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 neural, mesodermal and endodermal stem cells through a transient developmental structure known as the primitive streak (PS). We are studying a unique stem cell known as the neuromesodermal (NM) stem cell that gives rise to the spinal cord and musculoskeletal cells of the trunk and tail. NM stem cells reside in the epiblast adjacent to the PS, and remain in an undifferentiated, self-renewing state while under the influence of signals emanating from the PS. Progeny of NM cells that remain in the epiblast differentiate into neural cells, while cells destined for paraxial mesoderm ingress through the PS, undergo an epithelial-mesenchymal transition (EMT), and then mature and differentiate into musculoskeletal progenitors (which, collectively, are known as the presomitic mesoderm (PSM). The signals that maintain NM stem cells, and control their differentiation into neural or musculoskeletal progenitors are poorly understood. 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 Wnt3a maintains NM stem cells in the PS and promotes their differentiation into paraxial mesoderm. 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 PS 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. We have shown that Msgn1 is a direct target gene of the Wnt3a/bcatenin pathway and that it functions, at least in part, in a negative feedback loop to promote the differentiation of NM 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 PSM-specific markers, suggesting that Msgn1 promotes musculoskeletal cell fates. 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 therefore 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 NM 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 demonstrated that Msgn1 directly regulates EMT and musculoskeletal progenitor programs. 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 studying the zinc finger-containing transcription factor, Sp5, which 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. Recent studies have shown that Sp5 functions redundantly with the closely related family member Sp8 to regulate NM stem cells. We are currently elucidating the underlying mechanisms. We have also found that Sp5 alone is not required for spontaneous adenoma formation in the Apcmin mouse model of human intestinal cancer, suggesting that Sp5 may also have redundant functions during intestinal tumorigenesis. We are currently addressing the potential redundancy of Sp5 and Sp8 in intestinal cancer stem cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010345-13
Application #
8552675
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2012
Total Cost
$1,220,801
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Kumar, Amit; Chalamalasetty, Ravindra B; Kennedy, Mark W et al. (2016) Zfp703 Is a Wnt/β-Catenin Feedback Suppressor Targeting the β-Catenin/Tcf1 Complex. Mol Cell Biol 36:1793-802
Li, Ding; Sinha, Tanvi; Ajima, Rieko et al. (2016) Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment. Dev Biol 412:18-31
Cunningham, Thomas J; Kumar, Sandeep; Yamaguchi, Terry P et al. (2015) Wnt8a and Wnt3a cooperate in the axial stem cell niche to promote mammalian body axis extension. Dev Dyn 244:797-807
Garriock, Robert J; Chalamalasetty, Ravindra B; Kennedy, Mark W et al. (2015) Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation. Development 142:1628-38
Ajima, Rieko; Bisson, Joseph A; Helt, Jay-Christian et al. (2015) DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly. Dev Biol 408:126-39
Tsukiyama, Tadasuke; Fukui, Akimasa; Terai, Sayuri et al. (2015) Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling. Mol Cell Biol 35:2007-23
Dunty Jr, William C; Kennedy, Mark W L; Chalamalasetty, Ravindra B et al. (2014) Transcriptional profiling of Wnt3a mutants identifies Sp transcription factors as essential effectors of the Wnt/*-catenin pathway in neuromesodermal stem cells. PLoS One 9:e87018
Biris, Kristin K; Yamaguchi, Terry P (2014) Two-color in situ hybridization of whole-mount mouse embryos. Methods Mol Biol 1092:17-30
Garriock, Robert J; Mikawa, Takashi; Yamaguchi, Terry P (2014) Isolation and culture of mouse proepicardium using serum-free conditions. Methods 66:365-9
Tsukiyama, Tadasuke; Yamaguchi, Terry P (2012) Mice lacking Wnt2b are viable and display a postnatal olfactory bulb phenotype. Neurosci Lett 512:48-52

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