Abstract Book Oral Abstracts O-02 Subfunctionalization of cohesin components in The tumor microenvironment plays a critical role in zebrafish cell fate determination melanoma progression after its initial establishment. While relatively little is known about the identity of cells in the Sarada Ketharnathan1, Judith Marsman2, Jisha Antony1, 1 1 1 melanoma microenvironment, existing studies suggest Bridget Mackie , Amarni Thomas , Julia Horsfield it is primarily composed of keratinocytes, fibroblasts, 1 Department of Pathology, Dunedin School of Medicine, adipocytes, endothelial cells and immune cells. Our University of Otago, New Zealand 2Department of Surgical previous work has shown that keratinocytes and adipocytes Sciences, Dunedin School of Medicine, University of Otago, in the tumor microenvironment can supply critical factors New Zealand to melanoma cells. However, it is not known if melanoma cells themselves are capable of transferring biomolecules The cohesin complex was first known for roles in sister like protein/RNA to the microenvironment. Melanocytes, chromatin cohesion and DNA damage repair. Over the last from which melanomas arise, are highly secretory cells 15 years, new functions have been described for cohesin and studies have shown that specialized organelles called in gene expression and genome organization. Cohesin, melanosomes (which contain melanin) as well as exosomes in combination with transcription factors, or with the can be transferred from healthy melanocytes to neighboring insulator protein CCCTC-binding factor (CTCF), forms part stromal cells like keratinocytes. The functional consequence of a chromatin landscape that helps select cell type-specific of such transfer remains unknown. Using a zebrafish model genes for expression. Germline mutations in genes encoding of melanoma, we have engineered a Cre/Lox based donor/ cohesin subunits or cohesin regulators underlie a spectrum recipient system to identify the stromal cells that receive of human developmental disorders, the “cohesinopathies”, cargo from the melanoma cells. Zebrafish donor melanoma whereas somatic mutations in cohesin are frequently found cells expressing a Cre transgene were transplanted into in cancers, most notably, in acute myeloid leukemia (AML). recipient fish that ubiquitously express a GFP to mCherry We previously showed that zebrafish rad21 mutants lack “switch” cassette. Any cell which takes up Cre from the runx1 expression in the posterior lateral mesoderm in early donor cell will be permanently fate mapped and express somitogenesis, and we recently used this phenotype as an mCherry throughout the life of the animal. Using this assay for functionality of leukemicRAD21 mutations. system, we have found that the tumor cells can transfer mRNA into neighboring keratinocytes. RNA-seq and ATAC- Vertebrate cohesin comprises 4 core subunits: Smc1a, seq analysis of the recipient keratinocytes identifyMYCN as Smc3, Rad21 and either Stag1 or Stag2. In zebrafish, each the master regulator of this phenotype. Our studies thus stag gene is duplicated, giving rise to Stag1a and 1b, identify an unknown mechanism of microenvironment Stag2a and 2b. It is generally assumed that the cohesin reprogramming by melanoma cells which could aid both complex operates as a whole unit, and that mutation in tumor growth and metastasis. any one cohesin gene manifests through ablating the function of the entire complex. Our research in zebrafish Zebrafish provide an ideal system to study in vivo tumor shows that mutations in independent cohesin subunits and microenvironment crosstalk in melanoma due to the have distinct phenotypes. Individual germline or mosaic available genetic tools, ease of imaging and established Crispr/cas9-generated mutants of stag1a, 1b, 2a or 2b cell culture models. Ongoing work aims to delineate the did not phenocopy rad21 mutation, but rather, generated underlying mechanisms of this reprogramming and to alternative hematopoietic and/or novel developmental understand its clinical relevance. phenotypes. The hematopoietic phenotype included primitive myeloid skewing and downregulation of definitive runx1 expression, while developmental phenotypes O-04 Live-imaging coupled with single-cell RNA included generation of ectopic structures and mislocation sequencing identify zebrafish melanocyte stem cell- of cell types in the developing tail. derived subpopulations in development. Our results imply that individual cohesin subunits can Alessandro Brombin1, 2, Daniel J. Simpson1, Jana have independent functions in zebrafish development, Travnickova1, 2, Tamir Chandra1, E. Elizabeth Patton1, 2 and support observations in humans that distinct cohesin mutations have different consequences for cancers such as 1MRC Human Genetics Unit, MRC Institute of Genetics and AML, and in human development. Molecular Medicine, University of Edinburgh, Western general Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom, 2CRUK Edinburgh Centre, MRC Institute of O-03 Reprogramming of the microenvironment by Genetics and Molecular Medicine, University of Edinburgh, melanoma-derived cytoplasmic transfer Western general Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom Mohita M Tagore1, Richard M White1 Melanocytes develop either directly from the neural 1Cancer Biology and Genetics Program, Memorial Sloan- crest or via ERB-dependent melanocyte stem cells (MSCs) Kettering Cancer Center, New York, NY, 10021, United located at the dorsal root ganglia (DRG). During post- States embryonic development or melanocyte regeneration, the MSCs generate melanoblasts to form the adult stripes......

..... 1 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts Our goal is to understand the molecular mechanisms that formation using datasets, human tissue arrays, in vitro define MSC establishment, quiescence and activation. First, knockdowns, and transgenic zebrafish lines. High PRDM1 we used time-lapse imaging of live zebrafish expressing expression in melanoma patients is correlated with better Tg(crestin:mCherry; mitfa:GFP) that label the neural patient survival. In addition, whenPRDM1 is knocked down crest and the specified melanocyte lineage. We found in melanoma cell lines, there is an increase in cell growth crestin expressing cells at the DRG site, suggesting that a and colony formation. We used a stable transgenic zebrafish subpopulation of mitfa-expressing cells maintain neural line, Tg(mitfa:BRAFV600E);p53-/-, and found that when one crest identity in the stem cell niche. These cells are ERB- copy of prdm1a was mutated, melanoma onset occurred dependent and we observed that they produce a limited more quickly and the resulting tumors were more invasive. number of migrating melanoblasts before reducing crestin We then analyzed expression of a downstream target and and mitfa expression and become quiescent. Next, we well-established melanoma marker, SOX10, and found in performed single-cell RNA-seq (scRNA-seq; 10x Genomics) the human and zebrafish data, when PRDM1 expression is on 24hpf Tg(crestin:mCherry; mitfa:GFP) embryos with low, SOX10 is correspondingly high. Thus, the mechanism and without Erb inhibitor. We find crestin:mCherry by which PRDM1 functions as a tumor suppressor in and mitfa:GFP label subpopulations of neural crest melanoma is likely through regulation ofSOX10 expression. (characterised by the expression of either foxd3 or twist1a), xanthoblasts and melanoblasts. By building lineage trees, we discovered a small cell population that we defined as O-06 Nanoparticle-mediated low Z radiotherapy as a the MSCs. ERB inhibitor treatment leads to the loss of MSCs, potential treatment paradigm for radiosensitive cancers and surprisingly we found that these cells prematurely differentiate along the melanocyte lineage. Unexpectedly, Olivia Piccolo1, Nicole Melong 2, John D. Lincoln 3, we found that melanocytes derived from the MSC are Nicholas R. Fernandez 4, Michael Ha 5, Jason N. Berman transcriptionally distinct from the ones developing directly 3,6,7, James L. Robar 3,5,8 from the neural crest. Our work identifies molecular 1 Department of Biology, Dalhousie University, Halifax, signatures that define stem cell-derived subpopulations in 2 the melanocyte lineage and provides new opportunities for Nova Scotia, Canada, Department of Pediatrics, IWK Health Centre/Dalhousie University, Halifax, Nova Scotia, studying the MSC lineage in regeneration, adult tissues and 3 disease. Canada, Department of Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada,4 Department of Chemistry and Biochemistry, Mount Allison University, Sackville, New Brunswick, Canada,5 Department O-05 Loss of prdm1a accelerates melanoma onset and of Radiation Oncology, Dalhousie University, Halifax, Nova progression by regulating the melanocyte stem cell factor Scotia, Canada,6 Departments of Pathology, Dalhousie sox10 University, Halifax, Nova Scotia, Canada,7 Department 1, * 1,2, *, of Microbiology and Immunology, Dalhousie University, Ritsuko Iwanaga , Brittany T. Truong Rajesh Vyas6, 8 David Orlicky3, Yiqun G. Shellman4, Aik-Choon Halifax, Nova Scotia, Canada, Department of Medical Physics, Nova Scotia Health Authority, Halifax, Nova Scotia, Tan 5, Craig Ceol6 and Kristin Bruk Artinger1 Canada 1Department of Craniofacial Biology, University of Colorado Radiation is an effective treatment modality forthe Anschutz Medical Campus, Aurora, CO, USA,2Human management of cancer, but standard clinical radiation Medical Genetics & Genomics Graduate Program, often causes collateral damage to normal tissue in centrally University of Colorado Anschutz Medical,3Campus, Aurora, located tumours (e.g. pancreas) or tumours in close CO, USA 3Department of Pathology, University of Colorado proximity to important structures (e.g. head and neck). Anschutz Medical, 4Campus, Aurora, CO, USA 4Department Irradiation of gold and gadolinium nanoparticles (GNPs and of Dermatology, University of Colorado Anschutz Medical, GdNPs) produces bursts of photons via the photoelectric 5Campus, Aurora, CO, USA 5Department of Medicine, effect, which can sensitize tumour cells to clinical linear University of Colorado Anschutz Medical Campus, Aurora, accelerator (linac) generated radiation. We have developed CO, USA, 6Program in Molecular Medicine, University of a novel linac by replacing the standard copper/tungsten Massachusetts Medical School, Worcester, MA, USA target photon source with a sintered diamond target, producing a low Z beam. Computer models predict the *These two are considered joint first authors. diamond target to yield 400% more low energy photons, Melanoma is an aggressive and deadly skin cancer that resulting in a 7.7 fold dose enhancement in NP vicinity. The develops from melanocytes, a neural crest cell derivative. zebrafish platform has proven to be ideally suited for the Melanoma cells and neural crest cells share similar gene transplantation of human cancer cells and will be employed expression, behaviors, and cell mechanisms. Using cross- as a preclinical model to validate predicted effects. species oncogenomics, we identified genes recurrently Initial studies were performed in Panc1 (pancreatic deleted in both human and zebrafish melanomas, which adenocarcinoma) and FaDu (hypopharyngeal carcinoma) includes PRDM1/prdm1a, a neural crest developmental cell lines. Using transmission electron microscopy (TEM), regulator. We investigated its role in melanoma tumor cell uptake of GNPs was found to be cell specific with ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 2 Oral Abstracts increased retention in lysosomes in FaDu cells compared to mediate melanoma cell survival despite BCL2 inhibition. to Panc1 cells. Increased levels of reactive oxygen species As in the zebrafish model, the three-drug combination of (ROS) were observed in FaDu cells compared to Panc1 cells rapamycin, venetoclax and S63485 synergistically killed using flow cytometry. In vitro viability assays demonstrate these human melanoma cells. Our findings identify rapid decreased cell viability in low-Z beam irradiated FaDu upregulation of MCL1 as a mechanism of resistance to BCL2 cells in the presence of GNPs and GdNPs. NP labeled cells inhibitors in preclinical models, and the combination of were injected into casper zebrafish larvae and irradiated venetoclax and S63485 may effectively mediate tumor cell with the standard or low Z beam, then cell proliferation death in many molecular subtypes of human melanoma. was measured by live-cell fluorescence microscopy and quantified ex vivo. NP-labelled injected FaDu cells display decreased in vivo proliferation when irradiated with the low O-08 CRISPR lineage tracing in the zebrafish heart and Z beam. Increased cell death in GNP-labelled FaDu cells with brain low Z radiotherapy is consistent with higher intracellular ROS levels and may be attributable to improved retention Kimberly T. Truong1,2, Bushra Raj3, Aaron McKenna4, Jay of GNPs. Shendure4, Alexander F. Schier3, James P. Keener1, James A. Gagnon2,5 1 Department of Mathematics, University of Utah, Salt Lake O-07 Zebrafish models to optimize melanoma cell death City, UT 84112,2 Henry Eyring Center for Cell and Genome during therapy Sciences, University of Utah, Salt Lake City, UT 84112,3 1 1 1 Department of Molecular and, Cellular Biology, Harvard Shuning He , Hillary M. Layden , Mark W. Zimmerman , 4 1 1 1 University, Cambridge, MA, USA Department of Genome Julia Etchin , Alla Berezovskaya , Chang-Bin Jing , Grace 5 Thurston1, Megan W. Martel1, Ellen van Rooijen2, Charles Sciences, University of Washington, Seattle, WA, USA, K. Kaufman2, Scott J. Rodig3, Leonard I. Zon2, E. Elizabeth School of Biological Sciences, University of Utah, Salt Lake Patton4, Marc R. Mansour1,5, and A. Thomas Look1 City, UT 84112 1 Department of Pediatric Oncology, Dana-Farber Cancer How do embryonic cells contribute to growing organs such Institute, Harvard Medical School, Boston MA, 02115, as the heart or brain? The cell lineage tree is a structure USA, 2 Stem Cell Program and Division of Hematology/ that describes these relationships, but we lack adequate Oncology, Children’s Hospital Boston, Howard Hughes tools to record this tree in developing animals. Our strategy Medical Institute, Boston, MA, 02115, USA,3 Department for tracing the cell lineage tree uses CRISPR genome editing of Pathology, Brigham and Women’s Hospital, Boston, MA, to progressively accumulate mutations in a DNA barcode. 02115, USA, 4 MRC Human Genetics Unit, MRC Institute of These barcodes can be expressed and recovered along Genetics and Molecular Medicine, University of Edinburgh, with the transcriptome using single cell RNA sequencing. Crewe Road South, Edinburgh, EH4 2XR, UK,5 Department We have adapted tools from phylogenetics to reconstruct of Hematology, UCL Cancer Institute, University College branching trees that describe the lineage relationships London, WC1E 6BT, UK between tens of thousands of cells sampled from single animals. I will focus on our recent applications of this New targeted therapies are urgently needed for human method to understand the contributions of embryonic cells melanomas with PTEN mutations and loss of the NF1 to tissues and organs in the adult zebrafish. First, we have tumor suppressor or NRAS mutations. We have used a measured and modeled cardiomyocyte clonality within faithful zebrafish model to identify new drug combinations the developing heart, finding that a few embryonic clones that are active in melanomas with these mutant genes. dominate the population of this and other organs. Second, Surprisingly, inhibitors of MEK or PI3K showed little activity we have explored lineage relationships within the growing when given alone against these tumor cells, due to cross- zebrafish brain. We discovered prolonged contributions talk between the pathways. By contrast, inhibition of mTOR from regionally restricted pools of progenitor cells to with rapamycin induced autophagy with growth delay but distinct sets of cell types, including subsets of neurons no evidence of apoptosis. To convert growth delay to and oligodendrocytes. These approaches will enable new definitive melanoma cell death, we tested the BCL2 inhibitor studies of cell fate determination, stem cell population venetoclax in combination with rapamycin. Results with this dynamics and developmental noise. combination were identical to those with rapamycin alone. We found that venetoclax resistance was due to adaptation with rapid upregulation of MCL1, a transcriptionally labile O-09 Metformin rescues muscle function in preclinical pro-survival BCL2 family member. The addition of the MCL1 BAG3 myofibrillar myopathy models inhibitor S63485 to venetoclax and rapamycin induced extensive melanoma cell apoptosis without toxicity to Avnika A. Ruparelia1, Emily A. McKaige1, Caitlin Williams1, normal zebrafish tissues, leading to a dramatic increase in Keith E. Schulze2, Margit Fuchs3,4, Viola Oorschot5, survival of the tumor-bearing zebrafish. Studies in PTEN- Emmanuelle Lacene6, Meregalli Mirella7, Emily C. Baxter1, deficient human melanoma cell lines with either NF1-loss Yvan Torrente7, Georg Ramm5,8, Tanya Stojkovic9, Josée N. or mutant NRAS showed that MCL1 is rapidly upregulated Lavoie3,4,10, and Robert J. Bryson-Richardson1 ......

..... 3 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts 1 School of Biological Sciences, Monash University, Center, Department of Medicine, Section of Nephrology Melbourne 3800, Australia,2 Monash Micro Imaging, and Section of Endocrinology, Leiden, the Netherlands Monash University, Melbourne 3800, Australia,3 Centre de Recherche sur le Cancer de l’Université Laval, Ville * These authors contributed equally 4 de Québec, Quebec, Canada, Oncologie, Centre de The ability to enrich for different cell types from Recherche du Centre Hospitalier Universitaire (CHU) heterogeneous tissues underpins much of current biological de Québec-Université Laval, Ville de Québec, Québec, 5 and clinical research. Methods using FACS to enrich cells Canada, Monash Ramaciotti Centre for Structural Cryo- use reporter transgenes or fluorescent antibodies that Electron Microscopy, Monash University, Melbourne 6 are specific for the cell type of interest. However, limited 3800, Australia , APHP, Centre de Référence de availability of specific antibodies or - in case of reporter Pathologie Neuromusculaire Nord/Est/Ile-de-France, constructs - the need for genetic manipulation, limit this Institut de Myologie, Laboratoire de Pathologie Risler, approach. GH Pitié-Salpêtrière, Paris, France,7 Stem Cell Laboratory, Department of Pathophysiology and Transplantation, We developed GateID, an optimization algorithm that Università degli Studi di Milano, Fondazione IRCCS combines single-cell FACS and transcriptome information Ca’ Granda Ospedale Maggiore Policlinico di Milano, with a goal to predict FACS gates for cell types that Centro Dino Ferrari, via F Sforza, 35, 20122 Milan, Italy 8 were identified in an unbiased manner by the single- Biochemistry and Molecular Biology, Monash Biomedicine cell mRNA-sequencing. At the core of GateID is an Discovery Institute, Monash University, Melbourne, optimization algorithm that attempts to predict gates Victoria, Australia,9 Centre de référence des maladies to obtain the maximum number of desired cells while neuromusculaires, Hôpital Pitié-Salpétrière, Assistance- minimizing the number of undesired cells. It iterates this Publique Hôpitaux de Paris, Paris, France,10 Département procedure through all combinations of FACS channels and de Biologie Moléculaire, Biochimie Médicale et Pathologie, subsequently through combinations of gates to predict best Université Laval, Ville de Québec, Québec, Canada gates in terms of purity and yield. Ultimately, GateID allows purifying cell types based on general properties such as cell Dominant de novo mutations in the co-chaperone BAG3 size and granularity, nuclear staining, cellular proliferation, cause a severe form of myofibrillar myopathy, exhibiting and mitochondrial activity. progressive muscle weakness, muscle structural failure, and protein aggregation. To demonstrate the power of GateID, we enriched to high purity various cell types from the zebrafish hematopoietic To identify therapies we generated two zebrafish models, system (including hematopoietic stem and progenitors one conditionally expressing BAG3P209L and one with a cells) without resorting to antibodies or transgenes. nonsense mutation in bag3. Whilst transgenic BAG3P209L expressing fish display protein aggregation, modelling the early phase of the disease, bag3-/- fish demonstrate impaired autophagic activity, exercise dependent fibre O-11 Human genetic cancer mimicry in zebrafish thought disintegration, and reduced swimming activity, consistent somatic base editing. with later stages. Marion Rosello1, Viviana Anelli2, Céline Revenu1, Juliette Vougny1, Jean-Paul Concordet3, Marina Mione2, Filippo Having confirmed the presence of impaired autophagy in 1 patient samples we utilised the zebrafish model to screen Del Bene . a library of autophagy promoting compounds for their 1Institut Curie, PSL Research University, Inserm U934, effectiveness at removing protein aggregates, identifying CNRS UMR3215, Paris, France, 2Cibio, University of Trento, nine including Metformin. Further evaluation in our models Trento, Italy, 3Laboratoire Structure et Instabilité des demonstrated Metformin is not only able to remove the Génomes, Museum National d’Histoire Naturelle, INSERM protein aggregates in zebrafish and human myoblasts U1154, CNRS UMR7196, Paris, France but is also able to rescue the fibre disintegration and swimming deficit observed in the bag3-/- fish. Therefore, Nowadays zebrafish has become a powerful model to repurposing Metformin provides a promising therapy for study cancer genetics. Several models of cancer have been BAG3 myopathy. generated in zebrafish by combining transgenesis, chemical treatments, gene mutations using CRISPR/Cas9, and O-10 Cell type purification by single-cell transcriptome- xenotransplantation. A high number of cancers are modeled trained sorting in zebrafish and the vast majority are employing transgenic C.S. Baron1,2,4, A. Barve1,2,4, M.J. Muraro1,2,4, G. lines expressing mutated human oncogenes under the Dharmadhikari1,2, R. van der Linden1,2, E.J.P. de Koning1,2,3 control of tissue-specific promoters. A major limitation & A. van Oudenaarden1,2,* under these experimental conditions is the loss of the endogenous gene expression regulations, which is locus- 1 Hubrecht Institute-KNAW (Royal Netherlands Academy dependent and that is often critical in these diseases. In this of Arts & Sciences), Utrecht, The Netherlands,2 University context, being able to combine tumor suppressor knockout Medical Center Utrecht, Cancer Genomics Netherlands, and oncogene activation in an endogenous manner is an Utrecht, The Netherlands, 3 Leiden University Medical important challenge in the field. Here we have developed ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 4 Oral Abstracts an efficient method to recapitulate these human mutations visualized over time. For example, photoconversion cell directly in the genome of zebrafish to create more lineage-tracing identified migratory and proliferative physiological models of human cancers. First, using the cell states in human rhabdomyosarcoma, a pediatric very recent technology of base editors using CRISPR/Cas9, cancer of muscle. Additional experiments identified the we established a precise and tissue-specific base editing preclinical efficacy of combination olaparib PARP-inhibitor method in zebrafish. Secondly using this technique, we and temozolomide DNA-damaging agent as an effective are currently developing zebrafish cancer models in which therapy for rhabdomyosarcoma and visualized therapeutic we could efficiently recapitulate combinations of human responses using a four-color FUCCI cell cycle fluorescent cancer mutations by somatic single-base conversion in an reporter. These experiments identified that combination endogenous way. Finally, we are also expanding a toolbox of treatment arrested rhabdomyosarcoma cells in the G2 several base editors that recognize different PAMs working phase prior to induction of apoptosis. Importantly, this in zebrafish. This will broaden the editing possibilities to drug combination also exhibited remarkable efficacy using mimic human mutations in zebrafish, which could beof engraftment studies performed in NSG immune-deficient great use for the zebrafish community and that is essential mice and is likely to move forward for clinical evaluation for biomedical research. in patients. Additional studies have now extended the use of the zebrafish models to studying the pre-clinical efficacy of a wide array of cancer immunotherapies, including the O-12 Dynamic single cell imaging of human cancer dynamic imaging of CAR-T cell killing and visualizing the growth, immunotherapy responses, and stem cell effects of bispecific T cell engager antibodies in curbing pluripotency following engraftment into immunodeficient tumor growth, all of which can be imaged at single-cell zebrafish resolution. Finally, we will present data on the use of these models for assessing pluripotency of engrafted human Chuan Yan 1,2,3,4, Dalton C. Brunson1,2,3,4#, Qin Tang1,2,3,4#, ES and IPS cells. We envision our immune compromised Daniel Do1,2,3,4#, Nicolae A. Iftimia1,2,3,4 , John C. Moore1,2,3,4, zebrafish will be broadly useful for a wide array of cancer and Madeline N. Hayes1,2,3,4, Alessandra M. Welker1,2,3,4, Elaine regenerative medicine approaches, providing remarkable G. Garcia1,2,3,4, Taronish D. Dubash2, Xin Hong2, Benjamin imaging capability of single engrafted cells. J. Drapkin2, David T. Myers2, Sarah Phat2, Angela Volorio2, Dieuwke L. Marvin2, Matteo Ligorio2, Lyle Dershowitz1,2,3,4, Karin M. McCarthy1,2,3,4, Murat N. Karabacak5, Jonathan 7 1,2 1,2 O-13 Location, location, location! MMEJ real estate for A. Fletcher , Dennis Sgroi , John A. Iafrate , Shyamala functional genomics in zebrafish Maheswaran2, Nick J. Dyson2 , Daniel A. Haber2, 6, John F. Rawls8, David M. Langenau1,2,3,4 Stephen C Ekker1 1 Molecular Pathology Unit, Massachusetts General 1 Mayo Clinic, Rochester, MN 55906 Hospital Research Institute, Charlestown, MA 02129,2 Massachusetts General Hospital Cancer Center, Harvard The zebrafish (Danio rerio) is a powerful model system Medical School, Charlestown, MA 02129, 3 Center for for understanding the vertebrate genome using genetic Regenerative Medicine, Massachusetts General Hospital, applications. Conventional reverse genetic tools include Boston, MA 02114. 4 Harvard Stem Cell Institute, multi-generational, germline mutagenesis using gene Cambridge, MA 02139,5 Shriners Hospitals for Children- editing approaches such as CRISPRs/TALENs, or rapid but Boston, Center for Engineering in Medicine, Massachusetts short-duration knockdown approaches such as morpholinos. General Hospital and Harvard Medical School, Boston, Using CRISPR tools for F0 screening has been substantially Massachusetts, 02114, USA,6 Howard Hughes Medical limited by the inherent diverse outcomes due to NHEJ-based Institute, Bethesda MD 20815,7 Department of Pathology, DNA repair. We have harnessed Microhomology-Mediated Brigham and Women’s Hospital, Boston MA 02115,8 End-Joining (MMEJ) for precise gene editing applications Department of Molecular Genetics and Microbiology, Duke in the zebrafish. We previously developed a new algorithm University School of Medicine, Durham, NC 27710 (MENTHU) based on local microhomology competition and proximity post double-stranded break for identifying Xenograft cell transplantation into immune-deficient mice MMEJ-based alleles with predominant frameshift alleles has become the gold-standard for assessing regenerative (PreMA) after injecting in the F0 embryo. We show these capacity of stem cells and pre-clinical efficacy for new cancer PreMA alleles readily phenocopy known embryonic and drugs. Yet, mice are expensive and direct visualization larval phenotypes, are predictable and generate easily of engraftment at single-cell resolution is difficult. Here, sharable genomic edits precise to the nucleotide. We have we report optically-clear, prkdc-/-, il2rga-/- and rag2-/-, used our MENTHU algorithm to assess the availability of il2rga-/- casper-strains of zebrafish that lack adaptive and PreMA alleles in the zebrafish genome using a panel of natural killer immune cells. These fish can be reared at 37°C 56 test genes of different sizes to explore whether this and robustly engraft a wide variety of human and mice approach is suitable for genome-wide screening. We show cells, including cancer cell lines, patient-derived xenografts, that SpCas9-based out-of-frame PreMA alleles are found embryonic stem cells, and hematopoietic cells. Moreover, about 80% of the time in the first 20% of exons in zebrafish individual xenoengrafted cells can be dynamically test loci. For any genes where SpCas9 is not able to generate ......

..... 5 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts a PreMA allele, TALENs are a suitable gene editor for PreMA O-16 Modeling congenital disease in zebrafish: alleles due in part to the higher overall frequency of TALEN- RASopathy-like syndromes arise from novel mutations in based predicted PreMA alleles than either CRISPR systems. MAP4K4 We also explored how sites in the genome where MMEJ Victoria Patterson1, Li Dong2, Elizabeth Bhoj2, Rebecca is actively disfavored could serve as preferred locations for 1 targeted integration. Understanding DNA repair outcomes Burdine after a DSB impacts the precise editing for both knockin 1 Department of Molecular Biology, Princeton University,2 as well as knockout genetic approaches, with PreMA Center for Applied Genomics, Children’s Hospital of alleles representing a novel F0-based screening method in Pennsylvania zebrafish. In the USA, congenital malformations are the leading cause of infant mortality. While corrective surgeries can O-14 Personal Neoantigen Cancer Vaccines mitigate the impact in infancy, patients with congenital malformations are at significant risk of developing associated Catherine J. Wu, M.D.1 diseases in later life. Congenital malformations can occur

1 in isolation, but also arise within complex developmental Dana-Farber Cancer Institute and Harvard Medical syndromes such as RASopathies. RASopathies are caused by School, Boston, MA inappropriate activation of RAS signaling during embryonic With the recent availability of novel immunologic agents, development. While many RASopathy patients carry priority has shifted to understanding the mechanisms of mutations in components of the core signaling pathway, and predicting responses to each treatment. While the many others lack specific molecular diagnoses. Identifying search for immunogenic tumor antigens has been the new disease-associated variants can help direct patient subject of decades-long studies, multiple lines of evidence care, and extends our understanding of normal signaling have convincingly demonstrated tumor neoantigens and development. To detect novel variants, we performed as an important class of immunogenic tumor antigens. whole exome sequencing on a cohort of patients presenting Neoantigens arise from amino acid changes encoded two RASopathy-like symptoms: congenital heart defects and by somatic mutations in the tumor cell and have the craniofacial anomalies. Sequencing of a patient diagnosed potential to bind to and be presented by personal HLA with Noonan-like syndrome identified a novel truncating molecules. We can now systematically identify mutations variant in MAP4K4. We subsequently identified a further ten leading to amino acid changes that can be potentially families with distinct mutations in MAP4K4, all displaying recognized immunologically through the implementation some form of developmental disease. Using overexpression of neoantigen discovery pipelines. In recent studies, we approaches in zebrafish, we demonstrated that MAP4K4 have demonstrated that neoantigens can be safely and truncation causes a loss of protein function. Further analysis feasibly targeted to generate customized cancer vaccines. suggests that non-truncating variants may impede MAP4K4 We have been undertaking pilot clinical trials to develop protein function in a dominant negative manner. Moreover, personal cancer vaccines in melanoma and glioblastoma genetic mutations engineered to truncate endogenous that utilize synthetic long peptides as delivery approach Map4k4 lead to developmental abnormalities. We also for this therapy. Recent results and new directions will be tested whether MAP4K4 can modulate RAS signaling in discussed. the early embryo, and found that increasing MAP4K4 levels rescues the phenotypic consequences of hyperactive RAS signaling. MAP4K4 truncation abrogated this rescue. Together, our data suggest that MAP4K4 acts as a negative O-15 One beta-cell to rule them all? regulator of RAS signaling, and that loss of this function Nikolay Ninov1 leads to RASopathy-like disease in patients. 1 Technische Universität Dresden, Germany Using high-speed calcium imaging in the zebrafish O-17 Nicastrin deficiency induced Tyrosinase-dependent pancreas, we have collected evidence for the presence of acquired pigment loss and skin inflammation a critical sub-population of insulin-producing cells. These Chia-Hao Hsu1, Gan-Guang Liou2 and Yun-Jin Jiang1 cells, which we term Leaders, appear to orchestrate the function of the entire islet. Damaging or silencing Leaders 1Institute of Molecular and Genomic Medicine, National can impair the pan-islet response to glucose in vivo. Our Health Research Institutes, Miaoli, Taiwan,2 Institute of results indicate that the function of an entire islet can fail Molecular Biology, Academia Sinica, Taipei, Taiwan due to the loss of a small but critical sub-population of its constituent cells. This knowledge might become essential Skin depigmentation diseases, such as vitiligo, are when considering how to regenerate or replace beta-cells pigmentation disorders that often destroy melanocytes. in order to cure diabetes. However, their pathological mechanisms remain unclear and, therefore, promising treatment or prevention was lacking. Here we demonstrated that a zebrafish ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 6 Oral Abstracts insertional mutant showing a significant reduction of Taken together, these results demonstrate that our Crispr/ nicastrin transcript, possesses melanosome maturation Cas9-based F0 screen is a powerful approach to rapidly test defect, Tyrosinase-dependent mitochondrial swelling and the function of unknown genes and to study the molecular melanophore cell death. The depigmentation phenotypes mechanisms involved in congenital hypothyroidism in were proven to be a result of γ-secretase inactivation. humans. Using this approach already been able to shed light Furthermore, macrophages were recruited to and on the implication of nrf2a in thyroid dyshormonogenesis phagocytosed melanophore debris. Thus, we characterized which has never been done before. a potential zebrafish depigmentation disease model, nicastrinhi1384 mutants, which can be used for further treatment/drug development of skin depigmentation- or O-19 CRISPR screens in zebrafish identify regulators of inflammation-related diseases. left-right asymmetry Daniel T. Grimes1 O-18 A closer look upon thyroid functional maturation: a 1 Institute of Molecular Biology, Department of Biology, nrf2a story University of Oregon, Eugene, OR 97403, USA Pierre Gillotay1, B Dassy1, A. Trubiroha1, N. Giusti1, B. The internal organs of vertebrates are asymmetric between Haerlingen1, D. Gacquer 1, Panos G. Ziros3, Gerasimos P. left and right, an arrangement which is critical for their Sykiotis3, R. Opitz2 and Sabine Costagliola1 function. Many asymmetries originate in neurula-staged embryos when a cilia-driven fluid flow is generated in 1 Université Libre de Bruxelles (ULB), IRIBHM, 808 Lennik 2 midline-located left-right organizers (LROs). This flow is Street, 1070 Brussels, Belgium, Institute of Experimental asymmetric and so acts a directional signal that induces Pediatric Endocrinology Charité Universitätsmedizin Berlin downstream asymmetries in gene expression which then Campus Virchow-Klinikum Augustenburger Platz 1 Forum 3 drive asymmetric organ formation. Precisely how fluid flow 4, Haus 0037, PF 28 13353 Berlin / Germany, Service of induces asymmetric gene expression in LROs is unknown Endocrinology, Diabetology and Metabolism, Lausanne because very few factors that transduce the flow signal University Hospital, Ave de la Sallaz 8, 1011 Lausanne, have been found. We therefore undertook a reverse Switzerland genetic screen using CRISPR to identify novel regulators of left-right asymmetric patterning. Our approach allows Congenital hypothyroidism (CH) represents the most us to rapidly test the requirement of hundreds of genes in common congenital endocrine disorder in humans, affecting zebrafish patterning. I will report the results of our screen approximately 1 in every 3000 live births. However, to date, as well as validation work. Regulators of left-right patterning the cause of most case of CH remains unexplained. This discovered in this way will be candidates for roles in human situation is due to poor understanding of the molecular laterality disorders such as heterotaxy and will deepen our mechanisms regulating thyroid development and understanding of the transduction of flow-based signals. functional maturation. To tackle this problem we developed a thyroid specific zebrafish reporter line: tg(tg:nlsEGFP). By combining FACS of thyroid cells with RNA-seq, we provide a dynamic gene expression profile of the developing thyroid. O-20 Bouncer mediates species-specificity of fertilization These analyses revealed hundreds novel genes with Andrea Pauli1 strongly enriched expression during thyroid development. To identify the role of these novel genes in thyroid 1 Research Institute of Molecular Pathology, Austria development, we developed a F0 crispant screening assay that was successfully validated by targeting genes known to Fertilization is fundamental for sexual reproduction, yet the play different roles in thyroid development/function. Using molecular mechanism is poorly understood. We discovered this approach we identified nrf2a, a transcription factor Bouncer, a short, unannotated protein in zebrafish that is implicated in the cell’s oxidative stress response. Analysis of essential for fertilization. Bouncer is required for sperm-egg the functional maturation of the gland in our nrf2a stable binding and sperm entry into the egg. Remarkably, Bouncer mutant line generated by Crispr/Cas9 shows a strong defect functions as the gate-keeper of the egg by ensuring species- in thyroid hormone production. This suggests that nrf2a is specificity of fertilization: it allows conspecific spermto involved in the thyroid maturation. Indeed, quantitative enter while keeping heterospecific sperm out (Herberg et analysis of thyroxine (T4, the most abundant of the thyroid al., 2018). hormone) level indicates that homozygous mutants have While fish express Bouncer exclusively in the egg, the a complete absence of T4. This absence seems correlated mammalian orthologue of Bouncer, Spaca4, is restricted with the lack of iodinated thyroglobulin, the precursor to the male germline. Consistent with its testis-specific of thyroid hormones. Despite the defect in the thyroid expression in mammals, functional analyses in mice reveal function, folliculogenesis occurs normally suggesting that severely reduced fertilization rates in mutant male but not the phenotype is induced by a primary defect of the thyroid female mice. Thus, our study highlights Bouncer and Spaca4 hormone production machinery. as previously unknown fertilization factors that function either in female (fish) or male (mammalian) gametes......

..... 7 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts O-21 Reiterated BMP signaling in melanocyte the disease) are characterized by high-level amplification of development and melanoma chromosomal material derived from 12q. The presence of CDK4, HMGA2 and MDM2 were long presumed to drive Alec K. Gramann1,2, Arvind M. Venkatesan1,2, Craig J. 1,2 selection for these amplifications, but at least 24 genes are Ceol also consistently amplified in these tumors within the three 1 Program in Molecular Medicine, University of minimal amplicons on 12q. The lack of animal models Massachusetts Medical School,2 Molecular, Cell, and previously made it impossible to distinguish bona fide Cancer Biology, University of Massachusetts Medical oncogenes from non-pathogenic “passengers” within these School amplicons. We previously developed a zebrafish model of liposarcoma, the first animal model of the disease, in which Preventing terminal differentiation is important in the disease is driven by expression of an activated AKT allele by a development and progression of many cancers, including rag2 promoter construct that drives ectopic gene expression melanoma. Previous studies have determined that less in multipotent mesenchymal progenitors. We have now differentiated melanomas often acquire neural crest- screened all 10 genes within the most centromeric minimal like characteristics that promote aggressive phenotypes, amplicon by overexpressing these under the control of the such as metastasis and drug resistance. However, it is rag2 promoter, in order to access the ability of each gene unknown how these melanoma cells acquire and regulate to accelerate the onset of AKT-driven liposarcoma. This neural crest characteristics. Recently, we identified a revealed that 7 amplified genes had no effect on tumor novel melanoma oncogene, growth differentiation factor onset in this model (OS9, AGAP2, TSPAN31, CYP27B1, 6 (GDF6), a bone morphogenetic protein (BMP) ligand METT21B, TSFM, AVIL). By contrast, MARCH9, METTL1 and that promotes neural crest gene expression in melanoma CTDSP2 each accelerated tumor onset more strongly than cells. While previous studies have shown expression of the the positive controls (MDM2 and CDK4) overexpression. In zebrafish ortholog of GDF6, gdf6a, in the embryonic neural summary, our screen has identified novel oncogenes whose crest, the role of GDF6 and BMP signaling in pigment cell role in oncogenesis was not previously been suspected. We development is unknown. Here, we establish a loss of gdf6a are actively investigating the mechanism through which or BMP signaling during neural crest development disrupts these genes drive oncogenic transformation. normal pigment cell development and differentiation, leading to an increase in the number of melanocytes. We determine that this increase in pigment cell development O-23 The identification of SHP2 as a therapeutic target in is due to an increase in the number of neural crest cells RET rearranged thyroid cancers expressing mitfa, a key pigment cell regulatory factor. We determine these mitfa-expressing progenitors exhibit a Renuka Raman1, Timothy M. Ullmann1, Jacques corresponding increase in expression of mitfa, making Villefranc1, Viviana Annelli1, Theresa Scognamiglio2, Juan them more likely to differentiate to the melanocyte lineage. Miguel Mosquera2, Olivier Elemento3, Yariv Houvras1

BMP regulation of specification and differentiation in 1 melanocyte development parallels our previous findings Department of Surgery, Meyer Cancer Center, Weill Cornell Medical College,2 Department of Pathology and in melanoma and suggests a key role of BMP signaling the 3 pigment cell lineage. Together, these results indicate BMP Laboratory Medicine, Weill Cornell Medical College, signaling acts to suppress differentiation of melanocytes Institute for Computational Biomedicine - Englander during embryonic development. This further suggests the Institute for Precision Medicine oncogenic roles of GDF6 and the BMP pathway in supporting RET is a transmembrane receptor-tyrosine kinase required neural crest identity in melanoma may be a reiteration of for the development of the peripheral and enteric nervous their physiological roles during melanocyte development in system. RET signaling normally activates cellular proliferation, embryogenesis. survival, and migration pathways during development, however activating RET rearrangements and mutations are established drivers of oncogenesis. Multikinase inhibitors O-22 A zebrafish functional screen reveals novel driver targeting receptor tyrosine kinases (RTKs) are effective in oncogenes in liposarcoma patients with advanced papillary thyroid cancers driven by

1 1 1,2 RET rearrangements, but are limited by the development Raja Ali , Natsuko Yamagata , Gutierrez Alejandro of resistance. We developed a model of RET rearranged 1 Division of Hematology/Oncology, Boston Children’s thyroid cancer in zebrafish to study tumor initiation and Hospital, Boston, MA, USA,2 Department of Pediatric understand mechanisms of drug resistance. Using this Oncology, Dana-Farber Cancer Institute, Harvard Medical model, we show that expression of RET/CCDC6 in zebrafish School, Boston, MA, USA thyroid leads to excess thyroid proliferation, increased MAPK pathway activity, increased mitotic activity, and Liposarcoma, a mesenchymal tumor, is the most common sensitivity to small molecule RET inhibition. Using a human sarcoma of humans but remains incurable when it cannot thyroid cancer cell line harboring a RET rearrangement be fully surgically resected. Half of human liposarcomas (the we show that exposure to RET inhibitors leads to adaptive related well-differentiated and dedifferentiated subtypes of resistance within hours. Thyroid cancer cells that acquire ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 8 Oral Abstracts adaptive resistance are refractory to retreatment. We O-25 CDK13 mutations drive melanoma via accumulation identify SHP2 as a critical mediator of adaptive resistance of prematurely terminated transcripts

and show that SHP2 inhibition abrogates resistance. We 1, 2 3 4,5 demonstrate that combined inhibition of RET and SHP2 Megan L. Insco , Brian J. Abraham , Paul L. Boutz †, Sara J. Dubbury4,5†, Sofia Dust6, Kevin Y. Chen1, David Liu2, in transgenic zebrafish thyroid tumors expressing RET 1 1 1 rearrangments leads to enhanced tumor regression. To our Constance Wu , Calvin G. Ludwig , Tania Fabo , Telmo Henriques6, Karen Adelman6, Matthias Geyer7, Phillip A. knowledge this is the only vertebrate animal model of RET 4,5 3,5 1* rearranged thyroid cancer with an aggressive phenotype Sharp , Richard A. Young , and Leonard I. Zon that can be used to identify pharmacologic strategies to 1 Stem Cell Program and Division of Hematology/Oncology, prevent the acquisition of adaptive resistance. Boston Children’s Hospital, Howard Hughes Medical Institute, Boston, MA, USA, 2 Department of Medical Oncology, Dana- Farber Cancer Institute, Boston, MA, USA,3 Whitehead O-24 Heterogeneous cell clusters promote melanoma Institute for Biomedical Research, Massachusetts Institute metastasis via cell-cell cooperation of Technology, Cambridge, MA, USA ,4 The David H. Koch

1,2,3 3 Institute for Integrative Cancer Research, Massachusetts Nathaniel R. Campbell , Maomao Zhang , Apeksha Institute of Technology, Cambridge, MA, USA,5 Department Singh4, Malek Jacobs5, Maxime Deforet2, Joao B. Xavier2, 3 of Biology, Massachusetts Institute of Technology, Richard M. White Cambridge, MA, USA, 6 Department of Biological Chemistry 1 and Molecular Pharmacology, Harvard Medical School, Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional 7 MD-PhD Program, New York, NY, USA,2 Computational Boston, MA, USA, Institute of Structural Biology, University and Systems Biology, Memorial Sloan Kettering Cancer of Bonn, Bonn, DE. 3 Center, New York, NY, USA, Cancer Biology and Genetics, Transcriptional Cyclin Dependent Kinases (CDKs) modulate Memorial Sloan Kettering Cancer Center, New York, NY, 4 5 RNA Polymerase II (RNAPII) function to affect gene USA, University of California, Berkeley, Berkeley, CA, USA, expression and, consequently, represent potential targets Cornell University, Ithaca, NY, USA for the treatment of several cancers. Among the family of Metastasis is the primary cause of cancer death, yet we transcriptional CDKs, we find that CDK13 is mutated in 4% know little about what determines the number, size and of human melanomas and its mutation or downregulation aggressiveness of metastases. In melanoma, multiple is associated with poor overall survival. The amino acid studies have documented the co-existence of MITF+/ changes produced by mutation in melanoma are identical proliferative (PRO) and AXL+/invasive (INV) cell states, to mutations recently implicated in a congenital syndrome, but how these two states interact to promote metastasis underscoring the critical function of these residues. We find that CDK13 melanoma-associated mutations (CDK13mel) remains poorly understood. We utilized a zebrafish model mel of melanoma to longitudinally study these populations at inactivate kinase activity. Expression ofCDK13 in zebrafish single-cell resolution. From a BRAFV600E zebrafish melanoma leads to accelerated melanoma onset and downregulation of TNFa/NFKB pathway. Cdk13WT and CDK13mel localize to we isolated two melanoma cell populations, which are mel enriched for either proliferative (ZMEL1-PRO) or invasive the transcriptional start site of genes, while CDK13 is associated with abnormal RNAPII dynamics in zebrafish (ZMEL1-INV) phenotypes. Intravital imaging revealed that mel the INV population metastasized more aggressively, with melanomas. Upon CDK13 expression, RNA sequencing groups of INV cells extravasating and invading collectively. showed prematurely terminated transcripts and tandem In vitro mass spectrometry showed excess short proteins from , INV cells formed large tumor cell clusters, and WT unexpectedly the INV and PRO cells self-aggregated to form zebrafish melanomas. CDK13 bound CCNT1 in human heterotypic clusters, in which INV cells were surrounded by melanoma, and ccnT1 was required for the dominant negative tumorigenesis and localization of CDK13mel in PRO cells on the periphery of each cluster. Using intravital WT imaging, we observed co-extravasation of the PRO and INV zebrafish. CDK13 also bound the RNA stability factor ZC3H14. Interaction with ZC3H14 was disrupted by cells, with the INV cells as leaders and PRO cells as followers, mel suggesting that these two cell states may cooperate in the a CDK13 allele that changes the substrate binding domain, indicating ZC3H14 may be a CDK13 substrate. In formation of metastases. Consistent with this, multicolor mel imaging demonstrated that the PRO and INV cells co- a CDK13 expressing melanoma cell line, ZC3H14 lost metastasize, and that the less metastatic PRO lineage one phosphorylation and disassociated from the complex receives a cooperative benefit and metastasizes more when required for turnover of prematurely terminated RNAs in co-transplanted with INV. Mechanistically, RNA-seq and the nucleus. Our work identifies CDK13 as a melanoma chemical screening identified several transcription factors tumor suppressor which acts via a dominant negative and signaling nodes that may mediate the cooperation mechanism requiring CCNT1. Using zebrafish, we uncovered between these two cell states. Ongoing work aims to further a new mechanism of oncogenic transcriptional control elucidate the molecular mechanism of cooperation in where CDK13 is required for ZC3H14 phosphorylation and degradation of prematurely terminated RNAs. Melanoma metastasis via perturbation of these identified candidates. mel Interrupting mechanisms of cell-cell cooperation in clusters patients with CDK13 would benefit from aggressive may represent a novel approach to preventing metastatic monitoring and adjuvant trial inclusion. Our studies have implications for CDK13mut developmental disorder patients...... progression......

..... 9 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts O-26 Genotypic and phenotypic variables affect meiotic USA,4 School of Pharmacy, Qiqihar Medical University, cell cycle progression, tumor ploidy, and cancer- Qiqihr, Heilongjiang 161006, P.R.China,5 Department associated mortality in a brca2-mutant zebrafish model of Biostatistics, Boston University School of Public 6 1 1 1 Health, Boston, MA 02118, USA, Department of Leroy Mensah , Jordan L. Ferguson , Heather R. Shive Pharmacology, College of Medicine, University of 1 NC State University, College of Veterinary Medicine, Illinois at Chicago, Chicago, IL 60612, USA Raleigh, NC Triple-­‐negative breast cancer (TNBC) represents the most Successful cell replication requires both cell cycle completion aggressive disease among all breast cancer subtypes, yet and accurate chromosomal segregation. The tumor the molecular mechanisms underlying its aggressiveness suppressor BRCA2 is positioned to influence both of these remain incompletely understood. Here, we report outcomes, and thereby influence genomic integrity, during that hyperactive sphingosine 1-­‐phosphate receptor 1 meiotic and mitotic cell cycles. Accordingly, mutations in (S1P1) signaling promotes TNBC invasiveness. Compared BRCA2 induce chromosomal abnormalities and disrupt to luminal subtype of the disease, we detected a cell cycle progression in both germ cells and somatic cells. significant increase of phosphorylated S1P1 at Threonine Despite these findings, aneuploidy is not more prevalent 236 site rather than S1P1 itself in human TNBC cells. in BRCA2-associated versus non-BRCA2-associated human Overexpression of the phosphorylation-­‐defective S1P1 cancers. More puzzlingly, diploidy in BRCA2-associated mutant suppressed TNBC cell migration in vitro and cancers is a negative prognostic factor, unlike non-BRCA2- disease invasiveness in zebrafish xenografts. Compared associated cancers and many other human cancers. to other subtypes of breast cancer, AKT3 but not AKT1 or We used a brca2-mutant/tp53-mutant cancer-prone AKT2 is upregulated in TNBC patient samples and associated zebrafish model to explore the impact of BRCA2 mutation with the advanced disease stage. Treatment of TNBC cells on cell cycle progression, ploidy, and cancer-associated with an AKT inhibitor (MK2206) suppresses tumor cell mortality by performing DNA content/cell cycle analysis migration in vitro and disease invasiveness in zebrafish on zebrafish germ cells, somatic cells, and cancer cells. xenografts, suggesting a role of AKT3 in mediating First, we determined that combined brca2/tp53 mutations S1P1 phosphorylation. To understand how S1P1 receptor uniquely disrupt meiotic progression to cause both meiotic phosphorylation promotes TNBC migration, we examined arrest and suppression of spermatogonial differentiation. expression of a panel of epithelial-­‐mesenchymal transition Second, we determined that sex significantly influences (EMT) genes. Interestingly, S1P1 genetic inhibition or ploidy outcome in zebrafish cancers, as aneuploidy overexpression of the phosphorylation-­‐defective S1P1 was significantly more common in cancers from female mutant in TNBC cells led to downregulation of N-­‐cadherin zebrafish. Third, we determined that brca2 mutation and transcripts without affecting other EMT genes examined. female sex each significantly reduce survival time in cancer- These findings indicate that S1P1 receptor phosphorylation bearing zebrafish. Finally, we provide evidence to support induces N-­‐cadherin expression to promote TNBC migration a link between BRCA2 mutation, tumor diploidy, and poor and invasiveness, providing insights into molecular survival outcome. These outcomes underscore the utility mechanisms underlying disease aggressiveness and of this model for studying BRCA2-associated genomic implications for targeted therapy for this stubborn cancer. aberrations in normal and cancer cells.

O-28 Metabolic stress induces Ripk3- and macrophage- O-27 S1P1 phosphorylation promotes triple-­‐negative dependent beta-cell loss in zebrafish breast cancer invasiveness Lisette A Maddison1,2,#, Bingyuan Yang1,#, Linlin Yin1, and Wenbiao Chen1 Fabrice J.F. Laroche1, Sheng Li1,2, Xiaodan Qin1, Ning Shen1, Chen Khuan Wong3, Soo Kyung Hwang1, Hongxia 1 Department of Molecular Physiology and Biophysics, Cui1,4, Xiaoyu Zhang5, Gina Nguyen1, Wenling Yu1, Ryan J. Vanderbilt University School of Medicine, Nashville, TN Quinton1, Owen Tamplin6, Anurag Singh1, Neil J. Ganem1, 37232, 2 Current Affiliation: Washington State University Ching-­‐Ti Liu5, Sam Thiagalingam3, and Hui Feng1 College of Veterinary Medicine, Pullman, WA

1 Department of Pharmacology and Experimental # these authors contributed equally. Therapeutics, Section of Hematology and Medical Overnutrition-induced obesity and associated insulin Oncology, Department of Medicine, and Cancer resistance are a major risk factor for type 2 diabetes (T2D). Center, Boston University School of Medicine, Boston, The increased insulin demand is initially compensated MA 02118, USA,2 Institute of Agro-­‐Bioengineering and through increased β-cell number and activity. However, long- College of Life Sciences, Guizhou University, Guizhou, term increase of insulin demand can lead to β-cell failure Guiyang 550025, China,3 Biomedical Genetics Section, and death, and the development of T2D. The molecular Department of Medicine, Department of Pathology mechanisms underlying the β-cell dynamics in diabetogenic and Laboratory Medicine, Genetics and Genomics conditions are not well defined. With its genetic, chemical, Graduate Program and Cancer Center, Boston and anatomical tractability, zebrafish may help elucidate University School of Medicine, Boston, MA 02118, ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 10 Oral Abstracts these mechanisms. By expressing a dominant-negative diverse functions in homeostasis, disease progression, Igf1r in skeletal muscle, we generated a zebrafish muscle and resolution of the inflammatory response. Despite insulin resistance model (zMIR) that becomes diabetic in observations linking macrophages to liver regeneration adults due to β-cell death. In an overnutrition regimen, and homeostasis, the molecular and cellular mechanisms zMIR larvae exhibit an initial increase of β-cell number underlying those functions are largely unknown. We utilize as in sibling controls, but a significant β-cell loss and the fast and dynamic nature of embryonic development ensuing hyperglycemia after 3 days. This β-cell number to answer these questions. Using various genetic dynamics thus resembles that during pathogenesis of T2D manipulations of irf8 and spi1b, genes important for in humans. The β-cell loss is independent of apoptosis or macrophage differentiation, we assessed the impact of dedifferentiation. Screening of candidate compounds for macrophage ablation on liver organogenesis. Macrophage suppressors of β-cell loss identified anti-diabetic drugs, loss at the onset of embryonic development led to a necroptosis inhibitors, and anti-inflammatory drugs. decrease in hepatocyte number and reduced biliary tree Mechanistically, zMIR islets display increased levels of il1b complexity at 72 and 96 hours post fertilization (hpf). and il8a transcripts 2 hours prior to β-cell loss, followed Meanwhile, targeted ablation of macrophages between by macrophage infiltration that coincides with β-cell loss. 96-144hpf, using the macrophage-specific nitroreductase/ Genetic and pharmacologic depletion of macrophages also metrodinazole (NTR/Mtz) system, resulted in dysmorphic attenuates theβ -cell loss. Ablation ofil1b or il8a suppresses liver outgrowth. Moreover, time-lapse confocal imaging the β-cell loss and macrophage infiltration. Furthermore, revealed a direct interaction of macrophages with the il1b or il8a induction and macrophage infiltration requires growing liver throughout development. In particular, the β-cell activation of Ripk3, the key necroptosis regulator. location of physical contact between macrophages and Global disruption of ripk3 or β-cell specific expression biliary epithelium correlated to biliary tree sprouting. To of a dominant-negative form of human RIPK3, or its define the molecular mechanism by which macrophages upstream regulator RIPK1, inhibits il1b and il8a induction, instruct liver development, we focused on molecular macrophage infiltration, and β-cell loss. Taken together, signals known to be associated with macrophages: chemical these results suggest that overnutrition activates Ripk3 in or genetic perturbation of TGF-β signaling completely β-cells, causing islet inflammation, macrophage infiltration, recapitulated the phenotypes observed following and selective phagocytosis of damaged β-cells. Future macrophage ablation. Moreover, ligand independent studies will determine how metabolic stress activates Ripk3 activation of TGF-β pathway, using Tg(hsp70::caALK5) in β-cells. larvae, in the macrophage-ablated background lead to significant rescue of liver development. Liver dependence Key words. overnutrition, inflammation, necroptosis, β-cell, on macrophage activity persist throughout the life span of diabetes. the animal, as severe liver injury using hepatocyte-specific injury in a macrophage-ablated background resulted in a significant delay in liver recovery. In sum, we identified a O-29 Beyond metchnikoff’s phagocytosis novel and important role for macrophages in organogenesis theory: How macrophages regulate TGF-β and established a platform for probing macrophage-related signaling during zebrafish liver development. mechanisms in health and disease. Arkadi Shwartz1, Delaney Ingalls1, Lindsay N. Theodore2, Maryline Abrial3, Geoff Burns3,4, Caroline Burns3,4, Trista E. North2,4, Wolfram Goessling1,4-6 O-30 Mechanisms underlying synergy of DNA topoisomerase I and mTOR co-inhibition in malignant 1 Division of Genetics, Brigham and Women’s Hospital, peripheral nerve sheath tumors (MPNSTs) Harvard Medical School, Boston, MA 02115, USA, Dong Hyuk Ki 1, 2, Felix Oppel 1, 2, Adam D Durbin 1, 2, 3, A 2 Dept. of Hematology/Oncology, Boston Children’s Thomas Look 1, 2 3 Hospital, Harvard Medical School, Boston, MA 02115, 1 Dept. of Cardiology, Boston Children’s Hospital, Boston, Department of Pediatric Oncology, Dana-Farber Cancer 4 Institute, Harvard Medical School, Boston, MA 02115, MA 02115, Harvard Stem Cell Institute, Cambridge, 2 5 USA, Division of Pediatric Hematology/Oncology Boston MA 02138, USA, Dana-Farber Cancer Institute, Harvard 3 Medical School, Boston, MA 02115, USA,6 Broad Institute Children’s Hospital, Boston, MA 02115 USA , The Broad of MIT and Harvard, Cambridge, MA 02142, USA,7 Institute of MIT and Harvard, Cambridge, MA, 02142 Harvard-MIT Division of Health Sciences and Technology, 8 Malignant peripheral nerve sheath tumors (MPNSTs) Boston, MA 02115, USA, Division of Gastroenterology, are soft-tissue sarcomas that frequently arise in patients Massachusetts General Hospital, Harvard Medical School, with neurofibromatosis type 1 (NF1). Most MPNSTs Boston, MA 02114, USA are unresectable at diagnosis, lending urgency to the The liver is continuously exposed to antigens, microbial identification of new pathway dependencies and drugs products, and xenobiotics. To overcome this challenge, the with antitumor activities to treat these typically refractory liver harbors ~80% of the body’s residential macrophages, tumors. We therefore examined a series of candidate agents a heterogeneous population of immune cells, which fulfill for their ability to induce apoptosis in MPNST cells arising ......

..... 11 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts in nf1/tp53-deficient zebrafish. To facilitate simultaneous we found that intraluminal delivery of Trpa1 agonist AITC analysis of anti-tumor activity and toxicity to normal cells, or E.tarda also activated neurons in the vagal ganglia. Vagal we implanted fluorescent tumor cells into the pericardium activation in response to intraluminal Trpa1 activation was of pigmentless Casper zebrafish embryos and incubated blocked in zebrafish mutant that lacks enteric nervous the embryos in graded drug concentrations. Using this system (ENS), suggesting a requirement for enteric neurons strategy, we found that DNA topoisomerase I-targeted in this signal transduction pathway. Transgene-mediated drugs and mTOR kinase inhibitors were the most effective targeted ablation of EECs revealed that EECs are required single agents in eliminating MPNST cells at well-tolerated for this vagal response to intraluminal Trpa1 agonist and dosages. In addition, members of these classes of drugs, E.tarda stimulation. Collectively, these results indicate that irinotecan and AZD2014 or INK128, acted synergistically to an EEC-ENS-vagal signaling pathway is present in zebrafish, induce apoptosis when given in combination in vitro and in and that EECs are able to activate this pathway in response vivo. In mechanistic studies using human MPNST cells, we to distinct bacterial stimuli. This microbe-gut-brain axis found that irinotecan not only induces apoptosis by eliciting could be manipulated to to treat neurologic disorders which a DNA damage response, but also acts synergistically are associated with alterations in the intestinal microbiome. with the mTOR inhibitor AZD2014 to accentuate the hypophosphorylation of 4E-BP1. 4E-BP1 is a downstream target of mTORC1, which in its unphosphorylated form O-32 Palmitic acid-enriched diet induces hepatic binds and sequesters eIF4E eukaryotic translation initiation steatosis and injury in adult zebrafish factor. Profound hypophosphorylation of 4E-BP1 induced by the combination of these two drugs causes an arrest of 5’ Ki-Hoon Park1, Zhi-Wei Ye2, Jie Zhang2, Seok-Hyung Kim1, Cap-dependent protein synthesis, which potently induces 3, * tumor cell apoptosis. Our findings provide a compelling 1 Department of Medicine, Medical University of South rationale for clinical testing of the combination ofDNA 2 topoisomerase I-targeted drugs and mTOR kinase inhibitors Carolina, Charleston, SC, 29425, USA, Department of in patients with unresectable MPNSTs. Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, 29425, USA,3 Department of Regenerative Medicine and Cell Biology, Medical University of South O-31 Enteroendocrine cells sense gut bacteria and Carolina, Charleston, SC, 29425, USA activate a gut-brain pathway * Corresponding author Lihua Ye1,2, Rodger A. Liddle2, and John F. Rawls1,2

1 Palmitic acid (PA) is the most abundant saturated fatty acid in Department of Molecular Genetics and Microbiology, fast foods and is known to induce inflammation and cellular Duke Microbiome Center, Duke University School of 2 injury in various tissues. In this study, we investigated Medicine, Durham, NC, Division of Gastroenterology, whether a PA-enriched diet can induce hepatic steatosis Department of Medicine, Duke University School of and injury in adult zebrafish. The adult zebrafish exhibited Medicine, Durham, NC increased body weight, hyperlipidemia, hyperglycemia, The intestine harbors complex and dynamic microbial and steatosis and a hepatic injury phenotype after being communities that contribute significantly to host health fed with a PA-enriched diet for 6 weeks. The qPCR analysis and disease. However, mechanisms by which the intestine demonstrated that genes associated with hepatic injury perceives distinct microbial species and relays that were all significantly increased in the liver. Furthermore, information to the rest of the body remain unresolved. livers from the PA fed group showed an increased mRNA Enteroendocrine cells (EECs) are specialized sensory expression associated with oxidative stress and ER stress epithelial cells in the intestine that detect nutrients and responses. We also found significant upregulation of other chemicals in the intestinal lumen. To test the impact genes involved in lipid metabolism and triacylglycerides of bacteria on EECs, we developed a novel genetic system accumulation. Further, we found that the PA-enriched diet to record EECs activity in vivo following different bacteria increased sphingosine 1-phosphate (S1P), which may play a stimulation. In a small screen of zebrafish-associated critical role in hepatic injury. Ultrastructural analysis revealed bacteria, the only strain that significantly elicits EECs activity mitochondrial cristae injury and a dilated ER phenotype in was the fish pathogen,Edwardsiella tarda (E.tarda). E.tarda the PA-fed hepatocytes, which can be causes of hepatic stimulation of EEC activity required the Transient Receptor injury. In conclusions, PA-enriched diet induced steatosis Potential Ankyrin 1b (trpa1b) gene, a cation channel that and hepatic injury in adult zebrafish that recapitulated can be activated by a range of noxious chemicals including typical metabolic changes and pathophysiological changes allyl isothiocyanate (AITC). Photochemical activation of as well as increased oxidative stress and ER stress observed Trpa1 in EECs led to increased peristalsis, and trpa1b in patients with non-alcoholic steatohepatitis. mutant zebrafish were unable to clear E.tarda from the intestinal lumen. These data suggest chemical or E.tarda stimulation of Trpa1b promotes clearance of those noxious luminal stimuli via increased intestinal motility. In addition, ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 12 Oral Abstracts O-33 Intestinal dysmotility and reduced serotonin- is completely insulin resistant and unable to transport positive enteroendocrine cells in ashank3 loss-of- glucose during embryogenesis and larval development. function model of Autism Spectrum Disease Using a robotic high throughput method we have screened

1 1 1 for compounds that overcome insulin resistance. One of David James , Robert Kozol , Julia Dallman the compounds identified, NSC87877, appears to target the 1 University of Miami, Department of Biology non-receptor phosphatase Shp1. In this presentation we will provide further details on this screens and it relevance Using a zebrafish model of Autism Spectrum Disorder for understanding insulin resistance. (ASD), we explore gastrointestinal (GI) comorbidities which affect more than half of all ASD patients. To determine mechanisms behind ASD related GI distress, we explore how O-35 Contribution of sclerotome to the embryonic mutations in genes with high autism relatedness impact GI hematopoietic stem cell niche and vascular smooth function. Mutations in the syndromic ASD gene SHANK3 are muscle considered causal for Phelan-McDermid Syndrome (PMS), a disorder with associated GI distress including reflux, C.M. Kelley1, N.O. Glenn1, D. Gays2, M.M. Santoro2, and diarrhea, constipation, and/or cyclical vomiting. W.K. Clements1 We used CRISPR/Cas9 to make clinically-informed, 1 Department of Hematology, St. Jude Children’s Research C-terminal truncations (shank3abΔC) in both shank3a and Hospital, Memphis, TN 38105 USA,2 Department of shank3b paralogues as a means of generating a zebrafish Molecular Biotechnology and Health Sciences, Molecular model. By comparing shank3abΔC mutants with wild Biotechnology Center, University of Torino, Turin 10126, type (WT) larvae, we demonstrate significant changes in Italy GI structure and function. shank3abΔC mutants produce significantly slower rates of peristaltic contractions with Hematopoietic stem cells (HSCs) are a self-renewing correspondingly prolonged digestive passage time. Rescue population that ultimately sustains production of blood injections of mRNA encoding the longest human SHANK3 lineages throughout adult life. In vertebrates, these HSCs isoform into one-cell zebrafish mutants resulted in larvae are initially specified in the early embryo, arising from with gastric emptying similar to WT, but with remaining hemogenic endothelium in the ventral wall of the dorsal deficits in posterior intestinal motility. While enteric neuron aorta (DA). The cell types within this three-dimensional counts, and overall structure of the digestive tract epithelium specification environment and the molecular signals they was unaffected in shank3 mutant larvae, serotonin-positive provide to promote HSC development are not well defined. enteroendocrine cells (EECs) were significantly reduced in A better understanding of the native HSC specification shank3 mutants (James et al. Molecular Autism, Jan. 2019). niche could inform efficient directed differentiation of true HSCs--which is not currently possible--from induced Given the key role EECs play in serotonin production, GI pluripotent stem cells for clinical and research applications. motility, and general function of the GI system, our next We previously showed that wnt16 is required for the goal was to determine what causes reduced EEC numbers development of zebrafish HSCs, as well as normal patterning in the shank3abΔC mutants. EECs are endodermally of the ventral somite, the sclerotome. We hypothesized derived; reduced EECs in shank3abΔC mutants could be that normal sclerotome patterning is required for HSC due to alterations in endoderm or as a secondary effect specification by contributing to the cellular environment of alterations in brain regions that regulate gut, including that presents inductive signals. To follow sclerotome the hypothalamus and dorsal vagal complex. To distinguish development in living embryos, we have developed between these possibilities, we are generating genetically transgenic reporter zebrafish where expression of the mosaic zebrafish by transplanting embryonic WT cells into mNeonGreen fluorophore is driven by regulatory elements shank3abΔC mutants and vice versa to determine which of from the paradigmatic sclerotome gene, pax1a. We these tissues have the greatest impact on EEC numbers and show that sclerotome-derived cells migrate to and make GI motility. contact with hemogenic endothelium of the DA shortly before initiation of the definitive hematopoietic program, demonstrating that they are present at the right time and O-34 A high through-put screen for antidiabetic place to present specification signals. By live imaging and compounds in zebrafish embryos profiling of sorted cells, we show these cells also give rise to arterial vascular smooth muscle cells. Loss-of-function Herman P. Spaink1, Natalia Nowik1, Wouter Veneman1, Yi 1 1 2 analyses demonstrate a requirement for multiple sclerotome Ding , Fons Verbeek and Jan de Sonneville genes, including pax1a, for both proper patterning of the 1 Institute of Biology, Leiden University, The Netherlands,2 sclerotome itself, and subsequent specification of HSCs Life Science Methods, Leiden, The Netherlands and downstream definitive hematopoietic lineages. Our data indicate that sclerotomal cells contribute to the HSC We have shown that insulin signaling is already active during specification niche of the early embryo, and form a basis early zebrafish embryogenesis. This signaling is dependent for defining the niche architecture and signals that drive on the leptin protein. We have generated a mutant that definitive hematopoietic programming......

13 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts O-36 YAP regulates hematopoietic stem cell formation in ongoing work implicates the stretch-activated ion channel response to the biophysical forces of blood flow piezo1 and integrinB1 subunit genes as membrane proteins

1,3^ 1,2,3,4^ that may mechanistically link extracellular physical forces Wade W. Sugden , Vanessa Lundin , Lindsay N. from blood flow to YAP activation during the development Theodore1,3^, Patricia M. Sousa1,2,3, Areum Han1,2,3, Paul 3,5 3,5 4,6,7 of HSCs. Together, our study defines Yap1 as a transcriptional J. Wrighton , Andrew G. Cox , Donald E. Ingber , mediator that integrates hemodynamic forces and HSC Wolfram Goessling3,5,8,9, George Q. Daley1,2,3,8*, Trista E. 1,3* formation, which may be relevant to improving methods to North create or expand HSCs for therapeutic use. 1 Stem Cell Program, Division of Pediatric Hematology and Oncology, Boston Children’s Hospital, Boston, MA 02115, USA,2 Department of Biological Chemistry and O-37 Analysis of leukemia stem cells in a MYC-driven ALL Molecular Pharmacology, Harvard Medical School, Boston, zebrafish model 3 MA 02115, USA, Harvard Stem Cell Institute, Cambridge, 1 2 2 4 Jessica Burroughs-Garcia , Ameera Hasan , Gilseung Park MA 02138, USA, Wyss Institute for Biologically Inspired 1, 2, 3 Engineering, Harvard University, Boston, MA 02115, and J. Kimble Frazer 5 USA, Department of Medicine, Division of Genetics, 1 University of Oklahoma Health Sciences Center, Dept. Brigham and Women’s Hospital, Harvard Medical School, 6 of Pediatrics, Section of Pediatric Hematology-Oncology, Boston, MA 02115, USA, Vascular Biology Program and Oklahoma City, OK, USA,2 University of Oklahoma Health Department of Surgery, Boston Children’s Hospital and 7 Sciences Center Dept. of Cell Biology, Oklahoma City, OK, Harvard Medical School, Boston, MA 02115, USA, Harvard USA,3 University of Oklahoma Health Sciences Center John A. Paulson School of Engineering and Applied 8 Dept. of Microbiology & Immunology, Oklahoma City, OK, Sciences, Harvard University, Cambridge, MA 02138, USA, USA Dana-Farber Cancer Institute, Boston, MA 02115, USA,9 Harvard-MIT Division of Health Sciences and Technology, Acute lymphoblastic leukemia (ALL), is characterized by Harvard Medical School, Boston, MA 02115, USA the malignant transformation and proliferation of B- or T-lymphoid progenitors. Precursor-B (pre-B) ALL and T-ALL ^co-first author, *co-corresponding author are the two principal subtypes, together representing Hematopoietic stem cells (HSCs) arise from arterial over 25% of all pediatric malignancies. Although current endothelium in the ventral dorsal aorta during embryonic therapies are often curative, 10-20% of ALL patients development in a process termed endothelial to relapse. One cause of relapse is thought to be a failure of hematopoietic transition (EHT). HSCs give rise to all blood initial therapy to eliminate leukemia stem cells (LSC). LSC lineages of the adult organism. However, efforts in vitro to are rare, treatment resistant, quiescent, and epigenetically recapitulate EHT and reprogram induced pluripotent stem plastic. Despite their relative quiescence, LSC harbor self- cells (iPSCs) to HSCs via an endothelial intermediate yield few renewal capacity that can, ultimately, re-grow ALL from a functional HSCs that have limited long-term multilineage single cell. Complicating their study, LSC fates are at least potential, suggesting that available protocols fail to account partly governed by the microenvironment. An additional for necessary embryonic signals. We have previously shown challenge is that LSC are defined by their functional that embryonic blood flow is one such signal required for ability to regrow ALL, making identifying and isolating LSC effective HSC formation. How hemodynamic forces are cumbersome. transduced into gene expression changes in select vascular We have developed strategies to enrich B- and T-ALL LSC cells to drive HSC production remains unclear. from a transgenic rag2:hMYC zebrafish ALL model. We Here we present a microfluidics-based dorsal-aorta- found that dexamethasone (DXM) or gamma irradiation on-a-chip platform that enables the culture of human (IR) depletes most zebrafish B- or T- ALL cells and markedly iPSC-derived hemogenic endothelial cells (HECs) increasing LSC frequencies. In parallel studies, we identified exposed to hemodynamic forces. We identified the putative B- and T-ALL LSC using a flow cytometric “side transcription factor YAP as a potential candidate mediating population (SP) assay”. This method exploits the ability of mechanotransduction from blood flow in HSC development. stem cells to extrude fluorescent dyes like Hoechst 33342 Using loss- and gain-of-function genetic tools in zebrafish, via ATP-binding cassette (ABC) transporters. After dye we show that Yap-deficient animals have profound deficits exposure, Hoechst-positive cells are enriched for stem cells in HEC specification, while Yap overexpression increases or, in other systems, cancer stem cells. We found SP cells are runx1-specification and hematopoietic stem and progenitor increased in relapsed ALL from IR- or DXM-treated zebrafish. cell (HSPC) numbers in the embryo. Molecularly we show Moreover, allo-transplants using SP cells have higher that Rho-GTPase stimulation mimics Yap-overexpression, engraftment rates, indicating LSC enrichment. Overall, our inducing HSPCs in embryos lacking blood flow and results suggest that ALL SP cells from rag2:hMYC zebrafish increasing Runx1 expression and hematopoietic colony are significantly enriched for LSC by DXM or IR treatment. potential in iPSC-derived HEC grown in static culture. To Using these LSC-enriching methodologies, we next aim to understand how Yap regulation is induced by blood flow, we define the expression profiles of LSC vs. bulk ALL, in order to have investigated several potential cell surface networks: find LSC-specific markers to detect them, and LSC-specific pathway to target therapeutically......

12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 14 Oral Abstracts O-38 The endothelial niche detoxifies HSCs from ROS in the body. However, there is increasing evidence for the the caudal hematopoietic tissue. heterogeneity of tissue-resident macrophage populations,

1 1 and therefore many groups try to develop new tools to Pietro Cacialli , Julien Y. Bertrand discriminate each MPS subset from other hematopoietic 1 Department of Pathology and Immunology, School of lineages. Mpeg1.1 is an evolutionary conserved gene that Medicine, University of Geneva, Switzerland encodes perforin-2, a pore-forming protein associated with host defense against pathogens. The mpeg1.1:GFP Hematopoietic stem cells (HSCs) are responsible for and mpeg1.1:mCherry reporter lines have originally been sustaining hematopoietic homeostasis. In the adult, it has established to specifically mark macrophages. Since, more been well established that their functions can be affected than 300 peer-reviewed publications using mpeg1.1- by reactive oxygen species (ROS) that are produced driven fluorescent reporter lines have been published, endogenously through cellular metabolism or after exposure to observe macrophages from the embryo to the adult. to exogenous stress. An increase of ROS can inhibit HSC self- Although it is agreed that mpeg1.1 marks macrophages renewal and induce HSC senescence, resulting in premature only in the zebrafish embryo, very few data are available exhaustion of HSCs and hematopoietic dysfunctions. concerning mpeg1.1 expression in the adult immune Here we show that ROS similarly affect HSCs during their system. We therefore sought to investigate whether expansion phase in the embryo. We show that connexins mpeg1.1 constituted an adequate marker to isolate tissue- are important in regulating ROS levels in HSCs. Indeed, their resident macrophages in the adult zebrafish. By combining inhibition increases the level of ROS in HSCs and induces fluorescent reporter lines and gene-expression analysis, we their cell-death in the Caudal Hematopoietic Tissue (CHT) observed that, together with macrophages, a subpopulation niche of zebrafish embryos. The loss of HSCs after connexin of B-lymphocytes was marked by mpeg1.1 reporters in all inhibition can be rescued by reduced-Glutathione (GSH) adult zebrafish organs investigated (WKM, spleen, gut and treatment. In cells, GSH levels can be modulated by several skin). These lymphoid cells expressed the endogenous enzymes. Here, we show the importance of a new gene in mpeg1.1 transcript and could be accurately separated from this process: Gamma-interferon-inducible lysosomal thiol mpeg1.1-positive macrophages by flow-cytometry based on reductase (GILT/ifi30), an important enzyme for antigen light scatter characteristics. Remarkably, our analyses also presentation in the context of immunity, can rescue the revealed that B-lymphocytes, rather than macrophages, HSC loss resulting from ROS toxicity. Unexpectedly, we constitute the main mpeg1.1-positive population in irf8null found that ifi30 was highly expressed in endothelial cells myeloid-defective mutants, which were previously reported (ECs) from the CHT, but not in HSCs. Endothelial-specific to recover tissue-resident macrophages in adulthood. ifi30 overexpression increased HSCs expansion in the Overall, our results demonstrate the limitations of using CHT. Moreover, we found a high increase of ROS in ifi30- mpeg1.1-driven reporters and highlight the need for deficient embryos, resulting in a defect of HSC expansion in alternative macrophage-specific markers to study the MPS the CHT. This defect was rescued by several anti-oxidants: in adult zebrafish. GSH and N-acetyl-cysteine. Altogether, our data show that HSCs transfer ROS to the endothelial niche, where all the tools are expressed to detoxify the microenvironment. This O-40 A zebrafish model for Shwachman-Diamond new role of ifi30 seems to be conserved during human syndrome reveals dynamic p53-dependent changes embryogenesis as most of immature hematopoietic and suggests a p53-independent role in developmental progenitors are associated with IFI30/GILT expressing cells abnormalities in the human fetal liver. Usua Oyarbide1,2, Wilmer Amaya-Mejia1, Jacek Topczewski3,4, Seth J. Corey1,2 O-39 The macrophage-expressed gene (mpeg) 1.1 identifies a subpopulation of B cells in the adult zebrafish 1 Department of Pediatrics, Children’s Hospital of Richmond and Massey Cancer Center at Virginia Commonwealth Giuliano Ferrero1, Etienne Gomez2, Sowmya Lyer3,4, Mireia University, Richmond, VA,2 Departments of Pediatrics, Rovira1, Magali Miserocchi1, Dave M. Langenau3,4, Julien Cancer Biology, Translational Hematology and Oncology Y. Bertrand2, Valérie Wittamer1 Research, Cleveland Clinic, Cleveland, OH,3 Department of Pediatrics, Stanley Manne Research Center, Northwestern 1 Institut de Recherche Interdisciplinaire en Biologie University School of Medicine, Chicago, IL,4 Department of Humaine et Moléculaire (IRIBHM),ULB, Brussels, Belgium,2 Biochemistry and Molecular Biology, Medical University of Department of Pathology and Immunology, University Lublin, Poland of Geneva, School of Medicine, Geneva, Switzerland,3 Department of Pathology and Center for Cancer Research, Shwachman-Diamond Syndrome (SDS) is an autosomal Massachussets General Hospital Research Institute, recessive disorder characterized by pancreatic insufficiency, Boston, MA,4 Harvard Stem Cells Institute, Boston, MA, neutropenia, and an increased risk of leukemia. The disorder 02114, USA occurs in 1/75,000 births and in 90% of SDS patients biallelic mutations in the SBDS gene are found. SBDS is involved The mononuclear phagocytic system (MPS) consists of many in the assembly of the eukaryotic 80S ribosome. In mice, cells, in particular macrophages, scattered throughout ...... genetic ablation of Sbds results in early embryonic lethality ......

..... 15 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts (ED 6.5) hampering our efforts to understand of Sbds bone marrow phenotype but their phenotypic HSCs are function. Zebrafish provide an attractive, alternative model lower in frequency, more proliferative and transcriptionally organism to study SDS because the Sbds protein shares 90% more committed to differentiation compared to WT. identity to the human orthologue. Furthermore, they show increased DNA damage indicating these HSCs experience proliferative stress. Using genome editing, we created sbds zebrafish in/del strains that phenocopy the key futures of human SDS. In zebrafish, wo t orthologues of Gata2 exist: gata2a Survival of mutants was normal at 15 dpf but declined and gata2b, of which lineage analysis revealed that all significantly at 21 dpf. Western blot analysis showed a hematopoietic cells once expressed gata2b. We found that progressive decrease of Sbds during the first 8 dpf with total complete loss of gata2b results in a severe reduction in absence at 10 dpf. These results suggest that larval viability myeloid differentiation. Furthermore, gata2b+/- zebrafish is due to maternally deposited sbds transcript and translated develop dysplasia of several myeloid lineages as is found protein, which was confirmed by qRT-PCR and immunoblot in patients with GATA2 mutations. Using single-cell at early developmental stages (from 1-cell stage until 5 RNA sequencing we aim to identify early onset markers dpf). Zebrafish sbds mutants show neutropenia at 5 and of dysplasia and unveil the mechanism of malignant 15 dpf but normal macrophage and erythrocyte numbers. transformation. Organs such as the eye, pancreas, liver, and digestive tract display signs of atrophy at 21 dpf. RNAseq analysis indicated Our zebrafish model recapitulates characteristics of the that Sbds-deficiency decreased cholesterol biosynthesis GATA2 deficiency syndromes and it can give new insight (mvda) and activated fatty acid synthesis (fasn) and the into the pathophysiology of this disease. p53 pathway (e.g., p53, cdkn1a, bax, and puma). However, elimination of p53 activity is not sufficient to suppress sbds mutant lethality. O-42 Chemokine signaling is a driver of osteoclast recruitment in a medaka osteoporosis model We propose that the unique phenotype of SDS is a sum of several abnormally regulated molecular pathways, mediated Phan Quang Tien1, Tan Wen Hui1, Liu Ranran1, Nurgul only in part by the p53. We are currently determining which Imangali1, Anita Buettner1, Sudha Sundaram1, Susanne specific p53 targets are involved in the pathogenesis of SDS Kneitz2, Manfred Schartl2,3, Christoph Winkler1

and the identity of non-p53 pathways (metabolism defects) 1 involved in SDS. Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, Singapore 117543,2 Physiological Chemistry, Biozentrum, University of Würzburg, 97074 Würzburg, Germany,3 O-41 A zebrafish model for Gata2 haploinsufficiency Hagler Institute for Advanced Study and Department of mediated myeloid malignancy Biology, Texas A&M University, College Station, TX 77843, Emanuele Gioacchino, Cansu Koyunlar, Hans de Looper, USA Joke Peulen, Dennis Bosch, Remco Hoogenboezem, Bone is a highly dynamic tissue that requires a tightly Paulette van Strien, Eric Bindels, Kirsten L Gussinklo, coordinated balance of controlled bone resorption and bone Elaine Dzierzak, Ivo Touw and Emma de Pater formation to maintain its structural integrity. Imbalances Department of Hematology, Erasmus MC, Rotterdam, The caused by deficient intercellular communication between Netherlands bone-forming osteoblasts and bone-resorbing osteoclasts can lead to bone disorders such as osteoporosis. We Dept of Cell Biology, Erasmus MC, Rotterdam, The previously showed that transgenic expression of Receptor Netherlands activator of nuclear factor kappa-Β ligand (RANKL) leads to ectopic formation of osteoclasts and osteoporosis-like The Queen’s Medical Research Institute College of lesions in medaka embryos. Here, we show that these Medicine and Veterinary Medicine, Edinburgh, United RANKL-induced osteoclasts originate from mpeg1-positive Kingdom macrophages that migrate to the vertebral column where MonoMAC and Emberger syndromes are GATA2 they differentiate into osteoclasts. To identify signals that haploinsufficiency syndromes. These syndromes are induce macrophage recruitment and differentiation, we characterized by monocytopenia, neutropenia, B-, performed transcriptome profiling of various bone cell dendritic- and NK cell lymphopenia and up to 80% of patients types that were FACS-purified from RANKL-induced medaka with innate GATA2 mutations develop myelodysplastic embryos. Among the analyzed transcripts, we identified syndrome (MDS)/ acute myeloid leukemia (AML). Gata2 the chemokine Cxcl9-like (Cxcl9l), which was up-regulated level is a key factor in embryonic hematopoietic stem cell in osteoblasts, and its corresponding receptor Cxcr3.2, (HSC) generation and maintenance. To study the effect of which is strongly expressed in early osteoclasts. CRISPR/ Gata2 haploinsufficiency on the mechanism of malignant Cas9 mediated deletion ofcxcl9l did not affect macrophage transformation, we use both mouse and zebrafish Gata2 recruitment but severely impaired the differentiation of haploinsufficiency models. Gata2 heterozygous mutant macrophages into osteoclasts. On the other hand, knocking mice survive to adulthood without developing any visible out Cxcr3b blocked macrophage recruitment and osteoclast ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 16 Oral Abstracts differentiation in RANKL-induced medaka embryos cell engraftment in vivo: lysophosphatidic acid (LPA) and demonstrating that signaling through Cxcr3 is essential for niflumic acid (NFA). Using a bioluminescence imaging, osteoclast formation. In both mutants, osteoporotic lesions we further ascertained that both NFA and LPA enhance were absent after RANKL-induction. Finally, transgenic satellite cell engraftment in mouse (n=15, 1-way ANOVA, expression of cxcl9l in osterix (sp7, osx) positive osteoblasts p ≤ 0.05), indicating conservation of the promyogenic was sufficient to induce macrophage recruitment to bone activities of these compounds across vertebrate species. and their differentiation into osteoclasts in the absence Studies in sapje-like fish (dystrophin mutant) transplanted of ectopic RANKL. In conclusion, we suggest that Cxcl9l with NFA- or LPA-treated cells showed higher engraftment secreted from osteoblasts, likely together with other so efficiency and significantly better swimming performance far unknown ligands, recruits macrophages to bone matrix against a water current, compared to fish engrafted with through Cxcr3.2 activation. Also, while Cxcl9l is dispensable vehicle treated cells. Mechanistically, the promyogenic but sufficient for the recruitment of macrophages, itis activities of LPA and NFA appear to be associated with required for their differentiation into osteoclasts. This increased cytoplasmic Ca2+, and RNA sequencing analysis project is supported by a grant from the Singapore Ministry identified calcium binding dependent genes, including of Education (MOE2016-T2-2-086). Egfem1, Ndrg and Mep1a, that were regulated in NFA and LPA treated cells. The success of this cross-species approach to uncovering evolutionary conserved pathways regulating O-43 A cross-species biomolecule screen uncovers muscle regeneration suggests new potential opportunities chemical inducers of skeletal muscle engraftment for treating muscle disease by enhancing myogenic contributions of transplanted muscle progenitors. Sahar Tavakoli 1,2, Eric Gahwiler 1,3, Apoorva Rangan 1, Sara Ashrafi Kakhki1 , Victoria S. W. Chan 1,2, Margot E. Manning 1,2, Kathleen Messemer 1, Haleh Fotowat 4, Isaac 1 1,5,8 1,2,6,7,8* O-44 Focal neuroinflammatory signals drive spinal curve Adatto , Amy J. Wagers *, and Leonard I. Zon formation in zebrafish models of idiopathic scoliosis 1 Harvard Department of Stem Cell and Regenerative Jenica Van Gennip1,2, Curtis Boswell1,2, Brian Ciruna1,2 Biology, Cambridge, MA 02138, USA, 2 Division of Hematology/Oncology, Children’s Hospital Boston and 1 Program in Developmental & Stem Cell Biology, The Dana-Farber Cancer Institute, Boston, MA 02115, USA,3 Hospital for Sick Children, 686 Bay Street, Toronto, Ontario ETH Zurich, Institute for Regenerative Medicine – IREM, M5G 0A4, Canada,2 Department of Molecular Genetics, Zürich, Switzerland;4 Harvard Department of Molecular University of Toronto, Toronto, Ontario M5S 1A8, Canada and Cellular Biology, Cambridge, MA 02138, USA,5 Joslin Diabetes Center, Boston, MA 02115, USA,6 Howard Hughes Idiopathic scoliosis (IS) is a debilitating disease characterized Medical Institute, Harvard Stem Cell Institute, Boston, MA by three-dimensional curvatures of the spine that arise 02115, USA,7Harvard Medical School, Boston, MA 02115, in the absence of observable physiological or anatomical USA,8These authors contributed equally to this work defects. Although there has been investigation into genetic components of IS, the underlying cell biological and *Correspondence: [email protected] (A.J.W.), molecular mechanism for disease progression remains [email protected] (L.I.Z.) unknown. Consequently, treatment options are limited to bracing or surgery, which act more as preventative measures Genetic muscle disorders compromise muscle function rather than a cure. Our lab has previously described the through increased inflammation and impaired muscle first genetically defined developmental model of IS in ptk7 regeneration. Transplantation of exogenous muscle mutant zebrafish. Homozygous ptk7 mutant zebrafish progenitor cells could be an approach to enhance display hallmark phenotypes of the human disease, muscle function and repair; but this approach has been including 3-dimensional rotational spinal deformities, limited due to the typically poor engraftment efficiency and sexual dimorphic bias in phenotype severity. It was of cultured progenitors. To define critical regulators of found that ptk7 mutants display a late-onset motile cilia muscle engraftment, we developed a novel cross species defect that was directly responsible for the scoliosis platform, employing both zebrafish and mouse, to discover phenotype. To identify underlying mechanisms of disease, chemical compounds that promote muscle progenitor we employed next generation sequencing, lineage tracing engraftment in vivo. Muscle cells derived from zebrafish and conditional genetic methodologies to define the spatial blastomeres were treated for 4 hours with biomolecules and biological origins of spinal curve formation in ptk7 and transplanted into the flanks of adult zebrafish (15 mutant zebrafish. RNA-sequencing revealed an unexpected recipient flanks per compound). We focused our screening upregulation of immune factors. This, in conjunction with on a well-annotated library of 230 bioactive lipids; since previous work, suggests that scoliosis may be caused by a lipids are known to enhance cell migration and to regulate neuroinflammatory response due to defective CSF flow. We the homeostasis and regenerative function of muscle and demonstrate that focal activation of proinflammatory signals blood stem cells. Using limit-dilution assays, potential within the spinal cord is sufficient to initiate idiopathic- “hits” from the primary screen were identified and re- like spinal curvature, and that administration of anti- evaluated in replicate transplantation experiments. We inflammatory molecules to juvenile ptk7 mutant animals discovered two lipids that promote muscle progenitor ......

..... 17 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts significantly impacts the onset of scoliosis. Together, our NY11501,4 Wellcome Trust Sanger Institute, Wellcome data suggests that neuroinflammatory signals downstream Trust Genome Campus, Hinxton, Cambridge, CB101SA, UK of disrupted CSF homeostasis can initiate spinal deformity, and that modulation of these inflammatory signals could Plasma hyperlipidemia is a major risk factor for provide a treatment for this disease. cardiovascular disease. The microsomal triglyceride transfer protein complex (MTP) is essential for the assembly of plasma Apolipoprotein B (ApoB) containing lipoproteins (B-lps) in the intestine and liver. As ApoB is translated and O-45 On building and rebuilding the skeleton translocated into the endoplasmic reticulum, MTP transfers Joana Lopes1, Katrin Henke1, David Karasik2, Matthew lipids to ApoB to form primordial lipoproteins. Mutations Harris1 in the M-subunit (MTTP) gene cause Abetalipoproteinemia, which is characterized by intestinal lipid malabsorption 1Department of Genetics, Harvard Medical School; and an absence of plasma B-lps. As such, MTP has long Department of Orthopaedics, Boston Children’s been considered a target for lipid-lowering therapeutics.

2 While all currently described Abetalipoproteinemia disease Hospital, Azrieli Faculty of Medicine, Bar-Ilan University, mutations result in the complete loss of lipid transfer activity, Safed 1311502, Israel through forward genetic screening, we have discovered a The skeleton is a dynamic system, responding to changes zebrafish mutant (mttpc655) expressing a single C-terminal in the environment and internal physiology to shape the missense mutation (G863V), that only partially abrogates form and systemic signaling of musculoskeletal tissues. MTP function. In vitro, the G863V Mttp protein is Human diseases affecting this dynamic balance often lead defective at transferring triglycerides, but unexpectedly to debilitating disorders having different amount of bone of retains phospholipid transfer activity. The same effect was varied quality formed. We capitalize on the ease of genetics observed using a similarly mutated human mttp construct. in the zebrafish to establish defined models in which to The G863V mutation reduces the production and size of assess regulation of bone quality and remodeling. Using B-lps in vivo and results in the abnormal accumulation of specific mutants in known disease-causing genes, we show cytoplasmic lipid droplets in the yolk syncytial layer of the that the zebrafish nicely recapitulates phenotypes seen in zebrafish embryo. However, it only has mild effects on human patients. For example, we identify specific mutations lipid malabsorption in the intestine and no consequence in collagen1a1 that lead to an osteogenesis imperfecta on growth. In contrast, zebrafish mutants bearing the phenotype. For osteopetrosis, we show sensitivity of previously identified mttpstl (L475P mutation), which blocks the zebrafish model to detect changes in the balance of transfer of both triglycerides and phospholipids, exhibit osteoclast activity and provide a working model for coupling gross intestinal lipid accumulation and defective growth between osteoblasts and osteoclasts, a process at the core of due to a severe deficiency in lipoprotein production. Thus, remodeling. Using the zebrafish, we have devised screening the G863V point mutation provides the first evidence that methods in order to systematically assess function of genes the triglyceride and phospholipid transfer functions of a linked to loci associated with altered bone mineral density vertebrate MTP protein can be biochemically separated. in human populations. The identification of regulators Significantly, these data suggest that selective inhibition of of skeletal growth, differentiation, and remodeling using the triglyceride transfer activity of MTP may be an effective the zebrafish will provide substantial insight into how the strategy for treating hyperlipidemia, while preventing the skeleton forms and remodels, as well as provide potential known gastrointestinal side-effects associated with broad avenues into regulating this process. This work partially inhibition of MTP activity. supported through NSF/BSF Joint Funding grant 201720.

O-47 Optogenetic modulation of central and intracardiac O-46 A new zebrafish allele of microsomal triglyceride neural pathways controlling heart rhythm in zebrafish in transfer protein suggests that selectively inhibiting development, ageing, and disease triglyceride transfer would be effective to treat Matthew R. Stoyek1, Frank M. Smith2, Roger P. Croll1, dyslipidemia Thomas Brand3, T. Alexander Quinn1,4 1 1,2 3 Meredith H. Wilson , Aidan Danoff , Sujith Rajan , 1 4 1 Department of Physiology & Biophysics, Dalhousie Richard White , Monica R. Hensley , University, Canada,2 Department of Medical Neuroscience, 3 1,2 1,2 Dalhousie University, Developmental Dynamics, National Vanessa H. Quinlivan , James Thierer , Elisabeth M. 4 Busch-Nentwich4, M. Mahmood Hussain3, Heart and Lung Institute, Imperial College London, UK, School of Biomedical Engineering, Dalhousie University Steven A. Farber1,2 The heart’s beating rate and its adaptation to stress is 1Carnegie Institution for Science Department of regulated in part by the nervous system, including a Embryology, Baltimore, MD 21218,2 Johns Hopkins portion within the heart itself - the intracardiac nervous University Department of Biology, Baltimore, MD 21218,3 system (ICNS). Normal operation of the ICNS is key to New York University Winthrop Hospital, Mineola, supporting cardiovascular homeostasis, though neither its ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 18 Oral Abstracts development nor its remodeling with aging and in various rapidly develop severe aneurysmal dilatation of the cardiac cardiopathies is well understood. We have begun to utilize outflow tract (OFT), which is anatomically equivalent to the an optogenetic approach to extend our understanding of aortic root in humans, where TAAs commonly form in MFS the functional connectivity and network properties of the and LDS patients. Between days 3 and 5 post-fertilization, ICNS and its role in maintaining heart rhythm. A cation the diameter of the mutant OFT increases by greater than non-specific, light-activated ion channel (ChRWR) or 2 fold, and the OFT wall begins to exhibit several hallmarks intracellular Ca2+ indicator (GCaMP6) were expressed in all of TAA, which include elevated levels of TGFβ signaling and neurons (driven by HuC), or specifically in parasympathetic a characteristic molecular signature. Prior to aneurysm (ChAT) or sympathetic (TH) neurons. In intact zebrafish formation, the smooth muscle cells of the OFT pre-maturely larvae, the heart shows hormonal control at 3-4 days post adopt a “contractile” phenotype, which we hypothesize is a fertilization (dpf), vagal axons reach the heart at 7-9 dpf, primary driver of the disease phenotype. Current efforts are and the first neuronal somata arrive between 14-21 dpf. underway to determine whether elevated and/or reduced Experiments in HuC:ChRWR zebrafish have shown that TGFβ signaling are necessary and sufficient for instigating function is correlated with anatomy, as photo-stimulation TAA pathogenesis in wild-type and mutant animals. of distinct central and ICNS regions evoke graded heart rate Recent reports indicate that mutations in ltbp1 or ltbp3 responses that correlate with anatomical development. are linked to TAA susceptibility in humans, which validate We have shown in ageing zebrafish that although basal the relevance of ltbp1-/-, ltbp3-/- animals for investigating heart rate does not change with age, the total number TAA pathogenesis. Importantly, our new disease model will of ICNS neurons increases and the heart rate response to serve as a screening tool for identifying novel compounds vagal nerve stimulation decreases. We have also recently that prevent and treat aneurysmal disease. demonstrated ICNS dysfunction in zebrafish expressing the popdc1S191F mutation, as pharmacologic or vagal nerve stimulation evokes cardiac rhythm destabilization. In O-49 Hif-1alpha modulates neutrophil migration to ongoing work, we are using our optogenetic approach to inflammation and infection sites in a dual-model investigate how the structure, function, and connectivity of ICNS pathways are altered with ageing and disease. Overall, Abdirizak Mohamed1,2*, Yves Schild1,2*, Edward J results of this project will establish: i) the ontogeny of cardiac Wootton1,2*, Amy Lewis1,2 and Philip M. Elks1,2 control pathways in neurodevelopment; ii) physiologic ICNS 1 The Bateson Centre, University of Sheffield, Western functional control and neural network properties; and iii) 2 how these parameters change during normal ageing and in Bank, Sheffield, UK, Department of Infection and disease. Immunity and Cardiovascular Disease, University of Sheffield, Western Bank, Sheffield, UK *Joint authors O-48 Latent TGFβ binding proteins 1 and 3 function redundantly to protect zebrafish from aortic root Multi-drug resistant infections are a worldwide problem aneurysm and there is an urgent need for host-derived therapeutic targets. We have previously shown that hypoxia Maryline Abrial1,3, Asya Schwertner1,3, Spencer Jeffrey1, inducible-1α (Hif-1α) stabilisation helps the host to clear Caroline E. Burns1,2,3,4*, C. Geoffrey Burns1,2,3* mycobacterial infection via neutrophil antimicrobial nitric oxide production. However, Hif-1α stabilisation has also 1 Cardiovascular Research Center, Massachusetts General 2 been implicated in chronic inflammatory diseases, causing Hospital, Charlestown, MA 02129 , Department of delayed neutrophil inflammation resolution. Infection and Cardiology, Boston Children’s Hospital, Boston, MA 02115, 3 4 chronic inflammation are often found together in human Harvard Medical School, Boston, MA 02115, Harvard disease, so it remained unclear whether Hif-1α stabilisation Stem Cell Institute, Cambridge, MA 02138 would be beneficial in a holistic disease setting. *Co-senior authors Here, we set out to understand the effects of Hif-1α Thoracic aortic aneurysms (TAAs) are the leading cause stabilisation in a dual zebrafish disease model, by combining of death for individuals affected by the connective tissue two well-characterised in vivo models; TB infection disorders Marfan Syndrome (MFS) and Loeys-Dietz (Mycobacterium marinum) and sterile inflammation (tailfin Syndrome (LDS). In both cases, dysregulated transection). Our data show that infection led to neutrophil emergency haematopoeisis without changing the dynamics TGFβ signaling has been implicated in disease pathogenesis. of inflammation resolution. Sterile inflammation was However, the molecular and cellular mechanisms responsible detrimental to infection, with increased burden in larvae for TAA formation are controversial, and the search for novel with tailfin transections. We used variations of this dual- medical therapies remains an active area of investigation. model to investigate neutrophil migration to bacterial Moreover, despite the advantages of zebrafish for scientific infection and wound sites after Hif-1α modulation, and inquiry, a disease model of TAA has yet to be generated in assessed infection and inflammation outcomes. Hif-1α this species. We discovered that zebrafish larvae carrying stabilisation caused a delay in neutrophil inflammation null alleles of latent TGFβ binding protein (ltbp) 1 and ltbp3 resolution in the dual-model and modulated neutrophil ......

..... 19 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts migration to the sites. Despite these changes in neutrophil West Lafayette, IN 47907 USA,2 Medical College of China migration behaviour, the protective effect of Hif-1α Three Gorges University, Yichang, Hubei, 443002 PR China stabilisation against infection remained in the dual-model. ,3 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202 USA These data indicate that Hif-1α plays a role in neutrophil ,4 Collaborative Core for Cancer Bioinformatics, Indiana migration to infection and wound stimuli. The protective University Simon Cancer Center, Indianapolis, IN 46202 effect of Hif-1α in infection is maintained in a zebrafish USA dual-model, highlighting its potential as a host-derived 5 Department of Medical Microbiology and Immunology, target against multi-drug resistant infection in a disease University of Wisconsin-Madison, Madison, WI, USA,6 relevant setting. Department of Pediatrics, University of Wisconsin- Madison, Madison, WI, USA, 7 Center for Computational Biology and Bioinformatics, Indiana University School of O-50 Understanding the molecular diversification of Medicine, Indianapolis, IN 46202 USA, 8 Purdue Institute self recognition through ray-finned fish innate immune for Inflammation, Immunology, & Infectious Disease, receptor families. Purdue University, West Lafayette, IN 47907 USA Dustin J. Wcisel1, Jeffrey A. Yoder1,2,3, Lindsay Roupe- Neutrophil migration is essential for inflammatory responses Abrams4, Alex Dornburg4 to kill pathogens and cause tissue injury. To discover novel 1 Department of Molecular Biomedical Sciences, 2 therapeutic targets that modulate neutrophil migration, we Comparative Medicine Institute,3 Center for Human Health performed a neutrophil-specific microRNA overexpression and the Environment, NC State University, Raleigh, NC screen in zebrafish and identified eight microRNAs as USA,4 North Carolina Museum of Natural Sciences, Raleigh, potent suppressors of neutrophil migration. Among those, NC USA miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally The use of molecular markers of self-identity as a basis for differentiated neutrophils, miR-199 alters the cell cycle- immunity was a major evolutionary innovation in the early related pathways and directly suppresses cyclin-dependent history of vertebrates. The ability to discriminate between kinase 2 (cdk2), whose known activity is restricted to self and non-self has since spurred a coevolutionary cell cycle progression and cell differentiation. Inhibiting competition between hosts and pathogens driving high CDK2, but not DNA replication, disrupts cell polarity and levels of both inter- and intraspecific immune gene sequence chemotaxis of zebrafish neutrophils. Chemotaxis of primary diversity. Such diversification is essential for a species to human neutrophils are also reduced by CDK2 inhibition. survive new pathogens, yet the origin and evolutionary Furthermore, miR-199 overexpression or CDK2 inhibition dynamics of vertebrate self recognition remain poorly significantly improves the outcome of lethal systemic understood. A powerful system for understanding the inflammation challenges in zebrafish. Together, our results genetic and functional evolution of immune genes associated reveal previously unknown functions of miR-199 and with self recognition are ray-finned fishes (Actinopterygii) CDK2 in regulating neutrophil migration and provide new which constitute over half of all living vertebrates. Like all directions in alleviating systemic inflammation. vertebrates, fish possess certain core immune gene families, however they also encode a number of “fish-specific” immune gene families. Using a phylogenetic comparative framework, we integrate transcriptome and genomic O-52 Intravital imaging of fungal-innate immune sequence data from early diverging lineages of ray-finned interactions fishes to establish the evolutionary origins of fish-specific Allison K. Scherer1, Brittany G. Seman1, Jessica L. Moore1, immune receptor genes and test fundamental expectations Bailey A. Blair1, Linda S. Archambault1, Sony Manandhar1, of coevolution between markers of self and their candidate Joshua M. Jones1, Robert T. Wheeler1,2 receptors. Our work provides a new perspective on the 1 Department of Molecular & Biomedical Sciences, and 2 early history of the vertebrate immune system, overturning Graduate School of Biomedical Sciences and Engineering, assumptions of teleost specific innovations, while revealing University of Maine, Orono, ME 04469 novel molecular innovations to pathogen resistance. Candida albicans is a dimorphic fungus that lives benignly on mucosal surfaces of most individuals. Unfortunately, it can become a serious pathogen in immunocompromised O-51 Phenotypic microRNA screen reveals miR-199 and patients, causing a disseminated disease which leads to CDK2 as novel regulators of neutrophil migration and devastating organ damage and patient mortality. We have systemic inflammation shown using intravital imaging that fungal-innate immune Alan Y. Hsu1, Decheng Wang1,2, Sheng Liu3,4, Justice interactions and dynamics in the live host can differ Lu1, Ramizah Syahirah1, David A. Bennin5, Anna significantly from expectations based onin vitro challenges. Huttenlocher5,6, Jun Wan3,4,7, Qing Deng1,8 One important aspect of disease that is especially difficult to follow in murine infection models and in vitro 1 Department of Biological Sciences, Purdue University, is the dissemination of a localized infection through the ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 20 Oral Abstracts bloodstream. The mechanisms by which C. albicans spreads responses, whether they influence neutrophil bactericidal to cause disseminated infection remain poorly understood, activity, the most abundant immune cells that provides an so we have exploited the transparent zebrafish as a model indispensable first line of defence against bacteria, remains vertebrate host to characterize fungal and host determinants unknown. Here we reveal that neutrophils upregulate of infection spread. Quantitative intravital imaging was expression of the core clock gene per2 in response to used in conjunction with multiple genetic and non-genetic infection (Salmonella Typhimurium) that is essential for manipulations to determine that the two morphologies their antibacterial activity. Neutrophils deficient in per2 of this pleomorphic fungus have distinct and specialized cannot kill phagocytosed bacteria, even when transplanted roles in infection. Time-lapse and longitudinal imaging into WT recipients, and demonstrate decreased production have further demonstrated that the yeast form is best of two fundamental antibacterial weapons, reactive oxygen adapted for dissemination throughout the host. Through species (ROS) and complement factor properdin (Cfp, a a combination of non-invasive imaging and perturbation positive regulator of the alternative complement pathway). of specific host mechanisms, we have identified roles for Consistent with a role for per2 in driving neutrophil Cfp phagocytes in “Trojan Horse” dissemination of yeast and production,per2 mutant larvae are also more susceptible to also uncovered phagocyte-independent extracellular bacterial infections due to defective neutrophil recruitment spread. Morphological quantification in whole fish and neutrophil phagocytic ability, effector mechanisms of demonstrates that these divergent strategies represent complement activation. This discovery is significant asit redundant mechanisms utilized by C. albicans to travel to is the first time a core clock gene has been shown to cell- distant areas of the host and create new infection foci. Thus, autonomously control neutrophil antibacterial activity and while the host can aid in the dissemination of yeast through has been linked with the complement system, one of the immune responses and barrier disruption, this is only one most ancient components of innate immunity. component of a complex process utilized by a versatile fungal pathogen. Leveraging the zebrafish system, we will continue to interrogate the contributions of individual O-54 Pathogenic macrophage necrosis in Tuberculosis pathogen and host proteins in dissemination within a live vertebrate host. Lali Ramakrishnan1 1 University of Cambridge, UK O-53 Helping immunity run like clockwork: the core clock I will describe how a zebrafish forward genetic screen led us gene per2 contributes to neutrophil bactericidal activity to a human variant is associated with human susceptibility, in a cell-autonomous fashion how we have obtained the detailed mechanism of this

1 1 1 susceptibility by returning to the zebrafish and identifying Pramuk Keerthisinghe , Lucia Du , Hannah Darroch , a new pathway of pathogenic necrosis in the fish, and how Tanja Linnerz1, Kathy Crosier1, James Cheeseman2, Phil 1 2 1 to target this pathway with existing, inexpensive drugs to Crosier , Guy Warman , Chris Hall prevent necrosis. This work exemplifies the use of the 1 Department of Molecular Medicine and Pathology, zebrafish to uncover both new biology and personalized Faculty of Medical and Health Sciences, University of therapeutics that could be lifesaving in a disease that Auckland, New Zealand,2 Department of Anesthesiology, remains a major global scourge. Faculty of Medical and Health Sciences, University of Auckland, New Zealand O-55 Zebrafish with Noonan Syndrome develop The antibacterial immune response exhibits circadian myeloproliferative disease rhythmicity, with enhanced bacterial clearance during times of physical activity. This clever adaptation likely evolved to Jeroen den Hertog1,2 and Maja Solman1 synchronise elevated antibacterial immune responses with 1 Hubrecht Institute-KNAW and University Medical Center the increased threat of infection while active. Supporting 2 the conservation of this circadian response in zebrafish, Utrecht, Utrecht, the Netherlands, Institute Biology we have previously shown that larval zebrafish infected Leiden, Leiden, the Netherlands during the light/active phase demonstrate enhanced PTPN11 encodes SHP2, a modular protein with a protein- bacterial clearance compared to those infected during tyrosine phosphatase domain and two N-terminal SH2 the dark/inactive phase. Understanding the cellular and domains. Mutations inPTPN11 have been identified to cause molecular mechanisms behind this circadian-regulated Noonan syndrome (NS), which belongs to the RASopathies, a immune response promises to revolutionise when we treat group of human syndromes which display similar symptoms infections/inflammatory diseases and may reveal mays to and have in common that RAS-MAPK signaling is affected. boost the antibacterial immune response around the clock. We use zebrafish to study the molecular-genetic and cell Circadian rhythms are controlled at the molecular level by biological basis of NS. CRISPR/Cas9 technology facilitated a highly conserved set of core clock genes that function the generation of a point mutation in endogenous ptpn11 as a transcription-translation feedback loop. Although by homologous recombination, resulting in zebrafish these core clock genes are known to contribute to immune expressing a NS-variant (D61G) of Shp2. Characterization of ......

..... 21 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts this mutant zebrafish line indicated that the developmental targeting asxl1 independently lead to color-dominant defects in these fish morphologically resemble NS states at 8mpf. We successfully utilized the Zebrabow symptoms in humans. Strikingly, Shp2-D61G fish develop color barcoding system and mosaic mutagenesis in an hematopoietic disorders, assessed by classical blood cell endogenous and prospective model to show an important stainings and using transgenic lines expressing reporters in role for mutant ASXL1 in establishing clonal hematopoiesis. specific blood cell types. Single cell sequencing technology indicated that mutant fish develop myeloproliferative disease, which may phenocopy juvenile myelomonocytic O-57 Stat3 inhibition is a synthetic lethal vulnerability for leukemia observed in NS patients. Sf3b1-mutated hematopoietic cells Kathryn S. Potts1,2*, Rosannah C. Cameron1,2*, Varun Gupta3, Ana Leal Cervantes4, LaShanale Wallace4, Mia O-56 Color barcoding and mosaic mutagenesis 1,2 5 1,6 ASXL1 McKinstry , Xiaoying Bai , Gaurav Choudhary , Amit confirms mutant as a potent initiator of clonal 1,2,6 6 4 hematopoiesis Verma , Aditi Shastri , Esther Obeng and Teresa V. Bowman1,2,6 1 2 Serine Avagyan , Jonathan E. Henninger , William P. 1 3 4 5 Albert Einstein College of Medicine, Department of Mannherz , Margaret C. Weber , Jessica Moore , Leonard 2 I. Zon4,6 Developmental and Molecular Biology, Bronx, NY, USA, Albert Einstein College of Medicine, Gottesman Institute 1 Dana Farber/Boston Children’s Hospital Cancer and for Stem Cell Biology and Regenerative Medicine, Bronx, Blood Disorders Center, Boston, MA, 2 Whitehead Institute NY, USA,3 Albert Einstein College of Medicine, Department of Biomedical Research, Cambridge, MA, 3 Harvard of Cell Biology, Bronx, NY, USA,4 St. Jude Children Research Medical School, Boston, MA, 4 Department of Hematology- Hospital, Department of Oncology, Memphis, TN, USA,5 Oncology, Boston Children’s Hospital, Boston, MA,5 Yale University of Texas, Department of Obstetrics and University, New Haven, CT,6 Howard Hughes Medical Gynecology, Dallas, TX, USA,6 Albert Einstein College of Institute Medicine, Department of Medicine (Oncology), Bronx, NY, USA Clonal hematopoiesis of indeterminate potential (CHIP) is associated with mutations in single genes found in Hematopoietic stem and progenitor cells (HSPCs) maintain hematopoietic malignancies, with normal blood production, the hematopoietic system throughout the lifetime of an but an increased risk of developing myeloid cancers over organism. Malfunctioning of HSPCs can lead to hematologic time. The role of specific mutations in the pathogenesis disorders, such as myelodysplastic syndrome (MDS). of CHIP is largely inferred from large-scale human Mutations in spliceosomal components are prevalent sequencing data and only examined in transplantation- in MDS, but how splicing regulates HSPCs is still poorly based animal models. To study the role of CHIP-associated understood. Using a zebrafish loss-of-function mutant for mutations prospectively in a native setting, we used the the most commonly mutated splicing factor in MDS, splicing Zebrabow color barcoding system to tag and follow the factor 3b, subunit 1 (sf3b1), we demonstrated that impaired clonal activity of endogenous hematopoietic stem and splicing hindered HSPC specification during development. progenitor cells (HSPCs). We color labeled HSPCs using a To identify Sf3b1-regulated transcripts, we performed RNA- tamoxifen-dependent Cre recombinase under the tissue- sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 restricted promoter draculin (drl), producing stable color mutant and unaffected siblings at 24 hpf. Approximately 900 barcodes in stem cells and their progeny. CRISPR/Cas9 genes were mis-spliced with enrichment for STAT3 (Signaling technology was used to introduce mutations in 15 zebrafish Transducer and Activator of Transcription 3) pathway orthologs of CHIP-associated genes and 5 control genes, components. Stat3 is a transcription factor regulating mosaically mutating Tg(Zebrabow-M;drl:creERT2) embryos. several cytokine and inflammatory signal responses. Mis- Color labeling of HSPCs was performed at 24 hours post spliced variants in STAT3 signaling components observed fertilization. At various timepoints between 3 and8 in sf3b1 mutants were mostly predicted to dampen months post fertilization (mpf), we measured variant allelic levels or functionality. Consistent with this observation, frequency (VAF) of each targeted gene by next-generation STAT3 target genes were significantly downregulated in sequencing in peripheral blood and kidney marrow cells. sf3b1 mutants. Overexpression of a constitutively-active On average, 6 genes were targeted in individual fish with form of Stat3 in sf3b1 mutants suppressed their HSPC ≥5% VAF. Zebrafish injected with the 20 gRNAs pool had defect. Induced mis-splicing of stat3 using antisense increased incidence of clonal dominance as measured by splice-blocking morpholinos (MO) resulted in a significant the presence of a single-colored cluster contributing to decrease in runx1- and gata2b-expressing HSPCs in sf3b1 ≥30% of granulocytes compared to controls (Chi-square heterozygotes, but elicited no effect in wild-type embryos test, p=0.004). We identified gene mutations in sorted at 28hpf. Inhibiting Stat3 using the small molecule inhibitor dominant and non-dominant clusters, and correlated those STATTIC similarly resulted in reduced HSPC specification in with respective cluster size. We found that frameshift indels heterozygotes but not wild-type embryos. We next tested if in asxl1 and DNMT3A orthologs were highly associated STAT3 inhibition selectively affected SF3B1-mutated human with larger clusters (n=88, p<0.05). Singly injected gRNAs cells. K562 cells engineered to express the common SF3B1- ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 22 Oral Abstracts K666N mutation were more sensitive to STAT3 inhibition O-59 Inflammasome-mediated expansion of the than wild-type cells. Similarly, primary MDS patient cells hematopoietic system in the vertebrate embryo

carrying SF3B1 mutations produced fewer hematopoietic 1 1 colonies when treated with STATTIC than vehicle-treated Jenna M. Frame , Timothy L. Long , Caroline Schuster- Kubaczka1, Virginie Esain2, Sung-Eun Lim3, George Q. controls. These data demonstrate that Sf3b1-regulated 1,3,4 1,3,4 splicing impacts STAT3 signaling and STAT3 inhibition is a Daley , Trista E. North conserved synthetic lethal vulnerability for SF3B1-mutated 1 Stem Cell Program, Hematology-Oncology, Boston cells with potential to treat splicing factor-mutated MDS. Children’s Hospital, Boston, MA; 2 Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA; 3 Harvard Medical School, Boston, MA, 4Harvard Stem O-58 Deletion of a conserved gata2a enhancer Cell Institute, Cambridge, MA induces MonoMAC syndrome in zebrafish and reveals a requirement for gata2a in embryonic and adult Embryonic hematopoietic stem and progenitor cells haematopoiesis (HSPCs) have the unique capacity to rapidly expand while maintaining the capacity to differentiate into mature Tomasz Dobrzycki1, Monika Krecsmarik1,2, Cansu hematopoietic lineages. As efforts to derive HSPCs in vitro Koyunlar4, Rossella Rispoli1, Joke Peulen-Zink4, Kirsten for cell therapies are suboptimal, a better understanding Gussinklo4, Emma de Pater4, Roger Patient1,2 and Rui of mechanisms regulating HSPC formation and expansion Monteiro1,2,3,* during embryogenesis is needed. We previously reported

1 that sterile inflammatory signaling stimulates production MRC Molecular Haematology Unit, MRC Weatherall of embryonic HSPCs; however, it remains unclear how the Institute of Molecular Medicine, John Radcliffe Hospital, embryo initiates inflammatory cues to control HSPC output. University of Oxford, Oxford, OX3 9DS, United Kingdom, The NLRP3 inflammasome regulates interleukin-1-beta 2BHF Centre of Research Excellence, Oxford, United 3 (IL-1β) processing in response to sterile stimuli, including Kingdom, Institute of Cancer and Genomic Sciences, reactive oxygen species and metabolic state. Since we College of Medical and Dental Sciences, University of 4 previously demonstrated that embryonic glucose exposure Birmingham, UK, Department of Hematology, Erasmus expands HSPCs, we asked whether glucose expands HSPCs MC, Rotterdam, The Netherlands through inflammasome-mediated IL-1β signaling. Il1b *correspondence: expression was upregulated with glucose treatment, and il1b Email: [email protected] knockdown suppressed the inductive effects of glucose exposure on numbers of CD41+ HSPCs at 48hpf. Conversely, The transcription factor Gata2 is required to produce overexpression of the active form of il1b increased HSPC haematopoietic stem cells HSCs during embryonic numbers as assessed by flow cytometry and runx1/cmyb in development. To address whether Gata2 plays a role situ hybridization. As IL-1β is cleaved and activated by the in haemogenic endothelium programming prior to HSC inflammasome, we found that small molecule-mediated emergence, we took advantage of a partial genome inflammasome stimulation also increased runx1/cmyb duplication in zebrafish that lead to the appearance of expression and Flk1+cMyb+ HSPCs; this effect required two Gata2 genes, Gata2a and Gata2b, the latter thought NF-kB priming, and upregulated IL-1β targets IL-6 and to be the ‘haematopoietic’ Gata2 in zebrafish. Through IL-8, which we previously showed increases embryonic deletion of a conserved intronic enhancer (i4 enhancer) HSPCs. Loss of inflammasome components reduced HSPCs, in the gata2a locus, we show that reducing gata2a levels and prevented their expansion in response to glucose in the endothelium leads to decreased expression of stimulation. Inflammasome components are expressed in haemogenic endothelium markers gata2b and runx1, myeloid cells; knockdown and cell ablation studies indicate thus demonstrating that Gata2a activity is required that macrophages serve an important role in mediating for haemogenic endothelium programming. In adults, the effects of inflammasome stimulation on HSPCs. homozygous loss of the i4 enhancer lead to haematopoietic Significantly, inflammasome stimulation of human iPSC- hypocellularity in the marrow niche, as well as neutropenia derived hemogenic precursors increased the frequency of and propensity to develop AML, a phenotype that closely multilineage hematopoietic colony-forming units formed resembles disease progression in MonoMAC syndrome, a in culture. These studies identify the inflammasome as a Gata2 haploinsufficiency syndrome in humans. This mutant conserved metabolic sensor that expands HSPC production is a new model to study the pathophysiology of MonoMAC in vivo and in vitro, which may be useful to promote human syndrome, as existing Gata2 haploinsufficiency models do HSPC production for therapeutic application. not develop this syndrome. Importantly, further studies in this zebrafish model will also be invaluable to study disease progression towards AML, another characteristic that is O-60 Reprogramming of hematopoietic cell fate by unique to this model. cytokine switch Jana Oltova1, Ondrej Svoboda1, Olga Machonova1, Leonard I. Zon2 and Petr Bartunek1,2 ......

..... 23 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts 1 Laboratory of Cell Differentiation, Institute of Molecular embryonic haematopoiesis is known to occur. Because these Genetics AS CR v.v.i., 142 20 Prague 4, Czech Republic,2 studies lack spatial resolution, it remains debatable whether Stem Cell Program and Division of Hematology/Oncology, the mouse YS is the sole bona fide source for microglia, and Children’s Hospital and Dana Farber Cancer Institute, moreover, how microglial precursors born in peripheral Howard Hughes Medical Institute, Harvard Stem Cell tissues colonize the brain is completely undefined. Here, Institute, Harvard Medical School, Boston, MA, 02115, USA using an infrared light-induced temporal-spatial resolved fate mapping analysis, we challenge this single source view Blood development is a multistep process of formation by showing that microglia in zebrafish arise from multiple and turnover of blood cells and it is precisely regulated sources. We show that the embryonic/larval microglia by a variety of extrinsic and intrinsic factors. Historically, originate from the rostral blood island (RBI), the zebrafish reprogramming or transdifferentiation of cells from one counterpart of mouse YS for myelopoiesis, whereas the lineage to the other, was achieved by ectopic expression adult microglia arise from the ventral wall of dorsal aorta of single or multiple transcription factor(s). Here we (VDA), a tissue equivalent to the mouse aorta, gonads, show that the zebrafish committed erythroid cells can be and mesonephros (AGM) where hematopoietic stem cells reprogrammed into myeloid cells by granulocyte-colony emerge. Using in vivo imaging and genetic manipulation, stimulating factor (Gcsfa/CSF3a) only. Recently, we have we further show that the RBI-derived microglial precursors described the generation of immortalized erythropoietin enter the developing zebrafish brain via the lateral periphery (Epo)-dependent cell line ZEB. The cells require Epo for self- between the eyes and brain and the ventral periphery of renewal and proliferation and gradual decrease of Epo and the brain in a circulation-independent manner. Intriguingly, addition of increasing amount of Gcsf leads to complete the colonization of the brain by microglial precursors is myeloid reprogramming in less than two weeks. Initial dynamic and driven by apoptotic neurons through releasing experiments were performed using “mass” culture of ZEB lysophosphatidylcholine. cells, but later we have achieved the same efficiency using several independent clones excluding the possibility that the myeloid cells arise from “silent” multipotent progenitor O-62 Hedgehog signaling and the etiology of uveal cells. coloboma To understand the mechanisms underlying the Sarah Lusk 1, Hannah B. Gordon1 , Keith R. Carney1, Emily reprogramming of ZEB cells, we have performed extensive O. Wirick1, Brooke F. Murray1, Kristen M. Kwan1 transcriptome profiling followed by the analysis of the epigenetic landscape during the reprogramming process. 1 Department of Human Genetics, University of Utah To achieve that, we have employed ATAC-seq (Assay for During development, cells and tissues undergo dramatic Transposase-Accessible Chromatin using sequencing) and movements and rearrangements to assembly 3-dimensional ChIP-seq (Chromatin Immunoprecipitation) using H3K27Ac organs. Precise and stereotyped organ structures are and H3K27me3 to reveal activated and repressed loci and crucial for function, and disruptions to these structures their respective regulatory sequences. Correlating gene are a common cause of birth defects. In the vertebrate expression and chromatin accessibility (ATAC-seq peaks) eye, disruptions to morphogenetic movements result in together with transcription factor binding site enrichment structural defects which can cause visual impairment. For using HOMER show that multiple erythroid regulatory example, uveal coloboma results from failed development transcription factors (Gata1, Klf1, Hes) and myeloid master and morphogenesis of the optic fissure, a transient transcription factors (Cebpa/b, Spi1/Pu.1) are major factors structure in the embryonic eye. The genetic architecture regulating the process. Interestingly, Myb-binding sites underlying uveal coloboma is heterogeneous, with multiple were highly enriched in both erythroid and myeloid-specific pathways implicated. Mutations in the Hh receptor regulatory sequences. Patched result in overactive Hh signaling, and can cause In summary, we describe for the first time, that cell coloboma in both humans and zebrafish. We determined reprogramming of committed progenitor cell fate can be the cellular and molecular mechanisms by which overactive achieved by a single extrinsic factor. Hh signaling in the ptch2 mutant disrupts optic fissure morphogenesis. Using 4-dimensional imaging, cell tracking, and quantitative analysis, we pinpointed which cells are affected by overactive Hh signaling, and how exactly O-61 The Origin and Colonization of Microglia in Zebrafish cell movements and single cell behaviors are disrupted: Jin Xu1, Tienan Wang1, Sicong He1, Jianan Qu1 and Zilong overactive Hh signaling impairs processive cell motility and Wen1 elongated polarized cell morphology of prospective optic fissure cells. Using genetic approaches and transplantation 1 The Hong Kong University Science and Technology experiments, we found that overactive Hh signaling acts via Gli1-dependent transcription and via both cell-autonomous Microglia are central nervous system (CNS)-resident and non-cell-autonomous mechanisms to disrupt cell macrophages and play pivotal roles in neural development motility. These data suggest a model in which a specific and neural function. Previous studies in mice have suggested level of Hh signaling is crucial to regulate cell motility, and that microglia are derived from the yolk sac (YS) where we propose that overactive Hh signaling leads to increased ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 24 Oral Abstracts production of both cell-intrinsic and -extrinsic factors that Salt Lake City, Utah,2 Department of Neurobiology and disrupt cell elongation and orientation. We are actively Anatomy, University of Utah School of Medicine, Salt Lake pursuing candidate and unbiased approaches to identify City, Utah the downstream targets directly regulating cell motility and morphology in the optic fissure; these may represent novel The histone methyl-transferase Kmt2d is a critical regulator players in the uveal coloboma gene network. of the epigenome in vertebrates. Mutations in this gene cause Kabuki Syndrome (KS), a rare condition that includes craniofacial anomalies, developmental delay, O-63 Mimicking congenital disorders of glycosylation in cardiovascular defects and microcephaly. Little is known medaka unravels the role of N-glycosylation in central about the molecular pathways that cause microcephaly and nervous system development neurodevelopmental abnormalities in KS patients. Earlier work suggests that microcephaly in KS is due to a decrease in Sevinç Gücüm1,2, Thomas Thumberger1, Joachim neuronal differentiation. However, a molecular mechanism Wittbrodt1 by which Kmt2d regulates neuronal differentiation remains unclear. Here, we demonstrate that KS microcephaly is the 1 Centre for Organismal Studies (COS), Heidelberg result of a defect in the transition of neuronal progenitors University, Germany, 2 Heidelberg International Graduate to neurons, due to misregulation of the Notch signaling School of Molecular and Cellular Biology, Heidelberg pathway. University, Germany Our previous work generated zebrafish null mutants of Glycosylation is one of the most common forms of kmt2d, and found that Notch pathway is hyperactivated in posttranslational modifications. This complex sugar the cardiovascular system. Turning to brain development, branching pathway modulates folding, stability, Notch signaling has well-defined roles during neurogenesis transportation and, hence, overall protein function. but it is not known how this pathway is fine-tuned to Recessive mutations in enzymes catalyzing N-glycosylation strike a balance between progenitor cell maintenance vs. result in Congenital Disorders of Glycosylation (CDG), neuronal differentiation. We hypothesized that Kmt2d which are rare genetic and multisystemic disorders with regulates Notch signaling during neuronal differentiation, pronounced effects in the nervous system. Although the and loss of this regulation results in microcephaly. Using function of N-glycosylation enzymes is well characterized confocal imaging and fluorescent-activated cell sorting, we in yeast, studying their function during vertebrate found that microcephaly in kmt2d null mutants was not due development is not trivial due to embryonic lethality upon to alterations in apoptosis, proliferation, or neurovascular complete loss-of-function. One strategy to circumvent development, but due to altered Notch signaling and a this, is the generation of hypomorphs that have residual misbalance between neuronal progenitors and neuronal enzyme activity. As N-glycosylation enzymes are highly differentiation. Strikingly, inhibition of Notch signaling conserved across most vertebrates we introduced known was able to rescue neural differentiation defects in kmt2d human patient mutations into orthologous regions of mutants. medaka to generate novel disease models. Insertion of an alpha-1,3/1,6-mannosyltransferase (Alg2) C-terminal early Our results demonstrate that Kmt2d regulates neuronal stop codon (Thiel et al., 2003) via CRISPR/Cas9 in medaka differentiation during brain development in zebrafish and resulted in a multisystemic phenotype that closely reflects provide evidence of regulatory interactions between Kmt2d the human phenotype. Reduced white matter in the mid- and Notch signaling in the context of KS microcephaly. We and hindbrain of medaka directly correlates to the reduction are currently exploring a novel mechanism by which Kmt2d of white matter in the patient case, which hints towards a regulates the transition from neuronal precursors to mature reduced myelination of neurons. Both fish and the patient neurites in zebrafish. are suffering from heart arrhythmias and coagulopathies. Vision abnormalities of the patient with coloboma of the iris is correlating with eye phenotypes in medaka including O-65 Elucidating the role of Annexin A11 in ALS using rod and cone cell abnormalities. Our project overall showed over-expression and CRISPR Zebrafish Models that medaka is a good model organism to unravel the function of N-glycosylation in vertebrate neural system Bradley N Smith1,2, Valentina Marchica1,2, Nicholas development with a high clinical relevance for probing Nikolaou1 and Corinne Houart1 novel therapeutic avenues. 1 Department of Developmental Neurobiology, Kings College London, SE1 1UL, UK,2 Maurice Wohl Clinical Neuroscience Institute, Kings College London, SE5 9RT, UK O-64 Notch signaling hyperactivation is the leading cause of microcephaly in Kabuki Syndrome zebrafish Amyotrophic Lateral Sclerosis (ALS) is an incurable, late onset, relentlessly progressive disorder. 10% of ALS is Angie Serrano1, Bradley L. Demarest1, Callie Housden1, familial with 23% of the genetic basis caused by mutations Richard I. Dorsky2 and H. Joseph Yost1,2 in >20 genes. We recently identified novel mutations in 1 Molecular Medicine Program, University of Utah, Annexin A11 from exome sequencing of 751 familial ALS ...... cases. The most prevalent mutation, D40G segregated in ......

..... 25 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts two UK kindreds and was found in multiple familial cases lipid homeostasis, suggesting haploinsufficiency leads to a and a UK sporadic individual (Smith et.al Sci Trans Med, cascade of biochemical and cellular changes. 2017). Over-expression studies involved micro-injecting AB strain Zebrafish embryos at the 1cell stage with an equal mix of UAS:ANXA11-GFP WT or D40G mutant plasmid O-67 Developing zebrafish models to investigate the role and MNX1:GAL4 driver to obtain mosaic expression in of TCP1 in a rare neurological disease motor neurons. GFP positive primary motor neurons were imaged in 48hr larvae and were characterised for neuronal Kathryn I Adamson1 2, Ryan MacDonald2, Eamonn morphology and then fixed with 4%PFA for downstream Sheridan3, Andrew J Grierson1 2

immunohistochemistry and high-resolution confocal 1 imaging. Loss of function studies focussed on a CRISPR/ Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield,2 UK, Bateson Centre, Cas9- induced ANXA11A Zebrafish knockout and validated 3 by qPCR. F2 and F3 fish were characterised for phenotype, University of Sheffield, Sheffield, UK, School of Medicine, neuronal integrity and cellular localisation changes. Over- University of Leeds, Leeds, UK expression of Human UAS:ANXA11-GFP WT was non- Rare genetic diseases are collectively common and often disruptive however fish expressing the D40G protein chronically debilitating. Next-generation DNA sequencing manifested with multiple phenotypes at 48hrs. ANXA11- technology has accelerated the discovery of rare pathogenic GFP D40G positive motor neurons show loss of branching, gene variants, but their molecular mechanisms remain and a failure to extend their primary axon. Changes in underexplored, hindering therapeutic development. There subcellular ANXA11-GFP localization was observed in is therefore a growing demand for experimental models neuronal localisation in mutants versus WT, informing that enable investigation of rare disease mechanisms and putative disease mechanisms. Furthermore, the loss of therapeutic targets. These models will additionally deliver ANXA11A in F3 homozygous mutant fish larvae did lead to new insights into gene function, which could help us a low penetrant phenotype somewhat variable in severity, understand more prevalent conditions. including motor symptoms, indicating an importance of Annexin A11a to normal neuronal function Here, we are using the zebrafish to investigate a novel recessive variant (c.209C>T, p.P70L) in TCP1, which encodes part of an essential cytoplasmic chaperonin known as O-66 FAM57B ceramide synthase, a hub gene within the TCP1 Ring Complex. This variant was identified in a 16p11.2 deletion syndrome, maintains physiological lipid consanguineous pedigree by whole-exome sequencing and membrane composition segregates with a childhood-onset neurological phenotype featuring cerebellar hypoplasia, retinal dystrophy and Danielle L. Tomasello1, Jasmine M. McCammon1 and peripheral neuropathy. This is currently the only reported Hazel Sive1,2 pathogenic variant in TCP1; therefore, we aim to formally

1 demonstrate its pathogenicity and investigate the Whitehead Institute for Biomedical Research, Cambridge, mechanisms by which it causes disease. MA,2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA To this end, we have used CRISPR/Cas9 to create a knock- in zebrafish line harbouring a mutation equivalent to the The genetic basis for mental health disorders is complex, human variant (tcp1P73L), and two frameshift mutant lines making diagnostics unreliable and personalized treatments (tcp1I57fs, tcp1H72fs). We are now characterising these lines to complex. My studies are aimed to address this challenge determine whether they recapitulate the human phenotype, with a new approach to identify molecular changes in and to investigate neurodevelopmental roles fortcp1. Using 16p11.2 Deletion Syndrome. In this prevalent syndrome immunohistochemistry and in situ hybridisation, we are (occurring in 1 in 2000 individuals) part of chromosome examining the retinal, cerebellar and peripheral nervous 16 is lost, encompassing a core of 25 genes. Deletion system architecture of mutant larvae, as well as measuring leads to severe brain symptoms including autism, swimming activity as a read-out of neurological function. intellectual disability and epilepsy. We hypothesize that the metabolome of neurons is abnormal in individuals with Homozygous tcp1I57fs larvae display evidence of severe motor 16p11.2 Deletion Syndrome, providing a new approach dysfunction, retinal degeneration and axonal abnormalities to dissect this complex syndrome. I have concentrated on by 3 days post-fertilisation. Conversely, homozygoustcp1 P73L FAM57B, previously shown to interact with other enzyme- larvae exhibit no gross developmental abnormalities. Our encoded genes within the interval, encoding a ceramide data indicate that zebrafish tcp1 loss-of-function produces synthase. Consistent with altered metabolism in 16p11.2 neurological defects of relevance to human TCP1-related Deletion Syndrome, we see profound changes in lipids in disease. We are now analysing tcp1I57fs phenotypes in detail the zebrafish model and in 16p11.2 deletion fibroblasts. and characterising juvenile and adult tcp1P73L mutants to fam57b mutants show significant cellular and behavioral investigate whether a phenotype develops with age. phenotypes, including lipid membrane disruption, reduced firing in whole brain recordings and behavioral deficits. We propose FAM57B is a critical protein essential for membrane ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 26 Oral Abstracts O-68 Whole brain morphometric analysis to identify role of these immune cells in promoting successful spinal convergent functions of autism-risk genes cord regeneration in zebrafish has not been addressed. We have recently established a spinal cord lesion paradigm in Tripti Gupta1, Victoria Heigh1, Jennifer Sinclair1, and 1 3 day old larval zebrafish. These show axonal and neuronal Harold Burgess repair within 48 hours (Ohnmacht et al., 2016), with axons 1 Division of Developmental Biology, National Institute of actively navigating a complex extracellular matrix (Wehner Child Health and Human Development, National Institutes et al., 2017). Using this system, we have found that inhibition of Health, Bethesda, MD 20892 of inflammation reduces and promotion accelerates axonal regeneration in larval zebrafish. Peripheral macrophages, Autism spectrum disorders (ASD) is a set of complex, but not neutrophils or microglia, are necessary and heterogeneous neurodevelopmental disorders that are sufficient for full axonal regeneration and inflammation defined by persistent deficits in social communication is tightly and dynamically controlled by macrophages to and restricted, repetitive patterns of behavior. These promote functional spinal cord regeneration in zebrafish defining characteristics are also frequently accompanied (Tsarouchas et al., 2018). by a wide range of comorbidities, such as anxiety, seizures, and sleep disorders. Consistent with this heterogeneity Tsarouchas TM, Wehner D, Cavone L, Munir T, Keatinge of symptoms, variants in over 100 genetic loci have been M, Lambertus M, Underhill A, Barrett T, Kassapis E, Ogryzko strongly associated with increased risk of developing N, Feng Y, van Ham TJ, Becker T, Becker CG (2018) Dynamic autism. We expect that specific subsets of genes associated control of proinflammatory cytokines Il-1β and Tnf-α with autism-risk will have convergent neurodevelopmental by macrophages is necessary for functional spinal cord functions and that mutations in these genes will result in regeneration in zebrafish Nature Communications 9(1):4670 shared phenotypic outcomes. In order to determine the Wehner D, Tsarouchas TM, Michael A, Haase C, Weidinger shared biological mechanisms of these genes, we are using G, Reimer MM, Becker T*, Becker CG* (2017) Wnt signaling CRISPR gene editing to knock down expression of genes controls a pro-regenerative extracellular matrix in functional linked to ASD through exome sequencing studies, followed spinal cord regeneration, Nature Communications 8(1):126 by phenotypic analyses of mutant larvae. To this end, we have developed a novel method for confocal-based whole Ohnmacht J, Yang Y, Maurer GW, Barreiro-Iglesias A, brain morphometric analysis to identify changes in brain Tsarouchas TM, Wehner D, Sieger D, Becker CG*, Becker structure and inhibitory and excitatory neuron composition. T* (2016). Spinal Motor Neurons are Regenerated after Importantly, our method allows simultaneous identification Mechanical Lesion and Genetic Ablation in Larval Zebrafish. of global changes in brain size and localized changes in Development 143:1464-1474 brain morphometry and cellular composition, without a priori knowledge of what regions may be affected, making it a powerful method for unbiased whole brain phenotype O-70 Identification of tendon-promoting drugs for discovery. In addition to the morphometric analyses, we orthopaedic regenerative medicine are conducting behavioral assays for sensory deficits. To date, we have screened 20 genes and identified changes in Xubo Niu 1, Suyash Raj 2,Thorsten M. Schlaeger 2 , April M. brain size, regional morphometry, cellular composition, and Craft2 and Jenna L. Galloway 1

sensory processing behavior. We are currently screening 1 additional mutants and clustering the genes based on the Center for Regenerative Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, phenotypes to uncover groups of genes with convergent 2 functions. Harvard Stem Cell Institute, Harvard Medical School, Children’s Hospital Boston, Harvard Medical School Tendons and ligaments are essential components of the O-69 Successful regeneration in larval zebrafish musculoskeletal system and are necessary for transmitting movement and providing stability. Tendon and ligament Themistoklis M Tsarouchas1, Tahimina Munir1, Marcus 1 1 1 injuries affect a significant portion of population, and have Keatinge , Thomas Becker , Catherina Becker a slow and limited repair process, which can often lead to 1 Centre for Discovery Brain Sciences, University of the development of osteoarthritis and the need for joint Edinburgh, Edinburgh EH16 4SB, UK replacement surgery. Therefore, the development of drugs or stem cell-based tissue-replacement strategies represent In contrast to mammals, zebrafish can repair their spinal promising therapeutic strategies that could improve cord after injury and regain locomotor function. This injury outcomes. However, we currently have a limited recovery depends on the regrowth of severed axons and understanding of the pathways capable of inducing tendon coincides with significant de novo neurogenesis from and ligament fates, which can impede the development endogenous progenitor cells. Spinal cord injury also leads of directed differentiation strategies to generate tendon to a massive response of innate immune cells (microglia, tissues from pluripotent stem cells. To discover novel macrophages, neutrophils) both in non-regenerating regulators of tendon development, we took advantage of mammals and in successfully regenerating zebrafish and the the zebrafish system and performed a high-throughput ......

..... 27 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts chemical screen. Using a double transgenic zebrafish line O-72 Regulation and function of the Keap1-Nrf2 pathway that labels tendon (mcherry) and cartilage (eGFP) and a in zebrafish stress response blastomere cell culture system, we screened 7569 small molecules from 11 bioactive drug libraries. We identified Makoto Kobayashi1 111 hits that altered mcherry and eGFP expression. Among 1 these 111 hits, we selected and validated 16 with the highest Faculty of Medicine, University of Tsukuba tendon/ligament promoting abilities. qPCR analysis of the hit compounds in zebrafish blastomere cells and whole The Keap1-Nrf2 pathway is the major regulator of embryos revealed increased expression of tendon genes, cytoprotective responses to oxidative and electrophilic including the earliest progenitor marker scxa (scleraxis a). stress. Recently, its involvement in the aging and cancer To demonstrate a conserved role for these compounds growth has become a hot topic. We have been studying in mammalian cells, we treated mouse pluripotent ES the role of this Keap1-Nrf2 pathway using zebrafish as cells with the hit compounds, and we observed increased a model. In this talk, I will address about the regulation expression of the tendon markers, Scx, Mohawk, and and function of the Keap1-Nrf2 system in cytoprotection Col1a1, suggesting these drugs have conserved tendon against a variety of stresses, including oxidative stress, promoting activities in mammalian cells. We are currently heavy metal toxicity and endoplasmic stress. focused on testing the hits on human pluripotent stem cells and patient-derived bone marrow mesenchymal stem cells to determine if they can promote tendon and ligament O-73 Mechanisms of glial bridging during spinal cord differentiation. Taken together, the compounds that we regeneration discover will lead to the identification of novel pathways Lili Zhou1, Ryan McAdow1, Kevin Tran1, and Mayssa H. involved in tendon development as well as provide the 1 foundation for the development of new clinical treatment Mokalled strategies to improve tendon and ligament healing. 1 Department of Developmental Biology, Washington University School of Medicine, USA.

O-71 The circadian clock promotes spermatogonial Spinal cord injuries result in irreversible loss of sensory and differentiation and fertilization through retinoic acid moor functions in mammals. In contrast, adult zebrafish signaling naturally regenerate and reverse paralysis after complete spinal cord transection. Whereas glial scarring is a major Taole Liu 1,2 , Wei He 1,2 , Yingbin Zhong1,2 , Zhaomin Zhong roadblock for mammalian spinal cord repair, specialized 1,2 , Han Wang 1,2 glial cells in zebrafish form a bridge across severed spinal

1 cord tissue and facilitate regeneration. Using genome-wide Center for Circadian Clocks, Soochow University, Suzhou connective tissue 2 profiling, we have previously identified 215123, Jiangsu, China, School of Biology & Basic Medical growth factor a (ctgfa) as a pro-regenerative bridging Sciences, Medical College, Soochow University, Suzhou factor. ctgfa is induced in and around the glial cells that 215123, Jiangsu, China participate in initial bridging events. Genetic inactivation of The circadian clock generates and maintains 24-hour zebrafish ctgfa demonstrated its requirement during glial oscillations in almost all organs. However, the testis remains bridging and regeneration, while ctgfa overexpression or mysterious without a clear understanding of its circadian treatment with human CTGF peptide significantly enhanced function. Here we revisited the long thought exception in glial bridging, axon regrowth, and functional recovery. Here, the circadian system. Transcriptome analysis reveals more we generated a battery of CTGF-based genetic tools to than 1,000 rhythmically expressed genes in the zebrafish better understand glial bridging mechanisms. Our studies testis. Key circadian clock genes are expressed in both the determined the molecular identity of bridging glial cells by Sertoli cells and spermatogonia and regulate genes involved FACS-sequencing, and uncovered a yap-ctgf-twist signaling in retinoic acid (RA) signaling. Loss of Clock1a generally or axis that directs glial bridging and functional regeneration Sertoli-cell-specifically results in arrested spermatogonial in zebrafish. This work highlights novel cellular events and differentiation and reduced fertilization, which canbe gene networks that contribute to glial bridging in zebrafish, rescued by time-of-day-specific RA treatment. Conditional and opens new avenues to induce glial bridging as a means overexpression of clock1a shows that chronical perturbation to improve spinal cord repair in mammals. of circadian regulation leads to similar reproductive defects. The circadian clock acts through RA signaling to synchronize spermatogonial differentiation and to promote O-74 Single-cell analysis identifies a population of liver fertilization via izumo1. Hence, we demonstrate that the progenitor cells during regeneration

testis clock ticks in a cell-specific manner and contributes 1,2 1,2,3 to reproduction through RA signaling, highlighting circadian Isaac M. Oderberg and Wolfram Goessling roles in male fertility. 1 Brigham and Women’s Hospital, USA ,2 Harvard Medical School, USA,3 Massachusetts General Hospital, USA

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..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 28 Oral Abstracts The liver is an essential organ, responsible for key metabolic analysis shows that Hhex binds to the promoters of pdx1 functions, energy storage, blood detoxification, plasma and cdx1b genes to repress their expression. We therefore protein secretion, and bile production. It is composed of propose that Hhex, Cdx1b, Pdx1 and Sox9b form a genetic several different cell types and is capable of regenerating network governing the patterning and morphogenesis of by replacing hepatocytes lost due to toxic, physical or the HPD and digestive tract systems in zebrafish. inflammatory damage, primarily through division of other hepatocytes. However, there are limits to the capacity of the liver to compensate following sustained insults O-76 Targeting ligand-dependent BMP signaling in or injury. Liver failure is one of leading causes of death melanoma worldwide, and the only known cure for liver failure is organ transplantation. The development of a stem-cell Craig J. Ceol1

based therapy to regenerate the liver would provide a life- 1 altering treatment for patients with late-stage liver disease. Program in Molecular Medicine, University of While there is some evidence for a transient population Massachusetts Medical School, Worcester, MA, USA of liver stem cells that appear during regeneration, the Despite major advances in melanoma therapies, a majority origin, potency and regulation of these cells remains largely of patients with late-stage tumors will still die from their unknown. disease. To identify new drug targets in melanoma, we The zebrafish (Danio rerio) liver serves the same function screened candidate oncogenes and tumor suppressors and is thought to be composed of the same cell types as in that were shortlisted using integrative comparative mammals. To identify the response of the liver to acute injury oncogenomics. One of the new melanoma genes identified on a cellular and transcriptomic level, the livers of adult was GDF6, a BMP ligand normally expressed in the zebrafish were subjected to either drug-induced toxicity neural crest but absent in most adult tissues, including or genetic hepatocyte ablation, and were subsequently melanocytes. GDF6 is present in nearly 80% of human analyzed using single-cell RNA sequencing. This approach melanomas, where it activates BMP signaling. Knockdown resulted in the generation of transcriptomic information of GDF6 or inhibition of BMP signaling causes differentiation for ~25,000 single cells, inclusive of all known cell types and death of melanoma cells. Developmental studies in in the liver. Intriguingly, a population of cells that contains zebrafish indicate that GDF6-driven BMP signaling normally markers for both hepatocytes and biliary epithelial cells suppresses melanocytic differentiation of neural crest cells. appears early only after recovery from hepatocyte ablation. Together these studies show that ligand-dependent BMP These data suggest a progenitor population of biliary origin signaling promotes melanoma initiation and maintenance, is utilized during regeneration from extreme injury. Further and this activity is a reiteration of the normal function work will be needed to characterize the regulation and fate of GDF6-driven BMP signaling during development. The of the these cells during regeneration. dependence of melanomas on GDF6-driven BMP signaling presents an opportunity for therapeutic intervention, and we are currently developing inhibitors to target this activity. O-75 Zebrafish hhex null mutant develops an intrahepatic intestinal tube due to de-repression of cdx1b and pdx1 O-77 Interplay between retroelements and Mda5 Jinrong Peng, Zhejiang1 regulates developmental and regenerative hematopoiesis 1 University, China Eirini Trompouki1 The hepatopancreatic duct (HPD) system links the liver 1 Max Planck Institute of Immunobiology and Epigenetics, and pancreas to the intestinal tube and is composed of Freiburg, Germany the extrahepatic biliary duct, gallbladder and pancreatic duct. Haematopoietically-expressed-homeobox (Hhex) Hematopoietic stem and progenitor cell (HSPC) formation protein plays an essential role in the establishment of HPD, during development shares many features with adult however, the molecular mechanism remains elusive. Here hematopoietic regeneration. Even though these two we show that zebrafish hhex-null mutants fail to develop processes are widely studied and we know many coding the HPD system characterized by lacking the biliary marker genes that play a role in both, the role of the non-coding or Annexin A4 and the HPD marker sox9b. The hepatobiliary repetitive genome is not well understood. Using zebrafish duct part of the mutant HPD system is replaced by an as a model, we show that retroelements are expressed in intrahepatic intestinal tube characterized by expressing the embryonic hemogenic endothelial cells, suggesting their intestinal marker fatty-acid-binding–protein 2a (fabp2a). implication in HSPC formation. To investigate whether Cell lineage analysis showed that this intrahepatic intestinal upregulation of retroelements affects hematopoiesis, we tube is not originated from hepatocytes or cholangiocytes. treated embryos with the histone deacetylase inhibitor Further analysis revealed that cdx1b and pdx1 were valproic acid and showed by WISH for HSPC markers expressed ectopically in the intrahepatic intestinal tube that their formation is greatly enhanced. We reasoned and knockdown of cdx1b and pdx1 restored the expression that members of the RIG-I-like inflammatory receptor of sox9b in the mutant. Chromatin-immunoprecipitation family, which normally respond to viral RNAs, could ......

..... 29 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts act downstream of these elements. Indeed, minimal therapeutic window. To this end, we developed a cell-based, knockdown of Mda5 abrogated the effect of valproic acid high-throughput chemical screening assay capable of on HSPCs whereas severe knockdown led to decreased distinguishing between inhibitors of minor class and major HSPC numbers due to diminished inflammatory signaling. class splicing (required for splicing the remaining 96.5% of These results in development prompted us to investigate genes). This assay has been miniaturised and automated to the role of retroelements and MDA5 in the adult a 1,536-well plate format. To date, a pilot screen of 5,000 hematopoietic system of mouse, upon myeloablation with compounds has been successfully undertaken by the WEHI 5-fluorouracil (5-FU) treatment. We observed substantial Drug Discovery Centre, and a screen of >220,000 diverse, chromatin reorganization and upregulation of various drug-like compounds is currently underway. We propose retroelements as well as inflammatory signaling after one that minor class splicing provides an attractive target for day of treatment. The newly accessible regions harbor cancer therapy, worthy of future development. motifs for IRF transcription factors, indicating that, at least partly, inflammatory signaling is directly involved -/- in HSC activation. In similar experiments in Mda5 HSCs, O-79 Analyzing the activity of anti-fungal drugsin vivo in we observed upregulation of retroelements, but impaired larval zebrafish infected withA. fumigatus inflammatory signaling that is needed for HSPC activation. Indeed, Mda5-/- HSCs were significantly more quiescent than Emily E. Rosowski1,2, Nancy P. Keller2,3, Anna wild type counterparts and retained their fitness, as shown Huttenlocher2,4

by transplantation experiments. Our data reveal that during 1 development and regeneration, endogenous retroelements Department of Biological Sciences, Clemson University, SC, USA,2 Department of Medical Microbiology and act as ligands for the immune receptor Mda5 and this event 3 accounts partially for the inflammatory signaling activation Immunology, University of Wisconsin-Madison, WI, USA, that is required in both processes. Department of Bacteriology, University of Wisconsin- Madison, WI, USA,4 Department of Pediatrics, University of Wisconsin-Madison, WI, USA O-78 Tapping into zebrafish digestive organ development Fungal infections are estimated to kill more than 1.5 million to identify new targets for cancer therapy people annually, worldwide. There currently exist three main

1 1 1 classes of anti-fungal drugs, all of which have fungi-static Joan K Heath , Karen Doggett , Kimberly Morgan , in vitro 1 or fungi-cidal activity . In human patients, however, Stephen Mieruszynski these drugs often fail. For example, the survival rate of 1 Epigenetics and Development Division, Walter and patients infected with the fungusAspergillus fumigatus and Eliza Hall Institute of Medical Research (WEHI), 1G Royal treated with anti-fungal drugs is only ~50%. We therefore Parade, Parkville, Victoria 3052, Australia need to more thoroughly examine and understand the efficacy of anti-fungal drugs in live, intact host models. As a result of ENU-mutagenesis in zebrafish, we identified We have begun to analyze the activity of the anti-fungal a collection of genes that are indispensable for the huge drug voriconazole against A. fumigatus in infected larval expansion of the digestive organs during vertebrate zebrafish, using established models of immune deficiency, development. These genes encode proteins required for simple and consistent drug administration through RNA processing, transcription and nuclear pore formation. immersion, and multi-day live imaging of the fungus and Of these, rnpc3, which encodes an RNA-binding protein host immune cells. We find that the activity of voriconazole component of the minor class spliceosome, has emerged as in a live animal is different from its activity in vitro. In an essential gene for the rapid growth and proliferation of particular, while voriconazole prevents the germination cells during organogenesis. Minor class splicing is required of A. fumigatus spores into hyphae in vitro, it does not in for the proper expression of only a very small sub-set larval zebrafish. Instead, the drug kills A. fumigatus post- (3.5%) of human genes. Of interest, these genes participate germination, leading to the degradation and clearance of in processes that are essential for rapid rates of cell growth hyphae. The efficacy of voriconazole also depends on the and division, including MAPK signalling, DNA damage presence of macrophages, as these cells act to slow down repair, cycle cell progression and DNA replication; processes fungal germination and give voriconazole a longer time to that are also highly active in cancer cells. Using genetically act in vivo. These findings have significant implications for engineered zebrafish and mouse models of cancer, we have the deployment of anti-fungal drugs in patient populations demonstrated that disruption of minor class splicing reduces as well as for the anti-fungal drug development pipeline. tumour burden and/or prolongs survival in hepatocellular carcinoma, lung and gastric adenocarcinoma, follicular lymphoma and acute myeloid leukemia. These positive O-80 Chemical screening for anti-infectives in zebrafish outcomes are due in part to a Tp53-mediated reduction in reveals a novel role for iGluRs in innate immunity cell proliferation and increased apoptosis. Moreover, these anti-tumour effects produce no toxicity to normal tissues, Anne Clatworthy1,2,3, Margarita Parada-Kusz1,2,3, Senya suggesting that targeting minor class splicing may provide Combs1,2,3, Jenny S.W. Lee1,2,3, Stanley Shaw1,4, and a broad-spectrum approach to cancer therapy with a viable Deborah Hung1,2,3 ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 30 Oral Abstracts 1 Broad Institute of MIT and Harvard, Cambridge, MA by abnormalities in social interaction, communication, 02142, USA,2 Department of Molecular Biology and Center restrictive, repetitive behaviors, and sensory processing. for Computational and Integrative Biology, Massachusetts Advances in whole-exome sequencing have led to the General Hospital, Boston, MA 02114, USA,3 Department identification of a growing number of risk genes that are of Genetics, Harvard Medical School, Boston, MA 02115, strongly associated with ASD, yet how the disruption of USA,4 Center for Systems Biology; Center for Assessment these genes in the developing brain leads to alterations Technology &Continuous Health (CATCH), Massachusetts in neural circuits, predisposing to behavioral dysfunction General Hospital, Boston MA, 02114 USA remains unknown. Further, at present, there are no pharmacotherapies that target the core features of ASDs due to our limited understanding of the underlying There is an urgent need to consider novel ways of treating pathophysiology of these disorders. To gain insight bacterial infections. Traditional antibiotics have been into the function of ASD risk genes in the developing identified by their ability to kill or inhibit the growth of vertebrate brain, we generated zebrafish mutants of bacteria growing in vitro. This methodology ignores a 10 ASD risk genes, including: CHD8, CNTNAP2, CUL3, potentially wide array of small molecules that might have DYRK1A, GRIN2B, KATNAL2, KDM5B, POGZ, SCN2A and therapeutic utility that target the virulence of the infecting TBR1. First, we capitalized on the transparency of zebrafish bacterium, bacterial gene products that are required only in larvae to investigate the development of excitatory and the context of host infection, or host proteins themselves. inhibitory neurons in mutants using transgenic lines that With the goal of identifying small molecules that target this co-label these cell populations. Next, we performed high- wider array of host or bacterial ligands, we conducted a throughput quantitative behavioral profiling assays of rest- chemical screen for small molecules that rescued zebrafish wake and sensory processing behaviors across mutant embryos/ larvae from lethal bacterial challenge. We found lines. Finally, we are performing pharmacological screens one small molecule that rescued zebrafish from lethal of FDA-approved drugs to identify compounds that rescue Pseudomonas aeruginosa and Salmonella typhimurium behavioral phenotypes in mutants. Using these assays, we infection yet did not act like a typical antibiotic insofar found that zebrafish mutants of scn1lab, an ortholog of as it does not kill or inhibit the growth of bacteria in vitro, human SCN1A and SCN2A, display nighttime hyperactivity though it does act to control bacterial burden in vivo and and daytime hypoactivity, and that dyrk1a mutants exhibit this activity is dependent on the presence of immune cells decreased daytime activity and decreased responses to in the fish. Follow-up work to understand the mechanism dark flashes. Our long-term goal is to identify points of of action of this small molecule suggested that ionotropic convergence linking ASD risk genes, which can be used as glutamate receptors (kainate receptors) might be the target. the basis for pharmacological screens to identify novel drug Both pharmacologic and genetic data implicate kainate candidates with relevance to ASDs. receptors as being important for innate immunity, both in hematopoiesis and in immune defense against bacterial challenge, in part by altering immune cell numbers and O-82 Developing a model to screen for compounds that functions. This work should open new avenues and suggest protect against cisplatin-induced toxicities new potential targets to treat bacterial infections. Jaime Wertman1, Nicole Melong2, Stewart Langley2, Babak Razaghi2, Shelby Steele2, Meredith Irwin3,4, Jason N. Berman1,2,5 O-81 High-throughput behavioral characterization of 1 Microbiology & Immunology, Dalhousie University, zebrafish models of autism risk genes Halifax, NS, Canada ,2 Pediatrics, Dalhousie University/IWK Christina Szi1, Sundas Ijaz1, Catalina Sakai1, Brendan Health Centre, Halifax, NS, Canada ,3 Pediatrics, University Rooney1, Kristen Enriquez1, Sarah McLaughlin1, of Toronto, Toronto, ON, Canada,4 Hematology/Oncology, Sick Kids, Toronto, ON, Canada,5 Pathology, Dalhousie Jeffrey Eilbott1, Brent Vander Wyk3, Jason Rihel4, Ellen J. University, Halifax, NS, Canada,6 Hematology/Oncology, Hoffman1,2 IWK Health Centre, NS, Canada 1 Child Study Center,2 Program on Neurogenetics Yale Cisplatin is a chemotherapy used to treat several pediatric University School of Medicine, New Haven, CT 06510, malignancies, including neuroblastoma (NBL). NBL is the USA,3 Program on Aging, Geriatrics, Department of most common extracranial tumor in children, and survival Internal Medicine, Yale University School of Medicine, rates for metastatic disease are <50%. NBL survivors are New Haven, CT 06510, USA,4 Department of Cell and plagued by lifelong cisplatin-induced toxicities, including Developmental Biology, University College London, Gower kidney damage and ototoxicity (hearing loss). The Street, London, WC1E 6BT, UK zebrafish is an excellent model for studying toxicities due to conserved organ systems. Zebrafish have lateral line neuromasts, analogous to mammalian cochlear hair cells. Autism Spectrum Disorders (ASDs) are a group of Similar to hair cell death in humans, cisplatin exposure devastating neurodevelopmental disorders characterized causes decreases in neuromast viability, visualized with ......

..... 31 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Oral Abstracts YO-PRO1, a fluorescent dye. Larvae treated with cisplatin and HSPCs arrive in the new niche, akin to extramedullary (0.01-0.25mM) demonstrated a dose-dependent reduction hematopoiesis, are cuddled by endothelial cells and divide. in neuromast fluorescence. Zebrafish larvae also possess Our studies show that there is a transcriptional code for pronephros structures similar to the mammalian nephron. niche endothelial cells, and this cell fate is sufficient to Glomerular filtration rate (GFR) is measured in larvae create an artificial stem cell niche. by injecting FITC-tagged inulin into circulation. Inulin is renally-excreted, so decreases in vascular fluorescence approximates GFR. Cisplatin-treated larvae exhibited a reduction in GFR. We employed anin vivo larval neuromast screen to assess the effects of compounds from the Sigma LOPAC®1280 library on cisplatin-induced ototoxicity. We compared these results with an in vitro toxicity test of cisplatin +/- the compound library on human proximal tubule cells, and found 10 drugs that were effective in both assays. We validated the protective capacity of two of these adjuvants with confocal microscopy of the inner ear and the GFR assay. To determine if these protective compounds impact cisplatin’s cytotoxic effects, zebrafish (48 hpf) were xenotransplanted (XT) with fluorescently labeled SK-N- AS NBL cells. XT larvae were treated with 0.2mM cisplatin +/- the protective agents. Preliminary results suggest that neither compound interferes with the therapeutic efficacy of cisplatin. In summary, the zebrafish XT platform provided simultaneous evaluation of cancer cell proliferation and toxicity, revealing novel adjuvant therapies to enable safer administration of cisplatin.

O-83 Creating a new niche for blood stem cells using zebrafish Leonard I. Zon1 1 Stem Cell Program and Hematology/Oncology, Children’s Hospital and Dana-Farber Cancer Institute, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Stem Cell and Regenerative Biology Department, Harvard University, Boston, MA Hematopoietic stem cell transplantation involves the homing of stem cells to the marrow, an active process of engraftment, and the self-renewal of the blood stem cells. We have been using the zebrafish as a model to study the molecular biology of this process. Blood stem cells are born in the dorsal aorta of the developing embryo. By imaging RUNX1 GFP+ cells arriving in the next site of hematopoiesis (the caudal hematopoietic territory), engraftment can be visualized. The endothelial cells of the niche cuddle the hematopoietic stem cells, and the stem cells divide in the niche. We have used RNA tomography to find transcripts that are restricted in expression to the niche endothelial cells. After sorting these endothelial cells, we used ATAC seq to reveal regions of chromatin that are open. The regions function in enhancer assays to drive expression of GFP in the niche. By computing the binding sites in these regions, and examining RNA seq, we found transcription factors that may participate in niche endothelial development. Overexpression of three of these transcription factors is sufficient to reprogram embryonic cells to express the markers of the niche. New niches form in ectopic locations ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 32 Poster Abstracts P-001 Repressing to enhance: In vivo chemical screen the same gene set and overall genomic structure ranging identifies novel regulators of pancreatic alpha-cell fate from 16,596 base pairs (bp) in the teleost zebrafish (Danio and beta-cell regeneration rerio) to 16,569 bp in humans. Mitochondrial disorders are 1 1 * * amongst the most prevalent inherited diseases affecting ~1 Brijesh Kumar, Didier Stainier and Christian Helker in every 5000 individuals. Currently, few effective treatments 1Dept.III- Developmental Genetics, Max Planck Institute exist for those with mitochondrial ailments, representing for Heart and Lung Research, Ludwigstraße 43, 61231 Bad a major unmet patient need. Mitochondrial dysfunction Nauheim, Germany is also implicated to be a common component of myriad of other human illnesses ranging from neurodegenerative * co-corresponding authors disorders like Parkinson’s disease to autoimmune illnesses such as multiple sclerosis. The electron transport chain Glucose homeostasis is tightly regulated by insulin (ETC) of mitochondria is critical for mitochondrial biology production from beta-cells and glucagon production from and defects can lead to many mitochondrial disease alpha-cells. Changes in the balance of these hormones lead symptoms. Here we present a publicly available collection to Diabetes Mellitus (DM). DM is foreseen to be the 7th of genetic mutants created in highly conserved, nuclear- leading cause of death by 2030, warranting a high demand encoded mitochondrial genes in Danio rerio. The zebrafish to identify new therapeutics. DM is characterized bya system represents a potentially powerful new opportunity reduction in beta-cell mass and reduced insulin production for the study of mitochondrial biology and disease due to from beta-cells. Alpha-cell development and fate mainly the large number of orthologous genes shared with humans depend on the activity of the homeodomain-containing and the many advanced features of this model system from transcription factor Aristaless related homeobox (Arx). genetics to imaging. This collection includes 20 mutant lines Conditional loss-of-function of Arx in alpha-cells leads to in 17 different genes created by locus-specific gene editing their conversion into functional insulin-producing beta- to induce frameshift or splice-acceptor mutations leading cells and thus an expansion of beta-cell mass. Therefore, to predicted protein truncation during translation. Also inhibition of Arx is an interesting target for the expansion of included are 6 lines created by the random insertion of the beta-cells. The zebrafish model provides a fast, cost-effective gene-breaking transposon (GBT) protein trap cassette. All of and reliable translational platform for drug discovery in an these targeted mutant alleles truncate conserved domains in vivo setting. Here, we screened ~6217 small molecules of genes critical to the proper function of the ETC or genes on a transgenic zebrafish line (TgBAC(arxa:Luc2)) in which that have been implicated in human mitochondrial disease. the arxa promoter drives the expression of the luciferase This collection is designed to accelerate the use of zebrafish gene which allows a sensitive and quantitative readout of to study many different aspects of mitochondrial function promoter activity. Small molecule screening allowed us to with the goal of widening our understanding of their role in identify 36 candidate repressors of arxa promoter activity. biology and human disease. Furthermore, we started to validate these candidates in other assays. Preliminary results showed that DMAT, a potent CK2 inhibitor increases functional beta-cell regeneration. By lineage tracing alpha-cells during beta-cell P-003 A total synthetic approach to CRISPR/Cas9 genome regeneration, we could show that DMAT promotes alpha- editing and homology directed repair to beta-cell transdifferentiation. We intend to test further Sara E. DiNapoli1, Raul Martinez-McFaline1, Caitlin K. the other target molecules identified in the screen. Gribbin1, Paul J. Wrighton1, Courtney A. Balgobin1, Isabel Nelson1, Abigail Leonard1, Carolyn Maskin1, Darya Mailhiot3, Clara Kao3, Sean C. McConnell3, Jill L.O. de P-002 A primer genetic toolkit for exploring Jong3, Wolfram Goessling2, Yariv Houvras1

mitochondrial biology and disease using zebrafish 1 1 1,2 Departments of Surgery, Meyer Cancer Center, Weill 2 Ankit Sabharwal1 , Jarryd M. Campbell1 , Tanya L.1 Cornell Medical College, Brigham and Women‘s Hospital, Schwab1,2 , Zachary1,3 WareJoncas , Mark1 Wishman , Hirotaka1 Dana-Farber Cancer Institute, Harvard Medical School, 3 Ata , Wiebin Liu1 , Noriko Ichino1 , Danielle E. Hunter1 , Chicago Center for Childhood Cancer and Blood Disease, Jake D. Bergren1 , Mark D.1,3 Urban , Rhianna1,3 Urban , 1,2 University of Chicago Alex Cook , Yonghe1,2 Ding , Xiaolei Xu , Karl J. Clark , Stephen C. Ekker CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of 1Department of Biochemistry and Molecular Biology, Mayo loss of function mutations and the knock-in of specific Clinic, Rochester, MN 55905, USA, 2Center for Clinical and alleles using DNA templates and homology directed repair Translational Sciences, Mayo Clinic, Rochester, MN 55905, (HDR). We directly compared synthetic chemically modified USA, 3Division of Cardiovascular Diseases, Mayo Clinic, sgRNAs to in-vitro transcribed sgRNAs and demonstrate Rochester, MN 55905, USA the increased activity of synthetic sgRNAs. Using synthetic sgRNAs and dsDNA templates we optimized the efficiency Mitochondria are a dynamic eukaryotic innovation that of homology-directed repair (HDR) using a genetic assay play diverse roles in biology and disease. The mitochondrial in zebrafish. Utilizing these principles, we performed genome is remarkably conserved in all vertebrates, encoding ......

..... 33 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts precision knock-in of fluorophores at multiple genomic have analyzed drug sensitivities using zebrafish leukemias loci. We extended this approach to knock-in bacterial ex vivo by harvesting cells from leukemic zebrafishes and nitroreductase to a tissue specific gene, and demonstrate plating them into 96-well plates. After 30 hours, drugs were that the application of metronidazole leads to robust added into the wells in 4-fold dilution series and incubated tissue specific lineage ablation. Our studies demonstrate for 72h. Cell titer GLO-assay was used to measure cell the enhanced efficiency and utility of combining synthetic viabilities and IC50 values were estimated from dose- sgRNAs and DNA templates to perform gene knockout and response curves. Responses for 10 drugs (dexamethasone, homology directed repair in vivo. prednisolone, vincristine, doxorubicin, L-aspariginase, dasatinib, ruxolitinib, navitoclax, S63845, MK-2206) and several combinations of these drugs were studied in each P-004 A strategy for cell-specific CRISPR/Cas9 lesions leukemia. In total, 12 of 15 leukemias could be cultured ex vivo and assessed for drug sensitivities. As in primary Eric M. Walton1, Emily G. Hunt1, W. Jared Brewer1, and patient samples, zebrafish ALLs also showed heterogeneity David M. Tobin1 in their drug response profiles with the most variation

1 being seen for responses to dexamethasone, prednisolone Department of Molecular Genetics and Microbiology, and MK-2206. Two leukemias developed dexamethasone- Duke University School of Medicine resistance following tumor evolution and serial passaging, CRISPR/Cas9-based genome editing has expanded our providing potential new models to identify resistance ability for rapid and precise genetic manipulation. However, mechanisms using the zebrafish model. A subset of challenges still exist in creating cell-specific knockouts. leukemias were also responsive to a novel combination Other groups have achieved this in zebrafish by combining of tyrosine kinase inhibitor dasatinib and AKT inhibitor cell-specific Cas9 expression with ubiquitously expressed MK-2206, which is currently being assessed for efficacy in sgRNAs. Here we describe an alternative approach, in human T-ALL. In summary, we show that ex vivo screening which animals ubiquitously express Cas9, and sgRNAs are using zebrafish leukemia cells provides a facile and fast produced in specific cell types. The sgRNAs are generated approach for characterization of zebrafish leukemia drug after ribozyme processing of a Pol II transcript from cell- sensitivities. specific promoters, using a processing strategy described previously by the Ingham laboratory. We show that this method is effective for distinct cell types, including P-006 Exposure to Dithiopyr induces an abnormal macrophages in the context of mycobacterial infection. swimming patterns in zebrafish Jeonwon Lee1, Seungheon Lee1 P-005 Ex vivo screening of drug responses in the 1Faculty of Marine Biomedical Science, Jeju National zebrafish leukemia model University, Korea Saara Laukkanen1, Alexandra Veloso2,3,4,5, David The aim of this study to identify the effects of dithiopyr Langenau2,3,4,5, Olli Lohi1 (DTP) on behavior in zebrafish. DTP is a herbicide which

1 is often used on golf course or some agricultural land. Tampere Center for Child Health Research, Faculty of Toxicity studies about DTP were rarely investigated in fish. Medicine and Medical Technology, Tampere University and 2 Experimental fish were exposed to different concentrations Tampere University Hospital, Tampere, Finland, Department of DTP between range (10-20 µM) for 96 hours in test of Pathology, Massachusetts General Hospital, Boston, container for measurement of median lethal concentrations Massachusetts, 3Massachusetts General Hospital Cancer (LC50) value. We performed measurement of whole-body Center, Harvard Medical School, Boston, Massachusetts, cortisol level and behavioral experiments including the novel 4Harvard Stem Cell Institute, Cambridge, Massachusetts, 5 tank test (NTT) or the open field test (OFT) to assess stress Center for Regenerative Medicine, Massachusetts General responses. After exposure to DTP solution at concentration Hospital, Boston, Massachusetts of 2.5-10 µM or 0.9% NaCl solution for 6 min, we observed Leukemia is the most common pediatric cancer affecting behavior of zebrafish on behavioral tests for 6 min. In the approximately 3,500 children in US every year. Current acute toxicity test, LC50 value of DTP showed 14.63 µM chemotherapy protocols are able to cure over 85% of the on zebrafish. As a result of NTT, not moving duration and pediatric patients, but the prognosis of certain genetic velocity were significantly increased by the exposure at subtypes, including T- and B-acute lymphoblastic leukemia concentration of 5 µM of DTP compared with control group (ALL), and relapsed cases remain poor. Better understanding (p<0.05). However, compared with control group, DTP of the mechanisms behind the disease development and significantly decreased distance moved, frequency in top drug resistance is fundamental as we try to develop curative and significantly increased turn angle, duration in bottom treatments for these patients. To this end, zebrafish models in a concentration-dependent manner (p<0.05). In addition have been widely used to model acute lymphoblastic in OFT, the exposure to DTP decreased distance moved and leukemia, but in vivo drug studies are less suitable for high- velocity compared with control group significantly (p<0.05). throughput characterization of drug responses. Here, we And DTP significantly increased latency to first in center ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 34 Poster Abstracts zone, not moving duration, turn angle and meandering Rhabdomyosarcoma (ARMS) is characterized by genetic movement in a concentration-dependent manner (p<0.05). translocations while Embryonal Rhabdomyosarcoma (ERMS) In whole-body cortisol level, DTP did not affect on zebrafish. is characterized by Ras pathway activation. Treatment In conclusion, these results of study suggest that DTP for either RMS subtype requires surgical resection, induces toxic response and the stress response on behavior chemotherapy, and radiation with overall poor prognosis for but not whole-body cortisol in zebrafish. patients with high-risk features including metastasis. Thus, there is great interest in elucidating key molecular pathways and genetic factors that are involved in RMS progression. In P-007 Looking for adolescent idiopathic scoliosis the Langenau lab, we utilize a zebrafish model of kRASG12D- mechanisms in zebrafish induced ERMS and have recently developed a p53-/- (null) zebrafish model. More than of 1/3 of RMS patients have Diane Sepich 1, Lila Solnica-Krezel 1, Ryan Gray 2 mutations or Loss-of- Heterozygosity (LOH) of the p53 locus. Specifically, we performed a tumor evolution screen using 1 Dept. of Developmental Biology, Washington University 2 serial engraftment of ERMS followed by RNA sequencing to School of Medicine, St Louis MO, Department of discover novel pathways that are associated with elevated Pediatrics, Dell Pediatric Research Institute, The University tumor growth and metastasis. Plexin A1 was expressed at >8 of Texas at Austin, Dell Medical School, Austin, Texas, fold in metastatic lesions of the zebrafish and expressed in United States of America. >80% of human RMS. Plexins are a large family of receptors Adolescent Idiopathic Scoliosis afflicts 2-3% of the for semaphorin ligands. Knockdown of PLXNA1 decreased population. To analyze scoliosis in zebrafish, we are 1) human cell proliferation, increased differentiation and screening mutagenized adult fish for twists and bend in the impaired anchorage-independent growth. Collectively, my body, 2) identifying the mutated genes, and 3) testing genes data supports the hypothesis that PLXNA1 is an important reported to cause scoliosis and testing genes associated moderator of tumor growth in a large fraction of human with scoliosis from human studies. RMS and likely regulates local invasion and metastasis. This work is important because therapeutic targeting of the Screening starts at 21-35 dpf and is usually repeated at plexin/semaphorin signaling network by various inhibitors 2-week intervals. In two years, we have identified 18 are now in in clinical development. Dosing human RMS cells candidate mutations. The phenotypes recovered fall into with these inhibitors has shown decrease of migration and distinct categories. 1) Fish that fail to thrive. They first proliferation in vitro. I have also shown downstream cellular appear runted, and later, bent (stl427, stl430). 2) Fish mechanisms (small GTP-ases) by which PLXNA1 regulates appear to thrive, but as adults show multiple body bends; growth and metastatic progression. Following the success the time of onset varies by mutation (stl438; stl460, stl461). of this work, future experiments will focus on determining 3) Fish are bent at specific locations near head or tail, but additional molecular pathways regulated by PLXNA1, otherwise appear healthy (stl428; stl445). providing important clinical correlates for assessing on- target inhibition for selected drugs. Using Whole Genome and Exome Capture Sequencing, we identified SCO-spondin as a candidate gene (stl297). Crossing to a known allele of SCO-spondin confirms this gene. WGS identified premature stop codons in nlrc5 and P-009 MITF-low zebrafish melanomas reveal in alanine tRNA ligase in stl438. Both genes are being transcriptional signature subdivisions and cellular tested by creating new CRISPR/cas 9 induced mutations subpopulations of residual diseasein vivo and crossing to the stl438 mutation. WGS identified no Jana Travnickova1,2, Sonia Wojciechowska1,2, Ava premature stops in stl445. We are testing genes with amino Khamseh1, Philippe Gautier1, Amy Capper1,2, Marie E. acid substitutions, e.g. nlrc3 like. Mathers3, Chris Ponting1, E. Elizabeth Patton1,2 1 MRC Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit, University of Edinburgh, Crewe P-008 PlexinA1 is a driver of metastasis and growth in 2 rhabdomyosarcoma Road South, EH4 2XR, UK, CRUK Edinburgh Centre, MRC Institute of Genetics and Molecular Medicine, University Alessandra Welker1,2, Madeline Hayes1,2, Qin Tang1,2, of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, Sowmya Iyer1,2, David Langenau1,2 UK, 3Department of Pathology, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK 1Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital The MITF-low melanoma transcriptional signature is

2 predictive of poor outcome for patients and represented Research Institute, Boston, MA 02129, Harvard Stem Cell across all melanoma subtypes regardless of mutation Institute, Cambridge MA 02139 status, but little is known about the biological significance Relapse and metastasis are major clinical problems of this transcriptional class. Here we use genetic models facing patients with rhabdomyosarcoma (RMS), a of zebrafish with a conditional mitfa allele to show that devastating pediatric malignancy of the muscle. Alveolar the MITF-low state is causal of cancer progression and ......

..... 35 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts a predictor of melanoma biological subtype. MITF-low electron microscopy. No difference was observed in hair zebrafish melanomas resemble human TCGA and Lund cell morphology between wildtype and gmds mutant MITF-low melanomas, and MITF-low activity is sufficient zebrafish. Additional experiments utilizing vital stains and to promote melanoma with just a p53 mutation. BRAFV600E markers of synaptogenesis will be performed to determine accelerates MITF-low melanoma onset and alters melanoma potential neuromast-based defects that may lead to circular growth: melanomas with a nodular invasive growth swimming behavior in gmds mutants. pattern show very strong activation of the neural crest and melanocyte lineage, while melanomas with a superficial In summary, gmds mutation causes cerebral hemorrhage in invasive growth pattern reveal more mesenchymal and zebrafish, supporting human studies that identify this gene migratory characteristics. as contributing to stroke risk. This project will help to expand our understanding of vertebrate blood vessel development MITF-low melanomas regress following genetic inhibition and a potential genetic link to stroke, and may also serve as of MITF activity and recur following reactivation of a useful model for hearing loss studies based on potential MITF at the same site as the original tumour suggesting defects in neuromast hair cell function. that subpopulation(s) of melanoma cells persist during regression and may be capable of repopulating the tumour. Indeed, using fluorescent reporter transgenic lines we P-011 Using zebrafish larvae as a real-time and high- discover minimal residual disease with no MITF activity throughput in vivo platform for developing new at the regression site. Transcriptomic analysis reveals therapeutic regimens for epilepsy these residual cells are enriched in neural crest stem cell Teng-Yu Chiu1, Po-Yuan Chen2, Bing-Hung Chen3, and Tzu- and mesenchymal cell signatures. Critically, single cell Fun Fu1,2 RNA-seq analysis shows that these cells pre-exist in the primary tumour. Identification and characterization of 1 Department of Medical Laboratory Science and these subpopulations over regression and recurrence of Biotechnology, National Cheng Kung University, Tainan, melanoma may yield candidate targets for putative drug Taiwan, 2The Institute of Basic Medical Sciences, National resistant and tumor recurrent cell populations. Cheng Kung University, Tainan, Taiwan, 3Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan P-010 Understanding the role of GDP-fucose synthesis in blood vessel stability and circular swimming behavior Epilepsy is the second most common neurological disorders using zebrafish models in modern society. Approximately 50 million people

1 1 worldwide have active epilepsy with continuing seizures Gerissa Fowler , Curtis R. French that need treatment. Epilepsy is usually controlled with 1Discipline of Genetics, Memorial University, St. John’s, anti-epileptic drugs (AEDs) to ease or prevent seizure Newfoundland episode. However, 30-40% of epileptic patients are pharmacoresistant to the currently available AEDs and Blood vessel stability can be affected by many genes and remain uncontrolled. Furthermore, many patients receiving pathways. Mutations in such genes can increase stroke AEDs suffer from serious adverse effects including liver risk. Previous work has identified SNPs in the gmds gene toxicity, sedation and cognitive impairment. Therefore, as associated with cerebral small vessel disease, which new drug/strategies for treating epilepsy and tools assisting is a stroke factor. There is no information regarding the in developing the new therapeutic regimen are needed. biological function of gmds with respect to blood vessel In the current study, we characterized and compared development. several compound-induced epilepsy models established on zebrafish larvae of various stages. The swimming behaviors, The gmds gene encodes the enzyme GDP-Mannose including distance, activity, acceleration and turning angles, 4,6-Dehydratase that catalyzes the de novo synthesis of of larvae exposed to epilepsy inducers were recorded and GDP-fucose from mannose-based substrates. GDP-fucose analyzed. The responsiveness of epileptic larvae to AEDs can also be produced by a salvage pathway which utilizes were tested. The impact of several selected compounds, dietary fucose as a substrate. We have shown expression including micronutrients and 65 herbal extracts, to the of gmds in the pharyngeal arches, the site where the first compounds-induced epilepsy and the therapeutic effects of major arteries develop in zebrafish as well as lateral line AEDs were also screened and examined. Our results showed neuromasts. We demonstrate that knockdown or mutation that supplementing with multi-vitamins strengthened of gmds in zebrafish results in cerebral hemorrhages. In the therapeutic efficacy of AEDs but with significantly addition, we show that morpholino mediated knockdown varied effects depending on the AEDs used. In addition, of salvage pathway components also cause cerebral one of the tested herbal extracts, PL, displayed potential hemorrhage, and increase hemorrhages rates in gmds anti-epileptic activity. We concluded that multi-vitamins mutants. These results support a role for both de novo and supplementation could be a promising regimen to improve salvage pathway of GDP-fucose synthesis contributing to the therapeutic efficacy of AEDs. Our results also support blood vessel stability. gmds mutants also exhibit circular the use of zebrafish as an economical and efficient in vivo swimming behavior, and given its expression in neuromasts, platform for the development of anti-epileptic regimen. we assessed neuromast hair cell structure using scanning ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 36 Poster Abstracts P-012 Exploring the biological impact of grape seed Brisbane, Australia; Queensland Centre for Mental Health extract in vivo --- using zebrafish as a platform for Research, West Moreton Hospital and Health Service and compounds toxicity screening and evaluating the University of Queensland, Brisbane, Australia interplay among organisms Schizophrenia is a debilitating mental disorder of Hsin-Hsuan Yu1, Rui-Jun Liu1, Tzu-Fun Fu1,2, Bing-Hung neurodevelopmental origin. Although environmental Chen3 factors are implicated, it is now accepted that genetic

1 factors are responsible for most of the liability to disease. Department of Medical Laboratory Science and Identifying the genetics predisposing to this disorder could Biotechnology, National Cheng Kung University, Tainan, help us model the developmental pathogenic pathways Taiwan, 2The Institute of Basic Medical Sciences, National 3 underlying disease onset, progression and severity. These Cheng Kung University, Tainan, Taiwan, Department of models will in turn be of considerable value for both Biotechnology, Kaohsiung Medical University, Kaohsiung, understanding disease and discovering novel treatments Taiwan for schizophrenia. Grape seed extract (GSE) and its derived natural phenolic Recent years have seen rapid progress in discovering compounds have been known for their anti-inflammation schizophrenia-associated genetic variants., through the and anti-oxidation activities. We had previously shown that application of GWAS methodology. My group has been part GSE exhibited anti-allergic and anti-proliferative potential of this international effort in amassing essentially European against RBL-2H3 mast cells and Ca9-22 oral cancer cells, ancestry samples; in addition, we have studied two unique, respectively. In general, GSE is considered beneficial and non-European populations from India and Sarawak. We are has been routinely consumed as a nutrient supplement now reaching the exciting stage of employing the variants by the general public. However, we had shown that GSE discovered in these samples to establish stable genetic inhibited bacterial growth, raising the concern that it might models relevant for schizophrenia basic research and drug interfere with probiotics. In the current study, we examined discovery. the impact and potential side-effects of high-dose GSE, along with other 65 plant extracts, using zebrafish embryos We are taking advantage of the optical accessibility of as an in vivo high-throughput screening platform. The the zebrafish research model to first examine the loss- effects of plant extracts on the growth of both probiotic of-function effect of our schizophrenia candidate genes and pathogenic bacterial strains was also examined and using knockout (CRISPR) and knockdown (transgenic RNAi) compared. Several parameters pertaining to embryonic approaches. We have already established models through development, including larval survival rate, morphological which we have identified clues for the pathogenic role of characteristics (circulation, swim bladder, eyes, body the miRNA miR-137 and the gene NAPRT1. Specifically, curvature and pigmentation) and larval swimming behavior we have found that (a) miR-137 LOF strongly impacts the were recorded. Our results showed that GSE could be mechano-sensitivity of the zebrafish, presumably through a beneficial to zebrafish embryonic survival by alleviating key role in synaptogeneisis or synaptic function; (b) NAPRT1 DMSO-induced toxicity. In addition, GSE-mediated growth LOF is associated with abnormal brain development and inhibition exhibited significant variation among different folding. We are also working on additional candidates such bacterial strains and in zebrafish larval survival. Our data as NRXN1, for which we have just developed knockout lines. suggested that the observed difference in growth inhibition might be resulted from the varying impact of GSE on folate-mediated one-carbon metabolism among different P-014 Development of compound transgenic zebrafish organisms. Our results support the use of zebrafish embryos model to study the microglia function in glioblastoma as a measure for the screening of compounds activity/ toxicity and an applicable model for studies in probiotics- Suvarsha Sura1, 2 and Surendra K Rajpurohit2 related research. 1Department of Biology, 2College of Science and Mathematics, and Georgia Cancer Center, Augusta University, Augusta, GA. P-013 Investigating the neuro-developmental role of schizophrenia-associated genes using the zebrafish Glioblastoma is an aggressive cancer of the brain, causing

1 2 3 about 13,000 deaths annually in the United States. Jean Giacomotto , Alisha Tromp , Bryan Mowry Despite advances in treatment, the median survival 1 Queensland Brain Institute, The University of is only 15 months. Therefore, there is a dire need for Queensland, Brisbane, Australia; Queensland Centre for therapies that can improve outcome for patients with Mental Health Research, West Moreton Hospital and glioblastoma. Here we propose to utilize Zebrafish as a Health Service and University of Queensland, Brisbane, model to examine the role of NF-kB and microglia cells Australia, 2 Queensland Brain Institute, The University of in development of glioblastoma. Recently, zebrafish Queensland, Brisbane, Australia, been emerged as a powerful model organism for studying cancer because it is easy to introduce genetic 3Queensland Brain Institute, The University of Queensland, changes and to follow the phenotypic changes using ......

..... 37 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts microscopic imaging. We have established the zebrafish effects on intracellular ROS, as well as the reduction of transgenic model to develop automated high-throughput TUNAL reactions. No direct dug-drug interaction was found drug discovery platform and studied the pancreatic between both. Hence, the protective mechanisms of FA β-cell neogenesis and proliferation. In zebrafish and against aminoglycoside ototoxicity were elucidated in this mammals, mature microglia are present throughout the study. In conclusion, our study established a novel hair cell parenchyma of the brain and spinal cord. Generation of reporter line that may be of benefit in hair cell research new transgenic reporter lines and imaging tools further and screening applications of compounds for inducing, support the use of zebrafish in-vivo studies. In this study, preventing or therapeutic hearing loss. we are generating compound transgenic progeny of microglia; Tg(mpeg1:mCherry) with inflammatory NF-kB Tg(6xNFκB:EGFP) strain. For visualization and study of P-017 Investigating the role of tetraploid intermediates in cell death process in-vivo condition, this transgenic strain melanoma progression further cross breed with a transgenic line expressing Annexin-5-YFP fusion. The generated new compound Revati Darp1,2, Marc Vittoria3, Neil Ganem3, Craig Ceol1,2 transgenic line is of Microglia+/NF-kB+/Annexin-V+ strain. 1 University of Massachusetts Medical School, Program To get the transparent progeny, this newly generated 2 multiple transgenic fishes are crossbreeds with the in Molecular Medicine, Worcester, MA, USA , University transparent Mutant Caspe. Through these processes we of Massachusetts Medical School, Department of Molecular, Cellular and Cancer Biology, Worcester, MA, are developing the multifunctional microglia transgenic- 3 transparent mutant phenotype. Fluorescent microscopy USA, Departments of Pharmacology and Experimental is to be utilized to confirm the expression of fluorescent Therapeutics and Medicine, Division of Hematology and proteins in the newly generated transgenic progenies. Oncology, Boston University School of Medicine, Boston, Thus, we are generating and establishing a novel MA, USA transgenic model to study the role of microglia function Melanoma, the most lethal form of skin cancer arises from in brain tumor. This study will yield crucial new insights altered cells in the melanocyte lineage, but the mechanisms into NF-kB pathways in glioblastoma and result in the by which these cells progress to melanoma are unknown. development of novel therapeutic approaches. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in a melanoma-prone zebrafish strain expressing BRAFV600E, P-015 Using zebrafish model to elucidate the protective a mutant form of the BRAF kinase. We found that, unlike mechanisms of Ferulic acid (FA) against aminoglycoside wild-type melanocytes, melanocytes in transgenic ototoxicity BRAFV600E animals are binucleate and tetraploid. p53 1 Furthermore, melanocytes in -deficient transgenic Ju Chang-Chien, Jiann-Jou Yang BRAFV600E animals exhibit 8N and greater DNA content, 1Department of Biomedical Sciences, Chung Shan Medical suggesting bypass of a p53-dependent arrest that halts University, Taichung, Taiwan cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF Hair cells in the inner ear play essential roles in hearing. pathway activity as contributors to melanoma initiation. Genetic mutations and many environmental factors, such To gain insight into the mechanism by which BRAFV600E as acoustic trauma, age and ototoxic drugs, result in hair generates binucleate, tetraploid cells, we established cell malfunction and then hearing loss, which is a common a lentiviral-based in vitro model to induce BRAFV600E and irreversible sensory disorder in human populations. expression. Using live-cell imaging, immunofluorescence Mechanosensory hair cells in the inner ear are highly and flow cytometry approaches, we found thatBRAFV600E - conserved between zebrafish and mammals in terms of generated tetraploids arise via cytokinesis failure during development, morphology, and physiological function mitosis due to reduced activity of the small GTPase RhoA. so that we created a novel hair cell transgenic reporter Additionally, BRAFV600E activity in the prior G1/S phase of zebrafish (pvalb3b:TagGFP) using Tol2 system to study the cell cycle is essential in generating tetraploids. We are hearing impairment. Ototoxicity is an ignored side effect of currently determining the mechanism by which BRAFV600E aminoglycosides in clinical medicine, but its mechanisms activity in G1/S causes the cytokinesis defect in mitosis remain largely unknown. Ferulic acid (FA) derivatives have and whether BRAFV600E-generated tetraploids serve as been applied in the clinical trials for preventing and treating intermediates in melanoma initiation and progression. auditory dysfunctions in America, and in the present These are important questions as recent studies have study, effects of FA on neomycin-elicited ototoxicity are shown that aneuploid progeny of tetraploid cells can be demonstrated in the lateral line neuromasts of hair cell intermediates in tumor development and deep sequencing reporter transgenic line. In transgenic fish treated with data suggest at least one third of melanomas and other solid FA before and during neomycin exposure, FA conferred tumors have undergone a genome doubling event during sufficient protection against hair cell damage across a wide their progression. Overall, our melanoma-prone zebrafish range of neomycin concentrations by combining the uptake model and in vitro data suggest a role for BRAFV600E- block of neomycin into sensory hair cells, the antioxidant induced tetraploidy in the genesis of melanomas......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 38 Poster Abstracts P-018 ZeOncoTest: a new zebrafish xenotransplantation including human MYC-transgenic (rag2:hMYC) fish. model for drug discovery in cancer Until recently, robust B-ALL models in zebrafish had not

1 1 1 been described, but we and others discovered that both Carles Cornet , Sylvia Dyballa , Javier Terriente and rag2 mMyc rag2 hMYC 1 : and : fish actually develop both Valeria Di Giacomo B-ALL and T-ALL. Surprisingly, expression profiling showed 1 Zeclinics S.L., IGTP Edifici Muntanya (c/ de Can Ruti, Camí the seemingly-similar B-ALL of mMyc vs. hMYC fish are in de les Escoles s/n; 08916 BDN, Lab P0-8), Barcelona Spain. fact quite different, arising in distinct B cell lineages. T-ALL of mMyc and hMYC fish also show differences. Moreover, Zebrafish is a reliable and robust experimental tool for biphenotypic ALL expressing both B- and T-lineage genes cancer research: it develops tumors by exposure to was found in one rag2:mMyc case, and we have new carcinogens or by genetic mutations and can be host for evidence that hMYC fish also develop biphenotypic ALL. xenotransplants of human cancer cells. Together, this suggests at least 6 different types of ALL occur in mMyc/hMYC transgenic zebrafish, although both utilize In our assay, we use zebrafish larvae as hosts for the same rag2 promoter and near-identical MYC proteins. human cancer cells for drug discovery. In brief, cells are Potential explanations for these varying ALL phenotypes fluorescently labeled and injected into the larvae, which include activation of distinct mMyc vs. hMYC target genes are then incubated with candidate drugs. Injected larvae and pathways, rag2 (and thus MYC) expression by different are imaged individually before and few days after drug lymphocyte developmental stages, genetic polymorphisms incubation. Tumor volume and cell dispersion are analyzed in the strain backgrounds used, or perhaps all of these. at the two time points and the difference provides readout Overall, these data demonstrate that MYC can induce of tumor growth and metastatic capability. The comparison several forms of zebrafish ALL; we hypothesize each may between treated and untreated embryos determines the have a human counterpart. Deciphering the genetic and effect of tested drugs. Throughout the development of molecular details of these various ALL types and learning this project we have detected several discrepancies with how MYC fosters lymphocyte transformation in each of previous reports on zebrafish tumor xenotransplants them should be informative, particularly in light of the derived from technical differences in cell labelling methods, rapidly-expanding list of human ALL subtypes site of injection, imaging acquisition and analysis. After evaluating different methodologies, we have developed a robust and reproducible automated middle throughput assay, by treating MDA-MB-231, HCT116 and PC3 cell lines P-020 A Protein Tyrosine Phosphatase 4A3 (PRL-3)/ xenotransplants with drugs with known anti-cancer and Wnt signaling axis as a novel therapeutic target in Acute anti-metastatic effects. Lymphoblastic Leukemia (ALL) relapse Meghan G. Haney1, Kristin O’Leary1, Min Wei1, Jessica Our improved assay sets zebrafish as powerful tool between 1 in vitro and in vivo studies in mice, decreasing time and Blackburn costs of preclinical studies in the oncology field. 1University of Kentucky, College of Medicine, Department of Molecular and Cellular Biochemistry

P-019 Multiple types of zebrafish ALL induced by one Acute Lymphoblastic Leukemia (ALL) is the most common transgenic oncoprotein pediatric malignancy and 15-20% of patients experience relapse, which is frequently more aggressive and treatment Chiara Borga1, Jessica Burroughs-Garcia1, Clay Foster4, resistant than primary disease with unfavorable outcomes. Gilseung Park2, Ameera Hasan3, Arpan Sinha1, J. Kimble Relapse occurs because conventional chemotherapies are Frazer1,2,3 unable to reliably and completely eliminate leukemia stem

1 cells (LSCs), which have the ability to self-renew and form a Dept. of Pediatrics, Section of Pediatric Hematology- leukemia from a single cell. The Wnt signaling pathway has Oncology, University of Oklahoma Health Sciences Center, 2 emerged as having an important role in LSC self-renewal in Oklahoma City, OK USA, Dept. of Cell Biology, University T-ALL, but current Wnt inhibitors have unacceptable toxicity of Oklahoma Health Sciences Center, Oklahoma City, OK 3 in the clinic. I have found the Protein Tyrosine Phosphatase USA, Dept. of Microbiology & Immunology3, University 4A3 (PTP4A3 or PRL3) is highly expressed by ALL cells that of Oklahoma Health Sciences Center, Oklahoma City, OK 4 also express Wnt pathways genes, and is not expressed by USA, Dept. of Microbiology and Immunology, University normal cells. In a zebrafish Myc-induced ALL model, PRL3 of North Carolina, Chapel Hill, NC USA expression significantly enhanced LSC frequency, while Acute Lymphoblastic Leukemia (ALL) dominates pediatric inhibition of PRL3 reduced LSC numbers in vivo. In human oncology, representing >25% of all childhood cancer. ALL cells, I found that PRL3 activates the expression of Wnt can develop in either B or T lymphoblasts, with B-ALL pathway genes. I have created transgenic zebrafish models more common. The first zebrafish transgenic cancer of ALL that over-express both wild-type and mutant forms model used a zebrafish rag2 promoter to regulate murine of PRL3 and constitutively active beta-catenin to define Myc (rag2:mMyc), which potently induced T-ALL. Soon the role of PRL3 in Wnt signaling by assessing the effects after, other D. rerio lines prone to T-ALL were created, of PRL3 and PRL3 mutants on LSC self-renewal and the ......

..... 39 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts phosphorylation status of Wnt pathway components in cell culture and mouse xenograft models of RMS, we zebrafish models and human ALL cells. My research defines have shown that Six1 is necessary for the growth and a novel role for the phosphatase PRL3 in self-renewal survival of RMS, in that Six1 depletion abrogates RMS cell of cancer stem cells via activation of Wnt signaling, and proliferation and migration. Mechanistically, we discovered targeting PRL3, which is expressed specifically by leukemia that Six1 regulates RMS disease progression by inhibiting cells, represents a novel therapeutic strategy to inhibit WNT the differentiation of RMS cells into myogenin and myosin signaling in ALL. heavy chain expressing myotubes, thus maintaining the tumor cells in a more progenitor-like state. Our studies have thus demonstrated that understanding the role of Six1 in P-021 The Six1 Homeobox Transcription Factor basic skeletal muscle biology can provide mechanistic Regulates the Differentiation State of Embryonal insight on the pathophysiology of RMS and identified Six1 Rhabdomyosarcoma as a potential therapeutic target for RMS. Jessica Hsu1,2, Jenean O’Brien3, Jared Talbot4, Hope Eden3, Aaron Clark3, Oscar Yip, Theresa Sanborn, Sharon 4 2 1 P-022 Understanding tumor/microenvironment cell Amacher , Kristin Artinger , Heide Ford competition using a zebrafish model 1 University of Colorado Anschutz Medical Campus, Miranda V. Hunter1, Richard M. White1 Department of Pharmacology, 2University of Colorado Anschutz Medical Campus, Department of Craniofacial 1 Cancer Biology and Genetics Program, Memorial Sloan Biology, 3University of Wisconsin Superior, Department of Kettering Cancer Center, New York, NY 10065 Natural Sciences, 4Ohio State University, Department of Molecular Genetics The role of the tumor microenvironment (TME) is widely acknowledged to play a significant role in tumor growth and Skeletal muscle development requires exquisite coordination metastasis. Although the ‘seed and soil’ hypothesis, stating of gene expression for muscle to be specified, patterned and that cancer cells require a favorable microenvironment in to subsequently differentiate into contractile muscle fibers. order to grow and divide, was first proposed over a century Six1 is a homeobox transcription factor that is required for ago, it is still unclear how the tumor and its microenvironment multiple steps in muscle development, and importantly for interact to promote tumorigenesis. Specifically, it is muscle stem cell maintenance. In mature tissue, this muscle unknown how tumors cells reshape the TME to make space stem cell maintenance is critical to replenish damaged for the growing tumor mass. One likely possibility is that fibers, and as such the impairment of this coordinated tumors induce death of TME cells around them to allow process can manifest in the form of Rhabdomyosarcoma for tumor invasion into surrounding tissues, in a conserved (RMS), a pediatric cancer of the skeletal muscle. RMS is process known as cell competition. Cell competition occurs the most common soft tissue pediatric tumor with unique when faster growing (“winner”) cells kill the slower growing molecular characteristics that resembles embryonic skeletal (“loser”) cells around them, in order to facilitate winner muscle. Although the majority of RMS patients are cured cell growth. Tumor expansion could be enabled by direct with conventional chemotherapies, there have been few competitive interactions between the faster-dividing tumor advances in the treatment of high-risk metastatic RMS, in cells and slower-dividing TME cells, inducing cell death in the which surgery is difficult, chemotherapies are ineffective, TME. Although cell competition is known to be important and survival rates remain low (31% versus 67% for low/ for animal development and for maintaining tissue health, intermediate risk patients). Based on our understanding of it has not been studied in cancer in vertebrate animals. We Six1 in development, we hypothesized that the zebrafish are using a zebrafish model of melanoma to investigate Six1 ortholog, six1b was required for maintenance of a role for cell competition in tumor growth and invasion. muscle cell stem cells. Here, zebrafish six1b mutants were Using a combination of widefield and confocal microscopy examined for alterations in muscle differentiation gene approaches, we are examining whether tumors induce cell expression, however no difference in myoD or myogenin death in surrounding microenvironment cells. Preliminary expression was observed over the course of embryonic results show upregulation of the cell death markers cleaved myogenesis. Similarly, we observed that upon induction caspase-3 and Fas receptor in cells that contact tumors. We of skeletal muscle injury, no differences in activation of are currently manipulating apoptosis with pharmacological muscle satellite cells were seen between six1b mutant and and genetic approaches to examine the functional role of wildtype larvae, suggesting that thesix1b depletion alone is tumor-induced microenvironment apoptosis. Together, insufficient to impair skeletal muscle development. We next the results from these studies will clarify the role of cell explored the possible role of six1b in the context of RMS competition in vertebrate cancer, and will provide potential where skeletal muscle biology is heavily implicated in disease molecular targets to both identify tumor cells at early stages pathophysiology, and thus established a zebrafish RMS and develop therapies to prevent tumors from progressing. tumor model and found that despite six1b depletion being inconsequential to normal skeletal muscle development, RMS tumors in six1b mutants grew significantly slower compared to their wildtype siblings. Expanding to human ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 40 Poster Abstracts P-023 Characterizing neural crest epigenetic and adipocytes in the TME promote melanoma invasiveness transcriptional reactivation during melanoma initiation through direct transfer of lipids from adipocytes to cancer cells. RNA-seq of A375 melanoma cells cultured with 3T3L1 Eva T. Kramer1, Anna P. Zarov2, Paula M. Godoy3, Charles 4 adipocytes revealed a significant enrichment of ER stress K. Kaufman and melanoma gene invasion signatures. Consistent with 1 Washington University in Saint Louis, Division of Biology this, treatment of zebrafish melanoma cells with palmitic and Biomedical Sciences, Developmental, Regenerative, acid activated the ER stress pathway and induced genes and Stem Cell Biology Program, Saint Louis, MO, 2 associated with cancer invasiveness. Treatment with oleic Washington University in Saint Louis, Department of acid did not induce ER stress and adding oleic acid with Biology, Saint Louis, MO,3 Washington University in palmitic acid in a 1:1 ratio rescued cells from ER stress. Saint Louis, Division of Biology and Biomedical Sciences, Palmitic acid displayed toxicity to the zebrafish melanoma Computational and Systems Biology Program, Saint Louis, cells as measured by cell viability, but this was also rescued MO, 4 Washington University School of Medicine, Division through the mixture of oleic acid with palmitic acid in a of Medical Oncology, Saint Louis, MO 1:1 ratio. From this preliminary data, we hypothesize that adipocyte-derived lipids activate the ER stress pathway Pinpointing the critical events leading to cancer initiation which promotes melanoma invasiveness. We aim to from a precancerous field remains one of the biggest make novel discoveries on the molecular mechanisms of open questions in biology. To evaluate the steps leading to melanoma metastasis, and determine how the TME can melanoma initiation, we study a transgenic BRAFV600E / p53- support melanoma progression. /- zebrafish model which develops one to three separate melanomas during adulthood. The crestingene marks neural crest in zebrafish embryos up to 72 hours post fertilization; P-025 A new technique to biopsy benign and malignant however, it is reactivated in melanoma initiation indicating zebrafish lymphocytes a reemergence of a neural crest progenitor state. Utilizing fluorescent transgenes, we can visualize melanocytes with Gilseung Park1, Clay Allen Foster2, Megan Malone-Perez3 mitf:mCherry and melanoma cells from their initial stages and J. Kimble Frazer1,3,4 with a crestin:EGFP neural crest reporter. With earlier 1 Departments of Cell Biology, University of Oklahoma detection of melanoma, we can profile the earliest changes 2 in the epigenome and transcriptome using ATAC-Seq and Health Science Center, OK, USA, Departments of RNA-Seq. Analysis of melanoma cells compared to normal Microbiology and Immunology, University of North Carolina, Chapel Hill, NC USA, 3 Departments of Pediatrics, melanocytes identified upregulation of known differentially 4 expressed genes in melanoma, including sox10, dlx2a, University of Oklahoma Health Science Center, OK, USA, and mitfa, as well as genes previously uncharacterized in Departments of Microbiology & Immunology, University of melanoma such as etv4, etv5b, and sox4a. These genes Oklahoma Health Science Center, OK, USA are associated with proximal and distal open chromatin Over the last few decades, zebrafish (Danio rerio) have domains representing putative neural crest- and melanoma- emerged as a compelling model to study vertebrate associated enhancer elements, including regions with lymphocytes, as well as diseases of malignant lymphocytes. differential accessibility. Interestingly, we also identified Zebrafish have a similar adaptive immune system that downregulation ofsox9b , a SOXE transcription factor in the includes B and T cells, like humans, which has allowed same family as sox10, in melanoma cells. We have begun many zebrafish T-cell acute lymphoblastic leukemia (T-ALL) to dissect the underlying regulatory pathways driving models to be created, including human MYC (hMYC) alterations in etv4, etv5b, sox4a, and sox9b expression. transgenic fish. Recently, we described a new zebrafish Identifying the first molecular changes leading to melanoma B-cell ALL (B-ALL) model in double-transgenic rag2:hMYC, initiation and their underlying causes will allow earlier lck:GFP zebrafish. In these fish, human MYC (hMYC), an detection and more effective treatment for patients. oncogenic transcription factor, is regulated by a zebrafish rag2 promoter expressed by immature T and B cells, and the zebrafishlck promoter drives different levels of GFP in T and P-024 Adipocyte-derived lipids as drivers of endoplasmic B cells. Thus, in rag2:hMYC, lck:GFP fish, non-malignant T reticulum (ER) stress and invasion in melanoma and T-ALL cells are brightly fluorescent, whereas B and B-ALL D. rerio 1,2 2 cells are dimly fluorescent. Studies of lymphocytes Dianne Lumaquin , Richard M. White or zebrafish ALL typically require euthanizing animals to 1 Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional obtain lymphocytes. However, it would be advantageous MD-PhD Program, 2 Department of Cancer Biology and if we could obtain lymphocytes without euthanasia. To Genetics, Memorial Sloan Kettering Cancer Center permit this, we have developed techniques to biopsy living fish and obtain normal lymphocytes and ALL cells from The tumor microenvironment (TME) can influence various body regions, such as thymus, kidney-marrow, and cancer progression and metastasis but we have a limited even scales. In rag2:hMYC, lck:GFP fish, we have found that understanding of the mechanisms causing these changes both B and T cells can be obtained from thymus, and B cells in tumor phenotype. We have recently shown that are abundant in scales. We have also recovered ALL cells ......

..... 41 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts from scale biopsies of fish with T-ALL. Expression analyses Belgium, 2 Cancer Research Institute Ghent (CRIG), Ghent, of non-malignant lymphocytes and ALL cells from different Belgium, 3 Department of Human Structure and Repair, biopsy regions demonstrate that GFPhi cells from thymus Ghent University, Ghent, Belgium and scales express T cell genes, and GFPlo cells express B cell genes. These novel biopsy methods will now permit PARP inhibitors (PARPi) trap ‘Poly-ADP Ribose Polymerase’ new studies using ex vivo samples obtained from live fish (PARP) and force dividing cells into utilizing Homologous without euthanasia. Recombination (HR) to repair single stranded DNA breaks during replication. (Tumour) cells lacking HR repair are therefore sensitive to PARPi treatment. PARPi are being implemented as standard of care for specific tumour types. P-026 Determining the effects of nutrition in tumor New and more potent PARP inhibitors are constantly in initiation and growth during melanoma development development. Therefore, there is a great need to test novel Jonathan Spalding1,2,3,4, Vadim Grigura1,2, Gary Patti3,4, compounds in a fast and accurate manner. We investigated Charles Kaufman1,2 if zebrafish could be used to test PARPiin vivo. 1 Division of Medical Oncology, Department of Medicine We exposed zebrafish embryos to four different PARPi Washington University in Saint Louis, St. Louis, MO 63110, (Olaparib, Niraparib, Talazoparib, Veliparib) and measured USA, 2 Department of Developmental Biology, Washington their capacity to activate the HR pathway. This was University in Saint Louis, St. Louis, MO 63110, USA, 3 quantified by immunofluorescent staining of the number Department of Chemistry, Washington University in Saint of Rad51 foci in dividing cells of zebrafish embryos. Rad51 Louis, St. Louis, MO 63110, USA, 4 Division of General recruitment is a crucial event during HR and the number of Medical Sciences, Department of Medicine, Washington Rad51 foci allows measurement of HR efficiency. Olaparib, University in Saint Louis, St. Louis, MO 63110, USA Niraparib and Talazoparib treatment resulted in a 15 times increase of Rad51 foci compared to untreated controls. In Dietary restriction has long been observed to reduce the contrast, the weakest inhibitor Veliparib only induced a 4 incidence of a large number of chronic diseases, including times increased number of Rad51 foci. Prolonged exposure cancer. Epidemiological data has suggested that tumor to PARPi also causes cell cycle arrest, as indicated by higher onset is nutrition-dependent, and this correlation has been numbers of cells in S/G2 compared to controls. Combining experimentally confirmed in a variety of cancer models, PARPi treatment with irradiation did not increase Rad51 including for the first time in a zebrafish model of p53/ foci, suggesting that extra damage is accumulating or BRAFdependent melanoma by our group. However, the repaired with different pathways.

mechanisms by which diet controls cancer development -/- are not well understood. Increased food intake could speed brca2 embryos do not display Rad51 foci upon Olaparib cancer onset in at least two non-mutually exclusive ways: treatment, proving their sensitivity to this treatment. Interestingly, brca2+/- embryos harbour less Rad51 foci first, by driving faster growth in established tumors, thus +/+ decreasing the amount of time needed for them to become than brca2 embryos, indicative of haploinsufficiency in clinically observable; or second, by increasing the rate at these cells. There is an ongoing debate if haploinsufficiency which precancerous cells become transformed. Using a instigates tumorigenesis in patients with germline BRCA2 mutations, but our observations point towards a small crestin:EGFP reporter with our zebrafish melanoma model, +/- which allows us to visualize tumors from the first cancer decrease in HR efficiency in brca2 zebrafish. cell, we can determine the extent to which each of these In conclusion, zebrafish embryos can be used as a tool to two processes contributes to cancer development. We have accurately predict PARP inhibitor effectiveness. found that the incidence of preclinical melanoma patches of a few hundred cells or less is nutrition-dependent, suggesting that diet contributes to cancer development at the initiation stage. Building on this, we are measuring the P-028 Zebrafish xenografts for cancer therapy evaluation influence of food supply on postinitiation growth in already- Susana Pascoal1 , Fikret Rifatbegovic2 , Caterina Sturtzel1 established tumors. Together, these experiments will help , Ossia Eichhoff3 , Verena Paulitschke4 , Sabine Taschner- shed light on the specific aspects of cancer development Mandl2 , Martin Distel1 that are nutrition-dependent and, with the zebrafish model, will lay the foundations for future mechanistic genetic and 1 Innovative Cancer Models, Children’s Cancer Research biochemical studies. Institute (CCRI), Zimmermanplatz 10, 1090 Vienna (Austria), 2 Tumor Biology, Children’s Cancer Research Institute (CCRI), Zimmermanplatz 10, 1090 Vienna 3 P-027 Faithfully recapitulating PARP inhibitor (Austria), Department of Dermatology, Universityhospital effectiveness in zebrafish Zurich, Wagistrasse 14, 8952 Zürich-Schlieren (Switzerland), 4 Medical University Vienna, Department Jeroen Vierstraete1,2,3, Charlotte Fieuws1,2 , Andy Willaert of Dermatology, Division of General Dermatology, 1, Anne Vral 3, Kathleen BM Claes 1,2 Währingerguertel 19-20, 1090 Vienna (Austria) 1 Center for Medical Genetics, Ghent University, Ghent, ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 42 Poster Abstracts Current challenges in pediatric oncology arise from the development. Due to this abnormally fused phenotype, limited pool of available therapeutic compounds without we were intrigued to determine whether the abnormal side effects and the lack of cost-effective personalized cancer vasculature we observed in fzd4 mutant fish leads to visual models to identify „the right drug for the right patient“ in impairment. Therefore, we assayed visual acuity in fzd4 an in vivo pre-clinical setting. Recently, transplantation into mutant zebrafish by determining their optokinetic response zebrafish was discovered to be a promising alternative to (OKR), an involuntary eye movement in response to visual mouse xenograft models. Here, we investigate zebrafish stimuli. We found that fzd4 mutants have impaired OKR, larval xenografts for drug evaluation and compound requiring increased illumination. We have generated a screening. Proliferation and migration of several human visually impaired zebrafish FEVR model exhibiting abnormal cancer cell lines including melanoma, glioblastoma and retinal vasculature. These fish provide a tractable model for neuroblastoma cells were studied in zebrafish larvae. studying the vascular biology in retinovascular disorders, Subsequently, effects of selected compounds on these cells and serves as a baseline for evaluating future FEVR causing were investigated and a novel candidate compound from genes identified in humans. an in vitro screen could be confirmed. Current efforts to establish automated imaging and image analysis procedures for zebrafish xenografts will allow for higher throughput P-030 Knockout of Nr2e3 prevents rod photoreceptor compound evaluation in the near future. Ultimately, we aim differentiation and leads to selective for applying patient derived xenografts (PDX) in zebrafish larvae to identify the best available therapeutic compound Yuwen Huang1, Shanglun Xie1, Shanshan Yu1, Jiayi Tu1, for each patient within a short time period. Zhaohui Tang1, Fulton Wong2, Mugen Liu1,* 1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei P-029 Frizzled 4 regulates ventral blood vessel 430074, PR China, 2 Department of Ophthalmology, Duke remodeling in the zebrafish retina University School of Medicine, Durham, NC 27710, USA 1 1 2 Lucia Caceres , Sergey V. Prykhozhij , Harald Gjerde , *Correspondence to: M. Liu, College of Life Science 5 3 3 Nicole Marie Duff , Mike Ngo , Elizabeth Cairns , Gheyath and Technology, Huazhong University of Science and 5, 6 1, 3 K. Nasrallah , Christopher R. McMaster , Matthew Technology, Wuhan, Hubei 430074, PR China. E-mail 7 1, 2, 8 1, 4, 8* Litvak , Johane M. Robitaille Jason N. Berman addresses: lium@ hust.edu.cn 1 Departments of Pediatrics, IWK Health Centre/ Mutations in the photoreceptor cell-specific nuclear Dalhousie University, 2 Department of Ophthalmology and 3 receptor gene Nr2e3 increased the number of S-cone Visual Sciences, Dalhousie University, Department of photoreceptors in human and murine retinas and led to Pharmacology, Dalhousie University, retinal degeneration that involved photoreceptor and 4Department of Microbiology and Immunology, Dalhousie non-photoreceptor cells. The mechanisms underlying University, 5 Department of Biomedical Science, Qatar these complex phenotypes remain unclear. In the hope of University, 6 Biomedical Research Center, Qatar University, understanding the precise role of Nr2e3 in photoreceptor 7 Department of Biology, Mount Allison University, New cell fate determination and differentiation, we generated a Brunswick, Canada, 8 Department of Pathology, Dalhousie line of Nr2e3 knockout zebrafish using CRISPR technology. In University, Nova Scotia, Canada these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes Familial exudative vitreoretinopathy (FEVR) is a rare are not expressed and the outer segment (OS) fails to form. congenital disorder characterized by a lack of blood vessel Formation and differentiation of cone photoreceptors is growth to the periphery of the retina with secondary normal. Specifically, there is no increase in the number fibrovascular proliferation at the vascular-avascular of UV-cone or S-cone photoreceptors. Laminated retinal junction. These structurally abnormal vessels cause leakage structure is maintained. After normal development, L-/M- and hemorrhage, while the fibroproliferative scarring cones selectively degenerate, with progressive shortening results in retinal dragging, detachment and blindness. of OS that starts at age 1 month. The amount of cone Mutations in the FZD4 gene represent one of the most phototransduction proteins is concomitantly reduced, common causes of FEVR. Using transcription activator-like whereas UV- and S-cones have normal OS lengths even effector nucleases (TALENs), we have generated a loss of at age 10 months. In vitro studies show Nr2e3 synergizes function mutation resulting from a 10-nucleotide insertion with Crx and Nrl to enhance rhodopsin gene expression. into exon 1 of the zebrafish fdz4 gene. Characterization of Nr2e3 does not affect cone opsin expression. Our results the structural integrity of the retinal vasculature in control extend the knowledge of Nr2e3’s roles and have specific and fzd4 mutant fish using fluorescent microscopy reveals implications for the interpretation of the phenotypes an asymmetrical distribution of the vasculature along the observed in human and murine retinas. Furthermore, our dorsoventral axis, with active vascular remodeling on the model may offer new opportunities in finding treatments ventral surface of the retina throughout development. In for enhanced S-cone syndrome (ESCS) and other retinal addition, fzd4 mutants exhibit disorganized ventral retinal degenerative diseases. vasculature with discernable tubular fusion by week 8 of ......

..... 43 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts P-031 Identifying new drug combinations for the controllable temporally and spatially in transgenic fish, here treatment of rhabdomyosarcoma we conduct two approaches. First, we developed a system to express multiple sgRNAs efficiently in zebrafish using the Dalton Brunson1,2,3,4,5, Chuan Yan2,3,4,5, Qin Tang2,3,4,5, John 2,3,4,5 2,3,4,5, 2,3,4,5 endogenous tRNA processing machinery. We cloned nine Moore , Daniel Do Karin McCarthy , Madeline endogenous zebrafish tRNA genes, fused them to sgRNA, Hayes2,3,4,5, Alexandra Veloso2,3,4,5, Alessandra Welker2,3,4,5, 2,3,4,5, 2,3,4,5 and demonstrated that active sgRNA can be produced from laine Garcia David Langenau the tRNA-sgRNA fusion transcript. Then, we constructed 1 Harvard College, Cambridge, MA 02138, 2 Molecular transgenic fish expressing Cas9 ubiquitously (ubb:SpCas9) Pathology Unit, Massachusetts General Hospital Research and expressing a fusion transcript under the control of the Institute, Charlestown, MA 02129,3 Massachusetts U6 promoter that contained three distinct sgRNAs targeting General Hospital Cancer Center, Harvard Medical School, the slc45a2 (albino) gene fused to tRNAs (u6c:3xslc45a2- Charlestown, MA 02129, 4 Center for Regenerative sgRNA). We found that the Tg(ubb:SpCas9;u6c:3xslc45a2- Medicine, Massachusetts General Hospital, Boston, MA sgRNA) fish harbored mutations in all of the target sites 02114,5 Harvard Stem Cell Institute, Cambridge, MA 02139 and knocked out the albino gene efficiently, showing nearly complete albino phenotypes. We are now further Cure rates for pediatric rhabdomyosarcoma muscle cancers testing whether a PolII-transcribed tRNA-sgRNA fusion can have largely remained stagnant for the past decade. This provide functional sgRNA in vivo. Second, to test whether can be attributed to two main reasons: 1) the lack ofa Cas9 can be expressed in a tissue-specific manner via the high-throughput screening and in vivo drug validation Gal4-UAS system, we constructed the UAS:Cas9 transgenic platforms to identify clinically relevant drugs and 2) the fish. To show a proof of principle, we crossed them with insufficient understanding of how therapies target specific a Gal4 driver that expressed Gal4 maternally and injected heterogeneous RMS cell populations in vivo. To address gRNAs targeting the albino gene into fertilized eggs from these issues, we have recently generated optically clear, double transgenic females. By analyzing their offspring, we immune deficient prkdc-/-, il2rga-/- zebrafish that lack T, B demonstrated that maternally-provided Cas9 was capable and NK cells. Importantly, these fish models are viable, can of mutagenizing the target gene in the germ lineage. This be grown at 37C, and can engraft a wide array of human result opened up a possibility that tissue specific Cas9 cancers, including RMS. Remarkably, the growth kinetics and expression may be achieved via the Gal4-UAS system. histological features of RMS xenografts grown in immune- deficient zebrafish exhibit significant similarities to human tumors and those grown in immunodeficient mice. In fact, P-033 Neurotransmitter release impairment in zebrafish we uncovered potent additive effects of the PARP inhibitor, treated with chemotherapy agents Olaparib, and the DNA damaging agent, Temozolomide, on killing human RMS xenografts grown in prkdc-/-, il2rga-/- Michael A. Johnson1, Mimi Shin1, Thomas M. Field1, zebrafish, which was further validated in murine xenograft Chase Stucky1, Joseph Loomis1, Nicole Walker1

models using both RMS cell lines and PDXs. By mass 1 spectrometry, we next showed that pharmacokinetics of Department of Chemistry, University of Kansas, Lawrence, orally gavaged Olaparib and Temozolomide were similar KS 66045 USA in zebrafish, mouse and human using similar drug dosing. Chemobrain is an impairment in executive function that Transparency of these animals also allowed the visualization affects roughly one-third of cancer patients who have of therapy responses at single cell resolution using the undergone cancer chemotherapy. Rodents, including FUCCI4 fluorescent cell cycle reporter. Together, these pre- rats and mice, have often been used to study the clinical modeling experiments have identified PARPi and underlying mechanisms of chemobrain; nevertheless, DNA damaging agents as a new combination therapy that the use of rodents has limitations, including difficulty in has potential to move forward clinically for the treatment the administration of chemotherapeutic agents and low of human RMS. throughput. Previously, we have found that the release of neurotransmitters, especially dopamine, is impaired in rodents by the administration of carboplatin or P-032 Towards spatial and temporal control of gene 5-fluorouracil, agents commonly used in the treatment of knockout: transgenic approaches various cancers in humans, even though neurotransmitter

1 content remains unchanged. Here, we describe the use Koichi Kawakami of zebrafish to study the neurochemical mechanisms of 1National Institute of Genetics chemobrain. This model allows us to carry out studies with greater ease and higher throughput than in rodents The CRISPR/Cas9 system can be introduced into zebrafish as because we can add the agents to the water or food rather transgenes; namely, expression of single-guide RNA (sgRNA) than inject them intravenously. In our studies, we found that and expression of Cas9 from a tissue-specific promoter treatment with both 5-fluorouracil and carboplatin resulted in transgenic zebrafish enables the gene knockout in in impaired dopamine release, measured with fast-scan specific cell types (Ablain et al. Dev Cell 2015). To make the cyclic voltammetry at carbon-fiber microelectrodes (FSCV). CRISPR/Cas9-mediated gene knockout more efficient and Moreover, the route of treatment was important in how ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 44 Poster Abstracts quickly these impairments manifested, with oral treatments 1 Stem Cell Program and Division of Hematology/Oncology, affecting release faster than water treatment. Thus, these Boston Children’s Hospital and Dana Farber Cancer agents may be more readily absorbed through the gut than Institute, Howard Hughes Medical Institute, Harvard the gills. Collectively, our results indicate that zebrafish may Medical School, Harvard Stem Cell Institute, Stem Cell and be a useful model for the study of chemotherapy related Regenerative Biology Department, Harvard University, cognitive impairment. Boston, MA

Hematopoietic stem cells (HSCs) emerge in the embryo and P-034 Zebrafish modeling of intestinal injury, bacterial ultimately comprise adult blood system. Using a brainbow exposures and medications defines epithelialin vivo color barcoding system, we previously demonstrated responses relevant to human Crohn’s Disease that zebrafish produce 20-30 HSCs from the developing aorta. HSCs traffic to the embryonic niche where they Ling-shiang (Felix) Chuang 1, Joshua Morrison 1,3, Nai-yun exit circulation and divide. The embryonic niche is Hsu 1, Philippe Ronel Labrias 1, Shikha Nayar1, Ernie Chen thought to control the number of HSC clones that support 1, Nicole Villaverde 1, Jody Ann Facey 1, Jaime Chu 3, Judy hematopoiesis. Using spinning disk confocal microscopy, we Cho 2 imaged HSPCs in the niche and found surprising ‘grooming’

1 behavior by primitive mpeg1:GFP+ macrophages. Between Department of Genetics and Genomic Sciences, Icahn 48 – 72 hours post fertlization (hpf), ~20% of newly lodged School of Medicine at Mount Sinai, New York, United 2 HSPCs were contacted by a macrophage which scanned States, The Charles Bronfman Institute for Personalized their surface for 30-45 minutes. To evaluate a possible Medicine, Icahn School of Medicine at Mount Sinai, New 3 role of macrophages in attenuating HSC clone number, we York, United States, Department of Pediatrics, Icahn depleted macrophages in our brainbow barcoding system. School of Medicine at Mount Sinai, New York, United Unique color barcodes were induced in individual HSCs at States 24 hpf, just prior to stem cell emergence, and clodronate Inflammatory bowel disease (IBD) is a complex chronic loaded liposomes injected into circulation at 28 hpf. intestinal human disease composed of genetic, Macrophage-depleted embryos were raised to 3 months environmental and host immune factors. Genome-wide for adult marrow color analysis. On average, animals association studies (GWAS) have identified over 200 injected with clodronate liposomes had only 14 HSC color genomic loci associated to inflammatory bowel disease clones, compared to 24.6 HSC clones in sibling controls (p (IBD). High effect risk alleles define key roles for genes = 0.0002). A similar clone reduction was found with the involved in bacterial response, autophagy and innate macrophage-blocking irf8 morpholino, suggesting that this immunity. More robust and predictive high-throughput in is due to a specific loss of embryonic macrophages. Further vivo systems are required to rapidly evaluate therapeutic imaging studies of identify fluorescent protein exchanged agents. Dextran sodium sulfate (DSS) induces chemical from stem cells (runx1+23:mCherry) into grooming intestinal inflammation and has been extensively utilized to macrophages. FACS-purified macrophages exhibit punctate study intestinal injury. We compared the effects of single mCherry signal, and in some cases appear to have engulfed and repetitive DSS treatment in zebrafish and reported entire mCherry+ cells. Given direct interactions in the niche the mortality, lysosomal function and mucin production. and reduced numbers of stem cell clones in macrophage Repetitive injury induces dose-dependent mortality, depleted animals, niche macrophage grooming likely impaired recovery of intestinal barrier function, failure introduces pro-survival factors. Our barcoding studies to contain bacteria within the intestine, and impaired taken together with our imaging establish a role for autophagy. PGE2 administration protected against injury by macrophages in determining the number of HSC clones enhancing epithelial barrier function and limiting systemic during development and impacts our understanding of infection. We further validated the PGE2 protective clonal diseases in the adult hematopoietic system. effect with human enteroid-differentiated epithelium monolayers. Effects of IBD therapeutic agents were defined in our new disease model; mesalamine showed protective P-036 GPR182 is a novel regulator of hematopoietic stem features during injury while 6-mercaptopurine displayed cell specification during zebrafish development

marked induction of autophagy during recovery. Given the 1 1 highly conserved nature of intestinal barrier in zebrafish, Erich W. Damm and Wilson K. Clements it represents an ideal model system with which to test 1 Department of Hematology, St. Jude Children’s Research established and new IBD therapies targeted to epithelial or Hospital, Memphis, TN, 38105, USA innate immunity. Hematopoietic stem cells (HSCs) generate all hematopoietic lineages over the life of an animal and are the basic P-035 Embryonic macrophage-hematopoietic stem cell units of bone marrow transplants used in treatment of interactions determine adult HSC clone number in the hematologic diseases. However, the availability of this marrow therapy is limited by supply of immunologically compatible Samuel J. Wattrus1, Elliott J. Hagedorn1, Leonard I. Zon1 donor cells. The ability to generate autologous HSCs from ......

..... 45 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts induced pluripotent stem cells (iPSCs) in vitro for use in for its development. Our studies will provide a foundation regenerative therapies is a major biomedical objective, to define the in vivo cellular specification environment however challenges to generating transplantable HSCs and elucidate the complete set of signals involved in HSC capable of high level engraftment and multi-lineage specification, with the ultimate goal of informing clinical potential have yet to be overcome. HSCs are specified from efforts at directed differentiation of pluripotent stem cells hemogenic endothelium (HE) in the floor of the embryonic to an HSC fate. dorsal aorta (DA). Instructive signals originate from nearby cells, but the identity of these instructive signals remains Funding Sources: NHLBI: 5R00HL097150 and 1F32HL129819, unknown. Difficulties in generating HSCs in vitro likely March of Dimes #5-FY14-42 stem from lack of knowledge of key HSC specification signals. Reproducing the full set of in vivo HSC specification signals might help to overcome barriers to in vitro directed P-038 Low dose of chronic ethanol exposure in adult differentiation, but requires identifying the full set of zebrafish induces hepatic steatosis and injury

inductive signals. We have identified a novel role for the 1 1, 2, * orphan G-protein coupled receptor, Gpr182 in the control Ki-Hoon Park and Seok-Hyung Kim of HSC specification. We show that gpr182 is expressed in 1Department of Medicine, Medical University of South axial vasculature and that Gpr182 knockdown disrupts HSC Carolina, Charleston, SC, 29425, USA, 2Department of specification. Importantly specification of the DA and HE, Regenerative Medicine and Cell Biology, Medical University is unaffected. Our results suggest that Gpr182 is mediating of South Carolina, Charleston, SC, 29425, USA. an HSC specification signal that is required proximally for initiation of the hematopoietic program. We have recently * Corresponding author demonstrated that trunk neural crest cells (NCCs) control HSC specification through unknown signal(s). Similar to Chronic alcohol consumption is a major cause of chronic Gpr182, NCC derived signaling is not required for DA or the liver disease worldwide. Adult zebrafish have emerged as a HE specification, raising the possibility that Gpr182 could new vertebrate model of alcoholic liver disease. In previous be mediating a NCC derived signal. Interrogating the role of research, a high dose of chronic ethanol treatment induced Gpr182 in HSC specification will lead to novel insights into characteristic features of steatosis and hepatic injury in the extracellular signals and downstream regulation of HSC adult zebrafish, yet the ethanol concentration in that study specification in the vertebrate embryo. was significantly higher than the lethal dose in humans. In the current study, we examined whether a low dose of chronic ethanol treatment that approximated the average blood alcohol concentration (0.1 % and 0.2 %) in humans P-037 Somite patterning and its role during hsc niche may recapitulate the metabolic and pathological features formation of alcoholic liver disease. We found that chronic ethanol Nikki Glenn1 and Wilson Clements1 treatment for 4 weeks induced a significant elevation of serum glucose and triacylglycerol in adult zebrafish. 1 St. Jude Children’s Research Hospital, Memphis, TN In addition, serum alanine aminotransferase activity was significantly elevated in a dose dependent manner. Hematopoietic Stem Cells (HSCs) are born from endothelial Histological analysis revealed steatosis and hepatocyte cells in the floor of the primitive descending aorta, known ballooning phenotype. Gene expression analysis using as the dorsal aorta in zebrafish. A better understanding quantitative real-time PCR revealed that chronic ethanol of the cellular specification niche that regulates the birth exposure induced increased mRNA expression associated of these cells might inform attempts to instruct their with inflammation, apoptosis, and fibrosis. In addition, we specificationin vitro. Recently, Wnt16, a non-canonical Wnt, found significant increases in mRNAs involved in glucose was demonstrated to be required for HSC development in and lipid metabolism as well as mitochondrial biogenesis zebrafish. Wnt16 signals through a series of downstream and function. Importantly, expression of genes involved relay signals, but the final signal(s) most proximal to HSC in oxidative and endoplasmic reticulum stress, two major specification remain unknown. In Wnt16 loss of function stress signaling pathways underlying hepatic injury in animals, there is also an earlier defect in a compartment alcoholic liver disease, were highly upregulated in the of the somite, the sclerotome, which may house livers of adult zebrafish after chronic ethanol treatment. vascular smooth muscle cells. These results suggest that In conclusion, we found that 4 weeks of low dose ethanol sclerotome-derived cells might contribute to the regulatory exposure leads to typical ethanol-induced liver disease, environment, or “niche”, that directs HSC specification, with classic metabolic, pathological, and gene expression explaining the failure of HSC specification when this patterns. compartment develops incorrectly. We have found that loss of sclerotomal genes results in loss of HSCs and are further investigating the role of this somite compartment in HSC niche formation. Additionally, we are interested in understanding how the sclerotome is compartmentalized into its various fates and what proximal signals are required ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 46 Poster Abstracts P-039 The chromatin remodeler brg1 is required for The hematopoietic stem and progenitor cell (HSPC) niche hematopoietic stem cells specification is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial Jiayi Tu1, Xiliang Liu1, Shanshan Yu1, Fei Liu1, Tao Jiang1, 1 1 1 cells (ECs) that directly interact with HSPCs and promote Qunwei Lu , Mugen Liu , Zhaohui Tang * stem cell function. Utilizing spatial transcriptomics, in 1Key Laboratory of Molecular Biophysics of Ministry combination with tissue-specific RNA-seq, we identified of Education, College of Life Science and Technology, 29 genes selectively enriched in ECs of the zebrafish fetal Huazhong University of Science and Technology, Wuhan, hematopoietic niche. Using upstream regulatory sequences Hubei 430074, PR China for two of these genes, mrc1a and selectin E (sele), we generated GFP reporter lines that allowed us to selectively *To whom correspondence should be addressed at: isolate niche ECs for ATAC-seq. This analysis identified 6,848 Department of Genetics and regions of chromatin that were open in niche ECs but not ECs from other tissues. Several of these regions were associated Developmental Biology, College of Life Science and with the 29 genes. To evaluate whether these regions might Technology, 1037 Luoyu Road, Wuhan, P. R. China. Tel: 86 be enhancers we coupled them to GFP and injected them 27 87794549; Email: [email protected] into embryos. 12/15 sequences drove GFP in niche ECs. Hematopoietic stem and progenitor cells (HSPC) are Upon closer examination of the mrc1a and sele genes, we specialized cells that maintain blood production for the identified enhancer sequences as short as 125 bp and 158 life span of the organism. The transcriptional regulation bp, respectively, which drove niche EC-specific expression. in HSPC development has been well studied, whereas the A genome-wide motif enrichment analysis of the 6,848 epigenetic control has not been sufficiently elucidated. In uniquely open chromatin regions revealed that Ets, Sox and this study, we generated a brg1 knockout zebrafish model Nuclear Hormone sites were most enriched. Using mutant using TALEN technology and observed that the population of variants of the 125 bp and 158 bp enhancer sequences, we HSPC in mutant zebrafish was compromised. The decreased demonstrated that Ets, Sox and Nuclear Hormone (NR2F2/ number of HSPC can be partially rescued byklf2a mRNA and RORA/RXRA) sites were independently required for specific Nitric oxide (NO) donor. Molecularly, brg1 directly bound to transgene expression. We next injected pools of human the promoter of klf2, activate its expression. Moreover, an transcription factors containing at least one member from increasing apoptosis was observed in brg1 mutant at 48 hpf each of the three families. Strikingly, we found that a and it can also be rescued by NO donor. This study provides combination of ETV2, SOX7 and NR2F2 generated ectopic the first description of a chromatin remodeling factor brg1 patches of niche ECs that recruited runx1+ HSPCs and promoting klf2 expression in hemogenic endothelium and supported their division. Our results suggest these three highlights a novel role for chromatin remodeling in HSPC factors are sufficient to reprogram niche EC identifyin vivo, specification. which has important implications for designing a synthetic vascular niche for stem cells or for modulating the niche in a therapeutic context. P-040 Transcription factor induction of ectopic vascular blood stem cell niches in vivo Elliott J. Hagedorn1, Julie R. Perlin1, Clara Mao1, Inés P-041 GBA1 deficiency confers resistance to Fernández-Maestre1, Rebecca J. Freeman1, Madeleine mycobacterial infection 1 1 1 L. Daily , Christopher D’Amato , Samuel J. Wattrus , Jingwen Fan1, Francisco J Roca1, Lalita Ramakrishnan1 Tianxiao Han1, Raquel Riquelme1, Brian Li1, Shelby E. Redfield1, Samantha H. Collins1, Asher Lichtig1, Song 1Department of Medicine, University of Cambridge, MRC- Yang1, Yi Zhou1, Balvir Kunar2, Jesus Maria Gomez Laboratory of Molecular Biology, Francis Crick Avenue, Salinero2, Thanh T. Dinh3, Junliang Pan3, Karoline Holler4, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK Eugene C. Butcher3, Alexander van Oudenaarden5, Shahin Rafii2, J. Philipp Junker4, Leonard I. Zon1 Lysosomal storage disorders (LSD) are a group of about 50 rare inherited metabolic disorders that result from 1 Stem Cell Program and Division of Hematology/Oncology, defects in lysosomal function. The accumulation of Boston Children’s Hospital, Boston, MA, USA, 2 Ansary undigested lysosomal material in LSD macrophages impairs Stem Cell Institute, Division of Regenerative Medicine, their migration toward dying cells and causes buildup of Department of Medicine, Weill Cornell Medicine, New unengulfed cell debris. The hallmark of lysosomal storage York, NY, USA, 3 Veterans Affairs Palo Alto Health Care disorders is vacuolated macrophages. As we saw in our System, The Palo Alto Veterans Institute for Research zebrafish model, LSD macrophages, as well as GBA1- and the Department of Pathology, Stanford University, deficient macrophages, have much more vacuolation Stanford, CA, USA, 4 Berlin Institute for Medical Systems compared with WT macrophages. Biology, Max Delbruck Center for Molecular Medicine, Berlin, Germany, 5Hubrecht Institute, Royal Netherlands Because of this, it has been reported by our lab that Academy of Arts and Sciences and University Medical during mycobacterial infection, LSD macrophages fail to be Center Utrecht, Utrecht, the Netherlands recruited to infected macrophages undergoing apoptosis ......

..... 47 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts in the tuberculosis granuloma. The unengulfed apoptotic impact innate immune function at subteratogenic levels macrophages undergo secondary necrosis, resulting in and identifies two mechanistic routes by which xenobiotic granuloma breakdown and increased mycobacterial growth exposure may alter immune function. extracellularly. This unrestricted extracellular growth drives susceptibility to mycobacterial infection. Meanwhile, it has been repeatedly proposed and debated that resistance to tuberculosis for heterozygote carriers of LSD alleles among P-043 Novel alleles of antigen processing genes in ray- urban Ashkenazi Jews population. finned fishes Dustin J. Wcisel1, Alex Dornburg4, Andrew Thompson5, Gaucher’s disease is the most common LSD which is caused 5 1,2,3 by GBA1 mutation. We found thatGBA1 -deficient zebrafish Ingo Braasch , and Jeffrey A. Yoder larvae were resistant to Mycobacterium marinum (Mm) 1 Department of Molecular Biomedical Sciences, 2 infection even though LSD phenotype was observed. Comparative Medicine Institute,3 Center for Human Health and the Environment, NC State University, Raleigh, NC USA, 4 North Carolina Museum of Natural Sciences, P-042 Assessment of Immunotoxicity by Xenobiotic Raleigh, NC USA, 5 Department of Integrative Biology, Exposure Using Larval Zebrafish Michigan State University, Lansing, MI USA Drake W. Phelps1,2, Ashley A. Fletcher1, Ivan Rodriguez- The inducible proteasome subunit (PSMB) genes are part of Nunez1, Michele Balik-Meisner3, Debra A. Tokarz1,4, David the 20S proteasome complex, responsible for systematically M. Reif3,4,5, Dori R. Germolec6, and Jeffrey A. Yoder1,2,4 degrading proteins into small polypeptides for loading and display on major histocompatibility (MHC) proteins. In the 1 Department of Molecular Biomedical Sciences, 2 3 genomes of teleost species such as zebrafish, the PSMB Comparative Medicine Institute, Department of Biological genes are clustered together adjacent to their MHC Class I Sciences, 4 Center for Human Health and the Environment, 5 counterparts. Within a given species, alternative haplotypes Bioinformatics Research Center, NC State University, st 6 for these genes have been described. For example, the 31 Raleigh, NC USA, National Institute of Environmental amino acid residue of the PSMB8 mature protein is found Health Sciences, Research Triangle Park, NC USA to contain either an alanine or a phenylalanine (PSMB8A or Current assessment practices for immunotoxicity involves PSMB8F, respectively). The sequence differences may lead a tiered approach for hazard identification and mechanistic to functional changes in the types of proteins degraded studies, including observational studies, evaluation of and thus the antigens displayed. These alternative alleles immune function, and measurement of susceptibility to are present in Senegal bichir (Polypterus senegalus), and infectious and neoplastic disease. These studies generally numerous teleost species as well as many tetrapods (Xenopus use costly, low-throughput mammalian models. Zebrafish, tropicalis for example). This type of conserved haplotypic however, offer an excellent alternative due to their rapid variation is referred to as trans-species polymorphism. development, ease of maintenance, and homology to Recently, the genome sequencing of two holostean species, mammalian immune system function and development. spotted gar (Lepisosteus oculatus) and bowfin (Amia calva) Larval zebrafish also provide a convenient model to study were completed. Here, we present the annotation of the the innate immune system with no interference from PSMB loci in both species using the zebrafish genome as the adaptive immune system. In this study, we utilized a a reference point. We found the holostean PSMB8 gene, respiratory burst assay (RBA) to measure reactive oxygen while highly conserved with other fish species, encodes novel alleles containing either a threonine or serine at the species (ROS) production after xenobiotic exposure. ROS st are produced in macrophages and neutrophils in response 31 residue. Many additional polymorphisms were also to pathogens in order to eliminate them from the host. identified in the neighboring genes, suggesting holosteans Embryos were exposed to subteratogenic doses of chemicals experienced unique co-evolution of these genes at this from 6 hpf to 96 hpf with daily media changes, and at 96 hpf locus likely resulting in functional differences of antigen the ability to produce ROS was measured. Through the RBA, presentation. we identified five compounds that suppressed global ROS production: 17-β estradiol, benzo[a]pyrene, lead acetate, methoxychlor, and phenanthrene; these compounds have P-044 CRISPR/Cas9 genome editing to generate new also previously been identified as immunosuppressive in zebrafish models of centronuclear myopathy mammalian innate immunity assays. In order to evaluate whether the suppression of ROS by these compounds was E. Dupuis1, S. Coppens1, P. Gillotay1, V. Janssens1, D. Perez- due to a decreased number of neutrophils or macrophages, Morga2, A. Trubiroha1, S. Costagliola1 we combined flow cytometry with transgenic zebrafish 1 Institut de Recherche Interdisciplinaire en Biologie larvae to count the numbers of these cell types after 2 chemical exposure. With this assay, we found benzo[a] Humaine et Moléculaire, ULB, Brussels, Institut de pyrene altered macrophage number, but not neutrophil biologie et de médecine moléculaires, ULB, Gosselies number. Taken together, this work demonstrates the utility of zebrafish larvae as a tool for identifying compounds that ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 48 Poster Abstracts Centronuclear myopathies (CNM) are a group of rare myonuclei. Autosomal-dominant mutations in DNM2 and early-onset genetic muscle disorders for which no also cause Charcot-Marie-Tooth peripheral neuropathy. curative treatment is available. This fatal condition is The mechanism underlying how DNM2 mutations cause known to be due to mutations in several genes including myopathy, and why different mutations result in different myotubularin (MTM1). Mutations in this gene cause the phenotypes, are not well understood, and there are no X-linked myotubular myopathy (XLCNM), which is the most therapies to DNM2-related diseases. To study the impact frequent and severe form of CNM. In CNM, several defects of DNM2 mutations in skeletal muscle, and to uncover have been observed at the cellular level, such as abnormal novel therapies, we have developed humanized zebrafish triad structure and dysfunctional excitation-contraction models that allow systemic comparison between wildtype coupling, crucial for skeletal muscle contraction. In our and mutant DNM2. Our preliminary data show that CNM- lab, we generated a mtm1 zebrafish mutant using CRISPR/ causing mutant DNM2 mislocalized from the proximity of Cas9 technology. This mutant exhibits progressive fin fold T-tubule to the sarcolemmal membrane, suggesting the degeneration, defective motor behavior and early mortality. lack of DNM2 at the T-tubule can disrupt the structure and However, no defects in muscular ultrastructure were lead to muscle weakness. To test this hypothesis, we aim observed. Recent studies have shown that mutated mRNA to compare protein dynamics, subcellular localization, and can trigger genetic compensation through upregulation binding partners of wildtype and mutant DNM2. We will of a functionally redundant homologue. To highlight also capitalize on the suitability of the zebrafish model for potential genes presenting deregulated expression, we drug discovery to test targeted therapies and to perform a first performed a transcriptome analysis on mtm1 mutant large-scale unbiased drug screen. and WT fishes. This analysis did not show any obvious upregulation of other myotubularin-related family members while inflammation and apoptosis markers are upregulated, P-046 Dissecting UBA5 dysfunction in nervous system such as metalloproteinase (mmp) family. As these proteases disorders are involved in degrading the extracellular matrix proteins and in tissue remodeling, we investigated, in our mutant, Rita J Serrano1, Viola Oorschot2, Georg Ramm2, Robert J. a possible link between mmp upregulation and fin fold Bryson Richardson1

degradation. In parallel, to avoid genetic compensation 1 mtm1 School of Biological Sciences, Monash University, linked to mRNA decay, we are currently generating 2 mutants harboring large deletions encompassing promoter Ramaciotti Centre for Cryo-Electron Microscopy, Monash and exon 2 of the gene to avoid any active transcription of University the mutated mRNA. In conclusion, the investigation of the Mutations in the ubiquitin-like modifier activating enzyme effect of genetic compensation on zebrafish mtm1 mutant 5 (UBA5) gene were recently identified to cause childhood- phenotype could contribute to the generation of a new onset cerebellar ataxia and infantile-onset epileptic zebrafish model that recapitulates the human phenotype. encephalopathy, disorders predominantly associated It might constitute a great tool to better understand the with the central nervous system (CNS). Cerebellar ataxia physiopathology of the CNM and for development of more patients show cerebellar atrophy and motor dysfunction; effective therapeutic approaches. whereas, the clinical symptoms associated with epileptic encephalopathy include reduced motor skills, developmental delay, intellectual disability, microcephaly, delayed brain P-045 Investigation into disease pathogenesis and myelination, and recurrent seizures, ultimately resulting in identification of therapeutic targets for DNM2-related the death of most patients before the age of twenty. The centronuclear myopathy. function of UBA5 in the nervous system and how mutation in UBA5 results in disease remains to be elucidated. Mo Zhao1, James J. Dowling1, 2, 3, 4 To investigate the pathophysiology associated with UBA5 1 Genetics & Genome Biology Program, The Hospital for 2 mutation and to identify pathways that could reduce disease Sick Children, Toronto, ON M5G 0A4, Canada, Division progression we are using the zebrafish. We show that uba5 of Neurology, The Hospital for Sick Children, Toronto, ON loss of function in zebrafish leads to significant decrease in M5G 1X8, Canada, 3 Department of Pediatrics, University 4 locomotor activity, recapitulating the motor dysfunction of Toronto, Toronto, ON M5G 1X8, Canada, Department observed in UBA5 patients. Notably, the motor impairment of Molecular Genetics, University of Toronto, Toronto, ON is not preceded by damage to the overall neuronal network M5S 1A8, Canada and muscle morphology. However, electron microscopy Dynamin 2 (DNM2) belongs to a family of large GTPases reveals signs of degradation and aggregates in the nerves that act as molecular scissors during endocytic vesicle innervating the skeletal muscle of uba5 mutants. This release. Autosomal-dominant mutations in DNM2 finding suggests that there may be an early sub-clinical cause centronuclear myopathies (CNMs), a genetically phenotype in the peripheral nervous system that could heterogeneous group of congenital myopathies contribute to the pathology observed in UBA5 patients. We characterized by muscle weakness, myofibre atrophy, triad describe the identification of the pathways disrupted by the abnormalities, and an increased proportion of centralized loss of Uba5 leading, explaining the epileptic symptoms and neurodegeneration observed in patients......

..... 49 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts P-047 Exposure to copper (II) chloride alters the behavior from injured conspecific skin (“skin extract”). We find that and endocrine in zebrafish when mating, fish ignore this threatening stimulus. Water

1 1 conditioned by the mating fish (“mating water”) suffices Ji won Sung , Seungheon Lee to suppress much of the alarm response behavior. By 1 Faculty of Marine Biomedical Science, Jeju National 2-photon imaging of calcium transients, we mapped the University, Korea regions of the brain responding to skin extract and to mating water. In the telencephalon, we found regions where the Copper is an essential trace element in living organisms. responses overlap, and also one region (medial Dp), to be However, excessive exposure of copper might result in predominantly activated by skin extract, and another, Vs, to some toxic effects on living organisms. The aim of this study be predominantly activated by mating water. When mating

is to identify copper (II) chloride (CuCl2) effect in zebrafish. water and skin extract were applied simultaneously, the Zebrafish were exposed to different concentrations or alarm-specific response was suppressed, while the mating time for measurements of median lethal concentration water-specific response was retained, corresponding to

(LC50) value. We performed measurement of whole-body the dominance of mating over flight behavior. The choice cortisol level and behavioral parameters using the open made, for reproduction over escape, is opposite to that of field test (OFT) or the novel tank test (NTT) to evaluate mammals, presumably reflecting how the balance affects

stress responses. Zebrafish exposed to CuCl2 solution at species survival. concentration of 1.5 - 150 μg/l or vehicle 1 hour before

behavioral tests or colleting samples. In results, LC50 were 30.3, 25.3 and 14.8 μg/l in 24, 48 or 96 hours, respectively. As a result of NTT, distance moved, mobile and velocity were P-049 Developing a zebrafish gill model of respiratory significantly decreased by the exposures at concentration mucosal immune responses

of 15 or 150 μg/l of CuCl2 compared with control group. 1 1 2 Madina Wane , Fränze Progatzky , Akhilesh Jha , Ryan On the other hand, turn angle was significantly increased 3 2 S.Thwaites2, Robin J. Shattock , Peter J. Openshaw , by the exposure at concentration of 150 μg/l of CuCl2 1 2 1 compared with control group. In addition, in OFT, distance Laurence Bugeon , Trevor T. Hansel , Margaret J. Dallman moved, mobile and velocity were significantly decreased 1 by the exposures at all concentrations of CuCl2 compared Department of Life Sciences, Faculty of Natural Sciences, with control group. On the other hand, meandering and Imperial College London, SW7 2AZ, UK, 2National Heart turn angle was significantly increased by the exposure at and Lung Institute, Imperial College London, W2 1PG, UK., 3 concentration of 150 μg/l of CuCl2 compared with control Department of Infectious Diseases, Division of Medicine, group. Whole-body cortisol levels were significantly Imperial College London, W2 1PG, UK increased by the exposure at concentrations of 1.5 or 15 μg/l of CuCl compared with control group. In conclusion, Respiratory infections and inflammatory conditions remain 2 a major cause of mortality and morbidity globally. Tackling these results of study suggest that the exposure of CuCl2 induces the toxic response or the stress response on viral infections requires an improved understanding of the lethality, behavioral changes and whole-body cortisol level local molecular and cellular responses of the respiratory in zebrafish. mucosa. Zebrafish gills are functionally, and in some aspects, structurally analogous to mammalian respiratory systems, providing a novel opportunity to study respiratory immunology in vivo. Furthermore, the gills are easily P-048 Mating suppresses alarm response in zebrafish accessible, exposed to the environment and have been Carmen Diaz-Verdugo1, 3, Gerald J. Sun1, 3, Caroline H. shown to respond to inflammatory stimuli such as cigarette Fawcett1, Peixin Zhu1, Mark C. Fishman2,* smoke. In order to evaluate the use of gills as a model we need to improve our understanding of immune responses 1Novartis Institutes for Biomedical Research, Chemical in a variety of contexts. Biology & Therapeutics, 181 Massachusetts Avenue, Cambridge, MA 02139, U.S.A., 2 Harvard University, Dept. In this study we investigated the response to the viral ss-RNA of Stem Cell and Regenerative Biology, 7 Divinity Avenue, mimic and Toll-like receptor 7/8 agonist, R848 (resiquimod). Cambridge, MA 02138, U.S.A., 3 equal contributions Topical application of R848 to the gills elicited a strong cytokine response which was highly similar to responses in *corresponding author parallel human studies. We further dissected the zebrafish response, identifying changes in lymphocyte and neutrophil Mating and flight from threats are innate behaviors which localization using transgenic reporter lines and confocal enhance species survival. Stimuli to these behaviors often microscopy. Finally, we have started preliminary work on are contemporaneous and conflicting. How such conflicts establishing viral infections in the gills using influenza virus are resolved, and where in the brain such decisions are or infectious pancreatic necrosis virus. Compared to R848, made, are both poorly understood. For teleosts, olfactory neither virus has so far elicited major immune responses stimuli are key elements of mating and threat responses. in the gills, highlighting the need to elucidate pathogen For example, zebrafish manifest a stereotypical escape susceptibility and mechanisms of immune responses. response when exposed to alarm substance released ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 50 Poster Abstracts Payton Laurie1, Kristen Rueb1, Kallie Griffin1, David L Stachura1

P-050 A zebrafish JAK1-A634D gain-of-function model 1 provides new insights into the pathogenesis of familial California State University, Chico, Chico, CA, USA hypereosinophilia Hematopoiesis is a complex and highly regulated system Anna Cordeiro 1, Sergey Prykhozhij 1, Adam Deveau 2, where hematopoietic stem and progenitor cells (HSPCs) Kate Del Bel 3, Stuart Turvey 3, Jason N Berman1,2,4,5 differentiate into the numerous types of cells that make up mature blood. Understanding the genetic and molecular 1 Department of Pediatrics, Dalhousie University, Halifax, pathways involved in HSPC biology is an important step to NS, Canada, 2 IWK Health Center, Dalhousie University, treating many diseases associated with blood formation and Halifax, NS, Canada, 3 Department of Pediatrics, British differentiation. Due to the conservation of hematopoiesis Columbia Children’s Hospital, University of British and many of its genes in vertebrates we turn to zebrafish Columbia, Vancouver, BC, Canada, 4 Department of (Danio rerio) as a model organism. Three stromal cell Microbiology and Immunology, Dalhousie University, lines from known sites of hematopoiesis were generated Halifax, NS, Canada, 5 Department of Pathology, Dalhousie and shown to expand and differentiate hematopoietic University, Halifax, NS, Canada precursors. With the use of RNA sequencing we compared transcript expression of these three stromal cell lines and JAK/STAT signalling is of major importance for hematopoiesis generated a list of 447 genes that are likely important and immune function. A heterozygous de novo mutation regulatory factors in the hematopoietic system. A highly in JAK1 was identified in a mother and two sons. They expressed transcript from these cells was the chemokine presented a unique clinical phenotype of severe atopic (C-C motif) ligand 44 namedccl44. In order to test its effect dermatitis, elevated peripheral blood eosinophil counts on hematopoiesis, we performed knockdown experiments with eosinophilic infiltration of the liver and gastrointestinal using morpholinos (MOs). Transgenic zebrafish lines with tract, hepatosplenomegaly, and failure to thrive. Treatment fluorescently labelled myeloid, erythroid, and lymphoid cells with ruxolitinib, a JAK1/2 inhibitor, resulted in reduced were injected with ccl44 MO and a decrease in those cell eosinophilia and improved growth. However, how this JAK1 lineages was observed with flow cytometry, fluorescence mutation impacts hematopoiesis remains unclear. microscopy, and quantitative PCR. Co-injection of MO and ccl44 mRNA rescued these deficiencies, returning JAK1-WT/A634D zebrafish transgenic lines were generated, cell lineage numbers and hematopoietic gene expression but the survival rate for the mutant embryos was very low. to control levels. We are also generating CRISPR mutants Embryos with high expression of the transgene showed to confirm these findings. Elucidating the role of ccl44 in abnormal development which was rescued upon one day hematopoiesis will inform us about the evolution of the of exposure to ruxolitinib (70% reduced to 12%). Whole- vertebrate hematopoietic system and could have clinical mount in situ hybridization was used to assess changes in importance for treatment of human diseases like anemias hematopoietic stem and progenitor cells (HSPCs)/blood cell and leukemias. populations due to wild-type or mutant JAK1 expression. We observed increased HSPCs and myeloid progenitors, as well as neutrophils, granulocytes, macrophages and mast cells. We also observed an increase of early P-052 Foxo3 directed mitophagy plays a key role in erythromegakaryocytic cells and a decrease in haemoglobin protecting proliferating embryonic hematopoietic stem content. The embryos were analyzed by RNA sequencing, cells from oxidative stress which revealed a total 341 genes differentially expressed. In Ellie Meader1, Paul J. Wrighton2, Isaac M. Oderberg2, the JAK1-A634D fish we found enrichment for the JAK-STAT Jenna M. Frame1, Timothy L. Long1, Sung-Eun Lim3, pathway genes as well as for hematopoietic-related genes Virginie Esain4, Wolfram Goesling2,3,5, Trista E. North1,3 such has STAT1B, SOCS3A, SOCS3B, PIAS2 and THP1B. These results were validated using quantitative real time PCR. 1 Stem Cell Program, Hematology-Oncology, Boston Children’s Hospital, Boston, MA, 2 Department of In conclusion, we observed that JAK1-A634D plays Medicine, Division of Genetics, Brigham and Women’s an important role during zebrafish development as Hospital, Harvard Medical School, Boston, MA 02115, USA, hematopoietic regulator with a specific impact in the 3 Harvard Medical School, Boston, MA, 4Harvard Stem Cell myeloid lineage, from where the eosinophils arise. Institute, Cambridge, MA,4 Department of Pathology, Beth Moreover, we demonstrated that ruxolitinib can revert the Israel Deaconess Medical Center, Boston, MA, 5 Dana- arrested development phenotype. This study illustrates the Farber Cancer Institute, Boston, MA 02115, USA potential of the zebrafish to evaluate the molecular cause of a disease with implications for identifying and/or validating Hematopoietic stem cells (HSCs) in the developing embryo targeted therapies. are uniquely proliferative, simultaneously populating the growing organism with blood cells and generating an expanded pool of HSCs to sustain the organism throughout P-051 ccl44: a chemokine critical for normal its life. In contrast, adult HSCs are quiescent, they reside in a hematopoiesis hypoxic niche,nand use glycolysis as their primary metabolic ......

..... 51 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts process. Proliferative stress and the use of reactive embryos. Tumor-free survival and tumor volume change oxygennspecies (ROS) generating oxidative phosphorylation were assessed for RFP-positive tumor bearing-zebrafish. causes adult HSCs to differentiate, ultimatelynresulting We demonstrated robust activation of endogenous FBXW7 in loss of the HSC pool. Understanding how embryonic and BCOR expression in ERMS tumors. ERMS tumors with HSCs maintain stemness while bothnproliferating rapidly, activated expression of either FBXW7 or BCOR showed and undergoing oxidative phosphorylation, may provide significantly reduced tumor growth and increased overall important insights applicable to the expansion and tumor-free survival. To demonstrate conserved tumor maintenance HSCs in vitro for therapeutic use. Our data suppressor function in human ERMS, we showed that shows that HSC proliferation in the zebrafish embryo overexpression or dCas9-VP64-mediated activation ofBCOR is driven by an axis involving Insulin/IGF, PI3K/ERK or FBXW7 in human ERMS cell lines resulted in significant and mTOR, which can be stimulated by robust glucose inhibition of tumor growth. In further characterizing BCOR metabolism. However, high levels of ROS are a limiting function, overexpression ofBCOR in ERMS cell lines resulted factor in proliferation of these cells, suggesting a need for in G1/S cell cycle arrest and increased apoptosis. Structure/ mechanisms to balance proliferation with the control of function analysis of BCOR revealed the importance of the cellular stress. Our ssRNASeq data shows emerging HSCs Ankyrin and the BCL6 interacting domains for the function express foxo3b and its transcriptional target bnip3, both of BCOR. Overall, we have utilized the dCas9-VP64 gene key regulators of mitophagy, an essential process which activation platform in a zebrafish model of ERMSand removes damaged, ROS-producing mitochondria from the characterization in human cell lines to demonstrate novel cell. FOXO3 regulated mitophagy is essential for maintaining and conserved tumor suppressive role of BCOR and FBXW7 the adult HSC pool, but little is understood about its in RMS. mechanism or role in embryonic hematopoiesis. Using novel transgenic zebrafish expressing mitoQC, a fluorescent reporter for mitophagy, we see high levels of mitophagy in P-054 The adhesion molecule NECTIN1 is a novel cells in the caudal hematopoietic tissue (CHT), the niche for suppressor of metastasis in melanoma HSC proliferation at 120hpf. Pan-Foxo inhibitor AS1842856 reduces HSC marker expression in the CHT. In contrast, Julien Ablain1, Harriet Rothschild1, Michelle Dang1, exposure to D942, which increases mitophagy, enhances Leonard Zon1,2,3 myb cd41low both the expression of HSC markers ( and ) 1 and reduces measurements of ROS in those cells. We hope Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital and Dana Farber Cancer that further analysis will elucidate the mechanisms linking 2 mitophagy to HSC maintenance. Institute, Boston, MA 02115, USA, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA,3 Howard Hughes Medical Institute, Boston, MA 02115, USA P-053 Characterizing novel tumor suppressors in Metastasis is the leading cause of death in melanoma. A rhabdomyosarcoma better understanding of the molecular bases of cancer cell dissemination is critical to develop new therapies. GISTIC Michael Phelps1, Terra Vleeshouwer-Neumann1, Shivani 1 1 1 analysis of copy-number alterations in human melanoma Patel , Thao Pham , Eleanor Chen revealed that the third most significant focal deletion at 1 University of Washington, Department of Pathology, 1959 chr11q23.3 (overall frequency: 0.538, q=2.76E-21) only NE Pacific Street, Seattle, WA 98195 contained the gene NECTIN1. Biallelic loss of NECTIN1 was detected in 8% of cases. NECTIN1 is an adhesion Rhabdomyosarcoma (RMS) is one of the most common and molecule involved in adherens junctions. Human tissue devastating pediatric sarcomas. RMS is divided into two microarray staining showed that NECTIN1 levels were lower major histologic subtypes, embryonal (ERMS) and alveolar in melanoma metastases compared to primary tumors (ARMS). A recent genomic analysis of RMS demonstrates (average score: 4.8 vs 6.0, p=0.01). Using a subcutaneous a higher mutational burden in ERMS with many novel transplantation assay in adult zebrafish, we observed recurrent mutations. From the study, we identified several that nectin1 inactivation by CRISPR in primary zebrafish candidate tumor suppressors and some of which, including melanomas significantly increased tumor cell spreading in FBXW7 and BCOR, have unknown or poorly characterized secondary recipients compared to a control gene (62.8% roles in RMS pathogenesis. To characterize the role of vs 10.3%, p<0.001), demonstrating that nectin1 loss tumor suppressors in RMS, we performed a pilot study strongly promotes melanoma dissemination in vivo. This utilizing the dCas9-VP64 gene activation system to assess spreading capacity was associated with altered mechanical gain-of-function effects of TP53, a known tumor suppressor properties of nectin1-deficient tumors as measured by in ERMS, and two novel tumor suppressor candidates, atomic force microscopy (elastic modulus: 303 vs 492 in FBXW7 and BCOR, on tumor growth and progression in a control tumors, p<0.001). To investigate the function of zebrafish model of KRAS-induced ERMS. DNA constructs NECTIN1 at the molecular level, we established cell lines expressing dCas9-VP64, gene-specific or scrambled control from zebrafish melanomas. Surprisingly, nectin1 knockout dual gRNAs, activated mutant of KRAS (KRASG12D) and a clones appeared exquisitely sensitive to serum stress and fluorescent reporter (RFP) were co-injected into zebrafish failed to grow outside of rich media. Accordingly, in human ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 52 Poster Abstracts melanoma cell lines, NECTIN1 inactivation by shRNA or Sophia K. DeGeorgia1, Rebecca L. Cunningham1, Eva T. CRISPR reduced cell proliferation by 20% at low serum Kramer1, Vadim Gigura1, Charles K. Kaufman1

concentrations (p<0.001). It also impaired anchorage- 1 independent growth in semi-solid media and spheroid Washington University in St. Louis School of Medicine, formation in low-adhesion conditions, but significantly Department of Molecular Oncology, St. Louis, MO USA increased the migratory capacity of melanoma cells in a Melanoma is a devastating cancer of melanocytes. transwell assay (4 fold increase, p<0.001). Collectively, these Embryonic neural crest cells give rise to melanocytes, and data indicate that NECTIN1 controls a balance between during melanoma initiation, transformed melanocytes growth and migration. By combining analysis of human reactivate aspects of a neural crest transcriptional program. cancer genomics, functional testing in zebrafish tumors and Reemergence of a more proliferation-prone or invasive mechanistic studies in cell lines, we thus identifiedNECTIN1 state may be a key driver of melanoma initiation. Zebrafish as a novel metastasis suppressor gene in melanoma. harboring two cancer driving mutations BRAFV600E;p53-/- such that every melanocyte is poised to become cancerous. Strikingly, only 1-3 tumors form from thousands of P-055 Characterization of Leukemia stem cells and melanocytes in a given zebrafish after several months underlying mechanisms responsible for progression and of life. We aim to elucidate epigenetic mechanisms that relapse in acute lymphoblastic leukemia differentiate cancer-prone melanocytes from those that

1,2,3,4 5 become cancer, as detecting these earliest key events of Alexandra Veloso , Saara Laukkanen , Elaine cancer initiation is critical. Sox10, a neural crest transcription Garcia1,2,3,4, Alex Jin1,2,3,4, Sowmya Iyer1,2,3,4, Sara 1,2,3,4 6 1,2,3,4 factor is critical for neural crest identity, is upregulated Garcia , Siebe Loontiens , David M. Langenau in melanoma, and modulates melanoma onset rate. We 1 Department of Pathology, Massachusetts General hypothesize that sox10 regulatory elements are active in HospitalResearch Institute, Boston, MA 02114, USA melanoma initiation and that a subset of these putative Charlestown, MA 02129, USA, 2 Center for Cancer enhancers may be critical for regulating sox10 expression Research, Massachusetts General Hospital 3 Harvard in melanocytes to push back towards a neural crest Stem Cell Institute, Boston, MA 02114, USA,4 Center for identity. Our lab generated ATAC-seq data that revealed Regenerative Medicine, Massachusetts General Hospital, a cluster of recurrently open chromatin peaks upstream Boston, MA 02114, USA, 5 Tampere Center for Child Health of sox10 in multiple zebrafish melanoma tumors. Two Research, University of Tampere and Tampere University putative enhancers ~ 31.5 and ~35.2 kb upstream of the Hospital, 33520 Tampere, Finland, 6 Cancer Research sox10 promoter are active in melanoma as assessed via a Institute Ghent (CRIG) and Center for Medical Genetics, transgenic EGFP reporter assay. In order to comprehensively Ghent University, Ghent, Belgium study these enhancer elements, we will characterize their embryonic and adult expression patterns and then assess Acute lymphoblastic leukemia (ALL) arises from the malignant their roles in modulating melanoma onset by 1) generating transformation of lymphoid B- or T-lineage progenitor CRISPR/Cas9 mediated deletions of the endogenous loci and cells and its one of the most frequent malignancies in the 2) using nuclease inactive dCas9 variants fused to chromatin pediatric population. Even though considerable advances modifiers (e.g. KRAB repressor domain) to modulate their in the treatment of these diseases have occurred over native epigenetic state. Understanding the regulatory that past three decades, relapse following treatment is roles of each putative enhancer and the corresponding still a major clinical problem. Continued tumor growth and transcription factors mediating this activity will allow us to relapse are mainly driven by self-renewing leukemia stem understand and modulate the earliest initiating events of cells (LSC) that comprise <10% of all blast cells. Yet, the melanoma underlying mechanisms responsible for driving the self- renewal and survival of these LSCs remains largely unknown. To identify novel self-renewal mechanisms that drive ALL P-057 Unravelling the anti-tumor role of chemerin using growth, we are generating transgenic zebrafish models of T- zebrafish melanoma models and B-ALL by expressing c-Myc in developing lymphocytes Elodie Di Ruggiero1, Omid Ghandeharian1, Jennifer Pozo of zebrafish. By using high-throughput cell transplantation, Gomez1, Adam Hurlstone2, Marc Parmentier1 and Valérie dynamic cell imaging of fluorescent transgenic leukemias Wittamer1 and single-cell sequencing, our work will identify molecular pathways that regulate the self-renewal and overall number 1 Institut de Recherche Interdisciplinaire en Biologie of relapse-driving LSCs. Altogether, our experiments will Humaine et Moléculaire - Université Libre de Bruxelles, also provide new biological insights into the heterogeneity Campus Erasme, 808 Route de Lennik, B-1070 Brussels, found in B- and T-ALL LSCs, informing the next generation of Belgium, 2 Faculty of Life Sciences, University of treatments targeting for ALL. Manchester, C.4223 Michael Smith Building, Oxford Road, Manchester, England Melanoma is reported as the 19th most common cancer P-056 Characterization of melanoma initiation-specific worldwide and every year its incidence is rising faster than enhancer elements any other cancer type. The 5-year survival for patients ......

..... 53 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts diagnosed with metastatic melanoma, usually treated by or lose effectiveness due to development of resistance. a combination of targeted and immuno-therapies, is under Guided by our finding that activation of neural crest-related 10 %, principally due to tumor resistance mechanisms. New genes occurs in the first melanoma cell in zebrafish, we therapeutic targets are urgently needed in order to prevent combined functional datasets from human melanoma and melanoma progression and increase patient survival neural crest cells with whole-genome sequencing of 183 rate. The critical importance of chronic inflammation and melanomas from the ICGC dataset and identified mutation tumor microenvironment in the development, progression hotspots within putative regulatory regions. Importantly, and metastatic potential of this disease has been well our pipeline identified the known TERT hotspot as well established. In this context, chemoattractant molecules play as dozens of other candidate regions. Using transgenic a key role in the selective recruitment of specific leukocyte zebrafish reporters, we are functionally validating variants populations within the cancer niche. within hotspots by comparing relative and spatiotemporal differences in eGFP and mCherry fluorescence for the wild- Here, we investigate the role of a chemoattractant factor type and mutant hotspots, respectively, during neural identified in our group, chemerin, which displays anti- crest and melanoma development. Variants and their tumor properties in B16 melanoma skin graft and DMBA/ associated target genes will then be assayed for their role TPA skin carcinogenesis mouse models. Exploiting NRAS- in melanomagenesis in vivo. These findings may identify and BRAF/p53-driven zebrafish melanoma models along alternative mechanisms that modulate regulation of with zebrafish xenograft assays, we sought to obtain known melanoma drivers and novel pathways important new mechanistic insights by which chemerin modulates in melanomagenesis that are not dependent on protein- tumorigenesis and the metastatic process in vivo. Our coding mutations. group demonstrates that the chemerin axis in zebrafish is highly conserved across the vertebrate phylum. Taking advantage of our newly generated chemerin knock- out lines, we show that chemerin anti-tumor effect is P-059 Abnormal lymphopoesis precedes the onset of ALL also observed in zebrafish. Future work will focus on the in hMYC transgenic zebrafish thorough observation of the immune response dynamics Ameera Hasan1, Jessica Burroughs-Garcia3, Gilseung developed over the course of melanoma genesis, and the Park2, Megan Malone-Perez3, Clay A. Foster3, Chiara impact of chemerin on these processes. By providing a new Borga3 and J. Kimble Frazer1,2,3 level of precision in understanding the role of chemerin in melanoma pathogenesis, these approaches complement 1 Departments of Microbiology & Immunology, 2 Cell ongoing studies in mice and should help determining Biology2 and Section of Pediatric,3 Hematology-Oncology whether chemerin system constitutes an attractive target University of Oklahoma Health Science Center, Oklahoma for therapeutic intervention in human. City, OK, USA Acute lymphoblastic leukemia (ALL) is an aggressive P-058 Using zebrafish models to functionally characterize hematologic malignancy that afflicts children and adults. non-coding somatic variants from human melanoma ALL develops as a clonal expansion of an early precursor cell of either the B or T lineage, depending upon the specific Paula Godoy1, Charles Kaufman1 genetic aberrations that occur, and what lymphoblast lineage they occur in. Over 60% of ALL overexpress the 1 Department of Molecular Oncology, Washington myelocytomatosis (MYC) oncogene. Overexpressed MYC is University in St. Louis (WashU) known to drive leukemogenesis, but MYC alone is insufficient Melanoma, a cancer arising from melanocytes, is the to transform normal lymphoblasts into malignant form. deadliest skin cancer on a per case basis. The most Additional genetic/epigenetic changes have been linked common driver mutation, BRAFV600E, is not sufficient to with MYC in other cancers. These genetic events have induce melanoma, and the accumulation of additional not yet been discovered in ALL. Zebrafish develop highly- genetic and/or epigenetic lesions are required to drive penetrant MYC-driven ALL with short latency. Transgenic the conversion of a premalignant melanocyte to cancer. fish with murine or human MYC (mMyc, hMYC) driven by Similarly, in a zebrafish melanoma model driven by the rag2 promoters have been studied extensively with respect human BRAFV600E mutation and dependent on homozygous to T-ALL, and recently also B-ALL. Our lab, uses double- p53 loss-of-function, melanomas arise stochastically with transgenic rag2:hMYC, lck:eGFP fish, where T cells are a median onset rate of ~6 months, reinforcing the idea GFPhi and B cells are GFPlo. Lymphocyte protein tyrosine that additional pro-tumorigenic epigenetic and/or genetic kinase (lck), cell-specific fluorophore, was thought to be alterations are required. Interestingly, the most common only expressed by T lymphocytes. However, we found that mutation in cutaneous melanoma is in the TERT promoter a B cell subpopulation, innate lymphoid (ILCs and NK cells) and leads to increased TERT expression due to the creation and even myeloid cells also express lck. Thus, we can use of a de novo Ets1 transcription factor binding site. A need to lck:eGFP fish (with or without hMYC) to study abnormal discover and functionally assess other prevalent non-coding lymphopoiesis that precedes the onset of MYC-induced ALL. mutations in melanomagenesis is pressing, as current We hypothesize that MYC hyperactivity causes aberrant treatments sometimes fail to induce any tumor response proliferation of lymphoblasts before their malignant ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 54 Poster Abstracts transformation, setting the stage for additional genetic P-061 Employing a Zebrafish Angiosarcoma Model to changes to develop ALL. Our data show that overexpressed Study Tumor Heterogeneity and Metastasis MYC causes lymphoid hyperplasia of primary (thymus Amanda Lipsitt1,2, Kunal Baxi2,4, Nicole Hensch2,3,4, Myron and marrow) and secondary (spleen) lymphoid organs in 2,3,4 pre-leukemic stage. This include increase in thymic B cell, Ignatius a rare and poorly-understood population in WT fish, and 1 University of Texas Health San Antonio; Department in humans. Our current efforts seek to identify the MYC- of Pediatric Hematology Oncology, 2 University of Texas associated genes that drive the lymphoid hyperplasia to Health San Antonio; Greehey Children’s Cancer Research develop B- or T-ALL by transcriptomic analysis. Institute,3 University of Texas Health San Antonio; Graduate School of Biomedical Sciences, 4 University of Texas Health San Antonio; Greehey Children’s Cancer P-060 Mechanisms underlying synergy of DNA Research Institute, Department of Molecular Medicine. Topoisomerase I and mTOR co-inhibition in malignant peripheral nerve sheath tumors (MPNSTs) Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. This soft tissue sarcoma can arise in Dong Hyuk Ki 1, 2, Felix Oppel 1, 2, Adam D Durbin 1, 2, 3, A vascular or lymphatic tissues involving any part of the body, Thomas Look 1, 2 which creates difficulties with treatment and control of 1 Department of Pediatric Oncology, Dana-Farber Cancer the disease. With this neoplasm’s ability to aggressively Institute, Harvard Medical School, Boston, MA 02115, travel the roadways of the vessels and body cavities, USA, 2 Division of Pediatric Hematology/Oncology Boston these patients’ prognosis remains poor. Angiosarcoma Children’s Hospital, Boston, MA 02115 USA, 3 The Broad has a high propensity for aggressive metastatic multifocal Institute of MIT and Harvard, Cambridge, MA, 02142 disease. Little is known about the exact mechanisms of angiosarcoma metastasis and theories of hematologic Malignant peripheral nerve sheath tumors (MPNSTs) spread have been proposed. There is also lack of knowledge are soft-tissue sarcomas that frequently arise in patients about angiosarcoma metastasis to the bone marrow, the with neurofibromatosis type 1 (NF1). Most MPNSTs site of human hematopoiesis, and its effect on mortality. are unresectable at diagnosis, lending urgency to the Zebrafish tumor models have provided novel insights on identification of new pathway dependencies and drugs tumorigenesis. Recently we generated tp53 null zebrafish with antitumor activities to treat these typically refractory and found that it spontaneously develops angiosarcomas in tumors. We therefore examined a series of candidate agents addition to other tumors. The angiosarcomas histologically for their ability to induce apoptosis in MPNST cells arising and molecularly resemble the human disease. Moreover, we in nf1/tp53-deficient zebrafish. To facilitate simultaneous generated the tumors in syngeneic zebrafish thus enabling analysis of anti-tumor activity and toxicity to normal cells, transplantation of tumors cells in recipient animals. we implanted fluorescent tumor cells into the pericardium of pigmentless Casper zebrafish embryos and incubated The spontaneous angiosarcomas that arise in tp53 null the embryos in graded drug concentrations. Using this syngeneic zebrafish are also GFP+ thus enabling in vivo strategy, we found that DNA topoisomerase I-targeted visualization of tumor growth in recipient syngeneic larval drugs and mTOR kinase inhibitors were the most effective CG1 zebrafish hosts. We successfully transplanted this single agents in eliminating MPNST cells at well-tolerated angiosarcoma tumor and found that the transplanted dosages. In addition, members of these classes of drugs, tumors are extremely invasive and metastatic. We are irinotecan and AZD2014 or INK128, acted synergistically to able to quantify metastasis in vivo using two independent induce apoptosis when given in combination in vitro and in approaches with both primary and transplanted tumors. vivo. In mechanistic studies using human MPNST cells, we The transplanted tumors being GFP+ will help us determine found that irinotecan not only induces apoptosis by eliciting whether spread is via invasion or true metastasis through a DNA damage response, but also acts synergistically the vasculature. In summary, our overall goal is to define with the mTOR inhibitor AZD2014 to accentuate the the cellular mechanism of metastasis of angiosarcoma. hypophosphorylation of 4E-BP1. 4E-BP1 is a downstream Moving forward, we will use this model to complete a drug target of mTORC1, which in its unphosphorylated form study that will score for both growth and metastasis in binds and sequesters eIF4E eukaryotic translation initiation vivo to identify possible novel pathways for treatment for factor. Profound hypophosphorylation of 4E-BP1 induced by angiosarcoma that affect growth and self-renewal. the combination of these two drugs causes an arrest of 5’ Cap-dependent protein synthesis, which potently induces tumor cell apoptosis. Our findings provide a compelling P-062 Reactivation of neural crest progenitor cell fate in rationale for clinical testing of the combination ofDNA adult zebrafish is induced by transcription factors that topoisomerase I-targeted drugs and mTOR kinase inhibitors regulate neural crest specification and migration. in patients with unresectable MPNSTs. Alicia M. McConnell1, Erika Weiskopf1, and Leonard I. Zon1

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..... 55 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts 1 Stem Cell Program and Division of Hematology/Oncology, well demonstrated that MYCN-amplified neuroblastoma Children’s Hospital Boston, Howard Hughes Medical tumors have significantly lower number of cytotoxic tumor- Institute, Boston, MA 02115, USA. infiltrating lymphocytes (TILs), which are critical in control of tumor growth. However, it remains elusive how MYCN Melanoma arises due to a malignant transformation suppresses the infiltration of T cells to tumors and protects of melanocytes, which are embryonically derived from tumor cells from TIL-mediated cytotoxicity. Elucidation of the neural crest. Our lab developed a zebrafish model the mechanism by which MYCN suppresses anti-tumor of BRAF/p53-driven melanoma, which expresses neural response in the neuroblastoma microenvironment will lead crest progenitor markers, including the zebrafish specific to improved treatment outcomes through immunotherapy. gene crestin. crestin is expressed in the embryonic neural We applied a zebrafish model, in which lymphocytes and crest, off in adults, but is expressed again specifically in tumor cells were fluorescently differentially labeled. We tumors. Using the crestin promoter to drive EGFP, we can monitored the infiltration of EGFP and mCherry labeled CD4+ visualize melanoma initiation from the single cell stage in T cells in EGFP-positiveMYCN -overexpressing premalignant vivo. Despite the presence of oncogenic mutations, only neural crests on 7, 14 and 21 days post fertilization (dpf). certain cells undergo tumorigenesis, indicating that other We found that at day 14 and 21, MYCN-overexpressing pathways are required to drive tumor initiation. In order premalignant neural crests can attract a significantly higher to investigate the transcription factor networks driving number of lymphocytes, compared to control neural crests melanoma initiation, we performed RNA-Seq and ATAC- without MYCN overexpression. Interestingly, the increased Seq on sorted melanocytes from early patches of crestin number of infiltrated T cells is MYCN dose dependent. We positive cells. We found that many transcription factors are in the process of characterizing CD4+ T cells that are involved in neural crest development were upregulated in attracted to premalignant sites using fluorescence in situ crestin positive samples. Furthermore, we characterized hybridization and qRT-PCR assays and will present the data these early lesions using scRNA-Seq and found that there in the meeting. Our studies highlight the power of live are distinct subsets within the patches of crestinhigh/ imaging in zebrafish for studying immune and tumor cell mitotic cells and crestinlow/non-mitotic cells. Next, we interactions. The information gathered in this study may misexpressed various neural crest transcription factors guide the development of effective immunotherapy to in the melanocytes of BRAFV600E;p53-/-;crestin:EGFP treat children with high-risk neuroblastoma. zebrafish using the using the MiniCoopR system. We found that transcription factors driving neural crest specification and migration increased crestin patch formation at 4 and 6 weeks of age. When overexpressing a combination of P-064 Aberrant AKT activation drives ganglioneuroma

Sox10, Mitf, Tfap2a, and cMyc, crestin patch number was 1,* 1 2 1,3 significantly increased above Sox10 overexpression alone. Ting Tao , Hui Shi , Meng Wang , Adam D. Durbin , Antonio R. Perez-Atayde4, Wendy B. London1,3, Alejandro Additionally, overexpression of Sox10 alone induced tumors 1,3 1,* in 3.35% of animals at 6 weeks of age. When overexpressing Gutierrez , A. Thomas Look the Sox10, Mitf, Tfap2a, and cMyc combination, 66% of 1 Department of Pediatric Oncology, Dana-Farber Cancer animals had tumors at 6 weeks of age. Together these Institute, Harvard Medical School, Boston, MA 02115, results demonstrate that reactivation of the specification USA, 2 Department of Environmental Health & Molecular and migration neural crest transcriptional program is a key and Integrative Physiological Sciences program, Harvard event in melanoma initiation. T.H. Chan School of Public Health, Boston, MA 02115, USA, 3 Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA, P-063 Exploiting the zebrafish to study lymphocyte 4Department of Pathology, Boston Children‘s Hospital, infiltration inMYCN -driven neuroblastoma Harvard Medical School, Boston, MA 02115, USA Xiaodan Qin1, Andrew Lam1, Xike Zhang1, Adam *Correspondence: [email protected] (A.T.L.) Hurlstone2, and Hui Feng1 or [email protected] (T.T.) 1 Departments of Pharmacology and Medicine, Section Peripheral sympathetic nervous system tumors are the of Hematology and Medical Oncology, Cancer Research most common extra-cranial pediatric tumors in children and Center, Boston University School of Medicine, Boston, MA include neuroblastoma, ganglioneuroma and intermixed 02118, 2 Manchester Cancer Research Centre, Faculty of ganglioneuroblastoma. Activating mutations of the RET Biology, Medicine, and Health, School of Medical Sciences, proto-oncogene induce ganglioneuroma in murine models, Division of Molecular and Clinical Cancer Studies, The but pathogenic abnormalities have not been identified in University of Manchester, Manchester, England, UK human ganglioneuromas, and the etiology and molecular basis underlying this benign tumor remain elusive. Here we Neuroblastoma is one of the most common pediatric showed that expression of constitutively activated myr-mAkt2 solid tumors that derives from sympathetic nerve ganglia. induced ganglioneuroma in transgenic zebrafish, regardless Aberrant MYCN activity, resulting from gene amplification, of whether the fish were also transgenic for overexpression overexpression, or protein stabilization, is strongly associated of MYCN or not. Phosphorylated Akt was shown to be highly with highly aggressive disease and poor prognosis. It is ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 56 Poster Abstracts expressed in these tumors by immunohistochemistry. cancer treatment, particularly for rare and genetically This led us to compare phosphorylated AKT levels in complex cancers that otherwise would be difficult to human ganglioneuromas and poorly differentiated investigate. neuroblastomas arising in the adrenal medulla in young children. Consistently, our analysis documented activated AKT in 11 of 12 human ganglioneuromas, but only in 3 of 17 P-066 Single cell chromatin accessibility analysis of poorly differentiated neuroblastomas (p= 0.0001, Fisher’s zebrafish melanoma reveals heterogeneous epigenetic exact test). Histopathological and comparative genomic changes to MAPK inhibition analyses revealed that zebrafish ganglioneuroma highly resembles human ganglioneuroma at the levels of histology Jeffrey Mito1, Sai Ma2,3, Margaret Weber1, Jason and gene expression signature. Our results implicate Buenorostro2,3, Leonard Zon1,2

activated and phosphorylated AKT as a tumorigenic driver 1 in ganglioneuroma. Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, 2Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, 3The Broad Institute of MIT and Harvard, P-065 Using the zebrafish as a personalized medicine Cambridge, MA platform for melanoma Melanoma is the deadliest form of skin cancer. For patients Joshua Weiss1,2,3, Theresa Simon-Vermot1, Richard 1,2 with metastatic disease, treatments include immune White checkpoint blockade and, for patients with BRAFV600X 1 Cancer Biology & Genetics Program, Sloan Kettering mutations, targeted inhibitors of the MAPK pathway. Institute, New York, NY,2 Department of Cell and Although most patients show initial responses to small Developmental Biology, Weill Cornell Graduate School, molecule inhibitors, the majority will develop progressive New York, NY, 3 Weill Cornell/Rockefeller/Sloan Kettering disease, which is thought to arise from heterogeneous Tri-Institutional MD/PhD Program, New York, NY tumor cell populations with varying epigenetic states and intrinsic sensitivities to MAPK pathway inhibitors. The Acral melanoma is a rare subtype of melanoma that is single cell assay for transposase-accessible chromatin using characterized by its distribution on the palms and soles sequencing (scATAC-seq) has emerged as a powerful tool of the hands and feet. Because these regions are not sun- to define cell types and epigenetic states by identifying exposed, the genetic drivers of acral melanoma are distinct accessible chromatin regions at a scale and resolution from other cutaneous melanomas and its pathogenesis is previously unavailable. We utilize scATAC-seq to profile over still poorly understood. Currently there are no genetically 3000 tumor and stromal cells from zebrafish melanomas engineered animal models to specifically investigate driven by oncogenic BRAFV600E and inactivation of p53. acral melanoma. To address this, we have developed a Tumor bearing fish were treated with DMSO or dabrafenib, a platform for creating zebrafish avatars of rare types of acral BRAFV600E specific MAPK pathway inhibitor. Among tumor melanoma, which can then be used to identify potential cells, tSNE analysis demonstrated that dabrafenib treated therapeutics for that tumor. Compared with other models, cells predominantly clustered from controls and showed the zebrafish has the advantage of rapid transgenesis and distinctive chromatin accessibility patterns including high throughput drug testing. We identified a patient with enhanced accessibility of peaks containing sox10 motifs an unusual acral melanoma, which initially presented on her and relative loss of accessibility in peaks containing ets1 and foot and relapsed 8 years later with an in-transit metastasis mitf motifs, all of which have been previous associated with on her shin. Both her primary tumor and metastasis were the development of drug resistance. An unbiased analysis profiled by DNA and RNA-sequencing, which identified of sequences that correlate with chromatin accessibility a unique combination of genetic drivers never previously variation among all cells identified motifs associated with identified together in a single melanoma patient: NF1 epigenetic heterogeneity. These de novo sites closely deletion, CRKL amplification, GAB2 amplification, and matched known consensus motifs for FOS/JUN, ETS, and TERT promoter mutation. These specific genetic events are TEAD transcription factors, among others. Correlation with predicted to drive MAPK pathway activation. Using a new single cell RNA-sequencing on genetically identical zebrafish “single-shot” transgenic technique, we created a genetic melanomas suggests that transcription factors capable of replica of her tumor, in which the melanocyte specific MITF binding these motifs are variably expressed. These findings promoter drives expression of cDNA and CRISPR cassettes suggest that epigenetic heterogeneity is a hallmark of of these four lesions. Within 2 months, 42% of the fish tumor development and that external stimuli can readily developed hyperpigmented lesions that later progress to shift chromatin accessibility patterns, providing insight into melanoma. Using this personalized zebrafish avatar, we the early epigenetic events characterizing drug tolerance. are now screening drugs to identify the optimal targeted therapy combination for this patient and to understand the unique pathogenesis of acral melanoma more broadly. These studies demonstrate the power of the zebrafish as P-067 Genome editing and disease modeling at the a complementary animal model for informing personalized NHGRI Zebrafish Core Facility ......

..... 57 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts Blake Carrington 1, Kevin Bishop 1, Rachel Weinstein 1, and ear malformations. CHD7 is involved regulating neural Liesl Broadbridge 1, Erica Bresciani 1, Wuhong Pei 1, Paul crest development, ultimately impacting formation of Liu 1, and Raman Sood 1 skull and facial tissues, and components of the autonomic

1 nervous system. Due to complications arising from Translational and Functional Genomics Branch, National maldevelopment of neural crest cells (hearing defects, Human Genome Research Institute, NIH, Bethesda, MD, gastrointestinal abnormalities), individuals with CHARGE USA. undergo many surgeries and experience adverse events The NHGRI Zebrafish Core was established in 2005 to following anesthesia, increasing mortality in these patients. facilitate the use of zebrafish as a model system. The goal We examined response to anesthesia in a chd7 -/- zebrafish of the Core is to provide services aimed at using the model model of CHARGE syndrome generated using CRISPR system to understand the function of genesin vivo. Recent technology (Prykhozhij et al. DMM 2017; Cloney et al. advances in targeted mutagenesis using genome-editing FEBS J 2018). We are investigating the role of PHOX2B in nucleases have made it feasible to quickly and efficiently CHARGE syndrome. PHOX2b has been implicated in central generate loss of function mutant zebrafish lines. NHGRI congenital hypoventilation syndrome (CCHS), another Zebrafish Core has been at the forefront of developing neurocristopathy. Hypoventilation during sleep in CCHS and adopting these new technologies. To date, we have resembles hypoventilation observed during anesthesia in generated knockout mutants for over 200 genes resulting CHARGE. in many publications. We observed a baseline difference in heart rates between Here we will present a few vignettes from our currently on- wild type (chd7+/+) and homozygous mutant larvae (chd7- going projects. In the first project, we are characterizing /-), with significantly lower heart rates in homozygous the in vivo functions of ZRSR2, an essential splicing factor mutant larvae (p<0.05). that is commonly mutated in hematopoietic malignancies such as MDS. We have generated zrsr2 knockout zebrafish Exposure of adult zebrafish to tricaine revealed a difference lines using CRISPR/Cas9. The zrsr2-/- embryos have in time to reach anesthesia.chd7-/- fish took longer to reach normal emergence of HSC’s but fail to complete definitive anesthetic plane, and had higher respiratory rates during hematopoiesis. These embryos start to develop edema at the anesthetic recovery period compared withchd7+/+ fish 4dpf and die by 9dpf. In the second project, we are using (p<0.05). zebrafish to validate if a missense variant inTP63 in a patient with cleft palette is responsible for the phenotype. However, Zebrafish have two homologs of phox2B, phox2Ba and the tp63 knockout embryos display a severe phenotype and phox2Bb. chd7-/- larvae had lower levels of phox2Ba die by 3dpf before their jaw phenotype could be evaluated. expression than chd7+/+ or chd7+/- larvae, examined A knockin with the exact patient variant is desirable for this by whole mount in situ hybridization. Future work will project. Therefore, we have been testing various knockin investigate the effect of phox2Ba loss on respiratory and and base editing strategies to develop efficient methods heart rates, by knocking down phox2ba. to generate fish with exact missense variants as well as Our zebrafish model has provided new insights into insertion of tags and/or fluorescent reporters at the desired the symptoms experienced by CHARGE patients during loci. We will discuss the current status of the strategies we anesthesia, and allowed us to establish a link between CHD7 have tested, and lessons learned from them. knock out and clinical observations of CHARGE patients.

P-068 Investigating the response to anesthesia in a P-069 Novel pH-sensitive biosensor zebrafish enable zebrafish Danio( rerio) model of CHARGE syndrome the in vivo visualization and interrogation of mitophagy Jessica MacLean1, Jaime Wertman2, Emily Chedrawe1, during development and metabolic stress Stewart Langley1, Shelby Steele1, Sergey Prykhozhij1, Kim 1 1 2 Blake1, & Jason N Berman1,2,3,4 Paul J. Wrighton , Arkadi Shwartz , Jin-Mi Heo , Eleanor D. Quenzer1, Kyle A. LaBella1, J. Wade Harper2, Wolfram 1 Department of Pediatrics, Dalhousie University/IWK Goessling1,3,4,5,6,7 Health Centre, Halifax NS Canada, 2 Department of 1 Division of Genetics, Brigham and Women’s Hospital, Microbiology and Immunology, Dalhousie University, Harvard Medical School, Boston, MA 02115, 2Department Halifax NS Canada, 3 Department of Pathology, Dalhousie of Cell Biology, Harvard Medical School, Boston, MA University, Halifax NS Canada, 4 Department of Pediatrics, 02115, USA, 3Harvard Stem Cell Institute, Cambridge, IWK Health Centre, Halifax NS Canada MA 02138, USA, 4 Dana-Farber Cancer Institute, Harvard CHARGE syndrome is associated with mutations in the Medical School, Boston, MA 02115, USA, 5 Broad Institute chromodomain helicase DNA binding protein 7 (CHD7) of MIT and Harvard, Cambridge, MA 02142, USA, 6 gene. CHARGE is an acronym referring to major features Harvard-MIT Division of Health Sciences and Technology, of this syndrome; ocular coloboma, heart defects, choanal Boston, MA 02115, USA, 7 Division of Gastroenterology, atresia, retardation of growth/development, genitourinary Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 58 Poster Abstracts Mitophagy is a process by which damaged or unnecessary and spatial expression of cyp21a2 throughout development. mitochondria are degraded and recycled via autophagy. Through transcriptome analysis and real-time in vivo Defects in mitophagy are linked to aging and degenerative imaging of zebrafish interrenal steroidogenic cells, a specific diseases in the central nervous system (e.g., Parkinson’s gene expression signature has been identified, providing Disease) and skeletal muscle (e.g., sarcopenia). In vivo new insights into the disease mechanism. We performed an interrogation of the molecular and cellular mechanisms in vivo chemical-genetic screen to identify novel drugs for governing mitophagy could contribute to therapeutic insight. inducing cyp21a2 expression. Successful chemical-genetic We generated zebrafish mitophagy reporter lines expressing hits may translate to a novel treatment strategy for patients the pH-sensitive biosensors mito-mKeima or mito-QC. with CAH. These tools enable quantitative, in vivo, light microscopy analysis of mitophagy in real-time during embryonic development or in response to stress. Frequent mitophagy P-071 Vitamin D receptor regulates distinct phases of events can be observed during development in the heart, liver organogenesis vasculature, liver, kidney, brain, and spinal cord. In skeletal muscle, basal mitophagy is low, but nutrient deprivation Scott Freeburg1, Kristen Alexa2, Wolfram Goessling3

or hypoxia strongly induces mitophagy. Importantly, the 1 mito-mKeima signal in nutrient-deprived skeletal muscle Genetics Division, Brigham and Women’s Hospital, Boston, MA 02115, 2 Genetics Division, Brigham and transiently co-localizes with Lc3-positive autolysosomes. 3 This live-imaging analysis confirms the physiological validity Women’s Hospital, Boston, MA 02115, Genetics Division, of the reporter and reveals the trafficking dynamics of Brigham and Women’s Hospital, Boston, MA 02115 mitochondria to autolysosomes for the first time in a live The functions of Vitamin D/Vitamin D Receptor (VDR) vertebrate. These transgenic lines enable the interrogation signaling in liver development and disease are unknown, of molecular mechanisms governing hypoxia-induced but epidemiological and experimental evidence implicate mitophagy in skeletal muscle using chemical and genetic protective roles for VDR in liver pathologies such as cirrhosis modulation of hypoxia-inducible factor (HIF) signaling and and hepatocellular carcinoma. The hormone vitamin D3 known mitophagy receptors. Future work will leverage functions through VDR to drive transcriptional changes the advantages of the zebrafish model system to identify that modulate cell fate decisions and cell proliferation. novel chemical and genetic mitophagy modulators, probe Our objective is to examine the impact of VDR signaling the roles of mitophagy during embryonic development, in liver organogenesis and to define the roles of VDR organogenesis and aging, and define the role of mitophagy activity in hepatocellular function. We discovered that in heart, skeletal muscle, and liver regeneration. vitamin D exposure during liver progenitor specification and expansion dramatically elevates liver bud size as demonstrated by in situ hybridization. Enhanced liver P-070 A chemical-genetic approach to identify novel bud growth is potentiated by global vdra overexpression, drugs for treatment of congenital adrenal hyperplasia indicating that vitamin D regulation of liver bud growth is

1 1 2 3 Vdr dependent. Vitamin D exposure accelerates induction Jun Yao , Kiersten Craig , Dix Poppas , Yariv Houvras of the hepatic progenitor marker prox1a, suggesting that Vdr activity promotes hepatic specification or proliferation 1 Department of Urology, Weill Cornell Medical College, of nascent hepatic progenitor cells. To determine whether 2 The Comprehensive Center for Congenital Adrenal Vdr signaling also enhances later stages of liver growth, Hyperplasia, Komansky Children’s Hospital, New we administered vitamin D during hepatocyte outgrowth. 3 York-Presbyterian Hospital-Weill Cornell Medicine, Hepatocyte quantification demonstrated that vitamin D Departments of Surgery, Meyer Cancer Center, Weill exposure attenuates hepatocyte abundance by 28%. Genetic Cornell Medical College ablation ofvdra/b confirmed that vitamin D responsiveness Congenital adrenal hyperplasia (CAH) is an autosomal is vdra/b dependent. In situ hybridization revealed that recessive endocrine disorder caused by mutations in the mRNA expression of vdra and the Vdr transcriptional cytochrome P450 family 21 (CYP21A2) gene. Mutations target cyp24a1 is induced in the liver during hepatocyte in CYP21A2 lead to decreased activity of 21-hydroxylase outgrowth, highlighting endogenous Vdr activity during this (21OHD). The clinical manifestations of CAH may include developmental period. Finally, evaluation of hepatocyte cell virilization, and profound electrolyte abnormalities. cycle status suggests that vitamin D exposure diminishes Medical management of patients with CAH requires hepatocyte proliferation. These data demonstrate novel exogenous steroid and mineralocorticoid replacement, and opposing roles for Vdr signaling in distinct phases of and often surgery to address virilization. There is a need to hepatic organogenesis, promoting the expansion of hepatic understand the disease mechanism and identify novel drugs progenitor cells but diminishing hepatocyte proliferation. for CAH patients. Zebrafish interrenal organ steroidogenic Our work also sets the stage to identify the molecular cells and the adrenal biosynthesis pathway are evolutionally effectors of Vdr in hepatocellular function and to determine conserved with humans, including a CYP21A2 ortholog. We the functional implications of Vdr signaling in adult models developed a novel cyp21a2-mScarlet reporter zebrafish of liver carcinogenesis and regeneration. using CRISPR/Cas9 genome editing to study the temporal ......

..... 59 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts P-072 Loss of DNA methylation leads to inflammation Leigh Syndrome, French Canadian Type (LSFC) is a rare and derangement of conserved regional programs in the mitochondrial disease caused by allelic variations in the zebrafish intestine Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. LSFC has a worldwide prevalence of ~1 Gilberto Padilla Mercado 1, Colin R. Lickwar 1, Lindsay 2 2 1 in every 100,000, however it is far more common in the Marjoram , Michel Bagnat , John F. Rawls Saguanay-Lac-Saint-Jean Region of Quebec, Canada (up 1 Department of Molecular Genetics and Microbiology, to 1 in 25 individuals carrying a diseased allele) due to a Duke University, Durham, NC, 2 Department of Cell Biology, genetic founder effect. LRPPRC has native functions related Duke University, Durham, NC to mitochondrial mRNA polyadenylation and translation as well as a role in gluconeogenesis. Patients show symptoms of The intestine is organized into distinct regions along its mitochondrial disease including lactic acidosis, ataxia, liver anterior-posterior axis that display distinct physiologic dysfunction and developmental delay. Treatment options functions and gene expression programs. Derangement of for this disease focus primarily on symptom mitigation, this intestinal regionality is a hallmark of Crohn’s Disease however life expectancy of individuals with LSFC is tied to a (CD), one of the Inflammatory Bowel Diseases (IBD). series of metabolic crises that typically occurs around two However, the mechanisms by which intestinal regionality years of age. Using the Gene-Breaking Transposon (GBT) is established during development and perturbed by cassette, a revertible insertional mutagen that creates a intestinal inflammation remain unknown. To address these truncated fusion protein leading to >99% knockdown of gaps in knowledge, we identified regionspecific biomarker wild type transcripts, we have created a mutant zebrafish genes conserved in zebrafish and mammalian intestines, line in the LRPPRC gene enabling further investigation of its and developed transgenicreporters that permit in vivo role in this variation of Leigh Syndrome. LRPPRC zebrafish observation of these regions. For example, tnfa labels a mutants displayed impaired liver function and lowered distal colon-like region, fabp6 labels an ileal-like region, levels of mtDNA transcripts, similar to the clinical phenotype and fabp2 labelling a proximal jejunum-like region. In a observed in patients. To the best of our knowledge, this is previous forward genetic screen, we found that mutations the first zebrafish model of mitochondrial genetic disease. in DNA methylation factors uhrf1 and dnmt1 result in the Using engineered liver-specific rescue as a genetic therapy, expansion of the distal tnfa domain to proximal intestinal we demonstrate increased survival of the rescued mutants regions, accompanied by intestinal inflammation and barrier past the embryonic lethal stage, suggesting an important disruption. The objective of this project is to define the role for the liver in the pathophysiology of the disease. impact of DNA hypomethylation on specification of proximal Liver dysfunction is a common symptom in patients with regional programs and to identify underlying molecular mitochondrial and metabolic disease but has not been mechanisms. RNA-seq analysis of intestinal epithelial generally considered as a major system for therapeutic cells from wild-type and uhrf1 mutant zebrafish revealed development. Understanding the molecular mechanism reduction of proximal region markers like fabp6 and fabp2. of the liver-mediated genetic rescue underscores the Preliminary in vivo imaging results reveal that the ileal-like potential to improve the clinical diagnostic and therapeutic fabp6+ region is retained but disorganized in uhrf1 mutants developments for patients suffering from these devastating despite the expanded tnfa expression. We are currently disorders. testing the hypothesis that DNA hypomethylation leads to inflammation and alteration of regional identity through de- repression of endogenous retrotransposable elements and P-074 Mosaic expression of cxcl8 by hematopoietic stem activation of antiviral innate immunity pathways. This work and progenitor cells prolongs interactions with the niche is expected to provide new mechanistic insights into the in zebrafish embryos relationships between DNA methylation, antiviral innate immunity, and intestinal homeostasis which may contribute Donn L Calkins1, Jami Shaffer1, Emily Teets1, Leonard I to human IBD. Zon2, Bradley W Blaser1 1 The Ohio State University Comprehensive Cancer Center P-073 A genetic model therapy reveals a potential critical and Division of Hematology, 2 Boston Children’s Hospital role for liver dysfunction in mitochondrial disease Stem Cell Program, Division of Hematology/Oncology, and the Howard Hughes Medical Institute Roberto Lopez Cervera1, Weibin Liu1,2, MaKayla R. Serres1, Mark Wishman1, Hirotaka Ata1, Ankit Sabharwal1, Competition and clonal selection of cells with somatic Jennifer Anderson3, Yonghe Ding1,2, Steve Farber3, Karl J. genetic alterations are fundamental processes involved in Clark1, Xiaolei Xu1,2, Stephen C. Ekker1 the development of cancer, including myeloid neoplasia. However, it is challenging to directly observe these processes 1 Department of Biochemistry and Molecular Biology, in a living system. Cxcl8 is an angiogenic chemokine Mayo Clinic College of Medicine, Rochester, MN, USA, often expressed at high levels in patients with myeloid 2 Division of Cardiovascular Diseases, Department of neoplasia. We hypothesized that somatic mosaicism for Medicine, Mayo Clinic College of Medicine, Rochester, a cxcl8 transgene expressed by hematopoietic stem and MN, USA, 3 Carnegie Institute of Washington, Baltimore, progenitor cells (HSPCs) in zebrafish embryos would alter Maryland, USA ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 60 Poster Abstracts HSPC interactions with the niche and provide a competitive larval HSPC pool responds by expanding in number and advantage to cxcl8-expressing cells. Mosaic expression of skewing its commitment towards neutrophil production, cxcl8 was enforced in HSPCs by microinjecting Runx1:cxcl8 a process called emergency granulopoiesis (EG). Here we DNA constructs into embryos at the single-cell stage. show that neutrophils generated by EG in larval zebrafish Time-lapse fluorescence video microscopy was performed kill phagocytosed bacteria faster and also produce more on HSPCs within the caudal hematopoietic tissue (CHT) bactericidal reactive oxygen species than those generated between 48 and 96 hours post-fertilization (hpf) and CHT during steady-state (SS). Transplanting EG-generated residency time was quantified. Enforced expression cxcl8of neutrophils into infection-naïve recipients revealed these increased the amount of time HSPCs resided within the enhanced functions were cell-autonomous. Furthermore, CHT by 77% when compared to controls. To look directly at larvae populated with EG-generated neutrophils show a HSPC-endothelial niche interactions, kdrl:mcherry embryos survival advantage to subsequent infection, without an were microinjected with cxcl8 expression constructs. The increase in neutrophil recruitment, even in the absence of percent of time individual HSPCs spent closely interacting macrophages. These results add to the growing literature with a single group of CHT endothelial cells (endothelial around neutrophil heterogeneity and suggest that EG- cell cuddling) was quantified. Enforced expression of cxcl8 generated neutrophils may also contribute to trained by HSPCs increased HSPC-endothelial cell cuddling time by immunity downstream of ‘infection-experienced’ HSPCs. We 30% compared to controls. To directly observe competition are currently examining transcriptional changes between between HSPCs expressing cxcl8 and those not expressing SS- and EG-generated neutrophils to better understand cxcl8, zebrafish embryos were microinjected with a 1:1 their functional heterogeneity and are investigating how molar ratio of a Runx1:cxcl8-2A-mCherry DNA construct ‘infection-experienced’ HSPCs boost the innate immune and a Runx1:clover construct (competitor). Cells expressing response when transplanted into infection-naïve recipients. one or both constructs were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided within the CHT 61% longer than competitor HSPCs. P-076 AIBP-mediated cholesterol efflux instructs Taken together, these data support a model in which pre- hematopoietic stem and progenitor cellfate malignant HSPC clones aberrantly express cxcl8, acquire a selective advantage over normal clones through enhanced Qilin Gu1, Xiaojie Yang1, Jie Lv1, 2, Jiaxiong Zhang1, 5, Bo Xia1, interactions with the endothelial niche, and could progress 2, Jun-dae Kim1, Ruoyu Wang6, 7, Feng Xiong6, Shu Meng1, over time to a neoplastic state. Thomas P. Clements8, Bhavna Tandon8, Daniel S. Wagner8, Miguel F. Diaz9, Pamela L. Wenzel9, Yury I. Miller10, David Traver11, John P. Cooke1, 4, 13, Wenbo Li6, 7, Leonard I. Zon12, 1, 2, 4, 13 5 1, 3, 4, 13, P-075 Not all neutrophils are created equal: a new role Kaifu Chen , Yongping Bai , and Longhou Fang for neutrophils during trained immunity? 1 Center for Cardiovascular Regeneration, 2 Center for Bioinformatics and Computational Biology, Department of Hannah Darroch1, Lucia Du1, Pramuk Keerthisinghe1, Cardiovascular Sciences, 3 Department of Obstetrics and Anneke Prankerd-Gough1, Kathy Crosier1, Phil Crosier1, Gynecology, 4 Houston Methodist Institute for Academic Chris Hall1. Medicine, Houston Methodist Research Institute. 1 Molecular Medicine and Pathology, Faculty of Medical Houston Methodist, 6550 Fannin St, Texas 77030, USA, and Health Sciences, University of Auckland 5 Department of Geriatric Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China, The traditional view regarding innate immunity is that 6 Department of Biochemistry and Molecular Biology, it lacks memory, or the ability to generate an enhanced UTHealth McGovern Medical School, 7 Graduate School response following re-exposure to infectious stimuli. This of Biomedical Sciences, University of Texas MD Anderson view has been recently challenged by evidence showing Cancer Center and UTHealth Houston, Texas 77030, USA, 8 that haematopoietic stem and progenitor cells (HSPCs) are Department of BioSciences, Rice University, Houston, Texas able to undergo functional reprogramming in response 77005, USA, 9 Children‘s Regenerative Medicine Program, to pathogens providing a reservoir skewed towards the Department of Pediatric Surgery, Center for Stem Cell and production of innate immune cells with heightened activity. Regenerative Medicine, The Brown Foundation Institute This phenomenon, termed trained immunity, has been of Molecular Medicine University of Texas Health Science hypothesised as a potential mechanism explaining the non- Center at Houston, Texas 77030, USA, 10 Department of specific protection provided by some vaccinations. Trained Medicine, 11 Division of Biological Sciences, Department of immunity has been studied predominantly in macrophages, Cellular and Molecular Medicine, University of California, with little known about the contribution of neutrophils, the San Diego, La Jolla, California 92093, USA, 12 Stem Cell most abundant innate immune cell. Program and Division of Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, We have shown that trained immunity also operates in Howard Hughes Medical Institute, Harvard Stem Cell larval zebrafish wherein previously infected larvae, lacking Institute, Harvard Medical School, Boston, Massachusetts a fully functional adaptive immune system, have non- 02115, USA, 13 Department of Cardiothoracic Surgeries, specific protection against subsequent infection. We have Weill Cornell Medical College, Cornell University, New York also previously shown that in response to infections, the ......

..... 61 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts 10065, USA laser with peak emission at 410 nm and 475 nm, when bound and unbound to calcium, respectively, it is highly Hypercholesterolemia, the driving force of atherosclerosis, compatible with multicolor flow cytometry. Here, we accelerates the expansion and mobilization of demonstrate the feasibility of using flow cytometry with hematopoietic stem and progenitor cells (HSPCs). The Indo-1 labeling to assess zebrafish thymocyte activation in molecular determinants connecting hypercholesterolemia vitro at three temperatures (15°C, 25°C, and 37°C). To guide with hematopoiesis are underexplored. Here we report thymi dissection and facilitate the identification of T cells, that a novel somite-derived pro-hematopoietic cue, we employed lck:eGFP zebrafish in which the majority of AIBP, orchestrates HSPC emergence from the hemogenic eGFP-positive cells are T cells. Given the lack of zebrafish endothelium, a type of specialized endothelium manifesting antibodies, we use concanavalin A, a plant lectin capable hematopoietic potential. Mechanistically, AIBP-mediated of activating T cells, to stimulate thymocytes and analyze cholesterol efflux activates endothelial Srebp2, the master the kinetics of their response. We find that zebrafish T cells transcription factor for cholesterol biosynthesis, which flux calcium similarly at all three temperatures and that transactivates Notch and promotes HSPC emergence. mechanical stimulation may also play a significant role in Srebp2 inhibition impairs hypercholesterolemia-induced their responsiveness. Further, we note that the activation HSPC expansion. Srebp2 activation and Notch upregulation seems to be T cell specific, as eGFP-negative cells appear are associated with HSPC expansion in hypercholesterolemic non-responsive. Such data highlight the potential utility of human subjects. Genome-wide ChIP-seq, RNA-seq, and this approach for future investigations into calcium signaling ATAC-seq indicate that Srebp2 trans-regulates Notch in immune cells and other cell types. pathway genes required for hematopoiesis. Our studies outline a novel AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease. P-078 BCAS2 is essential for hematopoietic stem and progenitor cell maintenance during zebrafish embryogenesis P-077 Measuring calcium flux in zebrafish T cells by flow Shanshan Yu1, Yuwen Huang1, Jiayi Tu1, Zhaohui Tang1, cytometry Mugen Liu1 1,2 3 3,4 Sara A. Rubin, Wan-Lin Lo, Arthur Weiss, Leonard I. 1 1,2,5 Key Laboratory of Molecular Biophysics of the Ministry Zon of Education, College of Life Science and Technology, 1 Stem Cell Program and Division of Hematology/ Huazhong University of Science and Technology, Wuhan, Oncology, Boston Children’s Hospital and Dana-Farber Hubei 430074, PR China Cancer Institute; Program in Immunology, Harvard 2 Hematopoietic stem and progenitor cells (HSPCs) originate Medical School, Boston, MA 02115, USA, Harvard from the hemogenic endothelium via the endothelial-to- Stem Cell Institute, Harvard University, Cambridge, MA 3 hematopoietic transition, are self-renewing, and replenish 02138, USA, Division of Rheumatology, Rosalind Russell all lineages of blood cells throughout life. BCAS2 (breast and Ephraim P. Engleman Arthritis Research Center, carcinoma amplified sequence 2) is a component of the Department of Medicine, University of California, San 4 spliceosome and is involved in multiple biological processes. Francisco, San Francisco, CA 94143, USA, The Howard However, its role in hematopoiesis remains unknown. We Hughes Medical Institute, University of California, San 5 established a bcas2 knockout zebrafish model by using Francisco, San Francisco, CA 94143, USA, Howard Hughes transcription activator–like effector nucleases (TALENs). The Medical Institute, Boston Children’s Hospital and Harvard bcas2-/- zebrafish showed severe impairment of HSPCs and University, Boston, MA 02115, USA their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the CHT of Calcium is a critical second messenger involved in a variety -/- of cellular processes including T and B cell activation, bcas2 zebrafish, which may be rescued by suppression of cardiomyocyte contraction, and neuronal signaling. A variety p53. Furthermore, we show that the bcas2 deletion induces of methods have been developed to measure calcium flux an abnormal alternative splicing of Mdm4 that predisposes including the generation of genetically-encoded calcium cells to undergo p53-mediated apoptosis, providing a indicators in model organisms such as zebrafish. While mechanistic explanation of the deficiency observed in useful for many applications, imaging genetically-encoded HSPC. Our findings revealed a novel and vital role for BCAS2 transgenic lines to assess calcium signaling is limited in its during HSPC maintenance in zebrafish. ability to simultaneously provide read-outs of calcium flux across multiple cell types and may not be compatible with some stimulation assays and transgenic lines. With the P-079 Modeling MLL-rearranged infant leukemia for high development of cell-permeable, fluorescent, ratiometric, throughput drug screening calcium indicators such as Indo-1-AM, flow cytometry has become an attractive and convenient method to measure Keon Collett1, Benno Orr2, Todd Druley3 and Jason intracellular calcium across a wide range of cell types and Berman1,4,5 biological processes. Since Indo-1 is excitable off a UV 1 Department of Pathology, Dalhousie University, 2 ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 62 Poster Abstracts Department of Molecular Genetics, University of Toronto, variation at the lta4h locus has been implicated in 3 Departments of Pediatrics, Developmental Biology and tuberculosis (TB) susceptibility and severity. The enzyme 4 Genetics, Washington University St. Louis, Department encoded at this locus, leukotriene A4 hydrolase, acts in of Microbiology and Immunology, Dalhousie University, 5 the synthesis of a pro-inflammatory signaling eicosanoid,

Department of Pediatrics, IWK Health Center leukotriene B4 (LTB4). Both the loss of LTA4H function and overexpression of lta4h disrupt the balance of pro- and Reciprocal translocations involving the Mixed Lineage anti-inflammatory signaling, leading to hypersusceptibility, Leukaemia 1 gene (MLL1; or KMT2A) affect ~80% of infant excessive mycobacterial growth, and increased disease leukemia (IL) cases. IL is an aggressive disease with five- severity. In order to better understand the consequence year event-free survival less than 50%. Together MLL- of this imbalanced inflammatory state on the granuloma, rearrangements (MLL-r) MLL-AF9 (MA9) and MLL-ENL the hallmark structure of tuberculosis, we have developed (MENL), are present in ~36% of IL cases. The roles of MLL1 methods to extract and culture mature, adult granulomas and MLL-r in epigenetic regulation and hematopoiesis have from zebrafish. We find that granulomas from an lta4h been well characterized, including the master regulation deficient background show distinct granuloma morphology of homeobox (HOX) genes. However, the lack of targeted and dynamics with a decreased capacity to contain and therapies remains problematic, as outcomes for IL have restrict mycobacterial growth. These data extend findings not significantly improved since implementation of about TB susceptibility and disease progression from the hematopoietic stem cell transplantation. larval innate immune model to later stages of infection, We are developing transgenic zebrafish models of MA9- when granulomas have fully matured and in which adaptive and MENL-associated IL using a two-transgene tamoxifen- immunity is also present. inducible Cre recombinase system that allowing for control of timing and tissue-specific expression of MLL-r. We have created three blood cell lineage specific transgenic P-081 Zebrafish models for human Fibrodysplasia lines expressing CreERT2 under the control of promoters Ossificans Progressiva (FOP), a rare and debilitating runx1+23 (hematopoietic stem cells), cd45/ptprc (early progressive mineralized tissue disease. white blood cells), and pu.1/spi1 (myeloid cell populations), and two transgenic lines that contain MA9 and MENL within Robert Lalonde, Alex Cintolo, Melissa LaBonty and a ubiquitously expressed ‘floxed’ reporter. Crossing MLL-r Pamela C. Yelick and CreERT2 lines, and treating the double transgenic Fibrodysplasia Ossificans Progressiva (FOP) is a rare autosomal larvae with 4-hydroxytamoxifen will cause the excision dominant genetic disorder in humans characterized by the of a blue fluorescent protein and allow the expression of progressive onset of heterotopic ossification (HO) in fibrous the MLL-r and the red fluorescent protein mCherry within tissues, a condition exacerbated by injury. FOP is caused specific populations of blood cells. by mutations in the type I TGFb/BMP receptor ACVR1, We will evaluate the impact of MLL-r expression on which normally signals through BMPs and type II receptors differentiation and leukemic phenotype using fluorescence- to initiate an osteogenic signaling cascade through the activated cell sorting (FACS) to assess the abundance of phosphorylation of Smad1/5/8. ACVR1R206H, the classic different cell populations. Peripheral blood smears and variant present in ~97% FOP patients, remains permissive to whole kidney marrow (WKM) preparations will assess BMPs but also exhibits neofunctional activation by Activin morphology. RNA sequencing on whole transgenic larvae A, normally a repressor for ACVR1, to signal osteogenic and adult WKM will evaluate downstream targets regulated differentiation. Using the Hsp70 promoter, we created by increased MLL-r expression. We aim to develop this transgenic zebrafish lines that conditionally express either model as an effective platform for the rapid screening human ACVR1R206H or orthologous zebrafishacvr1l R203H. of novel therapies that restore normal blood cell ratios Heatshocked Tg(Hsp70:acvr1lR206H-mCherry) embryos by evaluating the abundance of florescent blood cells show severe ventralization and increased fluorescence in expressing MLL-r in transgenic larvae. Bre:GFP (BMP-Response Element) transgenic backgrounds, mirroring injection of hACVR1R206H mRNA. To avoid embryonic lethality, transgenic embryos were raised under normal conditions until 14dpf, and then were placed P-080 Host eicosanoid imbalance alters mycobacterial on an automated daily heat-shock system. By 45dpf, granuloma structure and dynamics Tg(Hsp70:acvr1lR203HmCherry) zebrafish showed spinal Erika J. Hughes1,2, Mark R. Cronan2, Charlie J. Pyle2, and lordosis and vertebral fusions similar to those observed in David M. Tobin1,2 human FOP patients. We are currently characterizing injury induced HO in these transgenic lines. Skeletal morphology 1 University Program in Genetics and Genomics, 2 and HO progression will be observed via developmental Department of Molecular Genetics and Microbiology, Duke Alizarin Red staining and micro-CT imaging. In addition, University School of Medicine we successfully have used CRISPR/cas9 homology directed repair to create endogenous acvr1lR203H zebrafish, which Human host genetics influence susceptibility to infectious we are currently characterizing. Once validated as robust diseases. In humans and in a zebrafish model of tuberculosis, models of FOP, we will pursue high throughput drug ......

..... 63 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts screening and mutagenesis approaches to identify potential Alzheimer’s disease (AD) is a progressive neurodegenerative new therapeutic targets to treat human FOP. These studies disorder developing silently over decades. When the were supported by NIH/NIAMS R21AR072085 (PCY). disease becomes symptomatic, damage to the brain is substantial and little can be done therapeutically. We need to understand the early pathological changes occurring P-082 The onset of Roberts syndrome correlates with in asymptomatic AD brains. However, we cannot easily defects in transcription of genes involved in neuronal investigate these changes in molecular detail as access to development asymptomatic human brains is limited. Valeria Mastrodonato1, Gianluca Deflorian1, Dana Interestingly, there is little overlap between the loci Branzei1 involved in familial, early onset Alzheimer’s disease (fAD) and those identified as influencing the risk of late onset 1 IFOM, Firc Institute of Molecular Oncology, Milan, Italy AD (LOAD). Mutations in only four genes have repeatedly been identified in fAD:AMYLOID β A4 PRECURSOR PROTEIN Cohesin is a multiprotein complex required for sister (APP), PRESENILIN 1 (PSEN1), PRESENILIN 2 (PSEN2) and chromatid cohesion, chromatin organization and gene SORTILIN-RELATED RECEPTOR 1 (SORL1). Uniquely, SORL1 is transcription regulation. Cohesin structural components also a locus for variation affecting risk of LOAD. However, include SMC1A, SMC3, RAD21 and SA1/SA2, which enclose SORL1 is the least understood of the fAD genes. DNA strands in a ring-like structure. During G1 phase, NIPBL and Mau-2 ensure cohesin loading onto chromatin, Our laboratory has undertaken a program to introduce while during mitosis cohesin is removed by WAPL, to fAD-like mutations into endogenous zebrafish genes to allow sister chromatids separation. A key role in cohesion analyse their effects. We have successfully introduced three establishment is played by ESCO1 and ESCO2 during S mutations into sorl1: 1) p.W1818* and 2) p.V1482Afster12 phase. In particular, ESCO2 is an acetyltransferase which mimic human fAD-mutations p.W1821* and p.C1478* promote sister chromatin cohesion establishment by Smc3 respectively. Mutations truncating the open reading frame residues acetylation. Point mutations in humanESCO2 gene (ORF) and causing fAD only occur in SORL1 (not in APP lead to Roberts syndrome (RBS), a rare autosomal recessive or PSEN1/2). We do not know whether these mutations disorder characterized by growth retardation, microcephaly, are, effectively, null mutations. Therefore, we have also craniofacial malformations, limb development defects and generated a putative null mutation 3) p.R122PfsTer118 for low rate survival after birth. An esco2 mutant generated comparison. by retroviral insertion in zebrafish (Percival et al., 2015) recapitulates phenotypic RBS traits, such as microcephaly, FAD-like mutations in SORL1 have not previously been fin development and cell division defects. According characterised in the endogenous gene of an animal model. with the role that cohesin plays in intra-molecular DNA We regard 6 month old fish as equivalent to early adulthood interactions and consequently in gene transcription, esco2 in humans. Therefore, analysis at this age may help define morphants show expression defects of genes involved in cell the early molecular pathological changes occurring in proliferation and apoptosis. Previous studies reported that human fAD mutation carrier brains. Analysing the function developmental defects observed upon esco2 depletion or of SORL1 may help us understand the similarities and downregulation are in part due to cell division defects, but differences between early onset fAD and LOAD. how gene transcription can affect cell lineage specification for different tissues still need to be determined. Preliminary qPCR analysis on 24 hpf esco2 mutants revealed a decrease P-084 Characterizing the role of CHD7 in GABAergic in crestin and pax2a expression suggesting potential network development problems in neural crest and brain development. To Jamadagni P 1, Breuer M 1, Kassa B 1, Samarut E 2, Patten investigate these potentials defects, we plan to analyse 1 Wnt pathway components expression as well specific K neuronal markers. Our project aims to understand how 1 Inst. Armand-Frappier, INRS, Laval, QC, Canada, 2 Esco2 depletion can affect the expression of genes required Neurosciences, CRCHUM, Montreal, QC, Canada to ensure correct organs development and to test potential drugs that could regulate gene transcription in order to Mutations in the ATP-dependent chromatin remodeller ameliorate RBS phenotype. chromodomain, helicase, DNA binding (CHD) 7 are the primary cause of CHARGE syndrome and have been associated with autism spectrum disorders (ASD). However, P-083 Identifying early molecular changes underlying the mechanisms by which mutations in CHD7 affect brain familial Alzheimer’s disease mutations insorl1. development and function are poorly understood. To address this question, we have developed a zebrafish chd7 Karissa Barthelson 1, Morgan Newman 1, Michael Lardelli CRISPR/Cas9 knockout model. Mutant chd7-/- zebrafish 1 larvae exhibit a small head phenotype, defects in craniofacial

1 cartilage development, heart defects and have no swim Alzheimer’s disease Genetics Laboratory. University of bladder. We also found that chd7-/- fish display aberrant Adelaide, Australia ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 64 Poster Abstracts axonal network development. Interestingly, the mutant fish University of Massachusetts Amherst, Amherst, MA displayed hyperactivity particularly during dark light cycle. 01003, 2 Biology Department, University of Massachusetts We thus next sought to perform a detailed analysis of the Amherst, Amherst, MA 01003, 3 Child Study Center, brain in our model. We observed a significant decrease in Program on Neurogenetics, Yale School of Medicine, New GABAergic cells in chd7 mutants. The decreased number of Haven, CT 06510 GABAergic cells in certain regions of the brain is due to a failure in the migration of these cells. To confirm GABAergic The branched-chain amino acids (BCAAs), leucine, signalling defects in our model, we performed a treatment isoleucine, and valine, are essential amino acids. While with the GABA-A receptor antagonist pentylenetetrazol crucial ingredients for protein biosynthesis, their controlled (PTZ) and showed that chd7-/- fish exhibit an increased degradation is critical for normal brain development. BCAA sensitivity to PTZ-induced seizure. Using an unbiased catabolism is performed by branched-chain alpha-keto acid whole transcriptomic approach, we identified many genes dehydrogenase (BCKD), a mitochondrial enzyme complex involved in cell proliferation, migration and cell adhesion whose activity is regulated by BCKD kinase (BCKDK). In that are dysregulated in the chd7 mutant. Together, our humans, loss of BCKDK reduces BCAA levels and leads to findings indicate loss of chd7 results in a deficit of inhibitory co-occurring epilepsy, autism, and intellectual disability. neurons and suggest an essential role of chd7 in GABAergic In BCKDK-deficient humans and rodent models, the network development in the zebrafish brain. phenotypic severity can be ameliorated by a high protein diet. However, the underlying mechanisms that cause the neurological symptoms are not well understood. Closing this knowledge gap could reveal novel cellular and P-085 AZBA: An adult zebrafish brain atlas for the digital molecular processes that enable diet and metabolism to age regulate brain development and epileptogenesis. 1,2 2 Justin W. Kenney , Patrick E. Steadman , Thomas To better understand the mechanisms by which BCAA Mueller3, Meng Ting Shi1, Sheena A Josselyn2, Paul W. 2 levels regulate nervous system development and function, Frankland we have established bckdk knockout zebrafish lines. 1 Department of Biological Sciences, Wayne State During early larval stages (4-7 dpf), homozygous mutants University, Detroit, MI 48202 USA, 2 Program in exhibit normal morphology and escape behavior, but Neuroscience and Mental Health, The Hospital for Sick subtle reductions in sleep duration. When maintained on Children, Toronto, ON, M5G 1X8, Canada, 3 Division of conventional diets, adult homoyzygous mutants are viable Biology, Kansas State University, Manhattan, KS, 66506, and fertile, although they exhibit reduced growth. Zebrafish USA consume large amounts of protein from their yolk during early development and conventional zebrafish diets are As adult zebrafish gain in popularity as a model organism extremely rich in protein (in many cases approaching 60% for studying neurological diseases, the need has arisen by weight), which likely explains the subtle phenotype for an adult digital brain atlas to facilitate whole-brain observed under these dietary conditions. To investigate functional and anatomical analysis. Here, we present our this possibility, custom zebrafish diets were formulated to progress towards translating the seminal atlas published in contain 15% or 45% protein while remaining isocaloric. book form by Wulliman, Rupp, and Reichert in 1996, into Using these special diets and bckdk mutants, we are a three-dimensional digital format. Our atlas is derived currently performing kinematic, micro-CT, and RNA-Seq from intact adult brains from that were subject to staining experiments. We will present these results, which will and tissue clearing using the iDISCO (immunolabelling illustrate the effects of reduced BCAA levels on zebrafish enabelled three-dimensional imaging of solvent cleared behavior, brain development, and gene expression. organs) protocol. Whole-mount images were acquired using a light-sheet fluorescent microscope. Nuclear stained images from multiple fish were registered into the same P-087 Ontogeny of calcium waves in cerebellar astrocytes space and averaged to generate the atlas. We have also of zebrafish generated images using antibodies targeting neurons, neurotransmitters, astrocytes, and other markers to help Elizabeth Pereida-Jaramillo1, Angeles Edith Espino- guide segmentation. We anticipate that this resource will Saldaña1 and Ataúlfo Martínez-Torres1

significantly enhance the utility of adult zebrafish to study 1 vertebrate brain function in both health and disease. Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus UNAM Juriquilla, Querétaro, Qro. CP76230, México P-086 Modeling branched-chain amino acid deficiency Functional interaction between astroglial cells occur by disorder in zebrafish calcium signalling, this process endows this kind of cells with a way of communication or excitability throughout Gregory Teicher1,2, Sundas Ijaz3, Sarah Woody3, Joseph M. 3 3 1,2 the nervous system. Initiation and transmission of Fernandez , Ellen J. Hoffman , and Gerald B. Downes calcium waves depend upon two known mechanisms: 1) 1 Graduate Program in Molecular and Cellular Biology, communication through gap junction channels that connect ......

..... 65 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts adjoining astrocytes, and 2) upon release of transmitters expression of the tagged member of the proteome while (ATP, dopamine, glutamate and acetylcholine) that target generating Cre-revertible genetic alleles. Over 1000 lines plasma membrane receptors in the neighbouring cells. have been generated as an initial approach to explore the These two mechanisms of calcium wave transmission are vertebrate codex. The mutagenicity of the GBT cassette well documented in different vertebrates, but the functional is very high as assessed using both forward and reverse consequences of the activation remain poorly understood. genetic approaches. Sampling over 150 lines for visible In addition, deregulation of the activity of calcium waves is phenotypes after 5dpf shows a similar rate of discovery of associated with pathologic states and aging. embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with The aim of this study was to determine the moment during previously described phenotypes; embryos homozygous ontogeny in which calcium waves appear in the cerebellum for each of the corresponding GBT alleles displayed strong of zebrafish. This study may help to infer whether the loss of function phenotypes comparable to published initiation of calcium wave transmission is associated with mutants using other mutagenesis strategies (ryr1b, fras1, the appearance of motor activity driven by the neuronal tnnt2a, edar and hmcn1). Using molecular assessment after component and/or with the proliferation of the vasculature. positional cloning, to date nearly all alleles cause at least Thus, transient expression of the calcium indicator GCaMP6s a 99+% knockdown of the tagged gene. Analyses of 183 under the control of the astrocyte-selective GFAP promoter molecularly characterized loci indicate a rich mix of genes was induced by microinjection and two-photon microscopy involved in diverse cellular processes from cell signaling was applied for imaging calcium activity from 1 to 7 days to DNA repair. This 1000+ locus collection represents postfertilization (dpf). novel expression data from the illuminated mRFP protein GCaMP6s expression was detected a few hours after trap, with 36% and 87% of the cloned lines showcasing injection; however, cells did not exhibit spontaneous to our knowledge the first described expression of these calcium activity. Cell coupling was observed until 4dpf genes at day 2 and day 4 of development, respectively. and the number of cell clusters that presented calcium Interestingly, over 35% of the cloned loci represent 68 elevations became more frequent by 7 dpf, the calcium mutants in zebrafish orthologs of human disease loci, wave propagation reached up to 80mm diameter. including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The The results suggest that astrocytes and Bergmann cells of GBT protein trapping system enabled the construction the cerebellum are differentiated in early development but of a comprehensive protein codex including expression cell coupling that permit calcium propagation or release of patterns and functional roles of the vertebrate genome transmitters is not evident until 4 dpf. As to which is the and generated a diverse collection of potential models of molecular component that permits establishing the calcium human disease. waves remains to be explored. This work was supported by CONACYT. We appreciate technical support of E. Nydia Hernández Ríos and Adriana P-089 Development of experimental model for ocular Gonzales. toxicity screening in zebrafish Youngsub Eom1, Mi Jeong Kim2, Soonil Koun2,3, Soo Youn Choi1, Cherry Kim4, Hae-Chul Park2,3, Jong Suk Song1, Hyo Myung Kim1 P-088 A codex of human disease models developed using 1 Department of Ophthalmology, Korea University College protein trapping in zebrafish of Medicine, Seoul, Republic of Korea, 2Biomedical

1 1 1 Research Center, Korea University Ansan Hospital, Noriko Ichino , Kyle Schaefbauer , MaKayla Serres , Gyeonggido , Republic of Korea, 3Department of Suzan El-Rass1, Melissa McNulty1, Mark Urban1, Rhianna 1 1 1 1 Biomedical Sciences, Korea University, Ansan, Gyeonggido, Urban , Camden Daby , Gaurav Varshney , Yonghe Ding , Republic of Korea, Weibin Liu1, Kimberly Skuster1, Darius Balciunas1, Lisa Schimmenti1, Maura McGrail1, Jeffrey J. Essner1, Sridhar 4Department of Radiology, Korea University College of Sivasubbu1, Matthias Hammerschmidt1, Steven Farber1, Medicine, Seoul, Republic of Korea Xiao-Yan Wen1, Shawn Burgess1,Xiaolei Xu1, Karl J. Clark1, Stephen C. Ekker1 Purpose : The purpose of this study was investigate the efficacy of novel experimental model for ocular toxicity 1 International Protein Trap Consortium, Mayo Clinic, 200 screening using advanced pattern of optomotor response First Street SW, Rochester, MN 55905 (OMR) assay in zebrafish. The zebrafish is a powerful model to explore the molecular Methods : Zebrafish larvae were raised in embryo medium genetics and expression of the vertebrate genome, containing various concentrations of gentamicin and including those loci that contribute to the over 7000 known digoxigenin from 4 days after fertilization. After 24 h, larvae cases of Mendelian disease. The gene break transposon were assessed using the OMR, an assay which quantifies (GBT) is a unique insertional mutagen that reports the locomotor responses to new visual stimulus in this study......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 66 Poster Abstracts The effects of drugs on retinal morphology were assessed P-091 Short-term high cholesterol diet induces meta- by histologic and immunohistochemical analyses inflammation and primes innate immune cell response towards tissue damage in zebrafish Results : Zebrafish larvae exposed to moderate and high levels of gentamicin and digoxigenin during early Sofia de Oliveira1 and Anna Huttenlocher 1,2

development had functional abnormalities of the eye 1 characterized by OMR. The advanced OMR test can Department of Medical Microbiology and Immunology, University of Wisconsin Madison, Madison, United States, distinguish ocular toxicity from low dose of drugs that could 2 not be distinguished in conventional OMR tests.The general Department of Pediatrics, University of Wisconsin- locomotor activity of low dose treated larvae showed no Madison, Madison, United States differences with respect to controls and no morphological The consumption of western-type calorically rich diets changes were observed. promotes a systemic low grade of chronic inflammation Discussion : The advanced OMR assay is a more sensitive often called meta-inflammation. Tissue and organ than conventional methods. These results suggest that the infiltrated innate immune cells are major cell players in the advanced OMR assay in zebrafish could be an efficient and development of non-communicable diet-induced diseases predictive preclinical approach to evaluate adverse ocular such as atherosclerosis or non-alcoholic fatty liver disease. effects of drugs on visual function in vivo. However, the effect of diet on neutrophils and macrophages and how it impacts their response towards a secondary inflammatory stimulus like tissue damage is largely unknown. Here, we developed a zebrafish model of meta- P-090 Novel valproate-associated cardiotoxicity and folic inflammation and studied the impact of a high cholesterol acid relief in zebrafish embryogenesis: 3D light-sheet diet (HCD) on innate immune cell response towards a tailfin assessment transection in two week-old zebrafish larvae. 1 2 1 Seoyoung Ki , Byung Joon Hwang , and Yun Kee A short-term feeding with HCD induced systemic 1 Division of Biomedical Convergence, College of inflammation, characterized by infiltration of both Biomedical Science, Kangwon National University, neutrophils and macrophages to different tissues and organs, Chuncheon, South Korea, 2 Department of Molecular including GI track, liver, heart and brain. At this stage, no Bioscience, College of Biomedical Science, Kangwon significant difference was found between HCD and normal National University, Chuncheon, South Korea diet (ND) in larvae development. Increased proliferation in hematopoietic tissues (2-fold) was associated with higher Precise in vivo toxicological assays to determine the total number of neutrophils (1.3-fold) and macrophages cardiotoxicity of pharmaceuticals and their waste products (1.6-fold) in larvae fed with HCD compared to controls. After are essential in order to evaluate their risks to humans challenging HCD larvae with a secondary inflammatory and the environment following industrial release. In stimulus of tailfin transection, an increase of neutrophil the present study, we aimed to develop the sensitive recruitment and accumulation (3.5-fold) as well as higher imaging-based cardiotoxicity assay and combined 3D number of TNFα positive macrophages (2-fold) at wound light-sheet microscopy with a zebrafish model to identify area was observed thru time. Resolution of inflammation hidden cardiovascular anomalies induced by valproic acid was not achieved at 24 hours-post-wounding in HCD larvae, exposure. The zebrafish model is advantageous for this but surprisingly tailfin regeneration at 3 and 6 days post- assessment because its embryos remain transparent. The wounding was improved. Finally, we observed a 4.5-fold 3D spatial localization of fluorescence-labeled cardiac increase in recruitment and accumulation of neutrophils cells in and around the heart using light-sheet technology that reverse migrated from primary inflamed liver/intestine revealed the novel cardiotoxicity phenotype, a second area to a wound in HCD larvae. Taken together, the findings heart field-derived cardiac defect, in two-day-old zebrafish show that a short term HCD promotes meta-inflammation in embryos treated with valproic acid. Quantitative analysis zebrafish and suggest that innate immune cells are primed of the light-sheet imaging demonstrated that folic acid at primary inflammatory sites, which ultimately leads to the supplementation significantly increased the numbers establishment of an exacerbated immune response towards of endocardial and myocardial cells (P < 0.05) and the a secondary inflammatory stimulus like tissue damage. accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate Grant Support: AH was funded by NCI CA085862; SdO was increased in response to the cellular changes occurring in supported by EMBO ALTF 620-2015 and Cancer Research embryonic heart development (P < 0.05). The present study Institute/Fibrolamellar Cancer Foundation disclosed the cellular mechanism underlying the role of folic acid in spontaneous cellular changes in cardiogenesis and in valproic acid-associated cardiotoxicity. The 3D light- sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicity in both humans and animals.

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... 67 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts P-092 Analysis of vertebrate vision in a 384-well imaging whole-brain immunohistochemical protocols allow post- system hoc activity labeling and the incorporation of numerous additional cell types and gene expression patterns. 3-D Robert J. Thorn 1, Amanda Dombroski 2, Kerry Eller 1, 1 1 1 images can be virtually sectioned in any plane to create a Tania M. Dominguez , Danielle E. Clift , Peter Baek , complete series of brain sections with cellular resolution, Renee J. Seto 1, Elizabeth S. Kahn 1, Sara K. Tucker 1, Ruth 3 2 1 facilitating the comparison of multiple genetically defined M. Colwill , Jason K. Sello , Robbert Creton markers and cell types. We are using these new imaging 1Department of Molecular Biology, Cell Biology and procedures to build the Virtual Zebrafish Brain (VZB) as a Biochemistry, 2Department of Chemistry, 3Department of community resource. The VZB will include reference brain Cognitive, Linguistic and Psychological Sciences, Brown images from throughout the zebrafish life span, providing University, Providence, RI 02912, USA the first vertebrate life-course atlas as a tool to examine changes in morphology, connectivity, and gene expression Visual impairment affects 253 million people worldwide associated with the aging brain. By providing a framework and new approaches for prevention and treatment are for organizing and standardizing zebrafish research urgently needed. As potential treatments are developed, resources, we hope the VZB will help expand the utility of a critical question needs to be addressed: how dowe zebrafish as a tool for understanding adult vertebrate brain monitor in vivo for functional success? The current study function while increasing the use of zebrafish as a surrogate introduces a novel imaging system for automated analysis model for human neurological disease, degeneration, and of visually-guided behavior in zebrafish larvae. The imaging regeneration. system is the first to image four 96-well plates with a single camera for automated measurements of activity in a 384- well format. In addition, it is the first system to project P-094 CRISPR/Cas9-induced hcn2b zebrafish mutant moving visual stimuli and analyze the optomotor response exhibit epileptic-like behaviors in the wells of a 96-well plate. We found that activity is affected by tricaine, diazepam and flumazenil. Surprisingly, Luis R. Rodríguez-Ortiz1 and Ataúlfo Martínez Torres2 diazepam treatments induce a loss of visual responses, 1 CONACYT- Institute of Neurobiology, National at concentrations that do not affect activity or induce 2 hyperactivity. Overall, our studies show that the developed Autonomous University of Mexico, Institute of imaging system is suitable for automated measurements of Neurobiology, National Autonomous University of Mexico vertebrate vision in a high-throughput format. Epilepsy is a chronic neurological disorder that affects 50 million people worldwide according to the World Health Organization, the most common epilepsy is idiopathic, P-093 The virtual zebrafish brain (VZB): A 4-D expression where most of the genetic defects of this type of epilepsy atlas of the adult brain occur in ion channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane Rolf Karlstrom1, Alyssa Lutservitz1, Shelby Mitchell1, 1 2 hyperpolarization and are mainly expressed in the heart Katlyn Gabriel , and Joseph Bergan and central and peripheral nervous systems. In humans four 1 Department of Biology, 2 Department of Psychological and HCN genes have been described and emergent clinical data Brain Sciences University of Massachusetts, Amherst, MA, evidence that dysfunctional HCN channels are involved in USA neurological disorders such epilepsy and transmission of pain. Recently, Danio rerio became a very convenient model The zebrafish research community has generated thousands organism to generate transgenic lines to model a wide variety of transgenic lines that are powerful resources to investigate of genetic diseases opening the possibilities to use it in drug neuroanatomy, monitor gene expression and neural discovery and translational medicine. Using CRISPR/Cas9 activity in the brain, and can form a framework for studying technology we generated the hcn2b mutants in zebrafish basic biological processes that underlie neurological and and we characterized them molecularly and behaviorally. neuropsychiatric disorders. The work characterizing these We obtained two different hcn2b mutant alleles with a zebrafish transgenic lines has been largely limited tothe 27nt or 89nt deletion, respectively. The first mutant allele developing (larval) animal, making it less useful for studies (hcn2b+/∆27) could be able to express an 865aa protein while of possible subtle adult brain phenotypes associated the second (hcn2b+/∆89) generates a premature stop codon, with the loss of human neurological disease genes. We producing an 32aa protein. To date, F1 heterozygous were are generating an anatomically annotated whole brain obtained and analyzed. Compared to controls, both mutant atlas of molecular expression for adult fish that can serve alleles exhibit high mortality rate during the first 15 days to catalogue and compare gene expression in different post-fertilization. Interestingly, while the controls can rise disease models. Using simplified tissue clearing strategies to adulthood easily, the mortality in the mutants continue and lightsheet microscopy we can capture high-resolution during larval-juvenile stages probably due to sudden death. images of an entire intact adult brain in less than 10 minutes. We did not detect heart malformations or cardiac defects Image registration allows alignment of gene expression from and heart rate is not altered in mutants. Hyperactivity, transgenic lines onto reference brain images, while new absence and moderate seizures were detected, and ......

... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 68 Poster Abstracts coincided with sudden exposure to light in a fraction of Steven Cassar1, Manon Beekhuijzen2, Bruce Beyer3, larvae. These moderate seizures rarely caused death. Robert Chapin4, Martina Dorau5, Alan Hoberman6, Eckart Acknowledgments: special thanks to all members of the Krupp5, Isabelle LeConte7, Don Stedman4, Christine zebrafish group at the Molecular and Cellular Neurobiology Stethem8, Daphne van den Oetelaar2, Belen Tornesi9 laboratory – INB-UNAM. 1AbbVie, North Chicago, IL, USA , 2Charles River, Den Bosch, Netherlands, 3Sanofi U.S. Inc., Bridgewater, NJ, P-095 Molecular diversity and expression pattern of the USA, 4Formerly of Pfizer, Groton, CT, USA,5 Sanofi, R&D, calcium binding Mctp proteins: putative modulators of Frankfurt, Germany, 6Charles River, Horsham, PA, USA, neural transmission 7Bayer SAS, Sophia Antipolis, France (Presently: Charles River Laboratories, France SA, SAS), 8Pfizer, Groton, CT, Angeles E. Espino-Saldaña1,2, Karina Duran-Rios1, Eduardo USA, 9AbbVie, North Chicago, IL, USA (Presently: Medicines Olivares-Hernandez1, Fausto Arellano-Carbajal2 and for Malaria Venture, Geneva, Switzerland Ataulfo Martinez-Torres1. Predicting embryotoxicity of pharmaceutical compounds or 1 Instituto de Neurobiología, Universidad Nacional industrial chemicals is crucial for public safety. Conventional Autónoma de México, Departamento de Neurobiología studies which monitor embryo-fetal development in rats Celular y Molecular, Laboratorio de Neurobiología and rabbits are costly and time consuming. Alternative Molecular y Celular, Juriquilla, Querétaro, 76230, México, assays which are simpler and less costly are being pursued. 2Universidad Autónoma de Querétaro, Facultad de The purpose of this research was to assess the capacity for Ciencias Naturales, Av. de las Ciencias S/N, Querétaro, the zebrafish development assay to predict mammalian México plasma levels that are embryotoxic. Previously published data on rat plasma levels associated with embryotoxicity Calcium ions trigger the release of neurotransmitters from were used to guide concentration ranges for each of 25 synaptic vesicles by activating a series of proteins that chemicals dissolved in the media bathing developing bear the calcium binding domains named C2. One of those zebrafish embryos. Embryotoxic media concentrations calcium sensor proteins are the MCTPs, which possess three were compared to embryotoxic rat plasma concentrations. C2 domains and two transmembrane regions. It has been Assays were conducted in parallel at multiple sites as a shown that MCTP proteins bind calcium in vitro, although consortium effort through the Health and Environmental they have not been well characterized. Recently, it has Sciences Institute (HESI). Considering results from all sites, been described that MCTPs are localized in endoplasmic the zebrafish embryo development assay predicted (within reticulum in motoneurons in Drosophila where they 1-log) the rat maternal exposure levels associated with stabilize homeostatic neuronal plasticity; whereas deletion embryotoxicity 75 % of the time. mutants of mctp in C. elegans exhibit changes in motor coordination related to altered presynaptic transmission. In this work, we aimed to characterize Mctp in zebrafish, first establishing their temporal and spatial expression pattern; P-098 Suppression of transcytosis is a conserved then, determining the intracellular localization of these mechanism of functional blood-brain barrier formation protein; and finally, analyzing the phenotype of zebrafish during zebrafish development after knocking down their expression. We found that the Natasha O’Brown 1, Chenghua Gu 1, Sean Megason 2 zebrafish genome has fourmctp genes (named mctp1a, 1b, 2a y 2b). Alternative splicing events were detected in these 1 Neurobiology Department, Harvard Medical School, genes, except for mctp2a. The four genes are expressed Boston, MA, 2Systems Biology Department, Harvard from early development. The expression is localized Medical School, Boston, MA mainly in the nervous system and through the muscular system. The four genes present a differential expression Cerebral blood vessels, composed of a single layer of pattern during the stages of development, but mctp2b endothelial cells, possess distinct functional properties that shows stronger expression. All four Mctp proteins have prevent unwanted entry of specific toxins and pathogens the same intracellular localization; mostly in endoplasmic into the brain, creating the blood-brain barrier (BBB). These reticulum but also in late and recycling endosomes. The BBB properties result from two main mechanisms: 1) tight effect of disrupting the mctp2b expression by CRISPR/Cas9 junction complexes between neighboring endothelial cells blocked embryo development. This suggests the relevance and 2) low rates of vesicular transport or transcytosis. of the functional participation of this gene, at least during Zebrafish have recently emerged as a powerful genetic tool early stages of development. This work was supported by to study the BBB. However the subcellular and molecular CONACYT Ciencia Básica A1-S-7659. mechanisms governing the formation and maintenance of the zebrafish BBB remain poorly characterized. We provide a spatio-temporal profile of BBB development in zebrafish using tracer leakage assays, with hindbrain barrier P-097 A multi-institutional study benchmarking the function preceding midbrain barrier function. Specifically, zebrafish developmental assay for prediction of we found that the midbrain BBB is immature at 3 days post embryotoxic plasma concentrations from rat embryo- fertilization (dpf), as tracers leak into the brain, but matures fetal development studies ......

..... 69 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts by 5 dpf. Our in vivo imaging of the immature BBB revealed P-100 Stat3 is necessary for the induction of Hif1a a steady increase in tracer uptake in the brain parenchyma transcriptional activity

that was not observed when the barrier became functional. 1 1 This tracer accumulation outside of cerebral blood vessels Alberto Dinarello , Giacomo Meneghetti , Margherita Peron 1, Chiara Cioccarelli 1, Riccardo Betto2 , Graziano results from elevated levels of transcytosis, and is gradually 2 1 suppressed during development. Tight junctions, on the Martello , Francesco Argenton other hand, were functional preceding BBB function. 1Department of Biology, University of Padua, Padua, Italy, We next investigated whether a key molecular regulator 2Department of Molecular Medicine, University of Padua, of transcytosis in the mammalian BBB, Mfsd2a, plays a Padua, Italy conserved role in regulating the zebrafish BBB. We used CRISPR to generate mutants in both paralogues (mfsd2aa Stat3 is a transcription factor involved in many biological and mfsd2ab) and performed functional tracer leakage processes like regeneration, stem cell pluripotency assays. While mfsd2aa mutants display increased BBB maintenance, metabolism, cell proliferation, inflammation permeability due to increased levels of transcytosis, and apoptosis. Many human malignancies are characterized mfsd2ab mutants show no permeability defects. Taken by alteration of the Jak2/Stat3 pathway, particularly, in together these results suggest that zebrafish use conserved colorectal cancer increased activation of Stat3 is associated cellular and molecular mechanisms to form the BBB, and with enhanced proliferation and tumour growth. This highlight the tractability of zebrafish as a model system for prominent activity of Stat3 in tumour progression has studying the BBB. recently been linked to the activity of other pathways playing pivotal roles in cancer. One of such pathways that might be regulated by Stat3 and is involved in the metabolic P-099 The zebrafishatf5a -KO, atf5b-KO and atf5a/b-DO switch of cancer cells and in many other phenomena to model neurodegenerative disorders that allow tumours to grow and proliferate is HIF1. Using zebrafish as an in vivo model, we are characterizing the Roberto Rodriguez1, Luis Colon1, Viveca Negron1, Aranza crosstalk between Stat3 and Hif1a pathways. We first Torrado1, Gaurav Varshney2, Martine Behra1 treated the stat3 mutants with 5% of oxygen and we saw that hypoxia affected heterozygote and homozygote 1University of Puerto Rico (UPR), Medical Sciences 2 mutants’ development. So we decided to analyse Hif1a Campus, Oklahoma Medical Research Foundation (OMRF) transcriptional activity in mutants using the Tg(4xHRE- The Activating Transcription Factor 5 (Atf5) is largely TATA:mCherry) reporter zebrafish line: we did not detect expressed in the developing rodent brain and was shown increase in fluorescence of mutants compared to wild type to be important for neuroprogenitors proliferation, while after hypoxic treatment. The same result was obtained its expression needs to be downregulated for neuronal after pharmacological inhibition of Stat3 using Ag490: and astroglia differentiation to occur, thus suggesting that interestingly whenever Hif1a was stabilized, the absence ATF5 has an important role in maintaining proliferation of of Stat3 blocked Hif1a transcriptional activity. Furthermore neuroprogenitors and preventing their differentiation during we demonstrate that Stat3 is necessary for nuclear activity development. However, in adult rodents Atf5 expression of Hif1a: the injection of a non-degradable form of hif1a was found in mature neurons throughout the brain as well mRNA in zebrafish 1 cell stage embryos was followed by as in the olfactory bulb where it appears to be important for the treatment with Ag490 and we observed with qPCR neurons’ stress survival. Taken together, this gene appears that Hif1a target genes expression could not be induced to be a potential key player in neurodegenerative disorders. after inhibition of Stat3. The characterization and the To elucidate further the functions of ATF5, we propose to dissection of this emerging crosstalk between these two study its role in zebrafish, where we will take advantage of important transcription factors will be useful in order to the presence of two paralogs: atf5aand atf5b. using CRISPR/ better understand how physiologically the organism is able Cas9 technology, we created loss of function alleles for both to adapt to different oxygen concentration, but also how genes and are currently establishing stable KOs as well as tumours can modulate their metabolism and cell functions double-KOs lines. We will present the detailed and dynamic for proliferate and expand in healthy tissues. expression pattern of bothatf5a and atf5bin the developing embryo and larva from 2 to 6 day-post-fertilization (dpf). Atf5a appears to be mostly restricted to the olfactory P-101 A novel function for the serotonin receptor HTR2B epithelia while atf5b mostly to the LL. This should allow us in CHARGE Syndrome to discriminate the tissue restricted function for each gene Breuer, Maximilian 1, Jamadagni, Priyanka 1, Patten, for proper development and regeneration to occur in those 1 sensory structures. The already established loss-of-function Kessen atf5aupr3is homozygote viable and will provide along with 1INRS – Centre Institut Armand Frappier, Laval, QC, Canada the other lines that we are currently establishing important tools for neurodegenerative disease modeling. CHARGE syndrome is a severe neurodevelopmental disorder that is most commonly caused by mutations in the ATP-dependent chromatin remodelling enzyme CHD7. To understand the function of CHD7, we generated a chd7 ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 70 Poster Abstracts mutant in zebrafish by CRISPR/Cas9 mediated mutagenesis. development and will assess the functional consequences This model has proven highly efficient in replicating the of disease-associated variants. We first disrupted both characteristics observed in CHARGE syndrome. Using an paralogues of the obligatory NMDAR subunit grin1. unbiased transcriptomic analysis (RNA-Seq), we identified Zebrafish with lesions in either grin1a or grin1b are viable, a significant downregulation of the 5-hydroxytryptamine although juvenile grin1a mutants have growth defects. receptor 2b (Htr2b) in these chd7 mutant zebrafish. grin1a mutant larvae display increased acoustic startle Interestingly, this member of the serotonin receptor responsiveness and reduced prepulse inhibition (PPI) at 500 family is closely associated with behavioral defects ms interstimulus intervals, but not at 50 ms or 100 ms. This including impulsiveness and aggressiveness, as well as data is consistent with a loss of glutamatergic modulation developmental defects in heart and jaw development, of the startle circuit. grin1a;grin1b double mutants also which are characteristics observed in CHARGE syndrome. show a decrease in habituation of the startle response, consistent with a disruption of NMDA signaling. . Both To gain more insights in the role of htr2b in CHARGE grin1a and grin1b show heightened responses to chemical syndrome pathogenesis, we assessed its function further seizure inducing treatments. Although grin1a;grin1b in zebrafish. We found that htr2b is expressed in the jaw, double mutants die at around 10 dpf, their nervous system heart and brain via whole-mount in situ hybridization. function is remarkably normal in light of the requirement Htr2b inhibition via the specific inhibitor RS-12744 partially for GluN1 in NMDA-mediated neurotransmission. The overt mimics the morphological phenotypes observed in chd7 spontaneous swimming behavior of the double mutants mutant fish namely the heart defects and abnormal jaw is similar to wild-type larvae at 6 dpf, but show atypical and swim bladder development. More specifically, the evoked responses. These mutants provide opportunities to inhibition if htr2b results in defective development of both study the function of NMDARs during nervous system the palatoquadrate in the jaw as determined by alcian development and assess the functional consequence of blue staining. Furthermore, inhibition of htr2b causes patient associated NMDAR lesions. a pericardial edema as well as failure to inflate the swim bladder, both characteristics were observed in the chd7 mutant. P-103 Analysis of leukemia-niche interactions to predict Finally, to investigate the link between CHD7 and HTR2B, clinical outcome in a patient-derived xenograft model we sought to attempt to compensate for the reduction Anja Arner1, Andreas Ettinger2, Irmela Jeremias1,3, Bettina of the receptor via the use of an agonist. We found 4 1 1 that treatment with the HTR2B agonist improved the Schmid , Tobias Feuchtinger , Vera Binder hyperactive behavioral phenotype in our chd7 mutant. 1Department of Pediatric Hematology/Oncology, Dr. Altogether, our data show an implication of dysregulated von Hauner Children’s Hospital, Ludwig Maximilians HTR2B functions upon loss of function of CHD7 in CHARGE University (LMU), Munich, Germany, 2Microscopy Core syndrome pathogenesis and suggest that HTR2B maybe a Facility, Helmholtz Centre Munich, German Research potential therapeutic target. Center for Environmental Health, Munich, Germany, 3Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Centre Munich, German Research Center for P-102 Modeling NMDAR-associated neurological Environmental Health, Munich, Germany, 4German Center disorders for Neurodegenerative Diseases (DZNE), Munich, Germany Josiah Zoodsma 1, Kelvin Chan1, Gabrielle Moody1, Acute lymphoblastic leukemia is the most common Ashwin Bhandiwad1, Harold Burgess1, Lonnie P. malignancy during childhood. Even though the prognosis Wollmuth1, Howard I. Sirotkin1, after initial diagnosis is good, patients suffering from 1Stony Brook University, USA relapse have poor outcome. It is crucial to identify patients at high risk upfront to prevent relapse. Leukemic stem cells Rapid communication between cells in the nervous system are thought to reprogram their surrounding niche cells depends on ion channels that are directly activated by a for protection from chemotherapy. We hypothesize that chemical neurotransmitter. In the human brain, glutamate relapse inducing cells utilize their niche more efficiently is the major excitatory neurotransmitter. One key mediator than cells that never caused relapse. of glutamatergic signaling is the N-methyl-D-aspartate receptor (NMDAR). These receptors impact nearly all forms To study distinct leukemia – niche cell interaction, we of nervous system function including circuit development, established larval zebrafish xenografts to compare primary higher brain functions such as learning and memory, patient samples from children that suffered from early and motor processing. Mutations in the genes encoding relapse (RS, relapse sample) versus no relapse (NRS, no NMDAR subunits (GRIN genes) are associated with an relapse sample). Upon transplantation of fluorescently array of neurological disorders, including epilepsy, autism, labeled cells into niche-transgenic reporter larvae, and schizophrenia. NMDARs are obligate heterotetramers we analyzed differential behavior in vivo in single cell composed of two GluN1 subunits and typically some resolution. Semi automated image analysis of time-lapse combination of GluN2(A-D) subunits. Zebrafish have retained movies guaranteed unbiased assessment. paralogues of all 5 of the principle NMDAR subunits. We are ...... studying the requirements for each of the subunits during ......

..... 71 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts Upon transplantation, human leukemic cells homed and not viable in mammalian species. These data also suggest engrafted in the caudal hematopoietic tissue, where they that other zebrafish fibrillin isoforms might compensate for divided and showed distinct distribution patterns. Cells of the loss of fbn1. We plan to generate a more severe model the RS extravasated and formed clusters in vascular pockets of MFS by generating additional compound mutations in next to the main artery which was not observed in the NRS. the fbn1 mutant lines, in order to obtain a reproducible By tracking cells within the niche we scored mean track phenotype in the embryonic or larval stage. This model will duration, speed and displacement. The RS migrated slower be useful for future screens of FBN1 variants, as well as for (0.50µm/min) than cells of the NRS (0.81µm/min, p<0.05). in vivo chemical compound screens to identify potential Tracked RS cells stayed longer within the niche (in average new treatment options. 85min vs. 55min, p<0.05) before going back to circulation. This shows that relapse inducing cells interact longer with niche cells indicating increased niche utilization. Their P-105 Identification of melanoma initiation-associated cluster formation suggests collective, targeted behavior sox10 enhancers and extensive internal communication. Rebecca L. Cunningham1, Eva T. Kramer1, Sophia K. Taken together, we developed a powerful model to study DeGeorgia1, Vadim Gigura1, Charles K. Kaufman1 distinct niche behavior. We aim to use this unbiased, short- term assay to systematically analyze and score the behavior 1Washington University in St. Louis School of Medicine, of pediatric patient samples at time of diagnosis to predict Department of Molecular Oncology, St. Louis, MO USA clinical outcome. Melanoma is a deadly cancer of melanocytes, pigment- producing cells derived from the neural crest. The re-emergence of a neural crest program, including P-104 Generating a new zebrafish model for Marfan upregulation of the neural crest transcription factor, sox10, syndrome is a key step in melanoma initiation in both humans and Patrick Sips1, Petra Vermassen1, Hanna De Saffel1, Andy zebrafish. Since sox10 is critical to neural crest identity, Willaert1, Anne De Paepe1, Paul Coucke1, and Julie De melanocyte development, and the maintenance of Backer1 melanoma, we hypothesize that epigenetic regulation of sox10 may be critical for re-establishing neural crest identity 1Center for Medical Genetics, Department of Biomolecular in melanoma initiation. Furthermore, the fundamental Medicine, Ghent University, Ghent, Belgium epigenetic mechanisms that drive the transformation of a melanocyte to melanoma remain largely unknown. Background: Marfan syndrome (MFS) is a heritable Thus, we are comprehensively studying the regulatory connective tissue disease caused by dominant mutations regions near sox10 in zebrafish for a role in melanoma in the gene coding for fibrillin-1 (FBN1). Patients with MFS initiation. Based on ATAC-seq analysis for recurrently open suffer from symptoms in different organ systems, including chromatin domains from zebrafish melanoma tumors, the skeletal, ocular, and particularly the cardiovascular reporter constructs for each putative regulatory element system. Although the prognosis of MFS patients has surrounding sox10 were injected into BRAFV600E/p53 fish improved in recent years, major challenges still exist for the and examined for enhancer activity in neural crest cells and management of patients with MFS. On one hand there is a melanoma. We have constructed stable transgenic lines need for reliable risk estimation based on genomic patient for top candidate sox10 regulatory elements to further data, and on the other hand there is a lack of disease- characterize embryonic neural crest expression patterns specific treatments besides symptomatic care. We set out and activity of these putative enhancer elements in to develop a zebrafish model for MFS to address these melanoma. One 669 bp element derived from a region ~23 problems. kb upstream of the sox10 transcriptional start site drives Methods: We used the CRISPR/Cas9 system to induce indel EGFP reporter expression in a subset of embryonic neural mutations in the zebrafish fibrillin-1 ortholog, fbn1. The crest as well as specifically in early melanoma patches and Tg(kdrl:GFP) transgenic line was used to visualize blood tumors. Within this regulatory element, we have identified vessels in embryos, and cardiac ultrasound was used to a region conserved across members of the Cyprinidae family characterize adult mutant zebrafish. that is required in the neural crest and hypothesize it may function as a critical enhancer in driving sox10 expression Results: We generated four novel independent fbn1 mutant during melanomagenesis. We are currently generating lines. In early development no robust cardiovascular CRISPR-mediated deletions of the endogenous element to phenotype was detected, and mutant fish survived normally test its necessity for melanoma initiation and will use dCas9- to adulthood. In adult homozygous fbn1 mutant zebrafish fusions to modulate its activityin vivo. Together, these data however a mild dilated cardiomyopathy was discovered. enhance our understanding of epigenetic changes that drive the re-emergence of neural crest identity in melanoma. Conclusions: Our initial data indicate that fbn1 is not essential for zebrafish development, but plays a role in adult cardiac function. MFS patients also show evidence of cardiomyopathy, although complete loss of fibrillin-1 is ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 72 Poster Abstracts P-106 SNAI1 enhances tumor initiation in Embryonal UMR 7241/INSERM U1050/Collège de France, 75231 Paris Rhabomyosarcoma by inhibitingTP53 expression Cedex 05, France, 4Department of Industrial Chemistry

1 1 “Toso Montanari”, Alma Mater Studiorum – University of Rodrigo Moreno-Campos , Long Wang , Myron S. 5 1 Bologna, Italy, Université Paris Diderot, Sorbonne Paris Ignatius Cité, Biology Department, 75205 Paris Cedex 13, France 1 University of Texas Health Science Center San Antonio, Reactive oxygen species (ROS) and downstream products Greehey Children’s Cancer Research Institute of lipid oxidation are emerging as important secondary Embryonal rhabdomyosarcoma (ERMS) is a pediatric messengers in tissue homeostasis. However their regulation malignancy of the muscle. ERMS is commonly driven and mechanism of action remain poorly studied in vivo by mutations in the RAS family that result in pathway during normal development. Here we reveal that the fine activation. We routinely generate ERMS in zebrafish with regulation of hydrogen peroxide (H2O2) levels through the morphological and molecular characteristics of the degradation by its scavenger Catalase is crucial to mediate human disease by expressing human oncogenic kRASG12D the switch from proliferation to differentiation in retinal under control of the rag2 promoter. In contrast to tumors progenitor cells (RPCs). We further show that altering the of epithelial origin, TP53 is often wild-type in RMS raising levels of downstream products of redox signaling can also the question of how these tumors are able to overcome affect this switch. Indeed, we identify9 -hydroxystearic acid TP53-effects on oncogene driven apoptosis or senescence. (9-HSA), an endogenous downstream lipid peroxidation SNAI1 and SNAI2 are transcription factors highly expressed product, as a mediator of this effect in the zebrafish retina. in ERMS and can reduce TP53 function in some cancers. In fact, embryonic and post-embryonic RPCs exposed to Therefore, we hypothesize a role for SNAI1 and SNAI2 in higher amounts of 9-HSA fail to differentiate and remain the inactivation of TP53 during tumor initiation. proliferative through the activation of Notch and Wnt pathways. We find that 9-HSA exerts its biological function Recently, we found that a NOTCH1/SNAI1 pathway is required in vivo by inhibiting the activity of histone deacetylase 1, to expand self-renewal and also block differentiation in which leads to defects in neurogenesis. We finally show

ERMS. Importantly, during development snai1a and snai2 that the local and temporal manipulation of H2O2 levels are expressed in the developing muscle in zebrafish at by catalase overexpression in RPCs is sufficient to trigger precisely the time that ERMS tumors arise. We tested the their premature differentiation while genetic disruption of effects of kRASG12D versus kRASG12D + SNAI1 or kRASG12D catalase leads to a significant increase in RPC proliferation.

+SNAI2 on tumor initiation. Our analysis finds that SNAI1 Therefore the amount of H2O2 in RPCs influences their ability significantly expands tumor initiation (p<0.001, Log-rank to switch from proliferation to differentiation. We propose

test). SNAI2 also expands tumor initiation, but the effect a mechanism that acts in RPCs to link H2O2 homeostasis is not statistically significant. We assessed Tp53 expression and neuronal differentiation via the modulation of lipid in tumors and find a significant reduction compared to peroxidation. control muscle. Additionally,SNAI1 expressing tumors grow faster in vivo compared to kRASG12D only tumors. To test whether SNAI1 effects on TP53 are transcriptional and/or P-108 Proteasome dysfunction as a mechanism for post transcriptional, siRNA for SNAI1 and inhibitor studies ethanol toxicity in fetal alcohol spectrum disorder using GN-25, an inhibitor of the SNAI1-TP53 proteinprotein interaction and ChIPseq experiments are underway in Olivia Weeks1,2, Brian Pepe-Mooney1,2, Isaac M. ERMS RH-18 cells that have high SNAI1 and wild-type TP53. Oderberg1,2, Matthew Wang2, Wolfram Goessling1,2,3,4

Our preliminary siRNA and inhibitor experiments support 1 2 TP53 Harvard Medical School, Boston, MA, USA, Brigham both transcriptional and postranslation effects on . In 3 summary, our data identify an important role for SNAI1 via and Women’s Hospital, Boston, MA, USA, Harvard-MIT inhibition of TP53 function in ERMS. Division of Health Sciences and Technology, Cambridge, MA, USA, 4Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, 02114, USA P-107 Redox signaling via lipid peroxidation regulates Prenatal alcohol exposure impacts at least 10% of the retinal progenitor cell differentiation global population, and 2 – 5% of U.S. school age children have a diagnosable fetal alcohol spectrum disorder Shahad Albadri1, Federica Naso1,2, Marion Thauvin3, 3 2 1 (FASD). The molecular mechanisms responsible for the Carole Gauron , Carola Parolin , Karine Duroure , Juliette teratogenicity of ethanol are largely uncharacterized, Vougny1, Jessica Fiori2, Carla Boga4, Sophie Vriz3,5, Natalia 2 1,6 and many signaling pathways implicated in FASD remain Calonghi , Filippo Del Bene unidentified. Identifying how ethanol disrupts normal cell 1Institute Curie, PSL Research University, INSERM, U 934, function during development is critical for understanding CNRS UMR3215, F-75005, Paris, France, 2Department FASD and potential therapeutic interventions. In this of Pharmacy and Biotechnology, Alma Mater Studiorum presentation, we describe the novel impacts of embryonic – University of Bologna, Bologna, Italy, 3Centre alcohol exposure (EAE) on the function of the proteasome, Interdisciplinaire de Recherche en Biologie (CIRB), CNRS a complex that is responsible for eliminating damaged or unnecessary proteins in the cell. Using a zebrafish model of ......

..... 73 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts FASD, we demonstrate that EAE causes an accumulation of formed mainly in the zebrafish yolk sac and perivitelline ubiquitinated proteins in developing organs and induces a space following injection in the yolk sac or the duct of compensatory up-regulation of proteasome components to Cuvier. Engraftment rate was 73 % and larvae survival rate deal with the resulting protein stress. Through proteasome was higher when they were injected at 48 hpf compared activity assays, we demonstrate that ethanol and to 24 hpf. Daily image analysis suggested cellular division, acetaldehyde, the primary metabolic product of ethanol as microtumors progressively grew with differentiated metabolism, specifically inhibit the chymotrypsin-like fluorescence intensity signals. At least three division cycles activity of the proteasome, preventing it from degrading for human GBM cells were identified in the dye dilution proteins. If proteasome inhibition plays a key role in ethanol’s assay by flow cytometry. Human cancer cells proliferate in teratogenicity, we would expect proteasome inhibition to zebrafish larvae, making it possible to assess cell divisions exacerbate FASD phenotypes. Pharmacological inhibition and tumor growth. With this approach, it should be of the proteasome with Bortezomib and MG132 treatment possible to test new therapies that alter human GBM cell sensitized fish to EAE, suggesting that the proteasome is proliferation in vivo, to better predict their clinical efficacy a critical regulator of FASD phenotypes. Fetal proteasome and individualize patient treatment. inhibition resulted in organ malformations and short stature phenotypes characteristic of FASD; however, several FASD phenotypes were not significantly impacted by proteasome P-110 The role of a novel microvascular network in cancer dysregulation. This suggests that proteasome inhibition progression and relapse following EAE may be responsible for some, but not all, of the teratogenic effects of ethanol. Fetal organoid modeling Sergei Revskoy1, Margaret Blair1, Jessica Blackburn1

has been deployed to confirm tissue-specific effects of 1 ethanol on proteasome inhibition during development. University of Kentucky, Department of Molecular and Taken together, these data point to the importance of Cellular Biochemistry, Lexington KY protein homeostasis in normal development and identify Angiogenesis provides tumor cells with oxygen and nutrients the proteasome as an important target of EAE. that are essential for cancer expansion and spread. The development of anti-angiogenesis drugs originally held great promise. Most of these target Vascular Endothelial Growth P-109 Xenotransplantation of human glioblastoma Factor (VEGF), which is secreted by the cancer cells, or it’s in zebrafish larvae:in vivo imaging and proliferation receptor VEGFR, thought to be expressed by all endothelial assessment. cells. Yet angiogenesis inhibitors have had limited success

1,3 1,2 in the clinic, and in some instances, cancers treated with Luis A. Vargas-Patron , Nathalie Agudelo-Dueñas , these drugs initially respond but surviving cells grow back Jorge Madrid-Wolff2, Juan A. Venegas3, John M. 3 2 1 more aggressively than before treatment. Why cancer cells González , Manu Forero-Shelton , Veronica Akle that rely on angiogenesis for growth can survive, and even 1Laboratory of Neurosciences and Circadian Rhythms, thrive, when treated with VEGF/VEGFR inhibitors is a cancer School of Medicine, Universidad de los Andes, Bogota, research paradox that has never been fully explained and Colombia, 2Biophysics Group, Department of Physics, has significant clinical implications. Using zebrafish models, Universidad de los Andes, Bogota, Colombia, 3Biomedical we have found a microvascular network that is comprised Sciences Laboratory, School of Medicine, Universidad de of endothelial cells but, surprisingly, is not VEGFR-positive. los Andes, Bogota, Colombia The vessels do not carry erythrocytes, so cannot oxygenate tissues, but are leaky and can deliver nutrients and Glioblastoma (GBM) is the most prevalent type of primary macromolecules that are essential for cell survival. Recent brain tumor. Treatment options include maximal surgical research in cancer stem cells has shown that hypoxia, or resection and drug-radiotherapy combination. However, the lack of oxygen, may create a metabolic program in stem patient prognosis remains very poor, prompting the search cells that promotes self-renewal over differentiation. We for new models for drug discovery and testing, especially hypothesize that this novel, deoxygenated vascular network those that allow assessment of in vivo responses to is an important component of the cancer stem cell niche, treatment. Zebrafish xenograft models have an enormous and anti-angiogenesis inhibitors that kill normal blood potential to study tumor behavior, proliferation and cellular vessels may expand this hypoxic vascular network and interactions. Here, it is introduced an in vivo imaging and increase the cancer stem cell population. The current focus proliferation assessment method of human GBM xenograft of our research is to define the role of this novel vascular in zebrafish larvae. Zebrafish larvae microinjected with network in cancer progression, determine how these fluorescently labeled human GBM cells were screened vessels differ from the canonical VEGFR-positive vessels, daily using a stereomicroscope and imaged by Light Sheet and identify FDA-approved drugs capable of eliminating Fluorescence Microscopy (LSFM); volumetric modeling and these vessels. Overall, this research will define a completely composite reconstructions was done in single individuals. new component of cancer tissue and has the potential to Larvae containing tumors were enzymatically dissociated, have a rapid and important impact in the cancer clinic. and the proliferation of human cancer cells was measured using dye dilution by flow cytometry. GBM microtumors ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 74 Poster Abstracts P-111 Functionalin vivo characterization of the P-112 APOBEC3B Cooperates with BRAFV600E to initiate ribonucleotide reductase subunit M2 (RRM2) as a novel malignant melanoma in the absence of UV

druggable copy number driven dependency gene in 1,2 1 1 neuroblastoma Maurizio Fazio , Erika Weiskopf , Ellen van Rooijen , Julien Ablain1, Leonard Zon1,2 Lisa Depestel1,2, Carolina Nunes1,2, Siebe Loontiens1,2, 1Howard Hughes Medical Institute, Stem Cell Program Emmy Dolman3, Aline Eggermont1,2, Ellen Sanders1,2, and the Division of Pediatric Hematology/Oncology, Givani Dewyn1,2, Christoph Van Neste1,2,4, Jan Molenaar3, Boston Children’s Hospital and Dana-Farber Cancer Kaat Durinck1,2, Frank Speleman1,2 Institute, Harvard Medical School, Boston, MA 02115, 2 1 USA, Department of Stem Cell and Regenerative Biology, Department of Biomolecular Medicine, Ghent University, Harvard Stem Cell Institute, Cambridge, MA 02138, USA Ghent, Belgium, 2Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium, 3Princess Maxima UV-mutagenesis induced by sun exposure is the major Center, Utrecht, The Netherlands, 4Computational environmental risk factor for developing cutaneous Bioscience Research Center (CBRC), King Abdullah melanoma, but more rare subtypes of melanoma, i.e. acral University of Science and Technology (KAUST), Thuwal, and mucosal melanoma, arise in non-sun exposed areas, Saudi Arabia have a worse prognosis and response to therapy, and have an overall lower mutation burden. While these tumors In neuroblastoma (NB), a pediatric cancer of the sympathetic lack a UV mutational signature, a ~6-fold increase in the nervous system, highly recurrent focal and large copy number proportion of APOBEC3-signature mutations is observed alterations affecting chromosome 2p and 17q segments are (1.703% vs. 9.458%, respectively; p<0.0001). APOBEC3- found in almost all high-risk cases. Combined transcriptome/ driven mutagenesis is a recognized driving force observed copy number/survival analysis revealed RRM2 (2p25.1) in over 15% of all human cancers, but its potential role as one of the highest ranked candidate driver genes in in non-UV driven melanoma has not been investigated. MYCN-amplified NB. Given that RRM2 depletion induces To investigate the role of APOBEC3-mutation in human replicative stress and senescence, we explored RRM2 as melanoma, we performed a TMA on 160 melanomas, novel drug target in NB. RRM2 dependency was evident and confirmed APOBEC3B expression in all melanoma by knock down experiments and upon pharmacological subtypes but not in normal skin (n=15), with ~27% (14/51) inhibition with triapine and gemcitabine. A further drug of Acral/Mucosal melanomas showing APOBEC3B protein screen identified CHK1 as synthetic lethal in combination expression. Zebrafish is an excellent model to study this with triapine through excessive replicative stress induced process since fish are not UV-exposed in captivity, and they DNA damage. We identified multiple regulatory pathways lack APOBEC3 cytidine-deaminases but their downstream converging to RRM2 regulation in NB cells, including MYCN, DNA repair pathways are well conserved, thus allowing LIN28B, BRCA1 and WEE1. studying the role of individual APOBEC3 family members. We To investigate the role of RRM2 in NB formation in vivo, we generated the first reportedin vivo model of APOBEC-driven used an established zebrafish model for MYCN-driven NB, in oncogenesis using the MASERATI zebrafish transgenesis which tumors develop from 10 weeks on with a penetrance approach, by testing whether APOBEC3B alone or in of 20% (S. Zhu, et al., Cancer Cell, 2012). Two independent combination with gain of a MAPK oncogene (BRAFV600E, transgenic lines expressing both RRM2 and mCherry were KITK642E), or loss of tumor suppressor tp53 could result created and outcrossed with the MYCN overexpression in tumor initiation. Melanocyte-specific expression of fish. RRM2 overexpression increased penetrance up to APOBEC3B together with BRAFV600E is sufficient to initiate 95% and accelerated NB formation. Increased tumor tumors in vivo (45% penetrance, n=156) with histologically aggressiveness is suggested by transplantation experiments invasive malignant melanoma forming as early as 5 weeks. in immunocompromised fish and observation of metastasis This newly established model offers functional proof that in two double positive fish. Ongoing experiments using APOBEC3B could contribute to the pathogenesis of non- TOL2 transposase-mediated mosaic transgenesis for RRM2 sun-exposed melanoma. Further, it provides a unique tool overexpression in MYCN driven tumors include monitoring to compare the oncogenic potential of different APOBEC3 of effects on DNA damage, apoptosis and transcriptome genes, and will help study the impact of the downstream by gH2AX, cleaved caspase-3 staining and single cell DNA damage repair pathways on APOBEC3B’s role in transcriptome profiling, respectively. In vivo drugging of oncogenesis. neuroblastomas in zebrafish for triapine, chk1 inhibitors and gemcitabine are ongoing while initiated for human PDX samples in mice. P-113 Establishing a model of Ewing Sarcoma in zebrafish In conclusion, this study presents for the first time RRM2 Sarah Grissenberger1, Anna Poetsch1, Monika Heinzl1, as a novel druggable DNA copy number driven dependency Luisa Morelli1, Niko Popitsch1, Nathan Sheffield2, Heinrich gene in NB. Kovar1 & Martin Distel1 1St. Anna Children’s Cancer Research Institute, Zimmermannplatz 10, 1090 Vienna, Austria, 2Center for Public Health Genomics, University of Virginia, ......

..... 75 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts Charlottesville, Virginia, USA melanoma bearing zebrafish. Genetic perturbation of Acetyl-CoA metabolism in the mitochondria via knockdown Ewing sarcoma (EwS) is a malignant bone and soft tissue of Hydroxy-acyl-CoA dehydrogenase (HADHA) or ATP-citrate tumor in children and adolescents. In 85% of all cases the lyase (ACLY) abolishes the octanoic acid-induced histone formation of EwS is caused by a chromosomal translocation, hyperacetylation. ChIP-seq analysis of octanoic acid treated leading to the expression of the oncogene EWS-FLI1. This melanoma cells reveals specific loci that are enriched for H3 aberrant transcription factor is the main driver of the acetylation. Ongoing work aims to test the importance of disease and leads to massive transcriptional deregulation. certain hyper-acetylated genomic loci and FAO regulatory Although the genetic mechanism that drives EwS is well genes using human tissues and a zebrafish melanoma understood, an animal model adequately mimicking the model to reveal new therapeutic targets. Taken together, disease is still lacking. One reason, why modeling attempts this study will increase our understanding of the interplay have remained difficult, is the elusive cell-of-origin in EwS. between lipid metabolism, epigenetic reprogramming and Several cell types including neural crest progenitor cells and melanoma progression, potentially leading to improved mesenchymal stem cells have been proposed, but none of treatment strategies for advanced melanoma. them have been confirmed as cell-of-origin of EwS yet. Zebrafish expressing human EWS-FLI1 in an untargeted way P-115 CK2 hyperactivation overcomes temporal restric- have been reported to develop EwS-like tumors, however tion of MYC-mediated lymphoblast transformation at very low frequency. We reason, that targeting EWS-FLI1 Yun Zhou1,2†, Haiwei Lian2,4†, Sovannarith Korm2, Hui- to the cell-of-origin cell type will greatly enhance tumor ting Leah2, Ning Shen2, Andrew Kwok Ping Lam2, Kevin formation. Towards this goal, we have already established Charles Tang2, David C. Seldin3, Esther Landesman-Bol- a Cre-inducible zebrafish effector strain, harboring human lag3, Hui Fu4, Li Hong1 and Hui Feng2,3 EWS-FLI1. By crossing this strain to different Cre-driver strains, we will target EWS-FLI1 expression to distinct cell 1Department of Gynaecology, Wuhan University Renmin types, including neural crest and mesenchymal stem cells. Hospital, Wuhan, Hubei, P.R. China, 2Department of Pharmacology & Experimental Therapeutics, Boston In addition, we are currently following a novel approach University School of Medicine, MA, USA, 3Department exploring the use of regulatory elements, identified to be of Medicine, Section of Hematology and Medical specifically active in EwS cells, to target EWS-FLI1 expression Oncology, Boston University School of Medicine, MA, in zebrafish to putative cell-of-origin cells. USA, 4Department of Anatomy and Embryology, Wuhan If successful, a zebrafish EwS model will help to understand University School of Basic Medical Sciences, Wuhan, tumor initiation and progression in EwS and furthermore, Hubei, P.R. China†equal contribution will be a valuable tool to develop novel therapeutic Protein kinase CK2 is a constitutively active serine/threonine strategies. kinase that is hyperactivated through gene overexpression in a broad spectrum of human cancers including various P-114 Fatty acids as mediators of epigenetic types of leukemia. Despite the demonstrated anti-leukemic reprogramming in melanoma efficacy of CK2 inhibitors, how CK2 contributes to leukemia development remains incompletely understood. Here we Ting-Hsiang Huang1, Joshua Weiss1,2 and Richard M. generated a zebrafish transgenic line that overexpresses White1 mammalian CK2α gene in developing T lymphocytes. We CK2α 1 found that overexpression of subunit alone failed to Department of Cancer Biology & Genetics, Memorial induce or maintain malignant transformation. However, Sloan Kettering Cancer Center, 417 E68th St., New York, 2 overexpression of wild-type but not enzyme- New York 10065, USA, Tri-Institutional MD-PhD Program, dead CK2α promoted both onset and progression of zebrafish Weill Cornell Medicine Graduate School of Medical MYC-induced leukemia through stabilizing MYC protein. Sciences, 1300 York Ave, New York, New York, 10065, USA Interestingly, MYC-induced leukemogenesis depends on Extrinsic fatty acid uptake is seen in various cancers including developmental stages of animals as single MYC transgenic melanoma. We recently found that melanoma cells directly fish failed to develop disease when MYC was activated take up lipids from stromal adipocytes to fuel proliferation at the later stage of animal development, whereas MYC and invasion. However, the mechanisms by which these activation alone at the embryonic stage was sufficient to extracellular lipids reshape tumor cell fate remains induce leukemic transformation. In CK2α;MYC double unknown. Here, we show that Acetyl-CoA, a metabolite transgenic fish, leukemia was rapidly induced regardless of produced during the process of fatty acid oxidation the animal developmental stage. Collectively, our findings (FAO), plays a key role in rewiring chromatin plasticity via demonstrated non-oncogene addition of CK2 can overcome changes in histone acetylation. Treatment of melanoma temporal restriction of MYC-mediated lymphoblast cells with octanoic acid, a medium chain fatty acid rapidly transformation, providing therapeutic implications. metabolized into Acetyl-CoA, leads to marked increases KEYWORDS: Protein kinase CK2; MYC; leukemia; in global H3 and H4 acetylation. This is accompanied by transformation an increase in tumor burden and shortened survival of ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 76 Poster Abstracts P-116 PHF6 loss drives IL7R oncogene addiction in TLX1 Andrew J. Coombs3, Andrew Young6, Chansey Veinotte5, driven T-ALL Carolyn-Ann Robinson1, Michael N. Ha7, Graham Dellaire4, Todd Druley6, Jason N. Berman1,3, 4, 5*, Craig McCormick1* Siebe Loontiens1, Kaat Durinck1 , Suzanne Vanhauwaert1, Lisa Depestel1, Mariana L. Oliveira2, Givani Dewyn1, 1Department of Microbiology and Immunology, Charles de Bock3, João T. Barata2, David Langenau4, Jan Dalhousie University, 2Beatrice Hunter Cancer Research Cools3, Tom Taghon5, Pieter Van Vlierberghe1, Frank Institute,3 Department of Pediatrics, IWK Health Centre, Speleman1 4Department of Pathology, Dalhousie University 1Center for Medical Genetics, Ghent University, Ghent, 5Faculty of Medicine, Dalhousie University, 6Division of Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Hematology and Oncology, Department of Pediatrics, Belgium, 2Instituto de Medicina Molecular, Faculdade de Washington University School of Medicine, 7Department Medicina, Universidade de Lisboa, Portugal, 3Laboratory of Radiation Oncology, Dalhousie University for the Molecular Biology of Leukemia, Center for Human Genetics, University of Leuven and Center for the Biology Kaposi’s sarcoma associated-herpesvirus (KSHV) is a human of Disease, Vlaams Instituut voor Biotechnologie, Leuven, gammaherpesvirus that establishes life-long latency in B Belgium, 4Molecular Pathology and Cancer Center, lymphocytes. KSHV infection is often asymptomatic but Massachusetts General Hospital, Boston, MA 02129, USA, has been associated with lymphoproliferative diseases and 5Department of Clinical Chemistry, Microbiology and cancers, such as primary effusion lymphoma (PEL), a rare Immunology, Ghent University, Ghent, Belgium HIV-associated lymphoma with poor outcome. The median survival for patients with PEL is approximately six months, The PHF6 gene is frequently targeted by loss-of-function with a one-year survival rate of approximately 40%. mutations or deletions in T-cell acute lymphoblastic Accumulating evidence indicates that KSHV oncogenesis leukemia (T-ALL), with the highest prevalence in TLX1 or requires both (i) aberrant proliferation of latently infected TLX3 rearranged T-ALLs. We investigated the effects of cells and (ii) the contribution of pro-inflammatory and pro- PHF6 knockdown in normal and malignant thymocytes. angiogenic factors elaborated by cells that succumb to lytic Notably, we observed broad effects on the investigated viral replication. There are currently no good preclinical transcriptomes suggesting an important role for PHF6 in animal models for KSHV-associated disease. Here, we gene regulation. Furthermore, IL7R, a known oncogene in report the development of a novel zebrafish xenograft T-ALL, was identified as a common transcriptional target model of PEL, designed to investigate both the utility of that was significantly upregulated upon PHF6 knockdown. this model for drug testing, and the impact of oxygen level Thus, loss of PHF6 might boost oncogenic addiction of on PEL cell proliferation. We demonstrate engraftment leukemic T-cell lymphoblast to IL7 signaling. and proliferation of KSHV+ PEL tumour cells transplanted into zebrafish larvae. Using a PEL cell line engineered to To explore the role of PHF6 inactivation in TLX1 driven produce the viral lytic switch protein RTA in the presence of leukemogenesis in vivo, we performed zebrafish modeling. doxycycline, we show drug-inducible reactivation from KSHV For this, we generated a stable tg(rag2:TLX1, rag2:GFP) latency in vivo. Digital droplet PCR was used as a sensitive overexpressing as well as a phf6 knock out zebrafish line. Thus and specific method to assess changes in viral and host gene far, no leukemia was detected in PHF6 mutated zebrafish. expression in xenografted larvae. Furthermore, to explore The T-ALLs were subjected to RNA- and ATAC –sequencing. the effects of the microenvironment in the larvae, we stably In addition, exome-sequencing was performed to identify knocked down the essential cap-binding protein of hypoxia somatic lesions that cooperated loss of Phf6 during TLX1 translation initiation machinery, eukaryotic initiation factor driven T-cell transformation. Furthermore, additional 4E2 (eIF4E2), and found that hypoxic translation initiation injections of TLX1 in combination with an activating IL7R is essential for engraftment of PEL cells in the yolk sac. This mutant into phf6 mutant zebrafish are currently ongoing has extensive implications, as it is well known that cancer to monitor additional effects on accelerated tumor cell proliferation, migration, induction of angiogenesis, formation. In conclusion, our data suggest that loss of PHF6 and response to drugs can all be influenced by low oxygen drives TLX1 mediated leukemogenesis, at least in part, by levels. This study provides innovative information on both increasing surface IL7R expression. Therefore, we believe the hypoxic microenvironment in host zebrafish and the that increased addiction to oncogenic JAK-STAT signaling biology and progression of KSHV. may render PHF6 mutant leukemic cells more sensitive to JAK inhibitors, a notion that we are currently investigating in our TLX1/PHF6 and TLX1/PHF6/IL7R zebrafish models. P-118 ARID1A loss promotes adrenergic to mesenchymal transition by regulating enhancer-mediated gene expression in neuroblastoma pathogenesis P-117 A novel zebrafish model of Kaposi’s Sarcoma- associated herpesvirus (KSHV) primary effusion Hui Shi1, Ting Tao1, Brian J. Abraham2, Adam D. Durbin1,3,4, lymphoma – a platform for understanding Mark W. Zimmerman1, Cigall Kadoch1,4, A. Thomas Look1,* pathophysiology and drug discovery 1Department of Pediatric Oncology, Dana-Farber Cancer

1,2 1 3 Institute, Harvard Medical School, Boston, MA 02115, USA, Eric S. Pringle , Jaime Wertman , Nicole Melong , 2Department of Computational Biology, St. Jude Children’s ......

..... 77 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts Research Hospital, Memphis, TN, 38105, USA, 3Division and accelerate tumor onset, we are currently focusing on of Pediatric Hematology/Oncology, Boston Children’s inducible Cre-lox based strategies using acinar (ela3l and Hospital, Boston, MA, 02215, USA, 4The Broad Institute, ptf1a) and ductal (ck18, anxa4 and nkx6.1) promoters. Since Cambridge, MA, 02139, USA inflammation enhances tumor formation in mice, weare also adding inflammatory signals through the expression *Correspondence: [email protected] of Il17a and Tgfα or intraperitoneal injection (IP) of the Gene mutations of components of SWI/SNF (mSWI/SNF cholecystokinin analogue ceruletide. Preliminary data or BAF) chromatin-remodeling complexes are found in show that inflammation induced by ceruletide can result approximately 20% of all human cancers, and ARID1A is in acinar-to-ductal-metaplasia, an important route to PDAC the most frequently mutated subunit. In neuroblastoma, initiation in rodents. Finally, we have performed a chemical sequence alterations of ARID1A were identified in6% screen to identify compounds that cause pancreatic damage of cases. In addition it is deleted on one allele in at least to accelerate tumor onset. We anticipate that combining 86.95% of cases with chromosome 1p deletions, which is the these approaches will further establish the zebrafish as a most common deletion found in high risk neuroblastomas. model in pancreatic cancer research. However, the mechanisms by which ARID1A depletion promotes neuroblastoma pathogenesis are unclear. Here, we show that zebrafish arid1aa or arid1ab deficiency P-120 A chemical genetic screen identifies a CBFβ accelerates the onset and increases the penetrance inhibitor as an expander of hematopoietic stem cells of MYCN-induced neuroblastoma by increasing cell during embryogenesis.

proliferation in the sympathoadrenal lineage. Depletion of 1 1 ARID1A in NGP neuroblastoma cells promotes adrenergic to Anne L. Robertson , Joseph Mandelbaum , Caroline Kubaczka2, Rebecca J. Freeman1, Megan C. Blair1, Rachel mesenchymal transition by regulating enhancer-mediated 1 2 3 gene expression through altering genomic occupancies E. Henderson , George Q. Daley and Leonard I. Zon

of two distinct mSWI/SNF assemblies, BAF and PBAF 1 complexes. Our work establishes the first in vivo ARID1A Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital and Harvard Medical School, deficiency zebrafish neuroblastoma model, which can be 2 used for synthetic lethality screen for drugs that specifically Boston, MA 02115, USA, Stem Cell Program and Division kill ARID1A deficiency neuroblastoma cells. of Hematology/Oncology, Boston Children’s Hospital, Dana Farber Cancer Institute, Harvard Stem Cell Institute and Harvard Medical School, Boston, MA 02115, USA, 3Stem Cell Program and Division of Hematology/Oncology, P-119 Modelling pancreatic ductal adenocarcinoma in Boston Children’s Hospital, Dana Farber Cancer Institute, zebrafish Howard Hughes Medical Institute, Harvard Stem Cell Thijs van Boxtel1, Theresa Simon-Vermot1, Gokçe Askan2, Institute and Harvard Medical School, Boston, MA 02115, Isabelle Kim1, Steven Leach3, Richard White1 USA 1Cancer Biology & Genetics, Memorial Sloan Kettering ESCs and iPSCs can be directed to differentiate in vitro Cancer Center, New York, NY, United States, 2David M. into many different cell types, but it is not yet possible Rubenstein Center for Pancreatic Cancer, Memorial Sloan to generate HSCs. To find new ways to expand HSCs, we Kettering Cancer Center, New York, NY, United States, developed a zebrafish embryo culture system in which 3Norris Cotton Cancer Centre, One Medical Center Drive, Runx1:GFP embryos are dissociated into single cells at 24 Lebanon, NH, United States hpf, prior to HSPC emergence. We screened 3,840 bioactive small molecules and identified 21 that increased Runx1:GFP Pancreatic ductal adenocarcinoma (PDAC) is currently cells after 48 hours in culture, including inhibitors of the the third leading cause of cancer-related deaths in the Runx1 cofactor, CBFβ. Using an in vivo imaging approach, United States. Due to the frequent late discovery of the we showed that treatment of zebrafish embryos from 24 disease the 5-year survival rate is only 9% with a median hpf with the CBFβ inhibitor Ro5-3335 led to expansion of survival of just six months. Improvements in detecting Runx1:GFP cells in the CHT by 54 hpf (mean number of and treating this disease will require models which allow Runx1:GFP cells = 11.45, compared to 4.85 in DMSO control for unbiased discovery of new pathways and drugs that treated embryos). Further analyses using other fluorescent target both tumor and the microenvironment. To improve reporter lines, in combination with in situ hybridization existing zebrafish PDAC models, we developed a pancreas for multiple markers of blood cell lineages, suggests that specific Crispr-Cas9 vector system to knock out the most continuous treatment of embryos with Ro5-3335 blocks the frequently mutated genes in human PDAC; tp53, cdkn2a/b differentiation of myeloid and lymphoid cells. Preliminary and smad4a. Mosaic knock out of these genes in pancreatic studies indicate that withdraw of Ro5-3335 releases the acinar cells, combined with expression of activated KRAS cells to enable differentiation to proceed. In human iPSC- (KRASG12V), induced mixed acinar- and ductal tumors at derived hemogenic endothelial cells, Ro5-3335 increases around 6 months with low efficiency. The ductal component CD34+ cells, while limiting acquisition of CD45 positivity. of the tumors showed a striking histological resemblance We also observed an increase in Runx1 expression in these with mouse- and human tumors. To improve penetrance cells using a fluorescent reporter line. Taken together, our ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 78 Poster Abstracts cross-species data suggest that Ro5-3335 functions to Siebe Loontiens1,2*, Lisa Depestel1,2*, Givani Dewyn1,2 maintain HSCs in a stem-cell state, allowing for additional , Niels Vandamme3,4 Volodimir Olexiouk1,2, Jo divisions to expand the stem cell pool. These studies will Vandesompele1,2, Kaat Durinck1,2 , Yvan Saeys3,4 Suzanne provide new insight towards the ultimate goal of generating Vanhauwaert1,2, Frank Speleman1,2

patient-specific HSCs from iPSCs for the treatment of blood 1 diseases and cancers. Center for Medical Genetics, Ghent University, Ghent, Belgium, 2Cancer Research Institute Ghent (CRIG), Ghent, Belgium, 3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, P-121 Development of a vestibular schwannoma xenograft zebrafish model for In vivo antitumor drug 4Molecular and Cellular Oncology Lab, VIB-UGent Center screening for Inflammation Research, Ghent, Belgium HyunJin Lee, MD, In Seok Moon, MD, PhD Neuroblastoma (NB) is a copy-number driven pediatric cancer arising from the sympathetic nervous system. Highly The development of a simple, reliable, and cost-effective aggressive NBs often present with large 1p deletions, animal model greatly facilitates disease treatment. We distal 2p gains in addition to MYCN amplification and 17q aimed to establish a rapid, simple, and reproducible live gain. A zebrafish model recapitulating the human disease zebrafish vestibular schwannoma xenograft model for was generated by the Look lab through overexpression antitumor drug screening. of MYCN in the zebrafish early sympathetic lineage with Methods: tumors arising in the interrenal gland from 10 weeks post fertilization (wpf) with a penetrance of around 20%. One We optimized each of the following conditions for tumor of the advantages of this tumor model is the relative easy cell xenografts in zebrafish larvae: larval stage, incu bation access to GFP-positive tumor cell throughout the different temperature, and injected cell number. We used NF2 mouse stages of tumor formation. We have previously established Schwann ( cells and generated mCherry fluorescent protein gene expression profiles on early hyperplastic lesions at expressing cells prior to injection into zebrafish larvae. 1 and 2 weeks after birth in mice which allowed to study SC4 cells were counted using a fluorescence microscope, the dynamic changes in the transcriptome during the early sus pended in 10% fetal bovine serum, and injected into steps of tumor formation. Here we present a validated the center of the yolk sac using a microinjection system. work-flow for dissection of early tumour formation by The injected embryos were transferred to E3 medium bulk and single cell RNA-sequencing. In addition to bulk (for zebrafish embryos), and subsequent tumor formation sequencing, we anticipate that single cell RNA sequencing was observed by fluores cence microscopy over a 5 day will yield further insights in the intratumoral heterogeneity period. To validate our model, xenografted embryos were arising in these tumours. GFP-sorted cells were obtained transferred into 6 well plates (5embryos per well) and from tg(dβh:MYCN;dβh:GFP) and tg(dβh:GFP) zebrafish treated with everolimus, a known antitumor drug. at different time points. Cells were collected 5 days and 6 weeks post fertilization. In addition, for the MYCN zebrafish Results: line, cells were also collected from full blown tumours. mCherry fluorescent protein expressing SC4 cells were Comparing the transcriptome of MYCN developing tumours successfully grafted into the yolk sacs of zebrafish at different time points during development with dβh:GFP zebrafish cells will aid in identifying genes and pathways embryos without any immunosuppressant treatment. At up or downregulated upon induced MYCN expression 2 days postinjection, the xenografted cells had grown into thus further expand our knowledge on the transcriptome tumor masses. The optimal speed of tumor formation changes during neuroblastoma development, progression depended on the larval stage (30 hpf), incubation and the heterogeneity within the arising tumour population temperature (31 8 C), and injected cell number (200 cells). and provide a powerful tool for cross species analysis of In preliminary tests, everolimus treatment yielded a 20% neuroblastoma gene expression data sets. reduction in the number of SC4 cells in the yolk. Conclusion: P-123 HMX1 interacts with UHRF1, an epigenetic Our in vivo model has the potential to greatly facilitate coordinator between DNA methylation and histone vestibular schwannoma Younes El Fersioui1, Gaetan1 Pinton, Nathalie Allaman- treatment because of its speed, simplicity, reproducibility, Pillet1, Daniel F. Schorderet1, 2,3 and amenability to live imaging. 1 Institute for Research in Ophthalmology,2 Faculty of Biology and Medicine, University of Lausanne, 3Faculty of Life Sciences, EPFL, Lausanne, Switzerland P-122 A validated work-flow for dissection of early tumour formation by bulk and single cell RNA-sequencing H6 family homeobox 1 (HMX1) belongs to the homeobox in a MYCN driven zebrafish model for neuroblastoma (HMX) family of transcription factors and is widely expressed ......

..... 79 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts in the eye, peripheral ganglia and branchial arches. In in regard to the difficulty to study the gene in mice due to Zebrafish eye formation, hmx1 expression is gradually the high prenatal lethality of severe mutations. In order to restricted to the nasal part of the ganglion cell layer. A further study the role of CWC27 in the course of RP and mutation in HMX1 is linked to a subset of ocular defected open up the opportunity of high throughput screening, we termed Oculo-auricular syndrome of Schorderet-Munier- developed several models of cwc27 mutants. Franceschetti (OAS) (MIM #612109). HMX1 presents a philogenetically conserved 60-amino acid homeobox Methods domain (HD) and two smaller domains called SD1 and SD2. We developed several zebrafish models using CRISPR- HMX1 contribution to the eye formation and generally Cas9 mediated mutation on CWC27 only ortholog. We to craniofacial development remains unclear, therefore performed visual acuity testing on different mutants we aimed at identifying the molecular mechanisms and using optokinetic reflex (OKR). We analysed the retina the biological processes in which HMX1 is involved. Using structure at 5dpf and in adult fish with chemical staining a predictive promoter model we were able to identify and immunohistochemistry labelling. We also analysed the Ubiquitin-like Containing PHD and RING Finger Domains 1 cartilaginous structure of the larvae. Finally we measured gene (UHRF1) as candidate gene common to mouse and cwc27 mRNA level in mutants. human genomes. UHRF1 acts as an epigenetic coordinator Results between DNA methylation and histone. UHRF1 and its partner proteins actively regulate chromatin modifications Here we present a new zebrafish model for retinal and cellular proliferation. degeneration with either a missense or a nonsense mutation in cwc27. Cwc27 mutant’s retinas are dystrophic at 5dpf To investigate whether HMX1 would affect human UHRF1 and in the adulthood in both models. OKR reveals that promoter activity, we placed a luciferase reporter under the 5dpf larvae have decreased visual acuity in the most severe control of the UHRF1 promoter and analyzed the interaction mutation case. Photoreceptors in mutants are altered; between HMX1 and UHRF1. UHRF1 promoter activity therefore they offer possibilities to further understand the was significantly inhibited by wildtype (WT) HMX1, while role of cwc27 in the development and physiology of the deleted HD, SD1 and SD2 regions, exhibited no inhibitory retina. effects on UHRF1 promoter. We then investigated the potential activity exerted in vivo by HMX1 on uhrf1 in a Hsp70-hmx1 Zebrafish transgenic line P-125 In vivo identification of active drugs for refractory carrying the human HMX1 coding sequence downstream of neuroblastoma using zebrafish models the hsp70 promoter. In response to the overexpression of Shuning He1, Megan W. Martel1, Monica Aves1, Daniel HMX1 we observed reduced expression of Uhrf1 at 1 dpf 1 1 1 confirming that is a target of HMX1. Debiasi , Mark W. Zimmerman , and A. Thomas Look 1 Uhrf1 and its partner, Dnmt1, are implicated in regulating Department of Pediatric Oncology, Dana-Farber Cancer lens development as well as regulating DNA methylation Institute, Harvard Medical School, Boston MA, 02115, USA process. Investigating the link between HMX1 and UHRF1 Neuroblastoma is a high-risk pediatric solid tumor that may help to better characterize the OA-SMF syndrome. accounts for 15% of childhood cancer deaths. Most children with neuroblastoma respond initially to chemotherapy, but a large proportion will experience therapy-resistant P-124 Zebrafish model for CWC27 mutation relapse. A recent international study found that in 80% of

1 1 1,2,3 cases of relapsed neuroblastoma, the tumors have somatic Linda Bapst-Wicht , Gaëtan Pinton , Daniel Schorderet mutations that activate RAS-MAPK signaling, including loss 1IRO-Institute for Research in Ophthalmology, Sion, of the NF1 tumor suppressor or activating mutations of Switzerland, 2EPFL-Faculty of Life Science, Swiss Federal the ALK, NRAS, HRAS, KRAS, BRAF or PTPN11 oncogenes. Institute of Technology, Lausanne, Switzerland, 3UNIL- Thus, upregulation of RAS-MAPK signaling through Dept. of Ophthalmology, University of Lausanne, multiple mechanisms poses a major barrier to the cure of Lausanne, Switzerland disseminated neuroblastoma. To identify novel effective therapies for these devastating diseases, we developed CWC27 also called SDCCAG10 is a recently characterized zebrafish models of refractory neuroblastoma, in which gene, coding for a protein member of the cyclophilin family RAS-MAPK signaling hyperactivation makes MYCN-driven and playing a role in the pre-mRNA splicing. In human, neuroblastomas resistant to isotretinoin. These zebrafish homozygous and compound heterozygous CWC27 mutations develop aggressive neuroblastoma with up to 80% tumor were identified in patients with retinitis pigmentosa with penetrance by 3 weeks of age, ideal for the rapid analysis or without skeletal anomalies (OMIM: 617170). In mouse, of small-molecule drugs by adding the drugs to fish water. we observed in the KO mice, significant embryonic lethality Using these faithful preclinical models of refractory and severe dystrophic retina in the only survivor. Milder neuroblastoma, we are analyzing libraries of FDA-approved mutation led to decreased ERG response and thinning of drugs and drugs currently in clinical trials by simultaneously the retina. We are interested in building a zebrafish model assessing drug toxicity to normal tissues and activity against ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 80 Poster Abstracts primary neuroblastoma tumors in juvenile zebrafish with P-127 Zbtb11 is required for liver development and is primary neuroblastoma. To our knowledge, this is the targeted by HNF4a first in vivo drug screen against primary tumors. Inour Shin Yi Tin1, Joly Kwek1, Mia de Seram1, Graham preclinical testing to date of small molecule inhibitors, the 1* 1,2* most active drug is entrectinib, a receptor tyrosine kinase Lieschke , M Cristina Keightley inhibitor that is active against NTRK1/2/3, ALK and ROS1. 1Australian Regenerative Medicine Institute, Monash We are currently testing this drug in combination with other University, VIC Australia, 2La Trobe Institute for Molecular drugs including isotretinoin and CDK4/6 inhibitors. Science, La Trobe University, VIC Australia Zbtb11 is a transcriptional repressor important for P-126 A new technique to biopsy benign and malignant haemopoiesis and is one of the most underexpressed zebrafish lymphocytes genes in hepatocellular carcinoma. In the zebrafish zbtb11 mutant, marsanne (mne), smaller liver primordia were Gilseung Park1, Clay Allen Foster2, Megan Malone-Perez3 observed compared to WT at 48 hpf. To determine when and J. Kimble Frazer1,3,4 these defects are first detectable in zbtb11 mutants we examined expression of hepatic markers by WISH. The 1Departments of Cell Biology, University of Oklahoma 2 earliest expressed genes associated with specification in Health Science Center, OK, USA, Departments of liver are hhex and prox1. In a mne heterozygous incross both Microbiology and Immunology, University of North 3 genes were expressed similarly in all embryos, indicating Carolina, Chapel Hill, NC USA, Departments of Pediatrics, Zbtb11 is not required for specification of hepatoblasts. University of Oklahoma Health Science Center, OK, USA, 4 Next, we examined expression of the differentiation marker, Departments of Microbiology & Immunology, University fatty acid binding protein, 10a liver (fabp10a), using the of Oklahoma Health Science Center, OK, USA Tg(fabp10a:dsRed, ela3l:GFP) line. Expression of fabp10a is Over the last few decades, zebrafish (Danio rerio) have significantly decreased at 48 and 56dpf and is almost absent emerged as a compelling model to study vertebrate by 96 hpf in mne compared to WT suggesting a defect in lymphocytes, as well as diseases of malignant lymphocytes. liver bud outgrowth. By 48 hpf mne undergoes organism- Zebrafish have a similar adaptive immune system that wide cell cycle arrest suggesting a proliferative defect includes B and T cells, like humans, which has allowed could underpin diminished liver bud outgrowth. By 96 hpf many zebrafish T-cell acute lymphoblastic leukemia (T-ALL) (prior to hepatic outgrowth at 5 dpf) foxa1 expression is models to be created, including human MYC (hMYC) decreased and hnf4a is virtually absent indicating there may transgenic fish. Recently, we described a new zebrafish also be a potential defect in differentiation. We examined B-cell ALL (B-ALL) model in double-transgenic rag2:hMYC, Zbtb11 expression by WISH and found that it is ubiquitously lck:GFP zebrafish. In these fish, human MYC (hMYC), an expressed during the onset of hepatic specification (22 oncogenic transcription factor, is regulated by a zebrafish hpf). To determine if Zbtb11 plays a direct role in liver rag2 promoter expressed by immature T and B cells, and the development, the major hepatic regulator HNF4a was co- zebrafishlck promoter drives different levels of GFP in T and expressed with a human -2.9kb-ZBTB11-luciferase reporter. B cells. Thus, in rag2:hMYC, lck:GFP fish, non-malignant T In hepatocellular carcinoma HepG2 cells but not in non- and T-ALL cells are brightly fluorescent, whereas B and B-ALL hepatic 293 HEK cells HNF4a represses ZBTB11 reporter cells are dimly fluorescent. Studies of D. rerio lymphocytes activity, placing ZBTB11 in the hepatic transcription factor or zebrafish ALL typically require euthanizing animals to hierarchy as a conserved direct target of HNF4a. This obtain lymphocytes. However, it would be advantageous suggests that despite its ubiquitous expression, Zbtb11 has if we could obtain lymphocytes without euthanasia. To a particular role in liver development. Studies are ongoing permit this, we have developed techniques to biopsy living to elucidate the mechanism underlying Zbtb11 action in the fish and obtain normal lymphocytes and ALL cells from liver and its contribution to hepatocellular carcinoma. various body regions, such as thymus, kidney-marrow, and even scales. In rag2:hMYC, lck:GFP fish, we have found that both B and T cells can be obtained from thymus, and B cells P-128 High fat diet induces microbiota-dependent are abundant in scales. We have also recovered ALL cells silencing of enteroendocrine cells from scale biopsies of fish with T-ALL. Expression analyses Lihua Ye1,2, Olaf Mueller1, Rodger A. Liddle2, and John F. of non-malignant lymphocytes and ALL cells from different 1,2 biopsy regions demonstrate that GFPhi cells from thymus Rawls lo and scales express T cell genes, and GFP cells express B 1 Department of Molecular Genetics and Microbiology, cell genes. These novel biopsy methods will now permit Duke Microbiome Center, Duke University School of new studies using ex vivo samples obtained from live fish Medicine, Durham, NC, 2 Division of Gastroenterology, without euthanasia. Department of Medicine, Duke University School of Medicine, Durham, NC Enteroendocrine cells (EECs) are specialized sensory cells in the intestinal epithelium that can sense and ......

81 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts transduce nutrient information. Consumption of dietary fat upon its interaction with LSD1. We are currently generating contributes to metabolic disorders, but EEC adaptations to lsd1 mutant zebrafish as well as heat-shock inducible Gfi1b high fat feeding were unknown. Here, we established a new animals that are able or unable to bind Lsd1 to assess the experimental system to directly investigate EEC activity in importance of this interaction in erythropoiesis in vivo. vivo using a zebrafish reporter of EEC calcium signaling. Our results reveal that high fat feeding alters EEC morphology and converts them into a nutrient insensitive state that is P-130 AIBP-mediated cholesterol efflux instructs coupled to endoplasmic reticulum (ER) stress. We called hematopoietic stem and progenitor cell fate this novel EEC adaptation “EEC silencing”. Gnotobiotic studies revealed that germ-free zebrafish are resistant to Qilin Gu1, Xiaojie Yang1, Jie Lv1, 2, Jiaxiong Zhang1, 5, Bo Xia1, high fat diet induced EEC silencing. High fat feeding altered 2, Jun-dae Kim1, Ruoyu Wang6, 7, Feng Xiong6, Shu Meng1, gut microbiota composition including enrichment of Thomas P. Clements8, Bhavna Tandon8, Daniel S. Wagner8, Acinetobacter species, and we identified an Acinetobacter Miguel F. Diaz9, Pamela L. Wenzel9, Yury I. Miller10, David sp. sufficient to induce EEC silencing. These results reveal Traver11, John P. Cooke1, 4, 13, Wenbo Li6, 7, Leonard I. Zon12, a new mechanism by which dietary fat and gut microbiota Kaifu Chen1, 2, 4, 13, Yongping Bai5, and Longhou Fang1, 3, 4, 13, modulate EEC nutrient sensing and signaling. 1 Center for Cardiovascular Regeneration, 2 Center for Bioinformatics and Computational Biology, Department of Cardiovascular Sciences, 3 Department of Obstetrics and P-129 Understanding the GFI1B-LSD1 axis in zebrafish Gynecology, 4 Houston Methodist Institute for Academic hematopoiesis Medicine, Houston Methodist Research Institute. 1 1 1 Houston Methodist, 6550 Fannin St, Texas 77030, USA, Mattie Casey , David McClellan , Rodney Stewart , 5 Michael Engel2 Department of Geriatric Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China, 1Department of Oncological Sciences, Huntsman Cancer 6 Department of Biochemistry and Molecular Biology, Institute, University of Utah, Salt Lake City, UT, USA, UTHealth McGovern Medical School, 7 Graduate School 2Department of Pediatrics, Huntsman Cancer Institute, of Biomedical Sciences, University of Texas MD Anderson University of Utah, Salt Lake City, UT, USA. Cancer Center and UTHealth Houston, Texas 77030, USA, 8 Department of BioSciences, Rice University, Houston, Texas In the United States, the annual cost of red blood cell (RBC) 77005, USA, 9 Children’s Regenerative Medicine Program, and platelet infusions exceeds 10 billion dollars. RBCs Department of Pediatric Surgery, Center for Stem Cell and and platelets are generated during hematopoiesis from Regenerative Medicine, The Brown Foundation Institute a megakaryocyte-erythrocyte progenitor (MEP), which of Molecular Medicine University of Texas Health Science activates a conserved transcriptional program that drives Center at Houston, Texas 77030, USA, 10 Department of the differentiation of MEPs into either an erythrocyte or Medicine, 11 Division of Biological Sciences, Department of megakaryocyte. Integral to this program is Growth Factor Cellular and Molecular Medicine, University of California, Independence (GFI)1B, a transcriptional repressor that San Diego, La Jolla, California 92093, USA, 12 Stem Cell recruits modifiers to regulate gene expression in MEP Program and Division of Hematology/Oncology, Boston cells to drive differentiation of one lineage over another. Children’s Hospital and Dana-Farber Cancer Institute, The mechanism(s) through which GFI1B regulates cell fate Howard Hughes Medical Institute, Harvard Stem Cell decisions remain incompletely understood, hampering Institute, Harvard Medical School, Boston, Massachusetts efforts to modulate this axis to produce RBCs and platelets 02115, USA , 13 Department of Cardiothoracic Surgeries, in vivo. We have taken a loss of function approach in Weill Cornell Medical College, Cornell University, New York zebrafish using CRISPR/Cas9 to generate a gfi1b mutant 10065, USA line. This gfi1b mutant line deletes a portion of the repressive (SNAG) domain that is required for lysine specific Hypercholesterolemia, the driving force of atherosclerosis, demethylase (LSD)1 binding, however gfi1b mutants do not accelerates the expansion and mobilization of display obvious erythroid or megakaryocyte defects, likely hematopoietic stem and progenitor cells (HSPCs). The because zebrafish have two other Gfi family genes, gfi1aa molecular determinants connecting hypercholesterolemia and gfi1ab. In order to assess potential compensatory with hematopoiesis are underexplored. Here we report mechanisms, we generated gfi1aa and gfi1ab mutant that a novel somite-derived pro-hematopoietic cue, zebrafish lines using CRISPR/Cas9 and are generating triple AIBP, orchestrates HSPC emergence from the hemogenic gfi1aa, gfi1ab and gfi1b mutant zebrafish to assess the role endothelium, a type of specialized endothelium manifesting of Gfi family members in promoting erythropoiesis and hematopoietic potential. Mechanistically, AIBP-mediated megakaryopoiesis in vivo. In addition to ourin vivo work, we cholesterol efflux activates endothelial Srebp2, the master have utilized a human cell line which mimics the bipotency transcription factor for cholesterol biosynthesis, which of MEPs to assess the dependency of GFI1B protein partners transactivates Notch and promotes HSPC emergence. to drive an erythrocyte fate as opposed to a megakaryocyte Srebp2 inhibition impairs hypercholesterolemia-induced fate. We have shown that GFI1B-mediated transcriptional HSPC expansion. Srebp2 activation and Notch upregulation repression, as well as erythroid differentiation depend are associated with HSPC expansion in hypercholesterolemic ......

12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 82 Poster Abstracts human subjects. Genome-wide ChIP-seq, RNA-seq, and subsequent role for these DCs during graft rejection. ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline a novel AIBP-regulated Srebp2-dependent paradigm P-132 Investigating the role of ezh1 in developmental for HSPC emergence in development and HPSC expansion hematopoiesis in atherosclerotic cardiovascular disease. Rebecca Soto1, Edroaldo Lummertz da Rocha2, Paul Wrighton3, George Q. Daley1,4, Wolfram Goessling3, Trista P-131 Distinct order of appearance of CD8+ dendritic cells E. North1 during T cell activation vs. T cell development 1Stem Cell Program, Boston Children’s Hospital, Aya Ludin1, Sahar Tavakoli1, Alexander Afeyan1, Song Harvard Medical School, Boston, MA, 2Laboratory of Yang2, Asher Lichtig2, James Sefton1, Wyatt D. McCall1, Immunobiology, Department of Microbiology, Immunology Meredith E. Stanhope1, Georgia L. Stirtz1, Louise and Parasitology, Federal University of Santa Catarina, Bonnelykke-Behrndtz1,3, Asaf Tal, Friedrich G. Kapp1,4, Florianópolis, Brazil, 3Genetics Division, Brigham and Douglas S. Richardson5, Yi Zhou2 and Leonard I. Zon1,2,6 Women‘s Hospital, Harvard Medical School, Boston, MA, 4Harvard Stem Cell Institute, Cambridge, MA 1Harvard Stem Cell Institute, Stem Cell and Regenerative Biology Department, Harvard University, Boston, MA, Understanding how hematopoietic stem cells (HSCs) 2Stem Cell Program and Division of Hematology/Oncology, are produced and maintained is essential to the goal of Boston Children’s Hospital and Dana-Farber Cancer generating patient-specific HSCs capable of multi-potent Institute, Boston, MA 02115, USA,3 Department of Plastic long-term function. HSCs are specified in select arterial and Reconstructive Surgery, Aarhus University Hospital, niches during embryonic development, however the Aarhus, Denmark, 4 Department of Pediatric Hematology pathways regulating their emergence remain incompletely and Oncology, Center for Pediatrics, Medical Center- defined. Recently, an in vitro loss-of-function screen in University of Freiburg, Faculty of Medicine, University of human hematopoietic progenitors identified the Polycomb Freiburg, Freiburg, Germany, 5Harvard Center for Biological group protein, Enhancer of Zeste 1 (EZH1), as a regulator Imaging, Department of Molecular and Cellular Biology, of definitive hematopoiesis. To evaluate whether this Harvard University, Cambridge, MA 02138, USA, 6Howard observation was conserved in vivo, functional knockdown Hughes Medical Institute, Harvard medical school, Boston techniques were employed in zebrafish. Initial analyses MA using in situ hybridization indicate that morpholino- mediated ezh1 deficiency promotes expression of the CD8+ dendritic cells (DCs) are potent antigen cross- conserved HSC markers runx1/c-myb in the ventral wall presenting cells that activate cytotoxic T cells. They have of the dorsal aorta (VDA) at 36 hours post fertilization; also been implicated in thymic central tolerance, but the these studies were confirmed and quantified by qPCR timing of their proliferation during T cell development has and FACS, indicating enhanced HSPC number. Strikingly, not been reported. Moreover, these cells have not yet been ezh1 knockdown increased hemogenic endothelium at the reported in the zebrafish. In this study, we identified CD8+ expense of arterial endothelium without disrupting initial DCs in our newly created CD8α:GFP transgenic zebrafish. arterial/venous specification, suggesting ezh1 expression These cells appeared in the myeloid population of the is spatiotemporally restricted within the endothelium. thymus and kidney of adult zebrafish. They had a dendritic As Notch signaling has been implicated in both arterial morphology and expressed ifr8 and batf3 that are essential specification and HSC emergence, we examined the for CD8+ DC development and function. The CD8+ DCs also potential role of Notch signaling in ezh1 knockdown- presented cross presentation abilities by ovalbumin uptake mediated HSPC expansion and observed differential and membrane presentation. We utilized this model to regulation of Notch ligands and receptors throughout study CD8+ T cell and DC population dynamics during T cell the endothelial-to-hematopoietic transition compared to development and graft rejection. During T cell development, wild-type siblings. Notably, the effect on Notch signaling CD8-GFP+ DCs first appeared in the thymus medulla at the was specific to ezh1 knockdown, as ezh2 knockdown peak of CD8+ T cell expansion, at 2 months of age. During shows a distinct pattern and temporal impact. This strong subsequent development, the DCs multiplied and created conservation of function will serve as a platform to identify nets throughout the medulla when the zebrafish reached chemical or genetic factors that regulate ezh1 expression adulthood at 4 months old. This was contrary to their and function to unlock multi-lineage HSC fate. appearance during graft rejection, where they participate in T cell activation. CD8+ dendritic cells were found in sites transplanted non-matched muscle cells in adult zebrafish, P-133 Metabolomic profiling of hematopoiesis in the 3 days post transplantation. There, they precede activated normal and hyperglycemic vertebrate embryo CD8+ T cells that expressed perforin and Interferon-gamma that appeared 5 days post transplantation. Our findings Timothy Long1, Jenna Frame1, Eleanor Meader1, Olivia reveal the existence of conserved CD8+ DCs in zebrafish and Weeks2, Isaac Oderberg2, Wolfram Goessling2,3,4, Trista sheds light on early T cell-dendritic cell dynamics during T North1,3,4 cell development in the juvenile thymus, and illustrate a ......

..... 83 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts 1Stem Cell Program, Boston Children’s Hospital, Boston, for isomerizing methylmalonyl-CoA to succinyl-CoA. MA, 2Brigham and Women’s Hospital, Boston, MA Using genome editing, we generated zebrafish carrying a 3Harvard Medical School, Boston, MA, 4Harvard Stem Cell loss-of-function variant in mut (c.326_335del, p.Y109*). Institute, Cambridge, MA Homozygous mut-/- zebrafish recapitulated several key clinical findings observed in patients with MMA, such as Hematopoietic stem cells (HSCs), which maintain all increased methylmalonic acid at 15 days post fertilization differentiated cell types of the blood system for the (dpf) (p<1.0e-4) and failure to thrive past 7 dpf (affected fish lifetime of a vertebrate organism, are first specified during were 62% the length of unaffected fish, p<1.0e-4). mut-/- embryogenesis. We have observed that embryonic glucose zebrafish presented with significant developmental delays levels increase precisely as HSCs are first being produced. in larval fin differentiation, swim bladder segmentation, Previous work by our laboratory has shown that elevations melanogenesis (z-score -4.95 as compared to unaffected in glucose metabolism accelerate the onset and increases clutchmates, p<1.0e-4) and exhibited 100% mortality the magnitude of HSC formation. Embryonic treatment of after 22 dpf, reflecting the physiological consequence zebrafish with glucose from 12-120 hours post fertilization of elevated metabolites and resultant mitochondrial results in expansion of HSC marker CD41+ progenitor cells dysfunction. Hepatocytes from the MMA zebrafish (15 dpf) in the adult kidney marrow by FACS analysis. Likewise, were studied using transmission electron microscopy and leptin receptor (lepr) mutants, which have physiological were noted to uniformly contain pale mitochondrial matrix, elevated levels of glucose during embryogenesis, have a which recapitulates the findings seen in liver biopsies larger proportion of cells in the lymphoid compartment, from mut MMA patients. To examine the contribution where HSCs also reside, compared to wildtype siblings. of skeletal muscle metabolism to the lethal phenotype To better characterize the specific energetic pathways observed in the mut-/- zebrafish, we generated a transgenic utilized during hematopoietic development, whole embryo zebrafish with muscle-specific expression of human polar metabolomics was performed on zebrafish treated MUT (hMUT) under control of muscle specific promoter with glucose at various timepoints coinciding with HSC 503unc Tg(503unc:hMUT;cmlc2:GFP). hMUT expression production, formation, and differentiation. Upregulated was demonstrated by RT-PCR performed on whole fish nucleotide metabolism was observed in glucose-treated lysates. Preliminary data on mut-/- zebrafish expressing embryos compared to wildtype controls. In addition, the Tg(503unc:hMUT+) demonstrated improved survival at 22 malate-aspartate shuttle, a key transporter of the reducing dpf when compared to mut-/- zebrafish lackinghMUT rescue agent NADH, was heavily altered in glucose-treated (p<1.0e-5). Zebrafish models of MMA provide a distinct and embryos during the time of HSC production. Consistent complementary set of tools to study the phenotypic and with these findings, single-cell RNA-sequencing on FACS- organ specific effects of perturbed organic acid metabolism sorted zebrafish HSPCs identified nucleic acid biosynthesis and should be useful for future small molecule and genetic and cell cycling as upregulated processes during embryonic modifier screens. HSC production. This research promotes the utility of polar metabolomics as a tool for classifying the energetic landscape of the developing embryo. We have identified nucleotide and electron donor synthesis as potential key P-135 Zebrafish as a model of tendon-bone attachment metabolic mechanisms, which serve to drive developmental development and regeneration HSC expansion. Marie-Therese Noedl1, Rishita Shah1, Jenna Galloway1,2 1Center for Regenerative Medicine, Department of 2 P-134 Rescue of methylmalonyl-CoA deficiency in Orthopaedic Surgery, MGH, HMS, Boston, MA, Harvard zebrafish using 503unc muscle specific promoter Stem Cell Institute, MA delineates role of muscle in disease condition. Tendon-bone attachment sites, named entheses, are KT Ellis1, J Pardo1, ML Arnold1, B Carrington1, K Bishop1, necessary to transfer muscle force to bone. Due to high JL Catlett1, V Hoffmann2, PM Zerfas3, NP Achilly1, RJ stress levels exerted on these structures, entheses are Chandler1, JL Sloan1, R Sood1, OA Shchelochkov1, CP prone to injury and require surgical intervention, which Venditti1 is only partially successful in recreating a functional interface. In testing the regenerative capacities of zebrafish 1National Human Genome Research Institute, National entheses, we found that zebrafish tendons are capable Institutes of Health, Bethesda, MD,2 Office of the Director, of autonomously reattaching to bone within a week National Institutes of Health, Department of Health after injury and fully regenerate within eight weeks. We and Human Services, Bethesda, MD, 3Office of Research therefore aimed at establishing the zebrafish as a model Services, OD, National Institutes of Health, Bethesda, MD of enthesis development and regeneration in order to investigate the cellular and molecular mechanisms guiding Methylmalonic acidemia (MMA) is a rare autosomal successful enthesis formation and repair. We particularly recessive disorder that impairs the oxidative metabolism focused on the role of the extracellular matrix component of valine, isoleucine, methionine, threonine, and odd-chain Tenascin C (Tnc) during enthesis healing, which has been fatty acids. Isolated MMA is mainly caused by mutations in associated with degenerative rotator cuff tears in humans. methylmalonyl-CoA mutase (mut), the enzyme responsible ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 84 Poster Abstracts Tnc is a glycoprotein involved in cell proliferation, adhesion partially rescue hair cell development in the sensory organs and migration and is expressed at the enthesis in the adult in our RD models, confirming our previous data on the zebrafish. Our results show that tnc mutant zebrafish hematopoietic system and pointing to the potential use were unable to remodel their tendon matrix and never of antioxidants as a supportive therapeutic modality for fully restored the attachment site. In trying to understand RD patients, not only to increase their chances of survival, whether the observed effect was due to an impaired healing but to prevent or ameliorate their sensorineural hearing response in the adult fish or indirectly caused by early deficits, as well. developmental abnormalities, we studied early enthesis formation during development. Our results indicate an aberrant innervation of the hypaxial musculature and a P-137 Modeling a human seizure disorder caused by delay in tendon development in tnc mutant zebrafish larvae PRUNE1 mutations in zebrafish when compared to wildtype controls. We therefore propose that the lack of Tnc may impact adult attachment repair Elizabeth Burke1, Kyle Reichard1, Ashrifia Adomako- via two mechanisms: aberrant muscle innervation during Ankomah1, Kevin Bishop2, Raman Sood2, Camilo development may lead to insufficient muscle function and Toro 3, Shawn Burgess2, William Gahl3, May Christine V abnormal and weakened tendon morphology, resulting in Malicdan3 impaired regeneration, and/or the absence of Tnc during 1NIH Undiagnosed Diseases Program Translational adult regeneration disrupts cell proliferation, migration, 2 or matrix deposition during the regenerative process. Laboratory, Bethesda, MD, Translational and Functional Genomics Branch, National Human Genome Research This research has the potential to reveal new mechanisms 3 underlying human enthesis pathologies. Institute, NIH, Bethesda, MD, Office of the Clinical Director, National Human Genome Research Institute, NIH, Bethesda, MD P-136 A model for reticular dysgenesis shows impaired The pathophysiology of seizures is complex and often sensory organ development and hair cell regeneration multifactorial. Several genes have been associated with linked to cellular stress seizure disorders, but the mechanisms by which the gene defects cause seizures often remain unknown due to the Alberto Rissone1, Erin Jimenez1, Kevin Bishop2, Blake 2 1 absence of a good cellular or animal model. An example of Carrington , Claire Slevin , this is the prune exopolyphosphatase 1 gene (PRUNE1) which Stephen Wincovitch3, Raman Sood2, Fabio Candotti4 and has recently been associated with neurodevelopmental Shawn Burgess1,* disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA). Patients with NMIHBA share a 1Translational and Functional Genomics Branch, National phenotypic spectrum of profound global developmental Human Genome Research Institute (NHGRI), National delay and intellectual disability, central hypotonia, spastic Institutes of Health (NIH), Bethesda, MD, USA,2 NHGRI quadriplegia, seizures and progressive brain atrophy. To Zebrafish Core, NHGRI, NIH, Bethesda, MD, USA,3 NHGRI study the pathophysiology of this disease, we utilized the Cytogenetics and Microscopy Core, NHGRI, NIH, Bethesda, CRISPR/Cas9 system to develop a prune knockout zebrafish MD, USA, 4Division of Immunology and Allergy, University line with a nonsense mutation in the DHH domain that Hospital of Lausanne, Lausanne, Switzerland is commonly affected in patients. As embryos, prune-/- showed delayed swim bladder inflation and a reduced Mutations in the gene AK2 are responsible for Reticular touch response. A combination of automated and manual Dysgenesis (RD), a rare and severe form of primary behavior analysis identified a handling-induced seizure immunodeficiency in children. RD patients have a severely phenotype in prune-/- adults. The total locomotion and shortened life expectancy and without treatment die a few seizure grading indicated a progressive nature of the weeks after birth. The only available therapeutic option seizures, from circular swimming and mild tonic seizures at for RD is bone marrow transplantation. To gain insight into the juvenile stage, to severe tonic seizures with loss of body the pathophysiology of RD, we previously created zebrafish posture and death as adults. Elevated c-fos expression, models for AK2 deficiency. One of the clinical features of RD which has been previously shown to indicate seizure activity is hearing loss, but its pathology and causes are unknown. in zebrafish and mice, was observed in the adult prune-/- In adult mammals, sensory hair cells of the inner ear do not fish but not in their clutchmates. Histological sectioning regenerate; however, their regeneration has been observed reveals cerebellar atrophy in prune-/- that progresses with in several non-mammalian vertebrates, including zebrafish. age as well. In addition to supporting the conclusion that Therefore, we use our RD zebrafish models to determine PRUNE1 mutations cause the NMIHBA phenotype, these if AK2 deficiency affects sensory organ development and/ results characterize the first spontaneously-seizing adult or hair cells regeneration. Our studies indicated that zebrafish model, which may prove useful in determining the AK2 is required for the correct development, survival mechanisms underlying the disease as well as identifying and regeneration of sensory hair cells. Interestingly, AK2 therapies for this and other seizure disorders in the future. deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in hair cells. Finally, we show that glutathione treatment can ......

..... 85 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts P-138 Reduced hindbrain activity underlies light-based of a response-contingent stimulus compared to untreated hyporeactivity in a zebrafishshank3 mutant model of and noncontingent control groups. We constructed a low- autism spectrum disorder. cost behavior monitoring system using a single-board

1 1 1 Raspberry PiR computer for imaging and a dedicated Robert Kozol , David James , Ivan Varela , Agustin TM 1 1 IntelR Movidius Neural Compute Stick for computer Tavares and Julia Dallman vision. We trained an artificial neural network to localize 1Department of Biology, University of Miami, Coral Gables, a free-swimming larval zebrafish and perform body-pose FL, 33146 estimation in real time. Using this novel system, we were able to deliver a visual stimulus as a function of the larva’s Sensory deficits are common in autism spectrum disorder location in a well (Contingent condition). Control larvae (ASD) and, as such, were recently added to the diagnostic received either no visual stimulus (Untreated condition) criteria. Hyporeactivity is a common sensory deficit found or matched (yoked to contingent) presentations of the in a syndromic form of ASD known as Phelan-McDermid visual stimulus delivered without regard to larval location Syndrome (PMS), caused by mutations in the geneSHANK3 . (Noncontingent condition). Differences were observed in To investigate PMS hyporeactivity, zebrafish shank3a and the swimming patterns between the contingent and the shank3b (shank3ab) mutant models were screened using two control groups during visual stimulus presentations the visual motor response (VMR) behavioral assay. The but not during a subsequent stimulus-free test period. We VMR is evoked by sudden changes in light intensity causing discuss the implications of our findings for the potential wildtype fish to transition from a less active (light-on) mechanisms of action of response-contingent events to a more active state (light-off). Compared to wildtype, and the application of our automated behavioral analysis shank3ab mutants were hypoactive during light-off system to the study of movement disorders using transgenic conditions and slower to react to the lights-off transition, zebrafish. or hyporeactive. In an attempt to normalize these phenotypes, we exposed shank3ab mutants to a commonly prescribed medication for ASD, the atypical anti-psychotic P-140 Effects of embryonic exposure to Aroclor 1254 on risperidone. Risperidone normalized hypoactivity but failed learning and emotional behavior in zebrafish larvae to normalize hyporeactivity. To identify circuits underlying VMR phenotypes, we used phosphorylation of ERK (pERK) Ruth M. Colwill1, Joseph Ellis1, Dylan Remick1, Lauren as a proxy for neuronal activity. Statistical differences in Chan1, Maria Perez1, Shireen Moshkelani1, Robbert pERK staining were calculated brain-wide, to produce Creton2, Sarah T. Gonzalez1,3 regional activity maps. Comparing wildtype and shank3ab 1Department of Cognitive, Linguistic & Psychological mutants, mutants had normal sensory perception but 2 reduced hindbrain activity during light-off conditions. To Sciences, Brown University, Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, functionally test the role of hindbrain in hyporeactivity, 3 presumptive hindbrain tissue was transplanted from Department of Psychology, University of California, Los wildtype to shank3ab mutant embryos. Wildtype derived Angeles hindbrains were sufficient to restore normal swimming Polychlorinated biphenyls (PCBs) are lipophilic toxicants behavior in shank3ab mutants, suggesting decreased that bioaccumulate and persist in measurable quantities neuronal activity in the hindbrain drives hypoactive and in human and wildlife tissues. Exposure to PCBs during hyporeactive phenotypes. This study is the first to suggest vulnerable developmental periods can disrupt cognitive that SHANK3 mutations impact sensory-motor circuits in and emotional development although the mechanisms the hindbrain to cause sensory hyporeactivity in individuals of action are as yet unknown. The zebrafish (Danio rerio) with PMS. is an excellent model system for a mechanistic analysis of the effects of toxicant exposure on behavior and neurodevelopment. In the present study, we examined the P-139 Effects of response-contingent events on behavior effect of sub-chronic embryonic exposure to Aroclor (A) in zebrafish larvae 1254, a commercial mixture of PCBs, on subsequent learning about a visual stimulus and anxiety-related behavior. From Lakshmi Govindarajan1, Thomas Serre1,2,3, Ruth M. 1,2 2 to 26 hours post-fertilization (hpf), larvae were exposed Colwill to 2 ppm, 5 ppm or 10 ppm A1254, egg water (untreated) 1Department of Cognitive, Linguistic & Psychological or vehicle (DMSO) control. At 7 days post-fertilization (dpf), Sciences, Brown University, 2Carney Institute for Brain larvae were given alternating presentations of a plain Sciences, Brown University, 3Center for Vision Research, white background for 5 min and either a red ‘bouncing’ Brown University disk (BD) or an identical but stationary disk (SD). This differential experience was followed by a common test in One hallmark of intelligent behavior lies in its sensitivity to which all larvae were presented with the red BD for 5 min response-contingent events. Here, we describe a series of preceded by a 5 min presentation of the white background. experiments demonstrating that the swimming behavior of Regardless of PCB exposure treatment, larvae previously one week old zebrafish larvae can be altered by presentations exposed to the red BD showed significantly less freezing ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 86 Poster Abstracts during testing than larvae exposed to the red SD. This result P-142 Bisphenol A, but not Bisphenol S, exposure indicates that PCB exposure did not prevent learning about increases lipid deposition by acting on the PI3K pathway the red BD. However, relative to the control larvae, the 5 in vivo

ppm and 10 ppm PCB-exposed larvae in the SD condition 1 1 displayed a muted response to the appearance of the red Prusothman Yoganantharajah , Faiza Basheer , Liam Hall 1, Natalie Mellet 2, Peter J. Meikle 2, Sean L. McGee 1 and BD in the test and exhibited a stronger edge preference 1,2 in the training phase. Developmental disorders including Yann Gibert anxiety, autism, and attention deficit hyperactivity disorders 1Deakin University, School of Medicine, Geelong, Australia, have been linked to PCB exposure. Our findings highlight 2Baker Institute, Melbourne , Australia, 3Dept of Cell the importance of continued monitoring of PCB levels in and Molecular Biology, University of Mississippi Medical the environment for human prenatal health care. Center, Jackson, USA Bisphenol A (BPA) is an endocrine disrupter present in P-141 Cell-cell signaling in zebrafish melanocyte many daily products. BPA exposure has been linked to many regeneration disorders in humans including cancer, neurological disorders, diabetes and obesity and has therefore been declared toxic William T. Frantz1,2, Craig J. Ceol1,2, and a partial ban is in effect in several countries. Recently the plastic industry has come with a bisphenol analog as a 1Program in Molecular Medicine, University of 2 substitute for BPA in BPA free products: BPS. However to Massachusetts Medical School, Department of Molecular, date, little is known regarding the toxicity of BPS in vivo. In Cell, and Cancer Biology, University of Massachusetts this study, we used the zebrafish as a vertebrate model to Medical School understand the mechanism of action of BPA and BPS in lipid Stem cells are important in maintenance of adult issues usage during embryogenesis. Zebrafish embryos exposed and their dysfunction can contribute to various diseases, to the extreme low dose 1 nM BPA (over 6,000-fold lower including cancer. Melanocytes, the cells that normally than the accepted human daily exposure), have a 65% pigment our skin, have their own dedicated pool of adult increase in lipid deposition. Interestingly, BPS exposure, at stem cells called melanocyte stem cells (MSCs). These the same concentration, has no effect on lipid deposition. MSCs replace damaged or exhausted melanocytes. Loss of BPA is classified as an estrogenic compound however, melanocyte or MSC function contributes to pigmentation pharmacological blockage of the nuclear estrogen receptors disorders like vitiligo and piebaldism. However, the does not suppress the induced lipid deposition. Lipidomic mechanisms that govern MSC activity during regeneration analysis revealed that BPA exposed embryos had a dramatic are largely unknown. To understand these mechanisms, increase of diacylglycerol (DAG) and Phosphatidylinositol (PI) we have conducted studies in zebrafish to identify lipids. Strikingly, lipidomic analysis of BPS exposed embryos genes and cells that regulate melanocyte regeneration. showed a decrease of DAGs and PIs. This is the first study Previously we discovered a novel mode of regeneration to demonstrate a phenotypical difference between BPA and that is used to replenish melanocytes in zebrafish; a BPS exposure at similar doses. Double exposure of BPA and subset of MSCs undergoes direct differentiation to LY294002, a strong inhibitor of phosphoinositide 3-kinases, generate new melanocytes and another subset of MSCs abolishes the BPA induced increase in lipid deposition and divides symmetrically to maintain the pool of MSCs. lipid usage. At the molecular level, BPA exposure increases Initial experiments have found that zebrafish harboring the expression of key enzymes of the PI3K pathway such deficiencies in kitlga/kita signaling, the zebrafish orthologs as: cdipt, plcb3, vps34p and p110. Our study allows us to of mammalian KIT ligand and receptor, are specifically propose a model in which BPA, but not BPS, at very low defective in the direct differentiation that occurs during concentration can activate the PI3K pathway leading to an melanocyte regeneration. Subsequent transcriptional accumulation of lipid during embryogenesis in an estrogen analyses in wild-type animals undergoing regeneration receptors independent manner. demonstrated an upregulation of kitlga as well as markers for macrophages (mpeg1) and neutrophils (mpx). In follow up experiments, genetic ablation of macrophages P-143 Zebrafish as a model of physiological and molecular produced a melanocyte regeneration defect. Together metabolic consequences of fetal hyperglycemia kit these results suggest a role for signaling and immune 1 1 1,2 cells in melanocyte regeneration. To further explore these Amitoj Singh , Karen Dwyer and Yann Gibert findings, we are currently investigating the interplay 1Deakin University, School of Medicine, Geelong, Australia, between kitlga/kita signaling and macrophages during 2Dept of Cell and Molecular Biology, University of melanocyte regeneration. An understanding of signals that Mississippi Medical Center, Jackson, USA regulate melanocyte stem cells during regeneration has the potential to reveal therapeutic targets important in One in seven births in the US are affected by gestational pigmentary disorders as well as melanoma. diabetes (GDM). In addition to the acute risk of death and complications for the fetus, GDM exposurein utero, leads to an increase in the development of cardiovascular diseases, ......

..... 87 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Poster Abstracts obesity, insulin resistance, type 2 diabetes and metabolic results using a stable homozygote-viable cannabinoid syndrome later in life. Furthermore, adults exposed in receptor 2 (CB2-KO) zebrafish line (cnr2upr1). In untreated utero to hyperglycemia also develop Non-Alcoholic Fatty CB2-KOs animals which are completely devoid of CB2, Liver Disease (NAFLD), with numerous lipid droplets the LL development was not significantly affected, but in hepatocytes. Although the health outcome of GDM HC regeneration was decreased. Furthermore, the PDR infants is well-documented, the cellular, molecular and in untreated CB2 animals was significantly different from physiological mechanisms underlying the link between fetal untreated wild type animals. Next, we will perform both hyperglycemia and diseases that develop later in life is still approaches in wildtype and CB2 mutant animals after poorly understood. We recently developed a model of fetal they have been treated with a range of environmental hyperglycemia in zebrafish embryo. This model offers the relevant glyphosate concentrations. Our results will unique advantage of allowing direct embryonic exposure to highlight how this dual neurotoxic-genetic approach can glucose alone. Our data show that adult zebrafish exposed quantify compounding effects of genetic sensitization and to high glucose solely during embryogenesis and then environmental neurotoxicity. This up-scalable approach can raised on a normal diet have a higher body mass index (BMI) eventually be expanded to additional muted/sensitizing and fat mass with lower lean mass (Echo MRI). Moreover, genes, while testing a wide range of putative pollutants, while these fish are normoglycemic, they experience thus providing a valuable disease modeling tool. hyperglycolysis based on our qPCR results. We propose that this hyperglycolysis leads to higher oxygen consumption rate in mitochondria exposed to high levels of glucose during P-145 Exploring the role of ASGR1 in lipoprotein embryogenesis leading to an increase in basal and maximal metabolism and cardiovascular disease in zebrafish mitochondrial respiration. We propose a model in which fetal hyperglycemia is forcing the glycolysis pathway at the James H. Thierer, Steven A. Farber, Tabea Moll expense of β-oxidation in embryos and that these effect are persistent in adults. Our model of controlled hyperglycemia High levels of plasma low density lipoprotein (LDL) are during embryogenesis will help us to understand how its correlated with increased risk for cardiovascular disease resulting adverse effects are present in adults. (CVD), the number one cause of death in the United States. Recently, a genome wide association study (GWAS) reported lower risk for CVD and reduced levels of LDL in individuals P-144 Homozygote viable mutant zebrafish used as with a mutation in the asialoglycoprotein receptor 1 sensitized neuro-environmental toxicological tools (ASGR1) gene. ASGR1 encodes for the major subunit of the hepatospecific asialoglycoprotein receptor (ASGPR), Alexis E. Santana-Cruz1, Luis R. Colón-Cruz1, Roberto E. which upon binding of its ligands, glycoproteins without Rodríguez-Morales1, Tiffany Tossas Deida1,2, and Martine terminal sial-groups, gets internalized by endocytosis. Behra1 Previous studies of ASGPR suggest that one of its ligands is LDL. Therefore it is paradox that loss of ASGR1 was 1University of Puerto Rico (UPR), Medical Sciences Campus 2 associated with lower plasma-LDL in the GWAS. However, (MSC), San Juan, PR, University of Puerto Rico (UPR), the reduction of LDL reported in the GWAS by itself was Cayey Campus, Cayey, PR not dramatic enough to account for the magnitude of the The severity of most genetic diseases can be enhanced by reduction in CVD risk. The apparent paradox may result environmental factors which have deleterious effects that from the influence of ASGR1 on LDL particle size distribution are difficult to model. Focusing on neurological disorders, rather than the total amount of LDL, as studies suggest that we propose to develop a combined neurotoxic-genetic small, dense LDL particles are more atherogenic. To address approach in which we will measure the effects of selected the missing mechanistic studies about the effects of ASGPR environmental neurotoxins on wild type and homozygote on lipoproteins and their uptake, I identified the zebrafish viable mutant zebrafish larvae. As proof of principle, we will ortholog of ASGR1 and demonstrated its robust liver study the effects of two water soluble long-lived herbicides expression using in situ hybridization and qPCR. Further, I that were previously described for their neurotoxicity and created zebrafish with a deleterious frameshift mutation consequently banned in the EU but are still heavily used in in ASGR1 by CRISPR/ Cas9 genome editing to phenocopy the US: glyphosate (the main compound of Roundup) and the human mutation. To examine the role of ASGR1 on LDL atrazine. Using CRISPR-Cas technology, we have generated levels and size distribution, and uptake, we are leveraging several loss-of-function mutant lines in several genes the many advantages of the zebrafish system. The Farber which when knocked-out, can potentially sensitize the lab recently developed the LipoGlo assay, a method that peripheral and/or central nervous system in the otherwise allows for the quantification of small amounts of ApoB- healthy and viable homozygote animals. The screening containing lipoproteins and their particle size by fusion of endpoints that we are monitoring are: (1) development apolipoprotein B (ApoB) to the luciferase Nanoluc. Using and regeneration of mechanoreceptors (hair cells, HCs) in these assays, ASGR1 mutant fish did not show significant the superficial sensory organ called the lateral line (LL), and changes in ApoB-containing lipoprotein levels or sizes in (2) photo-dependent swimming response (PDR) in 6day early and later stages of embryonic development. I plan post-fertilization (pdf) larvae. We will present preliminary to examine the effects of starvation on the mutant fish, as the lack of consumed fats will cause the zebrafish to utilize ......

..... 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 88 Poster Abstracts available lipoproteins. In addition, my analysis indicated that there may be other possible orthologs of ASGR1 in the zebrafish, thus I am exploring the possibilities of genetic compensation for the loss of ASGR1. Further, taking advantage of the optical clarity of the larvae, I created an additional ApoB fusion line with the photoconvertible protein Dendra2. Through whole-fish photoconversion in larvae, I will determine the turnover LDL particles, providing insight into how ASGR1 modulates LDL levels. Finally, to investigate the effects of ASGR1 on cardiovascular health, I will develop a method of inducing and quantifying CVD in zebrafish. Numerous studies in rodent models have identified a list of CVD markers, but surprisingly this has not been translated to zebrafish. I have identified several possible markers and plan to examine their expression in normal and high fat diet conditions on larval zebrafish and adult zebrafish.

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89 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA Author Index Ablain, Julien: P-54 Cunningham, Rebecca: P-105 Gupta, Tripti: O-68 Adamson, Kathryn: O-68 Damm, Erich: P-036 Hagedorn, Elliott: P-040 Akle, Veronica: P-109 Darp, Revati: P-017 Haney, Meghan: P-020 Albadri, Shahad: P-107 Darroch, Hannah: P-75 Harris, Matthew: O-45 Ali, Raja: O-22 De Oliveira, Sofia: P-91 Hasan, Ameera: P-59 Anderson, Nicole: P-016 de Pater, Emma: O-41 He, Shunning: P-125 Arner, Anja: P-103 DeGeorgia, Sophia: P-56 Heath, Joan: O-78 Artinger, Kristin: O-05 den Hertog, Jeroen: O-55 Hoffman, Ellen: O-81 Avagyan, Serine: O-56 Deng, Qing: O-51 Horsfield, Julia: O-02 Bacquelaine Veloso, Alexandra: P-55 Depestel, Lisa: P-111 Hsu, Jessica: P-021 Bapst-Wicht, Linda: P-124 Di , Valeria: P-018 Huang, Ting-Hsiang: P-114 Baron, Chloé: O-10 Di Ruggiero, Elodie: P-57 Hughes, Erika: P-80 Barthelson, Karissa: P-83 Diaz Verdugo, Carmen: P-048 Hunt, Emily: P-004 Bartunek, Petr: O-60 Dinarello, Alberto: P-100 Hunter, Miranda: P-022 Becker, Catherina: O-69 Dong, Duc: O-30 Hyuk Ki, Dong: P-60 Blackburn, Jessica: P-110 Dupuis, Eleonore: P-044 Ichino, Noriko: P-88 Bowman, Teresa: O-57 Ekker, Stephen: O-13 In Seok, Moon: P-121 Breuer, Maximilian: P-101 El Fersioui, Younes: P-123 Insco, Megan: O-25 Brombin, Alessandro: O-04 Elks, Philip: O-49 Jacks, Tyler: O-01 Brunson, Dalton: P-031 Eom, Youngsub: P-89 Jamadagni, Priyanka: P-84 Bryson-Richardson, Robert: O-09, Espino-Saldaña, Angeles: P-95 James, David: O-33 P-046 Fan, Jingwen: P-041 Ji Won, Sung: P-047 Burgess, Shawn: P-136 Fazio, Maurizio: P-112 Jiang, Yun-Jin: O-17 Burke, Elizabeth: P-137 Feng, Hui: O-27 Johnson, Michael: P-033 Burns, Geoff: O-48 Ferrero, Giuliano: O-39 Karlstrom, Rolf: P-93 Burroughs-Garcia, Jessica: O-37 Fowler, Gerissa: P-010 Kawakami, Koichi: P-032 Caceres, Lucia: P-029 Frame, Jenna: O-59 Kee, Yun: P-90 Cacialli, Pietro: O-38 Frantz, William: P-141 Keerthisinghe, Pramuk: O-53 Calkins, Donn: P-74 Frazer, Kimble: P-019 Keightley, Cristina: P-127 Campbell, Nathaniel: O-24 Freeburg, Scott: P-71 Kelley, Clair: O-35 Carrington, Blake: P-67 Fu, Tzu-Fun: P-011 Kenney, Justin: P-85 Casey, Mattie: P-129 Gagnon, James: O-08 Kim, Seok-Hyung: O-32 Cassar, Steven: P-97 Gibert, Yann: P-142, P-143 Kobayashi, Makoto: O-72 Ceol, Craig: O-76 Gillotay, Pierre: O-18 Korm, Sovannarith: P-115 Chen, Bing-Hung: P-012 Glenn, Nikki: P-037 Kozol, Robert: P-138 Chen, Wenbiao: O-28 Godoy, Paula: P-58 Kramer, Eva: P-023 Chuang, Ling-shiang: P-034 Gramann, Alec: O-21 Kumar, Brijesh: P-001 Clatworthy, Anne: O-80 Grimes, Daniel: O-19 Kwan, Kristen: O-62 Collett, Keon: P-79 Grissenberger, Sarah: P-113 Lalonde, Robert: P-81 Colwill, Ruth: P-139, P-140 Gu, Qilin: P-76, P-130 Laukkanen, Saara: P-005 Cordeiro, Anna: P-50 Gücüm, Sevinç: O-63 Laurie, Payton: P-51 ......

12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA 90 Author Index Lee, Jeongwon: P-006 Raman, Renuka: O-23 Wen, Zilong: O-61 Lipsitt, Amanda: P-61 Robertson, Anne: P-120 Wertman, Jaime: O-82 Liu, Mugen: P-030 Rodriguez, Roberto: P-099 Wheeler, Robert: O-52 Long, Timothy: P-133 Rodríguez-Ortiz, Luis: P-94 Wilson, Meredith: O-46 Look, A. Thomas: O-07 Rosello, Marion: O-11 Winkler, Christoph: O-42 Loontiens, Siebe: P-116 Rosowski, Emily: O-79 Wishman, Mark: P-73 Ludin Tal, Aya: P-131 Rubin, Sara: P-77 Wrighton, Paul: P-69 Lumaquin, Dianne: P-024 Sabharwal, Ankit: P-002 Wu, Catherine: O-14 MacLean, Jessica: P-68 Santana-Cruz, Alexis: P-144 Yan, Chuan: O-12 Maskin, Carolyn: P-003 Sepich, Diane: P-007 Yang, Jiann-Jou: P-015 Mastrodonato, Valeria: P-82 Serrano, Angie: O-64 Yao, Jun: P-70 McConnell, Alicia: P-62 Shi, Hui: P-118 Ye, Lihua: O-31, P-128 Meader, Ellie: P-52 Shive, Heather: O-26 Yoder, Jeffrey: O-50, P-042, P-043 Melong, Nicole: P-117 Shwartz, Arkadi: O-29 Yu, Shanshan: P-78 Meneghetti, Giacomo: P-96 Sips, Patrick: P-104 Zhao, Mo: P-045 Mito, Jeffrey: P-66 Sirotkin, Howard: P-102 Zon, Leonard: O-83 Mokalled, Mayssa: O-73 Smith, Bradley: O-65 Moll, Tabea: P-145 Soto, Rebecca: P-132 Monteiro, Rui: O-58 Spaink, Herman: O-34 Moreno-Campos, Rodrigo: P-106 Spalding, Jonathan: P-026 Mowry, Bryan: P-013 Stoyek, Matthew: O-47 Ninov, Nikolay: O-15 Sugden, Wade: O-36 Niu, Xubo: O-70 Tagore, Mohita: O-03 Noedl, Marie-Therese: P-135 Tang, Zhaohui: P-039 O’Brown, Natasha: P-098 Tao, Ting: P-64 Oderberg, Isaac: O-74 Tavakoli, Sahar: O-43 Oyarbide, Usua: O-40 Teicher, Gregory: P-86 Padilla, Gilberto: P-72 Tomasello, Danielle: O-66 Pardo, Joel: P-134 Travnickova, Jana: P-009 Park, Gilseung: P-025, P-126 Trompouki, Eirini: O-77 Park, Ki-Hoon: P-038 Tucker, Sara: P-92 Pascoal, Susana: P-028 Van Boxtel, Thijs: P-119 Patterson, Victoria: O-16 Van Gennip, Jenica: O-44 Pauli, Andrea: O-20 Vanhauwaert, Suzanne: P-122 Peng, Jinrong: O-75 Vierstraete, Jeroen: P-027 Pereida, Elizabeth: P-87 Wane, Madina: P-49 Phelps, Michael: P-53 Wang, Han: O-71 Piccolo, Olivia: O-06 Wattrus, Samuel: P-035 Qin, Xiaodan: P-63 Weeks, Olivia: P-108 Rajpurohit, Surendra: P-014 Weiss, Joshua: P-65 Ramakrishnan, Lali: O-54 Welker, Alessandra: P-008 ......

..... 91 12th Annual Zebrafish Disease Models Conference | July 15-18, 2019 | Boston, USA