RESUMO
Larval zebrafish show axonal regrowth over a complex spinal injury site and recovery of function within days after injury. Here we describe a simple protocol to disrupt gene function in this model using acute injections of highly active synthetic gRNAs to rapidly detect loss-of-function phenotypes without the need for breeding.
Assuntos
Traumatismos da Medula Espinal , Peixe-Zebra , Animais , Peixe-Zebra/genética , Fenótipo , Traumatismos da Medula Espinal/genética , Axônios , Larva/genéticaRESUMO
Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.
Assuntos
Histona Desacetilase 1/genética , Neurogênese/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Regeneração/genética , Medula Espinal/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Animais , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Macrófagos/citologia , Macrófagos/metabolismo , RNA-Seq , Transdução de Sinais/genética , Análise de Célula Única , Medula Espinal/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator de Transcrição AP-1/genética , Peixe-Zebra/genéticaRESUMO
Slit-Robo signaling has been characterized as a repulsive signal for precise axon pathfinding and cell migration during embryonic development. Here, we describe a role for Sox2 in the regulation of Robo1 in Schwann cells and for Slit3-Robo1 signaling in controlling axon guidance within the newly formed nerve bridge following peripheral nerve transection injury. In particular, we show that macrophages form the outermost layer of the nerve bridge and secrete high levels of Slit3, while migratory Schwann cells and fibroblasts inside the nerve bridge express the Robo1 receptor. In line with this pattern of Slit3 and Robo1 expression, we observed multiple axon regeneration and cell migration defects in the nerve bridge of Sox2-, Slit3-, and Robo1-mutant mice. Our findings have revealed important functions for macrophages in the peripheral nervous system, utilizing Slit3-Robo1 signaling to control correct peripheral nerve bridge formation and precise axon targeting to the distal nerve stump following injury.
Assuntos
Orientação de Axônios , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Regeneração Nervosa , Nervos Periféricos/metabolismo , Animais , Movimento Celular , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nervos Periféricos/fisiologia , Ratos , Ratos Wistar , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais , Proteínas RoundaboutRESUMO
Correct myelination is crucial for the function of the peripheral nervous system. Both positive and negative regulators within the axon and Schwann cell function to ensure the correct onset and progression of myelination during both development and following peripheral nerve injury and repair. The Sox2 transcription factor is well known for its roles in the development and maintenance of progenitor and stem cell populations, but has also been proposed in vitro as a negative regulator of myelination in Schwann cells. We wished to test fully whether Sox2 regulates myelination in vivo and show here that, in mice, sustained Sox2 expression in vivo blocks myelination in the peripheral nerves and maintains Schwann cells in a proliferative non-differentiated state, which is also associated with increased inflammation within the nerve. The plasticity of Schwann cells allows them to re-myelinate regenerated axons following injury and we show that re-myelination is also blocked by Sox2 expression in Schwann cells. These findings identify Sox2 as a physiological regulator of Schwann cell myelination in vivo and its potential to play a role in disorders of myelination in the peripheral nervous system.