Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

Abstract

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre ( iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X ( Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 ( Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice ( Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14- Cre/ Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Calcium Sets the Clock in Ameloblasts

“…with increased attrition and thinner enamel crystallites. This phenotype is not unsimilar to the enamel phenotype observed in other Stim1 cKO mice (Eckstein et al, 2017;Furukawa et al, 2017;Said et al, 2019), suggesting that TGF-β1 dysregulation might be partly involved in the development of Stim1 deletion-caused AI. On the other hand, Mapk14 expression was significantly upregulated in Stim1 deficient ameloblasts.…”

“…Stim1 cKO generation and characterization were previously described ( Said et al, 2019 ). Animals were housed in a pathogen-free facility in the Lab Animal Service Unit (LASU) at the University of Saskatchewan and all procedures for this study were authorized by the University Animal Care Committee (UACC) under an approved protocol (#20170014).…”

“…However, the molecular mechanisms involved in regulating the circadian clock during amelogenesis remain largely unexplored. In this study, we aimed to investigate the effects of calcium disruption on the ameloblasts molecular clock in vivo using a unique Stim1 fl/fl /Amelx-iCre +/+ (Stim1 cKO) model (Said et al, 2019). The significant impact of Ca 2+ and circadian clock in ameloblasts makes amelogenesis an excellent model system for deciphering the mechanistic links that may exist between intracellular calcium dynamics and molecular circadian clock.…”

“…The importance of SOCE in regulating calcium transport during enamel mineralization is clearly reflected in the fact that patients with mutations in either STIM1 or ORAI1 have severe dental enamel defects, characterized as hypocalcified Amelogenesis Imperfecta (AI) ( Eckstein and Lacruz, 2018 ; Feske, 2019 ). Moreover, several in vivo and in vitro studies have shown that SOCE components are robustly expressed by ameloblasts and served as key modulators of ameloblast’s differentiation and function ( Nurbaeva et al, 2015a , b ; Zheng et al, 2015 ; Eckstein et al, 2017 ; Furukawa et al, 2017 ; Said et al, 2019 ).…”

Principles of Genetic Engineering

“…Because of their size, BACs often carry all of the genetic regulatory elements to faithfully recapitulate the expression of genes contained in them, and thus can be used to generate BAC transgenic mice [19,20]. Recombineering can be used to insert reporters in BACs that are then used to generate transgenic mice to accurately label cells and tissues according to the genes in the BACs [21][22][23][24][25][26]. A panoply of approaches to genetic engineering are available for researchers to manipulate the genome.…”