Mechanisms of Neural Crest Cell Development

描述（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，其失败会导致多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基因突变可导致持续性动脉干、法洛四联症、覆盖主动脉和二尖瓣主动脉瓣。这些缺陷类似于在鸡神经嵴细胞消融时观察到的心脏异常。我们假设Pbx基因对小鼠神经嵴细胞功能至关重要，并建议利用这些小鼠模型研究Pbx1基因如何调节心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缺陷的发育机制提供新的见解。具体而言，我们将研究(1)Pbx1是否在神经嵴细胞内发挥内在作用，调节其功能；(2)Pbx1是否对神经嵴细胞的发育命运至关重要；(3)Pbx1下游在心脏发育中的分子途径。我们将使用遗传Cre-lox方法和几种在神经嵴细胞、心肌细胞和心内膜细胞中表达Ore重组酶的小鼠系来研究Pbx1可能调节心脏神经嵴细胞发育的细胞位点。目的2：确定Pbx1在神经嵴细胞发育中的作用。为了研究是否需要Pbx1来确定心脏神经嵴细胞的发育身份，我们计划采用RNA原位杂交和双形分化标记来研究Pbx1-/-小鼠胚胎的双形特征。由于心脏神经嵴细胞起源于菱形异构体，因此PbxT1胚胎中菱形异构体标记的表达将为心脏神经嵴细胞的命运规范提供见解。神经嵴细胞的其他标记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Pbx1缺失影响的分子途径。我们将使用标准的分子生物学和胚胎学方法来研究Pax3通路在心脏发育中是否位于Pbx1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.