Fig. 3

Wnt/β-Catenin Signaling in Preosteoblasts during Regeneration

(A–L) Immunostaining of Runx2 (red) and β-catenin (green) on fin sections at 24 (A–C), 32 (D–F), 48 (G–I), and 72 (J–L) hpa. Yellow arrows indicate Runx2⁺ cells with nuclear β-catenin, and the red arrows point to proximal osteoblasts at 72 hpa with membrane-localized β-catenin. For (L), regions bounded by dashed white boxes are shown in higher magnification in inset panels.

(M–R) Expression of Runx2 (M, white), sp7:EGFP (O, green), and localization of β-catenin (N, red) is shown and overlaid (P–R) on 96 hpa Tg(sp7:EGFP) section. Regions bound by dashed white boxes in (P) are shown at higher magnification in (Q) and (R). Yellow arrows highlight Runx2⁺ cells with nuclear-localized β-catenin, and green arrows indicate membrane-localized β-catenin in sp7:EGFP⁺ osteoblasts.

(S–U) A single optical section at 96 hpa fin showing immunostaining of Runx2 (red) and β-catenin (green) in distal preosteoblasts. Yellow arrows point to Runx2⁺ nuclei containing β-catenin, and the green arrow indicates exclusively membrane-localized β-catenin in the fin epidermis.

(V–X) Runx2 immunostaining (V, red) and tcf7 in situ hybridization (W, green) on a 72 hpa cryosection (X, overlay). The yellow arrow indicates distal Runx2⁺ preosteoblasts that coexpress tcf7; the red arrow indicates proximal osteoblasts lacking tcf7 expression.

(Y–AA) Runx2 immunostaining (Y, red) combined with axin2 in situ hybridization (Z, green) is shown on a 72 hpa cryosection and overlaid (AA). Yellow arrows point to distal preosteoblasts coexpressing axin2 and Runx2. The white arrow indicates distal nonosteoblast blastema cells that express axin2. The red arrow shows proximal osteoblasts without axin2 expression.

(BB–DD) Runx2 immunostaining (BB, red) and GFP in situ hybridization (CC, green) on a 72 hpa section from a Tg(TOP:GFP) fin shown in overlay (DD). Yellow arrows show Runx2⁺/GFP⁺ preosteoblasts. The white arrow indicates distal nonosteoblast blastema cells that express the reporter. Hoechst-stained nuclei are shown in blue. Scale bars represent 50 μm except in U, which represents 5 μm.