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First published online 11 February 2009
doi: 10.1242/dev.027979

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Primary cilia regulate Shh activity in the control of molar tooth number

¹ Department of Craniofacial Development, Dental Institute, King's College London, London SE1 9RT, UK.
² Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
³ Department of Craniofacial Development and Orthodontics, Dental Institute, King's College London, UK.
⁴ Department of Embryology, Carnegie Institution of Washington 3520 San Martin Drive, Baltimore, MD 21218, USA.
⁵ Department of Teratology, Institute of Experimental Medicine, Academy of Sciences of the CR, Prague, Czech Republic.
⁶ INSERM UMR977, Dental School, Strasbourg University, Strasbourg, France.

View larger version (78K): [in this window] [in a new window]   Fig. 1. Lower molar tooth phenotypes and Shh signalling in Tg737orpk homozygous mutant mice. (A-D) Whole teeth (A,B) and sagittal sections (C,D) show that supernumerary teeth develop mesial to the first molars (sn in B,D) in mutants. (E,F) Increase of Ptch1-lacZ staining in the mandible of Tg737orpk mutants with Ptch1-lacZ allele at E13.5. Ptch1-lacZ staining was expanded into the diastema region (arrowhead). Arrows indicate molar germs. (G-J) Radioactive in situ hybridisation on sagittal sections showing Gli1 (G,H), Gas1 (I,J) and Shh (G',H',I',J') expression in mandibles of embryo heads at E12.5. (G,H,I,J) Yellow circles represent positions of Shh expression in adjacent sections (blue arrowheads in G',H',I',J'). Gli1 expression (H) was expanded into the diastema and Gas1 (J) was downregulated in the diastema of Tg737orpk mutants (green arrowheads). (K-N) Sagittal sections (N) and three-dimensional reconstructions (K-M) of the dental epithelium of the mandible molar region at E15.5 (N) and E16.5 (K-M) in Tg737orpk mutants. The supernumerary tooth buds (arrows in K-M) were observed in the position of the mesial swellings (arrowhead in N). (O-T) SEM analysis shows that the lingual cusp of maxillary supernumerary tooth (red line in T) is more prominent in comparison with that of mandibular supernumerary tooth (red line in S). (U,V) Horizontal micro-CT sections of Tg737orpk show that maxillary supernumerary teeth have concave roots (circle in V) and in mandibles show round roots (circle in U). m1, first molar; m2, second molar; m3, third molar; mand, mandible jaw; max, maxillary jaw.

View larger version (73K): [in this window] [in a new window]   Fig. 2. Cilia, and Tg737 expression and localisation in tooth germs. (A-C) Acetylated -tubulin-positive cells (immunohistochemistry) are found in both tooth epithelium and mesenchyme of wild type (A,B). There are no significant differences between wild-type and Tg737orpk mice, or between first molar (m1) and supernumerary tooth (sn) in Tg737orpk mice (C). (D,E) -Tubulin-positive cells are observed in both tooth epithelium and mesenchyme of wild-type (D) and Tg737orpk mice (E). (F-I) TEM analysis of molar teeth show that cilia were found in both epithelium (G) and mesenchyme (F) of molar tooth germs in wild-type (F,G) and in molar tooth epithelium (H) and mesenchyme (I) in Tg737orpk (arrows). (J,K) β-Gal expression in molar of Tg7372-3β-gal show that Tg737 was expressed in both epithelium and mesenchyme. (L,M) Immunohistochemistry sections also show that polaris protein is localised in both dental epithelium (DE) and dental mesenchyme (DM). Frontal sections (A,B,D-J,L,M) and sagittal sections (C,K) of lower molars at E10.5 (A), E13.5 (B,D-J,L), E14.5 (C), E16.5 (M) and E18.5 (K) of wild-type littermates (A,B,D,F,G,J-M) and Tg737orpk mutants (C,E,H,I). Dots represent boundaries between epithelium and mesenchyme (A,M). Tooth epithelium is outlined in red (B-E) or green (L). Nuclei are shown in blue by DAPI (A'-E',L,M).

View larger version (116K): [in this window] [in a new window]   Fig. 3. Molar teeth of K5-Cre/polarisflox/flox mice and Wnt1-Cre/polarisflox/flox mice. (A,B) There are no supernumerary teeth in K5Cre (A) and K5-Cre/polarisflox/flox (B) mice. (C) Sagittal sections showing first molar (m1) and second molar (m2) in mandible. (D) Supernumerary teeth (sn) were observed in Wnt1-Cre/polarisflox/flox mice. (E-F') Radioactive in situ hybridisation on sagittal sections showing Gli1 (E,F) and Shh (E',F') expression in mandibles of embryo heads. (E,F) Yellow circles represent positions of Shh expression in adjacent sections (blue arrowheads in E',F'). Gli1 expression (E,F) was expanded into the diastema of Wnt1-Cre/Polarisflox/flox mice (green arrowheads). Images show lower mandibles of E11.5 (E-F'), newborn (C,D) and adult (A,B).

View larger version (90K): [in this window] [in a new window]   Fig. 4. Molar tooth phenotypes of Gas1 mutant mice. (A-F) Sagittal sections (A-D) and micro-CT analysis (E,F) show that supernumerary teeth develop mesial to the first molars (sn in C,F). Micro-CT analysis showed that supernumerary teeth were smaller than first molars and had fewer cusps (F). At the section level of the second molar, the supernumerary teeth were not visible (D). (G,H) Radioactive in situ hybridisation on sagittal sections, showing Gas1 expression in mandibles of embryo heads at E13.5. Gas1 was expressed in the diastema region (arrowheads), whereas it was absent in tooth regions (incisor in G; molar in H). (I,J) Whole-mount in situ hybridisation showing Ptch1 expression in the mandibular process of embryo heads at E12.5. Ptch1 expression was expanded into the diastema region in Gas1 mutants (arrowheads in J). (K,L) Radioactive in situ hybridisation on sagittal sections showing Gli1 expression in mandibles of embryo heads at E14.5. Gli1 expression was expanded into the diastema region of Gas1 mice (arrowhead in L). (N,P,R) Radioactive in situ hybridisation on sagittal sections showing Shh expression in maxillae of embryo heads at E14.5. Shh expression was found in both supernumerary (sn in R) and the first molars in Gas1 mutants (m1 in P). (M,O,Q) Haematoxylin and Eosin stained sections adjacent to N, P and R, respectively. Shh expression could not be seen in the first molar at the section level of the supernumerary tooth (R). (S-U) Immunohistochemistry on sagittal sections showing Shh protein localisation in mandibles of embryo heads at E12.5. Shh localisation was found in the diastema region of Gas1 mice (between arrowheads in U), as well as endogenous tooth germs (arrowheads in T); it was not observed in the diastema of wild type (between arrowheads in S). Shh protein deposition could not be seen in the incisors and molars at the section level of the diastema showing Shh expression (U). m1, first molar; m2, second molar; sn, supernumerary tooth. Lingual cusps (L1-L3) and extra cusp (ec). Scale bar: 500 µm.

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