Ultrastructure of the blastopore cells in the newt · Ultrastructure of the blastopore cells in the newt By MARGARET M. PERRY1 & C. H. WADDINGTON1 From the Institute of Animal Genetics,

J. Embryol. exp. Morph., Vol. 15, 3, pp. 317-330, June 1966 3 1 7With 3 plates

Printed in Great Britain

Ultrastructure of the blastopore cells in the newt

By MARGARET M. PERRY1 & C. H. WADDINGTON1

From the Institute of Animal Genetics, Edinburgh

INTRODUCTION

Invagination of the mesoderm through the blastopore in the amphibianembryo is one of the most impressive examples of the massive movement of acoherent sheet of cells from one region to another in a developing organism.The mechanisms by which the movement might be brought about have beenwidely discussed. Broadly speaking, three general types of active agent havebeen invoked: (1) relations between neighbouring cells of a kind comparableto differences in surface tension (Holtfreter, 19436, 1944); (2) more specificchemical affinities between neighbouring cells (Weiss, 1950); (3) the occurrenceof intra-cellular fibrils which bring about expansion, contraction, or both, atdifferent times (Waddington, 1940). Most authors have opted for some combina-tion of one, two or even all three of these factors. The most important points inthe older literature have been summarized by Waddington (1956, pp. 437 ff.).

All this older work was, of course, based on evidence derived from examina-tion with the light-microscope. This is not capable of resolving the fine structureswhich we now know to be so plentifully present in the cytoplasm of all cells.Even studies with polarized light (Waddington, 1940), although they show thatcertain 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 onthe gastrulating cells of amphibia. Balinsky (1961) has published a relativelyshort note describing the phenomena in two species of South African frogs, andwhile this work was in preparation Baker (1965) has given a longer account ofthe phenomena in another anuran, the Pacific tree frog, Hyla regilla. Both theseauthors used only osmic fixatives, and both seem to have confined their attentionto sections cut parallel to the longitudinal axis of the embryo. In the presentwork we have studied the urodele, Triturus alpestris, in which the major cellulartransformation in the blastopore region, the appearance of 'flask cells', is morehighly developed than in the anurans. We have used glutaraldehyde in combina-tion with osmic acid as a fixative and have studied transverse as well as longi-tudinal 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 thendecapsulated and fixed in 2-5% glutaraldehyde in 0-05 M phosphate bufferfor 4 h, rinsed overnight in the buffer, and post-fixed in buffered osmiumtetroxide for 2-3 h (Sabatini, Bensch & Barrnett, 1963). Pieces containing theblastopore region were excised from the embryos during glutaraldehyde fixation.The material was subsequently dehydrated in a graded series of alcohols, andembedded in Araldite. Sections of oriented blocks were cut on an L.K.B.Ultratome, mounted on carbon-formvar coated grids and stained with aqueousuranyl acetate, and lead citrate (Reynolds, 1963). They were examined with anA.E.I. EM6 electron microscope.

DESCRIPTION OF RESULTS

To assist in orientation, Holtfreter's well-known semidiagrammatic drawingof a newt gastrula is reproduced in Text-fig. 1. We shall distinguish three mainregions of the flask-like cells which line the blastopore (Text-fig. 2): (1) thetips, which actually abut on to the external surface which will later become thecavity of the archenteron; (2) the necks, which can be subdivided into a moredistal vesicular zone and a more proximal pigment zone; (3) the main body ofthe cell containing the nucleus, yolk platelets and large lipid droplets. Theproximal ends of the flask cells lie against the other cells of the endoderm. Thedescription 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 asickle 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 azone of dense, granular material with relatively little structure. At low magni-fications (Plate 1, fig. A) this granular zone appears to extend across the inter-cellular boundaries as a continuous sheet. However, closer examination revealsthat 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 andmicrovilli. It is bounded by an asymmetrical, triple-layered membrane, 100 Awide, the outer component of which is denser and thicker than the inner andappears to be continuous across the surface at the intercellular junction, whereit 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 sym-metrical, 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, toshow flask cells lining the blastopore and archenteron (from Holtfreter, 19436).

Text-fig. 2. Drawing from a montage of electron micrographs of a section of thecells lining the innermost extension of the blastoporal groove in the early gastrula ofTriturus alpestris. The elongated cells may be divided into three regions: the tipscontaining 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 ovoidyolk 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.


tinuous dense layer with its attached fibrillar material represents an extracellular 'coat' which is closely amalgamated to, or superimposed on, the outercomponent of the plasma membrane (Text-fig. 3). From its dimensions and thegeneral paucity of definite structure elements, it is unlikely that this continuouslayer corresponds to the elastic 'surface coat', believed by Holtfreter (1943a) tosurround the exterior of the embryo. It may be of similar composition to themucopolysaccharide surface coat which Bell (1960) demonstrated in embryos ofRanapipiens. Admittedly, the dimensions of the layer are such that it is unlikelyto 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, toillustrate the relative width of the plasma membranes at the cell tips and thecontinuous external layer. The outer component of the plasma membrane is repre-sented by a dotted line where it approaches the free surface, as its precise location inthis region is not clear. GZ, granular zone; // , intercellular junction; EL, externallayer; FM, fibrillar material.

PLATE 1

Fig. A. Longitudinal section of the necks of the blastopore cells. At the free surface thegranular zone (GZ) is thrown into folds. At a deeper level are the vesicular (VZ) and pigmentzones (PZ).Cytoplasmic fibrils (Fb) and longitudinal flanges (FT) containing granular material areevident. Y, yolk platelet, x 5000.Fig. B. Transverse section of the necks of the blastopore cells showing the complex inter-digitations of the longitudinal flanges (Fl). x 5000.

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J. Embryo/, exp. Morph., Vol. 15, Part 3 PLATE 2

M. M. PERRY & C. H. WADD1NGT0N facing p. 321


that during the preparative procedures some of this extracellular material isremoved and that the fuzzy material merely represents the remnants of a muchthicker layer.

Within the thickness of the accumulation of electron-dense material at thetips of the cell it is quite common to find odd pieces of apparently isolateddouble membranous material which are certainly not continuous with the generalintercellular boundary, although they could be elements of engulfed surfacemembrane. Balinsky (1961) has described large vacuoles in this region, formed,he suggests, by the pinching off and engulfing of the bottoms of the cavitiesbetween the microvillus projections at the cell surface. However, this vacuoliza-tion occurs at a later stage, in the mid-gastrula, than that examined in Triturus,where the process of pinocytosis is probably only beginning. The appearancessuggest that the whole of this dense material consists of molecular specieswhich can relatively simply become arranged into a laminar form, when it givesthe appearance of membranes. This seems not unreasonable since it is possiblethat the substance is in fact the accumulation, in this region of the cell, of materialthat originally spread out over a much more extensive surface. If this is so, nottoo much importance can be attached to the appearance of odd pieces ofmembranous structure within it.

The lateral plasma membranes of the cell tips are separated by a gap, varyingin width between 100 and 200 A, which contains material of low density (Plate2, fig. C). As the intercellular space at a deeper level is considerably wider, thesedistal cell junctions are probably areas of attachment, of a simple type.

n x T/ . ; The necks(1) Vesicular zone

Immediately proximal to the superficial, granular zone just described is aregion in which the cytoplasm is packed full of vesicles, which are embedded ina granular matrix similar to that in the cell tips. The depth of this zone dependson the extent of elongation of the cells; the more attenuated the necks, the longeris the vesicular zone, and the more closely packed the vesicles, which appearhollow 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 moreproximally 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 traverseareas 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.


Intermingled with the vesicles, and sometimes occurring in large numbers ata 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-layeredmembranes, and which encloses an area containing traces of material similarto that in the surrounding cytoplasm (Plate 3, fig. H). Occasionally, simplerod-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 stageof invagination than those already described, are the polyvesicular bodies (Plate3, fig. G). These contain traces of fibrillar material dispersed around the internalvesicles, and the triple-layered nature of their limiting membranes is ill-defined.

The three types of organelles should perhaps be given distinctive names. Wehave called them the alpha vesicles, appearing hollow with single membranes;the beta vesicles, sometimes multiple, with dense contents bounded by triple-layered membranes; and the gamma vesicles, which are polyvesicular. Thegamma vesicles could perhaps have been derived from the beta vesicles, whichare smaller in area, by, for instance, the granular regions becoming hydratedand swelling up.

Small vesicles bounded by a single membrane are a common feature of thecytoplasm in all the cells of the early amphibian embryo. In the cells of regionsother than the blastopore they are scattered rather thinly throughout the bulkof the cytoplasm, and never appear in such concentrated masses as they do inthe necks of the blastopore cells. However, it seems quite likely that their highconcentration in these cell necks is due more to the concentration of all thevacuoles of this order of size in that part of the cell than to a new formationof them. The blastopore cells have in general a zonation of contents accordingto 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 furtherproximally there is a concentration of yolk granules which are much largeragain. However, although this is the simplest hypothesis to account for thepresence 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), havedense, granular contents surrounding an internal cavity. Also evident are a stack of annulatelamellae (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 poly-vesicular bodies, the gamma vesicles (G). x 24000.Fig. H. Higher magnification of the beta vesicles (B). Triple-layered membranes delimit thedense, granular material from the internal cavities and the surrounding cytoplasm, x 100000.

/ . Embryol. exp. Morph., Vol. 15, Part 3 PLATE 3

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way from the more complex beta and gamma vesicles, which contain electron-dense material and which are, at least originally, bounded by double membranes.

Scattered amongst the vesicles of this zone, and, in fact, throughout the wholeof the cell, are a large number of dense granules, which from their size (300-400 A), their affinity for the lead stain, and their particulate substructure, maybe identified as glycogen (Revel, 1964). The glycogen granules are frequentlyseen in the intercellular spaces, and in spaces where, for instance, the cytoplasmhas contracted away from a yolk platelet. It seems most probable that undersome conditions of fixation these granules may be shifted in location within thetissue, and little can be safely inferred from their distribution.

Another type of structure which is sometimes encountered within the cyto-plasm 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 ofanimals. They are rare in adult cells, but have been seen in young cells activelyengaged in growth (Kessel, 1965). Owing to the resemblance in the pattern ofannuli to that seen on the nuclear envelope, they are often considered to bederived from that structure (Swift, 1956). We have seen them in other amphibianembryonic cells (mesenchyme derived from the endo-mesoderm) in the immedi-ate neighbourhood of the nuclear envelope (Waddington & Perry, 1966 a). Inthe flask cells, however, they lie at a considerable distance from the nuclei, asthey do in Drosophila oocytes (Okada & Waddington, 1959), and if originallyderived from them must have persisted for a considerable time since their origin.In these blastopore cells they are usually surrounded by rather electron-densematerial and it therefore seems rather probable that they are engaged in someform of synthesis, although nothing definite is known of their function.

The last constituents of the cytoplasm in the neck zone to require mentionare the microtubules. In longitudinal sections of the cells the tubules areoriented rather strictly in line with the main axes (Plate 2, fig. D). In trans-verse 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 areparticularly associated with long strands of granular cytoplasm. The fibrilswhich are visible in low-power micrographs (Plate 1, fig. A) can be resolved intothese microtubular and granular cytoplasmic components. Lengths of micro-tubules of up to 8 fi may be seen in a single section, which implies that they takean undeviating course, in spite of the presence around them of large numbersof vesicles. It appears as though the microtubules must, as it were, elbow thevesicles out of the way during their growth. At early stages in blastoporeformation, that is, when the cells are cuboidal in shape, scattered cross-sectionsof microtubules are found throughout the cytoplasm in longitudinal sections ofthe cells.

The overall shape of the neck regions of these cells is very interesting and atfirst sight unexpected. Longitudinal sections, it is true, reveal little more thanwas known already, namely that the cells are exceedingly long drawn out and


the necks very narrow and gradually tapering. One notices, however, the presenceof some spaces between the cells, which are not in close contact all along theirlength. Within these spaces there are sometimes protrusions containing granularcytoplasm. The nature of these outgrowths is much better revealed in transversesections (Plate 1, fig. B). These show that, in the neck region, the neighbouringcells are in general separated by inter-cellular spaces, but are closely involvedwith 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 withthe coverings lowered, but not rolled around their own supporting axes, butinstead allowed to become crumpled together with the covering of the neigh-bouring umbrellas. Such mutual involvement of the cells would presumably tiethem together into a rather firm longitudinal bundle, from which it would bedifficult to untangle the whole length of any single cell.

The granular cytoplasm in these tangled regions is very similar to that in thetip of the cell. The granular material within the tips can often be seen to extendproximally to fill the tangled lateral flanges. In general the flanges are moreextensive near the distal and thinner ends of the necks and become progressivelyreduced 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 thecytoplasm are the various types of vesicles described above, and there are veryfew pigment granules. Slightly further proximally there is a region in whichpigment granules are extremely frequent. They show the usual compositestructure common in Triturus (Plate 3, fig. F). A few mitochondria are evident,and are somewhat more numerous than in the vesicular zone. The othercomponents of the cytoplasm remain much as they were, although the concen-tration of beta and gamma vesicles is perhaps lower, and the longitudinalflanges become progressively reduced in dimensions. There is little sign offree ribosomes and no apparent endoplasmic reticulum in any of the zonesdescribed.

(3) The main body of the cell

More proximally still one comes to the bulbous region of the cell in which thenucleus is located. This region also contains the yolk platelets, and large dropletsof lipid, which usually appear rather pale in these preparations. The yolk plateletsmay extend some distance into the wider parts of the necks of the cells, and inthat case any platelets which are markedly ovoid in configuration are orientedwith the long axes parallel to the length of the cell. There is, however, noparticular orientation of the yolk platelets in the main body of the cells. Wehave not studied this region of the cell in any particular detail, since it doesnot appear to present any features of particular interest. It is, however, worth


mentioning that in the cells on the floor of the developing archenteron—thatis, in the ventral part of the region which is drawn out into flask cells—one cansometimes find a very peculiar type of solubilization of the yolk, involving theformation of arrays of tubular structures. These will be described elsewhere(Perry, 1966).

DISCUSSION

The observations recorded here throw considerable light on the processes bywhich 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 theexternal surface of the cells is not engaged in active contraction and cannot beproviding the main motive force for the original invagination. There is nothingcorresponding to the 'surface coat' to which Holtfreter (1943a) has attributedso much importance and which he supposes to form a continuous elasticmembrane-like structure spanning across cell boundaries. The dense layer onthe external plasma membrane of the cell, for which there are indications ofsome continuity, can certainly not be playing the part of Holtfreter's coat, andthe main bulk of the dense granular material in the tips of the cells is, as we haveseen, interrupted by intercellular boundaries and is thrown into numerous folds,suggesting that it has itself been forced to contract rather than that it exerts acontractile force of its own.

If the drawing together of the cells of the blastoporal groove is not due to acontraction by the material of the external tips of the cells, an explanation for itmust be sought somewhere else. Balinsky draws attention to the presence, inhis electron micrographs of the early neural groove, of an electron-dense layerlying some distance below the cell surface, which he interpreted as a contractileelement. We have also seen a dense layer in cells of the neural groove, but wewill 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 theblastopore. He therefore attributes the changes in shape of the blastoporal cellsto an active elongation within the cytoplasm.

Baker (1965) considers that the shape of the cells is changed by alternateexpansion and contraction in a peripheral layer of dense cytoplasm, which atthe period of maximum elongation occupies the entire neck region. However,she does not seem to have studied transverse sections of this tissue, and did notrealize the nature of the longitudinal flanges containing granular material, whichextend along the necks of the cells. If these were engaged in active elongation itis difficult to believe that in transverse section they would present the appearanceof loose flaccid bundles, as they do for instance in Plate 1, fig. B. One wouldrather expect to find solid ridges with a relatively simple external surface, butthe flanges in fact offer exactly the same evidence of a lack of contractility as doesthe similar substance at the external surface of the cells.

In our opinion the appearances of the granular zone and of the flanges suggest21 J E E M 15


that the blastopore cells are being caused to become elongated by some processoccurring within their internal cytoplasm. This elongation will result in a reduc-tion in the area of the external surface of the cells, and a narrowing of the regionswhich become the neck. If the material originally constituting the corticalcytoplasm in these regions is not able to move away rapidly enough, it is boundto become accumulated. Both the electron-dense material in the cell tips and thesimilar material forming the longitudinal flanges in the neck region can mosteasily be interpreted as accumulations of substances which originally formed thecell surface. The indications, which have already been mentioned, that thismaterial seems easily to form membranous structures on fixation is in fullaccordance with this. On this interpretation, this material is not likely to beexerting any particular forces assisting the process of gastrulation, though oneimagines 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 thedevelopment of a tendency to elongation within the internal cytoplasm. Thepresence of very many microtubules within the cytoplasm, which has beenrevealed in this investigation, provides a plausible mechanism. The possibilitythat cytoplasmic fibrils might play a role in such processes is one of the oldspeculative hypotheses in this field. They were, however, not visible with thelight-microscope. An attempt was made to detect their presence by examiningthe orientation of the ovoid yolk platelets, since it was argued that any fibrilspowerful enough to play a part in cell elongation might be expected to cause theyolk platelets to take up a preferred orientation (Waddington, 1942). However,it was found then that the yolk platelets only became regularly oriented in partsof the cell which were so narrow that there was no room for them to lie in anyother direction. The orientation might therefore have been imposed by theexternal cell membranes rather than by internal fibrils and no definite evidencefor the existence of such fibrils could be discovered. This observation on theorientation 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 ofmicrotubules, often associated in groups, embedded in strands of granularcytoplasm, which at low magnifications appear as fibrils. These may in factsucceed in orienting the small empty-looking vesicles in the way in which it wasearlier thought more coarse fibrils might act on the yolk granules.

Within the necks of the cells the microtubules are oriented rather accuratelyin the direction of the cellular long axis. They are therefore not only present inlarge numbers but in the right orientation to function as the agents of internalelongation, producing the effects which Balinsky thought must be present, butfor which he could identify no particular agents. Microtubular structures havebeen reported in many cells (Slautterback, 1963), and in asymmetrical metazoancells, where they take up a preferred orientation, they are thought to be impli-cated in the formation and maintenance of these asymmetries (Porter, Ledbetter& Badenhausen, 1964). For instance, Byers & Porter (1964) have described the


development of a microtubular system in cells of the chick lens rudiment whichcoincides precisely with the period of cellular elongation. They suggest a possiblemechanism whereby cytoplasmic movement along an array of microtubules,similar to the streaming in plant cells, produces elongation by gradual cyto-plasmic translocation, analogous to the translational motion of actin withrespect to myosin in myofibrils. As the microtubules in the necks appear to beparticularly associated with a granular cytoplasm, a similar process could beinvolved here. On the other hand Wolpert(1965) has suggested that microtubulesor micro-fibrils may be actively contractile by some mechanism of the slidingfilament type. He points out that quite small numbers of them could providethe forces required.

In early stages of blastopore formation microtubules can be found, as we haveseen, running in the plane tangential to the surface so that they are cut trans-versely in sections which are longitudinal as regards the whole embryo. Theyare, however, by no means so numerous as those found in the cell necks, andwhether these microtubules play a role in bringing about the initial formation ofthe blastopore pit is not clear. Further, it should be remarked that microtubulescan be found in almost all cells of the early newt embryo (Waddington & Perry,unpublished). They are often most frequent near and parallel to intercellularboundaries but are sometimes found running in rather haphazard directionswithin the general body of the cytoplasm. In cells not engaged in very activechange 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 theblastopore cells. It is perhaps only when we see them in very large concentrationsand in very regular orientation that, it is safe at present to attribute any importantmorphogenetic action to them.

Although the microtubules are present in considerable numbers in the necksof the cells it is improbable that they play an important role in producing thebirefringence which these cells show (Waddington, 1940). The large number ofmore or less orientated vesicles and the longitudinal cell flanges would providethe basis for a 'form birefringence' and it seems most likely that this was thephenomenon detected.

While the alpha vesicles are not unlike those found in most other cells of thenewt embryo, the beta and gamma vesicles are certainly peculiar, and it is to beexpected that they have precise and definite functions. Vesicles of a generally similarcharacter to these have often been referred to as lysosomes, and either inferred orshown to contain active enzymes (Novikoff, 1961). In some cases it has been re-ported that they originate in the Golgi region (Moe, Rostgaard & Benke, 1965).Nothing is yet known about any possible enzyme activity of the granules in theseblastopore cells although investigation of this problem is proceeding. It isperhaps noteworthy that these cells do not contain any identifiable Golgimaterial 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


have been seen in the main body of the cell in the neighbourhood of the nucleusand yolk granules. It seems most improbable however that the beta and gammavesicles are at all closely connected with Golgi and their relation to lysosomesin other forms remains uncertain. In this connexion Holtfreter (19436) hassuggested that the blastopore cells are relatively short-lived, and degenerate inthe larval period. It would not therefore be unexpected to find evidence oflysosomal particles within them.

SUMMARY

1. Longitudinal and transverse sections of the flask-shaped blastopore cellsin the early gastrula of Triturus alpestris have been examined in the electronmicroscope.

2. The cytoplasm may be divided into three main regions; a distal, super-ficial, granular zone; a vesicular and, more proximally, a pigment zone in thenecks; and the main body of the cell with nucleus, yolk platelets and lipiddroplets. A thin, continuous dense layer of extracellular material covers thecells at the free surface.

3. Numerous microtubules oriented parallel to the main axes are foundgrouped in the necks. Other prominent organelles are vesicles, which from theirstructure have been divided into three categories, the alpha, beta and gammavesicles. Longitudinal flanges extending along the necks are complexly inter-wound with those of adjacent cells and bind the cells firmly together in this region.

4. Neither the extracellular layer, nor the superficial granular layer, isconsidered to exert a contractile force which would cause the cells to becomeelongated; rather the change in shape is brought about by active elongationwithin the internal cytoplasm, in which the microtubular system plays animportant part.

5. The material in the cell tips probably results from the passive accumula-tions of substances which originally covered a larger surface area.

6. The alpha vesicles are common to all cells in the embryo, whereas thebeta and gamma vesicles are characteristic of the blastopore cells. The functionof these organelles is at present unknown, although there are some structuralsimilarities between the beta vesicles and lysosomal particles described in otherorganisms.

RESUME

Ultrastructure des cellules blastoporales chez le Triton

1. Des coupes longitudinales et transversales des cellules en bouteille dublastopore de la jeune gastrula de Triturus alpestris, ont ete examinees aumicroscope electronique.

2. Le cytoplasme peut etre subdivise en trois regions principales; une zonedistale, superficielle et granulaire; une zone vesiculaire, et, a un niveau plusproximal, pigmentaire dans les * cols' des bouteilles; ainsi que le corps principal


de la cellule avec son noyau, ses plaquettes vitellines et ses gouttelettes lipidiques.Une mince couche continue et dense de materiel extracellulaire recouvre lescellules au niveau de leur surface libre.

3. Dans les cols on a trouve de nombreux microtubules orientes parallele-ment au grand axe. Comme autre organites remarquables, il y a lieu de men-tionner des vesicules qui par leur structure peuvent etre classees dans troiscategories, les alpha, beta et gamma. Des franges longitudinales s'etendant lelong des collets sont entrelacees de fagon complexe avec celles des autres celluleset les solidarisent solidement a ce niveau.

4. Ni la couche extracellulaire, ni la couche granulaire superficielle peuventetre considerees comme exergant des forces contractiles pouvant provoquerl'elongation de cellules; le changement de forme est provoque par une elonga-tion active du cytoplasme interne, processus dans lequel le systeme micro-tubulaire joue un role important.

5. Le materiel situe dans les extremites retrecies des cellules provient prob-ablement d'une accumulation passive de substances qui a l'origine etaientdistributes 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 blasto-porales. La fonction de ces organites reste inconnue, toutefois il apparaitcertaines similitudes de structure entre les vesicules beta et les organites decritscomme lysosomes dans d'autres organismes.

The authors wish to thank Mr E. D. Roberts for his skilful drawing of Text-figs. 2 and 3

REFERENCESBAKER, 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 Inter-nationale d'Embryologie and Fondazione A. Baselli.

BELL, E. (1960). Some observations on the surface coat and intercellular matrix material ofthe amphibian ectoderm. Expl. Cell Res. 20, 378-83.

BYERS, B. & PORTER, K. R. (1964). Oriented microtubules in elongating cells of the develop-ing lens rudiment after induction. Proc. Natn. Acad. Sci. U.S.A. 52, 1091-9.

HOLTFRETER, J. (1943 a). Properties and functions of the surface coat in amphibian embryos./ . Exp. Zool. 93, 251-323.

HOLTFRETER, J. (19436). A study of the mechanics of gastrulation. Part I. / . exp. Zool. 94,261-318.

HOLTFRETER, J. (1944). A study of the mechanics of gastrulation. Part II. / . exp. Zool. 95,171-212.

KESSEL, R. G. (1965). Intra-nuclear and cytoplasmic annulate lamellae in tunicate oocytes./ . Cell Biol. 24, 471-87.

MOE, H., ROSTGAARD, J. & BENKE, O. (1965). On the morphology and origin of virginlysosomes in the intestinal epithelium of the rat. J. Ultrastruct. Res. 12, 396-403.

NOVIKOFF, A. B. (1961). Lysosomes and related particles. In The Cell, vol. n, pp. 423-82.Ed. J. Brachet and A. E. Mirsky. New York and London: Academic Press.


OKADA, E. & WADDINGTON, C. H. (1959). The submicroscopic structure of the Drosophilaegg. J. Embryol. exp. Morph. 7, 583-96.

PERRY, M. M. (1966). Tubular elements associated with amphibian yolk platelets. / .Ultrastruct. Res. (In the Press.)

PORTER, K. R., LEDBETTER, M. C. & BADENHAUSEN, S. (1964). The microtubule in cell finestructure as a constant accompaniment of cytoplasmic movements. In Third European-Conference on Electron Microscopy {Prague), p. 119.

REVEL, J. P. (1964). Electron microscopy of glycogen. / . Histochem. Cytochem. 12, 104-14.REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in

electron microscopy. / . Cell Biol. 17, 208-12.SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron micro-

scopy. The preservation of cellular ultrastructure and enzymatic activity by aldehydefixation. / . Cell Biol. 17, 19-58.

SLAUTTERBACK, D. B. (1963). Cytoplasmic microtubules. I. Hydra. / . Cell Biol. 18, 367-88.SWIFT, H. (1956). The fine structure of annulate lamellae. / . biophys. biochem. Cytol. 2

(Suppl.), 415-18.WADDINGTON, C. H. (1940). Organisers and Genes. Cambridge University Press.WADDINGTON, C. H. (1942). Observations on the forces of morphogenesis in the amphibian

embryo. / . exp. Biol. 19, 284-93.WADDINGTON, C. H. (1956). Principles of Embryology. London: Allen and Unwin.WADDINGTON, C. H. & PERRY, M. M. (1966a). Perinuclear structures in anaphase in some

amphibian mesenchyme cells. Expl. Cell Res. (In the Press.)WADDINGTON, C. H. & PERRY, M. M. (19666). A note on the mechanisms of cell deformation

in the neural folds of amphibia. Expl. Cell Res. (In the Press.)WEISS, P. (1950). Perspectives in the field of morphogenesis. Q. Rev. Biol. 25, 177-98.WOLPERT, L. (1965). Cytoplasmic streaming and amoeboid movement. Symp. Soc. Gen.

Microbiol. 15, 270-93.

{Manuscript received 5 December 1965)

Documents

Ultrastructure of the blastopore cells in the newt · Ultrastructure of the blastopore cells in the newt By MARGARET M. PERRY1 & C. H. WADDINGTON1 From the Institute of Animal Genetics,