ANG2 Blockade Diminishes Proangiogenic Cerebrovascular Defects Associated With Models of Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by arteriovenous malformations and blood vessel enlargements. However, there are no effective drug therapies to combat arteriovenous malformation formation in patients with HHT. Here, we aimed to address whether elevated levels of ANG2 (angiopoietin-2) in the endothelium is a conserved feature in mouse models of the 3 major forms of HHT that could be neutralized to treat brain arteriovenous malformations and associated vascular defects. In addition, we sought to identify the angiogenic molecular signature linked to HHT. METHODS: Cerebrovascular defects, including arteriovenous malformations and increased vessel calibers, were characterized in mouse models of the 3 common forms of HHT using transcriptomic and dye injection labeling methods. RESULTS: Comparative RNA sequencing analyses of isolated brain endothelial cells revealed a common, but unique proangiogenic transcriptional program associated with HHT. This included a consistent upregulation in cerebrovascular expression of ANG2 and downregulation of its receptor Tyr kinase with Ig and EGF homology domains (TIE2/TEK) in HHT mice compared with controls. Furthermore, in vitro experiments revealed TEK signaling activity was hampered in an HHT setting. Pharmacological blockade of ANG2 improved brain vascular pathologies in all HHT models, albeit to varying degrees. Transcriptomic profiling further indicated that ANG2 inhibition normalized the brain vasculature by impacting a subset of genes involved in angiogenesis and cell migration processes. CONCLUSIONS: Elevation of ANG2 in the brain vasculature is a shared trait among the mouse models of the common forms of HHT. Inhibition of ANG2 activity can significantly limit or prevent brain arteriovenous malformation formation and blood vessel enlargement in HHT mice. Thus, ANG2-targeted therapies may represent a compelling approach to treat arteriovenous malformations and vascular pathologies related to all forms of HHT.

Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors.

Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.

EHD2 modulates Dll4 endocytosis during blood vessel development.

OBJECTIVE: Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta-like ligand 4 (Dll4) endocytosis during Notch activation. APPROACH AND RESULTS: Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane-bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE-cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. CONCLUSIONS: Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.

Annexin A3 is necessary for parallel artery-vein alignment in the mouse retina.

BACKGROUND: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane-binding family of annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell-specific Anxa3 conditional knockout mouse model (Anxa3f/f ;Tie2-Cre). RESULTS: Anxa3f/f ;Tie2-Cre mice are born at Mendelian ratios and display morphologically normal blood vessels during development. However, loss of Anxa3 leads to artery-vein (AV) misalignment characterized by atypical AV crossovers in the postnatal and adult retina. CONCLUSIONS: Anxa3 is not essential for embryonic blood vessel formation but is required for proper parallel AV alignment in the murine retina. AV crossovers associated with Anxa3f/f ;Tie2-Cre mice are similar to AV intersections observed in patients with branch retinal vein occlusion (BRVO), although we did not observe occluded vessels. This new Anxa3 mouse model may provide a basis for understanding AV crossover formation associated with BRVO.

Angiopoietin-2 Inhibition Rescues Arteriovenous Malformation in a Smad4 Hereditary Hemorrhagic Telangiectasia Mouse Model.

BACKGROUND: Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFß) pathway members, including the central transcriptional mediator of the TGFß pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFß downstream effectors responsible for hereditary hemorrhagic telangiectasia-associated AVM formation are currently unknown. METHODS: To identify potential biological targets of the TGFß pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation-sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development. RESULTS: Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4-deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4. CONCLUSIONS: Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFß downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.

Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFß pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4f/f;Cdh5-CreERT2) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery-vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFß and VEGF signaling pathways in AVM pathogenesis.

Cdc42 is required for cytoskeletal support of endothelial cell adhesion during blood vessel formation in mice.

The Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain unclear. Here, we assess the cellular mechanisms by which Cdc42 regulates mammalian vascular morphogenesis and maintenance. In vivo deletion of Cdc42 in embryonic ECs (Cdc42(Tie2KO)) results in blocked lumen formation and endothelial tearing, leading to lethality of mutant embryos by E9-10 due to failed blood circulation. Similarly, inducible deletion of Cdc42 (Cdc42(Cad5KO)) at mid-gestation blocks angiogenic tubulogenesis. By contrast, deletion of Cdc42 in postnatal retinal vessels leads to aberrant vascular remodeling and sprouting, as well as markedly reduced filopodia formation. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. We also identify Cdc42 effectors Pak2/4 and N-WASP, as well as the actomyosin machinery, to be crucial for EC actin organization. This work supports the notion of Cdc42 as a central regulator of the cellular machinery in ECs that drives blood vessel formation.

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks.

BACKGROUND: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study's objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. METHODS: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. RESULTS: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. CONCLUSION: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks.

Wnt4 is essential to normal mammalian lung development.

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4(-/-) embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4(-/-) lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.

Integration of repulsive guidance cues generates avascular zones that shape mammalian blood vessels.

RATIONALE: Positive signals, such as vascular endothelial growth factor, direct endothelial cells (ECs) to specific locations during blood vessel formation. Less is known about repulsive signal contribution to shaping vessels. Recently, "neuronal guidance cues" have been shown to influence EC behavior, particularly in directing sprouting angiogenesis by repelling ECs. However, their role during de novo blood vessel formation remains unexplored. OBJECTIVE: To identify signals that guide and pattern the first mammalian blood vessels. METHODS AND RESULTS: Using genetic mouse models, we show that blood vessels are sculpted through the generation of stereotyped avascular zones by EC-repulsive cues. We demonstrate that Semaphorin3E (Sema3E) is a key factor that shapes the paired dorsal aortae in mouse, as sema3E(-/-) embryos develop an abnormally branched aortic plexus with a markedly narrowed avascular midline. In vitro cultures and avian grafting experiments show strong repulsion of ECs by Sema3E-expressing cells. We further identify the mouse notochord as a rich source of multiple redundant neuronal guidance cues. Mouse embryos that lack notochords fail to form cohesive aortic vessels because of loss of the avascular midline, yet maintain lateral avascular zones. We demonstrate that lateral avascular zones are directly generated by the lateral plate mesoderm, a critical source of Sema3E. CONCLUSIONS: These findings demonstrate that Sema3E-generated avascular zones are critical regulators of mammalian cardiovascular patterning and are the first to identify a repulsive role for the lateral plate mesoderm. Integration of multiple, and in some cases redundant, repulsive cues from various tissues is critical to patterning the first embryonic blood vessels.

Resolution of defective dorsal aortae patterning in Sema3E-deficient mice occurs via angiogenic remodeling.

BACKGROUND: Neuronal guidance cues influence endothelial cell (EC) behavior to shape the embryonic vascular system. The repulsive neuronal guidance cue, Semaphorin 3E (Sema3E), is critical for creating avascular zones that instruct and subsequently pattern the first embryonic vessels, the paired dorsal aortae (DA). Sema3E(-) (/) (-) embryos develop highly branched plexus-like vessels during vasculogenesis, instead of smooth paired vessels. Unexpectedly, despite these severe DA patterning defects, mutant mice are viable throughout adulthood. RESULTS: Examination of Sema3E(-) (/) (-) mice reveals that the plexus-like DA resolve into single, unbranched vessels between embryonic day (E) E8.25 and E8.75. Although fusion of Sema3E(-) (/) (-) DA occurs slightly earlier than in heterozygotes, the DA are otherwise indistinguishable, suggesting a complete "rescue" in their development. Resolution of the DA null plexuses occurs by remodeling rather than by means of changes in cell proliferation or death. CONCLUSIONS: Normalization of Sema3E(-) (/) (-) DA patterning defects demonstrates resilience of embryonic vascular patterning programs. Additional repulsive guidance cues within the lateral plate mesoderm likely re-establish avascular zones lost in Sema3E(-) (/) (-) embryos and guide resolution of mutant plexus into branchless, parallel aortae. Our observations explain how Sema3E(-) (/) (-) mice survive throughout development and into adulthood, despite severe initial vascular defects.

Zmiz1 is a novel regulator of brain development associated with autism and intellectual disability.

Neurodevelopmental disorders (NDDs) are a class of pathologies arising from perturbations in brain circuit formation and maturation with complex etiological triggers often classified as environmental and genetic. Neuropsychiatric conditions such as autism spectrum disorders (ASD), intellectual disability (ID), and attention deficit hyperactivity disorders (ADHD) are common NDDs characterized by their hereditary underpinnings and inherent heterogeneity. Genetic risk factors for NDDs are increasingly being identified in non-coding regions and proteins bound to them, including transcriptional regulators and chromatin remodelers. Importantly, de novo mutations are emerging as important contributors to NDDs and neuropsychiatric disorders. Recently, de novo mutations in transcriptional co-factor Zmiz1 or its regulatory regions have been identified in unrelated patients with syndromic ID and ASD. However, the role of Zmiz1 in brain development is unknown. Here, using publicly available databases and a Zmiz1 mutant mouse model, we reveal that Zmiz1 is highly expressed during embryonic brain development in mice and humans, and though broadly expressed across the brain, Zmiz1 is enriched in areas prominently impacted in ID and ASD such as cortex, hippocampus, and cerebellum. We investigated the relationship between Zmiz1 structure and pathogenicity of protein variants, the epigenetic marks associated with Zmiz1 regulation, and protein interactions and signaling pathways regulated by Zmiz1. Our analysis reveals that Zmiz1 regulates multiple developmental processes, including neurogenesis, neuron connectivity, and synaptic signaling. This work paves the way for future studies on the functions of Zmiz1 and highlights the importance of combining analysis of mouse models and human data.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Regulation of endothelial cell development by ETS transcription factors.

The ETS family of transcription factors plays an essential role in controlling endothelial gene expression. Multiple members of the ETS family are expressed in the developing endothelium and evidence suggests that the proteins function, to some extent, redundantly. However, recent studies have demonstrated a crucial non-redundant role for ETV2, as a primary player in specification and differentiation of the endothelial lineage. Here, we review the contribution of ETS factors, and their partner proteins, to the regulation of embryonic vascular development.

PRDM16 regulates arterial development and vascular integrity.

Proper vascular formation is regulated by multiple signaling pathways. The vascular endothelial growth factor (VEGF) signaling promotes endothelial proliferation. Notch and its downstream targets act to lead endothelial cells toward an arterial fate through regulation of arterial gene expression. However, the mechanisms of how endothelial cells (ECs) in the artery maintain their arterial characteristics remain unclear. Here, we show that PRDM16 (positive regulatory domain-containing protein 16), a zinc finger transcription factor, is expressed in arterial ECs, but not venous ECs in developing embryos and neonatal retinas. Endothelial-specific deletion of Prdm16 induced ectopic venous marker expression in the arterial ECs and reduced vascular smooth muscle cell (vSMC) recruitment around arteries. Whole-genome transcriptome analysis using isolated brain ECs show that the expression of Angpt2 (encoding ANGIOPOIETIN2, which inhibits vSMC recruitment) is upregulated in the Prdm16 knockout ECs. Conversely, forced expression of PRDM16 in venous ECs is sufficient to induce arterial gene expression and repress the ANGPT2 level. Together, these results reveal an arterial cell-autonomous function for PRDM16 in suppressing venous characteristics in arterial ECs.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of Zmiz1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis.

The (Pro)renin receptor ([P]RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell-specific (EC-specific) Atp6ap2-KO mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity, and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2-deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration, and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the ERK pathway. Furthermore, Atp6ap2-deficient retinas exhibited reduced revascularization in an oxygen-induced retinopathy model. Collectively, our results demonstrate a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.

The myocardin-related transcription factor, MASTR, cooperates with MyoD to activate skeletal muscle gene expression.

The myocardin family proteins (myocardin, MRTF-A, and MRTF-B) are serum response factor (SRF) cofactors and potent transcription activators. Gene-ablation studies have indicated important developmental functions for myocardin family proteins primarily in regulation of cardiac and smooth muscle development. Using Xenopus genome and cDNA databases, we identified a myocardin-related transcription factor expressed specifically in the skeletal muscle lineage. Synteny and sequence alignments indicate that this gene is the frog orthologue of mouse MASTR [Creemers EE, Sutherland LB, Oh J, Barbosa AC, Olson EN (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell 23:83-96]. Inhibition of MASTR function in the Xenopus embryo by using dominant-negative constructions or morpholino knockdown results in a dramatic reduction in expression of skeletal muscle marker genes. Overexpression of MASTR in whole embryos or embryonic tissue explants induces ectopic expression of muscle marker genes. Furthermore, MASTR cooperates with the myogenic regulatory factors MyoD and Myf5 to activate transcription of skeletal muscle genes. An essential function for MASTR in regulation of myogenic development in the vertebrate embryo has not been previously indicated.

ETS family protein ETV2 is required for initiation of the endothelial lineage but not the hematopoietic lineage in the Xenopus embryo.

Transcription factors of the ETS family are important regulators of endothelial and hematopoietic development. We have characterized the Xenopus orthologue of the ETS transcription factor, ETV2. Expression analysis shows that etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain-of-function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss-of-function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms.

Characterization of arteriovenous identity in the developing neonate mouse retina.

The murine retina has become an ideal model to study blood vessel formation. Blood vessels in the retina undergo various processes, including remodeling and differentiation, to form a stereotypical network that consists of precisely patterned arteries and veins. This model presents a powerful tool for understanding many different aspects of angiogenesis including artery and vein (AV) cell fate acquisition and differentiation. However, characterization of AV differentiation has been largely unexplored in the mouse retinal model. In this study, we describe the expression of previously established AV markers and assess arteriovenous acquisition and identity in the murine neonatal retina. Using in situ hybridization and immunofluorescent antibody staining techniques, we analyzed numerous AV differentiation markers such as EphB4-EphrinB2 and members of the Notch pathway. We find that at postnatal day 3 (P3), when blood vessels are beginning to populate the retina, AV identity is not immediately established. However, by P5 expression of many molecular identifiers of arteries and veins become restricted to their respective vessel types. This molecular distinction is more obvious at P7 and remains unchanged through P9. Overall, these studies indicate that, similar to the embryo, acquisition of AV identity occurs in a step-wise process and is largely established by P7 during retina development.