Fig. 4. Inhibition of FGFR4 signaling results in a block of limb bud myogenesis. Two-day-old embryos were injected in the prospective limb bud domain with cells infected with a secreted form of FGFR4. Four days later, infected embryos were processed for double in situ hybridization (A-E,G) or whole-mount immunohistochemistry with a monoclonal antibody directed against the embryonic form of the myosin heavy chain (MyHC, F). In a first round of in situ hybridization, a Fc-specific probe enabled us to determine which embryos had been efficiently infected (such an embryo is presented in A). These were then destained and a second in situ reaction was performed with probes specific for various stages of myogenic differentiation. Although none of the infected embryos displayed a variation in Pax3 expression (B), all muscle markers (Myf5, D; MyoD, E), the embryonic myosin heavy chain (F) and FGFR4 itself (C) were strongly downregulated. (G,H) To estimate the amplitude of the inhibition, embryos were separated in two parts after in situ hybridization, and then photographed (G); the stained dorsal muscle masses were delineated manually with Adobe Photoshop, and their surfaces were compared by pixel counting (H). In the case presented here, MyoD staining was decreased by 77%. When control embryos were counted in a similar manner, a difference of no more than 8% was observed between both limbs.