Cardiogenesis involves multiple biological processes acting in concert during development, coordination which is achieved via the regulation of diverse cardiac genes by a finite set of transcription factors (TFs). Forkhead (Fkh) TFs constitute one such family of regulators, with at least four Fkh TFs known to be required for proper cardiac development in mammals and three Fkh genes linked to human congenital heart defects. Our prior work demonstrated that two Drosophila Fkh genes, jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), determine cardiac cell fates, number and position by regulating a Polo-kinase pathway to mediate three distinct categories of cardiac progenitor cell divisions. Our genetic studies further revealed that these two genes also act in a partially redundant manner to regulate other, Polo-independent aspects of cardiogenesis. Finally, we found that Fkh TF binding sites were significantly enriched in combination with those of other known cardiogenic TFs in the enhancers of genes expressed in the heart. Collectively, these results suggested that these two Fkh TFs mediate multiple cardiogenic processes by regulating a large number of downstream effector genes. In order to identify these Fkh target genes and the distinct cardiogenic processes in which they are involved, we performed genome-wide gene expression profiling of flow cytometry-sorted cardiac mesodermal cells from wild-type embryos as well as from jumu and CHES-1-like gain- and loss-of-function embryos, including the double mutants. An initial analysis of these expression profiling-identified putative Fkh target genes based on significant enrichment of Gene Ontology (GO) attributes indicated that they are indeed involved in numerous cardiogenic processes, including those as diverse as initial specification of the cardiac mesoderm, establishment of the heart lumen, proper positioning of different heart cell types, sarcomere and myofibril formation, assembly of mitochondrial subunits and processes, and cardiac progenitor cell divisions. We are currently conducting genetic and RNAi-based functional investigations of these putative Fkh target genes in the heart in order to reveal the molecular basis of their cardiogenic activities. Of note, by combining classical genetic interaction and rescue assays with cis/trans-regulatory transgenic reporter studies, we demonstrated that the initial specification of the cardiac mesoderm is partly mediated by both Fkh genes regulating the expression of a Fibroblast growth factor receptor. Additionally, synergistic genetic interactions demonstrated that Myb, identified in the machine learning approach described in Project 1, functions in concert with both Jumu and CHES-1-like TFs to govern Polo-dependent cardiac progenitor cell divisions. Collectively, these studies are elucidating the molecular pathways used in multiple Fkh-mediated cardiogenic pathways, and are revealing how these cardiac subnetworks are orchestrated by a finite set of TFs.
| Ahmad, Shaad M; Busser, Brian W; Huang, Di et al. (2014) Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification. Development 141:878-88 |
| 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 |
| Busser, Brian W; Taher, Leila; Kim, Yongsok et al. (2012) A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis. PLoS Genet 8:e1002531 |
| 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 |
