Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: Cytoplasmic retention of SpOtx possibly mediated through an α-actinin interaction

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-vegetal axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities. Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained In the cytoplasm at early developmental stages we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be α-actinin. A yeast two-hybrid analysis showed a specific interaction between the proline-rich region of SpOtx and a putative SH3 domain of the sea urchin α-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to α-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate. © 1996 Wiley-Liss, Inc.     

STUDIES ON UNEQUAL CLEAVAGE IN SEA URCHINS I. MIGRATION OF THE NUCLEI TO THE VEGETAL POLE

It has been known for nearly a century that at the 16-cell stage of sea urchin embryos, the animal 4 cells divide equally and horizontally, whereas the vegetal 4 cells cleave unequally and practically vertically into macromeres and micromeres. Recently, more careful observations were made on the process of micromere formation and it has been revealed that a primary cause for the inequality lies in the migration of the 4 vegetal nuclei to the vegetal pole of the embryo which brings about excentricity of the mitotic apparatus. Records of this phenomenon are given in the present paper.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS 1. THE INHIBITION OF MICROMERE FORMATION AT THE FOURTH CLEAVAGE*

Eggs of the sea urchins, Hemicentrotus pulcherrimus, Temnopleurus toreumaticus and Pseudocentrotus depressus were used as materials. Embryos were exposed to the surfactants such as SLS, CTAB, digitonin, Tween 80, sodium deoxycholate and Lubrol P. If embryos are kept in the solutions of SLS, CTAB and digitonin, 4 vegetal cells of the 8-cell stage divide equally at the fourth cleavage and consequently 16 equal-sized blastomeres are formed at the 16-cell stage. In this case, micromere formation is inhibited by the equal cleavage. The minimum effective concentration of the surfactants for the equal cleavage gradually increases as the time performing the treatment is postponed. The continuous exposure to the surfactant is unnecessary for the inhibition of micromere formation. In the egg temporarily exposed during the earlier stage, the equal cleavage occurs at the fourth division in natural seawater. Micromere formation is strongly affected by the surfactant (SLS) at the mid 4-cell stage.     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3-D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non-radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12-cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs-Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28-and 32-cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56- and 60-cell stages.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.     

"Micromere" formation and expression of endomesoderm regulatory genes during embryogenesis of the primitive echinoid Prionocidaris baculosa

Blastomere composition and expression profiles of wnt8 and hox11/13b orthologues were examined in the primitive indirect-developing echinoid Prionocidaris baculosa. We found that blastomere composition in the 16-cell-stage Prionocidaris embryos was different from that of the indirect-developing echinoids belonging to Euechinoidea, a derived group of the echinoids. The sizes of the blastomeres in the 16-cell-stage embryo varied, and no embryos formed a "micromere quartet," a group of four equal-sized micromeres. The smallest blastomere was usually located around the vegetal pole. We also found significant differences in early expression profiles of wnt8 orthologues of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of wnt8 orthologue of Prionocidaris was not detected at the 16-cell stage; it began at the 32-cell stage in the broad area containing the vegetal pole. However, in later stages, the expression profiles of hox11/13b and wnt8 orthologues of Prionocidaris were similar to that of euechinoid orthologues. The present study suggests that there are considerable differences between Prionocidaris and euechinoids in early developmental mechanisms in the vicinity of the vegetal pole.     

The origin of skeleton forming cells in the sea urchin embryo

Summary In embryos of the modern sea urchin species, subclass Euechinoidea, primary mesenchyme cells are derived from the progeny of micromeres formed at the sixteen cell stage of embryogenesis. The micromeres reside within the vegetal plate epithelium and later ingress into the blastocoel as primary mesenchyme cells which form the larval skeleton. Embryos of Eucidaris tribuloides, a member of the primitive subclass Perischoechinoidea, exhibit several noteworthy differences from euechinoid primary mesenchyme cell lineage including variable numbers and sizes of micromeres, the absence of mesenchyme ingression, and the lack of any detectable primary mesenchyme although a larval skeleton forms. In the present study, the cell lineage of the spiculogenic mesenchyme has been studied in Eucidaris tribuloides and in the euechinoid Lytechinus pictus by microinjecting the fluorescent tracer, Lucifer Yellow, into individual blastomeres of the embryo. In addition, wheat germ agglutinin, a lectin which binds only to primary mesenchyme cells of the early euechinoid embryo, was injected into the blastocoel of embryos of both species in order to examine the distribution of cells which possess primary mesenchyme-specific cell surface markers. The results of these experiments demonstrate that the spiculogenic mesenchyme of both Lytechinus and Eucidaris arise from descendants of micromeres formed at the sixteen cell stage, although the temporal and spatial distribution of these mesenchyme cells varies considerably between species. Furthermore, the evidence obtained suggests that the information necessary for spicule formation is already segregated to the vegetal pole by the eight cell stage. The results also suggest that there are no gap junctions present between the blastomeres of the early sea urchin embryo.     

Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation

Sea urchin micromeres were isolated from the 16-cell stage embryos and cultured until they differentiated into spicule-forming cells. Electrophoretic analysis of proteins labeled with [³⁵S]-methionine showed that the differentiation accompanied the synthesis of five cell-specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso- and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn²⁺ -containing or serum-free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn²⁺, or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

Range and stability of cell fate determination in isolated sea urchin blastomeres

We have examined the developmental potential of blastomeres isolated from either the animal (mesomeres) or vegetal (macromeres-micromeres) half of 16-cell embryos of the sea urchin Lytechinus pictus. We have also examined the effects of two known vegetalizing agents on the development of isolated mesomeres; LiCl treatment and combination with micromeres, the small blastomeres found at the vegetal pole of the 16-cell embryo. The markers for differentiation used were both morphological (invaginations, spicules and pigment cells) and molecular (gut-specific alkaline phosphatase activity, and monoclonal antibodies against antigens specific for gut and oral ectoderm). Embryoids derived from isolated mesomeres expressed markers characteristic of vegetal differentiation only at very low levels. They did express an antigen characteristic of animal development, the oral ectoderm antigen, but with an altered pattern. Isolated macromere-micromere pairs expressed all markers characteristic of vegetal development, but did not express the marker characteristic of animal development. Increasing concentrations of LiCl caused isolated mesomeres to give rise to embryoids with an increasing tendency to express vegetal markers of differentiation, and it was found that expression of different vegetal markers begin to appear at different concentrations of LiCl. LiCl also caused the marker for oral ectoderm to be expressed in a more normal pattern. Combining micromeres with mesomeres also induced mesomere derivatives to differentiate in a vegetal manner. Micromeres were not completely effective in inducing a more normal pattern of expression of the marker for oral ectoderm. The treatment of isolated mesomeres with both LiCl and micromeres produces a synergistic effect resulting in embryoids expressing markers not induced by either treatment alone.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

Primary mesenchyme cell patterning during the early stages following ingression

Sea urchin primary mesenchyme cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-cell stage, and later the 2 unlabeled micromeres are removed at the 16-cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-cell stage shows a 2-step pattern of localization. At late mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.     

Cell lineage-specific inhibition of cytokinesis by concanavalin A in a molluscan embryo (Nassarius reticulatus,Gastropoda)

Summary The effects of the lectin concanavalin A (Con A) on cleavage were studied in early embryos of the gastropodNassarius reticulatus. Progression of the first cleavage furrow is inhibited by incubating eggs before the first cleavage with 0.3–20 μg/ml Con A. Treatment with 1.0–20 μg/ml Con A during first cleavage causes regression of the cleavage furrow. Treatment with low concentrations (0.3–1.0 μg/ml) during the same period does not affect first cleavage. However, when further development of such eggs is followed, one finds that second cleavage is inhibited typically in only one of the two blastomeres of the 2-cell stage, i.e. the CD-blastomere. As a result, a 3-cell embryo is formed. At third cleavage of such embryos, the CD-blastomere forms either one double-sized micromere (1cd-micromere) or two normal-sized micromeres (1c and 1d) simultaneously. Sometimes micromere formation in the CD-blastomere is inhibited. Con A binding does not affect karyokinesis, nor does it affect the division asynchronies typical for normal development. On the basis of these and other results it is argued that binding of Con A to sites located at the vegetal pole of the egg is responsible for the cell lineage-specific inhibition of cleavage by Con A. This effect is most probably mediated by changes in the organization of the egg cortex.     

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.     

A micromere induction signal is activated by beta-catenin and acts through notch to initiate specification of secondary mesenchyme cells in the sea urchin embryo

At fourth cleavage of sea urchin embryos four micromeres at the vegetal pole separate from four macromeres just above them in an unequal cleavage. The micromeres have the capacity to induce a second axis if transplanted to the animal pole and the absence of micromeres at the vegetal pole results in the failure of macromere progeny to specify secondary mesenchyme cells (SMCs). This suggests that micromeres have the capacity to induce SMCs. We demonstrate that micromeres require nuclear beta-catenin to exhibit SMC induction activity. Transplantation studies show that much of the vegetal hemisphere is competent to receive the induction signal. The micromeres induce SMCs, most likely through direct contact with macromere progeny, or at most a cell diameter away. The induction is quantitative in that more SMCs are induced by four micromeres than by one. Temporal studies show that the induction signal is passed from the micromeres to macromere progeny between the eighth and tenth cleavage. If micromeres are removed from hosts at the fourth cleavage, SMC induction in hosts is rescued if they later receive transplanted micromeres between the eighth and tenth cleavage. After the tenth cleavage addition of induction-competent micromeres to micromereless embryos fails to specify SMCs. For macromere progeny to be competent to receive the micromere induction signal, beta-catenin must enter macromere nuclei. The macromere progeny receive the micromere induction signal through the Notch receptor. Signaling-competent micromeres fail to induce SMCs if macromeres express dominant-negative Notch. Expression of an activated Notch construct in macromeres rescues SMC specification in the absence of induction-competent micromeres. These data are consistent with a model whereby beta-catenin enters the nuclei of micromeres and, as a consequence, the micromeres produce an inductive ligand. Between the eighth and tenth cleavage micromeres induce SMCs through Notch. In order to be receptive to the micromere inductive signal the macromeres first must transport beta-catenin to their nuclei, and as one consequence the Notch pathway becomes competent to receive the micromere induction signal, and to transduce that signal. As Notch is maternally expressed in macromeres, additional components must be downstream of nuclear beta-catenin in macromeres for these cells to receive and transduce the micromere induction signal.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Effect on Inhibition of Micromere Segregation on the Mitotic Pattern in the Sea Urchin Embryo

Detergent treatment of sea urchin eggs at the mid 4-cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16-cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished.
These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.