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.
| Busser, Brian W; Bulyk, Martha L; Michelson, Alan M (2008) Toward a systems-level understanding of developmental regulatory networks. Curr Opin Genet Dev 18:521-9 |
