that PDS5Bandthecohesincomplexhaveimportantfunctionsbeyondtheirroleinchromosomaldynamics. *Author forcorrespondence (e-mail:[email protected]) Avenue, StLouis,MO63110,USA. Neurological Disorders, Washington UniversitySchoolofMedicine,660SouthEuclid the discovery thatmutationsin developmental functionhasrecentlybeen extended tohumanswith (Dorsett etal.,2005;Rollins2004).Thisadditional blocking oflong-rangeenhancerandpromotercommunication developmental regulators through attenuatingcohesin-mediated B) regulate transcriptionof Drosophila (Benard etal.,2004;Seitan2006;Watrin etal.,2006).In guides cellmovement andaxonaloutgrowth duringdevelopment SCC4 example, its regulatory factors alsoplayimportantrolesindevelopment. For Haering, 2005).Inadditiontochromosomaldynamics,cohesinand PDS5, SCC2,SCC4andECO1(Hartmanetal.,2000;Nasmyth SCC3, andcohesinactivity isregulated byitsregulatory factors, complex consistsofcorecomponents,SMC1,SMC3, SCC1and conserved multi-proteincomplex, calledcohesin.Thecohesin segregation duringcelldivision andismediatedbyahighly phase untilonsetofanaphase,isimportantforaccuratechromosome Sister chromatidcohesion,thelinkageofsisterchromatidsfromS INTRODUCTION Accepted 22June 2007 Departments of PDS5,APRIN,SisterchromatiddeLangesyndrome, cohesion,Congenitaldefects,Cornelia Primordial germcells,Mouse KEY WORDS: anomalies of cells anddetectedhighPDS5Bexpressioninpost-mitoticneuronsthebrain.Theseresults,alongwithdevelopmental many ofwhicharesimilartoabnormalitiesfoundinhumanswithCdLS.Unexpectedly, wefoundnocohesiondefectsin limbs, distalcolonaganglionosis,abnormalmigrationandaxonalprojectionsofsympatheticneurons,germcelldepletion, shortly afterbirth.Theyexhibitedmultiplecongenitalanomalies,includingheartdefects,cleftpalate,fusionoftheribs,s delineate thephysiologicalrolesofPDS5Binmammals,wegeneratedmicelacking(APRIN). Mutations incohesionproteinsareassociatedwiththedevelopmentaldisorderCorneliadeLangesyndrome(CdLS)humans.To PDS5B isasisterchromatidcohesionproteinthatcrucialforfaithfulsegregationofduplicatedchromosomesinlowerorgani Bin Zhang Lange syndrome developmental abnormalitiesreminiscentofCorneliade Mice lackingsisterchromatidcohesionproteinPDS5Bexhibit (2007)doi:10.1242/dev.005884 Development 134,3191-3201 DEVELOPMENT ANDDISEASE arc Y. Jay Patrick division, Tonkin etal.,2004). (Deardorff etal.,2007;Krantz2004;Musio2006; growth delay, limb reduction,genitalanomaliesandcardiacdefects anomalies, includingdysmorphicfacial features,mentalretardation, (CdLS). CdLSpatientspresentwithavariety ofdevelopmental responsible for~50%ofcasesCorneliadeLangesyndrome , notonlyparticipatesinsisterchromatidcohesion,but also 4 Molecular BiologyandPharmacology, Mau-2 , mutantsofcohesionfactors (e.g.SMC1andNIPPED- 1,2 1 Genetics, Pds5B , SanjayJain 1,5 , arecentlyidentified metazoanhomologofyeast and Jeffrey Milbrandt –/– 2 Pathology andImmunology, mice, thepresenceofaDNA-bindingdomaininPDS5Bvertebratesanditsnucleolarlocalization,suggest 3 , HaengseokSong cut NIPBL and othergenesencoding 5 Pediatrics, and , SMC1A 3 Medicine andRenal 2,6, * 6 HOPE Centerfor and 2 , MingFu SMC3 are 4,5 , RobertO.Heuckeroth vertebrates, therearetwo homologsof SCC2) (Neuwald andHirano,2000;Panizza etal.,2000).In is presentinothercohesinsubunits andregulatory factors (i.e. repeats, amotifthatisinvolved inprotein-proteininteractionsand al., 1999).TheN-terminalregion ofPDS5containsseveral HEAT meiosis (Hartmanetal.,2000;Panizza etal.,2000;van Heemstet 2001) toensurepropersisterchromatidcohesionduringmitosisand SMC1, SMC3,SCC1andSCC3(Losadaetal.,2005;Tanaka etal., genetically orphysicallywiththecohesionfactors SCC2,ECO1, PDS5B during wingdevelopment (Dorsettetal.,2005).Inhumansbecause and SMC1,PDS5regulates transcriptionofthe developmental .For example, similartoSCC2/NIPPED-B PDS5 alsoappearstodirectlyregulate transcriptionof a numberof Interestingly, inadditiontoitsregulation ofchromatidcohesion, that bothcontribute tocohesiondynamics(Losadaetal.,2005). association ofCdLS withmutationsinseveral different cohesion itself isacriticalregulator ofmultipleaspectsorganogenesis. The these resultsindicatethatPDS5B andperhapsthecohesincomplex system formation,anddepletion ofprimordialgermcells.Together, nervous system(ENS) aganglionosis,abnormalautonomicnervous bone development defects,cardiacmalformation,distalenteric including dysmorphicfacies, cleftpalate,skeletal patterningand Loss of generated cohesion(Aguilaretal.,2005). PDS5 hascrucialfunctionsinyeastbeyond thoserelatedto compensate forthecohesiondeficit inthesemutants, indicatingthat overexpression oftopoisomeraseII,however, TOPII doesnot Finally, lethalityinyeast differentiation ofprostateepithelialcells(Gecketal.,2000). PDS5Bregulates androgen-induced 2000). Furthermore, tumors (13q12.3),itmayactasatumorsuppressor(Gecket al., a region wherelossofheterozygosityiscommonlydetectedin PDS5 isaregulatory componentofcohesinandinteracts To delineatephysiological rolesofPDS5Binmammalswe (also known as Pds5B Pds5B -deficient miceusinghomologousrecombination. results inanumberofdevelopmental defects, APRIN 4,5 Pds5 , JonathanM.Erlich in humanandmouse)islocated Pds5B- RESEARCH ARTICLE mutants canberescuedby PDS5 deficient micedied , PDS5A rspiacut Drosophila Pds5B 5 and , hort PDS5B –/– sms. 3191 ,

DEVELOPMENT . kb3 6.3 AAA; P3,ACCCTCCTGGAGTCAAGGAAA. genotypes weredeterminedbySouthernandPCRanalyses. Pds5B- Generation of first mousemodelresemblingCdLS,the pathogenesis ofCdLSandrelateddevelopmental disorders.Asthe development, andprovide apossiblemechanismfortheirroleinthe mice indicatethewidespreadroleofcohesionfactors innormal andtheconstellationofdevelopmental defectsin 3192 pRS315 weredigestedwith and cloning the5 reverse primersand filled byHerculaseDNA polymerase(Stratagene).For adapters. Two adaptersweregeneratedbyannealingadapterforward and repair byco-transformingyeastwithlinearized7.9kb5 et al.,2005).Thefinal targeting constructwas generatedusingyeastgap below forprimersequences)andclonedintopRS315byyeastgaprepair(Le downstream ofthefirst codingexon of coding exon of surrounding thefirst codingexon. A7.9kb5 MATERIALS ANDMETHODS developmental processes. be usefulindelineatinghow PDS5Bandcohesin regulate veracity ofthe recombination constructwas confirmed bysequencinganalysistoensure CTAGATGGAG; 2 ACCCTCCTGGAG TCAAGG- P2, ACCTGAAGAATTGGTGGAGGA; TAGCTCGAGCCCTTAA. CACACAAACTGCCCAGCTGTCTGCAGCATACACTGG GGCCCGC- AATTAAGGGCTCGAGCTAGCGGGCCC; Adapter2reverse primer: Adapter 2forward primer:ATACATTATACGAAGTTATAC TTTTCTTGCTTTAGGGGTACAGACATTTCCATCACGCGTGCTAGC.CGGTT - CGAGCTAGCACGCGTGATGGAAATG; adapter1reverse primer: yeast gaprepair: GCACA. CATGATTACGCCAACTCGAGCAGCCTGGTTTGCAGAGAGTTCTA - AGACAGCTGGGCAGTT; 3 TTAATGCGCCGCTACAGGGCGCGTCACGCGTCAGTGT ATGCTGC ATGTCTGTACCCCTAAAGCAAGAAAA; 3 ACAGCTATGACCATGATTACGCCAACTCGAGprimer: GATGGAA - ACGCGTCTGTGCTGGAGCTCACATGACCTGTCCT; 5 construct wereasfollows: used forPCRamplification ofthe5 sequences.) P1-P3 (450ntamplicon)primerpairs,respectively. (Seebelow forprimer type andmutantalleleswereamplified withP1-P2(390ntamplicon)and (95°C 30seconds,58°C6072°C90seconds;35cycles). Wild- were determinedusingSouthernblottingorPCRprimersP1,P2,P3 from amixed 129Sv/B6background.Thegenotypesof institutional approved protocols.Experimentswereperformedonmice similar. Allanimalprocedureswereperformedaccordingtoour independent lines(beforeorafterexcision ofthePGK-Neocassette)were toremove thePGK-Neocassette.Thephenotypesoftwo actin Cremice Southern blotting.Heterozygousmicegrew normallyandwerematedto successful germlinetransmissionofthetargeted allelewas detectedby blastocysts togeneratechimericmice.Thechimeraswerematedand the 3 were screenedbySouthernblothybridizationusingaprobedownstream of electroporation intoR1embryonicstemcells.Homologousrecombinants The The Primers usedtogenotypePds5B mutantmice:P1,CCAG - Adapter 1forward primer:GATCCCCCAAGCTTACT TAG ATCT - Primers usedtogenerateadaptersforthefinal targeting constructusing 5 Primers, containing Ј Xho Ј arm forward primer:TTAATGCGCCGCTACAGGG CGCG TC - Pds5B arm. Two properlytargeted EScellcloneswereinjectedinto deficent miceweregeneratedbyhomologousrecombinationand Ј Pds5B RESEARCH ARTICLE I. For cloningthefinal target construct,5 arm inpRS315,the Ј arm and3 targeting vector was linearizedwith Pds5B recombination constructencompassesgenomicregions Pds5B Pds5B arm regions. Pds5B knockout mice Ј containing 20ntof5 arm bygaprepair, pRS315was digestedwith ␤ Xho glPKNo( -gal-PGK-Neo genomic andassociated Ј I and arm reverse primer:ACAGCTATGAC - Mlu Ј Pds5B arm and3 I, respectively. Thefinal Pds5B were generatedbyPCR(See Ј ␤ Ј -gal UTR anda6.3kb3 Ј arm upstreamofthefirst Ј arm pRS315and3 Kpn arm forward primer: Ј - Neo) cassette,andtwo -deficient micewill arm ofthetargeting I andtransfectedby Pds5B pRS315 Ј arm inpRS315, Ј mutant mice arm reverse sequences, Pds5B Pds5B Ј Ј Sac arm arm –/– ␤ - I - (Molecular Probes)secondaryantibodies.Bisbenzimide(2 Cy3 (JacksonImmunoResearch),orgoatanti-mouseAlexa Fluor488 donkey anti-rabbitAlexa Fluor488(1:100;MolecularProbes),goatanti-rat visualized usingdonkey anti-sheepHRP(JacksonImmunoResearch), Medical Center, KansasCity, KS,USA),mouseBrdU(Roche),whichwere Covance), ratGCNA1 (George C.Enders, PhD,University ofKansas lysed in2 proteins derived fromembryonicday(E)14.5forelimbs.Thelimbswere was usedfornuclearstaining. 1 (TOH; Chemicon),rabbitneuron-specific classIII Primary antibodiesusedinthisstudyincludesheeptyrosinehydroxylase minutes priortoimmunostaining(Jainetal.,2004;Naughton2006). paraffinized inxylene,hydratedandboiled1mMEDTA solutionfor30 Bouin’s fixative. For antigenretrieval, paraffin-embedded sectionswerede- Tissues usedinimmunohistochemistrywerefixed inparaformaldehydeor blotanalysis Immunohistochemistry andwestern and usedforaffinity purification ofantibodiesfromserum. support byusingSulfoLinkcouplingGel(PierceBiotechnology, IL,USA) Immunology, CA,USA).Thesamepeptidewas crosslinked toasolid peptide immunogenNH2-CATKENDSSEEMDV-COOH (Pacific Rabbit anti-PDS5BpolyclonalantibodieswereraisedusingC-terminal Generation ofpolyclonalantibodyagainstmousePDS5B (Enomoto etal.,2001). using whole-mountTOH immunohistochemistryasdescribed previously et al.,2004). colonized byENSprecursorswas performedaspreviously described(Jain acetylcholinesterase andanalysis ofthepercentagedistalcolon anti-rabbit Cy3(1:500;JacksonImmunoResearch). P0gutstainingwith stained withanti-Tuj1 (rabbit;Covance; 1:500)andvisualizedusinggoat paraformaldehyde andembeddedinparaffin. Paraffin sectionswere sectional analysis,post-natalday(P)0gutwas fixed in4% antibody afterwashing threetimeswithTBST(Fuetal.,2006).For cross- anti-rabbit Alexa Fluor488(1:100;MolecularProbes) secondary anti-Tuj1, 1:100;Covance) stainingwas visualizedusingdonkey primary antibody(4°C,overnight) inTBST. Primaryantibody(rabbit 4%donkey seruminTBST(1hour, 25°C)beforeincubationwith with TBST (1mMTris, 150mMNaCl,0.2%Triton X-100)and blocked 25°C for30minutes.Afterfixation, explants werewashed threetimesin was dissectedfromthemouseandfixed with4%paraformaldehydeat For analysisoftheentericnervous system,theE12.5orE18.5wholegut Peripheral nervoussystemanalysis independently madethesamediagnosisforevery heartinthisstudy. every heartunder a5 for defects.Two people(J.M.E.andP.Y.J.) examined thesetofsectionsfrom were seriallysectionedat6 heart was dissectedfreeandembedded inparaffin. Entireneonatalhearts The thoraxes ofnewborn micewas fixed in4%formaldehydebeforethe Analysis ofcardiac malformations microscopically forevidence ofcleftpalate. sections werestainedwithHematoxylinandEosin(H&E)examined heads wereproperlyorientedinparaffin, coronalsectionswereprepared, heads werefixed in4%paraformaldehydeat4°Cfor16hours.Thefixed days, andstainedwithAlizarinRedSfor3days.Embryonicnewborn Alcian Bluesolutionfor3days,rehydratedandincubatedin1%KOH for2 were skinnedanddehydratedin95%ethanol.They werethenstainedin previously described(Wellik andCapecchi,2003).Briefly, newborn mice Alizarin RedSandAlcianBluestainingofnewborn micewas performedas Histological analysisofboneandpalate anti- using chemiluminescencesubstrate(Pierce).For aloadingcontrol,mouse rabbit anti-PDS5Bantibody(1:500dilution)andPDS5Bwas visualized at 1:20,000dilution. ϫ Western blotanalysiswas performedusingstandardtechniques The sympatheticnervous systemofembryos andnewborns was analyzed protease inhibitorcocktail;Roche).Thewesternblotswereprobedwith ␤ -tubulin antibody(DSHB,University ofIowa, IA,USA)was alsoused ϫ SDS proteinlysisbuffer (100mMTris-HCl, pH6.8,4%SDS, ϫ ␮ microscope objective. Thetwo examiners m thickness,stainedwithH&E,andinspected Development 134(17) ␤ -tubulin (TuJ1; ␮ g/ml; Sigma)

DEVELOPMENT at 45°Cfor4hoursinhybridizationbuffer containingthe 4%paraformaldehydeinPBS,acetylatedandhybridized slides, fixed incold weremountedonpoly-L-lysine-coated 2002). Frozensections(12µm) in situhybridizationwas performedaspreviously described(Songetal., (See below forprimersequences.) samples werenormalizedaccording tothelevels ofGAPDHor 18SmRNA. curves todeterminerelative levels ofgeneexpression. Individual RNA previously (Svaren et al.,2000).Target geneswereanalyzedusingstandard Biosystems) using qRT-PCR was performedusingaModel7700instrument(Applied transcriptase (Invitrogen) andoligo(dT)random hexamers asprimers. Probes). Reverse transcriptionwas performedusingM-MLV reverse (Invitrogen) andquantified bytheRibogreenfluorometricassay(Molecular RNA was preparedfrommouseadult andembryonictissuesusingTrizol Quantitative RT-PCR 35 Bam byRT-PCR andclonedintothe and probe2:nt4561-5098)weregenerated cDNA fragmentscorrespondingto Mouse In situhybridization banding. regardless ofthelengthchromosomesordifferences inthequalityof the first 22-25metaphasesencounteredwereincludedintheanalysis for precocioussisterchromatidseparation(PSCS).To avoid selectionbias, found tohave normalkaryotypes.Twenty-two to25cellswereexamined banding method.Two cellswerefullyanalyzedfromeachline,andall were wereanalyzedusingtheGeimsa-trypsin-Wrights(GTW) treatment andfixed inmethanol:aceticacid(3:1).Slideswerepreparedand 1 hourincolcemid(1 serum. Chromosomeswereharvested thedayafterplatinggrowth for mutant mice(passage3)wereculturedinDMEMwith10%fetalbovine Hsieh, PhD.Mouseembryonicfibroblasts fromwild-typeand Laboratory atUniversity ofSouthernCaliforniadirectedbyChih-Lin Chromosome analysiswas performed intheChromosomeAnalysis Metaphase spread analysis points. harvested andprocessedforhistologicalevaluation attheindicatedtime described (Honaramoozetal.,2002).Thegrafteddonortesteswere nude mice(Taconic No.NCRNUM,Germantown, NY, USA)aspreviously subcutaneously ontothebackand/orflankofcastrated4-to8-week-oldmale Testes fromE16.5mutantandwild-typemiceweretransplanted Testes transplantation al., 2004). ImmunoResearch). TUNELwas performedaspreviously described(Jainet (1:200) andgoatanti-ratimmunoglobulin labeledwithCy3(1:500,Jackson Molecular Probes)secondaryantibody, followed bystainingwithGCNA1 goat anti-mouseimmunoglobulin labeledwithAlexa Fluor488(1:500; incubated withBrdUprimaryantibody(1:200;Roche)andvisualizedusing (Jain etal.,2004).Paraffin-embedded sectionsofembryonictestiswere were injectedwith200mg/kgbodyweightBrdU2hourspriortosacrifice 2006). goat Sertolicellspolyclonal;SantaCruzBiotechnology)(Naughtonetal., whereas Sertolicellswereidentified usinganti-GATA4 antibody(1:200, identification, anti-GCNA1 antibody(1:200,ratpolyclonal)was used, histochemistry, orGCNA1 immunohistochemistry. For germcell germ cellswereexamined byH&Estaining,alkalinephosphatase 4°C, embeddedinparaffin, and6 Embryonic gonadsweredissected,fixed inBouin’s solutionovernight at Analysis ofgermcells necessaryfornormaldevelopment evda eaiecontrols. served asnegative Sectionshybridizedwiththesenseprobes detected byautoradiography. for20minutes.RNase A-resistanthybridswere RNase A(20µg/ml)at37°C sectionsweretreatedwith antisense cRNA probes.Afterhybridization,the S-labeled cRNA probesweregeneratedfromthese o rUandGCNA1 doubleimmunostaining,timedpregnant females For BrdU HI and Hin dIII sitesofpBluescriptIIKS+vector. Senseorantisense Sybr Green Ifluorescence(MolecularProbes) asdescribed ␮ g/ml) usingstandardhypotonic(0.075MKCl) ␮ m sectionswereprepared.Primordial Pds5B (probe 1:nt2903-3438 Pds5B fragments and 35 S-labeled Pds5B retardation. Newborn Pds5B E16.5, but thenthe mutantsbegan toshow signsofgrowth CAAGGAA andGCTGGAATTACCGCGGCT. GAGCCCTTCC; mouse18SribosomalRNA, CGGCTA CCA mouse GAPDH,TGCCCCCATGTTTGTGATGCATC andTGTGG - TCAT CCTGGAGTCAAGGAAA and CAGAGTCCTGGTCCAT - GT CCAT; , KIAA0979was digestedwith respectively. To generatethecarboxyl-terminalEGFP-PDS5Bfusion University, DC,USA)andKazusaDNA ResearchInstitute(Chiba,Japan), PDS5B Generation of RESULTS Flag-tagged human Plasmids andcellculture Wild-type and andcleftpalate abnormal skeletalpatterning Pds5B respiratory abnormalities(seebelow). died shortlyafterbirth,mostprobablyfromcardiacand/or intercostal andabdominalretractions,cyanosis andpallor. They all accessory musclesofrespirationasindicatedbyheadbobbing and signs ofrespiratorydistress,includinglaboredbreathing,use of mutant fetuses werebornalive andnoneofthemice homozygousforthe other cohesionproteins areassociatedwithCdLS, suggestedtous reported forCdLSpatients,along withthefact thatmutationsin Fig. 2A-C,K).Theseabnormalities, whicharesimilartothose facial dysmorphisms(short snout,shortlow chinandthinupperlip; type littermates,andhadshortstature, shortlimbs,asmallheadand However, onlyabout75%oftheexpected numberof were presentattheexpected Mendelianratiountil age E16.5. Pds5B supplementary material)inembryoshomozygousforthemutant did notdetect in was achieved usingLipofectamine-mediatedtransfection ofplasmids. bovine serum.Transient expression ofEGFP-taggedPDS5AandPDS5B Bgl Klenow, andclonedintotheblunted PDS5A EGFP-FLAG-PDS5A fusionproteinwas generatedby digesting the Pds5B homologous recombination(Fig.1E-G).To confirm theabsenceof physiological rolesinmice,wegenerated multiple processesinmammals.Therefore,todetermineits mitotic aswelldividing cells,suggestedthatitparticipatesin ubiquitously expressed atthecellularlevel (Fig.1D). (data notshown), andinsituhybridizationrevealed that embryo, in apatternconsistentwithimmaturegermcells(Fig.1B).Inthe expression was detectedattheperipheryofseminiferoustubules hippocampus anddentategyrus(Fig.1C).Inthetestis,highPDS5B experiments revealed high and testis(Fig.1A).Atthecellularlevel, insituhybridization the adultwefoundhighestlevels of mRNA expression levels inadultandembryonicmousetissues.In As aninitialinvestigation ofPDS5B functionwesurveyed Pds5B Primers usedforqRT-PCR analysis:mouse Pds5B The expression ofPDS5B,aregulatory factor ofcohesin,inpost- IsiteofpEGFP-C1.HeLacellswereculturedinDMEMwith10%fetal II allele, indicatingthatitisatruenullallele. mRNA inE14.5 expression inthehomozygous construct with Pds5B (KIAA0979) cDNA weregiftsfromKasidUsha(Georgetown deficiency results ingrowth retardation, Pds5B –/– limbs bywesternblottingusingPDS5Bantibodies.We Pds5B allele survived beyond P1.Thenewborn animalshad Pds5B expression was comparableinalltissuesexamined PDS5A Pds5B mRNA orprotein(Fig.1H,I;seeFig.S1inthe Pme –/– Pds5B mutant embryoswereofsimilarsizeuntil and insertingthisfragmentintotheblunted I in -deficient mice Pds5B pCR3.1 –/– –/– mice weresmallerthantheirwild- Xho brain andexamined PDS5Blevels expression inneuronsofadult I siteinpEGFP-C1(Clontech).The RESEARCH ARTICLE expression vector andthehuman Bam Pds5B Pds5B HI and Pds5B mice, wequantified expression inbrain Eco Pds5B Pds5B RI, bluntedwith null miceby –/– Pds5B , ACCCT - Pds5B embryos Pds5B 3193 Flag- was –/–

DEVELOPMENT type (WT):0/24; (Fig. 2G).Inaddition, thesternumsof unfused ossification centers orlackofossification ofthesternebrae that the 3194 scapula wereshorterinthe several bonesincludingthemandible,ulna, radius,humerusand Red SandAlcianBluetohighlighttheskeleton, andfound that patients, westainedwild-typeandmutantembryoswithAlizarin patients withCdLS. patients. All thatareobserved inmoreseverely affected CdLS (syndactyly) however, wedidnotobserve the limbtruncationsorfuseddigits limbs areassociatedwithmildcases ofCdLS(Irelandetal.,1993); other abnormalitiesin developmental syndrome.To substantiatethisidea,wesearchedfor To testforskeletal abnormalitiessimilartothoseinCdLS RESEARCH ARTICLE Pds5B Pds5B -deficient micecouldbeavaluable modelofthis Pds5B –/– mice exhibited sternalmalformations [wild Pds5B –/– Pds5B : 37/37]characterizedbyeither two –/– mice thatarecommonlyfoundin –/– ie(Fig.2D-F).Shortened mice Pds5B –/– mice wereshorter test. ( level. Error barsrepresent thes.e.m.* embryonic E14.5brainsusingqRT-PCR normalizedto from wild-type(+/+),heterozygous (+/–)andhomozygous(–/–) Pds5B antibodies toPDS5Band P1, P2,andP3indicatedinE.( genotyping analysisofthe hybridized withtheradiolabeledDNAprobe, indicatedinE.( homozygous (–/–)mice.TheDNAsampleswere digestedwith (+/+),heterozygous (+/–), genomic DNAsfrom tailsofwild-type analysis depictingsuccessfultargetingofthe indicate positionsofprimersusedforgenotyping.( Nhe hybridization (ISH)using cassette waslaterexcisedbymatingto Th, thymus).Error barsrepresent ±s.e.m.( stomach; Lu,lung;Sp,spleen;He,heart;Br, brain;Li,liver;Ki,kidney; replaced bya allele (bottom).Thecodingregion ofthesecondexon (top) andthetargetingconstruct(middle)togivemutant representation ofhomologousrecombination between with CdLSiscleftpalate.Anexamination ofthisregion in not shown). 0/10; shelves (PS)in PS ofE18.5 have normally fusedandthendegenerated (Fig.2P),however, the E18.5 wild-typeembryos,themedial-edge epitheliaofthetwo PS (Fig. 2N,Q),byE14.5they wereclearlyabnormal(Fig.2O,R).In Fig. 1.Expression of mouse tissuesnormalizedto the midline(Fig.2L,M;WT: 0/24; and failure oftheposteriorpalatalbonestoextend fullyandmeetat mice revealed acompletecleftofthesecondary palate(Fig.2J,K) Pds5B anlage atC7andthecostalcartilageofT1rib(WT: 0/10; and vertebrae (datanotshown), fusionofextensively ossified rib variable expressivity, includingdelayedossification ofdigitalbones abnormalities inskeletal patterning,withincompletepenetranceand than thoseofwild-typemice(Fig.2G).We alsofoundother apoptosis inpalatalshelves of we didnotobserve significant changesincellproliferationor 2S). UsingBrdUandTUNELanalysis onE13.5embryonicpalates, antisense (AS,left)andsensecontrol (S,right)probes. ( E13.5 mouseembryo(D).InD,thesagittalsectionsare probed with dentate gyrus(arrowhead inC),andwidespread expression inthe immature germcells(arrows inB),hippocampus(arrow inC)and of theseminiferous tubules(ST)consistentwith visualized bydarkfieldmicroscopy. Notethehighsignalatperiphery indicate adulttestis(B),brain(C),andE13.5embryo(D).Silvergrains WT vertebrae (WT: 0/10; or Pds5B mutant embryos,weexamined thepalatalshelves ofwild-typeand characterize thedysmorphogenesisofpalatalshelves in mice. Another developmental abnormalityobserved insomechildren Pds5B I; S, I –/– ) WesternblotonE14.5forelimb tissuelysateswasprobed with ( Pds5B A –/– –/– Sac Pds5B ) QuantitativeRT-PCR analysisof lysate. : 10/18;Fig.2H),lossorhypoplasiaofthe13thribs(WT: +/– embryos atE12.5,E14.5andE18.5.Whereaspalatal I. BlackbarswithnumbersindicateexonsandP1-P3 Pds5B embryos (seeFig. S2inthesupplementarymaterial). ␤ –/– mRNA. Thegreen background istheEosincounterstain -gal : 20/20),andunfusedossification centersinthe Pds5B /PGK-Neo –/– Pds5B Pds5B embryos wereshorterandfailed tofuse(Fig. –/– 35 ␤ S-CTP-labeled probes wasperformedusing -tubulin. NoteundetectablePDS5Bprotein in Pds5B embryos wereseeminglynormalatE12.5 18S ( and generationof ␤ –/– H Gal Neo)cassette.ThefloxedPGK-Neo ) Relativeamountsof : 5/18;Fig.2I)andhyoidbone(data Pds5B RNA. (Pr, prostate; Te, testis,St, mutation wasperformedwithprimers P <0.001, Student’s unpaired ␤ –/– Pds5B -actin Cre mice.K, Pds5B B embryos comparedtoWT , C Pds5B , D Development 134(17) –/– Pds5B mRNA levelsinadult ) Radioactiveinsitu : 25/33).To further Pds5B F using ) Southern blot ) Southern GAPDH Pds5B expression in E Pds5B ) Schematic -deficient Pds5B Kpn mRNA G Pds5B mRNA gene ) PCR Pds5B Nhe is I; N, Pds5B I and t - –/–

DEVELOPMENT Clearly, cleftpalateisadistinctive featureof mesenchymal transitionofmedialedgeepitheliaatalaterstage. abnormal reorientationofpalatalshelves orfailure ofepithelial- 12/24; tenperimembranous, onemuscular, oneinlet),anda common the secundumtype(ASD,6/24), ventricular septaldefects(VSD, patients (Jacksonetal.,1993).We identified atrialseptaldefectsof G). Thedefectsincludedthose thatarecommonlyfoundinCdLS compared towild-typemice(15/24 versus 0/14, The cleftpalateobserved in Cohesin necessaryfornormaldevelopment heart defectsofany type was significantly higherin searched forcardiacmalformations. Theincidenceofcongenital Pds5B Because childrenwithCdLSoftenhave congenitalheartdefectsand PDS5B isimportantforcardiac development contributing totheneonatallethalityofthesemice. could causeaspirationandsubsequentasphyxiation, thus –/– iesfe eprtr distressandperinatallethality, we mice suffer respiratory Pds5B –/– mice couldbetheresultof Pds5B P <0.001; Fig.3A- –/– mice and Pds5B –/– Pds5B with CdLS,encouragedustoexamine thenervous systemin neuronal migration,andtheprofoundmentalretardationassociated other cohesioncomponentssuchas mice islowerthanthatofwild-typelittermates(WT, palatal shelves.Scalebars:1mm. shelf; n,nasalbone;to,tongue.Arrow inSpointstotheunfused in bones are markedbydashedlines.Posteriorpartsofthepalatalbones Red SandAlcianBluestainingofneonateskulls.Theedgespalatal using H&Estainedcoronal sectionsofwild-typeand with wild-typelittermate(L).( The highexpression of nervous systemdevelopment PDS5B-deficiency causesabnormalperipheral mice. one atrioventricular canaldefect(1/24;Fig.3C-F)inthesemice.Ofnote, projections appearing normalin sympathetic chaingangliawere welldeveloped withneuronal examining thishypothesis. mice, Fig. 2. (arrowhead), andsmallheadin stature, facialdysmorphism,shortlimbs(arrow), shortsnout t and potentiallycontributing toearlypostnatallethalityof blood attheventricular level, thus resultinginrespiratorydistress pulmonary overcirculation andedemafromleft-to-rightshuntingof VSDs andcommonAV canaldefectswould beexpected tocause fruste oftetralogyFallot, acommonmalformationin CdLS.The right ventricular outflow tractobstructionandthushadtheforme VSD toward therightventricle (Fig.3C).Thismousedidnothave malalignment oftheoutletseptum,causingaortatoshiftover the ( Pds5B and arrowheads pointtothehypoplastic13thribsin and lumbar(L)vertebrae.Thearrow pointstotheunfusedL3vertebra centers. (H)Ribs.Thearrow pointstotheC7-T1fusion.(I)Thoracic(T) ossification centers;arrowheads, delayedormissingossification wild-type to wild-type(left).( mice. (C)Shortmandible(arrow) in E14.5, and E18.5 embryos. Wild-type (N-P); E14.5, andE18.5embryos.Wild-type lethality of neuronal functionorconnectivity. Unfortunately, theneonatal not playamajorroleinCNSdevelopment; instead,itmayinfluence Pds5B any grossormicroscopicstructuralabnormalitiesinthebrainof patients have relatively normalbrainanatomy, wedidnotobserve skeletal patterning andcleftpalate. skeletal patterning P radius andhumerusissignificantlyshorterin numbers (lengthsinmm±s.d.).Notethelengthofmandible,ulna, scapulaofforelimbs. Bonelengths(square bracket)are indicatedby (F) cartilage. (D)Mandibles;(E)ulna,radiusandhumerusofforelimbs; mouseskeleton.Redindicatesboneandblue newborn J,K -test. ( <0.001, Student’s unpaired Pds5B An examination oftheperipheral nervous systemrevealed thatthe ) Completecleftpalate(arrow) andthinupper-lip (arrowhead) ofa Pds5B n –/– –/– -deficient mice.Thissuggeststhatthecohesincomplex does B,C Pds5B =35). Error barsrepresent s.e.m.* –/– neonate (K)compared withwild-typecontrol (J).( . mice. Consistentwithclinicalreportsshowing thatCdLS Sample sizesforeachgenotyperangefrom 18to34. ) Morphologyofwild-typeandmutantmice.(B)Noteshort mutant failedtomeetatthemidline(arrows, M),compared –/– Pds5B mouse hadalarge perimembranousVSDwithanterior deficiency results ingrowth retardation, abnormal D-I –/– ) AlizarinRedSandAlcianBluestainingof mice currentlyprevents usfromfurther Pds5B t N-S -test. (G) Sternum. Arrows,-test. (G)Sternum. unfused Pds5B ) Palatogenesisdefectsillustrated in post-mitoticneurons,theroleof Pds5B Pds5B –/– RESEARCH ARTICLE Mau-2 ( compared towild-typeP0 A P –/– ) Theweightof <0.001, Student’s unpaired –/– Pds5B neonate (right)compared Pds5B mice (seeFig.S3 inthe in axonalguidanceand –/– –/– n Pds5B (Q-S). ps,palatal Pds5B =43 and compared to L,M Pds5B –/– –/– E12.5, neonate. Pds5B ) Alizarin Pds5B –/– 3195 P0 –/– –/–

DEVELOPMENT defects in right, leftventricle.Scalebar:1mm.( MV, aortic,tricuspidandmitralvalve;RA,LA,right,leftatrium;RV, LV, and ventricularseptum.Arrows indicatetherespective defects.AoV, TV, the singleatrioventricularvalveandcommondefectofatrial a muscularVSD;(F)acommonatrioventricularcanaldefect.Note (E) VSD withtheaortaridingoverbothventricles;(D)asecundumASD; Sections ofmutantmouseheartsshowing:(C)alargeperimembranous aortic, tricuspidandmitralvalves,(B)anintactatrialseptum.( demonstrating (A)theintactventricularseptumandrelationships ofthe normally reach the mid-colonbyE12.5(Fig.4H). Bycontrast,in proximal todistaldirectionalong thelengthofbowel and migrating neuralcrestcells(NCCs). TheNCCsmigrateina network ofneuronsandglia withinthebowel thatisderived from et al.,1993). likely causeoftheptosisobserved inmany CdLSpatients(Jackson 2002). TheseresultssuggestthatSCGinnervation defectsarethe commonly manifestunilateralorbilateralptosis(Honmaet al., observed inmicedeficient fortheneurotrophicfactor artemin the carotidnerve (Fig.4F,G). TheseSCGdefectsaresimilartothose and 30%(4/13)ofthesemicehadunilateralorbilateralabsence of ifrne nttlicdnebtenW and differences intotalincidencebetweenWT observed inCdLSpatients. in theSCGanditsprojectionstotarget organs inthe common conditioninCdLS.We foundanumberofabnormalities muscles. AbnormalitiesintheSCGareassociatedwithptosis, a ganglia (SCG),whichprovides sympatheticinnervation totheocular supplementary material).We alsoexamined thesuperiorcervical Fig. 3.Congenitalheartdefectsin 3196 Furthermore, all was locatedfar caudalofitsnormalposition (Fig. 4B,C). (Fig. 4A).For example, in40%(5/13)of We next examined theentericnervous system(ENS),acomplex RESEARCH ARTICLE Pds5B –/– Pds5B mice. P –/– <0.001 iscalculatedbyaZ-testforsignificant mice hadthincarotidnerves (Fig.4D,E) ( A,B ) Sectionsofwild-typemousehearts G Pds5B ) Summaryofcongenitalheart –/– Pds5B Pds5B mice mimicthose –/– homozygotes. mice, theSCG Pds5B –/– , which C-F mice ) encouraged ustoexamine germcelldevelopment in frequency ofgenitalanomaliesseeninpeoplewith CdLS, of PDS5inmitosisandmeiosislower organisms, andthehigh The highexpression of proliferation PDS5B regulates primordial germcell CdLS patients. hypoganglionosis mayunderliethegastrointestinalsymptomsof 1993). Theseobservations suggestthatdefectsintheENSsuchas 62% and77%,respectively, ofinfants withCdLS(Jacksonetal., persistent emesisandfeedingproblemsarecommon,occurringin reported, but whereothergastrointestinalproblemsincluding contrasts withCdLSwheredistalcolonicaganglionosishasnotbeen nervous systemthatresembleHirschsprungdiseaseinhumans.This the germcellsremainingatE16.5in immunohistochemistry (datanotshown). Thesestudiesindicatethat were morphologicallynormalasdeterminedbyGATA4 propagate E16.5testisofwild-typeand undergo spermatogenesis,weperformedtestistransplantationto (Fig. 5E,F,I). To determineiftheremaining germcellscould Pds5B investigate mechanismsunderlying germcelldepletion.AtE16.5, We thereforefocusedonmalegermcelldevelopment tofurther supplementary material)thatappearedtobemoresevere inmales. Pds5B Fig. S4inthesupplementarymaterial).The testes containedtesticularcordsthatonlySertolicells (see lower in the distalbowel colonizedbyNCCs,theneuronal densitywas (IC) junctionbythisage(Fig.4I).Furthermore,even inregions of grossly normalin colon (Fig.4K,M).Bycontrast,smallbowel innervation appeared degrees ofabnormalinnervation andganglionformationinthedistal 5G,H). By6weeksaftertransplantation,theexplanted spermatogonial stemcellsat2weeksaftertransplantation(Fig. immunohistochemistry, wefoundacompletedepletionof nude mice(Naughtonetal.,2006).UsingGCNA1 underlying thereducednumber of germcellsinmale until E13.5inmales(McLaren, 2000).To identifythe mechanism ridge byE11.5(Gompertsetal., 1994),andcontinueproliferating around E7.25(Ginsburg etal.,1990).Thesecellsmigratetothegenital cells intheextraembryonic mesodermposteriortotheprimitive streak development. PGCsarederived fromafounderpopulationof~45 suggested thatPDS5Bisimportantforprimordialgermcell(PGC) of sustainingspermatogenesis. (Fig. 4J,L),whereas70%(8/12)of neurons colonizetheentirelengthofwild-typeneonatalbowel see Fig.S3inthesupplementarymaterial).Asexpected, enteric form oftubulin, oracetylcholinesterase(AchE)staining(Fig.4J-M; E18.5 andP0usingtheTuj1 antibody, whichrecognizesaneuronal delayed migrationofNCCsintothecolon,weexamined theENSat shown). majority of ovaries ofnewborn We foundaseverely reducednumberofgermcellsintestesand the ridge, therewerealreadysignificantly fewer numbersofgermcellsin cell precursors.AtE12.5,atime pointwhenPGCsenterthegenital we evaluated themigration,proliferationandapoptosisofthesegerm The embryoniclossofgermcellsatE16.5in To determineiftheentericneurondefectsoccurbecauseof Pds5B –/– –/– Pds5B male micehadan80%reductioningermcellsthetestis mice, ENSprecursorsfailed tomigratepasttheileocecal –/– Pds5B mice. We hypothesizedthatovert migrationdefectsin –/– –/– mice comparedtowild-typeanimals(datanot Pds5B Pds5B mice hadsignificant defectsinthedistalenteric Pds5B –/– –/– in adultandembryonictestis,therole mice (Fig.5A-D;seeFig.S4inthe mice (datanotshown). Thus,the Pds5B Pds5B Pds5B –/– –/– mice exhibited variable Development 134(17) Pds5B –/– Pds5B mice wereincapable mice asexplants in –/– Pds5B Pds5B -deficient mice Sertoli cells Pds5B –/– –/– mice. mice, –/–

DEVELOPMENT mid-colon, whereas of entericplexuswithclustered neuronal cellbodies(arrowheads) inthe E10.5 gonadsto identifyPGCsrevealed nosignificant differences in genital ridgebyE11.5.However, alkaline phosphatasestainingof P0 paraffin sectionsofdistalcolonfrom wild-type(J) and cochlea. Scalebars:400 by arrows inDandE,bytwodashedlinesFG.ey, eye;co, in location ofSCG(C).Notethesevere reduction orabsenceofcarotid nerves type mice(SCG,arrows). Dashedcircle indicatestheexpectednormal Pds5B Cohesin necessaryfornormaldevelopment Fig. 4. junction in that theentericneuron migrationwavefront didnot pass theileocecal showing thelackofclustered entericneuron cellbodies in only afewthinnervefibers(arrows). Scalebar:100 wild-type mice(L)and neuron cellbodies.( colons. Scalebars:10 Scale bars:20 carotid nervemissing.* delayed migrationorabnormallocalization;TCN,thincarotid nerve;CNM, ganglion (SCG)abnormalitiesin nervous system. WT ( or largeintestine)successfullycolonized byneurons. corresponds tothepercentage oftherespective intestinalsegment(small representation ofENSdefectsin ( significant differences inincidencebetweenWTand muoitceity(,wildtype;I, (H, immunohistochemistry neurons inthedistalbowelofwholegutdemonstratedbyTuJ1 (B,C) AbnormalcaudallocationofSCGin B-G Pds5B ) TOHwhole-mountstainingofsympatheticneurons ofneonatemice. n =9); P0: Pds5B –/– –/– PGCs would resultinfailure oftheseprecursorstoreachthe Pds5B (E,G) compared toWTmice(D,F).Carotid nervesare indicated –/– ␮ Pds5B m. (Insets)HighmagnificationofTuJ1 stainingofP0distal mice displaymultipledefectsintheperipheral –/– ( A mice (I).Scalebars:10 L,M Pds5B –/– ) Summaryofcarotid nerveandsuperiorcervical ␮ Pds5B ( P m. Arrowheads pointtotheclustered enteric n ) Tuj1 whole-mountstainingofE18.5gutsfrom ␮ <0.05 and** =12) andWT( m (B-E);200 –/– –/– mice lackentericneuron cellbodiesandshow (M). Wild-type miceshownormal formation (M). Wild-type Pds5B Pds5B -deficient mice.SCGAML, P –/– Pds5B ␮ n <0.001 determinedbyaZ-testfor m (F,G). ( =13)]. Thescaleatthetop mice [E12.5: Pds5B ␮ m (H,I).( –/– ). *,ileocecaljunction.Note –/– H mice compared towild- , ␮ I Pds5B ) E12.5enteric J,K m. ( Pds5B ) TuJ1 stainingof Pds5B N Pds5B ) Schematic homozygotes. –/– ( –/– n –/– =7) and mice (K), mice. Student’s paired counted foreachanimal).Error barsrepresent s.e.m.* at E12.5( index measured byBrdU incorporationinGCNA1-positivegermcells in of E16.5wild-type(G)and represent SEM.* (G), notecompletelossofgermcellsin presence ofgermcellsintheperipheryWTseminiferous tubules and WT( cells. reduced proliferationisamajorfactor inthereducednumberofgerm stages indevelopment thatwedidnotexamine, itisclearthatthe depletion couldbepartiallydue to apoptosisordelayedmigrationat supplementary material).Although itispossiblethatgermcell and GCNA1 immunohistochemicalanalysisofE16.5germcellsinWT(E) embryogenesis. ( PGC locationbetweenwild-typeand Fig. 5. WT and bisbenzimide. ( *Non-specific auto-fluorescence signal;red, GCNA1;blue, Pds5B 30% reductioninthenumberof BrdU-positive germcellsintestesof proliferation inE12.5embryosusingBrdUincorporation,wefound a embryos (datanotshown). However, whenweexamined germcell in thenumberofapoptoticgermcellsE12.5orE16.5mutant relatively normal.Inaddition,TUNELanalysis revealed nochanges shown), indicatingthatPGCmigrationatthisearlystagemustbe staining ofgermcellsWT( Pds5B Pds5B –/– Pds5B –/– Pds5B C n -deficient (F)testesshowsareduction ingermcellsduring ) and =3 foreachgenotypeand100GCNA1-positivecellswere versus wild-typemice (Fig.5J;seeFig.S4inthe mice. Arrows pointtoemptytesticularcords inD.( -deficient micemanifestgermcelldefects. -deficient mice( I ) QuantitativeanalysisofgermcellsinE16.5testes Pds5B t G P -test. Scalebars:50 <0.001, Student’s unpaired , H ) GCNA1stainingoftwo-weektestestransplants –/– ( D Pds5B ) neonataltestesshowreduced germcells A n ) and =4 foreachgenotype).Error bars –/– mice (H).Incontrasttothe Pds5B ␮ RESEARCH ARTICLE m. Pds5B Pds5B –/– ( –/– t B -test. ( ) neonatalovaries, testis explant(H). –/– embryos (datanot J ) Proliferation P <0.05, GCNA1 E,F ) 3197

DEVELOPMENT cells perline(two WTandtwo the supplementarymaterial).We analyzedatotalof22-25metaphase were indeeddevoid ofboth western blotanalyseswereperformedandwefoundthatthesecells both fibroblast (MEF)chromosomes.Two MEFlineswereobtainedfrom GTW metaphasechromosomebandingofmouseembryonic development. chromatid cohesionthatareimportantformammalianembryonic cells suggeststhatPDS5Bhasnovel functionsotherthansister lack ofsisterchromatidcohesionabnormalitiesin cohesion defectsin Heemst etal.,1999;Wang etal.,2003;Wang etal.,2002).To testfor Hartman etal.,2000;Losada2005;Panizza etal.,2000;van PDS5 iscrucialforsisterchromatidcohesion(Dorsettetal.,2005; cohesion PDS5B lossdoesnotaltersisterchromatid 3198 abnormalities wereobserved inthe precocious sisterchromatidseparation(PSCS)orgrosschromosomal are available onrequest).Noobvious cohesiondefectssuchas Musio etal.,2006; Schuleetal.,2005;Tonkin etal.,2004;Vega et developmental anomalies(Deardorff etal.,2007;Krantz 2004; responsible forthebroadspectrum ofprenatalandpostnatal Roberts syndrome/SCphocomelia (RBS/SC),andarethoughttobe SMC3 andESCO2)have beenfoundinpatientswithCdLSand Mutations infourdifferent cohesionproteins(NIPBL,SMC1A, DISCUSSION than intherestofnucleus(Fig.6D). nucleus. Bycontrast,PDS5Awas lessabundant inthenucleolus to bemoreconcentratedinthenucleolusthanrestof the PDS5A andPDS5Bwerefoundinthenucleus,appeared interphase cellsusingfluorescencemicroscopy. Whereasboth proteins anddeterminedtheirsubcellularlocalization in proteins. We generated EGFP-taggedPDS5AandPDS5Bfusion part, fromdifferential subcellularlocalizationofthesetwo whether theirdistinctcellularfunctionscouldstem,atleast in least someofthephysiologicrolesPDS5B.We investigated deficiency clearlyshow thatPDS5A cannotcompensateforat their similarity, thesevere abnormalitiescausedbyPDS5B hook domainspresentintheC-terminalregion ofPDS5B. Despite HEAT repeatsandarecloselyrelated,but PDS5AlackstheAT 2005). TheN-terminalregions oftheseproteinsboth contain interactions mediatedbyPDS5B. regulate genetranscriptionthroughdirectcohesincomplex-DNA cohesion factor, indicatingthatPDS5Bandthecohesincomplex may recognizable DNA bindingmotifreported in asisterchromatid The identification ofAT hookdomainsinPDS5Bisthefirst AT tractsintheminorgroove ofDNA (MaherandNathans,1996). initially identified intheHMG-I/Yfamily asadomainthatbindsto in theyeast,worm andflyorthologs.TheAT hookdomainwas are presentinPDS5Bfromfish tohuman(Fig.6C),but arenotpresent IPB000116A). Furtheranalysisrevealed thattheseAT hookdomains match thehighmobilitygroupprotein(HMGY)signature(Block et al.,2000)andtwo AT hookdomainsintheCterminusthatclosely N terminus(Losadaetal.,2005;Neuwald andHirano,2000;Panizza scores. We identified thepreviously reportedHEAT repeatswithinthe PDS5B usingBlocksandPfam motiflibrarieswiththedefault cut-off To examine this possibility, weperformedmotif searchingofmouse PDS5B hasacquiredadditionalfunctionaldomainsduringevolution. The widerangingdeficits inthe There aretwo PDS5homologsinvertebrates (Losadaetal., Pds5B RESEARCH ARTICLE -deficent andwild-typemice.Quantitative RT-PCR and Pds5B- deficient mammaliancells,weperformed Pds5B Pds5B mRNA andprotein(seeFig.S1in Pds5B Pds5B –/– MEF lines;detailedresults –/– –/– MEFs (Fig.6A,B).The mice suggestedthat Pds5B -deficient from thenucleolus(arrows). Scalebar:5 (arrows), whereas EGFP-PDS5Aislocalizedtothenucleusbutexcluded PDS5B ispresent inthenucleusandhighlyenrichednucleolus HeLa cells;rightpanelsare imagesofGFPsignals.NotethatEGFP- EGFP signalswere examined.Leftpanelsare phase-contrastimagesof PDS5A andEGFP-PDS5BusingLipofectamine.At24hourslater, the and PDS5Bfusionproteins. HeLacellswere transfectedwithEGFP- localization ofhumanhomologsPDS5usingEGFP-taggedPDS5A from WTand tropicalis; Da,Daniorerio;Te, Tetraodon nigroviridis Ga,Gallusgallus;Xe,Xenopus norvegicus; musculus; Rn,Rattus Hs, Homosapiens;Pa,Pantroglodytes;Ca,Canisfamiliaris;Mm,Mus cohesion. ( ( patients, webelieve that mutations inothercohesionfactors have beenidentified inCdLS Fig. 6. Pds5B CdLS patients.Becauseofsignificant phenotypicoverlap in to thefrequentoccurrenceofptosisandgastrointestinalproblems in enteric andautonomicnervous systemsofthesemice mayberelated mice arenotviablepastP0.However, thestrikingdefectsin difficult toevaluate inthisanimalmodelparticularlysince mental retardation,hearinglossandgastroesophagealreflex are palate. OtherphenotypesthatareassociatedwithCdLSsuch as including abnormalskeletal patterning,cardiacanomalies andcleft developmental defectssimilartothosefound inhumanCdLS, chromatid cohesionfactor. Thesemicemanifestaspectrumof chromosome dynamicsorviaalternative functions. multicellular organisms, either throughabnormalitiesin cohesion factors areimportantforearlydevelopment of al., 2005).Theseobservations clearlyindicatethatsisterchromatid variable expressivity inthesemiceisalso a featuretypically broad spectrumofsymptoms withincompletepenetranceand model resemblinghumanCornelia deLangesyndrome.Infact, the A , In thiswork, wegeneratedmicedeficient in B Metaphases from mouseembryonicfibroblasts (MEFs)derived ) Pds5B –/– mice andpeoplewithCdLS(Table 1),andthefact that C ) Multi-speciesalignmentofAT hook domainsofPDS5B. –/– Pds5B MEFs lacksisterchromatid cohesiondefects. –/– mice shownodifferences insisterchromatid Pds5B –/– mice representthefirst mouse ␮ m. Development 134(17) . ( D Pds5B ) Subcellular , asister Pds5B –/–

DEVELOPMENT budding yeast,worm and (i.e. PSCS).Thisresultwas incontrasttothoseobtained cells didnotshow any abnormalitiesinsisterchromatidcohesion examination ofthechromosomesfrom between adjacentcohesincomplexes (Losadaetal.,2005).An a scaffolding proteinandpromoteprotein-protein interactions the dynamicsofcohesinring.Alternatively, PDS5mayactas shared bySCC2(KimuraandHirano,2000)therebymodulate act asaregulator oftheSMCATPase throughtheHEAT motifs for chromatidcohesion(Losadaetal.,2005).For example, itmay investigation, several organisms requirediverse functionofPDS5 hypomorphic. and becausethey mayresultinlethalityunlessthey are are dominantlyinherited(Krantzetal.,2004;Tonkin etal.,2004), may berarebecausethey would berecessive andmostCdLScases PDS5B that screeningCdLSpatientsfor association ofmutationsinothercohesionfactors andCdLSsuggest observed inCdLS.Thesestudiesandthepreviously known Cohesin necessaryfornormaldevelopment observed in lower organisms. For example, severe cohesiondefectshave been 2004). Supportforthisideacomesfromanumberofstudiesin expression (Krantzetal.,2004;Rollins1999;Tonkin etal., cis-elements andtranscriptionallyregulates developmental gene hypothesized thatthecohesincomplex directlybindsto regulatory Krantz etal.,2004;Tonkin etal.,2004).Inthisregard, ithasbeen functions otherthansisterchromatidcohesion(Dorsett,2007; diseases probablyresultfromabnormalcohesin-mediated the abnormalitiespresentinpatientswithCdLSandrelated 2005). Overall, theseresultsareconsistentwithourhypothesis that only mildcohesiondefectsin40%ofCdLSpatients(Kauretal., from CdLSpatients(Tonkin etal.,2004;Vrouwe2007)or reports ofthelacksisterchromatidcohesiondefectsincells (Losada etal.,2005).Furthermore,ourresultsareconsistentwith found inHeLacellswhich detected subtledefects.Indeed,onlymildcohesiondefectswere assay inwhichatotalof50cellswereexamined may nothave Hartman etal.,2000;Wang etal.,2003).Itispossiblethatour important roleinchromosomesegregation (Dorsett etal.,2005; important link in cohesinregulation of transcriptionthroughits Nathans, 1996).We proposethatPDS5Bmayconstitute an constitutes theDNA bindingdomainofHMGproteins(Maherand PDS5B hasidentified two AT hook domains,themotifthat the cohesincomplex or itsregulatory factors. Ouranalysisof DNA binding motifshave beenidentified inproteins thatcomprise directly regulate transcription hasremainedelusive becauseno do notundergo celldivision. How cohesincomplexes could to sisterchromatidcohesionasthesecellsarepost-mitoticand thus neurons, yetitsfunctioninadultneuronsisunlikely tobe related Watrin etal.,2006).PDS5B isalsoexpressed athighlevels in for neuronalmigrationandaxonalguidance(Seitanetal.,2006; expressed inthemammalianadultnervous systemandiscrucial et al.,2005).Furthermore,thecohesionprotein,MAU-2, ishighly the lethality, but notthecohesiondefectofthesemutants(Aguilar found inyeast et al.,2005).Yet anotherexample ofalternative Pds5Bfunctionis transcriptional regulation ofthedevelopmental gene sites betweenthewingmargin enhancerandpromoterleadto regard, directinteractionsofthecohesincomplex withmultiple (limb) development areobserved (Dorsett etal.,2005).Inthis heterozygotes, whereonlydefectsin Although themolecularfunctionofPDS5isstillunder mutations, unlessthey producedominantnegative mutants, Drosophila Pds5 Pds5 mutants, inwhichtopoisomeraseIIcanrescue Drosophila PDS5B homozygous mutants,but notin PDS5B cut was knocked down byRNAi gene expression andwing , wherePDS5playsan Pds5B mutations iswarranted. -deficient mouse cut (Dorsett Finally, normal development canbedisruptedbydefects inrRNA to maintainrDNA repeatcopy number(Kobayashi et al.,2004). complex isrecruitedby Sir2 tosilencedchromatinintherDNA loci (Bialkowska and Kurlandzka, 2002).Inaddition,thecohesin the defectscausedbymutation ofthecohesinproteinScc3 required for60Sribosomalsubunit maturation,isabletosuppress where anadditionalcopy of involvement inribosomalbiogenesiscomesfromstudiesyeast, Krogan etal.,2006).Furtherevidence ofcohesionprotein ribosomal RNA (rRNA) biosynthesis (Davierwala etal.,2005; interact with detected innucleoli(Andersenetal.,2005),andyeast other cohesionproteins(e.g.NIPBLandSMC1A)have alsobeen regulate rRNA metabolismandribosome biogenesis.Interestingly, that PDS5Band/orcohesincomplexes containing PDS5Bcould nucleolar localization.NucleolarlocalizationofPDS5Bindicates observation), suggestingthatthisdomainmaycontribute toits concentrated inthenucleolus(B.Z.andJ.M.,unpublished the secondAT hook motifwas localizedtothenucleusbut was not Interestingly, PDS5BlackingtheC-terminal142residuescontaining is localizedtothenucleuswithhigherlevels inthenucleolus. 2004; Maffini etal.,2002).Inthesestudies,wefoundthatPDS5B localized inthenucleusbyimmunohistochemistry(Kumar etal., these two closelyrelatedproteins. reflecting differences insubcellularlocalization(andfunction)of differential sensitivity toPDS5Blossinvarious organs orperhaps depletion; cohesion defectsin transcription. complex inproximitytopromoterelements regulate ability tobindAT richregulatory sequencesandbringthecohesin er eet 1 60 ND 14 60 75 + 70 + deLangesyndrome.2004); ND,notdetermined;CdLS,Cornelia – + + + feature hasnotbeenreported orobserved. ND 100 feature hasbeenreported orobservedmultipletimes and‘–‘indicatesthatthe The frequency ofthefeature isindicatedbynumbers; otherwise,‘+’indicatesthe 100 100 100 Sister chromatidcohesiondefects 27 93 + Heart defects Hearing loss 57 ~100 93 Gastrointestinal abnormalities Genital hypoplasia(arresteddevelopment) Limb-reduction defects/oligodactyly Micromelia (smallhandsandfeet) Cleft palate Micrognathia 84 Thin upperlip Ptosis + Microcephaly (smallhead) Mental retardation Growth retardation deLangesyndromebetween Cornelia andin + Table 1.Comparisonofclinicalandphenotypicfeatures 100 this willrequiregenerationof homolog PDS5A(Losadaetal.,2005).Physiologicalevidence for 1993); Pds5B found overlapping expression inanumberoforgan systemswhere expression (B.Z.andJ.M.unpublishedobservations). Indeed,we studies indicatesignificant overlap inPDS5AandPDS5BmRNA Both PDS5AandPDS5Bhave previously beenreportedtobe An alternative explanation forthe absenceofsisterchromatid b Superior cervicalgangliondefectsthatcanresult inptosis; –/– d Enteric nervoussystemdefects; mice manifestdevelopmental defects,suggesting NIP7 Pds5B and NOP7 –/– Pds5A mice couldberedundancy withits NOG2, , bothofwhichareinvolved in e a Data from Tonkin etal.(Tonkin etal., Data from Jacksonetal.(Jacksonal., RESEARCH ARTICLE –/– animals, but ourpreliminary which encodesaGTPase CdLS a e Pds5B (%) mice (%) c Germ cell –/– PDS5 mice Pds5B – 3199 d b can c –/–

DEVELOPMENT http://dev.biologists.org/cgi/content/full/134/17/3191/DC1 Supplementary materialforthisarticleisavailableat Supplementary material University SchoolofMedicine(HD001487). Research CenterofExcellenceinDevelopmentalBiologyatWashington Biology PathwayFellowship(toB.Z.).P.J. isaScholaroftheChildHealth Kidney DiseaseResearch (P30DK079333),andWashington UniversityCancer HD047396 (toS.J.),DK57038andDK6459201R.O.H.),O’BRIENcenterfor was supportedbyNIHgrantsCA111966,AG13730andNS039358(toJ.M.), We thankChih-LinHsiehforproviding thechromosomal analysis.Thiswork Deardorff, M.A.,Kaur, M.,Yaeger, D.,Rampuria,A.,Korolev, S.,Pie,J.,Gil- Davierwala, A.P., Haynes,J.,Li,Z.,Brost, R.L.,Robinson,M.D.,Yu, L., Bialkowska, A.andKurlandzka, Benard, C.Y., Kebir, H.,Takagi, S.andHekimi, Andersen, J.S.,Lam,Y. W., Leung,A.K.,Ong,S.E.,Lyon, C.E.,Lamond,A. Aguilar, C.,Davidson,Dix,M.,Stead,K.,Zheng,Hartman,T. and References cDNA, andMarkJohnstonfor Kasid Ushafor technical assistance.We are gratefultoGeorgeC.Enders forGCNA1antibody, Strickland, AmandaKnoten,Tatiana Gorodinsky andNinaPanchenkofor Simburger, ShellyAudrain,AmberNielson,JackShields,JenniferArmon,Amy review ofthemanuscriptand/orhelpfuldiscussionsdata.We thankKelli We are deeplyindebtedtoRobertH.BalohandJasonA.Gustinforcareful example, metabolism, aprocesslargely confined tothenucleolus.For 3200 Fu, M.,Vohra, B.P., Wind,D.andHeuckeroth, R.O. Enomoto, H.,Crawford, P. A.,Gorodinsky, A.,Heuckeroth, R.O.,Johnson, Dorsett, D.,Eissenberg,J.C.,Misulovin,Z.,Martens,A.,Redding,B.and Dorsett, D. Dixon, J.,Jones,N.C.,Sandell,L.L.,Jayasinghe,S.M.,Crane,Rey, J.P., related disorderswithmutationsincohesionfactors. functions ofthecohesincomplex andthepathogenesisofCdLS model tostudythemolecularmechanismsofdevelopmental cohesion. This via rolesunrelatedtoitsancientfunctioninsisterchromatid is acriticalregulator ofmultipleaspectsorganogenesis, probably observed inpatientswithCdLSorrelateddiseases. of cohesionproteinscouldalsocontribute totheabnormalities hypoplasia (Dixonetal.,2006).Deficits inthenucleolarfunctions craniofacial abnormalities,includingcleftpalateandmandibular in deficient neuralcrestcellmigrationandproliferationthatleadsto of craniofacial development (Dixonetal.,2006).Haploinsufficiency Treacher Collinssyndrome,anautosomaldominantdisorderof 80 de Langesyndrome withpredominant mentalretardation. cohesin complexmembersSMC3andSMC1Acauseamildvariantofcornelia M.,Loeys,B.,Kline,A.D.etal. Rodriguez, C.,Arnedo, interaction spectrumofessentialgenes. Mnaimneh, S.,Ding,H.,Zhu,Chen,Y. etal. cerevisiae. IST2 genessuppress thedeficiencyofcohesinIrr1p/Scc3pinSaccharomyces Development autonomously toguideaxonalmigrationsinCaenorhabditiselegans. I. andMann,M. cohesion. Saccharomyces cerevisiae pds5mutants,butnotthedefect insisterchromatid Guacci, V. regulates murineentericnervoussystemprecursor migration,neurite Development axonal growth andaxonguidanceofdevelopingsympatheticneurons. M., JrandMilbrandt,J. E. 4753. expression duringwingdevelopmentinDrosophila. McKim, K. abnormalities. cell formationandproliferation deficienciesthatcausecraniofacial Dixon, M.J.andTrainor, P. A. expression, development,and humansyndromes. In summary, wehave discovered thatthecohesionproteinPDS5B Tcof1 , 485-494. RESEARCH ARTICLE in micecausesareductionofmatureribosomesthatresults Tcof1 (2007). Rolesofthesisterchromatid cohesionapparatusingene Cell Cycle Acta Biochim.Pol. (2005). Topoisomerase IIsuppresses thetemperature sensitivityof (2005). Effects ofsisterchromatid cohesionproteins oncutgene PDS5A 131 128 Proc. Natl.Acad.Sci.USA Pds5B , whichencodesanucleolarprotein,ismutatedin , 5947-5958. , 3963-3974. (2005). Nucleolarproteome dynamics. 4 plasmid, KazusaDNAResearch InstituteforKIAA0979 , 1294-1304. -deficient mouseprovides thefirst mammalian (2001). RETsignalingisessentialformigration, 49 pRS315 , 421-425. (2006). Tcof1/Treacle isrequired forneuralcrest (2002). AdditionalcopiesoftheNOG2and plasmid andhelpwithyeastgaprepair. Nat. Genet. 103 , 13403-13408. Chromosoma (2004). mau-2actscell- (2005). Thesyntheticgenetic Development 37 (2006). BMPsignaling , 1147-1152. Nature Am. J.Hum.Genet. (2007). Mutationsin 433 116 132 , 77-83. , 1-13. , 4743- Honma, Y., Araki,T., Gianino,S.,Bruce,A.,Heuckeroth, R., Johnson,E.and Honaramooz, A.,Snedaker, A.,Boiani,M.,Scholer, H.,Dobrinski,I.and Hartman, T., Stead,K.,Koshland,D.andGuacci,V. Maffini, M.V., Geck,P., Powell,C.E.,Sonnenschein,andSoto,A.M. Gomperts, M.,Garcia-Castro, M.,Wylie,C.andHeasman,J. Geck, P., Maffini, M.V., Szelei,J.,Sonnenschein,C.andSoto,A.M. Losada, A.,Yokochi, T. andHirano,T. Le, N.,Nagarajan,R.,Wang, J.Y., Araki,T., Schmidt,R.E.andMilbrandt,J. Kaur, M.,DeScipio,C.,McCallum,J.,Yaeger, D.,Devoto,M.,Jackson,L.G., McLaren, A. Maher, J.F. andNathans,D. Jackson, L.,Kline,A.D.,Barr, M.A.andKoch,S. Ireland, J. M.,Donnai,D.andBurn, Ginsburg, M.,Snow, M.H.andMcLaren, A. Musio, A., Selicorni, A.,Focarelli,Musio, A.,Selicorni, M.L.,Gervasini,C.,Milani,D.,Russo, S., Kimura, K.andHirano,T. Jain, S.,Naughton,C.K.,Yang, K.,Encinas,M., M.,Strickland,A.,Vij, Kobayashi, T., Horiuchi,T., Tongaonkar, P., Vu, L.andNomura,M. Nasmyth, K.andHaering,C.H. Krantz, I.D.,McCallum,J.,DeScipio,C.,Kaur, M.,Gillis,L.A.,Yaeger, D., Kumar, D.,Sakabe,I.,Patel,S.,Zhang,Y., Ahmad,I.,Gehan,E.A., Krogan, N.J.,Cagney, G.,Yu, H.,Zhong,G.,Guo,X.,Ignatchenko,A.,Li,J., Naughton, C.K.,Jain,S.,Strickland,A.M.,Gupta,andMilbrandt,J. Panizza, S.,Tanaka, T., Hochwagen,A.,Eisenhaber, F. andNasmyth, K. Neuwald, A.F. andHirano,T. Rollins, R.A.,Korom, M.,Aulner, N.,Martens,A.andDorsett,D. Rollins, R.A.,Morcillo, P. andDorsett,D. developing sympatheticneurons. Milbrandt, J. Nature Schlatt, S. condensation inSaccharomyces cerevisiae. essential chromosomal protein required forbothsisterchromatid cohesionand mouse embryos. Interactions betweenprimordial germcellsplayarole intheirmigration mouse embryoduringgastrulation. AS3 asitsmediator. Androgen-induced proliferative quiescenceinprostate cancercells:therole of mediator ofproliferative arrest intheratprostate. (2002). Mechanismofandrogen actiononcellproliferation: AS3protein asa 118 cohesin-mediated cohesioninhumancellsandXenopuseggextracts. 299 viaalteredfasciculation, andpatterning Ncam1polysialicacidaddition. Sci. USA as aninhibitorofSchwanncelldifferentiation andmyelination. (2005). AnalysisofcongenitalhypomyelinatingEgr2Lo/LonervesidentifiesSox2 187-196. Mol. Cell.Endocrinol. deLangesyndrome.(PSCS) inCornelia Spinner, N.B.andKrantz,I.D. 5503-5513. clinical review of310individuals. Delineation oftheclinicalphenotype. HMG-1 proteins. 2714. to SMC1L1mutations. Vezzoni, P. andLarizza,L. condensin functions. disease anddelineateadirect role ofRetinspermatogenesis. expressing adominant-negativeRetmutationphenocopyhumanHirschsprung Golden, J.,Gupta,A.,Heuckeroth, R.,Johnson,E.M.,Jretal. recombination withinindividualrRNAgenesinyeast. SIR2 regulates recombination betweendifferent rDNArepeats, butnot kleisin complexes. homolog ofDrosophila melanogasterNipped-B. deLangesyndromeCornelia iscausedbymutationsinNIPBL,thehuman Jukofsky, L.,Wasserman, N.,Bottani,A.,Morris,C.A.etal. molecule, exhibitsreduced expression inhumanrenal carcinomas. Whiteside, T. L.andKasid,U. complexes intheyeastSaccharomyces cerevisiae. Pu, S.,Datta,N.,Tikuisis,A.P. etal. regulates spermatogonialstem cellfate. (2006). Glialcell-linederivedneurotrophic factor-mediated RETsignaling Genome Res. , andothercomplexesinvolved inchromosome-related functions. Curr. Biol. (2000). Pds5cooperateswithcohesinin maintainingsisterchromatid cohesion. enhancers inthecutandUltrabithorax genes. homologue ofchromosomal adherins,participatesinactivationby remote , 2133-2141. , 137-150. 418 102 10 (2000). Germandsomaticcelllineagesinthedevelopinggonad. , 778-781. (2002). Spermfrom neonatalmammaliantestesgraftedinmice. , 2596-2601. , 1557-1564. 10 (2002). Arteminisavascular-derived neurotropic factorfor , 1445-1452. Development Proc. Natl.Acad.Sci.USA Annu. Rev. Biochem. Proc. Natl.Acad.Sci.USA Proc. Natl.Acad.Sci.USA 163 Nat. Genet. (2000). Dualroles ofthe11Sregulatory subcomplexin , 3-9. (1996). MultivalentDNA-bindingproperties ofthe (2006). X-linked Cornelia deLangesyndrome(2006). X-linkedCornelia owing (2000). HEAT repeats associatedwithcondensins, 120 (2005). Thestructure andfunction ofSMCand (2004). SCC-112,anovelcellcycle-regulated Am. J.Med.Genet. Neuron (2005). Precocious sisterchromatid separation , 135-141. 38 Development (1993). Brachmann-deLangesyndrome. Am. J.Med.Genet. , 528-530. (2005). FunctionalcontributionofPds5to (2006). Globallandscapeofprotein Am. J.Med.Genet.A 74 Biol. Reprod. 35 (1999). Nipped-B,aDrosophila , 595-648. J. CellBiol. 93 , 267-282. (1990). Primordial germcellsinthe Genetics , 6716-6720. 97 97 Nat. Genet. , 10185-10190. Nature 110 Endocrinology , 11972-11977. (1993). deLangesyndrome: a Development 134(17) (2000). Pds5pisan Cell 47 , 521-528. 74 151 152 , 940-946. , 314-321. 440 117 47 , 613-626. , 577-593. , 959-964. 36 , 637-643. Development , 441-453. 138 (1994). Proc. Natl.Acad. , 631-635. (2004). 143 , 27-31. Gene (2004). Mice (2004). , 2708- (2004). J. CellSci. (2000). Dev. Biol. 328 131 , ,

DEVELOPMENT Cohesin necessaryfornormaldevelopment Song, H.,Lim,Paria,B.C.,Matsumoto,Swift,L.L.,Morrow, J., Schule, B.,Oviedo,A.,Johnston,K.,Pai,S.andFrancke,U. Tanaka, K.,Hao,Z.,Kai,M.andOkayama,H. Svaren, J.,Ehrig,T., Abdulkadir, S.A.,Ehrengruber, M.U.,Watson, M.A. Seitan, V. C.,Banks,P., Laval,S.,Majid,N.A.,Dorsett,D.,Rana,Smith,J., Tonkin, E.T., Wang, T. J.,Lisgo,S.,Bamshad,M.J.andStrachan,T. gene. stromalin/Scc3 cohesionfactortofacilitatelong-rangeactivationofthecut Drosophila nipped-Bprotein supportssisterchromatid cohesionandopposesthe implantation thatdirects subsequentdevelopment. crucial [correction ofA2alphadeficiencyiscrucial]for‘on-time’embryo Bonventre, J.V. andDey, S.K. e242. no phenotype-genotypecorrelation. Inactivating mutationsinESCO2causeSCphocomeliaandRobertssyndrome: mechanism. maintenance ofsisterchromatid cohesioninfissionyeastbyaunique identified bymicroarray analysis. and Milbrandt,J. link sisterchromatid cohesiontocellandaxonmigrationguidance. Bateman, A.,Krpic,S.,Hostert,A.etal. Genet. proteins deLangesyndrome. andflyNipped-B,ismutatedinCornelia NIPBL, encodingahomologoffungalScc2-typesisterchromatid cohesion Mol. Cell.Biol. 36 , 636-641. EMBO J. (2000). EGR1targetgenesinprostate carcinoma cells 24 20 , 3100-3111. , 5779-5790. (2002). CytosolicphospholipaseA2alphais J. Biol.Chem. Am. J.Hum.Genet. (2006). MetazoanScc4homologs (2001). Establishmentand 275 Development , 38524-38531. 77 , 1117-1128. (2005). 129 PLoS Biol. , 2879-2889. (2004). Nat. 4 , van Heemst,D.,James,F., Poggeler, S.,Berteaux-Lecellier, V. andZickler, D. Vega, H.,Waisfisz, Q.,Gordillo, M.,Sakai,N.,Yanagihara, I.,Yamada, M., Vrouwe, M.G.,Elghalbzouri-Maghrani,E.,Meijers,M.,Schouten,P., Wang, F., Yoder, J.,Antoshechkin,I.andHan,M. Watrin, E.,Schleiffer, A.,Tanaka, K.,Eisenhaber, F., Nasmyth,K.andPeters, Wang, S.W., Read,R.L.andNorbury, C.J. Wellik, D.M.andCapecchi,R. the mitoticandmeioticprograms. (1999). Spo76pisaconservedchromosome morphogenesisprotein thatlinks 37 that isessentialfortheestablishmentofsisterchromatid cohesion. syndrome iscausedbymutationsinESCO2,ahumanhomologofyeastECO1 van Gosliga,D.,Kayserili,H.,Xu,C.,Ozono,K.etal. repair. deLangesyndromeof Cornelia cells:evidenceforimpaired recombinational Mullenders, L.H.,Pastink,A.etal. Godthelp, B.C.,Bhuiyan,Z.A.,Redeker, E.J.,Mannens,M.M., meiosis andmitosis. elegans EVL-14/PDS-5andSCC-3are essentialforsisterchromatid cohesionin chromatid cohesion,andmitoticprogression. J. M. metaphase arrest. for accuratechromosome segregation andforsurvivalafterDNAdamageor to globally pattern themammalianskeleton. to globallypattern , 468-470. (2006). HumanScc4isrequired forcohesinbindingtochromatin, sister- Hum. Mol.Genet J. CellSci. Mol. Cell.Biol. . 16 , 1478-1487. 115 , 587-598. (2003). Hox10andHox11genesare required Cell 23 (2007). Increased DNAdamagesensitivity , 7698-7707. 98 , 261-271. RESEARCH ARTICLE (2002). FissionyeastPds5isrequired Science Curr. Biol. (2003). Caenorhabditis 301 16 , 363-367. (2005). Roberts , 863-874. Nat. Genet. 3201

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