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. Although it is well-established that transcriptional regulation is a key mechanism for establishing the coordinated and sequential expression of networks of genes whose products function together to execute a myriad of developmental programs during embryogenesis, the direct link between transcription factors and their target genes whose products orchestrate the numerous steps involved in organ formation remains poorly understood. Such biological processes include the determination of the correct numbers and types of cells that comprise an organ, as well as the proper differentiation, organization, morphogenesis and interactions of these cells in mature tissues. In a separate project, we assembled a detailed atlas of gene expression at cellular resolution across both spatial and temporal dimensions of heart development. To understand how this complex genetic network functions during cardiogenesis, we focused our further studies on the structure and activity of one subnetwork that we hypothesized acts early in heart formation under the direct control of two related genes, jumeau (jumu) and Checkpoint suppressor homolog (CHES-1-like), both of which encode Forkhead (Fkh) domain transcription factors (TFs) that are expressed in the initial heart-forming region of the embryo. RNAi-mediated knockdown of each of these Fkh proteins resulted in abnormal numbers and an uneven distribution of two distinct cell typescardial 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 in the mature heart revealed localized increases and decreases of CC and PC numbers in different regions of a given embryo that is homozygous mutant for either Fkh gene. 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, which was further accompanied by cell fate transformations. Fourth, abnormal karyokinesis occurred in some cells during cardiac progenitor cell divisions. Fifth, the defective asymmetric cell divisions associated with loss of Fkh function were found to be a consequence of failure of Numb protein localization in cardiac progenitors. Additional experiments enabled us to trace the molecular defect responsible for these abnormalities in heart development to the regulation by jumu and CHES-1-like of Polo, a kinase involved in multiple steps of mitosis. This conclusion was supported by multiple lines of evidence, including the finding that loss of polo function phenocopies the cardiac defects of jumu and CHES-1-like mutants, the occurrence of strong synergistic genetic interactions among polo, jumu and CHES-1-like, the failure of Polo protein to localize at centrosomes of dividing cardiac progenitors in jumu and CHES-1-like mutants, and the ability of either ubiquitously expressed or cardiac mesoderm-targeted polo to partially rescue both jumu and CHES-1-like mutant heart phenotypes. In contrast to the cardiac defects observed in single jumu and CHES-1-like mutants, double mutant embryos for these two Fkh TFs exhibited much more severe abnormalities in heart morphogenesis, a phenotype that could not be rescued by ectopic polo. These latter findings suggest that jumu and CHES-1-like control cardiac development by additional pathways that are independent of Polo-mediated processes. Collectively, this work exemplifies how studying the connectivity and functional relationships among upstream transcriptional regulators and their target effectors is crucial to obtaining an in depth view of how normal development occurs, and how mutations in key constituents of these pathways can lead to inherited abnormalities in the structures of organs such as the heart. To gain a more detailed understanding of how Fkh TFs regulate mesodermal gene expression, we conducted cis and trans studies of the involvement of multiple 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 the visceral musculature of late-stage embryos 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. Taken together, 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. A more detailed analysis revealed that the Fkh binding sites in this one enhancer represent two distinctively different DNA sequence specificities, thereby revealing an additional level of complexity for how a single gene is activated in multiple related but unique cell types of the developing embryo. Finally, a genome-wide computational scan demonstrated that both classes of Fkh binding sites are statistically over-represented in putative heart enhancers in combination with the motifs for five other known cardiac regulatory TFs, thereby providing evidence for the existence of a complex 7-way AND code of cis-regulatory factors that mediates the transcriptional control of a significant subset of heart genes.
| 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 |
