Determination of dorso-ventral axis in early embryos of the sea urchin, Hemicentrotus pulcherrimus

To elucidate a relationship between early cleavage planes and dorso-ventral (DV)-axis of sea urchin embryos, a fluorescent dye, Lucifer Yellow CH, was iontophoretically introduced into one blastomere at the 2-cell stage, and the location of the progeny cells was determined in the half-labeled prism larvae by examining the embryos from the animal pole. The boundary plane which divides the embryonic tissue into the labeled and nonlabeled parts was (1) coincident with, (2) perpendicular to, or (3) obliquely crossing the larval plane of bilateral symmetry. The oblique boundaries took only two angles mutually symmetrical with regard to the DV-axis of embryos. Combining these labeling patterns, the tissue of prism larvae could be divided into 8 sectors around the animal-vegetal axis. When the 2-cell stage embryos with different diameters of sister blastomeres were labeled with the dye, one end of the boundary plane was again found at one of the 8 boundary points noticed in equally cleaved embryos, while the other was observed to fall in the middle of a sector. These results indicate that the DV-axis of the embryo is established according to the spatial arrangement of blastomeres during the 5-6th cleavage stages when blastomeres align in 8 rows in meridional direction. It was also suggested that intercellular communication takes part in the determination of the fate of individual founder blastomeres during the two subsequent cleavages, i.e., 7-8th cleavage stages.     

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.     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

Role of specialized microvilli and the fertilization envelope in the spatial positioning of blastomeres in early development of embryos of the starfish Astropecten scoparius

In the eggs of a wide range of animal species, various factors that determine the blastomeres' presumptive fate are known to locate unevenly within the egg. In the embryos of these animals, cleavage occurs not just to increase cell numbers, but also to distribute the factors to the respective blastomeres, resulting in cell specialization at the later stages. In the early cleavage stages, before the establishment of a device such as desmosomes to directly join the blastomeres, some other means is needed to keep the blastomeres together and maintain the relative positions among them. In this study, we found that the embryos of the starfish Astropecten scoparius lack the hyaline layer seen in sea urchin embryos and that blastomeres adhere to the fertilization envelope (FE) via filamentous cellular projections (fixing processes). Electron microscopy revealed the fixing processes to be specialized microvilli formed, after the elevation of the FE, by the elongation of short microvilli that pre-exist in unfertilized eggs. After the first cleavage, the two blastomeres separate from each other and finally attach to the FE. In the subsequent cleavages, the blastomeres undergo repeated cell division without separating from the FE. Between the blastomeres and the FE, only shortened fixing processes were observed. Destruction of the fixing processes caused release of the blastomeres from the FE and disturbance of the relative positions of the blastomeres, resulting in abnormal development of the embryos. These observations suggest that the fixing process is a device to keep the egg placed centrally in the FE up to the first cleavage, and after the first cleavage and beyond to anchor the blastomeres to the FE so that the FE can be used as a scaffold for morphogenesis. Electron microscopy also suggests that the inner layer of the FE, which is derived from the contents of cortical granules, reinforces the adhesion of the fixing processes to the FE. Immuno-electron microscopy, using an antibody against sea urchin hyaline layer, showed that the inner layer of the FE of starfish eggs and the hyaline layer of sea urchin eggs, which are both derived from cortical granules, contain some common elements.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos.

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.     

The role of cleavage in the localization of developmental potential in the Ctenophore Mnemiopsis leidyi

This study defines the time period during which the cellular components that specify comb plates and photocytes become localized in different parts of blastomeres prior to their segregation to separate daughter cells. At the two-cell stage the factors which specify comb plates are localized at the aboral pole of the blastomeres. There is not a significant localization of the factors which specify comb plates and photocytes along the tentacular axis of the embryo. At the four-cell stage, the factors which specify comb plates become localized at one end of the tentacular axis of the blastomeres; however, the factors which specify photocytes have not yet become localized. At the eight-cell stage, the factors which specify these two cell types are segregated to different blastomeres.The role of cleavage in setting up these localized regions of developmental potential has been studied by reversibly inhibiting selected cleavages. After the first division, the pattern of cleavage that follows a period of cleavage inhibition corresponds to the pattern occurring in untreated embryos that began development at the same time. This situation is similar to the “clock” system, which controls many aspects of the pattern of cleavage in sea urchin embryos. The extent to which the factors that specify comb plates and photocytes become localized in a given region of a blastomere is correlated with the kind of cleavage which occurs after a block. Most of the activity involved in localizing developmental potential takes place during cleavage.     

Asynchronization of Cell Division is Concurrently Related with Ciliogenesis in Sea Urchin Blastulae

During the early development of the sea urchins, Temnopleurus toreumaticus, Temnopleurus hardwickii and Hemicentrotus pulcherrimus , the division synchrony in all blastomeres lasted only until the 4th cleavage and a regional synchrony or a graded activity of cell division appeared. In the midblastula stage prior to hatching, the regional synchrony vanished simultaneously with the formation of cilia, then the division proceeded asynchronously. The analysis of cell pedigrees confirmed that a variable extension of intercleavage times occurred after the ciliogenesis. In blastomeres derived from mesomeres of T. toreumaticus embryos, the mean intercleavage time extended from 48 min of the 8th cycle (pre-ciliated) to 115 min of the 9th cycle (ciliated), and the coefficient of variation increased from 15% to 39%. We attempted a kinetic analysis of cell proliferation on the basis of the transition probability model of cell cycle control. We concluded that the minimum time required for the completion of the cell cycle was the decisive factor in the cell cycle succession of pre-ciliated blastomeres, and that a sudden and sharp decrease in the transition probability of the ciliated blastomeres probably interpreted the abrupt slowing and asynchronization of the cleavage cycle at the time of ciliogenesis.     

Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

Injection of myo-inositol reverses the effects of lithium on sea urchin blastomeres

Lithium is known to cause sea urchin blastomeres destined to give rise to epithelium rather than to differentiate into gut or skeleton. While it has been proposed that lithium alters development by interfering with the inositol-tris phosphate-protein kinase C (IP₃-PKC) signaling pathway, the mechanism of action of lithium in sea urchins has remained elusive. Here we describe experiments that examine the hypothesis that lithium exerts its effect on sea urchin embryos via the IP₃-PKC pathway. We make use of methods developed to isolate epithelial precursor cells from the animal hemisphere of cleavage 16-cell stage embryos. Pairs of cells were isolated and one of each pair was injected with either myo-inositol or its inactive isomer, epi-inositol. Rhodamine dextran was co-injected as a lineage tracer to follow the fate of injected cells. We demonstrate that injected myo-inositol, but not epi-inositol, can reverse the effects of lithium on sea urchin blastomeres. This is direct evidence that lithium affects the IP₃-PKC pathway in sea urchins, and that this pathway plays an important role in cell fate determination.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

A “micromere model” of cell-cell interactions in sea urchin early embryos

It has been shown that isolation of sea urchin blastomeres before the post-division adhesion leads mainly to the formation of equal blastomeres at the stage of 4th cleavage division, whereas isolation after adhesion results in the formation of micromeres simultaneous with that in intact embryos. Similar results were obtained in five sea urchin species. It has been concluded that there exists a critical point in the cleavage process, when blastomeres exchange information that determines the further cleavage pattern. It has been shown with this “micromere model” that serotonin and its analogs influence the cleavage pattern of half-embryos. These data have served as a basis for the hypothesis of “protosynapse,” a bilaterally symmetric structure in which the blastomeres are not only source and target of the signal but also a passive obstacle to leakage of the signal substance from the interblastomere cleft to the milieu. Such a structure may also specify the primary asymmetry of the blastomeres. The micromere model may be useful in specific pharmacological screening.     

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus)

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.     

Timers in Early Development of Sea Urchin Embryos

To elucidate the timing mechanisms in the early development of sea urchin embryos, we measured the times of initiation of the first four cleavages, of ciliary movement, of primary mesenchyme cell ingression, and of gastrulation at four temperatures ranging from 11 to 20°C. The times of cleavage and of initiation of ciliary movement showed similar temperature dependency, indicating that these events may be controlled by a common timer (the first timer). Although batches of eggs often showed variation in the period between fertilization and the first cleavage, their subsequent cleavages were more regular. This indicates that the first timer may not start at fertilization. The ingression of mesenchyme cells and the onset of gastrulation showed similar temperature dependency that was higher than that of other events, suggesting the existence of a second timer. Temperature shift experiments indicate that the second timer starts at the mid-blastula (the 8–9th cleavage) stage when divisions of blastomeres become asynchronous.     

Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos.

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.     

The potency of the first two cleavage cells in echinoderm development: the experiments of Driesch revisited

A hundred years have passed since Driesch performed the classical experiment of separating sea urchin blastomeres from a two-cell-stage embryo, finding that each developed into a complete though smaller larva. The earlier studies of Roux using frogs showed that inactivating one of the two blastomeres by a heated needle resulted, during the early stages of development, in the formation of a half embryo. In this type of experiment, in which the two blastomeres are not separated, the live blastomere continues its development while it is still attached to an inactivated neighbour. In the work reported here, Roux's experimental design was used on two-cell-stage embryos of sea urchins. In contrast to the findings of Roux using amphibians, it was found (as claimed by Driesch) that the living blastomere developed as in the case of separated blastomeres.     

Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.