Figure 1.
BCL11B expression during incisor development.
BCL11B immunostaining in sections of wild-type mice at indicated developmental stages. The epithelium is outlined by white dots. Scale bars: (A-B) 100 µm; (C) 200 µm; (D-E) 500 µm. a, ameloblasts; an, anterior; cl, cervical loop; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; iee, inner enamel epithelium; lab, labial; lin, lingual; oee, outer enamel epithelium; pm, papillary mesenchyme; po, posterior; sr, stellate reticulum; vl, vestibular lamina.
Figure 2.
Epithelial invagination defect in Bcl11b−/− developing incisors between initiation and early bud stage.
(A-D) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined in black. (E-H) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (I-J) BrdU index of wild-type and Bcl11b−/− dental epithelium; *** denotes statistical significance at p ≤ 0.001, n = 3. (K-N) RNA ISH using a Bmp4 probe in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows denote epithelial staining. Scale bar, 100 µm.
Figure 3.
Alterations in Bcl11b−/− incisor development at cap stage.
(A-B) H&E staining in sections of wild-type and Bcl11b−/− mice at E14.5. The epithelium is outlined in black. (C-D) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice at E14.5. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (E) BrdU index of wild-type and Bcl11b−/− CLs; *** denotes statistical significance at p ≤ 0.001, n = 3. (F-G) RNA ISH using an Fgf10 probe in sections of wild-type and Bcl11b−/− mice at E14. Black arrows indicate mesenchymal staining; black asterisks denote the staining between dental epithelium and vestibular lamina. Scale bar, 200 µm. cl, cervical loop; ek, enamel knot; vl, vestibular lamina.
Figure 4.
Morphological defects in Bcl11b−/− incisor development at bell stage.
(A-H, L-Y) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined in black. Brackets indicate the posterior end of the incisors in A, E, L, N. Panels D, H, T, Y are higher magnification images of boxes in A, E, L, N, respectively. White arrows indicate lingual epithelium. I denotes the labial CL; II-IV denote stages of progression of ameloblast differentiation in L, N, P-S, U-X. The Bcl11b−/− labial CL was smaller than that of wild-type mice by 60% at E16.5 and by 69% at E18.5 (p ≤ 0.001, n = 11), whereas the mutant lingual CL was enlarged by 35% at E16.5 and by 64% at E18.5 relative to wild-type tissue (p ≤ 0.001, n = 11). (I-J) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice at E16.5. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (K) BrdU index of wild-type and Bcl11b−/− basal epithelium of labial and lingual CLs; ns, not significant; *** denotes statistical significance at p ≤ 0.001, n = 3. Scale bars: (A-S, U-X) 200 µm; other panels, 100 µm. a, ameloblasts; cl, cervical loop; lab, labial; lin, lingual.
Figure 5.
Altered ameloblast development in Bcl11b−/− incisors.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.
Figure 6.
Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Scale bars: (A-B, E-F, I-J, M-N, Q-R) 500 µm; other panels, 200 µm.
Figure 7.
Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Black and red asterisks denote staining in the posterior part of the dental follicle and epithelial tip of the incisor, respectively. Scale bars: (A-B, E-F, I-J) 500 µm; other panels, 200 µm.
Figure 8.
Ectopic lingual expression of ameloblast markers and signaling molecules in Bcl11bep−/− incisor.
(A-B) BCL11B immunostaining in sections of Bcl11bL2/L2 and Bcl11bep−/− mice at E16.5. The epithelium is outlined by white dots. (C-L) RNA ISH using the indicated probes in sections of Bcl11bL2/L2 and Bcl11bep−/− incisors at E16.5. The epithelium is outlined by red dots. Black and red arrowheads denote lingual mesenchymal and epithelial staining, respectively. Scale bar, 500 µm.
Figure 9.
Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/− mice at E16.5.
BCL11B immunostaining in sections of wild-type and Spry4−/−; Spry2+/− mice at E16.5. The epithelium is outlined by white dots. White arrowheads denote lingual epithelial staining. Scale bar, 500 µm.
Figure 10.
Model for a reciprocal, inhibitory circuit on the lingual side of the mouse incisor.
(A) Proposed model for integration of BCL11B into FGF signaling pathways in the lingual epithelium of wild-type incisors. This model suggests that BCL11B expression in the lingual epithelium is facilitated by SPRY2- and SPRY4-mediated inhibition of the FGF signaling pathways. The resulting, high levels of BCL11B directly or indirectly suppress expression of Shh and subsequently lead to disruption of pre-ameloblast differentiation and ameloblast formation. It is also proposed that BCL11B inhibits expression of Fgf9 in the lingual epithelium, and this serves to enforce disruption of the FGF signaling loop that is mediated by the Sprouty proteins. The repressive effect of BCL11B on Fgf3 and Fgf10 expression is most likely a secondary effect and is depicted by a dashed line. Therefore, BCL11B participates in the suppression of both FGF-mediated stimulation of lingual EpSC proliferation and differentiation of EpSC into Shh-positive pre-ameloblasts within the lingual IEE. (B) In the absence of Spry4 and one allele of Spry2, FGF signaling is enhanced, which extinguishes expression of BCL11B in the lingual IEE. Loss of BCL11B results in derepression of Fgf9 and Shh expression in the epithelium. FGF9 drives Fgf3 and Fgf10 expression in the lingual mesenchyme, whereas Shh expression is required for development of ectopic pre-ameloblasts within the lingual IEE.