The evolution of sensory and neurosecretory cell types in bilaterian brains DISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften Doctor rerum naturalium (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Dipl.-Biol. K. G. Kristin Teßmar–Raible aus Görlitz Marburg/ Lahn 2004 Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am angenommen. Erstgutachterin: Prof. Dr. Monika Hassel Zweitgutachterin : Prof. Dr. Renate Renkawitz-Pohl Tag der mündlichen Prüfung: The evolution of sensory and neurosecretory cell types in bilaterian brains To my parents and my husband for their encouragement and patience Acknowledgements I am very thankful to the following persons in Marburg and Heidelberg that made this study possible: Dr. Monika Hassel at Marburg University for the supervision of my PhD thesis and correction of the text; Dr. Detlev Arendt at the EMBL in Heidelberg for giving me the opportunity to spend time in his lab as a guest and for scientific advice; Dr. Monika Hassel, Dr. Renate Renkawitz-Pohl and the other members of my defense committee for reviewing this thesis. I want to acknowledge all members and guests of the Arendt lab, in particular Sebastian Klaus, Heidi Snyman, and Patrick Steinmetz, for for their help, their useful critical comments, and the interesting scientific discussions; in addition, I want to thank Heidi Snyman for very reliable practical help, especially with degenerated PCRs, minipreps, and restriction digests; and Dr. Detlev Arendt for minipreps of the Pdu-barH1 cloning. Moreover, I thank Dr. Florian Raible and Dr. Detlev Arendt for extensive critical feedback on previous versions of this text. There were certainly some difficult times when I was glad to be able to rely on the support and advise of more experienced people. For this, I am especially grateful to Dr. Florian Raible, Dr. Detlev Arendt, Dr. Oliver Hobert, Dr. Jochen Wittbrodt and Dr. Gary Ruvkun. I am thankful to Monika Beutelspacher and Dr. Herrmann Froehlich and the entire Boehringer Ingelheim Foundation not only for the financial support, that enabled me to perform this PhD study, but also for their encouragement and reliability. Much of my plannings and theoretical work depended on literature not easily avaible via the internet. I therefore thank the EMBL Szilárd librarians, especially David Wesley, who got many articles and books for me. I express my thanks for materials to Dr. Nicole Rebscher (anti-rabbit anti- RFamide(s) antibody), Alena Shkumatava and Dr. Carl Neumann (initial aliquots of cyclopamine). My special gratitude belongs to my husband Dr. Florian Raible, who is my truly best partner for both my scientific and personal life. Finally, I feel indebted to my parents and parents in law for their generous advice and help. Table of contents Acknowledgements ..........................................................................................................................i Table of contents ............................................................................................................................ii Index of Figures ...........................................................................................................................vii Index of tables .............................................................................................................................viii 1 INTRODUCTION ..........................................................................................................1 1.1 Defining homology relationships across Bilateria..........................................................2 1.2 Platynereis dumerilii – an ancestral organism for the comparative study of animal development and genome evolution.................................................................................4 1.3 The concept of homologous cell types .............................................................................8 1.4 The bilaterian nervous system........................................................................................12 1.4.1 The apical organ and its cell types...............................................................................................14 1.4.2 Neurosecretory cells.....................................................................................................................16 1.4.3 The vertebrate hypothalamus.......................................................................................................19 1.4.4 Cell types of photosensory structures..........................................................................................21 1.5 Aim of this thesis.............................................................................................................23 2 RESULTS ......................................................................................................................25 2.1 Indications for a conserved molecular organisation of Bilaterian brains ...................26 2.1.1 Pdu-nk2.1 is expressed in the median brain anlage complentary to Pdu-pax6..........................27 2.2 Molecular characterisation of the ciliary and rhabdomeric photoreceptor cell types in Platynereis dumerilii..................................................................................................31 2.2.1 Ciliary photoreceptor cell types...................................................................................................31 2.2.2 The opsin active in Platynereis ciliary PRCs closer relates to vertebrate rod and cone opsins than to canonical invertebrate opsins.................................................................................32 2.2.3 The Platynereis ciliary photoreceptor cells express rx, a transcription factor specific for the differentiating vertebrate rods and cones...........................................................................34 2.2.4 Rhabdomeric photoreceptor cell types ........................................................................................37 2.2.5 Platynereis atonal homolog/Pdu-ath is transiently expressed in the larval eye precursors .......38 2.2.6 Pdu-brn3 demarcates the larval eye region..................................................................................40 2.2.7 Cloning of Pdu-barH1 as additional rhabdomeric PRC marker gene ........................................42 2.2.8 Larval, but not adult eye regions are histaminergic ....................................................................44 2.3 Molecular characterisation of neuroendocrine regions and cell types in Platynereis .........................................................................................................................................44 TABLE OF CONTENTS iii 2.3.1 The Platynereis median episphere expresses homologs of transcription factors that are highly specific for the median/ventral forebrain in vertebrates ...................................................45 2.3.2 Orthologs of sim, pou3 and gsx transcription factors, essential for the formation of hypothalamic neuropeptidergic neurons, are also present in the apical organ region of Platynereis larvae................................................................................................................54 2.3.3 Analysis of markers that indicate specific differentiated cell types and exist in the median/ventral forebrain of vertebrates.............................................................................60 2.3.4 The lipophilic dye DiI stains a distinct set of cells in Platynereis larvae, including cells of the apical organ.........................................................................................................................76 2.4 A whole mount in situ hybridization screen uncovers additional genes involved in bilaterian brain development .........................................................................................78 2.5 The Hedgehog (Hh) pathway is involved in the development of the episphere in the Platynereis dumerilii trochophora.................................................................................81 2.5.1 Cloning and expression of members of the Hh-pathway in Platynereis dumerilii ....................83 2.5.2 Interfering with the hh-pathway using the drug cyclopamine ....................................................86 3 DISCUSSION................................................................................................................98 3.1 Equivalence between bilaterian photosensory and neurosecretory systems on different levels.................................................................................................................99 3.2 Common ancestry/homology is the most likely cause for the occurence of equivalent cell types across Bilateria ............................................................................................102 3.2.1 The master control gene concept: a valid alternative explanation?..........................................103 3.2.2 The molecular fingerprint is a useful criterium for cross-species comparisons.......................105 3.3 Is there conservation on the level of brain areas across Bilateria?...........................105 3.3.1 Robustness in molecular specification of regions within phyla as prerequisite for interphyletic comparison..................................................................................................107 3.3.2 No conservation of otx, bf-1, emx regions? ..............................................................................108