Cited 2 Action in Cardiac and Neural Development

描述(由申请人提供)：Cited2基因敲除小鼠的表型表明，Cited2，一种最近发现的转录激活因子，对圆锥干(心脏流出道)和脑神经管的形态发生至关重要。众所周知，这两种组织在发育过程中都处于缺氧状态。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-1α)竞争结合CBP/p300的CH1结构域，并负向调节低氧反应基因的表达。Cited2-/-胚胎中低氧诱导基因的上调支持这一假说。因此，在正常发育过程中经历最低氧分压的胚胎组织中，可能特别需要Cited2来调节缺氧反应。我们将在小鼠和鸡的胚胎中测试这一假说，我们主要关注心脏流出道(OFT)。目的1：确定正常发育所需的一种或多种细胞类型中Cited2的表达。我们将首先详细测定Cited2蛋白和mRNA在心脏和神经管形态发生关键阶段胚胎中的表达，并与缺氧和凋亡部位进行比较。我们将使用Cre-lox系统特异性地敲除神经脊细胞或心肌细胞中的Cited2，并分析其后果。目的2：利用体内系统验证Cited2需要与HIF-Ia竞争CBP/p300结合以下调心脏缺氧反应的假设。我们将减少Cited2的作用，特别是在禽类OFT的心肌细胞中，并观察其对形态发生和包括冠状动脉血管发育在内的缺氧反应的影响。我们将Cited2小鼠与HIF-Ia小鼠交叉，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心脏和神经的正常和异常发育提供关键信息，而且还将阐明体内转录因子和辅助因子之间的复杂相互作用。

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.