Figure 1.
Dact2 expression in dental and oral epithelia.
(A–F) Endogenous Dact2 protein was stained using a Dact2 antibody and secondary FITC conjugated antibody and nuclei were stained by DAPI on wild type mouse embryos. (A) E12.5 upper molar tooth germ, (B) E14.5 upper molar tooth germ, (C) E16.5 oral epithelium, (D) E12.5 lower molar tooth germ, (E) E14.5 lower molar tooth germ, and (F) E16.5 first lower molar all shows epithelial expression of Dact2. (G and H) molar germs at E14.5 stained without Dact2 primary antibody as negative controls. (I) LacZ staining with eosin counter staining on E14.5 Pitx2cre/+X Rosa26+/− mice showed Pitx2 highly expressed cell linages in the upper molar bud, indicating overlapping expression of Dact2 with Pitx2 at the same developmental stages. White dotted lines indicate the mesenchyme-epithelium boundaries. Scale bar represents 100 μm.
Figure 2.
Endogenous Pitx2 binds to a conservative region on the Dact2 promoter.
(A) Schematic of Dact2 10 kb promoter with six PITX2 binding motifs (TAATCC) indicated by arrowheads. The red arrowhead indicates the site verified by ChIP assay. The location of the sense primer and the antisense primer are shown for amplification of the immunoprecipitated chromatin. Blue arrowheads are putative binding sites with less conservation. The white arrowhead indicate a non-conserved Pitx2 binding motif that we tested in ChIP experiment as negative control shown in Figure S3. (B) Endogenous ChIP assay was performed in LS-8 cells. Lane 1 contains the PCR marker. Lane 2 shows the Dact2 primers-only control. Lane 3 is the immunoprecipitation using normal rabbit IgG and Dact2 primers. Lane 4 is the Pitx2 immunoprecipitated chromatin amplified using the specific Dact2 promoter primers. Lane 5 is the chromatin input amplified using the Dact2 primers. Lane 6 shows the Msx2 promoter primers-only control. Lane 7 is the immunoprecipitation using normal rabbit IgG and Msx2 primers. Lane 8 is the Pitx2 immunoprecipitated chromatin amplified using the specific Msx2 promoter primers. Lane 9 is the chromatin input amplified using the Msx2 primers. The amplified region of Msx2 promoter is −632 to −359 bp relative to transcription start site. All PCR products were sequenced to confirm their identity. (C) The PITX2 binding element on mouse Dact2 promoter verified by ChIP was mapped to a highly conserved (>70%) region among Mouse, Human, Chimpanzee, Rhesus macaque and Rat. The blue box indicates the PCR amplified region on Dact2 promoter in (B).
Figure 3.
PITX2 activates Dact2 expression.
(A) CHO cells were co-transfected with CMV-PITX2A expression plasmids and luciferase reporter driven by Dact2 10 kb promoter. Empty CMV-PITX2A expression plasmids were transfected in parallel as a negative control. All transfections included the SV-40-β-galactosidase reporter to control the transfection efficiency. Cells were incubated for 24 hrs and then assayed for luciferase and β-galactosidase activities. (B) Luciferase reporters driven by a duplicated 66 bp DNA segment of Dact2 promoter flanking the Pitx2 binding site in Fig. 2A at (−6172 to −6106) was co-transfected with or without CMV-PITX2A overexpression plasmid in CHO cells. A similar reporter with the mutated Pitx2 binding motif was transfected in parallel as control. All luciferase activities are shown as mean-fold activation compared with the Dact2 promoter plasmid co-transfected with empty CMV expression plasmid (±SEM from five independent experiments). (C) E14.5 Embryos from Pitx2−/−, Pitx2 transgenic and wild type mice were harvested to generate MEF cells. These MEFs were lysed and analyzed by Western blots to show endogenous Dact2 expression levels. GAPDH expression was probed as loading controls. Protein band intensities were quantified and shown as relative value ±SEM.
Figure 4.
Dact2 attenuates PITX2 transcription activity.
(A) CHO cells were transfected with CMV-PITX2A, CMV-Dact2 and luciferase reporter driven by Amelx 2.2 kb promoter. Empty CMV expression plasmids were transfected in parallel as a negative control. (B) CHO cells were transfected with combinations of CMV-PITX2A, CMV-β-catenin, CMV-Dact2 and luciferase reporter driven by Dlx2 3.2 kb promoter. Empty CMV expression plasmids were transfected in parallel as a negative control. The titration gradient of transfected Dact2 expression plasmids are from 0.5 μg to 8 μg in 2-fold increment. The luciferase activities were normalized by co-transfected β-galactosidase and ±SEM was from five independent experiments. (C) Dlx2 was transfected instead of PITX2A to show Dact2 attenuation is specific to PITX2 transcription activity. All luciferase activities were normalized by co-transfected β-galactosidase and ±SEM were from at least three independent experiments. (D) Dact2and PITX2A transfected CHO cells were lysed and analyzed by Western blot, probing for transfected PITX2A. β-catenin expression is shown by Western blot in transfected cells. β-tublin was probed as loading control.
Figure 5.
Dact2 is a potent inhibitor of Wnt/β-catenin signaling.
293FT cells were transfected with CMV-Lef-1, CMV-PITX2A, CMV-Dact2 overexpression plasmids and TOPFlash reporters. In parallel experiments FOPFlash reporter were transfected as negative control. All luciferase activities were normalized by co-transfected β-galactosidase and ±SEM were from five independent experiments.
Figure 6.
Dact2 protein localizes to the cytoplasm and nuclear compartments in LS-8 cells.
Endogenous immunofluorescence staining on LS-8 cells was performed. (A) Dact2 protein was probed by Dact2 primary antibody and labeled with FITC. (B) Nuclei were stained by DAPI. (C) Merged signals of FITC and DAPI. (D, E and F) Single cell was viewed under 100X objective. (G, H and I) Parallel experiments using normal rabbit IgG as mock primary antibody to show secondary antibody specificity. All scale bars represent 25 μm.
Figure 7.
Dact2 and Pitx2 physically interact.
(A) LS-8 cells were used for endogenous immunoprecipitation assays. Western Blots shows the high expression levels of Dact2 and Pitx2A in the 10 times diluted input LS-8 cell lysate. The right lane of the IP Western Blot shows Dact2 protein pulled down by Pitx2 antibody. Left lane shows the parallel IP performed using normal rabbit IgG as negative control. (B) Schematics of the gene structures of PITX2A truncations used in GST pull down assay. (C) Coomassie blue staining of the purified PITX2A truncated proteins fused with GST tag (bands with the correct sizes marked by *). (D) Dact2 Western blot of the GST pull-down assay. Dact2 pure protein was incubated with different truncated PITX2A in lane 1, 3, 5 and 7. Incubation of corresponding truncated PITX2A only controls were in lane 2, 4, 6 and 8. Lane 9 contains 10% Dact2 pure protein input. Results indicated Dact2 binds PITX2A through the homeodomain.
Figure 8.
Knock down of Dact2 activates endogenous Pitx2 target genes.
(A) Knocking down of endogenous Dact2 in LS-8 cells by shRNA was shown by Western Blot. Negative control shRNA transfected cells show no change. GAPDH was probed as loading controls. Protein band intensities were quantified and shown as relative value ±SEM. (B) mRNAs were extracted from LS-8 cells transfected with Dact2 shRNA or NC shRNA, and subjected to RT-PCRs and real-time PCRs. Relative expression levels of Dlx2 and Amelx were analyzed and correlated with Dact2 expression level. All Real-time PCRs were performed in triplicates and repeated six times.
Figure 9.
Dact2 down-regulates Wnt responsive proliferation markers.
(A) MEF cells from Dact2−/−, Dact2+/− and Dact2+/+ embryos were lysed and analyzed by Western blots. GAPDH was probed as loading controls. Protein band intensities were quantified and shown as relative value ±SEM. (B) mRNAs extracted from MEF cells were subjected to RT-PCRs and real-time PCRs. Specific primers for proliferation markers Ccnd1, Ccnd2 and c-Myc were used in the real-time PCRs to evaluate the relative expression level of these proliferation markers. All real-time PCRs were performed in triplicates and repeated five times.
Figure 10.
Dact2 represses cell proliferation.
(A) 96-hour cell proliferation assays were performed with Dact2−/−, Dact2+/− and Dact2+/+ MEF cells at passage 3. All cell counting were performed in triplicate. (B) Microscopic photos of seeded MEF cells at the beginning and end of the proliferation assay.
Figure 11.
β-catenin cellular localization is changed in the dental epithelium of Dact2 null mice.
(A and E) E18.5 WT and Dact2−/− lower incisors were examined by immunohistochemistry for β-catenin expression. Boxed region were examined under higher magnification. (B and F) Detailed views of the labial dental epithelium were shown. β-catenin was labeled with FITC. (C and G) Nuclei were stained with DAPI. (D and H) Merged signals of FITC and DAPI are shown. Arrowheads indicate the differentially localized β-catenin. MES, mesenchyme; OD, odontoblasts; AB, ameloblast; SI, stratum intermedium.
Figure 12.
Model for the mechanism of Dact2.
Dact2 is a direct downstream target gene of Pitx2 and Wnt signaling. Dact2 negatively feeds back and represses the transcriptional activity of Pitx2, and in turn inhibits Wnt/β-catenin signaling responsive proliferation. Dact2 also inhibits Wnt signaling responsive cell proliferation.