Animals were sacrificed in accordance with guidelines of the Animal Care and Use Committee at the Sanford Burnham Prebys Medical Discovery Institute and National Institutes of Health (NIH). Breeding, genotyping, and characterization of Phospho1^−/− mice has been described previously (Yadav et al., 2011; Zweifler et al., 2016). Eleven mandibles each from control and Phospho1^−/− mice were chosen for histology and microscopy studies. All research involving biohazards and toxins has been carried out according to Texas A&M institutional biosafety standards.

Thirty day old hemi-mandibles of control and Phospho1^−/− were fixed in 10% formalin and processed for ground sections. The mandibles were subjected to a series of different gradients of alcohol as well as mixture of ethanol/technovit as per the EXAKT company standard protocol for preparation of tissue samples for ground sections. Once the samples were in 100% light cure technovit (Technovit 7200, EXAKT), they were polymerized and embedded. The samples were grossly sectioned using a diamond bandsaw (EXAKT 300 CP), ground and polished to produce 30 μm thin sections (Liu et al., 2016). Two of the four ground sections were stained with alizarin red for 90 min and the other two for von Kossa staining.

Thirty day old hemi-mandibles were analyzed using a Faxitron MX-20 specimen radiography system (Faxitron X-ray Corp., IL) at 20 kV for 20 s.

Three WT and three Phospho1^−/− mouse mandibles were imaged individually and analyzed using a Micro-CT 20 Scanco Medical Scanner (Zürich, Switzerland). Specimens were scanned in standard resolution mode, with x-, y-, and z-axis resolution of ~10 μm. The level of X-ray exposure was 55 kVp energy and 800 ms exposure time. After the scanning was completed, 3-D data were generated by segmenting specimens at a threshold chosen to only include enamel tissue based on a morphological match with normal tooth histology. For each mandible, 70 slices were assembled and analyzed to perform a 3-D reconstruction of the incisors, and the total enamel volume fractions of the incisors were calculated and compared between the WT and Phospho1^−/− samples.

Thirty day old hemi-mandibles for control and Phospho1^−/− mice were fixed in 10% formalin and treated gently with 4.5% EDTA for 15 min. The etched samples were dried and sputter coated with Au/Pd alloy for 90 s. These mandibles were further analyzed using a scanning electron microscope (JEOL JSM-6010LA). For EDS analysis, the samples were fixed, dried, and analyzed by selecting five random points in the enamel region. The elemental mapping at the selected points was recorded and the mass percent amount of calcium and phosphorous was compared between the control and PHOSPHO1 samples.

E16 tooth organs were cultured for 12 days, and thereafter fixed in Karnovsky's fixative as previously described (Diekwisch, 1998), dehydrated and embedded in Eponate 12 (Ted Pella, Redding, CA). Following polymerization of the Eponate, ultrathin sections were cut on a Leica Ultracut UCT ultramicrotome. After drying, sections were contrasted using uranyl acetate and Reynold's lead citrate for 15 min each. Stained sections were examined using a JEOL 1220EX transmission electron microscope at the UIC Research Resources Center (Chicago, IL).

Three days old hemi-mandibles of three wild-type mice were fixed in 10% formalin, decalcified with 4.5% EDTA for 1 week, and then processed for regular paraffin sectioning after dehydrating through graded series of ethanol and xylene. Alternatively, a second set of 3-days old wildtype mandibles was fixed in Bouin's fixative and also processed into 5 μm thin paraffin sections. For antigen retrieval, the paraffin fixed samples were incubated in 10 mM sodium citrate buffer for 30 min at pH 6 and boiling temperature. Alternatively, the samples fixed in Bouin's fixative were treated with 8M guanidine HCl overnight at 4°C and pH 8 to unmask the PHOSPHO1 antigen sites as suggested by McKee et al. (2013). Following antigen retrieval, samples were incubated using human recombinant Fab monoclonal anti-PHOSPHO1 primary antibody (AbD Serotec, Morphosys AG) or polyclonal anti-PHOSPHO1 antibody (GeneTex, USA) for PHOSPHO1 identification. Other vesicle associated proteins such as amelogenin or HSP70 were labeled using a polyclonal anti-amelogenin antibody or a mouse monoclonal anti-HSP70 antibody (Abcam, Cambridge, MA). Immunohistochemistry was performed using a broad spectrum IHC staining kit and an AEC substrate kit (Life Technologies, Carlsbad, CA) and stained sections were analyzed using a Leica DMR light microscope (Nuhsbaum, IL).

Enamel organs were immediately harvested from unerupted mandibular molars of 3 months old pigs sacrificed at a local animal farm. The enamel organs were subjected to extraction with 0.5% sodium dodecyl sulfate (SDS) lysis buffer with subsequent extraction with 4M guanidine HCl (pH 7.4). Initially, the proteins from enamel organ were extracted using SDS lysis buffer for 5 days. The supernatant was collected after centrifugation at 2,400 g and 4°C. The residue after SDS extraction was subjected to extraction with 4M guanidine (Gu) HCl buffer for 5 days. The supernatant from Gu-based extract was collected and processed following the same procedure as the SDS based extract. The extracted protein lysate from SDS and Gu-based extracts were dialyzed against double distilled water for 1 week. Equal amounts of protein extracts were loaded for both groups in a 10% SDS polyacrylamide gel and was subjected to gel electrophoresis at 150 V for 58 min. A semi-dry transfer system was used to transfer proteins from the gel to a polyvinylidine difluoride (PVDF) membrane at 18 V for 40 min. The PVDF membrane was blocked for 1 h with 5% dry milk in tris buffered saline with tween 20 (TBST), incubated with human recombinant Fab monoclonal anti-PHOSPHO1 primary antibody (1:500, AbD Serotec, Morphosys AG) for 1 h, followed by washing in TBST three times for 15 min and incubation with anti-mouse IgG HRP conjugated secondary antibody (1:2,000, cell signaling) for 1 h. The signal was detected using a chemiluminescent substrate (Thermo Scientific, USA).

The width and the inter-prism space of enamel prisms was measured based on three control and three Phospho1^−/− samples. Measurement of prism width and interprismatic distance were obtained from scanning electron micrographs of etched mouse molar enamel cross sections. For our analysis, only mid-sagittal cross sections were chosen, and all samples were etched at identical times and concentrations, and rinsed for the same amount of time. Representative micrographs at 750x magnification from the distal interproximal enamel of the first molar were chosen for further analysis based on optimal prism pattern identification. Ten prisms were randomly chosen from each micrograph, and their width and the inter-prism space were measured using the ImageJ software. To analyze the silver grain density in von Kossa stained sections, micrographs at 400x magnification were imported into the ImageJ software package, and three 100 × 100 μm areas within the Phospho1^−/− and WT cuspal tip molar enamel region were randomly chosen and pixel density was analyzed. For EDS analysis, ten points were randomly chosen in the enamel region of three Phospho1^−/− and WT molars, and the values of calcium and phosphorus at the selected points was compared between both groups. All experiments were done in triplicates, the data analysis was conducted using student's T-test, and the significance value was set at p < 0.05.

Immunohistochemical and Western blot analysis were performed to verify whether the PHOSPHO1 protein was localized in the enamel organ. Our analysis of paraffin sections of 3 days old wildtype mice revealed PHOSPHO1 protein localization in the enamel layer, Tomes' processes, and in ameloblast secretory vesicles (Figure 1). Positive signals for PHOSPHO1 were obtained using both antibodies available to us (GTX119275 from Genetex in Figures 1A,C and AbD Serotec/Millán in Figures 1B,D) and independent from the type of fixative employed (Formalin in Figures 1A,C,E, and Bouin in Figures 1B,D). In general, PHOSPHO1 expression levels were stronger in the mineralized enamel layer and at the ameloblast secretory pole (Figures 1A,C), while there was no staining in the control section using the same technique without primary antibody (Figure 1E). High magnification images revealed positive staining for PHOSPHO1 at the periphery of circular organelles at the ameloblast secretory pole (Figure 1C,D). A Western blot using the AbD Serotec antibody and performed on guanidine or SDS extracts of enamel organs demonstrated a characteristic 32 KDa full-length PHOSPHO1 band and a 25 kDa cleavage product, confirming the presence of PHOSPHO1 in the enamel organ.

To determine how loss of PHOSPHO1 affects enamel quality, a number of physicochemical parameters such as total enamel volume, calcium, and phosphate ratios, level of mineralization based on von Kossa sections, and sharpness of incisal tips were assessed and compared between Phospho1^−/− and WT mice. Comparison of X-ray radiographs and alizarin red stained ground sections between 30 day old control and Phospho1^−/− mouse teeth revealed a rounded incisal tip in the PHOSPHO1 null incisors compared to the sharp incisal tips of their wild-type counterparts (Figures 2A–D). There was a significant 2-fold reduction in silver grain density on von Kossa stained ground sections (1.44 ± 0.11 vs. 0.71 ± 0.04 pixel units) indicative of reduced mineralization in the enamel layer of the Phospho1^−/− mice when compared to the WT mice (Figures 2E,F,J). Von Kossa staining also demonstrated a substantial reduction in dentin mineralization in the Phospho1^−/− mice vs. controls (Figures 2E,F). Total enamel volume as measured by micro-CT volumetric analysis was significantly higher in Phospho1^−/− incisors compared to WT controls (Figures 2G,I). EDS elemental analysis demonstrated a significant decrease in the amount of phosphorus in the Phospho1^−/− mice when compared to controls (p < 0.05), while calcium levels were only little affected by the loss of PHOSPHO1 (Figure 2H).

Scanning electron microscopy comparisons between 30 days old Phospho1^−/− and control mice demonstrated a complete loss of the prominent enamel prism “picket fence” structure on surface etched Phospho1^−/− mouse enamel preparations when compared to controls (Figures 3A–D). High magnification scanning electron micrographs at individual crystal resolution revealed a loss of individual prism boundaries as a result of a 45.3% reduction in the mineral-free inter-prism space between individual prisms (Figure 3F) in Phospho1^−/− mice when compared to controls (Figures 3C,D vs. Figures 3A,B). These micrographs also documented a loss of parallel crystal organization within prisms (Figure 3D). Moreover, there was a 1.56-fold increase in enamel prism width (Figure 3E) in Phospho1^−/− mutant mice (p < 0.0001).

Ameloblast secretory vesicles (sketch in Figure 4A) were long considered to be protein-rich intracellular entities within ameloblasts. Micrographs presented in this study illustrate that following 12 days of organ culture, these secretory vesicles also contained numerous mineral precipitates at the outside of and within the secretory vesicles (Figure 4B). Moreover, high magnification immunohistochemistry micrographs demonstrated the presence of half-moon shaped PHOSPHO1-positive signals in the walls of ameloblast secretory vesicles close to the developing enamel front (Figure 4C). Supportive of their involvement in secretory processes during amelogenesis, vesicles also stained positively for amelogenin and the vesicular marker protein HSP70 on paraffin sections of secretory ameloblasts (Figures 4D,E).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.