UNIVERSITY OF COPENHAGEN DEPARTMENT OF BIOLOGY Molecular Evolution of Peptide Signaling in Placozoa and Cnidaria THOMAS LUND KOCH PH.D. THESIS Department of Biology, Section for Cell and Neurobiology This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen THOMAS LUND KOCH – PH.D. THESIS Molecular evolution of peptide signaling in Placozoa and Cnidaria Main Academic Supervisor Professor Cornelis J.P. Grimmelikhuijzen Section for Cell and Neurobiology, University of Copenhagen, Denmark Co-supervisor Associate Professor Frank Hauser Section for Cell and Neurobiology, University of Copenhagen, Denmark – – – – – Chair of Assessment Committee Associate Professor Katrine Worsaae Marine Biological Section, University of Copenhagen, Denmark Assessment Committee Member (External) Senior Researcher Morten Schiøtt National Institute of Aquatic Resources, Technical University of Denmark, Denmark Assessment Committee Member (External) Professor Christian Wegener Neurobiology and Genetics, University of Würzburg, Germany Acknowledgement First, I would like to give a huge thank to my supervisor Cornelis Grimmelikhuijzen, who has been instrumental in supporting me though the three years of research. I cannot express how fortunate I feel for the opportunity and how much I have grown scientifically in the period. Thank you for your teachings, both in words and actions, our discussions and collaboration. I am thankful to all the people I have met at the Section of Cell and Neurobiology. It has been a great experience to meet you all and am grateful for the friendly, optimistic, enthusiastic, and inspiring environment you have created. In particular, I would like to thank my co-supervisor Frank Hauser for his intelligent and kind support; Gedske for the innumerable times she has helped with anything and everything; Kim for making me feel welcome in the section even though I do not know the ways of the fly; Kenneth for help with the confocal; Michael for hosting parties, funny references and scientific support; Takashi for football, workout and career advice; Christian for fishing garfish and crows; Xristina for our discussions; and Jeannette for answering all my emails with a smile J. A huge thank to the people of Marine Biological Section. I am grateful to Anders Garm for our collaborations, his enthusiasm for nature and for putting the interest of his students ahead of his own. Thanks to Sofie Dam Nielsen, who has become a scientific sister to me, for or joint work and always being ready to discuss life as a Ph.D. student. Anders and Sofie were instrumental in sparking my interest in jellies and our common work on Tripedalia, has truly been some of the most joyful part of my thesis. I would also like to thank Sofus for the fun we had coloring jellyfish and his help with Trix in the confocal. I would like to thank the group at Institut für Tierökologie at Tierärtzliche Hochschule Hannover, Germany for hosting me and teaching the ways of Trix. I especially want to thank Bernd Schierwater for welcoming me to the world of placozoan research; Kai Kamm and Haju Osigius for scientific discussions; Wolfgang Jacob for making the stay very fun and enjoyable outside of the lab as well; and Jutta and Nicole for the specifics on how to please placozoans. A huge thank you to Carolyn Smith and Tom Reese and their group at National Institute of Health, Maryland, USA for letting me joint their lab in the summer of 2019. It was a wonderful experience, both on a personal and professional level, and I hope we will be able to finish some of the projects in the future. Thanks to Independent Research Fund Denmark for the funding awarded to my supervisor. Finally, a huge thanks to my family for their support, being awesome and getting after it. Thank you to all my hunting, climbing, drinking, fishing, grappling, scienceing, training, and random friends … and to the D, Minds and LZ for playing while annotating peptides. 1 Table of contents Acknowledgment 1 Table of contents 2 Abstract 3 Sammenfatning (Danish abstract) 4 Abbreviation 6 Chapter 1: Introduction 7 Chapter 2: Neuropeptides in Placozoa 28 Chapter 3: Neuropeptide Processing Enzymes in Placozoa 75 Chapter 4: GPCRs in Placozoa 97 Chapter 5: Ionotropic Receptors in Placozoa 120 Chapter 6: Immunostaining in Placozoa 126 Chapter 7/ Paper 1: “De Novo Transcriptome Assembly of the Cubomedusa Tripedalia 141 cystophora, Including the Analysis of a Set of Genes Involved in Peptidergic Neurotransmission”, BMC Genomics (2019) 20:175 Chapter 8/ Paper 2: “Global Neuropeptide Annotations from the Genomes and 184 Transcriptomes of Cubozoa, Scyphzoa, Staurozoa (Cnidaria: Medusozoa), and Octocorallia (Cnidaria: Anthozoa)”, Frontiers in Endocrinology, (2019) 10:831 Chapter 9/ Paper 3: “A Comparative Genomics Study of Neuropeptide Genes in the 218 Cnidarian Subclasses Hexacorallia and Ceriantharia”, BMC Genomics (2020) 21:666 Chapter 10/ Paper 4: “A comparative genomics study of neuropeptide genes present in the 291 cnidarian classes Hydrozoa and Endocnidozoa”, in preparation Chapter 11/ Paper 5: “Neuropeptide Gene Expression in the Box Jellyfish Tripedalia 328 cystophora – New Insights into the Complexity of Its Nervous System”, in preparation Chapter 12: General discussion 380 2 Abstract Background: Nervous systems are found almost throughout the animal kingdom, but when and how the nervous system evolved are still some of the big, unanswered questions in biology. To answer these questions it is necessary to investigate the early-branching animal phyla: Porifera, Ctenophora, Placozoa and Cnidaria, of which placozoans and cnidarians are the closest relatives of bilaterians. Neuropeptides are some of the oldest and most conserved signaling molecules of the nervous system with key roles in both bilaterian and cnidarian biology. Few years ago neuropeptides were found to be present in placozoans as well – a surprise as placozoans are believed to be animals without a nervous system. Due to the knowledge gap in neuropeptide signaling systems of Placozoa and Cnidaria the aim of this thesis was to investigate the peptidergic systems of these two phyla and to compare them. This would better enable us to understand the role of neuropeptides in the evolution of the nervous system. Results: Using a novel search strategy I performed the first global neuropeptide annotation in all placozoan and cnidarian classes and found that their neuropeptide repertoires are much larger than previously thought. The set of cnidarian neuropeptides varies a great deal between the classes, even thought they almost all share a core set of primordial neuropeptides: GRFamides, GLWamides and XPRXamides. The three investigated placozoans, in contrast, express the same set of neuropeptides. Even though Placozoa and Cnidaria may be sister phyla there are no apparent orthology between neuropeptide families in the two phyla; and neither to any bilaterian neuropeptides. Only few antibodies have been used to localize cnidarian neuropeptides, which gives a less than optimal accuracy and resolution of their nervous system. In order to improve this we employed specific antibodies against novel Tripedalia cystophora neuropeptides and found a previously unrecognized complexity of the cubozoan nervous system. This might explain the behavioral complexity seen in this cnidarian subclass. Placozoans, on the other hand, don’t have neurons that could be stained by neuropeptides (pQFFNPamide and pQANLKSIFGamide) or neuropeptide processing enzymes (glutaminyl cyclase), which instead are expressed in different subpopulations of gland cells around the rim of the animal or scattered in the interior. Based on this finding I conclude that placozoans don’t have peptidergic neurons but endocrine cells. Staining of placozoan fiber cells however show that this cell type possess a morphology like nerve cells with processes. Additionally, I find that placozoan G protein-coupled receptors in Placozoa are more diverse than previously thought, in particular leucine rich repeat receptors. 3 Conclusion: Placozoans and cnidarians are situated at opposite borders of the presence and absence of a nervous system. Despite their large differences in both morphological and behavioral complexity, genetically they both possess the necessary molecular machinery for peptidergic signaling. Cnidarians employ neuropeptides primarily in nerve cells, whereas placozoan peptide signaling is based on endocrine gland cells. It is likely that neuropeptides were first employed by secretory gland-like cells and were only adapted by nerve cells at a later stage in evolution. Sammenfatning Baggrund: Nervesystemet er til stede i stort set hele dyreriget, men hvornår og hvordan nervesystemet udviklede sig er forsat nogle af de store, ubesvarede spørgsmål i biologien. For at undersøge dets oprindelse er det nødvendigt at undersøge de tidlige forgrenede rækker: Havsvampe (Porifera), Ribbegopler (Ctenophora), Placozoa og Nældedyr (Cnidaria), hvoraf placozoer og nældedyr er tættest beslægtede med bilaterale dyr. Neuropeptider er nogle af de ældste og mest konserverede signaleringsmolekyler i nervesystemet og har vigtige funktioner i både nældedyr og bilaterale dyr. For få år siden blev neuropeptider også identificeret i placozoer, hvilket var opsigtsvækkende da placozoer ikke menes at have et nervesystem. For at udfylde vores mangelfulde viden om neuropeptidsignalering i Placozoa og nældedyr er formålet med denne afhandling at undersøge de peptiderge systemer i disse to dyrerækker