Spatiotemporal characterization of Gpx4 expression and iron accumulation in tooth morphogenesis

(A) (i∼v) HE staining for tooth germ in E13.5 to P3, scar bars=200μm; (a∼e) Gpx4 expression detected by IHC, scar bars=200μm; (a’∼e’) Enlarged view of each Gpx4 staining, scar bars=50μm; epithelia versus mesenchyme ***P<0.001 (B) (a) Iron probe staining in incisor, scar bars=200μm; (b∼d) Enlarged view of selected region, scar bars=50μm, bright (gray) and high concentration of iron (red) are present; (C) IF staining for Gpx4 (red) and nucleus (blue) in selected region of incisor, scar bars=50μm. Epi=Epithelia, Mes=Mesenchyme; White dotted line outlines odontoblasts; Yellow dotted line outlines ameloblasts.

Erastin impairs tooth morphogenesis especially within dental mesenchyme

(A) Gross anatomy of tooth germ cultured ex vivo for five days, scar bars=500μm; (B) (a, c) HE staining for tooth germ in D5, scar bars=100μm; (b, d) High resolution of epi-mes junction papilla, scar bars=50μm; Black stars pointe out NLCs; (b’, d’) NLCs indicated by black stars. (C) (a∼e) Gross anatomy of tooth germs in different concentrations of erastin in D5, scar bars=500μm; (f∼j) HE staining of different concentration treated tooth germ in D5; (f’∼j’) High-resolution view of epi-mes junction region of each tooth germ, scar bars=50μm; (D) Rada graph for calculation of height, width, and area in each tooth germ. Black dotted line outlines ameloblasts, AM=ameloblast, DP=dental papilla.

Ferroptosis is activated in erastin-treated molar germ

(A) (a, b) High density of Fe3+ (red) in CTRL and Era-10μM of D5, white star pointed out strong fluoresce signal of Fe3+, scar bars=50μm; (a’, b’) Low density of Fe3+ (grey) and (a’’, b’’) merged view of iron probe staining; (c, d) Merged view of IF straining of 4-HNE (Magenta) and DAPI (blue), white star pointed out strong fluoresce signal of 4-HNE, scar bars=5050μm; (c’, d’) for DAPI and (c’’, d’’) for 4-HNE; AM=ameloblast, DP=dental papilla, HD=high density, LD=low density; (B) TEM scanning for CTRL and Era-10μM in D5; (a, d) Epi-Mes junction area of CTRL and Era-10μM in D5 are detected, scar bars=5μm; (b, c) representative view of cells in epithelia and mesenchyme for CTRL, scar bars=2μm; Black arrow pointed out typical mitochondria in each region (b’) for epithelia and (c’) of mesenchyme, outlined by white dotted line; (e, f) representative view of cells in epithelia and mesenchyme for Era-10μM, scar bars=2μm; Black arrow pointed out typical mitochondria in each region (e’) for epithelia and (f’) of mesenchyme, outlined by white dotted line; (C) Relative frequency of the mitochondrial size in both groups; (D) Fold changes of gene expression in CTRL and Era-10μM in D5, versus CTRL ***P<0.001.

Ferroptotic inhibitors partly rescue erastin-impaired tooth organogenesis

(A) (a∼e) Gross anatomy of tooth germs in differently treated group, scar bars=500μm; (f∼j) HE staining of differently treated tooth germ, scar bars=100μm; (f’∼j’) High resolution view of epi-mes junction region of each tooth germ; Black dotted line outlines ameloblasts, black star pointed out NLCs, AM=ameloblast, DP=dental papilla, scar bars=50μm; (B) Rada graph for calculation of height, width, and area in each tooth germ; (C) Average number of NLCs in each group, versus Era-1.5μM ***P<0.001, versus Era-1.5μM+Lip-1 ##P<0.01; (D) 3D reconstructed view of tooth germ in D5. (a∼d) CTRL from the front view, coronal plane, sagittal plane, and 45° side view; (e∼h) to Era-1.5μM and (i∼l) to Era-1.5μM+Fer-1 were viewed by the same way. Scar bars=100μm.

Ferroptosis is the dominant cell death type contributes to erastin-impaired tooth morphogenesis

(A) (a∼c) High density of Fe3+ (red) in CTRL, Era-1.5μM and Era-1.5μM+Fer-1 of D5, white star pointed out strong fluoresce signal of Fe3+, scar bars=50μm; (a’∼c’) Low density of Fe3+ (grey) and (a’’∼c’’) merged view of iron probe staining; (d∼f) Merged view of IF straining of 4-HNE (Magenta) and DAPI (blue), white star pointed out strong fluoresce signal of 4-HNE, scar bars=50μm; (d’∼ f’) for DAPI and (d’’∼ f’’) for 4-HNE; AM=ameloblast, DP=dental papilla, HD=high density, LD=low density; (B) TEM scanning for Era-1.5μM and Era-1.5μM+Fer-1 in D5; (a, d) Epi-Mes junction area of Era-1.5μM and Era-1.5μM+Fer-1 in D5 are detected, scar bars=5μm; (b, c) representative view of cells in epithelia and mesenchyme for Era-1.5μM, scar bars=2μm; Black arrow pointed out typical mitochondria in each region (b’) for epithelia and (c’) of mesenchyme, outlined by white dotted line; (e, f) representative view of cells in epithelia and mesenchyme for Era-1.5μM+Fer-1, scar bars=2μm; Black arrow pointed out typical mitochondria in each region (e’) for epithelia and (f’) of mesenchyme, outlined by white dotted line; (C) Relative frequency of the mitochondrial size in both groups. (D) (a∼c) Expression of CL-CASP3 (green) in CTRL, Era-1.5μM and Era-1.5μM+Fer-1 in D5, scar bars=200μm; (a’∼c’) Enlarged view of CL-CASP3 in each group, scar bars=50μm. (E) Schematic model illustrates the overburdened ferroptotic stress impaired tooth morphogenesis in ex vivo organ culture model.