Smad2/3a对斑马鱼神经嵴细胞标记基因crestin表达的影响

Effect of Smad2/3a on the expression of the neural crest cell marker crestin in zebrafish

目的 探讨Smad2/3a对脊椎动物神经嵴细胞发育的影响。方法 通过在斑马鱼胚胎单细胞时期显微注射smad2/3吗啉环修饰的反义寡核苷酸的方法,特异性敲降smad2/3基因的表达,至胚胎发育至6体节,利用整胚原位杂交检测神经嵴细胞特异性标记基因snail1b,sox10,foxd3和crestin的表达情况;通过casmad2 mRNA和smad3a mRNA显微注射的方法过表达smad2和smad3a,同样利用整胚原位杂交检测神经嵴细胞特异性标记基因crestin的表达情况;通过过表达casmad2及smad3a对下调smad2和smad3a的胚胎进行挽救。结果 smad2/3a被敲低后,crestin的表达量显著降低,而snail1b,sox10和foxd3的表达量无明显变化。smad3b被敲低后,crestin,snail1b,sox10和foxd3的表达量均无明显变化;过表达casmad2和smad3a均可导致crestin的表达量增高;过表达casmad2和smad3a可挽救由于smad2/3a敲降所造成crestin的低表达量。结论 Smad2和Smad3a对神经嵴细胞标记基因crestin的表达具有重要作用。

Objective To investigate the function of Smad2/3a on vertebrate neural crest cell development.Methods Knock-down of smad2/3 and overexpression of casmad2 and smad3a were achieved by microinjection of antisense morpholino(MO)and mRNA into one-cell stage embryos,respectively.Then,the expression of snail1b,sox10,foxd3,and crestin in MO-injected embryos and crestin in mRNA-injected embryos at the 6-somite stage were detected using whole-mount in situ hybridization.casmad2 mRNA and smad3a mRNA were used to rescue smad2 and smad3a morphants.Results Knockdown of smad2 and smad3a resulted in a remarkable downregulation of crestin,while there were no obvious changes in the expression of snail1b,sox10,or foxd3 in the smad2/3a morphants.Knockdown of smad3b did not inhibit the expression of crestin,snail1b,sox10,or foxd3.Overexpression of casmad2 and smad3a led to an upregulation of crestin.Overexpression of smad2 and smad3a restored the reduction of crestin expression caused by smad2 and smad3a depletion.Conclusions Smad2 and Smad3a play key roles in the regulation of expression levels of crestin during neural crest cell development.