SPATIALLY /TEMPORALLY REGULATED GENES DURING HEART DEVELOPMENT

哺乳动物心脏的形态发生涉及许多基本的过程，但在分子水平上尚不清楚，如中胚层细胞向心源性细胞的转化、心源性中胚层向管状心脏的融合、管状心脏的向右环、瓣膜和间隔的形成、腔室的规范、心肌的小梁形成、血管发生等。在发育中的心脏中具有空间和/或时间表达模式的基因预计在正常心脏的形成中发挥重要作用。因此，本提案的最终目标是确定心脏发育过程中空间和时间调控基因的子集。为了实现这一目标，研究人员建议建立一个大鼠心脏发育过程中表达基因的综合目录，并进行Cdna微阵列杂交实验，以鉴定空间和/或时间调控基因的子集。在本项目中鉴定的基因将根据其差异表达模式和/或定位于已知或推定包含先天性心脏病位点的染色体区域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，研究者建议(1)从发育中的大鼠心脏的三个区域生成单独标记的规范化文库：(2)通过一种有效的基于EST的策略，从这些心脏区域文库中鉴定出一组30,000个独特的大鼠cdna，该策略是研究人员开发的，包括对标准化文库进行连续减法，以最大限度地减少EST的冗余鉴定，同时增强在更随机的方法中可能遗漏的罕见转录本的代表。(3)构建全长克隆富集的小鼠心脏cDNA文库，以便对项目1、3和6中选定的克隆进行功能和人类遗传分析；(4)用本项目中鉴定的30,000个独特的大鼠心脏cDNA生成cDNA微阵列，并使用从心脏不同区域和不同发育阶段获得的mrna衍生的荧光标记探针进行杂交实验。从而确定了在心脏发育过程中受到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.