Fig 2: The images of H.E. staining and immunohistochemistry staining for prenatal rat dental germs. H.E. staining demonstrated that the rat dental germs entered the early cap stage at E14.5 d, the cap stage and early bell stage at E16.5 d, and the bell stage at E18.5 d. The separation between the pre-odontoblast and pre-ameloblast layers occurred in all E18.5 d species. Immunohistochemistry staining revealed that Clock, Per1, and Col1 were detected in the epithelial-mesenchymal interaction area, dental follicle, and dental papilla at E14.5 d, and became stronger at E16.5 d when p75NTR, Bmal1, and ALP were detected. All the factors were expressed at E18.5 d, but Cry1 showed the weakest expression. All experiments were repeated three times independently. op: oral epithelium; dp: dental papilla; iee: inner enamel epithelium; oee: outer enamel epithelium; sr: stellate reticulum. The scale bar represents 50 µm, respectively.

Fig 3: The in vivo assays for the effect of p75NTR knockout on the mRNA expression in dental germs at PN7d. As expected, p75NTR mRNA in the knockout mice was significantly lower than in THE wild-type mice (p < 0.05). Mage-D1, Bmal1, ALP, Col1, Msx1, Dmp1, and Dspp showed the same change in the p75NTR knockout mice, indicating a positive relationship with p75NTR. In contrast, Runx2 showed the reverse change, presenting a negative relationship with p75NTR. Clock, Per1, Per2, and Dlx1 showed no significant difference between p75NTR knockout and wild-type mice (p > 0.05). All experiments were repeated at least three times independently. Data are expressed as mean ± S.D. L. D. Condition group, n = 6; D. D. Condition group, n = 6.

Fig 4: The in vitro assays for the effect of p75NTR over-expression on the mRNA expression in immortalizing stem cells from dental apical papilla (iSCAP). p75NTR was expected to be significantly higher expressed in the over-expression group than that in the control group (p < 0.05). Mage-D1, Bmal1, Clock, Runx2, Col1, and Msx1 showed an apparent positive relationship when p75NTR was significantly up-regulated. In contrast, ALP, Dmp1, and Dspp showed a negative relationship with p75NTR. Per1, Per2, ALP, Dlx1, and Dmp1 showed no significant difference between the over-expression group and control group (p > 0.05) with lower than that in wild-type mice (p < 0.05). All experiments were repeated at least three times independently. Data are expressed as mean ± S.D. Overexpression negative control group, n = 3; p75NTR over-expression group, n = 3.

Fig 5: The in vivo observation of circadian rhythm dynamics in PN7 d dental germs of wild-type mice. p75NTR mRNA showed a significant difference between D.D. and L.D. conditions (p < 0.05), but the change tendencies at two sampling times were opposite: p75NTR mRNA expression in the L.D. condition was significantly higher at 7:30 a.m. and significantly lower at 7:30 p.m. in the D.D. condition (p < 0.05). The expression pattern of Mage-D1 was similar to that of p75NTR. Most factors (Bmal1, Clock, Per1, Per2, Runx2, ALP, Col1, and Dlx1) showed the same change tendency at the two sampling times: mRNA expression was significantly higher in the D.D. condition than that in the L.D. condition (p < 0.05). The change amplitudes of Clock, Per1, Per2, Runx2, and Dlx1 were significantly larger at the sampling time of 7:30 PM. Contrary to p75NTR, Msx1 mRNA expression in the L.D. condition was significantly lower at 7:30 a.m. and significantly higher at 7:30 p.m. compared with the D.D. condition (p < 0.05). Dmp1and Dspp mRNA expression in the D.D. condition was significantly lower than that in L.D. condition at both sampling times (p < 0.05). All experiments were repeated at least three times independently. Data are expressed as mean ± S.D. L. D. Condition group, n = 11; D. D. Condition group, n = 5.