A novel G protein-coupled receptor, related to GPR4, is required for assembly of the cortical actin skeleton in early Xenopus embryos

As the fertilized Xenopus egg undergoes sequential cell divisions to form a blastula, each cell develops a network of cortical actin that provides shape and skeletal support for the whole embryo. Disruption of this network causes loss of shape and rigidity of the embryo, and disrupts gastrulation movements. We previously showed that lysophosphatidic acid (LPA) signaling controls the change in cortical actin density that occurs at different stages of the cell cycle. Here, we use a gain-of-function screen, using an egg cDNA expression library, to identify an orphan G protein-coupled cell-surface receptor (XFlop) that controls the overall amount of cortical F-actin. Overexpression of XFlop increases the amount of cortical actin, as well as embryo rigidity and wound healing, whereas depletion of maternal XFlop mRNA does the reverse. Both overexpression and depletion of XFlop perturb gastrulation movements. Reciprocal rescue experiments, and comparison of the effects of their depletion in early embryos, show that the XLPA and XFlop signaling pathways play independent roles in cortical actin assembly, and thus that multiple signaling pathways control the actin skeleton in the blastula.     

G-protein-coupled signals control cortical actin assembly by controlling cadherin expression in the early Xenopus embryo

During embryonic development, each cell of a multicellular organ rudiment polymerizes its cytoskeletal elements in an amount and pattern that gives the whole cellular population its characteristic shape and mechanical properties. How does each cell know how to do this? We have used the Xenopus blastula as a model system to study this problem. Previous work has shown that the cortical actin network is required to maintain shape and rigidity of the whole embryo, and its assembly is coordinated throughout the embryo by signaling through G-protein-coupled receptors. In this paper, we show that the cortical actin network colocalizes with foci of cadherin expressed on the cell surface. We then show that cell-surface cadherin expression is both necessary and sufficient for cortical actin assembly and requires the associated catenin p120 for this function. Finally, we show that the previously identified G-protein-coupled receptors control cortical actin assembly by controlling the amount of cadherin expressed on the cell surface. This identifies a novel mechanism for control of cortical actin assembly during development that might be shared by many multicellular arrays.     

Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing

Early Xenopus embryos are large, and during the egg to gastrula stages, when there is little extracellular matrix, the cytoskeletons of the individual blastomeres are thought to maintain their spherical architecture and provide scaffolding for the cellular movements of gastrulation. We showed previously that depletion of plakoglobin protein during the egg to gastrula stages caused collapse of embryonic architecture. Here, we show that this is due to loss of the cortical actin skeleton after depletion of plakoglobin, whereas the microtubule and cytokeratin skeletons are still present. As a functional assay for the actin skeleton, we show that wound healing, an actin-based behavior in embryos, is also abrogated by plakoglobin depletion. Both wound healing and the amount of cortical actin are enhanced by overexpression of plakoglobin. To begin to identify links between plakoglobin and the cortical actin polymerization machinery, we show here that the Rho family GTPase cdc42, is required for wound healing in the Xenopus blastula. Myc-tagged cdc42 colocalizes with actin in purse-strings surrounding wounds. Overexpression of cdc42 dramatically enhances wound healing, whereas depletion of maternal cdc42 mRNA blocks it. In combinatorial experiments we show that cdc42 cannot rescue the effects of plakoglobin depletion, showing that plakoglobin is required for cdc42-mediated cortical actin assembly during wound healing. However, plakoglobin does rescue the effect of cdc42 depletion, suggesting that cdc42 somehow mediates the distribution or function of plakoglobin. Depletion of alpha-catenin does not remove the cortical actin skeleton, showing that plakoglobin does not mediate its effect by its known linkage through alpha-catenin to the actin skeleton. We conclude that in Xenopus, the actin skeleton is a major determinant of cell shape and overall architecture in the early embryo, and that plakoglobin plays an essential role in the assembly, maintenance, or organization of this cortical actin.     

Localization of nonerythroid spectrin and actin in mouse oocytes and preimplantation embryos

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembranous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembranous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double-staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The submembraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ectopic expression of N-cadherin perturbs histogenesis in Xenopus embryos

Xenopus embryos express N-cadherin in a pattern similar to that observed in other species, and cells expressing Xenopus N-cadherin can bind to cells expressing chicken N-cadherin in vitro. To investigate the developmental role of this molecule, we injected mRNA encoding chicken N-cadherin into one blastomere of 2-cell-stage Xenopus embryos and examined the effect of its expression on their development. The ectopic expression of N-cadherin occurred in various regions of the injected embryos and induced abnormal histogenesis, such as thickening, clumping or fusion of cell layers. These results suggest that the precise quantitative and qualitative regulation of the expression of cadherins is essential to embryonic morphogenesis.     

RhoA is required for cortical retraction and rigidity during mitotic cell rounding

Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.     

Role of c-myc protein in the early embryonic development of Xenopus

Microinjections of antibodies directed against the protein encoded by the c-myc protooncogene strongly inhibit or arrest the early cell cleavage stage of Xenopus laevis embryos. Injections in one blastomere of a two cell stage embryo inhibit the segmentation of this blastomere. The cleavage of the uninjected blastomere behaves normally. Injections of control rabbit immunoglobulins do not alter the embryonic development.     

Genetics and cell biology of lysophosphatidic acid receptor-mediated signaling during cortical neurogenesis

Lysophosphatidic acid (LPA) is a small lysophospholipid that signals through G-protein coupled receptors (GPCRs) to mediate diverse cellular responses. Two LPA receptors, LPA(1) and LPA(2), show gene expression profiles in mouse embryonic cerebral cortex, suggesting roles for LPA signaling in cerebral cortical development. Here, we review loss-of-function and gain-of-function models that have been used to examine LPA signaling. Genetic deletion of lpa(1) or both lpa(1) and lpa(2) in mice results in 50-65% neonatal lethality, but not obvious cortical phenotypes in survivors, suggesting that compensatory signaling systems exist for regulating cortical development. A gain-of-function model, approached by increasing receptor activation through exogenous delivery of LPA, shows that LPA signaling regulates cerebral cortical growth and anatomy by affecting proliferation, differentiation and cell survival during embryonic development.     

Regulation of Xenopus embryonic cell adhesion by the small GTPase,rac

TGF-beta family signalling pathways are important for germ layer formation and gastrulation in vertebrate embryos and have been studied extensively using embryos of Xenopus laevis. Activin causes changes in cell movements and cell adhesion in Xenopus animal caps and dispersed animal cap cells. Rho family GTPases, including rac, mediate growth factor-induced changes in the actin cytoskeleton, and consequently, in cell adhesion and motility, in a number of different cell types. Ectopic expression of mutant rac isoforms in Xenopus embryos was combined with animal cap adhesion assays and a biochemical assay for rac activity to investigate the role of rac in activin-induced changes in cell adhesion. The results indicate that (1) the perturbation of rac signalling disrupts embryonic cell-cell adhesion, (2) that rac activity is required for activin-induced changes in cell adhesive behavior on fibronectin, and (3) that activin increases endogenous rac activity in animal cap explants.     

Polar asymmetry in the organization of the cortical cytokeratin system of Xenopus laevis oocytes and embryos

We have used whole-mount immunofluorescence microscopy of late-stage Xenopus laevis oocytes and early embryos to examine the organization of their cortical cytokeratin systems. In both mature oocytes and early embryos, there is a distinct animal-vegetal polarity in cytokeratin organization. In mature (stage-VI) oocytes, the cytokeratin filaments of the vegetal region form a unique, almost geodesic network; in the animal region, cytokeratin organization appears much more variable and irregular. In unfertilized, postgerminal vesicle breakdown eggs, the cortical cytokeratin system is disorganized throughout both animal and vegetal hemispheres. After fertilization, cytokeratin organization reappears first in a punctate pattern that is transformed into an array of oriented filaments. These cytokeratin filaments appear first in the vegetal hemisphere and are initially thin. Subsequently, they form bundles that grow thicker through the period of first to second cleavage, at which point large cytokeratin filament bundles form a loose, fishnet-like system that encompasses the vegetal portion of each blastomere. In the animal region, cytokeratin filaments do not appear to form large fibre networks, but rather appear to be organized into a system of fine filaments. The animal-vegetal polarity in cytokeratin organization persists until early blastula (stage 5); in later-stage embryos, both animal and vegetal blastomeres possess qualitatively similar cytokeratin filament systems. The entire process of cytokeratin reorganization in the egg is initiated by prick activation. These observations indicate that the cortical cytoskeleton of Xenopus oocytes and early embryos is both dynamic and asymmetric.     

Contact-independent polarization of the cell surface and cortex of free sea urchin blastomeres

In a normal, intact sea urchin embryo blastomeres are structurally polarized so that all microvilli and cortical "pigment granules" are situated at the apical surfaces facing the hyaline layer and are absent from basolateral surfaces facing adjacent blastomeres and the internal embryonic cavity. To test the roles of intercellular contacts and the hyaline layer in the process of establishing this blastomere polarity, these two factors were experimentally eliminated; sea urchin eggs of four species were denuded of the nascent hyaline layer soon after fertilization and then cultured in calcium-free artificial seawater to prevent subsequent intercellular adhesion and contact. Such free blastomeres divided normally and still developed polarized distributions of microvilli and pigment granules resembling those of the corresponding blastomeres in intact embryos. These results indicate that the process of polarization is intrinsic to individual blastomeres (self-polarization) and that neither intercellular contacts nor adhesion of microvilli to the hyaline layer is necessary. The precise temporal and spatial coincidence of the patterns of polarization and the division cycles further suggests that a mechanistic link is maintained among cell division, blastomere polarization, and probably also a heritable component of the animal-vegetal axis.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

Essential role of MARCKS in cortical actin dynamics during gastrulation movements

Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

Pattern formation during early amphibian development: Embryogenesis in inverted anuran and urodele eggs

Early embryogenesis was monitored in Xenopus, Rana (anurans), and Ambystoma (urodele) eggs which were inverted at various times between fertilization and first cleavage. The pattern of cleavage furrow formation, site of involution, and extent of organogenesis were observed. In several instances, pattern formation was dramatically altered. The small/large blastomere pattern was, for example, reversed in some inverted embryos. Developmental arrest at early organogenesis usually followed pattern reversal. By employing a series of tissue transplantations, it was possible to establish that the activity of the primary embryonic organizer of inverted embryos was diminished drastically. The developmental competence of the prospective ectoderm of inverted embryos was, however, reversed. Incomplete organogenesis in inverted embryos is therefore probably due to either abnormal mesoderm formation or defective tissue interactions.     

Cell-autonomous and signal-dependent expression of liver and intestine marker genes in pluripotent precursor cells from Xenopus embryos

Early regulatory events in respect to the embryonic development of the vertebrate liver are only poorly defined. A better understanding of the gene network that mediates the formation of hepatocytes from pluripotent embryonic precursor cells may help to establish in vitro protocols for hepatocyte differentiation. Here, we describe our first attempts to make use of early embryonic explants from the amphibian Xenopus laevis in order to address these questions. We have identified several novel embryonic liver and intestine marker genes in a random expression pattern screen with cDNA libraries derived from the embryonic liver anlage and from the adult liver of Xenopus laevis. Based on their embryonic expression characteristics, these genes, together with the previously known ones, can be categorized into four different groups: the liver specific group (LS), the liver and intestine group A (LIA), the liver and intestine group B (LIB), and the intestine specific group (IS). Dissociation of endodermal explants isolated from early neurula stage embryos reveals that all genes in the LIB and IS groups are expressed in a cell-autonomous manner. In contrast, expression of genes in the LS and LIA groups requires cell-cell interactions. The regular temporal expression profile of genes in all four groups is mimicked in ectodermal explants from early embryos, reprogrammed by co-injection of VegT and beta-catenin mRNAs. FGF signaling is found to be required for the induction of liver specific marker (LS group) gene expression in the same system.