Förster S , Koziol U , Schäfer T , Duvoisin R , Cailliau K , Vanderstraete M , Dissous C , Brehm K .

107475/Z/15/Z Wellcome Trust

	Fig 1. Domain structure of E. multilocularis FGF receptors.Schematic representation of the domain structure of the E. multilocularis receptors EmFR1, EmFR2, and EmFR3 according to SMART (Simple Modular Architecture Tool) analyses. As a comparison, the structure of the human FGFR1 (HsFGFR1) is shown. Displayed are the location and size of the following predicted domains: TK domain (TKD) in purple and IG-domain (Ig) in orange. Predicted signal peptides are depicted in red and transmembrane domains are shown as blue bars. The numbers of amino acids in full length receptors are shown to the right.
	Fig 2. WMISH analysis of emfr1 expression.Left panel, antisense probe detecting emfr1 expression. Right panel, sense probe (negative control). WMISH signal is shown in green, nuclear DAPI staining in blue. gl, germinal layer; ps, developing protoscolex. Bar: 20 μm.
	Fig 3. WMISH analysis of emfr2 expression.Shown are WMISH analyses of emfr2 expression (A, B) and co-detection of emfr2 WMISH and EdU incorporation (C, D, E) during metacestode development. In C, D, and E, metacestodes were cultured in vitro with 50 μM EdU for 5 h and were then processed for WMISH and EdU detection. In all panels the WMISH signal is shown in green, EdU detection in red, and DAPI nuclear staining in blue. A, detail of the germinal layer. B, general view of a region of a metacestode with protoscoleces in different stages of development. C, early protoscolex development. D, late protoscolex development. E, Detail of a sucker, maximum intensity projection of a confocal Z-stack. r, rostellum; s, sucker. Bars are 20 μm for A, 50 μm for B, 10 μm for C, and 50 μm for D. Sense probe (negative control) was negative in all samples.
	Fig 4. Co-detection of emfr3 WMISH and EdU incorporation during metacestode development.Metacestodes were cultured in vitro with 50 μM EdU for 5 h and were then processed for WMISH and EdU detection. In panels displaying multiple channels, the WMISH signal is shown in green, EdU detection in red, and nuclear DAPI staining in blue. A, germinal layer. B, developing protoscolex. Arrowheads indicate Edu+ emfr3+ double-positive cells. Bars are 25 μm for A, 50 μm for B (main panel) and 10 μm for B (enlarged inset).
	Fig 5. Effects of host FGFs on E. multilocularis proliferation and development.A, effects of FGFs on metacestode proliferation. Axenically cultivated metacestode vesicles (eight per well) were incubated for 48 h in DMEM medium (without FCS), then BrdU (1 mM) and FGFs (10 or 100 nM) were added and incubation proceeded for another 48 h before cell lysis, DNA isolation, and BrdU detection. Bars represent percentage of BrdU incorporation with the cMEM control set to 100%. Statistical evaluation of four independent experiments (n = 4) which were conducted in duplicates is shown. Student’s t-test (two tailed): *p<0.05. B, effects of host FGFs on metacestode vesicle development. Single axenically generated metacestode vesicles were in vitro cultivated for four weeks in the presence of 10 nM FGF1 (aFGF) or 5 nM FGF2 (bFGF) with daily medium changes. Control vesicles were kept in cMEM. Growth (in ml) of vesicles was monitored. In each of three independent experiments (n = 3), four vesicles were examined for every single condition. C, effects of FGFs on E. multilocularis primary cell proliferation. Freshly isolated E. multilocularis primary cells were incubated for 48 h in 2 ml cMEM, then BrdU (1 mM) and FGFs (100 nM) were added and cells were incubated for another 48 h before DNA isolation and BrdU detection. Statistical analysis was performed as in A. D, effects of host FGFs on metacestode vesicle development. Freshly prepared E. multilocularis primary cells were cultured for 21 days in cMEM medium in the presence or absence of 100 nM FGF1 (aFGF) or FGF2 (bFGF). Half of the medium volume was renewed every second day. The number of newly formed metacestode vesicles at day 21 was analysed. The bars represent the percentage of formed vesicles with the cMEM control set to 100%. The statistical evaluation of three independent experiments (n = 3) which were conducted in duplicates is shown. Student’s t-test (two-tailed): *p<0.05.
	Fig 6. Effects of BIBF 1120 on E. multilcoularis metacestode vesicles and primary cells.A, axenically cultivated metacestode vesicles (8 per well in 2 ml volume) 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.
	Fig 7. Effects of host FGFs and BIBF 1120 on EmMPK1 phosphorylation in metacestode vesicles.A, axenically cultivated metacestode vesicles were incubated in cMEM (control) or in medium without FCS (0 s), upon which FGF1 (aFGF) or FGF2 (bFGF) were added at a concentration of 10 nM for 30 sec (30s), 60 sec (60s) or 60 min (60min). Protein lysates were subsequently separated by 12% SDS-PAGE and Western blots were analysed using polyclonal antibodies against Erk-like MAP kinases (anti-ERK) or double phosphorylated Erk-like MAP kinases (anti-p-ERK). B, axenically cultivated metacestode vesicles were incubated with DMSO (negative control), 5 mM or 10 mM BIBF1120 (30 min each) and cell lysates were subsequently analysed as described above. Both experiments were performed in triplicate with similar results.

Adalid-Peralta, Effect of Transforming Growth Factor-β upon Taenia solium and Taenia crassiceps Cysticerci. 2017, Pubmed

Adalid-Peralta, Effect of Transforming Growth Factor-β upon Taenia solium and Taenia crassiceps Cysticerci. 2017, Pubmed
Anthony, Hepatic stellate cells and parasite-induced liver fibrosis. 2010, Pubmed
Berriman, The genome of the blood fluke Schistosoma mansoni. 2009, Pubmed
Brehm, Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display. 2003, Pubmed
Brehm, Echinococcus-Host Interactions at Cellular and Molecular Levels. 2017, Pubmed
Browaeys-Poly, Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma. 2000, Pubmed , Xenbase
Cebrià, FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. 2002, Pubmed , Xenbase
Cheng, EGF-mediated EGFR/ERK signaling pathway promotes germinative cell proliferation in Echinococcus multilocularis that contributes to larval growth and development. 2017, Pubmed
Del Puerto, Negligible elongation of mucin glycans with Gal β1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus. 2016, Pubmed
Furdui, Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction. 2006, Pubmed
Gelmedin, Molecular characterisation of MEK1/2- and MKK3/6-like mitogen-activated protein kinase kinases (MAPKK) from the fox tapeworm Echinococcus multilocularis. 2010, Pubmed
Gottstein, Immunology of Alveolar and Cystic Echinococcosis (AE and CE). 2017, Pubmed
Gottstein, An intact laminated layer is important for the establishment of secondary Echinococcus multilocularis infection. 2002, Pubmed
Grassot, Origin and molecular evolution of receptor tyrosine kinases with immunoglobulin-like domains. 2006, Pubmed
Grohme, The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms. 2018, Pubmed
Hahnel, Gonad RNA-specific qRT-PCR analyses identify genes with potential functions in schistosome reproduction such as SmFz1 and SmFGFRs. 2014, Pubmed , Xenbase
Hanks, Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. 1995, Pubmed
Hemer, Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development. 2014, Pubmed
Hilberg, BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. 2008, Pubmed
Huang, FGF signaling in flies and worms: more and more relevant to vertebrate biology. 2005, Pubmed
Kan, Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration. 1989, Pubmed
Kapp, The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization. 2004, Pubmed
Kern, The Echinococcoses: Diagnosis, Clinical Management and Burden of Disease. 2017, Pubmed
Khayath, Diversification of the insulin receptor family in the helminth parasite Schistosoma mansoni. 2007, Pubmed
Konrad, Identification and molecular characterisation of a gene encoding a member of the insulin receptor family in Echinococcus multilocularis. 2003, Pubmed
Koziol, A Novel Terminal-Repeat Retrotransposon in Miniature (TRIM) Is Massively Expressed in Echinococcus multilocularis Stem Cells. 2015, Pubmed
Koziol, Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms. 2016, Pubmed
Koziol, The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis. 2014, Pubmed
Krishnapati, Identification and characterization of VEGF and FGF from Hydra. 2013, Pubmed
Livak, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 2001, Pubmed
Nagasaki, Liver contains heparin-binding growth factors as the major growth factor for cultured fibroblasts. 1991, Pubmed
Ogawa, Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions. 2002, Pubmed
Oliveira, Effect of human TGF-β on the gene expression profile of Schistosoma mansoni adult worms. 2012, Pubmed
Ornitz, The Fibroblast Growth Factor signaling pathway. 2015, Pubmed
Osman, Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene. 2006, Pubmed
Oulion, Evolution of the FGF Gene Family. 2012, Pubmed
Parkinson, A transcriptomic analysis of Echinococcus granulosus larval stages: implications for parasite biology and host adaptation. 2012, Pubmed
Ramachandran, The Schistosoma mansoni epidermal growth factor receptor homologue, SER, has tyrosine kinase activity and is localized in adult muscle. 1996, Pubmed
Rentzsch, FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. 2008, Pubmed
Roth, Nintedanib: from discovery to the clinic. 2015, Pubmed
Schubert, Targeting Echinococcus multilocularis stem cells by inhibition of the Polo-like kinase EmPlk1. 2014, Pubmed , Xenbase
Schumacher, Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors. 2016, Pubmed
Spiliotis, Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis. 2003, Pubmed
Spiliotis, Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions. 2004, Pubmed
Spiliotis, Molecular cloning and characterization of Ras- and Raf-homologues from the fox-tapeworm Echinococcus multilocularis. 2005, Pubmed
Spiliotis, Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor. 2006, Pubmed
Spiliotis, Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles. 2008, Pubmed
Spiliotis, Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures. 2009, Pubmed
Spiliotis, Echinococcus multilocularis primary cells: improved isolation, small-scale cultivation and RNA interference. 2010, Pubmed
Steiling, Fibroblast growth factor receptor signalling is crucial for liver homeostasis and regeneration. 2003, Pubmed
Tasaki, ERK signaling controls blastema cell differentiation during planarian regeneration. 2011, Pubmed
Thompson, Biology and Systematics of Echinococcus. 2017, Pubmed
Tsai, The genomes of four tapeworm species reveal adaptations to parasitism. 2013, Pubmed
Vicogne, Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni. 2004, Pubmed , Xenbase
Vuitton, Collagen immunotyping of the hepatic fibrosis in human alveolar echinococcosis. 1986, Pubmed
Wagner, Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis. 2012, Pubmed
Wang, Functional genomic characterization of neoblast-like stem cells in larval Schistosoma mansoni. 2013, Pubmed
Wang, Stem cell heterogeneity drives the parasitic life cycle of Schistosoma mansoni. 2018, Pubmed
Wendt, Flatworm-specific transcriptional regulators promote the specification of tegumental progenitors in Schistosoma mansoni. 2018, Pubmed
Witchley, Muscle cells provide instructions for planarian regeneration. 2013, Pubmed
You, Cloning and characterisation of Schistosoma japonicum insulin receptors. 2010, Pubmed
Zavala-Góngora, A member of the transforming growth factor-beta receptor family from Echinococcus multilocularis is activated by human bone morphogenetic protein 2. 2006, Pubmed