Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis

Abstract

The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2(-/-) mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2(-/-) mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.

Fig. 1.

Ctip2 expression profile during tooth development. (A–J) IHC in coronal sections of mouse embryonic heads at indicated developmental ages. Ctip2 staining appears green in all panels. (A) Expression of Ctip2 in the ectoderm of the 1st BA and future oe at E9.5. (B) Expression of Ctip2 in de at E10.5 and across the 1st BA. (C and D) Ctip2 expression in the oe, de, and cmes of a developing lower molar (C) and upper incisor (D) at E12.5. (E–G) Ctip2 expression in the developing lower molar at E14.5 (E), E16.5 (F), and E18.5 (G). (H–J) Double-label IHC of coronal sections of a lower incisor at E18.5 using antibodies against Ctip2 and amelogenin and counterstained with DAPI. The IHC data presented in this figure have been reproduced 4–6 times over the course of ≈2 years. [Scale bars: (A, B, D, G) 200 μm; (C, E, F) 100 μm; (H) 20 μm.] a, ameloblast; BA, branchial arch; cl, cervical loop; cmes, condensing mesenchyme; dc, dental cord; de, dental epilethium; iee, inner enamel epithelium; oe, oral epilethium; oee, outer enamel epithelium; p, papilla, si, stratum intermedium; sr, stellate reticulum.

Fig. 2.

Defects in tooth development in Ctip2^−/− mice. H & E staining in coronal sections of WT (A, C, E, G, I, K, M, O) and Ctip2^−/− (B, D, F, H, J, L, N, P) mice at E14.5 (A, B, K, L), E16.5 (C, D, G–J, M, N), and E18.5 (E, F, O, P). (G–J) Higher magnification of (C) and (D), respectively, highlighting the lingual (G and H) and labial (I and J) sides of a developing incisor. Note the elongated dental cord (B, L, N), reduced and disorganized ameloblast layer (D, F, J), and loss of lingual/labial asymmetry (G–J) in Ctip2^−/− mice. The black asterisk (J) indicates a reduced stellate reticulum on the labial side of a developing incisor; the red asterisks represent ectopic ameloblast-like cells on the lingual-side mutant incisors (H) and lack of these cells in WT incisors (G); and the green asterisks indicate the epithelial expansion on the lingual side of mutant incisors (H) and the corresponding cells in WT mice (G). All histology studies presented in this figure are representative of at least 4 independent mice of each genotype. [Scale bars: (A–F, K–P) 100 μm; (G–J) 200 μm.] de, dental cord; em, enamel matrix; m1, first molar; mes, mesenchyme; o, odontoblast.

Fig. 3.

Ameloblast defects in Ctip2^−/− mice. IHC on coronal sections of a mandibular incisor using indicated antibodies in WT (A, C, E, G, I, K, M, O, Q, U, W, Y) and Ctip2^−/− (B, D, F, H, J, L, N, P, R, V, X, Z) mice at E16.5 (U–Z) and E18.5 (A–R). All sections were counterstained with DAPI. Amelogenin- (A–F), Ameloblastin- (G–L), and Enamelin- (M–R) expressing ameloblasts were severely reduced in Ctip2^−/− mice. A secondary ameloblast-like layer was present on the lingual side of the Ctip2^−/− mice (white arrows in D, J, and P). Ameloblasts were reduced in size (white arrows in B, H, and N) with deformed or absent adhesion points on the labial side of incisors in the Ctip2^−/− mice (white stars in F, H, L, N, and R). (S) RT–quantitative PCR (RT-qPCR) analyses comparing levels of Amelogenin, Ameloblastin, Enamelin, and Laminin 5-3a expression in mandibular tissue of WT and Ctip2^−/− mice at E18.5. Expression levels of all 4 genes were significantly decreased in the mutants (P < 0.05 in all cases). (T) ChIP assays on proximal and distal promoter regions (as defined in Table S1) of the indicated genes conducted using qPCR. The ratio of amplification products present in immunoprecipitates from WT and Ctip2^−/− mice was determined to indicate the specificity of the ChIP signal in WT mice. Data shown in A–R and U–Z are representative of 3 and 5 similar experiments, respectively. RT-qPCR and ChIP data presented in (S) and (T) represent averages from studies using tissue from 3 (ChIP) to 6 (RT-qPCR) independent mice of each genotype. (U and V) Decreased expression levels of amelogenin in mutant ameloblasts at E16.5. The white dotted line represents the boundary between ameloblasts and odontoblasts in (V). (W and X) Immunostaining with an anti-ß-tubulin antibody and DAPI. Y and Z indicate the DAPI stained nuclei of the ameloblats indicated in the versions of (W) and (X), respectively. White dots in (W–Z) denote the ameloblast apical boundary. Nuclei of WT ameloblasts were predominantly found on the basal surface (position labeled “1” in Y) or in the middle of the cell (position “2”), but rarely on the apical aspect (position “3”). In contrast, nuclei were randomly distributed throughout the mutant ameloblasts (positions 1–3 in Z). [Scale bars: (A–R) 200 μm; (U–X) 100 μm; (Y and Z) 50 μm.]

Fig. 4.

Ctip2 acts upstream of Msx2 and epiprofin during ameloblast differentiation. (A–F) Double-label IHC on coronal sections of lower molars using indicated antibodies in WT mice at E14.5 (A) and E16.5 (D). Ctip2 and Msx2 are colocalized in the enamel knot, dental and oral epithelia. (Scale bars: 200 μm.) Images shown in panels (B) and (E) depict Msx2 staining in WT mice at E14.5 and E16.5, respectively. Msx2 expression in Ctip2^−/− mice at E14.5 (C), and E16.5 (F). (G) Comparative levels of expression of Msx2, epiprofin, and Sp3 in WT and Ctip2^−/− mandibles at E16.5, as determined by RT-qPCR. Expression of Msx2 and epiprofin, but not that of Sp3, was reduced in the mutants (P < 0.05). (H) ChIP assays on the proximal and distal promoter regions (as defined in Table S3) of the indicated genes from WT and Ctip2^−/− mandibles at E16.5. Data shown in (A–F) are representative of 4 similar experiments, whereas the studies presented in (G) and (H) represent averages of 3 to 5 mice of each genotype.