In vertebrates, skeletal muscles of the body arise from cells of somitic origin. Recently, somite culture experiments have identified a set of genes, including Pax3, Six1, Eya2, and Dach2, that appear to play an important role in early myogenesis during somite development (Heanue et al. [1999] Genes Dev. 13:3231-3243). In somite culture Pax3, Six1, Eya2, and Dach2 not only function to activate myogenesis, but they form a complex network regulating each other's transcription. We sought to examine whether this putative Pax3/Six1/Eya2/Dach2 network of regulation actually functions in vivo. In particular, we tested whether Pax3 and Dach2 participate in a positive regulatory feedback loop in vivo as they do in culture. To test in vivo Pax3/Dach2 interregulation, we took advantage of the known dependence of both factors on ectodermal signals. Somites isolated from the overlying ectoderm lose expression of Pax3 and Dach2. Therefore, we attempted to rescue Pax3 or Dach2 expression in somites isolated from the ectoderm by retroviral misexpression of the complementary factor. Indeed misexpression of Pax3 or Dach2 resulted in rescue of Dach2 or Pax3, respectively. These rescue experiments demonstrate that Pax3 and Dach2 positively regulate each other's expression in vivo and support the validity of the Pax3/Six1/Eya2/Dach 2 network in regulating myogenesis.