Akt is a mediator of artery specification during zebrafish development.

The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. Although vascular endothelial growth factor a (VEGF-A) is known to drive DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B (Akt) in zebrafish by generating a quadruple mutant (aktΔ/Δ), in which expression and activity of all Akt genes - akt1, -2, -3a and -3b - are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA-sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to re-establish normal arterial specification in aktΔ/Δ. The Akt loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.

Haematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic endothelium.

Definitive haematopoietic stem and progenitor cells (HSPCs) generate erythroid, lymphoid and myeloid lineages. HSPCs are produced in the embryo via transdifferentiation of haemogenic endothelial cells in the aorta-gonad-mesonephros (AGM). HSPCs in the AGM are heterogeneous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. Here we discovered that loss of microRNA (miR)-128 in zebrafish leads to an expansion of HSPCs in the AGM with different cell cycle states and a skew towards erythroid and lymphoid progenitors. Manipulating miR-128 in differentiating haemogenic endothelial cells, before their transition to HSPCs, recapitulated the lineage skewing in both zebrafish and human pluripotent stem cells. miR-128 promotes Wnt and Notch signalling in the AGM via post-transcriptional repression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b. De-repression of cskn1a1 resulted in replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in G2/M and lymphoid-biased HSPCs with long-term consequence on the respective blood lineages. We propose that HSPC heterogeneity arises in the AGM endothelium and is programmed in part by Wnt and Notch signalling.

Lactate-dependent chaperone-mediated autophagy induces oscillatory HIF-1α activity promoting proliferation of hypoxic cells.

Response to hypoxia is a highly regulated process, but little is known about single-cell responses to hypoxic conditions. Using fluorescent reporters of hypoxia response factor-1α (HIF-1α) activity in various cancer cell lines and patient-derived cancer cells, we show that hypoxic responses in individual cancer cells can be highly dynamic and variable. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, implicating reactive-oxygen-species-dependent chaperone-mediated autophagy of HIF-1α in a subset of cells. Furthermore, we show that oscillatory response is modulated by the abundance of extracellular lactate in a quorum-sensing-like mechanism. We show that oscillatory HIF-1α activity rescues hypoxia-mediated inhibition of cell division and causes broad suppression of genes downregulated in cancers and activation of genes upregulated in many cancers, suggesting a mechanism for aggressive growth in a subset of hypoxic tumor cells.

The N-glycome regulates the endothelial-to-hematopoietic transition.

Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

Epigenetic and Epitranscriptomic Factors Make a Mark on Hematopoietic Stem Cell Development.

PURPOSE OF THE REVIEW: Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. RECENT FINDINGS: Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between 'epi'-machineries. SUMMARY: Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Alk and Ltk ligands are essential for iridophore development in zebrafish mediated by the receptor tyrosine kinase Ltk.

Anaplastic lymphoma kinase (Alk) and leucocyte tyrosine kinase (Ltk) were identified as "orphan" receptor tyrosine kinases (RTKs) with oncogenic potential. Recently ALKAL1 and ALKAL2 (also named "augmentor-ß" and "augmentor-α" or "FAM150A" and "FAM150B," respectively) were discovered as physiological ligands of Alk and Ltk. Here, we employ zebrafish as a model system to explore the physiological function and to characterize in vivo links between Alk and Ltk with their ligands. Unlike the two ligands encoded by mammalian genomes, the zebrafish genome contains three genes: aug-α1, aug-α2, and aug-ß Our experiments demonstrate that these ligands play an important role in zebrafish pigment development. Deficiency in aug-α1, aug-α2, and aug-ß results in strong impairment in iridophore patterning of embryonic and adult zebrafish that is phenocopied in zebrafish deficient in Ltk. We show that aug-α1 and aug-α2 are essential for embryonic iridophore development and adult body coloration. In contrast, aug-α2 and aug-ß are essential for iridophore formation in the adult eye. Importantly, these processes are entirely mediated by Ltk and not by Alk. These experiments establish a physiological link between augmentor ligands and Ltk and demonstrate that particular augmentors activate Ltk in a tissue-specific context to induce iridophore differentiation from neural crest-derived cells and pigment progenitor cells.

Hematopoiesis Lineage Tree Uprooted: Every Cell Is a Rainbow.

Differentiation of hematopoietic stem cells into distinct cell types was thought to occur through a series of discrete, stable progenitor states. Work from Velten et al. (2017) now shows that hematopoietic cells differentiate via a mechanism of continuous lineage priming and thus represent a CLOUD-HSPC.

MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos.

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

A Dicer-miR-107 Interaction Regulates Biogenesis of Specific miRNAs Crucial for Neurogenesis.

Dicer controls the biogenesis of microRNAs (miRNAs) and is essential for neurogenesis. Recent reports show that the levels and substrate selectivity of DICER result in the preferential biogenesis of specific miRNAs in vitro. However, how dicer expression levels and miRNA biogenesis are regulated in vivo and how this affects neurogenesis is incompletely understood. Here we show that during zebrafish hindbrain development dicer expression levels are controlled by miR-107 to tune the biogenesis of specific miRNAs, such as miR-9, whose levels regulate neurogenesis. Loss of miR-107 function stabilizes dicer levels and miR-9 biogenesis across the ventricular hindbrain zone, resulting in an increase of both proliferating progenitors and postmitotic neurons. miR-9 ectopic accumulation in differentiating neuronal cells recapitulated the excessive neurogenesis phenotype. We propose that miR-107 modulation of dicer levels in differentiating neuronal cells is required to maintain the homeostatic levels of specific miRNAs, whose precise accumulation is essential for neurogenesis.

Endothelial cilia are essential for developmental vascular integrity in zebrafish.

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development.

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

Sphingosine-1-phosphate receptor-1 controls venous endothelial barrier integrity in zebrafish.

OBJECTIVE: Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P(1)) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. METHODS AND RESULTS: zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P(1) antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. CONCLUSIONS: The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

The synaptic proteins ß-neurexin and neuroligin synergize with extracellular matrix-binding vascular endothelial growth factor a during zebrafish vascular development.

OBJECTIVE: The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. METHODS AND RESULTS: In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas ß-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for ß-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and ß-neurexin 1a or neuroligin 1. CONCLUSIONS: Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.

miR-221 is required for endothelial tip cell behaviors during vascular development.

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.

MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

HIV-1 Tat and heparan sulfate proteoglycan interaction: a novel mechanism of lymphocyte adhesion and migration across the endothelium.

The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV(+) ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.

Fibroblast growth factor 2-induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay.

Angiogenesis plays a key role in tumour growth and metastasis. The teleost zebrafish (Danio rerio) represents a promising alternative model in cancer research. Here, we describe a zebrafish yolk membrane (ZFYM) angiogenesis assays based on the injection of 1-30 ng of human recombinant FGF2 (rFGF2) in the perivitelline space of zebrafish embryos in the proximity of developing subintestinal vein vessels (SIVs) at 48 hrs after fertilization. The rFGF2 induces a rapid and dose-dependent angiogenic response from the SIV basket, characterized by the ectopic growth of newly formed, alkaline phosphatase-positive blood vessels. These vessels are formed by proliferating cells that incorporate bromodeoxyuridine and express the endothelial cell markers vegfr2/kdr and fli1. Microangiography shows that rFGF2-induced vessels are patent and connected to the systemic circulation of the embryo. In keeping with these observations, fli1:EGFP(+) cells isolated from transgenic tg(fli1:EGFP)(y1) zebrafish embryos express the tyrosine kinase (TK) FGF receptor-1 (FGFR1) and activate extracellular signal-regulated kinase signalling when stimulated in vitro by rFGF2. The low molecular weight TK-FGFR1 inhibitor SU5402 and the high molecular weight FGF2 antagonist long-pentraxin 3 inhibit the angiogenic activity of rFGF2 when added to fish water or when co-injected with the growth factor, respectively. Moreover, similar to rFGF2, injection of the zebrafish form of vascular endothelial growth factor-A (VEGF-A) induces a significant angiogenic response in the ZFYM assay that is suppressed by the VEGF receptor-2/KDR TK inhibitor SU5416. The ZFYM assay represents a novel tool for testing the activity of low and high molecular weight inhibitors targeting a defined angiogenic growth factor in zebrafish. The assay may offer significant advantages when compared to other animal models.

alphavbeta3 Integrin-dependent antiangiogenic activity of resveratrol stereoisomers.

Angiogenesis is target for antineoplastic and chemopreventive therapies. The natural phytoalexin resveratrol is found in grapes and red wine as cis and trans stereoisomers. trans-Resveratrol shows antiangiogenic activity, but its mechanism of action is not fully elucidated. Recently, trans-resveratrol has been shown to interact with the beta3 integrin subunit, raising the possibility that inhibition of endothelial alphavbeta3 integrin function may concur to its angiosuppressive activity. To get novel insights about the antiangiogenic activity of resveratrol, we compared cis- and trans-resveratrol stereoisomers for their effect on the angiogenesis process and endothelial alphavbeta3 integrin function. trans-Resveratrol inhibits endothelial cell proliferation and the repair of mechanically wounded endothelial cell monolayers. Also, it prevents endothelial cell sprouting in fibrin gel, collagen gel invasion, and morphogenesis on Matrigel. In vivo, trans-resveratrol inhibits vascularization of the chick embryo area vasculosa and murine melanoma B16 tumor growth and neovascularization. In all the assays, cis-resveratrol exerts a limited, if any, effect. In keeping with these observations, trans-resveratrol, but not cis-resveratrol, inhibits alphavbeta3 integrin-dependent endothelial cell adhesion and the recruitment of enhanced green fluorescent protein-tagged beta3 integrin in focal adhesion contacts. In conclusion, stereoisomery affects the antiangiogenic activity of resveratrol, the trans isomer being significantly more potent than the cis isoform. The different antiangiogenic potential of resveratrol stereoisomers is related, at least in part, to their different capacity to affect alphavbeta3 integrin function. This may have profound implications for the design of synthetic antiangiogenic/angiopreventive phytoalexin derivatives.

Calcitonin receptor-like receptor guides arterial differentiation in zebrafish.

The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog-vegf-notch signaling cascade that controls arterial/venous fate.