Abstract 1241: Nkx2–5 Regulates Cell Fate Decisions in the Developing Embryo
The cardiovascular system is the first functional system to be established during embryogenesis. The cardiovascular system consists of the heart, a network of vessels and circulating blood cells. These constituents derive from presumptive regions of the splanchnopleural mesoderm that are genetically defined and distinctly localized in the embryo. The formation of the cardiovascular system relies on signal crosstalk between endoderm and mesoderm to regulate specific gene expression. Among these genes, Nkx2–5 and Gata1 have essential roles in the genesis of the cardiovascular system. Gene disruption studies have established that Nkx2–5 mutant hearts have perturbed cardiac morphogenesis while Gata1 deficiency results in impaired hematopoiesis. However, the direct interaction between these two specific transcription factors has not been defined. We combinatorially mated the Nkx2–5-EYFP transgenic mice (which directs reporter expression in the cardiac progenitors) into the Nkx2–5 wild type and null backgrounds. Using FACS and transcriptome analyses at two distinct stages of development (E7.75, E8.25) we observed a significant induction of transcripts associated with the erythroid program (Gata1, eKlf, Nfe2, Edr, etc.) in the Nkx2–5-EYFP+ null vs. the Nkx2–5-EYFP+ WT cardiac precursors. The upregulation of the erythroid program in the Nkx2–5-EYFP+ null cardiac precursors was confirmed by QRT-PCR analysis. Using EMSA, mutagenesis and ChIP assays we demonstrate that Nkx2–5 binds to an evolutionary conserved NKE located in the 3.9 Kb promoter of the Gata1 gene. To further confirm that Gata1 is a Nkx2–5 downstream target gene, we engineered an Nkx2–5 inducible ES/EB system and have shown that, upon induction, Nkx2–5 downregulates the endogenous Gata1 transcript to suppress hematopoiesis (i.e. complete absence of CD45+ cells in the Nkx2–5 overexpressing EBs analyzed by FACS) without affecting vasculogenesis (i.e. CD31+ cells are equally detected in the Nkx2–5 induced vs. the noninduced EBs). In conclusion, our data support a previously undescribed role for Nkx2–5 as a transcriptional repressor of Gata1 expression and further emphasize the notion that Nkx2–5, in combination with other factors, modulate mesodermal cell fate during embryogenesis.