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Fig. 3
Osteoblast dedifferentiation requires RA inactivation by Cyp26b1. RA and R115866 injections starting at 10hpa block osteoblast dedifferentiation. Inhibition of RA signaling in hsp70I:cyp26a1 fish positively affects dedifferentiation. (A) qPCR analysis of osteoblast markers at 10 and/or 24hpa. (B) EdU+/ZNS-5+ cells per section at 24hpa. (C) IHC for ZNS-5 combined with EdU labeling. Images show the stump region adjacent to the amputation plane. White arrow indicates EdU+/ZNS-5+ cell, yellow arrow denotes EdU+ fibroblast, arrowhead indicates EdU+ epidermal cell. (D) IHC for ZNS-5 and GFP in osc:gfp fish demonstrates absence of preosteoblasts in the blastema of R115866-treated fish at 2dpa. Arrowhead indicates GFP+ preosteoblast in control fish. (E) Model for Cyp26b1 function in osteoblast dedifferentiation. Data are represented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001. ns, not significant. Scale bars: 20µm in C; 100µm in D. Dashed lines indicate amputation planes. hs, heat shock.