Extended Data Fig. 6: Indications that hedgehog signalling controls stem cell renewal in the epiphyseal niche.

a, Shh-GFP reporter mice revealed Shh expression within the SOC at P30 (blue staining, DAPI). b, In situ hybridization for Gli2 mRNA at P30 (arrowheads point towards typical labelling). c, Gli1-CreERT2:tdTomato mice traced from P29 to P33 to reveal Gli1 activity. d, Col2-creERT:R26R-Confetti mice were traced at P30–P35, and received six injections of either DMSO or the SMO antagonist vismodegib (Vism, 100 mg per kg(body weight) per injection) between P31 and P34. Blue dashed lines indicate the top 50 µm, where the clone size was quantified (d′′′, mean ± s.e.m., n = 123 clones from 8 mice (vismodegib); n = 49 from 4 mice (DMSO)). When vismodegib treatment was extended by two extra injections, the growth plate fused at P37 (d′′′′; haemotoxylin and eosin staining). e–h, Growth plates from mice treated as in d′ and d′′ were analysed for proliferation within the top 50 µm (demarcated by blue dashed line) by means of EdU incorporation (e; injected daily P32–P34; n = 4 for each treatment) and KI67 immunolabelling (f; n = 4 (DMSO) or 3 (Vism) mice) and for expression of differentiation marker MEF2C (g) and stem cell marker CD73 (h). Arrowheads in e–g indicate typical positively-stained cells. i, Mice received six doses of either DMSO or SMO agonist (SAG, 25 mg per kg(body weight) per injection) between P31 and P34 and proliferation was analysed by Ki67 immunolabelling at P35 and quantified in the top 50 µm (depicted by blue dashed lines, n = 4 (DMSO) or 3 (SAG) mice). j, Mice at 7 ± 1 weeks of age received a single intra-articular administration of either SAG or DMSO followed by intraperitoneal EdU injection 4 h later. EdU incorporation was quantified 20 h later in the top 50 µm of the growth plate (n = 6). k, CD73 was immunodetected in mice treated as in i. CD73⁺CD49e⁺ cells were sorted by FACS as in Extended Data Fig. 4a and colony size (l) and number (m) were assessed following 10 days of culture in the presence of vehicle (DMSO (or ethanol for GANT61)), recombinant mouse sonic hedgehog (rmShh, 1 µg ml⁻¹), vismodegib (1 µM) or the Gli1 inhibitor GANT61 (5 µM). Data in l and m are mean ± s.e.m., n = 9 wells from 3 independent experiments for rmShh and Vism, and n = 3 wells from one experiment (GANT61). All white dashed lines demarcate the growth plate. The SOC, the resting (R), proliferating (P) and hypertrophic (H) zones are indicated for orientation. Data in a and j represent two independent experiments, data in b, c, d′′′′, e and k each derive from one experiment and those in d′–d′′′ are pooled from three independent experiments with the number of mice in each experiment indicated above. Plots represent mean ± s.e.m. and were analysed by unpaired two-tailed t-test, except the comparison between DMSO, rmShh and Vism groups (l and m), which was by one-way ANOVA with Tukey’s multiple comparisons.

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