Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina

Figures

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  Figure 1.. Angpt4 expression is temporally and spatially regulated at venous development sites in the peripheral retina. (A–G) Whole-mount retinas from Angpt4LacZ and Angpt4+/Cre; Rosa26mT/mG mice at the indicated postnatal days (P). (A) Angpt4LacZ allele visualized with X-Gal staining (blue) indicated numerous Angpt4 expressing cells in the P9 peripheral retina at the sites of developing vein (V, asterisks) as identified based on anatomical location, morphology and reticulocytes. (B) Angpt4LacZ allele revealed continued but lower Angpt4 expression (blue, arrows) around matured vein (V) in peripheral retina at P20. (C) In Angpt4+/Cre; Rosa26mT/mG mice, mTomato (red) is ubiquitously expressed until Angpt4-promoter-driven Cre-mediated loxP recombination induces GFP (green) expression. P12 retina shows GFP-positive cells preferentially locating in the peripheral part of retina close to the branching vein (arrowheads). Arrow indicates rare single GFP-positive cells that are apart from vein (asterisks). Angpt4-expressing cells in Angpt4+/Cre; Rosa26mT/mG mice are labeled permanently, that is they remain GFP positive even if Angpt4 expression is discontinued and may represent a progeny of…

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  Figure 1—figure supplement 1.. Generated Angpt4 mouse alleles. (A) Strategy used to insert Cre cassette into the murine Angpt4 locus. A targeting construct was generated by recombineering method. The flanking regions and position of used primers (black arrows) are shown and the primer sequences are provided in the Materials and methods section. The first exon of the Angpt4 gene was replaced by Cre/Neo cassette and Neo was removed by FRT sites and flippase enzyme. Black and red boxes represent generated homologous sequences for recombination. (B) A schematic representation of Angpt4LacZ allele. A splice acceptor (SA) site with LacZ cassette was inserted between exon III and exon IV in Angpt4 gene locus. Endogenous expression of Angpt4 resulted in a truncated Angpt4 fusion protein with LacZ revealing Angpt4 expression in X-Gal-stained tissues. (C) A fate mapping strategy to track Angpt4 expressing/expressed cells. Mouse line expressing Cre recombinase under endogenous Angpt4 promoter was crossed with Rosa26mT/mG mouse line. In resulting mice, constitutive tomato expression is replaced by Cre recombinase induced GFP when Angpt4 is expressed. In Angpt4+/Cre; Rosa26mTmG model, Angpt4-expressing cells are labeled…

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  Figure 1—figure supplement 2.. Controls of Angpt4 gene expression in mouse retina model. (A) Whole mount preparation showing entire adult mouse retina. αSMA staining indicates arteries and veins. Two major Y-shaped veins extending from optic nerve head (ON) forming branches in the periphery near ora serrata (OR) are highlighted by asterisks. (B) A cartoon indicating location of microscopic analysis (framed) shown in panels C–E. Blue line, vein (V); red line, artery (A); OR, ora serrata; ON, optic nerve head. (C) Angpt4LacZ allele visualized with X-Gal showed staining (blue, white arrows) in the peripheral part of retina near OR region at age P9 indicating Angpt4 expressing cells, whereas central retina is virtually negative. Asterisks indicate branching vein in the periphery of retina. (D) Retina from adult Angpt4+/+; Rosa26mT/mG mouse shows no GFP-positive cells (green) without Angpt4Cre. Only mTomato expressing cells (red) were seen as expected. (E) Peripheral retinal segment from Angpt4+/+ mouse stained with X-Gal showed no staining in P9 retina. Asterisks indicate a forming vein in the periphery of retina near OR.

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  Figure 2.. Angpt4 marks a specific subpopulation of retinal astrocytes and is induced in low oxygen. Angpt4 lineage cells (GFP, green) visualized in Angpt4Cre/+; Rosa26mT/mG reporter mice in adult (2 months) in (A-D) retina. Angpt4-negative cells express mTomato (red). (A) Similarly to developing retina (Figure 1), GFP-positive cells are enriched in the distal segment near ora serrata (OR) when compared to central retina. GFP positive cells are also less numbered near to arteries (A, arrowheads) than veins (V, arrows). Quantification of the image data is shown in (B). (C) Section through the adult retina (R) reveals Angpt4 lineage cells (arrowheads) locating in the superficial layer of retina, whereas other retina layers are negative (VB, vitreous body; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; C, choroid). (D) All GFP+ cells (arrowhead) express astrocyte marker GFAP (white). However, not all astrocytes are GFP positive (arrow). (E–G) Low oxygen increases number of Angpt4+ astrocytes, but does not induce Angpt4 expression in other cell types (in 1.5 months old mice). Quantification of the image data is shown in (G). (H) Induction of hypoxia-inducible…

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  Figure 2—figure supplement 1.. Induction of Angpt4 expression and degree of vasculopathy in OIR model in Angpt4-/- mice. In the oxygen-induced retinopathy (OIR) -model, P7 mice were exposed to 75% oxygen for 5 days and then returned to normoxia (21% O2) for 5 days, resulting in capillary dropout and retinal ischemia that are followed by capillary proliferation for neovascularization of avascular areas. (A) Angpt4 lineage cells (GFP, green) visualized in Angpt4Cre/+; Rosa26mT/mG reporter mice in flat mount retinas harvested at P17. (B) In OIR-model, number of GFP positive cells is increased compared to normoxia. (C–D) Whole mount retinal staining of OIR retinas, IB4 (green) and αSMA (red) staining visualize capillary-artery-vein hierarchy in the superficial layer. Avascular areas quantified in (E) are highlighted in yellow. Angpt4 deficiency does not affect neovascular tuft formation or capillary regeneration in avascular retina. (B) and (E) Quantification of image data; control n = 4 and OIR n = 2 mice (B); control n = 5 and Angpt4-/- n = 4 mice (E). Mean ±SD, **p<0.01 in t-test.

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  Figure 2—figure supplement 2.. Angpt4 expression in hyperoxia model. P7 mice were exposed to 75% oxygen for 5 days and number of GFP-positive cells were analyzed from P12 retinas. (A) Angpt4-lineage cells (GFP, green) visualized in Angpt4Cre/+; Rosa26mT/mG reporter mice in flat mount retinas. (B) Quantification of the image data. (C) Analysis of angiopoietins and Tie2 mRNA expression levels in normoxia (21% O2) and hyperoxia (75% O2) eyes in relation to β-actin. Fold change was calculated from Ct values. For verification, Angpt4 level was measured using two oligo pairs from exons 5–6 (Ex 5–6) and exons 8–9 (Ex 8–9). (B) n = 4 mice/group. (C) n = 9 mice/group. Mean ±SD, **p<0.01 and ***p<0.001 in t-test.

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  Figure 3.. mRNA expression of angiopoietins and Tie2 in retinal vein development. mRNA expression was detected in whole mount retinas from WT mice by in situ hybridization. (A) At P3, astrocytes expressing Angpt4 precede the front of the developing vasculature (red blood cells indicated by arrows). (B) At P12, Angpt4 expression is notable around the developing vein in the peripheral retina (V, asterisks) and colocalizes with the astrocyte marker GFAP (white overlay in insert). (C) Angpt1 expression is not detected at P12 by whole mount in situ hybridization in the peripheral retina. (D) Angpt2 is expressed in retinal neurons (arrowhead) in the intermediate retinal plexus. (E) Expression of Tie2 is detected in the endothelium of arteries (A), veins and capillaries at P12. OR, ora serrata.

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  Figure 3—figure supplement 1.. Controls of in situ hybridization staining. (A–F) Whole mount retinas from WT or Angpt4-/- mice. (A) Angpt4-/- retina at P12 shows no signal for Angpt4 antisense probe, as expected. (B–F) In situ hybridizations with sense control probes for Angpt4 at P3 (B) and P12 (C) and for Angpt1 (D) Angpt2 (E) and Tie2 (F) at P12 were performed in parallel with antisense probe hybridizations. All sense probes show only unspecific background staining. Asterisks indicate developing vein (V) OR, ora serrata. (G–J) Specificity of the Angpt1 antisense probe was confirmed by positive staining of retinal neurons at P4 (arrowhead) (G) and atriums at P2 (I). Corresponding sense controls (H, J) were negative.

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  Figure 4.. Retinal veins of Angpt4 deficient mice show SMC abnormality and smaller diameter. (A) P12 WT retina whole mount shows strong αSMA staining (red, arrows) in the developing annular vein in the peripheral retina. (B) αSMA staining is weak in Angpt4-/- retina especially in the peripheral location (red, arrows). Asterisks indicate developing Y-shaped annular peripheral vein in (A) and (B). (C–F) Whole mount retinal staining of nine months old mice. αSMA staining (red) (C, E) and merged image (D, F) with Col IV (green) indicate perivascular sleeves. In Angpt4-/- retina, αSMA level is low around annular veins (V, arrows) in the peripheral retina, but not around arteries (A). (G–I) αSMA immunofluorescence intensity (IF) in Angpt4-/- retina is reduced around annular peripheral veins (G), but not in veins close to optic nerve head (central) (H) or arteries (I) (n = 3 mice/group). (J–K) Peripheral annular vein diameter is reduced in Angpt4-/- retina. n = 8 WT and 6 Angpt4-/- mice, measured from the base of the peripheral bifurcation (J). n = 8 WT and 8 Angpt4-/- mice, measured from the central region close the optic nerve head (K). Mean ±SD, ***p<0.001 in t-test.

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  Figure 4—figure supplement 1.. Vascular structures and densities in the intermediate and deep vascular plexuses. (A-B) Periodic acid-Schiff staining (PAS) for polysaccharides counterstained with hematoxylin. PAS stains vascular basement membranes in superficial (black arrowheads) and intermediate/deep vascular plexuses (white arrowheads). PAS staining is also diagnostic for plasma leakage resulting in formation of exudates into the retina, which is not observed in either of the genotypes. (C) Quantification of the image data; n = 3 mice/group, average of 3–4 sections, mean ± SD. Peripheral sections representing microscopic fields are shown in (A) and (B). (D-E) Primary and deeper vascular plexuses at postnatal day 12. (F–G) Col IV staining illustrates superficial, intermediate and deep retinal vasculature in adult peripheral retina. V, bifurcation of peripheral vein. (H–I) TEM analysis of blood vessels locating in the intermediate/deep plexuses show no structural abnormalities. NS = not significant in t-test.

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  Figure 5.. Angpt4 deficiency results in defective SMC maturation in veins. TEM micrographs of peripheral annular vein in P8 (A) and adult (11 months old) mice (B). SMCs are marked by red line. In the developing veins, SMCs are covered by basement membranes (BM, arrowheads) and SMC cytoskeleton appears translucent with some myofilaments (arrow). Perivascular ECM contains no fibrillar collagen. In adult mouse, venous SMC cytoplasm is mostly filled with myofilaments and perivascular matrix contains fibrillar collagen (Col). TEM micrographs of peripheral vein in 2 (C, D) and 11 (E, F) months old WT and Angpt4-/- mice, respectively. In both genotypes, perivascular cells (red line) are closely aligned with ECs and surrounded by BM (arrowheads) that indicates SMC identity. In Angpt4-/- retina, SMC cytoplasm lacks myofibril-rich cytoskeleton and fibrillar collagen (Col) matrix. No ultrastructural abnormalities are observed in ECs. (G–J) Whole mount retinal staining of peripheral veins in 2 months old WT (G, I) and Angpt4-/- (H, J) mice. SM22 (red) (G–J) and IB4 (green) (G, H) staining. In Angpt4-/- retina, SM22 level is low around veins (arrows, V). (K) Tie2 expression in arterial (a) and venous…

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  Figure 5—figure supplement 1.. Smooth muscle cell proliferation, migration or apoptosis is not affected by ANGPT4. (A) ANGPT4 does not affect proliferation of aSMCs (n = 5 independent experiments) or vSMCs (n = 3) in BrdU ELISA. (B) ANGPT4 does not affect migration of aSMCs (n = 3) or vSMCs (n = 4) in scratch assay, bars represent wound closure time/wound area normalized to control. (C) ANGPT4 has no effect on aSMC (n = 3) or vSMC (n = 2) apoptosis in Caspase 3/7 ELISA.