Identification of a novel apoptotic pathway in the developing mammalian embryo

描述（由申请人提供）：细胞凋亡对于维持组织稳态和发育胚胎中各种器官的成熟至关重要。未能激活细胞死亡程序会导致胚胎死亡和严重的中枢神经系统（CNS）缺陷，包括CNS细胞增生、露脑畸形（脑物质从颅骨突出）和神经管闭合不全。虽然在某些哺乳动物细胞系中已经很好地表征了细胞凋亡途径，但是在原代胚胎干细胞（ESC）和CNS的神经前体细胞（NPC）中细胞死亡究竟是如何被调节的在很大程度上仍然未知。在这个提议中，我将探索一种新的凋亡途径，由ESC和NPC参与，使它们在发育中的胚胎中快速死亡。这项提议将检验这些细胞利用一种新的C. elegans样细胞凋亡途径激活半胱天冬酶和细胞死亡的线粒体独立。特别是，我假设，类似于在发育中的C。线虫胚胎、ESC和NPC表达以前未表征的促凋亡蛋白sApaf-1的短形式，sApaf-1与抗凋亡Bc-xL蛋白复合存在并被其抑制。在目的1中，我将研究sApaf-1/Bcl-xL相互作用如何调节ESCs和NPC的凋亡。具体来说，我将检查sApaf-1和Bcl-xL之间的物理相互作用，并确定Bcl-xL抑制或敲低是否足以使sApaf-1激活半胱天冬酶，并诱导两种细胞类型的快速凋亡。重要的是，我将测试ESCs和NPC中的这种新途径是否通过绕过线粒体诱导细胞凋亡，如在C。线虫胚胎目的2：研究sApaf-1/Bcl-xL凋亡通路在脑发育过程中的作用。这一目标将探讨在发育中的大脑中缺乏Bcl-xL的条件性敲除小鼠的细胞死亡表型增加，以及在这些动物中观察到的细胞死亡是否可以通过同时缺失Apaf-1来挽救。这项提议的结果对于揭示ESC和NPC生物学的关键特征将是重要的，这些特征决定了细胞死亡在早期哺乳动物胚胎发育中是如何调节的。

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.