DIFFERENTIALLY EXPRESSED GENES IN CARDIAC MORPHOGENESIS

（摘自申请人摘要）功能性哺乳动物的心脏需要成功的分离和瓣膜形成以形成四个腔室和完全分化的心肌。长期目标是了解分裂、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、差异表达的基因（DEGs），包括Xin。对Xin的初步研究表明，它可能在心脏循环中起重要作用。在一些小鼠模型中，不完全环与中隔错位/缺陷有关。因此，这些基因的小鼠同源物以及本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、房室管和室间隔缺陷病因学的相关性。目的1是确定deg的时空表达模式。将对项目2从发育中的大鼠心脏中克隆的deg进行大规模、全安装的原位筛选。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鉴定。例如，将使用详细的原位杂交和RNASE保护实验来确定Xin表达的时间、表达达到最大值的时间和位置。同时，全长小鼠克隆将被分离并用于蛋白质和抗体的生产。用特异性抗体进行免疫荧光检测。目的2是检查DEG蛋白的性质。预测的蛋白质序列将搜索已知的基序，以设计实验进行测试。例如，在Xin中发现的核定位信号已被证明是功能性的。一旦获得抗体，可以通过免疫荧光进一步确定Xin的定位。此外，还对Xin的DNA结合和转录活性进行了研究。我们将在Nkx2.5-、MEF2C-或dhand敲除小鼠中研究Xin与环化控制途径中其他基因的关系。目的3是评估deg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.