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PURPOSE: G protein-coupled receptor 155 (GPR155), which is significantly upregulated in maturation stage ameloblasts as compared to secretory stage ameloblasts, has not been previously explored in the context of amelogenesis. This study aims to determine the role of GPR155 in enamel maturation, and specifically its involvement in trans-ameloblast calcium transport.METHODS: To profile the expression pattern of Gpr155 in the progressively advancing ameloblasts, we collected enamel organ cells from P5, P9, P11, P12, and P13 wild type mouse first molars, providing ameloblasts at the secretory stage, transition stage, early maturation and late maturation stage of development. RNA was extracted from these ameloblasts and the expression of Gpr155 was quantified via RT-qPCR. The alteration of GPR155 expression in Nckx4-/- ameloblasts compared with Nckx4+/+ ameloblasts was analyzed by qPCR and immunostaining. Mice with loss-of-function of NCKX4 (potassium dependent sodium/calcium exchanger 4) developed hypocalcified and hypomature enamel, which resembles the defective enamel seen in humans harboring NCKX4 gene mutations. We further used RT-qPCR to characterize the expression of Orai2 and Trpm7, which have been previously characterized to contribute to the trans-ameloblast calcium transport. The correlation between the expression of GPR155 and enamel matrix calcification was also analyzed in the lactating mouse enamel organs.RESULTS: Among these progressively differentiating ameloblasts, Gpr155 expression started to increase in the transition stage, then reached to its climax in the middle of maturation stage, and slightly reduced at the end of maturation stage. There was a significant increase (p

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation, resulting in a hypomaturation AI phenotype, to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed that Wdr72−/− ameloblasts were shorter, and the enamel matrix was retained during maturation stage. H+/Cl− exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72−/− maturation-stage ameloblasts. Other markers along the endocytic degradative pathway were disrupted in Wdr72−/− mice, including clathrin, Dynamin II, and ANXA8. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72−/− mice as compared to wildtype and heterozygous controls. Moreover, Wdr72−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. In vivo and in vitro tracer studies with HRP and amelogenin showed delayed processing and loss of transport to lysosomes through the degradative pathway in our newly developed Wdr72−/− ameloblast-like cell line using CRISPR/Cas9. These cells enabled live cell mechanistic studies targeting WDR72 function in vesicle acidification and microtubule recruitment, revealing a complex relationship with a major vesicle acidifying protein, vacuolar H+-ATPase (v-ATPase), and a link to microtubule recruitment. Ultrastructure of Wdr72−/− ameloblasts also showed faulty vesicle formation and ruffled border formation that coincided with pH and calcium matrix defects. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins by regulating microtubule assembly and membrane turnover in the degradative pathway.

Family with sequence similarity 20 (FAM20) consists of three members: FAM20A, FAM20B and FAM20C. Mutations in FAM20 family have been linked to developmental disorders involving bones, cartilage and teeth. FAM20A mutations in humans are associated with amelogenesis imperfecta with gingival fibromatosis and enamel renal syndrome. Fam20a knockout (KO) mouse showed growth retardation. The aim of this study was to characterize the skeletal and dental phenotypes using Fam20a KO mouse. Our results showed that body size and bone length of KO mice were smaller than those of WT. The microcomputed tomography (μCT) analyses of trabecular and cortical bones in KO displayed lower bone volume, thinner trabeculae and thinner bone cortex as compared to WT. Histological examination of KO growth plate demonstrated disorganized chondrocyte zones and extended hypertrophic zone. qRT-PCR results showed downregulation of several osteoblast differentiation markers in KO long bone. Immunohistochemical examination demonstrated reduced chondrocyte proliferation, apoptosis and increased collagen X expression in KO growth plate. Our data showed a lower number of osteoblasts and osteoclasts in KO as compared to WT. In vitro study, Fam20a KO showed a lower number of bone marrow stromal cells and osteoprogenitors. In vitro mineralization was impaired in KO osteoblasts. Fam20a KO had hypoplastic enamel, delayed tooth eruption and gingival overgrowth. The µCT results demonstrated that enamel in Fam20a KO was not fully mineralized and enamel matrix was detached from dentin. Scanning electron microscopy displayed absence of decussation patterns in Fam20a KO enamel. Histological examination of maxillary first molar at differentiation stage showed no difference between WT and KO. At the secretory stage, Fam20a KO ameloblasts were short and non-polarized as compared to WT. Immunohistochemical analysis showed diffuse staining pattern of amelogenin in Fam20a KO first molar compared to WT. Western blot analysis demonstrated that amelogenin proteolytic process was impaired in KO and showed slower migration pattern of MMP20. In conclusion, endochondral ossification defects and reduced number of osteoblasts and their precursors led to the bone phenotype in Fam20a KO. Amelogenin processing defects caused amelogenesis imperfecta phenotype in KO. Our study indicated that Fam20a plays a role in skeletal development and amelogenesis.

For the past decades, tooth development has been extensively studied as a model for understanding organogenesis of ectoderm-derived structures. Much has been learned from the morphological patterning and molecular signaling of normal tooth development in model organisms, mainly rodents. On the other hand, human inherited dental anomalies also provide a valuable source for studying tooth development. Discerning the genetic etiology of these developmental defects not only enhances our understanding of normal tooth development but also provides a fundamental basis for developing potential therapeutic strategies for these disorders. Familial tooth agenesis (FTA) and amelogenesis imperfecta (AI) are the two most prevalent inherited dental defects in humans, characterized by failed tooth development and dental enamel malformations respectively. In this dissertation research, we described 7 FTA and 12 AI families and aimed to define the genetic etiology of the diseases through mutational analyses. By using target gene approaches and whole exome analyses, we successfully identified the disease-causing mutations in 2 FTA families and in all the AI cases. The results not only expanded the mutation spectrum of known disease-associated genes but also established novel candidate genes, revealing critical players in tooth and enamel development. Nevertheless, although human genetic studies of inherited dental and enamel defects have revealed many genes associated with the diseases, the functions of many of these genes and their roles in normal development and pathological conditions remain to be elucidated. Recently, mutations in FAM83H (family with sequence similarity 83, member H) were identified to cause autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). Although many disease-causing FAM83H mutations have been reported, the cellular functions of this gene and the pathogenesis of its associated enamel defects are completely unknown. In this study, we used a biochemical approach to study FAM83H protein-protein interactions and identified CK1 (casein kinase 1) and SEC16A as FAM83H interacting proteins, which indicated a potential cellular function of FAM83H in vesicle trafficking and protein transport. The results also provided an explanation for the high mutation homogeneity of FAM83H disease-causing mutations revealed by human genetic studies and suggested a potential pathological mechanism for FAM83H-associated enamel defects.

Purpose: The purpose of this study was to assess restorative treatment outcomes in the mixed dentition of patients with amelogenesis imperfecta (AI), and to determine post-rehabilitation oral health status and satisfaction of the patients. Methods: Seventy-four restorations placed in permanent incisors and molars in eight subjects with AI met the inclusion criteria. Clinical and radiographic examinations were performed to evaluate the integrity of the restorations and periodontal status post-treatment. Subjects completed a survey regarding esthetics, function, and sensitivity. Results: Among the 74 restorations evaluated, 7 restorations were lost and restorations present comprised of 31 posterior restorations and 36 anterior restorations. Ten were rated clinically unacceptable. Teeth with SSC had moderate gingival index (mean=2.3) and plaque index (mean=2.0) scores. Widening of the periodontal ligament and pulp canal obliteration were common radiographic findings. Subject satisfaction regarding esthetics (P=.002) and sensitivity (P=.03 brushing; P=.01 eating) showed a statically significant difference before and after restorative treatment. Conclusions: During mixed dentition, teeth with AI may be restored with conventional treatment modalities. Direct restorations should be considered “interim” and multiple repairs have to be expected. Post-treatment, gingival inflammation and plaque accumulation were observed. Subjects were satisfied with their appearance and reported a decrease in sensitivity.