Difference between revisions of "Paper - Contributions to the embryology of the marsupialia 4-2"

From Embryology
(Created page with "{{Hill1910 header}} ==Chapter II. - The Ovum of Dasyueus== 1. Structure of the Ovarian Ovum. The full-grown ovarian ovum of Dasyurus (PI. 1, fig. 1) appears as a rounded...")
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Caldwell into making the statement, now widely quoted in  
Caldwell into making the statement, now widely quoted in  
the text-books, that cleavage in Phascolarctus is of the  
the text-books, that cleavage in Phascolarctus is of the  
meroblastic type.
meroblastic type.
==Chapter III.  -  Cleavage and Formation op the Blastocyst==
1. Cleavage.
Cleavage begins in the uterus as in Didelphys, Phascolarctus, and no doubt Marsupials in general. The first externally visible step towards it consists, as already described,
in the elimination by abstriction of the deutoplasmic zone at
the upper pole. The yolk-body so formed appears as a
definitely limited, clear, rounded mass which lies in contact
with the slightly concave upper surface of the formative
remainder of the ovum. It is quite colourless and transparent
except for the frequent occurrence in it of a small, more or
less irregular opaque mass, representing probably a condensation product of its fluid material (cf. PI. , figs. 8, 14, y.h.).
Consisting as it does of a very delicate cytoplasmic reticulum
with fluid-filled meshes it is extremely fragile, and is seen to
advantage only in fresh material (figs. 14 and 19, y.h.). It
takes no direct part in the later developmental processes>
though during the formation of the blastocyst it becomes
enclosed in the blastocyst cavity and finally undergoes disintegration therein, its substance becoming added to the fluid
which Alls the same, so that it may be said, in this indirect
way, to fulfil, after all, its original nutritional destiny. Separa
tion of the yolk-body is rapidly followed by the completion
of the division of the formative remainder of the ovum into
the first two blastomeres, the plane of division being coincident with the polar diameter or egg-axis and at right
angles to the plane of separation of the yolk-body (PL 2, fig.
14). I obtained relatively little material between the stage
of the unsegmented ovum with two equal-sized pronuclei seen
in fig. 12 and the 2-celled stage (fig. 14), both of which
are well represented in my material, so that it would appear
that the separation of the yolk-body and the division of the
formative remainder of the ovum are effected with considerable
rapidity. Fig. 13 shows, however, a section of an unsegmented ovum in which the chromosomes of the metaphase
of the first cleavage figure are visible in the central region
of the formative cytoplasm, but situated, it is worthy of note,
rearer the future upper pole than the lower pole. The deutoplasmic zone {d.z.) still forms an integral part of the egg, and
there is no sign of commencing abstriction. I have also
sections of ova in a still more advanced stage of the first
cleavage, in which the daughter-nuclei have but recently been
constituted and are still quite minute, and the cleavage furrow
is well marked on the surface of the egg. In these ova the
yolk-body is already separated, so that we may conclude with
a fair degree of certainty that its elimination about coincides
with the first appearance of the cleavage furrow.
Figs. 14-16 show the 2-celled stage, respectively in side,
lower polar, and end views. The blastomeres are of approximately equal size and otherwise quite similar. Selenka also
found the same to be the case in Didelphys, though in the
single specimen of the 2-celled stage he had for examination
(Taf. xvii, fig. 3) the blastomeres are displaced and somewhat
shrunken. Each blastomere has much the shape of a hemisphere from which a wedge-shaped segment has been sliced
off, a form readily accounted for when we take account of the
effect of the elimination of the deutoplasmic zone. After
that event, the formative remainder of the ovum has the
form of a sphere from which a somewhat bi-convex lens
shaped piece lias been gouged out at the upper pole.
Consequently, when it divides along its polar diameter, the
resulting blastomeres will have the form of hemispheres with
obliquely truncated upper surfaces or ends, which will be proportionately thicker than the lower ends. In correlation
therewith we find the nucleus of each blastomere situated
slightly excentrically, rather nearer the upper than the lower
pole (fig. 18). The rounded yolk-body lies partly enclosed
betAveen the upper truncated surfaces of the blastomeres.
Two-celled eggs ai'e shown in vertical section in figs. 17
and 18. The cytoplasm of the blastomeres exhibits a wellmarked differentiation into two zones corresponding to that
ah'eady seen in the formative cytoplasm of the uusegmented
egg, only much more accentuated, viz. a dense, fine-grained
perinuclear zone, and a less dense, more vacuolated peripheral
zone, in which there is present a coarse, irregular network of
deeply staining strands, recalling the frameAvox'k of mitochondrial origin described by Van der Stricht ('04, '05) in
the human ovum and that of Vesperugo. We have here in
this differentiation of the cytoplasm, evidence of the occurrence of an intense metabolic activity Avhich has resulted in a
marked increase in the amount of deutoplasmic material
present in the blastomeres as compared Avith that found in
the ovarian egg or even in the unsegmented uterine egg.
The blastomeres consequently present a someAvhat dense
opaque appearance Avhen examined in the fresh state, their
nuclei being partially obscured from view. Amongst the
Eutheria, various observers (Sobotta, Van der Stricht, Lams
and Doorme) have described a similar inci'ease in the deutoplasmic contents of the egg after its passage into the
Fallopian tube or uterus.
The second cleavage plane is also vertical and at right
angles to the first. The resulting four equal-sized blastomeres
vieAved from the side (PI. 2, fig. 19) are seen to be ovalish in
outline, their loAver ends being slightly narroAver and more
pointed than their upper ends, Avhich diverge someAvhat to
enclose the lower part of the yolk-body. Seen from one of
tli 0 poles, in optical section (figs. 20, 21), they appeal triangular 'with rounded corners and centrally directed apices.
The space occupying the polar diameter, which they enclose
is the cleavage cavity. The blastomeres are now somewhat
less opaque than those of the 2-celled stage, so that their
nuclei, excentrically situated nearer their upper ends and
enclosed in the central granular zone of the cytoplasm, can
now be fairly distinctly made out in the fresh egg.
The arrangement of the blastomeres at this stage is
exceedingly characteristic, and is identical with that of the
blastomeres in the corresponding stage of Amphioxus or the
frog, but is quite different from that normal for the 4-celled
stage of the Eutheria. They lie disposed radially or meridionally around the polar diameter, occupied by the cleavage
cavity, their thicker upper ends partially surrounding the
yolk-body. Selenka figures a precisely similar arrangement
in his 4-celled stage of Didelphys, so that we may conclude
it holds good for the Marsupials in general.
Whilst, then, in Marsupials the first two cleavage planes are
vertical or meridional, and at right angles to each other, and
the first four blastomeres are arranged radially around the
polar diameter (radial type of cleavage), in the Eutheria
such is never the case, at all events normally, so far as is
known. In the Eutheria the first four blastomeres form, or
tend to form, a definite cross-shaped group, as the result
apparently of the independent division of thefii'st two blastomeres in two different planes at right angles to each other,
the division planes being meridional in the one, equatorial
in the other. ^ This pronounced diffei'ence in the spatial
relations of the fij'st four blastomeres in the Metatheria and
Eutheria is a feature of the very greatest interest and importance, since it is correlated with and in part conditions
the marked dissimilarity which we meet with in the later
developmental occurrences in the two groups, in particular
in the mode of formation of the blastocyst in the two.
* Compare in this connection Assheton's remarks ('09, pp. 232-233),
which have api>eared since this chapter was written.
Moreover, so far as the Eutheria are concerned, it affords us,
I believe, a striking and hitherto unrecognised example of a
phenomenon to which Lillie ('99) has directed attention, viz.
adaptation in cleavage.
Fig. 22 shows a horizontal section through the 4-celled
stage, and fig. 23 a vertical section of the same. The blastomeres in their cytoplasmic characters essentially resemble
those oE the 2-celled stage, but the peripheral deutoplasmic
network is here more strongly developed, and it is especially
worthy of note that it is more marked towards the lower
poles of the blastomeres (fig. 23), as also appears to be the
case in the 2-celled stage. The shell-membi'ane measures in
thickness '0072 mm.
The next succeeding (third) cleavages are again meridional, each of the four blastomeres becoming subdivided
vertically into two, not necessarily synchronously. Fig. 53.
PI. 6, shows a side view, and fig. 54 a view from the lower
pole of a 6-celled egg, two of the blastomeres of the 4-celled
stage having divided before the other two. The blastomeres have moved apart, and now form an' open ring
approximately equatorial in position, and surrounding the
central cleavage space, the upper opening of which is
occupied by the yolk-body. I have failed to obtain a
perfectly nonnal 8-celled stage, nevertheless the evidence
clearly shows that the first three cleavage generations in
Dasyurus are meridional and equal, and that the resulting
eight equal-sized blastomeres form an equatorial ring in
contact with the inner surface of the sphere formed by the
zona and shell-membrane.
Whilst, then, the first three cleavage generations are
meridional and equal, the succeeding divisions (fourth cleavage
generation), on the contrary, are equatorial and unequal, each
of the eight blastomeres becoming divided into a smaller,
more transparent upper cell, with relatively little deutoplasm,
and a larger, more opaque lower cell with more abundant
deutoplasmic contents. In this way there is formed an
exceedingly characteristic 1 6-celled stage, consisting of two
superimposed rings, each of eight cells. The upper ring of^
smaller and clearer cells partially encloses the yolk body, and
is situated entirely in the upper hemisphere of the sphere
formed by. the egg-envelopes. The lower ring of larger, more
opaque cells lies approximately in the equatorial region of the
said sphere. This 16-cell ed stage is.figm'ed in fig. 55, PI. 6,
as seen from the side, and in fi'g. 56 as seen from the upper ^
pole, both figures being taken from a spirit egg '37 mm. in
diameter. The marked' differences in the cells of the two
rings are well brought out in the micro-photographs reproduced
as figs. 24, 25, and 26, PI. â–  2. Pigs. 24 and 25 represent
horizontal sections of an egg '38 mm. in diameter, the former
showing the eight cells of the lower ring, and the latter the
eight cells of the upper ring. Pig. 26 shows a vertical section
through an egg also of a diameter of "38 mm., but with
seventeen cells, one of the original eight cells of the upper ring
having divided and one beinginprocessof division. Thesection
passes through the yolk-body [y.h.), which is seen as a faintly
outlined structure lying in contact with the zona between the
two cells of the upper ring (/.c.).
The shell-membrane in eggs of this 16-celled stage has
attained a thickness of -0075 mm., and the albumen layer has
been almost completely absorbed, so that the zona now lies
practically in apposition with the shell-membrane, the two
together forming a firm resistant sphere, to the inner surface
of which the blastomeres are closely applied. The separation
between the zona and shell-membrane seen in the figures is
largely, if not wholly, artificial. ,
The average^ measui'ements of the cells of the two rings ip
the '38 mm. egg, :^ured in figs. 24 and 25, are as follows :
Ppper ring cells. Lower ring cells.'
Diameter . : -06 x -058 mm. . -OOx-Ofidinm.^,'
Vertical height , -OSSimm. . -llS mm.
Nucleus . j . ‘0165 mm. ■ ■ • . •02 mm.
These measurements demonstrate at a glance the distinct
difference in. size which exists between the cells of the two
rings, whilst the cytoplasmic differences between them. ai-e
VOL. 56, PART 1.  -  NEW SERIES. ""S
equally evident from an inspection of the micro-photographs,
figs. 24-26. In the larger cells of the lower ring (fig. 24,
tr.ect.) the nucleus (rich in chromatin and nucleolated)
is surrounded by a perinuclear zone of clearer, coarsely
vacuolar cytoplasm, outside of which is a densely granular
deutoplasmic zone, which extends to within a short distance
of the periphery of the cell-body. In the smaller cells of the
upper ring (fig. 25, /.c.) the cytoplasm is coarsely reticular,
with a tendency to compactness round the nucleus, and its contained deutoplasmic material is spare in amount as compared
with that of the lower cells, being mainly located in a quite
narrow peripheral zone. The upper cells thus appear relatively
clear as compared with the dense, opaque-looking lower cells
(fig. 26).
It becomes evident, then, that we have to do here, in this
fourth cleavage generation, with an unequal qualitative
division of the cytoplasm of the blastomeres of the 8-celled
stage. Just such a division as this we should expect if the
deutoplasmic material were mainly aggregated towards the
lower poles of the dividing cells. The evidence shows that
this is actually the case. In the 2-celled and especially in the
4-celled eggs we have already seen that the deutoplasmic
network is already most strongly developed towards the lower
poles of the blastomeres. This polar concentration of the
deutoplasm reaches its maximum in blastomeres of the 8celled stage, and confers on these an obvious polarity.
Although I failed to obtain normal examples of the latter stage,
I have fortunately been able to observe the characters of the
blastomeres in sections of eggs with twelve, thirteen, and
fourteen cells respectively.
In the 12-celled egg (PI. 6, fig. 57), measuring '38 mm.
in diameter, four of the eight original blastomeres are still
undivided ; the remaining four have undergone division
unequally and qualitatively, one but recently, so that 4 +
(4 X 2) = 12. The undivided blastomeres are large (average
diameter, '11 x '076 mm.) and ovoidal in form, their lower
ends being thicker than their upper, and they exhibit a well
marked polarity. The nucleus lies excentrically in the
upper half of the cell, just above the equator, and is surrounded by a finely granular zone of cytoplasm, outside which
is a thin irregular ring of deutoplasmic material. The cytoplasm of the apical part of the cell is clear and relatively free
from deutoplasm ; that of the lower half, on the other hand,
is so rich in deutoplasm as to appear quite dense and opaque.
The conclusion is therefore justified that the blastomeres of
the 8-celled stage possess a definite polarity, which has beenj
acquired as the result of the progressive concentration of
deutoplasmic material at their vegetative poles during the>
cleavage process. Division, in the equatorial plane, of cells
so constituted must necessarily be unequal and qualitative, so
far at least as the cytoplasm is concerned.
In the 13-celled stage three of the original eight blastomeres are in process of division, and five have already divided
unequally and qualitatively, so that 3 -f- (5 x 2) = 13, and in
the 14-celled stage two of the original blastomeres are in
division and sis have already divided : 2 -t- (6 x 2) = 14.
The significance to be attached to this characteristic unequal
and qualitative division of the blastomeres of the 8-celled stage
to form two superimposed cell-rings, markedly differentiated
from each other, we shall presently consider. Meantime I
may categorically state the conclusions I have reached in
regard thereto. The wall of the blastocyst in Dasyurus is at
its first origin, and for some considerable time thereafter,
unilaminar throughout its entire extent, and I regard the
upper cell-ring of the 16-celled stage as giving origin to<
the formative or embryonal region of the unilaminar wall,
the lower cell-ring ae furnishing the extra-embryonal or nonformative remainder of the same. I shall therefore refer to
the upper cell-ring and its derivatives as formative or
embryonal, and to the lower, cell-ring and its derivatives
as non-formative or extra-embryonal.
The formative or embryonal region furnishes the embryonal
ectoderm and the entire entoderm of the vesicle, and I accordingly conclude that it is the homologue of the embryonal knot
or inner cell-mass of the EutHerian blastocyst. The nonformative or extra-embryonal region directly gives origin to
the outer extra-embryonal layer of the bilaminar blastocyst
wall, i.e. to that layer which in the Sauropsida and Prototheria is ordinarily termed the extra-embryonal ectoderm. I
regard it as such, and as the homologue of the so-called
trophoblast (or as I prefer to term it, the “ trophoblastic
ectoderm” or “ tropho-ectoderm ”) of the Eutherian blastocyst.
A word or two here before concluding this section by way
of summary, as to the condition of the enclosing egg-envelopes.
During the sojourn of the egg in the uterus the albumen is
gradually resorbed, and by about the 16-cell stage it has all
but completely disappeared, thus permitting the zona to come
into direct apposition with the inner surface of the shellmembrane. The shell-membrane itself increases very considerably in thickness during cleavage, and by the 16-celled
stage had practibklly reached its maximum, viz. '0075•008 mm., i.e. it is nearly five times thicker than that of the
ovum which has just entered the uterus. The thickened
shell-membrane by itself is firm and resistant, and it becomes-,
still more so by the application-of the zona to its inner surface,
the two together forming a spherical supporting case round
the segmenting egg, to the inner surface of which the blastomeres become closely applied. .
The existence of such a firm supporting envelope round
the Marsupial egg is, in my view, a feature of very great
ontogenetic significance, and one which must be taken into
account in any comparison of the early developmental occurrences in the Metatheria and Eutheria. As the sequel will
show, the mode of formation of the blastocyst in these two
sub-claisses is fundamentally different, and in my opinion
the explanation of this difference is to be found in the
retention by the Metatheria of a relatively thick resistant;
shell-membrane, and its complete disappearance amongst the
Eutheria. r j
2, Formation of the Blastocyst.
It is characteristic of the Marsupial that the cleavage-cells
proceed directly to form the wall of the blastocyst, without
the iuterventioii of a morula stage, as in' the Eutheria.
The fil'tli cleavages are meridional, each of the eight cells
of the two rings of the 16-celled stage becoming subdivided
vertically into two, so that there results a 32^celled stage
consisting of two rings, each composed of sixteen cells. As
might be expected, the smaller less- yolk-rich cells of the
upper ring tend to divide more rapidly than the larger yolkladen cells of the lower ring, but the difference in the rate of
division of the two is only slight. I have, for example,
sections of a 17-celled stage (that already referred to, hg. 26)
consisting of nine formative cells (= 6 + [1 x 2] + 1 in
division) and eight non-formative cells, and also of a 31-celled
stage (PI. 6, tig. 59, seen from lower pole; cf. also tig. 60,
showing a side view of another 31-celled egg, both eggs
"375 mm. in diameter), consisting of sixteen formative and
fifteen non-formative cells, of -which one is in process of
division. But I have also preparations of 32-celled eggs with
ail equal number of formative and non-formative cells,
showing that the latter may make up their leeway, the former
resting meantime. On the other hand, the cells of the two
rings may divide more irregularly, as evidenced by a stage
of about forty-two cells, consisting approximately of twentythree formative cells ( = 9 -f- [7 x 2J ) and nineteen nonformative (= 13 -f [3 X 2]). Whatever the rate of division,
the important point is that the division planes are always
radial to the surface, so that all the resulting blastomeres retain a superticial position in contact with the inner
surface of the supporting sphere formed by the zona and
shell-membrane. In apposition with the continuous surface
afforded by that, the blastomeres, continuing to divide,
gradually spread round towards the poles, the descendants of
the upper or formative cell-ring gradually extending towards
the upper pole marked by the yolk-body, whilst those of the
lower or uou-formative cell-riug similarly spread towards the
lower pole. As the blastomeres divide and spread they
become smaller and more flattened, and gradually cohere
together, and so in this way they eventually give origin to a
complete unilaminar layer lining the inner surface of the
sphere formed by the egg-envelopes. It is this unilaminar
layer which constitutes the wall of the blastocyst.
The just completed blastocyst of Dasyurus is a spherical
fluid-hlled vesicle measuidng about '4 mm. in diameter (PI. 3,
flgs. 27-29, PI. 6, figs. 61, 62), and invested externally by
the thin zona and the shell-membrane {â– 0075-0078 mm. in
thickness). The albumen layer has completely disappeared,
and the shell-membrane, zona, and cellular wall are from
without inwards in intimate apposition. The smallest complete vesicles which I have examined measure ‘39 mm. in
diameter (figs. 27, 61), and in one of these I find the cellular
wall consists approximately of about 108 cells. In four other
eggs of the same diameter and from the same female the wall
of the blastocyst is as yet incomplete at the lower pole (fig.
31, l.'p.), and in these, rough counts of the cells yielded the
following respective numbers  -  89, 93, 121, 128. In another
also incomplete blastocyst of the same batch, '41 mm. in
diameter (fig. 32), the cellular wall consists of about 130 cells.
'I'he largest complete blastocyst in this same batch measured
"49 mm. in diameter, so that we have a range of variation in size
of the just completed blastocyst extending from ‘39 to '49 mm.
The unilaminar wall of the blastocyst consists of a continuous layer of more or less flattened polygonal cells (figs.
27-29, 61, 62) lying in intimate contact with the zona, itself
closely applied to the shell-membrane. Over the lower hemisphere the non-formative cells are on the whole larger and
plumper than the formative cells of the upper hemisphere,
and in sui-face examination they appear somewhat denser
owing to the fact that they possess much more marked perinuclear zones of dense cytoplasm than do the formative cells
(cf. fig. 63, representing a ‘6 mm. vesicle). In sections,
however, this latter difference is much less obvious, indeed.
is hardly, if at all, detectable, so that one has to depend
partly on the relative thickness of the cells, partly, and,
indeed, mainly, on the yolk-body in determining which
hemisphere is which.
The blastocyst cavity is tensely filled by a coagulable fluid
derived from that poured into the uterine lumen through the
secretory activity of the uterine glands. Also situated in the
blastocyst cavity, in contact with the inner surface qf the
wall in the region of the upper pole, is the spherical yolkbody (fig. 29, y.h.). It becomes overgrown and enclosed in
the blastocyst cavity as the result of the completion of the
cellular wall over the upper polar region, much in the same
sort of way as the yolk in the meroblastic egg becomes
enclosed by the peripheral growth of the blastoderm. In the
majority of my sections of early blastocysts the yolk-body
has been dragged away from contact with the formative cells
through the coagulation of the albuminous blastocystic fluid,
and lies more or less remote from the wall enclosed by the
coagulum, except on the side next the upper hemisphere (fig.
31, y.h., c.g.). In two instances, one of which is shown in
fig. 32, 1 find the yolk-body had become so firmly attached to
one of the formative cells that the coag-ulum formed during
fixation failed to detach it, and only succeeded in drawing it
out to a pear-shape.
The yolk-body, it may here be mentioned, persists for a
considerable time in the blastocyst cavity; I have found it
shrunken indeed, but still recognisable, in relation to the
embryonal area in vesicles 4'5-6 mm. in diameter. And
there may even appear within it peripherally, irregular strands
which stain deeply wit^ iron-h<ematoxylin and which recall
those forming the periphei'al deutoplasmic network of the early
blastomeres. Eventually, however, it seems to disappear, its
substance passing into the blastocystic fluid, so that, as already
remarked, it fulfils in this indirect way its original destiny.
Normally the cavity of the just completed blastocyst contains no cellular elements whatever. In one otherwise
perfectly normal blastocyst ('39 mm. diam.) I find present.
j. p. hilL. ‘
however, a small spheroidal body *028 mm. in diameter,
composed of glasSy-looking cytoplasm enclosing a central
deeply staining granule. This I interpret as a cell or cellfragment which has been accidentally separated off from the
wall, and which has undergone degeneration. In later
blastocysts such cellular bodies exhibiting more or less
evident signs of degeneration are of fairly common occurrence. They are of no morphological significance.
Selenka^s Blastopo.re.” -  r-N'ormally the wall of the
blastocyst is first completed over the upper hemisphere, in
correspondence with the fact-that the formative cells not
only divide somewhat more rapidly than the non-formative
but have a smaller extent of surface to cover, since the upper
cell-ring from which they are derived lies about midway
between the upper pole of the sphere formed by the eggenvelopes and the equator of the same, whilst the lower cellring from which the noil-formative cells arise is approximately
equatorial in position. We thus meet with stages in the
formation of the blastocystic wall such as are represented in
surface view on PI. 3, fig. 30, and in section in figs. 31 and
32, in which the blastocystic cavity, prior to the completion
of the cellular walTbver the lower polar region, is more or less
widely open below. There can be no doubt, I think, but that
this opening corresponds to that observed by Selenka in his
42-celled “gastrula^' of Didelphys and regarded by him as
the blastopore, since he believed the entoderm arose from its
lips. My observations conclusively show that it has no
connection whatever â–  with the entoderm, this layer arising
from the formative region of the upper hemisphere, and that
it is a mere temporary opening of no morphological significance, blastoporic or other. Pi'ior to the completion of
the wall at the upper pole a corresponding opening is temporarily present there also. Both owe their existence to the
characteristic way in which the blastocyst wall is formed by
the spreading of the products of division of the two cell-rings
of the 16-celled stage towards opposite poles in contact with
the surface provided by the enclosing egg-envelopes.
I have met with one specimen, an incomplete blastocyst
•39 mm. in diameter (belonging to the same batch as the
other blastocysts referred to in this section^), in which the
lower hemisphei'e would appear to have been completed before
the upper, for the yolk-body lies in contact with the zona in
the region where the cellular wall is as yet absent, and that
the yolk-body has not been secondarily displaced is proved by
a micro-photograph of the specimen in my possession (taken
immediately after its transference to the fixing solution), in
which the yolk-body is seen to lie at the unclosed pole in
exactly the same position as in the sections.
In connection with this exceptional specimen, it may be
recalled that Selenka, in his 68-celled “ gastrula” of Didelphys
(fig, 10, Taf. xvii), figures the wall as complete at the lower
pole, the “blastopore” having alx'eady closed, but as still incomplete at the upper pole, there being present a small opening
leading into the blastocyst cavity. In the 42-celIed “gastrula”
(fig. 8, Taf. xvii) this same opening and the “blastopore” as
well are present. The occurrence of these openings at
opposite poles, and the general agreement in the constitution
of the blastocyst wall (larger, more yolk-rich cells at lower
pole, smaller, less yolk-rich cells at upper), in the corresponding stages in Didelphys, and Dasyurus justify the conclusion that the blastocyst of the former develops in the same
way as does that of the latter. It is worthy of remark,
however, that the just completed blastocyst of Didelphys
appears to be considerably smaller than that of Dasyurus.
Selenka unfortunately gives no measurements of his early
stages, but I have calculated from the figure, the magnificatiou
of which is given, that the ^8-celled blastocyst has a diameter
of about ‘IS? mm. The corresponding stage of Dasyurus
measures about '39 mm., and is therefore nearly three times as
• This batch, from female 2 b, 16 . vii . '01, comprised altogether
twenty-eight eggs, of which some eighteen were normal complete and
incomplete blastocysts mm. in diameter) and ten abnormal, four
of these being unsegmented ova.
.1. P. HILL.
Selenka's Urentoderinzelle.  -  Whilst the 42- and 68celled blastocjsts described by Selenka may be regarded
as normal so far as the occurrence of polar openings and
the constitution of their wall ai‘e concerned, I hold them to be
abnormal in respect of the presence in each of a single large
yolk-laden cell, regarded by Selenka as entodermal in significance. It is well to point out that Selenka was not able
actually to determine the fate of this cellj he merely presumed
that it took part in the formation of the definitive entoderm.
No such cell occurs in normal blastocysts of Dasyurus at any
stage of development, and in my opinion Selenka's “ urentodermzelle” is none other than a retarded and displaced
blastomere, i.e, a blastomere which has failed for some
reason to divide, and which has become secondarily enclosed
by the products of division ot its fellows, and I am
strengthened in this interpretation by the occurrence in
an abnormal blastocyst of Dasyurus of just such a large
cell as that observed by Selenka. The vesicle in question
is one of the batch already referred to, and measured '397 mm.
in diameter. The cellular wall (fig. 37) is apparently normal,
but is incomplete' at one spot, and the gap so left is occupied
by a large binucleated cell, i-ich in deutoplasm and measuring
•12 X '072 mm. (fig. 37, abn.). This cell corresponds in its
size and cytoplasmic characters with a non-formative blastomere of about the 16-celled stage, and I l egard it simply as
a blastomere which has failed to undergo normal division.
In another abnormal blastocyst (‘39 mm. diam.) from the
same batch, the cellular wall appears complete and normal,
but the blastocyst cavity contains a group of about sixteen
spherical cells averaging about ‘032 mm. in diameter, and in
yet another abnormal egg ot' the same diameter and batch
there is present an incomplete layer of flattened cells over
one hemisphere, and towards the opposite pole of the eggsphere there occurs a group of spherical cells of variable size
and some of them multinucleate. In this abnormal egg it
appears as if the formative cells had divided in fairly normal
fashion, whilst the nou-formative cells had failed to do so.
Chaptee IV.  -  Growth oe the Blastocyst and Differentiation OF THE Embryonal Ectoderm and the Entoderm.
1. Growth of the Blastocyst.
In the preceding chapter we have seen that the cleavage
process in Dasyurus results in the formation of a small
spherical vesicle, about '4 mm. in diameter, Avhich consists,
internally to the investment formed by the apposed zona and
shell-membrane, simply of a cellular wall, unilaminar throughout its entire extent, and enclosing a fluid-filled cavity
normally devoid of any cellular elements. The stage of the
just completed blastocyst is followed by a period of active
growth of the same, and it is a noteworthy featui'e in the
development of Dasyurus that during this time the blastocyst
undergoes no essential structural change, but remains unilaminar until it has reached a diameter of from 4'5 to 5'5 mm.
Even during cleavage, the egg of Dasyurus increases in
diameter, partly owing to the thickening of the shell membrane, partly, and, indeed, mainly, as the result of the accumulation of uterine fluid under pressure within the egg-envelopes,
but the increase due to these causes combined is relatively
insignificant, being only about '1 mm. As soon, however, as
the cellular wall of the blastocyst is completed, rapid growth
sets in, under the influence of the hydrostatic pressure of the
fluid, which tensely fills the blastocyst cavity, with the result
that the small relatively thick-walled blastocyst becomes
convei'ted into a large extremely thin-walled vesicle, but
beyond becoming very attenuated, the cellular wall during
this period of actjve growth uudei'goes no essential change,
and retains its unilaminar character until the blastocyst, as
already mentioned, has reached a diameter of from 4'5 to 5‘5
mm. In vesicles of about this size there become differentiated
from the formative cells of the upper hemisphei-e the embryonal ectoderm and the entoderm, and this latter layer then
gradually spreads round inside the non-formative (extraembiwonal ectodermal) layer of the lower hemisphere so as to
form a complete lining' to tlie blastocyst, whicli thereby
becomes bilaminar. Sucli a marked enlargement of the blastocyst prior to the differentiation of the embryonal ectoderm
and entoderm as is here described for Dasyurus does not
apparently occur, so far as known, in other Marsupials : in
Perameles, for example, the embryonal ectoderm and the
entoderm are in process of differentiation in vesicles a little
over 1 mm. in diameter (v. p. 77), in Macropus these two layers
are already fully established in a vesicle only *8 mm. in
diameter (v. p. 79), and much the same holds good for Ti'ichosurus and Petrogale. It is pai'alleled by the marked growth
which in the Monotremes follows the completion of the blastocyst and which precedes the appearance of embryonal diffei-entiatiou. It must be remembered, however, that the growing
blastocyst in the Monotreme is bilaminar and not unilaminar
as in Dasyurus, owing to the fact that the entoderm is established as a complete layer at a very much earlier period than
is the case in the latter. I am nevertheless inclined to regard
the attainment by the Dasyurus blastocyst of a large size,
prior to the differentiation of the embi'yonal ectoderm and the
entoderm, as a more primitive condition than that found in
other Marsupials. The pronounced hypertrophy which the
uteri of Dasyurus undergo during the early stages of gestation, an hypertrophy which appears to be proportionately
greater than that met with in other forms,^ is no doubt to be
correlated with the presence in them of such a considerable
number of actively growing blastocysts.
Selenka states (Heft 5, p. 180) that he examined seven
blastocysts of Dasyurus “-f mm.” in diameter, taken from a
female fifteen days after copulation. He describes their
structure as follows : “ Man unterscheidet (1) eine sehr
zarte aussere, homogene Haut (Granulosamembran), (2)
' Por example, the uteri of a female (5, 18 . vii . '01) from which 1
obtained twenty-one normal vesicles, 4'5-6 mm. in diameter, with the
embryonal area definitely established, measured as follows : Left uterus,
4'5 X 4'7 X 1'4 cm. (fourteen vesicles) ; right uterus, 4'5 X 4'2 x 1‘45 cm.
(seven vesicles and one shrivelled).
darmiter ein Lagei' von Ektodermzellen, welche im Gebiete
des Embryonalschildes prismafcich, am gegeniiberliegenden
Pole nahezu kubisch, im iibrigen abgeplattet erscbeinen, (3)
ein inneres zusammenliangeudes Lager von abgeflacbten Entodermzellen.” This description, apart from the reference to
the thin shell-membrane, is entii'ely inapplicable to blastocysts
of Dasyurus of the mentioned size which I have studied.
I have examined a practically complete series of vesicles of
Dasyinms ranging from '4 mm. to 4 mm. in diameter and all of
them without exception are unilaminar.
Of vesicles under 1 mm. diameter I possess serial sections
of more than two dozen, I'anging from '5 mm. to '8 mm. in
diameter, and obtained from three different females. These
differ structurally in no essential respect from the just completed blastocysts. A surface view of a blastocyst '6 mm. in
diameter is shown in fig. 63, PI. 6; in this the difference in
the cytoplasmic chai'acters of the cells of opposite hemispheres
is clearly brought out, the non-formative cells of the lower
hemisphere having much more marked perinuclear zones of
dense cytoplasm (deutoplasm) than the formative cells of the
upper hemisphere ; moreover, the former cells tend to be of
larger superficial extent than the latter. Pig. 34, PI. 3,
represents a section of a blastocyst '57 mm. in diameter, and
fig. 35 a section of one '73 mm. in diameter. These blastocysts differ in no essential way from the '43 mm. blastocyst
represented in fig. 33. As in the latter, the cellular wall is
unilaminar throughout, but both it and the shell-membrane
have undergone considerable attenuation. Moreover in these
blastocysts, apart from the clue afforded by the shrivelled
yolk^body, it is practically impossible to determine from the
sections which is morphologically the upper hemisphere and
which the lower. In fig. 36, from a '6 mm. blastocyst, on the
other hand, the cells of the hemisphere opposite the yolk-body
(y.b.) are larger than those of the hemisphei'e adjacent to
which that body is situated. In the '57 mm. blastocyst the
shell-membrane has a thickness of ‘0052 mm., in the -73 mm.
blastocyst it measures -0045 rnrh., and in a -84 mm. blastocyst
•0026 mm. The zona is now no longer recognisable as an
independent membrane. In blastocysts of this stage of
growth a variable number of small spherical cells or cellfragments are frequently met with in the blastocyst cavity,
usually lying in contact Avith the inner aspect of the cellular
wall (fig. 34, i.c.). In some blastocysts such structures are
absent, in others one or two may be present, in yet others
numbers of them may occur. They raa,y be definitely nucleated,
but this is exceptional; more usually they contain one or more
deeply staining granules (of chromatin ?), or are devoid of
such. They ai'e of no morphological importance, and I think
thei*e can be no doubt that they represent cells or fragments
of cells which have been separated off from the cellular wall
during the process of active growth. They are of common
occurrence in later blastocysts, and it is possible the so-called
“ yolk-balls ” observed by Selenka in Didelphys are of the
same nature.
If we pass now to vesicles from 1 to 3 or 3'5 mm. in
diameter, we find the wall still unilaminar, but considerably
more attenuated than it is in the blastocysts last referred to.
In a vesicle with a diameter of 1'24 mm. the shell-membrane
has a thickness of about '0015 mm., whilst the cellular wall
has a thickness of only '0045 mm. In a 3'5 mm. vesicle the
shell-membi'ane measures about •0012 mm., Avhilstthe cellular
wall ranges from •OOlS to •OOIS mm. in thickness. A small
portion of the wall of a vesicle, 2^4 mm. in diameter, is shown
in PI. 6, fig. 64. In these later vesicles I have failed to detect,
either in surface examination of the vesicles in to to or in
sections, any regional differences between the cells indicative
of a differentiation of the wall into upper or formative, and
lower or non-formative, hemispheres. Everywhere the wall
is composed of flattened, exti'emely attenuated cells, polygonal
in surface view, and all apparently of the same character. It
might therefore be supposed that the polarity, which is recognisable in early blastocysts, and which is dependent on the
pronounced differences existent between the cells of the
upper and lower rings of the 16-celled stage, is of no funda
mental importance, since it apparently becomes lost at an
early period during the growth of the blastocyst. Such an
assumption, however, would be very wide of the maxk, as I
hope to demonstrate in the next section of this paper, and,
indeed, in view of the facts already set forth, is an altogether
improbable one.
Reappearance of Polar Differentiation in the
Blastocyst Wall.  -  Following on the period of what may
be termed the preliminary growth of the blastocyst, in the
course of which the original polar differentiation in the
blastocyst wall apparently becomes obliterated, is an
extremely interesting one, during which that differentiation
again becomes manifest. In view of the fact (1) that the
fourth cleavage in Dasyurus is of the nature of a qualitative
cytoplasmic division, and (2) that approximately one half or
rather less of the unilaminar vosicle wall is formed from the
eight smaller and less yolk-rich cells of the upper ring of the
16-celled stage, and its remainder from the eight larger
more yolk-rich cells of the lower ring, it thus becomes a
question of the first importance to determine if we can the
significance of that differentiation.
Amongst the Eutheria, it has been conclusively shown by
various observers (Van Beneden, Heape, Hubrecht, Assheton,
and others) that there occurs during cleavage an early
separation of the blastomeres into two more or less distinctly
differentiated groups, one of which eventually, by a process
of overgrowth, completely encloses the other. The peripheral
cell-group or layer forms the outer extra-embryonal layer of
the wall of the later blastocyst (the trophoblast of Hubrecht,
or trophoblastic ectgderm as I prefer to term it). It therefore
takes no direct part in the formation of the embryo, and may
be distinguished as non-formative. The enclosed cell-group,
termed the inner cell-mass or embryonal knot, gives rise, on
the other hand, to the embryonal ectoderm as well as to the
entire entoderm of the vesicle, and may accordingly be distinguished as formative. May it not be, then, that we have
here at the fourth cleavage in Dasyurus a separation of the
blastomeres into two determinate cell-groups, respectively
foi'mative and non-formative in significance, entirely compar-,
able with, and, indeed, even more distinct than that which
occurs during cleavage in the Eutheria ? I venture to think
that the evidence brought forward in this paper conclusively
justifies an answer in the affirmative to that question.
If we assume that the upper cell-ring of the 16-celled stage
in Dasyurus is formative in destiny and the lower cell-ring
non-formative, then we might naturally expect to find in the
unilaminar wall of the later blastocyst some differentiation
indicative of its origin from two distinct cell-groups, and
indicative at the same time of the future embryonal and
extra-embryonal regions. Now just such a differentiation,
does, as a matter of fact, become evident in vesicles 3'5 to
4'5 mm. in diameter. We have already seen that the wall in
early blastocysts '4 to '8 mm. in diameter exhibits a wellmarked polar differentiation in correspondence with its mode
of oi'igin from the diffei-entiated cell-rings of the 16-celled
stage, its upper hemisphere or thereabouts consisting of
smaller cells, poor in deutoplasm, its remainder of larger
cells, rich in deutoplasm. .In later blastocysts, 1-3 mm. or,
more in diameter, it is no longer possible to recognise this
distinction  -  at all events I have failed to observe i't  -  but if
we pass to blastocysts 4-5 mm. in diameter, in which the wall
is still unilaminar, we find on careful examination of the
entire vesicle under a low power that there is now present a
definite continuous line^ which encircles the vesicle in theequatorial region so as to divide its wall into two hemi-,
spherical areas (PI. 4, fig. S8,j.L). If we remove and stain;
a portion of the wall of such a vesicle, including this line,)
and examine it microscopically (figs. ,42-46.), it becomes
apparent at once, from the .disposition of the cells on either
side of the bns, that we have to do with a sutural line or line
of junction produced by the meeting of twp sets- of cells,'
which are pursuing their .own, independent courses of growth
and division. ; The, cells never cross the demax'cation line
from the one side tn the other, but remain strictly confined
to their own territory, so that we are justified in regarding
the vesicle wall as composed of two independently growing
zones. Now tlj^ existence of two such independent zones in
the unilaminar wall is, to my mind, only intelligible on the
view that they are the products of two originally distinct,
predetermined cell'groups, and if this be admitted, then I
think we are justified in concluding, in view of the facts
already set forth, that tlie two zones in question are derived,
the one from the upper cell-ring of the 16-celled stage, the
other from the lower ring ; that, in other words, they represent respectively the upper and lower hemispheres of the
early blastocysts.
If, now, we find that the embryonal ectoderm and the entoderm arise from one of these two regions of the unilaminar
wall, whilst the other directly forms the outer extra-embryonal
layer of the later (bilaminar) vesicle, then we must designate
the former region as the upper or formative, and the latter as
the lower or non-formative. Further, bearing in mind the
characters of the cells of the two rings of the 16-celled stage,
T think we are justified in holding that the formative region
is derived from the ring of smaller, less yolk-rich cells, and
the non-formative region from the ring of larger, more yolkrich cells, even if it is impossible to demonstrate an actual
genetic continuity between the constituent cells of these two
rings and those forming the independently growing areas of
the later blastocyst. I have recently re-examined a series of
vesicles, measuring 1'5-1'8 mm. in diameter, obtained from a
female killed in 1906, and I have so far found it impossible,
either in the entire vesicle or in portions of the wall stained
and mounted on the flat, to distinguish between the cells over
opposite hemispheres. Thus the only actual guide Ave have
for the determination of the poles in such vesicles is the
yolk-body, and though the latter is liable to- displacement, it
is Avorthy of record that I have several times found it in
relation to the formative area in vesicles 4‘5-6 mm. in
diameter, but never in relation to the non-formative region.
This evidence is, therefore, so far as it goes, confirmatory of
VOL. 56, PART 1.  -  NEAV SERIES. 4
the conclusion reached above, viz, that the formative hemisphere is derived from the smaller-celled ring of the 16-celled
stage. On that conclusion is based my interpretation of the
poles in the unsegmented ovum, and of the two cell-rings o£
the 16-celled stage as respectively upper and lower.
Of vesicles ov'er 1 mm. in diameter, the smallest in which I
have been able to detect the sutural line above referred to
measure 3'25 mm. in diameter. In three lots of vesicles, 3'5
mm. in diameter from three different females, I have failed to
X'ecognise it, whilst in two other lots, respectively 3'75 mm.
(average) and 4 mm. in diameter, the line appears to be in
course of differentiation as in the 3'25 mm. vesicles. A
portion of the wall of one of the 3'5 mm. vesicles just referred
to is shown in PL 4, fig. 41, and a portion of the wall of the
3'25 mm. stage, including the sutural line, in fig. 42. Both
vesicles were fixed in the same fluid, viz. picro-nitro-osmic
acid. Comparison of the two figures reveals the existence, quite
apart from the presence of the junctional line in fig. 42, and its
absence in fig. 41, of certain more or less obvious differences
between them. In fig. 41 the cells are larger, and their cytoplasmic bodies are inconspicuous, being fairly homogeneous
and lightly staining. In fig. 42, on the contrary, the cellbodies are strongly marked, the cytoplasm being distinguishable into a lighter-staining peripheral zone, and a much more
deeply staining perinuclear zone, showing evidence of intense
metabolic activity. This latter zone is more or less vacuolated,
and contains, besides larger lightly staining granules, numerous
smaller ones of varying size, stained brown by the osmic acid
of the fixative. In the 4 ram. vesicles the cells show pi-ecisely
the same characters; in the 3'75 mm. vesicles, which were
fixed in a picro-corrosive-acetic fluid, the granules ax'e absent
from the cytoplasm, otherwise the cells are similar to those
of the other two. Mitotic figures are common. The sutural
line is recognisable in all three sets of vesicles (3'25, 3'75, and
4 mm.) (fig. but I cannot be certain that it runs con
tinuously round, and it appears to have a rather more sinuous
course than in later blastocysts. The cells of the two regions
of the bliistocyst wall, separated by the sutural line, differ
somewhat in tlieir characters. On one side of the line (fig.
42, tr.ect.) the cells appear to be on the whole slightly larger,
and of more uniform size than they are on the other, and they
also stain somewhat more deeply. Comparison with later
blastocysts shows that the region of more uniform • cells is
non-formative, that of less uniform, formative. At^this stage,
however, the differences between the cells of the two regions
are as yet so little pi'onounced that it is practically impossible
in the absence of the sutural line to say to which hemisphere
an isolated piece of the wall should be referred.
I am inclined to regard the sutui'al line in these vesicles as
being in course of differentiation, and judging from the disposition of the cells on either side of it, I think its appearance
is to be correlated with the marked increase in the mitotic
activity of the cells of the two hemispheres which sets in in
vesicles of 3-4 mm. diameter. The preliminary increase in
size of the blastocyst up to about the 3 mm. stage might be
described as of a passive character, i.e. it does not appear
to be effected as the result of the very active division of the
wall-cells, but is characterised rather by a minimum of mitotic
division and a maximum of increase in surface extent of the
cells, due to excessive stretching consequent on the rapid
imbibition of uterine fluid. Once, however, the requisite size
has been attained, the cells of the unilaminar wall commence
to divide activel}', and doubtless as the outcome of that
wave of activity, the sutural line makes its appeai-ance
between the two groups of independently growing cells.
On the inner surface of the blastocyst wall, especially in
the region of the formative hemisphere, there are present
in these vesicles numbers of small deeply staining cells of
spherical form, and containing osmicated granules similar
to those in the wall-cells. They may occur singly or in groups,
and appear to me to be of the same nature as the inteimal cells
of the earlier blastocyst. In addition to these cells, there are
present clusters of cytoplasmic spheres, staining similarly to
the spherical cells, and apparently of the nature of fragmeiita
tion products formed either directly from the â– \vall-cells or
from these internal cells.
2. Differentiation of the Embryonal Ectoderm and
the Entoderm.
After the preliminary growth in size of the blastocyst is
completed, the next most important step in the progressive
development of the latter is that just dealt with, involving
the appearance of the sutural line, with resulting re-establishment of polar differentiation in the blastocyst wall. Following
on that, we have the extremely important period during
which the embryonal ectoderm and the entoderm become
definitely established.
For the investigation of the earlier phases of this critical
period I have had at my disposal a large number of
unilaminar blastocysts derived from three females, distinguished in my notebooks as (3, 25 . vii . '01, with fifteen
vesicles of a maximum diameter of 4‘5 mm. ; 8 . vii . '99, with
twelve vesicles, 4‘6 .mm. in diameter ; and 6 . vii . '04, with
twfenty-two vesicles, 4‘5 and 5 mm. in diameter. These three
lots of vesicles may for descriptive purposes be designated
as '01, '99, and '04 respectively.
The '01 vesicles are distinctly less advanced than the
other two. The sutural line is now, at all events, definitely
continuous, and can readily be made out in the intact vesicle
with the aid of a low-power lens (PI. 4, fig. 38, j.L), but
the differences between the cellular constituents of the two
hemispheres which it separates are much less obvious than
they are in the '99 and '04 vesicles. Here, again, one
hemisphere forming half or perhaps rather more of the entire
vesicle is distinguished from the other by the greater uniformity and the slightly deeper staining character of its
constituent cells (figs. 43 and 44, tr. ecL). This hemisphere,
subsequent stages show, is the lower or non-formativ^
hemisphere. It is characterised especially by the striking
'uniformity in the size of its cells. Over the opposite hemisphere, the upper or formative one (figs. 43 and 44, the
cells are more variable iu size, the nuclei thus appearing less
uniformly and less closely arranged, and they stain,. on the
whole, somewhat less deeply than those of the lower hemisphere. The non-formative cells are on the average smaller
than the largest of the formative cells, but they are more
uniform iu size, and their nuclei thus lie at more regular
distances apart, and appear more closely packed. They are
also richer in deutoplasmic material, and so stain rather more
deeply than the formative cells. Sections show that the
cellular wall is unilaminar throughout its extent, and that,
whilst it is somewhat thicker than that of 3‘5 mm. vesicles,
it is still very attenuated, its thickness, including the shellmembrane, ranging-from ‘004 to '008 mm. I have examined
a number of series of sections taken through portions of the
wall known to include the sutural line, and find it quite
impossible to locate the position of the- latter; indeed, I
cannot certainly distinguish between the formative and nonformative regions.
In the blastocyst cavity, lying in contact with the inner
surface of the wall, and most abundant in the region of the
formative hemisphere, there are present numbers of deeply
staining spherical cells with relatively small nuclei similar to
those described in connection with the 3'25 mm. vesicles.
They occur singly or in groups, and may appear quite normal
or may show more or less evident signs of degeneration.
Their nuclei may stain deeply and homogeneously, or may be
represented by one or two deeply staining granules, vacuoles
may occur in their cytoplasm, and spherical cytoplasmic masses
of very variable size, with or without deeply staining granules
of chromatin) may occur along with them. In sections and
preparations of the wall of these, and other 4*5 mm. vesicles
there are to be found, in both the formative and non-formative
hemispheres, small localised areas from which such spherical
cells are being proliferated off in numbers together. PI. 5,
fig. 47, from the formative hemisphere of an ^04 vesicle shows
One of the most marked examples of such proliferative. activity
that I have encountered. A similar but smaller proliferative
J. ?. HILL.
area occurs on the non-formative hemisphere of the same
These spherical cells are, I am convinced, of no morphological importance, and are destined sooner or later to degenerate. They have certainly nothing to do with the
entoderm, the parent-cells of that layer arising exclusively
from the formative hemisphei'e and not from cells such as
these, which are budded ofE from both hemispheres. The fact
that they are, in unilaminar vesicles, more numerous over the
formative hemisphere may perhaps be taken as an indication
of the greater mitotic activity of the formative as compared
with the non-formative cells.
The Primitive Entodermal Cells.  -  Following closely
on the stage represented by these '01 blastocysts is the extremely important one constituted by the '99 and '04 vesicles
before referred to. This stage is the crucial one in primary
germ-layer formation, and marks the transition from the unilaminar to the bilaminar condition, since in it the entodermal
cells are not only distinctly recognisable as constituents of the
formative region, but are to be seen both in actual process of
separation from the latter and as definitely internal cells, frequently provided with, and even connected together by,
pseudopodial-like processes of their cell-bodies. Such cells
are already present in the '01 vesicles (fig. 71), and probably
also in the blastocysts in which the sutural line first makes
its appearance, but are much less conspicuous than in these
older blastocysts.
The '99 blastocysts are distinctly more advanced than the
'01 batch and are just a little earlier than the '04 lot. The
former measui'ed, as already mentioned, 4'5 mm. in diameter,
the latter 4'5 and 5 mm. (the majority being of the latter
size). In my notes, on the intact '99 vesicles I find it stated
that one hemisphere, forming rather less than half of the
entire extent of the vesicle wall, appeared somewhat denser
than the other, the sutural line marking the division between
the two. I naturally inferred at the time that the denser
hemisphere corresponded to the embryonal region of the
Eutherian blastocyst and the less dense to the extra-embryonal region of the same, but just the reverse proves to hold
true for the '04 vesicles, the formative hemisphere in these
appearing less dense than the non-formative. I cannot now
test my former inference by direct observation since I do not
appear to have any of the '99 vesicles left intact, but amongst
my in toto preparations of the vesicle wall I find one
labelled as from the “ lower pole ” which unmistakably
belongs to the formative hemisphere, hence I conclude that
the denser and slightly smaller region which I originally
regarded as formative is really non-formative, a conclusion
which brings the '99 vesicles into agreement with the '04
In these latter vesicles the sutural line and the two regions
of the wall can be quite readily made out on careful examination under a low power with transmitted light. The one
region appears slightly denser (darker) and has more closely
arranged nuclei (i. e. is composed of smaller cells) than the
other. On the average this denser region appeal's to be
rather the less extensive of the two ; the two regions may be
about equal ; on the other hand the denser may be the smaller.
Examination of stained preparations of the wall demonstrates
that the darker hemisphere is non-formative, the lighter,
formative. It would therefore seem that in certain of these
'04 vesicles the formative region has grown more rapidly than
the non-formative.
In stained preparations of the wall both of the '99 and '04
vesicles, the differences between the two hemispheres are now
so well marked that there is no diflBculty in referring even an
isolated fragment to its proper region. The non-formative
hemisphere differs in no essential way from that of the '01
vesicles, and as in these, is readily distinguishable from the
formative by the much greater uniformity in the size and
staining properties of its cells (fig. 45), as well as by the fact
that there are no primitive entodermal cells such as occur in
relation to the formative hemisphere, in connection with it.
Its constituent cells are on the average distinctly smaller than
the largest of the formative ; their nuclei lie nearer each other,
with the result that in surface examination of the blastocyst
the non-formative region appears rather denser than the
formative. In in toto preparations of the wall the former
usually stains darker than the latter (fig. 45), but this is not
always the case ; in fig. 46, from an '04 vesicle, there is
practically no difference in this respect between the two
regions ; in yet others of my preparations of '99 vesicles the
formative region has stained more deeply than the nonformative.
The formative hemisphere in the earlier blastocysts of this
particular developmental stage was described (ante, p. 51) as
differing from the non-formative in that its constituent cells
were much less uniform in chai*acter than those of the latter.
This same feature, but in much enhanced degree, characterises
the formative region of the vesicles under consideration, for it
can now be definitely stated that the latter I'egion is constituted by cells of two distinct varieties, viz. (1) moi*e lightly
staining cells which form the chief constituent of the formative region, its basis so to speak, and which are on the
average larger than those of the other variety, and (2), a less
numerous series of cells, distinctly smaller than the largest
cells of the former variety, and with denser, more granular and
more deeply staining cytoplasm, and frequently met with in
mitotic division (cf. PI. 6, fig. 65). The two varieties of cells are
intermingled promiscuously, the smaller cells occurring singly
and in groups but in a quite irregular fashion, so that here
and there we meet with patches of the wall composed exclusively of the larger cells.
The evidence presently to be adduced shows that the larger
cells furnish the embryonal ectoderm, and that the smaller
cells give origin to the primitive entodermal cells from which
the definitive entoderm arises. The smaller cells may therefore be regarded as entodermal mothei'-cells. Whether these
latter cells are progressively formed from the larger cells
simply by division, or whether the two vaifieties become
definitely differentiated from each other at a particular stage in
development, must for the present be left an open question. Of
the actual existence in tlie unilaminar formative region of these
'99 aud '04 blastocysts of two varieties of cells, respectively
ectodermal and entodermal in significance, there can be no
doubt. In preparations of the formative region, however,
whilst one can without hesitation identify certain cells as
being in all probability of ectodermal significance and others
as prospectively entodermal (cf. figs. 65, 66), it must be
admitted that one is often in doubt as to whether one is
dealing with small ectodermal cells or with genuine entodermal mother-cells. It is, therefore, hardly to be wondered
at that I have not yet been able to satisfactorily determine
at what precise period the entodermal mother-cells first
become differentiated, though judging from the facts that
in the eai-liest vesicles in which the sutural line is recognisable one region of the wall already differs from the other in
the less uniform size of its constituent cells, and that internally
situated entodermal cells are already present in small numbers
in the '01 vesicles (fig. 71), I incline to the belief that it
will probably be found to about coincide with the first
appearance of the sutural line. To this question I may
perhaps be able to return at some future time.
In addition to the presence of these entodei'mal mothercells, which enter directly into its constitution, the formative
region of the '99 and '04 blastocysts is. characterised by the
occurrence on its inner surface of definitely iuteimal cells,
which generally agree with the former cells as regards size
and staining properties and are evidently related to them. It
is these internally situated cells which directly give origin to
the definitive entoderm of the later blastocysts, and one need,
therefore, have no hesitation in applying to them the designation of primitive entodermal cells. They are exclusively found
in relation to the formative hemisphere, and appear in in toto
prepai'atious as flattened, darkly staining cells closely applied
to the inner surface of the unilaminar wall, and disposed quite
irregularly, singly, and in groups. They vary greatly in
number in blastocysts of even the same batch, but on the
J. r. HILL.
wholo are most abundant in the ^04 series, and they also
exhibit a remarkable range of variation in shape. They may
have a perfectly distinct oval or rounded outline (figs. 67, 71,
72), or, as is more frequently the case, they may lack a
determinate form and appear quite like amoeboid cells owing
to their possession of cytoplasmic processes of markedly
pseudopodial-like character (fig. 69). Frequently, indeed,
the cells are connected together by the anastomosing of these
processes, so that we have formed in this way the beginnings
at least, of a cellular reticulum (figs. 68, 69,70).
The question now arises. How do these primitive entodermal cells originate from the small, darkly staining cells of
the unilaminar formative region designated in the foregoing
as the entodermal mother-cells ? I can find no evidence that
the primitive entodermal cells are formed by the division of
the mother-cells in planes ta.ngential to the surface ; on the
contrary, all the evidence shows that we have to do here with
an actual inward migration of the mother-cells, with or without previous mitotic division, such inward migration being
the outcome of the assumption by the mother-cells, or their
division products, of amoeboid properties ; in other words, the
evidence shows that the formation of the entodei'm is effected
here not by simple delamination (using that term in the sense in
which it was originally employed by Lankester), but by a process involving the inward migration, with or without previous
division, of certain cells (entodermal mother-cells) of the unilaminar parent layer, a process comparable with that found in
certain Invertebrates (Hydroids) and distinguished by Metschnikoff as '^gemischte Delaminatiou.”
In this connection it has to be remembered that the cells of
the unilaminar wall of the blastocyst are under considei'able
hydrostatic pressure, and, in correlation therewith, tend to
be tangentially flattened, though the flattening in this stage
is much less than in the earlier blastocysts. From a series of
measurements made from an '04 vosicle, I find that over the
formative region the ratio of the breadth to the thickness of
the cells varies Horn 6 : 1 to 2 : 1, and even to 3 : 2. On the
whole cells of the type indicated by the ratio 6 : 1 predominate,
and we should hardly expect to find such cells dividing tangentially. In fact, the only undoubted examples of such division I
have met with occur in the single abnormal vesicle present in
the '04 batch. In this particular vesicle, which had a diameter
of 3 mm. and was thus smaller than the others, thei'e was
present on what appeared to correspond to the formative
hemisphere of the normal blastocyst a well-defined and conspicuous ovalish patch, 1'23 x '99 mm. in diameter.^ Sections
show that over this area the cells of the unilaminar wall are
much enlarged and , more or less cubical in form, their thickness varying from ‘012 to ‘019 mm. These cubical cells
exhibit distinct evidence of tangential division, both past and
in progress. But in normal vesicles, whilst mitotic figures are
quite commonly met with in the cells of the formative region
(in which, indeed, they are more numerous than in those of
the non-formative region), I have failed to find in my sections
after long-continued searching even a single spindle disposed
directly at right angles to the shell-membrane ; the mitotic
spindles lie disposed either tangentially to the surface or
obliquely thereto.
For the determination of the mode of origin of the
primitive entodermal cells, it is absolutely necessary to
study both in to to preparations of the formative region,
i.e. small portions of the unilaminar wall stained and
mounted on the flat, and sections of the same. Sections alone
are, on the whole, distinctly disappointing so far as the
question under discussion is concerned, and, indeed, give one
an altogether inadequate idea of the primitive entodeimial cells
themselves, seeing that practically all one can make out is that
1 Curiously enough, amongst the '99 vesicles there also occurred
a single small one, likewise 3 mm. in diameter, and with a thickened
patch 1-28 X 1 mm. in diameter, quite similar in its character to that
described in the text. I am as yet uncertain whether the thickened
area in these two vesicles represents the whole of the formative hemisphere of normal blastocysts or only a hypertrophied part of the same,
or whether, indeed, it may not represent the I'etarded non-formative
there are present, in close apposition with the inner surface of
the unilaininar wall, small, darkly staining cells, apparently
quite isolated from each other and usually of flattened form
(figs. 73, 74, 76, ent.). One has only to glance at a wellstained in to to preparation of the formative region (cf.
fig. 70) to realise how inadequate such a description of the
primitive entoderm cells really is.
Sections nevertheless do yield valuable information on
certain points. Besides affording the negative evidence of
the absence of tangential divisions and the positive evidence
that the primitive entodermal cells are actually internal (figs.
73, 74, 76), they show that growth of the wall iu thickness
has already set in, and that it is most marked over the
formative region, though the thickness attained by the cells
is as yet very unequal (figs. 73-76). Measurements takeu
from an '04 vesicle show that over the non-formative region
(fig. 77) the cells vary in thickness from *006 to '009 mm.,
whilst over the formative region the range of variation is
greater, viz. from ‘006 to ‘013 mm., so that we may conclude
that the latter region is on the average thicker than the
former (cf. figs. 73-76, with fig. 77 depicting a small portion
of the non-formative region). It is still impossible to determine the position of the sutural line, even in sections of
fragments of the wall known to contain it.
The entodermal mother-cells are not very readily recognisable in sections. In fig. 75, however, which is drawn
from an accurately transverse section through the formative
region of an '04 vesicle, there is depicted what is undoubtedly
an entodermal mother-cell {ent.). The interesting point
about this particular cell is that its cell-body, whilst still
intercalated between the adjoining cells of the unilaminar
wall, has extended inwards so as to directly underlie one of
the wall-cells. ' Division of such a cell as this would necessarily result in the production of an internally situated cell
with all the relations of one of the primitive entodermal type.
The inwardly projecting spheroidal cell situated immediately
to the left (in the figure) of the one just refeiTed to, I also
regard as an entodermal mother-cell. Cells of this type are
not infrequently met with in sections; they nsually stain
somewhat deeply, and are often found in mitosis.
The evidence obtainable from the study of in to to preparations conclusively proves that some at all events of the
primitive entodermal cells are actually derived from the entodermal mother-cells, much in the-way suggested above, whilst
others of the primitive entodermal cells are directly formed
from mother-cells which bodily migrate inwards.
Fig. 65, PI. 6, represents a small portion of the formative
region of an '04 vesicle viewed fPom the inner surface. In
the centre of the figure, surrounded by the larger, lighter
staining (ectodermal) cells of the wall, is a smaller cell in the
telophases of division, the cytoplasm of which is granular and
stains deeply. That cell unmistakably forms a constituent of
the unilaminar wall. I regard it as an entodermal mothercell. Fig. 66 shows another cell of the same character in the
anaphases of division, which likewise forms a constituent of
the unilaminar wall, but which differs from the corresponding
cell in fig. 65 in that its cytoplasmic body has extended out
on one side (lower in the figure), so as to directly underlie
part of an adjacent ectodermal cell. In other words we have
here a surface view of the condition represented in section in
fig. 75, only the entodermal mother-cell depicted therein is not
actually in process of division. Fig. 67, taken from the same
preparation as fig. 65, shows what I take to be the end result
of the division of such a cell as is i-epresented in the two
preceding figures. Here we see two small deeply staining
cells towards the centre of the figure, which from their disposition and agreement in size and cytological characters
are manifestly sister-cells, and the products of division of
just such an entodermal mother-cell as is represented in fig.
65, or, better, fig. 66. The one cell (upper in the figure) is
more angular in form and manifestly still lies in the unilaminar wall; the other (lower in the figure) is ovalish in form
and is no longer a constituent of the unilaminar wall, but is
on the contrary a free cell, definitely internal both to the
J. P. HILL. â–  . â–  :
latter and to its sister-cell. It is, in fact, a primitive entodermal cell, as comparison with fig. 68 proves, and that it has
been formed by the division of a mother-cell situated in the
unilaminar wall can hardly, I think, be doubted. Its sistercell, which is still a constituent of the wall, would presumably
have migrated inwai-ds some time later.
It is to be noted that the primitive entodermal cell referred
to above and those depicted in figs. 71 and 72 are definitely
contoured, ovalish and I'ounded cells, entirely devoid of processes. In these respects they differ markedly from the entodermal cells shown in fig^. 68-70, which are very variable in
form owing to their possession of more or less elongated
pseudopodial-like processes. It might thex'efore be inferred
that the formation of these processes only takes place after
the entodermal cells have become definitely internal. Such
an inference, however, would be incorrect, for I have abundant
evidence showing that such processes may be given off from
the entodermal mother- cells whilst they are still constituents
of the wall. In in toto preparations, it is often difficult to
determine with certainty whether a particular entodermal cell
still enters into the constitution of the unilaminar wall or not.
In the portion of the formative region of a '04 vesicle depicted
in fig. 70, however, I am satisfied that all the entodermal
cells therein shown (they are readily distinguishable by their
smaller size and more deeply staining character) are, with the
possible exception of the one on the extreme right, at least
partially intercalated between the larger ectodermal cells of
the wall. Some of them are entirely situated in the wall ;
others have extended inwards in varying degree so as to
partially underlie the ectodermal cells. It is these latter
entodermal cells in particular which exhibit the cytoplasmic
processes above referred to. As the figure shows, these processes have all the characters of pseudopodia,; they vary in
size, form, and number from cell to cell, individual processes
may be reticulate and their finer prolongations may anastomose with those of others, and they are formed of cytoplasm,
less dense and rather less, deeply staining than that of the
cell-bodies from which they arise. Attention may be specially
directed to the cell towards the left of the hgure (mai'ked ent.).
Here we have an entodermal cell whose cytoplasmic body is
evidently still partially intercalated between the cells of the wall,
but which is, at the same time, prolonged inwards (towards
the left) so as to underlie the adjoining ectodermal cell.
From this inward prolongation there are given off two slender
processes, one short and tapering, the other very much
longer ; this latter, after becoming vei'y attenuated, gradually
widens to form an irregular fan-shaped expansion, suckerlike in appearance, and produced into several slender
threads, which is situated adjacent to the nucleus of
the ectodermal cell on the extreme left. Then from the
right side of the same cell there is given off a small inwardly
projecting bulbous lobe which may well be the start of just
such another process as arises from the left side. Processes
of the peculiar sucker-like type just described, formed of a
slender elongated stem and a distal expanded extremity from
which delicate filamentous prolongations are given off, are
abundantly met with in preparations, and strikingly recall the
pseudopodia of various Ehizopoda. They are seen in connection with other entodermal cells in fig. 70, and with many
of those in fig. 68. I regard them as veritable pseudopodia.
Towards the right side of fig. 70 the two entodermal cells
there situated stand in direct protoplasmic continuity by
means of two slender connecting threads, whilst the upper of
these two cells is again joined by a very fine process to the
irregular pseudopodial expansion which arises from one of
the two entodermal cells situated nearer the middle of the
figure, and that same expansion is directly connected with the
second of the two entodermal cells just mentioned, so that we
have here established the beginning of a cell-network, prior
to the complete emancipation of its constituent entodermal
elements from the unilaminar wall. We have, then, clear
evidence that the entodermal elements in Dasyurus, prior to
their separation from the unilaminar formative region ai*e
capable of exhibiting amoeboid activity, since not only may
J. r. HILL.
they send lobose prolongations of their cytoplasmic bodies
inwards below the adjacent ectodermal cells, but they may
emit more or less elongated processes of indubitable pseudopodial character, which similarly lie in contact with the inner
surface of the wall-cells. Furthermore, we have evidence
that these pseudopodial processes may anastomose with each
â– other so as to initiate the formation of an entodermal reticulum,
whilst the cells from which they arise are still constituents of
the unilaminar wall  -  an especially noteworthy phenomenon.
Certain of the primitive entodermal cells, as we have seen,
are at first devoid of such processes, but since they all
eventually form part of a continuous reticulum, it is evident
that the entodermal elements are capable of emitting pseudopodial processes as well after as before their separation from
the formative region.
Finally, in view of the fact that the entodermal mothei'-cells
depicted in fig. 70 are not actually in process of division, and
therein differ from those of figs. 65 and 66, we may conclude
that the formation of the primitive entodermal cells is effected
either with or without the pi*evious division of the mother-cells.
If Ave admit, as I think on the evidence we must admit,
that the entodermal cells in Dasyurus are endowed with
amceboid properties, then Ave are relieved of any further
difficulty in regard to the mechanism of their inAvard migration
from the unilaminar Avail. Doubtless, in the case of those
entodermal mother-cells Avhich do not undergo division, the
precocious formation of the above-described pseudopodial
processes which spread out from the cells like so many
suckers considerably facilitates their direct detachment from
amongst the cells of the Avail. In the case of those primitive
entodermal cells Avhich originate as the direct products of
division of the mother-cells, it no doubt depends on a variety
of circumstances (e.g. actual form of the dividing cell,
direction of the spindle, etc.) whether they exhibit amoeboid
activity precociously (i.e. before their actual i separation), or
only at a later period.
The entoderm varies considerably in its degree of diffe
rentiation iu different vesicles of this stage, and even in
different parts of the formative region of one and the same
vesicle. In some vesicles there are relatively few primitive
entodermal cells, in othei*s they are much more abundant.
Fig. 68, from the formative region of an ^04 vesicle, shows a
typical patch of them and illustrates very well the highest
stage of differentiation which they attain in these vesicles. The
entodermal cells therein depicted all appear to be definitely
internal, and it is especially worthy of note that the portion
of the unilaminar wall in relation to them is composed exclusively of the larger, lighter staining cells. It is these cells
which directly form the embryonal ectoderm of the blastocysts
next to be described. The entodermal cells are obviously
amoeboid in character (obsei've especially the cells near the
middle of the figure), and are in active process of linking
themselves together into a cellular reticulum. In fig. 69 is
shown a small portion of the formative region of another ^04
vesicle. A single entodermal mother-cell in process of
division occurs in position in the unilaminar Avail, which is
otherwise composed of ectodermal cells, whilst internally there
are present three entodermal cells, already linked together by
their pseudopodial processes. ^Jfiie two lowermost cells afford
especially striking examples of amoeboid activity, the elongated
pseudopodial process of the cell on the left terminating iu a
well-marked reticulation in definite continuity Avith the corresponding, but shorter and thicker process of the cell on the
3. Establishment of the Definitive Embryonal
FolloAving directly on the stage represented by the '04
blastocysts described in the preceding section is one designated in my list as 5, 18 . vii . 01 and referred to here as 5, '01.
It comprises twenty-two blastocysts obtained from a female
killed fifteen days after coition and all normal, Avith the
exception of one Avhich Avas shrivelled, and all in precisely
VOL. 56, PAllT 1. NEW SERIES. 5
.T. P. HILL.
the same stag-e of development. They measured from 4‘5 to
6 mm. in diameter.
In this stage the formative region of the preceding blastocysts has become transformed into the definitive embryonal
area (embryonic shield, Hubrecht) as the result of the completion of that process of inward migration of the entodermal
mother-cells which we saw in pi-ogress in the vesicles last
described, and the consequent establishment of the entoderm
as a continuous cell-layer undeidying and independent of, the
embryonal ectoderm constituted by the larger passive cells of
the original unilaminar formative layer.
In the entii*e blastocyst (PI. 4, fig. 39) the embryonal area
is quite obvious to the naked eye as the more opaque, hemispherical region, forming rather less than half the entire
extent of the vesicle wall ; the larger remainder of the same
is formed by the much more transpai-ent, non-formative or
extra-embryonal region. Sections of the entire blastocyst
show (1) that the embryonal area is bilaminar over its entii-e
extent, its outer layer consisting of embryonal ectoderm,
already somewhat thickened, its much thinner inner layer
consisting of entoderm, partly still in the form of a cellular
reticulum, and (2) that the extra-embryonal region is still
unilaminar throughout and composed of a relatively thin
layer of flattened cells (extra-embryonal or trophoblastic ectoderm, trophoblast [Hubrecht])^ (PI. 8, fig. 78). The entoderm
is co-extensive at this stage with the embryonal ectoderm,
and terminates in a wavy, irregularly thickened, free, edge
(PI. 5, fig. 49), which over most of its extent either directly
underlies or extends very slightly beyond the line of junction
between the embryonal and extra-embryonal ectoderm. The
junctional line is thus not very easily seen. In fig. 48, however,
' In consonance witli my conviction that this layer is homologous
both Avith the so-called trophoblast of Eutheria and the exti-a-embryonal
ectoderm of Prototheria, and in view of the theoretical signification
which Hubrecht now insists should be attached to the term “ trophoblast.” and which I am wholly unable to accept, I venture to suggest as
an alteiTiative name for this layer that of “ tropho-ectoderni.
a small portion oP the line shows with sufficient distinctness, I
think, to demonstrate its identity with that of the preceding
The vesicle wall in all my sections of this stage appears
to be somewhat thinner than that of the '04 blastocysts, but
apart from this apparently variational difference the present
blastocysts are almost exactly intermediate between the latter
and those next to be described.
The embryonal ectoderm (fig. 78, emb. ect.) appears in
section fairly uniformly thickened, though its cells are still of
the flattened type. In surface view in in toto preparations
(cf . fig. 48), they exhibit the same polygonal form and lightly
staining qualities as the larger cells of the formative region
of the '04 blastocysts, which we have already identified as
prospective embryonal ectodermal cells. The junctional line
between the embryonal ectoderm and the extra-embryonal is
now for the first time readily distinguishable in sections
(fig. 78). The extra-embryonal ectoderm (tropho-ectoderm)
(PI. 5, figs. 48 and 49, PI. 8, fig. 78, tr. ect.) differs in no
essential respect from the corresponding layer in the '04
The entoderm in these blastocysts is exceedingly closely
adherent to the inner surface of the embryonal ectoderm and
cannot be removed therefrom by artificial means. It varies
slightly in its character in different vesicles and in different
parts of its extent in the same vesicle. Mostly it appears as
a continuous thin cell-layer (figs. 49 and 78, ent.), but here and
there patches occur in which the cells form a reticulum quite
similar to that shown in fig. 68 of the preceding stage.
The next stage (designated in my list as 8 . vi . 01), and the
last of Dasyurus that need be described in the present communication, comprises eleven vesicles (5-5'5 mm. in diameter),
in which the embryonal area is conspicuous and distinctly in
advance of that of the preceding vesicles, but is still devoid
of any trace of embryonal differentiation (PI. 4, fig. 40;
PI. 8, fig. 79).
The embryonal area is hemispherical in form (its greatest
diameter varying' from 3'5 to 4 mm.) in all except two of the
blastocysts, in which it is elongate, with longer and shorter
diameters. It occupies about a third or less of the entire
extent of the vesicle wall, and thus appears relatively smaller
than that of the preceding (.5, '01) vesicles. The entoderm now
extends for a distance of about 1 mm. beyond the limits of
the area, so that in the entire vesicle (fig. 40) three zones
differing in opacity are distinguishable, viz. the dense hemispherical zone at the upper pole, constituted by the embryonal
area; below that, a less dense, narrow annular zone, formed of
extra-embryonal ectoderm and the underlying peripheral
extension of the entoderm ; and finally, the still less dense
hemispherical area, forming the lower hemisphere of the
blastocyst and constituted, solely by extra-embryonal ectoderm. Thus approximately the upper half of the blastocyst
is bilaminar, the lower half unilaminar. Sections show that
the embryonal ectoderm (fig. 79, emh. ect.) is now a quite
thick layer of approximately cubical cells, whilst the extraembryonal ectoderm {tr. ect.) is formed of relatively thin
flattened cells. The line of junction between the two is perfectly obvious, both in sections (fig. 79) and in surface view
(PI. 5, fig. 50). The embryonal ectodermal cells, though
much thicker than the extra-embryonal, are of less superficial
extent; their nuclei therefore lie closer together than those
of the latter, moreover they are larger, stain more deeply, and
are more frequently found in division, all of which facts
testify to the much greater growth-activity of the embryonal
as compai'ed with the exti-a-embryonal ectoderm at this stage
of development (cf . fig. 50, emh. ect. and tr. ect.-, in the preparation from which this micro-photograph was made the entoderm
underlying the embryonal ectoderm has been removed, whilst
it is still partially present over the extra-embryonal ectoderm).
The entoderm (fig. 79, ent.) over the region of the embrvonal area is readily separable as a quite thin membrane,
and is then seen to consist of squamous cells, polygonal in
outline, and either in direct apposition by their edges or connected together by minute cytoplasmic processes. Beyond the
embryonal area, liowever, its peripliei'al extension below the
extra-embryonal ectoderm is much less easily separable in the
intact condition (cF. fig. 50), because oF its greater delicacy
due to the fact that it has here largely the form of a cellular
reticulum. In this extra-embryonal region the entodermal
cells are frequently found in mitosis. Ic would appear, then,
that the entoderm is first laid down in the region of the embi'yonal area as a cellular reticulum, which later becomes
ii'ansformed into a continuous cell-membrane, and that its
peripheral extension over the inner surface of the extraembryonal ectoderm is the result of the growth and activity
oF its own constituent cells.
This peripheral growth continues until there is formed
eventually a complete entodermal lining to the blastocyst
cavity. The rate of growth appears to be somewhat variable.
In a series of primitive streak vesicles (6-6'75 mm. in diameter)
the lower third oF the wall is, I find, still unilaminar. In
another series of vesicles of the same developmental stage
(4'5-6 mm. in diameter) a unilaminar area is present at the
lower pole, varying from I x ‘5 mm. in diameter to as much
as 4 mm. Even in vesicles 7-7'5 mm. in diameter a unilaminar patch may still occur at the lower pole, but in vesiqles
8'5 mm. in diameter (stage of fiat embryo) the entodermal
lining appears always to be complete.
The Origin of the Entoderm in Eutheria.  -  The
remai'kable facts relative to the origin of the entoderm in
Dasyurus which I have been able to place on record in the
jireceding pages, thanks to the large size attained by the
blastocyst prior to the differentiation of the formative germlayers and to the circumstance that the formative cells are
not arranged, as they are in Eutheria, in the form of a more
or less compact cell-mass, but constitute a thin unilaminar
cell-layer of relatively great extent which can easily be cut
up with scissors, and which, after staining and mounting on
the fiat can be examined under the highest powers, throw, it
seems to me, a new and unexpected light on the mammalian
entoderm, and at the same time help to fill the considerable
gap whicli has hitherto existed in our knowledge of its early
ontogenesis. Although the mode of origin of the entoderm
in Dasyurus would appear, in the present state of our knowledge, to find its closest parallel, not amongst vertebrates, but
in certain invertebrates (cf . the mode of origin of the entodermal cells from the wall of the blastula in Hydra as
described by Brauer^), the observations of Assheton ('94)
on the early history of the entoderm in the rabbit, when
viewed in the light of the foregoing, seem to me to afford
ground for the belief that phenomena comparable with those
hei'e recorded for Dasyui'us will eventually be recognised as
occurring also in Eutheria.
Hubrecht ('08), in his recent treatise on early Mammalian
ontogeny, deals very briefly with the question of the origin
of the entoderm in the latter group, merely stating that
“ from the inner cell-mass arises by delamination a separate
lower layer which we designate as the entoderm of the
embryo. These entoderm cells wander in radial direction
along the inner surface of the trophoblast, which in many
cases is thus soon transformed into a didermic structure.
. . . When the entoderm has separated off by delamina
tion from the embryonic knob, the remaining cells of the
latter form the ' embryonic ectodei'm,' which is thus situated
between the entoderm and the trophoblast.”
Assheton, in the paper just referred to, has given a careful
account of the first appearance of the entodermal cells in the
rabbit, and of what he believes to be the mode of their
peripheral extension below the trophoblastic wall of the
blastocyst. He shows that the inner cell-mass, at first
spherical, gradually, as the blastocyst enlarges, fiattens out
below the “ covering layer ” of the trophoblast until it forms
an approximately circular plate “ nowhere more than two
cells thick.” During the process of flattening, cells are seen
to jut out from the periphery of the mass; these eventually
separate, and appear as rounded cells scattered irregularly
over the inner surface of the trophoblast and ‘^extending
' ‘ Zeitschi'. f. wiss. Zool.,' Bd. Hi, 1891.
over an arc of about 60° from the upper pole in all directions.”
These “ straggling” cells, as Assheton terms them, as well as
the innermost cells of the now flattened inner cell-mass, are
regarded as hypoblastic and the outermost cells of the same
as epiblastic (embryonic epiblast). “The hypoblast, as a
perfectly definite layer, is formed by the time the blastodermic vesicle measures '5 mm. in diameter, that is, about the
102nd hour after coition. It is not, however, as yet by any
means a continuous membrane ; it is a network or fenestrated
membrane. For this reason, in section it appears to be
represented by isolated cells lying beneath the embryonic
disc (v. fig. 29, Sy.)” (cf. Dasyurus). In considering the
question how the peripherally situated (“ straggling ”) entodermal cells, which are undoubtedly derived from the inner
cell-mass, “apparently Avander round the inside of the blastodermic vesicle,” he I'eaches the conclusion that this is not the
result of amoeboid activity or growth “in the sense of migration ” on the part of these cells, but “ is only an apparent
growth round produced by the more rapid growth of a
zone of the [trophoblastic] wall of the vesicle immediately
surrounding the embryonic disc, in which zone the marginal
cells of the inner mass lie.” He is unable to find any
evidence of the production of pseudopodial processes by
these pei'ipheral entodermal cells, the majority of them
appearing at first to be quite isolated from each other and
approximately spherical. “Certain of the cells here and
there are connected by threads of protoplasm, but this, I
think, is not a sign of pseudopodic activity, but merely
indicates the final stage in division betAveen the tAvo cells.”
By the sixth day the hypoblast of the embryonic disc has
assumed the lorm of a continuous membrane, composed of
completely flattened cells, Avhilst the peripheral hypoblast
cells have become more numerous, and “many of them,
possibly all of them, are noAV undoubtedly connected by more
or less fine protoplasmic threads.” Such, in brief, is
Assheton's account of the early history of the entodenn in
the rabbit; it presents obvious points of agreement with my
own for Dasyunis, and I ventui'o to think the agreement is
even greater than would appear from Assheton's conclusions.
In adopting- the view that the more active growth of the
region of the blastocyst wall immediately surrounding the
inner cell-mass is the sole causal agent in effecting the separation and peripheral spreading of the entodermal cells, I cannot
but feel, in view of his own description and figures and of my
own results, that he has attributed a much too exclusive importance to that phenomenon and a much too passive role to the
entodermal cells themselves. In Dasyurus the inward migration and the later peripheral spreading of the entodermal
cells is effected without any such marked unequal growth of
tlie blastocyst wall as occurs, according to Assheton, in the
rabbit, as the direct outcome of their owu inherent activity,
and I believe the possession of a like activity characterises
the entodermal cells of the rabbit. The evidence of Assheton's
own fig. 40, which shows in surface view a portion of the
vesicle wall with the peripheral entodermal cells in relation
thereto, and which should be compared with my figs. 68 and
69, conclusively demonstrates, to my mind, the possession by
these cells of amoeboid properties, and thus support is
afforded for the belief that the separation of the entodermal
cells from the formative cell group (inner cell-mass) is here
also the expression of an actual migration. Whether or not
the strands of protoplasm which Assheton ('08, '09) describes
as present in the sheep, pig, ferret, and goat, connecting the
inner lining of the inner mass to the wall of the blastocyst,
and which he interprets as tending “ to show that the inner
lining of the inner mass is of common origin with the wall of
the blastocyst,” are of any significance in the present connection, I cannot certainly determine.
4. Summary.
The results and conclusions set forth in the preceding
pages of this chapter may be summarised as follows;
(1) The unilaminar wall of the blastocyst of Dasyurus con
sists of two regions distinct in origin and in destiny, viz. an
upper or formative region, derived from the upper cell-ring
of the 16-celled stage, and destined to furnish the embryonal ectoderm and the entoderm and a lower or nonformative region derived fi-om the lower cell-ring of the
mentioned stage, and destined to form directly the extraembryonal or trophoblastic ectoderm (tropho-ectoderrn) of the
bilaminar vesicle.
(2) 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.
(3) The entodermal mother-cells, either without or subsequently to division, bodily migrate inwards from amongst the
larger cells of the unilamiuar wall and so come to lie in
contact with the inner surface of the latter. Tkey thus give
origin to the primitive entodermal cells from which the
deKnitive entoderm arises. The larger passive cells, which
alone form the unilamiuar wall after the inward migration of
the entodermal cells is completed, constitute the embryonal
(4) The entodermal cells as well before as after their
migration from the unilamiuar wall are capable of exhibiting
amoeboid activity and of emitting pseudopodial processes, by
tlie anastomosing of which there is eventually formed a
cellular entodermal reticulum underlying, and at first coextensive with, the embryonal ectoderm.
(5) The definitive entoderm thus owes its character as a
connected cell-layer primarily to the formation of secondaiy
anastomoses between the pseudopodial processes emitted by
the primitive entodermal cells (or entodermal mothercells).
(6) The assumption by the entodermal cells of amoeboid
j^roperties whilst they are still constituents of the unilamiuar
wall affords an intelligible explanation of the mechaiiisin of
their inwai'd migration.
(/) 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 uuilaramar non-forrnative region so as to form eventually a
complete entodermal lining to the blastocyst cavity. In this
way the blastocyst wall becomes bilaminar throughout.
(8) The bilaminar blastocyst consists of two reguous, respectively embryonal and extra-embryonal. The embryonal
region (embryonal area) is constituted by an outer layer of
embryonal ectoderm and the underlying portion of the entoderm, and the extra-embryonal, of the extra-embrvonal or
trophoblastic ectoderm (tropho-ectoderm), which is separated
from the embryonal by a well-marked junctional line, together
with the underlying portion of the entoderm, which is perfectly continuous with that below the embryonal ectoderm.
(9) The formative or embryonal region of the blastocyst
in Dasyurus is from the first freely exposed, and at no time
daring the developmental period dealt with in this paper
does there exist any cellular layer externally to it, i. e. a
covering layer of trophoblast (Deckschicht, Kauber's layer)
is absent and there is no entypy of the primary germ-layers
(cf. p. 111).
Chapter V.  -  Some Early Stages op Perameles and
The early material of Perameles and Macropus at my
disposal comprises only a small number of stages, but is of
special importance, since it enables me to demonstrate that
so far as these particular stages are concerned, the early
developmental phenomena in these forms are essentially the
same as in Dasyurus, and thus affords ground for the belief
that there is one common type of early development throughout the series of the Marsupialia. Moreover, it is of interest
since it reveals the. existence of what might be termed
specific differences in the early development of these Marsupials, especially in regard to the time of appearance of the
entoderm. In Dasyurus, it will be remembered, the primitive
entoderm cells first become definitely recognisable as internally situated cells in vesicles 4‘5 mm. in diameter. In
Perameles they occur in vesicles just over 1 rnm. in diameter,
while in Macropus they are already present in a blastocyst
only -35 mm. in diameter, so that it would appear that the
entoderm is differentiated much earlier in the higher, more
specialised types than in the more generalised forms. This
difference in time of appearance of the entoderm is perhaps
to be correlated with a difference in size of the ovarian ova
in the three genera mentioned.
1. Perameles.
The earliest material of Perameles I possess consists of two
eggs of P. obesula, which I owe to the skill and enthusiasm
of my friend Mr. S. J. M. Moreau, of Sydney. Egg -Ameasures '23 mm. in diameter, and egg B, ‘24 x ‘23 mm.
The former consists of thirty-two cells, the latter of thirty. In
both the shell-membrane has partially collapsed, but the general
plan of arrangement of the blastomeres can still fairly readily
be made out. Fig. 51, PI. 3, represents a micro-photograph
of a section of egg B, the better of the two. It shows the
.shell-membrane (nearly '005 mm. thick) externally, considerable remains of the albumen between that and the
deeply stained zona, and then, closely applied to the inner
surface of the latter, the blastomeres arranged in the form of
an inverted D, so as to enclose a central space, open below
as the figure stands. This latter opening extends through
the series, and it seems probable that there was a corx*esponding one opposite to it in the intact egg. Evidently we
have hei'e a stage in the formation of the blastocyst, in which
the blastomeres are in course of spreading towards one or
both of the poles of the sphere formed by the egg-envelopes.
J. r, HILL,
â– just as liappeus in the corresponding' stage of Dasyurus (cf.
fig. 51 with fig. 31j though the latter represents a somewhat
older stage in Dasyurus). The blastocyst-wall here appears
relatively more extensive than in the 32-celled stage of
Dasyurus, an apparent difference which may perhaps be accounted for by the difference in size of the respective eggs
(•24 mm. as compared with '36 mm.) . The blastomeres situated
adjacent to the opening and those on the right side of the
figure tend to be more flattened and of greater superficial extent than the remainder, but I can recognise no
difference in the cytological characters of the cells. The
space or cleavage cavity enclosed by the blastomeres is partly
occupied by a granular coagulum, and towards the opening
there is present a lightly staining reticular mass, which
i*ecalls the yolk-body of Dasyurus, though I am not prepared
to affirm that it is of that significance. The fixation of the
specimen is not quite perfect.
My next stage of Perameles is constituted by a blastocyst
of P. nasuta, for which I am again indebted to Mr. Moreau
measuring in the preserved condition '29 x '26 mm. Pig. 52,
PI. 3, shows a section of this blastocyst. Structurally,
it corresponds in all essential respects with the '43 mm.
blastocyst of Dasyurus, figured on the same plate (fig. 33).
The blastocyst Avail is complete and unilamiuar throughout.
It is distinguishable into tAvo regions, a more extensive region
over Avhich the cells are large and flattened and a less extensive,
composed of smaller but thicker cells (left side of fig. 52).
In the early blastocysts of Dasyurus, it may be recalled, the
evidence showed that the region of more flattened cells is
formative in significance, that of more bulky cells, non-formative. It is possible the same holds good for this Perameles
blastocyst. On the other hand, the structural condition of
the stage next to be described rather supports the vieAv that
the smaller region, composed of plumper cells, is in this case
formative. That view seems to me the more probable of the
two, but there is a considerable difference in size betAveen the
present blastocyst and those next available, so that it is
impossible to decide this point witli certciinty. The blastocyst cavity is partly occupied by coagulnm. There are no
cells present in it, but the question of the presence of a yolkbody must remain open. The shell-membrane (‘0045 mm. in
thickness) and zona are in close apposition.
Following this early blastocyst, I have three vesicles of
P. nasuta, two of them measuring 1‘3 mm. in diameter,
the other PI mm. In their stage of development they
agree pretty closely with the 4'5-5 mm. vesicles of Dasyurus,
referred to in the preceding pages under the designation
6, '04, the entoderm being in process of differentiation. The
formative region was readily distinguishable in the intact
vesicles as a darker patch occupying about three eighths of
the surface extent of the wall. In section (PI. 8, figs. 80, 81)
it is characterised by its greater thickness as compared with
the non-formative or trophoblastic region, and by the
presence below it of numbers of primitive entodernial cells.
Compared with the corresponding stage in Dasyurus, the
chief difference consists in the relatively much greater thickness of the cells of the formative region in the Perameles
vesicle. The latter cells are here already more or less definitely cubical in shape, their thickness varying from '09
mm. to '015 mm., and altogether they form a layer of a much
more uniformly thickened character than that of the 6, '04
vesicles of Dasyurus. The trophoblastic ectoderm (figs. 80,
81, tr. ect.) is composed of somewhat flattened cells, .varying
in thickness from ‘005 to '008 mm.
The primitive entodermal cells (figs. 80, 81, ent.) are
present below the formative region in fair abundance, more
especially around the periphery of the same, which may thus
appear somewhat thickened (fig. 81). 4'he cells vary in size
from ‘01 X '007 mm. to '024 x '009 mm., and they stain on the
whole somewhat more deeply than the formative cells, to
whose under-surface they are closely applied. They occur
groups. Mitotic figures are frequently met
with in the cells of the formative ai'ea (observe the obliquely
disposed figure in one of the formative cells in fig. 81), and
J. r. HILL.
â– they also occur in the primitive entodermal cells. Examination of the sections leaves no doubt in one's mind as to the
source of the entodermal cells. They are undoubtedly derived
from the formative region of the vesicle wall. The 'shellmembrane has a thickness of about '0027 mm.
2. Macro pus.
Of Macropus the earliest stage I have examined is a blastocyst of M. ruficollis, -25 x *21 mm. in diameter. It is not
in a quite perfect state of preservation, but is in a sufficiently
good condition to enable me to say that the wall is complete
and unilaminar throughout, just as in the ‘29 x "26 mm.
blastocyst of Perameles. The shell-membrane has a thickness
of about -005 mm., and there are still remains of the albumen
between it and the zona.
My next stage (figs. 82-85) is a blastocyst of the same
species, *35 mm. in diameter. It unfortunately suffered in
preparation, but practically the whole of the formative area
of the blastocyst wall and part of the trophoblastic ectoderm
are comprised in the sections (PI. 9, fig. 82), so that it is still
possible to make out its chief structural features. In its stage
of development this blastocyst closely agrees with the last
described blastocysts of Perameles. The formative area of
the wall is perfectly distinct in the sections because of its
greater thickness and the presence below it of the primitive
entodermal cells. It attains its gi-eatest thickness (*027 mm.)
peripherally, whilst it is thinnest centrally (*006 mm.), so that,
taken as a whole, it is not quite such a uniformly thickened
layer as is that of the Perameles blastocysts. Primitive entodermal cells are present below it, but not in great abundance
(figs. 82, 84, 85, ent.). In fig. 83, a formative cell is seen in
division, the axis of the spindle being oblique to the surface.
The trophoblastic ectoderm (figs. 82, 83, tr. ect.) is composed
of the usual flattened cells, and varies in thickness from
*005 to *0067 mm.
In the blastocyst cavity, adjacent to the trophoblastic
ectoderm on the left side of fig. 82, there is visible a small
spherical cell similar to the degenerate cells met with in
blastocysts of Dasyurus.
My last stage of M. ruficollis comprises an excellently
preserved blastocyst, measuring '8 mm. in diameter, in which
the embryonal ectoderm and the entoderm are definitely
established. It thus corresponds to the 8, '01 stage of
Dasyurus (blastocysts o -  5'5 mm. diameter). The embryonal
area is circular and measures '468 mm. in diameter. Its
constituent cells are cubical and from '008 to ‘OlS mm. in
thickness, Avhilst the trophoblastic ectoderm is formed of
flattened cells, -006 ram. in thickness. The entoderm is
present as a continuous layer of attenuated cells below the
embryonal ectoderm, and it probably also forms a continuous
layer below the trophoblastic ectoderm. Entodermal cells are
certainly pi*esent over the lower polar region of the vesicle,
but it is difficult to be certain from the sections whether or not
they form a perfectly continuous layer. The shell membrane
has a thickness of •0026 mm.
I have a corresponding blastocyst of Petrogale penicillata •915 mm. in diameter, with an oval, embryonal area
•525 X ^45 mm. in diameter, and a later blastocyst of M.
ruficollis P46 mm. in diameter, with a circular embryonal
area '57 mm. in diameter.
Chapter VI.  -  General Summary and Conclusions.
The observations recoi'ded in the pi'eceding pages and the
conclusions deducible therefrom may be summarised as
follows ;
(a) Ovum.  -  The uterine ovum of Dasyurus is characterised
(1) by its large size relatively to those of Eutheria; (2) by
the presence externally to the zona of a layer of albumen and
a shell-membrane, both laid down in the Fallopian tube and
homologous with the corresponding structures in the Mouotreme ovum, the shell-membrane, like the shell of the latter,
inci'easing in thickness in the uterus; (3) by its marked
J. r. HILL.
polarity, its lower two thirds consisting of formative cytoplasm, dense and finely granular in appearance, owing to the
presence of fairly uniformly distributed deutoplasmic material,
and containing the two pronuclei, its upper third being
relatively clear and transparent, consisting as it does of a
delicate reticulum of non-formative cytoplasm, the meshes of
which are occupied by a clear deutoplasmic fluid. Study of
the pi'ocess of vitellogenesis in ovarian ova demonstrates that
this fluid represents surplus deutoplasmic material which has
not been utilised in the upbuilding of the formative region of
the ovum.
The fate of the clear non-formative portion of the ovum is
a very remarkable one. Prior to the completion of the first
cleavage, it is separated off from the formative remainder of
the ovum as a spherical mass or yolk-body, Avhich takes no
direct part in development, though it becomes enclosed iu the
blastocyst cavity on completion of the blastocyst wall at the
upper pole. Its contained deutoplasmic fluid is to be regarded
as the product of an abortive attempt at the formation of a
solid yolk-mass, such as is found in the Monotreme ovum.
By its elimination the potentially yolk-laden telolecithal ovum
becomes converted into a secondarily homolecithal, holoblastic
one. All the evidence is held to support the conclusion that
the Marsupials are descended from oviparous ancestors with
ineroblastic ova.
(b) Cleavage.  -  Cleavage begins in the uterus, is total, and
at first equal and of the radial type. The first two cleavage
planes are meridional and at right angles to each other.
The resulting four equal-sized blastomeres lie disposed radially
around the polar diameter like those of the Monotreme (not
in pairs at right angles to each other as in Eutheria), and
enclose a segmentation cavity open above and below, their
upper ends partially surrounding the yolk-body. The third
cleavage planes are again meridional, each of the four blastomeres becoming subdivided equally into two. The resulting
eight cells form an equatorial ring in contact with the inner
surface of the sphere formed by the egg-envelopes. They
contain deutoplasmic material, which is, however, located
mainly in their lower halves. The ensuing fourth cleavages
are equatorial, and in correlation with the just-mentioned
disposition of the deutoplasm, are unequal and qualitative,
each of the eight blastoraeres becoming subdivided into an
upper smaller and clearer cell, with relatively little deutoplasm fairly uniformly dispersed through the cytoplasm, and
a lower larger, more opaque cell Avith much deutoplasm,
mainly located in a broad zone in the outer portion of the
cell-body. A 16-celled stage is thus produced in which the
blastomeres are characteristically arranged in two superimposed rings, each of eight cells, an upper of smaller, clearer
cells next the yolk-body, and a lower of larger, denser cells.
The former is destined to give origin to the formative or
embryonal region of the blastocyst wall, the latter to the
non-formative or extra-embryonal region of the same.
(c) Formation of the Blastocyst.  -  There is in the
Marsupial no morula stage as in Butheria, the blastomeres
proceeding directly to form the wall of the blastocyst. The
cells of the two rings of the 16-celled stage divide at first
meridionally and then also equatorially, the division planes
being always vertical to the surface. The daughter-blastomeres so produced, continuing to divide in the same fashion,
gradually spread towards opposite poles in contact with
the inner surface of the fii-m sphere formed by the zona and
the thickened shell-membrane. Eventually they form a complete cellular lining to the said sphere and it is this which
constitutes the wall of the blastocyst. The latter is accordingly unilaminar at its first origin, and it remains so in
Dasyurus until it has attained, as the result of active gi'owth
accompanied by the imbibition of fiuid from the uterus, a
diameter of 4-5 mm. It consists of two parts or regions,
distinct in origin and in destiny, and clearly marked off from
each other in later blastocysts by a definite junctional line
approximately equatorial in position, viz. an upper, embryonal
or formative region derived from the upper cell-ring of the
16-celled stage, and a lower, extra-embryonal or nonVOL. 56, PART 1. NEW SERIES. 6
formative region derived from tlie lower cell-ring of the same
(d) Later History of the Two Region s of the Blastocyst Wall (for details see pp. 72-74). -  From the embryonal
region are derived the embryonal ectoderm and the entire
entoderm of the vesicle. I conclude^ therefore, that it is tlie
homologue of the inner cell-mass or embryonal knot of the
Eutherian blastocyst. The extra-embryonal region directly
furnishes the outer extra-embryonal layer of the vesicle wall,
i. e. the outer layer of the omphalopleure and chorion of later
stages. Assuming, as the facts of comparative anatomy and
palaeontology entirely justify us in doing, that the Mammals
are monophyletic and of reptilian origin, and further assuming
that the foetal membranes are homologous structures throughout the Amniotan series (also in my view a perfectly
justifiable assumption)^, then the homologies of this extraembryonal region of the Marsupial blastocyt are not far to
seek. It is clearly the homologue of the extra-embryonal
ectoderm of the Sauropsidan and Monotreme egg, and the
homologue also of the outer enveloping layer of the Eutlierian
blastocyst, to which Hubi'echt has given the special name of
“ trophoblast.” In my view the trophoblast is none other
than extra-embryonal ectoderm which in the viviparous
mammals, in correlation with the intra-uterine mode of
development, has acquired a special significance for the
nutrition of the embryo.
These, then, are my conclusions, and to me they seem on
general grounds perfectly obvious, viz. : (1) that the embryonal or formative region of the unilaminar Marsupial
blastocyst is the homologue of the inner cell-mass or
* How Assheton can maintain f 09, p. 266) “ that the amnion of the
rabbit is not more homologous to the amnion of the Sauropsidan than
the homy teeth of Ornithorhynchns ai-e homologous to the true teeth
of the mammal or reptile, which they have supplanted,” how he can
hold this view and yet proceed to utilise the presence of the amnion as
one of the leading charactei-s distinguishing the Amniota from the
Anamnia, I fail to comprehend. Surely the presence of a series of
purely analogous structures in a group is of no classificatory value.
imposed cell-viiigs, respectively and non-formative cell-rings of
formative (emlDryonal) and non- the Metatherian.
formative (extra-embryonal) in
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embryonal knob of tlie Eutberiaii blastocyst ; and (2) that the
extra-embryonal or noii-formative region of the same is the
homologne of the extra-embryonal ectoderm of the Sauropsida and Monotremata and of the trophoblast of the
As regards conclusion (1) there is not likely to be much
difference of opinion, but as regards (2), whilst perhaps the
majority of embryologists support the obvious, not to say
common-place view which I here advocate, it seems certain
that it will prove neither obvious nor acceptable to those
mammalian embryologists (I refer specifically to my friends
Professor A. A. W. Hubrecht and Mr. R. Asshetou) who, with
only Selenka^s account of eaidy Marsupial ontogeny before
them, have formulated other and quite divergent views as to
the morphological nature of the outer enveloping layer of the
Eutherian blastocyst. It is therefore necessary to discuss
this question further, though I would fain express my conviction that had the observations recorded in this paper been
earlier available, much vain speculation as to the phytogeny
of the trophoblast might possibly have been avoided.
Chapter VII.  -  The Early Ontogeny op the Mammalia in
THE Light op the Foregoing Observations.
In entering on a discussion of the bearings of the results
of my study of the early development of Marsupials on
current interpretations of early Mammalian ontogeny, and
especially of the homologies of the germ-layers, I desire at
the outset to emphasise my conviction that, specialised
though the Marsupials undoubtedly are in certain features of
their anatomy, e. g. their dentition, genital ducts, and mammary apparatus, the observations recorded in the preceding
pages of this paper afford not the slightest ground for the
supposition that their early ontogeny is also of an aberrant
type, devoid of signiffcance from the point of view of that of
other mammals. On the contrary, I hope to demonstrate
that the Marsupial type of early development not only readily
falls into line with that of Eutheria, and with what we know
of the early development of the Prototheria, but furnishes
ns with the key to the correct interpretation of that extraordinarily specialised developmental stage, the Eutherian
blastocyst. In particular I hope to show that the description
which I have been able to give of the mode of formation of
the' Marsupial blastocyst, bridges in the most satisfactory
fashion the great gap which has till now existed in our
knowledge of the way in which the transition from the
Monotrematous to the Eutherian type of development has
been effected.
1. The Early Development of the Monotremata.
Our knowledge of the early development of the oviparous
mammals is admittedly still far from complete. Nevertheless
it is not so absolutely fragmentary that it can be passed over
in any general discussion of early mammalian ontogeny, and
I certainly cannot agree with the opinion of Assheton ('08,
p. 227) that from it “we gain very little help towards the
elucidation of Eutherian development.” On the contrary, I
think that the combined observations of Semon ('94), and
Wilson and Hill ('07) shed most valuable light on the early
ontogenetic phenomena in both the Metatheria and Eutheria.
I propose therefore to give here a very brief resume of the
chief results of these observers,^ and at the same time to
indicate how the knowledge of early Monotreme ontogeny
we possess, limited though it be, does help us to a better
understanding of the phenomena to which I have just
The ovum, as is well known from the observations of
Caldwell. ('87), is Reptilian in its character in all but size.
It is yolk-laden and telolecithal, the yolk consisting of
discrete yolk-spheres, and it is enclosed outside the zona
(vitelline membrane) by a layer of albumen and a definite shell.
* In so doinff I have largely utilised the phraseology of Wilson and
Hill's paper ('07).
At the moment of entering the oviduct it has a diameter of
3‘5-4 mm. (2‘5-3 mm. according to Caldwell), and is therefore
small relatively to that of a reptile of the same size as the
adult Monotreme, but large relatively to those of other
mammals, being about twelve times larger than that of
Dasyurus, and about eighteen times larger than that of the
Cleavage is meroblastic. The first two cleavage planes are
at right angles to each other, as iii the Marsupial, and divide
the germinal disc into four approximately equal-sized cells
(Semon, Taf. ix, fig. 30). Each of these then becomes subdivided by a meridional furrow into two, so that an 8-celled
stage is produced, the blastomeres being arranged symmetrically, or almost symmetrically, on either side of a median line,
perhaps corresponding to the primary furrow (Wilson and Hill,
p. 37, text-figs. 1 and 2). Imagine the yolk removed and the
blastomeres arranged radially, and we have at once the open
ring-shaped 8-celled stage of Dasyurus. The details of the
succeeding cleavages are unknown. Semon has described a
stage of about twenty-four cells (Semon, Taf. ix, fig.31),inwhich
the latter formed a one-layered circular plate with no evidence
of bilateral symmetry, and this is succeeded by a stage also
figured by Semon (figs. 32 and 33, cf. also Wilson and Hill,
PI. 2, fig. 2), in which the blastoderm has become sevei'al
cells thick, though it has not yet increased in surface extent.
It is bi-convex lens-shaped in section, its lower surface being
sharply limited from the underlying white yolk. No nuclei
are recognisable in the latter, either in this or any subsequent
stage, nor is there ever any trace of a syncytial germ-wall,
features in which the Monotreme egg differs from the
The next available stage, represented by an egg of Ornithorhynchus, described by Wilson and Hill ('07, p. 38, PI. 2, fig.
4), and by an egg of Echidna, described by Semon ('94, p. 69,
figs. 22 and 33), is separated by a considerable gap from the
preceding, and most unfortunately so, since it belongs to the
period of commencing formation of the germ-layers. The
J. r. HILL.
cellular lens-shaped blastoderm of the preceding stage has
now extended in the peripheral direction so as to enclose
about the upper half of the yolk-mass, and in so doing it has
assumed the form, almost exclusively, of a unilaminar thin
cell-membrane, composed of flattened cells and closely applied
to the inner surface of the zona. At the embryonic pole,
however, in the region of the white yolk-bed, there are
present in the Ornithorhynchus egg a few plump cells,
immediately subjacent to the unilaminar blastoderm, but
separate and distinct from it, whilst in the Echidna egg
Semon's figure (fig. 33), which is perhaps somewhat schematic,
shows a group of scattered cells, similar to those in the
Ornithorhynchus egg but placed considei'ably deeper in the
white yolk-bed. Unfortunately we have no definite evidence
as to the significance of these internally situated cells. One
of two possible interpretations may be assigned to them.
Either they represent the last remaining deeply placed cells
of the blastodisc of the preceding stage, which have not yet
become intercalated in the unilaminar blastodermic membrane
believed by Semon to be the condition attained in eggs of
about this stage of development, or they are cells which have
been proliferated off from this unilaminar blastoderm, to
constitute the parent cells of the future yolk-entoderm. As
regards Echidna, Semon expresses a definite enough opinion ;
he holds that these deeply placed cells actually arise by a
somewhat diffuse proliferation or ingrowth from a localised
depressed area of the blastoderm at the embryonic pole, and
that they give origin to yolk-entoderm. This interpretation
of Semon seems probable enough in view of the mode of origin
of the entoderm in the Metatheria and Eutheria. Moreover
in the next available stage, an egg of Ornithorhynchus, just
â– over 6 mm. in diameter, described by Wilson and Hill, the
blastoderm is already bilaminar throughout its extent, so that
we .might veiy Avell expect to find the beginnings of the entoderm in the somewhat younger eggs.
In the 6 mm. egg just referred to, the peripheral portion of
the utjilaminar blastoderm of the preceding stage has grown
SO as to enclose the entive yolk-mass in a complete ectodeimal
envelope, whilst iiiteimally to that a complete lining of yolkentoderm has become established. As tlie result of these
changes, and of the imbibition of fluid from the uterus, the
solid yolk-laden egg has become converted into a relatively
thin-walled vesicle or blastocyst, possessed of a bilaminar
wall surrounding the partly fluid vitelline contents of the egg.
Throughout the greater part of its extent the structure of the
vesicle wall is very simple. It consists externally of an
extremely attenuated ectodermal cell - membrane closely
adherent to the deep surface of the vitelline membrane
(zona), and within that of a layer of yolk-entoderm, composed
of large swollen cells, containing each a vesicular nucleus,
and a number of yolk-spheres of varying size. Over a small
area, overlying the white yolk-bed, however, the ectodermal
layer of the wall presents a different character to that
described above. Its constituent cells are here not flattened
and attenuated, but irregnlai'ly cuboidal in form and much
more closely packed together; moreover they stand in proliferative continuity with a subjacent mass of cells, also in
process of division. The irregular superficial layer and this
latter mass together form a thickened lenticular cake, "5 mm.
in greatest diameter, projecting towards the white yolk-bed
but separated from it by the yolk-entoderm, which retains
its character as a continuous cell-membrane. This differentiated, thickened area of the wall, situated as it is at the upper
pole of the egg, as marked by the white yolk-bed, must be
held to represent a part of the future embryonal region.
Wilson and Hill incline to regard it as in some degree the
equivalent of the “primitive plate” of Eeptiles and as the
initial stage in the formation of the primitive knot of latex;
eggs. This question, however, does ixot closely concern us
here : the point I wish to emphasise is the relative inactivity
of the cells composing the embryonal region of the blastoderm
in the Monotreme as compared with the marked activity displayed by those constituting the peripheral (extra-embryonal)
region of the same. It is these latter cells which by their
rapid growth complete the envelopment of the yolk-mass and
so constitute the lower hemisphere of the blastocyst.
Ihe bilaminar blastocyst of the Monotreme, foi'nied in the
manner indicated above, is entirely comparable with the
Marsupial blastocyst of the same developmental stage. There
are differences in detail certainly (e.g. in the characters,
time of formation, and rate of spreading of the entoderm,
in the mode of formation of the blastocyst cavity and in its
contents, in the apparent absence in the Monotreme of any
well-marked line of division between the embryonal aud extraembryonal regions of the ectoderm, in the relatively earlier
appearance of differentiation in the embryonal region in the
Monotreme as compared with the Marsupial), but the agreements are obvious and fundamental ; in particular, I would
emphasise the fact that in both the embryonal region is
superficial and freely exposed, and forms part of the blastocyst wall just as that of the reptile forms part of the general
blastoderm. Moreover, should future observations confi^rm
the view of Semon that the primitive entodermal cells of the
Monotreme are proliferated off from the embryonal region of
the unilaminar blastoderm, then we should be justified in
directly comparing the latter with the unilaminar wall of the
Marsupial blastocyst, and in regarding it also as consisting
of two differentiated regions, viz. a formative or embryonal
region, overlying the white yolk-bed, and giving origin to
the embryonal ectoderm and the yolk-entoderm, and a nonformative region which rapidly overgrows the yolk-mass so
as to eventually completely enclose it, just as does the less
rapidly growing extra-embryonal ectoderm of the Sauropsidan blastoderm.^ Meantime I see no reason for doubting
that this rapidly growing peripheral portion of the unilaminar
blastoderm of the Monotreme is anything else than extraembryonal ectoderm homogenous with that of the reptile.
Indeed, I am not aware that any embryologist except Hubrecht
thinks otherwise. Even Asshetou is, I believe, content to
* We should further he justified in concluding that the entoderm is
similar in its mode of origin in all three mammalian sub-classes.
regard the outer layer of the Monotrerae blastocyst ns
ectodermal. Hubrecht's view is that the primitive eiitodermal
cells of Semon give origin, not to yolk-entoderm, but to the
equivalent of the embryonal knot of Eutheria, whilst the
uuilaminar blastodermic membrane itself is a larval layer
-  the trophoblast  -  that portion of it overlying the internally
situated cells representing the covering layer (Rauber's layer)
of the Eutherian blastocyst. ‘'For this view,” remarks
Assheton [^09, p. 283), “1 can see no reason derivable from
actual specimens described and figured by those four authors”
(Caldwell, Semon, Wilson and Hill), with which criticism I
am in entire agreement, as also with the following statement,
which, so far as the Metatheria are concerned, is based on
my own results: “Neither in the Prototheria [n ] or the
Metatheria is there really any tangible evidence of a trophoblast occui*ring as a covering layer over the definitive epiblast
as in Eutheria” (p. 234).
In connection with the peripheral growth of the unilaminar
blastoderm in the Monotreme, it is of interest to observe that
this takes place, not apparently in intimate contact with the
surface of the solid yolk, as is the case with the growing
margin of the extra-embryonal ectoderm in the Saui'opsidan
egg, but rather in contact with the inner surface of the
thickened zona, perhaps as the result of the accumulation in
the perivitelline space of tiuid which has diffused into the latter
from the uterus. In other words, the peripheral growth of
the extra-embryonal ectoderm to enclose the yolk-mass appears
to take place here in precisely the same way as the spreading
of the non-formative cells in Dasyurus to complete the lower
pole of the blastocyst. In my view the latter phenomenon
is none other than a recapitulation of the former ; on the
other hand, I regard the spreading of the formative cells in
Dasyurus towards the upper pole as a purely secondary
feature, conditioned by the loss of the yolk-mass and the
attainment of the holoblastic type of cleavage.
If it be admitted that the outer extra-embryonal layer of
the Monotreme blastocyst is homogenous with the extra
embryonal ectoderm of the Keptile, then it seems to me there
is no escape from tlie conclusion that these layers are also
homogenous with the non-formative region of the unilaminar
Marsupial blastocyst. I need only point out here that the
chief destiny of each of the mentioned layers, and I might
also add that of the outer enveloping layer of the Eutherian
blastocyst (the so-called trophoblast), is one and the same,
viz. to form the outer layer of the chorion (false amnion,
serous membrane) and omphalopleure (unsplit yolk-sac wall.
Hill ['97]),^ and that to deny their homogeny to each other
implies the nou-homogeny of these membranes and the amnion
in the Amniotan series, and consequently renders the group
name Amniota void of all moi'phological meaning.
The rapidity Avith which the enclosure of the yolk-mass
is effected, and the relative tardiness of differentiation in the
embryonal region are features Avhich sharply distinguish the
early ontogeny of the Monotremes from that of the Sauropsida,
and which, in my view, are of the very greatest importance,
since they afford the key to a correct understanding of the
peculiar coenogeuetic modifications observable in the early
ontogeny of the Metatheria and Eutheria. To appreciate the
significance of these featui-es it is necessary to take account
of the great difference which exists between the Sauropsidan
and Monotreme ovum in regard to size, as Avell as of the very
different conditions under Avhich the early development goes
on in the two groups. The Sauropsidan egg is large enough
to contain Avithin its OAvn confines the amount of yolk necessary for the production of a young one complete in all its
parts and capable of leading an independent existence
immediately it leaves the shell. Furthermore, it is also large
' In certain Ainniotes the layers in question appear also to participate
in the formation of the inner lining of the amnion (amniotic ectoderm)
(cf . Assheton ['09], pp. 248-9), but this does not affect the statement in
the text. In the Saxu'opsida and Monotremata I think I am coia-ect in
saying that no sharp distinction is recognisable between the embi'yonal
and extra-embryonal regions of the ectoderm, hence it is difficult, if not
imj)ossible, to determine with certainty their relative participation in
the formation of the amniotic ectoderm.
enough to provide room for tlie development of an embryo
without any secondary growth in size after it leaves the ovary.
Moreover we have to remember that after it has become
enclosed in the shelly it remains but a short time in the oviduct
and receives little or no additional nutrient material from the
oviducal walls. The yolk-mass in any case retains its solid
character; there is no necessity for its rapid enclosure, and
so enclosure is effected slowly, contemporaneously with the
differentiation of the embryo.
In the Monotreme the conditions are altogether different.
The ripe ovarian ovum when it enters the oviduct has a
diameter of about 3-5 to 4 mm., and is thns considerably
smaller than that of a Eeptile of the same size as the adult
Monotreme. The amount of yolk which it is capable of containing is not anything like sufficient to last the embryo
throughout the developmental period, and, moreover, it does
not provide the space essential for the development of an
embryo on the ancestral Reptilian lines. As Assheton ('98,
p. 251) has pointed out, “ the difference in size between
the fertilised ovum of a reptile or bird or of a mammal
is very great ; but the difference in size between the
embryo of, say, a bird with one pair of mesoblastic
somites and of a mammal of the same age is comparatively
small. This means that nearly the same space is required
for the production of the mammalian embryo as of the
Sauropsidan, and has to be provided.” In the Monotreme
not only is additional room necessary, but also additional
nutrient material, sufficient with that already present in the
egg to last the embryo throughout the period of incubation.
Both are acquired contemporaneously during the sojourn of
the egg in the uterine portion of the oviduct, wherein the egg
increases greatly in size. When it enters the uterus, the
Monotreme egg has a diameter, inclusive of its membranes, of
about 4-5 mm. ; when it is laid, it measures in Ornithorhynchus, in its greatest diameter, 16-19 mm., and somewhat
less in the case of Echidna. Prior to the enclosure of the yolk
the increase in diameter, due to the accumulation of fluid in
the perivitelliue space and between the zona and shell, is but
slight. But as soon as the yolk becomes suiTonnded by a
complete cellular membrane, i.e. as soon as the egg has
become converted into a thin-walled blastocyst, rapid growth
sets in, accompanied by the active imbibition of the nutrient
fluid, which is poured into the uterine lumen as the result of
the secretory activity of the abundantly developed uterine
glands. The fluid absorbed not only keeps the blastocyst
turgid, but it brings about the more or less complete disintegration of the yolk-mass, its constituent spherules
becoming disseminated in the fluid contents of the blastocyst
cavity. Although a distinct and continuous subgerminal
cavity, such as appears beneath the embryonal region of the
Sauropsidan blastoderm, does not occur in the Monotreme
egg, vacuolar spaces filled with fluid develop in the white
yolk-bed underlying the site of the germinal disc and appear
to represent it. As Wilson and Hill remark ('03, p. 317),
“ one can, without hesitation, homologise the interior of the
vesicle with the subgerminal cavity of a Saui'opsidan egg,
extended so as to include by liquefaction the whole of the
yolk itself.” In the Marsupial the blastocyst cavity has a quite
different origin, since it represents the persistent segmentation
cavity, whilst in the Eutheria the same cavity is secondarily
formed by the confluence of intra- or intei*-cellular vacuolar
spaces, but no one, so far as I know, has ever v^entured to
assert that, because of this difference in mode of origin, the
blastocyst cavity in the series of the Mammalia is a nonhomogenous formation.
To return to the matter under discussion, it appeal's to me
that the necessity which has arisen, consequent on the I'eduction in size of the ovum, for rapid growth of the same in
order to provide room for the development of an embryo and
for the storage of nutrient material furnished by the maternal
uterus, affords a satisfactory explanation of the much more
marked activity of the extra-embryonal I'egion of the blastoderm as compared with the embryonal, Avhich is such a striking
feature in the early ontogeny of the Monotremes, and not
only of them, but, as Assheton has pointed out ('98, p. 251),
of the higher mammals as well (cf. the process of epiboly and
the inertness at first displayed by the formative cells of
the embryonal knot as compared with the activity of the nonformative or tropho-ectodermal cells), an activity which
results in the rapid completion of that characteristically
mammalian developmental stage  -  the blastocyst or blastodermic vesicle.
The necessity for the early formation of such a stage,
capable of rapidly growing in a nutrient fluid medium
provided by the mother, has profoundly influenced the early
ontogeny in all three mammalian subclasses, and natui*ally
most of all that of the Eutheria, in which reduction of the
ovum, both as regards size and secondary envelopes, has
reached the maximum. And I think there can be little
doubt but that it is this necessity which has induced that
early separation of the blastomeres into two categories,
respectively formative and non-formative in significance,
which has long been recognised as occurring in Eutheria, and
which I have shown also occurs amongst the Metatheria.
This early separation of the blastomeres into two distinct
groups is not recognisable in the Sauropsida, and the idea
that it is in some way connected with the loss of yolk which
the mammalian ovum has suffered in the course ofphylogeny,
was first put forward, I believe, by Jenkinson. In his paper
on the germinal layers of Vertebrata ('06, p. 51) he writes:
“ Segmentation therefore is followed in the Placentalia by
the separation of the elements of the trophoblast from those
destined to give rise to the embryo and the remainder of its
foetal membranes, and this ^precocious segregation'
seems to have occurred phylogenetically during
the gradual loss of yolk which the egg of these
mammals has undergone.” Whether or not such a
precocious segregation ” has already become fixed in the
Monotremes,future investigation must decide (cf . ante, p.90).
Ihe loss of yolk, with resulting reduction in size which the
Monotreme ovum has suffered in the course of phylogeny, we
must assume to have taken place gi-adually and in correlation
with the longer retention of the egg in the oviduct, the
elaboration of the uterine portion of the same as an actively
secretory organ, and the evolution of the mammary apparatus.
The Monotremes thus render concrete to us one of the first
great steps in mammalian evolution so far as developmental
processes are concerned, viz. the substitution for intra-ovular
yolk of nutrient material furnished directly by the mother to
the developing egg or embryo. We see in them the beginnings of that process of substitution of uterine for ovarian
nutriment which reaches its culmination in the Eutheria with
their microscopic yolk-poor ova and long intra-uterine period
of development. The Marsupials show us in Dasyurus an
interesting intervening stage so far as the ovum is concerned,
in that this, though greatly reduced as compared with that
of the Monotreme, still retains somewhat of its old tendencies
and elaborates more yolk-material than it can conveniently
utilise, with the result that it has to eliminate the surplus
before cleavage begins. But as coucerns their utilisation of
intra-uterine nutriment, they have specialised along their
own lines, and instead of exhausting the possibilities implied
by the presence of that, they have extensively elaborated
the mammary apparatus for the nutrition of the young, born
in a relatively immature state, after a short period of intrauterine life (cf. Wilson and Hill [T7, p. 580]).
In view of the fact that the young Monotreme enjoys three
developmental periods, viz. intra-uterine, incubatory, and
lactatory, the question might be worthy of consideration
whether it may not be that the Marsupial has merged the
incubatory period in the lactatory, the Eutherian the same in
the intra-uterine.
2. The Early Development of the Metatheria and
It will have become evident Horn the foregoing that the
Metatherian mode of early development is to be regarded as
but a slightly modified version of the Prototherian, such
differences as exist between them being interpretable as coenogeuetic modifications, induced in the Metatherian by the
practically complete substitution of uterine nutriment for
intra-ovular yolk, a substitution which has resulted in the
attainment by the marsupial ovum of the holoblastic type of
cleavage. In tlie present section I hope to demonstrate how
the early ontogeny of the Metatlieria enables us to interpret
that of the Eutheria in terms of that of the Prototheria.
If we proceed to compare the early development in the
Metatlieria and Eutheria, we encounter, from the 4-celled
stage onwards, such obvious and profound differences in the
mode of formation of the blastocyst, and in the relations of
its constituent parts, that the differences seem at first sight
to far outweigh the resemblances. Nevertheless, apart from
their common possession of the same holoblastic mode of
cleavage, there exists one most striking and fundamental
agreement between the two in the fact that in both there
occurs, sooner or later during the cleavage process, a separation of the blastomeres into two distinct, pre-determined cellgroups, whose individual destinies are very different, but
apparently identical in the two subclasses. In tlie Marsupial,
as typified by Dasyurus, the fourth cleavages are, as we have
seen, unequal and qualitative, and result in the separation of
two differentiated groups of blastomeres, arranged in two
superimposed rings, viz. an upper ring of eight smaller, less
yolk-rich cells, and a lower of eight larger, more yolk-iuch
cells. The evidence justifies the conclusion that the former
gives origin directly to the formative or embryonal region of
the vesicle wall, the latter to tlie non-formative or extraembryonal region.
Amongst the Eutheria the evidence is no less clear. It has
been conclusively shown by various observers (Van Beneden,
Duval, Assheton, Hubrecht, Heape, and others) that, sooner
or later, there occui's a separation of the blastomeres into two
distinct groups, one of which eventually encloses the other
completely. The two groups may be clearly distinguishable
t.C.TTV. (€cj
emJb. ect.
Diagrams illustrating the mode of formation of the blastocyst
in Metatheria (a-d) and Eutheria (1-3). b.c. Blastocyst cavity.
i.c.m. Inner cell-mass, 'pr.amn.c. Primitive amniotic cavity.
r.l. Rauber's layer. s.c. Segmentation cavity. For other
reference letters see explanation of plates (p. 125).
in eai'lv cleavage stages, owing to diffecences in the characters
and staining reactions of their cells, and in such cases there
is definite evidence of the occurrence of a process of overgrowth
or epiboly, whereby one group gradually grows round and
completely envelops the other, so that in the completed
morula a distinction may be drawn between a central cellmass and a peripheral or enveloping layer (rabbit. Van
Beneden; sheep, Assheton). In other cases, where it has
been impossible to recognise the existence of these two
distinct cell-groups in the cleavage stages, we nevertheless
find, either in the completed moimla or in the blastocyst, that
a more or less sharp distinction may be drawn between an
enveloping layer of cells and an internally situated cell-mass
(inner cell-mass).
E. van Beneden, in his classical paper on the development
of the rabbit, published in 1875, was the first to recognise
definitely the existence of two categories of cells in the
segmenting egg of the Eutherian mammal. In this form he
showed how in the morula stage a cap of lighter blastomeres
gradually grows round and envelops a mass of more opaque
cells by a process of overgrowth or epiboly. In his more
recent and extremely valuable paper on the development of
Yespertilio ('99), he again demonstrated the existence of two
groups of blastomeres as well in the segmenting egg as in the
completed morula, but failed to find evidence of epiboly in all
cases. Nevertheless he holds fast to the opinion which he
expressed in 1875 : “ Que la segmentation s'accompagne, chez
les Mammiferes placentaires, d'un enveloppement progressif
d'une partie des blastomeres par une couche cellulaire, qui
commence a se differencier des le debut du developpement,”
and states that “dans tons les oeufs arrives a la fin de la
segmentation et dans ceux qui moutraient le debut de la
cavite Blastodermique j'ai constamment rencontre une couche
peripherique complete, eutourant de toutes parts un amas
cellulaire interne, bien separe de la couche enveloppante.”
The latter layer he regards as corresponding to the extraembryonal ectoderm of the Sauropsida, and points out that
“ chez tons les Choi'des les premiers blastomeres qui se
differencient et qui avoisinent le pole animal de I'oeuf sont
des elements epiblastiqnes. C'est par la couolie cellulaire qui
resulte de la segmentation ulterieure de ces premiers blastomeres epiblastiqnes que se fait, cbez les Sauropsides, benveloppement du vitellus. Dans Toeuf reduit a n'etre plus
qu'une sphere microscopiquej bepibolie a pu s'achever des la
fin de la segmentation, voire meme avant bachevement de ce
phenomene.” The “ amas cellulaire interne ” (embryonal
knot, inner cell mass). Van Beneden shows, differentiates
secondarily into “ un lecithophore et un bouton embryonnaire.'' The former is the entoderm of other authors, the
latter the formative or embryonal ectoderm. Hubrecht, in
the forms studied by him (Sorex, 'I'upaia, Tarsius^) finds
a corresponding differentiation. In Tupaia he describes the
morula stage as consisting of a single central lightly staining
cell, which he regards as the parent cell of the inner cell-mass
of later stages, and of a more darkly staining peripheral layer
which forms the unilaminar wall of the blastocyst. Here,
then, the parent cells of the two cell-groups would appear to
be separated at the first cleavage. Hubrecht, like Van
Beneden, holds that the inner cell-mass furnishes the
embryonal ectoderm and the entire entoderm of the blastocyst.
The peripheral layer he has termed the trophoblast ('88, p.
511), and in his paper on the placentation of the hedgehog
('89, p. 298) he defines the term as follows: “I propose to
confer this name to the epiblast of the blastocyst as far as it
has a dix'ect nutritive significance, as indicated by proliferating
processes, by immediate contact with maternal tissue, maternal
blood, or secreted material. The epiblast of the germinal
ai-ea  -  the formative epiblast  -  aud that which will take part
in the formation of the inner lining of the amnion cavity is,
ipso facto, excluded from the definition.” Thus the name
* In Erinacens the entoderm, from Hubrecht's observations, appears
to be precociously differentiated, prior to the separation of the embryonal
ectoderm fi'om the overlying trophoblast, but the details of the early
development in this form are as yet only incompletely known.
trophoblasb was originally employed by Hubrecbt as a convenient term designatory of what he at the time regarded as
the extra-embryonal ectoderm of the mammalian blastocyst.
In the course of his speculations on the oingin of this layer,
however, he has reached the conclusion that it is really of the
nature oP'a larval envelope, an Embryonalhiille (^08, p. 15),
inherited by the mammals, not from the reptiles (which have
no direct phylogenetic I'elationship to the latter), but from
their remote invertebrate ancestors ('Vermiform pi'edecessors
of coelenterate pedigree, provided with an ectodermal larval
investment [Laiwenhiille] ”).
Assheton, again, although he was unable to convince himself ('94) of the correctness of van Beneden's account of the
occurrence of a process of epiboly in the segmenting eggs of
the rabbit, finds in the sheep ('98) that a differentiation into
two groups of cells is recognisable “ perhaps as early as the
eight segment stage,” and that one of the groups gradually
envelops the other. “Let it be noted,” he writes ('98, p. 227),
“ that we have now to face the fact, based on actual sections,
that there is in certain mammals a clear separation of
segments at an early stage into two groups, one of which
eventually completely surrounds the other,” and instances
Van Beneden's observations on the rabbit (of the correctness
of which he, however, failed to satisfy himself, as noted above),
Duval's observations on the bat, Hubrecht's on Tupaia, and
his own on the sheep. Assheton thinks this phenomenon
“ must surely have some most profound significance,”
but finds himself unable to accept the interpretations of
either Van Beueden or Hubrecht, and puts forward yet
another view, “ based on the appearance of some segmenting
eggs of the sheep ” ('08, p. 233), “that in cases where this
differentiation does clearly occur, it is a division into epiblast
and hypoblast, the latter being the external layer” ('98, p. 227).
Assheton thus differs from all other observers in holding that
the inner cell-mass or embryonal knot of the Eutherian
blastocyst gives origin solely to the formative or embryonal
ectoderm, and I believe 1 am correct in stating that he also
J. p. mi,L.
differs from all other observers in holding that the outer
enveloping layer of the same is entodermald
The fact, then, of the occurrence amongst Eutheria of a
“precocious segregation ” of the blastomeres into two distinct
groups, one of which eventually surrounds the other completely, is not in dispute, though authorities differ widely in
the intei'pretation they place upon it. In the Eutherian
blastocyst stage, the enveloping layer forms the outer unilaminar wall of the vesicle, and encloses the blastocyst cavity
as well as the other internally situated group. This latter
typically appears as a rounded cell-mass, attached ac one spot
to the inner surface of tlie enveloping layer, but more or less
distinctly marked off from it. It is genei-ally termed the
inner cell-mass or embryonal knoc (“ amas cellulaire interne ”
of Van Beneden). For the enveloping layer Ilubrecht's name
of “ trophoblast ” is now generally employed, even by those
who refuse to adopt the speculative views with which its
originator has most unfortunately, as I think, enshrouded this
convenient term.
I have demonstrated the occurrence of an apparently comparable “precocious segregation^^ of the blastomeres into
two distinct groups in one member of the Metatheria which
there is no reason to regard as an abeirant type, and I have
shown beyond all shadow of doubt that from the one group,
which constitutes what I have termed the formative region
of the unilaminar vesicle-wall, there arise the embi*youal
ectoderm and the entire entoderm of the vesicle, both embryonal and extra-embryonal, and that the other group, which
constitutes the non-formative region of the vesicle-wall,
directly furnishes the extra-embryonal ectoderm, i.e. the
ectoderm of the omphalopleui'e and chorion."
* Assheton states ('08, p. 233, cf. also '98, p. 220) that his interpretation “ owes ranch also to the theoretical conclusions of Minot and
Robinson.” However that may be, both Minot and Robinson in their
most recent writings continue to speak of the chorionic ectoderm.
^ Whether or not it participates in the formation of the ainniotic
ectoderm future investigation must decide.
As resrards Eutheria, we have seen that Van Beneden and
Hubrecht, though their views in otlier respects are widely
divero-ent, both ag'ree that the inner cell-mass of the blastocyst furnishes the embryonal ectoderm (as well as the amniotic
ectoderm wholly or in part) and the entire entoderm of the
vesicle. That, in fact, is the view of Mammalian embryologists
generally (Duval and Assheton excepted),^ and if we may
assume it to be correct, then it would appear that the later
history of the formative region of the Marsupial blastocyst
and that of the inner cell-mass of the Eutherian are identical.
That being so, and bearing in mind that both have been
shown, at all events in certain Mammals, to have an identical
origin as a group of precociously segregated blastotneres,^ I
can come to no other conclusion than that they are homogenous formations. If that be accepted, then this fact by itself
renders highly probable the view that the so-called trophoblast of the Eutherian blastocyst is homogenous with the
non-formative region of the Metatherian vesicle, and v?hen
we reflect that both have precisely the same structural and
topographical (not to mention functional) relations in later
stages, inasmuch as they constitute the ectoderm of the chorion
and omphalopleure (with or without participation in the
formation of the amniotic ectoderm;, and that both have a
similar origin in those Mammals in which a precocious segregation of the blastomeres has been recognised, their exact
* The view of Duval ['95], based on the study of Vespertilio, that the
inner cell-mass gives rise solely to entoderm, and that the enveloping
layer furnishes not only the extra-embryonal but also the embryonal
ectoderm, is shown by Van Beneden's observations on the same form to
be devoid of any basis of fact. Assheton's views are referred to below
(p. 110).
- The fact that the phenomenon of the “ precocious segregation” of
the blastomeres into two groups with deteiminate destinies has already
become fixed in tlie Marsupial lends additional weight to the view of
Van Beneden that such a segregation will eventually be recognised as
occurring in all Eutheria without exception. Without it, it is difficult
to understand how the entypic condition, characteristic of the blastocysts of Ml known Eutheria, is attained, imless by differentiation in
situ, which .seems to me highly improbable.
J. r. HILL.
homology need no longer be doubted. In the preceding section
of this paper (ante, pp. 91, 92) I have shown reason for the
conclusion that the non-formative region of the Marsupial
blastocyst is the homologue of the extra-embryonal ectoderm
of the Monotreme and Reptile, and if that conclusion be
accepted it follows that the outer enveloping layer of the
Eutherian blastocyst, the so-called trophoblast of Hubrecht,
is none other than extra-embryonal ectoderm, as maintained
by Van Beneden, Keibel, Bonnet, Jenkinson, Lee, MacBride
and others, the homologue of that of Reptilia.
I am therefore wholly unable to accept the highly speculative conclusions of Hubrecht, set forth with such brilliancy
in a comparatively recent number of this Journal ('08), as
to the significance and phylogeny of this layer. These conclusions, on the basis of which he has proceeded to formulate
such far-reaching and, indeed, revolutionary ideas not only
on questions embryological, but on those pertaining to the
phylogeny and classification of vertebrates, have already
been critically considered by Assheton ('09) and MacBride
('09), also in the pages of this Journal, and found wanting,
and they are, to my mind, quite irreconcilable with the facts
I have brought to light in regard to the early development
of Marsupials. I yield to no one in my admiration for the
epoch-making work of Hubrecht on the early ontogeny and
placentation of the Mammalia, and I heartily associate
myself with the eulogium thereanent so admirably expressed
by Assheton in the cx'itique just referred to (p. 274), but
I am bound to confess that as concerns his views on the
phylogeny of this layer, which he has termed the “ trophoblast,” he seems to me to have forsaken the fertile field of
legitimate hypothesis for the barren waste of unprofitable
speculation, and to have erected therein an imposing edifice on
the very slenderest of foundations.
Before I proceed to justify this, my estimate of Hubrecht's
views on the phylogeny of the trophoblast, let me first set
forth his conception so far as I understand it. He starts
with the assumption that the vertebrates (with the exception
of Ainpliioxus, the CyclostoineSj and the Elasraobi'anclif!) are
descended from “vermiform predecessors of coelenterate
pedigree” possessed of free-swimming larvte, in which there
was present a complete larval membi'ane of ectodermal derivation, and of the same order of differentiation “as the outer
larval layer which in certain Nemertines, Gephyreans, and other
worms often serves as a temporaiy envelope that is stripped
off when the animal attains to a certain stage of development.”
When, for oviparity and larval development, viviparity and
embryonic development became established in the Protetrapodous successors of the ancestral vermiform stock, the
larval membrane did not disappear. On the contrary, it is
assumed that it merely changed “its protective or locomotor
function into an adhesive one,” and so, development now
taking place in utero, it is quite easy to understand how tlie
larval membrane could gradually become transformed into
a trophic vesicle, containing the embryo as before, and
functional in the reception of nutriment from the walls of
the maternal uterus. The final stages in the evolution of
this trophic vesicle constituted by the old larval membrane
are met with amongst the mammals, since in them it
became vascularised so as to constitute a “yet more
thorough system of nourishment at the expense of the
maternal circulatory system.” Such, then, is the phylogeny
of the trophoblast according to Hubrecht. The Eutheriau
mammals, which it is held trace their descent straight back to
some very early Protetrapodous stock, viviparous in habit and
with small yolk-poor, holoblastic eggs, exhibit the trophoblast in its most perfect condition. Hubrecht therefore starts
with them, and attempts to demonsti'ate the existence of a
larval membrane, or remnants of such, externally to the
embryonal ectoderm in all vertebrates with the exceptions
already mentioned. There is no question of its existence in
the Meta- and Eutherian mammals. “We may,” writes
Hubrecht ('08, p. 12), . . . “insist upon the fact that
. . . all Didelphia and Monodelphia hitherto investi
gated show at a very early moment the didermic stage out of
which the embryo will be built up enclosed in a cellular
vesicle (the troplioblast), of which no pai‘t ever enters into
the embryonic organisation.” The common possession by the
Metatheria and Eutheria of a larval membi'ane is after all
only what might be expected, “since after Hill's ('97)
investigations, we must assume that the didelphian mammals
are not descended from Ornithodelphia but from monodelphian
placental ancestors.” As concerns the Prototheria, although
they cannot in any sense be regarded as directly ancestral to
the other mammals, we nevertheless find the trophoblastic
vesicle “ compax'atively distinct.” “In many reptiles and
birds,” however, it is “.distinguished with great diflSculty
from the embryonic shield,” and this is explained bv the
fact that the Sauropsida which are assumed to have taken
their origin from the same Protetrapodous stock as the
mammals but along an entirely independent line, have
secondarily acquired, like the Prototheria, the oviparous
habit, with its concomitants, a yolk-laden egg and a shell, and
this latter acquisition has naturally tended “to relegate any
outer larval layer to the pension list” ('09, p. 5). “Concerning the yolk accumulation in the Sauropsidan egg, there
is no trouble at all to suppose that the vesicular blastocyst
of an early vivipai-ous ancestor had gradually become yolkladen. The contrary assumption, found in the handbooks,
that the mammalian egg, while totally losing its yolk, has
yet preserved the identical developmental featui-es as the
Sauropsid, is in ideality much more difiicult to reconcile with
sound evolutionary principles” ('09, p. 5).
Amongst the lower Vertebrates the larval membrane is
clearly enough recognisable in the so-called Deckschicht of
the Teleostomes, Dipnoans, and Amphibians. It is frankly
admitted that Amphioxus, the Cyclostomes, and the Elasmobranchs “ show in their early development no traces of a
Deckschicht” (larval layer, troiDhoblast), but there is no
difficulty about this, since it is easy enough to suppose, in
view of other characters, that “ the Selachians may very well
have descended from ancestors without any outer larval layer ”
{'08, p. 151), and ‘'for Cyclostomes tlie same reasoning holds
good” (p. 152).
The trophoblast, then, is conceived of by Hubrecht as a
larval membrane of ectodermal derivation, which invests the
embryonal ahlage in all Vertebrates with the exceptions
mentioned, 'which is subject to secondary reduction, and which
is homologous throughout the series. As I understand the
conception, what is ordinarily called extra-embryonal ectoderm in the Sauropsida is not trophoblast, otherwise Hubrecht
could hardly write  -  “in reptiles and birds traces of the
larval layer have in late years been unmistakably noticed”
('09, p. 5) ; nevertheless what other writers have termed
embryonal and extra-embryonal ectoderm in the Prototheria
is claimed by Hubrecht as trophoblast (at all events that is
my interpretation of his statement that a trophoblastic vesicle
is present in these forms), and yet some years ago Hubrecht
('04, p. 10) found it diflBcult “ to understand that the name
has been misunderstood both by embryologists and gynecologists.” My own feeling is that the more recent developments in his views have tended to obscure rather than to
clarify our ideas as to the trophoblast, especially if we must
now hold that the chorion or serosa of the Sauropsida is not
homologous with that of the Prototheria, which necessarily
follows if the extra-embi'yonal ectoderm of the Sauropsidan is
not the same thing as that of the Monotreme.
Assuming that we have formed a correct conception of the
trophoblast as a larval membrane, and bearing in mind that it
is best developed in the Metatheria and Eutheria, since these
alone amongst higher Vertebrates have retained unaltered
the viviparous habits of their Protetrapodous ancestors, let us
see what basis in fact there is for the statement of Hubrecht
('08, p. 68) that “before the ectoderm and the entoderm
have become differentiated from each other there is in
mammals a distinct larval cell-layer surrounding (as soon as
cleavage of the egg has attained the morula stage) the
mother-cells of the embryonic tissues.” Now that statement
as it stands, I have no hesitation in characterising as entirely
.T. P. HlIiL.
misleading, inasmuch as it is applicable not to the Mammalia
as a whole, but, so far as it refers to matters of undisputed
fact, to one only of the three mammalian subclasses, viz. the
Eutheria. So far as the latter ai'e concerned, practically all
observers, as we have seen, are agreed that there is present
during at least the early stages of development a complete
outer layer of cells which encloses the embryonal anlage
or inner cell-mass (that portion of it immediately overlying
the latter being termed the “ Deckschicht ” or “Rauber's
layer”). It is, of course, this envelojDing layer or trophoblast which Hubrecht interprets as a larval membrane.
It fulfils the conditions, and were the Eutheria the only
Vertebrates known to us, the idea might be plausible
Turning now to the Metatheria, and I'emembering that these,
according to Hubrecht, are descended from the Eutheria, we
should naturally expect to find the supposed larval membrane
fully developed, with all its ancestral relations ; and so we do
if we are content to accept Hubrecht's interpretation of
Selenka's results and figures in the case of Didelphys. The
“ urentodermzelle ” of Selenka is for Hubrecht “ undoubtedly
the mother-cell of the embryonic knob,” the ectoderm of
Selenka is manifestly the trophoblast  -  a complete larval
layer. It is no doubt unfortunate that Hubrecht had to rely
on the work of Selenka as his source of information on the
early development of Marsupials, but it must be remembered
that he reads his own views into Selenka's figures. On the
basis of my own observations on the early ontogeny of Marsupials, I have no hesitation in affirming that a larval membrane, in the sense of Hubrecht, does not exist in any of the
forms (Dasyurus, Perameles, Macropus) studied by me. The
observations recorded in the preceding pages of this paper
demonstrate, in the case of Dasyurus without the possibility
of doubt, the entire absence of any cellular layer external
to the formative region of the blastocyst, i.e. in a position
corresponding to that occupied by Rauber's layer in Eutheria,
whilst in the case of Perameles and Macropus, they yield not
the slightest evidence for the existence of any such layer.
The formative region of the Marsupial blastocyst, which is
undoubtedly the homologue of the inner cell mass of the
Eutheria, forms from the first part of the unilarninar blastocyst wall, and is freely exposed. The remainder of the latter
is constituted by a layer of non-formative cells, the destiny
of which is the same as that of the so-called trophoblast of
the Eutheria. I have therefore ventui'ed to suggest that they
are one and the same. If, then, the trophoblast is really a
larval membrane, we must assume, in the case of the Marsupial, either that its “ Deckschicht '' portion has been completely suppressed (but why it should have been I fail to
understand, unless, perhaps, it is a result of the secondary
acquisition by the Marsupials of a shell-membrane, these
mammals being even now on the, way to secondarily assume
the oviparous habit !), or that the non-formative region of the
Marsupials is not the homologue of the trophoblast, in which
case the Marsupials must be held to have entirely lost the larval
membrane, since there is no other layer present which could
possibly represent it. These considerations may well give us
pause before we calmly accept Hubrecht's conception of the
trophoblast as a larval membrane present in all mammals
without exception.
Coming now to the Prototheria, we find, according to
Hubrecht, the trophoblastic vesicle . . . yet compara
tively distinct,” and so it is if we accept the interpretation of
Hubrecht of the observations and figures of Semon, Wilson
and Hill. The unilarninar blastoderm of these authors is
unmistakably the trophoblast. The cells situated internally
to that in the region of the white yolk-bed are not entodertnal, as suggested by Semon, but constitute for Hubrecht
“ the mother cells of the embryonic knob.” I need only quote
again the opinion of Assheton thereanent and express my
agreement therewith; he writes (^09, p. 233) : For this view
I can see no reason derivable from actual specimens described
and figured by those four authors” (Caldwell, Semon, Wilson
and Hill). It would appear, then, that the assumption of
Hubreclit of the presence of a larval membrane of the nature
postulated in the Prototheria and Metatheria is devoid of
foundation in fact, so that there but remains the question of
the significance of the outer enveloping layer of the Eutherian
blastocyst. As regards that, I venture to think that the
alternative interpretation of E. van Beneden and other
investigators, which I have attempted to develop in the
pages of this paper, affords a simpler and more satisfying
explanation of its significance and phylogeny than that
advocated by Prof. Hubrecht, an interpretation, moreover,
which is more in accordance, not only with all the known
facts, but ''with sound evolutionary principles and with the
conclusions arrived at by the great majority of comparative
anatomists and palaeontologists as to the origin and intei-relationships of the Mammalia.
And I also venture to think that what has just been said
holds true with reference to the views advocated by Mr.
Assheton. These views owed their origin to certain appearances which he found in some segmenting ova of the sheep
(but, be it noted, not in all those he examined), and he has
attempted to re-intei pret not only his own earlier observations,
but those of other workers on the early ontogeny of the Eutheria
in the light of his newer faith, and not only so, he holds that it
is also possible to apply that in the interpretation of the early
ontogeny of Marsupials (v. '08, p. 235, and '09, p. 229). He
maintains that the inner cell-mass of Eutheria is purely ectodermal, aud that the enveloping trophoblast layer of the blastocyst arises in common with the entodermal lining of the same
and is therefore also entodei'mal. " On the theory I advocate,”
he writes ('09, p. 235), " the trophoblast is of Eutherian
mammalian origin only and is not homologous to any form of
envelope outside the group of Eutherian mammals.” These
views of Assheton are not only at variance with those of all
other investigators who have worked at the early ontogeny of
Eutheria, but they are quite irreconcilable with my observations on the development of Dasyurus herein recorded. I claim
to have shown in that Marsupial that the formative region, the
homologneof the inner cell-mass, gives origin not only to the
embryonal ectoderm, but to the entire entoderm, whilst tlie
non-formative region, whose homology to the trophoblast of
Eutheria is admitted by Assheton, arises quite independently
of the entoderm and a long time before the latter inakes its
appearance. There is, then, in Dasyurus no question of a
common origin of the entoderm and the non-forrnative or
trophoblastic region of the blastocyst wall. And exception
inay be taken to Assheton's views on quite other grounds
(e. g. the question of the homologies of the foetal membranes
in the series of the Amniota), as he himself is well awai'e, and
as Jenkinson ('00) has also emphasised. I feel, however, I can
leave further discussion of Assheton's views until such time
as my observations on Dasyurus are shown to be erroneous or
inapplicable to other Marsupials.
3. The Entypic Condition of the Eutherian
If, now, on the basis of the homologies I have ventm-ed to
advocate in the preceding pages, we proceed to compare the
Metatherian with the Eutherian blastocyst, we have to note
that, whereas in the latter the extra-embryonal or trophoblastic ectoderm alone forms the blastocyst wall in early
stages and completely encloses the embryonal knot, in the
former, the homologous parts, viz. the non-formative or exti'aembryonal and the formative or embryonal regions, both
enter into the constitution of the unilaminar blastocyst
wall, there being no such enclosure of the one by the
other as occurs in the Eutherian blastocy.st (Text-fig. 2, p. 98).
It is characteristic of the Marsupial as of the Monotreme that
the embryonal region is from the first superficial and freely
exposed. It is spread out as a cellular layer and simply
forms part of the blastocyst wall or blastoderm. It is equally
characteristic of the Eutherian that the homologous part, the
embryonal knot, has at first the form of a compact mass,
which is completely enclosed by the trophoblastic ectoderm.
The latter alone constitutes the unilaminar wall of the
blastocyst and has the embryonal knot adherent at one spot
to its inner surface. The formative cells which compose
the knot thus take at first no part in the constitution of
the outei wall of the blastocyst^ and may or may not
do so in later stages according as the covering layer of the
trophoblast (the Deckschicht or Rauber's layer) is transitory or permanent. This peculiar developmental condition, characterised by the internal position of the formative
or embryonal cells within the blastocyst cavity, has been
termed by Selenka (TO) “entypy” (Entypie des Keimfeldes).^ It is a phenomenon exclusively found in the
Eutheria and characteristic of them alone, amongst the
mammals. In the Marsupial, as in the Monotreme, the
formative cells are freely exposed, and constitute from the first
part of the blastocyst wall just as those of the Sauropsida form
a part of the general blastoderm. Limited as entypy thus
appears to be to the higher mammals, the probability is that
we have to do here with a purely secondary, adaptive feature.
If we proceed to inquire what is the significance of this
remarkable difference in the early developmental phenomena
of the lower and higher mammals, it seems to me that we have
to take account, in the first place, of the differences in the
structure of their respective eggs, and especially we have to
bear in mind that the Eutherian ovum is considerably more
specialised than even the Metatherian. It is on the average
smaller than the latter, i.e. it has suffered in the course of
phytogeny still further reduction in size, and has lost, to an
even greater extent than the Marsupial ovum, the store of foodyolk ancestrally present in it. Moreover, it has suffered a still
further i-eduction in respect of its secondary egg-membranes.
The Metatherian ovum still retains in its shell-membrane a
^ “ Unter Entypie des Keimfeldes mdcbte ich dalier verstanden
wissen : Die nicht dm-cli Bildung typischer Anmionfalten geschehende,
sondern durcli eine schon wiihrend der Gastrulation erfolgende Absclinurung des Keimfeldes ins Innere der Eiblasenbnlle (Oborion) ” ('00,
p. 203).
vestigial representative of the shell of the presumed oviparous
common ancestor of the Metatheria and Eutheria. The
Eutherian ovum, on the other hand, has lost all trace of the
shell in correlation with its more complete adaptation to the conditions of intra-nterine development. The albumen layer is
variable in its occurrence, being present in some (e.g. rabbit)
and absent in others (e.g. pig, Assheton), whilst the zona
itself, though always present, is variable both as to its thickness and the length of time it persists.
Strangely enough, although the prevaling opinion amongst
mammalian embryologists is that the Eutherian ovum has
been derived phylogenetically from an egg of the same telolecithal and shell-bearing type as is found in the Monotremes,
no one, so far as I am aware, has ever taken the shell into
account, and ventured to consider in what way its total disappearance from an ovum already greatly reduced in size,
might affect the course of the early developmental phenomena.
That is what I propose to do here, for iu my view it is just in
the complete loss of the shell by the Eutherian ovum that we
find the key to the explanation of those remarkable differences
which are observable between the early ontogeny of the
Eutheria and Metatheria, and which culminate in the entypic
condition so distinctive of the former. The acquisition of a
shell by the Proamniota conditioned the appearance of the
amnion. The loss of the shell in the Eutheria conditioned the
occui'rence in their ontogeny of entypy.
As we have seen, the mammalian ovum, already in the
Monotremes greatly reduced iu size as compared with that of
reptiles, and quite minute in the Metatheria and Eutheria,
contains within itself neither the cubic capacity nor the food
material necessary for the production of an embryo on the
ancestral reptilian lines. We accordingly find that the
primary object of the first developmeutal processes in the
mammals has come to be the formation of a vesicle with a
complete cellular wall, capable of absorbing nutrient fluid from
the maternal uterus and of growing I'apidly, so as to provide
the space necessary for embryonal differentiation.
,T. r. HILL.
In the Monotremes this vesiculai' stage is rapidly and
directly attained as the result, firstly, of the rearrangement
of the blastomeres of the cleavage-disc to form a unilaminar
blastodermic membi'ane overlying.tbe solid yolk, and, secondly,
of the rapid extension of the peripheral (extra-embryonal)
region of the same, in contact with the inner surface of the
firm sphere furnished by the egg-envelopes. During the
completion of the blastocyst embryonal differentiation remains
in abeyance, and practically does not start until after growth
of the blastocyst is well initiated.
In the Marsupial, notwithstanding the fact that the ovum
has become secondarily holoblastic, the mode of formation
of the blastocyst is essentially that of the Monotreme.
Cleavage is of the radial type, and owing to the persistence
of the shell, wliicb with the zona forms a firm resistant
sphere enclosing the egg, the radially arranged blastomeres ai'e able to assume the form of an open ring and to
proceed directly to the formation of the unilaminar wall of
the blastocyst. The enclosing sphere provides the necessary
firm surface over which the products of division of the upper
and lower cell-rings of the 16-celled stage can respectively
spread towards opposite poles, so as to directly constitute the
formative and non-formative regions of the blastocyst wall.
In my opinion it is the persistence of the resistant shellmembrane round the ovum which conditions the occurrence
in the Marsupial of this direct method of blastocyst formation.
As in the Monotreme, so here also embryonal differentiation
commences only after the blastocyst has gi'ovvn considerably
in size.
^ In the Eutheria, on the other hand, in the absence of the
shell-membrane, not only is the mode of formation of the
blastocyst quite different to that in the Marsupial, but
the relations of the constituent parts of the completed
structure also differ markedly from those of the homogenous parts in the latter. The cleavage process here leads
only indirectly to the formation of the blastocyst, and must be
held to be csenogeneticaily modified as compared with that of
lower mammals. In the cross-shaped arrangement of the
blastomeres in the 4-celled stage, in the occurrence of a
definite morula-stage and of the entypic condition, we have
features in which the early ontogeny of the Eutheria differs
fundamentally from that of the Metatheria. They are intimately correlated the one with the other, and are met "with in
all Eutheria, so far as known, but do not occur either in the
Prototheria or the Metatheria, so that we must regard them
as secondary features which were acquired by the primitive
Eutheria under the influence of some common causal factor
or factoi's, subsequent to their divergence from the ancestral
stock common to them and to the Metatheria. Now the crossshaped 4-celled stage and the morula-stage are undoubtedly
to be looked upon simply as cleavage adaptations of prospective
significance in regard to the entypic condition, so that the
problem reduces itself to this  -  how came these adaptations
to be induced in the first instance ? In view of the facts that
in the Metatheria, in the presence of the shell-membrane, the
formation of the blastocyst is the direct outcome of the cleavage
process, and is effected along the old ancestral lines without
any enclosure of the formative cells by the non-formative,
whilst in the Eutheria, in the absence of the shell-membrane, blastocyst formation results only indirectly from the
cleavage-process, is effected in a way quite different from
that characteristic of the Metatheria, and involves the
complete enclosure of the formative by the non-formative
cells, I venture to suggest that the cleavage adaptations
which I'esult in the entypic condition were acquired in the first
instance as the direct outcome of the total loss by the already
greatly reduced Eutlierian ovum of the shell-membrane.^
This view necessarily implies that the presence of a thick
zona such as occurs round the ovum in certain Eutheria is
secondary, and what we know of this membrane in existing
Eutheria is at all events not adverse to that conclusion.
This suggestion I first put foi'ward in a course of lectures on the
early ontogeny and placentation of the Mammalia delivered at the
University of Sydney in 1904.
Amongst tlie Marsupials the zona is quite thin (about -00] 6
imn. in Dasyurus), presumptive evidence that it was also thin
in the ancestral stock from which the Meta- and Eutheria
diverged, whilst amongst the Eutheria themselves the zona,
as Robinson ('03) has pointed out, is not only of very varying
thickness, but persists round the ovum for a very varying
period iu different species. It appears to be thinnest in the
mouse ('001 mm.), in most Eutheria it is considerably thicker
(•01 mm., bat, dog, rabbit, deer), whilst in Cavia it reaches
a thickness of as much as -02 mm. In those forms in which
the blastocyst early becomes embedded in, or attached to, the
mucosa, the zona naturally disappears early. In the rat,
mouse and guinea-pig it disappears before the blastocyst is
formed. Hubrecht failed to find it in the 2-celled egg of
Tupaia, and it was already absent in the 4-celled stage of
Macacus nemestrinus, discovered by Selenka and described by Hubrecht. On the other hand, it may persist for
a much longer period, up to the time of appearance of the
primitive streak (rabbit, dog, ferret). These facts sufficiently demonstrate the variability of the zona in the Eutherian
series, and its early disappearance in certain forms before the
completion of the blastocyst stage shows that it can have no
supporting function in i-egard to that.
Postulating, then, the disappearance of the shell-membrane
and the presence of a relatively thin, non-resistant zona (with
perhaps a layer of albumen) round the minute yolk-poor ovum
of the primitive Eutherian, and remembering that the ovum
starts with certain inherited tendencies, the most immediate
and pressing of which is to produce a blastocyst comprising
two differentiated groups of cells, the problem is how, in the
absence of the old supporting sphere constituted by the eggenvelopes, can such a vesicular stage be most easily and
most expeditiously attained ? The Eutherian solution as we see
it in operation to-day is really a very simple one, and withal a
noteworthy instance of adaptation in cleavage (Lillie, '99).
In the absence of any firm supporting membrane round the
egg, and the consequent impossibility of the blastomeres pro
ceecling- at once to forna the blastocyst wall, they are under
the necessity of keeping together, and to this end cleavage
has become adapted. For the ancestral radial arrangement
of the blastomeres in the 4-celled stage, characteristic of the
Monotreme and Marsupial, there has been substituted a
cross-shaped grouping into two pairs, and, as the outcome of
this adaptive alteration in the cleavage planes, there results
from the subsequent divisions, not an open cell-ring, as in tbe
Marsupial, but a compact cell-group or morula. In this we
again encounter precisely the same differentiation of the
blastomeres into two categories, respectively formative
(embryonal) and non-formative (trophoblastic) insignificance,
as is found in the 16-celled stage of the Marsupial, but, since
the two groups of cells are here massed together, and in the
absence of any firm enclosing sphere, cannot spread independently so as to form directly the wall of the blastocyst,
there has arisen the necessity for yet other adaptive modifications. Attention has already been directed to the tardiness
of differentiation in the embryonal region of the Monotreme
and Marsupial blastocyst, and here in the minute Eutherian
morula we find what is, perhaps, to be looked upon as a
further adaptive exaggeration of this same feature in the
inertness which is at tirst displayed by the formative cells,
and which is in marked contrast with the activity shown by
the non-formative ectodermal cells.^ It is these latter, it
* The inertness of the formative cell-mass is accounted for by Assheton
('98, p. 251) as follows : “ Now, as the epiblast plays the more prominent
part in the formation of the l^nlk of the embi-yo dui-ing the earliest
stages, it clearly would be useless for tlie embryonic part to exhibit
much energy of growth until the old conditions [in particular sufficient
room for embryonal differentiation] were to a certain extent regained ;
hence the lethargy exhibited by the embryonic epiblast in mammals
during the first week of develoxunent. No feature of the early stages of
the mammalian embryo is more striking than this inertness of the
embryonic eiriblast  -  or, as I should nowjrrefer to call it, simply epiblast
-  during the first few days.” Assheton, it should be remembered, holds
that the inner cell-mass of Eutheria furnishes only the embryonal
should be recollected, which exhibit the greatest growthenergy during the formation of the blastocyst in the Monotreme and Marsupial, and so their greater activity in the
Eutherian tnoi'ula is only what might be expected. Dividing
more rapidly than the formative cells, they gradually grow
round the latter, and eventually form a complete outer layer
enveloping the inert formative cell-group. This process oFovergrowth or epiboly is entirely comparable in its effect with the
spreading of the extra-embryonal region of the unilamiiiar
blastodermic membrane in the Monotreme to enclose the yolkmass, and with that of the non-formative cells in the Marsupial
to complete the lower hemisphere of the blastocyst, growlh
round an inert central cell-mass being here substituted for
growth over the inner surface of a I'esistant sphere constituted
by the egg-envelopes, such as occurs during the formation of
the blastocyst in the Monotreme and Marsupial. .Just as the
first objective of the cleavage process in the latter is to effect
the completion of the cellular wall of the blastocyst, so hei*e
the same objective recurs, and is attained in the simplest
possible way in the new circumstances, viz. by the I'apid envelopment of the formative by the, non-formative cells. Thus
at the end of the cleavage process in the EutheiJan we have
formed a solid entypic morula in which an inner mass of
formative cells is completely surrounded by an outer enveloping layer of non-formative or ti'opho-ectodermal cells, homogenous with the extra-embryonal ectoderm of the Sauropsidan
and Monotreme and the non-formative region of the unilaminar blastocyst of the Marsupial. Conversion of the solid
morula into a hollow blastocyst capable of imbibing fluid
from the uterus and of growing rapidly now follows. Intraor intercellular vacuoles appear below the inner cell-mass, by
the confluence of which the blastocyst cavity is established,
and the inner cell-mass becomes separated from the enveloping layer of tropho-ectoderm, except over a small area where
the two remain in contact.
The complete enclosure of the formative cells of the inner
cell-mass by the non-formative ectodermal cells of the
enveloping layer which produces this peculiar entypic condition in the Eutherian blastocyst, I would interpret, then, as
a purely adaptive phenomenon, which in the given circumstances effects in the simplest possible way the early completion
of the blastocyst wall, and whose origin is to be traced to
that reduction in size and in its envelopes which the Eutherian
ovum has suffered in the course of phylogeny, in adaptation
to the conditions of intra-uterine development. In particular,
starting with a shell-bearing ovum, already minute and
undergoing its development in utero, I see in the loss of
the shell such as has occurred in the Eutheria an intelligible
explanation of the first origin of those adaptations which
culminate in the condition of entypy. I am therefore wholly
unable to accept the view of Hubrecht (^08, p. 78), that " what
Selenka has designated by the name of Entypie is  -  from
our point of view  -  no secondary phenomenon, but one
which repeats very primitive featui*es of separation between
embryonic ectoderm and larval envelope in invertebrate
I see no reason for supposing that the intimate relationship
which is early established in many Eutheria between the
trophoblastic ectoderm and the uterine mucosa has had anything to do with the origination of the entypic condition. In
ray view such intimate relationship involving the complete
enclosui'e of the blastocyst in the mucosa only came to be
established secondarily, after entypy had become the rule.
On the other hand, the peculiar modifications of the entypic
condition met with in rodents with “^inversion” (e.g. i-at,
mouse, guinea-pig) are undoubtedly to be correlated, as Van
Beneden also believed ('99, p. 332), with the remarkably early
and complete enclosure or implantation of the germ in the
mucosa such as occurs in these and other Eutheria. Similar
views are expressed by Selenka in one of his last contributions
to mammalian embryology. He writes ('00, p. 205)  -  “Dass
die Entypie des Keimfeldes und die Blattinversion begiinstigt
wil'd durch die friihzeitige Yerwachsung der Eiblase mit dem
Uterus, ist nicht in Abrede zu stellen. Aber da dieser
Prozess auch in solclieu Eiblasen dei- Saugetiere vorkommen
kanii, die iiberhaupt nichb, odei- erst spiiter mifc dem Uterus
verwachsen, so kaiiu die Keimfeld-Entypie zwar durch die
frube Verwacbsung veraiilasst, aber nicht ausscldiesslich
liervorgerufeii werclen.” He goes on to remark that  -  “Die
Vorbedingimgeti zur Eutypie miissen in der Struktur der verwachseuden Eiblase gesucht werden/^ and expi-esses his
agreement with the views of Van Beneden as to tlie significance to be attributed to the early cleaviige phenomena in
The attitude of the illustrious Belgian embryologist whose
loss ws have so recently to deplore, towards this problem is
clearly set forth in the last memoir which issued from his
hand. “Je suis de ceux,^' he wrote (T9, p. 332), “qui pensent
que toute Pembryologie des Mammiferes placentaires temoigue
quTls derivent d'animaux qui, comme les Sauropsides et les
Mouotremes, produisaieut des oeufs meroblastiques. Je ne
puis a aucun point de vue me rallier aux idees contraires formulees eb defendues par Hubrecht. L^hypothese de Hubrecht
se heurte a des difiicultes morpliologiques et physiologiques
insurmontables : elle laisse inexpliquee Pexistence, chez les
Mammiferes placentaires, d'une vesicule ombilicale et dTne
foule de caracteres commnns a tons les Amniotes et distiuctifs
de ces auimaux.'^ Holding this view of tlie origin of the
Eutheria, Van Beneden based his interpretation of their early
ontogenetic phenomena on the belief that “ la reduction progressive du volume de Poeuf d'une part, le fait de son
developpement iutrauterin de hautre ont dii avoir une influence preponderante sur les premiers processus evolutifs.”
Balfour, in his classical treatise, had already some eighteen
years earlier expressed precisely the same view. “The
features of the development of the placental Mammalia,^' he
wrote (‘Mem. Edn.,^ vol. iii, p. 289), “receive their most
satisfactory explanation on the hypothesis that their ancestors
were provided with a large-yolked ovum like that of Sauropsida. The food-yolk must be supposed to have ceased to be
developed on the establishment of a maternal nutrition through
the uterus. . . . The embryonic evidence of the common
origin of Mammalia and Sauropsida, both as concerns the
formation of the layers and of the embryonic membranes is
as clear as it can be.'''
That view of tlie derivation of the Mammalia receives, I
venture to think, striking confirmation from the observations
and conclusions set forth in the preceding pages of this
memoir, and from it as a basis all attempts at a phylogenetic
interpretation of the early ontogenetic phenomena in the
Mammalia must, I am convinced, take their origin. Such an
attempt I have essayed in the foregoing pages, with what
success the reader must judge.
The memoir of Prof. 0. Van der Stricht, entitled “La structure de I'cBuf des Mammiferes (Chauve-souris, Vesperugo
noctula) : Troisieme Partie” (‘Mem. de PAcad. roy. de
Belgique,' 2nd ser., t. ii, 1909), came into my hands only
after my own paper had readied its final form, and therefore
too late for notice in the body of the text. In this extremely
valuable contribution, Van der Stricht gives a detailed
account of the growth, maturation, fertilisation, and early
cleavage-stages of the ovum of Vesperugo, illustrated by a
superb series of drawings and photo-micrographs. All I can
do here, however, is to direct attention to that section of the
paper entitled “ Phenomeues de deutoplasmolyse an pole
vegetatif de I'ceuf” (pp. 92 - 96), in which the author describes
the occurrence in the bat's ovum of just such a process of
elimination of surplus deutoplasmic material as I have
recorded for Dasyurus. Van der Stricht's interpretation of
this phenomenon agrees, I am glad to find, with my own.
He writes (pp. 92-93): “ Ce deutoplasme rudimentaire, i\
peine ebauche dans I'ovule des Mammiferes, parait etre
encore trop abundant dans I'oeuf de Chauve-souris, car ces
materiaux de reserve, en partie inutiles, sont partiellement
elimines, expulses de la cellule.”
.T. P. HILL.
To this pi'ocess of elimination of surplus deutoplasm he
applies the name deutoplasmolyse,” and states that Ce
phenomene consiste dans I'apparition de lobules vitellins
multiples, en nombre tres variable, a la surface du vitellus au
niveau du pole vegetatif. Ces bourgeons a peu pres tous de
meme grandeur, les uns etant cependant un peu plus volumineux que les autres, apparaissent dans le voisinage des globules
polaires et presentent la structure du deutophisme. 11s sont
formes de vacuoles claires, a I'interieur desquelles on aper^oit
parfois de petits grains vitellins, dont il a ete question plus
haut. . . . Ce processus de deutoplasmolyse devient
manifeste surtout apres I'expulsion du second globule polaire,
pendant la periode de la fecondation. 11 pent etre tres
accentue, au stade du premier fuseau de segmentation et au
debut de la segmentation de I'oeuf, notamment sur des ovules
divises en deux et en quatre (figs. 59, 61, 62, d).” It would
therefore appear that, whilst in Dasyurus the surplus deutoplasm is eliminated always prior to the completion of the
first cleavage and in the form of a single relatively large
spherical mass, in Vesperugo it is cast off generally, though
not invariably, before cleavage begins, and in the form of a
number of small separate lobules.
List op References.
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Development of the Rabbit,” ‘ Quart. Journ. Micr. Sci.,' vol. 34.
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‘ Quart. Journ. Micr. Sci.,' vol. 41.
'98. “ The Segmentation of the Ovum of the Sheep, with Obser
vations on the Hypothesis of a Hypoblastic Origin for the
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'08. “ The Blastocyst of Capra, with Some Remarks upon the
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'09. “Professor Hubrecht's Paper on the Early Ontogenetic
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'97. Bonnet. R.  -  “ Beitriige zur Embvyologie des Himdes,” ‘ Anatomische Hefte,' Bd. ix.
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'87. Caldwell, W. H. -  “ The Erabiyology of Monotremata and Marsnpialia,” Part I, ‘ Phil. Trans. Roy. Soc.,' vol. clxxviii B.
'95. Duval, M. -  “Etudes sur I'embryologie des Oliciropteres,” ‘ Joura.
de I'Anat. et de la Pliysiol.,' t. xxxi.
'86. Heape, W.  -  “ The Development of the Mole (Talpa Europea), the
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'00. “ On the Foetal Membranes, Placentation and Parturition of
theNative Cat(Dasyurus viverrinus),” ‘Anat. Anz.,'Bd.xviii.
'88. Hubrecht, A. A. W.  -  “ Keimbliitterbildung und Placentation des
Igels,” ‘ Anat. Anz.,' Bd. iii.
'89. “ Studies in Mammalian Embryology : (1) The Placentation
of Erinaceus europaeus, with Remarks on the Physiology of
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'95. “ Die Phylogenese des Amnions und die Bedeutung des
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'02. “ Fiirchung und Keimblattbildung bei Tarsius Spectrum,”
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'09. “The Foetal Membranes of the Vertebrates,” ‘ Proc.
Seventh Interaational Congress, Boston Meeting,' August 19th
to 24th, 1907.
'00. Jenkinson, J. W.  -  “A Re-investigation of the Early Stages of the
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'06. “ Remarks on the Germinal Layers of Vertebrates and on
the Significance of Germinal Layers in General,” ‘ Mem. and
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'01. Keibel, F.  -  “Die Gastrulation und die Keimblattbildung der
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“ Die Entwickelung der Rehes bis zui* Anlage des Meso
blast,” ‘ Arch, fiir Anat. u. Physiol. Anat. Abth.'
' 02 .
J. r. Hii,L.
0/. Lams, H., and Doonne, J.  -  “ Nouv^elles recheivhes sur la Maturation et la Fecondation de I'cenf des Maminiferes,” ‘ Arch de Biol.,'
t. xxiii.
03. Lee, T. Gr. ‘Implantation of the Ovum in Sf)ermoi)hilus
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and its bearing on the Interjiretation of the Eai'ly Ontogenetic
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03. Robinson, A. Lectures on the Early Stages in the Development
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“ La Structure de I'ceuf des Maminiferes. Premiere partie,
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Illustrating Prof. J. P. Hill's paper on “ The Early Development of the Marsupialia, with Special Reference to the
Native Cat (Dasyurus vi verrinus).”
[All figures are from specimens of Dasyurus, unless otherwise indicated. Drawings were executed with the aid of Zeiss's camera lucida,
except figs. 61-63, which were drawn from photographs.]
List of Common Reference Letters.
Ab7i. Abnormal blastomei'e, fig. 37. alh. Albumen, eg. Coagulum.
d. p. Discus proligerus. d. z. Deutoplasmic zone. emb. a. Embryonal
area. emb. ect. Embiyonal ectoderm, ent. Entoderm. /. ep. Follicular
epithelium. /. a. Formative area of blastocyst wall. /. c. Formative
cell. /. z. Formative zone. i. c. Internal cell, fig. 34. Z. eat. Limit of
extension of entoderm. Z. p. Incomjilete ai'ea of blastocyst wall at lower
pole. p. b'. First polar body. p. b'. s. First polar spindle, p. V. s.
Second polar spindle, p. s. Perivitelline space, s. m. Shell-membrane.
sp. Sperm in albumen. Zr. ect. Non-formative or trophoblastic ectoderm (tropho-ectoderm). y.b. Yolk-body. z. p. Zona.
Fig. 1.  -  Photo-micrograph (x 150 diameters) of the full-grown
ovarian ovum, '27 X ‘26 mm. diameter. The central deutoplasmic
zone (cZ. z.) and the peripheral formative zone (/. z.), in which the
J. 1>. HITiL.
vesicular nucleus ('QS X '03 mni. diameter) is situated, are clearly distinguishable. The zona (z. p.) measures •0021-'0025 mm. in thickness.
Outside it are the follicular epithelial cells of the discus proligerus
(d.p.), which is thickened on the upper side of the figure, where it
becomes continuous with the membrana granulosa. (D. v i v., 21 . vii .
'04, Hermann's fluid and iron-hsematoxylin.)
Fig. 2.  -  Photo-micrograph ( X. 150) of ripe ovarian ovum (in which
first polar body is separated and second polar spindle is present, though
neither is visible in figure), '29 X '23 mm. maximum diametei'. FoUicle
1'4 X IT mm. diameter. The ovum exhibits an obvious polarity.
Deutoplasmic zone {d. z.) in upper hemisphere ; formative zone (/. z.)
foi-ming lower. (D. v i v., 14, 26 . vii . '02, Flemming's fluid and
Fig. 3.  -  Photo-microgi'aph ( x 150) of ripe ovarian ovum ('28 x '24
mm. diameter) with first polar body (p. bK) and second polar spindle.
First polar body, •026-‘03 x '01 mm. Second polar spindle, '013 mm.
in length. (D. v i v., 14, 26 . vii . '02, Flemming's fli;id and ironhaematoxylin.)
Fig. 4.  -  Photo-micrograph (x 256) of ovarian ovum in process of
growth (“pseudo-alveolar” stage). Ovum, ‘26 X '20 mm. diameter.
Zona, •0017-‘002 mm. in thickness. (D. v i v., 14, 26 . vii . '02,
Hermann, iron-haematoxylin.)
Fig. 5.  -  Photo-microgi-aph (X 1250) of peripheral i-egion of ripe
ovarian ovum ('28 X T26 mm. diameter) with first polar spindle ('015
X '013 mm.). (D. v i v., 23 . vii . '02, Ohlmaicher's fluid, iron-haema
Fig. 6. -  Photo-micrograph (x 1250) of peripheral region of ripe
ovarian ovum ('26 X T8 mm.), showing first polar body (p. b'.) ('03 X
•006 mm.). (D. v i v., 14, 26 . vii . '02, Flemming, iron-hfematoxylin.)
Fig. 7. -  Photomicrograph ( X 1250) of periplieral region of ovum, fig.
3, showing portion of first polar body (p. 5'.), and the second polar
spindle. The dark body lying between p. 5'. and the surface of the
ovum is a displaced red blood-corpuscle.
Figs. 8 and 9. -  Photo-micrographs ( X about 84) of unsegmented ova,
respectively '33 mm. and '35 mm. in diameter, from the uterus, taken
immediately after their transference to the fixing fluid (picro-nitroosmic acid), showing the shell-membrane (s. m.), laminated albumen
{alb.), with sperms (sp.), the zona (z. p.), perivitelline space {p. s.), and
the body of the ovum, with its formative (/. z.), and deutoplasmic {d. z.)
zones. (D. v i v., 15, 19 . vii . '01.)
Fig. 10. -  Photo-micrograph ( X 150) of section of imsegmented ovum
almost immediately after its passage into the uterus, showing the veiy
thin sliell-inembvane externally (s. m.) (about '0016 mm. in thickness),
the albumen {alb.), zona (z-i?.), and the deutoplasmic {d. z.) and formative
(/. z.) zones of its cytoplasmic body. The male pronucleus is visible in
the formative zone. Diameter of entire egg about '29 mm. (D. viv.,
15, 19 . vii . '01, Picro-nitro-osmic and iron-hffimatoxylin.)
Fig. 11.  -  Photo-micrograph ( X 150) of section of unsegmented ovum
from the uterus, slightly older than that of fig. 10. Diameter of entire
egg in fresh state •34-'35 mm., of the ovum proper '3 X ‘28 mm. ; thickness of shell, -0024 mm. In the figure the female pronucleus is visible
near the centre of the formative zone (/. z.), and the male pronucleus
lies a little above it and to the right. The perivitelline space (jJ.s.)
is pai-tiaUy occupied by coagulum. (D . viv., 21 . v . '03, f. Hermann,
PLATE 2. •
Fig. 12.  -  Photo-micrograph ( X 150) of an unsegmented ovum from
the irterus, of the same batch as that of fig. 11, and '34 mm. in diameter.
The two pronuclei are visible in the central region of the formative
Fig. 13.  -  Photo-microgi-aph ( X 330) of uterine ovum. Stage of first
cleavage spindle. Diameter, '315 mm. (D. viv., 1, 15 . vii . '01, f.
Picro-nitro-osmic, iron-hiematoxylin.)
Fig. 14.  -  Photo-micrograph ( X about 78) of egg in the 2-celled stage,
taken immediately after its transference to the fixing fluid. Lateral
view. y. b. Yolk body. Diameter of entire egg about "34 mm. (D . viv.,
1, 15 . vii . '01. Picro-nitro-osmic.)
Fig. 15.  -  Photo-microgi'aph (x about 78) of another 2-celled egg,
seen from lower pole. Diameter, '35 mm. (D. viv., 4 B, 23 . vi . '02.
Perenyi's fluid.)
Fig. 16.  -  Photo-micrograph (x about 78) of another 2-celled egg,
of the same batch as preceding. End view, showing one of the two
blastomeres and the yolk -body (y. b.).
Fig. 17.  -  Photo-micrograph (x 150) of vertical section of 2-celled
egg, "34 mm. in diameter, showing the shell-membrane ('0064 mm. thick),
traces only of the albumen, the zona (z.p.), and the two blastomeres (the
left one measuring, from the sections, T6 x T8 x TO mm., its nucleus
‘031 X ‘027 mm. ; the right one, T6 x T9 X "09 mm., its nucleus, '03 x
•028 mm.). Note the differentiation in their cytoplasmic bodies.
(D . viv., 6, 21 . vii . '01, Picro-nitro -osmic and iron-hsematoxylin.)
Fig. 18.  -  Photo-micrograph (x 150) of vertical section of 2-celled
egg, '32 mm. in diameter, with shell-membrane '005 mm. thick, showing
the two blastomeres, and enclosed between their upper ends the yolk
J. r. Hii,L.
body {y. b.). (D . viv., 1, 15 . vii . '01, f. Picro-nitro-osmic, iron-htematoxylin.)
Figs. 19 and 20.  -  Photo-micrographs ( x about 70) of 4-eelled eggs
taken immediately after transference to Perenyi's fluid. Fig. 19, side
view, showing yolk-body (y. h.) ; fig. 20, polar view. Diameter of entire
egg about -35 mm. (D . viv., 14 b, 18 . vi . '02. Perenyi.)
Fig. 21. -  Photo- micrograph (x about 70) of another 4-celled egg,
from the same batch as the preceding, seen from lower pole.
Fig. 22. -  Photo-micrograph (x 150) of section of 4-ceUed egg of
same batch as those of figs. 19 and 20. The two right and the two
left blastomeres respectively form pairs, so that the plane of the first
cleavage is parallel with the sides of tlie plate, that of the second with
the top and bottom of the same. The two left blastomeres are still
connected by a narrow cytoplasmic bridge. Thickness of shell,
•0072 mm.
Fig. 23. -  Photo-micrograph ( x 150) of a vertical section through
a 4-celled egg. ‘35 mm. in diameter, showing two of the blastomeres
and a small portion of the yolk-body {y. b.). Note, as in fig. 22, the
marked diflierentiation in the cytoplasm of the blastomeres. (D. viv.,
4, 27 . vi . '01. Picro-nitro-osmic, iron-hsematoxylin.)
Figs. 24 and 25.  -  Photo-micrographs ( x 140) of horizontal sections
through a 16-celled egg, '38 mm. diameter, fig. 24 showing the eight
larger, more yolk-rich cells of the lower (non-formative) ring, and fig. 25
the eight smaller, less yolk-rich cells of the upper (formative) ring.
Shell ‘0075 mm. in thickness, yolk-body (not included in the figures)
'll X TO mm. in diameter. (D. viv., 3 b, 26 . vi . '01; 15, f and |.
Picro-nitro-osmic and iron-hsematoxylin.)
Fig. 26.  -  Photo-micrograph (x 140) of a vertical section of an egg
of the same batch and size as that represented in figs. 24 and 25, but
with seventeen cells  -  formative = 9 (6 + [1 X 2] + 1) in division ;
non-formative = 8. Two of the formative cells (/. c.) of the upper ring
are seen enclosing between them the faintly mai'ked yolk-body {y. b.),
and below them two of the much more opaque non-formative cells
{tr. ect.) of the lower ring.
Fig. 27.  -  Photo-micrograph (x about 76) of the just completed
blastocyst, '39 mm. in diameter. From a spirit specimen. The dark
spherical mass (eg.) in the blastocyst cavity is simply coagulum, produced by the action of the fixative (picro-nitro-osmic acid) on the
albuminous fluid which fills the blastocyst cavity. (D. viv., 2 b,
16 . vii . '01.)
Fig. 28. -  Plioto-anicrogi-apli ( X about 76) of a blastocyst of the same
batch as the preceding, •45 mm. in diameter. From a spirit specimen.
eg. Coagulum.
Fig. 29.  -  Photo-micrograph (x about 75) of another blastocyst,
•45 mm. diameter, of the same batch as the preceding, but taken
immediately after transference to the fixative. Viewed from the upper
pole. y. b. Tolk-body seen through the unilaminar wall.
Fig. 30.  -  Photo-micrograph ( X about 75) of a blastocyst of the same
batch as the preceding, about '39 mm. in diameter, in which the cellular
wall has not yet been completed over the lower polar region.
Fig. 31.  -  Photo-micrograph ( X 140) of a section of a blastocyst,
•39 mm. diameter, of the same batch as the preceding and at precisely
the same developmental stage, the cellular wall having yet to be completed over the lower polar region (l.p.). In the blastocyst cavity is
seen the yolk-body (y. b.) partially surroixnded by a mass of coagulum
(eg.). (D. viv., 2 B, 16 . vii . '01, m. = '39, Picro-nitro-osmic and
Fig. 32.  -  Photo-micrograph ( X 140) of another blastocyst, ^41 mm.
in diameter, of the same batch as the preceding, also 'with the cellular
wall still absent over the lower polar region. Shell-membrane ‘0075 mm.
in thickness, y. b. Tolk-body. c. g. Coagulum. The cellular wall
comprises about 130 cells.
Fig. 33.  -  Photo-micrograph ( X 140) of a blastocyst of the same batch
as the preceding, with a complete unilaminar cellular wall. y. b. Yolkbody, in contact with inner surface of wall, in the region of the upper
Fig. 34.  -  Photo-micrograph (x 100) of a section of a blastocyst
•57 mm. in diameter, i. c. Internal ceU. (D . vi v., 29 . vi . '04, y . Pici^onitro-osmic.)
Fig. 35.  -  Photo-micrograph (x 100) of a section of a blastocyst, '73
mm. diameter, of the same batch as the pi^eceding, shell, ^0045 mm.
Fig. 36.  -  Photo-micrograph (x 100) of a section of a blastocyst -66
mm. diameter, of the same batch as the pi-eceding. Lower hemisphere
opposite yolk-body {y. b.) formed of larger cells than upper. Hermann
Fig. 37. -  Photo-micrograph (x 140) of section of an abnormal
vesicle, 397 mm. diameter of the same batch as the normal vesicles
represented in figs. 27-33. abn. large binucleate cell, regarded as a
blastomere of the lower hemisphex^e which has failed to divide in noi^mal
fashion, cf . text, p. 42.
.1. P. HILL.
Fig. 38 -  Photo-micrograpli ( x 10) of entire blastocyst 4'5 mm. diameter to show the junctional line {j. 1.) between formative and nonformative regions. From a spirit specimen. (D . viv., /3, 25 . vii . '01.
Fig. 39. -  Photo-microgi-aph ( x about 10) of an entire blastocyst,
4'5 mm. diameter with distinct embryonal area {emh. a.). (D. viv., 5,
18 . vii . '01.)
Fig. 40.  -  Photo-micrograph { X 10) of entire blastocyst about 5 mm.
diameter showing embryonal area' {emh. a.), peripheral limit of entoderm (1. ent.), and the still unilaminar region of the wall {tr. ect.). (D.
viv., 8 . vi . '01.)
Fig. 41.  -  Photo-micrograph ( x 150) of an in toto preparation of the
wall of a blastocyst of 3'5 mm. diameter. (D . viv., 16, 21 . vii . '01.)
Fig. 42.  -  Photo-micrograph (x 150) of an in toto preparation of the
wall of a blastocyst of 3'25 mm. diameter, j. 1. Junctional line between
the formative (/. a.) and non-formative {tr. ect.) regions of the wall.
(D. viv., 24 . vii . '01.)
Figs. 43 and 44.  -  Photo-micrographs (x 150) of in toto preparations
of the wall of 4'5 mm. blastocyst showing the jimctional line between
the formative (/. a.) and non-formative {tr. ect.) regions. (D. viv.,
P, 25 . vii . '01. Picro-nitro-osmic and Ehrlich's hsematoxylin )
Fig. 45.  -  Photo-micrograph ( x 150) of a corresponding preparation
of the wall of a more advanced 4'5 mm. blastocyst ('99 stage), in which
the two regions of the wall are now clearly distinguishable. (D. viv.,
8.7. '99. Picro-nitro-osmic, Ehrlich's hsematoxylin.)
Fig. 46.  -  Photo -micrograph ( x 150) of a corresponding preparation
of a slightly more advanced blastocyst ('04 stage). (D. viv., 6 . 7 . '04.
Picro-nitro-osmic, Ehrlich's hsematoxylin.)
Fig. 47.  -  Photo-micrograph (x 150) of an in toto preparation of the
formative region of a 6 . 7 . '04 blastocyst, showing the proliferation
of spherical interaal cells refeiTed to in the text, p. 53.
Fig. 48.  -  Photo-micrograph ( X 150) of an in toto preparation of the
wall of a vesicle of the same batch as that represented in fig. 39, in
which a small part of the junctional line between the embryonal ectodenn and the extra-embryonal {tr. ect.) is visible, the free edge of the
entoderm {ent.) not having reached it. (D. viv., 5, 18 . vii . '01. Picronitro-osmic, Ehrlich's hsematoxylin.)
Fig. 49. -  Photo-micrograpli ( X 150) of a con-esponding preparation
of a vesicle of the same batch as the preceding, in which the wavy and
irregularly thickened free edge of the entoderm {ent.) practically
coincides with the junctional line and so conceals it from view.
Fig. 50. -  Photo-micrograph (x 150) of an in to to preparation of a
vesicle (8 . vi . '01 batch) viewed from the inner surface as in the corresponding preceding figures. The entoderm in the region of the embryonal
ax-ea has been removed, so that one sees the inner surface of the embryonal
ectoderm [emh. ect.) ; it is still in situ, though not in a quite intact condition over the adjoining portion of extra-embryonal ectoderm. The
entoderm has not yet extended over the region indicated by the reference
line to tr. ect., so that here the extra-embryonal ectoderm is cleai-ly
visible. The jimctional line is apparent. (D. viv., 8 . vi . '01. Picronitro-osmic. Ehrlich's hsematoxylin.)
Fig. 51 (Plate 3).  -  Photo-microgi-aph ( X 310) of a section of a 30celled egg of Perameles obesula; egg b, '24 X '23 mm. diameter,
showing the xinilaniinar layer formed by the blastomeres.
Fig. 52 (Plate 3).  -  Photo-micrograph (x 240) of a section of a
blastocyst of P. nasuta '29 X •26 mm. diameter, showing the shellmembrane {s.vi.), zona (z.p.), and the unilaminar celhxlar waU. The
portion of the latter adjacent to the reference lines is composed of
smaller but thicker cells than the remainder.
Figs. 53 and 54.  -  Drawings ( X 84) of a 6-celled egg '34 mm. diameter,
fig. 53 showing a side view and fig. 54 a view from the lower pole.
Observe the characteristic I'ing-shaped arrangement of the blastomeres.
y. b. Yolk -body, the shell-membrane, albumen layer with sperms included, and the zona are readily distinguishable. Outlines drawn with
the aid of the camera lucida immediately after transference of the egg
to the fixing fluid. (D . viv., 22, 16 . vii . '01.)
Figs. 55 and 56.  -  Drawings ( X about 88) of a 16-ceUed egg (about ‘37
mm. diameter) as seen fx'om the side and lower pole respectively, from
the same batch as the eggs represented in figs. 24, 25, and 26. The charactei'istic aii'angement of the blastomex'es in two sxxpex'imposed, open
x'ings (each of eight cells) and the diffex'ence in size between the cells of
the two riixgs are evident. The ix'x-egxxlar body (c.g.) seen ixx the cleavage
cavity in fig. 56 is a mass of coagxxluixx. Dx'aunx from a spix'it specimen.
The albumen layer as represented in fig. 56 is too thick. (D. viv.,
3 B, 26 . vi . '01.)
Figs. 57 and 58.  -  Drawings (x about 85) of a 12-celled egg (-38 xixm.
diameter) as seen from the side axxd lower pole respectively. Four of