Fig. 8.

Patterning of hedgehog responsive cells correlates with tidemark patterning. RNAscope detected by FastRed is visualized by confocal microscopy and overlaid with DIC imaging to visualize cell morphology and tidemark patterning. The blue dashed line in (A,B) represents the approximate level of the future tidemark in control AC and in (C,D) it follows the tidemark visualized by DIC. (A) Femoral condyle AC at 3 weeks. Control AC expresses Gli1 in multiple cell layers near the AC surface (Aa), Ptch1 in one to two cell layers around the presumptive tidemark (Ab) and Ihh in chondrocytes adjacent to the subchondral bone (Ac). Mutant AC displays decreased but patchy Gli1 (Ad, arrows) and Ptch1 (Ae, arrows) expression and normal Ihh in chondrocytes adjacent to the subchondral bone (Af). (B) Tibial plateau AC at 3 weeks. Control AC expresses Gli1 (Ba) and Ptch1 (Bb) in similar layers surrounding the presumptive tidemark and Ihh in chondrocytes adjacent to the subchondral bone (Bc). Mutant AC displays decreased Gli1 (Bd) and Ptch1 (Be) expression and normal Ihh in chondrocytes adjacent to the subchondral bone (Bf). (C) Femoral condyle AC at 8 weeks. Control AC expresses Gli1 in multiple cell layers near the AC surface (Ca), Ptch1 in one to two cell layers around the tidemark (Cb) and Ihh in chondrocytes just beneath the tidemark (Cc). Mutant AC displays decreased expression of Gli1 (Cd), decreased and patchy expression of Ptch1 that follows the irregular tidemark (Ce, arrows) and ectopic Ihh in nonmineralized chondrocytes (Cf, arrows). (D) Tibial plateau AC at 8 weeks. Control AC expresses Gli1 (Da) and Ptch1 (Db) in similar layers surrounding the tidemark and Ihh in chondrocytes just beneath the tidemark (Dc). Mutant AC displays increased and expanded expression of Gli1 (Dd) and Ptch1 (De) but normal Ihh just beneath the tidemark (Df). Scale bar = 100 μm.