Fig. 3: Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes.

a–c WT (control) and Ndp^KO male and female mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp^KO (PBS-treated). Right: Ndp^KO (PBS-treated) vs Ndp^KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value <0.05 and log₂ fold-change > 1 are indicated by red dots which represent upregulated genes. Genes with a p-value <0.05 and log₂ fold-change < −1 are indicated by blue dots, representing downregulated genes. b Left: Venn diagrams showing overlap in bulk RNA-seq differentially expressed genes following treatment with PBS or L6-F4-2 in WT or Ndp^KO mice. Right: Heat maps of the top expressed genes mapping to the indicated GO terms across treatment groups in the RNA-seq experiment. c qRT-PCR validation of differentially expressed genes identified by RNA-seq. Error bars represent mean ± SEM, triplicate from 5-7 pooled retina EC cells, *p < 0.05, **p < 0.01; one-way ANOVA. d–f Single-cell RNA-seq with the same treatment conditions as (a-c). d scRNA-seq UMAP plots of retinal EC sub-clusters from P8 retinas from all conditions merged (top) and from the WT, Ndp^KO, and Ndp^KO + L6-F4-2 conditions (bottom), 5–7 pooled retinas for each group. Alteration of the D-tip population is indicated by arrows. e Dot plot of NaRnEA enrichment for previously described Zarkada et al. gene sets⁵⁰. Dot size is determined by the absolute value of the normalized enrichment score (NES); large dots indicate more significant enrichment. Dot color is determined by the proportional enrichment score (PES), which indicates the sign of the enrichment. BRB, brain-retinal-barrier. f Master regulators of the Ndp^KO condition versus WT as identified by PISCES analysis of the scRNA-seq data with reduced or reverted activity profiles in the Ndp^KO + L6-F4-2 condition. g Master regulators of the Ndp^KO + L6-F4-2 condition versus WT without significant activity in the Ndp^KO condition as identified by PISCES analysis of scRNA seq data. Source data are provided as a Source Data file.