Single-cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair

Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity between 1 and 3 weeks post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair. Overall design: we performed complete SC transections on adult zebrafish and dissected 3 mm sections of SC tissues surrounding the lesion site at 1, 3, and 6 weeks post-injury (wpi) for nuclear isolation. Corresponding tissue sections were collected from uninjured controls. SC tissues were pooled from 50 animals per time point, and 2 pools of independent biological replicates were analyzed for each time point. Our dataset spans key regenerative windows including early injury-induced signals at 1 wpi, neuronal and glial regeneration at 3 wpi, and cellular remodeling at late stages of regeneration at 6 wpi (Mokalled et al., 2016). Isolated nuclei were sequenced using 10x genomics platform (3' v3.1 chemistry) and aligned to zebrafish genome GRCz11 with improved zebrafish transcriptome annotation (Lawson et al., 2020; Matson et al., 2018). Nuclei were subsequently filtered using the Decontx and DoubletFinder packages to exclude droplets that include doublet nuclei or a high proportion of ambient mRNA, respectively (McGinnis et al., 2019; Yang et al., 2020). A second round of filtering is performed by thresholding the covariates such as number of counts, genes and fraction of counts from mitochondrial genes per barcode. A total of 58,973 nuclei was obtained for downstream analysis using the Seurat package .