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. Acknowledgments

Professor Laurie Mather, Head Anaesthesiology and Pain Management

Laboratory Royal North Shore Hospital for the use of his laboratory.

Sonia Gu at the Anathesiology and Pain Management Laboratory Royal

North Shore Hospital for providing excellent assistance and advice on developing methods and analysis of HPLC chromatograms. Published papers from this thesis

A list of published papers from this thesis.

Visualization of the developing serotonergic nervous system in the larvae of the sea star, Patiriella regularis using confocal microscopy and computer generated 3-D reconstruction's. F. Chee and M. Byrne Invert. Reprod. Devel. 31: 1-3 151-158 1997

Serotonin-like expression in the gastrulae of Patiriella species (Asteroidea) F. Chee and M. Byrne Echinoderm Research 1998 183-184 Candia Carnevali and Bonasoro (Eds) 1999 Balkema, Rotterdam ISBN905809 102 3

Development of the larval serotonergic nervous system in the sea star Patiriella regularis as revealed by confocal imaging. F. Chee and M. Byrne Biol. Bull. 197: 123-131 1999

Serotonin-like immunoreactivity in the brachiolaria of larvae of Patiriella regularis. F. Chee and M. Byrne Invert. Reprod. Devel. 36: 1-3 1999

Serotonin depletion by para chlorophenylalanine (L-PCPA) on the larvae of Patiriella regularis: (Asteroidea): Effects on feeding and swimming behaviour. F. Chee and M. Byrne In Press Contents page

CONTENTS

GENERAL ABSTRACT...... i ACKNOWLEDGEMENTS...... ii LIST OF PUBLICATIONS...... iii TABLE OF CONTENTS...... iv-ix LIST OF ABBREVIATIONS x-xi

CHAPTER ONE: GENERAL INTRODUCTION 1.0 A brief history of nervous systems and neurotransmitters in the Echinodermata...... 1 1.1 Preneuronal/Prenervous serotonin expression during early development in echinoids and asteroids...... 2 1.2 The first serotonergic neurons...... 4 1.3 Expression of serotonin in a ’’nervous system”...... 4 1.4 Comparisons of serotonergic nervous systems in lecithotrophic larvae...... 6 1.5 Evolution of development in Patiriella (Echinodermata: Asteroidea...... 7 1.6 Experimental aims...... 8

CHAPTER TWO: GENERAL MATERIALS AND METHODS 2.1 Algal culturing...... 10 2.2 Larval culturing Patiriella regularis...... 10 2.3 Larval culturing P. calcar P. exigua_...... 11 2.4 Specimen fixation...... 12 2.5 Immunocytochemistry...... 12 2.6 Specimen preparation for confocal microscopy...... 13 2.7 Image acquisition...... 13 2.7.1 Scanning electron microscopy...... 13 2.7.2 Confocal microscopy...... 13 2.7.3 Image processing...... 14

IV Contents page

CHAPTER THREE: EXPRESSION OF SEROTONIN IN THE GASTRULAE OF PATIRIELLA REGULARIS, P. CALCAR AND P. EXIGUA (ASTEROIDEA):

EFFECTS OF p-CHLOROPHENYLALANINE (L-PCPA) ON GASTRULA AND

LARVAL MORPHOGENESIS 3.1 Abstract...... 16 3.2 Introduction...... 17 3.2.1 Definitions and aims...... 17 3.3 Materials and Methods...... 19 3.3.1 Larval culturing, specimen preparation and observation...... 19 3.3.2 Chromatography...... 19 3.3.3 Cation exchange purification...... 20 3.3.4 Qualitative analysis...... 20 3.3.5 Calibration curve...... 21 3.4 Pharmacology...... 21 3.4.1 Effects of L-PCPA on the planktotrophic feeder, Patiriella regularis: Serotonin and its role during morphogenesis...... 21 3.4.2 Effects of L-PCPA on the lecithotrophic species Patiriella exigua and P. calcar. Serotonin and its role during larval morphogenesis...... 22 3.5 Results...... 22 3.5.1 Reverse phase high performance liquid chromatography...... 22 3.5.2 Immunocytochemistry of P. regularis...... 23 3.5.3 Pharmacology P. regularis...... 23 3.5.4 Immunocytochemistry of P. exigua...... 24 3.5.5 Effects of L-PCPA on development in P. exigua and P. ca/car...... 25 3.6 Discussion...... 27 3.6.1 Preneuronal expression of 5-HT rpHPLC...... 27 3.6.2 Preneuronal expression of 5-HT immunocytochemistry...... 27 3.6.3 Possible developmental roles for serotonin in the planktotroph...... 28 3.6.4 Cytostatic effects of L-PC PA...... 29 3.6.5 Developmental abnormalities in P. exigua...... 30 3.6.6 l-PCPA affects hatching...... 31 3.6.7 Developmental abnormalities produced by L-PCPA...... 32 3.6.8 Comparative studies in vertebrate...... 34 3.6.9 Swimming activity in P. ca/car...... 34 3.6.10 Summary...... 34

v Contents page

CHAPTER FOUR: DEVELOPMENT OF THE BIPINNARIAL SEROTONERGIC NERVOUS SYSTEM IN THE SEASTAR PATIRIELLA REGULARIS AS REVEALED BY CONFOCAL IMAGING 4.1 Abstract...... 37 4.2 Introduction...... 38 4.3 Materials and Methods...... 39 4.4 Results...... 40 4.4.1 Gastrula...... 40 4.4.2 Bipinnaria...... 40 4.4.3 Adoral ciliated band...... 41 4.4.4 Preoral ciliated band...... 42 4.4.5 Postoral ciliated band...... 42 4.5 Discussion...... 43 4.5.1 The appearance of serotonergic neurons prior to a “nervous system”..... 43 4.5.2 Morphology of serotonergic neurons...... 44 4.5.3 Distribution of serotonergic immunoreactivity...... 44 4.5.4 Possible sensory function for serotonergic neurons...... 46 4.5.5 Comparisons with serotonergic ganglia in other echinoderms...... 46 4.6 Conclusion 47

vi Contents page

CHAPTER FIVE: SEROTONERGIC GANGLIA IN THE LARVAE OF

PATIRIELLA REGULARIS AND THE EFFECTS OF THE SEROTONIN DEPLETING DRUG p-CHLOROPHENYLALANINE (L-PCPA) ON FEEDING AND

SWIMMING 5.1 Abstract...... 49 5.2 Introduction...... 50 5.3 Materials and Methods...... 52 5.3.1 General larval protocols...... 52 5.3.2 Pharmacology...... 52 5.3.3 Imaging...... 53 5.3.4 Immunocytochemistry...... 54 5.3.5 Reverse phase high performance liquid chromatography...... 54 5.4 Results...... 56 5.4.1 General neuronal anatomy...... 56 5.4.2 The anterior ganglion...... 56 5.4.3 Preoral ganglion...... 56 5.4.4 The adoral ganglion...... 57 5.4.5 Effect of L-PCPA on feeding and swimming on Patiriella regularis...... 57 5.4.6 Effect of L-PCPA on swimming in Patiriella calcar...... 59 5.4.7 Unknown effects of L-PCPA...... 59 5.4.8 Immunostaining of L-PCPA treated bipinnariae...... 59 5.4.9 Chromatography...... 60 5.5 Discussion...... 60 5.5.1 Immunocytochemistry and confocal microscopy...... 60 5.5.2 The anterior ganglion...... 61 5.5.3 Comparisons of the anastomosing serotonergic processes in P. regularis with other asteroid bipinnaria...... 62 5.5.4 Imaging...... 63 5.5.5 Effects of L-PCPA on feeding...... 64 5.5.6 Effects of L-PCPA on swimming...... 64 5.5.7 Effects of L-PCPA on other biochemical pathways...... 65 5.5.8 Immunostained L-PCPA treated larvae...... 65 5.5.9 The decrease in swimming activity and the loss of feeding...... 66

5.5.10 A nervous system model to describe feeding behaviour in P. regularis,. 67 5.5.11 Reverse phase high performance liquid chromatography...... 68

VII Contents page

CHAPTER SIX: LOCALISATION OF SEROTONIN IN THE BRACHIOLARIA OF LARVAE OF PATIRIELLA REGULARIS, P. CALCAR AND P. EXIGUA 6.1 Abstract...... 70 6.2 Introduction...... 71 6.3 Materials and Methods...... 73 6.3.1 Immunocytochemistry...... 73 6.3.2 Histochemistry of P. calcar and P. exigua...... 74 6.3.3 Depletion of serotonin by p-chlorophenylalanine...... 75 6.4 Results...... 75 6.4.1 Serotonin-like immunoreactivity in the brachiolariae of Patiriella regularis 75 6.4.2 Advanced brachiolariae of P. regularis_...... 76 6.4.3 Serotonin-like immunoreactivity in the brachiolariae of P. calcar_...... 77 6.4.4 Histochemical staining of P. ca/car...... 78 6.4.5 Serotonin-like immunoreactivity in the early and hatched brachiolariae of P. exigua...... 78 6.4.6 Histochemical staining of P. exigua...... 79 6.4.7 Effects of p- chlorophenylalanine on P. exigua brachiolariae...... 80 6.5 Discussion...... 80 6.5.1 The planktotrophic developer...... 80 6.5.2 The lecithotrophic developers...... 83 6.5.3 Serotonin expression in the three species of seastar larvae...... 83 6.5.4 Speculative functional aspects of the serotonergic neurons in Patiriella calcar and P. exigua and suggested future directions...... 84 6.5.5 The nervous symmetry in the development of P. exigua brachiolariae...... 85 6.5.6 Comparisons of P. exigua and P. calcar to P. regularis...... 85

VIII Contents page

CHAPTER SEVEN: GENERAL DISCUSSION 7.1 What did this thesis set out to do?...... 87 7.1.2 Inhibition of preneuronal serotonin causes gross abnormalities during larval development...... 87 7.1.3 Hatching of P. exigua is affected by treatment with L-PCPA...... 89 7.1.4 The appearance of serotonergic neurons before the nervous system: The apical organ debate...... 90 7.1.5 The serotonergic nervous system in Patiriella regularis development..... 91 7.1.6 Serotonergic neurons in the brachiolar arms of: P. regularis, P. calcar and P. exigua...... 92 7.1.7 Regardless of the mode of larval development of the three Patiriella species, similar structures contain prenervous serotonin or serotonergic neurons.. 93 7.1.8 Addendum 93

LITERATURE CITED 94 List of abbreviations

List of abbreviations

0 diameter 3D Three dimensional 5-HT serotonin a anus A archenteron adcb adorai ciliated band adg adorai ganglion ag anterior ganglion axt axonal-like tract b blastopore be buccal cavity BSA bovine serum albumen CCD charged coupled device DIC differential interference microscopy EC enzyme commission FE fertilisation envelope FITC fluorescein isothiocyanate FSW (0.2 pm filtered seawater) 1 intestine Ic left coelomic pouch l-PCPA L-para chlorophenylalanine m mouth n neurite NA numerical aperture nm nanometre os oesophagus PBS 0.1 M phosphate buffered saline PC personal computer IBM compatible

v List of abbreviations continued

PC personal computer IBM compatible pocb postoral ciliated band procb preoral ciliated band rc right coelomic pouch rpHPLC reverse phase high performance liquid chromatography s stomach TPH tryptophan hydroxylase vn varicose neurite

VI Chapter one 1

General introduction

1.0 A brief history of nervous systems and neurotransmitters in the Echinodermata

Nervous systems were first described for adult echinoderms over 100 years ago by Hamann, (1887). Shortly after the advent of the transmission electron microscope (reviewed by Ruska, 1980) thin sections of biological material were first examined by Marton (1934a b). Almost 30 years later, the adult nervous systems of echinoderms were examined by electron microscopy techniques (Bargmann and Behrens, 1963) and various neuroanatomical structures have since been described in adult echinoderm tissue (reviewed by Pentreath and Cobb, 1972). One of the first neurotransmitters to be detected in echinoderms was serotonin. Serotonin has been shown to have numerous functions as a regulatory factor in morphogenetic events during early development in a diverse range of animal phyla (Brown and Shaver, 1989; Renaud, et al. 1983; Markova, et al. 1985; Hamalainen and Kohonen, 1989; see review by Lauder, 1993; Shuey, et al. 1993; see review by Buznikov, et al. 1996; Buznikov, et al., 1998; Shmukler, et al. 1999). Serotonin was found in the pre-gastrula stages of development (Buznikov, et al 1964). Based on behavioural studies, Strathmann, (1971, 1975) hypothesised that a possible nervous system could exist in planktotrophic larval echinoderms and these nervous systems may be responsible for the complex feeding and swimming behaviour associated with the ciliated bands in larvae. In a study by Toneby (1977), serotonin was detected in a developing asteroid larva, although neurons were not identified. Antibody labelling of echinoderm larvae in the 80's revealed that there were, in fact, neurons associated with the ciliated bands. Echinoderm embryos and larvae are used to study the location and function of neurotransmitters. The position of neurons expressing: serotonin-like; dopam inergic-like; GABA-like; cholinergic-like Chapter one 2 immunoreactivity; S1 neuropeptide; and unidentified catecholamines have been determined in the planktotrophic larval stages of: seastars; echinoids; holothuroids; crinoids and ophiuroids (Burke, 1983; Burke et al. 1986; Chia, et al. 1986; Bisgrove and Burke, 1987; Bisgrove and Raff, 1989; Nakajima, 1987a b; Nakajima, 1988; Nakajima, et al. 1993; Moss, et al. 1994; Chen et al. 1995; Byrne, et al. 1998; Falugi, et al, 1999). Functional aspects of neurotransmitters have been examined by pharmacological experimentation in the early embryonic stages of development (Gustafson and Toneby, 1970; Deeb, 1972; Markova, et al. 1985; Buznikov, 1991; Buznikov, et al. 1993; Buznikov, et al. 1998; Shmukler, et al. 1999) radio-labelling (Brown and Shaver, 1989). Other pharmacological studies of neurotransmitter influence on larval muscle contraction and ciliated band behaviour have focused on larval echinoplutei (Gustafson, et al. 1972; Soliman, 1983a).

1.1 Preneuronal/Prenervous serotonin expression during early development in echinoids and asteroids.

Thirty years ago Gustafson and Toneby (1970) examined the effect of numerous neuropharmacological and other chemical compounds on morphogenesis in early gastrulae of sea urchins. One of these compounds, para chlorophenylalanine, (L-PCPA) was used to interfere with serotonin biosynthesis and it was found that primary invagination was inhibited by the application of this drug. It is now known that classical neurotransmitters such as serotonin, dopamine, acetylcholine, noradrenaline, and gamma amino butyric acid are recognised as neurochemicals having multiple functions in both vertebrate and invertebrate development (reviews by: Lauder, 1988 and Buznikov, et al. 1996). Of these neurochemicals, serotonin has been the subject of numerous investigations into morphogenetic cellular events during pre- gastrulation in sea urchins and seastars, before the appearance of serotonergic neurons, and described as the “prenervous state” (Lauder, Chapter one 3

1983, Buznikov, et al. 1993, Shmukler, et al. 1999). The species used in the above studies have been exclusively those with planktotrophic development, where development progresses from a gastrula to a feeding echinopluteus (sea urchin) or bipinnaria and brachiolaria (seastar), eventually forming the juvenile rudiment and undergoing metamorphosis into an urchin or seastar. The presence of serotonin in non-neural tissue in the pre-gastrula stage, before the appearance of a serotonergic nervous system (prenervous/prenuronal), suggests that this neurochemical has a regulatory role during ontogenesis (Lauder, 1983; Buznikov, 1996). Prenervous expression of neurochemicals is thought to be an ancient function where several different neurotransmitters have been found in primitive organisms and early embryos that lack a nervous system (reviewed by Lauder, 1993). It is thought that the specialised roles of adult nervous systems may have evolved from primitive functions of intra- and intercellular signalling utilising prenervous neurochemicals in lower organisms (Lauder, 1993, Buznikov pers. comm.). The role of prenervous serotonin during post gastrulation events, such as larval development (see Chapter 1) have largely been ignored (Buznikov, et al. 1996). Using an undefined fluorescence detection method, Toneby, (1977) assessed the serotonin content during larval development of the seastar Pisaster ochraceus from the gastrula to the bipinnaria. Serotonin was not detected until the coelomic sacs had formed. In the same study, serotonin content in the bipinnaria of Pisaster ochraceus was quantified and found to be ten times greater than the serotonin content in the echinopluteus of Strongylocentrotus fransiscanus of an equivalent developmental age. This, however, was not a relevant comparison since it is comparing two different phylogenetically larval forms. Also, this assay did not distinguish between prenervous and nervous serotonin. In this thesis the term "prenervous neurotransmitter," describing the expression of serotonin before a recognisable serotonergic nervous Chapter one 4 system is present, is redefined as "preneuronal serotonergic expression", as the words "prenervous" and "neurotransmitter" appear somewhat contradictory.

1.2 The first serotonergic neurons and the apical organ

Several studies have shown a distinct cluster of serotonergic neurons described as a sensory apical organ (Lacalli, 1994) associated with the ciliary tuff occupying an apical position in the gastrula of the echinoids Heliocidaris tuberculata, Strongylocentrotus droebachiensis (Bisgrove and Burke, 1987, Bisgrove and Raff, 1989) and Hemicentrotus pulcherrimus (Nakajima, 1987b). It was suggested by Bisgrove and Raff (1989) that these neurons are similar to neurons seen in auricularia larvae of holothuroids (Burke, et al. 1986). This comparison is difficult to justify as the echinoid gastrula and the holothuroid auricularia are phylogenetically different larval forms and are also different larval stages. The presence of three serotonergic neurons as seen in figure 1 (Bisgrove and Raff, 1989) are referred to as a "nervous system arising shortly after gastrulation". These neurons have not been demonstrated to constitute a nervous system and the fate of these serotonergic neurons seen at the apical region of the echinoid gastrula was not determined (Bisgrove and Raff, 1989).

1.3 Expression of serotonin in a "nervous system"

A characteristic structure of all feeding larvae of the echinoderms is the ciliated bands (Strathmann, 1975). Using immunofluorescence and/or electron microscopic techniques, studies have shown serotonergic nervous systems to be associated with these ciliated bands (Nakajima, 1987a b, 1988, Bisgrove and Burke, 1987, Bisgrove and Raff, 1989, Moss, et al. 1994, see Chapters 2, 3 and 4). Chapter one 5

Only one study has shown serotonergic immunoreactivity in the neurons from an asteroid gastrula, which were present before the appearance of ciliated bands (Nakajima, 1988). However, the relevance of this serotonin expression in these neurons was not discussed. In a review on serotonergic apical organs by Lacalli, (1994), an apical organ was erroneously described for a seastar gastrula based on Nakajima’s 1988 study. In fact, the use of the term "apical organ", when referring to serotonergic cells first seen at the animal pole of asteroid and echinoid gastrulae, is quite confusing. For this thesis, the serotonergic cells seen at the anterior region of later larval stages, for example, the echinopluteus of echinoids, the bipinnaria of asteroids and the auricularia of holothuroids, be known as the "serotonergic anterior ganglion". Without cell fate studies to actually confirm that early appearing serotonergic neurons in the gastrula reposition themselves at the anterior region of the larvae, these neurons can only be assumed to be in a transitional state and be thus described as residing at the animal pole of the gastrula. As yet, the functions of serotonergic neurons in plutei and bipinnaria have not been elucidated. Based on the location of these neurons within the ciliated bands, and in structures known as the apical ganglion, apical organ (Lacalli, 1994) and anterior ganglion (see chapters 2 and 3), it has been speculated that serotonergic neurons may be involved in a sensory capacity implicated with metamorphosis (Bisgrove and Burke, 1987, Nakajima, 1988, Lacalli, 1994, Moss, et al. 1994). There is a paucity of knowledge surrounding the morphology and extent of serotonergic ganglia and/or serotonergic neurons in the ciliated bands of asteroid bipinnariae (Nakajima, 1988, Moss, et al. 1994). Ultrastructural examination of the bipinnaria of Asterina pectinifera revealed ciliated neurons which were suggested to be serotonergic sensory neurons (Nakajima, 1988). However, the cells described as neurons were not labelled for serotonin and can at best be described as putative serotonergic neurons. Serotonergic neurons have also been found Chapter one 6 associated with the adoral ciliated band (Nakajima, 1988) and this band is known to be involved in capturing food, and is thought to be under neuronal control (Strathmann, 1975). In a study based on the echinoplutei of Strongylocentrotus droebachiensis, serotonergic neurons were found in the lower lip ciliated band (Bisgrove and Burke, 1987), a similar region to that of the adoral ciliated band in an asteroid bipinnaria (Strathmann, 1975). However, Bisgrove and Burke (1987) speculated that these neurons function in metamorphosis. In many asteroids the brachiolaria represents the last stage of larval development prior to metamorphosis into a juvenile sea star. Brachiolariae have distinct structures, including the brachiolar arms and attachment disk, collectively forming the attachment complex (Hyman, 1955 and Barker, 1978). The attachment complex is used to help secure the brachiolaria to the substratum prior to metamorphosis. Ultrastructural studies have revealed that the attachment complex is highly innervated (Barker, 1978). To date the existence of a serotonergic nervous system in the asteroid brachiolaria, and its relationship to the bipinnarial serotonergic nervous system has not been determined. Chapter 4 provides a detailed description of serotonin-like immunoreactivity in the brachiolarial larval stage of Patiriella regulans and interprets its origins with respect to the bipinnariae of this species.

1.4 Comparisons of serotonergic nervous systems in planktotrophic and iecithotrophic larvae.

It is generally accepted that the feeding planktotrophic larva represents the ancestral larval form in echinoderms (Strathmann, 1978 and Cunningham, 1999). Lecithotrophic echinoid and asteroid larvae develop via a non-feeding larval stage which is associated with an evolutionary loss of the ciliated bands and functional gut. (Raff, 1987; Byrne, 1991, 1992, 1995; Byrne and Barker, 1991, Cerra and Byrne, 1995 a, b; Hart, 1997). One study has shown serotonergic neurons in the Chapter one 7 lecithotrophic larvae of Heliocidaris erythrogramma and compares this with the development of serotonergic neurons in the planktotrophic plutei of its congener H. tuberculata (Bisgrove and Raff, 1989). Changes in the expression of serotonergic neurons between these two larval forms were expected based on the different modes of larval development. It was found however, that the cellular location and morphology of serotonergic neurons were "putatively homologous" (Bisgrove and Raff, 1989). No studies have been done on congeneric asteroid species comparing the expression of serotonergic neurons between feeding and non-feeding larvae. This thesis details the expression of serotonin in the larvae of Patiriella species with planktotrophic and lecithotrophic developmental modes. Patiriella regularis larvae represent the typical planktotrophic asteroid bipinnaria, (Strathmann, 1975), having three distinct ciliated bands and a functional gut. In contrast P. calcar, a lecithotrophic planktonic developer and P.exigua, a lecithotrophic benthic developer lack ciliated bands and do not possess a functional gut.

1.5 Evolution of development in Patiriella (Echinodermata: Asteroidea)

In this thesis, the seastar genus Patiriella, was used as a model to explore serotonin expression between larval species with planktotrophic and lecithotrophic modes of development. This genus exhibits the greatest number of extant developmental modes seen for asteroids and has been extensively documented (Byrne, 1991, 1992, 1995; Byrne and Barker, 1991; Cerra and Byrne, 1995). The developmental modes, include planktotrophic, planktonic and benthic lecithotrophic, and intragonadal viviparity (Byrne, 1991; Byrne and Barker, 1991; Byrne and Cerra, 1996). The close taxonomic relationship between the planktotrophic and lecithotrophic species allows phylogenetic comparisons to be made with respect to evolutionary conservation and change in preneuronal and neuronal serotonin expression within this genus. Chapter one 8

This thesis also examines the development and function of preneuronal and neuronal serotonin from gastrula to bipinnaria and brachiolarial stages of the genus Patiriella. In line with advancements in microscopy techniques, confocal microscopy is used to non-destructively examine the three dimensional expression of serotonin in complex asteroid structures. The advantages of using an asteroid model are: 1 the ease of producing thousands of mature oocytes by intracoelomic or bath application of 1- methyladenine (Kanatani, 1969) over relatively short periods of time (several hours) compared with using a vertebrate model which can take weeks to develop. 2 the easy manipulation of the external environment to perturb normal behavioural functions is facilitated by their size and culture requirements. 3 their transparency (in most cases) facilitates ease of use for microscopical examination.

1.6 Aims of this thesis are:

1. To determine if preneuronal serotonin expression occurs in the gastrulae of Patiriella and if so to elucidate the role of serotonin in gastrula and larval morphogenesis by examining the consequences of pharmacological depletion of serotonin. Chapter 3. 2. To illustrate, by confocal immunofluorescence microscopy, a detailed temporal and spatial study of serotonin expression in the development of planktotrophic, planktonic lecithotrophic and benthic lecithotrophic asteroid larvae. Chapter 4. 3. To examine the functional consequences of depleting serotonin in developing bipinnaria and brachiolarial larvae. Chapter 5. 4. To determine what similarities in serotonin expression exist between three congeneric sea star species, in order to explore the possible evolutionary conservation or changes in serotonin expression that might be associated with different modes of larval development. Chapter 6. Chapter two 9

General Materials and Methods Chapter two 10

2.1 Algal culturing

Axenic cultures of several phytoplankton, Dunaliella tertiolecta (obtained from Dr. Ray Ritchie The Department of Biological Sciences The University of Sydney), Chaetoceros calcitrans CS-178, and Rhodomonas sp. Cryptophceae strain CS-202 (obtained from the CSIRO Division of Fisheries Hobart Tasmania Australia) were grown in 1000 ml_ of Guillards’ F2 media (Guillard 1983) in Pyrex flasks. The cultures were irradiated for a 12hr day length seven days per week with Coralife® 10,000K™ Super Daylight and Coralife® 6000K™ fluorescent tubes at a distance of 0.7 m at 19°C (±1 °C). Air was blown through Tygon® tubing via Millipore Millex-GV 0.22 pm inline filters into the media to aerate the cultures.(Fig. 1). Prior to feeding, algae were aseptically removed and spun at 1900 rpm at 4°C for 2 minutes to remove culture media for replacement with 0.2 pm filtered sea water (FSW).

2.2 Culturing of the planktotrophic species Patiriella regularis

Patiriella regularis adults were collected from the River Derwent Estuary, and Wrest point Casino Tasmania (Fig. 2A) in the months of November to January 1994-1998 and shipped via air cargo to the laboratory where they were kept at 19°C (± 1°C) in aquaria or plastic trays and fed a diet of live or frozen Donax deltoides (Lamark 1818) (Fig. 2B). Mature oocytes were obtained by intracoelomic injection of the starfish with 10"5 M 1-methyladenine (Sigma) (Kanatani 1969) in FSW. Spawning occurred within 2 to 3 hours at 23°C. Testes were dissected from mature males and a dilute solution of sperm was prepared by gently homogenizing the testes in FSW and approximately 100 pL was added to the eggs. Fertilisation success was checked with a stereo microscope by Chapter two

Figure I Erlenmeyer flasks containing axenic algal cultures. Note the in-line air filters (black arrow) and disposable pipettes used for outlets (white arrow).

Chapter two

Figure 2A Location of collection sites (circles) in Tasmania Australia for Patiriella regularis adults.

Figure 2B Patiriella regularis adults feeding on Donax deltoides (Lamark 1818) in the laboratory. ft

Hr*s Bridgewater

* Richmond Chapter two

Figure 3A Laboratory culture set up for culturing Patiriella regularis embryos and larvae. A yellow arrow indicates the cam driven perspex plate. Bulk culturing of larvae in 5 litre beakers, indicated by white arrow.

Figure 3B Map showing the location in Sydney where Patiriella exigua and P. calcar were collected.

Chapter two 11 the presence of an elevated fertilization envelope after 15 minutes. The fertilised eggs were washed in FSW to remove any remaining sperm and transferred to 600 mL of FSW in borosilicate beakers and gently stirred by a cam driven perspex plate containing polyethylene paddles (Fig. 3A) (Moreno, 1996). The water was changed every four days and the larvae fed every two days. Bulk culturing was also conducted using 5 L beakers with a rotating (10 rpm) 0.01 m wide polyethylene paddle (Fig. 3A). All submerged culturing equipment was washed with 75°C water. Larvae were raised at 19°C ±.1 °C.

2.3 Culturing of the lecithotrophic species Patiriella exigua and Patiriella calcar.

Adult Patiriella exigua and P. calcar were collected inter-tidally at Clovelly Bay and at Shelly beach Manly, Sydney (Fig. 3B) on the day of an experiment. Gonads were excised from P. calcar females (Fig. 4A-C) and placed in a solution containing approximately 10"5 M of 1-methyladenine in FSW. Eggs were released after approximately 4 hours. Testes were dissected from mature males and a dilute solution of sperm was added to the eggs for fertilisation. After 1 hour the eggs were removed to crystallising dishes containing FSW. Mature oocytes were also obtained from P. exigua by intra coelomic injection of both sea star species (Image not shown). Embryos were kept in 300ml of FSW in crystallising dishes at 23°C. The FSW was replaced every 2 days. Chapter two

Figure 4A-C (A) A generalised view of a dissection of a female seastar (Patiriella calcar). (B) Oocytes within the gonad (arrow). (C) Male gonads (arrow).

Chapter two

Figure 5 A schematic representation illustrating the confocal principle of optically sectioning a biological sample (after Michie 98). Photomultiplier (detector)

Confocal aperture Neutral density filter

Laser source Dichroic mirror

Out of focus rays

In focus Objective rays lens

Focal plane Specimen Chapter two 12

2.4 Specimen Fixation

Specimens were fixed in 4% paraformaldehyde (Probing & Structure, Prill grade 95.89% formaldehyde) in FSW at 22°C for 1-2 hr*, rinsed thoroughly in FSW and then placed into 0.1 M phosphate buffered saline pH 8.2-8.3 (PBS) prior to antibody labelling. (Note P. exigua were dissected from their fertilization envelopes using a 19 gauge hypodermic needle as a blade).

2.5 Immunocytochemistry

After fixation, specimens were immediately treated for immunocytochemistry and all steps were carried out at 19-23°C*. All antibodies were diluted with PBS containing 1 % bovine serum albumin (BSA, Boehringer Mannheim). Prior to antibody incubation, specimens were treated for 30 min in PBS containing 10% normal goat serum (Vector laboratories) and 0.3% Triton X 100 (BDH Chemicals) to reduce non­ specific staining and to aid antibody penetration. Following each incubation the specimens were washed with gentle agitation in three 15 min changes of PBS. Specimens were then incubated first in the primary antibody, rabbit anti-serotonin (Incstar/Dia Sorin) diluted 1 in 100, for 16 to 22 hr* and then in the secondary antibody, biotinylated goat anti rabbit IgG (H+L) (Vector laboratories) 1 in 50, for 2 hr*. The final incubation was in a 1 in 100 dilution of fluorescein (FITC) labeled streptavidin (Vector Laboratories) for 20 min* in the dark. Controls consisted of omitting the primary antibody, omitting the secondary antibody, using normal rabbit serum as a substitute for the primary antibody and checking for autofluorescence using only paraformaldehyde-fixed embryos and larvae. (*unless specified otherwise) Chapter two 13

2.6 Specimen preparation for Confocal Microscopy

Immunostained specimens were mounted in welled slides in an aqueous solution of either Vector shield (Vector laboratories) or Fluoroguard (Bio-Rad Laboratories) and the coverslips were sealed with nail polish. Specimens were viewed immediately or stored at 4°C in the dark. Slides stored for several months showed no sign of fading when examined.

2.7 Image Acquisition

Images were photographed on Kodak EPH colour slide film or digitally acquired using a Nikon E800 microscope fitted with a SensiCam 12 bit cooled digital camera (PCO computer optics GmbH Kelheim) and/or Zeiss Axiophot fluorescence microscopes.

2.7.1 Scanning electron microscopy

All scanning electron micrographs were digitally acquired on a Philips XL30 scanning electron microscope. Specimens were supplied by M. Byrne and A Cerra, The University of Sydney Department of the Anatomy and Histology.

2.7.2 Confocal Microscopy

All specimens were examined with a confocal laser scanner coupled to an epifluorescence microscope (a Bio-Rad MRC600 scanner and a Zeiss Axiophot microscope or an MRC1024 and an Olympus BX 60 microscope). The 488 nm line of the Krypton/Argon laser was used with a Chapter two 14

520DF32-nm filter block. The basic outline, of the optical pathways in a confocal microscope are shown in figure 5.

2.7.3 Image processing

Image projections (using maximum pixel intensities) and three- dimensional (3-D) stereo anaglyphs were created on a PC Pentium® II platform using Confocal Assistant (Todd Clark Brelje Software version 4.02). Computer animations were produced using a Silicon Graphics XS24 4000 Unix workstation with Voxel View Ultra volume rendering software and Silicon Graphics movieconvert software to produce QuickTime® and AVI videos (included on the CD ROM). Unless otherwise stated, sharpening algorithms were not used in the production of final images. Red/Green anaglyphs can be viewed preferentially with Red/Green or Red/Cyan stereo glasses respectively (red left eye, green right eye). All images are false coloured unless otherwise stated. QuickTime animations on CD ROM are included. Chapter three 15

Expression of Serotonin (5-HT) in the gastrulae of Patiriella regularis and P . exigua (Asteroidea): Depletion of Serotonin by p-chlorophenylalanine (L-PCPA): Effects on early and late morphogenesis. Chapter three 16

3.1 ABSTRACT

The early gastrulae of the sea stars Patiriella regularis and P. exigua exhibited a preneuronal expression of serotonin-like (5-HT-like) immunoreactivity in the apical cytoplasm of the ectoderm. The fluorescent signal was strongest adjacent to the base of the cilia. In contrast, the invaginating cells of the vegetal plate, and archenteron did not exhibit 5- HT-like immunoreactivity. The presence of serotonin (5-hydroxytryptamine, 5-HT) in these gastrulae was confirmed by reverse phase high performance liquid chromatography (rpHPLC) with electrochemical detection. Subsequently, as the embryos of P. regularis developed into the late gastrula and feeding bipinnarial stages, there was a visible decrease in non-neuronal expression of 5-HT as the nervous system developed. In contrast the preneuronal expression of 5-HT in the lecithotrophic embryos of P. exigua continued until the brachiolarial stage. The presence of serotonin in early Patiriella embryos suggests that this neurochemical is acting as a morphogen during early development of these seastars. To determine if the potential morphogenetic role of serotonin is similar across Patiriella species, the early gastrulae of P. regularis, P. exigua and P. calcar were treated with L- para chlorophenylalanine (L-PCPA), a potent serotonin biosynthesis inhibitor. Treatment with this drug during embyrogenesis induced teratogenic effects in P. regularis, including malformation of the gut, incomplete coelom development, cell lysis, arrested development, and death before larvae could reach a feeding state. Application of L-PCPA on the embryos of the lecithotrophic developers P. calcar and P. exigua also caused teratogenic effects, such as premature hatching of P. exigua, the benthic larva, and stunted brachiolar arm growth in both lecithotrophic species. In addition, drug treated P. calcar lost the ability to swim. These data support the theory that serotonin is directly involved in early morphogenesis at the preneuronal stage of embryogenesis and during later larval development. Chapter three 17

3.2 INTRODUCTION

Serotonin (5-hydroxytryptamine, 5-HT), in addition to its more familiar role as a classical neurotransmitter, (Frazer and Hensler, 1994) plays an important role in early development. Serotonin has been shown to have numerous functions as a regulatory factor in morphogenetic events during early development in a diverse range of animal phyla (Brown and Shaver, 1989; Renaud, et al. 1983; Markova, et al. 1985; Hamalainen and Kohonen, 1989; see review by Lauder, 1993; Shuey, et al. 1993; see review by Buznikov, et al. 1996; Buznikov, et al., 1998; Shmukler, et al. 1999). Planktotrophic echinoids have been studied for research into early morphogenetic events during and following blastulation and studies have shown that serotonin is involved in these early events (see reviews Buznikov, et al. 1996 and Buznikov, et al. 1998). Although there have been a number of studies showing the presence of serotonergic nervous systems in larvae of planktotrophic asteroids, (Moss, et al. 1994; Nakajima, 1988; Chapters 2 and 3), and similarly for echinoid larvae (Bisgrove and Burke, 1986; Bisgrove and Raff, 1989; Nakajima, 1993), little is known regarding the roles of preneuronal serotonin, in late and post gastrula stages of echinoderm development prior to the appearance of a serotonergic nervous system. There have been no comparative studies examining preneuronal serotonin expression during gastrulation and post gastrulation between planktotrophic and lecithotrophic congeners in either echinoids or asteroids. Chapter three 18

3.2.1 Definitions and Aims

In this thesis, the term "preneuronal" is defined as the detectable presence of 5-HT by both im mu nocytochemistry and high performance liquid chromatography prior to the formation of identifiable serotonergic neurons. This chapter explores the following questions: 1. What is the cellular location of preneuronal expression of serotonin in the gastrulae of a planktotrophic developer verses a lecithotrophic developing echinoderm? 2. Does the expression of preneuronal serotonin differ between different modes of larval development? 3. What (specific) events of post gastrula (larval) morphogenesis can be attributed to preneuronal serotonin? (For example, gut development, brachiolar arms etc). Generally the term "morphogen" has been used in connection with neurotransmitters to describe neurochemicals as regulators of morphogenetic processes (Lauder, 1988). In this thesis, serotonin is described as a "morphogen" where an inhibition of serotonin leads to a change in normal morphogenetic events. To explore questions 1 and 2, comparative studies based on preneuronal serotonin expression were made on P. regularis and P. exigua representing both planktotrophic and lecithotrophic benthic modes of development respectively. P. regularis is considered to represent the ancestral mode of echinoderm development (Hart, et al. 1997) and has 3 morphologically different developmental stages after gastrulation. These are the early bipinnaria, bipinnaria and brachiolaria. The comparative species Patiriella exigua exhibits an abbreviated form of development where a gastrula passes directly to a brachiolaria within a fertilisation envelope and has been suggested to represent the derived form of asteroid development within the Patiriella clade (Byrne, 1995 and Hart, et al. 1997). Serotonin presence in the gastrulae of P. regularis and P. exigua was qualitatively determined by reverse phase high performance liquid Chapter three 19 chromatography (rpHPLC), using electrochemical detection. The cellular location of serotonin was determined by immunofluorescence and visualised by confocal microscopy. To examine the morphogenetic events corresponding to preneuronal serotonin expression, L-para chlorophenylalanine (L-PCPA) was used to deplete serotonin at the gastrula stage. Para chlorophenylalanine is a potent inhibitor of the enzyme tryptophan hydroxylase (TPH) (Enzyme commission (EC) 1.14.16.4) (Jequier et al. 1967). Tryptophan hydroxylase catalyses the rate limiting step in the biosynthesis of 5-HT, converting the amino acid tryptophan into 5-hydroxytryptophan in the synthesis of 5- hydroxytryptamine (serotonin) (Frazer and Hensler, 1994). The effects of L- PCPA on larval morphogenesis were examined using P. exigua and the lecithotrophic planktonic congener P. calcar as a comparison. Preneuronal expression of serotonin is discussed with respect to the evolution of development of the three congeneric asteroid species.

3.3 MATERIALS AND METHODS

3.3.1 Larval culturing, specimen preparation and observation (See section 2)

3.3.2 Chromatography

Reverse phase high performance liquid chromotography (rpHPLC) was based on the techniques of Kulkarni and Fingerman (1992) and Hansson and Rosengren (1978). Gastrulae of P. régularisant P. exigua were pelleted in FSW in Eppendorf tubes, the FSW was removed and then covered with 1 mL of cold 0.4 M perchloric acid, sonicated and/or shaken by hand several times, then processed through three freeze/thaw cycles in liquid nitrogen. The samples were centrifuged at 3000 rpm for 15 minutes at 4°C and the supernatants removed and adjusted to pH 6.0 with 2 M potassium carbonate. After standing for several minutes in ice, the Chapter three 20 samples were centrifuged to sediment carbonate precipitate. This process was repeated until all carbonate deposits were removed. The resulting extracts were aliquoted to glass ampoules, lyophilized, sealed and stored at 20-23°C in the dark until required for cation exchange purification.

3.3.3 Cation Exchange Purification

Sepak® Accell Plus CM (Waters) weak cation exchange cartridges were preconditioned with 6-10 hold up volumes of 18 MQ water prior to being filled with 18 water rehydrated extracts. The cartridges were attached to an Alltech 12 port vacuum manifold and the flow rate was adjusted to approximately 50 pL per minute. A sample of five elutents was kept to inject into the column to check that the flow rate was appropriate. Once the flow rate was stabilized (50pL per minute), the remaining elutents were discarded. The 5-HT adsorbed onto the cation exchange resin was then eluted using 500 pL of 1.2 N HCI. The eluents were either frozen at -20°C or injected (25 pL) immediately into the column.

3.3.4 Qualitative Analysis

Analysis was conducted with a Varian 5000 HPLC liquid chromatograph fitted with a 100 pL injection loop. The mobile phase was prepared according to Kulkarni and Fingerman (1992) and automatically degassed using an in line X-ACT 4 channel degassing unit (Jour Research). A Waters™ Resolve® 5 pm spherical Ci8 3.9 x 150mm stainless steel column was used for the isolation. The column was also fitted with a small Sentry® (Waters) guard column containing the same stationary phase as above. A Waters™ 464 pulsed electrochemical detector fitted with a dual glassy carbon electrode was used for the detection of 5-HT. The current was set at 0.5 pA for the samples and 1.0 pA for the standards. Lower current Chapter three 21 values (up to 20 nA) for sample detection were required as the number of sample injections increased. Detection was carried out when the flow rate stabilized at 0.6 ml_ per minute. The extracts were sampled 12 times.

3.3.5 Calibration curve

Nine standards were prepared according to the method of Kulkarni and Fingerman (Kulkarni & Fingerman, 1992) ranging from 0 to 52.5 ng/mL of 5-HT creatinine sulphate monohydrate (Aldrich). Each standard contained

10"5M 3,4-dihydroxybenzylamine hydrobromide (DHBA Aldrich) as an internal standard. The samples were loaded onto the Sepak® cartridges and treated exactly as the larval extracts. Dopamine HCI (Sigma) was also injected as above onto the column to confirm the presence or absence of this catecholamine. The retention time for the 5-HT standard was 143 seconds.

3.4 Pharmacology The concentrations of para chlorophenylalanine (L-PCPA) used in these experiments were based on a pilot study to determine from a range of 0, 0.1,0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 7.0, 10, 20, 30, 40, 50, 60 pM L-PCPA, the lethal concentrations required for each species. Lethality was defined as rupturing of the embryo resulting in death within 24 hours of drug application. It was found that L-PCPA concentrations greater than 5.0 pM (for P. regulars and P . calcar) and 10 pM (for P. exlgua) caused immediate death.

3.4.1 Effects of L-PCPA on the planktotrophic feeder, Patiriella regularis: Serotonin and its role during morphogenesis.

Larval culturing (see general materials and methods sections 2.2.1-2.2.2) Patiriella regulars gastrulae (24 hours post fertilization) were treated with l-PCPA which had been dissolved in 0.2 pm FSW immediately prior Chapter three 22 to use. Experiments conducted on the gastrulae consisted of 2 treatments (2.5 pM and 5.0 pM L-PCPA) each with six replicates. Each replicate consisted of 20 randomly selected gastrulae placed into sterile six well Linbro® tissue culture plates (Flow Laboratories Inc.) with a well capacity of 9.6 mL. Cultures were raised at 23°C and the controls consisted of gastrulae raised in 0.2 pm FSW. Larvae were observed for morphological changes with a light microscope over a period of 48 hours.

3.4.2 Effects of L-PCPA on the lecithotrophic species Patiriella exigua and P. calcar. Serotonin and its role during larval morphogenesis.

Larval culturing (see general materials and methods sections 2.2.1-2.2.2)

Gastrulae of P. exigua and P. calcar (24 hours post fertilization) were randomly selected and placed into sterile six well Linbro® (Flow Laboratories Inc.) tissue culture plates with a well capacity of 9.6 mL. The experimental design was as described above. The treatments consisted of 2.5, 5 and 10 pM L-para chlorophenylalanine (L-PCPA) in 0.2 pm FSW. Morphogenesis was followed by microscopy of living embryos using an Olympus BHZ stereo dissecting microscope. The morphological features recorded were brachiolar arm formation for both species (stunting verses no stunting) and swimming behaviour for the planktonic P. calcar. Observations for both treated and control P. exigua and P. calcar were made at 51, 70.5, 75.5 and 120 hours and 52 hours post fertilization respectively. Chapter three 23

3.5 RESULTS 3.5.1 Reverse phase high performance liquid chromatography.

Patiriella regularis and P. exigua 24 hour post fertilized gastrulae samples injected onto the column gave consistent retention times of 143 seconds, the same as that for the 5-HT standards, confirming the presence of 5-HT (Figs. 1A and B). Dopamine was also detected in the gastrulae of P. exigua.

3.5.2 Immunocytochemistry P. regularis

On hatching from the fertilization envelope (FE), gastrulae are spherical and commence swimming immediately (Fig. 2A). Cells displaying 5-HT- like immunoreactivity were evident in the swimming gastrulae of P. regularis at the onset of invagination of the vegetal plate, approximately 24 hours post fertilisation (Figs. 2, Ai-Aii). The ectoderm was strongly fluorescent whereas the developing endoderm of the archenteron was not immunoreactive (Fig. 2Ai). Optical sections revealed that this immunofluorescence was localized to the apical region of the epithelial cells above the basally located unstained nuclei (Fig. 2Aii). This generalised immunoreactivity in the epithelium of the gastrula represents a preneuronal expression of 5-HT at the gastrula stage. As shown in Chapter 4, the first immunoreactive neuronal-like cells appear during late gastrulae (Fig. 2B Chapter 3). The gastrula subsequently elongates as the archenteron forms at the vegetal pole (Fig. 2B) developing into an early feeding bipinnaria (Fig. 2C lateral view) which has an ellipsoid shape and a prominent digestive system. After 48 hours the ciliated bands are distinct along with two lateral coelomic pouches either side of the digestive system. The larva broadens across the oral region and shape is no longer ellipsoidal (Fig. 2D ventral view). After approximately 10 to 12 weeks in culture, larvae develop to the brachiolarial stage (Fig. 2E) (see chapter 4). Chapter three

Figure 1 Reverse phase high performance liquid chromatograms of Patiriella regularis and P . exigua for gastrulae, 24hr post-fertilisation sample extracts. (A) P. regularis (B) P. exigua The first peak (from left to right) represents the solvent front, the second peak is unknown. Patiriella regularis gastrulae

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Archenteron, A; Blastopore, b; Brachia. Be; Intestine, I; Mouth, m; Oesophagus, o; Stomach, s; Preoral ciliated band, procb; Postoral ciliated band, poeb. 10-12 weeks Chapter three 24

3.5.3 Pharmacology

Effects of l-PCPA on development of P. regularis

Gastrulae raised in 2.5 pM L-PCPA for 2 days exhibited a range of developmental stages (Fig. 3 A-D). Partial gastrulation (Fig. 3A) and larval- like stages (Fig. 3B-D) were observed within the 48 hour period. Degeneration of the developing coeloms and the archenteron was evident with mesodermal cells detaching from the left and right coelomic pouches to move into the blastocoel (Fig. 3 B-D). In addition aggregations of celis appeared in the archenteron (Fig. 3B). A few larvae formed an archenteron but did not undergo coelomogenesis (image not shown). In these larvae, mesodermal-like cells detached from the archenteron and then formed aggregates around the archenteron (Fig. 3C). After 48 hours in 2.5 pM l-PCPA there was a complete absence of any differentiated digestive structures although a ring of cells surrounded the blastopore. (Fig. 3D). This was followed by developmental arrest and mortality as the larva disintegrated (image not shown). The experiment was terminated after 48 hours; however, it was noted that some larval-like morphs continued to swim for up to 5 days. Gastrulation in P. regularis was immediately arrested by immersion in 5 pM l-PCPA. After 48 hours in L-PCPA, the embryonic epithelium either remained intact with the embryos spinning as a ball of cells, or ruptured releasing cytoplasmic debris into the seawater (Fig. 3E). The presence of columnar cells at one end of the embryos suggested vegetal plate formation. Parallel control cultures had reached the feeding bipinnaria stage after 2 days (Fig. 1D). Chapter three 25

3.5.4 Immunocytochemistry of P. exigua

Confocal optical sectioning through immunostained 24 hour gastrula of P . exigua (Fig. 4A) revealed preneuronal serotonin-like immunoreactivity in the epithelium (Fig. 4B). Following on from the gastrula stage, the circular blastopore closes after approximately 51 hours post fertilization to form a slit, which gradually widens and two lateral folds formed at the animal region (image not shown). The FE changes colour from clear to translucent red, thus simultaneously becoming adhesive sticking tenaciously to the culture dish. The developing larva presses against the FE and after 70.5 hours, the formation of brachiolar arms had become visible as several small lines on the surface of the anterior end of the larva. (Fig. 4C). At 75.5 hours the larva has three distinct brachiolar arms tightly bunched within the FE (image not shown). A tear occurs in the FE next to the anterior region of the larva due to an enzymatic process as presumed by Byrne and Barker (1991), and the tripod-like brachiolaria emerges after approximately 120 hours (Fig. 4D). The tearing of the FE at the anterior region of the larvae was observed in 99% of the controls. Chapter three

Figure 3 Five different and simultaneous larval configurations observed after P. regularis gastrulae were treated exposed to 2.5 and 5.0 pM L-PCPA for 48 hours. (A) Partial gastrulation showing breakdown of the archenteron. (B) Degradation of the two coeloms (arrowheads) indicated by a black arrow, showing cells being lost from the coelom. (C) Cellular aggregations in the archenteron, ca. (D) A larval-like form, lacking a functional gut. Arrowhead indicates the ring of cells around the anus. (E) Release of cytoplasmic contents (arrow) from a rupturing 5.0 pM L-PCPA treated gastrula. Five different developmental morphs observed after P. regularis gastrulae were exposed to l-PCPA for 48 hours D

Key

2.5 pM L-PCPA

5.0 pM L-PCPA Chapter three 26

3.5.5 Effects of L-PCPA on development in P. exigua and P. calcar.

Patiriella exigua treated with 0.1 pM L-PCPA, were morphologically similar to the controls (image not shown). Concentrations of 2.5-10.0 pM L- PCPA caused a teratogenic effect on larval development (Figs. 4 E-H). After 120 hours 2.5 pM L-PCPA treated larvae had hatched with stunted or no brachiolar arm formation (Fig. 4E). Premature hatching occurred in every instance for larvae that had been treated with 5 pM L-PCPA after 70.5 hours (Fig.). A small region of the fertilization envelope (FE) opposite the blastopore became perforated and the larva expanded through the constricting opening (Fig. 4G). Prior to 120 hours, gastrulae, which had been treated with 10 pM L-PCPA, appeared no different to the control gastrulae. However, at 120 hours a tearing of the FE occurred with the extrusion of rapidly degrading embryonic contents (Fig. 4H). The gastrula of P. calcar develops inside a clear FE, which does not stick to the culture dish. The surface of the gastrula is ciliated however, no ciliated bands are present (image not shown). The un-hatched gastrula spins in a clockwise direction with the blastopore facing downwards. On hatching the blastopore closes and the gastrula begins to elongate. The developing larva still spins but its rotation is now observed to be anti­ clockwise. This apparent change in direction of rotation is merely a result of a re-orientation of the anterior/posterior axis from a vertical position to a horizontal position. Within 52 hours of fertilization, the gastrula elongates along the anterior posterior axis and well-defined brachiolar arms formed at the anterior end of the larva on either side of a median brachium. The larva is devoid of ciliated bands, and it swims ventral surface uppermost with the brachiolar arms trailing (Fig. 5A). (Also see Chapter 4). Patiriella calcar gastrulae which had been treated for 28 hours in either 1 or 2.5 pM l -PCPA had stunted brachiolar arms (Fig. 5 B, C), an incompletely closed blastopore (image not shown) and swam rotating around a horizontal anterior/posterior axis (image not shown). After 28 Chapter three 27 hours in 5.0 jiM L-PCPA, several developmental morphs were consistently seen (Fig. 5D-F), and larvae rotated in a fixed position about a vertical anterior/posterior axis (image not shown). Chapter three

Figure 4 Normal (Figs. A-D) and abnormal developmental events (Figs. E-H) for P. exigua. (A) A 24 hour gastrula showing the blastopore (arrow) and clear fertilisation envelope (red arrowhead). Scale bar = 400 pm. (B) Single confocal optical section through the gastrula epithelium showing a band of serotonin-like immunoreactivity. Scale bar = 70 pm. (C) Pre-hatched brachiolaria. Median brachium, blue arrow; lateral brachia, white arrows; Fertilisation envelope, red arrowhead. Scale bar = 400 pm. (D) Hatched tripod-like brachiolaria. Black arrow indicates the anterior region of the larva. White arrows indicate the brachiolar arms. Scale bar = 500 pm. (E) Hatched and emergent brachiolariae 96 hours post fertilisation (72 hours immersion in 2.5 L-PCPA), showing severely stunted brachiolar arms (black arrowheads). Scale bar = 500pm. (F) Premature hatching of a late gastrula occurring approximately 66 hours post fertilisation (48 hours immersion in 5.0 pM L-PCPA). Scale bar = 615 pm. (G) A schematic representation of the gastrula in (F), showing the small aperture in the FE. Scale bar = 330 pm. (H) Rupturing of a cytostatic embryo after 96 hours post fertilisation (72 hours immersion in 10 pM L-PCPA). Red arrowheads indicate the rupturing FE. Scale bar = 500 pm.

Chapter three

Figure 5 Stills from video sequences of live P. calcar brachiolaria taken at 52 hours post fertilisation of untreated (A) and L-PCPA treated larvae (B-F). (A) Normal elliptically shaped swimming brachiolaria, showing a well- developed median (white arrowhead) and lateral brachia (black arrows). (B-F) Teratogenic effects produced by treating P. calcar gastrulae with increasing concentrations of L-PCPA, resulting in stunted lateral brachia (black arrowheads) seen for 1-2.5 pM L-PCPA to the total absence of lateral brachia at 5.0 pM L-PCPA. Note the lateral widening of the larval bodies in (B) and (C). (D and E) Several different but characteristic deformed larval morphs (D) spherical, (E) slightly elongated and (F) globose were evident in 5.0 pM L-PCPA treated gastrula. Scale bar = 500 pm.

Chapter three 28

3.6 DISCUSSION

3.6.1 Preneural expression of 5-HT rpHPLC

Chromatographic analysis revealed that at 24 hours post fertilisation, both Patiriella regularis and P. exigua.gastrulae contained 5-HT. These results are in contrast to those of an earlier study of asteroid larval development, where serotonin was not detected until the formation of coeloms in a planktotrophic developer (Toneby, 1977). This discrepancy could be due to many variables, some of which might include differences in sensitivity of assay methods, experimental protocols and a different asteroid genus. The presence of 5-HT in the asteroid embryos studied here was not surprising as another study had also found serotonin present by rpHPLC in earlier stages of echinoid development (Renaud, et al. 1983). This is the first study to use rpHPLC to determine the presence of serotonin in asteroid gastrulae. Dopamine was also detected in the gastrulae of both P. regularis and P. exigua. The function of this catecholamine was not determined during gastrulation. However, unidentified catecholamines expressed before the appearance of a nervous system have also been found in the gastrulae of the sea star Archaster typicus using the glyoxilic acid method where it was speculated that catecholamines may also function in morphogenesis during gastrulation (Chen, et al. 1995).

3.6.2 Preneural expression of 5-HT Immunocytochemistry

Serotonin-like immunoreactivity in the epithelium of the 24 hour gastrulae of Patiriella regularis and P. exigua observed before the formation of any recognizable neuroblasts, suggested that the epithelial cells are involved in synthesis of 5-HT. These findings also corroborate other studies on the preneural expression of neurotransmitters in early Chapter three 29 development (Buznikov, 1984). The pattern of serotonin-like immunoreactivity in P. regularis is similar to that seen for several neurochemicals in the early embryos of a range of animal phyla (Lauder, 1993). In the Xenopus gastrula, the epithelium expresses serotonin while the archenteron does not (Rowe, et al. 1993), similar to that seen here for the gastrulae of P. regularis. These findings suggest that the endodermal tissue is not responsible for serotonin synthesis during gastrulation. It would also appear that the specialization of cell lineages in P. regularis arise during this early gastrula stage of preneuronal expression. Using confocal microscopy it was possible to determine what cells were expressing immunoreactivity in the epithelia of Patiriella regularis and P. exigua. It was not possible to section optically below the epithelium or through the invaginating blastopore of Patiriella exigua due to the density of this lecithotrophic gastrula and the wavelength (488 nm) used. At shorter wavelengths, such as 488 nm, light scattering becomes a problem using conventional confocal microscopy (Pawley, 1995). It is suggested that future fluorescence microscopy with lecithotrophic larvae be undertaken using multiphoton microscopy, where scattering effects would be less of a problem (Pawley, 1995). A general expression of serotonin in the gastrula epithelium suggests that serotonin may be functioning as a morphogen in early development of Patiriella regularis and P. exigua during gastrulation regardless of the different developmental modes of these two species. It appears that the preneuronal expression of serotonin found in this study adds to the growing body of evidence that “classical” neurotransmitters can play a number of roles during early development before formation of recognizable nerve cells (Buznikov, et al. 1996).

3.6.3 Possible developmental roles for serotonin in the planktotroph

The normal events associated with gastrulation for Patiriella regularis (Byrne and Barker, 1991) did not occur for embryos cultured with either

2.5 pM or 5.0 jliM L-PCPA. The development of embryos at the lower Chapter three 30 concentration of L-PCPA suggests an inhibition of serotonin synthesis to a level at which relatively normal morphogenetic events begin; such as partial formation of the left and right coelomic sacks, gut and anus; but then they are impeded before the embryo can develop into a bipinnaria. It thus appears that preneuronal serotonin is involved in the development of the gut, mouth and ciliated bands, none of which were functionally or fully present in the treated embryos of P. regularis. All of the above abnormalities pose the questions: what cells other than the epithelial cells are actually synthesizing serotonin? Are certain abnormalities an indirect consequence of serotonin depletion? Future studies using micro-injection of L-PCPA into specific cells might help answer these questions. However, in the case of incomplete coelomogenesis observed for Patiriella regularis, it may be the coelomic cells themselves, which are responsible for the production of serotonin, act as a trophic substance involved in their own development. This hypothesis is based on an observation in a different asteroid genus, in which non­ neuronal serotonergic-like immunoreactive cells were observed in the coelomic sacs, has been suggested that serotonin may be acting as a morphogen (Nakajima, 1988). This will be discussed further in chapter 4. Also, it cannot be ruled out that L-PCPA might be indirectly affecting larval morphogenesis by serotonin depletion affecting catecholamine levels (Yang and Pan, 1999).

3.6.4 Cytostatic effects of L-PCPA

As to why a cytostatic effect was observed for gastrulae of Patiriella regularis treated with 5 pM L-PCPA, it can be speculated that TPH enzyme activity was totally inactivated by a higher concentration of the drug, preventing any further development. Unfortunately, to quantify the level of TPH activity after L-PCPA treatment would prove impractical in this species by current enzyme assay methods (Nagai, et al. 1997) due to difficulty in rearing extremely large quantities of embryos for analysis. It has been Chapter three 31 suggested that “prenervous transmitters” could be involved in the transport of mRNA. Interfering with serotonin synthesis in P. regulans gastrulae may, in fact, affect intracellular transport of macromolecules. It has also been shown that disruption of serotonin synthesis leads to characteristic malformations in the early embryos of the chick and the newt (Païen, et al. 1979; Buznikov, 1990 and Hàmàlàinen and Kohonen, 1989). Concentrations of L-PCPA which were 200 times greater than those used in this study caused cytostatic effects and disintegration of embryos in the newt, similar to what was observed for the 5pM L-PCPA treated P. regularis gastrulae. It has also been suggested that preneuronal expression of neurochemicals may be necessary for maintaining normal cell membrane integrity (Buznikov, 1990) and this may explain why rupturing of the embryos of P. regulans were seen at the higher L-PCPA concentrations which may have been cytotoxic. There could be a myriad of reasons why depleting serotonin could be cytotoxic since it appears that preneuronal serotonin has a multifunctional role during early development and exactly which processes are being directly or indirectly effected is unknown at present. (Buznikov, 1990). It therefore appears that depleting serotonin during early development produces a characteristic set of malformations across several different animal phyla and this may indicate an evolutionary conservation of the function of serotonin during gastrulation.

3.6.5 Developmental abnormalities in P. exigua

Patiriella exigua gastrulae treated with L-PCPA displayed several interesting effects between the concentrations of 1 to 10 pM L-PCPA all of which produced deformed larvae similar to that seen for treated P. regularis.

Three specific and clearly identifiable developmental events were affected by depletion of 5-HT at L-PCPA concentrations above 1 pM: Premature Chapter three 32 hatching; stunting of brachiolar arms; and cytostatic events are now discussed. 3.6.6 l-PCPA affects hatching

Premature hatching of an underdeveloped embryo suggested that serotonin synthesis and the timing of hatching may be interrelated. Control and L-para chlorophenylalanine affected embryos of P. exigua always broke through the FE at the animal pole indicating that the location of a presumptive hatching enzyme is situated in cells at the animal pole of these larvae. These data corroborate those of Lepage, et al. (1992) who examined the spatial expression of the hatching enzyme gene in an echinoid embryo and suggested that the synthesis of a hatching enzyme was located at the animal pole of the embryo. Exogenous serotonin applied to the culture medium of the embryos of Paracentrotus lividus and Arbacia sp. resulted in delayed hatching, and it was suggested that the addition of serotonin might have blocked the synthesis of the hatching enzyme (Deeb, 1972). In this thesis, observations of premature hatching in P. exigua after serotonin depletion, could indicate that higher levels of serotonin may be necessary during early development while the gastrula develops and then prior to hatching serotonin levels drop. From a phylogenetic standpoint, preneuronal serotonin probably functions in a similar way between the echinoids and asteroids. However, further investigation is required to confirm a common role for preneuronal serotonin in the hatching process. The activity of a hatching enzyme isolated from an echinoid, has been shown to be Ca2+ dependent (Takeuchi, et al. 1979). Interactions between serotonin and calcium have been studied extensively and serotonin has been shown to affect calcium levels in numerous invertebrate models (Soliman, 1984a; Stommel and Stephens, 1985; Sanderson and Satir, 1985; Murakami, 1989; Laurienti and Blankenship, 1997; Siegman, et al. 1998 and Shmukler, et al. 1999). Chapter three 33

The possible involvement of serotonin in the hatching process is extremely significant as hatching enzymes have been found in many different animal phyla including both invertebrates and vertebrates (Sawada, et al. 1990). It could be hypothesized that interfering with serotonin synthesis affects calcium dynamics in the developing gastrula of P. exigua, subsequently disrupting the timing of hatching and hence the observation of early hatching. It would therefore be extremely interesting to follow calcium dynamics via confocal microscopy in L-PCPA treated larvae to confirm or reject this hypothesis. Future studies examining the levels of preneuronal serotonin by rpHPLC, prior to hatching and after hatching may determine if serotonin plays a direct role in the hatching process of an asteroid larva.

3.6.7 Developmental abnormalities produced by L-PCPA

The stunting of brachiolar arms occurred for both treated P. exigua and P. calcar, suggesting that serotonin synthesis is required for the development of the anterior region of the larva. Similar effects from the application of L-PCPA were seen during gastrulation in newt embryos, where malformations were prevalent at the anterior region of the embryo (Hamalainen and Kohonen, 1989). The results for these two Patiriella species were no different even though L-PCPA was applied to unhatched embryos of P. exigua and hatched embryos of P. calcar, suggesting that L- PCPA, being a relatively low molecular weight compound, passed easily through the FE of P. exigua. The amount of 5-HT in Patiriella calcar brachiolaria was below the detection limits of the fluorescence chromatographic system used in this study, the results of which confirmed that l-PCPA had actually lowered 5-HT levels. At an l-PCPA concentration of 10 pM, a cytostatic effect was observed for Patiriella exigua however embryos ruptured during hatching which might indicate a cytotoxic effect at this concentration. Chapter three 34

3.6.8 Comparative studies in a vertebrate

In a study by Hamalainen and Kohonen (1989), incubating gastrulae of a newt in L-PCPA over a 48 hour period, also produced teratogenic effects on development. Low doses (0.1 mM) produced partial gastrulation with several different larval morphs arising. Higher doses (1 mM, 100 times greater than the highest concentration of L-PCPA used in this thesis) caused the rupture of embryos. It should be noted that L-PCPA also delayed development in the newt, similar to that observed for P. exigua gastrulae treated with 2.5 jliM L-PCPA. Craniofacial morphogenesis in mouse embryos has been suggested to be regulated by blood borne serotonin (Shuey, et al. 1993) and it has also been suggested that serotonin might regulate cell migration via activation of receptors (Lee, et al. 1991).

3.6.9 Swimming activity in Patiriella Calcar

Interfering with serotonin synthesis in Patiriella calcar brachiolariae disrupted swimming patterns. Larvae were unable to swim in a posterior direction but instead spun in one position. While these larvae do not possess ciliated bands, clearly, co-ordination of ciliary beat was disrupted. These effects were not surprising as numerous studies have shown an interaction between serotonin and ciliary activity where the application of exogenous 5-HT causes an increase in beat frequency in echinoderms (Wada, et al.1997). This subject will be discussed further in Chapter 3. Chapter three 35

3.6.10 Summary

Based on the data presented above for the three congeneric seastar species, preneuronal expression of serotonin is involved in the development of both internal and external larval design: the gut and mouth; coeloms and ciliated bands; brachiolar arm development and gross larval morphology respectively. Behaviourally, serotonin also appears to play a role in the hatching process and swimming direction. These observations reinforce the multifunctional role of serotonin throughout the animal phyla. Aspects of serotonergic neurogenesis in asteroid larval development and behaviour will be discussed further in the following chapters. Chapter four 36

Development of the Bipinnarial Serotonergic Nervous System in the Sea Star Patiriella regularis as Revealed by Confocal Imaging. Chapter four 37

4.1 ABSTRACT

The distribution and development of serotonin-like immunoreactive cells in the larvae of the sea star Patiriella regularis was followed through larval development. Confocal optical sections revealed that serotonergic neurons with neurites first appeared in late gastrulae as a collection of neurons scattered across the animal pole. Subsequently these neurons gave rise to basal processes positioned along the basal lamina. Identification of the precise location and time of development of serotonergic neurons relative to subsequent development of larval features was made possible by the optical sectioning and image processing capabilities of the confocal microscope. Serotonergic neurons located in the stomodaeal region marked the beginnings of formation of the adoral ciliated band. Serotonergic neurons were also present in the mid-dorsal epithelium. Advanced bipinnaria had pyramidal serotonergic neurons within the adoral band and ovoid neurons within the pre- and postoral ciliated bands. Processes originating from neurons in the transverse region of the preoral ciliated band extended into the buccal cavity, suggesting a sensory role for these cells in feeding. In late bipinnaria an anterior ganglion formed, associated with the pre- and postoral ciliated bands. A complex network of varicose serotonergic processes linked the two bands. An immunopositive connection was also observed between the transverse preoral and adoral ciliated bands. This connection has not previously been described. It appears that the ciliated bands in the bipinnaria larvae of P. regularis communicate via serotonergic nerve tracts. Details of the development of the serotonergic nervous system in larval P. regularis were effectively resolved by confocal microscopy enabling comparisons to be drawn with respect to homologous features in the larvae of other echinoderms. Chapter four 38

4.2 INTRODUCTION

Development of the expression of serotonin-like immunoreactivity in echinoderms has been reported for the larvae of asteroids, echinoids and holothuroids (Bisgrove and Burke, 1986; Burke, et. al. 1986; Bisgrove and Burke, 1987; Nakajima, 1988; Bisgrove and Raff, 1989; Nakajima, et. al. 1993; Moss, et. al. 1994). In these studies serotonergic neurons have been localised to the ciliated bands of the larvae and it has been suggested that serotonin functions in a neuronal capacity with possible sensory roles associated with feeding and metamorphosis. The similarities between the serotonergic systems in the larvae of several echinoderm classes have been taken to suggest that these systems are homologous (Burke, et. al. 1986). In general, the increasing complexity in the development of the serotonergic nervous system in the feeding larvae of echinoderms (Nakajima, 1988) parallels the development of the ciliated bands (Burke, 1983). In particular, the number of serotonergic neurons appears greatest at the asteroid bipinnarial and echinoid pluteal stages of development where the ciliated bands are at their most developed structural state. There has been a paucity of high resolution data obtained regarding the nature of the development of the serotonergic architecture in bipinnariae at the light microscope level of resolution and detailed three dimensional studies of serotonergic neurogenesis have not previously been obtained. In Chapter 1, non-neuronal expression of serotonin was documented in the gastrulae of the three congeneric sea star species representing three different modes of development within the genus Patiriella. As a continued comparison of serotonergic neurogenesis between the three congeners, the next logical step to take was to investigate whether serotonin was expressed in subsequent larval stages for all three congeners. This chapter investigates the development of the serotonergic nervous system in the bipinnariae of Patiriella regularis. The neuronal expression of serotonin in the lecithotrophic developers, P. calcar and P. exigua is examined in Chapter 6. Chapter four 39

A serotonergic nervous system in this thesis is defined as groups of serotonin-like immunoreactive cells arranged in a distinct pattern, where each cell possesses a basal process. These immunoreactive cells are subsequently referred to as serotonergic neurons. The term "axon" is not used as it was not possible to identify axons at the light microscope level of resolution. Instead, the term neurite(s) is used to describe putative serotonergic axons in this thesis. The nomenclature used to describe the ciliated bands follows that established by previous authors (Strathmann, 1975; Moss, et. al. 1994). The aims here were twofold. Firstly to obtain a three dimensional picture of serotonergic neuronal development using confocal microscopy. Although previous studies have used conventional epifluorescence microscopy to follow the formation of the larval serotonergic nervous systems in echinoderms (Burke, 1983; Nakajima, 1988; Moss, et. al. 1994), insights into the three-dimensional nature of the nervous system were limited by these techniques. Secondly, the significance of the distribution of serotonergic neurons is assessed with respect to the functional morphology of the bipinnaria and the possible roles these neurons may play in larval behaviour. Parallels in the immunocytochemical expression of 5-HT with other neurotransmitters in the feeding larvae of other echinoderms are also discussed.

4.3 MATERIALS AND METHODS See Chapter 2 sections 2.1-2.6.4 for general protocols listed in materials and methods. Chapter four 40

4.4 RESULTS

4.4.1 Gastrula

Neurons exhibiting specific 5-HT-like immunoreactivity occurred in mid gastrulae (Fig. 1A), approximately 24 hours post fertilisation at the animal pole. As the gastrulae began to elongate, these neurons formed a partial dome-like array across the animal region and included mono, bipolar and tripolar cells (Fig. 1B). Varicosities were occasionally observed on neurites of these cells (Fig. 1C). Both the soma and the neurites of these neurons were immunopositive (Fig. 1C). These cells spanned the epithelium (Fig. 1D) and computer reconstructions and /or extended focus projections revealed they were pyramidal in profile (Fig. 1D). Control gastrulae (n=15), were non fluorescent.

4.4.2 Bipinnaria

Prior to the opening of the mouth, early bipinnariae had a distinct stomodaeal invagination while the blastopore was still located at the vegetal pole (Fig. 2A). The larvae could now be orientated according to their dorso-ventral axis (Fig. 2A). A 144 pm thick projection reconstructed from 32 optical sections showed that serotonergic neurons were abundant on both sides of the larva (Fig. 2A). On the ventral surface, the serotonergic neurons around the stomodaeum, approximately 10 in number, were mono-polar and marked the position where the adoral ciliated band will form (Fig. 2A). A collection of bipolar ovoid serotonergic neurons on the dorsal surface were positioned approximately opposite to the stomodaeal invagination (Fig. 2A). At approximately 48 hours post-fertilisation the mouth opened. The larvae were further elongated and the anus opened ventrally. With completion of the gut the larvae were able to feed. Serotonin-like immunoreactivity was conspicuous in the cells surrounding the mouth, Chapter four

Figure. 1. Confocal images showing serotonergic neurons and processes in early and advanced gastrula. (A) A projection from 16 optical sections taken at 4.5 pm intervals of a mid gastrula shows serotonergic neurons (arrowhead) scattered in the epithelium. C, Cilia; b blastopore Bar, 63 pm. (B) Advanced gastrula, projection created from 14 images at 4.5 pm depth intervals showing the concentration of serotonergic neurons (arrowheads) in the animal half. Bar, 95 pm. (C) 5-HT-like immunoreactivity in a tripolar nerve cell in a 34 hour gastrula. n(arrowheads), neurites; v, varicosities; neb, nerve cell body: Bar, 16 pm. (D) Advanced gastrula/early bipinnaria epithelium (e) showing nerve cell bodies (neb) and neurite (n) travelling along the basal lamina (arrowheads) in a single confocal section. Bar, 20

Chapter four

Figure 2. Confocal optical projections of early bipinnariae. (A) Early bipinnaria reconstructed from 32 optica! sections. Serotonergic neurons can be clearly seen around the stomodaeal invagination (si) and the dorsal surface (arrowheads) of the larva, a, anus. (B) Projection of five confocal sections from the ventral side of a 48 hr bipinnaria. 5-HT-like immunoreactivity is present in cell bodies (arrowheads) in the adoral ciliated band region (adcb) and a few immunoreactive cells are also present on the upper right hand side of the mouth (m). (C) Projection of 5 optical sections from the dorsal side of the larva in figure. B. Serotonergic neurons (neb) and neurites n(black arrowheads) form a band partially wrapping around the larva. Bars, 95 pm.

Chapter four 41 which marked the position of the developing adoral ciliated band (Fig. 2B). A few immunoreactive cell bodies were also observed on the upper right region of the buccal cavity (Fig. 2B). Serotonergic neurons and processes, on the mid-dorsal surface formed an incomplete ring wrapping partially around the larva but did not extend to the ventral surface (Fig. 2C). Neurites from these cell bodies extended towards the posterior end of the larva (Fig. 2C). Although the fate of this immunoreactive ring of neurons could not be followed, their mid-body position indicates that they may have been subsequently incorporated into the pre- and postoral ciliated bands. As the pre-, post- and adoral ciliated bands developed the oral hood was also beginning to form (Fig. 3A, B). Serotonin-like immunoreactivity was observed along the ciliated bands in the form of monopolar neurons following the path of these bands (Fig. 3A). By this stage a ganglion was evident at the anterior end of the larva. The cell bodies of the ganglion were situated amongst the columnar cells of the pre- and postoral ciliated bands at the anterior end of the larvae. Basal processes were seen to interconnect these bands (Fig. 3A). At this stage of larval development a serotonergic nervous system had formed in association with the ciliated bands. Advanced bipinnariae (approximately 18 days old) underwent a distinct shape change with the formation of an extension at the anterior end of the larva (Figs. 4A, B). The three ciliated bands were well developed in these larvae (Figs 4A, B). Internally, the larva had a well- developed gut and the right and left enterocoels had formed. The distribution of immunoreactive neurons in these bands, discussed below, was consistent in all larvae examined (n=100).

4.4.3 Adoral ciliated band

The adoral ciliated band was located along the posterior margin of the mouth and was characteristically paraboloid (Fig. 5A). The length of the adoral ciliated band were densely packed with serotonergic neurons with their apical ends extending externally from the ciliated epithelium. These cells were pyramidal and connected basally via a thick immunopositive Chapter four

Figure 3. Three-dimensional red/green anaglyph and a scanning electron micrograph (false coloured) of early bipinnariae. (A) A 3-D lateral view of an early bipinnaria showing serotonergic neurons and neurites following the ciliated bands. The anterior ganglion (ag) has formed and connects the pre- (procb) and postoral (pocb) ciliated bands, adocb, adoral ciliated band; o, oesophagus; s, stomach; i, intestine: Bar 95 pm. (B) Ventral view of an early bipinnaria at the same stage as Figure. 3A. The ciliated bands are developing but the anterior extension has not yet formed. Black arrowheads, preoral ciliated band; white arrowheads, postoral ciliated band; white arrow, adoral ciliated band; m, mouth; a, anus: Bar, 100 pm.

Chapter four

Figure 4. Scanning electron micrographs of a bipinnaria showing fully developed ciliated bands (arrowheads and arrows) (A) Ventral view of a bipinnaria with a flexed oral hood and mouth (m) open showing the position of the adoral ciliated band (adcb) (B) Lateral view of a bipinnaria showing the anterior extension of the oral hood, top right hand side. Double ended arrow indicates the anterior region where the anterior ganglion links the pre and postoral ciliated bands. Arrow heads, preoral ciliated band; arrows, postoral ciliated band: Bar, 200 pm. Figure 5. Bipinnaria: 3-D anaglyph and a high magnification confocal image projection of the anterior ganglion. Images were constructed from a series of optical sections covering a distance of 132 pm. (A) 3-D anaglyph of a bipinnaria detailing the serotonergic nervous system following the pathway of the ciliated bands. White arrow heads, serotonergic connection between pre- and adoral ciliated bands (also see Fig. 4A). s, stomach; m, mouth; o, oesophagus; arrows, immunoreactive coelomic cells: Bar 200 pm. (B) A projection of the anterior ganglion in a late bipinnaria. Parallel neurites on the opposing sides of the preoral ciliated band and the postoral ciliated bands interconnecting in a fine network of 5-HT-like immunoreactive processes forming the anterior ganglion (ag). n, neurites; neb, nerve cell bodies: Bar, 50 pm.

Chapter four 42 tract (Fig. 6A). Compared with the other ciliated bands, the adoral ciliated band had the highest concentration of serotonergic neurons, forming the adoral ganglion (adg). Confocal optical sectioning revealed that the apical region of these cells protruded to the exterior of the ciliated band epithelium. The adoral ganglion was also connected by serotonergic processes with the ganglion in the preoral transverse ciliated band via two thin (approximately 2.5 pm) lateral immunoreactive tracts as detected by computer animations (see CD ROM) and (Fig. 5A).

4.4.4 Preoral ciliated band

The preoral ciliated band was located on the ventral surface of the larva and outlined the oral hood (Fig. 4A). Where this band traversed the larva above the mouth (preoral transverse region), a large number of flask shaped neurons (x = 21, se = 0.1 n =10 larvae) were found in the epithelium (Fig. 6B). Confocal sectioning into the larva, from the ventral surface, revealed neurites arising from the basal portion of these cells extending inwards along the squamous epithelia towards the buccal cavity (Fig. 6B). The lateral regions of the preoral ciliated band contained a few serotonergic neurons scattered along its path. Occasionally, collections of cell bodies, forming a pair of lateral ganglia, were seen in the lateral region of the post oral ciliated band. These structures were not seen in all larvae and appear to be ephemeral. In the late bipinnaria, the previously seen structure of the anterior ganglion in the early bipinnaria increased in complexity. There were a greater number of anastomosing immunoreactive neurites present containing numerous varicosities. The anterior ganglion was a prominent neuroanatomical feature in the bipinnaria of Patiriella regularis (Fig. 5B).

4.4.5 Postoral ciliated band

The postoral ciliated band formed a continuous loop traversing the ventral surface adjacent to the mouth and continued laterally along either side of the larva (Fig. 4A, B). It then extended posteriorly crossing to the dorsal surface and then extending anteriorly where the anterior ganglion Chapter four 43

(ag) forms (Figs 5B). 5-HT-like immunoreactive cells and axonal-like processes occurred along the anterior postoral band (Figs 5A, B). In the transverse region adjacent to the mouth, a group of immunoreactive ovoid cells were found connected by a thin immunopositive tract (Fig. 5A). In general the immunoreactive cells and processes were more sparsely distributed in the post oral ciliated band compared with the other bands (Fig. 6B, C). Numerous cells of the left and right coelomic pouches exhibited 5-HT- like immunoreactivity (Fig. 5A). These cells were not neuronal in morphology but appeared to be exhibiting a general expression of serotonin. Immunoreactive non-neuronal cells were also concentrated around the anal opening (Fig. 7) and were observed in the intestinal epithelium, to a distance of up to 100 jim towards the stomach. In some instances the entire stomach was immunoreactive. This was not consistent however, and may have been due to incorporation of algal pigments (Fig. 7).

4.5 DISCUSSION

4.5.1 The appearance of serotonergic neurons prior to a "nervous system"

Serotonergic neurons were first seen at the animal pole of the gastrula of Patiriella regularis, similar to that found for other asteroids and echinoids with planktotrophic development (Bisgrove and Burke, 1986; Nakajima, 1988). As the gastrula elongated, these neurons formed a hemispherical array at the anterior end of the larva indicating the onset of migration and rearrangement of these cells. Cell fate studies would be required to determine this. The neuronal cells present in the gastrula included, monopolar, bipolar and tripolar cells. The cell bodies of the mono and bipolar neurons appeared to be flask shaped, similar to that described for neurons in the bipinnaria larvae of an asteroid (Lacalli, et. al. 1990). Chapter four

Figure 6. Confocal images detailing serotonergic neurons in ciliated bands. (A) Image from 15 optical sections (total 139 pm thickness) showing 5-HT-like immunoreactive nerve cell bodies (neb) and neurites forming an axonal-like tract (axt) in the adoral ciliated band. The entire band is immunoreactive. Note the apical region of the neuron extends to the edge of the epithelium of the ciliated band. Cilia projecting into the buccal cavity; arrow heads. Bar, 20 pm. (B) The preoral and postoral ciliated bands of a fully developed bipinnaria. Note the greater number of serotonergic neurons (neb) present in the preoral ciliated band (procb) compared with the postoral ciliated band (pocb). The preoral ciliated band has immunoreactive neurites (n) extending towards the buccal cavity, c, cilia; and arrowheads: Bar, 63 pm. (C) Nerve cell bodies (ncbl and arrowheads) in the right lateral postoral ciliated band. Bar 50 pm.

Chapter four

Figure 7. Confocal image showing immunoreactive cells (arrowheads) in the intestinal wall (i) and surrounding the anus, (a): Bar, 50 fim.

Chapter four 44

The development and arrangement of serotonergic neurons up until the feeding early bipinnaria, did not appear to form part of a nervous system. These cells were initially scattered over the gastrula and then eventually present around the developing mouth. The question arises, why were these neurons expressing serotonin-like immunoreactivity with no apparent function at this early stage of development? It could be speculated that these serotonergic neurons are influencing the growth of non-neuronal cells and hence the development of the larva via a trophic effect of 5-HT. Equally possible, is that these early serotonergic neurons may also influence their own growth by the synthesis of serotonin. Studies in molluscs have shown that serotonergic neurons auto-regulate their own growth via the endogenous synthesis of the neurotransmitter itself (Marcus, et al. 1994, Diefenbach, et al. 1995, Goldberg, 1998). For future studies, it would be interesting to test if similar processes are functioning in the early neurogenesis of serotonergic neurons in Patiriella regularis.

4.5.2 Morphology of serotonergic neurons

Flask-shaped nerve cells have been observed in an echinoid pluteus (Bisgrove and Burke, 1986; Bisgrove and Raff, 1989). Burke (1983b) noted that nerve cells in the bipinnaria of the asteroid Pisaster ochraceus had a fusiform profile. I did not observe any neuron-like cells in the gastrula or bipinnaria of P. regularis with a fusiform shape. Three- dimensional reconstructions of the tripolar cells revealed that they were pyramidal, a structure not previously reported. This observation however, would be dependent on the imaging technique employed. Monopolar cells were the most common types of immunoreactive cell in the pre- and postoral ciliated bands while pyramidal cells were the most common cell types in the adoral ganglion.

4.5.3 Distribution of serotonergic immunoreactivity

In the bipinnariae of Patiriella regularis serotonin-like immunoreactivity was conspicuous in the adoral ganglion (adg), in the pre- and postoral Chapter four 45 ciliated bands and in the anterior ganglion (ag). The adg was strongly fluorescent and connected to the neurons in the transverse preoral ciliated band by immunoreactive tracts. Detection of these connections was possible through generation of 3-D anaglyphs from confocal optical sections, making it possible to trace the complex immunostained network with respect to larval anatomy. On screen animations were also employed to view immunolabelled larvae to determine the structure and direction of the immunolabelled processes. Although serotonergic immunoreactivity associated with the adorai ciliated band has been described in several studies (Nakajima, 1988 and Moss et. al. 1994), the connections between the adorai and preoral ciliated bands have not been seen before. Conventional epifluorescence microscopy of these earlier studies would not have allowed resolution of this fine structure. Optical sections through the oral region revealed that the immunoreactive cells in the preoral ciliated band gave rise to basal immunoreactive processes which project into the squamous epithelia towards the buccal cavity. The high density of immunoreactive cells in the region of the preoral ciliated band along the buccal opening suggests that these cells may play a sensory role in feeding. Selection and rejection of particles during feeding is thought to be associated with sensory cells in the buccal cavity (Strathmann, 1975). The neurites in the roof of the buccal cavity in the larvae of Patiriella regularis may connect with receptor sites within the buccal cavity, which are involved in particle selection in feeding. The 5-HT immunopositive tracts connecting the adorai and preoral ciliated bands indicate a serotonergic link between the adorai ciliated band and the preoral ciliated band. This link could be important in feeding. The anterior ganglion (ag) is first seen in early bipinnariae prior to formation of the anterior extension. As this extension develops, the ganglion becomes more intricate and forms a highly complex network. In advanced bipinnariae the ag consisted of prominent strongly fluorescent tracts traversing the anterior region of the pre- and postoral ciliated bands. This was the only serotonergic connection between these ciliated bands. Chapter four 46

4.5.4 Possible sensory functions for serotonergic neurons

The presence of the apical regions of the serotonergic neurons of the adoral ganglion (adg) of the adoral ciliated band projecting towards the exterior of the ciliated band suggests that this ganglion may have a sensory role. These observations are similar to those of Komatsu et. al. (1991) where they define sensory neurons in the bipinnaria of Luidia senegalensis as "neurons whose apical surface contacts the external environment". Strathmann (1975) demonstrated that the cilia of the adoral ciliated band in bipinnariae are involved in carrying food particles into the oesophagus. Ciliated structures associated with the neurons of the adg were not found in this study. Although, it is possible that the adg of Patiriella regularis plays a gustatorial function under the influence of serotonergic activity. Further analysis of the oral region and the functional aspects of the adoral ganglion are examined in greater detail in the following chapter.

4.5.5 Comparisons with serotonergic ganglia in other echinoderms.

Observations of what appears to be a serotonergic anterior ganglion have been made for other echinoderms. Immunocytochemical labelling with anti-serotonin in the auricularia larvae of a holothuroid produced a structure described as an apical ganglion (Burke, et. al. 1986). This structure however, did not contain numerous immunoreactive tracts as seen in the anterior ganglion of Patiriella regularis. Serotonergic anterior ganglia differing structurally to those of seastars but still anterior in position have been extensively described for sea urchin plutei (Bisgrove and Burke, 1987; Bisgrove and Raff, 1989; Nakajima, et. al. 1993). The observation of an anterior ganglion in the bipinnaria of Patiriella regularis is similar to the anterior concentration of serotonergic neurons characteristic of many invertebrate larvae (Lacalli, 1994) and which are variously called apical organs or apical ganglia. These appear to be highly conserved structures in marine invertebrate larvae and are thought Chapter four 47 to have a sensory function (Lacalli, 1994; Marois and Carew, 1997). The function of the apical ganglion and the significance of the connection between the pre- and postoral ciliated bands of P. regularis is not known. Its position and the anterior direction of swimming suggests it may be involved in a sensory capacity in relation to directional swimming by the bipinnaria, similar to that suggested for the apical ganglion of other invertebrate larvae (Marois and Carew, 1997). Moreover, sensory cells were found in the preoral and postoral ciliated bands (Komatsu, et. al. 1991) in an ultrastructural study of the bipinnaria of Luidia senegalensis, in the region where the anterior ganglion is located in P. regularis. Immuno-electron microscopic examinations of thin sections from the anterior region of P. regularis would need to be undertaken to determine if similar cells are present in this species. The anterior ganglion (ag) in Patiriella regularis is also similar to non- serotonergic neuronal structures in other asteroids. Similar catecholaminergic anterior structures in the bipinnaria of Archaster typicus were described as a “fluorescent anastomosis” (Chen, et. al. 1995). Nakajima (1987) described a similar catecholaminergic structure as a “fibrous network” in the bipinnariae of Asterias amurensis. It is highly probable that confocal imaging would reveal that these structures are similar to the ag in P. regularis.

4.6 Conclusion

This chapter demonstrated the presence of an extensive network of serotonergic neurons and processes connecting all the ciliated bands. Serotonergic neurons may govern reactions to external stimuli and generate the behavioural patterns associated with feeding and swimming. The following chapter examines in detail the anterior and oral serotonergic ganglia in relation to larval behaviour associated with serotonin biosynthesis. Chapter five 48

Serotonergic Ganglia in the Larvae of Patiriella regularis and the Effects of the Serotonin Depleting Drug para- Chlorophenylalanine (L-PCPA) on Feeding and Swimming. Chapter five 49

5.1 ABSTRACT

Immunofluorescent labelling revealed a unique set of ciliated serotonergic neurons forming three distinct ganglia in the oral and anterior regions of the ciliated bands of the bipinnaria larva of the sea star Patiriella regularis. Three-dimensional confocal imaging showed that these ganglia contained cells that culminated in an apical cilium and basal varicose processes. These cilia were distinct from neighbouring locomotory cilia of the ciliated bands. The cilia of the oral ganglionic cells projected towards the inside of the buccal cavity and may have a sensory role in feeding. The larvae of P. regularis swim with their anterior end forward, therefore the location of ciliated serotonergic neurons in the anterior ganglion suggests that these cells play a sensory role in swimming and perhaps also in perception of food. Treatment of bipinnariae with para chlorophenylalanine (L-PCPA), a potent serotonin biosynthesis inhibitor, resulted in cessation of feeding and decreased swimming speed. Behavioural changes in swimming and feeding were evident in L-PCPA treated larvae. However, the number and staining intensities of neurons in L-PCPA treated larvae was similar to that observed in the controls. Behavioural changes due to L-PCPA were irreversible. Analysis of treated larvae by high performance liquid chromatography with fluorometric detection confirmed a 29% reduction in serotonin levels. Chapter five 50

5.2 INTRODUCTION

The planktotrophic feeding larva is considered to be the ancestral larval form of echinoderm development (Strathmann, 1978). The most prominent features of the planktotrophic feeding larva are the ciliated bands, which have been investigated with respect to the biomechanics of feeding, swimming behavior, neurogenesis and evolution of development (Strathmann, 1977; Barker, 1978; Nakajima, 1987; Hart, 1991; Lacalli and West 1993; Moss, et al. 1994; Pedrotti, 1995; Chapter 2 this thesis). Immunocytochemical and histofluorescent studies with asteroid and echinoid larvae have shown that several classical neurotransmitters (GABA, dopamine, serotonin), unidentified catecholamines and one asteroid specific neuropeptide GFNSALMFamide-1 (Gly-Phe-Asn-Ser- Ala-Leu-Met-Phe-NH2) are associated with the ciliated bands and brachiolarial attachment structures (Burke, 1983; Bisgrove and Burke, 1986; Bisgrove and Burke, 1987; Bisgrove and Raff, 1989; Nakajima, 1987; Nakajima, 1988; Nakajima, 1993; Moss, et al. 1994; Byrne et al. 1999; this thesis Chapter 2). The location of these neurotransmitters in neurons associated with the ciliated bands and attachment organs is indicative of possible functions in swimming, feeding and settlement behavior. As yet no other studies have demonstrated that these neurons associated with the ciliated bands might in fact control feeding and swimming. Serotonin (5-HT) is a ubiquitous neurotransmitter (Collier, 1958). In the feeding larvae of marine invertebrates including echinoderms, phoronids, brachiopods, nemerteans and molluscs, in each instance 5- HT-like immunoreactivity is associated with ciliated bands and structures which have been referred to as anterior or apical ganglia (Burke 1986; Nakajima 1988; Bisgrove and Raff 1989; Nakajima 1993; Moss, et al. 1994; Hay-Schmidt 1990 a, b; Hay-Schmidt 1995; Kempf et al., 1997; Marois and Carew 1997; Chapter 2 this thesis). In this chapter, the serotonergic ganglion located at the anterior region of a bipinnaria larva will be defined as an “anterior ganglion”. Serotonergic anterior ganglia Chapter five 51 appear to play an important role in sensing environmental stimuli (Kempt, et al. 1997; Marois and Carew, 1997). Serotonin has been the most studied neurotransmitter in asteroid larvae and the serotonergic nervous system is particularly developed in the planktotrophic bipinnariae of P. regularis (see Chapter 2). The biomechanics of ciliary action during feeding in echinoderm larvae has been extensively documented with several studies speculating that feeding is not an involuntary byproduct of ciliary action but is under nervous control (Strathmann, 1971, 1975; Hart, 1991). The aim of this chapter was to gain new insights into the functional role/s of serotonin based on further confocal microscopic analysis on the previously seen anterior and adoral ganglia of the serotonergic nervous system of Patiriella regularis. In particular, special attention was paid to the collection of neurons in the transverse preoral ciliated band. Based on the structure of the serotonergic ganglia, potential roles in modulation of larval behavior are discussed. If 5-HT is involved in the processes of feeding and swimming, then perturbation of 5-HT levels would be expected to interrupt these processes. Irrespective of the organisational mechanics of locomotion, this chapter investigates the possibility that serotonin might be involved in the modification of behaviour of ciliated cells. To assess the function/s of the serotonergic nervous system in Patiriella regularis, a pharmacological study based on a bipinnaria model examining feeding and swimming behavior was conducted, using the 5-HT depleting drug, L- para chlorophenylalanine (L-PCPA). Para chlorophenylalanine acts by selectively and irreversibly inactivating the enzyme tryptophan hydroxylase (TPH), the catalyst in the rate limiting step in the biosynthesis of 5-HT (Jequier, et al. 1967). The lecithotrophic planktonic larvae of the congener P. calcar were used as a comparative model to test the effects of 5-HT depletion on swimming behaviour. P. calcar provides an ideal model as this species does not possess ciliated bands (Chapter 4) but instead uses a evenly distributed covering of cilia for locomotion. It is proposed that irrespective of the mechanics of locomotion, ciliated bands vs no ciliated bands, serotonin is involved in the modification of behaviour of ciliated cells. Chapter five 52

The results of rpHPLC quantification of serotonin in L-PCPA treated larvae to confirm that L-PCPA application depleted serotonin levels in treated bipinnariae are also reported.

5.3 MATERIALS AND METHODS 5.3.1 General larval protocols see Chapter 2 section 2.1-2.2.1

Embryos were cultured to the bipinnaria and early brachiolaria stages at 15 C in FSW and were fed the alga Dunaliella tertiolecta (740 cells per ml) every two days. The cultures were cleaned every 3 to 4 days. Prior to the experimental treatments, bipinnaria were starved for 24 hours.

5.3.2 Pharmacology

Pharmacological Depletion of 5-HT in P. regularis

Three experiments were conducted in Pyrex® crystallizing dishes using freshly made 2.5 pM L-PCPA/filtered sea water (FSW) solution using the alga Dunaliella tertiolecta (740 cells per ml) as a food source. A concentration of 2.5 pM L-PCPA was chosen based on a pilot study in Chapter 3. Pooled bipinnariae of P. regularis were obtained from several 5 litre beakers and concentrated into 150 ml of FSW by aspirating excess FSW through a 60 pm nylon mesh filter. To calculate the approximate number of bipinnariae used in each of the three experiments, replicate 100 pL aliquots were taken and the mean number of larvae calculated. Addition of these bipinnariae to a 150 ml bath solution of 5.0 pM L-PCPA resulted in a final concentration of 3750 bipinnariae per 300 ml of 2.5 pM l-PCPA in each of the following experiments. All 3 experiments were replicated twice. Two hundred larvae were observed in each experiment using a stereo dissecting microscope to count the number of feeding and/or swimming larvae. Normal feeding was defined as larvae, which after one minute of contact with algal cells had full stomachs (greater than 240 algal cells counted in a 2 dimensional plane). Abnormal feeding was Chapter five 53 defined as larvae with less than 20 algal cells or no algal cells present after the equivalent time period.

The first experiment monitored the immediate effects (within the first minute up to 1 hour) of L-PCPA/ FSW on the feeding and swimming behaviour of larvae. One millilitre of algal suspension was added to the bipinnariae/L-PCPA/FSW and the larvae were observed using a stereo dissecting microscope. The second experiment examined effects on feeding and swimming after a 30 hour incubation in 2.5 pM L-PCPA/FSW prior to the introduction of 1 ml of algal cells. Finally, In the third experiment, residual drug effects and feeding recovery, were examined after a 24 hour incubation in L-PCPA/FSW (no food) followed by a 24 hour period in FSW, after which I ml of algal cells were introduced.

Pharmacological Depletion of 5-HT in P. calcar The lecithotrophic non-feeding larvae of P. calcar (4 day old swimming brachiolaria n = 1230) were subjected to a bath treatment of 2.5 jllM l-PCPA for 30 hours. Observations were conducted as above.

5.3.3 Imaging

Images of swimming P. regularis bipinnariae were captured on video using a SONY FIAD CCD color camera connected to a Leitz DMRB microscope. Images were recorded on a SONY U-matic SP videocassette recorder. The screen was calibrated for field width and straight line swimming velocity was calculated based on the distance covered in 1 second (25 frames = 1 second). Approximately 1230 Patiriella calcar larvae (4 day old) which had been treated with 2.5 jiM L-PCPA were captured using a Kyowa compound microscope with a C mounted Toshiba IK-M41A colour CCD video camera, using the X4 160 mm tube length objective. Swimming behaviour was recorded on a SONY Handy Cam Hi 8 XR NTSC video camera using SONY Hi 8 HME metal evaporated tape for recording. A calibration slide was also captured to allow size measurements to be taken. Images were dubbed from Hi 8 tape to a broadcast quality Fuji Chapter five 54

BETACAM SP™ format tape. A Pentium PC fitted with a Matrox Digisuite frame grabber board was used to capture individual frames. The digital editing software used was Speed Razor 4.7 (In-Sync USA). The BETACAM tape was dubbed to VHS format and played on a SONY SLV X835 stereo video recorder frame by frame to calculate the swimming speed of the larvae from a known field of view.

5.3.4 Immunocytochemistry of control and L-PCPA treated P. regularis P. regularis bipinnaria (n= 40) from the second pharmacological experiment and control larvae were prepared for immunocytochemistry as follows. Specimens were transferred to glass scintillation vials, and prepared according to the general materials and methods Chapter 2 sections 2.3-2.6.4. Immunocytochemistry was not performed on L-PCPA treated P. calcar larvae.

5.3.5 Reverse phase high performance liquid chromatography (rpHPLC)

The following chromatography method was developed by Chee and Gu (unpublished results).

Apparatus

The chromatographic system comprised of a Shimadzu LC 10 pump, CBM-10A communication bus module, SIL-10A auto injector and a RF-10A spectrafluorometric detector. An Alltech Alltima Cyano column (250 mm x 4.6 mm ID) was used. The optimum excitation and emission wavelengths 272 nm and 335 nm respectively, were determined by the 5- HT standard using the scanning mode in this system.

Mobile Phase

Isocratic mobile phase had the following composition: 5% acetonitrile (EM Science), 5 mM NH4COOH (Ajax Univar), and 0.02% formic acid (Ajax Univar) to give a pH of 4. All aqueous solutions were Chapter five 55 made with 18 MQ MilliQ water. The mobile phase was sonicated and degassed under vacuum immediately prior to use. The flow rate was 1 ml per minute.

Sample preparation

Larvae of Patiriella regularis were pooled for rpHPLC with approximately 17,000 larvae in FSW (control) and 17,000 larvae placed into 2.5 pM l-PCPA in 300 ml crystallising dishes. The duration of the drug treatment was for 31.25 hours at 22°C. Larvae were centrifuged at 4°C for 15 minutes at 3000 rpm and the seawater removed. The samples were then deproteinized with 0.4 M perchloric acid at 4°C alternating with sonication and 3 freeze thawing cycles in liquid nitrogen. The pH was adjusted to 6.0 with KC03 (Sigma), centrifuged several times to remove the precipitate and then frozen in liquid nitrogen and immediately lyophilized into pre-weighed tubes and dry weights calculated. Samples were then stored at -20°C and analyzed within 5 months. L-PCPA treated samples and controls were re-hydrated with ice cold 0.1 M L-ascorbic acid (Sigma), centrifuged and analyzed immediately at room temperature. Serotonin was added to some samples to confirm peak homogeneity. Patiriella calcar brachiolaria that were used as a comparison model for the effects of L-PCPA on swimming behaviour were also prepared as above for analysis of 5-HT content. Samples of the 2.5 pM L-PCPA were also frozen and assayed for the presence of L-PCPA (data not presented).

Calibration curve

A range of standards 0, 0.002, 0.005, 0.01, 0.05 pg/ml of serotonin (creatinine sulfate monohydrate complex; Aldrich) were prepared in 0.1M L-ascorbic acid and analyzed immediately. Two 40 pi samples (standards and controls) were injected into the column and a standard curve was obtained (y= 0.0457x (R2=1). The retention time for 5-HT was 6.27 minutes. A standard curve was generated and the peak heights used to calculate the amount of serotonin. The detection limit was 50 pg on the Chapter five 56 column. The amount of 5-HT (pg) was calculated per dry weight (mg) of larvae.

5.4 RESULTS

5.4.1 General neuronal anatomy Serotonergic neurons of the bipinnaria of Patiriella regularis are associated with the ciliated band epithelial cells (see also chapter 2) and are also found in three distinct ganglia situated at the anterior, transverse preoral and adoral ciliated bands (Fig. 1 A and B). Ganglionic cell bodies were located within the ciliated band epithelium. A network of processes arose from the base of these cells (Fig. 2A,B, D-G). High-resolution confocal imaging (X 60 1.4 NA objective) of the soma, revealed a characteristic apical protuberance from which a single cilium projected. These cilia were approximately 15 pm long and had a mean diameter of 0.8 pm (SE = 0.03 n= 10) (Fig. 2C). They were clearly larger than the adjacent locomotory cilia of the ciliated bands (Fig. 3) although it was not possible to quantify the size of the locomotory cilia at the light microscope level of resolution.

5.4.2 The anterior ganglion

The anterior ganglion (ag) is comprised of a thin sheet (~0. 7 pm thick) of anastomosing serotonergic processes positioned adjacent to the squamous epithelium and covering the anterior-most region of the larva. The ag, spans the anterior tip of the larval surface to connect the preoral (procb) and postoral (procb) ciliated bands. (Fig. 1B 2A).

5.4.3 Preoral ganglion

Serotonergic processes from the neurons of the preoral ganglionic cells (prg) were located in the squamous epithelia forming the roof of the buccal cavity. The cell bodies of these neurons were embedded within the ciliated band epithelia (see Chapter 2) along the transverse region of Chapter five

Figure 1 Schematic diagrams of the bipinnaria larva of Patiriella regularis in ventral (A) and lateral (B) view showing position of the preoral ciliated band (procb), postoral ciliated band (pocb) and the adoral ciliated band (adcb). The anterior (ag) is indicated in purple lines and a black arrowhead. The red V represents the position of the adoral ganglion (adg), and processes that extend along the ventral surface of the oesophagus (red lines). The purple shaded area illustrates the position where the preoral ganglion is located behind the transverse preoral ciliated band (yellow regions in Figs. A and. B). Scale bar = 25 pm. Atter Strathmann Chapter five

Figure 2 Serotonin-like immunoreactivity in the bipinnaria and early brachiolaria of Patiriella regularis. A, B and D-G are red/green stereo anaglyphs. (A) Anterior ganglion innervating the preoral and postoral ciliated bands of a bipinnaria of Patiriella regularis. The view looks end on to the ganglion with the ventral surface at the top of the page. The top row of immunoreactive cells lay beneath the preoral ciliated band and the lower row of immunoreactive cells lay beneath the postoral ciliated band. The ciliated bands are not visible in this image at the depth of confocal sectioning. Anastomosing (n) join the two rows of immunoreactive cells. Cilia (white arrowheads), arising from serotonergic neurons in the ganglion. Scale bar = 26 pm. (B) The oral region of a dorsally flexed bipinnaria with its oral region completely open. A white arrow indicates the ciliated neurons of the adoral ganglion. This 3-D image clearly shows that these cilia are located under the rim of the oral hood and face inwards into the buccal cavity (be). Numerous varicosities (vn) can be seen along the immunoreactive neurites of the adoral ganglion that extend towards the oesophagus (o) and towards the transverse preoral ganglion. Scale bar = 30 pm. (C) A high magnification confocal projection of several ciliated neurons in the transverse preoral ganglion (prg) seen in image B. The highly fluorescent immunoreactive cell bodies have a cilium (white arrowheads) emanating from an apical protuberance. Note varicose projections from the soma (yellow arrowheads). Scale bar = 8 pm. Figure 2 continued: Chapter five

(D) Detail showing ciliated neurons (white arrowheads) arising from the prg and adg associated with the adoral and the preoral ciliated bands respectively. Neurites (n) connect the cells of the preoral transverse (prg) and the adoral ganglion (adg). Scale bar = 26 pm. (E) A dorsally flexed bipinnaria shown in an oblique lateral view. The cell bodies in the prg show the characteristic anastomosing neurites (n) from several cell bodies merging into one nerve tract that projects towards the adoral ganglion (immunoreactive cells lower half of the image). Scale bar = 36 pm. (F) Ventral view of a grazing section through the orai hood and a portion of the procb (region between the white arrowheads) and associated locomotory cilia (yellow arrowheads) of an early brachiolaria. This image shows the position of the serotonergic cell bodies of the prg in relation to the ciliated band. Cilia arising from the serotonergic cells are not visible due to the angle of viewing, as they project into the page. Scale bar = 12 pm. (G) A planar ventral view through the transverse preoral ciliated band showing the immunoreactive cells of the preoral ganglion. This 3D image clearly shows cilia (arrowheads) from serotonergic neurons projecting directly into the buccal cavity. Scale bar = 19 pm.

Chapter five

Figure 3 Differential interference contrast (DIC) ventral view from a live bipinnaria, showing the transverse part of the preoral ciliated band (procb) with putative 5-HT neurons (black arrows) lying within the ciliated band epithelium. The red arrowhead indicates the locomotory cilia of the ciliated band epithelia. Postoral ciliated band (pocb). Scale bar = 16 pm.

Chapter five 57 the preoral ciliated band (Fig.1 A, B 2B, D-G). Typically, three varicose- like processes arise from the base of each neuron.

5.4.4 The adoral ganglion

The adoral ganglion (adg) is located at the posterior rim of the mouth (Fig. 1A, 2B, D E). The serotonergic nerve cell bodies of the adoral ganglion (adg) lie within the epithelium of the adoral v-shaped ciliated band (adcb) (Fig. 2B, D-E and 4A) (Also see Chapter 2). The adoral ganglion is comprised of a bundle of processes along the lateral edge of the mouth opening, and ciliated neurons located along the lower portion of the mouth and parallels the adoral ciliated band (Fig. 1A, 2B, D, E). Neurites from the adoral ganglion extend anteriorly on either side of the buccal cavity (be), to connect with processes arising from the preoral ganglion (Fig. 2B D, E). Varicose neurites from the adoral ganglion are also in close association with the ventral side of the oesophagus and extended towards the oesophageal sphincter (Fig. 4). Neurites were not seen in the dorsal side of the oesophagus. Neurites also extended posteriorly from the ganglion and terminate near the end of the larva (Fig. 4).

5.4.5 Effect of L-PCPA on feeding and swimming on Patiriella regularis

Normal swimming and feeding behaviour

Patiriella regularis bipinnaria larvae swam in mid-water either in straight lines or with an undulating motion. The average straight line normal swimming speed was 454 pm s'1 (n= 7 SE= 27) with the larval anterior end leading and ventral surface uppermost. The process of capturing algal food involved several steps. Algal cells that were caught at the anterior end of the larva where the current streamlined along the edges of the larval surface (Fig. 5A, B) were directed into the buccal cavity from the lateral regions of the transverse preoral ciliated bands. As a result, the algal particles passed under the transverse preoral band. They then rotated inside the buccal cavity for several seconds before Chapter five

Figure 4 A confocal projection showing varicose neurites (vn) innervating the ventral oesophageal cells and projecting towards the oesophageal sphincter (os). Neurites (n) also project from the adg past the stomach (s); cell bodies (cb). Scale bar is 22 pm.

Chapter five 58 being transported via the adoral ciliated band into the oesophagus. A combination of oesophageal peristalsis and ciliary beat propelled the cells towards the stomach sphincter, which opened by muscular activity, followed by an influx of cells into the expanding stomach. After approximately one minute of encountering algal cells the bipinnariae had full stomachs.

Experiment 1: Observations of feeding and swimming behaviour immediately after the addition of L-PCPA

Larvae captured algal cells encountered at their anterior end or anywhere along the preoral transverse ciliated band. These algal cells were transported into the oesophagus. All the larvae had approximately 16 algal cells in their oesophagi and the algal cells were kept in constant motion due to the movement of cilia. Although the oesophageal cilia continuously propelled the algae towards the sphincter, which periodically opened and closed, no peristaltic contractions were observed in the oesophagus and the stomach did not expand to accommodate the influx of water and algal cells. As a result, algal cells were not transferred to the stomach (images not shown). Bipinnarial swimming speed did not appear to be affected by 2.5 pM L-PCPA treatment for the duration of the experiment (observations taken after 1 minute up to 1 hour). Swimming speed was not recorded. Control larvae had greater than 240 algal cells in their stomachs throughout the duration of the experiment (Fig. 5 A and B).

Experiment 2: Effects of a 30 hour larval incubation in L-PCPA

In the second experiment, after 30 hours in L-PCPA, larvae did not swim or feed and remained on the bottom of the culture dish. Algal cells entrained by water currents from the ciliated bands moved in an anterior to posterior direction along the larvae (Fig. 5C and D). Subsequently, when these algal cells encountered the transverse preoral ciliated band, water currents generated by cilia of this band captured these cells in a circular water current for a few seconds until the algal cells were expelled Chapter five 59 over the pocb (Fig. 5D). All larvae exposed to 30 hours in L-PCPA exhibited impaired feeding and swimming. Control larvae exhibited normal swimming behaviour and had full stomachs within a few minutes of the introduction of algae.

Experiment 3: Residual drug effects from L-PCPA

Bipinnariae that had been placed in FSW for 24 hours after L-PCPA treatment did not recover the ability to feed. After seven days of treatment, larvae appeared no different in general morphology than the controls. The experiment was terminated at 7 days when the gut of the larvae started to atrophy from lack of feeding.

5.4.6 Effect of L-PCPA on swimming in Patiriella calcar Normal swimming behaviour

Untreated Patiriella calcar larvae swam at approximately 4.5 mm per second with their posterior end leading, p-chlorophenylalanine (2.5 pM) treated (31.25hr) larvae did not swim.

5.4.7 Unknown effects of L-PCPA It should be noted that behavioural effects observed for L-PCPA on the above species could also be a result of toxic effects of this drug on other biochemical pathways unrelated to serotonin synthesis.

5.4.8 Immunostaining of L-PCPA treated bipinnaria.

Confocal microscopy revealed no obvious differences in the distribution or intensities of immunoreactive neurons in the pre- and postoral ciliated bands in P. regularis larvae which had been incubated for 30 hours in 2.5 pM L-PCPA when compared with controls. In the region of the adoral ciliated band, however there appeared to be fewer neurons (images not shown), which exhibited a lower intensity of staining compared to control larvae. These differences were not quantified. Chapter five

Figure 5 Schematic ventral (A and C) and lateral (B and D) images illustrating the ciliated bands and feeding responses of control (A and B) and treated (C and D) bipinnaria of P. regularis. The large black vertical arrow represents the direction of swimming in control larvae (A and B). Green arrows represent the path of algal cells (green spheres) as they are captured by water currents generated by the transverse preoral (yellow region) and adoral (adcb) ciliated bands. Blue arrows show where algal cells are redirected from lateral positions of the transverse preoral ciliated band (yellow region) into the buccal cavity (be) and then subsequently transported to the oesophagus (os) by water currents generated by the adoral ciliated band (adcb) and then into the stomach (s), anus (a). (C and D) In larvae treated with 2.5 pM L-PCPA, algal cells were captured in the transoral groove. The red circular arrows show an anti-clockwise water current where algal cells remained in motion and were subsequently ejected (posterior pointing single red arrow) and streamline away from the larval body (green arrows). Scale bar = 25 pm. Treated After Strathmann Chapter five 60

5.4.9 Chromatography

Serotonin content in treated (31.25hr in 2.5 pM L-PCPA) verses untreated P. regularis larval samples were quantified from the standard curve (Figure not shown). Comparison of control larvae (44.31 pg of 5- HT/mg dry weight of larvae) and drug treated larvae (31.52 5-HT pg/mg dry weight of larvae) indicated that L-PCPA treatment affected a 29% decrease in serotonin levels (Fig. 6). It was not possible to quantify 5-HT in P. calcar brachiolariae, which had been treated with 2.5 jiM L-PCPA, as the amount of 5-HT found was below the lowest quantifiable value on the standard curve (chromatogram not shown).

5.5 DISCUSSION 5.5.1 Immunocytochemistry and confocal microscopy

The presence of ciliated serotonergic neurons in the three ganglia of Patiriella regularis provides strong evidence for a primary sensory role for these neurons. For P. regularis this is a significant observation because it provides direct evidence of an association between serotonin in sensory-like neurons associated with the ciliated bands of a bipinnaria. Ciliated serotonergic neurons, as seen in this chapter have not been previously found in other asteroid bipinnariae. Although it is unlikely that the cilia of the serotonergic neurons in P. regularis function as locomotory cilia within the ciliated bands as there are too few in number. It is suggested that future work involve freeze fracture and transmission electron microscopy to examine for the absence of dynein in the "9+2" microtubule assembly indicating a non-motile cilium (Nakajima, 1988). Previous studies on the bipinnaria of asteroids have identified serotonergic neurons in the ciliated bands by immunofluorescence microscopy and presumptive identification of serotonergic neurons has been made by ultrastructural examination of the ciliated band epithelia (Nakajima, 1988). It is not known whether the serotonergic neurons seen in P. regularis are neurotransmitter specific or whether the same neuron Chapter five

Figure 6 Chromatograms from rpHPLC representing the 2.5 pM L-PCPA treated and untreated (control) bipinnariae of P. regularis. The retention time for serotonin (5-HT) was 6.2 minutes. The peak preceding the 5-HT peak is the solvent front. 5 10 min 2.5 (jM L-PCPA treated larvae

5 10 min Control Chapter five 62

5.5.3 Comparisons of the anastomosing serotonergic processes in P. regularis with other asteroid bipinnaria.

The anastomosing serotonergic neurites shown in this study has never been described before in the Echinodermata. Interestingly, the location of unknown catecholamines by glyoxilic acid histochemistry in the bipinnariae of Pisaster ochraceus (Burke, 1983) revealed a morphologically similar nervous system as seen in Chapter 2 and this chapter. It would be of interest for future studies to determine if the serotonergic neurons visualised in P. regularis bipinnaria are capable of expressing more than one neurotransmitter by undertaking co­ localisation immunocytochemistry. It has been suggested for an echinoid pluteus that the same neurons are capable of sequentially expressing dopamine and serotonin, however co-localisation immunolabelling was not performed to confirm this suggestion (Bisgrove and Burke, 1987).

Non-fluorescent techniques

A nervous system, described as a “network of nerve cells” was described in an echinopluteus, based on observations using DIC microscopy (Ryberg, 1977). The neuronal-like structures described by Ryberg bear a striking similarity to the architecture of the immunofluorescent anterior ganglion described for P. regularis. The anastomosing 5-HT processes in the ganglia of P regularis may represent a collection of axo-axonic synapses similar to that seen in Aplysia (Greenberg et al. 1987). Electron microscopic analysis of these processes is required to confirm this suggestion.

The transverse pre-oral ganglion

The ciliated serotonergic neurons of the transverse preoral ganglion, which were found to project into the buccal cavity have not been previously described for the bipinnaria of other asteroids (see also Chapter 4). Chapter five 63

Adoral ganglion

The adoral ganglion is comprised of a set of serotonergic cell bodies embedded within the adoral ciliated band (also see Chapter 2) from which serotonergic neurites extend, in an anterior direction, either side of the mouth meeting in close proximity with processes arising from the transverse preoral ganglion. A posterior set of serotonergic varicose neurites, also originating from the adoral ganglion, extend intracellularly amongst the cells of the oesophagus towards the oesophageal sphincter. The serotonergic adoral ganglion of P. regularis was revealed, by confocal microscopy, to be a complex structure, extending between the oral and oesophageal region with serotonergic processes originating from cell bodies embedded within the adoral ciliated band. Previously, serotonergic immunoreactivity associated with neuronal cells in bipinnaria larvae have only been associated with the ciliated bands and the anterior/apical ganglion (Nakajima, 1988; Lacalli, 1994 and Moss, et al. 1994). The association of serotonergic cellular varicose processes within the oesophagus in a bipinnaria has not previously been demonstrated. It now appears that the serotonergic nervous system of a bipinnaria is more extensive than previously thought. Burke (1983) described axons based on ultrastructural morphology, associated with the basal region of the ciliated epithelial cells of the oesophagus in the bipinnaria of Pisaster ochraceus. These axons partially encircled the oesophagus and contained numerous "swellings" that could be varicosities not unlike the serotonergic processes found in the equivalent position in the bipinnariae of P. regularis.

5.5.4 Imaging

Computer generated imaging greatly assisted in the spatial interpretation of the complex neuronal network of the ganglia in the larvae of Patiriella regularis. This technique allows one to distinguish between structures, which are merely laying on top of one another from those which are actually joined together. Chapter five 64

5.5.5 Effects of L-PCPA on feeding

The bipinnaria of P. regularis captured algal cells in a similar manner to that described for other planktotrophic asteroids (Strathmann, 1971, 1975; Hart, 1991). Once algal cells entered the oesophagus, ingestion of these cells into the stomach required peristaltic contractions of oesophageal muscles combined with ciliary beat and periodic opening of the esophageal sphincter. Inhibition of peristaltic activity in L-PCPA treated larvae suggested that muscular contraction in the oesophagus is under serotonergic neuromodulatory control. Innervation of the oesophagus of P. regularis by serotonergic varicose neurites derived from the adoral ganglion supports this suggestion. The increased peristaltic contractions of coelomic and esophageal muscles in echinoplutei treated with exogenous serotonin also provides evidence for a role in serotonin modulating muscular activity (Gustafson, et al. 1972). Patiriella regularis larvae, which failed to recover and feed after a 24 hour L-PCPA-free period in FSW, as seen in the final pharmacological experiment, suggested the effect of L-PCPA on feeding is irreversible. This data confirms what is currently accepted for the action of L-PCPA in vertebrate serotonin biosynthesis (Jequier, et al. 1967).

5.5.6 Effects of L-PCPA on swimming

Suppression of swimming in L-PCPA treated P. regularis larvae indicates that this drug may have affected a down regulation of ciliary frequency and suggests that a flux of serotonin may be required for an increase or a decrease in ciliary activity. The lecithotrophic planktotroph, P. calcar treated with L-PCPA did not swim, which suggested that, irrespective of the lack of ciliated bands in this larva, serotonin depletion by l-PCPA appeared to modify the behaviour of ciliated cells. It has been suggested that serotonin is involved in modulation of ciliary activity in echinoderms and other marine invertebrates (Hay-Schmidt, 1990; Diefenbach et al. 1991; Wada, 1997). The increase in swimming speed of planktotrophic echinoderm larvae treated with exogenous 5-HT Chapter five 65 indicates that this neurochemical induced an up regulation of ciliary frequency (Mogami, et al. 1992). The question remains, what are the functions of the serotonergic neurons in the pre- and postoral ciliated bands? For future work, it would be of interest to determine whether the serotonergic neurons associated with the pre- and postoral ciliated bands in P regularis have a motor or sensory function. Specifically targeting individual neurons with microinjection of L-PCPA and examining the effects on ciliary movement may provide answers to the functions of the serotonergic neurons of the pre- and postoral ciliated bands.

5.5.7 Effects of L-PCPA on other biochemical pathways

Prior to 31 hours in L-PCPA (experiment 1), some behavioural changes were observed associated with an absence of peristaltic movement in the oesophagus. Since rpHPLC was not done during this period, it can not be ruled out that the observed behavioural changes were due to some form of L-PCPA toxicity. While the serotonin depleting effects of l-PCPA on TPH has been well documented in vertebrates, no studies have been done to determine whether L-PCPA influences other biochemical pathways in invertebrates. Further studies are needed to characterize the pharmacology of L-PCPA in invertebrates.

5.5.8 Immunostained L-PCPA treated larvae

Patiriella regularis larvae, which had been incubated for 30 hours in 2.5 pM and L-PCPA and immunostained for serotonin, showed no difference in fluorescent staining patterns from control larvae although swimming and feeding were effected. These observations raise two questions: 1. Following L-PCPA application, was there an immediate cessation of TPH activity and the serotonin detected by immunocytochemistry a result of un-catabolised serotonin? 2. Did only partial inactivation of TPH occur, resulting in a reduced serotonin biosynthesis, thereby suggesting a threshold level of serotonin required for normal larval behaviour? In order to answer question one, the mechanisms of the serotonin catabolism must be known for P. regularis Chapter five 66 and if l-PCPA might affect this pathway. Little is currently known regarding what enzyme/s are present in the catabolism of 5-HT in invertebrates and it has been suggested that monoamine oxidase (MAO), the enzyme present in vertebrate serotonin catabolism, is not present in the cockroach (Sloley and Downer, 1984). However, MAO has been found in adult seastars of Marthastarias glacialis (Nicotra, et al. 1986) and the sea urchin sperm cells and embryos of Paracentrotus lividus (Nicotra and Naccarato, 1982; Nicotra, et al. 1989). It would therefore be of future interest to elucidate the catabolic pathway of such a universal neurochemical as 5-HT in the asteroid bipinnariae of P. regularis. In addressing question 2, partial inactivation of TPH by L-PCPA application has been described for the rat (Lauder and Krebs, 1978) and it has also been suggested that a threshold level of serotonin is required to maintain pseudopregnancy in rats (Maekawa and Yamanouchi, 1996). Based on the presence of serotonin immunoreactivity in neurons after 30 hours in L-PCPA and the behavioural changes observed, it is suggested that a threshold level of serotonin is necessary for normal larval behaviour in P. regularis bipinnaria. To test the above suggestion the determination of TPH activity after treatment with L-PCPA would be required.

5.5.9 The decrease in swimming activity and the loss of feeding.

The reduction in serotonin level, caused by the application of L- PCPA in Patiriella regularis, may have affected intracellular cyclic AMP (cAMP) levels, resulting in a change in ciliary activity. Interactions between cAMP and serotonin and resultant changes in ciliary frequency have been demonstrated in echinoderms (see Soliman, 1984 a, b; Mogami, et al. 1991; Murakami, 1989), protists (see Hamasaki, et al. 1991) and in several gastropods (see Sanderson et al. 1985; Stephens and Prior, 1992). Chapter five 67

5.5.10 A nervous system model to describe feeding behaviour in P. regularis

If the ciliated serotonergic neurons of the three ganglia observed in the bipinnaria of Patiriella regularis are involved in feeding, what might their function be? Based on behavioural observations, the following model can be suggested. Algal cells encountered at the anterior end of the larvae are entrained in water currents generated by the ciliated bands and are transported into the buccal cavity by ciliary action of the transverse preoral ciliated band. As they enter the buccal cavity the algae pass by the ciliated serotonergic neurons of the preoral ganglion that lies within the transverse preoral ciliated band. These neurons are likely to function as a set of mechano- receptors and may send a signal via the serotonergic neurites lying on either side of the mouth to the adoral ganglion thereby stimulating the cilia of the adoral ciliated band to reverse their direction of beat. Reversal of ciliated beat is a characteristic of suspension feeding in echinoderm larvae (Strathmann, 1975, 1978). As algal cells pass the adoral band, their presence may be detected by ciliated serotonergic neurons lying within this band. These neurons in turn communicate with serotonergic neurites innervating the oesophagus and may thus influence peristaltic activity resulting in ingestion of the algal cells. If the serotonergic ciliated neurons of Patiriella regularis bipinnariae are involved in modulation of the correct sequence of ciliary beat for feeding, then interference of serotonin biosynthesis would be expected to cause a decrease in the ability of the larva to capture and ingest food as observed in the L-PCPA treated larvae. Hart (1991) speculated that a mechanism controlling the ciliated bands might be responsible for particle rejection in larval echinoderms. It was shown that mechanical stimulation of the cilia of the acron, a region of the ciliated band above the oral opening of the echinopluteus is involved in controlling muscular activity in the oesophagus (Gustafson, et al. 1972). GABAergic neurons were also found in the same region in a different species of echinoid by Bisgrove and Burke (1987), where it was Chapter five 68 suggested that these neurons are involved in sending signals via axonal tracts to the oesophagus and hence controlling "swallowing". In the same study, anti-serotonin labelling failed to reveal serotonergic neurons in the same region. It could be possible, that while the preoral and adoral ganglia of the bipinnariae of P. regularis may be involved in the ingestion of algal cells, serotonin may not be functioning in the same way in an echinopluteus.

5.5.11 Reverse phase high performance liquid chromatography

Reverse phase high performance liquid chromatographic analysis in l-PCPA treated bipinnariae of Patiriella regularis, confirmed a decrease in serotonin content, suggesting an inhibition of tryptophan hydroxylase (TPH) activity. These data suggest that serotonin biosynthesis in P. regularis probably follows the hydroxylation of tryptophan to 5-hydroxytryptophan pathway as seen for many vertebrates (Frazer and Hensler, 1994). The ubiquitous occurrence of 5-HT is well known (Collier, 1958). However, the presence of 5-HT across many phyla is not an indication of a universal biosynthetic pathway operating amongst the species. It appears, based on the rpHPLC and pharmacological data that the first step of 5-HT biosynthesis in P. regularis is regulated by TPH. Another experimental possibility may have been to identify the presence and activity of TPH. It is anticipated, based on limitations of culturing larvae for this thesis, enzyme purification, identification and activity using current methods for TPH assays (Nagai, et al. 1997 and Cash, 1998), would not be practically possible. Recently there has been a suggestion that L-PCPA is not, in fact, as specific in inactivating tryptophan hydroxylase as previously thought (Yang and Pan, 1999). Given this, it would be of interest to investigate immunocytochemical and biochemical techniques the effects of L-PCPA on other neurotransmitters, such as dopamine in P. regularis. Chapter six 69

Localisation of serotonin in the brachiolaria larvae of Patiriella regularis, P. exigua and P. calcar Chapter six 70

6.1 ABSTRACT Serotonin-like (5-HT-like) immunoreactivity was documented in brachiolaria larvae of the congeneric seastars Patiriella regularis, P. calcar and P. exigua. The brachiolariae of the planktotrophic developer P. regularis, had flask shaped 5-HT-like immunoreactive cells and processes innervating the preoral and postoral ciliated bands and in the brachiolar attachment complex. The first immunoreactive cells associated with this complex were seen in the early brachiolariae when the median brachia began to show as a bulge at the anterior of the larva. The transition from the bipinnaria to the early brachiolaria is seen with the extension of the anterior coelom to form the lumen of the central and lateral brachia, which did not contain immunoreactive cells at this early stage of development. As the central brachium developed, immunoreactive cell bodies were clustered in association with the papillae. In contrast to the bipinnarial stage, serotonin-like immunoreactivity was not detected in the adoral ciliated band of the mouth, suggesting a transitional change associated with the reorganisation of serotonergic neuronal activity in accordance with a change in developmental behavioural states, from feeding to settlement. The early brachiolaria of P. exigua exhibited a preneuronal expression of 5-HT within the epithelium of the entire larva. Upon hatching, fusiform 5- HT-like neurons were observed in the attachment complex only. A similar observation was made for the planktonic lecithotrophic brachiolaria of P. calcar. Regardless of developmental mode these seastar species, all possessed a brachiolar complex, which is involved in settlement, and it appears that the serotonergic innervation of this complex has been conserved. Application of the serotonin depleting drug para chlorophenylalanine on settled P. exigua brachiolariae caused the larvae to release their arms from the substrate, suggesting a role for serotonin in settlement behaviour. The presence of immunoreactivity in the ciliated bands and attachment complex suggests a multi-functional role of 5-HT in the larvae of P. regularis and P. calcar and P. exigua in modulation of swimming, settlement. Chapter six 71

6.2 INTRODUCTION

In most planktotrophic and lecithotrophic asteroids, the larvae develop through a brachiolarial stage prior to settlement (Hyman, 1955; Oguro, et al. 1987). The planktotrophic brachiolaria is characterised by three anterior brachiolar arms which differ in length: two short and one long. Central to the three brachiolar arms is an attachment disk (Hyman, 1955; Barker, 1978). In this chapter, the three brachia and central disk are defined as the attachment complex. There are exceptions where no brachiolarial stage is present (McEdward, 1992) or where brachiolaria develop arms of equal length, as in the case of the benthic larvae of Patiriella exigua (Byrne, 1995). Larval settlement in asteroids has been suggested to be cued by exogenous compounds, presumably acting on sensory nerves located within the attachment complex (Barker, 1977; Barker, 1978; Bisgrove and Burke, 1987; Byrne, 1991). Barker, (1978) has reported that the ventral surface of the brachia are covered with papillae, structures that contain an abundance of secretory cells. In the brachia of planktotrophic larvae, papillae provide temporary anchorage to a substratum during metamorphosis. Observations of brachiolariae “testing” the substratum prior to settlement suggests that site selection in the settlement process is under neuronal control (Barker, 1978; Byrne and Barker, 1991). The ultrastructure of neurons in the brachiolarial arms in two species of planktotrophic asteroids suggests a possible chemosensory role for these neurons (Barker, 1978). However, it is not known what neurotransmitters are present or the neuronal organisation of the attachment complex. As seen in Chapters 2 and 3, a serotonergic nervous system is present in the ciliated bands of the feeding bipinnaria of Patiriella regularis. This chapter describes the nervous system of the final larval stage of Patiriella regularis, the brachiolaria, with an emphasis on the serotonergic neuronal organisation of the attachment complex and the potential role of serotonergic neurons during larval settlement and metamorphosis. The serotonergic nervous system in these planktotrophic larvae is compared with that of larvae of two lecithotrophic species, P. calcar and P. exigua. Chapter six 72

Like P. regularis, P. calcar has a planktonic brachiolaria, while the brachiolaria of P. exigua is benthic. The following questions are addressed: 1. Does the attachment complex of P. regularis contain serotonergic neurons? 2. Is the nervous system seen in the bipinnaria of P. regularis continuous with a brachiolarial nervous system? 3. The brachiolaria is also a feeding larval stage, having the same ciliated band morphology as the bipinnaria. However, with the development of the attachment complex a behavioural change is seen where the brachiolaria is primarily concerned with finding a suitable substrate on which to complete metamorphosis (Byrne and Barker, 1991). Therefore, is there a change in neuronal organisation resulting from the shift from a feeding bipinnaria to the settlement stage brachiolaria in P. regularis? 4. In the evolution of the lecithotrophs, P. calcar and P. exigua, the ciliated bands and the bipinnarial stage was lost (Byrne and Barker, 1991). The brachiolariae of P. calcar and P. exigua were examined to determine if there were any vestiges remaining of the bipinnarial nervous system. The neuronal organisation of the attachment complex of these larvae is compared with that seen in P. regularis. 5. Do serotonergic neurons, if present in the attachment complex of these three congers, play a role in settlement? Answers to the above questions describe the comparative serotonergic putative nervous systems of the planktotrophic Patiriella regularis and the lecithotrophic P. calcar and P. exigua, and show what aspects of neurogenesis are conserved and what are modified in the evolution of development. Chapter six 73

6.3 MATERIALS AND METHODS

Larval culturing of the planktotrophic Patiriella regularis and lecithotrophic species P. calcar and P. exigua (see chapter 2 section 2.1-2.2.2).

6.3.1 Immunocytochemistry

Patiriella regularis Patiriella regularis brachiolaria at different stages of development were selected from 48 day old cultures according to the stages described in Byrne and Barker (1991). The selected stages included the following: 1) early brachiolaria, with the two lateral brachia just observable 2) larvae with a fully developed attachment complex, and 3) larvae with developing adult skeleton. Larvae were relaxed in 7% MgCI2 and then fixed in 4% paraformaldehyde in FSW at 4°C for 1-2 hours. Specimens were immunostained and prepared for confocal microscopy according to Chapter 2 sections 2.4- 2.6.4.

P. calcar Immunocytochemistry was performed on seven day-old Patiriella calcar brachiolaria as described in Chapter 2 sections 2.3-2.6.4. All times quoted are post-fertilisation.

P. exigua Unhatched (early) brachiolaria (66 hours) of P. exigua were excised from their fertilisation envelopes using 19 gauge hypodermic needles as blades, and were fixed in 4% paraformaldehyde in FSW from 3 to 12 hrs at 4°C for immunocytochemistry. Hatched and metamorphosing brachiolaria of P. exigua were relaxed as above. After fixation, the specimens were prepared for primary antibody incubation as described in Chapter 2 section 2.4. Gastrulae and larvae were then incubated in primary antibody for 17 hr to 27 hr at 4°C. The secondary antibody, biotinylated goat anti-rabbit was diluted at 1 in 50 in 0.1 M PBS and specimens incubated for 12 hrs at 23°C. The final Chapter six 74 incubation was with FITC streptavidin diluted at 1 in 100 in 0.1 M PBS for 20 minutes at 23°C in the dark. Controls (see section 2.4 general materials and methods).

6.3.2 Histochemistry of P. calcar and P. exigua

The yolky larvae of Patiriella calcar and P. exigua are not ideally suited for immunofluorescence microscopy, because the high yolk content interferes with antibody penetration (Chee personal observations). As a result the Faglu histochemical technique (Furness et al. 1977), which is not reliant on the penetration of antibodies, was used. Unlike the formaldehyde induced fluorescence (Falck, 1962) and the glyoxilic acid (Bjorklund, et al. 1973; Axelsson, et al. 1973; Lindvall and Bjorklund, 1974; and Sharpe and Atkinson, 1980) techniques of histochemical staining for biogenic amines, the Faglu reagent has been used to identify biogenic amines at room temperature under aqueous conditions. The Faglu reagent (0.5% glutaraldehyde and 4% paraformaldehyde, pH 7.0 in FSW) at 23°C, reacts with 5- hydroxytryptamine to produce water stable dihydroquinones. These fluorophores can be identified by the colour of the emission spectra on excitation with a specific wavelength using a suitable epifluorescent emission excitation filter set with an epifluorescence microscope. Hatched and metamorphosing brachiolariae of Patiriella exigua and seven day old P. calcar brachiolaria were immersed in a Faglu solution (0.5% glutaraldehyde in 4% paraformaldehyde) at pH 7 in FSW at 23C° for 2 days. Planktonic brachiolaria of P. calcar were treated as above. Controls for histochemistry consisted of fixing larvae in 4% paraformaldehyde for the same time. Histochemically stained larvae were prepared for microscopy as detailed in Chapter 2 sections 2.5 and 2.63. Larvae and metamorphosing larvae were examined for bright yellow fluorescence indicating the presence of biogenic amines, using a violet excitation filter block set (395-440 ex FT 460 LP 470) fitted to a Nikon E800 microscope, Neuronal cells fluorescing bright yellow were defined as “aminergic neurons”. Specimens were photographed using colour Chapter six 75 slide film. Slides were digitised using a Nikon LS-1000 slide scanner at 300-dpi resolution. Subsequent imaging with a confocal microscope was used to reduce fluorescent flare (Wijaendts van Resandnt et al. 1984 and White, et al. 1987). Various numbers of optical sections were collected at different depth intervals (depending on specimen thickness and opaqueness) using a range of objective lenses (x20, x40 and NA 1.4 x63 oil, x100 oil immersion lenses). All images of Patirieila calcar and P. exigua (unless otherwise stated) are displayed in an orientation, with the brachiolar complex down, attached to the substrata.

6.3.3 Depletion of serotonin by p- chlorophenylalanine

Patirieila exigua oocytes were fertilised and raised to the hatched brachiolaria stage (4 days) as described in (Chapter 2 section 2.2.2). The FSW was carefully removed so as not to disturb the settled brachiolaria and replaced with 300 mL of 5 pM L-PCPA in FSW at 22°C-23°C. Parallel cultures were raised in FSW and served as the control group. Observations were recorded at 5 and 20 minutes after treatment. At each time period, 10 larvae were randomly selected and the number of larvae counted raising 1-2 brachiolar arms from the substrate. The sampling was randomly repeated ten times and the data pooled. A Chi square analysis was performed on the data using JMP (SAS Institute Inc.) with a P <0.0001 to determine if there was a difference in treated verses untreated brachiolaria.

6.4 RESULTS

6.4.1 Serotonin-like immunoreactivity in the brachiolariae of Patirieila regularis.

In Patirieila regularis, the onset of brachiolarial development, coincided with the elongation of the anterior extension, involved the elongation of the anterior coelom in the bipinnaria (Figs. 1 A and A1). Externally, several small protuberances, the early median brachium and right and left lateral brachia began to develop on the anterior extension 6 .>< £ l & 0 .

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_>, X X X *0 X 4 O '0 X 03 4—* — -4 _C •4—* 'c -4—* 0 c 0 03 CL o X 3 0 0 3 > 0 C 1 > 0 0 _ k 0 0 0 o E E 0 03 0 0 0 _ i 0 E c 03 0 0 0 0 o E 0 Q. —> 0 0 0 c 03 0 — — 0 0 o o C 03 1 depth of 166 pm. Serotonergic neurons with anastomosing processes associated with the pre- and postoral ciliated bands (white arrows) are positioned laterally to the anterior coelom (ac). The anterior regions of the preoral and postoral ciliated bands are no longer immunoreactive. emb

ac Chapter six 76

(Fig. 1B). Confocal optical sectioning through the anterior extension of an early brachiolaria revealed the presence of serotonin-like immunoreactive cells in the sub epithelial region (Figs. 1C and D). The median brachium formed as a bulge on the anterior extension and immunostaining for serotonin revealed a collection of serotonergic cells, devoid of cellular processes, covering the emerging median brachium (Fig. 1C). The ventral side of the anterior extension had several serotonergic processes underlying the epithelium (Fig. 1C). At the same time as the median brachium emerged, two small lateral brachia developed at the base of the anterior extension (Fig. 1C). These lateral brachia did not contain serotonergic neurons at this early stage of development. The serotonergic anterior ganglion, previously seen in the anterior region of the bipinnaria connecting the preoral and postoral ciliated bands (Chapter 2), was no longer visible in the brachiolaria of P. regularis (Fig. 1D). However, two opposing lateral regions of serotonergic neurons with processes spanning the pre- and postoral ciliated bands on either side of the median brachium may be the remains of the anterior ganglion which were now being displaced as the brachium grew (Fig. 1D).

6.4.2 Advanced brachiolaria of P. regularis.

In the advanced brachiolaria of P. regularis, a group of papillae developed at the antero-ventral region of the median brachium (Fig. 2A) along with several papillae scattered over the surface of the brachium (Fig. 2A) Numerous papillae had also formed on the surface of the lateral brachia (Fig. 2B). The surface of papillae of the median brachium were covered in a clear mucus-like substance (Fig. 2C). Confocal serial sections of the advanced brachiolaria (Figs. 3A-D) revealed the presence of serotonergic neurons underlying the epithelia of the papillae of the brachia (Fig 3A and B). Serotonergic neurons were also associated with the dorsal epithelial surface of the median brachium and the anterior extension (Fig. 3C). The continuation of the bipinnarial Chapter six

Figure 2. (A-C) DIC images of the attachment complex of a live advanced Patiriella regularis brachiolaria. (A) Papillae (p) on the median brachium (mb). (B) Papillae on the right (rb) and left (lb) lateral brachia. Scale bars = 33 pm. (C) A lateral view of a median brachium, showing a clear mucus­ like substance (mis) covering the papillae. Anterior coelom; ac, cilia; c. Scale bar = 33 pm.

Chapter six

Figure 3. (A) 3-D anaglyph ventral view of an advanced brachiolaria of P. regularis created from 195 confocal optical sections representing a sectioning depth of 208 pm showing serotonergic neurons in the attachment complex and the pre- and postoral ciliated bands. Medium brachium (yellow arrow), postoral ciliated band (pink arrows), right brachium (rb), left brachium (lb), preoral ciliated band (white arrow), anus (red arrow), preoral transverse ciliated band (parenthesis). Scale bar = 158 pm. (B) A high magnification of the median brachium (yellow arrow) created from 44 confocal optical images from image (A) representing a sectioning depth of 39 pm. Brachial papillae are seen as protuberances on the ventral surface of the medium brachium. Serotonergic neurons are seen within the papillae (white arrows). The two lower left arrows indicate serotonergic neurons within the right lateral brachium. Adhesive disk (ad). Scale bar = 39 pm. (C) Dorsal view of (A) showing serotonergic neurons innervating the anterior dorsal surface of the median brachium. Scale bar= 158. (D) A 3-D anaglyph created from 89 images from the anterior region of a non- relaxed advanced brachiolaria, representing a sectioning depth of 95 pm. Serotonergic neurons and processes are associated with the median brachium (yellow arrow) and right (rb) and left (lb) lateral brachia. Scale bar = 34 pm.

Chapter six 77 serotonergic nervous system associated with the pre- and postoral ciliated bands was also evident (Fig. 3A and C). Image projections of several optical sections through the epithelium of the lateral and median brachia showed each serotonergic neuron with an intensely fluorescent apical region above a basally located non- fluorescent cell nucleus (Figs. 4A and B). Apical cell processes from the serotonergic neurons extended towards the surface of the epithelium of these brachia (Figs. 4A). Unlike the patterns of immunoreactivity observed for the adoral ganglionic cells in the bipinnaria (Chapters 2 and 3), there was an absence of immunoreactivity in the adoral region of the advanced brachiolaria (Fig. 4 C) and a decrease in immunoreactivity in the transverse preoral ganglion (Fig. 5A). Large immunoreactive cells (x 40 pm 0) which did not appear to be neuronal-like, (Fig. 5A), were approximately 4 times larger than the typical serotonergic neuron and were present in the ciliated bands at the same time as the appearance of the juvenile skeleton during rudiment formation (Fig. 5B). Control larvae were non-fluorescent.

6.4.3 Serotonin-like immunoreactivity in the brachiolariae of Patiriella calcar.

Patiriella calcar hatch at the gastrula stage and development proceeds directly to the planktonic brachiolaria without a bipinnaria stage. As the median brachium elongated, two lateral brachia began to form at the base of the median brachium on the ventral side of the larva forming an early attachment complex. This attachment complex developed directly from an extension of the animal pole in the gastrula. About seven days after fertilisation, the anterior region of the median brachium had curved towards the ventral side with the adhesive disk located between these brachia. The attachment complex of the advanced brachiolaria of P. calcar has one long arm, the median brachium, and two shorter lateral brachia similar to the brachiolarial complex of P. regularis. (Fig. 6A). Serotonergic neuronal-like cells covered the ventral side of the brachia of Patiriella calcar (Figs. 6 B-D) and were located within the epithelial cells (Figs. 6 C-D). Neurons were fusiform with a highly fluorescent Chapter six

Figure 4. (A) An image projection from several confocal optical sections through a left lateral brachium of an advanced brachiolaria. Serotonergic neurons with non-fluorescent nuclei extending through the brachial epithelium (arrowheads). Brachial coelomic space; cs. Scale bar = 21 pm. (B) An image projection from several confocal sections through a median brachium (yellow arrow) showing serotonergic neurons and processes (yellow arrowheads) within the epithelia around the lateral perimeter of this cross section. The density of the specimen prevents internal imaging. Scale bar = 25 pm. (C) A confocal projection created from the plane of the adoral ciliated band of an advanced brachiolaria, showing the absence of immunoreactivity in this advanced stage of larval development. Adoral ciliated band; adcb, mouth; m, right brachium; rb, left brachium; lb, median brachium; mb. Scale bar = 189 pm.

Chapter six

Figure 5. (A) Confocal optical section parallel to the ventral surface of an advanced P. regularis brachiolaria. This section cuts the preoral transverse ciliated band and shows the lack of immunoreactive neurons (yellow arrows), and the squamous epithelia at the top of the buccal cavity (white arrow). The red arrow indicates atypical serotonergic cells in the transverse preoral ciliated band. Scale bar = 47 jjm. (B) A DIC polarised light image of a live advanced brachiolaria showing the formation of the juvenile skeleton (black arrow) in the forming rudiment. The yellow and white arrows indicate similar regions as seen in (A). Preoral ciliated band; procb, postoral ciliated band; pocb, right brachium, rb, left brachium; lb. Scale bar = 65 jim.

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Chapter six 78 tapered apical end which projected towards the epithelium (Fig. 6 C). Confocal serial sectioning revealed that the cilia found covering the brachial arms were not associated with the neurons seen within the epithelium (Fig. 6 D). Serotonergic immunoreactivity was absent from the posterior region of the larvae. Control larvae were non-fluorescent.

6.4.4 Histochemical staining of P. calcar

Patiriella calcar brachiolaria treated with Faglu reagent and examined by conventional epifluorescence microscopy revealed numerous cells, which fluoresced yellow' within the brachia indicating the presence of biogenic amines (image not shown). Confocal microscopy of these cells revealed the presence of fusiform neurons (aminergic neurons) amongst the columnar epithelial cells of the brachia, forming a dome-like array at the distal region of the brachiolar arms (Figs. 7A and B). The apical region of the neurons were highly fluorescent and nuclei were non- fluorescent (Fig. 7 B). From the base of the aminergic neurons arose a branching and anastomosing network of fibres, which lined the coelomic epithelium of the brachiolar arms. These fibres were observed to connect the adhesive disk and brachiolar arms (Fig. 7 B). Aminergic neurons were not found in the poster region of the larvae of P. calcar.

6.4.5 Serotonin-like immunoreactivity in the early and hatched brachiolariae of P. exigua.

Early unhatched brachiolaria (66 hours) of P. exigua exhibited a general preneuronal expression of 5-HT-like immunoreactivity in the apical cytoplasm of the ectoderm (Figs. 8A-B). No serotonergic neuronal-like cells were identified in early brachiolariae. This early larva had a bilaterally symmetrical morphology with a long median brachium and two small lateral brachia (Fig. 9A) The larval surface was covered in microvilli with a sparse cover of cilia. Cilia were encircled by microvilli (Fig. 9B). Serotonergic neuronal-like cells were seen in the advanced hatched brachiolaria (4 days) (Figs. 10A-D). These larvae had a radially Chapter six

Figure 7. (A) 3D anaglyph created from 116 optical sections representing a 124 pm thick sectioning depth from a brachiolaria larva of Patiriella calcar. The oblique lateral view shows aminergic neurons innervating the bulbous tips of the brachiolar complex. Note the remaining larval body is devoid of aminergic cells. Scale bar = 172 jam. (B) A high magnification of a lateral view of the brachiolar complex illustrating the extensive anastomoses of aminergic neurites innervating the brachiolar arms (white arrow) and the adhesive disk (white arrow heads). Intensely fluorescent fusiform cell bodies project their apical region towards the exterior of the epithelium. Note the non-fluorescent nuclei (black arrowheads). Adhesive disk; ad. Scale bar = 47 pm. A Chapter six

Figure 8. (A) A projection created from 40 optical sections taken at 4.3 pm intervals of an early Patiriella exigua brachiolaria (66hr) excised from the fertilisation envelope. The general luminosity indicates 5-HT-like expression originating from the epithelia of the entire brachiolaria. Median brachium; white arrow, lateral brachia; yellow arrows. There is an absence of neurons at this stage of development. The larva when released from its’ fertilisation envelope is slightly convoluted as it would appear inside the fertilisation envelope and immersion in paraformaldehyde has fixed it as it would normally appear. Note the culture debris stuck to the distal regions of the brachiolar arms. Scale bar = 95 pm. (B) A High magnification projection from a section of the epithelia of a 66 hour unhatched brachiolaria of P. exigua showing 5-HT-like immunoreactivity. Note the absence of neuronal-like cell bodies. (Bi) Inset indicates the region of the columnar epithelial cells responsible for 5-HT expression. Scale bar = 8.2 pm.

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Chapter six

Figure 10. Three dimensional stereo anaglyphs of showing the serotonin­ like immunoreactivity in the brachiolaria (photomicrographic insert) of Patiriella exigua. (A). A low magnification image of a larval brachiolaria beginning to acquire juvenile features such as the pentamerous posterior region with a distinct ridge beginning to form (white arrowheads) where the tube feet will appear. The posterior region of the larva is devoid of serotonergic immunoreactivity. The bulbous tips of the brachiolar arms show numerous serotonergic cells (white arrows). (Note the black circle is a trapped air bubble). Scale Bar = 200 pm. (B) A high magnification projection of a bulbous tip produced from 74 optical sections comprising of a 65 pm thick section clearly showing the array of serotonergic-like neurons throughout the epithelium of a bulbous tip. Scale Bar = 145 pm. (C) A serotonergic-like dendritic-like cell, with a highly fluorescent apical end (white arrow) reconstructed from 23 optical sections. The view is from the inside of the larval bulbous tip looking out (into the page). White arrowheads are neurites. Scale Bar = 12 pm. (D) A sensory-like cell in the epidermis of a buibous tip showing a cilium (white arrow) projecting from the neuron. Scale bar = 12 pm.

Chapter six 79 symmetrical tripod-like shape, with the brachiolar arms being equal in length (Fig. 10A and insert). Serotonergic-like neurons were evenly distributed over the bulbous tip of the brachiolarial arms (Figs. 10A and B). The shape of these cells resembled those seen by Faglu staining of P. calcar (Section 4.32). The highest fluorescence intensity was seen at the apical region of the neuron (Fig. 10C). A few serotonergic cells were found to possess a single cilium (Fig. 10D) similar to those found in P. regularis (Chapter 2). Serotonergic Immunoreactivity was not seen in the posterior region of the larva (Fig. 10A).

6.4.6 Histochemical staining of P. exigua

Hatched brachiolaria of Patiriella exigua (Fig. 11 A) treated with Faglu reagent revealed fusiform-shaped aminergic neurons (Fig. 11 B). Aminergic neurons were found covering entire bulbous tip of the brachiolar arms (Fig. 12 A-C). Aminergic neurons were situated between the columnar epithelium cells (Fig. 12 A-C). The apical ends of these neurons were intensely fluorescent and projected towards the outer surface of the epithelium (Fig. 12 A-C). Each neuron gave rise to a single basal neurite below a non-fluorescent nucleus (Fig. 12 A). A complex anastomosing network of neurites lining the basal region of the epithelium encircled the axohydrocoelic cavity of the brachiolar arms (Fig. 12 B, C). Histofluorescence was absent from the posterior region of the brachiolaria (Fig. 12 A). As the brachiolariae metamorphosed, the posterior region of the larva became pentamerous and the developing juvenile formed functional tube feet (Fig. 13). The attachment complex is located central to the future oral region and contained numerous neurons. Aminergic neurons were not present in the developing juvenile body (Fig. 13). The distribution of serotonergic neurons in the brachiolaria, whether revealed by immunocytochemistry or histofluorescence, occurred at the same time as with the radial symmetry of the larva (Fig. 14A). An imaginary set of axes from the anterior to the posterior brachiolarial surfaces and through the centre of the adhesive disk (and internal structures) can be divided by three perpendicular planes intersecting at Chapter six

Figure 11. (A) Bright field image of a Faglu stained Patiriella exigua hatched brachiolaria. Remnants of the red fertilisation envelope (red arrowhead) can be seen covering the part of the brachiolar arms. Epithelium; yellow arrowheads, lateral brachia; black arrows. The median brachium is indistinguishable from the lateral brachia. (B) Epifluorescent image of the same specimen showing numerous yellow aminergic nerve cell bodies covering the distal region and parts of the brachiolar arms. Scale bars = 117 pm.

Chapter six

Figure 12. Three dimensional stereo anaglyphs of the bulbous tips of the brachiolar arms of P. exigua stained by Faglu reagent showing aminergic nerve-like cells at increasing depth into the larva. The viewing angle is from the distal most surface of the bulbous tip. (A) An anaglyph produced from 78 optical sections, each captured at a step size of 0.6 pm, representing a 41 pm thick view from just under the epithelium of the bulbous tip of the brachiolar arm. Highly fluorescent apical ends of nerve­ like cells can be seen covering this hemispherical portion of the bulbous tip. White arrow indicates a branching neurite. (B) A View directly underneath the epithelium, reconstructed from sections 24-58 illustrating the complex network of anastomosing aminergic fibres (white arrowheads). (C) Anaglyph showing aminergic fibres lining the remanent blastocoelic space (cs), reconstructed from sections 37-57. Epithelium; yellow arrowheads. Scale Bar = 17 pm.

Chapter six

Figure 13. View of the future oral surface of a metamorphosing juvenile of P. exigua represented by a 3-D anaglyph showing aminergic neurons innervating the attachment complex of a metamorphosing juvenile. Adhesive disk; ad. Note the absence of biogenic amine expression in the tube feet (white arrows represent non-specific autofluorescence of culture debris on the three tube feet). Scale bar = 126 pm.

Chapter six

Figure 14. (A) Schematic diagram of P. exigua brachiolaria showing the identical brachiolar arm length resulting in a radially symmetrical brachiolaria. (B) Schematic diagram representing the triradiate symmetry of the nervous system in the attachment complex of the brachiolaria. The black dots represent serotonergic neurons in the attachment complex. The 'Y" shaped arrowed marker represents an imaginary' plane where the larva can be divided into three evenly spaced regions from the anterior surface to the posterior surface. B Chapter six 80 the central axis (Fig 14B). Each plane bisects a brachiolar arm and hence divides the larva into three equal sections resulting in a radially symmetrical brachiolaria

6.4.7 Effects of p- chlorophenylalanine on P. exigua brachiolariae.

The distal region of the brachiolar arms of untreated Patiriella exigua brachiolaria flatten against the substrate and possessed a tenacious hold on the culture dish. (Fig. 15) On immediate application of L-PCPA solution, brachiolaria (n=100) released two arms, inverted themselves so that the adhesive disk faced upwards and moved their arms about. The frequency of arm detachment in treated larvae was significantly greater (P<000.1) than that seen in the untreated control larvae. Arm waving was observed for up to 20 minutes after application of L-PCPA. After 45 minutes the frequency of arm waving of the treated larvae was not significant (P< 0.05).

6.5 DISCUSSION

6.5.1 The planktotrophic developer

Serotonergic neurogenesis from the bipinnaria to the brachiolaria of Patiriella regularis involved several changes in the bipinnarial nervous system. 1. The anterior ganglion, which spanned the pre- and postoral ciliated bands in the bipinnaria (Chapters 2 and 3) was no longer visible at the tip of the anterior extension. However, similar serotonergic structures were located laterally and associated with the pre- and postoral ciliated bands. It might therefore be speculated that the anterior ganglion appears to have been pushed to both sides of the anterior extension. Future studies using cell fate markers would need to be done to confirm the above suggestions. 2. Coinciding with the apparent rearrangement of the anterior ganglion, a cluster of serotonergic neurons developed within the epithelium of the newly emerging median brachium. It is suggested that serotonin in these cells may act as a neurotrophic factor. Chapter six

Figure 15. Anterior view of a brachiolaria of Patiriella exigua attached to a glass surface by the distal regions of the brachiolar arms. Note the flattened region of two of the brachiolar arms (black arrows). Scale bar = 130 jim.

Chapter six 81

3. Protuberances previously described as papillae (Barker, 1978 and Byrne and Barker, 1991) formed on the ventral surface of the brachia. In Patiriella regularis, numerous serotonergic neurons were associated with the epithelia of the papillae. The origins of these serotonergic neurons may have been the initial serotonergic cells seen during early brachiolarial development. Cell migration studies would need to be conducted to confirm this hypothesis. The papillae of the median brachium in P. regularis were covered with a clear mucus-like substance that presumably is the adhesive material used in attaching the larvae to the substratum. Barker, (1978) suggested that adhesive substances are continually produced from papillae, and these compounds are used to secure the larva temporarily to the substrata prior to settlement. It has also been suggested that ciliated neurons within the attachment complex function as chemoreceptors for chemical stimuli arising from the substratum (Barker, 1978). While ciliated neurons were not found amongst the serotonergic neurons in the brachiolarial attachment complex in P. regularis, it is suggested that the serotonergic neurons seen in the papillae of P. regularis may be involved in reception/transduction of chemical stimuli originating from the substrata. Combined ultrastructural examination and pharmacological depletion of serotonin using L-PCPA at the settlement stage could be undertaken to confirm this suggestion. Unlike the transparent bipinnaria, the developing brachiolaria gradually loses its transparency as the median brachium forms. It was not possible to image internal structures by confocal microscopy. As a result, it was not possible to determine if there was a nervous-like connection between the serotonergic neurons seen in the attachment complex of the brachiolarial with the pre-existing bipinnarial nervous system. Future examination of the brachiolar complex using multi-photon microscopy techniques (Pawley, 1995) where longer wavelengths of light, which are less prone to scattering in dense specimens, may assist in determining the extent of serotonergic neurons in the attachment complex in P. regularis. The morphological transformation from the bipinnaria to the brachiolaria of Patiriella regularis parallels several distinct changes in 5- Chapter six 82

HT expression in other asteroid larvae (Nakajima, 1988; Moss, et al. 1994 and Chapter 2 this thesis). There is a net gain in serotonergic-like cells from the gastrula to the bipinnaria; however, it is not known if this trend is continued into the brachiolaria phase in P. regularis. Cell fate studies would be required to investigate this hypothesis. In summary, the following model is proposed which might explain serotonergic neurogenesis in brachiolaria development in P. regularis. During the bipinnaria phase, the primary activity of the larva is swimming in the plankton and capturing food. The bipinnaria has an extensive serotonergic nervous system associated with all ciliated bands (Chapter 2 and 3). With the development of the attachment complex in the brachiolaria, a shift in behaviour is seen from feeding to finding a suitable substrate on which to settle and metamorphose. Serotonergic neurons, which may have been involved in the modulation of capturing food, would no longer be required. The need for neuronal control over feeding will become less important as the larva settles and these ganglia (Chapters 2 and 3) would be “switched off during advanced brachiolaria stages of development. Immunoreactivity in the adoral ciliated band of the brachiolaria was absent and the transverse preoral ganglionic cell bodies appeared to degenerate. A similar observation was made for a metamorphosing echinoid plutei, where serotonergic immunoreactivity was also very faint in the lower lip ganglion (Bisgrove and Burke, 1987). The lower lip ganglion is analogous to the adoral ganglion in P. regularis brachiolaria. As suggested for P. regularis, the echinoid pluteus may down regulate serotonin synthesis in the oral region prior to metamorphosis. The origin and function of large non-neuronal serotonergic cells present in the pre- and postoral ciliated bands is not known. It is hypothesised that the disappearance and/or atypical cell shape of the adoral and preoral transverse ganglia during the advanced brachiolaria stage could be evidence of programmed cell death, which may be associated with the shift in larval behaviour from a feeding state to a settlement state. To test this hypothesis, it is suggested that future work be undertaken to determine the pyknotic profiles of the adoral and preoral transverse ganglionic cells observed during the transition of bipinnaria to Chapter six 83 brachiolaria. A suggested method would be to use a combination of Propidium iodide and YO-PRO-1 (Molecular Probes) to determine if apoptotic cells are present counter staining with anti-serotonin biotin/streptavidin FITC as described in this thesis. Serotonin-like immunoreactivity was not seen in the adhesive disk of P. regularis. Barker (1978) using both light and electron microscopy techniques, did not find neurons in the adhesive disks of the brachiolaria of Cosinasterias calamaria and Stichaster australis. Further microscopy studies using multiphoton techniques, is required to confirm the presence/absence of serotonergic neurons in the attachment disk of P regularis.

6.5.2 The lecithotrophic developers

Pre-neuronal expression of 5-HT in the unhatched brachiolaria of Patiriella exigua, is a continuation of the pre-neuronal expression of 5-HT first seen in the gastrula stage. The synthesis of 5-HT in the unhatched brachiolaria of P. exigua is suggested to be responsible for the morphogenesis of the brachiolarial arms (see Chapter 1). In contrast to P. regularis, where serotonergic neurons are seen in the gastrula stage before the presence of a nervous system, neurogenesis in the lecithotrophic larvae of P. exigua is delayed until the late brachiolarial stage. This delay in the onset of neurogenesis may represent a heterochronic change associated with the evolution of development in this species. There are no data for the onset of serotonin-like immunoreactivity in gastrulae of P. calcar but, based on results from its two congeners, a pre-neuronal expression of 5-HT would also be expected in this species.

6.5.3 Serotonin expression in the three species of seastar larvae.

In feeding asteroid larvae the presence of ciliated bands appears to be a prerequisite for serotonergic neurons (Nakajima, 1988; Komatsu, et al. 1991 and Moss, et al. 1994 and Chapters 2 and 3 this thesis) and/or a brachiolarial attachment complex as shown in this chapter. In the Chapter six 84 lecithotrophic developers Patiriella calcar and P. exigua, which have lost the bipinnarial stage and associated ciliated bands through evolution (Byrne, 1991 and Hart, 1997), serotonergic neurons were confined to the attachment complex. In the larvae of P. calcar and P. exigua, there was consistency between the two staining methods used to reveal the shape and distribution of serotonergic/aminergic neurons in the brachiolarial complex. Histochemical Faglu staining allowed a greater proportion of the nervous system to be visualised compared with antibody labelling. The lecithotrophic larvae of P. calcar and P. exigua, due to their dense nature, provide resistance to high molecular weight polyclonal antibody penetration, whereas the relatively low molecular weight Faglu reagent probably passes easily across the cell membranes, penetrating deeper in to larval tissue.

6.5.4 Speculative functional aspects of the serotonergic neurons in Patiriella. calcar and P. exigua and suggested future directions.

In Patiriella calcar, which has a swimming planktotrophic phase, it has been shown that settlement requires an exogenous cue from the red coralline alga, Amphiroa anceps (Byrne, unpublished results). Serotonergic neurons in the attachment complex in Patiriella calcar might function as chemoreceptors involved in settlement. It is suggested for future work that pharmacological depletion of 5-HT using L-PCPA during settlement could be used to determine a possible role for the serotonergic neurons. For Patiriella exigua the function/s of the serotonergic neurons is/are not know. It is speculated that since P exigua do not appear to be substrate selective (Chee personal observations), serotonergic neuronal-like cells are unlikely to function as chemoreceptors, at least in terms of receiving stimuli to induce settlement. Patiriella exigua brachiolaria are very difficult to dislodge from the substrata (Byrne, 1995) however, brachiolaria of P. exigua, which had been treated, with L-PCPA removed their brachiolar arms from the substrata at a significantly higher frequency (P<0.0001) than those of the controls. It may be that serotonin depletion effected the capacity of larvae Chapter six 85 to remain stuck to the glass. It is suggested that the synthesis, release and/or cessation of adhesive material at the distal regions of the brachiolar arms is under neuronal control, of which serotonin may be involved. Equally possible is that serotonin could be involved in the neuromuscular control of the brachiolar arms, since depletion of serotonin in the bipinnaria (see Chapter 5), where L-PCPA treated larvae were unable to undergo oesophageal peristalsis, indicating a role for serotonin in muscle contraction.

6.5.5 The nervous symmetry in the development of Patiriella exigua brachiolarial.

A unique difference between the brachiolaria larvae of Patiriella exigua and the brachiolariae of P. regularis and P. calcar is one of larval symmetry. The planktonic brachiolariae of Patiriella regularis and P. calcar have the bilateral symmetry characteristic of almost all of the asteroid larvae studied so far (McEdward, 1992) and this is also true of the early pre-hatched brachiolaria of P. exigua (Byrne, 1995). Hypertrophic growth of the two lateral brachiolar arms in P. exigua early brachiolaria results in the development of a tripod larval form (Byrne, 1995). As a result this larva has a unique tri-radiate symmetry centred on an anterior/posterior axis of symmetry through the adhesive disk, and the organisation of the serotonergic nervous system is reflected in this radial symmetry. The transition from a bilateral preneuronal expression of serotonin to a radial serotonergic nervous system in the brachiolariae of P. exigua has not previously been described for other seastar genera.

6.5.6 Comparisons of P. exigua and P. calcar to P. regularis

There were two differences observed between the brachiolaria of Patiriella exigua and P. regularis for the expression of serotonin. Firstly, the gastrulae (Chapter 1) and pre-hatched brachiolariae of P. exigua exhibited a preneuronal expression of 5-HT-like immunoreactivity. The observation of preneuronal expression of serotonin may indicate a Chapter six 86 morphogenetic role for serotonin in the development of the brachiolar arms in P. exigua, as the brachiolar arms are the only structures seen to increase in size while in the fertilisation envelope (Byrne, 1995). Secondly, Patiriella exigua larvae did not develop serotonergic neurons until the advanced tripod-like brachiolaria stage. If serotonergic neurons are involved in a sensory capacity then the absence of these cells during early brachiolaria development within the fertilisation envelope would not be unexpected. It could be hypothesised that the developing larva of P. exigua, whilst inside the fertilisation envelope, does not encounter the external environment and hence would not need any sensory neurons until hatching. This hypothesis is strengthened by data obtained from the immunostained hatched P. exigua which exhibited an abundance of serotonergic neurons at the distal region of the bulbous tips of the brachiolar arms. The bulbous tips contact the substrate and the serotonergic neurons may function in this contact behaviour. Changes in timing of 5-HT expression of the planktotroph P. regularis and the lecithotroph P. exigua are compared and are summarised in Figure 16. This schematic illustrates the heterochronies associated with the morphogenetic expression of 5-HT in a lecithotrophic larva (see chapter 1) with a longer preneuronal expression and late onset of development of serotonergic neurons. Chapter six

Figure 16. Schematic representation of morphogenetic events associated with serotonin expression in the brachiolaria of P. regularis and P. exigua. early bipinnaria early brachiolaria

gastrula radially symmetrical 5-HT nervous system General Discussion 87

General Discussion

7.1 What did this thesis set out to do?

This thesis set out to present a detailed spatial and temporal study, of serotonin expression in asteroid development. Patiriella regularis, P. calcar and P. exigua were chosen as they are closely related and exhibit divergent patterns of development ranging from the ancestral planktotrophic mode of development to the derived planktonic lecithotrophic and benthic lecithotrophic, respectively. The advantage of using closely related species in a study such as this is that comparisons can be drawn regarding the conservation of serotonin expression with respect to the evolution of development.

7.1.2 Inhibition of Preneuronal Serotonin causes Gross Abnormalities during Larval Development

It is commonly accepted that preneuronal serotonin plays a role in blastulation in both vertebrates and invertebrates (Lauder, 1993, Buznikov, et al. 1996). Investigations into the expression and consequences of preneuronal serotonin synthesis during gastrulation and post gastrulation, for asteroid development were undertaken in this study to elucidate the temporal extent of serotonin as a morphogen. Confocal serial sectioning of the ectoderm of the gastrulae of the planktotrophic developer Patiriella regularis, showed preneuronal serotonin-like immunoreactivity in the ectodermal cells but not in the developing archenteron. It is conceivable that serotonin might diffuse from ectodermal cells to act in a trophic role for gut development. Similarly, confocal sectioning of the gastrulae of the lecithotrophic developer P. exigua also revealed preneuronal serotonin in the ectoderm. General Discussion 88

This study is the first to demonstrate a role for preneuronal serotonin in larval development in the Echinodermata. Incubation of gastrulae in para chlorophenylalanine (L-PCPA), a potent inhibitor of tryptophan hydroxylase caused teratogenic effects in Patiriella regularis, and the lecithotrophic developers P. calcar and P. exlgua. In the L-PCPA treated embryos of P. regularis, a distinct archenteron did not form. Instead, a mass of cells migrated towards the anterior region of the larva and coelomogenesis did not go to completion. The ectoderm of the embryos of P. regularis appeared intact throughout the experimental period, which suggests that cohesion of ectodermal cells were not affected by L-PCPA. It is likely that the processes governing archenteron and coelom formation are more sensitive to the perturbation of serotonin levels. Uninhibited serotonin synthesis appears to be necessary for the integrity of distinct cell lineages in Patiriella embryos. The cells of the coelomic sacs in the bipinnaria (Chapter 2) showed serotonin-like immunoreactivity representing a stage of non-neuronal serotonin expression in the bipinnaria. There has only been one other study in an asteroid (Nakajima, 1988), where preneuronal expression of serotonin was seen in non-neuronal coelomic cells during the presence of a serotonergic nervous system. The function of these serotonergic cells was not known (Nakajima pers. comm.). It may be that the serotonergic coelomic cells in P. regularis exert some trophic effect on coelom growth. Although larval-like morphs of P. regularis developed after L-PCPA treatment, the ciliated bands did not form. During normal early larval development, of P. regularis, before a feeding state, has a distinctive "v" shaped grouping of serotonergic neurons surrounds the stomodaeum. It is therefore proposed that serotonin in these early neurons around the stomodaeum might have a trophic effect in the development of the adoral ciliated band in P. regularis bipinnariae. It might be speculated that depletion of serotonin disrupts ciliated band formation. The presence or absence of these deleterious effects produced by L-PCPA, on other planktotrophic asteroid gastrulae and bipinnariae, poses an intriguing General Discussion 89 question, as to whether preneuronal serotonin functions in a similar way irrespective of species. This warrants further study. Regardless of the differing modes of larval development between P. calcar (lecithotrophic planktonic) and P. exigua (lecithotrophic benthic), L- PCPA treated gastrulae developed to brachiolariae with stunted arms. These larval-like morphs were incapable of metamorphosing. It is suggested that brachiolar arm retardation was the result of a reduction in cell division brought about by decreased preneuronal serotonin. However, this suggestion still remains to be answered. Application of L-PCPA on P. calcar brachiolariae resulted in a loss of swimming direction, which may have been a consequence of a change in larval shape brought about by the teratogenic effects of L-PCPA. Treated P. exigua gastrulae exhibited developmental arrest at gastrulation with eventual rupturing of the undeveloped gastrula. It thus appears that uninterrupted serotonin biosynthesis is necessary for larval development in these lecithotrophic species.

7.1.3 Hatching of P. exigua is affected by treatment with L-PCPA.

Premature hatching of undeveloped P. exigua gastrulae from the fertilisation envelope always occurred at the animal pole of the gastrula. This indicates that a "hatching enzyme" may be located in this region. Similar findings achieved by different methods were obtained by Lepage et al. (1992) using immunofluorescence locating the site of the hatching enzyme secretion at the animal pole in an echinoid. While hatching enzymes have not been characterised for Patiriella or other asteroids, they have been found in echinoids and serotonin has been found to be a regulator of the secretion of the hatching enzyme (Deeb, 1972; Buznikov, et al. 1996). The activity of the hatching enzyme has been shown to be Ca2+ dependent and has been located at the animal pole of the gastrula in an urchin (Lepage, et al 1992). Further, it is known that serotonin antagonists can cause a fluctuation in intracellular calcium levels during General Discussion 90 cleavage divisions in early echinoid embryos (Shmukler, et al. 1999). Lowering serotonin levels by L-PCPA treatment in the gastrula of P. exigua might have resulted in the disruption of the timing of the release of a hatching enzyme. The application of L-PCPA to P. regularis and P. calcar bipinnariae and brachiolariae respectively, caused a reduction in endogenous serotonin which was confirmed by rpHPLC. This is the first time rpHPLC has been used to confirm a decrease in serotonin levels by L-PCPA and suggests that the first step in the synthesis of serotonin in planktotrophic and lecithotrophic asteroid larvae, probably involves TPH which is also the case for vertebrate synthesis of serotonin. Toneby (1977) also confirmed a decrease in serotonin in an echinoid after L-PCPA treatment.

7.1.4 The appearance of serotonergic neurons before the nervous system: The apical organ debate.

The existence of putative sensory serotonergic apical organs, described for echinoid gastrulae (Bisgrove and Burke, 1987, Nakajima, 1987b, Bisgrove and Raff, 1989) was not observed in the gastrulae of P. regularis or P. exigua. Serotonergic neurons in the gastrulae of P. regularis were located at the animal pole but not associated with any external ciliated structures that could be described as being sensory-like organs (Chapter 2). Preneuronal expression of serotonin was absent when serotonergic neurons first appeared in P. regularis gastrulae. The significance of serotonin in neurons observed shortly after the preneuronal phase in the gastrula and before a nervous system had formed was not determined. It is suggested that serotonin might be acting as a neurotrophic substance for the differentiation of the neurons expressing serotonin. Studies on fibroblasts in vertebrates by Seuwen et al (1988) and Lauder, (1993) have shown that serotonin is indirectly involved as a growth factor by stimulating DNA synthesis. The function of serotonin in asteroid gastrula neurons prior to the formation of a nervous system presents an intriguing field for future General Discussion 91 research. It would be of interest to examine possible growth factor similarities between an invertebrate such as Patiriella and a vertebrate model.

7.1.5 The serotonergic nervous system in Patiriella regularis development.

Until now, serotonergic nervous systems have been shown in the bipinnaria of asteroids where neurons are predominantly associated with the ciliated bands (Nakajima, 1987a, 1988, Bisgrove and Burke, 1987, Bisgrove and Raff, 1989, Moss, et al. 1994). Serotonergic ganglia, in P. regularis were located at the anterior region (anterior ganglion) of the bipinnaria between the pre- and postoral ciliated bands and at the oral region (transverse preoral and adoral ganglion). Unique ciliated serotonergic neurons were found within these ganglia and the association of these neurons with the ciliated bands suggest a sensory role in feeding and swimming behaviour. Serotonergic varicose neurites from the adoral ganglion were observed to project along the oesophagus. It is possible that these processes innervate the muscle cells of the oesophagus and thereby affect muscle control of the oesophagus. Pharmacological depletion of serotonin in the feeding bipinnariae of P. regularis by L-PCPA induced a loss of oesophageal peristalsis which prevented the larvae from feeding. It was shown (Chapter 3) that oesophageal peristalsis is necessary for ingestion of algal cells. The serotonergic anterior ganglion, a structure not previously described in asteroid development, did not develop until the feeding bipinnaria had formed with a full compliment of ciliated bands. This ganglion joined the pre- and postoral ciliated bands at the anterior region of the bipinnariae (Chapters 2 and 3). It is proposed that this ganglion is involved in the coordination of the ciliated bands. General Discussion 92

7.1.6 Serotonergic neurons in the brachiolar arms of: P. regularis, P. calcar and P. exigua.

As the bipinnariae of P. regularis developed into brachiolariae, the anterior ganglion appeared to be pushed aside as the brachiolarial attachment complex developed a separate set of serotonergic neurons (chapter 4). It seems likely that the anterior ganglion seen in the bipinnaria of P. regularis plays no role in metamorphosis. The brachiolar complex was shown to contain numerous serotonergic neurons underlying the adhesive papillae (Chapter 4). These serotonergic neurons may function in a sensory capacity during the settlement process. The lecithotrophic brachiolariae of P. calcar and P. exigua also contained numerous serotonergic and aminergic neurons and brachiolariae of P. exigua, when treated with para chlorophenylalanine, removed their brachiolar arms from the substratum, indicating a role for serotonin in settlement. This is the first time that serotonin has been found to be located in the brachiolar complex of an asteroid.

7.1.7 Regardless of the mode of larval development of the three Patiriella species, similar structures contain prenervous serotonin or serotonergic neurons.

The comparative examination of serotonin expression during the development of P. regularis, P. calcar and P. exigua provided vital insights into the morphogenetic influence of serotonin during gastrula and post gastrula development. The complex nature of the serotonergic nervous system in P. regularis larvae provided evidence linking the nervous system to feeding and swimming behaviour. Externally and internally, the lecithotrophic larvae, lacking ciliated bands and a functional digestive tract, are less complex than P. regularis larvae. Although the architecture of the serotonergic nervous system in the brachiolarial attachment complex of the lecithotrophic species studied here appears to be quite complex, the General Discussion 93 extent of the serotonergic cellular processes within these larvae is yet to be determined. At present, It is difficult to make evolutionary comparisons between species based on the nervous systems of the brachiolariae examined in this thesis, as the extent of the serotonergic nervous systems could not be determined by confocal microscopy. A further avenue of research, into 5-HT receptor sites within Patiriella, may provide important data on the phylogenetic ubiquity of 5-HT receptor sites across the animal phyla.

7.1.8 Addendum

While it has been demonstrated by pharmacological, biochemical and immunocytochemical techniques that serotonin is present in the gastrula and larvae in the three congeners studied in this thesis, serotonin has not yet been detected in the adult seastars of Patiriella (Chee, unpublished data). The detection of serotonin has also proved elusive in other sea star genera (Cottrell and Pentreath, 1970). Monoamine oxidase (MAO), the enzyme responsible for the catabolism of serotonin, has been detected in the asteroid Marthastarias glacialis (Nicotra, 1982) and this suggests the existence of serotonin in an adult seastar. The significance of the presence of MAO in an adult seastar requires further research to determine if serotonin is absent from the adult nervous system. Literature cited 94

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