Dkk2 Plays an Essential Role in the Corneal Fate of the Ocular Surface

ERRATUM

Development 133, 2595 (2006) doi:10.1242/dev.02462

Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium Mahua Mukhopadhyay, Marat Gorivodsky, Svetlana Shtrom, Alexander Grinberg, Christoph Niehrs, Maria I. Morasso and Heiner Westphal Development 133, 2149-2154.

An error in this paper was not corrected before the article went to press.

The ﬁnal sentence of the acknowledgements should read: ‘This research was supported by the Intramural Research Programs of the National Institute of Child Health and Human Development and of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.’

We apologise to the authors and readers for this mistake. ERRATUM

Development 133, 2447 (2006) doi:10.1242/dev.02434

An error in this paper was not corrected before the article went to press.

Morasso and Westphal are co-senior authors on this work, rather than equal contributors.

We apologise to the authors and readers for this mistake. Research Center, Heidelberg,Germany. Maria I.Morasso 1999) arestructurallysimilartotheextracellular domainsofFrizzled et al.,1997),WIF(Hsieh1999)andCerberus(Piccolo al., receptors. InhibitorsthatdirectlybindWnts,suchassFRP(Rattner regulate WntsignalingbybindingeithertoWntsortheir understanding development anddisease. Thus, investigating Wntsignalingpathways iscrucialfor pathways contributes toepithelialcancers(reviewed byWatt, 2004). a rangeofepithelia,andtheinappropriateactivation ofthese signaling alsoregulates stem-cellself-renewal anddifferentiation in syndromes andoculardisorders(reviewed byHeetal.,2004).Wnt gene homeostasis. ThisisillustratedbymutationsintheWntco-receptor embryogenesis but alsoforpostnataldevelopment andtissue Proper regulation ofWntsignalingisnotonlyessential for directly represstheE-cadherinpromoter(Jamoraetal.,2003). from theBMPinhibitorNoggin,whichinducesLef1expression, transcription. InputsfrombothWnt,whichstabilizes hair follicledevelopment requirestherepressionofE-cadherin affects celladhesion,whereformationofanepithelialbud during in coloncancercells(reviewed byMorin,1999).Wntsignalingalso cycle regulators Mycandcyclin D1aredirectWntsignalingtargets mutants (Megason andMcMahon,2002).Interestingly, thecell (Yamaguchi etal.,1999),andexpansion oftheCNSfails inWnt1 example, tissueoutgrowth isimpairedinlimbbuds lackingWnt5a phenotypes canoftenbeexplained byalossofcellproliferation. For commonly regulated byWntsignaling,and knockout (reviewed byLoganandNusse,2004).Cellproliferationis formation, generationofcellpolarityandspecificationfate during embryonicpatterning,includinganteroposterioraxis Wnt proteinsareimplicatedindiverse developmental processes INTRODUCTION Epidermis,Differentiation,KEY WORDS:Mouse,Dkk2,Cornea, Wnt/ the ocularsurfaceepithelium. achieved intheabsenceof stratified epitheliumthatexpressesepidermal-specificdifferentiation markersanddevelopsappendagessuchashairfolliclesis epithelial progenitorcellsintoanon-keratinizingstratifiedepithelium.Completetransformationofthecornealepitheliumint Here, weshowthatDkk2-mediatedrepressionoftheWnt/ The Dkkfamilyofsecretedcysteine-richproteinsregulatesWnt/ Mahua Mukhopadhyay surface epithelium Dkk2 playsanessentialroleinthecornealfateofocular Development 133,2149-2154(2006)doi:10.1242/dev.02381 Accepted 28March 2006 † 1 *These authorscontributedequallytothis work Health, Bethesda,MD20892,USA. Institute ofArthritisandMusculoskeletal andSkinDiseases,NationalInstitutesof Health andHumanDevelopment,Bethesda, MD20892,USA. Author forcorrespondence (e-mail:[email protected]) Laboratory ofMammalianGenesandDevelopment,NationalInstituteChild A variety ofsecretedWntinhibitorshave beenidentifiedthat Lrp5 that leadtofamilial osteoporosis,highbonedensity 3, * andHeinerWestphal 1 , MaratGorivodsky 3 Developmental SkinBiologyUnit,National Dkk2 gene function.We showthatDkk2isakeyregulatorofthecornealversusepidermalfate 2 German Cancer 1, * ␤ ,† -catenin, and 1 , SvetlanaShtrom ␤ -catenin pathwayisessentialtopromotedifferentiation ofthecorneal ␤ -catenin signalingbyinteractingwiththeWntco-receptorLrp5/6. ␤ -catenin GGC CCTCGA-3 ACC TCTCCTAA-3 AGC CTTTCAAGT CAG-3 GAC CAAACC TCTCCTAA-3 following primers. The genotypingwas performedbypolymerasechainreactionusingthe Genotyping animals, whichwereintercrossedtoproduceoffspring foranalysis. with aPGK-NEOcassette,preserving2.9kb(5 the targeting vector was constructedbyreplacingtheentire firstexon of Following ourpreviously publishedstrategies (Mukhopadhyayetal.,2001), Generation of Wnt/ inhibitors have beenimplicatedinrepression ofthecanonical of Bmp,NodalandWntsignals(Piccoloetal.,1999).These (Fz) family ofWntreceptors.Cerberusisamultifunctionalinhibitor selection. Thetargeting vector was linearizedataunique homologous sequencesandusingthethymidinekinasegenefordouble MATERIALS ANDMETHODS epithelial fate determination. uncovers anessentialrolefortheDkk2geneinocularsurface Dkk2 of themammalian et al.,1998;Mukhopadhyay2001).To investigate thefunction in headinductionandlimbdevelopment arewellestablished(Glinka diverse inductive processes. populations, suggestingthattheseligandproteinsareinvolved in andinhindbrain-derived neuralcrest 2000; Monaghanetal.,1999) expressed invarious neural,mesenchymaltissues(Hashimotoetal., mice. ChimericmaleswerematedwithC57BL/6femalestoproduce recombinant EScelllineswereinjectedintoblastocysts togenerate chimeric was confirmedbySouthernblotanalysis.Two independentlyderived selection. Targeted disruptionofthe transfection ofEScells.Transfected EScellsweresubjectedtodouble Niehrs, 2003).In Kremen classoftransmembraneproteins(Heetal.,2004;Maoand by interactingwiththeWntco-receptorLRP5/6and represent anotherclassofWntinhibitorsthatregulate Wnt pathways Mutant alleleprimerset:leftprimer, 5 Dkk2 The ␤ knockout micethroughhomologousrecombination.Ourstudy -catenin signalingpathway. 1 Dickkopf wild-type alleleprimerset:leftprimer, 5 , AlexanderGrinberg Dkk2 Ј (Dkk) family ofsecretedcysteine richproteins . Xenopus Dkk2 –/– Ј ; rightprimer, 5 mutant mice gene indevelopment wehave generated Ј . and mammals,Dkksaredifferentially Dkk1 Ј Dkk2 ; rightprimer, 5 1 is aWntantagonistwhoseroles Ј , ChristophNiehrs -GTA GAATTGACC TGCAGG Ј gene viahomologousrecombination RESEARCH ARTICLE -GGT CTCCTGGGTGAC CAA Ј ) and2.5kb(3 Ј Ј -GGT CTCCTGGGT -GAG GCCTTGGCT Not Ј I sitebefore ) offlanking 2 , Dkk2 o a 2149 Dkk2 +/–

DEVELOPMENT visible ontheocularsurfaceofa respectively, whendetected with a5 analysis: mutant alleles,respectively, when detectedwitha3 (neo) cassette.Theexpectedsizeof Dkk2 are indicated.( indicated (broken line).Scalebars:inC,1mm forC,D;inE,200 are present Theborder intheepithelial basalcellsofthemutantcornea. between epithelial(ep)andstromal (st)layers performed usingRiboGreenRNA Quantificationreagentandakitfrom RNA isolationkit’fromInvitrogen (Carlsbad,CA).RNA quantificationwas Total RNA was isolatedfromdissectedcorneasusingthe‘S.N.A.P. total RNA isolationandRT-PCR analysis in paraffin, sectionedandphotographed. using astandardprotocol(Hoganetal.,1994).Stainedeyes wereembedded ␤ Probes). Biotechnology) andrhodamine-conjugatedsecondaryantibodies(Molecular stained withcommerciallyavailable anti-Dkk2antibody(SantaCruz compound (Tissue Tek) and10 immunofluorescence, P2,P10andP15eyes wereembeddedinOCT secondary antibodies,BM-purplebluewas usedaschromogen(Roche).For with AquaPolyMount(Polysciences).For alkalinephosphatase-conjugated as chromogen(Zymed),counterstainedwithHematoxylin,andmounted ABC kit(Vector Laboratories).Thesectionsweredeveloped byusingAEC secondary antibodies(Vector Laboratories)andprocessedwithaVectastain incubation, sectionswerewashed inPBS,incubatedwithbiotinylated K1, anti-filaggrinandanti-Pax6 (Covance). Afterprimaryantibody antibodies usedareasfollows: anti-K12(SantaCruzBiotechnology),anti- primary antibodiesdilutedwithPBScontaining2%normalserum.The in a5mMsodiumcitratebuffer (pH6.0).We incubatedthesectionswith methanol for30minutes.Antigenretrieval was carriedoutbyboilingslides peroxidase activity byincubatingtheslidesin2.5%hydrogenperoxide removed byaseriesofxyleneandethanolrinses.We quenchedendogenous paraffin sectionscollectedfromE15,E17,P2,P5andP10eyes. Paraffin was base forhistologicalanalyses.Immunohistochemistrywas performedon Hematoxylin andEosin(H&E)orToluidine Blueorperiodicacid-Schiff paraffin andsectionedat5 Embryos werefixed in4%paraformaldehyde,dehydrated,embedded immunohistochemistry Histology and 2150 Fig. 1.Genetargetingatthe evident inthe e type.Corneal sections ofeyesstainedwithHematoxylin/Eosin, indicatingepitheliallayer(ep)andthestroma (st).(E)Wild ␤ -Galactosidase expression analysis -Galactosidase expression was visualizedbyX-galstainingofwholeeyes locus, thetargetingvectoranddisrupted RESEARCH ARTICLE Eco RI-digested genomicDNAisolatedfrom wild-typeandthree targetedEScelllines(lanes1-3)generated7kband5.2bands, Dkk2 H ) Periodicacid-Schiff basestaining ofthe –/– eye (F).(G)Highermagnificationview ofF;skinappendagessuchashairfollicles(arrow) andsebaceous glands(arrowhead) ␮ ␮ Dkk2 m frozensectionswerecut.Sections m. Sectionswereeitherstainedwith Dkk2 Eco Ј external genomicprobe.external locus and the cornea phenotypeof locus andthecornea –/– RI-generated fragmentsare 7and5.2kb.RI, mutant (D),whileanage-matched wild-typecontrol eyecontainsatransparent(C).( cornea Dkk2 Ј external genomicprobe.external ( allele. Exon1andpartoftheintron 1ofthe Dkk2 Apa –/– ␮ cornea demonstratespresencecornea of gobletcells(arrow). ( m forE,F;50 I-digested genomicDNAgenerated 7kband5bandsforwild-type set fromInvitrogen was usedforquantification. TGT TAC AAT AGC TGGATG AGG GAT-3 CTG GCTACA TAA TGATGCCCAATA-3 TAG TAG ACC CAG TCGAAA-3 ie(aas ta. 03.We alsoobserved presenceofpigment mice (Ramaeshetal.,2003). (Puangsricharern andTseng,1995)in invasion ofconjunctival tissuesasseeninseveral cornealdiseases localized preferentiallytotheperipheryofcornea,suggesting well asconjunctiva-specific gobletcells(Fig.1F-H).The Histological analysisrevealed thepresenceofsebaceousglandsas Dkk2 endothelium (Fig.1E).Bycontrast,theouterocularsurface ofadult a neuralcrest-derived collagenousstromaandasinglecelllayer consists ofanexternal stratifiednon-keratinizing epithelialcelllayer, elsewhere (Lietal.,2005). owing toabonedensitydefectthatisaddressedin studiesdescribed development, but aresmallerinsizethantheirwild-typelittermates after birth at birth,anddefectsincornealsurface morphologyappearbyP5 phenotype thataffects thecornea.Themutantcornea appearsnormal are viableandfertilebut arecharacterizedbyaconspicuouseye embryonic stem(ES)cells(Fig.1A,B).Homozygousmutantmice Dkk2 Corneal phenotypein Corneal RESULTS TAC AAG CAG TTTATT CCC-3 used forRT-PCR analysisareasfollows: leftprimer, 5 first-strand synthesiskit’fromInvitrogen. Thesequencesof Molecular Probes(Eugene,OR).For RT-PCR weusedthe‘ClonedAMV Dkk2 The outersurface oftheeye iscovered bythecornea,which C ␮ , D m inH;35 –/– ) Corneal phenotype.Anopaqueplaque andhairs(arrow)) Corneal are mutation was generatedviahomologousrecombinationin –/– Eco mutant mice. mice appearsopaqueandwenoticedhairgrowth. . RI; RV, Adult mutantmiceshow normalskinandpelage ␮ m inI. Eco RV; H3, ( A Dkk2 ) Partialrestriction mapsofthewild-type Hin Dkk2 allele were replaced withtheneomycin Ј dIII; Ap, Ј . and rightprimer, 5 Lef 1 –/– primers are:leftprimer, 5 mice Apa I Ј ) Ectopicpigment-granules Ј . A of a mutant cornea is of amutantcornea pithelial hyperplasiais I. ( and rightprimer, 5 Development 133(11) Pax6 B ␤ ) Southern blot ) Southern -actin controlprimer- +/– Ј Ј -CCA TTA GCC -TTG GAT TCA E-G heterozygote Shh ) Paraffin primers Ј Ј -AGG -ACT

DEVELOPMENT (broken lines).Scalebars:100 epithelial andstromal borderandthelimbusare ofthecornea indicated (A,arrow)layers inthecornea andlimbus(B,arrowhead). The antibody. Expression oftheDkk2protein islocalizedtotheepithelial Role ofDkk2inocularsurfacemorphogenesis ( Expression oftheDkk2protein inadultcornea. 2. Fig. i.3 Expression ofepithelialkeratinocytemarkers inwild-typeand Fig. 3. type corneas (K,M,P,R).type corneas Scalebars: 50 (G,I,J)atallpostnatal stages.Conversely,corneas K1( ( form interwovenandpartiallydesquamated scalylayers(arrows). ( fromepithelial markers(F-J,K-O,P-T)in wild-type andmutantcorneas P5(A,B,F,G,K,L,P,Q), P10(C,D,H,I,M,N,R,S)and6-month-o A A-E , B Paraffin sectionsfromare wild-typecornea stainedwithanti-Dkk2 ) ) Hyperplasia of the corneal epitheliallayer (ep)isapparentatallstages.Themore) Hyperplasiaofthecorneal inthemutantcorneas superficialcellso ␮ m. ␮ m. K-O ) andfilaggrin( F-J ) K12expression ispresent (F,H), inwild-typecorneas butnotinmutant P-T et al.,1999). and theopticstalk,notablytodeveloping cornea(Monaghan expression extends tomesenchymalcellscondensingaroundtheeye ventral diencephalonandintheoticvesicle andbyE11.5 observed atembryonicdayE9.5inneuralepithelialcellsofthe null mice. consistent withthedevelopment oftheobserved phenotypeinthe Pellegrini etal.,1999).Thus,theexpression patternofDkk2is the maintenanceofcornealepithelium(Cotsarelisetal.,1989; sclera isconsideredthesiteofprogenitorsthatarerequiredfor the limbus. Thelimbalepitheliumattheborderbetweencorneaand Its expression islocalizedtotheepitheliallayersofcorneaand cells andinthelimbus that takes placeduringthefirst2weeksafterbirth,anditisincorneal development (Angetal.,2004).Stratificationofthecornealepithelia The onsetof Expression of pattern eyes containcelltypesnormallyfoundinskin. These resultssuggestthattheocularsurface epitheliumof (Fig. 1I),reminiscentofsimilargranulesinepidermalkeratinocytes. granules incellsofthebasalepitheliallayermutantcornea ) expression (L,N,O,Q,S,T)butnotinwild- isinducedinmutant corneas Dkk2 –/– corneas. Dkk2 Dkk2 Hematoxylin/Eosin staining(A-E)and expression of expression inthecorneaismaintainedthroughout gene expression inthemouseembryohasbeen Dkk2 Dkk2 is prominentlyexpressed (Fig.2) RESEARCH ARTICLE f themutantcornea ld (E,J,O,T)mice. Dkk2 Dkk2 2151 –/– .

DEVELOPMENT and (A)butisabsentorexcludedfrom (B).( type cornea thenucleiofmutant corneas. K12expressioncorneas. ispresentbutabsent(K)ina (J)inwild-typecornea analysis. ofamutantey the centralregion(F),butdetectedinafewhairfollicleslocatedcornea ofawild-typecornea oaiaino h -a tiigi h toao h ibs h ibleihla-toa odri niae.AtP15, localization oftheX-galstaininginstroma ofthelimbus.Thelimbalepithelial-stromal border isindicated. (C).(E)Highermagnificationvie expression (D),butnotinthewild-typecornea isdetectedinthelimbusofmutantcornea development andmaintenance ofthecornea(Ramaeshet al.,2003; Pax6 Epithelial cellsofthecorneaderive fromectodermthatexpresses Molecular mechanismof transformed intoastratifiedepidermis. appendages, weconcludethat the hair folliclesandsebaceousglands,i.e.typicalepidermal expresses proteinscharacteristicofepidermis.Asitalsocontains marker K12(Fig.3G,I,J).Thus,theadultmutantocular surface 3L,N,O) andFIL(Fig.3Q,S,T)but lacksthecorneal epithelial absent in a mutant cornea (M).TOPGALexpressionabsent inamutantcornea isinducedthelimbalstroma region ofa Fig. 4.Molecularmechanismof 2152 the ocularsurfaceepitheliumof Expression ofepidermalkeratinocytemarkersin mutant corneas and absent in wild-type corneas. ( andabsentinwild-typecorneas. mutant corneas Fig. 3shows thattheadult information concerningthelineageoriginofepithelialcells. Hematoxylin/Eosin staining(Fig.3A-E)hasgenerateduseful al., 2005).Keratin andFILimmunostaining(Fig.3F-T),alongwith differentiation andaprecursorofthecornifiedenvelope (Candiet associated proteinfilaggrin(FIL)isamarker for terminal 2004). Inthekeratinizing epidermis,theintermediatefilament- keratins, respectively (Chaloin-Dufau etal.,1993;Wolosin etal., keratin5/keratin14 tothetissue-specificK3/K12andK1/K10 and epidermalcellsisassociatedwithaswitchfrombasal epidermal stratifiedepithelia.Initialdifferentiation inthecorneal The ocularsurface epitheliasharesbasicfeatureswiththe Scale bars:inA,50 Dkk2 , ahomeoboxgeneofprofound importanceforeye RESEARCH ARTICLE Lef1 –/– corneas ofmicecarryinga corneas mRNA expression issignificantlyupregulatedcompared inmutantcorneas withwild-typecorneas; ␮ m forA,B;inC,100 Dkk2 Dkk2 Dkk2 –/– ␤ -galactosidase ( gene function. cornea stainsforK1(Fig. Dkk2 ␮ m forC,D;inF, 100 gene function Dkk2 mutant corneawas J-M –/– ) Immunostainingand( lacZ mice ( A ) geneunderthecontrol of , B ) AtP10,Pax6protein isdetectedinthenucleiofepithelialbasalcellswild- ␮ m forF,G; inJ,50 sequence (DasGuptaandFuchs,1999).Upregulation oftheWnt/ et al.,1998;Huelsken etal.,2001).We examined appendages thatappearintheepidermis(Millar, 2003;St-Jacques in thecomplex seriesofevents thatdeterminetheectodermal Pax6 expression. fate changeinthe immunostaining (Fig.4A).Therefore,theobserved cornea-to-skin epithelial basalcellsofwild-typecorneasshow Pax6 surface ofthe 2003). We foundthat addition, conjunctival invasion (Ramaeshetal.,2003;Davis etal., null mutation,thereisdecreasedexpression ofkeratin K12and,in Davis etal.,2003).Inthecorneaofmiceheterozygousfora of catenin pathway was noticedfirstshortlyafterbirth(P2) intheform lacZ in the shows upregulation oftheWnt downstream gene mammalian eye camefrom resultsofourRT-PCR analysisthat wild-type corneas(Fig.4C,F). of thecentralcornea(Fig.4G).No hair follicleslocatedinregions correspondingtothestromallayer later stagesofdevelopment, weobserved occasional Dkk2 N Wnt andsonichedgehog(Shh)signalingpathways playkey roles Additional evidence foraWntinhibitoryroleofDkk2inthe ␤ , O -gal staininginthelimbalmesenchyme(stroma)(Fig.4D,E).At ) X-galstainingonparaffin-embedded sectionsfrom E15.5 expression iscontrolledbya –/– Dkk2 ␮ C-G cornea. Pax6expressioncornea. ispresent(L),but inawild-typecornea m forJ-M;inN,150 ␤ ) X-galstainingonparaffin sectionstakenfrom wild-type -null mutanteye usingtheTOPGAL transgeneinwhich -catenin-responsive elements.AtP2,significant Dkk2 Dkk2 Dkk2 mutant (Fig.4B),whereasthenucleiofcorneal Pax6 –/– -null mutanteye occursintheabsenceof cornea (O), but not in wild-type cornea (N). (O),butnotinwild-typecornea cornea gene expression isabsentintheocular ␮ m forN,O. Shh ␤ ␤ -gal stainingwas detectedin the -catenin-responsive promoter mRNA expression isinducedin lacZ expression isabsentin e (G).( Development 133(11) w ofD,showing Lef1 ␤ H -catenin activity , I ) RT-PCR lacZ in thecornea lacZ signals in Pax6 ␤ -

DEVELOPMENT The strikingcornealphenotypeofthe DISCUSSION adjacent stroma. regulator actsnon-autonomouslybyinhibitingsignaling inthe epithelial layerofthecornea,itstandstoreasonthatthisWnt the centralcornea.AsDkk2proteinisexpressed in theouter requires suppressionofWntpathways inthelimbus ratherthanin data suggestthatproperdevelopment ofthecornealepithelium in thewild-typecorneaatE15.5(compareFig.4Nwith4O).These expression inthelimbalstromaregion ofthemutantcorneabut not of thisnotion,weobserved aninductionofTOPGAL reporter may wellbeaconsequenceofincreasedWntsignaling.Insupport type corneas.TheabsenceofPax6 expression inmutantcorneas shown), adaybeforeK12expression isnormallyinducedinwild- Moreover, itsexpression was alreadyaffected atE14.5(datanot expression was absentatE15.5(compareFig. 4Lwith4M). (Ramaesh etal.,2003).InthemutantcornealepitheliumPax6 acts astheupstreamregulator ofcyokeratin-K12 geneexpression devoid ofany K12staining(Fig.4K).Inthedeveloping cornea,Pax6 corneal epitheliumatthisstage(Fig.4J),whilemutantcorneaswere We observed apunctatestainingpatternoftheK12geneinwild-type K12 isfirstinducedinthedeveloping corneaatembryonicday15.5. development. Expressionofthecornea-specifickeratinocyte marker the temporalrequirementfor these findings. appearance ofhairfolliclesinthemutantcorneaisconsistentwith during follicledevelopment (St-Jacquesetal.,1998).The morphogenesis, andWntShhpathways appeartointeract 4I). Shhplaysamajorroleatdiscretestagesofhairfollicle mutant corneaalsocoincideswithenhanced of Role ofDkk2inocularsurfacemorphogenesis mouse, ectopicexpression of implicated intheinitiationofhairfollicledevelopment. Inthe the mesenchyme(reviewed byMillar, 2003).Wntsignalinghasbeen requires reciprocalinteractionsbetweenthesurface epithelium and epithelia (Peartonetal.,2005). involves downregulation ofPax6 expression in basalcorneal that transdifferentiation ofcornealepitheliumintoepidermis the developing cornealepithelium.Thisagrees withrecentfindings expression isprecededbydownregulation ofPax6 expression in experiments indicatethatsuppressionofcornea-specific gene cutaneous appendages(Ferrarisetal.,2000).Furthermore,our mouse corneajuxtaposedwithembryonicdermiscanform reminiscent ofgraftingexperiments demonstrating that adult is suppressedandepidermalmarkers areinduced.Thisis place ofthecornea.We show thatcornea-specificgeneexpression and give risetoastratifiedskinandectodermalappendagesin In answertoincreasedWntsignaling,progenitorcellschangefate role ofthisWntinhibitorincornealepithelialfate determination. in thecorneal/limbal epitheliumisrequiredtomodulate Wntactivity appendages. Alltogether, thesedatasuggestthatDkk2expression with theformationofastratified epidermisandderived ectodermal mesenchyme, owing tolackofDkk2expression, isconcomitant study, weshow thattheactivation of the sebaceous glandsornew hairfollicles,dependingonthestrengthof epidermis canbereprogrammedtoforminterfollicularepidermis, activated formof development ofskinappendages(Andlet al.,2002).Usingan We next examined corneasfromembryonicstagestodetermine The development ofepidermalappendages, suchashairfollicles, Dkk2 ␤ ctnnsgaig(iv-agse l,20) Inthepresent -catenin signaling(Silva-Vargas etal.,2005). mutant mice(Fig.4H).Wntupregulation inthe ␤ -catenin, Silva-Vargas etal.showed thatadult Dkk1 Dkk2 ␤ in theskincompletelyblocks Dkk2 -catenin inthemutantlimbal gene functionincornea –/– Shh mutant pinpointsthe expression (Fig. Dkk2 null Ferraris, C.,Chevalier, G., Favier, B.,Jahoda,C.A.B.andDhouailly, D. Hashimoto, H.,Itoh,M.,Yamanaka, Y., Yamashita, S.,Shimizu,T., Solnica- Glinka, A.,Wu, W., Delius, H.,Monaghan,A.P., Blumenstock,C.andNiehrs, Davis, J.,Duncan,M.K.,Robison,W. G.,JrandPiatigorsky, J. Cotsarelis, G.,Cheng,S.Z., Dong,G.,Sun,T. T. andLavker, R.M. DasGupta, R.andFuchs,E. Chaloin-Dufau, C.,Pavitt,I.,Delorme,P. andDhouailly, D. pathways ofdifferentiation. signaling inthelimbus triggersepidermalandsuppressescorneal the integrity ofthecornea.Initsabsence,anincrease ofWnt Dkk2 triggered byWntsignalsemanatingfromthelimbus ofthenewborn we maynonethelesshypothesizethatasimilarseriesofevents is Although limbus tissuewas excluded fromthisinvitroexperiment, downregulation ofPax6 andtointeract withNoggintoinducehair. epidermis. Inthisprocess,Wntsarethoughttoberesponsibleforthe induction ofhairfolliclesandsubsequentformationinterfollicular dedifferentiation ofbasalepithelialcellsinthecornea,followed by change isbroughtaboutbyastepwiseprocessinvolving a The authorsshow datathatarecompatiblewithamodel wherebythe where centralrabbitcorneaisabutting embryonicmousedermis. transdifferentiation ofcornealepitheliumintoepidermis inasetting The secondstudybyPeartonetal.(Peartonal.,2005),described activation ofWntpathways, thoughhairfollicleswerenotobserved. hyperplasia andkeratinization ofthecornealepithelialcellsthrough gene functionleadstoepidermalhyperplasiaandextensive (Nicolas etal.,2003)reportthatconditionalablationofthe ocular surface aresuggestedbytwo recentreports.Nicolasetal. that bringabouttheepidermalcomponentsof Some ofthestepsinprofoundchangedifferentiation programs conjunctiva thatweobserve strong Wntsignalinginthemutant. transitional zonebetweenthetransparentcorneaandwhite likely, thatthey resideinthelimbus becauseitishere,inthe initial targets ofDkk2-mediatedepithelialtransformation.Itisvery development ofastratifiedepidermisandskinappendages. pathway thatleadstocornealepitheliumformationandprevents the in thelimbalstromaregion, andthatthisisarequiredstepinthe Candi, E.,Schmidt,R.andMelino,G. Ang, S.J.,Stump,R.J.W., Lovicu,F. J.and McAvoy, J.W. Andl, T., Reddy, S.T., Gaddapara,T. andMillar, S.E. References of Health. Institute ofArthritisandMusculoskeletalSkinDiseases,NationalInstitutes research wassupportedbytheIntramuralResearch Programs oftheNational embryonic stemcellcultures; andAliceSchindlerforhelpingenotyping.This in tissuepreparation andhistology;SingPingHuangforassistancewiththe suggestions; DrW. G.Robertsonandmembersofhislaboratoryforassistance We thankDrsJ.Smith,DavisandY. Zhaoforhelpfuldiscussionsand embryonic dermalstimuli. epidermal, pilosebaceousandsweatgland geneticprograms inresponse to epitheliumbasalcellspossessthecapacitytoactivate (2000). Adultcorneal 116 Requirement morphogenesis:arole forPax6incorneal inadhesion. Development transcription complexesduringhairfollicledevelopmentanddifferentiation. stimulated toproliferate: implications onepithelialstemcells. 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