/ . Embryol. exp. Morph. Vol. 41, pp. 175-188, 1977
Printed in Great Britain © Company of Biologists Limited 1977
175
Mesodermal expansion after arrest of the edge
in the area vasculosa of the chick
By J. M. AUGUSTINE 1
From the Department of Anatomy, Hahnemann Medical College
SUMMARY
To investigate whether mesodermal expansion in the area vasculosa is caused by tension
produced by outward migration of cells either in the somatic mesoderm or at the mesodermal edge on an ectodermal substratum, stage 18-20 embryos were transferred to a culture
dish. There mesodermal expansion proximal to an arrested edge could be compared with that
proximal to a moving edge by measuring the amount of vascular elongation occurring in
each. A proximo-distal gradient in vascular elongation rate was detected both in normal
embryos in ovo and in explants. This gradient was reversed following arrest of the edge, and
the rate of vascular elongation proximal to the arrested edge decreased to 60-70 % of that
proximal to a moving edge. Nearly all of the mesoderm producing this expansion was located
in the proximal two-thirds of the area vasculosa, where vascular elongation rate on the
stopped side of the explant was not significantly different from that on the moving side.
Similar results were obtained in the absence of the ectoderm, and when liquid culture medium
was used instead of semisolid medium. It is concluded that tensile force derived from mesodermal migration plays no role in expansion of the proximal two-thirds of the area vasculosa
mesoderm.
INTRODUCTION
In an earlier paper on the expansion of the mesoderm in the area vasculosa
of the chick (Augustine, 1970), the question was raised whether the ectoderm
plays a role by serving as a substratum on which the mesoderm migrates outward. It was thought that cells at the edge of the mesoderm might exert tension
on more proximal portions by such migration, providing force required for
their expansion, since special structural features and adhesive properties were
noted at the edge, and a wedge-shaped group of cells which might perform the
migratory function was found there. Similar observations have been reported
in work on the Fundulus embryo (Trinkaus, 1966) and on the chick blastoderm
(New, 1959), in both of which a mechanism of this kind has been postulated.
The aim of the present work has been to test this possibility by stopping
the outward movement of the mesodermal edge and observing the effect of its
arrest on the expansion of mesoderm proximal to the edge. This is facilitated
by the natural marking system which the pattern of blood vessels in the
1
Author's address: Department of Anatomy, Hahnemann Medical College, Two-thirty
North Broad Street, Philadelphia, Pennsylvania 19102, U.S.A.
176
J. M. AUGUSTINE
Ectoderm
Anterior vitellene vein
Area vascula
Basement
membrane
Sinus terminalis
Yolk sphere
Edge cells
Somatic
mesoderm
Area vitellina
Sinus terminalis
Posterior vitelline vein
Splanchnic
mesoderm
Entoderm
B
Fig. 1. (A) Stage-18-20 embryo on yolk sphere. (B) Schematic diagram of
structures at the edge of the area vasculosa.
mesoderm provides (Fig. 1). The edge of the mesoderm is marked by a roughly
oval or circular vein, the sinus terminalis. From each side of the embryo body a
vitelline artery, overlain by a vein, extends outward, bifurcating repeatedly and
filling the area vasculosa with a system of branching vessels. The branches can
be used as marks dividing the vessels into a series of segments extending outward from the body of the embryo to the sinus terminalis. Grodzinski (1934)
has pointed out that the area vasculosa does not expand by the production of
sprouts from the distal ends of these vessels, but by growth of the vessels along
their entire length. As the area vasculosa expands, all segments of the vessels,
from center to edge, grow longer. By stopping the outward movement of the
edge, the effect on mesodermal expansion proximal to the edge can be observed
by measuring the elongation of these segments.
The arrest of the edge has been accomplished by transferring the embryo
to a small culture dish. The area vasculosa continues to expand and the edge
of the mesoderm moves outward until it reaches the rim of the dish, where it
remains stationary during the remainder of the culture period, so that the
effect of its arrest on the elongation of vascular segments proximal to it can be
observed.
Grodzinski also noted that, as the area vasculosa expands, distal branches
move outward faster than proximal branches. He concluded that there must be
a difference in growth rate between proximal and distal regions, perhaps reflecting a difference in metabolic rate, but he made no measurements of rate of
elongation of proximal and distal vascular segments. This point is also investigated in the present paper.
MATERIALS AND METHODS
Embryos were explanted to an arrangement of three sterile dishes as shown
in Fig. 2. Dish 1 was filled with the culture medium made of thin albumen
Mesodermal expansion in area vasculosa of the chick
111
3
Fig. 2. Arrangement of three culture dishes and explanted embryo. Each number
designates dish, rim of which is below it. Dish 1 is the lid of a 35 mm plastic tissue
culture dish (Falcon), outside diameter 41 mm, depth 5 mm, thickness 0-7 mm;
dish 2 is a 55 mm plastic dish (Falcon) cut to inside depth of 6-5 mm; dish 3 is a
20 x 90 mm glass petri dish. Dishes 1 and 2 are fused at three points by hot metal
probe. Arrow points to body of embryo in central well of liquid medium. Thick
layer each side of body is area vasculosa; thin layer is area vitellina. Diagonal lines
= agar medium.
mixed with an equal volume of 2 % agar (Difco Bacto-Agar) in glucose Howard
Ringer solution, according to the method of DeHaan (1967) for semisolid
media. The resultant medium contains 50 % albumen, 1 % agar and 0-5 %
glucose. A cylinder of this semisolid medium approximately 12 mm in diameter
was removed from the center of the dish to provide a space for the body of the
embryo. This space was filled with liquid medium identical in composition to
the semisolid medium, with the omission of agar. Dish 2 was also filled temporarily with this liquid medium to a depth sufficient to cover the surface of the
agar medium in dish 1. During explantation this facilitated the positioning of the
extra-embryonic membranes.
Explantation was begun by depositing a yolk sphere bearing a White Leghorn
embryo at stage 18-20 of Hamburger and Hamilton (1951) into a bowl of
Howard Ringer solution warmed to 38 °C. The vitelline membrane was removed.
In two groups of embryos the ectoderm and its basement membrane were then
removed from one half of the area vasculosa by the method reported previously
(Augustine, 1970). The embryo was then removed from the yolk by cutting with
scissors through the area vitellina about one centimeter distal to the edge of the
area vasculosa. It was transferred in a spoon to a dish of fresh Ringer solution
to rinse off some of the yolk clinging to the entoderm, then transferred by spoon
to the slightly submerged surface of the agar medium with the entodermal
surface in contact with the medium. A small amount of Ringer solution was
unavoidably added to the liquid medium by this action.
After arranging the extra-embryonic portions in their approximately correct
position enough fluid was removed from dish 2 to bring its surface to a level
at or slightly below the surface of the agar in dish 1. By pulling the edge of the
area vitellina at appropriate points with forceps the size and shape of the area
vasculosa could then be adjusted to approximate that which it had in ovo. The
remaining fluid was then pipetted from dish 2. In the resultant preparation the
body of the embryo and part of the area pellucida floated on the surface of the
178
J. M. AUGUSTINE
liquid medium, in the center of dish 1 while the more peripheral portions rested
on the agar medium. At the edge of dish 1 the area vitellina turned sharply
downward and clung to the outer surface of the dish, serving to anchor the
explant in place. In one group of explants no agar was used, dish 1 being entirely filled with the liquid medium.
It was important during explantation to keep the embryo and all its membranes wet. Drying seemed to occur with surprising ease and was thought to
be the cause of foci of severe shrinkage which arose in the area vasculosa of
some explants. Such explants were subsequently discarded.
One group of explants was accompanied by controls incubated simultaneously in ovo. These were prepared by cutting a window in the shell, removing about
one third of the albumin, then enlarging the window by removing one third of
the shell. The eggs were sealed with cellophane tape which was removed only
for taking photographs and immediately replaced. All incubation was at 38-5 °C.
RESULTS
Three groups (1-3) of embryos were explanted to the agar medium and one
group (4) to the liquid medium. Results are given as the mean and standard
error of the mean.
Group 1. Explants and in ovo controls
In this group there were three aims: (1) to determine survival time of explants;
(2) to compare rate of expansion of the area vasculosa in explants with that of
controls incubated simultaneously in ovo; (3) to determine whether a difference
in growth rate exists between proximal and distal regions of the area vasculosa.
Twenty-one embryos were explanted. They were photographed immediately
before and after explantation and most of them at subsequent intervals of
approximately 0-5 h, 1 h, 6 h, 12 h and 24 h. After 24 h they were photographed
every 6 h until death. Nineteen of the explants were accompanied by simultaneously incubated in ovo controls.
Survival time. One explant died between the 12 h and 24 h observations. The
mean number of hours after explantation when the others were last seen alive
(heart rate normal - around 130/min) was 34-9, the minimum was 24, and the
maximum was 50-5. Explanted at stage 18-20, the embryos had reached stage
25-28 when found dead (heart rate zero). The stage of controls was not determined at this time since it would have been more advanced than at the time
(unknown) of the explanf s death. However, the rate of body development in
the explants appeared approximately normal, and no malformations were
observed.
Rate of expansion. Measurements of the diameter of the area vasculosa were
made in the photographs taken at 0-5 h, 12 h and 24 h after explantation. Points
on the sinus terminalis used for this were at its intersection with a line bisecting
Mesodermal expansion in area vasculosa of the chick
179
Fig. 3. Embryo of Group 1 before and at intervals after explantation. (A) Immediately before explantation. Stage 18. The scale at top is in mm. (B) Immediately
after explantation. (C) 35 min after explantation. (D) 6 h after explantation. (E) 12 h
after explantation. (F) 24 h after explantation. Arrow in (C) and (F) indicates the
most proximal of the four branches shown in Fig. 4.
the angle formed by the anterior and posterior vitelline veins. The mean increase in diameter per hour for the first 12 h, in %/h, was 1-06 ±0-08 in the
explants and 2-55 ± 0-15 in the controls; for the whole 24 h period it was 1-34 ±
0-07 in the explants and 2-70 ±0-13 in the controls. In the explants the area
vasculosa thus expanded at half the rate in controls during the 24 h period as a
whole, and slightly less than that during the first 12 h.
Of the 19 explant-control pairs only 18 were used for the 12 h period and
only 7 for the 24 h period. Pairs were excluded because either in the explant
one of the points needed for the measurement had reached the rim of the dish
prior to the end of the period, or, in the control, had passed too far around the
yolk sphere to be seen.
In the 18 pairs used for the 12 h period the mean initial (at 0-5 h) diameter
of the area vasculosa was 28-5 mm in the explants and 26-9 mm in the controls.
In the seven pairs used for the 24 h period it was 26-8 mm in the explants and
24-6 mm in the controls. Measurements of diameter were not corrected for
curvature of the surface of the area vasculosa in ovo; all visible portions of
its surface appeared to be nearly flat and to lie in the plane of the albumin
surface, which had been enlarged by the partial removal of albumin.
The rate of expansion in the explants was inconstant, as was shown by
measurement of the diameter of the area vasculosa at each time interval
180
J. M. AUGUSTINE
Table 1. Rate of expansion in explants in each period
No. of explants
Rate of expansion in
%/h (mean±S.E.)
Mean initial diameter
in mm
0-0 5 h
0-5 h-1 h
20
15
19-42+1-55 —1-39 + 1-58
26-3
28-4
1 h-6h
6h-12h
12h-24h
18
10
0-41 ±0-22
1-67±0-09
1 45±014
27-9
28-9
298
15
1
1
1
1
5 mm
Fig. 4. Arrows point to branches marking ends of four segments of a vessel in the
explant in Fig. 3. The most distal arrow is at the sinus terminalis, which marks the
end of the fourth segment. (A) 35 min after explantation. (B) 6 h after explantation.
(C) 12 h after explantation. (D) 24 h after explantation. Note that from 35 min to 6 h
sinus terminalis is nearly stationary relative to rim of dish, and that all four branches
move to left, away from rim. This may be related to left movement of left edge at this
time (Fig. 3C, D). Note also from 6 to 12 h, and 12 to 24 h sinus terminalis and all
branches move to right, toward rim of dish.
(Table 1 and Fig. 3). The rapid expansion in the 0-0-5 h period (Fig. 3B, C) was
thought to be an effect of rising temperature on tissues which had cooled and
contracted during explantation. This expansion was excluded in the preceding
comparison of explant with in ovo rates by using the 0-5 h photograph for the
initial measurement of diameter instead of the zero time photograph.
In determining the 12 h to 24 h rate of expansion, 8 explants were excluded
in which the area vasculosa reached the rim of the dish before 24 h. This was
at least partly due to a greater mean initial diameter in these 8 explants (34-0
mm) than in the 10 others (29-8 mm).
Growth rate of proximal and distal regions. One vessel in each of 16 explants
and 16 controls was chosen, and the distances between four of its branches,
and between the fourth (the most distal) and the sinus terminalis, were measured
in the 0-5 h and 24 h photographs (Fig. 4A, D). Measurements were made in
units inscribed on a disc placed in one ocular of a dissecting microscope, a unit
Mesodermal expansion in area vasculosa of the chick
181
O'UU
i
500
400 -
/
3 00 -
200
100
I——
T""'
i
Explant
i
1
Segment
Fig. 5. Mean rate of elongation of four contiguous segments of vessels in 16 explants and 16 in ovo controls incubated simultaneously for 24 h. Segments are
numbered in proximo-distal sequence.
representing 0-29 mm in the living embryo. From these measurements the
amount of elongation occurring during this period in four contiguous segments
of a vessel, from its proximal to its distal portion, was obtained. In proximodistal order the mean elongation rate of segments in control embryos, in %/h
was 1 -98 ± 0-24, 2-23 ± 0-26, 2-96 ± 0-32, and 4-76 ± 0-41, and in explants 0-93 ±
0-22, 1-37 ±0-25, 2-46 ±0-33, and 4-88 + 0-35. Thus in both controls and explants a proximo-distal gradient in rate of vascular elongation exists (Fig. 5).
Since the segments measured were located wherever the branches marking
their ends happened to arise, there was much variation in their length and in the
location of the proximal end of each series.
Group 2. Excentric explants
In this group embryos were placed excentrically in the culture dish so that the
edge of one side of the area vasculosa would reach the rim of the dish and be
stopped, while that of the other side would continue moving outward (Fig. 6).
Comparison of vascular elongation on the two sides would then indicate the
effect of edge arrest.
182
J. M. AUGUSTINE
Fig. 6. Embryo explanted excentrically. (A) 6 h after explantation. Part of edge
above upper arrow has arrived at rim of dish. Arrows point to branches nearly
equidistant from sinus terminalis on each side of area vasculosa. (B) 30 h after
explantation. Distance from branch to sinus terminalis on stopped side (upper) has
decreased, while that on moving side has increased.
Effect of edge arrest on proximo-distal growth rate gradient
Four vascular segments were measured as in Group 1. This was done on
both stopped and moving sides in the first photograph taken after arrest of
the closer edge (Fig. 6 A) and in the last photograph taken before the death of
the embryo (Fig. 6B). Eighteen explants were measured. The mean period
between measurements was 17-9 h. In proximo-distal order the mean rate of
elongation of segments on the moving side, in %/h, was 1-11 ±0-17, 0-84 ±0-13,
1-10 + 0-20, and 3-29±0-31, and on the stopped side l-34±0-17, 0-91 ±0-13,
0-20 + 0-11 and -2-14±0-21 (Fig. 7; minus sign means shortening occurred).
Thus the proximo-distal gradient in rate of elongation observed in Group 1 was
repeated on the moving side in Group 2, except in segment 1. On the stopped
side, however, the gradient was reversed.
The elongation measured in segment 1 on the stopped side was produced
partly by movement of its proximal end away from the rim on the stopped side
and toward that on the moving side in seven of the explants. This portion of the
elongation might have been caused by tension produced by the moving edge.
The mean elongation rate of segment 1 was therefore recalculated, after eliminating this portion. The resultant mean rate for segment 1 was 1-12 ±0-18
instead of 1-34 + 0-17.
Comparison of results on the two sides indicates that arrest of the edge had
no significant effect on elongation rate in segments 1 and 2. In segment 3,
however, the rate decreased nearly to zero on the stopped side, and segment
4 grew shorter rather than longer. The total length of the four segments on
Mesodermal expansion in area vasculosa of the chick
183
Moving side
300 -
Stopped side
200 -
/
/
100 0
%
I
I
^
I
1
2
3
4
Segment
100
\
\
200
\
X
Fig. 7. Mean rate of elongation of segments on stopped and moving sides of ] 8
explants in Group 2. Segments numbered in proximo-distal sequence. Negative
region below abscissa is rate of shortening instead of elongation.
the stopped side remained nearly unchanged. The elongation of segments 1 and
2 on the stopped side thus occurred at the expense of segment 4.
Proximo-distal location of segments. Since rate of elongation varies with
distance from the center of the explant, it was of interest to determine whether
the distances of the segments on the moving side were equivalent to those on the
stopped side. As an approximation of the center of the explant the midpoint
of the line connecting the proximal ends of the anterior and posterior vitelline
veins was chosen, and the distance was measured from it to the midpoint of
each segment on the stopped and moving sides in the photograph used for the
initial measurement above. The mean distances thus found for segments on the
moving side, in proximo-distal order, in mm was 8-9 ± 0-4, 11-1 ± 0-3, 13-6 ± 0-3
and 16-2 ± 0-3, and on the stopped side, 7-4 ± 0-3, 9-6 ± 0-3, 12-1 ± 0-3 and 14-8 ±
0-3. The segments on the moving side were thus more distal than those on the
stopped side, and their rates of elongation may be larger than those in segments
more nearly equivalent.
Location of proximal two segments on stopped side. Using the measurements
of distance made above and those of segment lengths obtained earlier, the mean
location of the distal end of segment 2 on the stopped side was found to be
67-2 ± 1-8 % of the distance from the center to the edge of the stopped side.
Since rates of expansion in segments 1 and 2 on the stopped side were not
significantly different from those on the moving side, the proximal 67 % of the
area vasculosa, where they are located, apparently expands by a mechanism
independent of outward movement of the edge.
184
J. M. AUGUSTINE
1 mm
Fig. 8. Edge of the area vasculosa on the stopped side of the explant in Fig. 6. The
portion shown is located just distal to the arrow on the stopped (upper) side in Fig.
6A. (A) 6 h after explantation. (B) 13-5 h (C) 19 h (D) 30 h. Arrows in (A) and (D)
point to same bifurcation. Note decrease in size of spaces between capillaries adjacent
to edge in successive photographs, and shortening and bending of large vessel
proximal to arrow.
Movement of single branches. As an additional method of determining the
effect of edge movement on mesodermal expansion two branches were chosen,
one on the stopped and the other on the moving side, which were very nearly
equidistant from the sinus terminalis on their respective sides (Fig. 6). The
distance moved by each branch toward the rim of the dish was then measured.
On the moving side this distance was the amount of expansion of all mesoderm
proximal to the branch in the direction of edge movement, while on the stopped
side it was the amount of expansion in the direction of the stopped edge. The
difference between the two distances moved was therefore regarded as the
amount of mesodermal expansion dependent on edge movement.
On both sides the branches were located in segment 3, with mean location
near its distal end. The mean distance from the branch to the sinus terminalis
on the moving side was 3-2 ±0-1 mm, and on the stopped side it was 3-1 ±0-2
mm. The mean distance moved by the branch on the moving side was 1-6 ± 0-1
mm, and on the stopped side it was 10 + 0 1 mm. Thus about 38 % of the mesodermal expansion proximal to an outwardly moving edge is dependent on that
movement, while 62 % can occur proximal to a stopped edge.
Marginal collapse. In the preceding section the branch on the stopped side
continued to move outward after arrest of the edge, decreasing the distance
between it and the sinus terminalis, while on the moving side this distance increased (Fig. 6). The mean increase on the moving side was 58-1 ± 7-7 % of the
Mesodermal expansion in area vasculosa of the chick
185
original distance, and the decrease on the stopped side was 34-7 ± 3-2 %. Fig. 8
shows the vascular pattern in the collapsing marginal region on the stopped
side at successive intervals after arrival of the sinus terminalis at the rim of the
dish. The photographs show that the vessels are shortening and becoming
packed together.
Group 3. Explants with ectoderm and basement membrane removed
In this group the ectoderm and its basement membrane were removed from
one half of the area vasculosa. The embryo was then placed excentrically in
the culture dish with the edge of the half lacking ectoderm closer to the rim
than that of the other half. The aim was to determine whether the mesodermal
expansion on the stopped side in Group 2 might have been caused by migration
of somatic mesoderm cells proximal to the sinus terminalis on an ectodermal
substratum. Adherence of these cells to the ectodermal basement membrane
and their connexion to subjacent vascular tissue was reported previously (1970).
Only 27 of the 44 explants made in this way survived long enough to provide
useful information.
Proximo-distal growth rate gradient. As in Groups 1 and 2 measurements
of four vascular segments were made. This was done on the stopped and moving
sides in nine explants, all of which survived a period of 24 h after arrest of
the edge. The mean rate of elongation of the segments in proximo-distal order
on the moving side, in %/h, was 0-89 ± 0-28, 0-71 ± 0-13, 1-24 ± 0-14 and 2-88 ±
0-45, and on the stopped side, 1-31 ± 0-22, 0-68 ± 0-19, 0-12 ± 0-20, and —1-83 ±
0-16. In two explants the proximal end of segment 1 on the stopped side moved
away from the rim on that side and towards the rim on the moving side, and in
two other explants the equivalent occurred on the moving side. Corrections
were made as in Group 2, resulting in a mean elongation rate for segment 1
on the moving side of 0-62 ±013, and on the stopped side 1-15 ±0-22 %/h.
The results are similar to those of Group 2. They indicate that migration of
somatic mesoderm cells on an ectodermal substratum plays no role in the
elongation of segments 1 and 2.
Movement of single branches. Expansion was also measured as the movement
of single branches equidistant from the sinus terminalis on the stopped and
moving sides toward the rim of the dish in 27 explants. The mean period between initial and final measurements was 19-4 h. The mean distance from the
branch to the sinus terminalis on the moving side was 3-6 ±0-2 mm, and on
the stopped side it was 3-5 ± 0-2 mm. The mean movement of the branch on the
moving side was 1-7 ±0-2 mm, and on the stopped side it was 1-2 ±0-1 mm.
These results are similar to those in Group 2 and show that about two-thirds
of the mesodermal expansion proximal to an outwardly moving edge can occur
in the absence of such movement and of an ectodermal substratum proximal
to the edge.
Histology of marginal collapse. In the preceding section the increase in
186
EC
J. M. AUGUSTINE
BM
ECT
lOO^m
Fig. 9. Edge of free and stopped sides of area vasculosa of Group 3 explant fixed
24 h after arrest of edge. Distal direction is to the left. Sections cut at 10 /im and
stained with PAS and hematoxylin. (A) Moving side. (B) Stopped side of the
same explant. Arrows point to a large vessel, or fusion of vessels, filled with blood
cells. There is a partial fold in the tissue; as the vessel passes distally it turns from
the horizontal, at top right, downward so that its distal end is at bottom left. The
entoderm is folded on itself and lies at the right side instead of the bottom of the
photograph. BM = basement membrane; CL = clumps of fuchsin-stained remnants of cut edge of ectodermal basement membrane; they lie on apparent line of
fusion of cut edge of ectoderm with mesoderm; EC = edge cells; ECT = ectoderm; EN = entoderm; ST = sinus terminalis.
distance between the branch and the sinus terminalis on the moving side was
51-2 ± 5-5 % of the initial distance, while the decrease on the stopped side was
37-5 ±5-1 %. This region was examined in sections of tissue from 11 explants
fixed 24 h after arrest of the edge (Fig. 9). On the stopped side vessels were
packed closely. In some cases there were vessels on top of each other, forming
two layers. They were full of blood cells. The cut edge of the ectoderm appeared
to have fused with the mesoderm; the cut edge of the basement membrane was
shown by clumps of fuchsin-stained material adjacent to the most distal vessel
(Fig. 9B). The densely packed vessels extended proximally about 1 mm. More
proximally the density decreased to that observed on the moving side.
Group 4. Explants on liquid medium
The experiment in Group 3 was repeated using liquid instead of semisolid
medium. The aim was to determine whether the vascular elongation obtained in
Group 3 might have been caused by outward migration of splanchnic mesoderm
on an entodermal substratum adhering to the semisolid medium, or by migration of the entoderm cells using this medium as a substratum. It has been
reported that entoderm grown in tissue culture becomes migratory (Romanoff,
1960).
Movement of single branches nearly equidistant from the sinus terminalis
Mesodermal expansion in area vasculosa of the chick
187
on the stopped and moving sides was measured in seven explants. The mean
period between measurements was 17-4 h. The mean distance from the branch
to the sinus terminalis on the moving side was 3-9 ± 0-3 mm, and on the stopped
side the same amount. On the moving side mean branch movement was 2-5 ± 0-3
mm, and on the stopped side 1-7 ±0-2 mm. Thus with liquid medium about
68 % of the mesodermal expansion occurring in the presence of an outwardly
moving edge and an ectodermal substratum can occur in their absence. Without
the support of the agar medium, the entoderm is probably not strong enough
to serve as a substratum for the production of tension by outwardly migrating
mesoderm cells.
DISCUSSION
In the present work expansion of mesoderm proximal to a stationary edge
occurred at a rate between 60 and 70 % of that proximal to an outwardly
moving edge, as indicated by the movement of single vascular branches. In
Group 2, growth-rate measurements of four vascular segments showed that the
expansion producing this movement on the stationary side is located almost
entirely in segments 1 and 2 in the proximal two-thirds of the area vasculosa.
Since rate of elongation in these two segments was not significantly different
on the stopped and moving sides, tension produced by outward movement of
the edge apparently does not contribute to the mechanism of expansion in this
portion of the area vasculosa.
Against this conclusion it might be argued that stopping movement of the
edge does not necessarily abolish tension produced by that movement. The
marked decrease in length of segment 4 after arrest of the edge might not result
in a complete loss of tension. Residual tension might suffice to cause a normal
rate of elongation in segments 1 and 2. However, to reach segments 1 and 2
such tension would have to pass through segment 3, in which rate of elongation
was reduced nearly to zero. This would contradict the elongation-producing
power of such tension.
The relation of tension to the elongation of segments 3 and 4 is less clear. It
seems likely that the very low elongation rate in segment 3 results from a positive rate at its proximal end changing gradually to a negative rate at the distal
end. In this case segment 3 may be a transitional zone in which a proximal mode
of elongation independent of tension gradually gives way to a distal mode
dependent on tension.
Alternatively, tension may play no role in the elongation of any of the segments. In this view the distal portions of vessels are shortened following arrest
of the edge by the elongation of the proximal portions which push them against
the rim of the dish. In the fixed space available the proximal portions elongate
at the expense of the distal portions because they are thicker and stronger. The
30-40 % decrease in expansion rate on the stopped side occurs not because the
arrested edge fails to exert tension, but because it fails to get out of the way,
188
J. M. AUGUSTINE
allowing compression to build up. Experiments of the type performed by
Beloussov, Dorfman & Cherdantzev (1975) and Downie (1975, 1976) on the
changes in size and shape of embryo parts freed from their attachments might
provide a means of testing this possibility. Portions of the area vasculosa cut
loose from adjacent portions might, by their changes in size and shape, indicate
the forces of compression or tension present in them.
REFERENCES
J. M. (1970). Expansion of the area vasculosa of the chick after removal of the
ectoderm. /. Embryol. exp. Morph. 24, 95-108.
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(Received 10 February 1977, revised 10 May 1977)