Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors

Abstract

TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1-/- mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1-/- mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.

Fig. 1

Patterns of Tbx1 expression in oral epithelium and in developing incisors. Whole-mount in situ hybridization (A–C) and in situ hybridization on cryosections (D–J) using a digoxigenin-labeled probe. Longitudinal (D–G, J) and frontal (H, I) sections. (A–B) Frontal (A, B) and lateral (C) views of E10 and E10.5–11 mouse embryos showing Tbx1 mRNA expression (violet color) in oral epithelium. Red and green arrows indicate the incisor and molar growing areas respectively. Note that Tbx1 expression is stronger in the incisor areas. (D–J) Tbx1 expression in developing incisors during the bud (D, E), cap (F–I) and bell (J) stages. Boxes in panel J represent high magnifications of selected areas of hematoxylin/eosin stained sections. The blue color indicates territories of Tbx1 expression. Abbreviations: An, anterior part; cl, cervical loop; d, dentin; de, dental epithelium; df, dental follicle; ide, inner dental epithelium; La, labial part; Li, lingual part; m, mesenchyme; md, mandible; mx, maxilla; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla; pa, preameloblasts; po, preodontoblasts; Po, posterior part.

Fig. 2

Incisors (intact and sectioned) isolated from skeletal preparations of E18.5 wild-type and Tbx1−/− mouse embryos. (A) Maxillary (left) and mandibular (right) E18.5 wild-type (wt) incisors (top panel) and Tbx1−/− incisors (three bottom incisors). Mineral matrix deposition in red color (alizarin red staining). The double-headed orange arrows indicate the difference in size between the normal incisors and the mutant incisors. Black arrowheads indicate the area of the incisor where minerals start to deposit (i.e. mineralization front), and green arrowheads show the layer of dental epithelium. The green line at the posterior end of the incisors represents the mineralization gradient on the labial and lingual sides. (B–F) Sections of the above mutant incisors showing variations in mineral deposition and organization (red color). (B) Section of an entire Tbx1−/− incisor. (C) High magnification of the lingual side. (D–F) High magnifications of the anterior parts of three mutant incisors. Abbreviations: An, anterior part; La, labial side; Li, lingual side; Po, posterior part.

Fig. 3

Histological examination of various Tbx1 mutant incisors. Histological preparations of Tbx1−/− (A–C, E–J, L) and Tbx1+/− (D, K) E18.5 incisors. Longitudinal sections. Hematoxylin-eosin (A–C), Sirius red (D–J), and periodic acid-Schiff and methylene blue-Azur II (K, L) stainings. (A) Maxillary and mandibular incisors (i) in Tbx1−/− mouse. (B) Higher magnification of the maxillary incisor. Note the thin cervical loop epithelium at the labial side (La) and the thick equivalent area at the lingual side (Li). (C) Higher magnification of the cytodifferentiation area. Only preameloblasts (pa) are visible. The enamel is also absent. (D) Maxillary incisor. Note the thick cervical loop epithelium at the labial side and the thin equivalent part at the lingual side. (E) Massive necrotic areas in all tissues of the craniofacial complex. (F) Mandibular incisor. The red frame represents the blow up area of the next figure. (G) High magnification showing the deposition of predentin and the existence of preameloblasts in the anterior part of the incisor. No enamel is visible. (H) Another mandibular incisor. Red frames represent blow up areas of the next two figures. (I) Start of predentin deposition by preodontoblasts (po) at the transition part of the incisor. Inner dental epithelial cells (ide), but not preameloblasts (pa), are visible. (J) Anterior part of the incisor. Odontoblasts (o) and predentin/dentin (pd) are visible. Note in the epithelium the existence of small round cells instead of inner dental epithelial cells/preameloblasts. (K) High magnification of the cervical loop area. (L) Disintegration of epithelial cells at the cervical loop of Tbx1−/− incisors. Red dots indicate the form of dental epithelium. Additional abbreviations: cl, cervical loop; d, dentin; de, dental epithelium; df, dental follicle; md, mandible; mx, maxilla; ode, outer dental epithelium; oe, oral epithelium; p, pulp.

Fig. 4

Transplantation and culture of incisor germs in kidney capsules. (A–C) Isolated incisor germs used for the culture in kidney capsules. Dotted red lines outline the epithelial part of the incisors. (D–G) X-rays of Tbx1−/− (D–F) and Tbx1+/− (G) incisors developed in kidney capsules for 28 days. (H–J) Macroscopic vu of incisors grown in kidney capsules for 28 days. Red arrows indicate the side where the enamel is normally formed (yellow color). No yellow color is seen in the Tbx1−/− (H) incisors, while in the Tbx1+/− incisors (I, J) the color is evident. Abbreviations: An, anterior part; La, labial side; Li, lingual side; Po, posterior part.

Fig. 5

Histological examination of incisors developed in kidney capsules. Histological preparations of Tbx1+/− (A; left side) and Tbx1−/− (B; right side) incisors grown for 28 days. Red color boxes represent high magnifications of the labial side where normally enamel forms. Green color boxes are high magnifications of the areas in the lingual side. (A) Ameloblasts (a) and enamel (e) is detected in the Tbx1+/− incisors. (B) Ameloblasts and enamel are absent in the Tbx1−/− incisors. Orange arrows indicate cementoblasts. Additional abbreviations: An, anterior part; b, bone; c, cementum; cl, cervical loop; d, dentin; dec, dental cementoblasts; df, dental follicle; es, enamel space; kt, kidney tissue; La, labial side; Li, lingual side; o, odontoblast; ode, outer dental epithelium; oe, oral epithelium; p, pulp; pd, predentin; Po, posterior part; sr, stellate reticulum; v, vessels.

Fig. 6

Structural alterations in the ameloblasts of Tbx1−/− incisors developed in kidney capsules. Light- (top row) and transmission electron micrographs (rows 2–4) of the forming enamel and ameloblasts of wild-type (left column), Tbx1+/− (middle column), and Tbx1−/− (right column) incisors grown in kidney capsules for 28 days. Red arrows point to vacuoles in the cytoplasm and at the secreting pole of ameloblasts. Blue arrows point to the enamel formation front. Abbreviations: a, ameloblasts; d, dentin; e, enamel; o, odontoblasts; p, pulp; si, stratum intermedium; wt, wild-type.

Fig. 7

Cell apoptosis in Tbx1−/− incisors. Frontal (A, B) and longitudinal sections (C, D). Tbx1+/− (A) and Tbx1−/− (B–D) E18.5 incisors. Apoptotic cells exhibit a brown color. (A) Very few apoptotic epithelial cells are detected in the Tbx1+/− maxillary incisor. Apoptosis is evident in cells of the alveolar bone (ab). (B, D) Apoptosis is not detected in epithelial cells of the maxillary (B) and mandibular (C) incisors. Note apoptosis in the alveolar bone. (D) Higher magnification of the previous figure focusing in the cervical loop and transition areas of the mandibular incisor. Abbreviations: cl, cervical loop; de, dental epithelium; o, odontoblasts; p, dental pulp; pa, preameloblasts.

Fig. 8

Altered epithelial cell proliferation in Tbx1−/− incisors. High magnifications of the cervical loop area on the labial side of Tbx1−/− (A, B) and wild-type (C, D) E18.5 incisors. Proliferating cells exhibit a brown color after PCNA immunostaining. (A) Histology of the cervical loop of a mutant incisor. (B) Proliferating epithelial cells are restricted in the cervical loop area of a mutant incisor. Red dots indicate the form of dental epithelium. Red arrows indicate cells of the inner and outer dental epithelium. Note that proliferation is seen in epithelial cells of the inner cell mass. No proliferation is seen in inner dental epithelium. (C) Histology of the cervical loop of a wild-type incisor. (D) Intense cell proliferation is detected in all epithelial cell layers of a wild-type incisor. Red arrows indicate cells of the inner and outer dental epithelium. Note massive cell proliferations in cells of the inner dental epithelium. Abbreviations: cl, cervical loop; de, dental epithelium; df, dental follicle; ide, inner dental epithelium; p, dental pulp; wt, wild-type.

Fig. 9

Patterns of amelogenin expression in developing and mature wild-type and Tbx1−/− incisors. In situ hybridization on paraffin sections. (A) Expression of amelogenin in maxillary and mandibular E18.5–E18.75 wild-type (wt) and Tbx1−/− mouse incisors. Note that expression is stronger in wild-type incisors than in mutant incisors. The hybridization signal is stronger in the mandibular mutant incisors when compared to that of the maxillary incisors. (B) Expression of amelogenin on Tbx1−/− (red box) and wild-type (red box) incisors cultured in kidney capsules for 28 days. Amelogenin expression is absent in the Tbx1−/− incisors. Abbreviations: a, ameloblasts; cl, cervical loop; d, dentin; de, dental epithelium; e, enamel; es, enamel space; ide, inner dental epithelium; La, labial side; Li, lingual side; o, odontoblasts; p, dental pulp; si, stratum intermedium; v, vessels.

Fig. 10

Patterns of Tbx1 and amelogenin expression in developing wild-type and Spry4−/−;Spry2+/− incisors. In situ hybridization on cryosections. (A) Expression of Tbx1 in an E16.5 wild-type incisor. (B) Expression of Tbx1 in an E16.5 Spry4−/−;Spry2+/− incisor. Note that Tbx1 is expressed in the lingual side and the labial side. (C) Amelogenin expression in a P2 Spry4−/−;Spry2+/− incisor. Note that amelogenin is expressed in both lingual and labial sides. Abbreviations: a, ameloblasts; An, anterior part; cl, cervical loop; d, dentin; de, dental epithelium; ide, inner dental epithelium; La, labial side; Li, lingual side; o, odontoblasts; p, dental pulp; wt, wild-type.

Fig. 11

Schematic representation of a model illustrating the effects of Tbx1 in the proliferation and maintenance of the ameloblast progenitors. Cells of the stem cell niche at the cervical loop of the rodent incisor that express Tbx1 (dark blue cells of the inner dental epithelium) will give rise to ameloblasts and form enamel. Tbx1 forms a regulatory loop with FGFs in teeth. Tbx1, in combination with FGF molecules (red arrows), promotes the proliferation and maintenance of ameloblast progenitors in incisors. Proliferation of cells of the inner dental epithelium (i.e. ameloblast progenitors), but not of cells of the inner cell mass (icm), is disrupted after Tbx1 deletion. As a consequence of this disruption ameloblast differentiation and enamel matrix production are severely affected. Additional abbreviations: ide, inner dental epithelium; ode, outer dental epithelium; p, dental pulp; si, stratum intermedium; sr, stellate reticulum.