Table_1_Nexmifa Regulates Axon Morphogenesis in Motor Neurons in Zebrafish.DOCX

Nexmif is mainly expressed in the central nervous system (CNS) and plays important roles in cell migration, cell to cell and cell-matrix adhesion, and maintains normal synaptic formation and function. Nevertheless, it is unclear how nexmif is linked to motor neuron morphogenesis. Here, we provided in situ hybridization evidence that nexmifa (zebrafish paralog) was localized to the brain and spinal cord and acted as a vital regulator of motor neuron morphogenesis. Nexmifa deficiency in zebrafish larvae generated abnormal primary motor neuron (PMN) development, including truncated Cap axons and decreased branches in Cap axons. Importantly, RNA-sequencing showed that nexmifa-depleted zebrafish embryos caused considerable CNS related gene expression alterations. Differentially expressed genes (DEGs) were mainly involved in axon guidance and several synaptic pathways, including glutamatergic, GABAergic, dopaminergic, cholinergic, and serotonergic synapse pathways, according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. In particular, when compared with other pathways, DEGs were highest (84) in the axon guidance pathway, according to Organismal Systems. Efna5b, bmpr2b, and sema6ba were decreased markedly in nexmifa-depleted zebrafish embryos. Moreover, both overexpression of efna5b mRNA and sema6ba mRNA could partially rescued motor neurons morphogenesis. These observations supported nexmifa as regulating axon morphogenesis of motor neurons in zebrafish. Taken together, nexmifa elicited crucial roles during motor neuron development by regulating the morphology of neuronal axons.

Nexmif主要表达于中枢神经系统（CNS），在细胞迁移、细胞间黏附及细胞-基质黏附过程中发挥关键作用，并可维持正常的突触形成与功能。然而，目前学界尚未明确Nexmif与运动神经元形态发生之间的调控关联。本研究通过原位杂交实验获得证据，显示Nexmif的斑马鱼旁系同源基因nexmifa定位于脑与脊髓，可作为运动神经元形态发生的重要调控因子。斑马鱼幼体中的nexmifa功能缺失会引发初级运动神经元（PMN）发育异常，具体表现为轴突截断以及轴突分支数目减少。值得注意的是，RNA测序结果表明，nexmifa缺失的斑马鱼胚胎出现了大量与中枢神经系统相关的基因表达改变。经京都基因与基因组百科全书（KEGG）通路注释分析，差异表达基因（DEGs）主要参与轴突导向及多种突触通路，包括谷氨酸能、γ-氨基丁酸能、多巴胺能、胆碱能及5-羟色胺能突触通路。尤为关键的是，在机体系统（Organismal Systems）通路分类下，轴突导向通路所含差异表达基因数量最多，共计84个。在nexmifa缺失的斑马鱼胚胎中，Efna5b、bmpr2b及sema6ba的表达水平显著下调。此外，过表达efna5b mRNA与sema6ba mRNA均可部分挽救运动神经元的形态发生异常。上述结果证实，nexmifa可通过调控斑马鱼运动神经元的轴突形态发生发挥功能。综上，nexmifa通过调控神经元轴突形态，在斑马鱼运动神经元发育过程中扮演了关键性角色。