schizoriza encodes a nuclear factor regulating asymmetry of stem cell di
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Current Biology 20, 452457, March 9, 2010 2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.01.018ReportSCHIZORIZAEncodesaNuclearFactorRegulatingAsymmetryofStemCellDivisionsintheArabidopsisRootColette A. ten Hove,1Viola Willemsen,1Wouter J. de Vries,1Anja van Dijken,1Ben Scheres,1and Renze Heidstra1,*1Molecular Genetics, Department of Biology,Faculty of Science, Utrecht University, Padualaan 8,3584 CH Utrecht, The NetherlandsSummaryCell divisionsgeneratingdaughter cellsdifferent insize,shape, identity, andfunctionareindispensablefor manydevelopmental processesincludingfatespecication, tis-sue patterning, and self-renewal. In animals and yeast,perturbationsinfactorsrequiredforwell-describedasym-metric cell divisions generally yield cells of equal fate. Herewereport onSCHIZORIZA(SCZ), asinglenuclear factorwith homology to heat-shock transcription factors that con-trolsthe separationof cellfateina setofstemcells gener-atingdifferent root tissues: root cap, epidermis, cortex,andendodermis. Loss-of-function, expression, andrecon-stitutionexperimentsindicatethat SCZactsmainlyfromwithin its cortical expression domain in the stem cell niche,exertingbothautonomousandnonautonomouseffectstospecifycortexidentityandcontrol theseparationof cellfates in surrounding layers. Thus, SCZ denes a novelpathway for asymmetric cell division in plants.Results and DiscussionAsymmetric cell division is a fundamental and universal mech-anismfor generating diversity and pattern in multicellularorganisms[1, 2]. Theradial organizationof theArabidopsisrootisderived fromstereotypedasymmetriccelldivisionsofdifferent stemcells, the initials. These cells and their daughtersproducedenedtissuelayers withdistinct cell fates (Fig-ure1A) [3]. Thestemcellssurroundasmall groupof rarelydividingcells, thequiescent center (QC), requiredfor theirmaintenance. The QC itself is formed early during embryogen-esis whenanasymmetricdivisionof thehypophyseal cellforms the lens-shaped QC progenitor cell and future columellaroot cap [4]. QC fate is specied in parallel by the PLETHORA(PLT), SHORTROOT(SHR), andSCARECROW(SCR) tran-scriptionfactors[57]. SHRandSCRarealsorequiredforground tissue patterning: shr and scr mutants lack the asym-metric periclinal division in the ground tissue stem cell daugh-ter, resulting in a single ground tissue layer. For shr, this layerlacks endodermal identity, but in scr, this layer displays mixedcortical/endodermal identity [810]. Several other reports haveappearedthatsuggestplantcellsmaypossessmixedfates[1113]. To our knowledge, the only known example of mixedfate phenotypes in animal development comes from extensivegenetic screening approaches in C. elegans, which haveuncoveredmutantsinwhichasingleneuronal fatedecisionisinappropriatelyexecuted,resultinginamixedfatepheno-type[14]. Here, wedescribetheSCHIZORIZA(SCZ) nuclearfactor, which is required for plant cell fate separation in severaltissues,actingbothcell-autonomouslyandnon-cell-autono-mously. Our datahighlight anovel mechanismof cell fateseparation in plants that is particularly relevant for asymmetriccell divisions within stem cell areas.SCZ Encodes a Member of the Heat-Shock TranscriptionFactor FamilyTo nd novel genes involved in QC specication and stem cellmaintenance, we performed a QC marker-based mutagenesisscreen. A line doubly homozygous for QC25 and QC46promoters [7] fused toERCFP andERYFP, respectively (Figures1Band1C), was mutagenized, andtheM2progeny wereanalyzedforalteredexpression.Fivephenotypicallyindistin-guishablemutantlinescombinedreducedQC25::ERCFPandQC46::ERYFP activity with retarded root growth and disorgani-zationof thestemcell niche(Figures1Dand1E; seealsobelow). Complementation tests showed that all ve linescarriedallelicmutations. Giventheequal allelestrength, wecontinued to use one allele, qc351, for further analysis.Compared to wild-type, qc351 roots developed more hairs,lacked the stereotypical pattern of alternating hair and nonhairles, and initiated root hairs fromsubepidermal tissue reminis-cent of the schizoriza (scz) mutant phenotype (see Figures S1AandS1Bavailableonline)[15].Complementationanalysisre-vealed that qc351 was allelic to scz. Accordingly, we renamedour mutant alleles scz-2 through scz-6.Wemolecularlycharacterizedthesczmutationviaamap-based approach. Fine mapping located SCZ to a single locusin an area of 70 kb on chromosome 1 (Figure 1G). One of thecandidate genes that we sequenced was the heat shock tran-scriptionfactor B4(HsfB4) locus(At1g46264), basedonitsstemcell-enrichedinsilicoroot expressionpattern(http://www.arexdb.org/;[1618].Comparisonwiththecorrespond-ingwild-typesequencerevealeddifferent mutationsintheHsfB4 gene for all scz alleles, including two additional TILLINGalleles (Figure 1G; Table S1). The identical phenotype indicatesthat they are likely HsfB4 mutant null alleles. For Arabidopsis,the 21 Hsfs are classied into three major groups, A (16 mem-bers), B(4 members, including SCZ), andC(1 member),according to the different exible linkers in their HR-A/B oligo-merizationregions(Figure1G,greenbar). Despiteconsider-ablediversicationinsizeandsequence,thebasicstructureof Hsfs is conserved among eukaryotes [19].SCZmRNAisrst detectedat triangular-stageembryosintheQCprogenitor cells. Fromheart stageonward, SCZmRNA accumulation expands into ground and vascular tissueprogenitorsandtheirimmediatedaughters(Figures2A2C).Thisexpressionpatternismaintainedinthepostembryonicroot with highest SCZ mRNA accumulation in QC and groundtissuestemcellsandtheirimmediatedaughters(Figure2D).Inthescz-2mutant, hybridizationsignal isabsent fromthesubepidermal layer (FiguresS2AandS2B). SCZpromoter-reporterfusionsessentiallycorroboratetheinsituhybridiza-tionexpressionpattern(FiguresS2CS2E). Consistent withits function as a putative transcription factor, the complement-ing35S::GFP:SCZtranslational fusion localizes tothe cellnucleus (see below). *Correspondence: [email protected] Asymmetric Cell Division from EmbryogenesisOnward in sczThe reduction in root growth together with altered expressionof the QC markers QC25::ERCFP and QC46::ERYFP in mutants(Figures 1D and 1E) might indicate a role for SCZ in QC/stemcell specication and/or function. However, scz-2 rootscontinue to grow in an indeterminate manner, with root lengthlaggingbyabouthalfbehindwild-type(Figure1F).Similarly,root meristemsizeisreducedbut maintainedinscz-2(Fig-ureS1C).TheseobservationsshowthatSCZisnotcriticallyrequired for stem cell maintenance.CellsatthepositionoftheQCandcolumellastemcellsinscz-2mutantrootsaremorphologicallyabnormal andaccu-mulate starch granules, which marks differentiated columellainwild-type(Figures2Fand2G). QC25, QC46, andQC184markers are displacedfromthe QCandexpress diffuse activityinthestarchgranule-containingcells. WOX5alsomarkstheQC,andthegeneproductisrequiredtomaintaintheunder-lyingcolumellastemcells. WOX5::ERGFPexpressionfadedfrom the position of the QC but did not appear in the columella(Figures2F2I; FiguresS2FS2I). Apparently, QCandcolu-mella fatesare presentbut notseparated inscz-2roots, andFigure 1. Identication and Cloning of SCZ(A) Schematicviewof theArabidopsisrootmeristem. Thefollowing abbreviations are used: En, endodermis; Co,cortex; Ep, epidermis; QC, quiescent center; Col, columella;LRC, lateral root cap.(BD) Four-day-old wild-type root expressing both QC46::ERYFP (B; arrowhead indicatesQC) andQC25::ERCFP (C)and4-day-oldqc351/scz-2root showingQC46::ERYFPexpres-sion (D; false green).(E) Eight-day-old wild-type and scz-2 through scz-6seedlings.(F)Rootlength(inmm)ofwild-typeandscz-2seedlingsattheindicatednumber of dayspostgermination(dpg). Foreachdatapoint,n R 57;errorbarsindicatestandarderrorof the mean.(G) Schematic representation of the identication of the SCZgenebypositional cloning. SCZ/HsfB4locationisshownrelativetoacontigof veBACclones; theproportionofrecombinant seedlings is shown in parentheses. Boxes indi-cate coding sequence. Conserved functional domainsaccordingto[19]areindicatedasfollows:gray,conservedDNA-binding domain; green, HR-A/Boligomerization region;orange, nuclear localization signal (NLS); blue, nuclearexport signal (NES); purple, R(R/KLFGV) motif [33]. The posi-tionsof thenucleotidesequencechangesareshownforeach mutant allele; scz-7 andscz-8 are TILLINGalleles[34]. See also Figure S1.Figure 2. SCZ Expression and Postembryonic Mutant StemCell Defects(AD) In situ hybridization with SCZ antisense probe in wild-type triangular- (A), heart- (B; black arrowhead marks groundtissueaccumulation), andtorpedo-stage(C) embryoand2-day-old seedling (D). White arrowheads in (A)(D), (F), (K),and (L) indicate QC.(E and J) Heart-stage wild-type (WT; E) and scz-2 (J)embryos. Inset showsmagnicationof normal anticlinallydivided ground tissue initial (E; green arrowhead) and aber-rant periclinallydividedgroundtissueinitial (J; redarrow-head).(F and G) QC184 expression (blue) in 6-day-old wild-type (F)shifts in the columella in scz-2 (G) roots. Red arrow indicatescolumella stem cells devoid of purple starch granules; blackarrowindicatesaccumulationofstarchgranulesinmutantQC region.(H and I) WOX5::ERGFP expression in 4-day-old wild-type (H)and scz-2 (I) roots.(KandL) Anilineblue-stainedwild-type(K) andscz-2(L)matureembryos. Whitearrowindicatesendoflateral rootcap layer, dashed line indicates root/hypocotyl boundary, #indicates ground tissue layers in hypocotyl, * indicatespericlinalgroundtissuedivisionsleadingto supernumerarymutant layers in scz-2. See also Figure S2.SCHIZORIZA Regulates Cell Fate Separation453the separation defect does not affect stem cell niche activity.Interestingly, thisobservationof mixedfatesisreminiscentoftheformationof roothairsfromsubepidermal tissuesre-ported when scz mutants were rst described [15].To gain more insight into the possible role for SCZ as a fatedeterminationand/orseparationfactor, wetracedthescz-2defectsbacktotheir embryonicorigin. Theinitial defect inscz-2occursinheart-stageembryos, wheregroundtissueinitials performan aberrant periclinal division (Figure 2J),resultingintheectopicgroundtissuelayerobservedattor-pedostage([15]; datanot shown). Theinner groundtissuedaughtercellscontinuetoperformadditional periclinal divi-sionsgeneratingmoreectopiclayers(Figure2L, asterisks).Theorganizationof QC, columella, pericycle, andvascula-tureappearsnormal throughout embryogenesis. Inmaturewild-typeembryos, anadditional cortical layer proximal tothe endof the lateral root capmarks the hypocotyl (Fig-ure 2K; [20]). Similarly, in scz-2, the hypocotyl possessesthreegroundtissuelayers(Figure2L). However, thelateralroot cap layer contains fewer cells, and epidermal cells belowthe root-hypocotyl junction appear long and at, morphologi-cally reminiscent of lateral root cap cells suggestive of mixedfates(Figure2L, proximal toarrow). TheseobservationsareFigure 3. SCZ Species Cortex and Segregates Cell Fates inthe Root NicheThe following abbreviations are used: WT, wild-tpe; ep,epidermis; lrc, lateral root cap; c, cortex; e, endodermis.* indicates mutant subepidermal layer, arrowhead indicatesQC, and square bracket indicates ectopic endodermallayers.(AH) WER[ERCFP (false red) and SMB:GFP expression inbent cotyledon-stage wild-type (Aand B) and scz-2 (Cand D)embryosandin4-day-oldwild-type(EandF) andscz-2(G and H) roots. Arrows in (G) and (H) indicate WER[ERCFPand SMB:GFP expression in distal subepidermal cells.(IL) Radial root tissue sections of 4-day-old wild-type (I andJ) andscz-2 (Kand L) roots stained for GL2::GUSexpression.scz-2 subepidermal cells express GL2::GUS (* in K) and formroot hairs (# in L).(MandN) Embryonicroot Co2::H2BYFPexpressioninlatetorpedo-stage wild-type embryo (M) is absent in scz-2embryo (bracket in N).(OandP) WOX5::ERGFPexpressioninlatetorpedo-stagewild-type (O) and scz-2 (P) embryos.(QV)Four-day-oldwild-type(Q,S,andU)andscz-2(R,T,and V) roots expressing SHR::SHR:GFP (Q and R),SCR::H2BYFP(SandT),andCo3::H2BYFP(UandV).FadingSCR::H2BYFPexpression in periclinally divided ground tissuestemcell daughter (arrowin S) is maintained in scz-2 subepi-dermal cell patch(arrowsinT). Co3::H2BYFPexpressionislost fromthemutant subepidermal layer inscz-2(* inV).See also Figure S3.consistent with a role for SCZ in cell fate separa-tion from heart-stage embryogenesis onward.Non-Cell-Autonomous Regulationof Epidermis/Lateral Root Cap Fate SeparationToprobetheidentityofthescz-2epidermis,weanalyzed the promoter activity of the WEREWOLFgene (WER; [21]) and accumulation ofSOMBRERO(SMB):GFPprotein[22] inembryosandroots.WERhasaroleinepidermal cell fatespecication,andWER[ERCFPisexpressedinlateral root cap and epidermis, including thestemcell inwild-type (Figures 3A, 3B, 3E, and3F). SMBrepresses stem cell-like divisions in the root cap, and accord-ingly, SMB::SMB:GFP is expressed in nuclei of root cap stemcelldaughtersandmaturingrootcaplayers(Figures3A,3B,3E, and 3F). In scz-2 embryos, SMB:GFP is also expressed inepidermal cells, and both markers even extend to the subepi-dermal cells (Figures3C and 3D). In the distal root meristem,theepidermal expressionoverlapismaintained,butsubepi-dermal expressionisobservedonlyinthedistalmost cells,includingthestemcells(Figures3Gand3H, arrows). Next,we combined GLABRA2 promoter expression marking devel-oping nonhair cells (GL2::GUS; [23]) with transverse rootsectionstoexaminecell numberandshapetypical fortheirdifferentiationstatus(Figures3I3L).Cross-sectionsofscz-2root meristemsreveal that vascular tissuesarenormal butthat adoublelayer of endodermis-likecellsispresent (Fig-ure3K). Epidermal cell number isreducedfromthenormalw22 rectangular-shaped cells to w12 ellipsoidal cells resem-bling the underlying cortex-like cells (Figures 3I and 3K).An additional layer with lateral root cap morphology surroundstheepidermis. GL2::GUSisweaklyexpressedinalmost allepidermal and in fewsubepidermal cells (Figure 3K). Strikingly,whereas epidermal cell les are tightly connected in theCurrent Biology Vol 20 No 5454wild-type, they separate as tissues mature in scz-2, a featurethat is restricted to root cap cells in the wild-type (Figure 3L).Our data indicate that scz-2 root epidermal and subepidermaltissues are compromisedin fate segregation. Importantly,thenearesttissueexpressingSCZisthecortex, suggestingthat SCZ acts non-cell-autonomously in epidermis/lateralroot cap fate separation.SCZ Is Required for Cortex Fate SpecicationInitial characterizationofsczrevealedectopicexpressionofepidermalmarkers into the groundtissue and misexpressionof a ground tissue marker [15]. To further investigate the fate ofthemutantgroundtissuelayers, weanalyzedexpressionofendodermal markers. SHR::SHR:GFPis expressedinstelecells, andtheproteinmovestoaccumulateinthenuclei ofQC, ground tissue stem cells, and endodermis, where it acti-vatesitstarget,SCR(Figure3Q;[10]).Uponpericlinalasym-metricdivisionofthegroundtissuestemcelldaughter,SCRpromoter activity is rapidly shut down in the outer cortex cell(Figure3S,arrow;[24]).InlinewiththeQCdefectsbywhichwe selected scz mutants, SHR:GFP and SCR::H2BYFP expres-sion is absent fromthe QC region (Figures 3R and 3T,arrowhead). In the scz-2 ground tissue, SHR:GFPis onlyobserved in the innermost layer adjacent to the stele(Figure3R), whereasSCR::H2BYFPexpressionextendsintothenext layer (Figure3T). Takingthesedatatogether withthecell morphologycharacteristics, weconcludethattheselayersrepresent endodermis. Inaddition, SCR::H2BYFP, likeFigure4. All SCZFateSeparationFunctionsAreEnabledfrom the Cortex Expression DomainArrowhead indicates QC.(AD) Five-day-old 35S::WOX5 (A) and scz-2 35S::WOX5 (B)roots expressingQC46::ERYFP, and4-day-oldsmb-3 (C)andscz-2smb-3(D) roots. Introductionof 35S::WOX5orsmb-3correctlysegregatesQC(BandD) andcolumellastem cell (blue arrow in D) fates.(EH) Four-day-old35S::SCZroots expressingQC25 (E),SMB::SMB:GFP(F), WER[ERCFP(G), andCo2::H2BYFP(H; false green).(I) Four-day-old 35S::GFP:SCZ roots showing nuclear accu-mulationof GFP:SCZfusionprotein. SCZoverexpressioninducesformationof additional columellastemcells(bluearrows in E and F), an ectopic epidermal layer (* in EI), andectopic Co2::H2BYFP expression (white arrow in H) inprogenyofsupernumeraryepidermis/lateral rootcapstemcells (white arrow in GI).(J) Wild-type N9094 root.(KandL)N9094[SCZ-complementedscz-2rootsdisplaynormal tissuearrangementandmarkerexpression(K) andcorrectly segregated QC and columella stem cell fates (L).(M) Role of SCZ in asymmetric division. See text for details.gtc indicates ground tissue stem cell. See also Figure S4.WER[ERCFPandSMB:GFP,evenextendsintothe distal subepidermal tissue (Figure 3T, arrows).Apparently, SCR::H2BYFP fails to segregate to theendodermis in a timely manner and is maintainedindependentlyof SHRpresence. Thissuggeststhat SCRpromoter downregulationinwild-typeisaidedbycorticallyexpressedSCZ. Similarly,the ectopic endodermis division can be explainedby coexpression of SHRand SCRresulting in divi-sionofthegroundtissue,asinwild-type,butintheabsenceof SCZ, theouter cell lefailstoadopt cortex fate.TofurthersubstantiateacausallinkbetweenSCZexpres-sion and cortex fate determination, we analyzed Co2::H2BYFPandCo3::H2BYFP, whicharehighlyexpressedinthecortexbut excluded from the QC and undivided ground tissue stemcellsfromembryogenesisonward(Figures3U; FigureS3A).Occasional weak expression in endodermal cells is observedinthewild-type. Importantly, inscz-2seedlingroots, bothmarkers are rarely expressed, and if so, onlythe weakendo-dermal expression is observed (Figure 3V; Figure S3B).Already during embryogenesis, Co2::H2BYFPexpression isexcluded from the root ground tissue but remains expressedin the hypocotyl ground tissue, consistent with the root-specicdefectsinscz-2(Figure3N). WeconcludethatSCZis necessary for the specication of root cortex cell identity.TotestwhetherSCZissufcienttodeterminecortexfate,weectopicallyexpressedSCZfromthe35Spromoter.DistaltotheQCin35S::SCZroots, thepresenceof anadditionalcolumella stem cell layer is apparent by the absence of starchgranulesandlackof SMB::SMB:GFPexpressionthatmarksdifferentiatedcolumella(Figures4Eand4F). Examinationofradial tissue markers reveals the formation of an ectopicepidermallayerthatshowsactivityoftheWER[ERCFPandGL2[ERGFP epidermal markers (Figures 4G and 4H; FiguresS4AandS4B). Cross-sectionsreveal occasional misexpres-sionof GL2[ERGFP(Figure S4C). Inaddition, the lateralrootcaplayerdoesnotsloughoffintheproximal meristem(FiguresS4EandS4F). WeconcludethatoverexpressionofSCZ introduces new cell fate separation defects.SCHIZORIZA Regulates Cell Fate Separation455Analysis of SCZ protein expression in 35S::GFP:SCZ overex-pressionlinesrevealsrelativelyhighlevelsinthecolumella,lateral root cap, and distal epidermal tissues correlatingtoregionsof ectopiccell division(Figure4I). Interestingly,WER[ERCFPexpressionislost fromtheepidermis/lateralrootcapstemcellregionwhereitisnormallyobserved(Fig-ure 4G; compare to Figures 3E and 3F), suggesting inhibitionofepidermal/lateralrootcapfate.WER[ERCFPandGL2[ERGFP are (re)expressed in outer layers proximally (Figure 4G;FigureS4B),suggestingtransientinhibitionofepidermalfatebyhighlevelsof SCZor, alternatively, higher sensitivityofthe stemcell regiontotheSCZeffect. Strikingly, ectopicexpressionof Co2::H2BYFPisobservedinthesehigh-GFP:SCZ-expressing root cap cells (Figure 4H), corroborating thatSCZexpressionissufcienttoinducecortexfateandinhibitepidermal and lateral root cap fates.Cortex-Expressed SCZ Rescues Mutant DefectsThe lack of distal QC and columella fate separation promptedus to examine embryonic QC25, QC46, QC184, SCR::H2BYFP,and WOX5::ERGFP marker expression. Surprisingly, allmarkerswereappropriatelyexpressedinscz-2throughoutembryogenesis, indicating that cell identities are correctly setupbutcannotbemaintained,whichcorrelateswithreducedstem cell activity and root growth (Figures 3O and 3P; FiguresS3CandS3D;datanotshown).Wetestedwhethercompro-misedstemcell activitymight causethefailuretoseparatecell fates by crossingscz-2 toWOX5-overexpressingandsmb-3knockout mutantsthat displayincreaseddistal stemcell activityandnumbers[22, 25]. Interestingly, scz-235S::WOX5andscz-2smb-3double-mutant rootsdisplaywild-typeQCmorphologyandpositioningasvisualizedbystrongre-expressionof QC46::ERYFPandSCR::H2BYFPintheQC(Figures4Aand4B; FigureS4D). Lackof starchstaininginscz-2 smb-3 reveals presence of columella stem cells, indica-tive of improved QC function (Figure 4D). However, the radialcell fateseparationdefectsremainandroot growthisnotrescuedinthesedouble-mutant combinations (Figures 4Band4D;FigureS4G). Ourdataindicatethatmixedcell fatesarenot dependent onQCfunction. Theremaininggrowthdefect is reminiscent of that observed when scr mutantsarecomplementedby QC-expressedSCR, whichrestoresafunctional stemcell nichebutnotcell fateseparationandgrowth [7].TodeterminewhereSCZactstopromotecell fatesep-aration, we reintroducedthe gene in specic tissues andexaminedcomplementationof thescz-2mutantdefect. TheGAL4VP16-UAStransactivationsystem[26] wasadoptedtodriveexpressionofSCZandtheERGFPreportervia(1)WOL(stele), WOX5(QC), andGL2(epidermis) promotersinscz-2and (2) scz-2 N9135 (endodermis and QC) and N9094 (cortex,endodermis, and occasional QC) enhancer trap crosses. Strik-ingly, thescz-2mutant couldbecompletelyrescuedintheN9094[SCZ scz-2 line, with restoration of growth, QC func-tion, groundtissue, andepidermis(Figures 4J-4L; FiguresS4G and S4H). None of the other drivers was able to comple-mentscz-2(FiguresS4IS4L), indicatingthatSCZactivityisrequiredinthecortextoexert itseffect onthecorrect fatesegregation in surrounding tissues.OurstudiesindicatethatSCZactsasafatedeterminationandseparationfactor(Figure4M). SCZactioninthegroundtissueinitial determinesitsfatefromembryogenesisonwardandsuppressesepidermisandlateral root capfateinthegroundtissue. Non-cell-autonomous SCZactionmaintainsQC fate and suppresses columella fate in the QC and in addi-tion segregates epidermis and lateral root cap fate, putativelythrough a ground tissue-derived factor X. After the SHR/SCR-induced periclinal division of the ground tissue stemcelldaughtertakesplace, SHRpromotesendodermal fate. SCZaction promotes cortex fate and suppresses endodermalfate, possiblybydownregulationof SHR/SCRexpressioninthematuregroundtissue. Furthermore, SCZcontinues itsnon-cell-autonomous suppressionof epidermis andlateralroot capfate in the mature ground tissue. The ability to expressdifferentiatedcharacteristicsof epidermis, lateral root cap,QC, andcolumellacell typesimpliesthat thedifferentiationpathwaysforthesetissuesarestill intactanddonotrequirea functional SCZ gene.Although SCZ belongs to the Hsf family, diverse microarrayanalyses show that SCZ hardly responds during stress situa-tions, suggestingthat it might beintegratedintosignalingpathways not directly relatedtothe heat-shock response[2729]. The shepherdmutant that harbors a mutation oftheER-specicHSP90producesoral andshoot meristemphenotypes closely resembling those of the clavata (clv)mutants,showingmore diverse rolesfor Hsfsand Hspsthansolely in stress signaling [30]. In addition to their role in adap-tationtostress, yeastandanimal HsfsandHspshavebeendemonstratedtobeinvolvedindifferentiationanddevelop-ment (reviewedin[31]. Our discoverythat theArabidopsisSCZgeneiscrucial forcellfateseparationsuggestsanovelmechanismof asymmetriccell divisioncontrol byHsfs, inwhich key determinants that are sequestered into bothdaughter cellsaredifferentiallydegraded. Recent workonthe membrane protein BASL in stomatal precursors cellsalsoindicatesthat noncanonical mechanismscontrol plantasymmetric cell division [32]. Future work will have to establishwhetherandhowsuchnovel factorscanbeintegratedwithwell-established mechanistic frameworks for asymmetric celldivision in other kingdoms of life.Supplemental InformationSupplemental Informationincludesfour gures, twotables, andSupple-mentalExperimentalProceduresandcanbefoundwiththisarticleonlineat doi:10.1016/j.cub.2010.01.018.AcknowledgmentsWe are grateful to M. Pernas and L. Dolan for sharing results prior to publi-cation. We thank A. van Aelst of the Wageningen University Electron Micros-copy Center for help with scanning electron microscopy.This project wasconancedbytheCentreforBioSystemsGenomics, whichispartoftheNetherlands Genomics Initiative/Netherlands Organisation for ScienticResearch.Received: November 25, 2009Revised: January 1, 2010Accepted: January 6, 2010Published online: February 18, 2010References1. Ten Hove, C.A., and Heidstra, R. (2008). Who begets whom? Plant cellfatedeterminationbyasymmetriccell division.Curr.Opin.PlantBiol.11, 3441.2. Knoblich, J.A. (2008). Mechanisms of asymmetric stemcell division. Cell132, 583597.3. Benfey, P.N., and Scheres, B. (2000). Root development. Curr. Biol. 10,R813R815.4. Ju rgens, G., and Mayer, U. (1994). Arabidopsis. In Embryos: Color Atlasof Development, J. Bard, ed. (London: Mosby-Year Book).Current Biology Vol 20 No 54565. Aida, M., Beis, D., Heidstra, R., Willemsen, V., Blilou, I., Galinha, C.,Nussaume, L., Noh, Y.S., Amasino, R., andScheres, B. 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