Figure 6.

Ligand-independent Met signaling mediates PAX3-FKHR activity in vivo. (A,B) In situ hybridization using a c-met probe shows ectopic and enhanced expression of c-met in occipital/cervical (green arrowhead) and thoracic (red arrowhead) somites of E10.5 Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos (B) compared with the Pax3^IRESnLacZ/+ control embryos, where it is expressed in the epaxial and hypaxial extremities of the dermomyotome (A). (A′,B′) Closeups of c-met expression in thoracic somites. In both cases, c-met is expressed in limb muscle precursor cells (red arrowhead). (C,D) SF/HGF, the Met ligand, is not expressed in thoracic somites of Pax3^IRESnLacZ/+ control embryos (C, black arrow), nor is it induced in Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos (D, black arrow). Normal expression in limb buds (red arrowheads) is also seen in Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos (D) compared with the control (C). (E,F) Pax3^{PAX3-FKHR-IRESnLacZ/+} animals were crossed with c-met^D/+ heterozygotes (Maina et al. 1996) to produce Pax3^{PAX3-FKHR-IRESnLacZ/+}/c-met^D/+ embryos (E) and Pax3^{PAX3-FKHR-IRESnLacZ/+}/c-met^D/D embryos (F). Reducing the number of c-met alleles suppresses in a dose-dependent manner the ectopic delamination gain-of-function phenotype (arrows) in Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos (cf. Fig. 3D′-E′), demonstrating that Met signaling is directly implicated in the phenotype. (G-N) Immunocytochemistry on sections of thoracic level somites (closeup of the dermomyotome) from Pax3^IRESnLacZ/+ control (G-J) or Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos (K-N) with DAPIstaining (G,K), using antibodies recognizing β-Gal (H,L) or the form of Met phosphorylated on tyrosine 1234/12235 (P-Met) (I,M). The merged images for β-Gal and P-Met are shown in J and N. β-Gal-positive cells that coexpress P-Met are detected in the dermomyotome of the Pax3^{PAX3-FKHR-IRESnLacZ/+} somites only, demonstrating that Met is activated in the presence of PAX3-FKHR. Cells that have delaminated from the dermomyotome (L-N, white arrowhead) in Pax3^{PAX3-FKHR-IRESnLacZ/+} embryos also colabel with β-Gal and P-Met antibodies. (O-Q) X-Gal staining of the cervical region of Pax3^IRESnLacZ/+ (O, 33 somites), Pax3^IRESnLacZ/+/c-met^D/D (P, 32 somites), and Pax3^{IRESnlacZIRESnLacZ/+} (Q, 34 somites) embryos at E10.5, shows maintenance of the full extent of the β-Gal⁺ dermomyotome and in particular the hypaxial domain, in the absence of Met (P, arrows), compared with the Pax3 mutant (Q, arrows). (HGC) Hypoglossal cord; (FL) forelimb.