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The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development

Christine A. Ferguson^1,*, Abigail S. Tucker^1,*, Kristiina Heikinheimo², Masatoshi Nomura³, Paul Oh³, En Li³ and Paul T. Sharpe^1,

¹ Department of Craniofacial Development, GKT Dental Institute, Kings College London, Floor 28 Guy’s Hospital, London Bridge, London SE1 9RT, UK
² Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of Turku, FIN-20520 Turku, Finland
³ Cardiovascular Research Centre, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, MA 02129, USA
^* Present address: MRC Centre for Developmental Neurobiology, 4th Floor, New Hunt’s House, Guy’s Campus, King’s College London, London Bridge, London SE1 1UL, UK

View larger version (97K): [in a new window]   Fig. 1. Expression patterns of Irx1 and activin ßA in wild-type heads as seen by DIG whole-mount (A-E) and radioactive section (F-M) in situ hybridisation analyses. (B,C) Frontal vibratome sections through the branchial arches of the embryo in A. (E) Frontal vibratome section through the branchial arches of the embryo in D. At E9.5, DIG-labelled Irx1 probe localises to the branchial arch region (A), labelling the epithelium of the branchial clefts and more posterior arches (C). It is excluded from the oral epithelium of the first branchial arch (B). By E10.5-E11, Irx1 is strongly expressed in the oral epithelium of the first branchial arch (white arrowhead, D; E). Radioactive in situ hybridisation on wax sections was used to analyse the Irx1 expression in the developing teeth at E11 (F,G), E12.5 (J), E13.5 (L) and E14.5 (M). Irx1 expression can be found in the dental epithelia of the incisors (J) and molars (F,G,L,M). Analysis of consecutive serial sections using a 35S-labelled activin ßA probe shows that the expression of these two genes is juxtaposed in the developing teeth. Activin ßA is expressed in the odontogenic mesenchyme underlying the Irx1-positive dental epithelia at E11 (white arrowheads, H) and E12.5 (K). (F,I) Corresponding brightfield illumination of the sections in G,H, respectively, shows the histological detail. ba1, first branchial arch (mandibular process); bc1, first branchial cleft; bc2, second branchial cleft; oe, oral epithelium; mand, mandible; max, maxilla; tg, tongue.

View larger version (67K): [in a new window]   Fig. 2. Irx1 expression is absent from tooth germs in activin ßA mutants. Radioactive in situ hybridisation was used to compare Irx1 expression in frontal sections of wild-type (+/+, A,E) and mutant (-/-, B,D) heads. Irx1 expression is lost specifically from the epithelia of all incisor (B) and molar tooth buds (F) in mutants. Expression in non-odontogenic tissues such as the superficial dermis of the skin is, however, normal (white arrows, A,B,E,F). The position of the incisor and molar tooth buds in the mutant tissue is indicated by broken orange lines on the darkfield photomicrographs (B,F), corresponding brightfield (bf) photomicrographs (C,G, black arrowheads) and mesenchymal Pax9 expression in consecutive serial sections (D,H), which underlies the oral epithelium and is unaffected in the activin ßA mutants. Note that bud stage tooth germs of wild-type (E13.5) and mutant heads (E14.5) are compared here.

View larger version (157K): [in a new window]   Fig. 3. Tooth phenotype in newborn heads of ActR[IIA+/-;IIB-/-] (A,D) and Smad2+/- (C,F) pups. Corresponding frontal sections of wild-type littermates are shown in B,E. Both maxillary (A,C) and mandibular incisors (data not shown) are missing from the heterozygotes. Mandibular molars are lost from the heterozygotes in D,F, whereas maxillary molars develop normally. mol, molar; nc, nasal cartilages; ns, nasal septum; oc, oral cavity; tg, tongue. Note that the oral cavity in the heterozygotes is grossly mis-shapen. Furthermore, remnants of bone (arrowheads, asterisk) and muscle masses take the place of the mandibular bone.

View larger version (35K): [in a new window]   Fig. 4. The procedure followed for treatment of maxillary and mandibular explants with soluble activin receptors. A frontal view of an E10.5 embryo is shown. Representative molars and a cyst retrieved from the kidney capsule grafts are shown in section stained with Alcian Blue/chlorontine Fast Red. The molars have multiple cusps and a characteristic red-stained dentine layer, which is secreted by the underlying layer of odontoblasts. The amorphous cysts lack cusps and are epithelial, keratinising in nature rather than having cytodifferentiated odontoblasts and ameloblasts. ba2, second branchial arch; fnm, frontonasal mass; mand, mandibular process; max, maxillary process; np, nasal process.

View larger version (79K): [in a new window]   Fig. 5. Radioactive in situ hybridisation analysis of mandibular explants after 2 days in culture in the absence or presence of soluble human activin receptors IIA and IIB. Adjacent serial frontal sections through tooth buds were analysed using bright field illumination to show histology (A,E,I), using Msx1 expression to determine the viability of the explants and to assist with orientation of the tooth germs within the explants (B,F,J), using Irx1 (C,G,K) and follistatin (Fst) expression (D,H,L) in the dental epithelia of the explants to assess the efficiency of repression of the activin signalling pathway. Explants treated with 0 mg/ml (A-D), 12.5 mg/ml (E-H) and 25 mg/ml (I-L) soluble receptors are shown. Fst expression is faint but detectable in the control explants and those treated with 12.5 mg/ml soluble receptors. It is missing from explants cultured in the presence of 25 mg/ml or higher concentrations of soluble receptors. Irx1 expression, however, appears to require less activin signalling, as it is maintained using concentrations of up to 25 mg/ml, and is lost at higher concentrations of soluble receptors. The hybridisation signal seen close to the non-oral surface of the explants corresponds to Irx1 expression in the superficial dermis in G and Fst expression in the tongue in L. Orange asterisks indicate the position of tooth germs.

View larger version (63K): [in a new window]   Fig. 6. Radioactive in situ hybridisation analysis of maxillary explants after 2 days in culture in the absence or presence of soluble human activin receptors IIA and IIB. Adjacent serial sections through presumptive molar regions were analysed as explained in Fig. 5. using bright field illumination to show histology (A,E,F,J,K,O), and dark field illumination to show mesenchymal Msx1 expression (B,G,L), and epithelial Irx1 (C,H,M) and Fst expression (D,I,N). Explants treated with 0 mg/ml (A-E), 12.5 mg/ml (F-J) and 25 mg/ml (K-O) soluble receptors are shown. Note that in order to assist the reader to assess Fst expression in the explants, dark field (D,I,N) and the corresponding bright field photomicrographs (E,J,O) of these samples are shown. Asterisks mark the same relative positions on adjacent serial sections.