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FTO promotes endothelial tip cell formation to facilitate neovascularization in mice and zebrafish. (A) Experimental scheme for (B–G) (B) Fluorescent staining of IB4 in retinal flat mounts originated from control, OIR mice without other treatment, and OIR mice intra-vitreal injected with AAV-blank/AAV-Fto at P17. n = 11 per group. Magnificent images are shown below to better visualize superficial and deep vascular plexuses. Gray dotted lines indicate the edge of the retina. Red lines suggest the avascular area. NVTs are represented by red dots. Representative images along with quantification results of NVT and avascular areas are shown. Scale bar: 2000 µm (up); 50 µm (below). (C) Fluorescent staining of EdU and IB4 in retinal flat mounts of control, OIR mice without other treatment, and OIR mice intra-vitreal injected with AAV-blank/AAV-Fto at P17. n = 4 per group. (D) Fluorescent staining of IB4 in retinal flat mounts of control, OIR mice without other treatment, and OIR mice intra-vitreal injected with AAV-blank/AAV-Fto at P17. n = 12 per group. Tip cells are indicated by yellow asterisks. Representative images along with quantification results of tip cell number per field are shown. Scale bar: 50 µm. (E, F) mRNA expression of tip cell markers Apln (E) and Esm1 (F) detected by qPCR in neural retinas of control, OIR mice without other treatment, and OIR mice intra-vitreal injected with AAV-blank/AAV-Fto at P17. n = 3 per group. (G) Immunofluorescence staining of PDGFRβ and IB4 in retinal flat mounts of control, OIR mice without other treatment, and OIR mice intra-vitreal injected with distinct virus at P17. n = 4 per group. (H) Experimental scheme for (I, J). (I) Truncal vasculatures demonstrated by endogenous EGFP and morphological structures of control and zebrafish injected with antisense or fto mRNA. n = 3 per group. (J) Ocular vasculatures shown by endogenous EGFP and morphological structures of control and zebrafish receiving indicated treatments. Ocular vasculatures are indicated by white circles. Representative images along with quantification results of ocular vessel density (n = 5 per group) and diameters (n = 8–10 per group) are shown. Scale bar: 100 µm. Data information: Data represent different numbers (n) of biological replicates. Data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni’s test is used. NS: not significant (p > 0.05); *p < 0.05; **p < 0.01; and ***p < 0.001. Source data are available online for this figure.
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