Fig. 7. The requirement of cell proliferation for Lbx1–Pax3-induced myogenesis cannot be overcome by tissues inducing myogenesis. Somites were dissected from HH10 embryos together with a segment of the adjacent NT and the notochord, and cultured in collagen gels. Tissues were infected with RCAS(A)–Lbx1 (lanes 1 and 3) and RCAS(A)–Pax3 (lanes 2 and 4) and cultured for 2 days. One set of tissues was left without additives (lanes 1 and 2), while the other set was treated with mitomycin C to inhibit cell proliferation (lanes 3 and 4). Four days after treatment, tissue explants were analyzed by RT–PCR. Similar to the experiment without muscle-inducing tissues, expression of Lbx1 (lane 3) and Pax3 (lane 4) did not result in an up-regulation of MyoD, myogenin and MyHC. To ensure that treatment of the NT–NC with mitomycin C or irradiation did not affect its ability to induce myogenesis in the PSM, the PSM was either cultured alone (lane 5) or with NT–NC that was not treated (lane 6), treated with mitomycin C (lane 7) or subjected to irradiation (lane 8). Myogenic marker molecules were induced in the PSM by the NT–NC (lanes 6–8), irrespective of a cell proliferation block in the NT–NC.