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 undergo an epithelial-mesenchymal transition (EMT), and then 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) every 2 hours, 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 shown 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 that Wnt2b does not have an essential role in embryogenesis but functions redundantly with the closely related Wnt2. In collaboration with Ed Morrissey, U. Pennsylvania (who generated Wnt2 mutants) we generated double mutants and found that embryos lacking both Wnt2 and 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 demonstrates that canonical Wnt2/2b signaling plays a fundamental role in lung organogenesis, functioning to specify lung endoderm progenitors. This work was 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 in stem cells 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. A comprehensive in situ hybridization screen to examine the expression of these putative target genes in embryos identified several important new targets. These studies have led us to focus, for the time being, on two particularly interesting transcription factors, Mesogenin (Msgn1) and Sp5. Msgn1 is a mesoderm-specific bHLH transcription factor that functions as a crucial mediator of Wnt3a/bcatenin signaling during mesoderm homeostasis. We have shown that Msgn1 is a direct target gene of the Wnt3a/bcatenin pathway and that it functions in a negative feedback loop to promote the maturation of mesoderm stem cells by suppressing Wnt3a. Overexpression of Msgn1 in zebrafish embryos (collaboration with B. Feldman, NHGRI), or in embryonic stem (ES) cells similarly represses Wnt3a, but also activates the expression of anterior PSM and segmentation markers, suggesting that Msgn1 promotes both the maturation and segmentation of PSM progenitors. Our microarray analysis has led to the identification of many putative Wn3a/bcatenin 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 ES cell lines carrying Doxycycline (Dox)-inducible epitope-tagged 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. Expression of Msgn1 in ES cells resulted in the rapid activation of early PSM-specific genes suggesting that it is a major determinant of the PSM lineage. To identify the target genes of Msgn1 and to characterize Msgn1 transcriptional activity, we have performed genome-wide microarrays and ChIP-Seq studies of Msgn1 activity in ES cells. Remarkably, we discovered that Msgn1 is a critical regulator of Notch signaling in the PSM, directly binding, and activating the transcription of, multiple genes in the Notch signaling pathway. Msgn1 also synergizes with the Notch pathway to directly trigger segmentation clock gene expression. Thus, we have demonstrated that Msgn is a new component of the segmentation clock, and have elucidated the molecular mechanisms underlying how Wnt3a controls the segmentation clock. This work was published in Nature Communications (Chalamalasetty et al., 2011). Continued integration of genomic data has also identified a role for Msgn1 in the regulation of Snai1 &EMT thus we are focusing on a possible role for Msgn1 in metastasis. Since gain of function mutations in the Wnt pathway cause colorectal cancer, we screened our embryonic Wnt target genes for expression in the murine 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. We have found that the zinc finger-containing transcription factor, Sp5, is regulated by Wnt3a/bcatenin signaling during gastrulation, and is expressed at multiple sites of Wnt activity, including normal and diseased embryonic and adult tissues, suggesting that Sp5 is a universal Wnt/bcatenin target gene. Genetic epistasis studies are consistent with Sp5 functioning downstream of Wnt3a in a genetic pathway that regulates somitogenesis and tail development. We found that Sp5 is not, however, required for spontaneous adenoma formation in the Apcmin mouse model of human intestinal cancer. We conclude that Sp5 is a mediator of canonical Wnt/bcatenin signaling during gastrulation and somitogenesis, but has a redundant role in intestinal tumorigenesis. We are currently addressing this potential redundancy by examining the function of the closely-related gene Sp8 in embryogenesis and intestinal cancer, and by making Sp5/Sp8 double mutants.
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