Genetic Pathways Directing Ventral Folding Morphogenesis in Mammalian Embryos

描述(由申请人提供)：本项目的总体目标是阐明介导腹侧折叠形态发生的遗传途径和细胞机制。人类胚胎在妊娠第四周经历腹侧折叠形态发生。尽管人类和小鼠早期胚胎的上胚层具有不同的拓扑结构(前者为平盘，后者为中空圆柱体)，但腹侧折叠形态发生的一些关键方面是保守的：肠道内化、线形心管的形成、腹侧体壁的关闭以及胎儿被包裹在羊膜中。腹侧形态发生异常是许多与体壁闭合不全有关的出生缺陷的基础(腹裂、腹壁外的肠道发育；心脏异位、心脏位于胸腔外)；这种腹侧折叠的异常发生在2000年的活产儿中。由于缺乏指导哺乳动物腹侧折叠形态发生的信号通路和细胞群的知识，使得对这一发育过程背后的遗传学和细胞生物学的系统研究成为可能。这些研究直接基于我们最近的发现，即前内脏内胚层(AVE)是前条纹(PS)和早期条纹(ES)小鼠胚胎中明确定义的信号中心，在启动和指导前腹侧折叠形态发生方面发挥着核心作用。小鼠组织特异性突变分析表明，骨形态发生蛋白(BMP)途径是前腹折叠形态发生的关键调节因子。在原肠形成过程中，AVE信号向上胚衍生物表达BMP2，以协调Ventra形态发生的初始阶段；包括前肠内陷的形成和心脏尾部向头部的放置。我们的中心假设是，AVE表达的BMP2通过向一种或多种不同的上胚层来源的细胞类型：肠道内胚层、前神经外胚层和/或心脏中胚层发出信号，指导前肠内陷和头褶形态发生。一旦收到AVE衍生的BMP2信号，靶组织就会经历细胞行为的形态发生变化，这种变化协调了前肠凹陷的产生和位置的吻尾移位 头脑和心灵。三个具体目标将调查这一假说。目的1利用谱系特异性的Cre转基因基因和BMPR1a的条件等位基因来确定AVE表达的BMP2信号是否可以表达到确定的内胚层、外胚层和/或中胚层，以启动腹侧折叠的形态发生。目的2研究BMP2的VE特异性零等位基因是否与Gata4和Hgs/HRs的零等位基因存在遗传上的相互作用，这两个突变体也可破坏腹侧折叠的形态发生。Aim3使用BMP2-LacZ BAC报告转基因鼠系将靶向转录的顺式作用元件映射到近端AVE；这将使我们能够开发表达载体，用于体内研究近端AVE中BMP2表达群体的功能特性。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the genetic pathways and cellular mechanism that mediate ventral folding morphogenesis. Human embryos undergo ventral folding morphogenesis during the fourth week of gestation. Despite the different topologies of the epiblast in early human and mouse embryos (a flat disc in the former, a hollow cylinder in the latter), a number of key aspects of ventral folding morphogenesis are conserved: internalization of the gut, formation of a linear heart tube, closure of the ventral body wall, and encasement of the fetus in the amniotic membrane. Abnormalities in ventral morphogenesis underlie a number of birth defects associated with incomplete body wall closure (gastroschisis, development of intestines outside the abdominal wall; ectopia cordis, location of heart outside the thoracic cavity); such aberrations of ventral folding occur in ~ 1/2000 live births. An absence of knowledge about the signaling pathways and cell populations directing ventral folding morphogenesis in mammals has precluded systematic study of the genetics and cell biology underlying this developmental process. The proposed studies build directly on our recent finding that the Anterior Visceral Endoderm (AVE), a well defined signaling center in the pre-streak (PS) and Early Streak (ES) mouse embryo, plays a central role in initiating and directing anterior ventral folding morphogenesis. Tissue-specific mouse mutant analyses have shown that the Bone Morphogenetic Protein (BMP) pathway is a key regulator of anterior ventral folding morphogenesis. BMP2 expressed by the AVE signals to epiblast derivatives during gastrulation to orchestrate the initial stages of ventra morphogenesis; including formation of the foregut invagination and placement of the heart caudal to the head. Our central hypothesis is that AVE-expressed BMP2 directs foregut invagination and head fold morphogenesis by signaling to one or more distinct epiblast-derived cell types: gut endoderm, anterior neuroectoderm, and/or cardiac mesoderm. Upon receipt of the AVE-derived BMP2 signal, target tissues undergo morphogenetic changes in cell behaviors that coordinate the generation of foregut invagination with a rostral-caudal shift in the positions of head and heart. Three specific aims will investigate this hypothesis. Aim 1 uses lineage-specific Cre transgenes and a conditional allele of Bmpr1a to determine if AVE-expressed Bmp2 signals to definitive endoderm, ectoderm and/or mesoderm to initiate ventral folding morphogenesis. Aim 2 investigates whether a VE specific null allele of Bmp2 genetically interacts with null alleles of Gata4 and Hgs/Hrs, mutants known to also disrupt ventral folding morphogenesis. Aim3 maps cis-acting elements that target transcription to the proximal AVE using Bmp2-LacZ BAC reporter transgenic mouse lines; this will allow us to develop expression vectors for in vivo studies on the functional properties of the BMP2-expressing population in the proximal AVE.