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胚胎发育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致命性和严重的中枢神经系统（CNS）缺陷，包括中枢神经系统细胞增生、脑外畸形（脑物质从颅骨突出）和神经管关闭不完全。虽然凋亡途径在某些哺乳动物细胞系中已被很好地表征，但细胞死亡究竟是如何在原代胚胎干细胞（ESCs）和中枢神经系统的神经前体细胞（npc）中调控的，在很大程度上仍然未知。在这一建议中，我将探索esc和npc参与的一种新的凋亡途径，使它们在发育中的胚胎中快速死亡。这一提议将验证这些细胞利用一种新的类似秀丽隐杆线虫的凋亡途径来激活半胱天冬酶和独立于线粒体的细胞死亡的假设。特别是，我假设，类似于在秀丽隐杆线虫胚胎发育过程中观察到的细胞凋亡，ESCs和npc表达一种以前未被表征的促凋亡蛋白的短形式sApaf-1，它与抗凋亡Bc-xL蛋白复合物存在，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，我将研究sApaf-1和Bcl-xL之间的物理相互作用，并确定Bcl-xL的抑制或敲低是否足以使sApaf-1激活半胱天蛋白酶，并在两种细胞类型中诱导快速凋亡。重要的是，我将测试esc和npc中的这种新途径是否通过在秀丽隐杆线虫胚胎中观察到的绕过线粒体诱导细胞凋亡。在Aim 2中，我将在体内研究发育中的脑中sApaf-1/Bcl-xL凋亡通路。本研究旨在探讨脑发育中缺乏Bcl-xL的条件敲除小鼠的细胞死亡表型增加，以及是否可以通过同时缺失Apaf-1来挽救这些动物中观察到的细胞死亡。这项提案的结果对于揭示ESC和NPC生物学的关键特征非常重要，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.