Notch signalling by the ligand Delta-like 4 (Dll4) is essential for normal vascular remodelling, yet the precise way in which the pathway influences the behaviour of endothelial cells remains a mystery. Using the embryonic zebrafish, we show that, when Dll4-Notch signalling is defective, endothelial cells continue to migrate and proliferate when they should normally stop these processes. Artificial overactivation of the Notch pathway has opposite consequences. When vascular endothelial growth factor (Vegf) signalling and Dll4-Notch signalling are both blocked, the endothelial cells remain quiescent. Thus, Dll4-Notch signalling acts as an angiogenic `off' switch by making endothelial cells unresponsive to Vegf.

Spinal cord injury (SCI) causes irreversible loss of sensory and motor functions in mammals. In contrast to mammals, adult zebrafish are capable of efficient, spontaneous recovery after SCI. Whereas glial scarring and neuron loss accentuate spinal cord (SC) damage and inhibit regeneration in mammals, zebrafish are capable of pro‐regenerative glial bridging and effective adult neurogenesis post‐injury. Emerging evidence suggests that in mammals these pro‐regenerative processes are either inefficient or overshadowed by anti‐regenerative effects. We propose that developing efficient SCI treatments will require a deeper understanding of endogenous SC regenerative capacity, and that adult zebrafish present a valuable model to uncover fundamental, evolutionarily concealed mechanisms of mammalian SC regeneration. Although glial bridging and neurogenesis are hallmarks of the robust SC repair observed in zebrafish, the mechanisms that direct these processes are underexplored. Using expression profiling and genetic loss‐of‐function, we screened for and identified a suite of molecular factors and cell populations that are activated after injury and required for SC regeneration. These studies revealed that discrete lineage‐restricted niches of progenitor cells differentially contribute to glial bridging and neurogenesis after SCI in zebrafish. Using a battery of genetic and molecular tools, we identified yap‐ctgfa‐twist1a as a central glial bridging signaling axis, generated the first transcriptional profiles of zebrafish bridging glia, and elucidated glial cell injury responses at the single cell level in zebrafish. We are currently using our understanding of the bridging glial cell fate in zebrafish to perform molecular comparisons with mammalian glial cells, and to reprogram mammalian glia into scarless, pro‐regenerative scaffolds that promote SC regrowth. Taken together, our studies elucidate fundamental mechanisms that direct innate SC regeneration in zebrafish, and use natural SC regeneration as a platform to enhance mammalian SC repair at the molecular and cellular levels. Support or Funding Information We acknowledge support from NIH (R01‐NS113915), the Missouri Spinal Cord Injury/Disease Research Program, the McDonnell Center for Cellular and Molecular Neuroscience, the Institute of Clinical and Translational Sciences, and from Washington University School of Medicine to M.H.M.

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences, and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show cells that direct expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1 kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration.

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury.Whereas scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue to facilitate regeneration. Here, we performed FACS sorting and genome-wide profiling to determine the transcriptional identity of purified bridging glia. We found that Yap-Ctgf signaling activates epithelial to mesenchymal transition (EMT) in localized niches of ependymal cells to promote glial bridging and regeneration.Preferentially activated in early bridging glia, Yap is required for the expression of the glial bridging factor Ctgfa and for functional spinal cord repair. Ctgfa regulation is controlled by an injury responsive enhancer element that drives expression in early bridging glia after injury. Yap-Ctgf signaling activates a mesenchymal transcriptional program that drives glial bridging. This study revealed the molecular signatures of bridging glia and identified an injury responsive gene regulatory network that promotes spinal cord regeneration in zebrafish.

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences, and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show that cells that direct expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1 kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration.