Figure 4. Fusion of the Incus with the petrosal in Echidna pouch young.

(A) Alcian blue/picrosirius red staining on the fusion between the incus and petrosal observed in the newly hatched echidna. (B) Immunofluorescence staining against the regulator of chondrogenesis Sox9 (red) (B,B’) and the marker of mature cartilage Collagen type 2 (green) (B,B”) demonstrates that the cartilaginous incus and petrosal bones are fully fused at post-hatching day 0/1 (P0/1). (C) Immunohfluorescence against β Catenin (green) shows no activity within the cartilages at this timepoint. Expression is observed in the neuroepithelium of the inner ear. (D) Alcian blue/picrosirius red staining on the fusion between the incus and petrosal observed in 3 day post-hatching echidna (P3) shows that the elements are now fused by fibrocartilage. (E) Immunofluorescence staining against the regulator of chondrogenesis Sox9 and the marker of mature cartilage collagen type 2 (E,E”). Sox9 is still continuously expressed between the elements (E,E’), but collagen type 2 is down regulated in the incus-petrosal and incus-malleus articulation region (E,E”). (F) Immunofluorescence against β Catenin shows nuclear localisation within the incus-petrosal and incus-malleus articulation regions, indicating active canonical Wnt signalling, an important step in suppression of chondrogenesis during joint formation. i. incus; m. malleus; ptr. petrosal.