Mechanism Of Adenylate Cyclase In Melanin Formation In Zebrafish

Melanin is a dark brown pigment widely found in animals and plants.The synthesis and pigmentation of melanin not only determines the color of hair and skin,but also serves as a key protective barrier for skin against ultraviolet radiation.When the body’s ability to synthesize melanin decreases,it becomes less protective against UV radiation,which increases the risk of melanoma and other skin cancers.The classical regulatory mechanism of melanin production is driven by changes in the expression of melanocortin 1 receptor（MC1R）induced pigment synthesis genes.In melanocytes of fish and amphibians,activation ofα-MSH-MC1R/cAMP-PKA signals promotes peripheral diffusion of melanocytes,while inhibition of this signal induces aggregation of melanocytes.The mammalian ADCY family consists of 10 members and is the central mediator of cAMP signaling cascade.Although in vitro studies have shown that the cAMP signaling pathway activated byα-MSH-MC1R plays a key role in melanocyte development,proliferation,differentiation,and melanin synthesis,but none of the ADCY isotype knockout mice showed pigmentation defects in skin and hair.Therefore,it remains unclear which ADCYs in vivo are involved in the regulation of melanocyte generation and melanin synthesis,and how these ADCYs regulate the transport of melanocytes in melanocytes.In this study,zebrafish were used as model animals to construct single mutants such as adcy3a^-/-,adcy5^-/-,adcy6a^-/-and double mutants such as adcy3a^-/-;adcy5^-/-,adcy3a^-/-;adcy3b^-/-and adcy6a^-/-;adcy6b^-/-using CRISPR/Cas9 gene editing technology.We analyzed the formation and differentiation of melanocytes and the spread and pigmentation of melanocytes in the development and growth stages of these mutants by tracking phenotypic changes and quantitative statistics of Image J.It was found that the single mutation of Adcy5 inhibited the spread of melanocytes in embryo,metamorphosis and adult stage,and melanin showed a state of aggregation.On this basis,mutant Adcy3a enhances melanin aggregation,indicating that Adcy3a is involved in Adcy5-mediated melanosomes transport.In addition,through embryo in situ hybridization,q PCR,exogenous small molecule drug treatment and other experiments,we found that the double mutation of Adcy3a and Adcy5 did not affect the melanocyte specialization and differentiation of zebrafish embryos,as well as the establishment of melanocyte stem cells and the regeneration of melanocytes.In the adult stage of zebrafish,double mutations of Adcy3a and Adcy5 inhibit the expression of transcription factor Mitfa and its three regulated melanin synthetases,Tyr,Dct and Tyrp1b,resulting in reduced melanin synthesis and pigmentation of mature melanocytes,thus unable to establish normal melanocyte streaks in adult zebrafish.Further,by exploring the effect of PKA signaling downstream of cAMP signaling pathway and related effectors on the dispersion of melanosomes in adcy3a^-/-;adcy5^-/-mutants,we found that Adcy3a and Adcy5 activate cell driver kinesin-1 activity through PKA,thus promoting the diffusion of melanosomes.However,the double mutation of Adcy3a and Adcy5 completely inhibited the activity of kinesin-1 and the diffusion of melanosomes,which were driven by dynein-1 to the perinuclear movement and finally showed a highly clustered state.In conclusion,our study not only identified the key adenylate cyclases Adcy3a and Adcy5 that regulate melanin synthesis,filling a gap in vivo research in this field,but also promoted our understanding of the most basic process of cAMP signaling in melanin synthesis and melansome transport.Moreover,it provides the best animal model for studying the role of cAMP signaling in the occurrence and development of melanoma.