Function of GATA Factors in Cardiogenesis

描述(由申请人提供)：GATA-4/5/6转录因子亚家族调节心脏发生的各个方面，从细胞规格到心管形态形成和瓣膜形成。这一建议的假设是，GATA-4/5/6基因参与了一个组合密码，该密码定义了心脏发生的多个步骤中的细胞和形态表型。重要的是要确定每个基因对心脏发生的相对贡献，以及上游调控程序如何建立它们的相对表达模式。需要创新的动物模型来克服当前进展的限制。我们建议使用非洲爪哇胚胎和转基因斑马鱼的新品系来了解GATA因子在心脏发育过程中的功能和调节。以下目标将检验控制GATA-4/5/6的功能和监管机制的特殊性： 1.明确GATA-4/5/6在心脏发生早期的功能特异性。非洲爪哇系统将被利用来有效地抑制每个基因单独或组合在胚胎的特定胚层中的表达，以及在外植体中的特定诱导试验中的表达。 2.明确调控因子在建立和维持GATA-4心源性表达模式中的作用。获得了受GATA-4调控序列控制表达绿色荧光蛋白的转基因斑马鱼新品系。利用反向遗传方法，转基因系统将被用于定义针对正常GATA-4模式的特定结构域进行表达所需的顺式元件，由于T-box结合位点调节已确定的上游增强子的活性，因此将进一步研究T-box因子的作用。 3.确定控制GATA-4和GATA-6表达的新的调控途径。这些关键调控基因的表达模式是重叠但不同的，因此可能受一些共同的途径控制，但也有一些是特定的。候选途径将在我们的记者FISH的背景下进行测试，正向遗传筛选将用于识别控制GATA-4和/或GATA-6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.