SMEDWI-2 Is a PIWI-Like Protein That Regulates Planarian Stem Cells

R EPORTS studied smedwi-1 and -2 because they encode proteins similar to the PIWI class of PIWI/ SMEDWI-2 Is a PIWI-Like Argonaute proteins (fig. S1) and are thus can- didates to regulate the adult somatic stem cells Protein That Regulates (neoblasts) of planarians (12, 13). Furthermore, smedwi-1 expression resembles the distribution Planarian Stem Cells of planarian neoblasts (14). Planarians present a promising venue for the study of PIWI-like Peter W. Reddien,* Ne´stor J. Oviedo,. Joya R. Jennings, genes because stem cells play prominent roles James C. Jenkin, Alejandro Sa´nchez Alvarado- in homeostasis and regeneration and because the genetic study of planarian stem cells is now We have identified two genes, smedwi-1 and smedwi-2,expressedinthedi- possible (12, 13, 15). viding adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. smedwi-1 and smedwi-2 are expressed in Both genes encode proteins that belong to the Argonaute/PIWI protein family small cells distributed like neoblasts—that and that share highest homology with those proteins defined by Drosophila is, posterior to photoreceptors and excluded PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though from the pharynx (Fig. 1, A to C). smedwi- neoblasts are present, irradiation-sensitive, and capable of proliferating in re- expressing cells, like neoblasts, reside in the sponse to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell parenchyma: mesenchymal tissue excluded turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that from the nervous system, pharynx, and gastro- SMEDWI-2 functions within dividing neoblasts to support the generation of cells vascular system (Fig. 1, D and E). Neoblasts that promote regeneration and homeostasis. are quickly and specifically eliminated after irradiation (12). Expression of smedwi-1 and Members of the PIWI/Argonaute family of proteins have been implicated in the regulation proteins fall into two main classes, one named of germ cells (9–11). Very little is known, how- Howard Hughes Medical Institute, Department of after Arabidopsis Argonaute and the other after ever, about how PIWI proteins regulate germ Neurobiology and Anatomy, University of Utah School Drosophila PIWI (1).Membersofthisprotein cells and whether these proteins typically pro- of Medicine, Salt Lake City, UT 84132, USA. family contain PAZ and PIWI domains and mote stem cell maintenance or differentiation. *Present address: MIT Biology, Whitehead Institute, mediate silencing via cleavage of mRNAs (2–4) Furthermore, the types of stem cells and de- Cambridge, MA 02142, USA. or by inhibition of translation (5, 6). These pro- velopmental events regulated by PIWI-class .Present address: Cytokine Biology Department, teins are found in plants (7), yeast (8), and proteins remain unclear. Forsyth Institute, Harvard Medical School, Boston, MA 02115, USA. throughout the animal kingdom—including at Here we report on the planarian S. medi- -To whom correspondence should be addressed. least eight in humans. Some of the PIWI-class terranea genes smedwi-1 and smedwi-2.We E-mail: [email protected]

Fig. 1. Dividing neoblasts express smedwi-1 and smedwi-2.(A) BrdU la- (I) FACS profile of S. mediterranea cells (e20 mm), Hoechst- and calcein- beling demonstrates neoblast distribution; (B and C) smedwi-1 and -2 labeled. X1 (blue) and X2 (pink) populations are irradiation-sensitive; Xins expression resembles neoblast distribution. Riboprobes are identified at (orange) population is not. (J) smedwi-1 (top), smedwi-2 (middle), and lower left; anterior is to the left. (D and E) Tissue cross sections, dorsal up. smedcyclinB [clone NBE.6.09E (AY967658); bottom] in situ hybrid- Green, nuclei; black, smedwi expression; e, epidermis; p, pharynx. (F to H) izations on FACS-isolated cells. Representative results are shown. Ex- Irradiation at 6000 rad. After irradiation, smedwi-1 signals (4/4 at 48 pression was typically in X1 but not X2 or Xins cells. Percentages are hours, 5/5 at 72 hours) and smedwi-2 signals (2/2 at 48 hours, 8/8 at 72 signal-positive cells (n 9 210 cells each). Scale bars, 0.2 mm [(A) to (C), hours) were undetectable. H.1.3B is an irradiation-insensitive control. (F) to (H)], 10 mm(J).

www.sciencemag.org SCIENCE VOL 310 25 NOVEMBER 2005 1327 R EPORTS -2 was markedly reduced by irradiation, con- cells (n 0 257) (Fig. 1J), which suggests that semble irradiated animals, smedwi-2 is likely sistent with their possible expression in neo- X1 cells are dividing. Together, our data in- needed for neoblast function. blasts (Fig. 1, F to H) (table S1). dicate that smedwi genes are expressed direct- smedwi-2(RNAi) defects progress through Flow cytometry has been used to separate ly and largely specifically within dividing temporal stages, ending in lethality (fig. S2B). planarian cells and identify neoblasts (16, 17). neoblasts. At Q8 days after exposure to smedwi-2 double- We used flow cytometry to isolate two pop- To investigate how SMEDWI proteins stranded RNA (dsRNA), animals were inca- ulations of irradiation-sensitive cells with neo- regulate neoblasts, we inhibited smedwi-1 pable of regeneration. Animals wounded earlier blast morphology, named X1 and X2, and and -2 with the use of RNA interference could regenerate small blastemas that re- examined smedwi-2 expression (Fig. 1I). In (RNAi). RNAi affected expression specifi- gressed (fig. S2C). Homeostasis defects after situ hybridizations and quantitative reverse cally (fig. S2A). RNAi of smedwi-1 did not smedwi-2 silencing arose with kinetics similar transcription polymerase chain reactions indi- cause robust defects, but RNAi of smedwi-2 to those in irradiated animals. For example, 9 cated that smedwi genes are predominantly resulted in 100% penetrant defects resem- days after irradiation, 15 of 30 animals dis- expressed within X1 cells, at low percentages bling those in irradiated animals lacking played head regression, whereas 9 days after in X2 cells, and perhaps absent from control neoblasts. Irradiated and smedwi-2(RNAi) smedwi-2 dsRNA feeding, 12 of 29 animals irradiation-insensitive Xins cells (Fig. 1J) (table animals displayed regression of tissue ante- did so. Because irradiation prevents new cell S2). Because neoblasts proliferate, we asked rior to the photoreceptors, were incapable of production within 24 hours, homeostasis is whether cell cycle differences between X1 regeneration, and curled around their ventral likely disrupted within a day after smedwi-2 and X2 cells exist by examining expression of surface (Fig. 2, A to C). Head regression inhibition (fig. S2B). SMEDWI-2 protein lev- an S. mediterranea gene homologous to the likely occurred because tissue in front of the els sufficient for neoblast function in tissue cell division marker cyclinB (smedcyclinB). photoreceptors lacks dividing neoblasts and turnover likely do not perdure after RNAi. This We observed smedcyclinB expression in is constantly replaced by neoblast progeny hypothesis is supported by the observation that 96% of X1 cells (n 0 325) and 0% of X2 (12). Because smedwi-2(RNAi) animals re- smedwi-2 is expressed in dividing cells, which

Fig. 2. smedwi-2(RNAi) animals display defects related to neoblast feedings, Q5 animals per time point. Times shown are days after first dysfunction. (A to C) Irradiation at 6000 rad. C. elegans unc-22, negative feeding. Right panel: One dsRNA feeding, Q6 animals per time point. control. Anterior is to the left; scale bar, 0.2 mm. Yellow arrowheads Control numbers decline with time because feedings boost mitotic show head regression. In (B) and (C); ventral is up; in (C), heads and tails numbers. *P G 0.001, t test. (E) Cell numbers were analyzed by FACS (see are amputated [dotted line, blastema (unpigmented) boundary]. White Fig. 1I for nomenclature). Control irradiations were done 5 days before arrows, blastemas; yellow arrows, healed wounds without blastema. (D) analysis. smedwi-2(RNAi) X1 and X2 irradiation-sensitive populations Mitotic numbers divided by animal length. Left panel: Two dsRNA were present 10 days after RNAi but were greatly reduced by 18 days.

1328 25 NOVEMBER 2005 VOL 310 SCIENCE www.sciencemag.org R EPORTS indicates that new protein is produced with new S2D). Fluorescence-activated cell sorting We asked whether smedwi-2(RNAi) neo- cell production. (FACS) analyses indicated that both X1 and blasts were capable of responding to wounds One simple explanation for the smedwi- X2 cells were present and irradiation-sensitive in animals incapable of regeneration. Specifi- 2(RNAi) phenotype would be absence of neo- in smedwi-2(RNAi) animals incapable of cally, we quantified mitotic numbers in pre- blasts. Neoblasts are the only known mitotic regeneration 10 and 12 days after RNAi but pharyngeal animal fragments that were or were cells in adult planarians (12). We observed that were greatly diminished by days 14 through not proximal to a wound produced 14 hours mitotic numbers in smedwi-2(RNAi) animals 18 (Fig. 2E) (fig. S3). earlier (Fig. 3A). We determined that smedwi- always eventually plummet (Fig. 2D). We These experiments revealed several impor- 2(RNAi) animals were capable of mounting an quantified this defect in intact RNAi animals. tant aspects of the smedwi-2 phenotype. First, apparently normal proliferative wounding re- As expected, at days 11 and 14, smedwi- and in contrast to irradiated animals, neoblasts sponse (Fig. 3A, P G 0.001). This finding 2(RNAi) animals had far fewer mitotic cells were present and proliferating at times after indicates that the primary defect that blocked than did controls (P G 0.001, t test). How- RNAi when animals could not regenerate. blastema formation in smedwi-2(RNAi) ani- ever, smedwi-2(RNAi) animals at day 9 were This suggests that failed neoblast mainte- mals was not a gross dysfunction in the ability defective for regeneration but had greater mi- nance was not the primary cause of failed of neoblasts to detect wounds. We therefore totic numbers than did the control (P G 0.001). regeneration and tissue turnover. Second, at propose that neoblast progeny function (e.g., We designed a second time course to carefully early time points after RNAi, a greater num- differentiation, migration) is the primary ab- observe this increase (Fig. 2D). Because ber of smedwi-2(RNAi) mitotic neoblasts were normality underlying the smedwi-2(RNAi) feeding boosts mitotic numbers, a single feed- present than in controls, suggesting a feed- phenotype. ing was used so that early time points could back regulation from loss of tissue homeosta- To assess whether neoblast progeny can be more easily observed. A clear and signif- sis. Because tissue turnover fails quickly after migrate, we labeled them with 5-bromo-2¶- icant increase in mitotic numbers above the RNAi of smedwi-2, mitotic numbers may in- deoxyuridine (BrdU) and examined head control was observed at days 7, 10, and 12 in crease during this period as a consequence of tips, a region devoid of proliferating cells smedwi-2(RNAi) animals (P G 0.001). In situ failed homeostasis. Finally, in late phases of and into which labeled cells migrate (18). hybridizations with the smedwi-1 riboprobe the smedwi-2 phenotype, neoblasts are not The progeny of smedwi-2(RNAi) neoblasts indicated that X1 cells sharply decline in maintained. Long-term neoblast maintenance did not grossly fail to migrate in front of the numbers rather than lose the capacity to abnormalities could be a secondary result of photoreceptors of regressing heads (Fig. 3B); divide at late time points after RNAi (fig. failed homeostasis. this result suggests that smedwi-2(RNAi)

Fig. 3. smedwi-2(RNAi) neoblasts respond to wounds but produce functionally abnormal progeny cells. (A) Two prepharyngeal fragment sets were compared: anterior surface generated 14 hours before fixation in one set and 45 min before fixation in the other. *P G 0.001, t test; Q7 fragments counted per time point. Day indicates day of analysis after first dsRNA feeding. Amputated smedwi-2(RNAi) animals regenerated abnormally by 5 days (8 of 22 animals amputated on day 7 had very small blastemas and 14 of 22 had no blastemas; 8 of 30 animals amputated on day 8 had very small blastemas and 22 of 30 had no blastemas). All controls were normal (day 7, n 0 26; day 8, n 0 20). (B) Cells in red are BrdU-positive 3 days after labeling. White arrowhead, tissue regression 10 days after dsRNA exposure; PR, photoreceptors. Proliferative neoblasts do not exist anterior to photoreceptors, which indicates that neoblast progeny migrated. Anterior is up; scale bar, 0.1 mm. (C) Postpharyngeal cross sections 13 days after RNAi and 6 days after BrdU labeling. White arrows, BrdU-positive cells in epidermis (outermost cells). Dotted lines are on both sides of the basement membrane of the single- cell epidermal layer. Ventral surface is up; scale bar, 10 mm.

www.sciencemag.org SCIENCE VOL 310 25 NOVEMBER 2005 1329 R EPORTS neoblasts produced cells capable of reaching How might a primary defect in smedwi- 3. J. Liu et al., Science 305, 1437 (2004). sites of tissue turnover. 2(RNAi) neoblast progeny relate to the second- 4. T. A. Rand, K. Ginalski, N. V. Grishin, X. Wang, Proc. Natl. Acad. Sci. U.S.A. 101, 14385 (2004). Next, we asked whether neoblast progeny ary defect in neoblast maintenance? Although 5. K. Okamura, A. Ishizuka, H. Siomi, M. C. Siomi, Genes were capable of producing differentiated cells. it is unknown how an aging differentiated Dev. 18, 1655 (2004). Neoblast progeny normally begin to replace cell population affects neoblast division pat- 6. R. S. Pillai, C. G. Artus, W. Filipowicz, RNA 10, 1518 (2004). aged epidermal cells within a week after terns, one possibility is that neoblasts are 7. K. Bohmert et al., EMBO J. 17, 170 (1998). labeling (18). We sectioned BrdU-labeled depleted because of their failure to quench 8. A. Sigova, N. Rhind, P. D. Zamore, Genes Dev. 18, smedwi-2(RNAi) animals after BrdU incor- an ever-increasing demand for differentiated 2359 (2004). 9. D. N. Cox et al., Genes Dev. 12, 3715 (1998). poration and determined that labeled cells en- cell replacement. 10. S. Kuramochi-Miyagawa et al., Development 131, tered the epidermis sooner than in the control Our results provide mechanistic insight into 839 (2004). and displayed a grossly abnormal morphology how specific cellular events of planarian re- 11. W. Deng, H. Lin, Dev. Cell 2, 819 (2002). 12. P. W. Reddien, A. Sańchez Alvarado, Annu. Rev. Cell (Fig. 3C) (fig. S4 and table S3). Planarian generation are controlled. PIWI-like proteins Dev. Biol. 20, 725 (2004). epidermal cells reside in a single-cell layer regulate molecular processes involving small 13. P. W. Reddien, A. L. Bermange, K. J. Murfitt, J. R. and display classic columnar epithelial mor- RNAs; SMEDWI-2 may regulate some aspect Jennings, A. Sańchez Alvarado, Dev. Cell 8, 635 (2005). 14. A. Sańchez Alvarado, P. A. Newmark, S. M. Robb, R. phology. BrdU-labeled smedwi-2(RNAi) cells of RNA metabolism within dividing neo- Juste, Development 129, 5659 (2002). that reach the epidermis, however, fail to blasts. Our data suggest that SMEDWI-2 is not 15. P. A. Newmark, A. Sańchez Alvarado, Nat. Rev. Genet. adopt a columnar morphology. For example, needed primarily for neoblast maintenance, but 3, 210 (2002). the average heights of BrdU-labeled epider- rather for the production of neoblast progeny 16. M. Asami et al., Zool. Sci. 19, 1257 (2002). 17. K. Ogawa et al., Dev. Growth Differ. 44, 191 (2002). mal cells were significantly different in smedwi- capable of replacing aged differentiated cells 18. P. Newmark, A. Sańchez Alvarado, Dev. Biol. 220, 2(RNAi) animals and control unc-22(RNAi) during homeostasis and missing tissues during 142 (2000). animals: 3.5 T 1.6 mm(n 0 18) and 10.0 T regeneration. Consistent with this suggestion, 19. A. K. Sharma et al., Blood 97, 426 (2001). 0 G 20. We thank K. Agata for FACS protocols, N. H. Oh for 1.9 mm(n 10), respectively (P 0.0001, t the mouse PIWI-related proteins MIWI and FACS assistance, P. Newmark and F. Cebria´ for dis- test). Regions posterior to the photoreceptors, MILI are needed for the completion of sper- cussions, B. Pearson for comments, and all Sańchez lab where epidermis could be clearly visualized, matogenesis but not for primordial germ cell members for support. P.W.R. is a Helen Hay Whitney Foundation fellow. A.S.A. is a Howard Hughes Medical were observed. In smedwi-2(RNAi) animals, formation (10, 11). Because human hiwi is ex- Institute Investigator. Supported by National Institute heads regress and the cells that reach the pressed in primitive hematopoietic cells, PIWI of General Medical Sciences grant RO-1 GM57260 epidermis in the body are morphologically proteins may regulate multiple types of stem (A.S.A.). abnormal; for this reason, we suggest that cells (19). We suggest that PIWI proteins may Supporting Online Material neoblast progeny cells migrate to sites of be universal regulators of the production of www.sciencemag.org/cgi/content/full/310/5752/1327/ tissue turnover but fail to function normally. stem cell progeny competent for performing DC1 Materials and Methods That the defect is within neoblast progeny is differentiated functions. Figs. S1 to S4 supported by the observations that smedwi- Tables S1 to S3 2(RNAi) animals resemble irradiated ani- References and Notes References 1. M. A. Carmell, Z. Xuan, M. Q. Zhang, G. J. Hannon, mals and that smedwi-2 is expressed in the Genes Dev. 16, 2733 (2002). 14 June 2005; accepted 26 October 2005 neoblasts. 2. G. Meister et al., Mol. Cell 15, 185 (2004). 10.1126/science.1116110

tive 1- VPC; it produces the lateral signal, LIN-12/Notch Activation Leads to which activates LIN-12 in P5.p and P7.p. When LIN-12 is activated, proteolysis releases its MicroRNA-Mediated Down- intracellular domain, which translocates to the nucleus and forms a transcriptional activation complex with the DNA binding protein LAG-1 Regulation of Vav in C. elegans (2). Transcriptional targets of LIN-12 in P5.p Andrew S. Yoo1 and Iva Greenwald2* and P7.p can be identified by the presence of LAG-1 binding sites (LBSs) in their 5¶ flanking Cell-cell interactions and cross-talk between signaling pathways specify regions and include genes that encode negative Caenorhabditis elegans vulval precursor cells (VPCs) to adopt a spatial pattern: regulators of EGFR-Ras-MAPK activity in P5.p a central ‘‘1-’’ VPC, in which epidermal growth factor receptor (EGFR)–mitogen- and P7.p, which inhibit the expression of 1- fate activated protein kinase (MAPK) activity is high and LIN-12/Notch activity is features in these cells (3). low, flanked by two ‘‘2-’’ VPCs, in which LIN-12/Notch activity is high and EGFR- Short regulatory microRNAs (miRNAs), MAPK activity is low. Here, we identify a microRNA gene, mir-61, as a direct first identified in C. elegans (4), mediate post- transcriptional target of LIN-12 and show that expression of mir-61 promotes transcriptional down-regulation of target genes. the 2- fate. We also identify vav-1, the ortholog of the Vav oncogene, as a The profound and pervasive roles that miRNAs target of mir-61, and show that down-regulation of VAV-1 promotes lin-12 play as critical regulators of developmental activity in specifying the 2- fate. Our results suggest that lin-12, mir-61,and gene expression are only now becoming fully vav-1 form a feedback loop that helps maximize lin-12 activity in the pre- appreciated. We obtained an indication that a sumptive 2- VPCs. 1Integrated Program in Cellular, Molecular, and 2 Six multipotential VPCs, numbered P3.p to P8.p, (Fig. 1A) (1). The inductive signal from the Biophysical Studies, Department of Biochemistry adopt an invariant pattern of fates termed 3--3-- gonad activates an EGFR-Ras-MAPK cascade and Molecular Biophysics, Howard Hughes Medical - - - - Institute, Columbia University College of Physicians 2 -1 -2 -3 (Fig. 1A). Two signaling events in a graded fashion in the underlying VPCs, and Surgeons, 701 West 168th Street, Room 720, specify this pattern: Binductive[ signaling, P5.p, P6.p, and P7.p. The centralmost VPC, New York, NY 10032, USA. mediated by an EGFR-Ras-MAPK pathway, P6.p, has the highest level of EGFR-Ras- *To whom correspondence should be addressed. and Blateral[ signaling, mediated by LIN-12 MAPK activation and becomes the presump- E-mail: [email protected]

1330 25 NOVEMBER 2005 VOL 310 SCIENCE www.sciencemag.org

www.sciencemag.org/cgi/content/full/310/5752/1327/DC1

Supporting Online Material for

SMEDWI-2 Is a PIWI-like Protein that Regulates Planarian Stem Cells

Peter W. Reddien, Néstor J. Oviedo, Joya R. Jennings, James C. Jenkin, Alejandro Sánchez Alvarado*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 25 November 2005, Science 310, 1327 (2005) DOI: 10.1126/science.1116110

This PDF file includes:

Materials and Methods Tables S1 and S2 Figs. S1 to S4 References

Supporting Online Material

Materials and Methods

GeneBank accession numbers for smedwi-1 and smedwi-2 are DQ186985 and

DQ186986.

In situ hybridizations

Animals were fixed in Carnoy’s fixative as described previously and stored in methanol.

Following re-hydration with a methanol:PBST (PBS, 0.1% triton X-100) dilution series, animals were incubated at 56°C for 2 hours in pre-hybridization solution (50% formamide, 5X SSC pH 7.0, 100µg/mL heparin, 0.05% tween-20, 0.05% triton X-100,

5mM DTT, 1mg/mL yeast RNA, 1X Denhardt’s). Hybridization with digoxigenin

(DIG)-conjugated riboprobes was done for 16 hours in hybridization solution (same as pre-hybridization solution, except plus 5% dextran sulfate). Animals were washed with wash Hybe (50% formamide, 5X SSC, 1X Denhardt’s), then transferred through a wash

Hybe dilution series into 2X SSC, then 0.2X SSC, and finally into MABT (100mM maleic acid, 150mM NaCl, 0.1% tween-20, pH7.5). Animals were blocked in MABT plus 1% BSA and 10% horse serum for one hour at room temperature (RT), and then labeled with a sheep anti-DIG antibody (1:2000, Roche) in MABT + BSA + 10% horse serum for four hours at RT. Animals were washed 8X with MABT and signal developed using NBT and BCIP. For in situ hybridizations on isolated cells, cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min. at RT, treated with 1µg/mL proteinase K at

RT for 10 min., and then post-fixed in 4% PFA for 15 min. at RT. Hybridization followed essentially the same protocol as described above.

Quantitative RT-PCR

RNA was extracted using the TriZol reagent (Invitrogen). Samples were DNase

(Promega) treated at 37 °C x 15 min and then 70 °C x 15 min and cDNA was obtained

from 1µg of total RNA by using SuperScriptTM II Reverse Transcriptase (Invitrogen).

Clone H.55.12e (accession number: AY068123) was selected as an internal control as a

constitutively expressed gene. Sets of specific smedwi-primers were used to evaluate

gene expression. Samples without reverse transcriptase served as the negative control

template. We used relative quantification to evaluate gene expression levels from control

and γ-irradiated planarians, as well as from different cell populations. Total RNA from

intact worms (2 µg) or from sorted cells (20 ng) was used to synthesize template cDNA.

PCR reactions were performed using the SYBR® Green RT-PCR kit (Applied

Biosystems) and reactions analyzed using an ABI Prism® 7900HT (Applied

Biosystems). Reactions were run in triplicate and at least two different experiments per condition were performed. Relative standard curves for each experiment were created from H.55.12e data. Data were analyzed using SDS 2.1 software (Applied Biosystems).

RNAi

The cDNAs H.2.12C and HB.14.6D were used for the RNAi of smedwi-1 and smedwi-2,

respectively. RNAi, unless stated otherwise, involved two dsRNA feedings four days

apart. dsRNA that was delivered by injections was prepared using T7 and T3 RNA

polymerases, and 32nL was injected 3X on each of three days. RNAi by feeding utilized

the pDONRdT7 vector and methods previously described (1).

FACS

Planarians were amputated into small pieces in cold calcium, magnesium-free medium

(CMF plus BSA) containing Trypsin Inhibitor Type II-0 (30µg/mL) and then placed into

CMF containing Trypsin (2 units/mL) and left to rock for 2 hours at 4°C. (CMF plus

BSA; 15mM Hepes, 400mg/L NaH2PO4, 800mg/L NaCl, 1200mg/L KCl, 800mg/L

NaHCO3, 240mg/L glucose, 1% BSA, pH7.3). Cells were collected by centrifugation, resuspended in CMF+BSA, then passed through 53µm and 20µm Nitex filters sequentially. Cells were then collected by centrifugation and re-suspended in fresh

CMF+BSA plus Hoechst33342 (7.5µg/mL) and Calcein (0.5µg/mL) and allowed to sit at

RT for 20min. Cells were collected, resuspended in CMF+BSA and 5µg/mL propidium iodide was added. Analyses and sorts were performed using a Becton Dickinson

FACSvantage.

BrdU labeling, antibody labeling, tissue sectioning, and microscopy

Animals were fed artificial planarian food containing 5mg/mL BrdU plus 0.6% ultra low gelling temperature agarose, 35% blended liver, and red food coloring to color. Animals were killed in 2% HCl for 30-60 seconds at RT, then fixed in Carnoy’s fixative for 3 hours at RT. Animals were rinsed in methanol and then bleached overnight in 6% hydrogen peroxide in methanol. Animals were re-hydrated through a methanol dilution series in PBST (0.3% triton X-100) and then treated with 2N HCl at RT for 45 min.

Followed by a rinse with 0.1M Borax in PBS, animals were rinsed 3X with PBST (0.3% triton X-100) then blocked at RT for 6h in PBST + 0.25% BSA. Animals were incubated

3 overnight in 1:10 rat anti-BrdU (Oxford Biotech), washed 7X over 6h the next day,

incubated overnight in 1:100 goat anti-rat conjugated to HRP (AbCam), then labeled with

tyramide conjugated to Alexa568 (Molecular Probes) as previously described (1).

Whole-mount confocal microscopy was carried out on BrdU-labeled animals using an

Olympus Fluoview confocal microscope. Animals were labeled with the anti-

phosphorylated histone H3 antibody as described previously (1). Numbers of mitoses per

length were determined by counting labeled cell nuclei and dividing the numbers by animal lengths in mm. For tissue sectioning, animals were incubated in 2% and then

10% Sucrose in PBST for 1 hour each, rocking at RT. Animals were then left to rock in

25% sucrose in PBST overnight at RT. Animals were embedded in OCT and 15µm sections created. Sections were observed using Nomarski optics.

Table S1. smedwi expression is greatly reduced by irradiation

Expression relative to control

Gene Wild-type animals Irradiated (6K Rad) animals smedwi-1 18.8 ± 1.4 1.9e-06 ± 7.6e-08 smedwi-2 6.9 ± 0.14 0.3 ± 0.001

Results represent mean ± SD of three experiments. Data are relative to expression detected for the ubiquitously expressed control H.55.12e. See methods for details.

Table S2. smedwi genes are predominantly expressed in X1 cells

Expression relative to control

Gene Xins cells X1 cells X2 cells smedwi-1 0.01 ± 0.002 8,498 ± 4,341 48.5 ± 22.5 smedwi-2 0.5 ± 0.1 1,017 ± 516 24.9 ± 14.0

Results represent mean ± SD of three experiments. Data are relative to expression detected for the ubiquitously expressed control H.55.12e. See

Fig. 1F for cell isolation details and nomenclature. See methods for details.

Fig. S1. SMEDWI-1 and –2 are homologous to the Drosophila proteins PIWI and AGO2.

AGO2 is a representative of the Argonaute class and PIWI of the PIWI class of proteins.

Top, PAZ domains. Bottom, PIWI domains. Amino acids (aa) labeled with a red circle beneath them indicate the putative RNase active site caboxylate residues (2). Yellow circles indicate candidate aa that create a binding pocket for the 5’-phosphate of RNA

(3). Blue circles indicate aa that can distinguish between the Argonaute and PIWI classes

(4). GeneBank accession numbers for smedwi-1 and smedwi-2 are DQ186985 and

DQ186986.

Fig. S2. Supplemental results related to the RNAi of smedwi-2. (A) smedwi-1(RNAi) eliminates detectable smedwi-1 message (animal is 13d post RNAi) and smedwi-2(RNAi)

(animal is 8 days RNAi) eliminates detectable smedwi-2 message. (B) Summary time course of the progression of the smedwi-2(RNAi) phenotype. See text for details. 7/7 animals cut eight days and 18/18 animals cut 10 days following smedwi-2 dsRNA exposure failed to generate a blastema. Multiple other experiments corroborated these example results. (C) smedwi-2(RNAi) animals cut early after exposure to dsRNA regenerated small blastemas that regressed. (e.g., 8/8 animals cut four or six days following exposure to dsRNA.) Animals were injected dsRNA 3X32nl per day for three days and then amputated on the following day. Mock treated animals were injected with water. Animals were decapitated and had their tails removed; blastemas are unpigmented. White dotted line indicates amputation site. White arrows point to blastemas. Yellow arrows point to regressed blastemas. Anterior, left. Bar, 0.2mm. (D)

6 To determine whether the presence of neoblasts or the capacity to undergo mitosis was affected by smedwi-2(RNAi), we first performed in situ hybridizations with the smedwi-1 riboprobe. By 12 days following exposure to dsRNA 9/9 smedwi-2(RNAi) animals had qualitatively normal smedwi-1 signal and by 15 days 9/9 smedwi-2(RNAi) animals had very low smedwi-1 signal. Anterior, left. Bar, 0.2mm. d, days following first feeding of dsRNA. αH3P, anti-phosphorylated histone H3 antibody.

Fig. S3. X1 and X2 cell populations decline at late time points following exposure to smedwi-2 dsRNA. Cell populations were analyzed by FACS. Though batch to batch variation in cell preparations make quantitative comparisons somewhat variable, trends are readily apparent. Cell populations were analyzed as described in the Figs. 1 and 2 legends. Y axis indicates the percentage of cells that were propidium iodide negative and

Hoechst 33342 positive and were within the gates for X1 and X2 as defined in Fig. 2G.

X axis indicates the number of days following the first of two dsRNA feedings of either smedwi-2 dsRNA or control unc-22 dsRNA.

Fig. S4. smedwi-2(RNAi) neoblast progeny cells that enter the epidermis are morphologically abnormal. Figure 3C displays an example at higher magnification of abnormal epidermal morphology. Post-pharyngeal cross-sections 13 days following

RNAi and 6 days post-BrdU labeling. Black arrowheads, BrdU positive cells intercalating in epidermis (outer-most cells). Ventral surface, left. Bar, 100µm.

1. P. W. Reddien, A. L. Bermange, K. J. Murfitt, J. R. Jennings, A. Sánchez Alvarado, Dev. Cell 8, 635 (2005).

7 2. J. J. Song, S. K. Smith, G. J. Hannon, L. Joshua-Tor, Science 305, 1434 (2004). 3. J. B. Ma et al., Nature 434, 666 (2005). 4. J. S. Parker, S. M. Roe, D. Barford, EMBO 23, 4727 (2004).

8 Figure S1

AGO2(601-740) S M P M I E Y L E R F S L K A K I N N T T N L D Y S R R F L E P F L - R G I N V V Y T P P Q S F Q S A P R V Y R V N G L S R A P A S S E T F E H D G K K V T I A S Y F H S R - N Y P L K F P Q L H C L N PIWI(261-395) T E T I Y D I M R R C S H N P ------A R H Q D E V R V N V L - D L I V L T D Y N N ------R T Y R I N D V D F G Q T P K S T F S C K G R D I S F V E Y Y L T K Y N I R I R D H N Q P L L I SMEDWI-1(209-356) L L Y L - E R I N R V L N E N - - - - N V V S V Y R R D Q I D Q L I G R D - - I I T K Y N N ------K T Y R I S D I K E M D V N D E - V Q L G D K K I S Y L N Y F K Q R Y N I N L K N D K Q P F V I SMEDWI-2(222-376) L L Y I - E R I N R V L N D N - - - S V M K A Y N R R - S I D Q L I G R D - - I I T K Y N N ------K T Y R I S E I K E M N V - D E K F E M G G R T L S Y A E Y F K E R Y N I R L T Q G D Q P F V L

AGO2(601-740) V G S S I ------K S I L L P I E L C S I E E G Q A L N R K D G A T Q V A N M I K Y A A T S PIWI(261-395) S - - - - - K N R D ------K A L K T N - - A S - E L - - V V L I P - E L C R V T G L N A E M R S N F Q L M R A M S S Y T R M N P SMEDWI-1(209-356) S R V K R S M V R P K E K ------T E N E P E G P T E T D Q - - S L S I P G E L C Y L C G F S D S E R S N I N L Q K N L G T V L K R E P SMEDWI-2(222-376) T R V K K P M R R E R K K K D E E G V - - - E K E K E K E A P E E K D M T L N I P G E L C F L C G F S D Q E K S N M D L Q K N L G C V L K R E P

AGO2(811-1214) E P K P K A H K C A V L Y C D P R S G R K M N Y T Q L N D F G N L I I S Q G K A V N I S L D S D V T Y R P F T D D E R S L D T I F A D L K R S Q H - - - D L A I V I I P Q F R - I S Y D T I K Q K A PIWI(468-844) L T T P S D G L D R W A V I A P Q - R - - - - - N S H E L R T L L D S L Y R A A S G M G L R I R S P Q E F I I Y D D R T G T Y V R A M D D C V R S D P - - - K L I L C L V P N D N A E R Y S S I K K R G SMEDWI-1(425-808) F D V P K G T A H R F G V L V V D - R - - - - - N P S H F N N F I E D V K R E I G R L R I N Y T M D S - - - I S S C R S N E V E D A L T D F I R V S K - - V H M A L V F I P D D - - K V Y A K V K N - F SMEDWI-2(446-833) F D I K R D R K H E I D V V I V D - R - - - - - N D F Q Y K N F M N D V E Q E L R N M R I D A R V G K - - - V N T C G P N D V E R C L N D A A R S G S G C A K M A L V F V P D D - - R V Y A K V K S - F

AGO2(811-1214) E L Q H G I L T Q C I K Q F - - T V E R K C N N Q T I G - N I L L K I N S K L N G I N H K I K D D P R L P M M K N T M Y I G A D V T H P S P D Q R E I P S V V G V A A S H D P Y G A S Y N M Q Y R L Q R PIWI(468-844) Y V D R A V P T Q V V T L K - - T T K K P Y S L M S I A T K I A I Q L N C K L G Y T P W M I E L P - - - - - L S G L M T I G F D I A K S T R D R K R A Y G A L I A S M D L Q - Q N S T Y F S T V T E C S SMEDWI-1(425-808) T M S T G L L T Q C V T Q R N G S N R D D R R R K T V A D K S V M Q M F S K L G Y D P W G I N L K - - - - - M A P T M I V G L D T F H S K T G K R S V Q A S V F S I S A K - - F S Q Y I S F V N S S - K SMEDWI-2(446-833) T M S T G L L T Q C V T T R N G T N R N D K R R K V V S S K T V M Q I F A K F G Y D P W T V E I K - - - - - L R P T M I V G M D T Y H N K S S K K S I Q A S V F S I N S T - - F T Q Y M S F V V N S P K

AGO2(811-1214) G A L E E I E D M F S I T L E H L R V Y K E Y R N A Y P D H I I Y Y R D G V S D G Q F P K I K N E E L R C I K Q A C D K V G - - - - - C K P K I C C V I V V K R H H T R F F P S G D V T T S N K F N N V PIWI(468-844) A F D V L A N T L W P M I A K A L R Q Y Q H E H R K L P S R I V F Y R D G V S S G S L K Q L F E F E V K D I I E K L K T E Y A R V Q L S P P Q L A Y I V V T R S M N T R F F L N G ------Q N P SMEDWI-1(425-808) G K N E F H E N L G K N F L T A L T T F Q N K F N T M P L R L I I Y R D G V G D S Q L A F T K K F E T D A V M K M I E K I Y E N Q - - T L P Q I I Y V V V K K R I S V K F F K D G ------A N P SMEDWI-2(446-833) G R Q E F H E T L G K N F N L A L E D F K K R Y D I L P Q R I L V F R D G V G D N Q L Q F T K N F E V D A M K P L I E N I Y K G N G F P V P Q I I Y V I V K K R V G T K L F N R G ------N N P

AGO2(811-1214) D P G T V V D R T I V H P N E M Q F F M V S H Q A I Q G T A K P T R Y N V I E N T G N L D I D L ------L Q Q L T Y N L C H M F P R C N R S V S Y P A P A Y L A H L V A A R G R V Y L T G T PIWI(468-844) P P G T I V D D V I T L P E R Y D F Y L V S Q Q V R Q G T V S P T S Y N V L Y S S M G L S P E K ------M Q K L T Y K M C H L Y Y N W S G T T R V P A V C Q Y A K K L A T L V G - - - - - T SMEDWI-1(425-808) N P G T V V D E K I V K P N F Y E F Y L V S Q K T T K G T A S P T N Y N V L M D T K F T N K K T N E V S V M S P S V L Q Q I T Y S L T H L Y F N W M G T I R V P V P T H Y A H R L A E L V G K I H R G V SMEDWI-2(446-833) N P G T V V D K E I V K P N F Y E F F L V S Q R T T K G T A T P T N Y N V L E D T R L L T K K G - T M D P M A P N E L Q K I T Y A L T H L Y F N W M G T I R V P V P V H Y A H R L A E L V G K V H K G S AGO2(811-1214) N R F L D L K K E Y A K R T I V P E F M K K N P M Y F V PIWI(468-844) N ------L H S I P Q N A L E K K F Y Y L SMEDWI-1(425-808) T ------P P A I N D R I R E R L F - Y L SMEDWI-2(446-833) N ------P V A I N E R I R N R L F - Y L Figure S2 A smedwi-1(RNAi) smedwi-1(RNAi)

smedwi-1 smedwi-2 smedwi-2(RNAi) smedwi-2(RNAi)

smedwi-1 smedwi-2 B RNAi Days 0, 4 SMEDWI-2 function perturbed Blastema formation fails Neoblast function in homeostasis Neoblasts present, proliferate, Neoblasts are lost, possibly fails immediately respond to wounding exhausted by failed homeostasis Neoblast overproliferation during Neoblast progeny fail to functionally Tissue integrity lost, animals die homeostasis replace aged tissues Days 0-6 Days 7-11 Days 12-21 Phenotype progression C wild type 5 dsmedwi-2(RNAi) 5 d MOCK regeneration regeneration

wild type 11 dsmedwi-2(RNAi) 11 d MOCK regeneration regeneration

ventral ventral

unc-22(RNAi) 15d D unc-22(RNAi) 17d

α-H3P smedwi-1 smedwi-2(RNAi) 17d smedwi-2(RNAi) 15d

α-H3P smedwi-1 Figure S3

s X2 unc-22(RNAi) l

l 14 e c )

+ 12 ( t s

h 10 c e o

H 8 e l b

a 6 X1 unc-22(RNAi) i v X2 smedwi-2(RNAi)

% 4

2 X1 smedwi-2(RNAi)

0 10d 12d 14d 16d 18d Days (d) following dsRNA feeding Figure S4

unc-22(RNAi) 13d smedwi-2(RNAi) 13d

6d BrdU 6d BrdU Cross Section Cross Section