Unequal cleavage and the differentiation of echinoid primary mesenchyme

DEVELOPMENTAL

BIOLOGY

109,

464-475

(19%)

Unequal

Cleavage and the Differentiation Echinoid Primary Mesenchyme

ROSALIE E. LANGELAN' Departmen,t

of Zoology, Friday Hnrbor

Received

November

University Labcwatties,

of

AND ARTHUR H. WHITELEY

of Washington, Seattle, Washin.qton 98195; and Friday Harbor, Wu.shin.qrtm 98250

14, 1984; accepted

in revised

fin-m

December

30, 1984

The role of unequal cleavage in echinoid micromere determination was investigated by equalizing the fourth and fifth cleavages with brief surfactant treatment. The surfactant sodium dodecyl sulfate was found to be effective in equalizing fourth cleavage when generally applied to 4-cell stage embryos of all species tested. Embryos of the sand dollar Den&aster excentricus developed normally when equalized at the fourth and fifth cleavages by surfactant treatment, as did untreated equally cleaving embryos of the sea urchin Strongylocentrotus droebachiensis. Embryos of the sea urchins Lytechinus pi&us and 5’. purpuratus were animalized by the treatment but were capable of forming spicules after treatments which equalized the fourth cleavage. In addition, orientation of the fourth division spindles was found to have no effect on differentiation of the primary mesenchyme in D. ezcentricus. The results confirm that micromere determination in echinoids does not depend upon a strict cleavage pattern at the l&cell stage. 0 1985 Academic

Press, Inc.

INTRODUCTION

micromeres, 26 ym (Mizuno et ah, 1974). Unequal fourth cleavage is characteristic of the euechinoid subclass of Echinoidea (Horstadius, 1973). Driesch (1892) and Boveri (1901) reported that the unequal cleavage responsible for micromere formation could be inhibited by mechanical disturbance without effect on later development. Tennent et al. (1929) carefully examined the relationship between the number of micromeres and the formation of mesenchyme in fertilized Lytechinus picks egg fragments and concluded that differentiation of the echinoid embryo is independent of micromere formation. However, Tanaka (1976) reported that when the fourth cleavage of sea urchins was equalized by brief treatment with surfactants such as sodium dodecyl sulfate (SDS),’ the embryos exhibited a variety of abnormalities in later development including inhibited gastrulation, reduced primary mesenchyme, and a lack of spicule growth. These defects are typical of animalized larvae and confirm previous reports that SDS has an animalizing influence on sea urchin embryos (Gustafson and Savhagen, 1949; Runnstrom, 1966; Lallier, 1973). SDS treatment prevents the normal migration of the four vegetal nuclei to an excentric position at the vegetal pole prior to the fourth division and has been shown to have a disruptive effect upon the cell cortex (Dan, 1979; Tanaka, 1979). Based on studies of

The 16-cell stage echinoid embryo consists of three cell types, macromeres, mesomeres, and micromeres, which differ in at least four major respects: cell size, cell division pattern, position along the animal-vegetal axis, and presumptive fate in the larva. The fact that the cell types can be separated on the basis of size has led to a large number of studies of this embryonic stage (for review see Harkey, 1983). Much interest has focussed on the micromeres at the vegetal pole of the embryo which follow their normal developmental pathway in vitro to produce skeletal spicules (Okazaki, 1971, 1975; Kitajima and Okazaki, 1980; Harkey and Whiteley, 1980, 1982, 1983; Harkey, 1983). Micromerespecific factors or determinants have not been identified, although there is evidence for factors that elicit micromere-specific transcription (Mizuno et ah, 1974) and for the segregation of unique-class RNA sequences among blastomere types (Rodgers and Gross, 1978; Ernst et al., 1980). The unequal fourth cleavage of echinoids alters the nucleocytoplasmic ratios among the cell types and nucleocytoplasmic ratio has been shown to affect the timing of developmental events in a variety of other systems (Smith and McClaren, 1977; Newport and Kirschner, 1982a,b; Mita, 1983). The average diameter of sand dollar (Dendraster excentricus) macromeres at the 16-cell stage is 55 grn, mesomeres, 44 pm, and ’ To whom 001%1606/85 Copyright All rights

correspondence

should

$3.00

Q 1985 by Academic Press, Inc. of reproduction in any form reserved.

‘Abbreviations used: SDS, sodium dodecyl sulfate; FSW, filtered seawater; SDS-SW, SDS dissolved in FSW; CTAB, cetyltrimethylammonium bromide.

be addressed. 464

LANCELAN

AND

WHITELEY

SDS-treated embryos, Dan has proposed that ultimate spicule differentiation in Hemicentrotus is dependent on the direction of the cleavage plane rather than on the size inequality of the fourth cleavage (Dan, 1978, 1979, 1984). In light of these recent reports, we have reexamined the development of surfactant-treated euechinoid embryos to clarify the relationship between unequal fourth cleavage and primary mesenchyme differentiation, extending the analysis to species not examined before. The importance of the vegetal cleavage plane in the development of equally cleaving embryos is also addressed. The results show that none of these factorsorientation of the fourth cleavage spindle, unequal fourth cleavage, or the resulting small size of the micromeres-is irrevocably related to the normal determination of cells to ‘differentiate the spicules in echinoids. MATERIALS

AND

METHODS

To control for species differences, we used one species of sand dollar (D. excentricus Eschscholtz) and three species of regular sea urchin (Strongylocentrotus purpuratus Stimpson, S. droebachiensis 0. F. Miiller, and L. pictus Verrill) as experimental material. D. ezcentricus were collected intertidally from San Juan Island and Seattle, in Washington and from Sidney Spit, British Columbia. S. purpuratus were collected intertidally along the Strait of Juan de Fuca, Washington. S. droebachiensis were obtained subtidally near Friday Harbor, Washington and L. pictus were obtained from Pacific Biomarine, courtesy of Dr. Merrill Hille. Gametes were obtained by intracoelomic injection of 0.55 M KC1 or by brief electric shocks with 60 V ac. Eggs were fertilized in filtered seawater and incubated at lo-12°C. Sodium dodecyl sulfate (BDH Ltd. specially pure) was dissolved in 0.5 MNalCl or FSW at a concentration of 500 pg/ml and diluted with FSW (SDS-SW). Controls were made using 0.5 M NaCl diluted with FSW. SDS treatments were carried out in 60-mm Falcon petri dishes by adding an equal volume of dilute embryo suspension to a twofold concentration of SDS-SW. Embryos were treated with fertilization membranes and jelly coats intact alth.ough the latter were usually lost during handling. Embryos were diluted from SDSSW by settling through 60-100 vol FSW or by gentle centrifugation and washes in FSW. Samples containing approximately 500 embryos were preserved at the 16or 32-cell stage with a drop of l-4% glutaraldehyde (Stevens Metallurgical Corp.) in 0.5-l ml FSW for overnight or longer. Uneiqual cleavage was scored in preserved embryos flattened between a glass microslide and a coverslip.

Unequal

Ckavage

in

465

Echinoids

Embryos were cultured to the pluteus stage in FSW at lo-12°C. Approximately 200 embryos were scored for normal development at various stages using a Zeiss microscope equipped with Nomarski differential interference optics or polarizing filters. Micrographs were taken on Kodak Panatomic-X film using a Nikon camera. Images were recorded on videotape using a video system composed of the following components: Dage MT1 Newvicon camera, RCA video time-date generator, Panasonic time-lapse VTR, and Sony black-white monitor. RESULTS

SDS Effect on Fourth

Cleavage

Treatment of D. excentricus embryos with 20 pug/ml SDS-SW for the entire 4-cell stage (approximately 60 min at 11°C) reproducibly results in equalization of the fourth cleavage. The size of the 16-cell stage blastomeres in equally cleaving embryos was relatively uniform, averaging 45 pm in diameter (Fig. lb). SDS is most effective in altering the fourth cleavage when administered during the mid four-cell stage of

FIG. 1. (a and b) Fixed l&cell stage D. ementricus embryos flattened to facilitate the scoring of micromeres. Cells from each embryo are retained within a fertilization membrane. (c and d) Live embryos photographed at the 32-cell stage. Control (a and c) embryos contain the normal complement of micromeres. (b and d) Embryos treated with 20 pg/ml SDS for 60 min at the 4-cell stage lack micromeres at the 16-cell stage (b) but cleave unequally to produce four small cells at the 32-cell stage (d). (mi) micromeres, (f) fertilization membrane. Bars = 25 pm.

466

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H. pulcherrimus

embryos (Tanaka, 1976). We obtained a similar result using D. excentricus embryos. The embryos were treated with either 30- or 60-min pulses of 25 pg/ml SDS-SW at various times prior to fourth cleavage and the percentage of embryos consisting of 16 equal-sized blastomeres was scored after the fourth cleavage. From the results (Fig. 2), it is apparent that the micromere-forming ability of Dendraster embryos is most sensitive to the treatment at the four-cell stage, with slight sensitivity at the two-cell stage. The SDS effect on fourth cleavage was dosage dependent (Fig. 3). Low concentrations of SDS (~1 pg/ml) had little or no effect on the fourth cleavage pattern. As the SDS concentration was increased to approximately 15 pg/ml, the percentage of embryos forming four micromeres decreased and the cleavage pattern became predominantly intermediate, with most embryos forming fewer than four micromeres. The intermediate class thus consisted of embryos containing one, two, or three micromeres, often of larger size than normal. The variation in the number of micromeres suggests that each blastomere responds to the SDS treatment independently of the response of the other blastomeres. The variation in micromere size suggests that within each blastomere the unequal fourth cleavage may be a graded phenomenon rather than an equal vs unequal event. Sixty-minute treatments of 20 to 25 pg/ml SDS were effective in completely eliminating unequal cleavage

100 *

c c

25pg/ml

h

SDS

w d

I

HRS

AFTER

I

\

I

FERTILIZATION

2. Sensitivity of D. excentticus embryos to pulse treatments with 25 fig/ml SDS before fourth cleavage. Data were obtained from a single batch of eggs treated for 30- (0) or 60-min (0) intervals. Each point marks the midpoint of a treatment period represented by horizontal bars. The percentage of equalized embryos (containing no micromeres) is based on counts of approximately 200 16-cell stage embryos in each sample. Arrows mark the time of 50% cleavage. FIG.

0

5

IO SDS

15

20

25

CONCENTRATION(ug/ml)

FIG. 3. The effect of SDS at various concentrations on D. excentrim fourth cleavage. Embryos were treated with SDS in seawater for 60 min at the 4-cell stage and scored for unequal cleavage at the 16. cell stage. Each point represents the mean of duplicate samples from a single batch of eggs. Open circles, solid line: embryos with no micromeres. Open circles, dashed line: embryos with one to three micromeres. Closed circles, solid line: embryos with four micromeres. Closed circles, dashed line: embryos with fewer than 16 cells.

from a majority of embryos (Fig. 3). The response varied slightly between egg batches. Higher SDS concentrations (a30 pg/ml) inhibited cytokinesis and the cells began to lyse. These data correspond closely to those reported by Tanaka (1976). All four species examined in the present study exhibited similar dosageresponse curves, with the exception of S. droebachiensis embryos which were subject to cytolysis at low SDS concentrations (<5 pg/ml). The cationic surfactant CTAB was effective in equalizing the fourth cleavage of D. excentricus and S. purpuratus embryos at concentrations of 2-3 pg/ml, but it frequently cytolyzed the cells and was not used in this study.

Development of D. excentricus Fourth Cleavage

with Equal

Table 1 presents data on the development of SDStreated sand dollar (D. excentricus) cultures. Embryos from a single batch of eggs were treated with 15 to 20 pug/ml SDS for 53 min during the 4-cell stage and embryos from each sample were fixed at the 16-cell stage to score micromere formation. Increasing concentrations of SDS decreased the percentage of embryos cleaving unequally at the fourth division (Table 1). However, the ability of the embryos to develop into normal larvae did not reflect the effect of these increasing SDS concentrations on micromere formation. Approximately 90% of the embryos in each SDS-treated culture formed normal larvae with well-developed guts and spicules, regardless of the percentage of embryos

LANGELAN

AND

WHITELEY

Unequal TABLE

RELATIONSHIP

BETWEEN

FOURTH

% l&Cell Cone. SDS (&ml) 0 15.0 17.0 18.5 20.0

CLEAVAGE

AND

Cleavage

in

467

Echinm’ds

1 LATER

DEVELOPMENT

OF Dendraster

excentricus % Normal

embryos

Micromeres

Intermediate

Equal

87.0 4.5 1.5 0 0

13.0 42.1 26.3 12.5 5.3

0 52.5 69.8 86.9 90.9

Abnormal cleavage 0 1.0 2.4 0.6 3.8

Mesenchyme blastula 96.5 82.0 72.0 71.5 72.5

development Early gastrula

Prism

97.5 86.0 89.0 89.5 89.0

93.0 89.0 86.5 86.0 90.5

Note. Embryos from a single spawning were immersed in SDS-SW for 53 min at the 4-cell stage and returned to seawater. The percentage values are based on counts of 200 embryos collected from cultures. Unequal cleavage was scored as described under Materials and Methods. The categories of micromeres, intermediate, and equal are based on the number of micromeres present at the 16-cell stage as described in the text, and the abnormal cleavage category represents the embryos with inhibited cytokinesis after SDS treatment. Mesenchyme blastula, 27-29 hr; early gastrula, 35 hr; prism, 56 hr. containing micromeres at the 16-cell stage. The increase in percentage of normal. development following the mesenchyme blastula stage (Table 1) may be indicative of “recovery” of a normal phenotype by some embryos.

The development of one of the of Table 1 (20 pg/ml) is shown percent of the embryos cleaved division, 5% had one or two

SDS-treated cultures in Fig. 4. Ninety-one equally at the fourth micromeres, and 4%

FIG. 4. Later development of D. ezcentricus embryos treated with 20 fig/ml SDS for 53 min at the 4-cell stage. (a) The 16-cell stage embryos equalized by the treatment, fixed at 5 hr. (b) Mesenchyme blastula stage, 27 hr after fertilization. Twenty-seven percent of the culture had abnormal numbers or sizes of cells in the blastocoel; 73% developed normally. (c) Same culture at the prism stage, 50 hr, showing early spicule formation. (d) Pluteus stage, 72 hr, showing normal development of larval spicules. Less than 10% of the larvae showed developmental aberrations (arrow). Skeletal spicules were scored and photographed in (c) and (d) using cross-polarized light. (a, b, and c) are shown at the same magnification. Bars = 100 pm.

468

DEVELOPMENTAL

BIOLOGY

showed aberrant cleavage patterns (Fig. 4a). At the mesenchyme blastula stage, 73% of the embryos developed primary mesenchyme that appeared normal both in the size and the number of cells, and in their distribution within the blastocoel (Fig. 4b). The remainder exhibited disorganized mesenchyme or an abnormal number of cells in the blastocoel. Ninety percent of the embryos in this culture gastrulated normally and formed spicules on schedule (Fig. 4~). Ten percent of the embryos had abnormal numbers or sizes of cells in the blastocoel, surrounding an archenteron normal in size and shape. At the pluteus stage, the culture was virtually indistinguishable from control cultures (Fig. 4d). This result is reproducible except for slight variation in SDS sensitivity between egg batches. To confirm the observation that SDS-treated D. excentricus embryos with 16 equal-sized cells differentiated into normal plutei, we isolated individual embryos at the 16-cell stage and observed their development. Embryos were selected on the basis of cleavage pattern and incubated at lo-12°C individually or in groups of four to five in covered 15-mm petri dishes containing FSW. Table 2 summarizes the results of these experiments. A substantial proportion (78%) of isolated embryos with 16 equal-sized cells after treatment with 15 hg/ml SDS developed normally. Fewer normal embryos were obtained from a higher SDS treatment (20 pg/ml); 65% of embryos with equal fourth cleavage developed into normal plutei. We have concluded from these studies that the formation of micromeres at the 16-cell stage is not

TABLE DEVELOPMENT

Treatment Control SDS 15 fig/ml SDS 20 pg/ml

2

OF SURFACTANT-TREATED

D. excentricus

Cleavage type

Total isolated

Number plutei

Micromeres Micromeres Intermediate Equal Intermediate Equal

111 31 89 80 70 138

93 27 82 62 59 90

EMBRYOS

Percentage normal 83.8 87.1 92.1 77.5 84.3 65.2

Note. Cultures were treated with SDS in seawater for 45-60 min at the 4-cell stage. Embryos were selected on the basis of their number of micromeres at the 16-cell stage and reared at 11°C in seawater until the controls reached the pluteus stage (72 hr after fertilization). A cell was considered to be a micromere if it was smaller than 27 wcm in diameter at the 16-cell stage. Intermediate embryos contained one, two, or three micromeres. Equal embryos contained no cells smaller than 27 Km in diameter at the 16-cell stage. Normal plutei contained no aberrations in the larval skeleton, digestive tract, or body form.

VOL~JME

required enchyme

109,

1985

for the differentiation of D. excentricus.

Equalization

of Fifth

of the primary

mes-

and Later Cleavages

The micromeres of echinoid embryos normally divide unequally at the fifth cleavage to produce four outer, large micromeres which give rise to primary mesenthyme and four inner, small micromeres which become incorporated into the pharynx (Endo, 1966). Thus, the definitive primary mesenchyme cell lineage actually appears at the fifth cleavage. Perhaps a determinative event occurs at the fifth division which segregates inner and outer micromeres of different developmental fate. In D. excentricus, the range of diameters of the large micromeres of the 32-cell stage is 24-26 pm and the range of diameters of the small micromeres is 14-17 pm (Figs. lc, 5a). D. excentricus embryos treated with 20 pg/ml SDS for 60 min at the 4-cell stage usually reverted to unequal division at the fifth cleavage (Fig. Id). The “micromeres” produced by unequal fifth cleavage of an SDS-treated embryo varied in number, and approached the size of micromeres of a normal 16-cell stage embryo (26 pm diameter). These “micromeres” formed at the 32-cell stage of an SDS-treated embryo usually underwent an additional unequal cleavage at the sixth division. That is, the normal pattern of unequal cleavage was delayed by one division cycle due to the SDS treatment. There was considerable variation in the pattern of later cleavages of SDS-treated D. excentricus embryos, and occasionally in control embryos. For example, an equalized cell or a control micromere could divide equally and meridionally at the fifth cleavage instead of unequally and horizontally. In these cases both daughter cells divided unequally at the sixth cleavage. SDS treatments of longer duration or higher concentration are effective in equalizing both fourth and fifth cleavages (Fig. 5e) (Tanaka, 1976). A minimal treatment of 25 pg/ml SDS for 45 min at the 4-cell stage equalized both fourth and fifth cleavages in a variable proportion of D. excentricus embryos. Embryos without micromeres at the 32-cell stage hatched on schedule. A cell was considered to be a “micromere” at the 32-cell stage if it was detectably smaller (under 34 pm) than the surrounding blastomeres (35 pm) when examined with 160X magnification. We selected individual embryos on the basis of number of “micromeres” at the 32-cell stage and observed them for the formation of primary mesenchyme (Fig. 6). Half of the embryos containing no “micromeres” at the 32-cell stage lacked primary mesenchyme when examined at the mesenthyme blastula stage, 27 hr after fertilization (Figs.

LANGELAN

AND

WHITELRY

Uwqual

Cleavapz

in,

Echinoids

469

FIG. 5. Development of SDS-treated D. excentricus equalized at both fourth and fifth cleavages. (a-d) Control embryos. (e-h) SDS-treated embryos at same magnification as controls. (a) Control embryo, fixed and flattened at the 3%cell stage. Note the presence of four small and four large micromeres. (b) Control mesenchyme blastula, 27 hr postfertilization, with primary mesenchyme cells in the blastoeoel. (c) Control gastrula, 31 hr, showing primary mesenchyme cells arranged on the outer blastocoel wall (arrowheads) as the archenteron invaginates. (d) Control pluteus, ‘75 hr. (e) SDS-treated embryo fixed and flattened after fifth cleavage. Note the absence of unequal cleavage. (f) Equalized embryo at 28 hr, lacking primary mesenchyme. (g) Equalized gastrula, 31 hr, showing mesenchyme cells clustered on the archenteron and missing from the outer blastocoel wall (arrowheads). (h) Equalized pluteus, 72 hr, showing abnormal spicules. (b, c, f, and g) are at the same magnification. Bars = 50 pm.

5f, 6). Those showing an absence of primary mesenthyme were usually abnormally elongated along the animal/vegetal axis of the swimming blastula. In these defective embryos, mesenchyme cells ingressed from the archenteron tip in the early to midgastrula stage, migrated down the sides of the archenteron, and began producing spicules with a delay of 5 to 15 hr when compared to controls (Fig. 5g). The archenteron frequently assumed an abnormal “pointed” shape and progressed slowly through gastrulation. The plutei which developed from these embryos showed only minor aberrations in the skeleton (Fig. 5h). The proportion of embryos with a deficient complement of primary mesenchyme prior to gastrulation varied in relation to the number of “micromeres” at the 32-cell stage (Fig. 6). It should be noted that 40% of embryos without “m.icromeres” at the 32-cell stage were capable of completely normal development to pluteus. We conclude from this study that (1) unequal

fifth cleavage is not essential for the determination of micromeres to form spicules, (2) the ingression of primary mesenchyme prior to gastrulation is not essential for the deposition of spicules, and (3) the number of micromeres at the 32-cell stage is correlated with the ingression of primary mesenchyme but not with the timing of hatching. D. excentricus embryos frequently deSDS-treated veloped abnormalities at SDS concentrations slightly higher than those needed to block unequal fourth cleavage (25 vs 20 pg/ml). A variety of developmental abnormalities were observed in some egg batches treated with 25 pg/ml for 60 min at the four-cell stage. The major abnormal morphology at the mesenchyme blastula stage was a pathological invasion of cells into the blastocoel. A substantial proportion of embryos showed the absence of primary mesenchyme described above or a displacement of mesenchyme cells toward the animal pole. Two types of abnormal larvae were

470

DEVELOPMENTAL

BIOLOGY

EQUALIZED

39

27

156

52

VOLIJME

109, 1985

division planes. These were classified as asymmetric and equal. Orientation of mitotic spindles prior to fourth cleavage was ascertained using video-enhanced polarization microscopy (Fig. 7~). SDS-treated D. excentricus embryos could maintain normal spindle orientations, to

4 NUMBER

OF MICROMERES

IN 32

CELL

STAGE

EMBRYO

FIG. 6. Relationship between the number of 3%cell micromeres and the percentage of embryos with primary mesenchyme in D. ezcenfricus. SDS-treated embryos were selected on the basis of their number of small cells at the 32-cell stage and scored at 27 hr for the presence of primary mesenchyme. The number of individuals (N) in each category is shown at the top of the figure. Open bars: proper number of primary mesenchyme cells. Closed bars: deficient in primary mesenchyme. Hatched bars: pathological number of blastocoel cells (stereoblastula).

a-

b-

observed: stereoblastulae which failed to gastrulate, and blocked gastrulae with rudimentary spicules. None D. excentricus larvae showed the of the SDS-treated H. animalized morphology described for SDS-treated pulchewimus embryos (Tanaka, 1976).

Cleavage Orientation

in SDS-Treated

D. excentricus

Dan (1978, 1979, 1984) suggested that spicule differis dependent on the dientiation in H. pulcherrimus rection of the vegetal cleavage plane at the fourth division. At the typical euechinoid fourth division, the animal blastomeres undergo equal, meridional cleavage to form a ring of eight blastomeres. In contrast, the vegetal blastomeres divide unequally and almost horizontally to form a subequatorial tier of four macromeres and a vegetal tier of four micromeres. Thus the normal 16-cell stage consists of three tiers of cells along the animal-vegetal axis, each tier containing cells of a single size class and a unique cytoplasmic composition. The micromeres are the only cells that include the vegetal pole cytoplasm. Many SDS-treated D. excentricus embryos treated with 20 pg/ml SDS for 60 min at the four-cell stage clearly contained three tiers of equal-sized blastomeres after fourth cleavage (Fig. ?‘a). Occasionally abnormal two-tiered embryos were produced as a result of vegetal blastomeres dividing in the same meridional direction as the animal cells (Fig. 7b). The majority of SDStreated embryos contained a loose blastomere arrangement from which it was difficult to ascertain the fourth

FIG. 7. Cleavage orientation of SDS-treated D. excentricus embryos. (a) Three-tiered cleavage orientation. (b) Two-tiered cleavage orientation. (c) Fourth division mitotic spindles viewed with videoenhanced polarization microscopy. Supposed vegetal cell spindles (V) are pointing toward one embryonic pole, in perpendicular orientation to supposed animal cell spindles (A). (d) The same embryos 9 min later, showing three-tiered orientation of the fourth cleavage. The daughter cells produced by cytokinesis of the labeled cells in (c) are indicated by letters A and V. Bars = 50 pm.

LANGELAN AND WHITELEY

give rise to three-tiered embryos (Figs. 7c, d). The developmental importance of the vegetal cleavage plane was investigated by separating SDS-treated embryos containing two tiers of cells from embryos that contained three tiers of cells or an asymmetric blastomere arrangement at the 16-cell stage (Table 3). A majority (72%) of isolated two-tiered embryos developed normally to 72 hr plutei. In these, the correct number of primary mesenchyme cells was formed on schedule, the embryos gastrulated and formed plutei with normal digestive tracts and normal spicules. The major abnormality at the pluteus stage consisted of the presence of one or two extra spurs or rods in the spicules (Table 3, abnormal spicules). Thus embryos consisting of two tiers of blastomeres at the 16-cell stage, presumably formed by meridional cleavage of all cells, were capable of normal morphogenesis. At the mesenchyme blastula stage (27 hr), a proportion (g-18%) of embryos in each of the three cleavage orientation categories was deficient in the formation of mesenchyme (Table 3). The embryos contained few or no primary mesenchyjme cells at the mesenchyme blastula stage (27 hr postfertilization). They began gastrulation without a full complement of primary mesenchyme cells (Fig. 5f) and subsequently formed mesenchyme from the tip of the archenteron at the early to midgastrula stage of these embryos (Fig. 5g), which migrated vegetally as described above. Nonetheless, these embryos formed plutei with normal or slightly irregular spicules. These observations indicate that the presence and ultimate differentiation of pri-

LATERDEVELOPMENTOFQURFACTANT-TREATED

D. excentricus

Cleavage

Unequal

in

471

Echinoids

mary mesenchyme in larvae does not depend on the plane of the fourth cleavage in SDS-treated D. excentricus embryos. Animalized Development of SDS-Treated L. pictus and S. purpuratus Embryos We have investigated the relationship between the SDS effect on fourth cleavage and the later development of two species of regular urchins, L. pi&us and S. purpuratus. Results obtained are essentially the same for both species. Therefore, the development of SDStreated L. pictus will be discussed. SDS-treated L. pictus embryos cleaved equally at the fourth division and exhibited animalized development similar to that reported for H. pulcherrimus (Tanaka, 1976). The embryos exhibited enlarged apical tufts, reduced archenterons, and little or no spicule development (Fig. 8~). The degree of animalization was dependent upon the concentration of SDS and varied only slightly between egg batches. Figures 8b and c show L, pictus larvae from different egg batches treated with 15 or 25 pg/ml SDS, respectively, at the four-cell stage. In both cultures, 87% of the embryos cleaved equally at the fourth division. At the lower SDS concentration, 15 pg/ml, the degree of animalization was not severe. The equalized embryos were capable of forming spicules, although spicule and gut development were reduced when compared with controls (Figs. 8a, b). Archenteron formation and spicule development were almost completely inhibited in the sample treated with 25 pg/ml (Fig. 8~).

TABLE3 EMBRYOSWITHRESPECTTOTHEPLANEOFTHEFOURTHCLEAVAGE Equalized

27 hr postfertilization Blastula with mesenchyme Blastula without mesenchyme Stereoblastula Total

embryos

isolated

72 hr postfertilization Normal pluteus larva Larva with abnormal Dense ovoid larva Total

embryos

isolated1

spicules

embryos

Controls

Z-Tiers

3-Tiers

77 (100%) 0 (0%) 0 (0%)

49 (86%) 6 (11%) 2 (4%)

39 (87%) 4 (9%) 2 (4%)

67 (80%) 15 (18%) 2 (2%)

77

57

45

84

72 (94%) 4 (5%) 1 (1%)

41 (72%) 15 (26%) 1(2%)

40 (89%) 5 (11%) 0 (0%)

65 (77%) 15 (18%) 4 (5%)

77

57

45

84

Note. Equalized embryos treated with 20-25 pg/ml SDS at the at the 16-cell stage and reared in seawater at 11°C. Larvae were hr postfertilization) and for the presence of normal spicules at the if they contained extra spurs or rods. Embryos were classified as poorly.

Asymmetric

4-cell stage were selected on the basis of the number of blastomere tiers scored for the presence of primary mesenchyme prior to gastrulation (27 pluteus stage (72 hr postfertilization). Spicules were considered abnormal stereoblastulae or dense ovoid larvae if they were opaque and swimming

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FIG. 8. Later development of normal and SDS-treated L. yicfus embryos. (a) Control larvae (5-day). (b) Larvae (72 hr) treated with I5 fig/ml SDS for 45 min at the 4-cell stage to produce equal-sized blastomeres at the l&cell stage and photographed with cross-polarized illumination. (c) Larvae (5 day) treated with 25 rg/ml SDS for 70 min at the 4-cell stage, showing reduced differentiation of spicules and digestive tract. (s) spicules, (g) gastrointestinal tract. Bar = 50 pm.

These observations suggest that although SDS animalizes the larvae of these two euechinoid species, spicule differentiation can occur from 16-cell stage embryos with equal-sized cells. We conclude that spicule formation in L. pi&us and S. purpurutus, like that in D. excentricus, does not require the formation of micromeres at the 16-cell stage. Normal

Development

of S. droebachiensis

Our cultures of S. droebachiensis made in the middle of several successive breeding seasons reproducibly contained substantial numbers of embryos that cleaved to produce blastomeres of equal size at the 16-cell stage with no experimental treatment. Other embryos contained one, two, or three micromeres that varied in size. Depending on the female, 5 to 53% of the control eggs cleaved equally at the fourth division (Fig. 9a). Eighty-one individual control embryos were selected on the basis of vegetal cell size at the 16-cell stage and followed through development. Of these, 95% of the isolated intermediate embryos (containing one to three micromeres) formed normal plutei and 90% of the isolated 16 equal embryos formed normal plutei (Fig. 9f). There was considerable variation in the number of large and small micromeres formed at the 32-cell stage of these control embryos. The embryos exhibited no defects in later development (Figs. 9d-f). Furthermore, equally cleaving S. droebachiensis cells appeared to cleave with either a typical horizontal orientation to form three tiers (Fig. 9b) or an altered meridional orientation to form two tiers (Fig. SC), without affecting

later development, Therefore, we conclude that primary mesenchyme formation and differentiation in S. droebachiensis, as in the sand dollar D. excentricus, is dependent on neither the formation of micromeres at the 16-cell stage nor the three-tiered orientation of the fourth cleavage. We cannot account for the variability in S. droebachiensis fourth cleavage pattern. It may be a natural cleavage variant, although equal fourth cleavage seems not to have been previously reported for this species. S. droebachiensis embryos are cytolyzed by SDS concentrations greater than 5 pg/ml. SDS treatment of 2.5 pg/ml for the entire four-cell stage results in equal fourth cleavage of the entire population. These embryos are severely animalized, with inhibited gastrulation and no spicule development. DISCUSSION

There are two morphological events associated with differentiation of the primary mesenchyme: ingression and spicule deposition. We have scored both of these events in embryos that have been equalized to prevent formation of micromeres. The experiments and observations reported here have confirmed that micromere formation at fourth cleavage can be prevented without effect on morphogenesis even in those species where it is customary. Ophiuroid embryos cleave equally at the fourth division yet form primary mesenchyme and spicules similar to those of echinoids. Moreover, unequal fourth cleavage is not universally conserved in echinoid development. A variable number of micromeres is

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FIG. 9. Development of S. droebachiensis embryos. (a) Fixed 16.cell stage control culture containing intermediate and equal cleavage types. (b) Lateral view of intermediate 16-cell embryo showing three-tiered cell orientation. (c) Vegetal view of intermediate 16-cell embryo showing meridional orientation of the equal-sized vegetal cells (arrow). (d) Late gastrula, 48 hr. (e) Prism larva, 60 hr. (f) Pluteus larvae from equally cleaving embryos, 72 hr. (b) and (c) are shown at the same magnification. Bars = 50 Wm.

formed at fourth cleavage in some species of the Perischoechinoidea subcl.ass of Echinoidea (Schroeder, 1981). Thus, the differentiation of primary mesenchyme in echinoids, like that in ophiuroids, is not dependent on the formation of micromeres at the 16-cell stage. embryos With respect to ingression, D. excentricus equalized at both fourth and fifth cleavages by SDS treatment exhibited a sltrong correlation between the number of small cells found in the 32cell stage embryo and the appearance of primary mesenchyme prior to gastrulation. It is clear that in equalized 32-cell embryos, 40% of the cases were capable of normal ingression. On the other hand, 48% of the cases failed to ingress at the proper time. From this we conclude that 32-cell micromeres are not required for primary mesenchyme ingression, but if present, ingression is more likely to occur. Whether size of the 32-cell stage micromeres affects primary mesenchyme ingression independently of SDS-treatment could be tested by preventing micromere formation by other means, such as flattening, stretching, shaking, or cytochalasin B (Boveri, 1901; Hiirstadius, 1928; Tanaka, 1979).

In the 48% of equalized embryos where ingression did not occur, it might be merely delayed in timing by some aspect of the treatment. The increasing nucleocytoplasmic ratios of developing embryos are reported to serve as a means of timing developmental events. Smith and McLaren (1977) suggested a role for the nucleocytoplasmic ratio in the timing of blastocoel formation of mouse embryos. Newport and Kirschner (1982a,b) demonstrated that a DNA-cytoplasm ratio establishes the timing of the midblastula transition of amphibian embryos and Mita (1983) found a similar result using starfish egg fragments. One possible interpretation of the absence of primary mesenchyme ingression in some equalized D. excentricus embryos is that attainment of a certain high-threshold nucleocytoplasmic ratio in the micromere descendents determines the onset of primary mesenchyme ingression into the blastocoel. There is evidence which speaks against this hypothesis: (1) The inner, small micromeres of the 32-cell embryo should achieve such a threshold at an earlier time than the outer, large micromeres but they do not contribute to the primary mesenchyme

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population (Endo, 1966). (2) Decreased vegetal cell size as a result of “precocious” micromere formation did not alter the timing of in vitro differentiation of Hemicentrotus micromeres (Kitajima and Okazaki, 1980). (3) Removal of as much as 50% of the endoplasm of an unfertilized sea urchin egg by micropipet produced no abnormalities in development except small size (Horstadius et al., 1950). According to an alternate interpretation, equalization of later cleavages could completely prevent ingression of the cells that normally would produce primary mesenchyme rather than merely delay the onset of their ingression. There is another source of mesenchyme which appears in the blastocoel, the secondary mesenchyme. Secondary mesenchyme cells, derived from Veg,, are known to assume the spicule-producing function in the absence of micromeres (Horstadius, 1928; Langelan and Whiteley, unpublished). In fact, the pattern of spiculogenesis in defective D. excentricus embryos resembles that reported in embryos which have had their micromeres mechanically removed (Horstadius, 1928) and in normal Eucidaris tribuloides (Schroeder, 1981). The two possibilities of spiculogenesis (1) by delayed primary mesenchyme or (2) by regulation of secondary mesenchyme are being examined through use of a monoclonal antibody specific for primary mesenchyme. Dan (19’78, 1979, 1984) suggested that the lack of spiculogenesis observed in SDS-treated Hemicentrotus embryos might be the result of the failure of micromere nuclei to segregate into vegetal cytoplasm at the fourth cleavage. An extension of this idea is that these nuclei might have to come into contact with vegetal cortex to achieve their determined state. The idea of vegetal segregation as a means for programming micromere nuclei is an attractive one. We have found that such segregation at the 16-cell stage is not essential for D. excentricus development. The redistribution of vegetal cytoplasm into 8 cells at the 16-cell stage did not alter the normal pattern of differentiation. If the vegetal region contains diffusible primary mesenchyme determinants active at the 16-cell stage, one might predict that the eight nuclei in contact with vegetal material in a two-tiered embryo would become committed to form primary mesenchyme. However, the correct number of primary mesenchyme cells was formed in embryos developing from two-tiered embryos. This result indicates that the inclusion of nuclei with vegetal cytoplasm at the 16-cell stage is not sufficient to program them to the primary mesenchyme lineage. Thus the commitment of micromeres to form primary mesenchyme requires neither the relatively high concentration of vegetal pole material nor the exclusion of macromere cytoplasm at the 16-cell stage, indicating

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that determination in D. excentricus micromeres is not dependent on strict cytoplasmic segregation at the 16cell stage. In summary, unequal fourth cleavage in echinoid development is not essential for the determination and differentiation of the primary mesenchyme. In addition, ingression of the primary mesenchyme is not essential for differentiation of skeletal spicules. D. excentricus embryos with equalized fourth and fifth cleavages can develop normally, indicating that unequal fifth cleavage is also not determinative. However, 48% of embryos equalized at both fourth and fifth cleavages failed to form primary mesenchyme prior to gastrulation, a result open to at least two interpretations-a delay in primary mesenchyme ingression or regulation of secondary mesenchyme. Finally, reorientation of the vegeta1 spindles, and the redistribution of vegetal cytoplasm into 8 cells at the 16-cell stage resulting from that orientation, did not alter the normal pattern of differentiation. We suggest that the vegetal pole does not contain active determinants for primary mesenthyme differentiation that must act at the 16-cell stage. Perhaps any such morphogenetic determinants in the vegetal region must reach a threshold level in the micromere lineage for effective activity. In an equalized 32-cell embryo, this could occur only at a later stage. R.E.L. was supported by NICHD Developmental Biology Training Grant HD-07183. We thank Dr. A. 0. D. Willows for the use of the facilities of the Friday Harbor Laboratories and Dr. Thomas E. Schroeder for insightful discussions and technical advice in preparation of the manuscript. REFERENCES BOVERI, T. H. (1901). Uber die Polaritat des Seeigel-Eies. Verb. Phys. Med. Ges. WzXrzb. 34, 145-176. DAN, K. (1978). Unequal division: Its cause and significance. In “Cell Reproduction: In Honor of Daniel Mazia” (E. R. Dirksen, et al, eds.), ICN-UCLA Symp. Mol. Cell Biol. Proc., 1978 Series. Vol. 12, pp. 557-561. Academic Press, New York. DAN, K. (1979). Studies on unequal cleavage in sea urchins I. Migration of the nuclei to the vegetal pole. Dev. Growth Difer. 21, 527-535. DAN, K. (1984). The cause and consequence of unequal cleavage in sea urchins. Zoo!. Sci. 1, 151-160. DRIESCH, H. (1892). Entwicklungsmechanische Studien III-VI. Z Wiss. Zool. 55, l-62. ENDO, Y. (1966). Fertilization, cleavage and early development. 1n “Biology of Today,” Vol. 4, “Development and Differentiation” pp. 1-61. Iwanami Shoten, Tokyo (in Japanese). ERNST, S. G., HOUGH-EVANS, B. R., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Limited complexity of RNA in micromeres of sixteencell sea urchin embryos. Dev. Biol. 79, 119-127. GUSTAFSON, T., and S~VHAGEN, R. (1949). Studies on the determination of the oral side of the sea urchin egg. I. The effect of some detergents on the development. Ark. Zool. 42, l-6. HARKEY, M. A. (1983). Determination and differentiation of micromeres in the sea urchin embryo. In: “Time, Space and Pattern in

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transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675686. NEWPORT, J., and KIRSCHNER, M. (1982b). A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687-696. OKAZAKI, K. (1971j. Spicule formation in sea urchin larvae; observations in vivo and in vitro. Symp. Cell Biol. 22, 163-171. OKAZAKI, K. (1975). Spicule formation by isolated micromeres of the sea urchin embryo. Amer. Zool. 15, 567-581. RODGERS, W. H., and GROSS, P. R. (1978). Inhomogeneous distribution of egg RNA sequences in the early embryo. Cell 14, 279-288. RIJNNSTRBM, J. (1966). The animalizing action of pretreatment of sea urchin eggs with thiocyanate in calcium-free sea water and its stabilization after fertilization. Ark. Zool. 19, 251-263. SCHROEDER, T. E. (1981). Development of a “primitive” sea urchin (Eucidaris tribuloides): Irregularities in the hyaline layer, micromeres and primary mesenchyme. Biol. Bull 161, 141-151. SMITH, R., and MCLAREN, A. (1977). Factors affecting the time of formation of the mouse blastocoele. J Embryol. Ezp. Morphol. 41, 79-92. TANAKA, Y. (1976). Effects of surfactants on the cleavage and further development of the sea urchin embryo. I. The inhibition of micromere formation at the fourth cleavage. Den Growth Difler. 18, 113-122. TANAKA, Y. (1979). Effects of surfactants on the cleavage and further development of the sea urchin embryo. II. Disturbance in the arrangement of cortical vesicles and change in cortical appearance. Dev. Growth Defer. 21, 331-342. TENNENT, D. H., TAYLOR, C. V., and WHITAKER, D. M. (1929). An investigation on organization in a sea-urchin egg. Carnegie Inst. Wu.shi?lgton Puhl. 391, l-104.