Supplementary Fig. 1 Schedule of Dox induction. (A) NTPDase2-rtTA transgenic mice were crossed with the TetO-DTA strain to produce double transgenic mice (NTPDase2-rtTA-TetODTA). In this system, Dox induction generates diphtheria toxin (DTA) in NTPDase2+ cells. DTA expression can ablate NTPDase2+ cells and NTPDase2+ lineage cells. (B) NTPDase2-rtTA transgenic mice were crossed with the TetO-GFP-ΔTgfbr2 (PTR) mice to produce double transgenic mice (NTDPase2-rtTA-TetO-GFP-ΔTgfbr2). In this system, Dox induction causes the disruption of TGF-β signalling in NTPDase2+ cells. The mice were sacrificed according to different treatment schedules, and the filiform papillae were examined in different planes of sectioning. (C) NTPDase2-rtTA transgenic mice were crossed with the TetO-Cre strain to produce the double transgenic mice NTPDase2-rtTA-TetOCre. In this system, Dox induction generates Cre+ cells in NTPDase2+ cells. The mice were sacrificed according to different treatment schedules, and the filiform papillae were examined in different planes of sectioning. This model was used as a control for the disruption of TGF-β signalling.

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Supplementary Fig. 2 In the double transgenic K14-rtTA-PTR mice, GFP+ cells firstly appeared at the basal cell layer, and then at the filiform papillae. Dox induction resulted in co-expression of EGFP and the dominant negative ΔTgfbr2 genes in K14+ cells. After 24 h of Dox induction, the GFP+ cells appeared at the basal cell layer (A,B). After 3 days of Dox induction, GFP+ cells appeared in both the filiform papillae and basal cell layer (C,D). After 35 days of Dox induction, GFP expression in filiform papillae (E) and lingual epithelia (F). Scale bar: A–D, 50 µm; E,F, 150 µm.

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Supplementary Fig. 3 Disruption of TGF-β signalling initially inhibited the formation of filiform papillae and then led to regeneration of filiform papillae over time. The dorsal (A) and ventral (B) surfaces in the control mouse without Dox induction. The dorsal (C) and ventral (D) surface surface after 24 h of Dox induction. Arrow, developmental papillae. Triangle, connective tissue core of filiform papillae. The dorsal (E) and ventral (F) surface after 3 days of Dox induction. Triangle, the connective tissue core of filiform papillae. The dorsal (G) and ventral (H) surface after 5 days of Dox induction. The dorsal (I) and ventral (J) surface after 18 days of Dox induction. The dorsal (K) and ventral (L) surface after 45 days of Dox induction. The ratio of nuclear/basal-suprabasal layer area was significantly increased after 24–72 h of TGF-β signalling disruption in the NTPDase2-rtTA-PTR model (M). Scale bar: A-L, 150 µm.

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Supplementary Fig. 4 Frontal section of tongue tip. (A) Control mouse. (B) After 24 h of Dox induction. (C) After 3 days of Dox induction. (D) After 5 days of Dox induction. (E) After 10 days of Dox induction. (F) After 18 days of Dox induction. Scale bar: 300 µm.

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Supplementary Fig. 5 Lingual epithelia near CV papillae invaded into connective tissue after 5 days of Dox induction. (A) Horizontal sections of posterior tongue in the control mouse. (B) Horizontal sections of posterior tongue after 24 h of TGF-β signalling disruption. (C) Horizontal sections of posterior tongue after 5 days of TGF-β signalling disruption (arrow, basal cells invading into connective tissues). (D) Horizontal sections of posterior tongue after 18 days of TGF-β signalling disruption. (E) After 5 or 18 days of TGF-β signalling disruption, the thickness of lingual epithelia significantly increased (a,b, p<0.05). (F) After 1 day of TGF-β signalling disruption, the ratio of cells to lingual epithelia was significantly increased (a,b, p<0.05). FP, filiform papillae; FuP, fungiform papillae; CT, connective tissue; EP, lingual epithelia; CV, circumvallate papillae. Scale bar: 200 µm.

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Supplementary Fig. 6 Dorsal surface of tongue. In saggital section from the control mouse, the whole dorsal surface was separated into nine parts from the tip to the posterior of the tongue (A–I). (A) Site 1 represents the tip of the tongue (~1 mm); (A–C) sites 1–3 represent the anterior one third (~3.6 mm); (D,E) sites 4–6 represent the middle tongue (~3.9 mm); (G–I) sites 7–9 represent the posterior tongue (~3.6 mm), which was separated from sites 1–6 by the sulcus terminalis. The transition between the lingual epithelia with and without filiform papillae was observed on the ventral surface (J). On the ventral surface of the posterior tongue, only the stratified epithelia were observed (K). The developmental papillae were observed only in a few sites (A and B, triangle). Thickened epithelia were observed in the middle tongue after 18 days of Dox induction (L, arrow, and M). Immunohistochemistry with anti-NTPDase2 revealed the expression of NTPDase2 in taste buds and connective tissues. Scale bar: 100 μm.

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Supplementary Fig. 7 After 24 h of TGF-β signalling disruption, AcH4+ cells are observed throughout the lingual epithelia. In the control (without Dox induction), cells with the higher acetylation levels of histone H4 (AcH4+ cells) were only observed in taste buds of fungiform papillae (A–C). After 24 h of TGF-β signalling disruption, AcH4+ cells were observed in all structures, including the basal cell layer, suprabasal cell layer, filiform papillae and fungiform papillae (D–F). A cell type with a hollow and flexible fishnet-like appearance was also observed in lingual papillae (D–F, arrow). After 60 h of TGF-β signalling disruption, AcH4+ cells were observed throughout the lingual epithelia, including filiform papillae and epithelia (G–I). After 3 days of TGF-β signalling disruption, AcH4+ cells were still seen throughout the lingual epithelia (J–L). A, D, G, J, dorsal surface; B, E, H, K, ventral surface; C, F, I, L, lateral surface of the tongue. CT, connective tissue core; FP, filiform papillae; FuP, fungiform papillae; sFP, secondary filiform papillae. Scale bar: 100 μm.

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Supplementary Fig. 8 Epithelial cells with higher acetylation levels of histone H4 were observed throughout the lingual epithelia after 18 days of TGF-β signalling disruption. After 5 days of TGF-β signalling disruption, the higher acetylation levels of histone H4 on the ventral surface (A) and lateral surface (B) of the tongue. In the control (NTPDase2-rtTA-TetOCre), the higher acetylation levels of histone H4 in the lingual epithelia after 18 days of Dox induction. (C) Dorsal surface, (D) ventral surface, (E) lateral surface of the tongue. The higher acetylation levels of histone H4 in the lingual epithelia after 18 days of TGF-β signalling disruption (F–I). (F) Dorsal surface, (G) ventral surface, (H) lateral surface and (I) ventral surface of the tongue. Scale bar: 150 µm.

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Supplementary Fig. 9 Disruption of TGF-β signalling in K14-rtTA-PTR mice rapidly results in tongue epithelia invasion into connective tissue. The dorsal surface (A) and ventral surface (B) after 5h of Dox induction. The dorsal surface (C) and ventral surface (D) after 9h of Dox induction. The dorsal surface (E) and ventral surface (F) after 24 h of Dox induction The dorsal surface (G) and ventral surface (H) after 3 days of Dox induction. The dorsal surface (I) and ventral surface (J) after 7 days of Dox induction. The dorsal surface (K) and ventral surface (L) after 35 days of Dox induction. Epithelial cells invade into connective tissue in ventral surface (L). Scale bar: 50 µm.

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Supplementary Fig. 10 TGF-β signalling and Jagged2/Notch1 cooperatively induce growth arrest in tongue epithelial cells. In normal tongue epithelia, TGF-β signalling induces growth arrest in NTPDase2+ cells (A). After disruption of TGF-β signalling, Jagged2 is activated by an unknown mechanism. Jagged2 then activates the Notch1 receptor, and cleaved Notch1 induces the proliferation of epithelial stem cells (B).

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