2.1. Detection clock RNAs using RT-PCR
Bmal1, Clock, Per1 and Per2 mRNAs were detected in PN1 whole tooth extracts by conventional RT-PCR (), as well as in brain (positive control). All the PCR products were sequenced to confirm clock RNA expression. In addition, quantitative real-time PCR (qRT-PCR) was used to evaluate relative expression levels between tooth and brain ().
Figure 1 Expression of clock genes RNAs in teeth and brain at post-natal (PN) day 1. Conventional RT-PCR showed that Bmal1, Clock, Per1, and Per2 RNAs are expressed in mouse teeth as well as in the brain. No amplification bands are detected in negative control (more ...)
2.2. Clock proteins expression in teeth at E13, E14 and E17
We have then characterized the protein expression patterns. At the bud stage (E13), the dental epithelium invades into presumptive dental mesenchyme. In this stage, clock proteins were undetectable in dental epithelium or condensed dental mesenchyme (). Similarly, no expression of clock proteins was detected in tooth germs of E14-E16 old mice ().
Figure 2 Expression of clock proteins in mouse teeth at E14, E15 and E17. None of the four clock proteins can be detected in teeth bud and cap stages as shown here for E14 (A) and E 15 (F) (B–E and G–J). Clock protein expression in teeth was first (more ...)
At the bell stage (E17), the neural crest-derived mesenchyme cells differentiate into dentin-secreting odontoblasts, while the jaw epithelium differentiates into the enamel-secreting ameloblasts. At this stage all four clock proteins showed similar expression patterns but with different intensities within the nucleus of ameloblasts and odontoblasts (). Bmal1 protein expression was relatively weak but clearly detected in ameloblasts and odontoblasts; Clock and Per2 showed stronger expression levels than Bmal1; and Per1 protein expression was the strongest among the four proteins characterized. Dental pulp cells, stratum intermedium (mainly for Per1) and osteoblasts (mainly for Bmal1) showed also variable levels of sporadic protein expression ().
2.3. Clock proteins localization in teeth at PN4
At PN4 stage, the ameloblast and odontoblast are well-differentiated. They are induced to synthesize enamel- and dentin-specific proteins and form enamel, pre-dentin and dentin. At this stage, Bmal1 was up-regulated in the nucleus of ameloblasts, odontoblasts (). Clock, Per1 and Per2 protein were continued to be strongly expressed in the nucleus of ameloblasts and odontoblasts (). At this stage, the staining of the dental pulp cells was down-regulated for Clock, Bmal1, and Per2 (). In contrast, Per1 was still strongly expressed in dental pulp cells (). All four clock proteins were also strongly expressed by the alveolar bone osteoblasts at this stage ().
Figure 3 Bmal1, Clock, Per1 and Per2 expression in mouse teeth at P4. Bmal1 was expressed in the nucleus of ameloblasts and odontoblasts (A and E). Bmal1 protein was not uniformly localized in ameloblasts and odontoblasts and some cells were devoid of staining (more ...)
2.4. Per2protein expression in teeth and bone at PN21
At PN21 stage, the first molars are being erupted. Interestingly, all clock genes showed distinct expression pattern at the crown-analogue side versus the root-analogue side of the incisor. Bmal1, Clock, Per1 and Per2 are expressed in incisor root odontoblasts strongly. In contrast, clock proteins expression was low/undetectable at the crown-analogue side odontoblasts (). Odontoblasts in first molars were also strongly stained for all four clock proteins but dental pulp cells were devoid of staining (). Periodontal dental ligament (PDL) cells showed weaker expression for clock proteins compared with odontoblast expression. In contrast, within the PDL space, epithelial rests of Malassez (ERM) showed strong expression of all the four clock proteins studied here. Clock proteins were also detected in the nucleus of the osteoblasts and osteoclasts in the alveolar bone (). No clock proteins expression could be detected in cementoblasts or osteocytes ().
Figure 4 Per2 expression in mouse incisor (A) and molar (B–C) at PN21 stage. Per2 showed distinct expression pattern at the root-analogue versus the root-analogue side in the incisor (A). Per2 was detected in root odontoblasts and ameloblasts (A, black (more ...)
Taken all together, we speculate that the expression of clock proteins in tooth directly correlates with cell differentiation and initiation of matrix production. Indeed, clock proteins expression was clearly up-regulated in ameloblasts and odontoblasts of PN4 teeth. At this stage (PN4), dental pulp staining was low further supporting a key role of clock genes in enamel and dentin formation. We are currently analyzing enamel and dentin formation in knock out models of clock genes to definitively elucidate the potential roles of clock genes in enamel and dentin development.
Of interest, at PN21 incisors a down-regulation of clock proteins expression was observed in the odontoblasts of the crown-analogue side. In contrast, root analogue odontoblasts continue to express clock proteins (). Comparison of the two dentin portions (crown versus root-analogue) at the ultra-structural level has revealed that differences occur in the morphology of the secretory granules of the odontoblast layer (Beertsen and Niehof, 1986
). Therefore, differences in clock proteins expression levels may reflect differences in the composition of the non-collagenous matrix between the two dentin portions. In molar teeth clock genes were expressed in both crown and root odontoblasts. Differential gene expression has been described in molar versus incisor odontoblasts (Xing et al., 2007
). Our study supports the idea that mouse incisors may have different root patterning mechanisms from molars, potentially due to different genetic signals. More studies are needed to clarify the potential role of clock genes differential expression in the root-analogue versus the crown-analogue of the mouse incisor and the differences of expression between molar and incisor odontoblasts.
In conclusion, our results show that the main clock genes known to control circadian rhythms and related functions in many tissues, Bmal1, Clock, Per1 and Per2 are expressed in teeth. Clock protein expression was found preferentially in matrix secreting cells i.e. ameloblasts, odontoblasts and osteoblasts but not in cementoblasts. These results suggest that the clock genes may be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin matrix protein secretion and biomineralization. However, the clock genes studied here may not play a role in early tooth development.