Charles E. Smith

Solute Carrier Family 13 Member 5 (SLC13A5) encodes the sodium-dependent citrate cotransporter NaCT, which mediates citrate transport across cell membranes. Pathogenic variants in SLC13A5 cause developmental and epileptic encephalopathy 25 with amelogenesis imperfecta, DEE25; OMIM #615905, a debilitating autosomal recessive disorder. To better define the role of NaCT in ameloblast function and enamel mineralization, we used CRISPR/Cas9 genome editing to generate Slc13a5^R337* knock-in mice that terminate NaCT translation at the Arg337 codon, which is homologous to the human SLC13A5^R333* variant associated with DEE25. We compared enamel phenotypes among wild-type, Slc13a5^+/+; heterozygous, Slc13a5^+/R337*; and homozygous, Slc13a5^R337*/R337* mice using light microscopy, in situ hybridization, immunohistochemistry, backscattered scanning electron microscopy (bSEM); and focused ion beam–scanning electron microscopy (FIB-SEM) with quantitative imaging of organelles and matrix. Citrate bioassays were performed on serum, long bones, such as the femur and tibia, and developing mouse first molars, including enamel organ epithelium, mineralized tooth matrix, and pulp mesenchyme, to assess citrate levels during the presecretory, secretory, and maturation stages of enamel formation. In addition, first molars collected at postnatal days 0, 3, 5, and 12 were analyzed to characterize glycolytic and TCA cycle-related metabolic signatures. Homozygous Slc13a5^R337*/R337* mice exhibited severe defects during the secretory and maturation stages of amelogenesis. Most notably, Slc13a5^R337*/R337* ameloblasts failed to develop a Tomes’ process, detached from the enamel matrix surface, and produced a thin, poorly mineralized crust on the dentin surface rather than organized enamel ribbons. Despite the absence of normal enamel deposition, ameloblasts initially appeared viable and did not become dysplastic until the late secretory stage. Cellular and subcellular analyses revealed increased secondary lysosomes and intracellular accumulation of enamel matrix proteins, consistent with impaired matrix processing or secretion. Citrate concentrations were elevated in serum and long bones at both 7 and 35 weeks of age. Citrate was elevated in secretory-stage Slc13a5^R337*/R337* molars at days 0 and 3, the enamel organ epithelium (including ameloblasts), the pulp mesenchyme (including odontoblasts), and mineralizing dentin and enamel matrices. These levels gradually declined at day 5 and into the enamel maturation stage (day 12). GC-MS-based analysis of central carbon metabolites revealed increased intracellular accumulation of citrate, malate, and pyruvate, suggesting altered energy metabolism and reduced metabolic efficiency in Slc13a5^R337*/R337* mice. Together, these findings indicate that loss of NaCT function in the ameloblasts causes citrate accumulation, which impairs hydroxyapatite formation. Consequently, only a thin, structurally defective mineral crust forms on the dentin surface, while mineral nodules develop ectopically within the maturation-stage enamel organ epithelium. We conclude that regulating citrate concentration is essential for proper appositional growth of enamel.

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.

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 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.

Smith CE, Hu Y, Richardson AS, Bartlett JD, Hu JC‐C, Simmer JP. Relationships between protein and mineral during enamel development in normal and genetically altered mice. 
Eur J Oral Sci 2011; 119 (Suppl. 1): 125–135. © 2011 Eur J Oral SciThe purpose of this study was to quantify and compare the amounts of volatiles (mostly protein) and mineral present in developing incisor enamel in normal mice and in those genetically engineered for absence of intact enamelin, ameloblastin, matrix metalloproteinase 20 (MMP20) or kallikrein‐related peptidase 4 (KLK4). Data indicated that all mice showed peaks in the gross weight of volatiles and a similar weight of mineral at locations on incisors normally associated with early maturation. Thereafter, the content of volatiles on normal incisors declined rapidly by as much as 62%, but not by 100%, over 2 mm, accompanied by increases of ∼threefold in mineral weights. Enamelin heterozygous mice (lower incisors) showed a decrease in volatile content across the maturation stage, yet mineral failed to increase significantly. Mmp20 null mice showed no significant loss of volatiles from maturing enamel, yet the amount of mineral increased. Klk4 null mice showed normal mineral acquisition up to early maturation, but the input of new volatiles in mid to late maturation caused the final mineralization to slow below normal levels. These results suggest that it is not only the amount of protein but also the nature or type of protein or fragments present in the local crystallite environment that affects their volumetric expansion as they mature.