Transgenic Stem Cells in Hydra Reveal an Early Evolutionary Origin for Key Elements Controlling Self-Renewal and Differentiation

Developmental Biology 309 (2007) 32–44 www.elsevier.com/developmentalbiology

Transgenic stem cells in Hydra reveal an early evolutionary origin for key elements controlling self-renewal and differentiation

Konstantin Khalturin 1, Friederike Anton-Erxleben 1, Sabine Milde 1, Christine Plötz 1, ⁎ Jörg Wittlieb 1, Georg Hemmrich, Thomas C.G. Bosch

Zoological Institute, Christian-Albrechts-University, Olshausenstrasse 40, 24098 Kiel, Germany Received for publication 28 March 2007; revised 15 June 2007; accepted 15 June 2007 Available online 22 June 2007

Abstract

Little is known about stem cells in organisms at the beginning of evolution. To characterize the regulatory events that control stem cells in the basal metazoan Hydra, we have generated transgenics which express eGFP selectively in the interstitial stem cell lineage. Using them we visualized stem cell and precursor migration in real-time in the context of the native environment. We demonstrate that interstitial cells respond to signals from the cellular environment, and that Wnt and Notch pathways are key players in this process. Furthermore, by analyzing polyps which overexpress the Polycomb protein HyEED in their interstitial cells, we provide in vivo evidence for a role of chromatin modification in terminal differentiation. These findings for the first time uncover insights into signalling pathways involved in stem cell differentiation in the Bilaterian ancestor; they demonstrate that mechanisms controlling stem cell behaviour are based on components which are conserved throughout the animal kingdom. © 2007 Elsevier Inc. All rights reserved.

Keywords: Hydra; Evolution of development; Stem cells; Stem cell niche; Wnt; Notch; Polycomb group proteins

Introduction relationships. Insects and vertebrates, however, all derive from the “Bilateria” clade of metazoans (Fig. 1A). Since several Understanding how adult stem cells are regulated is crucial animal phyla diverged before the origin of this clade, under- in learning how tissues are formed and maintained. Studies in standing the processes controlling stem cell behavior in basal insects, worms and vertebrates have shown that critical for the metazoans is essential for understanding how changes in basic maintenance, function and survival of adult stem cells is their developmental processes have enabled the evolution of the interaction with the supportive microenvironment (Nystul and diverse stem cell systems. Are stem cells in basal metazoans and Spradling, 2006; Ward et al., 2006). Wnt and Notch pathways vertebrates maintained and propagated by a common set of appear to mediate the integration of the extrinsic signals (Mikels regulatory pathways and molecular principles, or are there and Nusse, 2006; Chiba, 2006; Ehebauer et al., 2006). divergent solutions? Internally, epigenetic mechanisms involving members of the The sister group of the Bilateria are the Cnidaria (Fig. 1A). Polycomb group proteins that suppress activation of the The cnidarian Hydra has a simple body plan consisting of only differentiation program in stem cells play key roles (Ringrose two cell layers and a limited number of cell types derived from and Paro, 2004; Azuara et al., 2006; Schwartz and Pirrotta, three distinct stem cell lineages (Bosch, 2007a), the ectodermal 2007). These discoveries are beginning to show how stem cells and endodermal epithelial stem cells as well as the interstitial act to construct and maintain tissues, and to reveal evolutionary stem cells (Fig. 1B). Both the epithelial cells as well as the interstitial cells continuously undergo self-renewing mitotic ⁎ Corresponding author. Fax: +49 431 880 4747. divisions (Dübel et al., 1987). The main evidence for the stem E-mail address: [email protected] (T.C.G. Bosch). cell properties of interstitial cells comes from in vivo cloning 1 These authors contributed equally to the work. experiments which have shown that these cells are multipotent

Gierer, 1974, Heimfeld and Bode, 1984; Fujisawa, 1989; Teragawa and Bode, 1990, 1995; Technau and Holstein, 1996; Hager and David, 1997). In contrast to precursor cells, multipotent interstitial stem cells display very little migratory activity and grow as contiguous patches (Bosch and David, 1990; Fujisawa et al., 1990). (v) Transcription factors exclusively expressed in interstitial cells and/or their derivatives most likely play a key role in fate determination. Examples include the bHLH transcription factor gene achaete–scute homolog Cnash which is expressed in cell clusters that give rise to nematocytes (Grens et al., 1995) as well as in the differentiation pathway of sensory neurons (Hayakawa et al., 2004). Hyzic, a homolog of the Zn- finger transcription factor gene zic/odd-paired, is expressed in the early nematocyte differentiation pathway (Lindgens et al., 2004). Furthermore, two nanos (nos)-related genes, Cnnos1 and Cnnos2, are expressed in multipotent stem cells and germline cells, but not in somatic cells (Mochizuki et al., 2000). The vasa- related genes in Hydra, Cnvas1 and Cnvas2, are also strongly expressed in germline cells and less strongly expressed in multipotent interstitial stem cells and ectodermal epithelial cells (Mochizuki et al., 2001). (vi) Chromatin modification appears to Fig. 1. Interstitial stem cells in Hydra. (A) Relationships at the base of animal be involved in decision making during interstitial cell differentia- evolution. Cnidaria are often regarded as the closest outgroup of the Bilateria. tion. The embryonic ectoderm development (EED) homolog (B) The interstitial stem cell system in Hydra. (C) Scanning electron micro- scopic view of a longitudinal section through the ectoderm. m, mesoglea. Yellow HyEED, a member of the Polycomb Repressive Complex 2 arrows indicate the location of the mesoglea. (D) Scanning electron micrograph (PRC2) in adult Hydra is expressed in interstitial cells, showing interstitial cells within their niche. Note the close contact of interstitial nematoblasts and spermatogonia (Genikhovich et al., 2006). cells (green) to an ectodermal epithelial cell (red). (E) Distribution of interstitial Terminally differentiated interstitial cell derivatives such as cells in Hydra. Interstitial cells were stained with monoclonal antibody C41. nematocytes, gland cells or neurons do not express HyEED (F, G) Interstitial cells (stained by monoclonal antibody C41) follow positional cues and are absent in head (F) and foot (G) tissue. Arrows indicate the border (Genikhovich et al., 2006). Interestingly, HyEED is co-expressed at which interstitial cells disappear. with the Hydra homologue of EZH2 (Genikhovich et al., 2006) suggesting that, similar to mammals and Drosophila, the PRC2 complex may exist in Hydra. and capable of somatic and germ line differentiation, producing Taken together, the identification of a number of signaling neurons, nematocytes, secretory cells and gametes (David and molecules and genes which may act as regulators of interstitial Murphy, 1977; Bosch and David, 1986, 1987). stem cells point to the importance of spatial organization and What are the molecular mechanisms that control the ability of thus extrinsic influences on Hydra stem cells. However, the interstitial stem cells in Hydra to undergo self-renewal? Previous critical regulatory events and signal pathways that control the observations have revealed at least six strategies that underlie transformation of an interstitial stem cell into a differentiated interstitial stem cell behavior in Hydra: (i) Interstitial cells cell, the nature of the extrinsic signals as well as the in vivo interpret positional information and orient themselves towards dynamics of interstitial cells within the tissue, remain poorly spatial cues resulting in their presence exclusively in the gastric defined. So far, neither in Hydra nor in any other basal region and absence in head and foot tissue (Figs. 1E–G; David metazoan animal stem cells could be analyzed in vivo. and Plotnick, 1980). (ii) The self-renewal probability is controlled The advent of transgenics (Wittlieb et al., 2006) provides an by a negative feedback signal from interstitial cells and their opportunity to address these questions and to re-investigate the derivatives (Sproull and David, 1979; Bosch et al., 1991). (iii) Hydra interstitial stem cells in vivo. In this study, we generated Peptides emanating from surrounding epithelial cells contribute transgenic polyps expressing eGFP specifically in the interstitial to the regulation of interstitial cell differentiation. For example, stem cell lineage. We discovered that precise regulation of Wnt/ PW peptides such as Hym-33H directly affect interstitial cell β-catenin signalling is important for maintaining the balance of differentiation by inhibiting commitment or migration of nerve proliferation versus differentiation in the interstitial cell system. precursor cells (Takahashi et al., 1997, 2000). (iv) Interstitial cells Through a series of functional assays, we demonstrate that an and their derivatives transit between motile and sedentary modes. excess of Wnt signalling impaired interstitial cell differentia- Following differentiation, clusters of nematocyte precursors tion. In addition, our results suggest that the Notch pathway is break up into single cells that migrate towards the tentacles and involved to regulate the stem cell properties of interstitial cells. become mounted in specialized tentacle epithelial cells, termed We also describe here the generation of polyps in which the battery cells (David and Gierer, 1974). Similarly, after entering Polycomb group protein EED was expressed under control of the neuron differentiation pathway, about a half of the neuron the actin promoter in the interstitial cells. We report that precursor cells migrate toward the head and foot (David and terminal differentiation is correlated with absence of HyEED 34 K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 indicating that chromatin modification may have a critical Tissue grafting function in stem cell decision making. These data show that similar key signalling pathways appear to orchestrate stem cell Grafting of transgenic and non-transgenic tissue was done following standard procedures as previously described. behaviour throughout the animal kingdom from Hydra to man. Microscopical analysis of eGFP-expressing interstitial cells Materials and methods Fluorescent images were taken on a Zeiss Axioscope fluorescence Animals and culture conditions microscope with an Axiocam (Zeiss) digital camera. Confocal laser microscopy was done using a LEICA TCS SP1 CLS microscope. Experiments were carried out with Hydra magnipapillata (strain 105). Transgenic experiments were carried out with animals of the Hydra vulgaris AEP strain as described (Wittlieb et al., 2006). The animals where cultured Results according to standard procedures at 18 °C. Hydra has a distinct interstitial cell niche Generation of transgenic Hydra vulgaris expressing eGFP and – HyEED eGFP in their interstitial cells Interstitial cells grow and differentiate in the interstices between ectodermal epithelial cells. To visualize their precise Founder transgenic animals bearing the actin∷eGFP construct (hoT) were produced at the University of Kiel Transgenic Hydra Facility (http://www.uni- site of residence (referred to as niche) we did scanning electron kiel.de/zoologie/bosch/transgenic.htm) as previously described. For generation microscopy (Figs. 1C and D). Electron microscopy images of of actin∷HyEED–eGFP transgenics, a 1280bp fragment of HyEED coding for the ectodermal epithelium with part of the ectodermal cells full length protein was amplified from Hydra vulgaris AEP cDNA using peeled off show chains of interstitial cells and their derivatives Platinum High Fidelity polymerase (Invitrogene) and primers EED_F(28)Pst in close contact with the ectodermal epithelial cells (Fig. 1D). GAT AAC TGC AGC TAA TAG GAA GTT TCT GAT GGA TAC and EED_R (1326)Pst TTT TCT GCA GCT GTC TGC TTG TCC CAT CTC CAT AC. The Since the mesoglea (extracellular matrix) may present an cDNA was cloned into the modification of HoTG eGFP expression vector using additional important acellular component of the microenviron- PstI cutting site (see Fig. 8C). The resulting transfection construct (ligD) was ment, the interstitial cell niche in Hydra appears to contain a sequenced, plasmid DNA was purified using Quigen MidiPrep Kit and injected high level of structural complexity. into H. vulgaris AEP embryos as described earlier (Wittlieb et al., 2006). Embryos began to express the reporter gene 2–3 days after injection. Founder transgenic animals bearing the actin∷HyEED–eGFP construct started to hatch Generation of transgenic polyps expressing the eGFP marker 16 days after microinjection. Out of 72 embryos injected with the LigD selectively in the interstitial cell lineage construct, 60 hatched and out of them four transgenic lines were generated which expressed the transgene either in the epithelial cell lineage as well as in To study interstitial cell behavior in vivo in the context of the the interstitial cells or in the interstitial cell lineage only. Three of them showed native environment, we generated transgenic polyps expressing stable integration of actin∷HyEED–eGFP in ectodermal and endodermal epithelial cell lineages. One line showed integration of the construct in all three eGFP specifically in their interstitial stem cell lineage. We Hydra cell lineages, including interstitial cells. Initial founder transgenic microinjected 124 Hydra embryos with the eGFP expression animals were further expanded into a mass culture by clonal propagation by construct described earlier (Wittlieb et al., 2006). Of 20 hatched budding. polyps with eGFP+ cells, 18 expressed eGFP in their epithelial cells while two polyps contained both eGFP+ epithelial cells as DAPT, alsterpaullone, and MG132 administration well as cells that due to morphology and distribution were likely to be interstitial cells and their derivatives. From one of these DAPT (N-(N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl) ester; Sigma) treatment was performed as described (Geling et al., 2002; polyps, we selected buds, which contained only eGFP+ Käsbauer et al., 2006). DAPT was reconstituted with DMSO to make a stock interstitial cells and no eGFP+ epithelial cells. By clonal concentration of 10 mM. For experiments, aliquots were diluted to 10 μMin propagation, we generated a mass culture of animals in which hydra medium. Control embryos were incubated in an equivalent concentration most of the interstitial cells and their derivatives were eGFP+ (0.1%) of DMSO. Treatment with alsterpaullone was performed as described (Augustin et al., 2006). For treatment with the proteasome inhibitor MG123 (Fig. 2A). Transgenic polyps were healthy, exhibited normal (Sigma), animals were incubated in Hydra medium containing 5 μM of MG123 morphology and behavior and proliferated asexually by for up to 72 h. budding. When we examined these polyps by fluorescence and Gene expression analysis, TUNEL assay, and promoter confocal microscopy (Fig. 2), we observed eGFP expression characterization in all cell types of the interstitial cell lineage. eGFP+ interstitial cells (Fig. 2E) were found only in the gastric For assessment of gene expression, whole mount in situ hybridisation was carried out as described (Augustin et al., 2006). For TUNEL assay, polyps were region. Most of the bright eGFP+ cells were differentiating treated as described previously (Kuznetsov et al., 2001). The genomic sequences nematocytes (Figs. 2F, J) or nerve cells (Figs. 2F, H, I). of genes nb031 and nb35 as well as their 5′flanking sequences were hand- Clusters of differentiating nematocytes were detected in the assembled using single whole genome shotgun reads from the Hydra gastric region (arrows in Fig. 2C). Nerve cells were magnipapillata genome project deposited at NCBI trace archive. GenBank particularly abundant in the head (Fig. 2B) and foot (Fig. accession numbers are as follows: nb031, EU015879, xyz; nb035, EU015880 xyz (under submission). Tcf/Lef transcription factor binding sites were manually 2D) region where they formed a ring like structure. detected by BlastN pairwise alignments using previously described binding sites Additionally, eGFP expression was detected in gland cells (Brannon et al., 1997) as templates. (Fig. 2G) and male gametes inside testis (Figs. 2K, L). K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 35

Among the eGFP+ interstitial cells are multipotent cells which are able to self-renew

To determine whether these eGFP+ interstitial cells have functional characteristics of multipotent stem cells, we tested their ability to repopulate tissue which was free of eGFP+ cells. We produced two types of grafts. First, by axial grafting we allowed eGFP+ cells to enter unlabeled host tissue. Following separation from donor tissue after 3.5 days, we assayed the host for long-term presence of eGFP+ cells. Using this approach (schematically shown in Fig. 3A), we generated polyps (n=15) which contain all cells of the interstitial cell lineage (data not shown). Interestingly, tissue could get repopulated stably only when separation was close (cutting level I in Fig. 3A) to the donor tissue. When separation was done with some distance to the donor tissue (cutting level II in Fig. 3A), host tissue could not get repopulated with eGFP+ cells. We conclude that multipotent eGFP+ stem cells capable of repopulating the non- transgenic host tissue do not show motility over large distances. In the second approach shown schematically in Fig. 3B, we used lateral grafts to introduce small patches of eGFP+ cells into host tissue free of any transgenic cells. Again, we could generate polyps (n=10) which stably contain cells expressing eGFP in all cells of the interstitial cell lineage. As shown in Fig. 3C to E, these implanted cells could continuously self-renew and differentiate into all cell types of the interstitial cell lineage during the 6-month observation period. These functional assays are consistent with the eGFP+ population containing multipotent stem cells.

In vivo tracking of individual interstitial cells

Following differentiation of interstitial stem cells in the gastric region, nematoblasts and neuroblasts must traverse great distances to reach their final destination in the tentacle where most of them get incorporated in a “battery cell complex”. Since polyps are essentially transparent, eGFP+ transgenic interstitial cells enable live imaging of cell behaviour. Thus, to determine the intrinsic migratory behavior and to examine whether all cells of the interstitial cell lineage (see Fig. 1B) display high motility or whether migration is restricted to certain subpopulations, we grafted transgenic tissue to “naïve” tissue as shown schematically in Fig. 4A. Live imaging of eGFP+ cells (Figs. 4B–I) in 58 grafts demonstrated that migration of nematoblasts and Fig. 2. Transgenic interstitial cells expressing eGFP. (A) Whole mount neuroblasts occurs as individual cells and never as cluster of fluorescence analysis of eGFP expression in interstitial stem cells and cells. Surprisingly, migrating cells are capable of rapid (∼2 μm/ differentiated derivatives. (B) eGFP+ cells in the head. Note the numerous min) motility in vivo. Within 10 min after grafting, first cells eGFP+ neurons in the hypostome. Undifferentiated interstitial cells are absent in migrating towards the apical end could be observed (Fig. 4C). this region. (C) eGFP+ interstitial cells and derivatives in the gastric region. Migration of neuroblasts and nematoblasts occurred continu- Arrows indicate nests of developing nematoblasts. (D) eGFP+ cells in the foot – tissue. Note the eGFP+ ring of nerve cells. (E–L) Morphology of eGFP+ ously through a 24-h period of observation (Figs. 4F I). interstitial cells and differentiated derivatives. (E) Confocal analysis shows a Interestingly, nearly all migrating cells could be classified as pair of undifferentiated interstitial cells. Rhodamin–phalloidin was used to stain nematoblasts or neuroblasts whereas interstitial stem cells (large the muscle fibres. (F) Confocal analysis showing eGFP+ nerve cells in close 1+2s) were mostly residing at the transplantation edge and were contact with a nematocyte. Nc, nematocyte; nv, nerve cell. (G) Confocal analysis not actively motile. This in vivo observation supports our showing a eGFP+ gland cell. (H) Confocal analysis showing a eGFP+ sensory nerve cell. (I) Confocal analysis showing a eGFP+ multipolar ganglion cell. previous view (Bosch and David, 1990; Fujisawa et al., 1990) (J) Confocal analysis showing a nest of 8 developing nematoblasts. (K, L) Light that interstitial stem cells in Hydra in contrast to differentiating microscopy of a testis containing eGFP+ spermatogonia. interstitial cells show little if any migratory activity. 36 K. Khalturin et al. / Developmental Biology 309 (2007) 32–44

Fig. 3. Transgenic eGFP+ interstitial cells proliferate and differentiate in all cell types of the interstitial stem cell system. (A, B) Schematic diagrams of the grafting procedures used. (A) Horizontal grafting of transgenic tissue to “naïve” (i.e. eGFP−) tissue. I and II indicate different levels of separation. (B) Lateral grafting of small pieces of tissue containing eGFP+ cells into “naïve” (i.e. eGFP−) polyps. (C) Lateral grafting of eGFP+ interstitial cells in a non-transgenic host, 4 h after lateral implantation. (D) Implanted eGFP+ interstitial cells proliferate and differentiate; picture was taken 2 weeks after grafting. (E) 6 months after grafting all cell types of the interstitial stem cell system were eGFP+. Thus, the implanted cells included multipotent eGFP+ stem cells.

Activation of Wnt signalling alters interstitial cell and in the head. Since the absolute number of nb031 expressing differentiation nematoblasts is increased drastically in ALP-treated polyps (Figs. 6E and F) compared to control polyps, and since ALP To get insight into the molecular pathways controlling does not inhibit nematoblast migration per see (see below), we interstitial cell differentiation in Hydra, Wnt signalling was conclude that ALP treatment induces ectopic terminal differ- activated in transgenic polyps by the addition of alsterpaullone entiation of nematoblasts in the gastric region. Interestingly, as (ALP) (Broun et al., 2005). ALP is a specific inhibitor of GSK3- shown in Fig. 6J, in the promoter of the nb031 gene several Lef/ β and able to activate Wnt signalling in Hydra (Broun et al., Tcf consensus sites (Brannon et al., 1997) are located upstream 2005). Fig. 5 shows that addition of 5 μM ALP had drastic of the transcription initiation site supporting the view that effects on eGFP-expressing interstitial cells and their deriva- nb031 is a direct target for the β-catenin/Tcf transcription factor tives. In all animals examined (n=60), within 48 h upon complex. By analyzing the expression of neuron-specific gene treatment nests of differentiating nematoblasts break up into Hym355 (Takahashi et al., 2000) in ALP-treated tissue (Figs. single cells (Figs. 5E and F) indicating terminal differentiation 6G–I), we could not discover any effect of ALP on neuron of all nematoblasts present in the gastric region. In untreated differentiation. control polyps, nematoblast nests disaggregate progressively To determine whether the Wnt induced changes in interstitial within 5 to 48 h (see Supplementary Fig. S1) and new nests are cell behaviour reflect cell-intrinsic activity or rather a response produced continuously. The absence of new nematoblast nests towards the changed microenvironment, we grafted tissue with 48 h after ALP treatment (Fig. 5E), therefore, most likely eGFP+ interstitial cells to unlabelled host tissue which had been indicates that ALP directly inhibits the differentiation of treated with ALP for 48 h preceding transplantation (Fig. 4J). interstitial cells into nematoblasts. As shown in Figs. 4K and L, there is little if any migratory The expression of two genes (Fig. 6), which are markers for activity of interstitial cells and nematoblasts into ALP-treated nematocyte differentiation, supports this view. One of the genes, tissue. Control experiments demonstrate that ALP has no effect nb035, is expressed in nests of differentiating nematoblasts of on the migratory activity of interstitial cells per se. Fig. 4(M–P) all types in the gastric region (Fig. 6A). In ALP-treated polyps, (see also Supplementary Fig. S2) shows extensive migration of nests of nb035-expressing cells are largely undetectable (Figs. eGFP+ cells originating from donor tissue which was treated 6B and C). The second gene, nb031, is expressed in stenoteles with ALP for 48 h. Thus, interstitial cells appear to quickly at the base of tentacles (Fig. 6D). In ALP-treated tissue, this respond to changes in microenvironment. When confronted gene is ectopically expressed throughout the whole gastric with ALP-treated tissue, they behave as if they have reached region (Figs. 6E and F). The appearance of numerous nb031 their terminal destination in the head region. In sum, extrinsic expressing stenoteles in the gastric region of ALP-treated tissue signals from the microenvironment play a major role in (Fig. 6E) directly correlates with the disappearance of nb035- interstitial cell differentiation and migration, and may be positive nests of nematoblasts (Fig. 6B). Most likely due to β- mediated by the Wnt pathway. These observations demonstrate catenin stabilization in all cells of the animals, the gastric region the importance of the Wnt signalling pathway in interstitial cell changed its positional value and acquired environmental differentiation. They show for the first time that this pathway in qualities normally restricted to tissue at the base of tentacles adult Hydra fulfils two functions, one in patterning (Hobmayer K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 37

Fig. 4. Dynamics and motility of eGFP+ interstitial cells in normal and manipulated microenvironment. (A) Schematic diagram of grafting procedure. (B–I) In vivo tracking of eGFP+ interstitial cells and their derivatives entering “naïve” host tissue. Images were captured as indicated at ×50 magnification. Arrows point to migrating cells of the interstitial cell lineage. (J) Grafting procedure using host tissue (grey) in which either β-catenin signalling was activated by alsterpaullone (ALP) or Notch activity was blocked by DAPT. (K, L) In vivo tracking of eGFP+ interstitial cells which are confronted with ALP-treated host tissue. Note that nearly no eGFP+ cells enter the perturbated host tissue. There is massive migration into untreated control tissue (I). (M–P) Migration of cells of the interstitial cell lineage is not inhibited by ALP treatment. (M) Tissue treated for 48 h with ALP was grafted to untreated host tissue. (N–P) Extensive emigration of eGFP positive cells 24 and 48 h following transplantation. Arrows point to migrating cells (see also Supplementary Fig. S2). et al., 2000; Broun et al., 2005) and one in interstitial cell 48 to 96 h with DAPT which blocks Notch activity by inhibiting differentiation. the γ-secretase-dependent cleavage that releases the Notch intracellular domain (Geling et al., 2002; James et al., 2004). Fig. Inhibition of Notch signalling induces programmed cell death 7 shows that a block in Notch signalling prevents nematoblast in differentiating nematocytes differentiation. In polyps containing eGFP+ interstitial cells, following incubation in 10 μM DAPT the number of nests of Recent evidence (Käsbauer et al., 2006) suggested that the differentiating nematoblasts (Figs. 7A–F) was dramatically Notch pathway may be required to control interstitial cell decreased (∼5-fold compared to untreated control polyps) differentiation. To further dissect the molecular mechanisms that within 96 h (n =52). Strikingly, nerve cell differentiation are involved in interstitial cell differentiation and to investigate appeared not to be affected at all (Fig. 7F; see also Supplementary the function of the Notch pathway in vivo, we treated polyps for Fig. S3). In contrast to ALP (see Fig. 5C), administration of 38 K. Khalturin et al. / Developmental Biology 309 (2007) 32–44

How do the nematoblasts disappear so quickly after DAPT treatment? To investigate whether programmed cell death (PCD) is involved, we examined DAPT-treated polyps (n=32) by TUNEL. Figs. 7P to R shows that blocking of Notch activity is accompanied by drastic increase in TUNEL positive nuclei in the gastric region. Most of the nuclei belong to nematoblasts (see insert in Fig. 7R). Notch activity seems to operate at the level of transient amplifying cell proliferation or differentiation since PCD affects nematoblasts before the nests break up into single cells (see insert in Fig. 7Q). Thus, suppression of Notch signalling causes immediate death of differentiating nematoblasts. Inhibition of Notch does not affect neurons (Figs. 7M–O) nor interstitial cells as the nematocyte population quickly recovers after termination of DAPT treatment. We, therefore, assume that Notch is a permissive cue in nematoblast differentiation, rather than an instructive one. These observations support the previous study (Käsbauer et al., 2006) and strongly implicate Notch signalling as a key component in the acquisition of nematocyte fate. To investigate whether these responses to Notch blocking are determined in a cell-autonomous manner, in response to Fig. 5. Activation of Wnt/β-catenin signalling alters interstitial cell behaviour. instructive influences of the interstitial cell niche, or a All animals examined (n=60) behaved similarly. Observation of eGFP+ combination of both we grafted tissue with eGFP+ interstitial interstitial cells and their derivatives in control (A, D) and alsterpaullone-treated cells to host tissue which did not contain eGFP+ cells but which (B, C, E, F) tissue 48 and 96 h after treatment. Arrows in panels A and D point to was pretreated with DAPT for 48 h as shown schematically in nests of developing nematoblasts which disappeared upon ALP treatment. Fig. 4J. eGFP+ nematoblasts and neuroblasts entered this tissue and differentiated normally into nematocytes and neurons (data DAPT does not cause ectopic tentacle formation and appears not not shown). Thus, DAPT treatment seems to block Notch to affect the positional value system along the body column. signalling in cells of the interstitial cell lineage directly and not To analyze the inhibition of Notch signalling on interstitial through indirect interactions from the surrounding epithelial cell differentiation at the molecular level, we studied the cells. expression of several genes expressed in interstitial cells after entering either the nematoblast or the neuron differentiation Transgenic polyps expressing Polycomb group protein HyEED pathway. In the absence of active Notch signalling (n=20), gene in the interstitial cell lineage point to epigenetic control of nb035 which normally is expressed in nematoblast nests in the interstitial cell differentiation gastric region (Fig. 7G), is rapidly turned off after 48 h (Fig. 7H–I). Similar to that, gene nb031 which is normally expressed We previously have shown that in Hydra the gene encoding in stenoteles at the tentacle base, is also turned off in DAPT- Polycomb protein HyEED is specifically expressed in inter- treated tissue (Fig. 7J); 96 h following DAPT treatment no stitial cells and differentiating nematoblasts (Figs. 8A and B; nb031 transcripts can be detected anymore (Fig. 7L). The rapid Genikhovich et al., 2006). HyEED is not expressed at later disappearance of nb031 expressing stenoteles in the tentacles stages of differentiation and, therefore, absent in the head (see following DAPT treatment suggests that Notch activity is Fig. 8B) and foot regions. To explore whether epigenetic required by differentiated nematocytes, not just by the dividing histone modifications are important for differentiation of cells of the nematoblast nests. interstitial cells, we produced polyps which overexpress Since interstitial cells can differentiate both into neurons as HyEED in the interstitial cell lineage. Hydra embryos were well as nematoblasts and other cell types (Fig. 1B), our microinjected with an expression construct in which HyEED observation that Notch inhibition results in rapid disappearance was fused in frame to eGFP and under control of the Hydra of nematoblasts raised the possibility that interstitial cells in the actin promoter (Fig. 8C). 1 out of 60 hatched polyps expressed absence of Notch differentiate preferentially into nerve cells. To the HyEED–eGFP fusion protein in the interstitial cell lineage. test this premise, we examined the number of nerve cells Most strikingly, despite the fact that the construct is driven by expressing neuropeptide Hym355 (Takahashi et al., 2000)in the ubiquitously active actin promoter, cells expressing the control and DAPT-treated (n=20) polyps. As shown in Figs. 7M EED–eGFP fusion protein were observed only in the gastric to O, no differences could be observed in control and treated region and never in head or foot tissue (see Figs. 8D and E). Hydra tissue with regard to the number of Hym355-expressing Thus, unexpectedly, the localization of the fusion protein (Figs. cells present. Thus, there seems to be no trade-off between 8D and E) emulates the endogenous expression of HyEED nematoblast and nerve cell differentiation when Notch activity is (Figs. 8A and B). As shown above (Fig. 2), in transgenic polyps blocked. containing the same actin∷eGFP construct without the HyEED K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 39

protein appears to be located in both the nuclei (Fig. 8F) as well as in the cytoplasm. eGFP protein is a very stable protein (Ward, 2006). In Hydra cells, eGFP is stable for more than 2 weeks in the absence of transcription (Stefan Siebert and TCGB, personal observation). Thus, there are two possible explanations for the unexpected localization of HyEED–eGFP positive cells and their conspic- uous absence in head and foot tissue. One is the apoptotic death of fusion protein containing cells in head and foot tissue. The other possibility could be the active degradation of the fusion protein in cells entering terminal differentiation. There is no evidence for an increased level of apoptosis of eGFP+ cells in the Hydra head tissue (data not shown). However, in cells above the border between gastric and head tissue a progressively weaker eGFP signal can be observed (see Fig. 8D). To investigate whether the HyEED–eGFP fusion protein can be rapidly degraded in cells which experienced a rapid change in the positional value (from body tissue to head tissue), we performed two independent experiments. First, in a regeneration experiment, transgenic HyEED–eGFP animals (n=18) were decapitated at the border between head and gastric tissue as shown schematically in Fig. 8I. As shown in Fig. 8J, in such regenerates immediately after cutting eGFP+ cells are present at the most apical part of the animal. In all animals examined, 24 and 48 h following head regeneration some eGFP+ cells still can be observed in the most apical region (Figs. 8K and L). Strikingly, between 48 and 72 h when head structures such as tentacles start to develop (Fig. 8L) and interstitial cells undergo terminal differentiation into nematocytes, no or very few HyEED–eGFP positive cells can be detected anymore in the head tissue (Fig. 8M). To further examine whether the disappearance of the eGFP signal is correlated with terminal differentiation, we monitored HyEED–eGFP positive cells located in the gastric region. As shown in Figs. 8 (N–Q), the eGFP signal in nematoblasts of the lower gastric region disappears within 24 h after disaggregation of the nematoblast nests (see also Supplementary Fig. S4). This supports our view that HyEED–eGFP is actively degraded during the transition of a nematoblast into a mature nematocyte. If the disappearance of the fluorescent cells is due to proteolytic degradation of the fusion protein, the ubiquitin Fig. 6. Whole-mount in situ hybridization using two nematoblast-specific genes proteasome system should be involved. In the second experi- reveals that perturbation of Wnt/β-catenin signalling directly affects transcription of interstitial cell-specific genes. (A–C) Expression of nematoblast-specific mental approach, we tested this possibility by examining gene nb035 in control (A) animals and at successive stages after alsterpaullone whether inhibition of proteasome-mediated degradation affects treatment (B, C). (D–F) Expression of nematoblast-specific gene nb031 in the stability of the HyEED–eGFP fusion protein. Previous control (D) animals and at successive stages after alsterpaullone treatment (E, F). studies indicated that the MG132 molecule is a potent, (G–I) Expression of neuron-specific gene Hym355 in control (G) and ALP- ′ membrane-permeable proteasome inhibitor (Nencioni et al., treated (H, I) tissue. (J) Within the 1400 bp 5 flanking sequence of the nb031 μ gene there are seven putative Lef/ TCF binding sites indicating that nb031 may 2006). Transgenic polyps, therefore, were incubated in 5 M be a direct target gene for β-catenin. Lef/TCF binding sites are represented by MG132 for 72 h and monitored for the presence of eGFP+ cells. boxes: magenta, GAAACT; red, CTTTGT; yellow, CTTTGA; blue, CTTTGAA. Strikingly, as shown in Figs. 8G and H, inhibition of the proteasome system leads to presence of HyEED–eGFP+ sequence (see Fig. 8C), eGFP+ cells can be detected in all cells nematocytes in head and tentacle tissue whereas in control of the interstitial cell lineage all over the body column including polyps (Fig. 8D) eGFP+ cells are conspicuously absent from head and foot regions. Confocal microscopy (Fig. 8F) showed this region. It has been demonstrated (Higa et al., 2006) that HyEED–eGFP expression in interstitial cells as well as Polycomb-group protein EED is ubiquitinated and, thus, developing nematoblasts. Other derivatives of the interstitial directly targeted for proteolytic degradation. We, therefore, stem cells were not observed to express the fusion protein. The propose that nematoblasts entering head or foot territory (the 40 K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 region of terminal differentiation) abruptly loose the HyEED– differentiation and that – similar to cells in higher animals – eGFP fusion protein by proteolytic degradation to facilitate HyEED is likely to actively suppress final differentiation steps terminal differentiation. Our overexpression construct seems to in Hydra interstitial stem cells. be unable to override this endogeneous control mechanism. Taken together, the observations support the view that Discussion remodelling of chromatin structure is involved in interstitial cell Up to now, stem cells in basal metazoans were not approa- chable to cell tracking and direct molecular analysis. In this paper, we describe the generation of Hydra polyps containing transgenic stem cells to explore in vivo the mechanisms controlling stem cell behavior in an animal at the base of metazoan evolution. Importantly, we show that despite extensive evolutionary divergence, the molecular pathways driving stem cell differentiation have been highly conserved between Hydra and vertebrates.

The role of Wnt and Notch

Of particular significance was the observation that Wnt and Notch, which are known to be required for controlling stem cells in vertebrates and insects (Mikels and Nusse, 2006; Guder et al., 2006; Bejsovec, 2006; Clevers, 2006; Bray, 2006, Chiba, 2006), appear to mediate the integration of extrinsic signals from the microenvironment and are involved in regulating stem cell behaviour in Hydra. In adult Hydra, the Wnt signalling pathway was the first to be implicated in the control of the body axis (Hobmayer et al., 2000; Broun et al., 2005; reviewed in Guder et al., 2006). The data in this paper (Figs. 5 and 6) indicate that in adult polyps the Wnt pathway in addition is one of the key factors in controlling interstitial cell differentiation. Thus, while in higher organisms the Wnt signalling pathway regulates developmental processes such as patterning (Logan and Nusse, 2004) and stem cell homeostasis (Kirstetter et al., 2006; Crosnier et al., 2006), at different developmental stages successively, in adult Hydra Wnt appears to regulate these seemingly different processes simultaneously. This underscores the crucial role that Wnt genes play in stem cell differentiation throughout the animal kingdom. Similar to Wnt, Notch signalling seems to have pleiotropic effects on stem cells and their lineages in various organisms (Bray, 2006; Chiba, 2006; Ehebauer et al., 2006). In Hydra (Fig. 7), the blockage of the Notch pathway causes marked changes in

Fig. 7. Blockage of Notch signalling interferes with interstitial cell differentiation and induces nematocytes to initiate apoptosis. All animals examined (n=52) behaved similarly. (A–F) Observation of eGFP-expressing interstitial cells in control (A, D) and DAPT-treated (B, C, E, F) tissue 48 and 96 h after treatment. Higher magnification pictures for (E) and (F) are available in Supplementary Fig. S3. (G–L) Whole-mount in situ hybridization of two nematoblast-specific genes reveals direct effect at transcriptional level. (G–I) Expression of nematoblast-specific gene 035 in control (G) animals and at successive stages after DAPT treatment (H, I). (J–L) Expression of nematoblast- specific gene 031 in control (J) animals and at successive stages after DAPT treatment (K, L). (M–O) Whole-mount in situ hybridization of neuron-specific gene Hym355 reveals that administration of DAPT does not affect the nervous system. (M) Control, (N, O) 48 and 96 h after begin of DAPT treatment. (P–R) Upon DAPT treatment, developing nematoblasts in the gastric region undergo programmed cell death as monitored by TUNEL assay. (P), control, (Q, R), TUNEL assay in animals 48 and 96 h after begin of DAPT treatment. K. Khalturin et al. / Developmental Biology 309 (2007) 32–44 41

Fig. 8. Polycomb group protein HyEED is involved in interstitial stem cell differentiation. (A, B) In situ hybridization with HyEED probe labelled with digoxigenin shows that HyEED gene expression is restricted to interstitial cells in the gastric region. Bar in panel A indicates 1 mm. Note in panel B the absence of HyEED- expressing interstitial cells in the head tissue. (C) Structures of the reporter gene constructs used to assess the function of HyEED. eGFP, enhanced green fluorescent protein. (D) Transgenic polyp with interstitial cells expressing the HyEED–eGFP fusion protein. Note that in the head region in a few cells (indicated by arrows) a faint eGFP fluorescence can be detected. Strongly fluorescent HyEED–eGFP+ cells are absent in the head and tentacles. (E) Expression of HyEED–eGFP fusion protein parallels the expression of the HyEED gene with HyEED–eGFP+ cells located exclusively in the gastric region. (F) Morphology of HyEED–eGFP+ interstitial cells and differentiated derivatives. Confocal analysis shows single undifferentiated interstitial cells (i-cell, filled arrow) and differentiating nematoblasts (nb, open arrow). The eGFP signal in interstitial cells is always weaker than the one in nematoblasts. Note that the HyEED–eGFP fusion protein is located in both the cytoplasm as well as the nucleus (Nu, arrowhead). Bar indicates 50 μm. Cell nuclei were counterstained with DAPI. Rhodamin–phalloidin was used to stain the muscle fibres. (G, H) Inhibition of the ubiquitin proteasome system leads to presence of eGFP+ nematoblasts in head (G) and tentacle (H); h, hypostome; t, tentacle. (I–M) Tracking the fate of HyEED–eGFP-expressing interstitial cells during head regeneration. (I) Scheme of experimental procedure. (J) HyEED–eGFP+ cells at the cutting site directly after decapitation. (K) HyEED–eGFP+ cells 24 h and (L) 48 h after decapitation. (M) 72 hr after decapitation no HyEED–eGFP+ interstitial cells were detected in the regenerated tip, presumably because the cells have entered terminal differentiation into nematocytes and actively degraded the HyEED–eGFP fusion protein. (N–Q) Tracking for 47 h the fate of HyEED–eGFP positive nematoblast nests within the lower gastric region (see also Supplementary Fig. S4). Note the disappearance of the eGFP signal within 24 h following nematoblast nest disaggregation. the interstitial cell behaviour. As a result of Notch inhibition, complete their differentiation program or to enter it. Since one of differentiating nematoblasts undergo programmed cell death the roles for Notch in vertebrate development is to participate in (Fig. 7). Thus, Notch appears to be required for nematoblasts to neuronal subtype specification (Jadhav et al., 2006; Yaron et al., 42 K. Khalturin et al. / Developmental Biology 309 (2007) 32–44

2006; Yang et al., 2006), this supports the view (Hausmann and environments that are responsible for the proliferation and Holstein, 1985; Brinkmann et al., 1996; Grens et al., 1995; differentiation behaviour of interstitial cells. We show here that Fedders et al., 2004; Hayakawa et al., 2004) that Hydra Hydra interstitial cells reside in a specialized microenvironment nematoblasts should be considered a neuronal cell type. created by ectodermal epithelial cells (Fig. 1). Previous Taken together, in Hydra Wnt and Notch pathways play observations (Takahashi et al., 1997; reviewed in Bosch and major roles in interstitial cell differentiation. The challenge now Fujisawa, 2001; Bosch, 2007b) have indicated that interstitial is to understand not just the action of each type of signal cell differentiation is strongly affected by peptides produced by pathway individually, but how they operate as a system to both neighboring ectodermal epithelial cells as well as neurons. organize the distinct tissue architecture of the polyp and to Therefore, interstitial cells in Hydra – similar to bilaterian stem control the continuous patterning and renewal of the cell types. cells (Watt and Hogan, 2000; Fuchs et al., 2004) – appear to engage in a wide variety of interactions with their neighboring Epigenetic control at the base of animal evolution cells. This obviously intensive spatio-temporal dialog occurring between interstitial stem cells and epithelial cells (see also When cells become committed to differentiation, the epi- Takahashi et al., 2000, Bosch and Fujisawa, 2001) provides genetic status must be reprogrammed, and Polycomb proteins convincing support for the applicability of the niche concept mediate this process (Otte and Kwaks, 2003; Ringrose and Paro, (Schofield, 1978; Spradling et al., 2001; Moore and Lemischka, 2004; Nystul and Spradling, 2006; Azuara et al., 2006; Schwartz 2006) in the basal metazoan Hydra. and Pirrotta, 2007). Our observations in HyEED–eGFP- The results shown above suggest an early common origin of expressing transgenic Hydra (Fig. 8) indicate that members of the mechanisms that specify self-renewal and prevent differ- the Polycomb protein family are involved in interstitial cell entiation of stem cells. They provide compelling evidence for differentiation. The molecular mechanisms controlling the the existence of conserved mechanisms controlling stemness activity of the PRCs is largely unknown. Our study indicates a from Hydra to man. These elements include paracrine and rapid disappearance of HyEED–eGFP fusion protein in cells juxtacrine signalling pathways such as Wnt and Notch, negative undergoing terminal differentiation (Fig. 8). Since expression of feed back loops that limit the response to mitogenic signals this fusion protein is controlled by the actin promoter, and since (Takahashi et al., 2000; Bosch and Fujisawa, 2001; Bosch, we have shown (Fig. 2) that this promoter is active in tissue 2007b), and chromatin modifiers belonging to the Polycomb where terminal differentiation takes place, transcriptional group proteins. In Hydra and related cnidarians, these “pan- repression appears to be an unlikely reason for the disappearance metazoan” features together with the unexpected molecular of the protein in transgenic animals. Thus, both a loss of the equivalence to human cells (Kortschak et al., 2003; Technau et mRNA (see Figs. 8A and B) and protein degradation appears to al., 2005) is complemented by unique biological and experi- contribute to the absence of HyEED expression in the tentacles mental opportunities. Thus, studies in Hydra may provide a of normal Hydra. Interestingly, it has been recently shown (Ben- paradigm for the characterization and analysis of stem cells in Saadon et al., 2006) that in human cells the ubiquitin– other systems, and may reveal fundamental principles that proteasome system is involved in control of PRC activity and underlie all stem cell systems. that the proteasome system directly interacts with Polycomb- group protein EED (Higa et al., 2006). We, therefore, propose Acknowledgments that the rapid disappearance of HyEED–eGFP fusion protein may be caused by proteasomal degradation of the short-lived Sylvia Sassmann provided excellent help with analyzing the HyEED protein in cells undergoing terminal differentiation. transgenics. We thank Antje Thomas, and Birgit Belusa for The instructive role of HyEED in interstitial cell differentia- technical assistance, the members of the Bosch laboratory for tion remains to be elucidated. Most interestingly, Polycomb-like discussion, Roland Melzer (Munich) for help with scanning genes recently have been detected to play a role in neuronal electron microscopy, and Jan U. Lohmann (Tübingen) for specification in Caenorhabditis elegans (Yang et al., 2007). valuable comments on the manuscript. We also want to thank Since nematocytes in Hydra are considered as mechano- and/or the anonymous referees for their constructive comments on the chemosensory cells (Hausmann and Holstein, 1985; Brinkmann manuscript. This work is supported by the Deutsche For- et al., 1996) and express a number of neuron-specific genes schungsgemeinschaft (grants to T.C.G.B.). (Grens et al., 1995; Fedders et al., 2004; Hayakawa et al., 2004), it is tempting to speculate that in Hydra Polycomb proteins such Appendix A. Supplementary data as HyEED are involved in specifying certain nematocyte identities. We currently explore whether HyEED is involved in Supplementary data associated with this article can be found, regulating the nematocyte type by acting directly on genes in the online version, at doi:10.1016/j.ydbio.2007.06.013. linked to nematocyte formation. References Are there universal laws for all stem cell systems? 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