Transcriptional control of Drosophila heart development

果蝇心脏发育的转录控制

• 批准号: 6960214
• 负责人: ALAN M MICHELSON
• 金额: $ 39.15万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6960214
• 关键词: Drosophilidae; arthropod genetics; binding sites; biological signal transduction; cardiogenesis; developmental genetics; flow cytometry; gene expression; gene expression profiling; genetic regulation; genetic transcription; heart cell; in situ hybridization; invertebrate embryology; meta analysis; microarray technology; transcription factor

DESCRIPTION: Knowledge of the genetic networks regulating heart formation is essential for developing rational approaches to congenital heart disease in children and to cardiac regeneration for acquired heart disorders in adults. Given the well-established conservation of developmental control mechanisms between human and various model organisms, considerable insight can be derived from genetic and genomic methods that are uniquely applicable to the latter systems. The present proposal uses genome-wide strategies to dissect the intercellular signaling and intrinsic factors controlling gene expression during Drosophila embryonic heart development, with a focus on integration of individual regulators to generate combinatorial specificity in the emergence of distinct cellular identities. The specific aims of this project are: (1) to identify large sets of coexpressed genes in cardiac cell subtypes and to characterize their expression responses to multiple genetic perturbations of heart development; (2) to computationally analyze transcriptional models of cardiac gene expression and to identify candidate motifs that confer cell type expression specificity; and (3) to empirically evaluate the functional significance of predicted heart enhancers and binding site motifs in order to extend and refine regulatory models of cardiac gene expression. Cells from the heart-forming region of the Drosophila embryo will be purified by flow cytometry after targeted labeling with a fluorescent protein, and RNA isolated from these cells will be expression profiled using DNA microarrays. These experiments will be undertaken both with wild-type embryos and with embryos that are modified by informative gain- and loss-offunction genetic manipulations, including mutations in the GATA factor Pannier which either completely block or cause ectopic heart formation. Microarray results will be validated by extensive embryo in situ hybridizations, leading to the identification of large numbers of genes transcribed in the heart primordium and differentially expressed in mature heart cell subtypes. These findings will be combined with existing information for known cardiogenic TFs to computationally predict heart-specific cis regulatory modules and novel associated sequence motifs. Such predictions will be validated by transgenic reporter assays, enabling refinement of the initial transcriptional models. Collectively, these studies will provide a detailed understanding of the transcriptional regulatory mechanisms underlying cardiac development.

描述：调节心脏形成的遗传网络的知识对于开发儿童先天性心脏病的理性方法至关重要，以及成人心脏病的心脏再生。鉴于人类和各种模型生物之间对发展控制机制的良好保护，可以从遗传和基因组方法中得出相当大的见解，这些方法非常适用于后者系统。本提案使用全基因组策略来剖析果蝇胚胎心脏发育过程中控制基因表达的细胞间信号传导和内在因素，重点是整合单个调节剂，以在不同的细胞身份的出现中产生组合特异性。该项目的具体目的是：（1）确定心脏细胞亚型中的大量共表达基因，并表征其对心脏发育多种遗传扰动的表达反应； （2）计算分析心脏基因表达的转录模型，并确定赋予细胞类型表达特异性的候选基序； （3）凭经验评估预测的心脏增强子和结合位点基序的功能意义，以扩展和完善心脏基因表达的调节模型。用荧光蛋白靶向标记后，将通过流式细胞仪纯化来自果蝇胚胎心脏形成区域的细胞，并使用DNA微阵列表达从这些细胞中分离的RNA。这些实验将既有野生型胚胎，又可以通过通过信息性增益和损失官能办公遗传操作来修改的胚胎进行，包括GATA因子Pannier中的突变，这些突变完全阻止或引起异位心脏的形成。微阵列的结果将通过广泛的胚胎原位杂交来验证，从而鉴定出心脏原始中转录的大量基因并在成熟的心脏细胞亚型中差异表达。这些发现将与已知心源性TF的现有信息结合使用，以预测心脏特异性的CIS调节模块和新型相关序列基序。这些预测将通过转基因报告基因测定验证，从而使最初的转录模型进行完善。总的来说，这些研究将提供对心脏发展基础的转录调节机制的详细理解。