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

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Abstract

Amelogenesis is a tightly regulated process that culminates in the formation of mature dental enamel. The identification and targeting of the molecular pathways and mechanisms regulating ameloblasts’ proliferation and differentiation is an ongoing challenge in dental research. Progress in the field of enamel biology and regeneration has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated four transgenic Cre-driver mouse lines that expresses improve Cre (iCre)-recombinase from the locus of the mouse ameloblast specific gene Amelogenin-X (Amelx-iCre) using a large 250-kb bacterial artificial chromosome (BAC) DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to extensively characterize iCre developmental pattern by LacZ gene encoding β-galactosidase enzyme activity assay and CRE protein immunohistochemistry. Two of the transgenic lines expressing a high amount of CRE protein exclusively in ameloblasts and showing developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression pattern were selected for further validation. To test the functionality of the selected transgenic models we bred the two Amelx-iCre mice lines with Stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout (cKO) mice. STIM1 serves as the main calcium (Ca2+) sensor in ameloblasts and plays a major role in enamel mineralization and ameloblasts’ proliferation and differentiation. Amelx-iCre mice displayed exclusive cre-mediated recombination in both incisor and molar ameloblasts. Stim1 cKO mice showed severely abnormal enamel phenotype with pathological mineral content, concomitant with increased attrition and smaller teeth size. As such, the Amelx-iCre transgenic lines we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Keywords: Ameloblast, Enamel, Cre-Lox, Amelogenin, Conditional Knockout, Stim1, Calcium.

Introduction

Amelogenesis is a very complex embryological process that results in the development and mineralization of dental enamel (Simmer et al. 2010; Zheng et al. 2014). This process is initiated by a series of reciprocal epithelial-mesenchymal interactions that culminates in the differentiation of internal enamel epithelial cells of the enamel organ into ameloblasts. Throughout their lifecycle, ameloblasts modify their morphological features and genetic profile in a tightly regulated manner (Smith et al. 1998). Genetic manipulation tools have revealed several key regulators in enamel formation but the present models have several limitations. A major limitation has been the ubiquitous or near-ubiquitous expression of many genes in the ameloblast molecular toolkit and the resulting inability to target these genes specifically in ameloblasts. To accelerate research in the field of enamel formation there is an urgent need in developing new innovative models physiologically relevant to enamel development in humans.

Knockout mice are the tools of choice for studying mammalian gene function, but proper interpretation of results can be complicated by perinatal lethality and developmental compensation (Zurborg et al. 2011). These limitations can be addressed by generating tissue- or developmentally- specific knockout mice by means of the Cre-loxP system, where specific Cre-recombinase driver mice are crossed with mice containing loxP-flanked (‘floxed’) genes of interest. Indeed, generation of Cre mouse lines with specificity for ameloblasts would allow for a much more accurate investigation of the molecular mechanisms that regulate amelogenesis. To date, a very limited number of Cre mouse lines that specifically target ameloblasts are available. In fact,  most of the Cre-loxp cKO studies regarding amelogenesis use mice where Cre is under control of the locus of cytokeratin 14 (K14) (Furukawa et al. 2017).  A major shortcoming of such Cre lines is that K14 is profoundly expressed in multiple basal epithelial cells (Harnden and Southgate 1997; Yoshida et al. 2015). Consequently, besides ameloblasts, K14 Cre will target the genes of interest in all other cells of the enamel organ and in multiple non dental epithelial tissues including the epidermis, salivary glands (Li et al. 2015). This lack of specificity significantly complicates the interpretation of results. Moreover, it is unanimously accepted that K14 is mainly expressed by the undifferentiated internal enamel epithelial cells (pre-ameloblasts) prior to enamel matrix protein secretin (Tabata et al. 1996). Subsequently, K14 levels are greatly down regulated as pre-ameloblasts differentiate into secretory ameloblast (Tabata et al. 1996; Ravindranath et al. 2001). Such expression pattern further undermines the viability of K14 as a reliable Cre-promoter for investigating the complex pathways involved in enamel secretion and mineralization.

Amelogenin is a 20-kDa hydrophobic protein that constitutes about 90% of the total enamel matrix proteins (Bansal et al. 2012); it is synthesized almost exclusively by ameloblasts and plays a major role in the mineralization and morphogenesis of enamel. The human amelogenin gene is located on the human X (AMELX) and Y (AMELY) chromosomes (Butler and Li 2014). Most of amelogenin transcription occurs in the X-chromosome allele and deletion of this gene causes X-linked amelogenesis imperfecta, a genetic disorder affecting enamel formation (Kim et al. 2006). Due to its tissue specificity and relative abundance, amelogenin is considered the most useful postnatal differentiation marker of ameloblasts, particularly during the secretory stage (Gibson 2011). Hence, the Amelx gene appears to be the best suited candidate for the generation of a Cre-driver mouse line that specifically targets ameloblasts.

Herein, we report the generation and characterization of a 250-kb BAC-transgenic based mouse model expressing codon improved Cre-recombinase (iCre) under the regulatory elements of the Amelx gene (Amelx-iCre). We first characterized the pattern of transgene expression after crossing the Amelx-iCre mice with ROSA26 reporter mice to generate offspring Amelx-iCre+/+/ Rosa26 mice that bring the reporter gene LacZ under the control of the Amelx BAC transgene. To characterize transgene activity specificity, we used LacZ gene encoding β-galactosidase enzyme staining and immunohistochemistry to detect iCre protein expression pattern. We further confirmed the occurrence of specific Cre-mediated recombination by crossing Amelx-iCre+/- mice with mice containing a LoxP-flanked cassette of the Stromal interaction molecule 1 (Stim1) gene. STIM1 is an endothelial reticulum (ER) membrane protein that serves as the main Ca2+ sensor in multiple mammalian cells such as: lymphocyte, myoblasts and epithelial cells (Kiviluoto et al. 2011). Recently, several reports have shown that STIM1 plays a major role in enamel mineralization and ameloblasts proliferation (Wang et al. 2014; Zheng et al. 2015; Furukawa et al. 2017; Lacruz 2017) but a model of Stim1 deletion specific to functional ameloblasts has not yet been generated. The Stim1fl/fl/Amelx-iCre+/- (Stim1 cKO) model we generated is, to our knowledge, the only model with Stim1 ameloblast-specific deletion. Stim1fl/fl/Amelx-iCre+/- teeth displayed a severely defective enamel phenotype and our initial imaging analysis revealed the presence of mineralization defects associated with abnormal enamel structure in Stim1 cKO mice compared to control. Collectively, our data indicate that the Amelx-iCre mouse lines can serve as a valuable tool to specifically target and investigate the different molecular regulators of amelogenesis.

Materials and Methods

Generation of BAC transgenic iCre-driver lines

All of the animal protocols were approved by the University of Michigan Research Animal Resources Committee in accordance with the NIH Guideline for the Care and Use of Laboratory Animals. The targeting vector, Amelx-iCre mice were generated at the University of Michigan Transgenic Animal Core on the C57BL/6 background as described in Figure 1. Bacterial artificial chromosomes (BACs) was used to generate the iCre-driver mice line (Shimshek et al. 2002). Plasmids containing straight iCre along with a selectable marker flanked by flippase recognition target sites (FRT-Neo-FRT) were used for all subsequent targeting of BACs containing Amelx gene. Correct introduction into the BAC results in kanamycin drug resistance. DNA from kanamycin sensitive BACs were analyzed to identify correctly modified BACs. Flippase (Flp) recombination was then used to remove the neomycin selection marker from the BACs. The wild-type loxP with ampicillin resistance site present in the BACs backbone was removed before pronuclear injection. Linearized BAC DNA was injected at a concentration of 1 ng μl into C57BL/6 zygotes. The transgenic founder mice and their progeny were identified by PCR with iCre-specific primers (Forward, 5′-CTCTGACAGATGCCAGGACA-3′; and Reverse, 5′-TCTCTGCCCAGAGTCATCCT-3′). The expected PCR products were 394 bp with the Cre-specific primers. To evaluate the activity of iCre-recombinase, ROSA26 reporter was introduced to generate offspring Amelx-iCre; Rosa26 mice carrying the reporter gene LacZ (Soriano 1999). Mice were genotyped by PCR using tail extracted DNA and both iCre and Rosa26 primers.

X-gal Staining

X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) staining was performed for -galactosidase activity assay. In brief, heads collected from these mice postnatally starting at day 5 (P5) to day 21 (P21) were dissected and rinsed with phosphate buffered saline (PBS), fixed in 4 % Paraformaldehyde (PFA) for 20 min at room temperature, rinsed with PBS again, and then stained in X-gal solution containing 1 mg/mL X-gal (Invitrogen, Massachusetts, USA), 5 mM potassium ferricyanide, 2 mM MgCl2, 0.01 % sodium deoxycholate, and 0.02 % NP-40 at 37 C for overnight. Tissues were then fixed in 4 % PFA overnight, decalcified and further processed for paraffin embedding and sectioning. The X-gal staining was completed with an eosin counterstain.

Immunohistochemistry

Mice heads were sectioned in a frontal (coronal) plane at the incisor and molar tooth buds and immune-stained to assess the iCre protein cellular and tissue localization using a rabbit anti-Cre (1:1000, Novagen, Madison, WI, USA) antibody. Primary antibodies were diluted with PBS and 1% bovine serum albumin (BSA). Following several washes by PBS + 1% BSA, the sections were incubated for 30 min with a mouse anti-rabbit IgG biotinylated secondary antibody (Invitrogen, Massachusetts, USA). Secondary antibody incubation was followed by washes with PBS + 1% BSA, then incubation with an avidin-biotin-peroxidase complex. Visualization with 3-3′diaminobenzidine (DAB) was performed using the peroxidase substrate kit DAB (SK-4100, Vector Laboratories, Burlingame, CA) instructions, and the reaction was stopped before detection of nonspecific staining in control pre-immune serum-treated sections. Sections were then mounted and photographed with an Olympus microscope. Consecutive serial sections from the same mouse models incubated with rabbit serum instead of primary antibody were used as a control.

Generation of Stim1 cKO mice 

All mice were housed in a specific pathogen-free facility and fed ad libitum with a normal diet following an UACC approved animal use protocol (UAP). Amelx-iCre transgenic mice were mated with Stim1fl/fl obtained from Jackson Laboratory (strain 023350) and the progeny bred to generate Stim1fl/fl/Amelx-iCre+/- (Stim1 cKO colony). STIM1fl/fl/Amelx-iCre-/-mice were used as a control genotype. At P21, genotypes were determined by tail biopsy collected under 3% isoflurane anesthesia and by conventional polymerase chain reaction (PCR). The mice were euthanized at 3 months of age by an overdose of isoflurane. All animal procedures and breeding programs were approved by the University of Saskatchewan Animal Care and Use Committee and the Animal Research Ethics Board (protocol:  20170014).

Dissecting microscopy

Control and Stim1 cKO mice were anesthetized with isoflurane and then humanely sacrificed. The mandibles were subsequently removed and dissected free of soft tissues then fixed by immersion in 4% PFA. The teeth were cleaned with nonwoven gauze, displayed on the Nikon SMZ1000 (Nikon Corporation, Japan) dissection microscope and photographed using a Nikon digital camera DXM1200 (Nikon Corporation, Japan).

Scanning electron microscopy

Scanning electron microscopy (SEM) evaluation was performed at the Western College of Veterinary Medicine Image Centre at the University of Saskatchewan (Saskatoon, Canada). Ethanol dehydrated and air‐dried hemi‐mandibles and mandibular incisors from the control and Stim1 cKO mice were mounted on metallic stubs using conductive carbon cement and degassed in a vacuum desiccator overnight. The samples were imaged using a Hitachi SU8010 Scanning Electron Microscope (Hitachi High-Tec, Tokyo, Japan) operating at an accelerating voltage of 3.0 kV. For back scattered incisor imaging, the bony caps and soft tissues covering the mandibular incisors were carefully removed and the midpoint of the labial surface of the mandibular incisors was then examined at ×150 magnification in a Hitachi SU8010 scanning electron microscope using the backscatter mode at 3.0 kV. The hemi‐mandibles were then cross sectioned using a diamond saw at a level even with the buccal crest of the alveolar bone; this level was used to assess enamel thickness and mineralization under the SEM.

Results

Generation of Transgenic Mice and Comparative Analysis of Specific LacZ Expression Pattern

A total of 20 Amelx-iCre transgenic mice from four independent BAC founder lines (854, 863, 872 and 876) were collected (Fig. 1B). To confirm transgene inheritance, mice were backcrossed to C57B6 mice for six generations. The genotypes of the progeny were determined by PCR with iCre specific primers, which amplify a 394-bp fragment of the iCre gene. At postnatal day 21 (P21), functional ameloblasts in all the lines displayed iCre activity, as determined by X-gal staining (Fig 1 C-H). X-gal is an analog of lactose, and it is hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose allowing easy identification of an active enzyme for β-galactosidase (encoded by the LacZ gene). Among the four transgenic mouse lines tested, the strongest X-gal staining was detected in ameloblasts in the Amelx-iCre line 876 (Fig. 1C). However, unspecific β-galactosidase staining has also been noted in the osteoblasts (Fig 1E) and osteoclasts (Fig 1D) and therefore was excluded from further analyses. The mouse line 863 showed only weak β-galactosidase staining and it was also excluded from further analyses. In contrast, both the mouse Amelx-iCre lines 854 and 872 showed X-gal staining clearly noted in the ameloblasts at the P21. The mouse iCre line 854 showed also very light β-galactosidase staining activity in the bone tissue (Fig. 1G), in contrast to Amelx-iCre 872 where β-galactosidase staining activity was solely found in amelobalsts (Fig. 1H). Based on the above mentioned  β-galactosidase staining activity patterns, the mouse Amelx-iCre lines 854 and 872 were selected for subsequent studies due to strong ameloblast specific expression pattern mimicking endogenous Amelx expression.

Developmental Expression Pattern of β-galactosidase staining activity in Amelx-iCre Transgenic Mice during Enamel Development

To determine the onset of β-galactosidase staining activity during ameloblast cell development, we examined embryos at embryonic day (E) 17.5, newborn stage (NB) and mice from post-natal (P) days 5, 14 and 21, by using X-gal staining. No X-gal staining was observed in teeth at both early developmental points E17.5 and NB (data not shown). Strong X-gal positive cells were first observed in the secretory stage ameloblasts in both incisor and molars collected from P5 mice (Fig. 2A and B). At P14, X-gal staining has been also found in all three molar ameloblasts (Fig. 2C). In addition, a very strong expression of X-gal was noted in the secretory and maturation stage ameloblasts in incisors from P21 days mice (Fig. 2D).

To confirm the X-gal staining results, we also performed immunohistochemistry (IHC) staining by using an anti-iCre antibody. At P21, ameloblasts in both incisor and molars showed ameloblast-specific CRE protein positive signal similar to LacZ activity and the endogenous amelogenin expression (Fig. 3).

Enamel Phenotype of the Stim1fl/fl; Amelx-iCre mice

The gross morphologies of the fully erupted mouse molar and incisors (12 weeks old) were assessed under a dissecting microscope, by scanning electron microscopy (SEM) and by backscattered SEM (bSEM). Control mice (Stim1fl/fl/Amelx-iCre) incisors from the same litter displayed normal size with normal enamel translucency having normal yellowish pigmentation due to iron depositions. On the other hand, the erupted incisors in Stim1 cKO (Stim1fl/fl/ Amelx-iCre+) mice from the same litter showed chalky enamel with less iron deposition (Fig. 4 A, B). The molar cusps of control mice showed normal contours while the enamel surfaces were both smooth and reflective. In contrast, Stim1 cKO molars had thinner cusps with rough, dull surfaces and displayed severe attrition on their occlusal surfaces (Fig. 4 C, D). SEM analysis of the mandibular incisors revealed that enamel was thinner, irregular and coarse with dome‐like mineralization nodules protruding from the surface in STIM1 cKO mice (Fig. 5 A-D). Mandibular incisors from the control and Stim1 cKO mice were imaged using backscatter scanning electron microscopy (Fig. 5 E, F). Control incisors showed a smooth enamel layer covering the entire crown except in the enamel free zones at the cusp tips. Stim1 cKO incisors displayed rough enamel surfaces that were highly variable in their degree of mineralization.

 

Discussion

In this study, we report the generation of ameloblast specific iCre mouse lines driven by the Amelx gene expression which allows for realistic spatial control of the iCre activity. Our analysis revealed that iCre expression occurs robustly during both secretory and maturational ameloblast in the enamel organ mimicking the endogenous amelogenin expression pattern. No iCre activity was detected in pre-ameloblasts, dental mesenchymal cells and the other cellular subpopulations of the enamel organ (e.g. stratum intermedium, stellate reticulum and external enamel epithelium). We subsequently demonstrated the functional applicability of this Amelx-iCre model by generating a mouse model in which the vital calcium sensor gene, Stim1, was efficiently and selectively deleted, specifically in functional ameloblasts. Morphometric analysis of the Stim1 cKO mice showed that loss of the STIM1 protein had led to profound alterations of enamel secretion and mineralization without any systemic manifestations in other tissues. To our knowledge, this is the first report that comprehensively describes the successful generation of BAC-based Amelx-iCre mouse line and fully characterizes Amelx-iCre expression throughout all stages of the amelogenesis. Our results clearly indicate that these Amelx-iCre mice can serve as a powerful genetic tool to efficiently manipulate ameloblast-specific gene expression in vivo.

The primary objective of enamel research is to better understand normal and pathological enamel formation in order to better diagnose, categorize and potentially treat or prevent enamel defects caused by genetic diseases such as amelogenesis-imperfecta (AI) or by environmental factors. In addition, understanding the role of the complex interconnected systems that regulate amelogenesis is essential for setting up successful protocols of ex-vivo enamel regeneration (Mitsdiades and Papagerakis 2011). Generally, mice are considered to be an ideal model for enamel research due to the fact that in addition to the significant similarities between murine and human teeth, their genome can be easily modified (Pugach and Gibson 2014). Moreover, the continuously erupting mice incisors contain all the stages of enamel secretion and mineralization in one single section, which allows us to comprehensively examine the dynamic process of amelogenesis (Simmer et al. 2010).

The molecular toolkit of the Cre-loxP system has enabled researchers to conditionally recombine the mouse genome and thereby creating multiple disease models (Bouabe and Okkenhaug 2013). Two common strategies can be utilized to integrate transgenes (e.g. Cre) into the mouse genome; a targeted (knock in) and a non-targeted strategy. The most widely used technique of the latter is to clone transgenic DNA with its specific regulatory sequences (that are stored in bacterial artificial chromosomes or BAC) into the mouse genome (van Keuren et al. 2009). BACs can maintain large DNA fragments and are extensively used to generate transgenic Cre recombinase driver lines (Liu 2013). The common BAC length ranges from 150,000 base pairs (150 kb) to 350 kb, and it integrates randomly into the mouse genome (Zhao et al. 2001). A major advantage of BAC-based transgenes is their ability to direct gene expression within the same physiological levels and spatiotemporal patterns of endogenous genes (Poser et al. 2008). Moreover, generation of BAC-based models is less time consuming compared to knock-in models while showing similar end results (Zurborg et al. 2011). Based on the above, we opted to use a 250kb BAC transgenic-based approach to generate our Amelx-iCre transgenic mouse line as a strategy to generate a new model that allows to dissect the molecular determinants of enamel secretion and maturation. This new model was able to delete Stim1 in ameloblasts and it may be used to specifically delete any gene of interest in the future thus improving our understanding of enamel development.

Amelogenin is the most abundant protein in the developing enamel matrix. It’s mainly expressed by the ameloblasts and plays an essential role in enamel mineralization as it forms nanospheres that direct the initial mineral deposition and spacing of enamel crystallites (Dessombz et al. 2014). In addition to ameloblasts, previous reports have shown that amelogenin is also expressed by odontoblasts, root epithelial cells, and in some cells of non-dental origin (Mitsiadis et al. 2014).  However, it must be noted that amelogenein expression in the aforementioned sites occurs only transiently (Mitsiadis et al. 2014). The human Amelogenin gene is located on the X-chromosome at Xp22.1–p22.3 (AMELX) and on Y chromosome at Yp11.2 (Murphy et al. 2007). Approximately 90% of the transcripts are expressed from the X-chromosome and up to date the AMELX gene is considered the only causative gene of X-linked AI (Kim et al. 2017). Secreted amelogenins reside within the tooth germ until shortly before tooth eruption (Gibson et al. 2001). Their levels reach their zenith post-natal days 5-7 and thus it is considered the main marker of the secretory ameloblasts (Mitsiadis et al. 2014). Our X-gal staining results are consistent with this amelogenin temporal-spatial pattern of expression, as we were not able to detect X-gal Amelx-iCre expression in pre-ameloblasts at embryonic stages, while staining was clearly detectable in post-natal mice starting at P5.

Despite being the most characteristic enamel protein, amelogenin has been scarcely used as a Cre promoter for generating conditional knock out models. To the best of our knowledge, only two Amelx-cre mice have been hitherto reported the literature by Cho et al., 2013 and Fann et al., 2018 (Cho et al. 2013, Fan et al. 2018). However, neither of these models used a BAC-based transgenesis and Cho injected the mice embryo with a vector containing a bovine amelogenin promoter-cre transgene rather than a murine one. In fact, both studies used a very small part of Amelx promoter, as Cho used a using a 7.1 kb bovine amelogenin promoter fragment ligated to a 2.1 kb fragment containing the Cre recombinase gene, while Fan used a 3.5 kb promoter fragment. It must be noted that Cre mouse lines that only use small promoter constructs frequently show non-specific gene expression that does not mimic the target gene endogenous expression pattern and can be silenced over time (Lufino et al. 2016). On the other hand, the much larger and more stable BACs (250 kb) have the advantage of carrying the entire target gene locus in addition to its regulatory elements. The complex interactions between these elements create suitable expression properties that resemble those of the gene in its endogenous locus and thus BAC-based transgenesis can generate physiologically-relevant levels of the target gene and achieve its expression in a correct developmental and tissue-specific manner (Lufino et al. 2016). In addition, both reports did not comprehensively examine the developmental and adult Cre expression profile in their mice models as Cre expression was only examined during the early stages of amelogenesis. Finally, both of these models cloned the traditional Cre recombinase transgene while we choose to introduce the codon improved Cre gene (iCre). iCre was first designed by Shimshek et al., 2002 to reduce the high CpG content of the prokaryotic coding sequence in the Cre gene, thereby reducing the chances of epigenetic silencing in mammals and greatly enhancing Cre-expression.

Alternatively, the most commonly used mouse line for conditional gene deletion in the enamel organ is the K14 cre line despite its several limitations. Specifically, the main drawback of this line is its lack of specificity, as it deletes genes in all layers of the enamel organ including the stratum intermedium and papillary layer rather than targeting ameloblasts only (Klein et al. 2017). Moreover, and in contrast to amelogenin, the spatiotemporal distribution and expression pattern of CK14 in the dental epithelium during tooth formation is very complex and poorly delineated. Immunohistochemical studies showed that CK 14 is strongly expressed by the inner dental epithelium at early bell stage, then gradually wanes at the late bell stage when the ameloblasts become fully differentiated (Domingues et al. 2000; Chavez et al. 2013). Such expression pattern greatly undermines K14 cre lines reliability as suitable models for understanding the cellular complexity of amelogenesis, particularly during the secretory and maturation stage. Based on the above, it is safe to conclude that amelogenin has a clear advantage over the cytokeratins 14 as an ameloblast specific Cre-promoter. In fact, recent reports addressed the limitations of K14 models and recommended the use Amelx-Cre models to target gene deletion in ameloblasts from the secretory stage onwards (Klein et al. 2017).

STIM1 is an ER transmembrane protein that serves as the main intracellular calcium (iCa2+) sensor in several mammalian cells. Upon iCa+2 depletion in the ER, STIM1 and its analogue STIM2 activate the highly selective calcium release activated calcium channels (CRAC) in the plasma membrane that facilitate calcium entry into the cell to replenish ER Ca2+ levels and increase iCa2+ (Zheng et al. 2015).  This mode of Ca2+ transport is named Store-operated Ca2+ entry (SOCE) and it plays a major role in many essential physiological functions such as: immune cell activation, thrombus formation, muscle contractions, bone turnover and ameloblasts (Zheng et al. 2015). Humans with mutated STIM1 present with hereditary combined immunodeficiency, congenital myopathy and anhidrotic ectodermal dysplasia with severely defective enamel (AI) which suggests a critical role of STIM1 protein in Ca2+ influx into ameloblasts and subsequent enamel maturation (Lacruz and Feske 2015). In fact, recent reports revealed that STIM1 is highly expressed in ameloblasts of wild-type rodents (Simmer et al. 2014; Wang et al. 2014; Lacruz 2017). Moreover, in vitro studies investigating ameloblast-like cells suggested that in addition to its role in enamel mineralization, SOCE regulates the gene expression of several enamel matrix proteins such as: Amelogenin, Ameloblastin, and Mmp20 (Nurbaeva et al. 2015; Zheng et al. 2015). However, the mechanism by which SOCE regulates amelogenesis in vivo is largely unknown because animal models with impaired SOCE have been of limited use, as deletion of Stim1 genes results in perinatal lethality of mice (Lacruz and Feske 2015). Hence, two recent reports investigated SOCE role in ameloblasts by examining mice with conditional deletion of Stim1 in enamel cells using K14 Cre mice models (Furukawa et al. 2017, Eckstein et al. 2017). A major caveat in these models is that Stim1 mainly functions during the secretory and maturation stages of amelogenesis and the K14 Cre models are only suited to investigate the role of genes expressed by the Inner Enamel Epithelium cells up to the pre-secretory stage of enamel formation. In fact, both of these two reports did not identify any changes into ameloblast-specific genes in deleted Stim1 samples, suggesting that the described enamel phenotype of these models is rather indirect caused be early development epithelia cells defects. To address these issues, we generated Stim1 cKO mice by using our Amelx-iCre mice.  Our Stim1 cKO mice experienced a normal survival rate and did not show any systemic symptoms, while they exhibited severe mineralization defect in their fully erupted teeth. We also noticed that the enamel defects in our model were more pronounced in comparison to the K14 Cre mice, which further reflects Amelx suitability over K14 as an ameloblast Cre-promoter for studying the molecular mechanisms of ameloblast differentiation.

Our data provide key insights into the utility of the Amelx-iCre mouse models as tools to modulate gene expression in functional ameloblasts. Additionally, these models may contribute in designing therapeutic interventions to mitigate congenital enamel defects. Our results also clearly indicate that STIM1 plays a major role in enamel secretion and maturation by its ameloblast-specific deletion. Further investigations are still required to fully examine STIM1 exact role in enamel formation.

 

Acknowledgments: This work was partially funded by the University of Michigan start-up funds to Dr. Petros Papagerakis and by the University of Saskatchewan College of Medicine and Vice Provost Office start-up funds to Dr. Silvana Papagerakis. Raed Said is funded by Jordan University of Science and Technology (JUST) and received the University Of Saskatchewan College Of Medicine Travel Fund Award and the ISAAC U of S Student Travel Award to present part of these findings in the 2018 IADR Annual Meeting held in London, England.

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Figure Legends

Fig 1. Generation and characterization of Transgenic Amelx-iCre mice lines. (A) Structure of the Amelx-iCre transgene construct. The 250kb BAC contains the DNA sequence includes Amelx gene. The BAC clone and a synthetic donor DNA are combined in recombined competent bacteria. Correct introduction into the BAC results in kanamycin (kanr) drug resistance. DNA from kanr+ BACs are analyzed to identify correctly modified BACs. The kanr cassette has both prokaryotic and eukaryotic (mouse Pgk1) promoters. Then the kanr cassette is removed by induction of FLP recombinase in the BAC. A final recombination step replaces the BAC-backbone internal loxP site with ampicillin resistance. (B) PCR genotyping of the Amelx-iCre transgenic mice. Lane left to right represent different mouse lines. NC: negative control. (C-F) LacZ staining of different iCre mouse lines (876, 863, 854 and 872). Arrows in D and E show unspecific expression of Cre in mouse line 876. According to the Cre activity results, the mouse line 872 was selected for further analysis. ab, ameloblasts. Scale bars = 300 μm in C, 20 μm in D–H.

Fig 2LacZ staining of ameloblasts in Amelx-iCre mice at different developmental stages of enamel formation. (A) The secretory stage ameloblasts shows strong LacZ activity in both incisor and molars at postnatal day 5 (P5). (B) Enlarged picture shows LacZ positive staining in ameloblasts in incisor. (C) Strong LacZ expression levels can be detected in all three molars at postnatal day 14 (P14). (D) Maturation stage of amelobasts in incisor still shows strong positive signal of LacZ staining at postnatal day 21 (P21). ab, ameloblast. Scale bars = 300 μm in A, C and D; 10 μm in B.

Fig 3. iCre protein detection in ameloblasts at P21 by immunohistochemistry (IHC). (A) Positive signal can only be found in ameloblasts from the molars at P21. (B) Enlarged picture shows specific expression in molar amelobasts. (C and D) IHC staining can also be detected in ameloblast in incisors at P21. Scale bars = 300 μm in A, and C; 10 μm in B and D.

Fig 4. Teeth photographs of 12week old control and Stim1-cKO mice. Incisors in Stim1-cKO (Stim1fl/f/Amelx-iCre+/+) mice (B, D) show chalky enamel and their molars display significant attrition on the occlusal surfaces when compared to control (Stim1fl/fl /Amelx- iCre-/-) (A, C)  .

Fig.5 Scanning Electron Microscope and Backscattered (SEM-BSE) Imaging photographs of control and Stim1-cKO mice. (A, B) SEM images of mandibular incisor cross sections at the level of the buccal alveolar crest in control (A) and Stim1 cKO (B) mice. The enamel matrix is thinner, irregular and coarse with mineralization defects (asterisks) in Stim1 cKO mice teeth. (C, D) Higher magnification views (400x) of mid labial surface of control (C) and Stim1 cKO incisors (D). The enamel crust of Stim1 cKO incisors is irregular and shows dome-like mineral nodules of various sizes protruding from the surface. (E, F) Backscatter scanning electron microscopy of mid labial surface of control (E) and Stim1 cKO incisors (F). The Stim1 cKO incisors show severe enamel abrasion (asterisks) that exposes the underlying dentin. Bars: (A, B, C, D = 100 µm), (E, F = 300 µm).

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