Construction of a nicastrin gene-silenced zebrafish model and a primary study on the mechanism of abnormal pigmentation / 中华皮肤科杂志

ABSTRACT

Objective:To evaluate the effect of nicastrin (nct) gene on the biological functions of melanocytes in zebrafish.Methods:By using a morpholino oligonucleotide (MO) technology, a nct-MO sequence targeting the zebrafish nct mRNA was designed, so was a MO control (ctrl-MO) sequence. Then, the enhanced green fluorescent protein (EGFP) mRNA with MO target sequence at its 5′ end was synthesized, and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish. With wild-type zebrafish as a blank control group, real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of melanin synthesis-and notch signaling pathway-related genes, including mitfa, tyr, tyrp1a, tyrp1b, dct, pmela, notch1a, notch1b and hey1 genes. One-way analysis of variance was used for the comparison of means among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Eight hours after zebrafish fertilization, green fluorescence was observed in the zebrafish embryos in the ctrl-MO+EGFP mRNA group, but not in the nct-MO+EGFP mRNA group or blank control group. Forty-eight hours after fertilization, the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group (0.169 ± 0.083) than in the ctrl-MO group (0.258 ± 0.042, t=3.202, P=0.005) , and disorderly pigment distribution in the tails was observed in the nct-MO group. RT-PCR revealed significant differences in the mRNA expression of pmela, tyrp1a and hey1 genes among the nct-MO group, ctrl-MO group and blank control group (all P < 0.05) , but no significant difference was observed in the mRNA expression of mitfa, tyr, tyrp1b, dct, notch1a or notch1b genes among the 3 groups (all P>0.05) ; the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group (0.708 ± 0.028, 0.558 ± 0.136, respectively) than in the ctrl-MO group (1.023 ± 0.142, 1.016 ± 0.134, respectively, both P < 0.05) . Conclusion:The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.