Supplementary Material for: Zebrafish Minichromosome Maintenance Protein 5 Gene Regulates the Development and Migration of Facial Motor Neurons via Fibroblast Growth Factor Signaling

Minichromosome maintenance protein 5 (MCM5), a member of the microchromosomal maintenance protein family, plays an important role in the initiation and extension of DNA replication. However, its role in neural development in zebrafish remains unclear. Here, we used morpholino (MO) and CRISPR/Cas9 to knock down <i>mcm5</i> and investigated the developmental features of facial motor neurons (FMNs) in the hindbrain of zebrafish. We found that knockdown of <i>mcm5</i> using <i>mcm5</i> MO resulted in a small head, small eyes, and a blurred midbrain-hindbrain boundary, while MO injection of <i>mcm5</i> led to decrease in FMNs and their migration disorder. However, the mutant of <i>mcm5</i> only resulted in the migration defect of FMNs rather than quantity change. We further investigated the underlying mechanism of <i>mcm5</i> in the development of hindbrain using in situ hybridization (ISH) and <i>fgfr1a</i> mRNA co-injected with <i>mcm5</i> MO. Results from ISH showed that the fibroblast growth factor (FGF) signaling pathway was changed when the MCM5 function was lost, with the decrease in <i>fgfr1a</i> and the increase in <i>fgf8</i>, while that of <i>pea3</i> had opposite trend. FMN development defects were rescued by <i>fgfr1a</i> mRNA co-injected with <i>mcm5</i> MO. Our results demonstrated that FGF signaling pathway is required for FMN development in zebrafish. Specifically, <i>mcm5</i> regulates FMN development during zebrafish growing.

微小染色体维持蛋白家族成员微小染色体维持蛋白5（MCM5）在DNA复制的起始与延伸过程中发挥关键作用。然而，其在斑马鱼神经发育中的功能尚不明确。本研究借助吗啉代寡核苷酸（MO）与CRISPR/Cas9技术敲低*mcm5*基因，对斑马鱼后脑内面部运动神经元（FMNs）的发育特征展开探究。研究结果显示，通过*mcm5* MO敲低*mcm5*后，斑马鱼会出现小头、小眼及中脑-后脑边界模糊的表型；同时面部运动神经元数量减少且迁移出现紊乱。但*mcm5*基因的突变体仅会导致面部运动神经元的迁移缺陷，并未引发其数量改变。本研究进一步通过原位杂交（ISH）技术，以及将*fgfr1a*信使RNA（mRNA）与*mcm5* MO共注射的实验，探究了*mcm5*参与斑马鱼后脑发育的潜在分子机制。原位杂交结果表明，MCM5功能缺失会改变成纤维细胞生长因子（FGF）信号通路的活性：*fgfr1a*的表达量下调，*fgf8*的表达量上调，而*pea3*的表达趋势则与之相反。共注射*fgfr1a* mRNA可有效挽救*mcm5* MO诱导的面部运动神经元发育缺陷。本研究结果证实，FGF信号通路对斑马鱼面部运动神经元的发育不可或缺；具体而言，*mcm5*基因在斑马鱼发育进程中调控面部运动神经元的发育。