The molecular mechanism(s) by which a cell becomes polarized for directional migration remains poorly understood. The non-canonical Wnt pathway has recently been shown to play important roles in cell polarization and migration, which are required for gastrulation and neural fold closure during vertebrate embryogenesis. Importantly defects in non-canonical Wnt signaling are implicated human birth defects disorders including in neural fold closure disorders such as spina bifida and in cancer metastasis. To date, how the non-canonical Wnt signaling pathway regulates changes to the actin cytoskeleton remains at best poorly defined. Our studies from the prior funding period have established that the Formin protein Dishevelled- associated activator of morphogenesis (Daam1) provides a crucial link between Dishevelled (Dvl) and the small GTPase Rho for gastrulation. Importantly, our studies demonstrate Daam1 is required for Wnt-dependent cytoskeletal changes, although the underlying biochemical details are not known. To further identify factors required downstream of Daam1 for cytoskeletal changes, we performed a yeast two-hybrid screen and isolated two new proteins in addition to others we have published. Co-immunoprecipitation and GST-pulldown assays confirm that these proteins are bona-fide Daam1- interacting factors and their interaction and subcellular co-localization with Daam1 in mammalian cells is Wnt-regulated. Furthermore, over-expression or depletion of these proteins, similar to Daam1, blocks gastrulation in the Xenopus embryo and results in an open neural tube phenotype characteristic of spina bifida. We have further cloned and begun characterization of the second vertebrate Daam family member Daam2. Our preliminary studies show that similar to Daam1, Daam2 plays a functional role in non-canonical Wnt signaling but Daam2 in contrast to Daam1 regulates vertebrate neural tube closure. In building a model for how non-canonical Wnt signaling through the Daam proteins regulate cytoskeletal changes, we hypothesize Daam1 and Daam2 are key modulators of the actin cytoskeleton for cellular motility during gastrulation. In this competitive renewal proposal, we will investigate how the two new identified proteins that bind to Daam1 functions to mediate cytoskeletal changes for cell motility during gastrulation using mammalian cells, Xenopus and zebrafish embryos. Second, we will characterize the role of Daam2 as a regulator of non-canonical Wnt signaling during vertebrate gastrulation. We will further delineate whether Daam2 and Daam1 have non-redundant functions during gastrulation and well as defining effector proteins common and specific to Daam1 and/or Daam2. These studies together will significantly advance our understanding of how non-canonical Wnt signaling through the Daam family of Formin proteins regulate cell polarity and cell motility required during vertebrate gastrulation and neural fold closure. These studies will further provide additional insights into how defects in Wnt signaling contribute to birth defects such as spina bifida and cancer metastasis.

Public Health Relevance

Understanding the development of human cancers and birth defect disorders remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key signaling molecule that has been demonstrated to play causative roles in human cancer is the Wnt protein. Our studies are focused on understanding the role of the Dishevelled-associated activator of morphogenesis proteins Daam1 and Daam2 that transduces a branch of Wnt signaling termed non-canonical Wnt signaling. We propose that these studies can provide important new insights into the mechanisms regulating embryogenesis as well as gaining insights into how deregulated Wnt signaling contributes to cancer formation and birth defect disorders including spina bifida.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Development - 1 Study Section (DEV1)
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Hoodbhoy, Tanya
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Temple University
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Seo, Hwa-Seon; Habas, Raymond; Chang, Chenbei et al. (2017) Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis. Hum Mol Genet 26:2053-2061
Komiya, Yuko; Bai, Zhiyong; Cai, Na et al. (2017) A Nonredundant Role for the TRPM6 Channel in Neural Tube Closure. Sci Rep 7:15623
Overton, Jeffrey D; Komiya, Yuko; Mezzacappa, Courtney et al. (2015) Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis. Sci Rep 5:18395
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
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Beringer, Laura T; Kiechel, Marjorie A; Komiya, Yuko et al. (2015) Osteoblast biocompatibility of novel chitosan crosslinker, hexamethylene-1,6-diaminocarboxysulfonate. J Biomed Mater Res A 103:3026-33
Mezzacappa, Courtney; Komiya, Yuko; Habas, Raymond (2012) Activation and function of small GTPases Rho, Rac, and Cdc42 during gastrulation. Methods Mol Biol 839:119-31
Liu, Wei; Komiya, Yuko; Mezzacappa, Courtney et al. (2011) MIM regulates vertebrate neural tube closure. Development 138:2035-47
Miller, Rachel K; Canny, Sol Gomez de la Torre; Jang, Chuan-Wei et al. (2011) Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling. J Am Soc Nephrol 22:1654-64
Su, Li-Ting; Liu, Wei; Chen, Hsiang-Chin et al. (2011) TRPM7 regulates polarized cell movements. Biochem J 434:513-21

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