Fig. 3. The anteriormost part of the limb buds fails to develop in Msx1^-/-; Msx2^-/- double mutant embryos. (A,B) Double in situ hybridisation with Fgf8 (red) and Fgf4 (purple) probes on control (A) and Msx1^-/-; Msx2^-/- double mutant (B) embryos. At 11.5 dpc, in control embryos, the AER displays an anterior domain expressing Fgf8 (red staining) but not Fgf4 (purple staining), whereas in the double mutant the anterior expression limit is the same for both genes. Note the sharper aspect of the anterior border of the limb bud in the double mutant. White arrows indicate the anterior limit of Fgf8 and black arrows indicate the anterior limit of Fgf4 expression domains. (C,D) Left-side view of 10.5 dpc embryos showing both forelimbs (FL) and hindlimbs (HL). The Alx4 expression domain, corresponding to the anterior third of the limb bud mesenchyme, is normal at this stage in the double mutant. (E,F) At 10.75 dpc, the anterior Hoxd11-negative domain, corresponding to the anterior third of the normal limb bud (E), is virtually absent in the mutant (F), whereas the posterior Hoxd11-expression domain has globally the same size as in the control. (G,H) Analysis of Hoxd11 expression at 11.5 dpc confirms that the anterior domain is missing from the limb buds of double mutants. (I,J) Whole-mount analysis of apoptosis in limb buds at 10.5 dpc (ventral view). TUNEL staining shows no increase of apoptosis in the AER of the double mutant at E10.5. (A,B,E,F,I,J) Forelimbs. (G,H) Hindlimbs. Anterior is towards the right in all panels.