Review - Circulation Researchcircres.ahajournals.org/content/circresaha/112/9/1272.full.pdf · he...

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The formation of the circulatory system precedes that of all other organ systems during vertebrate development, born

out of a need to ensure rapid delivery of nutrients and oxygen to tissues of the growing embryo. Thus, as the developing organism continues to increase in size and undergo cellular differentiation and morphogenesis, the vasculature likewise expands, remodels, and becomes specialized to adapt to the requirements of the tissues it supplies. Endothelial cells that line the entire circulatory system are thought to emerge de novo from the mesoderm to form a primary vascular plexus. Further specialization of the endothelium to arterial, venous, hemogenic, and lymphatic subtypes is necessary to fulfill diverse functions of the vasculature. Disrupting this normal program of vascular development often results in disease phenotypes or even embryonic lethality. This underscores the need to understand the mechanisms that govern normal vascular development because it would not only allow us to better treat vascular pathologies but also provide insights needed to direct the differentiation of pluripotent human stem cells for tissue engineering and regenerative medicine strategies. In this review, we will the discuss present understanding of the extrinsic and intrinsic signals that regulate endothelial cell differentiation from their mesodermal progenitors, and the establishment of arterial, venous, hemogenic, and lymphatic endothelial cell (LEC) identities. We discuss insights derived from in vivo mouse, zebrafish, and avian models, as well as in vitro–based systems, including differentiated embryonic stem

(ES) cells, which recapitulate the earliest stages of mammalian lineage specification, events that have been difficult to study directly in the developing embryo.

Vasculogenesis: Differentiation of Endothelial Cells and Vascular Plexus Formation

Vasculogenesis, the de novo emergence of primordial endo-thelial cells and blood vessels, begins within the mammalian extraembryonic yolk sac soon after gastrulation, when signals from the visceral endoderm serve to pattern the underlying mesoderm.1,2 Development of the circulatory system is there-fore dependent on these early events during which mesoder-mal precursors are specified toward an endothelial cell lineage (Figure 1).

Signaling PathwaysFibroblast growth factor 2 (FGF2 or bFGF) and bone morpho-genetic protein 4 (BMP4) are 2 key signaling components that are not only important for specification of mesoderm3–5 but also for its differentiation toward endothelial and hematopoi-etic cell fates.6–8 BMP4 is sufficient to induce mesodermal dif-ferentiation in vitro, whereas its ablation results in a failure to generate mesoderm and leads to early embryonic lethality.9–11 Embryos deficient for downstream effectors of BMP4 signal-ing, such as Smad5, lack an organized yolk sac vasculature similar to Bmp4–/– mutant mice.6 Smad4 null mice display sim-ilar phenotypes and are also remarkably smaller in size, owing

Review

© 2013 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.113.300506

Abstract: The circulatory system is the first organ system to develop in the vertebrate embryo and is critical throughout gestation for the delivery of oxygen and nutrients to, as well as removal of metabolic waste products from, growing tissues. Endothelial cells, which constitute the luminal layer of all blood and lymphatic vessels, emerge de novo from the mesoderm in a process known as vasculogenesis. The vascular plexus that is initially formed is then remodeled and refined via proliferation, migration, and sprouting of endothelial cells to form new vessels from preexisting ones during angiogenesis. Mural cells are also recruited by endothelial cells to form the surrounding vessel wall. During this vascular remodeling process, primordial endothelial cells are specialized to acquire arterial, venous, and blood-forming hemogenic phenotypes and functions. A subset of venous endothelium is also specialized to become lymphatic endothelium later in development. The specialization of all endothelial cell subtypes requires extrinsic signals and intrinsic regulatory events, which will be discussed in this review. (Circ Res. 2013;112:1272-1287.)

Key Words: arterio-venous specification ■ endothelial cells ■ hemogenic endothelium ■ lymphatic endothelium

Regulation of Endothelial Cell Differentiation and Specification

Kathrina L. Marcelo, Lauren C. Goldie, Karen K. Hirschi

Original received January 9, 2013; revision received March 15, 2013; accepted March 29, 2013. In February 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.98 days.

From the Interdepartmental Program in Developmental Biology (K.L.M.), Department of Pediatrics (L.C.G., K.K.H.), Center for Cell and Gene Therapy (K.L.M., L.C.G., K.K.H.), and USDA/ARS Children’s Nutrition Research Center (K.L.M., L.C.G., K.K.H.), Baylor College of Medicine, Houston, TX (K.L.M., L.C.G., K.K.H.); and Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT (K.K.H.).

Correspondence to Karen K. Hirschi, PhD, Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, 300 George St, New Haven, CT 06511. E-mail [email protected]

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Marcelo et al Endothelial Cell Specification 1273

to severe cell proliferation defects.7,12 Meanwhile, gene dele-tion experiments demonstrate FGF2 signals via FGF receptor type I to induce and pattern the mesoderm.5,8,13 The hierarchy of these signals has not been clearly defined in vivo, and ES cell systems have yielded conflicting data. Experiments us-ing mouse ES (mES) cells suggest that BMP4 resides at the top of this signaling hierarchy, promoting mesoderm forma-tion and initiating a FGF2-dependent program to regulate the specification of angioblasts, which are thought to function as endothelial progenitors.9,10,14 In contrast, differentiation stud-ies in human ES cells, which require bFGF for survival and growth even in an undifferentiated state, do not support a role for this factor in the commitment of mesodermal cells to the endothelial lineage.15 Rather, in human ES cells, BMP4 is a critical regulator that functions downstream of Indian hedge-hog (IHH) to promote endothelial cell differentiation.15

IHH has also been shown to be a critical regulator of endothelial cell development during murine embryogenesis, although its role as a key inductive signal is not entirely clear. IHH is secreted by the yolk sac visceral endoderm during vas-culogenesis; its receptors are expressed within the underlying mesoderm. IHH has been shown to be sufficient to promote the formation of endothelial and hematopoietic cells in mouse embryo explant cultures that lack endoderm (Figure 1), and it can also respecify prospective neural ectoderm cells to adapt hematovascular fates. However, embryos deficient for IHH or its receptor smoothened16 do exhibit some degree of

endothelial cell differentiation, although less than normal. Thus, IHH may not be the only inductive signal in vivo, and the visceral endoderm may produce other signals required for endothelial cell development within the mesoderm.

Another visceral endoderm-derived factor that is a key regu-lator of vasculogenesis is vascular endothelial growth factor (VEGF); however, its cellular role in endothelial cell develop-ment in vivo is also not entirely clear. VEGF-A is the most extensively studied member of the VEGF family and is ex-pressed by the extraembryonic visceral endoderm in the mouse as early as embryonic day (E)7.5, coincident with blood island formation in the yolk sac.17 The requirement for VEGF-A is established early during vasculogenesis because heterozygous mutants are embryonic lethal due to failed development of the vasculature.18,19 Overexpression of VEGF-A also impairs car-diac development and causes embryonic lethality at midgesta-tion.20 These data reveal a precise dosage requirement for this growth factor for proper cardiovascular development.

VEGF-A signals through its main receptors, VEGFR1 (fms-related tyrosine kinase-1 [Flt-1]) and VEGFR2 (fe-tal liver kinase-1 [Flk-1] or kinase insert domain receptor), and also interacts with the coreceptors neuropilin-1 and -2. Although Flk-1 has a lower affinity for VEGF-A than Flt-1, it has stronger tyrosine kinase activity, and VEGF-A responses in endothelial cells and their precursors are usually attributed to Flk-1 activation. Mice lacking Flk-1 are embryonic lethal at E8.5 to 9.5 and lack blood island and vascular plexus develop-ment, despite normal formation of angioblasts.21 Consistent with this, Flk-1–/– mES cells can generate endothelial cells; however, they fail to propagate in vitro.22 Similarly, VEGF-A treatment of undifferentiated human ES cells does not promote their differentiation toward an endothelial cell phenotype.15

Collective in vivo and in vitro data from murine and human systems suggest that VEGF-A likely regulates the survival or propagation of endothelial cells, and not necessarily their differentiation. However, given that there are observed differences in the hierarchy of signals that controls endothelial cell differentiation/propagation in murine and human systems, it is critical to conduct more studies of vasculogenesis in human pluripotent stem cells, which may provide novel insights more directly applicable to the treatment of human pathologies.

Transcriptional RegulatorsE-twenty six (ETS) transcription factors are also known to play important roles in endothelial cell development. ETS family members share a highly conserved winged helix-turn-helix DNA-binding domain that binds to the ETS DNA-binding site 5′GGA(A/T)3′. There are ≈30 known mammalian ETS factors that generally serve as transcriptional activators, although some have been associated with repressor activity.23 Several members of this family, such as Ets1, Erg, Fli-1, and Etv2 (discussed below), have been demonstrated to play im-portant roles in vascular, as well as hematopoietic, develop-ment.23 Regulatory regions for almost all endothelial genes contain ETS binding sites, implying that ETS factors could potentially regulate most, if not all, endothelial-specific gene expression.23,24

Nonstandard Abbreviations and Acronyms

AGM aorta-gonad-mesonephros

BMP4 bone morphogenetic protein 4

Cdk cyclin-dependent kinase

COUP-TFII chicken ovalbumin upstream promoter–transcription factor II

Dll1/4 delta-like ligand 1 and 4

ES embryonic stem

ETS E-twenty six

FGF2 fibroblast growth factor 2

Flk-1 fetal liver kinase-1

Flt-1 fms-related tyrosine kinase-1

Foxc1/2 forkhead box C1 and C2

HSC hematopoietic stem cell

HSPC hematopoietic stem/progenitor cell

IHH Indian hedgehog

Kdr kinase insert domain receptor

LEC lymphatic endothelial cells

Lmo2 LIM domain only 2

Prox1 prospero homeodomain transcription factor 1

RA retinoic acid

RARα/β/γ RA receptors α, β, and γ

Scl/Tal-1 stem cell leukemia protein/T-cell acute lymphocytic leukemia protein-1

SP side population

TGF-β1 transforming growth factor β-1

VEGF vascular endothelial growth factor

VEGFR1 VEGF receptor type I


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1274 Circulation Research April 26, 2013

Although many ETS factors are thought to share redun-dant functions, recent studies have demonstrated a specific and critical role for Etv2(ER71/etsrp), or ETS variant 2, in the differentiation of mesodermal progenitors toward an endothelial cell fate. Etv2 expression is initially observed to be more widespread within the primitive streak meso-derm; however, it is soon restricted to developing vascular endothelial cells,25–28 and its critical function in their de-velopment is evolutionarily conserved.25–27,29–31 Morpholino knockdown25 of, or mutations30 within, the etsrp gene in zebrafish results in complete abrogation of circulation, to-gether with loss of expression of vascular endothelial cell markers, including VE-cadherin/Cdh5 and VEGFR2/Flk-1, in developing embryos.25,30 Likewise, Etv2–/– mice are em-bryonic lethal, and despite having intact mesoderm, they fail to generate angioblasts,25 a yolk sac primitive vascular plexus, dorsal aortae, or endocardium.26,32 Overexpression of Etv2 leads to ectopic expression of endothelial markers in multiple cell types in vivo,25,27,29,31 suggesting an expan-sion of the differentiation of endothelial cells. Thus, Etv2 seems to be unique among ETS domain transcription fac-tors in that it is both necessary and sufficient for in vivo vasculogenesis.25–27,30,32

Importantly, maintaining Etv2 expression within a narrow developmental window is critical for proper vasculogenesis. Transient Etv2 activation in the mesoderm is sufficient to initiate endothelial cell development,28,32,33 and Etv2 is rap-idly downregulated once endothelial cells begin to mature.28 Constitutive expression of Etv2 in endothelial cells under con-trol of the Tie2 promoter results in dilated yolk sac vessels and impaired remodeling of the extraembryonic vasculature, eventually leading to embryonic lethality.34 Interestingly, this

also results in perturbation of normal hematopoiesis attribut-able to endothelialization of hematopoietic progenitor cells that do not form properly.35

Molecular interactions between Etv2 and other signaling factors that regulate endothelial cell development are not yet clearly defined; however, Etv2 is closely associated with and known to precede Flk-1 expression during vasculogenesis.25,26,30 As previously mentioned, in vivo disruption of Etv2 or its orthologues results in loss of Flk-1–expressing cells,25,26,30,32 whereas forced expression of this ETS factor leads to ectopic Flk-1 expression.25,27,29,31 Flk-1:GFP transgenic zebrafish embryos injected with etsrp-directed morpholinos demonstrate loss of GFP+ cells in blood vessels.25 Etv2 expression is observed exclusively within Flk-1+ mesodermal cells in mES cell differentiation studies, and its induction within embryoid bodies results in de novo production of Flk-1+ cells.26 Thus, Etv2 seems to regulate expression of VEGF receptor Flk-1 within newly forming endothelial cells. Consistent with this, Etv2 also seems to be required for the generation of Flk-1 + platelet-derived growth factor receptor α–vascular progenitors from Flk-1 + platelet-derived growth factor receptor α + primitive mesoderm.36

Combining what is known about the role of ETS tran-scription factors and the VEGF-A/Flk-1 pathway, we can surmise that ETS transcriptional activity in mesodermal progenitors is necessary, if not sufficient, to induce Flk-1 expression, as well as to promote commitment to an en-dothelial cell fate. VEGF-A signaling via Flk-1 likely regulates the survival and propagation of newly forming en-dothelial cells, although it does not likely directly regulate their differentiation.

Mesoderm

RA

PrimordialEndothelial Cells

HemogenicEndothelial Cells

Arterial Endothelial Cells

VenousEndothelial Cells

Lymphatic Endothelial Cells

VASCULOGENESIS

DEFINITIVE HEMATOPOIESIS

Hematopoietic Progenitor Cells

ARTERIAL -VENOUS SPECIFICATION

Etv2

Runx1

Notch

COUP-TFII

Nrp1

ephrinB2

EphB4

COUP-TFII , Sox18

Prox1

LYMPHANGIOGENESIS

IHHBMP4FGF2

VEGFR2Figure 1. Major extrinsic and intrinsic factors that regulate endothelial cell specification throughout embryonic vascular development. BMP4 indicates bone morphogenetic protein 4; FGF2, fibroblast growth factor 2; IHH, Indian hedgehog; RA, retinoic acid; and VEGFR2, VEGF receptor type I.


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Control of Endothelial Cell Proliferation During Vascular Plexus Remodeling

Once endothelial cells are differentiated and coalesce into a vascular plexus, their proliferation must be tightly regulated to achieve proper remodeling of an expanding circulatory net-work. This regulation involves the coordination of multiple signaling pathways that either promote or inhibit endothelial cell cycle progression (summarized in Figure 2).

VEGF-AAs mentioned above, VEGF-A is required to stimulate en-dothelial cell proliferation and does so largely via signaling through Flk-1. Binding of VEGF-A to Flk-1 triggers its auto-phosphorylation, leading to complex formation with integrin αvβ3,35 an endothelial cell adhesion receptor, and αvβ3 auto-phosphorylation.37 Acting in a feedback loop, integrin αvβ3 is also capable of phosphorylating Flk-1, once it is bound by its ligand vitronectin.38 The cross-activation of these 2 receptors leads to the recruitment and activation of Src kinases,39 which mediate the mitogenic effects of VEGF-A40,41 by activating the mitogen-activated protein kinase intracellular signaling pathway.38

Flt-1/VEGFR1 also indirectly controls endothelial cell pro-liferation, by competing with Flk-1 for binding of VEGF-A. Flt-1–/– mutants42,43 and zebrafish flt-1 morphants44 exhibit en-dothelial cell hyperproliferation that leads to abnormal angio-genic development, suggesting that unlike Flk-1, Flt-1 plays a negative role in endothelial cell proliferation. Although VEGF-A has a higher affinity for Flt-1 than Flk-1, Flt-1 has a weaker kinase activity and is thought to act in part as a VEGF-A decoy receptor.45 In support of this idea, genetic studies have shown that the Flt-1 tyrosine kinase domain is dispensable for vasculogenesis,46 whereas the extracellular ligand–binding domain is essential to its ability to regulate proper VEGF-A signaling.45 Moreover, alternative splicing results in a soluble, catalytically inactive Flt-1 isoform that

acts as a molecular sink for VEGF-A and thereby limits Flk-1 ligation and activation.47,48

Retinoic AcidRetinoic acid (RA) signaling also controls endothelial cell proliferation as a negative regulator of cell cycle progression. RA is the biologically active derivative of dietary vitamin A or retinol. In circulation, retinol is complexed to retinoid-binding protein and can enter the cell via a specific receptor, stimulated by retinoic acid 6. Retinol is initially oxidized into retinaldehyde by either alcohol dehydrogenases or retinol dehydrogenases. Retinaldehyde dehydrogenases then catalyze the conversion of retinaldehyde to all-trans-RA, which is released and can be taken up by surrounding tissues. Cellular RA-binding proteins facilitate the uptake of RA in target cells, as well as its transport to the nucleus. RA then binds to nuclear RA receptors (RARα, β, and γ), which heterodimerize with retinoid X receptors (RXRα, β, and γ) to form active transcription factor complexes. These complexes occupy RA-response elements within target genes, altering the binding of transcriptional repressors and coactivators to modulate gene expression. In cells with inactive RA signaling, RA is oxidized for degradation by the cytochrome P450 enzyme, CYP26 (reviewed in Duester49).

In the murine and rat yolk sac, Raldh2 is expressed spe-cifically by the visceral endoderm at the onset of vasculogen-esis,50,51 whereas yolk sac endothelial cells within the adjacent mesoderm exclusively express RA receptors RARα1 and 2.51,52 Mice lacking the enzyme Raldh2 are RA-deficient and die in utero at approximately E10. One of the first morpohological defects observed in these mutants is lack of remodeling and blood vessel dilation within the yolk sac and embryo proper, as well as a marked decrease in blood cell production.51–53 Raldh2–/– mutant endothelial cells are hyperproliferative and exhibit reduced levels of p21(Cdkn1a) and p27(Cdkn1b), members of the Cip/Waf family of cyclin-dependent kinase

Figure 2. Retinoic acid (RA) is produced by the visceral endoderm (VE) and initiates a signaling cascade that regulates control of endothelial cell cycle proliferation. RA indicates retinoic acid; RAR, RA receptor; RBP, retinoid-binding protein; TGFβ, transforming growth factor β; and VEGF, vascular endothelial growth factor. (Illustration credit: Ben Smith).


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(Cdk) inhibitors. In vivo and in vitro studies collectively sug-gest that RA regulates endothelial cell cycle progression by upregulating the expression of p21 and p27, resulting in G1 arrest of endothelial cells52 (Figure 2).

Transforming Growth Factor-β and Extracellular MatrixRA signaling also suppresses endothelial cell proliferation by upregulating transforming growth factor β-1 (TGF-β1). Signal transduction via TGF-β family members is facilitated by heteromeric type I and type II serine/threonine kinase re-ceptors. TGF-β first binds to the constitutively active type II receptor dimer, leading to recruitment, phosphorylation, and activation of type I receptor dimers. Type I receptors in turn phosphorylate receptor-regulated Smad proteins, which func-tion as effectors of TGF-β signaling by binding to common partner Smad proteins (co-Smads), such as Smad4. These TGF-β precomplexes then shuttle to the nucleus, where they associate with other transcription factors to regulate target gene expression (reviewed in Lee and Bae54).

TGF-β signaling contributes to the control of endothe-lial cell proliferation in 2 important ways; first, by inducing the production and deposition of fibronectin, an extracellu-lar matrix protein. Endothelial-derived fibronectin not only promotes visceral endoderm survival and function, which is necessary for VEGF-A production, but also binds to integrins α5β1 and αVβ3 expressed on endothelial cells to elicit oppos-ing effects on cell cycle progression. Fibronectin signaling via α5β1 inhibits proliferation, whereas binding to αVβ3 induces endothelial cell proliferation via the Flk-1/mitogen-activated protein kinase pathway51 (Figure 2). TGF-β signaling also controls endothelial cell cycle progression via the regulation of key cell cycle control genes. As previously discussed, pro-gression through G1 into S phase of the cell cycle is regu-lated by cyclin–cdk complexes, such as cyclin D–Cdk4/6 and cyclin E–Cdk2. Conversely, interaction between CdK inhibi-tors (such as RA-induced p21 and p27) and Cdks promotes cell cycle arrest. TGF-β signaling increases expression and stabilization of p15, a member of the INK4 family of CdK inhibitors.55,56 Increased p15 protein levels in the cytoplasm allow it to bind cyclin D–Cdk4/6 complexes more readily than p27, and active cyclin D–Cdk4/6-p27 complexes are re-placed by inactive cyclin D–Cdk4-p15 complexes. Displaced p27 molecules are shuttled toward and interact with cyclin E–Cdk2 complexes rendering them inactive because p27 bind-ing prevents Cdk2 activation that leads to G1 arrest.57 TGF-β signaling also contributes to stabilization of p27 by blocking its phosphorylation,58 a prerequisite for its export from the nucleus and subsequent degradation.59 Thus, regulation of en-dothelial cell cycle progression and proliferation during vas-cular plexus remodeling requires a balance between multiple signaling effectors and downstream intracellular pathways.

Angiogenic Sprouting: Selection of Tip Versus Stalk Endothelial Cells

Remodeling of the vascular plexus to form a fully differenti-ated circulatory network also involves the sprouting of new blood vessels from preexisting vessels, a process referred to as angiogenesis. Sprouting involves the loss of cell–cell junction

contacts between endothelial cells, degradation of the base-ment membrane by proteases, and migration of tip cells in re-sponse to angiogenic stimuli. Meanwhile, stalk cells trail tip cells and undergo extensive proliferation to expand the grow-ing endothelial tube.60,61 Not all endothelial cells exposed to proangiogenic signals become sprouting tip cells, and tight regulation of tip cell formation is necessary to ensure integrity and proper patterning of the developing vasculature.

Notch and VEGF SignalingInterplay between VEGF-A/Flk-1 and Notch pathways is pri-marily responsible for the specification of tip versus stalk cell identity during sprouting angiogenesis. There are multiple vertebrate Notch genes; humans and mice express ≥4 iso-forms (Notch1-4), and Notch1 and Notch4 are expressed by vascular endothelial cells. Evolutionarily conserved across di-verse species, Notch-mediated signaling occurs over short dis-tances and is commonly implicated in cell fate determination. Single-pass transmembrane Notch receptors are activated by engagement of ligands presented by neighboring cells and are cleaved by metalloprotease enzymes that remove most of the receptor extracellular domain. An enzymatic complex known as γ-secretase then cleaves the Notch transmembrane domain, releasing a Notch intracellular domain which is capable of translocating to the nucleus and forming complexes with other DNA-binding proteins (reviewed in Bigas and Espinosa62).

Expression of the Notch ligand, delta-like ligand 4 (Dll4) by tip cells of growing vascular sprouts is VEGF-dependent.63–65 Thus, VEGF-A provides an angiogenic stimulus that guides sprouting behavior and subsequent initiation of endothelial cell proliferation.65–68 Dll4 activates Notch signaling in adjacent endothelial cells, reducing their ability to respond to VEGF-A stimulation by downregulating Flk-1 expression.65,67 Dll4 thus functions to suppress tip cell fate by effectively conferring stalk cell identity on these neighboring cells.67,69,70 Impairment of Dll4-Notch signaling results in dysregulation of tip versus stalk cell specification. Dll4 heterozygous mutant mice and dll4 zebrafish morphants display increased angiogenic sprouting because of increased tip cell formation.63,67–69,71 Loss of Dll4 in mice also results in increased proliferation of endothelial cells.65,68,69 Similarly, inhibition of Notch signaling results in increased sprouting, increased expression of tip cell–associated genes,67 hyperproliferative endothelial cells, and a denser and more highly interconnected vasculature.67,69,70 Endothelial-specific deletion of Notch1 is sufficient to increase tip cell numbers.71 Interestingly, when both VEGF-A and Notch signaling are impaired, excessive sprouting and tip cell formation are not observed.63,67,69 VEGF and Notch regulation of angiogenic sprouting is therefore clearly complex; VEGF-A provides proangiogenic stimulus and induces Flk-1 expressing cells to sprout and form new vessels, while simultaneously activating Dll4-Notch pathway signaling to maintain the necessary balance between tip and stalk cell specification.

Interactions between the VEGF and Notch pathways are further complicated by the involvement of other members of these signaling pathways. For example, VEGFR-3 (or Flt-4) becomes ectopically expressed throughout remodeling vessels when Notch signaling is blocked.69 Endothelial cell–specific deletion of Flt-4 in mice leads to excessive angiogenic


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sprouting and branching in retinal vessels, resembling loss of Notch signaling. VEGF-C binds to Flt-4 in tip cells, promot-ing stalk cell specification.72 Flt-1 also acts as a negative regu-lator of tip cell differentiation in a Notch-dependent manner. Loss of flt-1 function in zebrafish results in a phenotype con-sistent with loss of Notch signaling, whereas overexpression of the notch1a intracellular domain in flt-1 morphants restores proper vessel branching and patterning.44

Other Notch ligands, including Dll1 and Jag1, are also in-volved in angiogenic sprouting with reported proangiogenic effects.73,74 Heterozygous deletion of Dll1 leads to reduced vessel branching, although it is not expressed by endothelial cells themselves. Dll1 is expressed by cells peripheral to the vasculature and acts as an extrinsic signal to activate Notch signaling in presumptive tip cells, which reinforces VEGF-A action by further inducing Dll4 expression to promote tip cell specification.73 Endothelial-specific deletion of Jag1 also results in reduced vessel sprouting. Stalk cells are enriched for Jag1, which antagonizes Dll4 signaling by competing for Notch receptor binding, and reduces Dll4-Notch activation in adjacent tip cells.74

Although Notch ligands can exert opposing effects on en-dothelial cells, proteasomal degradation of Notch signaling components positively regulates angiogenesis and tip cell specification. Endothelial cell–specific deletion or morpholi-no knockdown of Fbxw7, an E3 ubiquitin ligase, results in in-creased stability of Notch signaling components and impaired tip cell formation.75 B-cell chronic lymphocytic leukemia/lymphoma 6–associated zinc finger protein, a component of the cullin3-based E3 ligase complex, exerts its proangiogenic effect by promoting degradation of Notch signaling factor, C-promoter binding factor 1, thereby curtailing Notch signal-ing and lateral inhibition of tip cell phenotype.76

The specification of tip and stalk cell identities among en-dothelial cells is dynamic in both in vivo and in vitro sys-tems.77,78 Arima et al77 speculated that, in addition to other key functions, tip cells create a localized microenvironment that facilitates further elongation by the successive tip cell. Interestingly, among the genes enriched in tip cells are ex-tracellular matrix–degrading enzymes and secreted proteins nidogen-1 and -2, which interact with extracellular matrix proteins, such as laminin and collagen, to stabilize basement membranes.79 Laminin and laminin-binding integrins have also been found to stimulate Dll4-Notch signaling during tip versus stalk cell selection.80,81

MicroRNAMicroRNAs (miRs) are emerging as new players in the regu-lation of tip versus stalk cell specification of endothelial cells. Primary microRNA transcripts are transcribed from intergenic or intronic genomic sequences and then cleaved by the RNase III endonuclease Drosha, allowing export to the cytoplasm. Once there, miR transcripts are processed through the endo-nuclease Dicer to yield mature miRs, small RNA fragments 18 to 25 nucleotides long. These bind to the 3′-UTRs of their respective target mRNAs to mediate posttranscriptional gene silencing.82 In vivo silencing of either Dicer or Drosha results in reduced sprouting of endothelial cells, demonstrating an es-sential role for miRs in angiogenesis.83

miR-126 is known to elicit proangiogenic effects by re-pressing endothelial expression of Sprouty-related protein 1, a known negative regulator of VEGF-A signaling, thereby enhancing the angiogenic response of endothelial cells to VEGF-A signaling.84,85 Another proangiogenic microRNA is miR-221, which is also expressed by embryonic endothe-lial cells. Among its targets is p27,86 previously implicated in regulating endothelial cell proliferation in response to RA signaling.52 Notch signaling represses expression of miR-221, thereby allowing p27 expression and subsequent regulation of stalk cell proliferation.86 miR-27b also promotes endothelial cell tip specification, as well as increased branching of the vascular plexus, by downregulating Dll4 and Sprouty homo-logue 2 (Spry2) expression.87

Arterial-Venous SpecificationIn a properly remodeled blood circulatory system, the vascu-lature is subdivided into 2 interconnected, yet structurally and functionally distinct, networks of arteries and veins. Unlike veins, arteries have to withstand high blood pressure and are surrounded by several layers of smooth muscle cells and con-nective tissues. It was originally thought that endothelial cell identity is determined during development by hemodynamic forces. For example, Le Noble et al88 demonstrated that rerout-ing of blood flow in chick yolk sac arteries causes downregu-lation of arterial markers and conversion of endothelial cells to a venous phenotype. However, other studies suggest that arterial versus venous endothelial cell specification may also be genetically determined.

The ephrin family of receptor tyrosine kinases constitutes the largest family of growth factor receptors, which use membrane-tethered ephrins as their ligands. EphrinB2 marks arterial endothelial cells, whereas its receptor EphB4 is expressed by venous endothelium (Figure 1). Their complementary and mutually exclusive expression is observed during early stages of vascular plexus remodeling before the establishment of systemic blood circulation, which supports a genetic basis for the specification of arteries and veins.89 Furthermore, ephrinB2 knockout mice display severe angiogenic defects and a general lack of vessel remodeling, implying that reciprocal ephrinB2–EphB4 signaling may be crucial for the establishment of arteries and veins.89 In further support of a genetic program for arterio-venous specification, endothelial cells surrounding blood islands of the chick chorioallantoic membrane express both the arterial marker neuropilin-1 (Nrp-1) and venous marker Nrp-2 before the onset of blood flow.90 Hemodynamic forces may therefore not be required to induce the expression of genes that dictate arterial and venous endothelial cell phenotype and function but may instead be required for their maintenance, perhaps, via regulation of soluble signals that modulate their expression.

The roles of specific molecular signaling pathways in arterio-venous specification are just beginning to be defined (Figure 1). Thus far, it seems that VEGF-A binding to Flk 1 and its coreceptor Nrp-191 activates Notch signaling,92 pro-motes EphrinB2 expression, and suppresses EphB4,93 thereby establishing (or maintaining) arterial identity, while suppress-ing a venous fate in endothelial cells. Forkhead box C1 and C2 (Foxc1/2) is thought to act downstream of VEGF-A to induce


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Dll494 and Hey295 expression by binding Foxc sites on their promoters.95 Mice deficient in either Foxc1 or Foxc2 present with an abnormal vasculature that lacks arteries but displays normal venous marker expression, suggesting a general fail-ure of arterialization.94 Consistent with this idea, inhibition of Notch signaling in vivo results in an arterial to venous fate switch.96 mES cell differentiation studies also demonstrate that high VEGF-A levels induce arterial endothelial cell phenotype, whereas low to intermediate concentrations are associated with a venous identity. Wnt and hedgehog signal-ing pathways are also involved in the specification of arte-rial endothelial cells. β-catenin, a transcriptional coactivator of Wnt signaling pathway, upregulates Dll4 expression and amplifies Notch signaling by forming a complex with Notch intracellular domain to induce arterial fate in vascular endo-thelial cells.97,98 In the mouse, Sonic hedgehog acts upstream of VEGF-A92 via the Smoothened receptor to promote arterial endothelial cell specification and repress venous fate. This is similarly observed in zebrafish smoothened mutants, which have disrupted hedgehog signaling and develop an expand-ed posterior cardinal vein at the expense of a dorsal aorta.99 Constitutive activation of hedgehog signaling in ptc1; ptc2 mutants also results in conversion of the posterior cardinal vein into an artery.100

Specification of venous endothelial cells is thought to be mediated primarily by chicken ovalbumin upstream promot-er–transcription factor II (COUP-TFII), a member of the orphan nuclear receptor superfamily (Figure 1). Endothelial cell–specific deletion of COUP-TFII results in arterialization of veins, whereas ectopic expression results in fusion of veins and arteries.101 COUP-TFII functions cell autonomously by suppressing Nrp-1 function and downstream Notch signal-ing to maintain vein identity.101 Thus, the establishment and maintenance of arterial and venous endothelial cell fates are likely to be dynamically regulated by a balance of signals within the milieu of the remodeling vasculature during de-velopment; the relevance of such signaling processes to the maintenance of the postnatal established vasculature is not yet clear.

Lymphangiogenesis: Specification of LECs From Venous Endothelial Cells

The lymphatic system is essential for returning extravasated proteins and cells to the bloodstream and for proper immune system surveillance. These roles are clearly distinct from those of blood vessels and require extensive specialization of LEC. LEC are derived from embryonic veins; thus, their develop-ment requires the further specification of a subpopulation of venous endothelial cells to a lymphatic fate via signals distinct from those that specify venous endothelium.

Prospero Homeodomain Transcription Factor 1Prospero homeodomain transcription factor (Prox1) is a ho-meobox transcription factor expressed by the subpopulation of venous endothelial cells that sprout and migrate to form the lymphatic system.102 A lineage-specific marker for LEC, Prox1, is essential for embryonic lymphatic development103,104 (Figure 1). Venous endothelial cells begin expressing Prox1 at E9.75,105 and by E11.5, clusters of Prox1-positive lymphatic

progenitor cells emerge along the anterioposterior axis of the cardinal vein.102,103,106 These clusters undergo ballooning to generate intermediate structures known as lymph sacs, from which lymphatic vessels emerge by a combination of sprout-ing102,106 and migration of streams of Nrp-2–positive LEC from the cardinal vein.106 Ablation of Prox1 leads to a general fail-ure of LEC specification and ultimately, a failure of lymphatic vascular development.103–105 Prox1-null endothelial cells are devoid of expression of LEC markers, such as lymphatic ves-sel hyaluronan receptor 1 and VEGFR-3, and instead express blood vessel endothelial cell markers.104 Conditional deletion of Prox1 in venous endothelium results in a severe reduc-tion in LEC numbers.102 Moreover, LEC were shown to de-differentiate into blood endothelial cells on postnatal Prox1 inactivation.105 Prox1 is, therefore, not only necessary for the specification of lymphatic lineage but also for maintenance of LEC identity.

Sox18Although Prox1 expression in venous endothelium is necessary for LEC specification, not all venous endothelial cells express Prox1; instead, its initial expression is clustered and polarized along the anterior-posterior axis of the cardinal vein,103,106 suggesting tight regulation of its expression. Both the SRY-related genes Sox18 and COUP-TFII, which is necessary for specifying venous endothelial cell fate, were shown to regulate Prox1 (Figure 1). Sox18 is expressed by cardinal vein endothelial cells that eventually express Prox1 and form the lymphatic vasculature. Ectopic Sox18 expression in blood endothelial cells induces Prox1 and LEC marker expression; conversely, Sox18-null embryos lack LEC specification from cardinal vein endothelium.107 Although Sox18 was demonstrated to induce Prox1 expression by direct binding to its promoter,107 it is not sufficient to control the distinct expression pattern exhibited by Prox1 because arteries also express Sox18.108

COUP-TFIICOUP-TFII potentially cooperates with Sox18 to tempo-rally and spatially regulate Prox1 expression. In addition to being expressed by venous endothelium, COUP-TFII is also expressed by LEC,109 and its conditional ablation in ve-nous endothelial cells either alone102 or ubiquitously during early development110 results in impaired lymphatic specifica-tion.102,110 Along with Sox18, COUP-TFII binds to the regula-tory regions of Prox1111 to cooperatively induce its expression in LEC progenitors. COUP-TFII also forms a stable complex with Prox1 itself109,111 to maintain Prox1 expression in LEC and regulate lineage-specific expression of markers, such as Flt4/VEGFR3109 and Nrp-2.110 Accordingly, siRNA-mediated knockdown experiments demonstrated a cell-autonomous re-quirement for COUP-TFII during maintenance of lymphatic identity and VEGFC-induced lymphangiogenesis.110

MicroRNAMicroRNAs have also been reported to regulate Prox1 expres-sion. For example, miR-181a binds to the 3′-untranslated re-gion of Prox1 mRNA to inhibit its expression, thereby acting as a negative regulator of lymphatic specification and develop-ment.112 Similarly, miR-31 is expressed in venous endothelial


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cells and inhibits lymphatic specification by modulating Prox1 transcript and protein levels.113

VEGF-CAfter specification of LEC via induction of Prox1 expres-sion in a subset of venous endothelium, LEC migrate out of the veins to form lymph sacs and then lymphatic vessels.106 VEGF-C is necessary for the initial steps of lymphangio-genic development, and, as discussed above, expression of its receptors Flt-4 and Nrp-2 by LEC enables VEGF-C re-sponsiveness.109,110 Homozygous and heterozygous deletion of VEGF-C in mouse embryos results in a failure of Prox1-positive LEC progenitors to sprout, suggesting that specifi-cation of lymphatic lineage can proceed without VEGF-C, but that subsequent morphogenesis requires precise dosage of this secreted factor. VEGF-C, but not VEGF-A, can res-cue the sprouting defect observed in these mutants, indicating specificity of Flt-4 function during lymphangiogenesis.114 In support of this, several studies have demonstrated a direct as-sociation between inhibition of Flt4 function and impairment of lymphatic system development.115–118 Several mechanisms for regulation of Flt-4 expression in LEC have been proposed. As previously mentioned, COUP-TFII induces expression of both Flt-4 and Nrp-2.109,110 Tbx1 binds to an enhancer el-ement in Flt-4 regulatory regions to induce VEGF-C recep-tor expression in LEC.119 Tbx1 itself is required for lymphatic vessel development.119 The ETS factor Ets-2 interacts with Prox1 to induce Flt-4 gene expression and effectively en-hance the lymphangiogenic response to VEGF-C.120 Finally, Notch has been shown to induce Flt-4 expression in endothe-lial cells by binding to its promoter in a complex consisting of C promoter–binding factor 1/suppressor of hairless/lag1, allowing these endothelial cells to increase their responsive-ness to VEGF-C.121 Interestingly, the Notch signaling pathway may also be active during the specification phase of lymphan-giogenic development. Knockdown of Dll4, or its receptors Notch1b or Notch6, in zebrafish severely impairs LEC sprout-ing and lymphatic system development.122

Hemogenic Endothelium: Specification of Hematopoietic Potential in Primordial

Endothelial CellsHemangioblast Versus Hemogenic Endothelium in Blood ProductionThe initial but transient phase of blood development known as primitive hematopoiesis involves the emergence of aggregates of mesoderm-derived cells at E7.0 to 7.5 within regions of the murine yolk sac referred to as blood islands (Figure 3A). Cells within these structures develop into nucleated primitive eryth-roblasts and megakaryocytes, whereas those along the border flatten and become endothelial cells.123,124 The close spatial and temporal development of blood and endothelial cells dur-ing this early phase of hematopoietic development led to the suggestion that a common bipotent progenitor known as the hemangioblast gives rise to both endothelial and hematopoi-etic cells. To date, however, the existence of hemangioblasts in any species and their relative contribution to hematopoietic development remain controversial.

A second wave of blood development known as definitive hematopoiesis involves the generation of all blood cell lineag-es. The extraembryonic yolk sac is the first site of definitive hematopoietic development (≈E8.25; Figure 3A); thereafter, multilineage hematopoietic stem/progenitor cells (HSPC) develop successively within the extraembryonic placenta (≈E9.5), as well as the embryonic aorta/gonad/mesonephros (AGM) region (≈E9.5–10.0), the fetal liver (E11.0–12.0), and the fetal bone marrow, where hematopoiesis persists postna-tally. It is during definitive hematopoiesis that a subset of the vascular endothelium, referred to as hemogenic endothelium, acquires blood-forming potential and gives rise to the first multilineage HSPC.

The first observations suggesting that endothelial cells give rise to blood cells came from studies of developing chick embryos, wherein endothelial cells of the aorta and yolk sac were found to reside in close proximity to hemoglobin-filled progeny, which seemed to project into the blood vessel lumen and bud off into the blood plasma.125,126 Many years later, in an attempt to prove this experimentally, the chick endothe-lium was labeled with Di-AcLDL before the onset of defini-tive hematopoiesis; dye-retaining hematopoietic clusters were demonstrated in direct physical association with the vascular endothelium of the aorta and within the blood circulation.127 Aortic endothelial-attached hematopoietic clusters were also noted by others in both mouse and human embryos, as well as in the lumens of murine yolk sac blood vessels.128–131 Phenotypic analysis of blood and endothelial cells revealed common expression of cell-surface markers during embryo-genesis,127,128,132 providing further evidence of their coordinat-ed development in vivo.

More recent lineage tracing and single cell–imaging studies have provided direct evidence that multipotent hematopoietic progenitors arise developmentally from vascular endothelium. Cells initially expressing the endothelial cell marker cluster of differentiation 31 (CD31) and then blood markers, CD41, stem cell antigen-1, and c-Kit, were shown to emerge from the ventral wall of explanted murine dorsal aortae.133 Endothelial lineage tracing also revealed hematopoietic cell clusters budding from reporter-labeled endothelium of the murine dorsal aorta, which were then found in the fetal liver and subsequently within the adult hematopoietic system.134 Similar studies in zebrafish demonstrated early hematopoietic cells emerging from the ventral wall of the dorsal aorta, expressing definitive hematopoietic genes and repopulating the adult hematopoietic organ.135,136 These studies provide direct evidence that specialized hemogenic endothelial cells generate definitive blood cells; however, we still know relatively little regarding mechanisms of their specification.

Phenotype of Hemogenic Endothelial CellsTo gain insight into the specialized functions of hemogenic endothelial cells and the molecular pathways that regulate their development, it is necessary to define their phenotype relative to non–blood-forming vascular endothelium. In vitro assays performed on sorted cell populations expressing both endothelial and hematopoietic markers have been used to try to define the phenotype of endothelial cells with hematopoietic potential. VE-cadherin+Ter119−CD45− endothelial cells that


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coexpress CD31, CD34, and Flk-1 were isolated from mu-rine yolk sac and AGM and shown to exhibit lymphopoietic potential in colony formation assays.137 CD34+CD31+CD45− endothelial cells from human yolk sac and AGM also display lymphomyelopoietic potential when cultured with supporting stromal cells.130 In other studies, Flk-1+VE-cad+CD45− endo-thelial cells were shown to generate Flk-1−CD45+ hematopoi-etic colonies more readily than those that were VE-cad−.138 Although these studies suggest that expression of VE-cadherin is necessary for hematopoietic potential, it was not found to be required for hemogenic endothelial cell function in the murine yolk sac.139 However, Hoechst dye-efflux capacity, characteris-tic of side population (SP) cells,139,140 was found to be an essen-tial characteristic of all blood-forming cells in the murine yolk sac and AGM. Furthermore, it was found that among SP cells within these tissues, Flk-1+c-Kit+CD45− SP cells specifically represent hemogenic endothelial cells that exhibit multilineage hematopoietic potential on a clonal level139 (Figure 3B). These cells are distinguishable from non–blood-forming vascular endothelial cells, which do not exhibit an SP phenotype and do not express c-Kit. It was further established that Flk-1+c-Kit+CD45− SP cells give rise to Flk-1−c-Kit+CD45+ SP cells, which represent multilineage HSPC within these tissues.139 HSPC are distinguishable from mature blood cell types, which neither exhibit an SP phenotype nor express Flk-1 or c-Kit, al-though they retain expression of CD45. Thus, flow cytometric analysis enables identification and isolation of these cell types for further investigation of function and molecular regulation.

Specification of Hemogenic Endothelial CellsOnly a small subset of the primordial endothelium within the yolk sac and AGM acquires hematopoietic potential and gives rise to HSPC. Thus, they are present in small numbers in these tissues, which limits the study of their development and function. Consequently, little is known about the molecular mechanisms that regulate hemogenic endothelial cell specification and their generation of HSPC; nonetheless, we review what is known thus far about factors implicated in these processes and summarize the impact of their deletion on murine hematopoietic development in the Table.

Retinoic AcidRA signaling has been found to be essential for normal he-matopoiesis in the developing embryo and in the adult. Early studies using vitamin A (retinol)-deficient quail embryos dem-onstrated impaired proliferation and survival of yolk sac prim-itive erythrocytes, despite their normal formation.142 Genetic approaches also demonstrated a requirement for RA receptors in these processes. Mice deficient for the RXRα exhibit im-paired fetal liver erythropoiesis.143 Ablation of RARγ results in reduced generation of bone marrow hematopoietic stem cell (HSC), whereas its overexpression in HSPC promotes an undifferentiated phenotype, suggesting a role for RA sig-naling in maintaining the balance between HSC self-renewal and differentiation.144 Finally, chromosomal translocations involving the RARα gene result in acute promyelocytic leu-kemia, wherein the resulting oncogenic fusion proteins block

Figure 3. Parallel development of blood and blood vessels in early vertebrate embryos. A, In the extraembryonic yolk sac (YS), aggregates of mesodermal cells form blood islands composed of primitive erythroid cells and angioblasts during the late gastrulation phases (E7.0). A primitive vascular plexus is soon formed by coalescence of blood islands, and by the time definitive hematopoietic development is initiated (E8.25), the vascular plexus begins to be remodel to a vascular network composed of large vitelline veins, as well as smaller, pruned vessels. Definitive hematopoiesis is marked by the generation of multiple, if not all, blood cells lineages from hemogenic endothelial cells, which give rise to blood clusters initially observed in the yolk sac tissues and later intraembryonically, on the ventral side of the aorta-gonads-mesonephros region (AGM; as early as E9.5). B, Intrinsic and extrinsic factors that regulate hemogenic endothelium specification and hematopoietic stem/progenitor cell development. Phenotypic markers that define relevant cell subpopulations during definitive hematopoiesis are also indicated. RA indicates retinoic acid; and RAR, RA receptor. (Illustration credit: Ben Smith).


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terminal differentiation and increase self-renewal of leukemic progenitor cells.145

RA-deficient (Raldh2–/–) embryos also exhibit impaired blood cell production, coincident with previously discussed endothelial cell hyperproliferation.51,52 Although primitive he-matopoiesis is not impaired in Raldh2–/– embryos, they do not undergo hemogenic endothelial cell development and exhibit a concomitant decrease in HSPC production.139 The expres-sion of key hematopoietic regulators, including c-Kit, Runx1, and c-Myb, is also significantly downregulated in Raldh2–/– embryos. Restoration of RA signaling in Raldh2–/– mutants in utero, via maternal feeding of active RA during the critical

developmental period encompassing the onset of definitive hematopoiesis, rescues the observed defects in hemogenic endothelial cell development and definitive hematopoiesis. RA signaling is therefore required for proper specification of hemogenic endothelial cells from primordial endothelium and for subsequent formation of multilineage HSPC;139 thus, de-fining the direct targets of RA during these processes is criti-cally important.

c-KitEndothelial cells with hematopoietic potential uniquely express c-Kit, which is downregulated in the absence of RA signaling. c-Kit/CD117 belongs to a family of growth factor receptors with tyrosine kinase activity. Binding of its ligand, stem cell factor, results in dimerization or oligomerization of receptors and activation of tyrosine kinase activity, leading to rapid phosphorylation of its cellular targets. The result is the activation of intracellular signal transduction pathways, which depending on the cellular context, include the Src kinase, Ras/mitogen-activated protein kinase, and phospholipase C-γ pathways. c-Kit can also signal via phosphatidylinositol-3 kinase, which is usually associated with control of cell proliferation, migration, and adhesion. Activation of the Janus kinase/signal transducer and activator of transcription pathway by c-Kit is most commonly associated with maintenance and self-renewal of HSC (reviewed in Sattler and Salgia146). Deletion of c-Kit results in early embryonic lethality in murine embryos,147 whereas adult mice harboring mutant c-Kit alleles demonstrate reduced HSC numbers (reviewed in Sharma et al148). Fetal and adult tissues carrying mutations in the c-Kit kinase domain also display decreased colony-forming activity in vitro, suggesting that c-Kit expression is required for HSC expansion. c-Kit is also frequently coexpressed with Flk-1 in vascular endothelial cells that are associated with hematopoietic clusters in the murine yolk sac and dorsal aorta,129 suggesting that c-Kit may be involved in hemogenic endothelial cell formation and function.

Hematopoietic Stem/Progenitor Cell Development and Propagation

Although the signaling pathways that regulate the specifica-tion of hemogenic endothelial cells from primordial endothe-lial cells have not been clearly defined, more is known about regulators of hematopoietic stem and progenitor cell genera-tion and function.

Notch SignalingSeveral studies demonstrate the involvement of Notch signal-ing in multiple stages of blood cell development or function. For example, Notch1–/– mutant explants exhibit significantly impaired hematopoietic colony formation, and transplanta-tion studies indicate that Notch1 is required for HSC activ-ity and definitive hematopoiesis in both mouse yolk sac and AGM. Thus, Notch1 seems to be required for the generation of HSC from hemogenic endothelial cells,149 as well as for the establishment of long-term, self-renewing HSC.150 Ablation of recombining binding protein suppressor of hairless-jκ, a co-factor for Notch signaling, also leads to loss of HSPC and loss of expression of the GATA2 transcription factor,151,152 which

Table. Loss-of-Function Phenotypes of Genes Associated With Hemogenic Endothelial Cell and HSPC Development

Gene Loss-of-Function Phenotype Reference(s)

Hemogenic endothelial cell specification

Raldh2 Embryonic lethal at E9.5–10.0; severe anemia and cardiovascular defects; impaired hemogenic endothelial cell and HSPC development

53,139

c-Kit Early to midgestational embryonic lethality; severe anemia and failure of hematopoietic stem cell, erythroid, and mast cell development; mutant, viable alleles display pleiotropic hematopoietic defects

146,147

HSPC formation and function

Notch1 Embryonic lethal at E9.5–10.0; severe vascular defects and impaired HSPC maintenance and self-renewal

148,152

RBPjκ Embryonic lethal at E10.5; failure to undergo embryonic turning, severe defects in somite development; complete absence of intraembryonic (AGM) hematopoiesis

150,151

Runx1 Embryonic lethal at E11.5–12.5; severe hemorrhaging and defects in definitive hematopoiesis

155

Scl/Tal1 Embryonic lethal at E9.5; severe anemia, edema, and impaired definitive hematopoiesis

163

Lmo2 Embryonic lethal at E10.5; failure of yolk sac hematopoiesis (erythropoiesis)

164

GATA1 Embryonic lethal at E10.5–11.5; severe anemia and defects in erythropoiesis

141

GATA2 Embryonic lethal at E10.5; severe anemia and impaired proliferation and survival of hematopoietic progenitors

165

c-Myb Embryonic lethal at E15.5; failure of erythropoiesis and maintenance of hematopoietic progenitor cells

178

AGM indicates aorta-gonad-mesonephros; and HSPC, hematopoietic stem/progenitor cell.


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is required for maintenance of HSC in an undifferentiated state. There may be earlier functions of Notch signaling in the specification of hemogenic endothelium because Notch1–/–

Notch4–/– mutants die at midgestation with defects in vascular remodeling and lack of establishment of specialized endothe-lial cell phenotypes.153

Runx1Also known as AML1, Runx1 has been shown to function downstream of Notch during developmental and adult hema-topoiesis.149,154,155 Runx1 encodes a transcription factor that het-erodimerizes with core binding factor β via its evolutionarily conserved Runt homology domain. Ablation of Runx1 results in embryonic lethality at E11.0 to E12.0 because of defects in definitive hematopoiesis.156 Runx1 expression by yolk sac and AGM endothelial cells is observed before the emergence of hematopoietic clusters131,157 and is necessary for HSPC for-mation.131,158,159 Once the endothelial-to-hematopoietic pro-genitor transition is completed, Runx1 function is no longer required.159 A novel role for Runx1 as a transcriptional repres-sor in hemogenic endothelial cells was suggested by mES cell differentiation studies, in which Flk-1–expressing cells were shown to initiate Runx1 expression, leading to silencing of the Flk-1 promoter and downregulation of receptor expression.160 This is consistent with a previous study demonstrating sup-pression of the Flk-1 regulatory region in Runx1-expressing hemogenic endothelial cells.161 Flk-1 gene silencing has been independently associated with the generation of HSPC from hemogenic endothelial cells.

Stem Cell Leukemia Protein/LIM Domain Only 2/Gata1/2 Transcription ComplexThe regulatory regions of hematopoietic genes, including the early hematopoietic marker CD41, are simultaneously bound by Runx1 and a transcription factor complex composed of stem cell leukemia protein (Scl)/T-cell acute lymphocytic leu-kemia protein-1 (Tal-1), GATA1, and GATA2.162,163 Mice de-ficient in Scl/Tal-1, LIM domain only 2 (Lmo2), or GATA1/2 all display midgestation lethality caused by severe hemato-poietic defects,164–167 suggesting that these transcription factors have closely related roles during embryonic hematopoiesis. Morpholino knockdown of scl and lmo2 in zebrafish results in similar phenotypes, including loss of intra-aortic hema-topoietic clusters and impaired expression of hematopoietic genes.168,169 ES cells deficient in Lmo2 also fail to contribute to blood cell production or to the endothelium of large ves-sels in adult chimeric mice.170,171 Both Scl/Tal-1 and Lmo2 are expressed by hematopoietic and vascular endothelial cells in midgestation embryos.169,170,172 Scl is necessary for the initial specification of hematopoietic lineage173,174 but dispensable for later fetal liver and adult HSC functions.173,175 Expression of GATA2, which promotes the expansion of hematopoietic progenitors, is enriched within blood islands during primi-tive hematopoiesis.166,176 This is followed by induction of GATA1 expression,176 which in turn represses GATA2, lead-ing to a developmental switch from proliferation to differ-entiation.177 Lmo2 acts as a bridging molecule between Scl/Tal-1 and GATA-binding proteins in a transcriptional trans-activating complex that has been shown to drive vertebrate

hematopoietic commitment.168,178 Although this complex is known to play a critical role in primitive hematopoiesis, it also likely regulates HSPC production and function during defini-tive hematopoiesis.

c-MybAnother transcriptional regulator thought to play a role in the regulation of HSPC self-renewal and propagation is c-Myb. Ablation of c-Myb results in embryonic lethality at E15.5 caused by failure of erythroid and myeloid development,179 whereas conditional deletion in adult bone marrow results in depletion and impaired self-renewal of the HSC pool.180 Loss of function studies in zebrafish also demonstrate impaired definitive hematopoietic development, despite intact primi-tive hematopoiesis.181 AGM explants from c-Myb–/– mutant mice also yield fewer HSPC,182 which usually express high c-Myb levels that are downregulated during differentiation. Consistent with this finding, overexpression of c-Myb results in inhibition of erythromyeloid differentiation.183

Blood FlowIn addition to soluble factors and transcriptional regulators, blood flow and hemodynamic forces also play a role in the regulation of HSC development. Endothelial cells lining blood vessels are known to respond to fluid forces through a complex of receptors capable of transducing biomechanical signals. Such signals initiate a cascade of downstream signal-ing events that precipitate changes in gene expression and cell behavior (reviewed in Li et al184). For example, shear stress regulates endothelium-dependent NO production, which is a known regulator of hematopoiesis. A decrease in hemato-poietic potential of the murine AGM is observed when the NO pathway is inhibited.185 In zebrafish, inhibition of NO synthase, the enzyme responsible for NO production, leads to a decrease in transplantable HSC; Nos3–/– knockout mice exhibit a similar phenotype.186 Recapitulating the hemody-namic environment of the murine AGM in an in vitro ES cell system significantly increases the generation of CD31-expressing endothelial cells, upregulates expression of HSC-associated genes, such as Runx1 and c-Myb, and increases the hematopoietic potential of differentiated CD41+c-Kit+ HSC.185 Zebrafish silent heart mutants, which lack a heartbeat and an established circulation, display impaired HSC generation.186 Ncx1–/–-null embryos, which also lack a heartbeat, properly initiate HSC development within the placenta, yet display fewer CD41-expressing hematopoietic cells than their wild-type counterparts.187 Collectively, these studies suggest that blood flow forces may not be required for hemogenic endothe-lial cell specification but may be necessary to promote HSC emergence or maintenance.

SummaryDuring vasculogenesis, multipotent mesodermal progenitors give rise to endothelial cells that form a primary vascular plexus. Tight control of endothelial cell proliferation and mi-gration, as well as proper specification of tip and stalk cells, enables endothelial cell sprouting and vascular plexus remod-eling. Endothelial cells become phenotypically and function-ally specialized because they acquire arterial, venous, and


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hemogenic fates. A subpopulation of venous endothelial cells later assumes a lymphatic endothelial identity. Precise regula-tion of these processes is not only necessary to ensure proper architecture and integrity of the circulatory system but also to guarantee that endothelial cells that line the blood and lym-phatic vessels are capable of performing their necessary func-tions. Perturbations of the molecular mechanisms that govern these events during development often results in embryonic lethality; defects in these processes postnatally contribute to the development of prevalent vascular, lymphatic, and hema-topoietic pathologies. An integrated understanding of how endothelial cells acquire distinct phenotypes, thus enabling specialized functions, not only extends our knowledge of the development of the circulatory system but also provides in-sights needed to optimize the treatment of prevalent diseases, as well as generate such cell types for tissue engineering and regenerative medicine applications.

Sources of FundingThese studies were supported by National Institutes of Health (NIH) grants R01-HL077675 and R01-HL096360 to K.K. Hirschi, and USDA/ARS CRIS 6250 51000 to K.K. Hirschi and L.C. Goldie.

DisclosuresNone.

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Kathrina L. Marcelo, Lauren C. Goldie and Karen K. HirschiRegulation of Endothelial Cell Differentiation and Specification

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