Correction

DEVELOPMENTAL BIOLOGY Correction for “FoxO is a critical regulator of main- tenance in immortal Hydra,” by Anna-Marei Boehm, Konstantin Khalturin, Friederike Anton-Erxleben, Georg Hemmrich, Ulrich C. Klostermeier, Javier A. Lopez-Quintero, Hans-Heinrich Oberg, Malte Puchert, Philip Rosenstiel, Jörg Wittlieb, and Thomas C. G. Bosch, which appeared in issue 48, November 27, 2012, of Proc Natl Acad Sci USA (109:19697–19702; first published November 12, 2012; 10.1073/pnas.1209714109). The authors note that they unintentionally omitted a reference to an article by Bridge et al., who first described the presence of a single FoxO in Hydra. The reference should be cited on page 19698, right column, first paragraph, after lines 9–10, “according to phylogenetic analyses (Fig. 2I and Fig. S1)”.Thecomplete reference appears below.

46. Bridge D, et al. (2010) FoxO and stress responses in the cnidarian Hydra vulgaris. PLoS ONE 5(7):e11686.

www.pnas.org/cgi/doi/10.1073/pnas.1221369110 CORRECTION

www.pnas.org PNAS | January 8, 2013 | vol. 110 | no. 2 | 797 Downloaded by guest on October 1, 2021 FoxO is a critical regulator of stem cell maintenance in immortal Hydra

Anna-Marei Boehm a,1, Konstantin Khalturin a,1, Friederike Anton-Erxleben a, Georg Hemmricha, Ulrich C. Klostermeierb, Javier A. Lopez-Quintero a, Hans-Heinrich Oberg c, Malte Puchert a, Philip Rosenstiel b, Jörg Wittlieba, and Thomas C. G. Bosch a,2 aZoological Institute, Christian-Albrechts-University, 24098 Kiel, Germany; bInstitute of Clinical Molecular Biology (IKMB), University Hospital Schleswig-Holstein, 24105 Kiel, Germany; and cInstitute of Immunology, University Hospital Schleswig-Holstein, 24105 Kiel, Germany

Edited by Cynthia Kenyon, University of California, San Francisco, CA, and approved October 19, 2012 (received for review June 11, 2012) Hydra’s unlimited life span has long attracted attention from nat- by being capable of giving rise not only to a number of somatic ural scientists. The reason for that phenomenon is the indefinite cell types but also to gametes (2, 5) (Fig. 1D). Although the self-renewal capacity of its stem cells. The underlying molecular mechanisms controlling self-renewal and differentiation of Hydra mechanisms have yet to be explored. Here, by comparing the tran- stem cells could reveal long-sought secrets about , until scriptomes of Hydra’s stem cells followed by functional analysis now such mechanisms have not been identified due to the lack of using transgenic polyps, we identified the factor unique markers and the absence of functional approaches (7). forkhead box O (FoxO) as one of the critical drivers of this contin- To uncover the regulatory events and signaling pathways that uous self-renewal. foxO overexpression increased interstitial stem control stem cells in an evolutionarily old animal that branched off cell and progenitor cell proliferation and activated stem cell as the bilaterian ancestor (Fig. 1A) almost 600 million years ago, we in terminally differentiated somatic cells. foxO down-regulation recently separated eGFP-labeled stem cells of all three stem cell led to an increase in the number of terminally differentiated cells, lineages by fluorescence-activated cell sorting and addressed the resulting in a drastically reduced population growth rate. In addi- commonalities and differences among the three stem cell lineages by tion, it caused down-regulation of stem cell genes and antimicrobial deep RNA sequencing and studies (8). This first- peptide (AMP) expression. These findings contribute to a molecular time characterization of the stem celltranscriptomesinananimalat BIOLOGY understanding of Hydra’s , indicate an evolutionarily the base of evolution revealed not only that stem cells in the DEVELOPMENTAL conserved role of FoxO in controlling longevity from Hydra to metazoan ancestors were multifunctional, but also that they bear humans, and have implications for understanding cellular aging. adefined molecular signature composedofdistinctsetsoftran- scription factors, signal transducers, and effector genes (8). In search | differentiation of transcription factors that are strongly expressed and shared by all three stem cell lineages and therefore that may play a role in the he unlimited life span of Hydra, due to the indefinite self- regulation of self-renewal and differentiation in all three stem cell Trenewal capacity of its stem cells, has long attracted attention lineages, we discovered FoxO, a member of the forkhead box (Fox) A from biologists, as it promises insights into the mechanisms family of (Fig. 2 ) (8). Recent work has implicated FoxO in fl controlling longevity in more advanced animals including conferring increased life span and stress resistance in ies and fi foxO3a humans. Analysis of the mortality patterns and reproductive worms and identi ed as an important component of the – rates of Hydra vulgaris could not detect any evidence for aging genetic signatures of human exceptional longevity (9 13). In addi- and no apparent signs of decline in reproductive rates (1).Thus, tion, FoxO3a was shown to play a role in maintenance of adult Hydra, a member of the phylogenetically old animal phyla Cni- hematopoietic stem cells (14, 15) as well as neural stem cells (16, 17), daria (Fig. 1A), has been suggested to not undergo whereas FoxO1 is important for embryonic stem cell maintenance and to be biologically immortal. In vivo tracking of individual (18). Thus, we hypothesized that the role of FoxO in controlling enhanced green fluorescent (eGFP)-expressing cells stem cell behavior and longevity might be evolutionarily highly provides experimental proof that both epithelial and interstitial conserved. Here we show by gain-of-function and loss-of-function Hydra analysis that FoxO indeed is a critical compo- stem cells in are continuously self-renewing and differ- Hydra entiating cells (2–4). Offspring of individual cells were tracked nent of the mechanisms controlling stem cell behavior in . for several years without obtaining evidence for apparent signs of Results decline in proliferation rates. The apparent lack of cellular se- Expression Correlates with the Undifferentiated Stem Cell State. nescence results in an adult polyp with indefinite proliferative foxO When analyzing the stem cell signatures of the three stem cell life span and adds support to the study by Martinez (1). The lineages in Hydra, foxO emerged as an obvious candidate for con- biological reason behind that seemingly unique potential to escape trolling stem cell self-renewal as it is highly expressed in all three mortality and senescence is simple: the animals have adopted a stem cell lineages (Fig. 2 A and B). To independently confirm life cycle in which proliferation and population growth occurs exclusively asexually by budding (2). This asexual mode of re- production not only demands that each individual polyp main- Author contributions: A.-M.B., K.K., and T.C.G.B. designed research; A.-M.B., F.A.-E., G.H., tains continuously self-renewing stem cells but also points to U.C.K, J.A.L.-Q., and J.W. performed research; G.H., H.-H.O., M.P., P.R., and T.C.G.B. con- a strong selective constraint that equips the adult polyp’s tissue tributed new reagents/analytic tools; A.-M.B., K.K., F.A.-E., J.A.L.-Q., and T.C.G.B. analyzed with cells that are capable of continuous self-renewal and dif- data; and A.-M.B., K.K., and T.C.G.B. wrote the paper. ferentiation. In Hydra, three stem cell lineages—ectodermal and The authors declare no conflict of interest. endodermal epitheliomuscular stem cells and interstitial stem This article is a PNAS Direct Submission. cells—contribute to the continuously self-renewing epithelium Data deposition: The sequences reported in this paper have been deposited in the Gen- (Fig. 1 B–D) (2, 5). The two epitheliomuscular stem cell lineages Bank database (accession nos. JX118843–JX118848). shape the diploblastic body of the polyps and are responsible for 1A.-M.B. and K.K. contributed equally to this work. all its morphogenetic properties (6). The multipotent interstitial 2To whom correspondence should be addressed. E-mail: [email protected]. stem cells located in the ectoderm of the gastric region have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a developmental potency much wider than that of epithelial cells 1073/pnas.1209714109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1209714109 PNAS Early Edition | 1of6 for this conclusion comes from mechanical separation of ecto- dermal and endodermal tissue followed by qRT-PCR (Fig. 2D) as well as by in situ hybridization (Fig. 2 F and G). Interestingly, foxO is down-regulated upon terminal differentiation in both head and foot tissue (Fig. 2 E and F) as well as in differentiating gametes within gonads (Fig. 2H), suggesting potential FoxO functions in stem cell regulation. Similar to all other invertebrate species, the Hydra magnipapillata genome contains only a single FoxO gene that, according to phylogenetic analyses (Fig. 2I and Fig. S1), groups in a basal position to all bilaterian FoxO genes, including human foxO3a, dauer-formation 16 (DAF-16)/foxO, and Drosophila dFOXO.

Overexpression of foxO in Interstitial Stem Cell Lineage Increases Stem Cell and Progenitor Cell Proliferation and Induces Expression of Stemness Genes in Terminally Differentiated Nematocytes. To examine whether FoxO plays essential regulatory roles in the processes that influence interstitial stem cell number and func- tion, we generated two independent lines of polyps containing an actin-driven foxO-eGFP transgene (Fig. 3A) expressed in in- terstitial stem cells and nematoblast precursors (Fig. 3 B–D). fi foxO Fig. 1. Stem cells in Hydra are continuously self-renewing. (A) Schematic phy- qRT-PCR results con rm that levels of transcripts are logenetic tree showing the main branches in metazoan evolution. (B)Schemeof significantly elevated in polyps of both foxO-eGFP transgenic the stem cell compartment in Hydra with sharp boundaries towards terminally lines, compared with eGFP-expressing control polyps (Fig. 3E). differentiated head and foot tissue. (C) The major cell types in Hydra.Stemcell Confocal microscopy of polyps overexpressing foxO in the in- lineages are colored, with derivatives of the interstitial cell lineage in gray. (D)The terstitial cell lineage (Fig. 3 B–D) and in ectodermal epithelial three independent stem cell systems in Hydra. Both epithelial cell lineages rep- cells (Fig. S2) shows that FoxO-eGFP fusion protein is loacalized resent unipotent stem cells whereas interstitial stem cells exhibit multipotent in both nucleus and cytoplasm of stem cells (Fig. 3B and Fig. features as they are able to differentiate into various derivatives. diff, differen- S2A). In terminally differentiated cells in tentacles and hypo- tiation ecto, ectoderm; ECM, ; ecto epi, ectodermal epithelial stome, FoxO-eGFP localization is mostly cytoplasmic (Fig. S2 B cell; endo, endoderm; endo epi, endodermal epithelial cell; gld, gland cell; i-cell, C interstitial stem cell; nv, nerve cell; nem, nematocyte. and ). To assess the role of FoxO in controlling interstitial cell proliferation, we determined the 5-bromo-2′-deoxyuridine (BrdU)- labeling index of interstitial stem cells and nematoblast precursors. foxO expression in all three stem cell lineages, we performed an Fig. 3F indicates that foxO overexpression in the interstitial cell additional cell-sorting experiment followed by quantitative real- lineage is accompanied by a significant increase in proliferation of time PCR (qRT-PCR) (Fig. 2C). Further independent support interstitial stem cells and nematoblast precursors, which results in

Fig. 2. foxO is expressed in all three stem cell types in Hydra.(A) Venn diagram of conserved transcription factors being expressed in all three stem cell types according to 454 transcriptome sequencing. (B) Reads from 454 sequencing for foxO, cnvas1,andcniwi.(C–E) qRT-PCR reveals foxO expression in (C) all three stem cell lineages, (D) both tissue layers, and (E) in the body column (n = 2 replicates). Expression in endodermal epithelial cells, endodermal tissue layer, and foot tissue was used as calibrator. (F–H) In situ hybridization shows that foxO is expressed in both tissue layers along the body axis and absent from terminally differentiated cells in head, foot, and (H) gonads. (I) Maximum-parsimony phylogram of the forkhead domain from selected FoxO proteins rooted using Mus musculus FoxA1 and Hydra FoxA2. Numbers at nodes are bootstrap support values calculated by 1,000 replicates of maximum parsimony. Bootstrap values under 50 are not shown. Aa: Aedes aegypti;Ad: Acropora digitifera;Am:Acropora millepora,Aq:Amphimedon queenslandica;Bf:Branchiostoma floridae;Ce:C. elegans;Ch:Clytia hemisphaerica;Ci:Ciona intes- tinalis;Dm:;Dr:Danio rerio;Gg:Gallus gallus;Hs:Homo sapiens; HvAEP: H. vulgaris strain AEP; Mm: M. musculus;Ms:Metridium senile;Nv: Nematostella vectensis;Sp:Strongylocentrotus purpuratus; Ta: Trichoplax adhaerens;Xl:Xenopus laevis;andXm:Xiphophorus maculates. (Scale bar in G:20μm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209714109 Boehm et al. BIOLOGY DEVELOPMENTAL

Fig. 3. foxO overexpression. (A) Construct used for foxO overexpression. (B–D) foxO overexpressing (B) interstitial stem cells and (C and D) groups of nematoblasts. + (E) foxO expression levels in ectodermal tissue layer of foxO polyps (A4 and C2 line), analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: A4 line = 0.014, C2 line = 0.0005. (F) BrdU-labeling index of interstitial stem cells (1s + 2s) and nematoblast progenitor cells (4s, 8s) + + in foxO (A4 line) and control polyps after 6 h exposure to BrdU (n1s 2s = ∼1,000, n4s = ∼200, n8s = ∼100 per replicate; n = 3 replicates). Asterisks indicate significant changes in cell numbers (t test); P values: 1s + 2s = 0.0265, 4s = 0.0034, 8s = 0.0136. (G) Numbers of interstitial stem cells (1s + 2s) and nematoblasts (4s, 8s) per 100 epithelial cells in foxO+ (A4 line) and control polyps (nepithelial cells = ∼1,000; n = 3 replicates). Asterisks indicate significant changes in cell numbers (t test); P values: 1s + + 2s = 0.0016, 4s = 0.0078, 8s = 0.0474. (H) Expression levels of stem cell genes in ectodermal tissue layer of foxO polyps (A4 and C2 line) analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: cnvas1A4 line = 0.0238, cnvas1C2 line = 0.0031. (I–P) Douple in situ hybrid- + ization of cniwi and mc17 in (I–L) control and (M–P) foxO (A4 line) polyps. (I and J) Mc17-positive nematoblasts and cniwi-positive interstitial stem cells. (K) Mc17- positive nematoblasts. (L) Cniwi-negative nematocyte. (M–O) Coexpression of mc17 and cniwi in nematoblasts. (P) Cniwi-positive nematocyte. (Scale bars: B–D, K, and O—10 μm; J and N—20 μm; L and P—5 μm.) cyt, cytoplasm; ic, interstitial stem cells; nb, nematoblasts; nc, nematocyst. an increased number of interstitial stem cells and nematoblast (Fig. 3H). As assessed by double in situ hybridization in polyps precursors per polyp (Fig. 3G). transfected with the control construct, nematocyte precursors As shown previously (19–22), interstitial stem cells express express minicollagen mc17 whereas interstitial stem cells express cniwi and cnvas1 whereas nematoblast precursors specifically cniwi (Fig. 3 I–K). Mature nematocytes never express cniwi (Fig. express the minicollagen mc17. We therefore asked next if foxO 3L). In transgenic polyps overexpressing foxO in the interstitial overexpression in interstitial cells could affect the expression of cell lineage, we observe not only a higher density of nematoblast stem cell genes. qRT-PCR results show that the level cnvas1 and progenitor cells (Fig. 3M), but also a large number of nemato- cniwi expression is substantially increased in polyps of both blast precursors expressing both cniwi and mc17 simultaneously transgenic lines overexpressing foxO in their interstitial stem cells (Fig. 3 M–O). Most strikingly, cniwi transcripts can even be

Boehm et al. PNAS Early Edition | 3of6 found in terminally differentiated nematocytes such as stenoteles assessed by qRT-PCR (Fig. 4B and Fig. S4). foxO silencing is or isorhizas in both transgenic lines (Figs. 3P and Fig. S3). Thus, associated with a severe enlargement of the foot and stalk region, overexpression of foxO appears to induce expression of stem cell as assessed by in situ staining with the stalk-specific Pedibin gene genes in terminally differentiated somatic cells. This finding (24) (Fig. 4 C–F and Fig. S5A). By examining the expression resembles earlier observations in C. elegans that ectopic expres- patterns of endodermal transcription factors known to be im- sion of DAF-16/foxO in somatic tissue also induces the ectopic portant for foot formation, we observed that both nk2 expression of germ-line genes (23) and may indicate that foxO (nk2)2 and erythroblast transformation-specific2() are strongly overexpression transfers stem cell character to terminally differ- expressed in the enlarged stalk structures (Fig. S5B). Measuring of entiated cells in the interstitial cell lineage. the expression field of pedibin revealed (Fig. 4G)thatallsix transgenic lines have an increased number of pedibin-expressing FoxO Silencing in Epithelial Cells Results in Enhanced Terminal Dif- cells, suggesting that silencing FoxO function results in an increase ferentiation and a Slow Growth Phenotype. To further investigate of terminally differentiated foot cells. The finding that mc17- functions of FoxO in Hydra stem cells, we knocked down its ex- expressing progenitor cells, which normally are never present in pression in ectodermal and endodermal epithelial cells using foot tissue, are absent from the enlarged stalks of transgenic a stable hairpin transcription approach (Fig. 4A). We have an- polyps (Fig. S5B) supports this view. foxO silencing and the as- alyzed six independent transgenic lines (three endodermal, two sociated increase in terminally differentiated foot cells are ac- ectodermal, and one ecto- and endodermal). Levels of foxO companied by defects in growth rate. Quantification of growth rate transcript are significantly and specifically reduced in all lines as under standard culture conditions shows that polyps with foxO-

− Fig. 4. foxO down-regulation. (A) Construct used for foxO down-regulation. (B) foxO expression levels in endodermal tissue layer of foxO endo polyps, − − ectodermal tissue layer of foxO ecto polyps, and total tissue of foxO ecto/endo polyps, analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: endo A6 line =<0.0001, endo D11a line = 0.0087, endo E1 line = 0.0032, ecto D11a line = 0.0123, and ecto/endo line = 0.0008. (C–F) In situ hybridization of pedibin in FoxO− endo, foxO− ecto, foxO− ecto/endo and control polyps. (G) Foot length compared with body length in foxO− and control polyps, as determined by pedibin labeling (ncontrol endo = 21, ncontrol ecto = 12, nendo A6 line = 17, nendo D11a line + nendo E1 line = 22, necto D11a line + necto D11b line = 18, and necto/endo line = 15). Asterisks indicate significant differences in foot length compared with control (t test); P values: all − <0.0001. (H) Growth curves (n = 10 replicates à five polyps each) and (I) time of bud development (n = 10 replicates) of foxO endo (A6 line) and control − polyps. Asterisks indicate significant differences in growth (t test); P value =<0.0001. (J) Expression levels of stem cell genes in foxO ecto/endo polyps analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: cnvas1 = 0.0021, cniwi = 0.0054. (K)Ex- pression levels of AMPs in foxO− endo polyps analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: hydramacinA6 line = 0.0001, periculin2bA6 line = 0.0316, hydramacinD11a line = 0.0031, hydramacinE1 line = 0.0011, and periculin2bE1 line = 0.0136.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209714109 Boehm et al. deficient endoderm have a reduced population growth rate, with a doubling time of 6 d compared with 4 d for the control trans- genic line (Fig. 4H) due to a 2-d prolonged bud attachment to the mother polyp (Fig. 4I). As indicated above (Fig. 2B), genes strongly expressed in all three stem cell lineages include cniwi and cnvas1. We therefore wondered if FoxO is required for the expression of these genes in epithelial tissue. As shown in Fig. 4J,inpolypswithfoxO-silenced ecto- and endoderm, both cnvas1 and cniwi are significantly down-regulated. These findings indicate once more that cnvas1 and cniwi are FoxO target genes and resemble earlier observations in C. elegans (23). Taken together, these observations raise the possibility that foxO- deficient epithelial cells are driven into terminal differentiation, indicating that FoxO function is necessary for stem cell self-renewal and continuous growth.

Silencing FoxO Activity in Epithelial Cells Causes Severe Changes in Fig. 5. Model of the role of FoxO in controlling longevity. Decline of foxO the Functionality of ’s Innate Immune System. fi expression results in aging and death. Mutations in DAF-16 and dfoxO re- Hydra Our ndings are duce life span in both C. elegans and flies. Increase of foxO expression delays intriguing in light of prior data showing that FoxO plays a key aging by maintaining stem cell self-renewal and functionality of the immune role in controlling life span and stress resistance in both C. ele- system. Universally expressed foxO in Hydra results in a continuous self- gans and flies (10, 11, 13). Because previous studies reported renewal capacity of stem cells and immortality. drastic changes of the immune system with age (“immuno se- nescence”; for review see ref. 25), we tested whether silencing of foxO might also cause changes in the immune status of polyps by Experimental evidence in flies, worms, and mice indicates that comparing the expression patterns of genes known to play im- changes in life span indeed can occur through changes in foxO portant roles in Hydra’s immune system, which is predominantly expression. Mutations in daf16/foxO in C. elegans prevent the – foxO located in the endoderm (2, 26 30). down-regulation in longevity effect whereas overexpression increases it (23). More- BIOLOGY endodermal epithelial cells of three independent lines reduces fi foxO3a fi over, speci c mutations in the sequence of signi cantly DEVELOPMENTAL the expression of the AMP gene arminin and causes severe increase human life span (9–13, 34). overexpression of the AMPs hydramacin and periculin2b (Fig. Our results also delineate a role for FoxO in innate immunity. We 4K). Analysis of the hydramacin, periculin2b, and arminin pro- show that silencing FoxO activity results in significant changes in the moters reveals the presence of numerous FoxO-binding sites expression of AMPs. AMPs in both plants and animals are activated (Fig. S6), indicating that FoxO can directly bind to these regu- through pattern recognition receptors in response to microbe- latory regions, acting either as repressor or as transcriptional associated molecular patterns. Although our observations do not elu- Hydra’ activator (31). Because AMPs are key players in s innate cidate the precise genetic network responsible for FoxO-mediated immunity and not only important defense molecules but also AMP activation, they are consistent with previous observations in – regulators of the host microbe interaction (28, 32), our obser- flies (33) and make a significant prediction: control of stem cell self- vations indicate that down-regulation of foxO results in signifi- Hydra’ renewal may be tightly coupled to innate immune pathways, and cant changes in the functionality of s innate immune aging may be the consequence of both reduced stem cell function system. This is consistent with prior results from Drosophila (33) and altered innate immune defense. This may be a common feature that showed that AMP genes are activated in response to nuclear in plants and animals because stem cells in the shoot apical meri- dFOXO activity. Thus, in addition to its importance in stem cell stem of plants express high levels of a peptide controlling immune homeostasis, FoxO appears to have an unexpected role in con- trolling innate immune genes in Hydra. signaling and microbe interaction (35). Taken together, studies of FoxO in Hydra have several im- Discussion portant implications. They not only reveal FoxO as a molecular By the following three lines of evidence we have established that factor that has contributed to the early evolution of stem cells, Hydra FoxO controls stem cell behavior in Hydra (summarized in Fig. but also highlight intriguing similarities between and other S7A and schematized in Fig. S7B): (i) foxO is strongly expressed multicellular organisms including humans, in the mechanisms in all three stem cell lineages; (ii) overexpression of foxO in in- that maintain stemness (14–17) and control life span (9, 12, 13, terstitial stem cells stimulates stem cell and progenitor cell 23, 36, 37). Thus, the work furthers our understanding of stem proliferation and confers stemness by activation of stem cell cell self-renewal at the beginning of animal evolution and also genes to terminally differentiated cells such as nematocytes; and has implications for regenerative medicine and cellular aging. (iii) silencing of foxO in epithelial cells increases the number of Materials and Methods terminally differentiated foot cells at the cost of growth rate and causes down-regulation of stem cell genes as well as changes in Animals and Culture Conditions. Experiments were carried out using H. vulgaris expression of genes controlling the functionality of the innate strain AEP Animals were cultured according to standard procedures (38). immune system. Based on the findings in Hydra we propose Generation of Transgenic Strain AEP. a general model in which FoxO plays a key role in controlling H. vulgaris foxO overexpression was longevity (Fig. 5). According to this model, in immortal Hydra achieved by generating transgenic polyps expressing foxO-eGFP transgene under the control of actin promoter. The stable knockdown of foxO was ach- the high expression of foxO in all three stem cell lineages is ieved by generating transgenic polyps expressing a foxO hairpin construct crucial for the continuous self-renewal capacity and unlimited under control of actin promoter. As control, transgenic lines expressing eGFP in life span as well as the continuous maintenance of the func- the endodermal and ectodermal epithelial cell and interstitial cell lineage, re- tionality of the innate immune system. In contrast, the aging spectively, were used (3, 4). SI Materials and Methods include more information. process of most organisms is caused by a reduction of foxO ac- tivity, which decreases functions of the immune system and stem BrdU Labeling and Detection. For analysis of cell proliferation, animals were cells. Aging in stem cell systems from flies to humans is associ- exposed for 6 h to BrdU (39), macerated (40), and subjected to BrdU de- ated with progressive loss of stem cell number and activity (23). tection (39) as described previously.

Boehm et al. PNAS Early Edition | 5of6 In Situ Hybridization. Gene expression analysis in foxO gain-of-function and Phylogenetic Analysis. Phylogenetic analyses were based on 31 amino acid loss-of-function strains was done by in situ hybridization and double in situ sequences of the forkhead domain, aligned using ClustalW (42). The most parsi- hybridization as described previously (3, 41). GenBank accession numbers of monious tree was found using MEGA 4 (43). Maximum-parsimony and Maximum- the analyzed genes can be found in SI Materials and Methods. likelihood bootstrap values were calculated based on 1,000 replicates. Bayesian posterior probabilities were calculated using MrBayes version 3.1.2 (44, 45) with qRT-PCR. Gene expression analysis in wild-type and foxO gain-of-function two independent runs of 3,500,000 generations each, sampled every 1,000 gen- and loss-of-function strains was analyzed by qRT-PCR using the Quanti- erations with four chains. SI Materials and Methods includes more information. Tect Probe RT-PCR Kit (QIAGEN) and the 7300 real-time PCR system (ABI) ’ according to the manufacturer s protocols (for primers see Table S1). GenBank ACKNOWLEDGMENTS. This work was supported by Grant Bo 848/15-1 from accession numbers of the analyzed genes can be found in SI Materials the Deutsche Forschungsgemeinschaft (DFG) and by several grants from the and Methods. DFG Excellence Initiative (to T.C.G.B.).

1. Martínez DE (1998) Mortality patterns suggest lack of senescence in hydra. Exp Ger- 24. Grens A, Shimizu H, Hoffmeister SA, Bode HR, Fujisawa T (1999) The novel signal ontol 33(3):217–225. peptides, pedibin and Hym-346, lower positional value thereby enhancing foot for- 2. Bosch TC (2009) Hydra and the evolution of stem cells. Bioessays 31(4):478–486. mation in hydra. Development 126(3):517–524. 3. Khalturin K, et al. (2007) Transgenic stem cells in Hydra reveal an early evolutionary 25. Pawelec G (2007) Immunosenescence comes of age. Symposium on Aging Research in origin for key elements controlling self-renewal and differentiation. Dev Biol 309(1): Immunology: The Impact of Genomics. EMBO Rep 8(3):220–223. 32–44. 26. Bosch TC, et al. (2009) Uncovering the evolutionary history of innate immunity: The 4. Wittlieb J, Khalturin K, Lohmann JU, Anton-Erxleben F, Bosch TC (2006) Transgenic simple metazoan Hydra uses epithelial cells for host defence. Dev Comp Immunol Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc Natl 33(4):559–569. Acad Sci USA 103(16):6208–6211. 27. Augustin R, et al. (2009) Activity of the novel peptide arminin against multiresistant 5. Bosch TC, Anton-Erxleben F, Hemmrich G, Khalturin K (2010) The Hydra polyp: human pathogens shows the considerable potential of phylogenetically ancient or- – Nothing but an active stem cell community. Dev Growth Differ 52(1):15–25. ganisms as drug sources. Antimicrob Agents Chemother 53(12):5245 5250. 6. Fujisawa T, Sugiyama T (1978) Genetic analysis of developmental mechanisms in Hy- 28. Augustin R, Fraune S, Bosch TC (2010) How Hydra senses and destroys microbes. – dra. IV. Characterization of a nematocyst-deficient strain. J Cell Sci 30:175–185. Semin Immunol 22(1):54 58. fi 7. Hemmrich G, Bosch TC (2008) Compagen, a comparative genomics platform for early 29. Augustin R, Siebert S, Bosch TC (2009) Identi cation of a kazal-type serine protease ’ branching metazoan animals, reveals early origins of genes regulating stem-cell dif- inhibitor with potent anti-staphylococcal activity as part of Hydra s innate immune – ferentiation. Bioessays 30(10):1010–1018. system. Dev Comp Immunol 33(7):830 837. 8. Hemmrich G, et al. (2012) Molecular signatures of the three stem cell lineages in hydra 30. Jung S, et al. (2009) Hydramacin-1, structure and antibacterial activity of a protein – and the emergence of stem cell function at the base of multicellularity. Mol Biol Evol from the basal metazoan Hydra. J Biol Chem 284(3):1896 1905. 31. Greer EL, Brunet A (2005) FOXO transcription factors at the interface between lon- 29(11):3267–3280. gevity and tumor suppression. Oncogene 24(50):7410–7425. 9. Flachsbart F, et al. (2009) Association of FOXO3A variation with human longevity 32. Augustin R, Fraune S, Franzenburg S, Bosch TC (2012) Where simplicity meets com- confirmed in German . Proc Natl Acad Sci USA 106(8):2700–2705. plexity: Hydra, a model for host-microbe interactions. Adv Exp Med Biol 710:71–81. 10. Jünger MA, et al. (2003) The Drosophila forkhead transcription factor FOXO mediates 33. Becker T, et al. (2010) FOXO-dependent regulation of innate immune homeostasis. the reduction in cell number associated with reduced insulin signaling. J Biol 2(3):20. Nature 463(7279):369–373. 11. Kenyon C (2010) A pathway that links reproductive status to lifespan in Caeno- 34. Zeng Y, et al. (2010) Effects of FOXO genotypes on longevity: A biodemographic rhabditis elegans. Ann N Y Acad Sci 1204:156–162. analysis. J Gerontol A Biol Sci Med Sci 65(12):1285–1299. 12. Li Y, et al. (2009) Genetic association of FOXO1A and FOXO3A with longevity trait in 35. Lee H, Chah OK, Sheen J (2011) Stem-cell-triggered immunity through CLV3p-FLS2 Han Chinese populations. Hum Mol Genet 18(24):4897–4904. signalling. Nature 473(7347):376–379. 13. Salih DA, Brunet A (2008) FoxO transcription factors in the maintenance of cellular 36. Greer EL, Brunet A (2008) FOXO transcription factors in and cancer. Acta homeostasis during aging. Curr Opin Cell Biol 20(2):126–136. Physiol (Oxf) 192(1):19–28. 14. Miyamoto K, et al. (2007) Foxo3a is essential for maintenance of the hematopoietic 37. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans mutant that – stem cell pool. Cell Stem Cell 1(1):101 112. lives twice as long as wild type. Nature 366(6454):461–464. 15. Tothova Z, Gilliland DG (2007) FoxO transcription factors and stem cell homeostasis: 38. Lenhoff HM, Brown RD (1970) Mass culture of hydra: An improved method and its – Insights from the hematopoietic system. Cell Stem Cell 1(2):140 152. application to other aquatic invertebrates. Lab Anim 4(1):139–154. 16. Renault VM, et al. (2009) FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell 39. Holstein TW, Hobmayer E, David CN (1991) Pattern of epithelial cell cycling in hydra. – 5(5):527 539. Dev Biol 148(2):602–611. 17. Paik JH, et al. (2009) FoxOs cooperatively regulate diverse pathways governing neural 40. David CN (1973) A quantitative method for maceration of Hydra tissue. Wilhelm – stem cell homeostasis. Cell Stem Cell 5(5):540 553. Roux’ Archiv 171:259–268. 18. Zhang X, et al. (2011) FOXO1 is an essential regulator of pluripotency in human 41. Hansen GN, Williamson M, Grimmelikhuijzen CJ (2000) Two-color double-labeling in – embryonic stem cells. Nat Cell Biol 13(9):1092 1099. situ hybridization of whole-mount Hydra using RNA probes for five different Hydra 19. Seipel K, Yanze N, Schmid V (2004) The germ line and somatic stem cell gene Cniwi in neuropeptide preprohormones: Evidence for colocalization. Cell Tissue Res 301(2): the jellyfish Podocoryne carnea. Int J Dev Biol 48(1):1–7. 245–253. 20. Mochizuki K, Nishimiya-Fujisawa C, Fujisawa T (2001) Universal occurrence of the 42. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of vasa-related genes among metazoans and their germline expression in Hydra. Dev progressive multiple sequence alignment through sequence weighting, position- Genes Evol 211(6):299–308. specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680. 21. Adamczyk P, et al. (2008) Minicollagen-15, a novel minicollagen isolated from Hydra, 43. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics forms tubule structures in nematocysts. J Mol Biol 376(4):1008–1020. Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599. 22. David CN, et al. (2008) Evolution of complex structures: Minicollagens shape the 44. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic cnidarian nematocyst. Trends Genet 24(9):431–438. trees. Bioinformatics 17(8):754–755. 23. Curran SP, Wu X, Riedel CG, Ruvkun G (2009) A soma-to-germline transformation in 45. Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP (2001) Bayesian inference of phy- long-lived Caenorhabditis elegans mutants. Nature 459(7250):1079–1084. logeny and its impact on evolutionary biology. Science 294(5550):2310–2314.

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