R740S MUTATION of Tcirg1 AFFECTS ENAMEL DEVELOPMENT in OSTEOPETROTIC MICE

R740S MUTATION OF Tcirg1 AFFECTS ENAMEL DEVELOPMENT IN OSTEOPETROTIC MICE

Lisa Elaine Johnson

A thesis submitted in conformity with the requirements for the degree of Oral Pathology and Medicine Graduate Department of Oral Pathology University of Toronto

R740S Mutation of Tcirg1 Affects Enamel Development in Osteopetrotic Mice

Lisa Johnson

Oral Pathology and Medicine

Graduate Department of Oral Pathology University of Toronto

2016

Abstract

Osteopetrosis is characterized by sclerotic bone due to impaired osteoclasts. Many cases are associated with defects in the TCIRG1 gene encoding the a3 subunit of vacuolar-ATPase (V-

ATPase). These patients have defects in bone, tooth eruption and enamel. To elucidate the role of V-ATPases in amelogenesis, we investigated the cellular distribution of a3, expression of enamel proteins, and quality of the enamel in a mouse with a mutation (R740S) in a3. In the homozygote (R740S/R740S), micro CT and SEM demonstrated a decrease in mineralization and thickness of the enamel. Using immunohistochemistry, the expression of the secretory stage protein, amelogenin remained in the R740S/R740S ameloblasts at day 9, concurrent with lower levels of the maturation stage proteins, amelotin and ODAM, suggesting a developmental delay. Low level expression of a3 was observed in the ameloblasts during the secretory stage.

Our results suggest that a3-V-ATPases may play a role in amelogenesis, possibly by affecting protein secretion.

Acknowledgements

This work is supported by TAS grant YI12_024 (IV) from the Arthritis Society.

Special thanks to Dr. Irina Voronov for her assistance in every aspect of this project and her moral support throughout my program. Thank you also to Dr. Ben Ganss for sharing his technical expertise, and to Dr. Grace Bradley for ensuring I succeeded in finishing this project in a timely manner. Many thanks to all the members of the Bone lab and the histology lab who assisted me greatly during this project.

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Table of Contents

Acknowledgements ...... iii

Table of Contents ...... iv

List of Figures ...... v

1 Introduction ...... 1

1.0 Osteopetrosis ...... 1

1.1 Physiology of the Osteoclast ...... 5

1.2 Vacuolar ATPase (V-ATPase) and TCIRG-1 ...... 9

1.3 Tcirg-1 Mouse models ...... 12

1.4 Dental Development and the Role of the Osteoclast ...... 14

1.5 Amelogenesis ...... 19

1.6 Amelogenesis and pH regulation ...... 25

1.7 Process of Enamel Crystallization ...... 29

1.8 Enamel Defects ...... 32

2 Rationale and Hypothesis ...... 34

3 Manuscript (under review) ...... 36

3.1 Abstract ...... 37

3.2 Introduction ...... 38

3.3 Materials and Methods ...... 41

3.4 Results ...... 45

3.5 Discussion ...... 54

4 Discussion ...... 57

5 Conclusion ...... 63

6 References ...... 64

Table of Figures

Figure 1. The Osteoclast ...... 6

Figure 2. V-ATPase Pump ...... 9

Figure 3. Tooth Germ ...... 15

Figure 4. Tomes Processes at the Dental Enamel Junction ...... 22

Figure 5. Maturation Stage Ameloblasts ...... 27

Figure 6. Quantification of the dental enamel...... 43

Figure 7. Odontogenesis is impaired in R740S/R740S osteopetrotic mice ...... 46

Figure 8. R740S/R740S mice have hypomineralized and hypoplastic enamel...... 48

Figure 9. Enamel rod organization is delayed in R740S/R740S enamel ...... 50

Figure 10. Enamel matrix protein expression is affected in R740S/R740S ameloblasts ...... 52

Figure 11. The a3 subunit of V-ATPase is expressed in day 5 but not in day 9 ameloblasts ...... 53

Figure 12. Macroscopic appearance of Day 9 mandible ...... 60

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1 Introduction

1.0 Osteopetrosis

Teeth are as critical to our existence as any other major organ in our body. They are essential for effective mastication and digestion. Teeth are necessary to maintain vertical dimension and appropriate spatial jaw relationships. They facilitate speech. Teeth play a key role in esthetics and psychosocial development. It is unsurprising, therefore, that odontogenesis is such an exquisitely complex process, combining tissues of ectoderm and neural crest origin to create a vital, highly mineralized structure [1]. Dental abnormalities, both in hard tissue development and eruption, are very common in the human population. These anomalies may be isolated or may be associated with an underlying systemic disease [2].

Studying these disease processes helps to further elucidate the underlying molecular events that are taking place during odontogenesis.

One disease that exemplifies this concept is osteopetrosis. Osteopetrosis refers to a group of diseases that are characterized by high bone density and often present with dental anomalies. The increase in bone density is due to either over-activity of osteoblasts or inactivity of osteoclasts. Commonly known as marble bone disease, osteopetrosis has an incidence of 1 in 100,000 individuals worldwide [3]. The sub-classifications of the disease are quite extensive and are based on their mode of inheritance and clinical phenotype; the two major forms of the disease are autosomal dominant osteopetrosis (ADO) and autosomal recessive osteopetrosis (ARO) [4]. Over 30% of osteopetrosis patients still have no identifiable genetic abnormality, making diagnosis and management a challenge [3].

Autosomal dominant osteopetrosis (ADO) generally affects the axial skeleton, while sparing the long bones. ADO presents in adulthood with symptoms ranging from bone pain, scoliosis, hip arthritis, bone fractures, and hearing loss [5]. Dental abnormalities include dental aplasia, retained primary teeth, delayed and unerupted teeth, and a tendency to develop osteomyelitis of the jaw [6]. There are two major adult variants: ADOI and ADOII. ADOI is associated with a gain of function mutation of the low-density lipoprotein receptor-related protein 5 gene (LRP5) resulting in osteoblast activation [7]. This form of the disease manifests with nerve compression and bone pain, but no noted increase in bone fracture rate [8]. ADOII, formerly known as Albers-Schonberg disease (named after the radiologist who originally described the disease in 1904 [9]), is the most common form of osteopetrosis, with an incidence of 5 in 100,000 people [8]. It is associated with a defect in the chloride channel 7 gene

(CLCN7) resulting in osteoclast dysfunction[10]. This form of the disease has an increased rate of bone fracture, while nerve compression is uncommon [5, 8, 11].

Autosomal recessive osteopetrosis (ARO) is the most severe form of the disease, with an incidence of 1 in 250,000 births in the general population [12]. It is characterized by dense, sclerotic bone with defects in the metaphyseal plates [12]. ARO presents in infancy, with the initial symptoms often being anemia and hepatosplenomegaly, as a result of bone marrow suppression and extramedullary hematopoiesis, respectively [6]. The increased bone mass and density causes children to develop head and facial deformities manifesting as macrocephaly, frontal bossing, hypertelorism, exophthalmos, and a flat nasal bridge [6, 12]. In the affected individuals who survive infancy, neurologic symptoms develop due to impingement of the cranial nerves as they pass through their bony foramina, resulting in blindness, deafness and facial paralysis [4-6]. These children are also at risk for developing hypocalcemia, a condition termed osteopetrorickets, and are thus prone to tetany, seizures and secondary

3 hyperparathyroidism [5]. Dental findings include eruption defects, primary tooth retention, dental aplasia, poor root development, severe dental caries, and a tendency to develop osteomyelitis [5, 13]. If untreated, ARO is fatal at a very young age due to severe anemia, bleeding, or infections [5, 6]. Current treatment modalities include hematopoeitic stem cell transplantation (HSCT) for appropriate candidates, interferon gamma 1b treatment to increase immune function, and other supportive therapies requiring multidisciplinary treatments [5].

ARO is certainly a candidate disease for the future development of gene therapy [14].

Many genes have been implicated in the pathogenesis of ARO and can be linked to specific disease presentations. Four of the genes are associated with osteoclast function and are characterized by osteoclast-rich osteopetrosis. These genes include the following:

TCIRG1, which encodes a component of a vacuolar-ATPase proton pump (V-ATPase);

CLCN7, which encodes a chloride exchange transporter; OSTM1 which encodes osteopetrosis- associated transmembrane protein, which plays a supportive role for the CLCN7 encoded chloride channel; and PLEKHM1, which encodes pleckstrin homology domain-containing M member 1, a protein that is thought to play a role in intracellular trafficking [5, 12]. SNX-10 is an additional gene, which encodes the protein Sorting nexin-10, which is thought to be involved in protein sorting and trafficking within the osteoclast; mutations in this gene are also found in osteoclast-rich osteopetrosis, although it is associated with a more benign disease presentation [12]. OSTM1 and CLCN7 are associated with a neuropathic subtype of ARO, which often manifests as seizures, developmental delay, hypotonia, and retinal atrophy [5].

CLCN7 plays a key role in lysosomal acidification accounting for the severe neurodegeneration that is seen in the central nervous system (CNS) and in the retina of this subset of osteopetrosis

[5]. TCIRG1 mutations are considered the more classic presentation of ARO, and often present with severe osteopetrorickets, characterized by a sclerotic skeleton accompanied by

4 hypocalcemia and skeletal mineralization defects. Hypocalcemia is thought to be due to a reduction in calcium uptake in the gut, resulting in an inability to fully mineralize newly forming hard tissue [12]. A rare form of osteoclast-poor ARO also exists and is associated with genes encoding receptor activation of nuclear factor-kappa B (RANK) and RANKL, two proteins involved in differentiation and formation of osteoclast during osteoclastogenesis [5].

A third variant of ARO, ARO with renal tubular acidosis (RTA), is characterized by a milder clinical course, including an increased frequency of bone fractures, short stature, dental abnormalities, cranial nerve compression and developmental delay [5]. RTA is linked to a mutation in the carbonic anhydrase II gene (CAII), which codes for an enzyme that catalyzes

+ the conversion of water (H20) and carbon dioxide (CO2) to hydrogen (H ) and bicarbonate ion

- (HCO3 ) [5].

An intermediate form of osteopetrosis also occurs in association with CLCN7 and

PLEKHM1. It presents during childhood with mild to moderate disease presentation [5].

Other forms of syndromic presentations of osteopetrosis do exist, but are not considered a classic presentation of the disease [5, 12]. For example, a mutation of the IKBKG gene on the

X-chromosome gives rise to osteopetrosis with ectodermal dysplasia and immune defect; a mutation in cathepsin K, results in pycnodyostosis; and a defect in KINDLIN3 gene results in leukocyte adhesion deficiency syndrome and osteopetrosis.

1.1 Physiology of the Osteoclast

Bone is a rigid, mineralized connective tissue that makes up our skeleton. It is critical to movement, protection of our viscera, hematopoiesis and mineral homeostasis [4]. It is continually maintained and remodeled in response to functional demands, by the co-ordinated activity of osteoblasts, laying down new bone, and osteoclasts, resorbing redundant, fatigued bone [15]. Many diseases, including osteoporosis, hyperparathyroidism, Paget’s disease and osteopetrosis, are characterized by an imbalance between these two processes [6]. The majority of osteopetrotic patients with a known genotype have been linked to a defect in one of the many genes associated with the osteoclast. Mouse models of diseases associated with osteoclast dysfunction have greatly contributed to our understanding of the physiology of this unique cell. In fact, early studies of osteopetrotic mice rescued by transfusion with spleens or bone marrow from healthy littermates, helped establish the myeloid lineage of the osteoclast

[16].

The osteoclast is a multinucleated cell of hematopoietic origin that is responsible for resorptive activity during physiologic bone metabolism [17]. It travels from the bone marrow to the bone in a pre-osteoclast form, and under the influence of several signaling pathways it forms a multinucleated cell [1]. These pathways include the interaction of RANKL, expressed by osteoblasts and other stromal cells, with its receptor RANK on the pre-osteoclast surface, as well as the binding of macrophage-colony stimulating factor (M-CSF), produced by osteoblasts, to its receptor on the progenitor cells [4]. Once activated, the osteoclast becomes polarized by undergoing extensive cytoskeleton remodeling, with one pole exhibiting deep folds creating a ruffled border [1]. The ruffle border is actually created by the fusion of lysosomal secretory vesicles with the cell membrane [18]. At the periphery of this ruffled

6 border, the osteoclast attaches via integrins to the underlying bone, creating a microenvironment, known as a lacunar space, as depicted in Figure 1 [3, 17].

Figure 1- The Osteoclast

Schematic diagram of the resorbing osteoclast. CAII = carbonic anhydrase II, Cat K = cathepsin K, TRAP = tartate resistant acid phosphatase, CLCN7 = chloride channel,OSTM1 = osteopetrosis associated transmembrane protein 1. Source: Author, adapted from Peruzzi, B. et al [3].

In order to accomplish bone resorption, the osteoclast first demineralizes the bone and then digests its organic matrix. An acidic environment is created when the osteoclast pumps

7 hydrogen ions, via a vacuolar ATPase pump (V-ATPase) (a component of which is encoded by

TCIRG1) into the lacunar space. In order to maintain electrical neutrality to ensure continued functioning of the V-ATPAse pump, a concurrent flow of chloride ions enters the lacuna via a chloride channel (coded by the CNCL7 gene) [3]. The stability and function of this channel is also thought to be supported by OSTM1 [19]. An overall pH of 4.5 is achieved within the discrete lacunar space, resulting in demineralization of the underlying bone, as well as providing an acidic environment conducive to proteolytic digestion of the remaining organic component of the bone [1, 20]. The required hydrogen and chloride ions are provided by the action of carbonic anhydrase II (CAII), which catalyzes the conversion of water and carbon dioxide to hydrogen and bicarbonate ion. The bicarbonate ion is then exchanged for a chloride ion on the membranous surface [3]. The final digestion of the organic matrix is achieved by an influx of proteolytic enzymes, including cathepsin K, matrix metalloproteinases and tartate- resistant acid phosphatase (TRAP) into the lacunar space [20]. Lastly, PLEKHM1 is a protein that is recruited to endosomes and lysosomes. It may play a role in the delivery of proteolytic enzymes to the ruffled border and may be associated with lysosomal trafficking and fusion at the ruffled border [12].

Multiple molecules from several organ systems are associated with osteoclast regulation. Stimulatory effects are mediated by activated Vitamin D3 and parathyroid hormone

[3]. They are thought to act indirectly by inducing RANKL expression in other cells, particularly osteoblasts, which in turn stimulate osteoclastic differentiation and activity [3].

Osteoblast signaling can also mediate an inhibitory effect on the osteoclast by the production of osteoprotegerin [21]. Osteoprotegerin acts as a decoy receptor, blocking the binding of

RANKL to RANK, thus down-regulating osteoclast differentiation [4, 18]. Inhibitory effects are also mediated by androgens and estrogens. These hormones increase the rate of apoptosis

8 of the osteoclast and decrease osteoclastogenesis [3, 18]. The osteoclast can also decrease its own activity through an autocrine type feedback loop, by sensing increased calcium levels via a calcium sensing receptor [3]. Additionally, calcitonin, a hormone produced by the thyroid parafollicular cells, is able to bind to osteoclasts via a calcitonin receptor, causing a cessation in osteoclastic activity in order to decrease circulating calcium levels [3, 22].

Although bone resorption is the primary function of the osteoclast, other secondary roles are being discovered. The osteoclast may play a part in the recruitment and stimulation of osteoblasts, the mobilization of hematopoietic stem cells from the bone marrow, and an indirect role in glucose metabolism [3].

Overall, the importance of the osteoclast as a bone resorbing cell in bone metabolism is exemplified by many diseases. Osteopetrosis is characterized by decreased osteoclast activity.

It may be associated with defects in genes that are responsible for differentiation of osteoclasts, namely RANK and its ligand RANKL, resulting in a decreased osteoclast population. It can also be due to mutations in genes that are associated with the ability of the osteoclast to acidify the resorptive lacunae and achieve bone resorption, those being CLCN7, OSTM1, CAII,

PLEKHM1, SNX-10 and TCIRG1.

1.2 Vacuolar ATPase (V-ATPase) and TCIRG1

Many of the genes that encode proteins intrinsic to the function of the osteoclast have been implicated in osteopetrosis. One gene that has been found to be associated with over 50% of the human cases of autosomal recessive osteopetrosis and has over 90 different mutations is the TCIRG1 gene [12, 23]. TCIRG1, through alternative splicing, codes two distinct proteins:

T-cell immune response cDNA-7, a protein involved in T-cell activation; and a3, an isoform of the ‘a’ subunit of a V-ATPase proton pump [24]. V-ATPase is a rotary proton pump composed of at least fourteen subunits [25]. It is involved in intracellular and extracellular acidification throughout the body [26]. V-ATPases maintain acidic pH levels that are necessary to mediate intracellular trafficking, hormone processing, endocytosis and protein degradation [26]. The V-

ATPase pump is divided into two functional domains: V1 and V0, as depicted in figure 2 [27].

Figure 2 V-ATPase Pump Schematic diagram of a vacuolar ATPase pump. Source: Author, adapted from Sun-Wada et al [27].

V1 is the peripheral cytoplasmic domain, composed of subunit A-H, and it is responsible for hydrolysis of ATP to generate energy to drive the proton pump, contained within the membrane bound V0 domain [27]. The V0 domain is composed of subunits a, c, cʹ, cʺ, d and e [25]. The

‘a’ subunit has been identified as containing the critical amino acid responsible for the translocation of a proton across a plasma membrane [28]. The ‘a’ subunit is also thought to contain information required for the targeting of the V-ATPase pump to different areas within the cell [29]. Several of the subunits have multiple isoforms, with the ‘a’ subunit having four isoforms that are expressed in a tissue specific manner [30]. a1 and a2 isoforms are ubiquitously expressed in endosomes and golgi bodies; a4 is present in renal cells, epididymis and optic cells; and a3 is found in all endocrine cells, including adrenal, thyroid, parathyroid, and pituitary glands [31], as well as macrophages, parietal cells, and osteoclasts [25].

Furthermore, the expression of a3 in the osteoclast is 100-fold higher in comparison to other cell types [23, 27]. Within the osteoclast, the a3 containing V-ATPase (a3-V-ATPase) is located within lysosomes and plasma membranes of the non-resorbing cell and within the ruffled border of the actively resorbing cell, thus playing a role in both intracellular lysosomal acidification and extracellular acidification [20].

Patients with osteopetrosis due to a defect in TCIRG1, have dysfunctional osteoclasts due to inactive a3-VATPase pumps, resulting in osteoclasts that are unable to acidify the lacunar space and resorb bone. To properly understand the complexities of this disease, it is critical to consider not only the osteoclast, but also the entire spectrum of cells that may be affected by a defective V-ATPase pump. For example, a defect in a3-V-ATPase affects the ability of parietal cells to properly acidify the stomach [32]. The resulting hypochlorhydria causes a decrease in calcium uptake in the gut, resulting in hypocalcemia, thus contributing to

11 the osteopetrorickets noted in this group of patients [33]. Further elucidation of the diverse role that a3-V-ATPase plays in various cell types is critical in the long term care of this substantial subset of ARO patients, given that HSCT is their primary mode of treatment. Donor marrow is able to repopulate the recipient with functional osteoclasts and restore normal bone metabolism

[3], but non-hematopoietic cells will be unaffected by this therapy.

1.3 Tcirg1 Mouse models

The role of a3 in V-ATPase function and osteoclastic activity is demonstrated by three mouse models: the oc/oc mouse, which is characterized by a truncated, non-functional a3 protein [32]; the a3 null mouse (Tcirg1-/-) which is completely lacking the a3 protein [34]; and lastly, the Tcirg1R740S/R740S, which contains a point mutation in a3, resulting in a protein that lacks the ability for proton translocation [35]. The homozygote of each of these models has severe osteopetrosis due to impaired osteoclast function, mirroring human disease [32, 34, 35].

Furthermore, similar to human osteopetrosis, the homozygote of each of these mutations lack tooth eruption [32, 34].

Osteoclasts from the oc/oc mouse and the Tcirg1-/- mouse models do not express an a3 subunit, thus resulting in osteoclasts that are unable to form a ruffled border and acidify the lacunar space [32, 34]. These findings suggest a3-V-ATPase may play a critical role in the fusion of lysosomal secretory vesicles and the plasma membrane during ruffle border formation

[35]. Interestingly, the acidification of lysosomes (measured in other cell types) are unaffected in these mutants, suggesting that a compensatory response of possibly other V-ATPases, with different functional ‘a’ subunits are able to maintain lysosomal pH in these mice [35]. Both these models have been shown to have a marked increase in osteoclastogenesis and are characterized as an osteoclast-rich osteopetrosis, likely due to a feedback mechanism related to the absence of bone resorption [35]. The dental phenotype of the oc/oc mouse is described as having a reduction in the length of the anterior incisors, an impairment of molar root formation and a lack of dental eruption [33]. Koehne et al. further demonstrated that the enamel of the oc/oc mouse molar was hypomineralized, using quantitative backscattered electron imaging

(BSE) [33]. This finding is in contrast to a study by Bronkers et al, who reported no effect on mineralization of the dental enamel in the oc/oc mouse, using micro computed tomography

(micro CT) quantification [36].

The Tcirg1 R740S/R740S osteopetrotic mouse model has a missense point mutation in

TCIRG1, resulting in the substitution of a highly conserved arginine by a serine at residue 740

(R740S) [35]. The mutated protein is able to become incorporated into the Vo subunit, but is unable to pump protons. It has been previously demonstrated that R740S is a dominant negative mutation, with the homozygous animal displaying severe osteopetrosis, hypocalcemia and a shortened lifespan, while the heterozygote displays mild osteopetrosis and a lifespan comparable to the wild-type littermates [35]. The osteoclasts of the heterozygote mice are able to form ruffled borders, unlike the oc/oc and Tcirg1-/- mice, supporting the concept that the presence of the a3-V-ATPase pump may be necessary for the formation of the ruffled border architecture, in addition to its role in lacunar acidification [35]. The dental phenotype of this mouse model has not been characterized.

1.4 Dental Development and the Role of the Osteoclast

The findings of delayed or lack of dental eruption and root malformations in many subtypes of osteopetrosis, support the accepted concept that osteoclasts play a critical role in tooth development and eruption.

Tooth morphogenesis begins in the tooth bud stage with an epithelial thickening in an area that is pre-programmed as odontogenic [2]. This event occurs as early as embryonic day

8.5 in mice, and the sixth week of human embryogenesis [37]. Prenatal tooth development proceeds through the cap and bell stage, resulting in the differentiation of the epithelial cells to enamel producing ameloblasts, and differentiation of the mesenchymal cells to dentin producing odontoblasts [37]. Although the development of the crown of the tooth is initiated in utero, the final morphogenesis, enamel mineralization, and eruption into the oral cavity are completed post-natally [38].

At birth, the enamel organ of the mouse molar is established and consists of four epithelial layers: the cuboidal, outer enamel epithelium (OEE); the star-shaped, glycosaminoglycan rich, stellate reticulum (SR); the more compressed stratum intermedium

(SI); and the inner enamel epithelium (IEE) with their centrally placed nuclei [38, 39]. The enamel organ, and the underlying dental papilla are encapsulated by the dental follicle, a layer of condensed ectomesenchymal cells in a fibrous stroma [1]. Together, all three components constitute the tooth germ [1]. Differentiation of the odontoblasts is already evident within the dental papilla at birth, with columnar odontoblasts lined up along the basal lamina of the enamel organ and laying down pre-dentin [38]. Furthermore, proliferation and folding of the inner enamel epithelium has completed, creating the shape of the crown of the mouse molar, as

15 depicted in figure 3 [1, 39, 40].

Figure 3 Tooth Germ

H&E of a tooth germ of a postnatal day 1 wild type (+/+) mouse molar. The (*) is highlighting predentin and early dentin formation. Source: Author.

By day 2, the inner enamel epithelium will undergo final differentiation to the ameloblast, and will begin to form enamel against the newly formed dentin [38]. The cervical loop, where the OEE and IEE join, will continue to proliferate and will give rise to Hertwigs epithelial root sheath (HERS) by day 4 or 5 [1, 38]. This bi-layered epithelium will stimulate the dental papilla cells to differentiate into odontoblasts and to lay down root dentin [1, 40].

Eventually HERS will disintegrate, with some cells undergoing apoptosis and others being incorporated into developing cementum or giving rise to the cell rests of Malassez [2]. With the onset of root formation, the crown of the tooth will begin its first eruptive movements away

16 from the base of the bony crypt, creating space for root formation, versus the developing roots growing into the underlying bone [1].

Postnatal tooth development is taking place within an intraosseous environment, thus critical to its success is the spatial regulation of bone around the tooth germ [2]. Both osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, play an important role in this process. Inhibition of osteoclastic bone resorption prevents tooth eruption [41], as does inhibition of bone deposition at the base of the crypt [2, 42]. Marks and Cahill demonstrated using scanning electron microscopy (SEM), a scalloped bone pattern characteristic of resorption present at the coronal area of tooth germ, and a trabecular bone pattern indicating bone deposition at the base of the crypt [43]. The dental follicle has been demonstrated to be the master controller of bone turnover surrounding the developing tooth [44]. The coronal portion of the dental follicle regulates bone resorption, while the basal portion regulates bone deposition [45]. In fact, the tooth itself plays no role in dental eruption, as was demonstrated by the successful eruption of an inert tooth replica transplanted into an intact dental follicle

[41]. A burst in osteoclastogenesis is noted around the rat and mouse molar with the recruitment of osteoclast precursors to the dental follicle on day 3 and day 5, respectively [38].

The resulting increased resorption is likely associated with early pre-eruptive movement of the tooth germ [46]. This event correlates with increased expression of macrophage-colony- stimulating factor-1 (M-CSF-1) and monocyte chemotactic protein-1 (MCP-1) in the dental follicle [45]. M-CSF-1 promotes osteoclastogenesis via upregulation of RANK expression in the osteoclast precursors cells, and by being chemotactic for mononuclear hematopoietic cells

(osteoclast precursors) [38, 44]. MCP-1 also acts as a chemokine for the precursor cells [38,

44]. Subsequently, during days 6 through 8, the mouse molar displays a rapid increase in tooth size, accompanied by resorption of the surrounding bone, particularly along the inner margins

17 of the alveolar crest [38]. A second wave of osteoclastogenesis is noted on day 9 or 10 (mouse and rat, respectively), in response to increased expression of RANKL, VEGF (which stimulates expression of RANK), and tumour necrosis factor alpha (TNF-α), another promoter of osteoclastogenesis [44, 45]. By day 10, in the mouse molar, adequate resorption of bone overlying the tooth has occurred such that it is able to begin its final movement towards the oral epithelium [38]. The enamel organ overlying the tooth undergoes significant programmed cell death (apoptosis), giving rise to the reduced enamel epithelium [1]. This reduced enamel epithelium will eventually re-establish an epithelial connection with the overlying oral mucosa, creating a canal through which the tooth will erupt [1]. Alveolar bone remodeling continues throughout the eruptive process, with the alveolar bone increasing in height and shape to accommodate the crown of the erupting tooth [46]. Bone deposition will be seen at the base of the crypt as it fills in the voids left by the erupting crown around the smaller diameter roots [1].

Continued bone resorption around the entire tooth germ will allow adequate space for the dental follicle to give rise to the periodontal ligament, a specialized connective tissue responsible for the attachment of the tooth to the alveolar bone; contraction of the myofibroblasts within the ligament are thought to be important to the eruption of the mouse molar as it emerges, on day 18, into the oral cavity [1, 38, 45].

The critical role that osteoclasts play in tooth development is demonstrated by the lack of dental eruption observed in the knockout-mouse devoid of RANKL [47] and the osteopetrotic mouse with non-functional M-CSF (the op/op mouse) [48], who both lack osteoclasts; as well as the oc/oc [32] and Tcirg1-/- mice [34], who lack a functional a3-V-

ATPase impairing osteoclast function. Furthermore, injection of M-CSF in the op/op rats was able to re-establish osteoclastogenesis and restore dental eruption [49], and hematopoeitic stem

18 cell transplantation (HSCT) in the oc/oc mouse was also able to restore dental eruption [50]. A case report of a patient, with non-genotyped osteopetrosis, also having undergone successful

HSCT at 6 weeks of age, displayed eruption of the primary teeth [51].

1.5 Amelogenesis

In addition to dental eruption problems, osteopetrotic patients also display aberrations associated with the dental enamel. Few studies characterizing the dental phenotypes of osteopetrotic mice have addressed the quality of the dental enamel. This fact is quite surprising since high dental caries is reported to be a significant finding in ARO patients [5], suggesting a possible defect in the integrity of the enamel. Given the prevalence of osteomyelitis in this population group, decay leading to infections and extractions may certainly be a contributing factor to this serious condition.

Enamel is the most highly mineralized tissue in the human body, being composed of

96% hydroxyapatite crystals, (or crystalline calcium phosphate) and 4% organic material and water [1, 52]. This high mineral content renders the surface of the tooth highly resistant to wear and demineralization at a pH greater than 5.5 [53]. The enamel is typically 2.5 mm at its thickness point along the cusps of the human molar and tapers to a feather edge at the base of the crown [1]. The crystalline structure is composed of cylindrical rod and interrod enamel crystals, orientated in different directions, imparting a distinct cross sectional pattern to the enamel when viewed with SEM [1, 54]. This pattern has been likened to a keyhole, with the enamel rod being the head of the keyhole and the interrod enamel being the neck and base [46].

A longitudinal view of the enamel rods demonstrates the undulating path they take from the dentin-enamel junction to the surface of the tooth [1, 46].

Mineralization of human primary teeth starts at 14-18 weeks in utero, while the permanent teeth begin to mineralize first with the molar at birth, followed by the incisors and canines during the first year of life, and the premolars and second molars between the second

20 and third year of life [51, 55]. Histological evidence of enamel formation in the mouse molar can be seen by day 2 of post-natal development [38].

Enamel formation is termed amelogenesis, and can be subdivided into 3 phases: differentiation, secretory and maturation phase. Differentiation of the ameloblasts begins at the tips of the tooth crown and proceeds cervically towards the cemental enamel junction (CEJ)

[1]. Specifically, the inner epithelial cell of the enamel organ increases in length, acquires a greater number of golgi bodies and more extensive rough endoplasmic reticulum, reverses the polarity of its nucleus, and develops a distal cytoplasmic extension termed a Tomes process; all giving rise to the secretory ameloblast, see figure 4A [1, 46]. The newly formed ameloblast creates a sealed enamel microenvironment along the enamel front by establishing tight junctional complexes, gap junctions and desmosomal attachments with adjacent ameloblasts and stratum intermedium cells [56]. The ameloblast is no longer able to replicate, but is capable of producing large amounts of enamel matrix proteins, including amelogenin, enamelin and ameloblastin, with amelogenin accounting for over 90% of the enamel matrix [1, 57].

As the ameloblast enters the secretory phase, the enamel proteins are packaged and secreted in secretory granules through the Tomes processes onto the newly forming dentin [1].

This phase is thought to correspond to approximately post-natal day 4 in the development of the mouse mandibular molar [58]. The secreted amelogenins are quickly cleaved into a wide range of smaller fragments by a tooth specific matrix metalloprotease, MMP-20 (enamelysin), also secreted by the ameloblasts [39, 59]. In vitro studies have demonstrated that MMP-20 activity can be modulated by calcium and phosphate concentrations within the enamel extracellular fluid [59]. MMP20 null mice have demonstrated the critical role MMP20 plays in amelogenesis, as mice lacking MMP20 activity have hypoplastic and hypomineralized enamel

[39]. The resulting cleaved amelogenin fragments undergo self assembly into nanospheres that act as scaffolding for the growing enamel crystals [39, 46]. The spatial position of amelogenin, ameloblastin and enamelin play a role not only in initiation of crystallization, but also in directional control of the growth of the crystal [46]. Interestingly, mice lacking amelobastin and enamelin are unable to form any true enamel, while mice lacking amelogenins are still able to form organized crystal structure, thus highlighting the critical role of these enamel matrix proteins in the initiation of crystallization [39]. The enamel matrix proteins are also thought to be important in buffering the hydrogen ions generated during the crystallization process [46].

Overall, the critical roles of both structural enamel matrix proteins and enzymatic proteins are demonstrated by human mutations in each of these proteins, (with the exception of ameloblastin), which give rise to the various forms of amelogenesis imperfecta [39].

The initial 20-40 microns of enamel is aprismic, as it lacks enamel rods; a similar crystalline structure is also noted in the outer enamel surface [46, 57]. As the enamel continues to be laid down, the ameloblasts begin to migrate away from the dentinal surface (dentin- enamel junction (DEJ)), maintaining contact with the aprismic layer via the distal ends of

Tomes processes, see Figure 4A [1]. Histologically, the Tomes processes give a saw tooth appearance to the junction between the enamel and the ameloblasts [1]. The unique enamel crystalline structure is established by a staggered secretory front along the Tomes process; enamel matrix proteins secreted from the base of the process give rise to the interrod enamel, and the proteins secreted at the tip give rise to the rod enamel, as depicted in Figure 4a [39].

The interrod enamel matrix that is laid down initially forms a “keyhole” outline and the enamel

(A)

(B)

Figure 4 Tomes Processes at the Dental Enamel Junction

(A) Schematic diagram of the secretory ameloblast and early enamel growth pattern.

(B) SEM, secondary electron image of +/+ enamel from the mesial cusp of the mandibular molar, demonstrating early crystals and cartoon representation of the positioning of the Tomes process of the secretory ameloblast. Source: Author.

23 rod fills in the cavity that is created [1, 46]. Essentially, the enamel rod represents mineralization of the path that the ameloblast takes as it migrates outward from the enamel front, see Figure 4b [46]. By the end of the secretory phase, the entire thickness of enamel has been deposited and has achieved a mineral content of 30% [1, 52].

In order to establish its final composition, the enamel must undergo a hardening process, resulting in significant growth and interlocking of the hydroxyapatite crystals, and a marked decrease in the organic content. The maturation phase is quite complex, taking up to four years to occur in human permanent dentition [52] and two weeks in the rat incisor [56].

This phase accounts for over 2/3 of the entire process of amelogenesis [1]. In the mouse, the first molar is primarily engaged in this maturation process by day 8 of development [58]. As the ameloblasts prepare to undergo the transition to the final maturation phase, approximately

25% of the cells undergo apoptosis [1, 46]. The overlying stellate reticulum becomes thinner, bringing the blood vessels within the dental follicle closer to the remaining ameloblasts [1, 52].

(This remaining layer of cells will eventually give rise to the reduced enamel epithelium that remains until the tooth erupts [1].) During the maturation process the proteinaceous enamel matrix is removed primarily by the proteolytic activity of serine protease kallikein 4 (KLK4), which is secreted by the maturation stage ameloblasts [59, 60]. Removal of the enamel proteins within the intercrystal space, allows for the incorporation of additional inorganic material, thus increasing both the thickness and the width of the enamel rods [1, 58]. Enamel matrix protein breakdown products are then reabsorbed via endocytosis and degraded within lysosomes [61]. Surprisingly, enamel protein secretion is still noted in the maturation phase.

Amelotin is a unique protein whose expression is primarily limited to the maturation stage; it is localized at the interface between the ameloblasts and the developing enamel surface [57, 62].

The expression of amelotin is accompanied by the expression of another enamel protein,

24 odontogenic ameloblast-associated protein (ODAM) [63]. ODAM appears at the ameloblast- enamel interface slightly in advance of amelotin [64]. Amelotin and ODAM are postulated to be associated with the attachment of junctional epithelium cells to the mineralized surface of the erupted tooth [64, 65]. Amelotin is also thought to play a role in the final, aprismic layer of surface enamel and in the removal of the organic enamel matrix [57, 66].

1.6 Amelogenesis and pH regulation

In order to succeed in generating a highly mineralized enamel product, the ameloblast must be capable of regulating water, modulating the concentrations of calcium, phosphate, bicarbonate and chloride ions, while maintaining an acid-base balance, and removing enamel matrix debris [58]. To accomplish these tasks, the maturation phase ameloblast becomes more cuboidal as the Tomes process recedes, and re-establishes a basal lamina along the outer enamel front [46]. Its apical pole modulates between periods of ruffled-ended appearance with deep infoldings and periods of smooth-ended appearance [1]. The rough ended morphology of the ameloblast is not unlike the morphology seen in the activated osteoclasts [36]. As the ameloblasts transition from one morphology to another, the tight junctions that exist between the ameloblasts fluctuate from a more apical position, close to the enamel surface in the rough- ended ameloblast (RA), to a more basal location in the smooth ended ameloblast (SA) [1, 46].

As the tight junctions relocate, there is a temporary breach in the permeability of the enamel microenvironment, as shown schematically in Figure 3 [1, 46, 56]. This cycling between two different morphologies can take up to 8 hours in the mouse, with the rough ended state being present for over half of the time [1, 53]. The significance of this modulation is not entirely understood and remains somewhat controversial, although most agree that it ties in closely with the regulation of pH within the enamel microenvironment.

The control of pH in the enamel microenvironment is not definitively understood. It has been proposed that the RA are associated with the pumping of bicarbonate ions into the enamel microenvironment to neutralize the hydrogen ions being released from the crystallization process [1]. Essentially, the bicarbonate ion makes up for the loss of the

26 buffering capacity of the enamel matrix proteins as they are being degraded, therefore, preventing demineralization of the newly formed mineral [1]. Yet, it has been shown that varying levels of pH can be detected in the maturing enamel, corresponding to zones beneath the cycling ameloblasts. The region beneath the RA is found to have a pH of approximately 6 versus a near neutral pH under the SA [52, 53]. Although this decrease in pH may be due to hydrogen ions being released from the growing crystals, and overwhelming the buffering capacity of the bicarbonate being secreted by the RA, it has also been proposed that this decrease in pH is actually due to the active pumping of hydrogen ions via a V-ATPase pump into the extracellular matrix by the RA (Figure 5) [52, 67]; a process not unlike the lacunar acidification observed in the activated osteoclast, [36] or the acidification of urine by the renal intercalated cells of the kidney [29]. In fact, the expression of V-ATPases within the ruffled border RA and within the cytoplasm of the SA is documented, with the ‘a’ subunit being identified in one study as a1 [52]. The finding of the a1 isoform on a plasma membrane surface is unexpected, as a1 is typically expressed in an intracellular location [27]. Evidence also exists of a 5-6 fold up-regulation of mRNA expression of Tcirg1 in genome wide profiling of the maturation stage ameloblast [68], suggesting that a3-V-ATPase plays a role in the ameloblast. The importance of a specific pH during the crystallization process is likely two fold. It is certainly required for the optimization of proteolytic digestion of enamel matrix proteins, and it may be important in the partial dissolution of the newly formed crystal to allow for more effective removal of enamel matrix proteins, adequate ion diffusion within the enamel microenvironment, and optimal re-positioning of the phosphate and calcium ions within the growing crystal [52, 53]. This tight regulation of pH is essentially acting as quality control for the enamel, ensuring only the most stable and pure crystal is formed [52, 53].

Figure 5 Maturation Stage Ameloblasts

Schematic diagram of proposed ions trafficking during the modulation of maturation stage - ameloblasts. CAII = carbonic anhydrase II, AE2 = HCO3/Cl anion exchanger, EMP = enamel matrix proteins. Source: Author, adapted from Josephsen, K. et al [52].

Another important factor in the regulation of pH is the activity of carbonic anhydrase II enzyme within the ameloblast. Similar to the osteoclast, carbonic anhydrase II activity is likely the source of the hydrogen ions for the V-ATPase pump [67]. Furthermore, the bicarbonate ions, generated from the CAII catalyzed reaction, are postulated to be pumped into the

- - intercellular space between the RA above the tight junction by an HCO3 /Cl anion exchanger

(Ae2), located in the basolateral membrane (as depicted in Figure 5) [69, 70]. The temporary change in permeability of the cycling ameloblasts will then permit the influx of these

28 bicarbonate ions (concurrently with the efflux of degraded enamel matrix proteins), thereby accounting for the abrupt rise in pH that is seen between the RA and the SA [53]. The importance of the buffering capacity created by Ae2 for proper enamel formation is demonstrated by Ae2 knockout mice, which has enamel that has less mineral and a higher protein content [69, 70]; again exemplifying the critical role of pH balance in successful enamel formation.

Chloride ions are also important in enamel formation and are thought to function as another regulator of pH during crystal growth [70]. Chloride channels including cystic fibrosis transmembrane conductance regulator (CFTR), chloride mammal channels (CLC) 1-7, chloride channel (CLCN) 3,5 and 7 have been described in association with the cells of the enamel organ [58, 69]. Interestingly, the same chloride channel encoded by CLCN7 found in osteoclasts is upregulated in the maturation stage ameloblast, although, unlike in the osteoclast, it appears to primarily function within the lysosomes of the ameloblast and does not localize to the ruffled border [54, 58]. Enamel defects have been noted in osteopetrotic patients genotyped with CLCN7 mutations [69], although recent studies of a Clcn7 knockout mouse reported no enamel aberrations [71]. Hypomineralization of the enamel of Cftr knockout pigs have also been described [72].

1.7 Process of Enamel Crystallization

The hydroxyapatite crystal that forms in enamel is composed of calcium, phosphate and hydroxide ions, in a ration of 10 to 6 to 2, respectively [1]. The phosphate and hydroxide ions are typically in a hydrogenated form in the enamel extracellular environment [73]. They must undergo deprotonation prior to being incorporated into the growing crystal, thereby creating an increasingly acidic environment during the crystallization process [73]. The enamel apatite is

-2) not a pure hydroxyapatite crystal, as ions including hydrogen phosphate (HPO4 , carbonate

-2 - + (CO3 ), fluoride (F ) and sodium (Na ) substitute into the enamel lattice [46, 73]. During the maturation process, the ameloblasts are situated along the outer enamel front ensuring only appropriate ion concentrations are achieved within the enamel fluid. Since the maturing crystal is increasing in width and size along the entire thickness of the enamel, the intercrystal space must be highly diffusible to these ions.

The high calcium requirement of the enamel apatite makes the growing crystalline structure particularly sensitive to calcium concentration [73]. The movement of calcium to the growing crystal front initially involves the transport of calcium from the circulation by cells within the papillary layer of the enamel organ, followed by the movement of the calcium through the ameloblast layer [56]. 45Ca tracer studies have suggested that both the intercellular movement of the calcium between the cycling ameloblasts as their junctional complexes temporarily uncouple, and the transcellular movement of calcium through the ameloblasts play an important role in modulating the calcium concentrations at the enamel front, with the latter mechanism being the most critical [53]. A 3-4 fold increase in calcium transport is noted in the maturation stage ameloblast, likely reflecting the increasing demands of the growing

30 crystal[56]. During the maturation stage, 45Ca tracer studies have demonstrated an increase in the uptake of calcium into the growing mineral beneath the ruffled border ameloblast versus the smooth ameloblast [56]. It is postulated that transcytosis of calcium through the ruffled ameloblast occurs initially with the movement of calcium from the extracellular fluid through calcium channels into the cytosol of the cell [56]. The calcium ion is actively taken up into the endoplasmic reticulum by Ca+2-ATPase pumps and bound to a calcium-binding protein such as calreticulin [56]. In this bound form, the calcium is then transported through the continuous tubules of the smooth ER and eventually excreted into the enamel fluid from the apical pole of the ameloblast via Ca+2-ATPase pumps, Ca+2/H+ and/or Na+-Ca+2 exchangers, as well as co- secreted with other enamel proteins [56, 74]. The concentration of unbound calcium ion needed to maintain saturation within the enamel fluid is tightly regulated and is actually 6-10 fold lower than serum calcium levels [56]. It is thought that over 85% of the calcium within the enamel microenvironment is bound to enamel matrix proteins; this bound calcium acts as a reservoir for calcium and a means of maintaining an appropriate calcium saturation to ensure only the most stable form of calcium phosphate is incorporated into the growing crystal [73].

The critical role of calcium and calcium channels in enamel formation is demonstrated by

Timothy disease; this disorder is characterized by a dysfunctional calcium channel and presents with cardiac, hand, foot, facial and neurological defects, as well as small teeth with hypoplastic enamel, prone to dental decay [69].

Phosphate ions are another critical component of the enamel extracellular fluid, but it is one of the more poorly studied molecules in amelogenesis. The stratum intermedium cells have been shown to have increased levels of alkaline phosphatase enzyme and are thought to be the primary source of phosphate ions [75]. At physiological pH, phosphate exists in three

-3 -2 -1 anionic forms, PO4 , HPO4 and H2PO4 , with the pH in the enamel fluid favouring the

-2 hydrogenated form [73]. It is thought that the HPO4 is initially incorporated into the growing

-3 crystal, followed by subsequent deprotonation to PO4 [73], the predominant form found within the mature crystal [53]. Overall, phosphate dynamics exemplify the critical importance of strict pH regulation within the enamel microenvironment. Furthermore, individuals suffering from hypophosphatemia, a disease associated with defective alkaline phosphatase activity, display varying degrees of enamel hypoplasia and lack of cementum [46].

1.8 Enamel Defects

Given the complexity of amelogenesis, it is unsurprising, therefore that aberrations in enamel formation can occur. The defects that may occur are dependent on the developmental stage of the ameloblast when the insult occurred. Defects are characterized by enamel hypoplasia, a decrease in the quantity of enamel, and enamel hypomineralization, a decrease in the mineral content of the enamel. Enamel hypoplasia is associated with dysfunction during the secretory phase of amelogenesis, while enamel hypomineralization is linked with problems arising during the maturation stage of enamel development [76]. Enamel hypoplasia has been strongly linked with hypocalcemia and vitamin A deficiency [46]. Hypocalcemia may be caused by a number of factors, including nutritional deficiencies, including low calcium diet and low vitamin D intake; hormone imbalances, such as hypoparathyroidism; and systemic diseases such as renal osteodystrophy and celiac disease [46, 77, 78]. Hypoplastic enamel may or may not be accompanied by hypomineralization, depending on the ability of the ameloblasts to recover from the insult [46]. For example, exposure to substances such as tetracycline and excessive levels of fluoride often result in both hypoplastic and hypomineralized enamel [46].

Hypomineralization of the enamel is due to an incomplete resorption of enamel matrix proteins during the maturation stage. Clinically, enamel hypomineralization presents as white-yellow or yellow-brown opacities that demonstrate a reduction in hardness and elastic modulus in comparison to normal enamel [79]. SEM studies have demonstrated that hypomineralized enamel contains more porous appearing prisms with wider gaps between prisms, although the overall, ordered crystal structure is still maintained [79]. Backscattered electron microscopy

(BSE) measurements are able to demonstrate a relative decrease in the mineralization of the

33 enamel [80]. Hypomineralization of the enamel has been linked to childhood illnesses associated with high fever, antibiotics, particularly tetracycline and macrolides and environmental toxins such as dioxins and polychlorinated biphenyls [81].

2 Rationale and Hypothesis

The dental phenotype in ARO patients includes eruption defects, primary tooth retention, dental aplasia, poor root development, a tendency to develop osteomyelitis and severe dental caries. The lack of functional osteoclasts for proper bone turnover around the developing tooth germ accounts for the eruption defects and poor root development noted in these patients. Osteoclasts are also required for normal tooth exfoliation, thus retention of primary teeth is also an anticipated clinical finding. Dental aplasia and osteomyelitis can be attributed to the compromised blood supply within the sclerotic gnathic bone of ARO patients.

Inadequate blood supply will prevent a dental follicle from developing [82]. It will also contribute to reduced immune surveillance and decreased healing, thus making the bone much more susceptible to infection. The connection between osteoclast dysfunction and a high dental decay rate is not readily apparent, but is suggestive of a defect in enamel formation. It is possible that amelogenesis is affected by the same mutations that give rise to osteopetrosis in

ARO patients. A case report [51] of a child, having undergone HSCT at 6 weeks of age for

ARO, showed that his complete primary dentition achieved eruption, presumably due to reconstitution of functional osteoclasts; however, the teeth were atypical in shape, with missing and abnormal roots, disturbances in enamel mineralization and multiple carious lesions. The child was followed until 8 years of age and was found to have several missing permanent teeth, abnormally shaped anterior teeth, and appropriately formed first permanent molars with hypoplastic and hypomineralized enamel. Since the primary teeth begin to develop and mineralize in utero, any defects in these teeth may be secondary to the disease process.

Comparatively, the enamel of the first permanent molar begins to mineralize at birth, and takes

35 a mean of approximately 3.5 years to complete, thus in this case, they were developing for many years post HSCT. Therefore, these findings of enamel abnormalties suggest that amelogenesis was affected in this patient and was not rescued by the transplant. Additionally, it has been shown that maturation stage ameloblasts express Tcirg1 mRNA; however the role of the TCIRG1 protein (a3) in ameloblast function has not been explored.

Based on the above evidence, I have hypothesized that ameloblasts share the same a3-

V-ATPase pump as osteoclasts and that this pump plays a critical role in amelogenesis.

To test my hypothesis, I propose the following objectives:

1. Describe and study the dental eruption pattern in the osteopetrotic Tcirg1R740S/R740S

mouse model.

2. Evaluate the effect of this mutation on enamel mineralization and spatial-temporal

expression of secretory and maturation stage enamel matrix proteins in the

Tcirg1R740S/R740S mouse model.

3 Manuscript

3.1 Abstract

Osteopetrosis is a rare disease characterized by sclerotic bone due to deficient osteoclast function. Over 50% of the cases of autosomal recessive osteopetrosis are associated with mutations in the a3 subunit of vacuolar H+-ATPase (V-ATPase). These patients often display dental anomalies, such as delayed eruption and enamel defects; however, little is known about enamel formation in osteopetrosis. We investigated enamel mineralization and spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development, using an osteopetrotic mouse model (R740S point mutation in the V-ATPase a3 subunit). Histological and micro CT data revealed aberrations in both crown and root development in homozygous

(R740S/R740S) animals, likely due to constraints imposed by sclerotic mandibular bone.

Scanning electron microscopy analysis demonstrated delayed enamel mineralization in homozygous R740S/R740S mice compared to wild type and heterozygous animals. Enamel thickness and mineralization were significantly decreased in R740S/R740S mice as determined by micro CT analysis. Spatiotemporal enamel matrix protein expression was assessed using immunohistochemistry. The expression pattern of the secretory stage protein amelogenin was comparable in all three genotypes at postnatal days 1 and 5, but displayed continued cytoplasmic staining in R740S/R740S ameloblasts at day 9 time point, suggesting delayed transition to the maturation stage. The maturation stage proteins amelotin and odontogenic ameloblast-associated protein (ODAM) showed a less intense and more diffuse staining pattern on day 9 in R740S/R740S ameloblasts, confirming a delay in transition to the maturation stage.

The V-ATPase a3 subunit expression was observed in ameloblasts at day 5, but not at day 9, in all three genotypes. These results demonstrate that osteopetrosis-causing R740S mutation in the V-ATPase a3 subunit affects amelogenesis by delaying transition to the maturation stage, resulting in hypomineralized and hypoplastic enamel.

3.2 Introduction

Osteopetrosis refers to a group of diseases characterized by high bone density due to either over-activity of osteoblasts or inactivity of osteoclasts. The most severe form of the disease is autosomal recessive osteopetrosis (ARO) [4, 5]. ARO is characterized by a dense, sclerotic skeleton due to the inability of the osteoclast to effectively resorb bone [23]. The resulting overabundance of dense bone causes facial deformities, bone marrow suppression, nerve impingement, and dental anomalies [5, 6]. Dental findings include eruption defects, primary tooth retention, dental aplasia, poor root development, severe dental caries, and a tendency to develop osteomyelitis [5, 13].

Dental enamel is the most highly mineralized tissue in the human body being composed of 96% hydroxyapatite crystals and 4% organic material and water [1, 52]. During the secretory stage of amelogenesis, ameloblasts secrete enamel matrix proteins (amelogenin, ameloblastin, enamelin) which act as a scaffolding for the crystallization of the enamel [1].

During the maturation phase of amelogenesis, the enamel matrix proteins are degraded allowing for incorporation of additional inorganic material[58, 83]. The maturation stage ameloblasts alternate their morphology between a smooth-ended and a ruffle-ended appearance of their apical membrane [1]. During this stage, the ruffle-ended ameloblasts are thought to be actively pumping hydrogen ions into the extracellular matrix [52, 67] via the vacuolar H+-

ATPase (V-ATPase) proton pump; a process not unlike the lacunar acidification observed in the bone resorbing osteoclasts [84, 85].

V-ATPases are ubiquitous, multi-subunit proton pumps responsible for acidification of intracellular and extracellular compartments, and are involved in numerous cellular processes,

39 such as protein degradation and vesicular trafficking [85]. The ‘a’ subunit of V-ATPase contains the amino acid critical to proton translocation across a membrane [28]. This subunit has four isoforms: a1, a2, a3 and a4. V-ATPases containing the a3 isoform are found in all endocrine cells [31], as well as macrophages, parietal cells, and osteoclasts [27]. The expression of a3 in the osteoclast is 100-fold higher in comparison to all other cell types [12,

19, 23]. The a3 subunit is encoded by the TCIRG1 (T-cell, immune regulator 1) gene and mutations in this gene have been implicated in over 50% of all genotyped ARO cases [3, 4, 51].

Bone marrow transplantation at a very early age is the only treatment for ARO [3]; it restores both osteoclast function and dental eruption; however, enamel formation and root development in the permanent dentition does not appear to be rescued by the procedure [51]. This observation suggests that a3-containing V-ATPases may also be expressed and play an important role in non-hematopoietic cells, such as enamel-forming ameloblasts. Furthermore, a

5-6 fold increase in a3 mRNA expression from secretory to the mid-to-late maturation stage ameloblasts was observed during genome wide profiling [58], suggesting that a3 may play a role in amelogenesis.

Two studies examined enamel mineralization in osteopetrotic animals using a3 knockout and oc/oc (a3 truncation) mouse models [33, 36]. Studies of the a3 knockout model showed that enamel formation in the continuously erupting incisor was normal. In the oc/oc model, dentin and enamel mineralization in the molar was decreased; however, the enamel thickness was unaffected. Neither study examined amelogenesis in these animals.

Based on the available evidence, we hypothesized that a3-containing V-ATPases are expressed in ameloblasts and play an important role in enamel formation and mineralization.

To elucidate this role, we focused on expression of secretory and maturation stage-specific

40 enamel matrix proteins using an osteopetrotic mouse model with a point mutation (R740S) in the a3 subunit of V-ATPase. The heterozygotes (+/R740S) have mild disease, while the homozygotes (R740S/R740S) have severe osteopetrorickets and a shortened lifespan compared to a3 knockout and oc/oc models [35, 86]. In the R740S model, the mutant a3 protein is expressed at wild type (+/+) levels, but is not functional, therefore eliminating the possibility of compensation by other ‘a’ isoforms.

Here we present evidence that R740S/R740S animals have hypoplastic and hypomineralized dental enamel due to delayed transition to the maturation stage. This suggests that V-ATPase function is necessary for proper enamel matrix protein secretion and timely transition through the stages of amelogenesis.

3.3 Materials and Methods

Animals

Heterozygous mice carrying the R740S mutation were generated as described previously [35].

Female and male +/R740S heterozygous mice were bred to produce homozygous

R740S/R740S animals. All experimental procedures received necessary approval and were conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Genotyping was performed on genomic DNA extracted from tail tissue using the PureLink

Genomic DNA kit (Invitrogen) using a custom TaqMan SNP Genotyping protocol. Mandibles were collected at days 1, 5, and 9 postpartum. Later time points were not used due to early lethality of R740S/R740S animals (between days 10-14).

SEM/µCT sample preparation

Day 5 (n=3/genotype) and day 9 (n=5/genotype) right hemi-mandibles were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.2) for at least 24 hours, cleaned of soft tissue, and then transferred into 70% ethanol. Specimens were dehydrated in

100% ethanol for a minimum of 2 hours, dried in a critical point drier (Polaron CPD 7501,

Quorum Technologies, Laughton, UK), embedded in epoxy resin blocks (West system 105/205 two part epoxy resin, Gougeon Brothers Inc., Bay City, MI, USA), and cured for 24 hours at

58.5oC.

Micro–computed tomography (µCT) of mouse mandibles

Micro-dissected and epoxy embedded, day 5 hemi-mandibles (n=3/genotype) and day 9 hemi- mandibles (n=5/genotype) were analyzed by µCT analysis. The specimens were scanned using

MicroCT40 system (Scanco Medical, Bruettisellen, Switzerland), at 70 kVp and 114 µA with resolution of 6 µm. The files were obtained in 1000 projections with 2048 samples per 180o of rotation, 300 milliseconds of integration time and 1 frame averaging. Each scan yielded an image data set of 2048 slice sections. Two-dimensional (2D) images were generated in both the coronal and sagittal plane focusing on the first molar.

Quantification of the enamel mineralization and thickness

After reconstruction, a region of interest (ROI), as depicted in Figure 6, was drawn outlining the cusp tip of the enamel in 20 representative samples of day 9 samples of the first molar in the coronal plane. The drawings were morphed into a 3D ROI and a relative density of a selected area of enamel was calculated with µCT40 analysis software supplied with the instrument and calibrated with a control disc of hydroxyapatite. To determine enamel thickness, 20 2D images were used. The region selected for the thickness measurement was the first area of level cuspal incline of the dental enamel junction (DEJ) as it extended down from the cusp tip. A line was drawn perpendicular to the DEJ to the outer edge of the enamel, as shown in Figure 6 and its length was measured using image J software on every 5th slice.

Statistical significance was tested using a one way ANOVA and post hoc Tukey and

Bonferroni tests (p< 0.05). Non-parametric tests were also performed using the Kruskal Wallis test.

Figure 6 Quantification of the dental enamel.

Enamel mineralization was calculated using the ROI highlighted in green. The region of the dental enamel used for the thickness calculation is marked in red. Representative slice. Source: Author.

Scanning Electron Microscopy (SEM)

Day 9 (n=5/genotype) resin embedded hemi-mandibles were ground using EXaKt grinding machine and Hermes sanding discs (1000 grit) to expose the mid point of the mouse first molar in the sagittal plane. A final polish was completed with a 1200/4000 silicon carbide disc. The samples were etched with 10% phosphoric acid for 15 sec, rinsed with distilled water, and dried for 24 hours. Samples were mounted on SEM stubs and coated with gold-palladium (DESK II denton vacuum sputter coater) following the operating procedure supplied with the machine.

The samples were processed for secondary electron images using the XL30 SEM system (FEI,

Hillsboro Oregon) operating at 10 kV as previously described [57, 87]. Images were captured using Quartz PCI-Image Management System (Quartz Imaging Corporation, Vancouver, BC,

Canada) at a scanning power of 50-60X and at 1200X.

Histology and Immunohistochemistry

Day 1, 5, and 9 mouse heads (n=3/genotype/time point) were fixed in 4% paraformaldehyde

(PFA) in phosphate-buffered saline (PBS; pH 7.2) for at least 24 hours. The right and left hemi-mandibles were micro-dissected and demineralized at 4°C in 12.5% EDTA (pH 7.0), with rocking for 5, 7 or 8 days, for day 1, 5 and 9 day old specimens, respectively, with daily change of solution. After washing overnight in PBS, tissues were dehydrated through graded ethanols, cleared with xylene, and embedded in paraffin. The tissue was sectioned at 5 microns thickness and stained with hematoxylin and eosin (H&E), and tartrate resistant acid phosphatase (TRAP) stain, as previously described [88].

Immunohistochemical staining was performed on deparaffinized sections using the EnVision+ system (DAKO, Carpinteria, CA, USA) as previously described [57, 58], and counterstained with light green. The following primary rabbit antibodies were used: anti-a3 [89] (1:1000 dil), anti-amelogenin (Abcam, Cat.# 59705, 1:500; Abcam, Cambridge, MA USA), anti-amelotin

(1:1,500) and anti-ODAM (1:10,000) [57].

Histological and immunohistochemical images were obtained using a Leica DM2500 microscope, equipped with a DFC320 camera and Application Suite 4.4.0 (Build:454) software.

3.4 Results

Dental formation and eruption is impaired in the R740S/R740S mouse

To characterize dental eruption in mice with the R740S mutation, demineralized mandibles were assessed by histology. H&E staining of coronal sections of day 1, 5, and 9 postnatal mouse molars (Figure 7) showed distortion and compression of the molar crown in the

R740S/R740S mouse and a blunting of root development. A significant disruption in the formation of the anterior tooth in the R740S/R740S mouse relative to the other two genotypes was also observed (Figure 7). Tartrate-resistant acid phosphatase (TRAP) staining showed a slight increase in the number of osteoclasts in +/R740S mandibles compared to +/+, and a dramatic increase in R740S/R740S animals. This is consistent with the osteoclast-rich osteopetrotic phenotype; similar findings were observed in the bones of oc/oc and a3 knockout animals.

Figure 7. Odontogenesis is impaired in R740S/R740S osteopetrotic mice.

H&E and TRAP staining of mouse mandibles on days 1, 5, and 9 postnatal. Sclerotic bone encases the tooth germ in the R740S/R740S constricting the growth and development of the molar and the anterior tooth (arrow), progressing from day 1 to day 9 when compared to the other genotypes. The red TRAP staining highlights the slight increase in the +/R740S, and considerable increase of osteoclast population in R740S/R740S mandibles. M = molar, A = anterior incisor.

The enamel in the R740S/R740S mouse is hypoplastic and hypomineralized

To characterize whether a mutation in the a3 subunit of V-ATPase had an effect on enamel thickness and mineralization, mandibles were analyzed using µCT. As expected, µCT showed increased trabecular volume in the R740S/R740S mandible (Figure 8A) consistent with the osteopetrotic phenotype [35]. Sagittal and coronal images of day 5 and 9 postnatal mouse first molars (Figure 8A) confirmed deformation and compression of the molar crown shape and blunting of root development in R740S/R740S animals. To quantify the enamel properties, day

9 samples (the most highly mineralized dental enamel in our samples) were used. Due to the aberrations present in the anterior tooth of the R740S/R740S animals, the analysis was performed using the first molar. The cusp tip of the molar was selected as an area of interest, since maturation of enamel begins in this region, and thus will represent the most highly mineralized site in our mice [46]. Quantification showed that the enamel mineralization in the

R740S/R740S animal was significantly decreased compared to the other two genotypes (Figure

8B).

To measure the thickness of the enamel, the inner cuspal plane of the molar was selected (this region should be unimpeded by the compressive effects of the bone in the R740S/R740S animals). The analysis showed that the enamel thickness was also significantly decreased in the R740S/R740S molar compared to the other two genotypes (Figure 8C). There was no statistical difference in enamel mineralization or enamel thickness between +/R740S and +/+ molars.

Figure 8. R740S/R740S mice have hypomineralized and hypoplastic enamel.

(A) Micro CT images of sagittal and coronal views of the molar region of the mandible. The R740S/R740S molar (arrows) is distorted and compressed within dense bone compared to the +/+ and +/R740S molars within the thin, expansive buccal and lingual plates of bone. (B,C) Quantification of enamel mineralization (B) and enamel thickness (C); region of interest used for quantification is highlighted in (A); * indicates significance, p<0.05.

To further evaluate the enamel, samples were examined using scanning electron microscopy

(SEM). If amelogenesis was significantly disrupted, a defect in the characteristic prismatic structure of the enamel should be easily observed. At low magnification, the sagittal view of the R740S/R740S mandible (Figure 9) showed the abundant sclerotic bone and severely deformed anterior tooth, confirming previous observations. High magnification day 9 enamel images demonstrated lack of any discernable mineral structures in the R740S/R740S animals compared to early signs of mineralization observed in both +/R740S and +/+ mice (Figure 9), therefore, qualitatively showing a decrease in enamel mineralization. Later time points were not possible to obtain due to early lethality of R740S/R740S animals (between postnatal days

10-14).

Figure 9. Enamel rod organization is delayed in R740S/R740S enamel.

SEM images of day 9 mandibles. (Left panels) Sagittal view; the presence and severe distortion of the R740S/R740S anterior tooth within the dense sclerotic mandibular bone can be easily observed; 50X magnification. (Right panel) 1200X magnification; early formation of ordered enamel rods in the +/+ and R740S/+ enamel is highlighted by the arrows, while R740S/R740S enamel lacks any signs of this organized enamel rod formation. M1 = first molar, M2 = second molar, A = anterior incisor, b = bone, e = enamel, d = dentin.

Expression of enamel matrix proteins is altered in R740S/R740S mice

SEM and µCT showed hypoplastic and hypomineralized enamel in the R740S/R740S animals.

Hypoplastic enamel is associated with the secretory phase of amelogenesis, while hypomineralization of the enamel is a result of a defect in the maturation stage. To determine if these two stages of amelogenesis were affected by R740S mutation, we examined expression of secretory and maturation stage enamel matrix proteins by immunohistochemistry (IHC). IHC evaluation of amelogenin (Figure 10A), demonstrated that all three genotypes expressed amelogenin at days 1 and 5. By day 9, amelogenin appeared to decrease in both the +/ + and

+/R740S ameloblasts, but remained detectable within the cytoplasm of the R740S/R740S cells, suggesting delayed transition to the maturation stage. Day 9 ameloblasts from all three genotypes expressed amelotin and ODAM, the enamel matrix proteins secreted during the maturation stage, although the R740S/R740S ameloblasts appeared to have less intense and more diffuse cytoplasmic staining (Figure 10B).

Figure 10. Enamel matrix protein expression is affected in R740S/R740S ameloblasts. Immunohistochemistry of day 1, 5, and 9 samples. (A) Amelogenin expression (occurring mainly during the secretory stage of amelogenesis). In +/+ and +/R740S samples, amelogenin was expressed at day 1 and day 5, and tapered off by day 9; in R740S/R740S ameloblasts, amelogenin expression was still present on day 9 (arrows). (B) Amelotin and ODAM expression (occurring mainly during the maturation stage of amelogenesis) in day 9 samples. Signals for both proteins in the R740S/R740S ameloblasts were less intense compared to the +/+ and +/R740S cells. Am = ameloblast.

Next, to verify whether the ameloblasts expressed the V-ATPase a3 subunit and if the aberrant enamel matrix protein secretion in R740S/R740S cells was related to the V-ATPase mutation, sections were stained using anti-a3 antibody. IHC confirmed the presence of a3 in secretory ameloblasts in all three genotypes (Figure 11), supporting previous observations that ameloblasts express low levels of a3 [33, 36].

Figure 11. The a3 subunit of V-ATPase is expressed in day 5 but not in day 9 ameloblasts.

Immunohistochemistry of day 5 and day 9 first molar ameloblasts of the mesial lingual cusp. Cytoplasmic a3 expression in the ameloblasts of all three genotypes can be observed in ameloblasts at day 5, but not at day 9. High intensity staining for a3 can be seen in the osteoclasts (arrows), confirming the specificity of the antibody. Am = Ameloblast.

3.5 Discussion

Severe dental anomalies have been observed in patients with osteopetrosis. If the disease- causing mutation affects the bone resorbing osteoclasts, hematopoietic stem cell transplantation

(HSCT) is the only available treatment. It restores osteoclast function as well as tooth eruption; however, the enamel defects present in these patients do not appear to be rescued by the HSCT.

This observation suggests that these mutations also affect non-hematopoietic cells, such as ameloblasts, which will not be restored by this form of therapy.

Ameloblasts are the cells responsible for enamel formation; a multi-stage process divided into presecretory, secretory, transition, and maturation stages. Any disruptions in the phases of amelogenesis result in hypoplastic and/or hypomineralized enamel. Here we present evidence that mouse ameloblasts express the a3 subunit of V-ATPase. Additionally, osteopetrotic mice homozygous for the a3 R740S mutation have hypoplastic and hypomineralized enamel due to delayed transition from secretory to maturation stage of amelogenesis.

The a3 containing V-ATPases are thought to be osteoclast-specific because of their high levels of expression in these cells, and are required for proper bone resorption. In the R740S mouse model, the a3 protein is expressed at +/+ levels but is not functional, resulting in severe osteopetrosis in R740S/R740S and mild disease in +/R740S animals. R740S/R740S mice have eruption defects in both the molar and anterior teeth as shown by histology, µCT, and SEM

(Figures 7-9), confirming the phenotype observed in the oc/oc (a3 truncation) and a3 knockout mouse models [33, 36]. It was interesting to note that the +/R740S animals displayed normal tooth formation and eruption, demonstrating that 50% of the V-ATPase function is sufficient for odontogenesis.

µCT and SEM showed that the enamel in R740S/R740S animals was hypoplastic and hypomineralized, supported by previous findings of reduced enamel mineralization in oc/oc mice [33]; however, enamel thickness was unaffected in the oc/oc animals. Analysis of spatiotemporal expression of enamel matrix proteins (amelogenin, amelotin and ODAM) demonstrated that the hypomineralized and hypoplastic enamel observed in R740S/R740S mice was due to a delay in transition to the maturation stage of amelogenesis. Ameloblasts also expressed a3 protein as determined by ICH, confirming previous observations by Lacruz et al.[58]. But the connection between a V-ATPase mutation and enamel protein secretion remains unclear.

V-ATPases are ubiquitous proton pumps present in every cell and are responsible for acidification of intracellular and extracellular compartments, including endosomes, lysosomes, and secretory vesicles. They are known to play key roles in numerous functions, such as protein degradation, receptor recycling, and intracellular vesicular trafficking [85]. In neurons, for example, V-ATPases maintain a specific secretory vesicle pH that is necessary for vesicle exocytosis [90]. Enamel matrix proteins are also secreted out in secretory vesicles [91], therefore, V-ATPases may also be involved in enamel protein transport and secretion during amelogenesis. Our results show that a3 protein expression could be detected in the ameloblasts of all three genotypes. Furthermore, the R740S mutation affects vesicular acidification [89]; therefore, it is possible that the increased vesicular pH in the R740S/R740S ameloblasts interfere with matrix protein transport and secretion, resulting in the observed hypoplastic and hypomineralized enamel.

Osteopetrosis due to a3 mutations have been traditionally described as a disease affecting only osteoclasts. It has been shown previously [92] that acid-producing parietal cells of the stomach

56 also express a3-containing V-ATPases and play a role in stomach acidification and calcium absorption. Here we show that the ameloblast is another cell type affected by the osteopetrotic

V-ATPase a3 R740S mutation. These results suggest that bone marrow transplantation, the only treatment for osteopetrosis, while rescuing osteoclast function and dental eruption, would not have a significant impact on enamel properties. Since currently there is no therapy for enamel regeneration or repair, preventative measures are the only treatment for caries control in patients with osteopetrosis and osteopetrorickets.

4 Discussion

Dental caries is a significant finding in patients who suffer from osteopetrosis.

Determining the etiology of this high caries rate is often difficult due to the rarity of the disease, the lack of case reports that adequately describe the status of the dental enamel, and the confounding variable of poor oral hygiene. Defects in enamel, including both hypoplasia and hypomineralization are thought to provide a favourable environment for cariogenic bacteria to adhere and colonize, thus contributing to the increased susceptibility to dental decay [93].

Therefore, the increased caries rate noted in these patients who suffer with osteopetrosis, may be associated with a deficiency in amelogenesis. In order to investigate the effects of osteopetrosis on amelogenesis, we used an osteopetrotic mouse model with a point mutation

(R740S) in the a3 subunit of a V-ATPase proton pump. This mutation results in decreased osteoclastic bone resorption [35]. We were able to demonstrate low levels of a3 expression in the secretory stage ameloblasts in all three genotypes, supporting our hypothesis that the a3 containing V-ATPase pump may play a role in amelogenesis. These findings do not agree with the a3 levels reported during the maturation stage in genome wide RNA profiling studies, by

La Cruz et al [68]. Although, given the early stage of maturation that was reached by day 9 in our mouse model, we may not have reached the stage of up-regulation noted in that study.

Using micro CT data, we were able to demonstrate quantitatively that the enamel in the

R740S/R740S mouse was both hypoplastic and hypomineralized relative to the +/+ and the

+/R740S molars. A finding of decreased mineralization is in keeping with the observation in the oc/oc mouse molar, although no decrease in enamel thickness was found in that study [32].

SEM evaluation of the enamel confirmed a lack of enamel crystals in the R740S/R740S enamel

58 relative to early crystallization at day 9 noted in the +/+ and +/R740S enamel (Figure 9).

Furthermore, continued presence of amelogenin in the homozygote ameloblasts, coupled with a decrease in staining intensity in the maturation stage proteins, ODAM and amelotin, indicate a delay in the transition to the maturation stage. This delay in amelogenesis could account for both the hypoplastic as well as the hypomineralized enamel found in the R740S/R740S mouse.

V-ATPases are involved in cellular processes such as protein transport and secretion. It has been shown that the internal pH of secretory granules, as well as the physical presence of the Vo portion of the V-ATPase pump, play an important role in membrane fusion during exocytosis [90]. More specifically, in neurons, V-ATPases containing the a1 subunit maintain a specific secretory vesicle pH that is necessary for vesicle exocytosis[90]. Since enamel matrix proteins are secreted via secretory granules along distinct sites of Tomes processes [91], a3-V-ATPase could play a key role in this process. A defective a3-V-ATPase could potentially affect the ability of secretory vesicles to fuse to the plasma membrane and secrete adequate quantities of enamel matrix proteins, resulting in hypoplastic enamel.

V-ATPases, containing a1 have already been shown to be present at the ruffled border of the maturation stage ameloblast, pumping hydrogen ions into the extracellular space, potentially playing a critical role in pH balance [53]. During the maturation stage, enamel matrix proteins are digested by proteases and replaced with more highly mineralized enamel.

The major proteolytic enzyme, Kallikrein 4 (KLK4) is likely sensitive to the pH in the extracellular environment. KLK-4 knock-out mice have enamel of appropriate thickness, but the normal digestion of the enamel matrix proteins is impeded, resulting in rod and interrod enamel unable to properly lock into its final crystalline structure [39, 60]. Also critical to the maturation process, is the final breakdown of the enamel matrix proteins. This is accomplished

59 by endocytosis of the proteinaceous debris, followed by lysosomal degradation within both the ameloblasts and papillary layer cells [58]. Lysosomes degrade internalized molecules within an acidic pH of 4.5, generated by V-ATPases [94]. The importance of lysosomes in amelogenesis is highlighted by the increase in expression of lysosomes and lysosomal enzymes during the maturation stage [61]. Our lab demonstrated that lysosomal pH is increased in the

+/R740S osteoclasts [35], therefore higher lysosomal pH in the maturation stage ameloblast may contribute to enamel hypomineralization by preventing intracellular degradation of the enamel matrix proteins.

Since amelogenesis is an extremely complex process requiring the co-ordination of enzymes, pH levels, and ion transport, it is possible that other aspects of the osteopetrotic phenotype affecting the R740S/R740S mouse may also be contributing to the observed enamel aberrations. For example, the R740S/R740S mouse suffers from hypocalcemia, likely due to a reduction in calcium uptake in the gut (secondary to hypochlorhydria caused by a defective a3-

V-ATPase pump within the acid producing parietal cells), as well as an imbalance in calcium homeostasis, with more calcium remaining sequestered within the bone. There is correlation between hypocalcemia and hypoplasia of the dental enamel. Studies of rats raised on a low calcium diet demonstrated decreased enamel volume, as well as increased levels of organic matrix noted within the intercrystal space, and a disruption of the basal lamina beneath the mature ameloblasts [95]. Furthermore decreased production of amelogenin was noted within the secretory ameloblasts [95]. Hypoplastic defects have also been reported in rats with severe hypocalcemia induced by thyro-parathyroidectomy [96]. Besides playing a role in mineral formation, calcium is important for proper enzyme function. During the secretory stage, amelogenin and the non-amelogenins are heavily processed in the enamel microenvironment by a specific matrix metalloprotease, MMP20. This protease has been shown to be sensitive to

60 calcium levels [59, 60]. It is possible that disruptions in MMP20 activity may affect post- translational processing of amelogenin, leading to an overall decrease in the thickness of the dental enamel, as is seen in the MMP20 null mouse [39]. There is much less correlation between hypocalcemia and hypomineralized enamel, despite the fact that there is a 3-4 fold increase in the calcium transport across and between the ameloblasts during the maturation phase [56]. The majority of the calcium within the enamel microenvironment is bound to enamel matrix proteins, acting as a calcium reservoir. The concentration of free calcium ion required for adequate enamel mineralization is actually 6-10 fold lower than the serum [73].

This calcium reservoir may account for the tolerance for low circulating calcium levels noted in the maturation stage. Therefore, the hypocalcemia present in the R740S/R740S mouse may contribute to the observed enamel hypoplasia. The resulting disruption in the secretory stage, may in turn contribute to a lag in the transition to the maturation stage, accounting for the lack of early mineralization in the R740S/R740S relative to the other two genotypes.

Another factor contributing to the enamel defects in the R740S/R740S mouse may be the effects of the sclerotic buccal and lingual alveolar bone encircling the developing tooth. The generalized pallor of the day 9 R740S/R740S mandible in comparison to the other genotypes

(Figure 12) highlights the lack of blood perfusing the sclerotic bone. Dental aplasia noted in osteopetrotic patients is thought to be due to a lack of nutrients from a compromised blood supply, resulting in the involution of the developing tooth germ [51, 82]. Therefore, a decrease in oxygen and nutrients due to an inadequate blood supply may also affect ameloblast efficiency and contribute to the observed delay in amelogenesis.

Figure 12 – Macroscopic appearance of Day 9 Mandible

Gross dissection of all three genotypes. The arrows highlight the anterior incisor in the +/+ and the +/R740S mandibles. Source: Author.

Further studies are required to demonstrate a stronger causal relationship between the presence of a mutation in the a3-containing V-ATPase and a defect in amelogenesis. Follow up work to further support our conclusions would be to quantify the expression of the enamel matrix proteins using Western Blot analysis [97] during both the secretory and the maturation stage. Using enhanced imaging techniques such as confocal microscopy and immunogold

TEM, would allow us to confirm protein expression within the ameloblast. Furthermore, co- localization studies of a3 with other enamel matrix proteins using immunogold TEM could help to elucidate a possible mechanistic role a3 containing V-ATPases in amelogenesis.

A significant challenge with the Tcirg1R740S/R740S mouse model is the early lethality of the R740S/R740S mouse, which prevents a thorough analysis of the maturation stage of amelogenesis and the quality of the mature enamel. To circumvent this problem, an ex vivo culturing technique could be employed. This technique involves the transplantation of mutant tooth germs, encased within their mandibular bone, into kidney capsules of wild type littermates. Using this model, these “explants” are able grow and produce mineralized material

62 equivalent in morphology and mineralization to dental enamel [98]. This technique would allow for evaluation of more mature dental enamel, as well as removing the effects of any systemic factors that may be confounding our results in the R740S/R740S mouse. Another strategy would be to create an ameloblast specific Tcirg1 conditional knock out mouse in order to study the role of a3-V-ATPase during each stage of amelogenesis. This advantage of this technique would be the presence of the anterior incisor, absent in the global Tcirg1 knockout, but present in the ameloblast-specific Tcirg1 conditional knock out. The continually erupting incisor is an established and ideal model for the study of amelogenesis, as it allows for the observation of each developmental stage of the ameloblast simultaneously.

Additionally, functional studies could further elucidate a potential role of a3-VATPase in amelogenesis. To decipher molecular pathways, an ameloblast cell line could be transfected with Tcirg1 carrying the R740S mutation. There are two ameloblast-like cell lines: the LS8 cell line, considered to be representative of the secretory stage of amelogenesis; and the ALC cell line, which has been used as a model of the maturation stage of amelogenesis [97].

Tranfecting these cell lines, with WT or R740S Tcirg 1 will allow us to examine the role of a3

V-ATPase in enamel matrix protein expression, secretion and trafficking.

5 Conclusion

In conclusion, aberrations in dental eruption of both the first molar and the anterior incisor were noted in the osteopetrotic Tcirg1R740S/R740S mouse model. Secondly, enamel hypoplasia and hypomineralization was noted in the day 9, Tcirg1R740S/R740S molar due to a delay in transition from the secretory to the maturation stage, possibly secondary to defective a3-V-ATPases noted in the secretory phase.

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