RESEARCH ARTICLE 1563

Development 137, 1563-1571 (2010) doi:10.1242/dev.049015 © 2010. Published by The Company of Biologists Ltd von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina Toshihide Kurihara1,2,3,4,*,†, Yoshiaki Kubota5,*, Yoko Ozawa1,2,3, Keiyo Takubo5, Kousuke Noda3,6, M. Celeste Simon7, Randall S. Johnson4, Makoto Suematsu8, Kazuo Tsubota2, Susumu Ishida3,6, Nobuhito Goda9, Toshio Suda5,‡ and Hideyuki Okano1,‡

SUMMARY In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In this study, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor as (HIF-as) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, we generated retina-specific conditional-knockout mice for VHL (Vhla-CreKO mice). These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1a gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina.

KEY WORDS: Angiogenesis, Circulatory system, Hypoxia-inducible factor 1, Mouse

INTRODUCTION Oxygen concentration is vital to nearly all forms of life on earth In early neonates, the fetal circulatory system undergoes rapid and via its role in energy homeostasis (Yun et al., 2002). Mammalian dramatic transition to the adult circulatory system to adapt to embryos develop in a low (~3%) oxygen environment inside the detachment from the maternal blood supply. Normally, embryonic maternal body (Rodesch et al., 1992). However, pups are abruptly connecting vessels such as ductus arteriosus, ductus venosus and the exposed to atmospheric oxygen levels (~21%) after birth. This foramen ovale close and regress (Merkle and Gilkeson, 2005). In the dramatic change in oxygen environment at the point of birth neonatal retina, it is generally known that the hyaloid vessels that indicates some crucial roles of oxygen concentration in triggering maintain blood flow in the embryonic retina regress, and that retinal the transition from the fetal to the adult circulatory system. vessels assume the role of supplying blood to the retina (Lang, 1997; The von Hippel-Lindau tumor suppressor protein (pVHL) is a Lobov et al., 2005). This process is regulated by the programmed substrate recognition component of an E3-ubiquitin ligase that cell death switch mediated by macrophages via Wnt and rapidly destabilizes hypoxia-inducible factor as (HIF-as) under angiopoietin 2 pathways (Lang and Bishop, 1993; Lobov et al., normoxic, but not hypoxic, conditions (Maxwell et al., 1999). 2005; Rao et al., 2007). Genetic inactivation of the Vhl gene results in the stabilization and increased activity of HIF-as, which includes HIF-1a, HIF-2a and Departments of 1Physiology and 2Ophthalmology, and 3Laboratory of Retinal Cell HIF-3a. It has been reported that Vhl-null embryos are lethal at Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo embryonic day (E) 11.5-E12.5 owing to abnormal placental 4 160-8582, Japan. Molecular Biology Section, Division of Biological Sciences, vascularization (Gnarra et al., 1997). Endothelial-specific deletion University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0366, USA. 5Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University of the Vhl gene using Tie2-Cre mice also results in intrauterine death School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. with hemorrhage at E12.5, and loss of HIF-1a does not rescue the 6 Department of Ophthalmology, Hokkaido University Graduate School of Medicine, lethality (Tang et al., 2006). Although the vascular endothelial Kita 8, Nishi 5 Kita-ku, Sapporo, Hokkaido, Japan. 7Abramson Family Cancer Research Institute and Howard Hughes Medical Institute, University of Pennsylvania growth factor (VEGF) gene is widely known to be a HIF-1a target School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104, USA. (Forsythe et al., 1996), HIF-2a is thought to be the main regulator 8 Department of Biochemistry and Integrative Medical Biology, Keio University School of the VEGF gene in tissues that express both HIF-1 and HIF-2 of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. 9Department a a of Life Science and Medical Bio-Science, School of Advanced Science and (Hu et al., 2003; Tang et al., 2006). These previous studies showed Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, that VHL is required for endothelial cells in a cell autonomous Japan. manner. However, surprisingly, it is still largely unknown how VHL *These authors contributed equally to this work contributes to vascular development, particularly how and when the †Present address: Department of Cell Biology, The Scripps Research Institute, well-known relationship between VHL and oxygen concentration 10550 North Torrey Pines Road, La Jolla, CA 92037, USA operates in vascular development. In humans, VHL disease is an ‡Authors for correspondence ([email protected]; [email protected]) autosomal dominant syndrome that causes the development of

Accepted 26 February 2010 various benign and malignant disorders (Nicholson et al., 1976). The DEVELOPMENT 1564 RESEARCH ARTICLE Development 137 (9) major disorders include retinal, brain and spinal cord angioma, (Sigma-Aldrich) was injected into the left cardiac ventricle and allowed to pheochromocytoma, renal cell carcinoma and pancreatic circulate for 2 minutes. After staining, samples were mounted using a cystadenoma. In the majority of VHL patients, retinal angioma is the Prolong Antifade Kit (Molecular Probes). first sign of the disease to appear. However, the molecular Assessment of tissue hypoxia mechanism of retinal angioma development in VHL disease still Detection of hypoxic cells in cryosections was performed using a remains unknown. Hypoxyprobe-1 Plus Kit (Chemicon). In brief, 60 mg/kg of pimonidazole In this study, by using retina-specific conditional-knockout was injected intraperitoneally 30 minutes before sacrifice, and samples were technology, we explore the precise in vivo function of VHL in retinal stained with Hypoxyprobe Mab1-FITC. vascular development, and show that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the RT-PCR analysis Total RNA was prepared from retinal tissues and reverse-transcribed using fetal to the adult circulatory system in the retina. Superscript II (Invitrogen). A quantitative PCR assay for Vegfa was performed on an ABI 7500 Fast Real-Time PCR System using TaqMan Fast MATERIALS AND METHODS Universal PCR master mixture (Applied Biosystems) and TaqMan Mice Gene Expression Assay Mix of Vegfa (Mm00437304_m1), Csf1r All animal experiments were conducted in accordance with the Association (Mm00432689_m1), and angiopoietin 2 (Mm00545822_m1). Mouse b- for Research in Vision and Ophthalmology (ARVO) Statement for the Use actin (Mm00607939_s1) assay mix served as an endogenous control. Data of Animals in Ophthalmic and Vision Research. In the expression analysis were analyzed with 7500 Fast System SDS Software 1.3.1. All experiments shown in Fig. 1, we used C57BL/6 mice (Clea Japan). Transgenic mice were done with four replicates. expressing Cre recombinase under control of the Pax6 retina-specific Intravitreous injections regulatory element -promoter ( -Cre) (Marquardt et al., 2001) were mated a a Injections into the vitreous body were performed using 33-gauge needles, with Vhlfloxed/floxed mice (Haase et al., 2001) (kind gift from Volker H. Haase, as described previously (Gerhardt et al., 2003; Kubota et al., 2008). Sterile University of Pennsylvania), HIF-1afloxed/floxed mice (Ryan et al., 2000), or PBS (0.5 ml) with or without 1 mg/ml Flt1-Fc chimera proteins (R&D HIF-2 floxed/floxed mice (Gruber et al., 2007). As control littermates for a Systems) was injected at postnatal day (P) 4. Vhla-CreKO mice, HIF-1aa-CreKO mice, HIF-1aa-CreKO;Vhla-CreKO mice a-CreKO a-CreKO floxed/floxed and HIF-2a ;Vhl mice, we used Vhl mice, HIF- Confocal microscopy and quantification floxed/floxed floxed/floxed floxed/floxed 1a mice, HIF-1a ;Vhl mice and HIF- Fluorescence images were obtained using a confocal laser scanning floxed/floxed floxed/floxed 2a ;Vhl mice without the a-Cre transgene, respectively. In microscope (FV1000; Olympus) at room temperature. Quantification of a preliminary study, we found no detectable difference in retinal vascular the cells or substances of interest was usually done in eight random structures between a-Cre+ and a-Cre– mice, confirming the validity of these 200 mm ϫ 200 mm fields just behind the sprouting edges of each retina. To -CreKO control littermates. To monitor Cre expression in a-Cre mice or Vhla construct three-dimensional projections, multiple slices horizontally mice, we mated these mice with CAG-CAT-EGFP transgenic mice imaged from the same field of view were integrated by FV10-ASW (Kawamoto et al., 2000). All mice used in this study were maintained on a Viewer (Olympus). C57BL/6J background. Retinal explant culture Preparation of whole-mount samples and cryosections of retinas Retinal explant culture was performed using P6 mouse neural retinas based Enucleated eyes were fixed for 20 minutes in 4% paraformaldehyde (PFA) on the protocol we previously described (Ozawa et al., 2007; Ozawa et al., in PBS and then dissected as previously described (Fruttiger et al., 1996; 2004). Briefly, eyes were enucleated and neural retinas were isolated and Gerhardt et al., 2003; Kubota et al., 2008). The obtained tissues were post- placed on a Millicell chamber filter (Millipore; pore size, 0.4 Am) with the fixed overnight in 4% PFA in PBS and stored in methanol at –20°C. ganglion cell layer facing up. The chamber was then placed in a 6-well Cryosections of retinas (10 mm) were prepared as previously described culture plate, containing 50% MEM (GIBCO), 25% HBSS (GIBCO), 25% (Kurihara et al., 2006), after eyeballs were immersed overnight in 4% PFA. horse serum (Thermo Trace), 200 mM L-glutamine, and 6.75 mg/ml D- glucose. Explants were incubated at 34°C in 5% CO . Twenty-four hours Immunostaining and in situ hybridization 2 after exposure of each concentration of oxygen, the explants were subjected Immunohistochemistry (IHC) for whole-mount retinas and other tissues was to immunostaining or RT-PCR analysis. The oxygen concentration in the performed as described previously (Fruttiger et al., 1996; Gerhardt et al., incubator was controlled with nitrogen. 2003; Kubota et al., 2008; Kurihara et al., 2006). The primary antibodies used were: monoclonal anti-PECAM1 (2H8, Chemicon), a-smooth muscle Statistical analysis actin (1A4; FITC-conjugated; Sigma-Aldrich), desmin (DAKO), F4/80 (A3- Comparison between the average variables of two groups was performed by 1; Serotec, Oxford, UK), GFAP (G-A-5; Cy3-conjugated; Sigma-Aldrich or two-tailed Student’s t-test. P-values less than 0.05 were considered to be Dako), polyclonal type IV collagen (Cosmo Bio), HIF-1a (originally statistically significant. established by immunizing purified fusion proteins encompassing amino acids 416 to 785 of mouse HIF-1a into guinea pigs), HIF-2a (Santa Cruz Biotechnology), glutamine synthetase (Molecular Probes), opsin (Cosmo RESULTS Bio) and pVHL (Santa Cruz Biotechnology). The secondary antibodies used HIF-1a expression is rapidly downregulated after were Alexa Fluor 488-conjugated IgGs (Molecular Probes) or Cy3/Cy5- birth conjugated IgGs (Jackson ImmunoResearch). Nuclei were stained with 10 As a first step in the investigation of the cellular responses in the mg/ml Hoechst bisbenzimide 33258 (Sigma-Aldrich) or DAPI (Molecular retina to the dramatic change in the oxygen environment at birth, we Probes). For whole-mount in situ hybridization (ISH), retinas were briefly examined the expression patterns of pVHL, HIF-1a and HIF-2a in digested with proteinase K and hybridized with digoxigenin (DIG)-labeled the developing retina. Expression of pVHL was detected antisense RNA probes. When ISH was combined with IHC, IHC was ubiquitously from the inner to the outer side in the retina, and did not performed after all the ISH procedures were completed. For the BrdU change between before and after birth (Fig. 1A-E). By contrast, incorporation assay, 100 mg per body weight (g) of BrdU (BD Pharmingen) dissolved in sterile PBS was injected intraperitoneally 2 hours before nuclear staining of HIF-1a was downregulated in the deep retinal sacrifice. Isolated retinas were stained using a BrdU immunohistochemistry layer [the neuroblastic layer (NBL) at this stage] after birth (Fig. 1H- system (Calbiochem) according to the manufacturer’s instructions. When J), despite its ubiquitous expression in embryonic stages (Fig. 1F,G). BrdU assays were combined with IHC, the application of the first antibody HIF-2a immunoreactivity was predominantly detected in

for each procedure was done simultaneously. FITC-conjugated Dextran endothelial cells of hyaloid vessels in the vitreal space (see Fig. DEVELOPMENT VHL regulates circulatory system transit RESEARCH ARTICLE 1565

Fig. 1. Expression patterns of pVHL and HIF-1a in the retina before and after birth. (A-E)pVHL staining (green), detected in the soma of cells in the whole retina, is unchanged after birth. (F-J) Nuclear staining of HIF-1a (red), detected ubiquitously in the embryo (F,G), is downregulated in the deep retinal layer (NBL at this stage) after birth (H-J). (K-O)In embryonic retina, hypoxic areas visualized by the hypoxic probe pimonidazole (yellow) spread all through the layers (K,L), although after birth the hypoxic signal was considerably downregulated in the retina, noticeably in the deep retinal layer (M-O) where HIF-1a expression has also declined (H-J). GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bar: 100 mm in A-O.

S1A-C in the supplementary material) and in retinal vessels in the us to examine the well-known mechanisms of hyaloid vessel ganglion cell layer (GCL) (see Fig. S1D,E in the supplementary regression: reduced perivascular macrophages or angiopoietin 2 material). In embryonic retina, hypoxic areas were visualized by a deficiency lead to persistence of the hyaloid vessels (Lobov et al., hypoxic probe, pimonidazole, through all layers (Fig. 1K,L), 2005; Rao et al., 2007). Macrophages around the hyaloid vessels although a relatively hypoxic area after birth was limited to the GCL were not significantly changed (Fig. 2K-M), and colony stimulating (Fig. 1M-O), which still expressed HIF-1a (Fig. 1H-J). factor 1 receptor (Csf1r) expression, which correlates with the number of macrophages (Kubota et al., 2009), showed no significant Vhla-CreKO mice show persistence of the hyaloid reduction in Vhla-CreKO mice (Fig. 2N). Angiopoietin 2 expression vessels independently of macrophages was rather increased in Vhla-CreKO mice (Fig. 2O). Collectively, To elucidate the function of pVHL in postnatal retinal Vhla-CreKO mice show persistence of the hyaloid vessels vascularization, we employed a retina-specific mouse Cre line, a- independently of macrophages and angiopoietin 2. Cre, which shows Cre expression in the deep retinal layer, including in retinal progenitor cell-derived neural cells, under the control of a Vhla-CreKO mice show poorly formed retinal vessels neural retina-specific regulatory element of murine Pax6 gene, but characterized by excessive vessel regression not in astrocytes and endothelial cells at P10 (Marquardt et al., As abnormal hyaloid vessel persistence could affect retinal 2001). We examined the past events of Cre expression in a-Cre mice vascularization, we examined the retinal vessels in Vhla-CreKO mice. until early postnatal age by crossing a-Cre mice with a reporter In these mice, the entire growth of the retinal vasculature spreading transgenic line, CAG-CAT-EGFP (Kawamoto et al., 2000). At P6, from the optic disc and the branching points were significantly GFP expression was detected in neural cells in the deep retinal layer, reduced compared with control mice (P=0.013 for spreading but not in astrocytes or endothelial cells of both retinal and hyaloid distance, P=0.00012 for branching points; Fig. 3A,B,D,E,M,N; see vasculature (see Fig. S2A-T in the supplementary material). Then, also Fig. S3A,B in the supplementary material). Endothelial tip cells we crossed Vhlfloxed/floxed mice (Haase et al., 2001) with a-Cre mice and their filopodia, which are controlled by VEGF and the Delta- and generated a-Cre-specific conditional-knockout mice for Vhl like 4/Notch pathway (Gerhardt et al., 2003; Hellstrom et al., 2007; (Vhla-CreKO mice). As control littermates for Vhla-CreKO mice, we Phng et al., 2009), were also significantly reduced in Vhla-CreKO mice used Vhlfloxed/floxed mice without the a-Cre transgene. At P6, blood (P=0.0012 for tip cells; P=0.011 for filopodia; Fig. 3C,F,O,P). A flow visualized by intracardiac injection of fluorescein significant decrease in endothelial proliferation (Fig. 3G,J,Q) and isothiocyanate (FITC)-dextran was detected in both hyaloid and excessive vessel regression (Fig. 3H,K,R), characterized by empty retinal vessels of control mice (Fig. 2A,B,E,G,I). In Vhla-CreKO mice, vascular sleeves (Phng et al., 2009), were also detected in Vhla-CreKO however, there was abundant blood flow in hyaloid vessels, poor mice (P=7.9ϫ10–5 for BrdU+ endothelial cells; P=0.0044 for empty flow in retinal vessels, and abundant collateral flow from hyaloid sleeves). Empty sleeves were abundantly detected not only in their vessels to retinal vessels (Fig. 2C,D,F,H,J). Nonetheless, despite the stalk cell area but also at their leading edge, suggesting that vessel impaired blood flow, the retinal vascular structure could be detected regression occurs in both tip cells and stalk cells in Vhla-CreKO mice. by immunohistochemistry for platelet/endothelial cell adhesion Although it is well known that pVHL regulates the expression of molecule 1 (PECAM1) in Vhla-CreKO mice (Fig. 2F,H). The various extracellular matrices (Ohh et al., 1998), Col IV expression a-CreKO a-CreKO persistent blood flow in hyaloid vessels in Vhl mice prompted was not impaired in Vhl mice. Although the number of DEVELOPMENT 1566 RESEARCH ARTICLE Development 137 (9)

Fig. 2. Vhla-CreKO mice show persistence of the hyaloid vessels independently of macrophages. (A-D) Representative images of retinas after intracardiac FITC injection (n=8). Vhla-CreKO mice show collateral flow from hyaloid vessels to retinal vessels (open arrowheads) in the P6 retinas. Blood flow was visualized by intracardiac FITC-conjugated dextran (green) injection. (E-H) Confocal images of FITC- (orange) PECAM1- (blue) labeled P6 retinas; G,H show sections. Poor blood flow is detected in retinal vasculature (asterisk) in Vhla-CreKO mice compared with control. Relatively abundant dextran was perfused into arteries (A) and veins (V) in control mice, and into hyaloid vessels (arrows) in Vhla-CreKO mice. (I,J) Schematics show abundant blood flow (red) in retinal vessels in control mice, and in hyaloid vessels in Vhla-CreKO mice. (K,L) Immunostaining with F4/80 (green) and PECAM1 (blue) on hyaloid vessels in P6 retinas. (M) Quantification of F4/80+ macrophages per 200 mm vessel length. Vessel length was calculated by FV10-ASW Viewer software (n=4). (N,O) Quantitative PCR of Csf1r (N) or angiopoietin 2 (O) for isolated RNA from P6 retinas (n=5). *P<0.05. All panels show P6 retina. Error bars indicate mean ± s.d. Scale bars: 500 mm in A-D; 100 mm in E-H,K,L.

pericytes associated with endothelial tubes was not changed in Vhla-CreKO mice (Fig. 4O). Because these expression patterns of Vhla-CreKO mice (P=0.395; Fig. 3I,L,S), detached pericytes were VEGF suggested that the vascular defects in Vhla-CreKO mice could frequently detected just beyond their sprouting edges. Previously, be attributable to ectopic and abundant VEGF expression, we injection of angiopoietin 2 into the eyes of normal rats was shown injected a potent VEGF inhibitor, FLT1-Fc chimeric protein to induce a dose-dependent pericyte loss (Hammes et al., 2004), (Gerhardt et al., 2003; Kubota et al., 2008), into the eyes of suggesting that upregulated angiopoietin 2 (Fig. 2O) might Vhla-CreKO mice. These FLT1-Fc injections reduced the amount of contribute to pericyte defects in Vhla-CreKO mice. Collectively, both collateral blood flow and vascular structures from hyaloid Vhla-CreKO mice showed poorly formed retinal vessels, presumably vessels to retinal vessels (Fig. 4P-U). As genetic inactivation of the owing to persistence of the hyaloid vessels. Vhl gene results in HIF-a stabilization (Kapitsinou and Haase, 2008) and the upregulation of Vegfa expression, which is widely known as -CreKO Vascular defects in Vhla mice are attributable a HIF-1a target gene (Forsythe et al., 1996), we examined the to ectopic VEGF expression expression of pVHL, HIF-1a and HIF-2a in Vhla-CreKO mice. We The intact macrophages and preserved angiopoietin 2 expression in found that pVHL immunoreactivity was greatly reduced in the deep Vhla-CreKO mice caused us to suppose that some other mechanism led layer of the peripheral retina (Fig. 4V,W), where the strong a-Cre- to the persistence of the hyaloid vessels. As retinal vascular growth mediated recombination is detected (see Fig. S2 in the depends on the appropriate spatial distribution of heparin-binding supplementary material), except for in astrocytes and endothelial VEGF within the retina (Gerhardt et al., 2003; Kubota et al., 2008; cells (see Fig. S4A-J in the supplementary material). HIF-1a Ruhrberg et al., 2002; Stalmans et al., 2002), we suspected that the immunoreactivity was increased in this area of Vhla-CreKO mice (Fig. vascular defects in Vhla-CreKO mice were attributable to the abnormal 4X,Y; see also Fig. S4K-O in the supplementary material). In expression pattern of VEGF. In control mice, VEGF expression was addition, strong HIF-2a immunoreactivity was detected in predominantly detected in astrocytes beyond the sprouting edge invaginating hyaloid vessels, but not in the area of reduced pVHL (Fig. 4A,B,E-H). In Vhla-CreKO mice, marked expression of VEGF expression (Fig. 4Z,AA; see also Fig. S4P-T in the supplementary was detected in the deep retinal layer (Fig. 4C,D,I-L), and was material). coincidental with the area of persistent hyaloid vessels. However, typical VEGF expression in astrocytes beyond the sprouting edge Increased VEGF expression in Vhla-CreKO mice is due was diminished in Vhla-CreKO mice, despite the fact that their to their impaired oxygen-sensing mechanism via astrocyte plexus was normal (Fig. 4M,N). This VEGF expression HIF-1a pattern in Vhla-CreKO mice was similar to normal VEGF expression As the VEGF expression pattern in postnatal Vhla-CreKO retina is in the embryonic retina (Saint-Geniez et al., 2006). By quantitative similar to normal VEGF expression in the embryonic retina (Saint- RT-PCR analysis, a more than 4-fold significant increase Geniez et al., 2006), we expected abnormal VEGF expression in the a-CreKO

(P=0.00071) in Vegfa expression was detected in the entire retina of Vhl retina to be attributable to an impaired oxygen-sensing DEVELOPMENT VHL regulates circulatory system transit RESEARCH ARTICLE 1567

Fig. 3. Vhla-CreKO mice show poorly formed retinal vessels characterized by excessive vessel regression. (A-F) PECAM1 staining of P6 retinas. Note delayed vascular growth (bidirectional arrows in A,D), decreased branching points and tip cells (B,E), and filopodia (C,F) in Vhla-CreKO mice. Persistent hyaloid vessels in the lower left lobe of Vhla–CreKO mice were removed mechanically before immunostaining (D). (G-L) Immunostaining with the indicated antibodies. Note the decreased BrdU+ endothelial cells (arrows, G,J), type IV collagen (Col IV)+isolectin– empty sleeves (arrowheads, H,K), and detached pericytes beyond sprouting edges (open arrowheads, L) in Vhla-CreKO mice, although most pericytes in control mice are closely contacted with vasculature (closed arrowheads, I,L). (M-S) Quantification of percentage spreading distance from optic discs (M), the number of branching points in the area behind sprouting edges (N), tip cells per field (O), filopodia per tip cell (P), BrdU+ endothelial cells (Q), Col IV+isolectin– vessels (R), and pericytes per 200 mm vessel length (S; n=6, in each genotype). *P<0.05, **P<0.01. All panels show P6 retina. Error bars indicate mean±s.d. Scale bars: 500 mm in A,D; 100 mm in B,E,G-L; 20 mm in C,F. mechanism and a lack of response to the atmospheric oxygen defects seen in Vhla-CreKO mice were suppressed in HIF-1a; concentration. As newborn mice do not tolerate oxygen levels below Vhla-CreKO mice (Fig. 6A,B,D-G,M-P). The abnormal expression of 10% (Claxton and Fruttiger, 2005), we employed an ex vivo retinal Vegfa in Vhla-CreKO was fairly normalized in HIF-1a; Vhla-CreKO culture system, which enabled us to expose the retina to variable mice (Fig. 6J,K; see also Fig. S5A in the supplementary material; oxygen concentrations between 1% and 21% (Fig. 5A). In the control 1.15±0.08-fold compared with control). Conversely, a-Cre- retina culture, strong nuclear HIF-1a expression was induced in 1% mediated VHL and HIF-2a (Gruber et al., 2007) double-knockout oxygen (Fig. 5D), but not in 21% oxygen (Fig. 5B). Consequently, mice (HIF-2a; Vhla-CreKO mice) showed similar vascular defects and Vegfa expression was significantly higher (P=2.4ϫ10–8) in 1% a similar VEGF expression pattern to Vhla-CreKO mice (Fig. oxygen than in 21% oxygen (Fig. 5J). In the Vhla-CreKO retina culture, 6C,H,I,L; see also Fig. S5B in the supplementary material). Taken strong nuclear HIF-1a expression was detected (Fig. 5H), even in together, HIF-1a, but not HIF-2a, plays essential roles in the 21% oxygen (Fig. 5F), and Vegfa expression in the Vhla-CreKO retina appearance of vascular defects in the Vhla-CreKO retina. in 21% oxygen was significantly higher (P=1.1ϫ10–5) than in the control in 21% oxygen; it was equivalent to the level observed in 1% Progressive retinal degeneration in adult oxygen (Fig. 5J). To determine the significance of the increased Vhla-CreKO mice nuclear HIF-1a level in hypoxic conditions in this culture system, we Next, we examined retinal function and circulation in adult generated a-Cre-mediated conditional-knockout mice for HIF-1a Vhla-CreKO mice. Increased apoptosis of photoreceptors and other (Ryan et al., 2000) (HIF-1aa-CreKO mice). In HIF-1aa-CreKO retina, neuronal cells was seen in Vhla-CreKO mice after P14 (Fig. 7A-H) hypoxia-induced HIF-1a stabilization (Fig. 5K,M) was not detected induced decrease of the outer nuclear layer (ONL; Fig. 7I,J), despite (Fig. 5O,Q) and, accordingly, increased Vegfa expression in 1% the normal development of photoreceptors at P7 (Fig. 7E,J). Gliosis, oxygen was significantly suppressed (P=0.00028) compared with in accompanied by vessels invaginating into the deep retinal layer, control retina (Fig. 5S). Consistent with the in vivo results (Fig. 1A- destroyed the construction of the ONL (Fig. 7K-T) at P14. As a E), the expression pattern of pVHL was stable in the deep retinal layer result, a significant (P=3.3ϫ10–6 for a-wave, P=1.3ϫ10–5 for b- during ex vivo culture, and was not affected by the oxygen wave) decrease in amplitude and a significant (P=0.046 for b-wave) concentration (Fig. 5C,E,G,I,L,N,P,R). These data in the ex vivo extension of implicit time in electroretinography (ERG) was culture system suggest that the increased VEGF expression in observed in Vhla-CreKO mice at P28 (Fig. 7U-Y). Circulation via Vhla-CreKO mice is due to their impaired oxygen-sensing mechanism hyaloid vessels in the Vhla-CreKO retina was detected at the age of 8 via HIF-1a. weeks (see Fig. S6A-D in the supplementary material) and at 18 months (see Fig. S6E-H in the supplementary material). Some of the Deletion of HIF-1a, but not HIF-2a, rescues vascular Vhla-CreKO mice developed pre-retinal hemorrhage, cataracts and iris defects in Vhla-CreKO mice neovascularization (Fig. 8A-C), similar to the phenotypes that The expression patterns of pVHL, HIF-1a, HIF-2a and VEGF in accompany ischemic retinopathy (Hayreh, 2007). Vhla-Cre KO mice (Fig. 4) and the data from the ex vivo culture system (Fig. 5) suggested that the increased activity of HIF-1a was DISCUSSION responsible for the vascular defects in Vhla-CreKO mice. Therefore, In the present study, we showed that retina-specific conditional- we generated a-Cre-specific double-knockout mice for VHL and knockout mice for the Vhl gene exhibit persistent hyaloid a-CreKO

HIF-1a (Ryan et al., 2000) (HIF-1a;Vhl mice). Vascular vessels independently of macrophage function, which are DEVELOPMENT 1568 RESEARCH ARTICLE Development 137 (9)

Fig. 4. Vascular defects in Vhla-CreKO mice are attributable to ectopic VEGF expression. (A-L) Whole-mount (A-D) or section (E-L) in situ hybridization for VEGF combined with immunostaining with the indicated antibodies. Although VEGF expression is detected in astrocytes located in the avascular area (arrows) of control mice, abundant VEGF expression is detected in the deep retinal layer (open arrowheads) where persistent hyaloid vessels invaginate in Vhla-CreKO mice. (M,N) Immunostaining with PECAM1 (green) and PDGFRa (red) on P6 retinas. Despite a normal astrocyte plexus (asterisks), vessel regression (open arrowheads) occurs in Vhla-CreKO mice. (O) Quantitative PCR of Vegfa RNA isolated from P6 retinas (n=6). (P-U) Fluorescence microscopic images in P6 retinas perfused with FITC-dextran (P-R), and confocal images labeled with PECAM1 (S-U). FLT1-Fc injection into the eyes of Vhla-CreKO mice reduces collateral flow (arrows, R) and vascular structures (arrows, U) that exist abundantly in Vhla-CreKO mice injected with vehicle (open arrowheads in Q,T). (V-AA) Immunostaining of P6 retinal sections with the indicated antibodies. Although pVHL expression is greatly reduced (open arrowheads, W), HIF- 1a immunoreactivity is increased (closed arrowheads, Y) in the deep retinal layer of Vhla–CreKO mice. HIF-2a staining is detected in invaginating hyaloid vessels in Vhla-CreKO mice (arrows, AA). **P<0.01. Error bars indicate mean ± s.d. Scale bars: 500 mm in A-L; 200 mm in P-AA; 100 mm in M,N.

sustained until adulthood. These vascular defects in Vhla-CreKO Considering that HIF-1a in the outer side of the retina is mice are rescued by either local VEGF inhibition or genetic dramatically downregulated after birth but that pVHL expression in deletion of HIF-1a, but not of HIF-2a. These results suggest not the retina does not differ between embryos and neonates (Fig. 1), the only macrophages but also tissue oxygen-sensing mechanisms change of environmental oxygen concentration must be important regulate the transition from the fetal to the adult circulatory for the role of pVHL in this transition (Rodesch et al., 1992). VEGF system in retina. expression in the deep retinal layer during embryonic but not

Fig. 5. Increased VEGF expression in Vhla-CreKO mice is due to their impaired oxygen-sensing mechanism via HIF-1a. (A) Schematics show the experimental procedure: isolated retinas from control or conditional-knockout mice were unfolded, placed on a chamber filter, and exposed to each concentration of oxygen. Retinal explants of control mice (B-E,K-N), Vhla-CreKO mice (F-I), or HIF-1aa-CreKO mice (O-R) were exposed to 21% oxygen (B,C,F,G,K,L,O,P) or 1% oxygen (D,E,H,I,M,N,Q,R). (J,S) Quantitative PCR of Vegfa RNA isolated from retinal explants (n=6, respectively). Note, strong HIF-1a staining (red) is induced by 1% oxygen (D,H,M) except in HIF- 1aa-CreKO retina (Q), and by genetic inactivation of VHL (F,H). pVHL expression was not detected in Vhla-CreKO retina (G,I). (J) Correlated with the expression of HIF-1a, Vegfa expression was upregulated even under normoxia conditions in Vhla-CreKO retina. (S) In HIF-1aa-CreKO retina, hypoxia-induced Vegfa expression was significantly suppressed. **P<0.01. Error bars indicate mean±s.d. Scale bars: 50 mm in B-I,K-R. DEVELOPMENT VHL regulates circulatory system transit RESEARCH ARTICLE 1569

Fig. 7. Vhla-CreKO mice develop retinal degeneration in adults. (A-H) TUNEL-positive cells (red) counterstained with Hoechst (blue) are Fig. 6. Deletion of HIF-1a but not HIF-2a, rescues vascular defects increased in the ONL in Vhla-CreKO mice (E-H) compared with in control Vhla-CreKO in mice. (A-I) Blood flow visualized by intracardiac FITC- mice (A-D) after P14. (I,J) ONL thickness is decreased in a time- dextran injection (A-C), or confocal images from mice of indicated dependent manner in Vhla-CreKO mice (n=3, respectively). (K-T) Merged genotypes labeled with PECAM1 at P6 (D-I). Vascular defects [collateral images for glial fibrillary acidic protein (GFAP), Hoechst and BrdU (K,P), flow from hyaloid vessels to retinal vessels (open arrowheads), delayed and IHC for PECAM1 (yellow, L,Q), glutamine synthetase (GS, green, vascular growth (bidirectional arrows), decreased branching points, tip M,R), opsin (red, N,S), and merged images for GS and opsin (O,T) in a-CreKO cells and filopodia] seen in Vhl mice are abolished by deletion of control (K-O) and Vhla-CreKO mice (P-T). Proliferating reactive glia are a-CreKO HIF-1a, but not HIF-2a, in Vhl mice. (J-L) Whole-mount in situ detected by BrdU (arrows, P), and gliosis destroys the construction of hybridization for VEGF (blue) combined with immunostaining with Col the ONL (open arrowheads, R-T) in Vhla-CreKO mice at P14. (U-Y)ERG a-CreKO IV (green). Ectopic VEGF expression seen in Vhl mice is abolished analysis at P28. (U) Representative wave responses from individual a-CreKO by deletion of HIF-1a, but not HIF-2a, in Vhl mice. control or Vhla-CreKO mice. Decreased amplitude (V,W) and prolonged (M-T) Quantification of percentage spreading distance from optic discs implicit time (Y) are detected in Vhla-CreKO mice (n=6). GCL, ganglion (M,Q), the number of branching points in the area behind sprouting cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. *P<0.05, edges (N,R), tip cells per field (O,S), filopodia per tip cell (P,T; n=6 for **P<0.01. Error bars indicate mean ± s.d. Scale bar: 100 mm in A-H,K-T. each genotype). *P<0.05, **P<0.01. Error bars indicate mean ± s.d. Scale bars: 500 mm in A-C,D,F,H; 100 mm in E,G,I,J-L. postnatal retinal stages (Saint-Geniez et al., 2006) supports the idea endothelial cells of hyaloid vessels and stimulating Wnt ligand that the abnormal VEGF distribution in postnatal Vhla-CreKO retina production by macrophages (Rao et al., 2007). Interestingly, the represents a defective VEGF expression switch from the embryonic macrophage/microglia number in Vhla-CreKO mice was not affected pattern to the postnatal one. Mice selectively expressing single (Fig. 2K-M), but angiopoietin 2 expression was increased (Fig. 2O), isoforms of VEGF (VEGF120 or VEGF188) or overexpressing VEGF which suggests that an oxygen-sensing mechanism mediated by the under the control of a lens-specific promoter show a persistence of VHL/HIF-1a/VEGF system operates hyaloid vessel regression hyaloid vessels as well as defective retinal vascularization, independently of macrophages, Wnt and angiopoietin 2. supporting the concept that spatial distribution of VEGF regulates A possible concern with our data might be the slightly, but the transition from the embryonic to the adult circulatory system in significantly, increased branching points observed in HIF-1a; the retina (Mitchell et al., 2006; Stalmans et al., 2002). Vhla-CreKO mice (Fig. 6G,N). Independently of HIF-a proteolysis, Previously, Lang and colleagues clearly demonstrated that pVHL is known to be involved in extracellular matrix assembly and macrophages mediate hyaloid vessel regression through the paracrine turnover (Ohh et al., 1998). Moreover, erythropoietin has been action of WNT7B (Lobov et al., 2005). They showed that a lack of reported to be regulated by VHL and HIFs (Chen et al., 2008; macrophages in PU.1–/– mice caused a significant delay in hyaloid Rankin et al., 2007). These previous findings suggest the vessel regression, and that intraocularly injected wild-type, but not involvement of multiple candidates in addition to HIF-1a and Wnt7b-mutant, macrophages ameliorated this delay. Moreover, VEGF, and might explain the minor vascular changes observed in angiopoietin 2 has also been shown to be involved in hyaloid vessel HIF-1a;Vhla-CreKO mice. However, the dramatic rescue effects

regression via the dual effect of suppressing survival signaling in the obtained by FLT1-Fc injection (Fig. 4P-U), or by the genetic DEVELOPMENT 1570 RESEARCH ARTICLE Development 137 (9)

Experiments using the anti-HIF-1a antibody were supported by the Japan Society for the Promotion of Science Grant-in-Aid for Creative Scientific Research 17GS0419. M.S. is the Leader of the Global COE Program for Human Metabolomic Systems Biology and H.O. is the Leader of the Global COE Program for Stem Cell Medicine supported by MEXT.

Competing interests statement The authors declare no competing financial interests.

Supplementary material Supplementary material for this article is available at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.049015/-/DC1

References Brown, D. M. and Regillo, C. D. (2007). Anti-VEGF agents in the treatment of neovascular age-related macular degeneration: applying clinical trial results to the treatment of everyday patients. Am. J. Ophthalmol. 144, 627-637. Chen, J., Connor, K. M., Aderman, C. M. and Smith, L. E. (2008). Erythropoietin deficiency decreases vascular stability in mice. J. Clin. Invest. 118, 526-533. Fig. 8. Vhla-Cre KO mice show phenotypes of hypoxic Claxton, S. and Fruttiger, M. (2005). Oxygen modifies artery differentiation and network morphogenesis in the retinal vasculature. Dev. Dyn. 233, 822-828. retinopathies. (A) Pre-retinal hemorrhage observed by fundus Fischer, C., Jonckx, B., Mazzone, M., Zacchigna, S., Loges, S., Pattarini, L., photography, (B) iris neovascularization (open arrowheads), and Chorianopoulos, E., Liesenborghs, L., Koch, M., De Mol, M. et al. (2007). (C) cataract formation (arrow) in Vhla-CreKO mice. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 131, 463-475. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D. inactivation of HIF-1a, but not HIF-2a (Fig. 6), show that the and Semenza, G. L. (1996). Activation of vascular endothelial growth factor VHL/HIF-1 /VEGF cascade plays crucial roles in the transition gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604-4613. a Fruttiger, M., Calver, A. R., Kruger, W. H., Mudhar, H. S., Michalovich, D., from the fetal to the adult circulatory system in the retina. Takakura, N., Nishikawa, S. and Richardson, W. D. (1996). PDGF mediates a Our current study has possible clinical implications. Adult neuron-astrocyte interaction in the developing retina. Neuron 17, 1117-1131. Vhla-CreKO mice show similar characteristics (Fig. 8) to human Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A., Abramsson, A., Jeltsch, M., Mitchell, C., Alitalo, K., Shima, D. et al. (2003). ischemic retinopathies, such as diabetic retinopathy, retinal vessel VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell occlusion, and retinopathy of prematurity (Hayreh, 2007), and could Biol. 161, 1163-1177. provide clues for exploring the mechanisms of these human Gnarra, J. R., Ward, J. M., Porter, F. D., Wagner, J. R., Devor, D. E., Grinberg, A., Emmert-Buck, M. R., Westphal, H., Klausner, R. D. and Linehan, W. M. diseases. It has been recently shown that systemic administration of (1997). Defective placental vasculogenesis causes embryonic lethality in VHL- a prolyl hydroxylase inhibitor protects from retinal vaso-obliteration deficient mice. Proc. Natl. Acad. Sci. USA 94, 9102-9107. in response to hyperoxia (Sears et al., 2008), suggesting that pVHL Gruber, M., Hu, C. J., Johnson, R. S., Brown, E. J., Keith, B. and Simon, M. C. functions in retinopathy of prematurity. (2007). Acute postnatal ablation of Hif-2alpha results in anemia. Proc. Natl. Acad. Sci. USA 104, 2301-2306. Retinal hemangioma in VHL patients and ischemic retinopathies Haase, V. H., Glickman, J. N., Socolovsky, M. and Jaenisch, R. (2001). Vascular are usually treated by laser photocoagulation that disrupts tumors in livers with targeted inactivation of the von Hippel-Lindau tumor photoreceptors and increases the oxygen supply for the other neural suppressor. Proc. Natl. Acad. Sci. USA 98, 1583-1588. Hammes, H. P., Lin, J., Wagner, P., Feng, Y., Vom Hagen, F., Krzizok, T., cells from the choroidal vasculature bed, although it may cause night Renner, O., Breier, G., Brownlee, M. and Deutsch, U. (2004). Angiopoietin-2 blindness and visual field defects (Yu and Cringle, 2001). Recently, causes pericyte dropout in the normal retina: evidence for involvement in various kinds of VEGF inhibitors (Brown and Regillo, 2007) have diabetic retinopathy. Diabetes 53, 1104-1110. become possible candidates for use against ophthalmic diseases, Hayreh, S. S. (2007). Neovascular glaucoma. Prog. Retin. Eye Res. 26, 470-485. Hellstrom, M., Phng, L. K., Hofmann, J. J., Wallgard, E., Coultas, L., although destruction of the physiological vasculature (Fischer et al., Lindblom, P., Alva, J., Nilsson, A. K., Karlsson, L., Gaiano, N. et al. (2007). 2007) and neuronal dysfunction (Saint-Geniez et al., 2008) caused Dll4 signalling through Notch1 regulates formation of tip cells during by long-term VEGF inhibition caution against the continuous angiogenesis. Nature 445, 776-780. Hu, C. J., Wang, L. Y., Chodosh, L. A., Keith, B. and Simon, M. C. (2003). administration of these reagents. Suppression of HIF-1a might be Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha an alternative strategy against these diseases in the future. in hypoxic gene regulation. Mol. Cell. Biol. 23, 9361-9374. Finally, all of the results presented suggest that oxygen-sensing Kapitsinou, P. P. and Haase, V. H. (2008). The VHL tumor suppressor and HIF: insights from genetic studies in mice. Cell Death Differ. 15, 650-659. mechanisms mediated by VHL/HIF-1a/VEGF regulate the Kawamoto, S., Niwa, H., Tashiro, F., Sano, S., Kondoh, G., Takeda, J., transition from the fetal to the adult circulatory system in the retina, Tabayashi, K. and Miyazaki, J. (2000). A novel reporter mouse strain that and could represent a theoretical basis for the retinal vascular expresses enhanced green fluorescent protein upon Cre-mediated abnormalities in human VHL disease and the development of recombination. FEBS Lett. 470, 263-268. Kubota, Y., Hirashima, M., Kishi, K., Stewart, C. L. and Suda, T. (2008). ischemic retinopathies. It will be interesting to determine whether Leukemia inhibitory factor regulates microvessel density by modulating oxygen- this oxygen-sensing system, the VHL/HIF-1a pathway, is involved dependent VEGF expression in mice. J. Clin. Invest. 118, 2393-2403. in the transition of the circulatory system in other parts, such as Kubota, Y., Takubo, K., Shimizu, T., Ohno, H., Kishi, K., Shibuya, M., Saya, H. and Suda, T. (2009). M-CSF inhibition selectively targets pathological ductus arteriosus, which would confirm its generality for future angiogenesis and lymphangiogenesis. J. Exp. Med. 206, 1089-1102. studies. Kurihara, T., Ozawa, Y., Shinoda, K., Nagai, N., Inoue, M., Oike, Y., Tsubota, K., Ishida, S. and Okano, H. (2006). Neuroprotective effects of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, via modulating AT1R and AT2R Acknowledgements signaling in retinal inflammation. Invest. Ophthalmol. Vis. Sci. 47, 5545-5552. We thank Ichie Kawamori, Rie Takeshita, Haruna Koizumi and Taiga Shioda Lang, R. A. (1997). Apoptosis in mammalian eye development: lens morphogenesis, (Keio University, Tokyo, Japan) for technical assistance. This study was vascular regression and immune privilege. Cell Death Differ. 4, 12-20. supported by the Japanese Ministry of Education, Culture, Sports, Science and Lang, R. A. and Bishop, J. M. (1993). Macrophages are required for cell death Technology (MEXT) [Grant-in-Aid for the 21st Century COE Program and and tissue remodeling in the developing mouse eye. Cell 74, 453-462. Global COE Program at Keio University, and Scientific Research (No. Lobov, I. B., Rao, S., Carroll, T. J., Vallance, J. E., Ito, M., Ondr, J. K., Kurup, S.,

21791710) to T.K.]. Y.K. was supported by Keio Kanrinmaru Project. Glass, D. A., Patel, M. S., Shu, W. et al. (2005). WNT7b mediates DEVELOPMENT VHL regulates circulatory system transit RESEARCH ARTICLE 1571

macrophage-induced programmed cell death in patterning of the vasculature. Rao, S., Lobov, I. B., Vallance, J. E., Tsujikawa, K., Shiojima, I., Akunuru, S., Nature 437, 417-421. Walsh, K., Benjamin, L. E. and Lang, R. A. (2007). Obligatory participation of Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F. macrophages in an angiopoietin 2-mediated cell death switch. Development and Gruss, P. (2001). Pax6 is required for the multipotent state of retinal 134, 4449-4458. progenitor cells. Cell 105, 43-55. Rodesch, F., Simon, P., Donner, C. and Jauniaux, E. (1992). Oxygen Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., measurements in endometrial and trophoblastic tissues during early pregnancy. Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R. and Ratcliffe, P. J. Obstet. Gynecol. 80, 283-285. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for Ruhrberg, C., Gerhardt, H., Golding, M., Watson, R., Ioannidou, S., oxygen-dependent proteolysis. Nature 399, 271-275. Fujisawa, H., Betsholtz, C. and Shima, D. T. (2002). Spatially restricted Merkle, E. M. and Gilkeson, R. C. (2005). Remnants of fetal circulation: patterning cues provided by heparin-binding VEGF-A control blood vessel appearance on MDCT in adults. Am. J. Roentgenol. 185, 541-549. branching morphogenesis. Genes Dev. 16, 2684-2698. Mitchell, C. A., Rutland, C. S., Walker, M., Nasir, M., Foss, A. J., Stewart, C., Ryan, H. E., Poloni, M., McNulty, W., Elson, D., Gassmann, M., Arbeit, J. M. Gerhardt, H., Konerding, M. A., Risau, W. and Drexler, H. C. (2006). Unique and Johnson, R. S. (2000). Hypoxia-inducible factor-1alpha is a positive factor vascular phenotypes following over-expression of individual VEGFA isoforms in solid tumor growth. Cancer Res. 60, 4010-4015. from the developing lens. Angiogenesis 9, 209-224. Saint-Geniez, M., Maldonado, A. E. and D’Amore, P. A. (2006). VEGF Nicholson, D. H., Green, W. R. and Kenyon, K. R. (1976). Light and electron expression and receptor activation in the choroid during development and in the microscopic study of early lesions in angiomatosis retinae. Am. J. Ophthalmol. adult. Invest. Ophthalmol. Vis. Sci. 47, 3135-3142. 82, 193-204. Saint-Geniez, M., Maharaj, A. S., Walshe, T. E., Tucker, B. A., Sekiyama, E., Ohh, M., Yauch, R. L., Lonergan, K. M., Whaley, J. M., Stemmer-Rachamimov, Kurihara, T., Darland, D. C., Young, M. J. and D’Amore, P. A. (2008). A. O., Louis, D. N., Gavin, B. J., Kley, N., Kaelin, W. G., Jr and Iliopoulos, O. Endogenous VEGF is required for visual function: evidence for a survival role on (1998). The von Hippel-Lindau tumor suppressor protein is required for proper muller cells and photoreceptors. PLoS ONE 3, e3554. assembly of an extracellular fibronectin matrix. Mol. Cell 1, 959-968. Sears, J. E., Hoppe, G., Ebrahem, Q. and Anand-Apte, B. (2008). Prolyl Ozawa, Y., Nakao, K., Shimazaki, T., Takeda, J., Akira, S., Ishihara, K., hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy. Hirano, T., Oguchi, Y. and Okano, H. (2004). Downregulation of STAT3 Proc. Natl. Acad. Sci. USA 105, 19898-19903. activation is required for presumptive rod photoreceptor cells to differentiate in Stalmans, I., Ng, Y. S., Rohan, R., Fruttiger, M., Bouche, A., Yuce, A., the postnatal retina. Mol. Cell. Neurosci. 26, 258-270. Fujisawa, H., Hermans, B., Shani, M., Jansen, S. et al. (2002). Arteriolar and Ozawa, Y., Nakao, K., Shimazaki, T., Shimmura, S., Kurihara, T., Ishida, S., venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Yoshimura, A., Tsubota, K. and Okano, H. (2007). SOCS3 is required to Invest. 109, 327-336. temporally fine-tune photoreceptor cell differentiation. Dev. Biol. 303, 591- Tang, N., Mack, F., Haase, V. H., Simon, M. C. and Johnson, R. S. (2006). pVHL 600. function is essential for endothelial extracellular matrix deposition. Mol. Cell. Phng, L. K., Potente, M., Leslie, J. D., Babbage, J., Nyqvist, D., Lobov, I., Biol. 26, 2519-2530. Ondr, J. K., Rao, S., Lang, R. A., Thurston, G. et al. (2009). Nrarp coordinates Yu, D. Y. and Cringle, S. J. (2001). Oxygen distribution and consumption within endothelial Notch and Wnt signaling to control vessel density in angiogenesis. the retina in vascularised and avascular retinas and in animal models of retinal Dev. Cell 16, 70-82. disease. Prog. Retin. Eye Res. 20, 175-208. Rankin, E. B., Biju, M. P., Liu, Q., Unger, T. L., Rha, J., Johnson, R. S., Simon, Yun, Z., Maecker, H. L., Johnson, R. S. and Giaccia, A. J. (2002). Inhibition of M. C., Keith, B. and Haase, V. H. (2007). Hypoxia-inducible factor-2 (HIF-2) PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a regulates hepatic erythropoietin in vivo. J. Clin. Invest. 117, 1068-1077. mechanism for regulation of adipogenesis by hypoxia. Dev. Cell 2, 331-341. DEVELOPMENT