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. 2015 Apr 15;24(8):2330-48.
doi: 10.1093/hmg/ddu750. Epub 2015 Jan 2.

TBX1 Protein Interactions and microRNA-96-5p Regulation Controls Cell Proliferation During Craniofacial and Dental Development: Implications for 22q11.2 Deletion Syndrome

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TBX1 Protein Interactions and microRNA-96-5p Regulation Controls Cell Proliferation During Craniofacial and Dental Development: Implications for 22q11.2 Deletion Syndrome

Shan Gao et al. Hum Mol Genet. .
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Abstract

T-box transcription factor TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS, DiGeorge syndrome/Velo-cardio-facial syndrome), whose phenotypes include craniofacial malformations such as dental defects and cleft palate. In this study, Tbx1 was conditionally deleted or over-expressed in the oral and dental epithelium to establish its role in odontogenesis and craniofacial developmental. Tbx1 lineage tracing experiments demonstrated a specific region of Tbx1-positive cells in the labial cervical loop (LaCL, stem cell niche). We found that Tbx1 conditional knockout (Tbx1(cKO)) mice featured microdontia, which coincides with decreased stem cell proliferation in the LaCL of Tbx1(cKO) mice. In contrast, Tbx1 over-expression increased dental epithelial progenitor cells in the LaCL. Furthermore, microRNA-96 (miR-96) repressed Tbx1 expression and Tbx1 repressed miR-96 expression, suggesting that miR-96 and Tbx1 work in a regulatory loop to maintain the correct levels of Tbx1. Cleft palate was observed in both conditional knockout and over-expression mice, consistent with the craniofacial/tooth defects associated with TBX1 deletion and the gene duplication that leads to 22q11.2DS. The biochemical analyses of TBX1 human mutations demonstrate functional differences in their transcriptional regulation of miR-96 and co-regulation of PITX2 activity. TBX1 interacts with PITX2 to negatively regulate PITX2 transcriptional activity and the TBX1 N-terminus is required for its repressive activity. Overall, our results indicate that Tbx1 regulates the proliferation of dental progenitor cells and craniofacial development through miR-96-5p and PITX2. Together, these data suggest a new molecular mechanism controlling pathogenesis of dental anomalies in human 22q11.2DS.

Figures

Figure 1.
Figure 1.
miR-96 targets Tbx1 and Tbx1 represses miR-96 expression. (A) Schematic depiction of the mouse LI. miR-96 is expressed at low levels in the LI LaCL, but increased in the differentiating ameloblast cells. Dn, dentin; En, enamel; Am, ameloblasts; Od, odontoblasts; LaCL, labial cervical loop; LiCL, lingual cervical loop. (B) Heat map of miR-96 differentially expressed between the LaCL (stem cell niche) and Am (differentiating cells) region. Five biological samples were assayed for each region, and one sample is shown. (C) The Tbx1 3′UTR miR-96 binding site is highly conserved among species. The sequence of this region that is mutated in the Mut Tbx1 3′UTR (miR-96 seed seq.; underlined) results in an inability to bind miR-96. (D) Normalized luciferase activity of the 3′-UTR Tbx1-luciferase reporter (WT Tbx1 3UTR) in the presence of empty plasmid (Vector) or CMV-miR-96 (miR-96) shows that luciferase activity is lost when miR-96 is expressed; this is not the case when the miR-96 seed sequence is mutated (Mut Tbx1 3′UTR). Error bars indicate ± SEM, five independent experiments (n = 5); P < 0.001. (E) Expression of miR-96 in LS-8 oral epithelial cells repressed endogenous Tbx1 expression. Real-time PCR experiments measured Tbx1 transcripts with and without miR-96 expression and normalized to control gene (N = 3). (F) Western blot analysis shows that Tbx1 levels decrease when miR-96 is over-expressed in LS-8 oral epithelial-like cells. GAPDH served as a loading control. (G) Tooth germ RNA isolated from WT and Tbx1K14COET mice mandibles demonstrated significantly decreased miR-96 expression. Real-time PCR was performed on three biological samples and each experiment was performed in triplicate (N = 3).
Figure 2.
Figure 2.
The Tbx1 N-terminus is required for PITX2 interaction and repression of PITX2 transcriptional activity. (A) A schematic of the Tbx1 truncated proteins used in the GST pull-down and transfection assays. The black shaded region is the T-box DNA binding domain. (B) GST pull-down using GST-Tbx1 truncated proteins to bind purified PITX2 protein. The PITX2 bound protein was resolved on 10% PAGE gel transferred to PVDF filters, immunoblotted and detected using PITX2ABCDE antibody (Capra Science, Sweden) and ECL reagents. PITX2 bound to Tbx1 full-length (FL), Tbx1ΔC (C-terminus deleted) and Tbx1ΔTC (T-box and C-terminus deleted). PITX2 did not bind to the Tbx1 T-box (Tbx1T-box) or Tbx1 C-terminus (Tbx1ΔNT), also PITX2 did not bind to Tbx1ΔN (data not shown). (C) Tbx1 truncations were tested in transfection assays to determine their activity and ability to repress PITX2 transcriptional activation of the Pitx2c promoter. As expected Tbx1 activated the Pitx2c promoter at low levels and the Tbx1ΔC and Tbx1ΔN proteins did not activate the promoter. However, deletion of the Tbx1 N-terminus (Tbx1ΔN) did relieve the repressive effect of Tbx1 on PITX2 transcriptional activation of the Pitx2c promoter. Luciferase activity is shown as mean-fold activation compared with activity in the context of the empty expression plasmid (Vector). All luciferase activities were normalized to β-galactose expression; five independent experiments were performed in LS-8 cells (N = 5). (D) Western blot of transfected cells to show expression in transfected LS-8 cells. Whole-cell lysates (30 μg) were resolved on 10% polyacrylamide gels, and PITX2 and Tbx1 truncated proteins were detected using an antibody against the Myc tag. All Tbx1 truncated proteins were expressed and denoted by an asterisk. (E) Real-time PCR experiments from cells transfected with Tbx1 or empty vector demonstrate decreased levels of endogenous Pitx2 transcripts by Tbx1 over-expression (N = 3).
Figure 3.
Figure 3.
TBX1 human mutants differentially regulate PITX2 transcriptional activity. (A) Schematic representation of human TBX1 isoforms and mutations. TBX1C, TBX1A and TBX1B are made by alternative splicing of the last exons and the T-box is shown. (B) TBX1 VC (variant C), TBX1 G310S (G-S) and TBX1 H194Q (H-Q) were transfected or cotransfected with PITX2 and the Pitx2c promoter. Each transfection used 0.1 µg reporter, 0.1 µg PITX2 and 0.25, 0.05, 0.1 µg of the TBX1 construct. To control for transfection efficiency, all transfections included the SV-40 β-galactosidase reporter (0.05 μg). Cells were incubated for 24 h and then assayed for luciferase and β-galactosidase activities. The activities are shown as mean-fold activation compared with the luciferase plasmid with empty vector and normalized to β-galactosidase activity ±SE from three independent experiments. TBX1 VC slightly activated the Pitx2c promoter (6-fold) and showed a dose-responsive repression of PITX2 transcriptional activity. TBX1 G-S did not activate the Pitx2c promoter and also showed a dose-responsive repression of PITX2 activity albeit at lower levels compared with WT TBX1 VC. However, TBX1 H-Q did not activate the Pitx2c promoter or repress PITX2 transcriptional activation of the Pitx2c promoter. (C) Western blots of transfected LS-8 cells to demonstrate expression levels of TBX1 mutant proteins. Whole-cell lysates (30 μg) were resolved on 10% polyacrylamide gels, and TBX1 mutant proteins were detected using an antibody against the Myc tag. GAPDH served as a loading control. TBX1 VC, TBX1 G-S and TBX1 H-Q transfected proteins were expressed in the cells.
Figure 4.
Figure 4.
Endogenous Tbx1 binds to the miR-96 chromatin. (A) Top panel is a schematic representation of the miR-96 5′-flanking region upstream of pri-miR-96. The Tbx1 binding site is shown and the primer regions used to amplify the chromatin as well as the control primers upstream of the Tbx1 binding site. Bottom panels, ChIP of endogenous Tbx1 binding to the T-box element upstream of the pri-miR-96 transcript in LS-8 cells, lane 7. IgG did not IP the Tbx1 binding site chromatin, lane 6. IgG and Tbx1 Ab did not IP the control region (non-specific primers) upstream of the Tbx1 binding sequence, lanes 2 and 3, respectively. Rabbit antisera was used as a control IP and Tbx1 antibody (Invitrogen) was used to IP Tbx1 binding to the chromatin. The input chromatin is shown as a positive control for the ChIP. (B) Expression plasmids containing the murine Tbx1, human TBX1 variant C, TBX1 G310S and TBX1 H194Q cDNAs were co-transfected into LS-8 cells with the miR-96 5 kb luciferase reporter plasmid. Luciferase activity is shown as mean-fold activation compared with that in the presence of empty mock expression plasmid. All luciferase activities were normalized to β-galactose expression, three independent experiments (N = 3). (C) Tbx1, PITX2 and miR-96 were co-transfected in LS-8 cells with the Pitx2c luciferase promoter and luciferase activity measured as in Figure 2. All luciferase activities were normalized to β-galactose expression, three independent experiments (N = 3).
Figure 5.
Figure 5.
Tbx1 expressing cells are in a unique region of the LaCL. (A) Schematic representation of depiction of the mouse LI. The LaCL contains dental stem cells that supply the continuously growing mouse incisor with replacement cells. The pre-secretory, secretory and mature ameloblasts are the components that produce enamel on the labial side of the incisor. The LiCL provides cells to the growing incisor but these cells do not form ameloblasts or make enamel. Od, odontoblasts. (B and C) H&E staining of the LIs of P0 WT, and Tbx1K14cKO mice to determine the role of Tbx1 in the LaCL. Mutant mice have smaller CLs and at P0, the cells of both the IEE and OEE are disorganized compared with their counterparts in WT mice. The SR contains the stem or progenitor cells. The SI is also thinner in the mutant mice. (DI) IHC experiments show that Tbx1 is highly expressed in the LaCL and LiCL regions of the E16.5 WT LI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining provides contrast. (J) The Tbx1Cre X Rosa26LacZ mouse was analyzed for LacZ staining and Tbx1 fate mapping experiments. LacZ-positive cells are derived from Tbx1 expressing cells. In panel J, the SR compartment of the LaCL has populations of Tbx1-negative cells and Tbx1-positive cells. The Tbx1-positive cells are incorporated into the OEE and IEE, as shown by LacZ staining (the arrows denote the direction of migration of the progenitor cells). (K) The secretory ameloblasts and SI contain cells that expressed Tbx1 and migrated to the distal end of the incisor. These experiments were repeated three times for each embryonic stage and genotype (N = 3). Abbreviations: t, tongue. Scale bar = 100 μm.
Figure 6.
Figure 6.
Tbx1K14cKO embryos have small incisors, abnormal molar cusping with defective ameloblast differentiation and amelogenin expression. H&E staining was carried out on WT and Tbx1K14cKO embryos at E16.5 (data not shown), E18.5 and P0. (A and B) At E18.5 the LI is small in the Tbx1K14cKO embryos (∼25% reduced compared with WT, black bar) and the portion that becomes the CL is underdeveloped (arrow). Scale bar = 100 μm. (C and D) At E18.5 the differences in Am and Od morphology compared with WT embryos is not as significant. (EH) Tbx1 mutant P1 LIs showed a decrease in amelogenin expression compared with WT (G, H, higher magnification of panels E and F, respectively). However, amelogenin is expressed in P4 Tbx1 mutant LIs albeit at low levels (data not shown), suggesting a delay in amelogenin expression the Tbx1K14cKO mice. (IL) H&E of sagittal sections of WT and Tbx1K14cKO mice at P0 show that the lower molar in the Tbx1 mutant undergoes abnormal cusping. The Tbx1K14cKO first molar (M1) has abnormal folding of the dental epithelium that gives rise to the molar cusps. Furthermore, the ameloblasts of the differentiating epithelium appears underdeveloped and undifferentiated (K and L, high magnification of panels I and J). (MP) Tbx1K14cKO mutant lower molars show decreased amelogenin expression at P4 compared with WT (O and P, higher magnification of panels M and N, respectively). These experiments were repeated three times for each genotype (N = 3). These experiments were repeated more than three times for each embryonic stage and genotype (N > 3). Abbreviations: De, dental epithelium; Am, ameloblasts; Od, odontoblasts; M1, first molar; M2, second molar. Scale bars: 100 μm.
Figure 7.
Figure 7.
Tbx1 regulates cell proliferation. (A) LIs from WT or Tbx1K14cKOP0 mice were processed, sectioned and stained for Ki67 to examine cell proliferation. Ki67 was decreased in Tbx1K14cKOtooth sections, and Ki67/DAPI ratio was quantified to determine that the decrease in cell proliferation was statistically significant (P < 0.05). (B) MEFs were collected from WT and Tbx1−/− embryos at E14.5. Growth was analyzed by seeding each well with 100 000 cells, and cells were counted after 24, 48 and 72 h. MEFs extracted from Tbx1−/− embryos showed decreased cell proliferation compared with WT. (C) MEF cell growth was quantitated revealing a decrease in proliferation of the Tbx1 mutant cells. Two different embryos were used for WT and Tbx1−/− and each cell count was done in triplicate. These experiments were repeated three times for each genotype. (D) LS-8 oral epithelial cells were transduced with a lentivirus expressing Tbx1 or empty vector. Cells were cultured for 48 h and cell growth/viability was measured every 12 h.
Figure 8.
Figure 8.
Tbx1K14cKO two-week-old mice have dental anomalies and developmentally advanced third molar eruption. Skeletal preparations were performed on WT and Tbx1K14cKO mice at P14. (A and B) μCT images of mandibles in parasagittal plane show the thinner enamel layer on the LIs (orange arrows), as well as advanced mineralization of the third molar crown in the Tbx1K14cKO mouse compared with WT (green arrows). (C and D) SEM of the lingual side of LIs reveal smaller incisors with more wear indicating a difference in biomechanical properties of enamel, compared with WT. Scale bar 100 μm. (E and F) SEM of fracture surfaces of the erupted portion of the LI show that Tbx1K14cKO enamel is significantly thinner as indicated by blue brackets of identical length. Scale bar 10 μm. (G and H) Mature enamel fractured at similar positions on the erupted incisor at higher magnification. Bundles of enamel crystallites (prisms) are packed densely in WT enamel but loosely with spaces between them in the Tbx1K14cKO mouse. Scale bar 10 μm. (IP) μCT scans at P14 show that mutant mice have a smaller first (M1) and second (M2) molars and a third (M3) molar that is unusually advanced in its development (green arrows). (I and L) Sections in coronal plane through the center of the distal root of the first molar, as indicated by the dotted blue line in image J and K, show differences in molar size an enamel development (less enamel mineralization on the incisor marked by orange arrow) (J and K) Maximum density projection through the mandible in transverse plane shows differences in tooth size. (M and P) Slices in parasagittal plane through the center of the distal root of the first molar and (N and O) maximum density projection in the sagittal plane show that in the mutant mice molar cusping is defective (purple arrow) and the third molar is advanced in development (green arrow). (Q and R) SEM images of whole mandibles show clearly that M2 lacks a distal cusp (purple arrow) and is smaller in the mutant mouse and that M3 has erupted and shows advanced development. Furthermore, the enamel of M1 and M2 shows signs of wear. Scale bar 100 μm. (S and T) SEM images of whole molars dissected out of the mandibles show differences in M2 cusp formation (purple arrows), similarities in root development and advanced M3 development in the mutant mouse. Scale bar 200 μm. Abbreviations: M1, first molar; M2, second molar; M3, third molar.
Figure 9.
Figure 9.
Tbx1 over-expression increases incisor size and dental stem cell proliferation. (AD) H&E staining of WT and COETK14Cre E16.5 LIs. The overall size of the incisor is similar in mice of the two genotypes at this early stage (A and B). Scale bar = 500 μm. However, the LaCL (boxed region) is larger (width and length) with an increase in cell number in the SR region of the CL and decreased ameloblast differentiation (outlined with white dotted line, higher magnification C and D). Scale bar = 100 μm. (EH) H&E staining of WT and COETK14Cre P1 LIs. At this later stage, the COETK14Cre LI is larger (width and length) than its WT counterpart and the LaCL remains increased in size and shape (the LaCL is elongated in the COETK14Cre incisor). Scale bar = 500μm. There are more cells in the SR region of the CL in the COETK14Cre incisor (outlined with white dotted line, higher magnification G and H). Scale bar = 100 μm. (IL) P1 LIs from WT or COETK14Cre mice were processed, sectioned and stained for Ki67 to assess cell proliferation. Ki67 expression was higher in the COETK14Cre LaCLs compared with WT, as established by quantifying the Ki67/DAPI ratio; 10 l of pups and 20 mutants were sectioned, all showed the same phenotype. Abbreviations: Am, ameloblast; t, tongue; LI, lower incisor; LaCL, labial cervical loop.
Figure 10.
Figure 10.
Tbx1 over-expression increases amelogenin, enamel formation and a delay in formation of the third molar. (AD) WT and COETK14Cre P1 UIs were sectioned and stained for the expression of amelogenin. Amelogenin expression was increased in the COETK14Cre UI (higher magnification, compare C, WT to D, COETK14Cre sections). (EH) Amelogenin expression was increased in the COETK14CreLI (higher magnification, compare G, WT to H, COETK14Cre sections). (IL) μCT imaging shows an increase in the size and shape of the P14 (2 weeks old) COETK14Cre incisor compared with its WT counterpart (I and J). The enamel on the labial side is thicker in the COETK14Cre incisor (blue arrow). Imaging of the P14 mandible (left and right sides) shows an increase in molar enamel formation (blue arrow) and a slight loss of alveolar and cortical bone formation in the COETK14Cre mandible (green color, K, L). (M and N) microCT analyses of P28 mandibles show an increase in enamel formation (blue arrow) in the incisor of the COETK14Cre mice, coincident with a decrease in alveolar bone formation (light green arrow and orange brackets). (O and P) microCT scans of the hemimandible show a developmental delay in formation of the third molar (yellow arrow), a decreased in formation of cortical bone (white arrow) and an increase in formation of incisor enamel in the COETK14Cre incisor (blue arrow).
Figure 11.
Figure 11.
Tbx1 loss-of-function and gain-of-function mice have cleft palate. (A and B) At E16.5 Tbx1K14cKO mice have a cleft palate shown by coronal sections of the anterior palate. (C and D) Coronal sections of COETK14Cre mice at E18.5 show that Tbx1 over-expression causes cleft palate. These experiments were repeated more than three times for each embryonic stage and genotype (N > 3). Abbreviations: LI, lower incisor; t, tongue; Ps, palatal shelves; p, palate. Scale bar = 500 μm.
Figure 12.
Figure 12.
Model for the role of Tbx1 in tooth and craniofacial development. PITX2 and TBX1 are two of the first transcription markers for dental development and both a co-expressed in the early dental epithelium, dental lamina and oral epithelium. PITX2 is a transcriptional activator, which activates a gene regulatory network required for dental development (62). TBX1 can repress PITX2 transcriptional activity, but also activate other genes required for cell proliferation. TBX1 is part of a negative feedback loop with miR-96. TBX1 represses miR-96 expression and miR-96 represses TBX1 expression. This feedback loop allows dental epithelial cells to differentiate and produce ameloblasts, which express amelogenin.

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