Download Ultrastructure of the blastopore cells in the newt

J. Embryol. exp. Morph., Vol. 15, 3, pp. 317-330, June 1966
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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.
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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
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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.
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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
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M. M. PERRY & C. H. WADDINGTON
facing p. 320
J. Embryo/, exp. Morph., Vol. 15, Part 3
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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.
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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
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PLATE 3
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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
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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
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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
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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
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