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Supplemental Figures
- 2. Epiprofin expression in molars
- 3. Epfn and Tbx1 expression in developing mouse molars 3 immunostaining Epfn Red : Perlecan Blue : DAPI Inner dental epithelium Inner dental epithelium Enamel knot Green: Epfn E13 Bud stage E14 Cap stage E17 Bell stage E10 Mandible in situ hybridization Tbx1 Inner dental epithelium Inner dental epithelium Green: Tbx1 E11 Dental placde E13 Bud stage E14 Cap stage E17 Bell stage Red : Perlecan Blue : DAPI
- 4. Reduced Tbx1 expression in Epfn KO teeth 4 Mesenchyme IDE Epfn (-/-)Epfn (+/-) E17 molars Tbx1 DAPI Cervical loop IDE Mesenchyme Dental epithelium mesenchyme ODE ode : outer dental epithelium Ide : inner dental epithelium Tbx1 DAPI P1 incisors Ameloblast How is Epfn expressed in Tbx1 cKO teeth? multiple incisors
- 5. 5 Sox2-expressing stem cells remain in Epfn KO teeth Sox2 Epfn DAPI 100nm Sox2 DAPI Epfn (-/-)Epfn (+/-) P1 incisors E17 molars IDE Cervical loop Dental papilla Cervical loop
- 6. in vitro analysis using PDE cells
- 7. Establishment of a dental epithelial stem cell line (PDE) 7 Does forced Tbx1 expression induce Sox2 and other factors involved in stem cell activity in PDE cells? Cervical Loop Sox2 DAPI Primary dental epithelial (PDE) cells (3-4 passages) P1 incisor x10 Sox2 CDy1 Hoechst 0 20 40 60 80 100 CDy1 Sox2 PDE cells Stem cell niche Marker-positivecells(%) PDE cells express stem cell makers stem cell makers Stem cell marker staining in PDE cells
- 8. Expression levels of Epfn and Tbx1 and their roles in dental epithelial stem cell differentiation into ameloblast 8 Proliferation status Stem cell Committed cell IDE cell Differentiated cell Very slow Slow (Low) Epfn (Low) Tbx1 (High)(Low) Tbx1 Epfn Epfn (TA cell) (progenitor) ExitRapid Tbx1 Epfn Tbx1 Epfn (High) (very High) EpfnTbx1 Sox2 Tbx1 Epfn Expression levels
- 9. Stem cell Committed cell IDE cell Differentiated cell (Low) Epfn (Low)(High)(Low) Tbx1 Epfn Epfn (TA cell) (progenitor) Tbx1 Epfn (High) (very High) Tbx1 Epfn Expression levels Sox2 CDy1 transfection Epfn Day1 Epfn Day3 transfection RNA-seq ① Stem cell model: cell sorting using CDy1 (& Sox2) ② Committed and proliferation stages: Epfn transfection Day1 ③ Cell cycle exit and differentiation stages: Epfn transfection Day3 Experimental design for RNA-seq ① ② ③ PDE cells
- 10. Epfn and Tbx1 are co-expressed in the proliferating inner dental epithelium Tbx1 expression is abolished at the preameloblast stage: Epfn expression continues during ameloblast development Tbx1 is downregulated in Epfn KO molars Epfn is an upstream molecule of Tbx1 Epfn and Tbx1 promote proliferation of CLDE cells Epfn binds the Tbx1 promoter region The Epfn protein physically interacts with the Tbx1 protein 10 Summary
- 11. mouse head sagittal section By Brittany Spivey
- 12. P1 Epfn+/- P1 Epfn -/-
- 14. WT 3-wks-old incisor Dental Epithelial Stem Cells Inner Dental Epithelium (Pre- Ameloblasts) Secretory Stage Ameloblasts Mature Ameloblasts Dentin Odontoblasts Pulp Enamel Tongue
- 15. Tbx1 Epfn Dental Epithelial Stem Cells Inner Dental Epithelium (Pre- Ameloblasts) Secretory Stage Ameloblasts Mature Ameloblasts Enamel Mature Odontoblasts
Editor's Notes
- Tbx1 expression is reduced in Epfn KO molars and P1 incisors. Next we examined Epfn expression in tbx1 cKO teeth.
- Sox2 is expressed in dental papilla of control E17 mouse molars and the cervical loop of control P1 incisors. However, Sox2 expressing cells remain in the IDE of Epfn KO molars and Epfn KO incisors. These results suggested that Epfn may regulate the commitment of stem cells to the ameloblast linage
- We isolated dental epithelial stem cells from the mouse cervical loop area and established a clonal line we called PDE cells. This showed immunostaining of stem cell markers, Sox2 and Cdy-1 in PDE cells with passage around 3-4. We found that more than 80% cells are positive for Sox2 and CDy1. We used this PDE cell line in the following our experiments. First we wanted to test whether Tbx1 induces Sox2 expression in PDE cells.
- In summary Tbx1 is expressed in the cervical loop and contribute stem cell maintenance by inducing Sox2 and other factors. Our result suggests that Epfn reduces Sox2 expression in part through inhibiting Tbx2 transcriptional activity and promotes stem cell commitment to the ameloblast lineage. In the proliferation stage, the Tbx1 expression level is highest. Epfn promotesTbx1 expression and contributes to this high level Tbx1 expression in the rapid proliferation stage. In the cell cycle exit and differentiation stages, Tbx1 expression is reduced, while Epfn expression is increased. Based on our previous work and other groups’ study, both high Epfn expression and low level Tbx1 expression contribute to promote cell cycle exit through different pathways.
- In summary Tbx1 is expressed in the cervical loop and contribute stem cell maintenance by inducing Sox2 and other factors. Our result suggests that Epfn reduces Sox2 expression in part through inhibiting Tbx2 transcriptional activity and promotes stem cell commitment to the ameloblast lineage. In the proliferation stage, the Tbx1 expression level is highest. Epfn promotesTbx1 expression and contributes to this high level Tbx1 expression in the rapid proliferation stage. In the cell cycle exit and differentiation stages, Tbx1 expression is reduced, while Epfn expression is increased. Based on our previous work and other groups’ study, both high Epfn expression and low level Tbx1 expression contribute to promote cell cycle exit through different pathways.
- In Summary We found Epfn and Tbx1 are co-expressed in the proliferating inner dental epithelium Tbx1 expression is abolished at the preameloblast stage: Epfn expression continues during ameloblast development Tbx1 is downregulated in Epfn KO molars Epfn induces Tbx1 transcription in PDE cells and is an upstream molecule of Tbx1 Epfn, not Tbx1, promotes progenitor proliferation: Epfn plus Tbx1 promote more Protein interaction between Epfn and Tbx1