CHARACTERIZING AQP9: A REGULATOR OF EPIPHYSEAL PLATE CHONDROCYTE PROLIFERATION, HYPERTROPHY, AND LONG BONE GROWTH by Pontius Pu Tian Tang A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Pontius Pu Tian Tang (2018) ii Abstract Characterizing Aqp9: a regulator of epiphyseal plate chondrocyte proliferation, hypertrophy, and long bone growth Pontius Pu Tian Tang Master of Science Institute of Medical Science University of Toronto 2018 Aquaporin-9 (AQP9) is a membrane channel protein suspected to regulate growth in the epiphyseal plate. As long bone defects often possess limited non-surgical options, novel factors underlying bone growth must be continuously explored to advance effective treatments. I hypothesized that Aqp9 is an important epiphyseal plate chondrocyte channel regulating the process of endochondral ossification. In this study, Aqp9 -/- mouse long bones compared to wildtype mouse long bones showed a neonatal hindlimb-specific acceleration of growth followed by reduced length in the juvenile age. Analysis of Aqp9 -/- epiphyseal plates and chondrocytes showed an early disposition for proliferation and aversion from hypertrophy, suggesting that Aqp9 may function similarly to genes such as Col10a1 and Mmp13. This study provides insight into chondrocyte membrane channel proteins and their regulation of the growing epiphyseal plate, demonstrating that Aqp9 may be a novel therapeutic target for the non-invasive intervention of leg length discrepancies. iii Acknowledgements I would like to take this opportunity to thank everyone who has helped me throughout my degree. Firstly, I would like to express my gratitude to my supervisor, Dr. Peter Kannu, for granting me the opportunity to dive into graduate work and explore a novel protein in a state-of- the-art facility. Secondly, I would like to thank my Program Advisory Committee members, Dr. Brian Ciruna and Dr. Marco Magalhaes, for their insight and constructive criticism. I would like to thank our associate Kashif Ahmed for guiding me through the basics of cell culture, our MSc candidate Liliana Vertel for assisting greatly with mouse management and dissection, our previous lab technician Angela Weng for establishing preliminary findings in the Aqp9 project, our colleague Raymond Poon for providing incredible guidance with genotyping and mouse work, previous summer students with the Alman lab for assisting with supporting research, our colleagues in the Alman lab for providing guidance with basic techniques, our summer students William Xie and Lisa Vi for assistance with cell culture and sectioning, members of the Wall lab for sharing equipment, and members of the Justice lab for sharing helpful reagents. I would also like to thank The Centre for Phenogenomics for assistance with cage maintenance and reminders for mouse weaning and health conditions. On a personal note, I would like to thank my family and friends for their continuous support and encouragement. I would also like to thank Michael Liang for guidance with data interpretation, Nicole Park for assistance with bioinformatic and literature searches, Mushriq Al-Jazrawe for introductory tips on laboratory work, and Neeti Vashi for guidance with qPCR analysis, and Erin Chown for guidance with writing and thesis defense. iv Table of Contents Acknowledgements........................................................................................................................ iii Table of Contents ............................................................................................................................ iv List of Abbreviations .....................................................................................................................vii List of Tables ................................................................................................................................... x List of Figures .................................................................................................................................. x Chapter 1: Introduction .................................................................................................................... 1 1.1 An overview of endochondral ossification and epiphyseal plate regulation ................. 1 1.2 Models of differential long bone growth ..................................................................... 19 1.3 Aquaporins and chondrocytes: expression, function, and regulation .......................... 25 1.4 Characterization of AQP9 and its novel role in chondrocyte and bone activity .......... 30 1.5 Aqp9 -/-: an accessible knockout mouse model for epiphyseal chondrocyte and bone length investigation ............................................................................................................ 43 1.6 Aqp9 has a novel function in cartilage and murine long bone growth ........................ 45 Chapter 2: Research Aims, Hypothesis, and Summary Plan ......................................................... 49 2.1 Rationale ...................................................................................................................... 49 2.2 Hypothesis ................................................................................................................... 49 2.3 Objectives .................................................................................................................... 50 2.4 Clinical significance .................................................................................................... 51 2.5 Cellular mechanisms .................................................................................................... 51 Chapter 3: Methods........................................................................................................................ 53 3.1 Mouse creation, maintenance, genotyping, and age selection ..................................... 53 3.2 In situ hybridization ..................................................................................................... 53 3.3 Skeletal staining ........................................................................................................... 54 v 3.4 Staining and immunohistochemistry of epiphyseal plates........................................... 54 3.5 Visualization and measurement of limbs and epiphyseal plates ................................. 55 3.6 Primary chondrocyte culture, qPCR, and RNA silencing ........................................... 56 3.7 Statistical analyses ....................................................................................................... 57 Chapter 4: Results .......................................................................................................................... 58 4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice.......................... 58 4.2 Histological analysis of Aqp9 expression in the developing epiphyseal plate ............ 63 4.3 Skeletal staining of P5 WT and Aqp9 -/- mice ............................................................ 65 4.4 Skeletal staining of P21 WT and Aqp9 -/- mice .......................................................... 72 4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates .................................. 79 4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates ............................ 84 4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates .................................................... 87 4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates ..................... 90 4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes ...................................................................................................................... 93 4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes ...................................................................................................................... 95 4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes ..................................... 97 Chapter 5: Discussion .................................................................................................................... 99 5.1 Aqp9 temporally influences hindlimb length ............................................................. 100 5.2 Epiphyseal plate irregularities underscore Aqp9-mediated bone length.................... 108 5.3 Aqp9 mutant chondrocytes show a differential phenotype ........................................ 116 5.4 A model for Aqp9 function in murine endochondral ossification ............................. 122 Chapter 6: Conclusions ................................................................................................................ 126 vi Chapter 7: Future Directions ....................................................................................................... 128 7.1 In situ hybridization of Aqp9 during mesenchymal condensation............................. 128 7.2 Histomorphometry of WT and Aqp9 -/- long bones .................................................. 129 7.3 Flow cytometry cell cycle analysis ............................................................................ 130 7.4 RNA-sequencing of WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes ...................................................................................................................