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Biology (Basel)
2022 Dec 20;121:. doi: 10.3390/biology12010001.
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MITF Contributes to the Body Color Differentiation of Sea Cucumbers Apostichopus japonicus through Expression Differences and Regulation of Downstream Genes.
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Melanin, which is a pigment produced in melanocytes, is an important contributor to sea cucumber body color. MITF is one of the most critical genes in melanocyte development and melanin synthesis pathways. However, how MITF regulates body color and differentiation in sea cucumbers is poorly understood. In this study, we analyzed the expression level and location of MITF in white, purple, and green sea cucumbers and identified the genes regulated by MITF using chromatin immunoprecipitation followed by sequencing. The mRNA and protein expression levels of MITF were all highest in purple morphs and lowest in white morphs. In situ hybridization indicated that MITF mRNA were mainly expressed in the epidermis. We also identified 984, 732, and 1191 peaks of MITF binding in green, purple, and white sea cucumbers, which were associated with 727, 557, and 887 genes, respectively. Our findings suggested that MITF contributed to the body color differentiation of green, purple, and white sea cucumbers through expression differences and regulation of downstream genes. These results provided a basis for future studies to determine the mechanisms underlying body color formation and provided insights into gene regulation in sea cucumbers.
No. 42106109 National Natural Science Foundation of China, ZR2020QD100 Natural Science Foundation of Shandong Province Youth Program, XDA24030304 Strategic Priority Research Program of the Chinese Academy of Sciences, 2018YFD0901602 National Key R&D Program of China, 2020LZGC015 Agricultural Seed Project of Shandong Province
Figure 1. Relative expression levels of MITF mRNA and protein in the body wall of white, green, and purple A. japonicus. (A) The MITF mRNA expression levels relative to β-actin mRNA levels were examined by qPCR, and the difference among different color morphs was analyzed. (B) Total protein (10 μg) was loaded in each line for Western blot analysis, and β-actin was used as a loading control. The relative protein abundance of MITF was calculated as relative expression values to the control group. Different letters (a, b, c) indicate significant differences among different color morphs (p < 0.05).
Figure 2. Fluorescence in situ hybridization analysis for MITF in the body wall of white, green, and purple A. japonicus. W1: hybridization signals of MITF in white A. japonicus; W2: negative control with nuclear dye DAPI in white A. japonicus; W3: merged signals in white A. japonicus; G1: hybridization signals of MITF in green A. japonicus; G2: negative control with nuclear dye DAPI in green A. japonicus; G3: merged signals in green A. japonicus; P1: hybridization signals of MITF in purple A. japonicus; P2: negative control with nuclear dye DAPI in purple A. japonicus; P3: merged signals in purple A. japonicus.
Figure 3. Peak analysis of MITF binding sequence in green, purple, and white A. japonicus. (A) Peak distribution across genome of green A. japonicus. (B) Peak distribution across genome of purple A. japonicus. (C) Peak distribution across genome of white A. japonicus. (D) KEGG pathway analysis of genes regulated by MITF in green A. japonicus. (E) KEGG pathway analysis of genes regulated by MITF in purple A. japonicus. (F) KEGG pathway analysis of genes regulated by MITF in white A. japonicus.
Figure 4. GO and KEGG analysis of genes differentially bound to MITF in purple and white sea cucumber, compared with those in green sea cucumber. (A) Gene Ontology classification in purple A. japonicus. (B) KEGG pathway analysis in purple A. japonicus. (C) Gene Ontology classification in white A. japonicus. (D) KEGG pathway analysis in white A. japonicus. “*” indicates significant differences with p < 0.05; “**” indicates significant differences with p < 0.01.
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