Augmented BMPRIA-mediated BMP signaling in cranial neural crest lineage leads to cleft palate formation and delayed tooth differentiation

Abstract

The importance of BMP receptor Ia (BMPRIa) mediated signaling in the development of craniofacial organs, including the tooth and palate, has been well illuminated in several mouse models of loss of function, and by its mutations associated with juvenile polyposis syndrome and facial defects in humans. In this study, we took a gain-of-function approach to further address the role of BMPR-IA-mediated signaling in the mesenchymal compartment during tooth and palate development. We generated transgenic mice expressing a constitutively active form of BmprIa (caBmprIa) in cranial neural crest (CNC) cells that contributes to the dental and palatal mesenchyme. Mice bearing enhanced BMPRIa-mediated signaling in CNC cells exhibit complete cleft palate and delayed odontogenic differentiation. We showed that the cleft palate defect in the transgenic animals is attributed to an altered cell proliferation rate in the anterior palatal mesenchyme and to the delayed palatal elevation in the posterior portion associated with ectopic cartilage formation. Despite enhanced activity of BMP signaling in the dental mesenchyme, tooth development and patterning in transgenic mice appeared normal except delayed odontogenic differentiation. These data support the hypothesis that a finely tuned level of BMPRIa-mediated signaling is essential for normal palate and tooth development.

Figure 1. Enhanced BMP activity in CNC-derived tissues via caBMPRIa causes complete cleft palate.

(A, C, E) Whole mount and coronal sections show normal palatal shelf of P0 wild type mice. Black lines in (A) indicate section levels shown in (C) and (E). (B, D, F) Whole mount and coronal sections show complete cleft (denoted by asterisk) of the secondary palate of P0 Wnt1Cre;pMes-caBmprIa mice. Note presence of ectopic cartilages (arrows) in craniofacial region. Black lines in (B) indicate section levels shown in (D) and (F). (G–J) Coronal sections of P0 control and Wnt1Cre;pMes-caBmprIa mice show comparable morphology of upper and lower incisors. Note enlarged nasal septal cartilage in transgenic animal. (K, L) Coronal sections of P0 control and transgenic mice show first molar structure with less differentiated odontoblasts and ameloblasts (inserts) in transgenic animal. T, tongue; AM, ameloblasts; LI, lower incisor; NS, nasal septum; OB, odontoblasts; PS, palatal shelf; UI, upper incisor. Scale bar = 500 µm.

Figure 2. Deformed structure and delayed elevation of palatal shelves in Wnt1Cre;capMes-caBmprIa mice.

(A–C) Coronal sections of E13.5 control and Wnt1Cre;pMes-caBmprIa embryos show deformed morphology of palatal shelves in transgenic animals. Note the presence of ectopic condensed cell masses (arrows) within in the posterior palatal shelves of the transgenic embryo (Fig. 2D). (E–H) Coronal sections of E14.5 wild type and Wnt1Cre;pMes-caBmprIa embryos show delayed elevation of palatal shelves in transgenic animal. M, Meckel's cartilage; T, tongue; PS, palatal shelf. Scale bar = 500 µm.

Figure 3. Reduced cell proliferation rate in the anterior palatal mesenchyme of Wnt1Cre;pMes-caBmprIa embryo.

(A–H) Coronal sections show BrdU-labeled cells in the palatal shelves of E12.5 (A–D) and E13.5 (E–H) control and Wnt1Cre;pMes-caBmprIa embryos. Square box in each panel indicates the area where total cells and BrdU-positive cells were counted. (I) Comparison of percentage of BrdU-labeled cells in the designated area of the palatal shelves in the control and transgenic animals. Standard deviation values were presented as error bars, and ** indicates P<0.01.

Figure 4. Altered BMP/Smad signaling activity and gene expression in Wnt1Cre;pMes - caBmprIa palatal shelves.

(A–D) Immunostaining shows pSmad1/5/8 signals in the palatal mesenchyme of E13.5 wild type (A, C) and transgenic embryos (B, C). Note in the anterior palatal shelf, pSmad1/5/8 signals were shifted to the future oral side (arrow) in the anterior palatal mesenchyme (B) and were ectopically activated (arrow) in the posterior palatal mesenchyme (D) of the transgenic palatal shelves. (E–H) In situ hybridization shows unaltered Shox2 expression in the anterior palatal mesenchyme (F) but an ectopic Shox2 expression domain (arrow) in the posterior palatal shelf (H) of E13.5 Wnt1Cre;pMes-caBmprIa embryo as compared to the counterpart of controls (E, G). (I–L) In situ hybridization shows a strong Msx1 expression domain (arrow) in the oral side of anterior palatal mesenchyme (J) and an ectopic Msx1 expression domain in the posterior palatal shelf (L) of E13.5 Wnt1Cre;pMes-caBmprIa embryo as compared to the controls (I, K). T, tongue; PS, palatal shelf.

Figure 5. Ectopic activation of BMP non-canonical signaling pathways in Wnt1Cre;pMes-caBmprIa palatal shelves.

(A, B) In situ hybridization shows ectopic expression of BmprIa in the palatal mesenchyme of E13.5 transgenic embryo (B), compared to BmprIa expression in wild type littermate (A). (C–H) Immunohistochemical staining shows expression of activated BMP non-canonical signaling mediators in E13.5 control and transgenic palatal shelves. Note ectopic expression (arrows) of P-p38 (D) and P-JNK (H) in the transgenic palatal mesenchyme. (I, J) Immunohistochemical staining shows expression of pSmad2/3 in E13.5 control (I) and transgenic palatal shelves (J).

Figure 6. Enhanced BMP signaling induces ectopic cartilage formation in the palatal shelves.

(A) In situ hybridization detects Col II expression in the Meckel's cartilage but not in the palatal shelf of an E13.5 wild type embryo. In situ hybridization shows an ectopic Col II-positive domain (arrow) within the palatal shelf of an E13.5 Wnt1Cre;pMes-caBmprIa embryo. (C) Alcian blue staining shows presence of an ectopic cartilage (arrow) within the palatal shelf of an E13.5 Wnt1Cre;pMes-caBmprIa embryo. (D) In situ hybridization shows a small ectopic Col II-positive cell mass (arrow) in the palatal shelf of an E13.5 Wnt1Cre;pMes-caBmprIa;BmprIa ^F/+ embryo. (E, F) Whole mount and section of P0 Wnt1Cre;pMes-caBmprIa;BmprIa ^F/+ mice show normal palate formation. Insert in (F) shows well differentiated ameloblasts and odontoblasts. T, tongue; Am, ameloblasts; Od, odontoblasts; PS, palatal shelf.

Figure 7. Unaffected early molar development and gene expression in Wnt1Cre;pMes - caBmprIa mice.

(A, B) Immunostaining shows enhanced pSmad1/5/8 signals in the molar germ of E13.5 Wnt1Cre;pMes-caBmprIa embrys (B) as compared to the control (A). (C–F) Coronal sections show comparable molar structures of E14.5 (C D) and E16.5 (E, F) wild type (C, E) and transgenic embryos (D, F). (G–L) In situ hybridization shows comparable expression levels and patterns of Msx1 (G, H), Shh (I, J) and Fgf4 (K, L) in the molars of E14.5 controls (G, I, K) and transgenic embryos (H, J, L). de, dental epithelium; dm, dental mesenchyme.

Figure 8. Enhanced BMP signaling activity does not affect size and cusp patterning but delays odontogenic differentiation.

(A, B) Whole mount images of P0 wild type (A) and Wnt1Cre;pMes-caBmprIa (B) molar shows comparable size and cusp patterns. (C–F) In situ hybridization shows strong expression of Amelogenin and Dspp in P0 wild type molar (C, E), but barely detectable expression of these two genes in P0 transgenic molar (D, F). (G, H) histological analyses show deposition of dentin and enamel in tooth grafts of wild type control (G) and Wnt1Cre;pMes-caBmprIa (H) molar after 2 weeks in kidney capsule culture. Inserts in (H) show Dspp and Amelogenin expression in the transgenic grafts. D, dentin; E, enamel; AM, ameloblasts, OB, odontoblasts. Scale bar = 200 µm.