京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21401号 / 生博第402号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 見学 美根子, 教授 上村 匡, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

During vertebrate gastrulation, an elaborate series of cellular motility events occur that define the three germ layers, and establishes the primary body axis. While it is known that non-canonical Wnt signaling plays a crucial role during this period of development, a complete picture of the molecular mechanisms controlling this pathway has yet to be established. Previous studies has shown that one essential component of the pathway linking the Wnt ligands to the cytoskeletal changes that occur during vertebrate development is Dishevelled-associated activator of morphogenesis (Daam1). Daam1 bridges the gap between Dishevelled (Dvl) and the small GTPase RhoA and is required for Wnt-dependent RhoA activation, but the biochemical details of this process are to date still undefined. To identify additional factors that might be involved in this process, a yeast two-hybrid screen using a C-terminal region of Daam1 was performed. During this screen SLIT-ROBO Rho GTPase-activating protein 2 (SRGAP2), was identified. The studies presented here were designed to establish a functional interaction between SRGAP2 and Daam1 during non-canonical Wnt signaling and to characterize the function of SRGAP2 during early vertebrate development. My studies uncover that SRGAP2 and Daam1 do indeed interact and that this interaction is positively influenced by Wnt stimulation. I also uncover via immunocytochemistry, that these two proteins share common sub-cellular localization patterns in HeLa cells that is responsive to Wnt stimulation. Further, I show that ectopic expression of SRGAP2 in HeLa cells has adverse effects on stress fiber formation in Wnt5a treated cells. And lastly, micro-injection experiments in Xenopus laevis show that over- or under-expression of SRGAP2 produces severe gastrulation defects in the developing embryos. These studies together demonstrate that SRGAP2 plays a critical role in regulating non-canonical Wnt signaling through its interaction with Daam1 and through regulation of the monomeric GTPases Rho. It also shows that SRGAP2 plays a functional role for gastrulation during early vertebrate development. / Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, has been implicated in the non-canonical Wnt mediated Planar Cell Polarity (PCP) signaling pathway. Although the studies in Drosophila Daam1 and Xenopus Daam1 generated inconsistent conclusions regarding the function of Daam1, the biological function of mammalian Daam1 was not evaluated. In this study, we used a mouse promoter trap technology to create Daam1 deficient mice to analyze the role of Daam1 in embryonic development and organogenesis. Daam1 is highly expressed in the developing heart. The majority of Daam1 mutant mice died between embryonic day 14.5 and birth, exhibiting a variety of heart defects, which include ventricular noncompaction, ventricular septal defects, and double outlet right ventricle. About 10% mutant mice survive to adulthood, and these survivors do not show significantly compromised heart function based on echocardiographic analyses. However, all of these mutant survivors have ventricular noncompaction with a range of severities. A conditional rescue experiment using a cardiac specific Cre mouse line, Nkx2-5Cre, confirmed that the cardiac defects are the primary cause of death in Daam1 mutants. Both in vivo and ex vivo analyses revealed that Daam1 is essential for regulating non-sarcomeric filamentous actin assembly in cardiomyocytes, which likely contributes to cardiac morphogenesis and ventricular wall maturation. Biochemical studies further suggested that Daam1 is not a key signaling component in regulating the activation of small GTPases, such as RhoA, Rac1 and Cdc42. In conclusion, our studies demonstrated that Daam1 is essential for cardiac morphogenesis likely through its regulation of cytoskeletal architecture in the developing cardiomyocytes. / indefinitely