Figure 1.
Slice organ culture of snake mandibles allows the formation of several generations of teeth.
(A–C) Newborn corn snake (Pantherophis guttatus). (A) Alcian blue-Alizarin red skeletal staining of the head. (B) Mandible microCT scan. (C) Oral view of erupted teeth. (A–B) White arrows indicate functional teeth. (B) Red arrow and arrowhead point to replacement teeth. The arrowhead indicates the younger replacement tooth. (D) 3-day cultured corn snake dental organ. Fibrillar-Actin (green) is used to show the outline of the developing tooth germs. (E) Schematic of the regions of the dental organ represented in D. (F) 30-days post-oviposition snake embryo. (G) Mandible from embryo in C. (G′) Magnification of the framed area in G, dental organs can be identified throughout the mucosa indicated by arrows and dotted circles in the inset. (H–K) 18-day culture period. Several generations of teeth develop over this period. (L) Histology of dental lamina after 10 days in culture. (H–L) Black arrows: tooth germs; 1 g, 2 g, 3 g, 4 g, are 1st, 2nd, 3rd, and 4th generations of teeth. Black arrowheads: successional lamina. White arrows: dental lamina. White arrowhead: origin of dental lamina from the oral epithelium. Scale bars: (A) 100 µm, (C,F) 1 cm, (G) 1 mm, (D,E) 500 µm, (G′,H–K) 250 µm, (L) 100 µm.
Table 1.
Pantherophis guttatus cDNAs cloned in this study.
Figure 2.
Fate and expression pattern of the dental lamina in the snake.
(A–D) 10-day culture of the snake dental organ labelled with DiI. (A, B, C) fluorescence microscopy and (D) confocal optical section. (A) 0-day culture. DiI labelling was performed at the region of the successional lamina (arrowhead). (B) 2-day culture. (C) 7-day culture. (B,C) The label remains in the region of the successional lamina (arrowhead) and in the forming second tooth (green asterisk). (D) Day 10. Magnification in D shows the framed area. Label is present in the second (green asterisk) and third (yellow asterisk) generation tooth germs and is retained in the successional lamina (arrowhead) and adjacent mesenchyme (white asterisk). (E–F) Pantherophis guttatus Lef1 mRNA is located in the successional lamina region (arrowhead), as observed by whole mount (E) and section (F) in situ hybridization. (G,G′,H) Sox2+ cells are found in the oral (asterisk) and aboral dental lamina (white arrows) and outer enamel epithelium (grey arrows), but are excluded from the successional lamina (yellow arrowhead in G′, H). (G′) magnification of the region indicated in G. (H) Lingual plane showing the connection of the dental lamina with the oral epithelium. Scale bars: 100 µm.
Figure 3.
GSK3β inhibitors induce Wnt/β-catenin overactivation and increase the number of tooth germs in the snake.
(A) RT-PCR analysis of Axin2 and Lunatic fringe (Lnfg) mRNA levels of the dental organ indicating Axin2 and Lnfg are up-regulated after LiCl treatment. EF1α was used as a loading control. (B–G) F-Actin detection after 3 days of culture in (B) Control, (D,F) LiCl, (C) DMSO and (E,G) BIO. Asterisks: dental lamina, White arrows: tooth germs; yellow arrowhead: successional lamina. (H) The volume of the dental organ does not change after GSK3β inhibitor treatment. (I) Number of new tooth germs that appeared during 3 days of treatment and (J) percentage of the number of tooth germs in 3-day snake cultures after treatment, n = number of slices analyzed. (K,L) Morphology of dental organs after 7 days of control culture (K) and LiCl culture (L). Asterisks: dental lamina, Black arrows: tooth germs. (M–Q) Histology of explanted dental organs after 10 days in culture: (M) control, (N) LiCl treatment. (O) DMSO control and (P,Q) BIO treatment. White arrows: tooth germs and epithelial projections. Yellow arrow: dysmorphic tooth germ. White asterisks: dental lamina. Yellow asterisks: acanthosis in dental lamina. Scale bars: 100 µm.
Figure 4.
Treatment with LiCl alters the expression pattern of the dental lamina.
(A–D) Lef1 expression in 3-day culture (A, B) and 10-day culture (C–D) assessed by in situ hybridization. (A,C) Polarized pattern of expression of Lef1 in control cultures. (B,D) loss of localized expression of Lef1 after treatment with LiCl. (E,F) Sox2 immunofluorescence of 10-day culture in (E) control and (F) LiCl treatment. Arrows highlight the positive signal. Scale bars: 100 µm.
Figure 5.
GSK3β inhibitors increase the proliferation of the dental lamina.
(A–D) Immunofluorescence against phospho-Histone H3 (pH3) (red) in explants after 3 days of culture. DNA (Hoechst) is in blue. LiCl increases the number of pH3+ cells in the dental organ and the dental lamina appears wider in treated cultures (asterisk in B,D compared to A,C). (E, F) Quantification of the samples represented in A–D showing (E) increase of pH3+ cells and (F) increase in mitotic index after LiCl and BIO treatment, n = number of slices analyzed. (G) Distribution of the number of pH3+ cells along the different regions of the dental organ represented in the scheme. Significant differences were found in the dental lamina after LiCl and BIO treatment. Scale bar: 100 µm.
Figure 6.
Working model for the emergence of tooth germs.
The Wnt/β-catenin pathway controls the proliferative and molecular patterning of the snake dental lamina allowing the ordered emergence of tooth germs. In control cultures, Wnt/β-catenin signalling via Lef1 is concentrated at the successional lamina (tip of the dental lamina). This region proliferates and elongates, regulating organized emergence of the tooth germs. In GSK3β inhibitor treatment Lef1 expression is expanded and proliferation increases in the rest of the dental lamina, which becomes wider and shorter. The dental lamina becomes odontogenic and ectopic tooth germs and epithelial projections appear.