DIFFERENTIALLY EXPRESSED GENES IN CARDIAC MORPHOGENESIS

（改编自申请人的摘要）功能性哺乳动物心脏需要成功的分隔和瓣膜形成以形成四个腔室和完全分化的心肌。 长期目标是了解分隔、瓣膜发生和分化的潜在机制。 研究人员先前克隆了几个新的差异表达基因（DEG），包括Xin。 对Xin的初步研究表明，它可能在心脏循环中发挥重要作用。在几种小鼠模型中，不完整的成环与间隔错位/缺损有关。 因此，这些基因的小鼠同源物以及在SCOR项目2中克隆的其他基因将进一步表征，以了解它们在心脏形态发生中的作用，并可能揭示它们与房间隔、AV管和室间隔缺损病因学的相关性。 目的1是确定DEG的时空表达模式。 将对最初由项目2从发育中的大鼠心脏中克隆的DEG进行大规模的整体原位筛选。 这些DEG克隆将被优先考虑，并将对选定的克隆进行进一步表征。 例如，详细的原位杂交和RNA酶保护试验将用于确定Xin表达的早期，何时达到最大表达以及在哪里。同时，将分离全长小鼠克隆并用于蛋白质和抗体生产。将使用特异性抗体进行免疫荧光。 目的2是研究DEG蛋白的性质。 将搜索预测的蛋白质序列中的已知基序，并设计实验对其进行测试。 例如，在Xin中发现的核定位信号已显示出功能性。 一旦抗体可用，Xin的定位可以通过免疫荧光法进一步确定。 并对Xin的DNA结合和转录活性进行了初步研究。 将在Nkx2.5-、MEF 2C-或dHAND-敲除小鼠中研究Xin与成环控制途径中的其他基因的关系。 目的3是进行分析，以评估DEGs在心脏形态发生中的作用，包括反义研究，上皮间充质细胞转化，基因敲除和转基因小鼠。

(Adapted from the Applicant's Abstract) Functional mammalian hearts require successful septation and valvulogenesis to form four chambers and a fully differentiated myocardium. The long-term goal is to understand underlying mechanisms for septation, valvulogenesis and differentiation. The investigators previously cloned several novel, differentially-expressed genes (DEGs) including Xin. Initial studies on Xin reveal that it may play important roles in cardiac looping. Incomplete looping has been implicated in septal misalignment/defect in several mouse models. Thus, mouse homologs of these genes together with additional genes to be cloned in Project 2 of this SCOR will be further characterized to understand their roles in cardiac morphogenesis and possibly to reveal their relevance to the etiology of atrial septal, AV canal and ventricular septal defects. Aim 1 is to determine temporal and spatial expression patterns of DEGs. Large-scale, whole-mount in situ screening on DEGs, initially cloned by Project 2 from developing rat hearts, will be carried out. These DEG clones will be prioritized and selected clones will be further characterized. For example, detail in situ hybridization and RNASE protection assay will be used to determine how early the Xin expresses, when its expression reaches maximum and where it is. Simultaneously, full-length mouse clones will be isolated and used for protein and antibody productions. Immunofluorescence will be performed with specific antibody. Aim 2 is to examine the properties of DEG proteins. The predicted protein sequence will be searched for known motifs for which experiments will be designed to test. For example, nuclear localization signal found in Xin has been shown to be functional. Once antibody is available, localization of Xin can be further determined by immunofluorescence. Studies on DNA binding and transcriptional activities of Xin are also proposed. Relationship of Xin to other genes in the controlling pathway for looping will be studied in Nkx2.5-, MEF2C-, or dHAND-knockout mice. Aim 3 is to perform assays to assess roles of DEGs in cardiac morphogenesis, including antisense studies, epithelial-mesenchymal cell transformation, and knockout and transgenic mice.