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Fig. 6
RA signaling promotes bone matrix synthesis while inhibiting degradation. (A) qPCR analysis. Inhibition of RA signaling in hsp70I:cyp26a1 fish at 4dpa causes downregulation of bone matrix genes. Conversely, RA injections barely affect expression. (B) Downregulation of RA signaling in hsp70I:cyp26a1 fish between 3 and 5dpa results in an enlarged noncalcified (Alizarin Red–) distal region (double-headed arrow) at 5dpa. (C) RA injections starting at 3dpa result in regeneration of irregularly shaped hemirays at 6dpa. Hematoxylin staining. Lower panel shows hemiray outlines. (D,E) TRAP staining demonstrates presence of osteoclasts (arrowheads in D) at the wound site, in the early blastema and at the inner (arrowhead in E) and outer surface (arrow in E) of newly formed bone during regenerative outgrowth. (F) RA injections starting at 3dpa result in fewer TRAP+ cells in the regenerate but not at the wound site (arrowhead) at 6dpa. (G) RA injections starting at 0hpa result in absence of TRAP+ cells at the wound site and in the early blastema. (H) Model for RA signaling function in bone matrix synthesis and degradation. Data are represented as mean±s.e.m. *P<0.05, ***P<0.01. ns, not significant. Dashed lines indicate amputation planes. Scale bars: 100µm.