Identification of a novel apoptotic pathway in the developing mammalian embryo

描述(申请人提供)：细胞凋亡对于维持组织内环境的稳定和发育中的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，包括中枢神经系统(CNS)细胞增殖、脑外脱出(脑组织从头骨突出)和神经管关闭不全。虽然在某些哺乳动物细胞系中已经很好地描述了细胞凋亡的途径，但在原代胚胎干细胞(ESCs)和中枢神经系统的神经前体细胞(NPC)中，细胞死亡是如何调控的仍然很大程度上未知。在这项提案中，我将探索一种由ESCs和NPC参与的新的凋亡途径，该途径使它们在发育中的胚胎中迅速死亡。这一提议将检验这样的假设，即这些细胞利用一种新的线虫样凋亡途径来激活caspase和细胞死亡，而不依赖于线粒体。特别是，我假设，与在发育中的线虫胚胎中观察到的经历细胞凋亡的细胞相似，ESCs和NPC表达一种以前没有特征的短促凋亡蛋白sApaf-1，它与抗凋亡的BC-xL蛋白形成复合体，并被抗凋亡的BC-xL蛋白抑制。在目标1中，我将研究sApaf-1/Bcl-xl相互作用如何调节ESCs和NPC的凋亡。具体地说，我将研究sApaf-1和Bclxl之间的物理相互作用，并确定Bclxl的抑制或敲除是否足以使sApaf-1激活caspase并在两种类型的细胞中诱导快速凋亡。重要的是，我将测试ESCs和NPC中的这一新途径是否像在线虫胚胎中观察到的那样，通过绕过线粒体而诱导细胞凋亡。在目标2中，我将在体内研究sApaf-1/Bclxl在发育中的脑内的凋亡通路。这一目的将探索发育中大脑中缺乏Bclxl的条件性基因敲除小鼠细胞死亡表型的增加，以及是否可以通过同时缺失APAF-1来挽救在这些动物中观察到的细胞死亡。这一提议的结果将对揭示胚胎干细胞和鼻咽癌生物学的关键特征具有重要意义，这些生物学特征决定了细胞死亡在哺乳动物早期胚胎发育中是如何调控的。

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.