Abstract 784: Nkx2–5 Is Transcriptional Activator Of Etsrp71, Which Promotes An Endocardial Fate In The Developing Heart
The overall goal of our study was to decipher transcriptional networks that promoted cardiac morphogenesis. We generated an Nkx2–5 enhancer-EYFP transgenic mouse and utilized FACS analysis to isolate wildtype and Nkx2–5 null cardiac progenitors from single, age-matched embryos at distinct developmental stages (E7.75, E8.25 and E9.5). Transcriptome and QRT-PCR analyses were used to identify Ets-related protein71 (Etsrp71), a transcript that was significantly downregulated in the Nkx2–5 null cardiac progenitors. The biological function of Etsrp71 during cardiac morphogenesis is unknown. Using molecular and biochemical techniques, we established that Etsrp71 is a novel downstream target of Nkx2–5. We observed an evolutionarily conserved Nkx2–5 responsive element (NKE) in the Etsrp71 promoter and verified the binding of Nkx2–5 to this site using electrophoretic mobility shift, mutagenesis and ChIP assays. Using RT-PCR and in situ hybridization techniques, we further established that Etsrp71 is transiently expressed in the endocardium of the developing heart (E7.75-E9.5) and is extinguished during the latter stages of development and in the adult heart. These spatial and temporal expression results were further supported using transcriptional assays. 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. We further engineered an Etsrp71 inducible ES/EB system and using FACS, QRT-PCR and immunohistochemical techniques, we demonstrate that induction of Etsrp71 promotes an endocardial fate. Furthermore, overexpression of Etsrp71 in the ES/EB system resulted in significant induction of a number of putative downstream target genes including brachyury. These results further extend the Nkx2.5-Etsrp71-brachury transcriptional network. Our results support the hypothesis that Nkx2–5 transactivates Etsrp71, which promotes an endocardial fate in the developing embryo by regulating its downstream target genes.