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. Author manuscript; available in PMC: 2009 Oct 27.
Published in final edited form as: Circ Res. 2008 Sep 12;103(6):573–579. doi: 10.1161/CIRCRESAHA.108.180745

Role of Cross Talk between PI3-Kinase and ERK/MAP Kinase Pathways in Artery-Vein Specification

Charles C Hong 1,2, Tsutomu Kume 1,3, Randall T Peterson 4
PMCID: PMC2768581  NIHMSID: NIHMS126759  PMID: 18796644

Abstract

Functional and structural differences between arteries and veins lie at the core of the circulatory system, both in health and disease. Therefore, understanding how artery and vein cell identities are established is a fundamental biological challenge with significant clinical implications. Molecular genetic studies in zebrafish and other vertebrates in the past decade have begun to reveal in detail the complex network of molecular pathways that specify artery and vein cell fates during embryonic development. Recently, a chemical genetic approach has revealed evidence that artery-vein specification is governed by cross talk between phosphoinositide-3-kinase (PI3K) and extracellular signal regulated/mitogen activated protein kinase (ERK/MAPK) signaling in artery-vein specification. We discuss recent findings on the signaling pathways involved in artery-vein specification during zebrafish development, and compare and contrast these results to those from mammalian systems. It is anticipated that the complementary approaches of genetics and chemical biology, involving a variety of model organisms and systems, will lead to a better understanding of artery-vein specification, and possibly to novel therapeutic approaches to treat vascular diseases.

Keywords: Artery-Vein Specification, Signaling Crosstalk, Shh, VEGF, Notch, Fox

Introduction

Arteries and veins are functionally and structurally distinct, and disturbances in artery and vein specialization are thought to be responsible for many of human vascular pathologies. The arterial system, with its thick outer layers of elastic fibers and smooth muscle, is a high-pressure conduit that is susceptible to atherosclerosis. The venous system, with its relatively thin supporting layers, is a low-pressure/high capacitance conduit that is relatively more prone to thrombosis. In the body, arteries and veins are typically found in close proximity to each other, but their distinct identifies are strictly maintained. Loss of this distinction due to improper establishment of artery-vein cell identity is thought to result in congenital arteriovenous malformations (AVMs)1 and potentially devastating diseases like hereditary hemorrhagic telangiectasia (HHT)2. Thus, a better understanding of how distinct artery and vein identities arise and are maintained may lead to future therapies for vascular disease.

Here, we review recent findings in zebrafish that reveal the genetic program for establishing artery-vein cell identity during embryogenesis. In addition, we discuss relatively novel chemical genetic studies of zebrafish vascular development that have provided evidence that a cross-talk between two ubiquitous signaling pathways, the phosphoinositide-3-kinase (PI3K) and the extracellular signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathways, plays a central antagonistic role in artery-vein specification during vasculogenesis.

Genetic control of artery-vein cell fates in the zebrafish embryo

The zebrafish, which provides a unique complement of embryologic and genetic tools for the study of vertebrate development, has had a major impact on the mechanistic understanding of artery-vein specification3,4. In zebrafish, endothelial progenitor cells, called angioblasts, first become evident in two parallel stripes of lateral plate mesoderm (LPM) by 12 to 14 hours post-fertilization (Figure 1)5. Within 2 hours of their formation, angioblasts begin their migration to the dorsal midline and, by 18 hpf, angioblasts coalesce at the midline to form the two major axial vessels, the dorsal aorta and the posterior cardinal vein (Figure 1)6,7. Prior to the commencement of circulation at 24 hours post-fertilization (hpf), dorsal aorta expresses arterial endothelial markers, such as Ephrin-B2a, Notch5 and Gridlock, while the posterior cardinal vein expresses venous endothelial markers, such as EphB4 and Flt4 (fms-related tyrosine kinase 4)8,9. In addition to the vessel-specific marker expression, the two axial vessels are clearly distinguishable before the onset of circulation by their spatial relationship within the developing embryo: the dorsal aorta always lies dorsal to the posterior cardinal vein (Figure 1)8. Remarkably, the arterial-venous cell fate decision appears to be made very early, even before the angioblasts migrate to the midline. Cell-tracing experiments with a fluorescent lineage tracer show that individual angioblasts in the LPM give rise to either arterial or venous endothelial cells, but not both10.

Figure 1. Schematic cross section of the trunk of zebrafish embryos showing endothelial development.

Figure 1

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(A) mid-somite stage embryo (12 to 14 hpf, 6 to 10 somites). At this stage, angioblasts are formed in the lateral plate mesoderm (LPM). In response to midline signaling by sonic hedgehog (Shh), secreted by notochord (NC), and VEGF, secreted by somites (S), the angioblasts migrate medially. (B) 24 hpf embryo. Upon completion of the migration, angioblasts situated dorsally adjacent to NC differentiate into the dorsal aorta (A) while ventral angioblast differentiate into the posterior cardinal vein (V). Dorsal is to the top. Schematic figure is based on findings presented in Gering and Patient.14

Investigations in the zebrafish have made seminal contributions in defining the molecular pathways required for the acquisition of the arterial cell fate during embryogenesis11. This pathway involves many of the signaling components known to play important roles in endothelial cell biology, including Sonic hedgehog (Shh), Vascular endothelial growth factor (VEGF), Notch, Phosphoinositide-3 kinase (PI3K) and Extracellular signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling12. Many of these have been shown to have pleiotropic effects essential to diverse aspects of vascular development. For example, Shh, which is expressed in the notochord (a dorsal midline structure)11, and VEGF, which is expressed in somites (future trunk muscles just lateral to the notochord), also serve as extracellular signals critical for the midline migration of angioblasts (Figure 1)13,14.

In a series of elegant zebrafish experiments using mRNA and morpholino antisense RNA injections, together with genetic mutants and transgenic embryos, Weinstein and colleagues revealed a genetic hierarchy of several signaling events that induce endothelial arterial fate (Figure 2)11. At the top of this hierarch is Shh, a member of the Hedgehog family of signaling molecules known to be an indirect angiogenic signal that regulates the expression of VEGF isoforms, and Angiopoietin-1 and Angiopoietin-2 in interstitial mesenchymal cells15. In zebrafish, loss of Shh signaling through genetic mutations in sonic-you (syu), encoding Shh, and you-too (yot), encoding its downstream activator Gli2a, or through pharmacological inhibition with cyclopamine results in the loss of the arterial marker Ephrin-B2a and the expansion of the venous marker Flt4 in the vasculature11. Conversely, overexpression of Shh caused formation of ectopic Ephrin-B2a–expressing presumptive arterial cells within the trunk vessels.

Figure 2. A model for molecular pathway for specification of artery and vein fates.

Figure 2

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This model is based largely on findings in zebrafish, plus Foxc1/2 and COUP-TFII results in mice (noted by dashed arrows). Shh, expressed in the notochord and hypochord (on top), induces VEGF expression in nearby somites. By a process yet to be fully determined, VEGF activates the PLCγ-ERK/MAPK pathway only in the dorsal angioblasts (pink circle), which will develop into aortic endothelial cells. ERK activation results in Notch activation (indicated by Notch and Dll4 expression) via the transcriptional factors Foxc1/2. Notch signaling induces arterial differentiation (indicated by the arterial marker EphrinB2 expression). In the arterial progenitor cells, Grl, induced by Foxc1/2, blocks venous differentiation (indicated by the venous marker EphB4 expression. In the ventral angioblasts (blue circle), VEGF activates the PI3K/AKT pathway, which inhibits the PLCγ-ERK/MAPK pathway, possibly by a direct inhibition of Raf by AKT. AKT signaling is hypothesized to induce expression of COUP-TFII, which blocks arterial differentiation (also EphrinB2 expression). In this model, a gradient of VEGF along the dorsal-ventral axis is postulated to govern whether VEGF receptor activates PI3K or ERK signaling. For simplicity, the artery-vein specification is depicted here as occurring following the midline convergence of endothelial progenitors, but evidence suggests that this occurs earlier in zebrafish embryos. Schematic figure is based on discussions presented in Lamont and Childs.12

Induction of the arterial fate by Shh is mediated by VEGF. VEGF expression in the somites is induced by and dependent on Shh signals from the adjacent notochord. Knockdown of VEGF using morpholino antisense RNA results in the loss of arterial marker Ephrin-B2a expression11. Conversely, overexpression of VEGF results in upregulation of artery-specific markers and can rescue arterial marker expression in the absence of Shh signals. While distinct combinations of multiple VEGF receptors are known to be required for development of different endothelial types in zebrafish, the VEGF receptor-2 homolog, Kdra, appears to play a dominant role in artery differentiation16. A forward genetic screen has revealed the involvement of phospholipase Cγ-1 (PLCγ1), a known downstream signaling component of various receptor tyrosine kinases including the VEGF receptors, in the arterial specification pathway. Like embryos lacking VEGF, PLCγ1 mutant embryos show specific defects in artery formation17. Overexpression of VEGF mRNA fails to restore arterial markers, suggesting that PLCγ1 is a key downstream transducer of VEGF signals for arterial development. Evidence in zebrafish indicates that Notch signaling acts downstream of both Hedgehog and VEGF signaling to induce the arterial differentiation by suppressing the venous fate. Disruption in Notch signaling through a mutation in mindbomb (mib), necessary for Notch activation, or via expression of dominant-negative suppressor of hairless [Su(H)], a downstream mediator of Notch signaling, abrogates the expression of arterial makers and induces ectopic expression of venous markers18. Conversely, ectopic activation of Notch signaling by the overexpression of constitutively active Notch intracellular domain [NICD] induces ectopic arterial marker expression and blocks vein marker expression. In addition, activation of Notch signaling can induce ectopic arterial marker expression in the absence of VEGF signaling. However, forced expression of VEGF in mib mutants does not restore artery marker expression, suggesting that Notch acts downstream of VEGF signaling.

Gridlock, a member of the Hairy-related family of transcription factors that act downstream of the Notch signaling, is also involved in artery specification in zebrafish embryos10. A hypomorphic mutation in the gridlock (grl; zebrafish hey2 homolog) gene results in a focal defect in the dorsal aorta. Gridlock is first expressed in the lateral plate mesoderm, but its expression becomes restricted to the dorsal aorta. Morpholino knockdown of gridlock led to progressive loss of dorsal aorta and concomitant enlargement of posterior cardinal vein. Consistent with Gridlock’s role in artery-vein specification, overexpression of Gridlock suppressed venous markers. However, Gridlock overexpression did not result in ectopic expression of artery markers, suggesting that it functions specifically to block the venous state. Whether Gridlock is directly downstream of Notch signaling is unclear since its vascular expression remains intact following expression of dominant-negative Su(H) or in mib mutants11.

Artery-vein Specification in Mammals: A Brief Comparative Overview

Studies in zebrafish have provided a crucial general framework for understanding the key aspects of mechanistic understanding of artery-vein specification during embryogenesis. Indeed, many signaling events in zebrafish are faithfully recapitulated in higher vertebrates19. Nonetheless, studies in mammalian systems have highlighted some differences between the two models as well as provided novel insights to artery-vein specification that are yet to be confirmed in zebrafish.

While the roles of Hedgehog and VEGF in artery-vein specification have been elegantly demonstrated in zebrafish, evidence for their involvement in mammalian artery-vein specification during development is less direct. In mice, disruption of Shh (Sonic Hedgehog), which is expressed in the notochord, does not result in obvious vascular defects20. Nonetheless, Hedgehog signaling may play a role in arterial specification in mice, as mice lacking Smoothened (Smo), a transmembrane protein that transduces Hedgehog signal, exhibit defective dorsal aorta formation, although this could be due a general defect in endothelial tube formation rather than a primary defect in artery-vein specification21. In addition, mice with disruptions in Ihh (Indian Hedgehog) or Smoothened (Smo) exhibit blocks in remodeling of the yolk sac vasculature, but it is unclear whether this is a consequence of a primary defect in artery-vein specification22. Mice lacking VEGF or PLCγ1 show such a severe disruption in overall vascular development that a definitive conclusion about the direct role of VEGF or PLCγ1 in artery-vein specification cannot be made23,24. However, a more selective perturbation of VEGF expression did result in defective arterial development in the mouse retina25.

In mice, Notch1 and Notch4, as well as Notch ligands Jagged1, Jagged2, Delta-like1 (Dll1) and Delta-like4 (Dll4) are selectively expressed in arterial endothelial cells26,27, suggesting that Notch signaling is required for arterial identity (reviewed in Roca and Adams28). In support of this notion, the Dll4 ligand is required for mouse artery development in a dose-dependent manner29. Studies in mice suggest that Gridlock/Hey2 and related Hey1 play an important and redundant role in mouse arterial specification. Although mice with a single knockout of Gridlock homolog Hey2 do not exhibit vascular defects, the compound Hey1:Hey2 double knockout mice show loss of the expression of arterial marker Ephrin-B230. Moreover, two members of the forkhead/Fox transcription factor family Foxc1 and Foxc2 have recently been shown to be required for artery specification in mice31. The compound Foxc1:Foxc2 knockout mice show various vascular defects including vascular fusions between the dorsal aorta and the anterior cardinal vein, and loss of expression of the arterial markers, such as Dll4. Importantly, Foxc proteins were shown to activate the expression of Dll4, along with other arterial markers such as Hey2, in endothelial cells in vitro. Moreover, Foxc proteins were shown to directly bind to the Dll4 promoter. These and more recent studies, discussed below, demonstrate that Foxc factors play a critical role in linking VEGF signals to the Notch/Hey2 pathway to direct arterial specification.

Collective evidence from zebrafish studies led to a picture in which the venous fate is the default state for bipotent angioblasts and Notch signaling induces arterial development8. In fact, very little is known regarding the signaling components involved in establishing venous fate identity. Recently, an orphan nuclear receptor COUP-TFII (chicken ovalbumin upstream promoter-transcription factor II) was found to be expressed specifically in venous endothelial cells32. Endothelial-specific knockout of COUP-TFII led to ectopic expression of arterial markers, and endothelial-specific overexpression of COUP-TFII led to loss of arterial markers. Since manipulations of COUP-TFII expression perturbed expression of arterial-specific Notch pathway markers, it was hypothesized that COUP-TFII functions to maintain venous identity by repressing Notch signaling in venous angioblasts. However, the precise step targeted by COUP-TFII remains unclear since ectopic COUP-TFII expression also disrupted the expression of neuropilin-1 (NP1), a VEGF co-receptor which is normally expressed in arterial cells33. COUP-TFII’s role in zebrafish artery-vein specification is yet to be determined.

Chemical Genetic Analysis of Artery-Vein Specification Reveals Opposing Effects of MAPK and PI3K on Artery-Vein Specification

While classic genetic analyses, such as those described above, have revealed much about the molecular pathways involved in artery-vein specification, such approaches have limitations when it comes to examining signaling pathways that function at multiple stages in development. With a typical non-conditional mutation, the primary role for a gene at later stages of development is often difficult to distinguish from indirect consequences of disrupting the gene at an earlier stage. The Hedgehog pathway, for example, plays a critical role in vasculogenesis, but is also involved in development of numerous other structures at various developmental time points34. Moreover, classic genetic approaches can be hampered by genetic redundancy35. A powerful alternative approach, chemical genetic analysis, can overcome the challenges posed by repeated utilization of a signaling pathway during development and by genetic redundancy. Recently, Hedgehog signaling inhibitor cyclopamine was instrumental in demonstrating that Hedgehog is required at three consecutive stages during vascular development: for medial migration of angioblasts, for arterial gene expression and for formation of intersomitic vessels36.

To complement the genetics studies, small molecule screens were recently performed to identify compounds that suppressed absent trunk circulation due to reduced artery formation in gridlock mutant embryos37,38. The central rationale of a chemical suppressor study is analogous to that of classic genetic modifier screen: namely, if impairment in a genetic pathway for arterial development can be suppressed by small molecule, the cellular target of the “chemical suppressor” must be relevant to arterial specification. These screens identified two classes of gridlock suppressors. The first class, represented by the compound GS4012, appears to function by activating VEGF signaling. GS4012 induces VEGF expression in zebrafish, and mimics the effects of VEGF in zebrafish and in endothelial tubule formation assays. The second class of gridlock suppressors, represented by GS4898, block the PI3K pathway. This was a surprising finding since PI3K is a well-known downstream component of VEGF signaling39.

The apparent paradox of rescue of the identical vascular phenotype by putative stimulators of VEGF signaling as well as inhibitors of a downstream VEGF signaling component was resolved with the recognition that two well known signaling pathways activated by the VEGF receptor, the PLCγ1-ERK/MAPK pathway and the PI3K–ATK pathways, could play competing or antagonistic roles (Figure 2). In human umbilical vein endothelial cells (HUVECs), the PI3K–AKT pathway has been shown to antagonize ERK/MAPK signaling40. Moreover, in a human breast cancer cell line, similar PI3K–AKT and ERK/MAPK crosstalk occurred through direct inhibitory phosphorylation of Raf, a MAPK signaling component, by AKT, a key kinase acting downstream of PI3K41. In zebrafish embryos, incubation with GS4898 or known P3K inhibitors, LY294002 and wortmannin, results in activation of the ERK/MAPK and expansion of the arterial fate38. Conversely, inhibition of mitogen-extracellular signal activated protein kinase kinase (MEK), an upstream activator of ERK, results in loss of arterial structures. To circumvent the pleiotropic effects of ERK/MAPK and PI3K/AKT in early development, mosaic transgenic expression of AKT in zebrafish were utilized to confirm chemical genetic findings. Expression of dominant negative AKT along with green fluorescent protein (GFP) resulted in preferential localization of GFP-positive cells in the dorsal aorta, whereas expression of constitutively active AKT resulted in localization of GFP-positive cells in the posterior cardinal vein. Together, these results suggest that ERK/MAPK signaling is required for the arterial cell fate, whereas PI3K signaling has an opposing effect of promoting the venous fate by inhibiting the ERK/MAPK pathway.

Importantly, during zebrafish development, activated/phosphorylated ERK is localized precisely to dorsal angioblasts that will develop into aortic endothelial cells but not ventral angioblasts destined to become venous endothelial cells (Figure 3)38. Pharmacological blockade of VEGF receptor-1 and −2 signaling, and of Hedgehog signaling, both of which block arterial specification, were shown to prevent ERK activation in endothelial progenitors. Conversely, high concentrations of PI3K inhibitors were shown to expand ERK activation within endothelial progenitors. These results show that Hedgehog and VEGF signaling are required upstream of ERK/MAPK pathway to activate it specifically in arterial progenitors, and that ERK activation is one of the earliest known markers and determinants of artery cell fate in zebrafish embryos. Of note, strikingly analogous aorta-specific ERK activation has also been observed in mouse embryos42, suggesting a similar role for ERK signaling in mouse arterial development.

Figure 3. ERK activation is an early marker and determinant of arterial fate.

Figure 3

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(A) Wholemount staining of 20-somite stage (ss) zebrafish embryo with antibody that recognizes di-phosphorylated (activated) ERK indicates high levels of ERK activation (indicated by arrows) in developing vasculature. (B) Longitudinal section and (C) cross section demonstrates the same. (D) In this cross section of a wild-type 20-ss embryo, ventral, venous angioblasts (V) are immunostained for vascular-specific GFP (green), and dorsal, arterial angioblasts (A) which immunostain for activated ERK are shown in yellow (green GFP merged with red phospho-ERK immunostaing). Nuclei are marked with DAPI (blue). (E) In embryos treated with a high dose of PI3K inhibitors, the proportion of angioblasts that immunostain for activated ERK (yellow) is greatly expanded. (F) In embryo treated with VEGF receptor inhibitor 676475, ERK activation in angioblasts is lost. (G) In this cross section of an in situ hybridization of 24-hpf wild-type embryo, the arterial marker ephrinB2a is clearly expressed in the dorsal aorta (A), but not in posterior cardinal vein (V). (H) In embryos treated with a high dose of PI3K inhibitors, the ephrin-B2a expressing dorsal aorta (A) is prominent, but the posterior cardinal vein is not observed. (I) In embryos treated with MEK inhibitor SL327, neither Ephrin-B2a expression nor dorsal aorta is observed. In A and B, the dorsal side is to the right. In C – I, the dorsal side is to the top. NC, notochord. Figures reproduced or adapted from Hong et al.38 with permission from Cell Press.

Interestingly, the earliest detection of ERK activation occurs even before the completion of angioblast migration to the midline (Figure 4, 15-hpf)38. During angioblast migration, activated ERK is preferentially localized to a distinct subset of angioblasts on the leading edge (Figure 4), which on time-lapse micrography appear to contribute to the nascent dorsal aorta (personal observations). Such early emergence of distinct arterial and venous angioblasts, which exhibit different migratory timing, is consistent with the findings in synectin deficient zebrafish, in which selective disturbance in angioblast migration precede the deficient artery differentiation43. These results are consistent with cell lineage tracing experiments which suggest that artery-vein fate is already established when angioblasts begin their midline migration from their origins in the LPM10. Lastly, activated ERK is not detected after the circulation is established, suggesting that ERK activation is not required for maintenance of the arterial phenotype in zebrafish.

Figure 4. Activated ERK is found in a subset of angioblasts prior to the completion of their midline migration.

Figure 4

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(A, B) Cross section of immunostain for activated ERK in 12-ss (15hpf) embryo. (A) Activated ERK can be found in angioblast in the process of midline migration. (B) Merged view of migrating angioblasts, marked by GFP (green), and by activated ERK (yellow). By 12-ss, activated ERK is found preferentially in the “leading edge” angioblast subpopulation (yellow) that reaches the midline earlier than the rest (green). NC, notochord. Dorsal side is to the top. Figure reproduced or adapted from Hong et al.38 with permission form Cell Press.

Crosstalk between PI3K and MAPK in Mammalian systems: Comparison to Zebrafish findings

In cultured mammalian endothelial cells, stimulation with VEGF results in immediate activation of PI3K and ERK/MAPK signaling39. An important distinction from in vivo findings in zebrafish embryos is that ERK/MAPK activation in cultured cells is transient (typically lasting only a few minutes)44, versus persistent detectable levels of activated ERK over several hours in zebrafish embryos. Such dramatic differences in signaling kinetics may have important functional implications when comparing biological responses in vitro and in vivo. In cultured cells, the VEGF receptor activates two downstream signaling branches, each of which elicit a distinct set of biological responses39. In the first branch, PI3K activation leads to AKT activation, which promotes endothelial cell migration, survival, and nitric oxide production. In the second branch, the VEGF receptor activates PLCγ, resulting in activation of protein kinase C (PKC) and Raf, which then triggers a kinase activation cascade leading to ERK/MAPK activation, which promotes endothelial cell proliferation. In most in vitro contexts, the two branches are stimulated by VEGF together and often act in a synergistic manner. Nonetheless, in certain endothelial culture systems, the PI3K branch has been shown to antagonize the PLCγ-ERK/MAPK branch, similar to the observations in zebrafish embryos40.

In vitro mammalian cell studies have uncovered important differences from in vivo zebrafish studies regarding interactions between VEGF, Notch and ERK signaling. In cultured human arterial endothelial cells, VEGF signal induces expression of Notch1 and Dll445. However, in contrast to the zebrafish data, VEGF-induced Notch activation is mediated by PI3K, rather than ERK/MAPK45. Additional studies in bovine aortic endothelial cells (BAECs) have demonstrated that Foxc transcription factors mediate VEGF signaling by directly activating Dll4 and Hey2 promoters46. Moreover, VEGF-activated PI3K and ERK pathways were found to modulate the transcriptional activation of Dll4 and Hey2 genes by Foxc proteins. Again in contrast to the zebrafish data, the PI3K pathway was found to be necessary for inducing Dll4 and Hey2 expression. Interestingly, the ERK pathway was found to repress Dll4 and Hey2 expression, supporting the presence of a functional ERK-PI3K cross talk in aortic endothelial cells. Reasons for the discrepancy between in vitro and in vivo data regarding the functional effects of ERK and PI3K crosstalk are unclear. One possible explanation is that these in vitro experiments were all done in endothelial cells with a well-established arterial cell identify, not in uncommitted bi-potent endothelial progenitor cells. In addition, as mentioned earlier, ERK activation in arterial progenitor cells is noted over the course of several hours, a far longer timeframe than the transient ERK activation observed in cultured endothelial cells following VEGF stimulation. Thus, there may be important fundamental differences in ERK activation between in vivo and in vitro models. Finally, in a mouse ES cell model of in vitro differentiation of artery and vein cell types, higher concentrations of VEGF promoted expression of arterial marker genes, whereas low and intermediate levels of VEGF preferentially induced expression of the venous marker COUP-TFII47. Moreover, this VEGF-dependent arterial development could be blocked by Notch signal inhibition. Whether graded VEGF signaling could govern preferential activation of either PI3K or ERK pathways was not tested in this model. In summary, while interactions between VEGF, Notch and ERK signaling in isolated endothelial cells in culture may not fully recapitulate complex multicellular interactions that occur in the developing embryo, recent evidence supports the existence of a crosstalk between PI3K and ERK, with functional implications for artery-vein specification.

Concluding Remarks

As discussed here, studies in zebrafish have made important contributions to the genetic control of artery-vein specification during development. Nonetheless, there remain numerous challenges to achieving a more complete understanding of this complex process, involving many cells types and signaling molecules characterized by both genetic redundancy and pleiotropic functions. For example, the detailed mechanistic understanding of how Shh and VEGF specify arterial cell fate is still a challenge, particularly because both Shh and VEGF modulate diverse cellular processes, from cell-fate decisions, cell division/arrest decisions, chemotaxis, and interpretation of positional information. Another important question involves specific components that link VEGF signaling and downstream Notch signaling in the artery-specification pathway.

The chemical genetic approach was recently employed to reveal previously unsuspected roles of PI3K and ERK, two well-known VEGF signaling branches, in artery-vein specification. These findings raise additional interesting questions. For example, what determines whether an angioblast activates ERK or PI3K signaling? Could particular VEGF receptor subtypes or VEGF gradient govern which signaling branch is activated? What are the downstream targets of the ERK and PI3K signaling involved in artery-vein specification? Another important question regards whether the crosstalk between PI3K and ERK also plays an analogous role in specification of artery-vein fates in mammals. Although studies in cultured mammalian cells have yielded contradictory results, an earlier finding of localized activation of ERK in the developing aorta in mouse embryos42 suggests a conserved role for ERK in mammalian arterial specification. Further studies, utilizing classical and chemical genetics in zebrafish as well as in vivo and in vitro mammalian models, will be necessary to resolve whether the discrepancy is due to a fundamental species difference, or to a difference between in vivo and in vitro models. Finally, while genetic programs play a critical role in artery-vein specification during development, local environmental factors such as shear stress are also known to modify artery and vein cell identity48–50. Curiously, in the setting of adaptation after femoral artery occlusion, sustained shear stress promotes collateral artery growth in part by activating the ERK pathway51. Thus, it will be interesting, and clinically relevant, to examine whether the influence of environmental factors on artery-vein plasticity involves the signaling components implicated in artery-vein specification during development.

Acknowledgments

Sources of Funding

This work was supported by NIH grants HL081535 (CCH), HL074121 (TK) and HL079267 (RTP), and a grant from the GlaxoSmithKline Research & Education Foundation for Cardiovascular Research (CCH).

Footnotes

Disclosures

None.

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