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Dissertation DISSERTATION Titel der Dissertation „Analysis of Tle genes in medaka embryonic development“ Verfasserin DI (FH) Gerlinde Jung angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr. rer. nat.) Wien, 2013 Studienkennzahl lt. Studienblatt: A 091 490 Dissertationsgebiet lt. Studienblatt: Molekulare Biologie Betreuerin / Betreuer: Univ. Prof. Dr. Erwin Heberle-Bors “The most exciting phrase to hear in science, the one that heralds new discoveries, is not 'Eureka!' but 'That's funny...' ” Isaac Asimov ABSTRACT Groucho/Transducine-like Enhancers of Split (Gro/TLE) belong to a conserved protein family of co-repressors. They mediate repression through interaction with various DNA- binding proteins by multiple molecular mechanisms, making them important players during developmental processes and in several diseases. The main part of this thesis focuses on their role in the embryonic development of medaka. For this, the function of Gro/TLE proteins was impaired by misexpressing their antagonist Amino-terminal Enhancer of Split (Aes) in heat shock inducible transgenic lines. The embryos developed smaller eyes that were tilted towards the midline where they fused to a cyclopic eye or were even lost in severe cases. To confirm these results individual members of this multigene family were knocked down. As expected, eye phenotypes like cyclopia appeared confirming the results of the misexpression experiments. Tcf3 is an interaction partner of Gro/TLE and an important determinant of anterior- posterior development. In zebrafish, lack of tcf3 function resulted in eye-less embryos. zThe single medaka tcf3 gene is expressed in a pattern comparable to its zebrafish orthologous headless and tcf3b. Loss of function experiments with PNA and morpholino antisense molecules resulted in anophthalmia and showed an anterior shift of the expression markers pax2, pax6 and gbx1. These results are in good agreement with zebrafish data. The phenotypes of gain of function experiments resembled those of aes- induced embryos. However, injection of tcf3 versions lacking the Gro/TLE interaction domain showed strongly reduced eye phenotypes, indicating a role of Gro/TLE corepressors in this process. The application of antisense molecules is an efficient way of gene inactivation in fish. Since microinjection generates varying results, methods with better reproducibility were explored. Compared to zebrafish, medaka have a substantially reduced sensitivity for chemicals (e.g. lithium chloride) and it is commonly assumed that this is due to the rigid chorion surrounding the embryo. Indeed, closer inspection of the chorion showed that larger molecules were not able to cross the chorionic barrier whereas small fluorescing tracer molecules readily passed into the perivitellin space. Interestingly, dechorionation only slightly improved the uptake into the embryos; instead inner membranes were identified as the limiting structure for diffusion. Adding detergents improved the diffusion effectively but also affected the internal distribution of the chemicals. Finally, electroporation could be established as a suitable method to enhance the uptake of small molecules into medaka embryos. i ii ZUSAMMENFASSUNG „Groucho/Transducin-like enhancer of split“ (Gro/TLE) Proteine gehören zu einer konservierten Familie von Korepressoren. Durch die Interaktion mit DNA gebundenen Proteinen hemmen sie die Transkription auf mehrere verschiedene Arten. Dadurch hat Gro/TLE eine Schlüsselrolle in verschiedensten entwicklungsbiologischen Prozessen, aber auch in der Entstehung von Krankheiten. Der Hauptteil dieser Arbeit konzentriert sich auf die Rolle von Gro/TLE in der frühen embryonalen Entwicklung von Medakafischen. Für diese Analyse wurde die Funktion durch ihren Antagonisten, “Amino-terminal Enhancer of Split“ (Aes) in einer Hitze induzierbaren transgenen Fischlinie gehemmt. Die Embryonen entwickelten kleinere Augen, deren anteriores Ende nach medial kippte, wo es zu einem zyklopischen Auge zusammen wuchs. In schweren Fällen wurde die Augenentwicklung zur Gänze gehemmt. „Knock-down“ von einzelnen Mitgliedern dieser großen Genfamilie bestätigten die Ergebnisse. Tcf3 ist einer der Interaktionspartner von Gro/TLE dem eine wichtige Rolle in der anterio-posterior Entwicklung zugeschrieben wird. Zebrafischembryonen ohne Tcf3 entwickelten keine Augen. Eine Expressionsanalyse des einzigen Medaka tcf3 Gens zeigte ähnliche Muster wie eine Kombination der zwei Zebrafisch Orthologe headless und tcf3. Embryonen, deren tcf3 Funktion durch PNA Moleküle oder Morpholino Oligonucleotide ausgeschaltet wurde, entwickelten kleine oder gar keine Augen. Zusätzlich war das Expressionsmuster der molekularen Marker pax2, pax6 und gbx1 anterior verschoben. Auch diese Beobachtungen waren vergleichbar mit Zebrafisch Resultaten. Die Überexpressionsphänotypen ähnelten wiederum denen der Aes-induzierten Embryonen. Injektionen mit tcf3 Versionen, deren Gro/TLE Interaktionsdomäne fehlte, zeigten eine deutlich reduzierte Zahl an Phänotypen, ein weiteres Indiz dafür, dass Gro/TLE eine Rolle in diesem Prozess spielt. Oft werden Antisensemoleküle für die Inaktivierung von Genen in Fischen verwendet. Sie werden mittels Mikroinjektion in den Embryo appliziert. Variierende Ergebnisse dieser Methode machen Alternativen mit höherer Reproduzierbarkeit interessant. Medakaembryonen reagieren weniger empfindlich auf Chemikalien (z.B. Litiumchlorid) als Zebrafische. Eine Erklärung dafür ist, dass die Embryonen durch eine starre Außenhülle, dem sogenannten Chorion, geschützt werden. Genauere Analysen des Chorions zeigten, dass große Moleküle diese Barriere wirklich nicht passieren konnten, kleine jedoch ungehindert bis in den Perivitellinraum vordringen konnten. Das Entfernen des Chorions brachte nur wenig Verbesserung. Es scheint deshalb, dass nicht, wie bisher angenommen, die harte Außenhülle, sondern ein weiteres Membransystem im Inneren des Embryos der Ausschlussgrund für viele Moleküle ist. Die Zugabe von Detergentien verbesserte die Durchlässigkeit wesentlich, hatte jedoch auch Auswirkungen auf die interne Verteilung der jeweiligen Chemikalien. Elektroporation dagegen steigerte nur deren Aufnahme, ohne ihre Verteilungseigenschaften zu ändern und stellt damit eine effiziente Methode für die Aufnahme kleiner Moleküle in Medaka Embryonen dar. iii iv ACKNOWLEDGEMENT No matter how much effort you put into your work, the success of any project depends largely on the suggestions and encouragement of many others. Therefore, I take this opportunity to express my deepest gratitude to the people who have been vital for the successful completion of this thesis. I would like to show my greatest appreciation to FH-Prof. Dr. Thomas Czerny. Thank you for giving me the possibility to work in such a wonderful team. Thank you for your guidance and support, for your suggestions and brilliant yet sometimes crazy ideas and most of all for all the endless discussions that helped putting the pieces together. Thank you for chocolate Easter bunnies in the waterbath, scientific Christmas treasure hunts and the exhilarating tales of Rambi and the three Ice-Ladies. I would also like to express my gratitude to the University of Applied Life Sciences FH Campus Wien for providing such a beautiful new laboratory and Univ. Prof. Dr. Erwin Heberle-Bors for being my advisor at the University of Vienna Thanks to all the “Czernys”: Viktoria, Lisi, Stefan, Laura, Adrian (Thomas), Adriane, Alfred, Andrea, Barbara, Baubi, Carmen, Chris, Christian, Christine, Fiona, Gemma, Kathi, Markus, Markus (Mäxchen), Markus Holzer, Narges, Noreen, Sebastian, Sylvia, Thomas H., Thomas P., Yvonne. Together we were a wonderful team. I would like to thank Baubi and Narges for introducing me to the topic and all the fish methods. To all my Diploma, Bachelor and Master students, I learned so much from you. Especially Laura, your motivation and enthusiasm were so inspiring. I cannot express how much I thank my fellow labmates Viktoria and Lisi. We have had such a great time. Thank you for all the fun we have had in the last six years, for inspiring discussions, for helping hands, for unwavering support, for keeping me harmonious during challenging times and for the big C’s (coffee, coke, cake and chocolate). No matter what, together we managed every project and every problem and made a lot out of so very little. Six years ago we were three labmates and now we are really great friends. GVL forever! An old proverb says. “True friends can be counted on one hand”. Well, luckily I need two hands to count those who were so (almost always) patient in all these years: Marion, Dani, Oliver, Manfred, Babsi, Mark, Sarah, Viktoria and Lisi. I would like to thank my parents who are always there for me, who help whenever I need them and encourage me to do everything my way. To Benny, my best friend and love of my life, who lived with this thesis as much as I did and who supported me in all these years without questioning. Now we will go on holidays, I promise! THANK YOU! v vi TABLE OF CONTENTS ABSTRACT ............................................................................................................... I ZUSAMMENFASSUNG ............................................................................................. III ACKNOWLEDGEMENT ............................................................................................. V TABLE OF CONTENTS ........................................................................................... VII LIST OF FIGURES .................................................................................................
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