Development of Dorsoventral Polarity and Mesentoblast Determination in Patella vulgata J. A. M. VAN DEN BIGGELAAR ' Station Biologtque, 29.21 1 Roscoff;France

ABSTRACT In Patella vulgata the 32-cell stage represents a pause in the mitotic activity prior t o the differentiation of the mesentoblast mother cell 3D. A t the onset of this stage, the embryo is radially symmetrical. Nevertheless, the plane of bilateral symmetry is indicated as it passes through the macromeres forming the vegetal cross-furrow. From the early beginning of the 32-cell stage, all four macromeres intrude far into the interior and touch the centrally radiating cells of the first quartet of micromeres. The two cross-furrow forming macromeres (3B and 3D) intrude the farthest and come into contact with the greatest number of micromeres. Finally, the contacts are extended significantly and maintained with only one of these macromeres. From that moment, this cell can be called the macromere 3D and the dorsoventral axis is determined. The evolution of the internal cell contacts between the micromeres of the first quartet and the macromeres indicates an essential role of the former in the determination of one of the latter as the mesentoblast mother cell. and thus in the determination of dorsoventral polarity.

A t the turn of the century, the origin of the primary mesoderm in molluscs, annelids and polyclads was studied and discussed extensively. It appears to arise from the macromere 3D after formation of the first three quartets of micromeres, which form the whole ectoderm. The entoderm is derived from the macromeres 3A, 3B and 3C and partly from 3D. After division, the macromere 3D differentiates into the entodermal component 4D and the mesentoblast 4d. Then, the 4d cell divides into two equal mesentoblasts a t either side of the plane of bilateral symmetry. Each mesentoblast may produce one or more enteroblasts and the two remaining mesoblasts give rise to the paired mesodermal bands. In species with an equal 4-cell stage, no landmarks are available to aid in denominating the four macromeres; and until the formation of the third quartet of micromeres it is not possible to indicate which macromere will form the mesentoblast. It is only possible to indicate which pair of macromeres will be 3B and 3D and which pair 3A and 3C, as the cross-furrow a t the vegetal pole develops between the B and D quadrants at second cleavage. Cytologically the four quadrants apJ. MORPH.. 154: 157-186.

pear to be equal. This situation changes rapidly as soon as the third quartet of micromeres has been formed. Then one of the cross-furrow macromeres is partly retracted from the surface of the vegetal hemisphere and is displaced into the interior, where i t makes contact with micromeres of the opposite animal pole. The next division of this macromere usually takes place after a certain interval of mitotic inactivity. Generally, the division is very unequal; the larger interior part is constricted off as the mesentoblast 4d, whereas usually the macromere 4D is a smaller cell a t the surface of the embryo. A t present, there are no arguments to assume that this typical behavior of one of the four macromeres is due to a preexisting pattern of differentiation or to a segregation of a special part of the unfertilized egg. On the contrary, the available experimental evidence is against the assumption of a definite relation between the developmental capacities of 3D and a prelocalization of morphogenetic substances (Guerrier, '70a,b; Morrill et al., '73). The analysis of the formation of the mesen-

' Author's present address: Zoaloglcal Laboratory, University of Utrecht. Transitorium 111. Padualaan H. Utrecht. The Netherlands 157

158

J . A. M. V A N DEN BIGGELAAR

toblast in Lyrnnaea (Biggelaar, '76a) is compatible with the concept of a gradual and epigenetic determination of 3D. The hypothesis has been presented that the differential behavior of one of the macromeres is not based upon intrinsic properties of that blastomere, but is determined epigenetically by means of an inevitable, but necessarily discriminating choice of one out of four equipotential macromeres made by the overlying micromeres of the first quartet as they limit their common cell borders with the opposite vegetal hemisphere to one macromere only. Initially, all four macromeres protrude far into the interior of the embryo. The cross-furrow forming macromeres have the lead, extend farther, and thus, first make contact with micromeres of the opposite pole. Finally, the contact area with only one of them is extended and is concomitantly decreased with the other. From that moment the former can be denominated as 3D. The above hypothesis on the determination of the macromere that will give rise to the mesentoblast is only based upon the analysis of the evolution of the cell contacts inside the embryo of Lymnaea (Biggelaar, '76a,b). Therefore, a comparable study has been performed on the limpet, Patella vulgata. The reason for this choice is t h a t it has been claimed t h a t in this species the mesoderm is not derived from 4d but from 4D (Smith, '351, which is very exceptional in spiralian morphogenesis. MATERIALS AND METHODS

The spawning season of Patella vulgata in the region of Roscoff was from October till the end of January. Once or twice a week, fresh animals were collected and kept in running sea-water in the laboratory. To obtain eggs and sperm the limpets were opened laterally. The ovary or testis was partly removed and immersed in a Boveri-dish with about 100 ml filtered sea-water. After having dissected a series of 15 animals, the ovaries were vigorously moved around by means of a strong current of water from a pipette. Usually, after ten ovaries had been treated in this way, the collected male gonads were treated similarly, giving a milky solution of sperm. After sedimentation of the gonad fragments, a diluted sperm suspension was made by pouring a certain volume of the original solution in a bowl with fresh sea-water, until this became slightly turbid. Finally, about five drops of this suspension were added to each of a series of

bowls with about 50 ml sea-water. After stirring, each bowl received a large amount of eggs from only one ovary. This procedure takes about 30 minutes. The percentage of fertilization may fluctuate between 0 and 100. A high percentage of fertilization was only observed after the chorion had been shaken off and after the eggs had lost their flattened appearance and become spherical. The diameter of the egg is about 180 p m . The first division occurred after 150 minutes, on a n average. Normally cleaving eggs were selected one by one to form synchronously dividing groups of embryos. During further division, each cleavage was followed until the 32-cell stage to ensure that no abnormal eggs were used. At successive intervals, groups of eggs were fixed for one hour in Zenker's fixative (2 gm potassium dichromate, 1gm sodium sulfate, 5 gm mercuric chloride, 100 ml distilled water, 5 ml acetic acid), washed briefly in tap-water and oriented in liquefied agar. After gelification, the egg containing pieces of agar were dehydrated via a series of alcohols and amylacetate and embedded in paraffin. Serial sections a t 3-pm thickness were stained with methyl green-pyronin. For a number of eggs each section was drawn on transparent paper and when superimposed upon each other the drawings permitted a reconstruction of the embryo. In addition, a large number of whole mounts were made after staining in gallocyanin. After fixation in Zenker's fluid as described above, the eggs were washed in tapwater and passed to the stain. This was prepared in the following way: 1 gm gallocyanin (Fluka, Bio Lyon) in 100 ml of a 4% solution of chromalumn in distilled water, according to Langeron ('49). Dissolution of the stain is very poor, even after boiling. The staining intensity of the solution gradually diminishes. Dilution of the staining solution and duration of the staining time must be determined by trial and error and may vary from one to two hours. After being washed for 10 minutes in distilled water, the eggs were dehydrated and mounted in Canada balsam, either with or without a thin ringlet of paper between the slide and the cover glass. Overstained eggs may be differentially destained by passing the eggs through 1 N HCl for two minutes and then through 50%acetic acid. In the acetic acid the eggs quickly lose color, and t h e y m u s t be controlled continuously. Mounted directly in glycerine, they give com-

DETERMINATION OF DORSOVENTRALITY IN PATELLA

pletely transparent and beautiful preparations. After dehydration and mounted in Canada balsam the embryos become a s dark as before. RESULTS

Division chronologV After fertilization, the first cleavage occurs 20 minutes. The after an interval of 150 blastomeres AB and CD are equal (fig. 1). After 32 minutes the second division partitions the egg into four equal quadrants (fig. 2). At the vegetal pole the two cells B and D form a distinct cross-furrow; a t the animal pole, the furrow is extremely short and usually present between A and C. After a n interval of another 32 minutes, the first quartet of micromeres is formed by a dexiotropic division (fig. 3). After 32 minutes, the egg passes into the 16-cell stage by laeotropic and synchronous divisions of l a - l d into lal-ld’ and la2-ld2,and of 1A-1D into 2a-2d and 2A-2D (fig. 4).The passage from the 16- into the 32cell stage takes place after 39 minutes, on an average. Again all blastomeres divide synchronously and, according to the rule of alternating cleavages, the daughter cells are formed dexiotropically (figs. 5-8). As described by Wilson (’04),“the 32-cell stage thus attained is a t first perfectly radially spirally symmetrical.” The plane of bilateral symmetry may be drawn through the macromeres forming the vegetal crossfurrow. In contrast to the earlier stages, the animal cross-furrow may be present between the micromeres of the A- and C- or the B- and D-quadrants. After having reached the 32-cell stage no further divisions occur for a period of 90 minutes. The first cells that resume mitotic activity are the primary trochoblasts laz1Id” and la’2-ld22.Almost in step with each other, they divide by a laeotropic and equal division (fig. 9). About five or ten minutes later, the animal cells lal’-ldll and both tiers of the second quartet, 2a1-2d’ and 2a2-2d2, divide in unison by a laeotropic division (figs. 10.11). In accordance with Wilson (‘041,it has been observed that the cells la”-ldI1 divide nearly equally, whereas the second quartet cells pass through unequal divisions. The upper cells 2a’-2d1 divide into the slightly smaller tip cells 2al1-2dl1and the larger cells 2a12-2d‘2.The lower micromeres 2a2-2d‘ have a characteristic unequal division; the upper cells 2a”-2d2’ are very large in comparison

*

159

with the lower sister cells 2aZ2-2dz2. Vegetally, the latter small cells lie upon the corresponding macromeres and are interposed between two successive micromeres of the third quartet (fig. 13). These small cells, 2aZ2-2dz2, are a useful landmark for the recognition of the individual blastomeres. About five to ten minutes later, the first quartet micromeres la12-ld12 divide. This division is synchronous, laeotropic and equal (fig. 12). At the same time, the third quartet cells 3a-3d divide synchronously. Whereas the divisions in the descendants of the first and second quartet are radially symmetrical, the first division of the third quartet cells adumbrates the appearance of dorsoventrality. The anterior cells divide unequally and laeotropically, with the upper cells 3a1 and 3b1distinctly smaller than the lower sister-cells 3a2 and 3b2 (figs. 14, 19). Again in accordance with Wilson (’041, it has been observed that the posterior cells, 3c and 3d, deviate from the radial type of cleavage. The lower poles of the spindles lie close against the mesentoblast mother cell 3D. Wilson was not able to observe the division and was not sure about the division inequality. From the figures 13, 15 and 21 of this study it is evident that a t least in Patella uulgata this division is bilaterally symmetrical and very unequal. In contrast to their anterior counterparts, the cells 3c’ and 3d1 are large, whereas 3c2 and 3d2 are small. The last division in the transition of the 32to the 64-cell stage is that of the macromeres 3A-3D. I t differs markedly from the description of Wilson (’04)for Patella coerulea. In both species it is markedly asynchronous. In Patella coerulea 3D would be in advance of the other macromeres; 4d would be the smaller peripheral and 4D the larger central cell. In Patella vulgata 3A, 3B and 3C divide about 25 minutes after the onset of the 60-cell stage, whereas 3D has an additional delay of 18 minutes, on an average (figs. 14, 15, 21). In 3A, 3B and 3C the division is slightly unequal, whereas 3D divides very unequally. As Wilson described, “at the time of its division it is only connected with the surface by a very narrow neck.” “The results of this division is to form a large rounded cell, that lies quite in the upper hemisphere and a more superficial cell.” Wilson was not sure about further development and he believed the large central cell to be 4D and the superficial cell to be 4d or M. He remarked, that it was extremely difficult to identify the individual cells after completion

160

J. A. M.

VAN DEN

of the fourth quartet, i.e., after completion of the 64-cell stage. The differential division in the third quartet of micromeres, the preceding partial retraction of 3D from the vegetal surface of the embryo and its peculiar division, definitely indicate the dorsoventral axis. From the 64-cell stage the course of the cleavages becomes less regular. As the main interest was focussed upon the formation and the first division of the mesentoblast 4d, the exact chronology during further development has not been studied with the same care as up to the 64-cell stage. Within 20 minutes after the formation of the mesentoblast, a large series of micromeres starts to divide. In 2aZ1-2dz1 the division is slightly unequal and laeotropic. As the preceding cleavage was also laeotropic, the alternation of sinistral and dextral cleavages is not continued (figs. 18, 19). The micromeres 2aI22d12 divide more or less equally by a dexiotropic division (figs. 18, 19). The tip cells 2a1I-2d" usually follow more or less in step with la121-ld121 and la122-ld12Z (fig. 17). The divisions of la"'- ldIz1are laeotropic and very unequal. The upper cells la1211-ld1211 are very small. In la'22-ld'22the division is dexiotropic and almost equal. In the third quartet of micromeres the anterior cells 3aZand 3b2 divide a t about the same time as the posterior cells 3c1 and 3d'. Meanwhile, the embryo has reached the 88-cell stage. At the vegetal pole the central 4d cell is in mitosis (fig. 18). The embryo is still immotile as the primary trochoblasts have not yet produced cilia. In embryos fixed about 25 minutes later, 4d appeared t o have divided by a bilateral division into the left and right mesentoblast (fig. 20). In embryos of the same age, i.e., about six hours after first cleavage, one can observe ciliated trochoblasts. This observation contradicts that of Smith ('341, who described the division of 4D into a left and right mesoderm mother cell in ciliated embryos 13 hours after the beginning of development in Patella vulgata.

BIGGELAAR

total number of common cell borders thus is 4 x 3 = 12.

At the 8-cell stage, the cross-furrow a t the animal pole is usually formed between l a and lc; a t the vegetal pole almost without exception between 1B and 1D. The micromeres l a and l c each border on three micromeres: on lb, Id and on each other. In addition, they have common boundaries with two macromeres: the one from which they arose and the one following in a clockwise direction (fig. 3). The micromeres l b and Id do not touch each other, but only l a and l c . For the macromeres 1A-1D there is an alternate situation. The cells 1B and 1D are in touch with three counterparts: the macromeres lA, 1C and with one another. The latter macromeres are mutually separated and only border on 1B and 1D. Each macromere is in touch with the micromere split off from it and with the one counter-clockwise (fig. 3). Thus, the total number of common cell borders is: (4 x 4) (4 X 5) = 36. Regardless of the size and the cytoplasmic composition of the egg, the disposition of the cells is completely alike, whether one starts at the animal or a t the vegetal pole. At the 16-cell stage the vegetal cross-furrow is still present between the macromeres of the B- and D-quadrant. A t the animal pole it is most frequently observed between lbl and Id'. The common cell borders, which can be distinguished a t the surface of the embryo, can easily be derived from in toto preparations (fig. 4). Besides these external contacts, serially sectioned embryos reveal additional common cell contacts in the center. The latter are internal common cell boundaries. These have been analyzed in three reconstructed embryos. Each blastomere superficially touches five other cells (tables 1-31, except the cross-furrow forming ones at either end of the egg axis, which are in touch with an additional cell from the opposite quadrant. Thus, the total number of external common partitions is: (12 X 5) + (4 X 6) = 84. In a surface view, all blastomeres are pentagonal, except the cross-furrow cells, which are hexagonal. Interiorly, the cell contacts are formed within Evolution of cell contacts the two central tiers of cells, l a 2 - l d 2and 2a-2d At the 4-cell stage a distinct cross-furrow (tables 1-3). In each of the three reconcan be seen at the vegetal pole between the structed embryos, the four superficially sepacells B and D. As both are exactly alike, the rated micromeres 2a-2d radiate toward the denomination is arbitrary. Usually, a very center of the embryo, where they touch each small furrow is present between A and C a t other. Although the cells la2-ld2also radiate the animal pole. As a consequence, each blas- far interiorly, only a few of them make additomere borders on all three counterparts. The tional contacts. Comparing the tables 1-3, it

+

161

DETERMINATION OF DORSOVENTRALITY IN PATELLA 'I ABLE 1

Internal and external common cell borders indicated by crosses and black squares, respectively. in an embryo at the 16-cell stage

1 a1

5

ib'

6

1 c'

5

6

1 d'

1 a2

5

1

ib2

5

1

1c 2

5

1

-

d2

5

1

2a

5

2

2b

5

2

2c

5

2

1

5

2d 2 A

5

2 6

6

2 c

5

2

6

2 D

I

I

I

I

I 8 4 12

The primary trochnblasts Ila'-ld'l and the secondquartet cells 2a.2ddo not huve mutual common cell burders visible at the sur face of the embryo. Protruding far interiorly. they meet one another centrally and make internal call contacts. The animal cross furrow IS present between lh' and Id'

will be evident that the second quartet cells, this succession is disturbed a t either end of together with a few of the primary trocho- the egg axis, as the cross-furrow cells have an blasts la2-ld2,in meeting centrally (figs. 22- additional common cell border (figs. 6-8; table 24), form a barrier between the vegetal mac- 4). After the duplication of the number of cells from the 16- to the 32-cell stage, the romeres and the animal micromeres. During the 32-cell stage the contacts were number of pentagonal cells has not been studied more extensively. Embryos were fixed changed and remains 12; the number of hexat successive intervals of 20 minutes. At the agonal cells has increased from 4 to 20. The animal pole the cross-furrow is usually pres- total of common cell boundaries is: (12 X 5) (20 X 6) = 180. This number is particularent between the micromeres lb" and Id". In the previous stages, it occurs most often be- ly constant. When deviations occurred, a reextween the micromeres of the A- and C-quad- amination of the contacts usually revealed rants. The first group of embryos was fixed ten erroneous observations. In comparison with the 16-cell stage, a reminutes after the beginning of the 32-cell stage. The internal and external common cell markable difference has been discovered in contacts of one representative embryo are the distribution of the internal cell contacts listed in table 4. The surface of the egg ap- (table 4). In contrast to the 16-cell stage, no pears to be partitioned in such a way that a common internal boundaries have been succession of pairs of pentagonal and hex- formed between micromeres of the second agonal cells is developed. The regularity of quartet, nor between the primary trocho-

+

162

J. A. M. VAN DEN BIGGELAAR TABLE 2

Internal and external common cell borders in a 16-cell embryo in which one of the cross-furrow macromeres (arbitrarilv denominated as 2B) in involved in the formatmn ofinternal common cell borders

1 a’ 1 b1

1c1 1 d’ 1 a2

5

1

1 b2

5

1

1c2

5

1

5

1

5

3

-

1 d2

za 2b

5

2

2c

5

2

5 2 -

2d 2 A

5

2 8

6

2 c

5

1

6

2D

1

1

blasts la2-ld2. The previously existing contacts have been broken by a centrifugal movement. Irregular blebs in the center of the embryo in the vicinity of the central apexes of the second quartet cells and the primary trochoblasts may be regarded as remnants of disrupted contacts (figs. 25,26). By the retraction of these micromeres from the center toward a more superficial position, a small cleavage cavity may be found initially (figs. 25, 26). From the vegetal side, the macromeres 3A-3D intrude into the free space. A t the peripheral borders of this cavity, they are in touch with the retracting tips of the micromeres of the second quartet, 2a1-2d1and 2a22d2, and with the apexes of the primary trochoblasts. In the center of the transient free space, t h e macromeres a r e placed opposite the internally touching cells la’*ldI2 (figs. 26, 27). In making internal contacts, these first quartet micromeres form a barrier between the intruding macromeres

1 8 4 14

and the most animal micromeres la1’-ld1l (figs. 27, 28, 34). The macromeres have internal common boundary walls with a few cells of the second quartet of micromeres, and with one or two of the most vegetal tier derived from the first quartet, i.e., la22-ld22. The macromere 3D has been denominated arbitrarily. Half an hour after the beginning of the 32cell stage, the second quartet cells are further retracted from the equatorial zone of the embryo. Simultaneously, the macromeres have intruded farther, enlarging the zones of contact with the above situated micromeres of the first quartet (fig.28). The consequence of this rearrangement can easily be derived from an analysis of the internal contacts found in four reconstructed embryos (table 5). The macromeres have almost lost the internal common boundaries with the micromeres 2a22d2. Generally, each macromere remains connected with that micromere of the tier 2a1-2d’ which is derived from it, and with a number of

163

DETERMINATION OF DORSOVENTRALITY IN PATELLA TAH1.E 3 Internal and externalcommon

cell borders in another Ibceliembryo Common cell borders

la'lb'lc'ld'la21b21c21d22Q2 b 2 c

2d2A282C2D

2'

$

-

1 a'

6

1 b'

5

1c '

6

-

1 d'

5

1 a2

5

2

1 b2

5

2

1c2

5

1

-

d2

5

1

20

5

2

2b

5

2

2c

5

2

i

5 2 -

2d

2A

5

2 8

6

2 c

5

2 0

6

I

I

I

I

I 8 4 14

This differs from the embryo of table 1 by the presence of the animal cross-furrow between la' and lc' and two uncertain addl tional contacts between la2 and Ib'.

micromeres of the first quartet. The lowest tier of micromeres of the first quartet (laz2l d 9 has the highest number of common cell contacts with the intruding macromeres. As the apexes of the cross-furrow macromeres occupy the most axial position, they necessarily have cell contacts in common with a greater number of micromeres than 3A and 3C. The free zones of the internal cell borders of the second quartet cells and the primary trochoblasts no longer have the above-mentioned irregular blebs. Apart from small remnants, the inner surfaces are smooth (fig. 29). This is in agreement with the assumption that the irregular protrusions, observed in the previous group, may be considered as remnants of disrupted contacts. In embryos fixed 50 minutes after the beginning of the 32-cell stage, the second quartet cells are still farther removed from the center. Five embryos have been recon-

structed. The number of internal contacts between the macromeres and the micromeres are listed in table 6. The lateral macromeres 3A and 3C still border on the animal second quartet cell of the corresponding quadrant (2a1on 3A etc.). The contacts between 3B and 2b1, on the one hand, and between 3D and 2d1 on the other, are strongly reduced or even absent. The persisting zones of contact are restricted to the vegetal parts of the second quartet cells. More animally, these micromeres are convincingly separated from the tips of the macromeres (figs. 32, 33, 38-40). This enables the macromeres to come into touch with the overlying micromeres of the first quartet and to extend the common cell borders. At the animal pole, the micromeres la1'Id" have enlarged their mutual contacts and are more flattened against each other. Concomitantly, they radiate farther interiorly,

164

J. A. M.VAN DEN BIGGELAAR TABLE 4

Internal and external common cell borders in a 32-cell embryo ten minutes after fifth cleavage

common cell borders ext. int. 1a’’ 1b’’ 1c ” 1d” 1a’’ 1d2 1Cl2 1d12 1a2’ 1b2’ 12’ 1d2’ 1aZ2 1b2’ 1CZ2 1d2‘ 2 a’ 2 b‘ 2c’ 2 d’ 2 a2 2 b2 2 c2 2 d2 3a 3b 3c

6 6 2 6 2 6 2 6 2 6 6

6 6 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 5 6 5 6 180

2 1 1 1 2 1 2 1 1

-

3d

3A 30 3c 30

1

2 4 1 6 34

-

I

I

I

I

I

1

I

I

1

The second quartet cells are completely separated from each other. The micromeres la’l-ld‘Ameet one another centrally. Each of the macro. meres 3A-3D borders on one or two second quartet cells and on a varying number of cells of the most vegetal tier of the first quartet, i.e..la”-ld‘*. Compare figures 25-27.

and wedge the centrally interconnected micromeres la12-ld1*apart (figs. 30, 34). In two embryos, l a ” - l d ” were found in touch with one of the cross-furrow forming macromeres (fig. 35). In the contact zones, the micromeres are poor in yolk and the composition of the cytoplasm differs from the rest of the cell (figs. 35,40), exactly as has been described for Lymn w u (Raven, ’74; Biggelaar, ’76a).

Considering the distribution of the internal contacts (table 5), it will be evident that in comparison with the previous stage, a progressive differentiation has taken place. The lateral macromeres seem to be pushed laterovegetally and become separated from the first quartet cells. The number of contacts with one of the cross-furrow macromeres is reduced, but increased significantly with the

165

DETERMINATION OF DORSOVENTRALITY IN PATELLA TABLE 5

Numbers of internal cell contacts between the macromeres 3A-3D(vertical column) and the micromeres of the first and second quartet, specified by quadrants a-d (horizontal rows), found in four embryos. 30 minutes after the beginning of the 32-cell stage Four embryos

Micromeres 1"

1"

12'

22

2'

121

Macromeres a

b

c

d

a

3A 3B

b

c

d

3

x

a

b

c

d

a

d

a

1

4 3 4 1 3 4 4

4

3 2 1

3D

2

1 3

Total contacts Subtotals found in 3A + 3C 3B+3D 3D

3 2

h

c

b

c

d

a

b

c

3 I

4

4

9

12

23

15

3

2 10 5

8 15

8

3

9

8

4

6

d

2

I

The lateral macromeres 3A and 3C make contact with fewer first quartet cells than the axial macromeres 3B and 3D. Of the latter two, 3D touches more first quartet cells than 38. 3A and X more frequently have internal common cell borders with the more latern-vegetally situated quartet cells. Compare figures 28. 29.

TABLE 6

Numbers of internal cell contacts between the macromeres 3A-3Dand the micromeres of the first and second quarted found in five embryos, 50 minutes after the beginning of the 32-cell stage Five embryos

Micromeres 1'2

1"

12'

2'

122

22

Macromeres a

3A 3B 3c 3D

b

c

d

a

b

c

d

a

b

c

d

a

b

c

d

h

c

d

a

h

c

2 1 1 1

5 5 5 5

2 2 1 2 4 5 4

Total contacts

5

24

20

19

12

1

Subtotals found in 3A 3C 3B+3D 3D

5 5

24 20

1 19 15

3 16 12

9 3 1

1

+

1 2 1

a

d

5

2

1 3 1 3 1 3 5

2 4 1

The macromeres 3 A and 3c' have lost the common cell horders with relatively more animal cells than with vegetal cells of the first quartet. The numhcr of internal contacts between the micromeres and 3D has increased, with 3 8 it is slightly diminished. For the first time contacts are realized with cells of the moat animal tier. la " -Id " Compare figures 30-35

other. The increase in the number of contacts is mainly realized in the tier la12-ld12.The apex of the macromere 3D is placed centrally between the interior cell tips of these micromeres (figs. 31, 34). In two embryos, the cells la12-ld12 had diminished mutual internal contacts and were partly separated from one another by the descending micromeres la"Id". A number of the latter then contact the tip of the central macromere (fig. 35). From this stage, the central blastomere can be denominated with certainty as 3D. Exactly as described for Lymnaea (Biggelaar, '76), dorso-

ventrality is expressed first in the center of the embryo. The differentiation of the macromeres becomes evident by the accumulation of internal contacts between the first quartet cells and the tip of the most central macromere. Almost without any exception, this is one of the cross-furrow macromeres. Only in one out of a large series of whole mounts and serially sectioned embryos, a lateral macromere occupied the pivot position in the embryo. In this exceptional case, however, the dorsoventral pattern was completely related to this macromere.

166

J. A. M. VAN DEN BIGGELAAR TABLE 7

Numbers of internal cell contacts between the macromeres 3A-3Dand the micromeres of the first and second quartet found in four embryos, 70 minutes after the beginning of the 32-cell stage Four embryos

Micromeres 1'2

1"

11'

12'

' 2

21

Macromeres a

3A 3B 3c 3D

b

c

d

a

b

c

d

a

b

c

d

a

b

c

d

a

b

c

d

a

1

2

b

c

d

4

3 4

1 4 2 2

4 4 4 4

4 4 4 4

3 3 3 3

Total contacts

9

16

16

12

Subtotals found in 3A 3C 3B + 3D 3D

9 9

16 16

16 16

12 12

+

12

2

a 4 1

2 2

The macromeres 3A and 3C are located farther away from the center toward a still more superficial position. They are separated from the first quark cells and touch a relatively high number of second quartet cells (2a'-2d'). This superficial position is shared more and more by 3B, which is also sepa rated from the first quartet cell8 and borders more frequently on a second quartet cell than 3D. From this stage, the first quartet cells only have commo cell walls with 3D. This macromere touches all four cells of the tiers la21-ld21 and la1a-ld12, an increasing number of the cells la"-ld", and less than th maximal number of the tier laZ2-ldz2. Compare figures 36-42.

Five embryos, fixed 70 minutes after the beginning of the 32-cell stage, have been reconstructed for the analysis of the internal contacts. In all five, the first quartet cells were in touch with 3D only. Macromere 3B was found at the same level as 3A and 3C. In four embryos each of the micromeres of the successive tiers la22-ld22, laZ1-ldz1 and la12Id" had an internal cell border in common with 3D (table 7). In the most animal tier, lall-ld'l, this was still limited to two or three cells. While the number of the micromeres that were in touch with 3D was increased, the surfaces of the common boundaries with the vegetal first quartet cells were reduced, and restricted to the animal parts (figs. 38,41, 42). In one embryo, the lowest micromeres (laz2ldZ2)were completely separated from 3D. The contact areas with the more animal cells, la12-ld12and lalL-ld" were significantly extended. A similar reduction of the affinity of the micromeres for the macromeres can be observed in the vegetal direction. In the middle region of the embryo, the central parts of the micromeres of the second quartet 2a1-2d1were separated from the intruded macromeres, and small cavities could be observed between them (figs. 37, 39). The zones of contact were limited to the more vegetal parts. The still more vegetally situated micromeres 2a2-2dz and 3a-3d of the third quartet had extended common cell boundaries with the neighbouring micro- and macromeres (figs. 36, 401, and

intruded far interiorly. In two embryos 3D was found in contact with one of the anterior third quartet cells (3a). After 90 minutes, the end of the resting stage is reached. First, the primary trochoblasts la2I-ldz1and lazz-ldz2 resume division activity. The cells lal'-ldl' and the second quartet micromeres follow almost immediately. Three embryos of this age have been reconstructed. The internal contacts between the micromeres and the macromeres can be found in table 8. The tendency of the most vegetal tiers of first quartet cells to separate from the central 3D blastomere is strengthened. The tiers 1a222-1d222 and la221-ld221 are almost or completely separated from 3D. In two embryos the next tier in animal direction ld2lZ)was already free from it. The common cell contacts with the more animal tiers la211ldZ1l,la12-ld12were still extended (figs. 43, 44). With the exception of one cell in one of the embryos, each of the micromeres la1l-ld1l bordered on the tip of 3D. Meanwhile, the pivot position of 3D was more prominent than in the previous stages (figs. 43, 45). About 110 minutes after the beginning of the 32-cell stage, the embryo reaches the 60cell stage. All blastomeres have passed through the second cleavage round, except the macromeres 3A-3D. Six embryos have been reconstructed for the analysis of the internal contacts. The results can be found in table 9. In these embryos, the first quartet was built

b

c

d

b

c

d

12

12 12

11 11

3 3 3 3

a

112

11

2 3 3 3

a

1" b

c

d

12 12

12

3 3 3 3

a

1"'

h

c

d

4 4

4

a

Micromeres

1 1 1 1

a

,>,> b

2 2

2

11" d

1 1

c

90 minutes after the begrnning of the 32-cell stage

a

2 2

2

1"' b

c

1

d

2

b

2'

c

d

12 10

6

18

3 1 2 3 3

3

a

a

b

2'

c

d

b

c

d

24 24

24

6 6 6 6

a

b

c

d

24 24

24

6 6 6 6

a

h

c

d

14

14

14

4 5 3 2

a

b

r

d

18 18

18

4 5 5 4

a

b

c

d

23 23

23

6 5 6 6

a

Micromeres

b

c

d

8 8

8

2 2 2 2

a

b

c

d

9

9

9

2 2 4 1

a

Numbers of internal cell contacts between the macromeres 3A-3Dand the micromeres of the first quartet in sir 60-cell embryos, 110 minutes after the beginning of the 32-cell stage

TABLE 9

a

b

2 2

2

d

1 1

c

The first quartet cells only border on 3D. In the tiers of trochoblasts. the gradual reduction of the contacts with 3D from vegetal to animal direction is evident again. A more or less comparable situation can be observed in the tiers llzL,I'll, 1"' and 1'". 3D only touches the maximal number of cells in the two most animal tiers. Compare figure 46.

+

found in 3A 3C 3B+3D 3D

Subtotals

Total contacts

3D

3c

3A 3B

Macromeres

six

43-45.

Only 3D has internal cell borders in common with first quartet cells. It is progressively separated from the derivatives of the primary trochoblests. This separation starts in the most vegetative parts and proceeds animally, from la'22-ld'Z2to la"'.ld"'. E xcept in one embryo, 3D has common cell boundaries with each of the four cells around the animal pole, i.e.. la".ld". Compare figures

Total contacts Subtotals found in 3A+X 3B + 3D 3D

3D

3c

3A 3B

Macromeres

Three embryos

Numbers of internal cell contacts between the macromeres 3A-3Dand the micromeres of the first and second quartet found an three 44-cell embryos,

TABLE 8

m

Q,

CL

E

2

2

*

E j

0 *I

rj

%

$

5%

*I

t3 M 4

TWO

b

4

4

4

Subtotals found in 3A + 3C 3B 3D

3D

d

b

c

d

4 4

4

1 1 1 1

a

1))'

a

c

2 2

2

1 1

h

1'1'

d

b

c

3

3

3

1 1 1

a

1"l

d

b

12"

c

3

3

3

1 1 1

a

Micromeres

d

a

b

1

c

1

1

I

1"2

d a

h

,225

c

d

a

h

,122

c

d

d

a

b

c

d

a

b

c

d

a

1

h

4d

d

a

Micromeres

h

1Z"

c

d

1

a

b

c

1

1

1"'

d

1

a

c

1 1

2

1

h

]??I

d

1

a

1 1

2

1

h

c

1I O Z

d

After division of 3D into the central mesentoblast 4d and the superficial macromere 4D. the former has lost Its internal cell contacts The micromeres 4a. 4h and 4c have common cell borders wlth a few of the lowest first quartet cells. Compare figure 48.

c

1'21

1

c

1'1'

Subtotals found in 4a +4c 4b+4d

b

I"?

1

a

1"'

Total contacts

4d

4c

4b

4a

Fourth quartet of micromeres

Two embryos

Numbers of internal cell contacts between the micromeres 4a-aand the micromeres of the first qunrtet in two 64-cell embryos, 150 minutes after the beginning of the 32-cell stage

TABLE 11

Cells 3A. 3B and 3C are in mitosis prior to t h e formation of the corresponding fourth quartet cell. 3D starts to lose contacts with cells around t h e animal pole (la"'-ld"'). Compare figure 47.

+

c

1 1 1 1

a

1"'

Total contacts

3A 3B 3c 3D

Macromeres

embryos

Numbers of internal cell contacts between the macromeres 3A-3Dand the micromeres of the first quartet in two 60-63-ce11embryos, 130 minutes after the beginning of the 32-cell stage

TABLE 10 CL

FG

3 n8

%

r'

?

4

00

Q,

DETERMINATION OF DORSOVENTRALITY IN PATELLA

up by eight tiers of four micromeres. Without exception each of the cells of the two most animal tiers, l a l l l - l d l l land la112-ld112, had extended common cell boundaries with 3D (fig. 46). In the more vegetal tiers, the contacts were largely reduced or lost. After 130 minutes, the macromeres 3A, 3B and 3C are in mitosis (figs. 14, 15). The division is laeotropic and more or less unequal. The micromeres 4a, 4b and 4c extend far into the center. The macromere 3D is still in interphase. The internal cell contacts between the first quartet cells and the mesentoblast mother cell have been traced in two reconstructed embryos (table 10). In one of them, the macromeres 3A, 3B and 3C were in prophase. In this embryo, 3D was in touch with each of the micromeres of the two animal tiers l a l L 1 - l d l land L la112-ld112; for the major part it was detached from the lower micromeres. In the other slightly older embryo, 3A, 3B and 3C had just divided; 3D was entirely separated from all first quartet cells and could be found as an isolated cone in the center (fig. 47). After 150 minutes, 3D is in mitosis and the embryo thus finishes its sixth cleavage round and is composed of 64-cells. The macromere 4D was small with a purely superficial position, whereas the micromere 4d was a huge cell, almost completely hidden inside the embryo. At the moment of division, 3D or its daughter-cell 4d is isolated from the micromeres with which it had internal common cell boundaries before. At the same stage, the micromeres 4a, 4b and 4c extend in an animal direction, where they touch a few of the lower first quartet cells (fig. 48). Finally, a group of embryos was fixed 170 minutes after the beginning of the 32-cell stage that is about 20 minutes after the onset of the 64-cell stage. In this group, only one embryo was reconstructed and analyzed in detail (table 11).The micromeres 4a, 4b and 4c were in contact with a number of the lower first quartet cells, whereas 4d was still separated from the majority of the surrounding micromeres and had maintained its isolated position. Up to the 64-cell stage no nucleoli were observed. At the transition of the 64- into the 88-cell stage, each nucleus usually had two small nucleoli.

169

uulgata has been described by Smith (’34). According to this author “the whole of the macromere in quadrant D gives rise t o mesoderm” and “entoderm is formed from the macromeres of the quadrants A, B and C alone.” If this description were correct, Patella uulgata would be an exception to the general rule that in spiralian development the mesoderm arises from the micromere 4d. Looking a t Smith’s figures, it will be evident that his first two illustrations are drawings of 13-hour larvae, which are already ciliated. In these embryos, 4D would divide into the paired mesoderm mother cells. While Smith’s observations were done on serial sections, it is doubtful t h a t the embryos were reconstructed. Only the large interior cells have been denominated in Smith‘s figures and even these have not been done without errors. In each of his figures the C-quadrant, instead of the B, is opposite the D-quadrant. In his description the division of 4D may very well coincide with the division of 4d found in the present study. There are two reasons to suppose an erroneous identification of 4D. First, 4d is the only large cell in the interior of the embryo; 4D is a small superficial cell. Second, 4d divides into the paired mesentoblasts at the moment at which the primary trochoblasts become ciliated; the macromere 4D remains undivided at that stage. With this correction in mind, the course of segmentation in Patella uulgata closely follows that of Patella coerulea described by Wilson (’04). However, the resemblance is lost a t the formation of the mesentoblast. According to Wilson, in Patella coerulea 3D would divide in advance of the other macromeres. He believed t h a t 4d would be the smaller superficial cell and 4D the “large rounded cell, that lies quite in the upper hemisphere.” If both species continue t o develop identically, then it may be expected that also in Patella coerulea, t h e macromere 3D divides significantly later than the other macromeres, and that 4d is the large central cbll and 4D the small superficial. I t must be added that Wilson remarked already, that after division of the macromeres, the cells undergo considerable shiftings, extend far in the embryo and, hence, it becomes difficult to identify them individually. After this introduction on the formation of the primary mesentoblast, attention must be DISCUSSION paid to its determination. Prior t o the onset of The formation of the mesoderm in Patella the 32-cell stage, it is only possible to distin-

170

J. A. M. VAN DEN BIGGELAAR TABLE 12

Percentages of realized internal common cell borders relative to the theoretically possible number (100%)between the macromeres 3A-3Dand the micromeres of the first quartet at regular intervals of 20 minutes during the 32-cell stage Macromeres

3A

Minutes after fifth cleavage

10 30 50 70

Micromeres la"-ld"

0 0 0

0

3B

10 30 50 70

0 0 0

3c

10 30 50 70

0 0 0 0

10 30 50 70

0 0

3D

0

25 60

la'2-ld'z

0 0

la" Id"

la".ld"%

0 0

0 0 0 0

25 31 10 0

0 25 20 0

0 31 25 0

40 44 30

0 8

0 6 5

0

0

0

13 25 5 0

0

25 31 75 100

40 44 75 80

0

31 75 100

Theoretically, each macromere may border on each side of the 16 micromeres, simultaneously excluding the other macromeres from forming common cell boundaries with the same number of first quartet cells.

guish the two lateral macromeres from the two axial ones which form the vegetal crossfurrow in the plane of bilateral symmetry of the future embryo. This furrow is present between the dorsal and ventral quadrants, which, at that stage, can only be denominated in an arbitrary way. In the beginning, the macromeres only differ in position. As one of the cross-furrow forming macromeres attains a central position during the relatively long 32-cell resting stage, the embryo gradually looses its radial symmetry. In this way the dorsoventral polarity first becomes apparent in the inside, and may be regarded as the result of a struggle for the pivot position. At the 16-cell stage, the macromeres are separated from the animal micromeres la'Id'. The micromeres la2-ld2and 2a-2d form a barrier between the macromeres and the animal micromeres (tables 1-3; figs. 22-24). At the beginning of the 32-cell stage, this barrier is broken, as the micromeres in the equatorial region of the embryo lose their mutual internal contacts. Seen from the interior, they retract toward a more superficial position. The free space in the center is occupied by the macromeres, of which all four intrude far into the embryo. At the same time, the animally located micromeres la'2-ld12extend centrally, where they contact each other, and come to border on the tips of the macromeres (figs. 2527). When the la12-ld12 micromeres meet each

other centrally, now a barrier is formed between the vegetal macromeres 3A-3D and the micromeres la"-ld" that are situated around the animal pole. About 30 minutes after the onset of the 32-cell stage, all four macromeres may have common cell borders with micromeres of the first quartet, except with la"Id". Theoretically, each macromere may touch first quartet cells. The frequency with which each of the macromeres 3A-3D has been found in contact with micromeres of the successive tiers of first quartet cells a t successive intervals during the 32-cell resting stage, has been summarized in table 12. In each embryo, the cross-furrow macromere with the highest percentage of contacts has been denominated as 3D. Initially, both crossfurrow macromeres are favored, exactly as has been found in the embryo of Lymnaea prior to the determination of 3D (Biggelaar, '76a). At ten minutes after the onset of the 32cell stage, the two cross-furrow macromeres 3B and 3D have realized 40% and 6391 of the theoretically possible contacts with the lowest tier of first quartet cells (la22-ld22). For 3A and 3C this is only 25% and 1391, respectively. With the exception of one of the cells of the next tier (laz1-ldZ1) in the animal direction, the other first quartet cells are still separated from the macromeres. Contacts with the micromeres laZ1-ld2'appear to be limited to the cross-furrow macromeres. Inci-

DETERMINATION OF DORSOVENTRALITY IN PATELLA

dently one of the lateral macromeres may contact one of the lower first quartet cells. With progression of time, the contacts shift toward one of the cross-furrow macromeres. Simultaneously, the other is found more vegetally. In the beginning the contacts with the more vegetal first quartet cells become extended. After a certain time these contacts are reduced again, and replaced by contacts with more animal micromeres. In this way, the macromere 3 D becomes attached to a progressively higher zone of the animal hemisphere and intrudes farther and farther into the interior, leaving behind the tips of the other macromeres (figs. 29, 31,34, 35,37).Finally, after 70 minutes, the central position of 3D is well established and the embryo is dorsoventralized. A simple hypothesis, adequately accounting for the evolution of the cell contacts during the 32-cell stage prior to the determination of the mesentoblast mother cell, is to assume a wave of high cell affinity starting in the equatorial zone and moving toward the animal pole of the egg axis. Initially, mutual internal cell contacts exist between the second quartet cells and a number of primary trochoblasts. Then, these bonds are broken, while the adhesivity between other cells, animally from the equator, increases. They extend their common cell boundaries and protrude centrally. As a result, the vegetal macromeres and the lower animal micromeres contact each other. It may be presumed, that an equilibrium in the adhesive strengths can only be reached by an enlargement of the contacts, limited to one of the macromeres. Automatically, this macromere has to move farther into the embryo. After passage of the wave of high cell adhesiveness, this macromere becomes detached again and is isolated inside the embryo. By the exclusive possession of the internal contacts with the micromeres of the animal hemisphere, and by its unique position, this central macromere is determined as the mesentoblast mother cell and follows a different developmental pathway than the other macromeres. With a term of Waddington (’62) this “canalization of differentiation” is brought about by a selection exerted by the micromeres of the first quartet. One consequence of this hypothesis is to suppose that the animal micromeres play a cardinal role in the determination of 3D and in the discrimination of the other macro-

171

meres, and thus in the process of dorsoventralization of the embryo. An alternative explanation might be t h a t a n equilibrium configuration with only one central macromere is the result of a higher affinity of one macromere for the micromeres. It may be expected that a decisive answer about the validity of both alternatives may be obtained by deletion experiments. ACKNOWLEDGMENTS

The author is greatly indebted t o the Centre National de la Recherche Scientifique of France (C.N.R.S., ATP, nr. A 655.9102), by which the stay a t the Station Biologique in Roscoff has been supported. The everlasting interest of Doctor P. Guerrier has been an essential stimulus in this study. Without the indispensable and skillful technical assistance of Mr. H. A. Wagemaker this study would almost have been impossible. Finally, the finishing touch in the outlay of the illustrations by Mr. J. J. van der Vlis, and the photographic assistance of Mr. H. van Kooten and Mr. E. van der Vlist, should be gratefully acknowledged. LITERATURE CITED Biggelaar, J. A. M., van den 1976a Development of dorsoventral polarity preceding the formation of the mesentoblast in Lymnaea stagnalis. Proc. Kon. Ned. Akad. v. Wetensch., Amsterdam, C 79: 112.126. 1976b The fate of maternal RNA containing ectosomes in relation to the appearance of dorsoventrality in the pond snail, Lymnaea stagnalis. Proc. Kon. Ned. Akad. v. Wetensch., Amsterdam, C 79: 421426. Guerrier, P. 1970a Les caracteres de la segmentation e t la determination de la polarite dorsoventrale dans le developpement de quelques Spiralia. I. Les formes a clivage egal. J. Embryol. Exp. Morph., 23: 61 1-637. 1970b Les caracteres de la segmentation et la determination de la polarite dorsoventrale dans le developpement de quelques Spiralia. 111. Pholas dactylus et Spisula subtruncata. J. Embryol. Exp. Morph., 23: 667692. Langeron. M.,ed. 1949 P r k i s de Microscopie. Masson e t Cie., Paris. Morrill, J. B., C. A. Blair and W. Larsen 1973 Regulative development in the pulmonate gastropod Lymnaea palustris, a s determined by blastomere deletion experiments. J. Exp. Zool., 183: 47-56. Raven, Chr. P. 1974 Further observations on the distribution of cytoplasmic substances among the cleavage cells in Lymnaea stagnalis.J. Embryol. Exp. Morph., 31: 37-59. Smith, F. G. W. 1935 The development of Patella uulgata. Phil. Transact. Roy. SOC.(London), B, 225: 95-125. Waddington, C. H. 1962 New Patterns in Genetics and Development, Columbia University Press, New York. Wilson, E. B. 1904 Experimental studies in germinal localization. J. Exp. Zool., 1: 197-268.

PLATE 1 EXPLANATION OF FIGURES

1 Drawing of 2-cell stage, showing AB and CD blastomeres.

2 Four-cell stage with a distinct vegetal cross-furrow between blastomeres B and D and a minimal animal one between A and C.

3 Eight-cell stage, viewed from the animal pole. showing t h e dexiotropically formed first quartet of micromeres, l a - l d , and their corresponding macromeres, 1A-1D. 4

Sixteen-cell stage, lateral view

5 Thirty-two-cell stage, animal view. All blastomeres synchronously divide through the fifth cleavage. 6

172

Thirty-two-cell stage, lateral view.

DETERMINATION OF DORSOVENTRALITY IN PATELLA J. A. M. van den Biggelaar

PLATE 2 EXPLANATION OF FIGURES

Age of the embryos is given in minutes after the beginning of the 32-cell stage. 7 Thirty-two-cell stage, animal view; 30 minutes.

8 Thirty-two-cell stage, vegetal view; 30 minutes. 9 Forty-cell stage, lateral view; 90 minutes. The primary trochoblasts la2'-ldZ1and la2*.1d22are divided. 10 Forty-cell stage seen from the animal pole; 100 minutes. The micromeres la "-ldLL are in prophase preceding the sixth cleavage. 11 Fifty-six-cell stage, animal view; 110 minutes. The micromeres la Lz -ldl* are in divi. sion.

12 Sixty-cell stage, animal view; 120 minutes.

174

DETERMINATION OF DORSOVENTKALITY IN PATELLA

PLATE 2

J . A. M. van den Biggelaar

175

PLATE 3 EXPLANATION OF FIGURES

Age of the embryos is given in minutes after the beginning of the 32-cell stage 13 Sixty-cell stage, dorso-vegetal view, 120 minutes. The dorsal cells of the third quartet, 3c and 3d, are divided very unequally. As a result 3D is surrounded by three small cells: 2d2*,3c' and 3d'. Compare figure 21. 14

Sixty-cell stage, ventro.vegeta1 view, 120 minutes. The unequal division of the ventral third quartet cells (3a and 3b) into 3a', 3a' and 3b' and 3b2 is shown.

15 Sixty-cell stage, vegetal view; 120 minutes. Note the division asynchronyof the macromeres 3A-3D. 16 Sixty-four-cell stage, vegetal view; 160 minutes. The larger cells of the third quartet, i.e.. ventrally 3a' and 3b', and dorsally 3c' and 3d', are in prophase preceding a following division. The previous differences in the division inequality are thus followed by a different division pattern.

17 Seventy-two-cell stage, animal view; 170 minutes. The dorsal tip cell 2d" and the micromeres la"'-ld"' and la'22-ld1'12 are in mitosis. 18 Eighty-eight-cell stage, seen from the vegetal pole; 190 minutes. At this stage the trochoblasts have developed cilia. The mesentoblast 4d is in prophase preceding the formation of the paired mesentoblast cells, MI and M,.

176

DETERMINATION O F UOHSOVENTHALITY IN PATELLA

PI , A T E :i

J A M van den Biggelaar

177

DETEHMlNATlON OF DORSOVENTRALITY IN PATELLA van den Biggelaar

J. A. M.

EXPLANATION OF FIGURES

19 Embryo of 190 minutes, lateral view. The tip cells in the quadrants A, B and C are divided too. 20 Embryo of 200 minutes seen from the vegetal pole. The primary mesentoblast 4d has divided into MI and MP 21

Micrograph of a living embryo a t the transition of the 60- into the 63-cell stage. Dorsoventrality is expressed in the division asynchrony of the macromeres 3A-3D and in the division pattern of the micromeres of the third quartet.

22 Animal cross-section of a 16-cell embryo. The animal cross-furrow is present between lbl and ldl. 23 Same embryo as in figure 22. Section through the aequatorial region, demonstrating the presence of internal cell contacts between the micromeres 2a-2d. 24 Same embryo as in figures 22 and 23.Section about 10pm more vegetal than the latter. The tips of the macromeres are wedged apart and held in a more superficial position by the inserted second quartet cells.

25 Thirty-two-cell stage, ten minutes after the beginning of the fifth cleavage. Sagittal section, near the plane of bilateral symmetry, indicating the remnants of the previously existing cell contacts between second quartet cells and between the lower micromeres of the first quartet. 26

178

Same embryo as in figure 25. Sagittal section, 3/rm nearer the plane of bilateral symmetry than in the latter figure.

PLATE 4

DETERMINATION OF DORSOVENTRALITY IN PATELLA J . A. M van den Biggelaar

PLATE 5

179

PLATE 6 EXPLANATION OF FIGURES

Age of the embryos given in minutes after the beginning of the 32-cell stage. 27 Same embryo as in t he figures 25 and 26, medial section. The macromeres 3B and 3D are placed opposite the centrally protruding micromeres la'2-ld'z. 28 Thirty-two-cell stage, 30 minutes. Sagittal section, about 9 Frn from the plane of bilateral symmetry. 3B and 3D (the axial or cross-furrow macromeres) laterally border on the centrally meeting group of micromeres la'z.ld'2. 29 Same embryo as in figure 28, medial section. Centrally, the macromeres are still separated from la12-ld12. The derivatives of the primary trochoblasts (la2'-ld2'and laz2IdZ') and of the second quartet of micromeres (2a1-2d' and 2az-2d2)have smooth inner cell surfaces. 30 Thirty-two-cell stage, 50 minutes. Slightly oblique cross-section through the major part of the centrally radiating micromeres of the first quartet. The animal cells la". The most vegetal trochoblasts, la22-ld22. are pushed Id" separate the cells la12-ld1Z. into a more superficial position than lazl-ld"'. 31 Same embryo as in figure 30, about 15 p m more vegetal. The tip of one macromere (3D) is surrounded by the centrally approaching cells la'z-ld'z. The cells laZ1-ldz1 lie The second quartet cells 2a'-2d1 are retracted further closer to 3D than laz2-IdZZ. superficially. 32 Same embryo as in figures 30 and 31, section a t a level of about 1 5 p m more vegetal than the latter. Note the advanced position of the two axial macromeres, and the absence of the lateral macromeres 3A and 3C. A t this lower section, 3B and 3D still border on t he micromeres lazz-ldz2,but are separated from the second quartet cells 2a'-2d1 and 2a2-2d2by means of large clefts.

180

DETERMINATION OF DORSOVENTKALITY IN PA7ELLA d A M van den Biggelaar

181

PLATE 7 EXPLANATION OF FIGURES

Age of the embryos is given in minutes after the beginning of the 32-cell stage. 33 Thirty-two-cell stage; 50 minutes. Nearly horizontal section. 3D definitely has a higher position than 3B, 3A and 3C, and is in touch with the most animal first quartet cells, la"-ld". The macromeres are separated from the major part of the second quartet cells. 34 Thirty.two-cell stage, 50 minutes. Nearly medial section, showing the central position of 3D, and the central contacts within the group of la"-ldlZ, which separate 3D from la"-ld". 35 Thirty-two-cell stage, 50 minutes. Nearly meridional section. The contacts of 3D are extended to the micromeres at the animal pole, la"-ld", stressing its pivot position. In the equatorial zone, small cavities indicate the absence of internal contacts.

36 Thirty-two-cell stage, 70 minutes. Meridional section. Extended common cell borders between t he macromeres 3 8 and 3D and the third quartet cells, 3a-3d. After a continued retraction of 3B, the micromere 3a may form a n internal common cell border with 3D.

37 Thirty-two-cell stage, 70 minutes. Nearly meridional section. Extended contact between 3D and 3d. By the extension of this common boundary wall and the one with Id", the trochoblasts will be pushed further superficially, farther away from 3D. 38 Same embryo as in figure 37, meridional section a t a distance of about 9 p m from t h a t represented in the latter.

182

DETERMINATIOK OF DORSOVENTHALITY IN PATELLA J A M van den Biggelaar

PLATE 7

.PLATE 8 EXPLANATION OF FIGURES

Age of the embryos is given in minutes after the beginning of the 32-cell stage. 39 Thirty-two-cell stage, 70 minutes. Nearly medial section. Extended common cell wall between 3B and 3b. and between 3D and 2d2. I n the equatorial zone, 2b’ and 2d’ are separated from the macromeres. 3D is the only macromere that borders on the animal micromeres. 40 Same embryo as in figure 39, medial section. Note the special cytoplasm in the tips of the micromeres along the common cell borders with 3D. 41 Thirty-two-cell stage, 70 minutes. Slightly oblique cross-section, showing the separation of 3D from the micromeres with an equatorial position. I n the C-quadrant it is evident that the intruding third quartet cells separate 2c’ and 1c2*from the macromeres 3C and 3D. 42 Same embryo as in figure 41, section a t a level about 3 p m higher than that of the latter. At this height 3D is in contact with first quartet cells. 43 Forty-cell stage, 90 minutes. Cross-section, demonstrating the connections between 3D and t he cells l a12-l d12 and its separation from lower cells. 44 Forty-cell stage, 90 minutes. Oblique cross.section, showing extreme axial position of 3D and its separation from the trochoblasts and second quartet cells, except those of the D-quadrant.

184

DETERMINATION OF DOKSOL ENI’HALITY IN PATELLA

J A M van den B1ggehd.r

1)E’l’k;KMINATlON O F 1)OHSOVENTHALI‘I’Y I N PATELLA

J. A. M

van drn Uiggelaar

EXPLANATION OF FIGURES

Age of the embryos is given in minutes after the beginning of the 32-cell stage.

186

45

Same embryo as in figure 44, section about 9 p m lower than that represented in the latter.

46

Sixty-cell embryo, 110 minutes. Meridional section. The tip of 3D rises far above the tips of 3B and 3C.I t has extended common cell borders with lb”’ and Id”’, whereas i t is separated from the majority of the lower first and second quartet cells.

47

Sixty-three-cellstage, 130 minutes. Oblique cross-section. showing the isolated axial position of 3D.

48

Sixty-four-cell stage, 150 minutes. Oblique cross-section, illustrating t h e isolated position of the primary mesentohlast 4d, and t h e contacts between the micromeres 4a and 4b with a cell of the first quartet and with a few animal second quartet cells.

PLATE 9