鱼类Ⅲ型Myosin基因家族成员的表达和功能研究  

Expression and Functional Analysis of Class Ⅲ Myosin in Zebrafish

作　　者：赵呈天[1] 陈哲[1] 冯东[1] ZHAO Cheng-Tian;CHEN Zhe;FENG Dong(Institute of Evolution&Marine Biodiversity,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋生物多样性与进化研究所

出　　处：《中国海洋大学学报（自然科学版）》2019年第10期126-132,共7页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(31772456)资助~~

摘　　要：Ⅲ型Myosin蛋白在哺乳动物的听觉毛细胞中发挥重要功能,其突变可导致内耳毛细胞功能异常,引起听力丧失,但在鱼类胚胎中的表达及功能仍不清楚。本研究以斑马鱼(Daniorerio)作为研究对象,研究了Ⅲ型Myosin基因家族成员myo3a及myo3b在斑马鱼早期胚胎中的表达及功能。整胚原位杂交结果显示:myo3a及myo3b在早期10体节前均无明显表达;在受精后24h胚胎中,myo3a基因的表达主要集中在脑部,并在端脑及间脑部位有强烈表达;而myo3b的表达主要集中在神经管腹部神经元细胞中。进一步双荧光原位杂交染色结果显示,myo3b主要在神经管腹部脑脊液接触神经元(CSF-cNs)中表达。此外,与人及小鼠类似,斑马鱼myo3a及myo3b基因在内耳中均有表达,暗示其功能在内耳发育中的保守性。为进一步研究其功能,本研究利用CRISPR/Cas9技术对myo3a和myo3b基因进行敲除,获得了相应的突变体。对突变体的初步研究表明,myo3a和myo3b的单独缺失对斑马鱼的内耳毛细胞以及神经管的发育均无显著影响,而双突变胚胎出现眼睛变小,鱼鳔不能形成以及毛细胞纤毛变短等表型,表明Ⅲ型Myosin基因家族在斑马鱼的胚胎发育中发挥一定的功能。Class Ⅲ Myosin is essential for the development of inner ear and auditory sense.This family contains two members,MYO 3 A and MYO 3 B,and mutations of MYO 3 A are related to human hearing loss.The roles of Class Ⅲ Myosin during fish hearing development have not been reported yet.Here we investigated the expression and function of myo 3 a and myo 3 b genes during early zebrafish embryonic development.Whole-mount in situ hybridization results showedthat the expression of myo 3 a and myo 3 b was undetectable at early stages,while both showed tissue specific expression at 24 hours post-fertilization.myo 3 a was mainly expressed in the brain with high enrichment in the telencephalon and diencephalon,while myo 3 b was highly expressed in the ventral spinal cord.Further double Fluorescence in situ Hybridization(dFISH)results suggested that myo 3 b was exclusively expressed in the ventral cerebrospinal-fluid-contacting neurons(CSF-cNs).Similar to those in mouse and human,both myo 3 a and myo 3 b were expressed in the otic vesicles,implying a conserved role of these proteins during inner cell development.Then,we generated myo 3 a and myo 3 b mutants by CRISPR/Cas9 technology.Unexpectedly,inner ear hair cells and eye developed normally in myo 3 a or myo 3 b mutants.In contrast,myo 3 a/myo 3 b double mutants displayed developmental defects,including small eyes and non-inflation of zebrafish swim bladder.Strikingly,the stereocilia in the hair cells of double mutants were also significantly shorter.These data suggest that Class Ⅲ Myosin plays a role during fish early embryonic development.

关 键 词：斑马鱼 myo3a myo3b 内耳 毛细胞 脑脊液接触神经元 

分 类 号：Q38[生物学—遗传学]