microRNA regulation of skin pigmentation in fish

35 Pages·2013·0.99 MB·English

Research Article 3401
microRNA regulation of skin pigmentation in fish
Biao Yan1,2, Ban Liu3, Chang-Dong Zhu2, Kang-Le Li1,2, Li-Jia Yue2, Jin-Liang Zhao1,2, Xiao-Ling Gong2 and
Cheng-Hui Wang1,2,*
1KeyLaboratoryofFreshwaterFisheryGermplasmResources,ShanghaiOceanUniversity,MinistryofAgriculture,Shanghai201306,China
2CollegeofFisheriesandLifeScience,ShanghaiOceanUniversity,Shanghai,201306,China
3DepartmentofCardiology,ShanghaiTenthPeople’sHospital,TongjiUniversity,Shanghai,200072,China
*Authorforcorrespondence([email protected])
Accepted17May2013
JournalofCellScience126,3401–3408
(cid:2)2013.PublishedbyTheCompanyofBiologistsLtd
doi:10.1242/jcs.125831
Summary
MicroRNAs(miRNAs)areendogenoussmallnon-codingRNAsthatplaycrucialrolesinnumerousbiologicalprocesses.However,the
role of miRNAs in skin color determination in fish has not been completely determined. Here, we identified that 13 miRNAs are
differentiallyexpressedbetweenredandwhiteskin.TheanalysisofmiRNAspatialandtemporalexpressionpatternssuggeststhatmiR-
429isapotentialregulatorofskinpigmentation.miR-429silencingresultsinanobviouschangeinskinpigmentation.Bioinformatics
analysis anda luciferase reporter assay showthat miR-429 directly regulates expression of Foxd3 by targeting its39-untranslated (39-
UTR)region.miR-429silencingleadstoasubstantialincreaseintheexpressionofFoxd3invivo,therebyrepressingthetranscriptionof
MITFanditsdownstreamgenes,suchasTYR,TYRP1orTYRP2.Thesefindingswouldprovideanovelinsightintothedetermination
of skin color in fish.
e
c
n Keywords:miRNA,Skincolor,Fish,Foxd3
e
ci
S
ell
C Introduction color formation and skin diseases. In a previous study, miR-203
Somefishusuallydisplayfascinatingcolorpatternsintheirskin, was found to define a molecular boundary between proliferative
f
o
which have important roles in numerous biological processes, basal progenitors and terminally differentiating suprabasal cells,
al such as mate choice, camouflage and the perception of ensuring proper identity of neighboring layers (Yi et al., 2008).
n
r threatening behavior (Kelsh, 2004; Protas and Patel, 2008). The TheexpressionlevelofmiR-137canaffectthebodycolorpattern
u
o determinationofskincolorisaverycomplicatedprocessinfish, in mice (Dong et al., 2012). In Drosophila, the loss of miR-8
J
which is associated with a series of cellular, genetic, shows a substantially decreased pigmentation of the dorsal
environmental and physiological factors (Aspengren et al., abdomen (Kennell et al., 2012). This evidence suggests that
2009). How skin color patterns form, however, is a miRNAs could be involved in the formation of body color.
longstanding question among biologists. The genetics of skin However, no miRNA has been reported in the process of the
pigmentation in vertebrates has been extensively studied in a determination of fish skin color.
range of laboratory animal models. To date, a series of genes The common carp (Cyprinus carpio) is a widespread
have been reported to be involved in the determination of skin freshwater fish of eutrophic waters in lakes and large rivers in
color, such as pro-opiomelanocortin (POMC), melanocyte- EuropeandAsia.Theyhavenumerousskincolorsinthenatural
stimulating hormone (MSH), microphthalmia-associated environment, such as red, white, orange or black (Wang et al.,
transcription factor (MITF), kit oncogene (KIT), tyrosinase 2009).Overthepasttenyears,wehavebuilttwopurestrainsof
(TYR), tyrosine related protein-1 (TYRP1) and tyrosine related commoncarp,whichhavewhiteorredskincolor.Moreover,the
protein-2 (TYRP2) (Hubbard et al., 2010; Kelsh, 2004). Yet trait of skin color is inherited in accordance with the law of
surely the majority of gene resources involved in the Mendelian inheritance, as shown in supplementary material Fig.
determination of fish skin color remain to be discovered. S1.Thus,theyofferanappealingmodelsystemtoinvestigatethe
Skin color pattern is governed by complex and well-balanced genetic basis of skin pigment patterns. However, the unclear
programs of gene activation and silencing. MicroRNAs genetic background hinders its application for basic biological
(miRNAs) are a set of single-stranded, non-coding RNA study. Tilapia is an important model for the research of fish
molecules with an average size of approximately 22 physiology, endocrinology and the evolutionary mechanisms in
nucleotides. They recognize and bind 39-untranslated regions vertebrates.Italsohasadiversityofcolorpatterns,includingred
(UTRs) of mRNA, blocking translation of the gene or inducing and white skin color. Scientists have successfully completed a
cleavage of the mRNA. The crosstalk between miRNAs and tilapia whole-genome sequencing project, providing relatively
mRNAsisimportantforthesteadinessofsignaltransductionand clear genome information (Guyon et al., 2012). Common carp
the transcriptional activities as well as the maintenance of and tilapia would be adopted as research subjects to investigate
homeostasisinmanyorgans,includingtheskin(Mo,2012;Sand the role of miRNAs in the determination of skin color.
et al., 2009; Yi and Fuchs, 2010). Accordingly, skin-expressed In this study, we used the RNA-Seq method to identify
miRNAs might have a crucial role in skin development, body skin-related miRNAs, and quantitive PCR to screen these
3402 Journal of Cell Science 126 (15)
differentiallyexpressedmiRNAsbetweenwhiteandredskin.We short oligonucleotide alignment program (SOAP) software. The
found that the miR-429 is a positive regulator of skin color in resultsshowthatapproximately76,595uniquesRNAs(7,976,459
fish, andthat loss of miR-429 affects the pigmentcontent in the totalsRNAs)werefoundtomatchthezebrafishgenomeperfectly
skin.Theseeffectsaremainlymediatedbythedirectinteraction (supplementarymaterialTableS1).Next,weperformedadatabase
betweenmiR-429andFoxd3,suggestingafunctionofmiRNAin search to find rRNA, tRNA, snRNA and snoRNA deposited at
the determination of skin color in fish. RfamandNCBIGenBankdatabase(Fig. 1).
To identify the conserved miRNA homologs in the skin of
Results commoncarp,BLASTnsearchwithanE-valuecutoffof10was
miRNA expressionsignatureinthe skinof commoncarp used to search for the remnant small RNAs with predicted
To obtain the miRNA expression signature in the skin of hairpin structures against the miRBase (http://microrna.sanger.
common carp, we constructed a small RNA library from RNA ac.uk/sequences/) (Kozomara and Griffiths-Jones, 2011). With
sample pools isolated from different colored skin. Total RNA thissimilaritysearch,73miRNAswerefoundtobethesameas
was size-fractionated through PAGE, and small RNAs at least one of the published miRNAs in the miRBase. All of
corresponding in molecular weight to the mature miRNA these conserved miRNA precursors were identified and had a
population (18–24 nt fraction) were extracted from the gel and hairpin structure (data not shown). On the basis of sequence
processed for reverse transcription and PCR amplification to similarity, these known miRNAs were divided into 37 families
createcDNAlibraries.SmallRNAsequencingusingtheIllumina (supplementary material Table S2).
Genome Analyzer II generated 15,505,485 raw reads. After
removing low quality sequences and adapter sequences, miRNAsshowdifferentialexpressioninwhiteandredskin
15,326,145 clean reads were left for further analysis. Of these, To gain insight into the function of miRNAs in physiological
60.06% of the small RNA sequences obtained were 20–24 nt in processes, it is essential to have precise information on their
size, which is the typical size range for Dicer-derived products temporal and spatial expression patterns. First, we detected the
(Fig. 1). Currently, the complete genome sequence of common expressionlevelsofskin-relatedmiRNAsindifferentskincolorin
e carpisstillunavailable.Weselectedthezebrafishgenomeasthe common carp. The results indicate that 14 distinct miRNAs are
c reference genome for subsequent analysis. The clean reads differentiallyexpressedbetweenwhiteandredskin.miR-25,miR-
n
e mentionedaboveweremappedtothezebrafishgenomebyusing 15a-3p, miR-146b, miR-184, miR-429 and miR-141 are
ci abundantly expressed in red skin, whereas miR-18a, miR-137,
S
miR-17a,miR-203a,miR-9-3p,miR-9-5p,miR-129-5pandmiR-
ell 204 are abundantly expressed in white skin (Table 1). In tilapia,
C
we also detected the expression levels of these differentially
f
o expressed miRNAs identified in common carp. The results show
al that excluding miR-141, miR-129-5p or miR-17a, other
n differentially expressed miRNAs have a similar expression
r
u
pattern between common carp and tilapia (supplementary
o
J materialTableS3).
The expression of miRNAs is tightly regulated in a time-
and space-dependent manner. Tissue-specific or high-abundance
miRNAs expressed in certain tissues imply that these miRNAs
havecrucialfunctionsinthemaintenanceoftissuefunction.The
observation that miR-25, miR-137, miR-203a and miR-429 are
stronglyexpressedintheskin,impliesanimportantroleforthese
miRNAsintheskinconstitutiveprocess(Table 2).Inaddition,we
examined the miRNA expression profile during five different
developmentalstagesincommoncarp,includingzygote,blastula,
gastrula, segmentation and larvae. Interestingly, we found that
miR-429showsadynamicexpressionpattern.ExpressionofmiR-
429isfirstdetectedatthebeginningofthegastrulaestage,andits
expressionissubstantiallyupregulatedinthesegmentationstage;
Table1. DifferentialmiRNAexpressioninwhiteandredskin
of common carp
Highlyexpressed Highlyexpressed
inwhiteskin inredskin
miR-18a miR-429
miR-137 miR-146b
miR-9-5p miR-184
miR-9-3p miR-25
Fig.1. OverviewofsmallRNAlibrarysequencing.(A)Therawreads miR-204 miR-15a-3p
wereprimarilyanalyzedtoobtaincleanreads.(B)Lengthdistributionof miR-129-5p miR-141
smallRNAsincommoncarp.(C)Thecleanreadswereblastedagainstthe miR-203a
miR-17a
GenBankandRfamdatabasestoannotaterRNA,tRNA,snRNAorsnoRNA.
miRNA regulates fish skin pigmentation 3403
Table 2. Overview of the expression pattern of these differential expressed miRNAs in different color skin
Temporalexpressionpattern
miRNA Strongexpressioninadulttissue zygote blastula gastrula segmentation larvae
miR-429 skin,gill,fin 0 0 1 2 2
miR-146b heart,gonad 0 0 0 0 2
miR-184 brain,gonad 0 0 1 1 1
miR-25 liver,muscle,skin 0 0 0 0 1
miR-15a-3p brain,gonad,heart 1 1 1 1 1
miR-18a kidney 2 2 2 2 2
miR-137 Intestine,skin,brain 0 0 2 2 2
miR-9-5p brain,gonad,heart 0 0 1 2 2
miR-9-3p brain,gonad 0 0 1 1 1
miR-204 gill,heart 0 0 0 1 1
miR-203a liver,skin,gill 1 1 2 2 2
0:noexpression;
1:weakubiquitousorbackgroundexpression;
2:strongubiquitousexpression.
this expression is then sustained through to the larvae stage engineered two luciferase reporters, which are the wild-type 39-
(Table 2; supplementary material Fig. S2). Given that pigment UTR of Foxd3 gene, or the mutant UTR of Foxd3 gene. One
cellsareinitiallyderivedfromtheneuralcrestduringthegastrula luciferase reporter with or without miR-429 mimic or a
stage (Betancur et al., 2010), we speculated that miR-429 is a
potentialregulatorofthepigmentationprocess.
e
c miR-429 silencingaffectsthe pigmentation process
n
e In previous studies, we used an miRNA antagomir method to
ci conductmiRNAloss-of-functionexperimentsinvivo(Yanetal.,
S
2012;Yanetal.,2013).Inthisstudy,wealsoobservedthatmiR-
ell 429expressioncouldbeefficientlyblockedbyitscorresponding
C
antagomir(miR-429antagomir)butnotthemismatchedmiRNA
of (miR-203antagomir)invivo.Furthermore,thesilencingeffectof
al the miRNA antagomir was detected at different time points
n (supplementarymaterialFig.S3).ToevaluatetheeffectofmiR-
r
u 429 silencing on skin pigmentation, we injected red tilapia
o
J weighing,5 gwithmiR-429antagomirfor30days.Expression
ofmiR-429isefficientlysilencedbythemiR-429antagomirbut
not by the mismatched antagomir (Fig. 2A). There is no
significant difference observed between the wild-type fish and
miR-429 silencing fish in survival rate (Data not shown). We
compared the melanin production between wild-type and miR-
429silencingfish.WefoundthattheredfishinjectedwithmiR-
429antagomirhasalowermelanincontentrelativetoitsmatched
red group(Fig. 2B). In red common carp, we also observed that
thetreatmentwiththemiR-429antagomirsignificantlydecreases
the melanin content in skin (supplementary material Fig. S4).
These data suggest that miR-429 expression levels could affect
the process of skin pigmentation in fish.
miR-429 actsdirectly atthe 39-UTR of Foxd3
To determine the role of miR-429 in the pigmentation process,
we performed an in silico functional annotation analysis of its
predicted target genes using the TargetScan and miRanda
program on the basis of the tilapia genome sequence (Chen Fig.2. miR-429silencingaffectsthepigmentationprocess.(A)Redtilapia
et al., 2005). We screened the KEGG pathway database (http:// weighing,5gwasreceivedmiR-429antagomir(ormismatchedantagomir)
atadoseof60mg/kgbodyweightorleftuntreatedfor30days.miR-429
www.kegg.jp/kegg/) (Kanehisa et al., 2008) for candidate genes
expressionwasdetectedbyusingreal-timePCR.18SrRNAexpressionwas
ofthemelanogenesissignalingpathway,andfoundthatFoxd3is
detectedastheinternalcontrol.Thedatawereexpressedastherelative
a potential target gene of miR-429. The sequence alignment
changecomparedwiththewild-typegroup.(B)Tilapiawastreatedasshown
betweenmiR-429andthe39-UTRsegmentofFoxd3isshownin
inFig.2A.Totalmelanincontentintheskinwasdetectedasdescribedin
Fig. 3A. Foxd3 has been shown to have a function in the MaterialsandMethods.Datarepresentthemean6s.e.m.fromthree
specification of various downstream neural crest derivatives. independentexperiments.Asterisk(*)indicatesasignificantdifference
To verify the interaction between Foxd3 and miR-429, we comparedwiththewild-typegroup(P,0.05).
3404 Journal of Cell Science 126 (15)
scrambled miRNA mimic were transfected into an HEK 293T vivo. Taken together, these data indicate that miR-429 regulates
cell. The scrambled miRNA mimic with no homology to the Foxd3 expression through targeting of the 39-UTR of Foxd3
tilapia genome was used to control the nonspecific effects. As gene.
shown in Fig. 3B, the transfection of scrambled miRNA mimic
doesnotaffecttheluciferaseactivityoftheFoxd339-UTRwild- Alteredexpression ofpigmentation genesinmiR-429
typereporter. However,miR-429 mimictransfection results ina silencing tilapia
significant decrease in the luciferase activity of Foxd3 39-UTR Skin color is primarily determined by the amount, type and
wildytpe reporter. By contrast, the luciferase activity of Foxd3 distributionofmelanin(Parra,2007).MITFhasbeenreportedas
39-UTRmutantreportersisnotrepressedbythemiR-429mimic akeygeneintheregulationofmelaninproduction.Itisinvolved
(Fig. 3B). In addition, we found that in vivo administration of in melanocyte development and melanogenesis. Foxd3 can bind
miR-429 antagomir but not NaCl results in a profound decrease the MITF promoter and repress MITF transcription, thereby
intheendogenousexpressionofmiR-429(Fig. 3C).Meanwhile, affecting the process of melanogenesis. Because miR-429
wedetectedasignificantincreaseinFoxd3expressionatmRNA silencing results in the upregulation of Foxd3 expression, MITF
level and protein level (Fig. 3D,E). The inverse expression wouldbeupregulatedduringthisprocess.Indeed,quantitivePCR
correlation between miRNAs and putative target genes also analysis reveals that miR-429 silencing results in a significant
suggests that miR-429 directly regulates Foxd3 expression in increase in Foxd3 expression, thereby upregulating MITF
expression (Fig. 4A,B). MITF is required for the expression of
TYR,TRP-1andTRP-2genesthatencodeenzymesimplicatedin
the production of melanin (Levy et al., 2006). Quantitive PCR
and western blot analysis indicate that the expression of MITF
downstream genes, TYR, TYRP1 and TYRP2, are markedly
downregulated in miR-429 silencing fish (Fig. 4C,D). Taken
together, these results show that miR-429 silencing can increase
e thelevelofFoxd3,whichrepressesthetranscriptionofMITFand
c
n its downstream genes.
e
ci
S miR-429-mediated pigmentationis distinct froma-MSH-
ell mediatedpigmentation uponUV radiation
C Ultraviolet (UV) radiation is a major environmental hazard that
can lead to skin inflammation, photoaging and skin cancer. The
f
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al
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u
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Fig.3. miR-429actsdirectlyatthe39-UTRofFoxd3.(A)Thealignment
betweenmiR-429andthe39-UTRsegmentofFoxd3.(B)Luciferasereporters
werelinkedwithFoxd339-UTRscontainingeitherputativemiR-429-binding
sites(39-UTRwt)ormutantmiR-429bindingsites(39-UTRmutant).miR-
429mimicorscrambledmimic(Scrmimic)werecotransfectedwitha
luciferase-UTRconstructintoHEK293Tcells,andthentheluciferase
activitywasdetermined.ThecellstransfectedwithScrmimicplus39-UTRwt Fig.4. miR-429silencingchangestheexpressionofMITFandits
wereusedasthecontrolgroup.Datarepresentthemean6s.e.m.fromthree downstreamgenes.Tilapiaweighing,5greceivedmiR-429antagomirata
independentexperiments.P,0.05.(CandD)Tilapiaweighing,5greceived doseof60mg/kgbodyweightorwereleftuntreatedfortheindicatedtimes.
atail-veininjectionofNaCl,ormiR-429antagomiratadoseof60mg/kg Theuntreatedgroupwastakenasthecontrolgroup.TherelativemRNA
bodyweightfortheindicatedtimes.Theuntreatedgroupwastakenasthe expressionofFoxd3(A),MITF(B),andMITFdownstreamgenes,including
controlgroup.TherelativeexpressionofmiR-429(C)andFoxd3(D)was TYR,TRP-1,andTRP-2(C)wasdetectedusingreal-timePCR.18SrRNA
detectedusingreal-timePCR.18SrRNAexpressionwasusedastheinternal expressionwasdetectedastheinternalcontrol.Thedatawereexpressedas
control.Thedatawereexpressedastherelativechangecomparedwiththe therelativechangecomparedwiththeuntreatedgroup.Asterisk(*)indicates
untreatedgroup.Asterisk(*)indicatesasignificantdifferencecomparedwith asignificantdifferencecomparedwiththecontrolgroup(P,0.05).(D)The
thecontrolgroup(P,0.05).(E)TilapiawastreatedasshowninFig.3D,and expressionofTYR,TYRP1andTYRP2proteinswasdetectedbywesternblot
theexpressionofFoxd3wasdetectedbyusingwesternblots.GAPDHwas analysis.GAPDHwasusedastheinternalcontrol.Arepresentativeresultof
detectedastheinternalcontrol.Showninrepresentativeimage. threerepeatedexperimentsisshown.
miRNA regulates fish skin pigmentation 3405
skin pigment can provide essential protection against UV in MITF expression at mRNA level and protein level (Fig. 5B–
radiation (Matsumura and Ananthaswamy, 2004). Here, we D).TodeterminewhethermiR-429isinvolvedinMSH-mediated
found that UV radiation significantly upregulates the level of pigmentation, we compared the expression profiling of a-MSH
miR-429 expression in the melanocyte. The change in miR-429 and MC1R between the miR-429 silencing group and the wild-
expression could be detected as early as 1 hour after UV typegroupinresponsetoUVradiation.Theresultsshowthatthe
treatment (Fig. 5A). UV treatment also could significantly miR-429leveldoesnotchangetheexpressionofa-MSHandthe
increase MC1R and a-MSH expression at the mRNA and level of MC1R under the same conditions, suggesting that miR-
protein level, which stimulates the keratinocytes to secrete to 429-mediated pigmentation is distinct from a-MSH-mediated
enhancethesynthesisofskinmelanin(Fig. 5A).Bycomparison, pigmentation (Fig. 5E,F,G).
we found that the change in UV-mediated miR-429 expression
occurs substantially earlier than that of UV-mediated gene and Discussion
protein expression, suggesting that miRNA regulation occurs miRNAs post-transcriptionally regulate gene expression by
earlier than most gene transcription responses (Fig. 5A). In promoting mRNA degradation or inhibiting mRNA translation.
addition, we found that miR-429 expression was significantly Theyhavepivotalrolesinavarietyofdevelopmentalprocesses,
increased in fish upon UV radiation. Meanwhile, we detected a and their dysregulations are linked to numerous skin diseases
significantreductioninFoxd3expressionandamarkedincrease (Mo,2012).WhenmiRNAsaregloballyablatedinskinepithelium
e
c
n
e
ci
S
ell
C
f
o
al
n
r
u
o
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Fig.5. miR-429-mediatedpigmentationisdistinct
froma-MSH-mediatedpigmentationupon
exposuretoUVradiation.(A)Themelanocytewas
exposedtoUVradiationorleftuntreatedforthe
indicatedtimes.TheexpressionsofmiR-429,MC1R
anda-MSHlevelsweredetectedbyreal-timePCRor
westernblotanalysis.Theresultswereexpressedas
thechangeintreatedgroupsrelativetotheuntreated
group.(B–D)TilapiawasexposedtoUVradiation
fortheindicatedtime.Theuntreatedgroupwastaken
asthecontrolgroup.Therelativeexpressionsof
MC1Randa-MSHlevelsweredetectedbyreal-time
PCRorwesternblotanalysis.18SrRNAexpression
(forreal-timePCR)orGAPDH(forwesternblot)
wasdetectedastheinternalcontrol.Thedatawere
expressedastherelativechangecomparedwiththe
untreatedgroup.(E,FandG)Themelanocyteswere
transfectedwithmiR-429inhibitororleftuntreated,
andthentheywereexposedtoUVradiationforthe
indicatedtimes.TherelativeexpressionsofMC1R
anda-MSHlevelsweredetectedbyreal-timePCR
andwesternblotanalysis.Eachsamplewasanalyzed
intriplicate.Asterisk(*)indicatesasignificant
differencecomparedwiththecontrolgroup
(P,0.05).
3406 Journal of Cell Science 126 (15)
byconditionallytargetingDicer1,anmiRNA-processing enzyme, Thomas and Erickson, 2009). Foxd3 can also control the
this operation could distort epidermal morphology, implying a lineage choice between neural or glial and pigment cells by
key role of miRNAs in skin development (Yi et al., 2006). To repressingMITFduringtheearlyphaseofneuralcrestmigration.
gain insight into the possible significance of skin miRNAs in In this study, we found that miR-429 expression begins in the
fish, we first identified the expression profile of miRNAs in the gastrulastage,whichleadstoagradualdecreaseinexpressionof
skin tissue of fish, and then compared the miRNA expression Foxd3. The ablation of MITF repression could contribute to the
pattern between white and red skin. We found that 11 miRNAs generation of pigment cells.
are differentially expressed in different colored skin, implying MITF is a member of the Myc-related family of basic helix-
that these differentially expressed miRNAs are involved in skin loop-helixleucinezipper(bHLH-Zip)transcriptionfactorsandis
pigmentation. The knowledge of tissue-specific and cell- highly conserved across different vertebrate species. MITF-
specific expression patterns of miRNAs can directly inform positivecellsarealsoobservedintheopticneuroepitheliallayer,
functional studies (Aboobaker et al., 2005). Previous studies the presumptive retinal pigment epithelium (RPE), as well as in
have revealed that miR-375 is specifically expressed in cells behind the optic cup that were probably derived from the
pancreatic islet cells, where it regulates the expression of neuralcrestandcoulddevelopintochoroidalandirispigmentcells
insulin secretion (Poy et al., 2004). miR-1 is expressed (Nakayama et al., 1998). Mutations of the MITF gene cause a
exclusively in muscle, where it regulates cardiomyocyte varietyofphenotypes,mostnotablyinpigmentedcells(Levyetal.,
proliferation in vertebrates and muscle physiology in flies 2010). This evidence suggests that MITF expression is tightly
(Sokol and Ambros, 2005; Zhao et al., 2005). Here, we found associated with the development of pigment cells. Expression of
that miR-429, miR-25 and miR-137 are highly expressed in fish MITFbeginsinneuralcrest-derivedcellsinmouseembryo(Hou
skin. In particular, miR-429 displays dynamic expression et al., 2000). Here, we found a similar result, which shows that
patterns during embryonic development. miR-429 is first miR-429isactivatedatthegastrulastage,whichdirectlyrepresses
detected at the gastrulae stage, and then its expression is Foxd3 expression, thereby releasing MITF inhibition by Foxd3.
gradually upregulated until the larvae stage. Because pigment MITFdirectlyregulatestheexpressionofmultiplegenesthatare
cells are initially derived from the neural crest during gastrula necessary for melanophore development, including tyrosinase,
e
c stage, we speculated that miR-429 has a key function in the tyrosinase-relatedprotein-1,tyrosinase-relatedprotein-2andsoon
n
e regulation of skin pigmentation. (Levyetal.,2006).Wealsoobservetheexpressionchangeinthe
ci Skin pigmentationinfish isa complexprocessthat involves a MITFdownstreamgenewhenthelevelofmiR-429expressionis
S
series of cellular, genetic and physiological factors (Colihueque, altered.
ell 2010).TheroleofmiRNAsinpigmentationhasbeenreportedin Ultravioletradiationisamajorenvironmentalhazardandbrings
C
somespecies,includingmouse,alpacaandDrosophila(Dongetal., harmful effect on skin tissue. Protection against UV-mediated
of 2012; Kennell et al., 2012; Zhu et al., 2010). In this study, we damageisaffordedbymelaninproductioninmelanocyte.Melanin
al revealedthatmiR-429isapotentialregulatoroffishpigmentation. absorbs UVradiationanddissipatestheenergyasharmlessheat,
n Our previous study found that miR-429 directly regulates the blockingtheUVfromdamagingskintissue.Akeycomponentof
r
u expression of OSTF1, an osmotic stress transcriptional factor, this process is the UV-induced a-MSH, pro-opiomelanocortin
o
J revealing a role of miR-429 in fish osmoregulation (Yan et al., (POMC)andMC1Rgeneexpression(MillerandTsao,2010).a-
2012). Here, we found that miR-429 is highly expressed in red MSH is the main physiological regulator of skin pigmentation,
skin, and inhibition of miR-429 function causes a substantial which is produced by the proteolytic cleavage of the large
decrease in skin pigmentation. This study further extends the precursor protein, POMC. a-MSH-bound MC1R can activate
biological role of miR-429 in fish. miR-429 is a member of the adenylyl cyclase, lead to phosphorylation of cAMP responsive-
miR-8 family, which has been predicted or experimentally element-binding protein (CREB) transcription factor family
confirmed in a wide range of species. Previous studies have members and in turn transcriptionally activate various genes,
identified miR-8 as a regulator of osmoregulation, growth, suchasMITF.OurresultsshowthatUVtreatmentinfishleadsto
apoptosis and neuronal survival by targeting multiple mRNAs theactivationofa-MSHandtheMC1Rgene.Theresultissimilar
(Hyun et al., 2009; Loya et al., 2009; Vallejo et al., 2011). In tothatpreviouslyreportedinkeratinocytes(Dongetal.,2012).By
Drosophila, miR-8 is required for proper spatial patterning of contrast,miR-429knockdownhasnoeffectonthelevelofa-MSH
pigment on adult female abdomens. Loss of miR-8 in the andMC1RexpressionwithorwithoutUVradiation.Onthebasis
developing cuticle results in cell-autonomous loss of of these results, we speculate that miR-429-mediated melanin
pigmentation (Kennell et al., 2012). Sequence alignment productionisdistinctfroma-MSH-mediatedmelaninproduction.
suggests that the miR-8 family is highly conserved between We proposed a model for the avoidance of UV injury in fish
invertebrates and vertebrates, which might indicate that its (Fig. 6). Once a fish is exposed to UV radiation, miR-429
function has been conserved. Our study provides an example expression is rapidly upregulated, and earlier than other
thatsuggeststheroleofmiR-8inpigmentationishighlyconserved pigmentation gene activation. miR-429 upregulation could
betweeninvertebratesandvertebrates. release Foxd3-mediated MITF inhibition, thus activating its
miRNAs control biological processes by regulating the downstream pathway and pigment synthesis (Segura et al.,
expression of their target genes. Here, we found a binding site 2009). If fish are exposed to sustained UV injury, the pituitary
of miR-429 in the 39-UTR region of Foxd3, and characterized and keratinocytes secrete a-MSH and induce the production of
theireffectsonFoxd3usingareporterassay.Foxd3isoneofthe MC1R in melanocytes, which activates the a-MSH pathway.
earliest molecular markers of the neural crest lineage; it is miRNA-mediated gene regulation operates earlier than most
expressed in many organisms in the premigratory and migrating transcriptional responses. The fast regulation of miRNAs after
neural crest, and its expression is downregulated as the cells UVtreatmentindicatesthatmiRNA-mediatedgenesilencingacts
differentiate into most derivatives (Abel and Aplin, 2010; earlierthanmostgenetranscriptionalresponsesafterUVdamage.
miRNA regulates fish skin pigmentation 3407
NCBI GenBank database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) by per-
forming a BLASTn search. Any small RNAs with exact matches to these
sequenceswereexcludedfromfurtheranalysis.Theremnantreadswerecompared
withthemiRBase(19.0)toannotateconservedmiRNAs.Thesecondarystructures
ofthepredictedmiRNAswereconfirmedusingRNAfold(http://rna.tbi.univie.ac.
at/cgi-bin/RNAfold.cgi).
Real-timeRT-PCRanalysesofmRNAsandmiRNAs
Total RNA was extracted using Trizol reagent (Invitrogen), and miRNAs were
extracted using the miRNeasy kit (Qiagen) according to the manufacturer’s
instruction.RNAintegritywasassessedbyelectrophoresisona1.0%agarosegel.
For mRNA quantification, reverse transcription was performed using a High
FidelityprimeScriptTMRT-PCRKitasinstructed(Takara,Dalian,China).Real-
timeRT-PCRwasperformedbyusingtheSYBRGreenReal-timePCRMaster
Mix(Toyobo,Shanghai,China)andtheStepOneReal-timePCRsystem(Applied
Biosystems Inc., Foster City, CA) according to the manufacturer’s protocol.
miRNAabundancewasdetectedusingthestem-loopPCRmethod,and18SrRNA
expression was detected as the internal control. The relative gene or miRNA
expressionwasdetectedusingthecomparativethresholdcycle(C )method,also
T
referredtoasthe2-DDC method.Allreactionswereperformedintriplicateonthe
T
MyiQ5Real-timePCRDetectionSystem(Bio-Rad,Hercules,CA,USA).
Luciferasereporterassay
The39-UTRofFoxd3wasamplifiedfromtilapiagenomicDNAandindividually
clonedintothepGL3vector(Promega)bydirectionalcloning.ThemutantFoxd3
Fig.6. ThebiologicaleffectsofmiR-429inthedeterminationofskin 39-UTRreporterswerecreatedbymutatingtheseedregionofthepredictedmiR-
color. 429 site. HEK293T cells were co-transfected with 0.4mg of firefly luciferase
reporter vector and 0.02mg of the control vector containing Renilla luciferase
(PRL-CMV, Promega) using lipofectamine 2000 (Invitrogen) in 24-well plates
FishhaveevolvednumerousstrategiesforeffectivelyescapingUV
(Costar).Eachtransfectionwasperformedinfourwells.Luciferaseassayswere
e injurythroughdistinctsignalingpathways.miRNAsareimplicated carriedout24hoursaftertransfectionusingtheDualLuciferaseReporterAssay
c
n in buffering developmental processes against the effects of System(Promega).FireflyluciferaseactivitywasnormalizedtoRenillaluciferase
e activity.
ci environmentalfluctuations.
S Insummary,werevealedanewregulatorymechanismforskin
Spectrophotometricassayofmelanin
ell pigmentation in fish from an miRNA viewpoint. We found that Skinsamplesuspensionsweresolubilizedin8Murea/1Msodiumhydroxideand
C miR-429 is differentially expressed between white and red skin, clearedbycentrifugationat10,700gfor10minutes.Chloroformwasaddedtothe
supernatantstoremovefattyimpurities.Skincontainingpheomelaninswerecleared
of and its silencing using antagomir leads to a substantial change bycentrifugationat10,700gfor10minutesandanalyzedforabsorbanceat400nm.
in skin melanin content. The post-transcriptional regulation of A400/mg5spectrophotometricpheomelanin(Sp.PM)(Dongetal.,2012).
al Foxd3 by miR-429 could affect the expression of MITF and its
n
r downstream genes, including TYR, TYRP1 and TYRP2, which Westernblotanalysis
u
o in turn affects the pigmentation process in fish skin. miRNA- Thesampleswerelysedinthelysisbuffer(20mMTris,pH7.4,150mMNaCl,
J 5mM EDTA, 1% Triton X-100) with 1mM phenylmethylsulfonyl fluoride,
mediated melanogenesis might provide an additional option for 10mMNaF,1mMNaVO andproteaseinhibitorcocktail.Theproteinlysates
3 4
fish to avoid UV injury. wereclearedbycentrifugationat4˚Cfor15minutesat12,000g,quantifiedusing
theBio-Radproteinassay,resolvedon8–12%SDS-PAGEgels,andtransferred
ontoPVDFmembranes(Millipore).Afterblocking,thePVDFmembraneswere
MaterialsandMethods
incubated with the primary antibody, and then the membrane was washed and
Experimentalfish incubatedwithgoatanti-mouseIgG(H+L)-HRPconjugateantibody(SantaCruz).
TilapiaandcommoncarpwereobtainedfromthefisheryfarmofShanghaiOcean Specificcomplexeswerevisualizedbyecho-chemiluminescence(Amersham).
University(Shanghai,China).Theywerekeptinawatercirculationsystemin200-
litertanks,andthewatertemperaturewaskeptat2662˚Cundera12-hourlight
Dataanalysis
and 12-hour dark photoperiod. All experiments were conducted under the
Data was expressed as mean 6s.e.m. unless otherwise stated. Statistical
Guidance of the Care and Use of Laboratory Animals in China. This research
significance was assessed by one-way ANOVA followed by Bonferroni’s
wasapprovedbytheCommitteeontheEthicsofAnimalExperimentsofShanghai
multiplecomparisontests.StatisticalsignificancewasdefinedasP,0.05.
OceanUniversity.
Acknowledgements
SmallRNAlibraryconstructionandsequencing
We are grateful to Xian-qiang Du and Ling Wang in Shanghai
RNA samples were harvested from different color skin of common carp, and
immediatelyfrozeninliquidnitrogen.SmallRNAlibrarieswereconstructedusing OebiotechCO.,LTDforadviceondataanalysis.Theauthorsdeclare
a Small RNA Cloning Kit (Takara). ,20mg of small RNA was submitted for thatthereisnoconflictofinterestthatwouldaffecttheimpartiality
sequencing.Briefly,theSolexasequencingwasperformedasfollows:RNAwas of thisscientific work.
purifiedbyPAGEtoenrichforthemoleculesintherangeof17–27nt,thenwas
ligatedwith59-and39-adaptors.Theresultingsampleswereusedastemplatesfor
Authorcontributions
cDNAsynthesisfollowedbyPCRamplification.Theobtainedsequencinglibraries
B.Y. and C.H.-W. designed the experiment and wrote the
weresubjectedtotheSolexasequencing-by-synthesismethod.Aftertherun,image
analysis,sequencingqualityevaluationanddataproductionsummarizationwere manuscript. B.Y., B.L. and C.D.-Z. carried out the experiments,
performedbyusingtheIllumina/Solexapipeline. data organization and statistical analyses. K.L.-L. and L.J.-Y.
provided the experimental samples. J.L.-Z. and X.L.-G.
Identificationofskin-relatedmiRNAs participated in the study design and discussed the manuscript. All
The sequencing data was pretreated to discard low quality reads, no 39-adaptor authors readand approvedthefinalmanuscript.
reads,59-adaptorcontaminantsandsequencesshorterthan18nucleotides.After
trimmingthe39-adaptorsequence,sequencetagsweremappedontothezebrafish
Funding
genomeusingtheSOAPsoftware(http://soap.genomics.org.cn)withatoleranceof
one mismatch. The matched sequences were then queried against non-coding This work was supported by Special Fund for Agro-scientific
RNAs from the Rfam database (http://www.sanger.ac.uk/Software/Rfam) and Research in the Public Interest [grant number 200903045 to
3408 Journal of Cell Science 126 (15)
C.H.W.]; the National Natural Science Foundation of China [grant Levy,C.,Khaled,M.,Robinson,K.C.,Veguilla,R.A.,Chen,P.H.,Yokoyama,S.,
number30972250toC.H.W.];theSpecializedResearchFundforthe Makino, E., Lu, J., Larue, L., Beermann, F. et al. (2010). Lineage-specific
transcriptionalregulationofDICERbyMITFinmelanocytes.Cell141,994-1005.
Doctoral Program of Higher Education of China [grant number
Loya, C. M., Lu, C. S., Van Vactor, D. and Fulga, T. A. (2009). Transgenic
20123104120005 to B.Y.]; the Shanghai Educational Development microRNA inhibition with spatiotemporal specificity in intact organisms. Nat.
Foundation [grant number 12CG56 to B.Y.]; and the Shanghai Methods6,897-903.
UniversityKnowledgeServicePlatform-ShanghaiOceanUniversity Matsumura, Y. and Ananthaswamy, H. N. (2004). Toxic effects of ultraviolet
radiationontheskin.Toxicol.Appl.Pharmacol.195,298-308.
aquaticanimal breedingcenter [grantnumberZF1206 toC.H.W.].
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