AX ANALYSIS O F THE FACTORS OF BILATERAL DETERMINATION IN CENTRIFUGED

ECHINODERM EMBRYOS

DAKIEL C. PEASE T h e Hopkins Marine Station, Stanfovd Uniwersity, Pacific Grove, California,

and Californin Instilute of Technology, Pnsadnin, California

THREE PLATES

INTRODUCTION

The following is a statistical study of the types of embryos that develop when the unfertilized eggs of Dendraster cx- centricus are centrifuged at high speeds in an ultracentrifuge. An analysis of the results indicates a probable type of mecha- nism involved in the determination of the bilateral symmetry of the pluteus. It becomes apparent, as others have preri- ously postulated, that the eggs are probably bilaterally pre- disposed before fertilization. The evidence is interpreted to mean that a ‘ventral determinant’ is present as a gradient, probably in the cortical layers of the vegetative hemisphere, which determines the presumptive ventral side at the region of the greatest concentration or activity. This ‘determinant ’ gradient is not shifted by centrifuging, even with an applied force of 45,000 g. However a ‘ snbs t i~~te’ is presumably present that is centrifuged toward the centripetal end of this egg. I t is concluded that the ventral side of the centrifuged eggs is determined Inp the region of the greatest interactivitJ- of these two factors causing a partial arid variable rotation of the presumptive dorsoventral axis.

226 DANIEL C. PEASE

I am particularly indebted to Dr. A. R. Moore of the Uni- versity of Oregon who has offered much helpful advice and placed his centrifuge at my disposal, and to Doctor Fisher, director of the Hopkins Station, who has made this work possible, as well as to Doctor Tyler of the California Institute of Technology who has critically read the manuscript.

TECHNIQUE

The unfertilized eggs of Dendraster excentricus were placed in about 40% sea water and 60% molar (isotonic) neutral sucrose. They were centrifuged in an air-driven ultracentri- fuge producing a calculated force of 46,900 g. with the air

. pressure used. This figure is only approximate inasmuch as the speed was calculated from the musical note. The force applied certainly lies between 45,000 and 50,000 g., based upon accurate calibrations of the centrifuge with a cathode ray oscillograph at slightly higher air pressures. The eggs were centrifuged for 6 to 8 minutes at terminal velocity unless otherwise specified in the results. The eggs elongate, becom- ing flask-shaped or dumb-bell shaped, and are well stratified after such treatment. The amount of centrifuging necessary to accomplish the same stratification varies with the eggs of different individuals. It was also found that the amount of elongation varies considerably with different lots of eggs after constant centrifuging, and this is not necessarily cor- related with the extent of stratification. After centrifuging the eggs were transferred to sea water and fertilized. Only those cultures in which the membranes lifted well were kept and studied.

When centrifuged for 12 minutes the eggs do not develop well, but after such hard centrifuging eight layers may be distinguished. Photographs of such eggs are shown on plate 3. At the centripetal end is the ‘oil cap’ marked layer 1. The second layer is clear cytoplasm, containing the nucleus but no granules. In the living egg it is not refractive under dark- field illumination and is but slightly so in the eggs fixed by osmic acid. Layer 3 makes up the largest bulk of the

ECHINODERM BILATERAL DETERMINATION 227

cytoplasm and is highly refractive and dense. After fixation by osmic acid it is colored a yellow-brown indicating the presence of some lipoids. Neutral red vital staining indicates that it is really a double layer since a thin band, just below the second layer of unstained and clear cytoplasm, is much more rapidly and heavily stained than the rest. Cresyl violet vitally stains this double layer blue, which, in conjunction with the neutral red staining, indicates protein. The layer 4 is clear in the living egg and but slightly refractive in the fixed eggs. Layer 5 is a thin layer not clearly visible in the photographs which can only be seen in the living egg under the highest magnifications. It is coarsely granular and not sharply dilineated toward the centripetal end. It is highly refractive in the fixed eggs, and is unstained by the osmic acid, but heavily stained vitally by the neutral red. Layer 6 is quite homogeneous and is stained darkly by neutral red and osmic acid. Cresyl violet stains it red. The extreme centri- fugal layer 7 is made up of highly refractive granules.

In the centrifuged eggs the first cleavage has a tendency to pass at right angles to the axis of centrifuging across the narrowest neck of the elongated egg. It is often more or less oblique. The following cleavages are very irregular, producing cells of quite unequal size. The second cleavages may also be more or less at right angles to the axis of centri- fuging producing, in extreme cases, four cells linearly ar- ranged. I n the following cleavages the centrifugal and middle portions of the egg divide but the extreme centripetal end, containing the first and second layers and part of the third, does not generally divide further; at most a single longi- tudinal furrow appears. The cells from the centrifugal and middle portions of the egg round up and form a blastula, but the centripetal end is left appended to this blastuIa as a lobe without further cleavage. This stage is well shown in figure 5 on plate 3, which, however, has an unusually large lobe following 12-minute centrifuging.

In these experiments this ‘centripetal lobe’ is used simply as a marker of the axis of centrifuging. Some blastulae

’PIIE JOURNAL OF EXPERIMENTAL ZOOLOOY, VOL. 80, NO. 2

228 DANIEL C. PEASE

pinch off this lobe, but in this report only those plutei with it still attached are considered. All such embryos in a single experiment were isolated in the early pluteus stage and the position of the lobe noted. It was found possible to group the embryos into six somewhat arbitrary categories according to the position of this centripetal lobe.

In the interpretation of the results the assumption is made that there is no orientation of the eggs in the centrifuge tubes. In respect to the polar axis there seems to be no good way of demonstrating this in the Dendraster egg. However E. B. Harvey ( '33) has demonstrated that the Paracentrotus lividus egg does not orient since the orange pigment band may bear any relation to the axis of centrifuging. This band is in the cortical layers and is undisturbed by large centrifugal forces. The results of the present experiments indicate that there can be no orientation in relation to the bilateral axis of the egg.

RESULTS

The position of the centripetal lobe in the pluteus was found to vary in the large majority of cases within very well-defined limits. I n those larvae which differentiated it generally oc- curred in the ventral region attached to ectodermal tissue normally derived from the original vegetative hemisphere of the egg. It rarely appeared above the junction of the anal and oral faces in ectoderm derived entirely from the animal hemisphere. The lobe was generally not found laterally further than the region of the abanal arms. Im relatiom to the polar azis 93.6% of the total number of

differentiated embryos recorded had the centripetal lobe in a region derived in whole or in part from the vegetative hemi- sphere of the egg. These data are included in terms of per cent in the first three vertical columns of table 2, which is based on the quantitative data of table 1. This type is repre- sented by figure 3a, plate 1. The lobe is at, or more vegetative than, the junction of the anal and oral faces. Thus it lies laterally at a region of an arm bud, or in the ventral region between them. The vertical columns 4 and 5 in table 2 show

ECHINODERM BILATERAL DETERMINATION 229

1 2 3 4 5 6 7 8 9

10

that only 3.970 of the recorded embryos had the lobe on the oral face above its juncture with its anal face in a region derived entirely from the animal hemisphere of the egg. Such types are represented by figures 5a and 6a, plate 1, as might be expected if there was no shift in the polar axis. This 3.9% probably represents eggs relatively too little centrifuged.

min.

6 8 8 10 12

TABLE 1 The position of the ceniripetal lobe in the young plutei is tabulated for ten groups

found in each lot with the lobe appended i n the various positions. Experiments 6 to 10 were all eggs taken from a single female

of centrifuged eggs. The figures give the total numbers of individuals

~

8 46 12 15 5 25 17 17 5 8

.~

168 Total for each

locus of lobe

28 90 26

115 14

226 46 31 25

2

603

15 41 18 43

7 55 24 16 13

1

233

3 6 1 1

3 1

1

.~

16

2 3 1 1 2

11

I 26

61 196 60

176 28

323 94 65 45 12

1061

I n addition to the recorded embryos, in all the cultures there was a large per cent which did not gastrulate or dif- ferentiate further than 'mesenchyme blastulae ' with the ap- pended lobe. Generally this group represented about 60% of all the eggs centrifuged, although with prolonged centri- fuging the relative number of such blastulae increased.

A large number of eggs were examined at the gastrulation stage. It was found that almost invariably, when gastrulation occurred, the point of gastrulation was less than 90" from

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230 DAXIEL C. PEASE

the centripetal pole, that is, never at the centrifugal end of the egg.

Coizclusiorzs relating the primary azis with the axis o f cerctrifuging may best be inserted here. Unless there has been some shift in the primary egg axis induced by the great centrifugal force, it appears that gastrulation does not occur when the centripetal end of the Dendraster egg lies in the animal hemisphere. The large proportion of the mesenchyme

TABLE 2 The data presented an table 1 are calculated in terms of perceiitages of the total

f o r each lot

1 2 J

4 5 6 7 8 9

10 Per rent of total Per cent of total Per cent of total

t 0 . Y

45.9 43.3 65.3 50.0 66.9 48.9 4 i . i 5*53 16.6 56.9

Z4.t)

20.9 30.0 24.4 25.0 17.0 25.5 24.6 28.8 8.3

22.0

.9 93.6

IJ .1

23.5 20.0

8.5 17.9

7.7 18.1 26.2 11.1 66.6 14.9 14.9

1.7 0.6

0.9 0.9 1.1 1.1

1.2 2.2 2.2

I

1.5 I 2.4 I

3.9 6.4

2.5

blastulae are probably mostly embryos resulting from such a relation between the axis of centrifuging and the primary egg axis, as are the 3.970 which did differentiate showing such a relation.

The only other apparent interpretation of the results is that the centrifuging has actually induced a rotation of the polar axis. All previous work has shown that the primary axis is undisturbed by centrifuging. Following the work of Lyon ( '06, ' O r ) , Morgan and Lyon ( '07) and 3forgan ( ' O S ) ,

ECHINODERM BILATERAL DETERMINATIOX 231

Morgan and Spooner ( '09) showed conclusively that the stratification induced no rotation of the polar axis in Arbacia when low centrifugal forces were used. E. B. Harvey ( '33), using much greater centrifugal forces, came to the same con- clusion with the Paracentrotus lividus egg. It was found that the eggs always gastrulated in the area of the orange pigment no matter what relation this bore to the axis of centri- fuging.

If there is no shift in the polar axis induced by the centri- fuging the fact that the Dendraster eggs do not gastrulate when the centripetal end lies in the animal hemisphere may possibly be due to the withdrawal of substances from the vegetative hemisphere which are necessary there for gastrula- tion and differentiation. Such substances may well be dis- placed in the endoplasm so that they are concentrated toward the centripetal end.

The data of Lindahl ('32 a) on Arbacia, in which the centri- fugal end influences the ventral determination, bears out the same conclusion. He reports 226 cases with the centrifugal pigment in regions derived from the vegetative hemisphere, and but forty-four are definitely specified as having the pig- ment in regions derived exclusively from the animal hemi- sphere. Re records forty-five more with the pigment in the ectoderm but not in the endoderm. His centrifuge developed less than 6000 g.

An occasional mesenchyme blastula was found that showed tiny spicules so oriented that it was apparent that the centri- petal lobe plugged the region of gastrulation. The bilateral organization of such an embryo was invariably very weak and the bilateral differentiation never progressed beyond the rudi- mentary stage. Runnstrom ('26) has shown that when the centrifugal end of the Psammechinus miliaris egg, which in this form influences the ventral determination, coincides with the vegetative pole very pathological plutei result, although normal plutei are formed if the centrifugal granules are in the endoderm but not in the mesoderm. Since the differenti- ation of the Dendraster eggs is so slight when the centripetal

232 DANIEL C. PEASE

end coincides with the vegetative pole it also seems justified to consider here that some factor is induced, either physical or chemical, that actively blocks the bilateral differentiation.

In relation to the dorsoventral asis it was found that 93.6% of the recorded embryos had the centripetal lobe in the ventral position, median or as lateral as an arm bud, in ectoderm ultimately derived, at least in part, from the vegetative hemi- sphere. Only 2.576 of the differentiated embryos had the lobe in the dorsal or abanal position as indicated in the small figures 8b to llb, plate 2. The embryos with the lobe in the ventral position have been divided into three somewhat arbitrary categories. Of the total, 14.9% had the lobe in a nearly median position as represented by figure 13a, plate 2. In 22% the lobe was in the intermediate position as shown by figure 12a. In 56.9% it was in the lateral position of an arm bud as shown in figvres 9a and 10a.

If the centrifuging had no effect on the ventral determination the centripetal lobe would be expected to have a random distribution around the polar axis in the pluteus and the types represented by the small figures 8b to l l b would be expected to be as common as those with the lobe in the ventral regions. Nor are the dorsal or abanal lobe types not differentiating. In that case the expected ratio between lateral and median types would be 2 : 1 instead of the 4: 1 ratio recorded. Also if the centripetal end directly determined the ventral side by, €or example, concentrating at that end some ‘ventral determinant’ the centripetal lobe would be expected at or near the median position in all cases. Hence it is apparent that the centri- fuging influences the determination of the dorsoventral axis but it cannot act directly through a single factor.

In experiments 6 to 10 the eggs of a single female were subjected to the same centrifugal force for varying periods of time. When centrifuged at terminal velocity for 6 minutes, 66.9% had the lobe in the lateral position and but 7.7% had it median. Experiments nos. 7 and 8 were both subjected to the centrifugal force for 8 minutes and a mean value of 48.4% had the lobe in the lateral position while- 22.1% (mean of the

ECHINODERM BILATEBAL DETERMIXATIOK 233

two experiments) had the lobe in the median position. These figures are statistically significantly different while those for the more prolonged centrifuging are not. From these figures it is apparent that the relative amount of centrifuging, and the consequent stratification of the endoplasm, plays a significant role in the determination of the dorsoventral axis. The greater the centrifuging the greater the per cent of the plutei with the lobe median and the smaller the per cent with the lobe lateral! This seems to be quite independent of the actual elongation of the egg.

COKCLCSIOKS

To interpret these data concerning dorsoventral determina- tion it seems necessary to postulate that two factors are involved. One factor, which may be designated the ‘ventral determinant,’ is to be regarded as being present as a gradient in the unfertilized egg. In normal development the pre- sumptive ventral side is in the region of its greatest concen- tration or activity. This factor can be but slightly, if at all, shifted by the centrifugal forces used, but maintains itself as the original gradient, probably in the cortical layers. To be effective, however, a second factor must be postulated, which may be termed the ‘substrate,’ and which in the normal egg may well be completely diffusely distributed in the endo- plasm. The centrifugal forces may be supposed to displace this substrate toward the centripetal end in the Dendraster egg, and in the centrifuged eggs the ventral side is determined by the region of the greatest interactivity between these two factors. Thus the centripetal end always is found in the ventral field and the harder the centrifuging the greater the centripetal displacement of the substrate, resulting in an in- crease in the relative per cent of the plutei found with the lobe in the median position.

This system is represented diagrammatically on plate 2. The polar axis of the egg and embryos is at right angles to the plane of the paper. Various possible axes of centrifuging around the polar axis are shown. The relative strength of the

234 DANIEL C. PEASE

‘ventral determinant’ gradient is represented by the solid black. The presumptive ventral side, at the region of the greatest ‘determinant’ concentration, is at x. The relative con- centration of the ‘substrate’ in the centrifuged eggs and plutei is represented by the differential stippling. In figure 11, for example, the axis of centrifuging is at right angles to the presumptive dorsoventral axis, y-x. The region of the greatest interactivity between the ‘determinant ’ and the ‘ substrate’ is at the region a, and the ventral side is determined there instead of at x, thus producing a rotation of the presumptive dorsoventral axis of approximately 45”. The amount of axial rotation is the angle included between the radii of x and z. If the substrate is packed more toward the centri- petal end by longer or harder centrifuging the angle is neces- sarily greater since the region of the greatest interactivity between the two factors is shifted more centripetally with the greater removal of substrate from the centrifugal end.

In figure 13 the centripetal end coincides with the pre- sumptive ventral side and there is no rotation of the pre- sumptive dorsoventral axis. The plutei have the lobe in the median position. Figure 12 shows the formation of the inter- mediate types. Figure 9 shows the formation of a lateral type by an axial rotation of about 90”. If the centripetal end is directly opposed to the presumptive ventral side, as shown in figure 8, probably one side generally has dominance over the other and an asial rotation of at least 90” occurs.

If a single factor determined the dorsoventral axis, centri- fuging might be expected either to have no effect or to dis- place it to one end of the axis of centrifuging. In the first case the centripetal lobe would be expected to have a random distribution around the polar axis and the ratio of ventral to lateral to dorsal types should be 1: 2: 1. The ratio found is almost 1: 4: 0. The dorsal types cannot, therefore, be re- garded as simply not differentiating. If a single factor was displaced the centripetal lobe would always be expected in the median position. Neither postulate agrees with the re- corded data which emphasize the only partial effect of great

ECHINODERM BILATERAL DETERMINATIOK 235

centrifugal forces in the determination of the dorsoventral axis. The assumption that two factors are involved, one un- disturbed by the centrifugal forces but normally present as a gradient, the other normally diffusely distributed but dis- placed by the centrifuging, accounts for the 14.9% of the recorded embryos with the lobe in the median position and the 78.9% with it more or less lateral. It also accounts for the greater relative per cent of median types as opposed to lateral following increased centrifuging. This would be ex- pected if the substrate which is displaced is sedimented with difficulty. Thus the amount of dorsoventral axial rotation is increased with greater centrifuging. This would be par- ticularly emphasized in those cases in which the presumptive ventral side is many degrees from the centripetal end.

This interpretation implies that the unfertilized Dendraster egg is already bilaterally determined inasmuch as the ‘ventral determinant’ is present at that time as a gradient which normally determines the ventral side at the region of its greatest concentration or activity. That there is a bilateral determination in the sea urchin egg before fertilization has been suspected for a long time. Horstadius and Wolslry (’36) have reviewed the evidence and added much of significance themselves. They have come to the conclusion that it is the ventral side that is first determined by some factor or factors maintained as a gradient, probably present in the unfertilized egg.

The data presented here seem to indicate that such a bi- lateral determination is present in the unfertilized egg. If the centrifuging had no effect on the bilateral determination, and such determination did not already exist in the unfertil- ized egg, the centripetal lobe would be expected to have a random distribution around the polar axis. Also if there was no bilateral predisposition in the unfertilized egg, but the centrifuging directly induced such a determination, the lobe would be expected in the median plane in all cases. The data do not agree with either postulate and there are only two ap- parent alternates. The Dendraster egg either has a bilateral

236 DANIEL C. PEASE

predisposition before fertilization, or the determination of the dorsoventral axis is related to the sperm entrance point. The results recorded here can, in fact, be adequately interpreted by the same system if the sperm entrance point established a gradient with the other characteristics I have postulated for the ‘ventral determinant’ gradient.

The latter snggestion, however, seems unlikely in view of the recent work of Lindahl and Stordal ( ’37). They have shown that in the Paracentrotus egg, in the large majority of cases, the sperm entrance point coincides nearly with the first cleavage plane. I n the same egg Horstadius and Wolsky ( ’36) have shown that the first cleavage plane bears no relation whatsoever to the dorsoventral axis. I t follows that in the Paracentrotus egg the sperm entrance point is unrelated to the bilateral symmetry. There is no reason for supposing that the relation is any different in the Dendraster egg. Hence it is concluded that in all probability these eggs have a bi- lateral predisposition before fertilization due t p the ‘ventral determinant ’ gradient .

That this is the case is also borne out by the work of Hor- stadius and Wolsky (’36) on the development of partial em- bryos and half eggs. Their conclusions on this point are entirely in agreement with mine and are derived by an entirely different method of approach. Horstadius seems to be of the opinion that it is the ventral side that is first established, and acts as the determinant of the bilateral axis. The demonstra- tion of this does not seem to be too convincing but I have accepted it and use the term ‘ventral determinant’ realizing that this theory may have to be completely reversed if later work should demonstrate that it is the dorsal side that de- termines the bilateral axis. This reversal of the theory would not in itself effect the underlying reasoning that these experi- ments have suggested.

The ‘ventral determinant’ gradient may best be regarded as being present in the cortical layers which are undisturbed by the centrifuging. E. B. Harvey (’33) has shown that the red piepent band of the Paracentrotus lividus egg is not

ECHINODERM BILATERAL DETERMIXATIOK 237

displaced by high centrifugal forces. Lindahl (’32 c) has claimed that the cortical layers of various eggs subjected to centrifugal forces become thinner a t the centripetal end and thicken at the centrifugal. I have most carefully checked this point in the Dendraster egg subjected to a force of 75,000 g. at terminal velocity for 4 minutes and find no visible trace of differential thickening or thinning anywhere in the cortical layers. A magnification of >( 2800 was used with an oil immersion apochromatic objective adapted specially for dark- field work, a 28x compensating occular, and the Leitz bicentric darkfield condenser. Under lower magnifications the differ- ential refraction of the different stratified endoplasmic layers, accompanied by the greater depth of focus, gives an illusion of thinning at the centripetal end and a thickening a t the centrifugal just as is found in the figures of Lindahl. I am convinced that this is an optical deception, and conclude that the stretching induced by the centrifuging is practically uniform over the surface of the egg, and that there is no displacement of the cortical layers.

The induction of the ventral side a t the front end of a sea urchin egg entering a capillary pipette, studied by Boveri ( ’ O l ) , and more extensively by Lindahl ( ’32 b), is probably a cortical effect. The latter author reports that a greater perme- ability is induced a t the front end. I have stretched eggs that have previously been locally stained with a spot of vital neutral red. There is little if any endoplasmic displacement or movement other than that induced by the simple com- pression. The cortical layers at the front are undoubtably stretched by the suction force. Since the ventral side is induced a t the front end of such an egg the ‘determinant’ may well be in those layers and is ‘activated’ by the stretching.

There is no reason for supposing that the ‘substrate’ is present as anything other than diffusely in the endoplasm, a t least of the vegetative hemisphere. Since the first two stratified layers, the oil cap and the clear layer, make up the bulk of the centripetal lobe it is probable that it is concen- trated in the third laper. This layer is the largest in the

238 DANIEL C. PEdSE

egg and seems to contain both lipoids and proteins. Toward the centrifugal end this layer has not a clearly defined border, even after prolonged centrifuging. Greater centrifuging, however, slowly concentrates it. It has been pointed out that the relative percentages with the centripetal lobe in the median and intermediate positions increases with longer centrifuging. This also slowly packs this layer more toward the centripetal end. The third layer certainly goes into the ventral field region and probably contains the ‘substrate.’

In conclusion it might be suggested that the ‘determinant’ may be an enzyme adsorbed in the cortical layers and that, for its action, an endoplasmic substrate is necessary that is concentrated in the third layer. The ventral side is deter- mined by the region of the greatest interactivity between the undisturbed enzyme gradient and the stratified substrate.

DISCUSSION

Runnstrom ( ’26) centrifuged Psammecliinus miliaris eggs at forces of about 9000 g. He found that in this form the centrifugal end influenced the ventral determination. How- ever, out of 435 cases reported, 133 had the centrifugal granules more or less lateral to the median line, and in 208 cases the granules are reported as diffusely distributed in the oral field. Only twenty-seven are described as being perfectly median. Although Runnstrom was of the opinion that the centrifugal end directly determined the ventral side, his data do not support the contention, but show, rather, the partial rotation I have reported for Dendraster.

Lindahl ( ’32 a) found that Psammechinus microtuberculatus had the ventral side determined in the region of the centripetal end. Arbacia had the ventral side induced near the centri- fugal end. I n the latter eggs only 64% are reported with the centrifugal granules symmetrically placed on the ventral side. He also found that the centripetal fragments developed with the extreme centripetal granules in the ventral field. A minority, sixteen, were found with the granules ventral sym- metrical. The majority, twenty-seven, had them ventral

ECHINODER,BT BILATERAL DETERMINATION 239

oblique or lateral. I n this case two substrates may be in- volved, the centrifugal one being dominant in the whole egg. When this is removed by fragmenting a centripetal substrate is substituted and bilateral differentiation occurs with the extreme centripetal end in the ventral field. Lindahl used forces of about 6000 g. His data on P. microtuberculatus are not precise enough to be analyzed in the way I have considered the other data.

SUMMARY

1. The unfertilized eggs of Dendraster, when centrifuged for 6 to 8 minutes at over 45,000 g., are sedimented into eight endoplasmic layers and, after fertilization, some develop into fairly normal plutei but with an uncleaved extreme centri- petal lobe attached.

2. It was found that 93.6% of the recorded embryos had the centripetal lobe in the ventral region at the angle formed by the junction of the anal and oral faces.

3. Of these 56.9% had the lobe in the lateral position of the arms. The lobe was nearly median and ventrally placed in 14.9% of the recorded embryos. In 2270 it mas found in an intermediate position. 4. From these data it is concluded that the egg is probably

bilaterally predisposed before fertilization, and that the centri- fuging does not directly determine the dorsoventral axis but induces a partial rotation OF the presumptive axis.

5 . The amount of rotation of the presumptive dorsoventral axis induced by the centrifuging is a function of the relative amount of centrifuging and hence the relative amount of displacement or stratification of endoplasmic constituents.

6. The amount of axial rotation is also a function o'f the angle between the presumptive ventral side and the centri- petal end of the axis of centrifuging.

7. Two factors are postulated to determine the dorsoventral axis in this egg.

8. In the normal egg the ventral side is determined by the region of the greatest concentration or activity of the 'ventral

240 DANIEL C . PEASE

determinant’ gradient. This factor is present as a gradient in the unfertilized egg and is but slightly, if at all, shifted by the centrifugal forces used. It is probably located in the cortical layers and it is suggested that it may be an enzyme adsorbed or bound in those layers in a gradient of unequal strength.

9. For its action a ‘substrate’ is needed that is probably diffusely distributed in the endoplasm of the normal egg, but which concentrates near the centripetal end of the Dendraster egg following centrifuging. This is probably in the third stratified layer.

10. I n the centrifuged eggs the ventral side is determined at the region of the greatest interactivity between these two factors inducing the observed partial rotation of the pre- sumptive ventral side, proportional to the amount of endo- plasmic stratification, and a function of the angle between the centripetal end and the presumptive ventral side of the normal egg.

11. Only 3.9% of the recorded embryos had the centripetal lobe of the pluteus in a region derived from the animal hemi- sphere of the egg. From this low per cent it is concluded that the centrifuging probably removes some substance (or substances) from the vegetative hemisphere to the animal, when the centripetal end is in the animal region, that is neces- sary f o r gastrulation and differentiation. The converse of animal hemisphere material driven into the vegetative hemi- sphere may inhibit the gastrulation.

12. When the centripetal pole coincides with the vegetative pole gastrulation cannot take place because of the mechanical plug. It is also probable that further differentiation is activdy inhibited by some physical or chemical factors.

13. The interpretation of these data presupposes that either the sperm entrance point gives the egg a bilateral disposition or that the egg has a bilateral predisposition before fertiliza- tion. Other evidence seems to eliminate the first postulate and support the latter.

ECHINODERM BILATERBL DETERMINATION 241,

LITERATURE CITED

BOYERI, TH. 1901 Uber die Polaritat des Seeigeleies. Verh. P1iys.-med. Ges.

HARVIY, E. B. Development of the parts of sea urchin eggs separated

H~~RSTADIUS, S., AND A. WOL~SKY 1936 Studien uber die Determination der Arch. f. Entw.-mech.,

LINDAHL, P. E. 1932 a Zur experimentellen Analyse der Determination der 11. Versuche init zentrifugierten

Wurzburg, Bd. 34. 1933

by centrifugal force. Biol. Bull., vol. 64, p. 125.

Bilateralsymmetrie des Jungen Seeigelkeimes. Bd. 135, 8. 69.

Dorsoventralachse beim Seeigelkeim. Eiern. Arch. f . 3:ntw.-meeh., Bd. 127, S. 323.

1932 b Idem. I. Versuehe mit gestreckten Eiem. Ibid., S. 300. 1932c h e r den feineren Bau der Rinde des Seeigeleies. Proto-

plasma, Bd. 16, S. 378. LINDAHL, P. E., AND A. STORDAL 1937 Uber die Determination der Xichtung der

ersten Furohe im Seeigelei. Arch. f . Entw.-mech., Bd. 136, 8. 286. LYON, E. P. 1906 Some results of centrifuging the eggs of Arbacia. Proc.

Am. Physiol. SOC., Am. J. Physiol., vol. 15, p. xxi. 1907 Results of centrifuging eggs. Arch. f . Entw.-inech., Bd. 23,

S. 151. MORGAN, T. H. 1908 The location of embryo-forming regions in the egg.

BJence, vol. 28, p. 287. MORGAN, T. H., AND E. P. L Y ~ N 1907 The relation of the substances of the

egg, separated by strong centrifugal force, to the location of the embryo. Arch. f . Ei1tw.-mech., Bd. 24, S. 147.

MORGAN, T. H., AND G. B. SPOONER 1909 The polarity of the centrifuged egg. Arch. f. Entw.-mech., Bd. 28, S. 104.

RUNNSTROM, J. 1926 Experimentelle Bestimmung der Dorso-Ventralachse bei dem Seeigelkeim. Arch. f . Zool., Bd. 18A.

PLATE I

EXPLANATION OF FIGURES

Various axes of centrifuging in relation to the polar axis are represented diagrammatically. Assuming that the polar axis remains constant, the centripetal lobe, c.L, appears in the early plnteus in the positions iiidiested by figures 3a, 40 and 5a.

242

ECHIXODERM BILATERAL DETERMINATION DAXIEI. C . PEASE

PLATE 1

243

PLATE 2

EXPLANATION OF FIGURES

Considering the axis of centrifuging to be in the position iiidieated by figures 3 and 38 (pl. l), the relation of the centripetal lobe to the bilateral axis around the p01a.r axis is shown. The presumptive dorsoventral axis is represented by y-x. The relative strength of the ‘ventral determiuant ’ gradient, which is not shifted by the centrifuging, is indicated by the solid black. The region of the maximum concentration of this ‘determinant’ is a t x, the presumptive ventral side. The concentration of the centripetal ‘substrate ’ is represented by the graded stippling. The region of greatest interactivity between these two factors where the ventral side is determined in the centrifuged eggs is marked by z.

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ECHINODERM BILATERAL DETERMINATION DANIEL 0. PEASE

PLATE 2

245

PLATE 3

EXPIANATION OF FIQUEES

Figure 1 ia the living egg stratified by the centrifuging. Figure 3 is the same figures 2 and 4 are of an egg fixed in osmie

Figure 5 is a live blastula With egg with darkfield illumination. acid viewed by transmitted and darkfield light. the centripetal lobe attached.

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ECHISODERM BILATERAL DETER;\IIXATIOK D A N I E L C. PEASE

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TIIH JOURNAL O F ESPERIJIEKTAL ZOOLOGY, TOL. 80, KO. 2

PLATE 3