| Body color and pigmentation patterns in animals are of great significance for defense against natural enemies,vigilance,reproduction and breeding,and also protection of the organism from UV irradiation.The body color of lower vertebrates is mainly determined by the differences in the number,distribution area and arrangement of various pigment cells,as well as the amount of pigment synthesis.Human and other mammals contain only one type of pigment cells,namely melanocytes,but there are many types of pigment cells in lower vertebrates.So far,at least six kinds of pigment cells have been identified in thermophilic animals like fishes,amphibians and reptiles,i.e.,melanophores,xanthophores,iridophores,erythrophores,leucophores and cyanophores.Generally,the first three types of pigme nt cells are more common.Studies on the three basic pigment cells in zebrafish provided important references for us to understand the body color and stripe formation from the molecular level,but different fish may have different types of pigment cells.Besides,the process of color patterning is also different.C ichlid is a large group of fishes with various body color and unpredictable patterns.A few studies have pointed out that they can be used as excellent materials to study the formation of vertebra te body color.As a representative,the N ile tilapia has the advantages of large size,rich protein content,fast growth,strong stress resistance,wide distribution（world-wide cultured fish）,published genome,short sexual maturity time（average ly 6 months）,short spawning cycle（average ly 14 days）,and it has four types of pigment cell types.The wild-type fish has erythrophores,hyper-pigmented bars and light-colored inter bars that zebrafish does not have.And there is also red tilapia,a natural mutant,suggesting that tilapia can be used as an ideal model to study the formation of body color in fish.Previous studies on body color of tilapia were mainly focused on cross breeding,transcriptome analysis and pigment cell distribution.In this study,four types of pigment cells in tilapia were identified,including melanophores,xanthophores,iridophores and erythrophores.And the ir first appearance and spatial distribution were defined.Using C RISPR/Cas9 gene editing technology,25genes involved in pigment cell differentiation and pigment biosynthesis were mutated.Some of the mutants were F0,and some of them were established to F1 and F 2.Obvious phenotypes in body color were observed in the F0 and F2 mutants in about half of the mutated genes.These results suggest that tilapia is an excellent model for studying teleost color patterns.Due to the third round of genome replication,many body color genes,such as mitf and pmel,have two copies.Homozygous lines of the key genes for melanophores differentiation and melanin biosynthesis,mitf,pmel and hps4,were constructed.Phenotypic analysis of the body color at different developmental stages was conducted.The main results are as follows:1.Body color formation and e stablishme nt of color gene mutant lines in tilapia.Four types of pigment cells:melanophores,xanthophores,iridophores and erythrophores were identified,and their first appearance was characterized in wild-type fish.It was found that melanophores appeared earliest（27.5 hpf）,followed by iridophores（2 dpf）,then yellow pigment cells（6 dpf）,and erythrophores appeared the latest（average 50 dpf）.A total of 25 genes involved in melanogenesis,pteridines metabolism,and the carotenoid s absorption and cleavage pathways were mutated through C RISPR/Cas9 gene editing.Among the 25 mutated genes,13 genes had a phenotype in both the F0 and F2 generations.None of F1 heterozygotes had phenotype.By comparing the body color of our mutants with that of red tilapia（Oreochromis spp）,a natural mutant,we found that the pigmentation of the trunk and eye is controlled by different genes.Color genes like mitf,kita/kitlga,pmel,tyrb,hps4,gch2,csf1ra,pax7b,and bco2b were proved to be of great significance for color patterning in tilapia.The results suggested that tilapia,a fish with four types of pigment cells and vertical bars,together with various natural and artificial color gene mutants,can serve as an excellent model for studying color patterning in vertebrates.2.Establishment and phenotype analysis of mitf mutants.In humans,mammals and birds,mitf mutation was responsible for different degrees of albinism,but has varying effects on the development and pigmentation of the retinal pigment epithelium（RPE）.The expression levels of mitfa and mitfb have been studied in zebrafish and medaka,but only in zebrafish the homozygous mutants have been reported.Homozygous mutation of mitfa leads to the absence of melanophores in the whole trunk;double mutation of mitfa and mitfb has no effect on the development and pigmentation of RPE.In this study,we found that mitfa and mitfb domains have high similarity,and both of them contain b HLH domain.Both mitfa and mitfb were expressed in skin and eyes in tilapia.mitfb was more widely expressed in various tissues,and the overall expression level was higher than that of mitfa.The expression levels of mitfa and mitfb in black tilapia were higher than that in red tilapia.To mutate the mitf genes,targets were designed in exon 3 of mitfa and exon 2 of mitfb,and Msp JI and Cac8I were selected for restriction enzyme digestion,respectively.F1 generation positive fish with-5 bp deletion were selected to establish homozygous mutant lines.The homozygous mutants were screened by PAGE,enzyme digestion and Sanger sequencing.mitfa-/-and mitfa-/-;mitfb-/-mutants were detected with melanophores reduction at early juvenile stage（12 and 60 dpf）.mitfb-/-mutants were detected with no obvious phenotype,only with slight hypo-pigmentation.mitfa-/-mutants were detected with light yellow plumage,together with weak vertical bars.mitfa-/-;mitfb-/-mutants were detected with pink-white plumage,with a few specified melanophores.No significant difference of body color was detected in mitfb-/-mutants,but loose round black spots were detected along the lateral line.Observation of the pigment cells in fins and scales showed that melanophores were significantly reduced in mitfa-/-and mitfa-/-;mitfb-/-mutants,which further explained the appearance of yellow and pink-white plumage.Disruption of mitf has no influence on RPE pigmentation,which was in consistent with the studies revealed in zebrafish.Both mitfa and mitfb disruption have resulted to significant reduction of melanophores,but melanophores were not totally absent even in mitfa-/-;mitfb-/-mutants,suggesting that there were at least two melanophores subtypes,which was also different with the studies in zebrafish.The homozygous mutation of mitfa and mitfb both led to the decrease of melanin in iris,but the pigmentation of RPE d id not change significantly.Besides,the phenotype of mitfa-/-mutants is more obvious than that of mitfb-/-mutants,which indicates that the mitfa plays a more important role in the maintenance of melanophores function than mitfb.3.Establishme nt and phenotype analysis of pmel mutants.pmel is the most downstream gene of melanogenesis pathway.It is directly regulated by transcription factor mitf in human and mouse,and plays an important role in melanin biosynthesis.pmela has only been knocked down in zebrafish,and the analysis was focused on the effects of pmela disruption on RPE development and pigmentation.In the studies of one African cichlid,the number and size of melanophores in vertical bars were significantly larger than those in inter-bars.The expression of pmela in vertical bars was significantly higher than that in inter-bars,suggesting that pmela may participate in the formation of the bars.In this study,both pmela and pmelb genes’promoters were found to have the binding site of mitf.pmela and pmelb domains have high similarity,and both of the m contain PKD and TM domains.pmela and pmelb were highly expressed in the eyes and skin of tilapia.The expression of pmela in the skin transcriptome of black tilapia was higher than that of red tilapia.To mutate the pmel genes,the targets were designed in exon 4 of pmela and exon 3 of pmelb,respectively.At the same time,F0 generation males with the highest positive rate were selected to establish F1 and F2 lines.The double mutants were obtained by mating of double heterozygous male and female fish.The homozygous single and double mutants were screened by PAGE,enzyme digestion and Sanger sequencing.It was found that the whole body of the pmela-/-mutants was pink,similar to the phenotype of red tilapia.The pmelb-/-mutants were also detected with hypopigmentation,but less effective than pmela-/-mutants.The pmela-/-;pmelb-/-mutants were detected with similar body color with pmela-/-mutants,but the pigmented melanophores were significant less in pmela-/-;pmelb-/-mutants than in pmela-/-mutants.Further analysis suggested that the development and pigmentation of eyes were significantly influenced in pmela-/-and pmela-/-;pmelb-/-mutants.The mutants were detected with significantly increased small sized melanophores with development,which made the fish a little hyper-pigmented.In this study,the two copies of pmel were found to participate in melanogenesis,the morphological maintenance and cell size regulation of melanophores in tilapia.And pmela played a more significant role in body color formation,RPE development and pigmentation in tilapia.4.Establis hment and phenotype analysis of hps4 mutants.In human and mouse studies,nine members of hps gene family have been confirmed to be associated with albinism,but the molecular mechanism inside is unknown.The homozygous mutation of hps5 resulted in white individuals and hypo-pigmented RPE in zebrafish.However,in fish,hps4 has only been reported to to be associated with natural albinism in channel catfish by GWAS mapping,and the specific mechanism is still unclear.In this study,we found that as a key gene for melanin synthesis,hps4 gene’s promoter region also contains the binding site of mitf.hps4 was widely expressed in many tissues of tilapia.The expression of hps4 in the skin transcriptome of black tilapia was higher than that of red tilapia.To mutate the hps4 gene,the target was designed in exon 2.The positive fish were screened and identified by PAGE,enzyme digestion and Sanger sequencing.The F0 generation male with the highest positive rate was selected to construct homozygous lines.XY male and XX female siblings of the heterozygous F1 generation that carried the same mutation（-7 bp）were mated to generate homozygous F2 mutants.hps4 was mutated in fish for the first time.The F0 chimeras showed different degrees of body albinism in different parts（trunk and fins）,but some other parts showed excessive hyper-pigmentation（melanin deposition）.A small number of F0 individuals were observed with systemic albinism.The homozygous mutants were observed with systemic albinism,and the phenotype was more and more obvious with development.RPE was detected with dark red color due to melanin shortage and blood color appearance.No melanophores,but some erythrophores were detected on the scales and fins of homozygous mutants.These results indicate that although the red pigment cells of tilapia have cluster branching structure and size similar to melanophores,they are not controlled by melanin synthesis genes.hps4 was detected with a more significant role in directing melanin biosynthesis than pmel in tilapia,different from the previous studies in mouse,in which hps4 was found to have the same function with pmel in melanin bosynthesis.5.Knock down of pmel in mitfa-/-mutants.The knock down of pmel in mitfa-/-mutants resulted in golden or incarnadine pink body color.No black vertical bars and black pigmented peritoneum were detected.Microscopic examination showed that most of the melanophores were in the state of dots,or fragment,the latter of w hich was a stage of apoptosis.The melanin contents in skin of the mutants were significantly lower than that of mitfa-/-mutants and wild-type fish.These results further confirmed the significant role of pmel in body color formation in tilapia by affecting melanin synthesis and melanophores size.6.Knock down of hps4 in mitfa-/-mutants.The knock down of hps4 in mitfa-/-mutants resulted in systemic yellowing,which was similar to the body color of the hps4-/-mutants,while the global color was a combination of the body color of mitfa-/-mutants and hps4-/-mutants.The mutants were absent of mature melanin-composed melanophores,but some of the not pigmented melanophores were still there,restricting the xanthophores to be round shaped instead of stretch like shaped as in the wild-type fish.Except for that,the mutants showed dark deep red and black color in the RPE.In conclusion,the homozygous mutants of mitfa,mitfb,mitfa;mitfb,pmela,pmelb,pmela;pmelb and hps4 were established in this study,among which tilapia pmelb,pmela;pmelb and hps4 were the first homozygous mutant lines in fish.Phenotypic analysis showed that mitf,pmel and hps4 played important roles in melanin biosynthesis and body color formation of tilapia as reported in other vetebrates.Among them,hps4has the strongest effect on melanin biosynthesis,which is characterized by systemic（including RPE）albinism and inability to synthesize melanin.pmel was followed by severe melanin reduction,insufficient melanin synthesis（including RPE）in the early stage and functional defects of melanophores（small size,small number,insufficient melanin synthesis）in the later stage.The role of mitf in the three genes is the weakest.The mitf mutants were detected with significant loss of melanophores,but still with melanophores in different developmental stages,leading to the formation of black spots on the trunk surface.This study will help to analyze the molecular mechanism of fish body color formation in theory,and provide a reference for vertebrate body color formation and various human pigment disordered skin diseases and eye diseases.In application,it is helpful to realize the large-scale creation of tilapia body color mutants through gene editing,and serve the aquaculture industry. |
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