Functional analysis of siRNA mediated knockdowns of fibroblast growth factors in Hydra vulgaris Dissertation zur Erlangung des Grades eines Doktor der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs Biologie der Philipps-Universitat¨ Marburg vorgelegt von Lisa Andrea Reichart aus Gelnhausen Marburg, November 2020 Die vorliegende Dissertation wurde von Mai 2015 bis November 2020 am Fachbereich Biologie der Philipps-Universitat¨ Marburg unter Leitung von Prof. Dr. Monika Hassel angefertigt. Vom Fachbereich Biologie der Philipps-Universitat¨ Marburg (Hochschul- kennziffer 1180) als Dissertation angenommen am Erstgutachter*in: Prof. Dr. Monika Hassel Zweitgutachter*in: Prof. Dr. Christian Helker Tag der Disputation: Abstract Fibroblast growth factor receptor (FGFR) signaling is crucial in animal development. Two FGFRs and one FGFR-like receptor, which lacks the intracellular domain, are known in the Cnidarian Hydra vulgaris. FGFRa, also known as Kringelchen, is an important factor in the developmental process of budding, as it controls the detachment of the bud. It is still unknown, which extracellular ligands are responsible for the start of the relevant signal transduction cascades in Hydra. This study gives first insights into the potential functions of five FGFs previously identified in Hydra. Analysis of the gene and protein expression patterns of different FGFs in several Hydra strains suggest that FGFs may comprise evolutionary conserved, multiple functions in bud detachment, neurogenesis, migration and cell differentiation, as well as in the regeneration of head and foot structures in Hydra. The electroporation of siRNAs into adult Hydra was used to analyze knockdown effects of FGFs and FGFRs in Hydra. This method was efficiently reproducing phenotypes obtained using the FGFR inhibitor SU5402 or, alternatively phosphorothioate antisense oligonucleotides or a dominant-negative FGFR mutant. Additionally, the siRNA-mediated knockdown showed a potential function of FGFRa in neuronal development and of FGFc in the differentiation of I-cells. In summary this work provides new insights into potential functions of FGFs and FGFRs in the model organism Hydra vulgaris and provides a basis for further studies investigating interactions of FGFRs and FGFs in this organism. Zusammenfassung Die Signaltransduktion durch Fibroblastenwachstumsfaktorrezeptoren (engl. fibro- blast growth factor receptors, FGFRs) ist wichtig fur¨ Wachstums- und Entwicklungspro- zesse von Tieren. Bei dem Sußwasserpolypen¨ Hydra vulgaris sind zwei FGFRs be- kannt, sowie ein weiterer den FGFRs ahnlicher¨ Rezeptor (FGFR-like) ohne intrazellulare Kinasedomane.¨ FGFRa, auch als Kringelchen bezeichnet, ist wegen seiner Rolle in dem Abloseprozess¨ der Knospe ein unabdingbarer Faktor in ihrer Entwicklung. Bisher ist allerdings unbekannt, welche extrazellularen¨ Liganden an FGFR von Hydra binden und diese relevante Signaltransduktionskaskade aktivieren. In der vorliegenden Arbeit wurden funf¨ zuvor identifizierte potenzielle FGFs auf ihre moglichen¨ Funktionen in Hydra untersucht. Die Analyse von Gen- und Protein- expressionsmustern unterschiedlicher FGFs in verschiedenen Hydra Stammen¨ weist auf evolutionar¨ konservierte, vielfaltige¨ Funktionen der FGFs in der Knospenablosung,¨ Neurogenese, Migration, Zelldifferenzierung und in der Regeneration von Kopf- und Fußstrukturen in Hydra hin. Mit Hilfe der siRNA-Elektroporation wurden die Effekte eines Knockdowns von FGFs und FGFR in Hydra untersucht. Dabei konnten die bereits zuvor beobachteten Phanotypen,¨ erzeugt durch pharmakologische Inhibition mit dem FGFR-Inhibitor SU5402, das Einbringen von Phosphorothioate-Antisense-Oligonukleotiden oder der Verwendung dominant-negativer FGFR Mutanten, erfolgreich reproduziert werden. Zusatzlich¨ zeigte der siRNA Knockdown eine mogliche¨ Funktion von FGFRa in der Neuronenentwicklung und von FGFc in der Differenzierung der I-Zellen. Zusammenfassend gewahrt¨ diese Arbeit neue Einblicke in mogliche¨ Funktionen verschiedener FGFs in Hydra und dient damit als Grundlage fur¨ weitere Untersuchungen zur Interaktion von FGFs und FGFRs diesem Organismus. Contents List of Abbreviations VII List of Figures VIII List of TablesX 1 Introduction1 1.1 The fibroblast growth factor receptor signaling pathway . .1 1.1.1 The molecular structure of FGFRs . .2 1.1.2 FGFs as ligands for the FGFR signaling . .4 1.1.3 The activation of the FGFRs and their downstream pathways .7 1.1.4 The role of FGFs during regeneration and wound healing .8 1.2 The model organism Hydra vulgaris .................... 10 1.2.1 Characteristics of Hydra ...................... 10 1.2.2 The reproduction of Hydra ..................... 12 1.2.3 Regeneration in Hydra ....................... 15 1.2.4 The Hydra nerve net and neurogenesis . 17 1.3 The FGFR signaling in Hydra ........................ 18 1.4 RNA interference as a tool for gene analysis . 19 1.4.1 The RNAi pathway . 20 1.4.2 RNAi in Hydra ........................... 22 1.5 Aim of the project . 22 2 Results 24 2.1 Analysis of the FGF transcript distribution in Hydra ........... 24 2.1.1 The FGFa gene was expressed ectodermally in the peduncle . 24 2.1.2 FGFb was transcribed endodermally in the tentacles, buds and around the foot pore . 25 2.1.3 The FGFc gene was expressed in a half-ring below the tentacle base and in the basal disc . 30 2.1.4 FGFe was transcribed in the peduncle and at the mouth opening 30 III Contents 2.1.5 FGFf was transcribed dynamically during budding . 31 2.1.6 FGFRa and FGFRb were co-localized from stage 4 onward in cells of the bud base . 35 2.1.7 FGFb and FGFf transcripts were excluded from cells carrying FGFR transcripts . 38 2.2 Analysis of the FGFf protein distribution in Hydra ............ 40 2.2.1 A Hydra-specific antibody detects FGFf in different Hydra strains 40 2.2.2 FGFf showed a dynamic pattern during bud development . 43 2.2.3 FGFf accumulated ectodermally at the bud base . 45 2.2.4 FGFf was not accumulated at the bud base after the treatment with SU5402 . 47 2.2.5 The FGFf gene was transcribed dynamically during regeneration 47 2.2.6 The FGFf protein was expressed dynamically during regeneration 51 2.3 A siRNA mediated knockdown of FGFs and FGFRs in Hydra ...... 51 2.3.1 The electroporation of FITC-Dextran was effective in all tested Hydra strains . 54 2.3.2 The siRNA mediated FGFRa knockdown induced a phenotype similar to the SU5402 inhibitor . 55 2.3.3 The FGFRa knockdown led to decreased detachment rates . 56 2.3.4 Statistical analysis of the siRNA detachment rates . 58 2.3.5 The siRNA treatment influenced the transcription patterns . 61 2.3.6 The siRNA mediated knockdown of FGFf was not detected at the protein level . 69 2.3.7 The siRNA mediated knockdowns partially influenced the cell type numbers . 69 2.3.8 Statistical analysis of the siRNA maceration . 74 3 Discussion 78 3.1 The function of FGFa remains unclear . 78 3.2 FGFb may promote bud detachment and cell differentiation . 79 3.3 FGFc may promote I-cell renewal and neuronal differentiation . 81 3.4 FGFe may function in the bud induction . 82 3.5 FGFf may provide many functions in the Hydra development . 83 3.5.1 FGFf may promote the boundary formation during the bud de- tachment . 83 3.5.2 FGFf may act in the regeneration of Hydra ............ 85 IV Contents 3.5.3 FGFf may be involved in the bud and tentacle evagination . 87 3.5.4 FGFf may be a potential migration factor . 88 3.6 The FGFRa siRNA experiments solidifies its role during the bud detach- ment and suggests a function during neurogenesis . 89 3.7 Conclusion and Outlook . 90 4 Methods 92 4.1 Model organism . 92 4.1.1 Cultivation of Hydra ........................ 92 4.1.2 Artificial seawater and Artemia salina preparation . 92 4.2 Hydra methods . 94 4.2.1 Fixation of Hydra .......................... 94 4.2.2 Regeneration series of Hydra ................... 94 4.2.3 Maceration of Hydra tissue . 94 4.2.4 Transplantation of Hydra ..................... 95 4.2.5 Microinjection of adult Hydra ................... 95 4.2.6 Pharmacological inhibition in Hydra ............... 95 4.2.7 Electroporation of adult Hydra .................. 96 4.2.8 siRNA mediated knockdown in Hydra .............. 96 4.3 DNA methods . 96 4.3.1 Polymerase Chain reaction (PCR) . 96 4.3.2 Insert PCR . 96 4.3.3 Agarose gel electrophoresis . 97 4.3.4 DNA restriction digest . 97 4.3.5 DNA ligation . 97 4.3.6 Chemical transformation of E. coli ................ 98 4.3.7 Plasmid DNA preparation . 98 4.3.8 DNA-sequence analysis . 98 4.4 RNA methods . 98 4.4.1 In vitro transcription of labelled RNA/ probe synthesis . 98 4.4.2 Dot Blot . 99 4.4.3 Northern Blot . 99 4.4.4 Antibody pre-absorption for ISH . 100 4.4.5 in situ hybridization (ISH) . 101 4.5 Protein methods . 103 4.5.1 Protein electrophoresis (SDS-PAGE) and western blot . 103 V Contents 4.5.2 Immunodetection . 104 4.6 Statistical analysis . 104 5 Materials 106 5.1 Hydra materials . 106 5.1.1 Hydra husbandry . 106 5.1.2 Hydra fixation . 106 5.1.3 Hydra maceration . 106 5.1.4 Hydra transplants . 107 5.1.5 Pharmacological inhibition of Hydra ............... 107 5.1.6 Microinjection of Hydra ...................... 107 5.1.7 Electroporation . 107 5.2 siRNA Duplexes . 108 5.3 RNA materials . 109 5.3.1 Pharmacological inhibition . 109 5.3.2 Northern Blot and Dot Blot . 109 5.3.3 In situ hybridization . 110 5.4 Protein materials . 112 5.4.1 Immunodetection . 112 5.4.2 SDS-PAGE and Western Blot . 112 5.5 Cloning materials . 114 5.5.1 Oligonucleotides . 114 5.5.2 Enzymes . 114 5.5.3 Bacteria . 114 5.5.4 Vectors . 115 5.5.5 Kits . 115 5.6 Additional chemicals, substances, and reagents . 115 5.7 Software and online tools . 116 Bibliography 117 A Supplements 148 B Danksagung 168 C Wissenschaftlicher Lebenslauf 169 D Eidesstattliche Erklarung¨ 170 VI List of Abbreviations ALP alsterpaullone. BMP bone morphogenetic protein. dsRNA double stranded RNAs.