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Fig. S5
Wnt/β-catenin signaling is required for fin regeneration by supporting proliferation of Runx2+ pre-osteoblasts. (A-D) Caudal fin regeneration of Tg(sp7:EGFP) zebrafish treated with DMSO (A and B) or 100 nM Wnt-C59 (C and D) from 0-8 dpa. Shown are representative Rotterman contrast (A and C) and epifluorescent images (B and D) from 1 of 3 animals in each treatment group. White arrows point to osteoblasts and the dashed yellow line indicates the amputation site. (E-L) Immunostaining to visualize expression of Runx2 (in red) and β-catenin (in green) on fin sections from control (E-H, DMSO 64-72 hpa) and Wnt-C59 treated fish (I-L, 100 nM 64-72 hpa). Yellow arrows point to Runx2+ pre-osteoblasts with accumulation of nuclear β-catenin; red arrows indicate Runx2+ cells with membrane-localized β-catenin. The scale bar represents 50 μm and the dashed line is the plane of amputation. (M-P) Trichrome staining of control fins (M and N, DMSO from 48-72 hpa) and IWP-2 exposed fins (O and P, 10 μM from 48-72 hpa) at 72 hpa. Blue staining (Aniline Blue) indicates collagen and other connective tissue (blue arrows). The regions bounded by boxes are shown in higher magnification in the adjacent panel. (Q-V) TUNEL staining on fin sections from control (Q and T, DMSO 24-48 hpa), IWP-2 (R and U, 10 μM 48-72 hpa), and BMPRi (S, 5 μM 24-48 hpa) treated fish. A section from an E9.5 mouse is a positive control that serves to demonstrate the distinct labeling of apoptotic cells detected by TUNEL assays. For M-U, a representative ray from one of three fish in each treatment group is shown, with at least three rays examined from each animal. Green arrows indicate TUNEL+ cells. The dashed yellow lines represent the amputation plane. Scale bars are 50 μm. (W and X) Scatter plots showing numbers of Runx2+, Runx2+/sp7+ and sp7+ cells in DMSO (W) or IWP-2 treated animals (X, 10 μM 64-72 hpa). Each point on the plots represents a single cell’s normalized expression levels for Runx2 (x-axis) and sp7 (y-axis). Scored cells are from at least 10 sections from 4 different animals for each treatment. Regions bounded by the dashed red ellipse are defined as Runx2+ cells and the percentage of Runx2+ cells within the bounded region is shown. (Y-DD) Runx2 expression (Y and BB) and EdU incorporation (Z and CC) in cultured primary fin osteoblasts in response to Wnt3a treatment. Overlay images are shown in AA and DD. White arrows indicate Runx2+/EdU+ cells. Results are representative of two independent cell preparations and three independent Wnt3a treatments. In each case p < 0.05 (one tailed Fisher’s test) comparing the fraction of EdU-incorporated Runx2+ cells in control vs. Wnt3a treated cells.