Mechanisms of Nural Crest Cell Development

描述（由申请人提供）：心脏流出道的形成是一个复杂的形态发生过程，其失败会导致各种临床上重要的先天性心脏畸形。我们发现Pbx基因家族编码的同源结构域转录因子是小鼠心脏流出道发育所必需的。Pbx基因突变导致永存动脉干、法洛四联症、主动脉瓣重叠和二叶主动脉瓣。这些缺陷类似于在鸡中观察到的神经嵴细胞消融时的心脏异常。我们假设Pbx基因对小鼠神经嵴细胞功能至关重要，并建议使用这些小鼠模型来研究Pbx 1基因如何调节心脏流出道发育所需的神经嵴细胞功能的机制。这些研究将为先天性心脏病的发生机制提供新的见解。具体而言，我们将研究（1）Pbx 1是否在神经嵴细胞内起细胞内在作用以调节其功能，（2）Pbx 1是否对神经嵴细胞的发育命运至关重要，以及（3）Pbx 1下游的分子通路在心脏发育中。目标一：确定心脏发育所需的Pbx 1作用的细胞位点我们将使用基因Cre-lox方法和在神经嵴细胞、心肌细胞和内皮细胞中表达Ore重组酶的几个小鼠品系来研究Pbx 1可能起作用以调节心脏神经嵴细胞发育的细胞位点。目的#2：确定Pbx 1在神经嵴细胞发育中的作用。 为了研究是否需要Pbx 1来指定心脏神经嵴细胞的发育身份，我们计划采用RNA原位杂交和菱形分化标记来研究Pbx 1-/-小鼠胚胎中的菱形身份。由于心脏神经嵴细胞起源于菱形体，因此菱形体标记物在PbxT 1 '胚胎中的表达将为心脏神经嵴细胞的命运特化提供见解。神经嵴细胞的其他标志物也将用于研究神经嵴细胞迁移出神经管时的分化。目标#3：定义受Pbx 1缺失影响的分子途径。 我们将采用标准的分子生物学和胚胎学方法研究Pax 3通路在心脏发育中是否位于Pbx 1的下游。

DESCRIPTION (provided by applicant): Formation of the cardiac outflow tract is a complex morphogenetic process, whose failure results in a variety of clinically important congenital heart malformations. We have found that the homeodomain transcription factors encoded by the Pbx gene family are required for development of the cardiac outflow tract in mice. Mutations in Pbx genes lead to persistent truncus arteriosus, tetralogy of Fallot, overriding aorta and bicuspid aortic valves. These defects resemble cardiac anomalies observed in chick when neural crest cells are ablated. We hypothesize that Pbx genes are essential for neural crest cell function in mice, and propose to use those mouse models to investigate the mechanisms of how Pbx1 gene regulates neural crest cell function required for cardiac outflow tract development. These studies will provide insights to the developmental mechanisms of congenital heart defects. Specifically, we will study (1) whether Pbx1 acts cell intrinsically within the neural crest cells to regulate their function, (2) whether Pbx1 is essential to program the developmental fates of neural crest cells and (3) the molecular pathways downstream of Pbx1 in cardiac development. Aim #1: Determine the cellular sites of Pbx1 action required for cardiac development We will use the genetic Cre-lox methods and several mouse lines that express Ore recombinase in neural crest cells, myocardial cells and endocardial cells to study the cellular sites where Pbx1 may function to regulate the development of cardiac neural crest cells. Aim #2: Determine the role of Pbx1 in neural crest cell development. To study if Pbx1 is required for specifying the developmental identities of cardiac neural crest cells, we plan to employ RNA in situ hybridization and rhombomeric differentiation markers to study the rhombomeric identities in Pbx1-/- mouse embryos. As cardiac neural crest cells originate from rhombomeres, expression of rhombomeric markers in the PbxT1' embryos will provide insights to the fate specification of cardiac neural crest cells. Additional markers of neural crest cells will also be used to study the differentiation of neural crest cells as they migrate out of the neural tube. Aim #3: Define the molecular pathways affected by the absence of Pbx1. We will use standard molecular biology and embryology methods to study if Pax3 pathway is downstream of Pbx1 in cardiac development.