Fig 1: 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 (v) SMC cell lines. HUVECs and fibroblasts are positive and negative controls, respectively. (L) qPCR analysis of venous specification marker genes at P12 retinas (Angpt4-/-n = 5 and WT n = 6). (M–N) Quantification of SMC maturation markers from TEM analyzed retinas (n = 6 WT and n = 7 Angpt4-/- mice). (O) Peripheral annular vein diameter is reduced in Angpt4-/- retina (n = 7 mice/group, the shortest diameter of peripheral branch measured from TEM cross-section). Data are presented as mean ±SD, ***p<0.001 and **p<0.01 Angpt4-/- vs. WT in t-test.
Fig 2: Neuronal cell swelling in adult Angpt4-/- mice.(A–B) Light microscopy images of peripheral retinal sections from control and Angpt4-/- mice stained with (A) toluidine blue (n = 7 mice/group) or (B) Masson's trichrome (n = 9 control and 5 Angpt4-/- mice). Framed area of inner nuclear layer (INL) is magnified on the right. Two different fixation, embedding and staining protocols indicate increased thickness of the INL in (A) and (B), data analyzed at least from four sections per mouse. (C–D) TEM micrographs from peripheral INL. (C) n = 8 control and 6 Angpt4-/- P13 mice and in adult mice (D) n = 8 control and 8 adult Angpt4-/- mice. Quantification (on the right) was done from cell somas locating in the interface between INL and inner plexiform layer that is composed mainly of amacrine cells (Jeon et al., 1998). Asterisks indicate cell swelling in (D) and black arrows Müller cell processes (C, D) that are displaced around swollen cells. A, amacrine cell; M, Müller glia; B, bipolar cell. (A–D) Median (line), average (square), 75th quartile (box), 5th and 95th percentile (whiskers) in whisker blots. *p<0.05 and **p<0.01 WT vs. Angpt4-/- in t-test. 10.7554/eLife.37776.017Figure 6—source data 1.Cell soma areas of individual neuronal cells in the interphase between INL and IPL.
Fig 3: Functional analysis of venous flow and neuronal activity in retina.(A-D) Filling of the retinal veins in fluorescein angiography. Fundus camera images show the same retinas in (A) and (B) and in (C) and (D) in early filling phase (A, C) and in the peak phase of fluorescence (B, D). Arrowheads highlight examples of veins extending peripheral retina. (E) Quantification of the early filling image data; symbols represent fluorescein intensity in individual veins/average arterial fluorescein intensity in the same eye from 7 WT and 5 Angpt4-deficient mice. (F–G) Retention of fluorospheres in the peripheral annular vein after carotid artery injection. Blood vessels were counterstained with Cy3-conjugated aSMA antibody and co-injected Evans blue. (H) Two different sphere concentrations were used, and results are expressed as number of microspheres normalized by the length of venous segment analyzed. Each symbol represents an individual vein from 5 WT and 5 Angpt4-deficient mice. (I) Amplitudes of the b-wave in aged (eight months) WT and Angpt4 deficient mice. ERGs were measured at six different flash intensities as indicated. Data are presented as mean ±SD (n = 12 WT and n = 8 Angpt4-/- eyes). **p<0.01, *p<0.05 in t-test.10.7554/eLife.37776.020Figure 7—source data 1.Electroretinogram measurements of individual eyes.
Fig 4: 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.
Fig 5: 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 Angpt4-expressing cells. (D) A schematic representation of mouse retina preparation. Peripheral venous circulation develops typically as two Y-shaped veins (thick arrows) starting after the first postnatal week and branching in the peripheral segment to form annular structure. Vein (V, blue line); artery (A, red line); OR, ora serrata; ON, optic nerve head; frame, location of microscopic analysis in Figure 1 panels. (E–G) At P23 (late maturation phase), Angpt4+/Cre; Rosa26mT/mG retina shows GFP-positive cells enriched in the peripheral segment when compared to the central retina. Magnifications (F) and (G) reveal a GFP-positive cell population (arrowheads) connected to veins and perivenous capillaries.
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