to three hours after fertilization, the time depending in part on
the temperature of the water. A rather wide furrow appears
in the flattened area near the black pole, and rapidly extends
over the upper surface of the egg, and then moves more
slowly over the lower or white surface. The sides of the
furrow are often wrinkled, probably a mechanical result of the

1 Roux believes the obliquity to be a usual phenomenon after fertilization
for some species ; in others the obliquity is only occasionally seen. Schultze
finds it to be as much as forty-five degrees in Rana fusca.

32

Ch. IV]

CLEAVAGE OF THE EGG

33

infolding of the outer harder crust of the egg*. These wrinklevS
are best seen in the upper hemisphere ; subsequently they dis-
appear. It will be found on cutting in two an egg in the
process of cleavage that the furrow is also extending throvgh

B

Ky^;:<^

D

G

H

I

Fig. 12. — Segmentation of esg and formation of blastopore (H, I). A. Eight-cell
stage. B. Beginning of sixteen-cell stage. C. Thirty-two-cell stage. D. Fortj'-
eight-cell stage (nnusually regnlar) . E, F. Two sides of same egg in later cleavage.
G. Still later cleavage. H. Dorsal lip of blastopore. I. Circular blastopore (with
lower pole toward observer).

the protoplasm of the egg, i.e. dividing the contents into two
parts. When the superficial furrow has encircled the egg., the
substance also has been divided.

D

34

DEVELOPMENT OF THE FKOG'S EGG

[Cii. IV

If a series of sections be made throiig-li the egg at different
stages ill the i)roeess of eleavage, we should see that prior to
the division o( cdvh bhistoniere the nneknis had divided into
two jiarts. This takes phice by the ordinary process of indirect

D

G H

Fir.. 13. — Seg'ineutation of egg (two, eight, sixteen, and thirty-two cell stages, after
^I. Sehultze). as seen from above. A. Two-cell stajre : Ijesiinning of second fur-
rows. B. Eiiiht-eell stage, with cross-furrow. C, D. F, G. Sixteeu-cell stages.
E. Eight-cell stage (regular type). H. Thirty-two-cell stage.

or karyokinetic division. Half of the chromatin passes to one
pole of the nuclear spindle, and the other half to the other pole.
As the spindle elongates, it carries with it the surrounding pig-

Cii. IV] CLEAVAGE OF THE EGG 35

ment. The first cleavage-plane always passes directly between
the separating lialves of the segmentation-nucleus.

There is an infinite number of possible planes through which
tlie first cleavage might divide the egg into equal portions.
What, then, determines the particular plane taken? We can
think of this plane as determined by external conditions, or
by the internal structure of the egg, or by a combination
of the two. In the first place, it seems probable that at the
first division of the segmentation-nucleus each resulting half
will sret half of the chromatin of the male and lialf of the chro-
matin of the female pronucleus. The first plane of division
must therefore j!?ass at right anfjlen to the plane of apposition of
the two pronuclei. That is to say, it will also pass through the
path of penetration of the spermatozoon (the male pronucleus),
and therefore approximately through the point at which the
spermatozoun has entered. This, according to Koux, is what
actually takes place. Moreover, since the egg lias rotated as a
whole in the direction of the point of entrance of the sperma-
tozoon, the first cleavage will pass exactly through tlie liighest
point of the white crescent, as seen from above.

On the other hand, there is no direct evidence U) show
that the two apposed pronuclei retain throughout subsequent
changes the position of first apposition, and there is much to
show that in the frog's egg, as well as in other eggs, the divid-
ing nucleus, or the direction of its spindle, is very susceptible
to modifications in the surrounding conditions.

There is also some evidence to sliow that the declination of
the axis of the frog's egg is not necessarily determined by the
entrance of the spermatozoon, but by the arrangement of the
internal constituents of the egg itself. If, therefore, it could
be sliown that the declination is present in unfertilized eggs,
and that in fertilized eggs the plane of first cleavage passes
more or less through the highest point of the white crescent,
then we should conclude that the plane of first cleavage is pre-
arrano-ed in tlie esfCf- It would follow as a corollary that the
nuclear spindle orients itself with respect to the egg.

There is direct evidence to show that in the newt some such
process as this does take place. Jordan ('9-3) has shown that
the spermatozoon may enter at any point of the surface of the

36 DEVELOPMENT OF THE FROG'S EGG [Ch. IV

upper hemisphere, yet the phxne of first division is always
across the long axis of the egg. Hence, it is fair to assume
that tlie segmentation-spindle does so orient itself after the
fusion of the male and female pronuclei that lialf of the
male and half of the female chromatin are carried apart in
the direction of the long axis of the egg., whatever may have
been at first the position of apposition of the two pronuclei. I
have dwelt on this point at some length because it is one of
great importance for our understanding of the relation between
egg and embryo ; and because it is much to be desired that the
present state of doubt should be cleared away.

After the protoplasm has divided into two equal parts, the egg
" rests " for a time. During the division-period the hemispheres
or blastomeres round up to some extent ; but as soon as the
division is completed they flatten against each other, so that the
cleavage-plane is not so distinctly seen on the surface of the egg.
The same process of flattening generally takes place also when
the dividing egg is brought into preserving fluids.

During the time of division we may speak of each blastomere
as tending to become itself a sphere, but, owing to the lack of
room, the rounding of the two parts is very imperfect. In other
eggs (e.g. the eggs of the sea-urchin), where it is possible to
remove the egg-membranes, it has been found that then each
of the blastomeres approaches more nearl}- the spherical form,
or even becomes a complete sphere. We see from this that the
external conditions may at least modify the form of cleavage
of the egg.

It is sometimes said that during the division the two new
parts or blastomeres tend to repel each other until after the
division is completed, and to attract each other after the divi-
sion is finished. Such a statement is, however, of little value, and
may convey an entirely wrong impression of the changes taking
place. One thing seems to be certain, that during the division
of the egg the spheres or cells have an influence on one another.
Whether unseen protoplasmic connections weld them together,
or whether it is merely a question of contact action., has not yet
been fully determined. ^

1 See Koux's experiments on cytotaxis ('90).

Ch. IV] CLEAVAGE OF THE EGG 37

The nucleus of each Mastomere durmg the resting-period
undergoes a series of changes, the so-called reconstructive pro-
cess taking place. The chromatin-granules or chromosomes
are again surrounded by a nuclear membrane, and the granules
fuse into a thread or network. At the next division of the
egg the nuclear chromatin is again set free in the protoplasm
by the absorption of the nuclear membrane. A spindle is
formed and the chromatin in each cell is again exactly halved.

The second cleavage-furrow appears about three-quarters of
an hour after the appearance of the first. Each of the two
blastomeres divides in a plane at right angles to the preced-
ing division. The furrows begin, generally simultaneously, in
the upper hemisphere of each of the first two blastomeres, and
})ush toward the lower pole (Fig. 13, A). The upper and
lower ends of these new cleavage-planes are sometimes exactly
opposite to each other, so that the effect is as though the whole
egg had been divided by a single furrow in a plane at right
angles to the first. In many cases, however, the new planes of
division are not quite opposite, but reach the upper and lower
poles of the egg at different points along tlie first plane
of division. A " cross-line " is thus formed. The same re-
sult may be brought about even subsequent to division by a
shifting or readjustment of the blastomeres on one another.
As a rule, when a cross-line occurs in the upper pole, another
one is formed in the lower pole, and the two stand in space at
right angles to each other, as is shown in the diagrammatic
reconstruction in Fig. 14, A. The same result can be obtained
by compressing four clay spheres together until a single sphere
results. It will be found in such a model that the cross-lines
above and below are generally at right angles to each other.

The third furrows come in at right angles to the preceding
planes of division, and are therefore horizontal (Fig. 12. A).
The third planes of division do not lie at the equator of tlie
egg, but, taken together, form a small circle in the black hemi-
sphere or on the border-line between the black and white areas.
Above there are four smaller dark blastomeres and below four
larger white blastomeres. The four upper blastomeres are of
approximately the same size, but in some species of frogs it
seems that one is a little smaller than the rest, one is somewhat

38

DEVELOPMENT OF THE FROG'S EGG

[Ch. IV

larsfer, and two are intermediate in size. The smallest blasto-
meres of the upper four ahvays lie nearest the summit of the tvhite
crescent; the largest is its vis-d-vis. If we think of the third
planes of cleavage as lying in a single plane not quite at right
angles to the first and second, but tilted a little, we get a clearer
conception of the conditions present. The fourth cleavage-
period comes in from a half to three-quarters of an hour later.
As an idealized form, we may think of the new planes as form-
ing two great circles at right angles to each other, and lying
vertically and between the planes of the first and second cleav-

B

Fig. 14.— a. Diagram of four-cell stage to show cross-line. B, C. Sixteen-cell stage

of two eggs. (After Rauber.)

ages (Fig. 13, C). This regular form is rarely if ever attained,
and the greatest amount of variation is found to exist.

Remak said that frog's eggs divide much more regularly
when carried in from places wher5 tbey were normally laid.
If allowed to stand quietly after being laid, they soon begin to
divide irregtdarly. Vogt has also observed that those eggs of
the salmon develop most regularly that have been kept in
motion. It does not seem probable, however, that the motion
itself could have anything to do directly with the matter ; but if
the egg be not supplied with a sufficient amount of fresh water.

Cir. IV] CLEAVAGE OF THE EGG 39

etc., it might no doubt segment irregularly, or it maj' be that
the motion equalizes the external conditions so that the eggs keep
a more nearly spherical shape and hence divide more regularl}-.

Max Schultze ('63) and Rauber ('82) have made the most
careful study of the variations in the planes of division of the
fourth cleavage. In Figs. 13, D, F, G, and 14, B, C, are shown
the upper hemispheres of several eggs. If we examine the
position of the planes of the last (fourth) divisions, we see that
in the upper hemisphere each new cleavage-plane fails generally
to reach the black pole of the egg, but passes to one or to the
other side. In the lower hemisphere tlie new planes fall far
short of the white pole.

Occasionally we find eggs in the upper hemisphere of which
one or more of the fourth planes reach the black pole itself,
and, therefore, lie more nearly radial in position (Fig. 13, C).
In other cases, however, one or more of the new fourth cleav-
age-lines may even be nearly in the horizontal plane. In the
lower pole also there is much variation, and occasionally a blas-
tomere is divided into a very small and a very large part, owing
to the sudden turning aside of the new cleavage-line, so that
it meets one of the first two cleavage-planes before it lias
extended far into the lower hemisphere. Other eggs at this
same stage show a strictly bilateral arrangement of the cells
in the upper hemisphere. In Fig. 13, D, F, G, we see that
the fourth cleavage-planes have met the same furrow (first or
second). Also in Fig. 14, B, a bilateral symmetry is present,
formed in a somewhat different way, as the figure shows ; and
in this egg the lower hemisphere also is symmetrically divided.

During the fifth cleavage-period the irregularities in the
division of the cells is generally so great that we cannot speak
definitely of any special direction of the new planes. Never-
theless there is a tendency for some of the new furrows to
come in at right angles to the last planes of division. There-
fore, many and occasionally all of the new fifth cleavage-planes
are horizontal (Fig. 12, C). The eight cells in the upper hemi-
sphere divide into equal or nearly equal parts, but the eight
blastomeres of the lower hemisphere divide unequallv into eight
upper smaller blastomeres containing pigment, and eight lower
blastomeres which are the white blastomeres around tlie lower

40

DEVELOPMENT OF THE FROG'S EGG

[Ch. IV

pole. The division of the lower eight blastomeres is sometimes
so regular that a circle of eight dark cells is formed around the
equator of the egg (Fig. 12, C). After this division thirty-
two cells are present. At this period the distribution of the
cells over the dark and light regions of the egg is such that
the cells on the side of the egg showing the light crescent above
are smaller than the corresponding cells on the opposite side.

B

Fig. 15. — Stages in the segmentation (A, B, C) of the egg, and the beginning of
gastrulation (D). AR. Beginning of archeuteron. SG. Segmentation-cavity.

Up to the present time all the divisions of the cells started
on the outside of the egg or blastomeres and progressed inward.
As early as the eight-cell stage, a cavity appeared between the
cells in the upper hemisphere of the egg. This is the "seg-
mentation-cavity." It is filled with an albuminous fluid which

Cii. IV] CLEAVAGE OF THE EGG 41

probably comes entirely, or in part, from the surrounding cells.
This cavity gets larger and larger as development proceeds
(Fig. 15, A). If an egg be cut open after the thirty-two-cell
stage, it will be found that many, perhaps all, of the cells or
blastomeres are undergoing division into outer and inner cells
(Fig. 15, B). We may speak of this process as a delamination.
Before the egg has divided into sixty-four cells, as seen on the
surface, this delamination into inner and outer cells has in most
cells taken place.

The cell-divisions now proceed more rapidly and with great
irregularity. The rln'thm also soon becomes lost, so that
while some cells are dividing others are resting. Not only
have the outer blastomeres continued to divide at the surface,
but also below the surface of the egg new blastomeres are beinsf
cut off from the outer cells; the inner blastomeres also continue
to divide. In the upper part of the egg a large segmentation-
cavity forms. Its roof is covered by several layers of small
deeply pigmented cells, its sides b}' larger cells, and its floor is
formed by the large whitish yolk-bearing cells (Fig. 15, C).

If the surface of the egg be carefullv examined during- these
later stages, it will be found that the cells over one side are dis-
tinctly smaller than those over the opposite side. We see that
the side of the egg containing the most pigment is made up of
larger cells. In Fig. 12, G, H, the opposite sides of an egg are
shown, and here the less pigmented cells are seen to be smaller
than the cells in the same position on the other side of the egg.
Sections show, moreover, that this difference in size is not onl}-
found on the surface of the egg, but also in the interior as well.
During the early periods of cleavage the egg has become
neither more nor less pigmented on its surface, and has retained
the same distribution of pigment as in the unsegmented egg.

Besides the variations in the cleavage noted above, others are
more rarely found that depart much further from the usual
typical forms. The first furrow, for instance, may divide the
egg into very unequal parts. The second furrow may appear
before the first has reached the lower pole. The third furrows
may stand vertically., passing from near the upper pole into the
lower hemisphere, i.e. the third furrows occupy the position of
the fourth furrows of the usual type of cleavage.

42 DEVELOPMENT OF THE FROG'S EGG [Ch. IV

Correspondence of the First Cleavage-plane and the
Median Plane of the Embryo

If the egg when in the two-cell stage be fixed ^ so that it can-
not rotate in a horizontal plane, and if such an egg be carefully
watched until the moment when the medullary folds have just
appeared, it will l)e found that the position of the plane of first
cleavage corresponds approximately, or even exactly, to the
median plane of the body of the embryo. This experiment
was first made by Newport in 1851, sul)sequently by PflUger
('83), and Roux ('85), and later by other workers.^ If, how-
ever, during the subsequent cleavage-periods, i.e. during the
eight and sixteen cell stages, etc., the positfon of this plane be
kept in mind, it will be found that the later blastomeres from
one or the other side often pass over the imaginary plane that
corresponds to the plane of first division. Striking, therefore,
as is the coincidence of the first plane of cleavage and the
middle plane of the embryo, it remains to be proved, I think,
that there is any direct causal connection between the first
cleavage-plane and the median line of the body. It may be
that the two phenomena are coincident because the internal
arrangement of the egg that determines one may also, but
independently, determine the other. In the newt Jordan ('93)
has shown that the first plane of cleavage corresponds approxi-
mately to the cross-plane of the body. That is, the first two
blastomeres correspond to the anterior and posterior parts of the
body respectively. He suggests that the shape of the egg-cap-
sule of the newt may be the cause determining the plane of first
division. Some other factor than that of the position of the
first plane of cleavage seems to determine the position of the
embryo on the egg, for in the teleost's egg, where the sym-
metry and bilaterality of the cleavage is even more sharply
marked than in the frog or newt, there seems to be no relation
at all between the first cleavage-planes and the planes of the
adult body. 3

1 For method, see Pfliiger ('83), Roux ('85), and Morgan ('91).
■^ Rauber ('86) has later contradicted these results, but it is probable that
there is an error in his experiment.
3 Clapp ('91), Morgan ('93).

Ch. IV]

CLEAVAGE OF THE EGG

43

Roux's Experiments with Oil-drops

The arrangement assumed bj the blastomeres after each
cleavage has attracted much attention. A system of soap-
bubbles, or of balls of clay compressed into a sphere, gives
somewhat similar figures. In this connection lioux has made
a most instructive series of experiments. A small wine-glass is
half filled with dilute alcohol and then sulhcient oil is poured
in to form a large drop. A stronger (lighter) alcohol is now
poured on top of the oil, which assumes a spherical or nearly
spherical shape. The drop lies suspended between the two

Fig. 16. — Systems of oil-drops. (After Roux.) In C, the lowest drops should be
marked A', B' ; and those next them A", B".

alcohols and its peripher}- just touches the walls of the glass. ^
It is possible to divide this sphere of oil into equal or unequal
parts by means of a glass rod and, if precautions are taken, the
drops will not for a time flow together. The drops tend each

1 Roux recommends olive or paraffiiie oil. I find that thick cotton-seed oil
gives as good or better results when suspended between fifty and seventy per
cent, alcohols. A smaller drop is to be used when more than two divisions are
to be made.

44 DEVELOPxMENT OF THE FROG'S EGG [Cii. IV

to become splierical, but the wine-glass holds them together
and their surface-tensions cause them to assume definite rela-
tions to one another. When the drop is divided into two
equal parts, the halves, if little compressed, arrange themselves
as shown in Fig. 16, A, and if much compressed, as shown in
Fig. 16, E.

If each drop is again divided,^ the resulting four drops
arrange themselves as shown in Fig. 16, B. A large central
cavity, similar to the segmentation-cavity of many segmenting
eggs, is present in the centre between the four drops.

If the drops had been first divided unequally, we should find
that the smaller drop, having a stronger tendency to become
round, caused the region of contact of the two drops to bend
in toward the larger drop. If each of these two drops is
again divided, the four parts arrange themselves as shown in
Fig. 16, D. The same result is brought about if we divide
the drop at first equally and then each of the products un-
equally (Fig. 16, D).

If we first divide a drop equally and then each of the two
unequally, but at different ends of each drop, as shown by the
dotted lines in Fig. 16, E, the resulting four drops arrange
themselves as shown in Fig. 16, F. Moreover, and this is a
point of much importance, it is a matter of indifference in
what direction the smaller drops are cut off. For instance,
if each of the first two drops is divided along the dotted lines,
a-«, a-a (Fig. 16, E), the result is the same as when the divi-
sion takes place along the line h-b, h-h. In either case the
drops arrange themselves as shown in Fig. 16, F. The two
larger drops come together at the centre of the system and
flatten somewhat against each other, producing a cross-line.
The two smaller drops are pushed out more toAvard the
periphery of the system.

If we adopt the method of lettering shown in Fig. 16, B,
we can follow more readily the further divisions. Divid-
ing equally two of the four drops of Fig. 16, B, we find the

1 In dividing the drops it is better to move the rod always from tlie centre
toward the periphery. The plane of the first division is indicated in the fig-
ures by the heavier line.

Ch. IV]

CLEAVAGE OF THE EGG

45

arrangement of the six resulting drops to be that shown in
Fis'. 17, A. We can write out the arrangement in the form
of equations: thus (in Fig. 16, B) a = A = B = b; and (in
Fig. IT, A) h' = h". a' = a".

Dividing unequally a and h so that a' is less than a" and V is
less than 5", the drops arrange themselves as shown in Fig.
17, B. A large central cavity is present in the centre of the
system.

If each of the four equal drops (Fig. 16, B) be equally
divided, the resulting eight drops arrange themselves as shown

/^

\ ^'

"' ;

N

' a"

>

^

/-

e.^

^

B

E F

Fig. 17.— Systems of oil-drops. (After Roux.)

in Fig. 16, C. A central cavity is present, but smaller than
when only four equal drops formed the system.

If we divide each of four equal drops (Fig. 16, B) unequally
so that a" is less than a' and h" is less than h\ also A" is less
than A' and B" is less than B' , the resulting eight drops
arrange themselves as shown in Fig. 17, C. The four larger
drops come together in the centre, pushing the smaller drops
more toward the periphery of the sj^stem.

If we divide four equal drops (Fig. 16, B) so that a" is less

46 DEVELOPMENT OF THE FROG'S EGG [Cii. IV

than a', b" is less tliaii 5', but A' is less than A" and B' is
less than B", then the drops assume the form shown in
Fig. 17, D.

If we divide four equal drops (Fig. 16, B) so that a' is less
than a", h" is less than h\ A" is less than A', and B' is less than
B", the resulting eight drops arrange themselves as shown in
Fig. 17, E. In this system it is instructive to note how far
the first division-plane is drawn out of its straight course as
a result of the shifting of the drops on one another. It is not
unusual for one of the drops to glide into the centre of the
system, as shown in Fig. 17, F. This produces a more stable
arrangement than when a large central cavity is present.

Most of these systems are found also in the segmenting frog's
eg^, as can be seen by comparing these figures of the oil-drops
with the figures of the segmenting frog's egg (Fig. 13) by ^Nlax
Schultze made in 1863. Rauber has also given figures showing
arrangements of the upper eight blastomeres (Fig. 14), like
the systems of oil-drops shown in Fig. 17, D and E.

A careful comparison between the systems of oil-drops and
the arrangement of the blastomeres of the frog's Qgg shows,
as Roux points out, that while in many cases the agreement
is perfect, yet occasionally the ]>lastomeres assume an arrange-
ment that oil-drops of the same size would not assume. For
instance, Roux figures an arrangement of the blastomeres like
that of Fig. 17, C, but here the blastomere corresponding to a'
is less than a" and A' is less than A" . In this egg the smaller
blastomeres meet in the centre, but this never occurs in the
system of oil-drops.

Roux removed a part of a blastomere so that it became sud-
denly smaller. A new arrangement ought now to have taken
place among the blastomeres if they conformed entirely to the
laws regulating the oil-drops. In one case where four blasto-
meres were present, the blastomere that had been reduced in
size did move out more toward the periphery of the system,
and the two neighboring blastomeres pushed in more toward
the centre to form a cross-line. In other experiments, how-
ever, the blastomeres did not rearrange themselves in con-
formity with the systems of oil-drops. For instance, in one
experiment in which material was drawn out of one of the first

Cii. IV] CLEAVAGE OF THE EGG 47

four blastomeres, the inner end of the reduced blastomere
retained its central position. In another instance material was
taken out of that one of the four blastomeres that had alread}-
made a broad cross-line with its vis-d-vis. Although this
blastomere was much reduced in size and made smaller than
any other blastomere of the system, yet it retained the same
cross-line as before ; i.e. it was not pushed out to the periph-
ery. Even when the experiment was made at the time of
appearance of the second cleavage, the newly fornung blasto-
meres did not in all cases adjust themselves in agreement with
the laws regulating the oil-drops.

These results show that the conditions present in the frog's
eo-e do not allow the blastomeres to assume always the arrange-
ment shown by the same number of oil-drops having the same
relative size. Roux points out several differences in the two
cases. The walls of neighboring blastomeres seem to stick
together, and this would prevent the blastomeres from gliding
freely over one another should any change take place to disturb
the equilibrium. Moreover, the blastomeres are living con-
tractile bodies, and through their own internal activity may
interfere with the mechanical tendencies of the system. The
nature of the surface of each blastomere and the sort of changes
taking place in the surface may also affect the arrangement.

It will be seen then, as has been said, that there may be fac-
tors present in the frog's egg that so influence the arrangement
of the blastomeres that the systems do not always conform to
those of the oil-drops. Nevertheless, the results from the latter
give us an ideal scheme showing the effect produced by one set
of factors, — that of surface-tension. It seems highly probable
that surface-tension is also an important factor in the segment-
ing egg, but other conditions present prevent its free play.

Historical Account of the Cleavage of the Frog's

Egg

The earliest observations on the segmentation of the animal-
ovum were made upon the frog's egg. Swammerdam ('37)
saw, but did not understand, the first cleavage-furrow of the
egg. Spallanzani, in 1785, observed the first two furrows cross-

48 DEVELOPMENT OF THE FROG'S EGG [Cii. IV

ing each other at right angles. Prevost and Dumas, in 1824,
gave for the first time a definite description of the cleavage of
the frog's egg. They described the first furrow beginning in
the black hemisphere and stretching out into the white hemi-
sphere. They saw, moreover, the small lateral creases or folds
along the edges of the first cleavage-furrow. The second fur-
row, they said, cuts the first at right angles. When the dark
hemisphere is divided into four segments, they saw that then
a third equatorial furrow forms near the boundary of the two
hemispheres. The next furrows, they said, appear parallel to
the first.

Rusconi ('26) observed that the furrows were not simply sur-
face-lines, but cut up the yolk into separate parts, producing
finally a large number of small pieces, which he believed were the
elements from which the different parts of the body developed.
Von Baer's description of the jDrocess, in 1834, is much more
exact than the accounts of his predecessors. His interpretation,
too, is much clearer and nearer to the truth. He said that the
advance of the first furrow into the lower hemisphere goes on
as though it were overcoming great difficulty. The tearing
apart of the yolk into halves is brought about as a result of a
living activity, and the power to divide the ovum does not reside
only in the surface of the ovum, but extends throughout the
whole mass. Von Baer noticed that after the division the
cross-diameter of the egg is greater than the vertical diameter
in the proportion of six to five, and he said that the difference
would be greater were it not for the egg-membrane. The ten-
dency, he said further, is to form two spheres which are, how-
ever, compressed against each other by the membrane. Since
the division of the white hemisphere progresses more slowly,
and since the third division is nearer to the upper hemisphere,
we can understand why the dark portions are always smaller
than the white portions. When the surface appears again
smooth (owing to the smallness of the portions into which the
ovum has divided), the egg is very distinctly larger than at
first. A^on Baer concluded that material is taken up from the
outside to form the albumen, and hence to enlarge the ovum.
He interpreted the process of cleavage as the self-division of the
individual to form innumerable smaller units. In the later

1

Cn. IVJ CLEAVAGE OF THE EGG 49

stages these smaller bodies fuse by a vital process into a new
whole, and a new individual is thus produced from the frag-
ments of the first.

Schwann and Schleiden promulgated the cell-theory in 1838-
1839. This produced an effect on all subsequent interpreta-
tions of the segmentation of the frog's egg. The main })oints
to settle were : first, wliether the process of cleavage is a pro-
cess of cell-division, i.e. whether the egg is a cell that divides ;
second, whether the bodies that result from the segmentation
of the egg pass over into the cells of the embryo. The search
for the nucleus, before and after the process, also occupied the
attention of workers on the subject. Bergmann ('-11) was the
first to treat the process of cleavage from the cell standpoint.
The first divisions of the egg did not produce true cells, he said;
yet as the results of these divisions went over directly into the
cells of the embryo, therefore the division of the batrachian
egg is the introduction of cell-formation into the yolk. Later,
he said that the yolk may be thought of as strongly disposed
to form cells, but that nuclei are wanting. Reichert's in-
terpretation ('46) was a step backwards. Kolliker, in 1843,
described the segmentation-spheres as without a membrane
and containing spore-like bodies which multiplied endoge-
nously. When these bodies are set free, he thought, they
become the cells from which the tadpole is built up. Cramer
('48) thought that the early cleavage-spheres formed mem-
branes (cell-walls) and were the progenitors of the true cells
of the body. Remak ('50-55) argued that the cleavage-pro-
cess was the beginning of cell-division, and that the products
resultino- from division formed the cells of the embryo. This
statement marked a distinct advance and is the standpoint
taken at the present time. ^Moreover, Remak thought it highly
probable that there was a continuity of the original egg-nucleus
with the cleavage-nuclei. Max Schultze, in 1863. described
admirably the process of cleavage of the frog's egg. He spoke
of the egg as a cell with protoplasm and nucleus, and of the
process of cleavage as cell-division. Ordinary cell-division
depends, he said, on the contractility of the protoplasm. The
same property belongs to the egg-yolk, since it divides like a
true cell.

N^-, :,y» ^o^ '

CHAPTER V

EARLY DEVELOPMENT OF THE EMBRYO

In the preceding chapter the cleavage of the egg has been
described to the period when the bhistopore is about to appear
on the surface. During the subsequent development the cells
continue to divide, so that at no time can the cleavage or the
cell-division be said to cease. At each successive stage the
number of cells is greater than in the preceding stage. This
statement does not inipl}^, however, that the formation of each
new structure is introduced by new cell-divisions in the region
where the change is about to begin, because many changes take
place in regions where cell-division is not more rapid than else-
where.

The spherical form of the " egg " or 3'oung embryo is soon
lost. In the present chapter we shall follow the changes that
can be seen taking place on the exterior of the living embryo ;
and in the following chapter we shall attempt to make out the
movements of cells and groups of cells that take place in the
interior of the embryo during this period.

The Blastopore

Un that side of the egg where the smaller cells are found, a
short horizontal line of pigment ^ appears amongst the white
cells below the equator of the egg (Fig. 12, I). This line marks
the beginning of the archenteron, and the cells bounding the
upper or darker side of the pigment-line form the dorsal lip of
the blastopore. The dorsal lip becomes crescentic in outline,
with the concavity of the crescent turned toward the white
hemisphere (Fig. 19, I, II). If the living egg be watched, it

1 There is a great deal of variation at first in tlie shape of the blastopore.

50

Ch. V] EARLY DEVELOPMENT OF THE EMBRYO 51

will be found that changes take place at this time in the blasto-
poric region with great rapidity.

Pfliiger has described ('83) these changes, and we may
follow his admirable account, subsequently adding other facts
that have since been discovered. The eggs Avhich Pfliiger
studied 1 were taken from the uterus at twelve o'clock midday,
and placed in a row on a thick glass mirror. The mirror was
then put into a dish, and water added to the depth of "2 mm.
In this way, owing to the reflection of the lower pole by the
mirror, both hemispheres of the egg could be watched. Dur-
ing the night, when the temperature was low, the eggs de-
veloped more slowly. At six o'clock in the morning the
thermometer stood at 16° C. At this time the eggs showed
on the lower hemisphere, and in the upper fourth of that region
and therefore just beneath the equator, the first trace of the
dorsal lip of the blastopore. By ten a.m. the long horizontal
split (dorsal lip of the blastopore) had become distinctly marked
as an indentation of the surface of the egg. At eleven a.m.,
the dorsal lip had moved somewhat further below the equator
of the egg, i.e. toward the lower pole. The "split" is now
broader, and its corners turned down so that it forms a cres-
cent, with the lower pole of the egg-axis as its middle point.
The diameter of the crescent is to the egg-diameter as 2 : 3.
From the corners of the crescent a furrow continues to extend
on each side around the white hemisphere. The progress of
the dorsal lip toward the lower pole is not due to a rotation
of the egg as a whole, but to the migration of the dorsal lip over
^the white hemisphere. At half-past twelve o'clock (twenty-
four hours after fertilization), the dorsal lip has progressed
further toward the lower pole. The crescent has at the same
time extended so as to form a half-circle whose diameter is
somewhat less than in the preceding stage. It stands now in
relation to the diameter of the egg as 1 : 2.

By one o'clock p.m., the semicircle forming the dorsal and
lateral lips of the blastopore has extended so as to form a
complete circle (Fig. 19, A, IV). The white yolk-cells pro-
trude from the centre of this circle and form the so-called

1 Bufo cinereus.

52

DEVELOPMEXT OF THE FROG'S EGG [Cir. V

B

Fig. 18. — Diagrams to show extent of movement of dorsal lip of blastopore and
rotation (F) of embryo. (After Ptliiger.)

yolk -plug. The diameter of the circle around the yolk-plug is
still smaller than before. At 2.15 p.m., the opening containing

Cri. V] EARLY DEVELOPMEXT OF THE EMBRYO 53

the Yolk-pkig- — the so-called opening- of Rusconi, or blastopore
— is still smaller. The periphery of this circular blastopore is
deeply pigmented. At 4.15, the opening is further reduced
and measures no more than one-eiglith of the diameter of the
egg. The blastopore will now be found to have progressed so
far that it again lies just beneath the equator of the egg, but on
the side of the egg opposite to that at which the dorsal lip first
appeared. We can summarize by saying that the dorsal lip of
the blastopore has moved over a meridian of the egg from a
point near the equator across nearly to the opposite point of the
equator. The movement takes place over the lower white hemi-
sphere, and during the process the position of the egg remains
unchanged. The arc traversed by the dorsal lip of the blasto-
pore is, however, not as much as 180 degrees, because it started
below the equator and does not quite reach the equator at the
opposite side. But the arc is certainly more than 90 degrees,
and varies in different eggs.

So far we have traced the history of the l)lastopore from six
o'clock in the morning to 4.15 in the afternoon of the same day.
Then a remarkable jjrocess begins. The blastopore moves back
as a whole in exactly the opposite direction until, at 7.45 in the
evening, it has come back to the point from which it started in
the morning. This reverse movement of the whole blastopore
is brought about by quite a different process from the first
movement of the dorsal lip. TIw whole egg rotates around a
horizontal axis.^

The overgrowth of the lower pole l)y the dorsal and lateral
lips of the blastopore has covered the lower hemisphere with
cells that do not contain as much pigment as do the cells that
lie around the upper pole, i.e. the original black hemisphere.
Hence when the egg rotates as a whole in the way just re-
corded, a somewhat lighter area Avill be carried into the new
upper hemisphere, while the original upper hemisphere will
now come to lie nearlv on the loAver side of the ego-. In the
lighter upper region, as we shall soon see, the central nervous
system develops. Tlie rotation of the whole egg appears to
take place through 180 degrees, although it is possible that the

1 All axis at right angles to the median plane of the later embryo.

64

DEVELOPMENT OF THE FROG'S EGG

[Cii. V

central nervous system may also grow forward somewhat so that
the actual rotation is not great.

Perhaps the whole process may be made clearer by reference
to a series of sections through the egg. These are taken through
the meridian that corresponds to the middle plane of the body ;
it therefore passes through the upper and lower poles (Fig. 18).
The arrows indicate the primary axis. The dorsal lip of the
blastopore has formed in Fig. 18, B, and in Fig. 18, C, D, the
migration of the lip has gone further over the lower hemisphere.
The ventral lip of the blastopore has also formed. Figure 18,
D, corresponds to a stage in which the blastoporic circle is com-
pleted. In Fig. 18, E, we see that the dorsal lip has travelled
further over the lower pole toward the ventral lip. Finally,

Fig. 19. — A. Diagram to illustrate overgrowth of dorsal lip of blastopore. I-IV rep-
resent different stages. B. Diagram of cross-section through Z-Y of A, to show
lateral lips of blastopore, and mesoderm (M), and (IW) inner wall of archeuteric
pit. OW. Outer wall.

in Fig. 18, F, the egg is represented as having rotated as a
whole to bring the embryonic portion above.

The changes that take place during the closure of the blasto-
pore are perhaps more clearly shown by the following experi-
ment. By means of a fine pointed needle it is easy to puncture
the egg slightly at any given point. When the outer surface
of the egg is pierced, there follows a protrusion of material as
soon as the needle is withdrawn. At other times when the
surface is only itidented (7iot pierced) by the needle, there
follows a blunt protrusion of material and the surface remains
unbroken. In the latter case the marks do not always last as

Cii. V] EARLY DEVELOPMENT OF THE EMBRYO 55

long as do those i^rocluced by the first method, but as less harm
is done to the egg, one can often get more satisfactory results.^

If wlien the first trace of the blastopore appears on the sur-
face of the egg (Fig. 19, A), a slight injury is made to the
surface of the white hemisphere at the side opposite the blasto-
poric lip, i.e. at a point 150 degrees from the dorsal lip of the
blastopore (Fig. 19, A, at x), we shall find that in the course
of four hours the blastopore will form a crescent, and that the
distance from the dorsal lip to the point of injury is much less
than at first (Fig. 19, III). A circular line of pigment in the
white hemisphere shows tlie line along which the lateral and
posterior lips of the blastopore will appear. It will be seen in
this case, that the point of injury lies therefore outside of the
yolk-plug, i.e. posterior to the ventral blastoporic lip.

In the course of four liours more it will be found that the
circular blastopore is much smaller than before, and that the
dorsal lip now lies much nearer to the point of injury (Fig. 19,
IV). The dorsal lip has travelled over more than two-thirds of
the original distance from its starting-point to the point of
injury. By making a new experiment with an egg that has
reached this stage of development, it will be found that when
the outlines of the blastopore have become sharply defined,
the later closure takes place at nearly an equal rate from all
points of the circumference, perhaps, however, still somewhat
more rapidly from the dorsal lip backwards.

Where the diameter of tlie circle representing the outline of
the egg equals 27 mm.,^ the distance between the blastopore
and the injury measures 21 mm. In the first four hours the
blastopore moves through 8 mm. In the next four hours it
travels through 7 mm., and is therefore now only 9 mm. from
the point of injury. By this time the blastopore is circular in
outline, and the injury lies just outside (2 mm.) of the circle.
The blastopore now measures 7 mm. in diameter. Assuming
that from this time forward the blastopore grows together at an
equal rate toward its centre, then the dorsal lip will pass over
about one-half of the diameter of the blastopore, or 4 mm.

1 This method can be used only with great caution.

^ The numbers refer to the measurement of the figure, and not to the egg itself.

56 DEVELOPMENT OF THE FROG'S EGG [Cii. V

The dorsal lip has passed then, in all, through 19 mm. of the
white area ; the ventral lip (from behind, forward) through
3 mm.

If the region overgrown by the dorsal lip be compared with
the length of the medullary folds which soon appear in the same
region of the embryo, it will be found that the latter, when they
first appear, are somewhat longer than the region overgrown.
If, however, we deduct from the length of the medullary folds
the thickness of the anterior connective that joins the right and
left sides of the nerve-plate, we shall find that the remaining
length of the medullary folds corresponds very closely with the
length of the region overgrown. We must, therefore, conclude
that the anterior connective lies just in front of the point at
which the first trace of the dorsal lip of the blastopore appeared.

We have assumed the point of injury to be a fixed point and
the overgrowth to be due to the progress of the dorsal lip. It
might equally well have been assumed that the overgrowth
was only apparent and was produced by the sliding forward
of the whole of the white area beneath the dorsal lip of the
blastopore. The end-result would be the same, but the process
different. There can be no question, however, that the move-
ment is really due to the progress of the dorsal lip. Other
experiments where two or more points of the surface are
injured show very conclusively that the movement is a back-
ward growth of the rim of the blastopore.

Comparing the statements made above with those of Pfltiger,
it will be found that they differ in three unimportant respects.
The rapidity of the overgrowth of the very early stages, before
the complete establishment of the crescent, was not noted by
Pfliiger. The distance travelled by the dorsal lip, as just
described, is somewhat less than that given by Pfliiger.
Pfliiger thought that the dorsal lip moved over about 180
degrees, but added that the amount of the movement differed
in different individuals, and was probably between 90 and 180
degrees. My own results make the region of overgrowth
about 120 degrees. From Pfliigfer's fiofures we are led to
believe that the whole blastopore after the establishment of
its ventral lip continues to move somewhat nearer to the
equator of the side nearest to the ventral lip. If this really

Ch. v] early dp:velopment of the embryo 57

does take place, in the way shown by Pfl tiger's figures,^ it can
only be due to a slight rotation of the ecror as a whole in this
direction, for experiments show that the entire movement of
the ventral lip is forward, i.e. toward the dorsal lip.

Tlie yolk-plug is finall}^ withdrawn into the interior of the eg^
and the blastopore remains as a round or often somewhat elon-
gated opening. Its subsequent changes we shall follow later.

External Changes after the Closure of the

Blastopore

Let us next examine the changes that appear at this time in
the region that now lies anterior to the blastopore and on the
upper surface of the egg. There is much variation in the early
stages of development of the embryos of a given species, and
in different species the variations are even greater. The dif-
ferences in level of different reg-ions are the result of move-
ments of the ectoderm. To see these to best advantage, the
living egg must be placed in the direct sunlight, and the surface
studied with low powers of the microscope.

An embrj'o in which the yolk is still exposed is shown in
Fig. 20, A. Passing forward from the yolk-plug over the
upper surface of the egg is a broad groove, the so-called
"primitive groove." At the anterior end of the primitive
groove is a circular elevation. On each side of the primitive
groove, at IM, the inner medullary folds are seen. Outside
of these we find a depression, and farther on each side, at
EM, the outer medullary folds. A sickle-shaped depression
lies just in front of the blastopore.

A later stage of the same embryo is shown in Fig. 20, B.
The primitive groove is narrower, the medullary folds are more
distinct, and anteriorly a continuation of the lateral folds has
formed. This will later be called the head-fold. Anteriorly
and laterally, there is formed on each side a lateral extension
of the medullary plate, the so-called "sense-plate."

The medullary plates now begin to roll in, producing a deep
furrow, the medullary furrow with the primitive groove at its

i Pfluger ('83), PI. IL, Figs. 4 and 5 (see Fig. 18, F).

58

DEVELOPMENT OF THE FROG'S EGG

[Ch. V

bottom (Fig. 20, C). The lateral seiise-jilate is split into an
anterior and posterior part on each side. The more anterior
part may still be called the sense-plate, SP, and the posterior,
the gill-plate, GP.

GP

SP

.GP

Fig. 20. — Surface views of young embryo of Rana. (After Schultze.) IM. Inner
medullary plate. EM. Outer medullary plate. GP. Gill-plate. SP. Sense-plate.
BP. Blastopore.

A later stage is shown in Fig. 20, D. Here the sense-plate
is fonnd to have extended laterally and forward, and the two

Ch. V] EARLY DEYELOPMEXT OF THE EMBRYO

59

sides have met iu front of the medullary folds. The gill-plate
is also seen, but the outer medullary folds are no longer con-
spicuous. The inner medullary folds are closing in to form
a tube. The blastopore is reduced to an elongated slit-like
opening,

A still later stage is drawn in Fig. 20, E. The outline of
the whole egg is now elliptical, with the long axis in the direc-
tion of the long axis of the embryo. The medullary folds are
also much longer, and have approached* each other in the
middle line. A deep furrow lies between the two halves. The

Fig. 21. — Embryos of Raua. (After Schultze.) Gs, Gs'. Two gill-slits. Gp. Gill-
plate. Sp. Sense-plate. S. Suckers. Ax. Anus.

folds have more nearly approached at the middle of their
length, and are more widely separated at the anterior and
posterior ends. At the posterior end the medullary folds are
overarching the small elongated blastopore. The sense-plates
and the gill-plates are distinctly visible.

The medullary folds now fuse along their whole length,
leaving, as we shall see, a central canal, which is the overarched
medullary furrow. The elongation of the embryo continues as

60

DEVELOPMEXT OF THE FROG'S EGG

[Cn. Y

seen in Fig. 21, A. The anterior end of the medullary tube
shows on each side a lateral protrusion, the eye-bulb. At the
anterior end of the body we can see the sense-i3late and on
each side the broad gill-plate. Lying in the sense-plate on
each side is a deeply pigmented area which is the forecast of
the "suckers," S. There is a depression in the middle line,
in the centre of the sense-plate. This depression marks the
mouth-depression, and indicates the point at which, later, the
stomodccal invagination will take place. (See Fig. 20, F.)

Fig.

-Development of embryo. Anterior view, showing sense-plate, uasal pits,
stomodauim, and gills. (After Ziegler.)

A later stage is shown in Fig. 21, B. The relation of the
parts is much as in the last figure. The anterior end of the
medullary tube is larger than before, and the protuberances of
the eye-evaginations are more apparent. In the gill-plate on
each side appears a vertical depression and later another de-
pression behind it, GS, GS'. These depressions mark the
external gill-slits. The anus has shifted to a more ventral
position. The suckers have each elongated ventrally, and have
fused into a pigmented V-shaped %ure. The outer medullary

Ch. y] early development of the embryo

61

plates take no part in ths rolling in to form the medullary tube,
but flatten out and seem to disappear.

Ziegler ('92) has made several excellent figures of living
embryos of Rana temporaria (Figs. 22, 23). The first of these
shows young embryos as seen from in front, so that the sense-
plate is turned toward the observer (Fig. 22). A longi-
tudinal groove appears in the middle of the sense-plate, and
subsequently a transverse groove develops across the sense-
plate (Fig. 22, D, E). The depression that later forms the
mouth lies at the crossing-point of the longitudinal and trans-

-r

B

■^.i.

D

Fig. 23. — Development of embryo, showing closure of blastopore and formation of

anus. (After Ziegler.)

verse grooves

There is present on each side above the mouth
a thickened ridge that forms the superior maxillary process.
Below and behind the mouth a pair of ridges appear that meet
in the middle line. These are the sub-maxillary processes
which later form the lower jaw. A pair of depressions of the
surface ectoderm below the mouth-area mark very early the

62 DEVELOPMENT OF THE FROG'S EGG [Ch. V

beginning of the "suckers," or adhesive ghmds (Fig. 22, I)).
The nasal pits appear above the mouth (Fig. 22, fV

The outlines of the three brain-vesicles can also be faintly
seen in surface view. A pair of swellings on each side of the
fore-brain shows the position of the eye-evaginations (Fig. 22,
D). In the pharyngeal region there first appears on each side
a vertical ridge, and later another ridge parallel to and behind
the first (Fig. 22, D, E). On these ridges gills appear as pro-
trusions of the surface, and later a third ridge. and gill are
formed behind and somewhat beneath the others. Some time
after hatching, the gill-slits break through to the exterior be-
tween the ridges or gill-arches, and at about the same time,
the mouth breaks through into the cavity of the pharynx.

In the head-region the beginnings of some of the spinal
ganglia may be seen, and a series of mesodermal blocks also
appear and may be dimly seen from the outer surface. These
structures do not, however, appear as distinctly in surface views
of the embryo of Rana temporaria as they do in the embryos
of some other species.

Soon after the nerve-tube has closed, the dorso-posterior end
of the body begins to extend backwards to form the tail (Fig.
23, D, E). The anal opening lies just behind and ventral to
this region of posterior growth. The anus seems to shift to a
more ventral position during the elongation of the tail. At
first the tail is a thick outgrowth of the posterior end of the
body, but as it grows longer it flattens from side to side, and
in later stages a thin fan-like border or fin develops on its
upper and lower margin (Fig. 38).

CHAPTER VI

FORMATION OF THE GERM-LAYERS

The period that we are now about to examine is marked by
extensive movements of parts of the segmented egg as a result
of which the organs are formed. During the segmentation-
period the cells retain, as we have seen, the position in which
they arise, but with tlie appearance of the blastopore a new
period is initiated in w'hich extensive movements of cells and
groups of cells take place.

HiS'S ExPERrSIENTS ^VTTH ELASTIC PlATES

His ('94), from his studies of the behavior of elastic plates,
has concluded that many of the phenomena of the develop-
ing embryo are the mechanical result of the tensions set up
in the different layers. In the embryo the shoving, compres-
sion, or extension is supposed to result from the unequal growth
of different parts. When a cell-plate lifts itself up into a fold,
as a result of more rapid growth in that region than elsewhere,
there is present on the concave side a positive tension ("Druck-
spannung"") and on the convex side a negative tension. Under
these conditions the cells become conical, i.e. they are small on
the concave side and broad on the convex side of the fold.
Each embr^^onic cell tends of itself to become spherical and only
the surrounding conditions, resulting from the growth of sur-
rounding parts, determine the shape of each cell at any period
of development. His has tried to explain many of the changes
taking place in the early embryo as the result of this simple
folding principle. The inrolling of the medullary plate, the
formation of the eye-outgrowth from this plate, the formation
of the mouth-cavity and the gill-slit-folds, etc., are examples of
some of these changes. His pointed out how closely the forms

63

61 DEVELOPMENT OF THE FROG'S EGG [Ch. VI

taken by many of these structures in the embryo resemble
the folds that can be produced mechanically by pulling out or
pushing in a thin elastic plate of rubber. If this interpretation
is true, it means that at different periods in the development,
regions of more rapid growth appear, now here, now there, and
as a mechanical result of the conditions present, such structures
as the medullary folds, the eye-outgrowths, etc., are produced.
The cells change their shape in response to surrounding con-
ditions, i.e. they do not by their individual activity or move-
ment change their shape to produce the successive changes of
the embryo, but the shape of many cells is changed as the
result of growth or increase in mass of certain reo-ions. For
Distance, a cell becomes conical not through its own initiative,
but because the surrounding pressure forces it into a conical
shape.

The Formation of the Embryo by Concrescence

The period of overgrowth of the blastopore when the so
called process of gastrulation is going on has been described in
Chapter \. We may now follow tlie clianges that take place
in the interior of the egg during that time.

When the dorsal lip of the blastopore appears, the cells have
shown little tendency to arrange themselves into sheets or
layers. However, even when the segmentation-cavity is covered
by a roof of small cells, the cells of the outer layer have begun
to flatten against one another and to form a thin layer of cells
over the outer surface of the black hemisphere. In the lower
hemisphere the larger white cells do not show such an arrange-
ment. In tlie equatorial region, where the black and white
cells meet, a careful examination of sections will show tliat
there exists a more or less dehned ring of cells stretching
around the embryo, forming a broad zone (Fig. 15, D). The
inner cells of this ring contain a good deal of pigment around
the nuclei. The yolk-granules of these inner cells are smaller
than the yolk-granules in the large white cells of the lower
hemisphere, and the cells of the ring seem to contain also a
larger amount of clear protoplasm. This inner zone of cells
passes, on the one hand, by insensible gradations into the cells
of the outer surface of the ring and internally it is continuous

Ch. VI]

FORMATIOX OF THE GERM-LAYERS

65

with, the inner region of large 3-olk-cells. This ring of cells,
as subsequent development shoivs, is the beginning of the embryo,
and the ring itself is composed of the material which subsequently
forms the central nervous system, the mesoderm, the notochord, and
a part of the endoderm. An understanding of the subsequent
development depends on a knowledge of the changes that take
j)lace in this ring.

The material of tlie rina: is intimately involved in the move-
ments that take place during the overgrowth of the lower
hemisphere by the lips of the blastopore. During this period,
we must picture to ourselves the ring as rising up and draAving
together over the lower white hemisphere, so that ultimately
it leaves its equatorial position and its halves come together to
form tlie embryo. (Fig. 24, A, B, C.)

A B C

Fig. 24. — Diagrams illustrating germ-ring and concrescence of lips of blastopore.

As the dorsal lip of the blastopore progresses over the white
hemisphere, its progress is due to the movement and fusion
along a meridian of the material of the equatorial ring. AVe
are to think of the material of the ring as moving toward the
middle line from the right and left sides (for with the estab-
lishment of the dorsal lip the ring becomes bilateral) and
fusing continuously in the dorsal lip (Fig. 2-1). The advance
of the blastopore is merel}^ the expression of the absorption
into its dorsal lip of the material of the two sides of the ring.
As soon as the material from the sides reaches the median line
in the dorsal lip of the blastopore, it remains stationary and
new material is added behind that just laid down. The mate-
rial of the equatorial ring is thus carried into a meridian of
the egg. With the disappearance of the yolk-plug below the

66 DEVELOPMENT OF THE FROG'S EGG [Cii. VI

surface, the final stages of overgrowth are completed. The
ventral lip of the blastopore has moved somewhat forward, as
previously explained, and this slight forward movement proba-
bly takes place by the growth toward the median line of tlie
material at the sides of the ventral lip.

There are other changes closely bound up with the preceding
phenomena and, although these changes take place simultane-
ously, it will be necessary first to consider them separately, and
then to try to combine them into a single statement. The
changes involve, 1) the formation of the archenteron, 2) the
progression of the blastoporic rim over the lower hemisphere,
3) the origin of the middle layer or mesoderm.

The Formation of the Archenteron

1) When the dorsal lip appears, certain cells pull away from
the surface, leaving their outer pigmented ends exposed for a time
(Fig. 15, D, Fig. 12, H). These cells are near the border-line
between the black and white regions, but lie distinctly amongst
tlie white cells. The next change involves the sinking in be-
neath the surface of the region in which these cells are present.
The dorsal lip of the blastopore now begins its movement over
the lower hemisphere. From the surface we can see that the
crescent becomes longer and longer, the horns extending out-
wards along the black-white border but well within the white.
The same changes that took place where the dorsal lip first
appeared, now take place also wherever the crescent extends.
First certain superficial cells pull into the interior of the egg
leaving only their pigmented ends at the surface, and then this
area of pigment sinks below the general surface. Simultane-
ously the edges of the blastopore roll over the inturned (invagi-
nated) cells. The same changes also take place at the posterior
or ventral lip of the blastopore, when the two horns of the lat-
eral lips have met there. It is necessary to examine sections
that have been cut in several planes in order to follow the
changes that take place during the further overgrowth of the
blastopore. If we examine a median longitudinal (sagittal)
section at the time when the dorsal lip has just begun to roll
over, we find (Fig. 25, A) that a narrow space is left between
the dorsal lip and the surface of the lower hemisphere over

Ch. VI]

FORMATION OF THE GERM-LAYERS

67

which the dorsal lip has begun to roll. We find, at the upper
end of this crevice, the pigmented ends of those cells that were
previously at the surface. During later stages the space, which
we may at once speak of as the archenteron, becomes longer,
due to a further progression of the dorsal lip over the white
hemisphere. If the
section were taken
somewhat to one side
of the median line,
the length of the ar-
chenteron would be
found to be less than
in the median line,
because the rollins: in
has been relatively
less. If we make a
section at right angles
to the last in the plane
Y-Z, in Fig. 19, A,
we cut the two Jiorns
or ends of the cres-
cent. The cavity on Fig. 25. — a (small figure inside B). Longitudiiical
each side is just be- se^-Jion through young embryo. B. Cross-section

J of last. (After Schultze.)

ginning, owing to the

smaller amount of closing in from the sides of the lateral lips

of the blastopore. (Fig. 19, B.)

A section at right angles to the last section in the plane of
the line in Fig. 25, A, is shown in Fig. 25, B. The archen-
teron is seen in the upper part of the section. Its upper or
dorsal wall is made up of small cells, while its floor is formed
of large cells filled with yolk. The segmentation-cavity fills
the centre of the section.

During the time when the yolk-plug is withdrawing from
the surface, the segmentation-cavity becomes smaller, owing,
without doubt, to the intrusion of the large yolk-mass into its
interior, and finall}", when the archenteron begins to open, the
segmentation-cavity is almost entirely obliterated. The seg-
mentation-cavity is thus utilized by the embrj^o, for into this
cavity is pushed the yolk-mass as the latter is overgrown by

68 DEVELOPMENT OF THE FROG'S EGG [Ch. VI

the blastopore-lijDs. This statement does not necessarily imply,
however, that the segmentation-cavity was prepared especially
in view of the subsequent changes.

It will be seen from the foregoing account that the walls of
the archenteron are formed as the blastopore closes in. The
floor of the archenteron (Fig. 25, B) is nothing more than the
surface of the lower white hemisphere that is overgrown. The
origin of the roof and sides of the archenteron is somewhat diffi-
cult to understand. We have seen that around the crescent of
the blastopore certain cells have pulled in, leaving a depression
on the surface. It is impossible to say just how far the cells
that pull in continue to be drawn inward, because simultane-
ously the lips of the blastopore roll over. This brings us to
a discussion of the second topic.

The Overgrowth of the Blastoporic Rim

2) There are at least two ways in which we may think of
the closing in of the lips of the blastopore, i.e. there are two
ways, either of which might explain the covering of the white
by the black cells. We may think of the free edye of the
blastopore as growing toward a middle point. Or we may
imagine that the lateral and dorsal edges actually roll in
toward the middle line. The latter process seems to be that
Avhich probably takes place, for Jordan ('93) has seen the outer
dark cells actually rolling over and into the archenteron in the
living Qgg.

The dorsal and lateral walls of the archenteron will then be
formed in part, or entirely, from those cells of the surface
that have rolled in and have come to lie beneath the surface.
These are the cells, therefore, that have been at one time
situated at the surface of the embryonic ring, and inasmuch
as the advance of the dorsal lip takes place very largely by
the fusion of the lateral lips, it follows that the material for
the greater part of the dorsal wall of the archenteron comes
from cells at one time on the outer surface of the qq-q-. I am
inclined to thiidc that at first there is also an actual in-pulling
of cells along the blastoporic rim so that cells at one time below
the outer surface come also to stand, later, at the sides of the
archenteron, i.e. where the dorsal and ventral walls meet.

Ch. YI] formation of the GERM-LAYERS 69

The Okigin of the Mesoderm

3) It is difficult to give an account of the method of de-
velopment of the mesoderm, because there are almost as many-
different descriptions of the process as authors who have de-
scribed it. I have without hesitation set aside those accounts
where the author has transparently sought to find his precon-
ceived theories demonstrated in his drawiuQ-s of the sections of
the embryo. In the second place, several of the more recent
accounts have started out, I think, with a false conception of
the position of the embryo on the egg and its method of for-
mation, hence in these accounts the method of the formation of
the mesoderm is likely to be erroneously described, although
in several cases the actual drawings of the sections have been,
I believe, accurately made. I have followed as far as possible
those interpretations that are in conformity with the experi-
mental results relating to the growth of the embryo. Certain
abnormal embryos, to be described later (Chapter VII), that
first appear as a ring around the egg throw, I think, also much
light on the subject.

The cells that are to form the mesodermal layer are present
at the time when the dorsal lip of the blastopore has first
appeared, and even just prior to that time. The innermost
of those cells forming the ring around the egg are the cells
that become the mesoderm (Fig. 19, B). These cells are
carried up to the median dorsal line of the embryo by the
closure of the blastopore (Fig. 24, A, B, C). They will then
be found forming a layer or sheet of cells (Fig. 25, B) that
separates itself on the outer side from the thick layer of small
ectodermal cells (that has been simultaneously lifted up) and
that is separated on the inner surface, but not very sharph^ if
at all, from the dors;d and dorso-lateral walls of the archen-
teron. A continuous sheet of tissue is formed in this way
over the dorsal surface stretching across the middle line.
According to some accounts, the fusion of this mesoblastic
sheet with the endoderm is much closer in the mid-dorsal line
than on each side. We may, however, think of the mesoder-
mal layer and endodermal layer as coming up together to the
median line from the sides, so that we are to think of the

70 DEVELOPMENT OF THE FROG'S EGG [Ch. VI

mesodermal and endodermal cells as being together from tlie
beginning.

Different Accounts of the Origin of the Archenteron

AND jNIesoderm

Before followinsr further the fate of these concentric coats
or layers of cells, the so-called "•germ-layers," we may for a
moment examine some other descriptions that have been given
as to the method of formation of the archenteron in the frog.
The most common view of the method of gastrnlation of the
frog has been that a process of invagination takes place at the
dorsal lip of the blastopore. This process is supposed to be
brought about by the drawing inwards and uj)wards of a fold
of the outer wall, so that a blind sac forms. As this presses"
forward into the yolk, the latter pushes before it and fills up
the segmentation-cavit}'. At the same time the mesoderm is
described as growing forward from the region of the blastojjore
over the dorsal surface of the embryo.

Other authors represent, however, the dorso-lateral edges
of the archenteron proliferating cells along the two sides to
form the mesoderm, while in the mid-dorsal line a solid block
of endoderm cuts off to form the notochord. Hertwig has gone
so far as to affirm that at the dorso-lateral edges of the archen-
teron there are traces of a pair of lateral pouches along each
side, and that these give rise to the cells that push in between
the ectoderm and endoderm to form the middle layer.

Robinson and Assheton ('91) assert that the old account of
the formation of the archenteron by invagination is entirely
erroneous, and that the cavity of the archenteron owes its exist-
ence to a process of progressive splitting or separation of the
large yolk-cells of the lower hemisphere, and that this splitting
extends up into the yolk beneath the upper hemisphere. The
dorsal lip of the blastopore remains approximately stationary
where it first formed, and the anus develops around this point. ^

^ In a later account Assheton ('04) has much altered his former view. He
describes only the anterior end of the archenteron as formed as a split amon.c:st
the endoderm- cells, while the posterior third of the archenteron is, he thinks, the
result of the overgrowth of the dorsal and lateral lips of the blastopore.

Ch. VI] FORMATION OF THE GERM-LAYERS 71

Both assumptions are, I think, erroneous, as a study of the
changes tliat take place in the dorsal lip will convince any one
who will take the trouble to follow in the living egg the
method by which the closure of the blastopore takes place.

Later Development of the Mesoderm and Origin of

THE NoTOCHORD

Schultze ('88), Avho has studied the formation of the middle
germ-layer of the frog, has given an accurate account of tlie
condition of the mesoblast in the embryo during the period of
overgrowth of the blastopore. He has done this, too, despite
the fact that he believes the embryo of the frog to be formed
over the upper or black hemisphere of the egg. This belief has
not, however, in my opinion, vitiated in any degree his descrip-
tion of the position of the mesoblast after its formation. I
have, therefore, reproduced his figures in Fig. 2(3, A-E.

If a cross-section be made through an embryo (in the plane
of the dark line of Fig. 25, A) at the time when the blastopore
has assumed a crescentic shape, we find over the surface of
the section a thick envelope of ectoderm. The ectoderm is at
this time composed of about four layers of cells (Fig. 25, B).
In. the outermost layer the cells are columnar in shape. In
the centre of the section there is a large segmentation-cavity
surrounded by large yolk-bearing cells. The archenteron, as
seen in cross-section, is a large, arched cavity, its lower Avail
formed by yolk- cells and its dorsal Avail covered by a layer of
small cells showing a tendency to become flattened against one
another. Above the upper Avail of the archenteron, and betAveen
it and the ectoderm, is a thick layer of cells. This layer
stretches out on each side of the embryo as a lateral sheet, but
the edges of the sheet merije insensibh" into the yolk-bearino-
cells at the sides. Where this middle layer (mesoderm) is
sharply defined, aa^c can easily distinguish its cells from those
of the endoderm, for the mesodermal cells are smaller and pig-
mented. At the free edge of the sheet it becomes, hoAvever,
impossible to distinguish between the cells of the mesoderm
and of the endoderm.

If Ave examine a complete series of sections through this

72

DEVELOPMENT OF THE FROG'S EGG

[Ch. VI

embryo, we find that the laj-er of mesoderm is inserted be-
tween ectoderm and yolk-cells over all the posterior half of
the embryo. There is a small antero-ventral region into
which the mesoderm does not extend. At a point posterior to
the section described above, we find the mesoderm extending
much farther ventral! i/^ so as to nearly encircle this region of
the embryo. The blastopore is completely encircled by the
sheet of mesoderm.

|^*^",_^''C^^ . ■ '■ "^ • ; ■ ■; '. ': V-';

D

Fk;. '2(>. — A. Lonoitiiilinal section througli a yoimg embryo of Rana. B, C, D,
E. Cross-sections of last in planes of lines in A.

Cross-sections through an older embr>"0 are drawn in Fig. 20,
B, C, D, E. The embryo has flattened along the mid-dorsal line.
The ectoderm has become thinner along this line, where a faint
groove can be seen on the surface of the living egg, — the primi-
tive groove. On each side of the mid-dorsal line, the ectoderm
is somewhat thicker than before, and the cells are more closely
packed together. The ectoderm over the surface of the embr3^o
consists of an outer layer and of several inner layers of cells.
The cavitj^of the archenteron has opened out and is very large.

Cn. VI] FORMATIOX OF THE GERM-LAYERS 73

As before, its ventral wall is composed of larger and yolk-bear-
ing- cells. Above and laterally the walls are formed of smaller
cells. The latter have now arranged themselves in a definite
layer, and have become somewhat flattened (Fig. 26, B, C, D).
This layer is also sharply separated from the mesoderm. The
mesoderm, as compared with its previous condition, has under-
gone important changes. It has extended further ventrally,
and has met from the right and left sides in the mid-ventral line
along most of the ventral surface. Over the dorsal and dorso-
lateral walls of the archenteron it forms a thinner layer of cells
than in the earlier embrj'o (Fig. 25, B).

There is still a ventral region of the embryo where the ecto-
derm and the yolk-cells are in contact, i.e. a region into which
the mesoderm has not extended (Fig. 26, C). The medullary
plate is seen in cross-section. It will be noticed that the plate
is much thinner in the mid-dorsal line than at the sides. On
each side the medullary plates show a differentiation into two
parts. The most lateral and ventral edge of the plate is formed
of cells less closely held together than those nearer the mid-
dorsal line. This mass of rounded cells is the beginning of the
neural crest.

The mesoderm in the mid-dorsal line is thickened in the
posterior sections. According to some writers, this median
mesoderm has always up to this time remained closely fused with
the layer of endoderm beneath it. It marks the beginning of the
notochord.

The formation of the notochord takes place from behind
forwards, so that in the same embryo different stages of its
development may be found (Fig. 26, D, E).

The account given above of the formation of the notochord
is not generally accepted, particularly since the formation of
the notochord from the endoderm is the method followed by
many, perhaps by all other vertebrates. That a median mass
of tissue stretches at first across the dorsal median wall of the
archenteron in the frog cannot be denied, but many embryolo-
gists have preferred an interpretation different from that which
I have followed. It is affirmed that there is always a closer con-
nection hetiveen the endoderm and the tissue lying above it in the
dorsal median line than between the endoderm on each side of

74 DEVELOPMENT OF THE FROG'S EGG [Cii. YI

the mid-dorsal line and the mesoderm. Further, it is said, that
the cord of cells in the median dorsal line remains for a longer
time connected with the mid-dorsal endoderm than does the
mesoderm at each side with the lateral endoderm, and that the
notochord separates from its lateral connections (right and
left) with the mesoderm, while it still remains for a time
closely fused in the mid-line with the endoderm.

In the newt and in other urodeles the endoderm in the mid-
dorsal line thickens and bends upward to form a longitudinal
fold. The fold pinches off from the endoderm and forms a cord
of cells, — the notochord. In the posterior end of the toad's
notochord the same method of development may be seen some-
times to take place. 1

With such clear e\ddence of the method of formation of the
notochord from endoderm in the newt, it is not surprising that
embryologists have attempted to interpret the changes that
take place in the frog in the same way. The main difficulty
arises from an unwillingness on their part to derive the noto-
chord from the so-called middle germ-layer, or mesoderm. The
question therefore turns, for them, on what tliey will call the
middle layer in the frog, and what not the middle layer.

Since, however, all the cells in this region have had a common
origin, the question is perhaps a trivial one ; for we cannot doubt,
I think, that had some of the cells in the middle line passed a
little to one side or the other of the median line, they would
have been capable of becoming mesoderm, and, vice versci, had
some of the lateral cells come to lie nearer to tlie middle line,
then they would have taken part in the formation of the
notochord.

The notochord separates entirely from the mesoderm and
endoderm, and becomes rounded in cross-section. On each
side of the notochord the mesoderm becomes thicker, as is
shown in Fig. 42. The final stage in the closure of the med-
ullary folds and the changes that take place in the mesoderm
will be described in a later chapter.

1 Field ('95).

CHAPTER VII

THE PRODUCTIOX OF ABNORMAL EMBRYOS WITH SPINA

BIFIDA

Embryos of the frog are occasionally found that differ
greatly from normal embryos. Roux, in 1888, first described
one of these embryos and showed that a knowledge of its
structure and method of development helped very much tow-
ard an understanding of the processes that take place in the

r^

B —

Fig. 27. — Two embryos formed as rings arouud equator of egg. A. Seen from iu
front (jjrodueed in salt solution). (Morgan.) B. Seen from side. (After Roux.)

normal development. An embryo described by Roux iis shown
in Fig. 27, B. Around the equator of the eg^ along the zone
between the white and black hemispheres is a thickened ridge.
A careful examination shows that this ridge is not uniform in
thickness, but is bilateral in form. Each half is somewhat
thickened at one end, and resembles half of the medullary plate
of the normal embryo. Cross-sections (Fig. 29, B) show that
these ridges around the equator of the egg are the two halves
of the medullary plate. Instead, however, of being in close

75

76

DEVELOPMEXT OF THE FROG'S EGG [Cir. VII

contact, the two lialf-plates are separated in the middle by the
diameter of the egg, but at the anterior and posterior ends the
half-phates unite to form the ring. In section, a cord of cells,
the notochord, is found beneath each half of the medullary fold;
and between the yolk-cells and the ectoderm there is also found
a sheet of tissue representing the mesoderm. Hertwig, in 1892,
described a large number of these embryos. One is shown in
surface view as seen from the white pole, in Fig. 28, A. The
embryo is at a later stage of development than that described
above. The exposed white yolk, turned toward the observer,

Fig. 2S. — Two '• spiua-bilida" embryos. (After Hertwig.) A. Earlier, B. older

stage (different embryos).

is surrounded by a groove, and outside of the groove there is a
bounding darker ridge. In the anterior portion of the white
is seen a crescent-shaped depression. A cross-section through
the middle of the body of an embryo similar to the last is
shown in Fig. 29, A. The exposed yolk is seen at Y. On
each side of this there is a depression, and beyond the depres-
sion a thickened ridge composed of ectoderm cells. Each ridge
passes over on its outer side into the ectoderm that covers
all the lower part of the embryo. Even in their present stage

Ch. VII] PRODUCTIOX OF ABNORMAL EMBRYOS 77

of development the ridges are clearly seen to be the widely
separated halves of the medullary plate. Beneath each half
of the medullary plate there is a cross-section of the notochord,
and lietween the yolk-cells, in the centre of the section, and the
ectoderm covering the lower surface, there is a thick sheet of
cells representing the mesoderm.

A longitudinal (sagittal) section of the embryo drawn in
Fig. 28, A, is shown in Fig. 29, C. The large exposure of yolk-
cells (Y) in the upper part of the figure is very conspicuous.
A deep and narrow depression, bounded for the most part by a
distinct layer of yolk-cells, is found near the anterior end.
This depression corresponds to the crescent-shaped opening
seen in surface view, and is supposed to correspond to a part
of the archenteron of the normal embryo. ^ Ectoderm covers
the lower (ventral) surface of this section, and at one point the
cells are thickened to form the adhesive glands of the larva.
At the posterior end of the embrj-o a small depression is pres-
ent, and, as later development shows, this corresponds to the
posterior portion of the archenteron of a normal embryo.

HertA\ig found that if male and female frogs of certain
species be separated and kept apart for several weeks, and the
eggs then be artificially fertilized, an abnormal segmentation
follows, and, although many of the eggs die, among those that
live a large number show this condition of spina bifida.

In 1893 I made a series of experiments attempting to pro-
duce artificially embrvos showing spina bifida, and found that
they could be made by two entirely different methods. If the
segmented egg, before the blastopore-lips appear, be placed in
water to which .6 per cent, of salt (XaCl) has been added, the
later development is modified. The dorsal lip of the blasto-
pore appears in its normal position but does not continue to
extend over the white hemisphere. The corners of the lips
gradually extend around the equator of the egg. A sharp
line or depression separates the black and white hemispheres,
and on the black side of the depression a circular ridge appears,
which marks the beginning of the medullary ring (Fig. 27, A).

Similar embrvos may also be produced if tlie dorsal lip of

1 Possibly it represents in part the liver-diverticulum.

78 DEVELOPMEXT OF THE FROG'S EGG [Cn. YII

the blastopore is injured with a needle at the moment of its
appearance, or if the yolk-mass in front of the dorsal lip is
injured so that the yolk protrudes from the general rounded
surface of the egg. The blastopore is thus prevented from
extending backward, and its material differentiates, in situ,
along the equatorial line. The lateral lips tend to approach
the middle line and to fuse, but the medullary folds may
appear before the fusioii has taken place. There is thus pro-

^i"^;f ■~;S'r'xi^' ^^. '''"•^ l"»s5t"fl"ial (C) sections through an embryo with spina
bitida. (After Hertwig.j M. Half medullary plate. N. Half uotochorcl. Y. Yolk.

duced an embryo Avith an exposure of yolk in the mid-dorsal
line. The exposure is more or less extensive, according to the
extent of fusion anteriorly of the blastopore, and to the extent
of fusion forwards of the lateral and ventral liiDs.

These embryos Avitli spina bifida show that the material for
the mid-dorsal surface of the embryos appears first as a ring
around the equator of the egg or a little below the equatoi^
If this material is prevented from reaching the mid-dorsal
surface, it differentiates in situ. Hence the production of a
ring-like medullary plate and a double notochord.

Ch. YII] PRODUCTIOX OF ABXORMAL EMBRYOS 79

It is important to know detinitelj the origin of the material
that forms the equatorial ring. We have seen that the ring
appears at the same time that the bhistopore-lips extend around
the equator of the egg. Does this material also extend out
laterally from the dorsal lip of the blastopore along the sides,
or is the material already present as a circular ring of tissue,
from which the lips of the blastopore differentiate ? A study
of the normal embryo combined with experiments gives, I
believe, a conclusive answer to these questions. In the first
place, if the dorsal Up be entirely destroyed, so that it cannot
advance, nevertheless the lateral lips still a2)pear and extend
backward. If a point of the surface be injured just in front of
one (or both) of the advancing corners of the dorso-lateral lips^
the advance of the latter would be stoj^ped if an actual transfer
of material were taking place; nevertheless, on the posterior side
of the point of injury, a depression of the surface, marking the
blastoporic rim, appears, and continues to extend backward.
The same thing happens if injuries be made at two consecutive
points in the direction of extension of the lateral lip. Now if
material were actually transferred backward from the dorsal
lip and around the equator of the egg, its movement would be
stopped when the dorsal lip M'as seriously injured, so that the
lateral lips of the blastopore, and, later, the medullary folds,
would not appear, or else their appearance would be delayed.
Further, if there were, in reality, any such transfer backward
of material around the equator, its progress would be stoj)ped
when the material reached the points of injury made along the
line of the lateral lip. On the contrary, the appearance of the
lateral lips, after the destruction of the dorsal lip, takes place
as though no hindrance were present.

The experiments point clearly to the conclusion that tliere is
no backward transfer of building material, but that the mate-
rial for the dorsal surface is already present as a ring around
or near tlie equator of the egg.

If the normal embrj- o be studied by means of sections at the
period of the extension of the lateral lips of the blastopore,
the material of the ring is found to be already present in the
region into which the lateral lips extend. The evidence from
these various sources proves that the j^^'oduction of the e7nbryos

80

DEVELOPMENT OF THE FROG'S EGG [Ch. VII

showing spina bifida is oiving to the differentiation in situ of cells
that in the 7iormal embryo are first carried to the dorsal suj'face
before they differentiate into their definitive oryajis.

Roux first pointed out tliat tlie embryo described by him
showed that the material for the two sides of the embryo is
laid down in a ring, and that by the growing together (con-
crescence) of this ring along the mid-dorsal line of the embryo,
the two halves of the body are brought together. The same
method of formation of the embryo by concrescence has been
described as taking place in other vertebrate embryos, and cer-
tain writers have even affirmed that this is the method by which
all embryos of vertebrates are formed. In the main, Roux's
conclusion for the frog seems to be correct,^ but in one respect
not an unimportant exception must be taken to his statement.
If the material be laid down as a ring of tissue around the equa-
tor, and if, by its coming together (apposition), the two halves
of the embryo result, it follows that the embryo should be at
least as long as one semicircle of the surface of the egg.
Further, we have seen that the anterior end of the medullary
plate lies somewhat above the point of appearance of the dorsal
lip of the blastopore, so that the embryo would be, on Roux's
supposition, even longer than a semicircle. But if we measure
the medullary plate of the embryo at the time of its first appear-
ance^ we tind that in length it is only about one-third of the
length of the circumference of the egg. It follows, then, that
as the material comes to the mid-dorsal line in the normal
embryo, it must also become more concentrated, so that the
length of the medullary plate is less than the length of the
material of its halves. There is an accrescence or concentration
of material combined with a concrescetice or union of material
from the two sides.

1 Although Roux did not foresee the possibility that material might grow
around the equator from the dorsal lip of the blastopore, my own experiments
show, I think, that such a transfer does not take place.

CHAPTER VIII

PFLUGER'S EXPERIMENTS OX THE FROG'S EGG

Ix order to discover how far the development depends on
the surrounding conditions to which the egg is subjected, we
must chancre those conditions and observe the result. In this
way we may hope to find out to what extent the phenomena of
development are dependent on conditions outside of the egg,
and how far they result from the egg itself.

Pfiiiger made, in 1883, a brilliant series of experiments that
have been the point of departure for much of the later work
on the frog's egg: therefore, in this chapter, I shall give a
somewhat detailed account of Pfiiiger's work. The results
are arranged in an order different from that followed bv
Pfiiiger, with the hope of making clearer a necessarily brief
abstract.

The following orientation of the egg will facilitate the de-
scription of the experiments. If the middle ^ point of the black
hemisphere of the frog's egg (the '' black pole ") is imagined
to be connected with the analogous point of the white hemi-
sphere (i.e. Avith the ^' white pole") by a straight line passing
through the centre of the egg, this line forms the primary diam-
eter or jyrimary axis of the egg. An imaginary primary equator
and a system of parallels and meridians belong to such a diam-
eter. When the frog's egg segments, the first two cleavage-
planes are found to be vertical in whatever position the egg
may lie. The line of intersection of these first two planes
passes through the centre of the egg, forming what we may

1 Pfliiger does not notice that in tlie normal egg at rest tliis " middle part " is
not necessarily the highest part of the egg. Correspondinglj*, the lower pole need
not be the lowest point of the egg. For the present, however, we must disregard
this distinction.

G 81

82

DEVELOPMENT OF THE FROG'S EGG [Cii. VIII

call the cleavage-axis or secondary axis. To this axis there also
belong an imaginary secondary equator, parallels, and meridians.
If the egg sliould be turned, after cleavage., so that neither the
primary nor the secondary axis is vertical, then the diameter
that stands at the time vertical may be spoken of as the tertiary
axis.

It will be seen, from what has been said, that the imaginary
primary and secondary axes (with their systems) turn with the
Qgg-^ i-e. may be thought of as constituent parts of the Qgg\
while the tertiary axis only corresponds to any diameter of tlie
esfcr that is for the moment vertical.

The Effect of Gravity on the Direction of the

Cleavage

In normal eggs the first and second cleavages are vertical,
the third horizontal. The question arises, "Does there exist a
causal relation between the cleavage-planes and the egg-axis, as
has always been assumed without question, or do the first two
cleavages go through the primary axis, only because the latter
coincides with the force of gravity ? " This can be tested by
preventing the normal rotation of the Qgg-, and Pfliiger found a
simple method by which this is possible.

When the frog's egg is removed from the uterus, it is covered
by a thin coat of gelatinous substance which quickly absorbs
water and, if sufficient water is present, a space appears after
fertilization between the egg and its innermost membrane.
If an egg is taken from the uterus and placed in a dry watch-
glass, and only a drop of water containing sperm is added,
then the membrane swells somewhat, and sticks firmly to the
glass ; if now the right amount of water is added, the surface
of the egg remains in contact with the egg-membranes and the
egg cannot rotate as it does under normal conditions. The
watch-glass containing the egg may be turned in any position,
and the egg turns with it, so that any desired point of the egg's
surface may be placed uppermost. Let us imagine an egg to
be so turned that the black pole lies on one side. In the
course of three hours the first division comes in, but now the
plane of the first cleavage may not correspond to the primary

Ch. VIII] PFLUGER'S EXPERIMEXTS 83

axis. It follows always the direction of the force of gravity,
i.e. it passes through the vertical diameter of the egg-

The second cleavage also is vertical, and its position is also
deterinined by the position of the egg, and by the position of
the plane of the first cleavage. The third cleavage-planes often
show irregularities. Generally they are at right angles to the
first two, and lie nearer the upper pole of the egg, or, in other
words, their position is also influenced by the force of gravit}^
for they lie nearer to the pole that stands uppermost at the
time. It is a remarkable fact that the subsequent cleavages are
more rapid in the upper than in the lower hemisphere, no matter
what region of the egg has been placed uppermost. Embryos
develop from these eggs that have been turned into abnormal
positions, and the embryos differ from normal embryos only in
the relative distribution of pigment over the surface of the
body. Many have the upper surface of the body a light brown
color with dark spots ; others have the head, the back, and
upijer surface of the tail almost free from pigment, and of a
whitish-yellow color. The belly in these embryos is more or
less deeply pigmented. In a few days, hoAvever, new pigment
develops over the dorsal surface of the embr3^o. It should be
noted that tliese paler embryos often show abnormalities, such
as bizarre excrescences, irregular movements, slower develop-
ment, and that after a few days they begin to die.

Pfiiiger concluded from his experiments that an egg may be
divided in all possible directions by the early cleavage-planes
according to the position in which the experimenter places the
egg, and from such an egg a normal tadpole may develop.

It is not, however, entirely a matter of indifference what angle
is made between the cleavage-planes and the primary axes. It
is certain that if the upturned hemisphere contains more white
than black, a normal embryo may develop ; but if the upturned
hemisphere be entirely white, i.e. if the egg has been rotated
through 180 degrees, embryos may occasionally develop, but
they are nearly always abnormal and soon die. It is difficult, in
fact almost impossible, to keep the white hemisphere upward ;
for in nearly every case Pfiiiger found that later a partial rota-
tion of the egg took place, so that a crescent of black appeared
above the horizon. One exceptional case is worth recording.

84 DEYELOPMEXT OF THE FROG'S EGG [Cii. YIII

An egg was observed that had its wliite hemisphere turned
exactli/ upwards until the first cleavage came in. More water
was then added, and the egg retained its reversed position and
continued to segment energetically and with wonderful regu-
larity. The upturned white hemisphere was soon divided into
many small cells, while the cells in the lower black hemisphere
were larger. A later examination of the egg showed that dur-
ing the cleavage the egg had rotated through about 45 degrees,
bringing a portion of the black hemisphere above the horizon.
Still later the egg seemed to rotate back again into its first re-
versed position. After a time the development stopped and the
egg died.

If, as the preceding experiments seem to show, there exists
a relation between the force of gravity and the position of the
first three cleavage-planes, it is important to know whether
gravity acts only at the moment of cleavage or whether the
action is a slow and continuous one. Pflhger found that if
the egg at the two-cell stage be rotated a few seconds before
the appearance of the seeond furrow, so that a new angle is made
by the primary axis with the direction of the force of gravity,
then the second furrow comes in as though the egg had not
been changed, and may therefore make any possible angle with
the direction of the force of gravity. The same experiment
can be made if more Avater is added to an egg that has already
segmented once in an abnormal position. The egg may then
rotate so that the first cleavage-plane is no longer vertical ;
nevertheless, the second furrow always comes in at right angles
to the plane of the first furrow, and, therefore, may make any
possible angle with the direction of the force of gravity.

A different result follows if the egg has been rotated one
hour after fertilization and therefore some time before the time
of the first cleavage. The plane of cleavage of the seco7id divi-
sion is then affected, and coincides with the direction of the
force of gravity. We must conclude that an interval of one
hour at least is necessary to produce any chans'e in the e2"2-.

What has just been said with regard to the second planes of
cleavage holds equally well for the third cleavage-planes. If
the egg be rotated through 180 degrees after it has divided
twice (into four parts), then the third furrows come in as

Ch. VIII] PFLUGER'S EXPERIMEXTS 85

thougli no change had taken phxce, i.e. nearer the former upper
pole. But if the egg had been rotated one hour after fertiliza-
tion (or even after the first cleavage), the third furrows would ap-
pear on the netv upper hemisphere, i.e. nearer the present upper
pole. In this last case the four upper cells resulting from the
third division are smaller than the lower four. Tliis shows that
the four upjjcr cells of the normal eight-cell stage are smaller,
not because they are black, but, according to Pflilger, on account
of their position in relation to the force of gravity. Embryos de-
velop from these eggs, but they shoAv many abnormal structures.
Pfliiger also rotated eggs through 180 degrees after the third
cleavage had come in. In four hours and a half the cells of the
new upper hemisphere were of the same size as those of the new
lower hemisphere. A normal egg at this time would have
shown a great difference in the size of the cells of the upper and
lower hemispheres. It follows from the last experiments that
gravity may affect not only the first, second, and third cleavage-
planes, but the later stages as well. " Gravity," Pfliiger said,
" according to some unknown law regulates the cleavage-planes.
A simple explanation of the phenomenon does not seem possible
in the light of the facts."

The Relation of the Planes of Cleavage to the Axes

OF THE EmBKYO

Pfliiger made other experiments to determine whether, under
normal conditions, there exists any relation between the planes
of cleavage and the axes of the embr3'o. He placed seventeen
eggs in as many watch-glasses and added water containing
sperm. The axes of the eggs were vertical.

The direction of the plane of first cleavage was noted and
marked by a line scratched on each glass. The beginning of
the nervous system appeared in about forty-eight hours. In
twelve eggs the median plane of the body coincided with the
first cleavage-plane, or at most the two planes did not differ
more than 10 degrees. In four eggs there was an angle of
30 to 60 degrees between the two planes, and in one egg one
of 90 degrees. Pfliiger concludes that it is highly probable
from this result that the plane of the first cleavage and the

86 DEVELOPMENT OF THE FROG'S EGG [Cii. VIII

median plane of the body coincide. The exceptions may be
due to the rough treatment of the eggs.^ jS^ewport ('51) had
previously made a similar experiment on normal eggs, i.e. eggs
not fixed artificially, and had reached the same conclusion as
Pfliiger, but Newport's results were unknown to Pfliiger when
he made liis experiments.

Pfliiger was led from certain results of his experiments to
observe carefully the position of the e^g at the time when the
normal embryo was developing. He found, as has been already
described, that the dorsal lip of the blastopore appeared in the
white below the equator of the egg. He noted in the living
Qgg that the blastopore slowly migrated over the white hemi-
sphere, and that it finally closed nearly 180 degrees from the
point of its first appearance. Subsequently the whole egg slowly
rotated, so that the small blastopore traced the same path (but
in a reversed direction) over which the dorsal lip of the blasto-
pore had passed. The results show that the nervous system
develops over the lower wdiite hemisphere of the egg. The
material for the nervous system comes from the substance of
the lips of the blastopore as they move over and cover the
loAver hemisphere. This material, from which the nervous
system is formed, is at first somewhat lighter in color than
the pigmented hemisphere of the egg. It is darker, however,
than the white material of the lower hemisphere.

If in normal eggs the first cleavage -plane corresponds to the
median plane of the body of the embryo, does the same relation
hold for eggs that have segmented in abnormal positions ? In
other words, does the median plane of the body in eggs that
have been turned so that the primary axis is no longer vertical
still correspond to one of the primary meridians of the egg., or to
one of the secondary (i.e. segmentation) meridians? Pfliiger's
observations showed that in eggs with oblique primary axes
tile plane of the first cleavage is not identical with the median
plane of the embryo, but forms different angles with it. In
forty-eight eggs there were thirty-three in which the median
plane of the embryo coincided with the lyrimary axis. In the

1 Or else to a very early rotation of the egg, either as it shifts around its cen-
tre of gravity during gastrulation, or from the action of surface-cilia. — T. H. M.

Ch. VIII] PFLtJGER'S EXPERIMENTS 87

remaining eggs there were eight in which the median plane of
the embryo made angles between 10 and 25 degrees with the
primary axis. In five cases the angle was between 25 and 45
degrees, and in two the angle was as great as 45 to 90 degrees.
Pilhger concluded that in abnormally turned eggs the median
plane of the embryo belongs to the system of meridians of the
primary axis of the egg, — as in normal eggs ; and that the
cleavage of the egg onl}^ breaks up the building material into
small building blocks, and it is of no importance in the subse-
quent stages of development how the splitting up has taken
place.

Conclusions from the Experiments

From the orientation of the embryo with respect to the pri-
mar}' axis in whatever position the egg may be, it might seem
that the material of tlie egg is not isotropic. That is to say,
the position of the embryo is fixed in the egg, and the embryo
assumes its predetermined position regardless of the method of
segmentation. A more careful examination will show however,
Pfliiger believes, that the egg is isotropic.

It is obvious that although in most cases when the egg lies
with an oblique primary axis, the median plane of the body
belongs to the system of primary meridians, yet there are theo-
retically an infinite number of these meridians any one of which
might happen to be uppermost and to coincide with the median
plane of the body ; and Pfliiger's tables show that there must
be a great many possible primar}- meridians any one of which
may become the median plane of the embryo.

In the second place, the dorsal lip of the blastopore never
develops on the upper hemisphere, however the egg may be
turned. Pfliiger says that, in all, he has probably examined a
thousand eggs, and never once found the blastopore above. It
appears always in the ivJiite heloiv the equator. Again, in an egg
abnormally placed the head always develops above and the body
heloiv. These relations could not exist for all positions of the
egg, if the position of the embryo were prefixed in its relation
to the primary meridians.

Pfliiger considered one other possibility ; namely, that the
semi-fluid contents of the egg may rearrange themselves in eggs

88 UEVELOPMEXT OF THE FROG'S EGG [Cii. VIII

abnormally turned, so that the j^redetermined material takes a
definite position, and the blastopore always appears in its proper
hemisphere. A rearrangement, Pfliiger believed, does not take
place, because the egg-, if set free, even after it has been turned
for two hours, will tend to rotate into its normal position. Such
an egg, set free in its membrane, places the primary axis ver-
tical, and this rotation will take j)kice even after the first and
second furrows have appeared ; and this would not be the case
had there been a rearrangement of the contents.

Pfliiger noticed, however, in eggs that had been turned into
abnormal positions, that the upper, white hemisphere is often
darker in the later stages than it was at first, and conversely,
the black hemisphere may appear lighter owing to the loss of a
part of its pigment. This is brought about, Pfliiger believed,
by a streaming of the pigment-granules of the egg, and is not
a result of the rotation of the contents as a whole.

The position of the dorsal lip of the blastopore is determined,
then, in part by the position of the primary axis, and in part
by the tertiary axis, since the blastopore is always in the lower
hemisphere, however the egg be turned. '' The pirimary axis
determines the meridian, and the tertiary axis the piarallel in
lohich the dorsal lip of the blastopore shall appear.''^

Since these statements are true for all possible positions of
the primary axis, it follows that all primary meridians are of
equal value. If we think of an egg with inclined primary axis
and imagine this egg rotated around such an axis, then all the
primary meridians of the egg Mill in tnrn come uppermost.
Whichever one is brought to rest in the vertical plane, that one
will symmetrically halve the opening of the blastopore when
the latter develops, and on that one the embryo will lie with its
head turned upwards. It is this vertical meridian that coin-
cides with the direction of the force of gravity. In this
meridian, every part is not of equal value, because the blastopore
appears only in a certain region, and the position of the embryo
is thus fixed. The appearance of the blastopore on the vertical
meridian below the equator marks the crystallization-point of
the whole organization. In other words, the egg-substance has
at this time one meridian polarized. Pfliiger says : " I think of
each half of the egg after this as polarized, for both halves are

Ch. YIII] PFLtJGER'S EXPERIMEXTS 89

then of equal value and are composed of equivalent molecular
rows. Gravity alone has determined which of all possible
meridians shall be the controlling one."

Pie adds : '• I imagine that the fertilized egg bears no more
relation to the later organization of the animal than the snow-
flakes bear to the size and structure of the glacier that develops
from them. From a germ there always arises the same struct-
ure because the external circumstances remain the same. The
glacier tliat develops out of the snowflakes has always the same
form, so long as the external conditions are unchanged."

CHAPTER IX

EXPERIMENTS OF BORN AND OF ROIJX

Pfluger, as we have seen, believes that when the frog's %g^
is rotated so that the white hemisphere is turned uppermost,
no rotation of the contents of the egg takes phxce. Born
('84, b) repeated this experiment of Pfliiger and sought, by
making actual sections of the eggs, to hnd out whether any
changes do take place in the interior of the reversed eo-o-.i

Sections through normal, fertilized or unfertilized frogs' eggs
show that there is a peripheral, darkly pigmented rind in the
form of a shell thickest at the black pole (30 to 40 microns)
and fading away at the white pole (Fig. 8). Beneath the
black rind in the upper hemisphere lies a brownish pig-
mented protoplasm. In the centre of this and just under the
black pole is found in cross-section a clearer spot containing
the nucleus. The yolk lies within the white hemisphere. The
yolk appears coarsely granular, while the protoplasm in the
dark hemisphere is finely granular.

Changes that take place in the Interior of the
Egg after Rotation

Born observed in the living Qg<g that when the white hemi-
sphere is kept upward, it gradually becomes darker in color,
owing to the appearance of a grayish-white area. Pfliiger had
noticed the same phenomenon. This area grows larger in pro-
portion to the length of time that the ^^g has been turned.

Examination of sections of an inverted ^gg shoAvs that fort}'-

1 After the first account of Bora's had appeared, other papers dealing with
the same subject by Roux, Rauber, and 0. Hertwig were also published. These
authors all agree with Born.

90

Ch. IX] EXPERiaiENTS OF BORX AND OF ROUX 91

live minutes after inversion a rearrangement of tlie contents
has begun. The heavier white yoUc has begun to sink down
on one side, taking the shortest path toward the bottom of
the inverted egg. As the heavier yolk sinks down in response
to the action of the force of gravity, the granuhir protoplasm
rises up on the opposite side. The two sorts of substances do
not mix during the interchange of position, but keep sharply
separated from each other. The pigment-rind remains fixed,
but loses something of its thickness. After an interval of
forty-five minutes to two hours, the finely granular protoplasm
has reached the highest point of the egg, and has spread out
under the surface of the white hemisphere. The yolk has
passed to the lower hemisphere of the inverted egg, and now
lies inside of the black rind.

This description of the movement of the contents of the egg
applies to all those cases in which the white pole does not stand
exactly upward, or, in other words, where the egg is turned
less than 180 degrees. When the egg is completely inverted
the force of gravity causes the contents of the egg to rearrange
themselves in a somewhat different way. The yolk sinks down
on all sides, while the lighter protoplasm rises up through the
centre of the egg, carrying with it the nucleus.

Pfliiger believed that eggs which had been rotated through
180 degrees, and kept in that position, did not segment because
of the covering up of a micropyle, where the black pole came
in contact with the lower surface of support. Born thinks
that such eggs do not segment, owing to the inability of the
spermatozoa to pierce the white rind which is uppermost.^

In the normal egg the path of the spermatozoon can be fol-
lowed by the trail of pigment passing in from the surface of
the egg, which marks the direction taken by the spermatozoon.
This pigment-line can also be followed in the partially inverted
egg, and it is seen that the male pronucleus is also carried
along in the streaming protoplasm.

Born found in eggs that have been partially inverted, that
the first cleavage-plane is generally vertical, passing through

^ The problem of the extrusion of the second polar body in these eggs should
be examined.

92 DEVELOPxAIENT OF THE FROG'S EGG [Ch. IX

the highest point of the egg. Sometimes, however, the pLane of
cleavage is oblique to the vertical, i.e. occasionally it does not
pass through the highest point of the egg. The position of
this vertical or nearly vertical plane of cleavage bears generally
some relation to the path, or meridian, of streaming of the
contents of the egg. The first plane of cleavage corresponded
with the streaming meridian in about one-third of one hundred
recorded cases. In nearly all of the remaining two-thirds, the
first cleavage-plane stood nearly at right angles to the stream-
ing meridian.!

Born's results throAV a new and important light on Pfliiger's
experiments. The force of gravity acts on the rotated "^ egg
only to bring about a rearrangement of the contents of the egg
in accordance with the specific gravity of the substances pres''-
ent. This is the only connection between the direction of the
force of gravity and the direction of the planes of cleavage.
We also see why a certain amount of time is necessary after
the reversal of the egg for the rearrangement to take place.

The Cleavage of the Egg in a Centrifugal Machine

Roux ('84) tested in another way the effect of gravity on
the segmentation of the frog's egg. "What would happen,"
he asked, "if an egg were so placed that at every moment a
new point was turned uppermost ? Further, if gravity acts
only so as to rearrange the contents of tlie egg, what would
take place if a centrifugal force were applied to the eggs before
cleavage ? " Such a centrifugal force ought to cause the egg
to orient itself in respect to the direction of that force, in the
same Avay that gravity causes the egg to turn.

A wheel rotating around a horizontal axis was used. To
this wheel were attached tin boxes into which the eggs were
put. A box could be placed at any point along a radius of the
wheel. When the machine made eighty-four revolutions a
minute, some of the boxes were so placed that the centrifuo-al

In this case the second cleavage-plane would correspond with the meridian
of streammg. Born states that the median plane of the embryos, developino-
from the rotated eggs, passes through the secondary meridian that cuts the
highest edge of the white iield in its partially inverted position.

Ch. IX] EXPERIMENTS OF BORX AXD OF ROUX 93

force was twice as great as the force of gravity. When the
box was at the lowermost point in a revolution, the centrifugal
force and the force of gravity acted together. When the box
was at the highest point in its revolution, then the centrifu-
gal force and the force of gravity acted against each other.
AVith the velocity of eighty-four revolutions a minute, the cen-
trifugal force was greater than the force of gravity, even at
the highest point of a revolution. At the intermediate points,
i.e. between the liighest and loAvest points, the conditions are
different for each point, and lie between the two extremes just
stated. Under the conditions of the experiment the eggs
rotated inside their membranes, so that the black pole turned
imcard, i.e. toward the axis of rotation, and the white pole
turned outward. In other words, the eggs now oriented
themselves with regard to the centrifugal force, and not with
regard to the force of gravity. Even when the centrifugal
force was only half that given above, the eggs still arranged
themselves with reference to that force. In the latter case the
force of gravity was barely overcome by the centrifugal force
at the higliest point of the revolution. If a still shorter radius
were used, so that at the highest point of the revolution the
force of gravity was three times as strong as the centrifugal
force, even then the eggs oriented themselves as before, i.e.
with the black pole turned toward the axis of rotation. In all
of these experiments it will be seen that the centrifugal force is
a constant force, while the action of gravity varies in direction
at each point in the revolution. If a still shorter radius of the
wheel was used, in which case the centrifugal force was still
less, then the eggs retained any position that they had when
first put into the box.

All of these different possibilities could be realized at the
same time by using a series of tin boxes placed at the proper
intervals along a radius of the wheel. " The apparatus,
laden with ten to eighteen freshly fertilized eg-gs, was set in
motion. I waited with great interest for the appearance of
the first cleavage. It appeared at the normal time and the
whole cleavage proceeded in exactly the normal way. A nor-
mal blastopore appeared, and the formation of the medullary
folds, the brain-folds, and closure of the neural tube, and later

94 DEVELOPMENT OF THE FROG'S EGG [Ch. IX

the formation of the suckers, gills, and tail, all took place nor-
mally. There Avas no difference in the time of development
between the eggs in the machine and the normal eggs outside,"
used to check the results.

In the eggs acted upon by the centrifugal force, the segmen-
tation-axis corresponded with the egg-axis, and showed no
relation to the direction of the force of gravity. The third
furrows appeared nearer to the black pole, and the black cells
always divided faster than the white cells, regardless of the
position of the egg in respect to the force of gravity. The
blastopore appeared in its usual position.

Roux concluded that Pfl tiger's interpretation of his experi-
ments in regard to the action of the force of gravity was in-
correct. Roux said that in his own experiment the localized
effect of the force of gravity had been done away with, when
the eggs were slowly revolved, i.e. in those eggs nearest the axis
of the machine, and still the cleavage appeared in these eggs
irrespective of their position. When the centrifugal force was
stronger, it replaced the force of gravity, and the eggs oriented
themselves in regard to the new force, and still the cleavage
and the subsequent development took place normally.

Roux pointed out that a possible objection might be made as
to the sufficiency of his results. Since the eggs were always
rotated in a constant plane, it might he affirmed that gravity,
acting vertically in the phxne of rotation, still acted upon the
egg. To meet this objection a new experiment was devised.
Single eggs were placed in a glass tube 6 cm. long. This tube,
only half filled with Avater, was closed and fastened to the cen-
trifugal machine. During each rotation of the apparatus, the
egg» and the water would fall from one end of the tube to the
other, so that the orientation of the eggs would be changed
during each revolution. Nevertheless embryos normal in
structure were produced. They were, however, small and
weak.

CHAPTER X

MODIFICATION OF CLEAVAGE BY COMPRESSIOX OF THE

EGG

In 1884 Pfliiger made the important and novel experiment
of compressing the unsegmented egg of the frog between paral-
lel plates of glass. In consequence, the cleavage was modified,
and there was found to be a direct relation established between
the planes of cleavage and the direction of tlie pressure applied.
The first three planes of division were at right angles to the
compressing plate. Pfliiger explained these results as due to
the position which the nuclear spindle would take during its
elongation. The long axis of the spindle, he thought, would
place itself in the direction of least resistance, i.e. in a plane
parallel to the glass plates ; and since the division of the cell is
at right angles to the long axis of the spindle, it will, therefore,
be at right angles to the compressing plates. Horn ("93) and
Hertwig ('93) simultaneously repeated Pflliger's experiment,
making also some modifications of the original experiment.
Horn's account is here followed, as it gives a more detailed
report of the results.

Effect of Compressing the Segmenting Egg between

Parallel Plates

The eggs of Rana fusca are on an average 1.5 mm. in diame-
ter. The distance between the two glass plates in the experiment
was 1.4 mm., for if less the eggs were burst by the pressure.
Since all of the jelly around the egg was not removed, the actual
diameter of the egg., as subsequent measurements showed, was
less than the distance between the two parallel plates (1.4 mm.).
For instance, a compressed egg (after it had been killed and

95

96

develop:ment of the frog's egg

[Ch. X

hardened) ^ measured in its longer diameter, parallel to the
two plates, 1.83 mm., while the shorter diameter, at right angles
to the plates, was 1.2 mm. The two axes, therefore, stand in
the relation of 2 : 8. These figures apply to eggs compressed
in the direction of the egg-axis, from above downward. When
the egg is compressed from side to side it will withstand
more pressure. AVith a distance of 1.37 mm. between the two
parallel plates, an egg compressed laterally measured in its
longer diameter 1.96 mm., while its shorter diameter, from
plate to plate, measured 0.91 mm. The two axes therefore
bear to each other the ratio of 1 : 2. The experiments may
be described in detail under the following categories.

1) ^(/[/8 compressed in the direction of the primary axis (Fig. 30,
A). The eggs taken from the uterus were placed on a dry plate

B

Fig. 30. — Diagram showing three positions of eggs under compression.

of glass, so that the white pole was exactly do^^ invard, i.e. the
egg-axis was vertical. Another glass plate was then ])laced
over the eggs and brought down into contact with two sup-
porting rods, so that the two glass plates were 1.1 mm. apart,
and the eggs correspondingly compressed. The eggs were then
fertilized and the whole apparatus put into a dish of water.
The primary axis of each egg was \^e\)t always vertical. When
the first furrow comes in, it is verticah i.e. at right angles to
the glass plates, and passes from the black to the white pole
(Fig. 31, A), dividing the egg into two symmetrical halves. The
second furrows come in at right angles to the first, and are also

1 The eggs contracted very little during the process of hardening.

Ch. X]

MODIFICATIOX OF CLEAVAGE

97

vertical, i.e. at right angles to the glass plates. The second
furrows may cross the first furrow in the middle of its upper
and lower surfaces, so that four cells of equal size result ; or the
second furrows may sometimes pass to one side of the middle
point, so that two cells may be larger than the other two (Fig.
31, A). The last result may be due to a slight obliquity in the
position of the axis of the compressed Q^g. The third furrou's
(which normally are horizontal and at right angles to the pre-

B

3 1

3 1

Fig. 31. — A, B. Egg compressed axially (Diagram A, Fig. 30). A. Above ; B. below,
C, D. Egg compressed laterally (Diagram B, Fig. 30). C. One side ; D. other side.

ceding furrows) are also vertical and at right angles to the
plates, and are generally parallel to the first furrow (Fig, 31,
A, B). The egg is now divided into eight cells, all lying in
one horizontal plane. In the black hemisphere the third fur-
rows abut against the second furrows (Fig. 31, A), but be-
low they as often run into the first furrows, as shown in the

II

98 DEVELOPMENT OF THE FROG'S EGG- [Ch. X

tigure (Fig. 31, B). The fourth furrows are also vertical {i.e.
at right angles to the plates) and generally run parallel to the
second planes of cleavage, as seen in the figure (Fig. 31, A, B).
There is no segmentation-cavity as yet present in these com-
pressed eggs.

It is possible to keep these eggs in position until the blasto-
pore appears, and then to follow its movements up to a time
when the medullary folds form. The blastopore appears on
the under side, i.e. on the white hemisphere near the edge of the
egfi. It closes at the opposite edge of the lower surface. The
medullary folds also appear on the lower surface of the Qgg-, and
remain there until the embryo begins to lengthen. The belly
is therefore turned upward.

2) Efifjs compressed latercdly, i.e. at right angles to the primary/
axis, zvith the black pole kept upward (Fig. 30, B). The eggs
were placed between glass plates so that, when the plates were
turned vertically, the axis of the eggs also stood vertical, and
the compression was from the sides. The hrst furrow is ver-
tical and at right angles to the two glass plates (Fig. 31, C, D).
The furrow passes through the middle of the egg., dividing it
into two equal parts. Deviations from this mode of division
often occur. The first division sometimes passes obliquely, i.e.
to one side from above downward, but keeps always at right
angles to the glass plates.

The second cleavage comes in also at right angles to the
plates, and at right angles to the first furrow, and therefore in
a horizontal position. It always lies nearer the upper (i.e. the
black) side of the agg, as shown in the figure (Fig. 31, C, D).
Two upper small cells and two lower large cells are formed.
The second furrows have come in where, normally, the third
furrows lie.

The furrows of the third order appear first in the upper
smaller cells. They are at right angles to the glass plates, and
parallel to the first furrow, near to which thej^ often lie (Fig.
31, C, D). Occasionall}^ a furrow of the third order may lie
parallel to the second, and not to the first furrow ; it may even
run along the edge of the compressed Qgg, and is then parallel
to the compressing plates. In the loAver cells the furrows of
the third order also come in vertically and at right angles to

Ch. X] MODIFICATION OF CLEAVAGE 99

the plates. They are generally more or less parallel to the
first furrow. The furrows of the fourth order come m as a
rule at right angles to the last furrows, and therefore vary
in i^osition according to the position of the third furrows
(Fig. 31, C).

The later development of tliese eggs is as follows : if the eggs
have been much compressed, the blastopore appears always at
the periphery of the flattened egg, i.e. at the edge, and in the
space between the two j)lates ; when the eggs are not so much
compressed, tlie blastopore appears near the edge but more or
less upon one or the other surface. Curiously enough, just
before the closure of the blastopore, its opening is found to lie
at the edge of the same side at ivhich itji)'st appeared. Born in-
terprets this result as due to a rotation of the whole egg during
the closure of the blastopore. The eggs, he believes, are able
to rotate in the space between the glass plates around an axis
at right angles to the plates. The medullary fold appears also
at the edge of the compressed egg.

3) Eggs compressed laterally and kept with the black ptole to
one side (Fig. 30, C). If the eggs, laterally compressed, are
kept after compression in a horizontal position, i.e. with the
primary axis horizontal, other phenomena appear. Under these
circumstances. Born says that a streaming of the contents of
the egg takes place. The cleavage of these eggs corresponds
in general to that of the laterally compressed eggs, Avith nor-
mally directed, i.e. vertical, axis.

4) Eggs compressed between two plates oblique to each other,
so that the eggs lie in a wedge-shaped space. The first two fur-
rows are at right angles to the compressing plates, which are
inclined 12 degrees to each other. The furrows of the third
order are in the smaller, dark and more compressed cells at
right angles to the plates, while in the yolk-cells, which are little
compressed, the furrows are horizontal. The details of these
experiments of cleavage have not been worked out by Born so
fully as in the cases where the compressing plates were parallel
to each other.

Hertwig ("83, b) has described the first cleavage of one of
these eggs compressed 1)y plates inclined 45 degrees to each
other. The first cleavage divides a smaller protoplasmic por-

100

DEVELOPMENT OF THE FROG'S EGG

[Ch. X

tion from a larger yolk-portion. It does not therefore divide
the egg, as in the preceding cases, symmetrically.

Hertwig found that when eggs were compressed from above
downward, i.e. flattened axially between parallel plates, there
was no agreement between the jjlane of the first cleavage and
the median plane of the embryo. Four times the two coin-
cided, approximately, with the first furrow, five times with tlie
second, and six times with neither. The blastopore closes in
these eggs, as Born had also shown, at a point of the white
hemisphere opposite to that at Avhich it first appeared. In the
eggs compressed from the sides and standing with the axis ver-
tical, the blastopore appeared generally at the edge between
the two plates, and closed at a point opposite to that at which
it had first appeared.^ Exceptionally in these eggs the blasto-
pore appeared on one of the flattened surfaces, i.e. against one
of the compressing glass plates.

Effect of Compressing the Egg in a Glass Tube

Koux has shown that if the frog's Qg^ be sucked up into a
glass tube of smaller diameter than the diameter of the Qgg,

Fig. 32. — Segmentation of egg enclosed in a tube. (After Hertwig.) A. Four-cell
stage. B, C. Eight-cell stage, above and below.

the Qgg will be drawn out into a barrel-shaped body and the
cleavage correspondingly modified. The results, however, are
not always alike. This is probably due to the presence of a
large amount of jelly surrounding the eggs, so that they do

1 Hert\Yig found that when unsegmented eggs compressed between parallel
plates were rotated so that the white pole was turned ttpward, the egg rotated

Cii. X] MODIFICATION OF CLEAVAGE 101

not always take the shape of the enclosing tube. In order to
avoid this inconstant element, Hertwig ('93) repeated the
experiment with eggs from which much of the jelly had been
cut away. The fertilized eggs were drawn up into cylin-
drical tubes in which they assumed a short cylindrical shape
(Fig. 32). The eggs hiy with the black hemisphere against
one side of the tube and this side was turned upward, and the
tube kept in a horizontal position. The first cleavage of such
an egg is vertical and at right angles to the long axis of the
tube (Fig. 32). The second furrows are also vertical and at
right angles to the first, therefore in the direction of the long
axis of the tube. The third furrows are also vertical and
parallel to the first. The result so far is the same as Avhen the
eggs are compressed from above downward between parallel
glass plates. The fourth furrows are horizontal and divide the
egg into eight black and eight white cells.

Conclusions from the Experiments

These experiments in Avhich the cleavage has been modified
by changing the shape of the egg have an important bearing
on the general problem of cleavage of the egg. In the first
place, the " induced " form of the cleavage may give us some
insight into the causes that determine the direction of the
normal cleavage-furrows. In the second place, we see that
when an egg is compressed, the sequence of the cell-division is
very different from the normal sequence. Since we get nor-
mal embryos from eggs modified in this way, it would seem to
follow, as Pfliiger was the first to point out, that the cleavage
simply divides the spherical egg into the building-blocks from
which the later embryo forms, and it is a matter of indifference

as a whole and tended to turn the white hemisphere downward. If, however,
the eggs were compressed after the two, four, or eight cell stage, they then lieid
their position much better when the white pole was turned upward. If the
compression was applied when the cleavage of the eggs had gone very far, but
before the blastopore appeared, it was again found that the rotation of the egg
as a whole takes place (as in the uusegmented egg). An egg that has been
turned with its white pole upward at the two or four cell stage and has kept its
position during the cleavage-period, no longer tends to rotate as a whole during
the later stages of cleavage.

102 DEVELOPMENT OF TPIE FROG'S EGG [Ch. X

as to the succession of divisions. The value of this statement
will be discussed later.

These experiments show clearly that by changing the form
of tlie egg., we change at the same time its method of cleavage.
Again, reasoning from these "induced" forms back to normal
forms of cleavage, we see also something of the forces at work
there. Pfliiger did not fail to see the importance of these
experiments. He believed, as we have seen, that the direction
of the cleavage-planes results from the direction of the pressure,
because when the nuclear spindle of the egg or of a blastomere
forms, the spindle elongates in the direction of least resistance,
that is, at right angles to the direction of the pressure.

The spindle in the egg axially compressed cannot lie at
right angles to the plates because of the resistance of the yolk
below, but it must elongate in a plane parallel to the plates.
Since the cleavage of the protoplasm takes place at right
angles to the long axis of the nuclear spindle, the division-
planes must appear at right angles to the plates. Born has
pointed out that this interpretation of Pfliiger cannot be the
true one, because the egg is not a solid elastic ball, but a fluid
globe with an elastic coat. The pressure, therefore, will be
quickly equalized in all directions, and cannot act during the
time of cleavage in an}^ given direction.

Sachs's law for the direction of new cleavage-planes seems to
apply to the compressed eggs. According to Sachs, the form
of the whole mass determines the position of the cleavage-
planes. Hertwig refers the processes of cleavage directly to
the changes that take place in the nucleus. He thinks that
the nucleus tends to assume the centre of its sphere of activity,
which is the centre of the j)rotoplasmic mass. This is not neces-
sarily the centre of an egg in which the yolk is unequally
distributed. Hertwig thinks that the nuclear spindle wall then
elongate in the direction of the greatest protoplasmic mass. If
we apply Hertwig's hypothesis to the segmenting frog's egg,
we see that it appears to explain in part the various phe-
nomena. In the egg compressed in the direction of its pri-
mary axis and with tlie primary axis vertical (category 1),
the greatest protoplasmic mass will be, for the first spindle, in
a horizontal plane ; similarly for the second spindle. Hence

Cn. X]

MODIFICATION OF CLEAVAGE

103

the cleavage-planes that come in at right angles to the cleav-
age-spindle must be vertical and at right angles to the plates.
The third cleavage-planes will be for the same reason vertical,
and even the fourth planes may be so. The number of con-
secutive divisions at right angles to the compressing plates must,
however, soon reach a limit, because the mass of protoplasm
in each cell will soon be thicker vertically than horizontally.
When this happens, the next cleavage comes in horizontally or
parallel to the plates.

Hertwig's hypothesis seems, therefore, in harmony witli the
})henomena of the compressed eggs. Whether it is of general
application may be doubted because cases have been recorded

D»

D^

D'

D"

D-

/5c^

S^

/

/c^

r\\

d\

Hc'

cWy

Di^

D'

G

H

Fir.. 33. — Diagrams to show the distrihutinn of nuclei in compressed (A-D) and
normal ejig (E-H). In the upper series (A-D) the black hemisphere is turned
toward the observer ; in the lower series (E-H) the egg is seen from the side and
in part from above the black hemisphere.

where the elongating spindle does not seem to take the direction
of the greatest protoplasmic mass. Further, in certain spheri-
cal eggs without yolk, all the axes are equal, and some other
cause must be present to determine the direction of the spindle.
Even in the compressed egg (category 1) the protoplasm must
be radially symmetrical. Finally, it is possible that the phe-
nomena of the greatest protoplasmic mass and the elongating
spindle ma}^ be only concomitant and not causal phenomena, for
the position assumed by the centrosomes, which come to lie at

104

DEVELOmiEXT OF THE FROG'S EGG

[Ch. X

the apices of the spindle, must also be considered. The centro-
somes determine the position of the poles of the nuclear spindle.
Moreover, the position of apposition of the tAvo pronuclei of the
egg may be a further factor in the first cleavage.

The Distribution- of the Nuclei in the Coinipressed

Egg

In the experiments recorded above, where the frog's egg is
compressed during the cleavage-period, the distribution of nuclei
in the protoplasm is different from that in the normal ep-o-.
This is illustrated in the accompanying diagrams (Fig. 33).
Let us call the segmentation-nucleus A, and its first products

Ch. X] MODIFICATIOX OF CLEAVAGE 105

A^-1j2. The products of these nuclei we may call B^-B^,
B'^-B*. The following division will give eight nuclei, C^-C^,
and at the sixteen-cell stage we may call the nuclei C^-D^,
C^-D^, etc., as shown in the accompanying diagram.

Now let us compare, using this nomenclature, a normal egg
(Fig. 33, I)) with an axially compressed egg (Fig. 33, A). In
the normal egg at the sixteen-cell stage, the nuclei around the
upper pole \^'ill be C^-B\ C^-D^, C^-D^, C'-Ds and those
around the lower pole, C2-D2, C^-DS C^-T)% C^-DS. On the
otlier hand, in a compressed egg that has been freed from the
compression after the eight-cell stage, so that the fourth fur-
row has come in horizontally (Fig. 33, A— D), we find that the
nuclei in the upper hemisphere are C^-C^ C^-C*, C^-C^, C'-C^,
and in the lower hemisphere, D^-D^ D^-D*, D^-D^, D'-DS.
Thus there is an entirely different distribution of the products
of the nuclear division in the two cases,^ j'et normal embryos
develop from both eggs.

The simplest and most obvious conclusion from this result is,
I think, that the sequence of nuclear division during the early
cleavage-period has no relation to the subsequent formation
of the embryo, and that at this time the nuclei are all equiva-
lent.

1 There are several otiier possible combinations of these sixteen nuclei, but in
no case is the distribution alike in the uomial and. in the compressed egg.

.<vCAl

CHAPTER XI

Ti/E EFFECT OF IXJURING ONE OF THE FIRST TWO

BLASTOMERES

We have seen that the plane of first cleavage corresponds as
a rule with the median plane of the fnture embryo, so that one
of the first two blastomeres gives rise to the cells that form one
side of the body of the embryo, and the other blastomere pro-
duces the cells of the other side. It would seem then that
even at the two-cell stage the axes of the future embryo are
definitely laid down. But the most fundamental question re-
mains unanswered; viz., has the egg after its first cleavage
divided its material into qualitatively different parts (^i.e.
has the material of the right side of the body been separated
qualitatively from that of the left side), or are the first-formed
blastomeres still undifferentiated, and their subsequent fate
dependent on the relative position they bear to each other as
a part of a tvhole f

Roux tried to answer this question Ijy the following ingenious
experiment.

Roux's Experiment of " Killing " One of the First

Two Blastomeres

As soon as the first furrow had passed through the ^gg, one
of the resulting blastomeres was pierced with a hot needle.
In order to carry out the experiment successfully, certain pre-
cautions must be taken. The eggs as soon as removed from
the uterus are scattered over a glass plate (under water) so
that they lie singly. Then Avater containing spermatozoa is
added. After ten minutes this water, clouded by the sperma-
tozoa, is poured off and fresh water is added. When the first
furrow in the eggs appears, the water is again poured off.
Each Q,gg is held by a pair of forceps and then pierced by a

106

Ch. XI] EFFECT OF IXJURIXG A BLASTOMERE 107

hot needle. The needle is carefully sharpened, and is re-
sharpened after each egg is operated upon. It is best to
pierce the blastomere in the black hemisphere near the first
cleavage-plane. The needle passes through about a half (or
more) of the blastomere. When the needle is withdrawn, a
greater or less amount of the contents of the blastomere pro-
trudes where the blastomere has been injured. The egg after
operation is returned to the water. It is necessary to keep the
eggs under careful observation, because sometimes the blasto-
mere has been onl}^ slightly injured and continues to develop
more or less irregularly. Such eggs should be removed.

A B

Fig. 34. — A. Hemiembrjo lateralis. B. Hemiembryo anterior. (After Roux.)

Roux found that in twenty per cent, of the eggs the unin-
jured blastomere lived and continued to develop. This blas-
tomere by continued division developed into a form that may
be called a " semimorula verticalis," since it is like the vertical
half of a normal "morula." "That is to say, it is a hemi-
spherical structure Avith small deeply pigmented cells above,
and with larger non-pigmented cells below." The segmenta-
tion-cavity is often absent ; sometimes it is represented by a
few loosely aggregated cells, and sometimes by a cavity bor-
dered in part by the injured half of the egg (Fig. 35, A). A
" semiblastula verticalis " then develops wdth a well-defined
segmentation-cavity. A " semigastrula " stage is next passed
through. " Hemiembryones laterales " develop from most of
these eggs, as seen in Fig. 34, A. This figure shows that

108

DEVELOPMENT OF THE FROG'S EGG

[Cii. XI

the right half of an embryo has developed from the uninjured
blastomere. Half a medullary plate is present along the line
of separation of the injured and uninjured halves. Near the
posterior end of the half plate, the yolk of the developed half
is exposed over a small region and surrounded by half of a
blastopore (?). A cross-section of such an embryo shoAvs
(Fig. 35, B) that the half plate has essentially the same form
as half of the normal medullary plate ; that beneath this half
plate a notochord is present forming a rod, round or slightly
oval in cross-section ; that a small archenteron is present in
the developing half, and that a mesodermal sheet is present
over the side of the hemiembryo. It is interesting to note
that while only half the medullary plate is present, yet the

Fig. 35. — Cross-sections through two half-embryos of different stages. (After Roux.)

notochord and archenteron, which are also median structures,
form whole structures but of smaller size than the correspond-
ing normal organs. Roux thought that the notochord was
very probably composed of only half the number of cells pres-
ent in the normal notochord, but, owing to a great amount of
variation in the latter, it was not possible to determine this
relation definitely.

Pfliiger, Roux, and Born have shown that sometimes in the
normal development the plane of the first cleavage corresponds
to the cross-plane of the body of the embryo, i.e. the plane of
the first cleavage separates the anterior from the posterior end
of the body. Under these circumstances, if one of the lirst two

Ch. XI] EFFECT OF IXJURING A BLASTOMERE 109

blastomeres had been killed, we should haA'e anticipated, Roux
says, that "hemiembryones anteriores"' or ''posteriores" would
have appeared. Koux claims that such forms do really appear.
The same result can be obtained, if, after the second cleavage
of the eg-g, two of the four cells be killed, i.e. those two that lie
on the same side of the second cleavage-plane. A heraiembrj^o
anterior (?) is shown in Fig. 34, B. It has the anterior end
of the medullary folds normally formed, also a normal chorda,
mesoderm, and archenteron in this anterior end. In every re-
spect it corresponds to the anterior end of a normal embryo,
except that the archenteric cavity is small, resulting, Roux
thinks, from the impossibility of ])ushing the yolk-mass poste-
riorly, as is done in the normal embryo when the archenteron
enlarges. Roux is uncertain whether he has seen any "hemi-
embryones posteriores,'' although one embryo that he found,
with thick and short blastoporic lips, may represent such a
form.^ Roux made some further experiments in which one of
the first four blastomeres was killed, and other experiments in
which three of the first four blastomeres were killed. In the
first case he obtained three-fourth nioruhe and three-fourth
blastuhne; in the latter case, one-fourth blastulse and one-fourth
embryos. Roux concluded from his experiments, "that the
development of the frog's gastrula and of the embryo immedi-
ately following the gastrula-stage is, after the second cleavage-
period, a mosaic work of at least four vertical self-developing
(or differentiating) parts." "How far this mosaic work is
changed by a change in the position of material in the later
development, cannot be determined."

In later stages in the development of the hemiembryos a new
series of phenomena appear, that result in the "reorganization"

1 ^ye should expect, foUowinrj Roux's argument, to get as many heniiem-
bryones posteriores as aiiteriores, yet such does not seem to be the case.
Hertwig ('93, A) has maintained that it is absurd to suppose the posterior
end of the blastopore could appear when there is no anterior end ; but this
supposition rests, I think, on an erroneous idea of the way in which the
blastopore forms, for I have shown in my experiments ('9-i) that the poste-
rior lips of the blastopore may appear when the anterior lip has been de-
stroyed. The experiment sliould be carefully repeated with the four-cell
stage, where it is possible to distinguish the two anterior and the two posterior
cells.

110 DEVELOPMENT OF THE FROG'S EGG [Ch. XI

of the half operated upon, and in the subsequent "postgenera-
tion" of the same.

Sections of eggs that have been successfully operated upon
show the kind of change that has taken place in the injured
blastomere as a result of the operation. The yolk is found much
vacuolated in places, and the protoplasm in the immediate path
of the needle has been killed, and much changed. After a time
it is found that scattered nuclei or nuclear-like structures are
also present in the injured half (Fig. 35, A). These have come
from the regular or irregular division of the nucleus of the
blastomere that has not in most cases been killed by the hot
needle. The developed half is somewhat larger than the injured
blastomere, and a sharp line of demarcation is at first present
between the two halves. Even in the early stages of some eggs
changes are found to take place that precede the "reorganiza-
tion " of the injured half. Roux describes three sorts of re-
organization-phenomeyia. The first of these changes involves the
formation of cells in the injured half. Nuclei, surrounded by
a finely granular protoplasm, appear in the injured blastomere.
These nuclei seem to arise from two sources, — from the nucleus
of the injured blastomere, and from nuclei (or cells) of the
developing half that have transmigrated. Around the nuclei
the yolk breaks up into cells. This cellulation of the yolk may
take place at very different times. It may be absent in some
cases in a semigastrula and be present in other cases in a semi-
morula or semiblastula. The cellulation of the injured half
begins always near the developing half, and extends thence
outward. The cells of the injured half are of various sizes,
but generally larger than the cells of the uninjured half.
N The cellulation of the yolk takes place only in the unchanged

non-vacuolated parts. Where the yolk has been much changed,
it is worked over by another method, i.e. by the second method
of reorganization. These parts are revived or reorganized by
the nuclei or the cells that have now appeared in the injured
half. Such parts are either actually devoured by wandering
cells or slowly changed under the influence of neighboring cells
or nuclei so that they become a part of these cells.

In addition to the two preceding modes, a third method of
reorganization takes place. When the yolk has been much

Ch. XIJ effect of injuring a BLASTOMERE 111

injured, the surface may be subsequently covered by ectoderm
that grows directly from the developing half over the injured
portions. '-^Postgeneration'''' now begins in the cellulated in-
jured half and ultimately the missing half of the embryo is
formed. The surface ectoderm is first postgenerated either b}^
direct overgrowth from the uninjured to the injured side, or
by the formation of ectoderm from the cells of the newly cellu-
lated yolk. The missing half of the medullary folds appears
very quickly. Half a day or a night is often sufficient to change
a hemiembryo lateralis into a whole embryo with a complete
medullary plate. The mesoblast grows over to the injured
half, but increases in length and breadth by the addition of
new cells from the cellulated yolk. The formation of new
mesoderm takes place only along the free edge of that already
formed. The growth is in a dorso-ventral direction.

The archenteron is postgenerated in a way very different
from the way in which the archenteron of the normal embryo
is formed. The lacking half of the archenteron arises in
connection with the lialf of the archenteron already present
in the hemiembryo. The yolk-cells of the injured half be-
come radially arranged and a slit appears in the postgenerated
half extending out from the archenteron of the hemiembryo.
The cells surrounding the slit arrange themselves into a lining
layer and the slit opens to form tlie missing half of the archen-
teron. In general we may say that in the postgeneration
of the organs of the injured half, the changes always proceed
from the already differentiated germ-layers of the hemiembryo,
and the postgeneration takes place where the exposed surfaces
of the germ-layers touch the newly cellulated yolk-mass of the
injured half.

Further Experiments

(By Hertwig, Entires and Walter, Schultze, Wetzel, Morgan)

We may next consider the work of others, who have, after
Roux, repeated the same experiment and made further varia-
tions of it. Lastly, before a final conclusion can be reached as
to the interpretation of the results, we must carefully examine
the evidence from similar experiments on other forms. We

112 • DEVELOPMENT OF THE FROG'S EGG [Cii. XI

shall l)e then in a position to understand more fully the results
of the experiments on the frog's egg.

Hertwig ('93, b) was the first to repeat Roux's experiment,
but reached results diametrically opposed to those of Roux.
At the two-cell stage, one of the blastomeres was stuck with
a hot needle,^ but unfortunately a detailed description of the
method employed is not given by Hertwig. After the opera-
tion ^ the egg so turns itself that the uninjured part rotates
upward, while the injured half is below. This is owing, Hert-
wig says, to the development of a blastula and gastrula cav-
ity, within the uninjured and segmented half. The cleavage-
stages of the egg are not described! Sections of the blastula
stage show that in the cellulated half a segmentation-cavity,
having a thin roof, has appeared. This cavity lies, in the
present case, in the centre of the developing half. In other
embryos, the cavity may lie excentrically, and in some cases a
part of the floor of the cavity may he hounded hy the yolk-substance
of the undeveloped half Hertwig interprets these results to
mean that when one of the first blastomeres is injured, the
method of development of the other blastomere is very much
altered. The injured half lying in contact with the active
half plays only a passive role in the further development.

The injured blastomere is closely applied to the developing
half, and in places passes continuously into the latter. Hertwig
thinks that the yolk of the injured blastomere exerts on the
developing half an influence similar to that which the food-
yolk of meroblastic eggs exerts on the protoplasmic portion
that forms the embr3^o. This injured yolk-material comes to
lie in the ventral and posterior portion of the embryo.

Hertwig ventures further to prophesy that if the injured
yolk-mass had been taken altogether out of the egg-coat (^i.e.
from its contact with the living half), then there would be
formed a normal embryo without defect and like the normal
embryo in every respect except its smaller size.

It is of importance to note that Hertwig describes other

1 In a few cases a galvanic stream was used to kill the blastomere.

2 How soon after is not stated.

Cri. XI] EFFECT OF IXJURIXG A BLASTOMERE US

embiyos that he obtained by Roux's methods, and contrasts
these with those described above. Some of the embryos showed
the condition of spina bifida, i.e. with both sides of the body
developed and witli a hirge yolk-exposure in the mid-dorsal
line.^ Others of the embryos Avere only slightly injured by
the operation and developed nearl}- normally. In these the en-
tire dorsal region was ^^•ell developed and the blastopore closed
to a small ring. Only on the ventral side was a small defect
found where the outer and middle germ-layers were absent.
In these latter embryos, and in those showing spina bifida,
Hertwig believes the injured blastomere was not killed or
even sufficiently injured to prevent its partial development.
That this is the true explanation cannot be doubted ; for it is
not at all unusual to find after the operation that the injured
blastomere may separate oft' small portions of itself as cells that
develop along with the cells from the uninjured half. Here, it
seems to me, is the uncertain part of Hertwig's work. He has
not observed, as far as stated, the segmentation of each egg on
which he has operated, and consequently his results are open to
the objection that in many cases, where he does not suspect it,
the injured cell has also continued to divide and to form a [)art
of the later embryo.

In nearly all of the embryos described by Hertwig the
medullary folds are unequally developed. ^ Hertwig's attempts
to meet this fact do not seem to me altogether satisfactory. A
large number of the embryos have developed unsymmetrically.
The ventral and posterior yolk-mass lies higher up on one side
than on the other. In consequence of this, one side of the
medullary fold lies nearer to the injured yolk than does the
other, and as a result the two sides of the body are unevenly
developed. The asymmetrical position of the blastopore on
the living part is assumed to be the underlying cause of the
later asymmetrical position of the medullary folds ; but for
the primary reason of the lack of symmetry of the blastopore
itself Hertwig gives really no explanation, and to state that it

1 Among these embryos Hertwig describes one that seems to have been an
excellent example of Roux's "hemiembryo lateralis."
- There are a few exceptions.

114 DEVELOPMENT OF THE FROG'S EGG [Cii. XI

is due to the "yolk lying higher up on one side" is only begging
the question. Roux has not failed to notice the incomplete-
ness of Hertwig's explanation, and has interpreted all of Hert-
Avig's results as due to a sudden postgeneration of the injured
half of the embryo; i.e. Roux believes a half-embryo to have
first formed, and then to have been quickly followed by an im-
perfect formation of the other half. Hence the asymmetry of
the embryos.

It is impossible to say how far postgeneration has played
a part in the development of the embryos described by Hert-
wig, but that postgeneration will explain all the difference
between the results of Roux and of Hertwig seems highly
improbable. Further, as I have said, it seems not unlikely
that many of the embryos described by Hertwig have come,
not only from the uninjured blastomere, but also from a part
of the injured hlastornere. If this latter supposition be true,
we can better understand why the injured yolk forms in many
cases an integral part of the developing embryo.

Hertwig has made a most formidable attaciv on Roux's expla-
nation of postgeneration of the embryo. The subject itself is of
secondary importance as compared with the main problem in-
volved in the experiment, and yet of sufficient interest to war-
rant careful examination. Roux describes the blastomere into
which the hot needle has been plunged as dead, and speaks of a
later revivification of the dead half of the &gg. Hertwig be-
lieves that all of that part of the operated blastomere that is
later divided up into cells (to be used in the development) is
not dead, but only more or less injured. Only a small portion
of the injured blastomere is really dead, and that is the por-
tion which has become coagulated by the hot needle. This
portion cannot later be broken up into cells, but may be either
thrown out by the living embryo or assimilated, owing to the
power of digestion of neighboring cells. The iujured blasto-
mere behaves in the same way that a portion of the body of an
animal would if a needle had been stuck into it. The place
injured might quickly heal, and the comparatively small region
that had been pierced and killed would be reabsorbed again.
\i the needle had been first heated, the region of injury would
only be larger, and the necrotic tissue would be either throAvn

Ch. XI] EFFECT OF IXJURING A BLASTOMERE II5

off or absorbed. It has been shoAvn by Roux that when a
blastomere has been pierced by a cold needle, there is a small
outflow of yolk, and the injured blastomere continues to divide
at the same rate as the uninjured cell. When the needle is
heated, the cleavage-process is delayed or prevented, while it
continues on the uninjured side ; but after a time the injured
blastomere may also begin to divide in an irregular way.
After two or three days one gets generally from such eggs
quite normal gastrulce and embryos, differing in little or no
respect from embryos from uninjured eggs.

The nucleus of the uninjured blastomere may continue to
divide, although the protoplasm, owing to its injury, may not
be able to do so for some time. The nuclei may scatter them-
selves through the protoplasm (and yolk), and subsequently
take part in the division of this into cells. In extreme cases
Hertwig admits that wlien the needle is very hot, the whole
of the protoplasm of the blastomere may be killed, and also
the nucleus. Furthermore, it is possible that occasionally the
heat may radiate from the one blastomere into the other and
partially kill this other one also. If the last condition is
brought about, the development of the partially injured blasto-
mere may take place only very slowly, if at all. In most cases,
therefore, Hertwig believes a "reorganization" of the injured
cell takes place, and not a " revivitication "' of the dead half.
In this reorganization, Hertwig thinks that the nucleus of the
injured cell itself plays the main part, while Roux believed
the process was brought about largely by an immigration of
cells (or nuclei) from the uninjured into the injured half.
Hertwig's conclusion here seems based rather on a priori
probability, while Roux's statements rest directly on his own
observations. Recently the same ground has been Avorked over
by Endres and Walter, whose results substantiate Roux in
every respect.

Endres and Walter ("95) have obtained the typical half-
blastulfe and lialf-gastruke and half-embryos which Roux has
described. They deny that tvJiole embryos develop from one of
the first two blastomeres, as Hertwig affirmed. Their figures
show in the most conclusive way that half-embryos do develop

116 DEVELOP.MENT OF THE EliOG'S EGG [Ch. XI

under tlie conditions of Roiix's experiment. The subsec.uent
postgeneration of the injured half of the egg has also been
studied by these authors. They confirm in every detail the
method of reorganization and postgeneration of tlie injured
half as described by Roux. The reorgamzing cells have migrated
Pom the wiinjured to the injured side, and there have caused the
protoplasm to break up into cells. The injured blastomere is
also overgrown directlg by the ectoderm of the uninjured and
developing side. In many of these embryos the right and
left side (one side has postgenerated) are separated from each
other by a protruding yolk-mass, forming spina-biiida embryos.
Ihe reorganization of tlie much changed mass of the injured
blastomere is brought about by being assimilated by the cells
that have migrated into that region, by the second and third
methods of reorganization described by Roux. When the mate-
rial of the mjure.l half is only incompletely reorganized, there
IS formed after postgeneration, a more or less pronounced spina
bihda. When the injured material is completely worked over
or reorganized and postgenerated, a perfect embryo mav be
formed.

Schultze ('94, b, d) has made an interesting modification of
one of the experiments of Pfltiger and obtained most unex-
pected results. The eggs of Rana fusca removed from the
uterus were placed singly upon slides. On each slide had
been stuck two thin glass rods from 1.65 to 1.35 mm. in thick-
ness Between these rods, which are separated from each other
by the width of the slide, an egg is placed with tlie ^ohite pole
uppermost. The egg is then fertilized in this position. After
three minutes the spermatozoa may be supposed to have entered
and a glass cover is placed over the egg and brought down into
conflict with the two glass rods above-mentioned, and there
fixed with rubber rings. The egg is by this means slightly
coinpressed and held more or less firmly in position. Each
slide IS then turned over, i.e. through 180 degrees, so that the
dark pole of the compressed egg is brought upward. The
eggs now m the^iormal position are put into a dish of water,
to remain m this position until the first furrow has appeared or
even until it has passed through the egg. Then the slide and

Ch. XI] EFFECT OF IXJURIXG A BLASTOMERE

11'

its egg are agaiyi rotated through 180 degrees^ so tliat tlie white
pole is once more turned uppermost. Owing to the compres-
sion, the eggs retain their inverted position.

After twenty-four hours at 17 degrees C, tlie gastrulation
begins. The rubber bands are then removed from the slide,

Fig. 36. — Double embryos. A. Section through segmentin.o; egg. (After Wetzel.)
B. Double embryos uuited veutrally. C, D. Double embryos united dorsally.
(After Schultze.) E. Cross-section through C. (After Wetzel.) F. Double
embryos united laterallj", and G, cross-section of same. (After Wetzel.)

the cover-slip carefully cut away from the jelly of the egg, and
the slide and egg returned to the water.

If eggs that have been inverted after the two-cell stage are

118 DEVELOPMENT OF THE FROCx'S EGG [Cii. XI

watched during the later cleavage-period, it will be found
that the upper Avhite surface disappears, and often a whitish
band IS found in the position of the first furrow. Continuous
observation also shows that the white hemisphere may slowly
snik to one side. At thirty hours the blastopore has appeared
m the normal eggs, while on the inverted eggs tivo gastrula-
invaf/uiations are found. From each half of the egg a more or
less complete embryo may develop (Fig. 36, B, C, D). The two
"double monsters" are united to each other in various ways,
often with the two ventral surfaces united in one common yolk-
mass, as shown in Fig. 36, B. Another of these double forms
is shown in Fig. 36, C, D, and a cross-section through the bodv
in Fig. 36, E. & y

The details of these experiments of Schultze have not yet
been published. The method of gastrulation of the halves is
not clearly explained, nor does Schultze explain the changes
that take place in the interior of the blastomere after the
rotation. The results show, however, in the clearest way that
each half of the egg, after the first division, has the power to
develop all the organs of a single embryo.

Wetzel ("95) has more recently studied the gastrulation-pro-
cess in some of these embryos and has given a fuller descrip-
tion than Schultze of the origin of the archenteron. A cross-
section through the blastula-stage of one of these eggs is shown
in Fig. 36, A. Two distinct segmentation-cavities are present
m the upper or white hemisphere of the egg. Tlie centre of the
double blastula is filled with large yolk-cells. The sides are
formed of smaller cells richer in protoplasm and pigment. The
structure of this double blastula shows that, in all probability,
the contents of each of the first two blastomeres have rotated
after the inversion of the egg so that the more protoplasmic
portions have come to lie at the outer and upper sides of each
blastomere ; while the heavier yolk has sunken down to the
lower surface along the cell-wall that separated the first two
blastomeres from each other.

At a later stage a depression appears on the surface of the
egg in the region of the plane that separated the first two blas-
tomeres from each other, i.e. approximately in the plane of the

Ch. XI] EFFECT OF INJURING A BLASTOMERE 119

first cleavage. This depression or groove on the surface may
divide at eitlier end into. two distinct and independent grooves.
Cross-sections through such an egg show tliat the groove on
the surface is the result of an invagination to form an archen-
teron in each half. This means that each half-blastula has
begun to invaginate along the common line of contact of the
halves. Since the halves are in contact, the overgrowth of
each blastopore is impossible. The lips of the blastopore of
each half, therefore, have extended around the equator of the
egg as in the spina-bifida embryos. A medullary fold appears
later along each blastoporic rim, and then it becomes apparent
that two embryos are present, each a spina bifida, and united
by a common central yolk-mass (Fig. 30, C, D, E). The open
dorsal surfaces of these two embryos are turned toward each
other (Fig. 36, E).

This seems to be the more common type of double monster
produced from these eggs. If, however, the blastoporic invagi-
nations begin at different regions of the two hemispheres, many
possible variations of the method described will be introduced ;
Schultze and Wetzel have in fact, as we have seen, described
several forms of these double monsters. (Fig. 36, B, F.)

It seemed to me not improbable that Schultze's results
explain in part the difference in the results of the experiments
of Roux and of Hertwig. If, on the one hand, the uninjured
blastomere retain its normal position after the operation,
i.e.. with the black pole turned upward, then there should
develop a half-embryo, in Roux's sense. On the other hand,
if, after the operation, the position of the egg be reversed so
that the white pole of the uninjured blastomere is turned
upward, then a whole embryo of half-size might develop. In
Roux's experiment it is probable (although not explicitly
stated) that the black hemisphere always remained upward
after the operation. Hertwig does not say in what position
the eggs lay in his experiments. He only says that in the blas-
tula and gastrula stage the heavier injured yolk was down, and
the lighter uninjured blastomere was above. If, immediately
after the operation, the eggs lay with the injured blastomere
below, we should expect some change to take place in the

120 DEVELOPMENT OF THE FROG'S EGG [Ch. XI

interior of the uninjured blastomere as a result of its oblique or
even inverted position; hence the uninjured blastomere might
develop differently than it would have done had it retained its
normal position (as in Roux's experiment). In this way we
might attempt to reconcile, in part, the different results of
Roux and Hertwig. I cannot but think, however, that the
main difference is due to the partial development of the injured
blastomere in many of Hertwig's experiments, so that cells split
off from the injured blastomere took part in the formation of
the embryo.

In 1894 I made the following experiments to determine
whether one of the first two blastomeres could give rise
to a half or to a whole embryo, according to the condi-
tions of the experiment. One of the first two blastomeres
was killed with a hot needle in the way described by Roux
('93, c).i

In some of the eggs the black pole remained upward after
the operation; other eggs were rotated after the operation,
so that the white pole was turned upward. The eggs were
closely watched for several hours, in order to ascertain with
certainty Avhether the injured half divided or not. In those
cases in which this happened, the eggs in question were elimi-
nated from the experiment.

The eggs were placed at first on a moistened glass plate and
kept for a time in a moist atmosphere, or else simply thrown
into water. The results seemed to be the same. When the
black pole of the uninjured blastomere remained up, the blas-
tomere developed, in all the cases observed, into a half-emhryo.
Conversely, those eggs in which the white pole was turned
upward, formed, in most cases, wliole embryos of lialf-size. In
the latter case the cleavage was modified in consequence of the
reversed position of the egg. The upturned white hemisphere
produced smaller cells than the lower black hemisphere, point-
ing unmistakably to a rotation of the fluid contents of the
blastomere.

1 The needle was heated each time before piercing an egg. This made a
greater injury to the blastomere much more certain. On the other hand, it
lowered the percentage of embryos obtained, because in many cases the other
blastomere was probably injured also by the heat.

Ch. XI] EFFECT OF INJURING A BLASTOMERE 121

The lialf -embryos and the ivliole embryos of half-size developed
independently of the yolk-mass of the inj-ared side. In this
respect my results differed very materially from the results of
Hertwig. ^Nlany of Hertwig's embr3'os developed in connection
with the injured blastomere ; mine, on the contrary, developed
independently of the injured blastomere. I suspect, as I have
said, that this difference may be in part due to this, tliat Hert-
wig did not carefully remove from his experiment those eggs
in which the injured blastomere continued to segment, and that
cells from the injured blastomere took a direct part in the sub-
sequent development.

In one of my experiments, in which the uninjured blasto-
mere had been reversed after the operation, it developed into a
half-embr3'o, and not into a whole embryo of half-size. More-
over, in this embryo the medullary folds appeared on the white
surface of the Qgg^ showing that a rotation of the contents of
the blastomere must have taken place. We must, therefore,
conclude that the simple fact of the rotation of the blastomere-
contents is not, in itself, the determining factor as to whether
a whole or a half-embryo will result, but probably the kind of
rotation determines this result. The result may also depend
in part, I think, upon how far the contents of the uninjured
blastomere have retained, after the operation, their organic
connection with the other injured blastomere.

In later papers Roux stated that he has often obtained in his
experiment other sorts of embryos than those he first described,
which he calls "hemiooholoplasten." These are ivliole embryos
that have come from the uninjured blastomere without the
postgeneration of the other injured blastomere. Roux inter-
prets these as embryos "completely postgenerated," with only
a partial use of material from the other side, or even with no
material from the injured side. Roux affirms that he has seen
all intermediate stages between those embryos that have used
all of the yolk-material of the injured side, those that have used
only a part of the material of the injured side, and those that
have not used any of this material. These embryos differ from
one another only in point of size. Roux does not call the em-
bryos that have developed entirely from the material of the

122 DEVELOPMENT OF THE FROG'S EGG [Cir. XI

non-injured side, whole embryos of half-size, but he believes
that at first there formed a half-gastrula, then a half-embryo.
Later this half-embryo completed itself without using material
from the injured side! That is to say, b}^ using "wandering
cells " the half-embryo has jjosfgenerated ^he other half of its
body !

CHAPTER XII

INTERPRETATIONS OF THE EXPERIMENTS; AND CON-
CLUSIONS

The results of the experiments of Pfliiger, of Roux, and of
others have given rise to much discussion in respect to the
relation existing between the unsegmented egg and the embryo.
The old questions of evolution and epigenesis have been once
more brought into the foreground, but divested of their historic
meaning.

The results of the experiments on the frog's egg are, how-
ever, in the first place, too insufficient in themselves, and in the
second place are as yet too uncertain on many points, to warrant
general conclusions based on these results alone. The experi-
ments can only be understood if considered in connection with
similar experiments on other groups of animals.

Roux's Mosaic Theory of Development

Roux's discussion of the problems of development is deserv-
ino- of most careful examination, for even in his earliest papers
we see foreshadowed many of the possible interpretations that
have later been accepted in one or another form. Roux pointed
out that the known facts of development showed that a certain
formal self-differentiation of many parts of the egg takes place
during development. This self-differentiation may result from
an unequal growth of different substances in the egg which
come into activity at different times ; and if so, it should be our
aim to discover the stimuli that bring these different substances
into action, and thus cause the consecutive series of eveftts.
The stimuli must come either from without at each stage of
development, or the egg may contain within itself the power of
progressive development as soon as it is once set into activity.
That the egg needs during its development certain things from

123

124 DEVELOPMENT OF THE FROG'S EGG [Ch. XII

its environment is self-evident; a certain amount of warmth
and of oxygen, etc., must be present. These, while necessary
for the development of the egg, do not necessarily determine
the sequence of events ; for under the same external conditions,
eggs of different animals develop very differently. The results
obtained by placing the frog's egg under different conditions
also show that the power of progressive development must lie
within the egg itself. Roux compared the egg, in this respect,
to a complicated piece of machinery which, when once set in
motion, would go through a long series of changes depending
on its internal structure.

If so much be granted, the next question to be answered is
this : do all the parts of the dividing egg work together, i.e. in-
teract to form the result, or have the parts of the egg separated
from one another by the cleavage the power to develop inde-
pendently? The first alternative Roux called the differentiat-
ing interaction of the parts, and the latter alternative, the self-
differentiation of the parts. With reference to the results of
the experiment in which one of the tirst two blastomeres of the
frog's egg was killed or injured, Roux concluded that each of
the first two blastomeres shows in this experiment the power
of self-development: i.e. each half is independent of the other
and we may legitimately infer that when both blastomeres are
alive, as in the normal development, the same self-differentia-
tion of each blastomere takes place. This independent devel-
opment goes on till the organs of the body begin to form.
Whether the limit of independent development is then reached
we do not know, for it is possible that in the complicated series
of movements that take place in the formation of some of the
organs, the power of independent development may be sup-
plemented or replaced by the action resulting from the cor-
relation of the parts to one another, i.e. by a mechanical
interaction of different parts. Each of the first two blasto-
meres contains not only the building-material for the corre-
sponding parts of the embryo, but also the dfferentiatiyig and
formative forces for those parts. The cleavage in the direct,
or normal development of the individual, divides qualitatively
the "germ-plasm," and, in particular, the nuclear material.
The development of the frog's gastrula and of the embryo

Cii. XII] INTERPRETATIONS AND CONCLUSIONS 125

immediately resulting" from the gastrula is, from the second
furrow on, a mosaic work of at least four vertical, independent
pieces. How far this mosaic work of four pieces is altered by
later changes in the position of the material, and by differentiat-
ing correlation, is not known.

Koux also stated clearly the relation that exists between the
method of self-differentiation, and the method of interaction
of the parts on one another, and the bearing of these questions
on the older problems of evolution and epigenesis. If many
portions of the egg are differentiated owing to their inherent
power, and produce in this way the manifold differentiations
seen in the embryo, then the egg must have been composed
in the beginning of many parts bound up together, and the
development is a metamorphosis or an unfolding of its pecu-
liarities ; i.e. the development is an evolution. Further, the
cleavage not only divides the egg into smaller parts, but at
the same time localizes the differentiated material, so that this
material is arranged definitely in relation to later development.
This result appears possible only through a qualitative sepa-
ration of the material during the course of the cleava<xe. If
this is true, we see that the development depends on the
molecular structure of the egg, and therefore further analysis
is beyond our reach. The seijmented egg would be then only
the sum of its independent parts, and during the period of the
self-differentiation of these parts, there has been no united
action to form a whole. Therefore the whole can have no
regulating or formative influence on the parts.

If this view be true, His's principle of germinal localization
in the egg has not only a descriptive worth, but also expresses
a causal relation, so that organs can be referred to parts of
the fertilized egg, and even to the unfertilized egg.'^

If, on the other hand, development takes place as a result
of the interaction of all or many parts on one another, then
the fertilized egg may be composed of a very few differentiated
parts, which by their interaction produce a greater and greater

1 "We could explain those exceptional cases in which two embryos arise fi-om
one egg, if we supposed that after the first cleavage there was a sort of doubling,
in each blastomere, of the primary constituents of the body (Roux).

126 DEVELOPMENT OF THE FROG'S EGG [Ch. XII

complexity. The development would then be due to the pro-
/duction of man}^ parts out of a few primary ones, i.e. the de-
velopment is a process of epigenesis. There would thus result
an ever-changing interaction of the parts to form the whole,
by which means there would be also brought into play a regu-
lating influence of the whole back again on the parts, i.e. corre-
lation of the parts under the influence of the whole. His's
principle of germinal localization would, therefore, have a
causal meaning only in so far as it points out the place in the
Q^g where the resulting formation of many-sided changes takes
place ; ajid it would be of only secondary value to be able to
refer the place of action of these changes to the undifferen-
tiated plasm or to the unfertilized Qgg.^

In conclusion, it should be noted, Roux said, that self -differ-
entiation of the parts and dependent differentiation of the parts,
i.e. evolution and epigenesis, may be combined in a many-sided
activity or union, and it would then be our duty, in order to
interpret these problems, to use a double foresight and a double
care, to make out the part played by each of these factors in the
development.

Theory of Deiesch and of Hertwig of the Equiva-

LEXCY OF the EaRLY BlASTOMERES

Studies on other forms show that great care must be taken
in interpreting the results of the experiments on the frog's Qgg.
In 1891 Driesch made a series of most important experiments
on the eggs of the sea-urchin. ^ The blastomeres were isolated
by shaking them apart, and it was found that although each
blastomere segmented as a part, i.e. as if still in contact with
the missing half, 3'et the open side of the blastula closed over
very soon, and a gastrula and embryo having the normal form
were produced. Driesch concluded from this and similar experi-
ments that all the blastomeres are equivalent, and that the posi-
tion of each blastomere in the segmenting Qgg determines in

1 The formation of two embryos from one egg would take place, on the theory
of interaction of the parts, at the time when the median axis of the body is formed.
Two such axes would be laid down instead of one.

2 Fiedler had made, in 1891, a somewhat similar experiment, but it was not
carried sufficiently far to be of great value.

Ch. XII] IXTERPRETATIOXS AXD COXCLUSIOXS 127

o-eneral the fate of the blastomere. If the bListomeres could be
interchanged as can the individual marbles of a heap, then the
fate of each would be determined by its new position in the
whole. This conclusion is directly opposed to Roux's theory
of a qualitative division of the blastomeres during the early
cleavage.

Hertwig had also stated shortly before Driesch that in his
opinion the egg divides quantitatively, and that Roux's experi-
ments did not touch the cardinal point of this problem, because
the other injured half of the egg remained in contact with the
developing half. Hertwig expressed his belief that if the hrst
two blastomeres of the frog's egg could be separated from eacli
other, each would develop into a whole embryo. Further, he
thought that the development of an organism is not a mosaic
work, but that tlie parts develop in relation to one another, i.e.
the development of a part is dependent on the development
of the whole. Wilson (T>3) also, from the results of a most
careful and important series of experiments on the egg of
Amphioxus, concluded that the division of the egg is not quali-
tative. He found that isolated blastomeres give rise to larvae
smaller in size than the normal, but having the normal form.
The differentiation of the blastomeres, Wilson thought, takes
place in the later periods of cleavage.

Roux's Subsidiary Hypothesis

Roux replied to the criticisms that Driesch, Hertwig, Wil-
son, and others have made of his theory, and attempted to show
that his view is fully compatible with the results that Driesch
and others obtained.

RoUx ('92, a, '9o, b) pointed out that the results of Chabry,i
Fiedler, and Chun show that in ascidians, sea-urchins, and
ctenophors a half-development takes place when one of the
first two blastomeres has been removed, and that the experi-
ments of Driesch also showed that an isolated blastomere
of the egg of Echinus cleaved as a half, and not as a whole,
and that a half-blastula also developed. These results indi-

1 Later experiments have shown that this statement is not true for ascidians,
as Chabry's work might seem, in part, to show.

128 DEVELOPMENT OF THE FROG'S EGG [Cn. XII

cate that a certain formal self-differentiation of many parts
of the segmenting egg has taken place. On the other hand,
the fact of postgeneration shows that in each of the first
blastomeres a power sufficient to complete the whole must
also be potentially present. In order to awaken this potential
power of a blastomere, a disturbance in the develoiDment must
occur. This latent activity may be only slowly awakened in
the development, sometimes sooner, sometimes later.

We have, therefore, to distinguish two sorts of development,
— the normal "direct" development, and an ''indirect" post-
generative (or regenerative) development. The first or direct
is the result of the self-differentiation of the early blastomeres,
and of the complexity of their derivatives. T'he second or
indirect is the result of a profound correlation which adds to
an imperfect whole the lacking parts. Should the postgenera-
tion set in immediately after the isolation of the blastomere
and so convert the blastomere at once iiito an actual whole,
then we should not have found out that each blastomere is
really a self-differentiating cell, but we should have erroneously
concluded that the first (four) cleavage-cells are qualitatively
equivalent. Into this error Roux believed Driesch and Hert-
wig to have fallen. In the frog, ascidian, and ctenophor each
of the first blastomeres is specifically different from the others,
but in respect to postgeneration we find that each blastomere
has the same potentiality, and each is in reality totipotent.
The '•'•idioplasm" in direct (i.e. normal) development, called
into activity by the process of fertilization, is divided qualita-
tively and unequally during the cleavage, while the material
which may later serve for postgeneration and regeneration
(which is not active during the normal development) is always
equally or quantitatively divided.

According to Roux, the nucleus represents the controlling
power of the cell, but the protoplasm acts as a stimulus to
the nucleus and hence may indirectly regulate the process of
cleavage. " In the telolecithal frog's egg the position of the
food-substances and formative substances stands in strict causal
relation to the position of the main axes of the embryo." The
nuclei of eggs in which the normal arrangement of the contents
has been disturbed will be influenced during the first cleavage-

Ch. XII] IXTERPRETATIOXS AXD COXCLUSIOXS 129

period, so that a qualitative division of the nucleus may result
different from the corresponding normal qualitative division.
The second cleavage, for instance, may come first (qualitatively)
as a result of the position of the nucleus in the protoplasm.

Roux further suggested that the consecutive series of nuclear
divisions must be different in kind in the normal and in the
compressed eggs, and that an " anachronism *' has taken ]ilace
in the latter case. By this '' anachronism '" Roux has tried to
save his theory of qualitative division of the nucleus during
the cleavage-period.

To sum up Roux's later position, we may say that in order
to vindicate his earlier theory of a qualitative division of the
nucleus and a resulting self-differentiation of the first-formed
blastomeres, he has been obliged in the first place to bring for-
ward his theory of postgeneration, assuming that along with the
qualitative division of the nucleus a parallel quantitative divi-
sion of the germ-material also occurs. Further, Roux assumes
that the kind of qualitative division of the nucleus is directly
influenced by the arrangement of the protoplasm, and, as we
have seen above, he is unable to explain satisfactorily the
results of the experiment of the compressed Qgg^ except as an
''anachronism." These complications into which Roux has
been forced are largely the outcome of the primary assump-
tion of a qualitative division of the nucleus. This Roux-Weis-
mann hypothesis of qualitative nuclear division has, however,
no known histological facts in its favor. On the contrary, all
we know of nuclear divisions speaks clearly in favor of an exact
division of the chromatin-material, and a most elaborate mech-
anism is present to bring about this result.

Experiments on Other Forms

The results obtained from a study of the development of
fragments of the unsegmented Qgg and of isolated blastomeres
of ctenophors^ have a direct bearing on our interpretation of the
experiments on the frog's Qgg- When the first two blastomeres
are separated from each other by a sharp needle or cut apart by
a pair of small scissors, each continues to cleave as a half. i.e.

1 Chun ('92). Driesch and Morgan ('95).

130 DEVELOPiMENT OF THE FlIOG'S EGG [Cn. XII

as though it were still in contact with its fellow-blastomere.
When the organs aj^pear in the larva, only half the full num-
ber of rows of swimming-paddles appear. Each row, hoAvever,
has its full complement of paddles. The invagination of ecto-
derm to form the " stomach " is very excentric in the half-larva,
but forms a closed tube running from the mouth-opening to the
excentric sense-plate. In several respects, therefore, the larva?
were distinctly half-larvte. But in another respect they were
more than half-larvse. The endodermal cells of the normal
larva arrange themselves into four hollow pouches, and the
" stomach " invagination passes in the central line of the four
pouches. In the half-larva, on the contrary, the endodermal
mass forms more than two pouches (i.e. more than half the
normal number in the whole larva). Two distinct pouches
are present and in addition, generally, a third smaller pouch is
formed. The latter lies excentrically. In the meeting-point
of the three pouches is the excentric " stomach " invagination.

The isolated one-fourth blastomere segments also as a part
of a whole, and develops in some cases into a one-fourth larva,
having only ttuo roivs of paddles (i.e. one-fourth the normal num-
ber), but with tivo endodermal pouches (i.e. wdth one more than
one-fourth the normal number). The three-fourth embryos
develop six rows of paddles (i.e. three-fourths of the normal
number) and four endodermal pouches. The problem is here
a complicated one, for while in one set of organs we find a half-
development, in otJier organs we find more than a half, but yet
not the whole development.

The results show, however, beyond question, that, even when
isolated from its fellow, the one-half blastomere may give rise
to a larva that is in many respects only one-half of the normal
larva.

There is yet to be described another series of experiments
that have a direct bearing on the interpretation of the preced-
ing results. Roux showed that if a part of the protoplasm be
removed from the U7isegmented frorf s egg, -the egg may continue
in many cases to develop into a normal embrvo. The eecs of
the sea-urchin lend themselves much more readily to this ex-
periment. They may be broken up into fragments of all sizes

Ch. XII] INTERPRETATIOXS AXD COXCLUSIOXS 131

if shaken in a small tube. Those fragments which contain the
egg-nucleus may be fertilized and will develop. If the pieces
are large enough a gastrula is formed, and still larger pieces
develop into normally formed larv?e.

When the unsegmented egg of the ctenopJior is cut into pieces,
there may result either a whole larva or a larAa lacking certain
parts, and, further, the study of the cleavage of these egg-
fragments shows that if the fragment cleaves like the whole
e^g (but with smaller blastomeres) then a whole larva results,
while if the cleavage is irregular the larva is also imperfect.
Presumably, in the first case tlie egg has been cut symmetri-
cally, but in the second case unsymmetrically. Or we might
assume that in the one case the egg-fragment rearranged its
protoplasm into a new whole, while in the second case it
was unable to do so. On either alternative we must conclude
that a defect in the protoplasm often brings about a modified
cleavage and also a defective embr3'o, and this takes place even
although the whole of the nuclear material of the unsegmented egg
remains present. There seems, therefore, no escape from the
conclusion that in the protoplasm and not in the nucleus lies the
differentiating power of the early stages of development.

General Conclusions

We have seen that one of the first two blastomeres of the
frog's Qg<g may develop into a half-embryo, or into a whole
embryo of half-size, according to the conditions of the experi-
ment. So long as the first tAvo blastomeres remain in contact,
without any disturbance of the cell-contents, each blastomere
develops its half of the body. On the other hand, if the proto-
plasm is disturbed by reversing the position of the ^gg after
the first cleavage, there generally results a whole embryo from
each blastomere. Unfortunatel}*, jt has not been found possible
to separate completely from each other the first two blasto-
meres of the frog's Qgg., so that we do not knoAv whether
a whole embryo of half-size or a half-embryo Avould result.
In other animals (Eehinodermata, Hydromeduste, Teleostei,
Amphioxus, Ascidia, and Salamandra) each of the first two
blastomeres, if separated from its fellow, develops into a whole
embryo, regardless of the means employed to separate the

132 Di: VELOPMFVT OF THE FROG'S ECU. [Cn. X\l

bLastomeres.i On the other hand, the isolated hlastomere of the
ctenophor-egg develops into a half-embryo. These experiments
show that the half-development of the frog's ego- need not be
the result of the presence of the other blastomere, as has been
suggested. This is also shown by Schultze's experiment, in
which, although both halves are present and in contact, each
blastomere develops into a whole embryo.

The results show that in general the first blastomeres are
totipotent, i.e. each has the power to produce a whole embryo
if separated from its fellow, even although it may under cer-
tain conditions produce only a half-embryo, as in the frog.
Nevertheless, in most forms each isolated blastomere continues
to segment as though still in contact with the other half. Tliis
latter phenomenon shows that the egg-protoplasm has a defi-
nite arrangement according to which the cleavage peculiar
to each kind of egg is brought about, and there is sufficient evi-
dence to show, I think, that this is a cytoplasmic phenomenon,
and is not the result of nuclear interference. We have also
seen that some of the isolated blastomeres that cleave as a
part may later develop into whole larvK (echinoderms), while
other blastomeres that cleave as a part may give rise to half-
larvce (ctenophors). That these phenomena too are dependent
directly on the cytoplasm is shoM'u by the experiment of cut-
ting a piece from the unsegmented egg. Under these circum-
stances, the nucleated fragment of the echinoderm-egg gives
rise to a whole embryo, although it segments as a part, while in
the ctenophor an imperfect embryo is generalhj formed. The
results in these two cases are nearly the same as when the blas-
tomeres of the respective eggs are isolated, although in the
latter experiment the entire segmentation-nucleus is present.
In the ctenophor the process of self-regulation seems to be

1 Roux ('95) has stated that the development of a half or a -whole embryo
may depend upon the method employed to separate the blastomeres. If shaken
apart, -ivhole embryos result; if cut apart, half-embryos. Zoja's results ('95)
refute such an interpretation. He cut apart echinoderm and hydroid egss and
yet got whole embryos. On the other hand, when the blastomeres of the cteno-
phor are cut apart, half-embryos result. It nuist. however, be admitted that
disturbance of the contents of an isolated blastomere might be favorable to
V, 'Vile <:i'. ■■ jlo^iment. .'i.- in liie

Ch. XII] IXTERPRETATIOXS AXD COXCLUSIOXS 133

largely absent, either because the blastomeres cannot be brought
into a new whole, or because the protoplasm is so fixed, so stiff,
that it cannot readily rearrange itself. If from either of these
conditions, or from some other, the blastomeres are not capable
of rearrangement or reconstruction, an imperfect embryo re-
sults.

How far does the totipotence of the blastomeres reach? Does
it end with the two-cell, four-cell, or later stages of cleavage ?
Probably this varies in different eggs. The one-fourth blasto-
mere of Echinus can form a perfect embryo, and even the one-
eighth blastomere may develop into a gastrula. The same is
true for the egg of Amphioxus. For the frog it is not yet
possible to say where the limit lies. In this connection the
following facts are of importance. The isolated blastomere
of the sea-urchin's egg runs through the same number of divi-
sions that it would have done had it remained in contact with
its fellows.^ Hence the half-embryo has only one-half the
number of cells of the normal embryo, and the one-fourth em-
bryo has only about one-fourth the full number. This seems
to give, in part, an explanation of the statement made above,
viz., that the one-half embryo develops further than the one-
fourth, and the latter further than the one-eighth, since the
smaller the isolated blastomere the fewer are the cells it pro-
duces from which the embryo is formed. The lack of power of
development of the small isolated blastomere is not, therefore,
dependent on its differentiation. This is also shown by the fol-
lowing experiment. In the blastula-stage of the sea-urchin's
egg, pieces may be cut or shaken from the blastula-wall, and,
if large enough, they develop into small larvae. Here also we
find that the large pieces can go further in the ontogeny than
the smaller pieces, probably owing to the presence of a sufficient
number of cells or of sufficient material to form the necessary
organs of the embryo. ^

If the early blastomeres are totipotent, what brings about the
later differentiation of these cells ? There are sufficient reasons.

1 Morgan ('95).

2 The same experiment cannot be made on the frog's blastula, because, if
cut, the pieces immediately disintegrate.

13J: DEVELOPMENT OF THE FROG'S EGG [Ch. XIl

I think, to conclude that the power of differentiation lies within
the egg itself, and does not depend directly on external stimuli.
We have seen that Roux and Weismann (particularly the latter)
explain this differentiation of the cells as a result of the quali-
tative division of the nucleus from the very beginning of the
cleavage. The nucleus, unravelling its qualities at each divi-
sion, sends into each cell the proper constituents, and the nuclei,
then acting on the cell-protoplasm, cause it to differentiate.
On the other hand, Hertwig contends that the early blastomeres
are equivalent, and that differentiation is brought about by the
interaction of the blastomeres. In other words, any blastomere
that has come to occupy a given position has its fate sealed, be-
cause in this position it bears a certain relation to the other
blastomeres of the whole ; the whole being simply the sum-total
of the blastomeres present. But it is impossible to imagine that
the interaction of strictly equivalent blastomeres could bring
about a self-differentiation. If it is assumed that the gross-
contents (such substances as yolk, etc.) determine the differ-
entiation of each part, still the hypothesis is obviously insuffi-
cient for all cases, because, as we have seen, fragments of ani/
part of the egg of echinoderms develop into whole embryos,
and fragments even of the blastula form new blastulae, gas-
trulce, and embryos. Some of these small blastulte represent
only the " animal " half of the original blastula, and the cells
will not, therefore, contain any of the protoplasm or yolk that
the cells usually contain that are invaginated, for all this por-
tion of the blastula has been cut off. And since these "ani-
mal" pieces gastrulate, we must infer that the gross-contents of
the blastomere, or collection of blastomeres, do not necessarily
cause the differentiation. If, then, neither qualitative division
of the nucleus, nor cellular interaction, nor the gross-contents
of the blastomeres can be the cause of differentiation of the
embryo, what does bring about the differentiation ? There are
certain facts of inheritance that also have a bearing on this ques-
tion. The characters of the male are known to be transmitted
by means of the spermatozoon. The latter carries into the egg
mainly the male nucleus. Therefore, many embryologists have
turned to the nucleus as the oris^inator of the differentiation of
the cell. Various suggestions have been offered as to the way

Cii. XII] INTERPRETATIONS AND CONCLUSIONS 135

ill wliieh such an influence could be transmitted from the nu-
cleus to the cytoplasm. Strasburger sujDposes the nucleus ex-
erts a dynamic influence on the cell-plasm. De Vries and others
imagine that organized particles, '"pangens," pass out of the
nucleus to transform the cytoplasm. Driesch suggests that the
nucleus secretes ferments which change the cell-plasm. These
hypotheses are purely imaginary, for at present we know almost
nothing of the function of the nucleus ; and even if we suppose
the differentiation comes in some unknown way from the nucleus,
still we do not know what could start the process in isolated
nuclei that are after the cleavage-period assumed to be equiva-
lent. There is, however, one series of experiments which seems
to throw some light on the present problem, although the inter-
pretation is extremely difficult and hazardous. I refer to the
experiment on the ctenophor-egg, in which a part of the cyto-
plasm was cut from the unsegmented egg, and the latter gave
rise in most cases to an imperfect embryo. Here, although the
entire segmentation-nucleus is present, yet by loss of cytoplasm
defects are produced in the embryo. The form, therefore, of
the early embryo Avould seem to result from the structure of
the protoplasm, or from the arrangement of the blastomeres
after cleavage. In either case the phenomenon is in the first
instance cytoplasmic. How can this conclusion be brought into
harmony with the facts, stated above, of inheritance of charac-
ters through the male pronucleus ? Let us assume an imaginary
case to show how this union of the two conceptions is possible.
If we had used the spermatozoon of one species (or variety) of
ctenophor and the egg of another species, and then after fertili-
zation had removed a part of the egg-cytoplasm, we should ex-
pect to find the embryo defective, but the organs that were
formed we should expect to show a combination of male and
female characters. In other words, the imperfect embryo would
have resulted from the arrangement of the protoplasm into an
imperfect form, but the kind of organ would have depended on
the structure of the nucleus in each cell. After cleavage, the
cytoplasm of each part differentiates into this or that organ,
but the kind of differentiation of each part is determined b}' the
nucleus of that part.

If the argument given above should prove true, then the

136 DEVELOPMENT OF THE FROG'S EGG [Ch. XII

orio-in of the differentiation is to be found in the ultimate struct-
ure of the cytophism of the egg or embryo, although even tlien
we do not know how this mechanism could be started. Whit-
man ('95) has stated his conviction that it is erroneous to think
of the embryo as only the sum-total of cells interacting upon
one another, but that the embryo itself is to be thought of as a
whole, which regulates its parts regardless of cell-boundaries.
According to this view, each portion of the embryo has its fate
sealed, not because the given portion forms a member of the
community of cells, but because the whole directs the fate of
each special part. Driesch has pointed out that the egg seems
to act like an intelligent being. If so, are the causes of dif-
ferentiation and of regeneration the same in kind as physico-
chemical causes, or do they belong to the category of intelligent
acts, and can these latter be accounted for by the knoivn princi-
ples of chemistry and physics? The plain answer is, we do not
know.

CHAPTER XIII

ORGANS FROM THE ENDODERM

We may now turn again to the history of the development
of the normal embryo.

The Closure of the Blastopore, and the Formation
OF the Neurenteric Canal

During the last stages of the closure of the blastopore its
lateral lips rapidly approach each other, and it then becomes
an elliptical and later a slit-like opening (Fig. 23). The pos-
terior edge of the blastopore also groAvs forward for a short
distance, and as a result a pocket-like continuation of the
archenteron is formed (Fig. 37, A). The depth of this pocket
corresponds to the extent of the forward growth of the poste-
rior edge or ventral lip of the blastopore. If the embryo be
examined in the region over which the posterior lip of the hlasto-
jyore has advanced, there will be found at first nothing on the
surface to mark the region closed over. Some observers have
described faint traces of a groove in this region, but such
appearances are probably exceptional. Later, however, when
the outlines of the medullary folds have appeared, a distinct
longitudinal groove appears in this region running posteriorly
from the small blastopore (Fig. 23, B). At the ventral end
of the groove a distinct depression or pit is soon formed
(Fig. 37), which marks the beginning of the anus. It lies
at a point opposite to the bottom of the posterior pocket of
the archenteron, and corresponds therefore approximately to
the reo-ion at which the first trace of the ventral lip of the
blastopore was found.

As the medullary folds close in to form the nervous system,
the blastopore is overarched by their posterior ends. The folds

137

138 DEVELOPMENT OF THE FROG'S EGG [Cii. XIII

meet above and posterior to the blastopore, so that the latter
can no longer be seen from the surface (Figs. 23, D, E, and
37, A). As a result the central canal of the nervous system

Fig. OK — Sagittal sections through two stages: A. when blastopore is overarched ;
a. when amis has formed. (After Marshall, with modifications in A.) A. 4uus
Fb. Fore-brain. Hb. Hind-brain. LV. Liver-divertirulum. Mb. Mid-brain.
N. Notochord. NT. Xeurenteric canal. PD. Proctodffium. PH. Pharynx
PN. Pineal body. PT. Pituitary body.

becomes continuous at its posterior end with the overarched
blastopore, and by means of the latter the so-called neurenteric

Ch. XIII] ORGAXS FROM THE EXDODERM 139

canal, the central canal of the nerve tube, is directly continued
into the archenteron (Fig. 37, A). At this time the archen-
teron is completely closed in from the exterior, since neither
the mouth nor the anus has as yet opened.

The posterior ends of the medullary folds close just behind
the blastopore. The groove lying behind the blastopore is not
overarched by the folds. During this period the posterior pit
of this groove has become much deeper. At first, the pit Avas
separated from the archenteron by a thick layer of cells con-
sistinof of ectoderm, mesoderm, and endoderm. The meso-
dermal cells begin to pull away from this region, and the pit,
in consequence, becomes deeper. Then the endodermal cells
pull away beneath the pit, and only a single layer of ecto-
dermal cells remains to separate the cavity of the archenteron
from the exterior. Finally the latter cells also draw away,
and the pit opens into the archenteron. The external opening
becomes the anus of the frog. It is at first almost on the
dorsal surface of the embryo, but it rapidly shifts ^ to a more
ventral position, and at the same time the region above it
elongates to form the beginning of the tail. The neurenteric
canal is only a temporary structure, and is soon obliterated by
the growing together of its walls, although its position may
be marked in sections for some time after its actual closure by
the irregular line of pigment in the region of the coalescence
of its walls.

In tlie Urodela the changes that take place during the
final stages of tlie blastopore are somewhat simpler. The
circular blastopore is reduced to an elongated slit-like open-
ing ; but there seems to be some variation in the details of the
method of its later reduction. The medullary folds arch over
only the anterior end of the elongated blastopore, leaving free
the posteiior end. The anterior end becomes the neurenteric
canal. The sides of the middle part of the slit-like blastopore
come together and fuse at the time of overgrowth of the med-
ullary folds. The posterior end of the blastopore always
remains open to the exterior, and forms the permanent anus.

1 The method by which the apparent change in position of the anal opening
takes place has not been clearly made out.

1-iO DEVELOPMENT OF THE FROG'S EGG [Cii. XIII

The main differences that exist between the methods of forma-
tion of neurenteric canal and anus in the frog and in urodeles
are these: In the frog the ventral lip of the blastopore grows
forward during the closure of the blastopore, and only subse-
quently a new opening forms at the point from which the for-

FiG. 38. — Embryo of Kaua tempo raria at time of hatching.

ward growth began (Fig. 37, A, B). In the urodeles (newt
and Amblystoma) the ventral lip of the blastopore remains
stationary, i.e. it retains its first position, and the anus forms
directly from its posterior end.

The Digestive Tract and the Gill-slits

The origin of the archenteron has been described in Chapter
VI. At the time when the yolk-plug is drawn in from the
surface, the archenteron has begun to enlarge (Fig. 26, A).
A series of cross-sections (Fig. 26, B-E) of an embryo at this
stage show that the dorsal and lateral walls of the archenteron
consist of a single layer of endodermal cells, wliile the floor of
the archenteron is formed by the upper surface of the yolk-
mass. The uppermost cells of the yolk-mass show, to some
extent, a tendency to arrange themselves in a single layer
bounding the archenteron.

Shortly after this period the embryo increases in length, and
the archenteron is correspondingly drawn out (Fig. 37). The
anterior end of the archenteron enlarges, and the yolk-mass is
pushed posteriorly. As a result the middle and posterior parts
of the archenteric cavity become smaller than they were in the
earlier stages (Figs. 39, 40). The walls of the anterior portion
of the archenteron are thin, and composed of a single layer of
cells. A blind diverticulum extending from this enlarged

Cii. XIII] ORGAXS FROM THE EXDODERM ' 141

anterior portion into the yolk-mass behind (Fig. 37, A, B)
forms the beginning of the liver.

The first gill-slits appear at a stage when the medullary folds
have rolled over and are about to fuse. At the jiresent stage,
the gill-slits are well marked. They appear along the lateral
walls of the enlarged anterior end of the archenteron as solid
outgrowths of its wall. At the posterior end of the archenteric
cavity the position of the blastopore, which has now closed,
is marked by a diverticulum, the so-called "post-anal-gut"
(Fig. 37). It is in this region that the neurenteric canal of
the embryo persists for a short time after the blastopore has
been covered over by the medullary folds. The pit-like invagi-
nation of ectoderm, the proctodceum, has opened into the pos-
tero-ventral portion of the archenteron (Fig. 37, B).

At the time when the tadpole is ready to emerge from the
jelly-capsule (Fig. 38), the anterior portion of the archen-
teron has become larger and longer (Fig. 39), and in the re-
gion Avhere the heart forms, ventral to the pharynx, an inward
projection of the endodermal wall is present. In the middle
region of the embryo tlie lumen of the archenteron is reduced
to a small cavity, as seen in cross-section (Fig. 40), and is now
longer from above downward than from side to side. The
yolk-mass as a whole is rounded and more compact than in the
earlier stages. At the posterior end of the embryo the archen-
teric cavity bends around the end of the yolk-mass, taking a
curved course to open on the ventro-posterior surface of the
body by the anus.

During the early stages of development the cells of the em-
brvo have been exceedingly active, but no food has been taken
as yet into the digestive tract, for the mouth does not open
until some time after the embryo has left the egg-membranes.
All the cells of the body contain yolk-granules, which serve in
part, beyond doubt, to supply the energy necessary for develop-
ment. A large amount of yolk is also stored up in tlie endo-
derm cells of tlie ventral yolk-mass, and must also long serve
as a source of nourishment for the young tadpole.

The changes in shape that the archenteron passes through
seem to be in part a result of the activity of the endodermal
cells, and in part the necessary result of the change in shape

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142

Cii. XIII]

ORGAXS FRO:\I THE EXDODERM

143

that the whole embryo assumes. The early enlargement of
the anterior archenteric cavity and the formation of a single-
layered wall at the anterior end, with the subsequent formation
of the gill-slits, would seem to be the result of the activity of
the endodermal cells of those regions. On the other hand,
some of the changes in shape that the lumen undergoes would
seem to be due to the change in shape of the whole embryo as
it elongates antero-posteriorly, and narrows from side to side.
Nevertheless, even in this case the cells do not seem to be

Fig. 40. — Cross-section through the iiiiddle of an embryo (."i mm.). AR. Archen-
teron. jMs. Mesoblastic somites. X. Notochord. Ns. Neural crest. M. ^Nledul-
lary tube. Pr. Pronephros. Sn. Subnotochordal rod. So, Sp. Somatic and
splanchnic mesoderm. (After Mai'shall.)

entirely passive, for the numljer of cells lining certain parts of
the early archenteron is, in cross-section, considerably larger
than the number lining the same region at a later stage.
Either certain of the cells have pulled awav from the surface
and have passed into the yolk, or else they have changed their
position relative to one another on account of the lengthening
of the archenteron. In the latter case the total number of

endoderm cells

lining

the archenteron would still be the

144

DEYELOPMEXT OF THE FROG'S EGG [Cii. XITI

same in the older and younger embryos, or greater in the
older embryo as a result of cell-division.

three

gill-slits

The first
pairs of

appear almost si-
multaneously ; the
first two, however,
before the third.
When the tadpole
leaves its capsule,
there are five pairs
of gill-slits ; the
two new pairs have
appeared succes-
sively behind the
third. A horizon-
tal section through
the larva (Fig. 41)
shows to best ad-
vantage the five
clefts at this stag-e.
'^ The gill-pouches
form vertical par-
titions radiating-
outwards from the
pharynx to the
surface - ectoderm,
Each pouch is
formed of a double
fold of endoderm,
the two layers of
which are in close
contact with each
other. The outer
ends of all five
pairs of gill-pouches reach the ectoderm and fuse with its
inner or nervous layer, "i The most anterior pouch or cleft

Fig. 41. — AR. Archenteron . BRi, BR2, BR3. Brancliial
arches. Hi, H\ m. Gill-clefts. HB. Hyoid cleft.
HM. Hyomaiulibular cleft. HY. Hyoid arch. IN.
Infundibulum. OF. Olfactory pit. OS. Optic stalk.
P. Pronephros. S. Segmeutal duct. (After Marshall.)

1 Marshall ('93).

Ch. XIII] ORGANS FROM THE EXDODERM 145

is the hyomandibular cleft, and this is followed successively
by the first, second, third, and fourth branchial clefts. The
last is the smallest and is often imijerfectly developed at this

time.

The visceral or gill-arches lie between the clefts. The first
arch between the hyomandibular and the first branchial clefts
is the hyoid arch (Fig. 41). Then follow the first branchial
arch (I>RO, second branchial arch (BR^), and third branchial
arch (1U13). Behind the fourth branchial pouch there is an
imperfectly defined fourth branchial arch.

When the tadpole leaves its jelly-capsule, the pouches are
still double-walled, solid partitions ; but about the time when
the mouth forms, the endodermal lamelhe of some of the
pouches separate and place the cavity of the pharynx in com-
munication with the exterior. The second and third brancliial
clefts open first. Later the first branchial cleft opens, and later
still the fourth.

The hyomandibular cleft is at first like the others, but it never
opens to the exterior. After its formation it separates from
its ectodermal connection, and recedes from the surface. The
lamelljB separate, and the cleft appears as a diverticulum of the
pharynx.

Two other structures arise from the walls of the pharynx
shortly before the hatching of the tadpole. " The lungs arise
as a pair of pouch-like diverticula of the walls of the oesophagus.
They are at first exceedingly small and have strongly pigmented
walls."

The thyroid body appears about the time of hatching as a
short median longitudinal groove along the wall of the pharynx.
" The groove is shallow anteriorly, but deepens at the hinder
end, where it leads into a small conical pit-like depression of
the endoderm, forming the pharyngeal floor, just in front of
the pericardial cavity. Soon after the mouth opens, the thyroid
separates completely from the floor of the pharynx, remaining
as a solid rounded mass of pigmented cells, in close contact
with the anterior wall of the pericardium." ^

Marshall ('93).

CHAPTER XIV

ORGANS FROM THE .MESODERM

The mesoderm appears as a distinct layer over the dorsal
surface of the embryo at the time when the dorsal lip of the
blastopore is moving over the white hemisphere (Fig. 25).
At first the mesoderm is in close contact witli the encfoderm,
particularly along the mid-dorsal line. The notochord soon
separates from the mesodermal sheets of each side by two verti-
cal furrows, so that from this time forward there are two lateral
sheets of mesoderm, separated in the mid-dorsal line by the
notochord (Fig. 26, E). Around the anterior and posterior
ends of the notochord, the two sheets of mesoderm are con-
tinued into each other.

These sheets of mesoderm now rapidly extend ventrally.
This down-growth is brought about by additions to the ven-
tral borders of the sheets. The new cells that are added
come, probably, from the yolk-cells along tlie free borders of
the mesoderm ; the yolk-cells in this region dividing rapidly
form smaller cells that are joined to the mesoderm. i At the
time when the medullary folds appear outlined upon the sur-
face, the lateral sheets of mesoderm have extended ventrally
and to a certain extent have fused in the mid-ventral line.
The cells of each sheet of mesoderm are arranged over the
greater part of their extent into two layers ; but on each side
of the notochord the mesoderm is somewhat thickened to form
the beginning of the segmental plate (Fig. 42) ; and in this
region there is, in the early stages of development, no distinct
arrangement of the cells into two layers.

1 According to some authors the ventral extension of mesoderm results from
a proliferation of the mesoderm that is first laid down over the dorsal region,
but It seems to me there is little ground for such an assumption.

146

Ch. XIV]

ORGANS FROM THE MESODERM

14"

Over the anterior end of the embryo and around the phar3aix
the mesoderm forms a thin layer of cells, loosely held together
(Fig. 26, B). The mesoderm over the dorsal surface of the
pharynx and beneath the brain plate is represented by only a
single layer of somewhat scattered cells. Around the blasto-
pore there is a thick layer of mesodermal cells which is thickest
on the dorsal surface. In general, in the posterior region of the
body the mesoderm is thicker than in the middle and anterior

regions.

The Mesodermic Somites

In the following stages of development of the embryo the
dorsal ectodermal plate is lifted up and rolled in to form the
central nervous system (Fig. 42). The mesoderm lying on

Fig. 42. — Cross-section through middle of embryo. M. Medullary plate. N. Noto-
chord. Nc. Neural crest. PS. Primitive segment-plate. SO, SP. Somatic and
sijlauchnic mesoderm.

each side of the notochord changes shape somewhat during this
time. It forms on each side a thick, nearly solid mass of cells,
the plate of the primitive segments or segmental plate (Fig.
42). The outermost cells of this mass, i.e. those lying nearest
to the dorsal surface, now show a tendency to arrange them-
selves into an epithelial layer. This layer is at first continu-
ous at the sides with the outer or somatic layer of cells of
the lateral mesodermal sheets. The two layers of cells of the
lateral mesodermal sheets (Fig. 42, SO and SP), the somatic
and splanchnic layers, often show a tendency to separate and
leave a cavity between them. This cavity filled with fluid

148

DEVELOPMENT OF THE FROG'S EGG [Ch. XIV

-M

is the coelom, or body-cavity, and is at first continued into the
segmental jDlate. Tlie cavity in the segmental plate lies be-
tween the outer epithelial layer and the inner solid mass of
cells.

When the medullary plate of the embryo begins to roll in
to form the nerve-tube, each segmental plate begins to break
up transversely into a series of blocks or mesodermic somites.

The process begins first in the region
anterior to the middle of the embryo
(Fig. 43). The mesodermic somites
are at first somcAvhat irregular in out-
line. The first well-marked somite lies
at about the level of the ganglion of
the vagus nerve. In front of this there
are traces of another somite wliich is
partially broken up into loose mesen-
chymatous tissue. Still further for-
ward, the series of somites is replaced
by loose mesenchyme. In the frog the
number of head-somites (or structures
■'"EoS.i^a'rrif'rSLl ™"-««P°'«l!ng to them) is uncertain.
MS. Mesobiastic somites. At first the primitive segments or
ml Si?"''''"- ''''• ""'"" «o"^ites are not separated from the

lateral sheets of mesoderm, but almost
immediately after the segmental plate has begun to break
up transversely into somites, these begin to separate also
from the lateral mesoderm. This separation appears first
in the intersegmental borders. At this time the medullary
folds have met to form a closed tube. Posterior to the
fourth segment, the segmental plate is beginning to break
up into blocks, but these have, as yet, no sharply marked
outer or ventral boundaries. The body-cavity of the lateral
mesodermal sheet is at first, as we have seen, sometimes con-
tinued into the cavity of the segmental plate, but when the
constriction of the plate from the lateral sheets takes place,
this communication (the communicating canal) is lost. Even
in the younger stages there is a differentiation of a peripheral
epithelial layer surrounding the dense central mass or kernel
of the somites.* This peripheral part is represented on the

Ch. XIV]

ORGANS FROM THE MESODERM

149

outer side of each somite by tlie entire somatic layer. Along
the ventral and median boundaries of the somites a layer
having a loose epithelial character (mesenchyme) is also to be
seen. Thus the central mass whicli is to develop into the
myotome lies on the median side of the coelom, and is wholly
surrounded by an epithelial layer. Frontal sections show that
this layer can also be traced inward for some distance between
successive somites over both their anterior and posterior sur-
faces (Fig. 44).

"Not merely is mesenchyme produced by the thin peripheral
layer of the somites, but in anterior regions considerable por-
tions of the kernels of the somites also undergo a metamor-
phosis in this direction. Thus, if I be not mistaken, a somite
immediately in front of somite 1 has been wholly converted
into mesenchymatic tissue. The kernel of the succeeding so-
mite (somite 1) has given rise to a considerable quantity of
mesenchyme, and the process has been manifested, though to a
less degree, even in succeeding somites." ^

At the time when fourteen pairs of somites are present 2
the cells of the more
anterior somites have
begun to differentiate
into muscle-fibres. The
cells of each somite elon-
gate in the antero-pos-
terior direction a n d
become cylindrical in
shape, and each extends
the whole length of its
somite (Fig. 44, B).
Each cylindrical cell has
at first but a single nu-
cleus. Around the wall
of the cell a layer of fine

fibrilke appears. The original nucleus divides and re-divides
into man}' nuclei, which lie scattered throughout the cell.

1 Field ('91).

- Four days after fertilization of the egg, when three pairs of gills have
appeared.

MS

B

Fig. 44. — Frontal sections through the anterior
end of Boml)inator. (After Gotte.) A. Shows
three gill-pouches (G), and mesoderm of
arches. B. Shows formation of mesodermic
somites (MS). PH. Pharynx.

150

DEVELOPMENT OF THE FROG'S EGG [Cii. XIV

The development of the musculature of the head, limbs, and
ventral body-wall takes place at a later stage. A description
of the origin and development of these structures is beyond
the limit of the present account.

The Heart and Blood-vessels

The heart appears at the time when the medullary folds have
rolled in, and have met along the mid-dorsal line ; it lies below
the pharynx, and anterior to the liver (Fig. 37, B). The meso-
derm in this region shows a tendency to split into two sheets
and, where the heart is about to develop, a cavity, a part of

P H

Fig. 45. -Three stages in development of heart. E. Endothelium. PE. Pericar-
dium. PH. Pharynx. W. Wall of heart.

the coelom, appears between the sheets. A cross-section of the
larva (Fig. 45, A) shows on each side of the mid- ventral line
in the region of the heart the somatic and splanchnic layers
widely separated from each other. The coelomic cavities of
the right and left sides are not continuous across the middle
line, but anterior and posterior to this section the crelomic
cavity is found to be continuous before and behind with the
general crelomic space on each side. A few scattered cells
lie in the middle line between the splanchnic layer and the
ventral wall of the pharynx (Fig. 45, A). These cells have

Ch. XTV] organs from the mesoderm 151

been described as originating from the ventral wall of the arch-
enteron, and if so, have had a different origin from the other
cells of the heart. ^

At a somewhat later stage of development the walls of the
coelomic cavities of the right and left sides separate further
(Fig. 45, B). The splanchnic layer thickens, and begins to sur-
round the proliferation of scattered "endodermal cells." These
endodermal cells arrange themselves into a thin-walled tube
stretching throughout the heart-region (Fig. 45, B). Subse-
quent development shows that this tube becomes the endothe-
lial lining of the heart. Around this endothelial tube the
thickened splanchnic layers now begin to push in from the sides
between the tube and the lower wall of the pharynx. The tube
becomes finally entirely surrounded by mesoderm (Fig. 45, C).
The mesoderm from the sides that has met beneath the pharynx
forms the dorsal mesentery of the heart. The mesoderm around
the tube continues to thicken, and forms later the musculature
of the heart.

At first the heart has also a ventral mesentery formed by the
union of the walls of the coelomic cavities below it (Fig. 45, B),
but later the mesentery is in part absorbed and the ccBlomic
cavities become continuous below from side to side, forming the
pericardial chamber. The onter layer of somatic mesoderm
gives rise to the pericardium itself. '

The tubular heart is attached at its posterior end to the
liver and anteriorly to the wall of the pharynx. It becomes
free ventrally and later also dorsally along the middle of its
course, and owing to an increase in length is bent on itself
into an (/)-shaped tube (Fig. 39).

When the tadpole is 4^- ram. in length, we find a vessel open-
ing into the posterior end of the heart, the sinus venosus,
formed by the union of two large vitelline veins. These veins
have appeared on each side of the liver-diverticulum and con-
tinue along the yolk-mass in a fold of the splanchnopleure.
They are supposed to carry to the heart the food-material ab-
sorbed from the yolk. Into the sinus venosus empty also two

1 At least these cells have arisen from the yolk-cells after the ventral meso-
derm has been split off.

EF* EF^'eF-^ EFl EH EM

B

AU

AF" AF- TA AF^

Fig. 46, A. — AF. Afferent branchial vessel. AR. Anterior cerebral artery. CA,
CP. Anterior and posterior commissural vessel. EFi, EF-, EF^, EF*4. Efferent
branchial vessels of the first, second, third, and fourth brancliial arches. EH. Ef-
ferent hyoid vessel. EM. Eft'ei-ent mandibular vessel, (i. Glomus. O. Aorta.
P. Pronephros. RT. Truncus arteriosus. S. Segmental duct. (After jNIarshall.)

B. — AFi, AF-^, AF3. Afferent branchial vessels. AU. Auricle. CV. Cuvierian
vein. EFl, eF^ EF3, EFi Efferent branchial vessels. EH. Efferent hyoid
vessel. EM. Efferent mandibular vessel. G. Glomus. HV. Hepatic veins.
MV. INIandibular vein. MY. Hyoidean vein. TA. Truncus arteriosus. V. Ven-
tricle. (After Marshall.)

152

Ch. xtv] orgaxs from the mesoderm 153

veins that have come down from the dorso-hiteral region of the
embryo. These are the Cuvierian veins formed on each side
by the union of the posterior and anterior cardinal veins. The
posterior cardinals bring back the blood from the head-kidneys.
Around tlie head-kidneys these veins form sinuses that are
enormously large. Each posterior cardinal also receives so-
matic veins from the posterior part of the body-wall. The
anterior cardinal veins bring back blood from the dorsal part
of the head-region.

In a larva 4.1- mm. in length, the blood-vessels of the branchial
region have also appeared. The anterior end of the heart, the
truncus arteriosus, divides into a right and left branch, which
pass forward and laterally toward the base of the gill-region. In
the mandibular arch no vessels are as yet present. In the hyoid
arch an irregular space appears in the mesoderm. In the first
Ijraiieliial arches two vessels appear, a large efferent vessel (Fig.
46, for an older embryo) connected with the dorsal aorta, and a
smaller afferent vessel. The latter is at present without con-
nection. In the second branchial arch the conditions are like
those in the first. In the third branchial arch only a small
efferent vessel has as yet appeared. Xo vessels are present
at this time in the fourth branchial arch. The dorsal aorta is
represented by a paired vessel in the dorso-pharyngeal region.
Opposite the hyoid arch each branch of the dorsal aorta di-
vides into a dorsal and into a ventral branch. The dorsal
branches meet each other behind the infundibulum, Avhile the
ventral branch passes forward to end blindly (Fig. 46). The
two aort?e unite posteriorly into a single vessel at the level of
the pronephros (Fig. 46, A).

The condition of the blood-vessels shortly after the tadpole
has left its envelopes (it is then 7 mm. in length) is illustrated
in Figs. 46 and 47. The heart has enlarged and is further
twisted on itself. The aortic bulb-portion and the auricuhir
and ventricular portions are distinctly' marked from each other
by constrictions of the tube. The right and left branches of
the aortic bulb have grown toward the gill-arches, and the
afferent vessels of the first and second branchial arches have
united with the ventral aortic branches AF^ and AF^. The
efferent branches, EF^ and EF^, of the first and second bran-

154

DEVELOPMENT OF THE FROG'S EGG [Ch. XIV

cliial arches have greatly enlarged, and the efferent and afferent
vessels are now also united to each other in each arch by small
vessels (Fig. 47) or capillary tubes. The efferent vessels of
these two arches are also in communication with the dorsal
aorta of their respective sides. There is thus established at
this time a circulation of blood from the heart to the dorsal
aorta by way of the first and second branchial arches.

In the third and fourth branchial arches the efferent vessels
have appeared. In the third arch the beginning of an affer-

FiG. 47. — AFi. Afferent branchial vessel. CV. Anterior cardinal vein. EFi. Effer-
ent branchial vein. G. Pneumogastric nerve. JV. Inferior jugular vein. L.
Capillary loop connecting afferent and efferent branchial vessels. N. Notochord.
O. Aorta. P. Pericardium. PH. Pharynx. SU. Suckers. V*. Fourth ven-
tricle. (After Marshall.)

ent vessel is seen (Fig. 46). In the hyoid arch blood-vessels
appear, as we have seen, at an early stage of development and
seem to correspond to those in the branchial arches, but after
developing to a certain extent, they begin to degenerate. In
the mandibular arch no vessels have appeared at the time when
the larva leaves its capsule. Soon after this time a vessel de-
velops in this arch, and a small diverticulum arises from the
dorsal aorta (Fig. 46, B, MV), and later the two vessels unite.
The origin of the heart has been described, but as yet the

Cn. XIV] ORGAXS FROM THE MESODERM 155

method by wliich the blood-vessels are formed has not been
fully considered. The dorsal aorta is the first vessel to arise.
A series of isolated lacunse appear in the mesoderm along the
roof of the pharynx, and by opening into one another form
a pair of longitudinal vessels. Vessels next apj^ear in the
first and second branchial arches. Similar vessels arise later
in the third and fourth branchial arches. In the hyoid and
mandibular arches the vessels appear, as we have seen, later
still. These branchial blood-vessels originate in part as iso-
lated lacuuic in the mesoderm, and in part as outgrowths of
already existing vessels. For instance, lacunar vessels appear
in the mesoderm of the gill-arches, two in each arch. One
of these is the efferent lacunar vessel, and later connects with
a corresponding diverticulum from the dorsal aorta, and the
other lacunar vessel is the afferent vessel of the same arch.
This latter vessel grows ventrally toward the diverticulum
from the truncus arteriosus and unites Avith it.

The walls of the blood-vessels are formed directly from the
mesodermal cells around the lacume. "The blood-corpuscles
are free cells that have been left in the lacuna-spaces, or more
usually are cells budded off at a later stage from the walls of
the vessels into their cavities." ^ At first the blood-corpuscles
are simply spherical cells containing yolk-granules. Only after
the embryo is hatched do many of the corpuscles begin to
acquire the shape and character of red blood-corpuscles.

The Phonephros

The excretory system of the young embryo is represented on
each side by the pronephros and the sei/mental duct. Whether
the pronephros and duct arise in part from an early ingrowth
of ectoderm or whether they develop in situ from the somatic
mesoderm is perhaps still open to doubt. Field ('91), who has
worked out most recently the development of the pronephros
and segmental duct in the frog, describes the organ as coming
entirely from the mesoderm. We shall follow closely Field's ac-
count. The pronephros appears at a stage when the medullary

1 Marshall ('93).

156 DEVELOPMENT OF THE FROG'S EGG [Cii. XIV

plate is first formed. It is well marked at the time when the
medullary folds have rolled in, but have not yet fused. A
thickening of the somatic layer of the lateral mesoderm near the
second mesoblastic somite marks the beginninir of the prone-
phros (Fig. 48, A). At a later stage, the mesodermic thick-
ening becomes larger, and the anterior end arches over toward
the coelomic cavity, to form the first 7iephrostome. The ventro-
posterior part of the nephrostomal thickening is continued
backward as a thickening of the somatic wall as far as the
seventh somite, to form the segmental duct. A canalization
now takes place in the nephrostomal portion and in the seg-
mental duct. Three short tubes or canals appear in the
pronephric mass running outward from the coilom (Fig. 41).
Constrictions appear between the first and second, and between
the second and third canalized tracts (Fig. 48, B), and short

B
Fig. 48. — Three stages in the formation of the pronephros. (A and C after Field.)

hollow Stalks are formed leading ventrally into the longitu-
dinal canal of the segmental duct.

A proliferation of cells from the somatic layer of the meso-
blastic somites, dorsal to the pronephros, gives rise to a cover-
ing of mesoderm for the pronephros, the pronephric capsule.
A little later a protrusion of the splanchnic wall opposite to
the funnels of the pronephros forms the glomus (Fig. 47, B).
The glomus" becomes filled with blood, and seems to have a
direct connection with the dorsal aorta. The bulging portion
of the glomus protrudes into the ccelom, and its cavity is sepa-
rated from the coelomic cavity by only a single layer of cells.

At the time when the embryo is hatched, the duct of the
pronephros, the segmental duct, has fused with the wall of
the cloaca, and the lumen of the duct opens into the digestive

Ch. XIV] ORGANS FROM THE MESODERM 157

tract (Fig. -11). Presumably the pronephros is functionally
active at this time. The arrangement of the tubes of the
pronephros, and their relation to the common tube or prone-
phric duct, is shown in Fig. 48, C. The three nephrostomes
open into three collecting tubules, and these tubules have
elongated independently of one another. The first collecting
tubule is short ; the second is thrown into several turns and
opens into the pronephric duct a short distance from the first.
The collecting tubule from the third nephrostome opens some
distance behind the point of opening of the second. The seg-
mental duct is thrown into a series of turns between the first
and second collecting tubules ; and as it leaves the pronephric
region it takes at first a tortuous course, and then runs as a
straight tube backward to the cloacal opening.

The posterior cardinal veins have appeared at this time, and
in the region of the head-kidneys these veins widen into a sinus
lying amongst the windings of the collecting tubules of the
pronephric duct. The glomus of each side reaching from the
region of the first to that of the third nephrostome, and lying
exactly opposite the nephrostomes, is well developed (Fig. 46).

So far the description of the development of the excretory
system has been that given by Field. The same author adds :
"According to the account which at present receives the most
general acceptance, the pronephros first aj^pears as an outfold-
ing of the somatopleure in the form of a longitudinal groove.
The anterior end of this groove is destined to become the prone-
phros, the remaining portion is constricted off to form the seg-
mental duct. Since the process of constriction advances from
before backward, stages may be found in which a completed
tube is continuous posteriorly with a mere groove of the soma-
topleure. In the anterior region the groove remains in com-
munication with the body-cavity, and grows down toward the
ventral surface of the embryo in the form of a broad pocket.
The slit-like peritoneal opening of this pouch closes through-
out the greater part of its length, leaving, however, two or
three regions of incomplete closure, the fundaments of the
nephrostomes."

" The nephrostomal tubules are formed by the fusion of the
Avails of the pouch between two nephrostomes. The regions of

158 DEVELOPMENT OF THE FROG'S EGG [Ch. XIV

fusion extend in vertical lines from the nephrostomal margin
of the pouch nearly to its ventral border, where there is feft
an unfused and therefore continuous longitudinal tract con-
stituting the canal which I have called the collecting trunk." i
Field continues, "In opposition to this view, I would maintain :
(1) That the first trace of the excretory system consists of a
solid proliferation of somatopleure, the pronephric thicken-
ing; (2) that the lumen of the system arises secondarily; and
(3) that the pronephric tubules do not appear in consequence of
the local fusion of the walls of a widely open pouch, but that
they are differentiated at an early stage from the hitherto
indifferent pronephric thickening."

The pronephric duct of the Amphibia arises, according to
one view, as we have seen above, from an evagination of soma-
topleure, its lumen being therefore a detached portion of the
body-cavity. A second view of the origin of the duct is, that
It arises from a solid proliferation of somatoj^leure. Field
agrees with the latter view. A third view maintains that the
duct is ectodermic in origin. Field has shown, however, that
in the Amphibia the excretory system develops most probably
without any participation of the ectoderm in its formation.

"This view of the development of the pronephros, although suggested by
Wilh. Miiller, was first described in detail by Goette for Bombinator, and was
later extended to other Amphibia by the researches of Furbringer. It has been
entirely confirmed by Wichmann, by Hoffmann, and still more recently by
Marshall and Bles." (Field, '91, page 281.)

CHAPTER XV
ORGANS FROM THE ECTODERM

The outer covering-layer of the embryo, the ectoderm, gives
rise to the nervous system and organs of special sense (eyes,
ears, nose). The Adhesive glands or "suckers" are also formed
by this layer ; and the anterior and posterior divisions of the
digestive tract, the stomodreum and proctodieum, have a lining
of ectoderm.

In the present chapter Ave shall follow the development of
these organs.

"O'

The Central Nervous System

The medullary plate appears on the surface of the young
embryo at the time when the yolk-plug is about to be drawn
in from the surface. It extends over about one-third of the
circumference of the e^^ and is, at first, quite broad. It is
slowly converted into a tube by the drawing together of its
material, and by a subsequent over-rolling of its sides to meet
in the mid-dorsal line. This change into a furrow, and then into
a closed tube, involves extensive movements of the material of
the plate. Whether the i^late moves as a whole, or whether
the movement is only the sum-total of changes in shape and
position of the individual cells, is not known (compare Figs. 26,
42, 50). While the medullary tube is developing, the embryo
as a whole is changing its spherical shape into a more elon-
gated form and the medullary tube is also drawn out.

The medullary plate is formed, for the most part, from a
thickening of the inner layer of the ectoderm (Figs. 26 and
42). It is continuous on each side with a broad flange or ridge
of thickened ectoderm (Fig. 42, Xc). This ridge of cells, the
neural crest or ridge, is also lifted up during the formation of

159

IGO DEVELOPMENT OF THE FROG'S EGG [Cii. XV

the tube, forming a broad sheet of cells on each side, continu-
ous with the dorsal edges of the closing tube. These lateral
sheets are very large and conspicuous at the anterior end of
the nerve-tube. The subsequent history of these structures
will be followed later.

The first part of the medullary tube to close, is the antero-
median portion, and from this point the closure of the tube
extends anteriorly and posteriorly. At the anterior end, the
tube remains open latest ; at the posterior end, the medullary
folds arch over the blastopore, as already described.

When the medullary folds have met along the mid-dorsal
line, the apposed edges fuse, and the outer layer of ectoderm
then becomes continuous over the outer surface of the embryo.
A part of the same layer has been cut off and lines the cavity
of the neural tube. The nerve-tube soon loses all connection
with the overlying ectoderm (Fig. 40).

The anterior end of the nerve tube is larger than the rest,
and this end is at first bent down nearly at right angles to the
long axis of the more posterior portion (Fig. 37, A). The
bending begins at the front end of the notochord. A slight
transverse infolding of the wall of the anterior end of the tube
takes place soon after its closure, and later another transverse
infolding occurs, still further forward. As a result three divi-
sions or vesicles of this region are produced. They correspond
to the fore-brain, mid-brain, and hind-brain respectively. The
fore-brain (Fig. 37, Fb) is the large anterior vesicle. From it
develops later the third ventricle, the pineal body, the infun-
dibulum, the optic vesicles, and the cerebral hemispheres. The
mid-brain (Fig. 37, B) is the smallest of the three divisions, and
gives rise to the optic lobes and to the Sylvian aqueduct. The
hind-brain is continued into the more posterior medullary tube.
It lies in the same plane with the medullary tube, and repre-
sents only a somewhat enlarged part of the tube. The hind-
brain becomes the medulla oblongata, and from its roof the
cerebellum is formed.

The roof of the fore-brain is very thin. Near the middle of
its upper margin an evagination is formed, wliich is, at first,
only a hollow diverticulum (Fig. 37, B), but when the tadpole
leaves its capsule, the peripheral end of the outgrowth forms a

Cn. XV] ORGANS FROM THE ECTODERM 161

small round knob. This knob, the pineal body, lies just below
the surface-ectoderm. Later the structure grows forward, and
becomes dilated at its distal end. The dilated end or bulb re-
mains connected with the brain by a stalk. White particles
develop in the bulb, so that it stands out in strong contrast to
the dark surface of the brain.

At the time of closure of the medullary folds, a mid-ventral
diverticulum forms from the floor of the fore-brain. This is
the infundibulum. It is in close contact with the anterior end
of the notochord (Fig. 38). The infundibulum is throughout
its subsequent history a wide sac with thin walls. It soon
comes into close connection with another structure, the pitui-
tary body (Fig. 39). The pituitary body arises very early,
even before the neural tube is closed, as a solid ingrowth, or
cord of cells, from the ectoderm, immediately in front of the
anterior end of the medullary plate (Fig. 37, A). Later, this
small, solid, tongue-like process projects inward from the
ectoderm beneath the brain and above the dorsal wall of the
pharynx. The inner end of the ingrowth expands into a flat-
tened mass of cells, which lies immediately beneath the anterior
end of the notochord. This mass becomes later the pituitary
body, while the rest of the process forms a slender stalk con-
nected at one end with the ectoderm.

The Eyes

The eyes develop in part from the walls of the fore-brain.
Even before the neural tube is closed, in the embryos of some
species of frogs, two pigmented areas mav be seen on the an-
tero-lateral walls at the anterior end of the infolding medullary
plate. These pigmented areas mark the region from whieli a
pair of evaginations of the fore-brain will develop to form the
optic vesicles. The hollow vesicles push out laterally toward
the sides of the head. Each tubular evagination then becomes
constricted, forming a distal hollow bulb and a proximal hollow
stalk (Fig. 49). The bulb gives rise to the retina and to the
pigment behind the retina, while, according to Marshall ('93),
the stalk forms a path along which the fibres of the optic
nerve pass from the eye to the brain. The outer hemisphere
of the optic bulb flattens and then pushes in so that the former

M

162 DEVELOPMENT OF THE FROG'S ECxG [Cii. XV

cavity of the vesicle is nearly obliterated (Fig. 49) ; and at
the same time the inturned wall becomes greatly thickened.
There is thus formed an open, cnp-shaped structure with the

opening of the cup turned
outward. The wall of this
optic cup lying toward the
brain remains thin, and pig-
ment soon appears in it.
The inturned wall becomes
the retina of the eye.

At the time when the
optic bulb turns in on it-
self, a thickening of the
inner layer of ectoderm op-
posite the optic cup takes
place. This thickening
forms a solid mass of cells
projecting into the open
mouth of the cup. It be-
comes hollow and then sep-
arates from the ectoderm
(Fig. 49), filling up the
opening of the optic cup,
and forms later the lens of
the eye. In the space left between the lens and the retinal
layer the vitreous body of the eye forms. The later stages of
the development of the eye take place after the embryo leaves
its capsule. The nerve-fibres that develop from the retina
and pass into the brain along the optic stalks have not yet
ajipeared.

The Ears

While the neural groove is closing, a pair of thickened circu-
lar patches of the imier Imjer of the ectoderm arises, one on
each side of the head near the hind-brain. • After the closure
of the neural tube each area forms a shallow pit with the con-
cavity turned outward, and each is covered by the outer layer
of the ectoderm. The pit deepens, the outer edges come
together, and a hollow vesicle is formed before the tadpole

Fic. 49. — Cross-sectiou through head and
eyes. F. Fore-brain. L. ^Lens of eye.
OP. Optic cup. OS. Optic stalk. PH.
Pharynx. FT. Pituitary body. ST. Sto-
modseum.

Ch. XV] ORGANS FROM THE ECTODERM 163

leaves the capsule. These auditory vesicles separate from the
surface ectoderm. "At the time of the separation the vesicle
is a closed sac somewhat pyriform in shape; its lower or
ventral portion being spheri-
cal and lying opposite the
notochord, and its dorsal
wall l)eing prolonged up-
wards into a short blind
diverticulum lying at the
side of the hind-brain. The
wall of the vesicle consists

of a single layer of cubical ' "^"^^ ,<tt^^7>>,

or colunniar cells." This

ectodermal sac becomes the Fig. so. — Cross-section through hind-
T . r ii • brain (H) and inner ear (E). N.

sensory lining of the inner Notochord.

ear (Fig. 50).

The Nerves

At the time when the medullary plate forms as a thickening
of the ectoderm, there also forms, as we have seen, on each side
of the plate a lateral neural ridge or jylate of ectoderm. Each
neural ridge appears at first as a continuation of one side of
the thickened medullary plate (Fig. 26). A slight constric-
tion on each side marks the line of demarcation between the
medullary plate and the neural ridge (Fig. 42). The neural
ridges are more conspicuous at the anterior end of the medul-
lary plate; they also develop somewhat earlier in this region.
After the medullary plate has rolled up to form the medullary
tube, the lateral neural ridges are also carried up, retaining for
a time their primitive connection with the outer (now dorsal)
part of the medullary tube (Fig. 40).

The neural ridges next become broken up into a series of
dorsal nerves, the cells collecting at certain regions, and thin-
ning out and disappearing in the intermediate regions. The
dorsal nerves grow down later between the myotomes and the
nerve-cord. Accumulations of cells occur at certain regions
on each dorsal nerve to form the ganglion of the dorsal root,
and nerve-fibres are spun out from the cells of the ganglion.
The ventral roots of the spinal nerves appear much later.

1G4 DEVELOPMENT OF THE FROG'S EGG [Cii. XV

Marsluill ('93) says the cranial nerves, '' which are iindoubtedlj
derived from the neural ridges, are the trigeminal, the facial
and auditory, and the sensory branches of the glosso-pharyn-
geal and pneumogastric nerves." These nerves, -although
arising from the neural ridges in the same way as the dorsal
roots of the spinal nerves, yet differ from these, and agree
amongst themselves in certain important features."

" I. The nerves in question, in place of growing downwards
like the spinal nerves, alongside the central nervous system,
grow outwards close to the surface of the embryo lietween the
epiblast and the mesoblast."

" II. Each of these four nerves acquires a new connection
with the surface epiblast some considerable distance beyond
the root of origin from the brain, and at about the horizontal
level of the notochord ; at this place and at any rate in part
from the surface epiblast itself, the ganglion of the nerve is
formed."

" III. The nerves have special relations to the gill-slits, each
nerve dividing into two main branches, which embrace between
them one of the gill-slits."

"IV. A special system of cutaneous nerves is developed
from the surface epiblast in connection with these four nerves,
forming the lateral line system of nerves."

The pneumogastric nerves are "wing-like" expansions of the
neural plate, extending more than half-way down the side of
the pharynx. At the time when the larva leaves the capsule,
a thickening of the ectoderm on each side opposite this nerve
and at the level of the notochord develops, and fuses with the
nerve. From this double origin arises the ganglion of the
pneumogastric. A lateral line thickening has appeared as a
solid cord of cells on each side, extending from the pneumo-
gastric backward along the side of the embryo.

It is not possible to enter here into the details of the develop-
ment of the other cranial nerves enumerated above. The
development of the first, third, fourth, and sixth nerves has
not as yet been fully worked out. Tlie origin of the optic
nerve has been described in connection with the development
of the eye.

Ch. XV]

ORGANS FR0:M THE ECTODERM

165

The Appearance of Cilia oisr the Surface of the

Embryo

If the living embryo be examined at the time when the
neural folds have appeared, it will be seen that the embiyo
slowly rotates within the jelly-capsule. This rotation is the
result of the activity of certain ciliated ectodermal cells. The
distribution of these cells over the surface of the body has been
recently described by Assheton ('96). Assheton states that
at the time when the medullary folds are first visible, and even
after they have begun to roll in, there are no traces of cilia on

Fig. 51. — Embryo of Rana. The arrows show the direction of currents of water
over the surface. A. Side view. B. Ventral view. (After Assheton.)

the surface of the embryo. Before the neural folds have met
in the middle line the ectoderm has become ciliated in certain
regions, as can be demonstrated by the streaming movement
of granules of carmine placed on the surface. The arrows in
Fig. 51 show the direction of the flow of granules over the sur-
face. The lateral edges of the anterior end of the medullary
folds seem to show the first traces of cilia, and a few hours
later (Fig. 51, A) cilia have also appeared along the sides of
the folds.

166 DEVELOPMENT OF THE FROG'S EGG [Ch. XV

As the medullary folds grow nearer together, the ciliation ex-
tends further back along the sides of the dorsal surface. When
the folds have just touched at the anterior end, cilia appear
on the antero- ventral surface of the embryo, in the region where
the mouth subsequently forms. The direction of the currents
set up is from before backward. The whole of the dorsal
surface next becomes ciliated. The ciliation spreads rapidly
and at the time when seven or eight mesoblastic somites are
present (when the embryo is 3 mm. in length) it has extended
over the whole surface of the embryo. The currents, however,
differ very much in intensity. Figure 51, A, B, shows the
direction of the flow, the larger arrows indicating stronger cur-
rents. The action of the cilia is strongest at the anterior end
of the body. A well-defined stream passes over the bases of
the gills, which have begun to appear at this time. Over the
ventral surface the currents move slowly and in eddies. At
the hinder end of the embryo the action of the cilia directs the
currents of water toward the blastopore and anus. When the
embryo measures 4 mm., the so-called "suckers" have appeared,
and the currents in that region have changed their direction.
These " suckers " are in reality mucous glands that secrete a
sticky substance by means of which the embryo can fix itself
to objects with which it comes in contact. The edges of the
glands have well-developed cilia, which direct a stream of
water over the stomoda^al depression, and thence backward
between the glands (Fig. 51, B). In older embryos, when
the glands have united to each other in the mid-ventral line,
the direction of the currents in this region is altered. The
central stream now turns outward around the anterior sides
of the glands and passes backward along the sides. In older
larvae (8 mm.) small special currents run over the edges of
the adhesive glands and into the depressions within the
glands.

The cilia that cause the flow of water over the surface
of the embryo are not developed by all the ectodermal cells.
Even where the currents are most active, the cilia-bearing
cells are slightly less abundant than the non-ciliated cells.
Each ciliated cell bears on its outer surface numerous short
cilia.

Ch. XV] ORGANS FROM THE ECTODERM 167

The gill-lilaments also carry cilia in the proportion of one
ciliated cell to two non-ciliated cells. The effect of the cilia
becomes less conspicnous after the larvae have reached 7 or 8
mm. in length, although even in much later stages the cilia are
still found over all parts of the bod}'. Their motion is suffi-
ciently strong to cause the embryo (6 or 7 mm. in length) to
move forward, if placed on a glass plate, at the rate of one
millimetre in from four to seven seconds.

CHAPTER XVI

EFFECTS OF TEMPERATURE AND OF LIGHT ON

DEVELOPMENT

It has been long known that the rate of development within
certain limits is dependent on temperature. The development
of the frog's egg is very much retarded, or even stopped, in
water at the freezing-point. In North America, liana tempo-
raria often lays its eggs so early in the spring that the water
is afterward frozen. The eggs that are caught in the ice are
generally killed, but those that lie in the water below often
remain alive and will subsequently develop normally.

Hertwig ('94) has shown that the maximum temperature
for normal development of the eggs of Rana fusca is about
25 degrees C. Eggs develop very rapidly at this tempera-
ture, and in twenty-four hours have reached a stage of ad-
vancement corresponding to that at the end of the second day
for the average temperature of 16 degrees. A temperature
of 25 to 30 degrees C. long continued, or a temperature of 30
to 35 degrees for a short time, injures the eggs ; their develop-
ment is arrested and many die. Eggs that have been partiall}^
injured by heat (after two or three hours at 30 degrees C.^ or
after three to eight hours at 26 to 28 degrees C. and then
brought into a normal temperature) continne to develop at a
slower rate than eggs under normal conditions. The yolk-
liemisphere of the egg is first affected, so that the cleavage-
furrows do not appear in it. The injured or dead half of the
egg lies below, and the segmented portion above.

Hertwig obtained similar results by cooling the eggs. Soon
after fertilization the eggs were placed in water at zero C. and
kept there for twenty-four hours. During that time they did
not segment, but when brought back to a higher (normal)
temperature, the egg divided into two, four, etc., blastomeres ;

168

Ch. XVI] EFFECTS OF TEMPERATURE AND LIGHT 169

nevertheless, as subsequent development showed, the egg had
been injured. Many of these eggs developed in the same way
as did those kept at a temperature of 25 degrees C, i.e. the
segmentation of the yolk-hemisphere was retarded.

Schultze ('95) has also made some experiments on the
eggs of Rana fusca in which the eggs were subjected to a
temperature of zero C. Embryos in the following stages of
development were used : sta(/e A, when the dorsal lip of the
blastopore had just appeared; sta^e B, at the end of the "gas-
trula "' period ; stage C, embryos with closed medullary folds.
Three days after these had been placed in a chamber at zero C.
they were examined and found in the same stage as when put
into the cold. Some of the eggs were then removed, and con-
tinued to develop normally at a higher temperature. After
fourteen days in the cold the remainino- eg'g-s were examined.
The eggs were still in the same stage as when put into the
cold chamber, but those of stage C had died. The others
developed normally when brought into a higher temperature.

Thus while Hertwig found that the eggs of liana fusca were
injured by only twenty-four hours at a temperature of zero C,
Schultze saw that certain stages, at least, were not affected by
fourteen days at the same temperature. It is to be noted that
Hertwig put the eggs into cold water soon after fertilization,
while Schultze used later stages of development.

Not only is the rate of development of the frog-embryo
affected by the temperature, but also by the kind of light in
which it develops. Schnetzler in 1874 compared the develop-
ment of Rana temporaria in white and in green light. The con-
ditions of the two sets of embryos were nearly the same except
as regards the kind of light. The embryos developed much
faster in the white light, and the tadpoles underwent sooner
their metamorphoses. Yung ('78) made a much more careful
and elaborate series of experiments in which the eggs and
embryos were subjected to a series of different lights. Instead
of colored glass, which is seldom monochromatic, Yung used
solutions of different sorts. The eggs were placed in a vessel
containing about 5 litres of water ; this vessel was then placed
in a larger vessel of the same form. A space of 5 to 10 mm.
was left between the two vessels. This space was filled with

170 DEVELOPMENT OF THE FROG'S EGG [Ch. XVI

a fluid that allows only certain parts of the spectrum to
pass through. The top of the dish containing the eggs was
covered by an opaque lid. An alcoholic solution of " fuchsine
cerise " was used to produce a monochromatic red light ; a
solution of potassium chromate for a yellow light (although
this allows a little red and green to pass through) ; a solution
of nitrate of nickel (which is perfectly monochromatic) for a
green light ; an alcoholic solution of aniline "bleu de Lyon"
for a blue light ; and an alcoholic solution of aniline violet for
a violet light. Parallel experiments took place in the daylight
("white light") and in the dark. The other conditions were
the same for all the aquaria ; they had the same amount of
water, the same extent of surface for aeration, the same tempera-
ture, and were placed in the same position before a window.
Eggs of R. esculenta and of R. temporaria were used.

At the end of seven days it could be seen that the embryos
in the violet and in the blue light were more vigorous and in a
later stage of development than the others. At the same time
the development in the red and in the green was retarded. At
the end of a month the tadpoles were in good condition, and the
following table shows their mean length in each aquarium.

Larv.e of Rana Esculenta at the End of One Month.

Violet.

Blue.

Yellow.

White.

Dark.

Red.

Green.

27

24

22.8

23

19.6

19.1

15.1

The breadth of the embryos shows the same differences. It
is interesting to see that in the red and in the green light the
tadpoles were even less developed than those in the white light
or even in the dark. The result of this series of experiments
on R. esculenta agrees with other experiments made by Yung
at different times, upon other species of frogs and upon other
animals.

APPENDIX

METHODS OF PRESERVATION, ETC.

For general purposes the eggs and embryos may be pre-
served in a saturated solution of picric acid in seventy per cent,
alcohol to which a little sulphuric acid has been added (as
in Kleinenberg's picro-sulphuric solution). The segmenting
eggs or the early stages of the embryo surrounded b}' the
jelly should be put directly into the fluid. Each egg should
have, however, the outer jelly-coats cut off with a pair of
scissors, and it is well to use an abundance of the preserv-
ing solution. Older embryos may be shelled out in the pre-
serving fluid with sharp needles. After from three to five
hours the eggs or embryos are transferred to seventy per
cent, alcohol, which is changed several times ; they should
be kept for several days in eighty per cent, alcohol. In
this alcohol (eighty per cent.) the inner egg-membrane slowly
separates from the egg., and can be easily removed, after which
the egg is preserved permanently in eighty-five per cent, to
ninety per cent, alcohol. Corrosive-acetic solution gives good
results with older embryos. For the early stages of fertiliza-
tion and of extrusion of the polar bodies the following solution
is to be recommended : one per cent, chromic acid, twenty-five
pairts ; water, seventy parts ; glacial acetic acid, five parts.
Boiling water also gives good results.

Dilficulty is often found in cutting the eggs on account of
the brittleness of the yolk-portion ; but if the following method
is carefully followed, there will be no trouble in this regard.
The preserved egg or embryo is put into absolute alcohol from
two to five hours, turpentine two to three hours, soft paraf-
fine a half-hour (change once), hard ^jara^ne a half-hour.
The melting-point of the hard parafiine should be from 56

171

172 DEVELOPMENT OF THE FROG'S EGG

to 58 degrees C. The egg must then be cut at a temperature
of seve7ity-five to eighty degrees Fahrenheit (24 to 26 degrees
C); one often succeeds best if the microtome is placed in the
sunlight during the cutting.

The segmentation-stages do not need to be stained. The
older embryos stain well in toto in borax carmine or in
heematoxylin on the slide. Fresh material cuts and stains
better than that long preserved.

Formalin preserves eggs and jelly most admirably for dem-
onstration. The segmentation-stages show particularly well
when preserved (permanently) in this solution.

LITERATURE

Assheton, R.

'9i, a. On the Phenomenon of the Fnsion of the Epiblastic Layers in

the Rabbit and in the Frog. Quart. Jour. Micr. Science, XXXVII, '94.
'94, b. On the Growth in Length of the Frog Embryo. Quart. Jour.

Micr. Science, XXXVII, '94.
'9G. Notes on the Ciliation of the Ectoderm of the Amphibian Embryo.

Quart. Jour. Micr. Science, XXXVIII, '96.
Von Baer, K. E.

'28. Ueber Entwickehnigsgeschichte der Thiere, '28 and '37.

'28. Geschichte des Froschembryo. Burdach, Die Physiologie als

Erfalirungswissenschaft, II, '28.
'34. Die Metamorphose des Eies der Batrachier von der Erscheinung

des Embryo und Folgerung aus ihr fiir die Theorie der Erzeugung.

:Muller's Archiv, '34.
Van Bambeke, Ch.

'68. Recherches sur le developpement du Pelobate brun. Memoires

couronnes de I'Acad. Roy. des Sc. de Belgique, XXXIV, '68.
'70. Sur les trous vitellins que presentent les wufs fecondes des

amphibiens. Bull, de I'Acad. Roy. d. Sc. de Belgique, (II), XXX, '70.
'80, a. Fractionnement de I'oeuf des Batraciens. Arch, de Biologie, I, '80.
'80, b. Xouvelles recherches sur I'embryologie des Batraciens. Arch.

de Biologie, I, '80.
'80, c. Formation des f euillets embryonuaires et de la notochorde chez les

Urodeles. Bull, de I'Acad. Roy. des Sc. de Belgique, (II), XLIX, '80.
Barfurth, D.

'93, a. Halbbildung oder Ganzbildung von halber Grosse. Anat. Anz.,

A^IIL '93.
'93, b. Esperimentelle rutersuchungen iiber die Regeneration der

Keimbliitter bei den Amphibien. Anat. Hefte, III, '93.
'93, c. Ueber organbildende Keimbezirke und kunstliche Missbildungen
des Amphibieneies. Anat. Hefte, III, '93.
Beddard, F. E.

•94. Xotes upon the Tadpole of Xenopus laevis (Dactylethra capensis).
Proc. Zool. Soc, London, '94.

173

174 DEVELOPMENT OF THE FROG'S EGG

Bellonci, G.

"SO. Siii nuclei palimorfii delle cellule sessuali degli anfibi, '86.
Benecke, B.

'80. Ueber die Entwickelung des Erdsalamanders. Zool. Anz., Ill '80.
Bergmann. ' '

'41. Die Zerkliiftung und Zellenbildung im Froschdotter. Muller's
Arcliiv, '11.
Bernard und Bratuschek.

'91. Der Nutzen der SchleimhuUen fur die Froscheier. Biol. Centralb
XI, '91.
Bertacchini, P.

'89. Sui fenomeni di divisione delle cellule seminali primitive nella
Raiia temporaria. Rassegna Sc. Med., IV, '89.
Born, G.

'81. Experinientelle Untersuchungen iiber die Entstehung der Ge-
schlechtsunterschiede. Breslauer iirztl. Zeitschr., No. 3, '81.

'82. Ueber Doppelbilduugen beim Froschund deren Entstehung. Bres-
lauer arztl. Zeitschr., No. 14, '82.

'83, a. Biologische Untersuchungen, I. Ueber den Einfluss der Schwere
auf das Froschei. Pfliiger's Archiv, XXXII, '83.

'83, b. Beitrage zur Bastardirung zwischen den einheimischen Anuren-
arten. Pfliiger's Archiv, XXXII, '83.

'84, a. Ueber die inneren Vorgjinge bei der Bastardbefruchtung der
Froscheier. Breslauer iirztl. Zeitschr., No. 16, '84.

'84, b. Ueber den Einfluss der Schwere auf das Froschei. Verh.
d. Med. Section d. Schles. Ges. f. vaterl. Cultur. April 4
'84. '■ '

'8.5. Biologische Untersuchungen iiber den Einfluss der Schwere auf

das Froschei. Archiv f. JMikr. Anat., XXIV, '85.
'86. Weitere Beitrage zur Bastardirung zwischen den einheimischen

Anuren. Archiv f. Mikr. Anat., XXVII, '86.
'87. Ueber die Furchung des Eies bei Doppelbildung. Breslauer iirztl.

Zeitschr., No. 15, '87.

'92. Die Reifung des Amphibieneies und die Befruchtung unreifer Eier

bei Triton ta^niatus. Anat. Anz., VII, '92.
'93. Ueber Druckversuche an Froscheiern. Anat. Anz., VIII, '93
'94, a. Die kiinstliche Vereinigung lebender Theilstucke 'von Amphibien-

Larven. Jahresb. d. Schles. Ges. f. vaterl. Cultur. Med. Section.

Juni 8, '94.

'94, b. Die Structur des Keimbliischens im Ovarialei von Triton tajuia-

tus. Archiv f . Mikr. Anat., XLIII, '94.
'94, c. Neue Compressionsversuche an Froscheiern. Jahresb. d. Schles.
Ges. f. vaterl. Cultur. Zool. Bot. Section. 10 Mai, '94.
Cramer, H.

'48 Bemerkungen uber das Zellenleben in der Entwickelung des
Froscheies. Muller's Archiv, '48.

LITERATURE 175

Cucati, G.

'90. Spermatogenesi nella Rana esculenta. Anat. Anz., V, '90.

Driesch, H.

'95. Zur Analysis der Potenzen enibryonaler Organzellen. Archiv f.

Eutwickelungsmechauik der Organismen, II, '95.
'96, a. Betrachtungen uber die Organisation des Eies und ihre Genese.

Archiv f. Entwickelungsmechanik der Organismen, IV, '96.
'96, b. Die Maschinentheorie des Lebens. Biol. Centralb., XVI,
'96.
Durham.

'86. Note on the Presence of a Neurenteric Canal in Rana. Quart.
Jour. Micr. Science, XXVI, '86.
Ecker, A.

'51. Icones physiologic^, '51-'59.
Endres, H.

'91. Ueber Anstichversuche an Froscheiern. Jahresb. d. Schles. Ges. f.

vaterl. Cultur. Zool. But. Section. Xov. 15, '91.
'95. Ueber Anstich- und Schnurversuche an Eiern von Triton tseniatus.
Jahresb. d. Schles. Ges. f. vaterl. Cultur, '95.
Endres, H., und Walter, H. E.

'95. Anstichversuche an Eiern von Rana fusca. Archiv f. Entwick-
elungsniechanik d. Organismen, II, '95.
Von Erlanger, R.

'91, a. Zur Blastoporusfrage bei den anuren Amphibien. Anat. Anz.,

VI, '91.
'91, b. Ueber den Blastoporus der anuren Amphibien, sein Schicksal

und seine Beziehungen zum bleibenden After. Zool. Jahrbiicher.

Abt. f. Anat. und Ontog., IV, '91.
Eycleshymer, A. C.

'92. Paraphysis and Epiphysis in Amblystoma. Anat. Anz., ^'1I,

'92.
'93. The Development of the Optic Vesicles in Amphibia. Jour. Morph.,

VIII, '93.
Fick, R.

'93. Ueber die Reifung und Befruchtung des Axolotleies. Zeitschr. f.

vriss. Zool., LVI, '93.
Field. H. H.

'91. The Development of the Pronephros and Segmental Duct in

Ampliibia. Bull. Museum of Comp. Zool., XXI, '91.
'93. Ueber die Geflissversorgung und die allgeraeine Morphologic des

Glomus. Anat. Anz., VIII, '93.
'94. Die Vornierenkapsel, ventrale Musculatur und Extremitatenan-

lagen bei den Amphibien. Anat. Anz., IX, '94.
'95. Bemerkungen uber die Entwickelung der Wirbelsaule bei den

Amphibien; nebst Schilderung eines abnormen Wirbelsegmentes.

Morph. Jahrbuch, XXII, '95.

176 DEVELOPMENT OF THE FROG'S EGG

Flemming, W.

'87. Neiie Beitrage zur Kenntniss der Zelle. Archiv f. Mikr Anat
XXIX, '87.
Fiirbringer, M.

'77. Zur Entwickelung der Amphibienniere, Dissertation, '77.
'78. Zur vergleichenden Anatomie uud Entwickelungsgeschichte der
Excretionsorgane der Vertebraten. Morpli. Jahrbuch, IV, '78.
Gurwitsch, A.

'95. Ueber die Einwirkung des Lithionchlorids auf die Entwickelung
der Frosch- und Kroteneier (R. fusca und Bufo vulg.). Anat Anz
XI, '95.

'96. Ueber die formative Wirkung des veriinderten chemischen Mediums
auf die embryouale Entwickelung. Archiv f. Entwickelungsme-
chanik d. Organismen, III, '96.
Gasser, E.

'82. Zur Entwickelung von Alytes obstetricans. Sitz-Ber d. Xaturf Ges
Marburg, Xo. 5, '82.
Gebhardt, W.

'94. Ueber die Bastardirung von Rana esculenta mit Rana arvalis. Dis-
sertation, Breslau, '94.
Goette, A.

'75. Die Entwickelungsgeschichte der Unke, '75,
Von Griesheim, A.

'82. Kiinstliche Befruchtung der Eier von Rana fusca. Dissertation,
'82. '

Von Griesheim, A., Kochs, W., Pfliiger, E.

'81. Beitrage zur Physiologie der Zeugung. Pfluger's Archiv, XXVI, '81.
Herlitzka, A.

'95. Contribute alio studio della capacita evolutiva dei due primi blas-
tomeri nell' novo di tritone (Triton cristatus). Archiv f. Entwick-
elungsmechanik d. Organismen, II, '95.
Heron Royer et Ch. van Bambeke.

'81. Sur les Caracteres fournis par la bouche des Tetards des Batraciens
anoures d'Europe. Bull. Soc. Zool. d. France, VI, '81.
Hertwig, 0.

'77. Beitrage zur Kenntniss der Bildung, Befruchtung, und Theilung

des thierischen Eies, II Theil. Morph. Jahrbuch, III, '77.
'82. Die Entwickelung des. mittlereu Keimblattes der Wirbelthiere.

Jena. Zeitschr. f. Xaturw., XV und XVI, '82-83.
'85, a. Das Problem der Befruchtung und der Isotropic des Eies, eine

Theorie der Vererbung. Jena. Zeitschr., XVIII, '85.'
'85, b. Welchen Einfluss iibt die Schwerkraft auf die Theilungen der

Zellen. Jena. Zeitschr., XVIII, '85.
'85, c. Ueber das Vorkommen Spindeliger Korper im Dotter junger

Froscheier. Morph. Jahrbuch, X, '85.
'92. Urmund und Spina bifida. Archiv f. Mikr. Anat., XXXIX, '92.

LITERATURE 177

Hertwig, 0. (continued).

'93, a. Experimentelle Untersuchungen iiber die ersten Theilungen des
'rroscheies mid ihre Beziehungen zu der Organbildung des Embryos.
Sitzungsb. d. k. Preuss. Akad. d. Wiss. zu Berlin, '93.
'93, b. Ueber den Werth der ersten Furchungszellen fur die Organ

bildung des Embryo. Archiv f . Mikr. Anat., XLII, '93.
'94. Ueber den Einfluss ausserer Bedingungen auf die Entwickelung
des Froscheies. Sitzungsb. d. k. Preuss. Akad. d. "\Yiss. zu Berlin,
XVII, '94.
'95. Beitrage zur experimentellen Morphologic und Entwickelungsge-
schichte, No. 1. Archiv f. :Mikr. Anat., XLIV, '95.
Higgenbotham, J.

'50. Influence of Physical Agents on the Development of the Tad-
pole of the Triton and the Frog. Phil. Trans. Roy. Soc, London,

'50.
'63. Influence des agents physiques sur le developpement du tetard de

la grenouille. Jour, de la Physiologic de I'lionnne et des animaux.

YI, '03.
Hinckley, Mary H.

'82. Xotes on the Development of Rana sylvatica. Proc. Boston Soc.

Xat. History, '82.
His, W.

74. Unsere Kbrperform, '74.

Hochstetter, F.

'94. Entwickelung des Yenensystemes der Wirbelthiere. Ergcbnisse
der Anatomic und Entwickelungsgeschichte, III, '94.

Houssay, F. * i • j

'90. Etudes d'embryologie sur les vertebres. L'Axolotl. Archiv de

Zool. exper. et gen., (Ill), YIII, '90.

De risle, A.

'73. De I'hybridation chez les amphibies. Ann. des sc. naturelles,

XVII, '73.

Johnson, A. ...

'84. On the Fate of the Blastopore and the Presence of a Primitive
Streak in the Newt (Triton cristatus). Quart. Jour. Micr. Science,
XXI Y, '84.
Johnson, A., and Sheldon, L.

'86. Notes on the Development of the Newt. Quart. Jour, ilicr.

Science, XXVI, '86.
Jordan, E. 0.. and Eycleshymer. A.

"92. The Cleavage of the Amphibian Ovum. Anat. Anz., VII, '92.

Jordan, E. 0.

'93. The Habits and Development of the Newt. Jour, of Morph., V 11 J,

'93.
Jordan, E. 0., and Eycleshymer. A. C

'94. On tlie Cleavage of Amphibian Ova. Jour. Morph., IX, '94.

N

178 DEVELOPMENT OF THE FROG'S EGG

Kolessnikow, N.

Kupffer, C.

•82. Ueber aktive Betheiligung de, Dotters am Befruchtungsakte bei

b Ik Td W- "; ""'f- '""■"«*• "• --'h-P'V-k Classe d k
D. Akad. d. \\ 18S. zu Miinchen, XII, '82.

Lataste, F.

78 Tentatives d'hybridation chez les Batraciens anoures et urodeles
Bulletin de la Societe zoalogique de France, III, '78
Lwoff, B. ; ) •

''^Chnid ^^^T/ ^7 ^";"""''" ^'^"^^'^^ ^nd die Enstehung der
Choida und des Mesoderms bei den Wirbelthieren. Bull, de la Soc
luiper. des iVaturalistes de Moscou, VIII '9i
Marshall, A. M.. and Bles, E. J. ' '

'""■^/^^p^r^''?'?"' °^ '^'' ^^'^''^y' ^"'^ F'-^t Bodies in the Fro-^
>Stud. Biolog. Lab. Owens College, IL '90

Marshall, A. M.

'90. The Development of the Blood Vessels in the Frog. Stud. Biolog.

Lab. Owens College, II, '90.
'93. Vertebrate Embryology, '93.
McDonnell, B.

'57. Expose de quelques experiences concernant I'influence des aoents
physiques sur le developpement du tetard de la grenouiUe °com-
mune. Jour, de la Physiologie de I'homme et des animaux. (I), II,

Massart, J.

\T\\f'"ff'''" ?'' «Pei™atoides dans l'c«uf de la grenouille.
Maur ^"' "^^ ^^^S'^^"^' C^^^)' ^VIII, '89.

■88. Die Kiemen und ihre Gefasse bei Anuren und Urodelen Amphibien.
jNIorph. Jahrbuch, XIV, '88.
Meves, F.

'91. Ueber amitotische Kerntheilung in den Spermatogonien des Sala-
manders. Anat. Anz., VI, '91. ^ =
'96. Ueber die Entwickelung der mannlichen Geschlechtszellen von
>Salamandra maculosa. Archiv. f. Mikr. Anat., XL VIII '96
Moquin-Tandon, G. '

•76. Recherche sur les Premiere Phases du Developpement des Batra-
ciens anoures. Ann. des sciences naturelles, (VI), III 76
Morgan. T. H. ^ '

•89. On the Amphibian Blastopore. Studies from the Biol. Lab. Johns
-Hoi^kins Univ., IV, '89.

'91. Some Notes on the Breeding Habits and Embryology of Froo-s.

American Naturalist, XXV, '91.
'94. The Formation of the Embryo of the Frog. Anat. Anz., IX, '9L

LITERATURE 179

Morgan, T. H. (continued).

'95. Half-Embryos and Whole-Embrj^os from one of the first two Blas-
tomeres of the Frog's Egg. Anat. Anz., X, '95.
Morgan, T. H., and Tsuda Ume.

'93. The Orientation of the Frog's Egg. Quart. Jour. :Micr. Science,
XXXV, '93.
Newport. G.

"51. On the Impregnation of the Ovum in the Amphibia. Phil. Trans.
Roy. Soc, London, '51.
Nussbaum.

'93. Zur Entwickelungsgeschichte der embryonalen Gefassendothelien
und der Blutkiirperclieu bei den Anuren (Rana temporaria). Abh.
Akad. der Wiss. in Krakau, XXII. (Reprinted in Biol. Centralblatt,
XIII, '93.)
Nussbaum. M.

'95. Zur Mechanik der Eiablage bei Ranafusca. Archiv f . Mikr. Anat.,
XLVI, '95.
Orr, H.

'88. Xote on the Development of Amphibians. Quart. Jour. Micr.
Science, XXIX, '88.
Von Perenyi, J.

'89. Die Entwickelung der Keimbliitter und der Chorda in neuer
Beleuchtung. Anat. Anz., IV, '89. (Page 587.)
Pfliiger, E.

'82. I. Ilat die Concentration des Samens einen Einfluss auf das
Geschlecht? II. Ueber die das Geschleclit bestimmenden Ursachen
und die Geschlechtverhaltnisse der Frdsche. III. Ueber die partheno-
genetische Furchung der Eier der Amphibien. IV. Wirkt der Saft
nicht briinstiger Miinnchen befruchtend? V. Die Bastardzeugung
bei den Batrachien. VI. Versuche der Befruchtung iil)erreifer Eier.
VII. Zur Entwickelungsgeschichte der Geburtslielferkrdte (Alytes
obstetricans). Pfliiger's Archiv, XXIX, '82.
'83. Ueber den Einfluss der Schwerkraft auf die Theilung der Zellen.

I, II, III, Theil. Pfliiger's Archiv, XXXL XXXII, '83.
'84. Ueber die Einwirkung der Schwerkraft und anderer Bedingungen
auf die Richtung der Zelltheilung. Pfluger's Archiv, XXXIV,
'84.
Pfluger, E., und Smith, W. J.

"83. Untersuchungen iiber Bastardirung der anuren Batrachier und die
Principien der Zeugung. Pfliiger's Archiv, XXXII, '83.
Ploetz. A. J.

'90. Die Vorgiinoe in den Froschhoden unter dem Einfluss der Jahres-
zeit. Archiv f. Anat. u. Phys., (Supplement-Band) '90,
Prevost et Dumas.

'24. Troisieme Memoire. De la gene'ration dans les Mammifers. Ann.
des sc. naturelles, II, '24.

180 DEVELOPMENT OF THE FROG'S EGG

Rabl. C.

■bG. Ueber die Bildung des Herzens der Amphibien. Morph. Jahrbuch
XII. '86. '

Vom Rath, 0.

'93. Beitrage zur Keiintniss der Spermatogenese von Salamander macu-
losa. Zeitschr. f. \yiss. Zool., LVII, '03.
Rauber, A.

'82. Xeue Grundlegungen zur Keuntnis der Zelle. Morph. Jalubucb

VIII, '82.

'83. Furchung und Achsenbildung der Wiebelthiere. Zool \nz VI
'83.

'86, a. Personaltheil und Germinaltbeil des Individimm. Zool Anz

IX, '86.

'86, b. Furchung und Achsenbildung. II. Zool. Anz., IX, '86.
Reichert, K. B.

'41. Ueber den Furchungsprocess der Batrachier-Eier. Muller's Archiv.
'41. '

'46. Der Furchungsprocess und die sogenannte Zellenbildung urn In-

haltsportionen. :Miiller's Archiv, '46.
'61. Der Faltenkranz an den beiden ersten Furchungskugeln des Frosch-

dotters und seine Bedeutung fur die Lehre von der Zelle. Mliller's

Archiv, '61.
Remak, R.

'55. I'ntersuchung iiber die Entwickelung der Wirbelthiere, '55.
Robinson, A., and Assheton, R.

'91. The Formation and Fate of the Primitive Streak, with Observa-
tions on the Archenteron and Germinal Layers of Rana temporaria.
Quart. Jour. INIicr. Science, XXXII, '91.
Rossi, U.

'90. Contributo alia maturazione delle nova degli Amfibii Anat
Anz., V, '90.
Roux, W.

'83. Ueber die Zeit der Bestimmung der Hauptrichtungen des Frosch-
embryo, '83.

'84-'91. Beitrage zur Entwickelungsmechanik des Embryo.

No. I. Zur Orientirung uber einige Probleme der embryonalen Ent-
wickelung. Zeitschr. f. Biologie, XXI, '85.

No. II. Ueber die Entwickelung der Froscheier bei Aufhel)ung
der richtenden Wirkung der Schwere. Breslauer arztl. Zeitschr"
'84.

No, III. Ueber die Bestimmung der Haiiptrichtungen des Frosch-
embryo im Ei und iiber die erste Theilung des Froscheies. Bres-
lauer iirztl. Zeitschr., '85.

No. IV. Die Bestimmung der Medianebene des Froschembryo dnrch
die Copulationsrichtung des Eikernesund des Spermakernes. Archiv
f. Mikr. Anat., XXIX, '87.

LITERATURE 181

Roux, W. (continued) .

No. V. Ueber die kiinstliche Hervorbringung halber Embryonen durch

Zerstorung" einer der beideu ersteu Furchungskugeln sowie iilier

die Nachentwickelung (Postgeneration) der fehlendeu Korperliiilfte.

Yirchow's Archiv, CXIV, '88.
Xo. VI. Ueber die morphologische Polarisation von Eiern und Embry-
onen durch den electrischen Strom. Sitzungsb. d. k. Akad. Wiss.

in Wien, CI, '01.
'88, a. Ueber die Lagerung des Materials des Medullarrohres im gefurch-

ten Froschei. Yerhandlungen d. Anat. Gesell. zu "Wiirzburg, '88;

also Anat. Anz., Ill, '88.
'88, b. Zur Frage der Axenbestimmung des Embryo im Froschei. Biol.

Centralb., VIII, '88.
'89, a. Die Entwickelungsmechanik der Organismen, eine anatomische

Wissenschaft der Zukunft. Festrede, '89.
'89, b. Ueber die Entwickekmg der Extraovates der Froscheier. Jahresb.

d. Schles. Ges. f. vaterl. Cultur, '89.
'92, a. Ziele und Wege der Entwickelungsmechanik. Merkel-Bonnet's

Ergebnisse der Anatomie iind Entwickelungsgeschichte, II, '92.
'92, b. Ueber das entwickelungsmechanische Vermogen jeder der beiden

ersten Fui'chungszellen des Eies. Verhandl. d. Anat. Gesellschaft

Wien, '92.
'93, a. Ueber Mosaikarbeit und neuere Entwickelungshypothesen.

Anat. Hefte, Merkel und Bonnet, '93.
'93, b. Ueber die Spezifikation der Furchungszellen und liber die bei

der Postgeneration und Regeneration anzunehmendeu Vorgange.

Biol. Centralb., XIII, '93.
'93, c. Ueber die ersten Theilungen des Froscheies und ihre Bezie-

hungen zu der Organbildung des Embryo. Anat. Anz., VIIT, '93.
'93, d. Ueber die Selbstordnung der Furchungszellen. Berichte des

naturw.-med. Vereins zu Innsbruck, XXI, '93.
'94, a. Die Methoden zur Hervorbringung halber Froschembryonen und

zum Nachweis der Beziehung der ersten Furchungsebenen des Frosch-
eies zur Medianebene des Embryo. Anat. Anz., IX, '91.
94, b. Ueber den Cytotropismus der Furchungszellen des Grasfrosches

(Rana fusca). Archiv f. Entwickelungsmechanik der Organismen,

I, '94.
'95. Gesammelte Abhandlungen iiber Entwickelungsmechanik der

Organismen, '95.
'96, a. Ueber die Selbstordnung (Cytotaxis) sich " beruhrender " Fur-
chungszellen des Froscheies durch Zellenzusammenfiigung, Zellentren-

nung und Zellengleiten. Archiv f. Entwickelungsmechanik der

Organismen, III, '96.
'96, b. Ueber die Bedeutung " geringer " Yerschiedenheiten der relativen

Grosse der Furchungszellen fiir den Charakter des Furchungsschemas.

Archiv f . Entwickelungsmechanik der Organismen, IV, '96,

182 DEVELOPMENT OF THE FROG'S EGG

Riickert, J., und Mollier, W.

'89. Resultate iiber die Entstehung des Vorniereiisystem bei Triton,
Rana und Biifo. Sitz. Ber. d. Ges. fiir Morph. u. Phys. in Miinchen
XIX, '89.
Rusconi, M.

'26. Developpement de la grenouille commune, '26.
'36. Zweiter Brief an E. H. Weber. Miiller's Archiv, '36.
'40. Ueber kiiiistliche Befruchtung von Fischen und iiber einige neue
Versuche in Betreff kiinstlicher Befruchtung an Frdschen. ^^liiller's
Archiv, '40.
'54. Histoire naturelle, developpement et metamorphose de la Sala-
mandre terrestre, '54.
Samassa, P.

'95. Studien iiber den Einfluss des Dotters auf die Gastrulation und die
Bildung der primaren Keimbljitter der Wirbelthiere. II. Amphibien.
Archiv f. Entw.-mechanik d. Organismen, II, '95.
Schanz, F.

'87. Das Schicksal des Blastoporus bei den Amphibien. Jena. Zeitschr.
f. Naturwissenschaft, XXI, '87.
Schmidt, V.

'93. Das Schwanzende der Chorda dorsalis bei den Wirbelthieren.
Anat. Hefte, II, '93.
Schnetzler, J. B.

'74. De I'influence de la lumiere sur le de'veloppement des larves de
grenouilles. Arch, des sciences physiques et naturelles, LI, '74.
Schultze, M.

'63. Observationes nonnullse de ovorum ranarum segmentatione, '63.
Schultze, 0.

'83. Beitriige zur Entwickelung der Batrachien. Archiv f. Mikr. Anat.,
XXIII, '83.

'86. Ueber Reifung und Befruchtung des Amphibieneies. Anat. Anz.,
I, '86.

'87, a. Zur Entwickelung des braunen Grasfrosches. Festschrift f.
Kblliker, '87.

'87, b. Die vitale Methylenblaureaction der Zellgranule. Anat. Anz.,

'87.
'87, c. Zurersten Entwickelung des braunen Grasfrosches (Ref. Roux).

Biol. Centralb., VII. '87.

'87, d. Ueber Axenbestimmung des Froschembryo. Biol. Centralb.,
VII, '87.

'87, e. Untersuchungen uber die Reifung und Befruchtung des Amphibi-
eneies. Zeitschr. f. wiss. Zool., XLV, '87.

'88. Die Entwickelung der Keimbliitter und der Chorda dorsalis von
Rana fusca. Zeitschr. f. wiss. Zool., XLVII, '88.

'89. Ueber die Entwickelung der Medullarplatte des Froscheies. Verb.
d. phys. med. Gesellschaft, A\'urzburg, XXIII, '89.

LITERATURE 183

Schultze, 0. (continued).

'94, a. Ueber die unbedingte Abhiingigkeit normaler thierischer Gestal-

tung von del- Wirkung der Schwerkraft. Verb. d. Anat. Ges., VIII, '94.
'94, b. Die kiinstliche Erzeugung von Doppelbildungen bei Froschlarven

mit Hilfe abnornier Gravitationswirkung. Archiv f. Entwickelungs-

mechanik der Organismen, I, '94.
'94, c. Ueber die Einwirknng niederer Teraperatur auf die Entwickelung

des Frosches. Anat. Anz., X, '94.
'94, d. Ueber die Bedeutung der Scliwerkraft fiir die organische Ges-

taltung sowie iiber die mit Hilfe der Schwerkraft niogliche kiinst-
liche Erzeugung von Doppelmissbildungen. Verh. phys. nied. Ges.

zu Wiirzburg, XXVIII, '94.
Schwink, F.

'88. Ueber die Gastrula bei Amphibieneiern. Biol. Centralb., VIII,

'88-'89.
'89. Ueber die Entwickelung des mittleren Keimblattes und der Chorda

dorsalis der Amphibien. Miinchen, '89.
'90. Ueber die Entwickelung des Herzenendothels der Amphibien.

Anat. Anz., V, '90.
'91. Untersuchungen iiber die Entwickelung des Endothels und der

Blutkorperchen bei Amphibien. iNIorph. Jahrb., XVII, '91.
Sidebotham, H.

'88. Note on the Fate of the Blastopore in Rana temporaria. Quart.

Jour. Micr. Science, XXIX, '88.
Spallanzani, L.

17^.'). Experience pour servir a I'histoire de la generation, 1785.
Spencer, W. B.

'85. On the Fate of the Blastopore in Rana tempoi'aria. Zool. Anz.,

VIII, '85.
'85. Some Xotes on the Early Development of Rana temporaria. Quart.

Jour. Micr. Science, XXV, '85.
Stahl, E.

'88. Pflanzen und Schnecken. (Jena,) '88.
Strieker, S.

'GO. Entwickelungsgeschichte von Bufo cinereus bis zum Erscheinen der

iiusseren Kiemen. Sitzungsb. d. k. Akademie der Wiss. zu Wien,

XXXIX, '60.
'62. Untersuchungen iiber die ersten Anlagen in Batrachier-Eiern.

Zeitschr. f . wiss. Zool., XI, '62.
Swammerdam, J.

1737. Die Bibel der Natur, 1737.
V. la Valette St. George.

'75. Die Spermatogenese bei den Amphibien. Archiv f . Mikr. Anat.,

XII, '75.
'86. Spermatologische Beitrage. Dritte Mittheilung. Archiv f . ]\Iikr.

Anat., XXVII, '86.

184 DEVELOPMENT OF THE FROG'S EGG

Vogt, K.

'42. Untersuchungen uber die Entwickelungsgeschichte der Geburts-
helferkiote, '42.
Wetzel, G.

'95. Ueber die Bedeutung der Cirkuliiren Furche in der Eutwickelung
der Schultzescheu Dopiielbildungeii von Rana fusca. Archiv f. ISlikv
Anat., XLVI, '95.
'96. Beitrag zuni Studium der kiinstlichen Doppelmissbildungen von
Rana fusca. Inaugural Dissertation. Berlin, '96.
Will, L.

'84. Ueber die Entstehung des Dotters und der Epithelzellen bei den
Aniphibien und Insecten. Zool. Anz., VII, '84.
Wilson, C. B.

'96. The Wrinkling of Frog's Eggs during Segmentation. American
Naturalist, XXX, '96.
Yung, E.

'78. Influence des differentes couleurs du spectre sur le developpement
des animaux. Arch, de zool. experimentale et generale, (I,) VII, '78,
and Arch, des sciences physiques et naturelles, '78.

'81. De I'influence des lumieres colorees sur le developpement des ani-
maux. Mittheil. a. d. zool. Station zu Neapel, II, '81.

'90. Propos scientifiques, '90.
Ziegler, F.

'92. Zur Kenntniss der Oberflachenbilder der Rana-Embryonen. Anat
Anz., VII, 92.

OTHER MEMOIRS REFERRED TO IN TEXT •

Boveri, Th.

'89. Ein geschlechtlich erzeugter Organismus ohne miitterliche Eigen-
schaften. Sitz. d. Ges. f. Morph. u. Physiol, zu Munchen, '89.
(Translated in American Naturalist, March, '93.)
Chun, B.

'92. Die Dissogonie der Rippenquallen. Festschrift f. Leuckart, '92.
Clapp, C. M.

'91 . Some Points on the Development of the Toad-fish (Batrachus Tau) .

Jour. Morph., V, '91,
Driesch, H.

'91-93. Entwickelungsmechanische Studien.

No. I. Der Werth der beiden Furchungszellen der Echinodermentwick-

elung. Zeitschr. f. wiss. Zool., LIII, '91.
No. II. Ueber die Beziehungen des Lichtes zur ersten Etapi:)e der

thierischen Fornibildung. Zeitschr. f. wiss. Zool., LIII, '91.
No. III. Die Verminderung des Furchungsmaterials und ihre Folgen.

Zeitschr. f. wiss. Zool., LV, '92.

LITERATURE 185

Driesch, H. {continued).

Xo. IV. Experimen telle Veranderungen des Typus der Fuvchung und

ihre Folgen. Zeitschr. f. wiss. ZooL, LV, '92.
Xo. V. You der Fuvchung doppeltbefruchteter Eier. Zeitschr. f. wiss.

ZooL, L\, '92.
No. VI. Ueber einige allgemeine Fragen der theoretischen Morphologic.

Zeitschr. f. wiss. ZooL, LV, '92.
No. VII. Exogastrula und Anenteria. Mittheil. a. d. zool. Station zu

Neapel, XI, '93.
No. VIII. Ueber Variation der ^Mikromerenbildung. Mittheil. a. d.

zool. Station zu Xeapel, XI, '93.
No. IX. Ueber die Vertretbarkeit der Anlagen von Ektoderm und

Endoderm. Mittheil. a. d. zool. Station zu X'eapel, XI, '93,
No. X. Ueber einige allgemeine entwickelungsniechanische Ergebnisse.

Mittheil. a. d. zool. Station zu Xeapel, XI, '93.
'92. Entwickelungsmechanisches. Anat. Anz., VII, '92.
'93, a. Zur Theorie der thierischen Formbildung. Biol. Centralb.,

Xin, '93.
'93, b. Zur Verlagerung der Blastomeren des Echinideueies. Anat.

Anz., VIII, '93.
'91. Analytische Theorie der Organischen Entwickelung, '94.
Driesch, H., und Morgan, T. H.

'95. Zur Analysis der ersten Entwickelungsstadien des Ctenophoreneies.
Archiv f. Entwickelungsmechanik d. Organismen, II, '95.
Hertwig, 0.

'90. Vergleich der Ei- und Samenliildung bei Xematoden. Archiv f.

Mikr. Anat., XXXVI, '90.
'91. Zeit- und Streitfragen der Biologie, '94.
Hertwig, 0., und Hertwig, R.

'87, Ueber den Befruchtungs- und Theilungs-vorgang des Thierischen
Eies unter deni Einfluss ausserer Agentien, Jena. Zeitschr. X^aturw.,
XX, '87,
His, W.

'94. Ueber mechanische Grundvorgiinge thierischer Formbildung,
Archiv f. Anat. u. Phys., '94.
Morgan, T, H,

'93. Experimental Studies on Teleost Eggs. Anat. Anz., VIII, '93,
'95. Studies of tlie "Partial" Larvaj of Sphrerechinus. Archiv f.
Entwickelungsmechanik d. Organismen, II, '95. .
Rabl, C.

"89. Die Theorie des Mesoderms. Morph. Jahvbuch, XV, '89.
Vom Rath. 0.

'92. Zur Kenntniss der Spermatogenese von Gryllotalpa vulgaris,

Archiv f. :Mikr. Anat., XL, '92.
'95. Xeue Beitriige zur Frage der Chromatinreduction in der Samen-
und Eireife, Archiv f . :Mikr, Anat., XL VI, '95,

186 DEVELOPMENT OF THE FROG'S EGG

Rauber, A.

'80. Formbildung und Formstorung. .Alorph. Jahibuch, VI, '80
Sachs.

'92. Die Aiiordnung- der Zellen in jiingsten Pflanzentheilen. Arbeiten
d. botan. Institute in Wiirzburg, II, '92.
Schwann, Th.

'39 Mikroskopische Untersuchungen iiber die Uebereinstimmung in
der Structur und Wachsthum der Thiere und Pflanzen, '39.
Weismann, A.

'92. Das Keimplasma. Fine Theorie der Vererbuno- '90
Whitman, C. 0. t^ --

'95. The Inadequacy of the Cell Theory of Development. Jour. Morph.,

Wilson, E. B.

'92. The Cell Lineage of Nereis. Jour. Morph., VI, '92.

■93. Amphioxus and the Mosaic Theory. Jour. Morph VIII '93
Zoja, R. ' 15,.

'95. Sullo svilluppo dei blastomeri isolati dalle uova di alcune meduse
(e di altri organismi). Archiv f. Entwickelungsmechanik d. Organ-
ismen, I, '95.

INDEX

Adhesive glands, 62, 165.
Afferent branchial vessels, 153-155.
Amphioxus, isolation of blastomere,
127, 131.

isolation of one-fourth and one-
eighth blastomeres, 133.
Anus, 60, 62.

beginning of, 136-139.

of Urodela, 139, 140.
Aorta, early stage, 153.
Aortic bulb, 153.
Archenteron, 66-68, 70.

of spina bifida, 77.

of heuiienibryo, 108, 109.

posterior pocket of, 136-140.

enlargement of, 140-143.
Ascidians, half-development, 127.

isolation of blastomere, 131.
Assheton, formation of archenteron,
70.

ciliation of embryo, 165.
Auditory nerve, 164.
Auricle of heart, 153.
Axis, primary, 81.

secondary, 82.

tertiary, 82.

von Baer, account of cleavage, 48.

Barock segmentation, 29.

Bellonci, direct division of germ-cells,

12.
Bergmann, account of cleavage, 49.
Bernard and Bratuschek, 20.
Blastomeres, injury to, 106-122.
Blastopore, 50-57.

overgrowth of, 50-57, 68.

injury to, 79.

position of dorsal lip, 88.

in compressed egg, 98-101.

closure of, 137-140.

of Urodela, 139, 140.
Blastula, double, 118.

Blood-corpuscles, 155.
Body-cavity, 148.
Bombinator, pronephros of, 158.
Born, cross-fertilization, 26-28.

sperm-fluid, 29.

experiments, 90-92.

compression of egg, 95-99.

conclusions from compressed egg,
102.

cleavage-plane and embryo-axis,
108.
Boveri, second maturation-division, 8,
9.

egg-fragments, 30, 31.
Brain-vesicles, 62.
Branchial arches, 145.
Branchial vessel, 153, 154.

development of, 155.
Brauer, second maturation-division, 8,

9.
Bufo, fertilization of egg, 22.

vulgaris, cross-fertilization, 26-28.

communis, cross-fertilization, 28.

Cardinal veins, 153, 157.
jjCellulation of yolk, hemiembryo, 110.
Centrifugal force, effect of, 92-94.
Centrifugal machine, 92-94.
Cerebellum, 160.
Cerebral hemispheres, 160.
Chabry, 127.
Chun, 127.

Cilia, on surface, 165.
Cleavage, 32-41.

of compressed egg, 96-101.
of egg in centrifugal machine, 92-94.
Cleavage-plane, relation to egg-axis,

82, 85-87.
Cloaca, opening of segmental duct into,

156.
Ccelom, 148, 150.

relation to pronephros, 156.
187

188

DEVELOPMENT OF THE FROG'S EGG

Collecting tubes of pronephros, 156,

157.
Communicating canal, 148.
Compression of egg, 95-105.
d Concrescence, 64, 65, 80.
Con-elation of parts, 124-126.
Cramer, account of cleavage, 49.
Cranial nerves, 164.
.- Cross-fertilization, 26-30.
Cross-line, 37, 39.
Ctenophor, half-development, 127.

isolated blastomere, 129, 130, 132.

half-larva, 130.

fragment of egg, 131.

imperfect embryo, 135.
Cutaneous nerves, 164.
Cuvierian^ veins, 153.

Delamination, 41.
Development, direct, 128.

indirect, 128.
Differentiation by protoplasm, 131.
Diverticula, from aorta, 155.

"from aortic bulb, 155.
Division (direct), of germ-cells, 11,

12.
Dorsal aorta, origin of, 155.
Driesch, sea-urchin egg, 126, 127.

action of nucleus on cell, 135.

theory of embryo, 136.
Dumas, account of cleavage, 48.

Ear, 162, 163.

Echinodermata, isolation of blasto-
mere, 131.
Echinus, isolation of one-fourth and

one-eighth blastomere, 133.
Ectoderm, 71, 72.

ciliation of, 165.

organs from, 159.
Efferent branchial vessels, 153-155.
Egg, separation from ovary, 15.

orientation of, 81.

rotation of, 83-85.

rotation of contents, 91, 92.

in centrifugal machine, 92-94.

of Rana fusca, size of, 95.

fragments of sea-urchin, 130, 131.

ctenophor, fragments of, 131.
Egg-axis, 32.

relation to cleavage-plane, 82, 85-87.
Egg-laying, Introduction.
Egg-membranes, 17, 19, 20.

Egg-membranes, absorption of heat
by, 20.
protection by, 19, 20.
Egg-nucleus, migration of, 13.
Elastic plates, 63, 64.
Embryo, on compressed egg, 98-101.
Embryonic ring, 64-66.
Embryos, double, 117-119.
Endoderm, 70, 73.
Endodermal cells of heart, 151.
Endothelium of heart, 151.
Endres and Walter, half-embryos, 115

116.
Epigenesis, 125, 126.
Evolution, 125, 126.
Eye, 60, 64, 161.

Facial nerve, 164.
Fiedler, 126, 127.
Field, H. H., origin of pronephros, 155-

158.
Fin, 62.
Flemming, spermatogenesis of Sala-

mandra, 5, 6.
Fore-brain, 62, 100.
Fiirbringer, pronephros, 158.

Ganglia, spinal, 62.
Germinal localization, 125, 126.
'iGerm-iDlasm, 124.
Gill-arches, 62.
Gill-plate, 68-60.
Gill-slits, 02.

formation of, 141, 144.

relation of nerves to, 164.
Gills, ciliation of, 167.
Glomus, 156, 157.
Glosso-pharyngeal nerve, 164.
Goette, origin of pronephros, 158.
Gryllotalpa, 2.

Half-blastula of Echinus, 127.
Ilalf-embryos of Echinus, 133.
Half-larva, ctenojjhor, 130.
Head-kidneys, 156-158.

veins from, 153.
Head-somites, 148.
Heart, 150-155.

bending of tube, 151.
ifHemiembryo, anterior, 109.

lateralis, 107, 108.

posterior, 109.
"/•^Hemiooholoplasten, 121.

INDEX

189

Hertwig, 0., second maturation-divi-
sion, 8, 9.

rotation of egg, 20.

cross-fertilization of sea-urchin, 28.

polyspermy, 30.

origin of mesoderm, 70.

formation of spina bifida, 77.

effect of gravity, 90.

compression of egg, 95, 99, 100.

egg in tube, 101.

lavr of cleavage, 102, 103.

injury to blastomere, 112-115.

methods of injury, 112.

hemiembryo lateralis, 113.

asymmetry of embryo, 113, 114.

criticism of Roux, 114, 115.

revivification of injured blastomere,
114, 115.

criticism of, 121.

quantitative division, 127.

interaction of blastomeres, 134.

effect of temperature, 168, 109.
[Heterotypic division, 5, 6.
Hind-brain, 160.
His, elastic plates, 63, 64.

germinal localization, 125, 126.
Hoffmann, pronephros, 158.
'Homreotypic division, 6, 7.
Hydromedusae, isolation of blastomere,

131.
Hyla, .spermatozoon of, 11.
Hyold arch, 145.

vessel, 153.
Hyomandibular-cleft, 145.

^- Idioplasm, 128.

Infundibulmn, 160, 161.

IiUeractiou of parts, 124, 125.

Invagination of archenteron, 70.
^ Isotropy, 87.

Jordan, E. O., entrance of spermato-

zoon, 35

30.

fertilization, 24.

median plane of embryo, 42.

overgrowth of blastopore, 68.

Kolliker, account of cleavage, 49.
Kupffer, fertilization, 22.

Lataste, cross-fertilization, 26.
Lateral line, 164.
Lens, 102.

Liver, origin of, 141.

relation of, to heart, 151.
Lungs, 145.

Mandibular vessels, 153, 154.
Marshall, origin of gill-slits, 144, 145.

account of lungs and thyroid body,
145.

origin of optic nerve, 101.

cranial nerves, 164.
Marshall and Bles, pronephros, 158.
Maturation-divisions, of Gryllotalpa,
2-4.

of Salamandra, 7, 9.

of frog, 10.
Maxillary process, inferior, 61.

superior, 61.
Medulla oblongata, 160.
Medullai-y folds, inner, 57-59.

outer, 57-59.
Medullary plate, 73, 158.

formation of, 80.

half, 108.

length of, 80.
--Mesenchyme, 148, 149.
Mesoderm, 69, 71-74.

early condition of, 146.

extension of, ventrally. 146.

around blastopore, 147.

in region of pharynx, 147.
Mesodermic somites, 148.
IMethods of preservation, Appendix.
Meves, spermatogenesis of Salaman-
dra, 5, 9.

direct division of germ-cells, 12.
Mid-brain, 160.
j\Iole-cricket, 2.

Morgan, T. H., formation of spina
bifida, 77.

injury to blastomere, 120, 121.

isolation of blastomeres by, 133.
Morula, 107.
Mouth, 60, 61.

Mliller, origin of pronephros, 158.
Muscle fibres, origin of, 149.

Nasal pits, 62.

Nephrostomes of pronephros, 156-158.

Nerves, 163, 164.

dorsal roots, 163.

ventral roots, 163.
Nerve-tube, bending of, 160.

closure of, 160.

190

DEVELOPMEXT OF THE FROG'S EGG

Nervous system, central, 159-161.
Neural crest, 159, 160.
Neural ridge or crest, 16.3, 164.
^Neurenteric canal, 1.38-140.
Newport, absorjition of water by egg-
membranes, 19.
entrance of egg into oviduct, 16.
median plane of embryo, 42.
Newt, fertilization of, 24.
Notochord, 70, 73, 74.
of spina bifida, 76-78.
half, 108.
origin of, 146.
Nuclei, distribution in compressed egg,

104, 105.
Nucleus, control of cell by, 128.

qualitative division of, 129.
Nussbaum, entrance of egg into ovi-
duct, 16.

Oil-drops, 43-47.
~ Oogenesis, 12.

and spermatogenesis, comparison
of, 13, 14.
Optic lobes, 160.
Optic stalk, 162.
Optic vesicles, 160, 161.
Orientation of egg, 81.

Pelobates, cross-fertilization, 26.

Pericardium, 151.

Pfliiger, cross-fertilization, 26-28.

median plane of embryo, 42.

blastopore, 51-53, 56.

account of experiments, 81-89.

methods, 82.

conclusions from experiments, 87-
89.

compression of egg, 95.

conclusions from compressed egg,
101, 102.

cleavage-plane and embryo-axis,
108.
Pharynx, 62, 145.
Pigment, distribution of, 15.

rotation of, 83.
Pineal body, 160, 161.
Pituitary body, 161.
Plane of embryo, median, 42.
Pneumogastric nerves, 164.
Polar body, first, extrusion of, 16-18.

second, 21.

in inverted egg, 91.

Polarization of egg, 88.
Poles of egg, 81.
Polyspermy, 30.
Post-anal-gut, 141.
j, Postgeneration, 110, 111, 116, 128,
129.

of archenteron. 111.

of medullary folds, 111.

of mesoderm, 111.

of ectoderm, 111.

of whole embryo, 122.
Provost, account of cleavage, 48.
Primitive gi-oove, 57, 72.
Primitive segments, origin of, 147.
Proctodaeum, 141, 158.
Pronephric capsule, 156.
-Pronephros, 155-158.
Pro-nucleus, union, 23.

apposition of, 35.

Rana arvalis, cross-fertilization, 27,

28.
Rana esculenta, spermatozoon of, 11.
cross-fertilization, 26-28.
effect of light, 170.
Rana fusca, extrusion of polar body,
21.
cross-fertilization, 26-28.
inversion after first cleavage, 116-

118.
effect of temperature, 168, 169.
Rana temiwraria, egg-laying, 17.
time of egg-laying, 168.
effect of light, 169, 170.
vom Rath, spermatogenesis of Gryllo-
talpa, 2-4.
spermatogenesis of Salamandra, 5,

7.
tetrad formation in Salamandra, 8.
spermatogenesis of frog, 10.
direct division of germ-cells, 12.
Rauber, interchange of nuclei, 30.
segmentation, 39.
effect of gravity, 90.
Reichert, account of cleavage, 49.
Remak, segmentation, 38.
account of cleavage, 49.
Reorganization, 109, 110.
Retiua, 161. ,
Robinson, formation of archenteron,

70.
Rotation of egg, 83-85.
Roux, artificial fertilization, 32.

INDKX

191

Roux {continued).

median plane of embryo, 42.

experiments with oil-drops, 43-47.

spina bifida, 75.

centrifugal machine, 92-94.

egg in tube, 100, 101.

methods, lOG, 107.

injury to blastomere, 106-111.

cleavage-plane and embryo-axis,
108.

mosaic theory, 109, 123, 126.

whole embryos, 121.

subsidiary hypothesis, 127-129.

anachronism in cleavage, 129.

part of egg removed, 130.

qualitative division of nucleus,
134.
Eusconi, cross-fertilization, 24.

account of cleavage, 48.

Sachs, law of cleavage, 102.
Salamandra, isolation of blastomere,

131.
Salt-solution, effect of, 77.
Schleiden, 49.

Schnetzler, effect of light, 169,
Schultzc, M., segmentation, 39.

account of cleavage, 49.
Schultze, O., formation of egg, 12.
rotation of egg, 20.
polar bodies, 21.
origin of mesoderm, 71.
experiments of, 110-118.
effect of temperature, 169.
Schwann, 49.

Sea-urchin, cross-fertilization, 30,
isolation of blastomeres, 120.
half development, 127.
fragments of egg, 130, 131.
Segmentation, variations of, 41.
. - Segmentation-cavity, 40, 41, 67, 71.

Self-differentiation, 123, 124, 126.
'fSemiblastula verticalis, 107, 108.
iVSemigastrula verticalis, 107, 108.
, (jSemimorula verticalis, 107, 108.
)1 Sense-plate, 57-60.
^rt Sex-cells, development of, 1.
Sinus venosus, 151.
Size of egg, 95.
■^ Somatic layer, of mesoderm, 147.

of heart, 151.
^ Somites, mesodermic, 148.
of head, 148,

Spallanzani, egg-laying, 17.

account of cleavage, 47,
Spermatid, 1.
Spermatocyte, 1.
Spermatogenesis, 1, 10.

.salamander 4, 5.
Spermatogenesis and oogenesis, com-
parison of, 13, 14.
Spermatogonia, 1.
Spermatozoon, of frog, 4, 5, 11,

inheritance through, 134.
Spina bifida, 75-80.
Splanchnic layer of mesoderm, 147.

of heart, 151.
Star-fish, cross-fertilization, 30.
Stomodseum, 60. 159.
Strasburger, action of nucleus on cell,

135.
Suckers, 60, 62, 166.
Swammerdam, passage of egg from
ovary to oviduct, 17.

account of cleavage, 47.
Sylvian aqueduct, 100.

Tail, 62.

Teleostei, isolation of blastomere, 131,

Temperature, effect of, 167-170.

Tetrad, 3, 4, 8.

Thyroid body, 145,

Toad, European, spermatozoon of,

11.
j^otipotence, 132, 133.
Trigeminal nerve, 164.
Triton alpestris, cross-fertilization, 26,

28.
tseniatus, cross-fertilization, 26, 28.
Truncus arteriosus, 153.

Urodela, anus of, 139, 140.

closure of blastopore, 139, 140.

Vagus, near first somite, 148.

V. la Valette St. George, terminol-
ogy, 1.

Ventricle of heart, 153.

Visceral-arches, 145.

Visceral-slits, 145.

Vitelline veins, 151.

Vitreous body, 162.

Vogt, segmentation, 38.

de Vries, action of nucleus on cell,
135.

192

DEVELOPMENT OF THE FROG'S EGG

Weismann, theory of heredity, 14.

qualitative nuclear division, 129.

qualitative division of nucleus,
134.
Wetzel, double embryos, 118, 119.
Whitman, theory of embryo, 136.
Wichmann, pronephros, 158.
Wilson, E. B., amphioxus, 127.

Wrinkles of egg, 33.

Yolk-granules, absorption of, 141.
Yolk-plug, withdrawal of, 140.
Yung, effect of light, 169, 170.

Ziegler, embryos, 61.

Zoja, isolation of blastomeres by, 132.

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4

ATLAS

OF THE

FERTILIZATION and KARYOKINESIS

OF THE OVUM

By EDMUND B. WILSON, Ph.D.,

Professor of Invertebrate Zoology hi Columbia College

WITH THE CO-OPERATION OF

EDWARD LEAMING, M.D., F.R.P.S.,
Instructor in Photography at the College of Physicians and Surgeons, Columbia College

WITH TEN PHOTOGRAPHIC PLATES AND NU.MEROUS DIAGRAMS

Extra 8vo. Cloth, pp. vii + 32. $4.00, net.

This work comprises forty figures, photographed from nature by Dr. Learning from the
preparations of Professor Wilson at an enlargement of one thousand diameters, and
mechanically reproduced by the gelatine process, without retouching, by Edward Bierstadt
of New York. The plates are accompanied by an explanatory text, giving a general intro-
duction to the subject for the use of students and general readers, a detailed description
of the photographs, and over sixty text-figures from camera-drawings.

It is the object of this atlas to place before students and teachers of biology a practically
continuous series of figures photographed directly from nature, to illustrate the principal
phenomena in the fertilization and eariy development of the animal egg. The new science
of cytology has in the course of the past two decades brought forward discoveries relating
to the fertilization of the egg and the closely related subject of cell-division (karyokinesis)
that have called forth on the part of Weismnnn and others some of the most important
and suggestive discussions of the post-Darwinian biology. These discoveries must in
some measure be dealt with by every modern text-book of morphology or physiology, yet
they belong to a region of observation inaccessible to the general reader or student, since
it can only be approached by means of a refined histological technique applied to special
objects not ordinarily available for practical study or demonstration. A knowledge of the
subject must therefore, in most cases, be acquired from text-books in which drawings are
made to take the place of the real object. But no drawing, however excellent, can convey
an accurate mental picture of the real object. It is extremely difficult for even the most
skilful draughtsman to represent in a drawing the exact appearance of protoplasm and the
delicate and complicated apparatus of the cell. It is impossible adequately to reproduce
the drawing in a black-and-white text-book figure. Ever>' such figure must necessarily be in
some measure schematic and embodies a considerable subjective element of interpretation.

The photograph, whatever be its shortcomings (and no photograph can do full justice
to nature), at least gives an absolutely faithful representation of what appears under the
microscope ; it contains no subjective element save that involved in the focussing of the
instrument, and hence conveys a true mental picture. It is hoped, therefore, that the pres-
ent work may serve a useful purpose, especially by enabling teachers of biology to place

7

before their students a series of illustrations whose fidelity is beyond question, and which
may serve as a basis for either elementary or advanced work in this direction.

Following is a partial list of the points clearly shown in the present series : The
ovarian egg, with germinal vesicle, germinal spot and chromatin-network ; the polar
amphiaster with the " Vierergruppen " or quadruple chromosome-groups ; the unfertilized
eoCT after extrusion of the polar bodies; entrance of the spermatozoon, the entrance-cone;
rotation of the sperm-head, origin of the sperm-aster from the middle-piece, growth of the
astral rays; conjugation of the germ-nuclei, extension and division of the sperm-aster;
formation of the cleavage-nucleus ; the attraction-spheres in the resting-cell ; formation of
the cleavage-amphiaster, origin of the spindle-fibres and chromosomes ; division of the
chromosomes, separation of the daughter-chromosomes; structure and growth of the
astrosphere ; degeneration of the spindle ; formation of the " Zwischenkorper " ; origin of
the chromatic vesicles from the chromosomes; reconstruction of the daughter-nuclei;
cleavage of the ovum ; the two-celled stage at several periods showing division of the
archoplasm-mass, " attraction-spheres " in the resting-cell, formation of the second cleavage-
amphiasters.

FROM THE PRESS

" A work which is an honor to American scholarship." — Philadelphia Evening Tele-
graph.

" Professor Wilson has rendered a great service to teachers and students in the publica-
tion of the splendid series of micro-photographs of these different processes. These are
accompanied by an admirably lucid text." — The Dial.

" It is not often that one is permitted to examine a piece of work which is dons, in all
respects, on an ideal standard, as this is. . . . It is safe to say that the whole area engaged
in the fertilization and division of the ovum has never been shown or the forces traced
with such precision before." — The Independent.

" Every biologist owes the greatest gratitude to the authors and publishers of this
beautiful volume; and only those who have labored themselves to make good photo-
graphic plates from specimens exhibiting karyokinesis, can appreciate the wonderful
delicacy of the results." — Natural Science.

" This work is of a very high order, and both by its merit and its opportuneness is a
noteworthy contribution to science. . . . The pictures obtained represent the highest
perfection of micro-photography yet reached, especially as applied to protoplasmic struct-
ures. . . . To the whole is prefixed an abundantly illustrated "General Introduction"
in which Professor Wilson gives a summary of our present knowledge of our history of
the ovum, so far as it has any bearing on the problems of fertilization. It would be very
difficult to surpass this introduction, owing to its felicitous combination of terseness,
clearness, and completeness. The work takes its place at once as a classic, and is certainly
one of the most notable productions of pure science which have appeared in America.
It will be valuable to every biologist, be he botanist or zoologist, be he investigator or
teacher." — Science.

THE MACMILLAN COMPANY,

66 FIFTH AVENUE, NEW YORK.

yUST HEADY

An Atlas of Nerve Cells

BY

M. ALLEN STARR, M.D., Ph.D.,

Professor of Diseases of the Mind and Nervous System, College of Physicians a7id Surgeons

Medical Department of Cohimbia College ; Consultitig Neurologist to the Presbyterian

and Orthopadic Hospitals, and to the New York Eye and Ear Infirmary

WITH THE CO-OPERATION OF

0. S. STRONG, Ph.D., and EDWARD LEAMING, M.D.
Extra 4to. Cloth. $10.00, net.

UNIFORM WITH DR. WILSON'S "ATLAS OF THE FERTILIZATION OF THE OVUM "

52 PLATES. 8 FIGURES.

It is the object of this atlas to present to students and teachers of histology a series of
photographs showing the appearance of the cells which form the central nervous system,
as seen under the microscope. These photographs have been made possible by the use
of the method of staining invented by Professor Camillo Golgi of Turin. This method
has revealed many facts hitherto unknown, and has given a conception of the structure and
connections of the nerve cells both novel and important.

In the most recent text-books of neurology and in the atlas of Golgi these facts have
been shown by drawings and diagrams. But all such drawings are necessarily imperfect
and involve a'personal element of interpretation. It has seemed to me, therefore, that a
series of photographs presenting the actual appearance of neurons under the microscope
would be not only of interest but also of service to students. The Golgi method lends
itself very readily to the photographic process, for the cell, with its dendrites and neuraxon,
is stained black'upon a light yellowish ground, and thus is capable of giving a sharp pict-
ure. In the preparation of this Atlas I have had the co-operation of Dr. O. S. Strong, who
has cut and stained the specimens, and of Dr. Edward Leaming, whose skill in photogra-
phy has made this work possible. Dr. Strong has been able to produce remarkably suc-
cessful sections of the various parts of the nervous system, both brain and spinal cord, and
has made some valuable modifications of Golgi's methods. He has contributed a section
upon the technique, containing many original and important suggestions. In the art of
photographing microscopic specimens Dr. Leaming has been particularly successful. It
can be readily imagined that the difficulties of obtaining a clear picture focussed in one
plane upon the photographic plate are at times almost insuperable, the microscopist
ordinarily bringing various planes into his vision by the aid of the fine adjusting screw of
the instrument. By care in the selection of specimens, by ingenious contrivances to
ensure a perfect focussmg, and by the use of various methods adapted to each emergency,
Dr. Leaming has succeeded where others have failed. He has contributed a section of
much value upon the photographic technique. The photographs have been reproduced
in a painstaking manner by Mr. Edward Bierstadt, whose process of autotyping has been
selected after a careful comparison with other methods of reproduction ; and it can be
justly said that they show every detail of the original photographs.

In presenting this Atlas I have not attempted to write an exhaustive account of nervous
histology, but rather to present a brief review of the essential facts so far as they can be
seen by the aid of the Golgi stain, and to show how these facts aid in the knowledge of
nervous action. I may be permitted, however, to point out that this atlas is based mainly
upon preparations from the human nervous system ; that it not only includes the spinal
cord, cerebellum, and brain cortex, which have been studied by Golgi, Cajal, Van Gehuch-
ten, Retzius, and Lenhossek, but also presents original studies of the corpora quadrigemina,
optic thalamus, and lenticular and caudate nuclei, and is thus quite complete in its scope.
It is my intention at some future time to issue another volume which will include the
peripheral nerves and their terminations and the organs of sense.

THE MACMILLAN COMPANY,

66 FIFTH AVENUE, NEW YORK.

A