Expression of Serotonin in the Development of P atiriella species (Echinodermata: Asteroidea) with Different Modes of Development Francis Craig Chee A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in the Department of Anatomy and Histology. The University of Sydney Australia. August 2000 The data contained in this thesis is the result of my own work except where specifically acknowledged. Francis Craig Chee Dedication This doctoral thesis is dedicated to Jaya Anne Paffard, my best friend and wife. Georgeous, as she was known to me, gave me unyielding love, support and encouragement in regards to everything in life. She made me realise how lucky I was to be able to achieve a childhood dream of becoming a scientist. Jaya shared her interest and enthusiasm for my research leading to this thesis for which I was and am forever grateful. I know that she was proud of me while we were together and I also know that she would be just as proud of my efforts now. Her spirit has kept me going and will continue to do so in the future. I wish you could be here to see this. I love you so much Georgeous and I will miss you forever. General Abstract General Abstract Serotonin expression was examined in the development of three species of seastars belonging to the genus Patiriella. These species represented three different modes of larval development, planktonic planktotrophic (P. regulars), planktonic lecithotrophic (P. calcar) and benthic lecithotrophic (P. exigua). Preneuronal serotonin-like immunoreactivity was detected in the ectoderm of the early gastrulae of P. regulars, and P. exigua up until the hatched brachiolarial stage. As serotonergic neurons appeared at the animal pole of the gastrulae of P. regulars, preneuronal serotonin expression was no longer evident in the ectodermal cells. These neurons were not associated with sensory-like structures such as an apical ciliary tuff. It is therefore suggested that serotonin in these neurons may be functioning as a trophic substance for their own growth and/or the development of the ciliated bands. These early serotonergic neurons also appeared to migrate to specific regions of the developing larva appearing to be the precursor neurons for the larval nervous system. Pharmacological depletion of serotonin, with the drug p- chlorophenylalanine (L-PCPA), caused developmental abnormalities during gastrulation in P. regulars, P. calcar and P. exigua. These data suggested a morphogenetic role for serotonin during gastrulation. Drug treated P. exigua gastrulae also hatched prematurely, indicating a role for serotonin in the hatching process. Confocal immunofluorescence revealed ciliated serotonergic neurons in ganglia associated with the ciliated bands at the anterior and oral regions of the bipinnaria of P. regulars. Based on the disruption to feeding and swimming behaviour in P. regulars, induced by l -PCPA treatment, it is suggested that these ciliated serotonergic neurons function as sensory-like neurons in feeding and swimming behaviour. Reduction of serotonin content by L-PCPA also resulted in disruption to larval swimming in the lecithotrophic developer P. calcar. General Abstract Serotonergic neurons were found in the brachiolarial arms and ciliated bands of P. regularis although immunoreactivity was absent from the adoral ciliated band of the mouth and the attachment disk. It is suggested that the absence of serotonergic neurons from the oral region reflects the changes in larval behaviour from a feeding state of the bipinnaria to the brachiolaria, which is primarily concerned with settlement behaviour. The brachiolarial arms and adhesive disk of the lecithotrophic developers, P. calcar and P. exigua contained numerous serotonergic neurons. The apical region of the serotonergic neurons of P. exigua and P. calcar contacted the exterior of the epithelium and it is suggested that these neurons function in sensing the substratum. The disruption of settlement behaviour in P. exigua by L-PCPA treatment also suggests a role for serotonin in attachment. Overall it appears that serotonin acts as a multi­ functional neurochemical during different phases of development from the gastrula through to metamorphosis in Patiriella. Depletion of serotonin by l -PCPA was confirmed by reverse phase high performance liquid chromatography (rpHPLC). Irrespective of the morphological differences between the brachiolaria of the three species in this study, it appears that serotonergic neurons are conserved in like structures. A functional conservation of the role of serotonergic neurons in Patiriella is suggested. Acknowledgments ACKNOWLEDGMENTS This section of the thesis was probably the most physically difficult thing to do. Difficult, because of a reluctance to get emotionally stressed by the thought of having to put pen to paper or rather fingers to keyboard! There are many people to thank who have helped along the way and it has been a long way. A PhD is tough, there is no doubt about it. Everyone who decides to undertake one should know this. If you don’t then you're fooling yourself. It becomes part of your life and is partially governed by the same sets of life's instructions (whatever the hell they are). So if you have been part of my life then you must have had a hand in this thesis some way and somewhere along the line! I thank my partner Jaya for providing me with emotional and financial support and having confidence in my abilities. I thank my family for their understanding and encouragement regardless of the situation. I thank all of my friends (and you know who you are) for their unquestioning support and encouragement through thick and thin, especially over the last two years. I could not have done this without your help. A special thank you to Kate Michie for her recent friendship, support and rational thought in times of sanity meltdowns, and arse kicking me Grand Turismo® style into action when I wandered off the track, 11 Acknowledgments Thank you to all the people who helped me along the way, there is no order to following list: Associate Professor Maria Byrne (my supervisor), for providing me with the opportunity to do a PhD on the extremely interesting group of sea stars, from the Patiriella genus. For her enthusiasm, financial assistance and laboratory equipment to conduct my research. Robert Kelly and family for putting up with somewhat random late night disturbances to family life. The Medical Faculty, The University of Sydney for providing financial assistance for journal publications and overseas travel to conferences. Australian Research Council for providing financial assistance. Associate Professor Cedric Shorey Head of Anatomy and Histology for providing financial assistance in times of need. Sophie Mc Cloy, Rod Williams, Paulina and Julian Selvakumaraswamy for feeding me after late nights in the lab and looking after me, a big thankyou. Dr. Justine O'Brien for supplying the best goat serum at the drop of a hat. Dr. Anne Constable, and Mrs Zophie Dreher for supplying helpful advice on immunocytochemistry. Dr. Gavin Dixon for proof reading the manuscript and late night unannounced dinners. Paulina Selvakumaraswamy, (best "brainstorming" sessions and proof reading the entire thesis, thanks so much, I owe you big time!), Christain Ramofafia, Anna Cerra, Suzanne Long, Paula Cisternas, Dr. Suzy Renn. Acknowledgments Franca Mazzone and Demian Koop for providing help with manuscripts, advice and all of the things that great lab buddies do, like mountain biking action. Franca Mazzone (Dr. Mazzone) for helping with the compilation of the entire thesis, for late nights/early morning chats, medicinal laughter with Christain in the lab tea room and insane rollerblading extravaganzas around campus late at night. Simon Prone for lending Dr. Mazzone 24-7! And educating me about HiFi Dr. Yvette Morcos for all the worlds greatest screen plays and help with reading manuscripts and "short coffee breaks". Nick and Cath Cook for being there, regardless of the distance! Alistair Merricks for providing me with a job that most people would say "you'd have to be crazy to do". Dr. Eleni Taylor-Wood for helping with the mapping. The Biological Bulletin Woods Hole for publishing my work within and on the front cover of their centenary journal issue. Associate Professor Ove Hoegh-Guldberg for supplying laboratory space and culturing facilities. Associate Professor Ron Dimmock, Alistair Simpson and Professor Paddy Patterson for supplying the use of vital video equipment and recording facilities. Chris Willing at Sydney Visualisation laboratory for helping with high end video editing. Dr. Tom Fiztgibbon for manuscript editing. Clive Jeffrey for your excellent photographic skills. Acknowledgments Professor David Cockayne , the Director of the Electron Microscope Unit for providing excellent microscopy facilities. All the staff at the Electron Microscope Unit, The University of Sydney for their help and encouragement. Tom Joyce for the things that count at the last minute-like computer problems! Associate Professor Esther Leise for invaluable manuscript advice at 1:00 am in the morning! John Cross for all of your nagging, support and proof reading the entire thesis for grammatical errors (what a job!). Margie Maurice for collecting seastars from the icy waters of Tasmania. Dr. Leila Blackman for providing the use of the only other "best" printer on campus. Dr. Valerie Morris for providing advice on manuscripts. Mike Speak and Dr. Guy Cox for getting me out of computer binds over the phone late at night. Craig Sowden and Chris Macdonald at Sydney Aquarium for an endless supply of salt water. Dr. Elizabeth Harry for putting me in touch with people who know about HPLC. Professor Michael Slater, Department of Biochemistry, The University of Sydney for the use of his HPLC laboratory facilities. Dr. Guillermo Moreno (aka "King Billy", you didn't know did you?) for supplying endless amounts larvae, algae, and advice for a great part of my experiments. Dr. Doug Chappell, Department of Biochemistry for helpful advice and assistance using the HPLC.