Roles of Bmp4 during tooth morphogenesis and sequential tooth formation

Abstract

Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas all tooth germs were arrested at the bud stage in Msx1(-/-) mice, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4(f/f);Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We found that expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, was significantly upregulated in the molar tooth mesenchyme in Bmp4(f/f);Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity partially rescued mandibular first molar morphogenesis in Bmp4(f/f);Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1(-/-)Osr2(-/-) mice, Osr2(-/-)Bmp4(f/f);Wnt1Cre compound mutant mice exhibited formation and subsequent arrest of supernumerary tooth germs that correlated with downregulation of Msx1 expression in the tooth mesenchyme. In addition, we found that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wild-type embryos and that Dkk2 expression was significantly upregulated in the molar mesenchyme in Bmp4(f/f);Wnt1Cre embryos, which correlated with the dramatic differences in maxillary and mandibular molar phenotypes in Bmp4(f/f);Wnt1Cre mice. Together, these data indicate that Bmp4 signaling suppresses tooth developmental inhibitors in the tooth mesenchyme, including Dkk2 and Osr2, and synergizes with Msx1 to activate mesenchymal odontogenic potential for tooth morphogenesis and sequential tooth formation.

Fig. 1.

Loss of Bmp4 gene activity in the developing tooth mesenchyme in Bmp4^f/f;Wnt1Cre mutant embryos. (A,B) Bmp4 mRNA expression in the tooth mesenchyme in E13.5 control (A) and Bmp4^f/f;Wnt1Cre mutant (B) mouse embryos detected using cRNA probes specific for exon-4 sequences. Signal is shown in blue. Dashed line marks the boundary between the dental epithelium and mesenchyme. (C) Relative amounts of exon-4-containing Bmp4 mRNAs in the E13.5 maxillary and mandibular molar mesenchyme in control and Bmp4^f/f;Wnt1Cre mutant embryos. Ctr-max, control maxillary; Ctr-Man, control mandibular; mt-max, mutant maxillary; mt-man, mutant mandibular. The double asterisks indicate significant difference for the pair-wise comparison (P<0.01). Error bars represent s.e.m. (D,E) Immunofluorescence detection of phospho-Smad1/5 (shown in red) in the first molar tooth germs in E13.5 control (D) and Bmp4^f/f;Wnt1Cre mutant (E) embryos. (F,G) Comparison of Dlx2 expression in the first molar tooth germs of E13.5 control (F) and Bmp4^f/f;Wnt1Cre mutant (G) embryos.

Fig. 2.

Tooth phenotype in Bmp4^f/f;Wnt1Cre mutant mice. (A,B) Skeletal preparations of P21 control (A) and Bmp4^f/f;Wnt1Cre (B) upper jaws. (C,D) Skeletal preparations of P21 control (C) and Bmp4^f/f;Wnt1Cre (D) lower jaws. M1, M2 and M3 mark the first, second and third molars, respectively. Arrows point to the incisor region.

Fig. 3.

Molar developmental defects in the Bmp4^f/f;Wnt1Cre embryos. (A-L) Hematoxylin and Eosin-stained frontal sections through the developing first molar tooth germs of Bmp4^f/+;Wnt1Cre control (A,C,E,G,I,K) and Bmp4^f/f;Wnt1Cre mutant (B,D,F,H,J,L) mouse embryos from E13.5 to E16.5 are shown.

Fig. 4.

Comparison of levels of Bmp4 mRNAs in the maxillary and mandibular molar tooth mesenchyme in control and Msx1^–/– mutant mouse embryos. (A) E13.5. (B) E14.0. Asterisk indicates significant difference between the sample pair (P<0.05). Error bars represent s.e.m.

Fig. 5.

Comparison of Msx1 mRNA expression in developing tooth mesenchyme in control and Bmp4^f/f;Wnt1Cre mutant mouse embryos. (A-C) Comparison of Msx1 mRNA expression in E13.5 control and Bmp4^f/f;Wnt1Cre mutant molar tooth mesenchyme by in situ hybridization (A,B) and real-time RT-PCR (C). Dashed lines mark the boundary between the dental epithelium and mesenchyme. Arrows point to maxillary first molar and arrowheads point to mandibular first molar tooth germ. (D-F) Comparison of Msx1 mRNA expression in E14.5 control and Bmp4^f/f;Wnt1Cre mutant molar tooth mesenchyme by in situ hybridization (D,E) and real-time RT-PCR (F). The double asterisks in F indicate significant difference between the sample pair (P<0.01). Error bars represent s.e.m. Ctr-max, control maxillary; Ctr-Man, control mandibular; mt-max, mutant maxillary; mt-man, mutant mandibular.

Fig. 6.

Reducing Msx1 gene dosage enhanced tooth developmental defects in Bmp4^f/-;Wnt1Cre mutant mice. (A-C) Skeletal preparations of P21 mice showing the maxillary molar regions. M1, M2 and M3 mark the first, second and third molars, respectively. (D-F) Hematoxylin and Eosin-stained frontal sections through the maxillary first molar tooth germs at E18.5. (G-I) Hematoxylin and Eosin-stained frontal sections through the maxillary second molar tooth germs at E18.5.

Fig. 7.

Relative levels of expression of Dlx6, Dkk2, Wif1 and Osr2 mRNAs in the maxillary and mandibular molar mesenchyme in E13.5 control and Bmp4^f/f;Wnt1Cre mouse embryos. Error bars indicate s.e.m. ^*P<0.05, ^**P<0.01. Ctr-max, control maxillary; Ctr-Man, control mandibular; mt-max, mutant maxillary; mt-man, mutant mandibular.

Fig. 8.

Osr2 heterozygosity partially rescued mandibular first molar development in Bmp4^f/f;Wnt1Cre mice. (A-C) Skeletal preparations showing P21 mandibular molar tooth region in control (A), Bmp4^f/f;Wnt1Cre (B) and Osr2^+/–Bmp4^f/f;Wnt1Cre (C) mice. M1, M2 and M3 mark the first, second and third molars, respectively. (D-F) Bmp4 mRNA expression in the maxillary (upper) and mandibular (lower) first molar tooth germs in E14.5 control (D), Bmp4^f/f;Wnt1Cre (E) and Osr2^+/–Bmp4^f/f;Wnt1Cre (F) embryos. (G-I) pSmad1/5 immunofluorescent staining in the first molar tooth germs in the control (G), Bmp4^f/f;Wnt1Cre (H) and Osr2^+/–Bmp4^f/f;Wnt1Cre (I) embryos. Dashed lines mark boundary between dental epithelium and mesenchyme.

Fig. 9.

Comparison of PEK molecular marker expression in control, Bmp4^f/–;Wnt1Cre and Osr2^+/–Bmp4^f/–;Wnt1Cre mutant mouse embryos at E14.5. (A-C) P21 mRNA expression in the maxillary (Max) and mandibular (Man) first molar tooth germs. (D-F) Lef1 mRNA expression. (G-I) Fgf3 expression. Dashed lines mark boundary between dental epithelium and mesenchyme.

Fig. 10.

Osr2^–/–Bmp4^f/f;Wnt1Cre double mutant mice showed initiation and subsequent arrest of supernumerary tooth germs. (A-D) Hematoxylin and Eosin-stained frontal sections through the posterior region of mandibular first molar tooth germs in Osr2^–/– (A,C) and Osr2^–/–Bmp4^f/f;Wnt-Cre (B,D) embryos at P0 (A,B) and E15.5 (C,D). Arrowheads point to the supernumerary tooth germ. (E,F) Expression of Pitx2 mRNAs in the tooth germs in E15.5 Osr2^–/– (E) and Osr2^–/–Bmp4^f/f;Wnt1Cre (F) embryos. Dashed line marks boundary between the supernumerary dental placode epithelium and mesenchyme. (G,H) Expression of Msx1 mRNAs in the tooth mesenchyme in E15.5 Osr2^–/– (G) and Osr2^–/–Bmp4^f/f;Wnt1Cre (H) embryos. Arrows point to the first molar tooth germ and arrowheads point to the supernumerary tooth germ. Dashed lines mark the boundary between epithelium and mesenchyme of the developing supernumerary tooth germs.

Fig. 11.

Summary of the effects of Bmp4, Msx1 and Osr2 mutations on sequential molar development. Each row shows the schematic representation of the molar tooth patterns in a maxillary (Max) and a mandibular (Man) quadrant for the indicated genotype. Cross indicates tooth developmental arrest at the bud stage.