the mi-2-like smed-chd4 gene is required for stem cell … · identified the smed-chd4 gene, which...

1231DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

INTRODUCTIONStem cells have diverse and important roles in animal developmentand are required for the maintenance of cell numbers during tissuehomeostasis in adult organisms. The hallmark properties of stemcells are long-term self-renewal and differentiation potential(Wagers and Weissman, 2004; Weissman et al., 2001). Regulationof these two processes under normal and abnormal conditions iscrucial, although still poorly defined.

Planarians are one of the simplest organisms known to haverobust regeneration capacities and extensive tissue turnover as adults(Reddien and Sánchez Alvarado, 2004); these properties requireactive dividing cells known as neoblasts. Neoblasts have beenidentified by their stem-cell-like morphology and their proliferativecapacity (Dubois, 1949; Reddien and Sánchez Alvarado, 2004).Although neoblasts were first described to have stem-cell-likeproperties more than 100 years ago, the mechanisms regulating theirproliferation and differentiation still remain largely unknown.Neoblasts are constantly dividing to maintain tissue turnover in theanimals. In addition, injury (e.g. amputation) stimulates neoblastproliferation. Neoblasts are required for the generation of ablastema, an unpigmented epithelial/mesenchymal bud at the site ofinjury, within which missing body parts are formed (Dubois, 1949;Reddien and Sánchez Alvarado, 2004).

Many newly developed resources and methodologies have madethe study of stem cells in planarians possible (Newmark andSánchez Alvarado, 2000; Newmark et al., 2003; Reddien et al.,2005a; Sánchez Alvarado et al., 2002). For example, whole-mountin situ hybridizations and high-throughput RNAi screens haveresulted in the identification of genes required for normal neoblast

function (Guo et al., 2006; Reddien et al., 2005a; Reddien et al.,2005b). Because neoblasts are the major category of proliferatingcells in the animal, they can be labeled with the thymidine analogbromodeoxyuridine (BrdU) and can also be isolated using a DNA-labeling dye by flow cytometry (Newmark and Sánchez Alvarado,2000; Hayashi et al., 2006; Reddien et al., 2005b). More recently, apanel of neoblast progeny markers have been identified (Eisenhofferet al., 2008). The abundance of neoblasts, the prominent role theyhave in regeneration and the newly available experimental tools forthe study of neoblasts, make these cells a powerful new setting forin vivo stem cell experimentation.

In several stem cell systems, transcription factors regulatedifferentiation towards a particular lineage (Bonifer et al., 2008;Fuchs and Horsley, 2008; Le Grand and Rudnicki, 2007; Orkin andZon, 2008). The accessibility of target genes to these transcriptionfactors can be dictated by chromatin structure; therefore, regulationof chromatin structure can contribute to changes in gene activity(Bibikova et al., 2008; Georgopoulos, 2002; Kingston and Narlikar,1999; Kouzarides, 2007; Li et al., 2007; Lunyak and Rosenfeld,2008; Narlikar et al., 2002). Changes in chromatin structure can beaccomplished by both post-translational modifications of histonetails and by ATP-dependent chromatin remodeling proteins (Beckerand Horz, 2002; Kouzarides, 2007).

The Mi-2 protein is a central component of the nucleosomeremodeling deacetylase (NuRD) complex (Tong et al., 1998; Wadeet al., 1998; Zhang et al., 1998). There are two Mi-2 proteins: Mi-2, also known as the chromodomain helicase DNA binding protein3 (CHD3) and Mi-2, also known as CHD4 (Seelig et al., 1996).The latter is the predominant Mi-2 protein associated with themammalian NuRD complex (Zhang et al., 1998). Chromatinremodeling by the NuRD complex usually results in transcriptionalrepression due to the histone deacetylase activity residing in thecomplex (Ng and Bird, 2000; Tong et al., 1998). However, Mi-2can also associate with histone acetyltransferases and methyltransferases, resulting in these cases in transcriptional activation(Denslow and Wade, 2007; Shimono et al., 2003; Williams et al.,2004). The NuRD complex can be recruited to a specific promoter

Development 137, 1231-1241 (2010) doi:10.1242/dev.042051© 2010. Published by The Company of Biologists Ltd

1Howard Hughes Medical Institute, Whitehead Institute, and 2Department ofBiology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge,MA 02142, USA.

*Author for correspondence ([email protected])

Accepted 4 February 2010

SUMMARYFreshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of theaction of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. Weidentified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, asrequired for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi),neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response ofneoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayeda dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of theseneoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cellscommitted to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins instem cell differentiation.

KEY WORDS: CHD4, Differentiation, Planaria, Regeneration, Stem cells

The Mi-2-like Smed-CHD4 gene is required for stem celldifferentiation in the planarian Schmidtea mediterraneaM. Lucila Scimone1, Joshua Meisel1 and Peter W. Reddien1,2,*

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directly by one of its subunits or indirectly by interaction with asequence-specific DNA-binding protein (Ahringer, 2000; Denslowand Wade, 2007; Kim et al., 1999). The CHD4 protein belongs toan evolutionarily conserved family and contains two planthomeodomain (PHD) zinc-finger motifs, two chromodomains andan ATPase/helicase domain of the SWI2/SNF family. The NuRDcomplex, and in particular Mi-2, is required for normal developmentand cell fate control in several organisms such as Caenorhabditiselegans, Drosophila melanogaster, Arabidopsis thaliana andmammals (Kehle et al., 1998; Ogas et al., 1997; Ogas et al., 1999;Unhavaithaya et al., 2002; von Zelewsky et al., 2000; Yoshida et al.,2008). The general role(s) for Mi-2 in adult stem cell biology hasremained unresolved.

Here, we show that Smed-CHD4 is essential for regeneration inthe planarian Schmidtea mediterranea. Many of the defectsobserved in intact Smed-CHD4(RNAi) animals are hallmarks ofneoblast dysfunction. Our experiments indicate that Smed-CHD4 isrequired for the ability of neoblasts to produce progeny cellscommitted to differentiation, suggesting that chromatin regulationby CHD4 is an important aspect of adult stem cell lineagespecification.

MATERIALS AND METHODSCHD4 and RNAi experimentsRT-PCR was used to amplify the CHD4 gene. Complete gene sequence wasdetermined using 5� and 3� RACE PCR (Ambion). CHD4 and the controlgene unc-22 from C. elegans were cloned into the pPR244 RNAi expressionvector using Gateway recombination reactions as described (Reddien et al.,2005a). RNAi experiments were performed by feeding the animals with amixture of liver and bacteria expressing CHD4 dsRNA (Reddien et al.,2005a). Ten milliliters of bacteria culture was pelleted and resuspended in100 l of liver or in 30 l of liver. Three feeding protocols were used:animals were fed on day 0, day 4 and day 7, or on day 0 and day 4, or on day0 only. For regeneration studies, animals were cut at day 8 (for the three-feedings protocol) or at day 5 (for the two-feedings protocol). Forhomeostasis studies, one extra feeding was added to the three-feedingsprotocol at day 14.

In situ hybridizationsIn situ hybridizations on whole animals, cell macerates or isolated cells wereperformed as described (Reddien et al., 2005b). CHD4 whole-mount in situhybridizations and fluorescence in situ hybridizations (FISH) wereperformed as described (Pearson et al., 2009). To quantify data from in situhybridizations on cells, the percentage of DAPI-positive cells with signalwas determined. To determine the number of Smed-AGAT-1-expressing cellspresent following irradiation, the number of Smed-AGAT-1+ cells within 0.3mm by 0.3 mm tissue from the dorsal side and anterior to the pharynx wasdetermined and normalized by animal length.

Antibody stainingsAnimals were treated with 10% N-acetyl-cysteine in PBS for 3-5 minutes atroom temperature (RT) and fixed in Carnoy’s solution. Animals were thenlabeled as previously described (Newmark and Sánchez Alvarado, 2000).Numbers of mitoses were divided by animal surface area. For SMEDWI-1antibody stainings, animals were fixed in a 4% formaldehyde, PBSTsolution following FISH with the Smed-AGAT-1 riboprobe, blocked andlabeled with 1:2000 -SMEDWI-1.The SMEDWI-1 polyclonal antibodywas raised in rabbits using the peptide previously described (Guo et al.,2006). Total numbers of Smed-AGAT-1+ cells, SMEDWI-1; Smed-AGAT-1double-positive cells, or SMEDWI-1-positive cells were determined inoptical sections.

BrdU labelingAnimals were fed with RNAi food at days 0 and 4, and injected at day 6following initial RNAi feeding with a solution of 5 mg/ml of BrdU (Fluka)in planarian water. Animals were then killed 4 days later in 5% N-acetyl-cysteine in PBS for 5 minutes at RT and fixed as described (Pearson et al.,

2009). FISH using the Smed-AGAT-1 riboprobe were performed asdescribed (Pearson et al., 2009). Following FISH, animals were fixed,treated with 2N HCl for 45 minutes at RT and labeled with a 1:100 rat anti-BrdU antibody (Oxford Biotech) as previously described (Newmark andSánchez Alvarado, 2000). Apotome images were obtained from BrdU-labeled and FISH animals using an Axiocam digital camera, a ZeissAxioImager with use of an Apotome, and Axiovision software. Totalnumbers of Smed-AGAT-1-expressing cells containing a BrdU-labelednucleus were determined in optical sections.

FACSPlanarians were cut into small pieces and incubated in calcium-free,magnesium-free medium plus BSA (CMFB) containing 1 mg/ml ofcollagenase for 45 minutes at RT as described (Reddien et al., 2005b). Cellswere centrifuged at 1250 rpm for 5 minutes and resuspended in CMFBcontaining Hoechst 33342 (10 g/ml) for 45 minutes at RT. Calcein (0.5g/ml) was incubated with the cells for 15 minutes at RT. Finally, 5 g/mlof propidium iodide was added to the cell suspension prior to flow cytometryanalysis or sorting. Analyses and sorts were performed using a MoFlo3FACS sorter.

CHD4 qPCRTotal RNA was isolated from wild-type or lethally irradiated animals andfrom isolated X1 and X2 cells. cDNA was prepared and quantitative PCR(qPCR) was performed using SYBR Green (Applied Biosystems). Datawere normalized to the expression of UDP (clone H.55.12e, accessionnumber AY068123). UDP was selected as an internal control as aconstitutively expressed gene. Sets of specific CHD4-primers were used toevaluate gene expression (5�-GTCAGCAAAATGTTTACACTGAGG-3�and 5�-ACGTCTTGCTTCATGTCTGATAGT-3�). Samples without reversetranscriptase served as the negative control template.

RESULTSCHD4 is required for planarian regenerationWe identified the S. mediterranea Smed-CHD4 gene(chromodomain helicase DNA-binding protein 4) in a screen forgenes expressed in neoblasts after wounding (data not shown).SMED-CHD4 is highly similar to proteins within the conservedCHD family of chromatin regulatory proteins; by phylogeneticanalysis it clustered to the CHD3/4/5 members of the family, andusing BLAST we found that it is most similar to the mammalianCHD4 (see Fig. S1 and Fig. S2 in the supplementary material).CHD4/Mi-2 is a central component of the nucleosome remodelinghistone deacetylase complex NuRD (Tong et al., 1998; Wade et al.,1998; Zhang et al., 1998). The Smed-CHD4 gene will be hereafterreferred to in the text in short as CHD4.

CHD4(RNAi) worms were unable to form a blastema followingamputation, demonstrating that CHD4 is required for planarianregeneration (Fig. 1A). The inability to form a blastema wasnoticeable as early as 5 days following initial double-stranded (ds)RNA delivery (see Fig. S3 in the supplementary material).Compared with other genes required for regeneration in planarians(Reddien et al., 2005a), the onset of regeneration failure inCHD4(RNAi) animals is very fast. The rapid appearance andstrength of the CHD4(RNAi) phenotype is consistent with thepossibility that CHD4 has a primary function in a crucial aspect ofneoblast function.

CHD4 was widely expressed in the planarian parenchyma (aregion containing neoblasts), brain and ventral nerve cords, asdetermined with in situ hybridizations (Fig. 1B). CHD4 mRNAwas reduced in CHD4(RNAi) worms following dsRNA feeding(see Fig. S4A in the supplementary material). A lethal dose of -irradiation (6000 rads) depletes planarians of neoblasts,eliminating the ability to regenerate and to replace aged cellsduring homeostasis (Bardeen and Baetjer, 1904; Dubois, 1949;

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Reddien et al., 2005b). Five days after lethal irradiation,expression of CHD4 in the nervous system persisted but theexpression in the parenchyma was reduced, suggesting that CHD4was expressed in neoblasts or in their immediate descendants(Fig. 1B). Expression of non-neoblast genes remained unchangedfollowing irradiation (Fig. 1B; see Fig. S4B in the supplementarymaterial).

Neoblasts are actively dividing cells that are required for theproduction of all cells of the adult organism (Reddien andSánchez Alvarado, 2004). Studies to uncover their lineagepotential are emerging (Eisenhoffer et al., 2008). Flow cytometryhas been used to isolate cells with neoblast properties (Hayashi etal., 2006; Reddien et al., 2005b). Using vital dyes, twopopulations of irradiation-sensitive cells can be purified. X1 cellsare actively dividing cells (in S and G2/M stages of the cell cycle)with a higher DNA content (>2n); X2 cells contain postmitoticneoblast descendants (Eisenhoffer et al., 2008) and might containneoblasts in the G1 stage of the cell cycle as well. A population ofirradiation-insensitive cells, Xins, has been used as a controlpopulation in flow cytometry experiments (Hayashi et al., 2006;Reddien et al., 2005b). To determine whether CHD4 wasexpressed in neoblasts, we performed in situ hybridizations withX1, X2 and Xins cells. CHD4 was indeed expressed in neoblastsas well as in presumptive differentiated cells: 53% of X1 cells,78% of X2 cells and 69% of Xins cells were labeled with theCHD4 riboprobe (Fig. 1C). We further confirmed that CHD4 isexpressed in neoblasts using quantitative PCR (qPCR; see Fig.S4C in the supplementary material). Moreover, CHD4 was highlyexpressed at wound sites 3 days following amputation and thisexpression was not detected in irradiated worms that lackneoblasts (see Fig. S4D in the supplementary material). It is notknown whether CHD4 expression was upregulated followingwounding or whether the high abundance of CHD4 expressionnear wounds was due to migration of cells that already expressedthis gene. Because irradiation eliminates neoblasts, this resultsuggests that neoblasts and/or their progeny near wounds expressCHD4 during regeneration.

CHD4 is required for tissue turnoverNeoblasts constantly proliferate in adult planarians in order toreplace aged tissues (Reddien and Sánchez Alvarado, 2004). Toinvestigate the role of CHD4 during normal tissue turnover, weperformed RNAi experiments in intact (i.e. non-amputated) animals.Head regression is a characteristic sign of neoblast dysfunction thatoccurs between 10 and 12 days following a lethal dose of irradiationthat eliminates all neoblasts. Head regression is also observed inanimals in which genes required for neoblast function have beeninhibited with RNAi (Guo et al., 2006; Reddien et al., 2005a;Reddien et al., 2005b). Intact CHD4(RNAi) worms started to displayhead regression 14 days after initial dsRNA feeding (Fig. 2A, middlepanel). Head regression in some intact CHD4(RNAi) animals startedin the center of the head instead of being initiated at the tip of thehead, as is typically the case for irradiated animals. Another well-documented defect of irradiated animals is curling around the ventralsurface of the animal, probably stemming from the failure ofneoblast progeny to replace normally aging differentiated tissue(Reddien and Sánchez Alvarado, 2004). Curling occurs byapproximately 14 days following irradiation. Following headregression, intact CHD4(RNAi) animals also displayed curlingaround their ventral surface (Fig. 2A, right panel). In addition tothese classic hallmarks of neoblast dysfunction, intact CHD4(RNAi)worms showed tissue protrusions after 16-18 days following the firstdsRNA feeding (Fig. 2A, right panel). These protrusions will bediscussed below. Shortly after curling, the CHD4(RNAi) animalsdied by lysis. Together, these observations demonstrate that CHD4is required for normal tissue homeostasis.

CHD4 is required for an increase in neoblastproliferation in response to feeding andwoundingOne simple hypothesis to explain the similarity of the CHD4(RNAi)phenotype to that of irradiated animals lacking neoblasts would be thatCHD4(RNAi) causes a decrease in the presence of neoblasts or in thecapacity of neoblasts to proliferate. To test this hypothesis, wequantified the number of mitotic neoblasts (Newmark and Sánchez

1233RESEARCH ARTICLEPlanarian stem cell differentiation

Fig. 1. CHD4 is required for planarian regeneration and is expressed in neoblasts. (A)As shown in comparison with a control animal, aCHD4(RNAi) worm did not regenerate 7 days following amputation (41/41 were similar). Worms were fed three times and amputated at day 8after initial RNAi. White dotted line represents the blastema boundary. Anterior is to the left. (B)Expression of CHD4 in wild-type and irradiated(6000 rads 5 days prior) animals, dorsal and ventral views (7/7 were similar). Expression of an irradiation-insensitive gene, PC2 (expressed in thenervous system) served as a control. Anterior is to the left. (C)Expression of CHD4 in neoblast subsets, X1 (1472 cells counted) and X2 (655 cellscounted), and radiation-insensitive cells, Xins (1117 cells counted). Scale bars: 0.1 mm.

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Alvarado, 2000) in intact animals using an RNAi feeding approach(see Materials and methods). Food stimulates neoblast proliferation,and proliferation returns to the previous steady-state levels a few dayslater (Baguñà, 1976). The elevation of mitotic numbers immediatelyfollowing feeding failed to robustly occur in CHD4(RNAi) animals(days 7 and 9 after RNAi; Fig. 2B). However, steady-state numbersof mitotic cells were comparable with those of control animals,including at time points after RNAi at which animals completelyfailed to regenerate (between days 12 and 16 following the first RNAifeeding), suggesting that neoblasts were maintained in CHD4(RNAi)animals (Fig. 2B). Much later, when severe head regression andcurling was occurring (approximately between 16-18 days followingthe first dsRNA feeding), a significant decrease in mitotic cells wasobserved (Fig. 2B). A drop in neoblast population numbers late in theprogression of an RNAi phenotype has also been observed followingRNAi of a different gene (smedwi-2) required for homeostasis but notfor neoblast maintenance (Reddien et al., 2005b). Therefore, this latemitotic decrease might be a consequence of, rather than cause of,failed homeostasis.

Because the number of steady-state mitotic neoblasts was normalat time points at which regeneration failed in CHD4(RNAi) animals,we sought to determine whether the total number of neoblasts was

unaffected. Indeed, approximately normal numbers of X1 and X2cells following RNAi were present at all time points examined,except near the time at which animals were dying (Fig. 2C). Atransient decrease in the number of X2 cells at day 11 following thefirst dsRNA feeding was observed. Because X2 cells appear toinclude non-dividing neoblasts as well as neoblast progeny(Eisenhoffer et al., 2008), a defect in neoblast proliferation followingfeeding could explain this slight decrease in X2 cells. Moreover, thegross expression of markers of X1 cells, smedwi-1 and Smed-histoneH2B (Smed-H2B), appeared normal in CHD4(RNAi) animals 10days after the initial dsRNA feeding (Fig. 2D; see Fig. S5 in thesupplementary material). Together, these observations indicate thatthe regeneration defect of CHD4(RNAi) animals is not aconsequence of a loss of neoblasts or a loss of their capacity toproliferate during homeostasis prior to injury.

In addition to the sustained neoblast proliferation in intactplanarians that is required for replacement of aging cells duringnormal turnover, neoblasts undergo an increase in proliferation afterwounding (Saló and Baguñà, 1984). We found that althoughneoblasts in CHD4(RNAi) worms were able to respond to woundingby elevating mitotic numbers at 48 hours and 96 hours followingamputation, the response was diminished compared with control


Fig. 2. CHD4 is required for homeostasis and normal stimulation of neoblast proliferation. (A)Control and CHD4(RNAi) animals duringnormal tissue turnover 14 and 18 days after initial RNAi. Yellow arrowhead, head regression; white arrowhead, lesions; red arrowhead, curling (10/10were similar). Anterior is to the left. (B)Numbers of mitoses labeled with an anti-phospho histone 3 (H3P) antibody were divided by animal surfacearea. Animals were fed three times and fixed at time points after initial RNAi. ‘+ food’ indicates the proliferation response following feedings. Dataare means ± s.e.m. (n2 experiments, >8 worms per time point). *, P<0.05; **, P<0.001, ***, P<0.0001, Student’s t-test. (C)Animals were fed threetimes with dsRNA and cell macerates were generated at multiple time points after initial RNAi. Cells were labeled with Hoechst 33342, calcein, andpropidium iodide; X1 and X2 cell percentages (in the total population of Hoechst-labeled cells) were determined by flow cytometry. Data are means ±s.e.m. (n3 experiments). *, P<0.05; **, P<0.001, Student’s t-test. (D)Fluorescence in situ hybridizations of animals fixed 10 days after multipledsRNA feedings using the smedwi-1 riboprobe (>12 worms per condition were similar). pr, photoreceptors. Anterior is up. (E)Numbers of mitoseslabeled with H3P were divided by animal surface area. Animals were fed three times, amputated 8 days after initial feeding and fixed at differenttimes following wounding. Data are means ± s.e.m.; n2 experiments, >8 animals per time point. ***, P<0.0001, Student’s t-test. Scale bars: 0.1 mm.

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RNAi animals (Fig. 2E). Because neoblast numbers in CHD4(RNAi)animals were normal in steady-state, a partially defectiveproliferative response to injury is probably not sufficient to explainthe complete absence of a blastema in CHD4(RNAi) worms. Forexample, prior RNAi screening indicates that animals with reduced,but not eliminated, mitoses can be capable of making smallblastemas (Reddien et al., 2005a). Thus, given the severe blastemafailure of CHD4(RNAi) animals, it is probable that an additional androbust defect exists to explain the regeneration phenotype. Wetherefore examined the ability of neoblasts in CHD4(RNAi) animalsto produce progeny cells.

CHD4(RNAi) animals die more rapidly than donormal animals following lethal irradiationLethally irradiated and intact wild-type worms develop the firstsigns of defects due to neoblast loss approximately 10-12 days afterirradiation (Reddien et al., 2005b). This phenotype involvesdarkening of the tip of the head, followed by head regression. A fewdays later, the worms curl around their ventral surface and finallythey lyse. Although actively dividing neoblasts completelydisappear within 1 day after irradiation, the initial 10- to 12-daywindow during which no phenotype is apparent might in part relyon the replacement of normally dying aged differentiated cells byexisting non-dividing progenitors (Reddien and Sánchez Alvarado,2004). Therefore, the time of survival after irradiation could beaffected by alterations in numbers or function of neoblast progenycells that can act as progenitors of differentiated cells. We lethallyirradiated control and CHD4(RNAi) worms 8 days after the initialdsRNA feeding. As expected, irradiated control(RNAi) wormsshowed head regression by 12 days following irradiation (Fig. 3A,B,left graph), followed by curling and lysis (Fig. 3A,B, right graph).By contrast, irradiated CHD4(RNAi) animals displayed headregression 6 days following irradiation, and curled and lysed 8 daysafter irradiation (Fig. 3). The rate of appearance of defects inirradiated CHD4(RNAi) animals was faster than that observed instarved or fed irradiated wild-type or control(RNAi) worms (Reddienet al., 2005b) (Fig. 3). Most of the non-irradiated CHD4(RNAi)

animals showed head regression by 12 days after the irradiation day(Fig. 3B, left graph) and they curled and lysed between days 20 and28 after the irradiation day (Fig. 3B, right graph). Similar resultswere obtained with CHD4(RNAi) animals irradiated at 10 days and12 days after the initial dsRNA feeding, times at which normalsteady-state neoblast numbers were reached following feeding (datanot shown). This indicates that the faster appearance of theirradiation phenotype in CHD4(RNAi) animals is not a consequenceof the failure to boost proliferation immediately after feeding. Therapid appearance of head regression following irradiation ofCHD4(RNAi) animals suggests a possible decreased number of non-dividing progeny cells that are neoblast descendants or impairedneoblast differentiation. We therefore directly examined neoblastprogeny cells in CHD4(RNAi) animals (see below).

CHD4(RNAi) animals have decreased numbers ofSmed-AGAT-1-expressing cellsPrevious studies identified three irradiation-sensitive, lineage-related cell subsets (Eisenhoffer et al., 2008) (Fig. 4A). Duringneoblast differentiation, Category 1 gene-expressing cellsdifferentiate into cells expressing Category 2 and 3 genes(Eisenhoffer et al., 2008) (Fig. 4A). Category 1 gene-expressingcells are self-renewing neoblasts (i.e. X1 cells) (Eisenhoffer et al.,2008), which are found in the parenchyma but excluded from theregion anterior to the photoreceptors and the pharynx (Newmark andSánchez Alvarado, 2000). Category 1 gene-expressing cellsdisappear 1 day after irradiation. Category 1 genes include theknown stem cell genes smedwi-1 and Smed-bruli-1, as well as cellcycle regulatory genes (Eisenhoffer et al., 2008; Guo et al., 2006;Reddien et al., 2005b). Genes from Categories 2 and 3 are expressedin X2 and Xins cells but not X1 cells, and are found anterior to thephotoreceptors and close to the animal margins (regions where nomitotic cells are found). Similar to the case for Category 1 genes, thecells expressing Category 2 genes (e.g. the two novel genes Smed-NB.21.11e and Smed-NB.32.1g) rapidly decrease in number 1 dayfollowing irradiation (Eisenhoffer et al., 2008). Cells expressingCategory 3 genes are lost within 7 days following irradiation.


Fig. 3. CHD4(RNAi) intact animalsdie faster following irradiation.(A)Control and CHD4(RNAi) animalswere lethally irradiated with 6000rads 8 days following initial RNAi.Irradiated control(RNAi) animalsshowed head regression by 12 daysfollowing irradiation (7/10 animals)and curling by 14 days (10/10),whereas irradiated CHD4(RNAi)animals showed head regression by 6days following irradiation (10/10) andcurling by 8 days (10/10). Yellowarrowhead, head regression; redarrowhead, curling. Anterior is to theleft. (B)Percentages of control orCHD4(RNAi) animals without headregression (left) and still alive (right)following irradiation are shown. Scalebars: 0.1 mm.

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Category 3 genes include those that encode L-arginine:glycineamidinotransferase (Smed-AGAT)-1, -2 and -3, a ras-related proteinfamily member (Smed-ras-related), a mitochondrial carrier protein1 (Smed-MCP-1) and a novel protein (Smed-NB.52.12f), amongothers. Based on BrdU labeling experiments, Category 2 and 3 gene-expressing cells are rapidly produced descendants of neoblasts(Eisenhoffer et al., 2008). Although the precise nature of these cellsis still unclear, they might either represent a population of non-mitotic progenitors that will generate terminal differentiated cells ora very short-lived population of differentiated cells. Regardless,these markers provide a useful tool to study neoblast progeny cellsand, therefore, neoblast differentiation.

We utilized expression of these genes as markers for distinct celltypes to determine if CHD4(RNAi) animals have a defect in theproduction or maintenance of neoblast progeny cells. We examinedthe expression of the Category 2 gene Smed-NB.21.11e and theCategory 3 gene Smed-AGAT-1 by in situ hybridizations.CHD4(RNAi) worms had approximately normal numbers of Smed-

NB.21.11e-expressing cells (Fig. 4B,C; see Fig. S6 in thesupplementary material). However, there was a marked decrease inthe number of Category 3 Smed-AGAT-1-expressing cells inCHD4(RNAi) worms, as early as 6 days after initial dsRNA feeding(Fig. 4B,C; see Fig. S6 in the supplementary material).

We quantified numbers of Category 1, 2 and 3 cell types byperforming in situ hybridizations on total cells from maceratedplanarians and counting the fraction of total cells that expressedsmedwi-1 (Category 1), Smed-NB.21.11e (Category 2) or Smed-AGAT-1 (Category 3). CHD4(RNAi) animals had significantlyreduced numbers of Smed-AGAT-1-expressing cells at every timepoint analyzed and largely normal numbers of Smed-NB.21.11e-expressing cells (Fig. 4C). There were lower numbers of Smed-NB.21.11e-expressing cells at 11 days following RNAi (Fig. 4C).The small defects in the numbers of Category 2-expressing cellsobserved were possibly due to defects in responses to feeding usedin the RNAi experiments. smedwi-1-expressing cells were presentin CHD4(RNAi) animals in approximately normal numbers initially,


Fig. 4. CHD4(RNAi) animals havereduced numbers of Smed-AGAT-1-expressing cells. (A)Lineage-related cellsand the markers expressed in each celltype. Other models are possible, seeEisenhoffer et al. (Eisenhoffer et al., 2008).(B)Head regions of whole-mount in situhybridizations using a Category 2 probeSmed-NB.21.11e (upper row) and aCategory 3 probe Smed-AGAT-1 (lowerrow) of control and CHD4(RNAi) animalsat several time points following initialRNAi (>4 worms/time point displayedsimilar results). Anterior is to the left.(C)Cell in situ hybridizations using aCategory 1 probe (smedwi-1; >823 cellsper time point), a Category 2 probe(Smed-NB.21.11e; >954 cells per timepoint) and a Category 3 probe (Smed-AGAT-1; >1157 cells per time point) ofcontrol and CHD4(RNAi) worm maceratesat multiple times after initial RNAi.Percentages of smedwi-1+, Smed-NB.21.11e+, and Smed-AGAT-1+ cellswithin the total DAPI-positive cellpopulation are shown (means ± s.e.m.,n3 experiments). *, P<0.05; **,P<0.001, Student’s t-test. (D)Whole-mount in situ hybridizations with Category1 probes (Smed-H2B and smedwi-1), aCategory 2 probe (Smed-NB.21.11e) anda Category 3 probe (Smed-AGAT-1) ofcontrol and CHD4(RNAi) animals 20 daysfollowing initial RNAi. At this late timepoint, CHD4(RNAi) animals displayed headregression, had lesions and began to curl(>7 worms displayed similar results).Anterior is to the left. (E)Whole-mount insitu hybridizations with the Smed-AGAT-1riboprobe of control and CHD4(RNAi)head, trunk and tail pieces fixed 3 daysfollowing amputation. Animals wereamputated 7 days following initial RNAi(>11 regenerating pieces displayed similarresults). Arrows indicate the site ofamputation. Anterior is to the left. Scalebars: 0.1 mm.

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elevated in number at day 11 after the first dsRNA feeding, and lowin number late following RNAi (Fig. 4C). The increased number ofsmedwi-1-expressing cells in CHD4(RNAi) animals at day 11,considered together with the associated decrease in Category 3-expressing cells, is consistent with a possible accumulation ofsmedwi-1-expressing cells because of failure to differentiate.Overall, the earliest, most prominent and most constant abnormalityobserved in CHD4(RNAi) animals was the pronounced low numberof Smed-AGAT-1-expresssing cells.

Twenty days after the initial dsRNA feeding, when theCHD4(RNAi) animals displayed head regression and were dying,the animals were mostly depleted of Smed-NB.21.11e-expressingcells and entirely lacked Smed-AGAT-1-expressing cells (Fig. 4D).At this late time point, similar to the observed decrease in mitoticcells (Fig. 2B), smedwi-1-expressing cells and Smed-H2B-expressing cells were greatly reduced as well (Fig. 4D). Because thisdefect occurred late following RNAi, it probably occurred as aconsequence of an earlier primary defect in the number of neoblastprogeny cells. At this 20-day time point, CHD4(RNAi) animals hadprotrusions; however, we saw no accumulation of dividing cells,Smed-NB.21.11e or Smed-AGAT-1-expressing cells into foci thatcould correspond to protrusions (Fig. 4D).

Smed-AGAT-1-expressing cells normally accumulate at thewound site and in blastemas after amputation, providing evidencefor an important role for this population in regeneration (Eisenhofferet al., 2008). Perhaps as a consequence of the reduced number ofSmed-AGAT-1-expressing cells, CHD4(RNAi) animals failed todisplay accumulation of Smed-AGAT-1-expressing cells at woundsites to the extent observed in the control (Fig. 4E). Our data suggestthat a failure to produce sufficient numbers of neoblast progeny cellscommitted to differentiation, such as Smed-AGAT-1-expressingcells, results in insufficient neoblast progeny for blastema formationin CHD4(RNAi) animals.

CHD4 is required for the formation of Smed-AGAT-1-expressing cellsThe decreased numbers of Smed-AGAT-1-expressing cells inCHD4(RNAi) animals suggests that CHD4 is required either for thenormal generation or maintenance of these cells. First, in order todetermine if the CHD4(RNAi) phenotype is explained by a defect inthe maintenance of these cells, we determined the rate at whichSmed-AGAT-1-expressing cells disappeared following irradiation.Irradiation kills neoblasts and therefore blocks the generation of newSmed-AGAT-1-expressing cells. Animals were fed only once withCHD4 dsRNA or control food and irradiated the same day (Fig. 5A,left graph), or fed 3 times over 7 days and irradiated 7 days followingthe initial feeding (Fig. 5A, right graph). The numbers of Smed-AGAT-1-expressing cells were determined at different time pointsfollowing irradiation. In both experiments, Smed-AGAT-1-expressing cells disappeared at similar rates following irradiation inboth control and CHD4(RNAi) animals, suggesting that theCHD4(RNAi) phenotype is not explained by a defect in themaintenance of these cells. Therefore, these data point to a defect inthe generation of Smed-AGAT-1-expressing cells.

To determine if CHD4 is required for the generation of Smed-AGAT-1-expressing cells directly, two different experiments wereperformed. First, the formation of Smed-AGAT-1-expressing cells inCHD4(RNAi) or control animals following one pulse with thethymidine analog BrdU was observed (see Fig. S7 in thesupplementary material). BrdU is incorporated into neoblasts withina few hours following injection (Newmark and Sánchez Alvarado,2000), and 4 days later, double-labeled BrdU-Smed-AGAT-1-

expressing cells are found (Eisenhoffer et al., 2008). Four days afterthe BrdU pulse, most of the Smed-AGAT-1-expressing cells werelabeled with BrdU (see Fig. S7 in the supplementary material). Wefound that the number of newly formed Smed-AGAT-1-expressingcells was significantly lower in CHD4(RNAi) animals comparedwith control animals 4 days after the BrdU pulse and 10 daysfollowing the initial RNAi feeding (see Fig. S7 in the supplementarymaterial).

Second, we determined the number of SMEDWI-1; Smed-AGAT-1 double-positive cells in CHD4(RNAi) and control animals. TheSMEDWI-1 protein has been demonstrated to be present not only inneoblasts, which also express smedwi-1 mRNA, but also in non-mitotic neoblast progeny that no longer express smedwi-1 mRNA(Guo et al., 2006). This is presumably due to perdurance of theSMEDWI-1 protein into immediate neoblast descendants.Moreover, three days following lethal irradiation, all SMEDWI-1-expressing cells disappeared from the animal, indicating that onlyneoblasts or their immediate progeny cells express this protein (Guoet al., 2006). We found that CHD4(RNAi) animals have a decreasednumber of SMEDWI-1; Smed-AGAT-1 double-positive cells at 7days following initial RNAi and an even greater defect at 10 daysfollowing the initial RNAi feeding (Fig. 5B). In summary, theseexperiments demonstrate that the CHD4(RNAi) phenotype isexplained by a defect in the generation of Smed-AGAT-1-expressingcells rather than by a defect in their perdurance.

If neoblasts are defective in production of Smed-AGAT-1-positivecells, where is the process of differentiation affected? We found atransient increase in the number of SMEDWI-1 protein-expressingcells in animal head regions in CHD4(RNAi) animals at day 7following RNAi (Fig. 5C). These SMEDWI-1 protein-positive cellsin head tips represent immediate non-mitotic neoblast progeny.Therefore, coincident with a decrease in Smed-AGAT-1-positivecells, there existed an increase in number of immediate neoblastprogeny. This observation indicates that immediate neoblastprogeny might accumulate because of a failure to differentiate.

CHD4(RNAi) animals have a decreased number ofneoblast progeny cellsTo more definitively determine the role of CHD4 in neoblastdifferentiation, and to exclude any potential role of CHD4 inspecifically regulating Smed-AGAT-1 expression, we examined theexpression of three additional Category 3 markers in CHD4(RNAi)animals (Eisenhoffer et al., 2008). CHD4(RNAi) animals also had arobust decrease in the number of cells expressing the Smed-ras-related, Smed-MCP-1 and Smed-NB.52.12f genes (Fig. 6A,B).However, the existing cells that expressed these genes displayed noobvious defect in expression levels for these genes. Previous datahave shown that not all Smed-AGAT-1-expressing cells co-expressthe Smed-ras-related gene (Eisenhoffer et al., 2008). In addition,almost no Smed-AGAT-1-expressing cells co-express the Smed-MCP-1 gene (Eisenhoffer et al., 2008), indicating that there is aheterogeneous population of neoblast progeny cells expressingCategory 3 genes. Our results demonstrate that CHD4 is required forthe generation of a heterogeneous population of neoblast progenycells committed to differentiation.

DISCUSSIONPlanarians are an emerging in vivo model system for the study ofmechanisms that regulate stem cell proliferation, maintenance anddifferentiation because of their remarkable regenerative capacitiesand extensive tissue turnover. These two processes are driven by thenumerous neoblasts that reside throughout the animal (Reddien and


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Sánchez Alvarado, 2004). The use of adult stem cells to replace agedor damaged cells and tissues is essential for most metazoans but isstill poorly understood at the molecular level.

To date, very few planarian genes have been described to have afunction crucial for neoblast biology. Two examples are the Smed-bruli-1 and smedwi-2 genes (Guo et al., 2006; Reddien et al., 2005b).Smed-bruli-1 is not needed for neoblast proliferation in response towounding and differentiation. Instead, Smed-bruli-1 has been shownto play a role in neoblast self-renewal. Smed-bruli(RNAi) animalsare gradually depleted of neoblasts and are therefore unable toregenerate and survive during homeostasis (Guo et al., 2006). Bycontrast, smedwi-2 is not required for neoblast maintenance but

instead is needed for the production of cells capable of replacingaged differentiated cells. In smedwi-2(RNAi) animals, ageddifferentiated cells are not replaced during homeostasis andregeneration, resulting in the incapacity of these animals to surviveand regenerate missing tissues (Reddien et al., 2005b). Few geneshave yet been described to have a crucial role in the production ofdifferentiation-committed neoblast progeny cells (Pearson andSánchez Alvarado, 2010).

The usage of RNAi and lineage markers helps to establish S.mediterranea as a powerful in vivo model system for the study ofstem cell proliferation and lineage differentiation during tissuehomeostasis and regeneration. Here, we show the existence of a


Fig. 5. CHD4 is required for the formation of Smed-AGAT-1-expressing cells. (A)Smed-AGAT-1+ cell numbers normalized by animal lengthfrom whole-mount in situ hybridizations are shown at time points following irradiation. Animals (>4 worms per group) were fed once and lethallyirradiated the same day (left) or fed three times and lethally irradiated at day 7 (right). Data are means ± s.e.m. (B)Percentage of double-labeledSMEDWI-1; Smed-AGAT-1-expressing cells in the total number of Smed-AGAT-1+ cells in control and CHD4(RNAi) animals (upper graph), and thepercentage of double-labeled SMEDWI-1; Smed-AGAT-1-expressing cells normalized by the area of the head region (lower graph). Data are means± s.e.m. (>5 worms per group). *, P<0.05; **, P<0.001; ***, P<0.0001, Student’s t-test. Animals were fed twice and fixed at time points followingthe initial RNAi. Fluorescence in situ hybridizations using Smed-AGAT-1 riboprobe (green) and SMEDWI-1 antibody (red) are shown. Arrowheadsindicate cells that co-express Smed-AGAT-1 and SMEDWI-1. (C)SMEDWI-1-expressing cell numbers in control and CHD4(RNAi) animals.pr, photoreceptors. Circled area, region counted. Data are means ± s.e.m. (>8 worms per group). **, P<0.001, Student’s t-test. A secondexperiment had similar results. Animals were fed twice and fixed at time points following initial RNAi. Anterior is up. Scale bars: 0.1 mm.DEVELO

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gene that is required for the transition of neoblasts from self-renewing stem cells into more committed non-dividing cells duringregeneration and homeostasis.

We found that a CHD4-like gene is required for planarianregeneration and neoblast differentiation. Numerous abnormalitieswere observed in CHD4(RNAi) animals. These included a robustblock in regeneration, failure in the maintenance of differentiatedtissues (which normally requires the neoblasts) and ultimatelylesions/protrusions in the body of the RNAi animals before animaldeath. The explanation for the protrusions is unknown. Our dataindicate that these protrusions are not explained by an accumulationof proliferating cells or any known neoblast progeny cells. Therefore,this defect might reflect a requirement for CHD4 in differentiatedtissue or the aberrant action of unidentified neoblast progeny cells.

CHD4(RNAi) animals cannot regenerate as early as 5 daysfollowing the initial dsRNA feeding (see Fig. S8 in thesupplementary material). This failure to regenerate occurs rapidlyfollowing RNAi when compared with worms where neoblastfunction has been disrupted by RNAi of other genes (Reddien et al.,2005b). The inability of CHD4(RNAi) animals to regenerate and toreplace normally aging differentiated tissue cannot be explained bya loss of neoblasts because these animals initially have normal orslightly higher numbers of actively dividing neoblasts (smedwi-1-expressing cells). By contrast, our data indicate that the failure toregenerate and to maintain normal tissue turnover is due to a markedreduction in numbers of neoblast progeny cells (such as cellsexpressing Category 3 genes). Furthermore, our results indicate thatthe reduced number of neoblast progeny cells found in CHD4(RNAi)animals was due to a defect in the generation of these cells asopposed to a defect in their maintenance. The neoblasts ofCHD4(RNAi) animals produced immediate progeny cells (notexpressing Category 3 genes) that transiently accumulated,presumably as a consequence of the differentiation failure. Becauseno other markers besides the Categories 2 and 3 markers used in thisstudy have been identified to be expressed in neoblast progeny cells,and no accumulation of germ cells has been observed inCHD4(RNAi) animals (not shown), it remains unresolved ifneoblasts in CHD4(RNAi) animals differentiate into a particularlineage different from the ones tested here. At least some neoblast

progeny cells have been shown to localize to the blastema(Eisenhoffer et al., 2008); however, perhaps because of the limitednumber of these cells in CHD4(RNAi) animals, neoblast progenycells did not localize to the wound sites in appropriate numbers.These observations, together with the finding that CHD4 isexpressed near the wound site in neoblasts or neoblast progeny cells3 days following amputation, suggest a crucial role for CHD4 earlyin the regeneration process.

Changes in chromatin structure have been implicated in theregulation and maintenance of many cell fate decisions. Chromatinmodifiers can be essential to keep stem cells in a pluripotent stageor to drive them into a differentiation pathway. For example, in thecase of mouse and human embryonic stem (ES) cells, componentsof the polycomb complex can silence developmental regulatorygenes that are preferentially expressed during differentiation (Boyeret al., 2006; Lee et al., 2006). These results suggest that polycombproteins maintain the pluripotent state of ES cells. Ezh, a componentof the PRC2 polycomb complex, is also active in epidermalprogenitors (Ezhkova et al., 2009). However, in general, how theregulation of differentiation in ES cells corresponds to what occursin adult stem cell types in vivo remains unresolved. In the case of theNuRD complex component CHD4, our data indicate a role inpromoting, rather than repressing, stem cell differentiation. TheNuRD complex has been shown to regulate cell fate decisions anddevelopment in multiple contexts. In D. melanogaster for example,Mi-2 can function with polycomb genes to maintain repression ofhomeotic genes during embryonic patterning (Kehle et al., 1998). InC. elegans, the CHD4 homolog let-418, is required for vulval cellfate determination and for maintenance of germline-somadistinctions (Unhavaithaya et al., 2002; von Zelewsky et al., 2000).Specifically, in let-418-defective animals, somatic cells expressgerm cell markers (e.g. the P granule component PGL-1 and Vasahomologs), indicating that a CHD4 protein in C. elegans is requiredto repress germline cell identity in differentiated cells. Interestingly,a CHD4 homolog in the plant A. thaliana, pickle, is required forrepression of embryonic-like characteristics after germination (Ogaset al., 1997; Ogas et al., 1999). In pickle mutants, the fatty acidcomposition of roots was shown to resemble that of seeds, and rootsformed callus growths and embryo-like physical structures.


Fig. 6. CHD4(RNAi) animals havedecreased numbers of severalCategory 3-expressing cells.(A)Whole-mount in situ hybridizationsusing Category 3 probes 10 daysfollowing initial RNAi (>4 worms perriboprobe were similar). Anterior is tothe left. (B)Fluorescence in situhybridizations of animals fixed 10 daysafter multiple dsRNA feedings usingthe Smed-NB.21.11e, Smed-AGAT-1,Smed-MCP-1 and Smed-NB.52.12friboprobes (>12 worms per conditionwere similar). The head region isshown. pr, photoreceptors. Anterior isup. Scale bars: 0.1 mm.

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Therefore, in both C. elegans and Arabidopsis, these data suggest afunction in repression of germ cell/embryonic-like features indifferentiated tissue. It will be interesting to determine in the futurehow these roles compare with the action of CHD4 in stem cells inother organisms. For instance, it is possible that CHD4 repressesstem cell/embryonic-like genes in differentiated tissues in theseorganisms and in planarian neoblasts to promote neoblastdifferentiation.

In the mouse, knockout of Mi-2 (CHD4) has complex impactson the functioning of hematopoietic stem cells (HSCs) (Yoshida etal., 2008). Mi-2-deficient HSCs displayed increased proliferationand were able to begin differentiation into erythroid, but not myeloidand lymphoid, lineages. Specifically, conditional knockout Mi-2animals showed depletion of granulocytes (myeloid) and newlyformed B cells (lymphocytes). Furthermore, although pro-erythroblasts were formed, the differentiation of these pro-erythroblasts into basophilic and other cells was aberrant in thesemice. Therefore, in the case of this stem cell type, Mi-2 appears tobe required for lineage decisions that could reflect a role indifferentiation. Expression analyses indicated that Mi-2 did notplay a global role in gene expression regulation but affected specifictranscripts. The gene expression defects in these Mi-2-deficientHSCs also indicated a potential additional role for Mi-2 in self-renewal. A different component of the NuRD complex, Mbd3, wasfound to be required for ES cells to differentiate in vitro (Kaji et al.,2006); Mbd3 was also required for proliferation of epiblast cells inculture and derivation of ES cells from inner-cell-mass cells (Kaji etal., 2007). The data from adult planarian neoblasts described here,taken in the context of the results describing the function of CHD4and other NuRD components in other organisms, suggest that amajor and broadly utilized role for this complex is in promoting stemcell differentiation.

AcknowledgementsWe thank Laurie Boyer, Ezequiel Alvarez-Saavedra, Daniel Wagner and allmembers of the Reddien lab for manuscript comments, support anddiscussions. We thank Danielle Wenemoser for collaboration with microarrayexperiments and the SMEDWI-1 antibody. This work was supported by NIHR01GM080639. We acknowledge support by the Keck, Smith, Searle and RitaAllen Foundations. M.L.S. was supported by a Jane Coffin Childs MemorialFund Fellowship. P.W.R. is an Early Career Scientist of the Howard HughesMedical Institute and holds a Thomas D. and Virginia W. Cabot CareerDevelopment Professorship. Deposited in PMC for release after 6 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042051/-/DC1

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