The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction

Background Alveolar echinococcosis (AE) is a lethal zoonosis caused by the metacestode larva of the tapeworm Echinococcus multilocularis. The infection is characterized by tumour-like growth of the metacestode within the host liver, leading to extensive fibrosis and organ-failure. The molecular mechanisms of parasite organ tropism towards the liver and influences of liver cytokines and hormones on parasite development are little studied to date. Methodology/Principal findings We show that the E. multilocularis larval stage expresses three members of the fibroblast growth factor (FGF) receptor family with homology to human FGF receptors. Using the Xenopus expression system we demonstrate that all three Echinococcus FGF receptors are activated in response to human acidic and basic FGF, which are present in the liver. In all three cases, activation could be prevented by addition of the tyrosine kinase (TK) inhibitor BIBF 1120, which is used to treat human cancer. At physiological concentrations, acidic and basic FGF significantly stimulated the formation of metacestode vesicles from parasite stem cells in vitro and supported metacestode growth. Furthermore, the parasite’s mitogen activated protein kinase signalling system was stimulated upon addition of human FGF. The survival of metacestode vesicles and parasite stem cells were drastically affected in vitro in the presence of BIBF 1120. Conclusions/Significance Our data indicate that mammalian FGF, which is present in the liver and upregulated during fibrosis, supports the establishment of the Echinococcus metacestode during AE by acting on an evolutionarily conserved parasite FGF signalling system. These data are valuable for understanding molecular mechanisms of organ tropism and host-parasite interaction in AE. Furthermore, our data indicate that the parasite’s FGF signalling systems are promising targets for the development of novel drugs against AE.


Methodology/Principal findings
We show that the E. multilocularis larval stage expresses three members of the fibroblast growth factor (FGF) receptor family with homology to human FGF receptors. Using the Xenopus expression system we demonstrate that all three Echinococcus FGF receptors are activated in response to human acidic and basic FGF, which are present in the liver. In all three cases, activation could be prevented by addition of the tyrosine kinase (TK) inhibitor BIBF 1120, which is used to treat human cancer. At physiological concentrations, acidic and basic FGF significantly stimulated the formation of metacestode vesicles from parasite stem cells in vitro and supported metacestode growth. Furthermore, the parasite's mitogen activated protein kinase signalling system was stimulated upon addition of human FGF. The survival of metacestode vesicles and parasite stem cells were drastically affected in vitro in the presence of BIBF 1120.

Conclusions/Significance
Our data indicate that mammalian FGF, which is present in the liver and upregulated during fibrosis, supports the establishment of the Echinococcus metacestode during AE by acting on an evolutionarily conserved parasite FGF signalling system. These data are valuable for understanding molecular mechanisms of organ tropism and host-parasite interaction in AE. Furthermore, our data indicate that the parasite's FGF signalling systems are promising targets for the development of novel drugs against AE. PLOS

Introduction
The flatworm parasite E. multilocularis (fox-tapeworm) is the causative agent of alveolar echinococcosis (AE), one of the most dangerous zoonoses of the Northern hemisphere. Infections of intermediate hosts (rodents, humans) are initiated by oral uptake of infectious eggs which contain the parasite's oncosphere larval stage [1,2]. After hatching in the host intestine and penetration of the intestinal wall the oncosphere gains access to the liver, where it undergoes a metamorphotic transition towards the metacestode larval stage [3]. The E. multilocularis metacestode consists of numerous vesicles which grow infiltratively, like a malignant tumor, into the liver tissue [1][2][3]. Due to the unrestricted growth of the metacestode, blood vessels and bile ducts of the liver tissue of the intermediate host are obstructed, eventually leading to organ failure [1]. Another hallmark of AE is extensive liver fibrosis which can lead to a complete disappearance of the liver parenchyma, and which most probably involves the activation of hepatic stellate cells during chronic infection [4,5]. Surgical removal of the parasite tissue, the only possible cure, is not feasible in the majority of patients leaving benzimidazole-based chemotherapy as the only treatment option. However, benzimidazoles act parasitostatic only and have to be given for prolonged periods of time (often life-long), underscoring the need for novel treatment options against AE [1]. We previously established that E. multilocularis development and larval growth is exclusively driven by a population of somatic stem cells, the germinative cells, which are the only mitotically active cells of the parasite and which give rise to all differentiated cells [6]. Using in vitro cultivation systems for metacestode vesicles and germinative cells [7][8][9][10], we also demonstrated that host insulin fosters parasite development by acting on evolutionarily conserved receptor kinases of the insulin receptor family that are expressed by the metacestode [11]. Evidence has also been obtained that host epidermal growth factor (EGF) stimulates Echinococcus germinative cell proliferation, most probably by acting on parasite receptor tyrosine kinases (RTK) of the EGF receptor family [12,13]. These studies indicate that the interaction of hostderived hormones and cytokines with corresponding receptors of evolutionarily conserved signalling pathways that are expressed by the parasite may play an important role in AE hostparasite interaction. Although the E. multilocularis genome project already indicated that the parasite expresses receptor TK of the fibroblast growth factor (FGF) receptor family in addition to insulin-and EGF-receptors [14], no studies concerning the effects of host FGF and their possible interaction with parasite FGF receptors have been carried out to date.
FGFs are an ancient group of polypeptide cytokines that are present in diploblastic animals, in deuterostomes and, among protostomes, only in ecdysozoa (with some distantly related members in lophotrochozoa) [15,16]. Humans express 22 different FGFs of which several (FGF11 -FGF14) are not secreted and act independently of FGF receptors in an intracrine modus only [15]. The remaining FGFs act in a paracrine fashion and are typically released via N-terminal signal peptides. Notable exceptions are the prototypic FGF1 (acidic FGF) and FGF2 (basic FGF) which are ubiquitously expressed in human tissues, are the most active members of the FGF family, and are released in a signal peptide-independent manner [15]. FGFs have a key role in metazoan embryonic development and, in adults, are typically involved in regeneration processes (angiogenesis, wound healing, liver regeneration, regeneration of nervous tissue) [15]. In the liver, particularly FGF1 but also FGF2 are present as proteins in significant amounts [17], are crucially involved in tissue regeneration upon damage [18,19], and are also upregulated and released during fibrosis [20]. Secreted FGFs act through surface receptor TK of the FGF receptor family, of which four isoforms, Fgfr1-Fgfr4, are expressed by humans [15]. The mammalian FGF receptors comprise an extracellular ligand-binding domain made up of three immunoglobulin (Ig)-like domains, a transmembrane domain, and a split intracellular kinase domain. FGF binding to the cognate FGF receptors typically results in receptor dimerization, transphosphorylation and subsequent activation of downstream signalling pathways such as the Ras-Raf-MAPK (mitogen-activated protein kinase) cascade or the PI3K/Akt pathway [15].
FGF signalling pathways have, in part, already been studied in flatworms. In the free-living planarian species Dugesia japonica, two members of the FGFR TK are expressed of which DjFGFR1 exhibits three immunoglobulin-like domains in the extracellular region whereas DjFGFR2 only contains two such domains [21]. Both receptors are expressed by X-ray sensitive planarian stem cells (neoblasts) and in cephalic ganglia and an important role of these FGFRs in planarian brain formation has been suggested [21,22]. Furthermore, similar FGF receptors were also detected in stem cells of the planarian Schmidtea mediterranea [23]. In the genome of the flatworm parasite species Schistosoma mansoni, two FGFR-encoding genes were identified of which fgfrA codes for a predicted protein with two extracellular immunoglobulin domains and a split TK domain whereas the fgfrB gene product only comprises one immunoglobulin domain in the extracellular region [24]. Expression of fgfrA and fgfrB in neoblast-like somatic stem cells has been shown and evidence was obtained for an important role of both receptors in schistosome stem cell maintenance [25][26][27]. Hahnel et al. [24] also demonstrated that both receptors are enzymatically active, are expressed in the gonads of schistosomes, and are upregulated following pairing, indicating a role in parasite fertility. Interestingly, these authors also showed that treatment of adult schistosomes with FGFR inhibitors leads to a reduction of somatic neoblast-like stem cells in both genders [24].
In the present work we provide a detailed analysis of three FGFRs in the cestode E. multilocularis and show that the expression patterns of these receptors differ from those in planaria and schistosomes. We also demonstrate that all three Echinococcus FGFRs are activated in response to human FGFs and that host FGF stimulates parasite development in vitro. Finally, we also show that inhibition of FGF signalling in Echinococcus larvae drastically reduces parasite development and survival.

Organisms and culture methods
FGF stimulation and inhibitor experiments were performed with the natural E. multilocularis isolate H95 [14]. Whole mount in situ hybridization was carried out using isolate GH09 which, in contrast to H95, is still capable of producing brood capsules and protoscoleces in vitro [14]. All isolates were continuously passaged in mongolian jirds (Meriones unguiculatus) as previously described [9]. The generation of metacestode vesicles and axenic cultivation of mature vesicles was performed essentially as previously described [7,9] with media changes usually every three days. Primary cell cultures were isolated from mature vesicles of isolate H95 and propagated in vitro essentially as previously described [8][9][10] with media changes every three days unless indicated otherwise. For FGF stimulation assays, 10 nM or 100 nM of recombinant human acidic FGF (FGF1) or basic FGF (FGF2)(both from ImmunoTools GmbH, Friesoythe, Germany) were freshly added to parasite cultures during medium changes. In the case of primary cells, cultivation was usually performed in cMEM medium which is host hepatocyte-conditioned DMEM (prepared as described in [10]). For inhibitor studies, specific concentrations of BIBF 1120 (Selleck Chemicals LLC, Houston, TX, USA) were added to parasite cultures as indicated and as negative control DMSO (0.1%) was used. The formation of mature metacestode vesicles from primary cells and measurement of metacestode vesicles size was performed essentially as previously described [11].
The full-length emfr1 cDNA was cloned using as starting material the partial sequence of a cDNA of the closely related cestode E. granulosus, which encoded a FGFR-like TK domain but which lacked the coding regions for transmembrane and extracellular parts [28]. Using primers directed against the E. granulosus sequence (5'-CTA CGC GTG CGT TTT CTG ATG-3'for first PCR; 5'-CCC TCT GAT CCA ACC TAC GAG-3'for nested PCR), the 3' end of the corresponding E. multilocularis cDNA was subsequently PCR amplified from a metacestode (isolate H95) cDNA preparation using primers CD3 and CD3nest as previously described [29]. 5'-RACE was performed using the SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions using primers 5'-ACC GTA TTT GGG TTG TGG TCG-3' (first PCR) and 5'-GAA CAG GCA GAT CGG CAG-3' (touchdown PCR) as previously described [30]. The presence of an in frame TAA stop codon 110 bp upstream of the emfr1 ATG start codon indicated that the correct 5' end had been identified. In a final step, the entire emfr1 cDNA was PCR amplified from metacestode cDNA using primers 5'-GAC ACA TCT CCT TGG CCG-3' and 5'-GCG AGT TGA TAC TTT ATG AGA G-3' and cloned using the TOPO XL PCR cloning kit (Invitrogen). The sequence is available in the GenBank TM , EMBL, and DDJB databases under the accession number LT599044.
For emfr2 cloning we first identified by BLAST analyses on the published E. multilocularis genome sequence [14] a reading frame encoding a FGFR-like TKD annotated as EmuJ_000196200. Transcriptome analyses [14] and 5'_RACE experiments, however, indicated that there is actually read-through transcription between gene models EmuJ_000196300 and EmuJ_000196200. We thus designed primers 5 '-ATG TGT CTC CGA GCT CTC TG-3', binding to the 5' end regions of gene model EmuJ_000196300, and primer 5'-TTA CTC GCT CGA  TCG TGG GG-3', binding to the reading frame 3' end of gene model EmuJ_000196200, to PCR amplify the entire reading frame from metacestode cDNA. The resulting PCR fragment was subsequently cloned using the TOPO XL cloning kit (Invitrogen) and fully sequenced. The sequence is available in the GenBank TM , EMBL, and DDJB databases under the accession number LT599045.
For emfr3 cloning and sequencing we used primers directed against the CDS 5' end (5'-ATG GCA CCT AAG GTT GTG TCA GGA-3') and 3' end (5'-GCA GAT GAG TAA GAA ACC CTC-3') of gene model EmuJ_000893600 [14] for direct PCR amplification of the reading frame from metacestode cDNA. The resulting PCR fragment was subsequently cloned using the TOPO XL cloning kit (Invitrogen) and sequenced. The sequence is available in the GenBank TM , EMBL, and DDJB databases under the accession number LT599046.

BrdU proliferation assays
Proliferation of E. multilocularis metacestode vesicles and primary cells was assessed by a bromodesoxyuridine (BrdU)-based method. Axenically cultivated metacestode vesicles (2-4 mm in diameter) were manually picked and incubated in 12-well plates (Greiner BioOne, Kremsmünster, Germany; 8 vesicles per well) in DMEM medium without serum for 2 days. Freshly isolated primary cells were plated on 12-well plates and grown for 2 days under axenic conditions in conditioned DMEM (cMEM) medium with serum [8]. BrdU (SigmaAldrich, taufkirchen, Germany) as well as recombinant human FGF1 and FGF2 were added at 1mM (BrdU) and 100 nM or 10 nM (FGF1, FGF2) final concentrations as indicated. Cultures were incubated for 48 h at 37˚C under 5% CO 2 for metacestode vesicles or under nitrogen atmosphere [7,8] in the case of primary cells. Samples were analysed in duplicates in three independent experiments. As controls, metacestode vesicles or primary cells were incubated in either DMEM without serum or conditioned DMEM, without addition of FGFs.
Primary cells and metacestode vesicles were then isolated for genomic DNA analysis. In detail, vesicles and primary cells were first washed with 1xPBS, pelleted, and subsequently transferred to lysis buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0; 50 mM EDTA, pH 8.0, 0,5% SDS) supplemented with 20 μg/ml RNAse A and 0,1 mg/ml proteinase K. Samples were then incubated at 50˚C for 4 h under constant shaking for complete lysis. DNA was isolated by two rounds of phenol/chlorophorm extraction (1 vol of phenol/chlorophorm/isoamylalcohol 25:24:1). DNA was then precipitated with 2 vol of 96% ethanol and 0,1 vol of LiCl (pH 4,5) after overnight incubation at -20˚C and centrifugation at 20.000 rcf for 30 min at 4˚C and washed with 70% ethanol. The pellet was then air dried for 15 min an resuspended in 1 x TE buffer (10 mM Tris, 1 mM EDTA, pH 8,0).
The DNA was then prepared for coating onto a 96-well plate (96 well optical bottom plates, Nunc, Langenselbold, Germany). To this end, 5 μg of DNA were combined with 1 vol of Reacti-Bind DNA Coating solution (Pierce Biotechnology, Rockford, IL, USA) and mixed for 10 min. The DNA mix was then added to the microplates in duplicates and incubated overnight at room temperature with gentle agitation. The TE/Reacti-Bind DNA coating solution mix served as a negative control. Unbound DNA was removed by washing three times with 1xPBS. After blocking with 5% nonfat dry milk in 1xPBS for 1 h at room temperature and extensive washing with 1xPBS, 100 μl of anti-BrdU-POD (Cell Proliferation ELISA, BrdU; Roche Applied Science, Mannheim, Germany) was added and incubated for 90 min at room temperature. After incubation, microplates were washed three times with 1xPBS buffer before substrate solution (Cell Proliferation ELISA, BrdU; Roche Applied Science, Mannheim, Germany) was added and the wells were incubated for 60 min. Stop-solution (25 μl of 1 M H 2 SO 4 ) was added and absorbance of the samples was measured using an ELISA reader at 450 nm.

In situ hybridization protocols
Coding sequences of FGF receptors from E. multilocularis were amplified by RT-PCR and cloned into the vectors pDrive (Qiagen) or pJET1.2 (Thermo Fisher). In the case of emfr2, the full length coding sequence was amplified using primers 5'-ATG TGT CTC CGA GCT CTC TG-3'(forward) and 5'-TTA CTC GCT CGA TCG TGG GG-3' (reverse), whereas partial coding sequences were amplified for emfr1 (using forward primer 5'-GCA GTG GGC GTC TTC TTT CAC-3' and reverse primer 5'-GTA AAT GTG GGC CGA CAC TCA G-3') and for emfr3 (using forward primer 5'-TTG CCC AGT CAT CCG CTA CAA G-3' and reverse primer 5'-GCA AGC GGT CAT GAG GCT GTA G-3'). The recombinant plasmids were used for in vitro transcription of digoxigenin-labelled RNA probes as previously described [6]. These probes were used for fluorescent WMISH of in vitro cultured E. multilocularis larvae as described in [6]. Control WMISH experiments using the corresponding sense probes were always negative.

Xenopus oocyte expression assays
For expression in the Xenopus system, the emfr1, emfr2, and emfr3 coding sequences without predicted signal peptide information were cloned into the pSecTag2/Hygro expression system (ThermoFisher Scientific, Germany) leading to an in frame fusion of the Igk leader sequence (provided by the vector system) and the E. multilocularis FGF receptor sequences under control of the T7 promoter. Capped messenger RNAs (cRNA) encoding EmFR1, EmFR2 and EmFR3 were then synthesized in vitro using the T7 mMessage mMachine Kit (Ambion, USA). Microinjection of EmFGFR cRNAs (60 ng in 60 μl) was performed in stage VI Xenopus laevis oocytes according to the procedure previously described [31]. Following 48h of receptor expression, human FGF1 or FGF2 (R & D systems, UK) were added to the extracellular medium at the final concentration of 10 nM. cRNA of Pleurodeles FGFR1 identified as homologous to human receptor [32] was a gift of Shi D.L. (CNRS UMR 722, Paris VI) and was used as a positive control.
In some experiments, BIBF1120 (stock solution 10mM in DMSO, Selleck Chemicals LLC) was added (0.1 to 20 μM final concentration) 1 h before the addition of 10 nM FGF1 or FGF2 on EmFR1, EmFR2, EmFR3 and Pleurodeles FGFR1 expressing oocytes. Following 15 h of FGF1 or FGF2 stimulation, oocytes were analyzed for their state of progression in the cell cycle. The detection of a white spot at the animal pole of the oocyte attested to G2/M transition and GVBD. Non-injected oocytes treated with progesterone (10 μM) were used as positive controls of GVBD. For each assay, sets of 20-30 oocytes removed from 3 different animals were used.
Dead kinase (TK-) receptors were obtained by site-directed mutagenesis of the EmFR1, EmFR2 and EmFR3 constructs. The active DFG sites present in EmFR1 (D 442 FG), EmFR2 (D 647 FG) and EmFR3 (D 701 FG) were replaced by an inactivating motif (DNA) as described in [31].

MAPK cascade activation assays
Axenically cultivated metacestode vesicles of about 0.5 cm in diameter were incubated in DMEM medium with or without 10% FCS for 4 days. Vesicles cultivated without FCS were subsequently incubated with 10 nM FGF1 (aFGF) or 10 nM FGF2 (bFGF) for 30 sec, 60 sec or 60 min. Immediately after stimulation, vesicles were harvested, cut by a scalpel to remove cyst fluid and then subjected to protein isolation as described previously [33]. Isolated protein lysates were then separated on a 12% acrylamide gel and analysed by Western blotting using a polyclonal anti-Erk1/Erk2 antibody (ThermoFisher Scientific; #61-7400), recognizing Erk-like MAP kinases in phosphorylated and non-phosphorylated form, as well as a polyclonal antibody against phospho-Erk1/Erk2 (ThermoFisher Scientific; #44-680G), specifically directed against the double-phosphorylated (activated) form of Erk1/Erk2 (Thr-185, Tyr-187). We had previously shown that these antibodies also recognize the Erk-like MAP kinase EmMPK1 of E. multilocularis in phosphorylated and non-phosphorylated form [33]. As secondary antibody, a peroxidase-conjugated anti-mouse IgG antibody (Dianova, Hamburg, Germany) was used. In inhibitor experiments, axenically cultivated metacestode vesicles were incubated with either 5 μM or 10 μM BIBF 1120 for 30 min and then processed essentially as described above.

Quantitative reverse transcription PCR (qRT-PCR)
qRT-PCR experiments have been performed as previously described by del Puerto et al. [34] with several modifications. Briefly, RNA samples were prepared with TRI-reagent and the DirectzolTM RNA Mini Prep Kit (Zymo Research, USA). Prior to RNA-isolation, in vitro cultured metacestode vesicles were deprived of stem cells by treatment with hydroxyurea (HU) and the Polo-like kinase inhibitor BI 2536 essentially as previously described by Koziol et al. [6] and Schubert et al. [35], respectively. Reverse transcription was performed with Omniscript RT Kit (Qiagen, Germany) from 500 ng of RNA using oligo dT23 as primers according to the manufacturer's instructions. qRT-PCRs were then performed using primers Forward F_emfr1_qPCR (5'-CCG TAT GAA GGG AAA TGG TCG TGT T-3') and Reverse R_emfr1_qPCR (5'-TGG TGA ATC GCC AAG GCT GAA A-3') for emfr1, Forward F_emfr2_qPCR (5'-GGG AAT TTC CAA GGT CAT CAG GGA C-3') and Reverse R_emfr2_qPCR (5'-ATC GTG GGG GCA CAA CAT AAT TGC-3') for emfr2, as well as Forward F_emfr3_qPCR (5'-GTC TAC CTT GAG GAA ATT GCT GTG GTC-3') and Reverse R_emfr3_qPCR (5'-CGT GAG GAA TGA CGC AGG C-3') for emfr3. As a control gene for normalization, the constitutively expressed gene elp was used with primers as described previously [34]. qRT-PCR conditions and primer amplification efficiency control experiments were performed essentially as previously described [34]. The relative expression of emfr genes in each sample was estimated using the delt-delta-Ct method [36], normalising with elp expression levels using the StepOne software (Thermo Fisher). All experiments were carried out as technical triplicates.

Computer analyses and statistics
Amino acid comparisons were performed using BLAST on the nr-aa and swissprot database collections available under (https://www.genome.jp/). Genomic analyses and BLAST searches against the E. multilocularis genome [14] were done using resources at (https://parasite. wormbase.org/index.html). CLUSTAL W alignments were generated using MegAlign software (DNASTAR Version 12.0.0) applying the BLOSUM62 matrix. Domain predictions were carried out using the simple modular architecture research tool (SMART) available under (http:// smart.embl-heidelberg.de/) as well as PROSITE scans available under (https://prosite.expasy. org/scanprosite/). Two-tailed, unpaired student's T-tests were performed for statistical analyses (GraphPad Prism, version 4). Error bars represent standard error of the mean. Differences were considered significant for p-values below 0.05 (indicated by � ).

Ethics statement
All experiments were carried out in accordance with European and German regulations on the protection of animals (Tierschutzgesetz). Ethical approval of the study was obtained from the local ethics committee of the government of Lower Franconia (permit no. 55.2 DMS 2532-2-354).

The E. multilocularis genome encodes FGF receptor kinases but no canonical FGF ligands
By cDNA library screening and mining of the available E. multilocularis genome sequence we identified a total of three Echinococcus genes encoding members of the FGFR family of RTK. A partial cDNA for a gene encoding a TK with homology to FGFRs was previously cloned for E. granulosus [28] and by RT-PCR amplification of metacestode cDNA as well as 5'-RACE, the entire cDNA of the E. multilocularis ortholog, designated emfr1 (E. multilocularis fibroblast growth factor receptor 1), was subsequently cloned. As shown in Fig 1, the encoded protein, EmFR1, contained an N-terminal export directing signal peptide, followed by one single Iglike domain, a transmembrane region, and an intracellular TK domain (Figs 1 and S1). In the recently released E. multilocularis genome information [14], this gene was correctly predicted on the basis of genome and transcriptome data (E. multilocularis gene designation: EmuJ_000833200). In the upstream genomic regions of emfr1, no information encoding potential Ig-like domains was identified which, together with the presence of a signal peptide A second gene encoding a TK with significant homology to known FGFRs was identified on the available E. multilocularis genome sequence [14] under the annotation EmuJ_000196200. The amino acid sequence of the predicted protein only contained an intracellular TK domain, a transmembrane region, and one extracellular Ig-like domain, but no putative signal peptide. We therefore carried out 5'-RACE analyses on a cDNA preparation deriving from protoscolex RNA and identified the remaining 5' portion of the cDNA, which contained one additional Iglike domain and a predicted signal peptide. In the genome annotation, these remaining parts were wrongly annotated as a separate gene under the designation EmuJ_000196300. Hence, the second FGFR encoding gene of E. multilocularis, emfr2, encoding the protein EmFR2, actually comprises the gene models EmuJ_000196300 and EmuJ_000196200 of the genome sequence. EmFR2 thus comprises a signal peptide, two extracellular Ig-like domains, a transmembrane region, and an intracellular TKD (Figs 1 and S1). The TKD contained all residues critical for TK activity (S2 Fig) and, in SWISS-PROT BLASTP analyses, showed highest similarity to two FGF receptor kinases of the flatworm Dugesia japonica (45% identical, 65% similar residues), to the S. mansoni receptor FGFRB (55%, 68%) and to human FGFR3 (48%, 62%).
The third FGF receptor encoding gene of E. multilocularis, emfr3, was identified under the designation EmuJ_000893600 and was originally listed as an ortholog of the tyrosine protein kinase Fes:Fps [14]. However, unlike the Fes:Fps kinase which contains FCH and SH2 domains, the EmuJ_000893600 gene product, EmFR3, comprised an N-terminal signal peptide, two extracellular Ig-like domains, a transmembrane region, and an intracellular TKD (Figs 1 and S1), in which 22 of 30 highly conserved residues of TK are present at the corresponding position (S2 Fig). Furthermore in SWISS-PROT BLASTP analyses the EmFR3 TKD displayed highest similarity to several vertebrate FGF receptors and to human FGFR2 (32%, 47%). We thus concluded that EmuJ_000893600 actually encoded a third Echinococcus FGF receptor TK.
Apart from emfr1, emfr2, and emfr3, no further genes were identified in the E. multilocularis genome which displayed clear homology to known FGFR TKDs and which contained characteristic IG domains in the extracellular portions. In vertebrates, structural homology has been described between the TKDs of the receptor families of FGF receptors, the vascular endothelial growth factor (VEGF) receptors, and the platelet-derived growth factor (PDGF) receptors, which also contain varying number of Ig domains in the extracellular parts [37]. Furthermore, VEGF receptor-like molecules have also been described in invertebrates such as Hydra [37]. We therefore carried out additional BLASTP searches on the E. multilocularis genome using human VEGF-and PDGF-receptors as queries, but only obtained significant hits with the above mentioned TKDs of EmFR1, EmFR2, and EmFR3. These data indicated that members of the VEGF-and PDGF-receptor families are absent in Echinococcus, as has already been described for the closely related schistosomes [24].
Genes encoding canonical FGF ligands have so far neither been identified in genome projects of free-living flatworms [38], nor in those of trematodes [39] or cestodes [14]. In vertebrates [15] as well as several invertebrate phyla [40][41][42], however, canonical FGF ligands are clearly expressed. To investigate the situation more closely, we carried out BLASTP and TBLASTN analyses against the predicted proteins and E. multilocularis contig information, respectively, using FGF ligand sequences of insect, nematode, and cnidarian origin as queries.
The product of only one E. multilocularis gene, EmuJ_000840500 (annotated as 'conserved hypothetical protein') showed certain similarity to these FGF ligands and according to SMART protein domain analyses could contain a FGF-ligand domain between amino acids 166 and 258, although this prediction was clearly below the prediction threshold and of low probability (E-value: 817). No export directing signal peptide was predicted for the EmuJ_000840500 protein, as would be typical for FGF ligands. Furthermore, although EmuJ_000840500 had clear orthologs in the cestodes Taenia solium (TsM_000953800) and Hymenolepis microstoma (HmN_000558500), none of these gene models had any prediction of an FGF-ligand domain in SMART analyses (nor predicted signal peptides). We thus considered it highly unlikely that EmuJ_000840500 encodes a so far not identified flatworm FGF-ligand.
Taken together, our analyses indicated that E. multilocularis contains genomic information for three members of the FGFR family of RTK with either one (EmFR1) or two (EmFR2, EmFR3) extracellular Ig-like domains, of which one, EmFR3, showed higher divergence within the TKD as it contained only 22 of otherwise 30 highly conserved amino acid residues of TK. On the other hand, no members of the VEGF-and PDGF-receptor families are encoded by the E. multilocularis genome, nor does it contain genes coding for canonical FGF-ligands.

Expression of FGF receptors in E. multilocularis larval stages
To investigate gene expression patterns of the Echinococcus FGF receptors in parasite larvae, we first inspected Illumina transcriptome data for parasite primary cells after 2 and 11 days of culture (PC2d, PC11d, respectively), metacestode vesicles without or with brood capsules (MC-, MC+, respectively), as well as protoscoleces before or after activation by low pH/pepsin treatment (PS-, PS+, respectively), that had be produced during the E. multilocularis genome project [14]. According to these data, emfr1 was moderately expressed in primary cells, metacestode vesicles, and protoscoleces (S3 Fig). Likewise, emfr2 was expressed in all stages, but very lowly in primary cells, somewhat more in metacestode vesicles, and highest in protoscoleces. emfr3, on the other hand, was low to moderately expressed in primary cells, low in metacestode vesicles, and highest in protoscoleces (S3 Fig). Since primary cell preparations are characterized by a much higher content of germinative (stem) cells than metacestode vesicles [6], these expression patterns could indicate that emfr3 is stem cell-specifically expressed. We therefore carried out quantitative RT-PCR experiments on cDNA preparations from in vitrocultivated metacestode vesicles (MV) versus metacestode vesicles after treatment with hudroxyurea (MV-HU) or the Polo-like kinase inhibitor BI 2536 (MV-BI), in which the stem cell population had been selectively depleted [6,40]. While emfr1 expression levels were equal in MV when compared to MV-HU and MV-BI, both emfr2 and emfr3 transcripts were significantly reduced in MV-HU and MV-BI (S4 Fig). This indicated that emfr1 does not have a typical stem cell-specific expression pattern whereas emfr2 and emfr3 could be expressed in the parasite's stem cells or their immediate progeny (since HU-and BI 2536-treatment has to be carried out for at least one week [6,34]).
To clarify the situation we carried out whole-mount in situ hybridization experiments on metacestode vesicles according to recently established protocols [6,43,44]. In these experiments, proliferating parasite stem cells were labeled by incorporation of the nucleotide analog EdU, which was combined with detection of gene transcripts by using fluorescently labeled probes. According to these experiments, emfr1 was expressed at low levels throughout the germinal layer and the signal was somewhat higher during the development of protoscoleces (Fig  2), maybe due to higher cell density in the protoscolex. The intensity of the signal was heterogenous, but no clear pattern could be discerned and it was too low to clearly determine the percentage of positive cells.
emfr2, on the other hand, was specifically expressed in a population of small sized cells, which comprised 1.7% to 6.3% of all cells in the germinal layer (Fig 3A). None of these emfr2 + cells were also EdU + , indicating that they were post-mitotic [6]. During initial protoscolex development, emfr2 + cells accumulated in the peripheral-most layer of cells, as well as in the anterior-most region (which will form the rostellum), and in three longitudinal bands of cells in the interior of the protoscolex buds ( Fig 3B). Again, practically none of the emfr2 labelled cells was EdU + (less than 1% of emfr2 + cells were EdU + ; Fig 3C). Later during development, some emfr2 + cells were found in the protoscolex body, but most accumulated in the developing rostellum and the suckers (Fig 3D). Importantly, emfr2 expression was restricted to the sucker cup, where cells are differentiating into em-tpm-hmw + muscle cells [6], and not the sucker base, where EdU + germinative cells accumulate [6] (Fig 3D). Taken together, these data indicate that emfr2 is expressed in post-mitotic cells, of which many are likely to be differentiating or differentiated muscle cells.
Finally, emfr3 was expressed in very few cells in the germinal layer (less than 1% of all cells, although the number is difficult to estimate since they were absent in most random microscopy fields) (Fig 4A). emfr3 + cells accumulated in small numbers during brood capsule and protoscolex development (Fig 4B). The emfr3 + cells had a large nucleus and nucleolus and, thus, had the typical morphology of germinative cells (S5 Fig). At the final stages of protoscolex development, few emfr3 + cells were present in the body region and some signals were also present in the rostellar region (S5 Fig). In the developing protoscolex and in the germinal layer, emfr3 + EdU + double positive cells were found (Fig 4). These data indicated that emfr3 is expressed in a very small number of proliferating cells with the typical morphology of germinative cells.
In summary, the three E. multilocularis FGF receptor genes showed very different expression patterns in metacestode and protoscolex larval stages. While emfr1 was lowly expressed in cells that are dispersed throughout the germinal layer, emfr2 displayed an expression pattern indicative of differentiating/differentiated muscle cells. emfr3, on the other hand, appeared to be expressed in a minor sub-population of germinative cells.

Host FGF stimulates E. multilocularis larval proliferation and development
Since E. multilocularis larvae do not express canonical FGF ligands (see above), but usually develop in an environment in which FGF1 and FGF2 are abundant [17], we next tested whether host-derived FGF ligands can stimulate parasite development in vitro. To this end, we  employed two different cultivation systems which we had previously established [7][8][9][10]. In the axenic metacestode vesicle cultivation system, mature metacestode vesicles of the parasite are cultivated in the absence of host cells under reducing culture conditions (e.g. low oxygen) [7,9]. In the primary cell cultivation system [8,10], axenically cultivated metacestode vesicles are digested to set up cell cultures which are highly enriched in parasite germinative (stem) cells (~80% [6]), but which also contain certain amounts of differentiated cells such as muscle or nerve cells. In the primary cell cultivation system, mature metacestode vesicles are typically formed within 2-3 weeks, which is critically dependent on proliferation and differentiation of the germinative cell population [6] in a manner highly reminiscent of the oncosphere-metacestode transition [3].
As shown in Fig 5A, the exogenous addition of 10 nM FGF1 to mature metacestode vesicles already resulted in an elevated incorporation of BrdU, indicating enhanced proliferation of parasite stem cells, which was even more pronounced in the presence of 100 nM FGF1. In the case of FGF2, addition of 100 nM resulted in enhanced BrdU incorporation in a statistically significant manner (Fig 5A). Likewise, metacestode vesicles cultured for 4 weeks in the presence of 10 nM FGF1 or FGF2 displayed a considerably larger volume (about two-fold) when compared to non-FGF-stimulated vesicles (Fig 5B).
In the primary cell cultivation system, 100 nM concentrations of host ligands had to be added to observe statistically significant effects. Again, the incorporation of BrdU by primary cell cultures was stimulated in the presence of host-derived FGF ligands (Fig 5C), as was the formation of mature metacestode vesicles from primary cell cultures (Fig 5D).
Taken together, these results indicated that host-derived FGF ligands, and in particular FGF1, can stimulate cell proliferation and development of E. multilocularis primary cell cultures and mature metacestode vesicles.

The E. multilocularis FGF receptors are activated by host-derived FGF ligands
Having shown that host-derived FGF ligands can stimulate parasite proliferation and development in vitro, we were interested whether these effects might be mediated by one or all three of the parasite's FGF receptors which are expressed by the metacestode larval stage. To this end, we first made use of the Xenopus oocyte expression system in which the activity of heterologously expressed protein kinases can be measured by germinal vesicle breakdown (GVBD). This system has previously been used to measure the activities of the TKD of schistosome FGF receptors [24], as well as the host-EGF (epidermal growth factor) dependent activation of a schistosome member of the EGF receptor family [31]. We, thus, expressed Pleurodeles FGFR1 (as a positive control), which is highly similar to human FGFR1 [32], and all three parasite FGF receptors in Xenopus oocytes, which were then stimulated by the addition of 10 nM FGF1 or FGF2. As negative controls, we also expressed kinase-dead versions of all Echinococcus FGF receptors in Xenopus oocytes.
As can be deduced from Table 1, control (non-stimulated) oocytes were negative for GVBD, whereas progesterone-stimulated oocytes displayed 100% vesicle breakdown. Expression of FGFR1 did not yield GVBD but, after stimulation with 10 nM FGF1, 100% of oocytes underwent GVBD, indicating stimulation of the Pleurodeles receptor by FGF1 (as expected). Upon expression of any of the parasite receptors in Xenopus oocytes, no GVBD was observed when no ligand was added. The addition of 10 nM FGF1 to these receptors, however, resulted in 100% GVBD for EmFR1 and EmFR2, as well as to 80% GVBD in the case of EmFR3. In the case of human FGF2 (10 nM), 90% GVBD was observed for EmFR1 and EmFR3, and 85% for EmFR2. No GVBD was observed upon addition of 10 nM FGF1 or FGF2 when the kinase-dead versions of the parasite receptors were expressed (S6 Fig). These data clearly indicated that all three parasite receptors were responsive to host derived FGF ligands (albeit to somewhat different extent) and that the kinase activity of the parasite receptors was essential to transmit the signal. We also measured the phosphorylation state of the parasite FGF receptors upon addition of exogenous FGF1 and FGF2 (10 nM each) to Xenopus oocytes and obtained significantly induced levels of tyrosine phosphorylation in all three cases (S6 Fig). Taken together, these data indicated that all three parasite FGF receptors were activated by binding of host-derived FGF1 and FGF2, which was followed by auto-phosphorylation of the intracellular kinase domain and downstream transmission of the signal to the Xenopus oocyte signaling systems which induce GVBD. Host FGF stimulates Echinococcus larval development

Inhibition of the E. multilocularis FGF receptors by BIBF 1120
The small molecule compound BIBF 1120 (also known as Nintedanib or Vargatef) is a wellstudied and highly selective, ATP-competitive inhibitor of mammalian members of the FGF-, VEGF-, and PDGF-receptor families with very limited affinity to other RTK [45,46]. As a possible agent to selectively inhibit FGF RTK activities in the parasite, we measured the effects of BIBF 1120 on EmFR1, EmFR2, and EmFR3 upon expression in the Xenopus oocyte system. As can be deduced from Table 1, a concentration of 1 μM of exogenously added BIBF 1120 already diminished the activity (after stimulation with 10 nM FGF1) of FGFR1 to 40%, and led to a complete block of kinase activity upon addition of 10 μM BIBF 1120. In the case of EmFR1 and EmFR2, 1 μM BIBF1120 also led to a marked decrease of receptor kinase activity, although to a somewhat lower extent than in the case of FGFR1. In the presence of 10 μM BIBF 1120, on the other hand, the activities of EmFR1 and EmFR2 were completely blocked. In the case of EmFR3, exogenous addition of 1 μM BIBF 1120 had only slight effects on GVBD, whereas 10 μM BIBF 1120 reduced the activity to less than 50% and 20 μM BIBF 1120 completely blocked TK-dependent GVBD (Table 1). Upon addition of 20 μM BIBF 1120, TK activity of all receptors tested was completely inhibited.
Taken together, these data indicated that all three Echinococcus FGF RTK were affected by BIBF 1120, although in all three cases higher concentrations of the inhibitor were necessary to completely block TK activity when compared to FGFR1. BIBF 1120 treatment had the lowest effects on the activity of EmFR3.

BIBF 1120 inhibits E. multilocularis larval development
We next tested the effects of different concentrations of BIBF 1120 on parasite development and survival in the primary cell and metacestode vesicle culture systems. As shown in Fig 6A, the addition of 1-10 μM BIBF 1120 had clear concentration-dependent effects on mature metacestode vesicle survival which, after cultivation for 18 days, led to about 20% surviving vesicles in the presence of 1 μM BIBF 1120, 10% surviving vesicles in the presence of 5 μM BIBF 1120, and no survival when 10 μM BIBF 1120 was applied. To test whether the metacestode vesicles were indeed no longer capable of parasite tissue regeneration, we set up primary cell cultures from microscopically intact vesicles which had been treated with 5 μM BIBF 1120 for 5 days (90% intact vesicles) and let the cultures recover in medium without inhibitor. In these cultures, however, we never observed the formation of mature vesicles, indicating that either the parasite's stem cell population, and/or other cell types necessary for parasite development in the primary cell culture system, were severely damaged after BIBF 1120 treatment. We then also tested the effects of BIBF 1120 on fresh primary cell cultures from previously untreated metacestode vesicles. As shown in Fig 6B, a concentration of 1 μM BIBF 1120 had no effect on the formation of metacestode vesicles from these cultures, whereas vesicle formation was completely blocked in the presence of 5 μM or 10 μM BIBF 1120.
Altogether, these results clearly indicated detrimental effects of BIBF 1120 on parasite development already at concentrations as low as 5 μM. Since the parasite does not express known alternative targets for BIBF 1120, such as VEGF-or PDGFR-receptors, we deduced that these effects are due to the inhibition of one or more of the parasite's FGF RTK.

Host FGF ligands stimulate Erk signaling in E. multiocularis
One of the major downstream targets of FGF signaling in other organisms is the Erk-like MAPK cascade, a complete module of which we had previously identified in E. multilocularis [33,47,48]. In particular, we had previously shown that the phosphorylation status of the parasite's Erk-like MAP kinase, EmMPK1, can be measured by using antibodies against the phosphorylated form of the human Erk-kinase [33]. To investigate whether exogenously added host FGF can affect the E. multilocularis Erk-like MAPK cascade module, we first incubated mature metacestode vesicles for 4 days in serum-free medium (which has no effect on vesicle integrity [29]) and then stimulated these vesicles for 30 sec, 60 sec, and 60 min with 10 nM FGF1 and FGF2. As shown in Fig 7A, FGF1 treatment had a clear effect of EmMPK1 phosphorylation already after 30 sec of exposure. In the case of FGF2, the effect was still measurable, but clearly less pronounced than in the case of FGF1 (Fig 7A). We then also measured the effects of BIBF 1120 treatment on EmMPK1 phosphorylation. To this end, metacestode vesicles were incubated in hepatocyte-conditioned medium and were then subjected to BIBF 1120 treatment (5 μM, 10 μM) for 30 min. As shown in Fig 7B, this led to diminished phosphorylation of EmMPK1 when 10 μM BIBF 1120 was used.
Taken together, these data indicated that, like in mammals and other invertebrates, the E. multilocularis Erk-like MAPK cascade can be activated through FGF signaling, initiated by exogenously added, host-derived FGF ligands.

Discussion
An important difference between parasitic helminths and all other infectious agents (excluding viruses) is that these organisms are evolutionarily relatively closely related to their vertebrate or invertebrate hosts with which they share an ancestor that has lived around 500 to 600 million years ago. Since all metazoans share evolutionarily conserved signalling systems for cellcell communication, this opens the possibility for host-parasite cross-communication involving evolutionarily conserved cytokines of one partner (e.g. the host) and cognate receptors of the other (e.g. the parasite), which would be of particular relevance for systemic helminths that infect host organs. Several previous studies indicated that this type of host-pathogen interaction is indeed important in helminth infections. In the E. multilocularis system, which develops in close association with host liver tissue, we previously demonstrated that host insulin stimulates parasite development and growth via acting on evolutionarily conserved insulin-receptor TK that are expressed by the parasite [11]. A similar type of cytokine receptor interaction appears to involve epidermal growth factor (EGF)-like cytokines and cognate parasite receptors, of which three (EmER, EmERb, EmERc) are expressed by E. multilocularis larvae [3,12]. were incubated for 18 days in the presence of 0.1% DMSO or BIBF 1120 in different concentrations (as indicated) and the structural integrity of the vesicles was monitored. Structurally intact vesicles (B) and damaged vesicles (C) are shown to the right. The experiment was repeated three times in duplicates. D, freshly isolated E. multilocularis primary cells were cultured for 21 days in cMEM medium in the presence of DMSO (0.1%) or BIBF 1120 at different concentrations (as indicated). After 21 days, newly formed metacestode vesicles were counted. The DMSO control for each of the three independent experiments was set to 100%. Microscopic images (25x magnification) of cultures after 2 weeks with DMSO (E) or 10 μM BIBF 1120 (F) are shown to the right.
As recently shown by Cheng et al. [13], host-derived EGF is able to stimulate germinative cell proliferation in in vitro cultivated parasite larvae and can stimulate at least one of the parasite EGF-receptors, EmER, when heterologously expressed in Xenopus oocytes. Although the parasite itself expresses several EGF-like molecules [14], which likely act on its EGF receptors, these data indicate that host-EGF could act as an additional stimulus, particularly in response to liver tissue damage as it is inflicted upon entry of the parasite into the host liver [4]. Apart from insulin-and EGF-signalling systems, host-parasite interactions in larval cestode infections might also involve the family of transforming growth factor (TGF)-β/bone morphogenetic protein (BMP)-family of cytokines since host TGF-β has very recently been shown to stimulate larval growth of the cestode Taenia crassiceps in vitro and was found to interact with parasite TGF-β receptors [49]. In a similar way, human BMP2 was reported by us to interact with an E. multilocularis BMP receptor [50], although no direct effects of host BMP on parasite development were yet observed. Like in cestodes, the occurrence of insulin-and EGF-receptor TK as well as TGF-β/BMP serine/threonine kinases with the capability of interacting with respective human hormones/cytokines was reported for schistosomes [31,[51][52][53][54][55], and stimulatory effects of host EGF on the development of schistosome snail stages were observed [31]. In the present work, we extend the list of respective host-parasite cross-communication systems to the FGF-family of host cytokines and cognate FGF RTK which, to our knowledge, have never before been addressed in this context.
We herein clearly show that mammalian FGF1 and FGF2 stimulate the development of E. multilcularis metacestode vesicles from cultivated parasite primary cells, which are highly enriched in germinative (stem) cells [6]. Furthermore, we show that human FGF also stimulates proliferation and growth of mature metacestode vesicles. Both FGF1 and FGF2 are abundantly present in mammalian liver tissue where they are mostly released upon liver cell damage and during regeneration processes [17][18][19][20]. Although the precise amounts of host FGF in periparasitic lesions of E. multilocularis infected mice has not been measured to date, it is very likely that the early establishing metacestode is encountering considerable amounts of these cytokines since extensive damage to liver tissue is observed not only in chronically infected mice but also in early stages of the infection [4]. The early infectious stage is critical in the establishment of the parasite since the invading oncosophere is not yet producing the laminated layer (LL), an important structure that protects the parasite from direct actions of immune cells [56]. The laminated layer surrounds mature metacestode vesicles (established after 1-2 weeks after invasion) in the chronic phase of the disease. The stimulation of parasite development from stem cells towards mature metacestode vesicles by host FGF, as observed in our cultures, could thus help the parasite to abridge this critical phase and to successfully establish within the liver. Chronic AE is characterized by extensive liver fibrosis, particularly in the peri-parasitic area [57] and is thought to be mediated by hepatic stellate cells [5], which greatly upregulate FGF release during liver regeneration and fibrosis induction [20]. Hence, not only in the initial phase of parasite establishment, but also in the chronic phase of AE, the E. multilocularis metacestode should be in contact with elevated levels of host FGF that can continuously support parasite growth and proliferation.
Using the Xenopus expression system we clearly showed that all three identified FGF receptors of E. multilocularis are functionally active kinases that are capable of inducing GVBD when properly stimulated. We also demonstrated that all three Echincoccus kinases are responsive to human FGF1 and FGF2, albeit to somewhat different extent. While 10 nM FGF1 fully stimulated both EmFR1 and EmFR2, EmFR3 was less activated than the other receptors by both FGFs. This does not necessarily indicate, however, that human FGFs bind less well to EmFR3 than to the other two receptors. Instead, EmFR3 might be activated to a lesser extent since two tyrosine residues, Y653 and Y654, which in human FGFR1 are necessary for full activation of the receptor [58], are conserved in EmFR1 and EmFR2 but absent in EmFR3 (S2 Fig). In any case, our data clearly show that particularly human FGF1, but also human FGF2, are capable of activating the parasite receptors. Since the parasite apparently does not produce intrinsic FGF ligands, the only canonical FGFs it encounters during liver invasion are host derived. It is, thus, logical to assume that the effects of FGF1 and FGF2 on parasite growth and development are mediated by one, two or all three Echinococcus FGF receptors. In line with this assumption is the stimulation of the parasite's Erk-like MAPK cascade module upon exogenous addition of host FGF to metacestode vesicles. In mammals, the two prominent downstream signalling pathways of FGF receptors are the Ras-Raf-MAPK cascade and the PI3K/Akt pathway, while two others are STAT signalling and the phospholipase γ (PLCγ) pathway [15]. The STAT signalling pathway is absent in Echinococcus [14] and PLCγ involves binding to human FGF receptors at tyrosine residues that are not conserved in the Echinococcus receptors. We had previously demonstrated that the PI3K/Akt pathway exists in Echinococcus [11] but we could not measure differential phosphorylation of EmAKT in response to exogenous FGF or after metacestode vesicle treatment with BIBF 1120, indicating that in contrast to human cells, Echinococcus mainly involves the MAPK cascade pathway for downstream FGF signalling. In the related planaria, an important role of the Erk-like MAPK cascade in the differentiation of stem cells into multiple cell lineages has been reported [59]. In this system, inhibition of the Erk-like MAPK cascade resulted in reduced differentiation of neoblasts which retained their ability to proliferate and produced new neoblasts [59]. In a recent study on E. multilocularis stem cell dynamics, on the other hand, inhibition of the Erk-like MAPK cascade resulted in strongly diminished cell proliferation whereas activation of Erk signalling by exogenous addition of EGF stimulated stem cell self-renewal and clonal expansion, at least under conditions of low stem cell densities [13]. Furthermore, these authors showed that stimulating EGF signalling can activate quiescent stem cells in the protoscolex to re-enter the cell cycle [13]. Unfortunately, in the Echinococcus system no markers are known so far to distinguish between self-renewing stem cells from transit amplifying cells. It will thus in future experiments be interesting to study whether host FGF stimulates Echinococcus stem cell renewal, including activation of quiescent stem cells, or the differentiation of transit amplifying stem cells towards differentiated cell lineages. Furthermore, since apparently both FGF and EGF signalling in Echinococcus converge on the Erk-like MAPK cascade pathway, it will be worthwhile to study differential or additive effects of both stimuli on E. multilocularis stem cell dynamics.
Concerning cellular expression patterns we detected significant differences between the Echincoccus FGF receptors and those of the related schistosomes and planaria. In planaria, the expression of FGF receptors is a hallmark of neoblast stem cells and also occurs in cephalic ganglions [21,23]. In schistosomes, one of the two FGF receptors, fgfrA, is a marker for a prominent subset of somatic stem cells [25][26][27] and, like fgfrB, is also expressed in the reproductive organs [24]. In Echinococcus, we found only one receptor, EmFR3, which is expressed in germinative cells, but only in a very tiny subpopulation. EmFR2 was also only expressed in a few cells which were, however, clearly post-mitotic and most probably represented muscle cells. Likewise, the third Echinococcus FGF receptor, EmFR1, was expressed throughout the parasite's metacestode tissue without specific association with the germinative cells. In qRT-PCR analyses on metaces-tode´vesicles which were specifically deprived of stem cells after treatment with hydroxyurea [6] or the Polo-like kinase inhibitor BI 2536 [35] we also never observed diminished levels of emfr1 expression (S4 Fig), which further supports that the gene is not exclusively expressed in germinative cells. Taken together, these data indicate that the close association of FGF receptor expression with stem cells as observed in planaria and schistosomes is at least highly modified in the Echinococcus system, in which at least EmFR1 and EmFR2 are clearly expressed in postmitotic cells. This adds to stem cell-specific gene expression differences which we had previously observed between planaria and cestodes e.g. concerning argonaute or histone deacetylaseorthologs [6], and also indicates clear differences between stem cell-specific gene expression patterns in the parasitic flatworm lineages of trematodes and cestodes.
We observed clear inhibitory effects of the TK inhibitor BIBF 1120 on the enzymatic activity of all three Echinococcus FGF receptors upon heterologous expression in the Xenopus system and also demonstrated that this compound can profoundly affect parasite survival and development in vitro. Since in the Xenopus system concentrations of 20 μM BIBF 1120 fully inhibited all three Echinococcus FGF receptors we cannot clearly state whether the detrimental effects on parasite development were due to the specific inhibition of one of the three Echinococcus FGF receptors, or on combined activities against all three enzymes. However, based on the fact that EmFR3 is only expressed in less than 1% of the cells of the metacestode, that emfr3 expressing cells only accumulate during the formation of protoscoleces, and that EmFR3 showed lowest levels of inhibition upon expression in Xenopus cells, we do not consider this receptor as a prominent candidate for mediating the BIBF 1120 effects on primary cells and the metacestode. We also consider it unlikely that the inhibition of EmFR2 had produced these effects since the respective gene is only expressed in a small subset (2-5%) of all metacestode cells. Based on the expression of emfr2 in muscle cells or muscle cell progenitors, however, we cannot completely rule out that EmFR2 inhibition might have led to the depletion of specific parasite cells that are necessary to form a stem cell niche for the parasite's germinative cells. At least in planaria it has already been shown that muscle cells provide important positional information on the stem cell population [60] and our recent studies on the wnt signalling pathway in Echinococcus clearly demonstrated that this is also the case for cestodes [44]. However, based on the fact that emfr1 is expressed throughout the metacestode and that in both primary cells and metacestode vesicles emfr1 is the highest expressed FGF receptor gene we propose that most of the effects of BIBF 1120 on Echinococcus development are mediated by EmFR1 inhibition. On the cellular level, BIBF 1120 could either affect emfr1-expressing germinative cells or differentiated cells of the germinal layer or both. Since metacestode vesicles treated with stem cell-specific drugs such as hydroxyurea or BI 2536 still remain intact for 2-3 weeks [6,35], but BIBF 1120-treated vesicles already loose structural integrity after 5 days (Fig  6), we consider it highly likely that EmFR1 inhibition largely damages germinal layer cells which form an important niche for the germinative cells to survive and proliferate.
BIBF 1120 has originally been described as a multi-kinase inhibitor with specificity against members of the VEGF-, FGF-, and PDGF-families, but also with activity against members of the Src-family of non-receptor TK [45]. Our genomic analyses revealed that, similar to schistosomes [24], no genes encoding VEGF-or PDGF-receptors are present on the E. multilocularis genome. So far, no Src-like kinase has been biochemically characterized in cestodes. In the related trematode Schistosoma mansoni, however, at least one clear Src family kinase, SmTK3, has been described [61] and a close ortholog, EmuJ_000598200, is encoded on the E. multilocularis genome and, at least to a certain level, expressed in primary cells and metacestode vesicles. We thus cannot completely exclude that some of the effects of BIBF 1120 on these cell systems were due to an inhibition of the Echinococcus SmTK3 ortholog. On the other hand, we never observed clear effects of Src-kinase specific inhibitors such as PP2 or herbimycin A on Echinococcus development in vitro which indicates that the observed effects of BIBF 1120 on Echinococcus larval development are rather due to the inhibition of other kinases, most probably EmFR1 and/or EmFR2. For the development of novel chemotherapeutics against AE, EmFR1 and EmFR2 would thus be highly interesting target molecules although, of course, BIBF 1120 as originally developed against human FGF receptors showed somewhat higher activities against FGFR1 in the Xenopus expression system than against the parasite FGF receptors. Nevertheless, and using the activity assays developed in this work, it should be possible to identify compounds which are related to BIBF 1120 but which show higher affinities against the parasite enzymes than against human FGF receptors.

Conclusions
In the present work we provide clear evidence that human FGF ligands are capable of activating evolutionarily conserved TK of the FGF receptor family that are expressed by the larval stage of E. multilocularis and that the parasite's Erk-like MAPK cascade is stimulated upon exogenous addition of human FGFs to metacestode vesicles. We also showed that human FGF1 and FGF2 are stimulating the development of metacestode vesicles from parasite primary cell cultures and that they accelerate metacestode vesicle proliferation and growth in vitro. Since FGF1 and FGF2 are expressed in considerable amounts within the host liver, the primary target organ for the establishment of the E. multilocularis metacestode, and since FGF ligands are also constantly produced during liver regeneration and fibrosis, which are consequences of parasite growth within the intermediate host, we consider the observed in vitro effects on parasite FGF signalling and metacestode development also of high relevance in vivo. Liver-specific activities of host FGF could thus support the development of metacestode vesicles from stem cells that are delivered to the liver by the oncosphere larva, and could constantly stimulate asexual proliferation of the metacestode during an infection. We finally showed that at least one compound that inhibits the activities of mammalian FGF receptors, BIBF 1120, also inhibits the parasite orthologs, leads to metacestode inactivation, and prevents parasite development of stem cell-containing primary cell cultures. This opens new ways for the development of anti-Echinococcus drugs using the parasite FGF receptors as target molecules. The insert region of the split TK domain is indicated by a grey bar. Amino acids known to be highly conserved among tyrosine and serine/threonine kinases [62] are indicated by asterisks above the alignment. The two tyrosine residues known to be important for full activation of the human FGF receptor [58] are marked by black triangles. The TKD DFG motif which was modified to DNA to generate kinase-dead FGF receptors is indicated by black stars below the sequence. Transcriptome data have been generated during the E. multilocularis genome project and were mapped to the genome as described in [14]. (TIF)

S4 Fig. qRT-PCR analysis of emfr1-3 expression in stem cell-free metacestode vesicles.
Metacestode vesicles cultivated for 7 days in the presence of DMSO (control, black) or in the presence of hydroxyurea (HU, yellow) or BI 2536 (BI, red) were used for RNA isolation and cDNA preparation. Expression levels of emfr1, emfr2, and emfr3 (as indicated) were measured using the constitutively expressed gene elp as a normalization control (set to 1.0). All experiments were carried out in triplicates.

S6 Fig. Phosphorylation of E. multilocularis FGF receptors upon expression in Xenopus
oocytes. E. multilocularis EmFR2 (EmFR2) and a kinase-dead version of EmFR2 (EmFR2 TK-) were expressed in Xenopus oocytes and stimulated with either 10 nM FGF1 or 10 nM FGF2 as indicated. After stimulation, cell lysates were generated, separated by SDS-PAGE and analysed by Western blot using antibodies against the myc-tag (Anti-myc; loading control) or phosphorylated tyrosine (Anti Ptyr). Depicted are the results for EmFR2. Results for EmFR1 and EmFR3 were comparable. (TIF)