/ . Embryol. exp. Morph. Vol. 24, 1, pp. 95-108, 1970
Printed in Great Britain
95
Expansion of the area vasculosa of the chick after
removal of the ectoderm
By J. M. AUGUSTINE 1
From the Department of Anatomy, Hahnemann Medical College
SUMMARY
The role of the ectoderm in the expansion of the mesoderm in the area vasculosa of the
chicken embryo was studied.
The basement membrane of the ectoderm was found to constitute a substratum for the
expansion of both layers of mesoderm, since (a) the somatic mesoderm, particularly at its
margin, adheres to the basement membrane, and (b) the somatic and splanchnic mesoderm
adhere to each other throughout most of the area opaca.
Following removal of the ectoderm from the outer surface of the basement membrane,
movement of the underlying mesoderm along its inner surface stopped. Mean expansion of
the mesoderm in these cases was zero.
Following removal of both ectoderm and basement membrane, expansion of the underlying mesoderm was normal in amount.
Experimental changes in the ectodermal substratum can thus stop movement of the associated mesoderm, but the role which the substratum normally plays in mesodermal expansion
remains unclear.
INTRODUCTION
During tht .rst two weeks of development in the chicken embryo the mesoderm of the area /asculosa spreads outward from the embryo between the ectoderm and endoderm to surround the yolk. It consists of two layers, somatic and
splanchnic, lining the ectoderm and endoderm respectively. The somatic layer is
mesothelial, while the splanchnic layer is vascular and hemopoietic.
The work reported here is concerned with the role of the ectoderm in this
mesodermal expansion. Grodzinski (1934) has shown that in the early days of
mesodermal expansion the ectoderm is also expanding, but much more slowly
than the mesoderm. The mesoderm thus moves outward relative to the surface
of the ectoderm, which may therefore serve as a substratum for its movement,
provided the two are in contact. The fine structure of this region was reported by
Bellairs (1963), whose figure R shows points where the somatic mesoderm
approaches material apparently attached to the basement membrane of the
ectoderm, but the question of contact between the two is not dealt with.
If the ectoderm serves as a substratum for the expansion of the somatic
1
Author's address: The Hahnemann Medical College and Hospital of Philadelphia,
235 North Fifteenth Street, Philadelphia, P.A. 19102, U.S.A.
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J. M. AUGUSTINE
mesoderm it may also affect the splanchnic mesoderm, for the two are connected
to each other. They are fused at their margin. Proximal to the margin the extraembryonic coelom intervenes between them as a potential space, but here also
occasional junctions occur between them. They remain coextensive throughout
their expansion and therefore expand at equal rates. For these reasons it seems
likely that the two mesodermal layers are not independent of each other in their
expansion, but that the movement of one layer may affect that of the other.
Movement of the somatic layer, for example, may by means of its connexions to
the splanchnic layer transmit to it a tensile force contributory or essential to its
expansion, or movement of the common mesodermal margin may exert such
force on both layers. In either case the ectoderm, as substratum for this movement, may be important to the expansion of the splanchnic as well as the somatic
mesoderm.
These possibilities were investigated by observing the development of the
area vasculosa following removal of the ectoderm.
MATERIALS AND METHODS
White Leghorn embryos were used throughout. Eggs incubated unturned for
3 days at 38 °C were opened by cutting a window in the shell. Approximately
half of the albumen was then removed by pipette. Following operations the
window was sealed with cellophane tape and the egg replaced in the incubator.
To expose the ectoderm a hole was cut in the vitelline membrane. Ectoderm
or ectoderm and basement membrane were then removed from the right half of
the area vasculosa, the left half remaining intact as a control. To remove the
ectoderm from its basement membrane it was grasped in the area opaca by
watchmaker's forceps (Dumont no. 5), the tips of which were bent toward each
other to meet in a point. The ectoderm was then lifted by the forceps from its
basement membrane and pierced by a flat probe, and the probe was passed
Fig. 1 (A) Section of dissected portion of area vasculosa. Proximo-distal sequence
is from left to right. Arrow on left shows point where attachment of somatic mesoderm to splanchic mesoderm was broken (also shown in Fig. 1 C). Below it and to
the right loops of somatic mesoderm (som) are still attached to splanchnic mesoderm.
Arrow on right shows point where basement membrane becomes separated from
ectoderm (also shown in Fig. IB). Pieces of Millipore filter were inserted between
components immediately after their separation to identify the level reached by the
procedure of separation used, bm, basement membrane; ect, ectoderm; end, endoderm; fil, Millipore filter; som, somatic mesoderm. x 100. (B) Ectoderm with its
basement membrane in left half of figure, and separated from its basement membrane in right half, x 440. (C) The point at which the attachment between somatic and splanchnic mesoderm was broken is in the middle. To the left the somatic
mesoderm remains adherent to the ectoderm, but to the right the ectoderm and
basement membrane have been lifted clear of the somatic mesoderm, which remains
attached to the splanchic mesoderm. x 440.
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Expansion of the area vasculosa
fit
EM B 2 4
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J. M. AUGUSTINE
between it and the basement membrane to separate the two. Pieces of ectoderm
thus separated were cut free with scissors and fixed in Bouin's fluid for later
identification.
Removal of the ectoderm with its basement membrane was begun by inserting the probe into the extra-embryonic coelom near the body of the embryo
and passing it distad. Near the border between the areas pellucida and opaca the
two mesodermal layers adhered strongly to each other and could not be separated. At this point the probe pierced the somatic mesoderm and passed distad
between it and the basement membrane of the ectoderm, separating them. The
separated tissue thus contained somatic mesoderm near its proximal edge, but
only ectoderm and basement membrane elsewhere. As before, it was cut free and
fixed.
Three groups of embryos were used: group A, consisting of control embryos,
untreated except for a hole made in the vitelline membrane; group B, of embryos
in which the ectoderm was removed from its basement membrane in the right
half of the area vasculosa; and group C, of embryos in which the ectoderm and
its basement membrane were removed in the right half of the area vasculosa.
Embryos from these three groups were photographed just prior to the operations.
Following operations they were incubated simultaneously for 16 h, photographed
again and fixed. The embryos were between stages 17 and 19 (Hamburger &
Hamilton, 1951) at the beginning of the period. Tissues were sectioned at 10/A
and stained with PAS and hematoxylin.
RESULTS
Identification of removed tissue
To aid in identifying the tissue removed by the procedures just described,
dissections of the area vasculosa were prepared. This was done by using the
two procedures of separation in sequence on the same embryo, such that
ectoderm, basement membrane, and somatic mesoderm were separated from
each other in the distal part of the dissection, but unseparated proximally
(Fig. 1). Eight of these dissections were made. They showed that the ectoderm
has a characteristic appearance after removal from its basement membrane.
Approximately half of its cells are pulled out of alignment with the others and
form a second layer beneath them (Fig. 1 B). These cells may be confused with
somatic mesoderm. However, in the dissections the separated basement membrane and somatic mesoderm may be seen below them (Fig. 1 A, B), ruling out
this possibility. The appearance of tissues in these dissections was used to
identify the tissues removed from the area vasculosa in the experiments reported
below; that is, to establish whether ectoderm, basement membrane, and somatic
mesoderm were present in them or not (Fig. 1B, C).
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Expansion of the area vasculosa
Adhesion between basement membrane and somatic mesoderm
The following observations, made during the removal of the ectoderm and its
basement membrane, indicate that the latter is in contact with the somatic
mesoderm, and is a substratum for its expansion.
Fig. 2. Margin of the area vasculosa of an untreated embryo. Proximo-distal
sequence is from right to left. Note thickening of ectodermal basement membrane
above and just distal to sinus terminalis (st), and the wedge-shaped group of cells (me)
below it. coel, extra-embryonic coelom; me, marginal cells; end = endoderm. x 440.
Prior to stage 17 a strong adhesive force exists between the basement membrane
of the ectoderm and the somatic mesoderm, preventing their separation except
in restricted locations. At a variable time, but usually between stages 17 and 19,
this force decreases, and the ectoderm with its basement membrane may be
lifted and removed from the somatic mesoderm throughout most of the area
opaca. However, as this is done a temporary dilatation and hemostasis occur in
the underlying blood vessels. This may be due to mechanical disturbance
transmitted to the vessels by the connexions between them and the somatic
mesoderm; Lange (1930) produced a similar reaction in these vessels by disturbing them mechanically with a glass rod. In contrast to this effect, removal of
the ectoderm from its basement membrane has no discernible effect on the
underlying blood vessels.
At the margin of the mesoderm the adhesive force remains strong at stage 19.
Special features are observed in sections of the marginal region (Fig. 2). A
wedge-shaped group of cells is present, attached proximally to the sinus terminalis and extending distally about 200/t between the ectodermal basement
membrane and the endoderm. These cells comprise the bourrelet mesodermique
of Duval (1884), which takes part in the closing of the yolk-sac umbilicus at a
later stage. The basement membrane over these cells and over the sinus terminalis is thickened. Duval reported a thickening in the cellular portion of the
ectoderm in this location, but this was not constantly observed in the present
7-2
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J. M. AUGUSTINE
work. However, when the ectoderm is removed from the basement membrane a
white streak, which may represent this thickening, is faintly visible in it at this
location. These features apparently represent a temporary response of the ectoderm and its basement membrane to the passage of the marginal portion of the
mesoderm, since they are always present above the margin as it moves outward.
The force required to separate the basement membrane from the somatic
mesoderm in the area opaca and the vascular reaction to this separation indicate
that the somatic mesoderm, especially at its margin, adheres to the basement
membrane of the ectoderm and moves outward in contact with it. The basement
membrane may therefore be regarded as a substratum for its movement.
Adhesion between somatic and splanchnic mesoderm
Although the somatic mesoderm is said to be separated from the splanchnic
mesoderm by the coelomic space, it was found to be impossible to separate them
with the probe except near the body of the embryo.
Fig. 3. Portion of area vasculosa fixed 16 h after removal of ectoderm from its basement membrane. Note connexions between somatic and splanchnic mesoderm
(arrows). x440.
The structural basis for the adhesion of the two mesodermal layers may be
the connexions between them proximal to the margin. These are not easy to see
in sections of normal material but are prominent in some of the experimental
material (Fig. 3). They probably represent a persistence in scattered loci of the
unseparated condition of the mesoderm which exists prior to coelom formation.
Figures of such connexions at earlier stages have been shown by Sabin (1920,
figs. 21, 28, 29) and by Hamilton (1952, fig. 74C).
Expansion following removal of ectodermal components
While the adhesive forces described in the preceding sections indicate that the
basement membrane of the ectoderm is, in effect, a substratum for both layers of
Expansion of the area vasculosa
101
mesoderm, the question remains whether its role as such is important in the
mechanism of their expansion or not. Experiments related to this question are
reported below, and are summarized in Fig. 4.
Ectoderm
Amniotic cavity
Somatic mesoderm
Coelom
Sinus terminalis
Basement membrane
16 hours
Group B
16 hours
Grouo C
Fig. 4. Results of ectoderm removals. Group B,0h: ectoderm has been removed
from its basement membrane in the right half of the area vasculosa. 16 h: the left half
has expanded a norma I amount, but mean expansion of the right half is approximately
zero. The splanchnopleure is wrinkled in 8 of the 13 cases. The basement membrane
is visible proximally anddistally, but indistinct elsewhere. Group C,0 h: ectoderm and
basement membrane have been removed in the right half of the area vasculosa.
16 h: mean expansion in both halves is normal. Somatic mesoderm has disappeared
from the right half.
Group A: untreated controls
To determine the amount of expansion normally occurring in each half of the
area vasculosa during the 16 h experimental period the distance was measured
between the aorta, at the origin of the vitelline arteries, and the sinus terminalis
in the photographs taken before and after the 16 h period. The points on the
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J. M. AUGUSTINE
sinus terminalis used for this measurement were at its intersection with the line
bisecting the angle formed by the anterior and posterior vitelline veins. The
expansion during this period was expressed as a percentage of the distance
measured before the period
^ x l O O ,
where D2 is the distance measured after the period and D1 the distance measured
before it. The results for the group are given as the mean and standard error of
the mean. Thirteen embryos were used. In eight embryos the right half expanded
more than the left and in five the left expanded more than the right, but the
means for the two halves were virtually equal: 46-9 ± 4-0% for the left half and
46-8 ± 3-9% for the right.
Group B: removal of ectoderm from its basement membrane
In thirteen embryos the ectoderm was removed from its basement membrane
in the right half of the area vasculosa. The boundaries of the area from which it
was removed were the anterior and posterior vitelline veins and a line approximately 1 mm distal to the sinus terminalis (Fig. 4).
In all 13 embryos the left half expanded more than the right. In five embryos
the right half shrank during the 16 h period (Fig. 5 A, B), in two it remained unchanged, and in six it expanded. The mean change for the right half was a
shrinkage of 0-2 ± 5-2 %, but for the left an expansion of 52-4 ±3-9 %. Thus for
the group as a whole removal of the ectoderm from its basement membrane
stopped expansion in the right half of the area vasculosa. In contrast the left half,
in which the ectoderm remained intact, expanded a normal amount compared
to untreated controls.
In all cases in the right half, regardless of its change in size, the sinus terminalis
was located immediately beneath the cut edge of the ectoderm at the end of the
period (Figs. 4, 6). Thus movement of the mesoderm relative to the ectodermal
substratum was practically nil following the removal. It is possible that removal
of the ectoderm from the outer surface of the basement membrane in these
embryos so altered the membrane that its inner surface no longer provided a
substratum suitable for mesodermal movement.
Fig. 5. (A) Embryo of group B at stage 18, immediately prior to removal of the ectoderm from its basement membrane in the right half of the area vasculosa. (B) Same
embryo 16 h after the removal. The right half of the area vasculosa has shrunk.
Arrows indicate anterior and posterior vitelline veins in both pictures.
Expansion of the area vasculosa
103
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J. M. AUGUSTINE
Group C: removal of ectoderm with its basement membrane
In thirteen embryos the ectoderm and its basement membrane were removed
from the right half of the area vasculosa. The somatic mesoderm was also removed in a region approximately corresponding to the area pellucida in this
half. The boundaries of the area of removal were the same as those in group B,
except that the basement membrane was cut just proximal, instead of distal, to
the sinus terminalis (Fig. 4). It was necessary to leave the basement membrane
attached to the mesodermal edge in this way, since if it is removed only endoderm
remains, which breaks down in about 2 h. The area vasculosa then becomes
flooded with yolk.
Fig. 6. Marginal portion of area vasculosa of embryo in group B, fixed 16 h after
removal of ectoderm from its basement membrane. Note wrinkle in splanchnopleure
and enlargement of coelom. st, sinus terminalis. x 200.
In all thirteen embryos the right half of the area vasculosa expanded during the
16 h period after the removal (Fig. 7). The mean expansion for the right half
was 51 -7 ± 6-4%. In the left half, where the ectoderm and its basement membrane
remained intact, expansion occurred in all cases also; the mean expansion was
44-2 ±3-3%. The difference between these two means is not statistically significant. Thus removal of the basement membrane with the ectoderm does not
significantly affect the expansion of the underlying mesoderm.
As in the preceding experiment the sinus terminalis was found immediately
beneath the cut edge of the ectoderm in all cases at the end of the 16 h period
(Figs. 4, 8 A), indicating the same absence of movement of the mesoderm
relative to the ectoderm. In sections of the right half fixed at the end of the 16 h
period the splanchnic mesoderm was readily visible, but few traces of somatic
Expansion of the area vasculosa
105
mesoderm were observed. However, small nodules of tissue were present on the
surface of the splanchnic mesoderm (Fig. 8B). Their origin is not known. They
may represent remnants of somatic mesoderm.
Fig. 7. Embryo of group C 16 h after removal of ectoderm and its basement
membrane from right half of area vasculosa. Expansion of the right half is comparable to that of the left.
DISCUSSION
The preceding work was done to investigate the role of the ectoderm in the
expansion of the mesoderm of the area vasculosa. It was thought that the ectoderm might serve as a substratum for the movement of the somatic mesoderm,
enabling it to exert tension on the splanchnic mesoderm to which it is attached.
Tension exerted on blood vessels by tissues attached to them has been cited as a
factor causing their growth during embryonic development (Thoma, 1893), and
it was thought that the vessels of the splanchnic mesoderm might be caused to
grow partly by such tension. A second possibility was that expansion of both
somatic and splanchnic mesoderm might be dependent on tension produced by
the outward movement of their common margin on an ectodermal substratum.
New (1959) and Bellairs & New (1962) have shown that the expansion of the
chick blastoderm is dependent on tension produced by the outward movement
of specialized marginal cells, utilizing the vitelline membrane as a substratum.
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J. M. AUGUSTINE
Fig. 8. (A) Marginal portion of area vasculosa of embryo in group C fixed 16 h after
removal of part of ectoderm {ect) and basement membrane (6m). Note absence
of somatic mesoderm. x 200. (B) More proximal portion of section shown in A.
Three nodules of cells are attached to the surface of the splanchnic mesoderm.
x440.
In support of these possibilities it was found that the basement membrane of
the ectoderm is a substratum for the somatic mesoderm in the area vasculosa,
that the somatic mesoderm adheres to the splanchnic mesoderm, and that special
features are present at the margin of the mesoderm. It remains to consider
whether the outward movement of the somatic mesoderm, or of the mesodermal
margin, upon this substratum produces tension essential to vascular expansion
or not.
In group B outward movement of the mesoderm upon the basement membrane was practically stopped, as shown by the superposition of the cut edge of
the ectoderm over the sinus terminalis 16 h after the operation. The mean
expansion of the mesoderm in this group was also virtually zero. This suggests
that mesodermal movement relative to the ectoderm is essential for mesodermal expansion. It is possible, however, that the mesoderm failed to expand
because it was held back by the basement membrane proximal to the edge, to
which it became, in effect, fused (Fig. 4).
In group C, in which the basement membrane proximal to the edge was
removed, a normal amount of mesodermal expansion occurred. This expansion
could not have been produced by mesodermal movement relative to the ectoderm
since the edge of the mesoderm again became fused to the cut edge of the ecto-
Expansion of the area vasculosa
107
derm and the two moved outward at the same rate. It is possible, however, that
the mesoderm was pulled by the ectoderm as it moved outward. Since the rate
of this movement was more than twice that normal for ectoderma\ expansion
(Grodzinski, 1934), it seems more likely that the ectoderm was pushed outward
by the mesoderm. Nevertheless, the former possibility cannot be ruled out.
With respect to the role of tension in mesodermal expansion, the experiments are
therefore inconclusive and the function of the ectodermal substratum remains
unclear.
RESUME
Expansion de Vaire vascuiaire du poulet apres ablation de Vectoderme
On a etudie lerole jouepar l'ectoderme dans l'etalement du mesoderme dans l'aire vascuiaire
de I'embryon de Poulet.
On a constate que la membrane basale de l'ectoderme constitue un substratum pour
Petalement des deux couches de mesoderme, puisque (a) le mesoderme somatique, particulierement dans sa partie marginale, adhere a la membrane basale, et (b) le mesoderme somatique
et le mesoderme splanchnique adherent I'un a l'autre dans la majeure partie de l'aire
opaque.
Apres ablation de l'ectoderme situe a la partie externe de la membrane basale, le mouvement du mesoderme sousjacent le long de sa surface interne s'est arrete. L'expansion moyenne
du mesoderme etait, dans ce cas, nulle.
Apres ablation de l'ectoderme et de la membrane basale, l'expansion du mesoderme sousjacent etait normale.
Des modifications experimentales du substratum ectodermique peuvent done arreter le
mouvement du mesoderme associe, mais le role joue normalement par le substratum dans
l'expansion du mesoderme reste obscur.
Some of the methods used in this work were developed during a post-doctoral fellowship
1963-5, in the Department of Anatomy, Albany Medical College of Union University, in
which the author was supported by a training grant, GM-155, from the Division of General
Medical Sciences, U.S. Public Health Service. Subsequently, portions of the work were supported by U.S. Public Health Service general research grant 5-S01 FR05413 to Hahnemann
Medical College.
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(Manuscript received 3 October 1969, revised 20 December 1969)