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 1 week 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.

Elevated phagocytic capacity directs innate spinal cord repair.

Immune cells elicit a continuum of transcriptional and functional states after spinal cord injury (SCI). In mammals, inefficient debris clearance and chronic inflammation impede recovery and overshadow pro-regenerative immune functions. We found that, unlike mammals, zebrafish SCI elicits transient immune activation and efficient debris clearance, without causing chronic inflammation. Single-cell transcriptomics and inducible genetic ablation showed zebrafish macrophages are highly phagocytic and required for regeneration. Cross-species comparisons between zebrafish and mammalian macrophages identified transcription and immune response regulator ( tcim ) as a macrophage-enriched zebrafish gene. Genetic deletion of zebrafish tcim impairs phagocytosis and regeneration, causes aberrant and chronic immune activation, and can be rescued by transplanting wild-type immune precursors into tcim mutants. Conversely, genetic expression of human TCIM accelerates debris clearance and regeneration by reprogramming myeloid precursors into activated phagocytes. This study establishes a central requirement for elevated phagocytic capacity to achieve innate spinal cord repair.

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

The E3 ubiquitin ligase mindbomb1 controls planar cell polarity-dependent convergent extension movements during zebrafish gastrulation.

Vertebrate Delta/Notch signaling involves multiple ligands, receptors and transcription factors. Delta endocytosis - a critical event for Notch activation - is however essentially controlled by the E3 Ubiquitin ligase Mindbomb1 (Mib1). Mib1 inactivation is therefore often used to inhibit Notch signaling. However, recent findings indicate that Mib1 function extends beyond the Notch pathway. We report a novel Notch-independent role of Mib1 in zebrafish gastrulation. mib1 null mutants and morphants display impaired Convergence Extension (CE) movements. Comparison of different mib1 mutants and functional rescue experiments indicate that Mib1 controls CE independently of Notch. Mib1-dependent CE defects can be rescued using the Planar Cell Polarity (PCP) downstream mediator RhoA, or enhanced through knock-down of the PCP ligand Wnt5b. Mib1 regulates CE through its RING Finger domains that have been implicated in substrate ubiquitination, suggesting that Mib1 may control PCP protein trafficking. Accordingly, we show that Mib1 controls the endocytosis of the PCP component Ryk and that Ryk internalization is required for CE. Numerous morphogenetic processes involve both Notch and PCP signaling. Our observation that during zebrafish gastrulation Mib1 exerts a Notch-independent control of PCP-dependent CE movements suggest that Mib1 loss-of-function phenotypes should be cautiously interpreted depending on the biological context.

Animal embryonic development involves producing an entire animal from a single starting cell, the zygote. To do this, the zygote must divide to make new cells, and these cells have to arrange themselves into the correct body shape. This requires a lot of cells to move in a coordinated fashion. One of these movements is called 'convergent extension', in which a typically round group of cells rearranges into a long, thin shape, for example, to increase the distance between the head and the tail of the animal. In order to coordinate this movement, cells need to communicate with each other. One of the signaling pathways cells use to guide them to the right positions is the planar cell polarity (PCP) pathway. Zebrafish are used to study PCP in convergent extension because they are transparent, making it easy to track their cell movements under the microscope. Interestingly, when a protein called Mindbomb1 (Mib1) is inactivated in zebrafish embryos, convergent extension is reduced. Mib1 helps control the activity of other proteins by attaching a chemical marker called ubiquitin to them, which tags these proteins to be relocated from the cell surface to small vesicles within the cell. The protein is known to be involved in the formation of neurons ­ the cells that make up the brain and nerves ­ but its links to cell movement and the PCP pathway had not been explored. Saraswathy et al. used a technique called Crispr/Cas9 mutagenesis to genetically modify zebrafish and then used observations under the microscope to determine the role of Mib1 in PCP and convergent extension. Their experiments show that Mib1 helps internalize a protein called Ryk from the cell surface into the cell. This internalization of Ryk is required to relay signals through the PCP pathway. When Mib1 is missing, Ryk stays on the surface of the cell, instead of moving to the inside, blocking PCP signaling between cells and therefore blocking convergent extension. Understanding the role of Mib1 in PCP signaling sheds light on how cell movements are coordinated during the embryonic development of zebrafish. Future research will involve determining whether Mib1 plays the same role in other animals, offering further insights into embryonic development. Additionally, PCP is known to have a role in disease, including the spread of cancer. It will be important to determine whether Mib1 is involved in this process as well.

Localized EMT reprograms glial progenitors to promote spinal cord repair.

Anti-regenerative scarring obstructs spinal cord repair in mammals and presents a major hurdle for regenerative medicine. In contrast, adult zebrafish possess specialized glial cells that spontaneously repair spinal cord injuries by forming a pro-regenerative bridge across the severed tissue. To identify the mechanisms that regulate differential regenerative capacity between mammals and zebrafish, we first defined the molecular identity of zebrafish bridging glia and then performed cross-species comparisons with mammalian glia. Our transcriptomics show that pro-regenerative zebrafish glia activate an epithelial-to-mesenchymal transition (EMT) gene program and that EMT gene expression is a major factor distinguishing mammalian and zebrafish glia. Functionally, we found that localized niches of glial progenitors undergo EMT after spinal cord injury in zebrafish and, using large-scale CRISPR-Cas9 mutagenesis, we identified the gene regulatory network that activates EMT and drives functional regeneration. Thus, non-regenerative mammalian glia lack an essential EMT-driving gene regulatory network that reprograms pro-regenerative zebrafish glia after injury.

Notch-mediated inhibition of neurogenesis is required for zebrafish spinal cord morphogenesis.

The morphogenesis of the nervous system requires coordinating the specification and differentiation of neural precursor cells, the establishment of neuroepithelial tissue architecture and the execution of specific cellular movements. How these aspects of neural development are linked is incompletely understood. Here we inactivate a major regulator of embryonic neurogenesis - the Delta/Notch pathway - and analyze the effect on zebrafish central nervous system morphogenesis. While some parts of the nervous system can establish neuroepithelial tissue architecture independently of Notch, Notch signaling is essential for spinal cord morphogenesis. In this tissue, Notch signaling is required to repress neuronal differentiation and allow thereby the emergence of neuroepithelial apico-basal polarity. Notch-mediated suppression of neurogenesis is also essential for the execution of specific morphogenetic movements of zebrafish spinal cord precursor cells. In the wild-type neural tube, cells divide at the organ midline to contribute one daughter cell to each organ half. Notch signaling deficient animals fail to display this behavior and therefore form a misproportioned spinal cord. Taken together, our findings show that Notch-mediated suppression of neurogenesis is required to allow the execution of morphogenetic programs that shape the zebrafish spinal cord.