THE LONG WAY TO ECHINOCOCCUS NEOBLAST CULTURES

During the golden age of ultrastructural studies on cestodes in the 1950–1980s, the presence of undifferentiated ‘germinal cells’ has frequently been described in a variety of adult and larval tissues. Morphologically, these cells could be clearly distinguished from their differentiated neighbours in having a large nucleus with a prominent nucleolus, surrounded by scant, basophilic cytoplasm, that contained few mitochondria as the only organelles (Heath and Lawrence Reference Heath and Lawrence1976; Mehlhorn et al. Reference Mehlhorn, Eckert and Thompson1983; Swiderski, Reference Swiderski1983; Rybicka, Reference Rybicka1966; Sakamoto and Sugimura, Reference Sakamoto and Sugimura1970; Slais, Reference Slais1973; Sakamoto, Reference Sakamoto1981, Reference Sakamoto1982). Mostly by studying the cellular dynamics of proliferating cells through a combination of electron microscopy and the uptake of radioactively labelled thymidine, a relatively clear picture emerged that ‘germinal cells’, like their neoblast counterparts in planarians, are the only mitotically active cells in cestodes that give rise to all other differentiated cells (reviewed by Reuter and Kreshchenko, Reference Reuter and Kreshchenko2004). According to these early studies, neoblasts are usually located in the neck region of the adult's scolex and initiate the formation of proglottides from the germinative area. Within proglottides, neoblasts are then involved in the formation of the genital anlagen and the genital apparatus (see references in Reuter and Kreshchenko, Reference Reuter and Kreshchenko2004). After gamete formation and fertilization, neoblasts arise early during embryogenesis, actively synthesize RNA during the entire process of oncosphere formation and are usually located at one pole of the late bilateral symmetrical oncosphere in differing numbers (between 6 and 12), depending on the species (Rybicka, Reference Rybicka1966; Slais, Reference Slais1973; Swiderski, Reference Swiderski1983; Mlocicki et al. Reference Mlocicki, Swiderski, Miquel, Eira and Conn2006). Studies on the early development of E. multilocularis in experimentally infected mice then showed that the neoblasts are the only oncosphere cells that contribute to metacestode formation and that hooks and muscles completely disappear within a few days after infection (Sakamoto and Sugimura, Reference Sakamoto and Sugimura1970; Vogel, Reference Vogel1977; Mehlhorn et al. Reference Mehlhorn, Eckert and Thompson1983). Hence, the transition from oncosphere to metacestode in Echinococcus spp. is, as in other tapeworms, a true metamorphosis. Although only very few investigations on the formation of protoscoleces within metacestode vesicles and hydatid cysts of E. multilocularis and E. granulosus have so far been undertaken, it can be expected that the development of this larval stage is also induced by neoblasts that are part of the germinal layer. At least preliminary evidence that this is indeed the case has been obtained for E. granulosus (Galindo et al. Reference Galindo, Paredes, Marchant, Mino and Galanti2003).

Due to the decisive role of neoblasts in driving the life-cycle of Echinococcus and other cestodes, several attempts toward their cultivation in vitro have already been undertaken in the 1980s (reviewed in Brehm and Spiliotis, Reference Brehm and Spiliotis2008a). However, these early studies were confounded by the fact that parasite material from experimentally infected mice had been used as a source for parasite cells, and that it was not known at that time that Echinococcus cells are highly sensitive towards reactive oxygen species that are typically produced during in vitro culture. As a consequence, the isolated parasite cells were frequently overgrown in culture by contaminating host cells (Brehm and Spiliotis, Reference Brehm and Spiliotis2008a). First steps towards a solution of this problem have been made by Hemphill and Gottstein (Reference Hemphill and Gottstein1995) as well as Jura et al. (Reference Jura, Bader, Hartmann, Maschek and Frosch1996) through the development of in vitro cultivation systems for Echinococcus metacestode vesicles. Although these systems still relied upon co-cultivation of Echinococcus tissue with host feeder-cells (Caco-2 colon carcinoma cells or primary rat hepatocytes), they provided the first systems by which parasite vesicles could be produced that, after vigorous washing, were almost free of host-cell contamination. A decisive step was then made by Spiliotis et al. (Reference Spiliotis, Tappe, Sesterhenn and Brehm2004) who established the first system for axenic cultivation of E. multilocularis vesicles. This study showed that parasite vesicles can be kept in the absence of host cells, provided that the medium contained reducing substances, and that the cultures were maintained under a nitrogen atmosphere, thus eliminating the formation of reactive oxygen species (Spiliotis et al. Reference Spiliotis, Tappe, Sesterhenn and Brehm2004; Brehm and Spiliotis, Reference Brehm and Spiliotis2008a). Since, even under these conditions, parasite vesicles only proliferated when host cell-conditioned medium was applied, the axenic cultivation system was also the first to demonstrate unequivocally that host cell-produced growth factors support parasite development and proliferation (Spiliotis et al. Reference Spiliotis, Tappe, Sesterhenn and Brehm2004). One of the most important outcomes of this study was that the axenic vesicles now provided an excellent source to set up parasite cell cultures essentially free of host contamination. After modifying the axenic cultivation system towards the so-called ‘large-scale liquid culture system’, by which significant numbers of axenic vesicles can be produced, Spiliotis and Brehm (Reference Spiliotis and Brehm2009) were able to isolate, through tryptic digestion, primary parasite cells in sufficient quantity to establish in vitro cultures. Using flow cytometry, Spiliotis, M. et al. (Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008) showed that at least 30% of freshly established primary cells were in the S- and G2-phase of the cell cycle and, thus, represented neoblasts. Furthermore, the typical cestode neoblast morphology of a large fraction of Echinococcus primary cells was demonstrated in this study by transmission electron microscopy (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008). Under ideal growth conditions, the Echinococcus neoblasts proliferated in culture and formed cell aggregates which later developed small internal cavities (Fig. 1). After 5 weeks of incubation, the young vesicles enlarged and the parasite tissue surrounding the cavities contained both neoblasts and differentiated cells. At this time, the acellular laminated layer (LL), which is the typical distal border of mature metacestode vesicles acting as a physical barrier to separate host and parasite tissue in the later phase of the infection, was not yet formed. After 6 weeks of incubation, the LL was present, indicating the formation of fully mature metacestode vesicles. When injected into the peritoneal cavity of mice, these vesicles yielded high loads of parasite material, including metacestode tissue and protoscoleces, indicating totipotency of the cells that were initially set up in the primary culture system (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008). Hence, the enormous regenerative potential of free-living flatworms can also be observed for parasitic species and, in the case of E. multilocularis, the complete regeneration of metacestode vesicles was obtained from dispersed cells that were not even part of an intact tissue.

Fig. 1. In vitro regeneration of E. multilocularis metacestode vesicles from primary cells. (A) Displayed is a typical culture after 3–4 weeks of cultivation. Note the extensive cell aggregates in which metacestode tissue is formed around central cavities. A complete, young metacestode vesicle is present close to the middle (arrow). (B) Scanning electron microscopic image of regenerating cell-aggregate (detail). During fixation, a developing vesicle was ruptured, thus revealing the central cavity surrounded by the growing germinal layer. Arrows indicate small cells that most probably represent stem cells. Double pointed arrow indicates a differentiating cell that fuses with the growing germinal layer. For further images of E. multilocularis primary cells, please refer to Spiliotis et al. (Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008).

On the one hand, the E. multilocularis neoblast culture system should be a highly useful tool to study metastasis formation which often occurs during prolonged infections of the intermediate host (Mehlhorn et al. Reference Mehlhorn, Eckert and Thompson1983). On the other hand, the pattern of parasite tissue formation from neoblasts in the in vitro regeneration system (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008) closely resembles the different phases of metacestode development as observed after infecting laboratory mice with oncospheres (Rausch, Reference Rausch1954; Sakamoto and Sugimura, Reference Sakamoto and Sugimura1970; Vogel, Reference Vogel1977). Hence, despite the different origin of Echinococcus neoblasts in the regeneration system (metacestode-derived) and in natural infections (oncosphere-derived), there seems to be no qualitative difference in how they react to comparable environmental conditions. I therefore suggest that metacestode-derived Echinococcus neoblasts are useful tools to study a wide variety of developmental transitions and processes, including the formation of metastases, the oncosphere-metacestode-metamorphosis, the formation of protoscoleces from the germinal layer, or even the production of proglottides by adult worms. The main challenge for the future will be to find suitable experimental in vitro conditions for each of these different contexts and settings in order to obtain data that are applicable to the in vivo situation.

ECHINOCOCCUS NEOBLASTS – WHAT NEXT?

The establishment of the neoblast regeneration system is, of course, only the first step into hypothesis-driven research on Echinococcus development. Next, it will be important to identify stem cell markers in order to track the parasite's neoblast population during vesicle generation by immunohistochemical methods and flow cytometry. This is especially important as studies on planarians have already shown that several sub-types of neoblast exist, some of which might be ‘true’ totipotent stem cells whereas others probably represent cells that are in a transition state and bound to differentiate (Eisenhoffer et al. Reference Eisenhoffer, Kang and Sanchez-Alvarado2008; Higuchi et al. Reference Higuchi, Hayashi, Hori, Shibata, Sakamoto and Agata2007; Rieger et al. Reference Rieger, Legniti, Ladurner, Reiter, Asch, Salvenmoser, Schürmann and Peter1999). The situation could be similar in Echinococcus since ultrastructural studies have regularly revealed the co-occurrence of so-called ‘light-stained’ and ‘dark-stained’ undifferentiated cells in developing metacestode tissue (Sakamoto and Sugimura, Reference Sakamoto and Sugimura1970; Sakamoto, Reference Sakamoto1981, Reference Sakamoto1982). Dark-stained undifferentiated cells apparently contain numerous polyribosomes in the cytoplasm and, according to Sakamoto, T. and Sugimura, M. (Reference Sakamoto and Sugimura1970), are differentiating into so-called ‘asteroid transforming cells’ or fuse with the syncytial germinal layer during asexual multiplication, whereas light-stained undifferentiated cells appear to be precursors of the dark-stained type. Hence, like in the neoblast populations of planarians, one type of Echinococcus neoblasts, the light-stained cells, might represent the true stem cell lineage that is self-sustained during larval development and that, by asymmetrical cell division, gives rise to dark-stained cells that either directly fuse with the growing syncytial tissue, or undergo limited rounds of self-amplification prior to differentiation into other cell types.

Classical methods of staining neoblasts in planarians involved labelling of S-phase cells through incorporation of the thymidine analogue 5′-bromo-desoxy-uridine (BrdU) or the utilization of anti-phospho-histone H3 antibodies in immunohistochemistry (Saló and Baguna, Reference Saló and Baguna2002; Nimeth et al. Reference Nimeth, Mahlknecht, Mezzanato, Peter, Rieger and Ladurner2004). The first method has already been employed by us to identify proliferating cells in E. multilocularis primary cell populations (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008) and, at least in preliminary Western blot experiments, we could detect phospho-histone H3 in Echinococcus metacestode vesicles (Hemer, S. and Brehm, K., unpublished results). While these techniques will be suitable for detecting proliferating cells in the Echinococcus neoblast regeneration system, it will not be possible to differentiate between different subpopulations of stem cells. Towards this end, it will be necessary to employ antibodies against stem cell-specific markers, of which several have been identified in planarian neoblasts (Table 1). The list includes the VasA orthologue DjvlgA (a DEAD-box family RNA helicase; Shibata et al. Reference Shibata, Umenoso, Orii, Sakurai, Watanabe and Agata1999), a member of the minichromosome maintenance protein family, DjMCM2, that is involved in the formation of pre-replication complexes during G1-S transition (Salvetti et al. Reference Salvetti, Rossi, Deri and Batistoni2000), and the proliferating cell nuclear antigen DjPCNA (Ito et al. Reference Ito, Saito, Watanabe and Orii2001). Interestingly, BLAST analyses against the first draft version of the genome predict that close orthologues to all those factors are also expressed by E. multilocularis (Table 1) and that, due to highly conserved domains, antibodies directed against the planarian proteins might also cross-react with the cestode factors. Using such antibodies in combination with anti-phospho-histone H3 antibodies and BrdU labelling in immuno-histochemical studies will most probably reveal a picture of Echinococcus stem cell dynamics that cannot be obtained by ultrastructural investigations alone. Mostly by using RNA interference (RNAi) techniques, a series of additional proteins have been identified that, by different cellular and molecular mechanisms, are involved in planarian stem cell maintenance (Table 1). Although not exclusively restricted to neoblasts, and thus not suitable as direct markers, it can be expected that their functions are also conserved in stem cell populations of parasitic flatworms. In the first draft version of the Echinococcus genome, orthologues of the majority of these factors are present (Table 1) and, by adapting RNAi and transgenic techniques to the Echinococcus neoblast regeneration system (see below), it should be possible to discern how far the molecular mechanisms of stem cell renewal and differentiation overlap, or differ, in planarians and cestodes.

Table 1. Possible stem cell markers and regulators encoded by the E. multilocularis genome.

Homology searches against the publicly available, first assembly of the E. multilocularis genome have been performed to identify orthologues of known stem cell regulatory proteins of free-living flatworms. Indicated are the protein designations (protein), the species where they have been identified as well as homologies and putative functions. Expression patterns refer to somatic stem cells (SSC), germline stem cells (GSC) and differentiated cells (diff. cells). ‘CB’ indicates that the protein specifically locates to chromatoid bodies in planarians. ‘Contig’ indicates the contig number (first assembly version) where the closest orthologue is located on the E. multilocularis genome. Dashes (–) indicate that no orthologue is present in the first assembly version. Question marks indicate that proteins with TUDOR-domains are encoded but that overall e-values are low.

One striking morphological feature that differs between flatworm neoblasts appears to be the occurrence of so-called ‘chromatoid bodies’ that are typically present in stem cells of free-living species but have, so far, never been reported for parasitic species (Reuter and Kreshchenko, Reference Reuter and Kreshchenko2004). Chromatoid bodies are composed of amorphous material and locate close to the nucleus of undifferentiated and differentiating planarian neoblasts as well as germ cells. Since chromatoid body-like structures are also present in germ cells of vertebrate and other invertebrate species such as D. melanogaster or C. elegans, an involvement of these structures in establishing and maintaining an undifferentiated, totipotent state of stem cells has already been suggested in early ultrastructural studies on planarians (Hori, Reference Hori1982). Various RNA-binding proteins such as DEAD-box RNA helicases (e.g. VasA) or members of the PIWI/Argonaute family map to the chromatoid body of many different species, including planarians (Table 1), and there is accumulating evidence that micro-RNA and RNA-decay pathways converge to exactly this structure in germ cells (Kotaja and Sassone-Corsi, Reference Kotaja and Sassone-Corsi2007). It is presently hard to understand why structures like chromatoid bodies, conserved in totipotent stem cells across phyla as different as arthropods, chordates, nemathelminths, and (free-living) flatworms should not be present in stem cells of the parasitic flatworm lineages, particularly since proteins that are typically located to these structures appear to be conserved in these organisms (Table 1). It might be that chromatoid body-like structures have so far merely been overlooked in ultrastructural studies on parasitic flatworms since their particular role in stem cell maintenance has only recently been established. Alternatively, since the formation of the chromatoid body appears to be linked to the cell cycle (Yokota, Reference Yokota2008), the neoblasts of proliferating parasite tissue might preferentially be in a state that is free of chromatoid bodies whereas ‘resting’, non-proliferating stem cells are equipped with these structures. At least in his ultrastructural studies on Echinococcus oncospheres, Swiderski (Reference Swiderski1983) described numerous ‘chromatin aggregates beneath the nuclear envelope’ in undifferentiated cells that were ‘often surrounded by small spherical granules of high electron density’. Whether these structures represent the Echinococcus type of chromatoid bodies still remains to be elucidated.

A highly interesting observation of potential relevance for chromatoid body-like structures in cestodes has been made by Fernandez et al. (Reference Fernandez, Gregory, Loke and Maizels2002) during the establishment of cDNA libraries from E. granulosus protoscoleces and metacestode tissue. These authors reported that a considerable part (>60%) of cDNA clones from conventional libraries represented polyadenylated, mitochondrial (mt) transcripts coding for ribosomal subunits of the 55S (mt) type. During a sabbatical in my laboratory, C. Fernandez later made the same observation for E. multilocularis cDNA libraries (Fernandez, C. and Brehm, K., unpublished observation). Interestingly, polyadenylated ribosome-encoding mt transcripts are also present in D. melanogaster germ plasm which is inherited by cell lineages that give rise to germ cells (Kobayashi et al. Reference Kobayashi, Sato and Hayashi2005). Furthermore, they have been identified in germinal granules of Xenopus embryos (Kashikawa et al. Reference Kashikawa, Amikura and Kobayashi2001), and they are used in ejaculated mammalian sperm for protein translation during the final maturation steps prior to fertilization (Villegas et al. Reference Villegas, Araya, Bustos-Obregon and Burzio2002; Gur and Breitbart, Reference Gur and Breitbart2008). In Drosophila, the TUDOR protein was found to be essential for the transport of mt polyA-RNAs to polar granules (Amikura et al. Reference Amikura, Hanyu, Kashikawa and Kobayashi2001a; Kobayashi et al. Reference Kobayashi, Sato and Hayashi2005) and RNAi treatment against the planarian TUDOR orthologue, Spoltud-1, resulted in an efficient depletion of neoblasts, indicating that similar mechanisms are also relevant for stem cell maintenance in free-living flatworms (Solana et al. Reference Solana, Lasko and Romero2009). Indeed, polyadenylated mt RNAs have already been specifically localized to chromatoid bodies in stem cells of planarian polyclad embryos (Sato et al. Reference Sato, Sugita, Kobayashi, Fujita, Fujii, Matsumoto, Mikami, Nishizuka, Nishizuka, Shojima, Suda, Takahashi, Himeno, Muto and Ishida2001). Taken together, these data point to an important role of mt polyA-RNAs in stem cell maintenance in different animal phyla and, due to the predominance of respective transcripts in the Echinococcus polyA transcript pool, they are probably also relevant for cestodes. This offers an excellent opportunity to identify Echinococcus neoblasts by using the characterized mt polyA-RNAs (Fernandez et al. Reference Fernandez, Gregory, Loke and Maizels2002) as probes in in situ hybridization experiments, as previously carried out in planarians (Sato et al. Reference Sato, Sugita, Kobayashi, Fujita, Fujii, Matsumoto, Mikami, Nishizuka, Nishizuka, Shojima, Suda, Takahashi, Himeno, Muto and Ishida2001). Moreover, not only are polyadenylated mt RNAs present in cytoplasmic structures of Drosophila and mammalian germ cells, they also produce 55S ribosomal subunits that are located in germinal granules and mediate the translation of a subset of nuclear mRNAs (Amikura et al. Reference Amikura, Kashikawa, Nakamura and Kobayashi2001b, Reference Amikura, Sato and Kobayashi2005; Gur and Breitbart, Reference Gur and Breitbart2008). Hence, by using antibodies against mt 55S ribosomes in immuno-histochemistry, not only neoblasts but also chromatoid body-like structures could be located in Echinococcus cell preparations. Finally, since antibiotics directed against prokaryotic (and mt) ribosomes, such as chloramphenicol or the aminoglycoside kasugamycin, have already been successfully used to affect stem cell function in Drosophila (Amikura et al. Reference Amikura, Sato and Kobayashi2005), they might also be used to study Echinococcus neoblast function in vitro, and even to inhibit parasite development in vivo during an infection. In view of these data, it might well be that the highly deleterious effects of the macrolide antibiotic clarithromycin on in vitro cultivated E. multilocularis metacestode vesicles, recently reported by Mathis et al. (Reference Mathis, Wild, Boettger, Kapel and Deplazes2005), were not only due to an inhibition of ribosomes in mitochondria, but also to stem cell-specific effects on cytosolically active mt ribosomes. In a similar way, the specific depletion of neoblasts of the germinative area could account for the profound anthelminthic effects of several different aminoglycoside antibiotics on strobilar stages of Taenia spp. (Botero, Reference Botero1970) and Hymenolepis nana (Maki and Yanagisawa, Reference Maki and Yanagisawa1985).

THE ECHINOCOCCUS METACESTODE CYTOKINE MICROENVIRONMENT AND HOST-PARASITE CROSS-COMMUNICATION

While stem cell maintenance in planarians is mainly regulated by cell-autonomous factors such as VasA-like RNA helicases and other components of chromatoid bodies, neoblast proliferation and differentiation during the regeneration process is also governed by surrounding planarian cells that provide an appropriate cytokine microenvironment to ensure correct pattern formation and integration of the newly formed tissues into established body structures. Respective signals involve, for example, cytokines of the bone morphogenetic protein (BMP)-family that are required for proper formation of the dorsoventral axis during planarian regeneration (Molina et al. Reference Molina, Saló and Cebrià2007) or ligands of the wingless-related (Wnt)-family that are involved in determining anterior-posterior polarity (Tanaka and Weidinger, Reference Tanaka and Weidinger2008). As already mentioned, the situation is somewhat different for the developing E. multilocularis metacestode that is embedded in liver tissue of the host and surrounded by immune effector cells (Fig. 2). On the one hand, it is expected that the proliferating Echinococcus tissue secretes parasite-derived cytokines and hormones into the freshly forming cavities and into the surrounding host medium. In this context, we are currently studying three members of the transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP)-family that are encoded by the E. multilocularis genome and are all expressed in the early developing metacestode (Table 2). On the other hand, in addition to Echinococcus cytokines the primary site of infection should also contain host-derived cytokines and hormones in high concentrations (Fig. 2). BMP-cytokines are, for example, regularly present in liver tissue where they regulate extracellular matrix production, regeneration and iron homeostasis (Xu et al. Reference Xu, Ji, van den Brink and Peppelenbosch2006: Babitt et al. Reference Babitt, Huang, Xia, Siodis, Andrews and Lin2007; Kinoshita et al. Reference Kinoshita, Iimuro, Otogawa, Saika, Inagaki, Nakajima, Kawada, Fujimoto, Friedman and Ikeda2007; Sugimoto et al. Reference Sugimoto, Yang, LeBleu, Soubasakos, Giraldo, Zeisberg and Kalluri2007; Truksa et al. Reference Truksa, Peng, Lee and Beutler2007). EGF- and FGF-family cytokines are strongly expressed and secreted by hepatocytes during regeneration processes (Fausto, Reference Fausto2000), which might be induced once damage has been inflicted on liver tissue either by the oncosphere or by the ongoing immune response. Insulin concentrations in mammalian hosts are highest where the portal vein meets the liver parenchyma (Shojaee-Moradie et al. Reference Shojaee-Moradie, Powrie, Sundermann, Spring, Schüttler, Sönksen, Brandenburg and Jones2000), and this is exactly the site where the oncosphere gains entry to its preferred target organ. TGF-β and related activin- and inhibin-cytokines as well as several interleukins (IL-10, IL-6, IL-4, IL-5 in the case of echinococcosis) are secreted in significant quantities by host cells in the vicinity of the developing parasite (Wellinghausen et al. Reference Wellinghausen, Gebert and Kern1999; Harraga et al. Reference Harraga, Godot, Bresson-Hadni, Mantion and Vuitton2003). This is similar to the situation in cancer initiation, where the immune response around cancer stem cells and the released cytokine milieu stimulate cell proliferation (Tysnes and Bjerkvig, Reference Tysnes and Bjerkvig2007); thus, the parasite-induced immune response around establishing metacestode vesicles, supported by hepatocyte-derived cytokines and hormones, could have an influence on parasite neoblast proliferation and tissue formation.

Fig. 2. The hormone- and cytokine-microenvironment for metacestode growth. Within the liver, young metacestode vesicles are subject to regulation by parasite-derived cytokines but also to host-derived signalling factors present in the liver or generated during the immune response. Not displayed are lipophilic hormones that are present at the site of infection and might bind to nuclear hormone receptors of the parasite.

Table 2. Receptor tyrosine- and serine/threonine-kinase signalling pathways expressed by the E. multilocularis metacestode.

* intracellular signal transducers

For this to occur, the parasite has to be equipped with signalling systems that are able to respond to host-derived cytokines, which in fact seems to be the case. During recent years, we were able to characterize a variety of evolutionarily conserved signalling systems in E. multilocularis that are structurally and functionally closely related to insulin-, EGF-, FGF-, and TGF-β/BMP-signalling systems of mammals (Table 2; Brehm et al. Reference Brehm, Spiliotis, Zavala-Góngora, Konrad and Frosch2006; Brehm and Spiliotis, Reference Brehm and Spiliotis2008b). The list includes insulin- and EGF-receptor-like receptor tyrosine kinases (RTKs) (Konrad et al. Reference Konrad, Kroner, Spiliotis, Zavala-Gongora and Brehm2003; Spiliotis et al. Reference Spiliotis, Kroner and Brehm2003) as well as a series of downstream-acting factors of the MAP kinase cascade such as orthologues to Ras-like small GTPases, a Raf-like mitogen activated protein kinase kinase kinase (MAPKKK), an Erk-like MAPK, and a member of the p38-family of MAPKs (Spiliotis and Brehm Reference Spiliotis and Brehm2004; Spiliotis et al. Reference Spiliotis, Tappe, Brückner, Mösch and Brehm2005, Reference Spiliotis, Konrad, Gelmedin, Tappe, Brückner, Mösch and Brehm2006; Gelmedin et al. Reference Gelmedin, Caballero-Gamiz and Brehm2008). Encoded by the parasite's genome are an additional insulin-receptor-like RTK, EmIR2, an FGF-receptor-like RTK, EmFR, a JUN-kinase orthologue, EmMPK3, and two MAPKKs, EmMKK1 and 2, that are currently investigated in our laboratory (Table 2). Regarding TGF-β/BMP-signalling, we have identified and characterized a type I receptor serine/threonine kinase of the Alk1 family, EmTR1 (Zavala-Gongora et al. Reference Zavala-Gongora, Kroner, Bernthaler, Knaus and Brehm2006), three receptor-activated Smad (R-Smad) transcription factors, EmSmadA-C, a common-mediator Smad (Co-Smad), EmSmadD, and a member of the SNW/Skip family of transcriptional co-regulators that interact with several of the Echinococcus Smads (Zavala-Gongora et al. Reference Zavala-Gongora, Kroner, Wittek, Knaus and Brehm2003, Reference Zavala-Gongora, Derrer, Gelmedin, Knaus and Brehm2008; Gelmedin et al. Reference Gelmedin, Zavala-Gongora, Fernandez and Brehm2005). This list will soon be complemented by a type II – family RSTK, two type I RSTKs, and an additional BR-Smad (Epping, K., Bernthaler, P. and Brehm, K., unpublished results). Evidence that the Echinococcus signalling systems are indeed able to respond to corresponding host signals has been obtained in three cases. First, we demonstrated that the insulin-receptor-like RTK EmIR can interact with host-derived insulin (Konrad et al. Reference Konrad, Kroner, Spiliotis, Zavala-Gongora and Brehm2003) which is especially interesting as we already observed significant effects of insulin on the formation of metacestode vesicles from primary cells in vitro (Konrad, C., Hemer, S. and Brehm, K., unpublished results). Second, the RSTK EmTR1 interacted with mammalian BMP2 upon expression in HEK-293 cells (Zavala-Gongora et al. Reference Zavala-Gongora, Kroner, Bernthaler, Knaus and Brehm2006). Third, exogenous addition of human EGF to in vitro cultivated metacestode vesicles resulted in a stimulation of the parasite's MAPK cascade, as demonstrated by the induced phosphorylation of the Erk-like MAPK, EmMPK1, and this was most probably mediated by the EGF-receptor-like RTK EmER (Spiliotis et al. Reference Spiliotis, Konrad, Gelmedin, Tappe, Brückner, Mösch and Brehm2006). Preliminary evidence that host-derived FGF and TGF-β also stimulate corresponding receptors in E. multilocularis has recently been obtained in our laboratory (Epping, K., Schäfer, T. and Brehm, K., unpublished results). Intense biochemical cross-communication between cytokine–cytokine receptor systems of invertebrates and vertebrates is not restricted to Echinococcus since it has already been observed for other organisms such as the trematode S. mansoni (EGF-, insulin-, and TGF-β-signalling; Beall and Pearce, Reference Beall and Pearce2001;Vicogne et al. Reference Vicogne, Cailliau, Tulasne, Browaeys, Yan, Fafeur, Vilain, Legrand, Trolet and Dissous2004; Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007) and the nematode Brugia malayi (TGF-β-signalling; Gomez-Escobar et al. Reference Gomez-Escobar, Gregory and Maizels2000). Indeed, a picture is currently emerging in which hormonal cross-communication between tissue-dwelling helminth parasites and host signalling-systems appear to be the rule, rather than an exception.

Using the neoblast regeneration system and transgenic techniques (see below), one of the major challenges of the next years will be to determine the contribution of each of the characterized RTK- and RSTK-signalling systems, and corresponding host cytokines, to stem cell maintenance and pattern formation in Echinococcus larvae. In order to obtain a comprehensive picture, components of three additional evolutionary conserved signalling systems, Delta/Notch-, Hedgehog (Hh)-, and Wnt-signalling, should be included since these have also been demonstrated to play important roles in stem cell maintenance, body patterning, and cell fate determination (Guo and Wang, Reference Guo and Wang2009). In the available E. multilocularis genome information, genes encoding respective ligands, receptors and intracellular signal transducers were identified (Table 3), indicating that all three systems are operative in the parasite. Unlike the situation with insulin-, EGF- and TGF-β/BMP-like cytokines, it has not yet been established whether vertebrate ligands of the Delta/Notch, Hh and Wnt-signalling pathways are capable of interacting with corresponding receptors of invertebrates. Furthermore, it is not known whether significant amounts of these ligands are expressed in adult liver, the primary site of Echinococcus infection, or whether they contribute to the cytokine microenvironment for parasite development. However, due to considerable intracellular cross-interaction with MAPK cascade components and Smad transcription factors, as regularly observed in other stem cell systems (Guo and Wang, Reference Guo and Wang2009), the parasite's Delta/Notch-, Hh- and Wnt pathways may significantly modify the processing of host-signals that are transmitted through RTKs and RSTKs. Furthermore, through homologous interactions with the Echinococcus frizzled-, Notch-, and patched-like receptors, these ligands are of course expected to contribute to metacestode pattern formation and differentiation.

Table 3. Predicted Wnt, Delta/Notch and Hh signalling components in the E. multilocuaris genome.

* e-value in comparison with the closest human orthologue.

To my knowledge, it has not yet been investigated whether cytokines of the adaptive or the innate immune system of the host can influence Echinococcus development in vitro. Particularly Th2-cytokines such as interleukin 4 (IL-4) as well as anti-inflammatory IL-10 are secreted in considerable amounts during active echinococcosis (Wellinghausen et al. Reference Wellinghausen, Gebert and Kern1999), and IL-10 has been demonstrated to be produced by host immune cells in the vicinity of the developing parasite (Harraga et al. Reference Harraga, Godot, Bresson-Hadni, Mantion and Vuitton2003). Although direct effects of host-derived interleukins or interferons on Echinococcus cells cannot be excluded, the situation will surely be different from the above outlined mechanism of hormonal host-parasite cross-communication involving insulin-, EGF, FGF- and TGF-β-like cytokines. In contrast to the latter hormones and cytokines (and corresponding receptors) which have arisen very early in animal evolution and are thus still present in metazoans of all phyla, ILs and IL-receptors of the adaptive immune system have first evolved in jawed vertebrates and are therefore absent in invertebrates (Kaiser et al. Reference Kaiser, Rothwell, Avery and Balu2004). At least in the present version of the E. multilocularis genome, I could find no indication for the presence of IL-or IL-receptor orthologues. Furthermore, components of the Jak/STAT (Janus-kinase/signal transducers and activators of transcription)- or the NFκB (nuclear factor kappa-light chain enhancer of activated B cells)-pathways which, in vertebrates, are collectively mediating IL-, tumor necrosis factor- and interferon-signalling (Guo and Wang, Reference Guo and Wang2009), appear to be completely absent in the Echinococcus genome. Hence, on the basis of these results, it is highly unlikely that E. multilocularis stem cells directly respond to cytokines other than TGF-β, which are released as part of innate or adaptive immune responses, or that they release interleukin orthologues in order to affect the immune system.

RELATED QUESTIONS OF INTEREST

E. MULTILOCULARIS GENOMICS AND GENETIC MANIPULATION

As befits an experimental model system, a whole genome sequencing project for E. multilocularis is currently being carried out in co-operation between the Parasite Sequencing Unit of the Wellcome Trust Sanger Centre (Hinxton, Cambridge, UK), led by Matt Berriman, and my group. After initiation of the project in 2004, extensive BAC (bacterial artificial chromosome) libraries were generated from genomic DNA that derived from the natural parasite isolate JAVA (Tappe et al. Reference Tappe, Brehm, Frosch, Blankenburg, Schrod, Kaup and Mätz-Rensing2007) and, after determination of the parasite's genome size by flow cytometry (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008), capillary sequencing to ∼4-fold coverage was carried out. After a first round of assembly, ∼19,000 sequence contigs with an average length of 15 kb were obtained which are publicly available under http://www.sanger.ac.uk/Projects/Echinococcus/. BLAST analyses of 50 randomly picked cDNAs from previously generated spliced-leader libraries (Brehm et al. Reference Brehm, Jensen and Frosch2000a) against the available contigs revealed that sequence information for all cDNAs was present although, in some cases, the cDNAs were only partially covered by the corresponding contig (Brehm, K., unpublished results). Hence, although several prominent gaps are still present, at least protein coding information appears to be covered to a great extent by the presently available sequence contigs.

On the basis of extensive sequence variations in alleles encoding antigen B (see above), it has previously been suggested that the genome of the closely related dog tapeworm E. granulosus might undergo gene conversion (Haag et al. Reference Haag, Alves-Junior, Zaha and Ayala2004). If also active in E. multilocularis, such a mechanism could severely hamper all attempts to properly assemble the genome data. I therefore carried out a preliminary analysis of the distribution of antigen B genes in the available sequence information and found a total of 7 gene copies clustered on 5 adjacent contigs (5712–5716; Fig. 3). In all cases, the genes comprised two exons, separated by introns of 66 to 130 nt, of which the first exon consistently encoded an export directing signal sequence. Two of the gene loci of the antigen B cluster apparently encoded the previously described (Mamuti et al. Reference Mamuti, Sako, Xiao, Nakaya, Nakao, Yamasaki, Lightowlers, Craig and Ito2006) isoform EmAgB8/3 (100% amino acid sequence identity). Another three encode proteins identical to EmAgB8/5, EmAgB8/4 and EmAgB8/1, while two loci display imperfect matches to previously published sequences for EmAgB8/2 (95% identical, 97% similar residues) and, again, EmAgB8/3 (92%/94%). Although further genomic analyses, supported by EST (expressed sequence tag) data, are surely necessary to assign each of the identified gene loci to reported antigen B isoforms from E. multilocularis (Mamuti et al. Reference Mamuti, Sako, Xiao, Nakaya, Nakao, Yamasaki, Lightowlers, Craig and Ito2006) and E. granulosus (Haag et al. Reference Haag, Alves-Junior, Zaha and Ayala2004), the sequence data present in the first assembly of the E. multilocularis genome indicate that the antigen B isoforms are encoded by a cluster of 7 genes, that is not interrupted by other predicted genes. Furthermore, the contig information does not contain any indication that antigen B encoding E. multilocularis genes are subject to gene conversion, as previously suggested for E. granulosus (Haag et al. Reference Haag, Alves-Junior, Zaha and Ayala2004). It should be noted that clustering of Echinococcus gene families seems not to be the rule since BLAST analyses concerning the eg95 family, which encodes host-protective antigens (Chow et al. Reference Chow, Gauci, Cowman and Lightowlers2001), revealed at least 12 family members that are dispersed over the entire genome (Brehm, unpublished observation). Taken together, the above mentioned analyses as well as the data present in tables 1 and 2, demonstrate that the presently available, preliminary genome information of E. multilocularis already serves as a highly useful tool in gene identification.

Fig. 3. The E. multilocularis antigen B cluster. Database mining with known antigen B sequences against the first E. multilocularis genome assembly version revealed the presence of 7 antigen B isoform genes located on 5 adjacent contigs. Two identical (within ORF) copies encoding EmAgB8/3, separated by a gene encoding EmAgB8/5, are present. An additional, imperfectly matching copy is designated EmAgB8/3*. Arrows indicate AgB reading frames and direction of transcription. Numbers below indicate the (minimal) distance between each reading frame on the genome. Double slashes (//) indicate gaps in the genome sequence.

Following capillary sequencing, additional rounds of unpaired and paired 454 reads as well as extensive sequencing runs using Solexa technology have meanwhile been carried out, amounting to ∼140-fold coverage. In the present (unpublished) assembly version, the sequence information is present within 1841 contigs and 597 supercontigs, with 50% of the parasite's genome assembled into 17 scaffolds of more than 1·6 Mb. Final assembly is expected by the end of 2009. Together with the sequence data from the ongoing T. solium project (http://www.taeniasolium.unam.mx/advisory.htm), which is also in an advanced stage, a detailed picture of taenid cestode genes and genomes should therefore be available in 2010. Supported by extensive EST data from both E. multilocularis and E. granulosus (Fernandez et al. Reference Fernandez, Gregory, Loke and Maizels2002), available under http://www.nematodes.org/NeglectedGenomes/Lopho/LophDB.php and http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus, genome annotation is currently ongoing in the parasite sequencing unit of the Sanger Centre and in my laboratory. Furthermore, we are currently carrying out transcriptome sequencing for a variety of different developmental stages such as early and late metacestode vesicles (with and without brood capsules) as well as dormant and activated protoscoleces, which will eventually be complemented by transcriptome analyses of oncospheres and adult worms. Taken together, these investigations will provide valuable information on developmental stage-specific gene expression patterns covering the entire life-cycle.

The major aim of the E. multilocularis genome project is to provide new ideas and concepts for hypothesis-driven research into parasite development, immunological aspects of alveolar echinococcosis, and drug design. To make effective use of this information platform it is necessary to establish robust and reliable technologies for targeted genetic manipulation. We have recently reviewed the approaches that have so far been undertaken towards this aim (Brehm and Spiliotis, Reference Brehm and Spiliotis2008a), so I will just give a brief outline of what might be achieved in short time. Transient transfection of in vitro cultivated cells using liposome-based transfection reagents has been successfully carried out (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008) and should soon be improved in a way that Echinococcus proteins can be expressed in primary cells for functional assays. Furthermore, we have demonstrated that the facultative intracellular bacterium Listeria monocytogenes, that has already been successfully used to introduce foreign DNA into mammalian cells, can also be used to effectively infect Echinococcus cells, thus providing an elegant alternative to lipofection in future attempts to manipulate parasite cells (Brehm and Spiliotis Reference Brehm and Spiliotis2008a; Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008). RNAi – approaches that have recently been established as a useful tool for targeted knock-down of genes in S. mansoni (Pearce and Freitas, Reference Pearce and Freitas2008) should also be applicable to E. multilocularis since the genome contains all necessary components (such as the DICER protein; Kiss and Brehm, unpublished results) and first protocols to effectively knock down gene expression in primary cells are already at hand (Spiliotis, M., personal communication). Finally, successful attempts into stable transfection and genomic integration of foreign DNA have been carried out in my laboratory using pantropic virus-constructs (Spiliotis, Kiss, Brehm, unpublished results) in a way very similar to previously reported DNA integration strategies for S. mansoni (Kines et al. Reference Kines, Morales, Mann, Gobert and Brindley2008). In combination with the in vitro system for parasite regeneration from primary cells (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008), stable integration by virus-based systems should, in principle, produce entirely transgenic parasite strains, and this approach is currently being used in our laboratory. As a result of these first successful attempts, a very positive prognosis can be given that E. multilocularis genes and mRNAs can be effectively manipulated for a variety of experimental approaches in the near future.

CONCLUDING REMARKS

All parasitic flatworms have their evolutionary roots in the free-living species (Olson, Reference Olson2008) and, as outlined in this review, it is expected that basic developmental mechanisms such as the maintenance and differentiation of totipotent stem cells, pattern formation, and signal transmission are largely shared between the parasitic and free-living members of the phylum. Research into the developmental biology of trematodes and cestodes can thus profit immensely from the wealth of data that has already been gathered concerning genetic control of regeneration, asexual multiplication and fecundity in planarians and other free-living flatworm model systems. Furthermore, elaborate methodological approaches such as systematic RNAi-screens of genes involved in stem cell control (Reddien et al. Reference Reddien, Bermange, Murfitt, Jennings and Sánchez-Alvarado2005), or the utilization of flow cytometry to characterize stem cell populations in planarians (Higuchi et al. Reference Higuchi, Hayashi, Hori, Shibata, Sakamoto and Agata2007), should be directly applicable and very helpful in cestode and trematode neoblast research.

In turn, can the free-living flatworm research community take profit from investigations on trematodes and cestodes? I truly think so. As exemplified by the E. multilocularis cell culture system (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008), neoblast populations of asexually multiplying larval stages such as cyclophyllidean metacestodes or schistosome sporocysts can be more advantageous for isolated cultivation than their counterparts deriving from free-living species (see Schürmann and Peter, Reference Schürmann and Peter2001), thus facilitating the establishment of cell lines that are of use for functional analyses on both parasitic and free-living flatworms. The steadily improving methods of genetic manipulation on parasitic flatworm species through virus-based transduction systems (Brehm and Spiliotis, 2008 a; Kines et al. Reference Kines, Morales, Mann, Gobert and Brindley2008), ballistic gene transfer (Wippersteg et al. Reference Wippersteg, Kapp, Kunz, Jackstadt, Zahner and Grevelding2002) or intracellular bacteria (Spiliotis et al. Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008) are principally also applicable to free-living species and can broaden the spectrum of methods for functional analyses of planarian genes. Finally, it also appears clear that comparative genomics on model species such as Schmidtea mediterranea, Schistosoma mansoni and E. multilocularis, including analyses of the gain and loss of genes between parasitic and free-living species, should be highly useful in defining the ‘core’ elements of the flatworm genome responsible for neoblast function.

At present, there is little interaction between the research communities interested in molecular and cellular biology of free-living and parasitic flatworms, which is reflected by the fact that planarians have already been used as model systems for genotoxicity testing (Lau et al. Reference Lau, Knakievicz, Prá and Erdtmann2007) or for the development of neuropharmacological (Buttarelli et al. Reference Buttarelli, Pellicano and Pontieri2008) and anti-cancer drugs (Pearson and Sanchez-Alvarado, Reference Pearson and Sanchez-Alvarado2008; Oviedo and Beane, Reference Oviedo and Beane2009) but, at least to my knowledge, never for the development of anthelminthic drugs against trematode- and cestode-borne diseases. Just as in the case of the nematode research field, where close interactions between the C. elegans- and ‘parasitic nematode’-research communities are nowadays commonplace (Gilleard Reference Gilleard2004), I therefore encourage similar networks among flatworm researchers interested in molecular genetics and developmental control of their organisms. By closing ranks, we significantly broaden our methodological repertoire, our comparative reference base, and our chain of arguments for doing research on flatworms.