Some New Data concerning the Formation of the
Definitive Endoblast in the Chick Embryo
by
L. VAKAET 1
From the Department of Anatomy and Comparative Anatomy, University of Ghent
WITH ONE PLATE
T H E origin of the primary deep layer in the chick blastoderm is a difficult
problem, mainly because its formation commences during intra-uterine life.
Yet this layer is not complete when the egg is laid. The process of completion
is not yet uniformly interpreted. Some workers propose a concentric ingrowth
from the germ-wall towards the centre of the blastoderm; Disse (1878) was the
first to mention this. Others describe an anteriorly directed ingrowth exclusively
from the posterior germ-wall—among them Duval (1888), Patterson (1909), and,
more recently, Lutz (1955). Chen (1932) concludes from a study of the orientation of mitotic spindles that cell-proliferation, mainly taking place in the
posterior germ-wall, is responsible for the completion of the endophyll. Isolated
but multiple imagination of cells has been held partly responsible for the completion of the lower germ-layer. Nowack (1902) was the first to announce this
opinion clearly; these cells were described as joining the endophyll, having
descended from the ectophyll. Peter (1938), whose description of the stages of
endophyll formation has become a classic, believes cell-proliferation to be the
main factor in its completion. However, he admits addition of cells from the
ectophyll. He moreover denies categorically that there is any relative movement
of cells in the endophyll with respect to the ectophyll.
Another question to answer is whether, at the closure of the endophyll, the
endoblast may be considered as definitively established, differentiation being
the only process still due to occur. This has been generally accepted until now.
Yet we must not omit to mention that a few authors accept an origin of the
fowl endoblast through gastrulation. The most important of these is Hunt (1937
a, b), who concludes from grafts of blastoderm fragments on chorio-allantoic
membranes and from vital staining experiments that cells from the ectophyll
are the probable origin of the definitive endoblast.
This brief survey of opinions concerning the problem in which we are interested must include the vital marking experiments on the endoblast of young
1
Authors address: Laboratorium voor Menselijke en Vergelijkende Anatomie der Rijksuniversiteit,
Bijlokekaai, 1, Ghent, Belgium.
[J. Embryol. exp. Morph. Vol. 10, Part 1, pp. 38-57, March 1962]
L. V A K A E T — F O R M A T I O N OF CHICK ENDOBLAST
39
blastoderms cultivated in vitro. About the same time Bellairs (1953) and Fraser
(1954) published studies on the endoblast of the chick embryo. Bellairs, however,
is almost exclusively interested in the destination of the definitive endoblast,
whilst Fraser is only slightly interested in its formation. A complete lower layer
is already present in the youngest stages studied, as Fraser himself mentions
(p. 362). The principal reason for this apparent lack of interest is to be found in
the techniques available. The more recent technique of New (1955) is in fact up
to now the only possible way of culturing previously unincubated blastoderms
in vitro. Fraser indeed mentions only 6 cases of survival out of 62 explantations
of unincubated chick blastoderms, adding, moreover, that these 6 were 'not
strictly normal by in ovo standards'.
MATERIAL AND METHODS
Brown Leghorn eggs recently laid from commercial stock, fresh or incubated
up to the desired stage, were used. Only reliable blastoderms served as experimental material: eggs presenting considerable deviations from the expected
stage have systematically been eliminated.
Zenker's solution was used as fixing fluid for histological examination. Staining was performed, either in toto or on sections cut l\x thick, according to the
technique of Unna-Brachet or with Harris's haematoxylin counterstained with
eosin.
The cultures were made as described by New. This technique provides a
fundamental improvement in the possibilities of explantation of whole blastoderms, even when using the youngest stages found in laid eggs. It makes possible
normal reproduction in vitro of the first 60 hours of incubation. For technical
details, see New (1955). The greatest limitation of the method is the necessity
of keeping the germs with their endoblast upwards. However, in our own
investigations, this circumstance was more an advantage than a hindrance. The
results of experiments obtained with the new technique strongly diverge from
those obtained with Spratt's culturing technique (1947). We therefore also
mention the latter.
The vital marks consisted of pulverized charcoal which was applied under
saline to the endophyll or endoblast. The exact position of these marks is described together with the results. This marking technique has often been critically
examined. Our experience allows us to conclude that it is absolutely reliable
for incubations not exceeding 24 hours. However, an essential precaution in
scoring the results consists in checking that each mark is firmly attached to the
blastoderm. Reference marks outside the blastoderm have been systematically
applied.
Elimination of endophyll and endoblast was carried out with steel needles
under Tyrode solution.
40
L. VA.KAET—FORMATION OF CHICK ENDOBLAST
Classification of Blastoderms
The normal stages of Hamburger & Hamilton (1951) are generally accepted.
Yet it appeared to us that they provide too few stages of the gastrulation period.
TABLE 1
Mean
Criteria
Stage
Appearance
{Stages of Hamburger & Hamilton
between clamps)
Length of
primitive streak
(mm.)
Duration
of inc.
(hr.)
No polarity
Area opaca and area pellucida: indistinct separation. Polarity may
be visible
(1)
Differentiation of germ-wall: yolk endoderm
and margin of overgrowth are drawn
Dotted double line indicates variability in
width of area opaca which is here minimal
Broad primordium of primitive streak
(2)
O
0-3
Short primitive streak, straight, ungrooved
From this stage on the area opaca is omitted
(-3)
0-5
Longer primitive streak with shallow
groove
(3)
0-7
12
Nearly full primitive streak with welldeveloped groove
(3+)
1-2
15
Full primitive streak
(4)
1-6
18
(5~)
1-7
20
Head-process primordium
Clear-cut head process
Shortening of primitive streak
started
(5)
Head-fold
(6)
22
24
Instead of following the suggestion of the authors to use plus and minus signs,
we have designed a new series of stages for this short, but very important,
developmental period (Table 1). It practically corresponds to the series of
L. VAKAET—FORMATION OF CHICK ENDOBLAST
41
Dalton (1935, fig. 1); Chen (1932) also illustrates his description of the duck
blastoderm with the same series of stages.
In Table 1, a diagram of the blastoderm in toto is given next to the stage
number. Some criteria, other than histological ones, which are given below, are
also listed. As an indication we mention the mean primitive-streak length in our
material at the different stages, as well as the normal incubation time for each
stage. These figures are based on observations of more than 200 living blastoderms, incubated at 38° C.
RESULTS
In our results three main groups can be distinguished:
The first consists of histological observations. We do not, however, intend
to give again a full histological description of the young fowl blastoderm.
The second group contains the results of marking experiments on the deep
layer of the chick blastoderm. A distinction has to be made between blastoderms showing a primitive streak and pre-streak stages.
In the last group we mention some observations on blastoderms cultivated
in vitro after elimination of the deep layer at stages 2 and 3.
Histological examination
In this section we want only to stress some facts and peculiarities which will
permit us to make our classification precise, and enable us to give a more
accurate description of our experiments. On the other hand, it brings forth some
facts bearing especially on the lower layer of the blastoderm.
Stage 0, which is the normal stage in fresh duck-eggs, cannot be found in
most fresh chicken eggs. We did find it in some cases, however. The complete
separation of the blastoderm from the yolk is noteworthy at this stage. This
situation is a normal one, as duck and pigeon are also entirely free from the
underlying yolk during the corresponding period of their development. This
does not mean that there would exist a visible cleft between the two. We only
want to stress that there is neither macroscopically nor microscopically any
morphological continuity between blastoderm and yolk.
The microscopical preparations show that in whole blastoderms there is no
separation of an inner clearer and an outer darker zone, and no symmetry.
The youngest chick blastoderms normally found (stage 1) do, on the contrary,
exhibit some symmetry in toto, although it is not always clear. It is most easily
recognizable when the posterior part of the area opaca is larger than the anterior.
A sickle-shaped condensation may be present at the posterior border of the
area opaca and the area pellucida. This is Roller's sickle, whose existence has
often been denied. To find it regularly, or in about 90 per cent, of the chick
blastoderms, it suffices to keep the eggs for a week or more at room temperature.
On opening these unincubated eggs, nearly all show a very clear sickle, the
middle of which corresponds to the posterior side of the area pellucida.
42
L. VAKAET—FORMATION OF CHICK ENDOBLAST
According to Roller (1882), the sickle has a small furrow at this place. We found
this structure much less regularly than the sickle itself. This is in accord with the
literature, in which the existence of a furrow is discussed, and denied, more
often than the existence of the sickle. This may be due to the fact that Roller
(1882), in immediate connexion with his summary, exhaustively discusses the
eventual phylogenetic significance of the small furrow. His description shows
clearly, however, that morphologically the most important structure is the sickle.
Orientation may also be possible at this stage by the presence of numerous
folds in the posterior part of the ectophyll, one of which may be larger than
the others and run parallel to the sickle of Roller. Although Merbach (1935)
believed that this posterior folding corresponds to the 'Sichelrinne' of Roller,
we cannot agree with this view.
Sometimes the symmetry of the blastoderm is manifested by the endophyll,
which is mainly concentrated posteriorly and is only there continuous, whilst
anteriorly it is still lacunar. Fig. A of the Plate shows some of these characteristics.
We consider as very important the absence of yolk endoderm in stage 1. It
explains how easily the blastoderm can be taken from the yolk. On the other
hand, there is no question of any fixation of the blastoderm to the yolk membrane. At the most there is a weak adherence. We therefore make a distinction
between the germ-wall and the zone of junction, characterized by the presence
of yolk endoderm.
In section, the blastoderm shows a more or less biconcave shape. Below a onelayered cubical ectophyll, the deeper cells are mostly crowded towards the
margins of the blastoderm. These peripheral accumulations of cells, the germwalls, give rise to the appearance in toto of the area opaca. In the germ-walls
the cells of the endophyll are linked together rather closely, but in the central
area they are more loosely scattered. Here the endophyll is distinctly separated
from the ectophyll by a cleft, which ends at the margins of the blastoderm where
ectophyll and endophyll cells are not separated. As was already visible in toto,
the endophyll is not yet growing into the yolk at this stage. The posterior germwall is always better developed than the anterior one. Crowded against the
ectophyll, and partly caught between its cells, some cells are regularly found,
often gathered in a cluster of three to five (Plate,fig.B). When they are grouped,
these cells may be found in the depth of a small furrow, probably corresponding
to one of those we mentioned at the surface of the ectophyll. Roller's sickle
cannot be attributed to one definite structure. The thicker germ-wall as well as
a thickening of the ectophyll in this region contribute to its appearance.
The germ-wall may differentiate at stage 1 or during stage 2. According to
our experience this variability is mainly determined by the duration of the preincubation period and the environmental temperature. It has in fact been known
since the work of von Baer (1837) that development may proceed slowly when
eggs are kept some time at room temperature (22-23° C ) . The synchronization
L. VAKAET—FORMATIO.N OF CHICK END;OBLAST
43
of development, however, is not exactly the same as in normal incubation. The
appearance under these circumstances of a sickle of Koller (see above) is an
example of this. Another is the differentiation of the germ-wall, which is more
hampered by subnormal incubation temperature than the evolution within the
area pellucida. At normal incubation temperature, differentiation of the germwall begins after 5-6 hours' incubation, rather independently of the development
which occurs in the area pellucida prior to incubation. For this reason we
describe the differentiation of the germ-wall separately.
After 5-6 hours' incubation the blastoderm begins to be fixed to the yolk membrane, a very interesting situation when culture according to New's method is
intended. Simultaneously, one may observe the appearance of yolk endoderm,
constituting the zone of junction, at the under side of the area opaca. The
formation of this yolk endoderm does not always happen synchronously
throughout the germ-wall. In some cases we have been able to observe that
a zone of junction was lacking at the posterior part of the area opaca. This
description strikingly resembles the drawings of Patterson (1909), illustrating
what he calls the closure of the blastopore (Plate, fig. C).
In sections, it was found that the yolk endoderm does not extend to the
margin of the blastoderm. There exists, indeed, a free margin built up of ectoblast cells forming a layer 2-3 cells thick. These cells strongly adhere to the yolk
membrane, and it is their presence which explains the fixation of the blastoderm
to the yolk membrane. This margin may be called the 'area of overgrowth', as
this old name describes the situation and function of the structure rather
accurately. The zone of junction is situated more ventrally and more centrally
than the area of overgrowth. It forms a syncytium, which penetrates into the
underlying yolk. It is continuous with the endoblast. In about the anterior half
of the blastoderm this connexion is realized through a transitional zone. This
zone of transition is generally called the germ-wall, but we prefer not to use
this term. The structure here designated at the most corresponds to only a small
part of the primary germ-wall. We therefore prefer the circumlocution 'margin
of the endoblast'.
At stage 2 there are no more problems about the polarity of the blastoderm
/// toto. The first indication of the primitive streak allows an easy orientation.
At this stage the area pellucida is still circular, and the streak is situated at the
border of the area opaca and the area pellucida. This point corresponds to
the middle of the sickle of Koller. At the lower surface of the area pellucida
the coarsely cellular endophyll is situated in the anterior half. It forms a broad
crescent open posteriorly. The membrane constituting the posterior part of the
lower layer is strikingly smooth. We prefer to use the term 'endoblast' exclusively
for this structure, to distinguish it from the coarser endophyll.
Transverse sections differ markedly according to whether they are made in
front of or through the primitive streak. In anterior sections the endophyll
consists of tall cells and forms a somewhat folded layer. The ectophyll increases
44
L. VAKAET—FORMATION OF CHICK ENDOBLAST
in thickness towards the centre of the blastoderm. No free cells are present
between these two layers. At most a rare cell, rich in yolk, can be seen half-way
between the ectophyll and the endophyll, pinched between the cells of the ectophyll. The ectophyll in the posterior sections is thickened in the middle line.
On its underside it is not as clearly delimited as in anterior sections. Small cells
are indeed diffusely scattered between the ectophyll and the endoblast in the
posterior quadrant of the blastoderm. These cells, by their position, can only be
mesoblast cells. The production of these cells at this stage is apparently not
strictly limited to the midline. The whole posterior half of the blastoderm shows
traces of this process, which culminates, however, in the midline.
After stage 2 the area pellucida enlarges rapidly. The circumference of the
area of overgrowth increases and the zone of junction, which is situated between
this margin and the area pellucida, broadens accordingly. The surface increase
of the germ and area pellucida is indeed not proportional to the intensity of
epiboly.
Stage 3 germs exhibit a distinct primitive streak. It has grown longer: its
anterior end is nearer to the centre of the area pellucida, and its posterior end is
farther from it than at stage 2. It does not always suffice to look at the germ in
toto in order to see extension of the primitive streak in the posterior direction,
but it is in most cases necessary to eliminate the yolk endoderm forming the zone
of junction, which covers this area. In front and at the anterior sides of the
primitive streak the ectophyll appears thickened; this can be most easily seen
with transmitted light. We avoid the term 'Scheibe', introduced by Wetzel (1929)
for this area, as we avoided the term 'primitive shield' at younger stages. It
seemed preferable to indicate an appearance, as much as possible, by the structure
to which it is due. In the deeper layer the endophyll is better marked off from the
endoblast than it was at stage 2.
In sections through the blastoderm in front of the primitive streak the central
ectoblast is highly cylindrical and apparently pluristratified. Close to the anterior
end of the primitive streak some scattered cells are free between ectoblast and
endoblast. The coarse deep endophyll cells are somewhat smaller than before,
though still noticeably taller than those of the endoblast. This difference is due
to their content of yolk granules.
Frontal sections through the primitive streak show a conspicuous median cell
condensation. The ectophyll is built of very tall cylindrical cells. At first sight
it bears a close resemblance to the structure of the ectoblast in front of the streak.
It is not delimited on its under-surface, however. Numerous small cells fill the
space between ectophyll and endoblast. Below the primitive streak these cells are
arranged radially, pointing towards the middle of it. The primitive streak has
no groove yet. The free mesoblast cells show no tendency to migrate laterally.
They seem to stay at their site of origin.
The mesoblast reaches the area opaca only posteriorly. This mesoblastic layer
is bordered underneath by a thin endoblast layer which at its sides is in
L. VAKAET—FORMATION OF CHICK ENDOBLAST
45
continuity with the yolk endoderm. This margin of the endoblast is most evident
in frontal sections, half-way along the area pellucida, as a layer of taller and
more crowded cells. Anteriorly it is obscured by the endophyll, posteriorly it
can hardly be found. (For some details, see Plate, fig. D.)
From stage 4 a description of the evolution in the deeper layer will suffice
for the purposes of this paper. Often at this stage, and always from stage 5, the
endophyll wall is established. It is situated just within the anterior border of
the area pellucida and area opaca. It is a sickle-shaped, hollow structure, consisting of tall cells containing numerous yolk inclusions. Analogous cells may
be found all over the endoblast, but they are conspicuously concentrated in and
about the endophyll wall.
200 p
TEXT-FIG. 1. Scheme of the elongation of the primitive streak. The blastoderms are viewed from the
dorsal side. We consider the posterior border of the area opaca and pellucida as the site of origin of
the primitive streak at stage 1. The mid-point of the primitive streak at stages 3, 5, and 7 respectively is
drawn purposely at the same level. In this way we have integrated the descriptive data related above
with our previous conclusions from experimental work on the elongation of the primitive streak. It
was concluded that the elongation of the primitive streak takes place in an area situated on the border
between area opaca and area pellucida at stage 1, and in the mid-point of the primitive streak during
subsequent development.
On sections of the blastoderm at the primitive-streak level the ecto-mesoblast
elements are always limited underneath by a thin endothelial layer of cells—
the definitive endoblast. This layer is in continuity with the yolk endoderm.
From stage 7 the small cell endoblast underlies the whole embryo. Anteriorly
a clear-cut endophyll wall is found and from stage 9, and often earlier, free
germ-cells bud from it. It is situated close to the yolk endoderm, but separate
from it.
To end this description we mention a morphological relation which until
now has not received the attention it deserves. The primitive streak appears at
the border of the area opaca and area pellucida. As this border moves posteriorly
with the developing streak, this reference point is soon lost. But it may be easily
reconstructed by drawing the outlines of the anterior part of the area pellucida
through the streak (Text-fig. 1). When we do so the point of section appears to
divide the streak very nearly into two equally long segments, throughout the
46
L. VAKAET—FORMATION OF CHICK ENDOBLAST
period of elongation of the primitive streak. This way of representing the facts
is not pure fantasy. There are reasons indeed (Vakaet, 19606) to believe that
the elongation of the streak is due to an active stretching taking place in its
midst. We might therefore describe the elongation of the primitive streak as the
consequence of an active pushing forwards and backwards of cells. These cells
passively stream to a relatively immobile area. This area would be the border of
the area opaca and area pellucida at stage 1, and its theoretical reconstruction
in the later stages of elongation.
Experiments with vital marks on the deep layer
Marks on the germ-wall at stage 1
The experiment consists of marking with charcoal the inner germ-wall rim of
these very young unincubated blastoderms. The criteria we mentioned in the
histological description permit in most cases the orientation of the germ. Sometimes this is not the case. We have always made four marks, trying to place them
TEXT-FIG. 2. Blastoderms at stages 1, 2, and 7 viewed from their ventral side. With reference marks
outside the germ the original centre of the blastoderm has been found to stay almost immobile
during normal development. Four marks are outlined, two of which, the anterior and the posterior,
are more specially followed. At stage 7 the endophyll wall is drawn in dotted lines.
on the most anterior, posterior, and lateral parts of the inner germ-wall rim. We
repeated this experiment 16 times. In 12 cases the supposed orientation was
correct. In 4 cases orientation was impossible or was found to be incorrect. In
spite of this, the results from all 16 cases were in perfect agreement.
After a few hours incubation all marks have moved somewhat towards the
centre of the blastoderm. The caudal mark shows the most pronounced centripetal displacement.
L. VAKAET—FORMATION OF CHICK ENDOBLAST
47
After 8 hours the posterior mark has moved in such a way that it is regularly
situated between the two lateral marks, nearly on the same transverse line. No
further displacement seems to have occurred in the anterior and lateral marks.
After 20 hours a primitive streak of normal dimensions has formed. The yolk
endoblast is recognizable in spite of the absence of the normal quantity of yolk.
All carbon marks are situated in the endophyll wall, in front of the area opaca.
The initially lateral marks are situated near the posterior horns, the marks on
the axis of the germ lie near the middle of the crescent very close to each other
(Text-fig. 2).
The absolute regularity of these results and the possibility offered by the
technique of following the marks during their movements, together with the
normal development of all blastoderms, confirm that the closure of the endophyll
is operated by a centrally directed ingrowth from the germ-wall. This ingrowth,
however, is predominant in the posterior part of the germ-wall. The integration
of these movements results in a displacement of the endophyll and germ-wall
material towards a sickle-shaped structure: the endophyll wall.
Marks on the primitive streak at stages 2, 3, 4
Longitudinal marks
In these experiments a series of individually recognizable marks was placed
on the endoblast of the primitive streak. At stage 2, two to three marks can be
realized; at stage 4, up to five marks. After deposition of the marks, the germs
were practically continuously observed during 8 hours, after which period they
were further incubated in order to observe their final development. This was
normal in 17 out of the 18 experimental blastoderms of this series.
200jj
TEXT-FIG. 3. Evolution of carbon marks on the deep layer of stage 2 blastoderms through stage 5.
See text for comments.
(a) Marks at stage 2 (6 germs). The marks were placed on the endoblast, close
to, but within, the extremities of the young primitive streak. After 8 hours they
5584.10
E
48
L. VAKAET—FORMATION OF CHICK ENDOBLAST
had strongly diverged, but were nearly at the same distances from the extremities of the primitive streak. The rate of separation was 100 to 120 /u/hour
(Text-fig. 3).
(b) Marks at stage 3 (7 germs) and at stage 4 (4 germs). Two marks were placed
as at stage 2: close to, but within, the ends of the primitive streak. One or two
more marks were made between these two.
The evolution of these marks is in perfect agreement with those from the
former group. The outermost marks diverge at a rate which again is nearly
120/x/hour.The intermediate marks also separate. This separation is consistently
most pronounced in the anterior half of the primitive streak during the first 4
hours of incubation, and stronger in the posterior half during the next 4 hours.
When stage 7 is attained, the elongation in the endoblast is about the same in the
anterior and in the posterior half of the primitive streak (Text-fig. 4).
200JJ
TEXT-FIG. 4. Evolution of carbon marks on the streak endoblast of stage 3 blastoderms through
stage 7. See text for comments.
From the evolution of these longitudinal marks we may deduce that the endoblast underlying the primitive streak grows in length in accord with the primitive
streak. A reasonable interpretation seems to be that cells invaginating through
the primitive streak migrate between the endoblast cells and are thus responsible
for the rapid growth. This opinion is discussed later on.
Transverse marks (12 germs). In each experiment five marks were made transversely across the primitive streak. One mark was put on the primitive streak,
two just right and left of it, and two more laterally left and right. The level of
this transverse series of marks differed in each experiment.
(a) At stages 2-4. The evolution of these marks corresponds to that described
by Fraser (1954). Transverse marks situated in front of the middle of the primitive streak form, after 8 hours' incubation, a crescent with posterior concavity.
We want to stress the fact that very little laterally directed movement can be
observed. Marks through the most posterior part of the primitive streak
L. VAKAET—FORMATIONOFCHICK ENDOBLAST
49
describe, after 8 hours, a crescent with anterior concavity. No appreciable
lateral displacement of these marks can be noticed, at least during the stages of
elongation of the streak (Text-fig. 5).
TEXT-FIG. 5. Combined outline of the behaviour of transverse carbon marks on a stage 2 endoblast,
through stage 6.
(b) At stages 6-7. The evolution of marks at stages 6-7 does not differ from
the description given by Bellairs and Fraser. Both detected a strong regression
of the endoblast in the middle line. We may add that
it is at these stages that the divergence of marks situated besides the streak is initiated. This divergence
only becomes obvious when the shortening of the
streak begins (after stage 7). From that moment it is
greatest at posterior levels of the streak. The movement of regression in the endoblast, on the other
hand, is strongest at Hensen's node and diminishes
towards the hind end. A mark on the endoblast
underneath Hensen's node regresses with the node
at the same rate. A mark on the posterior end. of
the primitive streak at stage 7 is found much more
TEXT-FIG. 6. Outline of the
future movements in the endoblast of a stage 6 blastoderm.
The transverse arrows illustrate the movements of divergence. In the median line the
arrows represent the movements of regression which are
synchronized with the former.
In the oblique arrows the regression and the divergence
movements are vectorially
combined.
posteriorly than this hind end after a few hours. The
endoblast under the posterior extremity of the streak
thus behaves in a way radically different from that of
the overlying ectophyll (Vakaet, 1960a).
An outline of the future movements in the endoblast of a stage 6 blastoderm, as visualized with
carbon marks, is given in Text-fig. 6. The vectors
obtained in this way give a rough approximation of
the movements at work, and correspond to those
which may be observed. They are very similar to
those on the map of Bellairs (1953). We have not
50
L. VAKAET—FORMATION OF CHICK ENDOBLAST
attempted to calculate speeds for these movements, owing to the impossibility
of establishing exact figures.
Development of blastoderms after elimination of the deep layer at
stages 2-3
Results obtained with Spratfs technique (8 experiments)
After elimination of the endophyll, 8 stage 2-3 blastoderms have been
cultured with ectoblast-side down, using Spratt's technique. A curious phenomenon, consisting of a massive polyinvagination of cells, takes place within
the first hours of incubation. On the whole ventral surface of the ectoblast
numerous large cells appear. After 6 to 8 hours they cover the whole ectophyll.
The diameter of the area pellucida has already considerably shrunk by then. This
is possible because in Spratt's technique most of the area opaca is trimmed. After
this period the phenomenon seems to cease. After 18 hours the mass of loose
cells is gathered in a crescent around the anterior end of a rudimentary primitive
streak. This primitive streak can be easily distinguished from the loose cells as,
after washing these, they have disappeared whilst the primitive-streak cells
cling together better. These results confirm those of Fraser, who, using the same
technique, mentions that after removal of the deep germ-layer at analogous
stages only very defective development occurs.
Results with New's technique (10 experiments)
After the same intervention but cultivation according to New, only traces of
polyinvagination are visible. We observe the appearance of some isolated cells
at the ventral surface of the ectophyll. Moreover, development of the germ is
fairly normal up to 18 hours after the intervention. At that stage a defective
formation of the central nervous system, a hypertrophy of the chorda, and an
impression of regeneration of a very great part of the endoblast are the most
noteworthy facts.
Histologically these observations are confirmed. A defective morphogenesis
of the nervous system which seems incapable of closure, a very massive notochord, and extensive regeneration of the endoblast are conspicuous. This endoblast is sufficiently normal to be capable of forming an anterior intestinal portal
(Plate, fig. E). The regenerated endoblast, however, is limited to the anterior
part of the germ and does not extend beyond half-way along the primitive streak
at stage 9 (Plate, figs. F, G).
These results fit in with those obtained by Waddington (1932). At stages
corresponding to ours, this author found isolated and cultivated ectophyll to be
capable of yielding notochord, neural groove, and somites. He was less affirmative, however, as to its capacity to form endoblast under these circumstances.
L. VAKAET—FORMATION OF CHICK ENDOBLAST
51
DISCUSSION
Mechanism of closure of the endophyll
From our marking experiments on the endophyll at stage 1 we drew the immediate conclusion that the closure of the endophyll proceeds at least to an important extent through cell movements originating from the inner margins of
the germ-wall. These movements are outlined
in Text-fig. 7. This interpretation is in direct
contradiction to the opinion of Peter (1938),
who denies all relative movement between
endophyll and ectophyll during completion
of the lower layer. It corresponds with the
data from the experiments of Lutz (1955).
A direct visualization of movements in the
endophyll, however, only became possible
with New's technique, which was for the
first time combined with carbon marking
at this stage.
2 0 0 >J
This interpretation does not, however,
TEXT-FIG. 7. Outline of a stage 1 blasto- refute the other possible ways of closure
derm, lying endoblast-side upwards. The of the endophyll. It is most probable that
thin arrows inside the area pellucida
correspond to the movements of the cell-divisions play an important role, as suginner germ-wall ridge. Their combination gested by the careful observations of Chen
explains the situation of the definitive
endophyll wall. These movements precede (1932) on duck-eggs. We must add, however,
those of the definitive endoblast, under- that, contrary to this author, we found a
lying the elongating streak, and indicated
by the big double arrow. The latter move- random orientation of the mitotic axes in the
ments come to an end at stages 6-7. The endophyll. Pasteels (1940) warned against
thin arrows pointing eccentrically repreexplanation of morphogenetic events by
sent the overall growth of the area
pellucida throughout stages 1-9. Integra- means of proliferation centres. According to
tion of the movements outlined in this this author the number of mitoses all over
scheme can explain the morphogenesis
the germ is very high, but he was not able to
of the deep layer up to stage 7.
detect any special centre. Moreover, Bellairs
(1955) was not able to explain the morphogenetic movements in the endoblast
at later stages by the distribution of mitoses.
On the other hand, the possibility persists that cells from the ectophyll may
contribute to the completion of the lower layer before the primitive streak
appears. Histological examination did not permit a definitive proof. Numerous
investigators have observed cells between ectophyll and endophyll at pre-streak
stages (Nowack, 1902; Pasteels, 1937,1945; Peter, 1938) and have drawn the most
varied conclusions, always confirming their own opinions at the time. We do the
same (cf. Plate, fig. B). The development of blastoderms, cultivated on Spratt's
medium after elimination of the endophyll-endoblast, seems to plead in favour
of a contribution of the ectophyll to the endophyll by means of polyinvagination.
52
L. V A K A E T — F O R M A T I O N OF C H I C K E N D O B L A S T
In any case, it suggests the possibility that ectophyll cells may migrate towards
the deeper side. When cultivated according to New's technique, however, endoblast-free blastoderms no longer show this large number of invaginating cells.
Hence the question arises whether the isolation of ectophyll cells is determined
by the elimination of the lower layer or by the lack of radial tension on Spratt's
medium. Maybe both factors intervene, as no abnormal endophyll formation
occurs when whole blastoderms are cultivated on Spratt's medium, endoblastside up.
Formation of the definitive endoblast
It has been generally accepted hitherto that the definitive endoblast is formed
by a progressive transformation of the coarsely cellular endophyll into the
endothelial endoblast. This opinion is difficult to accept, as we believe we have
demonstrated that the posterior germ-wall, in a broad movement, brings about
the closure of the endophyll. This makes us wonder, indeed, where the cells
forming the definitive endoblast really do come from. It is improbable that mere
cell proliferation would be responsible for the remarkable extension of the area
situated behind and within the crescentic endophyll wall. Nobody, indeed, has
proved the existence of a proliferation centre in the endoblast. We did not find
any ourselves.
On the other hand, the primitive streak arises in the area which we might call
the enlarged posterior germ-wall. We might wonder if the formation of this very
important structure has any relation to the formation of the endoblast. In the
nineteenth century, before von Kolliker (1882), there had often been consideration of the formation of mesoblastic elements from the endophyll, but hitherto
very little attention has been given to the possibility of an ectophyll origin of the
endoblast itself. Yet we believe this to be the only possible interpretation of our
results, in spite of the conclusions of Fraser (1954), who, from very similar results,
concluded that the forming streak has nothing to do with the underlying endoblast. We, on the contrary, think it to be suggestive of a very close correlation
between primitive streak and endoblast. Indeed, the observation of two marks on
the endoblast separating at exactly the same rate as the rate of elongation of the
overlying streak appears very convincing. We hold the definitive endoblast to be
a result of gastrulation.
Another argument is furnished by the strong regenerative power of this
endoblast. What origin could be ascribed to the latter if—after excision of the
endophyll and the already-formed endoblast—the primitive streak were not
at least capable of forming endoblast cells? Why should this endoblast not be
complete if it is merely a transformed lower mesoblast layer?
In the literature there are indications of a gastrular origin of the definitive
endoblast. Hunt (19376), after application of Nile blue marks at one side of the
primitive streak in ovo, detected blue endoblast cells at the other side of the
streak. He explains this by invagination of these cells through the streak and
L. VAKAET—FORMATION OF CHICK ENDOBLAST
53
ensuing lateral migration, but towards the opposite side from that of the mark.
It is true that the use of colour marks in these experiments is not fully reliable
owing to the possibility of diffusion artifacts. Hunt also found formation of
endoblastic organs after chorio-allantoic grafts of mesectoderm (ectophyll).
Grafts from young stages yielded intestine and such endoblast derivatives as
pancreas, liver, thyroid gland, and lungs. In older ectophyll the potency towards
formation of these structures strongly diminishes in the anterior part of the
blastoderm, but in post-node fragments the capacity to form gut is conserved
much longer. He considered the endoblast derivatives not to be products of
regeneration but direct derivates from the ectophyll.
Significance of the germ-wall material
At stage 1 the germ-wall material, and certainly the inner rim of it, is descended from the deep layers of blastomeres, visible at stage 0. This is the conclusion which Kionka (1894) drew and which still seems the most probable,
though it has not yet been confirmed experimentally. According to our experiments their fate seems to be to take an important part in the formation of the
endophyll wall. In addition to vital staining we made other observations pointing to the same conclusion. Silver impregnations analogous to those Bounoure
(1934) used for the demonstration of the germ plasma in anuran embryos, as
well as enzyme histochemical reactions such as the peroxidase technique according to Wu-Hsien (see Slonimski, 1927) and the dehydrogenase technique using
neotetrazolium chloride (Spratt, 1958), enabled us to stain rather selectively the
germ-wall material at stage 1. Blastoderms mounted in toto show the migration
stages we have been able to follow with carbon marks. The same more or less
specific colorations stain preferentially the endophyll wall, at least up to the
moment of differentiation of primary gonocytes in this structure. We therefore
suggest that in the young germ-wall the 'germ-cell plasm' is to be localized. The
typical situation of the germ-cells in the endophyll wall would thus be only
a secondary one, contrary to the classical opinions of Swift (1914) and Willier
(1937).
Some observations of Matsumoto (1932), describing primary gonocytes in
the posterior germ-wall at stages not exceeding our stages 1 or 2, fit in with our
conception. The earliest stage, however, in which we could detect cytologically
differentiated gonocytes was stage 4 (Plate, fig. D), and they were not yet isolated at this moment, so that the final proof of their nature is lacking.
The mechanism by which, according to our conception, the endoblast originates, explains readily why all gonocytes are not strictly situated in the endophyll
wall. The cells of the definitive endoblast indeed do not replace as a sheet or
massively the cells of the posterior germ-wall, but through interstitial immigration. Thus it becomes easy to understand that some primary gonocytes 'stick'
within the definitive endoblast.
54
L. VAKAET—FORMATION OF CHICK ENDOBLAST
Finally, we wonder if the distribution of the germ-wall material and of the
gonocytes which we have found in the chick is comparable with that in other
germs, and especially in those with telolecithal eggs. We have no personal
experience from this point of view with germs of other classes. Yet in reptiles
the distribution of the gonocytes at the border of the intra- and extra-embryonic
areas may be explained in a way comparable to the one we propose for birds.
Moreover, in reptiles there is also a striking histological difference between
an endophyll, in which subsequently primary gonocytes appear, and a very thin
definitive endoblast whose gastrular origin seems established (Pasteels, 1953,
1957). It seems worth while to consider whether analogous relations exist
between an early germ plasma and the gastrulating endoblast in selachians. It
would not be surprising if this were the case, as there is a striking analogy
between anurans and birds when the influence of the yolk is taken into consideration.
SUMMARY
1. An histological and experimental study of the very young chick blastoderm
was made. The experiments consisted in marking with carbon the deep layer of
blastoderms subsequently cultivated in vitro with New's technique. In some
experiments the lower layer had been either rotated or excised.
2. Our histological data on the formation of the primitive streak correspond
to our previous experimental data and make it possible to consider that the
elongation of the primitive streak is due to an active stretching taking place in its
middle.
3. Moreover, our data stress the differences between the two parts of the lower
layer and the persistence of the endophyll cells after the appearance of the endoblast. It is therefore held to be improbable that the endoblast should arise from
the endophyll.
4. From marks on the inner germ-wall rim of unincubated blastoderms it is
concluded that the closure of the endophyll occurs by an ingrowth from the
germ-wall, predominantly taking place in its most posterior part. These movements bring forth the formation of the endophyll wall, apparently originating
from the inner germ-wall cells. The origin of the primary gonocytes from these
cells is held to be probable.
5. The displacements of marks placed along the length of the young primitivestreak endoblast are explained by an origin of the endoblast layer through
gastrulation.
6. Extensive regenerative capacities of young streak endoblast, but not of
endophyll, were found to be present. These data support the theory of the origin
of the endoblast from the ectophyll from which it passes during gastrulation.
L. VAKAET—FORMATION OF CHICK ENDOBLAST
55
RESUME
Quelques donnees nouvelles sur la formation de Vendoblaste definitif
du blastoderme de Poulet
1. Nous avons entrepris une etude histologique et experimentale du tres jeune
blastoderme du Poulet. Nous avons applique des marques de carbone sur le
feuillet inferieur. Les blastodermes ont ete cultives ulterieurement in vitro par
la technique de New. Dans certaines experiences, le feuillet inferieur avait subi
une rotation; dans d'autres il a ete enleve en entier.
2. Nos resultats histologiques concordent parfaitement avec certains de nos
resultats experimentaux publies precedemment (Vakaet, 19606). Nous arrivons
a une interpretation de l'elongation de la ligne primitive qui attribue ce phenomene a une extension active au niveau du milieu de la ligne.
3. Nous avons d'autre part mis l'accent sur les differences qui existent entre
les deux parties du feuillet inferieur, et notamment sur la persistance de cellules
entophylliques apres l'apparition de l'endoblaste. Nous croyons qu'il est peu
probable que l'endoblaste prenne son origine dans l'endophylle.
4. L'evolution de marques posees sur le bord central du rempart germinatif
nous a permis de deceler une croissance centripete dans ce rempart. Cette
croissance est predominante dans la partie posterieure. Ces mouvements, tout
en operant la fermeture de l'endophylle, menent a la formation du croissant
entophyllique anterieur. Ce croissant prendrait done son origine dans le bord
central du rempart germinatif, qui serait en outre le lieu d'origine des gonocytes
primaires.
5. Les deplacements de marques posees sur l'endoblaste de la ligne primitive en
extension sont expliques par une origine gastruleenne du feuillet endoblastique.
6. Une large capacite de regeneration de l'endoblaste — qui manque entierement dans l'endophylle—a pu etre demontree. Elle nous fournit un autre argument en faveur de l'origine de l'endoblaste definitif dans l'ectophylle dont il
descendrait par gastrulation.
ACKNOWLEDGEMENTS
I should like to thank Professor J. Fautrez for his continual interest and helpful criticism. I am grateful to Professor M. Abercrombie who suggested the
use of New's technique. I am also indebted to Mrs. L. van Strijthem for most
valuable technical assistance, to Mr. H. van Ooteghem for the photography,
and to Mr. G. Lenney for reading the manuscript.
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L. V A K A E T — F O R M A T I O N OF C H I C K ENDOBLAST
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Vol. 10, Part 1
L. VAKAET
L. V A K A E T — F O R M A T I O N OF C H I C K ENDOBLAST
57
E X P L A N A T I O N OF PLATE
FIG. A. Stage 1 blastoderm, fixed in Zenker, stained in Unna mixture. The egg had been kept for
a week at room temperature, hence the beginning of differentiation of the germ-wall. Note the numerous grooves, the most important of which is situated at the posterior border of the area pellucida.
The most posterior part (p) of the area pellucida is darker owing to the presence of more endophyll
in this region than in the anterior part (an), where it is more loosely scattered.
FIG. B. Stage 1 blastoderm, fixed in Zenker, Azan stained. A section through a fold in the ectophyll
is shown. This fold does not look artificial: the cells visible at its bottom are wedge-shaped and
apparently normal. The presence of cell detritus in the groove (a frequently observed phenomenon
favours the interpretation of a downward migration of ectophyll cells, the most superficial part of
which might be strangulated by the neighbouring cells. The presence of this material would be
difficult to interpret by the upward migration of cells from below.
FIG. C. View from below of a stage 1 blastoderm in which differentiation of the germ-wall has
started in the anterior two-thirds (an). The posterior one-third (p) of the germ-wall is larger than its
other part and does not show any yolk endoderm yet. Subsequent cultivation verified the orientation
we accept here.
FIG. D. Longitudinal section through a stage 4 blastoderm lateral to the primitive walls. The
median thickening of the ectophyll is conspicuous. The mesoblast is in continuity with its posterior
part (p) and extends anteriorly. The two types of cells we distinguish in the deep layer are very well
marked. From the posterior zone of junction to the anterior limit of the mesoblast a very thin endoblast is present. Note the absence of a 'germ-wall' posteriorly. The anterior part of the deep layer is
constituted by larger endophyll cells.
Figs. E, F, G. Frontal sections through a blastoderm from which all of the deep layer was removed
at stage 2. Fig. E shows a defective head development, a very big chorda, and a quite normal foregut.
No primary gonocytes are to be found. Fig. F estimates the extent of the endoblast layer which
obviously is not regenerated from the yolk endoderm. Fig. G is a more posterior section of an analogous blastoderm showing the presence of mesoblast without underlying endoblast.
{Manuscript received 6: iv: 61)