3rd Medaka Strategic Meeting
and
17th Australia & New Zealand Zebrafish Meeting
Feb 1 – 6, 2016 Flinders Golf Club, Flinders, Victoria AUSTRALIA
Courtesy of Dominik Paquet, The Rockefeller University, New York, USA
TABLE OF CONTENTS
Sponsor Pages Tecniplast Pentair Viewpoint Transnetyx Intavis GeneTarget Solutions Merck The Aquarium Vet Flinders maps…………………………………………………………………………………………………1 Meeting Organisers……………………………………………………………………………………2 Medaka Schedule at a glance……………………………………………………………3 Zebrafish Schedule at a glance……………………………………………………4 Medaka Detailed Schedule……………………………………………………………………5 Zebrafish Detailed Schedule……………………………………………………………7 Plenary Speaker Biographies & Abstracts Hiroyuki Takeda…………………………………………………………………………………12 Joachim Wittbrodt……………………………………………………………………………13 Elzabeth Patton…………………………………………………………………………………14 Hideaki Takeuchi………………………………………………………………………………15 Medaka Oral Abstracts……………………………………………………………………………16 Medaka Poster Abstracts………………………………………………………………………23 Zebrafish Oral Abstracts……………………………………………………………………30 Zebrafish Poster Abstracts………………………………………………………………77 Delegate List…………………………………………………………………………………………………85
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MAP OF FLINDERS
Agribusiness Conference February 2016 Treehouse Accommodation Map 4 Rest Dve
Deckhouse 1 St Annes Rd
Villa Flinders Halks Nest 8 Hurst St 18b Barker St
Vlinders Hedge House 80 Cook St 68 Cook St Flinders Cove Motel Cook St
Flinders Hotel Cook St Combermere 11 Double Creek rd
Flinders Golf Club 1 Bass St
For Breakfast:
3. Coffee Supreme Cook St 4. Flinders Bakehouse Café 60 Cook St 5. Pier Provodore 38 Cook St 6. Flinders Hotel Cook St 10. Flinders Village Café 49 Cook St
1 ORGANISERS
Dr. Mirana Ramialison Australian Regenerative Medicine Institute Monash University, Clayton, Victoria
e-mail: [email protected] Tel: +61 (0)3 99029645 @ramialison_lab
Dr. Yann Gibert Deakin School of Medicine Metabolic Genetic Diseases Laboratory Geelong, Victoria
e-mail: [email protected] Tel: +61 (0)3 5227 1197
Dr. Jan Kaslin Australian Regenerative Medicine Institute Monash University, Clayton, Victoria
e-mail:[email protected] Tel: +61 (0)3 99029613
Dr. Cristina Keightley Australian Regenerative Medicine Institute Monash University, Clayton Victoria
e-mail: [email protected] Tel: +61 (0)3 99029738
2 SCHEDULE AT A GLANCE MEDAKA
Monday 1st Feb 9:00 am Pick up from Melbourne Airport / CBD 10:00 – 12 noon Visit ARMI / Monash University Aquatic Facilities 12:00 – 1:00 pm Lunch at Monash University 1:00 – 2.30 pm Travel to conference venue (Flinders) 2.30 pm – 3:30 pm Hotel check-in and registration 3.30 pm – 3:45 pm Welcome address 3:45-4:45 pm Keynote speaker: Hideaki Takeuchi 4:45 – 6:00 pm Posters session with drinks and canapés 7:00 pm Welcome dinner (BBQ at Flinders)
Tuesday 2nd Feb 9:30-10:30 am Keynote speaker: Joachim Wittbrodt 10:30 – 11:00 am Morning tea 11:00 – 12:30 pm Medaka Talks – Session I 12:30 – 2:00 pm Lunch 2:00 – 4:00 pm Medaka Talks – Session II 4.30 pm onwards Wine Tasting / Conference Dinner at Merricks
Wednesday 3rd Feb 9:30 – 9:45 am Sponsors presentation 9:45 – 11:00 am Community meeting
3 SCHEDULE AT A GLANCE ZEBRAFISH
Wednesday 3rd Feb 12:30 – 1:45 pm Registration / Meeting materials pick-up 1:45 – 2:00 pm Welcome 2:00 – 3:00 pm (Session 1) Blood and Vessels (Chair: Chris Hall) 3:00 – 4:00 pm (Plenary 1) Hiroyuki Takeda (Chair: Mirana Ramialison) 4:00 – 4:30 pm Afternoon tea 4:30 – 6:15 pm (Session 2) Neurobiology (Chair: Patricia Jusuf) 6:15 – 8:00pm Welcome Reception
Thursday 4th Feb 9:30 – 10:45 am (Session 3) Development and Muscle Biology – I (Chair: Ben Hogan) 10:45 – 11:15 am Morning tea 11:15 – 12:30 pm (Session 4) Emerging Technologies (Chair: Mirana Ramialison) 12:30 – 2:00 pm Lunch 2:00 – 3:00 pm 10 minutes presentation of platinum sponsors 3:00 – 4:00 pm (Plenary 2) Joachim Wittbrodt (Chair: Jan Kaslin) 4:00 – 4:30 pm Afternoon tea 4:30 – 5:45 pm (Session 5) Regeneration and Stem Cells (Chair: Nicholas Cole) 6:30 pm Conference Dinner (Yacht Club)
Friday 5th Feb 9:30 – 11:15am (Session 6) Disease Models – I (Chair: Ethan Scott) 11:15 – 11:45 am Morning tea 11:45 – 12:45 pm (Plenary 3) Hideaki Takeuchi (Chair: Yann Gibert) 12:45 – 2:00 pm Lunch 2:00 – 3:15 pm (Session 7) Infection and Immunity (Chair: Cristina Keightley) 3:15 – 4:15 pm (Plenary 4) Liz Patton (Chair: Cristina Keightley) 4:30 – 6:30 pm (Session 8) Posters and Canapes 7:00 pm BBQ + dancing (Flinders Golf Club)
Saturday 6th Feb 9:15 – 10:30 am (Session 9) Disease Models - II (Chair: Dan Hesseslson) 10:30 – 11:30 am (Session 10) Development and Muscle Biology – II (Chair: Rob Bryson-Richardson) 11:30 – 12:30 pm Morning Tea 11:40 – 12:20pm Community meeting 12:20 – 12:30pm Awards and Meeting Close
4 DETAILED SCHEDULE MEDAKA Monday 1st February
9:00 am Pick up from Melbourne Airport / CBD 10:00 – 12 noon Visit ARMI / Monash University Aquatic Facilities
12:00 – 1:00 pm Lunch at Monash University
1:00 – 2.30 pm Travel to conference venue (Flinders) 2.30 pm – 3:30 pm Hotel check-in and registration 3.30 pm – 3:45 pm Welcome address
3:45-4:45 pm Keynote speaker: HIDEAKI TAKEUCHI
Exploring the neural geography of the social brain using medaka
4:45 – 6:00 pm Posters session with drinks and canapés 7:00 pm Welcome dinner (BBQ at Flinders)
Tuesday 2nd February
9:30-10:30 am Keynote speaker: JOACHIM WITTBRODT The memory of retinal stem cells: How to shape form and function
10:30 – 11:00 am Morning tea
11:00 – 12:30 pm Medaka Talks – Session I 11:00 Kiyoshi Naruse Medaka as a model animal and the current status of medaka biological resources 11:30 Manfred Schartl Gene expression in the medaka melanoma model 12:00 Tomonori Deguchi Interaction between lymphatic vasculature and peripheral nerves during development of medaka
12:30 – 2:00 pm Lunch
2:00 – 4:00 pm Medaka Talks – Session II 2:00 Felix Loosli A food-on-demand paradigm to study medaka behaviour 2:30 Mu-Yun Wang Your face looks familiar: multimodal individual recognition in medaka fish and the individual difference in discrimination ability
5 DETAILED SCHEDULE
MEDAKA (cont’d)
Tuesday 2nd February, cont’d
3:00 Mirana Ramialison Medaka project down-under 3:30 Reina Watanabe Expression analysis of novel transcription factor, Mbo-1 and behavioral analysis of mho-1 mutants in Oryzias latipes, Medaka
4.30 pm onwards Wine Tasting / Conference Dinner
Wednesday 3rd February
9:30 – 9:45 am Sponsors presentation 9:45 – 11:00 am Community meeting
6 DETAILED SCHEDULE
ZEBRAFISH Wednesday 3rd February
12:30 –1:45 pm Registration / Meeting materials pick-up 1:45 – 2:00 pm Welcome 2:00 – 3:00 pm Session 1: Blood and Vessels (Chair: Chris Hall) 2:00 Tiffany Eng, Pauline Misa, Kazuhide S. Okuda, Kathryn E. Crosier,Philip S. Crosier and Jonathan W. Astin Characterisation of the zebrafish facial lymphatic network 2:15 Neil I. Bower, Cathy Pichol-Thievend, Stephen J. Bent, Cas Simons, Mathias Francois and Benjamin M. Hogan Characterisation of lymphatic development in different vascular beds using zebrafish mutants 2:30 Faiza Basheer, Clifford Liongue, Alister C. Ward Zebrafish as genetic model for leukemia and other hematopoietic disorders 2:45 Jessica E De Angelis, Anne K Lagendijk, Huijun Chen, Alisha Tromp, Neil Bower, Carol Wicking, Mathias Francois, Jeroen Bakkers, Alpha Yap, Benjamin Hogan, Kelly Smith Tmem2 regulates developmental Vegf-signaling by controlling Hyaluronic acid turnover 3:00 – 4:00 pm Plenary 1: HIROYUKI TAKEDA Deciphering the genetic code for the vertebrate pluripotent epigenome --- Insights from the medaka genome (Chair: Mirana Ramialison) 4:00 – 4:30 pm Afternoon tea 4:30 – 6:15 pm Session 2: Neurobiology (Chair: Patricia Jusuf) 4:30 Thompson, Adrian, Gasperini, Robert, Young, Kaylene, Foa, Lisa Store-operated calcium entry (SOCE) is important for axon guidance in vivo 4:45 Lucy A Heap, Gilles C. Vanwalleghem, Andrew W. Thompson, Itia A. Favre-Bulle and Ethan K. Scott Anatomical and Functional Analysis of Tectal Afferents in Zebrafish 5:00 Itia Favre-Bulle, Halina Rubinsztein-Dunlop, and Ethan Scott Exploring Vestibular Circuits in Zebrafish 5:15 Andrew W. Thompson, Gilles C. Vanwalleghem, Lucy A. Heap, and Ethan K. Scott Functional profiles of visual, auditory, and water flow responsive neurons in the zebrafish tectum 5:30 Yuxi Zhang, Ellen Olzomer, David Nguyen, Daniel Hesselson Phenotypic identification of novel neuroprotective small molecules 5:45 Mu-Yun Wang and Hideaki Takeuchi Your face looks familiar: multimodal individual recognition in medaka fish and the individual difference in discrimination ability 6:00 Benjamin Lindsey, Viola Oorschot, Georg Ramm, Jan Kaslin Near and Far: The Cellular Response of the Adult Zebrafish Brain to Injury 6:15 – 8:00pm Welcome Reception
7
Thursday 4th February
9:30 – 10:45 am Session 3: Development and Muscle Biology – I (Chair: Ben Hogan) 9:30 Mo Zhao, Caitlin Williams, Robert J Bryson-Richardson Myomesin-1 stabilises sarcomeric structure acting as a shock absorber in skeletal muscle 9:45 A.J.Wood, C.H.Lin, V.Joshi, A.Siegel, L.B.Miles, S.Dudczig, P.R.Jusuf, N.Packer, P.D.Currie Glycosylation in the Maintenance of Myotendinous Junction Integrity 10:00 Inken G Huttner, Louis W Wang, Celine Santiago, Scott Kesteven, Wolfgang Linke, Michael Feneley, Daniel Hesselson, Diane Fatkin Zebrafish with truncating mutations in titin develop late-onset cardiac function defects. 10:15 Hakan Tarakci, Joachim Berger, Peter D Currie Thin filament capping in nemaline myopathy 10:30 Emily Claire Baxter, Raquel Vaz, Tamar Sztal, Caitlin Williams, Robert Bryson- Richardson Investigation of Small Heat Shock Proteins in Muscle Disease
10:45 – 11:15 am Morning tea
11:15 – 12:30 pm Session 4: Emerging Technologies (Chair: Mirana Ramialison) 11:15 Pei Jean Tan, Kazuhide Shaun Okuda, Mei Fong Ng, Norazwana Samat, Vithya Velaithan and Vyomesh Patel Zebrafish as a tool for targeted natural product drug discovery 11:30 Markus Tondl, Michael Eichenlaub, Lauren Bottrell, Mirana Ramialison BiTS and pisces: generating a repertoire of cardiac cell type specific regulatory elements 11:45 Manjula Algama, Caitlin Williams, Edward Tasker, Adam C. Parslow, Robert J. Bryson-Richardson, Jonathan M. Keith Genome-wide identification of ncRNAs using a Bayesian segmentation approach 12:00 Thomas E. Hall, Nicholas Ariotti, Zherui Xiong, Harriet Lo, James Rae, Charles Ferguson, Kerrie-Ann McMahon, Nick Martel, Robyn E. Webb, Richard I. Webb, Rohan D Teasdale, Robert G. Parton New methodology for in vivo visualisation of GFP-Labeled Proteins by Electron Microscopy 12:15 Wang LW, Huttner IG, Santiago C, Kesteven S, Feneley MP, Fatkin D. Evaluation of cardiac function in adult zebrafish using high frequency echocardiography.
12:30 – 2:00 pm Lunch 2:00 – 3:00 pm Sponsor presentations 3:00 – 4:00 pm Plenary 2: JOACHIM WITTBRODT The memory of retinal stem cells: How to shape form and function Chair: Jan Kaslin
8 Thursday 4th February, cont’d
4:00 – 4:30 pm Afternoon tea
4:30 – 5:45 pm Session 5: Regeneration and Stem Cells (Chair: Nicholas Cole) 4:30 N.D.D. Ratnayake and P.D. Currie Defining the zebrafish skeletal muscle stem cell compartment during regeneration 4:45 Brandli, A, Dudczig, S and Jusuf, P. Glial derived regeneration of the outer retina: a new method 5:00 Subhra P Hui, Delicia Sheng, Kotaro Sugimoto, Kazu Kikuchi A pro-regenerative T cell subset promoting adult tissue regeneration 5:15 P.D. Nguyen, C. Sonntag, L. Hersey, P.D. Currie Examining Clonal drift of muscle stem cells during growth 5:30 Daniel Colquhoun, Benjamin Lindsey, Kit Tang, Jan Kaslin The role of HMGA proteins in Regulating Neural Stem Cell Activity in vivo
6:30pm Conference Dinner
Friday 5th February 9:30 – 11:15 am Session 6: Disease Models – I (Chair: Ethan Scott) 9:30 Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Robert J. Bryson-Richardson Investigating of the Pathobiology of Myofibrillar Myopathies and Potential Therapies using Zebrafish 9:45 Kimberly Morgan, Karen Doggett, Lachlan Whitehead, Stephen Mieruszynski & Joan K. Heath Investigating novel non-oncogene targets for cancer therapies 10:00 Emily K Don, Serene Gwee, Sharron Chow, Ian Blair, Julie Atkin, Garth Nicholson, Nicholas J Cole A zebrafish model of the mnd linked c9orf72 hexanucleotide repeat expansion. 10:15 Karen Doggett, Andrew P Badrock, Yeliz Boglev, Tanya A de Jong-Curtain, M. Cristina Keightley, Laure Lam Hung, Derek Stemple, Dan Fraher, Yann Gibert, Graham J Lieschke and Joan K Heath To what extent do zebrafish models of impaired ribosomal biogenesis recapitulate human ribosomopathies? 10:30 Giacomotto Jean, Mowry Bryan, Becker Thomas miR-mediated knockdown to recapitulate Spinal Muscular Atrophy 10:45 Alison L. Hogan, Emily K. Don, Serene Gwee, Isabel Formella, Kelly L.Williams, Garth A. Nicholson, Nicholas J.Cole, Ian P.Blair Generation of novel zebrafish models of amyotrophic lateral sclerosis. 11:00 Jack J. Stoddart, Marco Morsch, Isabel Formella, Andrew P. Badrock, Emily K. Don, E., Roger S. Chung, Nicholas J. Cole An in vivo study of the localization and function of ALS-associated protein, TDP-43 in transgenic zebrafish
11:15 – 11:45 am Morning tea
9 Friday 5th February, cont’d
11:45 – 12:45 pm Plenary 3: HIDEAKI TAKEUCHI Exploring the neural geography of the social brain using medaka fish Chair: Yann Gibert 12:45 – 2:00 pm Lunch 2:00 – 3:15 pm Session 7: Infection and Immunity (Chair: Cristina Keightley) 2:00 Lucia Du, Lisa Lawrence, James Cheeseman, Guy Warman, Kathy Crosier, Phil Crosier, Chris Hall Circadian regulation of the innate immune cell response following infection 2:15 Tarannum Taznin, Yann Gibert, Clifford Liongue, Alister C. Ward Understanding the role of cytokine receptor like factor 3 (crlf3) in zebrafish 2:30 Marco Morsch, Rowan Radford, Jack Stoddart, Emily Don, Serene Gwee, Isabel Formella, Andrew Badrock, Thomas Hall, Nicholas Cole, Roger Chung Features of a good meal: Microglia engulfment in the spinal cord 2:45 Vahid Pazhakh, Felix Ellett, Stefan Greulich, Alex Andrianopoulos, Graham Lieschke Leukocyte/pathogen interactions during fungal infection establishment 3:00 Celia Vandestadt, Daniel Colquhoun, Timo Friedrich, Jan Kaslin Inflammation orchestrates neural regeneration in the spinal cord 3:15 – 4:15 pm Plenary 4: ELIZABETH PATTON Chemical-genetics in the melanocyte lineage reveals new targets in melanoma Chair: Cristina Keightley 4:30 – 6:30 pm Session 8: Posters and Canapes 7:00 pm BBQ and dancing
Saturday 6th February
9:15 – 10:30 am Session 9: Disease Models - II (Chair: Dan Hesselson) 9:15 Delfine Cheng, Gerry Shami, Marco Morsch, Roger Chung, Filip Braet High-resolution mapping the Microanatomy of the Zebrafish Gastro Digestive System by mean of correlative and combined x-ray, light and electron microscopy. 9:30 M. Li, M. Andersson-Lendahl, T. Sejersen, A. Arner Functional characterization and drug screening on zebrafish muscular dystrophy models 9:45 E. Hortle, E. Don, S. Chow, J. Stoddart, G. Nicholson, N. Cole Creating an Inducible Zebrafish Model of TDP-43 Mislocalisation 10:00 Yagiz Alp Aksoy, Daniel Hesselson and Nicholas Cole Telling TALEs to Recode Zebrafish Genome to generate novel ALS models 10:15 Maxinne Watchon, Kristy Yuan, Nick Mackovski, Thomas Becker, Garth Nicholson and Angela Laird Protein cleavage and motor dysfunction in a transgenic zebrafish model of spinocerebellar ataxia-3
10 Saturday 6th February, cont’d
10:30 – 11:30 am Session 10: Development and Muscle Biology – II (Chair: Rob Bryson-Richardson) 10:30 D. Grassini, J. De Angelis, S. Capon, S. Paterson, G. Baillie, C. Simons, R. Taft, B. Hogan and K. Smith Myosin Vb-mediated endosomal trafficking of N-cadherin is required for heart chamber form and function 10:45 Joachim Berger, Silke Berger, Mei Li, Patricia R. Jusuf, Arie S. Jacoby, Anders Arner and Peter D. Currie In vivo function of the chaperonin TRiC/CCT complex 11:00 L. Miles, K. Doggett, J. Kaslin, J. K. Heath, S. M. Jane, S. Dworkin Grainyhead-like 3 (Grhl3) regulates gastrulation through its role in formation and maintenance of the EVL 11:15 Prusothman Yoganantharajah, Alister Ward, Yann Gibert The implications of BPA usage on developmental obesity
11:30 – 12:30 pm Morning Tea
11:40 – 12:20pm Community meeting 12:20 – 12:30pm Awards and Meeting Close
11 Plenary 1 HIROYUKI TAKEDA
Hiroyuki Takeda earned his Ph.D. from the University of Tokyo, Japan in 1987. Just prior to receiving his Ph.D. he worked for one year as a visiting scholar at the Strangeways Research Laboratory in Cambridge, England, and then in 1989, spent another year as a visiting scholar in the Urological Department of Chicago University to study androgen-dependent organogenesis such as the prostate in rodent embryos. He returned to Japan in 1990 to work as a Research Associate at RIKEN and later moved to Nagoya University to take a position of Assistant Professor in 1993. During this period, he introduced zebrafish for the first time in Japan as a model to study axis formation and organogenesis in vertebrate embryos. In 1999, he was appointed Professor at the National Institute of Genetics, where he remained until 2001. In 2000, he started using medaka, a Japanese killifish for genetic analysis in addition to zebrafish. Since 2001, he has served as Professor in the Graduate School of Science at the University of Tokyo. His group was one of the core laboratories involved in the medaka genome project, which was successfully complete in 2007. Dr. Takeda’s group has addressed how the segmentation clock functions and which tissues are responsible for mesoderm induction and neural patterning in zebrafish. His group currently works with both medaka and zebrafish to study the mechanisms underlying vertebrate axis formation and organogenesis, and also goes into the epigenetic regulation of key developmental genes in medaka.
Deciphering the genetic code for the vertebrate pluripotent epigenome --- Insights from the medaka genome Embryonic cells at the blastula stage are pluripotent, and their genome (referred to as pluripotent genome) is under the control of a network of specific transcription factors (TFs) such as the Yamanaka factors, and exhibits a characteristic pattern of epigenetic modifications and transcriptional activity (1). The epigenome of pluripotent cells ensures active transcription of pluripotency-related genes and repression of developmental genes. However, the DNA sequences that define the pattern of the epigenome are not well understood. We are examining those crucial DNA sequences in the medaka genome (2). By using a supervised machine learning algorithm, support vector machine (SVM), we demonstrate that DNA hypomethylated domains (HMDs) and their histone modification states can be predicted from DNA sequence (Figure), indicating the presence of the sequence-basic logic. We further characterized these predictable DNA sequences by DNase I-seq, and extended their generality among vertebrate genomes. We will discuss the conserved logic and sequence variations among vertebrate genomes. References: 1. R. Nakamura et al. Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates. Development 141, 2568-2580 (2014). 2. H. Takeda and A. Shimada. The art of medaka genetics and genomics: what makes them so unique? Ann Rev Genet, 44, 217 - 241. (2010)
12 Plenary 2 JOACHIM WITTBRODT
The lab is studying neuronal cell proliferation and differentiation in the developing, growing and regenerating eye and brain of fish (zebrafish, medaka) as model system. We are combining genetic, molecular and cell biological approaches with advanced imaging approaches to decipher the basic mechanisms that govern the balance of cell proliferation and differentiation in vivo. Special emphasis is given to follow the fate of proliferating and differentiating cells in the context of the fish retina and brain and to establish tools that allow visualizing those processes in vivo. We take advantage of the life-long proliferation of retinal stem cells from the ciliary marginal zone (CMZ) that facilitates the continuous study of cells exiting the stem cell niche at the CMZ and their subsequent stereotypic differentiation. Novel tools developed in the lab allow clonal analysis in 4D by the induction of the expression of genes of interest at physiological levels in individual retinal cells of any cell type. The combination of these approaches with the systematic analysis of transcription factors that control the expression of key genes will contribute to a functional understanding of the molecular processes that govern the proliferation and differentiation of retinal stem cells. The memory of retinal stem cells: How to shape form and function. Erika Tsingos, Lazaro Centanin, Burkhard Hoeckendorf, Joachim Wittbrodt In the continuously growing eyes of fish, individual stem cells coordinate their proliferation life-long to maintain organ structure and function. Studies in Medaka (Oryzias latipes) revealed that the neural retina and the surrounding retinal pigmented epithelium (RPE) share a bipartite stem cell niche in a ring-shaped domain apposed to the lens - the ciliary marginal zone (CMZ). The bipartite stem cell niche in the CMZ gives rise to new differentiated cells in homogeneous concentric rings, both in the retinal pigmented epithelium as well as in the neural retina. Here, lineages of differentiated cell types originate from individual stem cells in the CMZ. Those give rise to heterogeneous stripes (ArCoS) composed of all neuroretinal cell types and span the retinal radius. Intriguingly, the shape and pattern of ArCoSs differs between neural retina and RPE, and, despite the common niche, each lineage exclusively feeds into only one of these two tissues. How does the heterogeneous individual stem cell behavior translate to homogeneous tissue growth? The molecular machinery of cells in the CMZ remains obscure. Furthermore, other, yet unaddressed factors such as physical constraints by surrounding tissue may also regulate cellular decisions. An in silico model of the growing eye allows to explore potential mechanisms, thus guiding targeted experimental design. We use a three-dimensional agent-based computational approach to model the eye as a radially growing hemisphere populated by spherical cells. Thus, we recapitulate the growth of the entire population of stem cell lineages. In this virtual playground, we test and quantitatively validate hypotheses on stem cell behavior using the existing lineage tracing data. Beyond this, we aim to use the model to explore how stem cells transition to fate-restricted progenitors, how tissue function constrains cell proliferation, and how cells may contribute to eye shape, a crucial parameter for vision.
13 Plenary 3 ELIZABETH PATTON
Dr Patton received a BSc (Honours) degree from King’s College at Dalhousie University, and worked with Professor Gerry Johnston to study the initiation of the budding yeast cell cycle. She then received a PhD from the University of Toronto, working with Mike Tyers to discover how E3 ubiquitin ligases control cell division. Following this, Liz received a Human Frontier Science Programme Postdoctoral Fellowship to work with Len Zon at Harvard Medical School, where she developed a zebrafish model for melanoma. She continued her training with a MRC Career Development Award at The University of Oxford and the MRC Human Genetics Unit, and is currently a Group Leader at the MRC Institute for Genetics and Molecular Medicine in Edinburgh, in addition to manager of the MRC IGMM zebrafish service facility. Dr. Patton has recently been elected to the Young Academy of Scotland at the Royal Society of Edinburgh, and to the Council of the European Society of Pigment Cell and Melanoma Research. She serves on the Editorial Board for Pigment Cell and Melanoma Research (Wiley) and is a Monitoring Editor for Disease Models and Mechanisms (The Company of Biologists).
Chemical-genetics in the melanocyte lineage reveals new targets in melanoma Melanoma (cancer of the melanocyte) kills over 20,000 Europeans each year and incidence continues to rise rapidly. BRAFV600E inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance, or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance. An important and new therapeutic concept for melanoma is to target the melanocyte lineage. Recent evidence reveals that a melanocyte lineage specific programme maintains melanoma survival, and we have engineered the first animal model in zebrafish to demonstrate that targeting the master melanocyte lineage transcription factor MITF leads to rapid melanoma regression. Thus, understanding and targeting the melanocyte lineage is directly relevant to melanoma, and reveals therapeutically targetable processes. We are using live-imaging of the melanocyte lineage as the basis for phenotypic chemical screens in zebrafish to find drugs/leads and identify targetable processes that might elucidate pathways for cancer therapy. Screening for targets of the melanocyte lineage is highly relevant to melanoma because melanocytes are the melanoma cell of origin, and genes that specify the melanocyte stem cells and the lineage during embryogenesis are the same genes that play fundamental roles in cancer. We are using innovative chemical-biology to capture and validate targets in vivo, and perform chemo-preventative and -therapeutic trials in zebrafish melanoma models. Ultimately, we aim to translate our most promising drug/leads and targets into the mammalian system, to establish the basis for clinical trials.
14 Plenary 4 HIDEAKI TAKEUCHI
To clarify both molecular/neural basis and brain information processing underlying social interactions in vertebrates, we have focused on Medaka fish and established novel behavior systems to assess social interactions (schooling behavior, individual recognition, group-escape and group-learning). Using these systems we are planning to identify internal factors (genes, neural networks, and brain regions) essential for vertebrate social interactions using transgenic medaka systems. On the other hand, to estimate behavior rules underlying social interactions, we developed a hypothesis-independent data mining, which could explain actual fish movement. Our eventual purpose is to how the internal factors (genes, neural networks, and brain regions) influences animal behaviors, which can serve as the basis for the emergence of sociality. Furthermore I have investigated the molecular/neural basis underlying invertebrate social behavior (honeybee) and compare neural/ social mechanisms between the vertebrate and invertebrate.
Exploring the neural geography of the social brain using medaka fish
Within group-living animals, individuals appropriately tailor attitudes and responses to other group members according to the social context and external environment. At the simplest level, the behavioral output can be described as approach and affiliation (positive response) versus agonistic behavior and avoidance (negative response). The neural substrate that works between sensory input and behavioral output, or the integrative circuits underlying decision- making processes, however, is vast and mysterious. To address this issue, we have focused on medaka fish, a model animal used mainly in the field of molecular genetics. Previously, we demonstrated that medaka females recognize familiar males following prior visual exposure, and social familiarity influences female mating receptivity. Medaka females exhibit a positive response (high receptivity) to familiar males, and a negative response (low receptivity) to unfamiliar males. Further, we demonstrated the essential role of a subpopulation of gonadotropin-releasing hormone-producing neurons (GnRH3 neurons) in switching from low to high female receptivity. Recently we established a new methodology for heat-inducible Cre/LoxP recombination in the medaka brain. Using the IRLEGO system, heat shock induced in a very small area of the developing brains leads to spatially controlled recombination of progeny cells in adult medaka fish, which allows for genetic modulation and/or visualization of neuronal populations of interest. Using this system, medaka may serve as an ideal model animal to understand how the “social brain” works at molecular and neuronal levels.
15 MEDAKA – ORAL ABSTRACTS
Medaka as a model animal and the current status of medaka biological resources
Kiyoshi Naruse*, Takao Sasado, Shin-ichi Chisada, Kazunori Yamahira and Yusuke Takehana
National Institute for Basic Biology Laboratory of Bioresources, Aichi, Japan
Medaka has been used as a model animal worldwide. Medaka has the similar characters with zebrafish as an experimental animal and also have species specific features. As medaka is fish in temperate zone, spawning is influenced by photoperiod and circadian rhythms. Medaka can adapt to lower temperatures in winter and higher temperature in summer. Adaptability to high salinity is also prominent. Medaka has been supported as important biological resources with National BioResource Project (NBRP) since 2002. Now NBRP medaka is a central repository of medaka resources established in medaka research community and have provided several resources like fish, cDNA/BAC/fosmid, hatching enzyme and several database useful for the community and recently provided screening system of TILLING library as well as the genome editing platform by CRISPR-Cas9. NBRP medaka continue to support medaka research community to enhance the productivity, continuity and reproducibility for each researcher.
16 Gene expression in the medaka melanoma model
Barbara Klotz1, Rasmi Mishra1,2, Janine Regneri1, Susanne Kneitz1, Michael Hausmann1, Wesley Warren3, John Postlethwait4, Ron Walter5, Manfred Schartl1
1 University of Wuerzburg, Department of Physiological Chemistry, Biocenter, Am Hubland, 97074 Wuerzburg, Germany 2 Bilkent University, Department of Molecular Biology and Genetics, Faculty of Science, TR- 06800 Bilkent, Ankara, Turkey 3McDonnell Genome Institute Washington University School of Medicine 4444 Forest Park Ave., Campus Box 8501 St Louis, MO 63108, USA 4 University of Oregon, Institute of Neuroscience, 324 Huestis Hall, 1425 E. 13th Avenue, Eugene OR 97403, USA 5 Texas State University, Department of Chemistry & Biochemistry, Molecular Biosciences Research Group, 419. Centennial Hall, 601 University Drive, San Marcos, TX 78666, USA|
Aberrations in gene expression are a hallmark of cancer cells. Differential tumour- specific transcript levels of single protein-coding genes and genes that give rise to non-coding regulatory RNAs or whole sets of genes (transcriptional disease signatures, TDS) may be critical for the neoplastic phenotype and are important for therapeutic considerations or useful as biomarkers. In an approach to filter out the relevant expression differences from the plethora of changes noted in global expression profiling studies, we focus on a certain type of highly malignant skin cancer, melanoma and use transgenic medaka models, which we compare with other fish melanoma models and human melanoma. With this strategy we have identified a novel set of dysregulated non-coding small RNAs (piRNAs, miRNAs) and a so far overlooked long non-coding RNA (lncRNA) in fish melanoma. We find similar expression changes in human melanoma cell lines. Experimental modulation of transcript levels identified changes in relevant melanoma signalling pathways and resulted in altered growth of tumour cells. A selection of TDS protein coding genes was made that allows to follow-up with short time-windows the effects of drug treatments and high-throughput drug screening.
17 Interaction between lymphatic vasculature and peripheral nerves during development of medaka
Tomonori Deguchi
Health Research Institute (HRI), National Institute of Advanced Industrial Science and Technology (AIST)
Although the lymphatic vasculature is a very important organ as well as the blood vasculature, its development has been poorly understood for a long time. In this study, we speculated a hypothesis that lymphatic vessels were guided by peripheral nerves as same as blood vessels, and started to research the interactions between lymphatic vessels and peripheral nerves. For that, we developed a transgenic medaka in which the lymphatic vessels and peripheral nerves were made visible in vivo. Here, we show that in the medaka skin, some lymphatic vasculatures run along the peripheral nerves.
18 A food-on-demand paradigm to study medaka behaviour
Felix Loosli*, Nicholas Foulkes*, Jose Lopez Olmeda#, Haiyu Zhao*, Ravindra Peravali*, Markus Reischlˆ, Christian Pylatiukˆ
* Institute of Toxicology and Genetics, KIT, Karlsruhe; Germany ^ Institute of Applied Informatics, KIT; Germany # Department of Physiology, University of Murcia; Spain
Genetic teleost models are increasingly used to study complex traits, including behaviour. Medaka offers a unique advantage in that isogenic inbred lines are available that allow the genetic basis of such complex traits to be addressed. We have exploited this to study the feeding behaviour of small groups of medaka. Associative learning in task-reward paradigms has been shown for many fish species. Commercial fisheries exploit this to use food-on-demand (self feeding) systems where fish can manipulate a mechanical device to get a reward in the form of food. Pozo et al have adapted this system for use with small fish species using infrared emitter-receiver devices that couple a visit to a defined spot in an aquarium with a food reward (Ana del Pozo et al., 2011). Zebrafish (Ana del Pozo et al., 2011) as well as medaka (our study) learn within a few days to associate the visit with a food reward. Thus, medaka can learn how to feed themselves. In addition to activation of the feeding device we also monitor overall locomotor activity. In this paradigm, fish are self sufficient and can thus be kept in isolation for several weeks. We will present our results obtained using small groups of medaka maintained in this associative learning paradigm.
References del Pozo, A., Sánchez-Férez, J. A., & Sánchez-Vázquez, F. J. (2011). Circadian rhythms of self-feeding and locomotor activity in zebra sh (Danio Rerio). Chronobiology International, 28(1), 39–47. http://doi.org/10.3109/07420528.2010.530728
19 Your face looks familiar: multimodal individual recognition in medaka fish and the individual difference in discrimination ability
Mu-Yun Wang and Hideaki Takeuchi
Okayama University, Tokyo, Japan
The ability to discriminate different conspecifics is the first step for many social interactions, such as mate choice, hierarchies and recognition of offsprings, nestmates, fellow groups and neighbours. Few studies have look at how fish use olfactory, acoustic and electric cues for individual recognition, and to the best of our knowledge, none of them have investigate the cognition of visual recognition and how different modalities work together. Medaka females prefer familiar males and decrease the latency to mate after familiarization. We found that both visual and olfactory cues were used for mate discrimination, but only visual cue is adequate for shorten the latency to mate. Fish showed individual differences in discrimination ability: once exchanged the familiar male to an unfamiliar one, some female can recognise the change and extend the latency to mate. Those females succeeded to condition one male with electric shock from another, while all females learned to condition electric shock with black or white colour signals. We found that fish used only appearance but not motion cue for individual recognition, and signals around the head might be important; however, when we painted the male face with black ink after familiarisation, the female could still recognise the male. When flip the fish horizontal with prism during familiarisation, females recognised him as the same male, while when the image was upside-down the female failed to recognise the male. Medaka opens the opportunity to investigate the function of specific brain areas with cognitive abilities, which can benefit human studies.
20 Medaka project down-under
Michael Eichenlaub, Marc Drvodelic, Jeannette Hallab, Markus Tondl, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Every day in Australia, 8 babies are born with heart defects, of which, 7 cases are of unknown origin. It is a heavy burden for the children as the only effective treatment available is invasive surgery in the first year of life. Hence, finding the causes of congenital heart disease (CHD) is not only crucial for its early diagnosis but most importantly, it provides answers to distressed families in search for an explanation why this disease is striking their children.
There is only a limited number of genes known to cause CHD, however there is growing evidence that mutations in regulatory regions of the genome (such as enhancers and promoters) account for genetic malformations. In fact, genome-wide association studies revealed that 88% of sequence variants linked to disease fall in non-coding regulatory regions and only a minority in protein-coding genes. Despite the burden CHD places on families, the contribution of regulatory mutations towards CHD remains neglected and poorly understood.
Here we are addressing this lack of knowledge by identifying regulatory regions that are specifically active during the formation of the human heart and that lead to CHD if altered. Using bioinformatics and comparative genomics approaches, we identified enhancers or promoters that play a specific role in heart formation. The function of regulatory elements needs to be tested in an in vivo system to assess whether they specifically activate gene expression in the heart. We aim to establish a new, fast and cost-efficient screening pipeline for validating the in vivo function of human regulatory elements in heart tissues, by utilizing the medaka model organism for the first time in Australia.
21 Expression analysis of novel transcription factor, Mbo-1 and behavioral analysis of mho-1 mutants in Oryzias latipes, Medaka
Reina Watanabe, Saori Yokoi, Yasuko Isoe, Satoshi Anzai, Masato Kinoshita, Atsushi Ugajin, Teruhiro Okuyama, Takeu Kubo, Hideaki Takeuchi
Department of Biological Science, Graduate school of Science, University of Tokyo, Japan
Various animals change their behaviors in a developmental stage-dependent manner. Although remodeling of neural circuits is supposed to be related to this behavioral change, the relationship between neural circuit remodeling and behavioral change remains largely unknown. To approach this issue, I focused on homologues of the transcription factor, Mblk-1, as a candidate factor involved in the neural remodeling. Mblk-1 was originally identified from the honeybee, as a gene expressed preferentially in a higher-order center of the brain (mushroom body). In nematode, Mblk-1 homologue (mbr-1) functions in the pruning of excessive neural circuits, which occurs during larval development. In the present study, I performed expression analysis of a medaka homologue of Mblk-1, mbo-1 in the medaka brain. mbo-1 was selectively expressed in a brain-region preferential manner: in the telencephalon, preoptic area and dorsal habenula. Furthermore, I generated mbo-1 mutant stains and revealed that females of mbo-1 mutants had a defect in mating preference for familiar males. These findings suggest that mbo-1 functions in controlling mating behavior in a restricted brain regions in Medaka fish. In addition, I revealed that mho-1 mutants showed higher velocity in startle response to a visual stimulus and this finding suggests that mbo-1 functions also in controlling fear responses.
22 MEDAKA – POSTER ABSTRACTS
Analysis of the neural/molecular basis of mate-guarding behavior in medaka.
Saori Yokoi, Teruhiro Okuyama, Yasuhiro Kamei, Yoshihito Taniguchi, Satoshi Ansai, Masato Kinoshita, Larry J Young, Nobuaki Takemori, Takeo Kubo, Hideaki Takeuchi
National Institute for Basic Biology, Laboratory of Bioresources, Aichi, Japan
In various animal species from insects to vertebrates, males exhibit mate-guarding behavior to prevent other males from mating a potential or former mate, which is one of the forms of male-male competition for mates. Although the mate-guarding behavior has been studied extensively in behavioral ecology and phylogeny, its neural/ molecular basis remains unknown. We previously demonstrated that medaka (Oryzias latipes) fish, a model animal for molecular genetics, robustly exhibited mate-guarding behavior under laboratory condition. In addition, we also suggested a possible involvement of vasotocin (non-mammalian homolog of vasopressin) in this behavior based on pharmacologic methods. Here, we report that we generated mutants of vasotocin and its receptors (V1a1 and V1a2) genes by TILLING methods and revealed that two genes, vasotocin and V1a2, are required for normal mate- guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within an all-male group revealed that vasotocin mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Therefore, impaired mate-guarding in vasotocin homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.
23 Further investigating the role of Elk1 during cardiogenesis
Jeannette C. Hallab, Romaric Bouveret, Daniel Hesselson, Richard .P. Harvey, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The imperative role of transcription factors (TFs) in the temporal and spatial regulation of gene expression and ultimate tissue patterning and organogenesis is well established. Nevertheless, the genetic regulation of cardiogenesis remains poorly understood and only a handful of TFs are known to play an essential role in the process. Recent evidence suggests that the ETS Class I transcription factor, Elk1, which has not previously been implicated in heart development, may in fact, be important in the context of cardiogenesis. Morpholino studies targeting elk1 in zebrafish embryos have implied perturbations in the expression of genes known to be important in patterning in the anterior lateral plate mesoderm and normal cardiogenesis, and heart looping defects. The present study utilizes TALEN- modified zebrafish in an attempt to further investigate the morphant phenotype using more robust gene editing technology. We have identified four mutant Elk1 alleles among these fish and are currently investigating possible phenotype in the F4 generation for a mutant carrying a large (543bp) genomic deletion between exon and intron 2. Preliminary results indicate high mortality within 24 hpf among the F4 (homozygous) offpspring of homozygous F3s parents compared to the F4 offspring generated by INX of WT and heterozygous F3 parents. We plan to interrogate this putative, mutation-dependent lethality on a molecular level, firstly by determining the status of Elk1 protein using western blot. We then hope to acquire a deeper understanding of the RNA status of Elk1 itself and possible responses of the cardiac gene regulatory network to Elk1 defect in mutants and morphants using QPCR. Overall, we hope to gain a deeper understanding of the possible role of Elk1 during embryogenesis and the molecular mechanisms underlying the observed phenotypes and the similarities or differences between the mutant and morphant scenarios.
24 BiTS and pisces: generating a repertoire of cardiac cell type specific regulatory elements
Markus Tondl, Michael Eichenlaub, Lauren Bottrell, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Regulatory elements (REs; promoters, enhancers) are essential for the spatio- temporal control during development and hence as important as the genes they regulate. Although many genes involved in heart development have already been identified, little is known about the REs that control their expression in cardiac tissues. REs can be identified by chromatin marks, that is, post-translational modifications of histones. Data from chromatin immunoprecipitation followed by deep sequencing experiments (ChIP-seq) for some chromatin marks are publicly available for whole zebrafish larvae as well as whole mouse hearts. However, these datasets may not reflect the specific REs activate in specific cell types. In order to identify cardiac REs that are specific to cardiomyocytes, we established a protocol to isolate zebrafish cardiomyocytes by FACS and sequential ChIP-seq for histone modifications. Hence, we modified the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) [BONN et al., Nat Protoc, 2012]. Our preliminary data show that REs identified in cardiomyocytes regulate genes enriched in “heart development” functions which validates our data as a cardiac-specific repertoire. This is the first cardiomyocyte-specific regulatory repertoire in a developing heart. Our findings will not only be useful for cross-species comparisons to answer questions about heart evolution but will also be advantageous for clinical studies to identify ultra-conserved regulatory elements from human to fish as candidate regions for mutations in congenital heart diseases. Moreover, our dataset is a great resource for researchers to create cardiac-specific reporter lines.
25 In silico investigation of the evolution of a cardiac-specific repertoire of regulatory elements
Mark Drvodelic, Michael Eichenlaub, Markus Tondl and Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The heart is an ancient organ which has undergone significant changes during vertebrate evolution. It has evolved from a simple tube in invertebrates to a pump with two chambers in fish, three in amphibians and four in birds and mammals. Explaining this increase in complexity is hindered because the genes that drive cardiac development are extremely well-conserved across these species and hence during evolution. Consequently, we hypothesise that structural changes in the heart result from changes in expression of cardiac genes, which are triggered by changes in the cis-regulation of these genes.
Initially, we obtained the cardiac repertoire of cis-regulatory elements in zebrafish by identifying heart-specific regulatory regions genome-wide using tissue-specific chromatin immunoprecipitation followed by deep-sequencing. This provides an extensive atlas of regulatory regions controlling gene expression in heart development of the zebrafish; a widely used model organism for the study of cardiogenesis, but whose cardiac regulatory landscape has been left mostly unexplored.
Secondly, we will assess sequence conservation of these regulatory regions in evolution and perform cross-species comparisons of cardiac cis-regulatory elements between teleosts (zebrafish, medaka, stickleback), birds (chicken) and mammals (mouse, marmoset and human). We expect to identify regulatory regions that are either conserved, gained or lost during heart evolution and investigate changes in their composition of binding sites for cardiac regulators.
By deriving the cis-regulatory landscape driving zebrafish cardiogenesis; an essential missing link into the understanding of heart evolution, and then further comparing the resulting regulatory regions with other species, we hope to determine the cis- regulatory variations responsible for formation of the complex organ that is, the heart.
26 Behavioral alterations in medaka caused by serotonin deficiency in the raphe neurons
Satoshi Ansai, Hiroshi Hosokawa, Shingo Maegawa, Youhei Washio, Kenji Sato, Masato Kinoshita
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Serotonin (5-hydroxytryptamine; 5-HT) is a bioactive monoamine that acts as a neurotransmitter in the central nervous system as well as a hormone in the peripheral organ systems. Teleost fish species have serotonergic neurons in the raphe nuclei of the brainstem, which project to a number of brain regions similar to mammals; however, the role of the raphe serotonergic neurons in behaviors of teleost fishes remains largely unknown. Therefore, we generated a medaka mutant that was disrupted tph2 gene, encoding the tryptophan hydroxylase involved in the 5-HT synthesis in the raphe, using targeted mutagenesis using transcription activator-like effector nucleases (TALENs). At first, we showed that the mutant fish were deficient in 5-HT of serotonergic neurons in the raphe nuclei by immunohistochemical and mass spectrometric analysis. Subsequently, the behavioral alterations in the mutant fish were assessed using five behavioral paradigms (diving, open-field, light-dark transition, mirror approaching, and social interaction). The tph2 mutant fish exhibited decreased locomotor activity and reduced the number of entries to the top area in the diving test. The tph2 mutant female also showed a decreased mirror biting time in the mirror biting test and an increased contact number in social interaction test. In addition, the tph2 mutant fish exhibited longer duration of freezing for the first several minutes of each test in all examined paradigms. These results indicate that central serotonergic systems will modulate anxiety/fear responses and social behaviors in medaka.
27 3D reconstruction of genome-wide cardiac gene expression and regulation
Nathalia M. Tan, Jose M. Polo, Matthias Klapperstueck, Falk Schreiber, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
An understanding of spatial and temporal gene expression and regulation is key to uncovering developmental and physiological processes, and accordingly, disease processes. Numerous techniques exist to gain gene expression and regulation information, but very few utilise intuitive true-to-life imaging methods to analyse and present their results. Mapping gene expression and regulation information onto a three-dimensional model will greatly aid in the visualisation of gene expression patterns and allow researchers to see which areas in an organ or organism express which genes in the genome, and vice versa. Furthermore, analysing this data using cluster analysis may uncover novel genes relating to development and disease. We hypothesise that finding the three-dimensional position and regulation of every gene at a given time during development will reveal the specific subset of genes that play an essential role in specific sub-compartments. We aim firstly to build a 3D model of the adult murine heart that can incorporate gene expression and regulatory patterns. Secondly, we wish to derive a 3D transcriptome and epigenome map according to sub-anatomical compartments. Lastly, we want to perform cluster analysis on 3D gene expression patterns to reconstruct gene networks involved in heart development and disease at a sub-anatomical resolution. Results from this study will contribute to the discovery of key genes epigenetic changes responsible for driving heart development and disease. Additionally, this study will act as a proof of concept in applying similar techniques to other tissues and organs. We aim to apply this technology to investigate the gene regulatory networks defining cardiac boundaries in the zebrafish lateral plate mesoderm.
28 Development of Trawler, a web based motif discovery tool
Louis T. Dang, Henry M.H. Chiu, Jerico Revote, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Identification of enriched motifs in downstream analysis of DNA binding assays is one of many biological computational problems. As a result, a variety of motif discovery tools and algorithms have been developed to accurately identify the correct binding motif present in the sequenced regions. We have previously developed Trawler, a fast motif-discovery tool that searches for de novo binding motifs against a biological background. To make Trawler user friendly, we aim to develop a web version with the option of using bed files as input and automation of the background data.
Firstly, the user inputs a bed file used to retrieve sequences. A “bedtofasta” script has been designed to extract FASTA sequences from locally stored chromosome data. Second, generation of the random background takes calculations of each input region to the nearest transcription start site. A gene list and chromosome lengths are required to retrieve the corresponding sequences for the background data. In addition, Trawler displays conservation scores for every instance of the motif identified in the sample sequences, a feature unique to Trawler.
Overall, the process of generating a random background has been streamlined and automated into the current version of software while allowing the use of bed files as input, which is not available with other motif discovery tool. Trawler is able to analyse data from a range of model organisms including mouse, human, zebrafish and medaka. It is hoped that this new implementation of Trawler will help researchers to not only detect the primary binding motif, but also accurately identify secondary motifs.
29 ZEBRAFISH – ORAL ABSTRACTS
Characterisation of the zebrafish facial lymphatic network.
Tiffany Eng, Pauline Misa, Kazuhide S. Okuda, Kathryn E. Crosier,Philip S. Crosier and Jonathan W. Astin.
University of Auckland, NZ
In our lab we focus on using the zebrafish to understand mechanisms of lymphatic vessel development. The formation of the thoracic duct (the major trunk lymphatic vessel) in the zebrafish embryo has been established as a model of lymphangiogenesis. However, the remaining lymphatic networks in the zebrafish embryo are poorly characterised and little is known about the development of lymphatic vessels outside the trunk. We have begun to characterise how the facial lymphatics, a complex network of lymphatic vessels in the zebrafish head, are formed during development.
We have used live imaging and lineage tracing studies to demonstrate that the facial lymphatics initially develop by the migration of a single lymphatic vascular sprout from the common cardinal vein at 1.5 dpf. Following this, two additional populations of lymphangioblasts, one from the primary head sinus at 1.75 dpf and another from the ventral aorta region at 2.25 dpf, fuse to the growing vascular tip. Following each fusion event, the migration of existing lymphatic cells slows and the new population drives vessel growth. These data describe a new “relay-like” mechanism of lymphatic vessel development in which sequential addition of lymphangioblasts is required for migration.
Finally, we have started to characterise the how the developing facial lymphatic vessels are patterned through interactions with other tissues. While blood vessels are required for the guidance of lymphangioblasts in the zebrafish trunk, we will present preliminary data suggesting that both cartilage and sensory neurons are also required for the guidance of facial lymphatic vessels.
30 Characterisation of lymphatic development in different vascular beds using zebrafish mutants
Neil I. Bower, Cathy Pichol-Thievend, Stephen J. Bent, Cas Simons, Mathias Francois and Benjamin M. Hogan
University of Queensland Institute for Molecular Bioscience, QLD, Australia
Perivascular cells regulate the blood brain barrier and maintenance of neural stem cells. Using transgenic markers, we identified endothelial cells that are perivascular, surrounding blood vessels within the zebrafish meninges. These cells express markers of lymphatics and produce vascular endothelial growth factors. They develop from a continuous endothelial loop into a dispersed, non-lumenized, perivascular lineage. These cells form by lymphangiogenesis, identifying an unexpected lineage and developmental mechanism.
31 Zebrafish as genetic model for leukemia and other hematopoietic disorders
Faiza Basheer, Clifford Liongue, Alister C. Ward
Deakin University, Geelong, Australia
Cytokines play a major role in regulating hematopoiesis and immunity. Perturbed cytokine signalling has been implicated in a variety of myeloproliferative disorders, including leukemia. Unravelling the molecular mechanisms involved in the pathogenesis of these hematological malignancies can provide major insights into their mechanism of action and contribute to the development of novel therapeutics. Indeed cytokine receptors and the associated tyrosine kinases are attractive targets for oncogenic therapy, with dysregulation often leading to constitutive activation that can be successfully inhibited by pharmacological agents. Zebrafish is well suited to model hematopoiesis and leukemogenesis, providing information into underlying genetic causes, as well as providing a platform for therapeutic testing. This project aims to generate mutants of relevant cytokine signalling components by genome editing to create zebrafish models of hematologic malignancies and other disorders. The first of these targets the jak3 gene, encoding a key tyrosine kinase downstream of the interleukin 2 receptor family.
32 Tmem2 regulates developmental Vegf-signaling by controlling Hyaluronic acid turnover
Jessica E De Angelis, Anne K Lagendijk, Huijun Chen, Alisha Tromp, Neil Bower, Carol Wicking, Mathias Francois, Jeroen Bakkers, Alpha Yap, Benjamin Hogan, Kelly Smith
Institute of Molecular Bioscience, QLD, Australia
Sprouting angiogenesis is responsible for the majority of tissue vascularization. We have identified a novel modulator of angiogenesis and Vegfa signaling in tmem2. Zebrafish tmem2 mutants have impaired angiogenesis, with reduced Vegf- dependent Erk-signaling. The C-terminal domains of Tmem2 function in an extracellular location and using a novel biosensor, we show that Tmem2 is needed to reduce perivascular levels of the space filling glycosaminoglycan Hyaluronic acid (HA). Importantly, degradation of HA with exogenous hyaluronidase dramatically rescues the angiogenesis defect in tmem2 mutants. Injection of enzymatically digested HA (o-HA) also rescues angiogenesis, revealing that stalled angiogenesis is not due to accumulation of macromolecular HA around vessels but rather that o-HA is permissive in angiogenesis. Finally, we show that o-HA regulates angiogenesis in a Vegf-receptor dependent manner, suggesting an extracellular role, enhancing Vegfa/Kdrl function. These findings identify a role for Tmem2 and the turnover of extracellular HA in vertebrate embryonic angiogenesis and Vegf-signaling.
33 Store-operated calcium entry (SOCE) is important for axon guidance in vivo
Thompson, Adrian, Gasperini, Robert, Young, Kaylene, Foa, Lisa
School of Medicine, University of Tasmania, TAS, Australia
Wiring of the nervous system relies on guidance of axons to their synaptic targets, a process regulated by neuronal growth cones. Calcium signaling is a major determinate of growth cone guidance. As such, deciphering the calcium regulatory mechanisms that control growth cone guidance is necessary to understand nervous system development.
Recent studies have identified store-operated calcium entry (SOCE) as a source of calcium during growth cone guidance. SOCE is triggered by depletion of calcium from the endoplasmic reticulum. A process regulated by the ER-localised calcium sensory protein stromal interacting molecule 1 (STIM1). We previously demonstrated that STIM1 expression is necessary for correct growth cone guidance in vitro. However there is limited evidence for the requirement of STIM1 and SOCE for axon guidance in vivo.
We used the zebrafish as an experimental model to investigate the importance of STIM1 and SOCE for axon guidance in vivo. We demonstrate ubiquitous expression of STIM1 during nervous system development, including developing motor neurons. We show the importance of STIM1 expression for axon guidance in vivo, with axon length at 24 hours post fertilization and the angle of axon outgrowth away from the spinal cord both significantly reduced in STIM1 morphants. Furthermore, we reveal the presence of SOCE in motor neurons in vitro, and demonstrate that reduced STIM1 expression perturbs the frequency of calcium transients during axon pathfinding in vivo. These findings demonstrate that STIM1 expression is important for correct axon pathfinding, and that STIM1 regulates calcium dynamics within pathfinding axons in vivo.
34 Anatomical and Functional Analysis of Tectal Afferents in Zebrafish
Lucy A Heap, Gilles C. Vanwalleghem, Andrew W. Thompson, Itia A. Favre-Bulle and Ethan K. Scott
School of Biomedical Sciences, The University of Queensland, QLD, Australia
The teleost optic tectum has been historically described only in terms of the visual system. In contrast to this, the mammalian equivalent of the optic tectum the superior colliculus, has been described as receiving input from a multitude of sensory systems. This study has used a combination of anatomical and functional experiments to describe three areas of the brain that do not receive direct retinal input (the thalamus, hypothalamus and cerebellum) that send efferent projections into the tectal neuropil. Optogenetic excitation of these regions revealed that the cerebellum sends purely excitatory output to the tectum, while the thalamus sends a combination of excitatory and inhibitory output, and the hypothalamus sends purely inhibitory output. This study provides evidence that the tectum of teleosts is not a solely visual structure, and hints towards it being more conserved with its mammalian counterpart than previously thought.
35 Exploring Vestibular Circuits in Zebrafish
Itia Favre-Bulle, Halina Rubinsztein-Dunlop, and Ethan Scott
School of Biomedical Sciences, The University of Queensland, QLD, Australia
The mechanisms by which the brain perceives and processes vestibular information are poorly understood. This is because popular approaches for observing brain activity (MRI, electrophysiology, and microscopy) are poorly suited to moving subjects. We have developed an experimental preparation in which we use an infrared laser to perform optical trapping on the otoliths of intact, behaving, immobilised zebrafish larvae. Using this preparation, we can apply forces to the otoliths, simulating physical acceleration of the animal, although it remains stationary. By tracking tail movements of head-embedded larvae in this preparation, we have begun to explore the relationship between forces placed on the otoliths and behavioural responses to the perceived vestibular stimuli. We find that trap strength correlates with the magnitude of postural changes in the tail, and that stronger traps also trigger forward swimming. We will now perform functional imaging using a genetically encoded calcium indicator and a house-build selective planar illumination microscope to map out the circuit dynamics that lie between our vestibular stimulation and the behaviours that it elicits.
36 Functional profiles of visual, auditory, and water flow responsive neurons in the zebrafish tectum
Andrew W. Thompson, Gilles C. Vanwalleghem, Lucy A. Heap, and Ethan K. Scott
School of Biomedical Sciences, The University of Queensland, QLD, Australia
The tectum has long been known as a hub of visual processing, and recent studies have elucidated many of the circuit-level mechanisms by which tectal neurons filter visual information. Here, we use population-scale imaging of tectal neurons expressing a genetically-encoded calcium indicator to characterize tectal responses to non-visual stimuli in zebrafish. We identify ensembles of neurons responsive to stimuli for each of three sensory modalities: vision, audition, and water flow sensation. These ensembles possess one of several consistently represented response profiles to our stimuli, each with a preferred stimulus and salient feature to which it is most responsive. Each sensory modality drives a unique spatial profile of activity in the tectal neuropil, suggesting that the neuropil’s laminar structure functionally subserves multiple modalities. The positions of the responsive neurons in the periventricular layer are also distinct across modalities, and very few neurons are responsive to multiple modalities. The cells contributing to each ensemble are highly variable from trial to trial, but ensembles contain “cores” of reliably responsive cells, suggesting a mechanism whereby they could both maintain consistency in reporting salient stimulus features, while retaining flexibility to report on similar stimuli.
37 Phenotypic identification of novel neuroprotective small molecules
Yuxi Zhang, Ellen Olzomer, David Nguyen, Daniel Hesselson
Garvan Institute of Medical Research, NSW, Australia
Despite intensive investigation the mechanisms underlying the loss of dopamine- producing neurons from the substantia nigra in Parkinson’s disease remain unclear hampering target based drug discovery. Mutations in the kinase Pink1 cause early- onset familial Parkinson’s disease and have been associated with defective mitochondrial quality control, a process that targets defective mitochondria for autophagic degradation (mitophagy). We generated Pink1-/- knockout zebrafish and developed a high throughput phenotypic screen for neuroprotective small molecules. We have identified several lead compounds that rescued mitochondrial function and prevented dopaminergic neuronal loss. These hits appear to function by stimulating Pink1-independent mitophagy suggesting that pharmacological activation of latent mitochondrial quality control pathways may be a viable therapeutic strategy.
38 Your face looks familiar: multimodal individual recognition in medaka fish and the individual difference in discrimination ability
Mu-Yun Wang and Hideaki Takeuchi
Okayama University, Tokyo, Japan
The ability to discriminate different conspecifics is the first step for many social interactions, such as mate choice, hierarchies and recognition of offsprings, nestmates, fellow groups and neighbours. Few studies have look at how fish use olfactory, acoustic and electric cues for individual recognition, and to the best of our knowledge, none of them have investigate the cognition of visual recognition and how different modalities work together. Medaka females prefer familiar males and decrease the latency to mate after familiarization. We found that both visual and olfactory cues were used for mate discrimination, but only visual cue is adequate for shorten the latency to mate. Fish showed individual differences in discrimination ability: once exchanged the familiar male to an unfamiliar one, some female can recognise the change and extend the latency to mate. Those females succeeded to condition one male with electric shock from another, while all females learned to condition electric shock with black or white colour signals. We found that fish used only appearance but not motion cue for individual recognition, and signals around the head might be important however, when we painted the male face with black ink after familiarisation, the female could still recognise the male. When flip the fish horizontal with prism during familiarisation, females recognised him as the same male, while when the image was upside-down the female failed to recognise the male. Medaka opens the opportunity to investigate the function of specific brain areas with cognitive abilities, which can benefit human studies.
39 Near and Far: The Cellular Response of the Adult Zebrafish Brain to Injury
Benjamin Lindsey, Viola Oorschot, Georg Ramm, Jan Kaslin
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The ability to discriminate different conspecifics is the first step for many social interactions, such as mate choice, hierarchies and recognition of offsprings, nestmates, fellow groups and neighbours. Few studies have look at how fish use olfactory, acoustic and electric cues for individual recognition, and to the best of our knowledge, none of them have investigate the cognition of visual recognition and how different modalities work together. Medaka females prefer familiar males and decrease the latency to mate after familiarization. We found that both visual and olfactory cues were used for mate discrimination, but only visual cue is adequate for shorten the latency to mate. Fish showed individual differences in discrimination ability: once exchanged the familiar male to an unfamiliar one, some female can recognise the change and extend the latency to mate. Those females succeeded to condition one male with electric shock from another, while all females learned to condition electric shock with black or white colour signals. We found that fish used only appearance but not motion cue for individual recognition, and signals around the head might be important however, when we painted the male face with black ink after familiarisation, the female could still recognise the male. When flip the fish horizontal with prism during familiarisation, females recognised him as the same male, while when the image was upside-down the female failed to recognise the male. Medaka opens the opportunity to investigate the function of specific brain areas with cognitive abilities, which can benefit human studies.
40 Myomesin-1 stabilises sarcomeric structure acting as a shock absorber in skeletal muscle
Mo Zhao, Caitlin Williams, Robert J Bryson- Richardson
School of Biological Sciences, Monash University, Clayton, VIC, Australia
Skeletal muscle consists of bundles of myofibrils that act in concert to generate movement. Each myofibril is composed of repeating contractile units, known as sarcomeres, which produce force by the sliding of the myosin-rich thick filaments along the actin-rich thin filaments, reducing sarcomere length. Thin filaments are anchored to the Z-disks that flank the sarcomere, whereas thick filaments anchor to the M-band at the centre of the sarcomere. In contrast to the extensive research into Z-disks and the identification of skeletal myopathies that result from their disruption, little is known about the M-band. Myomesins (MYOMs) are a family of M-band- restricted highly-elastic proteins that crosslink thick filaments with MYOM1, MYOM2, and MYOM3, each occupying distinct locations within the M-band. To investigate the roles of Myoms and the M-band, we utilized the zebrafish model system. Electron microscopic analysis showed the absence of Myom1, but not other Myoms, leads to contraction-dependent loss of the M-band, thick filament misalignment, and occasional Z-disk thickening, showing that Myom1/M-band is essential for sarcomeric integrity. We show that loss of Myom1/M-band does not affect force generation but significantly decreases swimming activity, suggesting the M-band may be important for force transmission. Our results demonstrate, for the first time, the role of Myomesins and the M-band in skeletal muscle and suggest the M-band acts as a shock absorber at the centre of the sarcomere that is crucial in maintaining the structural stability of the sarcomere.
41 Glycosylation in the Maintenance of Myotendinous Junction Integrity
A.J.Wood, C.H.Lin, V.Joshi, A.Siegel, L.B.Miles, S.Dudczig, P.R.Jusuf, N.Packer, P.D.Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The myotendinous junction (MTJ) is a critical structure for force transmission, during muscle contraction. MTJ strength is derived from a highly orchestrated deposition of components during development. The establishment of a fibronectin mesh around the somites provides the initial framework, for the main basement membrane structural components laminin and collagen. Using zinc fingers nucleases we have generated a complete loss of function mutant line for the putative glycosyltransferase fukutin related protein (FKRP). In the fkrp-/- fish we find a fibre crossing phenotype highly reminiscent of the natter mutant, a fibronectin null fish. Wholemount staining showed no observable loss of fibronectin in early somite boundary formtion. However, we did find a very significant loss of loss of fibronectin binding partner collagen at the basement membrane in 5dpf fish. To determine if the loss of collagen is due to a glycosylation defect in fibronectin, we employed negative ion mass spectroscopy analysis of fibronectin, pulled down from human cultured FKRP deficient patient cell lines. Finding that there are indeed sialyation and fucosylation changes in the N-glycans within the collagen-binding region of the fibronectin. Further analysis of collagen at 7dpf both on TEM and wholemount staining shows collagen does not form fibrils within the basement membranes of the fkrp-/- fish. Ultimately leading to fibre detachment at 7dpf. Our findings represent a novel mechanistic axis for FKRP deficiency related muscular dystrophy.
42 Zebrafish with truncating mutations in titin develop late-onset cardiac function defects.
Inken G Huttner, Louis W Wang, Celine Santiago, Scott Kesteven, Wolfgang Linke, Michael Feneley, Daniel Hesselson, Diane Fatkin
Victor Chang Cardiac Research Institute, NSW, Australia
Truncating mutations in the TTN gene, encoding the giant sarcomeric protein, titin, have been proposed to be the most common genetic cause of dilated cardiomyopathy (DCM), but whether these variants are sufficient alone to cause DCM or primarily modify disease susceptibility is unclear. To address this, we have generated zebrafish harboring truncating mutations in four different regions of zebrafish titin (ttna004, ttna103, herzschlag (heltg287) and ttna199). Analyses of embryonic heart and skeletal muscle function, as well as adult heart function were performed to assess phenotype onset and the influence of mutation position on phenotype severity. Using video microscopy, homozygous mutants embryos from all four lines showed severe cardiomyopathy, whereas only heltg287 and ttna199 showed skeletal muscle abnormalities and paralysis. Heart function in adult heterozygous mutation carriers was examined using the Visualsonics Vevo2100 high- frequency ultrasound system. Adult heterozygous ttna004 and ttna103 carriers were indistinguishable from wild-type siblings at 3 and 6 months of age, whereas heltg287 and ttna199 showed reduced contractile function (ejection fraction: WT, 37.8% +/- 5.4% vs. heltg287, 31.2% +/- 5.2% (p=0.001) WT, 36.1% +/- 4.1% vs. ttna199 30.9% +/- 2.6% (p=0.003)). Interestingly, contractile dysfunction in normal ttna103 carriers was unmasked after adrenalin stress. Evaluation of titin expression and localization using quantitative RT-PCR, Western Blot and immunohistochemistry is underway to determine whether truncated titin protein is produced and correctly localized, and whether titin levels or isoform ratios are differentially regulated. Our study describes the first zebrafish models of titin-related DCM with spontaneous left ventricular dysfunction in C-terminal truncation carriers.
43 Thin filament capping in nemaline myopathy
Hakan Tarakci, Joachim Berger, Peter D Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Skeletal muscle is the most abundant tissue in humans and makes up approximately 40% of our bodyweight. Due to the important roles that skeletal plays, diseases of skeletal muscle can therefore be debilitating for patients. Identifying the molecular and genetic basis of muscle diseases is crucial for the development of cures and treatments. From an ENU mutagenesis, the zebrafish mutant träge was identified by its reduction in birefringence, a measure of muscle damage. Following genetic mapping, this mutant was found to carry a nonsense mutation in tropomodulin-4, making tropomodulin-4 a novel candidate gene to screen for genetically unresolved nemaline myopathy. Interestingly, leiomodin-3 has recently been shown to cause nemaline myopathy in patients. Analysis of leiomodin3 zebrafish mutants, as well as generation of compound mutants, can be utilized to gain novel insight into the role of these thin filament capping proteins during de novo assembly of sarcomeres.
44 Investigation of Small Heat Shock Proteins in Muscle Disease
Emily Claire Baxter, Raquel Vaz, Tamar Sztal, Caitlin Williams, Robert Bryson- Richardson
School of Biological Sciences, Monash University, Clayton, VIC, Australia
Small Heat Shock Proteins (HSPBs) are molecular chaperones that target mis-folded and denatured proteins for repair or degradation. If left unchecked, mis-folded proteins can form insoluble protein masses (aggregates) that can draw other proteins, including native binding-partners, into the aggregate. Consequently, aggregate- prone proteins can disrupt cellular functions and contribute to disease states. Homologues of HSPBs have previously been described in zebrafish and we examined these to confirm that a subset have expression in skeletal and cardiac muscle. Given the role of HSPBs in protein quality control and their location in muscle, we hypothesise that they will be important as a direct cause, or as modifiers, of muscle disease. Analysis of HSPBs in several muscle disease models suggests that they can form part of a cellular response to pathology.
To further examine the function of HSPBs in muscle maintenance and disease we are utilising CRISPR/Cas9 genome editing technology to generate knock-out lines and characterising their phenotype. We are also utilising a modification of the CRISPR/Cas9 technology to regulate their transcription. We will use CRISPRi and CRISPRon in existing zebrafish muscle disease models and examine the ability of HSPBs to modify the phenotype of the models. These experiments will give us a better understanding of the role of HSPBs in muscle diseases as direct causes, and their potential to act as disease modifiers, both of which may form the basis of future therapies.
45 Zebrafish as a tool for targeted natural product drug discovery
Pei Jean Tan, Kazuhide Shaun Okuda, Mei Fong Ng, Norazwana Samat, Vithya Velaithan and Vyomesh Patel
Cancer Research Malaysia, Selangor, Malaysia
Natural products remain a remarkable rich source of bioactive agents that holds value for developing novel personalized anti-cancer therapies. However, despite representing a rich resource of bio-diverse drug candidates, only a hand-full have been found to be effective as targeted therapies largely due to the incompatibility of natural products to current high-throughput screening methods. As an alternative, we have developed and optimized a zebrafish developmental phenotypic assay to search for natural compounds with bioactivity that can target key conserved signaling pathways that are essential in zebrafish embryo development and causal in several human cancers. The resultant bioactive compound that able to perturb the normal developmental of zebrafish embryos can likely indicate an excellent opportunity for developing as promising cancer therapy with the potential targeted a dysregulated human cancer signaling pathway. This phenotypic assay is highly amenable for a high-throughput approach, whereby several hundred embryos from a single mating can be arrayed in a 96 well plate format and subsequently exposed to natural products prior to observations of developmental defects after 72 hours. As a result from our efforts to search for target specific anti-cancer compounds from our Malaysian bio-diverse resources, we have identified several prospective hits that can be potentially developed as cancer signaling pathway inhibitors. To this end, further validation in human cancer cell lines together with the mechanism of action are currently under investigation. Targeted anti-cancer drug discovery from natural products is made feasible by integrating the zebrafish embryo developmental phenotypic assay into our pipeline.
46 BiTS and pisces: generating a repertoire of cardiac cell type specific regulatory elements
Markus Tondl, Michael Eichenlaub, Lauren Bottrell, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Regulatory elements (REs; promoters, enhancers) are essential for the spatio- temporal control during development and hence as important as the genes they regulate. Although many genes involved in heart development have already been identified, little is known about the REs that control their expression in cardiac tissues. REs can be identified by chromatin marks, that is, post-translational modifications of histones. Data from chromatin immunoprecipitation followed by deep sequencing experiments (ChIP-seq) for some chromatin marks are publicly available for whole zebrafish larvae as well as whole mouse hearts. However, these datasets may not reflect the specific REs activate in specific cell types. In order to identify cardiac REs that are specific to cardiomyocytes, we established a protocol to isolate zebrafish cardiomyocytes by FACS and sequential ChIP-seq for histone modifications. Hence, we modified the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) [BONN et al., Nat Protoc, 2012]. Our preliminary data show that REs identified in cardiomyocytes regulate genes enriched in “heart development” functions which validates our data as a cardiac-specific repertoire. This is the first cardiomyocyte-specific regulatory repertoire in a developing heart. Our findings will not only be useful for cross-species comparisons to answer questions about heart evolution but will also be advantageous for clinical studies to identify ultra-conserved regulatory elements from human to fish as candidate regions for mutations in congenital heart diseases. Moreover, our dataset is a great resource for researchers to create cardiac-specific reporter lines.
47 Genome-wide identification of ncRNAs using a Bayesian segmentation approach
Manjula Algama, Caitlin Williams, Edward Tasker, Adam C. Parslow, Robert J. Bryson-Richardson, Jonathan M. Keith
Monash University, Clayton, VIC, Australia
Non-coding RNAs (ncRNAs) play crucial roles in transcriptional and post- transcriptional regulation. The identification of ncRNAs in genome sequences however, can be challenging as they lack the characteristic identifiers of protein coding-genes. In this study, we describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species, human, mouse and zebrafish. These elements include ncRNAs, or domains of ncRNAs, and regulatory sequences. We identified 655 such intronic sequences, which are significantly over-represented in the introns of transcription factors. We also performed a pathway-focussed analysis using a set of genes involved in muscle development, detecting 27 intronic elements in 7 transcription factors. The expression of 26 of these elements was experimentally validated using strand specific RT-PCR, providing confirmation that they include expressed ncRNAs. The identification of ncRNAs is a critical step in understanding the role of the transcripts in development and disease and this study demonstrates a novel, highly effective, Bayesian approach to identifying putative ncRNAs.
48 New methodology for in vivo visualisation of GFP-Labeled Proteins by Electron Microscopy
Thomas E. Hall, Nicholas Ariotti, Zherui Xiong, Harriet Lo, James Rae, Charles Ferguson, Kerrie-Ann McMahon, Nick Martel, Robyn E. Webb, Richard I. Webb, Rohan D Teasdale, Robert G. Parton
Institute for Molecular Bioscience, University of Queensland, QLD, Australia
Until recently, the only straightforward method for localizing proteins at the EM level was immunogold labelling. However, immunogold possesses significant limitations such as i) only a small pool of the protein of interest is labelled ii) non-specific background is common and inevitable iii) it is inherently dependent upon reliable cross-reacting antibodies; one of the long standing bugbears of zebrafish biologists. Horseradish peroxidase (HRP) conjugates, as well as gold, have been used by electron microscopists to produce a black precipitate upon exposure to the chemical DAB, but since HRP requires glycosylation it cannot be functionally expressed within cells. The recent development of a genetically encoded soybean peroxidase (APEX) which does not require glycosylation has allowed direct genetic tagging of proteins of interest in tissue culture. However, its utility in vivo has been limited until now because new, APEX-tagged transgenic lines must be made for each protein of interest. We have fused the APEX sequence with a small, genetically encoded, anti- GFP antibody known as a “GBP-nanobody”, and have generated both constitutive and heat-shock inducible transgenic zebrafish lines. Remarkably, APEX-GBP binds GFP with such high affinity that when an APEX-GBP line is crossed with a GFP-fusion line, in vivo protein localization can be examined by EM using a simple DAB reaction.
49 Evaluation of cardiac function in adult zebrafish using high frequency echocardiography.
Wang LW, Huttner IG, Santiago C, Kesteven S, Feneley MP, Fatkin D.
Department of Cardiology, St Vincent's Hospital, Victor Chang Cardiac Research Institute, NSW, Australia
Introduction: Zebrafish are popular for studies of heart function, with embryonic models predominantly used because optical transparency facilitates cardiac evaluation. Given that many human heart diseases are late in onset, adult zebrafish models are required. The recent development of high-frequency ultrasound technology allows the evaluation of adult zebrafish heart function, and we are rigorously studying the reliability of this method.
Methods: We developed a protocol to evaluate the performance and reliability of echocardiography in adult zebrafish underwater using the VisualSonics Vevo®2100 ultrasound system. We first compared cardiac function in three different wildtype strains at 3, 6 and 9 months of age (n=10-15 each). We compared the effect of tricaine and 2-phenoxy-ethanol on heart rate and contractile function, and evaluated the utility of adrenaline as a pharmacological stress agent. Lastly, we evaluated the ability of this method to detect changes in cardiac function due to chronic anaemia or toxic cardiomyopathy.
Results: There were no significant differences in ejection fraction due to gender or age. Anaesthesia with 0.02% tricaine (125±9.6bpm) provided a more physiological heart rate than 0.04% tricaine (89.2±13.1bpm) or 0.04% 2-phenoxy-ethanol (89.2±13.1bpm), P<0.001. Echocardiography detected changes in systolic function due to acute adrenaline exposure (EF 0.31±0.06 vs 0.35±0.04 at 100µM adrenaline, P<0.05), chronic anaemia (hyperdynamic volume overload model; EF 0.33±0.05 vs 0.44±0.08 post treatment, P=0.01), and induced toxic cardiomyopathy (EF 0.35±0.05 vs 0.17±0.06 post treatment, P<0.001).
Conclusion: Echocardiography allows serial cardiac function analysis in adult zebrafish, but study design needs to take into account the limitations of this imaging modality.
50 Defining the zebrafish skeletal muscle stem cell compartment during regeneration
N.D.D. Ratnayake and P.D. Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Regeneration is an important process to be carried out in order to recover following injury. Skeletal muscle possesses a robust regenerative capacity as demonstrated by the formation of myofibres following injury. In vivo time-lapse microscopy was utilised by our laboratory to observe regeneration in a newly defined muscle injury model. Our findings reveal that zebrafish skeletal muscle utilises a regeneration-specific stem cell source to recover from injury. Furthermore, we have discovered that this regeneration-specific stem cell compartment is marked by cmet, a tyrosine kinase receptor. The current study aims to functionally validate these cmet-expressing cells by examining the mechanisms of activation, self-renewal and stem cell interactions at the site of injury. Stem cell function will be validated via serial transplantations and zebrafish carrying null mutations in genes believed to regulate muscle stem cells will be utilised to dissect stem cell regulation. Manipulation of the resident fibres surrounding the site of injury at a genetic level will enable us to define new roles of resident fibres in directing stem cell function.
51 Glial derived regeneration of the outer retina: a new method
Brandli, A, Dudczig, S and Jusuf, P.
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Photoreceptors are light detecting retinal nerve cells, and loss of photoreceptors leads to blindness, which occurs in the majority of degenerative visual disorders. New photoreceptors can be produced by the transplantation of stem cells and progenitors, or by activating existing stem-cell-like (SCL) cells in the retina. In zebrafish, the formation of new photoreceptors is favoured by activation of glial cells that become SCL. In mammals, retinal glia can become activated following injury, but do not revert to a SCL state. Recently, adenosine triphosphate (ATP) has been shown to induce photoreceptor loss and potent activation of glia in mammals, but has not been tested in zebrafish.
This study aims to establish a model of photoreceptor damage and activation of retinal glia cells by using ATP. Validation of the ATP model in zebrafish will enable comparable studies of molecular signalling involved in regeneration between mammal and teleosts.
The effects of differing ATP concentrations were assessed in the zebrafish retina using markers for apoptosis (TUNEL) and proliferation (PCNA). 11 mM ATP injections caused an increase in TUNEL labelling, localised photoreceptor death and an increase in PCNA labelling in glial and photoreceptor layers. A study to confirm whether glia are the source of proliferation, and are producing new photoreceptors is currently being undertaken.
Thus, the photoreceptor toxin ATP also causes selective photoreceptor loss and regeneration in zebrafish. Further studies using this model will enable comparative cross-species studies to identify reprogramming genes and improve the production of glial-derived photoreceptors in mammals.
52 A pro-regenerative T cell subset promoting adult tissue regeneration
Subhra P Hui, Delicia Sheng, Kotaro Sugimoto, Kazu Kikuchi
Victor Chang Cardiac Research Institute, NSW, Australia
Zebrafish have a remarkable capacity to regenerate their various body parts in adulthood, providing a model to understand how regeneration capacity might be induced in humans. In this study, we investigated the mechanism of adult tissue regeneration regulated by immune cells, an aspect of restorative processes that has been poorly explored. Characterizing the immune cells in various regenerating tissues, we have identified a novel T cell subset in zebrafish, termed zTregs. After the injury, zTregs markedly infiltrated the damaged tissues and persisted throughout the regeneration period. To address the regenerative function of zTregs, we established a genetic model in which zTregs could be ablated conditionally. Using this model, we specifically depleted zTregs in several regeneration models, in which zTregs otherwise actively infiltrates in response to injury, and found a severe impairment in the regeneration of multiple tissues. We investigated the mechanism underlying this regeneration defect and found that zTregs provide tissue context- specific growth factors and directly stimulate tissue neogenesis in damaged organs. Our results reveal a novel pro-regenerative role of T cells during natural tissue regeneration in adult vertebrates, a finding that may have a significant therapeutic implication for future regenerative medicines.
53 Examining clonal drift of muscle stem cells during growth
P.D. Nguyen, C. Sonntag, L. Hersey, P.D. Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Organ growth requires a careful balance between cell commitment and stem cell self renewal to maintain tissue growth trajectories. While the processes that regulate resident stem cells during regeneration and disease have received much attention, the basis of stem cell deployment during organ growth remains poorly defined. This study characterized the mechanisms that control stem cell behaviours during skeletal muscle growth in zebrafish. We identifed a lifelong stem cell pool that exhibits extensive clonal drift, shifting from the random deployment of a large population of stem cells during larval growth, to the reliance on a small number of dominant stem cell clones to fuel adult muscle growth. We further reveal that self renewal and clonal drift of growth specific muscle stem cells requires the activity of the homeobox gene meox1. This study defines a distinct mechanism of regulation for stem cells required for organ growth and in the process provides the first molecular understanding of the mechanisms underlying clonal drift in vivo.
54 The role of HMGA proteins in Regulating Neural Stem Cell Activity in vivo
Daniel Colquhoun, Benjamin Lindsey, Kit Tang, Jan Kaslin
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Increased cell plasticity is hallmark of many regenerating tissues, for example, during cellular de-differentiation to a stem or progenitor-like state or activation of quiescent stem cells. The underlying genetic networks controlling cell plasticity in vivo are not well known. We have recently identified the High Mobility Group A proteins (HMGA) as key candidates for neural stem cell regulation and regeneration in zebrafish. They are relatively small and have motifs that enable binding to DNA and proteins, allowing them to regulate gene expression via direct binding to promoters and through opening of transcriptionally dormant chromatin. We explored the expression profiles of HMGA1 proteins during development and found that HMGA1 proteins are highly expressed during development but rapidly declines and in adults expression is restricted to stem cell populations. To determine the function of this protein in vivo, a mutant line was generated which displays a pygmy phenotype and reduced regenerative potential. Gene expression studies carried out in mutant regenerates have shown that HMGA1 regulates components of the Insulin-like Growth Factor (IGF) signalling pathway, a seminal pathway in regulating organismal growth as well as neural stem cell activity. More-over, Chip-PCR experiments show that HMGA1 binds to the promoters of these genes. Herein we present data that shows that HMGA1 regulates stem cells throughout development and regeneration by regulating the expression of multiple stem cell associated genes as well IGF signalling components.
55 Investigating of the Pathobiology of Myofibrillar Myopathies and Potential Therapies using Zebrafish
Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Robert J. Bryson-Richardson
School of Biological Sciences, Monash University, Clayton, VIC, Australia
Myofibrillar myopathies exhibit progressive muscle weakness and are characterized by structural failure of the muscle and the formation of cytoplasmic protein aggregates. All the proteins known to be mutated in myofibrillar myopathies localize to the Z-disk where they have a diverse range of functions. Two such proteins are FLNC, an actin binding protein, and BAG3, a co-chaperone involved in regulating autophagic degradation of Z-disk proteins. To understand how dominant mutations in these two functionally different Z-disk proteins results in a highly consistent MFM pathology we examined the effect of either knocking down zebrafish BAG3 or FLNC or ectopically expressing MFM causing variants of both genes. Utilizing the advantages of the zebrafish model system we demonstrate that whilst expression of mutant protein results in protein aggregation, it is the loss of functional protein that results in fibre disintegration. We identify a common mechanism of disease whereby protein insufficiency due to sequestration and subsequent myofibrillar disintegration cause muscle weakness in MFM. Furthermore, using a combination of knockdown experiments and drug treatments, we investigate the role of autophagy in promoting protein aggregation and examine the upregulation of autophagy as a potential therapy for MFM. We will present this data, that combined with detailed characterization by immunolabelling and confocal and electron microscopy, has allowed us to determine the mechanistic basis of disease and suggest potential therapeutic approaches.
56 Investigating novel non-oncogene targets for cancer therapies
Kimberly Morgan, Karen Doggett, Lachlan Whitehead, Stephen Mieruszynski & Joan K. Heath
Development & Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic.
The activation of an oncogenic transformation can result in secondary stresses on cells to sustain the higher mitotic and metabolic demands of a tumour. This can make them more dependent on fundamental processes for survival. This dependency on non oncogenes can be exploited to provide novel therapeutic targets to selectively kill malignant cells without perturbing healthy cells in the process of synthetic lethality.
Using a doxycycline inducible zebrafish hepatocellular carcinoma (HCC) model, we identified synthetic lethal interactions between krasG12V and three of our endoderm ENU mutants. Heterozygous mutations in genes participating in the fundamental cellular processes of U12-type splicing, ribosome biogenesis and nuclear pore formation, namely: rnpc3, nol8 and ahctf1, respectively, specifically decreased HCC growth whilst not impacting on normal liver growth. Since heterozygous mutations do not effect normal development, this creates the promising opportunity to develop novel targeted therapies to improve existing cancer treatments.
57 A zebrafish model of the MND linked c9orf72 hexanucleotide repeat expansion.
Emily K Don, Serene Gwee, Sharron Chow, Ian Blair, Julie Atkin, Garth Nicholson, Nicholas J Cole
Macquarie University, NSW, Australia
Recently, the most common genetic cause of ALS was identified as a hexanucleotide (GGGGCC) repeat expansion in a non-coding region of the gene C9ORF72. While healthy individuals have 2-23 repeats, ALS patients can have up to 700-1600 repeats. This repeat expansion accounts for 20-80% of familiar and 5-15% of sporadic ALS in North American and European populations. However, it is currently unknown how this repeat expansion leads to the loss of motor neurons and the development of MND. As the repetitive nature of the C9ORF72 hexanucleotide repeat expansion causes inherent difficulties when generating animal models, the high-throughput capabilities and quick turnaround time of the zebrafish makes the zebrafish an ideal system in which to test several different approaches to model the MND linked C9ORF72 hexanucleotide repeat expansion. We therefore plan to make several different stable transgenic zebrafish lines of the MND linked C9ORF72 hexanucleotide repeat expansion in order to examine the mechanisms by which the repeat expansion causes MND. We will generate zebrafish expressing short or long repeat expansions either ubiquitously or specifically in the motor neurons, in addition to generating lines which allow for inducible expression of the repeat associated proteins. It is hoped that the flexibility of this approach will generate a clinically relevant model of the MND linked C9ORF72 hexanucleotide repeat expansion to allow for observation and characterisation of disease progression at a molecular and behavioural level.
58 To what extent do zebrafish models of impaired ribosomal biogenesis recapitulate human ribosomopathies?
Karen Doggett1, Andrew P Badrock2, Yeliz Boglev1, Tanya A de Jong-Curtain1,2, M. Cristina Keightley3, Laure Lam Hung4, Derek Stemple4, Dan Fraher5, Yann Gibert5, Graham J Lieschke3 and Joan K Heath1,2
1) Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 2) Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, VIC 3) Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 4) Wellcome Trust Sanger Institute, Hinxton, UK; 5) School of Medicine, Deakin University, Geelong campus, VIC
Multiple human syndromes have been linked to abnormal ribosome biogenesis (ribosomopathies), and several predispose to cancer, including Dyskeratosis Congenita and Diamond-Blackfan Anaemia. Tp53-mediated cell cycle arrest and/or apoptosis have been suggested as potential mechanisms linking abnormal ribosome biogenesis to the congenital abnormalities associated with human ribosomopathies. The zebrafish mutant seteboss453, mutated in nucleolar protein 8 (nol8), is impaired in the development of many tissues that develop abnormally in ribosomopathies, including the craniofacial cartilages, exocrine pancreas, eye and brain. Yeast and mammalian orthologues of zebrafish Nol8 are required for processing of ribosomal RNA (rRNA).
Northern blot analysis demonstrates that setebos mutants exhibit defective rRNA processing that preferentially impairs 28S biogenesis and the formation of the 60S ribosomal subunit. In response to this nucleolar stress, Tp53 is activated in setebos, resulting in elevated transcription of several pro-cell cycle arrest genes, including p21, mdm2, cyclinG1 and Δ113p53, encoding an N-terminally truncated isoform of Tp53. WISH analysis of Δ113p53 mRNA demonstrates that Tp53 is activated as early as 21 hours post fertilization (hpf) in setebos, specifically in regions of the embryo associated with high proliferative activity in wild-type. To gain molecular insights into the events that are triggered by abnormal ribosome biogenesis in setebos, we conducted genome-wide mRNA expression analysis using DETCT (differential expression transcript counting technique). Ingenuity Pathway Analysis revealed disruption to multiple gene networks, including several encoding proteins involved in mitochondrial function. To examine this further, we measured mitochondrial bioenergetics with a Seahorse extracellular flux analyser and found that oxygen consumption and extracellular acidification rates were significantly reduced in setebos compared to wild-type. We propose that setebos provides an ideal in vivo model to interrogate the mechanisms underlying the abnormalities that arise in ribosomopathies.
59 miR-mediated knockdown to recapitulate Spinal Muscular Atrophy
Giacomotto Jean, Mowry Bryan, Becker Thomas
Psychiatric Genomics Queensland Brain Institute. The University of Queensland, QLD, Australia
Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. We recently investigated the efficiency of transgenic miRNA- mediated knockdown and showed that this system is a valuable tool to manipulate gene function in zebrafish.
Using this approach, we reproduced Spinal Muscular Atrophy (SMA) in zebrafish by targeting the smn1 gene. We generated different transgenic lines, with severity and age of onset correlated to the level of smn-1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish.
These new models open new avenues to study this disease as for example using early onset animals (severe form) to set up simple drug screening experiments.
One important question that remains unsolved about SMA is why ubiquitous low levels of SMN affects specifically the neuromuscular system despite its function being critical for the viability of all cells? To help responding to this question, we also took advantage of this new system to test the effect of motor neuron or muscle-specific smn1-silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function, and premature death. Interestingly, smn1-knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1-silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish.
It is noteworthy that this research is going beyond SMA as this versatile gene- silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism.
60 Generation of novel zebrafish models of amyotrophic lateral sclerosis.
Alison L. Hogan, Emily K. Don, Serene Gwee, Isabel Formella, Kelly L.Williams, Garth A. Nicholson, Nicholas J.Cole, Ian P.Blair
Faculty of Medicine and Health Sciences, Macquarie University, NSW, Australia
Amyotrophic lateral sclerosis (ALS) is a late-onset, rapidly progressive and fatal neurodegenerative disease. The pathophysiology of ALS is poorly understood and effective therapeutics are yet to be identified. Animal models are essential for studies into disease biology and for preclinical testing of potential therapeutics. However, established models of ALS fail to adequately reflect disease pathology and there has been repeated failure of translation from animal models to clinical trials.
Our laboratory is developing ALS models based on mutations in novel ALS genes. Altered protein homeostasis has been identified as a major pathological pathway in ALS. Several recently identified ALS molecules, including ubiquilin 2 and and an E3 ubiquitin ligase, play a role in protein degradation pathways. These provide an opportunity to develop novel zebrafish models (Danio rerio) and assess the pathological role of dysfunctional protein homeostasis in ALS. The phenotypic accuracy of each model will be established and the most suitable used in functional studies and preliminary trials of potential therapeutics.
We initially used the Tol2 transgenic system to overexpress a human E3 ubiquitin ligase or the zebrafish orthologue either ubiquitously or specifically within motor neurons from fertilisation. However, overexpression of both the mutant and wildtype genes proved toxic in embryos, impeding further development of the models. A doxycycline inducible system is now being trialled, which will allow delayed overexpression of the transgene, potentially overcoming the toxicity observed during development. This approach also has the potential to better reflect adult-onset diseases, such as ALS.
61 An in vivo study of the localization and function of ALS-associated protein, TDP- 43 in transgenic Zebrafish
Jack J. Stoddart, Marco Morsch, Isabel Formella, Andrew P. Badrock, Emily K. Don, E., Roger S. Chung, Nicholas J. Cole
MND Research Centre, Macquarie University, NSW, Australia
Amyotrophic lateral sclerosis (ALS) is a progressive muscle wasting disorder, characterized by the degeneration of upper and lower motor neurons. There is currently no cure and the disease is fatal 3-5 years post symptom onset. An improved understanding of ALS at a cellular level is essential in order to identify disease pathways, leading to motor dysfunction. Zebrafish (Danio rerio) have emerged as a unique model organism to study the cellular and molecular mechanisms of disease in vivo and in real-time, as well as possible resulting musculoskeletal phenotypes. The DNA/RNA binding protein, Transactive Response DNA-binding protein 43kDa (TDP-43) is a component of ubiquitinated cytoplasmic inclusions, present in around 97% of ALS cases. A number of causative mutations in the TARDBP gene have also been identified in both familial and sporadic ALS patients. We have developed a stable transgenic Zebrafish specifically expressing the Zebrafish ortholog of TDP-43 (zTDP-43) in motor neurons. In this study, overexpression (OE) of a conserved mutation associated with sporadic ALS, Q331K (Tg zTDP-43Q331K), was compared to OE of wild-type TDP-43 (Tg zTDP-43WT) and non-transgenic controls (TAB WT). We conducted cellular analysis of these transgenic Zebrafish, assessing the localization of zTDP-43 in motor neurons, as well as examining motor phenotypes. Tg zTDP-43Q331K larvae showed increased cytoplasmic mislocalization compared to Tg zTDP-43WT and reduced locomotion under oxidative stress when compared to Tg zTDP-43WT and TAB WT controls. This suggests a difference in functionality between TDP-43WT and sporadic- associated TDP-43Q331K at the cellular level, resulting in a musculoskeletal phenotype. Further elucidating this link may better our understanding of ALS and take a step toward unveiling the complex neurobiology underlying the disease.
62 Circadian regulation of the innate immune cell response following infection
Lucia Du, Lisa Lawrence, James Cheeseman, Guy Warman, Kathy Crosier, Phil Crosier, Chris Hall
School of Medical Sciences, University of Auckland, Auckland, New Zealand
Circadian regulation of the immune system enables organisms to anticipate daily changes in the environment that are associated with the risk of infection and host- tissue damage. This is driven at the molecular level by a complex network of clock proteins that include Period, Bmal and Clock. Circadian disruption significantly alters the immune response through mechanisms that are poorly understood. By taking advantage of the live imaging potential of the zebrafish model, we can investigate circadian-regulated pathways that underlie the innate immune cell response to infection.
A circadian model was established by conditioning larvae to a 14-hour light/10-hour dark (LD) cycle. This was validated by expression analysis of the clock-controlled genes, period2 (per2) and arylalkylamine N-acetyltransferase2 (aanat2), and a locomotor assay. Expression of per2 and aanat2 were restricted to the light and dark phases respectively, while the larval circadian period under the LD cycle was closely aligned to the 24-hour cycle. These results indicate a robust light-entrained circadian rhythm. Larvae reared under LD and constant light conditions displayed greater neutrophil recruitment and a higher survival rate following infection with Salmonella enterica serovar Typhimurium when compared with larvae maintained under constant darkness. These results show that, as in mammalian systems, the larval zebrafish response to bacterial infection is circadian-dependent. We are currently characterising mutant knockouts of two core clock genes, per2 and clock1, that were generated with CRISPR-Cas9. These mutants will provide further insight into the role of clock genes in the innate immune cell response.
63 Understanding the role of cytokine receptor like factor 3 (crlf3) in zebrafish
Tarannum Taznin, Yann Gibert, Clifford Liongue, Alister C. Ward
Deakin University, Geelong, VIC, Australia
Cytokine receptor-like factor 3 (CRLF3) is a protein that consists of almost entirely a classical cytokine homology domain (CHD) with unknown function. Bioinformatic analysis revealed that a single CRLF3 homologue is present in early Placozoa (Trichoplax adhaerens), and likely represents the precursor of the CHD found in all cytokine receptors. Zebrafish possess a single crlf3 gene expressed during embryogenesis. Transcripts of crlf3 gene were first evident in the adaxial mesoderm and later in the anterior and posterior lateral plate mesoderm and anteriorly in the third brain ventricle, retina and thymus. To study the role of the crlf3 gene in embryogenesis TALEN and CRISPR-based genome editing approaches have been designed to target the gene. This has generated several potential knockout alleles for further analysis.
64 Features of a good meal: Microglia engulfment in the spinal cord
Marco Morsch, Rowan Radford, Jack Stoddart, Emily Don, Serene Gwee, Isabel Formella, Andrew Badrock, Thomas Hall, Nicholas Cole, Roger Chung
Faculty of Medicine and Health Sciences, Macquarie University, NSW, Australia
Microglia are the resident macrophages of the CNS and play crucial roles in mediating immune-related functions. Microglia patrol the entire vertebrate nervous system, where they can detect the presence of apoptotic and damaged neurons, and consequently engulf these cells to minimize the spread of neuronal debris. This microglial activity requires fast-acting communication between the two cell types, such that microglia are primed for rapid response to a variety of stimuli (such as dying neurons). However, many of the fundamental mechanisms that regulate the detection of injured neurons and subsequent microglial activation during phagocytosis still remain elusive. Understanding these processes is critical as microglial clearance is symptomatic for many neurodegenerative diseases including motor neuron disease, where glial activation has been shown to contribute to the death of motor neurons. We describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance and will further advance our knowledge of microglia activation and homeostasis.
65 Leukocyte/pathogen interactions during fungal infection establishment
Vahid Pazhakh, Felix Ellett, Stefan Greulich, Alex Andrianopoulos, Graham Lieschke
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
When microbial pathogens breach host epithelial barriers, the immediate host innate immune response is critical in determining the outcome of the infective challenge. Optical transparency and proven utility in infection modelling make zebrafish embryos an excellent model for studying host/pathogen interactions in vivo. We have examined two opportunistic fungal pathogens (Penicillium marneffei, Aspergillus fumigatus) in this model. As fungal spores are a large particle, leukocyte/pathogen interactions can be studied in one-on-one detail.
P. marneffei spores are preferentially phagocytosed by macrophages and neutrophils are fungicidal in a myeloperoxidase-dependent manner. In contrast to previous reports, we observed A. fumigatus spores phagocytosis predominantly by neutrophils.
High-resolution intravital imaging of leukocyte/spore interactions revealed a previously undescribed phenomenon: neutrophil-to-macrophage spore shuttling between living leukocytes during infection establishment. Shuttling is directed from neutrophils towards both naive and spore-laden macrophages. Multiple observations collected from >20 examples impose morphological constraints on the molecular mechanisms involved in shuttling. As both P. marneffei and A. fumigatus spores are shuttled, it is not pathogen-specific. To examine shuttling further and help determine its mechanism and purpose, we are trying to develop an in vitro system for studying it and for monitoring individual spore viability, which has presented experimental challenges.
Whether shuttling is a pathogen-driven mechanism evolved to facilitate their transfer to a more hospitable leukocyte or a mechanism evolved by the immune system to transfer the pathogen to an antigen-presenting cell is not yet established, but either scenario is significant for understanding the process of infection establishment.
66 Inflammation orchestrate neural regeneration in the spinal cord
Celia Vandestadt, Daniel Colquhoun, Timo Friedrich, Jan Kaslin
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
In mammals spinal cord injury results in devastating loss of function and lifelong disability. Zebrafish have a remarkable capacity to regenerate their central nervous system (CNS) following injury. The regenerative response of CNS cell types following injury is beginning to be unravelled, however, the contribution of immune cells and inflammation in mediating the regenerative response is not yet fully understood. Here we have developed a larval spinal cord lesion assay within which we can study CNS regeneration at the cellular level with confocal imaging in vivo. Our results showed that in agreement with other injury models, glial regeneration precedes the reestablishment of neural cell types. We showed that glial bridging occurs prior to vascular regeneration and, with mutant experiments, showed that the vasculature played redundant role in early spinal cord regeneration. Macrophages and neutrophils are initially rapidly recruited following injury followed by a return to homeostasis. Cellular ablation experiments demonstrated that spinal cord regeneration did not require leukocyte infiltration, while mutant experiments suggest a functional acute immune response is required for complete neuronal regeneration. Furthermore, we found that immune suppression by glucocorticoid treatment reduced regeneration in wildtype larvae. Collectively we have demonstrated that inflammatory factors relating to the acute immune response are important for the regenerative response following spinal cord injury in larval zebrafish.
67 High-resolution mapping the microanatomy of the zebrafish gastro digestive system by mean of correlative and combined X-ray, light and electron microscopy.
Delfine Cheng, Gerry Shami, Marco Morsch, Roger Chung, Filip Braet
The University of Sydney, NSW, Australia
The present work aims to fill in the missing histological pieces of the Zebrafish (ZF) Gastro Digestive System (GDS) at high-resolution using correlative and combined x- ray, light and electron microscopy. To-date, the ZF model has been employed in a diverse range of diseases research such as neural defects, development disorders and cancer. However, the ZF is still not well studied in the fields of gastroenterology and hepatology, to-date, mainly due to the limited literature available on ZF GDS subcellular structure-function properties.
In order to allow combined and correlative imaging across different microscopy platforms, a workflow/specific sample preparation protocol was designed. A series of combinatorial heavy metal staining agents were used to improve preservation and contrast. Following micro-CT scanning, serial sections throughout the entire ZF were then cut, collected using array tomography and stained. Histological stains were used to identify different tissue types under LM, which could then be correlated with back- scattered scanning EM or TEM for high-resolution ultrastructural details.
3D models of the ZF GDS were generated from micro-CT and LM. In addition, EM data provided 2D/3D information such as total surface area and volume, of sub- cellular structures, which can be compared to more familiar models (rodents). In particular, the exchange sites within the different digestive organs (e.g. brush border and hepatocyte sinusoids) and circulatory systems were analysed in more details.
The knowledge gathered assesses the extent to which the ZF can be used as a gastrointestinal experimental model for future drug delivery studies in cancer and adverse lipid transport.
68 Functional characterization and drug screening on zebrafish muscular dystrophy models
M. Li, M. Andersson-Lendahl, T. Sejersen, A. Arner
Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia.
Muscular dystrophy is a heterogeneous group of inherited disorders manifested by progressive skeletal muscle wasting and degeneration. Mutations in dystrophin and laminin α2 gene are the genetic causes for Duchenne muscular dystrophy (DMD) and Merosin-deficient congenital muscular dystrophy type 1A (MDC1A). Sapje and Candyfloss zebrafish are null in dystrophin and laminin α2-chain respectively, resulting in severe dystrophic phenotype. They display several of the structural characteristics of the DMD and MDC1A in human. However, the functional impairment in these mutants remains to be determined. We have established muscle physiology techniques, enabling high resolution of contractile function in zebrafish larvae. We aim to provide mechanical characterization of Sapje and Candyfloss larval muscle and examine effects of potential therapeutic drugs for muscular dystrophy. We found that the Sapje and Candyfloss mutant larvae exhibited contractile defects with 50 % decrease in active tension generation at 4dpf, and with about 60% decay in force at 6dpf, revealing the progressive nature of the disease. Ataluren (PTC124), a small molecular compound, is proposed to cause read-through of premature stop-codons, and has been introduced as a potential treatment for DMD. We performed the ataluren treatment on Sapje mutant larvae, and found that the contractile function was significantly improved with a maximum of about 60 % increase of force at optimal dose. We thus conclude that both Sapje and Candyfloss mutant larvae are functionally relevant models for muscular dystrophy, and can be utilized for drug screening with high resolution.
69 Creating an Inducible Zebrafish Model of TDP-43 Mislocalisation
E. Hortle, E. Don, S. Chow, J. Stoddart, G. Nicholson, N. Cole
Macquarie University, NSW, Australia
Amyotrophic lateral sclerosis (ALS), is a fatal neurodegenerative disease which affects an increasing number of Australians every year. Treatment is limited, and in many cases the cause of disease is unknown. However, recent studies show that almost all cases of ALS share a common neuropathology characterized by the mislocalistation of TAR-DNA binding protein (TDP-43) to the cytoplasm of cells, and the deposition of TDP-43-positive protein inclusions. Here we aim to gain further insight into the mechanisms behind ALS, by creating a zebrafish model of this pathology. We have created transgenic fish that ubiquitously express an inducible form of either WT human TDP-43, or human TDP-43 with a mutated nuclear localisation sequence, which causes the protein to be mislocalised to the cytoplasm. Further development of this model will allow us to study the ways in which TDP-43 mislocalisation is involved in neurodegeneration, and may provide an invaluable model for testing future ALS therapeutics.
70 Telling TALEs to Recode Zebrafish Genome to generate novel ALS models
Yagiz Alp Aksoy1, 2, Daniel Hesselson2 and Nicholas Cole1
1 Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia 2 Garvan Institute of Medical Research, Sydney, NSW 2010, Australia
As next-generation sequencing costs continue to fall, whole-genome and exome- genome sequencing and genome-wide association studies are generating more candidate human disease genes than ever. However, the function and role of these genes in the human disease remain to be discovered. Model organisms are crucial for efficient and robust investigation of candidate human disease genes in vivo. Zebrafish provides a powerful model system for investigating these genes however inability to alter the genome with ease and accuracy has been a major challenge. Genome editing tools such as CRISPR-Cas9 and TALENs have recently become available allowing introduction of heritable and precise sequence modifications in the genome. Both of these tools induce highly precise, locus-specific double strand breaks in the genome to generate multiple target-specific gene knock-ins and knock-outs in vivo. These exciting genome editing tools redefine the boundaries of the medical research by allowing scientists to generate various human disease models in model organisms much more quickly than before. We can now, investigate the functional role of individual genes much faster and readily alter multiple genes in cells at once to reveal their interactions. Here we discuss the application of these powerful genome editing tools to generate amyotrophic lateral sclerosis (ALS) models in Zebrafish.
71 Protein cleavage and motor dysfunction in a transgenic zebrafish model of spinocerebellar ataxia-3
Maxinne Watchon, Kristy Yuan, Nick Mackovski, Thomas Becker, Garth Nicholson and Angela Laird
ANZAC Research Institute, Concord Repatriation Hospital, Sydney, NSW, Australia
Spinocerebellar ataxin-3 (SCA-3) is a fatal hereditary neurodegenerative disease affecting neurons of the brain and spinal cord, disrupting the ability to control movement of muscles. MJD is caused by a long trinucleotide (CAG) repeat region within the gene ATXN3. This CAG repeat region encodes a poly-glutamine (Q) tract containing greater than forty glutamines, compared to 1Q-40Q in healthy subjects. Characterisation of our transgenic SCA-3 zebrafish has revealed that they develop progressive motor dysfunction, and a sensitive behavioural test (escape response during darkness) detected slower swimming speeds in ataxin-Q84 zebrafish than ataxin-Q19 at 6dpf. We have also found through immunoblot analysis that protein lysates extracted from our transgenic zebrafish contain ataxin-3-positive cleavage fragments. Similar sized cleavage fragments (50kDa and 30kDa) have been identified in human SCA-3 patient samples. Here we have investigated whether cleavage of ataxin-3 protein is important in the development of disease phenotypes such as the motor dysfunction found in our transgenic zebrafish. We found that treating our zebrafish with the calpain inhibitors calpeptin and MDL28170 prevents formation of the ataxin-3 fragments and also improves the motor function of the SCA-3 zebrafish. However we also found that treatment with these inhibitors results in removal of the full-length ataxin-3, suggesting that protein quality control pathways (e.g. autophagy) may be induced by the treatment. Further investigation is underway to determine whether the beneficial effect of calpain inhibition on the SCA-3 zebrafish is preventing the formation of ataxin-3 cleavage fragments or clearance of the full- length protein by a protein quality control pathway.
72 Myosin Vb-mediated endosomal trafficking of N-cadherin is required for heart chamber form and function
D. Grassini, J. De Angelis, S. Capon, S. Paterson, G. Baillie, C. Simons, R. Taft, B. Hogan and K. Smith
IMB / University of Queensland, QLD, Australia
The heart is a complex organ made up of different tissues and compartments that ensure it functions correctly. The different components are shaped and “assembled” early in development in a process called cardiac morphogenesis, during which finely regulated cellular and genetic interactions occur. To identify new genes important for cardiac morphogenesis, we have performed a forward genetic screen in the zebrafish and isolated the 60-1 mutant. The 60-1 mutant has small, dismorphic heart chambers that beat poorly. This is accompanied by a characteristic epithelial defect, whereby the cells round up and are extruded from the epithelium. Closer analysis of the cardiac phenotype revealed small, round cells in mutant ventricles as opposed to enlarged, elongated cells in the siblings. F-actin staining shows this is mislocalized in mutant cells, suggesting that the cytoskeleton fails to undergo the remodeling necessary for correct morphogenesis.
We mapped the 60-1 mutation to a region on chromosome 21, containing the myosin Vb (myoVb) gene. A mutant for myoVb has previously been described that also manifests the characteristic epithelial phenotype, although no description of the heart was reported. MyoVb is an atypical myosin, involved in the transport of recycling endosomes back to the plasma membrane. To try to understand how disruption of the vesicular trafficking causes specific heart defects, we performed genetic interaction experiments between the 60-1 and a range of cardiac mutants with analogous phenotypes. Excitingly, we found a genetic interaction with the n- cadherin/glass-onion mutant, by which compound mutants have a phenotype distinct from either single mutant phenotype alone. Finally, we have promising preliminary data, using a mutant form of n-cadherin lacking the internalization signal, which suggests that when N-cadherin persists at the plasma membrane, it can rescue the 60-1 phenotype. These data lead us to a model whereby, in the heart, N-cadherin is a MyoVb cargo-protein and diminution of MyoVb activity results in decreased N- cadherin at the plasma membrane and, therefore, compromised cell-cell adhesion and ability of the actin cytoskeleton to properly rearrange during morphogenesis.
73 In vivo function of the chaperonin TRiC/CCT complex
Joachim Berger, Silke Berger, Mei Li, Patricia R. Jusuf, Arie S. Jacoby, Anders Arner and Peter D. Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
To enable the evolution into multi-cellular organisms, eukaryotic cells developed amongst other features the cytoskeleton that mainly consists of actin filaments and tubulin-containing microtubules. But actin and tubulin have also other crucial functions, including muscle contraction, chromosome segregation, and extension of neuronal axons. Biogenesis of actin and tubulin is enabled by chaperonin (TRiC) that is considered as absolutely required for the folding of these proteins (Llorca et al. 1999 Munoz et al. 2011, Lopez et al. 2015). On the other hand, Anfinsen showed that the amino acid sequence of proteins contain all the information necessary for their folding into native conformation (Anfinsen 1973). Whereas TRiC has extensively been studied in vitro, its in vivo analysis is hampered due to the scarcity of animals carrying mutations in the cct genes that encode for the TRiC subunits and null mutations in yeast resulting in a lethal phenotype (Stoldt et al. 1996).
We will present our analysis of zebrafish mutants with various TRiC mutations that confirms the folding of tubulin and skeletal actin by TRiC in vivo. However, novel insights into the in vivo function and significance of TRiC and its role in sarcomere assembly will be presented.
74 Grainyhead-like 3 (Grhl3) regulates gastrulation through its role in formation and maintenance of the EVL
L. Miles, K. Doggett, J. Kaslin, J. K. Heath, S. M. Jane, S. Dworkin
Monash University, Prahran, VIC, Australia
The highly conserved grainyhead-like (grhl) transcription factor family regulate numerous processes in animals, particularly formation and maintenance of epithelial barriers. Recently, we have shown that family member grhl3 is strongly activated during early embryogenesis in zebrafish, during the time point where the embryo begins to synthesise its' own RNA, a period termed the maternal-zygotic transition. Both loss-of function and gain-of-function experiments highlight a critical role for grhl3 during gastrulation, and our recent work suggests that this is due to the role played by grhl3 in the formation, maintenance and growth of the outermost (enveloping) layer of the embryo (the EVL). Our current focus is to delineate the precise cellular and molecular mechanisms regulated by grhl3 in this context.
75 The implications of BPA usage on developmental obesity
Prusothman Yoganantharajah, Alister Ward, Yann Gibert
Deakin University School of Medicine, Geelong, VIC, Australia
Bisphenol A (BPA) is a chemical compound commonly found in polycarbonate plastics. It has been banned by many countries, especially in the production of baby products, due to potential metabolic effects but no it in Australia. Currently, the involvement of BPA in lipid deposition and adipogenesis during early development is unknown. We investigated if BPA exposure at physiological doses could alter lipid deposition or composition in zebrafish embryos. We also determined the effect of BPA on the expression of the lipidogenic and adipogenic markers lipoprotein lipase, CCAT/enhancer-binding protein alpha and liver fatty acid binding protein. The quantity of lipid deposition was observed via Oil-Red-O staining (ORO), a stain that specifically binds to neutral lipids and triglycerides, which was quantified using a new technique we developed based on optical density. We demonstrated that BPA exposure led to a 2-fold increase in ORO staining. To measure the changes in lipid species, lipidomics analysis using tandem mass spectrometry was performed enabling the analysis of changes across 24 lipid classes, with significant changes observed following BPA exposure. BPA also caused a significant increase in expression of lipidogenic and adipogenic markers. Since BPA exerts a substantial effect on lipid deposition and adipogenesis during embryogenesis, we believe its use in Australia should be reconsidered.
76 ZEBRAFISH – POSTER ABSTRACTS
Function of melanopsin in mechanosensation
Marcus P.S. Dekens and Kristin Tessmar-Raible
University of Vienna, Austria
What was previously known? Hair cells, the primary receptors of auditory and lateral line sensory systems, are neuroepithelial cells with protruding bundles of stereocilia that are deflected by pressure. Displacement of these stereocilia stretches linkages at their tips, thereby gating mechanotransduction channels and inducing electrical currents. The molecular details underlying this transduction process are only partially understood. A recent Drosophila screen for auditory organ genes revealed mechanical amplification by Johnston's neurons to be severely impaired by mutations in rhodopsin, demonstrating its requirement for mechanotransduction. Rhodopsin is a pigment in the photoreceptor cells of the fruit fly'ss retina that is responsible for the first step in the perception of light. We discovered two melanopsins, photoreceptors known for their function in photosensitive retinal ganglion cells, to be expressed in the mechanosensory hair cells of the zebrafish lateral line. The expression of orthologous opsins in invertebrate and vertebrate mechanosensory cells strongly implies that zebrafish melanopsin plays a role in vertebrate mechanosensation.
What is the conceptual advance? The direct use of the force provided by a mechanical stimulus in opening a transducer channel is considered to be the hallmark of the mechanical senses. In contrast, light cannot exert pressure and thus the light sensory system is thought to rely on indirect second messenger dependent transducers. Recently, however Drosophila rhodopsin has been shown to induce a mechanical force on the cell membrane upon activation. To date photoreceptors have not been implied in vertebrate mechanosensation or its modulation. I aim to reveal the role of melanopsins in zebrafish hair cells, and have for this purpose generated mutants by reverse genetics. Melanopsin could either have a direct role in mechanotransduction, which is light independent, or modulate the mechanosensory signal in a light dependent manner. These hypotheses will be tested by analysis of the melanopsin mutant on the molecular, electrophysiological and behavioural levels.
What is the significance of this study for the advancement of science? 1) This study breaks down the molecular and conceptual boundaries between the mechanical and non- mechanical senses. 2) This study will lead to a conceptual advance in our understanding of vertebrate mechanosensation, which is so far not associated with a light receptive molecule. 3) This study will provide substantial new mechanistic insight into the function of melanopsins.
77 Characterisation of Nr4a2a during neurogenesis of the zebrafish retina
S. Dudczig, L. Goodings, P. Nguyen, P.D. Currie, P.R. Jusuf
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Nr4a2 plays an integral role during dopaminergic neurons (DA) development and maintenance in multiple central nervous system (CNS) regions. In mice, Nr4a2 loss leads to the absence of DA neurons in specific CNS regions. In zebrafish two paralogues exists, however nr4a2a is closer in expression, phylogeny and sequence to the mammalian gene compared to nr4a2b.
This study examined the role of Nr4a2a through morpholino knockdown and newly generated CRISPR mutants in modulating DA neuron generation using the retina as a simplified model of the CNS.
We found that DA neurons loss following nr4a2a knockdown was due to a neuron conversion to form a non-dopaminergic neuron type, as cell death was not observed. Moreover, we used CRISPR/Cas9 technology to generate a number of germline transmitting alleles for nr4a2a that resulted in a truncation of the protein. Using the DA neuron marker tyrosine hydroxylase (TH), we found an absence of DA neurons in the retina. Additionally, these mutants are homozygous viable and are now undergoing a full spectrum of phenotypic characterisation with a focus on the subtype changes we observed using the transient morpholino.
This study has established the re-specification of neurons at the expense of DA neurons following loss of Nr4a2a. Future work will focus on the functional consequences the retina may adopt with this neuron conversion. Furthermore, this study reveals a potential strategy to generate de novo DA neurons as a neuron replacement therapy in diseases with DA neuron loss by targeting converted neurons with Nr4a2.
78 Examination of asph function in zebrafish embryonic development
Quoc Tran, Ivana Mirkovic, Peter Currie
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
Hedgehog (HH) signalling has been intensively studied due to its important role in the development. HH proteins (Hh) are secreted morphogens although they are strongly hydrophobic due to post-translational lipid modifications. The underlying molecular mechanisms of HH serection are not completely understood.
As part of an ongoing research in our group, which focuses on the role of Scube in HH secretion, we describe the zebrafish orthologue of asph, one of the Scube- interacting proteins discovered in our biochemical screen. We hypothesise that ASPH has a role in HH secretion by facilitating interactions between Scube2 and other factors involved in HH secretion. Here we describe expression patterns of two asph genes in zebrafish embryos. This second zebrafish asph orthologue has not been described previously. Also, we examined whether asph expression is regulated by HH signalling in mammalian cell culture and in the zebrafish embryo. As previously described, asph/C971 (the fully annotated asph gene) is expressed in a pattern that partially overlaps with domains of HH expression. The gene that is more similar to the mammalian asph, the asph/X7 appears to be expressed specifically in the HH-receiving site, adaxial cells. Our results suggest that asph/C971 expression is sensitive to HH signalling activity. In cell culture, HH signalling appears to influence levels of ASPH protein. In conclusion, my results allow a possibility that ASPH function in HH signalling in zebrafish and contribute to the model that HH is secreted as soluble monomer, without lipid residues.
79 Behavioral alterations in medaka caused by serotonin deficiency in the raphe neurons
Satoshi Ansai, Hiroshi Hosokawa, Shingo Maegawa, Youhei Washio, Kenji Sato, Masato Kinoshita
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Japan
Serotonin (5-hydroxytryptamine 5-HT) is a bioactive monoamine that acts as a neurotransmitter in the central nervous system as well as a hormone in the peripheral organ systems. Teleost fish species have serotonergic neurons in the raphe nuclei of the brainstem, which project to a number of brain regions similar to mammals however, the role of the raphe serotonergic neurons in behaviors of teleost fishes remains largely unknown. Therefore, we generated a medaka mutant that was disrupted tph2 gene, encoding the tryptophan hydroxylase involved in the 5-HT synthesis in the raphe, using targeted mutagenesis using transcription activator-like effector nucleases (TALENs). At first, we showed that the mutant fish were deficient in 5-HT of serotonergic neurons in the raphe nuclei by immunohistochemical and mass spectrometric analysis. Subsequently, the behavioral alterations in the mutant fish were assessed using five behavioral paradigms (diving, open-field, light-dark transition, mirror approaching, and social interaction). The tph2 mutant fish exhibited decreased locomotor activity and reduced the number of entries to the top area in the diving test. The tph2 mutant female also showed a decreased mirror biting time in the mirror biting test and an increased contact number in social interaction test. In addition, the tph2 mutant fish exhibited longer duration of freezing for the first several minutes of each test in all examined paradigms. These results indicate that central serotonergic systems will modulate anxiety/fear responses and social behaviors in medaka.
80 Temporal and spatially resolved in vivo proteomics to find novel interactors of GFP tagged proteins.
Zherui Xiong, Robert G. Parton, Thomas E. Hall
Institute for Molecular Bioscience, University of Queensland, QLD, Australia
An engineered ascorbate peroxidase, APEX, has been developed for EM and proximity labelling in vitro. In the presence of hydrogen peroxide, APEX catalyses oxidation of biotin-phenol to generate a short-lived radical, which tags proteins proximal to APEX, allowing the proteins closest to the genetically labelled protein to be identified by mass spectrometry. We are pioneering methodology to apply APEX labelling in zebrafish for in vivo proteomics. By genetically fusing the APEX enzyme with a protein of interest in zebrafish, we will identify potential interactors of the tagged protein. In order for the APEX labelling system to be effective in zebrafish, biotin-phenol must permeate through the epidermis and spread into the tissue. Furthermore, the biotin-phenol must be accessible to a short burst of hydrogen peroxide to provide temporal control of proximity labelling. We have generated a transgenic zebrafish ubiquitously expressing the APEX enzyme. We have tested a variety of proximity labelling conditions and evaluated the efficiency of in vivo biotinylation using streptavidin labelling. Our results indicate that APEX-mediated proximity labelling is promising tool for in vivo proteomics in zebrafish.
81 In silico investigation of the evolution of a cardiac-specific repertoire of regulatory elements
Mark Drvodelic, Michael Eichenlaub, Markus Tondl and Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The heart is an ancient organ which has undergone significant changes during vertebrate evolution. It has evolved from a simple tube in invertebrates to a pump with two chambers in fish, three in amphibians and four in birds and mammals. Explaining this increase in complexity is hindered because the genes that drive cardiac development are extremely well-conserved across these species and hence during evolution. Consequently, we hypothesise that structural changes in the heart result from changes in expression of cardiac genes, which are triggered by changes in the cis-regulation of these genes.
Initially, we obtained the cardiac repertoire of cis-regulatory elements in zebrafish by identifying heart-specific regulatory regions genome-wide using tissue-specific chromatin immunoprecipitation followed by deep-sequencing. This provides an extensive atlas of regulatory regions controlling gene expression in heart development of the zebrafish; a widely used model organism for the study of cardiogenesis, but whose cardiac regulatory landscape has been left mostly unexplored.
Secondly, we will assess sequence conservation of these regulatory regions in evolution and perform cross-species comparisons of cardiac cis-regulatory elements between teleosts (zebrafish, medaka, stickleback), birds (chicken) and mammals (mouse, marmoset and human). We expect to identify regulatory regions that are either conserved, gained or lost during heart evolution and investigate changes in their composition of binding sites for cardiac regulators.
By deriving the cis-regulatory landscape driving zebrafish cardiogenesis; an essential missing link into the understanding of heart evolution, and then further comparing the resulting regulatory regions with other species, we hope to determine the cis- regulatory variations responsible for formation of the complex organ that is, the heart.
82 Further investigating the role of Elk1 during cardiogenesis
Jeannette C. Hallab, Romaric Bouveret, Daniel Hesselson, Richard .P. Harvey, Mirana Ramialison
Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia
The imperative role of transcription factors (TFs) in the temporal and spatial regulation of gene expression and ultimate tissue patterning and organogenesis is well established. Nevertheless, the genetic regulation of cardiogenesis remains poorly understood and only a handful of TFs are known to play an essential role in the process. Recent evidence suggests that the ETS Class I transcription factor, Elk1, which has not previously been implicated in heart development, may in fact, be important in the context of cardiogenesis. Morpholino studies targeting elk1 in zebrafish embryos have implied perturbations in the expression of genes known to be important in patterning in the anterior lateral plate mesoderm and normal cardiogenesis, and heart looping defects. The present study utilizes TALEN- modified zebrafish in an attempt to further investiage the morphant phenotype using more robust gene editing technology. We have identified four mutant Elk1 alleles among these fish and are currently investigating possible phenotype in the F4 generation for a mutant carrying a large (543bp) genomic deletion between exon and intron 2. Preliminary results indicate high mortality within 24 hpf among the F4 (homozygous) offpspring of homozygous F3s parents compared to the F4 offspring generated by INX of WT and heterozygous F3 parents. We plan to interrogate this putative, mutation-dependent lethality on a molecular level, firstly by determining the status of Elk1 protein using western blot. We then hope to acquire a deeper understanding of the RNA status of Elk1 itself and possible responses of the cardiac gene regulatory network to Elk1 defect in mutants and morphants using QPCR. Overall, we hope to gain a deeper understanding of the possible role of Elk1 during embryogenesis and the molecular mechanisms underlying the observed phenotypes and the similarities or differences between the mutant and morphant scenarios.
83 Elucidating the Biology of Motor Neurone Disease/Frontotemporal Dementia
Nicholas Cole, Emily Don, Marco Morsch, Jack Stoddart, Yagiz Alp Aksoy, Rowan Radford, Alison Hogan, Jennifer Fifita, Isabel Formella, Andrew Badrock, Dasha Sayal, Gilles Guillemin, Julie Atkin, Ian Blair, Roger Chung, Garth Nicholson.
Macquarie University, NSW, Australia
Our approach is to use zebrafish, an established research model organism, to generate animal models that develop features of ALS/MND/FTD in order to understand the biology of the disease. Zebrafish offer some advantages for human disease research that compliment mammalian models. Transgenic technologies allow us to express known ALS/MND causing human genes in the fish in vivo whilst at the same time observing biology in the living organism in real time. In addition the zebrafish provides many elegant tools in order for us to investigate the biology of MND/FTD. An overview of our zebrafish projects and research strategy is presented.
84 DELEGATE LIST
LAST NAME FIRST NAME EMAIL ALP AKSOY Yagiz [email protected] ANSAI Satoshi [email protected] ASTIN Jonathan [email protected] BASHEER Faiza [email protected] BAXTER Emily Claire [email protected] BERGER Joachim [email protected] BOWER Neil [email protected] BRANDLI Alice [email protected] BRYSON-RICHARDSON Robert [email protected] CHAUDHURY Smrita [email protected] CHENG Delfine [email protected] COLE Nicholas [email protected] COLQUHOUN Daniel [email protected] CURRIE Peter [email protected] DANG Louis [email protected] DE ANGELIS Jessica [email protected] DEGUCHI Tomonori [email protected] DEKENS Marcus [email protected] DOGGETT Karen [email protected] DON Emily [email protected] DRVODELIC Mark [email protected] DU Lucia [email protected] DUDCZIG Stefanie [email protected] DWORKIN Seb [email protected] EICHENLAUB Michael [email protected] FENYES fruzsina [email protected] GIACOMOTTO Jean [email protected] GIBERT Yann [email protected] GRASSINI Daniela [email protected] GRIMM Lin [email protected] HALL Chris [email protected] HALL Thomas [email protected] HALLAB Jeannette [email protected] HEAP Lucy [email protected] HEATH Joan [email protected] HERSEY Lucy [email protected] HESSELSON Dan [email protected] HOGAN Alison [email protected] HOGAN Ben [email protected] HORTLE Elinor [email protected] HUI Subhra [email protected] HUTTNER Inken [email protected] JUSUF Patricia [email protected] KASLIN Jan [email protected] KEIGHTLEY Cristina [email protected]
85 LAIRD Angela [email protected] LEE Brendan [email protected] LI Mei [email protected] LIESCHKE Graham [email protected] LINDSEY Benjamin [email protected] LOOSLI Felix [email protected] MIERUSZYNSKI Stephen [email protected] MORGAN Kimberley [email protected] MORSCH Marco [email protected] MOWRY Bryan [email protected] NARUSE Kiyoshi [email protected] NGUYEN Phong [email protected] PATTON Elizabeth [email protected] PAZHAKH Vahid [email protected] RAMIALISON Mirana [email protected] RATNAYAKE Nilakshi [email protected] RUPARELIA Avnika [email protected] SANTIAGO Celine [email protected] SCHARTL Manfred [email protected] SCOTT Ethan [email protected] SERRANO Rita [email protected] SONNTAG Carmen [email protected] STODDART Jack [email protected] TAKEDA* Hiroyuki [email protected] TAKEUCHI Hideaki [email protected] TAN Pei Jean [email protected] TAN Nathalia [email protected] TARAKCI Hakan [email protected] TAZNIN Tarannum [email protected] THOMPSON Adrian [email protected] THOMPSON Andrew [email protected] TONDL Markus [email protected] TRAN Quoc [email protected] VANDESTADT Celia [email protected] WANG Mu-Yun [email protected] WANG** Louis [email protected] WARD Alister [email protected] WATANABE Reina [email protected] WILLIAMS Caitlin [email protected] WITTBRODT Jochen [email protected] WOOD Alasdair [email protected] YOGANANTHARAJAH Prusothman [email protected] YOKOI Saori [email protected] ZHANG Yuxi [email protected] ZHAO Mo [email protected]
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