SOX9 regulates endothelial tip cell specification to promote cerebral and neuroretinal vascularization

Background: Tissue vascularization relies on the organotypic specification of endothelial tip cells to interpret local cues and guide angiogenic sprouts into specific tissue compartments. However, the molecular regulators controlling brain and retinal endothelial tip cell identity remain poorly understood. Methods: Endothelial-specific Sox9 loss-of-function mouse was combined with single-cell, spatial, and bulk RNA sequencing analyses of developing mouse brain and retinal vasculature, as well as experimental ischemic stroke. Transcriptomic findings were validated using in situ hybridization, immunofluorescence, and functional angiogenesis assays. Results: Transcription factor activity analysis of single-cell RNA-sequencing datasets identified SOX9 as a candidate regulator selectively enriched in developing brain and retinal endothelial tip cells. Endothelial-specific deletion of Sox9 impaired brain and neuroretina vascularization and disrupted the tip cell transcriptomic program, resulting in reduced sprouting angiogenesis and matrix-remodeling pathways. Conversely, SOX9 overexpression in HUVECs promoted neuro-tip-like signatures and enhanced endothelial invasion and sprouting. Following ischemic stroke, single-cell and spatial transcriptomic analyses identified a transient angiogenic endothelial population within the ischemic area. However, these cells failed to express Sox9 and lacked key developmental brain tip cell features. Conclusions: SOX9 is a key regulator of endothelial tip cell identity and neuronal angiogenesis. These findings reveal fundamental differences between developmental and injury-induced vascular responses and identify SOX9 as a potential therapeutic target to promote functional vascular regeneration.