Fig. 5. Impaired mammary line formation due to reduced somitic Fgf10 expression precedes the absence of mammary bud 3 in Gli3^Xt-J/Xt-J embryos. (A,B) Whole-mount in situ hybridization for Wnt10b reveals impaired mammary line formation (between arrows), the presence of placodes 2 and 4 (numbered), but not placode 3 in an E11.5 (44s) Gli3^Xt-J/Xt-J embryo. (C,D) X-gal staining for TOPGAL expression indicates that WNT signaling in an E11.5 Gli3^Xt-J/Xt-J embryo is reduced in a pattern similar to Wnt10b expression. (E,F) Whole-mount in situ hybridization for Lef1 reveals the absence of bud 3 in an E12.5 Gli3^Xt-J/Xt-J embryo. (G,H) Hematoxylin/Eosin-stained sections through the plane indicated in Fig. 2E reveals the absence of the stratum intermedium at the mammary line of an E11.5 (45 somites) Gli3^Xt-J/Xt-J mutant. (I-K) Radiolabeled in situ hybridization for Gli3 (red) on a section of an E11.0 (41 somite) wild-type embryo (I) through the plane indicated in Fig. 2E, and whole-mount in situ hybridization for Gli3 on a control (J) and Fgf10^-/- (K) embryo shows unchanged somitic Gli3 expression in the Fgf10^-/- mutant. (L,M) Whole-mount in situ hybridization for Fgf10 reveals that somitic Fgf10 expression (between black arrowheads) is restricted to the hypaxial domain in the Gli3^Xt-J/Xt-J embryo. (N,O) Whole-mount in situ hybridization for Wnt10b on flanks of Gli3^Xt-J/Xt-J embryos, cultured with an implanted bead (circle) soaked in BSA (N) or rFGF10 (O). Arrows indicate the mammary placodes or the expansion of a Wnt10b expression domain around the bead in O. (P,Q) Nuclear Fast Red stained section through the planes indicated by lines through the flank in L. Broken black lines outline the mammary epithelium. Abbreviations: s, somite stage; p, periderm; sg, stratum germinativum; si, stratum intermedium; pl #3, mammary placode 3; nt, neural tube; st, stomach; fl, forelimb; hl, hindlimb; rFGF10, recombinant FGF10. Scale bars: 10 µm in G-H,P-Q; 100 µm in A-F,I-M; 1 mm in N,O. Panel I is not to scale.