Vaccinia as a tool for functional analysis in regenerating limbs: Ectopic expression of Shh

Axolotls, with their extensive abilities to regenerate as adults, provide a useful model in which to study the mechanisms of regeneration in a vertebr ate, in hopes of understanding why other vertebrates cannot regenerate. Alt hough the expression of many genes has been-described in regeneration, tech niques for functional analysis have so far been limited. In this paper we d emonstrate a new method for efficient overexpression of foreign genes in ax olotls. Using vaccinia virus expressing beta-galactosidase microinjected in to regenerating limbs, we show that vaccinia can infect both dividing and n ondividing limb cells. The site of infection remains discrete and there is no secondary spread of infection to nearby cells. beta-Gal is expressed at high levels in blastema cells for about a week and in differentiated cells for longer. Blastemas that have been injected with vaccinia at different st ages regenerate normally. As a test of the utility of vaccinia for function al analysis in regeneration, we constructed a virus expressing Shh and inje cted it into the anterior of regenerating limbs. Ectopic Shh expression cau sed extra digits, carpals, and tarsals in the hands and feet of regeneratin g limbs, suggesting that despite differences in the timing of expression an d the eventual pattern, the function of Shh appears to be similar to that i n the developing limbs of other vertebrates. Our results demonstrate that v accinia virus is an excellent vector for ectopically expressing genes for s ecreted proteins and is a useful tool to study the function of signaling mo lecules during the process of regeneration in urodeles. (C) 2000 Academic P ress.