Sphingosine 1-phosphate receptor signaling establishes AP-1 transcriptional factor gradients and permits retinal endothelial specialization

Transcriptional mechanisms that drive angiogenesis and organotypic endothelial specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors. Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent VE-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this transcription factor. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of ß-catenin-dependent genes such as TCF/LEF1 and ZIC3 transcription factors, transporters and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 transcriptional factor gradient and enables organotypic endothelial specialization of the vascular network. Individual biological replicates represents retinas from 4-6 P6 pups pooled for FACS isolation of retinal endothelial cells for each experiment (ATAC-seq and RNA-seq). N=5 for RNAseq, N=3 for ATAC-seq.