Daxx在斑马鱼胚胎发育中的作用及其机制研究

Function and mechanism of death domain-associated protein in zebrafish embryonic development

目的明确死亡结构域相关蛋白(Daxx)在斑马鱼胚胎发育过程中的时-空表达谱,并研究其作用与机制。方法利用原位杂交技术检测Daxx的表达谱;利用吗啉反义寡核苷酸介导的基因敲减技术敲低Daxx的表达,观测斑马鱼胚胎发育情况,并用pH3和TUNEL染色检测Daxx敲低后细胞增殖及凋亡的改变;通过Real-Time PCR实验研究Daxx影响细胞凋亡和胚胎发育的分子机制。结果敲低Daxx后斑马鱼胚胎细胞凋亡和畸形率显著增加,这一表型能被p53蛋白共敲低所恢复;在分子机制上,Daxx敲低可不同程度特异性激活bax、mdm2、p21和cyclin G1等p53下游多个基因的表达,Daxx富含酸性氨基酸的功能区介导了上述过程。结论 Daxx利用其自身结构域与p53作用,并特异调控其下游关键基因的表达,参与调节斑马鱼胚胎细胞凋亡和形态发育。

Objective To explore the spatio-temporal mRNA expression pattern of death domain-associated protein （Daxx） in the whole process of zebrafish embryonic development, and to study the physiological role and possible molecule mechanism of Daxx in the process. Methods Whole-mount mRNA in situ hybridization （WISH） was performed to ascertain the spatio-temporal expression pattern of Daxx in the process of zebrafish embryonic development. Daxx expression was knocked down by using morpholino mediated gene knockdown. TUNEL and pH3 staining assay were applied to check cell apoptosis and proliferation in Daxx deficient embryos. The molecule mechanism of Daxx was explored by using Real-time PCR. Results Knock-down Daxx activated p53 dependent cell apoptosis pathway, thus caused increasing rate of apopotosis and malformation, whereas those could be rescued by p53 co-knockdown. Moreover, the acid-enriched domain ~f Daxx mediated the interaction between Daxx and p53 and regulated the expression of p53 target genes bax, mdm2, p21, anti cyclin G1. Conclusion Daxx could bind to p53 via its specific motif and regulate the expression of key genes downstream of p53, which may be the key mechanism of Daxx during zebrafish embryonic development.