Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis

Abstract

ABSTRACT The wound epidermis is a transient secretory epithelium that apposes the mesenchymal blastema of a regenerating urodele limb, and is required for regeneration. Previous studies have shown that the positional identity of the blastema is respecified by retinoic acid (RA; Maden, M. (1982)Nature 295, 672-675), that the blastema contains RA (Scadding, S. R. and Maden, M. (1994) Dev. Biol. 162, 608617), and that an RA-reporter gene introduced into the blastema is differentially activated along the proximodistal axis (Brockes, J. P. (1992)Proc. Natl. Acad. Sci. USA 89, 11386-11390). The newt limb wound epidermis has been explanted with minimal mesenchymal contamination and cultured under conditions where it retains expression and inducibility of marker antigens. We have assayed for the release of retinoids from the wound epidermis by coculture with cells transfected with an RA-responsive reporter gene. The reporter was activated to a level corresponding to stimulation by 0.1–1 nM RA, and this activation was substantially conferred by medium conditioned by the wound epidermis. No significant activation was observed for cells transfected with mutated reporter plasmids and analysed in parallel co-cultures. Wound epidermis from contralateral proximal and distal blastemas were compared for reporter activation, and gave a P/D activation ratio significantly greater than 1.Wound epidermis explants were cultured in the presence of tritiated retinol, and extracts were analysed by HPLC on three different columns. Radioactivity was detected in peaks corresponding to didehydroretinol, 9-cis RA and other unidentified metabolites. Analysis of conditioned media samples, some after pulse chase experiments, detected significant release of retinol, 9-cis RA and other metabolites. Although all-trans RA was detectable, the predominant acidic metabolite was 9-cis RA. These experiments establish the wound epidermis as a source of RA for local cellular interactions in the blastema.