nd Zebrafish Workshop Virtual Meeting

March 18th 2021

Izmir Biomedicine and Genome Center Izmir International Biomedicine and Genome Institute, Dokuz Eylul University

2nd Zebrafish Workshop (Virtual Meeting) 18 March 2021

Izmir Biomedicine and Genome Center Izmir International Biomedicine and Genome Institute, Dokuz Eylul University ORGANIZING COMMITTEE

Güneş Özhan, PhD Izmir Biomedicine and Genome Center & Dokuz Eylul University Izmir International Biomedicine and Genome Institute

Gülçin Çakan Akdoğan, PhD Izmir Biomedicine and Genome Center

Pervin Dinçer, PhD Hacettepe University 2nd Zebrafish Workshop (Virtual meeting) Turkey, 18 March 2021 Izmir Biomedicine and Genome Center & Izmir International Biomedicine and Genome Institute, Dokuz Eylül University

PROGRAM GMT+3 (Istanbul Time) Opening Remarks 10:50-11:00 Pervin Dinçer, PhD

Session 1: Development and Regeneration 11:00-12:45 Moderator: Güneş Özhan, PhD

Erez Raz, PhD Center for Molecular Biology of Inflammation, Institute of Cell Biology “Cellular mechanisms controlling the polarisation and directed migration of zebrafish germ cells”

Gilbert Weidinger, PhD Ulm University, Institute of Biochemistry and Molecular Biology “Regulation of cellular dedifferentiation during zebrafish regeneration”

Didier Stainier, PhD Max Planck Institute for Heart and Lung Research “Transcriptional adaptation and genetic compensation”

Lunch Break 12:45-13:30

Session 2: Disease Modeling 13:30-15:15 Moderator: Gülçin Çakan Akdoğan, PhD

Nathalie Jurisch-Yaksi, PhD Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology “Diversity and function of motile ciliated cell-types within ependymal lineages of the zebrafish brain”

Adam Hurlstone, MA, PhD University of Manchester “Fat uptake and fat storage promote melanoma”

Miguel A. Moreno-Mateos, PhD Andalusian Center for Developmental Biology “Understanding early vertebrate development: a CRISPR-Cas13d view”

Coffee Break 15:15-15:30 Session 3: Selected Short Talks 15:30-17:45 Moderator: Güneş Özhan, PhD 15:30-15:45 Saltuk Buğrahan Ceyhun “Effect of diet-induced obesity on anxiety and offspring in zebrafish: high fat vs. high carbohydrate” 15:45-16:00 Özgün Özalp “Nradd acts as a negative feedback regulator of Wnt/β-catenin signaling and promotes apoptosis” 16:00-16:15 Özge Çark “A possible regulatory role of γ- in Wnt/β-catenin signaling” 16:15-16:30 Aysu Şevval Alkiraz “Proliferation of regenerative neuronal progenitor cells in the zebrafish olfactory epithelium is controlled by the EGFR- JAK/STAT axis” 16:30-16:45 Esra Katkat “Comparative analysis of the molecular mechanisms of melanocyte regeneration and melanoma using the zebrafish model and manipulation of expression profiles in melanoma cells using the CRISPR/Cas9 to induce a regeneration-like progress” 16:45-17:00 Merve Başol “Generating a zebrafish model for macular corneal dystrophy” 17:00-17:15 Gülsüm Kayman Kürekçi “Characterization of zebrafish desmin orthologs and incomplete penetrance in CRISPR/Cas9-generated stable knockouts” 17:15-17:30 Fulya Yaylacıoğlu-Tuncay “Creating rare disease specific CRISPR- Cas9 platforms in zebrafish and ensuring their sustainability in Hacettepe University Zebrafish Research Laboratory: Challenges in mimicking missense variants” 17:30-17:45 Seniye Targen “Development and ongoing curation of the ZenoFish Database: ZenoFishDb v1.2”

Coffee Break 17:45-18:00

Session 4: Good Zebrafish Practices/Zebrafish health monitoring 18:00-18:50 Moderator: Gülçin Çakan Akdoğan, PhD Chereen Collymore, Director Veterinary Care and Services at Charles River Laboratories “Zebrafish health monitoring” Abstracts Abstracts

Comparative analysis of the molecular mechanisms of melanocyte regeneration and melanoma using the zebrafish model and manipulation of gene expression profiles in melanoma cells using the CRISPR/Cas9 to induce a regeneration-like progress

Esra Katkat, Yeliz Demirci, Güneş Özhan

İzmir Biomedicine and Genome Center, İzmir International Biomedicine and Genome Institute, Dokuz Eylül University

The cellular pathways required for proliferation during regeneration are also common in tumorigenesis. Yet, tumors differ from regenerating cells in various aspects. The difference between regeneration and tumorigenesis happens as regenerating cells decelerate proliferation and acquire differentiation. We will investigate the molecular mechanisms at the transcriptional level by comparing the early/late stages of melanocyte regeneration and melanoma in adult zebrafish. We created a melanocyte regeneration model and determined early/late stages based on the expression levels of proliferation and melanocyte markers. We used tg(kita: GFP- RASG12V) line to generate zebrafish melanoma. We compared DEGs of melanoma to those of regenerating melanocytes. This comparison yielded critical insights into the different molecular mechanisms. Next, we will direct the gene expression profile (GIP) of melanoma cells to the GIP of melanocytes undergoing regeneration by gene activation/knock-down. These manipulations on cancer progression will be investigated in zebrafish xenografts.

Keywords : Melanocyte, Regeneration, Melanoma, Transcriptome

1 Abstracts

Effect of diet-induced obesity on anxiety and offspring in zebrafish: high fat vs high carbohydrate

Medine Türkoğlu 1,2, Alper Baran 3, Ekrem Sulukan 2,4, Atena Ghosigharehagaji 2, Serkan Yildirim 5, Hacer Akgül Ceyhun f, İsmail Bolat 5, Murat Arslan 4, Saltuk Buğrahan Ceyhun 2,4,*

1.Department of Nanoscience, Graduate School of Natural and Applied Science, Atatürk University, Erzurum, TURKEY 2.Aquatic Biotechnology Laboratory, Fisheries Faculty, Atatürk University, Erzurum, TURKEY 3.Department of Food Quality Control and Analysis, Erzurum Vocational School, Atatürk University, Erzurum, TURKEY 4.Aquaculture Department, Fisheries Faculty, Atatürk University, Erzurum, TURKEY 5.Department of Pathology, Faculty of Veterinary, Atatürk University, Erzurum, TURKEY 6.Department of Psychiatry, Faculty of Medicine, Atatürk University, Erzurum, TURKEY

Anxiety and obesity; two current phenomena. They are among the important public health problems with increasing prevalence worldwide. Although it is claimed that there are strong relations between them, the mechanism of this relationship has not been fully clarified yet. On the other hand, the effect of this relationship on the offspring has been another research subject. In this study, obese zebrafish were obtained by feeding two different diets, one containing high amount of lipid (HF) and the other containing high amount of carbohydrate (HK), and their anxiety levels were evaluated. In order to establish a relationship between these two phenomena, in addition to histopathological and immunohistochemical analysis in the brain tissues of fish, the transcription levels of some related to lipid and carbohydrate metabolisms were determined. In addition, offspring were taken from obese zebrafish and studied to examine the effect of parental obesity on offspring.

Keywords : Neurodegeneration, PPARs, Appetite-Related Genes, Brain

2 Abstracts

Nradd Acts as a Negative Feedback Regulator of Wnt/ β-Catenin Signaling and Promotes Apoptosis

Ozgun Ozalp 1,2, Ozge Cark 1,2, Yagmur Azbazdar 1,2, Betul Haykir 1,3, Gokhan Cucun 1,2, Ismail Kucukaylak 1,4, Gozde Alkan-Yesilyurt 1, Erdinc Sezgin 5,6 and Gunes Ozhan 1,2

1.Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul University Health Campus, Inciralti-Balcova, 35340 Izmir, Turkey; [email protected] (O.O.); [email protected] (O.C.); [email protected]. edu.tr (Y.A.); [email protected] (B.H.); [email protected] (G.C.); ikuecue1@uni- koeln.de (I.K.); [email protected] (G.A.-Y.) 2.Izmir International Biomedicine and Genome Institute (IBG-Izmir), Dokuz Eylul University, Inciralti-Balcova, 35340 Izmir, Turkey 3.Institute of Physiology, Switzerland and National Center of Competence in Research NCCR Kidney, University of Zurich, CH-8057 Zurich, Switzerland 4.Institute of Zoology-Developmental Biology, University of Cologne, 50674 Cologne, Germany 5.Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institutet, 17177 Stockholm, Sweden; [email protected] 6. MRC Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, University of Oxford, Oxford OX39DS, UK

Wnt/β-catenin signaling regulates several biological processes for the generation of proper tissue size, organization and function during development and homeostasis. Thus, mutations in the Wnt pathway components and modulators lead to diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases requires detailed understanding of the regulatory mechanisms that operates Wnt signaling. In our study, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/β-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly inhibits Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/β-catenin signaling.

Keywords : Wnt/β-catenin, Neurotrophin, Death Receptor, Apoptosis

3 Abstracts

A possible regulatory role of γ-tubulin in Wnt/β- catenin signaling

Özge Çark 1,2, Esra Katkat 1,2, Yavuz Oktay 1,2, Güneş Özhan 1,2

1.Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul University Health Campus, Inciralti-Balcova, 35340 Izmir, Turkey 2.Izmir International Biomedicine and Genome Institute (IBG-Izmir), Dokuz Eylul University, Inciralti-Balcova, 35340 Izmir, Turkey

γ-Tubulin is a tubulin superfamily member which is required for the nucleation of . Several variants of the γ-Tubulin gene (Tubg1) have been implicated with abnormal neurogenesis observed in patients with lissencephaly and microcephaly. In addition, it was reported that the localization of γTubulin for nucleation is regulated through interactions with several canonical Wnt signaling , especially Axin. Since it is known that disrupted Wnt/β-catenin signaling regulation leads to abnormal neurogenesis, we investigated how neurogenesis and Wnt/β-catenin signaling affected in zebrafish Tubg1 G0 Knockouts (KO) generated by CRISPR/Cas9 gene editing. We observed a significant reduction in neurogenesis-related genes and Wnt-target genes in Tubg1 G0 KO having decreased level of γ-Tubulin. Upon these results, we hypothesized that the interaction between γ-Tubulin and Axin may regulate Wnt/β-catenin signaling by limiting the formation of the β-catenin destruction complex, which may result in transcriptional activation of Wnt/β-catenin target genes.

Keywords : γ-Tubulin, Wnt/β-Catenin, Signaling, Neurogenesis, CRISPR/Cas9

4 Abstracts

Proliferation of regenerative neuronal progenitor cells in the zebrafish olfactory epithelium is controlled by the EGFR-JAK/STAT axis

Aysu Şevval Alkiraz, Yiğit Kocagöz, Kardelen Güler, Şiran Şireci, Stefan H. Fuss

Bogazici University, North Campus, K-Park 216

The olfactory epithelium (OE) provides an insightful model to study the cellular and molecular mechanisms of adult neurogenesis and nervous tissue regeneration. Olfactory sensory neurons undergo continuous turnover in the intact OE but also regenerate rapidly in response to injury. Maintenance and repair neurogenesis are distinct processes that depend on unique neuronal progenitor cells and different inter- and intracellular signals. Transcriptome analysis of the OE during experimental de- and regeneration revealed rapid and transient upregulation of hb-egfa, a member of the epidermal growth factor family, and related signaling components. Exogenous stimulation of the OE with HB-EGF results in mitotic activation of regenerative progenitors, whereas pharmacological inhibition of HB-EGF activation or EGFR signaling severely impairs OE regeneration. JAK/STAT signaling downstream of the EGFR appears to be critical for progenitor cell activation. Together, our results identify HB-EGF as a key molecular signal to stimulate regenerative neurogenesis through the EGFR-JAK/STAT axis.

Keywords : Neurogenesis, Stem Cell, Signaling, Zebrafish

5 Abstracts

Characterization of zebrafish desmin orthologs and incomplete penetrance in CRISPR/Cas9- generated stable knockouts

Gülsüm Kayman Kürekçi 1, Ecem Kural Mangıt 1,2, Cansu Koyunlar 1,3, Şeyda Ünsal 1, Berk Sağlam 4, Bora Ergin 4, Merve Gizer 5, Ismail Uyanık 6, Niloufar Boustanabadimaralan Düz 1, Petek Korkusuz 5, Beril Talim 7, Nuhan Puralı 4, Simon M. Hughes 8, Pervin R. Dinçer 1,2

1.Department of Medical Biology, Hacettepe University Faculty of Medicine, Ankara, Turkey. 2.Laboratory Animals Research and Application Centre, Zebrafish Unit, Hacettepe University, Ankara, Turkey. 3.Current address: Department of Hematology, Erasmus MC, Rotterdam, The Netherlands. 4.Department of Biophysics, Hacettepe University Faculty of Medicine, Ankara, Turkey. 5.Department of Histology and Embryology, Hacettepe University Faculty of Medicine, Ankara, Turkey. 6.Department of Electrical and Electronics Engineering, Hacettepe University Faculty of Engineering, Ankara, Turkey. 7.Department of Pediatrics, Pathology Unit, Hacettepe University Faculty of Medicine, Ankara, Turkey. 8.Randall Centre for Cell and Molecular Biophysics, New Hunt’s House, Guy’s Campus, King’s College London, London, United Kingdom.

Desmin is a muscle-specific intermediate filament supporting structural integrity and mechanotransduction in muscle cells. In human and mouse, desmin mutations cause progressive skeletal myopathy and cardiomyopathy. Characterization of the two desmin paralogs (desma and desmb) in zebrafish revealed differential spatiotemporal expression during embryonic development. Both genes were expressed in striated muscle until 72 hpf while desmb expression shifted to gut smooth muscle afterwards. In contrast to other reported loss-of-function mutants, CRISPR/Cas9-mediated knockout mutant lines exhibited an alleviated phenotype with no overt muscle degeneration but only altered calcium signaling. In conclusion, despite having high and functional overlap, desma and desmb did not compensate for each other in single gene mutants, suggesting the activation of different adapting genes or mechanisms. This study was supported by TÜBİTAK Project no. 214S174 to P.R.D.

Keywords : CRISPR/Cas9, Muscle development, Genetic compensation

6 Abstracts

Creating Rare Disease Specific CRISPR-Cas9 Platforms in Zebrafish and Ensuring Their Sustainability in Hacettepe University Zebrafish Research Laboratory: Challenges in mimicking missense variants

Fulya Yaylacıoğlu Tuncay1,2, Ecem Kural Mangıt1, Gülsüm Kayman Kürekçi1, Şeyda Ünsal1,3, Gizem Önal1, Burcu Kesikli1,4, Nilüfer Düz1, Sezen Güntekin Ergün1, Zeynep Çınar1, Pervin Rukiye Dinçer

1.Department of Medical Biology, Hacettepe Medical Faculty, Ankara 2.Department of Medical Biology, Gülhane Medical Faculty, Ankara 3.Faculty of Health Sciences, University of Southern Denmark, Copenhagen 4.Department of Pathophysiology, Ankara Medical Faculty, Ankara

Zebrafish is an attractive vertebrate model organism for rare disease research. Introduction of point mutations in the zebrafish genome would be necessary to model diseases however this has been still a challenge in zebrafish. As a part of our project‘’Creating Rare Disease Specific CRISPR-Cas9 Platforms in Zebrafish and Ensuring Their Sustainability’’, we aimed to introduce a target missense mutation in zebrafish genome and optimize our protocol for this work package. Our target gene for this aim is tgfbi. Despite using nine different injection protocols including standard ones and novel different combinations from literature we could not achieve knock- in of the precise mutation in the target sequence Therefore, introduction of precise missense mutations to zebrafish genome is still a challenge in our laboratory as it is the case for many other zebrafish researchers. However, we will continue to try and optimize protocols via using novel methods such as base editing and prime editing. This project was funded Hacettepe University (Grant reference:TAY-2017-12735).

Keywords : Zebrafish, Knock-in, CRISPR/Cas9

7 Abstracts

Development and Ongoing Curation of the Ze- noFish Database: ZenoFishDb v1.2

Seniye Targen1,2, Tuğberk Kaya3,4, Ender Mehmet Avci5, Damla Güneş3, Ayşe Gökçe Keşküş3, Rüya Tombuloğlu2, Elif Oymalil2, İlayda Fırlar2, Fatma Betül Metin2, Mehr-Un-Nisa Usman2, Ece Urani2, Ozlen Konu1,3,6

1.Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey. 2.Department of Molecular Biology and Genetics, Eastern Mediterranean University, TRNC. 3.Interdisciplinary Program in Neuroscience, Bilkent University, Ankara, Turkey. 4.Institute of Neuronal Cell Biology, Technical University Munich, Munich, Germany. 5Izmir Biomedicine and Genome Center, Dokuz Eylul University, Izmir, Turkey. 6UNAM-Institute of Materials Science and Nanotechnology, Bilkent University, Ankara, Turkey.

Successful human cell transplantation into zebrafish model organism has paved the way for standardising use of zebrafish xenografts in cancer research. Fast-paced evolvement of the zebrafish xenograft literature highlighted the need for an easy- access repository and search tool for the latter. ZenoFishDb v1.1 database (https:// konulab.shinyapps.io/zenofishdb), a manually curated database based on R shiny platform, has been developed for depicting individual cell-line transplantation and PDX study details and projecting statistical information on these aspects through DataTable, Visualisation, PDX Details and PDX charts modules. In the current version of ZenoFishDb v1.1 we mainly focused on the molecularly-modified cell injections, patient-derived xenografts and modified host strains. In the upcoming version, ZenoFishDb v1.2, we also aim to include naive cell injections, detailed drug-dose applications and biological assessment-specific record timings.

Keywords : Zebrafish, Xenograft, Patient-Derived Xenograft, Database

8 Abstracts

Generating a Zebrafish Model for Macular Corneal Dystrophy

Merve Başol1,2, Esra Ersöz1,2, Khalid Abdalla1, Gülçin Çakan-Akdoğan1*

1.Izmir Biomedicine and Genome Center 2. Izmir Biomedicine and Genome Institute, Dokuz Eylül University

Corneal dystrophies are a group of rare diseases, characterized by formation of vision blocking aggregates in the cornea tissue. The resemblance of human and zebrafish cornea structure and genetic homology indicates that zebrafish can be used to model these diseases. We aimed at development of the first in vivo model of Macular Corneal Dystrophy (MCD), which is one of the more common CDs in Turkey. Human CHST6 gene encodes the corneal N-acetylglucosamine-6-O-sulfotransferase enzyme, mutations of which are linked to reduced sulfation and aggregation of KSPGs in MCD patients. We showed that the zebrafish CHST6 gene is expressed in cornea, among with other tissues. We successfully generated several mutant alleles of CHST6 using CRISPR/Cas9 method. The mutants that are homozygous viable, and display cartilage specific phenotype in the larval stages. We will present our findings as well as ongoing work on the characterization of corneal phenotypes in the adult mutants.

Keywords : Macular Cornea Dystrophy, CRISPR/Cas9, Rare Disease, Keratan Sulfate

9

www.ibg.edu.tr Dokuz Eylul University Health Campus Mithatpasa Ave. 58/5 35340 Balcova, Izmir/TURKEY