Effect on Black Stripes Formation in Zebrafish (Danio rerio) by CRISPR/Cas9 Editing on Mlpha Gene
HU Xu-Wen, CHEN Hong-Lin, YU Lu-Wei, XU Xi-Dan, CHEN Xiao-Wen, WANG Jun, WANG Cheng-Hui*
Key Laboratory of Freshwater Aquatic Genetic Resources certificated by the Ministry of Agriculture and Rural Affairs/National Demonstration Center for Experimental Fisheries Science Education/Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China
Abstract：Melanophilin (Mlph) gene plays fundamental roles in regulating the transportation of mature melanosomes in vertebrates, and its mutations can lead to hair or feather color fading in animals. However, the molecular function of Mlpha gene in aquatic animals need to be explored. In this study, spatial and temporal expression of Mlpha gene were investigated during different developmental stages in zebrafish (Danio rerio), and Mlpha gene was also knocked-out by using the CRISPR/Cas9 technology to exam body color changes in the F2 homozygous mutants in zebrafish. The results of in situ hybridization and real-time quantitative PCR (qRT-PCR) expression indicated that Mlpha gene expressed at all the collected developmental stages, including 6, 12, 24, 30, 36, 48, 60 hpf (hours post fetilization) and 1, 2, 3, 4, 5,10, 15 dph (days post hatching) and the expression level increased with the increase of dendritic melanocytes in zebrafish. Meanwhile, the highest expression level was identified on the abdomen of zebrafish at 5 dph when obvious melanin band appeared. Similarly, the in situ hybridization results also showed the detectable signals and the strongest signal at 5 dph. After Mlpha gene was knocked-out by using CRISPR/Cas9 technology, the larval individuals of F1 mosaic mutant zebrafish displayed slow expansion of melanin in juveniles and fragmented black stripe in adults. Under observation using the steroscope, the numbers of melanocytes and their coloration were not significantly reduced at the stripe fragments, but their morphology was reduced and dendritic structure was degenerated. Furthermore, the development speed of Mlpha knockout zebrafish was slower than that of wild-type zebrafish, and the diffusion speed of melanin was also significantly slower than that of wild-type zebrafish, indicated that melanosome dispersion was affected if Mlpha gene was knocked-out. In the F2 homozygous mutant zebrafish, the Mlpha protein could not be completely translated owing to a terminator codon production in the second exon of this gene, and the adults appeared small melanocyte in skin. Moreover, in situ hybridization and qRT-PCR expression analysis revealed that the expression levels of Rab27a and myosin Va (MyoVa) which interacted with Mlpha were significantly down-regulated in the F2 homozygous mutants (P<0.05). It is speculated that the mutation of Mlpha gene may lead to its inability to bind to MyoVa, resulting in the slow aggregation and transportation of melanosomes. It may also be that the mutant can't bind Rab27a, which makes melanosomes unable to transport along actin filament. In addition, there was no dimming of melanin pigments in adult zebrafish, indicating that the mutation of Mlpha gene did not affect the synthesis process of melanin, but may affect the transport process of melanin. Our study indicated that Mlpha gene played an essential role in regulating the transportation process of melanin, and its mutation could affect black stripe formation in zebrafish. The results of this study provide a reliable reference for studying the genetic basis and mechanism of melanoma pigmentation or formation in fish. However, the mechanism of Mlpha gene regulating body color in fish needs to be further studied.
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