SELF-NOnsELF RECOGNITION GENOMIC AND TRANSCRIPTOMIC INSIGHTS FROM THE SPONGE AGGREGATION FACTORS LAURA FRANCES ELIZABETH GRICE B.SC. (HONS) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Biological Sciences S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R EG ATIO N F A CTO rs Abstract The multicellular condition cannot be maintained without safeguards protecting the integrity of the individual. Tissue contact and fusion with other conspecific individuals may threaten this integrity, as genetically non-identical cells may shirk their somatic duties and gain disproportionate access to the germ line. As sessile invertebrates that commonly inhabit crowded benthic environments, sponges are particularly reliant on a molecular self-nonself defense system in order to resist loss of habitat space, chimerism and possible germ line parasitism by neighbouring conspecific sponges. Sponge allorecognition appears to be, at least in part, under the control of extracellular proteoglycans called aggregation factors (AFs), which were first discovered based on their role in the species-specific reaggregation of dissociated sponge cells. Although the AFs have been extensively studied for over fifty years, the majority of this work has involved biochemical, rather than genetic approaches, and has focussed on the role of the glycan subunits associated with the AFs. In the present work, I investigate the genetic properties underlying the AF protein backbone, to better understand the functions and evolution of these putative allorecognition molecules. Using newly-available genomic and transcriptomic data, I surveyed the phylum Porifera for novel putative AF sequences, to explore the evolutionary origins of this gene family. I conclude that the AFs are a demosponge and hexactinellid-specific innovation. I then performed an in-depth characterisation of the six AF genes from the model demosponge species, Amphimedon queenslandica. The six genes display a highly modular intron/exon organisation. However, as expected of putative allorecognition genes, the AFs are greatly diversified between individuals, with nucleotide polymorphism (and possible positive selection) and intron retention events distributed across the six genes. The AFs are very highly expressed across sponge development and in response to alloimmune challenge, and undergo a particular spike in gene expression levels after the onset of sponge metamorphosis. The AF genes also exhibit expression patterns across development that are significantly correlated with those of other, developmentally important genes with roles in various cell signalling pathways. I conclude that the AFs play a novel developmental role, in addition to their putative allorecognition capabilities. ii S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R E G AT I O N F ACTO rs 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. iii S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R EG ATIO N F A CTO rs Publications during candidature Peer-reviewed paper Grice LF, Degnan BM. 2015. The origin of the ADAR gene family and animal RNA editing. BMC Evol Biol 15: 4. Book chapter Grice LF, Degnan BM. 2015. How to build an allorecognition system: A guide for prospective multicellular organisms. In Evolutionary Transitions to Multicellular Life (eds. I. Ruiz-Trillo and A.M. Nedelcu), Springer, Dordrecht Heidelberg New York London. Conference abstracts Grice LF, Gauthier MEA, Fernandez-Valverde S, Degnan BM. 2013. Genomic and trancriptomic profiling of the aggregation factor genes of the demospongeAmphimedon queenslandica. Ninth World Sponge Conference. Fremantle, WA, Australia, 4 – 8 Nov 2013. Grice LF, Gauthier MEA, Degnan BM. 2012. Seeking diversity: Genomic characterisation of the aggregation factor genes of the demosponge Amphimedon queenslandica. Twelfth Congress of the International Society of Developmental and Comparative Immunology (ISDCI). Fukuoka, Japan, 9 – 13 July 2012. iv S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R E G AT I O N F ACTO rs Publications included in this thesis Grice LF, Degnan BM. 2015. How to build an allorecognition system: A guide for prospective multicellular organisms. In Evolutionary Transitions to Multicellular Life (eds. I. Ruiz-Trillo and A.M. Nedelcu), Springer, Dordrecht Heidelberg New York London. This work was incorporated in the Abstract and Chapter 1 CONTRIBUTOR STATEMENT OF CONTRIBUTION Conducted the literature review (100%) Laura F. Grice (Candidate) Wrote and edited the chapter (90%) Bernard M. Degnan Wrote and edited the chapter (10%) Grice LF, Degnan BM. 2015. The origin of the ADAR gene family and animal RNA editing. BMC Evol Biol 15: 4. This work was included as Chapter 5 CONTRIBUTOR STATEMENT OF CONTRIBUTION Designed study (60%) Laura F. Grice (Candidate) Conducted analyses (100%) Wrote and edited the paper (90%) Designed study (40%) Bernard M. Degnan Wrote and edited the paper (10%) v S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R EG ATIO N F A CTO rs Contributions by others to the thesis Bernard M. Degnan contributed to the conception and design of this research, advised on methods and analysis, and provided critical comments on the thesis and on the associated publications in Chapters 1 and 5. Selene Fernandez Valverde advised on methods and analysis, and produced several of the datasets analysed across this thesis (these contributions are specifically acknowledged in the relevant chapters). William Hatleberg produced several of the datasets analysed across this thesis (these contributions are specifically acknowledged in the relevant chapters). Transcriptome sequencing was conducted by Macrogen Inc., South Korea. Statement of parts of the thesis submitted to qualify for the award of another degree None. vi S ELF -N O ns ELF R ECOG N ITIO N : S PO N GE A GG R E G AT I O N F ACTO rs Acknowledgements I have been a member of the Degnan labs in various capacities since 2008, so it is with great fondness (and just a tinge of sadness) that I write these acknowledgements at the end of this endeavour. First, of course, to my remarkable supervisor Bernie Degnan. Your enthusiasm and insight has kept this project rolling, when the craziness that is the AF locus seemed impenetrable. Thank you for your clarity, for pretending not to notice when I felt I hadn’t done enough, for the ‘Degnan Gold’ you can sprinkle on a piece of writing to give it life. To my co-supervisor and joint lab Head, Sandie Degnan. Sandie, your wisdom and integrity - regarding not only my project, but also science as a field and as a lifestyle - have been a source of inspiration. Thank you also to Bernie and Sandie for the opportunity to develop additional skills in the more administrative side of science, in the role of Lab Manager in 2013. I also wish to thank my other co-supervisor, Andy Barnes, for his advice regarding this project. As the chair of my thesis examining committee, I am grateful to Sassan Asgari for his advice regarding my project at each milestone. Thanks are also due to Gail Walter, postgraduate administrator for the School of Biological Sciences, for her endless patience. Bernie and Sandie have fostered an ongoing lab culture that is nothing but welcoming and helpful. To all the past and current Deglabbers who have overlapped with my period in the lab: your assistance, advice, and generosity with your time - not to mention friendship, competitive baking skills, milkshakes and Spice Girls puzzles - have made the lab a wonderful environment to both do and avoid science. Particular thanks must go to Andrew Calcino, Bryony Fahey, Melanie Havler and Carmel McDougall for their advice in the lab and the field, and to Melanie Havler and Kerry Roper for running a tight ship during their respective glorious reigns as Lab Manager. On the computational side of things, thanks to Ben Woodcroft for providing bioinformatics assistance from afar in the early days of my PhD, and Timothy Lamberton for on-call statistical, mathematical and programming consulting. In 2012, I fell down the rabbit hole into ‘black screen’ (command line) data analysis. Most of this project would not have been possible without Selene Fernandez Valverde, whose help and patience went above and beyond - thank you Selene.