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. In a separate project, we used a computational approach to reveal that binding sites for Forkhead (Fkh) family transcription factors (TFs) are highly represented in enhancers having muscle founder cell (FC) and/or cardiac specificities. To further evaluate the functions of Fkh TFs in mesodermal gene regulation, we conducted cis and trans studies of the involvement of Fkh family members in controlling one particular enhancer whose activity can be monitored in multiple heart and muscle cell types. Three evolutionarily conserved Fkh motifs were mapped in this enhancer, and systematic mutagenesis revealed that each site participates differentially in the cell type-specific activity of this regulatory element. For example, blocking Fkh TF binding causes derepression in fusion-competent somatic myoblasts and cardiac cells that do not normally express the associated gene, whereas normal activity of the enhancer in visceral muscle is extinguished by ablating Fkh sites. We also used a combination of classical genetic and RNAi knockdown methods to determine which Fkh TFs regulate this enhancer in several of the relevant mesodermal cell types. Collectively, these results indicate that different tissue-specific Fkh TFs mediate distinct gene expression responses through combinations of the same binding sites in a single enhancer, and support a role for these factors in determining the unique genetic programs that characterize different subtypes of mesodermal cells. Having identified specific gene regulatory roles for Fkh TFs in different subsets of mesodermal cells, we also investigated the broader functions of two of these factors in development of the embryonic heart. RNAi knockdown of each of two Fkh proteins that are expressed in the cardiac mesoderm resulted in abnormal numbers and an uneven distribution of both cardial cells (CCs) and pericardial cells (PCs) in the fully differentiated heart tube. To further investigate this phenotype, we studied loss-of-function mutations in each of the corresponding Fkh genes, which revealed multiple classes of developmental defects. First, careful quantitation of cells revealed localized increases and decreases of CC and PC numbers. Second, misalignment of CCs frequently occured in apposed hemisegments on either side of the dorsal midline resulting in localized deformation of the heart tube. Third, the cardiac dysmorphology occurring in these mutants was found to be due to defects in both symmetric and asymmetric cardiac progenitor cell divisions accompanied by transformations of cell fate. Fourth, abnormal karyokinesis occurs in some cells during progenitor divisions. Fifth, the defective asymmetric cell divisions are a consequence of failure of Numb protein localization in cardiac progenitors. This latter observation, together with both the cell and nuclear division defects associated with each of the Fkh TF mutants, suggested that these genes might act through a pathway that converges on Polo, a key cell cycle kinase that is known to be required for the asymmetric localization of Numb. Consistent with this hypothesis, polo ranked highly in a genome-wide cardiac gene expression profiling study conducted in an independent project, and was found by in situ hybridization to be expressed in the cardiac mesoderm coincident with progenitor cell divisions. Most importantly, loss of polo function caused the identical heart phenotypes that occur in both of the Fkh gene mutants. As further evidence that these genes are involved in a common pathway, we found that the Fkh genes and polo all exhibit synergistic genetic interactions. Moreover, ectopic expression of polo is capable of partially rescuing the Fkh mutant heart phenotypes, strongly suggesting that polo acts downstream of Fkh TFs during the division of cardiac progenitor cells. Interestingly, the Fkh TF double mutant embryos exhibit more severe heart defects than either Fkh TF or polo single mutants, indicating that together these TFs likely regulate aspects of cardiogenesis other than those involving Polo-dependent cell division.

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National Heart, Lung, and Blood Institute
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Zhu, Xianmin; Ahmad, Shaad M; Aboukhalil, Anton et al. (2012) Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors. Development 139:1457-66
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