Editor's index

Abstract

In amphibian limb regeneration memory for position in the proximal-distal axis can be respecified by retinoic acid. The favoured candidates to mediate this effect are the retinoic acid receptors (RARs) and of the RARs identified in the regeneration blastema, the δ receptor is the most abundant. The presence in blastemal mesenchyme of at least two δ receptor isoforms, δ1 and δ2, alternatively spliced at the A-B junction, was demonstrated in expression studies and by PCR cloning. The δ1 receptor is abundant in regenerative structures such as the limb and tail, whereas the δ2 and a receptors show a more uniform pattern of expression across adult newt tissues. Full-length cloning of the δ1 receptor established the presence of an unusally long open reading frame and N-terminal sequence that appears unique among vertebrate retinoic acid receptors. Transient transfection of expression constructs into COS cells followed by Western blotting confirmed the existence of at least three potential initiation sites for δ1 translation. The possibility that δ1 RAR expression may specify positional memory directly was tested in RNase protection experiments. δ1 receptor message is increased on amputation, but does not exhibit a pronounced differential distribution along the proximal-distal axis in normal and regenerating limbs, nor does it show a persistent alteration in expression levels following a dose of retinoic acid sufficient to respecify position. The possibility that the morphogenetic effects of RA may be mediated through receptor interactions is raised by the finding that single mesenchymal blastemal cells in culture can express multiple RAR subtypes (δ1, and α) and isoforms (δ1 and δ2).