Fig. 3. Mammary placode formation in cultured embryos. (A-C) X-gal-stained E10.5 TOPGAL embryos after 0 hours (A), 24 hours (B) or 48 hours (C) culture in control medium. Arrows indicate developing placodes. (D-F) X-gal-stained E11 TOPGAL embryos after 24 hours culture in control-conditioned medium (D), WNT3A-conditioned medium (E) or LiCl-supplemented medium (F). WNT3A treatment causes the formation of placodes that are larger and more distinct than in the littermate control (arrows in D,E). LiCl treatment causes activation of TOPGAL transgene expression along the lateral aspect of the embryo, and formation of multiple intensely blue-staining clusters of cells (F, arrows). (G-I) Histological sections (counterstained with eosin) through the mammary placodes of E11 X-gal-stained embryos cultured for 24 hours in control medium (G), WNT3A-conditioned medium (H) or LiCl (I). (J-K) Whole mount in-situ hybridizations for Wnt10b on E11 embryos cultured for 48 hours (J,K) or 24 hours (L) in control medium (J), WNT3A-conditioned medium (K) or 50 mM LiCl (L). LiCl treatment was limited to 24 hours, as we found 48 hours' exposure to LiCl to be toxic. Arrows indicate Wnt10b expression in placodes (J-L) and ectopic placode-like structures (L). Scale bar: 0.65 mm in A; 0.75 mm in B; 0.9 mm in C; 0.5 mm in D-F, 33 µm in G-I; 0.6 mm in J-L.