Odd-Skipped Related 1 Is Required for Development of the Metanephric Kidney and Regulates Formation and Differentiation of Kidne

Home , Kidney development, Mesonephric tubules, Mesonephros, Ureteric bud

plays animportantroleinestablishingkidneyprecursorcells,andregulatingtheirdifferentiation intokidneytubulartis in chickmesonephricprecursorcellsinhibitsdifferentiation oftheseprecursorsintokidneytubules.Thesedataindicatethat embryo, differentiation. Micelacking to mesenchymalprecursorswithinthemesonephricandmetanephrickidneyissubsequentlydownregulatedupontubule these processes. mesenchyme atitstips.Thecondensedisthought to branching oftheuretericbud andcondensationof metanephric derivative ofthe nephric ductknown astheuretericbud resultsin Reciprocal signalingbetweenthemetanephricmesenchymeand a and Sainio,1997;Schultheissetal.,2003;Vainio andMuller, 1997). metanephric zonefromamoreanteriorregion oftheembryo(Sariola IM, andthenephricduct,anotherIMderivative thatmigratesintothe mesenchyme, whichisderived fromthemost posteriorregion ofthe differentiates astheresultofinteractionbetweenmetanephric mouse metanephros,whichisthebeststudiedexample, kidney tissue the secondstage:differentiation ofnephrogenicmesenchyme.Inthe numerous studiesover many years,muchhasbeenlearnedconcerning differentiation ofthatmesenchymeintofunctionalnephrons.Through stages: establishmentofnephrogenicmesenchymewithintheIM,and mesonephros hasmerelytwelve (Sainio,2003). contain upto11,000nephrons(Yuan etal.,2002),whilethe of thesekidney typesisvery different: themousemetanephroscan fundamental functionalunit(thenephron),thesizeandorganization kidney). Whileallkidney typesarecomprisedofthesame reproductive system)andthemetanephros(thedefinitive adult functional embryonickidney, whichalsocontributes tothemale pronephros (atransientembryonicstructure),themesonephros(the three typesofkidney tissue,called(fromanteriortoposterior) the developing embryo(Saxen, 1987). Inamniotes,theIMgives riseto (IM), astripoftissuelocatedadjacenttothesomitesin Vertebrate kidney tissueisderived fromtheintermediatemesoderm INTRODUCTION KEY WORDS:Kidney, Metanephros, Mesonephros, Chickembryo,Mouse,Osr1,Odd1 functional filtrationunitofthekidney. Herewereportthatthegene Formation ofkidneytissuerequiresthegenerationprecursorcellsandtheirsubsequentdifferentiation intonephrons Richard G.James differentiation ofkidney precursorcells metanephric kidneyandregulatesformation Odd-skipped related1isrequiredfordevelopmentofthe Development 133,2995-3004(2006)doi:10.1242/dev.02442 DEVELOPMENT ANDDISEASE Accepted 15May 2006 † 1 metanephric kidneyformation,including Brookline Ave, Boston,MA02215,USA. RW-663, Biomedical Sciences,Harvard MedicalSchool,Boston,MA,USA. *These authorscontributedequallytothis work Medicine andDentistry, Rochester, NY, USA. Biology andDepartmentofBiomedicalGenetics, UniversityofRochesterSchool Author forcorrespondence (e-mail:[email protected]) Molecular andVascular Medicine Unit,BethIsraelDeaconessMedicalCenter, 330 Kidney formationcanbeconceptualizedasconsisting oftwo Odd1 can promoteexpressionofthemesonephricprecursormarkers Odd1 1,2, is theearliestknownmarkerofintermediatemesoderm,precursortoallkidneytissue.Itlocalized *, CaramaiN.Kamei Odd1 do notformmetanephricmesenchyme,andexpressseveralotherfactorsrequiredfor 2 Program inBiologicaland Eya1, Six2,Pax2,Sall1 1,2, 3 Center forOral *, QingruWang and MATERIALS ANDMETHODS precursor populationfromwhich thekidney isderived. establishment andmaintenanceof thenephrogenicmesenchyme, studies, weproposethat the productionofdifferentiated kidney tubules. Onthebasisofthese Odd1 formation. Gain-of-functionstudiesinthechickembryorevealed that a setoftheearliestknown genesexpressed during metanephrickidney establishment ofthemetanephricmesenchymeandinactivation of studies inthemouserevealed arequirement for confined toundifferentiated kidney precursor tissue.Loss-of-function previously describedkidney regulatory genes,anditsexpression is pair rulegene zinc finger-containing transcriptionfactor related tothe exception of Genome Informatics)(Batesetal.,2000).However, withthe paralogous group(Wellik etal.,2002)and 2005), RNA probesweregeneratedfor chicken In situhybridization odd-skipped related 1 required foritssubsequentdifferentiation and/orsurvival. initial formationofthemetanephricmesenchyme,andareinstead 1993), kidney, including of thesearerequiredforproperdifferentiation ofthemetanephric specifically intheundifferentiated metanephricmesenchyme;many understood. Multiplegeneshave beenidentified thatareexpressed development –formationofnephrogenicmesenchymeareless precursors ofthekidney tubules (ChoandDressler, 2003). differentiate intopretubular aggregates andrenalvesicles, the surrounding non-condensedmesenchyme)andgives off cellsthat of theuretericbud (viaproliferationand/oradditionfromthe form aprecursorcellpopulation,whichbothmaintainsitselfatthetips al., 1994)andmouse Gdnf 3 odd-skipped related1 , RulangJiang The currentstudycharacterizestheroleduringkidney formation of The mechanismsthatregulate theearlierphaseof kidney . Intransientectopicexpressionexperimentsinthechick can activate earlymarkers ofkidney precursorcellsbut inhibits Pax2 Eya1 Pax2 (Burrill etal.,1997;Herbrand 1998), (Xu etal.,1999), Eya1 odd skipped and Pax2 , alloftheabove genesaredispensableforthe Pax2 3 Lim1 ( and ThomasM.Schultheiss Odd1 (Torres etal.,1995), (Dressler etal.,1990), ( . Finally, persistentexpressionof Odd1 Odd1 )( ( Osr1 Odd ) playsanimportantroleinboth Six1 plays animportantroleinthe ). – MouseGenomeInformatics),a RESEARCH ARTICLE Odd1 (Xu etal.,2003),the Odd1 is expressed beforeall N-myc (James andSchultheiss, Lim1 Wt1 (Kreidberg etal., (Fujii etal.,1994), Lim1 ( Mycn Odd1 1,2,† Drosophila (Tsuchida et sue. – Mouse

Odd1 Odd1 Hox11 in the 2995 , the

DEVELOPMENT washed andthecolorwas visualizedusingNBTandBCIP. (Roche, 1:2000,inPBT+10%sheepserum)overnight. Lastly, sectionswere 100), thesectionswereincubatedinalkalinephosphataseantibodysolution (65°C). AfterblockinginPBT(PBSwith2mg/mlBSA,0.1%Triton X- incubated withRNAse (1 probe (1 ␮ paraformaldehyde-fixed cryostatsectionswerepost-fixed, treatedwith10 (Schultheiss etal.,1995).For insituhybridizationonsections, mount insituhybridizationwas performedasdescribedpreviously standard methodsdescribedpreviously (Schultheissetal.,1995).Whole- (Nishinakamura etal.,2001)and Plasmid andretroviral expression vectors the firstcodingexon ofthe Generation ofmicecarryingatargeted in-framefusionofa Odd1 (Lobe etal.,1999). instructions. Stainingfor were detectedbyTUNELassay(Sigma)asperthemanufacturer’s Probes) andmousemonoclonalanti-Wt1(1:100,Dako). Apoptoticcells 1:250), mousemonoclonalandrabbitpolyclonalanti-Gfp(1:100,Molecular anti-Odd1 [1:750(JamesandSchultheiss,2005)],rabbitanti-Pax2 (BabCo, 2003). Thefollowing primaryantibodieswereused:polyclonalguineapig sections was performedaspreviously described(JamesandSchultheiss, Immunofluorescence microscopy onparaformaldehyde-fixed cryostat Immunohistochemistry and Eya1 2996 ␮ wire (positive electrode).Throughasmallholeinthevitellinemembrane,1 suspended inTyrode’s saline,ventral sidedown, above a1mmgaugeplatinum attached toadoughnut-shapedpaperring(P5,Fisher).Theembryo was were collectedatStage5oryounger(Hamburger andHamilton,1951) described (JamesandSchultheiss,2003;Wilm etal.,2004).Briefly, embryos Electroporation andcultureofchicken embryos was performedaspreviously Electroporation andinfectionofchickenembryos For retroviral expression studies,full-length promoter/enhancer, andwhichalsoexpresses al.,2001), whichdrives expression fromaCMV/chicken et Schultheiss, 2005)was clonedintothe For electroporationstudies,full-lengthchicken similar manner( RCAS-Gfp and titeredusingpreviously describedmethods(Fekete andCepko, 1993). retroviral particleswereharvested fromculturesupernatant,concentrated RCAS-Odd1 site ofthe above. situ hybridization,immunofluorescenceor 2005). Embryoswereharvested attheindicatedtimesandprocessedforin entering theegg, Tyrode’s was continuouslyadded tothehole;any eggs that subsequently removed using a microscalpel.Inordertoprevent airfrom Tyrode’s salinewas placedontopoftheshell membrane,whichwas 965A Fisher),whileleaving theunderlying membraneintact.Adropof 5 mmindiameter)was groundintotheshellusingabluntneedle(cat.#08- temperature for2hoursbeforemicroinjection. Asmallhole(approximately International (Elizabethtown, PA), wereplaced ontheirsidesatroom Unincubated fertilizedwhite-leghorn chicken eggs, obtained fromHy-Line were adaptedfromprevious work (Andachtetal.,2004;Sang,2004). NaCl (SundinandEichele,1992)]at38°Cfor24-48hours. side uponalbumin-agar culturedishes[50%Albumin, 1.5%Glucose,0.9% porator BTX-830(BTX).Subsequently, embryoswereincubatedendoderm- embryos werepulsedthreetimesfor25msat12Vusinganelectro-square was lowered above theembryountilitwas submerged inTyrode’s andthe embryo andthemembrane.A20 l ofDNA solution(0.6 g/ml proteinaseK(Roche),andhybridizedovernight withlabeledRNA Methods forinfectingunincubatedchicken eggs withretroviral vectors (Xu etal.,1999), knockout mice RESEARCH ARTICLE ␮ RCAS g/ml) at70°C.Thesectionswerethenwashed in2 retroviral particlesforcontrolexperiments wereproducedina vector was transfectedinto chickembryofibroblasts,and RCAS-Gfp expression vector (Fekete andCepko, 1993).Theresulting Six2 ␮ ␮ lacZ (Xu etal.,1999), g/ml, 2 plasmid was akindgiftfromCliff Tabin). g/ Odd1 ␮ lacZ l) was injectedintothespacebetween ␮ expression was adaptedfrom Lobeetal. m gaugetungstenwire(negative electrode) ϫ gene hasbeendescribed(Wang etal., histochemistry Wt1 SSC, 37°C)andwashed in0.2 pMES (Armstrong etal.,1993)using lacZ Gdnf Odd1 Gfp histochemistry asdescribed expression vector (Swartz (Pichel etal.,1996), was clonedintothe from anIRESelement. Odd1 ϫ lacZ (James and SSC (70°C), gene into ␤ ϫ -actin Sall1 SSC Cla I significance atthe0.05level bythetwo-sample Student’s standard error. Differences betweenthemeanvalues weretested forstatistical 7 shows themeanofallembryos analyzedandtheerrorbarrepresentsone standardize thetubular tissueandductareameasurements.ThegraphinFig. control forvariability ofembryosize,thesizenotochordwas usedto summed them.Ductareawas determinedinthesamemanner. Finally, to (groups ofcellscontainingpolarizednuclei)andlumenizedtubules and we calculatedtheareaofindividual Pax2-positive epithelialcondensates pixels. To calculatethetotalareaofalltubular tissuefromthesecondimage, borders, andthemeasurecommandwas executed tocalculateitsareain freehand drawing toolwas usedtotracealinethatencircledthenotochord calculate thetwo-dimensional areaofthenotochordfromfirstimage.The antibody. For standardization,the ImageJsoftware packagewas usedto and (2)a20 microscope withaSPOT camera:(1)10 For eachembryo,two imageswerecollectedusingaZeissAxiophot Quantification ofmesonephros, tubuleandductsize micromanipulator wereusedtoinject1-2 glass capillary(100-200 developed airbubbles werediscarded.Afterlocatingtheembryo,apulled mesenchyme adjacenttothetubules (Fig.1G). tubules (Mauchetal.,2000), is detectableinthedifferentiated epitheliumofthenephricductand complementary expression patternsinthemesonephros:while mesonephric tubule differentiation (Fig.1F,G). of nephricductdifferentiation (Fig.1E),andlaterduring during thedevelopment oftheurogenitalregion: initiallyattheonset mesenchymal components.Thisistrueatseveral different stages of kidney morphogenesisrevealed that expression isinitiated.Analysisofsectionsduringthe earlystages 2000; Obara-Ishiharaetal.,1999). pattern (Fig.1D)(seeJamesandSchultheiss,2003;Mauchetal., expressed later(HH9,Fig.1C),andinamorespatially restricted markers ofkidney differentiation, suchas mesoderm andthemediallateralplate(Fig.1B,E).Bycontrast,other thereafter, itsexpression patternbroadens toincludetheintermediate Hamilton, 1951)justlateraltoHenson’s node(Fig.1A).Shortly gastrulation atHamburger Hamilton(HH)Stage5(Hamburger and is initiatedintheintermediatemesodermimmediatelyafter before previously characterizedkidney genes.Transcription of embryos revealed that chicken andmouseembryos.Examinationofdeveloping chicken Odd1 cells Odd1 RESULTS glue-side down at38°Cuntilthey wereharvested foranalysis. The gluewas allowed tohardenfor5minutes,andtheeggs wereincubated The holewas thensealedwithhotgluefromaSuperbondergun(FPC). particles/ml) throughthecenterofembryointosubgerminalspace. 1M). Odd1protein isdetectableonlyinPax2-expressing cells that Pax2-expressing cellsthatmigratetoward theectodermallayer (Fig. nephric ductrudimentisformed, asevidenced byasubsetofthe medial partoftheurogenitalregion (Fig.1J).Subsequently, the before nephricductformation, Pax2 expression isinitiatedinthe differentiation was examined atsinglecellresolution.Immediately investigate thisideafurther, Odd1expression duringkidney tissue downregulated asthey undergo epithelialmorphogenesis. To that differentiated epitheliumatseveral stagesofdevelopment, suggested That Cellular morphogenesisbegins shortlyafterkidney-specific gene Odd1 expression inthedeveloping nephric systemwas examined in is expressed specificallyinkidneyprecursor Odd1 ϫ is expressed inkidney precursorsandsubsequently fluorescent imageoftheurogenitalridgestainedwithanti-Pax2 is presentinkidney mesenchyme,but absentin ␮ m indiameter),PicospritzerIIinjectorandLeitz Odd1 Odd1 is expressed innephrogenictissue ϫ ␮ is limitedtosmallregions of l ofretrovirus (10 Odd1 DIC imageoftheentiresection; Development 133(15) is expressed onlyinthe Pax2 Pax2 t -test. and , areinitially 7 -10 Odd1 8 infectious Odd1 Pax2 have

DEVELOPMENT mesenchyme and isabsentintheuretericbud (Fig.2D).Shortly left). Atthisstage, which bulges intothe metanephricmesenchymeprimordia(Fig.2C, Initially, theuretericbud formsasathickening ofthenephricduct, metanephric mesenchyme(reviewed byBard,2003;Saxen, 1987). develops via reciprocalinteractionbetweentheureterand kidney (Fig.2B).Starting atE10.5,themousemetanephrickidney mesonephric mesenchymethaninthetubules ofthe mesonephric embryos (Fig.1). Odd1 isrequired forkidneyformation similar tothelocalizationof localized totheintermediateandlateralplatemesoderm(Fig. 2A), locus (Wang etal.,2005). Atembryonicday(E)9.5, examined inembryosheterozygousfora that have organized intotubules (Fig.1O,P). Pax2-expressing nephrogenicmesenchyme,but isabsentincells Similarly, inthemesonephrosOdd1expression isdetectablein that Odd1isdownregulated incellsthathave begun todifferentiate. have notmigrateddorsally(compareFig.1I,Jwith1L,M),indicating Expression of LacZ Odd1 expression ismuchhigherinundifferentiated Odd1 during mousekidney development was is expressed inthemetanephric Odd1 transcript describedinchicken lacZ knock-in atthe lacZ Odd1 is the ureter(Fig.2Cmiddle,and thereafter, signalsfromthemesenchymeinduce branchingwithin the the kidney, micewereexamined thatcarryatargeted disruptionof In ordertodeterminewhether formation Odd1 and downregulated upontheinitiationofepithelialdifferentiation. metanephros formation, and chickembryos,duringnephricduct,mesonephros and comma/S-shaped bodies(arrow, Fig.2I).Insummary, inbothmouse cells thathave formedpre-tubular aggregates (arrow, Fig.2H)and I). Asinthemesonephros, the tubular tissuethatdifferentiates fromthismesenchyme(Fig.2E- mesenchyme thatsurroundsthebranchinguretericbud andnotin ureteric bud branching, ureteric bud trunks(yellow circles,Fig.2C,right).Atallstagesof tubular condensatesbegin toepithelializeadjacentthebranching Odd1 is required formetanephric mesenchyme locus (Wang etal.,2005).Themajority ofmicethatare Odd1 lacZ tubules. Atembryonicday5(G), but notinepithelializednephricductor derivatives, includingthedorsalmesentery, tubules andalsoinsplanchnicmesoderm mesenchymal cellsadjacenttomesonephric Stage 13(F), but isnotfoundinthenephricduct.At mesoderm andextendsintothelateralplate (E) (arrow). ( and intherudimentarysomaticgonad expressed inthecoelomiclining(arrowhead) in situhybridizationfor Hensen’s node;t,tubules. intermediate mesoderm;lp,lateralplate;n, detail from OandP. d,nephricduct;im, tubules themselves.InsetinPshowsmerged the tubules,butnotinPax2-expressing expressed inmesenchymalcellsadjacentto developing mesonephros, Odd1protein is merged detailfrom LandM.( no longerexpresses Odd1.InsetinMshows (arrow) andcontinuestoexpress Pax2but rudiment hasbeguntobulgedorsally and J.ByStage10(K-M),thenephricduct Odd1. InsetinJshowsmergeddetailfrom I Pax rudiment ismorphologicallydetectable,most rudiment. AtStage9(H-J),before theduct protein duringformationofthenephricduct respectively. ( indicate nascent posterior directions. Arrows inAandC broadly than hybridization for hybridization for earlier than Stage 11(D).Notethat Fig. 1.Expression of Stage 9(B)chickembryos.( embryo. Odd1 2 Odd1 -expressing cells(arrow) alsoexpress is detectableonlyinthecondensing is expressed inkidney precursorcells Odd1 lacZ H-M is expressed intheintermediate ( A expression isdownregulated in RESEARCH ARTICLE Pax2 , localization Fig.2E,F).Lastly, is requiredfordevelopment of B E-G Odd1 Pax2 Expression ofOdd1andPax2 ) ) Whole-mountinsitu , andthatitextendsmore Odd1 ) Sectionsofwhole-mount Pax2 Odd1 in lateral,anteriorand is expressed in at Stage9(C)and Odd1 and in HHStage5(A)and Odd1 Odd1 Pax2 C , . AtStage11 in thechick D N-P is expressed ) Insitu expression, Odd1 ) Inthe is 2997

DEVELOPMENT ureter epithelium;m,mesonephricmesenchyme;mm,metanephrics,ureteric stalk;t,mesonephrictubules;u,ureteri bodies (I,arrow). The At E13.5( adjacent totheventralaorta(arrow inE). Odd1 stalk, butnotinureteric budderivatives. mesenchyme surrounding theureteric of themetanephros andatlowerlevelsin undifferentiated condensedmesenchyme ( mesenchyme butnotintheureteric bud. Odd1 (yellow). Seetextfordetails.( and formationofepithelialvesicles continued branchingoftheureteric bud branching oftheureteric bud;(right), mesenchyme (blue);(center)initial bud (white)intothemetanephric formation: (left)invasionoftheureteric mesonephros ( mesoderm ( intermediate andpartofthelateralplate locus. AtE9.5, galactosidase geneinsertedintothe ( absent from themesonephrictubules. is absentfrom thenephricductandlargely the coelomicliningandmesenchyme,but embryos heterozygous forthe mouse embryo. Fig. 2.Expression of 2998 1987). Asuretericbud defectswereobserved in The uretericbud formsasanoutcroppingofthenephric duct(Saxen, mutant embryos Nephric ductandmesonephros defectsinOdd1 required specificallyformetanephricmesenchymeformation. epistatic toEya1andastheearliestexpressed factor known tobe (Sajithlal etal.,2005).ThedatareportedhereimplicateOdd1 as mutants have similardefectsinmetanephricmesenchymeformation 3 anddatanotshown). Previous reports have shown thatEya1 of Six2,Eya1,Gdnf,Pax2 andSall1inthemetanephricregion (Fig. et al.,2001).EmbryosmutantforOdd1completelylackexpression (Dressler etal.,1990;Torres etal.,1995);andSall1(Nishinakamura et al.,1996;PichelSanchez1996);Pax2 (Fig.3H) Eya1 (Fig.3E)(Kalatzisetal.,1998;Xu1999);Gdnf(Moore ureteric bud outgrowth, including:Six2(Fig.3C)(Xuetal.,1999); development areexpressed inthemetanephric mesenchymebefore homozygous mutantembryos(Fig.3A,B). metanephric mesenchymecondensationarecompletelyabsentinthe type and already invaded thecondensedmetanephricmesenchymeinwild- abnormalities (Wang etal.,2005).AtE11.5theuretericbud has homozygous forlossof 4A,B), nephric ductprimordia(Fig.4A,B). In nephric duct(Kobayashi etal.,2005),isaspecificmarker forthe analyzed. AtE9.5, embryos (Fig.3B),theearlierdevelopment ofthenephricductwas sections of heterozygous controlembryos (Fig.4C,D).Analysisofserial at E9.5relative tothenumber seenincontrolembryos(Fig. 4B,D), E C , Schematicofeventsinmetanephros ) F In wild-typeembryos,several factors requiredforkidney ) AtE11.5, is alsoexpressed inmesenchyme is expressed inthemetanephric RESEARCH ARTICLE Lim1 G-I Odd1 Odd1 A ), ). Inasectionthrough the is expressed, but atsignificantlylower levels thanin B Odd1 Odd1 Odd1 ), heterozygous embryos,but theuretericbud and mutants demonstratedthatfewer cellsexpress LacZ lacZ Lim1, continues tobeexpressed inthecondensedmesenchyme,butnotpretubular aggregates (H,arrow) or comma-shaped is expressed inthe is expressed inthe lacZ Odd1 staining ispresent in staining ofmouse Odd1 staining intheureter epitheliuminGisnon-specific.a,aorta;cm,condensedmesenchyme;d,nephricduct;du, which isrequiredforformation ofthe in the D ␤ ) AtE10.5, function dieatE11.5duetocardiac - Odd1 Odd1 mutant embryos(Fig. Odd1 mutant Lim1 performed onsectioned mouseembryos.AtE8.5, thenephricduct in thedeveloping meso-andmetanephros,TUNELassayswere In ordertodeterminewhetherapoptosis was contributing tocellloss Odd1 Gene expression defectsprecede apoptosisin significantly smallerthanincontrol embryos. tubule (Fig.4H),althoughthenumberandsizeof tubules was regionalization ofPax2 andWt1expression withintheductand Wt1 (arrow Fig.4G). Pax2 (Fig.4G),whiletheproximaltubule coexpresses Pax2 and and Pax2. Normally, the ductandthedistaltubule express only immunofluorescence was usedtolocalizetheexpression ofWt1 tubules in Sainio etal.,1997).Inordertodeterminewhetherthemesonephric tubules formviadifferent mechanisms(Kobayashi etal.,2005; supporting thesuggestionthatanteriorandposteriormesonephric and whole-mountinsituhybridizationfor mesonephric tubules formin separate fromtheduct(Sainioetal.,1997).Onlyanterior thenephricduct,andposteriortubules, whichremain to fused tubule forminmouseembryos:anteriortubules, whichare mutants (Fig.4F, arrowhead). Normally, two typesofmesonephric does inwild-typeembryos(Fig.4E,arrow pointstocloaca). metanephric mesenchymein showed thatthenephricductdoesnotmigratetoward thecloacaand formation. Visualization ofembryosstainedfor nephric ductarepresentfromclosetotheinitiationof was alsoseenatE8.5(datanotshown), indicating thatdefectsinthe diameter ofthenephricductasassessedby gaps inthe A smallnumberofmesonephrictubules differentiate in mutants Odd1 Odd1 mutant nephricducts(Fig.4C,D).Decreased null embryosexhibit normalpatterning, Odd1 Odd1 mutant embryosexhibited normal Odd1 mutants (Fig.4F)asitnormally mutant mice(Fig.4F), Lim1 Lim1 Development 133(15) and revealed significant Pax2 Pax2 at E10.5also expression c bud. Odd1

DEVELOPMENT nephric duct;mm,metanephricmesenchyme;u,ureteric bud. outlined withadottedline(D,F,I). ThefluorescenceinJisautofluoresc;ence intheupperrightcorner from bloodcellsinthe the metanephricmesenchymeregion of the metanephricmesenchymeof immunofluorescence (G-J)fortheindicatedgeneproducts (DapinuclearstainsinGandIcorrespond tosectionsinHandJ,resp the often present (onrightsidebutnotleftinB),smallerthannormal.Theregion betweentheaortaandnephricductisalso metanephric mesenchymeare seen.Homozygousnullembryos(B)completelylackaureteric budandcondensedmesenchyme(arrow). A heterozygous andhomozygousnullembryosstainedwithhematoxylineosin.Inheterozygotes (A),anephricduct,ureteric bud gene expression defects observed in that celldeathcannotexplain theinitialmetanephricmesenchyme mesenchymal Pax2 inFig. 5Dand mesenchyme geneexpression isnormallyinitiated(seeabsenceof Odd1 Therefore, apoptosisbegins in in metanephric mesenchymeprecursorcellsatE9.5aresimilarly low is initiatedbyE9.5(seealsoFig.5C,E).Thelevels ofapoptosisin Odd1 isrequired forkidneyformation (Bouchard etal.,2002;Dressler1990) mutant embryos. contributor totheobserved mesonephricsizedecreasesin indicating thatincreasedprogrammedcelldeathisaprobable surrounding themesonephrictubules (compare Fig.5A,B), exhibit significantinappropriateapoptosisinthemesenchyme mesonephric mesenchyme(Fig.5A,arrowhead). some normalapoptosisattheborderofproximaltubule andthe to thoseobserved incontrols(datanotshown). AtE9.5,thereis apoptosis inthemesonephricregion ofmutantembryos aresimilar is alreadythinnerthanincontrolembryos,whilethelevels of of Fig. 3.Themetanephricmesenchymefailstoformin 2002) Expression ofthemetanephricmesenchymegenes Odd1 Odd1 Odd1 , Eya1 is requiredautonomously fornormalgeneexpression inthe mutant embryos,asassessedbyLim1andPax2 expression, null embryos.( mutant andcontrolembryos (compareFig.5C,D). Sjtlle l,20)and (Sajithlal etal.,2005) C-J ) Sectionsthrough themetanephricregion ofmouseembryosstainedbyinsituwhole-mounthybridization(C-F)or Odd1 Odd1 Odd1 Eya1 mutant miceaftermetanephric heterozygous embryosexpresses Odd1 Wt1 mutant embryos,andthat in Fig.5F).Thisimplies (Armstrong etal.,1993) null mutants(D,F,J). Themutantnephricductdoesexpress Pax2(J).Theductinmutantembryosis , Pax8 (Bouchard etal., Odd1 Odd1 mutants –/– Odd1 Pax2 embryos. Six2 and thedetectionofmany nephrogenic genes,asevidenced byexpression ofWt1(Fig.5I,J) metanephric region of E10.5, therearestillmany mesenchymalcellsinthemeso/ the levels ofapoptosisincreaseinthemetanephricmesenchymeat metanephric sizedecreasesobserved in mesenchyme byE10.5(Fig.5G,H),andthismaycontribute tothe metanephric mesenchyme.Apoptosisdoesincreasein (8/16 embryos;seeFig.6B,C,E). Ectopic Morphologically, ectopic embryos, specificallytargeting kidney andsomite precursors. Gfp promoteexpression ofkidney-specific genes,an to mesenchyme-specific factors. To testif precursor cellsandisrequiredforexpression ofmany metanephric As describedabove, Odd1 promoter activity (Fig.5K,L). embryos; Fig.6A,B)and expression ofthekidney transcriptionfactors Pax2 (11/14 Lim1 didnot occur inallcellsthatwere electroporated. In cells within24hoursofelectroporation. Upregulation ofPax2 and (C), ( A , B plasmid was electroporatedintoHH4(gastrula)chick Eya1 ) Sectionsthrough themetanephricregion ofE11.5 promotes kidneyprecursor geneexpression (E) andPax2(H).Allthree geneproducts are absent from Odd1 Lim1 lacZ Odd1 Odd1 is expressed innon-epithelialkidney -positive cellsexhibiting intact (4/5 embryos;Fig.6D,E)insomitic often disruptedsomiteformation mutant embryosthatexpress RESEARCH ARTICLE Odd1 aorta.a,aorta;d, Odd1 significantlysmallerin mutants. Even after ectively). AtE10.5, Odd1 could function andcondensed Odd1 Odd1-IRES- promoted ductis mutant Odd1 2999

DEVELOPMENT well asinthemesenchyme andcoelomiclining.d, nephricduct;t,tubules. control embryos,Pax2isexpressed inthenephricductandtubules,whileWt1isexpressed intheproximalmost partofthe immunofluorescence against theindicatedantigens.Tubules are smallerinmutant(H) compared withcontrol (G)embryos.Inboth plate where absence ofkidney geneexpression inthemedialpartoflateral (arrows inFig.6B,C) express Pax2 particular, Gfp-labeledcellspresentinthelateralplatedidnot 3000 normally toward themidline attheposteriorend(arrows inE,F).( the nullmutants(F, arrowhead), but fewerthanintheheterozygotes (E,arrowheads). In region. Inhomozygousnull embryos,thenephricductisthinnerandofteninterrupted(C,left arrowhead inD).Afewmesonephr Fig. 4.Effects ofloss with the Odd1 expressing chicken mesonephroiwereinfectedwithRCASretrovirus of structures (Figs1,2).Inordertodeterminewhetherdownregulation intermediate mesodermderivatives differentiate intoepithelial We have foundthat Odd1 expression iscontext-dependent. served ascontrols. aggregates withpolarizednuclei. as Pax2-positive cellseitherinepithelialized tubules orincellular distinct lumen,whilethebroaderterm‘tubular tissue’was defined tubules’ weredefinedasPax2-positive cellsarrangedarounda for Pax2 byimmunofluorescence.For analysis,‘epithelialized adjacent sectionswerestainedfor (C,D,F) mouseembryosstainedby whole-mountinsituhybridizationfor indicates thattheabilityof Odd1 . Unincubated(pre-gastrula)chicken embryoswereinfected RESEARCH ARTICLE represses kidneytubuledifferentiation RCAS-Odd1 is requiredfornormalkidney tubule differentiation, Odd1 Odd1 , whichallows forthestableectopicexpression of is normallyexpressed (Fig.1E,I,L) retrovirus and,afterincubationfor4-5days, Odd1 Odd1 on nephricductandmesonephric tubuledevelopment. Odd1 is normallydownregulated as Odd1 RCAS-Gfp . This isconsistentwiththe to promotekidney gene by insituhybridizationor -infected embryos G , , H and ) Sectionsthrough themesonephric region ofE10.5embryosstainedwith Lim1 reflects areducedabilityof epithelial cellscannotbeduetoinfectionartifact andprobably duct andepithelialtubules (Fig.7A),thelackof 7D, outline).As typically formedfromadjacentnon- duct orepithelializedtubules (Fig.7D),whileductandtubules cells thatexpress ectopic tubules. organization ofprecursorsintotubular tissueandepithelialized Odd1 not shown). Together, thesedataindicatethatectopicexpression of RCAS-Odd1 indicating thatthedecreaseinareaoftubular epitheliumin seen inthemesonephricregions of embryos (Fig.7H).Increasedlevels ofTUNELstainingwerenot (Fig. 7G, Odd1 7B,C). Theaverage total‘tubular tissue’areapersectionin Odd1- immunostaining showed thattherewas lesstubular tissueoverall in tubules. Second,visualizationofurogenitalridgesbyDICorPax2 epithelium was similarincontroland ( Several observations emerged fromtheseexperiments. First, A-D -infected embryoswas only49%ofthatcontrolembryos can promotekidney precursorgeneexpression, but inhibits ) or infected embryosthanincontrols(compareFig.7E,Fwith Pax2 P Odd1 <0.005). Interestingly, thesize ofthenephricduct -infected embryoswas notcausedbyapoptosis(data ( E Odd1 , mutants, thenephricductalsodoes notturn RCAS-Gfp F ). BandDshowsectionsthrough themesonephric heterozygous (A,B,E)andhomozygous null Odd1 Odd1 efficiently infectedthemesonephric did notdifferentiate intonephric -expressing cellstoformductand RCAS-Odd1 Odd1 -expressing cells(Fig. Development 133(15) RCAS-Odd1 tubule (arrow), as infected embryos, ic tubulesformin mutant and Odd1 -infected infected RCAS-

DEVELOPMENT mutant embryos( shown in anterior mesonephrictubules,levels ofapoptosisare signiﬁcantlyelevatedin with heterozygotes (G).At E10.5, expression isnormal( axial level,Pax2(D)and contrast, levelsofapoptosisinE9.5 metanephricmesenchymeprecursor cellsare lowin both null ( al., 1993)and et al.,1996), et al.,1995), in mutationsof been previously describedinotherloss-of-function studies,suchas defect inthedevelopment ofthemetanephricmesenchymethanhas and Pax2 mesenchyme rudimentarenever expressed, including Odd1 ureteric bud andthemetanephricmesenchyme.Inabsenceof The metanephrickidney develops frominteractionsbetweenthe formation Odd1 DISCUSSION Odd1 isrequired forkidneyformation kidneys (Wang etal.,2005). beyond E11.5have nomorphologicallydetectablemetanephric consistent withthereportthatrare formation ofthemetanephricmesenchyme.Theseﬁndingsare These dataimplicate metanephric mesenchyme(Sajithlaletal.,2005;Xu1999). which hasalsobeenshown tobecrucialforthe formation ofthe Additionally, wehave shown herethat and expression ofatleastasubsetmetanephricgenes. which show someevidence ofmetanephricmesenchymeformation Fig. 5.Timingofapoptosisandlossnephricgeneexpression in B , many ofthegenesthatmarkearlymetanephric , D is required formetanephricmesenchyme , H K ) embryoswere analyzedby TUNELtodetectapoptoticcellsandstainedwithantibodiesagainst Pax2.Inthemesenchymesurround (Fig. 3).Absenceof ). a,aorta;d,duct; m,nephricmesenchyme;mm, metanephric mesenchyme;t,tubule; u,ureter. Gdnf Sall1 Wnt4 Six1 L ) exhibitsubstantiallevelsof (Moore etal.,1996;PichelSanchez (Nishinakamura etal.,2001), E (Kispert etal.,1998;Stark1994),allof (Li etal.,2003;Xu2003), , F Eya1 ). ByE10.5, Odd1 (F) expression inprospective metanephricmesenchymeare absentinmutantembryos, whiledermamyotomal as oneoftheearliestregulators of Wt1 Odd1 Odd1 is expressed inthemesenchyme ofbothmutant( Odd1 results inamoreprofound null mutants(H)haveincreased levelsofapoptosisinthemetanephricmesenchyme(asterisk) compared Odd1 null embryossurviving Odd1 is epistaticto Wt1 promoter activity, asdetected by (Kreidberg et Pax2 Eya1, Six2 (Torres Eya1 , Odd1 and after thetimethatearlymetanephricmesenchymemarkers region in from thisexplanation. Whilethereisapoptosisinthemetanephric mesenchyme precursorcells.Several linesofevidence pointaway expression, andthatsuchapoptosiseliminatesallmetanephric apoptosis beforetheonsetofmetanephricmesenchymegene would give risetothemetanephricmesenchymeundergoes expression. explain theeffects of where mesenchyme, asseenforexample inthe is notrequiredforinitialexpression ofgenesinthemetanephric metanephric mesenchyme(Saxen, 1987).However, thenephricduct problems intheinductive interactionsbetweenuretericbud and duct anditsderivative, theuretericbud, whichwould leadto expression aresecondarytodefectsintheformationofnephric One possibilityisthatthedefectsinmetanephricmesenchymegene of metanephricmesenchymegeneexpression inthe Thus theeffects of into thisregion oftheembryo(Groteetal.,2006;Sainio,2003). the metanephriczonedespitefailure ofthenephricducttomigrate Odd1 Another possibilityisthat,intheabsenceof There areseveral possiblealternative explanations fortheabsence mutant embryos. Eya1 Pax2 mutant (B,asterisk)compared withcontrol embryos(A).By Odd1 begin tobeexpressed (Fig.5).Also,abundant cellswith lacZ J -expressing metanephricmesenchymecellsarefoundin , arrowhead) andwild-type ( expression drivenbythe null mice,theapoptosisbegins afterE9.5,whichis Odd1 Odd1 Odd1 mutants (D)andcontrols (C). Atthisstageand Odd1 heterozygous ( on nephricductdevelopment cannot loss onearlymetanephricgene RESEARCH ARTICLE Odd1 I ) embryos.Similarly, A Gata3 , C promoter (heterozygote , G Odd1 ) andhomozygous knockout mouse, Odd1 , thetissuethat null mice. Odd1 Eya1 3001 ing Pax2

DEVELOPMENT expressing cellsarepresentinthemetanephricregion of normally express the initiationofapoptosis(Fig.5L),indicatingthatmany cellsthat Odd1 3002 nevertheless significantthat afew anteriortubules formin mutants isgreatlyreduced compared withwildtype,itis pathways. apoptosis alsooccursinmouselinesmutantfor proceed toexpress otherkidney markers. Metanephricmesenchyme potential exist intheabsenceof mice (Fig.5J),furtherindicatingthatcellswithnephrogenic suggesting aninteractionof consistently moresevere ontheleftsidethanright, (Wang etal.,2005)have reportedthat thenephricductdefectsare nephric ductmaintenance(Obara-Ishiharaetal.,1999).Wang etal. Odd1 precursor cells,ortodefectsininteractionsbetweentheduct and seen in in thenephricductitself(Fig.1).Thus,defects in nephricductprecursorsandnephrogenicmesenchyme,but not Pax2/8 nephric ductitself,suggestingacell-autonomousrequirement for tubule formationin Defects innephricductandpro/mesonephric of metanephricmesenchyme. these datasupportanessentialrolefor as oftheseothermetanephricmesenchymegenes.Taken together, E9.5 couldbeduetoacombinationoftheabsence et al.,2003).Theapoptosisseeninthe Eya1 those ofdoublemutantsfor However, Nishinakamura etal.,2001;Xu1999;2003). morphologically normalducts(Kreidberg etal.,1993; including severe thanthatseeninmutantsforanumberofotherkidney genes, (Fig. 4).Thenephricductphenotypein duct andsomepro/mesonephrictubules doformin By contrasttothecompleteabsenceofmetanephros,nephric all (Bouchardetal.,2002). While thenumberofmesonephric tubules thatformin (Kreidberg etal.,1993;Torres etal.,1995;Xu 1999;Xu -expressing mesenchyme,whichmaybenecessaryfor promoter activity arefoundin RESEARCH ARTICLE Odd1 activity inthenephricduct.Bycontrast, Odd1 Eya1 mutant micecanbeattributed eithertodefectsinduct , mutant nephricductdefectsarelesssevere than Odd1 Six1 , survive intheabsenceof Wt1 Odd1 Odd1 Pax2 Pax2 and Odd1 and activity withlaterality regulation null nice and Odd1 Sall-1 Odd1 Odd1 Pax8 , even ifsuchcellsdonot Pax8 null miceatE10.5,after Odd1 , whichhave noductat , allofwhichhave null metanephrosafter in theinitialformation are expressed inthe Pax2 Odd1 Odd1 mutants ismore Odd1 , Wt1 Odd1 . Many Wt1- is expressed Odd1 , null mice Six1 as well Odd1 Odd1 null and but do notadoptanepithelialmorphology. Indeed, region (Fig.7). expression istheinhibition oftubule formationinthemesonephric seen. Rather, thepredominanteffect oflong-termectopic 4 to5days,relatively littleectopicexpression of ectopically viaaretroviral vector, andembryosareexamined after mesoderm (Fig.6).However, when transiently expressed ectopicallybyelectroporationintheparaxial Odd1 a laterstageintubule differentiation orinductformation. in (Dressler etal.,1990).Thus,thelackofallpro/mesonephrictubules Odd1 (Brophy etal.,2001;Torres etal.,1995).However, than thatseenin Odd1 of themetanephros. such as both mesonephricandmetanephrictubule formation,whilegenes et al.,1997).In duct andposteriortubules thatremainunfusedwiththeduct(Sainio tubules develop inmice:anteriortubules thatfusewiththenephric mutant mice.Ithasbeenreportedthattwo typesofmesonephric suggests that Sajithlal etal.,2005;Xu1999;2003).This normal mesonephrictubule formation(Nishinakamuraetal.,2001; and posterior mesonephrictubule formation.Mutantsfor mesenchyme (Fig.4),whichwould explain thesevere defectsin mesenchymal Pax2 expression orformationofthisnephrogenic nephric duct.In of pro/mesonephric tubules. function isnotrequiredfortheformationofthisanteriorgroup Nakamura, 2003;Sainioetal.,1997).Ourdataindicatethat or bydirectextension fromthedeveloping duct(Hirumaand simultaneously withthenephricductfromcommonprecursorcells suggestedthattheanterior, fusedtubules differentiate been al., 1997),onlytheanterior, fusedtubules differentiate. Ithas Pax2 The By contrast,posteriormesonephrictubules develop fromaband Pax2 Sall1 , isexpressed intubules themselves and inthenephricduct can activate thetranscriptionof Odd1 may promote anephric precursor state Eya1 mutants mayreflectanautonomousrequirementfor -expressing nephrogenicmesenchymeadjacenttothe , whicharerequiredformetanephrosdevelopment, have , Odd1 mutant mesonephrictubule phenotypeislesssevere Six1 Odd1 Odd1 Pax2 and is requiredforcommonprocessesthatunderlie Odd1 -expressing mesonephric cellsexpress mutants, aswellin mutants, whichhave nomesonephrictubules Sall1 mutants, thereisnoevidence for are requiredspecificallyforformation hybridization for whole-mount insitu Pax2 (A,B)orGFP(C),by immunofluorescence against stained with neural tube;s,somite. intermediate mesoderm;nt, not express ectopicPax2.im, the lateralplate(arrows) do B andC,notethatGFPcellsin region (arrowheads inB,E).In Lim1 Ectopic cellsexpressing Pax2or expression vector( electroporated with Fig. 6. embryos ( somites. ectopically expressed inthe early nephricmarkerswhen Odd1 are foundinthesomite Pax2 Development 133(15) Wt1 Odd1 is stablyexpressed A Control chicken , mutants (Sainioet D and Pax2 can induce ) andembryos Lim1 Pax2 Lim1 B Eya1 , or Mes-Odd1 C , (D,E). E , unlike Lim1 Pax2 ) were Odd1 , Odd1 when Pax2 Six1 at is -

DEVELOPMENT standard error of8 significantly smallerthanincontrol embryos( Pax2-expressing cellsarrangedeitheraslumenizedepitheliaoraggregates withpolarizednuclei)in detection conditionswere calibratedtodetectonlyectopicandnotendogenous RCAS-Odd1 E,F), butdocontainsignificantnumbersofPax2-expressing non-epithelializedcells(arrow inF).Whenepithelializedtubulesa immunofluorescence forPax2(F).Themesonephroi of analyzed bysectioninsituhybridizationfor 0 andharvestedatday5,showingextensiveinfection(A)robust mesonephrictubuleformation(A-C).( degree ofectopic it, therebyblockingsubsequenttubule differentiation. Thelow fate. promotes theestablishmentofa Odd1 isrequired forkidneyformation expression pattern,as longer-term ectopicexpression of (Fig. 7).Oneway toreconciletheresultsofshort-term versus expressing cellsareselectively excluded fromepithelialtubules Fig. 7.Expression of mesoderm. maintain kidney geneexpression inregions outsidetheintermediate many genecopiespercell)ortotheabsenceoffactors neededto one copy ofthegenepercell,whileelectroporationcan introduce to levels ofexpression (theRCASretroviral vector integrates only embryos comparedwiththeelectroporatedcouldbe due work, itwill beimportanttounderstandthe molecularmechanisms but isdownregulated upontubule formation(Figs1, 2). Infuture structures. Sucharolefor necessary fordifferentiation ofnephrictissueintoepithelial of earlykidney geneexpression, downregulation of These datasuggestthatwhile Odd1 may beabletobothproducesuchastateandmaintain infected embryos,thetubulestendtobeuninfected(D),whereas tubulesare readily infectedbycontrol RCAS-Gfp Pax2 Odd1 Odd1 expression intheretrovirus-infected inhibits kidneytubuleformation. and 10 is expressed inallnephric precursors Odd1 Odd1 Pax2 Odd1 RCAS-Odd1 is consistentwiththe is necessaryfortheactivation -positive nephricprecursor is topostulatethat Odd1 G ), butthenephricductsare approximately thesamesize( to documentextentofinfection(D),byNomarskimicroscopy (E)andwith samples. The RCAS-Odd1 Odd1 may be y Odd1 Odd1 -axis isinarbitraryunits.d,nephricduct;t,tubule. ( A-C infected embryoscontainfewerepithelializedtubules(compare B,Cwith ) Representative control chickenembryosinfectedwith Andacht, T., Hu, W. andIvarie,R. References expression plasmids,andD. Herzlingerforproviding animproved Kreidberg, R.Maas,Nishinakamura andC.Tabin forsharingprobes and We gratefullyacknowledge A.Bober, P. Danielian,M.Goulding, T. Jessell,J. expression andinhibitsepithelialdifferentiation. well asthemechanismsthroughwhich by which Armstrong, J.F., Pritchard-Jones, K.,Bickmore, W. A.,Hastie,N.D.andBard, (NIDDK) toT.M.S., andR01DE013681(NIH/NIDCR)toR.J. was supportedbygrantsR01DK59980andDK71041from theNIH Predoctoral Fellowshipaward from theNationalScienceFoundation.Thiswork from theHoward HughesMedicalInstitute,andC.N.K.issupportedbya discussions andsuggestions.R.G.J.wassupportedbyaPredoctoral Fellowship and SashaPetrova forcritical reading ofthemanuscriptandformanyhelpful protocol. We alsothankMozhganAfrakhte,IainDrummond,DorisHerzlinger Bard, J. B. L. windowing eggsaccessingthestageX chicken embryo. mammalian embryo. J. B. 31-34. (1993). The expression of the Wilms’ tumourgene,WT1,inthedeveloping (1993). Theexpression oftheWilms’ Odd1 Odd1 (2003). TheMetanephros. In message). Thearea oftotaltubulartissue(definedas is downregulated duringepitheliumformation,as Mech. Dev. RCAS-Odd1 40 H (2004). Rapidandimproved methodfor ). Bargraphsshowmeanand , 85-97. D-F The Kidney:FromNormal Development ) RESEARCH ARTICLE RCAS-Odd1 infected embryosis Odd1 RCAS-Gfp regulates kidney gene Mol. Reprod.Dev. infected embryos re present in RCAS-Gfp virus (A)(insitu lacZ staining at day 3003 69 ,

DEVELOPMENT Fekete, D.M.andCepko,C.L. Hamburger, V. andHamilton,H.L. Dressler, G.R.,Deutsch,U.,Chowdhury, H.O.andGruss,P. K.,Nornes, Bates, C.M.,Kharzai,S.,Erwin,T., Rossant,J.andParada,L.F. Kreidberg, J.A.,Sariola,H.,Loring,M.,Maeda,Pelletier, J.,Housman, 3004 Hiruma, T. andNakamura,H. Herbrand, H.,Guthrie,S.,Hadrys,T., Hoffmann, H.H.,Rinkwitz- S.,Arnold, Grote, D.,Souabni,A.,Busslinger, M.andBouchard, M. Fujii, T., Pichel,J.G.,Taira, M.,Toyama, R.,Dawid,I.B.andWestphal, H. Cho, E.A.andDressler, G.R. Burrill, J.D.,Moran,L.,Goulding,M.D.andSaueressig, H. Brophy, P. D.,Ostrom, L.,Lang,K.M.andDressler, G.R. Bouchard, M.,Souabni,A.,Mandler, M.,Neubuser, A.andBusslinger, M. Lobe, C.G.,Koop,K.E.,Kreppner, W., Lomeli,H., Gertsenstein,M.and Li, X.,Oghi,K.A.,Zhang,J.,Krones, A.,Bush,K.T., Glass,C.K.,Nigam,S. Kobayashi, A.,Kwan,K.M.,Carroll, T. J.,McMahon,A.P., Mendelsohn,C.L. Kispert, A.,Vainio, S.andMcMahon,A.P. Kalatzis, V., Sahly, I.,El-Amraoui,A.andPetit,C. James, R.G.andSchultheiss,T. M. 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