斑马鱼侧线神经丘毛细胞发育的研究

The Zebrafish Model for the Study on Hair Cell Development

目的 研究斑马鱼后部侧线系统神经丘细胞早期迁移及增殖分化过程。方法 选取不同发育阶段的野生型斑马鱼胚胎,采用免疫组织化学染色和原位杂交方法,观察斑马鱼后部侧线原基的迁徙以及神经丘的沉积过程,Brd U 标记原基增殖细胞,观察原基细胞团的增殖情况;选择不同发育时期的转基因斑马鱼（Brn3c ：mGFP ）胚胎,统计绿色荧光蛋白（GFP ）阳性表达的神经丘毛细胞数目;采用活细胞染料F M1 43FX标记神经丘中的功能性毛细胞并进行统计计数,Brd U 标记神经丘增殖细胞,观察神经丘细胞的增殖情况。结果 受精后20 小时的斑马鱼侧线器基板沿水平肌隔开始向后迁移并形成后部侧线神经丘,迁徙速度约为1 .7 肌节/小时;受精后48 小时,初级后部侧线系统基本发育完全,包括躯干部的5～6 组神经丘细胞团,尾部的2～3 组神经丘细胞团;受精后3 5、7、天时的斑马鱼神经丘毛细胞数量分别为5 .68±1 .46、10 .10±0 .99、12 .45±1 .32 个;F M1 43FX阳性毛细胞数量分别为3 .68±1 .11、8 .18±1 .86、10 .22±1 .24 个。结论 受精后3～7 天是斑马鱼侧线神经丘毛细胞发育的活跃时期。

Objective This study aims to examine the development of the posterior lateral line of the zebrafish and establish ant model to study the process of hair cell differentiation and regeneration. Methods We observed the posterior lateral line system formation by DAPI immunohistochemistry and whole mount in situ hybridization. We further evaluated hair cells differentiation within neuromast by using Transgenic Tg （Brn3c..mGFP） zebrafish and stained the functional hair cells by the mechanotransduction marker FM1-43FX. We labelled proliferating cells in primordium and neuromast by addition of BrdU to the system water. Results The posterior lateral line primordium originated from a sensory placode and started its journey at around 20 hours post fertilization to migrate along the horizontal myoseptum to the tail-tip with a constant speed （1.7somite/hour）. The primordium depositd five or six neuromasts spaced along the body, and two or three terminal neuromasts at the tail-tip at 48 hours post fertiliza-tion. At 3,5 and ？ days post fertilization, zehrafish contained 5.68±1.46, 10.1±0.99, and 12.45±1.32 hair cells per neuromast, respectively. Furthermore, the average number of FM1--43FX stained hair ceils within each neuro- mast were 3.68± 1.11, 8.18 ± 1.86, and 10.22 ± 1.24, respectively. Conclusion We establish the development model of hair cells in zebrafish lateral line neuromast and suggest that 3 to 7 days post fertilization is an important period for lateral line neuromast differentiation. This study would be useful for underlying the mechanisms of hair cell differentiation and regeneration.