One-month-old male mice lacking Cx43 cells expressing DPM1 (Cx43^{flox/flox;DMP1-8kb-Cre}, N = 4) and control littermates (Cx43^flox/flox, N = 5) were generated and genotyped as previously described (Bivi et al., 2012). Other mouse strains and procedures for this study have been described before (Bronckers et al., 2010; Jalali et al., 2014). For each genotypic mouse strain, teeth, and enamel organs from three wild type mice and three null mutant mice were analyzed and compared. All animal procedures were approved by the Committee for Animal Care using standards for treatment of animals (University of Amsterdam), the Animal Care and Use Committee of the National Institute of Dental and Craniofacial Research, National Institutes of Health (ASP 13-686), and the Institutional Animal Care and Use Committee of Indiana University School of Medicine.

Upper and lower jaws of wild type, Cx43^{flox/flox;DMP1-8kb-Cre}, Cx43^flox/flox, Nbce1 null, and Cftr- null mice were fixed by immersion in 5% paraformaldehyde in 0.1 M phosphate buffer pH 7.3. Hemimandibles were then decalcified in 4% EDTA, pH 7.3 for 2 weeks at 4°C and processed into paraffin. Sagittal serial sections in thickness of 5–7 μm were prepared and mounted on polylysine coated glass slides.

Dewaxed sections were rinsed in phosphate buffered saline (PBS) and subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0) either at 60°C overnight, or for 20 min at 95°C in a microwave oven. After antigen retrieval, endogenous peroxidase was blocked with a peroxidase block solution (Envision kit, Dakocytomation) for 5 min. Sections were washed in TBST (3x). Non-specific staining was blocked for 30 min with 2% BSA. Sections were then incubated overnight at 4°C with rabbit anti-Connexin-43 (Cx43) (Abcam, Catalog No. Ab11370). Matched non-immune IgG (1:200 to 1:300) or normal serum (same concentration as primary antibodies) served as controls. After overnight incubation at 4°C with primary antibodies, sections were washed three times in TBS and incubated with goat anti-rabbit IgG antibodies conjugated with peroxidase (EnVision Kits) for 1 h at room temperature and counterstained with hematoxylin. Otherwise, rabbit anti-Cx43 was visualized by goat anti-rabbit Alexa Fluor 488 (5 μg/mL; Invitrogen) after 1 h incubation at room temperature and counterstained with DAPI (Vector Laboratories, Burlingame, CA, United States). Immunohistochemistry images were acquired with Leica EL6000 or Axio Zoom V16 microscope.

To determine the degree of mineral content, hemi-mandibles from Cx43^{flox/flox;DMP1-8kb-Cre}, Cx43^flox/flox mice were scanned at a resolution of 8 μm voxel using a μCT-40 high resolution scanner (Scanco Medical, AG, Bassersdorf, Switzerland). Mineral density was determined at sequential stages of development. Cross-sectional virtual images were collected from the most developed (incisor tip) to the least developed (cervical) area. The most incisal slice containing the most mineralized enamel was identified visually. Measurements were made at 500 μm intervals (50 slices at 10 μm interval) and slices at the same developmental stage from three mice per group were averaged and plotted as function of stage (slice number). Independent t-test was used to compare the groups. Statistical significance was set at p < 0.05 level.

From freeze-dried wild type and Cftr null mutant mandibular incisors early maturation stage enamel organs were micro dissected incisally based on a reference line projected between the first and second molars (M1 and M2), to separately dissect secretory and maturation stage ameloblasts, as described before (Schmitz et al., 2014). The apical, secretory, half of the enamel organ was placed in SDS loading buffer (from Nucleospin Triprep kit, Macherey-Nagel, supplied by Bioke, Leiden, Netherlands) and protein was measured using the BCA protein assay (Bio-Rad, Hercules, CA, United States). Twenty 20 μg of enamel organ denatured protein and 10 μg of molecular weight markers (Novex^® Sharp Pre-stained Protein Standard (# LC5800) or SeeBlue^® Plus2 Pre-stained Protein Standard (#LC5925) were subjected to electrophoresis in a 3–8% Tris acetate Nupage gel with Tris acetate running buffer for 60 min at 150 V or 4–12% Bis-Tris Protein Gels with MOPS buffer for 35 min at 200 V. Subsequent electro blotted by an iBlot device (Invitrogen) on nitrocellulose membrane was performed according to the manufacturer’s instructions. Membranes were blocked with BSA 2% for 1 h at room temperature and incubated overnight with the primary antibodies. Blots were then washed three times in TBST and incubated in with IRDye secondary antibodies (LI-COR). Visualization and quantification was carried out with the LI-COR Odyssey scanner and software (LI-COR Biosciences). Red color (for actin) was detected at 680 nm wavelength and a green color (other primary antibodies and tubulin) was detected at 800 nm wavelength. Quantification was performed using Odyssey software. Intensity values of the bands were normalized for actin or tubulin and expressed as percentage of wild type (100%). These primary antibodies were used: rabbit anti-Cx43 (Abcam, see above), and anti-tubulin rabbit antibody (ab, 59680). Secondary antibodies: IRDye 800CW conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed (LI-COR; Product No. 926-32211). Dilutions: anti-tubulin (1:1000); anti-connexin 43 (1:250); secondary antibodies [antibodies (1:10000)].

Paraffin embedded incisor sections were dewaxed in xylene, rehydrated in descending grades of ethanol and stained with 1% hematoxylin (15 min) and eosin (5 min). Sections were dehydrated in ascending grades of ethanol and xylene and mounted in Depex mounting medium. The enamel organs were imaged under 40x objective lenses. Images of secretory and maturation stage of enamel organ were selected based on the anatomical position in the tooth. The number of GJ plaques were counted as previously described (Toth et al., 2010). The investigator was blinded, and GJs (defined as a discernable Cx43-labeled bright fluorescent structure of at least 0.5 μm in length, seen in membranes of adjacent cells) were counted per field of view and normalized to the number of the cells in the area (number of DAPI-stained nuclei) as described before. Stratum intermedium (SI), papillary layer (PL), and ameloblasts were counted separately and combined for a total enamel organ assessment. Statistical analysis was performed by independent t-test (p < 0.05).

Histological assessment of enamel organs from Cx43^flox/flox control mice revealed a tall, columnar ameloblast cell layer in both the secretory and maturation stage of enamel formation (Figures 1a,c). However, both secretory and maturation stage ameloblasts from Cx43^{flox/flox;DMP1-8kb-Cre} mice revealed no evidence of cell polarity and the columnar profile of this epithelial cell layer was lost (Figures 1b,d; Cx43^{flox/flox;DMP1-8kb-Cre}) and Figure 1e (Cx43^flox/flox stained with IgG serum antibody) shows the specificity of the antibody.

To investigate whether absence of CX43 in dental epithelium influences enamel mineralization micro-CT analysis was carried out on jaws of conditional Cx43 ko mice as compared to controls. In upper incisors disruption of the CX43 gene reduced mineral density by ∼10% at late maturation (p < 0.0001) but not at secretory and early maturation phase (Figures 2A–D). The fact that in the mutant teeth enamel was hypomineralized at late maturation stage suggested that GJs are required to fully mineralize enamel at late maturation.

To examine the possible role of GJs in directing the transport of ions from the papillary layer to ameloblasts, tissue sections of mouse mutants with hypomineralized enamel as result from disruption in local pH regulation (Cftr- null and Nbce1- null mice) were immunostained for Cx43. In wild type mouse secretory ameloblasts and stratum intermedium strong positive staining for Cx43 was seen in the basolateral plasma membranes; weaker staining was observed in stellate reticulum cells located between the stratum intermedium and outer enamel epithelium (Figures 3e,k). Wild type maturation ameloblasts and papillary layer stained strongly for Cx43 (Figures 3a–d,h,n).

In Nbce1 null mice (Figures 3f,i) and Cftr null mice (Figures 3l,o) the distribution pattern of immunostaining for Cx43 in enamel organs was largely similar as in wild type mice. Protein expression levels of Cx43 in enamel organs by Western blotting showed that Cx43 was increased by 65% (p = 0.06) in Cftr null mice (Figures 3q,r).

Immunohistochemical staining with anti-Cx43 showed that in both Nbce1 null and Cftr null mice the total number of GJs (number of fluorescent Cx43 positive plaques in the enamel organ), was not different from wild type controls in secretory stage (Figures 3g,j,m,p) but had increased by in maturation stage but significantly increased in the papillary layer in maturation stage (Cftr null mice, p < 0.0001; for Nbce1 null mice p = 0.005 respectively; Figures 3g,j,m,p).