A gap junction connexin is required in the vertebrate left-right organizer

Early patterning of vertebrate embryos involves the generation of asymmetric signals across the left-right (L-R) axis that position and are required for the proper function of internal organs. This patterning is directed by a conserved nodal/lefty signaling cascade on the left side of the embryo, thought to be asymmetrically directed by ciliary beating that generates a leftward fluid flow in the mammalian node and in Kupffer's vesicle (KV), the related structure in zebrafish. Following morpholino knockdown of Cx43.4, asymmetric gene expression and global organ distribution are randomized, consistent with the expression of Cx43.4 in KV. Randomization is recapitulated in mosaic embryos in which Cx43.4 is depleted preferentially in KV cells, showing that Cx43.4 is specifically required in KV for proper L-R axis formation. The mechanistic basis for the laterality anomalies in Cx43.4-deficient embryos is a primary morphogenesis defect during lumen formation in KV. Additionally, the role of Cx43.4 appears to be conserved given that its ortholog, human Cx45, is able to functionally compensate for zebrafish Cx43.4 during L-R patterning. This is the first report linking connexin function in the ciliated, node-like cells of KV with normal L-R axis development.     

Neural induction in the absence of organizer in salamanders is mediated by MAPK

Research on the mechanisms of embryonic induction had a great setback in the 1940s when Barth discovered and Holtfreter confirmed that ectoderm of Ambystoma maculatum salamander embryos could form brain tissue when cultured in a simple saline solution. We have revisited this classical experiment and found that when cultured animal cap ectoderm attaches to a glass substratum, it can self-organize to form complex organs such as brain vesicles, eyes, lens and olfactory placodes. Only anterior neural organs were generated. Under these culture conditions ERK became diphosphorylated, indicating a sustained activation of the Ras/MAPK pathway. Using sand particles as an example of a heterologous neural inducer similar results were obtained. Addition of U0126, a specific antagonist of MEK, the enzyme that phosphorylates ERK/MAPK, inhibited neural differentiation. The closely related control compound U0124 had no effect. We conclude that neural induction in the absence of organizer in A. maculatum requires Ras/MAPK-activation. These findings provide a molecular explanation for the activity of heterologous neural inducers that dominated thinking in amphibian experimental embryology for many decades.     

The vertebrate organizer: structure and molecules

Since the identification of the first organizer gene, goosecoid, more than 15 organizer-specific genes have been characterized. Here, we present our current understanding of the roles of these molecules in amphiabians, fish and amniotes and show how their identification has confirmed Spemann's original proposition that the vertebrate organizer is subdivided into separate domains: the head, trunk and tail organizers.     

Repression of CD2 gene expression is mediated by an AP-2 related factor

Tissue specific and developmental expression of the CD2 gene is tightly regulated during T cell development. DNase I hypersensitivity analysis has revealed the presence of two sites (DHS1 and 2) located 5' to the CD2 gene which have been reported to be implicated in the developmental regulation of expression of CD2. The location of DHS2 marks the position of the minimal promoter whereas DHS1 is located approximately 1800 bp upstream. We show that repressor and derepressor activities are contained within the region of DNA marked by DHS1. The repressor is capable of regulating homologous and heterologous promoters regardless of orientation. This activity is entirely dependent upon the presence of an AP-2 binding site as mutation of this site resulted in a loss of repressor activity. A nuclear factor found in Jurkat cells specifically binds this site but was shown to be serologically distinct from AP-2.     

Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor

Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.     

The establishment of Spemann's organizer and patterning of the vertebrate embryo

Molecular studies have begun to unravel the sequential cell-cell signalling events that establish the dorsal-ventral, or 'back-to-belly', axis of vertebrate animals. In Xenopus and zebrafish, these events start with the movement of membrane vesicles associated with dorsal determinants. This mediates the induction of mesoderm by generating gradients of growth factors. Dorsal mesoderm then becomes a signalling centre, the Spemann's organizer, which secretes several antagonists of growth-factor signalling. Recent studies have led to new models for the regulation of cell-cell signalling during development, which may also apply to the homeostasis of adult tissues.     

Regulation of convergent extension in Xenopus by Wnt5a and Frizzled-8 is independent of the canonical Wnt pathway

The Wnt signaling pathway is increasingly recognized as a highly branched signaling network. Experimental uncoupling of the different branches of this pathway has proven difficult, as many single components are shared downstream by multiple, distinct pathways. In this report, we demonstrate that the upstream Wnt antagonists Xwnt5a and Nxfz-8, which inhibit normal morphogenetic movements during Xenopus gastrulation, act independently of the canonical Wnt signaling pathway. This finding is important, as it highlights the promiscuity of upstream Wnt signaling components and further establishes an important role for non-canonical Wnt signaling in Xenopus morphogenesis.     

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.     

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.     

The mouse frizzled 8 receptor is expressed in anterior organizer tissues

Wnt signaling has been shown to be important for axis formation in vertebrates. However, no Wnt ligand or receptor has been shown to be specifically expressed in all the organizer tissues in the mouse embryo. Here we report that the mouse frizzled 8 (mfz8) gene, a Wnt receptor, is expressed in the anterior visceral endoderm (AVE) and the anterior primitive streak, which have been shown to possess organizer activity. mFz8 is also expressed in the descendents of the anterior streak that comprise the anterior mesendoderm (AME) at midgastrulation, with subsequent expression in the anterior neurectoderm, which is specified and patterned by the AVE and AME. Thus, mfz8 is specifically expressed in the organizer tissues that establish the anterior-posterior axis in the mouse embryo.