Circadian Rhythm Regulates Development of Enamel in Mouse Mandibular First Molar

Abstract

Rhythmic incremental growth lines and the presence of melatonin receptors were discovered in tooth enamel, suggesting possible role of circadian rhythm. We therefore hypothesized that circadian rhythm may regulate enamel formation through melatonin receptors. To test this hypothesis, we examined expression of melatonin receptors (MTs) and amelogenin (AMELX), a maker of enamel formation, during tooth germ development in mouse. Using qRT-PCR and immunocytochemistry, we found that mRNA and protein levels of both MTs and AMELX in normal mandibular first molar tooth germs increased gradually after birth, peaked at 3 or 4 day postnatal, and then decreased. Expression of MTs and AMELX by immunocytochemistry was significantly delayed in neonatal mice raised in all-dark or all-light environment as well as the enamel development. Furthermore, development of tooth enamel was also delayed showing significant immature histology in those animals, especially for newborn mice raised in all daylight condition. Interestingly, disruption in circadian rhythm in pregnant mice also resulted in delayed enamel development in their babies. Treatment with melatonin receptor antagonist 4P-PDOT in pregnant mice caused underexpression of MTs and AMELX associated with long-lasting deficiency in baby enamel tissue. Electromicroscopic evidence demonstrated increased necrosis and poor enamel mineralization in ameloblasts. The above results suggest that circadian rhythm is important for normal enamel development at both pre- and postnatal stages. Melatonin receptors were partly responsible for the regulation.

Fig 1. Expression of MT1, MT2 (A) and amelogenins (B) by qRT-PCR in developing enamel of the mandibular first molar of mouse at different ages.

The results of the qRT-PCR are depicted as means ± SD (n = 30 in each group) * p<0.05, **p<0.01.

Fig 2. Immunocytochemistry of melatonin receptors in developing first molar tooth germ of mice at different ages (n = 6 in each age group).

A, microphotograph with 10x objective; B, microphotograph with 40x objective. Scale bar, 10μm. Labels in photos: A, ameloblast; AB, alveolar bone; I, inner enamel epithelium; M, mineralization layer; O, odontoblast; P, dental papilla cell; SI, stratum intermedium.

Fig 3. Immunocytochemistry of amelogenins proteins in developing first molar tooth germ of mice at different ages (n = 6 in each age group).

A, microphotograph with 10x objective; B, microphotograph with 40x objective. Scale bar, 10μm. Labels in photos: A, ameloblast; I, Inner enamel epithelium; M, mineralization layer; O, odontoblast; P, dental papilla cell; SI, stratum intermedium.

Fig 4. Messenger RNA by qRT-PCR of MT1, MT2 and amelogenins in developing enamel of the mandibular first molar in mice experienced all dark or all light since birth.

D3, animals deprived daylight or darkness for 3 days since birth. D4, animals deprived daylight or darkness for 3 days followed by one day of normal day-night cycle. The results of the qRT-PCR are depicted as means ± SD (n = 3 in each group).*, p<0.05, **, p<0.01.

Fig 5. Immunocytochemistry of melatonin receptors in the first molar tooth germ of mice raised in either night-deprived or daylight-deprived conditions (D3) as well as mice experienced three day circadian rhythm derivation followed by one day-night cycle (D4) (n = 6 in each group).

A, microphotograph with 10x objective; B, microphotograph with 40× objective. Scale bar, 10μm. Labels in photos: A, ameloblast; I, inner enamel epithelium; M, mineralization layer; O, odontoblast; P, dental papilla cell.

Fig 6. Immunocytochemistry of amelogenins in the first molar tooth germ of mice raised in either night-deprived or daylight-deprived conditions (D3) as well as mice experienced three day circadian rhythm derivation followed by one day-night cycle (D4) (n = 6 in each group).

A, microphotograph with 10x objective; B, microphotograph with 40x objective. Scale bar, 10μm. Labels in photos: A, ameloblast; I, inner enamel epithelium; M, mineralization layer; P, dental papilla cell; O, odontoblast.

Fig 7. Expression of MT1, MT2 and amelogenins by qRT-PCR in developing enamel of the mandibular first molar of newborn mice from mothers with deprived circadian rhythm.

The results of the qRT-PCR are depicted as means ± SD (n = 8 in each group). * p<0.05, ** p<0.01.

Fig 8. Immunocytochemistry of amelogenin and MT in the tooth germ of newborn mice (D0) from mothers night-deprived or melatonin receptor blocked.

A, microphotograph with 10x objective; B, microphotograph with 40x objective. Scale bar, 10μm. Labels in photos: I, inner enamel epithelium; O, odontoblast; P, dental papilla cell; SI, stratum intermedium.

Fig 9. Average heights of ameloblasts (% of control animals) of postnatal day 3 mice of all-day raised, all-night raised and born from 4P-PDOT treated mothers.

The data are depicted as means ± SD (9 sections from 3 animals for each group).* p<0.05.

Fig 10. Electromicroscopic photographs of ameloblasts of mandibular first molar tooth germ in baby mice of postnatal day 7 and 10 from mothers treated with 4P- PDOT, a melatonin receptor blocker.

A, ultrastructure of ameloblasts, 13500x; B, hydroxyapatite matrix and enamel rod, 24500x. Labels in photos: E, enamel rod; M, mitochondria; N, nucleus. Arrows point to rough surfaced endoplasmic reticulum.