Building a light-sheet microscope to study the early development of the polyclad flatworm Maritigrella crozieri (Hyman, 1939) Johannes Girstmair Research Department of Genetics, Evolution and Environment University College London Submitted for the Degree of Doctor of Philosophy April 2017 1 Declaration of ownership I, Johannes Girstmair, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The Lophotrochozoa is an evolutionary interesting clade comprising many and mostly marine phyla. Despite their diverse morphology, these animals often have a biphasic life- cycle in form of a free-swimming, ciliated larval stage, the trochophore, and seemingly retained a highly conserved developmental pattern called spiral cleavage. This opens the door for comparative studies to better understand the shared mechanisms of the spiralian developmental program, its deviations and the evolution of lophotrochozoan body plans. Here I studied the early development and larva formation in the polyclad flatworm Maritigrella crozieri and compared it with other members of this evolutionarily diverse group of animals. In order to conduct this research, I first built a light-sheet microscope (OpenSPIM) that would allow me to follow the development of the polyclad embryo from the zygote into the larval stage and to acquire sophisticated 3d-reconstructions of fixed embryonic stages. I then used this and other techniques to characterise in detail the early development of M. crozieri and its conserved spiral cleavage pattern. Precise volume measurements of 3d- reconstructed early blastomeres and the investigations of associated cleavage patterns indicate that this polyclad worm may not follow a strictly equal spiral cleavage type, as was previously thought. I investigated the cleavage pattern and fate of micromere 4d, which in M. crozieri gives rise to mesoderm and generates bilaterally symmetric embryos at a cellular level. A first cell lineage analysis of this organism involved long-term live imaging recordings and point to a conserved fate of blastomeres that, like in other spiral cleavers, 3 give rise to ectodermally derived structures, specifically the larva’s locomotion system (ciliary band) and a sensory organ at the apical point (apical organ). These findings strengthen the idea that these structures may be homologous to those found in other trochophore larvae. This work increased the current knowledge of the early development of the polyclad flatworm M. crozieri, which facilitates evolutionary comparisons of the development of different flatworms and lophotrochozoans more broadly and can contribute to addressing the homology of marine larvae. 4 Statement of impact With so many marine animal phyla that undergo the highly conserved developmental pattern called spiral cleavage, it is of great interest for evolutionary biologists to study the spiralian developmental program in as many representatives as possible. This allows to understand the fundamentals of the evolutionary mechanism that generated an astonishingly diversity of marine invertebrate species in the largest clade of bilaterally symmetric animals: the Lophotrochozoa. The polyclad flatworms are a member of the Lophotrochozoa and represent an evolutionary particularly important group, as they are the only platyhelminths that have retained the conserved spiral cleavage mode, while embryogenesis in most other flatworms is highly derived. Additionally, many polyclads have a larval stage. Studying the early spiral cleavage mechanism, embryogenesis and polyclad larvae, contributes not only to a better understanding of the evolution of the Platyhelminthes but is interesting for the relationships of the Lophotrochozoa as a whole. 5 Acknowledgements During my time as a PhD student I was surrounded by extraordinary people, and met many more, who were very much interested in my project and contributed to this work. Foremost I would like to thank my supervisor Max Telford, who immensely cared about me and my wellbeing during my time in his lab and who was a constant source of advice, encouragement and new exciting ideas. I am very grateful that the door to his office was always widely open for me and for his optimism and confidence in me and my work. I also thank Paola Oliveri for her endless advice, which was very helpful and for introducing me to her microinjection setup. It truly is a sacred place. I cannot thank enough my dear colleagues from the Telford Lab and the Oliveri Lab for being more than just colleagues. It was so much fun to have you around. Particularly I want to thank Anne Zakrzewski, Helen Robertson, Steven Müller and François Lapraz, who all directly contributed to my work in terms of SEM microscopy, WISH and python programming, OpenSPIM microscopy and in many other ways. I am very grateful to Kate Rawlinson for her advice, reading suggestions and encouraging words throughout my work. Furthermore, I thank our lab technician, Fraser Simpson, who made my work a lot easier and who joined forces with me on several collection trips to Florida, where his incredible endurance was very much needed to find all the worms. I want to thank the people involved in the Neptune Marie Curie Initial Training network for their excellent and demanding work that made my life as a Neptune student so much more 6 enjoyable and allowed me to constantly gain new experience during our conferences and practical courses. I also take this opportunity to thank the people at the MPI-CBG in Dresden, in particular Pavel Tomancak, for allowing me to visit his lab and learn everything needed to establish an OpenSPIM in the Telford lab; and Peter Pitrone who helped with the design and got me started in terms of laser alignment and all the little tricks. I am especially grateful to Mette Handberg-Thorsager, who is one of the most dedicated researchers I know. Her interest in the spiral cleaving Maritigrellas and the initial trials on microinjecting the embryos were crucial for this project. I also want to thank Bernhard Egger for introducing me to the world of flatworms and the initial time of my PhD we spend together in London, which was full of advice, discussion about flatworms and FTL. Finally, I want to thank Anna Czarkwiani for being there for me literally all the time and never getting tired of it. It is impossible to imagine the last three years without this extraordinary person and fantastic young researcher on my side, and without her support. I am the luckiest man to have you in my life. Let’s continue the fun! 7 Meiner Familie in großer Dankbarkeit, besonders meinen lieben Eltern, Elisabeth und Hermann 8 Table of Contents Declaration of ownership ........................................................................................................ 2 Abstract .......................................................................................................................................... 3 Statement of impact ................................................................................................................... 5 Acknowledgements .................................................................................................................... 6 Table of Figures ......................................................................................................................... 14 List of Abbreviations ................................................................................................................ 19 CHAPTER 1 Introduction ...................................................................................................... 21 1.1 History of the phylogeny of the Lophotrochozoa...................................................... 21 1.2 Phylogeny of the Platyhelminthes ................................................................................. 23 1.3 Platyhelminthes: body simplicity with complex life-cycles .................................. 25 1.4 Summary of spiral cleavage in the Lophotrochozoa ............................................... 26 1.5 The spiralian trochophore larva .................................................................................... 35 1.6 Polyclad flatworms and their larval types .................................................................. 39 1.7 Larval evolution in polyclad flatworms ....................................................................... 42 1.8 Maritigrella crozieri: a new organism for evo-devo studies ................................. 44 1.9 Innovative ways of performing cell lineage studies in polyclad flatworms .... 48 1.10 Light-sheet microscopy ...................................................................................................... 50 1.11 Hypothesis and aims addressed in this thesis ........................................................... 53 CHAPTER 2 Methods .............................................................................................................. 56 2.1 Building a single plane illuminated light-sheet microscope (OpenSPIM) ....... 56 2.2 Animal culture and embryo collection ......................................................................... 60 2.3 Plasmid preparations ......................................................................................................... 63 9 2.4 Microinjections...................................................................................................................... 65 2.5 Standard