Function of GATA Factors in Cardiogenesis

描述（由申请人提供）：转录因子的加塔-4/5/6亚家族调节心脏发生的各个方面，从细胞特化到心管形态发生和瓣膜形成。该建议的假设是，加塔-4/5/6基因有助于在心脏发生的多个步骤中定义细胞和形态表型的组合密码。确定每个基因对心脏发生的相对贡献以及它们的相对表达模式如何通过上游调控程序建立将是重要的。需要创新的动物模型来克服目前进展的局限性。我们建议使用非洲爪蟾胚胎和新品系的转基因斑马鱼，以了解心脏发育过程中的加塔因子的功能和调节。以下目标将测试控制加塔-4/5/6的功能和调节机制的特异性： 1.定义加塔-4/5/6在早期心脏发生过程中的功能特异性。非洲爪蟾系统将被开发用于有效地抑制每个基因单独或组合的表达，在特定的胚层内的胚胎，并在特定的诱导试验在外植体。 2.确定调节因子在建立和维持加塔-4心源性表达模式中的作用。产生了在加塔-4调控序列控制下表达GFP的转基因斑马鱼的新品系。使用反向遗传学方法，转基因系统将被用来定义的顺式元件，所需的正常加塔-4模式的特定结构域的靶向表达，和T-盒因子的作用将被进一步研究，因为T-盒结合位点调节已经确定的上游增强子的活性。 3.鉴定控制加塔-4和加塔-6表达的新调控途径。这些关键调控基因的表达模式是重叠的，但不同的，因此可能是由一些共同的途径，但也有一些是特定的控制。候选途径将在我们的报道鱼的背景下进行测试，并将使用正向遗传筛选来鉴定控制加塔-4、加塔-6或两者表达的基因。

DESCRIPTION (provided by applicant): The GATA-4/5/6 subfamily of transcription factors regulates various aspects of cardiogenesis, from cell specification through heart tube morphogenesis and valve formation. The hypothesis of this proposal is that the GATA-4/5/6 genes contribute to a combinatorial code that defines cellular and morphological phenotypes during multiple steps of cardiogenesis. It will be important to determine the relative contribution of each gene to cardiogenesis, and how their relative expression patterns are established by upstream regulatory programs. Innovative animal models are needed to overcome limitations to current progress. We propose to use Xenopus embryos and novel lines of transgenic zebrafish to understand the function and regulation of GATA factors during heart development. The following aims will test the specificity of function and regulatory mechanisms controlling GATA-4/5/6: 1. Define the specificity of function for GATA-4/5/6 during early cardiogenesis. The Xenopus system will be exploited to effectively inhibit expression of each gene alone or in combination, within specific germ layers of embryos, and during specific induction assays in explants. 2. Define the role of regulatory factors to establish and maintain the GATA-4 cardiogenic expression pattern. Novel lines of transgenic zebrafish were generated that express GFP under the control of GATA-4 regulatory sequences. Using a reverse genetic approach the transgenic system will be used to define the cis-elements that are required to target expression to specific domains of the normal GATA-4 pattem, and the role of T-box factors will be further investigated since T-box binding sites regulate activity of an already identified upstream enhancer. 3. Identify novel regulatory pathways that control expression of GATA-4 and GATA-6. The expression patterns for these key regulatory genes are overlapping but distinct and are likely therefore to be controlled by some common pathways, but also some that arc specific. Candidate pathways will be tested in the context of our reporter fish and a forward genetic screen will be used to identify genes that control expression of GATA-4, GATA-6, or both.