Search

Enamel knot (EK) is known to be a central organ in tooth development, especially for cusp patterning. To trace the exact position and movement among the inner dental epithelium (IDE) and EK cells, and to monitor the relationship between the EK and cusp patterning, it is essential that we understand the cell cycle status of the EK in early stages of tooth development. In this study, thymidine analogous (IdU, BrdU) staining was used to evaluate the cell cycle phase of the primary EK at the early casp stage (E13.0) and the gerbil embryo (E19) in a developing mouse embryo. The centerpiece of this study was to describe the cell cycle phasing and sequencing during proliferation in the IDE according to the expression of IdU and BrdU following their injection at calculated time points. The interval time between IdU injection and BrdU injection was set at 4 h. As a result, the cell cycle in the IDE of the mouse and gerbil was found to be synchronous. Conversely, the cell cycle in primary EKs of mice was much longer than that of the IDE. Therefore, the difference of cell cycle of the IDE and the EK is related to the diversity of cusp patterning and would provide a new insight into tooth morphogenesis.

Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE.

We examined the detailed in situ expression pattern of thymosin beta 4 (Tbeta4) in the developing mouse mandibular first molar. Tbeta4 mRNA was expressed in the presumptive dental epithelium at embryonic day 10.5 (E10.5) and in the thickened dental epithelium at E12. An in situ signal was observed in the invaginated epithelial bud at E13, in the enamel organ at E14 and E14.5, and in the primary enamel knot (PEK) at E14.5. The signal was localized in the epithelial cells of the outer layer of the enamel organ at E15 and E15.5. No signal was found in the PEK at these stages. Tbeta4 mRNA was expressed in the inner enamel epithelium, cervical loop and dental lamina at E16 and E17. The expression of Tbeta4 mRNA was observed in the polarized inner epithelial cells at E18, newborn day 1 (N1) and N2. However, the signal intensity decreased markedly at N3. We herein report for the first time that Tbeta4 is distinctly expressed in developing tooth germ, and it may also play functional roles in the initiation, growth and differentiation of tooth germ.

We analyzed the subcellular distribution of beta-catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated beta-catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, beta-catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear beta-catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, beta-catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of beta-catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.