Nkx2-5 transactivates the Ets-related protein 71 gene and specifies an endothelial/endocardial fate in the developing embryo

Abstract

Recent studies support the existence of a common progenitor for the cardiac and endothelial cell lineages, but the underlying transcriptional networks responsible for specification of these cell fates remain unclear. Here we demonstrated that Ets-related protein 71 (Etsrp71), a newly discovered ETS family transcription factor, was a novel downstream target of the homeodomain protein, Nkx2-5. Using genetic mouse models and molecular biological techniques, we demonstrated that Nkx2-5 binds to an evolutionarily conserved Nkx2-5 response element in the Etsrp71 promoter and induces the Etsrp71 gene expression in vitro and in vivo. Etsrp71 was transiently expressed in the endocardium/endothelium of the developing embryo (E7.75-E9.5) and was extinguished during the latter stages of development. Using a gene disruption strategy, we found that Etsrp71 mutant embryos lacked endocardial/endothelial lineages and were nonviable. Moreover, using transgenic technologies and transcriptional and chromatin immunoprecipitation (ChIP) assays, we further established that Tie2 is a direct downstream target of Etsrp71. Collectively, our results uncover a novel functional role for Nkx2-5 and define a transcriptional network that specifies an endocardial/endothelial fate in the developing heart and embryo.

Fig. 1.

Transient expression of Etsrp71 in the endocardial/endothelial lineage is downregulated in Nkx2–5 null cardiac progenitors. (A) Six-kilobase Nkx2–5-EYFP Tg:Nkx2–5± and Nkx2–5± mice were mated to generate EYFP⁺ Nkx2–5+/+ and EYFP⁺ Nkx2–5−/− littermates at E8.0 and E9.5. Note that the WT and Nkx2–5 mutant embryos are indistinguishable at E8.0 while at E9.5 the Nkx2–5−/− embryo has severe growth retardation compared to the WT littermate. (B) Semiquantitative RT-PCR in 6-kb Nxk2–5-EYFP Tg:Nkx2–5 null vs. 6-kb Nxk2–5-EYFP Tg:WT cardiac progenitors. Note decreased Etsrp71 transcript expression in the Nkx2.5−/− cardiac progenitors vs. the WT controls. (C) In situ hybridization techniques for Etsrp 71 expression at E8.5. The bright (Top) and dark (Bottom) fields of the transverse section of an E8.5 heart are shown and arrowheads indicate endocardium. Note an absence of Etsrp71 expression in the E11.5 embryo. (D) RT-PCR analysis of Etsrp71 expression in the developing heart and embryo. RNA was extracted at the indicated stages from the developing heart and embryo to analyze the expression of Etsrp71. ATP synthase (ATPSyn) was used as a loading control. (E) Coexpression of Etsrp71 and Nkx2–5 in endothelial progenitor cells of developing embryos. Tie2-GFP⁺ cells were isolated from developing embryos using FACS for semiquantitative RT-PCR (25 cycles). Actin was used as a loading control and was analyzed after 15 cycles.

Fig. 2.

Nkx2–5 is a transcriptional activator of Etsrp71. (A–C) Schematic of the construct used to generate an Etsrp71 transgenic mouse line. Within the developing transgenic heart, β-galactosidase expression was observed in endocardial precursors at E7.75–E8.5 and was extinguished at E11.5. (D and E) Etsrp71-LacZ Tg mice were mated with Nkx2–5± mice and their progeny analyzed. Transverse sections of the looping E8.5 heart from Nkx2–5+/+ and Nkx2–5−/− embryos are shown. Arrowheads denote the β-galactosidase-positive endothelium/endocardium. Note that β-galactosidase expression was severely attenuated in the Nkx2–5 null heart. (F) Alignment of the mouse and human Etsrp71 promoter sequence revealing conservation of the Nkx2–5 response element. (G) Single-strand sequence of the Etsrp71 promoter harboring the WT (shown in box) and mutant (Mut) NKE (shown in boldface type) were used as radiolabeled probes. Electrophoretic mobility shift assay (EMSA) revealed the formation of a stable Nkx2–5-DNA complex (lane 2) that could be competed with WT but not with Mut probe (lanes 2–4) and supershifted (lane 5). Note that heat denaturation (+HD) (control for the supershift assay) of the myc antibody is unable to supershift the complex (lane 6). (H) Schematic of the upstream promoter region of Etsrp71 containing the evolutionary conserved NKE (black bar). ChIP assay for in vivo binding of Nkx2–5 to the Etsrp71 promoter used the myc-tagged full-length (FL) and mutant (lack of DNA-binding domain, ΔHD) Nkx2–5. DNA purified before immunoprecipitation (IP) was used as input. Note IP of Etsrp71 promoter harboring the NKE only from cells expressing FL Nkx2–5. (I) Nkx2–5 transcriptionally activates Etsrp71 gene expression in C2C12 myoblasts. Overexpression of the CMV-myc-Nkx2–5 plasmid results in a dose-dependent activation of the 2-kb-Etsrp71-luc promoter construct. Mutation of NKE in the 2-kb Etsrp71 promoter significantly attenuated trancriptional activation by Nkx2–5 (n = 6 for each sample; data presented are mean ± SEM; *, P < 0.001). Fold change represents luciferase activity normalized to lacZ expression.

Fig. 3.

Etsrp71 is essential for cardiac morphogenesis and specification of the endocardial/endothelial lineage. (A) Schematic of the Etsrp71 gene and the targeted disruption. (B) (Top) Morphological appearance of E8.5 +/+ and Etsrp71 −/− embryo littermates. Note that the whole-mount WT and Etsrp71 mutant embryos are indistinguishable at E8.5. (Middle) Histological analyses of the WT and null embryo demonstrating an absence of the endocardial/endothelial lineage (End) and vasculature (PHV, primary heart vein; DA, dorsal aorta) in the null embryo compared to WT littermate. (Bottom) Immunohistochemical analysis of α-endomucin in the WT and Etsrp71 null embryo further demonstrating an absence of the endocardial/endothelial lineage (arrowhead shown in WT) and an absence of the vasculature in the null embryo. A higher magnification field reveals an absence of endocardium in the null heart compared to the WT littermate (α-endomucin expressing endocardium indicated by an arrowhead). (C) qRT-PCR analyses using purified RNA from individual Etsrp71 WT (black) and null (white) E8.5 hearts (performed in triplicate). Transcript level in the WT heart was considered to be 1 and Etsrp71 transcript was not detectable in the mutant heart (*). Note the significant downregulation of Flk1 transcripts and the essential absence of Tie2 transcripts in the Etsrp71 mutant heart (**, P < 0.001). Each assay was analyzed in triplicate and repeated twice.

Fig. 4.

Etsrp71 transcriptionally activates the Tie2 gene. (A) Tie2-lacZ Tg and Etsrp71± mice were mated to analyze β-galactosidase expression in WT (+/+) and Etsrp71 null (−/−) littermate embryos at the indicated developmental stages. Note the absence of β-galactosidase expression in the Etsrp71 null embryos. (B) Etsrp71 binds to the upstream promoter fragment of the Tie2 gene. Promoter occupancy of Etsrp71 in the upstream promoter fragment (UPF) and enhancer modules of the Tie2 gene was evaluated using a ChIP assay. Note the Etsrp71 binding to the UPF only (not the intronic enhancer of the Tie2 gene). (C) Etsrp71 transactivates Tie2 expression from UPF. Schematic of a 2.1-kb UPF fused to the luciferase (Luc) reporter is shown. Transcriptional assays in C2C12 myoblast cells reveal a dose-dependent induction of luc activity by Etsrp71 (black). Mutation of the Ets-binding site completely abolished the transcriptional activity (hatched) (*, P < 0.001). Each assay was analyzed in triplicate and repeated twice.