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 which when mutated are associated with 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. Additionally, synergistic genetic interactions showed that Myb, identified in the machine learning approach described in Project 1, functions in concert with both Jumu and CHES-1-like TFs to regulate two of the three types of Polo-dependent cardiac progenitor cell divisions. Our genetic studies further revealed that the two Fkh 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 that are 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 utilized both microarrays and RNA-seq to perform 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 analysis of these expression profiling results identified putative Fkh target genes based on significant enrichment of Gene Ontology (GO) attributes that are consistent with involvement in numerous cardiogenic processes, including those as diverse as the 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 also have been 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. We initially showed that CHES-1-like and jumu play a mutually redundant role in specifying the cardiac mesoderm since eliminating the functions of both Fkh genes in the same embryo results in defective hearts with missing hemisegments. Subsequent experiments involving putative Fkh target genes identified by expression profiling demonstrated that this process is mediated at the molecular level at least in part by the Fkh TFs regulating the fibroblast growth factor receptor Heartless (Htl) and the Wnt receptor Frizzled (Fz), with both of these signaling pathways known from previous studies to function in cardiac progenitor specification. In further support of this regulatory model, CHES-1-like and jumu exhibit synergistic genetic interactions with htl and fz in cardiac mesoderm specification. These findings imply that the Forkhead TFs and the identified signaling receptors function through the same genetic pathways such that this class of TFs transcriptionally activates the expression of both receptor-encoding genes. Furthermore, ectopic overexpression of either htl or fz in the entire mesoderm partially rescues the defective cardiac mesoderm specification phenotype seen in embryos doubly homozygous for mutations in jumu and CHES-1-like. When targeted exclusively to the cardiac mesoderm using even more restrictive Gal4 drivers to mediate Fkh gene RNAi, we were able to show that the cardiogenic functions of both CHES-1-like and jumu are autonomous to cells comprising the heart-forming region of the embryo. Taken together, these data demonstrate that functional redundancy of the Forkhead TFs leads to robustness in the specification of cardiac progenitors at least in part by regulating the expression of two well-known cardiogenic signaling pathway receptors. 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 |
