von hippel-lindau protein regulates transition from the ...with hemorrhage at e12.5, and loss of...

1563RESEARCH ARTICLE

INTRODUCTIONIn early neonates, the fetal circulatory system undergoes rapid anddramatic transition to the adult circulatory system to adapt todetachment from the maternal blood supply. Normally, embryonicconnecting vessels such as ductus arteriosus, ductus venosus and theforamen ovale close and regress (Merkle and Gilkeson, 2005). In theneonatal retina, it is generally known that the hyaloid vessels thatmaintain blood flow in the embryonic retina regress, and that retinalvessels assume the role of supplying blood to the retina (Lang, 1997;Lobov et al., 2005). This process is regulated by the programmedcell death switch mediated by macrophages via Wnt andangiopoietin 2 pathways (Lang and Bishop, 1993; Lobov et al.,2005; Rao et al., 2007).

Oxygen concentration is vital to nearly all forms of life on earthvia its role in energy homeostasis (Yun et al., 2002). Mammalianembryos develop in a low (~3%) oxygen environment inside thematernal body (Rodesch et al., 1992). However, pups are abruptlyexposed to atmospheric oxygen levels (~21%) after birth. Thisdramatic change in oxygen environment at the point of birthindicates some crucial roles of oxygen concentration in triggeringthe transition from the fetal to the adult circulatory system.

The von Hippel-Lindau tumor suppressor protein (pVHL) is asubstrate recognition component of an E3-ubiquitin ligase thatrapidly destabilizes hypoxia-inducible factor as (HIF-as) undernormoxic, but not hypoxic, conditions (Maxwell et al., 1999).Genetic inactivation of the Vhl gene results in the stabilization andincreased activity of HIF-as, which includes HIF-1a, HIF-2a andHIF-3a. It has been reported that Vhl-null embryos are lethal atembryonic day (E) 11.5-E12.5 owing to abnormal placentalvascularization (Gnarra et al., 1997). Endothelial-specific deletionof the Vhl gene using Tie2-Cre mice also results in intrauterine deathwith hemorrhage at E12.5, and loss of HIF-1a does not rescue thelethality (Tang et al., 2006). Although the vascular endothelialgrowth factor (VEGF) gene is widely known to be a HIF-1a target(Forsythe et al., 1996), HIF-2a is thought to be the main regulatorof the VEGF gene in tissues that express both HIF-1a and HIF-2a(Hu et al., 2003; Tang et al., 2006). These previous studies showedthat VHL is required for endothelial cells in a cell autonomousmanner. However, surprisingly, it is still largely unknown how VHLcontributes to vascular development, particularly how and when thewell-known relationship between VHL and oxygen concentrationoperates in vascular development. In humans, VHL disease is anautosomal dominant syndrome that causes the development ofvarious benign and malignant disorders (Nicholson et al., 1976). The

Development 137, 1563-1571 (2010) doi:10.1242/dev.049015© 2010. Published by The Company of Biologists Ltd

Departments of 1Physiology and 2Ophthalmology, and 3Laboratory of Retinal CellBiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo160-8582, Japan. 4Molecular Biology Section, Division of Biological Sciences,University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0366,USA. 5Department of Cell Differentiation, The Sakaguchi Laboratory, Keio UniversitySchool of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.6Department of Ophthalmology, Hokkaido University Graduate School of Medicine,Kita 8, Nishi 5 Kita-ku, Sapporo, Hokkaido, Japan. 7Abramson Family CancerResearch Institute and Howard Hughes Medical Institute, University of PennsylvaniaSchool of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104, USA.8Department of Biochemistry and Integrative Medical Biology, Keio University Schoolof Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. 9Departmentof Life Science and Medical Bio-Science, School of Advanced Science andEngineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480,Japan.

*These authors contributed equally to this work†Present address: Department of Cell Biology, The Scripps Research Institute,10550 North Torrey Pines Road, La Jolla, CA 92037, USA‡Authors for correspondence ([email protected]; [email protected])

Accepted 26 February 2010

SUMMARYIn early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonicconnecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vesselsmaintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. Thisprocess is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In thisstudy, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the pointof birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligasethat rapidly destabilizes hypoxia-inducible factor as (HIF-as) under normoxic, but not hypoxic, conditions. To examine the role ofoxygen-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 vesselsand poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelialgrowth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1agene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from thefetal to the adult circulatory system in the retina.

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

von Hippel-Lindau protein regulates transition from the fetalto the adult circulatory system in retinaToshihide 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,‡

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major disorders include retinal, brain and spinal cord angioma,pheochromocytoma, renal cell carcinoma and pancreaticcystadenoma. In the majority of VHL patients, retinal angioma is thefirst sign of the disease to appear. However, the molecularmechanism of retinal angioma development in VHL disease stillremains unknown.

In this study, by using retina-specific conditional-knockouttechnology, we explore the precise in vivo function of VHL in retinalvascular development, and show that not only macrophages but alsotissue oxygen-sensing mechanisms regulate the transition from thefetal to the adult circulatory system in the retina.

MATERIALS AND METHODSMiceAll animal experiments were conducted in accordance with the Associationfor Research in Vision and Ophthalmology (ARVO) Statement for the Useof Animals in Ophthalmic and Vision Research. In the expression analysisshown in Fig. 1, we used C57BL/6 mice (Clea Japan). Transgenic miceexpressing Cre recombinase under control of the Pax6 retina-specificregulatory element a-promoter (a-Cre) (Marquardt et al., 2001) were matedwith Vhlfloxed/floxed mice (Haase et al., 2001) (kind gift from Volker H. Haase,University of Pennsylvania), HIF-1afloxed/floxed mice (Ryan et al., 2000), orHIF-2afloxed/floxed mice (Gruber et al., 2007). As control littermates forVhla-CreKO mice, HIF-1aa-CreKO mice, HIF-1aa-CreKO;Vhla-CreKO miceand HIF-2aa-CreKO;Vhla-CreKO mice, we used Vhlfloxed/floxed mice, HIF-1afloxed/floxed mice, HIF-1afloxed/floxed;Vhlfloxed/floxed mice and HIF-2afloxed/floxed;Vhlfloxed/floxed mice without the a-Cre transgene, respectively. Ina preliminary study, we found no detectable difference in retinal vascularstructures between a-Cre+ and a-Cre– mice, confirming the validity of thesecontrol littermates. To monitor Cre expression in a-Cre mice or Vhla-CreKO

mice, we mated these mice with CAG-CAT-EGFP transgenic mice(Kawamoto et al., 2000). All mice used in this study were maintained on aC57BL/6J background.

Preparation of whole-mount samples and cryosections of retinasEnucleated eyes were fixed for 20 minutes in 4% paraformaldehyde (PFA)in PBS and then dissected as previously described (Fruttiger et al., 1996;Gerhardt et al., 2003; Kubota et al., 2008). The obtained tissues were post-fixed overnight in 4% PFA in PBS and stored in methanol at –20°C.Cryosections of retinas (10 mm) were prepared as previously described(Kurihara et al., 2006), after eyeballs were immersed overnight in 4% PFA.

Immunostaining and in situ hybridizationImmunohistochemistry (IHC) for whole-mount retinas and other tissues wasperformed as described previously (Fruttiger et al., 1996; Gerhardt et al.,2003; Kubota et al., 2008; Kurihara et al., 2006). The primary antibodiesused were: monoclonal anti-PECAM1 (2H8, Chemicon), a-smooth muscleactin (1A4; FITC-conjugated; Sigma-Aldrich), desmin (DAKO), F4/80 (A3-1; Serotec, Oxford, UK), GFAP (G-A-5; Cy3-conjugated; Sigma-Aldrich orDako), polyclonal type IV collagen (Cosmo Bio), HIF-1a (originallyestablished by immunizing purified fusion proteins encompassing aminoacids 416 to 785 of mouse HIF-1a into guinea pigs), HIF-2a (Santa CruzBiotechnology), glutamine synthetase (Molecular Probes), opsin (CosmoBio) and pVHL (Santa Cruz Biotechnology). The secondary antibodies usedwere Alexa Fluor 488-conjugated IgGs (Molecular Probes) or Cy3/Cy5-conjugated IgGs (Jackson ImmunoResearch). Nuclei were stained with 10mg/ml Hoechst bisbenzimide 33258 (Sigma-Aldrich) or DAPI (MolecularProbes). For whole-mount in situ hybridization (ISH), retinas were brieflydigested with proteinase K and hybridized with digoxigenin (DIG)-labeledantisense RNA probes. When ISH was combined with IHC, IHC wasperformed after all the ISH procedures were completed. For the BrdUincorporation assay, 100 mg per body weight (g) of BrdU (BD Pharmingen)dissolved in sterile PBS was injected intraperitoneally 2 hours beforesacrifice. Isolated retinas were stained using a BrdU immunohistochemistrysystem (Calbiochem) according to the manufacturer’s instructions. WhenBrdU assays were combined with IHC, the application of the first antibodyfor each procedure was done simultaneously. FITC-conjugated Dextran

(Sigma-Aldrich) was injected into the left cardiac ventricle and allowed tocirculate for 2 minutes. After staining, samples were mounted using aProlong Antifade Kit (Molecular Probes).

Assessment of tissue hypoxiaDetection of hypoxic cells in cryosections was performed using aHypoxyprobe-1 Plus Kit (Chemicon). In brief, 60 mg/kg of pimonidazolewas injected intraperitoneally 30 minutes before sacrifice, and samples werestained with Hypoxyprobe Mab1-FITC.

RT-PCR analysisTotal RNA was prepared from retinal tissues and reverse-transcribed usingSuperscript II (Invitrogen). A quantitative PCR assay for Vegfa wasperformed on an ABI 7500 Fast Real-Time PCR System using TaqMan FastUniversal PCR master mixture (Applied Biosystems) and TaqManGene Expression Assay Mix of Vegfa (Mm00437304_m1), Csf1r(Mm00432689_m1), and angiopoietin 2 (Mm00545822_m1). Mouse b-actin (Mm00607939_s1) assay mix served as an endogenous control. Datawere analyzed with 7500 Fast System SDS Software 1.3.1. All experimentswere done with four replicates.

Intravitreous injectionsInjections into the vitreous body were performed using 33-gauge needles,as described previously (Gerhardt et al., 2003; Kubota et al., 2008). SterilePBS (0.5 ml) with or without 1 mg/ml Flt1-Fc chimera proteins (R&DSystems) was injected at postnatal day (P) 4.

Confocal microscopy and quantificationFluorescence images were obtained using a confocal laser scanningmicroscope (FV1000; Olympus) at room temperature. Quantification ofthe cells or substances of interest was usually done in eight random200 mm � 200 mm fields just behind the sprouting edges of each retina. Toconstruct three-dimensional projections, multiple slices horizontallyimaged from the same field of view were integrated by FV10-ASWViewer (Olympus).

Retinal explant cultureRetinal explant culture was performed using P6 mouse neural retinas basedon the protocol we previously described (Ozawa et al., 2007; Ozawa et al.,2004). Briefly, eyes were enucleated and neural retinas were isolated andplaced on a Millicell chamber filter (Millipore; pore size, 0.4 Am) with theganglion cell layer facing up. The chamber was then placed in a 6-wellculture plate, containing 50% MEM (GIBCO), 25% HBSS (GIBCO), 25%horse serum (Thermo Trace), 200 mM L-glutamine, and 6.75 mg/ml D-glucose. Explants were incubated at 34°C in 5% CO2. Twenty-four hoursafter exposure of each concentration of oxygen, the explants were subjectedto immunostaining or RT-PCR analysis. The oxygen concentration in theincubator was controlled with nitrogen.

Statistical analysisComparison between the average variables of two groups was performed bytwo-tailed Student’s t-test. P-values less than 0.05 were considered to bestatistically significant.

RESULTSHIF-1a expression is rapidly downregulated afterbirthAs a first step in the investigation of the cellular responses in theretina to the dramatic change in the oxygen environment at birth, weexamined the expression patterns of pVHL, HIF-1a and HIF-2a inthe developing retina. Expression of pVHL was detectedubiquitously from the inner to the outer side in the retina, and did notchange between before and after birth (Fig. 1A-E). By contrast,nuclear staining of HIF-1a was downregulated in the deep retinallayer [the neuroblastic layer (NBL) at this stage] after birth (Fig. 1H-J), despite its ubiquitous expression in embryonic stages (Fig. 1F,G).HIF-2a immunoreactivity was predominantly detected inendothelial cells of hyaloid vessels in the vitreal space (see Fig.

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S1A-C in the supplementary material) and in retinal vessels in theganglion cell layer (GCL) (see Fig. S1D,E in the supplementarymaterial). In embryonic retina, hypoxic areas were visualized by ahypoxic probe, pimonidazole, through all layers (Fig. 1K,L),although a relatively hypoxic area after birth was limited to the GCL(Fig. 1M-O), which still expressed HIF-1a (Fig. 1H-J).

Vhla-CreKO mice show persistence of the hyaloidvessels independently of macrophagesTo elucidate the function of pVHL in postnatal retinalvascularization, we employed a retina-specific mouse Cre line, a-Cre, which shows Cre expression in the deep retinal layer, includingin retinal progenitor cell-derived neural cells, under the control of aneural retina-specific regulatory element of murine Pax6 gene, butnot in astrocytes and endothelial cells at P10 (Marquardt et al.,2001). We examined the past events of Cre expression in a-Cre miceuntil early postnatal age by crossing a-Cre mice with a reportertransgenic line, CAG-CAT-EGFP (Kawamoto et al., 2000). At P6,GFP expression was detected in neural cells in the deep retinal layer,but not in astrocytes or endothelial cells of both retinal and hyaloidvasculature (see Fig. S2A-T in the supplementary material). Then,we crossed Vhlfloxed/floxed mice (Haase et al., 2001) with a-Cre miceand generated a-Cre-specific conditional-knockout mice for Vhl(Vhla-CreKO mice). As control littermates for Vhla-CreKO mice, weused Vhlfloxed/floxed mice without the a-Cre transgene. At P6, bloodflow visualized by intracardiac injection of fluoresceinisothiocyanate (FITC)-dextran was detected in both hyaloid andretinal vessels of control mice (Fig. 2A,B,E,G,I). In Vhla-CreKO mice,however, there was abundant blood flow in hyaloid vessels, poorflow in retinal vessels, and abundant collateral flow from hyaloidvessels to retinal vessels (Fig. 2C,D,F,H,J). Nonetheless, despite theimpaired blood flow, the retinal vascular structure could be detectedby immunohistochemistry for platelet/endothelial cell adhesionmolecule 1 (PECAM1) in Vhla-CreKO mice (Fig. 2F,H). Thepersistent blood flow in hyaloid vessels in Vhla-CreKO mice prompted

us to examine the well-known mechanisms of hyaloid vesselregression: reduced perivascular macrophages or angiopoietin 2deficiency lead to persistence of the hyaloid vessels (Lobov et al.,2005; Rao et al., 2007). Macrophages around the hyaloid vesselswere not significantly changed (Fig. 2K-M), and colony stimulatingfactor 1 receptor (Csf1r) expression, which correlates with thenumber of macrophages (Kubota et al., 2009), showed no significantreduction in Vhla-CreKO mice (Fig. 2N). Angiopoietin 2 expressionwas rather increased in Vhla-CreKO mice (Fig. 2O). Collectively,Vhla-CreKO mice show persistence of the hyaloid vesselsindependently of macrophages and angiopoietin 2.

Vhla-CreKO mice show poorly formed retinal vesselscharacterized by excessive vessel regressionAs abnormal hyaloid vessel persistence could affect retinalvascularization, we examined the retinal vessels in Vhla-CreKO mice.In these mice, the entire growth of the retinal vasculature spreadingfrom the optic disc and the branching points were significantlyreduced compared with control mice (P=0.013 for spreadingdistance, P=0.00012 for branching points; Fig. 3A,B,D,E,M,N; seealso Fig. S3A,B in the supplementary material). Endothelial tip cellsand their filopodia, which are controlled by VEGF and the Delta-like 4/Notch pathway (Gerhardt et al., 2003; Hellstrom et al., 2007;Phng et al., 2009), were also significantly reduced in Vhla-CreKO mice(P=0.0012 for tip cells; P=0.011 for filopodia; Fig. 3C,F,O,P). Asignificant decrease in endothelial proliferation (Fig. 3G,J,Q) andexcessive vessel regression (Fig. 3H,K,R), characterized by emptyvascular sleeves (Phng et al., 2009), were also detected in Vhla-CreKO

mice (P=7.9�10–5 for BrdU+ endothelial cells; P=0.0044 for emptysleeves). Empty sleeves were abundantly detected not only in theirstalk cell area but also at their leading edge, suggesting that vesselregression occurs in both tip cells and stalk cells in Vhla-CreKO mice.Although it is well known that pVHL regulates the expression ofvarious extracellular matrices (Ohh et al., 1998), Col IV expressionwas not impaired in Vhla-CreKO mice. Although the number of

1565RESEARCH ARTICLEVHL regulates circulatory system transit

Fig. 1. Expression patterns of pVHL and HIF-1ain the retina before and after birth. (A-E) pVHLstaining (green), detected in the soma of cells in thewhole retina, is unchanged after birth. (F-J) Nuclearstaining of HIF-1a (red), detected ubiquitously in theembryo (F,G), is downregulated in the deep retinallayer (NBL at this stage) after birth (H-J). (K-O) Inembryonic retina, hypoxic areas visualized by thehypoxic probe pimonidazole (yellow) spread allthrough the layers (K,L), although after birth thehypoxic signal was considerably downregulated inthe 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: 100mm in A-O.

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pericytes associated with endothelial tubes was not changed inVhla-CreKO mice (P=0.395; Fig. 3I,L,S), detached pericytes werefrequently detected just beyond their sprouting edges. Previously,injection of angiopoietin 2 into the eyes of normal rats was shownto induce a dose-dependent pericyte loss (Hammes et al., 2004),suggesting that upregulated angiopoietin 2 (Fig. 2O) mightcontribute to pericyte defects in Vhla-CreKO mice. Collectively,Vhla-CreKO mice showed poorly formed retinal vessels, presumablyowing to persistence of the hyaloid vessels.

Vascular defects in Vhla-CreKO mice are attributableto ectopic VEGF expressionThe intact macrophages and preserved angiopoietin 2 expression inVhla-CreKO mice caused us to suppose that some other mechanism ledto the persistence of the hyaloid vessels. As retinal vascular growthdepends on the appropriate spatial distribution of heparin-bindingVEGF within the retina (Gerhardt et al., 2003; Kubota et al., 2008;Ruhrberg et al., 2002; Stalmans et al., 2002), we suspected that thevascular defects in Vhla-CreKO mice were attributable to the abnormalexpression pattern of VEGF. In control mice, VEGF expression waspredominantly detected in astrocytes beyond the sprouting edge(Fig. 4A,B,E-H). In Vhla-CreKO mice, marked expression of VEGFwas detected in the deep retinal layer (Fig. 4C,D,I-L), and wascoincidental with the area of persistent hyaloid vessels. However,typical VEGF expression in astrocytes beyond the sprouting edgewas diminished in Vhla-CreKO mice, despite the fact that theirastrocyte plexus was normal (Fig. 4M,N). This VEGF expressionpattern in Vhla-CreKO mice was similar to normal VEGF expressionin the embryonic retina (Saint-Geniez et al., 2006). By quantitativeRT-PCR analysis, a more than 4-fold significant increase(P=0.00071) in Vegfa expression was detected in the entire retina of

Vhla-CreKO mice (Fig. 4O). Because these expression patterns ofVEGF suggested that the vascular defects in Vhla-CreKO mice couldbe attributable to ectopic and abundant VEGF expression, weinjected a potent VEGF inhibitor, FLT1-Fc chimeric protein(Gerhardt et al., 2003; Kubota et al., 2008), into the eyes ofVhla-CreKO mice. These FLT1-Fc injections reduced the amount ofboth collateral blood flow and vascular structures from hyaloidvessels to retinal vessels (Fig. 4P-U). As genetic inactivation of theVhl gene results in HIF-a stabilization (Kapitsinou and Haase, 2008)and the upregulation of Vegfa expression, which is widely known asa HIF-1a target gene (Forsythe et al., 1996), we examined theexpression of pVHL, HIF-1a and HIF-2a in Vhla-CreKO mice. Wefound that pVHL immunoreactivity was greatly reduced in the deeplayer of the peripheral retina (Fig. 4V,W), where the strong a-Cre-mediated recombination is detected (see Fig. S2 in thesupplementary material), except for in astrocytes and endothelialcells (see Fig. S4A-J in the supplementary material). HIF-1aimmunoreactivity was increased in this area of Vhla-CreKO mice (Fig.4X,Y; see also Fig. S4K-O in the supplementary material). Inaddition, strong HIF-2a immunoreactivity was detected ininvaginating hyaloid vessels, but not in the area of reduced pVHLexpression (Fig. 4Z,AA; see also Fig. S4P-T in the supplementarymaterial).

Increased VEGF expression in Vhla-CreKO mice is dueto their impaired oxygen-sensing mechanism viaHIF-1aAs the VEGF expression pattern in postnatal Vhla-CreKO retina issimilar to normal VEGF expression in the embryonic retina (Saint-Geniez et al., 2006), we expected abnormal VEGF expression in theVhla-CreKO retina to be attributable to an impaired oxygen-sensing


Fig. 2. Vhla-CreKO mice show persistence of thehyaloid vessels independently of macrophages.(A-D) Representative images of retinas afterintracardiac FITC injection (n=8). Vhla-CreKO miceshow collateral flow from hyaloid vessels to retinalvessels (open arrowheads) in the P6 retinas. Bloodflow was visualized by intracardiac FITC-conjugateddextran (green) injection. (E-H) Confocal images ofFITC- (orange) PECAM1- (blue) labeled P6 retinas;G,H show sections. Poor blood flow is detected inretinal vasculature (asterisk) in Vhla-CreKO micecompared with control. Relatively abundant dextranwas perfused into arteries (A) and veins (V) incontrol mice, and into hyaloid vessels (arrows) inVhla-CreKO mice. (I,J) Schematics show abundantblood flow (red) in retinal vessels in control mice,and in hyaloid vessels in Vhla-CreKO mice.(K,L) Immunostaining with F4/80 (green) andPECAM1 (blue) on hyaloid vessels in P6 retinas.(M) Quantification of F4/80+ macrophages per200mm vessel length. Vessel length was calculatedby FV10-ASW Viewer software (n=4).(N,O) Quantitative PCR of Csf1r (N) or angiopoietin2 (O) for isolated RNA from P6 retinas (n=5).*P<0.05. All panels show P6 retina. Error barsindicate mean ± s.d. Scale bars: 500mm in A-D;100mm in E-H,K,L.

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mechanism and a lack of response to the atmospheric oxygenconcentration. As newborn mice do not tolerate oxygen levels below10% (Claxton and Fruttiger, 2005), we employed an ex vivo retinalculture system, which enabled us to expose the retina to variableoxygen concentrations between 1% and 21% (Fig. 5A). In the controlretina culture, strong nuclear HIF-1a expression was induced in 1%oxygen (Fig. 5D), but not in 21% oxygen (Fig. 5B). Consequently,Vegfa expression was significantly higher (P=2.4�10–8) in 1%oxygen than in 21% oxygen (Fig. 5J). In the Vhla-CreKO retina culture,strong nuclear HIF-1a expression was detected (Fig. 5H), even in21% oxygen (Fig. 5F), and Vegfa expression in the Vhla-CreKO retinain 21% oxygen was significantly higher (P=1.1�10–5) than in thecontrol in 21% oxygen; it was equivalent to the level observed in 1%oxygen (Fig. 5J). To determine the significance of the increasednuclear HIF-1a level in hypoxic conditions in this culture system, wegenerated a-Cre-mediated conditional-knockout mice for HIF-1a(Ryan et al., 2000) (HIF-1aa-CreKO mice). In HIF-1aa-CreKO retina,hypoxia-induced HIF-1a stabilization (Fig. 5K,M) was not detected(Fig. 5O,Q) and, accordingly, increased Vegfa expression in 1%oxygen was significantly suppressed (P=0.00028) compared with incontrol retina (Fig. 5S). Consistent with the in vivo results (Fig. 1A-E), the expression pattern of pVHL was stable in the deep retinal layerduring ex vivo culture, and was not affected by the oxygenconcentration (Fig. 5C,E,G,I,L,N,P,R). These data in the ex vivoculture system suggest that the increased VEGF expression inVhla-CreKO mice is due to their impaired oxygen-sensing mechanismvia HIF-1a.

Deletion of HIF-1a, but not HIF-2a, rescues vasculardefects in Vhla-CreKO miceThe expression patterns of pVHL, HIF-1a, HIF-2a and VEGF inVhla-Cre KO mice (Fig. 4) and the data from the ex vivo culture system(Fig. 5) suggested that the increased activity of HIF-1a wasresponsible for the vascular defects in Vhla-CreKO mice. Therefore,we generated a-Cre-specific double-knockout mice for VHL andHIF-1a (Ryan et al., 2000) (HIF-1a;Vhla-CreKO mice). Vascular

defects seen in Vhla-CreKO mice were suppressed in HIF-1a;Vhla-CreKO mice (Fig. 6A,B,D-G,M-P). The abnormal expression ofVegfa in Vhla-CreKO was fairly normalized in HIF-1a; Vhla-CreKO

mice (Fig. 6J,K; see also Fig. S5A in the supplementary material;1.15±0.08-fold compared with control). Conversely, a-Cre-mediated VHL and HIF-2a (Gruber et al., 2007) double-knockoutmice (HIF-2a; Vhla-CreKO mice) showed similar vascular defects anda similar VEGF expression pattern to Vhla-CreKO mice (Fig.6C,H,I,L; see also Fig. S5B in the supplementary material). Takentogether, HIF-1a, but not HIF-2a, plays essential roles in theappearance of vascular defects in the Vhla-CreKO retina.

Progressive retinal degeneration in adultVhla-CreKO miceNext, we examined retinal function and circulation in adultVhla-CreKO mice. Increased apoptosis of photoreceptors and otherneuronal cells was seen in Vhla-CreKO mice after P14 (Fig. 7A-H)induced decrease of the outer nuclear layer (ONL; Fig. 7I,J), despitethe normal development of photoreceptors at P7 (Fig. 7E,J). Gliosis,accompanied by vessels invaginating into the deep retinal layer,destroyed the construction of the ONL (Fig. 7K-T) at P14. As aresult, a significant (P=3.3�10–6 for a-wave, P=1.3�10–5 for b-wave) decrease in amplitude and a significant (P=0.046 for b-wave)extension of implicit time in electroretinography (ERG) wasobserved in Vhla-CreKO mice at P28 (Fig. 7U-Y). Circulation viahyaloid vessels in the Vhla-CreKO retina was detected at the age of 8weeks (see Fig. S6A-D in the supplementary material) and at 18months (see Fig. S6E-H in the supplementary material). Some of theVhla-CreKO mice developed pre-retinal hemorrhage, cataracts and irisneovascularization (Fig. 8A-C), similar to the phenotypes thataccompany ischemic retinopathy (Hayreh, 2007).

DISCUSSIONIn the present study, we showed that retina-specific conditional-knockout mice for the Vhl gene exhibit persistent hyaloidvessels independently of macrophage function, which are


Fig. 3. Vhla-CreKO mice show poorly formedretinal vessels characterized by excessivevessel regression. (A-F) PECAM1 staining of P6retinas. Note delayed vascular growth (bidirectionalarrows in A,D), decreased branching points and tipcells (B,E), and filopodia (C,F) in Vhla-CreKO mice.Persistent hyaloid vessels in the lower left lobe ofVhla–CreKO mice were removed mechanically beforeimmunostaining (D). (G-L) Immunostaining withthe indicated antibodies. Note the decreased BrdU+

endothelial cells (arrows, G,J), type IV collagen (ColIV)+isolectin– empty sleeves (arrowheads, H,K), anddetached pericytes beyond sprouting edges (openarrowheads, L) in Vhla-CreKO mice, although mostpericytes in control mice are closely contacted withvasculature (closed arrowheads, I,L).(M-S) Quantification of percentage spreadingdistance from optic discs (M), the number ofbranching points in the area behind sproutingedges (N), tip cells per field (O), filopodia per tipcell (P), BrdU+ endothelial cells (Q), Col IV+isolectin–

vessels (R), and pericytes per 200mm vessel length(S; n=6, in each genotype). *P<0.05, **P<0.01. Allpanels show P6 retina. Error bars indicatemean±s.d. Scale bars: 500mm in A,D; 100mm inB,E,G-L; 20mm in C,F.

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sustained until adulthood. These vascular defects in Vhla-CreKO

mice are rescued by either local VEGF inhibition or geneticdeletion of HIF-1a, but not of HIF-2a. These results suggest notonly macrophages but also tissue oxygen-sensing mechanismsregulate the transition from the fetal to the adult circulatorysystem in retina.

Considering that HIF-1a in the outer side of the retina isdramatically downregulated after birth but that pVHL expression inthe retina does not differ between embryos and neonates (Fig. 1), thechange of environmental oxygen concentration must be importantfor the role of pVHL in this transition (Rodesch et al., 1992). VEGFexpression in the deep retinal layer during embryonic but not


Fig. 4. Vascular defects in Vhla-CreKO mice are attributableto ectopic VEGF expression. (A-L) Whole-mount (A-D) orsection (E-L) in situ hybridization for VEGF combined withimmunostaining with the indicated antibodies. Although VEGFexpression is detected in astrocytes located in the avasculararea (arrows) of control mice, abundant VEGF expression isdetected in the deep retinal layer (open arrowheads) wherepersistent 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 retinasperfused with FITC-dextran (P-R), and confocal images labeledwith 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 injectedwith vehicle (open arrowheads in Q,T). (V-AA) Immunostainingof P6 retinal sections with the indicated antibodies. AlthoughpVHL expression is greatly reduced (open arrowheads, W), HIF-1a immunoreactivity is increased (closed arrowheads, Y) in thedeep retinal layer of Vhla–CreKO mice. HIF-2a staining is detectedin invaginating hyaloid vessels in Vhla-CreKO mice (arrows, AA).**P<0.01. Error bars indicate mean ± s.d. Scale bars: 500mm inA-L; 200mm in P-AA; 100mm in M,N.

Fig. 5. Increased VEGF expression in Vhla-CreKO mice is dueto their impaired oxygen-sensing mechanism via HIF-1a.(A) Schematics show the experimental procedure: isolatedretinas from control or conditional-knockout mice wereunfolded, placed on a chamber filter, and exposed to eachconcentration 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 isolatedfrom retinal explants (n=6, respectively). Note, strong HIF-1astaining (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 expressionwas upregulated even under normoxia conditions in Vhla-CreKO

retina. (S) In HIF-1aa-CreKO retina, hypoxia-induced Vegfaexpression was significantly suppressed. **P<0.01. Error barsindicate mean±s.d. Scale bars: 50mm in B-I,K-R.

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postnatal retinal stages (Saint-Geniez et al., 2006) supports the ideathat the abnormal VEGF distribution in postnatal Vhla-CreKO retinarepresents a defective VEGF expression switch from the embryonicpattern to the postnatal one. Mice selectively expressing singleisoforms of VEGF (VEGF120 or VEGF188) or overexpressing VEGFunder the control of a lens-specific promoter show a persistence ofhyaloid vessels as well as defective retinal vascularization,supporting the concept that spatial distribution of VEGF regulatesthe transition from the embryonic to the adult circulatory system inthe retina (Mitchell et al., 2006; Stalmans et al., 2002).

Previously, Lang and colleagues clearly demonstrated thatmacrophages mediate hyaloid vessel regression through the paracrineaction of WNT7B (Lobov et al., 2005). They showed that a lack ofmacrophages in PU.1–/– mice caused a significant delay in hyaloidvessel regression, and that intraocularly injected wild-type, but notWnt7b-mutant, macrophages ameliorated this delay. Moreover,angiopoietin 2 has also been shown to be involved in hyaloid vesselregression via the dual effect of suppressing survival signaling in the

endothelial cells of hyaloid vessels and stimulating Wnt ligandproduction by macrophages (Rao et al., 2007). Interestingly, themacrophage/microglia number in Vhla-CreKO mice was not affected(Fig. 2K-M), but angiopoietin 2 expression was increased (Fig. 2O),which suggests that an oxygen-sensing mechanism mediated by theVHL/HIF-1a/VEGF system operates hyaloid vessel regressionindependently of macrophages, Wnt and angiopoietin 2.

A possible concern with our data might be the slightly, butsignificantly, increased branching points observed in HIF-1a;Vhla-CreKO mice (Fig. 6G,N). Independently of HIF-a proteolysis,pVHL is known to be involved in extracellular matrix assembly andturnover (Ohh et al., 1998). Moreover, erythropoietin has beenreported to be regulated by VHL and HIFs (Chen et al., 2008;Rankin et al., 2007). These previous findings suggest theinvolvement of multiple candidates in addition to HIF-1a andVEGF, and might explain the minor vascular changes observed inHIF-1a;Vhla-CreKO mice. However, the dramatic rescue effectsobtained by FLT1-Fc injection (Fig. 4P-U), or by the genetic


Fig. 6. Deletion of HIF-1a but not HIF-2a, rescues vascular defectsin Vhla-CreKO mice. (A-I) Blood flow visualized by intracardiac FITC-dextran injection (A-C), or confocal images from mice of indicatedgenotypes labeled with PECAM1 at P6 (D-I). Vascular defects [collateralflow from hyaloid vessels to retinal vessels (open arrowheads), delayedvascular growth (bidirectional arrows), decreased branching points, tipcells and filopodia] seen in Vhla-CreKO mice are abolished by deletion ofHIF-1a, but not HIF-2a, in Vhla-CreKO mice. (J-L) Whole-mount in situhybridization for VEGF (blue) combined with immunostaining with ColIV (green). Ectopic VEGF expression seen in Vhla-CreKO mice is abolishedby deletion of HIF-1a, but not HIF-2a, in Vhla-CreKO mice.(M-T) Quantification of percentage spreading distance from optic discs(M,Q), the number of branching points in the area behind sproutingedges (N,R), tip cells per field (O,S), filopodia per tip cell (P,T; n=6 foreach genotype). *P<0.05, **P<0.01. Error bars indicate mean ± s.d.Scale bars: 500mm in A-C,D,F,H; 100mm in E,G,I,J-L.

Fig. 7. Vhla-CreKO mice develop retinal degeneration in adults.(A-H) TUNEL-positive cells (red) counterstained with Hoechst (blue) areincreased in the ONL in Vhla-CreKO mice (E-H) compared with in controlmice (A-D) after P14. (I,J) ONL thickness is decreased in a time-dependent manner in Vhla-CreKO mice (n=3, respectively). (K-T) Mergedimages for glial fibrillary acidic protein (GFAP), Hoechst and BrdU (K,P),and IHC for PECAM1 (yellow, L,Q), glutamine synthetase (GS, green,M,R), opsin (red, N,S), and merged images for GS and opsin (O,T) incontrol (K-O) and Vhla-CreKO mice (P-T). Proliferating reactive glia aredetected by BrdU (arrows, P), and gliosis destroys the construction ofthe ONL (open arrowheads, R-T) in Vhla-CreKO mice at P14. (U-Y) ERGanalysis at P28. (U) Representative wave responses from individualcontrol or Vhla-CreKO mice. Decreased amplitude (V,W) and prolongedimplicit time (Y) are detected in Vhla-CreKO mice (n=6). GCL, ganglioncell layer; INL, inner nuclear layer; ONL, outer nuclear layer. *P<0.05,**P<0.01. Error bars indicate mean ± s.d. Scale bar: 100mm in A-H,K-T.

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inactivation of HIF-1a, but not HIF-2a (Fig. 6), show that theVHL/HIF-1a/VEGF cascade plays crucial roles in the transitionfrom the fetal to the adult circulatory system in the retina.

Our current study has possible clinical implications. AdultVhla-CreKO mice show similar characteristics (Fig. 8) to humanischemic retinopathies, such as diabetic retinopathy, retinal vesselocclusion, and retinopathy of prematurity (Hayreh, 2007), and couldprovide clues for exploring the mechanisms of these humandiseases. It has been recently shown that systemic administration ofa prolyl hydroxylase inhibitor protects from retinal vaso-obliterationin response to hyperoxia (Sears et al., 2008), suggesting that pVHLfunctions in retinopathy of prematurity.

Retinal hemangioma in VHL patients and ischemic retinopathiesare usually treated by laser photocoagulation that disruptsphotoreceptors and increases the oxygen supply for the other neuralcells from the choroidal vasculature bed, although it may cause nightblindness and visual field defects (Yu and Cringle, 2001). Recently,various kinds of VEGF inhibitors (Brown and Regillo, 2007) havebecome possible candidates for use against ophthalmic diseases,although destruction of the physiological vasculature (Fischer et al.,2007) and neuronal dysfunction (Saint-Geniez et al., 2008) causedby long-term VEGF inhibition caution against the continuousadministration of these reagents. Suppression of HIF-1a might bean alternative strategy against these diseases in the future.

Finally, all of the results presented suggest that oxygen-sensingmechanisms mediated by VHL/HIF-1a/VEGF regulate thetransition from the fetal to the adult circulatory system in the retina,and could represent a theoretical basis for the retinal vascularabnormalities in human VHL disease and the development ofischemic retinopathies. It will be interesting to determine whetherthis oxygen-sensing system, the VHL/HIF-1a pathway, is involvedin the transition of the circulatory system in other parts, such asductus arteriosus, which would confirm its generality for futurestudies.

AcknowledgementsWe thank Ichie Kawamori, Rie Takeshita, Haruna Koizumi and Taiga Shioda(Keio University, Tokyo, Japan) for technical assistance. This study wassupported by the Japanese Ministry of Education, Culture, Sports, Science andTechnology (MEXT) [Grant-in-Aid for the 21st Century COE Program andGlobal COE Program at Keio University, and Scientific Research (No.21791710) to T.K.]. Y.K. was supported by Keio Kanrinmaru Project.

Experiments using the anti-HIF-1a antibody were supported by the JapanSociety for the Promotion of Science Grant-in-Aid for Creative ScientificResearch 17GS0419. M.S. is the Leader of the Global COE Program for HumanMetabolomic Systems Biology and H.O. is the Leader of the Global COEProgram for Stem Cell Medicine supported by MEXT.

Competing interests statementThe authors declare no competing financial interests.

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

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