Spatiotemporal characterization of Gpx4 expression and iron accumulation in tooth morphogenesis. (A) (i∼v) HE staining for tooth germ in E13.5 to PN3, 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) (i∼v) Iron probe staining in for tooth germ in E13.5 to PN3, scar bars=200μm; (a∼e) Enlarged view of selected region, scar bars=100μm, low concentration (a’∼e’, blue) and high concentration of iron (a’’∼e’’, red) are present, scar bars=100μm. Epi=Epithelia, Mes=Mesenchyme.

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 partially 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.

(A) Negative control of different developmental stage of molar germ in Gpx4 IHC staining. (B) Ex vivo culture of tooth molar germ from D0 to D7. Scar bars=20μm.

Original bar graphs of height, width, and area of erastin-treated tooth germ. *P<0.05, **P<0.01***P<0.001

Results for iron accumulation and 4-HNE expression in Era-10μM of D1 and D3. Scar bars=50μm

Results for iron accumulation and 4-HNE expression in Era-1.5μM, Era-5μM, Era-10μM, and Era-20μM of D5. Scar bars=50μm

Original bar graphs of height, width, and area of tooth germ in rescue assay. *P<0.05, **P<0.01***P<0.001

Source sequential HE slides for 3D reconstruction of CTRL

Source sequential HE slides for 3D reconstruction of Era-1.5μM

Source sequential HE slides for 3D reconstruction of Era-1.5μM+Fer

(A) Mean influence intensity of ROI of CL-CASP3 in CTRL, Era-1.5μM, and Era-1.5μM+Fer-1. Both Era-1.5μM and Era-1.5μM+Fer-1 showed slightly increased CL-CASP3 activation than that in CTRL but showed no statistical differences. (B) Results of TUNEL assay in each experimental group. DNA damage detected by TUNEL assay is similar to that of CL-CASP3, which indicated that apoptosis is not significantly activated in erastin treated tooth germ. Scar bars=200μm