Regulation of Endothelial and Hematopoietic Development by the ETS Transcription Factor Etv2

REVIEW

CURRENT OPINION Regulation of endothelial and hematopoietic development by the ETS transcription factor Etv2

Kelly Lammerts van Bueren and Brian L. Black

Purpose of review Vasculogenesis and hematopoiesis are essential for development. Recently, the ETS domain transcription factor Etv2 has been identified as an essential regulator of vasculogenesis and hematopoiesis. Here, we review the recent studies that have established the critical role of Etv2 in the specification of mesoderm to blood and endothelial cells. Recent findings Loss and gain-of-function studies have demonstrated the conserved role of Etv2 in endothelial and hematopoietic development. Recent studies have placed Etv2 at or near the top of the hierarchy in specification of these lineages and have begun to dissect the upstream regulators and downstream effectors of Etv2 function using multiple model organisms and experimental systems. Summary Etv2 is essential for the specification of endothelial and hematopoietic lineages. Understanding the mechanisms through which Etv2 specifies endothelial and blood cells by defining upstream transcriptional regulators and cofactors will lead to greater insight into vasculogenesis and hematopoiesis, and may help to identify therapeutic targets to treat vascular disorders or to promote or inhibit vessel growth. Keywords endothelial, ER71, ETS, etsrp, Etv2, hematopoietic, mesoderm, vasculogenesis

INTRODUCTION colony-forming cell (BL-CFC), characterized by The vascular system is essential for embryogenesis expression of Brachyury and Flk1, which gives rise and is one of the first organ systems to develop. to both endothelial and hematopoietic cells and Blood vessels initially form through the process of represents the in vitro equivalent of the hemangio- vasculogenesis, which first occurs in the extra- blast [5,6]. Within the embryo, hemangioblasts have embryonic yolk sac and subsequently in the embryo been shown to arise within the posterior primitive proper [1]. After primitive vessels have formed, the streak [7], and recent studies established that heman- vascular plexus expands by endothelial sprouting gioblasts give rise to hematopoietic cells through a and intussusceptive vessel growth in a process hemogenic endothelial intermediate [8,9]. known as angiogenesis. Maturation of the vascular Several major signaling pathways, including network also involves recruitment of mural cells for vascular endothelial growth factor (VEGF), bone stabilization, and remodeling of blood vessels into morphogenetic protein (BMP), Notch, Wnt, and arteries, veins and capillaries [2,3]. fibroblast growth factor (FGF) are required for induc- During vasculogenesis, the initial formation of tion of endothelial and hematopoietic lineages [7], endothelial progenitors (angioblasts) from the and much has been learned about the nonauto- extra-embryonic mesoderm and hematopoietic cells nomous signals involved in vasculogenesis and also appear. These angioblasts and hematopoietic cells coalesce into blood islands, which fuse and Cardiovascular Research Institute and Department of Biochemistry and form the primitive vascular plexus [4]. The close Biophysics, University of California, San Francisco, California, USA appearance of hematopoietic cells and endothelial Correspondence to Brian L. Black, Cardiovascular Research Institute progenitors in the extraembryonic mesoderm led to and Department of Biochemistry and Biophysics, University of California, the hypothesis that they develop from a common San Francisco, CA 94158-2517, USA. Tel: +1 415 502 7628; e-mail: progenitor, the hemangioblast. In embryonic [email protected] stem cell differentiation experiments, researchers Curr Opin Hematol 2012, 19:199–205 identified a mesodermal precursor called the blast DOI:10.1097/MOH.0b013e3283523e07

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Recently, the essential role of Etv2 in endo- KEY POINTS thelial and hematopoietic development has been The ETS domain transcription factor Etv2 is essential for clearly demonstrated in multiple vertebrate species. endothelial and hematopoietic development. In contrast to other members of the ETS family, mouse knockouts for this gene die early in gestation Etv2 is sufficient to induce ectopic endothelial gene and fail to develop any endothelial or hemato- expression in zebrafish, frogs, and embryonic poietic cells [18,19]. Fli1 has been proposed to act stem cells. upstream of Etv2 [15], but other studies show that Etv2 cooperates with the Forkhead transcription factor Fli1 is downstream of Etv2 in mice and zebrafish FoxC2 to activate numerous endothelial genes through [20&,21,22]. Additionally, Etv2 overexpression is suf- a composite binding motif, the FOX:ETS motif. ficient to up-regulate fli1 expression in zebrafish Etv2 promotes endothelial and hematopoietic [23,24] (discussed below). Etv2-null mouse embry- þ development and inhibits cardiac development, onic stem cells fail to differentiate into Flk1 vas- possibly through an antagonistic relationship with the cular mesoderm from which endothelial and blood cardiac-restricted homeodomain protein Nkx2–5. cells are derived [20&]. Mutation or knockdown of the zebrafish or Xenopus orthologs of Etv2 also causes severe vascular and endocardial defects angiogenesis. By comparison, much less is known [22,25–27,28&,29&,30&&,31]. In contrast to the about the transcription factors involved in vascular requirement for Etv2 during early hematopoiesis development. Recent studies have identified the ETS in mice, only the myeloid lineage is affected in (E26 transformation specific) domain transcription zebrafish Etv2 mutants, whereas erythroid lineage factor Etv2 as an essential regulator of vasculogen- development occurs normally [22,25,26,30&&,31]. esis and hematopoiesis. In this review, we highlight However, Ren et al. [21] have shown that Etv2 is the role of Etv2 in the development of endothelial also required for the initiation of hematopoietic and blood lineages and discuss what has been stem cell (HSC) development from hemogenic learned about this transcription factor’s function, endothelium, demonstrating a requirement for lineage contributions, targets and cofactors. Etv2 in definitive hematopoiesis in zebrafish. In the mouse, definitive HSCs arise from hemo- THE ETS TRANSCRIPTION FACTOR Etv2 IS genic endothelium at E10.0 [32–34]. However, ESSENTIAL FOR ENDOTHELIAL AND Etv2/ mouse embryos die prior to this stage HEMATOPOIETIC DEVELOPMENT [18,19], precluding an analysis of hemogenic endo- Members of the ETS family of transcription factors thelium formation in Etv2 germline-null mice. play important roles in endothelial development Recent work by Lee et al. [35&] demonstrated that [1,10]. There are approximately 30 members of when Etv2 function is removed exclusively from the the ETS family in vertebrates [11]. All ETS proteins hematopoietic system in adult mice, there was a share a highly conserved winged helix–turn–helix significant decrease in HSCs. The authors of that ETS DNA-binding domain and bind to a consensus study found that Etv2 was required for maintenance GGAA/T-binding motif [11]. Many ETS proteins are of HSCs, that HSC progenitors isolated from Etv2/ expressed within hematopoietic and endothelial bone marrow exhibit reduced capacity to differen- cells and are important in the development of these tiate into granulocyte-monocyte progenitors, and tissues. For example, erg is essential for definitive that the number of myeloid lineage cells in the bone hematopoiesis in mice and is required for angio- marrow was significantly reduced [35&]. These genesis in zebrafish [12,13]. The ETS transcription results are consistent with the role of Etv2 in zebra- factor Etv6 controls angiogenic sprouting [14], and fish and Xenopus myeloid development. Thus, in Fli1 acts early in hemangioblast development by addition to its requirement for endothelial cell spec- functioning upstream of many early endothelial ification, Etv2 is also required for HSC development, and hematopoietic genes, including Flk1, Scl and differentiation and maintenance (Fig. 1), although Gata2 [15]. Loss of Fli1 function in Xenopus causes a further work needs to be done to clarify its role severe decrease in the number of hemangioblasts during mouse hematopoietic development. [15]. However, in mouse and zebrafish, loss of Fli1 does not result in severe vascular defects [15–17], suggesting that other ETS factors may compensate Etv2 IS SUFFICIENT TO INDUCE or that Fli1 is not absolutely required for hemangio- ENDOTHELIAL AND HEMATOPOIETIC blast specification. It is plausible that other ETS GENE EXPRESSION factors may function redundantly during embryo- In addition to the loss-of-function experiments des- genesis to compensate for the loss of Fli1. cribed above, which demonstrate the requirement

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Etv2 expression persists in early progenitors and in Primitive mesoderm Paraxial mesoderm newly formed endothelial cells, but expression Flk1+, PDGFRα+ Flk1−, PDGFRα+ rapidly fades and is essentially absent by E11.5 [18,19,20&,36&&]. In zebrafish, the Etv2 ortholog − etsrp is also expressed transiently within angioblasts Cardiac mesoderm Etv2 Flk1−, PDGFRα+, Nkx2-5+ and the early vasculature but is down-regulated as + blood vessels mature [22,37,38&]. Etv2 is enriched in embryonic stem cell-derived hemangioblast-like Etv2 BL-CFCs and is only briefly expressed during embry-

Endothelial/endocardial cells onic stem cell differentiation to mature endo- Vascular mesoderm thelium and hematopoietic lineages, similar to α− Flk1+, PDGFR the early and transient expression seen in mouse and zebrafish embryos [19,20&].

Hematopoietic cells The early expression of Etv2 in the mesoderm at sites where endothelial and hematopoietic progen- FIGURE 1. The role of Etv2 in mesodermal differentiation. itors arise, combined with the critical requirement Etv2 promotes the differentiation of primitive mesoderm into for Etv2 during development, suggests that Etv2 is vascular mesoderm and endothelial and hematopoietic expressed and functions in the progenitors that give lineages, while inhibiting differentiation into paraxial and rise to the mature vasculature and to the hemogenic cardiac mesoderm. endothelium from which hematopoietic stem cells arise. Indeed, recent lineage tracing studies using an Etv2 enhancer to direct expression of Cre recombi- of Etv2 for endothelial and hematopoietic develop- nase demonstrated that Etv2-expressing progenitors ment, gain-of-function experiments performed in contribute to the vasculature of the developing zebrafish and Xenopus embryos demonstrate that embryo and to hematopoietic cells including Etv2 is also sufficient to induce ectopic endothelial CD45þ nonerythroid and Ter119þ erythroid cells and myeloid marker expression [22–24,27,29&,31]. [36&&]. Similarly, Flk1þ, Etv2þ cells isolated from Overexpression of the ortholog of Etv2 in zebrafish E7.5 or E8.5 embryos are enriched for cells that embryos induces expression of endothelial markers differentiate into hematopoietic lineages, including flk1, scl and fli1 and the myeloid marker pu.1 Mac1þ, CD45þ granulocytes and Ter119þ erythroid [22–24,31]. Likewise, injection of Etv2 mRNA into cells [20&]. It is still unclear whether Etv2-expressing Xenopus embryos at an early stage into cells nor- progenitors give rise to the entire vasculature or to mally fated to be avascular endoderm results in what extent hematopoietic stem cells and hemato- ectopic induction of endothelial-specific markers poietic lineages are derived from Etv2-expressing flk1 and cd31 and the myeloid marker runx1 cells. A more detailed characterization of the line- [27,29&]. This activity of Etv2 is profoundly ages derived from Etv2þ cells is still needed to gain a enhanced by co-injection of mRNA encoding the complete understanding of the origins of the vas- Forkhead transcription factor FoxC2 ([27]; discussed cular system. below). Similarly, overexpression of Etv2 in embry- Rasmussen et al. [36&&] also described a contri- onic stem cells strongly induces the formation of bution of Etv2þ cells to other mesodermal compart- hematopoietic and endothelial progenitor cells, and ments, suggesting the possibility that Etv2 may play Etv2 expression is sufficient to rescue formation of additional roles outside of the cardiovascular sys- Flk1þ mesoderm after inhibition of BMP, Notch, and tem. These extra-mesodermal contributions by Wnt signaling [19]. Etv2-expressing cells need further investigation and confirmation using additional techniques, but raise the intriguing possibility of additional devel- EXPRESSION AND LINEAGE ANALYSES opmental roles for Etv2. DEMONSTRATE AN EARLY ROLE FOR Etv2 The differentiation of multipotent mesodermal IN ENDOTHELIAL AND HEMATOPOIETIC progenitors requires tight control of cell fate, which SPECIFICATION recent studies demonstrate is achieved through Etv2 is essential for normal hematopoietic and reciprocal control of cardiac versus hematopoietic endothelial development, but it is only expressed and endothelial fate (Fig. 1). In zebrafish, the car- transiently in early progenitors in the mesoderm. In diac-restricted homeodomain protein Nkx2–5 the mouse, Etv2 is expressed at the earliest site of [39,40] acts downstream of FGF signaling to restrict endothelial cell development within the extraem- hemangioblast fate, causing down-regulation of bryonic mesoderm in E7 mouse embryos [19,20&]. Etv2 and Scl, while promoting cardiac fate [41].

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Similarly, in mice, Nkx2–5 acts to repress mesoder- of zebrafish [30&&], suggesting that a distinct Etv2- mal differentiation to hematopoietic lineages dependent transcriptional program may function in and to promote cardiomyocyte development [42], the endocardium compared to the vascular endo- suggesting an antagonistic relationship between thelium. cardiomyocyte and endothelial/hematopoietic lin- Another interesting outcome of recent genome- eage commitment. In support of this notion, Etv2 wide transcriptional analyses performed in Etv2-null has recently been shown to modulate these meso- cells and embryos is the observation that expression dermal cell fate decisions in favor of hematopoietic of genes associated with other mesodermal lineages, and endothelial differentiation at the expense of the including the cardiac lineage, is increased in the cardiomyocyte lineage [30&&,36&&]. absence of Etv2, further supporting the notion that In Etv2-null mouse embryos, mesodermal cells Etv2 function promotes mesodermal specification normally fated to become endothelial and hemato- to the endothelial/hematopoietic lineage and poietic cells differentiate into cardiac and other represses cardiomyocyte specification [30&&,36&&]. mesodermal lineages [36&&]. These studies in mice This raises an interesting question as to whether are complemented by studies in zebrafish demon- cardiac genes are repressed directly or indirectly strating that loss of Etv2 causes down-regulation of by Etv2. Further work needs to be carried out to endocardial markers and expansion of myocardial determine whether Etv2 may act as a transcriptional gene expression [30&&]. Furthermore, induction of repressor, potentially through interactions with Etv2 both in embryonic stem cells and zebrafish cofactors. is sufficient to inhibit cardiac gene expression As an alternative approach for identification of [19,36&&]. Together, these observations emphasize Etv2 targets, our lab identified a novel FOX:ETS the importance of Etv2 in the specification and motif [27], which is strongly associated with endo- differentiation of endothelial and hematopoietic thelial genes and provides predictive power for cells from multipotent mesodermal progenitors, endothelial enhancers (Fig. 2). The FOX:ETS motif and suggest that it acts as a master regulator of is bound by Etv2 in combination with Forkhead mesoderm specification to the endothelial/hemato- transcription factors, including FoxC2 [27]. Etv2 poietic lineage, possibly in an antagonistic relation- and FoxC2 bind to the FOX:ETS motif and cooper- ship with the cardiac regulatory factor Nkx2–5 atively activate endothelial enhancers containing (Fig. 1). that motif (Fig. 2, [27]). Regardless of the experimental or bioinformatic methodology used, the identification of Etv2 targets TRANSCRIPTIONAL TARGETS OF Etv2 represents an important starting point for under- To better understand how Etv2 controls develop- standing the genetic networks and mechanisms ment of hematopoietic and endothelial lineages, it governing endothelial and hematopoietic develop- is essential to identify the direct targets of Etv2 ment. during early mesoderm development, and several studies have sought to identify Etv2 targets using genome-wide expression analyses. Microarrays have been carried out on zebrafish embryos and mouse ? Activation, chromatin modifications embryonic stem cells overexpressing Etv2 and on þ þ ?Post-translational Etv2::YFP, Flk1 , Pdgfra cells isolated from Etv2- modifications & && null mouse embryos [20 ,23,24,36 ]. A common Cofactor conclusion that can be drawn from these studies is the important role that Etv2 plays in inducing the Forkhead Etv2 expression of essential endothelial and hemato- FOX : ETS poietic genes. Some of the direct targets of Etv2, Endothelial which may mediate its downstream effects, include and hematopoietic genes Flk1, Scl, Fli1, Gata2 and Tie2 [18,19,20&,27,35&]. Importantly, all of these targets are required for FIGURE 2. A model for the synergistic activation of normal hematopoietic or vascular development endothelial and hematopoietic genes by Etv2 and Forkhead [1,43]. These microarray analyses also led to the transcription factors. Binding of Etv2 and Forkhead factors, identification of novel vascular and hemato- such as FoxC2, to the FOX:ETS motif could allow recruitment poietic-restricted genes [23,24]. Additionally, nfatc1, of cofactors which cause posttranslational modifications to which encodes a REL domain transcription factor Etv2 and Forkhead factors. Alternatively, cofactors could important in endocardial development, is also a induce histone modifications and alter DNA methylation to transcriptional target of Etv2 in the endocardium activate downstream gene targets.

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REGULATION OF Etv2: SIGNALING down-regulation of Etv2 and other hemangioblast PATHWAYS AND TRANSCRIPTIONAL markers in zebrafish and affects hematopoietic REGULATION development in embryonic stem cells [41,42]. As described in this review, Etv2 plays an essential In addition to transcriptional regulators, some role in endothelial and hematopoietic development of the signaling pathways that induce Etv2 expres- and numerous studies have suggested that it acts at sion have been identified. BMP, Notch and Wnt or near the top of the hierarchy in the specification signaling are required for induction of Etv2 expres- of those lineages [19,20&,30&&,36&&]. Furthermore, sion in BL-CFCs and the subsequent formation of Etv2 exhibits very dynamic and transient expression Flk1þ mesoderm and endothelial and hemato- during endothelial development ([18,19,20&,36&&]; poietic cell differentiation [19]. Importantly, other K.L. and B.L.B., unpublished observations). Thus, studies have demonstrated that Etv2 is not essential it is important to understand how Etv2 is activated for the generation of all Flk1þ mesoderm. Rather, in the early mesoderm. The rapid extinction of Etv2 Etv2 is essential for the generation of Flk1þ, Pdgfra expression and function also suggests a highly vascular mesoderm from primitive Flk1þ, Pdgfraþ regulated transcriptional shut off as well. Transcrip- mesoderm, and VEGF signaling was shown to be a tional regulators, post-translational modifications, robust inducer of Etv2 expression within primitive and epigenetic mechanisms appear likely to play mesoderm [20&]. The SHH–VEGF–Notch signaling major roles in Etv2 regulation. axis has also been reported to be important in the To understand how Etv2 is transcriptionally proliferation of Fli1þ, Etv2(etsrp)þ hematopoietic/ regulated during development, several groups have endothelial progenitors in zebrafish [37]. However, identified conserved Etv2 promoter and enhancer it is unclear whether this pathway is required for elements that are active at sites of endogenous Etv2 specification or only for proliferation of these expression, leading to the identification of trans- progenitors. acting factors that bind to and regulate Etv2 expression. Recently, Veldman and Lin [38&] identified a conserved cis-acting element in zebrafish Etv2 (etsrp) Regulation of Etv2 function by cofactors that directs expression to angioblasts and the devel- Transcription factors often interact with other oping vasculature. They showed that FoxC1a and proteins to increase target specificity or modulate FoxC1b bind to this enhancer region and are function. As previously mentioned, Etv2 interacts required for Etv2 expression in endothelial progen- with members of the Forkhead family, including itors [38&]. Although Etv2 expression is reduced, FoxC2, to synergistically activate endothelial- some expression is retained, suggesting that other specific gene expression [27]. Etv2 and FoxC2 act factors are required for the initial induction of Etv2. combinatorially to induce ectopic endothelial gene Furthermore, mouse Foxc1 and Foxc2 mutants do expression in Xenopus embryos, but the mechanism not exhibit as severe defects as Etv2 mutants [44], by which these two factors promote synergistic suggesting that critical upstream regulators are activation is still unknown. It is possible that still unknown. Etv2–FoxC2 interaction facilitates recruitment of A 3.9-kb promoter/enhancer element from the additional proteins that provide strong activation mouse Etv2 locus has also been identified. This or induce post-translational modifications. Interest- element directs expression to the developing vascu- ingly, Etv2 also interacts with the histone demethy- lature and endocardium [18]. Nkx2–5, which is a lase Jmjd1a to repress transcriptional activation of critical regulator of myocardial and endocardial the Etv2 target gene, Mmp1 [45]. Interestingly, development [39,40], acts upstream of Etv2 via the Jmjd1a alters the self-renewal of embryonic stem 3.9-kb promoter/enhancer in the endocardium [18]. cells through demethylation and positive regulation However, it is unclear whether Nkx2–5 regulates the of pluripotency-associated genes [46]. Thus, Jmjd1a expression of Etv2 within endothelial progenitors represents a good candidate for the modification of in the early mesoderm. Notably, the expression of Etv2 function during vasculogenesis, possibly by Etv2 precedes expression of Nkx2–5 in the early affecting the balance between progenitor main- mesoderm ([36&&]; K.L. and B.L.B., unpublished tenance and differentiation, but its effect on Etv2 observations), suggesting that factors other than targets during endothelial and hematopoietic devel- Nkx2–5 are likely to be responsible for the initial opment are unknown. activation of Etv2 in mesodermal progenitors. Chromatin modifications play an integral role Furthermore, loss of Nkx2–5 in mice or over- in the regulation of embryonic stem cell differen- expression in embryonic stem cells does not affect tiation to multiple lineages. Further studies should endothelial differentiation [42]. On the other determine how histones at the Etv2 locus are modi- hand, ectopic overexpression of Nkx2–5 causes fied during its induction and repression. Other ETS

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