Antagonistic interaction between IGF and Wnt/JNK signaling in convergent extension in Xenopus embryo.

The homeobox gene Otx2 is expressed during gastrulation in the anterior domain of the vertebrate embryo and is involved in neural and head induction during Xenopus early development. It also prevents convergent extension movements in trunk and posterior mesoderm. Insulin-like growth factors (IGFs) were shown to have similar function. However, whether they interact and the mechanism by which they affect convergent extension remain unclear. We show that IGF pathway specifically induces the expression of Otx2 in the early gastrula and blocks convergent extension of neuroectoderm and mesoderm through the transcriptional activation of Otx2 gene. Otx2 represses the expression of Xbra and Xwnt-11, and the effects of IGF on gastrulation movements can be partially rescued by antisense Otx2 morpholino oligonucleotide. These indicate that IGF pathway interacts with Otx2 to restrict Xbra and Xwnt-11 expression in the trunk and posterior regions. Consistent with this, we show that inhibition of IGF signaling or Otx2 function induces Xbra and Xwnt11 expression and convergent extension in ectodermal cells. Furthermore, the blockade of convergent extension by IGF-I and Otx2 can be rescued by coexpression of Xwnt-11 or a constitutively active Jun N-terminal kinase (JNK). Because Xbra and Xwnt-11 are required for convergent extension movements and Xwnt-11 activates the non-canonical Wnt-11/JNK pathway, our results reveal a mutually exclusive function between IGF and Wnt-11/JNK pathways in regulating cell behaviours during vertebrate head and trunk development.

Identification of distinct genes with restricted expression in the somitic mesoderm in Xenopus embryo.

We have used whole-mount in situ hybridisation to identify genes expressed in the somitic mesoderm during Xenopus early development. We report here the analysis of eight genes whose expression pattern has not been described previously. They include the Xenopus homologues of eukaryotic initiation factor 2beta, methionine adenosyltransferase II, serine dehydratase, alpha-adducin, oxoglutarate dehydrogenase, fragile X mental retardation syndrome related protein 1, monocarboxylate transporter and voltage-dependent anion channel 1. Interestingly, these genes exhibit very dynamic expression pattern during early development. At early gastrula stages several genes do not show localised expression pattern, while other genes are expressed in the marginal mesoderm or in ectoderm. As development proceeds, the expression of these genes is gradually restricted to different compartments of somite. This study thus reveals an unexpected dynamic expression pattern for various genes with distinct function in vertebrates.

Expression of Three Gastrula Cell Surface Glycoproteins during Embryonic and Larval Development in the Amphibian Pleurodeles waltlii: (amphibian/antibodies/cell surface glycoproteins/development).

We have previously reported the identification of cell surface glycoproteins in Pleurodeles waltlii gastrulae. In an attempt to study the expression of three of these cell surface glycoproteins (proteins referred to 1, 11 and 14), we have produced monoclonal and polyclonal antibodies by immunizing mice with the spots of the three selected glycoproteins excised from 2D-gels. Expression of the three glycoproteins was detected on the surfaces of all cells during embryonic development. Before hatching, proteins 1, 11 and 14 become expressed in a limited number of tissues.

FGF8, Wnt8 and Myf5 are target genes of Tbx6 during anteroposterior specification in Xenopus embryo.

The T-box transcription factor Tbx6 is required for somite formation and loss-of-function or reduced activity of Tbx6 result in absence of posterior paraxial mesoderm or disorganized somites, but how it is involved in a regulatory hierarchy during Xenopus early development is not clear. We show here that Tbx6 is expressed in the lateral and ventral mesoderm of early gastrula, and it is necessary and sufficient to directly and indirectly regulate the expression of a subset of early mesodermal and endodermal genes. Ectopic expression of Tbx6 inhibits early neuroectodermal gene expression by strongly inducing the expression of posterior mesodermal genes, and expands the mesoderm territory at the expense of neuroectoderm. Conversely, overexpression of a dominant negative Tbx6 mutant in the ventral mesoderm inhibits the expression of several mesodermal genes and results in neural induction in a dose-dependent manner. Using a hormone-inducible form of Tbx6, we have identified FGF8, Xwnt8 and XMyf5 as immediate early responsive genes of Tbx6, and the induction of these genes by Tbx6 is independent of Xbra and VegT. These target genes act downstream and mediate the function of Tbx6 in anteroposterior specification. Our results therefore identify a regulatory cascade governed by Tbx6 in the specification of posterior mesoderm during Xenopus early development.

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions.

Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus.

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.

Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate beta-catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/beta-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/beta-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/beta-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/beta-catenin signal.

Synthesis of laminin-related polypeptides in oocytes, eggs and early embryos of the amphibian Pleurodeles waltlii.

In the amphibian Pleurodeles waltlii, lamininrelated polypeptides (amphibian-LN) are present in the extracellular matrix underlying the blastocoel roof of gastrulating embryos. Immunoprecipitation with affinity-purified anti-laminin antibodies demonstrated that amphibian-LN is synthesized in oocytes (from stage III onward), eggs and throughout early development. At the late blastula stage, when experiments were carried out with animal and vegetal halves, there were no regional differences in the pattern of amphibian-LN synthesis. The results obtained with transcription inhibitors suggest that throughout pregastrula stages, amphibian-LN is a translation product of stored maternal mRNA. Finally, having compared amphibian-LN and fibronectin synthesis, it is concluded that both extracellular glycoproteins have a common pattern of synthesis.

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation.

A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.

Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation.

Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.

Lithium induces dorsal-type migration of mesodermal cells in the entire marginal zone of urodele amphibian embryos.

The effect of lithium (Li+) on gastrulation movements was investigated during the development of the urodele amphibianPleurodeles waltl. Attention was focused on mesodermal cell migration. Under conditions of Li+ treatment providing a maximal enhancement of dorsoanterior structures, it was found that the dorsoventral polarity of gastrulation was abolished. In particular, vital staining and scanning electron microscopy observations on embryo fractures showed that mesodermal cells migrated radially after Li+ treatment, which led to the formation of rounded embryos. Epiboly movements thus were accelerated. Nevertheless, contrasting with the precocious disappearance of the early-formed yolk plug, archenteron invagination was constantly retarded and commenced with a delay of several hours as compared to control gastrulae. Cell-lineage analysis of the progenies from ventral or dorsal equatorial blastomeres of 32-cell-stage embryos provided evidence that both dorsal and ventral mesoderm contributed to notochordal tissue after Li+ treatment. Dorsalization of the entire marginal zone was confirmed by the ability of the entire mesoderm rudiment to behave as a dorsal organiser after Li+ treatment. Comparison of the migratory behaviour of isolated animal hemispheres from Li+-treated or control embryos cultured on fibronectin-coated substrate indicated that all marginal cells acquired the autonomous capacity for migration of dorsal marginal cells under the action of lithium.