The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein highly expressed by odontoblasts in teeth. DSPP mutations in humans may cause dentinogenesis imperfecta (DGI), an autosomal dominant dentin disorder. We recently generated a mouse model (named “ Dspp^P19L/+ mice”) that expressed a mutant DSPP in which the proline residue at position 19 was replaced by a leucine residue. We found that the Dspp^P19L/+ and Dspp^P19L/P19L mice at a younger age displayed a tooth phenotype resembling human DGI type III characterized by enlarged dental pulp chambers, while the teeth of older Dspp^P19L/+ and Dspp^P19L/P19L mice had smaller dental pulp chambers mimicking DGI type II. The teeth of Dspp^P19L/+ and Dspp^P19L/P19L mice had a narrower pulp chamber roof predentin layer, thinner pulp chamber roof dentin, and thicker pulp chamber floor dentin. In addition, these mice also had increased enamel attrition, accompanied by excessive deposition of peritubular dentin. Immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction analyses showed that the odontoblasts in both Dspp^P19L/+ and Dspp^P19L/P19L mice had reduced DSPP expression, compared to the wild-type mice. We also observed that the levels of DSPP expression were much higher in the roof-forming odontoblasts than in the floor-forming odontoblasts in the wild-type mice and mutant mice. Moreover, immunohistochemistry showed that while the immunostaining signals of dentin sialoprotein (N-terminal fragment of DSPP) were decreased in the dentin matrix, they were remarkably increased in the odontoblasts of the Dspp^P19L/+ and Dspp^P19L/P19L mice. Consistently, our in vitro studies showed that the secretion of the mutant DSPP was impaired and accumulated within endoplasmic reticulum. These findings suggest that the dental phenotypes of the mutant mice were associated with the intracellular retention of the mutant DSPP in the odontoblasts of the DSPP-mutant mice.

Background: Ameloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel‐specific with respect to its essential functions. Biallelic AMBN defects cause non‐syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin.Methods: We characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS‐lacZ (Ambn^lacZ/lacZ) knockin mice.Results: No AMBN protein was detected using immunohistochemistry in null mice. ß‐galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. Ambn^lacZ/lacZ 7‐week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the Ambn^lacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the Ambn^lacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS‐lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X‐gal.Conclusions: Ameloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

Mutations in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta. After synthesis, DSPP is proteolytically processed into NH₂- and COOH-terminal fragments. The NH₂-terminal fragment of DSPP is highly glycosylated but not phosphorylated, whereas the COOH-terminal fragment (named “dentin phosphoprotein” or “DPP”) is highly phosphorylated but not glycosylated. These two fragments are believed to perform distinct roles in dentin formation. To analyze the functions of DPP in dentinogenesis, we created “Dspp^-/-;DPP Tg mice”, which expressed transgenic DPP driven by a Type I collagen promoter but lacked the endogenous Dspp gene. We characterized the dentin of the Dspp^-/-;DPP Tg mice using X-ray radiography, histology, scanning electron microscopy, double fluorochrome labeling, immunohistochemistry and in situ hybridization. Micro-computed tomography analyses revealed that at postnatal 6 months, the transgenic expression of DPP increased the dentin thickness of the Dspp-null mice by 97.1% and restored the dentin material density by 29.5%. Histological analyses showed that the Dspp-null mice manifested an abnormal widening of the predentin while the predentin in Dspp^-/-;DPP Tg mice was narrower than in the Dspp-null mice. Scanning electron microscopy analyses showed that the dentinal tubules in the Dspp^-/-;DPP Tg mice were better organized than in the Dspp-null mice. The double fluorochrome labeling analyses demonstrated that the dentin mineral deposition rate in the Dspp^-/-;DPP Tg mice was significantly improved compared to that in the Dspp-null mice. These findings indicate that the transgenic expression of DPP partially rescued the dentin defects of the DSPP-null mice, suggesting that DPP may promote dentin formation and that the coordinated actions between DPP and the NH₂-terminal fragment of DSPP may be necessary for dentinogenesis.

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5’ mutations affecting targeting and trafficking, and 3’ − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of Dspp^P19L and Dspp^−1fs mice that replicate the two categories of human DSPP mutations. In Dspp^P19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp^−1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of Dspp^P19L and Dspp^−1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp^−1fs mice may be valuable for the study of autophagy and ER-phagy.