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。尽管早期人类和小鼠胚胎的上胚层具有不同的拓扑结构（前者是扁平的圆盘，后者是中空的圆柱体），但腹侧折叠形态发生的一些关键方面是保守的：肠道的内化，线性心管的形成，腹侧体壁的闭合以及羊膜中胎儿的包裹。腹侧形态发生异常是许多与体壁闭合不全相关的出生缺陷的基础（腹裂，肠在腹壁外发育;心脏异位，心脏位于胸腔外）;这种腹侧折叠异常发生在约1/2000例活产婴儿中。 缺乏有关哺乳动物腹侧折叠形态发生的信号通路和细胞群的知识，阻碍了对这一发育过程的遗传学和细胞生物学的系统研究。提出的研究直接建立在我们最近的发现，前内脏内胚层（AVE），一个明确的信号中心，在前条纹（PS）和早期条纹（ES）小鼠胚胎，起着核心作用，在启动和指导前腹折叠形态发生。组织特异性小鼠突变体分析表明，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。由AVE表达的BMP 2在原肠胚形成期间向上胚层衍生物发出信号，以协调腹形态发生的初始阶段;包括前肠内陷的形成和心脏在头部尾侧的放置。 我们的中心假设是AVE表达的BMP 2通过向一种或多种不同的外胚层来源的细胞类型（肠内胚层、前神经外胚层和/或心脏中胚层）发出信号来指导前肠内陷和头褶形态发生。在接收到AVE来源的BMP 2信号后，靶组织经历细胞行为的形态发生变化，其协调前肠内陷的产生和位置的头-尾移位 的头和心。三个具体目标将调查这一假设。目的1使用谱系特异性Cre转基因和Bmpr 1a的条件等位基因来确定AVE表达的Bmp 2是否向定形内胚层、外胚层和/或中胚层发出信号以启动腹侧折叠形态发生。目的2调查是否VE特定的无效等位基因Bmp 2基因与无效等位基因Gata 4和Hgs/Hrs，突变体也破坏腹折叠形态发生。Aim 3映射顺式作用元件，靶向转录到近端AVE使用Bmp 2-LacZ BAC报告基因转基因小鼠系;这将使我们能够开发表达载体，用于近端AVE中BMP 2表达群体的功能特性的体内研究。

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.