Fig. 9. FGF10 restores FgF8 expression in Gas1^-/- limbs. Mouse trunk fragments containing forelimbs were isolated from E9.75-E11.5 control (con; B-D) and mutant (-/-; E-G) embryos. (A) Diagram of FGF10 injection in anterior tip domain of the mesenchyme in the right limb. The injected embryo fragments were cultured for 16 hours and then subjected to whole-mount ISH to assay for Fgf8 expression in the AER. (B,E) PBS-injected, (C,D,F,G) injected with recombinant human FGF10 protein: (C,F) 9.2 pg and (D,G) 230 pg. Note that in D and G, the color was developed for 20 minutes to reveal the upregulation of Fgf8 in the injected wild-type limb (open arrowhead in D). The others were developed for 90 minutes. (J) Mutant E9.75 forelimb injected with 560 pg of FGF8 did not show any rescue of Fgf8 expression in the AER. (H,I) Mutant E11.5 forelimbs were injected with PBS (H) or 230 pg of FGF10 (I). Fgf8 expression in the AER was rescued in the FGF10-injected samples (arrowheads in F,G,I). Owing to the angle of the limbs relative to the body at this stage, the image was taken in a ventral view with the anterior pointing down. The control E11.5 limbs retained Fgf8 expression in the AER after culture (not shown).