The Cell Signaling in Vertebrate Development Section is taking genetic and molecular approaches to understand the cellular and molecular targets of Wnt signaling molecules during early embryogenesis and tumorigenesis. The laboratory is focused on a complex morphogenetic process known as gastrulation, during which the mesoderm and endoderm germ layers are formed and the body plan is established. Gastrulation begins with the formation of the primitive streak (PS), a transient developmental structure that forms at the posterior terminus and converts pluripotent epiblast stem cells into mesoderm and endoderm progenitors. We have found that seven of the nineteen mammalian Wnt genes are expressed in the PS, suggesting that Wnts may function there to regulate stem cell self-renewal or differentiation. Several pathways are known to transduce Wnt signals, including the Wnt/bcatenin and Wnt/Planar Cell Polarity (PCP) pathways. This project specifically addresses the role that the Wnt/bcatenin pathway plays in gastrulation and stem cell development. Wnt2b is one of the seven Wnt genes expressed in the PS. To examine its function there we generated a null allele and found 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 early lung progenitors. To test whether Wnt2b functions redundantly with the closely related Wnt2a, we collaborated with Ed Morrisey at Penn to generate double mutants. Remarkably, we found that embryos lacking Wnt2a/2b do not develop lungs, and do not express Nkx2.1, the earliest marker of lung endodermal progenitors. In contrast, activation of canonical Wnt/beta-catenin signaling results in expansion of lung endoderm progenitors into the posterior gut. Our data reveal that canonical Wnt2a/2b signaling is uniquely required for specification of lung endoderm progenitors but not endoderm progenitors of other foregut derived tissues. This work is currently under review. We have previously generated a transgenic reporter of Wnt/bcatenin signals in vivo (the BATlacZ mouse) and shown that the pathway is highly active in the PS, primarily functioning to transduce Wnt3a signals (Nakaya et al., 2005). By crossing the T-Cre mouse (Perantoni et al., 2005), which expresses Cre recombinase specifically in the PS and posterior mesoderm, with mice carrying conditional loss and gain of function alleles of beta-catenin, we have shown that Ctnnb1 (which encodes beta-catenin) transduces Wnt3a signals in the PS and is necessary for mesoderm formation and segmentation (Dunty et al., 2008). These and other results suggest that Wnt3a and bcatenin are necessary for the specification or maintenance of an embryonic mesodermal stem cell. Interestingly, constitutive activation of bcatenin in the PSM inhibits progenitor maturation, suggesting that Wnt/bcatenin signaling maintains PSM progenitor self-renewal by inhibiting their differentiation. How Wnt signaling balances stem/progenitor cell renewal and differentiation during the homeostatic maintenance of the embryonic mesoderm is poorly understood. 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. Thus 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 identified 150 differentially expressed genes including several previously characterized direct Wnt/bcatenin target genes (Dunty et al., in prep). Remarkably, over a third of these genes encode transcriptional regulators. We have performed a comprehensive in situ hybridization screen to examine the expression of these putative target genes in embryos and have identified several novel 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. Molecular genetic studies demonstrate that 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 similarly represses Wnt3a, but also activates the premature expression of somitic markers, suggesting that Msgn controls the commitment of nascent PSM progenitors to the somitic lineage. Remarkably, Msgn also regulates the expression of Snail, a transcriptional repressor necessary for the epithelial-mesenchymal transitions that occur during mesodermal morphogenesis and segmentation. Thus, Msgn functions as a critical activator of mesodermal maturation by blocking the renewal of mesodermal progenitors, and promoting their morphological transition and commitment to the somitic lineage. Our transcriptional profiing studies have also led to the identification of a novel transcriptional corepressor, Ripply2, which is transcriptionally regulated by Wnt3a and bcatenin during mesodermal segmentation (Biris et al., 2007), suggesting a potential mechanism for the control of segmentation by Wnts (Dunty et al., 2008). 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'. Although other groups have previously shown that the Zn finger transcription factor Sp5 is an embryonic Wnt target, we have shown that Sp5 is highly expressed in adult gastrointestinal tumors. Our preliminary functional analyses suggest that Sp5 is an important effector of Wnt signaling during tumorigenesis. The loss of Sp5 in animal models of familial adenomatous polyposis (FAP) caused by mutation in tumor suppressors in the Wnt/bcatenin pathway led to the formation of fewer GI tumors. To test whether Sp5 is sufficient to cause intestinal tumorigenesis independent of Wnt signaling, we are currently using the well-characterized GI-specific Villin promoter to overexpress our target genes in the intestinal epithelium of transgenic mice, specifically in the intestinal stem cell niche or crypts. Another interesting stem cell-associated gene functioning downstream of Wnt3a is the Tgf-beta family member Nodal. Nodal is known to regulate pluripotent stem cells in the early embryo and has also been associated with melanoma agressiveness. Nodal was identified as a putative downstream target gene of Wnt3a/bcatenin signaling in the early embryo and is expressed in the adult intestinal crypt s [summary truncated at 7800 characters]

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Basic Sciences
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Tsukiyama, Tadasuke; Yamaguchi, Terry P (2012) Mice lacking Wnt2b are viable and display a postnatal olfactory bulb phenotype. Neurosci Lett 512:48-52
Joesting, Margaret S; Cheever, Thomas R; Volzing, Katherine G et al. (2008) Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate. Dev Biol 317:161-73
Warming, Soren; Suzuki, Takeshi; Yamaguchi, Terry P et al. (2004) Early B-cell factor-associated zinc-finger gene is a frequent target of retroviral integration in murine B-cell lymphomas. Oncogene 23:2727-31