J. Embryol. exp. Morph. Vol. 32, 3, pp. 603-617, 1974 6 0 3

Printed in Great Britain

On the mechanism of determination of embryonicpolarity in Parascaris and Rhabditis

By YANAGI TADANO1 AND MASASHI TADANO2

From the Department of Anatomy, Nagoya University,and Biological Laboratory, Gifu University

SUMMARYIn 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 Parascarisequorum, 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 secondcleavage embryos become T-shape. Along with streaming of ectoplasm at the animal side ofS2-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 rhomboidalin shape and their axes are established. The extended side of S2-cell points roughly to theventral side of the embryo, and the other to the dorsal.

In the centrifuged embryos, extension and contraction of cell-surfaces and turning ofP2-cell take place also accompanied by streaming of the ectoplasm at the centrifugal sideof S2-cell.

It is concluded from these facts that the determination of embryonic polarity depends onthe turning of the P2-cell by the extension and the contraction of the surfaces of S2-celland that the direction of this turning depends on that of the primary streaming ofectoplasm in S2-cell. It is assumed that the direction of the streaming is due to themigration of the nucleus, and that the extension and contraction of cell-surfaces is basedon the behaviour of the E.R. and microtubules in the ectoplasm. The tetrahedral embryo iscaused by a change in the streaming of ectoplasm. The formation of a rhomboidal embryoin Rhabditis without a preceding T-stage is discussed in connection with the behaviour ofthe ectoplasm.

INTRODUCTION

Embryonic polarity is a very important feature of all embryos, since thedirection of subsequent development is marked out by the axis of polarity.

Although many studies have been made on the embryonic development ofParascaris we have as yet little information on the fundamental nature ofits 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 Univer-sity, Gifu-shi, Japan.

604 Y. TADANO AND M. TADANO

1924; Bonfig, 1925; Schleip, 1929). Particularly, it seems that the mechanism ofdetermination of embryonic polarity is still an open question.

Concerning polarity in ripe eggs of Parascaris, it has been previously indicatedby us that both animal-vegetal polarity and embryonic polarity could beinverted by centrifuging in such a way that the centrifugal end came to thevegetal side, and it has been concluded that polarity is based on the gradientin the distribution of ectoplasm (Tadano & Tadano, 1961; Tadano, 1961, 1962).Guerrier (1964, 1967) confirmed the inversion of the animal-vegetal polarityin Parascaris eggs by means of centrifuging.

In ripe eggs there is an animal-vegetal gradient represented by a characteristicpattern in the distribution of ectoplasm. Each of the blastomeres which arosefrom successive division also shows this pattern corresponding to the prospec-tive fate of respective blastomeres. At the four-cell stage the embryo changesfrom a T-shape into a rhomboidal shape as a result of the horizontal turning ofP2-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 earlycleavage active streaming, consumption and formation of ectoplasm are seen,and movements of the cells almost always occur at the same time as this stream-ing. These phenomena lead us to postulate that turning of the vegetal cell isclosely related to the behaviour of the ectoplasm and that the explanation ofboth phenomena has a deep significance in the determination of embryonicpolarity.

For this reason an attempt has been made to explain the relation between thebehaviour of the ectoplasm and the turning of the P2-cell.

MATERIALS AND METHODS

Fertilized eggs of Parascaris equorum Goeze, Rhabditis sp. and Rhabditisikedai Tadano were used. Only ripe eggs after completion of the perivitellinespace were separated from the uterus and used. The egg-membranes of Para-scaris from the first outer layer to the outer part of the inmost fifth layer wereusually 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 ofyolk substance, it was difficult to trace the behaviour of ectoplasm. Thereforesome of these eggs were centrifuged with a force of 10000-20000 g for 1-2 hat a temperature of 10 °C. In the Parascaris eggs centrifuged at the one- or two-cell stage many of the stratifications of cell substance recovered normal dis-tribution before the T-stage. Therefore Parascaris embryos were centrifugedat the three-cell stage or in the T-stage. In Rhabditis some embryos from un-centrifuged eggs do not show a sharp three-cell stage or T-stage and eggscentrifuged at the one-cell stage show quick recovery of normal distribution of

Embryonic polarity in Parascaris and Rhabditis 605cell substances. Therefore, to follow behaviour of the ectoplasm in the S2 cell,Rhabditis embryos were centrifuged at the two-cell stage. The observationson both uncentrifuged and centrifuged eggs were carried out at a temperaturebetween 25 and 30 °C.

RESULTS

The first and second cleavage of uncentrifuged eggs ofParascaris and Rhabditis

(a) Parascaris

The ripe egg of Parascaris shows a visible gradient in the distribution ofectoplasm. 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 ofthe 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° withdevelopment of the mitotic figure. Finally it lies along the polar axis and at thistime furrow formation begins. This is accompanied by streaming of the ecto-plasm 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, andthe Pl-cell more of the endoplasm. In the former the ectoplasm is rich at theanimal side, while in the latter at the vegetal (Fig. 1).

In the later phase, extension and contraction of the opposing faces and theirvicinity occur accompanying the streaming of the ectoplasm. Thus, the twoblastomeres elongate in the direction of the egg axis and then, they contract inthe same way. The elongation alternates between the two blastomeres.

In the early phase of the second cleavage, the mitotic spindle of the Sl-cellappears on the polar axis and then turns through 90° to lie perpendicular to thepolar axis. During this turning ectoplasm streams from the animal side to thespindle poles, so that it shows an uneven distribution, with most at the spindlepoles and least in the equatorial region. After this, streaming of ectoplasm fromthe 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 thepolar axis cuts the SI into A and B cells. In most cases the A-cell contains a littlemore 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 thepolar axis. Turning of the spindle causes migration of ectoplasm into both theanimal and the vegetal region (Fig. 27). When the cleavage furrow appearstransversely to the polar axis, the Pl-cell divides into a larger S2-cell and asmaller P2-cell (Fig. 3). The S2-cell contains larger amount of the ectoplasmthan the P2 (Fig. 28). With the appearance of the second cleavage furrow thesefour cells come to T-shape (Fig. 4).

In the later phase of this cleavage migration of nuclei is seen, accompanyingthe 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 ofuncentrifuged 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 Aand 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 ofP2-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 ofP2-ceIl before division of Sl-cell.

contraction and extension of the surfaces are more delicate than those in thefirst cleavage. Accompanying the streaming of ectoplasm these four cells beginto shift. These events appear first in the A and B cells, while in the S2-cellstreaming of the ectoplasm, and marked extension and contraction of the lateralfree 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, thenucleus in the S2-cell migrates towards the right side, and the ectoplasm at theanimal side streams counterclockwise. Accompanying this streaming, the leftlateral cell surface extends, while the right surface contracts. Thus the S2-cellalways turns counterclockwise. Similarly, the P2-cell turns in the direction of theprimary streaming of ectoplasm in the S2-cell, that is, from the extending sideof the cell surface of the S2-cell to the contracting side. It gradually approachesthe B-cell which also moves closer to the P2. Finally the P2-cell adheres to theB-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 andthe P2 cells adhere to one another. Gradually the embryo becomes a rhomboidalshape.

Until this stage the ectoplasm locally disappears, accompanying the streamingto the periphery. Ultimately the free surface of cells is surrounded by the ecto-plasmic zone, and yolk granules are comparatively dense near the fused faces ofcells. At this time the swellings on the opposing faces gradually disappear, andthe embryo becomes a regular rhomboidal shape (Figs. 11, 36-38).

During turning of the cells, Golgi bodies disperse markedly, indicatingvacuolation. At the rhomboidal stage, the A and the B cells point to the dorsalside of the embryo, and the S2 and the P2 cells to the ventral; the P2-cell points

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Y. TADANO AND M. TADANO

Figs. 14-17. Photographs of rhomboidal formation and behaviour ofuncentrifuged 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, Centri-fugal side; c.p., centripetal side.

Embryonic polarity in Parascaris and Rhabditis

19 , _"•/>••

609

' v.p

a.p.

r.p.

Figs. 19-26. Centrifuged embryos of Rhabditis. Figs. 19-23. A-type of rhomboidalformation. Fig. 19. Two-cell stage. Fig. 20. Beginning of cleavage furrow forma-tion of Sl-cell. Fig. 21. Rotation of spindle in Pl-cell. Fig. 22. Turning of P2-cellfrom the centrifugal side to the centripetal. Beginning of furrow formation in Pl-cell. Fig. 23. Rhomboidal stage. Figs. 24-26. Rhomboidal formation throughpassing T-shape (B-type).

610 Y. TADANO AND M. TADANO

27

Figs. 27-38. Diagrams showing the behaviour of the ectoplasmic zone in cells ofuncentrifuged embryos of Parascaris during the second cleavage. Stippled area ineach cell indicates the ectoplasmic zone, and arrow the direction of ectoplasmicstreaming. All figures are frontal views. Fig. 27. Just before T-stage. a.p., Animalpole; v.p., vegetal pole. Fig. 28. T-stage. Figs. 39-30. Extension of right lateralfaces and contraction of left ones of S2 and P2 cells. Beginning of counterclockwiseturning of P2-cells. Figs. 31-34. Approach of P2-cell to B, and extension ofA and B cell. Fig. 35. Adhesion of P2-cell to B. Figs. 36-38. Formation ofrhomboidal embryo.

to the posterior portion, and the A-cell to the anterior. In this way the embryonicaxis 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 andcontraction of the cell surface in S2-cell appear on the polar axis prior to thisturning 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 perpendicularto the longitudinal axis of the egg shell, the embryo assumes a tetrahedralshape temporarily, and then goes to a rhomboidal shape. In eggs of the one-celland two-cell stage stored at a temperature of 5 °C, the ectoplasmic region oftenbecomes indistinguishable from the endoplasmic region after a sudden exposureto a temperature of 25 °C; these eggs also result in tetrahedral embryos, which

Embryonic polarity in Parascaris and Rhabditis 611only later become rhomboidal. However, when eggs were repeatedly exposed tothis sudden change of temperature, the ectoplasm became loose and yolkgranules dispersed to the periphery. These eggs also gave rise to the tetrahedralembryos, which did not become rhomboidal, and finally resulted in variousmalformed embryos. When the eggs kept at low temperature were slowlyexposed to room temperature, the distribution of cell substance did not change;these eggs resulted in normal embryos. When the Pl-cell divided earlier thanthe SI-cell, the P2-cell turned horizontally (Fig. 13), and the four cells resultingafter 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 essentiallysimilar to Parascaris eggs. In ripe eggs of Rhabditis, however, the boundarybetween ectoplasm and endoplasm was comparatively indistinct. Because ofthis, 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-cellappears perpendicular to the polar axis, and then becomes oblique because of itsrotation. During this process ectoplasm of SI-cell streams rapidly from thepolar region to the equatorial. Accompanying this, the free cell surface extends,while the cell surface contacting the Pl-cell contracts. Consequently, the Sl-cellnoticeably elongates towards the spindle poles, and then cleaves into A and Bcells (Fig. 14). On the other hand, the spindle of Pl-cell appears on the polaraxis and rotates in a similar fashion to that of Sl-cell. Ultimately it runs parallelto 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 slightlyearlier than that of the Pl-cell, but elongation of both cells finishes almostsimultaneously.

The higher the temperature, the more rapid is the streaming of ectoplasmand the greater is the elongation of the spindle. When the cell divides at a lowtemperature, the streaming of the ectoplasm is inactive and the embryo forms aT-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 inverseorder; the P2-cell turns horizontally before division of the Sl-cell. Turning ofthe 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 isstratified into five layers. After the treatment, each of these layers recovers itsprimary distribution in a characteristic way. During this process, a large amountof cytoplasm is always secondarily formed at the centrifugal side from the heavybrown granular layer, the mitochondrial layer and the hyaloplasmic layer.

612 Y. TADANO AND M. TADANO

When the intensely centrifuged egg or embryo begins to divide soon afterbeing centrifuged, a large amount of the secondarily formed ectoplasm remainsat the centrifugal side. If division takes place a considerable time after centri-fugation, a greater part of the ectoplasm recovers its normal distribution. Inmost cases stratification at the centrifugal side disperses towards the centripetalside, and vice versa.

(a) Parascaris

The centrifuged embryos were classified into the following five cases, accord-ing to the developmental stages of embryos and to the direction of the centri-fugal axis to the polar axis.

(1) Centrifugation axis perpendicular to the polar axis. Fig. 39 represents theParascaris embryo soon after centrifugation at the three-cell stage. The unbrokenarrow shows the direction of the centrifugal axis, and a broken arrow (a) thedirection of horizontal turning of the vegetal cell. The centrifugal end is on theright side and the centripetal on the left. The stippled area indicates the heaviestlayer of cell substance in the centrifuged embryo.

After centrifuging, the spindle of Pl-cell always lies on the polar axis. Thecleavage plane formed transversely cuts the Pl-cell into S2 and P2 cells and thefour cells form T-shape. At this stage a large amount of the ectoplasm remainsat the centrifugal side in S2 and P2 cells, though some of it streams along thecell-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 fromthe Pl-cell. They turn clockwise, and the embryo becomes a rhomboidalshape (Fig. 44). The free cell-surfaces often show varying expansions, butthey gradually become spherical shape.

(2) Centrifugation at the T-stage (Fig. 40). Centrifugation direction was thesame as in the first case (Fig. 39). The behaviour of the ectoplasm, the cell-surface and the cells in this case were similar to those in the first case. Aftercentrifugation, the streaming of ectoplasm appeared simultaneously in the fourcells.

In the S2-cell, the cell-surface at the centrifugal side extends and swells alongwith streaming of the ectoplasm, whereas the cell-surface at the centripetal sidecontracts. The S2-cell turns clockwise as broken arrow (a) in Fig. 40 shows, andthe P2-cell turns similarly, pushed by the S2-cell. When the ectoplasm of theS2-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 andits vicinity show bulges of various sizes. The four cells form a rhomboidal shape(Fig. 44), and swelling of the cells gradually disappears and the opposingsurfaces become flattened. Yolk granules are distributed near the opposingsurfaces, 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 ofParascaris. Figs. 39-43. The embryos soon after centrifugation, and unbrokenarrows show the centrifugal axis, the head being on the centrifugal side. Brokenarrows show the direction of turning of P2-cell, and stippled areas show the firstlayers stratified at the centrifugal side. Other unbroken arrows indicate the embryothat 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 ofembryos streams either clockwise or counterclockwise. The lateral cell-surface,where the ectoplasm in the S2-cell began to disperse earlier, shows remarkableextension, whereas the opposite cell-surface contracts. The S2-cell turns hori-zontally from one side, where the previously dispersed ectoplasm is concentrated,to the other, where there is little ectoplasm. In other words, it turns from theextending side to the contracting. In Fig. 41, broken arrow (a) shows only aclockwise 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 centripetalside along the polar axis, while a small amount of ectoplasm remains at thecentrifugal end. During this dispersion, one side of the cell-surface of the S2-cellextends along the polar axis, while the other side contracts. The P2-cell graduallymoves 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 counter-clockwise. 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

614 Y. TADANO AND M. TADANO

those of the S2-cell. If the initial streaming of ectoplasm in the S2-cell is clock-wise, the right cell-surfaces of the S2-cell swell remarkably and the S2-cellturns clockwise. A little later the P2-cell turns similarly, as shown by the brokenarrow (a) in Fig. 42, producing a rhomboidal shape (Fig. 44).

On rare occasions the ectoplasm of the S2 and the P2 cells streams verticallyalong the polar axis, as shown by the broken arrow (b), and the embryo becomesa 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 endextended accompanied by streaming of the ectoplasm. The broken arrow (a) inFig. 43 shows the clockwise turning of P2-cell, and Fig. 46 is the embryo whoseP2-cell is now at the end of its turning.

In S2 and P2 cells the behaviours of the ectoplasm and the cell surfaces arealso 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 itcleaves vertically and the four cells resulted in the tetrahedral shape (Figs. 18,45). Formation of the rhomboidal or the tetrahedral embryo is affected by thedirection of the mitotic spindle in the Sl-cell during centrifugation.

(b) Rhabditis

The Rhabditis embryo was centrifuged so that the centrifugal axis cameperpendicular to the polar axis. Figs. 19-23 show the embryo of Rhabditiscentrifuged at the beginning of the second cleavage. The centrifugal axis is thesame as in the first example of centrifuged embryos of Parascaris, but in thepresent case the centrifugal end is opposite to that in the first case. In Fig. 19the first layer stratified at the centrifugal side is seen at the left side, and thefifth layer stratified at the centripetal at the right side.

In some cases as in Fig. 20, the spindle of SI becomes oblique to the polaraxis, because of its rotation. The cell itself becomes oblique to the polar axis, andthe ectoplasm at the centrifugal side streams along the cell surface. The cellsurface at the centrifugal end extends, and in the Sl-cell furrow formationbegins. Subsequently the spindle of the Pl-cell rotates and runs parallel to thatof the Sl-cell (Fig. 21). In the Pl-cell which has arranged itself oblique to thepolar axis, furrow formation begins (Fig. 22). The four cells assume a rhomboidalshape instead of a 'T ' as in Figs. 14-16 (Figs. 22, 23). Formation of the rhom-boidal 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 otherembryos. As a result of this rotation, the spindle of Sl-cell lies perpendicular tothe polar axis, and that of Pl-cell on the polar axis. Successively, ectoplasmstreams along the polar axis, and furrow formation begins. Namely, the Sl-cellis divided into A and B cells by a cleavage plane parallel to the polar axis. Inthe Pl-cell the cell-surface at the centrifugal side extends along with streaming of

Embryonic polarity in Parascaris and Rhabditis 615ectoplasm along the polar axis, while the cell-surface at the centripetal contracts.Accordingly, the Pl-cell gradually turns counterclockwise and the cleavageplane formed transversely divides the Pl-cell into S2 and P2 cells. The four cellsresulting from the second cleavage form a sharp T-shape (Fig. 26). Finally theP2-cell contacts the B-cell, and the embryo becomes a rhomboidal shape. Forma-tion 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 incentrifuged embryos of Rhabditis in the cases other than that stated above wereessentially similar to those in corresponding cases of Parascaris.

DISCUSSION

Since the Sl-cell divides earlier than the PI, and the B-cell shifts horizontallytowards the P2-cell before it starts turning, it is possible that the B-cell causesP2 to turn. Strassen (1903) assumed that in the B-cell there existed a region thatattracts 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 in-dispensable factor in the turning of the P2-cell. Schleip (1929) attached animportance to the role of the egg shell in forming a rhomboidal embryo. How-ever, in the present experiments a rhomboidal embryo developed without a shell.

In the early cleavage of Parascaris and Rhabditis eggs, extension and contrac-tion of the cell surfaces were always accompanied by streaming of ectoplasm,followed by movement of the cells (Tadano, 1962). In the later phase of thesecond cleavage, the embryo changes from a T-shape into a rhomboidal shapethrough turning of the P2-cell. In this respect uncentrifuged embryos wereessentially consistent with centrifuged ones. Streaming of the ectoplasm in theS2 and P2 cells began before extension and contraction of the surfaces. It ispossible that this streaming induced the extension and contraction of the sur-faces and consequently the turning of the P2-cell.

In uncentrifuged embryos of both Parascaris and Rhabditis, the P2-cellturned in the direction of the initial streaming of ectoplasm at the animal sideof the S2-cell. In centrifuged embryos, the P2-cell turned in the direction of theinitial stream of the ectoplasm at the centrifugal side of S2-cell. Parascaris eggs,whose ectoplasm was distributed abnormally after a sudden change of tempera-ture, developed into tetrahedral embryos. They became rhomboidal embryosonly after the recovery of the normal distribution of ectoplasm.

In view of these facts, turning of the P2-cell may be attributable directly tothe behaviour of the S2-cell surface induced by the streaming of ectoplasm in theS2-cell. In the case of uncentrifuged embryos, it seemed that the direction of theinitial streaming of ectoplasm is related to the migration of the nucleus. However,streaming of the ectoplasm in intensely centrifuged embryos bore no relation tothe behaviour of the nucleus, and extension of the S2-cell surface always beganfrom the region where a large amount of the ectoplasm had accumulated. Thesefacts imply that the determination of embryonic polarity is due to the behaviour

616 Y. TADANO AND M. TADANO

of the ectoplasm. Arrangement of the blastomeres, however, relates primarily tothe direction of mitotic figure. Accordingly, there is a close interrelation betweenthe behaviours of the mitotic figure and the ectoplasm in the determinationprocess of polarity.

According to Bonfig (1925), the direction of turning of the cell is determinedaccidentally. However, he gives no explanation as to the mechanism of thedetermination based on the behaviour of the cytoplasm. He also stated that thetetrahedral embryo was caused by an injurious condition. Therefore, a changein the streaming of the ectoplasm may have resulted from this condition.

In the previous electron microscopy on the Parascaris eggs, endoplasmicreticulum (E.R.) and often microtubules were seen in the ectoplasm, and manyelongated rows of E.R. and microtubules were observed in the centrifuged eggsduring their recovery from the stratification (Tadano & Tadano (1963),unpublished). From these facts it seems possible that the active behaviour ofcell surfaces depends on contractility of the ectoplasm which may reside in theE.R. and microtubules.

Guerrier (1964, 1967) reported that the animal-vegetal polarity of Parascariseggs was inverted by means of centrifugation, and that the determination ofpolarity depended on the establishment of the cortical differentiation precedingthe first cleavage, which agreed with our findings (Tadano & Tadano, 1961;Tadano, 1961, 1962). According to his report, however, the egg substance wasstratified into only three layers by centrifugation and this continued untilcompletion of the first cleavage. There is not much explanation given on thebehaviour of each stratification and the mitotic spindle. On the other hand, wehave indicated previously that the egg substance was densely stratified into fivelayers at the one cell stage, and that each of the stratifications dispersed in acharacteristic way (Tadano, 1961, 1962). It seems that these differences fromGuerrier's results are mainly due to the difference in temperature duringcentrifugation, as it is difficult to obtain such dense stratification at a hightemperature even under a strong force. Also the stratification recovers itsnormal distribution rapidly, and consequently the inversion of the polaritydecreases in rate.

Ziegler (1895) pointed out that embryos of Diplogaster did not show theT-shape at the four-cell stage, as the Rhabditis eggs of type-A in the presentpaper (Figs. 15-16, 22-23). Comparing type A with B, therefore, the differencebetween them may be derived from the change in behaviour of the ectoplasmand the mitotic figure in the change of temperature (Figs. 5-10, 26).

Although there are some differences in the distribution of cytoplasm betweenthe two daughter cells derived from SI-cell, their fates altered according to thedirection 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 atT-stage, and their fates are determined with the turning of P2-cell. This conclusionagrees with Bonfig (1925).

Embryonic polarity in Parascaris and Rhabditis 617The 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 prepara-tion of the manuscript. This study was supported in part by a Grant in Aid for FundamentalScientific Research from the Ministry of Education of Japan.

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BOVERF, TH. (1910). Die Potenzen der /4sctfm-Blastomeren bei abgeanderter Furchung. InFestschrift}. R. Her twig 3, 131-214. Jena: G. Fisher.

GUERRIER, P. M. (1964). Fixation de la polarite chez Parascaris equorum. C.r. hebd. Seanc.Aead. Sci., Paris 258, 3566-3568.

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(Received 26 February 1974)