Fig. 1. Initiation of secondary chondrocyte formation. (A-M) quadratojugal/quadrate joints at stage: e10 (A-G), e11 (H-J) and e13 (K-M). (A,D,H,K) Alcian Blue/Chlorantine Fast Red stained controls. (B) Cbfa1 expression is found only in the membranous periosteum of the quadratojugal (QJ). (C) Sox9 expression is only found in the cartilaginous quadrate (Q). (D-G) Comparison of Cbfa1, Gdf5 and Col2 expression. Arrowheads (D-J) indicate future sites of secondary chondrocytes (SCs). (E) Cbfa1 expression is maintained in the periosteum prior to SC formation. Gdf5 (F) is expressed around the quadrate and Col2 (G) is expressed within the quadrate, but neither is expressed within the quadratojugal. (H,I,J) Cbfa1 and Sox9 expression on adjacent sections after formation of the first SCs, adjacent to the germinal region (GR). (I) Cbfa1 is expressed by the periosteum, and by the nascent SCs (arrowed and circled). (J) Sox9 is expressed only by the SCs of the QJ (arrowhead). (K,L,M) Cbfa1 and Sox9 expression at e13. (L) Cbfa1 continues to be expressed in the periosteum and is also expressed by SCs (asterisk). (M) Sox9 is expressed only by SCs (arrowed), not in the periosteum. GR, germinal region; PO, periosteum; Q, quadrate; QJ, quadratojugal; SC, secondary chondrocytes. Scale bars: 100 µm.