J. Embryol. exp. Morph. Vol. 32, 3, pp. 603-617, 1974
Printed in Great Britain
603
On the mechanism of determination of embryonic
polarity in Parascaris and Rhabditis
By YANAGI TADANO 1 AND MASASHI TADANO 2
From the Department of Anatomy, Nagoya University,
and Biological Laboratory, Gifu University
SUMMARY
In an attempt to explain the determination mechanism of embryonic polarity, the relation
between the behaviour of ectoplasm and the turning of the P2-cell in embryo sof Parascaris
equorum, Rhabditis ikedai and Rhabditis sp., has been studied by means of centrifugation.
During cleavage of uncentrifuged eggs, extension and contraction of the cell-surface occur.
These are accompanied by streaming of the ectoplasm. In the early phase of the second
cleavage embryos become T-shape. Along with streaming of ectoplasm at the animal side of
S2-cell in the later phase, the surface of S2-cell extends on one side and contracts on the other.
Successively, P2-cell turns from the extending side of S2-cell to the contracting one, that is,
in the direction of the primary streaming of ectoplasm. Thus, the embryos become rhomboidal
in shape and their axes are established. The extended side of S2-cell points roughly to the
ventral side of the embryo, and the other to the dorsal.
In the centrifuged embryos, extension and contraction of cell-surfaces and turning of
P2-cell take place also accompanied by streaming of the ectoplasm at the centrifugal side
of S2-cell.
It is concluded from these facts that the determination of embryonic polarity depends on
the turning of the P2-cell by the extension and the contraction of the surfaces of S2-cell
and that the direction of this turning depends on that of the primary streaming of
ectoplasm in S2-cell. It is assumed that the direction of the streaming is due to the
migration of the nucleus, and that the extension and contraction of cell-surfaces is based
on the behaviour of the E.R. and microtubules in the ectoplasm. The tetrahedral embryo is
caused by a change in the streaming of ectoplasm. The formation of a rhomboidal embryo
in Rhabditis without a preceding T-stage is discussed in connection with the behaviour of
the ectoplasm.
INTRODUCTION
Embryonic polarity is a very important feature of all embryos, since the
direction of subsequent development is marked out by the axis of polarity.
Although many studies have been made on the embryonic development of
Parascaris we have as yet little information on the fundamental nature of
its embryonic polarity (cf. Boveri, 1899; Strassen, 1903; Boveri, 1910; Schleip,
1
Author's address: Department of Anatomy, Nagoya University, School of Medicine,
Nagoya, Japan.
2
Author's address: Biological Laboratory, Faculty of General Education, Gifu University, Gifu-shi, Japan.
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Y. TADANO AND M. TADANO
1924; Bonfig, 1925; Schleip, 1929). Particularly, it seems that the mechanism of
determination of embryonic polarity is still an open question.
Concerning polarity in ripe eggs of Parascaris, it has been previously indicated
by us that both animal-vegetal polarity and embryonic polarity could be
inverted by centrifuging in such a way that the centrifugal end came to the
vegetal side, and it has been concluded that polarity is based on the gradient
in the distribution of ectoplasm (Tadano & Tadano, 1961; Tadano, 1961, 1962).
Guerrier (1964, 1967) confirmed the inversion of the animal-vegetal polarity
in Parascaris eggs by means of centrifuging.
In ripe eggs there is an animal-vegetal gradient represented by a characteristic
pattern in the distribution of ectoplasm. Each of the blastomeres which arose
from successive division also shows this pattern corresponding to the prospective fate of respective blastomeres. At the four-cell stage the embryo changes
from a T-shape into a rhomboidal shape as a result of the horizontal turning of
P2-cell. Thus, the embryonic polarity is established.
If the P2-cell turns vertically, the resulting embryo forms a tetrahedral shape,
and then the polarity is either temporarily or permanently disturbed. In early
cleavage active streaming, consumption and formation of ectoplasm are seen,
and movements of the cells almost always occur at the same time as this streaming. These phenomena lead us to postulate that turning of the vegetal cell is
closely related to the behaviour of the ectoplasm and that the explanation of
both phenomena has a deep significance in the determination of embryonic
polarity.
For this reason an attempt has been made to explain the relation between the
behaviour of the ectoplasm and the turning of the P2-cell.
MATERIALS AND METHODS
Fertilized eggs of Parascaris equorum Goeze, Rhabditis sp. and Rhabditis
ikedai Tadano were used. Only ripe eggs after completion of the perivitelline
space were separated from the uterus and used. The egg-membranes of Parascaris from the first outer layer to the outer part of the inmost fifth layer were
usually removed by shaking in a 10-20 % solution of Sodium hypochlorite.
After repeated washing, these eggs were immersed in Ringer's solution.
Because the S2-cell of embryos of these species contains a large amount of
yolk substance, it was difficult to trace the behaviour of ectoplasm. Therefore
some of these eggs were centrifuged with a force of 10000-20000 g for 1-2 h
at a temperature of 10 °C. In the Parascaris eggs centrifuged at the one- or twocell stage many of the stratifications of cell substance recovered normal distribution before the T-stage. Therefore Parascaris embryos were centrifuged
at the three-cell stage or in the T-stage. In Rhabditis some embryos from uncentrifuged eggs do not show a sharp three-cell stage or T-stage and eggs
centrifuged at the one-cell stage show quick recovery of normal distribution of
Embryonic polarity in Parascaris and Rhabditis
605
cell substances. Therefore, to follow behaviour of the ectoplasm in the S2 cell,
Rhabditis embryos were centrifuged at the two-cell stage. The observations
on both uncentrifuged and centrifuged eggs were carried out at a temperature
between 25 and 30 °C.
RESULTS
The first and second cleavage of uncentrifuged eggs of
Parascaris and Rhabditis
(a) Parascaris
The ripe egg of Parascaris shows a visible gradient in the distribution of
ectoplasm. This gradient is largest at the animal side and smallest at the vegetal.
In the ectoplasm, heavy brown granules and mitochondria occur at the base of
the hyaloplasm whereas in the endoplasm there are a number of yolk granules.
In the early phase of the first cleavage, the spindle axis turns through 90° with
development of the mitotic figure. Finally it lies along the polar axis and at this
time furrow formation begins. This is accompanied by streaming of the ectoplasm and extension of the cell surface from the poles to the equatorial region.
The first cleavage plane cuts the egg into a somewhat larger animal cell (SI)
and a smaller vegetal cell (PI). The Sl-cell contains more of the ectoplasm, and
the Pl-cell more of the endoplasm. In the former the ectoplasm is rich at the
animal side, while in the latter at the vegetal (Fig. 1).
In the later phase, extension and contraction of the opposing faces and their
vicinity occur accompanying the streaming of the ectoplasm. Thus, the two
blastomeres elongate in the direction of the egg axis and then, they contract in
the same way. The elongation alternates between the two blastomeres.
In the early phase of the second cleavage, the mitotic spindle of the Sl-cell
appears on the polar axis and then turns through 90° to lie perpendicular to the
polar axis. During this turning ectoplasm streams from the animal side to the
spindle poles, so that it shows an uneven distribution, with most at the spindle
poles and least in the equatorial region. After this, streaming of ectoplasm from
the spindle poles to the equatorial region occurs and furrow formation begins.
Usually Sl-cell begins to divide earlier than PI. The cleavage plane formed on the
polar axis cuts the SI into A and B cells. In most cases the A-cell contains a little
more of the ectoplasm than the B-cell (Fig. 2).
On the other hand, the spindle of the Pl-cell turns through 90° to lie on the
polar axis. Turning of the spindle causes migration of ectoplasm into both the
animal and the vegetal region (Fig. 27). When the cleavage furrow appears
transversely to the polar axis, the Pl-cell divides into a larger S2-cell and a
smaller P2-cell (Fig. 3). The S2-cell contains larger amount of the ectoplasm
than the P2 (Fig. 28). With the appearance of the second cleavage furrow these
four cells come to T-shape (Fig. 4).
In the later phase of this cleavage migration of nuclei is seen, accompanying
the streaming of ectoplasm. The opposing surfaces of the cells bulge out.
Because of this enlarging of the opposing faces, the streaming of ectoplasm and
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606
Y. TADANO AND M. TADANO
Photographs on rhomboidal formation and behaviour of P2-cell of
uncentrifuged embryos of Parascaris.
Fig. 1. Two-cell stage. Earlier phase of the first cleavage, a.p.. Animal pole; v.p.,
vegetal pole (frontal view). Fig. 2. Three-cell stage. Division of Sl-cell into A
and B cells. Fig. 3. Division of Pl-cell into S2 and P2 cells. Fig. 4. T-stage.
Earlier phase of the second cleavage. Figs. 5-9. Counterclockwise turning of
P2-cell. Fig. 10. Adhesion of P2-cell to B. Fig. 11. Rhomboidal stage. Fig. 12.
Vertical turning of P2-cell (Lateral view). Fig. 13. Counterclockwise turning of
P2-ceIl before division of Sl-cell.
contraction and extension of the surfaces are more delicate than those in the
first cleavage. Accompanying the streaming of ectoplasm these four cells begin
to shift. These events appear first in the A and B cells, while in the S2-cell
streaming of the ectoplasm, and marked extension and contraction of the lateral
free surface occur (Fig. 5). The migration of the nucleus is always accompanied
Embryonic polarity in Parascaris and Rhabditis
607
with streaming of ectoplasm in the opposite direction. As seen in Fig. 5, the
nucleus in the S2-cell migrates towards the right side, and the ectoplasm at the
animal side streams counterclockwise. Accompanying this streaming, the left
lateral cell surface extends, while the right surface contracts. Thus the S2-cell
always turns counterclockwise. Similarly, the P2-cell turns in the direction of the
primary streaming of ectoplasm in the S2-cell, that is, from the extending side
of the cell surface of the S2-cell to the contracting side. It gradually approaches
the B-cell which also moves closer to the P2. Finally the P2-cell adheres to the
B-cell, showing accumulation of the ectoplasm at the leading edge (Figs. 5-10,
29-35). During turning of the cells, the opposing surfaces in the B, the S2 and
the P2 cells adhere to one another. Gradually the embryo becomes a rhomboidal
shape.
Until this stage the ectoplasm locally disappears, accompanying the streaming
to the periphery. Ultimately the free surface of cells is surrounded by the ectoplasmic zone, and yolk granules are comparatively dense near the fused faces of
cells. At this time the swellings on the opposing faces gradually disappear, and
the embryo becomes a regular rhomboidal shape (Figs. 11, 36-38).
During turning of the cells, Golgi bodies disperse markedly, indicating
vacuolation. At the rhomboidal stage, the A and the B cells point to the dorsal
side of the embryo, and the S2 and the P2 cells to the ventral; the P2-cell points
39-2
Y. TADANO AND M. TADANO
Figs. 14-17. Photographs of rhomboidal formation and behaviour of
uncentrifuged embryos o/Rhabditis.
Figs. 14-16. Rhomboidal formation without forming T-shape (A-type). Fig. 14.
Two-cell stage. Rotation of spindles in SI and PI cells. Fig. 15. Three-cell stage.
Beginning of cleavage furrow formation of Sl-cell. Fig. 16. Rhomboidal stage.
Fig. 17. Counterclockwise turning of P2-cell before division of Sl-cell.
Fig. 18. Centrifuged embryo ofParascaris.
Vertical turning of P2-cell from the centrifugal to the centripetal side, c.f, Centrifugal side; c.p., centripetal side.
19
Embryonic polarity in Parascaris and Rhabditis
, _"•/>••
609
' v.p
a.p.
r.p.
Figs. 19-26. Centrifuged embryos of Rhabditis. Figs. 19-23. A-type of rhomboidal
formation. Fig. 19. Two-cell stage. Fig. 20. Beginning of cleavage furrow formation of Sl-cell. Fig. 21. Rotation of spindle in Pl-cell. Fig. 22. Turning of P2-cell
from the centrifugal side to the centripetal. Beginning of furrow formation in Plcell. Fig. 23. Rhomboidal stage. Figs. 24-26. Rhomboidal formation through
passing T-shape (B-type).
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Y. TADANO AND M. TADANO
27
Figs. 27-38. Diagrams showing the behaviour of the ectoplasmic zone in cells of
uncentrifuged embryos of Parascaris during the second cleavage. Stippled area in
each cell indicates the ectoplasmic zone, and arrow the direction of ectoplasmic
streaming. All figures are frontal views. Fig. 27. Just before T-stage. a.p., Animal
pole; v.p., vegetal pole. Fig. 28. T-stage. Figs. 39-30. Extension of right lateral
faces and contraction of left ones of S2 and P2 cells. Beginning of counterclockwise
turning of P2-cells. Figs. 31-34. Approach of P2-cell to B, and extension of
A and B cell. Fig. 35. Adhesion of P2-cell to B. Figs. 36-38. Formation of
rhomboidal embryo.
to the posterior portion, and the A-cell to the anterior. In this way the embryonic
axis is established.
In rare cases the P2-cell turns vertically on the polar axis instead of horizontally,
and rides over the other three cells. Streaming of ectoplasm and extension and
contraction of the cell surface in S2-cell appear on the polar axis prior to this
turning of P2-cell. Consequently the embryo forms a tetrahedral shape (Fig. 12).
When the vertical beam of the T of the embryo at the T-stage was perpendicular
to the longitudinal axis of the egg shell, the embryo assumes a tetrahedral
shape temporarily, and then goes to a rhomboidal shape. In eggs of the one-cell
and two-cell stage stored at a temperature of 5 °C, the ectoplasmic region often
becomes indistinguishable from the endoplasmic region after a sudden exposure
to a temperature of 25 °C; these eggs also result in tetrahedral embryos, which
Embryonic polarity in Parascaris and Rhabditis
611
only later become rhomboidal. However, when eggs were repeatedly exposed to
this sudden change of temperature, the ectoplasm became loose and yolk
granules dispersed to the periphery. These eggs also gave rise to the tetrahedral
embryos, which did not become rhomboidal, and finally resulted in various
malformed embryos. When the eggs kept at low temperature were slowly
exposed to room temperature, the distribution of cell substance did not change;
these eggs resulted in normal embryos. When the Pl-cell divided earlier than
the SI-cell, the P2-cell turned horizontally (Fig. 13), and the four cells resulting
after the division of SI into A and B cells assumed the rhomboidal shape.
(b) Rhabditis
Events from the first to the second cleavage in Rhabditis eggs were essentially
similar to Parascaris eggs. In ripe eggs of Rhabditis, however, the boundary
between ectoplasm and endoplasm was comparatively indistinct. Because of
this, it is not easy to trace the behaviour of ectoplasm during cleavage.
At a temperature between 28 and 32 °C, the mitotic spindle of the SI-cell
appears perpendicular to the polar axis, and then becomes oblique because of its
rotation. During this process ectoplasm of SI-cell streams rapidly from the
polar region to the equatorial. Accompanying this, the free cell surface extends,
while the cell surface contacting the Pl-cell contracts. Consequently, the Sl-cell
noticeably elongates towards the spindle poles, and then cleaves into A and B
cells (Fig. 14). On the other hand, the spindle of Pl-cell appears on the polar
axis and rotates in a similar fashion to that of Sl-cell. Ultimately it runs parallel
to the spindle of the Sl-cell, and the Pl-cell divides into S2 and P2 cells. Thus,
these four cells assume the rhomboidal shape without first forming the T-shape
(Type A) (Figs. 15-16). The spindle of the Sl-cell begins to elongate slightly
earlier than that of the Pl-cell, but elongation of both cells finishes almost
simultaneously.
The higher the temperature, the more rapid is the streaming of ectoplasm
and the greater is the elongation of the spindle. When the cell divides at a low
temperature, the streaming of the ectoplasm is inactive and the embryo forms a
T-shape before assuming the rhomboidal shape as in Parascaris embryo
(Type B). On rare occasions, division of the SI and PI cells occurs in the inverse
order; the P2-cell turns horizontally before division of the Sl-cell. Turning of
the P2-cell accompanies the streaming of ectoplasm in the S2-cell as in Parascaris
(Fig. 17).
Development of the centrifuged eggs of Parascaris and Rhabditis
When eggs of Parascaris and Rhabditis are centrifuged, the cell substance is
stratified into five layers. After the treatment, each of these layers recovers its
primary distribution in a characteristic way. During this process, a large amount
of cytoplasm is always secondarily formed at the centrifugal side from the heavy
brown granular layer, the mitochondrial layer and the hyaloplasmic layer.
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Y. TADANO AND M. TADANO
When the intensely centrifuged egg or embryo begins to divide soon after
being centrifuged, a large amount of the secondarily formed ectoplasm remains
at the centrifugal side. If division takes place a considerable time after centrifugation, a greater part of the ectoplasm recovers its normal distribution. In
most cases stratification at the centrifugal side disperses towards the centripetal
side, and vice versa.
(a) Parascaris
The centrifuged embryos were classified into the following five cases, according to the developmental stages of embryos and to the direction of the centrifugal axis to the polar axis.
(1) Centrifugation axis perpendicular to the polar axis. Fig. 39 represents the
Parascaris embryo soon after centrifugation at the three-cell stage. The unbroken
arrow shows the direction of the centrifugal axis, and a broken arrow (a) the
direction of horizontal turning of the vegetal cell. The centrifugal end is on the
right side and the centripetal on the left. The stippled area indicates the heaviest
layer of cell substance in the centrifuged embryo.
After centrifuging, the spindle of Pl-cell always lies on the polar axis. The
cleavage plane formed transversely cuts the Pl-cell into S2 and P2 cells and the
four cells form T-shape. At this stage a large amount of the ectoplasm remains
at the centrifugal side in S2 and P2 cells, though some of it streams along the
cell-surface. Accompanying this, the cell surface at the centrifugal side extends,
while the cell-surface at the centripetal contracts.
As the broken arrow (a) in Fig. 39 shows, S2 and P2 cells are derived from
the Pl-cell. They turn clockwise, and the embryo becomes a rhomboidal
shape (Fig. 44). The free cell-surfaces often show varying expansions, but
they gradually become spherical shape.
(2) Centrifugation at the T-stage (Fig. 40). Centrifugation direction was the
same as in the first case (Fig. 39). The behaviour of the ectoplasm, the cellsurface and the cells in this case were similar to those in the first case. After
centrifugation, the streaming of ectoplasm appeared simultaneously in the four
cells.
In the S2-cell, the cell-surface at the centrifugal side extends and swells along
with streaming of the ectoplasm, whereas the cell-surface at the centripetal side
contracts. The S2-cell turns clockwise as broken arrow (a) in Fig. 40 shows, and
the P2-cell turns similarly, pushed by the S2-cell. When the ectoplasm of the
S2-cell approaches that of P2-cell, the former streams in accord with the latter,
as if they were connected together. During this process, the cleavage-plane and
its vicinity show bulges of various sizes. The four cells form a rhomboidal shape
(Fig. 44), and swelling of the cells gradually disappears and the opposing
surfaces become flattened. Yolk granules are distributed near the opposing
surfaces, and the ectoplasm is seen only at the free cell-surfaces.
Embryonic polarity in Parascaris and Rhabditis
613
a.p.
Fig. 39-46. Diagrams showing the behaviour of P2-cell in centrifuged embryo of
Parascaris. Figs. 39-43. The embryos soon after centrifugation, and unbroken
arrows show the centrifugal axis, the head being on the centrifugal side. Broken
arrows show the direction of turning of P2-cell, and stippled areas show the first
layers stratified at the centrifugal side. Other unbroken arrows indicate the embryo
that can be derived from each centrifuged embryo of the respective cases (Fig. 44-46).
(3) Centrifugation at the T-stage with the vegetal side at the centrifugal end
(Fig. 41). After centrifugation the ectoplasm of the S2-cell in a small number of
embryos streams either clockwise or counterclockwise. The lateral cell-surface,
where the ectoplasm in the S2-cell began to disperse earlier, shows remarkable
extension, whereas the opposite cell-surface contracts. The S2-cell turns horizontally from one side, where the previously dispersed ectoplasm is concentrated,
to the other, where there is little ectoplasm. In other words, it turns from the
extending side to the contracting. In Fig. 41, broken arrow (a) shows only a
clockwise turning of the P2-cell. Such embryos result in a rhomboidal shape
(Fig. 44).
In other cases a large amount of the ectoplasm disperses to the centripetal
side along the polar axis, while a small amount of ectoplasm remains at the
centrifugal end. During this dispersion, one side of the cell-surface of the S2-cell
extends along the polar axis, while the other side contracts. The P2-cell gradually
moves on top of the S2-cell through a vertical turning as broken arrow (b) shows,
and finally rides over the other three. The embryo becomes a tetrahedral shape
(Fig. 45).
(4) Centrifugation at the T-stage with the animal pole at the centrifugal end
(Fig. 42). In the S2-cell the ectoplasm streamed either clockwise or counterclockwise. If it streams clockwise, the right side of the cell becomes rich with it,
and vice versa. The cell-surface of the 'rich' side extends, while that of the 'poor'
contracts. The ectoplasm and the surface of the P2-cell behave similarly to
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Y. TADANO AND M. TADANO
those of the S2-cell. If the initial streaming of ectoplasm in the S2-cell is clockwise, the right cell-surfaces of the S2-cell swell remarkably and the S2-cell
turns clockwise. A little later the P2-cell turns similarly, as shown by the broken
arrow (a) in Fig. 42, producing a rhomboidal shape (Fig. 44).
On rare occasions the ectoplasm of the S2 and the P2 cells streams vertically
along the polar axis, as shown by the broken arrow (b), and the embryo becomes
a tetrahedral (Fig. 45).
(5) Centrifugation at the three-cell stage, consisting of SI, S2 and P2 cells
(Fig. 43). Direction of the centrifugal force is the same as that of Figs. 39 and 40.
As in the former cases, the surfaces of S2 and P2 cells at the centrifugal end
extended accompanied by streaming of the ectoplasm. The broken arrow (a) in
Fig. 43 shows the clockwise turning of P2-cell, and Fig. 46 is the embryo whose
P2-cell is now at the end of its turning.
In S2 and P2 cells the behaviours of the ectoplasm and the cell surfaces are
also the same as those in the second case. If the SI-cell cleaves horizontally,
the four cells form the rhomboidal shape (Fig. 44). On very rare occasions it
cleaves vertically and the four cells resulted in the tetrahedral shape (Figs. 18,
45). Formation of the rhomboidal or the tetrahedral embryo is affected by the
direction of the mitotic spindle in the Sl-cell during centrifugation.
(b) Rhabditis
The Rhabditis embryo was centrifuged so that the centrifugal axis came
perpendicular to the polar axis. Figs. 19-23 show the embryo of Rhabditis
centrifuged at the beginning of the second cleavage. The centrifugal axis is the
same as in the first example of centrifuged embryos of Parascaris, but in the
present case the centrifugal end is opposite to that in the first case. In Fig. 19
the first layer stratified at the centrifugal side is seen at the left side, and the
fifth layer stratified at the centripetal at the right side.
In some cases as in Fig. 20, the spindle of SI becomes oblique to the polar
axis, because of its rotation. The cell itself becomes oblique to the polar axis, and
the ectoplasm at the centrifugal side streams along the cell surface. The cell
surface at the centrifugal end extends, and in the Sl-cell furrow formation
begins. Subsequently the spindle of the Pl-cell rotates and runs parallel to that
of the Sl-cell (Fig. 21). In the Pl-cell which has arranged itself oblique to the
polar axis, furrow formation begins (Fig. 22). The four cells assume a rhomboidal
shape instead of a ' T ' as in Figs. 14-16 (Figs. 22, 23). Formation of the rhomboidal embryo in this case corresponds to the A-type of the uncentrifugal ones.
Figs. 24 and 25 indicate rotation of the spindles in SI and PI cells in other
embryos. As a result of this rotation, the spindle of Sl-cell lies perpendicular to
the polar axis, and that of Pl-cell on the polar axis. Successively, ectoplasm
streams along the polar axis, and furrow formation begins. Namely, the Sl-cell
is divided into A and B cells by a cleavage plane parallel to the polar axis. In
the Pl-cell the cell-surface at the centrifugal side extends along with streaming of
Embryonic polarity in Parascaris and Rhabditis
615
ectoplasm along the polar axis, while the cell-surface at the centripetal contracts.
Accordingly, the Pl-cell gradually turns counterclockwise and the cleavage
plane formed transversely divides the Pl-cell into S2 and P2 cells. The four cells
resulting from the second cleavage form a sharp T-shape (Fig. 26). Finally the
P2-cell contacts the B-cell, and the embryo becomes a rhomboidal shape. Formation of the embryo in this case corresponds to the B-type of uncentrifuged eggs.
The developmental state, the behaviour of ectoplasm, the turning of cells in
centrifuged embryos of Rhabditis in the cases other than that stated above were
essentially similar to those in corresponding cases of Parascaris.
DISCUSSION
Since the Sl-cell divides earlier than the PI, and the B-cell shifts horizontally
towards the P2-cell before it starts turning, it is possible that the B-cell causes
P2 to turn. Strassen (1903) assumed that in the B-cell there existed a region that
attracts the P2-cell. But, as even when the Pl-cell divides earlier than the Sl-cell,
the P2-cell turns as usual, the behaviour of the animal cell cannot be an indispensable factor in the turning of the P2-cell. Schleip (1929) attached an
importance to the role of the egg shell in forming a rhomboidal embryo. However, in the present experiments a rhomboidal embryo developed without a shell.
In the early cleavage of Parascaris and Rhabditis eggs, extension and contraction of the cell surfaces were always accompanied by streaming of ectoplasm,
followed by movement of the cells (Tadano, 1962). In the later phase of the
second cleavage, the embryo changes from a T-shape into a rhomboidal shape
through turning of the P2-cell. In this respect uncentrifuged embryos were
essentially consistent with centrifuged ones. Streaming of the ectoplasm in the
S2 and P2 cells began before extension and contraction of the surfaces. It is
possible that this streaming induced the extension and contraction of the surfaces and consequently the turning of the P2-cell.
In uncentrifuged embryos of both Parascaris and Rhabditis, the P2-cell
turned in the direction of the initial streaming of ectoplasm at the animal side
of the S2-cell. In centrifuged embryos, the P2-cell turned in the direction of the
initial stream of the ectoplasm at the centrifugal side of S2-cell. Parascaris eggs,
whose ectoplasm was distributed abnormally after a sudden change of temperature, developed into tetrahedral embryos. They became rhomboidal embryos
only after the recovery of the normal distribution of ectoplasm.
In view of these facts, turning of the P2-cell may be attributable directly to
the behaviour of the S2-cell surface induced by the streaming of ectoplasm in the
S2-cell. In the case of uncentrifuged embryos, it seemed that the direction of the
initial streaming of ectoplasm is related to the migration of the nucleus. However,
streaming of the ectoplasm in intensely centrifuged embryos bore no relation to
the behaviour of the nucleus, and extension of the S2-cell surface always began
from the region where a large amount of the ectoplasm had accumulated. These
facts imply that the determination of embryonic polarity is due to the behaviour
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Y. TADANO AND M. TADANO
of the ectoplasm. Arrangement of the blastomeres, however, relates primarily to
the direction of mitotic figure. Accordingly, there is a close interrelation between
the behaviours of the mitotic figure and the ectoplasm in the determination
process of polarity.
According to Bonfig (1925), the direction of turning of the cell is determined
accidentally. However, he gives no explanation as to the mechanism of the
determination based on the behaviour of the cytoplasm. He also stated that the
tetrahedral embryo was caused by an injurious condition. Therefore, a change
in the streaming of the ectoplasm may have resulted from this condition.
In the previous electron microscopy on the Parascaris eggs, endoplasmic
reticulum (E.R.) and often microtubules were seen in the ectoplasm, and many
elongated rows of E.R. and microtubules were observed in the centrifuged eggs
during their recovery from the stratification (Tadano & Tadano (1963),
unpublished). From these facts it seems possible that the active behaviour of
cell surfaces depends on contractility of the ectoplasm which may reside in the
E.R. and microtubules.
Guerrier (1964, 1967) reported that the animal-vegetal polarity of Parascaris
eggs was inverted by means of centrifugation, and that the determination of
polarity depended on the establishment of the cortical differentiation preceding
the first cleavage, which agreed with our findings (Tadano & Tadano, 1961;
Tadano, 1961, 1962). According to his report, however, the egg substance was
stratified into only three layers by centrifugation and this continued until
completion of the first cleavage. There is not much explanation given on the
behaviour of each stratification and the mitotic spindle. On the other hand, we
have indicated previously that the egg substance was densely stratified into five
layers at the one cell stage, and that each of the stratifications dispersed in a
characteristic way (Tadano, 1961, 1962). It seems that these differences from
Guerrier's results are mainly due to the difference in temperature during
centrifugation, as it is difficult to obtain such dense stratification at a high
temperature even under a strong force. Also the stratification recovers its
normal distribution rapidly, and consequently the inversion of the polarity
decreases in rate.
Ziegler (1895) pointed out that embryos of Diplogaster did not show the
T-shape at the four-cell stage, as the Rhabditis eggs of type-A in the present
paper (Figs. 15-16, 22-23). Comparing type A with B, therefore, the difference
between them may be derived from the change in behaviour of the ectoplasm
and the mitotic figure in the change of temperature (Figs. 5-10, 26).
Although there are some differences in the distribution of cytoplasm between
the two daughter cells derived from SI-cell, their fates altered according to the
direction of turning of the P2-cell - that is, by the displacement of its ectoplasm.
Accordingly, it is certain that the daughter cells of SI-cell are still equivalent at
T-stage, and their fates are determined with the turning of P2-cell. This conclusion
agrees with Bonfig (1925).
Embryonic polarity in Parascaris and Rhabditis
617
The authors wish to express their deep sense of gratitude to Professor D. R. Newth,
Department of Zoology, University of Glasgow for valuable suggestions made in the preparation of the manuscript. This study was supported in part by a Grant in Aid for Fundamental
Scientific Research from the Ministry of Education of Japan.
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(Received 26 February 1974)