Open Research Online The Open University’s repository of research publications and other research outputs Study of the Evolutionary Role of Nitric Oxide (NO) in the Cephalochordate Amphioxus, “Branchiostoma lanceolatum” Thesis How to cite: Caccavale, Filomena (2018). Study of the Evolutionary Role of Nitric Oxide (NO) in the Cephalochordate Amphioxus, “Branchiostoma lanceolatum”. PhD thesis The Open University. For guidance on citations see FAQs. c 2017 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000d5d2 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk Study of the Evolutionary Role of Nitric Oxide (NO) in the Cephalochordate Amphioxus, “Branchiostoma lanceolatum” A thesis submitted to the Open University of London for the degree of DOCTOR OF PHILOSOPHY by Filomena Caccavale Diploma Degree in Biology Università degli studi di Napoli Federico II Affiliated Research Center (ARC) STAZIONE ZOOLOGICA “ANTON DOHRN” DI NAPOLI Naples, Italy November 2017 This thesis work has been carried out in the laboratory of Dr. Salvatore D’Aniello, at the Stazione Zoologica “Anton Dohrn” di Napoli, Italy. Director of studies: Dr. Salvatore D’Aniello (BEOM, Stazione Zoologica “Anton Dohrn” di Napoli, Italy) Internal Supervisor: Dr. Anna Palumbo (BEOM, Stazione Zoologica “Anton Dohrn” di Napoli, Italy) External Supervisor: Prof. Ricard Albalat Rodriguez (Departament de Genètica, Microbiologia i Estadística and Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Spain) “Find a job you enjoy doing, and you will never have to work a day in your life.” Mark Twain ABSTRACT Nitric Oxide (NO) is a gaseous molecule that acts in a wide range of biological processes. NO can be produced by enzymatic and non-enzymatic pathways and, in this scenario, the enzyme Nitric Oxide Synthase (NOS) has a great importance for its exclusive role in de novo synthesis of NO. In the present study, the role of the NO in the embryonic development was investigated in the cephalochordate Branchiostoma lanceolatum (amphioxus) with the purpose of acquiring further knowledge on the ancestral role of animal Nos and the acquisition of new NO functions during evolution. Amphioxus has three different Nos genes (NosA, NosB and NosC) that are not orthologues of the three Nos of mammals (NosI, NosII and NosIII) deriving from an independent duplication occurred in the common ancestor of cephalochordates. The three amphioxus Nos genes showed a different temporal and spatial expression during development and a different susceptibility to be induced after immune stimulation. The study of the promoter regions of these genes can be very useful to identify possible diversities in regulation that can lead those peculiar expression features. In amphioxus larva, NO was mainly detected in the developing nervous system and in the pharyngeal area, before and after the mouth opening. The inhibition of NOS activity, and as consequence the enzymatic NO production, during amphioxus neurulation, resulted in the alteration of pharyngeal structures formation in the larvae, in particular opening of the mouth resulted compromised. Moreover, an alteration in larva locomotion was observed. Further studies will be necessary to reveal the exactly molecular mechanisms and the pathways in which I NO acts for establishment of the pharyngeal structures and the neuromuscular junctions early in development. For this purpose, a differential transcriptomic analysis of NOS- inhibited embryos was performed but the results are still very preliminary. II ACKNOWLEDGEMENTS I am very thankful to my director of studies Dr. Salvatore D’Aniello (SZN) and my supervisors Prof. Ricard Albalat (Universitat de Barcelona) and Dr. Anna Palumbo (SZN) for excellent leadership, their time spent in our discussions, for their support and encouragement to pursue new ideas. Next, I thank all my labmates for friendly and stimulating atmosphere, for all their advices, nice experiences and refreshing moments we could spend together. I would like to acknowledge Dr. Hector Escrivà (UPMC) and Dr. Stéphanie Bertrand (UPMC) for their hospitality and support during the period that I spent in their laboratory at the Oceanographic Observatory in Banyuls sur-Mer to performing experiments that are part of this work. I thank The Company of Biologists for found that travel. I am grateful to the Molecular biology service, the MaRe unit and the AMOBIO unit of the SZN for technical assistance. Last but not least, special thanks to my parents and family and to Luciano for their constant support and encouragement during my studies. III TABLE OF CONTENTS ABSTRACT .......................................................................................................................... I ACKNOWLEDGEMENTS ............................................................................................ III TABLE OF CONTENTS ................................................................................................. IV LIST OF FIGURES ......................................................................................................... VII LIST OF TABLES ........................................................................................................... XII CHAPTER 1: INTRODUCTION ..................................................................................... 1 1.1 NITRIC OXIDE: SIGNALING, BIOLOGICAL FUNCTION AND BIOSYNTHESIS ............................................................................................................... 1 1.2 THE EXPERIMENTAL MODEL SYSTEM: AMPHIOXUS ............................... 11 1.2.1 History of amphioxus and its anatomy .............................................................. 11 1.2.2 Phylogeny............................................................................................................ 17 1.2.3 Reproduction and embryology: early and late amphioxus development ........ 20 1.3 NITRIC OXIDE AND NITRIC OXIDE SINTHASES IN AMPHIOXUS ........... 33 1.4 NEURONAL NOS GENE TRANSCRIPTIONAL REGULATION ..................... 39 1.5 TRANSCRIPTIONAL GENE REGULATION ...................................................... 42 1.5.1 Conserved non-coding regions and their gene regulatory function ................. 42 1.5.2 Ciona robusta as model organism for transgenic experiments ........................ 45 CHAPTER 2: MATERIALS AND METHODS ........................................................... 50 2.1 ANIMALS CARE AND GAMETES COLLECTION ........................................... 50 IV 2.1.1 Branchiostoma lanceolatum sampling and ethics statement ........................... 50 2.1.2 Branchiostoma lanceolatum care ...................................................................... 51 2.1.3 Branchiostoma lanceolatum spawnings ............................................................ 52 2.1.4 Ciona robusta sampling, care and gametes collection ..................................... 54 2.2 IN VIVO EMBRYONIC MANIPULATION ........................................................... 54 2.2.1 TRIM and L-NAME treatments in amphioxus ................................................. 54 2.2.2 LPS treatment in amphioxus .............................................................................. 55 2.2.3 Transgenesis in Ciona robusta .......................................................................... 56 2.3 AMPHIOXUS EMBRYOS COLLECTION AND FIXATION ............................. 57 2.4 BASIC MOLECULAR BIOLOGY PROCEDURES ............................................. 57 2.4.1 RNA purification ................................................................................................ 57 2.4.2 Reverse transcription of RNA............................................................................ 58 2.4.3 DNA and RNA gel electrophoresis ................................................................... 58 2.4.4 Genomic DNA preparation ................................................................................ 59 2.4.5 Molecular cloning ............................................................................................... 59 2.4.6 Isolation of plasmidic DNA from Escherichia coli .......................................... 61 2.4.7 Polymerase chain reaction (PCR) ...................................................................... 61 2.4.8 DNA sequencing ................................................................................................. 62 2.4.9 Oligonucleotides ................................................................................................. 62 2.5 QUANTITATIVE GENE EXPRESSION ............................................................... 64 2.5.1 Quantitative real-time PCR (qPCR) .................................................................. 64 2.5.2 Digital droplet PCR (ddPCR) ............................................................................ 65 2.6 CONSTRUCTS PREPARATION FOR TRANSGENESIS ................................... 66 2.6.1 Amphioxus enhancers ........................................................................................ 66 2.6.2 DNA constructs for transgenesis in Ciona robusta .......................................... 70 V 2.7 NO QUANTIFICATION AND LOCALIZATION ...............................................