Analysis of silicatein gene expression and spicule formation in the demosponge Amphimedon queenslandica Aude Gauthier BMarSt (Hons) A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2014 School of Biological Sciences Abstract The skeletal elements in most sponges are siliceous spicules These are fabricated into species-specific sizes and shapes Demosponges, in particular, have specialised cells called sclerocytes that possess the unique ability to synthesise biosilica and these spicules Underlying the diversity of demosponge spicules morphology is a conserved protein, called silicatein This thesis aims to investigate the process of spiculogenesis in the different developmental stages of the demosponge Amphimedon queenslandica, and the evolution and developmental expression of the silicatein gene family in relation to spicule formation A queenslandica is the only sponge species to have its genome fully sequenced, assembled and annotated, and currently is one of the best models to study sponge development (Srivastava et al 2010) This species broods embryos year-round, facilitating the access to embryological and larval material (Leys and Degnan 2001) This combination of logistical advantages means that I was able to trace the expression of silicatein genes through A queenslandica embryonic, larval and postlarval development Spicule formation starts in the early embryogenesis in A queenslandica during, gastrulation or the brown stage Spicule number increases throughout embryonic development until the pre-hatching larval stage, with the emerging larvae having about 1000 spicules No detectable increase in spicule number was recorded during larval and postlarval development Spicule number varied remarkably between different individual embryos and larvae of the same stage of development I initially identified six silicatein β like genes in the genome of A queenslandica, among which four can be categorised as non-conventional by the absence of the serine in the catalytic triad of the protein These genes not have direct orthologues in other sponge species and appear to have evolved by a lineage-specific gene duplication A comprehensive phylogenetic analysis of this gene family in sponge indicated that silicatein α arose from silicatein β by gene duplication and that silicatein β gene share traits with both cathepsin L and silicatein Conservation of gene structure and exon length in silicatein and cathepsin L genes suggests that these genes have preserved an ancestral gene structure common to both gene families in both marine and freshwater sponges Using in situ hybridisation, I demonstrated that silicatein genes are expressed during A queenslandica early embryonic development, with genes being expressed exclusively in sclerocytes Analysis of gene expression levels through embryogenesis and metamorphosis, using RNA-Seq performed on a pool of same stage individuals, revealed that all silicatein-like genes are differentially expressed throughout development, and the expression of silicatein genes occurs prior to spicule formation However, some silicatein-like gene expression levels and spicule number not appear to be tightly correlated Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis Publication during candidature No publications Publication included in this thesis No publications included Contributions by others to the thesis My supervisor, Professor Bernard Degnan, contributed to the conception and design of the research and critically revised and proofread all sections of this thesis My co-supervisor, Dr Nagayasu Nakanishi performed technical work required for the RNA-Seq data Statement of parts of the thesis submitted to quality for the award of another degree None Acknowledgements I am extremely grateful to my supervisor Bernie Degnan for accepting me into his group and introducing me to the world of sponges and research, and for his continuous support and invaluable advice through this project Additionally, I would like to thank my committee members, Dr Nagayasu Nakanishi and Dr David Merritt, for their encouragement and interest in my work I would also like to thank everyone in the Degnan lab, both past and present members, for their help, support, coffee breaks, stimulating discussions, and fun times in and out of the office throughout the years This includes in no particular order, Carmel McDougall, Andrew Calcino, Laura Grice, Kerry Roper, Maely Gauthier, Jo Bayes, Jaret Bilewitch, Nobuo Ueda, Felipe Aguilera, Jabin Watson, Simone Higgie, Federico Gaiti, Katia Jindrich, Kevin Kocot, Sunsuke Sogabe, Selene Fernandez Valverde, Ben Yuen, Tahsha Say, Rebecca Fieth and William Hatleberg I am most particularly thankful to Carmel McDougall and Kerry Roper for their invaluable help and guidance in the laboratory A special thanks to Sandie Degnan for her support and encouragement through the different milestones of this project, Nagayasu Nakanishi for providing help and suggestions with my in situ hybridization approach, and Katia Jindrich and Ben Yuen for going through some troubleshooting with me in the lab I also wish to thank Maely Gauthier, Kevin Kocot, Laura Grice, Simone Higgie and William Hatleberg for taking the time to proofread parts of my thesis During the course of this project, I was lucky to participate in field trips to Heron Island So, I would like to acknowledge staff members of Heron Island Research Station, which are always available and understanding of our last minute experimental set-up, for their technical help and great working environment atmosphere Thanks to the sponge people who made those trips worth remembering for their assistance in the field, the great times and sunset drinks, as well as for going through those endless night with me fixing material and dissecting brood chambers I would like to thank friends and family who supported me during my time here Above all, my parents for supporting my choice and giving me the opportunity to undertake my studies in Australia A special thanks ‘au Nain’ (Arnault Gauthier) for listening to my complaints when things were not working to plan and checking for the numerous grammatical mistakes Keywords Porifera, silicatein, silicatein gene expression, spicules Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060309 Phylogeny and comparative analysis 50% ANZSRC code: 060808 Invertebrate biology 50% Fields of Research (FoR) Classification FoR code: 0603 Evolutionary biology 50% FoR code: 0608 Zoology 50% Table of Contents Chapter 1: General Introduction 1.1 Sponge biosilica structure…………………………………………………………………………………………….2 1.1.1 Spicule morphology………………………………………………………………………………………………3 1.1.2 Spicule formation…………………………………………………………………………………………………4 1.2 Silicatein……………………………………………………………………………………………………………………….7 1.2.1 Catalytic mechanism of silicatein………………………………………………………………………….7 1.3 Amphimedon queenslandica as a study model………………………………………………………………8 1.4 Aims of this study………………………………………………………………………………………………………….9 Chapter 2: Identification of silicatein genes in the demosponge Amphimedon queenslandica and their evolutionary relationship to other sponge silicatein and cathepsin genes……………………………………………… 11 2.1 Abstract…………………………………………………………………………………………………………………… 11 2.2 Introduction……………………………………………………………………………………………………………….12 2.3 Materials and Methods………………………………………………………………………………………………14 2.3.1 Identification of silicatein genes in Amphimedon queenslandica……………………… 14 2.3.2 Gene architecture analyses……………………………………………………………………………… 15 2.3.3 Molecular phylogenetic analyses……………………………………………………………………… 15 2.4 Results……………………………………………………………………………………………………………………… 16 2.4.1 Identification and genomic organisation of A queenslandica silicatein genes…… 16 2.4.2 Conservation in the genomic structure of A queenslandica silicatein genes……… 20 2.4.3 Phylogenetic relationship of sponge silicateins……………………………………………………23 2.4.4 More detailed analysis of silicatein protein sequences……………………………………… 27 2.4.5 Presence of the CY motif in other metazoan cathepsin L sequences……………………28 2.5 Discussion………………………………………………………………………………………………………………… 33 Chapter 3: Analysis of spicule formation and silicatein gene expression in the demosponge Amphimedon queenslandica……………………………………………38 3.1 Abstract…………………………………………………………………………………………………………………… 38 3.2 Introduction……………………………………………………………………………………………………………….38 3.3 Materials and Methods………………………………………………………………………………………………41 3.3.1 Sponge collection and fixation of biological materials…………………………………………41 3.3.1.1 Brooded embryos………………………………………………………………………………………41 3.3.1.2 Larval and postlarval stages……………………………………………………………………….42 3.3.2 Spicule preparation…………………………………………………………………………………………….43 3.3.3 Gene expression analysis – RNA-Seq protocol…………………………………………………….43 3.3.3.1 RNA extraction and cDNA synthesis………………………………………………………… 44 3.3.3.2 Samples processing………………………………………………………………………………… 44 3.3.4 Gene expression analysis – qRT-PCR………………………………………………………………… 44 3.3.4.1 Primer design for genes of interest…………………………………………………………….45 3.3.4.4 qRT-PCR analyses………………………………………………………………………………………46 3.3.5 Whole-mount in situ hybridisation…………………………………………………………………… 47 3.3.5.1 Probe synthesis…………………………………………………………………………………………48 3.3.6 Statistical analyses…………………………………………………………………………………………… 48 3.4 Results……………………………………………………………………………………………………………………… 49 3.4.1 Spicule number increases during embryogenesis but not in larvae and early postlarvae………………………………………………………………………………………………….49 3.4.2 Temporal expression of A queenslandica silicatein genes during development…50 3.4.3 Expression pattern of A queenslandica silicatein genes, Aqu2.41046, Aqu2.41047 and Aqu2.42494…………………………………………………………………………… 54 3.4.4 Localised expression of Aqu2.42494 during development………………………………… 54 3.4.5 Individual variation in gene expression and spicule number……………………………….57 3.5 Discussion………………………………………………………………………………………………………………… 60 Chapter 4: General Discussion…………………………………………………………………… 64 4.1 Does silicatein expression correlate with spicule number? …………………………………………65 4.2 Why does variation in silicatein expression and spicule number occur? .66 4.3 Conclusions and future directions……………………………………………………………………………….67 References………………………………………………………………………………………………….69 Appendices…………………………………………………………………………………………………81 …A1 GeneBank (NCBI) accession numbers of protein sequences 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Discodermia japonica Ephydatia fluviatilis Ephydatia muelleri Ephydatia sp n.1 PW-2008 Ephydatia sp n.2 PW-2008 Euplectella aspergillum * Geodia cydonium Halichondria okadai Hymeniacidon perlevis Silicatein isoforms/cathepsin sil-a sil-b cath-L cath-L Sil-like cath-L1 cath-L2 sil-a1 sil-a2 sil-a3 sil-a4 sil-a1 sil-a1’ sil-a4 sil-a4’ cath-L1 cath-L2 sil cath-like1 cath-like2 cath-like3 cath-like4 cath-like5 sil sil sil-2 sil-a4 sil-G1 sil-G2 sil-M1 sil-M2 sil-M3 sil-M4 sil-a2 sil-a3 sil-a4 sil-a4 sil-a2 sil sil-a cath sil sil-a sil-1 81 GenBank accession numbers ACH92669.1 ACH92668.1 ACJ02498.1 CAI91577.1 ACU86976.1 ACU86972.1 ACU86974.1 AEO36958.1 AEO36959.1 AEO36960.1 ADQ74580.1 ACO51493.1 CAQ54043.1 ACO51494.1 CAQ54044.1 ACU86973.1 ACU86975.1 CAP49202.2 CAP17584 CAP17585.1 CAP17586.1 CAP17587.1 CAP17588.1 CBY80151.1 CAJ44453.1 CAJ44454.1 ACO51487.1 BAG74346.1 BAG74347.1 BAE54434.1 BAG74343.1 BAG74344.1 BAG74345.1 CAQ54046.1 CAQ54047.1 CAQ54048.1 CAQ54050.1 CAQ54049.1 CBY80150.1 CAM57981.1 CAA71554.1 BAB86343.1 ABC94586.1 ABM47424.1 Comments Partial sequence “ Partial sequence “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Partial sequence “ “ “ Partial sequence Partial sequence Latrunculia oparinae Lubomirskia baicalensis Lubomirskia incrustans Monorhaphis chuni Petrocia ficiformis Pheromena raphanus Spongilla lacustris Suberites domuncula Swartschewskia papyracea Tethya aurantium Tethya aurantium red variant Tethya aurantium yellow variant sil-2 cath-L sil-a1 sil-a1’ sil-a2 sil-a3 sil-b cath sil sil-a sil-a2 sil-a2’ sil-a3 sil-a4 cath-L cath-L’ cath-L2 sil-a1 sil-a4 sil sil-b cath-L1 cath-L2 sil-a3 sil-a4 sil-a4’ sil-a sil-b sil-b’ cath-L sil-a4 sil-a sil-b sil sil ABM47425.2 ABM47423.1 ACG63793.1 ACH47999.1 ACH48000.1 ACH48001.1 ACH48002.1 ACH48003.1 CAH10753.1 CAI43319.1 ADQ74585.1 CAI91571.1 CAI91572.1 CAI91573.1 CAI43320.1 CAH10752.1 CAI91575.1 ACO51492.1 ACO51491.1 CAZ04880.1 AAO23671.1 ACU82389.1 ACU82390.1 CAQ54051.1 ACO51489.1 CAQ54052.1 CAI46305.1 CAI46304.1 CAH04635.1 CAH04632.1 CAQ54053.1 AAC23951.1 AAF21819.1 CBY80148.1 CBY80149.1 82 “ “ Partial sequence Partial sequence “ Partial sequence Only amino acid differ between a2 and a2’ Partial sequence Partial sequence “ “ Partial sequence Only amino acids differ between sil-b and sil-b’ Partial sequence A2 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of Amphimedon queenslandica silicatein and cathepsin L genes Conserved amino acid are highlighted in black (100% similarity) and similar amino acid (>60%) are highlighted in grey Arrows indicate the catalytic amino acids, Cysteine (Cys/C) or Serine (Ser/S), Histidine (His/H) and Asparagine (Asn/N) Key conserved silicateins residues are underlined The Cys residues involved in the formation of disulphide bonds are represented by dots and the serine cluster by a box 83 A3 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of silicatein-like sequences in different animals 84 ... Identification of silicatein genes in the demosponge Amphimedon queenslandica and their evolutionary relationship to other sponge silicatein and cathepsin genes 2.1 Abstract Silicatein genes are involved in. .. investigate the process of spiculogenesis in the different developmental stages of the demosponge Amphimedon queenslandica, and the evolution and developmental expression of the silicatein gene family in. .. identify and verify the conserved region position of domains in proteins and genes 2.3.2 Gene architecture analyses Silicatein and cathepsin L genes in A queenslandica, S domuncula (silicatein α and