Abstract 897: Etsrp71 is a Novel Downstream Target of Nkx2–5 which Promotes Cardiac Morphogenesis
Ets-related protein71 (Etsrp71) is a novel transcription factor that has extensive conservation with pointed, a Drosophila ETS-domain factor that acts downstream of the GATA factor. Although transcriptional regulation of other ETS family members Ets1 and Ets2 in the developing chick heart have been implicated in epicardial migration and coronary vessel remodeling, transcriptional regulation or the biological function of Etsrp71 in embryogenesis and/or cardiogenesis is poorly understood. Here we show that the Etsrp71 is a novel downstream target of Nkx2–5. The homeodomain transcription factor, Nkx2–5, is one of the earliest markers of the cardiac lineage and mice lacking this transcription factor are nonviable and die early (~E9.5) during development. We have utilized transgenic technology to isolate and characterize cardiac progenitor cells (CPCs) from the developing mouse heart. Transcriptome analyses of CPCs from wildtype and age-matched Nkx2–5 null hearts were undertaken to identify potential Nkx2–5 downstream target genes. We observed that Etsrp71 was significantly downregulated in the Nkx2–5 deficient CPCs at E8.25 and E9.5 using transcriptome and RT-PCR analyses. We observed an evolutionarily conserved Nkx2–5 responsive element (NKE) in the Etsrp71 promoter and verified the binding of Nkx2–5 to this site by using electrophoretic mobility shift and ChIP assays. We also fused the 2kb upstream fragment of the Etsrp71 gene, which harbors the NKE to the luciferase reporter and undertook transcriptional assays in C2C12 myoblasts. We observed that Nkx2–5 activated the Etsrp71 gene in a dose-dependent fashion and mutation of the NKE significantly abrogated the transcriptional activation. To further confirm that Etsrp71 is an Nkx2–5 direct downstream target we engineered an Nkx2–5 inducible ES/EB system and have shown that upon induction, Nkx2–5 can upregulate the endogenous Etsrp71 expression and promote cardiogenesis. Our results support the notion that Etsrp71 acts downstream of the homeodomain transcription factor, Nkx2–5 and promotes cardiac morphogenesis in the developing embryo.