J. Embryol exp. Morph. Vol. 67, pp. 27-35, 1982
Printed in Great Britain © Company of Biologists Limited 1982
27
Scanning electron microscopy of gastrulation in a
sea urchin (Anthocidaris crassispina)
By SHONAN AMEMIYA,1 KOJI AKASAKA2 AND
HIROSHI TERAYAMA2
From the Misaki Marine Biological Station,
University of Tokyo
SUMMARY
Gastrulation in Anthocidaris was investigated by observing the inside and the outside of
embryos by scanning electron microscopy.
Furrows which possibly reflect changes in intercellular interactions were observed on
the outer surface (hyaline layer side) of embryos twice in development: firstly at the time
of primary mesenchyme cell formation, and secondly at the time of vegetal plate indentation.
In the latter case, the cells within and surrounding the vegetal plate appeared to change
their shapes differently; the former (within the plate) having broader surfaces on the blastocoel side whereas the latter (surrounding the plate) having broader surfaces on the hyaline
layer side. This suggests that the first phase of indentation may be mediated by the autonomous change of cell shape and intercellular adhesiveness, accompanied by an autonomous
cell movement in the vegetal pole region.
Although some pseudopodial linkages were observed between secondary mesenchyme
cells on the top of the invaginating archenteron and the animal pole in the mid-gastrula and
later stage embryos, they were thinner and smaller in number as compared to those in the
Pseudocentrotus embryos. The rate of invagination appeared rather constant throughout
gastrulation in contrast to the accelerated invagination in other embryos with larger blastocoel cavities. Moreover, the number of columnar cells on the dissected surface of embryos
remained unaltered.
These findings suggest that the secondary mesenchyme cells may act as a linker between
the archenteron tip and the animal pole, but they may not generate major motive forces
for archenteron invagination at least in the Anthocidaris embryos.
INTRODUCTION
The early morphogenetic processes involved in gastrulation in developing
sea-urchin embryos have been investigated by means of light microscopy and
transmission electron microscopy. Gastrulation was reported to consist of
two phases (Gustafson & Wolpert, 1961); the first and slow phase is characterized by active pulsation and altered intercellular adhesiveness of cells in the
1
Author's address: Misaki Marine Biological Station, Faculty of Science, University of
Tokyo, Miura-shi, Kanagawa-ken 238-02, Japan.
2
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113, Japan.
28
S. AMEMIYA, K. AKASAKA AND H. TERAYAMA
vegetal plate following the emigration of primary mesenchyme cells (Gustafson
& Wolpert, 1961), and the second and fast phase seems to be implemented by
the action of pseudopodia of the secondary mesenchyme cells on the top of
invaginating archenteron (Dan & Okazaki, 1956) or of tip cells of the archenteron
itself (Gustafson & Kinnander, 1956; Kinnander & Gustafson, 1960). Based
on their observations, Gustafson & Wolpert (1962) proposed a hypothetical
model for gastrulation.
In the preceding paper (Akasaka, Amemiya & Terayama, 1980) we observed
the inside of developing sea-urchin embryos {Pseudocentrotus depressus) with
large blastocoel cavities by means of scanning electron microscopy, in the
present study, we carried out observations of both the inside and the outside
of developing Anthocidaris crassispina embryos with much smaller blastocoel
cavities, finding that there may be some principal differences in the gastrulation
processes between the two types of sea-urchin embryos.
MATERIALS AND METHODS
Sea urchins, Anthocidaris crassispina, were caught near the Misaki Marine
Biological Station. Eggs and sperm were collected by artificial spawning using
a KC1 solution. Batches of eggs of which more than 95 % could be fertilized
were used. Eggs were cultured in filtered sea water at 20 °C with gentle stirring.
Preparation of embryos for scanning electron microscopy was described in
the earlier paper (Akasaka et al. 1980). A Hitachi HHS-2R scanning electron
microscope was used for observing the surfaces of outside and inside of
embryos.
RESULTS
Figure 1 shows the interior surface of an Anthocidaris embryo (blastula, at
10-5 h after insemination) dissected along the animal-vegetal axis. It should
be noted that the length of columnar cells (or the depth of ectodermal wall) is
rather long and the size of blastocoel cavity is extremely small. The intercellular contact in the vegetal plate appears to be loosened. A few cells with
their heads somewhat protruded into the blastocoel, which may correspond to
the basal lobe cells (Be) (Gibbins, Tilney & Porter, 1969), are seen in the
periphery of the vegetal plate.
Moreover one can see some tadpole-like (Tc) or bottle-shaped cells in the
vegetal plate in accordance with the observation by Katow & Solursh (1980).
They appear to be presumptive primary mesenchyme cells emigrating from the
vegetal plate into the blastocoel. Some gaps apparently remaining after the
emigration of tadpole-like cells (primary mesenchyme cells) are also seen.
Probably reflecting the alteration of cell shape and intercellular adhesion
in the vegetal plate and its vicinity, furrows (a big circular furrow surrounding
the vegetal plate with long radial ones in the vicinity) were seen on the outer
SEM observations on developing sea-urchin embryos
29
surface. The number of cells remaining in the vegetal plate after primary
mesenchyme cell emigration appears to be about eight as judged from the
small furrows apparently surrounding each cell in the vegetal plate (Fig. 2)
in accord with the description of Endo (1966) and Katow & Solursh (1980).
At this stage, the cells surrounding the vegetal plate appear to expand their
outer cell surfaces (on the hyaline layer side) towards the vegetal plate as
judged from the apparently reduced density of micro villi in the area surrounding
the vegetal plate as also shown in Fig. 2.
At 13-5 h after fertilization, the big circular furrow surrounding the vegetal
plate as well as small ones surrounding each of the vegetal plate cells becomes
less distinct as shown in Fig. 3 A. The dissected surface of an embryo (Fig. 32?)
shows that the intercellular adhesion in the vegetal plate region is reestablished.
At 15 h after fertilization, small furrows surrounding each cell reappeared
in the vegetal plate and its vicinity (Fig. 4 A), suggesting that the intercellular
contact was again loosened at this stage. At the same time, some cells in the
vegetal pole region started to protrude their heads into the blastocoel (Fig. 4B).
The phenomenon seems similar to the one observed at the time of primary
mesenchyme cell formation, suggesting that active cellular movement is taking
place.
The cells with surrounding furrows are not restricted to the vegetal plate and
the number appears to increase continuously in the vegetal pole region (Fig. 5 A).
Some of the cells near the centre of vegetal plate appear to show a characteristic
deformation (Fig. 5B), suggesting that the cellular mass is transferring towards
the blastocoel side. As a result the cell surface on the hyaline layer side became
smaller and smaller while increasing on the blastocoel side.
At 18 h after fertilization, the outer surface area with furrows expanded
further, and the furrows in the centre of the vegetal plate now became very
small, leaving big furrows or gaps (Fig. 6 A). Accordingly the dissected surface
of an embryo at this stage (Fig. 6B) clearly indicated that most of the cells
in the vegetal plate were transformed into tadpole-like forms with their heads
on the blastocoel side and their smaller tails on the hyaline layer side (or a
little inside the vegetal plate wall) in contrast to the surrounding cells which
had heads on the hyaline layer side. Some cells in the middle of vegetal plate
appear to be translocated inwardly (the first sign of indentation).
At 19-5 h after fertilization, the indentation of the archenteron is clearly
seen not only from the outside of an embryo (Fig. 1 A) but also from the
inside (Fig. IB). The cellular adhesion in the vegetal region appears to be
reestablished (Fig. IB) and furrows are no longer seen on the outer surface
of embryos (Fig. 1A).
At 21 h after fertilization, the embryos seem to be in the mid-gastrular
stage; and some secondary mesenchyme-like cells are seen on the tip of invaginating archenteron. Some pseudopodia from these cells appear to extend to the
animal pole, but others are linked to the primary mesenchyme cells (Fig. 8).
2
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30
S. AMEMIYA, K. AKASAKA AND H. TERAYAMA
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SEM observations on developing sea-urchin embryos
31
These pseudopodia are much thinner and their number was smaller compared
to those in the corresponding-stage embryos of Pseudocentrotus (Akasaka et al.
1980). The cells on the tip of the invaginating archenteron (Fig. 8) are smaller
in size and irregular in shape as compared to those in the other parts of
archenteron, apparently forming a bud-like structure.
At 22-5 h after fertilization, the tip of the invaginating archenteron nearly
reached the animal pole (Fig. 9). The cells on the tip of archenteron now appear
to be fully developed and the bud-like structure is no longer seen.
The columnar cells (or the depth of ectodermal wall), except those in the
animal pole region, were found to contract up to the late gastrula stage. The
tendency appeared to be more marked after the onset of gastrulation. The
shortening of columnar cells may contribute to the enlargement of embryonic
size or the blastocoel cavity. However, the number of columnar cells (except
archenteron cells) on the dissected surfaces of embryos (Figs. 1, 8, 9) remained
almost constant (about 50) in spite of the increase in embryonic size, the size
of archenteron and the number of archenteron cells.
DISCUSSION
The invagination (or indentation) of the archenteron in sea-urchin embryos
has been extensively investigated by Gustafson and his colleagues (Gustafson
& Kinnander, 1956; Kinnander & Gustafson, 1960; Gustafson & Wolpert,
1961; Gustafson & Wolpert, 1962), and the two-phase mechanism was presented.
The inward indentation of archenteron rudiment in the vegetal pole region
Fig. 1. S.E.M. of an A. crassispina embryo dissected along the animal-vegetal axis
10-5 h after insemination. Ap, Animal pole. Vp, vegetal pole. Be; basal lobe cells,
being localized around the vegetal plate and supposed to be in the active pulsation.
Tc, Tadpole-shape cell emigrating into the blastocoel. Scale, 10 /xm.
Fig. 2. S.E.M. of the outer surface in the vegetal pole region of an A. crassispina
embryo at 12 h after insemination. Many furrows are seen. Microvilli are poorer
in the area surrounding the vegetal plate. Scale, 10 /«n.
Fig. 3. S.E.M. of A. crassispina embryo at 13-5 h after insemination. {A) Outer
surface near the vegetal pole region, showing the disappearance of furrows. Scale,
10 fim. (B) Vegetal pole region of embryo dissected along the animal-vegetal axis.
The intercellular adhesion is reestablished, and the cells in the vegetal region
are now elongated. Scale, 10 fim.
Fig. 4. S.E.M. of A. crassispina embryo at 15 h after insemination. (A) Outer surface
in the vegetal pole region, showing the reappearance of many furrows. Scale; 10 /*m.
(B) Vegetal pole region of an embryo dissected along the animal-vegetal axis,
showing soms of the vegetal plate cells with heads (Ph) protruded into the blastocoel. Scale, 5 /*m.
Fig. 5. S.E.M. of A. crassispina embryo at 16-5 h after insemination. (A) Outer surface
in the vegetal pole region, showing many furrows spreading outwardly. Scale,
10/tm. (B) Vegetal pole region of an embryo dissected along the animal-vegetal
axis, showing the cellular mass translocating towards the blastocoel side. Scale,
32
S. AMEMIYA, K. AKASAKA AND H. TERAYAMA
SEM observations on developing sea-urchin embryos
33
(the first phase) is supposed to be due to the reduced intercellular adhesiveness
without changing the adhesiveness to the hyaline layer, that may allow the
pulsating activity of cells which leads to indentation. On the other hand, the
elongation of the archenteron (the second phase) is supposed to be implemented
by secondary mesenchyme cells (or archenteron tip cells) and their pseudopodia.
The possible involvement of pseudopodia in the gastrulation processes has in
general been accepted (Dan & Okazaki, 1956; Trinkaus, 1969; Akasaka et al.
1980), mainly based on the characteristic situation of secondary mesenchyme
cells between the archenteron tip and the animal pole, their well-developed
pseudopodia and the accelerated indentation rate after the development of
pseudopodia. Alternative mechanisms have not been completely eliminated.
In the present study, using Anthocidaris embryos with small blastocoel
cavities, we showed that furrows are observed twice on the outer surface
(hyaline layer side) of embryos; the first occurrence is at the time of primary
mesenchyme cell emigration and the second is at the onset of indentation
of archenteron rudiment. The occurrence of furrows on the outer surface of
the vegetal pole region seems to suggest that the reduction of intercellular
adhesion extends even up to the hyaline layer in contrast to the earlier hypothesis (Gustafson & Wolpert, 1963).
The reduction of intercellular adhesion in the vegetal pole region may allow
the deformation of cells (appearance of tadpole-shaped cells) and following
cellular emigration (primary mesenchyme cell formation) or sliding (first sign
of archenteron indentation, Fig. 6B). It should be noted that the furrow
formation at the onset of gastrulation appears to be only temporary, and the
intercellular adhesion appears to be reestablished already in the early gastrula
(Fig. 7 A, B).
The observations on the Anthocidaris embryos question the positive role of
Fig. 6. S.E.M. of A. crassispina embryo at 18 h after insemination. (A) Outer surface
in the vegetal pole region, showing many furrows with irregular shapes propagating
from the vegetal pole. Deep furrows or gaps are seen in the presumptive blastopore
region. Scale, 10/tm. (B) Vegetal half of an embryo dissected along the animalvegetal axis, showing early invagination. Cells in the vegetal plate are taking
tadpole-like forms with broader heads facing to the blastocoel and their tails
to the hyaline layer. Soms cells are seen to have slided inwardly. Scale, 10 //m.
Fig. 7. S.E.M. of A. crassispina embryo at 19-5 h after insemination. (A) Outer surface
in the vegetal pole region, showing that the vegetal plate has already indented. Scale,
10 fim. (B) Vegetal half of an embryo dissected along the animal-vegetal axis, showing that the intercellular adhesion has been reestablished. Scale, 10 (im.
Fig. 8. S.E.M. of A. crassispina embryo dissected along the animal-vegetal axis 21 h
after insemination. Some thin pseudopodia from secondary mesenchyme cells are
seen. The cells on the tip of archenteron form a bud-like structure. Scale, 10 ptm.
Fig. 9. S.E.M. of A. crassispina embryo dissected along the animal-vegetal axis 22-5 h
after insemination. The archenteron tip nearly reaches the animal pole, and cells
on the tip of archenteron now appear to be fully developed. Scale, 10 fim.
34
S. AMEMIYA, K. AKASAKA AND H. TERAYAMA
pseudopodial contraction in the further elongation of archenteron. The number
of pseudopodia was much smaller as compared to Pseudocentrotus (Akasaka
et ah 1980). The pseudopodial linkage between the animal pole and the
archenteron tip was observed only at stages later than the mid-gastrula, and
the distance between the animal pole and the tip of archenteron did not change
so much in gastrulation as in Pseudocentrotus. Moreover the rate of archenteron
elongation appeared to be constant (about 4-6/*m/h) as roughly calculated
from the figures at various stages (Figs. 6B,7B, 8, 9). Even though the pseudopodia may not contribute to the forces for archenteron elongation, the role of
pseudopodia as a specific linker between the archenteron tip and the animal
pole seems likely in Anthocidaris.
The shortening of columnar cells in the ectodermal wall in gastrulation
was observed much more markedly in the Anthocidaris as compared to Pseudocentrotus embryos (Akasaka et al. 1980). The shortening of columnar cells
may give rise to the enlargement of the outer surface of embryos, and henceforth the blastocoel cavity. In the case of Anthocidaris, the enlargement of
embryonic size proceeds coherently with the elongation of the archenteron into
the blastocoel cavity, so that the blastocoel always remains small. However,
the number of ectodermal wall cells on the dissected surfaces of embryos at
various stages (Figs. 1, 8, 9) remained almost unchanged, arguing against the
possibility that a portion of columnar cells in the ectodermal wall near the
vegetal pole may continuously be translocated into a developing archenteron.
The bud-like structure of the archenteron tip in the mid-gastrula (Fig. 8) as
well as the increase in the number of archenteron cells (in contrast to the
constant number of ectodermal wall cells) appears to suggest that the proliferation
of tip cells may be involved in the gastrulation process. The elongation of the
archenteron after the mid-gastrular stage appeared to be due to the development
and rearrangement of archenteron cells not to increase in cell number.
Whether or not some of these findings in Anthocidaris (for instance, the
occurrence of furrows or apparent constancy of columnar cells) may be true
for other sea-urchin embryos with larger blastocoel cavities is under investigation.
REFERENCES
K., AMEMIYA, S. & TERAYAMA, H. (1980). Scanning electron microscopical study
on the inside of sea urchin embryos {Pseudocentrotus depressus); effects of aryl /?-D-xyloside,
Tunicamycin and deprivation of sulfate ions. Expl Cell Res. 129, 1-13.
DAN, K. & OKAZAKI, K. (1956). Cyto-embryological studies of sea urchins. III. Role of
the secondary mesenchyme cells in the formation of the primitive gut in sea urchin larvae
Biol. Bull. mar. Biol. Lab., Woods Hole 110, 29-42.
ENDO, Y. (1966). Development and differentiation. Iwanami Shoten, Tokyo, pp. 1-61 (in
Japanese).
GIBBINS, J. R., TILNEY, L. G. & PORTER, K. R. (1969). Microtubules in the formation and
development of the primary mesenchyme in Arbacia punctulata. J. Cell Biol. 41, 201-226
AKASAKA,
SEM observations on developing sea-urchin embryos
35
T. & KINNANDER, H. (1956). Microaquaria for time-lapse cinematographic
studies of morphogenesis in swimming larvae and observations on sea urchin gastrulation.
Expl Cell Res. 11, 36-51.
GUSTAFSON, T. & WOLPERT, L. (1961). Studies on the cellular basis of morphogenesis in
the sea urchin embryo. Expl Cell Res. 22, 437-449.
GUSTAFSON, T. & WOLPERT, L. (1962). Cellular mechanisms in the morphogenesis of the
sea urchin larva. Change in shape of cell sheets. Expl Cell Res. 27, 260-279.
GUSTAFSON, T. & WOLPERT, L. (1963). The cellular basis of morphogenesis and sea urchin
development. Int. Rev. Cyt. 15, 139-214.
KATOW, K. & SOLURSH, M. (1980). Ultrastructure of primary mesenchyme cell ingression
in the sea urchin Lytechinus pictus. J. exp. Zool. 213, 231-246.
KINNANDER, H. & GUSTAFSON, T. (1960). Further studies on the cellular basis of gastrulation
in the sea urchin larva. Expl Cell Res. 19, 278-290.
TRINKAUS, J. P. (1969). Echinoderm invagination. In Cells into Organs. The Forces that
Shape the Embryo, pp. 136-145. New Jersey: Prentice-Hall.
GUSTAFSON,
{Received 24 March 1981, revised 29 June 1981)