L. Préau (1), F. le Noble (1)

1. Department of Cell and Developmental Biology, Karlsruhe Institute of Technology (KIT), Fritz Haber Weg 4, 76131 Karlsruhe, Germany

Tissue regeneration involves a complex interplay between immune cells, activation of stem cells, and vascular innervation. In the nervous system, promoting vascular innervation is considered as therapeutic approach to enhance spinal cord regeneration. In zebrafish, genetically ablating Vegfa trapping receptor Flt1 creates a Vegfa gain of function scenario and spinal cord hypervascularization. We thus hypothesized that spinal cord regeneration should be enhanced in flt1-/- mutants. Spinal cord injury was inflicted by surgical transection   on zebrafish embryos at day 3. Using in vivo imaging substantiated by single-cell sequencing, the innate immune response, neural stem cell behavior, neuron migration, glia and axonal bridging were investigated during spinal cord regeneration. Surprisingly, while loss of flt1-/- increased spinal cord vascularization, regeneration was severely impaired. Loss of regeneration associated with disturbed dynamics of neutrophil recruitment toward the injured area. Using single-cell sequencing followed by cell-cell communication analysis, we identified that Vegfa gain of function promoted endothelial-derived cxcl12a expression, activating Cxcr4b receptor in neutrophils. Increased Cxcr4 signaling in neutrophils inhibited reverse migration, resulting in prolonged neutrophil presence at the lesion site. Genetic and pharmacological Cxcr4 inhibition in flt1-/- mutants reduced neutrophil number and rescued spinal cord regeneration defects. In addition, anti-inflammatory treatment restored spinal cord regeneration, suggesting that prolonged neutrophil presence promotes inflammation and inhibits neural regeneration. Conclusion: we identified a hitherto unknown role for VEGF signaling in regulating an angiocrine process determining regeneration by titrating immune cell behavior during spinal cord regeneration. These findings which may open novel therapeutic avenues for treating neural injuries.