CHARACTERIZATION OF TWO HAIR CELL PROTEINS IN THE ZEBRAFISH LATERAL LINE BY ROBIN WOODS DAVIS Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Brian M. McDermott Jr. Department of Biology CASE WESTERN RESERVE UNIVERSITY August 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Robin Woods Davis candidate for the degree of Master of Science *. Committee Chair Ryan Martin Committee Member Brian M. McDermott Jr. Committee Member Hillel Chiel Committee Member Ruben Stepanyan Date of Defense May 23, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables ..................................................................................................................... 2 List of Figures .................................................................................................................... 3 Acknowledgments ............................................................................................................. 4 Abstract .............................................................................................................................. 5 Introduction ....................................................................................................................... 6 Hearing and the Cochlea ........................................................................................................... 6 The Hair Cell .............................................................................................................................. 8 The Mechanotransduction Channel ...................................................................................... 12 Potential Channel Proteins ..................................................................................................... 14 Zebrafish as a Model Organism ............................................................................................. 24 Materials and Methods ................................................................................................... 29 Zebrafish strains and husbandry ........................................................................................... 29 Microphonic potential recording ........................................................................................... 29 Results .............................................................................................................................. 31 Tmc2b is differentially required in lateral line neuromasts ................................................ 31 Microphonic potentials in the lateral line neuromasts of Piezo1 mutants ......................... 37 Discussion ........................................................................................................................ 40 Appendix .......................................................................................................................... 46 Model for Piezo1 Effect on Mechanotransduction ............................................................... 46 Microphonic Potential Example Traces ................................................................................ 47 Creation of Tmc1 CRISPR mutant ........................................................................................ 51 References ........................................................................................................................ 54 1 List of Tables Table 1…………………………………………………………………………...53 2 List of Figures Figure 1….………………………………………………………………………..7 Figure 2………………………………………………………………………….10 Figure 3………………………………………………………………………….11 Figure 4………………………………………………………………………….16 Figure 5………………………………………………………………………….20 Figure 6………………………………………………………………………….22 Figure 7………………………………………………………………………….26 Figure 8………………………………………………………………………….27 Figure 9………………………………………………………………………….32 Figure 10………………………………………………………………………...33 Figure 11………………………………………………………………………...34 Figure 12………………………………………………………………………...35 Figure 13………………………………………………………………………...36 Figure 14………………………………………………………………………...37 Figure 15………………………………………………………………………...39 Figure 16………………………………………………………………………...46 Figure 17………………………………………………………………………...47 Figure 18………………………………………………………………………...47 Figure 19………………………………………………………………………...48 Figure 20………………………………………………………………………...48 Figure 21………………………………………………………………………...49 Figure 22………………………………………………………………………...49 Figure 23………………………………………………………………………...50 Figure 24………………………………………………………………………...52 Figure 25………………………………………………………………………...53 3 Acknowledgments First and foremost, I would like to thank my advisor Dr. Brian M. McDermott Jr. for his mentorship and support, for his enthusiasm, and for letting me work on such an awesome project. I would also like to thank my former lab members and friends Victoria (Shih- Wei Chou), for her guidance and insightful thoughts (even from California), Jiaqi Hu, Nick Sarn, and Lana Pollock for their sheparding me through my early days of learning CRISPR and zebrafish work, and Nilay Gupta, for his artistic talents and moral support; as well as Carol Fernando, our ever-knowledgeable lab manager; and current lab members Sara (Shaoyuan) Zhu and Kevin (Zongwei) Chen for their support and bonhomie. I also must thank Dr. Ruben Stepanyan for sharing with me his expertise in electrophysiology and for his very patient troubleshooting when things went wrong. And finally, my completing this program would not have been possible without the love and encouragement from my husband, Zach Davis, who steady presence and support as I went from a theater administrator to scientist was invaluable. 4 Characterization of Two Hair Cell Proteins in the Zebrafish Lateral Line ROBIN WOODS DAVIS Abstract Mechanotransduction is an important mechanism found in several sensory systems. It is the act of transforming a mechanical stimulus into an electrical response, the language of the nervous system. In the auditory, vestibular, and lateral line system, the sensory receptor of mechanosensation is the hair cell, a cell so named because of the bundle of stereocilia on its apical surface. When the stereocilia are deflected as a result of the movement of the fluid surrounding them, ion channels located at their tips open and cations enter, creating an electrical current. This thesis examines the function of two proteins found in hair cells, using gene-specific knockout zebrafish lines. The experiments performed help to elucidate the complex diversity of proteins involved in the hair cell. 5 Introduction Hearing and the Cochlea At about 20 weeks’ gestation, human hearing begins to develop, giving us one of our first interactions with the outside world (Hepper and Shahidullah 1994). Our hearing develops, and we are able to perceive sound from 20 Hz to 20 kHz. As we grow older, our hearing begins to decline; many of us losing the highest range of pitches beginning at around 20 years old (National Institute of Deafness and Other Communication Disorders). One in three adults over 65 has disabling hearing loss, and one in eight people over 12 have some hearing loss (NIDCD). Hearing loss can be genetic, due to a mutation of one of the many genes that are involved in hearing, but can also be caused by such triggers as loud noise or illness. One of the goals of hearing research is to determine the proteins involved in the auditory system so that therapies to treat or prevent deafness can be developed. The job of the auditory system is to convert sound waves — compressions and rarefactions of air — into the electrical signals understood by the brain. This is done through the process of mechanotransduction. Sound waves enter the outer ear and transfer their energy to vibrations of the eardrum and middle ear bones, which amplify the sound (Figure 1a). The last of these bones, the stapes, contacts the oval window, 6 sending vibrations into the fluid-filled inner ear. It is here, in the cochlea, where mechanotransduction takes place. The cochlea is divided into three fluid filled compartments: the scala tympani, the scala media, and the scala vestibuli (Figure 1b). Inside the scala media which, as the name suggests, lies between the scala tympani and vestibuli, is found the organ of Corti (Figure 1c). The organ of Corti contains around 16,000 hair cells arranged in four rows surrounded by supporting cells, all of which rest on a basilar membrane. B A C Figure 1. (A) The mammalian ear showing the outer ear, or pinna, the middle ear, consisting of the tympanum, malleus, incus, and stapes, and the inner ear, which is the snail-shaped cochlea and circular vestibular labyrinth. (B) A cross-section of the cochlea showing the three compartments, the scala vestibuli, scala media, and scala tympani. (C) The scala media showing the locations of the inner and outer hair cells, as well as the tectorial and basilar membranes. Reprinted from Principles of Neural Science 5th Ed. Kandel et al. (Hudspeth 2013). 7 Hair cells are named after the bundles of hundreds of stereocilia — tall, thin actin- based processes — found on their apical ends. In the ear, there are two groups of hair cells. The outer hair cells, found closer to the outside of the cochlea, lie in three rows, and their hair bundles are embedded in the tectorial membrane, a gelatinous membrane which rests over the top of the organ of Corti. The inner hair cells form one row, and their hair bundles are free-standing. When the basilar