The Cell Signaling in Vertebrate Development Section is taking genetic and molecular approaches to understand the mechanisms of Wnt signaling during early embryogenesis and tumorigenesis. The laboratory is primarily focused on two related developmental processes, gastrulation and somitogenesis, as they provide an excellent opportunity to study how signaling pathways regulate stem cell homeostasis in vivo. Gastrulation converts pluripotent epiblast stem cells into mesoderm and endoderm stem cells through a transient developmental structure known as the primitive streak (PS). Mesodermal stem cells remain in an undifferentiated, self-renewing state while in the PS, and generate progeny that mature and differentiate into different mesodermal lineages after they have moved away from the PS. One of the major mesodermal lineages arising during gastrulation is the somite lineage. Somite progenitors leave the PS to form the presomitic mesoderm (PSM). Somites are blocks of mesoderm that bud off of the anterior end of the presomitic mesoderm (PSM) at 2 hour intervals, eventually giving rise to 65 pairs of somites that differentiate into the vertebrae, ribs, muscles, and dermis of the trunk and tail. Somitogenesis therefore relies upon the maintenance of the PSM, which in turn depends upon maintaining a balance of mesodermal stem cells and lineage-committed PSM progenitors. The rhythmic formation of somites is controlled by a molecular oscillator, or segmentation clock, that involves the periodic activation of the Notch, Wnt and Fgf pathways in the PSM. The best molecular evidence for the existence of a clock comes from the analysis of Notch target genes (ex. Lunatic fringe (Lfng)) which are expressed in a caudal-rostral wave that sweeps across the PSM once during each somite formation. A gradient of Wnt3a/bcatenin signals are thought to drive the segmentation clock however the precise molecular mechanisms remain elusive. We have found that 7 of the 19 mammalian Wnt genes, including Wnt2b, Wnt3a and Wnt5a, are expressed in the PS. Null mutations in Wnt3a generate phenotypes that suggest that it functions to maintain stem/progenitor cells in the PS. To examine the role of Wnt2b in the PS, we generated a null allele and found to our surprise that Wnt2b does not have an essential role in embryogenesis. Interestingly, Wnt2b is co-expressed with other Wnt genes in several stem cell niches in the embryo, including the lung progenitors. To test whether Wnt2b functions redundantly with the closely related Wnt2, we generated double mutants (collaboration with Ed Morrissey, U. Pennsylvania who generated Wnt2 mutants). Remarkably, we found that embryos lacking Wnt2/2b display complete agenesis of the lung, and do not express markers of lung endoderm progenitors. In contrast, activation of canonical Wnt/beta-catenin signaling results in expansion of lung endoderm progenitors. Our data demonstrate that canonical Wnt2/2b signaling plays a fundamental role in lung organogenesis, functioning to specify lung endoderm progenitors. This work was just published in Developmental Cell (Goss et al., 2009). Activation of the Wnt/bcatenin signaling pathway stabilizes bcatenin, which interacts with members of the Lef/Tcf family of DNA-binding factors to transcriptionally activate target genes. A major goal of the laboratory is to elucidate the target genes and transcriptional networks activated by Wnt3a during early embryogenesis. We have generated genome-wide transcriptional profiles of wildtype (wt) and Wnt3a-/- embryos and have identified 133 differentially expressed genes, including several previously characterized direct Wnt/bcatenin target genes. We have performed a comprehensive in situ hybridization screen to examine the expression of these putative target genes in embryos and have identified several important targets. These studies have led to the demonstration that Mesogenin (Msgn), a mesoderm-specific bHLH transcription factor, is a crucial mediator of Wnt3a/bcatenin signaling during mesoderm homeostasis. Msgn is a direct target gene of the Wnt3a/bcatenin pathway that functions in a negative feedback loop to promote mesoderm maturation by suppressing Wnt3a. Overexpression of Msgn in zebrafish embryos (collaboration with B. Feldman, NHGRI) similarly represses Wnt3a, but also activates the expression of anterior PSM and segmentation markers, suggesting that Msgn promotes the maturation and segmentation of PSM progenitors. Remarkably, we showed that Msgn synergizes with the Notch pathway to directly regulate transcription of the oscillating segmentation clock gene Lfng. Thus, we have demonstrated that Msgn is a new component of the segmentation clock, and have identified a mechanism for how the Wnt and Notch pathways crosstalk. Msgn appears to function as a critical activator of mesodermal maturation by blocking the signals that maintain nascent PSM progenitors, while activating the segmentation clock and promoting the transition of PSM to somites. A manuscript describing these studies has just been submitted for publication. Our microarray analysis has led to the identification of many putative Wn3a/βcatenin target genes, but it remains a significant challenge to identify the genes that convey the mesoderm inducing activity of Wnt3a. We have generated a series of embryonic stem (ES) cell lines carrying Doxycycline (Dox)-inducible transgenes, to develop a functional screen for Wnt target genes that regulate mesodermal stem cell development. Since stimulation of ES cells with Wnt3a rapidly induces mesoderm, we are overexpressing putative Wnt3a target genes in ES cells (in the absence of exogenous Wnt3a) and examining the expression of mesodermal stem cell markers by qPCR. Using an epitope-tagged Msgn as our prototype, we have inducibly expressed Msgn in ES cells and shown that Msgn rapidly represses the expression of genes that maintain pluripotency, while simultaneously activating the expression of PSM-specific genes such as Tbx6. The ability of Msgn to inhibit pluripotency and activate the expression of lineage-specific genes suggests that Msgn is a major determinant of the PSM lineage. We are now transcriptionally profiling ES cells +/- Msgn induction, and performing ChIP-seq experiments to comprehensively identify the Msgn target genes that regulate stem cell development. By transcriptionally profiling Wnt mutants that display deficits in stem cell populations in the early embryo, we have identified a large number of new stem cell-associated Wnt target genes. Since gain of function mutations in the Wnt pathway cause colorectal cancer in humans and gastrointestinal tumors in mice, we screened our embryonic Wnt target genes for expression in the adult intestine and in intestinal tumors. Remarkably, many of our genes are expressed in the stem cell compartment of the GI tract, and are highly expressed in adenomas caused by Wnt gain of function mutations. These results demonstrate that our Wnt target genes are expressed in unrelated embryonic and adult stem cell compartments suggesting that their expression may be associated with 'stemness'. We are currently focused on the potential roles that several of these stem cell-specific Wnt target genes play in intestinal tumorigenesis. To test if these genes are sufficient to cause intestinal tumorigenesis, independent of Wnt signaling, we are overexpressing them in the intestinal epithelium of transgenic mice using the well-characterized GI-specific Villin promoter. 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