Fig. 3. Impaired mammary line formation precedes the absence of mammary placodes in Fgf10^-/- and Fgfr2b^-/- embryos. (A-F) Hematoxylin/Eosin-stained sections through a wild-type (A-D), Fgf10^-/- (E) and Fgfr2b^-/- embryo (F) around E11.5, through the plane indicated by the line in Fig. 2E. Boxed areas in A are magnified in B-F. First, the stratum germinativum consists of squamous cells (B) that become cuboidal and covered with a layer of periderm (C) on the Wolffian ridge or flank. Next, they become cylindrical and form an additional layer (stratum intermedium) of cylindrical cells on the ventral area of the flank (D). This indicates mammary line formation, which does not occur in either mutants (E,F). (G-I) In situ hybridization for Wnt10b expression shows formation of the mammary line (between arrows) and mammary placodes (numbered), restricted to the area of placode 4 in the mutants. (J-L) X-gal staining visualizing TOPGAL expression indicates that WNT signaling occurs in a pattern similar to Wnt10b expression. (M-O) In situ hybridization for Lef1 reveals that all placodes (numbered) except 4 are absent in the mutants. Abbreviations: s, somite stage; wt, wild type; ct, control; sg, stratum germinativum; dm, dermal mesenchyme; p, periderm; si, stratum intermedium. Scale bars: 10 µm in B-F; 100 µm in G-O; 1 mm in A.