The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. (1/1724)

Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut.  (+info)

Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation. (2/1724)

Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation.  (+info)

Rac1 is required for the formation of three germ layers during gastrulation. (3/1724)

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.  (+info)

alphaSU2, an epithelial integrin that binds laminin in the sea urchin embryo. (4/1724)

At gastrulation in the sea urchin embryo dramatic cell adhesion changes contribute to primary mesenchyme cell ingression movements and to cell rearrangements during archenteron invagination. At ingression, quantitative adhesion assays demonstrated previously that primary mesenchyme cells (PMCs) change their affinity for neighboring cells, for a fibronectin-like substrate, and for the hyaline layer. To investigate the molecular basis for these and other differential cell affinities at gastrulation, we have identified an integrin that appears to be responsible for specific alterations in cell-substrate adhesion to laminin. During early cleavage stages blastomeres adhere poorly to laminin substrates. Around hatching there is a large increase in the ability of blastomeres to bind to laminin and this increase correlates temporally with the expression of an integrin on the basal surface all blastomeres. PMCs, after undergoing their epithelial-mesenchymal transition, have a strongly reduced affinity for laminin relative to ectoderm cells and, correspondingly, do not stain for the presence of the integrin. We identified the alpha integrin cDNA from Lytechinus variegatus by RT-PCR. Overlapping clones were obtained from a midgastrula cDNA library to provide a complete sequence for the integrin. The composite cDNA encoded a protein that was most similar to the alpha5 subgroup of vertebrate integrins, but there was not a definitive vertebrate integrin homolog. Northern blots and Western immunoblots showed that the sea urchin integrin, which we have named alphaSU2, is present in eggs and during all stages of development. Immunolocalization with specific polyclonal antibodies showed that alphaSU2 first appears on the basal cell surface of epithelia at the midblastula stage, at a time correlating with the increase in adhesive affinity for laminin. The protein remains at high levels on the basal surface of ectoderm cells but is temporarily reduced or eliminated from endoderm cells during their convergent-extension movements. To confirm integrin binding specificity, alphaSU2 was transfected into an alpha-integrin-deficient CHO cell line. alphaSU2-expressing CHO cells bound well to isolated sea urchin basal lamina and to purified laminin. The transfected cells bound weakly or not at all to fibronectin, type I collagen, and type IV collagen. This is consistent with the hypothesis that alphaSU2 integrin functions by binding epithelial cells to laminin in the basal lamina. In vivo, modulation of alphaSU2 integrin expression correlates with critical adhesive changes during cleavage and gastrulation. Thus, this protein appears to be an important contributor to the morphogenetic rearrangements that characterize gastrulation in the sea urchin embryo.  (+info)

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification. (5/1724)

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.  (+info)

The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development. (6/1724)

Fertilized oocytes of the brine shrimp Artemia franciscana undergo either ovoviviparous or oviparous development, yielding free-swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Encystment is followed by diapause, wherein metabolism is greatly reduced; the resulting cysts are very resistant to extreme stress, including desiccation and long-term anoxia. The synthesis of p26, a small heat shock/alpha-crystallin protein produced only in oviparously developing Artemia, is shown in this paper to be transcriptionally regulated. A p26 mRNA of about 0.7 kb was detected on Northern blots in the second day after oocyte fertilization. It peaked as embryos encysted and declined rapidly when activated cysts resumed development. The appearance of p26 protein, as indicated by immunoprobing of Western blots, followed mRNA by 1 day; it also increased as encystment occurred but remained constant during postgastrula development of cysts. However, p26 underwent a marked reduction during emergence of nauplii and could not be detected in cell-free extracts of second-instar larvae. p26 entered nuclei of encysting embryos soon after synthesis and was localized therein as late as instar II, when it was restricted to a small set of salt gland nuclei. First-instar larvae derived from cysts were more thermotolerant than larvae that had developed ovoviviparously, but synthesis of p26 was not induced by heat under the experimental conditions employed. Additionally, transformed bacteria synthesizing p26 were more thermotolerant than bacteria that lacked the protein. The results support the proposal that p26, a developmentally regulated protein synthesized during embryo encystment, has chaperone activity in vivo and protects the proteins of encysted Artemia from stress-induced denaturation.  (+info)

Delta-mediated specification of midline cell fates in zebrafish embryos. (7/1724)

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.  (+info)

LvNotch signaling mediates secondary mesenchyme specification in the sea urchin embryo. (8/1724)

Cell-cell interactions are thought to regulate the differential specification of secondary mesenchyme cells (SMCs) and endoderm in the sea urchin embryo. The molecular bases of these interactions, however, are unknown. We have previously shown that the sea urchin homologue of the LIN-12/Notch receptor, LvNotch, displays dynamic patterns of expression within both the presumptive SMCs and endoderm during the blastula stage, the time at which these two cell types are thought to be differentially specified (Sherwood, D. R. and McClay, D. R. (1997) Development 124, 3363-3374). The LIN-12/Notch signaling pathway has been shown to mediate the segregation of numerous cell types in both invertebrate and vertebrate embryos. To directly examine whether LvNotch signaling has a role in the differential specification of SMCs and endoderm, we have overexpressed activated and dominant negative forms of LvNotch during early sea urchin development. We show that activation of LvNotch signaling increases SMC specification, while loss or reduction of LvNotch signaling eliminates or significantly decreases SMC specification. Furthermore, results from a mosaic analysis of LvNotch function as well as endogenous LvNotch expression strongly suggest that LvNotch signaling acts autonomously within the presumptive SMCs to mediate SMC specification. Finally, we demonstrate that the expansion of SMCs seen with activation of LvNotch signaling comes at the expense of presumptive endoderm cells, while loss of SMC specification results in the endoderm expanding into territory where SMCs usually arise. Taken together, these results offer compelling evidence that LvNotch signaling directly specifies the SMC fate, and that this signaling is critical for the differential specification of SMCs and endoderm in the sea urchin embryo.  (+info)