MicroRNAs play a critical role in tooth development

Abstract

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

Figure 1.

Absence of enamel and formation of supernumerary incisors in Pitx2-Cre/Dicer1 cKO. (a) Two yellow-brown lower incisors (LI) and 2 yellow-brown upper incisors (UI) in one-month-old wild-type (WT) mice. (b) Supernumerary white incisors (asterisk) in one-month-old Pitx2-Cre/Dicer1 cKO mice. (c,d) Trichrome staining of two-week-old WT and Pitx2-Cre/Dicer1 cKO incisor. A complete absence of enamel (E, staining for red) in Dicer1 cKO incisor. Dentin (D, staining for blue) is present at both sides of the incisor in Dicer1 cKO, the same as in WT. Note that in Dicer1 cKO, odontoblast (Od) differentiation is relatively unaffected compared with ameloblasts (Am). UI, upper incisors; LI, lower incisors; LM, lower molars; Od, odontoblasts; Am, ameloblasts; D, dentin; E, enamel; Epi, epithelium.

Figure 2.

Expanded cervical loop region of incisors and repressed ameloblast differentiation in Pitx2-Cre/Dicer1 cKO. (a,b) Sagittal sections of E14.5 lower incisors, demonstrating smaller incisors in the Pitx2-Cre/Dicer1 cKO mice. (c,d) At P0, the expanded cervical loops and reduced epithelial cell differentiation were clearly observed in the mutant. Note that the cervical loop regions (CL, where tooth epithelial stem cells are located) of Pitx2-Cre/Dicer1 cKO are expanded compared with those in WT mice (lines bracket the CL). (e,g) In the WT lower incisor, tall and polarized ameloblast cells are present on the labial side. (f,h) In the Dicer1 cKO lower incisor, ameloblast cells are short and poorly polarized. (e,f) Higher magnification of (c,d). (g,h) Higher magnification of (e,f) boxed region. CL, cervical loop; Mes, mesenchyme; Epi, epithelium; Od, odontoblasts; Am, ameloblasts.

Figure 3.

Ameloblast differentiation is repressed in the Pitx2-Cre/Dicer1 cKO. (a-f) Ameloblast differentiation markers observed by immunohistochemistry of newborn (P0) wild-type and Pitx2-Cre/Dicer1 cKO lower incisors. (a-d) Secretory-stage ameloblast marker: Amelogenin and Ameloblastin. Note the dramatic decrease of amelogenin and ameloblastin in Pitx2-Cre/Dicer1 cKO compared with WT. (e,f) Pre-secretory-stage ameloblast marker: DSPP. DSPP was expressed in odontoblasts; however, it was expressed only in pre-secretory-stage ameloblasts. In Pitx2-Cre/Dicer1 cKO, DSPP was clearly expressed in odontoblasts. There are also a few cells in the epithelium compartment that express DSPP (arrows). Higher-magnification photos are shown in Appendix Fig. 6.