黑色素干细胞及mitf基因在花鲫体色形成和成体体色维持中的作用

Colourful fish provides a material for studying the mechanism of pigment development. By hybrid breeding and gynogenesis, we have established a population of improved colour crucian carp with beautiful body shapes and fantastic skin colours. The change of skin colour from black to red/white takes place in the juvenile fish of this strain of crucian carp.It found that there is no melanocyte in the skin tissue of the adult colour crucian carp.However, the mitf gene,a label of the melanoma stem cells(MSCs), is still expressed in the skin of the adult fish. To address these issues, we shall first establish the function of MSCs and mitf gene in the development of pigment cell lineage in the colour crucian carp. Then research on MSCs and mitf gene in regulation in change of skin colour from black to red/white. And then identify effect of MSCs in maintained the adult body color. Finally, we should prove whether mitf gene are directly or indirectly involved in developmental regulation of adult non-melanoma cells in the colour crucian carp. As a result,this project will further reveal the molecular mechanisms of pigment formation and maintenance in fish. Moreover, the study can also provide theoretical guides for breeding of improved ornamental fish strains.

本实验室利用杂交选育和雌核发育技术创制了新型观赏鱼高背花鲫。高背花鲫个体发育中存在着"青转花"的体色变迁，黑色素干细胞标签基因mitf在无黑色细胞表型的成体花鲫皮肤组织中仍有表达。本项目以高背花鲫为材料，在鉴定色素细胞谱系和建立基于CRISPR-Cas9技术的高背花鲫mitf基因突变体的基础上，重点研究：1）黑色素干细胞和mitf基因在花鲫色素细胞谱系建立中的作用；2）黑色素干细胞和mitf基因在花鲫"青转花"体色变迁中的功能；3）鉴定黑色素干细胞在花鲫成体体色维持中的作用；4）确定mitf基因是否直接或间接参与花鲫成体非黑色素细胞的发育调控。本项目研究旨在解析高背花鲫体色形成的发育机制，进一步揭示鱼类体色发育和成体体色维持的分子调控机制，同时也为观赏鱼类的分子遗传选育和品质改良提供重要的理论指导。

本项目针对花鲫体色形成过程的“褪黑”现象，研究mitfa基因在鱼类体色发育中的功能，旨在揭示鱼类体色形成和成体体色维持的分子调控机制。通过对不同体色鲫和非黑色表型鲤体色发育的比较观察，证实红鲫体色发育具有与花鲫相同的“褪黑”现象，建立了鲫鲤体色遗传回归模型；克隆了mitfa等几种重要体色基因并分析了其在不同体色鲫中的表达；建立了Mitfa-EGFP荧光报告体系并发现鲫尾鳍基部“浅色素带”具有色素干细胞龛室特性；利用RNA-Seq技术对红鲫“褪黑”过程的皮肤组织进行了全转录组学分析，获得了大量的差异表达基因和SNP位点信息，证实mitfa作为主控基因通过调节黑色素合成代谢途径参与调控鲫“青转红”体色转变; 利用RNA-Seq和MethylRAD技术比较分析了成体红鲫与白鲫皮肤组织中差异表达基因与DNA甲基化位点信息，证实了黑色素合成关键酶tyr基因的甲基化水平与体色密切关联; mitfa基因敲除与过表达分析进一步证实了mitfa基因对鱼类黑色素细胞发育的重要调控功能。相关研究成果为进一步解析鱼类体色形成的发育机制奠定了基础，也为经济观赏鱼类的选育和品质改良提供理论指导。