CHARACTERIZING AQP9: A REGULATOR OF PROLIFERATION, HYPERTROPHY, AND LONG 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 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 . In this study, Aqp9 -/- mouse long 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 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.

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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 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 ...... 131

7.5 Therapeutic strategies ...... 132

Appendix...... 159

Statement of Contributions ...... 159

vii

List of Abbreviations

A Adenine Adam A disintegrin and metalloproteinase Akt Protein kinase B Aqp Aquaporin AQPap Human aquaporin adipose ARE Androgen response element Atf AMP-dependent transcription factor Bgp Osteocalcin Bmp Bone morphogenetic protein Bsp Bone sialoprotein C Cytosine CACNA1H Calcium Voltage-Gated Channel Subunit Alpha1 H CD Cluster of differentiation CDH2 Cadherin-2 Cdk Cyclin-dependent kinase cDNA Complementary DNA CHIP Channel-forming integral protein CHO Chinese hamster ovary Col10a1 Collagen, type X, alpha 1 Col1a1 Collagen, type 1, alpha 1 Col2a1 Collagen, type II, alpha 1 DAB Diaminobenzidine DEPC Diethyl pyrocarbonate DIG Digoxigenin Dkk Dickkopf-related protein DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSLR Digital single-lens reflex E. coli Escherichia coli ECM Extracellular matrix EDNRB Endothelin B receptor EDTA Ethylenediaminetetraacetic acid EMSA Electrophoretic mobility shift assay ERE Estrogen response element Fgf Fibroblast growth factor G Guanine GDF Growth/differentiation factor GH Growth hormone GlpF Glycerol uptake facilitator protein GLUT4 Glucose transporter type 4 H&E Hematoxylin and Eosin H2O2 Hydrogen peroxide HB Helix B HE Helix E HIF Hypoxia-inducible factors Hox Homeobox Hp1bp3 Heterochromatin protein 1, binding protein 3 HRP Horseradish peroxidase HSC Hematopoietic stem cell IGF-1 Insulin-like growth factor 1 Ihh Indian hedgehog IL Interleukin IRE Insulin response element viii

KCNB1 Potassium voltage-gated channel, Shab-related subfamily, member 1 KOH Potassium hydroxide Lepr Leptin receptor LLD Leg Length Discrepancy MAPK Mitogen-activated protein kinase Matn Matrilin MCDS Metaphyseal Chondrodysplasia Schmid Type MEM Minimum Essential Medium MKRN3 Makorin ring finger protein 3 Mmp13 Matrix metallopeptidase 13 mRNA Messenger RNA MSC Mesenchymal stem cell mTOR Mammalian target of rapamycin AMPK AMP-activated protein kinase N-CAM Neural cell adhesion molecule Nfat Nuclear factor of activated T-cells NPA Asn-Pro-Ala Npr Natriuretic peptide receptor OARSI Osteoarthritis Research Society International Osx Osterix Panx Pannexin PBS Phosphate-buffered saline PCR Polymerase chain reaction PEPCK Phosphoenolpyruvate carboxykinase PF4V1 Platelet factor 4 variant 1 PFA Paraformaldehyde pH Power of hydrogen PI3K Phosphoinositide 3-kinase PKC Protein kinase C Prx1 Paired related homeobox 1 PTHrP Parathyroid hormone-related protein PTP Protein tyrosine phosphatase qPCR Quantitative polymerase chain reaction RANK-L Receptor activator of nuclear factor kappa-Β ligand RNA Ribonucleic acid ROI Region of Interest ROS Reactive oxygen species Rspo R-spondin RT-PCR Reverse transcription polymerase chain reaction Runx Runt-related transcription factor SDS Sodium dodecyl sulfate Shh Sonic hedgehog Shox Short stature homeobox siRNA Silencing RNA Smad "small" "Mothers Against Decapentaplegic" Sox Sry-related HMG box Spred2 Sprouty-related, EVH1 domain-containing protein 2 SSC Saline-sodium citrate Stat1 Signal transducer and activator of transcription 1 T Thymine Tak1 Transforming growth factor beta-activated kinase 1 TBST Tris-buffered saline, polysorbate 20 Tbx T-box TCP The Centre for Phenogenomics TEA Triethylamine Tgf Transforming growth factor ix

TNE Tris, EDTA, NaCl TonEBP Tonicity-responsive enhancer binding protein TRPM7 Transient receptor potential cation channel subfamily M member 7 TRPV4 Transient receptor potential cation channel subfamily V member 4 VEGF Vascular endothelial growth factor VILO Variable input, linear output Wnt Wingless-related integration site WT Wildtype Yap Yes-associated protein

x

List of Tables

Table 1.1 Models of differential bone growth ...... 24

Table 5.1 Primary epiphyseal plate chondrocyte proliferation rates (48-96 hours) ...... 117

List of Figures

Figure 1.1 Mesenchymal condensation ...... 5

Figure 1.2 Early ...... 8

Figure 1.3 Chondrocyte proliferation, columnar formation, and pre-hypertrophy...... 11

Figure 1.4 Chondrocyte hypertrophy ...... 14

Figure 1.5 Ossification and long bone growth ...... 18

Figure 1.6 AQP9 protein structure...... 33

Figure 1.6 cont...... 35

Figure 1.7 Expression and regulation of AQP9 ...... 38

Figure 4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice ...... 59

Figure 4.2 Histological analysis of Aqp9 expression in the developing epiphyseal plate ...... 65

Figure 4.3 Skeletal staining of P5 WT and Aqp9 -/- mice ...... 67

Figure 4.4 Skeletal staining of P21 WT and Aqp9 -/- mice ...... 74

Figure 4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates ...... 82

Figure 4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates ...... 86

Figure 4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates ...... 89

Figure 4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates ...... 92

Figure 4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes ...... 95 xi

Figure 4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes ...... 97

Figure 4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes ...... 99

Figure 5.1 A model for Aqp9 function in murine endochondral ossification ...... 125 1

1 Introduction

The epiphyseal plate is a region of growing tissue near the ends of long bones in children and adolescents (Mirtz, Chandler, & Eyers, 2011). It is an exceptional model for understanding genetic mechanisms that underlie long bone development because it is:

1) The foremost location where long bone grows, 2) Easily visualized and accessible, 3) Regulated by a myriad of genes, and 4) Organized in specific zones with remarkable cell function and morphology.

Numerous studies have provided information on the mechanisms governing long bone growth. Sectioning of multispecies epiphyseal plates and histological analyses paved the way for several of the basic structures recognized today, such as the epiphyseal plate zones. The characterizations of simple elements—such as chondrocytes; the primary cellular unit of epiphyseal plate cartilage—were critical towards a richer understanding of long bone development (Brighton, Sugioka, & Hunt, 1973). These studies further led to the investigation of spatiotemporal markers at each zone during skeletal morphogenesis, including the expression of now-familiar genes Ihh and Bmp-6 (Iwasaki, Le, & Helms, 1997). This process has been accelerated by the advent of mutant mouse models, which have revealed the functional role of specific genes contributory to long bone development. Currently, the challenge is to understand the interaction of novel genes and pathways that coordinate in the epiphyseal plate to control cell proliferation, progression into cellular hypertrophy, and ultimately extend long bone length. In particular, transmembrane residents of the channelome—the diverse set of ion channels and pores on the chondrocyte membrane—are emerging as targets to further understand cartilage and bone biology (Barrett-Jolley, Lewis, Fallman, & Mobasheri, 2010).

1.1 An overview of endochondral ossification and epiphyseal plate regulation

Osseous tissues in the mammalian skeleton are formed through two different processes— intramembranous ossification and endochondral ossification (Berendsen & Olsen, 2015). In the 2 former, derived from mesenchymal cells during embryonic skeletal development secrete that eventually calcify and form mature bone matrix (Lefebvre & Bhattaram, 2010). This process gives rise to flat bones, the majority of cranial bones, and the clavicles. In contrast, endochondral ossification creates the long bones of the body by replacing (Mackie, Ahmed, Tatarczuch, Chen, & Mirams, 2008). Here, mesenchymal cells condense and differentiate into chondrocytes that secrete matrix, proliferate, undergo hypertrophy, and expire sequentially to allow and vascular invasion. Bone matrix is formed at the epiphyseal plate until the primary and secondary centres of ossification join and prevent further growth (Ortega, Behonick, & Werb, 2004).

This process is tightly regulated and dictates the skeletal structure of the individual, impacting their appearance, health, and quality of life. Examining the structures and regulatory markers present during each step of murine endochondral ossification will entrench an understanding of the known factors important to long bone growth—as well as provide insight into novel contributory genes.

1.1.1 Mesenchymal condensation (see Figure 1.1)

Endochondral ossification commences with the migration of mesenchymal cells from the lateral plate mesoderm and subsequent condensation at the respective limb field (Long & Ornitz, 2013). Initiating limb outgrowth requires T-box transcription factors such as Tbx5 in the forelimb and Tbx4 in the hindlimb to establish fibroblast growth factor (FGF) gene expression and eventual chondrogenesis (Nishimoto & Logan, 2016). This embryonic process occurs as early as E9.5- 10.5 in the murine and sets the foundation for later chondrogenic differentiation and production of early chondrocyte-specific extracellular matrix (ECM) products such as Col2a1 and aggrecan (Hata, Takahata, Murakami, & Nishimura, 2017). At this stage, the mesenchymal cells are tightly packed and rely on body-plan genes such as Hox to direct their condensation position. Expression of Hox10 guides growth of the proximal long bone—such as the femur— and expression of Hox11 guides the distal long bone—such as the tibia (Rux & Wellik, 2017). A member of the Hedgehog signalling pathway, Shh, is expressed in the posterior margin of the limb bud—named the zone of polarizing activity—to guide appropriate anterior-posterior 3 patterning of the developing limb (Yang, Andre, Ye, & Yang, 2015). While the evolutionary background of appendicular skeleton development requires further investigation, genes important to future chondrogenic maturation also play a role in driving initial mesenchymal condensation.

Bone morphogenetic proteins (BMPs) are part of the transforming growth factor-β (TGF- β) family and play fundamental roles in embryonic skeletal development (Beederman et al., 2013). BMPs bind to serine/threonine kinase receptors, inducing their phosphorylation, which then phosphorylate SMAD proteins that can complex with SMAD4 and enter the nucleus to perform transcriptional activation of genes such as CDH2 to synthesize N-cadherin (Massagué, Seoane, & Wotton, 2005; Wang, Zhao, & Zhang, 2017). Both N-cadherin and N-CAM are proteins responsible for cell-to-cell adhesion during mesenchymal condensation, as driven by the BMP- SMAD pathway (DeLise, Fischer, & Tuan, 2000). The modern consensus is that BMP signalling is required for mesenchymal cells to form necessary small aggregates and cluster into condensations with definite boundaries for later chondrogenic differentiation (Barna & Niswander, 2007). BMP2 and BMP4 are two such explored genes that are required for proper long bone formation (Bandyopadhyay et al., 2006). Later genes such as Sox9 are necessary for chondrogenesis but rely on BMP signalling to maintain the condensation aggregate, as Sox9 alone is insufficient to rescue chondrocyte formation in Smad4-deficient embryos (Akiyama, Chaboissier, Martin, Schedl, & de Crombrugghe, 2002; Lim et al., 2015).

Amidst the importance of BMPs, fibroblast growth factors (FGFs) are also necessary precursors to condensation formation. FGFR1-4 in both mice and humans are cell surface tyrosine kinase receptors that bind FGF and phosphorylate important downstream pathways involving MAPK, PI3K, STAT1, and PKC (Turner & Grose, 2010). Primarily, FGFR1 and 2 are expressed widely in limb bud mesenchyme prior to condensation and their deletion from the limb bud produces diminished skeletal products (Peters, Werner, Chen, & Williams, 1992; Yu et al., 2003). FGF signalling in the mesenchyme provides essential cell survival signals during pre-condensation, as well as in subsequent chondrogenic differentiation (Sun, Mariani, & Martin, 2002).

Condensation may ultimately rely on mechanical factors such as ion channels, cilia, and the cytoskeleton for mesenchymal cells to communicate (Hughes et al., 2018). The presence of 4 mechanosensitive calcium channels such as TRPM7 and potassium channels such as KCNB1 in mesenchymal cell membranes highlight specific proteins through which condensation may be initiated (Xiao, Chen, & Zhang, 2016; Pillozzi & Becchetti, 2012). Hence, even the earliest stage of long bone growth—at the limb bud—may require channelome members to progress toward chondrogenesis. At this stage, mesenchymal cells have formed the cartilage anlage necessary for further chondrocytic differentiation and eventual ossification.

Figure 1.1. Mesenchymal condensation 5

Limb bud

Mesenchymal cell Developing embryo Condensing BMPs mesenchymal SMADs cells > N-cadherin > N-CAM Encourage TRPM7 survival, KCNB1 promote cell-to- Zone of Other channelome proteins… cell adhesion, polarizing and guide condensation activity FGFs Lateral plate > MAPK mesoderm Shh > PI3K > STAT1 > PKC

Tbx5

Developing Forelimb

Tbx4

Developing Hindlimb

Developing digits Hox10 Hox11 6

1.1.2 Early chondrogenesis (see Figure 1.2)

After condensation, mesenchymal cells in the core of the anlage can differentiate into early chondrocytes by committing to a chondrogenic fate (Somoza, Welter, Correa, & Caplan, 2014). These pre-chondrocytes cease production of adhesion molecules and begin secreting cartilage matrix including collagen types II, IX, and XI into the extracellular space. As these cells migrate towards the anlage periphery, a portion commit to the osteogenic fate and form the that surrounds the developing epiphyseal plate (Lefebvre & Bhattaram, 2010).

Three main proteins drive early chondrogenesis—Sox5, Sox6, and Sox9 (Smits et al., 2001; Akiyama et al., 2002). This trio performs cooperative binding at the promoter regions of the aforementioned matrix genes to drive transcription and protein synthesis. Sox5 and Sox6 share similar promoter-binding capacities and act in concert, but Sox9 is expressed upstream and acts earlier than the two in chondrogenesis. In fact, homozygous deletion of murine Sox9 in early chondrocytes completely prevents chondrocytic differentiation, whereas deletion of Sox5 and Sox6 only diminishes differentiation (Bi, Deng, & Zhang, 1999). Overall, early chondrogenesis relies on the Sox triad greatly—but other factors are necessary as Sox does not function uniquely in the chondrocytic lineage (Han & Lefebvre, 2008).

BMP signalling is necessary for the Sox trio to function (Yoon et al., 2005). Without BMP1 receptors and their downstream SMAD modules, chondrogenic cells fail to activate Col2a1 and differentiate, resulting in severe dysplasia—a dramatic dysregulation of skeletal stature (Retting, Song, Yoon, & Lyons, 2009). FGF is also a transcriptional enhancer of Sox9, where FGFR1-4 are capable of increasing its expression (Murakami, Kan, McKeehan, & de Crombrugghe, 2000). However, members of the Wnt/β-catenin pathway inhibit differentiation and overexpression of β-catenin signaling can a) reroute chondrogenic cells to soft connective tissue formation, b) inhibit Sox9 activity, and c) result in achondrodysplasia, or dwarfism (ten Berge, Brugmann, Helms, & Nusse, 2008; Hill, Spater, Taketo, Birchmeier, & Hartmann, 2005; Akiyama et al., 2004). Furthermore, ectopic Notch signalling suppresses chondrogenesis through binding of the Notch Intracellular Domain and suppression of Sox9 in the budding limb (Mead & Yutzey, 2009). Evidently, Sox9 is an important transcription factor in early chondrogenesis. 7

In relation to the channelome, cation channels such as TRPV4 can be pharmacologically activated to artificially raise Sox9 reporter activity (Muramatsu et al., 2007). Removal of calcium channels such as CACNA1H in cartilage is also able to attenuate Sox9 expression (Lin et al., 2014). As pre-chondrocytes gradually differentiate into primordial chondrocytes, their membrane proteome likely evolves in channel expression to reflect the impending need for robust proliferation, volume expansion, and calcification.

Figure 1.2. Early chondrogenesis 8

Developing anlage

Condensing mesenchymal Cartilage anlage cells

Pre-chondrocytes Secreting collagens II, IX, XI Pre-chondrocytes at the periphery envelop the anlage

Pre-chondrocyte

Sox5, 6, 9 BMPs SMADs FGFs Prepare for Wnt/β-catenin signalling chondrocyte proliferation TRPV4 CACNA1H Other channelome proteins…

Developing Perichondrium epiphyseal plate

Developing bone 9

1.1.3 Chondrocyte proliferation, columnar formation, and pre-hypertrophy (see Figure 1.3)

By E11.5, chondrocytes have begun to proliferate and orient into a long shaft—the — surrounded on either end by globular masses—the epiphyses. Diaphysis growth occurs through the epiphyseal plate, where layers of phenotypically different chondrocytes exist as a spectrum toward eventual long bone development. While a portion of chondrocytes remain round and randomly distributed throughout the ECM—deemed resting chondrocytes—another population begins to proliferate, flatten, and organize into columns towards the diaphysis (Abad et al., 2002; Long, Schipani, Asahara, Kronenberg, & Montminy, 2001). BMP1/BMP2 receptors signal here with SMAD1, 5, and 8 to secrete type II collagen and proceed with cyclin-dependent kinase (Cdk) activation for cell division (Sherr & Roberts, 2004; Schmidl et al., 2006).

The matrilins, such as Matn1 and Matn3, are ECM proteins expressed at the proliferating stage (Yang et al., 2014). Matn3 is able to bind to Bmp2, suppressing its Col10a1 activation and delaying hypertrophy. Col10a1 encodes type X collagen, a marker of chondrocyte hypertrophy that is suppressed at the proliferating stage. Also, FGFR3 acts as a negative regulator of proliferation and accelerates hypertrophy through mitogen-activated protein kinase phosphorylation of Stat1 (Murakami et al., 2004). Furthermore, the Sox genes are still required: deletion of Sox5 and Sox6 block proliferation, and Sox9 delays hypertrophy (Smits, Dy, Mitra, & Lefebvre, 2004; Huang, Chung, Kronenberg, & de Crombrugghe, 2001). Sox9 protein degradation is a requisite for hypertrophy, and Runx2—a gene that will become more active in hypertrophy—is activated during pre-hypertrophy by Atf-3 to inhibit Sox9, cyclins, and Cdks (James, Woods, Underhill, & Beier, 2006; Yamashita et al., 2009).

However, as the columnar proliferating chondrocytes enter the pre-hypertrophic stage and prepare themselves for hypertrophy, the Ihh/PTHrP negative feedback loop plays one of the most critical roles (Kronenberg & Chung, 2001). As Kronenberg (2003) describes, PTHrP, the parathyroid hormone-related protein, is first secreted from the perichondrium and chondrocytes near the . It binds to proliferating chondrocytes and helps them retain their phenotype, promoting division and delaying hypertrophy. As these chondrocytes divide, form columns, and 10 eventually distance themselves sufficiently from the top of the epiphyseal plate—in a paracrine fashion—the level of circulating PTHrP is low enough for Ihh production. At this time, the chondrocytes have become pre-hypertrophic and cease with proliferation. Ihh, a member of the Hedgehog family, acts on Ihh receptors in the preceding resting and proliferating chondrocytes and increases their division rate (Chau et al., 2011). Simultaneously, Ihh signals to the chondrocytes near the epiphysis and stimulates PTHrP production, essentially retaining the proliferative phenotype and preventing hypertrophy in a negative feedback fashion. This way, the Ihh/PTHrP duo reduces possible fluctuations in hypertrophic maturation and promotes stability. Modulation of this gene pair therefore controls the spatial boundaries that the proliferative and hypertrophic zones can consume in the epiphyseal plate.

Similar to previous stages of endochondral ossification, the chondrocyte membrane induces diverse channel proteins to support differentiation. Ihh is a mechanosensitive gene that promotes the proliferative phenotype by receiving mechanical stress signals through membrane stretch- activated channels (Nowlan, Prendergast, & Murphy, 2008; Wu, Zhang, & Chen, 2001). Interestingly, aquaporin-5, an integral membrane protein and transporter of water, is expressed in human proliferating chondrocytes as well as the surrounding mesenchyme (Shimasaki, Kanazawa, Sato, Tsuchiya, & Ueda, 2018). As columnar chondrocytes exit the cell cycle, their channelome must begin to reflect the demand for hypertrophic differentiation by expressing membrane channel proteins that help increase intracellular volume. Here, water-selective pores such as aquaporins place their stake.

Figure 1.3. Chondrocyte proliferation, columnar formation, and pre-hypertrophy 11

Epiphysis Diaphysis Epiphysis

Developing epiphyseal plate

Pre-hypertrophic Proliferating chondrocytes Proliferating chondrocytes chondrocytes Resting Eventual direction of long bone growth Resting chondrocytes chondrocytes

Eventual direction of long bone growth

BMPs SMADs Pre-hypertrophic Proliferating Cdks chondrocyte chondrocyte Col2a1 Matns FGFR3 Prepare for pre- Ihh Prepare for Sox 5, 6, 9 hypertrophy hypertrophy Runx2 PTHrP

AQP5 Secreting collagens II, IX, XI Other channelome proteins . . . 12

1.1.4 Chondrocyte hypertrophy (see Figure 1.4)

By the time the developing embryo reaches E12.5-E13.5, the first wave of proliferating chondrocytes leave the cell cycle and begin differentiating into hypertrophic chondrocytes. A volume increase occurs prior to terminal differentiation. Chondrocyte volume increase contributes to growth rate and the final long bone length primarily through accumulation of water (Wilsman, Farnum, Leiferman, Fry, & Barreto, 1996; Buckwalter, Mower, Ungar, Schaeffer, & Ginsberg, 1986). However, whether the expansion occurs through imbalanced fluid uptake or through a matched growth of intracellular organelles was initially unclear. Cooper et al. (2013) explain that murine tibial epiphyseal plate chondrocytes undergo three specific phases of hypertrophy.

Using P5 mice, it was observed that chondrocytes first increase 3-fold from ~600 femtolitres (fl) to 2000 fl in volume—the internal macromolecules also grow proportionately and the dry mass density is therefore retained. In the second stage, a 4-fold increase in volume occurs from 2000 fl to ~8000 fl—but without an increase in internal dry mass, effectively quartering the dry mass density. In the third and final stage, the dry mass is permitted to stabilize before the chondrocyte enlarges 2-fold to ~14000 fl. The dry mass grows proportionately with this increase at this stage and the final density remains as approximately ¼ of the initial density. Cooper et al. (2013) show in a linear regression that without disproportional growth, the chondrocyte volume would not even reach 10000 fl if dry mass grew linearly. This process of hypertrophy, like endochondral ossification as a whole, is henceforth sequential and tightly regulated by a variety of factors.

BMPs generally promote hypertrophy as explored in a variety of cell culture experiments, but results can vary from stimulation to delay (Kobayashi, Lyons, McMahon, & Kronenberg, 2005). For example, mice overexpressing Bmp4 have enhanced chondrocyte hypertrophy but BMP7 addition to ATDC5 cell-line chondrocytes suppresses their hypertrophy (Tsumaki et al., 2002; Caron et al., 2013). As mentioned before, the Ihh/PTHrP duo tightly controls the rate of hypertrophy. Transcription factor Mef2c binds and activates Col10a1 as a marker of hypertrophy (Arnold et al., 2007). The hypoxia-inducible factors (HIFs) are heterodimers that can also bind the Col10a1 promoter potently to transition chondrocytes into hypertrophy (Saito et al., 2010). 13

Not surprisingly, the most common markers of hypertrophic chondrocytes are type X collagen as well as Mmp13, an enzyme responsible for collagen matrix restructuring for bone formation (Nurminskaya & Linsenmayer, 1996). Runx2 and Runx3 are both expressed during hypertrophy as requirements for chondrocyte maturation, albeit in a redundant manner (Inada et al., 1999). Runx2 directly binds and activates Col10a1, Mmp13, and Ihh to maintain the hypertrophic phenotype (Zheng et al., 2003; Selvamurugan, Kwok, Alliston, Reiss, & Partridge, 2004; Yoshida et al., 2004). Proteins important for impending bone formation and blood vessel invasion, such as osteopontin, osteocalcin, and VEGF, are also expressed during hypertrophy (Lian, McKee, Todd, Gerstenfeld, 1993; Horner et al., 1999). As for the Sox trio genes, they are likely turned off by Runx2 activity through reciprocal inhibition (Cheng & Genever, 2011).

As chondrocyte hypertrophy relies on water accumulation, members of the channelome capable of water transport likely play an important role. Passive water intake through aquaporin channels in articular chondrocytes surrounding the epiphyses have been well documented (Liang, Feng, & Ma, 2008; Mobasheri et al., 2004). Furthermore, Aquaporin-1 is expressed throughout rat epiphyseal plates and promotes hypertrophy (Claramunt et al., 2017). Other aquaporin isoforms may be present to conduct water intake but have not been largely explored in the epiphyseal zones. Channels such as calcium ion transporters can import calcium to activate calmodulin, RUNX2, and maintain the hypertrophic phenotype (Chen, Fu, Cong, Wu, & Pei, 2015). As well, channels that can import reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) may generate hypoxic environments necessary to increase HIF activity and promote hypertrophy (Lennicke, Rahn, Lichtenfels, Wessjohann, & Seliger, 2015). Members of the channelome, cellular volume expansion, and specific gene expression work together to prepare hypertrophic chondrocytes for terminal differentiation, ossification, and ultimately long bone growth.

Figure 1.4. Chondrocyte hypertrophy 14

Diaphysis

Epiphysis (not fully Epiphysis (not fully shown) shown)

Hypertrophic chondrocytes

Eventual direction of long bone growth

Hypertrophic Phases of chondrocyte chondrocyte hypertrophy

Pre-hypertrophic chondrocyte 1 Volume 3x, dry mass 3x

2 Volume x2, dry mass x2

Volume 4x

Result: 3 Volume ~24x Dry mass ~6x BMPs Aqp1 Dry mass density = ¼ initial density Mef2c Calcium ion transporters Prepare for Col10a1 Other channelome proteins . . . ossification HIFs Mmp13 Runx2, Runx3 15

1.1.5 Ossification and long bone growth (see Figure 1.5)

Thus far, the entire process of endochondral ossification has been occurring in the centre of the diaphysis; the primary centre of ossification. As Salazar, Gamer, & Rosen (2016) describe, the centre is dominated by hypertrophic chondrocytes and chondrocytes are sequentially less differentiated towards either of the epiphyseal ends, reflecting the spectrum of chondrocyte phenotypes. By E14.5, hypertrophic chondrocytes in the centre core undergo apoptosis and blood vessels invade to deliver hematopoietic stem cells (HSCs) that differentiate into . Osteoclasts are cells that perform matrix resorption, whereas osteoblasts—which are required after resorption—originate from MSCs similarly to chondrocytes and perform bone matrix formation instead (Caetano-Lopes, Canhão, & Fonseca, 2007). The collagen matrix left behind by the hypertrophic chondrocytes is excavated by osteoclasts and forms the cavity. Here, the remaining hypertrophic chondrocyte population is bisected by the vasculature and osteoclasts, forming a longitudinal growth axis with one population of chondrocytes pointing towards one epiphysis and the second population heading toward the other. As the populations grow further in distance appositionally from the marrow cavity, osteoblasts arrive with the invading blood vessels to synthesize osteoid for bone mineralization in the spaces where hypertrophic chondrocytes have died and osteoclasts have cleared.

At postnatal days 5-7 (P5-P7), vasculature invades the globular ends of each epiphysis and chondrocytes in their centre undergo the entire aforementioned process of differentiation towards hypertrophy (Xing, Cheng, Wergedal, & Mohan, 2014). These secondary centres of ossification ossify in the same manner, with the chondrocytes spreading to the ends of the bone becoming articular cartilage, and the chondrocytes approaching either one of the primary centre populations forming a union known as the epiphyseal plate. Eventually, ossification from either end of the plate permeates into the chondrocyte population, causing the plate to narrow until the diaphysis meets the epiphysis and long bone growth ceases. The primary centre of ossification eventually becomes the bone marrow cavity, while the secondary centre of ossification is filled with trabecular bone. Trabecular bone is a porous, spongy tissue found at the epiphyses and is the main load-bearing bone in vertebrates (Oftadeh, Perez-Viloria, Villa-Camacho, Vaziri, & Nazarian, 2015). The outer layer surrounding the bone is hard cortical bone, the compact tissue 16 enveloping the diaphysis (Eriksen, 2010). The process of ossification therefore requires many steps of regulation as the entire long bone structure lengthens. Here, osteoblastogenesis is critical to promote bone growth.

Lefebvre & Bhattaram (2010) succinctly describe the roles of osteoblasts: 1) to secrete non- mineralized osteoid for later mineralization, 2) to secrete alkaline phosphatase and provide inorganic phosphate for mineralization, and 3) to produce osteocalcin and other bone-specific proteins to help mineralize the osteoid matrix. Osteoblasts primarily rely on Runx2 as a master factor to differentiate from mesenchymal stem cells (MSCs); lack of Runx2 at the ossification stage completely ablates bone formation (Otto et al., 1997). Osx is another transcription factor that activates several bone genes—Col1a1 for type 1 collagen, Bgp for osteocalcin, and Bsp for bone sialoprotein—in MSCs and transforms them into functional osteoblasts (Nakashima et al., 2002; Sinha, Yasuda, Coombes, Dent, & de Crombrugghe, 2010). However, osteoblasts are not only derived from MSCs. Hypertrophic chondrocytes in the epiphyseal plate are also able to transdifferentiate into osteoblasts as an alternative to the apoptotic fate (Tsang, Chan, & Cheah, 2015). Here, Runx2 and Osx can stimulate them to re-enter the cell cycle, secreting Col1a1 and osteocalcin as newly differentiated osteoblasts. This optional ‘chondro-osteoblastic’ lineage highlights that osteogenic differentiation of hypertrophic chondrocytes is yet another point of control in ossification.

In the case of rare metaphyseal chondrodysplasias—diseases that affect skeletal stature—such as MCDS, a failure of chondro-osteoblastic differentiation is responsible for delayed ossification, shorter long bones, and bowed legs (Ho et al., 2007). Nevertheless, general osteoblastogenesis control still relies on many key factors from previous steps of endochondral ossification. Wnt/β- catenin signalling is required in osteoblasts after Osx expression for osteoblasts to mature and produce osteocalcin (Day & Yang, 2008). FGFR3 is a promoter of MSC-osteoblast differentiation, but inhibitor of mature osteoblast mineralization (Su, Jin, & Chen, 2014). BMP2 also upregulates Runx2 and Osx expression during osteoblastogenesis (Matsubara et al., 2008).

During ossification, gene dysregulation can lead to abnormal length in specific bones (Panda, Gamanagatti, Jana, & Gupta, 2014). Unnatural shortening of the major long bones can be 17 rhizomelic—affecting the proximal limbs; the humerus and femur—or mesomelic—affecting the distal limbs; the radius, ulna, tibia, and fibula. Aside from mutations in the main players of endochondral ossification, genes located on the sex chromosomes can also cause dysplasias. Mutation of SHOX, the Short Stature Homeobox-containing gene, can cause idiopathic short stature (Marchini, Rappold, & Schneider, 2007). Interestingly, the malformation of singular or multiple individual bones can also occur; however, these are classified as dysostoses instead of dysplasias (Offiah & Hall, 2003). Overall, ossification may not necessarily proceed at the same rate in chiral limbs. Furthermore, irregular ossification and abnormal skeletal stature may arise from mutations not necessarily related to the major genes in the epiphyseal plate.

In the channelome, aquaporin-5 enhances MSC apoptosis in the bone marrow through high water permeability and decelerates femoral , where osteoblasts play a critical role (Yi et al., 2012). In the osteoblast nuclear membrane, vitamin D receptors receive the 1,25D hormone to stimulate bone-specific protein production and matrix secretion through chloride and calcium channel activation (Wang, Zhu, & DeLuca, 2014; Zanello & Norman, 2004). Overall, a myriad of genes, transcription factors, and protein channels dictate the cellular profile and ossification status of the growing long bone. To further examine their specific contributions, the generation of mutant mouse models is paramount to understanding effectors of bone elongation. The deletion or overexpression of important aforementioned genes—such as Sox9 or Ihh—has helped provide insight into the known and unknown processes affecting endochondral ossification.

Figure 1.5. Ossification and long bone growth 18

Epiphysis Epiphysis Cortical bone

Diaphysis

Epiphyseal plate Epiphyseal plate Growing bone Growing bone tissue Bone tissue Secondary centre Secondary centre (osteoblasts, marrow (osteoblasts, Developing of ossification Developing of ossification osteoclasts, cavity osteoclasts, articular (developing articular (developing trabecular bone) trabecular bone) cartilage trabecular bone) cartilage trabecular bone)

Growing Narrowing Narrowing Growing Primary centre of secondary epiphyseal epiphyseal secondary ossification centre plate plate centre

Direction of long bone growth

Terminal hypertrophic Osteoblast (forms bone matrix) (resorbs bone matrix) chondrocyte Osx Aqp5 Col1a1 Vitamin D receptors & Bgp chloride/calcium channels Prepare for Bsp Other channelome proteins . . . apoptosis or Runx2 transdifferentiation FGFR3 BMP2 HSCs MSCs 19

1.2 Models of differential long bone growth

The house mouse, Mus musculus, is an invaluable model organism to understanding human biology due to strikingly similar genomes and physiology (Perlman, 2016). Using homologous recombination or Cre-Lox recombination, genes implicated in development and disease can be knocked out globally or in specified sites throughout the mutant. However, both knockout and overexpression models are useful in determining the effect of a gene in absence or abundance. Here, murine mutants of the most critical transcription factors in endochondral ossification and their skeletal phenotypes are described in detail. Their human mutation counterparts, if known, are also described (see Table 1.1).

1.2.1 Sox mutants

As previously mentioned, the Sox protein trio is important towards chondrogenesis and early differentiation, but delay hypertrophy. Sox9flox/flox; Prx1-Cre embryos, which have targeted limb bud deletion of Sox9 prior to mesenchymal condensation, are unable to form either cartilage or bone (Akiyama et al., 2002). In the same study, Sox9flox/flox; Col2a1-Cre embryos, which have Sox9 deletion after condensation, present with severe chondrodysplasia. Akiyama et al. (2004) also examined Sox9 overexpression with Col2a1/Sox9 knock-in embryos, observing decreased chondrocyte proliferation, delayed hypertrophy, and diminished long bone formation. Interestingly, Smits et al. (2001) found that Sox5 and Sox6 single-null mice presented with mild skeletal abnormalities. However, Sox5; Sox6 double-null fetuses present with severe chondrodysplasia, suggesting that Sox5 and Sox6 are redundant but essential together for bone growth. Sox5 and Sox6 help secure Sox9 to the Col2a1 enhancer for type II collagen production (Han & Lefebvre, 2008).

Overall, the Sox trio carefully regulates endochondral ossification and fluctuations in expression—up or down—can severely dysregulate the final skeletal structure. In humans, any mutations in the SOX9 coding region can lead to campomelic dysplasia: a typically lethal disorder resulting in long bone bowing and other skeletal defects (Mansour et al., 2002).

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1.2.2 Bmp mutants

Bmp signalling is present in all stages of endochondral ossification and primarily functions through Smad pathways (Retting, Song, Yoon, & Lyons, 2009). Limb bud deletion of Bmp2 and Bmp4 from MSCs severely impairs osteogenesis (Bandyopadhyay et al., 2006). However, Bmp4flox/flox; Col2a1-Cre embryos alone do not display a striking cartilage phenotype—only when Bmp2 is deleted alongside do severe chondrodysplasias occur (Shu et al., 2011). In the study, it was concluded that Bmp4 alone is insufficient to affect chondrocyte differentiation but Bmp2 alone is. Also, Bmp6 -/- mice at 10 weeks of age have smaller long bones and reduced longitudinal growth rate following estrogen treatment (Perry, McDougall, Hou, & Tobias, 2008). The BMP6 promoter is transcriptionally regulated by estrogen receptor α, suggesting that Bmp6 may be hormonally stimulated (Ong, Colley, Norman, Kitazawa, & Tobias, 2004). Yet, not all Bmp mutants with bone phenotypes have diminished growth. GDF-7 is the murine homologue of human BMP12 and GDF-7 -/- mice have accelerated endochondral bone growth, due to a shorter hypertrophic phase duration (Mikic, Ferreira, Battaglia, & Hunziker, 2008). Mutations in specific Bmp isoforms do not all result in long bone defects, but many known isoforms are important in embryogenesis. In humans, dysregulation of BMP signalling can lead to osteogenesis imperfecta—through BMP1—and osteoarthritis—through BMP5 and BMP14 (Wang et al., 2014).

1.2.3 Fgfr mutants

The Fgfs mediate many endochondral cellular responses by binding to the four unique Fgfrs (Su, Jin, & Chen, 2014. Since activated Fgfrs then phosphorylate and activate pathways downstream, modulating Fgfr expression greatly influences the efficacy of Fgf signalling (Ornitz, 2005). Fgfr1flox/flox; Col2-Cre embryos have osteo-chondroprogenitor cells with delayed osteoblast differentiation, but show increased bone mass by adulthood (Jacob, Smith, Partenen, & Ornitz, 2006). In humans, activating mutations of FGFR1 leads to osteoglophonic dysplasia and rhizomelic dwarfism, where proximal limbs are shortened (White et al., 2005). Fgfr2 mutants have bone defects which are mainly cranial rather than endochondral. However, deletion of the 3c alternative of Fgfr2 results in premature loss of growth and diminished long bones 21

(Eswarakumar et al., 2002). Human gain-of-function mutations of FGFR2 cause a variety of craniosynostes, which are premature fusions of skull sutures (Wilkie, 2005). In Fgfr3 mutants, activating mutations result in shortened long bones, disorganized proliferating zone columns, and smaller body size overall (Ornitz & Marie, 2002). Deletion of the 3c isoform of Fgfr3 results in overstimulation of the proliferating zone and dramatic skeletal overgrowth with reduced bone mineral density (Eswarakumar & Schlessinger, 2007). In humans, Ornitz & Marie (2002) also review that a variety of different FGFR3 point mutations result in many dysplasias, such as achondroplasia and hypochondroplasia. On the contrary, Fgfr4 -/- mutants are conversely normal in development—but when deleted alongside Fgfr3 in a double knockout, they are dwarfed in overall size (Weinstein, Xu, Ohyama, & Deng, 1998). Overall, Fgfrs control balance among the cellular players of skeletal growth.

1.2.4 Ihh & PTHrP mutants

Indian hedgehog and PTHrP are responsible for controlling the rate at which chondrocytes undergo hypertrophy, meaning that their modulations should critically alter the long bone phenotype in mutants. Indeed, Ihh -/- mutants show severe shortening of limbs at all endochondral ossification embryonic timepoints with almost completely reduced appendicular bones by the newborn stage (St-Jacques, Hammerschmidt, & McMahon, 1999). These mutants have drastically reduced chondrocyte proliferation, as well as delayed chondrocyte maturation into hypertrophy. In humans, homozygous mutations of IHH result in acrocapitofemoral dysplasia: a rare autosomal recessive disorder featuring short limbs and irregular-shaped hands and hips (Hellemans et al., 2003). As the partner of Ihh, PTHrP mutations display similar phenotypes. The epiphyseal plates of PTHrP -/- mutants have markedly shorter proliferating zones as well as advanced hypertrophy, apoptosis, and terminal mineralization (Lee et al., 1996). Without surprise, human mutations of PTHrP result in chondrodysplasia (Nissenson, 1998).

1.2.5 Runx2 mutants

Runx2 plays a pivotal role in the hypertrophic and mineralization stages of endochondral ossification. Runx2 -/- mutants die shortly after birth due to a complete lack of ossification 22

(Komori et al., 1997). Takarada et al. (2013) show that even Runx2flox/flox; Col2-Cre mutants are unable to prevent this perinatal lethality, suggesting that Runx2 is a requirement for proper endochondral ossification in both embryonic and postnatal stages. However, they also show that Runx2flox/flox; Col1-Cre present with no observable skeletal abnormalities. Runx2 may therefore be important but redundant by the time committed osteoblasts arrive to build bone, where Col1a1 is expressed. Using the same type 1 collagen promoter, Runx2 was overexpressed in osteoblasts (Liu et al., 2001). Surprisingly, these transgenic mutants are stunted in growth, prone to fractures, and have diminished osteoblast mineralization capacities. Runx2 is likely a negative regulator of bone growth in late osteoblast development to control bone mass, but normally promotes mineralization at physiological levels of expression. In humans, the importance of RUNX2 is also evident. RUNX2 haploinsufficiency causes cleidocranial dysplasia, where the clavicles, teeth, and overall stature are underdeveloped (Xu et al., 2017).

1.2.6 Mutant models with membrane protein and channelome modulation

Of the aforementioned critical players of endochondral ossification, proteins such as Fgf, Bmp, and Ihh bind their receptors on the plasm membrane for downstream signalling. However, the chondrocyte membrane is not static; its protein expression must change to reflect every stage of its transformation during the long bone growth process. The channelome is home to a variety of membrane proteins that are gaining explorative value as more mutant mice are generated to investigate their skeletal outcomes.

Pannexin-3, or Panx3, is one such transmembrane gap junction channel with robust expression in skin and cartilage (Penuela et al., 2007). As Panx3 is also expressed in pre-hypertrophic chondrocytes, hypertrophic chondrocytes, and osteoblasts, Oh et al. (2015) generated Panx3 -/- mice to observe their skeletal phenotypes. Panx3 -/- embryo epiphyseal plates showed delayed hypertrophy, delayed osteoblast differentiation, delayed mineralization, and ultimately shortened long bones that persisted till adulthood. Indeed, the Panx3 promoter region contains binding sites where Runx2 may act and promote bone growth (Bond et al., 2011). Adam17 is a membrane protein that functions as a metalloproteinase, releasing factors critical to hypertrophy such as tumor necrosis factor alpha (Hall & Blobel, 2012). Hall & Blobel (2012) generated 23

Adam17flox/flox; Col2a1-Cre mutants and showed that newborn mice have expanded hypertrophic zones with retarded long bone growth. Concerning hypertrophy, it should be reiterated that fluid uptake—primarily water—is the root mechanism for chondrocyte enlargement. Investigating the channelome members that transport water, the aquaporins, may be valuable to deciphering how ubiquitous membrane proteins contribute to bone growth. However, mutant mice with modulated aquaporin isoform genes are not well explored, and neither are the examination of their chondro- osseous physiologies. Previously, Wu et al. (2007) ventured to measure the femoral of aquaporin-1-null mice and found that 2-month old mutants had reduced density, calcium, and phosphorous in the bones. A focus on the precursors to ossification—the different chondrocyte phenotypes—will characterize how these bone irregularities might arise. Specifically, the aquaporins show promise as effectors of chondrocyte gene expression, differentiation, and potentially long bone growth.

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Table 1.1. Models of differential bone growth

Mutation / Affected gene(s) Hallmarks Disorder (if described) BMP1 Fragile bones Osteogenesis imperfecta BMP14 Articular cartilage damage Osteoarthritis Bmp2C/C ; Bmp4C/C Severe chondrodysplasia - Bmp4flox/flox; Col2a1-Cre Mild cartilage abnormalities - BMP5 Articular cartilage damage Osteoarthritis Bmp6 -/- (w/ estrogen) Smaller long bones, reduced growth rate - Decreased chondrocyte proliferation, delayed Col2a1/Sox9 knock-in - hypertrophy, decreased long bone formation Craniofacial abnormalities, dwarfism; proximal Osteoglophonic dysplasia; FGFR1 (activating) limb shortening rhizomelic dwarfism Delayed osteoblast differentiation, increased Fgfr1flox/flox; Col2-Cre - adult bone mass FGFR2 Premature fusions of skull sutures Craniosynostis Fgfr2 (3c) Chondrodysplasia - Skeletal overgrowth, reduced bone mineral Fgfr3 (3c) -/- - density Dwarfism, disorganized epiphyseal plate Fgfr3 (activating) - proliferating zone columns GDF-7 -/- (murine homologue Accelerated endochondral bone growth - of BMP12) IHH Shortened long bones, irregular hands and hips Acrocapitofemoral dysplasia Ihh -/- Severe chondrodysplasia - PTHrP Chondrodysplasia - Shorter epiphyseal plate proliferating zones, PTHrP -/- advanced hypertrophy and mineralization Short stature, underdeveloped clavicles and RUNX2 (haploinsufficiency) Cleidocranial dysplasia teeth Fragile bones, diminished mineralization, Runx2 (transgenic) - stunted growth Runx2 -/- Absence of ossification, perinatal lethality - Runx2flox/flox; Col2-Cre Perinatal lethality - Sox5 -/- Mild skeletal abnormalities - Sox5; Sox6 -/- Severe chondrodysplasia - Sox6 -/- Mild skeletal abnormalities - Lethality, long bone bowing, generalized SOX9 Campomelic dysplasia skeletal defects Sox9flox/flox; Col2a1-Cre Severe chondrodysplasia - Sox9flox/flox; Prx1-Cre Absence of cartilage and bone - 25

1.3 Aquaporins and chondrocytes: expression, function, and regulation

Aquaporins (AQPs) are a subfamily of integral membrane proteins that facilitate water and solute conductance in a wide variety of physiological structures (Agre, 2006). AQPs are distinguished from other integral transporters by a series of conserved hydrophobic residues and a signature ‘NPA’ motif (Kruse, Uehlein, & Kaldenhoff, 2006). Functionally, AQPs are defined as the isoforms that display selectivity in water and small solute transport across a membrane (Takata, Matsuzaki, & Tajika, 2004). Thirteen mammalian isoforms (AQP0—AQP12) exist throughout the human body with tissue-specific patterns and a higher concentration in organs that constantly process water, such as the kidneys. However, homeostatic fluid transport is an integral function to all healthy cells in maintaining basic tissue upkeep and controlled growth. In particular, chondrocytes undergo notable water transport during a) volume changes in resting and loaded cartilage tissue at the articular region, and b) phased volume changes for hypertrophy in the epiphyseal plate that underlie skeletal growth (Oswald, Chao, Bulinkski, Ateshian, & Hung, 2008; Cooper et al., 2013). The chondrocyte-aquaporin relationship has been explored among a variety of isoforms and substantiates the importance of the channelome in chondrocyte proliferation, growth, and apoptosis.

1.3.1 AQP1

Aquaporin-1 (AQP1) was initially discovered as a tetrameric 28-kDa integral membrane protein in human red blood cells when co-purified with the 32-kDa subunit of the Rh polypeptides (Agre, Saboori, Asimos, & Smith, 1987). Originally named CHIP28 (channel-forming integral protein of 28 kDa), expression of the gene in the X. laevis oocyte expression system exhibited swollen oocytes with higher osmotic water permeability coefficients than their control counterparts when placed in osmolarity-tempered solution (Agre et al., 1993). This demonstrated that an abundant, archetypal membrane channel was capable of specific water transport and confirmed its existence in a variety of mammalian organs as the newly termed ‘aquaporin’. The wide distribution of AQP1 in ordinary human tissue was first illustrated using tissue microarray analysis and visualized with immunohistochemistry, where the protein was found to be strongly localized in chondrocytes within the deep zone of articular cartilage (Mobasheri & Marples, 26

2004). This presence supported a role of AQP1-mediated water transport across the chondrocyte membrane, suggesting that compressive and osmotic forces finely balanced by the protein channel may be necessary in the maintenance of healthy cartilage. Water content and type II collagen collectively contribute to articular cartilage load bearing, where the loss of either component can strain the other with a greater weight burden (Mow, Kuei, Lai, & Armstrong, 1980; Fox, Bedi, & Rodeo, 2009). As dysregulated water content in articular chondrocytes may promote pathophysiological degradation of cartilage collagen fibers, these findings were extrapolated to investigations in the possible contribution of AQP1 to osteoarthritis—a degenerative disease featuring breakdown of cartilage in articular regions.

Meng, Ma, Li, & Wu (2007) simultaneously measured Aqp1 and Aqp3 mRNA expression in adult rats after inducing osteoarthritis in their temporomandibular , observing that Aqp3— but not Aqp1—mRNA was highly upregulated in the dissected cartilage groups. Gao et al. (2011) then focused on determining a relationship between Aqp1 expression and osteoarthritis, albeit in the long bone joints. After amputation of knee ligaments and partial damage to the medial menisci in different test groups, aged Sprague-Dawley rats presented with significantly elevated Aqp1 mRNA, Caspase-3 mRNA, and Caspase-3 protease activity several weeks post- operation. As caspases execute cell apoptosis, the concomitant rise of Aqp1 expression suggested a positive co-regulatory relationship between the two genes in chondrocyte death and henceforth the onset of osteoarthritis.

Musumeci et al. (2013) further clarified the relationship between Aqp1 and osteoarthritis by performing medial and lateral meniscectomies on thirty-six rats, observing that Aqp1 expression was increased in the test groups via immunochemistry and Western blot analysis. In human chondrocytes, AQP1 mRNA is also significantly higher in osteoarthritic cartilage and is further elevated through IL-1B treatment, hinting at an inflammation-controlled regulation of AQP1 in osteoarthritis progression (Haneda et al., 2018). Thus, the breadth of AQP1-osteoarthritis investigations suggests that the aquaporin may exacerbate osteoarthritic swelling in the articular regions and compromise the cellular morphology and metabolism of chondrocytes—although a purported mechanism has not been elucidated thus far. Overall, AQP1 plays a functional role in 27 chondrocyte water uptake and its expression is implicated as a contributor to the onset of osteoarthritis.

1.3.2 AQP2

The concrete ability of aquaporins to perform bidirectional transport of water in cartilage further led to investigations of their presence in chondrocyte-like cells. Aquaporin-2 (AQP2) expression was initially measured in the nucleus pulposus and annulus fibrosus of human intervertebral discs, both consisting of hyperosmotic responsible for absorbing daily compressive loads in bodily movement (Richardson, Knowles, Marples, Hoyland, & Mobasheri, 2008). The study sought to discover if AQP1, AQP2, and AQP3 were localized in discs and controlled cell volume and extracellular matrix metabolism. While their immunohistochemistry was negative for AQP2 protein in all fibrocartilage region cells, Gajghate et al. (2009) found AQP2 protein expression in vivo in both rat and humans nucleus pulposus tissue, regulated by the tonicity- enhancer binding protein TonEBP. Using mouse embryonic fibroblasts—a precursor of the murine chondrocytic lineage—they demonstrated that TonEBP/NFAT5-null mutants experience ~50% decreases in Aqp2 promoter activity. AQP2 is largely unexplored in chondroskeletal studies; yet its capacity to control water balance in chondrocyte-like resident cells by the osmosensitive transcription factor TonEBP suggests that other aquaporin isoforms are also transcriptionally regulated for cellular volume control. In chondrocytes, the diverse membrane channels and porins rely on these signalling molecules to control not only disease—such as osteoarthritis in the articular region—but also their natural differentiation in strictly-regulated processes such as endochondral ossification. In chondrocyte hypertrophy, water accumulation is a defining characteristic (Buckwalter et al., 1986). Therefore, aquaporins—the only known membrane proteins that directly transport water—likely contribute to hypertrophy during endochondral ossification (Kozono, Yasui, King, & Agre, 2002).

1.3.3 AQP3

Several aquaporin family members—deemed ‘aquaglyceroporins’—are also capable of glycerol and uncharged solute transport in addition to water. Aquaporin-3 (AQP3) was the first 28 mammalian aquaglyceroporin to be cloned and was explored in chondrocytes to determine if equine articular cartilage expressed aquaporins as a means of volume regulatory behavior (Mobasheri & Marples, 2004). AQP3 and AQP1 were two isoforms expressed in the articular region, suggesting their roles in the transport of metabolites and water respectively across the chondrocyte membrane. Indeed, AQP3 was later found to be expressed in human articular chondrocytes through human tissue microarrays (Mobasheri et al., 2005). Due to the presence of both AQP3 and AQP1 in the articular region, both members were investigated during the chondrogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Graziano et al., 2018). Chondrogenic markers including SOX9, aggrecan, and type II collagen were measured during a four-week period alongside AQP3 and AQP1. Graziano, Avola, Pannuzzo, & Cardile (2018) discovered that while AQP1 protein levels decreased after 21 days, AQP3 protein persisted at high levels by 28 days. This suggests that while AQP1 may play important roles at the earlier stages of differentiation, AQP3 is necessary throughout the process and its alteration may lead to chondrogenic death and cartilage damage. As AQP3 is also expressed in other immature cell types such as murine bone-marrow derived dendritic cells and bovine blastomeres, aquaporins may be responsible for important developmental aspects in the embryonic stages in addition to postnatal conditions such as osteoarthritis (Song et al., 2011; Zhao et al., 2015). In particular, the importance of AQP3 expression during chondrogenesis highlights a critical role of aquaglyceroporins in chondrocyte generation. Other members of the aquaglyceroporin family—AQP7, AQP9, and AQP10—may play special functional roles during prenatal processes, although their chondrogenic significance has yet to be delineated.

1.3.4 AQP4

Aquaporin-4 (AQP4) is largely explored in brain disorders, with a particular focus on its involvement in astrocyte activity and the central nervous system (Verkman, Smith, Phuan, Tradtrantip, & Anderson, 2017). However, Aqp4 is expressed in rat articular chondrocytes and its siRNA inhibition during IL-1B-induced apoptosis reduces chondrogenic death by reducing p38 MAPK activity (Cai et al., 2017). Similar to the AQP1 findings of Haneda et al. (2018), Aqp4 likely contributes to chondrocyte apoptosis in diseases such as osteoarthritis and rheumatoid arthritis. The presence of all aquaporins isoforms in varying tissues has not yet been 29 clearly characterized, and Aqp4 appears to have therapeutic value as a knockdown target in organs aside from the brain. Due to the ubiquitous nature of aquaporins in a wide range of mammalian cells, even uninvestigated isoforms may emerge as promising candidates as meta- analyses continue revealing relationships between the channelome and diseases. The rising recognition of aquaglyceroporins in metabolic syndrome and obesity-related pathologies substantiates this phenomenon (da Silva, Rodrigues, Rebelo, Miranda, & Soveral, 2018).

1.3.5 Other aquaporins in chondrocyte activity

The remaining mammalian aquaporin isoforms in relation to chondrocytes have been largely uninvestigated. Aquaporin-0 (AQP0) expression has been discovered in osteoarthritic articular cartilage, but is mostly explored in its essential homeostatic role in the lens of the eye (Haneda et al., 2018; Schey, Petrova, Gletten, & Donaldson, 2017). Aquaporin-5 (AQP5) is regulated by BMP6—an inducer of cartilage and bone growth—in the salivary gland of Sjögren's syndrome- like mice, yet its relationship to chondrocyte function is tangential (Lai et al., 2016). Aquaporin- 6 (AQP6) is expressed in Meckel’s cartilage in human orofacial tissues, but there are no studies in its function in articular and epiphyseal plate chondrocytes (Wang et al., 2003). Aquaporin-7 (AQP7) is expressed in adipose tissue, and its deficiency results in increased glycerol kinase activity, triglyceride accumulation, and ultimately obesity and a Type-2 diabetic phenotype (Iena & Lebeck, 2018). Interestingly, these phenotypes are reminiscent of metabolic syndrome symptoms, which include inflammation and osteoarthritis. Beyond cartilage damage from obesity-added compressive forces, hyperglycemia from insulin resistance triggers production of mitochondrial reactive oxygen species, inflammatory mediators, and metalloproteinases in chondrocytes (Courties, Sellam, & Berenbaum, 2017). It is possible that Aqp7-null mice chondrocytes are abnormally prone to degradation in the articular region and hypertrophy in the epiphyseal plate due to these factors, although a definitive study has not been made.

The other aquaporin isoforms (8, 10, 11, and 12), aside from Aquaporin-9 (AQP9), have not been explored in fields related to cartilage, bone, or pathologies that influence cartilage or bone development. AQP11 and 12 are ‘super-aquaporins’ that are more recent discoveries and have been briefly explored in major murine organs without attention to chondrocytes or skeletal 30 phenotypes (Ishibashi, Tanaka, & Morishita, 2014). As a whole, the majority of aquaporin investigations focus on isoforms in physiological structures that unambiguously require water transport due to the nature of their functions. The chondrocyte-aquaporin relationship is delineated in several isoforms, but characterization of previously unexplored aquaporins in chondrocytes will highlight the importance of the channelome in both the articular and epiphyseal zones. In particular, AQP9 is a strong candidate for investigation in chondrocyte function due to its indiscriminate conductance nature and supporting literature for its relationship with hypertrophy-inducing mediators. It has previously been explored in bone, osteoclasts, and synoviocytes. A definitive study of AQP9 in chondrocytes may unveil an aquaporin isoform with the capacity to control chondrocyte proliferation, hypertrophy, death, and even biogenesis.

1.4 Characterization of AQP9 and its novel role in chondrocyte and bone activity

AQP9 was initially discovered in a systematic gene analysis of human adipocytes which revealed a unique sequence encoding for a 342-amino-acid membrane protein (Kuriyama et al., 1997). This channel was named ‘Aquaporin-9’ and was tested via expression in X. laevis oocytes, demonstrating a potent 7-fold increase in water permeability and the ability to conduct glycerol. Over a decade of literature has revealed its capacity to facilitate transport of urea, purines, pyrimidines, arsenic, hydrogen peroxide, and a variety of other small uncharged solutes (Viadiu, Gonen, & Walz, 2007; Liu et al., 2002; Watanabe, Moniaga, Nielsen, & Hara-Chikuma, 2016). The indiscriminate substrate specificity of AQP9 makes it unique among other aquaporin isoforms, where it may conduct a wider range of solutes that regulate cellular metabolism. Given that endochondral ossification relies on harmony between transcription factors, gene expression, and channelome activity, the liberal solute conductance of AQP9 shows promise as an important aquaporin in chondrocytes as they differentiate and extend bone length.

1.4.1 General aquaporin protein structure

All aquaporin isoforms are tetramers and emit a positive electrostatic field that prohibits cations from passing through (Rothert, Rönfeldt, & Beitz, 2017). Each monomer forms an independent 31 pore, and all aquaporins share a common intramembranous protein fold comprised of two amino acid tandem repeats in opposing directions. These repeats largely define aquaporin function and ultimately the identity of solutes that chondrocytes may receive during regulated processes, such as differentiation in the epiphyseal plate. In the first repeat, 3 helices aptly named ‘helices 1-3’ are present alongside a reentrant loop named ‘B’. Yan & Luo (2010) characterized that reentrant loops are important structural motifs in alpha-helical transmembrane proteins that penetrate halfway into the membrane and then return to their side of origin. These loops feature low hydrophobicity and may serve to limit the amount of water permeated through aquaporins, as more hydrophilic residues in the inner pore constriction can attract water molecules to the channel walls and raise the energy cost of passage (Murata et al., 2000). In the second repeat, 3 more helices named ‘helices 4-6’ are present alongside a reentrant loop named ‘E’. Loops B and E form the short pore-lining alpha-helices HB and HE respectively, which contain important residues located midway into the channel. These loops also contain the hallmark motifs of aquaporins, the Asn-Pro-Ala (NPA) motifs. These two motifs are co-localized and attract passing water molecules to the same side, where the helices HB and HE are nestled midway into the membrane. Murata et al. (2000) discovered that as water molecules pass through the pore in single-file due to efficient hydrogen-bond arrangement, Asn residues 76 and 192 from HB and HE respectively will use their amido groups to force every incoming water molecule to switch their hydrogen-bonding partners to themselves instead. Having abandoned the hydrogen bonds formed with the molecule ahead and the molecule above, the targeted water molecule hydrogen- bonds to the Asn residues and is contorted perpendicularly to the direction of water flow. With the target’s hydrogen atoms facing 90° from the single-file water chain, the efficient stream is broken. However, Murata et al. (2000) further explained that the pore walls feature exclusively hydrophobic residues at the Asn constriction site, promoting exit and total permeation of water molecules through the channel with an ultimately low energy barrier. For cations such as protons that rely on a stable ‘proton wire’ built on the hydrogen bonds of the single-file water chain, the chain breakage prohibits them from passage through the channel (Pomès & Roux, 1996). The relatively tight intramembranous constrictions and wider membranous openings of aquaporins creates a high dielectric barrier to the majority of ions. Hence, neutral solutes are favored for passage among all aquaporin isoforms.

32

1.4.2 AQP9 protein structure (see Figure 1.6)

Despite their shared structural features and resulting permeation capacities, AQP9 is distinguished by having the widest range of substrate specificity among the aquaporins. Viadiu et al. (2007) created an AQP9 projection map by reconstituting glycosylated rat AQP9 into two- dimensional crystals with aid from magnesium and calcium ions. The crystals were then embedded in glucose, frozen, and processed at 7 angstrom resolution to view the protein structure. Interestingly, the projected AQP9 tetramer resembled the E. coli-derived glycerol facilitator protein GlpF with greater fidelity than pure water-specific aquaporins, such as AQP0 and AQP1. AQP9 and GlpF monomer projections presented with a more square-shaped phenotype than those of AQP0, which are more wedge-shaped in characteristic. Furthermore, GlpF boasts a round pore diameter of 7 angstroms while AQP9 has a more oval-shaped pore of approximately 7x12 angstroms in dimension. Viadiu et al. (2007) identified a region of lower electron density existing in both GlpF and AQP9 specifically, which may explain their capacities to conduct larger solutes. AQP9 not only boasts a larger pore area, but also has ‘tripathic pores’ where each channel has a hydrophobic corner, a hydrogen-bond donor corner, and a hydrogen- bond acceptor corner (Stroud et al., 2003). These designated areas allow for substrates to be oriented by region, increasing their likelihood of reaching an optimal spatial configuration and passage. Viadiu et al. (2007) analyzed this characteristic and identified seven amino acid residues conserved among all AQP9 homologues, but different from other aquaporins and GlpF. These residues are Phe 64, Gly 81, Met 91, Val 176, Phe 180, Leu 209 and Cys 213. As most of these substitutions occur at the hydrophobic corner near the entrance of the pore, the residues themselves may be responsible for the lower density and easier substrate access. Overall, AQP9 is an isoform that features larger pores, a lower density barrier, and unique residues compared to other aquaporin members. Describing substances that the channel conducts will provide insight into AQP9 function in chondrocyte differentiation and long bone growth.

Figure 1.6. AQP9 protein structure 33

Pore size (~7Å x 12Å)

12Å Aqp9 monomer

7Å

Conducts:

Water Urea Purines Pyrimidines Arsenic Hydrogen peroxide Glycerol Lactate Selenite

Tripathic pore components and unique residues Projection map of AQP9 tetramer 34

1.4.3 AQP9 protein function (see Figure 1.6. cont.)

The elucidated pore size and residue composition of AQP9 grants the ability to transport both orthodox and unorthodox molecules. In the chondrocyte channelome, many membrane proteins are multifunctional and can be ion channels, receptors, and signalling elements all in one (Mobasheri et al., 2018). The diversity of substrates that a channel can tolerate may parallel the amount of control it has over highly regulated and sequential processes, such as endochondral ossification. Aside from water, AQP9 is permeable to glycerol, urea and smaller molecules including purines and pyrimidines (Tsukaguchi, Weremowicz, Morton, & Hediger, 1999). Lactate is also transported by AQP9 (Badaut & Regli, 2004). Chondrocytes rely heavily on glycolysis in culture and glycerol may be imported by AQP9 as gluconeogenic fuel (Jackson, Huang, & Gu, 2012). Conversely, lactate is a by-product of glycolysis and is produced heavily during chondrocyte growth (Hossain, Bergstrom, & Chen, 2015). AQP9 may be important in exporting excess lactate to maintain physiological pH and cell growth rate. As a neutral solute conductor, AQP9 is also permeable to arsenic and even selenite (Liu et al., 2002; Geng et al., 2017). Interestingly, arsenic contributes to telomere attrition and apoptosis by creating ROS, which is an inducer of chondrocyte hypertrophy (Liu, Trimarchi, Navarro, Blasco, & Keefe, 2003; Morita et al., 2007). It is sensible to question if AQP9 in the chondrocyte channelome may be an important modulator of chondrocyte differentiation under arsenic exposure. Interestingly, arsenic exposure has been shown to decrease hypertrophic zone height in rats during endochondral ossification (Aybar Odstrcil, Carino, Ricci, & Mandalunis, 2010).

In relation to ROS transport, one of the most striking permeants of AQP9 is H2O2 (Watanabe et al., 2016). It was previously discussed that the HIF family of transcription factors induce hypertrophic differentiation in the epiphyseal plate under hypoxic conditions. As H2O2 accumulates in eukaryotes in response to hypoxia, AQP9 H2O2 transport may therefore be critical toward differentiation as well (Vergara, Parada, Rubio, & Pérez, 2012). Overall, AQP9 channels conduct a broad spectrum of substrates—some of which have clear connections to chondrocyte growth and possibly ossification. Their distribution and transcriptional control should be further described to support a role in chondrocyte activity.

Figure 1.6. cont. 35 AQP9 monomer side view

Selenite Glycerol Purines Urea Arsenic Water Lactate Hydrogen peroxide Pyrimidines

HE Membrane Hydrophobic H-bond corner Gly corner 81 Phe 64

Asn Met 91 Selective Residues passage Hallmark Val through Pro NPA unique to 176 AQP9 interaction motif with residues

Phe 180 Ala

Cys 213 Leu H-bond 209 corner HB

Other helices (H1-H6) 36

1.4.4 Expression and regulation of AQP9 (see Figure 1.7)

Aqp9 protein is expressed in a variety of tissues, such as rat Leydig cell membranes, spleens, brains, and murine spinal cords (Nicchia, Frigeri, Nico, Ribatti, & Svelto, 2001; Elkjaer et al., 2000; Oshio et al., 2004). In mice, Aqp9 is expressed developmentally in the trophoblast (Barcroft, Offenberg, Thomsen, & Watson, 2003). Human placentas and peripheral leukocytes from the blood also express AQP9, although the function has not been fully explored (Damiano, Zotta, Goldstein, Reisin, & Ibarra, 2001; Ishibashi et al., 1998). AQP9 is also expressed in liver hepatocytes, where hepatic gluconeogenesis occurs (Rodríguez et al., 2014). In fact, their study found that AQP9 was the most abundantly expressed aquaporin isoform in the human liver. This coincides with the function of AQP9 as a glycerol transporter useful for energy metabolism. Aqp9 is also localized within mitochondrial membranes, where lactate import can influence the mitochondrial matrix pH and regulate the formation of ROS which may be critical to chondrocyte hypertrophy (Amiry-Moghaddam et al., 2005).

Concerning regulation, the promoter sequence of Aqp9 features the insulin response element (IRE) sequence TGTTTTC (-496/-502), sharing homology with core negative IREs found in the promoters of genes including PEPCK and AQPap/7 (Kuriyama et al., 2002). In their study, insulin downregulated Aqp9 mRNA expression in cultured hepatocytes and it is expected that Aqp9 in other organs is transcriptionally suppressed by insulin at the same putative site. Insulin is a known inducer of chondrogenic differentiation, although conflicting results have been found (Phornphutkul, Wu, & Gruppuso, 2006; Torres, Andrade, Foncesa, Mello, & Duarte, 2003). Nevertheless, transcriptional regulation of Aqp9 in varying tissues has been largely unexplored. Aqp9 expression in hepatocytes is decreased by estrogen in a concentration-dependent manner, as well by estrogen receptor agonists (Lebeck et al., 2012). As well, Vitamin-D receptor deficient mice present with lower estrogen levels, higher Aqp9 mRNA expression, and higher estrogen receptor alpha expression—likely due to compensation for the hormone deficiency (Zanatta et al., 2017). Estrogen is a gonadal steroid that can promote epiphyseal growth and slow it as well, and it may use AQP9 as a signalling intermediate to regulate growth during puberty (Nilsson et al., 2014). Testosterone is also able to rescue Aqp9 expression after estrogen treatment in the developing rat epididymis, although the effect of steroid hormones on Aqp9 may depend strongly 37 on the cell type Aqp9 is expressed in (Pastor-Soler et al., 2010). Hence, the Aqp9 promoter is known to contain androgen response elements (AREs) and estrogen response elements (EREs) where hormones can bind to regulate transcription (Joseph, Shur, & Hess, 2011).

A natural phenol, phloretin, is a known inhibitor of AQP9 and has been used as a channel blocker in a variety of studies (Tsukaguchi et al., 1998; Dibas, Yang, Bobich, & Yorio, 2007; Calamita et al., 2012). However, phloretin is not AQP9-specific and also affects other proteins like anion channels (Sabirov, Kurbannazarova, Melanova, & Okada, 2013). AQP9 is a viable candidate for control of endochondral ossification; however, it needs to be further explored in both chondrocytes and bone. Here, the expression and function of AQP9 in chondro-osseous tissues and pathways is described in detail.

Figure 1.7. Expression and regulation of AQP9 38

AQP9 protein expression has been shown in the mammalian…

Testicles (Leydig cells) Spleen Brain Spinal cord Trophoblast Placenta Liver (hepatocytes) Leukocyte Mitochondria

Known AQP9 gene regulation

Regulatory region Transcribed region

IRE ERE ARE Introns and exons

Insulin Estrogen / estrogen receptor agonists Testosterone

Downregulates Aqp9 in Downregulates Aqp9 in hepatocytes May upregulate Aqp9 in the hepatocytes through the through the estrogen response epididymis through the androgen insulin response element (IRE) element (ERE) response element (ARE)

Known AQP9 protein regulation

Inhibitory effect Phloretin

Downregulates Aqp9 activity 39

1.4.5 H2O2, an inducer of chondrocyte hypertrophy, is transported by AQP9

AQP8 was among the first aquaporin isoforms to be implicated in general ROS transport (Bienert et al., 2007). It was demonstrated that expression of AQP8 in yeast cells increased their sensitivity to exogenously supplied H2O2, through observing increased fluorescence in AQP8- transformed yeast cells supplied with fluorescent-dyed H2O2. Then, AQP3 was shown to import

H2O2 in keratinocytes when stimulated with TNF-α in psoriasis development (Hara-Chikuma et al., 2015). Watanabe et al. (2016) eventually demonstrated that AQP9 expression in CHO-K1 cells increased H2O2 import with exogenously added H2O2, and that AQP9 silencing in HepG2 cells reduced extracellular import. Their study further used Aqp9 -/- mice to observe suppressed uptake of H2O2 in knockout erythrocytes and mast cells compared to their WT (wildtype) counterparts, highlighting a third aquaporin isoform capable of ROS conductance.

The developing epiphyseal plate is typically regarded as hypoxic, where ROS expression has been shown to increase as well as decrease in separate studies (Schipani et al., 2001; Bell, Klimova, Eisenbart, Schumacker, & Chandel, 2007; Frandrey, Frede, & Jelkmann, 1994). To complicate matters, HIF-1α, an oxygen-sensitive component of the transcription factor HIF-1, does not appear to be expressed linearly with ROS formation (Qutub & Popel, 2008). Nevertheless, the HIF homologue HIF-2α is important for chondrocyte hypertrophy and its encoding gene, Epas1, increases in expression alongside other important genes such as Col10a1 and Mmp13 (Saito et al., 2010). As well, AQP9 may be responsible for controlling intracellular

H2O2 levels in the epiphyseal plate chondrocyte mitochondria, where HIF-1α can accumulate in response to ROS generation (Chandel et al., 2000). Describing the function of AQP9 in chondrocyte-oriented processes such as endochondral ossification or cartilage degradation would further clarify its role in chondrocyte activity, possibly through controlled H2O2 uptake.

1.4.6 AQP9 expression is upregulated in osteoarthritis

To suggest that AQP9 affects chondrocyte activity, solely highlighting its conductance of a known chondrocyte hypertrophy modulator is insufficient—evidence of its gene expression and functional protein activity in physiological or disease processes is necessary. In patients with 40 osteoarthritis and rheumatoid arthritis, AQP9 was detected in their synovial tissues at the RNA and protein levels (Nagahara et al., 2010). More importantly, osteoarthritic tissues with hydrarthrosis—irregular fluid accumulation in the knee—featured significantly higher AQP9 mRNA expression than in tissues without. This suggests that in chondrocyte diseases such as osteoarthritis, AQP9 may not only contribute to abnormal water homeostasis but may also be a genetic marker of the disorder. Osteoarthritis involves elevated chondrocyte production of proteolytic enzymes such as MMP13 which subsequently cause cartilage damage and diminished joint function (van der Kraan & van den Berg, 2012). Given that AQP9 expression parallels the expression of degradative genes in chondrocyte disease—and that these same genes are necessary in physiological hypertrophy of chondrocytes during endochondral ossification—the function of AQP9 in chondrocytes appears to mimic a hypertrophic or inflammatory phenotype.

Another study investigating upregulated genes in human osteoarthritic synovial tissues found that AQP9 expression was significantly higher among other angiogenic-classified genes when compared to normal tissues, such as PF4V1 and EDNRB (Lambert et al., 2014). Angiogenesis and osteogenesis in endochondral ossification are post-hypertrophic processes, suggesting that AQP9 may favor a non-proliferative chondrocyte phenotype. Whether AQP9 contributes to hypertrophy in the articular cartilage or epiphyseal plate chondrocytes through water conductance, H2O2 uptake, an uninvestigated metabolic pathway, or a combination of the aforementioned is currently unknown. Nevertheless, AQP9 is associated with hypertrophic chondrocyte gene expression. It may underlie chondrocyte disorders that induce a hypertrophic phenotype, such as osteoarthritis where type X collagen expression is increased (Walker, Fischer, Gannon, Thompson, & Oegema, 1995). If AQP9 plays a role in dysregulating hypertrophy at the articular cartilage, it may also function in chondrocyte regions that undergo natural hypertrophy—such as the epiphyseal plate. However, a functional study of AQP9 in the epiphyseal zones has yet to be performed. The identification of AQP9 in the terminal products of endochondral ossification may highlight a gap in the literature for the channel’s function in the epiphyseal plate. In particular, it would support that AQP9 is important in the exterior of bone— at the articular cartilage—as well as in bone tissue itself—in cells or matrix—but is unexplored in between the two regions. Investigating AQP9 in the epiphyseal plate may describe a novel gene potentially important to long bone formation. 41

1.4.7 Aqp9 expression rises during osteoclast biogenesis

To further support a role of AQP9 in regulating chondrocyte activity and prospective bone growth, a function of the channel in bone cells would connect these two phases of endochondral ossification and suggest its importance in the process.

Aharon & Bar-Shavit (2006) hypothesized that Aqp9 would be critical in osteoclast differentiation due to a significant volume change—like chondrocyte hypertrophy—from their murine bone marrow macrophage precursors. When differentiated via RANK-L, the subsequent osteoclasts presented with higher Aqp9 and Mmp9 expression. Mmp9 is a well-known matrix metallopeptidase that is elevated in rheumatoid arthritis just as Aqp9 is, suggesting the two genes are active in concert during both chondrocyte dysregulation and osteoclast differentiation. The RANK-L-stimulated macrophages were then treated with phloretin to investigate if an Aqp9 inhibitor would diminish the differentiation process. The macrophages were unable to survive the differentiation process, suggesting that Aqp9 plays a critical role in bone cell formation.

However, Liu et al. (2009) compared osteoclasts between Aqp9 WT and -/- mice, discovering that -/- osteoclasts did not differ in morphology and resorption ability in comparison to their WT counterparts. Due to phloretin’s capacity to block other channels aside from Aqp9, the -/- mouse model is more representative of the importance of Aqp9 in osteoclast differentiation. Nevertheless, they also observed increased Aqp9 expression during differentiation of the pre- osteoclast cell line, RAW264.7. While Aqp9 -/- chondrocytes have not been explored, the presence and rise of Aqp9 expression in osteoclasts suggests that Aqp9 may be important at the culminating steps of endochondral ossification. The two main osteoclast resorption enzymes, MMP9 and MMP13, are present during osteoarthritic cartilage damage as well as Aqp9 expression. If Aqp9 and Aqp9-affiliated genes play functional roles at the articular and bone matrix regions respectively, Aqp9 is likely to act in the structure adjoining the two as well—the epiphyseal plate. Describing roles of Aqp9 in macroscopic bone metabolism would further clarify its importance throughout the entire endochondral ossification process and resultant bone tissue formation.

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1.4.8 AQP9 is a possible target for bone loss attenuation and bone length modulation

AQP9 has been described as a target for the prevention of bone loss, suggesting its activity may span anywhere from the cartilage anlage to post-endochondral ossification structures. Since endochondral ossification underlies long bone development, it is likely that Aqp9 functions in the epiphyseal plate and affects bone health. In postmenopausal women, a single nucleotide polymorphism in intron 1 of AQP9 is associated with increased bone mineral density at the femoral neck (Chanprasertyothin, Saetung, Rajatanavin, & Ongphiphadhanakul, 2010). Subjects with a thymine nucleotide rather than an adenine nucleotide at the rs2414539 A/T position had the highest estimated odds ratio for differential density, suggesting the role of AQP9 in human bone metabolism. Although the polymorphism is located in an intronic region, it may affect splice variance and result in AQP9 protein isoforms that behave according to their host cell type (Wang & Cooper, 2007). Therefore, AQP9 may play an important role in the cells upstream of bone formation. Interestingly, Aqp9 -/- mice are able to retain more bone tissue after microgravity-induced bone loss when compared to their WT counterparts (Bu, Shuang, Wu, Ren, & Hou, 2012). Femoral Aqp9 expression was also elevated during the simulated microgravity, suggesting that Aqp9 is at least partly responsible for the bone loss observed. The study also captured postmenopausal effects on Aqp9 expression by checking for femoral bone mineral density in ovariectomized mice; the lack of estrogen did not change expression and hints that Aqp9 may be reactive to stressful bone conditions. Aqp9 may therefore play important roles throughout endochondral ossification and mature .

Previously, the osteoclast findings of Liu et al. (2009) had included comparisons of femur and tibia lengths between one-year old female Aqp9 WT and -/- C57BL/6 mice; they revealed no significant difference in bone length between the two genotypes. If Aqp9 is important in bone development, its functional role may not be apparent in mature bone tissue but rather in the earlier murine stages before longitudinal growth has slowed. However, a definitive study of Aqp9 in early postnatal chondrocytes and bone has not yet been made. Examining the long bone lengths of Aqp9 WT and -/- mice at a time before the juvenile P21 age may determine if chondrocytes active in endochondral ossification are functionally different in the knockout 43 mutation. The use of a mutant model with inactivated Aqp9 will clarify the role of Aqp9 in epiphyseal chondrocyte activity and long bone growth.

1.5 Aqp9 -/-: an accessible knockout mouse model for epiphyseal chondrocyte and bone length investigation

Rojek et al. (2007) initially created the Aqp9 -/- mouse to investigate the role of Aqp9 in type 2 diabetes mellitus. In obese type 2 diabetic patients, hepatic glucose production is elevated in part from the gluconeogenic properties of glycerol. Given that Aqp9 is a glycerol conductor, the expectation was that inactivation of Aqp9 might reduce the import of the gluconeogenic substrate and decrease blood glucose levels. Aqp9 -/- mutants feature different metabolic characteristics in comparison to their heterozygous counterparts that may support the role of Aqp9 in chondrocyte differentiation. Furthermore, the mutants do not experience any embryonic or early postnatal mortality, easing dissection and examination of bone and cartilage phenotypes.

1.5.1 Aqp9 -/- mutants have abnormal glycerol metabolism

In comparison to +/- mutants, Aqp9 -/- mutant mice present with increased plasma glycerol and triglyceride levels after 24 hours of fasting (Rojek et al., 2007). This seemingly contradictory observation may be credited to compensatory glycerol uptake by the other aquaglyceroporin isoforms to stabilize glycerol blood levels. Glycerol is a gluconeogenic precursor vital for phospholipid synthesis, entry into the glycolytic pathway, and conversion to glucose for metabolic homeostasis and skeletal growth. While the knockdown of Aqp9 would then appear to dysregulate metabolism, Rojek et al. (2007) further demonstrated that administration of exogenous glycerol in both genotypes did not result in significantly differential glucose levels between the two. Hence, the capacity for -/- mutants to generate glucose despite the blockage of a gluconeogenic precursor conductor suggests they are sufficient in maintaining a relatively normal whole-body metabolic phenotype. However, the plasma measurements were implicitly performed in mature mice several weeks past the young pup/neonatal age; the early postnatal phenotypes were not characterized. The availability of glucose and gluconeogenic precursors greatly influence the capacity for early bone growth. Maor & Karnieli (1999) demonstrated that GLUT4, IGF-1, and insulin receptors play critical roles in preventing retardation of skeletal 44 growth in P6-P8 mice. Indeed, glucose transporters and insulin-related receptors are critical components of anabolism in bone (Klein, 2014). Furthermore, epiphyseal plate chondrocyte proliferation is dependent on the presence of glucose intake, as insulin and IGF-1 are both capable of promoting chondrogenic differentiation (Zhang et al., 2014). Given that the building blocks of anabolism in skeletal growth—energy-generating precursors such as glycerol—were unexplored in the early age Aqp9 -/- mutants where they would have the greatest influence on bone development, the mouse model presents as a prime candidate for discovering the effects of Aqp9 in the epiphyseal plate. The uninvestigated glycerol defect in early postnatal mutants suggests that Aqp9 deletion may induce a possible unique skeletal phenotype.

1.5.2 Mature Aqp9 -/- mutants show superficially normal physical characteristics

In comparison to age-matched WT and +/- littermate controls, the Aqp9 -/- mutants presented with no apparent differences in body weight and physical appearance. Rojek et al. (2007) implicitly performed all observations at mature timepoints, as referenced by immunostaining for Aqp9 protein in 6-week-old littermates, 15-week periods of waiting for development of type 2 diabetes in Leprdb and Aqp9 knockout mice, and 2-week periods of waiting post-shave to prepare for skin regeneration experiments. Indeed, the body weight measurements that were presented were reported from 6-14 weeks of age, and only in obese (Leprdb/Leprdb) Aqp9 +/- and -/- mutants. If physical measurements were indeed made at early postnatal timepoints for Aqp9 -/- mutants—before the P21 timepoint—it is unlikely they were statistically analyzed due to their absence of presentation. However, it cannot be excluded that measurements may have been statistically analyzed but omitted from presentation due to a lack of significant differences. In the case of epiphyseal plate and long bone length differences, dissection and extraction of the structures would be necessary for accurate measurement.

Liu et al. (2009) investigated osteoclast function in Aqp9 -/-mutants and extensively characterized Aqp9 WT femurs and tibias in comparison to their -/- mutant counterparts. They show that in one-year-old female mice, both genotypes do not differ in femur length, tibia length, whole-body and long bone density, and osteoclast cell density. In femur and tibia length, Aqp9 - /- mutants show minor decreases in comparison but without statistical significance. Bone density 45 was also measured at 4, 6, and 10 weeks of age in both genotypes and showed no significant differences, suggesting that mature WT and -/- mice share physically similar bones phenotypes. However, if Aqp9 plays an important functional role in bone development, it may be apparent only during timepoints where epiphyseal plate chondrocytes are actively differentiating to lengthen bone—before approaching maturity, partial epiphyseal plate fusion, and dramatic retardation of longitudinal growth. To best capture the influence of Aqp9 in epiphyseal plate chondrocyte activity, experiments should be performed at postnatal timepoints close to the onset of bone development at E14.0 as endochondral ossification begins. Timepoints ranging between P0 and P21 would serve as candidates for long bone measurement, where Aqp9 may be functioning without apparent influence from pubertal skeletal mediators including sex steroids and growth hormone (Courtland et al., 2011). There are currently no characterizations of early postnatal Aqp9 -/- mutant mouse epiphyseal plate chondrocytes and long bone length measurements. Observed differences in long bone length between -/- mutants and their WT or heterozygous littermates at an appropriate postnatal timepoint—such as P5—would support Aqp9 as a regulator of chondrocyte activity in the epiphyseal plate.

1.6 Aqp9 has a novel function in cartilage and murine long bone growth

In the developing cartilage anlage, joint-site associated MSCs cease expression of early chondrocyte markers such as Col2a1 to form the joint menisci (Hyde, Boot-Handford, & Wallis, 2008). MSCs that retain Col2a1 expression in the center of the premature joint—the interzone— form articular cartilage (Jiang & Tuan, 2015). Although articular cartilage is hyaline, simple, and generally non-proliferative, these interzonal cells reside where chondrocytes once occupied and are henceforth undifferentiated chondrocytic descendants (Nalin, Greenlee, & Sandell, 1995). Articular and epiphyseal plate chondrocytes are then discriminated by their location, function, and microenvironment despite their common origin. These cell types share conducting pathways that affect their development and dysregulation. In particular, the Wnt/β-catenin signaling pathway has been implicated in osteoarthritis development where genes such as Wnt7b and Dkk1 expression levels correlate with osteoarthritic severity (Nakamura, Nawata, & Wakitani, 2005; Honsawek et al., 2010). This canonical pathway is complex and not only also regulates skeletal phenotypes—such as Wnt3a and Wnt5a +/- mutants which exhibit lower bone mineral density— 46 but may also target lesser known genes and pseudogenes such as those from the aquaporin families (Okamoto et al., 2014). Here, the Kannu lab has demonstrated the merit of investigating aquaporin/β-catenin interactions in osteoarthritis and also skeletal bone length.

1.6.1 Inhibition of Aqp9 protects against cartilage damage in humans and mice

Previous work in the Kannu lab identified AQP7P1 as a β-catenin target gene in resected femoral articular cartilage from patients undergoing total knee replacement for osteoarthritis (Ma, Vi, Whetstone, Kannu, & Alman, 2014). AQP7 presented with a -2 fold change through ChIP-sequencing and was not known to be functional in osteoarthritis or a known target of β- catenin at the time. Dkk1, a target of the β-catenin pathway and ameliorator of osteoarthritis, was then used to treat articular chondrocyte samples in culture from 5 patients undergoing knee replacement surgery (Kannu, Weng, Poon, Ali, & Ma, 2015). Interestingly, a microarray showed that AQP9 was differentially regulated by Dkk1 with a greater than 2-fold change; this supported that aquaporins from the aquaglyceroporin family appeared to be critical in osteoarthritis pathogenesis. Aqp9 -/- mice aged to 18 months were then sacrificed and measured for knee cartilage damage, revealing less osteoarthritis severity in the mutants in comparison to the WT mice, as determined through the recommended method of OARSI scoring (Glasson, Chambers, Van den berg, & Little, 2010). Kannu et al. (2015) also demonstrated that 18 month Aqp9 -/- mice were protected against age-induced cartilage damage in the knee, as measured by decreased Col10a1 immunohistochemistry staining. With the previous literature regarding Aqp9 in osteoarthritis, osteoclast biogenesis, and bone mineral density, Ma et al. (2014) and Kannu et al. (2015) were able to further highlight the importance of aquaporins such as Aqp9 in the articular region and possibly the growing epiphyseal plate as well. However, preliminary evidence of Aqp9 function in a mouse model would be required to substantiate this postulation.

1.6.2 Early-aged Aqp9 -/- pups appear to be larger than their WT littermates

During examination of P5 WT and Aqp9 -/- pups, the Kannu lab also noticed that the -/- pups appeared to be slightly larger than their WT counterparts. These differences were occasionally observable by eye but complicated by the skin appearance and arrangement of the limbs which 47 may have biased interpretations. Initial weight measurements of both genotypes did not reveal any significant differences, as relayed by colleagues of the Kannu lab. However, even mice that exhibit skeletal overgrowth defects—such as Fgfr3c -/- mutants—have lower mean body weights, suggesting that any long bone differences in Aqp9 -/- mice may not necessarily correlate with their weight measurements (Eswarakumar & Schlessinger, 2007). Instead, performing skeletal preparations and epiphyseal plate measurements would be necessary in delineating any particular long bone phenotype. Previous work with the Kannu lab performed preliminary measurements of Aqp9 -/- Col10a1-stained epiphyseal plate regions at unidentified timepoints but did not return any significant differences (Yang et al., 2014). Preliminary measurements of Col2a1-staining in P14 Aqp9 -/- epiphyseal plates also returned no significant differences, although the proliferative and hypertrophic zones appeared to be partially shorter in height (Shao et al., 2016). Overall, appropriate replicates of epiphyseal regions would be required alongside skeletal preparations at various timepoints to identify Aqp9 as a regulator of epiphyseal chondrocyte function. Furthermore, characterizing any remarkable transport function of Aqp9 -/- chondrocytes at the in vitro or in vivo level would support its role in modulating the epiphyseal plate and long bone growth.

1.6.3 Aqp9 may be a critical regulator of chondrocyte proliferation, hypertrophy, and long bone growth

With the Kannu lab, Shao et al. (2016) also demonstrated that Aqp9 -/- knee epiphyseal chondrocytes treated with 1 mM H2O2 were more viable than their WT counterparts after 24 hours. This result substantiates that H2O2 transport in Aqp9 -/- chondrocytes is reduced and is able to elicit an anti-apoptotic effect. Furthermore, examination of intracellular H2O2 concentrations after treatment showed a >0.01 mM reduction in the Aqp9 -/- chondrocytes. The proportion of apoptotic cells was then measured after treatment through Annexin V and propidium iodide gating in flow cytometry, showing a >20% reduction in the Aqp9 -/- group. If these epiphyseal plate chondrocytes are capable of resisting apoptosis induced by exogenous means, they may also be resistant in physiological environments where a reduction of intracellular H2O2 may affect gene expression, chondrocytic phenotype, and ultimately long bone growth. Indeed, Shao et al. (2016) found that Mmp13 expression in Aqp9 -/- chondrocytes was 48

>6-fold less in comparison to WT chondrocytes post-treatment. The expression of Sox9 was conversely >1.5 fold greater, suggesting that Aqp9 deletion supports chondrocytic retention of the proliferative phenotype. Taken together, the Kannu lab has demonstrated that Aqp9 may be important in regulating epiphyseal plate chondrocyte gene expression and directing their fate toward long bone growth. H2O2 may also be the main substrate blocked to resist the hypertrophic phenotype. Nevertheless, scrutiny of the long bones, epiphyseal plates, and chondrocyte gene expression of Aqp9 -/- mutants at pup, juvenile, and old age timepoints are required to test this hypothesis. My Master’s thesis project is to further characterize any purported long bone phenotypes in Aqp9 -/- mice and study their essential structures: epiphyseal plate zones and chondrocyte gene profiles. 49

2 Research Aims, Hypothesis, and Summary Plan

Investigating the epiphyseal plate and chondrocyte behavior can provide insight into new mechanisms by which long bones grow. Specific experiments must be planned to measure the long bones, epiphyseal plate zones, and chondrocyte activity in both control and test groups.

Aqp9 is a transporter of H2O2 and expressed in articular cartilage and osseous tissues, but unexplored in the epiphyseal plate. I hypothesized that Aqp9 is a regulator of epiphyseal plate chondrocyte proliferation, hypertrophy, and long bone growth. Here, the significance and structure of the investigation are described.

2.1 Rationale

Chondrocyte hypertrophy, where massive fluid uptake is performed, is the largest contributor to long bone growth during endochondral ossification (Cooper et al., 2013). Given that the channelome comprises the set of membrane ion channels and porins that dictate chondrocyte function, aquaporins may be responsible for governing how chondrocytes differentiate in the epiphyseal plate (Mobasheri et al., 2018). Aqp9 is an isoform capable of transporting diverse substrates across the membrane and potentially influencing chondrocyte gene expression (Tsukaguchi et al., 1999). Aqp9 is important in articular cartilage and in the bone shaft where osteoclasts function (Kannu et al., 2015; Liu et al., 2009). However, the function of Aqp9 at their structural intermediary—the epiphyseal plate—is unclear.

2.2 Hypothesis

Preliminary findings in the Kannu lab have shown that Aqp9 deletion is protective against chondrocyte hypertrophy, but analysis of the Aqp9 -/- skeletal and epiphyseal plate phenotype has not been completed. I hypothesized that Aqp9 is a regulator of epiphyseal plate chondrocyte proliferation, hypertrophy, and long bone growth. As Aqp9 is a transporter of H2O2, an inducer of chondrocyte hypertrophy, Aqp9 -/- mice epiphyseal plates may present with dysregulated hypertrophy.

50

2.3 Objectives

2.3.1 Objective 1: Long bone measurements of WT and Aqp9 -/- mice To continue the preliminary findings of the Kannu lab, the first aim of this study was to determine if long bone differences existed between WT and Aqp9 -/- littermate mice. Full-body dissections of mice at the pup age (P5) and male mice at the juvenile age (P21) were performed to liberate whole skeletons for skeletal preparation staining (Mitchell, Gould, Smolik, Koek, & Daws, 2013; Rigueur & Lyons, 2014). Finished skeletons were photographed, then further dissected to isolate long bones for manual and digital measurement. Measurements were analyzed to observe for any statistical significance.

2.3.2 Objective 2: Epiphyseal plate analysis of WT and Aqp9 -/- mice The second aim of the study was to examine the long bone epiphyseal plate zones of WT and Aqp9 -/- mice to determine if differences in zone heights existed. In situ hybridization of Aqp9 to the WT epiphyseal plate was performed to confirm gene expression. H&E staining of P5 epiphyseal plates was performed to observe any differences in zone heights. Col10a1 immunohistochemistry of P21 epiphyseal plates was performed to observe if Col10a1, a marker of hypertrophy, stained differently between the WT and Aqp9 -/- mice. Toluidine blue staining of 18 month old mice was performed to observe any differences in epiphyseal line fusion. To observe if Aqp9 deletion affected the epiphyseal plate at the developmental stage, E16.5 embryo epiphyseal plates were stained with Safranin-O to visualize the chondrocyte distribution. All epiphyseal plate zone heights were quantified and analyzed for statistical significance.

2.3.3 Objective 3: Primary chondrocyte analysis of WT and Aqp9 -/- mice The third aim of the study was to determine the proliferation rate and gene profile of WT and Aqp9 -/- epiphyseal plate chondrocytes. P5 WT and Aqp9 -/- primary chondrocytes were seeded at 100,000 cells and counted daily over 96 hours. Cell counts were analyzed for statistical significance to determine if Aqp9 -/- chondrocytes had a proliferation defect. Whole RNA from P5 WT and Aqp9 -/- primary chondrocytes was also extracted to synthesize cDNA for qPCR analysis with probes for Aqp9 and other markers of proliferation and hypertrophy. P5 WT 51 primary chondrocytes were also cultured with an Aqp9-siRNA, harvested for whole RNA extraction, and used for cDNA synthesis for qPCR analysis with the aforementioned probes.

2.4 Clinical significance

Leg length discrepancies (LLDs) are inequalities in the length of the lower limbs that can arise from anatomical or functional means (Murray & Azari, 2015). Anatomical LLD is characterized by actual bone asymmetry in the lower limbs, whereas functional LLD is characterized by abnormalities in other structures—such as the lower spine or pelvis—that result in apparent shortening of one leg (Gurney, 2002; Subotnick, 1981). LLDs are universal and affect up to 90% of the population with a mean length inequality of 5.2mm between the lower limbs (Knutson, 2005). However, deviations from this discrepancy can alter the weight-bearing capacities of the lower extremities and contribute to osteoarthritis, scoliosis, lower back pain, and gait disruption (Golightly, Allen, Helmick, Renner, & Jordan, 2009; Raczkowski, Daniszewska, & Zolynski, 2010; Kaufman, Miller, & Sutherland, 1996). Currently, the only interventions are physical methods—such as shoe lifts for mild cases—or surgery—such as bone resection, mechanical lengthening, or guided growth in severe cases (Brady, Dean, Skinner, & Gross, 2003; Hasler, 2000). These procedures are complex, invasive, and often depend on extensive rehabilitation. Hence, there is a continuous need to determine the mechanisms underlying long bone growth in order to identify therapeutic targets and create effective treatments.

2.5 Cellular mechanisms

LLDs can be congenital—through disorders such as dysplasias—or acquired through trauma, and the root mechanism lies in dysregulated long bone growth (Murray & Azari, 2015). Long bone growth occurs at the epiphyseal plate through the process of endochondral ossification. Here, chondrocytes proliferate, undergo hypertrophy, and undergo apoptosis or transdifferentiate into osteoblasts to allow for bone formation (Mackie et al., 2008). Each of these stages are highly regulated by expression of key marker proteins including SOX9, BMPs, RUNX2, and FGFs (Long & Ornitz, 2013). Among others, these transcription factors dictate chondrocyte division capacity, shape, and expression of specific cartilage matrix collagens. Deletion of these critical genes often results in dysregulation of chondrocyte activity, malformed epiphyseal plates during 52 development, and ultimately shortened and disfigured long bones. However, the contribution of each protein isoform is variant: in FGFs, Fgfr3 -/- mice have longer bones than their WT counterparts (Su et al., 2010). This suggests that Fgfr3 negatively regulates long bone growth. Overall, investigating the expression of unique marker genes at each stage of epiphyseal plate chondrocyte differentiation may provide insight into factors which may be controlled to ameliorate LLDs.

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3 Methods

3.1 Mouse creation, maintenance, genotyping, and age selection

The Aqp9 mutant mouse model was generated by substituting 55 nucleotides of exon 2 in Aqp9 with a neomycin phosphotransferase expression cassette, resulting in correct translation of the first 47 amino acids, then 100 randomized amino acids, and then a stop codon. RT-PCR with customized primer pairs confirmed the mutated Aqp9 gene and western blotting with the AQP9A-1 primary antibody confirmed the absence of Aqp9 protein in the mutants. Aqp9 +/- mutants were crossed with a C57BL/6 mouse background and initial heterozygote crosses produced offspring expected of Mendelian ratios, suggesting that -/- mutants can be bred without difficulty. No embryonic or early postnatal mortality is present in the mutants. The Aqp9 mutants were a generous gift imported by Dr. Soren Nielsen from Aarhus University, Denmark. Mice used in this study were housed in standardized cages at The Centre for Phenogenomics (TCP) in Toronto, ON according to their guidelines. General mouse maintenance was performed by employees at TCP. For the genotyping of embryonic and pup stage mice, yolk sacs and tail clips were harvested respectively and digested in 50ul of QuantaBio Extraction Reagent for 30 minutes. Then, 50ul of QuantaBio Stabilization Buffer was added to neutralize the mixture. 2uL of DNA was used for PCR. For genotyping of juvenile and old mice, ear notches were made by employees at TCP and sent to Transnetyx, Inc. for DNA analysis.

For specific experiments, the P5 age was selected to investigate endochondral ossification at the pup stage where pubertal growth has not yet occurred. The P14 age was selected to best visualize the discrete zones of the epiphyseal plate. The P21 age was selected to investigate endochondral ossification at the juvenile stage where pubertal growth and early adolescence has begun. The 18 month age was selected to investigate a timepoint where the epiphyseal plate is senescent.

3.2 In situ hybridization

RNA digoxigenin-dUTP-labeled riboprobes were created from linearized template DNA plasmids for Aqp9. Riboprobes were synthesized using a DIG-labeling mix (Roche) according to 54 the manufacturer’s instructions and precipitated in 5M LiCl DEPC, 100% ethanol at -80°C for 2 hours. The riboprobes were resuspended in 75ul sterile DEPC-water, 75ul formamide and stored at -80°C until usage. In brief, mouse lower limbs were harvested in cold DEPC-PBS, embedded in paraffin blocks, and sectioned. Slides were prepared through a xylene/DEPC-ethanol/DEPC- PBS/PFA/TEA solution series and then air dried. Hybridization buffer (distilled formamide, 20x DEPC-SSC pH 4.5, 10% DEPC-SDS, and 20mg/ml heparin) was warmed at 85°C prior to addition of the riboprobes. Riboprobes were then added and hybridization was performed overnight at 55°C. After a post-hybridization SSC/formamide/TNE/TBST wash series, slides were blocked with blocking reagent (Roche) for 1 hour. Anti-DIG antibody (1:2000, Roche) was then added for room temperature incubation, for 1 hour. BM purple colour substrate (Roche) was used for colour development over 8-20 hours. The colour reaction was stopped with a distilled water/100% ethanol/xylene rinse series. Slides were then mounted in Permount and covered in coverslips for air drying.

3.3 Skeletal staining

For staining of bone and cartilage, whole skeletons were dissected from mice and fixed in 95% ethanol for 48 hours in room temperature. Then, skeletons were submerged in Alcian blue staining solution (2.5ml 0.3% Alcian Blue SGS (Sigma), 10ml glacial acetic acid, and 40ml ethanol) for 48 hours at 37°C. Alcian blue staining solution was replaced with 95% ethanol daily over 3 days. Skeletons were then submerged in Alizarin red staining solution (0.5ml 0.2% Alizarin Red S (Sigma), 5ml 10% KOH, and 45ml distilled water) for 24 hours in room temperature. Alizarin red staining solution was then replaced with 20% glycerol, 1% KOH for 3 days in room temperature and then 50% glycerol, 1% KOH until muscle and fat tissue was dissolved. The dissolving step varied in time depending on the mouse size. Skeletons were then stored in 80% glycerol for 24 hours and then 100% glycerol for long-term storage.

3.4 Staining and immunohistochemistry of epiphyseal plates

Mouse limbs were harvested in PBS and stored in 70% ethanol prior to paraffin embedding and sectioning. Limbs were then paraffin embedded and sectioned. Slides were rehydrated in a 100%/90%/70%/50% ethanol series, then washed twice in distilled water for 3 minutes. For 55

H&E staining, slides were then immersed in 0.3% ammonium hydroxide for 20 dips and rinsed twice in water for 1 minute, followed by immersion in Eosin Y certified biological stain (Fisher Scientific) for 10 dips. For Toluidine blue staining, slides were then immersed in 1% Toluidine Blue O (Sigma) for 10 minutes. For Safranin-O staining, slides were then immersed in Weigert’s iron hematoxylin solution (Sigma) for 10 minutes and rinsed in distilled water followed by immersion in 1% Safranin O (Sigma) for 5 minutes. After all staining procedures, slides were washed in water thrice for 1 minute, dehydrated in a 95%/100% ethanol and xylene series, then mounted and covered. For immunohistochemistry, slides were then bleached in 6% H2O2 for 6 hours, followed by rehydration in 1% Tween-20 in PBS. Blocking was performed with sheep serum:DMSO 4:1 and then incubated in antibody solution containing 1:100 anti-phosphorylated H3 antibody (Cell Signaling Technology) for 24 hours at 4°C. After a wash series of 1% Tween- 20 in PBS, slides were incubated in an antibody solution containing 1:200 HRP-conjugated secondary antibody. Slides were then covered in DAB and incubated for 20 minutes for the colour reaction to occur. Then, slides were mounted and covered.

3.5 Visualization and measurement of limbs and epiphyseal plates

Skeletal staining preparations of mice were photographed with a Canon DSLR over a backlight and self-made cardboard aperture to maintain the same magnification. A ruler was placed in each shot for measurement references, and images were rotated in ImageJ and PowerPoint. Measurements of limbs were performed using a Mastercraft digital caliper. The measurements were performed blinded and each bone was measured twice with its averaged value reported for statistical analyses.

For P5 mice, humerus bones were measured from the most proximal staining point (at the articulation with the distal glenoid cavity) to the most distal staining point (at the articulation with the proximal ulnar aspect). Femur bones were measured from the most proximal staining point (at the articulation with the distal acetabulum) to the most distal staining point (preceding the knee femoral cartilage). Tibia bones were measured from the most proximal staining point (proceeding the knee tibial cartilage) to the most distal staining point (at the articulation with the talus bone). For P21 mice, humerus bones were measured from the most proximal staining point 56

(at the head of the humerus) to the most distal staining point (at the condyle). Femur bones were measured from the most proximal staining point (the femoral head and greater trochanter) to the most distal staining point (at the femoral ). Tibia bones were measured from the most proximal staining point (the head and tuberosity) to the most distal staining point (at the malleoli). Skull condylo-basal lengths were measured from the most anterior aspect of the nasal bone to the most posterior aspect of the braincase.

Epiphyseal plates were visualized and photographed microscopically, then transferred to ImageJ and PowerPoint for cropping. Measurements of epiphyseal plate zones and cellularity were performed using ImageJ, then recorded using the Region of Interest (ROI) Manager. The measurements were also performed blinded and each replicate was measured twice with its averaged value reported for statistical analyses.

3.6 Primary chondrocyte culture, qPCR, and RNA silencing

Epiphyseal cartilage tissue was excised from mouse limb knee joints in PBS and digested in 1mg/ml Pronase powder (Sigma) for 1 hour at 37°C. The tissue contained all cartilage normally stained by Alcian blue in the knee region. The tissue was digested in 0.5mg/ml Collagenase P powder (Sigma) for 24 hours at 37°C. Chondrocytes were re-suspended in PBS and plated in Dulbecco’s Modified Eagle Medium F/12 (Gibco) with 5% anti-anti and 10% fetal bovine serum. Cell counting was performed using a manual counter and ThermoFisher Scientific Countess® Automated Cell Counter. RNA was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. RNA was then reverse transcribed into cDNA using a TM TM SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to manufacturer’s instructions. Gene probe primers were designed using Primer3 Version 4.0.0 to target the exon- exon junctions of murine mRNA. Design parameters included primer sizes of 18-30bp, Tm of 58- 60°C, and GC content of 40-60%. Reactions were performed using Fast Evagreen® qPCR Master Mix (Biotium) according to manufacturer’s instructions, and run on the ViiA 7 Real- Time PCR System (Agilent Technologies) with actin and GAPDH controls. RNA silencing was performed using the Lipofectamine® 2000 Reagent (Invitrogen) kit according to manufacturer’s instructions. Primary chondrocytes were cultured until ~50% confluency in 6-well plates for 57 transfection. 100pmol Aqp9 siRNA oligomer was diluted in 250ul Opti-MEM® Medium. 5ul Lipofectamine® 2000 was diluted in 250ul Opti-MEM® Medium and incubated for 5 minutes at room temperature. The diluted mixture was incubated for 20 minutes at room temperature. The mixture was made for each chondrocyte sample and added to each well for 48 hours of incubation at 37°C. An equimolar diluted control siRNA (Santa Cruz Biotechnology) was added to control wells. RNA extraction, cDNA synthesis, and qPCR were then performed.

3.7 Statistical analyses

Data (mouse weights, bone measurements, epiphyseal plate measurements, chondrocyte experiments, etc.) were analyzed using the Student’s two-tailed heteroscedastic t test by comparing all test groups (heterozygous, knockout, silenced, etc.) to corresponding control groups (wildtype, wildtype with control, etc.). The ANOVA test was not utilized as multiple group comparisons were not performed in any analyses. Significance was defined as the p-value (P), with *P <0.05, **P <0.01, ***P <0.001. Means and error bars were graphed using Microsoft Excel chart and error bar formatting tools.

58

4 Results

4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice

Mice were selected at the pup/neonatal age (P5), juvenile age (P21), and old age (18 months old) for imaging and mean body weight measurements. This would serve to determine if any superficial body size differences existed across the Aqp9 -/- mouse lifespan. At the P5 timepoint, the mice were not sexed due to sex determination unreliability. Whole body differences due to sex are typically unobservable at this age (Schlomer et al., 2013; Bouleftour et al., 2014). At the P21 and 18 month timepoints, male mice were used to prevent any sex-specific body weight differences.

At P5, decapitated WT and Aqp9 -/- pups do not appear to differ significantly in body size or average body weight (Figure 4.1A, 4.1D). At P21, WT and Aqp9 -/- juvenile mice also appear superficially similar and do not differ significantly in average body weight (Figure 4.1B, 4.1E). 18 month old WT and Aqp9 -/- mice display the same characteristics (Figure 4.1C, 4.1F). The measurements at the three timepoints charted over time show consistent body weights between WT and Aqp9 -/- mice.

59

A

WT Aqp9 -/- 5cm

Figure 4.1. Body weight and superficial comparisons of WT and Aqp9 -/- mice

Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at P5 to observe any superficial differences at the pup stage (A). n=6. 60

B

WT Aqp9 -/- 10cm

Figure 4.1. cont.

Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at P21 to observe any superficial differences at the juvenile stage (B). n=6. 61

C

WT Aqp9 -/- 10cm

Figure 4.1. cont.

Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at 18-months of age to observe any superficial differences at the mature stage (C). n=3. 62

P5 P21 18 months

5.00 D 14.00 E 40.00 F N.S N.S N.S 12.00 35.00 4.00 10.00 30.00 25.00 3.00 8.00 20.00 6.00 2.00 15.00 4.00 10.00 1.00 2.00 5.00 Average weight (g) weight Average (g) weight Average (g) weight Average 0.00 0.00 0.00 WT Aqp9 -/- WT Aqp9 -/- WT Aqp9 -/-

40 G 35

30

25

20 WT 15 Aqp9 -/-

Average weight Average weight (g) 10

5

0 P5 P21 5 mos. Mouse age

Figure 4.1. cont.

Body weight measurements of WT and Aqp9 -/- mice at P5 (D), P21 (E), and 18 months of age (F). n=6, 6, and 3 respectively. Average body weights of WT and Aqp9 -/- plotted over time (G). Student’s two-tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05. 63

4.2 Histological analysis of Aqp9 expression in the juvenile epiphyseal plate

Aqp9 expression was checked in the P14 WT mouse epiphyseal plate to confirm that Aqp9 is normally present in the area where endochondral ossification occurs. In situ hybridization of Aqp9 in the proximal tibia epiphyseal plate from dissected lower limbs was performed using an Aqp9 DIG-labeled RNA probe (Figure 4.2A).

The transcript was detected in specific regions of the epiphyseal plate using the antisense probe. Aqp9 is expressed in chondrocytes in the proliferating zone, as observed by the violet columns of stacked cells. Furthermore, Aqp9 is strongly expressed in chondrocytes in the pre-hypertrophic zone, as observed in the dark violet lower portions of the columns as chondrocytes begin to deviate from their columnar organization. In the hypertrophic zone, Aqp9 staining is not robust and suggests that Aqp9 is not expressed there. The sense probe produced less prominent staining of Aqp9 in the epiphyseal plate, suggesting that the antisense probe is transcript specific (Figure 4.2B).

64

Figure 4.2. Histological analysis of Aqp9 expression in the juvenile epiphyseal plate In situ hybridization of Aqp9 using the Aqp9 antisense probe in the WT P14 proximal tibia (A) and with its corresponding sense probe (B). The gene can be detected in the proliferating and pre-hypertrophic zones of the epiphyseal plate. R = resting zone, P = proliferating zone, PH = pre-hypertrophic zone, H = hypertrophic zone, OC = ossification centre. 65

4.3 Skeletal staining of P5 WT and Aqp9 -/- mice

P5 mice were sacrificed and dissected for whole-mount skeletal staining. This experiment served to identify if any long bone phenotype existed in neonatal mice with Aqp9 deletion. The staining protocol was used to digest excess muscle and fat tissue, stain cartilage with Alcian blue, and stain bone with Alizarin red to identify skeletal regions for accurate measurement. No gross body differences are observable between WT and Aqp9 -/- mice (Figure 4.3A). Isolation of the upper limb and scapula show no significant difference between the average Aqp9 -/- humerus bone length in comparison to the WT humerus bone (Figure 4.3B, 4.3F, 4.3G). Isolation of the lower limb shows that the average Aqp9 -/- femur bones also show no significant difference compared to their WT counterparts, but the tibia bones are significantly different (Figure 4.3C, 4.3H, 4.3I, 4.3J, 4.3K).

To determine the overall percent differences between P5 WT and Aqp9 -/- long bones, the right and left humerus, femur, and tibia bones from each biological replicate were grouped according to bone type. The average Aqp9 -/- long bone length was divided by the average WT long bone length to determine the percent change. The Aqp9 -/- humerus (Figure 4.3L) and femur (Figure 4.3M) bones do not show any significant percent differences compared to their WT counterparts on average. The Aqp9 -/- tibia bones (Figure 4.3N) are significantly longer compared to their WT counterparts on average. To test if the Aqp9 -/- long bone observation was specific to bone formation and not affecting other organs, excised spleens from P5 WT and Aqp9 -/- mice were weighed (Figure 4.3E). No gross differences in appearance or weight were observed, suggesting that the mutation does not cause full body overgrowth (Figure 4.3O).

Furthermore, to test if the Aqp9 -/- long bone observation was specific to endochondral ossification, P5 WT and Aqp9 -/- skulls were measured for their condylo-basal lengths (Figure 4.3D). The condylo-basal length is the length of the skull. No gross differences in skull length or size were apparent, suggesting that the rate of intramembranous ossification—how skulls form— may not be affected by the mutation (Figure 4.3P).

66

A WT Aqp9 -/-

50mm

B WT Aqp9 -/-

10mm 67

C WT Aqp9 -/-

10mm

D WT Aqp9 -/-

25mm 68

E WT Aqp9 -/-

10mm

Figure 4.3. Skeletal staining of P5 WT and Aqp9 -/- mice

Images of WT and Aqp9 -/- whole body lengths (A), humerus bones (B), femur and tibia bones (C), and condylo-basal lengths (D) stained with Alcian blue and Alizarin red for cartilage and bone identification. Green lines indicate the observable ossified regions. Excised spleens are shown as well (E). 69 Right Humerus Left Humerus F G 6 N.S 6 N.S 5 5

4 4

3 3

2 2 Average Average length (mm) Average length (mm) 1 1

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Right Femur Left Femur

H 6 I 6 N.S N.S 5 5

4 4

3 3

2 2 Average Average length (mm) Average Average length (mm) 1 1

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Right Tibia Left Tibia J 6 * K 6 * 5 5

4 4

3 3

2 2 Average Average length (mm) Average Average length (mm) 1 1

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype 70 Humerus Femur L M 125% 125% N.S N.S 120% 120% 115% 115% 110% 110% 105% 105% 100% 100% 95% 95% 90% 90% 85% 85% Average percent difference Average Average percent difference 80% 80% 75% 75% WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Tibia

N 125% 120% * 115% 110% 105% 100% 95% 90% 85% Average Average percent difference 80% 75% WT Aqp9 -/- Mouse genotype

Figure 4.3. cont.

Measurements of WT and Aqp9 -/- right humerus bones (F), left humerus bones (G), right femur bones (H), left femur bones (I), right tibia bones (J), and left tibia bones (K). Average percent differences of Aqp9 -/- humerus bones (L), femur bones (M), and tibia bones (N) compared to their WT counterparts. n=5. Student’s two-tailed t test was performed for statistical analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05. 71

O 10 9 N.S 8 7 6 5 4 3 Average Average spleen weight (mg) 2 1 0 WT Aqp9 -/- Mouse genotype

P 20 N.S

15 basal length (mm)

- 10

5 Average Average condylo

0 WT Aqp9 -/- Mouse genotype

Figure 4.3. cont.

Measurements of WT and Aqp9 -/- excised spleen weights (O). n=3. Measurements of WT and Aqp9 -/- condylo-basal lengths (P). n=5. Student’s two-tailed t test was performed for statistical analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05. 72

4.4 Skeletal staining of P21 WT and Aqp9 -/- mice

P21 male mice were sacrificed and dissected for whole-mount skeletal staining. This experiment served to identify if the P5 bone phenotype persisted from the neonatal stage to the juvenile stage. The same Alcian blue-Alizarin red staining protocol was used to identify the skeleton elements for accurate measurement. Gross body differences are not apparent between the WT and Aqp9 -/- mice (Figure 4.4A). Isolation of the upper limb and scapula show no significant difference between the average Aqp9 -/- humerus bone length in comparison to the WT humerus bone (Figure 4.4B, 4.4F, 4.4G). Isolation of the lower limb shows that the average Aqp9 -/- tibia bones also show no significant difference compared to their WT counterparts, but the femur bones are significantly different (Figure 4.4C, 4.4H, 4.4I, 4.4J, 4.4K).

To determine the overall percent differences between P21 WT and Aqp9 -/- long bones, the right and left humerus, femur, and tibia bones from each biological replicate were grouped according to bone type. The average Aqp9 -/- long bone length was divided by the average WT long bone length to determine the percent change. The Aqp9 -/- humerus (Figure 4.4L) and tibia (Figure 4.4N) bones do not show any significant percent differences compared to their WT counterparts on average. The Aqp9 -/- femur bones (Figure 4.4M) are significantly shorter compared to their WT counterparts on average.

To test if the P21 Aqp9 -/- bone observation was specific to bone formation and not affecting other organs, excised spleens from P21 WT and Aqp9 -/- mice were weighed (Figure 4.4E). No gross differences in appearance or weight were observed, suggesting that the mutation does not cause full body dwarfism (Figure 4.4O). Furthermore, to test if the Aqp9 -/- observation was specific to endochondral ossification, P5 WT and Aqp9 -/- skulls were measured for their condylo-basal lengths (Figure 4.4D). No gross differences in skull length or size were apparent, similar to the P5 mice. This suggests that the rate of intramembranous ossification— how skulls form—may not be affected by the mutation at this timepoint either (Figure 4.4P).

73 A WT Aqp9 -/-

100mm

B 74

C

D 75

E

Figure 4.4. Skeletal staining of P21 WT and Aqp9 -/- mice

Images of WT and Aqp9 -/- whole body lengths (A), humerus bones (B), femur and tibia bones (C), and condylo-basal lengths (D) stained with Alcian blue and Alizarin red for cartilage and bone identification. Green lines indicate the observable ossified regions. Excised spleens are shown as well (E). 76

Right Humerus Left Humerus

F 10 G 10 N.S N.S 8 8

6 6

4 4 Average Average length (mm) Average Average length (mm) 2 2

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Right Femur Left Femur

H 12 I 12 N.S * 10 10

8 8

6 6

4 4 Average Average length (mm) Average Average length (mm) 2 2

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Right Tibia Left Tibia

J 16 K 16 14 N.S 14 N.S 12 12 10 10 8 8 6 6

Average Average length (mm) 4 Average length (mm) 4 2 2 0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype 77

Humerus Femur

L 125% M 125% 120% N.S 120% 115% 115% * 110% 110% 105% 105% 100% 100% 95% 95% 90% 90% 85% 85% Average Average percent difference Average Average percent difference 80% 80% 75% 75% WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Tibia

N 125% 120% N.S 115% 110% 105% 100% 95% 90% 85% Average Average percent difference 80% 75% WT Aqp9 -/- Mouse genotype

Figure 4.4. cont.

Measurements of WT and Aqp9 -/- right humerus bones (F), left humerus bones (G), right femur bones (H), left femur bones (I), right tibia bones (J), and left tibia bones (K). Average percent differences of Aqp9 -/- humerus bones (L), femur bones (M), and tibia bones (N) compared to their WT counterparts. n=6. Student’s two-tailed t test was performed for statistical analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05. 78

O 20 19 N.S 18 17 16 15 14 13 Average Average spleen weight (mg) 12 11 10 WT Aqp9 -/- Mouse genotype

P 25 N.S

20

15 basal length (mm) - 10

5 Average Average condylo

0 WT Aqp9 -/- Mouse genotype

Figure 4.4. cont.

Measurements of WT and Aqp9 -/- excised spleen weights (O). n=2. Measurements of WT and Aqp9 -/- condylo-basal lengths (P). n=6. Student’s two-tailed t test was performed for statistical analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05. 79

4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates

P5 WT and Aqp9 -/- littermate distal femur epiphyseal plates were sectioned and stained with H&E to visualize their chondrocyte distributions. This would help support if dysregulation in the epiphyseal plate influenced the P5 bone phenotype. The femur was selected rather than the tibia due to the availability of sections. Furthermore, all mouse models previously described with long bone phenotypes—including those with phenotypes in specific bones only—have spatial irregularities in those epiphyseal plate zones. Investigating the tibia epiphyseal plate was determined to be repetitious. As the long bone phenotypes observed at both P5 and P21 were isolated to the hindlimb only, the hindlimb remained of interest. Hence, the adjoining epiphyseal plate at the distal femur was selected for histological analysis.

An overview of the epiphyseal plates show subtle spatial differences near the top of the proliferating zone in Aqp9 -/- mice in comparison to WT mice (Figure 4.5A, 4.5B). Image enlargement shows the chondrocyte distribution ranging from the resting zone to the hypertrophic zone (Figure 4.5C, 4.5D). In the WT epiphyseal plate, the proliferating zone is separated from the resting zone by a clear boundary where chondrocytes are different in shape and distribution. The proliferating chondrocytes are flat and columnar, while resting chondrocytes are rounder and randomly dispersed with less organization. In the Aqp9 -/- epiphyseal plate, chondrocytes at the top of the proliferating zone become gradually rounder but retain columnar organization transitioning into the resting zone.

The Aqp9 -/- proliferating zone appears expanded in comparison to the WT proliferating zone. The pre-hypertrophic zones of WT and Aqp9 -/- epiphyseal plates are demarcated by where chondrocytes at the bottom of the proliferating zones begin to lose their columnar organization and have enlarged cytoplasmic regions. No differences in pre-hypertrophic zone height are apparent. The hypertrophic zones are demarcated by where the chondrocytes are engulfed in round and pale cytoplasm and lose their H&E staining, up until the heavily stained bone matrix at the bottom of the epiphyseal plate.

80

Proliferating and hypertrophic zone heights were measured by drawing five vertical lines across each zone per biological replicate and averaging their heights. Statistical analysis of the proliferating and hypertrophic zone heights show that proliferating zones in P5 Aqp9 -/- epiphyseal plates are significantly taller than their WT counterparts (Figure 4.5E). No significant differences in hypertrophic zone height were observed.

The average number of chondrocytes per column in proliferating and hypertrophic zones were quantified by counting the number of chondrocytes that passed through each vertical line and averaging them. Aqp9 -/- proliferating zones have significantly more chondrocytes per column in comparison to their WT counterparts (Figure 4.5F). No significant differences were observed in the hypertrophic zone.

The cellular density of proliferating and hypertrophic zones, in cells/um2, was also measured by counting the individual chondrocytes in each zone and dividing the number by the total area of the zone. Neither Aqp9 -/- proliferating or hypertrophic zones were significantly denser than their WT counterparts (Figure 4.5G).

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WT A Aqp9 -/- B

200um 200um

WT C Aqp9 -/- RZ D RZ

RZ

PZ PZ

PH PH

HZ HZ

100um 100um

Figure 4.5. Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates

H&E staining of P5 WT and Aqp9 -/- distal femurs (A, B) with their respective image enlargements (C, D). Dotted black lines demarcate the observable zone boundaries. Yellow and red lines indicate the observable proliferating and hypertrophic zones respectively (RZ = resting zone, PZ = proliferating zone, PH = pre-hypertrophic zone, HZ = hypertrophic zone). n=3. 82

500 N.S E 450 400 350 * 300 250 N.S WT 200

Height Height (um) Aqp9 -/- 150 100 50 0 PZ HZ Total Epiphyseal plate zone

30 * F

25

20

N.S 15 WT

10 Aqp9 -/- Average Average cells/column 5

0 PZ HZ Epiphyseal plate zone

Figure 4.5. cont.

Quantification of the proliferating and hypertrophic zone heights (E) and average cells/column (F) of the WT and Aqp9 -/- distal femurs (PZ = proliferating zone, HZ = hypertrophic zone). n=3. Student’s two-tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05. 83

4 G N.S

N.S 3 ) 3 - (10 2 2 WT Aqp9 -/- Cells/um 1

0 PZ HZ Epiphyseal plate zone

Figure 4.5. cont.

Quantification of the cellular density of the proliferating and hypertrophic zones (G) of the WT and Aqp9 -/- distal femurs (PZ = proliferating zone, HZ = hypertrophic zone). n=3. Student’s two-tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05. 84

4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates

P21 WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were sectioned and probed with a primary antibody against Col10a1 to qualitatively visualize their zone arrangements and chondrocyte distributions, with a focus on the hypertrophic zone. The tibia was selected rather than the femur due to the availability of sections. As mentioned previously, all mouse models described with long bone phenotypes—including those with phenotypes in specific bones only— have spatial irregularities in those epiphyseal plate zones. Therefore, investigating the femur epiphyseal plate was determined to be repetitious. As the long bone phenotypes observed at both P5 and P21 were isolated to the hindlimb only, the hindlimb remained of interest. The adjoining epiphyseal plate at the proximal tibia was selected for histological analysis.

An overview of the epiphyseal plates do not show any apparent differences in zonal height between WT and Aqp9 -/- mice (Figure 4.6A, 4.6B). The image enlargements clearly indicate the height of the entire epiphyseal plate, as well as their zonal compartments (Figure 4.6C, 4.6D). In the upper portions, the resting-proliferating zones were measured due to difficulty in distinguishing between the chondrocyte types. The resting zone regions, where chondrocytes are more randomly dispersed, are narrow in both WT and Aqp9 -/- mice. The proliferating zone regions clearly end where the dark staining for Col10a1 begins, indicating the upper portion of the hypertrophic zone. The hypertrophic zone is dense with chondrocytes outlined in the dark stain, where Col10a1 protein is robustly expressed.

Resting-proliferating and hypertrophic zone heights were measured by drawing five vertical lines across each zone per biological replicate and averaging their heights. Hypertrophic zone heights were divided by resting-proliferative zone heights to measure the hypertrophic zone – resting- proliferative zone ratio. Statistical analysis of the ratios show no significant difference between the P21 WT and Aqp9 -/- mice (Figure 4.6E). Total epiphyseal plate heights were also measured and show no significant difference (Figure 4.6F).

85

400um 400um

R-PZ R-PZ

HZ HZ

400um 400um

Figure 4.6. Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates

Col10a1 staining of P21 WT and P21 proximal tibias (A and B) with their respective image enlargements (C and D). Blue bar sections indicate the observable epiphyseal plate, green sections indicate the observable resting and proliferating zones combined, and red sections indicate the observable hypertrophic zones (C and D) (R-PZ = resting-proliferative zone, H = hypertrophic zone). 86

1 E 1000 F 0.9 N.S 0.8 N.S 800

0.7 PZ ratio - 0.6 600 0.5 0.4 400 0.3

0.2 200

R HZ / Proximal tibia 0.1 Proxminal tibia epiphyseal plate height (um) height plate epiphyseal tibia Proxminal 0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Figure 4.6. cont.

Quantification of WT and Aqp9 -/- hypertrophic zones as a ratio to their respective resting- proliferating zones (E). Measurement of the total epiphyseal plate heights of WT and Aqp9 -/- mice (F). n=2. Student’s 2-tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05. 87

4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates

18 month old male WT and Aqp9 -/- femoral heads were stained with Toluidine blue to highlight the cartilaginous epiphyseal plates. This would help determine if Aqp9 affects epiphyseal plate narrowing into an old age timepoint. An overview shows the epiphyseal plates stained a deep blue as a thin line traversing across the diameter of the femoral head (Figure 4.7A, 4.7B). Image enlargement shows that in the WT head, the epiphyseal plate reaches the right side toward the greater trochanter and makes contact with the cortical bone (Figure 4.7C). However, in the Aqp9 -/- head, the epiphyseal plate does not reach the cortical bone and is instead ablated (Figure 4.7D). The observation was initially thought to be due to sectioning inconsistencies that caught the Aqp9 -/- epiphyseal plates at a particular depth, but statistical analysis shows the same trend across all Aqp9 -/- biological replicates and significantly reduced width compared to the WT replicates (Figure 4.7E).

The femoral heads were also stained with H&E to highlight the bone marrow, presence of hematopoietic cells, and the overall trabecular bone. The epiphyseal plate is visible as a line running in between the bone marrow and trabecular bone. In the WT femoral head, the bone marrow is white in colour (Figure 4.7F). In the Aqp9 -/- femoral head, the bone marrow is not white but rather stained with the H&E stain (Figure 4.7G). Furthermore, the presence of hematopoietic cells were more visible in the Aqp9 -/- bone marrow than in WT bone marrow. The epiphyseal plate in the Aqp9 -/- femoral head does not appear to fully contact the cortical bone. The region of trabecular bone in the Aqp9 -/- femoral head also appears smaller than the one in the WT femoral head.

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EP EP

500um 500um

C B C B

250um 250um

Figure 4.7. Analysis of adult WT and Aqp9 -/- epiphyseal plates

Toluidine blue staining of 18 month old WT and Aqp9 -/- femoral heads (A and B) with their respective image enlargements (C and D). Red-dotted lines (C and D) indicate how far the epiphyseal line traverses the entire femoral head diameter. Yellow-dotted line (D) indicates the area where the epiphyseal plate ceases to exist. EP = epiphyseal plate, CB = cortical bone. 89

100.00 E *

80.00

60.00

40.00

20.00

Epiphyseal Epiphyseal line length femoral / head diameter (%) 0.00 WT Aqp9 -/- Mouse genotype

Figure 4.7. cont.

Quantification of each epiphyseal plate length of the WT and Aqp9 -/- mice as a percentage of the entire femoral head diameter (E). n=3. Student’s two-tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05.

WT F Aqp9 -/- G

500um 500um

Figure 4.7. cont.

H&E staining of 18 month old WT and Aqp9 -/- mouse femoral heads (F & G). Black arrows indicate the region of observable bone marrow and its respective colour. Yellow arrows indicate locations where observable hematopoietic cells reside. CB = cortical bone, EP = epiphyseal plate, BM = bone marrow, TB = trabecular bone. 90

4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates

E16.5 WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were sectioned and stained with Safranin-O to visualize their zone arrangements and chondrocyte distributions. This would help determine if Aqp9 affects endochondral ossification during development. An overview of the epiphyseal plates show similar zonal heights and total height between WT and Aqp9 -/- mice (Figure 4.8A, 4.8B). Image enlargement shows that the Aqp9 -/- resting zone appears to cave in towards the top of the epiphyseal plate, whereas the WT resting zone forms a straight boundary with the proliferating zone perpendicular to the long bone (Figure 4.8C, 4.8D). The Aqp9 -/- proliferating zone chondrocytes appear to retain columnar structures further up the epiphyseal plate than their WT counterparts.

Resting, proliferating, and hypertrophic zone heights were measured by drawing five vertical lines across each zone per biological replicate and averaging their heights. The hypertrophic zone included the pre-hypertrophic zone to aid measurement boundaries. The heights were summed and averaged to determine the total epiphyseal plate height. Then, the ratio of each zone compared to the total height was determined by dividing each zone height by the total height.

Statistical analysis of the ratios show that the E16.5 Aqp9 -/- resting zones are significantly shorter than those of their WT counterparts (Figure 4.8E). No significant differences were observed in the proliferating and hypertrophic zones (Figure 4.8F, 4.8G). The sum of the proliferating and hypertrophic zone heights divided by the total plate height are also shown, to demonstrate that the non-resting portions of Aqp9 -/- epiphyseal plates are taller than those of WT mice (Figure 4.8H).

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A B

RZ RZ

PZ PZ

HZ HZ

WT 100um Aqp9 -/- 100um

C D

RZ RZ

PZ

PZ

100um 100um WT Aqp9 -/-

Figure 4.8. Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates

Safranin-O staining of E16.5 WT and Aqp9 -/- proximal tibias (A and B) with their respective image enlargements (C and D). Dotted-line regions indicate the observable resting zone of the epiphyseal plate (RZ = resting zone, PZ = proliferating zone, HZ = hypertrophic zone). 92

0.5 E 0.5 F * N.S 0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1 PZ height (um) / EP height(um)/ (um)PZ height RZ height (um) / EP height(um)/ (um)EP RZ height

0 0 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

0.5 G 0.8 H

0.4 N.S * 0.7 0.3

0.2 0.6

0.1 HZ height (um) / EP height(um)/ (um)EP HZ height PZ + HZ height (um) / EP height (um)/ (um)EP heightHZ height PZ + 0 0.5 WT Aqp9 -/- WT Aqp9 -/- Mouse genotype Mouse genotype

Figure 4.8. cont.

Quantification of each epiphyseal plate zone height (E, F, G) of the WT and Aqp9 -/- embryos as a ratio of the entire epiphyseal plate height (EP = epiphyseal plate). Ratio of the proliferating zone and hypertrophic zone combined to the entire epiphyseal plate height (H). n=3 (2 WT biological replicates and 1 technical replicate, 3 Aqp9 -/- biological replicates). Student’s two- tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05. 93

4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes

P5 WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes were subjected to a cell proliferation experiment to determine if deletion of Aqp9 resulted in any proliferation defect. Any proliferation difference observed would help support the epiphyseal plate zone dysregulation observed. Epiphyseal plate chondrocytes from P5 WT, Aqp9 +/-, and Aqp9 -/- mice were excised and digested in a pronase and collagenase protocol, then seeded at 1 x 105 primary chondrocytes per well in a 6-well plate. Chondrocyte numbers per well were counted every 24 hours over a 96 hour period (Figure 4.9).

WT, Aqp9 +/-, and Aqp9 -/- chondrocytes reach confluency by the 96 hour timepoint. A lag in growth is observed in Aqp9 +/- and Aqp9 -/- chondrocytes at the 48 hour timepoint, followed by a jump in the cell number at the 72 hour timepoint. Overall, Aqp9 +/- and Aqp9 -/- chondrocytes appear to recover from an abnormally low cell number to match the WT cell number at the 96 hour timepoint.

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12 WT Aqp9 +/- 10 Aqp9 -/- ) 5 8 *** N.S *** N.S 6 N.S N.S N.S 4 N.S Cell Cell number(1e 2

0 0 24 48 72 96 Time (hours)

Figure 4.9. Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes

Cell count of WT (black), Aqp9 +/- (orange), and Aqp9 -/- (red) epiphyseal plate chondrocytes seeded at 100,000 cells from 0-96 hours. n=3. Student’s 2-tailed t test was performed for statistical analysis comparing Aqp9 +/- and Aqp9 -/- to WT at each timepoint, with the level of significance set at *P < 0.05, **P < 0.01, ***P < 0.001. 95

4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes

P5 WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes were subjected to gene expression analysis to observe if their individual gene profiles would reflect the P5 tibia bone phenotype and femur epiphyseal plates. The experiment would also help explain the irregular proliferation pattern observed with the cell proliferation experiment. Epiphyseal plate chondrocytes from P5 WT, Aqp9 +/-, and Aqp9 -/- mice were excised and digested. RNA was extracted and then reverse transcribed to cDNA for qPCR gene expression analysis. Five gene probes were used: Col10a1, Mmp13, Runx2, Sox9, and Aqp9. Col10a1, Mmp13, and Runx2 are known aforementioned markers of chondrocyte hypertrophy. Sox9 is a known aforementioned marker of early chondrogenesis, proliferation, and hypertrophy delay. Aqp9 was used to observe if its expression levels would parallel the expression of the other probes in a remarkable manner or trend.

Charting of the gene probe fold changes presented with significant differences in Mmp13, Sox9, and Aqp9 (Figure 4.10). For the Mmp13 probe, Aqp9 +/- chondrocytes presented with a significant fold change less than 1. Here, Aqp9 -/- chondrocytes did not present with a significant change. For the Sox9 probe, Aqp9 -/- chondrocytes presented with a significant fold change greater than 1. Here, Aqp9 +/- chondrocytes did not present with a significant change. For the Aqp9 probe, Aqp9 -/- chondrocytes presented with a significant decrease while Aqp9 +/- chondrocytes did not. Significant fold changes were not observed in either Aqp9 +/- or Aqp9 -/- chondrocytes for the Col10a1 and Runx2 probes.

Overall, the fold change levels of hypertrophic markers in the Aqp9 -/- chondrocytes were not significantly different relative to the WT control. The fold change of the proliferation marker Sox9 was increased. Aqp9 expression was significantly decreased. In Aqp9 +/- chondrocytes, the fold change level of the hypertrophic marker Mmp13 was significantly decreased relative to the WT control.

96

5 **

N.S 4

3 N.S N.S WT *** 2 N.S N.S N.S Aqp9 +/- Foldchange N.S ** Aqp9 -/- 1

0 Col10a1 Mmp13 Runx2 Sox9 Aqp9 qPCR gene

Figure 4.10. Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes qPCR of WT (white), Aqp9 +/- (blue) and Aqp9 -/- (orange) epiphyseal plate chondrocytes with gene probes for markers of proliferation and hypertrophy in chondrocyte differentiation. n=3. Student’s 2-tailed t test was performed for statistical analysis comparing Aqp9 +/- and Aqp9 -/- to WT for each probe, with the level of significance set at *P < 0.05, **P < 0.01, ***P < 0.001. 97

4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes

P5 WT primary epiphyseal plate chondrocytes were subjected to an Aqp9-siRNA silencing protocol to observe if the Aqp9 +/- or Aqp9 -/- gene profile could be simulated. The experiment would also help determine if the Aqp9 +/- or Aqp9 -/- gene profile was cell autonomous to the chondrocytes. Epiphyseal plate chondrocytes were excised from P5 WT mice, digested, and plated for transfection with the Aqp9-siRNA. qPCR using the five aforementioned probes Col10a1, Mmp13, Runx2, Sox9, and Aqp9 was performed after 24 hours of incubation time. Charting of the gene probe fold changes did not present with any significant differences (Figure 4.11).

98

3 N.S 2.5

2 N.S N.S 1.5 N.S N.S WT-control

Fold change Fold WT-siRNA 1

0.5

0 Col10a1 Mmp13 Runx2 Sox9 Aqp9 qPCR gene

Figure 4.11. Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes qPCR of WT epiphyseal plate chondrocytes with an Aqp9-siRNA (yellow) and with a control siRNA (white), with gene probes for markers of proliferation and hypertrophy in chondrocyte differentiation. n=3. Student’s 2-tailed t test was performed for statistical analysis comparing WT-siRNA to WT-control, with the level of significance set at *P < 0.05. 99

5 Discussion

The process of long bone growth is dependent on endochondral ossification, where chondrocytes in the epiphyseal plates proliferate, undergo hypertrophy, and eventually ossify (Mackie, Tatarczuch, & Mirams, 2011). The biology underscoring this process is complex and the clinical management of LLDs relies on continuous endeavours in deciphering its unknown factors (Killion, Mitchell, Duke, & Serra, 2017). Hence, identifying any novel regulators of endochondral ossification is valuable for non-invasive therapies that can influence long bone growth. The chondrocyte channelome is a popular area of focus that investigates the ion channels and porins on the chondrocyte membrane that affect cartilage activity and pathogenesis (Mobasheri et al., 2018). In particular, Aqp9 is an aquaporin isoform that indiscriminately conducts solutes through a larger-than-usual pore (Viadiu et al., 2007). Interestingly, Aqp9 is one of the few aquaporin species that is capable of transporting H2O2, a known inducer of chondrocyte hypertrophy (Watanabe et al., 2016; Morita et al., 2007). Kannu et al. (2015) initially identified that in human articular cartilage samples, those cultured with the WNT pathway antagonist DKK1 had a greater than 2-fold change in expression of AQP9. As Dkk1 transcripts regulate embryogenesis and are restricted to the murine forelimb and hindlimb buds at E10.5, Aqp9 may also play a role in guiding (Grotewold, Theil, & Rüther, 1999; Lieven, Knobloch, & Rüther, 2010). However, the study of Aqp9 in chondrocytes and bone has been limited to articular cartilage, osteoclasts, and bone density (Nagahara et al., 2010; Liu et al., 2009; Bu et al., 2012). To study Aqp9 in endochondral ossification, the use of the Aqp9 -/- mutant model developed by Rojek et al. (2007) allowed for a specific focus on its function in the epiphyseal plate.

In this study, the Aqp9 -/- mutation was shown to be involved in differential long bone length, epiphyseal plate zone dysregulation, and chondrocyte gene expression. Aqp9 is expressed in the murine epiphyseal plate at the critical proliferating and pre-hypertrophic zones. The Aqp9 -/- mice have longer tibia bones at the P5 age and shorter femur bones at the P21 age, suggesting that Aqp9 may be important in hindlimb development. Analysis of the P5 Aqp9 -/- femur epiphyseal plates show irregular expansion of the proliferating zone, suggesting that even long bones not significantly different may have dysregulated endochondral ossification. Analysis of the P21 Aqp9 -/- tibia epiphyseal plates do not show any significant differences in epiphyseal 100 zone height or hypertrophy marker staining. At the old age stage, Aqp9 -/- femoral heads show unnatural remission and narrowing of the epiphyseal plate, suggesting that the mutants may experience faster partial epiphyseal closure. In development, E16.5 Aqp9 -/- tibial heads show diminished epiphyseal plate resting zone lengths, suggesting that their chondrocytes may attain a proliferative and differentiated state faster than WT counterparts. Both Aqp9 +/- and Aqp9 -/- chondrocytes have different proliferation rates than WT chondrocytes. Aqp9 -/- chondrocytes also presented significantly increased expression of the proliferation marker Sox9. RNA silencing of Aqp9 in WT chondrocytes did not present with any significant differences in expression of the gene probes used. In this study, I hypothesized that Aqp9 is a regulator of epiphyseal plate chondrocyte proliferation, hypertrophy, and long bone growth. The data provide characterization of the Aqp9 -/- mutation at the anatomical, histological, cellular, and gene expression levels. In future studies, analysis of Aqp9 -/- chondrocytes through transcriptome sequencing may provide a richer understanding of aquaporins in endochondral ossification.

5.1 Aqp9 temporally influences hindlimb length

5.1.1 Aqp9 -/- mutants do not differ in appearance and weight

To determine if WT and Aqp9 -/- mice were comparable in terms of physical appearance and body weight, mice at P5, P21, and 18 months of age were culled for imaging and body weight measurements. These timepoints served to examine gross phenotypes before pubertal growth, during pubertal growth, and during old age respectively (Mitchell et al., 2013; Jackson et al., 2017). WT and Aqp9 -/- mice at each timepoint were comparable in terms of overall appearance and weight, as determined by photography and weighing with a precision scale. As mentioned previously, male mice were used at P21 and 18 months to prevent any sex-specific body weight differences. P5 mice were not sexed due to difficulty in determining anogenital differences.

Rojek et al. (2007) reported that no detectable differences in physical appearance or body weight were observed between age-matched WT and Aqp9 -/- littermate mice. This was confirmed across the murine lifespan at the pup, juvenile, and old age stages. The average body weights charted over time appear to match lifelong body weight measurements of WT male mice (List, 101

Berryman, Wright-Piekarski, Jara, & Kopchick, 2013). Rojek et al. (2007) also reported increased plasma glycerol in Aqp9 -/- mice after starvation, suggesting that Aqp9 may play a role in repressing gluconeogenesis without significantly affecting weight. It is also possible that deletion of a functional aquaporin isoform is compensated by the activity of other isoforms, as shown in human pancreatic duct cell function and even plant water regulation (Burghardt et al., 2003; Cohen et al., 2013). Hence, aquaporin redundancy may rescue any superficially observable defect that Aqp9 deletion would have caused. Specifically, Aqp9 -/- mice may be expected to have defected H2O2 transport, but AQP3 and AQP8 are also H2O2 transporters (Watanabe et al., 2016). The role of these aquaporins have not been explored in epiphyseal plate chondrocytes or bone and their contributions are unknown. Nevertheless, deletion of a channel capable of transporting a known hypertrophy inducer may relay an effect on endochondral ossification.

In light of the literature and previous data from the Kannu lab, Aqp9 may affect long bone growth. These differences may not be observable without skeletal dissection and analysis of the long bones and their histology.

5.1.2 Aqp9 is expressed in the P14 mouse epiphyseal plate

Prior to long bone analysis, Aqp9 expression in the murine epiphyseal plate was checked to confirm a possible spatiotemporal role in endochondral ossification. P14 WT mice were selected as endochondral ossification is prominent at this timepoint (Williams, 2014). Furthermore, the discrete zones of the murine epiphyseal plate are best visualized by in situ hybridization at P14 due to their visibility at this timepoint (Belluoccio, Bernardo, Rowley, & Bateman, 2008). In situ hybridization of the proximal tibia epiphyseal plate with an Aqp9 riboprobe showed robust expression of the Aqp9 transcript in the proliferating and pre-hypertrophic zone chondrocytes.

The presence of Aqp9 in the maturing epiphyseal plate suggests that it may be involved in endochondral ossification. The use of in situ hybridization to confirm the expression of Col2a1, Sox9, Mmp13, Col10a1, and other currently well-known chondrocytic markers is well reported (Hall et al., 2013; Hattori et al., 2010). However, the demonstration of aquaporin expression in the epiphyseal plate is limited with the exception of Aqp1 in the rat hypertrophic zone 102

(Claramunt et al., 2017). Here, staining of Aqp9 to the proliferating and pre-hypertrophic zones suggests that Aqp9 regulates chondrocyte activity prior to hypertrophy. The absence of Aqp9 expression in the hypertrophic zone supports that Aqp9 may function to maintain the proliferative state and prepare chondrocytes for hypertrophy. Then, chondrocytes that attain hypertrophy cease expression of Aqp9 and may rely on other markers to maintain hypertrophy and prepare for ossification.

In the epiphyseal plate, Aqp9 appears to mimic the expression gradient of Col2a1 and Sox9, both of which are known markers of the proliferating and pre-hypertrophic zones (Gómez-Picos & Eames, 2015). However, specific Aqp9 expression in these zones does not necessarily mean Aqp9 functions to maintain those states. The murine epiphyseal plate is a host to extremely diverse factors that can promote certain chondrocytic phenotypes without being expressed where those chondrocytes would be. Ihh is expressed at the pre-hypertrophic junction but serves to maintain the proliferative state through PTHrP (Kronenberg, 2003). Wnt4, a Wnt family member that signals through β-catenin, is also expressed in the pre-hypertrophic zone but instead accelerates hypertrophy (Später et al., 2006; Hartmann & Tabin, 2000; Lee & Behringer, 2007). Rspo2, an activator of Wnt/ β-catenin signalling, is expressed in the cartilage primordium and embryonic long bone at the proliferating and pre-hypertrophic zones; yet it suppresses Col2a1 and Sox9 to promote hypertrophy (Nam, Turcotte, & Yoon, 2007; Takegami et al., 2016). It is possible that Aqp9 imports H2O2 in proliferating chondrocytes to induce hypertrophy and then ceases in expression, fulfilling its moonlighting role as a channelome member. However, Aqp9 is a bidirectional transporter and may instead export H2O2 to delay hypertrophic differentiation. Regardless, Aqp9 may function under the spatiotemporal guidance of unexplored transcription factors within the epiphyseal plate.

Overall, Aqp9 expression in the epiphyseal plate suggests that Aqp9 may be important in endochondral ossification and long bone growth. In situ hybridization at the proximal tibia suggests that Aqp9 regulates hindlimb growth, although it is likely that Aqp9 is expressed in other appendicular epiphyseal plates as well.

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5.1.3 P5 Aqp9 -/- mice have longer tibia bones

To determine if Aqp9 -/- mice have differential long bones at the pup stage, P5 WT and Aqp9 -/- littermates were dissected for whole-mount skeletal staining. There were no apparent gross body differences between the two genotypes. Long bone measurements revealed that Aqp9 -/- mice had significantly longer tibia bones than their WT counterparts. All humerus and femur bone measurements did not present with significant differences. Excised spleens did not appear to differ in weight, nor did the condylo-basal lengths of their skulls.

A long bone phenotype is observed in Fgfr3c -/- mice, where deletion of the Ffgf3 isoform results in dramatic skeletal overgrowth of all long bones (Eswarakumar et al., 2007). Fgfr3 is a known negative regulator of bone growth, as Aqp9 may also be as both their knockouts result in bone overgrowth (Deng, Wynshaw-Boris, Zhou, Kuo, & Leder, 1996). Other knockout mutations are reported to result in skeletal overgrowth. Deletion of a natriuretic peptide gene through Npr3 -/- mice result in skeletal overgrowth in 2 month old mutants (Matsuwaka et al., 1999). Conditional deletion of the cell proliferation activator Yap1 through Yap1c/c; Col2a1-Cre mutants result in skeletal overgrowth at E16.5 and E18.5 (Deng et al., 2016). However, the observations suggest that Aqp9 deletion is tibia-specific at P5 in bone length modulation. There are also certain knockout mutations that are bone-specific in their phenotype. In skeletal development, the highly regulated ECM contains proteoglycans of which the chondroitin sulfate proteoglycans are a large proportion (Wilson et al., 2012). In mice lacking chondroitin sulfate synthase-2, femur and tibia bone lengths are significantly reduced whereas humerus and ulnar lengths are unchanged (Ogawa et al., 2012). Nevertheless, tibial-specific overgrowth defects are mostly reported in physiological compensatory responses instead of mutations, such as in human fractures and canine femoral shortening (Taylor, 1963; Schaefer, Johnson, & O’Brien, 1995). At development, Aqp9 may normally signal with hindlimb-specific genes such as Tbx4 albeit in a mesomelic fashion at the tibia (Rodriguez-Esteban et al., 1999; Isidor et al., 2010). Interestingly, gene expression profiling of muscle invasive bladder cancer samples identified AQP9 and TBX4 as upregulated genes together in connective tissue disorders (Hussain et al., 2017). Hence, Aqp9 and Tbx4 may interact in the developing hindlimb. In mesomelic syndromes such as Langer mesomelic dysplasia, shortening of the distal limbs involves inactivation of the short-stature 104 gene Shox2, Runx2, and Ihh (Cobb, Dierich, Huss-Garcia, & Duboule, 2006). Interestingly, use of the ISMARA (Integrated System for Motif Activity Response Analysis) public dataset ‘Illumina Body Map 2’ shows that the AQP9 promoter is a top ten target for SHOX (Balwierz et al., 2014). It is possible that the Shox transcription factor regulates endochondral ossification partially through Aqp9. Deletion of Aqp9 may therefore repress a natural repressor of tibial growth and resolve in abnormally lengthened tibia bones at P5. It is also possible that Aqp9 is expressed differently throughout all long bone epiphyses and is under unexplored transcriptional control. Conversely, the use of more biological replicates may reveal that the tibial overgrowth is actually total long bone overgrowth. Overall, Aqp9 deletion does not severely affect skeletal stature at the pup stage and may be dispensable or redundant in effect. However, the lack of a difference in spleen and skull measurements between WT and Aqp9 -/- pups suggest that the mutation targets endochondral ossification—and does not affect intramembranous ossification or general organ development.

The early tibial overgrowth in P5 Aqp9 -/- mice appears reminiscent of precocious puberty. Human precocious puberty is characterized by the early development of sexual characteristics and is often idiopathic (Bourayou, Giabicani, Pouillot, Brailly-Tabard, & Brauner, 2015). However, the cause of precocious puberty has been linked to deficiencies in MKRN3, a zinc finger motif gene that normally helps inhibit activation of the hypothalamic-pituitary-gonadal axis (Shin, 2016). Defects in MKRN3 lead to accelerated growth, early bone maturation, early height gain, but ultimately reduced stature (Carel, Lahlou, Roger, & Chaussain, 2004). At P5, Aqp9 deletion appears to mimic the early growth though tibial elongation. Interestingly, absence of MKRN3 in Prader-Willi syndrome—a genetic disorder that includes short stature—is accompanied by a 1.52 fold change decrease of AQP1 expression and 2100.84 fold change decrease in AQP3 expression in tissue mitochondria (Yazdi et al., 2013). Since AQP1 and AQP3 are also aquaglyceroporins and can transport H2O2 like AQP9, it is possible that AQP9 plays a role here to influence skeletal stature but was not detected (Plourde et al., 2015; Laforenza, Bottino, & Gastaldi, 2016; Almasalmeh, Krenc, Wu, & Beitz, 2014). If Aqp9 deletion is involved in precocious puberty, a shorter skeletal stature relative to WT littermates closer to adulthood would support its role.

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The role of estrogen in Aqp9 regulation should also be considered. Estrogen receptors are expressed in newborn mice, where basal levels of estrogen bind and gradually stimulate long bone growth through growth hormone activation (Zuloaga, Zuloaga, Hinds, Carbone, & Handa, 2014; Cutler, 1997; Avtanski et al., 2014). During postnatal development, estrogen promotes chondrocyte proliferation and type II collagen expression in the epiphyseal plate (Shi, Zheng, Li, & Liu, 2017). Aqp9 is upregulated by estrogen in rat epididymal ductules but downregulated in rat hepatocytes (Oliveira, Carnes, França, Hermo, & Hess, 2005; Lebeck et al., 2012). The role of estrogen in chondrocyte Aqp9 expression has not been explored. Normally, estrogen binds alpha and beta estrogen receptors expressed in epiphyseal plate chondrocytes to promote proliferation (Li, Wang, Jiang, & Dai, 2012). Epiphyseal plate chondrocytes can endogenously produce 17β-estradiol to stimulate proliferation, protect against apoptosis, and promote longitudinal growth (Chagin, Chrysis, Takigawa, Ritzen, & Sävendahl, 2006). In Aqp9 -/- mice, Aqp9 is not available as a target and may free available estrogen to bind growth-promoting receptors, such as growth hormone receptors (Slootweg, Swolin, Netelenbos, Isaksson, & Ohlsson, 1997). It is also possible that if Aqp9 is a negative regulator of bone growth, estrogen regularly binds to its enhancer to prevent rampant bone growth. Without Aqp9 as a limiter, the P5 long bone phenotype may occur. This is similar to what is observed with insulin, which promotes chondrocyte proliferation and long bone growth (Zhang et al., 2014). Kuryiama et al. (2002) have shown that Aqp9 is downregulated by insulin addition via the negative insulin response element. If Aqp9 limits bone growth, insulin may bind to the element and instead promote bone growth. Estrogen and insulin may work together with Aqp9 as a downstream target, as combined estradiol and growth hormone treatment has been shown to elevate IGF levels in primates (Wilson, 1998).

Overall, Aqp9 may be a negative regulator of bone growth in early murine endochondral ossification. It is a possible intermediate in hormonal signalling that subtly impacts tibial growth, and its deletion in the Aqp9 -/- mutation may involve an estrogenic spurt that induces a precocious overgrowth phenotype. This hypothesis would be supported by hastened epiphyseal closure and shortened long bone lengths closer to the adult stage (Nilsson et al., 2014).

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5.1.4 P21 Aqp9 -/- mice have shorter femur bones

To determine if long bone abnormalities persisted between WT and Aqp9 -/- littermates at the juvenile stage, P21 male mice were dissected for whole-mount skeletal staining. There were no observable gross body differences between the two genotypes. Measurement of the long bones revealed that Aqp9 -/- mice had significantly shorter femur bones than their WT counterparts. All humerus and tibia bone measurements did not present with significant differences. Similarly to the P5 mice, excised spleens did not appear to differ in weight and condylo-basal skull lengths did not differ either. This reiterates that Aqp9 deletion targets endochondral ossification and may be independent of other growth processes.

A short bone phenotype observed in knockout mouse models is common. Sox9 +/- mice die perinatally with premature mineralization and shorter, distorted bones (Bi et al., 2001). Runx2 -/- mice also die shortly after birth without proper mineralization and subsequently shorter, nearly nonexistent limbs (Otto et al., 1997). Ihh -/- mice that survive till birth display severe dwarfism of the limbs (St-Jacques et al., 1999). Although these genes play different proliferative- hypertrophic roles in endochondral ossification, it is evident that they are all indispensable to long bone growth: their deletion, regardless of which chondrocytic phenotype they promote, severely diminishes the final bone length. However, certain gene mutations can induce shortened limbs without lethality. In Hp1bp3 -/- mice that lack the binding protein and IGF-1 modulator Hp1bp3, mutants display reduced femoral length and overall dwarfism (Garfinkel et al., 2015). Cobb et al. (2006) previously explored Shox2C/- mice with conditional Shox2 deletion via the limb-specific Prx1-Cre transgene. The mutants present with virtually nonexistent humerus bones, severely shortened femurs, and shorter tibias with bowing. At P21, Aqp9 deletion also appears to shorten long bones without inducing lethality. However, the observations suggest that the mutation is femur-specific at P21. There are few reported mouse mutants to model this phenotype. In Mmp13 -/- mutants, where Mmp13 is a critical enzyme for chondrocyte hypertrophy, a significant ~8% reduction in long bone length occurs only at the juvenile timepoint and only in femur bones (Inada et al., 2004). Murine deletion of Spred2, an FGFR3/MAPK intermediate, also results in bone-specific shortening in the hindlimb but at the tibia instead (Bundschu et al., 2005). Femoral length defects are mostly reported in human 107 conditions such as Down’s syndrome and skeletal dysplasia (Morales-Roselló & Llorens, 2012). In early puberty, Aqp9 may continue to normally signal with Tbx4 to affect hindlimb growth. Interestingly, the short bone phenotype here appears to be rhizomelic (Panda et al., 2014). In rhizomelic disorders such as rhizomelic chondrodysplasia punctata, shortening of the proximal limbs involves mutation of FGFR1 (White et al., 2005). Interestingly, Col2a1-Cre-driven deletion of Shox2 also results in rhizomelia due to precocious chondrocyte maturation and hypertrophy (Bobick & Cobb, 2012). As mentioned previously, AQP9 is a target of SHOX regulation and the two may function in concert at P21 to drive normal bone growth. Here, deletion of Aqp9 appears to reverse the long bone phenotype seen at the P5 timepoint. Aqp9 expression may naturally vary throughout the hindlimb epiphyseal plates temporally. Repeating the skeletal staining with more biological replicates may reveal that the femoral shortening actually affects the entire hindlimb significantly, or possibly all limbs. Overall, the Aqp9 -/- mutation does not drastically affect skeletal stature at the juvenile stage.

In comparison to the P5 bone phenotype, a striking observation is the reduced femur bone length at P21. In skeletal overgrowth models such as Fgfr3 -/-, the long bone defect persists into early adulthood without any apparent change in growth rate (Xie et al., 2017). Here, an early, specific long bone phenotype has transformed into a juvenile, specific short bone phenotype within the hindlimb. This observation is similar to precocious puberty, where mice can exhibit its characteristics as early as 7 days of age (McGee & Narayan, 2013). As mentioned previously, Aqp9 is also a target of estrogen and its deletion in the Aqp9 -/- mutation at P5 may promote estrogenic binding to growth receptors. By P21, excess estrogen may lead to earlier narrowing of the epiphyseal plate, slower growth, and shorter femurs (Weise et al., 2001). Deletion of Aqp9 appears to encourage growth through accelerated endochondral ossification but later discourages growth through a mechanism involving dysregulated hormonal and sexual development. Aqp9 may function alongside transcription factors that affect secondary sexual characteristics at P21, where a significant portion of sexual differentiation has occurred (Schlomer et al., 2013). In comparison to male mice, female mice have higher plasma estrogen levels at the juvenile stage (Saito et al., 2009). If female mice were used, the femoral shortening may be more pronounced.

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The effects of chondrocyte senescence in the epiphyseal plate should also be considered. Lui, Nilsson, & Baron (2011) describe that as the number of cell divisions accumulate, resting chondrocytes may gradually lose their replicative capacity. This would translate into proliferating chondrocytes with diminished proliferation and then growth deceleration. It is possible that every proliferating chondrocyte is affected by an intracellular or extracellular ‘cell cycle counter’ factor that progressively changes with each cell division. Interestingly, estrogen has been suggested to increase the loss of proliferative capacity per cell cycle (Schrier et al., 2006). P5 Aqp9 -/- mice likely exhibit accelerated proliferation that underscores their elongated tibias, whereas P21 mutants likely exhibit subsequent decelerated proliferation that captures their shortened femurs. Both observations would be supported by epiphyseal plate examination and chondrocyte gene profiling.

Overall, Aqp9 -/- mice appear to experience subtle hindlimb defects at P5 and P21. Aqp9 may have differential distribution on the chondrocyte membrane depending on the specific bone and stage of endochondral ossification. Aqp9 may also be under the control of factors such as estrogen, and its deletion may elicit the early bone elongation and later bone shortening through accelerated but ultimately impeded growth. The observations suggest that Aqp9 may be a highly plastic channelome gene that plays inconsistent roles in regulating long bone growth when deleted in a mouse model.

5.2 Epiphyseal plate irregularities underscore Aqp9-mediated bone length

5.2.1 P5 Aqp9 -/- mice have expanded distal femur proliferating zones

To determine the cause of the P5 bone phenotype, WT and Aqp9 -/- littermate distal femur epiphyseal plates were examined with H&E. Although the overgrowth phenotype occurred significantly in the tibias only, femurs were selected to investigate if any epiphyseal plate dysregulation could be observed in the adjoining hindlimb long bone. In mouse models with long bone phenotypes, spatial irregularities in those epiphyseal plate zones are predominantly guaranteed. Hence, investigating the tibia histologically was determined to be repetitious as 109 epiphyseal plate growth is predominantly coupled with bone formation (Jones et al., 2010). Additionally, the P5 knee sections used in this study provided the best visual clarity at the distal femur. The femur was therefore investigated; any defect observed in the Aqp9 -/- femur epiphyseal plate would support not only how the tibial overgrowth occurs, but also the possibility of the mutation affecting the whole hindlimb.

In the P5 Aqp9 -/- distal femur epiphyseal plate, the average height of the proliferating zone is significantly expanded compared to the WT epiphyseal plate. No significant differences are observed in the hypertrophic zone. The proliferating zone also features a significantly higher number of chondrocytes per column. The cellular density in Aqp9 -/- proliferating and hypertrophic zones did not significantly differ from that of WT littermates.

Based on the observed long bone phenotype and literature supporting accelerated proliferation, the proliferating and hypertrophic zones were focused upon to determine if their morphology would reflect the overgrowth. This was also supported by the fact that Aqp9 is unavailable to likely transport H2O2 into the chondrocyte and induce hypertrophy, which may allow chondrocytes to retain a proliferative phenotype for longer than usual in the epiphyseal plate. The resting zones were more dispersed and difficult to border, and were not measured in this figure. The Aqp9 -/- proliferating zone appears to have more acellular matrix surrounding the chondrocytes, possibly due to more synthesis of type II collagen and aggrecan than in the WT epiphyseal plate. However, a greater area of matrix and chondrocytes may have leveled off the cellular density in comparison to the WT proliferating zone. The hypertrophic zones in WT and Aqp9 -/- mice feature chondrocytes of varying shape and size that may influence the number of cells permitted in the zone. Increasing the sample size of epiphyseal plates may provide a more accurate representation. Importantly, the higher number of chondrocytes per column in the Aqp9 -/- epiphyseal plate is a hallmark of accelerated proliferation (Lui et al., 2011).

The expansion of the proliferating zone in P5 Aqp9 -/- mice supports the longer bone phenotype observed. Many knockout mouse models that feature long bone phenotypes, such as Fgfr3floxneo/floxneo mice, have increased expansion of proliferating and hypertrophic zones (Su et al., 2010). However, knockout mouse models such as Stat1 -/- present with early longer bones 110 and a proliferating zone-specific expansion, as observed with the P5 Aqp9 -/- mice (Sahni, Raz, Coffin, Levy, & Basilico, 2001). Interestingly, STAT1 is a negative regulator of chondrocyte proliferation and promoter of chondrocyte apoptosis through upstream FGFR3 signalling (Sahni et al., 1999). This coincides with the observation that chondrocyte deletion of Aqp9 results in improved cell viability after H2O2 treatment (Shao et al., 2016). As STAT1 is also activated through H2O2 induction, the two genes may normally share a hypoxic method of promoting physiological apoptosis in the epiphyseal plate (Burova, Grudinkin, Bardin, & Gamaleĭ, 2001). Their deletion, however, may lead to expansion of the proliferating zone and subsequently longer bones. Aqp9 may function in a similar proliferation-specific manner as Stat1 in the epiphyseal plate to regulate bone growth.

Histological examination of the P5 Aqp9 -/- distal femur epiphyseal plate indicates that an irregular enlargement of the proliferating zone underscores not only the tibial long bone phenotype, but the possible enlargement of other hindlimb bones as well. Deletion of Aqp9 may promote a proliferation-leaning, anti-apoptotic phenotype that accelerates early growth and is highlighted by expansion of the epiphyseal plate proliferating zone.

5.2.2 P21 Aqp9 -/- mice present with typical epiphyseal plate zones

To determine the cause of the P21 bone phenotype, WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were examined with a primary antibody against Col10a1. Col10a1 is a marker of chondrocyte hypertrophy and was expected to stain robustly in the hypertrophic zones. In particular, the hypertrophic zone was of interest as a shorter bone phenotype is often rooted in defected chondrocyte hypertrophy (Shu et al., 2011). Using a marker of hypertrophy served to investigate if any hypertrophy defect would be apparent by P21 to explain the Aqp9 -/- short bone phenotype. Although the short bone phenotype occurred significantly in the femurs only, proximal tibias were selected for the aforementioned reason of redundancy in epiphyseal plate- bone growth coupling. Furthermore, only tibial sections were available. The tibia was therefore investigated histologically as an alternative. Any irregularity in Col10a1 staining of the Aqp9 -/- proximal tibia epiphyseal plate would support not only how the femoral shortening occurs, but also the possibility of defected hypertrophy throughout the hindlimb at the juvenile stage. 111

In the P21 Aqp9 -/- proximal tibia epiphyseal plate, the ratio of the hypertrophic zone to the resting-proliferative zone is not significantly different from that of WT littermates. Similarly, the observable epiphyseal plate heights did not significantly differ either. As mice age, the rate of longitudinal growth also slows and is reflected by an increasing ratio of hypertrophic zone height to proliferating zone height (Lee, Song, Pai, Chen, & Chen, 2017). It was postulated that in mice with comparatively lower rates of growth and shorter bones—as expected in P21 Aqp9 -/- mutants—this ratio would be higher.

Overall, the P21 Aqp9 -/- proximal tibia epiphyseal plate is comparable to that of P21 WT mice. Interestingly, the knockout of genes contributory to endochondral ossification do not always result in drastic adult skeletal phenotypes. MATN3 is an aforementioned gene that encodes non- collagenous ECM and is mutated in many epiphyseal dysplasias (Klatt et al., 2000). Embryonic Matn3 -/- mice have hypertrophic zones 47% larger than that of embryonic WT mice, but no long bone phenotype is observed during development or adulthood in the mutants (van der Weyden, 2006). In the case of Aqp9 -/-, a juvenile short bone phenotype is observed but may not necessarily be due to irregularities of the epiphyseal plate. In P15 Stat1 -/- mice, the epiphyseal plate differences underlying the early long bone phenotype are attenuated (Sahni et al., 2001). Hence, it possible that temporary expansions of epiphyseal plate zones do not persist into later development. However, it is more probable that due to the small sample size of biological replicates, the analysis is not statistically powerful. Repetition of the experiment with a greater sample size may find that the P21 Aqp9 -/- hypertrophic zone consumes more area in the epiphyseal plate, and that the entire epiphyseal plate is significantly shorter.

5.2.3 18 month old Aqp9 -/- mice show prominent epiphyseal plate narrowing and reduction of marrow adipose tissue

Previously, Liu et al. (2007) had isolated femurs and tibias from one year old female WT and Aqp9 -/- mice and found no significant difference in length between the WT and Aqp9 -/- bones. This was not consistent with the short bone phenotype observed in the male P21 Aqp9 -/- mice. The Aqp9 -/- mice were derived from C57BL/6 mice in both studies, suggesting that strain- 112 specific differences cannot be implicated. This suggests that the bone phenotype may not persist into adulthood, or that sex differences complicate measurement consistency. Indeed, female C57BL/6J mice experience greater age-related declines in femoral bone volume and length than males over time (Glatt, Canalis, Stadmeyer, & Bouxsein, 2007). The absence of Aqp9 in old male mutants was therefore unexplored, and may present with different long bone observations than those of female mice.

Observing the old epiphyseal plate is a strong indicator of whether or not long bone growth has been arrested. Epiphyseal plate narrowing and closure into the epiphyseal line indicates that chondrocyte proliferation has stopped and that bone growth can no longer proceed (Shim, 2015). However, longitudinal bone growth does not cease in mice at sexual maturity like humans; the rate of growth dramatically slows instead (Jilka, 2013). In mice, total epiphyseal plate fusion does not occur but narrows as the growth velocity approaches zero (Kilborn, Trudel, & Uhthoff, 2002; Emons, Chagin, Sävendahl, Karperien, & Wit, 2011). Unusually narrow old epiphyseal plates or striking abnormalities would then indicate a defect in bone growth and provide insight into the final long bone length. Hence, male WT and Aqp9 -/- mice were aged to 18 months for epiphyseal plate analysis to observe if they would reflect a shorter bone phenotype, as observed in the male P21 mutants.

18 month old male WT and Aqp9 -/- femoral heads were examined with Toluidine blue to analyze their cartilaginous epiphyseal plates. Femoral heads were selected due to their availability from previous experiments in the Kannu lab. In the Aqp9 -/- mice, the epiphyseal plate does not appear to reach the cortical bone on the right-side and is instead ablated. The observation was initially thought to be due to sectioning inconsistencies that caught the Aqp9 -/- epiphyseal plates at a particular depth, but statistical analysis shows the same trend and significantly reduced width across all Aqp9 -/- biological replicates compared to WT replicates.

Overall, analysis of old male WT and Aqp9 -/- mice suggest that 18 month old male Aqp9 -/- mice not only experience a more rapid disappearance of the epiphyseal plate, but that it may translate into subtle long bone defects. A quicker narrowing of the epiphyseal plate suggests that the long bone growth rate is reduced, and that the final bone length may be shortened in 113 comparison. This is consistent with precocious puberty, where an accelerated period of growth— as observed at P5—ultimately results in diminished growth by adulthood. Although this phenotype is observed as early as in P21, the 18 month old observations here suggest that this irregular narrowing may occur as early as the juvenile stage and into the geriatric stage. However, epiphyseal plate analyses at each of the hindlimb bones would be required to identify if Aqp9 deletion continues to affect specific long bones. Nevertheless, the irregular epiphyseal plate narrowing observed here in the old age Aqp9 -/- femur is consistent with the femoral short bone phenotype in the P21 mice. In other mutant mouse models—such as deletion of Ahsg, an inhibitor of skeletal mineralization—discontinuities along the old epiphyseal plate also underscore accelerated ossification and shortened long bones (Seto et al., 2012).

Interestingly, the Aqp9 -/- primary bone marrow also presented with a drastic reduction of white adipose tissue in comparison to WT littermates. This reduction of fat was accompanied by an increase of visible HSCs, as shown in the small, stained spots distributed throughout the bone marrow. Typically, fat content in the femoral bone marrow increases with age (Tuljapurkar et al., 2011). However, a lack of adipose tissue is not necessarily suggestive that the bone is immature. When MSCs decide on a cellular lineage, they can differentiate into chondrocytes and fibroblasts, pre-osteoblasts, or pre-adipocytes (Rosen, Ackert-Bicknell, Rodriguez, & Pino, 2009). However, the presence of marrow adipocytes may be self-promotive and induce the differentiation of MSCs into adipocytes, preventing other cellular lineages from being attained (Duque, 2007). In the Aqp9 -/- bone marrow, a severely diminished amount of adipose tissue suggests that the majority of MSCs have likely committed to other lineages instead—such as chondrogenesis or osteoblastogenesis—without coercion by adipocytes. Without fatty bone marrow, it is possible that old Aqp9 -/- mutants proceeded with endochondral ossification at a faster rate than WT mice because the available MSCs were condensed for long bone growth instead. This would support the hypothesis that Aqp9 -/- mutants experienced accelerated growth at a younger age. Furthermore, bone marrow adipocytes function to release fatty acids and provide energy for bone growth, remodelling, and hematopoiesis (Veldhuis‐Vlug & Rosen, 2017). A reduction of adipose tissue may also suggest that during early endochondral ossification, Aqp9 -/- chondrocytes rely on fatty reserves for accelerated growth. Examination of the bone marrow at various timepoints would be required to verify this possibility. 114

It is also possible that the reduced bone marrow adipose tissue is reflective of protection against through Aqp9 deletion. Bone quality declines with aging and subcutaneous fat redistributes to sites such as the bone marrow, heart, and liver (Tchkonia et al., 2010; Rodríguez, Catalán, Gómez-Ambrosi, & Frühbeck, 2006). A reduction of bone marrow adipose suggests that the 18 month Aqp9 -/- mice may be protected against age-related biasing of adipogenesis and bone loss. This suggests that Aqp9 may continue to be a negative regulator of bone growth in adulthood. This also appears to parallel the findings of Bu et al. (2012), where ten week old Aqp9 -/- mice were protected against microgravity-induced bone resorption. In aging bone marrow stromal cells, ROS accumulation prevents their ability to maintain HSCs (Khatri et al., 2016; Anthony & Link, 2014). Simultaneously, this invites adipocyte accumulation in the bone marrow that further impairs hematopoiesis and bone regeneration (Ambrosi et al., 2017). In Aqp9 -/- mice, a lack of functional Aqp9 may prevent endogenous cytoplasm H2O2 accumulation or intercellular H2O2 transport in the bone marrow. Hence, the Aqp9 -/- bone marrow microenvironment may favor HSC maintenance instead of adipogenesis, resulting in the reduced bone marrow adipose observed at 18 months. This mechanism may suggest that reduced bone marrow adipose in 18 month Aqp9 -/- mice is not age-oriented, but rather a functional consequence of Aqp9 absence. Nevertheless, the irregular epiphyseal plate narrowing suggests that Aqp9 deletion induces a diminished long bone growth rate in adulthood.

5.2.4 E16.5 Aqp9 -/- mice have irregular epiphyseal plate resting zones

To determine if Aqp9 affects endochondral ossification during development, E16.5 WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were examined with Safranin-O. Proximal tibias were selected due to their availability from previous experiments in the Kannu lab. At E16.5, the developing embryo is at the halfway point between when endochondral ossification commences—at approximately E10.5—and when birth occurs—at approximately E21.0. Hence, E16.5 is a prime timepoint to examine endochondral ossification in development. Here, the average Aqp9 -/- resting zone height is significantly shorter than that of WT littermates. No significant differences were observed in the proliferating and hypertrophic zones. Consequently, the fraction of the whole epiphyseal plate that contains proliferating and hypertrophic 115 chondrocytes is significantly greater in the Aqp9 -/- mice than in WT mice. Although the proliferating and hypertrophic zones are a popular focus in mutant mouse models, the height of the resting zone has been scrutinized in Smad4 mutants. SMAD4 is a member of the SMAD family and signals under BMP and TGF-β as early as in mesenchymal differentiation (Wu, Chen, & Li, 2016). In Col2a1-Cre; Smad4Co/Co mice, the conditional deletion of Smad4 results in newborn dwarfism characterized by a drastically expanded resting zone (Zhang et al., 2005). In E16.5 Aqp9 -/- mice, a diminished resting zone may suggest the opposite. Skeletal staining at E16.5 would be required to verify long bone lengths in comparison to WT littermates.

The resting zone is host to chondrocytes that are stem-like; they can divide and differentiate into proliferating chondrocytes but with a limit reflecting a senescence factor (Abad et al., 2002). A depletion of resting chondrocytes is indicative of proliferative capacity expenditure, which is suggested to underlie the natural decline in long bone growth rate with age (Schrier et al., 2006). The irregular central ‘caving’ of the proliferating zone into the resting zone in the Aqp9 -/- embryos suggests that a greater proportion of resting chondrocytes are differentiating during development. Hence, the resting zone is diminished in height while the proliferating and hypertrophic zones together are expanded in height. This observation reflects the hypothesis that Aqp9 deletion results in an early accelerated phenotype at the epiphyseal plate, which may occur in development before P5. In epiphyseal plate senescence, Lui et al. (2011) discuss that during accelerated growth, resting chondrocytes sacrifice proliferative capacity to produce more proliferating chondrocyte columns in the short term. This comes at a cost of the resting zone cell density and lifespan, and may require spatial decline as the proliferating zone consumes a greater area. Here, an irregularly diminished resting zone in E16.5 Aqp9 -/- mice reflects the theory. Although the P5 epiphyseal plate resting zones were not measured in this study, the observation coincides with the P5 Aqp9 -/- proliferating zone expansion. Therefore, the tibial overgrowth observed in Aqp9 -/- mutants may commence in development as early as E16.5.

Overall, shortened proximal tibia resting zones in developing Aqp9 -/- mice suggest that their epiphyseal plates host chondrocytes that may differentiate faster. This phenotype may persist postnatally into the P5 age where Aqp9 -/- mice show expanded tibial proliferating zones and longer tibias than their WT littermates. However, accelerated differentiation likely leads to early 116 epiphyseal plate senescence and slower growth rate, as observed in the shortened P21 Aqp9 -/- femurs. By adulthood, the epiphyseal plate shows more prominent narrowing than WT littermates, suggesting that the growth rate has rescinded more rapidly into maturity due to the precocious overgrowth observed.

5.3 Aqp9 mutant chondrocytes show a differential phenotype

5.3.1 P5 Aqp9 +/- and Aqp9 -/- epiphyseal plate chondrocytes proliferate irregularly

To support the role of Aqp9 as a regulator of chondrocyte differentiation and long bone growth, the investigation was continued at the cellular level. The P5 timepoint was chosen because:

1) Early long bone phenotypes in mutant mouse models are infrequent, and 2) Epiphyseal plate chondrocytes at the P5 timepoint are excised without complication.

A cell proliferation experiment was performed by seeding WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes for cell counts over 96 hours. Over the experiment duration, WT chondrocytes did not differ significantly in cell number from Aqp9 +/- or Aqp9 -/- chondrocytes during any timepoints except at the 48 hour timepoint. By the final timepoint, all samples approached the expected confluency of 1.2 x 106 cells.

From 0-96 hours, the WT plot appears relatively linear and deviates from an expected exponential rate of growth in culture. While exponential growth is observed from 0-48 hours, the slope plateaus greatly by the 72 hour timepoint before approaching confluency. This may be attributed to cellular density or natural declines in chondrocyte proliferation that may be triggered by culture conditions (Lui & Baron, 2011). The Aqp9 -/- plot shows a striking difference at the 48 hour timepoint in comparison to the WT plot; rather than experiencing exponential growth, the cell number was only marginally higher than that of the 24 hour timepoint. At the 48 hour timepoint, the Aqp9 -/- number is ~4 x 105 lower than that of the WT. However, at the 72 hour timepoint, the Aqp9 -/- number jumped to ~1 x 106. The slope declines greatly by the 96 hour timepoint, likely due to cellular density as plate confluency is approached. 117

The Aqp9 +/- plot parallels the Aqp9 -/- plot, although the jump from 48-72 hours is not as drastic. Nevertheless, the Aqp9 +/- chondrocytes matched the WT chondrocyte cell number at the 72 hour timepoint despite a lower count by comparison at the 48 hour timepoint. As the most striking change occurs between the 48-96 hours, the rates there can be calculated as follows:

Table 5.1. Primary epiphyseal plate chondrocyte proliferation rates (48-96 hours)

(Cell number at 96 hours – cell number at Sample genotype Rate (cells/hour) 48 hours) / (96 hours – 48 hours)

WT (~1x106 – ~5.5x105) / 48 ~9375

Aqp9 +/- (~9.5x105 – ~2x105) / 48 ~15625

Aqp9 -/- (~1.5x106 – ~2.5x105) / 48 ~26042

Here, both Aqp9 +/- and -/- chondrocytes appear to have accelerated proliferation following an apparent pause in growth, in comparison to WT chondrocytes. However, their rates from 0-96 hours do not differ as they start and end at approximately the same cell number counts. In the growing rat epiphyseal plate, this phenomenon of ‘catch-up growth’ is observed in proliferating chondrocytes immediately after cessation of dexamethasone, a growth inhibitor (Chagin, Karimian, Sundström, Eriksson, & Sävendahl, 2010). Deletion of Aqp9 may promote cell proliferation after suppression through an unexplored mechanism. The pause in growth at the 48 hour timepoint may also be caused by an unexplored mechanism. Interestingly, the error bars for all chondrocyte counts at this timepoint appear narrower than those of the other timepoints. This may be due to a small sample size resulting in a statistical anomaly. Nevertheless, the error bars for the Aqp9 -/- chondrocyte count at 72 hours are remarkably wide and suggest that further repetition of the experiment would be supportive. Increasing the sample size would be beneficial to reduce the variance.

In normal epiphyseal plate chondrocytes, H2O2 is involved in a variety of signal transduction pathways. Intercellular H2O2 transport is only reported among aquaporin homologues (Bienert,

Schjoerring, & Jahn, 2006). However, the cellular production of H2O2 is mainly derived from the mitochondrial matrix and intermembrane space (Boveris & Cadenas, 2000). Hence, the transport of H2O2 from the mitochondria to the cytoplasm likely requires Aqp9 function, or other aquaporin isoforms that are capable such as Aqp3 and Aqp8. AQP9 protein localization in 118 mitochondria has been described, although aquaporin transport functionality in mitochondria has been argued against due to a lack of physiological consequence in several aquaporin mutants (Lindskog, Asplund, Catrina, Nielsen, & Rützler; 2016; Yang, Zhao, & Verkman, 2006). It is also possible for H2O2 to alternatively exit the mitochondria through initial enzymatic conversion to superoxide, and then passage through the electron transport chain proteins and voltage- dependent anion channels (Han, Canali, Rettori, & Kaplowitz, 2003). Nevertheless, Aqp9 is a channelome member and is likely expressed on the chondrocyte mitochondrial membrane, allowing passage of H2O2 into the cytoplasm. Further study such as Aqp9 immunostaining of the murine chondrocyte mitochondria would confirm its membrane expression. Outside the mitochondria, members of the HIF family are stimulated through mTOR signalling (Land & Tee, 2007; Mohlin et al., 2015). mTOR signalling is repressed by AMPK signalling, which in turn is repressed by H2O2 (Lennicke, Rahn, Lichtenfels, Wessjohann, & Seliger, 2015). Hence, H2O2 is a stimulator of HIF activity. In particular, HIF2α is a HIF homologue stabilized by H2O2 oxidative stress and induces chondrocyte hypertrophy by binding to the COL10A1, MMP13, and VEGFA promoters in the nucleus (Diebold et al., 2010; Saito et al., 2010). From mitochondria to nuclear transcription, the transport of H2O2 to the cytoplasm likely relays stimulus for chondrocyte hypertrophy. When extra-mitochondrial transport of H2O2 is diminished, hypertrophy may be delayed. In Aqp9 -/- chondrocytes, it is possible that a proliferative phenotype is attained this way. This would support the irregular proliferation rate observed in this experiment, the expanded proliferating zone, and early long bone phenotype observed at P5.

It is also possible that deletion of Aqp9 induces a ‘mitotic wave’. Rabbit articular chondrocytes treated with TGF-β1 show increased DNA replication rates, a sequestering of chondrocytes in the G2/M checkpoint, and a subsequent wave of mitosis (Vivien et al., 1993). This would require a temporary increase in proliferation rate as shown in the Aqp9 -/- chondrocytes. The catch-up growth between 48-96 hours of culture may be reflective of this pattern. However, the relationship between TGF-β and Aqp9 in the epiphyseal plate has not been explored. Overall, Aqp9 may be important in chondrocyte proliferation in the early pup stage. From the observations, Aqp9 may be a negative regulator as its deletion induces a different proliferation trend compared to WT chondrocytes. Deletion of Aqp9 may induce a proliferating-leaning 119

chondrocytic phenotype by barring physiological levels of H2O2 from stimulating hypertrophy at a regular rate.

5.3.2 P5 Aqp9 +/- and Aqp9 -/- epiphyseal plate chondrocytes have differential gene expression profiles

To clarify the proliferation rate difference observed in P5 Aqp9 +/- and -/- chondrocytes— however limited to specific experimental timepoints—their gene expression profiles were investigated. P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes were subjected to qPCR gene expression analysis. The fold change levels of several hypertrophic markers in the Aqp9 +/- and -/- chondrocytes were not significantly different relative to the WT control. In Aqp9 -/- chondrocytes, the fold change of the one marker of proliferation, Sox9, was significantly increased. Aqp9 expression was significantly decreased. The Aqp9 +/- chondrocytes did not follow these changes as consistently. There was a significant reduction of Mmp13 expression in the Aqp9 +/- chondrocytes compared to the WT chondrocytes.

In Aqp9 -/- chondrocytes, the increase of Sox9 is indicative that their gene profiles lean towards a proliferative phenotype. In Aqp9 +/- chondrocytes, the significant decrease of Mmp13 is indicative that they are resisting the hypertrophic phenotype, where Mmp13 would be increased. While the fold changes of the remaining hypertrophic markers—Col10a1 and Runx2—were not statistically significant in both Aqp9 -/- and +/- chondrocytes, their direction of change is noteworthy. Since Aqp9 expression changes in the same direction as the hypertrophic marker genes and also opposes the fold change direction of the proliferative marker Sox9, Aqp9 may be a contributor to chondrocyte hypertrophy upstream of Col10a1, Mmp13, and Runx2. It is also possible that the mutant chondrocytes invoke surveillance mechanisms to ensure appropriate mRNA quality, influencing the gene profile readout observed. Mammalian cells are capable of using the Nonsense-mediated decay pathway, a RNA surveillance mechanism that targets mRNAs with premature stop codons for deletion to prevent production of truncated proteins (Hug, Longman, & Cáceres, 2016). If this pathway is activated in the mutant chondrocytes, the significant fold change decrease of Aqp9 may not be reflective of reduced hypertrophy but of Nonsense-mediated decay checking the Aqp9 mutation instead. Furthermore, the non-significant 120 change of Col10a1 and Runx2 in both Aqp9 -/- and +/- chondrocytes relative to the WT chondrocytes may be analytical error due to a low number of biological replicates. Repetition with a greater number of replicates may produce a more consistent gene profile.

Other mutant mouse models with deletion of genes critical to chondrogenic differentiation have similar patterns of chondrocyte marker expression. MAP3K7 is a gene encoding TAK1, an enzyme critical to the TGF-β and BMP pathways (Le Goff et al., 2016). In chondrocyte-specific deletion of Tak1 in P7 mice, there is an accompanying significant decrease in expression of Sox9 and Col2a1 while Mmp13 expression increases (Gao et al., 2013). In Aqp9 -/-, the same pattern of proliferative and hypertrophic marker expression is observed in the opposite fold change direction. Hence, deletion of Aqp9 appears to be deletion of a functional hypertrophy inducer.

It is possible that certain signalling molecules transported among epiphyseal plate chondrocytes in culture by Aqp9 influence the expression of hypertrophic genes. If functional Aqp9 is ablated as in Aqp9 -/- chondrocytes, then a reduction of those transported molecules may trigger non- physiological pathways leading to reduced transcription factor-promotion of genes like Col10a1, Mmp13, and Runx2. Epiphyseal plate chondrocytes can exchange micro RNAs among one another through extracellular vesicles to elicit chondrogenic differentiation (Lin et al., 2018). However, the function of Aqp9 in chondrocyte matrix vesicle communication has not yet been explored. The hypothesized blockage of endogenous H2O2 from exiting the chondrocyte mitochondria may also be responsible for the decreased expression of hypertrophic genes.

Without Aqp9 to transport H2O2 and stabilize Hif2α, binding to Col10a1 and Mmp13 promoter regions is likely reduced, resulting in diminished hypertrophy. It is also important to consider that cellular hypertrophy is maintained by the cytoskeleton. This cellular network of microtubules, actin, and other diverse filaments provides the physical structure for chondrocyte shape and henceforth governs—at least partially—chondrocyte phenotype (Benjamin, Archer, & Ralphs, 1994). Interestingly, actin-containing cytoplasmic projections known as filopodia were upregulated in murine fibroblasts when transfected with a human aquaporin-9 (AQP9) overexpression vector (Loitto et al., 2007). It was hypothesized that a specific water/solute transporter increased hydrostatic pressure within the membrane-cytoskeleton space to permit outward filament elongation and form extracellular protrusions. Karlsson et al. (2013) confirmed 121 this with a model for AQP9-induced membrane projection in embryonic kidney cells, demonstrating that local membranous accumulation of AQP9 initiates protrusion and subsequent extension of the actin cytoskeleton. In Aqp9 -/- chondrocytes, lack of functional Aqp9 may inhibit the cytoskeleton plasticity necessary to initiate and maintain the hypertrophic shape.

In summary, P5 Aqp9 -/- epiphyseal plate chondrocytes express gene profiles that appear to oppose hypertrophy and favor proliferation, as noted by the significant expression increase of Sox9—a promoter of chondrocyte proliferation and inhibitor of hypertrophy. This may explain the differential proliferation rate of Aqp9 -/- chondrocytes, leading to expansion of the proliferating zone and accelerated long bone growth at the pup stage. A chondrocyte proliferation increase in the fetal metatarsal epiphyseal plate via insulin and IGF-1 treatment results in increased proliferating zone length and total bone length (Zhang et al., 2014). Deletion of chondrocyte Aqp9 may mimic this mitogenic effect.

5.3.3 Aqp9 knockdown in P5 WT epiphyseal plate chondrocytes induces a gene profile similar to Aqp9 -/- chondrocytes

In the Aqp9 +/- and Aqp9 -/- chondrocytes, it is possible that their gene expression profiles were not induced within the chondrocyte but were rather a consequence of signalling from other organs, such as the bone. Hence, P5 WT chondrocytes were excised and transfected with an Aqp9-siRNA to determine if a similar gene profile could be adopted. If so, any Aqp9 -/- cellular phenotypes can be accredited to processes occurring specifically in the chondrocytes in a cell autonomous manner.

Overall, there were no significant differences in gene expression after Aqp9 silencing. However, the direction of change of each gene probe is noteworthy. The direction of change of the hypertrophic markers—Col10a1, Mmp13, and Runx2—was downward, and the proliferative marker—Sox9—was upward. The direction of change of Aqp9 expression was also downward. Importantly, the overall gene profile of WT-siRNA reflected the gene profile of the Aqp9 -/- chondrocytes. This supports that Aqp9 knockdown alone is able to influence the expression of chondrocyte proliferative and hypertrophic markers, and that the similar gene profile observed in 122

Aqp9 -/- chondrocytes was not attributed to external factors such as the generation of the Aqp9 -/- mutant mouse itself. The lack of observed statistical significance is likely due to a small sample size. The incubation time post-transfection may also be on the shorter end of the protocol recommendation. The Aqp9 siRNA oligomer concentration may also be insufficient. The error bars for all gene probes are wide, hinting at deviant fold change values among the biological replicates.

In this experiment, knockdown of Aqp9 in WT chondrocytes was able to simulate the Aqp9 -/- chondrocyte gene profile. A natural inhibitor of Aqp9 named phloretin is able to decrease expression of MMP13 and prevent the degradation of type II collagen, albeit in human articular chondrocytes (Zheng, Chen, Zhang, Cai, & Chen, 2018). The use of phloretin to target Aqp9 in epiphyseal plate chondrocytes is unexplored. Nevertheless, the concomitant downregulation of hypertrophy-related genes following Aqp9 inhibition is well-reported.

5.4 A model for Aqp9 function in murine endochondral ossification

In this study, analysis of the literature and findings support a speculative model for the function of Aqp9 in murine long bone growth. In Figure 5.1, the impact of Aqp9 deletion is described from the chondrocytic level to the long bone at both P5 and P21. The steps synthesize the aforementioned literature and results, and are described as follows:

1) The pathway starts in the developing epiphyseal plate. The ATP-synthesizing process of oxidative phosphorylation in the epiphyseal plate chondrocyte mitochondrial matrix generates endogenous superoxide as a waste product, which is then

converted into H2O2.

2) H2O2 attempts to leave through H2O2 transporters along the mitochondrial membranes, but Aqp9 is not available for

transport due to the Aqp9 -/- mutation. Here, H2O2 may be sequestered in the mitochondria.

3) H2O2 can exit the mitochondria through alternative means, such as enzymatic conversion back to superoxide and then

transport through voltage-dependent anion channels. The rate of H2O2 departure is therefore diminished in Aqp9 -/- chondrocytes.

4) Superoxide that is transported into the cytoplasm can be reconverted into H2O2. H2O2 can function to repress AMPK,

which is a repressor of mTOR. H2O2 is then an activator of mTOR. However, loss of Aqp9 suggests that less-than-

physiological H2O2 will be present in the cytoplasm, so mTOR activation may be diminished. 123

5) H2O2 can repress PTP, which is a repressor of Akt (Lennicke et al., 2015). Akt is a factor that ultimately activates mTOR.

Once again, H2O2 is an activator of mTOR—but without physiological concentrations of H2O2 in the cytoplasm due to Aqp9 deletion, mTOR activation may be repressed.

6) mTOR is an activator of Hif2α, a hypertrophy inducer in chondrocytes. Oxidative stress conferred by cytoplasmic H2O2

normally stabilizes Hif2α. However, Aqp9 deletion suggests less cytoplasmic H2O2 and therefore less Hif2α. 7) Normally, Hif2α binds to the promoters of hypertrophy genes in the nucleus to promote the hypertrophic phenotype. Without physiological amounts of Hif2α, fewer hypertrophy genes are transcribed and the proliferative phenotype is more likely to be retained. 8) In the P5 Aqp9 -/- mouse, more proliferation allows endochondral ossification to occur at a faster rate than usual. Circulating estrogen finds that there is no Aqp9 target to bind and instead binds available growth hormone receptors, further accelerating growth at this stage. The growth phenotype becomes localized in the hindlimb, which Tbx4 governs in development. Furthermore, the Shox transcription factor finds there is no Aqp9 promoter to bind, possibly inducing a rhizomelic effect in the tibia along with Runx2 and Ihh. Ultimately, a long bone phenotype is observed in the tibia bones. 9) As the Aqp9 -/- mouse ages, it continues to undergo endochondral ossification as the epiphyseal plate narrows. Due to the precocious overgrowth, the epiphyseal plate has become more senescent than usual. The resting chondrocytes have lost more replicative capacity than usual. The rate of long bone growth has become gradually retarded relative to WT mice. 10) By P21, the Aqp9 -/- mouse experiences the consequences of accelerated growth. Circulating estrogen now hastens epiphyseal plate narrowing, further resulting in slower growth rate. The shortening phenotype is still localized in the hindlimb, likely through Tbx4. However, the Shox transcription factor now induces a mesomelic effect in the femur, possibly through dysregulation of Fgfr1. Finally, the femur bones are shorter.

11) In WT mice, epiphyseal plate chondrocytes maintained physiological levels of cytoplasmic H2O2 due to functioning Aqp9 transporters. Chondrocyte hypertrophy occurred naturally without a proliferative-leaning phenotype. As a result, P5 WT mice have shorter tibia bones compared to their Aqp9 -/- littermates. By P21, a regular rate of growth has allowed for the WT mice to have longer femur bones instead. Throughout growth in the WT mice, Aqp9 is able to interact normally with its transcription factors.

Figure 5.1. A model for Aqp9 function in murine endochondral ossification 124

ECM / Extracellular space

Chondrocyte cell membrane

H2O2

Chondrocyte cytoplasm H2O2 5 Akt Hif2α PTP H2O2 6

mTOR AMPK

H2O2 4 7

3 Col10a1 Aqp9 -/- Mitochondria Alternative Mmp13 exit

2 1 Vegfa Oxidative H O phosphorylation 2 2

Mitochondrial matrix Etc…

Intermembrane space Nucleus (not to scale) Figure 5.1. cont. 125

8 Tibia 10 Femur P5 P21

9

Aqp9 -/- Aqp9 -/-

Tbx4 Shox, Runx2, Ihh Tbx4 Shox, Fgfr1

Estrogen (+growth) Estrogen (-growth) WT growth scenario

Femur P5 Tibia P21

11

Aqp9 Aqp9

Regular TF-promoter interaction Regular TF-promoter interaction 126

6 Conclusions

To address whether Aqp9 is important in endochondral ossification, Aqp9 -/- mice were examined at the long bone, epiphyseal plate, and chondrocyte levels from the pup stage to old age. At P5, Aqp9 -/- mice have significantly longer tibia bones than their WT littermates. At P21, Aqp9 -/- mice have significantly shorter femur bones than their WT littermates. These defects suggested that Aqp9 deletion may induce accelerated neonatal growth that stunts skeletal length by the juvenile age, albeit in specific hindlimb bones. Hindlimb epiphyseal plates were then examined to see if dysregulation in the epiphyseal plate zones would underscore the bone phenotypes. Analysis of the P5 Aqp9 -/- femur epiphyseal plate showed expansion of the proliferating zone, supporting a long bone phenotype throughout the hindlimb. However, analysis of Col10a1 staining in the P21 Aqp9 -/- tibia epiphyseal plate did not show any remarkable dysregulation. Examination of 18 month Aqp9 -/- femur heads showed irregular narrowing of the epiphyseal plate and reduced adipose tissue in the bone marrow, suggesting that old age Aqp9 -/- mice experience irregular growth earlier in their lifespan and resist bone marrow adipose accumulation. E16.5 Aqp9 -/- embryo epiphyseal plates were also examined to determine if Aqp9 is important in development; they showed significantly reduced resting zone heights, indicating an early acceleration of chondrocyte differentiation. Finally, P5 Aqp9 +/- and Aqp9 -/- epiphyseal plate chondrocytes were examined to see if their behaviour and gene expression could explain the P5 bone phenotype and epiphyseal plate dysregulation. Aqp9 -/- chondrocytes displayed an irregular rate of proliferation characterized by a lag in growth and then subsequent recovery. Gene expression analysis of Aqp9 -/- chondrocytes showed a significant increase of proliferative marker expression. Silencing of Aqp9 in P5 WT chondrocytes did not yield significant expression differences, but was able to simulate the same directions of change observed in the Aqp9 -/- chondrocyte gene probes. This suggests that Aqp9 -/- chondrocytes may favour a proliferative phenotype in differentiation, supporting the expanded proliferating zone in the P5 epiphyseal plate.

Aqp9 may be a negative regulator of neonatal bone growth. Deletion of Aqp9 appears to induce chondrocyte proliferation and accelerate the process of endochondral ossification, leading to bone-specific elongation in the hindlimb at P5. However, the accelerated growth may trigger earlier senescence of the epiphyseal plate chondrocytes and ultimately delay growth, leading to 127 the bone-specific shortening in the hindlimb at P21. The accelerated differentiation may start as early as in development at E16.5, but the latent phenotypes are also observable at 18 months of age. Aqp9 appears to function similarly to hypertrophic genes and its deletion promotes a proliferative-leaning gene profile. Aqp9 is also a target of estrogen and SHOX, suggesting another avenue by which Aqp9 deletion may dysregulate long bone growth. Aqp9 may normally function with Tbx4 and unexplored factors related to mesomelic and rhizomelic disorders in order to regulate growth in the hindlimb bones. At the chondrocyte level, Aqp9 may normally shuttle endogenous H2O2 to the cytoplasm to activate hypertrophic genes and regulate the rate of differentiation. Deletion of Aqp9 would then delay the hypertrophic phenotype.

The limitations of this study include methodological sample size, data measurement, and prior available research of Aqp9. Regarding sample size, the immunohistochemistry and Safranin-O staining experiments in this study did not meet a minimum of three biological replicates. Using a larger sample size for each experiment would improve the statistical power of all analyses and how representative they are of the Aqp9 mutation. Regarding data measurement, long bone lengths and epiphyseal plate parameters were collected manually. Despite using digitally-assisted calipers and software in a blinded setting, fully computerized scanning of long bones and epiphyseal plates would improve the accuracy of all measurements. Regarding prior research, investigations in Aqp9 are limited (<500 PubMed search results as of August 2018). It is possible that a greater number of prior studies would help form a more comprehensive literature review and discussion. Nevertheless, this limitation may describe a need for further biomedical research of Aqp9.

Overall, this study demonstrated that Aqp9 plays a role in endochondral ossification. Deletion of Aqp9 showed significant differences at the long bone, epiphyseal plate, and chondrocyte levels as hypothesized. As a transporter of a known hypertrophy inducer, subject of regulation by estrogen and Shox, and a chondrocyte channelome member, Aqp9 does not appear completely dispensable in function. Aqp9 stands out as a unique aquaporin isoform and sheds light on how the channelome can critically regulate chondrocyte differentiation in the epiphyseal plate. Further investigation of Aqp9 chondrocyte function may unveil a novel therapeutic target for combatting LLDs before resorting to invasive surgical intervention. 128

7 Future Directions

7.1 In situ hybridization of Aqp9 during mesenchymal condensation

In this study, in situ hybridization of Aqp9 to the P14 murine WT epiphyseal plate revealed robust expression in the proliferating and pre-hypertrophic zones. This suggests that Aqp9 functions postnatally to modulate long bone growth. However, endochondral ossification commences during development at approximately E10.5. Here, mesenchymal condensation occurs as the initiating step. To determine if Aqp9 is present at the embryonic level, in situ hybridization could be performed to visualize Aqp9 RNA in the developing murine limb buds. A multi-probe fluorescent in situ protocol targeting other genes important in condensation could also be used to determine if Aqp9 is co-localized with them. During mesenchymal condensation, the well-known Bmps and Fgfs are present as essential factors toward chondrogenesis (Hata et al., 2017). However, limb outgrowth is also dependent on the Hox and Tbx genes, which coordinate the spatial and temporal development of the limb (Guy & Clarke, 2008). Hence, genes can be differentially expressed in the forelimb and hindlimb. The Hox genes contain promoter regions that will only activate if a specific combination of transcription factors bind. In general, Hox genes create positional memory in MSCs and influence their ability to differentiate (Seifert, Werheid, Knapp, & Tobiasch, 2015). A variety of Hoxb isoforms and Hoxc4 have been identified in murine MSCs, making them distinct markers of condensation (Phinney, Gray, Hill, & Pandey, 2005). The Tbx genes determine limb identity, where Tbx5 is localized to the forelimb only and Tbx4 is localized to the hindlimb only (Logan, 2003). If Aqp9 functions in the developing limb, it may regulate these genes. A preliminary whole-mount in situ hybridization of Aqp9 to WT E10.5 embryos can be performed to identify its expression, following the protocol mentioned in this study with minor modifications for embryos (Koyama et al., 1996). If Aqp9 can be visualized in the developing forelimbs and hindlimbs, then a multi-plex assay can be performed to check if Hox and Tbx are expressed in the same patterns. A multi-plex fluorescent in situ assay can detect molecules for up to four RNA targets simultaneously, where Aqp9, Tbx4, Tbx5, and Hoxc4 can be selected and designed as riboprobes. If Aqp9 fluorescence is co- localized with that of the Hox or Tbx genes, it suggests that Aqp9 may help coordinate limb outgrowth. Its deletion in Aqp9 -/- mutants would then dysregulate how endochondral 129 ossification is initiated, possibly relaying into differential long bone growth. An EMSA (electrophoretic mobility shift assay) could then be performed to confirm if Hox or Tbx proteins bind to the Aqp9 promoter. If they do not appear to regulate Aqp9, then promoter bashing could be performed to specifically identify which region of the Aqp9 gene promoter controls transcription the most. The new region can be sequenced, matched to other proteins that regulate limb patterning through databases such as BioGRID, and an EMSA can be repeated.

7.2 Histomorphometry of WT and Aqp9 -/- long bones

In this study, the long bone length comparisons of WT and Aqp9 -/- mice showed a differential phenotype. The long bones were measured and superficially analyzed to suggest that endochondral ossification occurs differently in the Aqp9 -/- mutants. However, the role of Aqp9 may go beyond bone length and also affect bone mineral density and structure. Liu et al. (2009) show that one year old female Aqp9 -/- mouse bones do not significantly differ from that of WT mice. Therefore, the contribution of Aqp9 to long bone characteristics at younger timepoints has not been explored. To determine if Aqp9 affects bone in vivo, long bones can be isolated from P5, P21, and one year old WT and Aqp9 -/- mice for histomorphometric analysis. Male mice can be used exclusively to compare to the findings of Liu et al. (2009) and determine if Aqp9 functions in a sex-specific manner. Humerus, femur, and tibia bones can be dissected for micro- CT analysis. Both trabecular and cortical bone characteristics can be analyzed for bone mineral density and percent bone volume. The P5 and P21 skeletons stained in this study may be isolated for this experiment, as the soft tissue has been removed and the fixation process is similar. In P5 Aqp9 -/- mice, their long bones may exhibit decreased bone mineral density as in Fgfr3 null mice (Su et al., 2010). At P21, Aqp9 -/- long bones may also present with differential bone density, although even Bmp2fl/fl; OSX-Cre mice—which would be expected to have defected endochondral characteristics due to the importance of Bmp2—have comparable bone density to WT control mice (McBride-Gagyi, McKenzie, Buettmann, Gardner, & Silva, 2015). Hence, any differences observed may be minimal. In this study, the 18 month old mice Aqp9 -/- mice exhibited an unusual narrowing of the epiphyseal plate. It is possible that in one year old mice, the narrowing phenotype is also present and may be indicative of shorter bone length in comparison to WT mice. Micro-CT or 3D skeletal reconstruction would identify the pertinent 130 parameters of bones at this age which could be charted for comparison to the P5 and P21 characteristics. Overall, histomorphometric analysis of the long bones would provide deeper insight into the endochondral characteristics of Aqp9 -/- mice beyond length. By analyzing long bones from pups to old mice, the role that Aqp9 plays in murine skeletal development may be better understood.

7.3 Flow cytometry cell cycle analysis

The cell proliferation experiment performed in this study revealed an irregular proliferation phenotype among Aqp9 +/- and Aqp9 -/- chondrocytes. Chondrocytes were cultured and counted at specific timepoints to measure their growth rate. However, the variance, weak statistical power, and time consumption of the experiment design suggests that other methods can be employed to accurately measure proliferation rates. To determine if Aqp9 affects the cell cycle profile of epiphyseal plate chondrocytes, WT, Aqp9 +/-, and Apq9 -/- chondrocytes can be isolated and subjected to flow cytometry cell cycle analysis. With flow cytometry, propidium iodide can be used to bind DNA inside the chondrocyte samples and correlate with the amount they contain. Therefore, the fluorescent intensity measures the number of cells at any one time that are in each cell cycle phase. A high percentage of cells in the G2/M phase would suggest an accelerated proliferation phenotype, as cells have just doubled their DNA content in the preceding S phase. Initially, WT and Aqp9 -/- epiphyseal plate chondrocytes from P5 and P21 mice can be compared first. If they present with distinct cell cycle profiles, then Aqp9 +/- chondrocytes can be checked as well to determine if heterozygous Aqp9 deletion has an intermediary profile. Then, chondrocytes from embryonic and old age timepoints can be harvested to compare profiles from development till adulthood. Chondrocytes can be sorted according to their phenotype to ensure that only specific chondrocytes are analyzed. Belluoccio et al. (2010) describe that the cluster of differentiation antigen CD200 is a cell surface marker restricted to chondrocytes from the pre-hypertrophic and hypertrophic zones. If the analysis is to be performed only on proliferating chondrocytes, the CD200-positive chondrocytes can be sorted out. Furthermore, the cell proliferation experiment in this study showed accelerated proliferation in Aqp9 mutant chondrocytes only between the 48-72 hour timepoints. If chondrocytes are prepared and permeabilized for flow cytometry immediately after pronase/collagenase treatment, 131 a distinct profile may not be observed. Samples can be cultured for 48 hours and then subjected to the analysis. The analysis would determine whether the G2M:G1 ratios reflect the proliferation phenotype observed among Aqp9 -/-chondrocytes.

7.4 RNA-sequencing of WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes

In this study, the P5 Aqp9 -/- mice exhibited tibial overgrowth and the P21 Aqp9 -/- mice exhibited femoral shortening. Furthermore, qPCR of P5 Aqp9 -/- epiphyseal plate chondrocytes revealed a proliferation-leaning gene profile. To determine if Aqp9 functions in any pathways affecting endochondral ossification, epiphyseal plate chondrocytes isolated in this study could be RNA-sequenced to identify their gene expression both differentially and temporally. Additional chondrocytes from different timepoints would also be required to assess expression throughout endochondral ossification. WT, Aqp9 +/-, and Aqp9 -/- chondrocytes were investigated in this study at the P5 timepoint through a cell proliferation assay, gene expression analysis, and Aqp9 silencing. While Mmp13 was significantly reduced and Sox9 was significantly increased in the qPCR experiment among the mutant chondrocytes, siRNA knockdown of Aqp9 only showed a gene expression trend without significance. Furthermore, the experiments were only performed at one timepoint whereas Aqp9 deletion appears to affect skeletal stature differently depending on age. By isolating WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes at E16.5 and P21, their RNA could be sequenced alongside the P5 samples to show how the Aqp9 mutation may affect pathways involving proliferation, hypertrophy, and ossification over time. At E16.5, embryos have commenced with endochondral ossification for 5-6 days and would capture any function of Aqp9 in early long bone development. At P21, mice are juvenile and would represent any function of Aqp9 during early pubertal bone growth. The use of 5 biological replicates per genotype and timepoint would improve the statistical power of the analysis. However, excision of epiphyseal plate cartilage from embryos may be manually challenging due to size. To isolate chondrocytes, whole embryo limbs may be roughly dissected and then digested prior to flow cytometry sorting. The use of the aforementioned chondrocyte-associated antibodies would improve the isolation of cells from only the developing limb buds. Altogether, RNA-seq of WT, Aqp9 +/-, and Aqp9 -/- chondrocytes at the developmental, pup, and juvenile stages can establish 132 how Aqp9 affects differential and temporal gene expression during endochondral ossification. The RNA-seq may reveal a temporal method by which Aqp9 deletion influences chondrocyte proliferation and hypertrophy. Disrupted pathways that link Aqp9 to bone defects may also be identified. Validation of the RNA-seq output could be performed at the protein expression level through Western blotting with antibodies for AQP9 and other markers differentially expressed. The frozen P5 Aqp9 -/- epiphyseal plate chondrocyte protein samples saved in this study could also be used for Western blotting, then compared to the biopsy blot results through band signal quantification on ImageJ. In standard RNA-seq, all RNA submitted is ultimately reverse transcribed into double stranded cDNA. However, this means that one of the cDNA strands represents sense mRNA while the other cDNA strand represents antisense mRNA. Antisense transcripts may be non-coding but play critical roles in transcriptome regulation (He, Vogelstein, Velculescu, Papadopoulos, & Kinzler, 2008). With standard sequencing, the PCR amplification step cannot differentiate between sense and antisense strands and devalues the output. This can lead to an inaccurate representation of the transcriptome (Mills, Kawahara, & Janitz, 2013). As Aqp9 deletion appears to relay subtle differences in endochondral ossification, a cleaner transcriptome analysis may be required. Hence, creating a strand-specific cDNA library through strand marking or using strand-specific RNA-seq should be considered.

7.5 Therapeutic strategies

The current treatment for LLDs are primarily limited to invasive surgical interventions. Aside from resecting bone from the longer limb or applying a lengthening fixator to the shorter limb, bone growth can be temporarily or permanently arrested through stapling the epiphyseal plate. Epiphysiodesis is the process of guiding bone growth through inhibition of the epiphyseal plate until a deformity has been corrected (Gottliebsen et al., 2013). Traditionally, stapling the plate longitudinally prevents that area from ossifying properly and can be used to correct angular deformities and LLDs (Blount & Clarke, 1949). Stapling is well-established and has been used for many decades as a safe intervention for inhibiting growth (Raab, Wild, Seller, & Krauspe, 2001). This process has also been developed into tension band plating, where the staple is replaced with a non-rigid plate-and-screw apparatus that better shifts epiphyseal load and improves correction time (Stevens, 2007). Permanent epiphysiodesis can also be achieved 133 through the archaic Phemister technique, where a portion containing epiphyseal plate and surrounding bone are resected and reinserted into the joint with the ends reversed (Gottliebsen, Shiguetomi-Medina, Rahbek, & Møller-Madsen, 2016). Nevertheless, the discovery of non- invasive therapies has not been well-explored. Epiphysiodesis was achieved in rabbits through radiofrequency delivery to their epiphyseal plates with minimal pain and postoperative complication (Ghanem et al., 2009). Recombinant human BMP2 has also been used in orthopedic settings to induce bone formation through biomaterial delivery (Wang et al., 1990; Khan & Lane, 2004; Agrawal & Sinha, 2017). Consuming recombinant human growth hormone can also rescue the effects of idiopathic short stature in children (Sotos & Tokar, 2014). As Aqp9 may be a negative regulator of long bone growth at the early stages of life, children diagnosed with dysplasias may benefit from its targeted knockdown. Phloretin is a dihydrochalcone derived from apples, known AQP9 inhibitor, and protector against osteoarthritis (Zheng, Chen, Zhang, Cai, & Chen, 2018; Geng et al., 2017). Previously, the Kannu lab has shown that mice that underwent a 60-day phloretin oral gavage treatment exhibited significantly reduced osteoarthritic severity (Xie et al., 2017). However, the use of phloretin as an epiphyseal plate deliverable has not been investigated. To test if Aqp9 can be ameliorated and affect endochondral ossification, a fracture healing experiment can be performed in young mice using phloretin in the treatment group. Radiography can be used to capture healing rates over a 60-day timespan. qPCR can be performed on resected epiphyseal plates to measure chondrogenic, angiogenic, and osteogenic gene expression. In this study, skeletal staining of Aqp9 -/- mice at P5 and P21 suggests that phloretin would modulate the long bone growth rate.

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References

Abad, V., Meyers, J. L., Weise, M., Gafni, R. I., Barnes, K. M., Nilsson, O., & Baron, J. (2002). The role of the resting zone in growth plate chondrogenesis. Endocrinology, 143(5), 1851-7.

Agrawal, V., & Sinha, M. (2017). A review on carrier systems for bone morphogenetic protein-2. J Biomed Mater Res Part B Appl Biomater, 105(4), 904-925.

Agre, P. (2006). The aquaporin water channels. Proc Am Thorac Soc, 3(1), 5–13.

Agre, P., Preston, G. M., Smith, B. L., Jung, J. S., Raina, S., Moon, C., . . . Nielsen, S. (1993). Aquaporin CHIP: the archetypal molecular water channel. American Journal of Physiology, 265, F463–476.

Agre, P., Saboori, A. M., Asimos, A., & Smith, B. L. (1987). Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. Journal of Biological Chemistry, 262, 17497–17503.

Aharon, R., & Bar-Shavit, Z. (2006). Involvement of aquaporin 9 in osteoclast differentiation. J Biol Chem, 281(28), 19305-9.

Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A., & de Crombrugghe, B. (2002). The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev, 16(21), 2813-28.

Akiyama, H., Lyons, J. P., Mori-Akiyama, Y., Yang, X., Zhang, R., Zhang, Z., . . . de Crombrugghe, B. (2004). Interactions between Sox9 and β-catenin control chondrocyte differentiation. Genes Dev, 18, 1072–1087

Almasalmeh, A., Krenc, D., Wu, B., & Beitz, E. (2014). Structural determinants of the hydrogen peroxide permeability of aquaporins. FEBS J, 281(3), 647-56.

Ambrosi, T. H., Scialdone, A., Graja, A., Gohlke, S., Jank, A., Bocian, C., . . . Schulz, T. J. (2017). Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration. Cell Stem Cell, 20(6), 771-784.e6.

Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., . . . Ottersen, O. P. (2005). Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane. FASEB J, 19(11), 1459-67.

Anthony, B. A., & Link, D. C. (2014). Regulation of hematopoietic stem cells by bone marrow stromal cells. Trends Immunol, 35(1), 32-7.

Arnold, M. A., Kim, Y., Czubryt, M. P., Phan, D., McAnally, J., Qi, X., . . . Olson, E. N. (2007). MEF2C transcription factor controls chondrocyte hypertrophy and bone development. Dev Cell, 12, 377–389.

Avtanski, D., Novaira, H. J., Wu, S., Romero, C. J., Kineman, R., Luque, R. M., . . . Radovick, S. (2014). Both estrogen receptor α and β stimulate pituitary GH gene expression. Mol Endocrinol, 28(1), 40-52. 135

Aybar Odstrcil, A. C., Carino, S. N., Ricci, J. C., & Mandalunis, P. M. (2010). Effect of arsenic in endochondral ossification of experimental animals. Exp Toxicol Path, 62(3), 243-9.

Badaut, J., & Regli, L. (2004). Distribution and possible roles of aquaporin 9 in the brain. Neuroscience, 129(4), 971-81.

Balwierz, P. J., Pachkov, M., Arnold, P., Gruber, A. J., Zavolan, M., & van Nimwegen, E. (2014). ISMARA: automated modeling of genomic signals as a democracy of regulatory motifs. Genome Res, 24(5), 869–884.

Bandyopadhyay, A., Tsuji, K., Cox, K., Harfe, B. D., Rosen, V., & Tabin, C. J. (2006). Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet, 2(12), e216.

Barcroft, L. C., Offenberg, H., Thomsen, P., & Watson, A. J. (2003). Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol, 256(2), 342-54.

Barham, G., & Clarke, N. M. (2008). Genetic regulation of embryological limb development with relation to congenital limb deformity in humans. J Child Orthop, 2(1), 1-9.

Barna, M., & Niswander, L. (2007). Visualization of cartilage formation: insight into cellular properties of skeletal progenitors and chondrodysplasia syndromes. Dev Cell, 12(6), 931-41.

Barrett-Jolley, R., Lewis, R., Fallman, R., & Mobasheri, A. (2010). The emerging chondrocyte channelome. Front Physiol, 1, 135.

Beederman, M., Lamplot, J. D., Nan, G., Wang, J, Liu, X., Yin, L., Li, R., . . . He, T. (2013). BMP signaling in mesenchymal stem cell differentiation and bone formation. J Biomed Sci Eng, 6(8A), 32–52.

Bell, E. L., Klimova, T. A., Eisenbart, J., Schumacker, P. T., & Chandel, N. S. (2007). Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia. Mol Cell Biol, 27(16), 5737-45.

Belluoccio, D., Bernardo, B. C., Rowley, L., & Bateman, J. F. (2008). A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage. Biochim Biophys Acta, 1779(5), 330-40.

Belluoccio, D., Etich, J., Rosenbaum, S., Frie, C., Grskovic, I., Stermann, J., . . . Brachvogel, B. (2010). Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status. J Bone Miner Res, 25(6), 1267-81.

Benjamin, M., Archer, C. W., & Ralphs, J. R. (1994). Cytoskeleton of cartilage cells. Microsc Res Tech, 28(5), 372- 7.

Berendsen, A. D., & Olsen, B. R. (2015). Bone development. Bone, 80, 14-18.

Bi, W., Deng, J. M., Zhang, Z., Behringer, R. R., & de Crombrugghe, B. (1999). Sox9 is required for cartilage formation. Nat Genet, 22(1), 85-9. 136

Bi, W., Huang, W., Whitworth, D. J., Deng, J. M., Zhang, Z., Behringer, R. R., & de Crombrugghe, B. (2001). Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 98(12), 6698-703.

Bienert, G. P., Møller, A. L., Kristiansen, K. A., Schulz, A., Møller, I. M., Schjoerring, J. K., & Jahn, T. P. (2007). Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J Biol Chem, 282(2), 1183-92.

Bienert, G. P., Schjoerring, J. K., & Jahn, T. P. (2006). Membrane transport of hydrogen peroxide. Biochim Biophys Acta, 1758(8), 994-1003.

Blount, W. P., & Clarke, G. R. (1949). Control of bone growth by epiphyseal stapling; a preliminary report. J Bone Joint Surg Am, 31A(3), 464-78.

Bobick, B. E., Cobb, J. (2012). Shox2 regulates progression through chondrogenesis in the mouse proximal limb. J Cell Sci, 125(Pt 24), 6071-83.

Bond, S. R., Lau, A., Penuela, S., Sampaio, A. V., Underhill, T. M., Laird, D. W., & Naus, C. C. (2011). Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J Bone Miner Res, 26(12), 2911-22.

Bouleftour, W., Boudiffa, M., Wade-Gueye, N. M., Bouët, G., Cardelli, M., Laroche, N., . . . Malaval, L. (2014). Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass. PLoS ONE, 9(5), e95144.

Bourayou, R., Giabicani, E., Pouillot, Brailly-Tabard, S., & Brauner, R. (2015). Premature Pubarche before One Year of Age: Distinguishing between Mini-Puberty Variants and Precocious Puberty. Med Sci Monit, 21, 955–963.

Boveris, A., & Cadenas, E. (2000). Mitochondrial production of hydrogen peroxide regulation by nitric oxide and the role of ubisemiquinone. IUBMB Life, 50(4-5), 245-50.

Brady, R. J., Dean, J. B., Skinner, T. M., & Gross, M. T. (2003). Limb length inequality: clinical implications for assessment and intervention. J Orthop Sports Phys Ther, 33(5), 221-34.

Brighton, C. T., Sugioka, Y., & Hunt, R. M.( 1973). Cytoplasmic structures of epiphyseal plate chondrocytes. Quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to the fate of hypertrophic cells. J Bone Joint Surg Am, 55(4), 771-84.

Brougham, D. I., & Nicol, R. O. (1987). Valgus deformity after proximal tibial fractures in children. J Bone Joint Surg Br, 69(3), 482.

Bu, G., Shuang, F., Wu, Y., Ren, D., & Hou, S. (2012). AQP9: a novel target for bone loss induced by microgravity. Biochem Biophys Res Commun, 419(4), 774-8.

Buckwalter, J. A., Mower, D., Ungar, R., Schaeffer, J., & Ginsberg, B. (1986). Morphometric analysis of chondrocyte hypertrophy. J Bone Joint Surg Am, 68(2), 243-55. 137

Bundschu, K., Knobeloch, K. P., Ullrich, M., Schinke, T., Amling, M., Engelhardt, C. M., . . . Schuh, K. (2005). Gene disruption of Spred-2 causes dwarfism. J Biol Chem, 280(31), 28572-80.

Burghardt, B., Elkaer, M. L., Kwon, T. H., Rácz, G. Z., Varga, G., Steward, M. C., & Nielsen, S. (2003). Distribution of aquaporin water channels AQP1 and AQP5 in the ductal system of the human pancreas. Gut, 52(7), 1008-16.

Burova, E. B., Grudinkin, P. S., Bardin, A. A., & Gamaleĭ, N. N. (2001). H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2. Tsitologiia, 43(12), 1153-61.

Caetano-Lopes, J., Canhão, H., & Fonseca, J. E. (2007). Osteoblasts and bone formation. Acta Reumatol Port, 32(2), 103-10.

Cai, L., Lei, C., Li, R., Chen, W., Hu, C., Chen, X., & Li, C. (2017). Overexpression of aquaporin 4 in articular chondrocytes exacerbates the severity of adjuvant-induced arthritis in rats: an in vivo and in vitro study. J Inflamm (Lond), 14, 6.

Calamita, G., Gena, P., Ferri, D., Rosito, A., Rojek, A., Nielsen, S., . . . Svelto, M. (2012). Biophysical assessment of aquaporin-9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol. Biol Cell, 104(6), 342-51.

Carel, J. C., Lahlou, N., Roger, M., & Chaussain, J. L. (2004). Precocious puberty and statural growth. Hum Reprod Update, 10(2), 135-47.

Caron, M. M., Emans, P. J., Cremers, A., Surtel, D. A., Coolsen, M. M., van Rhijn, L. W., & Welting, T. J. (2013). Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2 but suppressed by BMP-7. Osteoarthritis Cartilage, 21(4), 604-13.

Chagin, A. S., Chrysis, D., Takigawa, M., Ritzen, E. M., & Sävendahl, L. (2006). Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis. J Endocrinol, 188(2), 193-203.

Chagin, A. S., Karimian, E., Sundström, K., Eriksson, E., & Sävendahl, L. (2010). Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones. J Endocrinol. 2010, (1), 21-9.

Chandel, N. S., Mcclintock, D. S., Feliciano, C. E., Wood, T. M., Melendez, J. A., Rodriguez, A. M., & Schumacker, P. T. (2000). Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia- inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem, 275(33), 25130-8.

Chanprasertyothin, S., Saetung, S., Rajatanavin, R., & Ongphiphadhanakul, B. (2010). Genetic variant in the aquaporin 9 gene is associated with bone mineral density in postmenopausal women. Endocrine, 38(1), 83-6.

Chau, M., Forcinito, P., Andrade, A. C., Hegde, A., Ahn, S., Lui, J. C., . . . Nilsson, O. (2011). Organization of the Indian hedgehog--parathyroid hormone-related protein system in the postnatal growth plate. J Mol Endocrinol, 47(1), 99-107.

Chen, S., Fu, P., Cong, R., Wu, H., & Pei, M. (2015). Strategies to minimize hypertrophy in cartilage engineering and regeneration. Genes Dis, 2(1), 76-95. 138

Cheng, A., & Genever, P. G. (2011). SOX9 determines RUNX2 transactivity by directing intracellular degradation. J Bone Miner Res, 26(2), 439.

Claramunt, D., Gil-Peña, H., Fuente, R., García-López, E., Hernández, F. O., Ordoñez, F. A., . . . Santos, F. (2017). Effects of growth hormone treatment on growth plate, bone, and mineral metabolism of young rats with uremia induced by adenine. Pediatr Res, 82(1), 148-154.

Cobb, J., Dierich, A., Huss-Garcia, Y., & Duboule, D. (2006). A mouse model for human short-stature syndromes identifies Shox2 as an upstream regulator of Runx2 during long-bone development. Proc Natl Acad Sci U S A, 103(12), 4511–4515.

Cohen, D., Bogeat-Triboulot, M. B., Vialet-Chabrand, S., Merret, R., Courty, P. E., Moretti, S., . . . Hummel, I. (2013). Developmental and environmental regulation of Aquaporin gene expression across Populus species: divergence or redundancy? PLoS ONE, 8(2), e55506.

Cooper, K. L., Oh, S., Sung, Y., Dasari, R. R., Kirschner, M. W., & Tabin C. J. (2013). Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature, 495(7441), 375-8.

Courties, A., Sellam, J., & Berenbaum, F. (2017). Metabolic syndrome-associated osteoarthritis. Curr Opin Rheumatol, 29(2), 214-222.

Courtland, H. W., Sun, H., Beth-On, M., Wu, Y., Elis, S., Rosen, C. J., & Yakar, S. (2011). Growth hormone mediates pubertal skeletal development independent of hepatic IGF-1 production. J Bone Miner Res, 26(4), 761-8.

Cutler, G. B. (1997). The role of estrogen in bone growth and maturation during childhood and adolescence. J Steroid Biochem Mol Biol, 61(3-6), 141-4. da Silva, I. V., Rodrigues, J. S., Rebelo, I., Miranda, J. P. G., & Soveral, G. (2018) Revisiting the metabolic syndrome: the emerging role of aquaglyceroporins. Cell Mol Life Sci, 75(11), 1973-1988.

Damiano, A., Zotta, E., Goldstein, J., Reisin, I., & Ibarra, C. (2001). Water channel proteins AQP3 and AQP9 are present in syncytiotrophoblast of human term placenta. Placenta, 22(8-9), 776-81.

Day, T. F., & Yang, Y. (2008). Wnt and hedgehog signaling pathways in bone development. J Bone Joint Surg Am, 90 Suppl 1, 19-24.

Delise, A. M., Fischer, L., & Tuan, R. S. (2000). Cellular interactions and signaling in cartilage development. Osteoarthritis Cartilage, 8(5), 309-34.

Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A., & Leder, P. (1996). Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell, 84(6), 911-21.

Deng, Y., Wu, A., Li, P., Li, G., Qin, L., Song, H., & Mak, K. K. (2016). Yap1 Regulates Multiple Steps of Chondrocyte Differentiation during Skeletal Development and Bone Repair. Cell Rep, 14(9), 2224-2237.

Dibas, A., Yang, M. H., Bobich, J., & Yorio, T. (2007). Stress-induced changes in neuronal Aquaporin-9 (AQP9) in a retinal ganglion cell-line. Pharmacol Res, 55(5), 378-84. 139

Diebold, I., Flügel, D., Becht, S., Belaiba, R. S., Bonello, S., Hess, J., . . . Görlach, A. (2010). The hypoxia-inducible factor-2alpha is stabilized by oxidative stress involving NOX4. Antioxid Redox Signal, 13(4), 425-36.

Duque, G. (2007). As a matter of fat: New perspectives on the understanding of age-related bone loss. BoneKEy- Osteovision, 4, 129–140.

Elkjaer, M., Vajda, Z., Nejsum, L. N., Kwon, T., Jensen, U. B., Amiry-Moghaddam, M., . . . Nielsen, S. (2000). Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. Biochem Biophys Res Commun, 276(3), 1118-28.

Emons, J., Chagin, A. S., Sävendahl, L., Karperien, M., & Wit, J. M. (2011). Mechanisms of growth plate maturation and epiphyseal fusion. Horm Res Paediatr, 75(6), 383-91.

Eriksen, E. F. (2010). Cellular mechanisms of bone remodeling. Rev Endocr Metab Disord, 11(4), 219-27.

Eswarakumar, V. P., & Schlessinger, J. (2007). Skeletal overgrowth is mediated by deficiency in a specific isoform of fibroblast growth factor receptor 3. Proc Natl Acad Sci U S A, 104(10), 3937–3942.

Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M., & Lonai, P. (2002). The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development, (16), 3783-93.

Fandrey, J., Frede, S., & Jelkmann, W. (1994). Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem J, 303(Pt 2), 507-10.

Fox, A. J. S., Bedi, A., & Rodeo, S. A. (2009). The basic science of articular cartilage: structure, composition, and function. Sports Health, 1(6), 461-8.

Gajghate, S., Hiyama, A., Shah, M., Sakai, D., Anderson, D. G., Shapiro, I. M., & Risbud, M. V. (2009). Osmolarity and intracellular calcium regulate Aquaporin2 expression through TonEBP in nucleus pulposus cells of the intervertebral disc. J Bone Miner Res, 24(6), 992–1001.

Gao, H., Ren, G., Xu, Y., Jin, C., Jiang, Y., Lin, L., . . . Gui, L. (2011). Correlation between expression of aquaporins 1 and chondrocyte apoptosis in articular chondrocyte of osteoarthritis. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 25(3), 279-84.

Gao, L., Sheu, T. J., Dong, Y., Hoak, D. M., Zuscik, M. J., Schwarz, E. M., . . . Jonason, J. H. (2013). TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular . J Cell Sci, 126(Pt 24), 5704-13.

Garfinkel, B. P., Arad, S., Le, P. T., Bustin, M., Rosen, C. J., Gabet, Y., & Orly, J. (2015). Proportionate Dwarfism in Mice Lacking Heterochromatin Protein 1 Binding Protein 3 (HP1BP3) Is Associated With Alterations in the Endocrine IGF-1 Pathway. Endocrinology, 156(12), 4558-70.

Ge, X., Tsang, K., He, L., Garcia, R. A., Ermann, J., Mizoguchi, F., . . . Aliprantis, A. O. (2016). NFAT restricts osteochondroma formation from entheseal progenitors. JCI Insight, 1(4), e86254.

Geng, X., Mcdermott, J., Lundgren, J., Liu, L., Tsai, K. J., Shen, J., & Liu, Z. (2017). Role of AQP9 in transport of monomethyselenic acid and selenite. Biometals, 30(5), 747-755. 140

Ghanem, I., El Hage, S., Diab, M., Saliba, E., Khazzaka, A., Aftimos, G., . . . Kharrat, K. (2009). Radiofrequency application to the growth plate in the rabbit: a new potential approach to epiphysiodesis. J Pediatr Orthop, 29(6), 629-35.

Glasson, S. S., Chambers, M. G., Van den berg, W. B., & Little C. B. (2010). The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthr Cartil, 18 Suppl 3, S17-23.

Glatt, V., Canalis, E., Stadmeyer, L., & Bouxsein, M. L. (2007). Age-related changes in trabecular architecture differ in female and male C57BL/6J mice. J Bone Miner Res, 22(8), 1197-207.

Golightly, Y. M., Allen, K. D., Helmick, C. G., Renner, J. B., & Jordan, J. M. (2009). Symptoms of the knee and hip in individuals with and without limb length inequality. Osteoarthr Cartil, 17(5), 596-600.

Gottliebsen, M., Rahbek, O., Hvid, I., Davidsen, M., Hellfritzsch, M. B., & Møller-Madsen, B. (2013). Hemiepiphysiodesis: similar treatment time for tension-band plating and for stapling: a randomized clinical trial on guided growth for idiopathic genu valgum. Acta Orthop, 84(2), 202-6.

Gottliebsen, M., Shiguetomi-Medina, J. M., Rahbek, O., & Møller-Madsen, B. (2016). Guided growth: mechanism and reversibility of modulation. J Child Orthop, 10(6), 471-477.

Graziano, A. C. E., Avola, R., Pannuzzo, G., & Cardile, V. (2018). Aquaporin1 and 3 modification as a result of chondrogenic differentiation of human mesenchymal stem cell. J Cell Physiol, 233(3), 2279-2291.

Gurney, B. (2002). Leg length discrepancy. Gait Posture, 15(2), 195-206.

Hall, K. C., & Blobel, C. P. (2012). Interleukin-1 Stimulates ADAM17 through a Mechanism Independent of its Cytoplasmic Domain or Phosphorylation at Threonine 735. PLoS One, 7(2), e31600.

Hall, K. C., Hill, D., Otero, M., Plumb, D. A., Froemel, D., Dragomir, C. L., . . . Blobel, C. P. (2013). ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes. Mol Cell Biol, 33(16), 3077-90.

Han, D., Canali, R., Rettori, D., & Kaplowitz, N. (2003). Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria. Mol Pharmacol, 64(5), 1136-44.

Han, Y., & Lefebvre, V. (2008). L-Sox5 and Sox6 drive expression of the aggrecan gene in cartilage by securing binding of Sox9 to a far-upstream enhancer. Mol Cell Biol, 28(16), 4999-5013.

Haneda, M., Hayashi, S., Matsumoto, T., Hashimoto, S., Takayama, K., Chinzei, N., . . . Kuroda, R. (2018). Depletion of aquaporin 1 decreased ADAMTS-4 expression in human chondrocytes. Mol Med Rep, 17(4), 4874–4882.

Hara-Chikuma, M., Satooka, H., Watanabe, S., Honda, T., Miyachi, Y., Watanabe, T., & Verkman, A. S. (2015). Aquaporin-3-mediated hydrogen peroxide transport is required for NF-κB signalling in keratinocytes and development of psoriasis. Nat Commun, 6, 7454. 141

Hartmann, C., & Tabin, C. J. (2000). Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development, 127(14), 3141-59.

Hasler, C. C. (2000). Leg length inequality. Indications for treatment and importance of shortening procedures. Orthopade, 29(9), 766-74.

Hata, K., Takahata, Y., Murakami, T., & Nishimura, R. (2017). Transcriptional network controlling endochondral ossification. J Bone Metab, 24(2), 75–82.

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., . . . von der Mark, K. (2010). SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development, 137(6), 901-11.

He, Y., Vogelstein, B., Velculescu, V. E., Papadopoulos, N., & Kinzler, K. W. (2008). The antisense transcriptomes of human cells. Science, 322(5909), 1855-7.

Hellemans, J., Coucke, P. J., Giedion, A., De Paepe, A., Kramer, P., Beemer, F., & Mortier, G. R. (2003). Homozygous mutations in IHH cause acrocapitofemoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips. Am J Hum Genet, 72(4), 1040-6.

Hill, T. P., Spater, D., Taketo, M. M., Birchmeier, W., & Hartmann, C. (2005). Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell, 8, 727–738

Ho, M. S., Tsang, K. Y., Lo, R. L., Susic, M., Mäkitie, O., Chan, T. W., . . . Chan, D. (2007). COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid. Hum Mol Genet, 16(10), 1201-15.

Honsawek, S., Tanavalee, A., Yuktanandana, P., Ngarmukos, S., Saetan, N., & Tantavisut, S. (2010). Dickkopf-1 (Dkk-1) in plasma and synovial fluid is inversely correlated with radiographic severity of knee osteoarthritis patients. BMC Musculoskelet Disord, 11, 257.

Horner, A., Bishop, N. J., Bord, S., Beeton, C., Kelsall, A. W., Coleman, N., & Compston, J. E. (1999). Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage. J Anat, 194, 519–524.

Hossain, M. S., Bergstrom, D. J., & Chen, X. B. (2015). Modelling and simulation of the chondrocyte cell growth, glucose consumption and lactate production within a porous tissue scaffold inside a perfusion bioreactor. Biotechnol Rep (Amst), 5, 55–62.

Huang, W., Chung, U. I., Kronenberg, H. M., & de Crombrugghe, B. (2001). The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones. Proc Natl Acad Sci U S A, 98, 160–165.

Hug, N., Longman, D., & Cáceres, J. F. (2016). Mechanism and regulation of the nonsense-mediated decay pathway. Nucleic Acids Res, 44(4), 1483-95.

Hughes, A. J., Miyazaki, H., Coyle, M. C., Zhang, J., Laurie, M. T., Chu, D., . . . Gartner, Z. J. (2018). Engineered tissue folding by mechanical compaction of the mesenchyme. Dev Cell, 44(2), 165-178.e6. 142

Hussain, S. A., Palmer, D. H., Syn, W. K., Sacco, J. J., Greensmith, R. M. D., Elmetwali, T., . . . James, N. D. (2017). Gene expression profiling in bladder cancer identifies potential therapeutic targets. Int J Oncol, 50(4), 1147-1159.

Hyde, G., Boot-Handford, R. P., & Wallis, G. A. (2008). Col2a1 lineage tracing reveals that the meniscus of the knee joint has a complex cellular origin. J Anat, 213(5), 531-8.

Iena, F. M., & Lebeck, J. (2018). Implications of Aquaglyceroporin 7 in Energy Metabolism. Int J Mol Sci, 19(1), 154.

Inada, M., Wang, Y., Byrne, M. H., Rahman, M. U., Miyaura, C., López-Otín, C, & Krane, S. M. (2004). Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A, 101(49), 17192-7.

Inada, M., Yasui, T., Nomura, S., Miyake, S., Deguchi, K., Himeno, M., . . . Komori, T. (1999). Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev Dyn, 214(4), 279-90.

Ishibashi, K., Kuwahara, M., Gu, Y., Tanaka, Y., Marumo, F., & Sasaki, S. (1998). Cloning and functional expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem Biophys Res Commun, 244(1), 268-74.

Ishibashi, K., Tanaka, Y., & Morishita, Y. (2014). The role of mammalian superaquaporins inside the cell. Biochim Biophys Acta, 1840(5), 1507-12.

Isidor, B., Pichon, O., Redon, R., Day-Salvatore, D., Hamel, A., Siwicka, K. A., . . . Le Caignec, C. (2010). Mesomelia-Synostoses Syndrome Results from Deletion of SULF1 and SLCO5A1 Genes at 8q13. Am J Hum Genet, 87(1), 95–100.

Iwasaki, M., Le, A. X., & Helms, J. A. (1997). Expression of indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis. Mech Dev, 69(1-2), 197-202.

Jackson, A. R., Huang, C. Y., & Gu, W. Y. (2011). Effect of endplate calcification and mechanical deformation on the distribution of glucose in intervertebral disc: a 3D finite element study. Comput Methods Biomech Biomed Engin, 14(2), 195-204.

Jackson, S. J., Andrews, N., Ball, D., Bellantuono, I., Gray, J, Hachoumi, L., … Chapman, K. (2017). Does age matter? The impact of rodent age on study outcomes. Lab Anim, 51(2), 160-169.

Jacob, A. L., Smith, C., Partanen, J., & Ornitz, D. M. (2006). Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev Biol, 296(2), 315-28.

James, C. G., Woods, A., Underhill, T. M., & Beier, F. (2006). The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription. BMC Mol Biol, 7, 30.

Jiang, Y., & Tuan, R. S. (2015). Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol, 11(4), 206-12. 143

Jilka, R. L. (2013). The relevance of mouse models for investigating age-related bone loss in humans. J Gerontol A Biol Sci Med Sci, 68(10), 1209-17.

Jones, D. C., Schweitzer, M. N., Wein, M., Sigrist, K., Takagi, T., Ishii, S., & Glimcher, L. H. (2010). Uncoupling of growth plate maturation and bone formation in mice lacking both Schnurri-2 and Schnurri-3. Proc Natl Acad Sci U S A, 107(18), 8254-8.

Joseph, A., Shur, B. D., & Hess, R. A. (2011). Estrogen, efferent ductules, and the epididymis. Biol Reprod, 84(2), 207-17.

Kannu, P., Weng, A., Poon, R., Ali, S. A., & Ma, H. (2015). Knee deep in water: aquaporin 9 and cartilage damage. Poster session presented at the Matrix Dynamics Group Seminar, Toronto, ON.

Karlsson, T., Bolshakova, A., Magalhães, M. A., Loitto, V. M., & Magnusson, K. E. (2013). Fluxes of water through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane protrusions. PLoS ONE, 8(4), e59901.

Kaufman, K. R., Miller, L. S., & Sutherland, D. H. (1996). Gait asymmetry in patients with limb-length inequality. J Pediatr Orthop, 16(2), 144-50.

Khan, S. N., & Lane, J. M. (2004). The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in orthopaedic applications. Expert Opin Biol Ther, 4(5), 741-8.

Khatri, R., Krishnan, S., Roy, S., Chattopadhyay, S., Kumar, V., & Mukhopadhyay, A. (2016). Reactive Oxygen Species Limit the Ability of Bone Marrow Stromal Cells to Support Hematopoietic Reconstitution in Aging Mice. Stem Cells Dev, 25(12), 948-58.

Kilborn, S. H., Trudel, G., & Uhthoff, H. (2002). Review of growth plate closure compared with age at sexual maturity and lifespan in laboratory animals. Contemp Top Lab Anim Sci, 41(5), 21-6.

Klatt, A. R., Nitsche, D. P., Kobbe, B., Mörgelin, M., Paulsson, M., & Wagener, R. (2000). Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development. J Biol Chem, 275(6), 3999-4006.

Klein, G. L. (2014). Insulin & bone: recent developments. World J Diabetes, 5(1), 14–16.

Knutson, G, A. (2005). Anatomic and functional leg-length inequality: a review and recommendation for clinical decision-making. Part I, anatomic leg-length inequality: prevalence, magnitude, effects and clinical significance. Chiropr Osteopat, 13, 11.

Kobayashi, T., Lyons, K. M., McMahon, A. P., & Kronenberg, H. M. (2005). BMP signaling stimulates cellular differentiation at multiple steps during cartilage development. Proc Natl Acad Sci U S A, 102(50), 18023-7.

Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., . . . Kishimoto, T. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 89(5), 755-64. 144

Koyama, E., Yamaai, T., Iseki, S., Ohuchi, H., Nohno, T., Yoshioka, H., . . . Pacifici, M. (1996). Polarizing activity, Sonic hedgehog, and tooth development in embryonic and postnatal mouse. Dev Dyn, 206(1), 59-72.

Kozono, D., Yasui, M., King, L. S., & Agre, P. (2002). Aquaporin water channels: atomic structure molecular dynamics meet clinical medicine. J Clin Invest, 109(11), 1395-9.

Kronenberg, H. M. (2003). Developmental regulation of the growth plate. Nature, 423(6937), 332-6.

Kronenberg, H. M., & Chung, U. (2001). The parathyroid hormone-related protein and Indian hedgehog feedback loop in the growth plate. Novartis Found Symp, 232, 144-5.

Kruse, E., Uehlein, N., & Kaldenhoff, R. (2006). The aquaporins. Genome Biol, 7(2), 206.

Kuriyama, H., Kawamoto, S., Ishida, N., Ohno, I., Mita, S., Matsuzawa, Y., . . . Okubo, K. (1997). Molecular cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability. Biochem Biophys Res Commun, 241(1), 53-8.

Kuriyama, H., Shimomura, I., Kishida, K., Kondo, H., Furuyama, N., Nishizawa, H., . . . Matsuzawa, Y. (2002). Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes, 51(10), 2915-21.

Laforenza, U., Bottino, C., & Gastaldi, G. (2016). Mammalian aquaglyceroporin function in metabolism. Biochim Biophys Acta, 1858(1), 1-11.

Lai, Z., Yin, H., Cabrera-Pérez, J., Guimaro, M. C., Afione, S., Michael, D. G., . . . Chiorini, J. A. (2016). Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice. Proc Natl Acad Sci U S A, 113(20), 5694-9.

Lambert, C., Dubuc, J. E., Montell, E., Vergés, J., Munaut, C., Noël, A., & Henrotin, Y. (2014). Gene expression pattern of cells from inflamed and normal areas of osteoarthritis synovial membrane. Arthritis Rheumatol, 66(4), 960-8.

Land, S. C., & Tee, A. R. (2007). Hypoxia-inducible factor 1alpha is regulated by the mammalian target of rapamycin (mTOR) via an mTOR signaling motif. J Biol Chem, 282(28), 20534-43.

Le Goff, C., Rogers, C., Le Goff, W., Pinto, G., Bonnet, D., Chrabieh, M., . . . Cormier-Daire, V. (2016). Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome. Am J Hum Genet, 99(2), 407-13.

Lebeck, J., Gena, P., O'neill, H., Skowronski, M. T., Lund, S., Calamita, G., & Praetorius, J. (2012). Estrogen prevents increased hepatic aquaporin-9 expression and glycerol uptake during starvation. Am J Physiol Gastrointest Liver Physiol, 302(3), G365-74.

Lee, H., & Behringer, R. R. (2007). Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in Mice. PLoS One, 2(5), e450. 145

Lee, K., Lanske, B., Karaplis, A. C., Deeds, J. D., Kohno, H., Nissenson, R. A., . . . Segre, G. V. (1996). Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology, 137(11), 5109-18.

Lee, Y. C., Song, I. W., Pai, Y. J., Chen, S. D., & Chen, Y. T. (2017). Knock-in human FGFR3 achondroplasia mutation as a mouse model for human skeletal dysplasia. Sci Rep, 7, 43220.

Lefevbre, V., & Bhattaram, P. (2010). Vertebrate skeletogenesis. Curr Top Dev Biol, 90, 291-317.

Lennicke, C., Rahn, J., Lichtenfels, R., Wessjohann, L. A., Seliger, B. (2015). Hydrogen peroxide - production, fate and role in redox signaling of tumor cells. Cell Commun Signal, 14, 13:39.

Li, X. F., Wang, S. J., Jiang, L. S., & Dai, L. Y. (2012). Gender- and region-specific variations of estrogen receptor α and β expression in the growth plate of spine and limb during development and adulthood. Histochem Cell Biol, 137(1), 79-95.

Lian, J. B., McKee, M. D., Todd, A. M., & Gerstenfeld, L. C. (1993). Induction of bone-related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro. J Cell Biochem, 52(2), 206-19.

Liang, H. T., Feng, X. C., & Ma, T. H. (2008). Water channel activity of plasma membrane affects chondrocyte migration and adhesion. Clin Exp Pharmacol Physiol, 35(1), 7-10.

Lim, J., Tu, X., Choi, K., Akiyama, H., Mishina, Y, & Long, F. (2015). BMP-Smad4 signaling is required for precartilaginous mesenchymal condensation independent of Sox9 in the mouse. Dev Biol, 400(1), 132–138.

Lin, S. S., Tzeng, B., Lee, K., Smith, R. J. H., Campbell, K. P., & Chen, C. (2014). Cav3.2 T-type calcium channel is required for the NFAT-dependent Sox9 expression in tracheal cartilage. Proc Natl Acad Sci U S A, 111(19), E1990–E1998.

Lin, Z., McClure, M. J., Zhao, J., Ramey, A. N., Asmussen, N., Hyzy, S. L., . . . Boyan, B. D. (2018). MicroRNA Contents in Matrix Vesicles Produced by Growth Plate Chondrocytes are Cell Maturation Dependent. Sci Rep, 8(1), 3609.

Lindskog, C., Asplund, A., Catrina, A., Nielsen, S., & Rützler. M. (2016). A Systematic Characterization of Aquaporin-9 Expression in Human Normal and Pathological Tissues. J Histochem Cytochem, 64(5), 287- 300.

List, E. O., Berryman, D. E., Wright-Piekarski, J., Jara, A., & Kopchick, J. J. (2012). The effects of weight cycling on lifespan in male C57BL/6J mice. Int J Obes (Lond), 37(8), 1088–1094.

Liu, L., Trimarchi, J. R., Navarro, P., Blasco, M. A., & Keefe, D. L. (2003). Oxidative stress contributes to arsenic- induced telomere attrition, chromosome instability, and apoptosis. J Biol Chem, 278(34), 31998-2004.

Liu, W., Toyosawa, S., Furuichi, T., Kanatani, N., Yoshida, C., Liu, Y., . . . Komori, T. (2001). Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol, 155(1), 157–166. 146

Liu, Y., Song, L., Wang, Y., Rojek, A., Nielsen, S., Agre, P., & Carbrey, J. M. (2009). Osteoclast differentiation and function in aquaglyceroporin AQP9-null mice. Biol Cell, 101(3), 133-40.

Liu, Z., Shen, J., Carbrey, J. M., Mukhopadhyay, R., Agre, P., & Rosen, B. P. (2002). Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9. Proc Natl Acad Sci U S A, 99(9), 6053-8.

Logan, M. (2003). Finger or toe: the molecular basis of limb identity. Development, 130(26), 6401-10.

Loitto, V. M., Huang, C., Sigal, Y. J., & Jacobson, K. (2007). Filopodia are induced by aquaporin-9 expression. Exp Cell Res, 313(7), 1295-306.

Long, F., & Ornitz, D. M. (2013). Development of the endochondral skeleton. Cold Spring Harb Perspect Biol, 5(1), a008334.

Long, F., Schipani, E., Asahara, H., Kronenberg, H., & Montminy, M. (2001). The CREB family of activators is required for endochondral bone development. Development, 128, 541–550.

Lui, J. C., & Baron, J. (2011). Mechanisms limiting body growth in mammals. Endocr Rev, 32(3), 422-40.

Lui, J. C., Nilsson, O., & Baron, J. (2011). Growth plate senescence and catch-up growth. Endocr Dev, 21, 23-9.

Ma, H., Vi, L., Whetstone, H., Kannu, P., & Alman, B. (2014). Identifying β-catenin targets in osteoarthritis. Poster session presented at the 2014 Clinical and Metabolics Research Day, Toronto, ON.

Mackie, E. J., Ahmed, Y. A., Tatarczuch, L., Chen, K. S., & Mirams, M. (2008). Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol, 40(1), 46-62.

Mansour, S., Offiah, A. C., McDowall, S., Sim, P., Tolmie, J., & Hall, C. (2002). J Med Genet, 39(8), 597-602.

Maor, G., & Karnieli, E. (1999). The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor. Endocrinology, 140(4), 1841-51.

Marchini, A., Rappold, G., & Schneider, K. U. (2007). SHOX at a glance: from gene to protein. Arch Physiol Biochem, 113(3), 116-23.

Massagué, J., Seoane, J., & Wotton, D. (2005). Smad transcription factors. Genes Dev, 19(23), 2783-810.

Matsubara, T., Kida, K., Yamaguchi, A., Hata, K., Ichida, F., Meguro, H., . . . Yoneda, T. (2008). BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem, 283(43), 29119-25.

Matsukawa, N., Grzesik, W. J., Takahashi, N., Pandey, K. N., Pang, S., Yamauchi, M., & Smithies, O. (1999). The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system. Proc Natl Acad Sci U S A, 96(13), 7403–7408. 147

McBride-Gagyi, S. H., Mckenzie, J. A., Buettmann, E. G., Gardner, M. J., & Silva, M. J. (2015). Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice. Bone, 81, 533-543.

McGee S. R., & Narayan, P. (2013). Precocious puberty and Leydig cell hyperplasia in male mice with a gain of function mutation in the LH receptor gene. Endocrinology, 154(10), 3900-13.

Mead, T. J., & Yutzey, K. E. (2009). Notch pathway regulation of chondrocyte differentiation and proliferation during appendicular and axial skeleton development. Proc Natl Acad Sci, 106, 14420–14425

Meng, J. H., Ma, X. C., Li, Z. M., & Wu, D. C. (2007). Aquaporin-1 and aquaporin-3 expressions in the temporo- mandibular joint condylar cartilage after an experimentally induced osteoarthritis. Chin Med J (Engl), 120(24), 2191-4.

Mikic, B., Ferreira, M. P., Battaglia, T. C., & Hunziker, E. B. (2008). Accelerated hypertrophic chondrocyte kinetics in GDF-7 deficient murine tibial growth plates. J Orthop Res, 26(7), 986-90.

Mills, J. D., Kawahara, Y., & Janitz, M. (2013). Strand-Specific RNA-Seq Provides Greater Resolution of Transcriptome Profiling. Curr Genomics, 14(3), 173-81.

Mirtz, T. A., Chandler, J. P., & Eyers, C. M. (2011). The effects of physical activity on the epiphyseal growth plates: a review of the literature on normal physiology and clinical implications. J Clin Med Res, 3(1), 1-7.

Mitchell, N. C., Gould, G. G., Smolik, C. M., Koek, W., & Daws, L. C. (2013). Antidepressant-like drug effects in juvenile and adolescent mice in the tail suspension test: Relationship with hippocampal serotonin and norepinephrine transporter expression and function. Front Pharmacol, 4, 131.

Mobasheri, A., & Marples, D. (2004). Expression of the AQP-1 water channel in normal human tissues: a semiquantitative study using tissue microarray technology. Am J Physiol Cell Physiol, 286(3), C529-37.

Mobasheri, A., Matta, C., Uzielienè, I., Budd, E., Martín-Vasallo, P., & Bernotiene, E. (2018). The chondrocyte channelome: A narrative review. Joint Bone Spine, pii: S1297-319X(18), 30015-0.

Mobasheri, A., Trujillo, E., Bell, S., Carter, S. D., Clegg, P. D., Martín-Vasallo, P., & Marples, D. (2004). Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes. Vet J, 168(2), 143-50.

Mobasheri, A., Wray, S., & Marples, D. (2005). Distribution of AQP2 and AQP3 water channels in human tissue microarrays. J Mol Histol, 36(1-2), 1-14.

Mohlin, S., Hamidian, A., Von stedingk, K., Bridges, E., Wigerup, C., Bexell, D., & Påhlman, S. (2015). PI3K- mTORC2 but not PI3K-mTORC1 regulates transcription of HIF2A/EPAS1 and vascularization in neuroblastoma. Cancer Res, 75(21), 4617-28.

Morales-Roselló, J., & Llorens, N. P. (2012). Outcome of fetuses with diagnosis of isolated short femur in the second half of pregnancy. ISRN Obstet Gynecol, 2012, 268218. 148

Morita, K., Miyamoto, T., Fujita, N., Kubota, Y., Ito, K., Takubo, K., . . . Suda, T. (2007). Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification. J Exp Med, 204(7), 1613-23.

Mow, V. C., Kuei, S. C., Lai, W. M., & Armstrong, C. G. (1980). Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments. J Biomech Eng, 102(1), 73-84.

Murakami, S., Balmes, G., McKinney, S., Zhang, Z., Givol, D., & de Crombrugghe, B. (2004). Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev, 18, 290–305.

Murakami, S., Kan, M., McKeehan, W. L., & de Crombrugghe, B. (2000). Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci U S A, 97(3), 1113-8.

Muramatsu, S., Wakabayashi, M., Ohno, T., Amano, K., Ooishi, R., Sugahara, T., . . . Matsuda, A. (2007). Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation. J Biol Chem, 282(44), 32158-67.

Murata, K., Mitsuoka, K., Hirai, T., Walz, T., Agre, P., Heymann, J. B., . . . Fujiyoshi, Y. (2000). Structural determinants of water permeation through aquaporin-1. Nature, 407(6804), 599-605.

Murray, K. J., & Azari, M. F. (2015). Leg length discrepancy and osteoarthritis in the knee, hip and lumbar spine. J Can Chiropr Assoc, 59(3), 226-37.

Musumeci, G., Leonardi, R., Carnazza, M. L., Cardile, V., Pichler, K., Weinberg, A. M., & Loreto, C. (2013). Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: an in vivo and in vitro study. Tissue Cell, 45(2), 145-52.

Nagahara, M., Waguri-Nagaya, Y., Yamagami, T., Aoyama, M., Tada, T., Inoue, K., . . . Otsuka, T. (2010). TNF- alpha-induced aquaporin 9 in synoviocytes from patients with OA and RA. Rheumatology (Oxford). 2010;49(5):898-906.

Nakamura, Y., Nawata, M., & Wakitani, S. (2005). Expression profiles and functional analyses of Wnt-related genes in human joint disorders. Am J Pathol, 167(1), 97-105.

Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J. M., Behringer, R. R., & de Crombrugghe, B. (2002). The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell, 108, 17–29.

Nalin, A. M., Greenlee, T. K., & Sandell, L. J. (1995). Collagen gene expression during development of avian synovial joints: transient expression of types II and XI collagen genes in the joint capsule. Dev Dyn, 203(3), 352-62.

Nam, J. S., Turcotte, T. J., & Yoon, J. K. (2007). Dynamic expression of R-spondin family genes in mouse development. Gene Expr Patterns, 7(3), 306-12.

Nicchia, G. P., Frigeri, A., Nico, B., Ribatti, D., & Svelto, M. (2001). Tissue distribution and membrane localization of aquaporin-9 water channel: evidence for sex-linked differences in liver. J Histochem Cytochem, 49(12), 1547-56. 149

Nilsson, O., Weise, M., Landman, E. B., Meyers, J. L., Barnes, K. M., & Baron, J. (2014). Evidence that estrogen hastens epiphyseal fusion and cessation of longitudinal bone growth by irreversibly depleting the number of resting zone progenitor cells in female rabbits. Endocrinology, 155(8), 2892-9.

Nishimoto, S., & Logan, M. P. (2016). Subdivision of the lateral plate mesoderm and specification of the forelimb and hindlimb forming domains. Semin Cell Dev Biol, 49, 102-8.

Nissenson, R. A. (1998). Parathyroid hormone (PTH)/PTHrP receptor mutations in human chondrodysplasia. Endocrinology, 139(12), 4753-5.

Nowlan, N. C., Prendergast, P. J., & Murphy, P. (2008). Identification of mechanosensitive genes during embryonic bone formation. PLoS Comput Biol, 4(12), e1000250.

Nurminskaya, M., & Linsenmayer, T. F. (1996). Identification and characterization of up-regulated genes during chondrocyte hypertrophy. Dev Dyn, 206, 260–271.

Offiah, A. C., & Hall, C. M. (2003). Radiological diagnosis of the constitutional disorders of bone. As easy as A, B, C?. Pediatr Radiol, 33(3), 153-61.

Oftadeh, R., Perez-Viloria, M., Villa-Camacho, J. C., Vaziri, A., & Nazarian A. (2015). Biomechanics and mechanobiology of trabecular bone: a review. J Biomech Eng, 137(1), 0108021–01080215.

Ogawa, H., Hatano, S., Suguira, N., Nagai, N., Sato, T., Shimizu, K., . . . Watanabe, H. (2012). Chondroitin Sulfate Synthase-2 Is Necessary for Chain Extension of Chondroitin Sulfate but Not Critical for Skeletal Development. PLoS One, 7(8), e43806.

Oh, S. K., Shin, J. O., Baek, J. I., Lee, J., Bae, J. W., Aknamerddy, H., . . . Lee, K. Y. (2015). Pannexin 3 is required for normal progression of skeletal development in vertebrates. FASEB J, 29(11), 4473-84.

Okamoto, M., Udagawa, N., Uehara, S., Maeda, K., Yamashita, T., Nakamichi, Y., . . . Kobayashi, Y. (2014). Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis. Sci Rep, 4, 4493.

Oliveira, C. A., Carnes, K., França, L. R., Hermo, L., & Hess, R. A. (2005). Aquaporin-1 and -9 are differentially regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis. Biol Cell, 97(6), 385-95.

Ong, D. B., Colley, S. M., Norman, M. R., Kitazawa, S., & Tobias, J. H. (2004). Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha. J Bone Miner Res, 19(3), 447-54.

Ornitz, D. M. (2005). FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev, 16(2), 205-13.

Ornitz, D. M., & Marie, P. J. (2002). FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev, 16(12), 1446-65.

Ortega, N., Behonick, D. J., & Werb, Z. 2004. Matrix remodeling during endochondral ossification. Trends Cell Biol, 14(2), 86-93. 150

Oshio, K., Binder, D. K., Yang, B., Schecter, S., Verkman, A. S., & Manley, G. T. (2004). Expression of aquaporin water channels in mouse spinal cord. Neuroscience, 127(3), 685-93.

Oswald, E. S., Chao, P. G., Bulinkski, J. C., Ateshian, G. A., & Hung, C. T. (2008). Dependence of zonal chondrocyte water transport properties on osmotic environment. Cell Mol Bioeng. 1(4), 339–348.

Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Glimour, K. C., Rosewell, I. R., . . . Owen, M. J. (1987). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell, 89(5), 765-71.

Panda, A., Gamanagatti, S., Jana, M., & Gupta, A. K. (2014). Skeletal dysplasias: A radiographic approach and review of common non-lethal skeletal dysplasias. World J Radiol, 6(10), 808-25.

Pastor-Soler, N. M., Fisher, J. S., Sharpe, R., Hill, E., Van Hoek, A., Brown, D., & Breton, S. (2010). Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones. Reproduction, 139(3), 613– 621.

Penuela, S., Bhalla, R., Gong, X. Q., Cowan, K. N., Celetti, S. J., Cowan, B. J., . . . Laird, D. W. (2007). Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. J Cell Sci, 120(Pt 21), 3772-83.

Perlman, R. L. (2016). Mouse models of human disease. Evol Med Public Health, 2016(1), 170–176.

Perry, M. J., McDougall, K. E., Hou, S. C., & Tobias, J. H. (2008). Impaired growth plate function in bmp-6 null mice. Bone, 42(1), 216-25.

Peters, K. G., Werner, S., Chen, G., & Williams, L. T. (1992). Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development, 114(1), 233-43.

Phinney, D. G., Gray, A. J., Hill, K., & Pandey, A. (2005). Murine mesenchymal and embryonic stem cells express a similar Hox gene profile. Biochem Biophys Res Commun, 338(4), 1759-65.

Phornphutkul, C., Wu, K. Y., & Gruppuso, P. A. (2006). The role of insulin in chondrogenesis. Mol Cell Endocrinol, 249(1-2), 107-15.

Pillozzi, S., & Becchetti, A. (2012). Ion channels in hematopoietic and mesenchymal stem cells. Stem Cells Int, 2012, 217910.

Plourde, M., Ubeda, J. M., Mandal, G., Monte-Neto, R. L., Mukhopadhyay, R., & Ouellette, M. (2015). Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major. Mol Biochem Parasitol, 201(2), 108-11.

Pomès, R., & Roux, B. (1996). Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. Biophys J, 71(1), 19-39.

Qutub, A. A., & Popel, A. S. (2008). Reactive oxygen species regulate hypoxia-inducible factor 1alpha differentially in cancer and ischemia. Mol Cell Biol, 28(16), 5106-19. 151

Raab, P., Wild, A., Seller, K., & Krauspe, R. (2001). Correction of length discrepancies and angular deformities of the leg by Blount's epiphyseal stapling. Eur J Pediatr, 160(11), 668-74.

Raczkowski, J. W., Daniszewska, B., & Zolynski, K. (2010). Functional scoliosis caused by leg length discrepancy. Arch Med Sci, 6(3), 393-8.

Rampersad, S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel), 12(9), 12347-60.

Retting, K. N., Song, B., Yoon, B. S., & Lyons, K. M. (2009). BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation. Development, 136(7), 1093-104.

Richardson, S. M., Knowles, R., Marples, D., Hoyland, J. A., & Mobasheri, A. (2008). Aquaporin expression in the human intervertebral disc. J Mol Histol, 39(3), 303-9.

Rigueur, D., & Lyons, K. M. (2014). Whole-mount skeletal staining. Methods Mol Biol, 1130, 113-121.

Rodríguez, A., Catalán, V., Gómez-Ambrosi, J., & Frühbeck, G. (2006). Role of aquaporin-7 in the pathophysiological control of fat accumulation in mice. FEBS Lett, 580(20), 4771-6.

Rodríguez, A., Gena, P., Méndez-Giménez, L., Rosito, A., Valenti, V., Rotellar, F., . . . Frühbeck, G. (2014). Reduced hepatic aquaporin-9 and glycerol permeability are related to insulin resistance in non-alcoholic fatty liver disease. Int J Obes (Lond, 38(9), 1213-20.

Rodriguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K., & Izpisua Belmonte, J. C. (1999). The T- box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature, 398(6730), 814-8.

Rojek, A. M., Skowronski, M. T., Füchtbauer, E. M., Füchtbauer, A. C., Fenton, R. A., Agre, P., . . . Nielsen, S. (2007). Defective glycerol metabolism in aquaporin 9 (AQP9) knockout mice. Proc Natl Acad Sci U S A, 104(9), 3609-14.

Rosen, C. J., Ackert-Bicknell, C., Rodriguez, J. P., & Pino, A. M. (2009). Marrow fat and the bone microenvironment: developmental, functional, and pathological implications. Crit Rev Eukaryot Gene Expr, 19(2), 109-24.

Rothert, M., Rönfeldt, D., & Beitz, E. (2017). Electrostatic attraction of weak monoacid anions increases probability for protonation and passage through aquaporins. The Journal of Biological Chemistry, 292, 9358-9364.

Rux, D. R., & Wellik, D. M. (2017). Hox genes in the adult skeleton: Novel functions beyond embryonic development. Dev Dyn, 246(4), 310-317.

Sabirov, R. Z., Kurbannazarova, R. S., Melanova, N. R., & Okada, Y. (2013). Volume-sensitive anion channels mediate osmosensitive glutathione release from rat thymocytes. PLoS One, 8(1), e55646.

Sahni, M., Ambrosetti, D. C., Mansukhani, A., Gertner, R., Levy, D., & Basilico, C. (1999). FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev, 13(11), 1361-6. 152

Sahni, M., Raz, R., Coffin, J. D., Levy, D., & Basilico, C. (2001). STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2. Development, 128(11), 2119-29.

Saito, T., Ciobotaru, A., Bopassa, J. C., Toro, L., Stefani, E., & Eghbali, M. (2009). Estrogen contributes to gender differences in mouse ventricular repolarization. Circ Res, 105(4), 343-52.

Saito, T., Fukai, A., Mabuchi, A., Ikeda, T., Yano, F., Ohba, S., . . . Kawaguchi, H. (2010). Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development. Nat Med, 16(6), 678-86.

Salazar, V. S., Gamer, L. W., & Rosen, V. (2016). BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol, 12(4), 203-21.

Sarabipour, S., & Hristova, K. (2016). Mechanism of FGF receptor dimerization and activation. Nat Commun, 7, 10262.

Schaefer, S. L., Johnson, K. A., & O’Brien, R. T. (1995). Compensatory tibial overgrowth following healing of closed femoral fractures in young dogs. Veterinary and Comparative Orthopaedics and Traumatology, 08(03), 159-162

Schey, K. L., Petrova, R. S., Gletten, R. B., & Donaldson, P. J. (2017). The role of aquaporins in ocular lens homeostasis. Int J Mol Sci, 18(12), 2693.

Schipani, E., Ryan, H. E., Didrickson, S., Kobayashi, T., Knight, M., & Johnson, R. S. (2001). Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev, 15(21), 2865-76.

Schlomer, B. J., Feretti, M., Rodriguez, E., Blaschko, S., Cunha, G., & Baskin, L. (2013). Sexual differentiation in the male and female mouse from days 0 to 21: a detailed and novel morphometric description. J Urol, 190(4 Suppl), 1610-7.

Schmidl, M., Adam, N., Surmann-Schmitt, C., Hattori, T., Stock, M., Dietz, U., . . . von der Mark, K. (2006). Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo. J Biol Chem, 281(42), 31790-800.

Schrier, L., Ferns, S. P., Barnes, K. M., Emons, J. A., Newman, E. I., Nilsson, O., & Baron, J. (2006). Depletion of resting zone chondrocytes during growth plate senescence. J Endocrinol, 189(1), 27-36.

Seifert, A., Werheid, D. F., Knapp, S. M., & Tobiasch, E. (2015). Role of Hox genes in stem cell differentiation. World J Stem Cells, 7(3), 583-95.

Selvamurugan, N., Kwok, S., Alliston, T., Reiss, M., & Partridge, N. C. (2004). Transforming growth factor-beta 1 regulation of collagenase-3 expression in osteoblastic cells by crosstalk between the Smad and MAPK signaling pathways and their components, Smad2 and Runx2. J Biol Chem, 279, 19327–19334.

Seto, J., Busse, B., Gupta, H. S., Schäfer, C., Krauss, S., Dunlop, J. W. C., . . . Jahnen-Dechent, W. (2012). Accelerated growth plate mineralization and foreshortened proximal limb bones in fetuin-A knockout mice. PLoS ONE, 7(10), e47338. 153

Shao, Z., Poon, R., Vertel, L., Yang, J., Weng, A., Wei, Q., & Kannu, P. (2016). Closing the floodgates: the role of aquaporin-9 in osteoarthritis. Poster session presented at the SickKids Summer Research Program Annual Summer Student Symposium, Toronto, ON.

Sherr, C. J., & Roberts, J. M. (2004). Living with or without cyclins and cyclin-dependent kinases. Genes Dev, 18(22), 2699-711.

Shi, S., Zheng, S., Li, X. F., & Liu, Z. D. (2017). The Effect of Estradiol on the Growth Plate Chondrocytes of Limb and Spine from Postnatal Mice in vitro: The Role of Estrogen-Receptor and Estradiol Concentration. Int J Biol Sci, 13(1), 100-109.

Shim, K. S. (2015). Pubertal growth and epiphyseal fusion. Ann Pediatr Endocrinol Metab, 20(1), 8-12.

Shim, Y. (2016). An update on the genetic causes of central precocious puberty. Ann Pediatr Endocrinol Metab, 21(2), 66–69.

Shimasaki, M., Kanazawa, Y., Sato, K., Tsuchiya, H., & Ueda, Y. (2018). Aquaporin-1 and -5 are involved in the invasion and proliferation of soft tissue sarcomas. Pathology - Research and Practice, 214(1), 80-88.

Shu, B., Zhang, M., Xie, R., Wang, M., Jin, H., Hou, W., . . . Chen, D. (2011). BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development. J Cell Sci, 124(Pt 20), 3428-40.

Sinha, K. M., Yasuda, H., Coombes, M. M., Dent, S. Y., & de Crombrugghe, B. (2010). Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J, 29, 68–79.

Slootweg, M. C., Swolin, D., Netelenbos, J. C., Isaksson, O. G., & Ohlsson, C. (1997). Estrogen enhances growth hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like cells. J Endocrinol, 155(1), 159-64.

Smits, P., Dy, P., Mitra, S., & Lefebvre, V. (2004). Sox5 and Sox6 are needed to develop and maintain source, columnar and hypertrophic chondrocytes in the cartilage growth plate. J Cell Biol, 164, 747–758.

Smits, P., Li, P., Mandel, J., Zhang, Z., Deng, J. M., Behringer, R. R., de Crombrugghe, B., & Lefebvre, V. (2001). The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell, 1(2), 277-90.

Somoza, R. A., Welter, J. F., Correa, D., & Caplan, A. I. (2014). Chondrogenic differentiation of mesenchymal stem cells: challenges and unfulfilled expectations. Tissue Eng Part B Rev, 20(6), 596–608.

Song, M. G., Hwang, S. Y., Park, J. I., Yoon, S., Bae, H. R., & Kwak, J. Y. (2011). Role of aquaporin 3 in development, subtypes and activation of dendritic cells. Mol Immunol, 49(1-2), 28-37.

Sotos, J. F., & Tokar, N. J. (2014). Growth hormone significantly increases the adult height of children with idiopathic short stature: comparison of subgroups and benefit. Int J Pediatr Endocrinol, 2014(1), 15.

Später, D., Hill, T. P., O'Sullivan, R. J., Gruber, M., Conner, D. A., & Hartmann, C. (2006). Wnt9a signaling is required for joint integrity and regulation of Ihh during chondrogenesis. Development, 133(15), 3039-49. 154

Stevens, P, M. (2007). Guided growth for angular correction: a preliminary series using a tension band plate. J Pediatr Orthop, 27(3), 253-9.

St-Jacques, B., Hammerschmidt, M., & McMahon, A. P. (1999). Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 13(16), 2072-86.

Stroud, R. M., Miercke, L. J., O'connell, J., Khademi, S., Lee, J. K., Remis, J., . . . Akhavan, D. (2003). Glycerol facilitator GlpF and the associated aquaporin family of channels. Curr Opin Struct Biol, 13(4), 424-31.

Su, N., Xu, X., Li, C., He, Q., Zhao, L., Li, C., . . . Chen, L. (2010). Generation of Fgfr3 conditional knockout mice. Int J Biol Sci, 6(4), 327-32.

Su. N., Jin M., Chen, L. (2014). Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models. Bone Res, 2, 14003.

Subotnick, S. I. (1981). Limb length discrepancies of the lower extremity (the short leg syndrome). J Orthop Sports Phys Ther, 3(1), 11-6.

Sun, X., Mariani, F. V., & Martin, G. R. (2002). Functions of FGF signalling from the in limb development. Nature, 418(6897), 501-8.

Takarada, T., Hinoi, E., Nakazato, R., Ochi, H., Xu, C., Tsuchikane, A., . . . Yoneda, Y. (2013). An analysis of skeletal development in osteoblast-specific and chondrocyte-specific runt-related transcription factor-2 (Runx2) knockout mice. J Bone Miner Res, 28(10), 2064-9.

Takata, K., Matsuzaki, T., & Tajika, Y. (2004). Aquaporins: water channel proteins of the cell membrane. Prog Histochem Cytochem, 39(1), 1-83.

Takegami, Y., Ohkawara, B., Ito, M., Masuda, A., Nakashima, H., Ishiguro, N., & Ohno, K. (2016). R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β- catenin signaling in endochondral ossification. Biochem Biophys Res Commun, 473(1), 255-264.

Tchkonia, T., Morbeck, D. E., von Zglinicki, T., van Deursen, J., Lustgarten, J., Scrable, H., . . . Kirkland, J. L. (2010). Aging cell, 9(5), 667-84. ten Berge, D., Brugmann, S. A., Helms, J. A., & Nusse, R. (2008). Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development. Development, 135, 3247–3257.

Torres, E. S., Andrade, C. V., Fonseca, E. C., Mello, M. A., & Duarte, M. E. (2003). Insulin impairs the maturation of chondrocytes in vitro. Braz J Med Biol Res, 36(9), 1185-92.

Tsang, K. Y., Chan, D., & Cheah, K. S. (2015). Fate of growth plate hypertrophic chondrocytes: death or lineage extension? Dev Growth Differ, 57(2), 179-92.

Tsukaguchi, H., Shayakul, C., Berger, U. V., Mackenzie, B., Devidas, S., Guggino, W. B., . . . Hediger, M. A. (1998). Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem, 273(38), 24737-43. 155

Tsukaguchi, H., Weremowicz, S., Morton, C. C., & Hediger, M. A. (1999). Functional and molecular characterization of the human neutral solute channel aquaporin-9. Am J Physiol, 277(5 Pt 2), F685-96.

Tsumaki, N., Nakase, T., Miyaji, T., Kakuichi, M., Kimura, T., Ochi, T., & Yoshikawa, H. (2002). Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis. J Bone Miner Res, 17, 898–906.

Tuljapurkar, S. R., Mcguire, T. R., Brusnahan, S. K., Jackson, J. D., Garvin, K. L., Kessinger, M. A., . . . Sharp, J. G. (2011). Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging. J Anat, 219(5), 574-81.

Turner, N., & Grose, R. (2010). Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer, 10, 116–129

Van der kraan, P. M., & Van den berg, W. B. (2012). Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration? Osteoarthr Cartil, 20(3), 223-32.

Van der weyden, L., Wei, L., Luo, J., Yang, X., Birk, D. E., Adams, D. J., . . . Chen, Q. (2006). Functional knockout of the matrilin-3 gene causes premature chondrocyte maturation to hypertrophy and increases bone mineral density and osteoarthritis. Am J Pathol, 169(2), 515-27.

Veldhuis-Vlug, A. G., & Rosen, C. J. (2018). Clinical implications of bone marrow adiposity. J Intern Med, 283(2), 121-139.

Vergara, R., Parada, F., Rubio, S., & Pérez, F. J. (2012). Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene. J Exp Bot, 63(11), 4123-31.

Verkman, A. S., Smith, A. J., Phuan, P. W., Tradtrantip, L., & Anderson, M. O. (2017). The aquaporin-4 water channel as a potential drug target in neurological disorders. Expert Opin Ther Targets, 21(12), 1161-1170.

Viadiu, H., Gonen, T., & Walz, T. Projection map of aquaporin-9 at 7 A resolution. (2007). J Mol Biol, 367(1), 80-8.

Vivien, D., Petitfrère, E., Martiny, L., Sartelet, H., Galéra, P., Haye, B., & Pujol, J. P. (1993). IPG (inositolphosphate glycan) as a cellular signal for TGF-beta 1 modulation of chondrocyte cell cycle. J Cell Physiol, 155(3), 437-44.

Walker, G. D., Fischer, M., Gannon, J., Thompson, R. C., & Oegema, T. R. (1995). Expression of type-X collagen in osteoarthritis. J Orthop Res, 13(1), 4-12.

Wang, B., Zhao, J., & Zhang, P. (2017). Gene signatures in osteoarthritic acetabular labrum using microarray analysis. Int J Rheum Dis, 20(12), 1927-1934.

Wang, E. A., Rosen, V., D’Alessandro, J. S., Bauduy, M., Cordes, P., Harada, T., . . . LaPan, P. (1990). Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A, 87(6), 2220-4. 156

Wang, G. S., & Cooper, T. A. (2007). Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet, 8(10), 749-61.

Wang, K., Yamamoto, H., Chin, J. R., Werb, Z., & Vu, T. H. (2004). Epidermal growth factor receptor-deficient mice have delayed primary endochondral ossification because of defective osteoclast recruitment. J Biol Chem, 279(51), 53848-56.

Wang, R. N., Green, J., Wang, Z., Deng, Y., Qiao, M., Peabody, M., . . . Shi, L. L. (2014). Bone Morphogenetic Protein (BMP) signaling in development and human diseases. Genes Dis, 1(1), 87-105.

Wang, W., Hart, P. S., Piesco, N. P., Lu, X., Gorry, M. C, & Hart, T. C. (2003). Aquaporin expression in developing human teeth and selected orofacial tissues. Calcif Tissue Int, 72(3), 222-7.

Wang, Y., Zhu, J., & DeLuca, H. F. (2014). Identification of the vitamin D receptor in osteoblasts and chondrocytes but not osteoclasts in mouse bone. J Bone Miner Res, 29(3), 685-92.

Watanabe, S., Moniaga, C. S., Nielsen, S., & Hara-Chikuma, M. (2016). Aquaporin-9 facilitates membrane transport of hydrogen peroxide in mammalian cells. Biochem Biophys Res Commun, 471(1), 191-7.

Weinstein, M., Xu, X., Ohyama, K., & Deng, C. X. (1998). FGFR-3 and FGFR-4 function cooperatively to direct alveogenesis in the murine lung. Development, 125(18), 3615-23.

Weise, M., De-Levi, S., Barnes, K. M., Gafni, R. I., Abad, V., & Baron, J. (2001). Effects of estrogen on growth plate senescence and epiphyseal fusion. Proc Natl Acad Sci U S A, 98(12), 6871-6.

White, K. E., Cabral, J. M., Davis, S. I., Fishburn, T., Evans, W. E., Ichikawa, S., . . . Econs, M. J. (2005). Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation. Am J Hum Genet, 76(2), 361-7.

Wilkie, A. O. (2005). Bad bones, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor mutations. Cytokine Growth Factor Rev, 16(2), 187-203.

Williams, G. R. (20140. Role of thyroid hormone receptor-α1 in endochondral ossification. Endocrinology, 155(8), 2747-50.

Wilsman, N. J., Farnum, C. E., Leiferman, E. M., Fry, M., & Barreto, C. (1996). Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics. J Orthop Res, 14(6), 927-36.

Wilson, D. G., Phamluong, K., Lin, W. Y., Barck, K., Carano, R. A., Diehl, L., . . . Solloway, M. J. (2012). Chondroitin sulfate synthase 1 (Chsy1) is required for bone development and digit patterning. Dev Biol, 363(2), 413-25.

Wilson, M. E. (1998). Effects of estradiol and exogenous insulin-like growth factor I (IGF-I) on the IGF-I axis during growth hormone inhibition and antagonism. J Clin Endocrinol Metab, 83(11), 4013-21.

Wu, M., Chen, G., & Li, Y. P. (2016). TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4, 16009. 157

Wu, Q., Ma, Q., He, C., Wang, C., Gao, S., Hou, X., & Ma, T. (2007). Reduced Bone Mineral Density and Bone Metabolism in Aquaporin-1 Knockout Mice. Chemical Research in Chinese Universities, 23(3), 297-299.

Wu, Q., Zhang, Y., & Chen, Q. (2001). Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation. J Biol Chem, 276(38), 35290-6.

Xiao, E., Chen, C., & Zhang, Y. (2016). The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton? Stem Cell Res Ther, 7, 140.

Xie, W., Tang, P., Ahmed, K., Vertel, L., Vi, L., Lawrence, M., Poon, R., & Kannu, P. (2017). An Apple a Day Keeps The Osteoarthritis Away: Identifying Regulators of Osteoarthritis. Poster session presented at the SickKids Summer Research Program Annual Summer Student Symposium, Toronto, ON.

Xie, Y., Luo, F., Xu, W., Wang, Z., Sun, X., Xu, M., . . . Chen, L. (2017). FGFR3 deficient mice have accelerated fracture repair. Int J Biol Sci, 13(8), 1029-1037.

Xing, W., Cheng, S., Wergedal, J., & Mohan, S. (2014). Epiphyseal chondrocyte secondary ossification centers require thyroid hormone activation of Indian hedgehog and osterix signaling. J Bone Miner Res, 29(10), 2262–2275.

Xu, W., Chen, Q, Liu, C., Chen, J, Xiong, F., & Wu, B. (2017). A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia. BMC Med Genet, 18, 13.

Yamashita, S., Andoh, M., Ueno-Kudoh, H., Sato, T., Miyaki, S., & Asahara, H. (2009). Exp Cell Res, 315(13), 2231–2240.

Yan, C., & Luo, J. (2010). An analysis of reentrant loops. Protein J, 29(5), 350-4.

Yang, B., Zhao, D., & Verkman, A. S. (2006). Evidence against functionally significant aquaporin expression in mitochondria. J Biol Chem, 281(24), 16202-6.

Yang, J., Andre, P., Ye, L., & Yang, Y. Z. (2015). The Hedgehog signalling pathway in bone formation. Int J Oral Sci, 7(2), 73-9.

Yang, J., Safavi, A., Ma, H., Vi, L., Poon, R., Kannu, P., & Alman, B. (2014). Knee deep in water: aquaporin 9 and cartilage damage. Poster session presented at the SickKids Summer Research Program Annual Summer Student Symposium, Toronto, ON.

Yang, X., Trehan, S. K., Guan, Y., Sun, C., Moore, D. C., Jayasuriya, C. T., & Chen, Q. (2014). Matrilin-3 inhibits chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist. J Biol Chem, 289(50), 34768–34779.

Yao, Y., & Wang, Y. (2013). ATDC5: an excellent in vitro model cell line for skeletal development. J Cell Biochem, 114(6), 1223-9.

Yazdi, P. G., Su, H., Ghimbovschi, S., Fan, W., Coskun, P. E., Nalbandian, A., . . . Kimonis, V. E. (2013). Differential gene expression reveals mitochondrial dysfunction in an imprinting center deletion mouse model of Prader-Willi syndrome. Clin Transl Sci, 6(5), 347-55. 158

Yi, F., Khan, M., Gao, H., Hao, F., Sun, M., Zhong, L., . . . Ma, T. (2012). Increased differentiation capacity of bone marrow-derived mesenchymal stem cells in aquaporin-5 deficiency. Stem Cells Dev, 21(13), 2495-507.

Yoon, B. S., Ovchinnikov, D. A., Yoshii, I., Mishina, Y., Behringer, R. R., & Lyons, K. M. (2005). Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci U S A, 102(14), 5062-7.

Yoshida, C. A., Yamamoto, H., Fujita, T., Furuichi, T., Ito, K., Inoue, K., . . . Komori, T. (2004). Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog. Genes Dev, 18, 952–963.

Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., . . . Ornitz, D. M. (2003). Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth. Development, 130(13), 3063-74.

Zanatta, A. P., Brouard, V., Gautier, C., Goncalves, R., Bouraïma-Lelong, H., Silva, F. R. M. B., & Delalande, C. (2017). Interactions between oestrogen and 1α,25(OH)2-vitamin D3 signalling and their roles in spermatogenesis and spermatozoa functions. Basic Clin Androl, 27, 10.

Zanello, L. P., & Norman, A. W. (2004). Rapid modulation of osteoblast ion channel responses by 1α,25(OH)2- vitamin D3 requires the presence of a functional vitamin D nuclear receptor. Proc Natl Acad Sci U S A, 101(6), 1589–1594.

Zhang, F., He, Q., Tsang, W. P., Garvey, W. T., Chan, W. Y., & Wan, C. (2014). Insulin exerts direct, IGF-1 independent actions in growth plate chondrocytes. Bone Res, 2, 14012.

Zhang, J., Tan, X., Li, W., Wang, Y., Wang, J., Cheng, X., & Yang, X. (2005). Smad4 is required for the normal organization of the cartilage growth plate. Dev Biol, 284(2), 311-22.

Zhao, Y. P., Liu, B., Tian, Q. Y., Wei, J. L., Richbourgh, B., & Liu, C. J. (2015). Progranulin protects against osteoarthritis through interacting with TNF-α and β-Catenin signalling. Ann Rheum Dis, 74(12), 2244-2253.

Zheng, Q., Zhou, G., Morello, R., Chen, Y., Garcia-Rojas, X., & Lee, B. (2003). Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo. J Cell Biol, 162, 833– 842.

Zheng, W., Chen, C., Zhang, C., Cai, L., & Chen, H. (2018). The protective effect of phloretin in osteoarthritis: an in vitro and in vivo study. Food Funct, 9(1), 263-278.

Zuloaga, D. G., Zuloaga, K. L., Hinds, L. R., Carbone, D. L., & Handa, R. J. (2014). Estrogen receptor β expression in the mouse forebrain: age and sex differences. J Comp Neurol, 522(2), 358-71. 159

Appendix

Statement of Contributions

My supervisor—Dr. Peter Kannu—and my Program Advisory Committee members—Dr. Brian Ciruna and Dr. Marco Magalhaes— contributed to the experimental design, data interpretation, thesis revision, and approval. Liliana Vertel, Kashif Ahmed, Michael Liang, Raymond Poon, Mushriq Al-Jazrawe, Qingxia Wei, and Chunying Yu contributed to the experimental design and data interpretation. Dr. Eric Campos, William Scott, Stephanie Tran, Erin Chown, William Xie, Lisa Vi, Marc Lawrence, Tarimobo Otobo, Archita Srinath, Carlos King, Anh Chu, and members of the University of Toronto Collaborative Program in Musculoskeletal Science provided supporting data interpretation and advice.

The Kannu lab and Alman lab organized all mouse cages and maintenance with The Centre for Phenogenomics (TCP). Genotyping of weaned mice was performed by TCP and Transnetyx, Inc. Liliana Vertel performed in situ hybridization. TCP employees performed H&E and Toluidine blue staining. Marco Magalhaes performed H&E staining. Angela Weng performed immunohistochemistry and Safranin-O staining. Kashif Ahmed assisted with cell culture troubleshooting. Joseph Yang, Ziyi Shao, and members of the Alman lab performed qPCR and RNA silencing. Additional dissection tools, reagents, and miscellaneous equipment were provided by the Wall and Justice labs.

I contributed to the experimental design, genotyped all pups/neonatal mice, performed superficial mouse measurements, performed skeletal staining, photographed and processed all images, harvested whole knees and primary chondrocytes, performed cell culture, performed all statistical analysis, interpreted data, hypothesized a working model, and wrote the thesis.

This study is funded by the Sickkids Restracomp Scholarship and the CIHR CGS-M Program.