Cited 2 Action in Cardiac and Neural Development

描述（由申请人提供）：Cited2基因敲除小鼠表型表明，Cited2是一种最近发现的转录激活因子，对颈干（心流出道）和颅神经管的形态发生至关重要。这两种组织在发育过程中都是缺氧的。在体外系统中，Cited2已被证明能有效地与低氧诱导因子1- α (hif -1 α)竞争，结合到CBP/p300的CH1结构域，并负向调节低氧反应基因的表达。在Cited2-/-胚胎中，缺氧诱导基因的上调支持了这一假设。因此，在正常发育过程中经历最低氧张力的胚胎组织中，Cited2可能特别需要调节缺氧反应。我们将在小鼠和鸡胚胎中测试这一假设，我们的主要重点是心脏流出道（OFT）。目的1：确定哪种细胞类型或哪种细胞类型需要表达Cited2才能正常发育。我们将首先详细确定胚胎在心脏和神经管形态发生的关键阶段Cited2蛋白和mRNA的表达，并与缺氧和凋亡位点进行比较。我们将使用Cre-lox系统特异性敲除神经嵴细胞或心肌细胞中的Cited2，并分析其结果。目的2：使用体内系统验证Cited2需要与HIF-Ia竞争CBP/p300结合以下调心脏缺氧反应的假设。我们将在禽类OFT的心肌细胞中特异性地降低Cited2的作用，并观察其对形态发生和缺氧反应的影响，包括冠状血管的发育。我们将把Cited2小鼠与HIF-Ia小鼠杂交，以确定HIF-Ia基因剂量是否可以调节Cited2的作用，反之亦然。这些研究不仅将为我们理解正常和异常的心脏和神经发育提供关键信息，而且还将阐明体内转录因子和辅助因子之间复杂的相互作用。

DESCRIPTION (provided by applicant): The Cited2 knockout mouse phenotype indicates that Cited2, a recently discovered transcriptional activator, is critical for morphogenesis of the conotruncus (cardiac outflow tract) and the cranial neural tube. Both of these tissues are known to be hypoxic during their development. Cited2 has been shown in in vitro systems to effectively compete with hypoxia-inducible factor 1-alpha (HIF-1alpha) for binding to the CH1 domain of CBP/p300 and to negatively regulate hypoxia-responsive gene expression. Up regulation of hypoxia inducible genes in the Cited2-/- embryo supports this hypothesis. Thus, Cited2 may be especially required for modulating the hypoxia response in those embryonic tissues that experience the lowest oxygen tension during normal development. We will test this hypothesis in mouse and chicken embryos with our main focus on the cardiac outflow tract (OFT). Aim 1: Determine in which cell type or cell types Cited2 expression is required for normal development. We will first determine in detail the expression of the Cited2 protein and mRNA in the embryo at critical stages in heart and neural tube morphogenesis and compare to hypoxic and apoptotic sites. We will specifically knock out Cited2 in the neural crest cells or cardiomyocytes using the Cre-lox system and assay the consequences. Aim 2: Test using in vivo systems the hypothesis that Cited2 is required to compete with HIF-Ia for CBP/p300 binding to down-regulate the hypoxia response in the heart. We will reduce Cited2 action specifically in the cardiomyocytes of the avian OFT and observe the consequences to morphogenesis and the hypoxia response including coronary vascular development. We will cross the Cited2 mice with HIF-Ia mice to determine whether the HIF-Ia gene dosage can modulate Cited2 action and vice versa. These studies will not only provide information critical to our understanding of normal and abnormal heart and neural development but also elucidate the complex interplay between transcription factors and co-factors in vivo.