Transcriptional control of Drosophila heart development

果蝇心脏发育的转录控制

• 批准号: 7072342
• 负责人: MARTHA L BULYK
• 金额: $ 38.23万
• 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/7072342
• 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：了解调节心脏形成的遗传网络对于开发治疗儿童先天性心脏病和成人获得性心脏病的心脏再生的合理方法至关重要。鉴于人类和各种模式生物之间发育控制机制的完善保守性，可以从特别适用于后一种系统的遗传和基因组方法中获得相当多的见解。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，重点是个体调节因子的整合，以在不同细胞身份出现时产生组合特异性。该项目的具体目标是：（1）鉴定心脏细胞亚型中大量共表达基因，并表征它们对心脏发育的多种遗传扰动的表达反应； (2) 通过计算分析心脏基因表达的转录模型，并识别赋予细胞类型表达特异性的候选基序； （3）根据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控模型。来自果蝇胚胎心脏形成区域的细胞在用荧光蛋白进行靶向标记后，将通过流式细胞术进行纯化，并使用 DNA 微阵列对从这些细胞中分离的 RNA 进行表达谱分析。这些实验将使用野生型胚胎和通过信息获得和功能丧失基因操作进行修饰的胚胎，包括完全阻止或导致异位心脏形成的 GATA 因子 Pannier 突变。微阵列结果将通过广泛的胚胎原位杂交进行验证，从而鉴定出大量在心脏原基中转录并在成熟心脏细胞亚型中差异表达的基因。这些发现将与已知的心源性 TF 的现有信息相结合，以计算预测心脏特异性顺式调节模块和新的相关序列基序。此类预测将通过转基因报告基因检测进行验证，从而能够完善初始转录模型。总的来说，这些研究将提供对心脏发育背后的转录调控机制的详细了解。