DIFFERENTIALLY EXPRESSED GENES IN CARDIAC MORPHOGENESIS

(改编自申请人的摘要)功能正常的哺乳动物心脏需要成功的隔膜和瓣膜形成，才能形成四个心室和一个完全分化的心肌。长期目标是了解间隔、瓣膜形成和分化的潜在机制。研究人员此前克隆了包括XIN在内的几个新的差异表达基因(Deg)。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，不完全环路与间隔不对齐/缺陷有关。因此，这些基因的小鼠同源基因以及将在本SCOR项目2中克隆的其他基因将被进一步鉴定，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、房室管和室间隔缺陷的病因学相关。目的1是确定DEGS的时间和空间表达模式。由Project 2最初从发育中的大鼠心脏克隆的DEGS将进行大规模、整体安装的原位筛选。将对这些DEG克隆进行优先排序，并对选定的克隆进行进一步表征。例如，详细的原位杂交和核糖核酸酶保护试验将被用来确定Xin表达的早期时间、何时达到最大值以及它在哪里表达。同时，将分离出全长的小鼠克隆，用于生产蛋白质和抗体。用特异性抗体进行免疫荧光检测。目的2是研究DEG蛋白的性质。将在预测的蛋白质序列中搜索已知的基序，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被证明是起作用的。一旦获得抗体，Xin的定位可以通过免疫荧光进一步确定。并对XIN的DNA结合和转录活性进行了研究。将在NKX2.5-、MEF2C-或D基因敲除小鼠中研究XIN与环路控制途径中其他基因的关系。目的3是评估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.