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Home , Ectoderm, Lateral plate mesoderm, Mesoderm, Somite

/. /, exp. Morph. Vol. 51, pp. 227-243, 1979 227 Printed in Great Britain © Company of Biologists Limited 1979

The mechanism of in the chick embryo

By RUTH BELLAIRS1 From the Department of and , University College London

SUMMARY The segmentation of in the chick embryo has been studied by transmission and scanning electron microscopy (stages 8—14). The segmental plate consists of loosely arranged mesenchymal cells, whereas the newly formed somites are composed of elongated, spindle-shaped cells arranged radially around a lumen, the myocoele. The diameter of each somite is thus two cells plus the myocoele. Two major factors appear to be responsible for the change in cell shape at segmentation: (1) Each prospective somite cell becomes anchored at one end to the adjacent epithelia (i.e. the , the , the , the or the ) by means of collagen fibrils. These fibrils are already present in the segmental plate before the somites begin to form. (2) A change in cell-to-cell adhesiveness causes the free ends of these cells to adhere to one another. (Bellairs, Curtis & Sanders, .1978). This adhesion is then supplemented by the development of tight junctions proximally in the somite. Because it is anchored at both ends, each somite cell is under tension in much the same way as a cell in culture is under tension. Each somite cell therefore becomes elongated and the somite as a whole accommodates its general shape to that of the space available between the adjacent tissues. The arrangement of the cells in the more differentiated somites (stages 17-18) has also been examined and it has been found that the chick resembles Xenopus in that the cells undergo rotation and become orientated in an antero- posterior direction.

INTRODUCTION One of the most lively topics in developmental is that of how the mesoderm becomes segmented into somites. This problem has been tackled in four main ways. The first aims at disturbing the relationship between the prospective somite mesoderm and the adjacent tissues to determine which of these are necessary for segmentation. This type of experiment was originally based on the idea that the somites formed as the result of a specific embryonic induction by another tissue (e.g. by the notochord or neural tissue), but recently that idea has been modified and a more modern concept is that there is a pro- gramming of the mesoderm at an earlier stage and that the role of the notochord may be to help in 'stabilizing' the somites once they are formed (Lipton & Jacobson, 1974 a, b; Menkes & Sandor, 1977). 1 Author's address: Department of Anatomy and Embryology, University College, Gower Street, London WC1 6BT, U.K. 228 R. BELLAIRS The second approach is essentially a theoretical one and aims to solve such problems as that of how the number of somites is determined by the embryo. The most recent of these is the application of the mathematical' Catastrophe Theory' to by Cooke & Zeeman (1976), which has received support from the experiments of Elsdale, Pearson & Whitehead (1976). Catastrophe Theory has also been applied to chick by Zeeman (1976). The third approach has been to compare the properties of unsegmented and segmented mesoderm to gain some idea of the changes that occur in the cells at segmentation. Bellairs et ah (1978), showed that as the mesoderm became segmented, its cells became more adhesive to one another. Similarly it was found that when pieces of unsegmented mesoderm were explanted in tissue culture, they behaved differently from segmented mesoderm (Bellairs & Portch, 1977; Bellairs, Sanders & Portch, 1980). The fourth approach has been to study the morphological changes that take place in somite segmentation. Although these were described by many of the earlier authors (e.g. Duval, 1889; Williams, 1910), the only recent major analysis was by Lipton & Jacobson (1974a,b) who used both light and transmission electron microscopy, but these authors were concerned with the first six pairs of somites only. No detailed account of segmentation by scanning electron microscopy has been given previously. The present paper is therefore concerned with an SEM study of somite segmentation. Particular attention will be paid to the role of the extracellular materials in somitogenesis.

MATERIALS AND METHODS Hens' eggs were incubated for periods between 30 and 72 h so that the embryos were between about stages 8 and 18 of Hamburger & Hamilton (1951). Because the process of segmentation begins at the anterior end and spreads posteriorly, there is a gradient of developmental stages along the body axis, the anterior regions being in advance of the posterior ones. Four different developmental stages of somitic mesoderm (Fig. 1) were therefore distinguishable: (a) Unsegmented mesoderm, which is a thick band of tissue, the segmental or paraxial plate, which runs longitudinally down either side of the neural tube and notochord in the trunk region. (b) Transitional mesoderm, which is partly segmented; for example, two pairs of partly formed somites are present at stage 12. (c) Newly formed somites, which lie just anterior to the transitional mesoderm, and which have not yet begun to form dermo- and sclerotomes. (d) Mature somites, which are the most anteriorly situated somites and which have begun to form dermo-myotomes and sclerotomes. At stage 12, 16 pairs of somites are present but by stage 18 a further 20 or more pairs have differentiated and there is little unsegmented mesoderm Somite segmentation in the chick embryo 229

Differentiated somites

Newly segmented somites

Transitional region

Lateral plate

Segmental plate

Fig. 1. Diagram to show the different regions of somitic mesoderm and lateral plate in a stage-14 embryo. remaining. The differentiation of most of the somites into dermo-myotomes and sclerotomes is well advanced in these older embryos. Fourteen specimens were prepared for transmission electron microscopy (TEM) and sixteen for scanning electron microscopy (SEM). Specimens for TEM were fixed in 2-5 % glutaraldehyde in 0-1M sodium cacodylate at a pH of 7-2 for 1-4 h and then washed three times in 0-1 M sodium cacodylate contain- ing 0-333 g CaCl2 for a total of l|h. They were treated with 1 % osmium tetroxide in phosphate buffer (pH 7-2 for 1 h at 4 °C then rinsed in phosphate buffer. After dehydration in graded ethanols, followed by two changes of propylene oxide, they were embedded in Araldite. Sections were stained with 2 % uranyl acetate at 38 °C for 2 min then counterstained with lead citrate. Twenty-seven specimens were fixed for periods of 4-24 h in 3 % glutaraldehyde in cacodylate buffer, at pH 7-2. After washing in cacodylate buffer, the specimens were immersed in 1 % osmium tetroxide for 30 min, washed again in buffer and dehydrated in graded ethanols. They were dried in a Polaron critical point drying apparatus from liquid CO2, mounted on stubs with UHU glue (Fishmar, Ltd, Waterford, Eire) and coated with gold. 230 R. BELLAIRS Somite segmentation in the chick embryo 231

RESULTS Figure 2 shows a recently formed somite cut transversely and viewed by SEM. It is triangular in section and comparable to the specimen illustrated with a light micrograph by Lipton & Jacobson (1974a; their fig. 8). Those cells which lie on the dorso-medial side of the somite and beneath the are arranged in a columnar manner, but the cells of the more ventral region are less well organized. There are many contacts between the somite and the neural plate and these consist mainly of extracellular materials (Fig. 3). Similar materials are present between the somite and the endoderm. A large part of these extracellular materials consists of fibrils which are probably collagenous (see Discussion). Similar fibrils are also present between the and the underlying endoderm. It is well known that soon after the first somites have formed, the neural folds rise up towards one another, and the dorso-medial wall of each somite rises with it (Lipton & Jacobson, 1974a) eventually becoming the vertically orientated medial wall of each somite (Williams, 1910). In this way these first formed somites change shape and acquire the rosette shape which is generally considered to be a characteristic of all somites. When seen in longitudinal section a somite is typically rosette-shaped (Fig. 4) though in transverse section it is box or wedge-shaped (Fig. 5). Lipton & Jacobson (1974a) have already pointed out that only the first three pairs of somites are formed in association with a flat neural plate, and that these are

FIGURES 2-7 Fig. 2. SEM micrograph of part of a stage-8 embryo. The specimen has been fractured across the 3rd somite. Note the collagen fibrils (/) which lie between the dorsal side of the somite (s) and the neural plate (/?). e, Endoderm; Ip, lateral plate, x 650. Fig. 3. Enlargement of part of Fig. 2 to show the collagen fibrils. Fig. 4. SEM micrograph of a longitudinal section through a somite of a stage-1.1 embryo. Spindle-shaped cells are arranged around a lumen (/) which contains other cells. Extracellular materials cover the surface of the somite, n, Neural plate; no, Notochord. x 585. Fig. 5. SEM micrograph of a transverse section through a somite and the associated lateral plate mesoderm of a stage-] 2 embryo. The aorta (a) and endoderm (e) are visible but other tissues have been removed, x 780. Fig. 6. SEM micrograph of the centre of a transversely broken somite from a stage-14 embryo. The myocoele is packed loosely with mesenchymal cells (arrowed), which make contact with the spindle-shaped cells of the somite (sp). x 1820. Fig. 7. SEM micrograph of a stage-10 embryo which has been broken transversely caudal to the somites. The ectoderm has been removed and a meshwork of fibrils can be seen on the dorsal side of the segmental plate mesoderm (sp). Many fibrils lie between the segmental plate and the endoderm (e). Some of the most dorsally situated cells are already elongated (arrowed), x 600. 232 R. BELLAIRS Somite segmentation in the chick embryo 233 therefore the only ones to pass through the triangular phase. Furthermore, these first three pairs of somites differ from the succeeding ones in that they are not formed from segmental plate but from a direct aggregation of mesoderm cells. By the time that the remainder of the somites develop, the neural plate has already rolled up into a tube, so that each new somite now takes on the rosette shape right from its first appearance. This is illustrated in Figs. 8, 9 and 11, which are all from the same embryo. Figure 8 is a low power view to show part of a stage-12 embryo. The ectoderm and neural tube have been removed from the posterior trunk region so that the notochord may be seen as a dark band running down the mid-line. Figures 10, 12 and 13 show details of the segmental plate region, whilst Figs. 9 and 11 are enlargements of one of the most recently formed pair of somites. The segmental plate is covered dorsally with extracellular material. Most of this consists of fibrils (see Fig. 10) which are probably collagen (see Discussion). A mat of finer material has also been seen in some specimens and in Fig. 13 this has been pulled aside from the underlying collagen. The collagenous fibrils which are associated with the segmented somites (Figs. 3, 9 and 11) increase in number as differentiation proceeds. They attach the somite to the neural tube (Fig. 14) the notochord, the ectoderm (Fig. 17), the endoderm (Fig. 7) and when it has developed, to the aorta (Fig. 5). Figure 14 shows the dorsal view of a somite at stage 15 which is attached to the neural tube by strands of collagen fibrils and some cell processes. Collagen fibrils also pass across the furrows from one somite to the next. Eventually, each somite becomes encapsulated with collagen fibrils (Figs. 16 and 22). Segmentation is accompanied by a marked change in the shape of the cells. This can be seen by comparing the dorsal view of the unsegmented (Fig. 10) and the segmented (Fig. 11) mesoderm. The cells of the unsegmented mesoderm

FIGURES 8-13 Fig. 8. SEM micrograph of part of a stage-12 embryo from which the ectoderm has been removed, s, Completely segmented somites; t, transitional zone; u, un- segmented region. The posterior part of the neural tube has been removed, x 100. Figs. 9 and 11. Higher magnification of recently segmented somites shown in Fig. 8, to illustrate the distal ends of the elongated somite cells, x 250 and x 500, respectively. Fig. .10. Higher magnification of the unsegmented mesoderm shown in Fig. 8, to illustrate the flattened cells. Note the meshwork of collagen fibrils, x 1000. Fig. 12. SEM micrograph to show the unsegmented mesoderm caudal to the region shown in Fig. 8 (same specimen). The neural tube has been removed. Note the concavity of the mesoderm where it lay in association with the neural tube. x250. Fig. 13. SEM micrograph of the dorsal surface of the unsegmented mesoderm to show the meshwork of fibrils (/) and patches of less structured material (m). x1000. 234 R. BELLAIRS Somite segmentation in the chick embryo 235 are flat and packed loosely on top of one another. They appear to be in contact by short processes and localized contacts, although there are many spaces between them. In many ways they possess the typical shape. By contrast the cells of the most recently segmented somites appear rounded on their dorsal surfaces and more tightly packed together; in comparison with the unsegmented cells, they present a smaller area when seen from above. This change in shape which accompanies segmentation is seen in a slightly less advanced stage in the transitional (i.e. part segmented mesoderm). It is associated with the elongation of the cells and their rearrangement into an . The cells at the dorsal side appear to become elongated before those at the ventral (Fig. 2) and this may be related to the fact that segmentation starts dorsally. Even in the segmental plate (Fig. 7) some of the most dorsally situated cells show signs of elongation as if in preparation for segmentation. Soon after its formation, each somite becomes arranged as an epithelium surrounding a small, central lumen, the myocoele, which is filled with an irregu- larly arranged group of cells (Fig. 4). The cells of the epithelium are long and narrow and extend the full width of the wall. Transmission electron micrographs show that they are attached to one another proximally by desmosomes and distally by tight junctions. The cells lying in the myocoele appear to be orientated at random and do not have the regular arrangement of the epithelial cells. Many appear to adhere to the inner wall of the epithelium surrounding the myocoele (Fig. 6). There is very little extracellular material visible in the myocoele. Similarly, when light microscope sections were stained with Alcian blue, using the critical electrolyte concentration technique (Pearce, 1972), very little extracellular material was visible in the myocoele. The epithelial cells of the somite make contact with one another along their length; the regions of contact are visible in the SEM micrographs as short processes. The epithelial cells tend to be narrowest at their luminal ends and are frequently widest at their distal regions, though the widest part at any time probably depends on the location of the nucleus; like the nuclei of other epithelial

FIGURES 14-17 Fig. 14. SEM montage of a stage-15 embryo, after removal of the ectoderm. Note the collagen fibrils which run from the somite (s) to the neural tube («) as well as to the lateral plate mesoderm (Ip). nc, ; wd, Wolffian duct, x 780. Fig. 15. SEM micrograph of part of a stage-11 embryo, whose neural tube has been removed to expose the medial surface of two somites. Many fibrils are present running from the somites to the ectoderm (ect), and others run from one somite to the next, x 780. Fig. 16. SEM micrograph of longitudinally cut somites from a stage-13 embryo showing that each somite is encapsulated by a meshwork of fibrils, x 1300. Fig. 17. SEM micrograph showing somite cells and fibrils making contact with the basal lamina of the overlying ectoderm (ect) in a stage-14 embryo, x 1300. R. BELLAIRS Somite segmentation in the chick embryo 237 cells, those of the somites undergo inter-kinetic migration, dividing at the luminal surface (Langman & Nelson, 1968). The general shape of a newly formed somite at stage 12 is such that it fits closely against adjacent tissues (Fig. 5). It is concave on its inner edge where it lies against the neural tube, and there is also an indentation where it is associated with the . The concavity on the inner edge can be seen even in the unsegmented mesoderm (Fig. 12). Figure 18 is an SEM micrograph of an anterior somite of a stage-14 embryo. The dorsal wall of the somite has become the , the cells being arranged in a columnar epithelium. Each cell extends the full thickness of the dermatome and is in especially close contact with its neighbours on the inner, apical, wall. Transmission electron micrographs show that these contacts are primarily desmosomes. The small intercellular spaces visible in the scanning electron micrographs have probably been exaggerated during preparation. Rounded cells are often present at the apical edge of the dermatome and these are considered to be undergoing mitosis. Transmission electron micrographs show that micro- filaments are present in these cells. The section shown in Fig. 18 passes through the lateral part of the somite and so the myotome is not included in it. The myotome appears first along the dorso- medial border of the somite. The free edge of the myotome extends progressively further and further toward the lateral edge of the dermatome until it eventually extends the whole way along its inner margin. The myocoele becomes obliterated, thus allowing close contact between the two layers of dermo-myotomic plate. The myotome cells resemble the dermatome cells in being elongated, but they lie at right angles to the dermatome cells so that the two tissues are orientated at right angles to one another. The myotome cells thus run along the long axis of

FIGURES 18-22 Fig. 18. SEM micrograph of part of a stage-14 embryo which has been broken across a somite. Because of the angle of the section very little of the myotome is visible. cl, Dermatome; in, myotome; scl, sclerotome. x650. Fig. 19. SEM micrograph to show the dorso-medial wall of a somite from a stage-17 embryo. The myotome cells (m) lie at right angles to the dermatome (d) x 910. Fig. 20. Higher power view of dermatome and myotome from a stage-17 embryo. The myotome cells appear to be granular because the break has passed through their cytoplasm, whilst the dermatome cells are smoother because they have not been broken, x 3250. Fig. 21. SEM micrograph to show the lateral part of a differentiating sclerotome of a stage-12 embryo. The dermatome (d) is a simple columnar epithelium, whilst the sclerotome (sc) is mesenchymatous. The ectoderm has been removed and a mass of fibrils lies on the surface of the dermatome. x 910. Fig. 22. TEM micrograph to show the junction between two somites of a stage-14 embryo. Many collagen fibrils (c) lie between the two somites, x 4500. 238 R. BELLAIRS the body in an antero-posterior direction, whilst the dermatome cells lie perpendicular to it. Figures 19 and 20 show the broken edges of myotome cells which have been fractured across their long axes to reveal granular cytoplasmic contents. The myotome cells have extensive areas of contact with both the dermatome and the sclerotome. Many of the contacts are fine ones and probably consist of extracellular materials, but others are more substantial and therefore probably cytoplasmic extensions. These deductions are supported by TEM studies which have shown that the myotomes have many cellular contacts with both the dermatome and sclerotome. The sclerotome is much larger than either the dermatome or myotome (Fig. 21). It consists of mesenchyme cells with irregular shapes and orientations, many cellular contacts and larger intercellular spaces than exist in the derma- tome or myotome. Each somite is in close contact with the lateral plate mesoderm prior to and soon after segmentation (Fig. 5). The cells of the lateral plate are flatter and more irregularly shaped than those of the early somites. Figure 8 shows the view from the dorsal surface. Like the segmental plate, the lateral plate mesoderm has many collagen fibrils associated with it.

DISCUSSION The first point for discussion is the role of the extracellular materials in the process of segmentation. The SEMs show that two types or extracellular materials are present between the segmental plate mesoderm and the overlying ectoderm. The more conspicuous component is the meshwork of fibrils which are also present in the region of the formed somites and of the lateral plate mesoderm. These fibrils are visible by TEM and appear to consist of collagen (Cohen & Hay, 1971; Hay, 1973). According to Hay (1973), they are secreted by the neural and other epithelial tissues; indeed there is biochemical evidence that collagen begins to be laid down by the as early as stage 4 (Trelstad, Hay & Revel, 1974). We have seen that once a somite is segmented, a meshwork of fibrils becomes wrapped around it. A similar observation was made by Cohen & Hay (1971), and Hay (1973), using transmission electron microscopy. Strands from this meshwork pass from the somite to the neural tissue, as Lipton & Jacobson (1974a), have also shown. A significant new finding however in the present investigation, is that collagen fibrils also pass from the somite to the ectoderm and to the endoderm and to the aorta. Eventually, some even extend from the somite to the lateral plate. Lipton & Jacobson (19746) suggested that the function of the collagen fibrils was to 'stabilize' the somites once they had formed, by anchoring them to the notochord; if they were not anchored in this way they tended to regress Somite segmentation in the chick embryo 239 because the cells migrated away from the somite. Support for this idea may be found in the fact that when somites are isolated and explanted in vitro, the cells migrate away from them (Bellairs & Portch, 1977). It seems likely however that the role of the collagen fibrils is even more fundamental and that they play a part in the segmentation process itself. The argument, which has several steps, is as follows: (a) Segmentation is related to a change in cell adhesiveness. In a recent paper, Bellairs et al. (1978), showed that the cell to cell adhesiveness was significantly higher in cells from newly formed somites than in those from segmental plate mesoderm. They suggested that this increase of adhesiveness did not take place uniformally over the surface of each cell but was localized largely at one end, the result being that clusters of cells aggregated together at their regions of high adhesiveness, and so formed a clump of cells, a somite. At the same time the cells separated from one another at regions of low adhesiveness so that the developing somites became separated from one another by furrows and also became marked off from the adjacent tissues. (b) Segmentation is associated with an elongation of the cells. Two morpho- logical facts may be expected to follow from such a change in adhesiveness. First, the number of cells which can adhere together in this way depends partly on the shape of the cells. Thus, if each cell is a sphere then only a few cells can join together, but if each cell is spindle-shaped, then the number in each somite can be increased. In the present investigation it has been shown that segmentation is indeed accompanied by such a change in cell shape. Furthermore, the small lumen which appears in the early stages of formation of each somite permits a further increase in the numbers of cells which may be assembled. The second morphological fact is that if all the cells adhere medially during segmentation, each newly formed somite is essentially two cells only in antero- posterior length. (We ignore the small cell population of the lumen.) Such an arrangement is clearly visible in Figs. 4 and 5. An accompaniment of this cell change is the appearance of desmosomes at the centre of each somite. These have been described by others (e.g. Hay, 1968, Lipton & Jacobson, 1974a) and have therefore not been illustrated here. It has been suggested that these desmosomes supplement the adhesiveness of the cell membranes in that region and help the cells to remain attached (Bellairs et al., 1978). (c) The elongated cells are under tension. We may now continue our argument by enquiring as to the factors that promote elongation of the cells during seg- mentation. A clue may be obtained by considering the changes in shape that take place in and other cells when migrating in vitro. When these cells are in their elongated phase they are attached to the substrate by each end and are under considerable tension. This can be seen by the way they retract to a rounded shape if one end is released. In the same way, each somite cell appears to be under tension and its shape appears to be related to the degree of this tension. 240 R. BELLAIRS Thus, in Fig. 5, those somite cells which are extended out to the neural folds are long and thin and are therefore probably under greater tension than those which lie adjacent to the medial part of the neural tube and which are shorter and fatter. Similarly, if a somite is dissociated with trypsin treatment, the individual cells become rounded (Bellairs et al., 1978). Again, cells in culture which are under tension possess long microtubules (Abercrombie, Dunn & Heath, 1977) and in the same way, the extended somite cells are rich in microtubules. Further­ more, fibroblast cells which are extended in culture usually possess bundles of microfilaments; there is evidence that these are composed largely of actin (Abercrombie et al, 1977; Vasiliev & Gelfand, 1977). By contrast these bundles disappear in fibroblast cells which are not under tension. Similar bundles of microfilaments are a characteristic of segmented but not of unsegmented mesoderm. (d) Collagen fibrils help to produce the tension. Like a fibroblast, a somite cell must be anchored at each end if it is to achieve this tension. We have seen that it is anchored proximally by a relatively high degree of cell to cell adhesion which is supplemented by desmosomes. It is now suggested that the distal end is anchored by the collagen fibrils which extend from the somite in all directions, not just to the neural tube and notochord, and which act like guy ropes. It seems likely therefore that the tension exerted on the somite cells by the collagen fibrils has the following effects: (e) Collagen affects the formation, size and shape of each somite, (i) It plays an essential role in the formation of each somite. Thus, if there is no tension the cells remain spherical and stick together in small clusters of three or four, and no somite is able to form; (ii) it has the 'stabilizing' action described by Lipton & Jacobson (1974«) which is necessary to keep the somite from dissociating; and (iii) it affects the size and shape of each somite. Thus the greater the tension exerted, the greater the number of cells which may be accommodated in each somite, and hence the bigger the somite. Again, the tension on the cells may change during development and so the shape of the somite changes, thus the first few pairs of somites which lie beneath the neural plate are wide and rectangular, but as the neural folds rise up these somites acquire 'the cuboidal configuration typical of the later somites' (Lipton & Jaconbson, 1974a). It seems most probable that as the neural folds rise up they drag on the collagen fibrils thus increasing the tension on certain cells. In this way the entire somite adapts its shape to the new space available to it. More collagen fibrils run from the somites to the neural tube and notochord than to any other tissue. This is of significance because many authors have suggested that the neural tube and/or notochord are responsible for inducing the somites (Waddington, 1935; Fraser, 1960; Butros, 1967; Nicolet, 1971; Lipton & Jacobson, 19746), though this concept has been shown to be improbable by others who have demonstrated that development of somites can occur in the absence of the neural and notochordal tissue (Bellairs, 1963; Christ, 1970; Somite segmentation in the chick embryo 241 Menkes & Sandor, 1977). It appears therefore that whatever the role of the neural tube, it can be played instead by other tissues. Christ, Jacob & Jacob (1972), showed that presumptive somite mesoderm would develop after removal of the neural tissue only if ectoderm, endoderm or the aortic arch were present, and they concluded that the presence of an epithelium was necessary though its action was not an inductive one. In the present investigation, it has been shown that the somites are connected by collagen fibrils to these epithelia as well as to the neural plate and notochord. It is suggested therefore that the segmental plate must be in contact with some epithelial tissues that can produce collagen fibrils, if it is to form somites. Sometimes in explants lacking neural tissue the somites that form are ab­ normal in morphology, and consist of thin walls surrounding an unusually large lumen (Packard & Jacobson, 1976). Here it would seem that the pull exerted by the collagen fibrils exceeds the adhesiveness of the proximal ends of the cells so that the somite has become distorted. Under normal conditions the two would probably be in balance with one another. It is possible therefore that the neural tissue normally also plays some role in maintaining the high adhesiveness of the somite cells. The second type of extracellular material is the amorphous type illustrated in Fig. 13 which is especially conspicuous on the dorsal aspect of the mesoderm. This material has not been analysed in this investigation, but probably includes glycosaminoglycans since these are found in the extracellular spaces at this time. Shur & Roth (1973), have demonstrated that galactosyl transferase has a high concentration in the extracellular materials of the segmental plate mesoderm but is low in the region of the somites. These authors have suggested that it may be an important factor in causing cells to migrate as well as to recognize and communicate with other cells. It seems possible therefore that this extracellular material may play a role in preparing the mesoderm cells for the segmentation process, though we do not know the nature of this preparation. One possibility is that the amorphorous material is responsible for causing the mesoderm cells to aggregate loosely together in the segmental plate, and for maintaining them together as a block of tissue ready for segmentation. Another possibility is that the amorphous material may initiate some more fundamental change in the cells so that they become programmed for segmentation. Indeed there is evidence that the presumptive somite mesoderm becomes determined to form somites at about the time it becomes arranged as segmental plate (Packard & Jacobson, 1976; Packard, 1978). This determination is not however one that fixes the precise positions of the individual somites and follows, since consider­ able regulation of the tissue is possible even after it has been stirred with a needle (Menkes & Sandor, 1977). The second and final point for discussion concerns the rearrangement of the cells to form the dermatome, myotome and sclerotome. In a recent experimental analysis, Christ, Jacob & Jacob (1978) showed that the 242 R. BELLAIRS dermatome gave rise to the myotome. It is of interest therefore that in the present investigation the myotome cells were found to be orientated at right angles to the dermatome cells. This is reminiscent of the situation in Xenopus, although there the dermatome cells remain unsegmented and the myotome cells rotate at the time of segmentation and come to lie parallel to the notochord (Hamilton, 1969). Thus, the chick resembles Xenopus in that it is only the myo- tome that rotates, but the two species differ in that whereas rotation occurs after segmentation in the chick, it coincides with it in Xenopus.

I wish to express my gratitude to Dr Mary Bancroft for her kindness in providing me with some of the scanning electron micrographs, and to Miss E. Maconochie who helped me to prepare the remainder. I wish to thank Dr Alan Boyde for the use of his scanning electron microscope facilities which were partly provided by the Medical Research Council. I am also most grateful to Miss Doreen Bailey for her skilled technical assistance, and to Mrs J. Astafiev for drawing Fig. 1. Funds towards supporting the work were generously provided by the University of Kuwait.

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(Received 4 December 1978)