EXPRESSION AND ACTIVITY OF THE NEWT MSX-1 GENE IN RELATION TO LIMB REGENERATION

The Msx-1 homeobox gene is expressed in various contexts during verteb rate development, including the progress zone of the avian and mouse l imb bud. Expression of mouse Msx-1 in a cultured myogenic cell line co nferred a transformed phenotype and inhibited fusion into myotubes. It has been proposed that Msx-1 expression is required to maintain certa in cells in a proliferating and undifferentiated state and may be asso ciated with the ability to regenerate limbs. Urodele amphibians such a s the newt regenerate their limbs by formation of a growth zone or bla stema, and we have isolated and sequenced newt Msx-1 (NvMsx-1) from a limb blastemal cDNA library. NvMsx-1 expression was detectable in RNA preparations from both limb and tail and their regeneration blastemas, although cultured cells established from limb blastemal mesenchyme ga ve negative results. When either COS cells or cultured newt blastemal cells were cotransfected with an expression vector for NvMsx-1 and rep orter plasmids containing multiple homeobox protein binding sites, NvM sx-1 repressed reporter expression. If NvMsx-1 was expressed together with a marker enzyme in cultured newt blastemal cells, no significant difference in DNA synthesis was observed relative to control transfect ants. When myogenic mononucleate cells were transfected with NvMsx-1 a nd subsequently exposed to low serum to promote fusion, the fraction o f Msx-1 positive cells in myotubes was comparable to a control transfe cted population analysed in tile same culture. These results indicate that although Msx-1 expression could be important for limb regeneratio n, it does not exert a cell-autonomous effect on proliferation or myog enic differentiation of cultured blastemal cells.