The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. As a first step toward understanding the structure and function of a developmental regulatory network, the component parts must be identified and assembled into coherent groups. To accomplish this goal, we have undertaken extensive profiling of the genetic programs expressed by various subtypes of mesodermal cells using a combination of genome-wide and in situ hybridization approaches. The integrated strategy that we developed for identifying sets of co-expressed genes in various mesodermal cell types involves the following steps: (i) targeting green fluorescent protein (GFP) to the entire mesoderm or to subsets of mesodermal cells in both wild-type (WT) embryos and embryos from strains in which gain- and loss-of-function genetic manipulations perturb mesoderm development and gene expression patterns in predictable and informative ways;(ii) using flow cytometry to purify GFP-positive and negative cells from strains of each genotype;(iii) profiling levels of mRNA transcripts using Affymetrix GeneChips;(iv) identifying transcripts that are enriched in the WT mesoderm;(v) pooling the microarray data across genotypes to yield a compendium of expression profiles;(vi) performing a statistical meta-analysis of the combined datasets based on expected trends in gene expression and the relative contribution of each genotype to the detection of appropriate training sets of co-expressed genes;and (vii) validating the genome-wide data by whole-embryo in situ hybridization in both WT and selected mutant backgrounds, thereby obtaining an accurate spatiotemporal assessment of gene expression during development. Using this approach, we initially characterized large sets of genes having restricted expression in somatic muscle founder cells (FCs) and fusion-competnt myoblasts (FCMs). We then added microarray data from purified WT dorsal mesodermal cells, and performed independent meta-analyses for genes expressed in the cardiac mesoderm (CM). In situ hybridization of over 260 candidate CM genes revealed a total of 111 genes not previously known to be expressed in either the CM or differentiated heart. Temporal and spatial details of the in situ hybridization patterns were also highly informative, yielding 56 genes expressed in both the CM and mature heart, 24 genes expressed in the heart but not the CM, 27 genes expressed in the CM but not the heart, and numerous genes expressed in specific subsets of differentiated heart cells. We also targeted mesodermal overexpression of three FC identity homeodomain (FCI-HD) transcription factors (TFs), Slou, Msh and Eya, the latter being a Six4 co-activator whose ectopic expression serves as a surrogate for activity of this FCI-HD protein. We found that FC gene expression is differentially regulated in a cell-specific manner by HD TFs, and responsiveness correlates with TF co-expression in WT embryos. In addition, HD TFs activate genes uniquely expressed in FCMs, which do not normally contain these TFs, suggesting that FCI-HDs regulate two distinct temporal waves of myogenic gene expression, one in the developing FC and a second in the corresponding mature myotube. Collectively, these results provide an essential and substantive framework for our more recent, in-depth computational and experimental studies of both the structure and function of myogenic and cardiogenic transcriptional regulatory networks.
|Ahmad, Shaad M; Tansey, Terese R; Busser, Brian W et al. (2012) Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway. Dev Cell 23:97-111|
|Busser, Brian W; Shokri, Leila; Jaeger, Savina A et al. (2012) Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity. Development 139:1164-74|