CAPSL point mutations in two families with familial exudative vitreoretinopathy (FEVR).

(A-B) FEVR pedigrees (patients are denoted with black symbols) and Sanger sequencing of two heterozygous mutations identified in two families. Black arrows indicate the proband of each family and red arrows indicate the changed nucleotides. (C) Alignment of amino sequences surrounding the CAPSL variants in different species and all mutated sites are highly conserved. Altered amino acid residues are highlighted in red. (D) Fundus fluorescein angiography (FFA) (left panel) and fundus photography (right panel) of a normal individual and FEVR-affected patient (I:1) in family 3036. (E-G) Western blot (E-F) and RT-qPCR analysis (G) of CAPSL expression of WT and mutant plasmids. An empty vector with GFP tag was used as a negative control. GFP was used as an internal reference. Error bars indicate the SD. **P < 0.01, ****P < 0.0001, ns: no significance, by Student’s t test (n = 3).

EC-specific inactivation of Capsl impairs retina angiogenesis.

(A) Flat-mounted retinas obtained from P5 Ctrl and littermate CapsliECKO/iECKO mice were stained with Isolectin-B4 (IB4) to visualize blood vessel. Dashed circle mark the edge of the developing retina vessel in CapsliECKO/iECKO mice. Scale bar: 250 μm. (B) Low magnification images (top panels) and high magnification images (bottom panels) in boxed areas of IB4-stained angiogenic front of Ctrl and CapsliECKO/iECKOmice, respectively. Red cross mark tip cells at the angiogenic growth front. Scale bar: 100 μm (top panels), 25 μm (bottom panels). (C) High magnification images of filopodia-extending cells at the edge of retinal angiogenic growth front from Ctrl and CapsliECKO/iECKO mice. Red arrowheads indicate the sprouts at the angiogenic growth front. (D-H) Quantification of retinal vascular development parameters, including vascular progression, vessel density, branchpoints, number of tip cells, and number of filopodia. Error bars indicate the SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s t test (n = 6).

Loss of Capsl results in delayed hyaloid regression and deep retinal blood vessel growth.

(A) Hyaloid vessels stained with DAPI in the eyes of Ctrl and CapsliECKO/iECKO mice at P10. Scale bar: 250 μm. (B) Retina sections from P10 Ctrl and CapsliECKO/iECKO mice were costained with IB4 (greed) and DAPI (blue). Scale bar: 100 μm. (C) Flat-mounted retains stained with IB4 at P14 Ctrl and CapsliECKO/iECKO mice. Optical sections of z-stacked confocal images were divided to represent the nerve fiber layer (NFL), inner plexiform layer (INL), and outer plexiform layer (ONL). Dashed lines mark the edge of the developing retina. Scale bar: 100 μm

Deletion of Capsl impairs EC proliferation and migration.

(A) Retina endothelial cell proliferation of Ctrl and CapsliECKO/iECKO mice at the vitreal surface was measured with EdU and ERG labeling at P5. Images captured at higher magnification are shown at right. Dashed lines mark the edge of the developing retina, and dashed circles represent both EdU+ and ERG+ cells. EC proliferation ability was measured by the ration of EdU+ and ERG+ cells per vascular area. Scale bar: 200 μm and 50 μm (enlarged insets). Error bars indicate the SD. ***P < 0.001, by Student’s t test (n = 6). (B) Representative images of retinal vessels at the periphery plexus and inner plexus of Ctrl and CapsliECKO/iECKO mice at P5 costained with IB4 (green) and Collagen IV (red). Arrowhead point to empty Collagen IV sleeves. And quantification of ratio of Collagen IV positive vessel segments to IB4 labeling-negative vessel segments. Scale bar: 50 μm. Error bars indicate the SD. ***P < 0.001, ****P < 0.0001, by Student’s t test (n = 6). (C) Magnified images of IB4+ vessels and ERG+ nuclei of ECs at angiogenic front of Ctrl and CapsliECKO/iECKO mice at P5. Quantification of tip cell nuclear ellipicity at the angiogenic growth front. Scale bar: 50 μm. Error bars indicate the SD. ***P < 0.001, by Student’s t test (n = 14).

Depletion of CAPSL in HRECs compromises in vitro EC proliferation and migration.

(A) Representative images of in vitro tube formation after transfection of HRECs with shRNA. Scale bar: 200 μm. Error bars indicate the SD. ***P < 0.001, ****P < 0.0001, by Student’s t test (n=6). (B) Incorporation of EdU in shRNA transfected HRECs. Representative confocal images and quantification of proliferating HRECs both in number per field and proportion of EdU-positive cells. Scale bar: 50 μm. Error bars indicate the SD. ****P < 0.0001, by Student’s t test (n=10). (C-D) Cell cycle analysis of shCtrl-ECs and shCAPSL-ECs by flow cytometry. Error bars indicate the SD. ****P < 0.0001, by Student’s t test (n=4). (E) Representative images of phalloidin actin cytoskeleton (green) and GM130 (red) showing polarity angles of shCtrl-ECs and shCAPSL-ECs at the edge of scratch wound. The arrow points toward the wound. Colored arrowheads represent different migration state. Scale bar: 200 μm (left panel) 50 μm (right panel). (F) Quantification of nuclear ellipticity of HRECs at the margin of wound scratch. Error bars indicate the SD. ****P < 0.0001, by Student’s t test (n = 14). (G) Schematic pictures showing the define of polarity axis of each cell. Polarity axis was measured with the angle (α) between the scratch edge and the vector drawn from the center of nucleus to the center of the Golgi apparatus. (H) Polar plots showing Golgi apparatus polarization. The bold lines represent 120° region centered on the vector, which is perpendicular to the wound scratch. The dots represent the angle (α) of each cell and the numbers indicate the frequency of dots within the 120° region of the bold line of shCtrl-ECs (n=243) and shCAPSL-ECs (n=244). (I) Images of phalloidin-stained actin cytoskeleton and comparisons of indicated parameters in shCtrl-ECs and shCAPSL-ECs at the edge of scratch wound. The dashed boxed region is shown at higher magnification at the bottom panel. Scale bar: 50 μm (top panels), 25 μm (bottom panels). (J) Representative images of wound scratch assay at 0h, 12h, and 16h after wound was made. And the quantification of covered area at different time point. The dashed line indicates the gap of the wound after wound scratch at different time point. Scale bar: 200 μm. Error bars indicate the SD. ****P < 0.0001, ns: no significance, by Student’s t test (n = 4). (K) Immunoblot and quantification analysis of expression of small GTPase proteins and a key regulator of contractile force MYL9 in shCtrl-ECs and shCAPSL-ECs. Error bars indicate the SD. *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t test (n = 3).

CAPSL suppresses MYC signaling axis.

(A) Differential gene expression information of shCAPSL-ECs versus shCtrl-ECs group and LentiOE-ECs versus shCtrl-ECs group. (B-E) Gene set enrichment analysis (GSEA) analysis on the RNA sequencing data of HRECs. Top 10 ranked up or down regulated signaling axis were listed (B) and top 4 down-regulated gene sets were listed (C) in comparison of shCtrl-ECs versus shCAPSL-ECs. Top 10 ranked up or down regulated signaling axis were listed (D) and top 4 up-regulated gene sets were listed (E) in comparison of LentiOE-ECs versus shCtrl-ECs. (F-I) Gene set enrichment analysis (GSEA) analysis on the proteomic profiling data of HRECs. Top 10 ranked up or down regulated signaling axis were listed (F) and top 4 down-regulated gene sets were listed (G) in comparison of shCtrl-ECs versus shCAPSL-ECs. Top 10 ranked up or down regulated signaling axis were listed (H) and top 4 up-regulated gene sets were listed (I) in comparison of LentiOE-ECs versus shCtrl-ECs. (J) Correlated RNAs and proteins enriched in nine quadrants of shCtrl-ECs versus shCAPSL-ECs. (K) Gene set enrichment analysis (GSEA) analysis on the genes/proteins in quadrants 3 and 7, and top 5 ranked up or down regulated signaling axis were listed. (L) Clustered heat map of the expression fold changes of several MYC signature genes of in both RNA profiling and proteomic profiling of shCtrl-ECs and shCAPSL-ECs.

Loss of CAPSL led to similar transcriptional regulatory patterns to the loss of c-MYC in HUVECs.

(A-B) Gene set enrichment analysis (GSEA) analysis on the RNA sequencing data in comparison of shCtrl-HUVECs versus shc-MYC-HUVECs. Top 10 ranked up or down regulated signaling axis were listed (A) and top 4 down-regulated gene sets were listed (B). (C) Venn diagram analysis of down-regulated genes in CAPSL-depleted HRECs and c-MYC-depleted HUVECs. (D-E) Gene set enrichment analysis (GSEA) analysis on the shared down-regulated genes in CAPSL-depleted HRECs and c-MYC-depleted HUVECs. (F) Heatmap of MYC signature genes of shared genes based on Venn analysis. (G) c-MYC expression level in shCtrl-ECs and shCAPSL-ECs was quantified by western blot and RT-qPCR. Error bars indicate the SD. ****P < 0.0001, ns: no significance by Student’s t test (n = 3). (H) Western blot analysis of expression of MYC targets. (I) Quantification analysis of MYC targets. Error bars indicate the SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s t test (n = 3)