SPATIALLY /TEMPORALLY REGULATED GENES DURING HEART DEVELOPMENT

（改编自申请人的摘要）哺乳动物心脏形态发生涉及许多在分子水平上还知之甚少的基本过程，例如中胚层细胞向心源性细胞的定型、心源性中胚层融合成管状心脏、管状心脏的成环、瓣膜和隔膜的形成、腔室特化、心肌的小梁形成、血管发生、在发育中的心脏中具有空间和/或时间表达模式的基因预期在正常心脏的形成中起重要作用。 因此，该建议的最终目标是确定心脏发育过程中空间和时间调节基因的子集。 为了实现这一目标，研究人员建议生成一个全面的目录中表达的基因在发育过程中的大鼠心脏，并进行cDNA微阵列杂交实验，以确定一个子集的空间和/或时间调节基因。 本项目中鉴定的基因将根据其差异表达模式和/或定位于已知或推测包含先天性心脏病基因座的染色体区域进行选择，以进行进一步的功能分析（项目3和6）和人类遗传分析（项目1）。 更具体地说，研究人员建议（1）从发育中的大鼠心脏的三个区域生成单独标记的标准化文库：心房、心室和房室管，从胚胎发生的第12、14.5和17天获得，（2）鉴定一组30个，000个独特的大鼠心脏区域cDNA文库，研究人员开发的一种基于序列减法的策略，涉及标准化文库的连续减法，以最大限度地减少EST的冗余识别，同时增强稀有EST的代表性。转录本可能会错过更随机的方法，（3）构建小鼠心脏cDNA文库富集全长克隆，以促进功能和人类遗传分析的选择克隆进行项目1，3，和6，和（4）产生cDNA微阵列与独特的一套30，000个大鼠心脏cDNA，并与来自心脏不同区域和不同发育阶段的mRNA的荧光标记探针进行杂交实验，从而鉴定出在心脏发育过程中受空间和时间调控的基因子集。

(Adapted from the Applicant's Abstract) Mammalian cardiac morphogenesis involves a number of fundamental processes which are yet poorly understood at the molecular level, such as the commitment of mesodermic cells to cardiogenic cells, the fusion of cardiogenic mesoderm into a tubular heart, the rightward looping of tubular heart, the formation of valves and septa, the chamber specification, the trabeculation of myocardium, the vasculogenesis, etc. Genes with a spatial and/or temporal pattern of expression in the developing heart are anticipated to play an important role in the formation of a normal heart. Thus, the ultimate goal of this proposal is to identify a subset of spatially and temporally regulated genes during heart development. Towards this goal, the investigators propose to generate a comprehensive catalogue of genes expressed during development of the rat heart, and to conduct Cdna microarray hybridization experiments to identify a subset of spatially and/or temporally regulated genes. Genes identified in this project will be selected on the basis of their differential pattern of expression and/or localization to a chromosomal region known or presumed to encompass a congenital heart disease locus for further functional analysis (Projects 3 and 6) and human genetic analysis (Project 1). More specifically, the investigators propose (1) to generate individually tagged normalized libraries from three regions of the developing rat heart: atrium, ventriculum and atrioventricular canal, obtained from days 12, 14.5 and 17 of embryogenesis, (2) to identify a set of 30,000 unique rat cDNAs from these heart region libraries by an efficient EST- based strategy that the investigators have developed involving serial subtraction of normalized libraries to minimize redundant identification of ESTs while enhancing the representation of rarer transcripts likely to be missed in more random approaches, (3) to construct mouse heart cDNA libraries enriched for full-length clones to facilitate functional and human genetic analyses of selected clones to be performed in Projects 1, 3, and 6, and (4) to generate cDNA microarrays with the unique set of 30,000 rat heart cDNAs identified in this project and conduct hybridization experiments with fluorescently labeled probes derived from mRNAs isolated from different regions of the heart and obtained from different stages of development, thus identifying a subset of genes that are spatially and temporally regulated during heart development.