斑马鱼Pax3a结构域过表达对瓦登伯革氏症候群疾病影响

Effect of the overexpression of Pax3a domains on modeling Waardenburg syndrome in zebrafish

目的:探究斑马鱼PAX3直系同源基因Pax3a的结构域在多种组织细胞中的复杂功能.进一步了解哺乳类肌肉间质先祖细胞标志基因PAX3在胚胎发育、组织再生和肿瘤发生过程中发挥的重要作用.方法:构建斑马鱼Pax3a4个保守结构域编码的过表达质粒,包括p-Paired box domain 3(PD3)、p-Homeobox domain (HD)、p-G protein-pathway-suppressor domain (GPD)和p-Paired box domain 7 (PD7),使用蛋白免疫印迹,细胞免疫荧光和实时荧光定量PCR方法验证体内外mRNA和蛋白水平表达,并进行体内胚胎显微注射和体外转染细胞.结果:胚胎发育变异表型的统计分析结果显示:每个结构域会引起斑马鱼胚胎产生较高比例的特异瓦登伯革综合征表型,比如白斑症、内眦外移、瞳孔变大等症状.在细胞学水平显示,每个结构域过表达对细胞迁移有不同影响.结论:Pax3a每个结构域有各自的作用通路,其功能增溢与PAX3基因突变均可产生瓦登伯革氏症候群.

Objective:To explore multifunctions of PAX3 in various tissues,zebrafish and PAX3 ortholog ( Pax3a ),and deeply understood PAX3 which was considered as a marker gene of myogenic precursors and played an important role in embryonic development,tissue regeneration and tumorigenesis. Methods:Overexpression vectors encoding four conserved domains of zebrafish Pax3a were constructed,including p-Paired box domain 3 (PD3),p-Homeobox domain (HD),p-G protein-pathway-suppressor (GPD) and p-Paired box domain 7 (PD7).Western blot,cell immunofluorescence and Real-time Quantitative Polymerase Chain Reaction were applied to verify expression level of mRNA and protein.Embyro microinjection in vivo and cell transfection in vitro was used for study.Result:Statistical analysis of pheotypes of embryonic development variation revealed that overexpression of each domain caused high proportion of variant phenotypes similar to human Waardenburg Syndrome in zebrafish embryos,such as leukoplakia,dystopia canthorum,and pupil enlargment.In cell level,overexpression of each domain altered cell migration differently.Conclusion:The results suggested that each domain of Pax3a protein has its specific fucntional pathways.Similar to PAX3 gene mutation,the gain of fucntion of the domains also causes Waardenburg Syndrome.