RESEARCH ARTICLE STEM CELLS AND REGENERATION

WOX13-like genes are required for reprogramming of leaf andprotoplast cells into stem cells in the moss Physcomitrella patensKeiko Sakakibara1,2,3,*,‡, Pascal Reisewitz4,5,‡, Tsuyoshi Aoyama1,6, Thomas Friedrich4, Sayuri Ando1,2,7,Yoshikatsu Sato1,2,8,9, Yosuke Tamada1,6, Tomoaki Nishiyama1,2,10, Yuji Hiwatashi1,6,11, Tetsuya Kurata1,2,12,Masaki Ishikawa1,2,6, Hironori Deguchi3, Stefan A. Rensing4,13, Wolfgang Werr5, Takashi Murata1,6,Mitsuyasu Hasebe1,2,6,§ and Thomas Laux4,§

ABSTRACTMany differentiated plant cells can dedifferentiate into stem cells,reflecting the remarkable developmental plasticity of plants. In themoss Physcomitrella patens, cells at the wound margin of detachedleaves become reprogrammed into stem cells. Here, we report thattwo paralogous P. patens WUSCHEL-related homeobox 13-like(PpWOX13L) genes, homologs of stem cell regulators in floweringplants, are transiently upregulated and required for the initiation of cellgrowth during stem cell formation. Concordantly, Δppwox13l deletionmutants fail to upregulate genes encoding homologs of cell wallloosening factors during this process. During themoss life cycle, mostof the Δppwox13lmutant zygotes fail to expand and initiate an apicalstem cell to form the embryo. Our data show that PpWOX13L genesare required for the initiation of cell growth specifically during stem cellformation, in analogy to WOX stem cell functions in seed plants, butusing a different cellular mechanism.

KEY WORDS: Stem cell, WOX, Physcomitrella

INTRODUCTIONDifferentiated cells can dedifferentiate under both natural andartificial conditions. In mice and humans, artificial expressionof transcription factors, for example, can cause transformation ofdifferentiated somatic cells into pluripotent stem cells (reviewed byMasip et al., 2010). Stem cell regeneration from differentiated cells

occurs more readily in plants, reflecting the remarkable plasticity ofplant cell identity (Birnbaum and Sanchez Alvarado, 2008). It canoccur under natural conditions (Steeves and Sussex, 1989) but alsoafter wounding or addition of phytohormones (Skoog and Miller,1957). In seed plants, experimentally induced regeneration caninvolve the formation of a mass of disorganized tissue, called thecallus, from which a complete plant can subsequently be obtained,but regeneration without an intermediate callus stage has also beenreported (Williams and Maheswaran, 1986; Vogel, 2005; De Smetet al., 2006; Garces et al., 2007; Sena et al., 2009). In non-seedplants, differentiated cells can generate stem cells without therequirement of exogenously supplied hormones and withoutforming a callus (Chopra and Kumra, 1988; Raghavan, 1989). Inthe moss Physcomitrella patens (Physcomitrella), differentiatedcells at the wound margin of detached gametophore leaves arereprogrammed into apical stem cells, which subsequently producethe filamentous chloronemata by asymmetric divisions (Prigge andBezanilla, 2010; Ishikawa et al., 2011). Although severaltranscription factors involved in the induction of reprogramminghave been characterized in flowering plants (Banno et al., 2001;Gordon et al., 2007; Sugimoto et al., 2010; Iwase et al., 2011), noregulators have been reported in non-seed plants.

Seed plants contain three subclades of WUS-related homeobox(WOX) genes (Haecker et al., 2004). First, several members of theWUSCHEL (WUS) subclade are involved in stem cell maintenance:WUS in the shoot meristem (Laux et al., 1996), WOX5 in the rootmeristem (Kamiya et al., 2003; Sarkar et al., 2007), WOX3 in leafmarginal meristems (Shimizu et al., 2009; Nakata et al., 2012) andWOX4 in the vascular meristem (Hirakawa et al., 2010; Ji et al.,2010). Expression ofWUS promotes pluripotent stem cell fate in theshoot apical meristem of Arabidopsis thaliana (Arabidopsis) from asmall underlying group of cells, named the organizing center(Mayer et al., 1998; Schoof et al., 2000). In addition, WUS andWOX5 are involved in the de novo formation of meristemsand somatic embryos from differentiated cells (Zuo et al., 2002;Gallois et al., 2004; Gordon et al., 2007; Chen et al., 2009; Sugimotoet al., 2010). Second, two members of the WOX9 clade, WOX8 andWOX9, redundantly regulate zygote development and embryo axisformation (Breuningeret al., 2008;Ueda et al., 2011) andWOX9has anadditional role in shoot meristem regulation (Wu et al., 2005). Finally,recent reports show that members of the WOX13 clade affect replumdevelopment, and dehiscence, flowering time, lateral root formation,and fertility (Deveaux et al., 2008; Romera-Branchat et al., 2012).

The WOX13 clade appears to contain the evolutionarily basalmembers, whereas the WUS and WOX9 clades diverged duringthe seed plant lineage. Concordantly, all WOX genes found in thePhyscomitrella genome group into the WOX13 clade (Deveauxet al., 2008; van der Graaff et al., 2009; Nardmann et al., 2009). TheReceived 11 April 2013; Accepted 13 February 2014

1National Institute for Basic Biology, Okazaki 444-8585, Japan. 2ERATO, JapanScience and Technology Agency, Okazaki 444-8585, Japan. 3Department ofBiological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan. 4BIOSS Centre for Biological Signalling Studies,Faculty of Biology, Albert-Ludwigs-Universitat Freiburg, Freiburg 79104, Germany.5Institut fur Entwicklungsbiologie, Universitat Koln, Koln 50674, Germany. 6Schoolof Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585,Japan. 7Department of Plant Sciences, University of Oxford, South Parks Road,Oxford OX1 3RB, UK. 8Graduate School of Science, Nagoya University, Furo-cho,Chikusa-ku, Nagoya, Aichi 464-8602, Japan. 9JST, ERATO, HigashiyamaLive-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi464-8602, Japan. 10Advanced Science Research Center, Kanazawa University,Kanazawa 920-0934, Japan. 11National Plant Phenomics Centre, Institute ofBiological, Environmental and Rural Sciences, Aberystwyth University, AberystwythSY23 3EB, UK. 12Plant Global Education Project, Graduate School of BiologicalSciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.13Faculty of Biology, University of Marburg, Marburg, Germany.*Present address: Graduate School of Science, University of Tokyo, 7-3-1 Hongo,Tokyo 113-0033, Japan.‡These authors contributed equally to this work

§Authors for correspondence (mhasebe@nibb.ac.jp; laux@biologie.uni-freiburg.de)

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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absence ofWUS-clade WOX genes in mosses raises the question ofhow stem cells are regulated. In contrast to the complex meristemsof seed plants, growth of mosses depends on single, apical stemcells that undergo asymmetric cell divisions (Kofuji and Hasebe,2014). In the gametophytic phase of Physcomitrella, chloronemaand caulonema stem cells form filaments (Prigge and Bezanilla,2010) and the gametophore stem cell gives rise to a leafy shoot(Harrison et al., 2009). In the sporophytic phase, the division of thezygote generates an apical stem cell that gives rise to the embryo byseveral asymmetric divisions, and a basal cell that forms part of thefoot structure (Kofuji et al., 2009). Here, we report that loss-of-function mutants of the Physcomitrella WOX13-like genes showaberrations during stem cell formation from the zygote, protoplastsand detached leaves. Phenotypic and molecular analysis implies acrucial role for mossWOX13-like genes in the activation of cellulargrowth programs.

RESULTSPpWOX13-like genes are upregulated during stem cellformation in detached leavesThree loci containingWOX-related sequences in the Physcomitrellagenome (Nardmann and Werr, 2007; Richardt et al., 2007) clusterwith the Arabidopsis WOX13 clade (Deveaux et al., 2008; van derGraaff et al., 2009). Given that ArabidopsisWOX10 andWOX14 inthis clade represent Brassicaceae-specific duplications of anancestral WOX13-like gene (Deveaux et al., 2008), we named themoss homologs PpWOX13-like (PpWOX13L) genes (Fig. 1A). Wedetected transcripts of PpWOX13LA [DDBJ/EMBL/GenBankaccession number: AB699867, formerly PpWOXA (Nardmannand Werr, 2007) and PpaWOX02 (Deveaux et al., 2008)] andPpWOX13LB [accession number: AB699868, formerly PpWOXB(Nardmann and Werr, 2007) and PpaWOX01 (Deveaux et al.,2008)] by RT-PCR throughout the moss life cycle, whereas notranscript for PpWOX13LC was detectable at any developmentalstage examined (Fig. 1B).To investigate the spatiotemporal expression patterns of

PpWOX13LA and PpWOX13LB proteins, we generated severalknock-in reporter lines by inserting a fluorescent protein codingsequence just before the stop codon of each gene (supplementarymaterial Fig. S1A-H). Three different plants each of at least twoindependently generated reporter lines were studied. All differentPpWOX13LA and PpWOX13LB reporter lines displayedindistinguishable expression patterns (Fig. 1D-M; supplementarymaterial Fig. S1I-R) and no aberrant phenotypes were detected,suggesting that the observed expression patterns are authentic. Forbrevity, we focus on the PpWOX13LA-Citrine and PpWOX13LB-Citrine reporter lines. Fluorescent signals of both fusion proteinswere detected in nuclei of all tissues examined: protonemata, youngand mature gametophores, antheridia, archegonia, unfertilized eggcells, zygotes, sporophyte cells, and spores (Fig. 1D-M;supplementary material Fig. S1I-R). Notably, the signal wasstronger in the apical stem cells of chloronemata and caulonematacompared with subapical cells (Fig. 1D,E; supplementary materialFig. S1I,J, arrows), and in egg cells and zygotes compared withsurrounding cells (Fig. 1I,J; supplementary material Fig. S1N,O,red arrows).In order to investigate PpWOX13L expression during the induced

formation of stem cells, we used a detached leaf assay, in whichchloronema stem cells are established from reprogrammeddifferentiated leaf cells (Ishikawa et al., 2011). We observed thatwhole-leaf PpWOX13LA and PpWOX13LB transcript levelstransiently increased after detachment (Fig. 1C). To investigate

protein levels at a cellular level, we used the reporter lines and foundthat the expression of both reporters slightly increased in most leafcells until 12 h after detachment and then specifically in the cellsfacing the cut from which stem cells were generated (Fig. 1N;supplementary material Fig. S1S and Movie 1). During subsequentchloronema growth, the signal remained stronger in the apical stemcell than in its descendants.

PpWOX13-like genes are required for outgrowth anddevelopment of the zygoteTo investigate the role of PpWOX13L genes, we generated severalindependent deletion mutants (supplementary material Fig. S2A-I),replacing each gene via homologous recombination either with acassette of a GUS reporter gene and a selection marker(Δppwox13la1 and Δppwox13lb1) or with only a selection markercassette (Δppwox13la2 and Δppwox13lb2). Both single mutants didnot display any robust differences compared with thewild type at anystage of the life cycle (data not shown). Although we observedattenuated fertilization, zygote expansion, and subsequent celldivisions in the single deletion lines (Δppwox13la2 andΔppwox13lb2; supplementary material Table S3), they eventuallyexhibited normal sporangium formation rates and furtherdevelopment compared with wild type (supplementary materialTable S2). In the double mutant (Δppwox13lab), chloronemata,caulonemata and gametophores were also indistinguishable fromwild type (supplementary material Fig. S2J-O). Expression ofPpWOX13LC was not detected by RT-PCR in the double mutant asin the wild type (supplementary material Fig. S3). In contrast to wildtype, we observed fewer sporophytes in two independent doubledeletion mutant lines (Fig. 2A,B; supplementary material Table S2).A detailed confocal microscopic analysis revealed that the doublemutant is able to form sperm and an egg cell, and that it displayedsperm in the archegonium cavity (Fig. 2D), a brownish archegoniumneck (supplementary material Table S2) and stronger auto-fluorescence in the nucleus than in the cytoplasm (Tanahashi et al.,2005), and in these respects was indistinguishable from the wild type(Fig. 2C-F; supplementary material Table S3). The last three criteriaare indicative of fertilization in wild type (Tanahashi et al., 2005).Therefore, although we cannot exclude alternative explanations,these findings strongly suggest that fertilization has taken place in thedouble mutant.

After fertilization, the wild-type zygote expands by dispersedgrowth, reaching the size of the archegonial cavity before undergoingan asymmetric cell division into an apical stem cell and a basal cell(Fig. 2G,H). Subsequently, ∼60% of brown-necked archegoniacontain an embryo with more than two cells 4 weeks after inductionof gametangia (Fig. 2H; supplementary material Table S3). Bycontrast, in the double mutant, we observed only unexpandedzygotes of a size similar to an unfertilized egg (Fig. 2F), but did notfind expanded or divided zygotes as in the wild type (Fig. 2G,H).After 2 months of incubation under gametangium-inducingconditions, no normal sporangia were found (supplementarymaterial Table S2). Several gametophores of the double mutantproduced malformed sporangia without normal spores, reminiscentof sporangia formed by parthenogenesis (Tanahashi et al., 2005)(Fig. 2B, inset).

To confirm further that the double mutant can form normal, fertilesperm and egg cells, it was crossed with the wild type. To this end,gametophores were submerged with distilled water, which increasedthe rate of sporophyte formation. Sporangia were formed on 92.5±3.9% (mean±s.d.; n=10) of 100 gametophores of self-crossed wildtype, and on 5.6±5.0% of self-crossed double deletion line (n=10).

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We observed that 8.9±3.6% of 100 gametophores of the doubledeletion line that had been crossed with the wild type developedsporangia (n=10). Among the sporangia on the crossed mutantgametophores, we arbitrarily selected three sporangia with normalmorphology and sowed the spores on plates. Spores germinated

normally and we collected all germinated chloronemata from eachline for genotyping for the PpWOX13L genes by PCR. In one of thethree lines, we identified both the wild type and the mutant alleles ofPpWOX13LA and PpWOX13LB, indicating that the Δppwox13labegg cell was fertilized with a wild-type sperm. In another two lines,

Fig. 1. Amino acid sequences and expression of PpWOX13L. (A) ClustalW alignment of full-length amino acid sequences of the WOX13-clade proteinsin Arabidopsis thaliana (At), Oryza sativa (Os), Physcomitrella patens (Pp) and Ostreococcus tauri (Ot). The homeodomain and the WOX13-specificN-terminal domain (Deveaux et al., 2008) are highlighted. (B) Semi-quantitative RT-PCR of PpWOX13LA and PpWOX13LB expression during the life cycleof Physcomitrella. RanD (Phypa55364) is used as RNA control. 7d, 7-day-old tissue containing only protonemata; 14d, 14-day-old tissue containingprotonemata and gametophores; 14dai, gametophores 14 days after gametangia induction; 1mai, gametophores with sporophytes one month aftergametangia induction. PCR reactions ran for 28 cycles with identical template concentrations. (C) PpWOX13LA and PpWOX13LB transcript levels (tags permillion, TPM) after leaf detachment determined by 5′-DGE (Nishiyama et al., 2012). Abscissae indicate the time after leaf detachment. Results of threeindependent experiments are shown. (D-N) Localization of PpWOX13LA-Citrine fusion protein. (D-G) Bright-field images (upper images in D-F and left-handimage in G) and fluorescent images (lower images in D-F and right-hand image in G) of chloronema (D), caulonema (E), young gametophore (F) andgametophore (G). Arrows in D and E indicate nuclei of apical stem cells. (H-M) Laser scanning microscopy images of antheridia (H), an archegonium with aventral canal cell (yellow arrow) and an unfertilized egg cell (red arrow) (I), an archegonium with an unfertilized egg cell (red arrow) after degeneration of theventral canal cell (J), an archegonium containing a young sporophyte (K,L), and a spore (M). (N) Snapshots of Citrine fluorescence at indicated time afterleaf excision taken from a time-lapse movie (supplementary material Movie 1). Scale bars: 100 μm (D,E,H,N); 50 μm (F,I-L); 200 μm (G); 25 μm (M).

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the analyzed chloronemata contained only mutant alleles. Thisindicates that the double mutants can fertilize, at least under thesubmerged crossing conditions. Together, these results indicate thatPpWOX13L genes are redundantly required for the outgrowth of thezygote and the initiation of embryogenesis.

PpWOX13-like genes are required for initiation of cell growthspecifically during stem cell formationBecause of the upregulation of PpWOX13L reporters during stemcell formation from detached leaves, we analyzed whether thisprocess requires PpWOX13 activity. In the wild type, live imagingof detached leaves revealed that the majority of leaf cells at thewound margin that had undergone division subsequently initiatedtip growth, giving rise to stem cells that produced chloronemafilaments by further divisions (Fig. 3A,C, red arrowheads). Aproportion of the divided cells, however, failed to initiate tip growthand to undergo any further divisions within 72 h after detachment(Fig. 3A,C, yellow arrowheads).In contrast to the wild type, the Δppwox13lab double mutant

displays a strongly decreased frequency of divided cells thatinitiated tip growth (Fig. 3B,C; supplementary material Movie 2).

We observed the same phenotype albeit at low frequency in someΔppwox13la single mutant lines (data not shown), whereas nodifference from the wild type was detected for Δppwox13lb. Forty-eight hours after leaf detachment, expression of the protonema-specific reporters RM09 and RM55 (Ishikawa et al., 2011) waspresent in all cells that underwent divisions in both thewild type andthe double deletion mutant line Δppwox13lab#186 (Fig. 3D),regardless of whether tip growth was initiated or not. This suggeststhat cells at the wound margin of detached leaves, which enterproliferation, acquire at least some aspects of chloronema identityeven if they are unable to initiate cellular growth. In summary,during reprogramming of leaf cells into chloronema stem cells,PpWOX13LA and PpWOX13LB are redundantly required for theinitiation of cellular growth, but not for entry into cell division orexpression of two chloronema identity markers.

As an independent test for PpWOX13L function in stem cellformation, we analyzed the initiation of chloronema stem cellsfrom protoplasts. In the wild type, protoplasts cultivated underhigh osmolarity conditions to prevent rupture firstly initiate tipgrowth and secondly divide, producing a primary chloronemastem cell, along with synthesizing a new cell wall (Jenkins and

Fig. 2. Deletion of PpWOX13L genes blocks zygoteoutgrowth. (A-H) Gametophores able to form sporangia (A,B),an unfertilized egg (red arrow) and sperms (yellow arrowheads)(C,D), a zygote (red arrow) (E,F), an expanded zygote (G) and atwo-cell stage sporophyte (H) are shown. Inset in B: malformedsporangium. Criteria for fertilization are a brownisharchegonium neck (supplementary material Table S2) andstronger auto-fluorescence in the nucleus than the cytoplasm(Tanahashi et al., 2005). Outlines of cells and nuclei areindicated in the images at the right (C-H). Bright field images(A,B) and fluorescent images taken with a confocal laserscanning microscope (C-H) of the wild type (A,C,E,G,H) andΔppwox13lab#186 line (B,D,F). Scale bars: 5 mm (A,B);100 μm (B, inset); 25 μm (C-H).

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Cove, 1983). To investigate this process, we scored a primarystem cell as established when the protoplast had elongated anddivided (supplementary material Fig. S3A). Subsequently, theprotoplast-derived stem cells form chloronema filaments whilestill cultured on high osmolarity medium (Fig. 3E,G). Duringside branch formation, subapical cells of these filaments formlateral bulges, which become new chloronema stem cells by adivision roughly aligned to the filament surface (Fig. 3E). In thedouble mutant, the formation of the primary stem cells fromprotoplasts and of side branch stem cells was delayed comparedwith the wild type (Fig. 3G, red dashed lines). In addition, weobserved chloronema subapical cells that failed to form a sidebulge like the wild type but rather divided in oblique directionswithout formation of a side branch (Fig. 3E,F), reminiscent of thedefective stem cell formation in the leaf detachment assay(Fig. 3B). Notably, when shifted to normal medium, the doublemutant formed side branches that were indistinguishable from thewild type (data not shown), suggesting that a higher turgor isrequired for cell outgrowth in the absence of PpWOX13Lactivity, exposing a potential role of PpWOX13L in cell wallexpansion.

PpWOX13-like genes are required for upregulation of cellwall loosening genesCellular outgrowth in plants requires the coordination of severalactivities. First, the cell must have sufficient turgor pressure to

overcome the cell wall resistance (Cosgrove, 2005; Taiz and Zeiger,2010). Second, the cell wall becomes loosened at the site of growthby enzymatic activities that disrupt cross-linkages between cellulosemicrofibrils (Cosgrove, 2005; Taiz and Zeiger, 2010). In order toinvestigate whether insufficient turgor might contribute to theimpaired outgrowth of reprogrammed leaf cells in the doublemutant, we determined the osmolarity required for plasmolysis ofthe cells at the wound margin. When using 15 (supplementarymaterial Fig. S4B,C) or 45 (data not shown) minutes of incubation,complete plasmolysis of wild-type cells requires 0.55 M mannitol,whereas 0.6 M mannitol is required for the double mutant (n=50),suggesting an even higher turgor in the double-mutant cells. Thus,a reduced turgor is not the reason for the blocked outgrowth duringstem cell formation in the double mutant.

To analyze whether cell wall loosening might requirePpWOX13L function during stem cell formation, we comparedtranscriptomes of wild-type and the double-mutant lines after leafdetachment. To this end, we performed digital gene expressionprofiling with mRNA 5′-end tags (5′-DGE) (Nishiyama et al.,2012). We analyzed the expression profiles in intact gametophoreleaves (t=0) and gametophore leaves at 1.5, 3, 6, 12 and 24 h afterdetachment. Comparison of expression between wild-type and themutant lines revealed <60 genes differentially expressed at eachtime point until 6 h, and 115 and 254 genes were differentiallyexpressed at 12 and 24 h, respectively (supplementary materialTable S4). Notably, we found that cell wall-related genes are

Fig. 3. PpWOX13L genes are required for initiation oftip growth during stem cell formation. (A) A wild-typeleaf cell that divided giving rise to one daughter acquiringtip growth (red arrowheads). A divided cell without tipgrowth is also indicated (yellow arrowheads). (B) In theΔppwox13labmutant, most leaf cells that divided did notacquire tip growth. Yellow arrowheads in A and Bindicate septa of cells that divided after leaf detachment.(C) Frequency of cells initiating tip growth at the woundmargin. n=16, corresponding to 223 and 297 cells.Asterisks indicate significant deviation from the wild type(P<0.01, Student’s t-test). Standard deviation given insquare brackets. (D) Expression of protonema-specificreporter genes RM09 and RM55 are unaffected inΔppwox13lab. Arrows indicate examples ofcorresponding cells expressing GFP signal. (E-G) Stemcell formation from protoplasts. (E,F) Bright-field imagesand DAPI-stained filaments (right-hand side, bottom) ofregenerated protoplasts 13 days after plating.Arrowheads and arrows indicate initiated chloronemaapical stem cells and the original protoplast cell,respectively. In Δppwox13lab#186, several chloronemacells have divided but did not initiate outgrowth (red orwhite arrows in F). (G) Time course of stem cell formationfrom protoplasts (n=50). Circles, triangles and crossesindicate percentages of regenerated chloronemata fromprotoplasts with at least one primary chloronema apicalstem cell, with at least one primary chloronema apicalstem cell and one side branch stem cell, and with at leastone primary chloronema apical stem cell and two sidebranch stem cells, respectively. Bars indicate s.d. ofthree independent experiments. Blue solid linesand red dashed lines indicate the wild type andΔppwox13lab#186, respectively. Asterisks indicatesignificant differences in the numbers of apical cellsbetween the wild type and Δppwox13lab#186(Mann–Whitney U-test, **P<0.01 in all threeexperiments; *P<0.05 in all the three experiments).Scale bars: 50 μm (A,B,D); 100 μm (E,F).

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enriched in the set of genes with reduced expression in the mutantcompared with the wild type at 12 and 24 h after leaf detachment(supplementarymaterial Table S5). Among them, we found that 12mRNAs predicted to encode cell wall loosening enzymes,including expansins, xyloglucan-specific endo-β-1,4-glucanases(XEG), xyloglucan endotransglucosylases/hydrolase (XET) andpectin methylesterases (PME), are upregulated in the wild typeafter leaf detachment, whereas this is not the case to the same extentin the double mutant (Fig. 4A-L). For eight of these genes, thelower expression levels in the mutant compared with the wild typewere confirmed by quantitative RT-PCR (supplementary materialTable S6). Furthermore, we found that during stem cell formationfrom protoplasts, transcript levels of three β-expansin genes aresignificantly lower in the double mutant than in the wild type,although those of one α-expansin and XET are not (supplementarymaterial Table S6). By contrast, we did not detect differenttranscript levels between the wild type and the double mutant forgenes associated with cellular tip growth, such as actincytoskeleton- or polar membrane transport-related functions(supplementary material Table S4).In summary, PpWOX13L activity is required for the upregulation

of specific cell wall loosening enzymes in protoplasts and cells ofdetached leaves that regenerate stem cells.

Expression of cell wall loosening genes enhances stem cellformation in chloronemataWe hypothesized that cell wall loosening genes might be involvedin stem cell formation. Because transformation of the doublemutant was not successful for unknown reasons, we tested thishypothesis in wild-type protoplasts. To this end, we expressedthree predicted β-expansin genes (Fig. 4) driven by the rice actinpromoter (Zhang et al., 1991), which provides high expressionlevels in protoplasts and protonemata (Horstmann et al., 2004).We analyzed stem cell formation from protoplasts comparingchloronemata with similar expression intensities based on aco-transformed p35S:GFP reporter. Transformation with each oftwo different β-expansin genes, denoted as 146965 and 96876,resulted in a significant increase of stem cell formation (Fig. 4N)compared with the untransformed control, whereas transformationwith β-expansin 150257 had no significant effect. We concludethat in the wild type, β-expansin activity is a limiting factor forstem cell formation, at least in chloronemata regenerated fromprotoplasts.

Arabidopsis WOX13 and WOX14 are not essential duringregeneration of root meristem stem cellsThe flowering plant Arabidopsis encodes three members in theWOX13 clade, namely WOX13, WOX14 and the putative pseudo-geneWOX10 (Haecker et al., 2004; Deveaux et al., 2008). We foundthat WOX13 and WOX14 promoters have transcriptional activity inroot meristems (Fig. 5E,F). In order to analyze whether ArabidopsisWOX13 clade genes might play a role in regeneration of stem cellsafter wounding, in similarity to stem cell regeneration from detachedleaves in moss, we performed root regeneration assays usingT-DNA insertion mutants (Fig. 5A-D). wox13-2 carries an insertionin exon 1, and transcript could not be detected by RT-PCR(Romera-Branchat et al., 2012), indicating that this mutant is astrong loss-of-function or a null allele. wox14-1 was previouslydescribed as pst13645 and has the homeodomain deleted (Deveauxet al., 2008). We found that wox13-2 and wox14-1 single mutantsand the wox13-2 wox14-1 double mutant regenerated rootmeristems at a similar efficiency as the wild type (Fig. 5G). Thus,

in contrast to Physcomitrella, we did not detect a regeneration-related function for Arabidopsis WOX13-clade genes.

DISCUSSIONStem cell formation in seed plants is tightly associated with thefunction ofWOX genes. Here, we addressed whether this associationmight be conserved outside of the seed plants by studying WOXfunctions in the moss Physcomitrella. We show that bothPpWOX13L proteins accumulate in all cells examined (Fig. 1), andthat the double knockout mutants had defects in stem cell formationfrom cut leaves and in protonemata derived from protoplasts as wellas in zygotes (Figs 2, 3). In all cases, cell wall expansion was arrested(Figs 2, 3). Expression of cell wall loosening genes, including β-expansin genes, was reduced compared with wild type in the cutleaves of the double mutant (Fig. 4), and β-expansin induction inwild-type protoplasts enhanced the stem cell formation.

Together, these results indicate that the two moss PpWOX13Lgenes are redundantly required for the formation of stem cells in cutleaves and from protoplasts. The failure to initiate cell growth is thefirst phenotypic manifestation during stem cell formation in thedouble mutant, and the upregulation of genes encoding cell wallloosening enzymes is reduced. Because normal growth ofchloronema and caulonema is not impaired, we propose thatmossWOX13L activity is involved in cellular growth specifically inthe process of stem cell formation at least in protoplasts anddissected leaves.

The role of WOX13L in PhyscomitrellaDuring stem cell formation from differentiated leaf cells, severalcellular changes, including the re-entry into cell cycle and theinitiation of tip growth, occur (Ishikawa et al., 2011). Our resultsindicate that during this process: (1) the expression levels ofPpWOX13LA and PpWOX13LB genes are upregulated, (2) bothgenes are redundantly required to initiate cellular outgrowth but notfor entry into the cell cycle, and (3) both genes are required forupregulation of genes predicted to encode cell wall looseningfactors. Likewise, stem cell formation from protoplasts was delayedin the double mutant. Furthermore, the initial outgrowth of a lateralbulge during establishment of side-branch forming stem cells fromchloronema subapical cells was impaired. Curiously, this defect wasseen only on high but not on low osmolarity medium. We reasonthat owing to the reduced cell wall loosening in the double mutant, ahigher turgor pressure is required for cell expansion. Finally, most ofthe zygotes in the Δppwox13l double mutant neither elongated norentered cell division to form an apical stem cell, unlike the wildtype. Although we cannot exclude that in the zygote, PpWOX13Lgenes are involved in cell outgrowth and division separately, aplausible hypothesis in analogy to the effects in induced stem cellregeneration is that the initiation of cell growth is primarily regulatedby WOX13L function in the zygote, and that the failure to undergocell division is a consequence thereof.

Is there a common function of PpWOX13L underlying all theobserved defects? We propose that the phenotypes observed inthe leaves, protoplasts and zygotes of the double mutant can beexplained by an impaired activation of cell growth due to insufficientupregulation of cell wall loosening activities. Although zygotes andstem cells employ different growth modes (diffuse cell growthand tip growth, respectively) the requirement of cell wall looseningenzymes is likely to be key in both processes. Further studies ongene regulatory networks in reprogramming leaf cells andprotoplasts as well as on zygotes are necessary to examinewhether PpWOX13L genes function similarly in these cells.

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Notably, all mutant defects are restricted to formation of apicalstem cells, whereas growth of the established filaments by theirapical stem cells is unaffected. The broad expression of PpWOX13Lgenes suggest yet unknown functions in non-stem cells.Alternatively, the global expression of PpWOX13L genes could

contribute to the general competence for stem cell formation. Stemcell regulators that are not restricted to the stem cell niche include,for example, WUS andWOX5 in Arabidopsis, which, in addition totheir stem cell niches, are expressed in ovules and cotyledons,respectively (Groß-Hardt et al., 2002; Sarkar et al., 2007). Inconclusion, we propose that PpWOX13L activity is required for theonset of cell growth programs specifically during stem cellformation, but not for maintaining them.

Comparison with ArabidopsisThe significance of cell growth regulation in stem cells has not beenstudied yet in seed plants, where the formation of stem cells andzygote development are also regulated byWOX genes. First, severalmembers of the WUS clade in seed plants function in maintainingpluripotent stem cells (Aichinger et al., 2012). In Arabidopsis,these includeWUS in the shootmeristem (Laux et al., 1996),WOX5in the root meristem (Kamiya et al., 2003; Sarkar et al., 2007),WOX4 in the cambium (Hirakawa et al., 2010; Ji et al., 2010) andWOX1 together with PRS (WOX3) in the leaf margins (Shimizuet al., 2009; Nakata et al., 2012). Notably, it is the formation of theprimary shoot meristem in the embryo that is most sensitive toreduction of WUS activity, whereas maintenance of stem cells

Fig. 4. PpWOX13 L activity regulates cell wallloosening genes. (A-L) Accumulation patternsof transcripts encoding cell wall loosening genesafter leaf excision in 5′-DGE analysis. Protein IDsare shown above each graph. Horizontal axesindicate the time after leaf detachment andvertical axes indicate tags per million (TPM)values. Blue and red lines indicate expressionlevels in the wild type and the Δppwox13lab#186double mutant, respectively. Results of threeindependent experiments are shown.(M,N) Transient expression of β-expansins(146965 and 96876) increases apical stem cellformation during protoplast regeneration in thewild type, whereas β-expansin 150257 had nosignificant effect. Primary chloronemal apical celland secondary chloronemal apical cells derivedfrom subapical cells are indicated by arrows andarrowheads, respectively, and the number ofsecondary apical cells is shown in the upper right(M). The total number of secondary chloronemaapical stem cells was counted at the fifth day afterprotoplast regeneration on the high osmolaritymedium. The total number of observed protoplastregenerations is 60 (divided among threebiological replicates) for every construct.Significantly different distributions from thecontrol are shown with P-values (Mann–WhitneyU-test). Scale bars: 50 μm.

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during inflorescence meristem growth is less sensitive (Graf et al.,2010). Furthermore, both WUS and WOX5 are essential inregenerating stem cells via tissue culture (Su et al., 2009;Sugimoto et al., 2010). The requirement for WUS and WOX5specifically during formation of stem cells but to a weaker extentduring further development is similar to that for PpWOX13L inmoss stem cell formation reported here. However, there are alsonotable differences. Whereas in moss, we found that the activationof the cell growth program is an essential part of PpWOX13Lfunction, in Arabidopsis, WUS and WOX5 genes may have arepressive effect on cell enlargement based on their cellular mutantphenotypes (Laux et al., 1996; Sarkar et al., 2007). One interestingpossibility to be tested in the future is whether these oppositeeffects could be explained by the acquisition of the transcriptionalrepressive WUS box in the WUS-clade genes (Lin et al., 2013).Second, severalWOX genes are required for normal development

in the Arabidopsis zygote (Breuninger et al., 2008; Ueda et al.,2011), which superficially parallels the function of PpWOX13Lgenes in Physcomitrella. However, also here there are markeddifferences: in Arabidopsis, it is the asymmetry of the zygoticdivision that is under control of WOX2, 8 and 9 genes, whereas aneffect on zygote expansion, as seen in the moss for PpWOX13Lgenes, has not been reported. Zygote expansion in Arabidopsis israther under the control of the YODA pathway (Lukowitz et al.,2004), which acts in parallel toWOX genes at later stages of embryopatterning. Expression of a moss YODA ortholog is induced afterleaf detachment independently of PpWOX13L (Nishiyama et al.,2012), but its function remains to be investigated.Our mutant analysis of the only two expressed members of the

WOX13-clade in Arabidopsis,WOX13 andWOX14, did not reveal

an obvious stem cell regeneration defect. Of course, possible stemcell- or regeneration-related functions of WOX13 and WOX14could be masked by the existence of yet unknown redundantfactors. We consider it unlikely, however, that ArabidopsisWOX13 genes function redundantly with WUS clade genes,given the sequence differences in their recognition helix of thehomeodomain that mediates DNA contact (Deveaux et al., 2008;Nardmann and Werr, 2012). Thus, the function of WOX13 cladegenes in Arabidopsis appears to be distinct from that ofWUS cladegenes. WOX13-type genes evolved in an ancestor of green algaeand land plants (Deveaux et al., 2008) and in light of the earlyseparation of the two lineages in plant evolution, PpWOX13Lfunction in moss could be either close to the ancestral trait or aderived character. The Δppwox13l deficiencies in activating celloutgrowth are consistent with ancestry, as cell expansion is a sinequa non condition for cell divisions in uni- or multicellularorganisms, and this view is compatible with the PpWOX13Lphylogenetic position in the ancestral branch of the WOX family(Deveaux et al., 2008; van der Graaff et al., 2009; Nardmannet al., 2009). However, the Δppwox13l phenotypes associate thePpWOX13L function with stem cell formation, which isreminiscent of the function of WUS clade members that evolvedafter the separation of moss and vascular plant lineages(Nardmann and Werr, 2012). The PpWOX13L function in mossthus exemplifies either convergent evolution or recruitment ofWOX13 genes to the interface of cell enlargement/division andcellular differentiation in the last common ancestor of mosses andvascular plants.

It is noteworthy in this regard that expression of WOX13-cladegenes in Physcomitrella and Arabidopsis is not restricted to

Fig. 5. Root tip regeneration inArabidopsis thaliana. (A-D) Regenerationassay with wild-type roots carrying thequiescent center (QC) specific markerQC184 stained for GUS and with Lugol’ssolution that stains starch grains specific fordifferentiated columella cells. Five daysafter germination, roots were cut at adistance of 100 μm from the root tip (blackbar in A). One day after cutting, cells withcolumella identity are detected by Lugolstaining (B, arrow). Two days after cutting,the roots form a typical root morphology (C),and 3 days after, restore expression ofQC184 (D, arrowhead). (E,F) pWOX13:erYFP (E) and pWOX14:erYFP (F) wereexpressed in the root meristem. Inset in E:enlarged view of boxed area. (G) Efficiencyof root tip regeneration 2 days after cutting:no significant differences between the wildtype and mutants are apparent. Valuesshown represent averages of two siblinglines and four biological replicates, errorbars represent s.d.

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small signaling centers, and there is no indication of a non-cell-autonomous function. By contrast, WUS clade function in stemcell regulation appears to involve spatially restricted expressionin stem cell organizers and the organization of multicellularniches via non-cell-autonomous mechanisms (Schoof et al.,2000; Sarkar et al., 2007; Hirakawa et al., 2010), whichplausibly could have been an acquisition during evolution ofincreasingly complex stem cell systems typical for angiosperms.

MATERIALS AND METHODSPlant materials and growth conditionsPhyscomitrella patens Cove-NIBB (Nishiyama et al., 2000) or Gransden 2004(Rensing et al., 2008) lines were used as a wild-type strain and cultured onBCDAT medium supplemented with 1 mM CaCl2 and 0.8% (w/v) agar(BCDAT agar medium) at 25°C in continuous light or long-day conditions(16 h/8 h) (Nishiyama et al., 2000). Gametophore preparation for transcriptomeanalysis, quantitativeRT-PCRanalysis, andplasmolysis experiments (Ishikawaet al., 2011), protoplast preparation (Nishiyama et al., 2000), and gametangia/sporophyte observation (Sakakibara et al., 2008) were previously described.

Transformation and genetic crossesThe primer sequences used for plasmid construction are provided insupplementary material Table S1. The constructs used for transformation areshown in supplementary material Fig. S1A-D and Fig. S2A-D. Transformationwas performed as described previously (Nishiyama et al., 2000; Knight et al.,2002). Stably transformed lines were screened by DNA gel-blot analyses. Toexclude polyploid lines, flow cytometry analysis was performed (Okano et al.,2009).Crosseswereperformedas previously described (Tanahashi et al., 2005).

MicroscopyImages were captured using SZX16 and BX51 (Olympus). Citrinefluorescent images were obtained using fluorescence microscopes with aU-MNIBA3 filter unit or using confocal laser scanning microscope LSM510 confocal system (Carl Zeiss) with a detecting fluorescence emissionbetween 530-600 nm under excitation with 514 nm.

To quantify reprogramming of leaf cells in wild-type and the Δppwox13ldeletion lines, 10-15 leaves of Δppwox13la1 and Δppwox13lb1 lines wereplaced on a layer of BCDATmedium with 0.8% agar on a microscope slide.Pictures were taken every 24 h and subsequently evaluated for division andelongation events at the wound margin, with all cells directly adjacent to adead cell scored as margin cells. Three repetitions were carried out. Forobservation of reprogramming in the wild-type and Δppwox13lab doubledeletion lines, the third to fifth leaves from a gametophore tip were cut in halfand the detached leaves were laid on a glass-base Petri dish (Iwaki 3911-035,Asahi Techno Glass, Shizuoka, Japan) and were then overlaid with BCDATagar medium. Images of leaf reprogramming were recorded at 10- or 20-minintervals with a digital camera ORCA-AG (Hamamatsu Photonics K. K.)using an inverted microscope IX81 (Olympus). The images werereconstructed to create a movie with Metamorph software (MolecularDevices).

For observation of nuclei, regenerated protoplasts at 6 days after platingwere fixed with 1% formaldehyde in 50 mM sodium phosphate buffer (pH7.0) and stained with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole). Toobserve egg cells and embryos, shoot apices with gametangia and severalyoung leaves were collected 3-4 weeks after the induction of gametangiaand fixed in a solution of 4% (v/v) glutaraldehyde and 1 μg/ml DAPI in12.5 mM sodium phosphate buffer (pH 7.0) overnight at 4°C. The fixedmaterials were then dehydrated in a graded ethanol series and cleared in a 2:1mixture of benzyl benzoate and benzyl alcohol for observation on a LSM510 confocal system. Fluorescence between 420 and 480 nm was detectedunder excitation with 405 nm beams.

Digital gene expression profiling withmRNA 5′-end tags (5′-DGE)analysisTranscriptome comparisons between the wild-type and theΔppwox13lab#186 double deletion lines were performed as previously

described (Nishiyama et al., 2012). Gene Ontology enrichment analyseswere performed using the BinGO plugin (version 2.44) on Cytoscapeversion 2.8.3 (Maere et al., 2005) with the table (Rensing et al., 2008)available at (ftp://ftp.jgi-psf.org/pub/JGI_data/Physcomitrella_patens/v1.1/Phypa1_1_goinfo_FilteredModels3.tab.gz). For each set of up- ordownregulated genes at each time point or the union of such sets for anytime point, overrepresented GO categories were searched. Hypergeometrictests with Benjamini and Hochberg FDR were carried out with thesignificance level of 0.01. Corrected P-values for significantlyoverrepresented GO terms were obtained.

Transient expression assay of β-expansinsPlasmids for transient expression assay of β-expansins were constructed usingpTFH22.4 (accession number: AB758445), which contains a rice actinpromoter (McElroy et al., 1990), amultiple-cloning site (SalI-AscI-SmaI-ApaI-SfiI), the pea rbcS-3A polyadenylation signal, the cauliflower mosaic virus(CaMV) 35S promoter, sGFP, and the polyadenylation signal of the nopalinesynthase gene. cDNA of each β-expansin coding region was amplified by PCRwith primers shown in supplementary material Table S1 and cloned into theAscI-ApaI site of pTFH22.4. The original pTFH22.4 was used as a control.Transient transformation was performed using 10 μg of circular plasmid DNA(Nishiyama et al., 2000). The protoplasts were left on high osmolarity plates[6% (w/v) mannitol] after transformation. Twenty protoplasts or regeneratingchloronemata with GFP signal were arbitrarily chosen on the second, third,fourth and fifth days after plating using theOlympusBX51microscopewith theGFP filter set. Each regenerating plant was scored for the number of apical cellsshowing tip growth, and the total number foreach classwas obtained from threerepeat experiments. The difference in the number of the acquired apical stemcells was tested by the non-parametric Mann–Whitney U-test, because thenumber of apical stem cells is not considered to follow the normal distributionbut is an ordered variable. The calculations were performed using the Rprogram (R Development Core Team, 2012).

Observation of plasmolysisTo examine plasmolysis in cut leaves, ten leaves were cut and incubated in10 mM MES buffer (pH 6.5) for 24 h, and then incubated in mannitolsolutions of different concentrations for 15 min or 45 min. Five arbitrarilyselected wound margin cells in each leaf were examined for plasmolysis.Three biological replicates were analyzed for each experiment. Statisticalanalysis was performed by Fisher’s exact test using the R program(R Development Core Team, 2012).

RNA preparation and quantitative RT-PCR analysisExcised leaves for quantitative RT-PCR analysis were prepared in the samemanner as samples for 5′-DGE analysis and collected 24 h after leaf excision.Regenerated protoplasts for quantitative RT-PCR analysis were preparedfollowing protoplast regeneration. After overnight incubation, protoplastswere collected, suspended in PRM/Tmediumwithout agar (BCDATmediumsupplemented with 10 mM CaCl2 and 0.044 M mannitol), and plated on thePPM/B plate (BCDAT medium supplemented with 10 mM CaCl2, 6%mannitol and 0.8% agar) covered with sterile cellophane. Regeneratedprotoplasts were collected 4 days after plating.

Total RNA preparation and cDNA synthesis were described previously(Ishikawa et al., 2011). Quantitative RT-PCR (qRT-PCR) was performedusing an ABI PRISM 7500 (Life Technologies) with the SYBR GreenERqPCR Super mix Universal (Life Technologies) and appropriate primersshown in supplementary material Table S1. Results were analyzed using thecomparative critical threshold method (Livak and Schmittgen, 2001). Thetranscript levels were normalized against α-tubulin gene (TUA1) transcriptlevels using TUA1 primers (Ishikawa et al., 2011). The quantification ofeach sample was performed in triplicate. Three biological replicates wereanalyzed for transcript accumulation.

Generation of Arabidopsis reportersPromoter fragments including 4.4 kb ( pWOX13) and 1.4 kb ( pWOX14)were amplified with primers containing adapter sequences for ligationindependent cloning (LIC) (Li and Elledge, 2007; Eschenfeldt et al., 2009),

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and were subsequently inserted by the LIC procedure in front of an erYFPand 35S-terminator within a binary vector. Plants were transformed usingthe floral-dip method. Primer sequences are provided in supplementarymaterial Table S1.

Arabidopsis root regeneration assayThe QC184 marker (Sabatini et al., 1999) was introduced by crossing withthe wox13-2 mutant, and the selected wox13-2 QC184 plants weresubsequently crossed to wox14-1. Because these crosses generated amixture of three ecotypes (QC184: WS; wox13-2: Ler; wox14-1: No), wild-type controls were selected from the progeny of these crosses. Theregeneration assay was carried out as previously described (Sena et al.,2009) and regeneration was scored two days after cutting (four biologicalreplicates for each background, n=22-48 for each replicate).

AcknowledgementsWe thank Yohei Higuchi for the technical support to establish 5′-DGE indexing;Nagisa Sugimoto for the time-lapse observation; Kazuhiko Nishitani for suggestionson physiological experiments; Tomomichi Fujita for providing the plasmid pTFH22.4and technical advice for the experiment; Tomoko Masuoka, Mika Hiramatsu andIkumi Kajikawa for genetic crosses and Physcomitrella genotyping; ThomasMunster for providing plasmids and plant material; Eric van der Graaff for providingplasmids; and Minako Ueda, Miyuki Nakata, Masaki Shimamura, Edwin Groot andJudith Nardmann for helpful discussions.

Competing interestsThe authors declare no competing financial interests.

Author contributionsK.S., P.R., T.N., W.W., S.A.R., H.D., M.H. and T.L. designed and discussedexperiments. K.S. and P.R. performed Physcomitrella mutant and expressionanalysis. T.A. established thewox13l single and double mutant lines. T.F. performedArabidopsis experiments. S.A. performed Southern hybridization of wox13l linesand confocal microscopy. Y.S. performed the time-lapse observation analysis. Y.T.crossed Physcomitrella. Y.H. transformed Physcomitrella. T.N. and T.K. performedtranscriptome analysis. M.I. performed the protoplast transient assay. T.M.conducted plasmolysis experiments. K.S., P.R., M.H. and T.L. wrote the paper.

FundingThisworkwas fundedby theYoungResearcherOverseasVisitsProgram for VitalizingBrain Circulation (K.S., Y.H., M.I., Y.T. and M.H.); the Japan Science and TechnologyAgency ERATO program (M.H.); the Ministry of Education, Culture, Sports, ScienceandTechnology [grant no. 24113521 toY.T., no. 25291067and22128002 toM.H.]; theDeutsche Forschungsgemeinschaft [WE1262/7-1, SFB 680 to W.W.]; the EuropeanResearch Area in Plant Genomics program (T.L.); the EU-cofunded INTERREG IVUpper Rhine Project 692 A17 (T.L.); the Excellence Initiative of the German FederalandStateGovernments [BIOSS,EXC294 toT.L.andS.A.R.]; and theGermanFederalMinistry of Education and Research (Freiburg Initiative for Systems Biology) [FKZ0313921 to S.A.R. and T.L.]. Deposited in PMC for immediate release.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.097444/-/DC1

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RESEARCH ARTICLE Development (2014) 141, 1660-1670 doi:10.1242/dev.097444

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