OVOL2 is a critical regulator of ER71/ETV2 in generating FLK1⁺, hematopoietic, and endothelial cells from embryonic stem cells

It is widely accepted that blood cells are generated from 2 distinct cell populations during embryogenesis: hemangioblast and hemogenic endothelium.^1,2  The blood islands (BIs) in the extraembryonic yolk sac as early as embryonic day (E) 7.5 is the first site of hematopoiesis. Both blood and endothelial cells in the BIs are generated from the hemangioblast. On the other hand, the hemogenic endothelium, a specialized endothelial cell population within the aorta-gonad-mesonephros, generates hematopoietic stem cells, which can populate hematopoietic organs including bone marrow. Despite the unsettled controversy regarding the hemangioblast and the hemogenic endothelium, it is evident that the generation of FLK1(VEGFR2)⁺ cells in the developing embryos is an essential step for both hematopoiesis and vessel development.^3,4  In this regard, we and others have demonstrated that Er71/Etv2, a member of the ETS transcription factor family, is indispensable for specification of mesodermal precursors into endothelial and hematopoietic lineages during mouse embryogenesis.^5-8  ER71 activates genes critical for both hematopoiesis and vessel development including Flk1, VECadherin (VE-Cad), Scl, and Lmo2 through direct binding to gene-associated promoters/enhancers.^5,8-10  Studies in zebrafish and Xenopus also reported er71’s potent function,^11,12  indicating an evolutionally conserved and essential role for ER71 in the establishment of the cardiovascular system.

The specificity and function of the majority of ETS-domain transcription factors are dependent upon their interacting proteins.^13-15  However, very few studies have examined the role of ER71 interacting proteins.^9,16  In this study, we show that ER71 interacts with OVOL2, a C₂H₂ zinc finger (ZF) transcription factor essential for early embryogenesis,¹⁷  and that this interaction plays an important role in the generation of FLK1⁺, as well as endothelial and erythroid cells.

Full-length complementary DNA of FLAG-ER71, HA-OVOL2, or bi-cistronic FLAG-ER71-P2A-HA-OVOL2 in p2lox, respectively, was inserted into the tet-responsive locus of A2Lox ESCs with nucleofection (Lonza), followed by G418 selection and western blot analysis as described previously.^18,19 

GST or GST-ER71 incubated overnight with 1 mg of whole cell lysate of day (D) 3.5 embryoid bodies (EBs) was washed, and the resulting beads were then subjected to liquid chromatography/mass spectrometry (MS) analysis. For complete details, see the supplemental Methods on the Blood Web site.

In this study we investigated whether ER71 could interact with other regulatory proteins to regulate FLK1⁺ cell generation in mouse ESC differentiation. To this end, a GST-ER71 fusion protein was incubated with lysates prepared from D3-3.5 EBs, a time at which the expression of ER71 reached its peak.⁵  Pull-down fractions were then subjected to liquid chromatography-MS/MS proteomic analysis. Among candidates, priority was given to OVOL2 (Figure 1A and supplemental Table 1), a member of the ZF transcription factor family, because Ovol2-deficient mouse embryos showed several developmental defects, including abnormal vessel formation.^17,20  Subsequent pull-down assays between GST-ER71 and EB-cell lysates identified OVOL2 (Figure 1B). The interaction of ER71 and OVOL2 was further validated by coimmunoprecipitation in 293T cells (Figure 1C and supplemental Figure 1). Interestingly, OVOL2 did not interact with ETS1 or ETS2 (Figure 1C), which have been reported to activate the Flk1 promoter.^21,22  This finding indicates that the binding of OVOL2 is specific to ER71. We also confirmed the colocalization of ER71 and OVOL2 in the nucleus of 293T cells by immunostaining (Figure 1E and supplemental Figure 2). To further characterize the interaction between ER71 and OVOL2, a series of deletion mutant forms of OVOL2 (Figure 1A) were subjected to in vitro pull-down with the GST-ER71 fusion protein. As shown in Figure 1F-G, in vitro translated wild-type (WT) OVOL2 (full-length, WT) were precipitated with GST-ER71, indicating direct interaction. Interestingly, OVOL2 mutants lacking ZF domains, especially Δ6 and Δ8, showed reduced binding to GST-ER71. The pull-down experiment with a GST protein control failed to precipitate OVOL2 (supplemental Figure 3). In agreement with these results, a binding inhibition assay showed that the peptides corresponding to each ZF domain of OVOL2 efficiently inhibited binding between ER71 and OVOL2 (Figure 1H-I). Collectively, these results suggest that ER71 can directly bind with OVOL2 partly through the ZF domains.

As reported previously,⁵  the expression of Er71 reached its peak at D3, followed by a sharp decrease in ESC differentiation, whereas that of Ovol2 increased steadily up to D6 (Figure 2A). The Flk1 message was detectable after the induction of Ovol2 or Er71. Further, Ovol2, as well as Flk1 message, was enriched in ER71-VENUS⁺ cells and ER71-VENUS⁺FLK1⁺ from E8.5 mouse embryos, in which VENUS expression was controlled by the endogenous Er71 locus (Figure 2B and supplemental Figure 4).⁶  Taken together, with the finding that ER71 and OVOL2 are coexpressed in the BIs at E8.5 (Figure 2C), these results suggest a functional significance of ER71-OVOL2 interaction in regulating FLK1⁺ cell generation and differentiation. To further test this, we performed a luciferase-based promoter assay and found that coexpression of OVOL2 and ER71 doubled the Flk1 promoter activity compared with ER71 alone (Figure 2D). OVOL2 itself did not increase the transcriptional activity of the Flk1 promoter used in this assay. Next, we generated doxycycline (DOX) inducible ESCs expressing: 1) FLAG-tagged ER71 (iER71), 2) HA-tagged OVOL2 (iOVOL2), and 3) both FLAG-tagged ER71 and HA-tagged OVOL2 (iER71-P2A-OVOL2) (supplemental Figure 5).^18,19  We first confirmed the interaction between ER71 and OVOL2 in iER71-P2A-OVOL2 ESCs by coimmunoprecipitation (Figure 1D). Next, upon differentiation in a serum-free media,⁵  overexpression of ER71 significantly induced the generation of FLK1⁺ cells (Figure 2E). However, such de novo generation of FLK1⁺ cells was not observed in iOVOL2, iETS1, or iETS2 (Figure 2E and supplemental Figure 6). Consistent with the analysis of the Flk1 promoter (Figure 2D), the percentage of FLK1⁺ cells was higher in iER71-P2A-OVOL2 than in iER71 (73.4 ± 3.35% vs 50.2 ± 4.08%; Figure 2E-E’). We also found such a cooperative effect under differentiation conditions in the presence of serum (Figure 2F-F’). Interestingly, we found that the levels of ER71 were increased in cells overexpressing OVOL2-HA, which may also contribute to such a cooperative effect (supplemental Figure 7). Intriguingly, OVOL2 induction alone did not stimulate the Flk1 promoter nor did it generate FLK1⁺ cells in a serum-free differentiation condition (Figure 2E-E’), whereas in a serum condition, OVOL2 generated FLK1⁺ cells to a level comparable to that induced by ER71 (Figure 2F-F’). This indirectly suggests that OVOL2 might depend on other transcriptional regulators (eg, ER71) present in a serum condition to generate FLK1⁺ cells. Consistent with this, Er71 message was approximately fivefold higher in serum, compared with serum-free differentiation conditions (supplemental Figure 8). In a subsequent study, we found that the generation of endothelial cells (CD31⁺VE-Cad⁺) was increased by ER71-OVOL2 coexpression (11.3 ± 1.35%), compared with iER71 (6.1 ± 1.90%) or iOVOL2 ESCs (2.0 ± 1.69%) (Figure 2G-G’). Also, coexpression of ER71 and OVOL2 led to a further increase in the number of erythroid colonies, compared with iER71 or iOVOL2 (Figure 2H). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis (Figure 2I) showed that the expression level of the genes critical for endothelial cell development (Flk1, VE-Cad, Cd31, Mef2c, and Tie2)^5,9  were significantly higher when ER71 and OVOL2 were co-induced, compared with cells induced with either ER71 or OVOL2. Gata1, a critical gene for erythropoiesis,²³  was also upregulated in iER71-P2A-OVOL2, compared with iER71 or OVOL2. In contrast, both Islet1 and Gata4, critical genes for cardiac development,²⁴  were downregulated in iER71-P2A-OVOL2. Pdgfrβ and Rgs5 (smooth muscle cell markers) were further reduced in iER71-P2A-OVOL2 cells. MyoD and Myogenin (skeletal muscle markers) and Runx2 and Bglap (osteteoblast markers) were not significantly changed in the 3 cell lines. While other germ layer markers such as NeuroD and Pax6 (neuroectoderm) were similar or slightly enhanced upon coexpression of ER71 and OVOL2, Hinf4, Sox17, and Sox7 (endoderm) were similar in iER71 and iER71-P2A-OVOL2 cells and were reduced in iOVOL2 cells. The expressions of posterior primitive streak genes (T,Fgf8) were also not significantly changed in any of the cell lines.

To further determine whether ER71-OVOL2 interaction is functional in FLK1⁺ cell generation, we infected Ovol2 lentiviral short hairpin RNA (shRNA) particles into iER71 cells, and found that the FLK1⁺ cells induced by ER71 were significantly decreased in Ovol2 shRNAs-infected EBs compared with the Gfp shRNA-infected control (Figure 2J-K and supplemental Figure 9). Also, qRT-PCR analysis showed that Ovol2 knockdown led to a greater reduction of expression of the genes known to be induced by ER71 (Figure 2L). Other markers representing non-hemo/endo mesoderm derivatives (Isl1, Gata4, Pdgfrb, Rsg5, MyoD, Myogenin, Runx2, and Bglap) were slightly upregulated, or not affected in Ovol2 shRNA-infected EBs. While the expression of neuroectoderm markers (NeuroD and Pax6) appeared augmented in Ovol2 shRNA-infected EBs, messages of endodermal markers (Hinf4a and Sox17) were decreased. Thus, these results suggest that ER71-mediated FLK1⁺ cell generation is partly dependent upon OVOL2.

In summary, we demonstrate that ER71 interacts with OVOL2, and that such interaction cooperatively enhances the generation of FLK1⁺ cells, as well as endothelial and hematopoietic cells. A recent study reported that OVOL2 acting downstream of bone morphogenetic protein signaling can expand mesendoderm at the expense of neuroectoderm.²⁵  However, our analysis clearly showed that OVOL2 in a serum-free condition failed to induce FLK1⁺ cell generation, suggesting that Ovol2 is not sufficient for FLK1⁺ mesoderm formation. Rather, OVOL2, in collaboration with ER71, directly activated the FLK1 promoter activity and was required for optimal endothelial and hematopoietic cell generation without altering the formation of the mesoderm.

The authors thank Dr Novina for providing lentiviral packaging and envelope plasmids, and Emory+Children's Pediatric Research Flow Cytometry Core.

This study was supported by grants from the March of Dimes Foundation (5-FY12-44) (C.P.), the American Heart Association (11SDG7390074) (C.P.), National Institutes of Health, National Heart, Lung, and Blood Institute (HL119291) (C.P.) and (HL105732) (D.M.O), and the National Research Foundation Stem Cell Program, Republic of Korea (NRF-2014M3A9B4043056) (J.-I.C.).

Contribution: J.Y.K., R.H.L., and T.M.K., performed experiments and analyzed data; D.-W.K., Y.-J.J., S.-H.H., and S.-Y.O. performed experiments; M.K., H.K., K.C., and D.M.O. provided experimental materials; J.-I.C. conceived, designed research, analyzed data, and wrote the manuscript; C.P. conceived, designed, and performed experiments, analyzed and interpreted data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Current address for D.-W.K. is Department of Microbiology, Immunology, and Cancer Biology, School of Medicine, University of Virginia, Charlottesville, VA.

Correspondence: Jung-Il Chae, 664, 1Ga DeokJin, Jeonju 561-756, Republic of Korea; e-mail: jichae@jbnu.ac.kr; and Changwon Park, 2015 Uppergate Dr, #208B, Atlanta, GA 30322; e-mail: cpark23@emory.edu.