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. 2023 May 16;12:e86066. doi: 10.7554/eLife.86066

Figure 9. Normal choriocapillaris formation in zebrafish deficient for Wnt/β-catenin signaling, and conserved expression of Vegfa paralogs in retinal pigment epithelium (RPE).

Figure 9.

(A) Dissected eye from 10 days post fertilization (dpf) Tg(plvap:EGFP);Tg(glut1b:mCherry) zebrafish immunostained for GFP and DsRed shows strong Tg(plvap:EGFP) and absent Tg(glut1b:mCherry) expression in the choriocapillaris (choroidal vascular plexus, CVP). (B–D) Transmission electron microscopy images of 10 dpf wild-type (WT) outer retina focused on the CVP and RPE layer. Magnified images of the boxed areas in (B) show the presence of fenestrae (F) in the vascular endothelial cells (vECs) comprising the CVP (orange arrows, C, D). (E) Schematic diagram of 3D confocal CVP imaging from the back of dissected eyes. (F–I) WT (F), gpr124-/- (G), reck-/- (H), and wnt7aa-/- (I) CVP visualized by Tg(kdrl:EGFP) expression at 6 dpf. Confocal z-stack images of dissected eyes were taken after immunostaining for GFP. (J) Quantification of vECs that comprise the CVP at 6 dpf (n=15 for WT, n=16 for gpr124-/-, n=10 for reck-/-, and n=11 for wnt7aa-/- fish). No significant difference was observed across these genotypes. Each data point shown in magenta represents individual animal’s quantification. Refer to Figure 9—source data 1 for the precise cell counts of individual larvae. (K–R”) Lateral views of 54 hours post fertilization (hpf) TgBAC(vegfaa:EGFP) (K), TgBAC(vegfab:EGFP) (M), TgBAC(vegfc:EGFP) (O), and TgBAC(vegfd:EGFP) (Q) embryos immunostained for GFP and ZO-1, a tight junction marker for RPE. Magnified images of the boxed areas in (K), (M), (O), and (Q) are shown in (L–L”), (N–N”), (P–P”), and (R–R”), respectively. TgBAC(vegfaa:EGFP) and TgBAC(vegfab:EGFP) expression was broadly co-localized with ZO-1 immunoreactivity in RPE (asterisks in L”, N”). Sparse EGFP+ cells were observed in TgBAC(vegfc:EGFP) and TgBAC(vegfd:EGFP) eyes, some of which were co-localized with ZO-1 immunoreactivity (asterisks in P”, R”). (S–Z”) Cryosections of 50 hpf TgBAC(vegfaa:EGFP) (S), TgBAC(vegfab:EGFP) (U), TgBAC(vegfc:EGFP) (W), and TgBAC(vegfd:EGFP) (Y) embryos that carried the Tg(kdrl:ras-mCherry) transgene. Sections were immunostained for GFP and DsRed, and counterstained for DAPI. Magnified images of the boxed areas in (S), (U), (W), and (Y) are shown in (T–T”), (V–V”), (X–X”), and (Z–Z”), respectively. TgBAC(vegfaa:EGFP) and TgBAC(vegfab:EGFP) expression was broadly observed in the RPE layer directly adjacent to the CVP (white arrows in T”, V”). Sparse EGFP+ cells on TgBAC(vegfc:EGFP) and TgBAC(vegfd:EGFP) sections resided in close proximity to the CVP (white arrows in X”, Z”). NL: inner nuclear layer, ONL: outer nuclear layer. Scale bars: 500 nm in (B); 200 nm in (C), (D); 50 µm in (A), in (I) for (F–I), in (Q) for (K), (M), (O), in (Y) for (S), (U), (W); 30 µm in (R”) for (L–L”), (N–N”), (P–P”), (R–R”); 25 µm in (Z”) for (T–T”), (V–V”), (X–X”), (Z–Z”).

Figure 9—source data 1. Quantification of the number of endothelial cells that comprised the choroidal vascular plexus (CVP) in wild-type (WT), gpr124-/-, reck-/-, and wnt7aa-/- at 6 days post fertilization (dpf).