Diphtheria toxin (DT) administration

Grem1 cells within the AC are abundant at 2 weeks of age, therefore, we selected mice at this age for initial validation of Grem1 cell ablation within the AC. Diphtheria toxin (#BML-G135, Enzo) at a concentration of 250 ng in 10 µL of PBS was injected intraperitoneally into 2-week-old Grem1-Td-DTR and mice sacrificed 2 days later. Anti-RFP immunohistological staining was performed to validate the distribution and ablation of Grem1 cells in the Grem1-Td-DTR AC compared to aged matched Grem1-TdT mice (Supplementary Fig. ^3g). For OA pathology, mice 5–7 weeks of age were administered two doses of 250 ng DT intra-articularly in 10 µL of PBS and limbs collected 3 days later for analysis and blinded scoring of OA pathology. Grem1-Td-DTR mice administered with DT developed a fatal small intestinal phenotype at day 4 for homozygous mice and at day 7 for heterozygous mice after DT injection. A similar phenotype has been previously reported⁵¹. Ablation of Grem1-lineage cells in Grem1-creERT;DTR mice and Acan-lineage cells in Acan-creERT;DTR mice was performed by administering 100–125 ng of DT (#D0564, Sigma) intra-articularly 3 times weekly for 2 weeks into 8 week old mice that were given tamoxifen prior at 4–6 weeks of age. Mice were then sacrificed at 26 weeks of age for analysis and unblinded OARSI scoring.