Fig. 6. Local inhibition of FGFR4 signaling does not modify muscle progenitor proliferation. S-FR4-Fc-expressing cells were injected into the limb buds of E6 embryos. These cells serve as a source of protein, which locally perturbs the signaling of endogenous FGFR4. After overnight incubation, the embryos were exposed to BrdU for 1 hour and analyzed for FGFR4 expression. The choice of the probe enabled us to examine the expression of endogenous FGFR4 and the position of the injected cell pellet (P). Two independent experiments are shown. (A,B,E) The first embryo; (C,D,F) the second embryo. (A,C) General views of the injected limbs. (B,D) Close up of the views presented in A,C. Broken lines in B,D represent the outline of the muscle bundles as they are observed in the contralateral, uninjected side. In B,D, a muscle bundle crosses the cell pellet. We observed that the muscles immediately adjacent to the injected cells display a strong downregulation of FGFR4 expression (red arrowheads), indicative of an efficient inhibition of muscle differentiation. (E,F) Sections of the embryos presented in B,D. Around the cell pellet, light, but clearly visible, blue staining (red arrowhead) enabled us to identify the position of affected muscle progenitors, which we delineated with a broken line. At 10-20 cell diameters away from the injected cells, FGFR4 expression was normal (dark blue, green arrowhead). BrdU counting was carried out by arbitrarily choosing a similar surface in the affected and unaffected regions and comparing the number of BrdU-positive nuclei in each. This was done in four embryos and in at least two adjacent sections each. No differences in BrdU-positive cells were observed between the two regions, demonstrating that an arrest of cell division cannot explain the disappearance of myogenic markers.