The regenerating newt limb blastema expresses a number of molecular markers which are not expressed in the developing limb. Two antibodies which stain most if not all of the mesenchymal cells of the blastema are LPIK and RGE53. In other species these identify keratin 8 and keratin 18, which are usually restricted to simple epithelia. The newt homologues of these keratins, NvK8/18 (Notophthalmus viridescens keratin 8/18) have been cloned. It is shown that whilst NvK8/18 are down-regulated by retinoic acid in the liver, this regulation is not seen in the blastema. However, in cultured limb cells obtained from normal limb muscle (TH4B), RA clearly down-regulates NvK8/K18, there is a decrease in their proliferation and differentiation into myotubes takes place. In contrast NvK8/18 are not directly regulated by RA in cultured blastemal cells (BlHl). In addition retinoic acid neither has a direct effect on the proliferation or differentiation of the BlHl cells. Myogenesis of the BlHl does occur at high cell density. Therefore it is proposed that there are at least two mechanisms of forming muscle in the newt, one is used for repair myogenesis the other for regeneration of muscle. It is further shown that NvK8/18 are only expressed in the regenerating limb when the nerve has entered the limb bud. The nerve does not control NvK8/18 expression, it appears to define a developmental stage whereby the blastemal cells have to express NvK8/18 if regeneration is to proceed. The link between NvK8/18 expression and proliferation appears to be direct. Antisense oligomers targeted against NvK8/18 expressed in either limb or blastemal cells causes a decrease in their proliferation and differentiation is observed. Therefore, it is suggested that one of the first steps in blastemal cell differentiation is the down- regulation of NvK8/18. A model is proposed how NvK8/18 could regulate transcription
