Regional specification in the early embryo of the brittle star Ophiopholis aculeata

Early embryogenesis has been examined experimentally in several echinoderm and hemichordate classes. Although these studies suggest that the mechanisms which underlie regional specification have been highly conserved within the echinoderm + hemichordate clade, nothing is known about these mechanisms in several other echinoderm classes, including the Ophiuroidea. In this study, early embryogenesis was examined in a very little studied animal, the ophiuroid Ophiopholis aculeata. In O. aculeata, the first two cleavage planes do not coincide with the animal-vegetal axis but rather form approximately 45 degrees off this axis. A fate map of the early embryo was constructed using microinjected lineage tracers. Most significantly, this fate map indicates that there is a major segregation of ectodermal from endomesodermal fates at first cleavage. The distribution of developmental potential in the early embryo was also examined by isolating different regions of the early embryo and following these isolates though larval development. These analyses indicate that endomesodermal developmental potential segregates unequally at first, second, and third cleavage in O. aculeata. These results provide insight into the mechanisms of regional specification in O. aculeata and yield new material for the study of the evolution of echinoderm development.     

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.     

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.     

Morphogenetic tissue movement and the establishment of body plan during development from blastocyst to gastrula in the mouse

In many animal species, the early development of the embryo follows a stereotypic pattern of cell cleavage, lineage allocation and generation of tissue asymmetry leading to delineation of the body plan with three primary embryonic axes. The mammalian embryo has been regarded as an exception and primary body axes of the mouse embryo were thought to develop after implantation. However, recent findings have challenged this view. Asymmetry in the fertilised oocyte, as defined by the position of the second polar body and the sperm entry point, can be correlated with the orientation of the animal-vegetal and the embryonic-abembryonic axes in the preimplantation blastocyst. Studies of the pattern of morphogenetic movement of cells and genetic activity in the peri-implantation embryo suggest that the animal-vegetal axis of the blastocyst might presage the orientation of the anterior-posterior axis of the gastrula. This suggests that the asymmetry of the zygote that is established at fertilisation and early cleavage has a lasting impact on the delineation of body axes during embryogenesis.     

Regulation and the modification of axial properties in partial embryos of the nemertean, Cerebratulus lacteus

 Embryos acquire axial properties (e.g., the animal-vegetal, dorsoventral and bilateral axes) at various times over the course of their normal developmental programs. In the spiral-cleaving nemertean, Cerebratulus lacteus, lineage tracing studies have shown that the dorsoventral axis is set up prior to the first cleavage division; however, blastomeres isolated at the two-cell stage will regulate to form apparently perfect, miniature pilidium larvae. We have examined the nature of axial specification in this organism by determining whether partial embryos retain the original embryonic/larval axial properties of the intact embryo, or whether new axial relationships are generated as a consequence of the regulatory process. Single blastomeres in two-cell stage embryos were injected with lineage tracer, and were then bisected along the second cleavage plane at the four-cell stage. Thus, the relationship between the plane of the first cleavage division and various developmental axes could be followed throughout development in the ”half-embryos”. While some embryo fragments appear to retain their original animal-vegetal and dorsoventral axes, many fragments generate novel axial properties. These results indicate that axial properties set up and used during normal development in C. lacteus can be completely reorganized during the course of regulation. While certain embryonic axes, such as the animal-vegetal and dorsoventral axes, appear to be set up prior to first cleavage, these axes and associated cell fates are not irreversibly fixed until later stages of development in normal intact embryos. In C. lacteus, the process whereby these properties are ultimately determined is apparently controlled by complex sets of cell-cell interactions. Received: 11 October 1996 / Accepted: 21 February 1997     

Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal–vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.     

The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata

Summary In hydrozoans the sperm will fuse with the egg only at the site of polar body formation. The primary oocyte and maturing oocytes which have produced the first polar body cannot be fertilized even though maturing oocytes which have produced the first polar body attract sperm. These eggs do not acquire the ability to be fertilized until after second polar body formation. If either first or second polar body formation is inhibited or if first and second polar body formation do not take place in close proximity to each other, the fertilization site is not set up. Under normal circumstances the site of polar body formation takes place at the region on the maturing oocyte surface nearest the site where the germinal vesicle resided in the primary oocyte. When maturing oocytes are centrifuged prior to polar body formation, the site of polar body formation is frequently shifted so that it does not correspond to the site where it would be given off under normal circumstances. Under these conditions the shifted site of polar body formation is the only site where the egg can be fertilized, indicating that the fertilization site is selected during oocyte maturation.Oocyte maturation in these hydrozoans is mediated by a hormone released by the somatic cells of gonophores as a consequence of bringing dark adapted gonophores into the light. The hormone acts directly on the oocyte to induce maturation. The oocyte only has to be exposed to the hormone for the first few minutes of the maturation process in order to complete the process of maturation.Dedicated to Professor N.H. Verdonk of the Rijksuniversiteit Utrecht on his 65th birthday     

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster. Received: 29 September 1999 / Accepted: 24 November 1999     

Regional specification during embryogenesis in the inarticulate brachiopod Discinisca.

The process of embryogenesis is described for the inarticulate brachiopod Discinisca strigata of the family Discinidae. A fate map has been constructed for the early embryo. The animal half of the egg forms the dorsal ectoderm of the apical and mantle lobes. The vegetal half forms mesoderm and endoderm and is the site of gastrulation; it also forms the ectoderm of the ventral regions of the apical and mantle lobes of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal, vegetal, or lateral regions at different times from the 2-cell stage through gastrulation. Animal halves isolated at the 8-cell and blastula stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from blastulae they formed the cell types typical of apical and mantle lobes. Vegetal halves isolated at all stages gastrulated and formed a more or less normal larva; the only defect these larvae had was the lack of an apical tuft, which normally forms from cells at the animal pole of the embryo. When lateral isolates were created at all developmental stages, these halves gastrulated. Cuts which separated presumptive anterior and posterior regions generated isolates at the 4-cell and blastula stages that formed essentially normal larvae; however, at the midgastrula stage these halves formed primarily anterior or posterior structures indicating that regional specification had taken place along the anterior-posterior axis. The plane of the first cleavage, which predicts the plane of bilateral symmetry, can be shifted by either changing the cleavage pattern that generates the bilateral 16-cell blastomere configuration or by isolating embryo halves prior to, or during, the 16-cell stage. These results indicate that while the plane of the first cleavage predicts the axis of bilateral symmetry, the axis is not established until the fourth cleavage. The development of Discinisca is compared to development in the inarticulate brachiopod Glottidia of the family Lingulidae and to Phoronis in the phylum Phoronida.     

Mechanism of First Cleavage Specification in the Mouse Egg: Is Our Body Plan Set at Day 0?

In most animals the body axis is specified in the egg. Because of their highlyregulative capacity after experimental manipulations,1-4 mammalianpreimplantation embryos have long been thought to be an exception to this rule,lacking polarity until the blastocyst stage. However, it has recently been suggested5-7 that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst arisesperpendicular to the first cleavage plane. Considering the second polar body (2pb)as a stationary marker for the “animal pole (A-pole)” during preimplantationdevelopment,5,6 the authors concluded that the polarity of the mouse embryo isalready specified in the egg, as is the case for most non-mammalian animals.5-7However, the results of our recent time-lapse recordings have shown8 that in 50 %of the embryos the first cleavage occurs at a considerable distance from the“animal-vegetal (A-V) axis” and that the 2pb moves towards the first cleavageplane, in contrast to the previous claims. Thus, there is no predetermined axis in themouse egg. We also presented a novel model for specification of the first cleavageplane: this is defined as the plane separating the two apposing pronuclei that havemoved to the center of the egg. In this review we will elucidate the discrepancybetween the previous model and our model, and discuss the possible causes.     

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.     

The role of polarity in the development of the hydrozoan planula larva

Summary These experiments were done in order to define the role that polarity plays during embryogenesis in hydrozoans.Parts of hydrozoan embryos isolated at different developmental stages from early cleavage to postgastrula will regulate to form normal planulae. During this process, the original anterior-posterior axis of the part is conserved. In normal embryos the posterior pole of the anterior-posterior axis is congruent with the site where the polar bodies are given off and with the site where the first cleavage is initiated. By centrifuging fertilized eggs, it is possible to create embryos in which the first cleavage initiation site does not correspond to the site where the polar bodies are given off. In these embryos the posterior pole of the anterior-posterior axis corresponds to the first cleavage initiation site. When parts of these embryos are isolated at different stages they also regulate to form normal planulae. The axial properties of these planulae are determined by the site of first cleavage initiation.The interactions between regions of the embryo with different axial properties were studied by grafting together parts in such a way as to create embryos with abnormal axial arrangements. Following gastrulation interactions take place between the grafted parts leading to the formation of normal planulae with a new set of axial properties.Blastula stage embryos can be dissociated into single cells and the cells can be reaggregated. These reaggregates form normal planulae. Polarity can be entrained in the reaggregates by grafting a small piece of tissue from any part of an intact blastula to the reaggregate. These cells organize the formation of an axis of symmetry with an appropriate orientation with respect to the graft.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

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.     

WAVE2 regulates meiotic spindle stability,peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.     

Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.