Transcription factors regulating vasculogenesis and angiogenesis

Abstract Transcription factors (TFs) play a crucial role in regulating the dynamic and precise patterns of gene expression required for the initial specification of endothelial cells (ECs), and during endothelial growth and differentiation. While sharing many core features, ECs can be highly heterogeneous. Differential gene expression between ECs is essential to pattern the hierarchical vascular network into arteries, veins and capillaries, to drive angiogenic growth of new vessels, and to direct specialization in response to local signals. Unlike many other cell types, ECs have no single master regulator, instead relying on differing combinations of a necessarily limited repertoire of TFs to achieve tight spatial and temporal activation and repression of gene expression. Here, we will discuss the cohort of TFs known to be involved in directing gene expression during different stages of mammalian vasculogenesis and angiogenesis, with a primary focus on development.


| INTRODUCTION
In the early mammalian embryo, endothelial cells (ECs) are formed de novo through a process known as vasculogenesis, canonically defined as the differentiation of specific mesodermal precursors into endothelial progenitor cells known as angioblasts.This may occur via multipotential blood islands, although the existence of a shared progenitor population for hematopoietic and endothelial lineages remains subject to debate.Hemato-endothelial progenitor cells can be detected in the mouse from embryonic days (E) 6.75-7.0,2][3][4][5][6] By E8.0 angioblasts have begun to form the first vascular structures, organizing into two parallel tracts to form the primitive paired dorsal aorta, and along the cardiac crescent as a precursor to the endocardium. 5Vein primordia form soon after at the yolk sac/embryo interface, presaging the future sinus venosus, and by E8.5 the first intra-embryonic veins coalesce and a primitive vascular plexus can be found throughout the embryo. 5nce the vascular plexus has been established, subsequent growth of blood vessels in the embryo and in the adult primarily occurs via angiogenesis.While angiogenesis by definition refers to the formation of blood vessels from existing ones via any mechanism, the best described and apparently most abundant form of angiogeniesis is sprouting angiogenesis.8][9] In turn, tip cells use DLL4/JAG-Notch signaling to suppress migratory behavior in neighboring ECs, which instead become highly proliferative stalk cells, enabling vessel elongation and lumen formation. 10Further, tip cells with high Notch activity can also migrate toward, and differentiate into, arterial ECs. 9 In addition to VEGF-VEGFR2, CXCL12-CXCR4, and NOTCH signaling, multiple additional growth factors and signaling pathways provide both positive and negative regulatory inputs to control angiogenesis, as do cell adhesion interactions with the extracellular matrix.Thus sprouting angiogenesis allows a vascular bed to expand in response to hypoxia and insufficient nutrients, both during development and during physiological processes in the adult such as wound healing and changes in the lining of the endometrium.Pathological angiogenesis can also contribute to and aid the development of conditions such as cancer, atherosclerosis, and age-related macular degeneration.
The transition from mesodermal progenitors into a differentiated, functional endothelium depends on the activity of lineage-defining genes.Similarly, the coordinated response of ECs to the complex signals directing sprouting angiogenesis is dependent on the tight spatiotemporal expression and repression of specific cohorts of genes.Transcription factors (TFs) play a key role in this dynamic gene regulation.These DNA-binding proteins influence transcription by binding to specific motifs within cis-regulatory elements (enhancers and promoters), in combinations that often include both lineagespecifying factors (e.g.ETS factors for ECs) and transcriptional effectors of different signaling pathways (e.g., RBPJ for Notch, SMADs for TGFβ/BMP).In this way, multiple different environmental cues can collectively influence enhancer/promoter activity and subsequent gene expression. 11,12While most TFs have a defined "consensus" DNA binding motif, usually established using either ChIP-seq (chromatin immunoprecipitation combined with sequencing) or HT-SELEX (high-throughput systematic evolution of ligands by exponential enrichment), TFs are also often able to bind slightly alternative (nonconsensus) sequences, a process that can be influenced by other bound or accessory proteins and that likely also contributes to the differing patterns of gene activation downstream of different enhancers.In this review, we aim to summarize what is currently understood about the complex network of TFs that orchestrate the formation of a functional circulatory network in the mammalian embryo through the processes of vasculogenesis and sprouting angiogenesis.In addition, we provide three tables covering specific aspects of this regulation, including the known binding motifs for each endothelial TF (Table 1), the expression patterns of each TF during mammalian embryonic development and available ChIP-seq data sets (Table 2), and a detailed assessment of all in vivo-characterized mammalian endothelial enhancers and their cognate binding TFs (Table 3).
This review will focus on data from mouse models of vasculogenesis and angiogenesis, which predominantly involves analysis of vascular development during embryonic and early postnatal growth.Vasculogenesis research focuses primarily on the E7.5-E9.5 embryo, whereas a variety of later embryonic timepoints have been used to study angiogenesis (with the E10-E12 hindbrain providing a useful angiogenic-only vascular bed 7,96 ).Additionally, the post-natal retina (from post-natal days (P)4-7) has been a favored model for many angiogenic studies, 96,97 while angiogenesis can also be studied in the adult after insertion of Matrigel plug or tumor cells.These models are often used in combination with different Cre-driver transgenes to delete floxed genes-of-interest, permitting endothelial specific (e.g., Tie2-Cre), induced (e.g., RosaCreER) or induced endothelial specific (e.g., CDH5-CreERT2 and PDGFB-iCreERT2) patterns of gene deletion (reviewed by 98), although constitutive gene deletion has also been a useful approach for those genes largely specific to ECs.Because the efficacy of Cre and CreERT2 transgenes can vary considerably (both between different types of drivers, and between different versions of the same Cre transgene 98 ), and tamoxifen administration alongside Cre can itself result in endothelial phenotypes, 99 analysis of results should always consider the Cre driver utilized, the evidence of gene knockdown provided and the quality of controls.In vitro models can also provide powerful information (e.g., analysis of embryonic stem cell differentiation to study vasculogenesis 100 ; angiogenesis assays using cultured ECs to investigate sprouting from EC spheroids 101 ) and are included alongside animal studies where relevant.

| ETS
ETS proteins comprise a large family of TFs characterized by a common DNA-binding domain (the ETS domain) which mediates binding to a core GGA A / T motif (reviewed by 102 and summarised in Table 1).While ETS factors are not specific to ECs and contribute to the differentiation of other cell types (e.g., PU.1 in hematopoiesis, 103 ELK1 in neurogenesis, 104 see Table 2), it is increasingly appreciated that ETS factors play a key role in EC identity.In particular, ETS binding motifs are a defining and essential feature of all known endothelialexpressed promoters and enhancers (e.g., 45,48,57,58,76  and Table 3).However, analysis of the direct role(s) of ETS TFs within the vasculature has been complicated by the sheer number of ETS family members expressed in ECs.Adult ECs express at least 13 different ETS factor genes (Ets1, Ets2, Erg, Fli1, Etv3, Etv6/Tel, Elk1, Elk3/Net, Elk4/Sap1, Elf1, Elf2/Nerf, Elf4, and Gabpa), although consistent high expression across adult aorta, lung and brain ECs is principally restricted to Ets1, Ets2, Erg, Fli1, Elk3, Elf1, Elf2, and Gabpa, the latter three of which are also highly expressed in many other cell types. 102,105,106f these adult ETS, all but Elk1 are also expressed in angioblasts in E8.0 embryos. 2 Additionally, Etv2 (Etsrp71) is strongly expressed in hemato-endothelial progenitors in the early mouse embryo, 107 although little expression is reported after mid-gestation.Similarly, Fev, Etv4, Etv5, and Erf are expressed just after Etv2 in the same progenitor population, 2,108 although none are strongly expressed in adult ECs.Consequently, analysis of phenotypes after deletion of a single ETS factor must always consider the potential for functional redundancy and compensation.Nonetheless, gene deletional studies in both mouse and zebrafish have repeatedly found significant endothelial defects downstream of ETS factor deletion, strongly supporting a key role for ETS factors in the regulation of vasculogenesis and angiogenesis. 102,109It is, however, unlikely that ETS factors regulate their gene targets alone: gene enhancers with entirely different patterns of EC expression (e.g., arterial-specific, angiogenic-specific, and vein-specific) can all be robustly bound and activated by ETS factors (e.g., 32,48-51,57,69,85; see also Table 3) indicating that ETS-driven regulation of many aspects of endothelial behavior is likely to involve combinatorial interaction with other families of TFs to achieve the required spatio-temporal specificity.

| ETV2
Of all the ETS TFs investigated in endothelial knock-out mouse models, only loss of ETV2 (ETSRP71/ER71) ablates vasculogenesis, resulting in a complete absence of both endothelial and blood cell lineages and subsequent lethality by E9.0. 61,107Conversely, prolonged activation of Etv2 leads to an overly dense capillary bed, hemorrhage, and an absence of hematopoietic cell differentiation. 110redating these discoveries, knockdown of the zebrafish orthologue, etsrp/etv2, was also shown to result in cessation of embryonic circulation. 109,111tv2 is expressed within a narrow time window during mouse development between E6.75 and E9.5 (Table 2), a transience that is crucial for endothelial maturation.It is first observed in a subset of cells in the posterolateral mesoderm at E6.75-7.0, 2,108,112with the strongest Etv2 expression occurring in hematoendothelial progenitor populations rather than    differentiated ECs by E8.5. 2,19,113While it is possible that the uniquely early expression pattern of Etv2 contributes to the severe consequences of deletion, overexpression of Fli1, Erg and Ets1 in ETV2-null embryoid body culture systems could not fully rescue the formation of the endothelial lineage. 1 Alternatively, the importance of ETV2 in the specification of ECs may, at least in part, be attributed to its crucial role in regulating the expression of Flk1 (also known as Kdr and encoding the VEGFR2 receptor).In the mouse embryo, loss of ETV2 markedly reduces the number of VEGFR2+ cells, 114 likely due to direct regulation of Flk1 through ETV2 binding at Flk1 enhancer elements 69,115,116 (Table 3).While not all Flk1 expression is lost, those VEGFR2+ cells that persist have a cardiac rather than EC lineage. 117This is supported by studies in embryonic stem cell cultures, which also found that cells differentiate into cardiac lineages in the absence of ETV2. 112,114,118ETV2 is therefore not essential for the expression of Flk1 per se, but instead up-regulates expression levels to create a subpopulation of high VEGFR2+ cells driven towards an endothelial fate. 1 VEGFA signaling through VEGFR2 is a potent activator of MAPK/ERK signaling (reviewed in 119), which in turn phosphorylates ETS factors and increases their binding affinity (reviewed in 120,121).Therefore, VEGFA-VEGFR2 signaling enhances Flk1 gene transcription through an ETV2-dependent positive feedback loop.
In addition to Flk1, ETV2 is implicated in the direct activation of many other key endothelial lineage specifying genes, including Cdh5, Tek, Tal1(SCL), Notch4, Nfatc1, and Sox7, 45,114,122,123 often but not always in combination with Forkhead TFs (see below and Table 3).Additionally, ETV2 can play a role in the activation of other endothelial ETS factors, including Fli1, Ets1, Ets2, Erg, and Fev. 1,108It has been hypothesized that ETV2 may act as a pioneer factor, creating an endothelial lineage-specific epigenetic landscape. 124Supporting this, ETV2 binding can directly result in demethylation of its binding sites, and this hypomethylation can be maintained in blood and vascular systems as an epigenetic memory. 1,125Further, this role may be facilitated by complexing with TET1/TET2 enzymes and thereby directly promoting locus-specific reversal of methylation marks. 126ChIP-seq data show that binding sites at regulatory elements previously occupied by ETV2 become occupied by other ETS factors at later stages of development. 1Cultured amniotic cells can also transition into immature ECs through transient Etv2 expression, while co-expression with Fli1 and Erg1 is required for maturation. 118It is therefore clear that ETV2 plays a unique role in establishing EC fate during vasculogenesis, both specifying the endothelial epigenetic landscape and directly establishing the expression of lineage-specifying genes that will maintain EC fate once ETV2 itself is gone.

| FLI1
FLI1 is expressed in hemato-endothelial progenitors soon after ETV2 and remains highly expressed in ECs throughout development (see Table 2).However, unlike Etv2, constitutive deletion of Fli1 in mice does not affect vasculogenesis, although Fli1 null mice die at E11 due to cerebral hemorrhage and loss of vessel integrity.Instead, FLI1 may play a role in the differentiation of angioblasts into a functional vascular network, 127,128 and has been implicated in the direct regulation of many genes involved in maintaining vascular homeostasis (including Cdh5, Cd31, Col4a1, Mmp9, Pdgfb, and S1pr1). 129However, endothelial-specific deletion of Fli1 mediated by Tie2-Cre results in a milder phenotype and mice are born at expected mendelian ratios. 129While this could indicate an EC-independent role for FLI1 in vascular assembly, it is also possible that critical EC functions of FLI1 occur before the onset of Tie2-Cre activity.Further, the limited phenotype may be explained by functional redundancy between FLI1 and the homologous ETS factor ERG. ERG shares over 70% amino acid sequence similarity and a near-identical ETS domain with FLI1, 130 but is expressed slightly later during development. 2,19,131Of note, it has recently been shown that induced endothelial-specific compound knockdown of Fli1 and Erg together in adult mice results in rapid lethality alongside transcriptional silencing of core EC genes, strongly indicating both significant functional redundancy and a shared yet crucial role for FLI1/ERG in EC identity. 132

| ERG
ERG is the most highly expressed ETS factor in the mature mouse vasculature, with robust EC expression beginning at E7.75-8.0 and maintained throughout the lifespan across all EC subtypes 2,133-135 (Table 2).Although highly expressed in ECs, ERG is not endothelial-specific, with expression also observed in some blood cell lineages, in osteoblasts and in chondrocytes (reviewed by 136).Endothelial-specific deletion of Erg leads to growth defects, cardiovascular abnormalities, hemorrhage, and embryonic lethality at E10-E12.5, indicating a crucial and independent role for ERG in vascular development. 137,138Analysis of ERG binding patterns in cultured ECs suggests that it promotes endothelial homeostasis via directly binding a majority of active endothelial enhancers and super-enhancers. 33Such genome-wide enhancer occupancy underscores the critical role ERG plays in the regulation of endothelial function, although unlike ETV2, ERG enhancer occupancy requires a pre-specified chromatin landscape. 33RG is also known to directly regulate many proangiogenic genes, including those involved in EC migration, apoptosis, and vascular stability (reviewed in 136).VEGF-VEGFR2 signaling during angiogenesis can induce phosphorylation and activation of ERG via the MAPK/ ERK pathway, resulting in increased ERG binding and co-factor recruitment at regulatory elements driving angiogenic gene expression (e.g., Dll4 and Hlx 49 ), although this VEGF-enriched ERG binding is non- specific and is equally found at arterial and venousspecific enhancers. 57Additionally, siRNA-mediated knockdown of ERG reduces vascularization of Matrigel plugs, a phenotype linked to reduced Hdac6 expression. 139,140ERG-deficient ECs also have reduced WNT signaling, with ERG influencing angiogenesis by promoting β-catenin stability via the Wnt receptor FZD4 and CDH5 (VE-Cadherin). 137

| ETS1/2
Ets1, the founding member of the ETS family, is first seen in hemato-endothelial progenitors from E7.0 to E7.25 and is strongly expressed in mature endothelium as well as in other tissues 2,141,142 (Table 2).Like several other non-EC ETS factors, ETS1 exhibits autoinhibition (meaning the full-length protein has less affinity for DNA than the binding domain alone), which is reinforced by phosphorylation and counteracted by protein partnerships. 130TS1 is expressed in ECs at the sites of both developmental and tumor angiogenesis, [143][144][145] and is up-regulated by both pro-angiogenic VEGF-VEGFR2 and hypoxia signaling pathways. 146Although constitutive deletion of Ets1 results in few vascular defects, ETS1 shows significant functional redundancy with its close homolog ETS2, with which it shares a PNT domain targeted by Raf/Mek/Erkmediated phosphorylation. 130While single knockout of Ets2 also does not result in significant vascular phenotypes, compound Ets1; Ets2 knockout mice are embryonic lethal by E12 with dilated vessels, failed blood vessel branching, edema, and hemorrhage, suggesting a key but redundant role for ETS1/2 in angiogenesis. 113Dominant negative ETS1 also inhibits retinal angiogenesis during proliferative retinopathy, 145 while antisense oligonucleotides directed against ETS1 inhibit EC migration and VEGF-induced EC proliferation. 1448][149][150] ETS1 binding is enriched at gene promoters, and strongly correlates with transcription 23 : in cultured ECs, VEGFA stimulation results in increased ETS1 occupation at both P300-bound enhancers and promoters of activated angiogenic genes. 23,151High expression of ETS1 in EC lines can also drive the switch from a quiescent to angiogenic state, which is attributed to increased expression of matrix metalloproteinases. 113,1525 | ELK3 Among the ETS factors expressed strongly and early in ECs, the function of ELK3 (NET) is probably the least understood.ELK3 is part of the ternary complex factor (TCF) subfamily of ETS alongside ELK1 and ELK4 (SAP1), all of which contain an additional B-box domain to mediate interaction with serum response factor (SRF).
Elk3 expression is first seen in hemato-endothelial progenitors from E7.0 (concurrent with Fli1 but after Etv2) and persists in ECs throughout development and in the adult 2,153,154 (Table 2).In the absence of MAPK activation (particularly isoforms ERK2, JNK, or p38), ELK3 strongly inhibits transcription, 130 and the switch from inhibitor to activator has been implicated in the role of ELK3 in angiogenesis. 155However, while tumor cells with reduced ELK3 form fewer vessels, and Elk3 downregulation inhibits VEGFA expression 155 and modulates HIF1 stability, 156 constitutive Elk3 deletion results in viable mice with only mild vascular defects. 1579][160] While no true Elk1;Elk3;Elk4 triple knockout mice have been studied, neither Elk1 or Elk4 are strongly expressed during early endothelial development, and consequently the precise role of ELK3, and of the TCF ETS subfamily, in vasculogenesis and angiogenesis is still not fully defined.

| FORKHEAD
Forkhead (FOX) TFs are characterized by a winged-helix Forkhead box DNA binding domain which binds a core C / T AAA C / T A motif (Table 1).The 44 different FOX proteins found in mice and humans can be divided into 22 subclasses (denoted as FOXA through to FOXS) according to sequence similarities, 161 with members of the FOXC and FOXO subfamilies the principal FOX factors implicated in the regulation of vasculogenesis and angiogenesis.

| FOXC1/2
Both Foxc1 (Mf1) and Foxc2 (Mfh1) are expressed in ECs from early in embryonic development, detected by scRNA-seq from around E8.0 and by in situ hybridization from E9.5 2,162 (Table 2).Compound Foxc1;Foxc2 deletion results in a failure of blood vessel development and lethality by E9.5.Although ECs are specified and an initial vascular plexus forms in these mutant mice, the plexus does not remodel into a functional vascular network, 162,163 indicating important functions for FOXC factors in endothelial differentiation and angiogenesis.In particular, FOXC factors have been implicated in angiogenesis via direct induction of Itgb3-mediated EC adhesion and migration, 164 and of Dll4-mediated Notch signaling downstream of VEGF. 165,166Analysis of endothelial enhancers also identified a shared role for FOXC and ETS factors in endothelial specification.FOXC1/2 and the ETS factor ETV2 combinatorially bind compound FOX:ETS motifs within gene enhancers 167 and promoters to synergistically activate the transcription of crucial endothelial lineage-identity genes, including those for Flk1, Flt4, Tal1, Cdh5, Tie2, Notch4, and Pdgfrβ 45 (see Table 3).Further, FOXC factors are also important regulators of endothelial patterning in the maturing vasculature.Embryos in which only a single FOXC allele remains show severe arterio-venous malformations and lack expression of arterial/angiogenic-associated genes including Notch1, Dll4, and Jag1, 163 while lymphaticspecific compound deletion of Foxc1;Foxc2 results in increased lymphatic EC proliferation and abnormal lymphatic vessel morphogenesis. 168

| FOXO1/3/4
Mammalian Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx) encode the evolutionarily conserved FOXO subfamily, which act as key nuclear effectors of the PI3K/ AKT pathway.3][174] It is also able to bind the compound FOX:ETS motifs found in many early EC enhancers. 45Foxo3a null and Foxo4 null mice do not die during embryogenesis; however, post-natal angiogenic capacity is increased in Foxo3a knockout mice. 175FOXO1 is also required to direct angiogenesis in the post-natal retina, with deletion resulting in uncoordinated vascular growth, increased endothelial number, density, and vessel diameter. 174Mechanistically, FOXO factors function by coupling changes in metabolism with changes in gene transcription and cell activity.
In ECs, the transcriptional targets of FOXOs include antioxidants, cell cycle inhibitors, and metabolic regulators, and they act as potent negative regulators of MYC activity. 174FOXO activity itself is also regulated by energy-sensing post-transcriptional modifiers. 176Therefore, in ECs, FOXO1 activity reduces metabolic activity, reduces MYC-induced glycolysis and mitochondrial respiration, thus mediating angiogenesis in response to changes in metabolism. 174

| Other FOX factors
Foxm1 is ubiquitously expressed in the early embryo but may play a role in vascular growth.Although EC-specific deletion of Foxm1 results in no overt phenotype, pulmonary vascular injury in mice lacking or overexpressing Foxm1 revealed a role for EC FOXM1 in the restoration of endothelial barrier function. 177,178Additionally, both Foxp1 and Foxp4 are expressed during early endothelial development, 2 with Foxp1 most highly expressed in ECs in culture, and up-regulated in ECs during injury-induced neovascular growth.Knockdown of Foxp1 in ECs in culture inhibits EC proliferation, migration, and tube formation, 179 while constitutive Foxp1 knockout mice die at E14.5 with complex cardiovascular defects including vascular hemorrhage. 180However, the vessel defects are thought to be secondary to severe cardiac and valve defects and were not seen after EC-specific Foxp1 deletion. 181

| GATA
The GATA family consists of six TFs characterized by a highly conserved zinc finger domain binding a core GATA motif (reviewed by 182; Table 1).GATA1, GATA2, and GATA3 have well-established roles in the specification of hematopoietic cells from the hemogenic endothelium (a specialized subset of ECs that give rise to the hematopoietic stem and progenitor cells, reviewed by 183), but are also implicated in EC specification and development.

| GATA2
Gata2 is expressed in hemato-endothelial progenitors from E7.0 (Table 2) and has been implicated in the regulation of EC lineage specification via interaction with ETV2. 184Motif analysis of endothelial enhancers also supports a role for GATA2 in the expression of angiogenic genes, with all characterized Flk1 EC enhancers containing essential GATA-binding motifs 69,185 (Table 3).GATA2 binding at the Flk1 promoter also increases expression in response to changes in matrix stiffness, 186 while expression of Emcn (Endomucin), a type I integral membrane glycoprotein important in EC signaling and angiogenesis, requires GATA2-mediated chromatin remodeling. 187Furthermore, GATA2 binds a unique set of gene loci in ECs compared to other cells lines, 28 dynamic GATA2 binding has been observed at key EC enhancers in response to VEGFA stimulation, 31 and siRNA-mediated GATA2 knockdown in the post-natal retina inhibits angiogenic sprouting. 186However, constitutive deletion of Gata2 had no clear effect on early vascular formation before embryonic lethality at E10.5 due to hematopoietic defects, 188 although it is possible that the deletion strategy used to generate these mice (which removes only the C-terminal zinc finger of GATA2) did not ablate all GATA2 DNA binding. 189Alternatively, the early hematopoietic lethality may predate overt EC phenotypes.Supporting this, both constitutive deletion of the GATA2+9.5 enhancer (strongly active in developing embryonic ECs) 72 and endothelial-specific deletions of Gata2 result in lethality at E13-16.5 with vascular abnormalities including hemorrhage, edema, and anemia.However, important roles for GATA2 in lymphangiogenesis underpins many of these defects. 85,190ATA3 and GATA6 are also expressed in ECs in culture and have been implicated in the regulation of Tie2. 191Although it is possible that endothelial GATA2 may be functionally redundant to some degree with these other GATA factors, there is little evidence that GATA3 or GATA6 are robustly expressed in the developing endothelium (Table 2).Consequently, the precise roles of GATA factors in vasculogenesis and angiogenesis remain unclear.

| GATA/TAL1
Combinatorial binding of GATA alongside the TAL1/ SCL TF at a composite GATA-E-box binding motif plays a key role in hematopoietic development. 192,193][194] GATA/TAL1 binding may also play a role in maintaining endothelial identity in the early embryo: constitutive Tal1 knockout embryos display irregular yolk sac vasculature, 195,196 while EC-specific deletion of Tal1 results in edema, hemorrhage, and defective vascular remodeling in the yolk sac. 197These defects have been attributed to dual roles for TAL1, in both activating hemogenic endothelium and repressing ectopic cardiomyogenesis in the yolk sac endothelium and endocardium. 198,199ChIP-seq experiments that assess chromatin states of enhancer elements suggest that TAL1 exploits a pre-established epigenetic landscape, likely generated by ETV2, to bind and repress enhancers of the cardiac lineage, while binding together with GATA to activate endothelial/hematopoietic lineage specification. 200

| SMAD
SMAD proteins are the transcriptional effectors of the TGFβ superfamily and can be subdivided into receptorregulated SMADs (R-SMADs, consisting of SMAD1, 2, 3, 5, and 8), common SMAD (SMAD4), and inhibitory SMADs (I-SMADs, SMAD6 and 7). 201,202R-SMADs act as the primary signal transducers, forming complexes with SMAD4 after phosphorylation and subsequently translocating to the nucleus to directly regulate gene transcription. 202R-SMADs can be further subdivided into those primarily downstream of canonical BMP signaling (SMAD1/5/8) and those primarily downstream of canonical TGFβ signaling (SMAD2-3).Both BMP and TGFβ signaling pathways play complex and sometimes contradictory roles in angiogenesis.TGFβ signaling via ALK1 and endoglin can stimulate EC activation, proliferation, and migration, whereas TGFβ signaling via ALK5 can inhibit proliferation and promote vascular maturation (reviewed by 203).Similarly, BMP signaling is implicated in both the promotion of angiogenesis and the maintenance of endothelial homeostasis via interactions of different ligands, receptors, and co-factors (reviewed by 202).Most SMADs are expressed early during EC development but also widely in non-ECs (Table 2).While endothelial-specific deletion of Smad2/3 did not affect vasculogenesis and early angiogenesis (although defects in vascular maturation and mural cell assembly resulted in hemorrhage and lethality by E12.5), endothelialspecific deletion of either Smad4 or Smad1/5 led to defective angiogenic sprouting and embryonic lethality by E10.5. 204,205Analysis of SMAD1/5 binding in ECs identified numerous direct targets, including Id1, Notch pathway genes Hey1, Hey2, Hes1, and Jag1, and venous identity genes Ephb4 and Coup-TFII (Nr2f2) 20,58,204 (Table 3).These targets have also been verified in knockout models, with endothelial-specific deletion of Smad4 resulting in embryos with reduced Ephb4 and Coup-TFII expression and defective venous differentiation, while endothelial-specific deletion of Smad1/5 resulted in embryos with impaired DLL4-Notch signaling.The inhibitory SMAD6 may also play a role in this process downstream of Notch and upstream of target gene activation. 206[209] 6 | HYPOXIA-INDUCIBLE FACTOR Hypoxia-inducible factor (HIF) TFs are master regulators of oxygen homeostasis 210 and therefore have significant direct and indirect influences on vascular growth.HIF1α and HIF2α (EPAS) subunits are stabilized in low oxygen conditions, permitting them to translocate to the nucleus and bind DNA at consensus A / G CGTG motifs (Table 1) as a heterodimer with HIF1β (ARNT), inducing gene programs that respond to the hypoxic environment (reviewed by 211).This adaptive response includes the activation of physiological and pathological angiogenesis through both cell autonomous and non-autonomous mechanisms. 212,213While Hif1a is ubiquitously expressed, Hif1b is predominantly EC-specific from E8.5 (see Table 2) where it is up-regulated by hypoxia 214,215 (Table 2).Fewer than 20% of HIF target genes are regulated by both isoforms in ECs indicating predominantly non-overlapping functions, with HIF2α inducing a larger and more diverse transcriptional response. 216Overexpression of HIF isoforms in cultured ECs increases expression of a range of pro-angiogenic genes, including Vegfa, Angpt2, and Pdgfb (by HIF1α) [217][218][219] and Flt1 (by HIF2α). 220However, studies into the role of HIFs in ECs during developmental angiogenesis in mice have produced conflicting results.EC-specific expression (driven by a Flk1 enhancer/promoter) of a dominant negative HIF mutant (which inhibits transcriptional responses by both HIF1α and EPAS1/HIF2α) results in embryonic lethality by E11.5 alongside severe cardiovascular defects and loss of vascular sprout formation attributed to reduced Tie2 (Tek) expression. 221Embryonic lethality and severe vascular defects (with reduced Tie2) were also seen after constitutive deletion of Hif2a. 222owever, Cdh5-Cre-driven EC-specific deletion of Hif2a alone or in combination with Hif1a resulted in no major developmental vascular abnormalities, 223,224 although increased vascular permeability and pulmonary hypertension were seen in the adult. 224This discrepancy in phenotype may be due to an early role for HIFs before robust Cdh5-Cre activity or incomplete Cre-mediated deletion (in part because the Cdh5-Cre driver used here is known to be only weakly and sporadically expressed before E14.5, 98,225 whereas the Flk1 driver of dominant negative HIF is active more robustly and much earlier), or may instead indicate a key function for HIF proteins in the activation of Vegfa in non-EC lineages. 226,227Alternatively, off-target effects of the dominant negative form of HIF may have contributed to the severity of phenotype seen in the dominant negative mutant mouse.

| RBPJ
The RBPJ (CSL) TF acts as the nuclear effector of the Notch signaling pathway, forming a complex with Notch intracellular domain (NICD) and the MAML1 coactivator to promote transcription via direct binding to Notch target genes at a consensus TGGGAA motif (reviewed by 228 and Table 1).Analysis of many deletional models strongly indicates that Notch signaling via RBPJ plays an essential role in vasculogenesis, angiogenesis, and arteriovenous differentiation. 8,9,229,230Loss of multiple different Notch pathway components results in early lethality in mice associated with severe vascular defects including aberrant arteriovenous specification (e.g., compound Notch1/Notch4 deletion, 231 deletion of ligand Dll4 232,233 ), while ablation of Notch signaling in the post-natal retina results in significant defects in sprouting angiogenesis (reviewed by 8,10).Loss of RBPJ largely recapitulates defects downstream of Notch receptor/ligand deletion, 9,232,234 and the Notch signaling components Hey1 and Dll4 are both direct targets of RBPJ via arterial/angiogenic-specific enhancers. 50,75Further, multiple different signaling pathways can converge upon, and influence, RBPJ binding.For example, Ang1/Tie2 signaling induces Dll4 via AKT-mediated activation of β-catenin, which complexes with RBPJ to bind and activate an intronic enhancer, 52,235 whereas KLF4 can inhibit Dll4 expression during angiogenesis by interfering with RBPJ binding to the same element 53 (Table 3).In the absence of NICD, RBPJ can also act as a transcriptional repressor, and may repress EC gene expression in some circumstances.For example, RBPJ binding to an Flk1 arterial enhancer represses its activity in veins, 69 while RBPJ binding to a Vegfa promoter negatively regulates its expression. 9However, although it was long assumed that Notch-RBPJ influenced vascular patterning by directly regulating key arteriovenous genes, postnatal deletion of RBPJ does not ablate arterial gene expression, 234,236 and ECs lacking MYC (a key driver of metabolism and proliferation) require neither RBPJ nor Notch for correct arteriovenous gene expression. 237Consequently, it is now thought that Notch-RBPJ regulates arteriovenous specification by reducing metabolism and cell cycle rather than via direct activation or repression of arteriovenous identity genes. 237Given that the signaling processes involved in angiogenic sprouting are often coupled to arterial formation, 9 it is still unclear to what degree RBPJ directly versus indirectly regulates gene expression in arterial and angiogenic ECs.

| HEY FACTORS
The Hey family of transcriptional repressors are basic helix-loop-helix proteins that act downstream of Notch signaling and are directly activated by RBPJ 75,238 (Table 3).Hey1 and Hey2 are expressed in ECs from early during mammalian development (see Table 1) and directly bind DNA at E-box motifs. 238While the orthologue of Hey2 in zebrafish (gridlock) is essential for arterial morphogenesis, 239 deletion of either Hey1 or Hey2 separately does not result in gross embryonic vascular phenotypes, although Hey2 null mice die soon after birth and Hey1 null mice show anomalies of the thoracic great vessels. 240,241However, compound deletion of both Hey1 and Hey2 results in embryonic lethality and significant endothelial defects including in arteriovenous differentiation. 242,243[244] 9 | HLX HLX1 is a homeobox TF with a currently undefined DNA binding motif expressed in hemato-endothelial progenitors from E7.75 (Table 2).HLX1 was first implicated as a key regulator of sprouting angiogenesis in zebrafish (as the orthologue hlx), where it maintains endothelial stalk-cell fate in a cell-autonomous manner. 245In mammalian EC culture, HLX1 regulates expression of cell guidance molecules including Unc5b, Plxna1, and Sema3g downstream of VEGF-VEGFR2 signaling, 246 and is a direct transcriptional target of both ERG and MEF2 TFs downstream of VEGFA-VEGFR2 signaling. 49,51However, although both overexpression and knockdown of Hlx1 in cultured ECs disrupt sprouting angiogenesis, 246,247 constitutive Hlx1 deletion in mice causes only a mild vascular remodeling phenotype. 247onsequently, the precise role and direct transcriptional targets of HLX1 in angiogenesis are yet to be clearly elucidated.

| MEF2
The MEF2 family of TFs is characterized by their highly conserved MADS-box and MEF2 domains which mediate dimerization, co-factor interactions, and DNA binding to a consensus C / T TA A / T A / T A / T A / T TA A / G motif. 248They are found in multiple cell lineages and play key roles in a diverse range of developmental processes. 248While constitutive Mef2c deletion results in both cardiac and vascular defects, 249,250 endothelial-specific Mef2c deletion has no effect on embryonic vascular development regardless of Cre driver, 251,252 although subtle defects are detected in pathological angiogenesis. 251However, Mef2a, Mef2c, and Mef2d are all expressed in the early endothelium 2,51 (Table 3), and studies from other cell types show strong functional redundancy between the three proteins. 253nalysis of direct MEF2 targets in ECs alongside combinatorial gene deletion has strongly implicated MEF2 factors in angiogenesis downstream of VEGFA-VEGFR2 signaling, and in endothelial homeostasis downstream of blood flow. 51,254Supporting this, induced EC-specific deletion of Mef2a alongside Mef2c reduces sprouting angiogenesis and Dll4 expression in the postnatal retina, while MEF2 proteins directly bind and activate many angiogenic-specific genes including Dll4 and Hlx1. 515][256] MEF2 factors also directly regulate the expression of Mmp10 to regulate vascular integrity. 257It is, however, unclear how the widely expressed MEF2 factors are able to regulate such disparate vascular behavior.Although MEF2 factor activity can itself be regulated by both ERK5 and by complexing with class IIa HDACs, it is likely that additional transcriptional cofactors are required to enable MEF2 factors to achieve their diverse gene expression patterns in the endothelium.

| AP1
AP1 proteins are made up of a ubiquitously expressed family of transcription complexes most commonly defined as a collection of dimers from the Jun and Fos families, although it can also be considered to include members of the ATF and MAF subfamilies. 258AP1 TFs bind a consensus TGA C / G TCA motif, although the ATF and MAF subfamilies have slightly differing preferences (see Table 1).

| JUNB
Induced EC-specific deletion of Junb in the retina leads to reduced angiogenic vascular growth and diminished expression of neurovascular guidance genes including Sema3a, likely via direct binding at AP1 motifs within enhancer elements. 259Although often considered ubiquitous, Junb expression is enriched in hemato-endothelial progenitors and angioblasts in the E8.5 embryo. 2 Further, JUNB is spatially restricted at the angiogenic front by a combination of VEGFA-VEGFR2 and S1P-S1PR signaling: induction of Junb by VEGFA-VEGFR2 is countered by the circulating vascular maturation factor sphingosine 1-phosphate (S1P), which restricts Junb expression in the perfused vasculature via S1PR-dependent VE-cadherin assembly. 259The related factor JUN/c-JUN may also play a role in angiogenesis in some contexts and is implicated in the regulation of expansion of the vasa vasorum downstream of extracellular ATP signaling. 260

| MAFB
MAFB, a member of the large MAF TF subfamily, has been implicated in the regulation of both lymphangiogenesis and sprouting angiogenesis. 261,262Analysis of the actively translated transcriptome at different stages of postnatal retinal angiogenesis combined with promoter analysis identified MAFB as a key regulator of postnatal angiogenesis. 261Mafb is enriched at the angiogenic front in postnatal retinas, while induced EC-specific deletion results in defective angiogenic expansion.MAFB expression up-regulates Git1 and down-regulates Arhgdib expression, two Rho GTPase modulators that activate and inhibit Rac1/Cdc42 signaling, respectively, leading to cytoskeletal changes and EC migration during sprouting angiogenesis. 261However, constitutive deletion of Mafb in mice results in no vascular-related lethality and embryonic angiogenesis is unaffected, 263 suggesting organotypic specificity of its angiogenic role or potential redundancy.In lymphatic ECs, Mafb expression is strongly up-regulated by VEGFC, while Mafb overexpression results in increased levels of many lymphatic EC genes including Prox1. 264Additionally, although viable both constitutive and lymphatic EC-specific Mafb deletion results in impaired lymphatic patterning, further supporting a role for MAFB in lymphatic ECs. 264,265

| Small MAFs
Analysis of the epigenetic and transcriptional changes in cultured ECs following VEGFA stimulation identified small MAF proteins MAFF, MAFG, and MAFK as key regulators of the angiogenic transcriptional response alongside ETS1, ERG, MEF2C, and FOXO1. 31This is supported by in vitro sprouting angiogenesis assays, which indicate partially redundant but collectively critical functions for small MAFs in EC migration, proliferation, and tube formation. 31However, these observations have yet to be validated in animal models.

| TEAD
The TEAD/TEF TF family consists of four highly homologous proteins (TEAD1-4), all containing the conserved TEA DNA-binding domain recognizing a consensus GGAATG motif 266 (Table 1).TEAD proteins are the mediators of YAP/TAZ-dependent gene regulation downstream of the highly conserved Hippo signaling pathway.In their active state, YAP and TAZ translocate to the nucleus and form YAP/TAZ-TEAD complexes, which are associated with the expression of genes controlling cell proliferation, migration, and apoptosis. 267YAP/TAZ activity is limited through phosphorylation by Hippo pathway components, leading to cytoplasmic retention and destabilization. 268C-specific deletion of Yap/Taz in mice leads to embryonic lethality associated with severe vascular defects throughout the embryo and yolk sac. 269,270Similarly, induced EC deletion of Yap/Taz in the postnatal retina results in reduced vessel growth, blunted-ended tip cells with fewer filipodia and defective lumen formation. 269Lowered levels of tip cell-associated ANGPT2 and ESM1 are observed, the number of ERG-positive ECs is reduced, and fewer actively proliferating ECs are detected. 269Conversely, overexpression of a stabilized TAZ protein results in a dense and hyperplastic vascular network with increased EC proliferation. 271Notably, induced EC-specific compound deletion of Tead1, Tead2, and Tead4 together results in a similar phenotype to that seen after Yap/Taz deletion, validating TEADs as crucial transcriptional effectors of endothelial YAP/TAZ signaling. 271Potential mechanisms for YAP/TAZ/TEAD involvement in angiogenesis include activation of actin cytoskeleton remodeling downstream of VEGF-VEGFR2, 269,270 modulation of MYC signaling, 269 activation of the small GTPase CDC42, 272 and fuelling nutrient-dependent mTORC1 signaling via transcriptional activation of cell-surface transporters. 271

| SOXF
The SOXF proteins, comprising SOX7, SOX17, and SOX18, are the only members of the SOX TF family strongly expressed in the developing endothelium, and recognize a consensus A / T CAA A / T DNA motif. 2735][276] While SOX17 has been specifically implicated in arterial differentiation 277 and SOX18 plays a key role in the initiation of lymphangiogenesis, 278 SOXF factors also play crucial, although redundant, roles in vasculogenesis and angiogenesis.Although Sox7 deficiency does not impact the emergence of ECs, vasculogenic defects to dorsal aorta formation are visible by E8.5, impaired angiogenesis is seen from E9.5, and both constitutive and EC-specific deletion of Sox7 results in significant growth retardation, severe vascular defects, and lethality by E10.5-E11.5. 273,274EC-specific deletion of Sox17 results in a similar loss of angiogenic sprouting in some mouse backgrounds, 279 as does compound heterozygous deletion of Sox7 alongside Sox17. 273Complicating analysis, genetic levels of compensation between SOXF factors can vary depending on mouse background. 280This is evident in analysis of the role of SOXF factors in postnatal retinal angiogenesis, with the severity of single or compound deletion of Sox7, Sox17, and Sox18 varying between mouse models.Notably, however, the resultant phenotypes are similar, resulting in ablation of tip and stalk cell identity, reduced vascular outgrowth and branching, and compromised tube formation and perfusion. 273,281Overexpression of Sox17 also promotes tumor angiogenesis and vascular abnormalities, while Sox17 deletion in ECs reduces tumor angiogenesis and normalized tumor vessels, inhibiting tumor growth. 282he known direct SOXF target genes align with the range of vascular functions associated with these TFs.SOX17 directly binds arterial enhancers regulating Dll4, Notch1, and Ece1, while SOX18 directly targets a Prox1 enhancer/promoter. 50,56,277,278,283SOX7 and/or SOX17 can also directly up-regulate angiogenic Flk1, 69,273 Lef1, and β-catenin 284 expression, while enforced expression of Sox17 in EC-like cells increases expression of Col18a1 and Cd31 as well as Flk1. 285However, given the wide and overlapping expression patterns of each SOXF factor both within and beyond the endothelium, it is apparent that additional co-factors must be involved in the regulation of genes downstream of SOXF, while expression of the SoxF genes is themselves likely to be controlled by multiple upstream inputs.

| FUTURE DIRECTIONS
Our understanding of the array of TFs involved in vasculogenesis and angiogenesis has greatly increased in the last ten years, as has our ability to link these factors both to specific aspects of EC biology and to their direct target genes.Progress in these areas will continue as new technologies increasingly provide information of gene expression patterns, enhancer marks, and protein binding patterns at a single-cell resolution, and as computational pathways are developed to process and analyze such complex information simply and efficiently.Alongside this progress sit innovations in our ability to experimentally examine the role of novel EC transcriptional regulators, including the increasing ease of genetic manipulation, the generation of more diverse and specific methods to alter gene expression selectively in certain ECs, and higherthroughput methods to validate and interrogate enhancer elements directly in animal models.However, while such approaches will improve our knowledge of the roles of known vascular TFs and identify new factors, a better understanding of gene regulation within the vasculature also requires a greater appreciation of the manner in which the limited cohort of TFs work together to achieve different outputs.As is made clear in this review, ECs contain no single lineage-defining TF.Instead, the vast majority of endothelial transcriptional regulators are both expressed outside of ECs and involved in activating genes with more than one type of endothelial expression pattern and/or in response to more than one stimulus.Consequently, while improved genetic information and animal models will provide a more complete picture of the TF repertoire coordinating vasculogenesis and angiogenesis, this must be coupled with a greater understanding of the different combinatorial, synergistic, and antagonistic ways in which these factors work together to enable this limited number of proteins to achieve such complex and responsive patterns of gene expression.

T A B L E 1
Consensus binding motifs for common EC transcription factors (TFs) JASPAR refers to JASPAR 2022 unless specified, logos from primary publications are adapted by authors from the original paper.ChIP-seq refers to chromatin immunoprecipitation combined with sequencing; HT-SELEX refers to high-throughput systematic evolution of ligands by exponential enrichment, bold denotes that ECs were experimental model used in motif designation.Note that consensus motifs should be viewed as only a guide to potential TF binding locations, as TFs can also bind non-consensus motifs (often similar to consensus) and conversely do not always bind sequences containing the exact consensus motif.T A B L E 2 Expression dynamics of common endothelial TFs and information on publicly deposited ChIP-seq data sets 39 in vivo-validated EC enhancers alongside approximate location and associated regulatory TFs ETS: mo, ch (FLI1) MU 60T A B L E 3 (Continued)RBPJ: mo, em, MU, oe 75(Continues)T A B L E 3 (Continued)