Mesodermal expansion after arrest of the edge in the area vasculosa of the chick

Transcription

1 /. Embryol. exp. Morph. Vol. 41, pp , Printed in Great Britain Company of Biologists Limited 1977 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 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 % 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.

2 176 J. M. AUGUSTINE Anterior vitellene vein Area vascula Basement membrane Ectoderm Sinus terminalis Yolk sphere Edge cells Somatic mesoderm Posterior vitelline vein Area vitellina Sinus terminalis Splanchnic mesoderm B Entoderm Fig. 1. (A) Stage 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

3 Mesodermal expansion in area vasculosa of the chick 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 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

4 178 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 Explanted at stage 18-20, the embryos had reached stage 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

5 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

6 J. M. AUGUSTINE Table 1. Rate of expansion in explants in each period h 0-5 h-1 h 1 h-6h 6h-12h 12h-24h No. of explants Rate of expansion in %/h (mean±s.e.) ± ± ±014 Mean initial diameter in mm mm 1 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 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

7 Mesodermal expansion in area vasculosa of the chick 181 O'UU 500 i / I 100 T""' Explant i 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 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.

8 182 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, , and 3-29±0-31, and on the stopped side l-34±0-17, 0-91 ±0-13, 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 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

9 Mesodermal expansion in area vasculosa of the chick Moving side Stopped side / / % I 1 I ^ 2 3 Segment \ \ \ I 4 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.

10 184 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 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

11 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 ± 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

12 186 J. M. AUGUSTINE EC 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

13 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 % 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,

14 188 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 AUGUSTINE, J. M. (1970). Expansion of the area vasculosa of the chick after removal of the ectoderm. /. Embryol. exp. Morph. 24, BELOUSSOV, L. V., DORFMAN, J. G. & CHERDANTZEV, V. G. (1975). Mechanical stresses and morphological patterns in amphibian embryos. J. Embryol. exp. Morph. 34, DEHAAN, R. L. (1967). Avian embryo culture. In Methods in Developmental Biology (ed. F. H. Wilt & N. K. Wessels), pp New York: Crowell. DOWNIE, J. R. (1975). The role of microtubules in chick blastoderm expansion - a quantitative study using colchicine. /. Embryol. exp. Morph. 34, DOWNIE, J. R. (1976). The mechanism of chick blastoderm expansion. /. Embryol. exp. Morph. 35, GRODZINSKI, Z. (1934). Zur Kenntnis der Wachstumvorgange der Area vasculosa beim Huhnchen. Bull. Int. Acad. Pol. Sci. Lett. B HAMBURGER, Y. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, NEW, D. A. T. (1959). The adhesive properties and expansion of the chick blastoderm. /. Embryol. exp. Morph. 7, ROMANOFF, A. L. (1960). The Avian Embryo., pp New York: MacMillan. TRINKAUS, J. P. (1966). Morphogenetic cell movements. In Major Problems in Developmental Biology (ed. M. Locke), pp th Symp. Soc. Devi Biol. New York, London: Academic Press. (Received 10 February 1977, revised 10 May 1977)