Two Forkhead transcription factors regulate cardiac progenitor specification by controlling the expression of receptors of the fibroblast growth factor and Wnt signaling pathways

Abstract

Cardiogenesis involves the coordinated regulation of multiple biological processes by a finite set of transcription factors (TFs). Here, we show that the Forkhead TFs Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu), which govern cardiac progenitor cell divisions by regulating Polo kinase activity, play an additional, mutually redundant role in specifying the cardiac mesoderm (CM) as eliminating the functions of both Forkhead genes in the same Drosophila embryo results in defective hearts with missing hemisegments. This process is mediated by the Forkhead TFs regulating the fibroblast growth factor receptor Heartless (Htl) and the Wnt receptor Frizzled (Fz): CHES-1-like and jumu exhibit synergistic genetic interactions with htl and fz in CM specification, thereby implying that they function through the same genetic pathways, and transcriptionally activate the expression of both receptor-encoding genes. Furthermore, ectopic overexpression of either htl or fz in the mesoderm partially rescues the defective CM specification phenotype in embryos lacking both Forkhead genes. Together, these data emphasize the functional redundancy that leads to robustness in the cardiac progenitor specification process, and illustrate the pleiotropic functions of Forkhead TFs in different aspects of cardiogenesis.

CHES-1-like, the ortholog of a non-obstructive azoospermia-associated gene, blocks germline stem cell differentiation by upregulating Dpp expression inDrosophilatestis

“…In flies, CHES-1-like, together with another fork head transcription factor Jumu, governs cardiac progenitor cell division and specification by regulating Polo kinase activity, as well as the expression of fibroblast growth factor and Wnt signaling pathway receptors [31, 32]. Inactivation of FOXN3 in Xenopus and mice led to craniofacial defects and was sometimes lethal [33, 34].…”

Three distinct mechanisms, Notch instructive, permissive, and independent, regulate the expression of two different pericardial genes to specify cardiac cell subtypes

“…Su(H) binding sites were identified in the Him and zfh1 enhancers by scanning their sequences for any matches to YGTGDGAA but not TGTGTGAA as reported previously [ 14 – 18 ]. Both wild-type enhancer regions (sequences in S1 and S2 Files ), as well as Him and zfh1 enhancer regions with single base-pair mutations at these binding sites to eliminate Su(H) binding ( S3 and S4 Files) [ 15 ], were synthesized in vitro (Integrated DNA Technologies, Coralville, IA, USA), sequence verified, and subcloned into pWattB-nlacZ [ 3 , 19 , 20 ]. Details of this cloning vector are presented in S1 Fig and its complete sequence is available from GenBank with the accession number MT747949 and in S6 File .…”

“…Whole embryo fluorescent immunohistochemistry followed standard protocols [ 3 , 20 ]. The following primary antibodies were used: rabbit anti-Mef2 (1:1000, gift from B. Paterson; 1:7500, gift from J. Jacobs) [ 29 , 30 ], guinea pig anti-Zfh1 (1:1000, gift from J. Skeath) [ 31 ], guinea pig anti-Eve (1:100, gift from D. Kosman and J. Reinitz) [ 32 ], rat anti-Odd (1:100, gift from D. Kosman and J. Reinitz) [ 32 ] and mouse anti-β–galactosidase (1:500, Promega, Madison, WI, USA).…”

Conserved signaling mechanisms in Drosophila heart development

“…One illustration of this involves the cells that will become specified as the cardiac mesoderm by the later FGFR‐mediated signaling process once mesoderm migration is completed (Michelson et al, ). These localized cells must continue to produce the FGFR Heartless, a feat mediated by a heartless enhancer driving expression specifically in the presumptive cardiac mesodermal cells (Ahmad et al, ).…”