Genome-wide expression profiling and phenotypic analysis of downstream targets identify the Fox transcription factor Jumeau as a master regulator of cardiac progenitor cell division

Forkhead box (Fox) transcription factors (TFs) mediate multiple conserved cardiogenic processes in both mammals and Drosophila. Our prior work identified the roles of two Drosophila Fox genes, jumeau (jumu) and Checkpoint suppressor 1-like (CHES-1-like), in cardiac progenitor cell specification and division, and in the proper positioning of cardiac cell subtypes. Fox TF binding sites are also significantly enriched in the enhancers of genes expressed in the heart, suggesting that these genes may play a core regulatory role in one or more of these cardiogenic processes. We identified downstream targets of Jumu by comparing transcriptional expression profiles of flow cytometry-sorted mesodermal cells from wild-type embryos and embryos completely lacking the jumu gene and found that genes with functional annotation and ontological features suggesting roles in cell division were overrepresented among Jumu targets. Phenotypic analysis of a subset of these targets identified 21 jumu-regulated genes that mediate cardiac progenitor cell division, one of which, Retinal Homeobox (Rx), was characterized in more detail. Finally, the observation that many of these 21 genes and/or their orthologs exhibit genetic or physical interactions among themselves indicates that Jumu is a master regulator acting as a hub of a cardiac progenitor cell division-mediating network. In order to identify the genes regulated by the Fox TFs Jumu and CHES-1-like, FACS was used to isolate mesodermal cells (GFP-positive cells) from Stage 11-early Stage 12 Drosophila embryos of the following six genotypes: 1: twi-GAL4 UAS-2EGFP/+ 2: twi-GAL4 Df(3R)Exel6157/UAS-2EGFP Df(3R)Exel6157 3: Df(1)CHES-1-like1; twi-GAL4 UAS-2EGFP/+ 4: Df(1)CHES-1-like1; twi-GAL4 Df(3R)Exel6157/UAS-2EGFP Df(3R)Exel6157 5: twi-GAL4 UAS-2EGFP/UAS-jumu 6: twi-GAL4 UAS-2EGFP/UAS-CHES-1-like These corresponded respectively to mesodermal cells that (1) were wild type, (2) were homozygous for a jumu null deficiency, (3) were homozygous for a CHES-1-like null deficiency, (4) were doubly homozygous for both the jumu and CHES-1-like null deficiencies, (5) overexpressed jumu, and (6) overexpressed CHES-1-like. Four biological replicates were obtained for each genotype. Gene expression profiling was performed using data obtained from RNA-seq for each replicate of these six genotypes. Comparative gene expression profiling analysis was then used to identify genes regulated by Jumu and/or CHES-1-like.