Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.

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.

The human laminin family is composed of five α, four β, and three γ chains. Laminins are heterotrimers of α, β, and γ chains. Laminins play critical roles during organogenesis, mostly as basement membrane components. The expression of all and the localization of most laminin chains were characterized in mouse developing teeth. Primary laminin isoforms in basement membranes along the inner enamel epithelium before the secretory stage and outside of the outer enamel epithelium were laminins 111 (α1β1γ1) and 511. The mouse laminin α3 chain has two variants, α3A and α3B. Although a basement membrane structure is absent, laminin 3A32 was localized along the secretory surface of the secretory stage ameloblast Tomes’ processes. Laminin 3A32 was localized along the atypical basement membrane of maturation stage ameloblasts and the specialized basement membrane of junctional epithelium facing the enamel surface. The endothelial basement membrane in the dental papilla and outside of the enamel organ contained laminins 411 and 511. Laminin 332 was detected in the extracellular matrix but not the basement membrane of the apical loop. Laminin 111 was localized in the extracellular matrix of the apical dental papilla without forming a visible basement membrane. These findings suggest the multifaceted functions of laminins in tooth development and set the foundation for functional investigations.

Research on how a stratified oral epithelium gained the capability to create the hardest hydroxyapatite-based mineralized tissue produced biologically to protect the surfaces of teeth has been ongoing for at least 175 years. Many advances have been made in unraveling some of the key factors that allowed the innermost undifferentiated epithelial cells sitting on a skin-type basement membrane to transform into highly polarized cells capable of forming and controlling the mineralization of the extracellular organic matrix that becomes enamel. Genetic manipulation of mice has proven to be a useful approach for studying specific events in the amelogenesis developmental sequence but there have been pitfalls in interpreting loss of function data caused in part by conflicting literature, technical problems in tissue preservation, and the total amount of time spent on tooth development between different species that have led to equivocal conclusions. This critical review attempts to discuss some of these issues and highlight the challenges of characterizing amelogenesis in gene-targeted mouse models.

The sodium–citrate cotransporter (NaCT) plays a crucial role in citrate transport during amelogenesis. Mutations in the SLC13A5 gene, which encodes the NaCT, cause early infantile epileptic encephalopathy 25 and amelogenesis imperfecta. We analyzed developing pig molars and determined that the citrate concentrations in secretory- and maturation-stage enamel are both 5.3 µmol/g, with about 95% of the citrate being bound to mineral. To better understand how citrate might enter developing enamel, we developed Slc13a5^Flag reporter mice that express NaCT with a C-terminal Flag-tag (DYKDDDDK) that can be specifically and accurately recognized by commercially available anti-Flag antibodies. The 24-base Flag coding sequence was located immediately upstream of the natural translation termination codon (TAG) and was validated by Sanger sequencing. The general development, physical activities, and reproductive outcomes of this mouse strain were comparable to those of the C57BL/6 mice. No differences were detected between the Slc13a5^Flag and wild-type mice. Tooth development was extensively characterized using dissection microscopy, bSEM, light microscopy, in situ hybridization, and immunohistochemistry. Tooth formation was not altered in any detectable way by the introduction of the Flag. The Slc13a5^Flag citrate transporter was observed on all outer membranes of secretory ameloblasts (distal, lateral, and proximal), with the strongest signal on the Tomes process, and was detectable in all but the distal membrane of maturation-stage ameloblasts. The papillary layer also showed positive immunostaining for Flag. The outer membrane of odontoblasts stained stronger than ameloblasts, except for the odontoblastic processes, which did not immunostain. As NaCT is thought to only facilitate citrate entry into the cell, we performed in situ hybridization that showed Ank is not expressed by secretory- or maturation-stage ameloblasts, ruling out that ANK can transport citrate into enamel. In conclusion, we developed Slc13a5^Flag reporter mice that provide specific and sensitive localization of a fully functional NaCT-Flag protein. The localization of the Slc13a5^Flag citrate transporter throughout the ameloblast membrane suggests that either citrate enters enamel by a paracellular route or NaCT can transport citrate bidirectionally (into or out of ameloblasts) depending upon local conditions.