ONTOGENETIC CHANGES IN THE SKELETAL MUSCLE FUNCTION OF ZEBRAFISH (DANIO RERIO) LARVAE: IDENTIFICATION OF BIOCHEMICAL MARKERS FOR SLOW AND FAST CONTRACTILE BEHAVIOR

ONTOGENETIC CHANGES IN THE SKELETAL MUSCLE FUNCTION OF ZEBRAFISH (DANIO RERIO) LARVAE: IDENTIFICATION OF BIOCHEMICAL MARKERS FOR SLOW AND FAST CONTRACTILE BEHAVIOR Zebrafish is an important model species to study vertebrate development. One particularly interesting aspect of fish ontogeny, which zebrafish larvae exhibit, is the high tail beat frequency up to 10 times higher than that of adult fish. Larvae therefore require a different muscle fiber type than adult fish. This study identifies the changes in muscle protein composition that might underlie this change in muscle contraction frequency. This question is interesting because contraction frequencies above 80 Hz require the so-called superfast muscles, a muscle type so far only been identified in non-locomotory muscles not required to generate significant forces. Superfast muscles are known to differ from normal fast muscles in several proteins, including myosin heavy chain. In this study we will focus on myosin, the largest protein fraction of a muscle that constitutes 4050% of total muscle. This study identifies and validates a protocol for myosin protein extraction and identification of myosin isoforms. Fast myosin heavy chains (210 kD) were identified in hatchlings (not yet swimming actively) and in adult Zebrafish (tail beat frequency 10 Hz). In 6-d old larvae (swimming actively, tail beat frequency 100 Hz), we found an isoform (160kD) that was identified in cat masseter muscle, which is a muscle with superfast contraction characteristics. We identified isoforms by probing Western blots with primary antibody (MYH1/2/3) against myosin heavy chains. BCA and ImageJ analysis were performed to quantify the amount of total protein and the myosin content. Christina Joyce Benjamin May 2011 ONTOGENETIC CHANGES IN THE SKELETAL MUSCLE FUNCTION OF ZEBRAFISH (DANIO RERIO) LARVAE: IDENTIFICATION OF BIOCHEMICAL MARKERS FOR SLOW AND FAST CONTRACTILE BEHAVIOR by Christina Joyce Benjamin A thesis submitted in partial fulfillment of the requirements for the degree of Master of Biotechnology in the College of Science and Mathematics California State University, Fresno May 2011 APPROVED For the Department of Biology: We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree. Christina Joyce Benjamin Thesis Author Ulrike Müller (Chair) Biology Joy June Goto Chemistry Alejandro Calderon Urrea Biology For the University Graduate Committee: Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS x I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship. Permission to reproduce this thesis in part or in its entirety must be obtained from me. Signature of thesis author: ACKNOWLEDGMENTS I would like to express my deepest gratitude to Dr. Ulrike Müller (Department of Biology) and Dr. Joy J Goto (Department of Chemistry), my mentors, for their intense guidance and support throughout my time here. It was a privilege to work for them. I am so grateful to them for giving me the opportunity to learn and for all their encouragement. I also want to thank Dr. Alejandro Calderon Urrea for all the suggestions and guidance with my work I extend my thanks to Dr. Larry Riley, Dr. Jason Bush for allowing me to use the instruments in their labs, Dr. Alice Wright and Dr. Shirley Kovacs, for their continuous guidance and valuable suggestions to help me with my degree. I express my heart-felt thanks, to my colleagues for sharing their expertise and helping me with the research. Also I would like to thank Preethi, Sara and the Friesen’s for their friendship and support throughout my time here at Fresno state. With great pride I would like to express my respect and deep sense of gratitude to my family and all my Intervarsity and North Fresno friends. Their amazing support and faith has been a huge motivation towards this success. I thank them for their constant moral boost and encouragement every step along the journey towards my goal. TABLE OF CONTENTS Page LIST OF TABLES . vii LIST OF FIGURES . vii