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1 3 13 Polyembryonic Development in
Tatusia Novemcincta

J. T. IPATTERSON

Reprinted from the JOURNAL OP MORPHOLOGY, Vol. 24,
No. 4, December, 1913

Reprinted from JOURNAL OF MORPHOLOGY, VOL. 24, No. 4
December, 1913

POLYEMBRYONIC DEVELOPMENT IN TATUSIA
NOVEMCINCTA.1

J. T. PATTERSON

THIRTY-FIVE TEXT FIGURES AND ELEVEN PLATES

CONTENTS

Introduction 560

Methods 561

1. Technique 561

2. Method of securing early stages 561

Notes on breeding habits 563

1. General statement 563

2. Available material 566

Development of the blastocyst 569

1. The monodermic blastocyst 569

2. The didermic stage 574

Attachment of the blastocyst 585

Formation of the ectodermic vesicle 587

Origin of the extraembryonic mesoderm or mesothelium 593

Comparison with other forms 595

Origin of the primary buds 598

Origin of the secondary buds 603

Extension of the secondary buds and organization of the embryos 610

Implantation of ovum and placentation 617

General discussion 625

1. Theories of polyembryony 625

a. Theory of polyovular follicles 625

b. Theory of blastotomy 629

c. Theory of budding , 631

2. Origin of polyembryony in Dasypodidae 636

3. Polyembryony and duplicate twins and double monsters 641

4. Polyembryony and sex 643

Summary 645

Bibliography 648

1 Contributions from the Zoological Laboratory of the University of Texas.
No. 115.

559

560 J. T. PATTERSON

INTRODUCTION

The development of more than one individual from a single egg
while not a rare phenomenon among animals, is nevertheless of
much biological interest. It has become customary to classify
such types of development as asexual or agamic reproduction; but
obviously this term has come to include a variety of developmental
phenomena, which are exhibited in animals ranging from the
Protozoa to the mammals. Among the more common types of
agamic reproduction are ordinary binary fission, budding, cyclical
parthenogenesis, paedogenesis, and polyembryony.

Upon the basis of certain evidence which has been brought for-
ward from a study of comparatively late embryonic stages, it has
been correctly concluded that the type of agamogenesis which has
become habitual in the Texas armadillo is that of polyembryony;
but so far no one has succeeded in demonstrating the validity of
this conclusion. The writer has in his possession a series of young
stages which covers the period of early embryonic differentiation,
and which represents the material upon which this paper is based.
An outline of the more general features of the work has already
been given in a preliminary paper (Patterson '12). The facts to
be presented in detail are not without a certain interest and
significance, not only because they raise to the dignity of an ob-
served fact the claim for polyembryonic development in the arma-
dillo, but also for the reason that they throw a great deal of light
upon related phenomena in other mammals. It is unusual to find
agamic reproduction in the highest class of animals, and a detailed
study of the history of this process is greatly to be desired. Fur-
thermore, there has been considerable speculation as to how 'iden-
tical twins' and similar types of development have arisen, and I
believe that these studies on the development of the armadillo
will at least indicate how these phenomena may have come about.2

2 It is a pleasure to acknowledge here my indebtedness to my friend Mr. F. L.
Whitney of the School of Geology for his able assistance in connection with the
photographic work. I am also grateful to Mr. F. Pfeiffer, who has greatly facili-
tated this work by his many successful efforts in obtaining material.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 561

METHODS

1. Technique

For all of the stages described in this paper, I have found Zen-
ker's fluid to be the most useful fixing reagent. Kleinenberg's
picrosulphuric acetic acid works well on attached stages, notwith-
standing the fact that many embryologists have regarded this
fluid as poor for preserving the finer structures. All of the mate-
rial has been imbedded in paraffin, and the sections cut either 5 or
7 micra thick. Most of the preparations have been stained with
acid hematoxylin, although Heidenhain's iron-hematoxylin has
been used for some of the earliest stages. Whole mount prepara-
tions of the early embryonic vesicles, which were made by the
glycerin jelly method, were found to be very useful in the interpre-
tation of certain changes occurring in these young stages.

2. Method of securing early stages

In order to understand the methods employed for securing the
young vesicles it is necessary to give a brief account of the structure
of the uterus. The uterus of this armadillo is of the simplex type,
like that of the primates, and gives no evidence, in its external ap-
pearance, of being in any way adapted to accomodate a litter of
four. Viewed from either the dorsal or ventral sides, the uterus
proper resembles a rather blunt spear-head, with the distal or fun-
dus part representing the tip and the proximal or cervex part
the shaft end of the instrument. The fallopian tubes, which lie
in the plane of the broad ligament, enter the uterus at points lying
approximately two-thirds the distance between the cervix and the
tip of the fundus.

The external appearance of the non-pregnant uterus varies
greatly, both in the virgin and in the old female; but in this con-
nection we are concerned primarily with the condition of the inter-
nal surface. In the young female, two-thirds to three-fourths
grown, the suface of the mucosa is rather smooth, and gives only
faint indications of a folded condition, but in the adult virgin fe-
males of two years, and especially in the -old females, the uterine
mucosa becomes greatly folded. The folds have a general distribu-

562 J. T. PATTERSON

tion over the lining of the uterus, except at the tip of the fundus,
where there is a four-pointed, cross-shaped area of rather smooth
mucosa. The arms of this cross meet each other at approximately
right angles, and their common area is the extreme tip of the
fundus. This cross also indicates the orientation of the uterus; for
two of the arms mark the dividing line between the upper and
lower halves of the fundus, and two form a rather broad, shallow
groove extending from the mid-dorsal point around to the mid-
ventral point of the uterine cavity. These facts are most clearly
brought out in an everted uterus (fig. 21).

Each one of the arms forms a distinct furrow leading from the
tip of the fundus to the uterine opening of the fallopian tube. As
already indicated, the other two arms lead from the center of the
fundus to the middle of the upper and lower surfaces, respectively.
These usually form shallow furrows which end distally among the
folds of the mucosa. For convenience we shall speak of the first
pair as the right and left horizontal grooves, and of the second pair
as the dorsal and ventral vertical grooves. On account of the fact
that the uterine openings are situated slightly nearer the fundus
than the cervix end, the horizontal grooves are somewhat shorter
than the vertical grooves.

To the student of the early development of the armadillo, the
significance and importance of this cross-shaped area can scarcely
be over emphasized. The right and left horizontal groove forms
the pathway along which the embryonic vesicle passes on its way
from the fallopian tube to the tip of the fundus; and the center of
the cross, or the central area of the fundus, is the attachment zone
or placental area for the vesicle. The discovery of these facts has
greatly facilitated the collecting of the early stages. Prior to 191 1
attempts were made to obtain the young stages, and various
methods were tried, such as that used with great success by Eng-
lish workers in collecting the early stages, namely, that of inject-
ing the uterine cavity full of some killing fluid, and later examining
the contents for the embryos. While this method works well where
one is dealing with an animal which gives off several eggs at each
ovulation, yet in the case of a single egg at each pregnancy the
chances of its discovery are few. Furthermore, to any one who

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 563

has endeavored to study the fragile mammalian vesicle, the diffi-
culty of preserving the delicate structure even after discovery, is
indeed very great.

Early in the fall of 1911, however, the real significance of the
cross-shaped grooves, and especially of the horizontal one, was
first fully realized, and since then not much difficulty has been ex-
perienced in obtaining the free uterine or early attached vesicles.
In the living or fresh uterus it can easily be demonstrated that the
•horizontal groove of each side is virtually a tube, and is therefore,
a continuation of the fallopian tube. This is brought about by
the depth of furrow and by the depressed condition of the uterus
dorso-ventrally.

The best method to follow in seeking to obtain a young vesicle
is to cut the uterus along each side in the plane of the broad liga-
ment, beginning at the cervix and extending to a point lying a
short distance from the opening of the fallopian tube, and then
slowly to evert the fundus portion over the end of the small finger.
By this procedure the horizontal furrows are spread wide open,
and the vesicle, if present, will be revealed. The tip of the everted
uterus is then applied to the killing fluid and the vesicle, if not
attached, will float out on the surface of the fluid, and after a few
seconds slowly sink to the bottom without distortion. In the case
of attached vesicles, the everted uterus is placed in the fluid, and,
after fixation and hardening is affected, a properly oriented block
containing the vesicle is cut out of the mucosa. The process of
obtaining these early free stages may be greatly facilitated by
first determining which ovary holds the corpus luteum, and confin-
ing one's search to the corresponding horizontal groove.

NOTES ON BREEDING HABITS
1. General statement

During the past five years a considerable amount of informa-
tion concerning the habits of this animal has been secured by the
writer, and it may be worth while eventually to publish these ob-
servations; but here I wish merely to make a few remarks on cer-
tain phases of the breeding habits.

564 J. T. PATTERSON

The breeding season extends over a considerable portion of the
months of October and November, and thus any lot of embryos
taken at a given time during the period of gestation will present
much variation in development. This makes the determination
of the length of gravidity difficult and quite uncertain. An ex-
act determination could only be made by breeding animals in
captivity. Since a majority of the young are born in the months
of March and April, gestation is probably about one-hundred and
forty days.

The old females breed first, mating in most cases before October
fifteenth, while the second year virgin females continue to breed
for some time after this period. Females of one year do not breed
except in rare instances. My records for the past five years show
just three cases of pregnancy among these young animals, out of at
at least two-hundred examinations.

In connection with the collecting of material and this incidental
study of habits, I have discovered a 'period of quiescence' of
the embryonic blastocyst. The fact was first made apparent in
1911, when, after I had started collecting two weeks earlier than
in the preceding year, I failed to obtain the cleavage stages, al-
though judging from the condition of development in the vesicles
collected in previous years, one would naturally expect to find
these early stages during the period of my first collections in 191 1,

Again in 1912, I began collecting material two weeks earlier
than in 1911, and much to my surprise obtained blastocysts in al-
most exactly the same condition as those secured during the pre-
ceding fall. Practically all of these vesicles lie free within the
uterine cavity, either in the horizontal groove or in the region of
the attachment zone (placental area).

It is evident from these data that the embryonic vesicle remains
for some tune lying free within the uterine cavity. Just how long
this period lasts, I am unable to state; for practically every old
female taken at the earliest date (October 15) at which I have
collected, possesses a free blastocyst. How long such blastocysts
have been in the uterine cavity it is, of course, impossible to
determine; but I should judge not very long, because two vesicles

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 565

taken from the fallopian tubes show a development almost as far
advanced as that of some vesicles taken from the proximal -parts
of the horizontal grooves.

Taking all the facts into consideration, I estimate the 'period of
quiescence' to last about three weeks; that is from about the mid-
dle of October to the third or fourth of November. There are ex-
ceptions to this, but not sufficient in numbers to modify the gen-
eral conclusions. Of the thirty-four free blastocysts obtained in
1911 and 1912, twenty-eight of them were secured within this
period.

There is another line of evidence which more or less supports the
above conclusion. I refer to the condition of the corpus luteum.
In all the females from which the free blastocysts were taken the
corpus has attained approximately its maximum size. Unless we
attribute a phenomenal rate of growth to the corpus, it is neces-
sary to assume that quite a long period has elapsed since ovulation
took place, in order to account for its large size. The short fal-
lopian tube of the armadillo precludes the suggestion that the egg
has spent a great while in traversing this passage, so that we must
conclude that any arrested development in the egg takes place
after it enters the uterine cavity.

So far as the writer is aware, the only other mammal in which a
similar quiescent period in the development of the blastocyst oc-
curs is the deer. According to Assheton ('98) Bischoff ('54) states
that in this animal the embryo, upon reaching the so-called morula
stage, enters upon a 'period of quiescence,' and remains unaltered
for some weeks.

It is scarcely correct, however, to state that the blastocyst of
the armadillos, during the entire period of quiescence, is in a state
of arrested development, because it undergoes certain progressive
changes. So far as one can tell from a study of sections, no mi-
totic divisions occur, but the vesicle increases in size, due to the
accumulation of fluid within its cavity, accompanied by the atten-
uation of the trophoblastic cells. Furthermore, the differentia-
tion of the ectoderm and entoderm from the inner cell-mass is com-
pleted while the blastocyst lies free within the uterine cavity.

566 J. T. PATTERSON

2. Available material

The desirability of having at one's disposal a close series of
early stages for a study of this kind is self-evident; but from what
has been stated in the foregoing pages it is clear that many
obstacles stand in the way of securing such a series. This is parti-
cularly true with reference to the cleavage stages. In another
season this much desired material can probably be obtained by be-
ginning to collect at a period still earlier than that of the preceding
year. During the season of 1912 about a dozen uteri, with attach-
ed fallopian tubes and ovaries, taken from females in which signs
of recent pregnancy were evident, were preserved. The study of
some of these has led to the discovery of early blastocysts lying
within the lumen of the fallopian tube. It is therefore highly prob-
able that if a complete series of cleavage stages is to be had, it
will be necessary to pursue the laborious method of making sec-
tions of the fallopian tubes from females showing signs of recent
fertilization.

In this connection I should like to point out a possible source of
error, and one that must be carefully guarded against. In sec-
tioning the ovaries of females well started in pregnancy one occa-
sionally finds undivided eggs in that part of the fallopian tube
which is situated close to the ovary. Such cases are to be attrib-
uted to ovulations that have occurred after normal ovulation and
fertilization have taken place. The fact that the nucleus in these
eggs may undergo division does not signify anything of unique
importance, since it must be regarded simply as an expression of
the same tendency to parthenogenetic development which fre-
quently is seen in matured ova still confined within the ovarian
tissues of this animal.

In table 1 is given a list of all the free blastocysts which have
been secured during the seasons of 1911 and 1912, including the
two taken from the fallopian tubes. The first vertical column
gives the catalogue number of the specimen, arranged chronologi-
cally; the second, the date at which the vesicle was taken; the
third, the diameter of the vesicle in millimeters, measured in 70
per cent alcohol; the fourth, the ovary, right R or left L, from

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 567

which the egg came; the fifth, the age of the mother; the sixth,
the number of the figure, in case the specimen is illustrated in
the paper; the seventh, remarks. Unless otherwise stated, under
remarks, the vesicle was taken from the right to left horizontal
groove of the uterine cavity. In most instances differences in
size indicate differences in the degree of differentiation among the
several blastocysts, although there is some variation in vesicles
showing corresponding differentiations.

The table brings out several interesting points, and to those that
will not be specifically treated in subsequent sections we must
direct a few remarks.

In twenty^nine of the thirty-two cases listed the ovary from
which the egg came was determined; and the record shows that
fourteen were derived from the right ovary and fifteen from the
left, thus indicating that there is no tendency for one ovary to
function more frequently than the other. In two 'of the remaining
cases I failed to make a record on this point, but in the third (No.
297) both ovaries were enlarged. This would indicate that occa-
sionally the two ovaries function simultaneously, although in this
particular case I was not able to find a second egg. There is of
course the possibility that the one ovary gave off an egg which
failed to become implanted, and that a second ovulation, involving
the other ovary, followed immediately.

It was stated above that the 'old' females, that is females which
have previously borne young, breed first, and that the second year
virgin females come on later. The table brings these facts out
clearly. Thus, of the eighteen cases with complete records, which
were taken in October, fourteen (Nos. 230, 287, 288, 291, 292, 295,
297, 300, 304, 305, 309, 320, 325, 326) were from old females, while
only three were from virgin females, that is females that had never
before borne young. Of the eleven cases involving second year
females, eight vesicles (Nos. 243, 244, 245, 251, 258, 259, 261, 335)
were taken between November first and eighth; while two of the
remaining three were secured late in the month of October, both
on the 26th (Nos. 311, 315). Finally, of the three vesicles
obtained from the first year females, that is females that had
been born during the previous winter or spring, two were taken

568

J. T. PATTERSON

TABLE 1
Free embryonic vesicles

NO. OF

SPECIMEN

DATE

SIZE IN
MM.

OVARY

AQE OP
MOTHER

FIGURES

REMARKS

230

10-30-11

0.319

L

Old

242

11- 1-11

0.307

R

Old

243

11- 1-11

0.495

?

2dyear

244

11- 1-11

0.263

L

2d year

9

From left fal. tube

245

11- 3-11

0.324

R

2d year

249

11- 3-11

0.429

R

Old

14,41

Lying free on pla-

cental area

251

11- 3-11

0.420

L

2d year

• .

253

11- 3-11

0.306

R

Old

258

11- 8-11

0.384

L

2d year

259

11- 8-11

?

L

2d year

261

11- 8-11

9

R

2d year

268

11-14-11

0.330

L

1st year

284

12-12-11

?

?

1st year

287

10-15-12

0.312

L

Old

6

288

10-15-12

9

L

Old

291

10-18-12

0.404

R

Old

Lying free on pla-

cental area

292

10-18-12

0.480

L

Old

Lying free on pla-

cental area

295

10-18-12

0.380

R

Old

296

10-18-12

0.376

L

2d year

10,37

From left fal._tube

297

10-19-12

9

R=L

Old

300

10-19-12

0.656

L

Old

12,42

Lying free on pla-

cental area

304

10-22-12

?

R

Old

305

10-22-12

0.348

L

Old

309

10-26-12

0.432

L

Old

310

10-26-12

0.360

R

?

7,36

311

10-C6-12

0.504

L

2d year

11,47

Lying free on pla-

cental area

315

10-26-12

0.380

R

2d year

318

10-26-12

0.336

R

1st year

320

10-28-12

0.387

L

Old

13,39,40

325

10-28-12

0.290

R

Old

326

10-28-12

0.339

R

Old

335

11- 2-12

0.220

R

2d year

8,38

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 569

very late in the breeding season, one (No. 268) on November 14,
and the other (No. 284) on December 12.

In table 2 are listed all the early attached or implanted vesicles
which are directly referred to in the subsequent parts of the paper.
They are arranged, so far as possible, in the order of their degree
of development. Vesicle No. 311 of the preceding table should
probably be included in this list, and No. 300 should almost cer-
tainly be included. They were both lying free upon the placental
area when first observed, and for that reason are included in the
list of unattached specimens; but inasmuch as the early attached
vesicles are so easily dislodged, it might well be that these two
vesicles were loosened during the process of everting the uterus.
Their size and structure most certainly point to this conclusion.

In determining the size of those vesicles which had gained a
firm attachment to the mucosa it was found best to measure the
base of the vesicle, as this gave the most trustworthy results. In
everting the uterus, however, the mucosa of the placental area
may become slightly stretched or otherwise distorted, thus spread-
ing the base of the vesicle, and consequently giving a measurement
that is abnormally large. This is the case in No. 256. Further-
more, all of the vesicles listed beyond No. 332 were measured from
the microscopic preparations, and consequently after the shrink-
ing effects of dehydration, clearing, and imbedding had come in.
The sizes indicated for these vesicles are too small as compared
with those listed above No. 332 (cf. 233 and 307).

DEVELOPMENT OF THE BLASTOCYST
1. The monodermic blastocyst

The transformation from the so-called morula stage to the hol-
low sphere, or 'monodermic blastocyst/ has not been observed in
the armadillo. The youngest normal stage which I have at my
disposal already shows the finished product of this transforma-
tion. However, I possess representative stages throughout the
period of development which extends from this point on until the
'didermic blastocyst' is reached. In the present section we shall
describe three blastocysts, which, although all of the monodermic

570

J. T. PATTERSON

TABLE 2
Attached embryonic vesicles

NO. OP

SPECIMEN

DATE

SIZE IN
MM.

OVARY

AGE OF

MOTHER

FIGURES

REMARKS

307

10-22-12

0.516

L

2d year

Came off after 2

minutes in fixing

fluid

328

10-28-12

0.468

R

Old

Came off immedi-

ately in fixing fluid

339

11-15-12

0.425

L

Came off after 2-3

mins. in fixing fluid

340

11-15-12

0.438

L

2d year

15,48

Came off immedi-

ately in fixing fluid

316

10-26-12

0.449

L

Old

16,31

43-46

332

11- 2-12

0.500

L

2d year

17,49,50

Measurement across

widest points

233

10-30-11

0.340

R

Old

18,32,51

52

329

11- 2-12

0.312

R

Old

53

289

10-15-12

0.336

L

Old

54,55

298

10-19-12

0.440

L

Old

56,57

234

11- 1-11

0.598

•R

Old

19,33,58

256

11- 4-11

0.944

L

Old

20,59

247

11- 3-11

0.722

R

Old

1,21,22

34,60,61

255

11- 4-11

1.000

L

2d year

290

10-15-12

1.490

R

Old

2,23-26

62-65

175

11-14-10

0.931

R

Old

27-29,35

66-69

257

11- 4-11

0.909

L

2d year

3,30

70-74

170

11-18-11

1.533

L

2d year

4,75-77

226

10-30-11

1.367

L

Old

80-84

276

11-21-11

3x5

L

2d year

5,78

216

1-11-11

90

L

Old

79

Advanced stage, con-

taining embryos 10

cm. long.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 571

type, show interesting and significant differences. These will be
described in the order of their development.

Blastocyst No. 287. This specimen measures 0.312 mm. in diam-
eter, and is therefore larger than several others, but in point of
development it is the youngest specimen in the collection. The
inner cell-mass appeared both in the living and in the preserved
condition as an opaque spot, the inner surface of which showed
many elevations, caused by the protruding embryonic cells. In
section the mass is seen to be made up of embryonic cells with rel-
atively large, distinct nuclei (fig. 6). For the most part each cell
is delimited by a cell wall ; but here and there may be found one in
which part of the wall has disappeared, or at least the membrane
can not be made out with certainty. Another point of interest is
the fact that there is a considerable difference in the size of the
nuclei. Thus of the eleven nuclei shown in the median section
(fig. 6) six are decidedly larger than the remaining five. I am
unable to detect any difference in the staining properties of these
two types of nuclei, although the cytoplasm of some of the cells
which possess the larger nucleus takes a much paler tint than that
of the cells with the small nucleus.

The embryonic knob measures 0.026 mm. deep by 0.055 mm.
wide. If we regard the trophoblast of the median section of the
blastocyst as the circumference of a circle whose diameter is equal
to the diameter of the blastocyst, and using the width of the inner
cell-mass as a chord, then we may express the size of the mass in
the terms of degrees and minutes, as measured on the circumfer-
ence by the subtended arc. In this particular specimen the diam-
eter was 0.312 mm., and since the diameter of the inner cell-
mass was 0.055 mm., it covered an arc on the circumference equal
to 20° 18'. The embryonic mass is composed of about 75 cells,
as determined by a count of the number of nuclei. They are
tightly pressed up against the trophoblast, from which, however,
they are sharply cut off by the under surfaces of the trophoblastic
cells.

Blastocyst No. 310 measured 0.360 mm. in diameter, and is there-
fore larger than the preceding, and that it is also further developed
is evidenced by the fact that there are 136 cells in the embryonic

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

572 J. T. PATTERSON

knob. In the whole condition the knob appeared as a conical-
like elevation extending up from the trophoblast. The surface of
the mass was uneven, due to the projecting embryonic cells (fig.
36).

In detail, the embryonic knob is composed of a central core of
protoplasm surrounded by nuclei (fig. 7). Some of the nuclei ly-
ing towards the free surface of the mass are completely delimited
by the cell-membranes, but in the majority of cases that portion
of the cell membrane which is directed towards the center of the
mass has faded out. Some of these incomplete cells have two
nuclei. In the region of the trophoblast the cells have lost all
traces of membranes, but Rauber's portion of the trophoblast re-
tains its distinctiveness from the underlying embryonic mass.

While the embryonic knob of the preceding blastocyst presented
when viewed from above, a perfectly sharp contour, the knob of
this specimen, on the contrary, showed many ray-like protusions
extending out from the base of the cone along the trophoblast.
Thus the picture revealed in the upper view of such a mass is that
of a many pointed star. The spreading of the inner cell-mass
would seem to be accomplished by the migration of the cells from
the base. They creep out with pseudopod-like processes, and can
frequently be seen in the -sections. On the left side of the median
section (fig. 7) one of these processes is seen pushing out along the
under side of the trophoblast, and on the right of the same section
another cell is in the act of beginning a similar migration. In con-
sequence of this spreading, the diameter of the embryonic mass
has increased to 0.090 mm., as against 0.055 mm. in blastocyst
No. 287; and the arc on the circumference covered by it is 28°, 57'.

Blastocyst No. 335. In many respects this is one of the most
interesting of all the early blastocists in the collection. It is
easily the smallest, since it measures but 0.220 mm. in diameter;
and as compared with No. 310, is less than two-thirds the size.
Nevertheless, the differentiation of the embryonic knob is dis-
tinctly more advanced than that of the preceding blastocyst.
What explanation are we to offer then for this apparent disparity
in size? Is this variation in size of the vesicles to be correlated
with a difference in the size of the undivided egg? I think not.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 573

The explanation is rather to be sought in the condition of the troph-
oblast. If the median sections of blastocysts Nos. 310 and 335,
which have been photographed at the same magnification (figs. 36,
38) be compared, it is at once evident that, while the trophoblastic
cells in 335 are unchanged, remaining relatively thick, as they
must have been at the close of cell division, those of 310 are very
much attenuated. The nuclei have become widely separated by
the stretching of the wall, which in turn has become so thin in
places that it appears as a delicate line in the photograph. Evi-
dently this enormous increase of the blastocyst is the result of the
accumulation of fluid within the cavity of the vesicle, and the con-
sequent stretching of the wall. It is a fact worthy of note that one
never finds the trophoblastic cells undergoing division while the
blastocyst is free within the uterine cavity. The conclusion to
which we must come is that the accumulation of fluid in the cavity
of the vesicle must, at least in some cases, go on quite independ-
ently of the differentiation of the embryonic mass.

Turning to the detailed structure of the embryonic region one
finds that the inner cell-mass is flattened out until it has become a
lens-shaped structure measuring 0.024 by 0.115 mm., and cover-
ing an arc on the circumference equal to 63°, 1'. There has also
been some increase in the number of embryonic cells, the knob
now showing 166 cells.

The point of greatest significance and interest concerns the two
types of cells of which the embryonic mass is composed. In the
two preceding blastocysts there were two sizes of nuclei observable
and here not only does this same disparity still exist, but there is
also a corresponding difference in the cells themselves. Thus
there are clearly two types of cells, a large cell in which the nucleus
is large, and a smaller cell with a comparatively small nucleus. In
this particular specimen the small cells have a general distribution
throughout the mass, and appear either singly, or in groups of two
or more. The ratio of the small cells to the larger is about as one is
to three. Thus in the median section there are fourteen large cells
and five smaller ones. The latter are in three groups: a single
cell lying on the surface at the extreme left (fig. 8 a), a pair situ-
ated some distance to the right of this (6 and c), and a second pair

574 J. T. PATTERSON

lying about one-third the distance from the right end of the sec-
tion (d and e). In the first pair one of the cells borders on the
under surface of the mass, while the other is wedged in between
two large cells. I A the other pair one of the components also
comes to the under surface and the other is in contact with the
trophoblastic cells.

I interpret the condition here to mean that the nuclei of the
small cells are identical with the smaller nuclei of the preceding
stages, or else the progeny of such nuclei.

The size relation of the different cells is not the only evidence
which indicates that the embryonic mass is gradually differentiat-
ing into two types of cells; for the small cells have begun to take on
a slightly deeper tint of stain than do their larger fellows, and this
difference in the staining capacity of the two types of cells becomes
more and more evident as development progresses, until finally
it becomes one of the most striking features of the armadillo
blastocyst.

2. The didermic stage

The changes which we have just observed forshadow the trans-
formation of the monodermic blastocyst into one of the didermic
type; that is to say, the differentiation of the inner cell-mass into
its two primary components, the embryonic ectoderm and ento-
derm. We have seen that the inner cell-mass gradually differen-
tiates into two rather distinct types of cells, which differ from each
other both in size and in staining properties. The bulk of the
mass is composed of large, faintly staining cells which are more
numerous in that part of the mass which is situated towards the
trophoblast. The other type of cell is much smaller than the pre-
ceding, takes the stain much more readily, and has a sharply de-
fined outline. These smaller cells are at first evenly distributed
among the larger (except in a few cases to be noted later), but
later become collected toward that side of the inner cell-mass
which borders on the cavity of the vesicle. Subsequently they
become split off from the lower side of the mass to form the ento-
derm. In speaking of these cells in my preliminary paper I make
the following statement.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 575

There are, however, two distinct types of cells. . The bulk of the mass
consists of rather large, ill-defined cells, the cytoplasmic and nuclear por-
tions of which do not stain well. Scattered among these are smaller
cells, which are found much more abundantly hi that part of the mass
lying towards the cavity of the vesicle. The smaller cells are character-
ized by their sharply denned outlines and by the ease with which they
take the. stain. There is considerable evidence to indicate that the
smaller cells are gradually undergoing a segregation from their larger
fellows to form the hypoblastic layer of the vesicle. At any rate the
hypoblast upon its completion possesses cellular elements that very
closely simulate the smaller cells of the earlier stages.3

This statement was made before I had had an opportunity of
examining Hill's ('10) excellent paper on the early development of
the 'native cat/ Dasyurus viverrinus. In this paper Hill shows
in the clearest possible way that the entoderm arises in a manner
quite similar to that in the blastocyst of Tatusia, and I shall there-
fore briefly state his general results on the point under discussion.
It will be recalled that in the 16-cell stagfe the blastomeres are ar-
ranged in two superimposed rings of eight cells each. The eight
upper cells, which are smaller than the eight lower, are destined
to produce the formative or embryonal region of the blastocyst
wall, while the eight lower cells will give rise to the non-formative
or extra-embryonal region. In accordance with the characteristic
mode of development in the marsupial, no morula stage is formed
in the egg of Dasyurus, but the blastomeres proceed directly to
form the wall of the blastocyst. This is brought about through
the division of the blastomeres of each ring and their gradual
spreading toward the opposite poles, on contact with the inner
surface of the sphere formed by the zona and the shell-membrane.
The daughter blastomeres continue to divide and eventually pro-
duce a complete cellular lining to the zona sphere, constituting
the unilaminar wall of the blastocyst. The wall remains in this
unilaminar condition until the blastocyst attains a diameter of
4 to 5 mm.

Hill here draws the most fundamental conclusions of his paper,
pointing out that the formative or embryonal region, which from
the first possesses no covering of trophoblast (i.e., Rauber's layer),

3 Loc. cit., pp. 369-370.

576 J. T. PATTERSON

is the homologue of the inner cell-mass of the eutherian blastocyst ;
and that the non-formative region is the homologue of the hypo-
blast of the eutheria and of the extra-embryonal ectoderm of the
sauropsida and monotremata.

After the blastocyst has reached a diameter of from 4 to 5 mm.,
two distinct varieties of cells can be recognized in the unilaminar
wall of the formative region. Hill regards this as the crucial stage
in the formation of the primary germ layers, as it marks the tran-
sition from the unilaminar to the bilaminar condition. We may
quote from Hill's summary such paragraphs as give a resume of
his account of the development of the entoderm.

The formative region, unlike the non-formative, is constituted by
cells of two varieties, viz. : (i) a more numerous series of larger, lighter-
staining cells destined to form the embryonal ectoderm, and (ii) a less
numerous series of smaller, more granular, and more deeply staining
cells, destined to give origin to the entoderm and hence distinguishable
as the entodermal mother-cells.

The entodermal mother-cells, either without or subsequent to division,
bodily migrate inwards from amongst the larger cells of the unilaminar
wall and so come to lie in contact with the inner surface of the latter.
They thus give origin to the primitive entodermal cells from which the
definitive entoderm arises. The larger passive cells, which alone form
the unilaminar wall after the inward migration of the entodermal cells is
completed, constitute the embryonal ectoderm.

The entodermal cells as well before as after their migration from the
unilaminar wall are capable of exhibiting amoeboid activity and of
emitting pseudopodial processes, by the anastomosing of which there is
eventually formed a cellular entodermal reticulum underlying, and at
first coextensive with, the embryonal ectoderm.

The entoderm is first laid down below the formative or embryonal
region of the blastocyst; thence it extends gradually by its own growth
round the inner surface of the unilaminar non-formative region so as to
form eventually a complete entodermal lining of the blastocyst cavity.
In this way the blastocyst wall becomes bilaminar throughout.

Thus it will be seen that, contrary to the generally accepted view
of mammalian embryologists, the entoderm of Dasyurus does not
arise by delamination, but through an inward migration of differ-
entiated entodermal mother-cells from among the ectoderm cells
of the embryonic region. The independent discovery of a similar
method of entodermal formation in the armadillo is of more than
ordinary interest, and calls for a detailed account of the process in

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 577

this eutherian mammal. Before taking up this account, it should
be pointed out that the blastocyst of Dasyurus presents an unusu-
ally favorable opportunity for the study of the origin of the ento-
derm. The large size of the vesicle, together with the unilaminar
condition of its wall, makes possible the preparation of the embry-
onic region as whole mounts, by the means of which detailed ob-
servations can easily be made. The eutherian blastocyst, on the
contrary, does not admit of satisfactory whole mount preparations,
owing to the fact that the embryonic region is more than a cell
thick. In the blastocyst of the armadillo this is particularly true,
as the wall of the formative or embryonal region is about four cells
thick. Nevertheless, the evidence gathered from the study of
sections, which we shall now present, is convincingly in favor
of the view that the entoderm arises by the means of migrating
cells, and does not favor the commonly accepted idea that it arises
by a delamination, that is, by the splitting off of the lower layer of
cells from the embryonic knob.

Blastocyst No. 244 is next to the smallest blastocyst secured,
measuring but 0.263 mm. in diameter, and, when taken, was on the
point of entering the uterine cavity from the left fallopian tube.
At this time it was distinctly spherical in outline, with an em-
bryonic spot, having a slightly irregular margin and covering an
arc 52° 58'. The specimen is described here, not only because it
shows a continuation of the changes observed above, but also for
the reason that it is remarkably well preserved, and consequently
will give more than ordinary confidence to any interpretations
which may be based upon its structures.

The embryonic spot, which measured 0.025 mm. deep by 0.100
mm. in diameter, was too thick to permit the determination of the
details of structure from a study of the living specimen; but in the
sections the details stand out brilliantly, and prove beyond a per-
adventure that there are two types of cells composing the embry-
onic mass. The entodermal cells, as in the case of specimen No.
335, are apparently quite generally distributed throughout the
inner cell mass. The section passing through the center of the
mass is typical in that it shows all of the essential features, and
especially the relation which exists between the two elements con-

578 J. T. PATTERSON

stituting the embryonic spot (fig. 9). There are six entodermal
and thirteen ectodermal cells, or rather ectodermal nuclei, for in
some cases two or more of these nuclei are included within a single
cytoplasmic mass. No entodermal cells have been found which
show more than one nucleus.

The entodermal cells in the section are located in three regions,
similarly to those of the preceding figure. On the extreme left
the single cell (fig. 9, a) is easily distinguishable from its adjacent,
binucleated, ectodermal fellow. It gives evidence of beginning to
spread out beneath the binucleated cell. To the right of this is a
group of three entodermal cells (b, c, and d), °ne of which comes to
the lower surface, while the other two lie one above the other, well
within the mass. These cells are darker than the neighboring ec-
todermal cells, but the difference in the size of the nuclei that was
so striking a feature of the preceding stage is here not so marked.
In fact, the nuclei of some of the ectodermal cells are smaller than
those of the entodermal ones. It must be kept in mind, however,
that in these thin sections only the tip or at least a small portion
of a large nucleus may be visible in a given section. A study of the
preparation shows that on the average the entodermal nuclei are
smaller than the nuclei of the ectodermal cells. The remaining
entodermal cells constitute a pair situated about in the middle of
the right half of the section (fig. 9, e and/). Both of these cells
are on the surface of the mass. Their cytoptasmic and nuclear
portions do not stain so deeply as in the other entodermal cells,
but still more deeply than in the case of the ectodermal cells.

The affinity of the entodermal cells for the stains evidently lies
in the nature of the protoplasm itself. In the ectodermal cells,
both in the cytoplasm and nucleus, the structural configuration of
the protoplasm is of a more open mesh-like character than that of
the entodermal cells, in which it may assume a finely granular
appearance.

The conditions observed in this and the other sections of the
series is interpreted to mean that the more deeply staining cells are
entodermal, all of which will eventually migrate to the lower sur-
face of the embryonic mass to form the characteristic entodermal
layer of the mammalian blastocyst. The cells marked c, e, and /

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 579

are in the act of migrating to the surface. The cells lying within
the mass (b and d) are rounded in outline, while those in the act of
coming to the lower surface are invariably elongated in the direc-
tion of migration.

The evidence obtained from a study of these sections indicates
that each group of two or more entodermal cells has, in all proba-
bility, arisen from a single primary cell, or entodermal mother-
cell (using Hill's terminology). Futhermore it suggests that the
primary cell may, from the first, be situated on the surface; and
consequently will undergo no migration; or it may migrate to the
surface without having undergone division; or again, divisions
may have come in before the migration occurs.

Blastocyst No. 296 came from the proximal part of the left fallo-
pian tube, and measured 0.376 mm. in diameter. The embryonic
spot had a very even outline and measured 0.161 mm. across.
It is distinctly thicker than the last specimen, averaging about 4
cells, exclusive of the trophoblast (fig. 10). The arc covered by
the embryonic spot is 50°, 42'.

In every way the blastocyst is more advanced than any we have
so far described, It is not only larger, but also shows a higher
state of differentiation. One feature in particular, although not
entirely unique since it has been observed in one or two other cases,
is nevertheless worthy of mention. This is the presence of faint
structures on the outer surface of the trophoblast (fig. 37). These
were observed in the preserved egg and were naturally taken to be
the follicle cells, which sometimes adhere to the ovulated mamma-
lian egg and persist for some time; but in section they are seen to
be protrusions or exudations from the trophoblastic cells, and are
probably formed at the time the egg is fixed.

The number of entodermal cells in the median section is thirteen
as against sixteen ectodermal cells. This would seem to indicate
that there has been a great increase of entodermal cells, but in
several of the other sections they are very much less numerous,
owing to the fact that the median section passes through the prin-
cipal groups of entodermal cells.

The difference in size between the ectodermal and entodermal
cells is very obvious (fig. 10), and, as compared with the preceding
figures, stands in sharp contrast.

580 J. T. PATTERSON

The distribution of the entodermal cells is interesting and in-
structive. Except for a single cell (fig. 10m) the entire upper half
of the embryonic mass is free from them; and certainly this suggests
that these cells are gradually passing towards the lower surface.
Already nine of the remaining twelve cells have reached the lower
surface. The position of the twelve lower cells indicates that they
probably came from five different sources (five entodermal mother-
cells) as follows: cell a is in the act of migrating to the surface;
cells b-e have had a common origin ; likewise cells i-k have proba-
bly come from a single mother-cell; and finally, cell I gives evidence
of once having occupied a position against the trophoblast between
the two ectodermal cells situated farthest to the right in the sec-
tion. It is now clearly in the act of migrating out from between
these two cells.

I have already stated that cell m is the only entodermal element
situated in the upper half of the mass. There is no way of deter-
mining whther this cell will also migrate to the lower surface. A
pseudopodial-like process from its lower border is pushed in
between two ectodermal cells, and suggests at least that it is about
to move down. Furthermore, in the later stages, when the ento-
derm is completed as a distinct layer, no such cell as m is found
within the ectoderm.

The cell which lies just above e has caused me considerable
difficulty in attempting to determine to which of the two catego-
ries it belongs. Its relatively small, deeply staining nucleus closely
resembles those of the entoderm, but its faintly tinted cytoplasm,
together with its square-like outline, are sufficient to place it
among the ectodermal cells.

A word here as to the changes occuring in the size and thickness
of the inner cell-mass may be said. These changes can be seen by
comparing figures 6 to 1 1 . At first the inner cell-mass is composed
of a group of spherical cells, but, as development progresses, the
mass becomes flattened out against the trophoblast, until finally
it forms a circular plate of cells about two deep. This period is
then followed by one in which there is a distinct increase in the
thickness of the mass, due evidently to the multiplication and
growth of the cells. Growth and multiplication of the cells con-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 581

tinues until the maximum thickness is reached in such specimens as
No. 296 (fig. 10), when the embryonic spot again begins to spread
and continues to increase in diameter until finally the mass is re-
duced to about two cells in depth (fig. 11).

This process of migration of the entodermal cells to the lower
surface of the embryonic knob and their subsequent peripheral
movement along the inner surface of the trophoblast must take
place by amoeboid activity. Indeed, the evidence for this con-
clusion is irresistible. If one examine in the living condition a
stage somewhat more advanced than No. 296, one finds that
numerous pseudopod-like processes are radiating out in all direc-
tions from the embryonic spot. In many instances the connection
of these processes with some of the outlying entodermal cells is
clearly discernible.

In order that a photographic record of this phenomenon might
be made, glycerin jelly mounts were prepared of several unstained
blastocysts which exhibited it. A photograph of such a prepara-
tion is shown in figure 40. In the enlarged view of the embryonic
spot of the same specimen (fig. 39), the pseudopodia are particu-
larly clear and striking. The high power of the microscope was
focused so as to bring the entoderm as sharply as possible into
view and while the rather thick ectoderm of the central area ob-
scured the true condition of the entoderm liere, yet the periph-
eral portions are brought out sharply. A small area of these
anastomosing processes from the lower border of the embryonic
area is sketched in figure 13. Some of the pseudopodia have
sharply pointed ends, others have rather blunt ends, and still
others have flattened terminations. The processes from two or
more cells frequently anastomose and form a fenestrated struc-
ture. In sections also the pseudopodia can be demonstrated,
especially when the section happens to cut one of them length-
wise (fig. 14) . However, it is from the study of the living mate-
rial and glycerin jelly preparations that one obtains the most con-
vincing evidence of these pseudopodia, and of the role they play
in the formation and migration of the entoderm.

The segregation of the entodermal cells from the ectodermic
mass is completed by the time the vesicle has attained a diameter

582 J. T. PATTERSON

of about 0.430 mm. At this time the embryonic spot has not
reached its maximum expansion, that is, it has not completely
flattened out. Consequently in section the entoderm forms a
slightly curved line, due to the bulging out of the mass of embry-
onic ectoderm (fig. 14). The ectodermal cells are large, relatively
clear, and sharply cut off from the overlying trophoblastic cells.
On the inner surface of the mass they are less sharply separated
from the entodermal cells, which here and there send processes up
between some of the bordering ectodermal cells. Such processes
are undoubtedly the last remnants of the migrating entodermal
cells to be withdrawn from the embryonic mass.

The condition of the entoderm is of much interest. It does not
as yet form a complete sheet of cells underlying and coextensive
with the embryonal ectoderm. In places the entoderm may be
wanting for more than the width of a cell. Furthermore, on the
right side of the sections of specimen No. 249 the entoderm is fre-
quently wanting, indicating that it must have taken its origin from
the left portion of the embryonal mass. In some of the earlier
blastocysts this same fact was observed. The smaller, deeply
staining cells, which give rise to the entoderm, were found to ex-
tend over not more than two-thirds of the embryonic mass.
Finally, in the largest free blastocyst secured (No. 300) and one in
which the entoderm is completely segregated, we find this same
excentricity of the entoderm. It was so evident in this specimen
that I have gone to the trouble of making a special preparation
for the purpose of demonstration by a photograph. The vesicle
in question was slightly stained in eosin and imbedded in paraffin.
Under the high power of the binocular microscope the non-em-
bryonic hemisphere was carefully pared away with a sharp razor.
The remaining hemisphere was dissolved out of the paraffin,
stained in hematoxylin, and mounted in balsam, with the cut sur-
face uppermost. Thus it was possible to get an unobstructed
view of the entoderm, and since the embryonic mass had become
completely flattened, the entodermal layer lies in a single plane.
The exposed sheet of entoderm was then studied and pho-
tographed (fig. 42).

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 583

The center of the embryonic spot lies about five millimeters be-
low the center of the circular figure, and, since the entodermal
cells are practically all in focus, it can be seen that, as a continu-
ous layer, the entoderm covers only about the lower three-fourths
of the embryonic spot. Over the other fourth only a very few
entodermal cells are found, and these lie for the most part slightly
to the left of the center. The other cells in this area which are
slightly out of focus represent the exposed ectoderm, which is here
on-ly about one cell deep.

What does the excentric position of the-entoderm mean? The
observation of this phenomenon in some four or five blastocysts
doubtless furnishes too meager evidence upon which to base any
fundamental conclusion. Nevertheless one can not resist the
temptation to suggest that we may have here a key to the much
mooted question of gastrulation in eutherian mammals. For if
it could be shown that the origin of the entoderm is confined to a
definite area of the embryonic mass, the center of such an area
might be regarded as corresponding to the region of a blastopore,
regardless of whether or not this spot later became perforated by
an actual opening or evanescent blastopore. Hubrecht ('02 '05
'08) has argued that the didermic stage of the mammalian blasto-
cyst is to be regarded as a 'gastrula'. He further states that we
must separate the phenomena of notogenesis from the phenom-
enon of gastrulation. He expresses himself very clearly and
concisely on this point in the last of the three contributions
mentioned above in which he makes the following statements:

As soon as we separate the phenomena of notogenesis, such as we have
found in all vertebrates — Amphioxus included — -from the phenomenon
of gastrulation, recognizing that the former follow upon the latter and
bring about the formation of the notochord and the mesoblastic somites,
the difficulties are considerably simplified.

Gastrulation is thus terminated in the mammalia when the didermic
stage of the embryonic shield has come into existance. We have seen
that this takes place not in consequence of any process of invagination
but by means of a most unmistakable delamination of the entoderm, out
of the embryonic knob.

This delamination gastrula of the mammalia generally enters upon the
latter phases of ontogeny which will be described hereafter without the
appearance of a distinct blastopore.4

4 Loc. cit., p. 13.

584 J. T. PATTERSON

Concerning the idea of separating the processes of gastrulation
and notogenesis I believe that in the main we must agree with
Hubrecht, although the close genetic continuity of these two proc-
esses must never be lost sight of, even in mammals, else the ap-
pearance of an evanescent blastopore in such forms as the hedge-
hog, Tarsius, rabbit, mole, shrew, and opossum would have little
significance. But the unexpected discovery of migrating cells to
form the entoderm in the blastocyst of the armadillo must deter
us from speaking of the process of entoderm formation as one of
delamination, for this term as used in mammalian embryology im-
plies that the entoderm is differentiated from those cells of the
inner cell-mass which happen to border on the cavity of the vesicle.
So to regard the origin of the entoderm would be equivalent to ac-
cepting Driesch's ('93) aphorism; for it would amount to saying
that the prospective value of one of these bordering cells "is a
function of its position. " Delamination is not therefore the cor-
rect term to employ in describing the -mode of entoderm forma-
tion in the armadillo, for while it is true that the entoderm as a
distinct layer is split off from the embryonic ectoderm, as we shall
see later, yet prior to this so-called delamination there is an un-
mistakable migration of primary entodermal cells to the lower
surface of the inner cell-mass, and this it seems to me is the funda-
mental step in the whole process of entoderm formation.

The final steps in the differentiation of the entoderm in the arma-
dillo may be considered here, as we shall not have occasion again
to refer to them. Following the conditions such as we have seen
in figure 41 the entodermal cells spread out until they have formed
a continuous sheet of cells beneath and coextensive with the em-
bryonic ectoderm (fig. 47). During this change the entodermal
cells become much flattened against the ectoderm. There is no
evidence here, such as we have observed in the pseudopodia of
earlier stages, to indicate that the entodermal cells at the margin
of the embryonic area are pushing out further beneath the tropho-
blast. Even in the specimen shown in figure 42 the pseudopodia
have practically all disappeared, except in the case of the upper
marginal cells that border on the area of ectoderm still not covered
by entoderm (fig. 12).

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 585

Measurements show that the entoderm has practically reached
its maximum extension in such specimens as No. 311, in which the
embryonic spot measures 0.020 mm. by 0.157 mm., and covers an
arc on the circumference of 36°, 18'. It does not here reach much
beyond the extreme limits of the ectoderm (fig. 11). This does
not mean that the entoderm may not cover an actually great
area, for it becomes attenuated through the expansion of the
trophoblastic wall; but what is meant is that the entodermal cells
do not push out any further along the trophoblastic wall. Con-
sequently that part of the wall which lies beyond the limits of
the embryonic area never becomes didermic, or bilaminar. Per-
haps then it would be better to confine the use of the term dider-
mic to the embryonic spot, and not apply it to the blastocyst as
a whole.

It may not be amiss in conclusion to draw attention again to the
very close similarity between the mode of origin of the entoderm
in the armadillo and that of Dasyurus, as given by Hill. The
similarity is indeed striking, especially if one consider the funda-
mental differences in the character of the walls of the two types
of blastocysts. The thin wall of the formative region of the blast-
ocyst of Dasyurus stands in sharp contrast to its homologue, the
relatively thick inner cell-mass of the armadillo blastocyst, and
yet in all essential features their modes of entoderm formation are
practically identical.

ATTACHMENT OF THE BLASTOCYST

The implantation of the embryonic vesicle is a subject of much
importance, and is treated elsewhere as fully as the material at
hand warrants. At this point it is desirable to discuss briefly only
the first step in implantation, or the attachment of the blastocyst.

A great deal of effort has been put forth to obtain the earliest
attached stages, and, to date, four clear cases have been observed.
Two other doubtful cases were seen. Unfortunately in each case
the vesicle became detached from the mucosa upon placing the
uterus in the fixing fluid or very shortly thereafter. In two of
these the separation took place immediately, while in the other two
from two to three minutes elapsed between the immersion of the

586 J. T. PATTERSON

uterus and the loosening of the vesicle. However, these stages
were studied under the high and low powers of the binocular mi-
scroscope before the uterus had been placed in the fixing fluid, and
it was therefore possible to make out most of the details of
structure.

In general appearance these vesicles are not unlike the largest
of those which were found lying free within the uterine cavity.
The large polygonal trophoblastic cells are clearly discernible,
and the embryonic area appears as a whitish spot lying directly in
contact with the mucosa. In size, too, they are not larger than
the more advanced free- vesicles.

These four vesicles were sectioned for microscopic examination,
and in three of them a detailed study reveals nothing different
from what we have already seen in the free stages ; but in the fourth,
which was one of the two that remained attached for the longest
period, an important difference was observed. The change to
which I refer involves the most essential part of the vesicle, the
embryonic ectoderm, and consists of a thickening of that structure
(fig. 48) . It is certain that this increase is not due alone to a mul-
tiplication of cells, since mitotic figures are rarely found, but to a
distinct rounding up of the entire embryonic ectoderm. In fact
this change is the beginning of a process that will eventually trans-
form the lens-shaped ectodermal mass into a ball-like structure.

In the study of these four vesicles particular attention has been
paid to the area of trophoblast (Rauber's layer) which directly
overlies the embryonic ectoderm, and which forms the seat of at-
tachment. The trophoblastic cells of this area do not as yet be-
tray any evident changes looking to the formation of the 'Trager.'
We must conclude therefore that for a short time at least the vesi-
cle is held to the mucosa by adhesion. It would seem that it sim-
ply 'sticks' to the uterine lining. Nor is there any evidence
to show that there is a localized area within the attachment zone
of the fundus to which the vesicle migrates before becoming at-
tached. Any spot on the entire attachment zone may furnish a
foothold for the vesicle, if one may judge from the collected date
on the distribution of about twenty young attached stages.
Apparently the vesicle adheres to the mucosa very soon after it

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 587

passes from the horizontal groove to the attachment zone; for it is
almost invariably the rule that the vesicle is located on that half
of the zone lying adjacent to the groove along which it had passed
from the fallopian tube. Thus, in a pregnancy in which the left
ovary holds the corpus luteum, the vesicle will be found to be
attached at some spot on the left side of the attachment zone.

FORMATION OF THE ECTODERMIC VESICLE

In the preceding paragraphs we have referred to the change in-
volving the embryonic ectoderm which takes place shortly after
the vesicle becomes attached to the mucosa. This change is per-
haps anticipated in the more advanced of the free blastocysts in
which a few divisions of the ectodermal cells are taking place (fig.
47) ; but evidently does not express itself clearly until after the egg
becomes quite well anchored to the uterine wall. As already in-
timated, the change consists in the transformation of the flat, cir-
cular plate of ectodermal cells into a spherical or ball-like mass
(fig. 15).

The entoderm in this stage is recognized as a distinct layer,
and, although no longer connected with the ectodermic mass by
protoplasmic strands, yet is still in close contact with it. The
entodermal cells which lie directly beneath the ectoderm become
distinctly thicker than before, and accompanying this change is
the appearance of mitoses (fig. 15).

The entodermal cells which lie beyond the limits of the embry-
onic ectoderm, differ from those lying beneath the ectoderm in two
important respects. First, the nuclei of the cells remain small,
thus indicating that the cells are not in an active state of division ;
and second, these cells are flattened out against the trophoblast,
with which they are in very close contact. These cells form a nar-
row ring or annular zone around the margin of the ectoderm.
This zone is of especial interest because it forms the axis about
which the so-called inversion of germ layers revolves.

The transformation of the plate of ectoderm into a spherical
mass results eventually in its entire separation from the tropho-
blast and its inclusion within an entodermal sac, thus leaving a

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

588 J. T. PATTERSON

cavity between the Trager and the ectoderm. Material with
which to follow the steps through which the blastocyst passes
during this is not at hand, for there is here a slight gap in the
series. However, I have been fortunate enough to obtain two
blastocysts which show the condition immediately following the
inclusion of the ectoderm. The two specimens are nearly of the
same age, one being slightly more advanced than the other.

The younger blastocyst (No. 316) was found attached to a
small leaf-like outgrowth from one of the folds of the placental
mucosa. The outgrowth lay horizontal to the surface of the mu-
cosa, and the vesicle had gained attachment to its under side,
close to the edge. One side of the trophoblastic wall caved in
during fixation, otherwise the vesicle is in an excellent state of
preservation (fig. 16).

That portion of the trophoblastic wall which has caved in pre-
sents nothing different from what was observed in earlier stages,
except that the cells are more attenuated, but the opposite wall of
the vesicle has undergone a marked thickening, in addition to a
great increase in its cellular elements. The cells have, therefore,
changed in shape from a flattened squamous type to a distinctly
columnar epithelium. All the thickened portion of the wall was
originally in contact with the mucosa, but during the process of
fixation the leaf -like fold of the mucosa has undergone a great deal
of contraction and has shrunk away from the trophoblast, as is
evident from its folded appearance. Evidently the thickened
trophoblast owes its existence to contact with the mucosa, which
in some way specifically stimulates the cells to a striking activity,
as shown by the rate of division and change in shape. The spe-
cific reaction between the trophoblast and the mucosa must be
limited to that portion of the uterine epithelium which covers the
placental area, otherwise the wall of a free vesicle would give evi-
dence of response before the blastocyst had reached this area.

It is also a point worthy of note that the columnar cells send
out pseudopod-like processes, which eat into the mucosa, giving
it a serrated appearance in section. That the placental tropho-
blast has therefore a specific action on the trophoblastic cells can

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 589

not be doubted, and this effect seems to be limited entirely to the
region in direct contact.

The sections are cut somewhat obliquely, and hence the section
passing through the middle of the embryonic ectoderm does not
cut the region of the primitive placenta, or point of attachment
(fig. 16). Consequently the exact relation of the entoderm to the
other parts of the blastocyst can only be made out by referring to
several of the sections of the series. In plate 2 is shown a series
of four photographs which will demonstrate this relation.

In figure 43, which represents the section from which the draw-
ing was made (fig. 16), the entoderm is a well organized layer pass-
ing around the sphere of ectoderm. On approaching the low-
er side of the ectoderm the entoderm at each side passes laterally
and upwards to join on to the inner surface of the trophoblastic
wall (fig. 16, x). The layer of entoderm lying directly beneath
the ectoderm belongs to that portion which connects the entoder-
mal sac with the left wall of the trophoblast. A few sections to the
right in the series the lower layer of entoderm disappears, except
a small group of cells (fig. 44) . In this section the entoderm, on
leaving the ectoderm, passes outwards and upwards as before,to
join the inner surface of the trophoblast. Aside from the group of
entodermal cells lying directly beneath it, the embryonic ectoderm
is open below to a cavity which is bounded beneath by the placental
portion of the trophoblast, and which represents what is later to
become an extraembryonic cavity. In sections still farther to the
right, at a point where the section cuts just the tip of the ecto-
derm (fig. 45), the extraembryonic cavity is almost free from cells
of any kind. This section passes through the center of the Trager
(fig. 31). Beyond the limits of^the ectoderm (fig. 46) we see noth-
ing but a line of entodermal cells, which represents the right lat-
eral portion of the entodermal layer as it passes outwards to unite
with the trophoblast on this side of the blastocyst.

To sum up: The conditions revealed in the series of photo-
graphs shows that the ectoderm, upon assuming a spherical form
pushes up into the cavity of the blastocyst, carrying before it the
well established layer of entoderm, and creating behind a cavity
which gives rise to the extraembryonic cavity, and which will

590 J. T. PATTERSON

subsequently become lined with a layer of mesoderm. The con-
dition here presented reminds one somewhat of that found in
a corresponding stage of the blastocyst of Pteropus edulis
(Selenka and Gohre '92), except that in the case of the latter the
entoderm continues around the inside of the trophoblast, forming
a complete, inner layer to the blastocyst.

In the older of the two blastocysts (No. 332) the sections pass
exactly parallel to the median axis of the vesicle, and consequently
the relation of the different parts of the embryo is clearer than
in No. 316. The general conditions of the trophoblast are much
the same in the two specimens, except that at one point on No.
332 there is a knot of cells (fig. 17, k) which, in the living condi-
tion, fitted into a corresponding crypt in the mucosa. In later
stages we shall see further evidence of similar knots, which rep-
resent points on the trophoblast that have been specifically
stimulated to cell proliferation.

The relation of the entoderm to the embryonic ectoderm is re-
markably clear in this preparation (fig. 17). In contrast with
the preceding blastocyst, the entoderm of this specimen has under-
gone one important change, in that it has folded in beneath the
ectoderm, forming all but a closed entodermal sac. Only a small
pore-like opening (fig. 17) remains to place the extraembryonic
cavity in communication with the cavity of the entodermal sac.
On the right-hand side (or lower side, owing to the inclination to
the right of the blastocyst) a fusion has taken place between the two
layers of the entoderm and a portion of the Trager; but this fu-
sion, especially with the Trager, covers a very small area, as it no
longer exists in the sections a short distance to either side of this
one. The loop of entoderm which lies between the fused area and
the pore (fig. 49) is probably comparable to the group of cells sit-
uated in a similar position in the other specimen. The loop of
cells is especially clear in figure 17.

Returning to a fuller consideration of the ectodermal sphere,
we see that even in so young a stage as that of No. 316 it is no
longer a solid mass of cells, as must have been the case at first, but
in the central portion there are three relatively large and distinct
besides several smaller, less distinct vacuoles. In fact, the

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 591

entire core of the ectodermal sphere becomes honeycombed by
these vacuoles, which later unite. In blastocyst No. 332 the
union of the vacuoles has already made considerable progress
(figs. 49, 50). Eventually there is produced a large distinct cav-
ity within the ectodermal mass, thus transforming it into a true
vesicle. *

I obtained one vesicle which clearly represents a further advance
in the progress of vacuolization, and yet one in which the com-
pleted stage of the vesicle has not been attained. Unfortunately
the specimen became slightly crushed in the course of transporta-
tion from the field to the laboratory, after it had been fixed and
partially hardened. I therefore deem it unsafe to base any def-
inite conclusions upon its structures; but simply give a photo-
graph of one of the sections (fig. 53), which, in a measure at least
can be understood after we have considered a normal specimen of
a little later stage.

In figure 51 is shown a section of a stage at the completion of
the ectodermic vesicle. The specimen consists of the following
structures: (1) an outer layer of trophoblast, which on the lower
side has become modified into the primitive placenta; (2) an in-
complete entodermal sac which is connected laterally with the
trophoblast; and (3) an ectodermic vesicle.

The trophoblast is composed of a single layer on the upper or
free surface (fig. 18), but towards the base it thickens into two
layers of cells, especially on the right side of the figure. The tro-
phoblast, now in contact with the mucosa, has greatly extended
its area, as compared with that of specimens Nos. 316 and 332.
Since it is intended to devote an entire chapter to the subject of
placentation, we shall not give here any further consideration
to the lower layer of trophoblast, which is of course concerned
with placenta formation.

The entodermal sac is, as already staged, incomplete, remaining
open on the side turned towards the mucosa. The cells at the
point where the layer turns out to join the trophoblast are rela-
tively thick, while those of that portion of the entoderm which
passes over the ectodermal vesicle have undergone no important
change. In the extraembryonic cavity are found a few scatter-

592 J. T. PATTERSON

ed cells which are, for the most part, of entodermal origin, but
which will soon disappear.

The entodermic vesicle is completely developed. Its upper or
embryonic side is two or three cells thick, while its lower side is
but a single cell deep. On this side is a small opening or pore
which places the amniotic cavity in communication with the
extraembryonic cavity. This pore is found in but four sections,
and no other vesicle shows it, thus leading one to suspect that its
existence is more or less accidental, due to the manner in which
the vacuolization took place. In blastocyst No. 332 the vacuo-
lization occurs towards that side of the ectodermal mass which is
nearest to the mucosa, and in the present specimen we may sup-
pose that the excentric position of the vacuolization has resulted
in perforating the lower side of the vesicle.

There is, of course, another very plausible explanation, namely,
that the opening appears in all of the vesicles, but soon closes
thus accounting for the fact that it is never seen in the older
stages. This view receives support from the work of Fernandez
('09) on Mulita. The youngest stage secured by Fernandez is
about in the same state of development as this specimen of the
Texas armadillo, and presents the same structural relations. Fer-
nandez states that the cavity of the ectodermal vesicle and what
he calls the 'Trager cavity' are connected by a small pore which
he compares to the 'Verbindungsrohre' in the mouse (Melissinos
'07). His 'Trager cavity' represents the same space that I have
called the extraembryonic cavity in T. novemcincta. I have so
named this cavity because later, when it becomes lined with meso-
derm, it is recognized as a true exocoelome. It should be pointed
out here that what Fernandez terms the Trager cavity in his sec-
ond youngest state (Fernandez '09, text figure 2) I have regarded
as an artifact (for reasons to be presented later) and consequently
it can not be compared with the similarly named cavity of his
youngest stage.

In this mode of producing a vesicle of the ectodermal sphere is
to be recognized an amnion formation through a process of vac-
uolization; for the entire subsequent history of development
demonstrates that the cavity thus formed is an amniotic cavity.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 593

The cavity has been previously termed the 'common amniotic
cavity' (Fernandez '09, and Newman and Patterson '10), because
it is common to the amniotic connections of the four embryos
which later take their origin from the ectodermal vesicle.

It is evident from this that in the mode of amnion formation the
armadillo is to be classed with that group of mammals in which
the amniotic cavity is from the first an enclosed space, and never
has a free communication with the space outside the trophoblast.
The cavity is always intra-trophoblastic (Hubrecht '08), and
consequently no folds ever arise to deliminate it, for this is not
necessary. The armadillo can therefore be added to Hubrecht's
('08) compiled list of mammals, in which the amniotic cavity arises
within the ectoderm and remains a closed vesicle. His list is as
follows": "Cavia and other rodents, Pteropus, Galeopithecus,
Erinaceus, Gymnura, monkeys and man.5

ORIGIN OF THE EXTRAEMBRYONIC MESODERM OR
MESOTHELIUM

The origin of the mesoderm in mammals is a problem which has
given rise to much difference of opinion among embryologists, but
it is not necessary here to enter into this controversy. The first
appearance of the mesoderm in the armadillo blastocyst is seen in
connection with the cavity which lies between the ectodermal
vesicle and the placenta, or what has been termed above, the extra-
embryonic cavity.

The entoderm, at the angle where it parts company with the
ectodermal sphere to join the trophoblast (fig. 51), gives rise to a
few cells which frequently become scattered throughout the extra-
embryonic cavity. There is good reason for believing that most
of these cells undergo disintegration pari passu with the develop-
ment of the ectodermal vesicle. At any rate there comes a tune
when the exocoelomic space is essentially free from such ento-
dermal cells (fig. 54) . In some of the sections of this series a few
of these cells are still found within the cavity. Thus in figure 55
there are four of them, two lying some distance below the ectoderm
and slightly to the left of the center, and two situated against the

5 Loc. cit., p. 71.

594 J. T. PATTERSON

under side of the ectodermal vesicle, at a point where the embry-
onic and amniotic portions of the vesicle meet on the left side. All
such straggling entodermal cells show signs of decadence, and
without doubt play no role in the development of the mesoderm.

The first evidence of mesoderm formation is also found in this
same vesicle, and is indicated by the beginning of a process of pro-
liferation involving the ectodermal cells which lie at the point or
angle where the entoderm parts from the ectodermal vesicle. In
figure 55 one of the proliferated cells, which has just been set free,
is seen on the right, slightly removed from the angle. Only a very
few such cells are found about the edge of the vesicle, but the
presence of mitotic figures in this general region of the ectoderm
indicates the approach of a rather profuse proliferation of meso-
dermal cells.

In another vesicle, somewhat larger than the preceding, the
formation of the mesoderm has made rapid progress. The cells
are proliferated in clusters, and soon develop into small vesicular
structures (figs. 56, 57) . In this particular specimen there are about
eight small mesodermal vesicles, but it was not possible to deter-
mine whether their origin was confined to one or more localized
regions of the ectodermal vesicle, or centers of active prolifera-
tion. The preparation shows every stage in the formation of ves-
icles. In most instances the cluster of cells is set free from the
ectoderm before a cavity appears within their midst; in others, the
cavity arises while the cells still retain a connection with the ecto-
derm. In all cases the smaller mesodermal vesicles gradually fuse
together to form larger and larger cavities, until finally the entire
space lying below the ectoderm is lined with a mesodermal layer.

While I was unable to determine any definite localized regions
of mesoderm proliferation in this specimen, in all the older stages
the mesodermal vesicles fuse in such a way as to produce two main
vesicles. For example, specimen No. 234 has two well differen-
tiated mesodermal vesicles, which are unequal in size (fig. 19).
The larger one on the left-hand side is composed of a typical meso-
dermal layer. In this region it is free from any connection with
the ectodermal vesicle, but in other sections it not only has such
connections, but is also united to other smaller mesodermal vesi-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 595

cles, with which it is undergoing fusion. The smaller vesicle is
similar in its general structure to the larger one, but retains a
distinct conection with the ectodermal vesicle.

The relation of the mesodermal vesicle to the rest of the chor-
ionic vesicle is of great interest, and can be illustrated by explain-
ing the manner in which the sections have been cut. In each of
the specimens represented in figures 51 to 61 the sections have been
cut so that their plane is parallel to the plane passing through the
'horizontal grooves' of the uterus and perpendicular to the surface
of the mucosa (fig. 21). We thus see that the right and left me-
sodermal vesicles lie on those sides of the blastocyst which are
turned towards the right and left openings of the fallopian tubes,
respectively.

In figure 19 the mesodermal vesicles lie to the right and left of
the center of the blastocyst, and, as we shall see later, hold the
same orientation as do the two primary buds of the embryos.

Figure 59 shows a median section of a vesicle in which the two
mesodermal vesicles have expanded until they occupy the entire
extraembryonic cavity; but the double partition made by the ap-
proach of their adjacent sides does not allow a communication of
the two cavities (fig. 20) . However, this condition does not exist
throughout the entire series of sections, for in many places a por-
tion of the partition has already broken down. Eventually it
will entirely disappear. This final condition in the formation of
the extraembryonic mesoderm is soon reached, and the single
layer of flattened mesodermal cells then completely lines the space
below the ectoderm, conforming to the various irregularities of its
bounding walls (fig. 22). The new cavity thus formed and lined
with the mesodermal epithelium may now be called the extraem-
bryonic coelome.

COMPARISON WITH OTHER FORMS

i

At this point it is well to compare briefly the armadillo blasto-
cyst with that of other forms. The early development of the
armadillo parallels most closely that of the mammals in which the
so-called inversion of germ layers is found. Fernandez ('09) has
pointed out that certain stages of the South American armadillo

596 J. T. PATTERSON

Mulita are comparable to corresponding stages of the mouse, as
figured by Melissinos ('07) ; and, while a similar comparison may
be made between the mouse and the Texas armadillo, neverthe-
less, a closer similarity exists between the early stages of the fru-
giverous bat Pteropus and this armadillo. Thus one of the
youngest stages of Pteropus figured by Selenka and Gohre ('92
pi. 41, fig. 4) is strikingly like the blastocyst shown in figure 17 of
this paper; for in each case the embryonic ectoderm has separated
from the trophoblast to form a spherical mass, which has become
included within the entoderm.

The principal feature in which they are dissimilar is seen in the
extension of the entoderm. In Pteropus the entoderm completely
lines the cavity of the blastocyst, forming an epithelial lining for
the yolk-sac. In the armadillo the entoderm extends out along
the inner side of the trophoblast for only a short distance from the
ectoderm, and at most never covers an area of over 80° on the cir-
cumference of the blastocyst. Consequently a closed epithelial
sac of entoderm is not formed, and the yolk-sac cavity is bounded
on the non-embryonic side by a single layer of trophoblastic cells,
or chorionic ectoderm (fig. 19).

The similarity between Pteropus and the Texas armadillo is not
confined to this early period, but is also seen in later stages; es-
pecially is this true with reference to the formation of the amniotic
cavity. In each, the solid sphere of ectoderm becomes hollowed
out through the disintegration or vacuolization of the core to form
the primary amniotic cavity (cf. fig. 18 with fig. 6 of Selenka and
Gohre). Finally, in the condition of the mesoderm the two forms
show severarpoints of similarity.

In the blastocysts of the mouse and of the armadillo are also
to be seen many points of similarity, though the resemblance is
here less striking than in the preceding case. The figures of
Melissinos ('07) are very similar to several of the stages §hown in
this paper. His figure 31 shows a stage directly comparable to
our specimen No. 311 (fig. 11) ; and his figures 33 and 34 illustrate
the manner in which the embryonic ectoderm is pushed out into
the general cavity of the blastocyst, carrying before it the visceral
layer of entoderm. In the armadillo I have not succeeded in ob-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 597

taining a stage which corresponds exactly to the stage of the mouse
shown in figure 33 of Melissinos, although specimen No. 340 (fig.
15) may be compared with it. 'Figure 34 of Melissinos and my
figure 19 can be directly compared, especially if it be kept in mind
that the parietal layer of yolk-sac entoderm is incomplete in the
armadillo. In the mouse the embryonic ectoderm is borne upon
a mass of cells conecting it with the ectoplacental plate, and the
separation of these two embryonic structures does not take place
for some time. In the armadillo, on the contrary, the ectodermal
mass early parts company with the Trager or ectoplacental re-
gion, thus giving rise to an extraembryonic cavity at a very early
stage. It is this difference in the time of appearance of the extra-
embryonic cavity which renders difficult an exact comparison
between the two forms throughout the subsequent history of
development; and in support of this view we may cite the case of
mesoderm formation.

We have seen that the mesoderm does not make its appearance
in the armadillo blastocyst until after the extraembryonic cavity
has arisen, and that upon arising from that portion of the ectoder-
mal vesicle which is turned toward the placental region, it imme-
diately develops into an epithelial lining membrane for this cavity,
which is thereby transformed into a true extraembryonic coelome.

In the mouse, according to the account of Melissinos, the meso-
derm is very early recognized as a mass of cells lying in a position
somewhat similar to that of the mesoderm in the armadillo; that
is, immediately ventral to the ectodermal mass, at the point of
constriction between the embryonic ectoderm and the group of
cells connecting it with the ectoplacental plate. Later, when the
embryonic ectoderm and the ectoplacental plate become entirely
separated, a cavity lined with mesoderm appears between these
two embryonic structures. This cavity becomes then an extra-
embryonic body cavity (the 'mittlere Hohlung' of Melissinos).
When this process is completed we are presented with a condition
quite similar to that of a relatively late but corresponding stage of
the armadillo (c/., fig. 43 of Melissinos and my fig. 22). The es-
sential difference lies in the fact that in the armadillo blastocyst
there is no ectoplacental cavity; but even this difference later be-

598 J. T. PATTERSON

comes lessened upon the formation in the armadillo of a distinct
Trager epithelium, with a potential cavity lying between it and
the invaded mucosa (fig. 23).

Other comparisons might be drawn between the armadillo
blastocyst and those of other mammals, but this is not necessary.
Sufficient evidence has been presented, I believe, to establish the
fact that the early stages of the armadillo give a history corre-
sponding in its general outlines to the development of an egg which
in other forms produces but a single individual. The differences
noted are no greater than would be expected to exist between
mammals as widely separated as the armadillo and the bat or
mouse.

ORIGIN OF PRIMARY BUDS

In the late stages of development it has been demonstrated
(Newman and Patterson '10) that the four embryos of an arma-
dillo litter are arranged within the single chorion in two pairs,
which hold a very definite orientation with reference to the uter-
ine axes. Thus it was found that one embryo always occupied that
portion of the chorionic cavity lying adjacent to the dorsal wall of
the uterus, one holds a ventral position, and the other two lie to the
right and left sides, respectively. The heads of the embryos are
always directed toward the cervix end of the uterus, and conse-
quently point in a direction exactly opposite to that of the head
of the mother. It was further found that the ventral embryo
(I) is paired with the right-lateral (II) and that the dorsal (III)
and left-lateral (IV) embryos are members of the other pair.6
This relation was apparent, not only from the very close heredi-
tary similarity existing between the two individuals of a pair, but
also from certain foetal connections, notably the union of the
amniotic canals of each pair in comparatively early stages.

The question now arises as to how such a striking relationship
between the embryos has come into existance; and in seeking

6 The terms 'right-lateral' and left-lateral' refer to the position of the embryos
within the blastocyst, and not to the right and left sides respectively of the uterus.
For example, the right-lateral embryo lies on the left side of the uterine cavity.
Throughout the paper I use the Roman numerals (I to IV) to designate the em-
bryos, so that the reader will find no difficulty in locating any embryo to which
reference is made.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 599

•

answer to this question we are at once confronted with the much
more important problem of the origin of the multiple embryos
from the single fertilized egg. In the paper cited above the
position was taken that the embryos belonging to a pair were
probably derived from one of the blastomeres of the two-celled
stage, and that each embryo could therefore be looked upon as
a lineal descendent of one of the blastomeres of the four-celled
stage. In the present contribution the view is held that the
four embryos do not owe their origin to a spontaneous blas-
totomy, but rather that they are the product of a form of
agamogenesis belonging to the general category of budding.

It was considered then of the utmost importance to determine
at just what point in the development of the armadillo blasto-
cyst evidence of its quadruplicity first appeared. Consequently
a sharp lookout was maintained in the study of all the early
stages for signs of the first expression of polyembryony. The
earliest observed evidence which could be interpreted as repre-
senting the beginning of multiple embryos comes in the forma-
tion of the mesothelium — not in the manner in which the ele-
ments of this layer arise, for localized centers of proliferation
were not found, but in the early formation of the two large
mesodermal vesicles through the fusion of several smaller ones.
The development of two mesodermal vesicles would not in it-
self be so significant, as it might be merely an expression of a
bilateral arrangement of mesoderm similar to that of many other
vertebrate embryos, were it not for the fact that they hold a
position corresponding exactly to the two primary ectoderma.1
buds; that is, they lie on the sides of the vesicle which are di-
rected towards the openings of the fallopian tubes. However,
it may be that these two mesothelial vesicles have no general
significance with reference to polyembryonic development, for it
must be kept in mind that they have arisen by the fusion of
numerous smaller vesicles, and later they in turn fuse to form a
single vesicle.

Whatever may be the significance of the position of the two
mesodermal vesicles, certain it is that the first indisputable evi-
dence of the differentiation of the four embryos from the blasto-

600 J. T. PATTERSON

cyst, appears in the formation of two diverticula from the ecto-
dermal vesicle. In the preliminary paper these diverticula were
termed the 'primary buds/ and I shall continue so to designate
them. The buds appear on the opposite sides of the vesicle,
and, with respect to the orientation of the blastocyst within the
uterus, on the right and left sides, respectively, that is, on the
sides of the vesicle that face the openings of the fallopian tubes.
In accordance with the statement made above, the right primary
bud faces the left fallopian tube opening, and similarly the left
primary bud faces the right opening.

The primary buds do not develop for some time after the
completion of the ectodermal vesicle, although their appearance
is anticipated soon after this period by certain easily detectable
changes in the walls of the vesicle. It will be recalled that
immediately after the ectodermal sphere has become transformed
into a vesicle, that portion of the wall of the vesicle which is
turned toward the free pole of the blastocyst is of a relatively
uniform thickness (fig. 51). Very shortly thereafter one can
detect a tendency in this region of the wall to become less thick
(figs. 55-58). The thinning out may be due in part to an in-
crease in size of the vesicle by the accumulation of fluid within
its cavity, but undoubtedly in the main it is brought about
through the shifting of cells from here to the lateral portions of
the wall, for these show an increase in thickness (fig. 59).

The shifting of the cells from the pole of the vesicle results in
the formation of a thickened zone adjoining the thin or endo-
thelial-like portion of the ectodermal vesicle (figs. 59, 61). The
zone is not uniformly thick, but is thickest at the two regions
corresponding respectively to the right and left sides of the vesi-
cle. One can therefore correctly speak of these thickened areas
as lateral plates.

The primary buds arise from these lateral plates, and appear
as two broad, blunt processes protruding from the sides of the
ectodermal vesicle (fig. 21). Each bud involves the greater por-
tion of the side of the vesicle, covering an arc of approximately
80 degrees on the circumference. These points can be made out
in specimen No. 247, which will now be described.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

601

In the preserved condition, the chorionic vesicle measured
0.722 mm., from right to left, at the base, and the ectodermal
vesicle 0.443 mm. in the same plane; while in the antero-poste--
rior plane it measured 0.866 mm., and the vesicle 0.312 mm.
The ectodermal vesicle is, therefore, approximately a third wider
in the right-left 'plane than in the antero-posterior plane, and
this difference is due to the presence of the primary buds. In

Fig. 1 Outline reconstruction of specimen No. 247. This shows the well
formed 'primary buds,' situated on the right and left hand sides of the vesicle.
The broken line shows the plane of the two sections illustrated in plate 6, figures
60,61. X 95.

preparing the specimen for microscopical study the sections were
cut so as to pass parallel to the long axis of the ectodermal vesi-
cle (fig. 1).

In the median sections of the series the primary buds are in-
stantly recognized on the right and left sides of the ectodermal
vesicle. They appear as outgrowths from the vesicle, and their
cavities (fig. 22, R and L) are seen to be extensions of the com-
mon amniotic cavity. The wall of each bud is three or four
cells thick, while the roof of the vesicle has thinned out to a
single layer in thickness. Furthermore, the cells in the walls

602 J. T. PATTERSON

of the buds are undergoing a rapid increase, as is evident from
the numerous mitotic figures seen here.

On the lower side of the ectodermal vesicle the wall is composed
of a single, thin layer of cells, and below this is the layer of
mesoderm. It will be recognized that these two layers together
constitute the true amnion, out of which the amnion of each
embryo will eventually arise.

The mesoderm of the amnion is a portion of the mesothelium,
the origin of which was described in a preceding section. The
extraembryonic body cavity occupies the entire space lying be-
tween the amnion and the mucosa, and only traces (fig. 22) of
the partition of the two original mesodermal vesicles remain to
tell the history of this large cavity.

The outer (upper) surface of the chorionic vesicle consists of
entoderm. This unique condition has been brought about
through the disappearance of the chorionic ectoderm (hinfalliges
Ectoderm of Fernandez '09), which has sloughed off. Its dis-
appearance may occur prior to this period (fig. 20), or it may
persist even until the four embryonic rudiments are established
(fig. 23). The ectoderm breaks off just beyond the point where
the entoderm unites to its inner surface (fig. 22, x), and thus
exposes the entire outer surface of the entoderm to the cavity
of the uterus. This condition persists throughout the entire
period of gestation, in a manner to be described later.

Prior to the formation of the primary buds, the ectoderm and
the adjacent entoderm remain distinct from each other, as
seen in such specimens as those shown in figures 58 and 59; but,
upon the development of the buds, these two layers are brought
into intimate contact, which at certain points amounts practi-
cally to a fusion between the two layers. These points are situ-
ated just above the central area of each primary bud, and
therefore mark the general region of the primordia of the future
embryos.

No important changes in the extraembryonic mesoderm appear
to be taking place at this time. It retains its epithelial-like
character and no cell proliferations are found. A few mesoder-

POLYEAIBRYONIC DEVELOPMENT IN TATUSIA 603

mal cells are found just beyond the outer margin of the embry-
onic ectoderm, in the space lying between the entoderm and the
extraembryonic mesoderm, but such cells are undoubtedly given
off from the marginal cells of the ectoderm.

ORIGIN OF THE SECONDARY BUDS

The formation of the secondary buds immediately follows the
establishment of the primary diverticula, and three or four
specimens in the collection show the main steps in the process.
However, it will be necessary to secure a closer series through
this period of development before a detailed account can be
given of the origin of the secondary buds. Each primary bud
gives rise to two secondary buds, and consequently there are
four secondary diverticula. Each secondary bud carries the
rudiment or primordium of an embryo. The first step leading
to the development of the secondary diverticula consists in the
formation of two thickenings in the wall of each primary bud.
One of these areas lies at the tip t>f the bud, while the other
appears slightly to the left (as viewed from above) of the tip.
The secondary buds then arise from these areas as blind diver-
ticula, which extend down along the inner surface of the yolk-
sac entoderm. In specimen No. 247 the beginning of the second-
ary buds can be seen in the left-hand primary bud (fig. 1). At
the point marked 5 is seen a slight protrusion which will form
secondary bud No. III.

The secondary buds soon become recognizable in surface views
of living specimens, and appear as four blunt processes from the
sides of the ectodermal vesicle. Upon the upper surface of each
bud an embryonic rudiment appears, in the form of a white,
opaque spot. It is somewhat difficult to make out the exact
limits of the different parts of the ectodermal structures, owing
to the fact that the entoderm (and the chorionic ectoderm,
if still present) tend to obscure the view. This difficulty was
obviated by making an outline reconstruction from the series
of sections of the blastocyst.

JOURNAL OF MORPHOLOGY, VOL. 24, XO. 4

604

J. T. PATTERSON

The reconstruction of the specimen, which in point of devel-
opment comes closest to No. 247 (fig. 1), is represented in figure
2, • in which the outer, circular line marks the margin of the

Fig. 2 Outline reconstruction of specimen No. 29Q. The outer circular line
marks the margin of the blastocyst; the broken line indicates the upper limit of the
Trager; and the diamond-shaped central figure is the outline of the ectodermal
vesicle. The four oval figures lying within the ectodermal vesicle are the embry-
onic areas. The parallel broken lines represent the planes of the sections shown
in plate 7. X 63.

blastocyst, the broken line the upper limit of the Trager, the
central irregular line the limits of the ectodermal vesicle.

Our attention must naturally be centered on the ectodermal
structure. Its general outline is much like that of a diamond,
in which the points are occupied by the secondary buds, or em-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 605

bryos. The 'right-lateral' (II) and 'left-lateral' (IV) embryos
lie at the acute angles of the diamond, while the 'dorsal' (III)
and 'ventral' (I) embryos occupy the obtuse angles. The same
condition prevails in two specimens but slightly older than this
one; hence we may conclude that it is the normal arrangement
of the secondary buds.

It is certain from the evidence that Embryos I and II are the
product of the right-hand primary bud, while Embryos III and
IV have come from the left-hand primary bud. It might appear
from an examination of figure 2 that the secondary buds at II
and IV had alone come from the two primary buds of such a
specimen as No. 247 (fig. 1), while those at III and I had
arisen de novo from the dorsal and ventral sides, respectively,
of the ectodermal vesicle. But the entire genesis of these sec-
ondary buds argues against such an interpretation, as will be-
come clear after a complete account of their development has
been given. Nevertheless, it 'is an interesting fact, and one I
believe not to be without significance, that the secondary buds
II and IV occupy positions on the periphery similar to those of
the two primary buds. Furthermore, it is probably correct to
regard Embryos I and III as having arisen as outgrowths from
the left sides (assuming that the observer is situated hi the cen-
ter of the free surface of the blastocyst) of the right and left
primary buds, respectively.

The oval figure in each bud indicates the area over which the
ectoderm and entoderm have come into intimate contact or have
fused, while the solid line within each oval area shows the posi-
tion and extent of the primitive groove. The head of the future
embryo will, in each case, be directed towards the center of the
vesicle.

The plane of the sections is indicated by the broken parallel
lines, which also show the position of the four sections from
which the photographs of plate 7 were made.

The section passing through the plane e to f will be taken for
detailed account of the different parts of the chorionic vesicle.
The outer membrane of the chorionic ectoderm (fig. 23) shows
signs of breaking away. It has usually disappeared before this

606 J. T. PATTERSON

time; thus exposing the entoderm to the uterine cavity. The
entoderm is connected at the margin to a thickened portion of
the chorionic ectoderm, which is recognized as the Trager (2V.).

The mesothelium which lines the extraembryonic cavity (E.
E. B. (7.), has undergone no important changes; but there has
been organized just beneath it a distinct Trager epithelium
(Tr. Ep.), which in its relation to the uterine mucosa is particu-
larly clear in the photograph (fig. 64).

The most important changes involve the ectodermal vesicle,
and chief among these is the one affecting the organization of
the embryonic primordia. On account of the inversion of germ
layers, the lower side of each embryonic primordium is upper-
most in the figure, that is, the lower side of the future embryo
will be on the outside of the ectodermal vesicle. The section
under discussion shows the rudiments of Embryos I and II, which
are cut across at a slightly oblique angle (fig. 2). Each embry-
onic spot is characterized by (1) a fusion between the ectoderm
and the entoderm, and (2) by a rather shallow depression, or
primitive groove, which is situated on the lower or inner side of
the embryonic ectoderm (fig. 23, P. G.). Between the two em-
bryos the entoderm is entirely free from that portion of the ecto-
derm which joins the adjacent sides of the two embryonic rudi-
ments. The cavity lying below the embryos is the bay of the
original right-hand primary bud, and is a portion of the general
amniotic cavity. The thin lower wall of this cavity is, there-
fore, the ectodermal layer, which, together with the adjacent me-
sodermal epithelium, constitutes the true amnion (fig. 23, Am.).

The primitive groove of each embryo measured about 0.095
mm., and is rather shallow throughout the greater part of its
length. There is, however, a pit-like depression at one point
which undoubtedly corresponds to a primitive pit. This is es-
pecially distinct in Embryo I, in which it lies about 0.032 mm.,
from the anterior end of the groove (fig. 24, P. P.).

Beneath the primitive groove there is the typically early prim-
itive streak region, from which cells are being proliferated later-
ally to form the true embryonic mesoderm (fig. 24, E. Mes.).
Throughout the extent of the primitive streak there is no

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 607

evidence that the underlying entoderm takes part in this pro-
liferation of the mesodermal cells, but directly anterior to the
primitive streak the entoderm is found to be actively dividing to
form a group of cells, which can be traced throughout successive
stages to mesodermal tissue. Undoubtedly this center of pro-
liferation corresponds to the protochordal plate of Hubrecht
(fig. 25, P. PL).

The conditions which we have here described for the pair of
Embryos I and II also hold for the other pair, or Embryos III
and IV. The relation of the various parts is identical in the two
pairs. The only point in which they do differ is the fact that
the two secondary buds III and IV are more widely separated
than I and II, and hence the ectodermal vesicle appears much
wider (cf. fig. 62 with fig. 64).

Following the sections through from the anterior limits of
either pair of embryos towards the center of the vesicle, two
important changes are noticeable: First, the entoderm becomes
entirely separated and distinct from the ectoderm; that is, these
two layers have never become fused; and second, the roof of the
ectodermal vesicle thins out to a single layer, while to either side
its wall remains from two to three cells thick (fig. 63). These
lateral thickenings are found prior to the appearance of the em-
bryonic "rudiments, when the roof of the vesicle undergoes the
general reduction in thickness.

Toward the posterior ends of the embryos the posterior
grooves fade out, completely disappearing before the sections
which cut the tip of the primary buds are reached (fig. 26). In
the case of Embryos III and IV the posterior ends of the em-
bryos extend well back into the primary buds, which are seen
to be sharply separated from each other, especially in the last
three or four sections which cut the ectodermal vesicle (fig. 26).

The embryonic entoderm or gut-en toderm of each embryo is dif-
ferentiated from the primary 3'olk-sac entoderm. Apparently any
region of the yolk-sac where the embryonic buds happen to im-
pinge against its inner surface will differentiate into gut-entoderm.

The conditions which we have just recorded may be further
chronicled by a brief account of specimen No. 175, which is the

608 J. T. PATTERSON

next oldest blastocyst in the collection. In the entire blastocyst
the primary buds with their accompanying embryonic rudiments
stood out clearer than in the preceding specimen (No. 290),
mainly because the chorionic ectoderm had already sloughed off
from the upper side of the vesicle, thus giving a better view of
the ectodermal vesicle.

The general arrangement of the secondary buds is identical in
these two specimens (cf. fig. 2 and 28), but No. 175 is distinctly
further developed. This becomes especially evident in a de-
tailed study of the sections. In the section which passes
through the place a to 6, figure 29, bud IV is seen to be well
defined, and the accompanying embryonic rudiment extends
down into the tip of the bud, showing a well defined primitive
groove (fig. 66). Anteriorly the groove becomes very pronounced,
due ill a large measure to the elevation of the medullary folds
(fig. 67).

In the central region of the ectodermal vesicle (fig. 68) the
lateral walls have become distinctly thinner, and will soon reach
a state in which the entire wall, exclusive of the embryonic por-
tions, will become but a single layer thick. When this condi-
tion is once attained the ectodermal vesicle undergoes no further
growth or expansion, but remains a small inconspicuous structure
(common amniotic vesicle) to which the embryos remain con-
nected by means of amniotic canals or tubes. These canals are
developed through the extension of the secondary buds, which
rapidly push outward and then downward along the under side
of the entoderm, carrying with them the embryonic rudiments.
At this point we are concerned with the beginning only of the
tubes, and this can be seen not only in figure 66, but also in
figure 69, especially in the case of Embryo II.

It remains to say a word about the cavity lying just beneath
the Trager epithelium in blastocyst No. 175. This cavity ex-
tends throughout the greater part of the left side of the chori-
onic vesicle (fig. 27, Cav.). If these figures are compared with
text figure 1 of Fernandez ('09) it will be found that there is
much similarity between them as regards this cavity. Fernan-
dez designates it the Trager cavity (or Ectoplacentarplatten-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 609

hohle) in Mulita; but in the Texas armadillo the enlarged space
is clearly an artifact. One can easily observe in the sections that
it has been produced by lifting up the Trager epithelium from
the subjacent emaciated mucosa, probably through the action of
the fixing and hardening reagents. It has not been observed in
any other specimen, either older or younger, and this leads one
to suspect that it is likewise an artifact in the blastocyst of
Mulita, especially as it does not appear in the older vesicles of
this animal.

In plate 9 is shown a series of five sections from a chorionic
vesicle presenting a further advance hi the development of the
secondary buds and their accompanying embryos. The speci-
men is one of the finest in my collection, not only because of
its excellent state of preservation, but also for the reason that
it remained turgid while undergoing fixation, and thus gives us
a picture in the sections which most closely resembles that of
the living vesicle.

Figure 3 is an outline reconstruction of this series, and shows
that the buds have made considerable progress. Buds II and
IV are still larger than I and III, but this inequality gradually
grows less and less as the buds extend outward and downward
beneath the entoderm.

In the section passing through plane e to / of figure 3, the
general relation of the various parts is well shown. The large
extraembryonic cavity, lined with mesoderm, is conspicuous.
Above this, and separated by the thin amnion, is the amniotic
cavity of the ectodermal vesicle (fig. 72). The section passes
a little to the left of the center of the left-lateral bud, which
appears in section as a prolongation of the left side of the ecto-
dermal vesicle. The outer covering of the chorionic vesicle is
the entoderm, the chorionic ectoderm having already disap-
peared. The point at which it has broken off close to the base
of the vesicle is clearly seen in this and the other photographs.

The other sections illustrated in the plate present special
parts of the several embryos. Thus figures 70 and 74 show
transverse sections of Embryo III and Embryo I, respectively.
In each case the primitive groove is distinct. In figure 71 the

610

J. T. PATTERSON

Fig. 3 Outline reconstruction of the left half of specimen no. 257. The five
sections from this specimen are illustrated in plate 9. X 83.

section passes through line c to d of figure 3, and thus cuts
across the angle lying between Embryos II and III. Finally
figure 73 passes a little to the left of the center of Embryo IV,
and shows the structure of that embryo.

At the tips of the buds the cells are in active division (fig.
30), indicating that the extension of the buds, during their early
existence, is to be accounted for in this way. Their subsequent
extension is due to other factors, which will be considered in
the next sections.

EXTENSION OF THE SECONDARY BUDS AND ORGANIZATION
OF THE EMBRYOS

The further extension of the secondary buds from the sides of
the ectodermal vesicle very rapidly follows such conditions as we
have described in connection with specimen No. 257. A rapid
growth or increase in size of the vesicle also follows or accompanies
this extension of the buds. Each bud grows down along the inner
side of the wall of the vesicle, between the entoderm and the meso-

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 611

thelium, eventually establishing a placental connection with the
extending Trager, thus forming a sort of 'belly-stalk' through
which the placental blood vessels run, and into which the rudi-
mentary allantois later extends. In their growth from the vesicle
the buds do not pass out as four distinct rays from a common
center, the common amniotic vesicle, but extend out in pairs, the
individuals of each pair retaining a common connection with the
ectodermal vesicle. The paired condition is but a further expres-
sion of the same relation which was noted in connection with
the account of the origin of the secondary buds.

The earliest phase of this condition is very clearly brought
out in one of the specimens of the series. This specimen has
almost completely collapsed and is inclined to the left. Hence
Embryos I and II are in part folded beneath the wall of the
blastocyst, making their study difficult.

In plate 10 are shown photographs of three sections which
pass through Embryos III and IV at different levels. Figure
75 is taken across the common bay of the two embryos, about
half way between the common amniotic vesicle (remains of the
old ectodermal vesicle) and the point of departure of the two
secondary buds. The chorionic ectoderm has disappeared and
consequently the entoderm is the uppermost layer. It is en-
tirely distinct from the embryonic ectoderm.

Figure 76 represents a section which passes through the point
where the secondary buds arise and diverge. The bud on the
left contains Embryo IV, and its width is almost twice that of
its paired mate on the right, or number III.7 The difference
between the two buds exists throughout their entire length.
Thus hi the section cutting the middle of the buds (fig. 77) the
difference in size is particularly striking. Each embryo consists
of the following parts: (1) the entoderm, which is in contact
with the primitive streak mesoderm; (2) the primitive streak
mesoderm, which is being proliferated from the ectoderm; (3)

7 In the preliminary paper (figs. 8 and 9) these embryonic buds were incor-
rectly labeled. This was due to the fact that the sections had inadvertently been
reversed in mounting them on the slide, a fact not discovered until after plate 10
of the present paper had been made up.

612

J. T. PATTERSON

the thick embryonic ectoderm, curved upward on each side; (4)
the thin amniotic ectoderm above; and finally, (5) the meso-
derm of the false "amniotic or extraembryonic cavity.

Briefly stated then, each embryo consists of a tube-like out-
growth from the ectodermal vesicle, with which it retains a

Fig. 4 Outline reconstruction of the left half of specimen No. 170. Three sec-
tions from this specimen are shown in plate 10, figures 75 to 77. X 62.

connection in the form of the proximal part of the secondary
bud. As a result of the manner in which the secondary buds
arise from the primary ones, this connection is common to two
embryos, which always constitutes a pair.

Aside from the differentiation or organization of the embryos,
the final stages in the extension of the secondary buds presents
very little of special interest. We may therefore refer to them
briefly. First of all, it should be stated that following a stage

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 613

such as we have just considered the vesicle grows rapidly in
size, quickly filling up the entire lumen of the uterus. The first
noticable change in growth affects the free or distal portion of
the wall, and the chorionic vesicle soon becomes bulb shaped
(fig. 5).

The expansion and growth of the wall of the blastocyst grad-
ually carries distally the common amniotic vesicle, which by this
time has ceased to grow. As a result, the embryonic rudiments
are gradually separated from the amniotic vesicle, and their or-
ganic connections with the vesicle are drawn out into small
tube-like structures, the amniotic connecting canals. These
canals lie against the under or inner surface of the entoderm,
and each consists of an inner layer of ectoderm, surrounded by
a layer of mesoderm.

In plate 11 are shown five sections, taken at different levels,
from a specimen in which the amniotic vesicle was just begin-
ning to be drawn away from the embryonic rudiments, through
the expansion of the wall. The sections are not quite transverse
to the axes of the four embryos.

The section represented in figure 80 cuts the tip of the chori-
onic vesicle and passes through the lower portion of the common
amniotic vesicle. The anterior parts of three of the embryos
are seen in the section. These are the dorsal, right-lateral,
and ventral embryos. The position of the other embryo, or
the left-lateral, is indicated by the evagination at the left side
of the vesicle.

In figure 81, which is three sections further down, the same
relation with reference to the embryonic rudiments still exists,
but the section passes through the extreme lower limit of the
amniotic vesicle, and the right-lateral embryo becomes entirely
separated from the others.

In figure 82, which is five sections lower down on the chorionic
vesicle than figure 81, the dorsal and right-lateral embryos are
both free from any connections with the amniotic vesicle. In
section each embryo appears as a section of a tube. The chief
interest in this section lies in the condition of the ventral and
left-lateral embryonic rudiments. Only the anterior tips of

614 J. T. PATTERSON

these two embryos are shown, and they lie each at the end of
a common bay by means of which their amniotic cavities are
placed in communication with the cavity of the common amni-
otic vesicle. This condition is of the greatest significance, since
it indicates the common origin of the pair of embryonic tubes
from the left-hand primary bud.

Figure 83 shows all four of the embryonic rudiments in sec-
tion lying on the sides of the wall of the blastocyst, and facing
on the inner side of the large extraembryonic cavity.

Figure 84 is taken about half way between the posterior ends
of the embryonic tubes and the base of the chorionic vesicle.
Here the wall of the blastocyst is composed of the typical
structures, entoderm on the outside and mesoderm within. The
latter is rapidly becoming vasculated, especially in the regions
lying directly posterior to the embryonic tubes.

Soon after the stage just referred to, the chorionic vesicle
begins a very rapid expansion, and, in doing so, first assumes a
bulb-like shape (fig. 5). The vesicle is united to the mucosa by
an annular zone of thickened trophoblastic ectoderm, the so-
called Trager (fig. 23), and from the edge of this the yolk sac
entoderm extends upwards to form the outer layer of the chori-
onic wall, the chorionic ectoderm having long since disappeared.
The inner layer of the chorion everywhere consists of mesoderm.

The embryonic portion of the vesicle consists of the relatively
small, common amniotic vesicle (fig. 5, C A. V.), from the right
and left sides of which spring a pair of small connecting canals.
Each canal places the cavity of the amniotic vesicle in direct
communication with the amniotic cavity of the embryo. It
will be recognized that these canals are the elongated proximal
parts of the original ' secondary buds', and that the embryonic
rudiments are the distal portions of such buds. This is the
reason why the canals spring in pairs from the vesicle. How-
ever, there is considerable variation in the relation of the pair
of canals to the vesicle. The one presented in figure 5 is rather
unusual, in that the amniotic vesicle is greatly elongated, instead
of spherical, and the two canals of a pair arise very close to-
gether from the end of the vesicle. The usual condition shows

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

615

the pair of canals united at a very short distance from the
vesicle, and thus entering it as a single short tube. Variations
from this are seen in those cases in which the union takes place
further and further away from the amniotic vesicle. In one of
the most extreme cases observed the two canals on one side
were united for a distance of about three millirneters from the
vesicle, or more than half way between the anterior end of the
embryo and the amniotic vesicle.

Fig. 5 A free hand drawing of specimen No. 276, in which the embryonic
rudiments are well started. Each embryo is connected with the common amniotic
vesicle (C.A.V.} by means of a slender tube-like canal. In the living condition
the vesicle measured about 5 mm. high by 3 mm. at the widest point. X 11.

The important point is that in all of these cases the paired
condition of the embryos is unmistakably clear, and I know of
no other way to account for this except to assume that it rests
upon the manner in which the pair of secondary buds arises
from the primary buds. If the secondary buds start soon after
the primary buds are formed, than a condition similar to that
seen in figure 5 might readily result; but if the primary bud has
made considerable progress in its extension from the vesicle
before the secondary buds arose from it, then we should expect

616 J. T. PATTERSON

to find a common canal for the two embryos developing out of
the proximal part of the primary bud.

Each embryonic rudiment is a slipper-shaped structure lying
at the terminus of a canal (fig. 5), and is in a relatively late
primitive streak stage. At the anterior end of the embryo the
medullary plate is well formed, although as yet the medullary
folds have not become elevated. Posteriorly the embryo ends
as an irregular, blunt process of mesodermal tissue.

Fig. 78 shows a transverse section of one of the embryonic
tubes from specimen No. 276. The section passes through the
embryo at the level of the extreme anterior tip of the primitive
streak, and hence cuts the thickened medullary plate, which
curves upward and inward to become the thin ectodermal layer
of the amnion. Beneath the medullary plate, and between it and
the entoderm, are seen the scattered mesodermal cells which
have been proliferated from the primitive streak.

Lateral to the embryo is a loose mass of mesodermal cells
which lie between the entoderm and epithelial-like mesoderm
of the extraembryonic cavity. In the whole condition of the
chorionic vesicle the mesoderm was seen to fringe each side of
the embryonic tube, extending throughout the entire length of
the embryo proper. It already shows a rudimentary net-work
of blood-vessels, which represents the beginning of the area vas-
culosa. I have not worked out the detailed history of the vas-
cular area, but undoubtedly it arises in each embryo in a man-
ner similar to that in the typical mammalian ovum from which
but a single embryo develops.

The embryonic mesoderm is thickest at the posterior end of
the embryo, where it gives rise to a series of enlargements which
extend for some little distance behind the extreme posterior tip
of the embryonic tube. This is the portion of the mesoderm
into which the umbilical vessels and the rudimentary divertic-
ulum of the allantois later extend; that is, it forms the basis
for the belly-stalk.

The further development of each embryo is very similar to
that of the ordinary mammal, and therefore calls for a brief
description only. The reader is referred to an earlier paper *in

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 617

which is found an account of the late stages of development
(Newman and Patterson '10).

The differentiation of the embryo within the tube is soon
made evident by the elevation of the neural folds to form the
neural tube, and by the cutting off of paired mesodermic so-
mites. As these changes in the embryo are progressing, the tube-
like amnion is rapidly undergoing modification. First, it devel-
ops a finger-like prolongation which extends back beyond the
posterior limit of the embryo. Then there follows a rapid ex-
pansion of that portion of the amniotic tube which is occupied
by the embryo, the anterior portion of the tube remaining small,
and, together with the common amniotic vesicle, is destined to
degenerate and disappear.

During the expansion the amnion at first assumes a cigar-like
shape, and the amnia of the four embryos soon come to fill the
entire cavity of the chorionic vesicle, each embryo and its mem-
branes occupying one quadrant of the vesicle. In the mean
time, each embryo has become constricted from the extra-em-
bryonic parts by the development of the characteristic head,
tail, and lateral folds, and retains its connections with the
chorion by means of a typical umbilicus. It thus floats quite
freely within the amniotic fluid.

During all of these changes the paired condition of the embryos
is perfectly distinct, and, in the final stages of development,
expresses itself in the arrangement of the embryos into right-
hand and left-hand pairs, as is evidenced by the umbilical con-
nections with the placenta (fig. 79).

IMPLANTATION OF THE OVUM AND PLACENTATION

The more general features of the late phases of placentation
have been presented in another paper, but most of the material
for the study of the early development of the placenta has but
recently been secured. The nature of this paper does not call
for a detailed account of this process, and even though it did,
it would not be possible to write it in full at this time, since one
or two of the critical stages have not yet been been obtained.

618 J. T. PATTERSON

Particularly is this true of the changes which immediately follow
the attachment of the blastocyst. It is therefore proposed
briefly to describe some of those changes involved in the early
history of implantation and placentation which are essential
to a clear understanding of the embryology of the armadillo.
This account has been deferred until now, because it will be
.somewhat easier to follow after the description of the develop-
ment has been read.

A statement as to the manner in which the embryonic vesi-
cle migrates along the horizontal groove of the uterus and even-
tually attaches itself has been given above. The principal
facts are as follows: After the blastocyst has reached the placen-
tal area, which lies at the extreme anterior tip of the fundus,
it adheres to the mucosa, apparently for some time before the
actual placentation or intimate union with the uterine mem-
brane is consumated. Four clear and two somewhat doubtful
cases of early adhesion between the ovum and the mucosa have
been observed. In every instance the ovum freed itself either
immediately or very shortly after the application of the fixing
fluid, thus indicating that the implantation proper had not
really taken place. Fortunately, these vesicles were studied in
salt solution under the binocular microscope before fixation and
it was possible to make out several important points. It was
observed that the ovum always comes to rest upon the uterine
surface in such a way that the embryonic spot or germinal area
is turned toward the mucosa. Hence, the foetal contribution
to the early placenta arises from that portion of the trophoblast
which overlies the inner cell-mass; that is, from the so-called
Rauber's layer.

It is to this region of the trophoblast then that one must look
to detect the first indications of placentation. In five of the
blastocysts to which we have just called attention no evidence
of importance in this connection was observable, but in the sixth
unmistakable indications of early placentation are present. In
this specimen Rauber's layer is seen to have undergone impor-
tant modifications. Several of the cells are in mitosis, and all
of the cells of this region have lost their original attenuated

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 619

appearance, and now show a decided increase in thickness (fig.
11). There is also a distinct tendency for some of the cell
walls to disappear, thus transforming Rauber's layer into a
syncytium. Furthermore, the surface of Rauber's layer presents
a 'fuzzy' appearance, the outer wall of some of the cells actually
being broken as though ruptures had resulted when the blasto-
cyst was freed from its insecure moorings to the mucosa.

That all of these facts are evidence of the beginning of im-
plantation is clearly indicated by the act that the trophoblastic
cells which lie beyond Rauber's area are still unchanged, and
show the mosaic-like arrangement of polygonal cells so charac-
teristic of all of the free blastocysts. Unfortunately, there is
here a slight break in the series, so that we are not able to fol-
low up this clew through the obviously critical period of implan-
tation. We are therefore obliged to pass directly to an ac-
count of the modifications which are occurring both in the
mucosa epithelium and in the wall of the blastocyst in a vesicle
which has already become firmly anchored to the maternal
tissue.

Blastocyst No. 316 represents the youngest firmly attached
stage that has been secured. A series of sections from this
specimen is shown in plate 2. In the living vesicle it could be
seen that, in addition to the small area which had established
an intimate union with the mucosa, almost the entire left side
was lying in contact with the uterine wall, and that as a result
the trophoblastic cells here had. greatly increased in thickness
(fig. 16). Whatever may be the nature of the stimulus which
causes the trophoblastic cells to react whenever brought into
close relation with the uterine epithelium, it is certain that the
influence- is not confined to Rauber's layer, but any portion of
the trophoblast, upon coming in contact with the mucosa, will
respond. It does not necessarily follow that such thickened
trophoblast establishes eventually a fusion or placental union
with the uterine wall, although a study of the stages more ad-
vanced than this one, suggests that some of it may so unite.
However, it should be pointed out that only in rare instances
does a blastocyst become attached in a manner such that an

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

620 J. T. PATTERSON

extensive area of the trophoblast is brought into close contact
with the mucosa. Whenever the blastocyst happens to become
anchored at the bottom of a furrow or on the side of a fold, as
in the case of No. 316, it follows that a larger area of contact
is possible than when its union is established on the top of a
fold or on a perfectly smooth surface.

Although there is an extensive area of contact in the speci-
men under discussion, yet the place of fusion is indeed small,
and does not occupy a space any greater than that previously
covered by the Rauber's layer. The chorionic vesicle rests
upon a mass of trophoblastic tissue which is in the form of a
concave disc, with the concave side directed upwards, and with
the upper margin of the disc passing insensibly into the free
trophoblastic portion of the vesicle (fig. 31).

The disc or primitive placenta is comparable to the attach-
ment mass of rodents, and has been termed the Trager. In
the armadillo the Trager arises through the formation of the
syncytium in Rauber's portion of the trophoblast, followed by
a fusion of this syncytium with the surface layer of the mucosa.
The fused mass thus forms a bridge across which the embry-
onic nuclei can pass from the syncytium into the maternal
tissues, portions of which are soon destroyed by these invading
nuclei, doubtless as a result of their phagocytic or histolytic
action.

In specimen No. 316 the fusion is firmly established and sev-
eral embryonic nuclei have already penetrated well into the
mucosa epithelium (fig. 31, Em. N.). Several other nuclei are
on the point of passing into the mucosa. Evidences of the
histolytic properties of these foreign nuclei are everywhere
present in the maternal portion of the fused region, - and the
nutritive substances which result from the breaking down of the
maternal tissues must serve as an embryotrophe. The embryo-
trophic phase of placentation must last throughout a relatively
long period of the early development, because neither the ma-
ternal nor foetal circulation is established in the placenta until
the embryonic rudiments are well formed.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 621

In the next stage, which is but slightly older than No. 316, a
somewhat larger number of nuclei have passed over into the
uterine wall. They have almost completely destroyed the epi-
thelium, and have also affected the sub-epithelial layers (fig. 17).
The syncytium and adjacent thickened trophoblast together form
a layer which is coextensive with the germinal spot, that is, they
extend to the margin of the entoderm. In addition there is
found on the left wall, and somewhat removed from the rest of
the thickened extragerminal ectoderm, a distinct knot of cells
which must have resulted from the contact of a very small spot
of the trophoblast with the mucosa (fig. 17, K).

The next important change is a spreading of the base of the
vesicle. Instead of having but a small area of contact or point
of union, as in specimen Nos. 316 and 332 (figs. 31 and 17), the
blastocyst soon acquires a base, often greater in width than that
of the entire free portion of the vesicle (fig. 18). As a result,
the wall of the chorion, upon approaching the mucosa, instead
of curving inward, now slopes gradually outward, and merges
into the uterine tissue with which it has established a very
intimate union. The base forms an annular zone of thickened
trophoblast, which in width extends from the margin of the en-
toderm to the surface of the uterine wall. In its more distal
part the annular zone consists of thickened cells (two or three
cells thick), but its proximal part is much thicker and one finds
here a tendency to form a syncytium, continuous with the
syncytial-like mass lying directly beneath the chorionic vesicle.

In the latter region are found numerous embryonic nuclei,
which have destroyed the mucous epithelium, the connective-
tissue stroma, and even portions of the uterine glands. These
nuclei may be divided into two groups, one of which occupies a
superficial position; that is, its elements border on the extra-
embryonic cavity and apparently are inactive in so far as the
destruction of the mucosa is concerned; and the other consti-
tutes the deeper lying nuclei which are instrumental in dissolv-
ing the mucosa. At first this distinction is not very evident,
(figs. 18 and 32), but as development progresses the protoplasm
surrounding the surface nuclei becomes more and more dense

622 J. T. PATTERSON

(fig. 33), and there is gradually evolved a distinct epithelial
layer (figs. 33, 34, 64). Following the suggestion of Fernandez
('09) in his paper on Mulita, we may designate it the 'Trager
Epithelium/ on contrast to the Trager zone, or thickened an-
nular portion of the trophoblast which forms the base of the at-
tached chorionic vesicle. The Trager epithelium, together with
the layer of extraembryonic mesoderm which directly overlies it,
forms the lower wall of the chorionic vesicle.

In the meantime the deeper-seated nuclei continue to migrate
farther into the uterine tissue, which, as already stated, soon
becomes destroyed. During these changes the upper portion of
the syncytium begins to degenerate, as is evidenced by the appear-
ance of numerous vacuoles (figs. 34, 35). The vacuoles flow
together and eventually produce a shallow cavity which lies
just beneath the now well-organized Trager epithelium. The
final condition in the development of this 'sub-Trager cavity' is
well illustrated in the series of photographs in plate 7. All of
these show the condition just referred to, but figure 64 is par-
ticularly clear. Here the sub-Trager cavity is sharply defined
above by the Trager epithelium, but on the lower side it is con-
nected with numerous spaces which lie between the remains of
the partly dissociated mucosa.

Occasionally during the process of fixation the Trager epithe-
lium becomes pushed up into the extraembryonic cavity, carry-
ing before it the layer of mesoderm. Under such conditions it
appears as though the Trager epithelium was normally arched
(figs. 66-69). I am inclined to believe that a similar artificial
condition in the chorionic vesicle of Mulita has lead Fernandez
('09) to conclude that the Trager epithelium is normally pushed
up into the extraembryonic cavity, and that consequently the
cavity lying just below it is to be regarded as a true Trager cav-
ity. In view of the fact that Fernandez had only a few scatter-
ed stages at command, it was easy for him to be mislead into
drawing this conclusion. But the evidence which I have just
presented from a study of the development of this cavity makes
it doubtful whether it should be regarded as a Trager cavity, in
the sense in which that term is used in rodents. For that

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 623

reason I have preferred to call it by the name of sub-Trager
cavity.

A further examination of the figures on plate 7 will show that
at this stage of development the blastocyst is held to the muc osa
by the Trager zone alone, so that in order to free it from the
uterine wall it is merely necessary to cut this band of tissue at a
short distance beyond the edge of the vesicle.

The transition from the Trager stage or primary placenta to
that of the secondary placenta, or a condition in which distinct
villi are present, is very gradual. The first villi arise from that
portion of the chorionic wall which corresponds to the Trager
zone; that is at the attached margin of the chorionic vesicle.
From here they successively appear further and further toward
the central area of the Trager epithelium, until its entire surface
becomes studded with them. However, the villi in the central
region do not become so long or so highly developed as those that
are situated towards its peripheral parts.

Each villus starts as a thickening in the Trager epithelium,
and soon becomes a mass of cells protruding from the epithelial
surface. At first it is flat or disc-shaped and seems to serve as an
adhesive pad, but later it elongates into a true Trager cord,
which may become very much branched. These cords later
become invaded by a stroma-like mesenchyme, developed from
the mesodermal epithelium which directly overlies the Trager
epithelium.8

In order to understand the changes which take place in the
upper free portion of the chorionic wall it is necessary to recall
that all of the trophoblast lying above the margin of the ento-
derm sooner or later sloughs off, leaving only a fragment-like
base, which in section can often be seen protruding from tne
side of the vesicle at a short distance above the uterine surface.
This fragment is clearly shown in all of the figures of plate 9.

The portion of the trophoblast which thus breaks away cor-
responds to the chorionic ectoderm in the vesicle of other mam-

8 For a more detailed account, and for figures of the structure of the villi, see
Newman and Patterson, '10.

624 J. T. PATTERSON

mals, but in the blastocyst of the armadillo its loss results in
bringing the yolk-sac entoderm to the outer surface. From now
on the increase in size of the blastocyst is evidently brought about
through the growth of this entodermal portion of the wall of
the ovum, as can be determined by comparing successive
stages. But after the vesicle has attained a diameter of from
4 to 5 mm. (fig. 5) the entodermal portion does not expand to
any great extent, and the chorionic vesicle owes its further growth
to the extension of the Trager or placental portion of the cho-
rionic wall.

As the Trager area increases in extent the yolk-sac is carried
farther and farther toward the cervix end of the uterus, and in the
advanced stages of gestation it forms a cap at the tip of the cer-
vix end of the chorionic vesicle. However, it becomes partly
covered by an overgrowth of Trager tissue, which arises at the
boundary line between the yolk-sac and the Trager, and extends
posteriorly as a free margin which later becomes fused with the
wall of the cervix. In vesicles which contain 30 to 35 mm.
embryos, the yolk-sac portion of the chorion protrudes through
the thickened ring-like, placental overgrowth as a clear trans-
parent membrane.

If a chorionic vesicle from which the chorionic ectoderm has
already disappeared be examined, it is found that the entoderm
is attached to the upper edge of a mass of Trager tissue which
lies just inside the basal fragment of trophoblast (figs. 70-74).
In tracing back the origin of this mass one finds that it arises as
a thickening on the inner surface of the Trager zone, just below
the margin of the entoderm. It can be recognized in very early
stages as a small mass of actively dividing cells which lie in the
angle formed by the Trager zone and the potential Trager epithe-
lium (fig. 55, on left). The thickening gradually increases in
volume (figs. 33, 34, m), and apparently involves the entire inner
surface of the Trager zone, and thus comes to form at its crest a
fusion with the margin of the entoderm (fig. 23, on right). At
the same time the outer layer of cells of the Trager zone becomes
split off from the mass (fig. 23, on left), and thus forms the basal
fragment when the chorionic ectoderm breaks awa;y.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 625

The mass of Trager tissue constitutes the material upon which
the further growth of the chorionic wall depends, and after the
4 to 5 nri nn. stage is reached it rapidly extends upward, becomes
thinner, and carries before it the cap of the yolk-sac entoderm,
in the manner already described. It also forms the basis for the
formation of the villi which appear upon this region of the cho-
rionic wall. At first the villi have a general distribution over the
surface of the modified Trager zone, but hi blastocysts which have
attained a diameter of 25 to 30 mm., the long branched villi become
restricted to four distinct areas or patches. These patches are
situated just below the boundary line between the Trager and the
yolk-sac, and each villous area lies opposite the point at which an
umbilical cord arises from the inner surface of the chorionic wall.
The four villous areas give rise to the four placental discs of later
stages, and, corresponding to the position of the embryos, are ar-
ranged into two pairs. In the final stages of gestation the two
discs belonging to a pair become closely associated together, thus
forming two large double-discoidal placenta, which occupy respec-
tively the right and left sides of the chorion.

In the formation of the placenta, as well as hi the general devel-
opment of the blastocyst of the armadillo, there are many
opportunities for comparison with the developmental stages of
other mammals, but such comparisons can be more safely drawn
after 'we have had a chance to work out a detailed history of
placentation.

GENERAL DISCUSSION
1. Theories of polyembryony

A great many different views have been expressed in explana-
tion of polyembryonic development. Most of these are pure
conjectures, and as such hold no place in any serious attempt at
a scientific treatment of the subject. Exceptions are made here
to those theories only which seem to hold a grain of truth and
which have gained a certain number of adherents.

a. Theory of polyovular follicles. An attempt has been made
to account for polyembryony on the basis of polyovular follicles.

626 J. T. PATTERSON

Since it was known that the mammalian ovary occasionally
possesses such follicles, it was natural to suppose that this fact
might furnish a clew to the problem of polyembryony. In fact,
Rosner ('01) not only made this claim, but he also attempted to
prove his point by a study of the ovaries of the South American
variety of the very animal with which this paper deals, namely,
the nine-banded armadillo. Rosner studied a single pair of
ovaries from a pregnant female sent him by von Jhering. He
found fifty-two polyovular follicles, as follows: one with seven
ova, one with five, two with four, seven with three, and eleven
with two. Rosner believes that the condition of multiple em-
bryos in the armadillo was to be explained by assuming that
four young, adjacent follicles fused together, and that the four
ova thus brought within a single cavity were later ovulated and
fertilized, and held together in such a way as to produce event-
ually the quadruplex foetal structure characteristic of the arma-
dillo pregnancy.

Aside from the fact that only two out of the fifty-two poly-
ovular follicles observed by Rosner possessed the requisite number
of ova to account for the four embryos of each armadillo litter,
it has since been abundantly proved (Cuenot '03, and Newman
and Patterson '10) that the pair of ovaries studied by Rosner was
quite exceptional. Further, sufficient literature has accumu-
lated to show that the phenomenon of polyovular follicles occurs
in several widely separated species of mammals, and consequently
can have nothing to do with polyembryonic development.

Schron ('63) seems to have been one of the first to record the
occurrence of polyovular follicles. He observed them in the
ovaries of the cat. Since then they have been reported in various
Eutheria, as follows: in the human by Nagel ('88), Schott-
lander ('93), Stoeckel ('98), Rabl ('99), and Arnold ('12); in dogs
by Waldeyer (70), Wagener (79), Bouins ('00) and Smyth ('08);
in cats by Rabl ('99) ; in bats by Van Beneden ('80) ; in rabbits
by Wagener (79) and Honore ('00) ; in the armadillo by Rosner
('01). In addition to these records on the Eutheria, O'Donoghue
('12) has very recently added the Marsupial, Dasyurus viver-
rinus.

POLYEMBRYOXIC DEVELOPMENT IN TATUSIA 627

These citations will suffice to indicate how widespread is the
occurrence of polyovular follicles among mafrimals; but, although
they have a distribution among widely separated forms, their
occurrence in a given species seems to be rare. Thus O'Donoghue
('12) found them but twice in forty-five individuals of Dasyurus;
and Schron ('63) only twice hi the ovaries of four hundred cats
and but once in eighty dogs. The writer has examined in all
probably more than fifty pairs of ovaries from the Texas arma-
dillo without finding a single case.

In the light of these data it is impossible to associate the occur-
rence of polyovular follicles with the causation of polyembryonic
development in mammals. The fact that Rosner has found a
single armadillo with such follicles can have no greater impor-
tance than have the similar sporadic cases in certain other mam-
mals. The significant fact is, rather, that hi the ovaries of fifty
individuals belonging to a species which reproduces by specific
polyembryony alone not a single case of polyovular follicles
was found.

It is well to emphasize the fact that the polyovular follicles do
not lie at the basis of polyembryony, for to accept Rosner's
theory would be equivalent to a denial altogether of the phenom-
enon of pohrembryonic development hi the Mammalia. A
multiple gestation from ova which have accidentally become
associated together during a part of the ovarian history, through
the fusion of .adjacent follicles, may have no more interest or
significance than a similar gestation resulting from ova from
uniovular follicles, but simultaneously ovulated, as occurs in
many mammals that are normally multiparous. One cannot
hope to throw much light on such fundamental biological problems
as those of sex determination, the limits of hereditary control,
and others, by the study of this type of development. It is only
to those cases in which the several embryos of a multiple preg-
nancy have taken their origin from a single fertilized egg that we
must look for facts with which to elucidate these problems.

It is not intended here to underestimate the biological impor-
tance of polyovular follicles and of multiple gestations other
than those of polyembryony; but rather to point out that these

628 J. T. PATTERSON

phenomena have an interest lying along a different line. In
fact there is considerable evidence to indicate that polyovular
follicles and such multiple gestations are to be correlated as cause
and effect. This applies not only to cases of multiple pregnan-
cies among forms that are normally uniparous, but also occa-
sionally to cases in animals that are normally multiparous. Ac-
cording to Wilder ('04) von Franque ('98) was the first to start
the discussion which has led up to the conclusion that polyovu-
lar follicles bearing two eggs might result in the origin of twins—
not compound monsters or duplicate twins, but to fraternal twins,
as Wilder points out, since the eggs must be fertilized by different
spermatozoa.

The theory that polyovular follicles may account for 'fraterni-
ties' of this sort receives considerable support in the case of a dog
reported by Smyth ('08). In 1906 he obtained a young setter
pup from a litter of fourteen pups (four dogs and ten sluts) born
to a Gordon setter. When the pup was ten months old she was
spayed for reasons of convenience, and upon preparing the ovaries
in sections it was discovered that not a few of the ripe follicles
held double and triple ova. The ovaries from one of the other
pups were also sectioned, and they too possessed polyovular
follicles, one containing as high as seven ova. In 1907 one of
the sluts from this same litter gave birth to nine pups, three
dogs and six sluts. It is a great pity that the ovaries from this
bitch were not studied, as well as those from her mother. But,
even though the data are not as complete as might be desired,
still they point unmistakably to the fact that a tendency to poly-
ovular follicles was inherited in this family of dogs; and, further
more, they suggest that the unusually large litters of the mother
and her daughter might in part be accounted for on the basis of
compound follicles.

It is a well-known fact that other normally multiparous animals
sometimes show a tendency to bring forth very large litters.
While it is of course possible that in such cases all of the ova
belonging to a given litter may come from simple follicles, yet
it is not improbable that some of them may come from polyovular
follicles. There is but one way in which it would be possible to

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 629

settle this question satisfactorily, and that is to study histologi-
cally the ovaries of several generations in the family of some
animal belonging to a species normally bearing a single young,
but showing a tendency to multiple births. If polyovulated fol-
licles were present in those individuals which had had multiple
births and if the number of corpora present in the ovaries after
a given pregnancy were found to be fewer than the number of
young in the litter, it would be reasonably certain that the mul-
tiple gestations were the result of ova from a compound follicle.
Some of the cases in cattle recently cited by Pearl ('12) would
have made excellent material for such a test.

I have discussed the topic of polyovular follicles somewhat in
detail in order to emphasize the necessity of keeping it entirely
distinct from the subject of polyembryony, for not to do so will
most certainly lead to great confusion. This applies not only to
the problem of sex heredity, but also to other questions in hered-
ity that are capable of elucidation through the study of poly-
embryonic development, particularly the one dealing with the
limits of hereditary control. It is here essential, as I have already
pointed out, that all of the individuals of a litter of embryos to be
studied should have the same germinal constitution; but this
condition would never be fulfilled in multiple gestations resulting
from ova from a polyovular follicle, even if this could be proved
beyond question, and even though all the ova came from a single
mother cell in oogenesis; because in that event each egg must be
fertilized by a different spermatozoon.

b. Theory of blastotomy. According to this theory each embryo
is looked upon as the lineal descendant of one of the early blasto-
meres. In the case of two embryos arising from a single egg
(identical twins) it has been supposed that each individual is the
product of one of the blastomeres of the two-celled stage, while
in the case of four embryos (armadillo quadruplets) it has been
assumed that each embryo arises from one of the blastomeres of
the four-celled stage. And so it has been argued for those cases
in which even a greater number than four come from one egg.

The idea of an early spontaneous blastotomy lying at the basis
of polyembryony has been very persistently urged by a number

630 J. T. PATTERSON

of investigators, and in the earlier work on this armadillo the same
view was held. This idea undoubtedly has its inception in, and
has received most of its support from the results of certain ex-
perimental studies involving the mechanical or semi-mechanical
separation of early blastomeres. For it has been demonstrated
that an early isolated blastomere of the two-celled or four-celled
stage of the eggs of the echinoderm (Driesch '92), medusa
(Zoga '95-6), Amphioxus (Wilson '93), and teleost (Morgan '93)
may develop into a complete but small larva, and even in the egg
of the amphibian a blastomere of the two-celled stage, under
certain conditions (Schultze '95, Morgan '95, and Herlitzka '97)
may also develop into a complete organism. What then seemed
more logical then to conclude that in the case of polyembryonic
development the early blastomeres had in some way become
displaced or isolated, and that each cell thus separated formed
the center for a single individual. Moreover, this inference
seemed all the more plausible in the light of certain studies on
twins in the human species. Wilder ('04) in particular, in his
extensive paper on duplicate twins and double monsters, advo-
cates the theory that each member of a pair is the product of
one blastomere.

The evidence that has been presented in the first part of this
paper makes it certain that polyembryonic development in the
armadillo cannot be explained on the basis of a spontaneous
blastotomy, in the sense that each embryo is the lineal descen-
dant of a single blastomere of the four-celled stage, and it causes
one to view with some doubt the conclusions of this same nature
that have been drawn by those who have worked on other poly-
embryonic forms. In this connection it should be kept in mind
that, although an equipotentiality seems well established for
the early blastomeres of the eggs of the echinoderms, medusa,
amphioxus, teleost, and others, yet there are many forms in
in which a blastomere does not have the power to develop into
a whole individual. Crampton ('96) on gasteropods, and Chabry
('87) and Conklin ('05, '06) on ascidians have conclusively demon-
strated that a blastomere of the two-celled or four-celled stage

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 631

in these forms develops essentially in the same manner as though
producing a part of the whole embryo.

It is not intended to deny that influences of a mechanical nature
may not, in certain cases, lie at the basis of multiple-embryo
formation. Any one advocating such a theory may bring to
his support not only the facts of artificial blastotomy, but also
those derived from experimental studies on later development,
like those of the pioneer work of Haeckel ('69) on the blastulae
of Crystallodes and of the more recent and well-known studies
of Spemann ('01, '03) on the triton egg. This rather simple
mechanical or semi-mechanical explanation might hold in the
sporadic cases of polyembryony, like those of duplicate twins
and double monsters, but what evidence have we that blastotomy
operates in the case of specific polyembryony in higher forms?
As yet we know very little about the details of the early develop-
ment in such cases. It is a significant fact that such evidence
as we do possess does not support the theory, and it is certainly
true that these studies on the armadillo — a form in which we
have a most striking case of specific polyembryony — have not
revealed any evidence which tends to support the blastotomy
theory.9 On the contrary, the evidence points unmistakably to
a different explanation, namely, that a type of budding lies at
the basis of polyembryony in this form.

c. The theory of budding. The process of budding is a very
common method of reproduction among organisms. In plants
it is practically universal, and in animals it is frequently met with,
especially among the lower forms. In many cases asexual repro-
duction by budding occurs late in the life cycle, as for example
among coelenterates. In such forms as the common Hydra it
is customary to regard the organism as an adult when budding
begins. But the appearance of budding is by no means confined
to adult organisms, or even to late stages of development, for it
may appear \iery early in the life cycle.

9 The term 'spontaneous blastotomy' has been used by Bugnion and Marchel
to describe the process of polyembryony in the parasitic Hymenoptera, but not
in the sense that each embryo can be traced to a single blastomere. Brandes ('98)
has suggested the term 'Germ'mogonie' in lieu of polyembryony.

632 J. T. PATTERSON

In the earthworm, Kleinenberg ('79) has described a gemelli-
parous development occurring in the gastrula stage and initiated
by a sort of fission or budding. In the Cyclostomatous Bryzsoa
(Harmer '93, Robertson '03) a. primary embryo, prior to the for-
mation of germ layers, buds off a large number of secondary em-
bryos which differentiate into larvae. In the parasitic Hymen-
optera (Marchal '04, and Silvestri '06) the differentiation of the
embryos occurs relatively still earlier and consists in the disso-
ciation of the egg into embryonic masses, which vary in number
according to the species, and which later form larvae. In the
light of artificial blastotomy it is possible, theoretically, to have
the process of dissociation carried still further forward into the
cleavage stages — even to the two-celled stage; but so far as the
writer knows, the ocurrence of blastotomy as early as the two-
or four-celled stage has never been observed as a natural phenom-
enon in development.

From among the several forms having polyembryonic develop-
ment, one can select a series in which embryonic fission or budding
is carried farther and farther toward the adult stage, and as
Marchal has observed, one can pass insensibly from these cases of
budding in the egg to the more frequent and well-known phenome-
non in which the budding occurs after the individual has already
come from the egg, as, for example, in the coelenterates, Ortho-
nee tida, Dicyemida, platyhelminthes and tunicates. We may ask
then, below what point in the developmental cycle must one
cease to speak of asexual reproduction as budding, and refer to
it as polyembryony? Evidently a sharp line cannot be drawn
between the two.

It is best to regard polyembryony as a precocious type of bud-
ding; and this, perhaps, only in the sense that it occurs early in
the embryonic life, and without the implication that it has push-
ed forward in the life cycle or superseded a budding which in the
ancestral froms occurred at a late period of development. This
would seem to be the case at least in the Polyzoa, in which the
embryonic budding is followed in the sessile larval stage by the
typical budding to produce the colony.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 633

In the armadillo the facts of development which we have pre-
sented in the descriptive part of this paper are fully in accord
with the theory of budding. We have seen that the early phases
of differentiation are similar to those of other mammals in which
the ovum produces but one embryo. Prior to the appearance of
the embryos, the ectoderm, the entoderm, and the exocoelomic
mesoderm are differentiated, and later all three of these layers are
concerned in the formation of the embryonic buds. The initial
step in budding apparently occurs in the embryonic ectoderm, but
the entodermal layer is soon involved in the process. It is there-
fore entirely correct to say that the seat of budding in the arma-
dillo is to be found in the blastoderm, that is, the blastoderm i$
the budding organ. It may be possible to extend this same con-
ception to accidental or sporadic cases of polyembryony occurring
in the lower forms which lay yolk-laden eggs.

The most important point brought out in this study is the fact
that polyembryonic development in the armadillo can be inter-
preted as a type of budding; and, while to show that polyembry-
ony is a budding process does not solve the question as to the
determining cause of the division of the blastoderm, yet it is a
distinct step toward the solution of that important problem.

It is perhaps premature to attempt an explanation of the ulti-
mate cause of polyembryony. We first need a comprehensive
study of each of the forms in which it occurs. Such investiga-
tions, followed by well-directed experiments, may yield results
that will reveal at least some of the factors which control poly-
embryonic development. At present only a few suggestions
need be made; and, first, we may briefly consider the ideas that
have been expressed by some of those who have worked on the
subject.

Harmer ('93), in his excellent paper on embryonic fission in
Bryozoa, points out several interesting comparisons that can be
drawn between the process of multiple-embryo formation in
Crisia and budding in many other organisms. He calls atten-
tion to the fact that, in at least some of these forms, embryonic
fission is connected with the deviation from the normal type of

634 J. T. PATTERSON

segmentation of the egg. Furthermore, he points out that the
early blastomeres of the egg of Crisia are separated from each
other by follicle-tissue, and that they are surrounded by a rich
nutritive material, which is obtained through the protoplasmic
strands connecting the several units of the colony with the ovi-
cell. He believes that the production of numerous larvae from
the primary embryo in Crisia is a process comparable to arti-
ficial blastotomy in Echinus eggs, as shown by the experiments of
Driesch ('92). His general conclusion is clearly stated in the
closing paragraph of his paper. He says :

TJie cases already quoted may be taken as showing that some of the
abnormalities in the development of Crisia may be due to the nutritive
conditions in which the development takes place. Just as the presence
of food-yolk within the egg modifies the character of the segmentation
and the formation of the layers, so the presence of copious stores of nu-
trient material in the maternal tissues outside the egg may also effect the
early developmental processes. Thus the large number of relatively
large larvae which develop from the minute egg of a Crisia could not be
produced if the egg were not supplied with nutriment from outside it-
self. While some of the irregularity in the segmentation of the egg may
be due to this cause, the extreme independence of the blastomeres at an
early stage may be connected with the acquirement by the embryo of a
habit of forming buds in the embryonic condition.10

Marchal ('04) has expressed somewhat similar views, as may
be gathered from the following quotation.11

As to the determining cause of the division of the germ, Marchal
thinks that it is from the sudden surrounding with more dilute liquids in
the interior of the nourishing mass and in a concomitant modification of
the osmotic exchanges in the interior of the cellules. One sees, in
fact, with Encyrtus that polyembryony reaches its greatest intensity at
the moment when the larvae of the Hyponomeuta commences to feed
(in the early days of April), and for the Polygnotus at the period when
the young larva of the Hessian fly engorges itself with sap. Now, the
production of the rapid changes bringing about osmotic pressure con-
stitutes precisely the procedure employed to bring about the separation
of the blastomeres and their evolution into several distinct individuals,
as has been shown by the experiments already mentioned of Loeb and
Bataillon.

10 Loc. cit., p. 236.

11 From Howard's ('06, p. 816) clear translation of Bugnion's ('06) review of
Marchel's ('04) paper.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 635

Moreover, in connection with his study on Polygnotus, Mar-
chal observed that the polygerm is moved back and forth in the
digestive tract as a result of contractions of the wall of the host.
He believes that this movement is analogous to the shaking of
Echinus eggs, and has a similar influence upon the division of
the germ.

In the preliminary paper (Patterson '12) similar views were
given, but expressed in a somewhat different way. It was stated
that in all of the well known cases of polyembryony the cleavage
of the egg is of the 'indeterminate' type, so that it was impossible
to trace out a 'cell-lineage' for any particular embryo. It was
also stated that the primary embryo or polygerm led a sort of
parasitic existence, and that as a consequence it was surrounded
an abundance of nutritive substances.

The cleavage of the mammalian egg is generally regarded as
belonging to the indeterminate type, and, although the cleavage
stages of the armadillo have yet to be studied, still we have no
reasons for believing that they will be found to differ from those
of other mammals; and if we may judge from the conditions of the
earliest stages of the blastocyst that have been examined, there
is no evidence to show that the early blastomeres have been
separated by foreign nutritive substances. The development of
the embryonic vesicle until the germ layers are differentiated can
be compared to that of certain other mammals. However, it
is a significant fact that at the close of the period of germ layer
formation the embryotrophic phase of placentation, which is par-
ticularly striking in the armadillo, becomes well established. It
may be that the nutritive substances produced by the action of
the embryonic nuclei upon. the maternal tissue furnish the stim-
ulus which excites the blastoderm to bud off the embryonic tubes,
just as the engorged sap of the Hessian fly is suggested by Mar-
chal to be the determining cause of the division of the germ of
Encyrtus. If this point be well taken, it is evidently not neces-
sary to assume that the time of stimulation to polyembryonic
development dates back to the early cleavage stages.

In this*connection it might be well to call attention to another
suggestion that has been made. It is generally believed that in

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

636 J. T. PATTERSON

coelenterates, ascidians, and Polyzoa the germ cells antedate the
formation of the buds in which they are found; and Montgomery
('06) has gone so far as to suggest that perhaps in all cases the
products of asexual reproduction contain germ cells. He sug-
gests that "If this were so, it might then be the case that the
incapacity of any part of the body of an animal to reproduce
asexually or even to regenerate, would be due to the absence of
germ cells in it. " It may be possible to explain in this way the
appearance of rudimentary embryos that have been observed in
both species of armadillo, or even, indeed, to account for the £o-
called asexual larvae in Litomastix. Such abortive attempts to
produce normal organisms may be the result of a failure on the
part of a bud to receive germ cells.

2. Origin of polyembryony in the Dasypodidae

One of the interesting problems which presents itself in con-
nection with this study pertains to the question of the origin of
specific polyembryony among the Dasypodidae. The question
becomes all the more interesting, from the standpoint of the physi-
ology of reproduction, in view of the fact that the uterus of the
armadillo is of the simplex type. The same type of uterus in
other mammals is adapted to the function of receiving and nour-
ishing a single ovum, although occasionally it may receive and
nourish two or more ova at the same time, in which event a multi-
ple birth results ('frajternal twins/ 'triplets/ etc.). In the arma-
dillo the uterus also receives a single ovum at a time, but instead
of but one embryo arising from the egg, polyembryonic develop-
ment comes on and increases the number of offspring to several
individuals. Here we have a clear case of adaptation, in which
the productivity of the ovum of a uniparous mammal is increased
several fold.

Two quite distinct problems are presented. One of these is the
most fundamental question of the whole problem of polyembry-
ony, namely: What are the causal factors underlying the forma-
tion of two or more individuals from a single egg? The other
problem concerns the phylogeny of specific polyembryony in

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 637

the group of armadillos. It must be stated frankly that both of
these questions are wholly unanswered. But a few suggestions
are given in the hope that they may point the way to their solu-
tion. In the remaining part of this section we shall discuss the
question of phylogeny as the other problem was discussed in the
preceding section.

So far as I am aware, the only armadillos in which specific poly-
embryony is definitely known to occur are the North and South
American varieties of T. novemeincta, and T. hybrida, which
inhabits the southern part of the South American continent.
Judging from the description of Fernandez ('09) on comparatively
late stages of T. hybrida, the two species agree very closely in
many details of development; but there are evidently important
differences, one of which relates to the number of offspring in a
litter. In T. novemeincta a litter consists typically of four indi-
viduals, while the number in T. hybrida varies from seven to
twelve, with a strong tendencey to produce eight or nine.

In T. novemeincta, out of 219 pregnant females that have been
studied carefully, 176 had embryonic vesicles far enough advanc-
ed to permit a determination of the number of embryos, and of
these all but four, or about 98 per cent, possessed four embryos
each. Of the four exceptions, three had five-embryo sets and
one a two-embryo set.

There is some doubt in the latter case, since the embryos were
born in the laboratory and therefore an opportunity to study them
in utero was not offered. But all of the five-embryo sets were
carefully studied and the relations of the different parts of the
blastocyst were determined. In each case two members of the
litter (either on the right or on the left side) showed the normal
paired condition, while the other three presented an asym-
metrical arrangement, one of the primary placental discs being
about twice the usual size and bearing the umbilical cords of two
embryos.

This arrangement of the members of a five-embryo set is signi-
ficant, in that it suggests that the two embryos arose in the nor-
mal fashion from one of the primary buds, and that the three
embryos of the opposite side have come from the other primary

638 J. T. PATTERSON

bud, either directly, through the formation of three secondary
buds from it, or through a further division of one of the two
secondary buds, after they had been formed in the normal way.

It is to be regretted that the rare case of a two-embryo set,
referred to above, was not studied before birth occurred, as it
would doubtless have been found that the embryos occupied the
right and left sides of the chorionic vesicle, thus indicating that
each primary bud had been directly transformed into a single
individual. In that event, we should have had very strong evi-
dence in support of our contention that specific polyembryony in
the Dasypodidae began by the formation of a set of twins, per-
haps at first as sporadic cases of gemelliparous development such
as probably occurs in the production of duplicate twins in the
human species.

However that may be, I have recently descovered certain evi-
dence in the early development of the Texas armadillo which
strongly supports this view concerning the origin of specific poly-
embryony in the Dasypodidae. It was pointed out in an earlier
section that when the secondary buds first appear, Nos. II and
IV apparently arise from the tips of the primary buds, as though
they were merely prolongations of these buds; while each of the
other secondary buds evidently arises slightly to one side of tip
of a primary bud. That is to say, that Embryo I always arises
to the left of its paired mate No. II, and likewise Embryo III to
the left of IV (fig. 2). This may be expressed in another way
by saying that buds I and III are outgrowths from the primary
buds, and that consequently they follow chronologically the
development of buds II and IV. The evidence upon which this
interpretation is based is to be seen in several of the young
blastocysts.

It has been pointed out elsewhere that the first sign of a secon-
dary bud appears on the right side of the left-hand primary bud
in blastocyst No. 247 (fig. 1). The other embryonic bud is the
result of a prolongation of the extreme tip of the primary diver-
ticulum. In figures 2, 3, 4, and 28 is seen further evidence of
this same difference in the size of the two members of a pair of
embryos, and it is also evident in the sections (figs. 67, 77).

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 639

Furthermore, it was observed in at least three living specimens, so
that there can be no doubt but that during the early history
of the embryonic buds, Nos. II and IV are more highly devel-
oped than their mates I and III. And what else can this mean
than that phylogenetically the former pair should precede the
latter.

This obvious difference in size does not last indefinitely, and
indeed is no longer discernable after the embryonic rudiments
have become slipper-shaped (fig. 5) .

That primary buds may also be precursors of secondary buds
hi Mulita is, I believe, to be inferred from the work of Fernandez
('09) on that form. Unfortunately, he has but a few scattered
stages at his command, and therefore was not able to give us a
full history of the development of the embryonic rudiments.
His specimen 46, which is the one next to his youngest stage
(corresponding to Specimen .233), already has embryonic rudi-
ments fully as well developed as those in Specimen No. 226 (plate
11). Nevertheless, in speaking of the relation which exists be-
tween the ectoderm of the common amnion and that of the
embryonic diverticula in this specimen, he makes the following
significant statement, "Der Cervix uteri zu, d.h. tiber dem Anfang
der Medullarplatte, ist das Amnion keines Tieres geschlossen, as
steht vielmehr durch eine sehr weite Offnung mit dem Amnion
eines oder mehrerer Nachbartiere in vollkommener Kommunika-
tion. ' '12 Again in the same paragraph he says, ' ' Das Amnion eines
Einzelembryo kann sich auch direkt in diese Blase offnen, ohne
vorher mit den Amnia andrer Embryonen in Verbindung getreten

zu sein. "

If we pass from these two statements to an examination of his
figures 1 and 2 (plate 17), which are photographs of vesicles show-
ing well-formed embryos, we shall find further evidence of this
same nature. The specimen shown in figure 1 is from a vesicle
containing 11 embryos, six of which appear in the photograph.
On account of the advanced stage of development, the evidence
that two embryos have come from a common diverticulum must
be sought in the relation of the amniotic canals to the common

12 Loc. cit., p. 314.

640 J. T. PATTERSON

amniotic vesicle. Curiously enough it is found in two or three
instances that the canals of two embryos unite and enter the vesi-
cle as a single tube, and in one case at least three canals so unite.
His figure 2 presents a still more striking case. It contains 9 em-
bryos, and the embryo lying at the top of the figure, and slightly
to the right of the center, sends its canal directly to the vesicle,
while the canals from the four embryos on the right enter the vesi-
cle very close together, two of them by a common tube. Like-
wise, the canals from the four embryos lying on the left enter the
vesicle at a common point. Indeed, they apparently unite just
before reaching the vesicle.

In view of the fact that we have shown that the union of two
canals in T. novemcincta is a certain indication of their common
origin from a primary bud, I believe we are justified in drawing a
similar conclusion from the conditions to which we have just
called attention "in T. hybrida. And I venture to predict that
when Fernandez shall have secured intermediate stages, he will be
able to confirm this conclusion.13

It does not follow, of course, that in Mulita only two primary
buds will be found -to appear, for while in T. novencincta the poly-
embryonic process in the ovum is* extremely stable, as expressed
by the constancy with which litters of quadruplets appear, in T.
hybrida, on the contrary, variability characterizes this process.
Hence, there are no good reasons why in this species, regions on
the ectodermal vesicle which correspond to those unoccupied by
the two primary buds in the vesicle of novemcincta might not
give rise to new primary diver ticula. If this be found to be the
the case it would in no wise nullify our conclusion regarding the
origin of polyembryony among the armadillos; that is that it
began in the ancestors by the formation of twins. Whether all
of the species which might show such a primitive condition are

13 After the above was written my attention was called to a report, in the Jour-
nal of the Royal Microscopical Society, June 1913, page 279, of Fernandez's commu-
nication at the Ninth International Congress of Zoologists. From the brief state-
ment given it seems clear that Fernandez has observed exactly the same method of
embryo formation in Mulita that I have described for T. novemcincta, that is, the
embryonic primordia arise as diverticula. He states that the diverticula "become
the primordia of embryos, either directly or after division.'' (Italics my own).

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 641

now extinct is, of course, not known; but it can be stated that
some of the living species, other than T. novemcincta and T.
hybrida, are known to give birth sometimes to a single young,
and at other tunes to two individuals.

3. Polyembryony and duplicate twins and double monsters

Another interesting problem with which polyembryony is con-
cerned is that of the origin of duplicate twins in the human species.
There is a vast literature bearing on duplicate twins and various
types of double monsters, and several different theories have been
advanced to account for their production. The reader is referred
to Wilder's ('04) extensive paper in which the more important
references are cited, and hi which the leading theories are dis-
cussed.

It is well at first to distinguish between the different kinds of
twins, 'duplicate twins' and ' fraternal twins', understanding by
the former those cases in which the two members of a pair are
supposed to have come from a single egg, and by the latter those
supposed to be the product of two distinct eggs. This distinction
is by no means an artificial one, but is based upon a considerable
amount of data. It is supported not only by the general physi-
cal appearance of the members of a pair, but also by the intra-
uterine relationships of the two members. In fraternal twins
the two individuals do not resemble each other any more closely
than do the several individuals of a litter belonging to a normally
multiparous animal, and the intra-uterine relationship of two
chorions indicate that their origin is from two distinct eggs. In
duplicate twins the individuals are enclosed within a single cho-
rion, and their close resemblance to each other is often so striking
that it has gained for them the name of ' identical twins.' Fur-
thermore, duplicate twins are invariably of the same sex, while
fraternal twins may be of the same or of different sex.

Wilder points out that this fundamental principle upon which
the distinction between duplicate and fraternal twins is based,
also holds in multiple births involving more than two individuals,
and that it can be extended to include cases of duplicate twins
and similar combinations in other forms.

642 J. T. PATTERSON

As to the origin of duplicate twins, Wilder advocates the
blastotomy theory, believing that it is the result of a total sepa-
ration of the first two blastomeres of the single egg. In case the
blastomeres fail to separate completely, symmetrical double
monsters (dislopagi) result. In this connection he says, "The
double monsters of which we have authentic record are sufficiently
numerous and diverse to represent every stage from that of the
otherwise normal individual with a doubling of certain of the
median parts, to that of two complete duplicate twins with a
slight connection between them."14 Finally, be believes that
unequal duplicate monsters (autosite and parasite) are the result
of a secondary fusion (due to the great contiguity) of two embryos
which were at first duplicate twins.

The contention that duplicate twins and double monsters arise
from a single egg is undoubtedly sound, but* the conclusion that
their origin is the result of a complete or a partial separation of the
first two blastomeres, is, I believe, open to question. However,
in the absence of any study in the early stages of these sporadic
cases of polyembryony, and in view of the results from experiments
on artificial blastotomy, this conclusion seemed both natural
and logical. In the s-tudy of the embryology of the armadillo we
have a most excellent opportunity to put the blastotomy theory
to test; for here is a mammal with a simplex type of uterus, and
one which habitually reproduces by polyembryony. I am there-
fore bold enough to suggest that the conclusions which I have
drawn concerning the origin of the embryos in this mammal may
also apply to cases of duplicate twins and double monsters in the
human species. And I am encouraged to make this suggestion be-
cause of the recent discoveries in the human ovum (e. g. the Bryce
and Teacher ovum, '08), in which the condition of the ectodermal
vesicle is shown to be such as to require no great stretch of the
imagination to picture how diverticula might arise from it, and
thus initiate the development of two or more embryos. Nor is
there any greater mental strain in accounting for the origin of
composite monsters in this way than is required in the hypothet-

14 Loc. cit., p. 462.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 643

ical juggling of blastomeres to account for the various relation-
ships and positions assumed between the components of these
monsters.

4. Polyembryony and sex

One of the obvious biological bearings that the study of poly-
embryonic development has revealed concerns heredity, includ-
ing the heredity of sex. The illuminating studies of McClung
('03), Stevens ('06), Wilson ('05-'12), Morgan ('09) and many
others, including the recent excellent paper on Ancyracanthus by
Mulsow ('12), have shown that in a large number of animal forms
the heredity of sex is in some way bound up with certain co-called
sex chomosomes, and that, as a consequence, the sex of a given
individual is irrevocably fixed at the time of fertilization, or in the
case of an. unfertilized or parthenogenetic egg, not later than the
time the e'gg starts to develop.

It is in this connection that polyembryonic development fur-
nishes a very strong confirmation of the modern cytological view
of sex heredity. For in every authenic case of polyembryony
among dioecious species all of the individuals arising from one
egg are invariably of the same sex; that is they are either all males
or all females, never mixed. The most logical conclusion that
can be drawn from these facts is that the sex character is stamped
upon the egg prior to the origin of the several individuals to which
it gives rise. That the sex of the egg is determined as early as
the time at which development begins, seems certain in the case
of parasitic hymenoptera. It will be recalled that Silvestri ('06)
has shown in Litomastix (and he thinks that is is almost cer-
tainly true in Ageniaspis) that the fertilized egg produces females
only, while the unfertilized egg gives rise to males only, exactly
as in the well known case of the bee. Here fertilization or the
lack thereof determines the sex of the offspring, and, no matter
how many individuals the egg may produce by polyembryonic
development — and in Litomastix the astonishingly large number
of about 3000 may develop — they are all of -the same sex.15

15 Except, of course, the so-called asexual larvae — the origin and development
of aaefe needf further study.

644 J. T. PATTERSON

' It might be argued that the identity of sex among the several
individuals of a polyembryonic litter is the result of similarity of
environment. But here again the facts of fertilization in the
development of Litomastix completely disproves the idea that
external influences are in any way causal factors in sex deter-
mination, at least in this parasitic insect. Furthermore, in the
case of the development of the armadillo the four embryonic
primordia early become separated from each other, each embryo
becoming enclosed within its own amniotic envelope, and in a
great measure acquiring its own individual environment. All of
this takes place long before the sexual organs develop, indeed,
long before the so-called ' hermaphroditic stage' of the embryo
appears. If external factors play any role in sex determination,
it is difficult to understand, under the conditions obtaining in the
armadillo, why litters containing both male and female individ-
uals are never produced.

The study of polyembryonic development in Litomastix also
calls to mind another important fact, viz., that polyembryony
has nothing to do with the actual determination of the sex of the
egg. This conclusion becomes evident if one considers the differ-
ent sexual conditions which exist in the several species exhibit-
ing polyembryony. It occurs in the typical dioecious species,
like the armadillo, in which the fertilized egg produces all females
or all males, it occurs in the parasitic hymenoptera, in which the
fertilized egg produces females, and the unfertilized egg males;
and finally, it occurs in hermaphroditic forms, like the earth
worm, and also (probably) certain cyclostomatous Bryozoa.16

Polyembryonic development may obtain, therefore, no matter
whether the egg be fertilized or not, or whether it is destined to
bring forth a progeny of unisexual or one of bisexual individuals.
Let us repeat, then, that polyembryonic development is not to be
considered as a causal factor in sex determination. The facts
of polyembryonic development adds, rather strong corrobora-
tive evidence to that of cytology, namely, that the sex poten-
tiality of the egg is fixed at a very early stage of development —
doubtless in all ordinary cases, at the time of fertilization.

16 Robertson ('03) states that Crisia geniculata and C. cornuta (or edwardsiana,
according to her later identification, '10) are probably monoecious.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 645

SUMMARY

The main facts brought out in the descriptive part of the paper
may be summarized as follows:

1. The arrangement of the folds of the uterine mucosa is such
that there is formed a distinct cross-shaped area of compara-
tively smooth mucosa at the tip of the fundus. The center of
the cross is the attachment zone for the embryonic vesicle, and
its right or left arm, depending on which ovary functions, forms
the pathway along which the vesicle travels from the Fallopian
tube to the point of attachment (fig. 21, pp. 561-563).

2. The armadillos breed during October and the early part of
November. A large majority of the old females are pregnant
before October 15, but the second year females may continue to
breed for some time after this period. The young are born in
March and April, but an occasional litter may appear in Febru-
ary. The period of gestation is estimated at one hundred and
forty days (p. 564).

3. Except in very rare instances, young females born in March
or April do not breed during the succeeding fall (p. 564).

4. A 'period of quiescence7 of the blastocyst was discovered.
This period lasts for at least three weeks, and is probably similar
to the quiescent period of the blastocyst of the deer described by
Bischoff (pp. 564-565).

5. The right and left ovaries function with equal frequency
(p. 567).

6. In no case has more than one ovum been found in the uterus
(p. 567).

7. The youngest specimen obtained was a typical mammalian
blastocyst, consisting of an outer trophoblastic layer of polygonal
cells, and an inner cell-mass of embryonic cells (fig. 6, pp. 571-
572).

8. The entoderm differentiates before implantation occurs,
and arises through a migration of entodermal mother-cells from
among the embryonic ectodermal cells of the inner cell-mass.
These entodermal cells, migrate, either directly or after having
undergone division, to the under surface of the mass, where they

646 J. T. PATTERSON

first form a fenestrated structure which later is transformed into
a continuous layer (figs. 8-14, 36^2, pp. 572-585).

9. After the entoderm becomes a continuous layer, it splits
from the ectoderm, and its free margin passes beyond the limits
of the ectodermal mass until the area covered by the entoderm
equals an arc of about 80 degrees on the circumference of the
blastocyst. The margin of the entoderm now unites with the
trophoblastic wall. The remaining 280 degrees of trophoblas-
tic wall never becomes lined with entoderm (figs. 15, 47, 48;
p. 585).

10. The blastocyst becomes attached at the embryonic pole
to the uterine wall. (figs. 16, 17, 43-46, 49, 50; pp. 585-587).

11. After the entoderm is split from the embryonic ectoderm,
the latter rounds up into a. spherical mass, which upon parting
company with the trophoblast, pushes into the cavity of the ves-
icle and becomes included in the entodermal layer (figs. 15-17,
43-46, 48-50; pp. 587-590).

12. The ectodermal vesicle is formed by a vacuolization proc-
ess, which results in disintegrating the core of the ectodermal
sphere. When complete, the ectodermal vesicle consists of a
single layered pole turned toward the Trager,and a uniformly
thick pole, which faces the now inverted entoderm or yolk-sac
(figs. 16, 17, 18, 43-46, 49-51; pp. 590-593).

13. During the process of inclusion of the ectodermal mass,
there is created an extraembryonic cavity, which lies between the
Trager and the endothelial-like pole of the ectodermal vesicle
(figs. 17, 18, 49-51; pp. 592-593).

14. The extraembryonic mesoderm arises through cell prolifer-
ations from the ectodermal vesicle. These proliferations occur
around the entire vesicle, at the angle formed by the vesicle and
the entoderm, where the latter turns out to join the trophoblast.
The mesoderm cells are given off in groups, which are quickly
transformed into vesicular-like structures that fuse together to
form a continuous lining or mesothelium for the extraembryonic
cavity (figs. 19, 20, 55-60; pp. 593-595).

15. On the right and left sides of the ectodermal vesicle, the
primary diverticula or buds appear from thickened areas that

POLYEMBRYONIC DEVELOPMENT IN TATUSIA 647

have arisen through the shifting of cells from the thick pole of the
vesicle (figs. 1, 21, 22, 59-61; pp. 598-603).

16. Soon after its origin, each primary diverticulum gives rise
to two secondary diverticula or buds. One of these buds appar-
ently is but an extension from the tip of the primary diverticu-
lum, while the other takes its origin from the left-lateral portion
of the tip of the diverticulum. The embryonic buds extend
toward the Trager down along the inner side of the entodermal
portion of the blastocyst wall as tube-like processes, which in-
volve not only the thickened lateral plates of ectoderm, but also
portions of the thin endothelial-like wall of the vesicle (figs. 23-
29, 62-74; pp. 603-610).

17. The part of the ectodermal vesicle which remains after the
embryonic tubes are given off becomes the common amniotic
vesicle. It retains for some time connections with the embryonic
tubes by means of the amniotic canals, which are differentiated
from the proximal parts of the original diverticula. The charac-
teristic paired condition of litters of T. novemcincta is the result
of the method by which two secondary buds arise from each of
the primary diverticula. The common amniotic vesicle eventu-
ally degenerates and disapppears (figs. 5, 80-84; pp. 611-613).

18. Each embryo differentiates within the secondary diverti-
culum, deriving its ectoderm from a portion of the lateral plate
which was carried down into the diverticulum, and its entoderm in
loco from the primitive entodermal sac or yolk-sac. The embry-
onic mesoderm arises from a typical primitive streak region in
each embryonic primordium (figs. 75-84; pp. 614-617).

19. The region of the so-called Rauber's layer forms the. seat
of attachment of the blastocyst. This region is soon trans-
formed into a syncytium, from which the embryonic nuclei pass
over into the mucosa, destroying it by their phagocytic or his-
tolytic action (figs. 31-33; pp. 617-619).

20. The more superficially situated nuclei of the embryonic
syncytium organize the Trager epithelium, which, together with
the overlying mesothelial layer, forms the lower portion of the
chorionic wall. The Trager proper, which forms a thickened
annular zone about the base of the attached vesicle, gives rise

648 J. T. PATTERSON

to the villi of the definitive placental discs. Villi also appear
on the Trager epithelium but they remain more or less rudimen-
tary (figs. 34, 35, 62-65; pp. 619-625).

Marine Biological Laboratory

Woods Hole, Massachusetts

August 10, 1913

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POLYEMBRYONIC DEVELOPMENT IN TATUSIA

651

Fig. 6 Median section of the inner cell-mass of specimen No. 287. X 913.
Fig. 7 Median section of the inner cell-mass of specimen No. 310. X 913.
Fig. 8 Median section of the inner cell-mass of specimen No. 335. The letters
a i o e indicate the entodermal mother cells. X 728.

Fig. 9 Median section of inner cell-mass of blastocyst No. 244. X 728.

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

652

J. T. PATTERSON

a b 0 d e f g b i J k ' 1

En

11

1 2

Fig. 10 Median section of the inner cell-mass of blastocyst No. 296. This
specimen shows with special clearness the formation of the entoderm. X 728.

Fig. 11 A section taken slightly to the side of the center of the inner cell-mass
of blastocyst no. 311. X 596.

Fig. 12 Outline of the marginal entodermal cells from blastocyst No. 300.

Fig. 13 Marginal entodermal cells from blastocyst No. 320 (see plate 1).

Fig. 14 Median section of the inner cell-mass of blastocyst No. 249. X 1190.

Fig. 15 Median section of the embryonic spot of blastocyst No. 340. X 1190.

Fig. 16 A section passing through the center of the ectodermal mass of speci-
men No. 316. The sections are not quite perpendicular to the surface of the mu-
cosa, and consequently the point of attachment does not show in this section
(see plate 2). X 218.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

653

15

y-

Tro

654

J. T. PATTERSON

K- —

Tro

17

-Tro

G C-

Fig. 17 Median section of the ectodermal mass of specimen No. 332. This
specimen is just a little older than the preceding, and shows with great clearness
the manner in which the ectodermal mass is included within the entoderm. At
k is a knot-like growth from the trophoblast. Note that the ectodermal mass is
undergoing vacuolization to form a vesicle. X 245.

Fig. 18 Median section of specimen No. 233. This shows the condition just at
the close of the formation of the ectodermal vesicle (see text for description). X
162.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

655

Tro

Tr Ep

19

Fig. 19 Median section in a right-left plane of specimen No. 234. The devel-
opment of the mesoderm has made considerable progress, a^wfc^JMMBtattezsspfr^be
MM* V?the point where the entoderm joins the chorionic ecto-

derm (trophoblast). X 142.

Fig. 20 A. section similar to the preceding, from specimen No. 256. The vesicle
has collapsed and thus appears very much flattened in the figure. The chorionic
ectoderm has sloughed off and disappeared. The mesodermal vesicles have ex-
panded until they now fill up the entire extraembryonic cavity. X 113.

656

J. T. PATTERSON

21

Fig. 21 Drawing of the inner surface of the fundus end of the uterus, from
specimen No. 247. It shows the cross-shaped area, in the center of which is an
attached vesicle. The vesicle has come from the right ovary and is attached on
the right side of the placental area. X 14.5.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

657

24

26

Fig. 22 A median section through plane a to 6, figure 1. The right and left
bays (It and L) of the primary buds are well shown in the figure. A remnant of
the partition which previously separated the two mesodermal vesicles is seen at
par. X 130.

Fig. 23 The section through e to / of figure 2. It shows the transverse section
across Embryos I and II. This specimen shows a rather rare condition for a
blastocyst so far advanced in development, in that the chorionic ectoderm has not
as yet disappeared. However, it shows many signs of degenerating. X 71.

Fig. 24 Enlarged view of the embryonic portion of the ectodermal vesicle of
the preceding figure. The figure is inverted in order to bring the dorsal sides of
the embryonic primordia uppermost. The primitive streak region of each embryo
is proliferating mesodermal cells (E. Mes.}. In the case of Embryo I the section
cuts across a pit-like depression (P.P.) of the primitive groove. X 153.

Fig. 25 A portion of the section passing through the anterior end of Embryo
II. It shows the entoderm beginning to thicken preparatory to a proliferation of
mesodermal cells. This region of the entoderm corresponds to the protochordal
plate of Hubrecht. X 153.

Fig. 26 A section across the tips of the secondary buds of Embryos III and IV.
X 153.

658

J. T. PATTERSON

n-

27

28

Fig. 27 A section through g to h of figure 29. The buds of Embryos I and II
are shown in section. The enlargement of the cavity (marked Cay.) lying below
and to the left of the extraembryonic cavity is an artifact, caused by the lifting up
of the Trager epithelium from the mucosa. X 79.

Fig. 28 Sketch of the blastocyst No. 175, showing the four embryonic rudi-
ments. X 14.5.

Fig. 29 Diagram of the same specimen. The broken parallel lines show the
planes of the sections illustrated in plate 10. X 29.

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

659

Vl-'TJ £"C

'm H

Fig. 30 Detailed drawing of a transverse section across bud I of specimen No.
•257. X 312.

Fig. 31 Section through the Trager region of blastocyst No. 316. X 271.

660

J. T. PATTERSON

Em N

D C

Tr Ep

Fig. 32 A portion of the left side of the section shown in figure 51. X 306.

Fig. 33 A similar region from the section shown in figure 58. D.C., cells in
mitosis; G.C., giant cell, which is frequently found at the base of the blastocyst
At mis the beginning of a mass of Trager cells which later extends up to receive the
entoderm. X 306.

Fig. 34 A portion of the left side of the section shown in figure 60. The mass
at m now carries the entoderm, while the outer layer of Trager cells is seen as the
basal fragment (&./.). Note that the vacuoles are beginning to appear in the
syncytium. X 319.

Fig. 35 A portion of the right side of a section from specimen No. 175,
plate 8. The Trager epithelium (Tr.Ep.) is quite well organized, and root-like
growths of Trager tissue are seen extending into the mucosa at Em. X 485.

Ew //.

ii

^yj^fs -- ft^sx - "^

$^i^^^fe^;,'::*

661

DESCRIPTION OF PLATES

All of the photographic figures shown in these plates were made directly from
the negative without retouching. The photographs were made by the use of the
Bausch and Lomb combination micro-photographic and drawing apparatus.
Various combinations of Zeiss objectives and oculars were used in the work. In
practically every case the magnification is given after the description of the figure.

ABBREVIATIONS

C.A.V., common amniotic vesicle M.V., mesodermal vesicle

EC., ectoderm P.O., primitive groove

En., entoderm P.P., primitive pit

E.Mes., embryonic or primitive streak Tr., Trager

mesoderm Tr.Ep., Trager epithelium

Em.N., embryonic nuclei Tro., trophoblast

Ec.Ves., ectodermal vesicle /, ventral embryo

Ec.Cav., ectodermal cavity 77, right-lateral embryo

G.C., giant cell ///, dorsal embryo

Mes., mesothelium IV, left-lateral embryo

PLATE 1

EXPLANATION OF FIGURES

36 Median section of blastocyst No. 310. This shows the knob-like inner cell-
mass lying against the thin trophoblast. X 186.

37 Median section of blastocyst No. 296. The deeply staining entodermal
cells are seen in the act of migrating to the lower or inner side of the cell-mass.
X 186.

38 Median section of blastocyst No. 335. Note that the trophoblast has not
yet become thinned out or attenuated. X 186.

39 Enlarged view of the inner cell-mass of the blastocyst shown in the
next figure. It clearly shows the pseudopod-like processes extending out from
the marginal entodermal cells. X 225.

40 Upper view of blastocyst No. 320. From an unstained glycerin jelly prep-
aration. X 90.

41 Median section of blastocyst No. 249. The section does not lie flat on the
side, and since the microscope was focused on the trophoblastic cells, the inner
cell-mass is slightly out of focus. Note how sharply Rauber's layer stands out
from the mass. X 417.

42 Enlarged view of the inner surface of the entoderm of blastocyst No. 300.

662

POJ.YKMBRYONIC DEVELOPMENT IN TATUSIA

J. T. I'ATTBKSON

PLATE 1

39

II

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 2

EXPLANATION OF FIGURES

43 to 46 A series of four sections from specimen No. 316 (see text for descrip-
tions). X 148.

664

POLYEMBIIYOMC DEVELOPMENT IX TATUSIA

J. T. I'ATTERSON

PLATE 2

it

:<„

JOURNAL OK MORPHOLOGY, VOL. 24, NO. 4

PLATE 3

EXPLANATION OF FIGURES

47 Portion of a section from blastocyst No. 311. It shows the well-organ-
ized layer of entoderm, which takes a deeper stain than does the overlying ecto-
derm. X 417.

48 Similar section from specimen No. 340. This shows the first step in the
rounding up of the ectoderm to form a ball-like mass. X 416.

49 Median section of blastocyst No. 332. The ectodermal mass has become
included within the entoderm. X 149.

50 This section is taken four sections to the right of the preceding, and
shows with especial clearness the vacuolization of the ectodermal mass to form a
vesicle. X 149.

666

POLYEMBRYOXIC DEVELOPMENT IN TATUSIA

J. T. I'ATTERSON

PLATE 3

50

JOURNAL OF MORPHOLOGV, VOL. 24, NO. 4

PLATE 4

EXPLANATION OF FIGURES

51 Median section of specimen No. 233. The ectodermal mass has become trans-
formed into a vesicle, the lower wall of which has a small pore. X 167.

52 This section lies ten sections to the right of the preceding. X 167.

53 Section from blastocyst No. 329. This specimen was unfortunately
crushed in transportation from the field to the laboratory, but the general rela-
tions of the different parts can be made out. X 167.

54 Section from specimen No. 289. The relation of the different parts is par-
ticularly clear in this section. Note that the large extraembryonic cavity, which
lies between the ectodermal vesicle and the mucosa, is entirely free from cells.
X 85.

068

POI.YEMBRYOXIC DEVELOPMENT IX TATUS1A
j. T. I-ATTERSON

PLATE 4

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 5

EXPLANATION OF FIGURES

55 Median section of specimen No. 289. The two deeply-staining cells lying
at about the center of the extraembryonic cavity are degenerating entodermal cells
the single cell situated toward the right side of this cavity is of ectodermal origin,
and indicates the beginning of a cell proliferation which will give rise to the extra-
embryonic mesoderm. X 106.

56 Median section from specimen No. 298. It shows an early stage in the
development of the mesoderm, which appears in the form of small vesicles. X 85.

57 A section lying four sections to the left of the preceding, and showing well-
formed but small mesoderm vesicles. X 106.

58 Median section, taken in a right-left plane, of No. 234. This shows two rel-
atively large mesodermal vesicles, which have been formed by a fusion of smaller
vesicles, such as appear in the preceding figure. X 106.

070

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. I'ATTERSON

55

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 6

EXPLANATION OF FIGURES

59 Median section, taken in a right-left plane, of specimen No. 256. The two
mesodermal vesicles have expanded until they now fill up the entire large extra-
embryonic cavity; but their adjacent walls still remain, forming a double-walled
partition between the cavities of the vesicles. X 95.

60 Median section of specimen No. 247. It passes through plane a to 6 of figure
1. X 90.

61 A section taken through plane c to d of figure 1 . X 90.

672

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. PATTERSON

PLATE 6

JOCRNAI. OF MORPHOI-OGY, VOI-. 24, NO. 4

PLATE 7

EXPLANATION OF FIGURES

62 to 65 A series of four sections from specimen No. 290; all X 46.

62 Taken through plane a to b, figure 2.

63 Taken through plane c to d, figure 2.

64 Taken through plane e to /, figure 2.

65 Taken through plane g to h, figure 2.

674

POLYEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. I'ATTKKSON

PLATE 7

(53

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 8

EXPLANATION OF FIGURES

66 to 69 A series of four sections from specimen No. 175; all X 56.

66 Taken through plane a to 6, figure 29.

67 Taken through plane c to d, figure 29.

68 Taken through plane e to /, figure 29.

69 Taken through plane g to h, figure 29.

676

POI.YEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. PATTERSON

PLATE

69

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 9

EXPLANATION OF FIGURES

70 to 74 A series of five sections from specimen No. 257; all X 63.

70 Taken through plane a to b, figure 3.

71 Taken through plane c to d, figure 3.

72 Taken through plane e to /, figure 3.

73 Taken through plane g to h, figure 3.

74 Taken through plane i to j, figure 3.

678

J. T. PATTERSON

PLATE 9

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73

74

PLATE 10

EXPLANATION OF FIGURES

75 to 77 A series of three sections from specimen No. 170; all X 75.

75 Taken through plane e to /, figure 4.

76 Taken through plane c to d, figure 4.

77 Taken through plane a to b, figure 4.

78 Transverse section through the middle of one of the embryos from specimen
No. 276 (fig. 5).

79 Litter of four embryos attached to the placentae of the chorion, which has
been cut open on the ventral side. The paired arrangement of the embryos is very
clearly shown. X 3.

680

FOLYEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. PATTERSON

PLATE 10

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

PLATE 11

EXPLANATION OF FIGURES

80 to 84 A series of five sections from specimen No. 226. The sections are cut
almost transversely to the long axes of the embryos, that is, parallel to the surface
of themucosa (see text for descriptions). Figures 80 to 81, X 46; figures 82 to 83;
X 42; and figure 84, X 37.

682

POI.YEMBRYONIC DEVELOPMENT IN TATUSIA

J. T. I'ATTBRSON

PI.ATE 11

80

si

JOURNAL OF MORPHOLOGY, VOL. 24, NO. 4

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