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First published online January 13, 2009
doi: 10.1242/10.1242/dev.025064

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Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction

Developmental Biology Program, Institute of Biotechnology, PO Box 56, University of Helsinki, FIN-00014, Helsinki, Finland.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Sostdc1 expression is mostly confined to the mesenchyme, and Sostdc1-deficient mice have extra incisors and expanded expression patterns of Shh. (A-D) Radioactive and whole-mount in situ hybridization show Sostdc1 expression becoming localized to the underlying mesenchyme of lower incisor region between E12 and E13 (C,D). The incisor and molar placodes are outlined in B and D. (E) At E14 cap stage, Sostdc1 expression is intense in the mesenchyme at the lingual side of the incisor and in dental papilla, and it can be detected in the incisor epithelium, whereas it is absent from the mesenchyme under the enamel knot. (F) Bmp4 colocalizes with Sostdc1 but shows additional expression in the enamel knot and dental papilla at E14. (G,H) The extra incisors are in the Sostdc1-deficient lower jaw, mesiolingual to the main incisors. (I,J) During development, Sostdc1-deficient jaws have enlarged expression of Shh in the epithelium. (K,L) The epithelially restricted Shh expression is visible in histological sections. e, dental epithelium; ek, enamel knot; dp, dental papilla; vl, vestibular lamina; la, labial; li, lingual. Scale bars: 0.2 mm in A-F,I-L; 0.5 mm in G,H.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Apoptosis, seen in wild-type odontogenic epithelium at E14 stage, is absent from Sostdc1-deficient jaws and the site correlates with the epithelial Wnt activity seen at E13.5 stage. (A,B) Sagittal sections show apoptotic cells (detected using TUNEL staining) in regions of the epithelium of control teeth, the enamel knot and a site near the oral surface (A), whereas apoptosis is absent in the Sostdc1-deficient teeth (B). (C,D) Sagittal sections from control (C) and Sostdc1-deficient (D) incisor region in the TOPgal-Wnt reporter mouse (at E13.5) show epithelial Wnt activity in the forming enamel knot and at the site that correlates with the extensive epithelial apoptosis in the E14 control teeth (A). Comparable, albeit weaker, Wnt activity was detected in BATgal mice (not shown). Scale bars: 0.2 mm in A,B; 0.1 mm in C,D.

View larger version (53K): [in this window] [in a new window]   Fig. 3. 3D-reconstruction reveals the origins of the extra incisor in relation to the main incisor. (A) Sostdc1-deficient and heterozygous controls possessing the TOPgal-Wnt reporter gene show differential epithelial Wnt activity in a sagittal projection of the 3D reconstruction. In addition to the forming enamel knot, an additional epithelial Wnt activity site was observed inside the Sostdc1-deficient incisor (circle). (B) 3D reconstructions of E16 and E17 Sostdc1-deficient and wild-type jaws show the site of extra incisor formation in relation to the main incisor. This location is at the mesiolingual aspect of the main incisor and corresponds to the site of ectopic Wnt activity seen at E13 and E14 (A). oe, oral epithelium; vl, vestibular lamina; CL, cervical loop; ek, enamel knot. Scale bars: 0.2 mm.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Extra incisors develop in cultured wild-type incisors after minimizing the amount of tooth-surrounding mesenchyme. (A,B) A dissected E13 incisor with a large amount of tooth-surrounding mesenchyme (A) exhibits more Sostdc1 expression than an incisor from which most surrounding mesenchymal tissue has been removed (B). The broken lines show the approximate border between epithelium and mesenchyme. (C,D) In vitro culture experiments show that extra incisors develop in wild-type explants when most of the surrounding mesenchyme is removed before culture (C). When the surrounding mesenchyme is present in wild-type incisor explants, no extra incisors develop (D). Scale bars: 0.5 mm in A,B; 1 mm in C,D.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Additional de novo incisors develop in cultured Sostdc1-deficient incisors after minimizing the amount of tooth-surrounding mesenchyme. (A-D) De novo incisors form from the lingual epithelium of the Sostdc1-deficient incisors after the removal of most of the incisor-surrounding mesenchyme. The larger white asterisks show the extra incisor that appears first and the smaller asterisks indicate de novo incisors (A-D). (E-H) Sostdc1-deficient incisor explants with the surrounding mesenchyme intact show only formation of one extra incisor (asterisks) (I) Schematic drawings summarize the possible ways to produce extra incisors in in vitro cultures. Pink indicates Sostdc1 expression in the surrounding mesenchyme and in the lingual cervical loop epithelium. Scale bars: 1 mm in A-H.

View larger version (80K): [in this window] [in a new window]   Fig. 6. Fluorescent DiI label reveals the different origins of extra and de novo incisors in Sostdc1-deficient mouse. (A,B) DiI was injected in the in vitro cultured E13 Sostdc1-deficient (A) and wild-type incisor explants (see Fig. S3 in the supplementary material) that expressed GFP under the Shh promoter. The dye was injected at the epithelial site where the extra incisor starts to form. The dye became incorporated into the forming extra incisor (large asterisks), but not into the main incisor. The de novo incisor (small asterisks) forms from or close to the lingual cervical loop epithelium of the main incisor and lacks DiI label (B). (C) Schematic drawings summarize the bright-field (A) and fluorescent (B) pictures; green is the GFP expression and red is the injected DiI. Asterisk indicates extra incisors. Scale bar: 0.5 mm.

View larger version (58K): [in this window] [in a new window]   Fig. 7. Acceleration of extra incisor formation by BMP4. (A,B) Incisors from Sostdc1-deficient mice, that express GFP under the Shh promoter, have wider enamel knot area after BMP4 treatment. (C) Cumulative percentage curves show the appearance of extra incisor. Sostdc1-deficient explants (n=18/18), Sostdc1-deficient explants with BMP4-soaked bead (n=16/16), trimmed Sostdc1-deficient explants (n=24/24) (of which 80% formed de novo incisors), trimmed wild-type explants n=17/17. (D-F) In vitro culture experiments show that Sostdc1-deficient tissue has increased sensitivity to BMP4; a BMP4-soaked bead (blue) placed on top of the lingual incisor surrounding mesenchyme accelerates the development of extra incisors in the Sostdc1-deficient incisors (E) but not in the control (F). Arrow indicates the extra incisor. Scale bars: 0.25 mm in A,B; 1 mm in D-F.

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