Studies on Unequal Cleavage in Sea Urchins III. Micromere Formation under Compression

When sea urchin embryos at 2-cell stage are flattered between agar plates, the direction of cleavage is rotated by 90° in each division in reference to the preceding cleavage and no micromere is formed. But under this condition, micromeres are formed in 2 cases; 1) When the egg axis is parallel to the plane of flattening, 2 micromeres are formed on one side of a square 16-cell stage. 2) when the egg axis is perpendicular to the plane, 4 micromeres are formed at the center of the square.
When put into a groove, a string of 4 cells is formed showing that the spindle direction is further deflected by the groove. In the following 16-cell stage in the groove, which consists of 2 layers of 8 cells, cases with 2 micromeres on one side and 4 micromeres at the center are still found. If the 2-cell stage is introduced into a groove after the formation of mitotic apparatus, the spindle direction can no longer be changed and the 4-cell stage becomes like 4 pancakes stuck in 2 layers, indicating that 2 asters are holding the ends of a spindle in fixed positions.     

Unequal cleavage and the differentiation of echinoid primary mesenchyme

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 Dendraster 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 pictus and S. 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. excentricus. The results confirm that micromere determination in echinoids does not depend upon a strict cleavage pattern at the 16-cell stage.     

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.     

Cleavage and differentiation in the sea urchin embryo transplantation studies of micromeres

Summary Micromeres isolated from the 16-cell stage were implanted on mesomeres or macromeres from the same larva. The process of coalescence and the cleavage pattern of the transplanted micromeres were studied by means of light and electron microscopy.The transplanted micromere shows the same cleavage pattern as the micromerein situ. A close contact is established between the micromere and the host cell and cytoplasmic bridges are found between the cells.The micromere is dependent on its adjoining blastomere(s) and the rate of cleavage is slowed down when the micromere is isolated. Macromeres and mesomeres are not subjected to a similar change in rate of cleavage when isolated from the rest of the embryo.The ratio mitochondria/yolk in micromeres is different from that observed in macro- or mesomeres and the possible consequences of this fact are discussed.     

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2-cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10-15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.     

Subequatorial cytoplasm plays an important role in ectoderm patterning in the sea urchin embryo

To gain information on the process of ectoderm patterning, the animal halves of sea urchin embryos were isolated at various stages, and their morphology was examined when control embryos developed into pluteus larvae. The animal halves separated at the 8-cell stage developed into 'dauerblastula', without showing any conspicuous ectoderm differentiation. In contrast, some of the animal halves isolated at the 60-cell stage (after the sixth cleavage) formed a ciliated band and oral opening, suggesting that some patterning signal was transmitted from the vegetal to animal hemisphere during early cleavage. Further patterning of the animal hemisphere did not seem to occur until hatching, since both the animal halves isolated at the 60-cell stage and hatching stage showed the same degree of ectoderm patterning. After hatching, the later animal halves were isolated, the more patterned ectoderm they formed. The animal halves isolated just prior to gastrulation differentiated well-patterned ectoderm. It is of note, however, that the level of separation was a more crucial factor than the timing of separation; even the animal fragments of newly hatched embryos differentiated well-patterned ectoderm if they had been separated at a subequatorial level. This suggests that the signal for ectoderm patterning is transmitted over the equator after hatching, and once the cells in the supra-equatorial region receive the signal, they, in turn, can transmit the signal upwardly. Interestingly, if the third cleavage plane was shifted toward the vegetal pole, the isolated animal pole-side fragments developed into 'embryoids' with fully patterned ectoderm. These results indicate that not the micromere descendants but the subequatorial cytoplasm plays an important role in ectoderm patterning.     

The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.     

Induction of achromosomal cleavage by hydroxyurea in starfish embryo and the reversal by a combination of deoxyadenosine and deoxycytidine: possible involvement of salvage pathway for deoxynucleoside triphosphate biosynthesis in the presence of hydroxyurea

Effects of hydroxyurea, an inhibitor of ribonucleotide reductase, on cleavage of starfish embryos were studied. In the presence of 1 mM hydroxyurea, fertilized eggs of the starfish, Asterina pectinifera, cleaved up to the 256-cell stage and decomposed before blastulation. Before the 16-cell stage, each blastomere contained a normal nucleus or chromosomes with mitotic apparatus. The cleavage after the 16-cell stage was slow compared to the control embryos, and not all blastomeres contained a nucleus or normal chromosomes. During the fifth cell division (between 16-cell- and 32-cell-stage embryos), chromatin mass unassociated with the mitotic apparatus remained near the cleavage furrow. When hydroxyurea was removed before the 16-cell stage, the embryos developed to normal bipinnalia larvae via normal blastulae. However, the embryos were disintegrated before blastulation when hydroxyurea was removed after the 32-cell stage. DNA synthesis was normally observed before the 16-cell stage but not after the 16-cell stage, but dNTP contents in the embryos remained low throughout development in the presence of hydroxyurea. The achromosomal cleavage observed in the presence of hydroxyurea was reversed by the combination of extracellular dAR and dCR. Therefore, it is assumed that the synthesis of dNTPs required for DNA synthesis in the presence of hydroxyurea occurs via the salvage pathway using deoxynucleosides (dNR) (dNR to dNTP via dNMP and dNDP).     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

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.     

Cleavage and gastrulation of the euphausiacean<Emphasis Type="Italic"> Meganyctiphanes norvegica</Emphasis> (Crustacea,Malacostraca)

According to the Articulata hypothesis the cleavage of arthropods must be derived from spiral cleavage. However, arthropods show a great variety of cleavage modes with a widespread occurrence of superficial cleavage. In the Malacostraca, holoblastic cleavage occurs in some taxa such as Amphipoda, Euphausiacea and Dendrobranchiata. In particular, the cleavage of euphausiaceans has been proposed to be a modified spiral cleavage. The cell lineage of early stages up to blastoderm formation of the euphausiacean Meganyctiphanes norvegica is reconstructed using recent methods of fluorescent staining. Only the oblique angle of the mitotic spindles during the transition from the 2- to the 4-cell stage resembles the spiral cleavage mode. At the 8-cell stage, four cells each form a pattern of two interlocking bands which is preserved until the 122-cell stage. One blastomere is delayed in division and shows an oblique division from the fourth cleavage on. It is the precursor cell of two enlarged and cleavage-arrested cells at the 32-cell stage. At the 62-cell stage, these two cells are surrounded by eight cells following a specific cell division pattern during the subsequent division cycles. The cleavage pattern of M. norvegica occurs in two mirror images. A comparative approach reveals distinct similarities between the early cleavage patterns of Euphausiacea and Dendrobranchiata which are suggested to be homologous. Furthermore, the relationships to non-malacostracan cleavage patterns are discussed. It is shown that the early cleavage pattern of M. norvegica does not offer an example of a spiral cleavage within arthropods.     

Development of Microtubule-Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos

Animal-cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M-) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%. From the 12th to 15th cycles, control cycles become asynchronous, their period gradually increases 2 to 3 times, and the mitotic index declines to 10%. In cells treated after 12th cleavage with taxol, the mitotic index declines, similarly to control cells. However, in nocodazole-treated cells, it increases steadily, and exceeds 70% at 2 hr of treatment, but gradually declines to 40% at 6 hr. Therefore, while inhibition of microtubule activities does not significantly alter the timing of chromosome condensation cycles during synchronous cleavage, inhibition of microtubule assembly can prolong M-phase during asynchronous cleavage after the midblastula transition.     

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage.

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.     

Control of first cleavage in single-cell reconstituted mouse embryos

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.     

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.     

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.     

Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro

Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E199 + 10%GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1-8-cell embryos in Medium E199 + 10%GS led to cleavage arrest at the 8-16-cell stage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8-16-cell stage. In co-culture, however, a high percentage (77% and 96%) of 1-2-cell and 4-8-cell embryos, respectively, developed beyond the 16-cell stage. In co-culture, 1-2-cell embryos maintained cleavage rates equivalent to those in vivo until the 8-cell stage, but thereafter cell numbers lagged behind those in vivo, and by 168 h after ovulation, cell numbers (+/- s.e.) in vitro were 47.6 +/- 7.9 compared to 238 +/- 27.2 in vivo (t = 6.93, P less than 0.001). The results demonstrate that co-culture of embryos with oviduct cells allows a high percentage of embryos to develop through the period of cleavage arrest, providing a favourable environment for development through the 1-16-cell stages but a less adequate environment for development to the blastocyst stage.