LOCALIZATION STUDY OF SUPERVILLIN IN ZEBRAFISH HAIR CELLS USING IMMUNO-FLUORESCENCE ASSAY & IDENTIFICATION OF SMALL MOLECULES THAT IMPACT THE INNERVATION OF THE LATERAL LINE SYSTEM OF DEVELOPING ZEBRAFISH NILAY GUPTA Submitted in partial fulfilment of the requirements for the degree of Masters of Science Department of Biology CASE WESTERN RESERVE UNIVERSITY MAY 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Nilay Gupta Candidate for the degree of Master of Science. Committee Chair Hillel Chiel, Ph.D. Committee Member/Research Advisor Brian M. McDermott, Ph.D. Committee Member Stephen Haynesworth, Ph.D. Committee Member Susan Burden-Gulley, Ph.D. Date of Defense March 18th, 2016 We also certify that written approval has been obtained for any proprietary material contained therein. 2 Dedicated to my little brother, Kush 3 Table of Contents Contents Page number List of Figures 7 List of Abbreviations 9 Acknowledgements 13 Abstract 15 Background The Sense of Hearing 17 Information The Hair Cells 19 Hearing in Zebrafish 21 Advantages of using zebrafish as a model 23 organism Part I - Localization study of supervillin in zebrafish hair cells using 25 immuno-fluorescence assay Introduction Hair cell cuticular plate 26 Known proteins that constitute cuticular plate 29 Supervillin 31 Supervillin in the vertebrate hair cells 33 Materials and Zebrafish strains and husbandry 37 Methods Generation of zebrafish Svila antibody 37 4 Zebrafish whole-mount immunofluorescence 38 Imaging under confocal microscope 38 Results Supervillin localization in GFP-fascin 2b 39 transgenic zebrafish Supervillin localization in Gt(macf1a-citrine)ct68a/+ 40 transgenic zebrafish Discussion 42 Part II – Identification of small molecules that impact the innervation of 47 the lateral line system of developing zebrafish Introduction Lateral line system in zebrafish 48 Development of the posterior lateral line (PLL) 52 system in zebrafish Innervation of the lateral line system in zebrafish 54 The HGn39D transgenic zebrafish 56 In vivo chemical screening in zebrafish 58 Materials and Zebrafish strain and husbandry 60 Methods Chemical library 60 Dechorionation of zebrafish embryos 61 Primary chemical screen and rescreen protocol 61 Imaging and phenotype analysis 62 Results Phenotype 1 68 5 Phenotype 2 69 Phenotype 3 71 Phenotype 4 72 Phenotype 5 73 Phenotype 6 75 Phenotype 7 76 Phenotype 8 76 Phenotype 9 77 Phenotype 10 78 Phenotype 11 79 Phenotype 12 80 Discussion 83 Appendix 88 References 110 6 List of Figures Figure Description Page Number number Figure 1 The structure of the human ear 18 Figure 2 Schematic of a hair cell 20 Figure 3 Ear and lateral line system in the larval zebrafish 23 Figure 4 Presence of linker proteins in the cuticular plate 28 Figure 5 Schematic showing supervillin functional domains 32 Figure 6 Images from whole mount in situ hybridization of 4 dpf 34 zebrafish Figure 7 Supervillin localization in the mouse vestibular and 36 cochlear hair cells as observed under confocal microscope Figure 8 Confocal images of a 4 dpf GFP-fascin 2b transgenic 39 zebrafish immunolabeled with anti-Svila Figure 9 Confocal images of a 4 dpf Gt(macf1a-citrine)ct68a/+ 41 transgenic zebrafish immunolabeled with anti-Svila Figure 10 Schematic for supervillin localization in the organ of Corti 44 and models for its function Figure 11 Schematic for supervillin localization in the zebrafish 45 macula Figure 12 Schematic showing zebrafish lateral line system and 50 individual neuromast Figure 13 Schematic to depict planar cell polarity and mirror 51 symmetry in the hair cells of a nueromast Figure 14 Posterior lateral line primordium migration in a Zebrabow 54 transgenic line Figure 15 Schematic depicting the ALL and PLL ganglia 55 7 Figure 16 Expression of EGFP by the HGn39D transgenic line of 57 zebrafish in the afferent neurons of the lateral line system Figure 17 Schematic to show the design of the chemical screen 63 Figure 18 Phenotype 1 - Phenotype with poor afferent neuronal 69 development Figure 19 Phenotype 2 - Phenotype with hair cell-like structures at the 70 end of dendritic arbors Figure 20 Confocal micrographs of the phenotype exhibiting the hair 71 cell-like structures on the dendritic arbors Figure 21 Phenotype 3 - Phenotype with the wavy sensory afferent 72 axon in the lateral line system Figure 22 Phenotype 4 - Phenotype wherein the sensory afferent axon 73 in the lateral line system terminates prematurely Figure 23 Phenotype 5 - Phenotype where the sensory afferent axon 74 in the posterior lateral line system appears to be thinner Figure 24 Phenotype 6 - Phenotype where the sensory afferent axon 75 in the posterior lateral line system is wavy and ends prematurely Figure 25 Phenotype 7 - Phenotype where the zebrafish embryos 76 show abnormal morphology Figure 26 Phenotype 8 - Phenotype in HGn39D zebrafish embryos 77 where they show GFP expression/green fluorescence in the somites Figure 27 Phenotype 9 - Phenotype wherein zebrafish embryos 78 exhibit an abnormal morphology of the heart Figure 28 Phenotype 10 - Phenotype showing fluorescent cell-like 79 structures in zebrafish circulation Figure 29 Phenotype 11 - Phenotype showing fluorescent patches on 80 the body surface Figure 30 Bar-graph depicting the number of hits obtained in this 82 small molecule screen Figure 31 Schematic showing different approaches for target 84 identification after a small molecule screen Table 1 ‘Hits’ obtained from the small molecule screen on the 64 HGn39D transgenic zebrafish and their phenotypes 8 List of Abbreviations ac anterior crista ACF7 actin crosslinking factor 7 ALL anterior lateral line AP anterior-posterior axis bp base pairs CaCl2 calcium chloride cDNA complementary DNA Cntnap2a contactin associated protein-like 2/Caspr2 CP cuticular plate CWRU Case Western Reserve University Da Dalton DC Deiters’ cells DMSO dimethylsulfoxide DNA deoxyribonucleic acid Dpf days post fertilization EB embryo buffer EGFP enhanced green fluorescent protein F-actin filamentous actin FCH FER-CIP4 homology FCHSD1 FCH domain and double SH3 domains containing protein 1 FDA US food and drug administration G-actin globular actin GEF GTP exchange factor 9 GFP green fluorescent protein GST glutathione S-transferase GTP guanosine triphosphate h hours HNK-1 human natural killer 1 hpf hours post fertilization HTS high throughput screening IACUC Institutional Animal Care and Use Committee IgG gamma immunoglobulin IHC inner hair cells IPC inner phalangeal cells KCl potassium chloride kDa kilodalton KH2PO4 monopotassium phosphate lc lateral crista MDBK Madin–Darby bovine kidney MgSO4 magnesium sulfate mM millimolar Mm millimeter MOA mechanism of action mRNA messenger RNA Na2HPO4 disodium phosphate NaCl sodium chloride NaHCO3 sodium bicarbonate 10 ngn1 neurogenin-1 NIDCD National Institute on Deafness and Other Communication Disorders NLS nuclear localization signal oC degree centigrade OHC outer hair cells OP outer pillar cells PBS phosphate buffered saline pc posterior crista PCDH15 protocadherin 15 PCP planar cell polarity PCR polymerase chain reaction PLL posterior lateral line PLL posterior lateral line PTU 1-phenyl 2-thiourea RNA ribonucleic acid RNA-seq high throughput RNA sequencing RPKM reads per kilobase per million mapped reads RT-PCR reverse transcription PCR SAR structure-activity relationship SDF1 stromal-derived factor 1 sm saccular macula SNX9 sorting Nexin 9 SVIL supervillin TALEN transcription activator-like effector nuclease 11 um utricular macula XIRP2 Xin-actin binding repeat containing 2 l microliter m micrometer 12 Acknowledgements I owe these research projects and this thesis to my research advisor, Dr. Brian M. McDermott. I cannot thank him enough for his constant guidance and support. He plays a big role in shaping me as a researcher and has always been encouraging during the hard times. Apart from research, he has also encouraged me to reignite my interest in art, which has helped me to develop skills as an illustrator. My deep sense of gratitude goes to my thesis committee members, Dr. Stephen Haynesworth, and Dr. Susan Burden-Gulley who have been excellent teachers and have helped me develop a deeper understanding of cell and developmental biology. Dr. Burden-Gulley’s guidance during independent research course and her valuable suggestions have truly helped me shape this project. I would also like to thank Dr. Hillel Chiel for his guidelines pertaining to defense and for serving as my CGA representative. Words are inadequate to thank all my lab members who continue to make a great work atmosphere in the lab. I specially want to thank Carol Fernando for teaching me the basics about the zebrafish and helping me maintain them, without which this project would not have been possible. I owe my success in the supervillin project to Dr. Lana M. Pollock, who has been a great mentor, teacher, and a friend. Thanks to Robin Wood Davis for always providing an honest opinion, and for numerous texts during this thesis- writing time, which kept me going. I also extend my thanks to other lab mates, including Shih-Wei (Victoria) Chou, Nicholas Sarn, Shaoyuan (Sara) Zhu, Kevin Chen, Wenbo Chen, Bona Ko, Tracy Chen, and Karthik Mohanarangan. I would also like to thank my friends outside of the lab including everyone from International Student Fellowship. I thank Sharoon Hanook for being an elder brother 13 and my family away from home. Thank you Megan Stonebraker for being the nicest friend who always takes my side; Soumili Chatterjee, Wayne Gibson, Richard Nii Larte Lartey, Thomas Atta-Fosu, Sneha Bandi, Nandini Puttarudraiah, and Bok Chew for making my study abroad a memorable experience. A special thanks to Robert McMurray and his dog Lulu, who has been exceptionally helpful as a stress-reliever during this time. I am truly thankful to God for His blessings. I am indeed lucky to have parents who have always shown trust in me and have stood by me through thick and thin. My little brother has been my constant motivation during this time and it is his strength that has ultimately helped me to successfully complete my masters.