Organizing centers are sources of secreted signaling molecules and morphogens which diffuse to establish morphogen gradients that control the fates of cells in neighboring embryonic tissues. Wnts are secreted signaling molecules that are expressed in the primitive streak and the node, two important organizing centers that function to coordinate anterior-posterior (AP) and left-right (LR) axis patterning, mesoderm formation and somitogenesis, to elicit the development of the trunk and tail. We have primarily focused our studies on Wnt3a, since it is expressed in the primitive streak and is a critical regulator of trunk and tail development. We recently published a manuscript showing a novel function for Wnt3a in the regulation of LR patterning and proposed that Wnt3a functions as an organizer of trunk and tail development by linking the segmentation clock and AP axis elongation with the specification of the LR axis. Towards achieving our specific goals, we have generated a loss of function (LOF) allele of Wnt2b (which is expressed in the streak), and shown that animals lacking Wnt2b are viable and fertile. Potential phenotypes in animals doubly mutant for Wnt2b, and the closely related Wnt2 gene are currently being examined. We have also generated conditional loss (LOF) and gain of function (GOF) alleles of beta-catenin in the primitive streak using T-Cre, and have shown that the beta-catenin LOF has a more severe phenotype than the Wnt3a LOF phenotype, demonstrating that Wnts are partially redundant in the primitive streak. A detailed phenotypic analysis of conditional beta-catenin mutants has shown that Wnt3a and beta-catenin function during somitogenesis to position the expression domains of segment boundary determination genes. To understand the molecular mechanisms underlying Wnt-mediated control of segment boundary determination gene expression, we have identified and started to validate a large number of target genes of Wnt3a and beta-catenin. The Affymetrix microarray screen was very successful, identifying previously characterized direct target genes, as well as several novel or previously unknown Wnt target genes including a new oscillating segment boundary determination gene. We have shown that this novel gene likely functions as a transcriptional repressor, and that it is itself repressed by Wnt signaling. This work has been written up and is about to be submitted for publication. We are currently characterizing the regulatory elements that drive expression of this gene in the anterior presomitic mesoderm (psm) and the transcription factors that bind to these regulatory elements. We are also continuing to systematically validate the remaining target genes (150).We have previously shown that Wnt5a is required for the morphogenetic movements that extend the trunk and tail and for the growth of the limbs, face, gut and genitalia. We have obtained strong genetic evidence that indicates that Wnt5a signals through an alternative pathway termed the Wnt/Planar Cell Polarity (PCP) pathway to regulate body axis extension and cardiovascular development (manuscript in prep). Transcriptional profiles of Wnt5a mutant embryos have been performed, as described previously for Wnt3a mutants. We have also identified mouse homologues of Daam (Dsh-associated activator of morphogenesis), a Formin protein that promotes actin polymerization and which interacts directly with Dsh and Rho to control convergent extension in frogs. We have published the expression patterns of Daam during embryogenesis. We have generated numerous tools including targeted conditional loss-of-function alleles, LacZ-tagged alleles, tagged cDNAs and antibodies to understand how the Daam proteins function in the Wnt pathway to regulate cell and tissue polarity during mouse embryogenesis. A manuscript describing the cell polarity defects in Daam1 mutant preimplantation embryos is in preparation.
| Nakaya, Masa-aki; Biris, Kristin; Tsukiyama, Tadasuke et al. (2005) Wnt3a links left-right determination with segmentation and anteroposterior axis elongation. Development 132:5425-36 |
