SYNTHESIS AND RELEASE OF 9-CIS RETINOIC ACID BY THE URODELE WOUND EPIDERMIS

The wound epidermis is a transient secretory epithelium that apposes t he mesenchymal blastema of a regenerating urodele limb, and is require d for regeneration, Previous studies have shown that the positional id entity 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, 608-617), and that an RA-re porter gene introduced into the blastema is differentially activated a long the proximo-distal axis (Brockes, J. P. (1992) Proc. Natl. Acad. Sci. USA 89, 11386-11390). The newt limb wound epidermis has been expl anted with minimal mesenchymal contamination and cultured under condit ions 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 gen e. The reporter was activated to a level corresponding to stimulation by 0.1-1 nM RA, and this activation was substantially conferred by med ium conditioned by the wound epidermis. No significant activation was observed for cells transfected with mutated reporter plasmids and anal ysed in parallel co-cultures. Wound epidermis from contralateral proxi mal and distal blastemas were compared for reporter activation, and ga ve a P/D activation ratio significantly greater than 1. Wound epidermi s explants were cultured in the presence of tritiated retinol, and ext racts 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 r etinol, 9-cis RA and other metabolites. Although all-trans RA was dete ctable, the predominant acidic metabolite was 9-cis RA. These experime nts establish the wound epidermis as a source of RA for local cellular interactions in the blastema.