Retinoic acid impairs shh and ptc1 expression in regenerating caudal fins. (A-C) Zebrafish were treated with 10-6 M all-trans-RA for a period of 1 day, starting 1 day after amputation. Shh expression was determined (A), 1 day; (B), 3 days; (C), 6 days after the end of the RA treatment. The curve in the caudal fin in A and B is due to the fusion of rays. Shh expression is abolished in treated fins at 1 or 3 days but gradually re-established at 6 days. (D-F) Alcian blue and Alizarin red stainings showing the lepidotrichial matrix and the mineralized bones of the stump, respectively, in half-fins treated with RA (D,E) or in untreated fins (F). Three days after the end of the treatment, the regenerate is developing but there is little lepidotrichial matrix deposition at the tip of each native fin rays (D); in situ hybridization on the second half of this fin shows a weak shh expression (not shown). Five days after the end of the treatment (E), the lepidotrichial matrix does not extend in the regenerate as far as in the control fin (F). Expression of shh (G-I) or of ptc1 (J-L) is abolished by short RA treatments. Zebrafish were treated with 10-6 M RA for 1 hour (H,K), or 4 hours (I,L), starting 3 days after amputation. Expression of shh and ptc1 was determined immediately before RA treatment (G,J); or immediately after the end of the treatment (H,I,K,L). Three days after amputation, shh (G) and ptc1 (J) transcripts are clearly detectable in fin rays, but markedly decreased (H,K) or undetectable (I,L) after 1 and 4 hours of RA treatment, respectively. Arrows indicate the level of amputation; dotted lines in D-F indicate the distal limit of the regenerate. Scale bars, C-L, 80 μm.
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