Identification of a novel apoptotic pathway in the developing mammalian embryo

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DESCRIPTION (provided by applicant): Apoptosis is critical for the maintenance of tissue homeostasis and for the maturation of various organs in the developing embryo. Failure to activate the cell death program results in embryonic lethality and severe central nervous system (CNS) defects, including hyperplasia of cells in the CNS, exencephaly (protrusion of brain matter from the skull), and incomplete neural tube closure. Although the apoptotic pathway has been well characterized in certain mammalian cell lines, exactly how cell death is regulated in primary embryonic stem cells (ESCs) and in the neural precursor cells (NPCs) of the CNS remains largely unknown. In this proposal, I will explore a novel apoptotic pathway engaged by ESCs and NPCs that primes them for rapid death in the developing embryo. This proposal will test the hypothesis that these cells utilize a novel C. elegans-like apoptotic pathway for activating caspases and cell death independently of the mitochondria. In particular, I hypothesize that, similar to what is observed in cells undergoing apoptosis in the developing C. elegans embryo, ESCs and NPCs express a previously uncharacterized short form of the pro-apoptotic protein, sApaf-1, that exists in complex with, and is inhibited by, the anti-apoptotic Bc-xL protein. In Aim 1, I will investigate how the sApaf-1/Bcl-xL interaction regulates apoptosis in ESCs and NPCs. Specifically, I will examine the physical interaction between sApaf-1 and Bcl-xL and determine whether Bcl-xL inhibition or knockdown is sufficient to allow sApaf-1 to activate caspases and induce rapid apoptosis in both cell types. Importantly, I will test whether this novel pathway in ESCs and NPCs induces apoptosis by bypassing the mitochondria as observed in the C. elegans embryo. In Aim 2, I will investigate the sApaf-1/Bcl-xL apoptotic pathway in the developing brain in vivo. This aim will explore the increased cell death phenotype of conditional knockout mice that are deficient in Bcl-xL in the developing brain, and whether the cell death observed in these animals can be rescued by simultaneous deletion of Apaf-1. The results from this proposal will be important for uncovering key features of ESC and NPC biology that determine how cell death is regulated in early mammalian embryonic development.