The Mechanism of Chick Blastoderm Expansion

/. Embryol. exp. Morph. Vol. 35, 3, pp. 559-575, 1976 559 Printed in Great Britain

The mechanism of chick blastoderm expansion

By J. R. DOWNIE1 From the Department of Zoology, University of Glasgow

SUMMARY At the time of laying, the domestic fowl blastoderm measures 4 mm across. After 4 days* incubation, the extra-embryonic yolk-sac tissues have expanded to encompass the whole yolk mass. This expansion involves the migration over the inner surface of the vitelline membrane of a specialized band of 'edge cells' at the blastoderm periphery. As they move, they pull out the blastoderm behind them, setting up a considerable tension. Expansion also involves cell proliferation and changes in cell shape. This paper attempts to show how locomotion, tension, proliferation and changes in cell shape all contribute to the orderly process of expansion. As a simplification, only the extra-embryonic epiblast is considered here. The findings are: 1. Expansion does not occur at a constant rate, but starts slowly, rises to a peak (over 500 /*m/h) at around 3 days, and then slows as coverage of the yolk mass nears completion. 2. During the first day of incubation, edge-cell migration produces a tension in the blastoderm. This rises to a peak at 20-24 h, then declines. This tension may be due to an imbalance between expansion by migration and expansion by proliferation. 3. Migration of edge cells can be affected by tension in the blastoderm, i.e. very high tension may hold them back. However, the tension level normally found in the blastoderm seems not to do so. The low rate of expansion in the first day is therefore not due to the high level of tension. It may instead be due to changes in edge-cell organization. 4. Proliferation occurs throughout the extra-embryonic epiblast during the expansion period. It is not restricted to the blastoderm periphery. After the yolk has been covered, the epiblast continues to grow, with proliferation restricted largely to a band just distal to the advancing edge of the area vasculosa. 5. Cell shape and arrangement change considerably during expansion. The epiblast of the unincubated embryo is a monolayer of tall cells. During expansion, these become considerably flattened so that each contributes a larger amount to yolk-sac surface area.

INTRODUCTION One of the most impressive features of early avian development is blastoderm expansion. At laying, the domestic fowl blastoderm measures about 4 mm across. After 4 days' incubation, the extra-embryonic yolk-sac tissues have totally encompassed the yolk mass, a more than 200-fold increase in tissue area. The mechanism of blastoderm expansion has been investigated several times. Schlesinger (1952) believed that the periphery was a syncytium, throughout 1 Author's address: Developmental Biology Building, Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland U.K. 560 J. R. DOWNIE expansion, and that expansion was the result of the growth of this syncytium across the yolk surface, with individual cells being formed behind as the syncytium spread outwards. Rather similarly, Haas & Spratt (1968) envisaged a 'ring blastema' zone just within the blastoderm periphery, where cells proliferate very rapidly and move out centrifugally. However New (1959), without ignoring the importance of cell proliferation, emphasized the role of active cell migration. He noted that cells in a narrow band round the blastoderm margin attach to the overlying vitelline membrane and, using this membrane as a substrate for locomotion, move out centrifugally until the yolk mass is surrounded. The properties of these specialized blastoderm 'edge cells' have been the subject of several studies (Bellairs & New, 1962; Bellairs, 1963; Bellairs, Boyde & Heays- man, 1968; Downie & Pegrum, 1971; Downie, 1975). New (1959) also noted that the blastoderm during expansion is under a tension, presumably generated by the activity of the edge cells. He believed this tension essential for expansion, the main effect being to maintain the sheet-like arrangement of the cells, and to prevent their piling up. This paper investigates the changing pattern of locomotion, tension and proliferation in blastoderm expansion. The details of individual experiments are described with the results. Hens' eggs used were White Leghorn or De Kaalb.

EDGE CELL LOCOMOTION AND SHEET TENSION IN THE EXPANDING BLASTODERM (1) Rate of expansion This was determined by measuring how far round the circumference of the yolk mass the blastoderm edge had travelled after different times. This was not an entirely simple matter since (1) there is considerable individual variation, but any attempt to follow the whole expansion process in a single specimen involves interference with the egg; and (2) it is difficult to make accurate measurements on the surface of a soft wet sphere. The results shown in Fig. 1 were obtained by taking samples from a single large batch of eggs at regular time intervals, remov- ing the yolks and assessing the expansion distance by comparison with known standards. These were drawings of the yolk mass as circles of 3 cm diameter (close to the usual yolk-mass diameter) with the expanding blastoderm drawn from above and from the side. Expansion was divided into 20 equal stages (in terms of sphere surface area). It was always possible to assign any actual embryos to one of these standards. Each standard is easily converted into blastoderm radius as a proportion of the circumference of a circle of diameter 3 cm. Each embryo is plotted in this way in Fig. 1. The broad pattern of results is consistent with those from more direct measuring methods, and shows several points of interest. (1) Expansion does not start immediately, but after about 10 h. This period is Mechanics of chick blastoderm expansion 561

20 30 40 50 60 70 80 90 Incubation time (h) Fig. 1. Blastoderm expansion rate in ovo, plotted as blastoderm radius after different incubation times. Each spot represents a different embryo. The line is drawn through the mean radius for each incubation time. much longer than the warming-up time of around 2 h. This pre-expansion period has been previously reported (Downie, 1974). (2) The rate of expansion is not constant. There is an initial slow phase to 16 h, when the rate is 200 /*m/h. From 24-84 h, the average rate is much faster (555 /tm/h) and this slows again over the final phase, to 96 h at 292 /^m/h. The exact time of completion of expansion is very variable, the last little patch of vitelline membrane often taking a long time to cover.

(2) Blastoderm retraction No method was found of measuring directly the tension generated by the edge cells. The problem lies in holding the thin cell sheet without tearing it. Only occasionally, as in the work of James & Taylor (1969), has this problem been overcome in simple cell sheets. An indirect method was, however, success- ful in giving relative measurements. Attached blastoderms of known incubation time were set up as New (1955) cultures, using a glass ring of 25 mm internal diameter, staged (Hamburger & Hamilton, 1951) and an outline drawing made using a Wild drawing tube and microscope. The edge of each embryo was then detached from the vitelline membrane and, after allowing a few seconds for any retraction to occur, a new outline drawing was made. Since it is the centrifugal movement of the edge cells which stretches the blastoderm, a comparison of the stretched and retracted blastoderm size gives a relative measure of the tension exerted by the edge cells. A difficulty in relating retraction directly to tension exerted by the edge cells is that the tension required to stretch a cell varies with the mechanical properties 562 J. R. DOWNIE

Up to 30 31-3-8 3-9-4-5 4-6-6-0 61-7-5 7-6-90 91 and over Initial blastoderm radius (mm) Fig. 2. Blastoderm retraction after loosening of the edge. Retraction is given as the percentage area reduction from the original (attached) area. Embryos are grouped according to their original radius. The number of embryos in each group is given in brackets. The histograms represent the mean retraction ± standard deviation for each radius group. of the cell. This may not alter much over the rather short incubation time covered by these measurements, but is at present an indeterminate factor. The results, involving measurements of 68 different embryos, are given in Fig. 2. Only embryos between 12 and 36 h incubation were used. Before 12 h, embryos are rarely reliably attached, and after 36 h, embryos are too large and too curved to be measured by this method. From Fig. 2, we see that newly attached blastoderms retract little; retraction rises to a peak when the blastoderm has a radius of around 6 mm (after 20-24 h incubation), then falls again to a low level when the blastoderm radius passes 9 mm (after about 36 h incubation). A blastoderm area retraction of 30 % - found when the radius is in the range 4-6-6-0 mm - implies that the cells are each occupying 30/70 x 100 = 43 % more than their resting area when the edge is attached. It is impossible to say what happens to tension later in development but, as the blastoderm becomes more curved, it is difficult to imagine how a tension generated at the edge could be transmitted throughout the cell sheet.

(3) The relationship between locomotion and tension What might cause the changes in rate of movement of the edge cells during the expansion period? There are two general possibilities: (1) external constraints, (2) changes in edge-cell organization. Mechanics of chick blastoderm expansion 563

Incubation time (h) Fig. 3. Plots of expansion distance (given as blastoderm radius) against time, comparing expansion by 'migration' (r = vt + r^ with expansion by 'growth' tlT (r = /*! «Je ). Values used are T = 10 h, v = 0-3 ram/h, rx = 2 mm.

(a) External constraints The most obvious external constraint (there could be others such as energy supply, or the state of the substratum: one possible substratum effect is considered later) is the tension generated by the edge cells themselves. As they stretch the sheet, this tension must act against further centrifugal movement. Blastoderm spreading is the result of two processes: edge-cell migration and growth of new yolk-sac tissue. The radius r of an expanding cell sheet (initial radius r±) where the edge is moving with a velocity v is given by r = vt + rt at any time t. Similarly, the radius of a flat cell sheet proliferating with a generation time T (cell size constant and all maintaining their arrangement as a cell mono- tlT layer) is given by r = rx Je . Given constant v and T, these processes are in imbalance; indeed, using the values of v, T and rx found in the 1-day chick 564 J. R. DOWNIE blastoderm, expansion by migration runs ahead of expansion by growth for about 38 h (see Fig. 3). This theoretical analysis could explain the observed tension and expansion rate results. At the start, migration is expanding the sheet faster than can be accommodated by the increase in cell numbers. The result is that cells are stretched to occupy more space. This generates a tension in the sheet which holds back the migrating edge, reducing expansion rate. As expansion proceeds, growth catches up, allowing edge-cell migration to proceed up to its maximal rate. When this happens, tension in the sheet drops as the cells cease to be stretched. The plausibility of this explanation is enhanced by the close coincidence of the theoretical time when growth catches up with migration (38 h) and the actual time when tension appears to drop to a low level (around 36 h). This explanation of the changes in tension and edge-cell migration rate rests on two ideas: (1) edge-cell migration can be restrained by tension within the blastoderm and (2) it is so restrained in the first 36 h of development, leading to a reduction in the potential expansion rate. The first idea receives support from experiments already reported (Downie, 1975). Colchicine slows down and may stop blastoderm expansion, but the effect is not on the locomotory abilities of the edge cells, but rather on the remaining cells of the blastoderm. Dissolution of their microtubules by colchicine reduces their ability to maintain a flattened shape, thereby increasing the tension on the attached edge cells and halting migration. Indeed, this tension may be so great that the whole blastoderm retracts, with its edge cells still attached to the vitelline membrane. This does not necessarily mean that the tension normally generated during expansion restrains the edge-cell migration rate. If it does, then reduction of the normal tension should increase the rate. Two attempts were made to reduce the tension: Experiment 1. Eleven 1-day incubated embryos were set up as New cultures and most of the blastoderm (about 80 %) excised, leaving a ring of tissue with the edge cells intact and attached. The expansion rate of these edge cells over a 4 h period was compared with 14 controls. With most of the blastoderm removed, tension on the edge should be considerably reduced, but controls and experi- mentals expanded at indistinguishable rates. Experiment 2. As we have seen, when the edge of a 1-day embryo is detached from the vitelline membrane, the blastoderm retracts by as much as 30 % of its original area (Fig. 2), releasing all the tension in the blastoderm. If edge-cell migration is slowed down by tension, the edges of these blastoderms, on re- attaching, should move quickly until they approach their previous attached position. Since re-attachment time is variable, these embryos, again set up as New cultures, were recorded by means of time-lapse filming. The results, shown in Table 1, were precisely the opposite of the prediction. On re-attaching, expansion rate was at first slow, increasing later and then stabilizing. Mechanics of chick blastoderm expansion 565

Table 1. Expansion during the period immediately after edge re-attachment Distances moved calculated from time-lapse films.

Distances (>m) moved in successive time periods Incubation time (min) after start of expansion before expansion Embryo re-starts (min) 0-25 25-50 50-75 75-100 100-125 125-150 150-175 1 5 153 204 214 214 228 238 224 2 18 82 109 133 150 160 156 156 3 18 163 224 211 228 245 245 228 4 53 52 61 61 70 87 87 104 5 27 87 104 104 113 113 113 131 Mean 24 107 140 145 155 167 168 169

The results of expts 1 and 2 suggest that though edge cells may be responsive to tension within the blastoderm, the actual tension generated during early expansion is not responsible for the low initial expansion velocity. The work of Curtis & Buultjens (1973) makes this result less unexpected. They showed that very large differences in surface adhesiveness had no effect on the rate of cell (fibroblast) movement and suggested a bulldozer analogy - that within rather wide limits of environmental conditions, rate of movement remains constant. This may well be the case for blastoderm edge cells. Over the range of tension normally experienced, rate of movement of edge cells is unaffected; but when tension is drastically increased (by treating with colchicine (Downie, 1975)) movement is inhibited. This argument may also apply to another possible external constraint - the tautness of the substrate. The vitelline membrane of the unincubated egg is rather slack. During the period of blastoderm expansion it becomes taut, due to pump- ing of water into the sub-blastodermal space (New, 1956). Bellairs, Bromham & Wylie (1967) noticed that in New cultures, blastoderms cannot expand on a very loose vitelline membrane. This factor needs further investigation, but could well be an all-or-none one: once the edge cells can get a grip, they move at their maximal rate. The most likely alternative explanation of the changes in the migration rate of edge cells is a change in the edge cells themselves.

(b) Edge-cell organization It seems possible from the above that the changes in the rate of blastoderm expansion in ovo reflect real changes in the capabilities of the edge cells. To test this, edge-cell fragments with attached vitelline membrane were isolated from 1-, 2- and 3-day incubated embryos, set up as New cultures and the expansion rate assessed over a 5-h incubation period. The results (Table 2) indicate that 36 EMB 35 566 J. R. DOWNIE

Table 2. Expansion rates in New culture of blastoderm edges, isolated from embryos at different stages Expansion is given as the distance covered in a 5-h test period.

Distance (/*m) moved Incubation Proportion of Number of in 5 h test period time in ovo circumference edge pieces (mean ± standard) (h) covered (approx.) tested deviation) 24 I 11 169-3 ±38-8 48 i 5 285-7 ±43-7 66 ±-f 5 383-8 ±41-7

3-day edge cells are capable of moving faster than 1-day edge cells, with 2-day cells intermediate. Downie & Pegrum (1971) found that the attached blastoderm edge in 1-day incubated embryos was a band of cells between 90 and 130 /im wide. Preliminary results from embryos of different ages suggest that this is variable, from around 70 jam when the edge cells first attach to the vitelline membrane, to as much as 260 (im at 3 days' incubation, when the blastoderm is expanding at its fastest. Though the details of this change must await further work, it seems likely that recruitment to the population of attached edge cells, making the edge a more powerful motile unit, is responsible for the increasingly rapid rate as expansion proceeds.

TISSUE GROWTH IN THE EXPANDING BLASTODERM Blastoderm expansion involves tissue growth as well as edge-cell migration. Growth is analysed here in terms of cell proliferation, shape and size.

(1) Cell proliferation - the pattern ofmitotic index During blastoderm expansion, proliferation rate might vary (i) in different parts of the expanding tissue, (ii) at different developmental stages. Both New (1959) and Haas & Spratt (1968) envisaged a peripheral ring of rapidly proliferating cells, with mitosis absent, or at a low rate elsewhere. Classically, comparison of proliferation rates in embryos has been by means of the mitotic index, i.e. the proportion of cells in mitosis at any one time. This is used here to compare proliferation in different parts of the extra-embryonic epiblast at different stages. Though mitotic index is usually calculated from serial sections, this is not easy since a correction factor is necessary for nuclear size (Abercrombie, 1946; Simnett, 1968). Fortunately, extra-embryonic yolk-sac can be stripped of its yolk and endoderm, and the epiblast fixed as a flat sheet, allowing counting of the cells as if in a monolayer culture. Extra-embryonic tissue was stripped of hypoblast and yolk by means of a fine Mechanics of chick blastoderm expansion 567

Table 3. The mitotic index (%) in the extra-embryonic epiblast at different stages and in different positions Stages are given as defined by Hamburger & Hamilton (1951) (HH); incubation time in hours (inc) and yolk-sac coverage proportion as a fraction (cp). Positions are given as distances (mm) distally from either the area pellucida margin (apm) or area vasculosa margin (avm).

No. of Stage embryos Mean mitotic index (%) at different positions

HH2-3 All counts made near the blastoderm margin inc 15 5 (see text) 4-2 cp start HH4-6 apm 2 A\ position inc 24 5 cpi 2-7 2-7 3-2 mitotic index HH 12 avm 2 4 6 8 inc 48 cpi 20 2-2 2-6 2-6 2-4 HH 17-18 avm 2 4 6 10 12 inc 72 cpi 1-2 1-8 21 30 21 1-6 21 HH 19-23 avm 2 4 6 8 10 12 14 16 18 20 22 24 inc 90 cp complete 0-4 1-4 1-7 1-7 10 11 10 0-8 0-3 0-2 01 01 0 HH 25-26 avm 2 4 6 8 10 12 14 16 inc 120 >. cp complete 0-4 0-9 1-6 1-2 0-6 0-2 0 0 0 jet of chick saline (Britt & Herrmann, 1959). To keep the large pieces of tissue flat, they were fixed in Bouin on a coverslip, with the edges held by the corners of the coverslip. They were then stained in Ehrlich's haematoxylin and mounted in Canada balsam. Cell counts were made at a 400 x magnification, using a counting grid that divided the field into 180 x 900 /tm2 squares. The mitotic index for each position in each embryo is the mean number of mitotic cells found in five complete microscope fields involving a total cell population of 103-1-5 x 104, depending on cell density. Table 3 gives the means of these mitotic indices for each embryonic stage examined. In early embryos (15 h, Hamburger & Hamilton stage 2-3) there is no clear distinction between intra- and extra-embryonic tissue. Counts were made near the margin. In 24-h embryos (stage 4-6), the densely packed cells of the area pellucida give a boundary, and counts were made at 2 mm intervals outwards from the area pellucida/area opaca margin. In later embryos, the area vasculosa mesoderm remains attached to the epiblast when the hypoblast is removed. The area vasculosa/vitellina margin was therefore used as a starting point, with

36-2 568 J. R. DOWNIE counts again made at 2 mm intervals outwards, to near the blastoderm edge. After most of the yolk has been covered by the yolk-sac, the vitelline membrane ruptures. The yolk-sac continues to grow in total volume, and the area vitellina eventually becomes obliterated by the invasion of area vasculosa mesoderm. Although no longer relevant to the inter-relationship of active edge cell migration and yolk-sac tissue growth, it was of interest to discover whether tissue growth continued in the epiblast after rupture of the vitelline membrane. One group of embryos (stage 25/26) represents the post-rupture situation. Prolifera- tion clearly continues but in a restricted band. Since most of the epiblast at this stage is in fact in the area vasculosa, an estimate of mitotic index in this area was made. Serially-sectioned material from a single stage-25/26 embryo, where the area vasculosa epiblast was cut obliquely, laying it out flat, gave a mitotic index of 1 % (on 2 x 103 cells). Proliferation obviously still continues in the area vasculosa epiblast. Two general points emerge from the counts. (1) Mitotic index is highest at the earliest stages examined: a mean of 4-2% at stage 2-3, declining to a maximum of 2-1 % in the latest embryos examined (stage 25-26). (2) The mitotic index remains fairly uniform throughout the epiblast until stage 17-18. Thereafter, mitosis becomes progressively restricted with two low points - near the area vasculosa, and towards the edge - and a broad high band between. Mitotic index towards the periphery becomes low only around the time blastoderm expansion is completed. After this, mitosis is largely restricted to an area between 2 mm and 10 mm from the margin of the area vasculosa. This pattern is unlike the 'ring blastema' of Haas & Spratt (1968), envisaged as a narrow peripheral ring of ' undifferentiated' and rapidly dividing cells. During the expansion period, mitosis occurs at a high level throughout the extra-embryonic epiblast, rather than being restricted to a peripheral ring.

(2) Cell proliferation {doubling time) Though mitotic indices are the easiest data to collect on proliferating tissues, they may be difficult to interpret, since they can vary with both proliferation rate and mitotic time (the time spent in the process of mitosis). A low mitotic index may therefore indicate a low proliferation rate or any unusually short mitotic time. Because of this, some workers, (e.g. Woodard, 1948; Dondua, Efremov, Krichinskaya & Nikolaeva, 1966) have suggested that mitotic index is a useless measure, at least in comparing different tissues. A better measure of proliferation rate is doubling time (the time for the cell population to double in numbers), equivalent to the generation time in a population where all cells are dividing with cell cycles of equal duration. Doubling time can be calculated from the proportion of the cell population entering a fixed point in the cell cycle in a specific time. In practice, this proportion is often Mechanics of chick blastoderm expansion 569

Left Right

Posterior B

Edge 109 ±1-2

Outer 12-2 ±0-4

|;-Vv;"'!f| Middle 11-6 ±10

Inner 12-3 ±0-9

Fig. 4. Metaphase index (%) after 2 h Colcemid block in different parts of 1-day epiblast. (A) for regions anterior, posterior, left and right. (B) for zones edge, outer, middle and inner. Each index is given as the mean ± standard deviation of 11 embryos. determined from the number of cells entering metaphase during a specific period of treatment with colchicine or one of its derivatives. This has often been done to chick embryos (see for example Woodard & Estes, 1944; Emanuelsson, 1961; Pearce & Zwann, 1970) but usually to investigate embryonic rather than extra- embryonic tissues, and with rather variable success. The problem lies in treating all the cells at as near the same time as possible. Preliminary experiments in which the drug was injected into either the yolk, the albumen, or both together gave very variable results. The most reliable method was to set up embryos as New cultures, adding the drug in a few drops of medium 199 (Biocult) both to the albumen and directly on top of the embryo. Unfortunately, this worked well with 1-day embryos only. All attempts to obtain reliable metaphase blocking with later (3-day to 5-day) material failed, with 2-h metaphase indices sometimes lower than simple mitotic indices for the same tissue. The reasons for this are unknown. For the metaphase blocks on 1-day embryos, Colcemid (Ciba) was used at a concentration of 0-l^gm/ml (2-7X10~7M), the lowest concentration to give 570 J. R. DOWNIE reliable results. The blocking period was 2 h since after longer periods, cells sometimes appeared abnormal. After 2 h incubation, the embryos were cooled, the hypoblast removed as before and the epiblasts prepared for examination. The epiblast of 11 embryos treated in this way was divided into zones as shown in Fig. 4, and each zone counted separately as described before. The zone marked 'edge' does not include the 'edge cells', but is a distinctive zone, about 90/*m wide, of densely packed cells adjacent to the edge. The 'edge cells' themselves do not appear to divide at all. Colcemid treatment for as long as 6 h failed to reveal any mitotic figures amongst them. The results (Fig. 4) showed no significant differences in metaphase index (using Student's t test) between any of the epiblast zones, confirming that in the 1-day embryo, the whole of the extra-embryonic epiblast is proliferating at about the same rate. The overall mean 2-h-blocked metaphase index was 11-6 ± 0-7 % (± standard deviation). This can be converted to doubling time (T) using the formula ln 2 x block duration (h) T = e metaphase index (as a fraction)' giving a doubling time of 11-9 ±0-7 h. Mitotic index is higher at 15 h (4-2%) than at 24 h (2-7-3-2 %) and to deduce from this a higher proliferation rate would fit in with Wylie (1972) and Emanuelsson (1965), who found a doubling time in the earliest stages (after laying) of 7-8 h. After 1 day, mitotic index declines and cell division becomes restricted in area. The precise effect of this on proliferation rate depends on what happens to mitotic time. Few seem to have studied this in detail. Dondua et ah (1966) found variation from 34-90 min in different regions of stage 3-5 chick embryos, but did not study later ones. Perhaps more suggestively, Fujita (1962) found a mitotic time in 1-day neural tube of 24 min, lengthening to 60 min in the same tissue at 6 days. If a similar lengthening occurs in chick epiblast, then the drop in mitotic index may actually underestimate the drop in proliferative activity. In summary, at the start of expansion, all cells of the extra-embryonic epiblast, apart from the edge cells, are proliferating at a rapid rate. As expansion proceeds, proliferation becomes progressively restricted in area, and probably very reduced in rate.

(3) Cell size and shape in the expanding blastoderm The yolk-sac epiblast stretches from the area pellucida to the blastoderm edge. It is generally a single epithelial layer, though at some points, e.g. the epiblast of the area vasculosa and its periphery, there is enough overlapping to consider it a bilayer. Cell packing density was determined from the Bouin-fixed material used for mitotic index calculations. Wax sections turned out to be unsuitable for epiblast height determinations. Instead, yolk-sacs were prepared as for transmission Mechanics of chick blastoderm expansion 571

Table 4. Cell diameter (/*m) in the extra-embryonic epiblast at different stages and in different positions Diameters are calculated from cell density counts: where there is significant overlapping (marked *), these figures do not accurately reflect the yolk-sac surface area occupied by each cell. Abbreviations as in Table 3.

No. of Stage embryos Mean cell diameter (/tm) at different positions HH 1 unincubated 7 Counts made near the blastoderm margin 13-4 cp start HH 1 inc 6-5 3 Counts made near the blastoderm margin 10-2 cp start HH2-3 inc 15 5 Counts made near the blastoderm margin 14-6 cp start HH4-6 apm 2 4 position inc 24 cpi 16-3 19-7 16-6 cell diameter HH 12 avm 2 4 6 8 inc 48 cp| 14 0 15-9 17 6 18-9 16-: HH 17-18 avm 4 8 12 inc 72 cpi 11-1* 12-3* 16-3 200 HH 17-23 avm 4 8 12 16 20 inc 90 cp complete 114* 130* 16-2 18-9 21-6 21-8 HH 25-26 avm 4 8 12 inc 120 ^ cp complete 10-8* 12-0* 14-6 180 electron microscopy, sections cut at 1 /*m and stained with methylene blue. Epiblast height was then measured from camera lucida drawings. The mean area of epiblast surface occupied by individual cells (ignoring overlapping) at different stages and in different positions is given in Table 4. Some of these figures, along with epiblast height determinations, are used in Table 5 to calculate cell volume at three stages. Figure 5 shows camera lucida drawings from epiblast transverse sections to indicate the appearance of the cells at different positions and stages. Two general points emerge from this analysis: (1) Extra-embryonic epiblast cell volume is halved during the first day's incubation, and thereafter remains fairly constant. A reduction in cell size has been noted by Bancroft & Bellairs (1974), though they give no measurements. (2) The shape taken up by the epiblast cells shows a definite pattern of 572 J. R. DOWNIE

Table 5. Derivation of extra-embryonic epiblast cell volume at different stages and in different positions Cell area (/*m2) calculated from density counts on Bouin-fixed flat-mounted epiblasts. Epiblast height (/im) measured from 1 [im araldite sections.

Stage (incubation Mean cell Mean epiblast Mean cell time) Position area Om2) height (/*m) volume (/Mm3) Unincubated Near edge 141-9 19-8 2809-6 24 h Near edge area opaca 221-3 5-3 1172-9 Not near edge 248-2 4-8 1191-4 72 h Area vitellina near edge 186-6 7-5 1399-5 Area vitellina near area vasculosa* 106-5 12-3 13100 Area vasculosa* 465-5 3-3 1536-2 * Positions with significant overlapping. development. In the unincubated blastoderm, they are close-packed and tall. Soon after the onset of expansion, they become considerably flattened. This persists throughout expansion, though flattening seems less pronounced in the 3-day than in the 1-day epiblast. A new feature in the later stages (continuing after the yolk mass is covered) is the appearance of a zone of high cell overlap in the epiblast around the periphery of the area vasculosa. Epiblast cells within the area vasculosa are in a bilayer and are very flattened. We can perhaps regard the unincubated blastoderm partly as a reservoir of tissue, compactly stored as tall cells. Once expansion begins, these cells assume a shape that covers a large area. This flat shape is partly intrinsic, maintained to some degree by microtubules (Downie, 1975), and partly produced by the stretching effect of edge-cell tension, at least during the early stages of expansion. Later, just before vitelline membrane rupture, the yolk-sac is very tightly opposed to the vitelline membrane, due to the large volume of sub-blastodermal fluid (New, 1956). Any edge-cell tension that persists until then could hardly be transmitted throughout the yolk-sac. This may partly account for the considerable variations in cell density and arrangement that are then apparent.

The role of tension in blastoderm expansion New (1959) first noticed the tension in early blastoderms, his observation being confirmed by Bellairs et al. (1967); but in neither study was there any attempt to measure changes in blastoderm tension as expansion proceeds. New found that edge-cell attachment is essential if expansion is to occur, and suggested that this is because the tension created by the edge cells is in some way essential for expansion. It is difficult, however, to imagine how blastoderm expansion could occur in any way other than by active outgrowth, involving cell migration at the Mechanics of chick blastoderm expansion 573

50 urn

Fig. 5. Camera lucida drawings of epiblast cells, all to the same scale and all from 1 /Am araldite sections. (A) Unincubated embryo, near the blastoderm periphery, (B) 23-h incubated embryo, area opaca. (C) 74-h embryo, near blastoderm periphery. (D) 74-h embryo, area vitellina near the area vasculosa; a region of high cell overlap, (E) 74-h embryo, area vasculosa. periphery. Alternative mechanisms such as appositional growth or cell spreading are not real alternatives since they are likely also to require peripheral attachment and cell migration. How essential then is the tension created by the edge cells ? New suggested that tension is necessary to maintain the epiblast as a flattened monolayer. His test was, however, not entirely unequivocal. He showed that when yolk-sac epiblast is prevented from expanding by anchoring its edge, it continues to grow, inevitably forming a multilayer. This does not show that the 574 J. R. DOWNIE rather high tension found in the 1-day blastoderm is necessary to prevent this multi-layering. Epiblast cells are well able to maintain a flattened shape (Downie, 1975) and post-mitotic cells would have little resistance to overcome in assuming this shape: they would certainly not require the assistance of a tension capable of stretching them to 40 % more than their resting area. I would like to suggest that the high level of tension around 20-24 h is the result of an imbalance between active migration and proliferation. At the start of expansion, cells are columnar and tightly packed, giving considerable 'slack' that the edge cells pull out in the first few hours. Tension in the sheet is therefore rather low. Around 20-24 h, this 'slack' has been used up and, as expansion continues, tension increases. Thereafter, the amount of new tissue produced by proliferation catches up with edge-cell migration (as suggested in Fig. 3) and tension is reduced. It seems likely that the fall in proliferation activity in the later stages of expansion is correlated with this 'catching-up' process.

I should like to thank Professor D. R. Newth and Professor Ruth Bellairs for reading the manuscript and suggesting several improvements. This work was started while I was in receipt of an S.R.C. studentship and under the guidance of Professor M. Abercrombie at University College London, and continued in the Department of Zoology, University of Glasgow.

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{Received 19 November 1975; revised 23 January 1976)