Function of the Apela-APJ signaling axis in mammalian development.

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 DESCRIPTION (provided by applicant): Proper embryo formation and organogenesis relies on molecular signaling pathways that are used in a reiterated manner throughout development, and are essential for viability. The Apela-APJ signaling axis is a new pathway that was only recently discovered in zebrafish. Loss of apela in zebrafish causes early defects in germ layer formation as well as later malformations of various organs. Notably, these mutants often fail to form a heart. It is not clear whether defects in organogenesis are a consequence of earlier deficiencies in endoderm and mesoderm, or if Apela-APJ also functions in particular organs, such as the developing heart. Since the discovery of Apela as a signaling molecule occurred so recently, its role in mammalian development is yet to be determined, though evidence suggests conservation of the Apela-APJ signaling pathway among vertebrates. The aims of this project are designed to uncover and define the function and requirement for Apela-APJ signaling in mouse embryonic development and organogenesis. To accomplish this, the sponsor's lab has recently created mice lacking Apela. The phenotype of Apela null mutants will be characterized by analyzing morphology of vital organs as well as expression of various developmental markers important for heart development, endoderm/mesoderm specification, and left-right patterning. It has been shown that Apela signals via binding to the G protein-coupled receptor APJ. Another non-homologous molecule, Apelin, also activates APJ. Discrepancies between Apelin and APJ mutant phenotypes suggest that Apela may play a critical role in mouse development. Therefore overlapping roles between Apela versus Apelin mediated activation of APJ will be investigated using Apelin and APJ mouse mutants. Defects in endoderm and mesoderm progenitors will be assessed in Apela mutants using primitive streak explants, motility assays, and live imaging of fluorescent reporter mice. To test the requirements for Apela-APJ signaling with respect to the specification and behavior of endoderm and cardiac progenitors, mouse embryonic stem cells (mESCs) from Apela;Apelin, and APJ mutants will be derived for in vitro experiments. Apelin-APJ signaling promotes the differentiation and maintenance of mESC-derived cardiovascular progenitors, suggesting that Apela may exert similar effects. The role of Apela-APJ signaling in directed differentiation of mESCs into endoderm and cardiac progenitor cell fates will be investigated, thereby establishing its relevance to future therapeutic applications. To determine cell autonomous requirements for APJ-mediated signaling, mutant and control mESCs will be assayed in co-cultures, and will also be used to make chimeric embryos. Collectively, these studies will shed light on the critical functions of a novel signaling axis in mammalian development, and they will provide fundamental knowledge for building new genetic pathways that function in development or disease.