Figure 1.
The in situ expression of Itm2a mRNA in the tooth germ during the fetal stages.
The in situ Itm2a expression was temporally and spatially stage-specific during the tooth germ development. A & B. The in situ Itm2a signals were not detected in either the epithelial or ectomesenchymal cells corresponding to the predicted lower first molar region before or after oral epithelium thickening. C & D. At the bud stage, the in situ expression of Itm2a was not observed in the tooth germ. E & F. At the cap stage, the in situ expression of Itm2a was observed in outer enamel epithelial cells (red arrows), inner enamel epithelial cells (black arrows) and in the enamel knot (asterisk). G & H. At the early bell stage, the in situ expression of Itm2a was found in inner enamel epithelial cells (black arrows), epithelial cells in the tip of the cervical loop (green arrows) and in the outer enamel epithelial cells (red arrows). Li; lingual side, Bu; buccal side. Scale bars: 100 µm (A–D), 200 µm (E–H).
Figure 2.
The in situ expression of Itm2a mRNA in the developing tooth germ at the late bell stage.
A. On PN0, the in situ expression of Itm2a was detected in the inner enamel epithelial cells (black arrow). B. The boxed area in A is shown at a higher magnification. The in situ expression of Itm2a was detected in the inner enamel epithelial cells (black arrow) and odontoblasts (white arrowhead). C. An HE-stained section of the tooth germ on PN1 is shown, corresponding to the region shown in B. D. On PN1, the initiation stage of matrix formation, in situ Itm2a signals were observed in the ameloblasts (white arrow) and odontoblasts (white arrowheads). E & G. The red- and blue-boxed areas in D are shown at a higher magnification, respectively. The in situ expression of Itm2a was detected in the ameloblasts (white arrow) and odontoblasts (white arrowheads). An in situ Itm2a signal was also observed in preameloblasts (blue arrow). F & H. HE-stained sections of the tooth germ on PN1 are shown, corresponding to the regions shown in E and G, respectively. Li; lingual side, Bu; buccal side. Scale bars; 200 µm (A, D), 100 µm (B, C, E–H).
Figure 3.
The in situ expression of Itm2a mRNA in the developing tooth germ at the matrix formation stage and the initiation stage of root formation.
A–C. During matrix formation, an in situ Itm2a signal was detected in the ameloblasts (white arrows), preameloblasts (blue arrows), odontoblasts (white arrowheads) and preodontoblasts (black arrowheads). The signal was also detected in the osteoblasts (red arrowheads) of the bone tissue surrounding the tooth germ. D. The in situ Itm2a signal was observed in the ameloblasts (white arrow), odontoblasts (white arrowhead) and preodontoblasts (black arrowhead). E. The boxed area in D is shown at a higher magnification. The in situ expression of Itm2a was detected in the ameloblasts (white arrow) and odontoblasts (white arrowhead). Itm2a expression was also detected in the HERS (yellow arrow). F. An HE-stained section of the tooth germ on PN5 is shown. Li; lingual side, Bu; buccal side. Scale bars; 200 µm (A–D), 100 µm (E, F).
Figure 4.
The in situ expression of Itm2a mRNA in the developing tooth germ at the tooth root formation and the tooth eruption stages.
A. An HE-stained section shows the tooth germ on PN10 at the tooth root formation stage. B. The in situ signal of Itm2a was observed in the tooth germ on PN10. C. The boxed area in B is shown at a higher magnification. The in situ expression of Itm2a was detected in the ameloblasts (white arrow), odontoblasts (white arrowhead) and HERS (yellow arrow). D. An HE-stained section shows the tooth germ on PN20 at the tooth eruption stage. On PN20, the root formation is almost completed, and the crown is exposed. E. The Itm2a expression was observed in the odontoblasts and HERS. F. The boxed area in E is shown at a higher magnification. The in situ expression of Itm2a signal was detected in the odontoblasts (white arrowhead) and HERS around the root apex (yellow arrow). Li; lingual side, Bu; buccal side. Scale bars; 200 µm (A, B, D, E), 100 µm (C, F).
Figure 5.
The Itm2a protein expression in the tooth germ on E10.5–E14.
A–D. The expression of the Itm2a protein was not observed in the epithelial or ectomesenchymal cells corresponding to the predicted lower first molar region either before or after oral epithelium thickening, or at the bud stage. The dotted lines indicate the contours of the oral epithelial layer and/or tooth bud. E & F. The boxed areas in B and D are respectively shown at a higher magnification. Li; lingual side, Bu; buccal side. Scale bars; 100 µm (A–D), 50 µm (E, F).
Figure 6.
The Itm2a protein expression in the tooth germ on E15–E18.
A. At the cap stage (E15), the inner enamel epithelium (black arrows) exhibited the expression of the Itm2a protein, but the signal was weak. A strong signal was observed in the outer enamel epithelium (red arrows) and dental sac (blue arrowheads). B. At the cap stage (E16), the inner enamel epithelium (black arrows) exhibited a strong signal for the Itm2a protein, as did the outer enamel epithelium (red arrows). C & D. At the early bell stage (E17–18), the Itm2a protein was detected in the outer enamel epithelium cells (red arrows), the inner enamel epithelium cells (black arrows) and dental sac (blue arrowheads). E & F. The yellow- and green-boxed areas in B are respectively shown at a higher magnification. G. The boxed areas in D are shown at a higher magnification. Li; lingual side, Bu; buccal side. Scale bars; 100 µm (A, B), 200 µm (C, D), 50 µm (E–G).
Figure 7.
The Itm2a protein expression during tooth germ development on PN0–PN2.
A. Itm2a protein expression was observed in the inner (black arrows) and outer (red arrows) enamel epithelial cells of neonatal mice. The protein signal was also detected in the dental sac (blue arrowheads). B. After starting matrix formation, a strong protein signal was observed in the ameloblasts (white arrows) and odontoblasts (white arrowheads). C. In addition to the ameloblasts (white arrows) and odontoblasts (white arrowheads), the osteoblasts (red arrowheads) of the bone tissue surrounding the tooth germ exhibited a strong protein signal. D & E. The yellow- and green-boxed areas in B are respectively shown at a higher magnification. Itm2a protein expression was observed in the preameloblasts (blue arrow). Li; lingual side, Bu; buccal side. Scale bars; 200 µm (A–C), 50µm (D, E).
Figure 8.
The Itm2a protein expression during tooth germ development on PN3-PN5.
A & B. The ameloblasts (white arrows), odontoblasts (white arrowheads) and the osteoblasts (red arrowheads) of the bone tissue surrounding the tooth germ exhibited a strong protein signal. C. At the initiation stage of root formation, a strong Itm2a signal was observed in the odontoblasts (white arrowheads) and the osteoblasts (red arrowheads) of the bone tissue. The Itm2a signal was also detected in the ameloblasts (white arrows) and HERS (yellow arrows). D–F. The yellow-, green- and orange-boxed areas in C are respectively shown at a higher magnification. Li; lingual side, Bu; buccal side. Scale bars; 200 µm (A–C), 50 µm (D–F).
Figure 9.
The subcellular localization of the Itm2a protein expression in cultured dental epithelial cells.
The subcellular localization of the Itm2a protein in murine dental epithelial cells, mDE6 cells, which had been transfected with a pcDNA3.1(+)/Hygro vector encompassing Itm2a-EGFP cDNA (Itm2a-EGFP-mDE6 cells) was determined. The Itm2a in the Itm2a-EGFP-mDE6 cells was detected to have a punctate cytoplasmic distribution, which was shown as a green signal (left panels). The Itm2a-EGFP-mDE6 cells were stained with antibodies for cell organelle markers to indicate the cell organelles as follows: (A) Golgi apparatus, (B) lysosome, (C) endoplasmic reticulum (ER) and (D) mitochondria. These organelles were represented by red signals (second panels from the left). The nucleus was stained with DAPI (second panels from the right). The green signals indicating Itm2a, and red signals for each organelle and DAPI- counterstained nuclear images were merged, and are shown in the right panel. The yellow arrowheads indicate the colocalization of the organelle marker signal and Itm2a protein signal. Scale bars; 10 µm.