Extended Data Fig. 2 |. In vivo depletion of CTSK⁺ cells using an inducible DTR (iDTR) system.

a, Schematic representation of the experimental design and gross appearance of ROSA26iDTR and iDTR^Ctsk mice before and after DT administration. The red arrow in image indicates an abnormal dome-shaped skull. b, Immunostaining for Cleaved Caspased-3 (CC3) in the sutures of iDTR^Ctsk;mTmG mice with PBS or DT. n=3 independent experiments. Green, CTSK⁺ (mGFP) cells; Red, non-CTSK lineage (tdTomato) cells; Magenta, CC3. Far-right, enlarged view of the box. c, FACS analysis of the absolute amount of CTSK⁺ cells and CTSK⁻ pCSCs in the sutures of iDTR^Ctsk mice with PBS (n=4) or DT (n=4) administration (i). Normalized absolute quantification of the indicated cell types by analyzing the same number of cells passing the FSC/SSC, doublet, and live gates in all specimens (ii). d, μCT scan 3D images of the skull of ROSA26iDTR and iDTR^Ctsk mice at P11, P21, and P35 after DT administration. Red arrows indicate suture defects including abnormal fusion and hypomineralization (ROSA26iDTR, n=5; iDTR^Ctsk, n=5). e, μCT scan 2D images of the skull with an enlarged view of the region outlined in blue of ROSA26iDTR and iDTR^Ctsk mice at P21 and P35. f, Confocal immunofluorescence images of Ki-67 and quantification of the number of tdTomato and Ki-67 double-positive cells per high-power field of the OIP and LAM sutures in iDTR^Ctsk;mTmG mice at P14 after PBS (n=3) or DT (n=3) administration. Dots in graphs represent an individual mouse. g-i, Representative μCT images of the cortical (g), trabecular bone (h), and tibias (i) in ROSA26iDTR and iDTR^Ctsk mice after DT administration. Arrows show cortical defects including uneven periosteal surfaces and a double cortex. Quantification of bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of trabecular bones in ROSA26iDTR (n=4) and iDTR^Ctsk (n=4) mice at P21 after DT administration (h). j, Representative confocal fluorescent images of CTSK-expressing osteoclasts in iDTR^Ctsk;mTmG mice at P35 after PBS or DT administration. Red arrows indicate CTSK⁺ osteoclasts in the interparietal bones. Quantification of the number of osteoclasts per high-power field (HPF) of the sutures iDTR^Ctsk;mTmG mice after PBS or DT administration. Two or three regions from each group (n=3) were measured for quantification. k, Experimental scheme for functional inhibition of osteoclast activity using alendronate (1 mg/kg) and μCT scan images of the long bones and skulls of C57BL/6J mice at P42 after alendronate treatment. (Vehicle (PBS), n=3; Alendronate, n=5). ****P < 0.0001, P values are shown. Mean ± s.d., unpaired, two-tailed Student’s t-test (c, f, h, j). All images and FACS plots are representative of at least three independent experiments. Scale bars are denoted in images.