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J. Embryol. exp. Morph., Vol. 15, 3, pp. 317-330, June 1966 With 3 plates Printed in Great Britain 317 Ultrastructure of the blastopore cells in the newt By MARGARET M. PERRY 1 & C. H. WADDINGTON 1 From the Institute of Animal Genetics, Edinburgh INTRODUCTION Invagination of the mesoderm through the blastopore in the amphibian embryo is one of the most impressive examples of the massive movement of a coherent sheet of cells from one region to another in a developing organism. The mechanisms by which the movement might be brought about have been widely discussed. Broadly speaking, three general types of active agent have been invoked: (1) relations between neighbouring cells of a kind comparable to differences in surface tension (Holtfreter, 19436, 1944); (2) more specific chemical affinities between neighbouring cells (Weiss, 1950); (3) the occurrence of intra-cellular fibrils which bring about expansion, contraction, or both, at different times (Waddington, 1940). Most authors have opted for some combination of one, two or even all three of these factors. The most important points in the older literature have been summarized by Waddington (1956, pp. 437 ff.). All this older work was, of course, based on evidence derived from examination with the light-microscope. This is not capable of resolving the fine structures which we now know to be so plentifully present in the cytoplasm of all cells. Even studies with polarized light (Waddington, 1940), although they show that certain regions of some of the invaginating cells exhibit double birefringence, cannot reveal the nature of the structural organization on which this depends. A completely new insight is, of course, made possible by the electron microscope. Surprisingly enough, not many studies with this instrument have been made on the gastrulating cells of amphibia. Balinsky (1961) has published a relatively short note describing the phenomena in two species of South African frogs, and while this work was in preparation Baker (1965) has given a longer account of the phenomena in another anuran, the Pacific tree frog, Hyla regilla. Both these authors used only osmic fixatives, and both seem to have confined their attention to sections cut parallel to the longitudinal axis of the embryo. In the present work we have studied the urodele, Triturus alpestris, in which the major cellular transformation in the blastopore region, the appearance of 'flask cells', is more highly developed than in the anurans. We have used glutaraldehyde in combination with osmic acid as a fixative and have studied transverse as well as longitudinal sections. 1 Authors' address: Institute of Animal Genetics, West Mains Road, Edinburgh, 9, Scotland. 318 M. M. PERRY & C. H. WADDINGTON MATERIALS AND METHODS Fertilized eggs were collected from a laboratory stock of Triturus alpestris, and reared at room temperature until the onset of gastrulation. They were then decapsulated and fixed in 2-5% glutaraldehyde in 0-05 M phosphate buffer for 4 h, rinsed overnight in the buffer, and post-fixed in buffered osmium tetroxide for 2-3 h (Sabatini, Bensch & Barrnett, 1963). Pieces containing the blastopore region were excised from the embryos during glutaraldehyde fixation. The material was subsequently dehydrated in a graded series of alcohols, and embedded in Araldite. Sections of oriented blocks were cut on an L.K.B. Ultratome, mounted on carbon-formvar coated grids and stained with aqueous uranyl acetate, and lead citrate (Reynolds, 1963). They were examined with an A.E.I. EM6 electron microscope. DESCRIPTION OF RESULTS To assist in orientation, Holtfreter's well-known semidiagrammatic drawing of a newt gastrula is reproduced in Text-fig. 1. We shall distinguish three main regions of the flask-like cells which line the blastopore (Text-fig. 2): (1) the tips, which actually abut on to the external surface which will later become the cavity of the archenteron; (2) the necks, which can be subdivided into a more distal vesicular zone and a more proximal pigment zone; (3) the main body of the cell containing the nucleus, yolk platelets and large lipid droplets. The proximal ends of the flask cells lie against the other cells of the endoderm. The description to be given will deal only with fairly early stages of gastrulation, from the early appearance of the blastopore to the time when it has acquired a sickle shape; that is to say, before the archenteron cavity has become very deep, and slightly earlier than the stage illustrated (Text-fig. 1). The cell tips Throughout the whole of this period the tips of the cells are occupied by a zone of dense, granular material with relatively little structure. At low magnifications (Plate 1, fig. A) this granular zone appears to extend across the intercellular boundaries as a continuous sheet. However, closer examination reveals that intercellular junctions continue from the depths of the tissue right up to1 the surface. At the free surface the zone is thrown into many corrugations and microvilli. It is bounded by an asymmetrical, triple-layered membrane, 100 A wide, the outer component of which is denser and thicker than the inner and appears to be continuous across the surface at the intercellular junction, where it becomes indented (Plate 2, fig. C). It commonly bears a number of thin, hair-like projections which gives the surface a somewhat fuzzy appearance. The lateral plasma membranes in the region of the granular zone are symmetrical, triple-layered structures, 75 A in width. It is possible that the con- Newt blastopore cells 319 Text-fig. 1. Semi-diagrammatic section through an advanced urodele gastrula, to show flask cells lining the blastopore and archenteron (from Holtfreter, 19436). Text-fig. 2. Drawing from a montage of electron micrographs of a section of the cells lining the innermost extension of the blastoporal groove in the early gastrula of Triturus alpestris. The elongated cells may be divided into three regions: the tips containing granular material (GZ), the necks consisting of a vesicular zone (VZ) and a pigment zone (PZ); and proximally the main cell body. Note that the ovoid yolk platelets are mostly oriented in the direction of the long axis of the cells. Y, yolk platelet; L, lipid droplet; P, pigment granule; N, nucleus. 320 M. M. PERRY & C. H. WADDINGTON tinuous dense layer with its attached fibrillar material represents an extra cellular 'coat' which is closely amalgamated to, or superimposed on, the outer component of the plasma membrane (Text-fig. 3). From its dimensions and the general paucity of definite structure elements, it is unlikely that this continuous layer corresponds to the elastic 'surface coat', believed by Holtfreter (1943a) to surround the exterior of the embryo. It may be of similar composition to the mucopolysaccharide surface coat which Bell (1960) demonstrated in embryos of Ranapipiens. Admittedly, the dimensions of the layer are such that it is unlikely to be detected by the methods used by Bell. On the other hand it is probable 75 A 75 A Text-fig. 3. Diagram of part of the electron micrograph shown in Plate 2, fig. C, to illustrate the relative width of the plasma membranes at the cell tips and the continuous external layer. The outer component of the plasma membrane is represented by a dotted line where it approaches the free surface, as its precise location in this region is not clear. GZ, granular zone; //, intercellular junction; EL, external layer; FM, fibrillar material. PLATE 1 Fig. A. Longitudinal section of the necks of the blastopore cells. At the free surface the granular zone (GZ) is thrown into folds. At a deeper level are the vesicular (VZ) and pigment zones (PZ). Cytoplasmic fibrils (Fb) and longitudinal flanges (FT) containing granular material are evident. Y, yolk platelet, x 5000. Fig. B. Transverse section of the necks of the blastopore cells showing the complex interdigitations of the longitudinal flanges (Fl). x 5000. J. Embryo!, exp. Morph., Vol. 15, Part 3 PLATE 1 M. M. PERRY & C. H. WADDINGTON facing p. 320 J. Embryo/, exp. Morph., Vol. 15, Part 3 M. M. PERRY & C. H. WADD1NGT0N PLATE 2 facing p. 321 Newt blastopore cells 321 that during the preparative procedures some of this extracellular material is removed and that the fuzzy material merely represents the remnants of a much thicker layer. Within the thickness of the accumulation of electron-dense material at the tips of the cell it is quite common to find odd pieces of apparently isolated double membranous material which are certainly not continuous with the general intercellular boundary, although they could be elements of engulfed surface membrane. Balinsky (1961) has described large vacuoles in this region, formed, he suggests, by the pinching off and engulfing of the bottoms of the cavities between the microvillus projections at the cell surface. However, this vacuolization occurs at a later stage, in the mid-gastrula, than that examined in Triturus, where the process of pinocytosis is probably only beginning. The appearances suggest that the whole of this dense material consists of molecular species which can relatively simply become arranged into a laminar form, when it gives the appearance of membranes. This seems not unreasonable since it is possible that the substance is in fact the accumulation, in this region of the cell, of material that originally spread out over a much more extensive surface. If this is so, not too much importance can be attached to the appearance of odd pieces of membranous structure within it. The lateral plasma membranes of the cell tips are separated by a gap, varying in width between 100 and 200 A, which contains material of low density (Plate 2, fig. C). As the intercellular space at a deeper level is considerably wider, these distal cell junctions are probably areas of attachment, of a simple type. The necks n x T/ . ; (1) Vesicular zone Immediately proximal to the superficial, granular zone just described is a region in which the cytoplasm is packed full of vesicles, which are embedded in a granular matrix similar to that in the cell tips. The depth of this zone depends on the extent of elongation of the cells; the more attenuated the necks, the longer is the vesicular zone, and the more closely packed the vesicles, which appear hollow in these preparations and are delimited by a single membrane (Plate 2, fig. D). PLATE 2 Fig. C. Longitudinal section of the distal tips of two adjacent cells. A dense external layer (EL) with attached fibrillar material is continuous across the intercellular junction (//). Here the intercellular space is narrow and contains material of low density, while more proximally the space becomes wider. The cells are bounded by triple-layered membranes. Glycogen granules (Gl), granular zone (GZ). x 100000. Fig. D. Longitudinal section of the vesicular zone. Note the microtubules (M) which traverse areas of dense, granular cytoplasm, and the electron-transparent alpha vesicles (A), x 32000. Fig. E. Transverse section of the vesicular zone, to show cross-sections of microtubules (M) and beta vesicles (B). x 48000. 322 M. M. PERRY & C. H. WADDINGTON Intermingled with the vesicles, and sometimes occurring in large numbers at a deeper level, are irregularly shaped cytoplasmic organelles (Plate 3, fig. F). They are composed of a peripheral ring of relatively dense granular material, which is bounded internally and externally by well-defined triple-layered membranes, and which encloses an area containing traces of material similar to that in the surrounding cytoplasm (Plate 3, fig. H). Occasionally, simple rod-shaped bodies with similar dense granular contents are seen (Plate 2, fig. E). It is likely that these simple and the more complex bodies are interrelated. Still other organelles, which are found predominantly at a slightly later stage of invagination than those already described, are the polyvesicular bodies (Plate 3, fig. G). These contain traces of fibrillar material dispersed around the internal vesicles, and the triple-layered nature of their limiting membranes is ill-defined. The three types of organelles should perhaps be given distinctive names. We have called them the alpha vesicles, appearing hollow with single membranes; the beta vesicles, sometimes multiple, with dense contents bounded by triplelayered membranes; and the gamma vesicles, which are polyvesicular. The gamma vesicles could perhaps have been derived from the beta vesicles, which are smaller in area, by, for instance, the granular regions becoming hydrated and swelling up. Small vesicles bounded by a single membrane are a common feature of the cytoplasm in all the cells of the early amphibian embryo. In the cells of regions other than the blastopore they are scattered rather thinly throughout the bulk of the cytoplasm, and never appear in such concentrated masses as they do in the necks of the blastopore cells. However, it seems quite likely that their high concentration in these cell necks is due more to the concentration of all the vacuoles of this order of size in that part of the cell than to a new formation of them. The blastopore cells have in general a zonation of contents according to size. The hollow vesicles are fairly small and lie at the distal end of the neck; they are succeeded by a zone with a very high concentration of pigment granules, which have two or three times the diameter of the alpha vesicles; and still further proximally there is a concentration of yolk granules which are much larger again. However, although this is the simplest hypothesis to account for the presence of these vesicles, it cannot be excluded that they are derived in some PLATE 3 Fig. F. Vesicular-pigment zone. Numerous organelles, here termed 'beta vesicles' (B), have dense, granular contents surrounding an internal cavity. Also evident are a stack of annulate lamellae (AL), composite pigment granules (P) and contorted pieces of membranous material (Me), x 25000. Fig. G. Vesicular-pigment zone, at a later stage of gastrulation than fig. F, to show the polyvesicular bodies, the gamma vesicles (G). x 24000. Fig. H. Higher magnification of the beta vesicles (B). Triple-layered membranes delimit the dense, granular material from the internal cavities and the surrounding cytoplasm, x 100000. /. Embryol. exp. Morph., Vol. 15, Part 3 i v PLATE 3 '. !V*''i' M. M. PERRY & C. H. WADDINGTON facing p. 322 Newt blastopore cells 323 way from the more complex beta and gamma vesicles, which contain electrondense material and which are, at least originally, bounded by double membranes. Scattered amongst the vesicles of this zone, and, in fact, throughout the whole of the cell, are a large number of dense granules, which from their size (300400 A), their affinity for the lead stain, and their particulate substructure, may be identified as glycogen (Revel, 1964). The glycogen granules are frequently seen in the intercellular spaces, and in spaces where, for instance, the cytoplasm has contracted away from a yolk platelet. It seems most probable that under some conditions of fixation these granules may be shifted in location within the tissue, and little can be safely inferred from their distribution. Another type of structure which is sometimes encountered within the cytoplasm of the vesicular zone is a stack of annulate lamellae (Plate 3, fig. F). Structures of this kind are, of course, well known in oocytes of many groups of animals. They are rare in adult cells, but have been seen in young cells actively engaged in growth (Kessel, 1965). Owing to the resemblance in the pattern of annuli to that seen on the nuclear envelope, they are often considered to be derived from that structure (Swift, 1956). We have seen them in other amphibian embryonic cells (mesenchyme derived from the endo-mesoderm) in the immediate neighbourhood of the nuclear envelope (Waddington & Perry, 1966 a). In the flask cells, however, they lie at a considerable distance from the nuclei, as they do in Drosophila oocytes (Okada & Waddington, 1959), and if originally derived from them must have persisted for a considerable time since their origin. In these blastopore cells they are usually surrounded by rather electron-dense material and it therefore seems rather probable that they are engaged in some form of synthesis, although nothing definite is known of their function. The last constituents of the cytoplasm in the neck zone to require mention are the microtubules. In longitudinal sections of the cells the tubules are oriented rather strictly in line with the main axes (Plate 2, fig. D). In transverse sections they appear as hollow circular profiles, about 300 A in diameter (Plate 2, fig. E). They are especially common in the neck regions, where they are particularly associated with long strands of granular cytoplasm. The fibrils which are visible in low-power micrographs (Plate 1, fig. A) can be resolved into these microtubular and granular cytoplasmic components. Lengths of microtubules of up to 8 fi may be seen in a single section, which implies that they take an undeviating course, in spite of the presence around them of large numbers of vesicles. It appears as though the microtubules must, as it were, elbow the vesicles out of the way during their growth. At early stages in blastopore formation, that is, when the cells are cuboidal in shape, scattered cross-sections of microtubules are found throughout the cytoplasm in longitudinal sections of the cells. The overall shape of the neck regions of these cells is very interesting and at first sight unexpected. Longitudinal sections, it is true, reveal little more than was known already, namely that the cells are exceedingly long drawn out and 324 M. M. PERRY & C. H. WADDINGTON the necks very narrow and gradually tapering. One notices, however, the presence of some spaces between the cells, which are not in close contact all along their length. Within these spaces there are sometimes protrusions containing granular cytoplasm. The nature of these outgrowths is much better revealed in transverse sections (Plate 1, fig. B). These show that, in the neck region, the neighbouring cells are in general separated by inter-cellular spaces, but are closely involved with one another by the development of what appear to be longitudinal flanges, which become wrapped around one another, forming a set of longitudinal folds. They might be likened to a pile of umbrellas lying in parallel orientation with the coverings lowered, but not rolled around their own supporting axes, but instead allowed to become crumpled together with the covering of the neighbouring umbrellas. Such mutual involvement of the cells would presumably tie them together into a rather firm longitudinal bundle, from which it would be difficult to untangle the whole length of any single cell. The granular cytoplasm in these tangled regions is very similar to that in the tip of the cell. The granular material within the tips can often be seen to extend proximally to fill the tangled lateral flanges. In general the flanges are more extensive near the distal and thinner ends of the necks and become progressively reduced as one proceeds proximally into thicker regions of the cell body. (2) The pigment zone In the more distal regions of the neck the most striking components of the cytoplasm are the various types of vesicles described above, and there are very few pigment granules. Slightly further proximally there is a region in which pigment granules are extremely frequent. They show the usual composite structure common in Triturus (Plate 3, fig. F). A few mitochondria are evident, and are somewhat more numerous than in the vesicular zone. The other components of the cytoplasm remain much as they were, although the concentration of beta and gamma vesicles is perhaps lower, and the longitudinal flanges become progressively reduced in dimensions. There is little sign of free ribosomes and no apparent endoplasmic reticulum in any of the zones described. (3) The main body of the cell More proximally still one comes to the bulbous region of the cell in which the nucleus is located. This region also contains the yolk platelets, and large droplets of lipid, which usually appear rather pale in these preparations. The yolk platelets may extend some distance into the wider parts of the necks of the cells, and in that case any platelets which are markedly ovoid in configuration are oriented with the long axes parallel to the length of the cell. There is, however, no particular orientation of the yolk platelets in the main body of the cells. We have not studied this region of the cell in any particular detail, since it does not appear to present any features of particular interest. It is, however, worth Newt blastopore cells 325 mentioning that in the cells on the floor of the developing archenteron—that is, in the ventral part of the region which is drawn out into flask cells—one can sometimes find a very peculiar type of solubilization of the yolk, involving the formation of arrays of tubular structures. These will be described elsewhere (Perry, 1966). DISCUSSION The observations recorded here throw considerable light on the processes by which the invagination of the blastopore is brought about in these embryos. In the first place, it is clear, as Balinsky (1961) has already pointed out, that the external surface of the cells is not engaged in active contraction and cannot be providing the main motive force for the original invagination. There is nothing corresponding to the 'surface coat' to which Holtfreter (1943a) has attributed so much importance and which he supposes to form a continuous elastic membrane-like structure spanning across cell boundaries. The dense layer on the external plasma membrane of the cell, for which there are indications of some continuity, can certainly not be playing the part of Holtfreter's coat, and the main bulk of the dense granular material in the tips of the cells is, as we have seen, interrupted by intercellular boundaries and is thrown into numerous folds, suggesting that it has itself been forced to contract rather than that it exerts a contractile force of its own. If the drawing together of the cells of the blastoporal groove is not due to a contraction by the material of the external tips of the cells, an explanation for it must be sought somewhere else. Balinsky draws attention to the presence, in his electron micrographs of the early neural groove, of an electron-dense layer lying some distance below the cell surface, which he interpreted as a contractile element. We have also seen a dense layer in cells of the neural groove, but we will leave discussion of it to a later occasion (Waddington & Perry, 19666), since Balinsky admits that no such structure can be found in the region of the blastopore. He therefore attributes the changes in shape of the blastoporal cells to an active elongation within the cytoplasm. Baker (1965) considers that the shape of the cells is changed by alternate expansion and contraction in a peripheral layer of dense cytoplasm, which at the period of maximum elongation occupies the entire neck region. However, she does not seem to have studied transverse sections of this tissue, and did not realize the nature of the longitudinal flanges containing granular material, which extend along the necks of the cells. If these were engaged in active elongation it is difficult to believe that in transverse section they would present the appearance of loose flaccid bundles, as they do for instance in Plate 1, fig. B. One would rather expect to find solid ridges with a relatively simple external surface, but theflangesin fact offer exactly the same evidence of a lack of contractility as does the similar substance at the external surface of the cells. In our opinion the appearances of the granular zone and of theflangessuggest 21 J E E M 15 326 M. M. PERRY & C. H. WADDINGTON that the blastopore cells are being caused to become elongated by some process occurring within their internal cytoplasm. This elongation will result in a reduction in the area of the external surface of the cells, and a narrowing of the regions which become the neck. If the material originally constituting the cortical cytoplasm in these regions is not able to move away rapidly enough, it is bound to become accumulated. Both the electron-dense material in the cell tips and the similar material forming the longitudinal flanges in the neck region can most easily be interpreted as accumulations of substances which originally formed the cell surface. The indications, which have already been mentioned, that this material seems easily to form membranous structures on fixation is in full accordance with this. On this interpretation, this material is not likely to be exerting any particular forces assisting the process of gastrulation, though one imagines that the flanges serve to hold the necks of the cells together laterally. The key factor in the invagination process would therefore seem to be the development of a tendency to elongation within the internal cytoplasm. The presence of very many microtubules within the cytoplasm, which has been revealed in this investigation, provides a plausible mechanism. The possibility that cytoplasmic fibrils might play a role in such processes is one of the old speculative hypotheses in this field. They were, however, not visible with the light-microscope. An attempt was made to detect their presence by examining the orientation of the ovoid yolk platelets, since it was argued that any fibrils powerful enough to play a part in cell elongation might be expected to cause the yolk platelets to take up a preferred orientation (Waddington, 1942). However, it was found then that the yolk platelets only became regularly oriented in parts of the cell which were so narrow that there was no room for them to lie in any other direction. The orientation might therefore have been imposed by the external cell membranes rather than by internal fibrils and no definite evidence for the existence of such fibrils could be discovered. This observation on the orientation of the yolk platelets has been confirmed in the present investigation, but as we have seen, the necks of these cells contain very large numbers of microtubules, often associated in groups, embedded in strands of granular cytoplasm, which at low magnifications appear as fibrils. These may in fact succeed in orienting the small empty-looking vesicles in the way in which it was earlier thought more coarse fibrils might act on the yolk granules. Within the necks of the cells the microtubules are oriented rather accurately in the direction of the cellular long axis. They are therefore not only present in large numbers but in the right orientation to function as the agents of internal elongation, producing the effects which Balinsky thought must be present, but for which he could identify no particular agents. Microtubular structures have been reported in many cells (Slautterback, 1963), and in asymmetrical metazoan cells, where they take up a preferred orientation, they are thought to be implicated in the formation and maintenance of these asymmetries (Porter, Ledbetter & Badenhausen, 1964). For instance, Byers & Porter (1964) have described the Newt blastopore cells 327 development of a microtubular system in cells of the chick lens rudiment which coincides precisely with the period of cellular elongation. They suggest a possible mechanism whereby cytoplasmic movement along an array of microtubules, similar to the streaming in plant cells, produces elongation by gradual cytoplasmic translocation, analogous to the translational motion of actin with respect to myosin in myofibrils. As the microtubules in the necks appear to be particularly associated with a granular cytoplasm, a similar process could be involved here. On the other hand Wolpert(1965) has suggested that microtubules or micro-fibrils may be actively contractile by some mechanism of the sliding filament type. He points out that quite small numbers of them could provide the forces required. In early stages of blastopore formation microtubules can be found, as we have seen, running in the plane tangential to the surface so that they are cut transversely in sections which are longitudinal as regards the whole embryo. They are, however, by no means so numerous as those found in the cell necks, and whether these microtubules play a role in bringing about the initial formation of the blastopore pit is not clear. Further, it should be remarked that microtubules can be found in almost all cells of the early newt embryo (Waddington & Perry, unpublished). They are often most frequent near and parallel to intercellular boundaries but are sometimes found running in rather haphazard directions within the general body of the cytoplasm. In cells not engaged in very active change of shape, such as those of the animal hemisphere of the early gastrula, the microtubules are never in as high concentration as in the cell necks of the blastopore cells. It is perhaps only when we see them in very large concentrations and in very regular orientation that, it is safe at present to attribute any important morphogenetic action to them. Although the microtubules are present in considerable numbers in the necks of the cells it is improbable that they play an important role in producing the birefringence which these cells show (Waddington, 1940). The large number of more or less orientated vesicles and the longitudinal cell flanges would provide the basis for a 'form birefringence' and it seems most likely that this was the phenomenon detected. While the alpha vesicles are not unlike those found in most other cells of the newt embryo, the beta and gamma vesicles are certainly peculiar, and it is to be expected that they have precise and definite functions. Vesicles of a generally similar character to these have often been referred to as lysosomes, and either inferred or shown to contain active enzymes (Novikoff, 1961). In some cases it has been reported that they originate in the Golgi region (Moe, Rostgaard & Benke, 1965). Nothing is yet known about any possible enzyme activity of the granules in these blastopore cells although investigation of this problem is proceeding. It is perhaps noteworthy that these cells do not contain any identifiable Golgi material in the neck region where the beta and gamma vesicles are so frequent. Golgi material is in fact very sparse in these cells, though a few patches of it 328 M. M. PERRY & C. H. WADDINGTON have been seen in the main body of the cell in the neighbourhood of the nucleus and yolk granules. It seems most improbable however that the beta and gamma vesicles are at all closely connected with Golgi and their relation to lysosomes in other forms remains uncertain. In this connexion Holtfreter (19436) has suggested that the blastopore cells are relatively short-lived, and degenerate in the larval period. It would not therefore be unexpected to find evidence of lysosomal particles within them. SUMMARY 1. Longitudinal and transverse sections of the flask-shaped blastopore cells in the early gastrula of Triturus alpestris have been examined in the electron microscope. 2. The cytoplasm may be divided into three main regions; a distal, superficial, granular zone; a vesicular and, more proximally, a pigment zone in the necks; and the main body of the cell with nucleus, yolk platelets and lipid droplets. A thin, continuous dense layer of extracellular material covers the cells at the free surface. 3. Numerous microtubules oriented parallel to the main axes are found grouped in the necks. Other prominent organelles are vesicles, which from their structure have been divided into three categories, the alpha, beta and gamma vesicles. Longitudinal flanges extending along the necks are complexly interwound with those of adjacent cells and bind the cellsfirmlytogether in this region. 4. Neither the extracellular layer, nor the superficial granular layer, is considered to exert a contractile force which would cause the cells to become elongated; rather the change in shape is brought about by active elongation within the internal cytoplasm, in which the microtubular system plays an important part. 5. The material in the cell tips probably results from the passive accumulations of substances which originally covered a larger surface area. 6. The alpha vesicles are common to all cells in the embryo, whereas the beta and gamma vesicles are characteristic of the blastopore cells. The function of these organelles is at present unknown, although there are some structural similarities between the beta vesicles and lysosomal particles described in other organisms. RESUME Ultrastructure des cellules blastoporales chez le Triton 1. Des coupes longitudinales et transversales des cellules en bouteille du blastopore de la jeune gastrula de Triturus alpestris, ont ete examinees au microscope electronique. 2. Le cytoplasme peut etre subdivise en trois regions principales; une zone distale, superficielle et granulaire; une zone vesiculaire, et, a un niveau plus proximal, pigmentaire dans les * cols' des bouteilles; ainsi que le corps principal Newt blastopore cells 329 de la cellule avec son noyau, ses plaquettes vitellines et ses gouttelettes lipidiques. Une mince couche continue et dense de materiel extracellulaire recouvre les cellules au niveau de leur surface libre. 3. Dans les cols on a trouve de nombreux microtubules orientes parallelement au grand axe. Comme autre organites remarquables, il y a lieu de mentionner des vesicules qui par leur structure peuvent etre classees dans trois categories, les alpha, beta et gamma. Des franges longitudinales s'etendant le long des collets sont entrelacees de fagon complexe avec celles des autres cellules et les solidarisent solidement a ce niveau. 4. Ni la couche extracellulaire, ni la couche granulaire superficielle peuvent etre considerees comme exergant des forces contractiles pouvant provoquer l'elongation de cellules; le changement de forme est provoque par une elongation active du cytoplasme interne, processus dans lequel le systeme microtubulaire joue un role important. 5. Le materiel situe dans les extremites retrecies des cellules provient probablement d'une accumulation passive de substances qui a l'origine etaient distributes sur une surface plus etendue. 6. Les vesicules alpha sont communes a toutes les cellules de l'embryon, tandis que les vesicules beta et gamma sont caracteristiques des cellules blastoporales. La fonction de ces organites reste inconnue, toutefois il apparait certaines similitudes de structure entre les vesicules beta et les organites decrits comme lysosomes dans d'autres organismes. The authors wish to thank Mr E. D. Roberts for his skilful drawing of Text-figs. 2 and 3 REFERENCES BAKER, P. C. (1965). Fine structure and morphogenic movements in the gastrula of the tree-frog, Hyla Regilla. J. Cell Biol. 24, 95-116. BALINSKY, B. I. (1961). Ultrastructural mechanisms of gastrulation and neurulation. In Symposium on Germ Cells and Development (Pallanza, 1960), pp. 550-63. Institut Internationale d'Embryologie and Fondazione A. 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