The Etv2-miR-130a Network Regulates Mesodermal Specification

Abstract

MicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.

Keywords: Etv2; dicer; lineage specification; miR-130a; microRNA.

Figure 1. Etv2 modulates miR-130a expression in the endothelial progenitors

(A–F) Representative FACS profiles (A, D) and quantification (B, C, E, F) of mesodermal populations in Etv2-EYFP:Etv2-Cre;Dicer^L/L embryos at E7.5 from EYFP⁺ and EYFP⁻ compartments. (G) qPCR analysis of miR-130a in CD31⁺ cells sorted from Dicer^L/L and Etv2-Cre;Dicer^L/L embryos at E9.5. (H) Evolutionary conservation of the 5.0 kb upstream fragment of the miR-130a gene. (I) Top: Schematic of the 2.8 kb upstream region of the miR-130a promoter. Bottom: ChIP analysis of d4 Dox-inducible HA-Etv2 EBs using an HA antibody. ChIP assay for the Gapdh promoter (Control) and a non-specific locus (miR-130a 3′ UTR region: 3′ Flank) are shown as controls. (J) EMSA showing Etv2 bound to the Ets binding site in the miR-130a promoter region. (K) Luciferase reporter constructs using the miR-130a promoter (-1.0 kb) harboring wild-type (wt; open box) or mutant (mut; crossed box) Etv2 binding sites. (L) qPCR analysis of miR-130a using EYFP⁺ sorted cells from Etv2 wild-type and mutant embryos at E7.5. Error bars indicate SEM (n = 4; *p<0.05; **p < 0.005) (see also Figure S1).

Figure 2. miR-130a promotes the endothelial lineage

(A) Left panel: phase contrast images of wild-type E14 (ESCs) and Dox-inducible miR-130a ES cell (miR-130a iES) colonies. Right panel: qPCR analysis of miR-130a in the absence (−Dox) and presence of Dox (+Dox). (B) qPCR analyses for endothelial and hematopoietic markers using −Dox and +Dox EBs at d6 of differentiation (ratio shown as +Dox/−Dox). (C–F) FACS profiles (C, E) and quantification (D, F) of endothelial [Tie2 and CD31 (C, D)]; and hematopoietic [CD41 and CD45 (E, F)] markers in −Dox and +Dox conditions. (G–J) Representative FACS profiles (G, I) and quantification (H, J) of endothelial (G, H) and hematopoietic (I, J) markers in WT and miR130a^−/− EBs. (K) Sections of −Dox and +Dox miR-130a iES/EBs immunostained with CD31 (red) and cTnT (green) at d10 of EB differentiation. Boxed regions are shown in panel K′ and K″. (L) Quantitative analysis of cTnT⁺ EBs in −Dox (n = 123) and +Dox (n = 142) EBs. (M) Contractility assay using −Dox (n = 146) and +Dox (n = 135) EBs. (N) qPCR analyses of a cardiac marker (cTnT) from −Dox and +Dox miR-130a iES/EBs. (O) Cardiogenic assay using WT and miR-130a^−/− embryoid bodies. Error bars indicate SEM (n = 5; *p < 0.05). Scale bar: 200 μm, (see also Figure S2).

Figure 3. miR-130a targets Pdgfra and miR-130a-Pdgfra pathway modulates mesodermal progenitors

(A) ClustalW multiple sequence alignment of Pdgfra 3′ UTR and miR-130a. (B) Luciferase activity of Luc-Pdgfra-3′-UTR reporter constructs in the presence of miR-130a mimic and miR-130a antagomir. (C) Whole-mount in situ hybridization images of control and miR-130a morphants using pdgfra probes at 48 hpf. (D) qPCR analysis of pdgfra transcripts using fli1-EGFP⁺ sorted cells from control and miR-130a morphants at 48 hpf. (E–G) qPCR analysis of CD31, Tie2 and CD45 transcripts from control and Pdgfra neutralizing antibody-treated EBs. (H–K) Representative FACS profile (H, J) and quantification (I, K) of endothelial lineages (H, I) and hematopoietic lineages (J, K) in control and Pdgfra neutralizing antibody-treated EBs. (L–O) Representative FACS profile (L, N) and quantification (M, O) of endothelial lineages (L, M) and hematopoietic lineages (N, O) using d6 EB in control and Pdgfra over-expression conditions. Error bars indicate SEM (n = 5; *p < 0.05). Scale bar: 200 μm (see also Figure S3).

Figure 4. miR-130a targets Pdgfra in vivo and promotes lateral plate mesodermal lineage

(A–C) FACS profile (A) and quantification (B, C) of mesodermal populations in miR-130a iES/EB differentiation in −Dox and +Dox conditions. (D) qPCR analysis of Pdgfra in −Dox and +Dox conditions using d4 EBs. (E, F) FACS profile and quantification (F) of endothelial (Flk1 and CD31) markers during miR-130a iES/EB differentiation in −Dox and +Dox conditions. (G) Schematic of the experiment to determine the ability of miR-130a to re-specify mesoderm. (H, I) FACS profiles (H) and quantification (I) of endothelial markers at d6. (J, K) Hematopoietic colony forming assay from Flk1⁺/Pdgfra⁻ (J) and Flk⁺/Pdgfra⁺ (K) sorted cells using d3.5 miR-130a iES/EB system. Error bars indicate SEM (n = 3; *p < 0.05).