Fig. 6. Mammary bud 4 from Fgf10^–/– mutant female embryos is maintained and competent to ramify in wild-type stroma. Whole-mount in situ hybridization on Fgf10^–/– and wild-type embryos at E13.5 with dig-labeled antisense riboprobe for Lef1 and transplantation of wild-type and mutant mammary glands in cleared fat pads. B and D are higher magnification views of the boxed areas in A and C, respectively. (A) Lef1 expression is detected in the hair follicles (small dots) and in the mammary buds (dilated dots) in wild-type embryos at E13.5. (B) Note that the mammary bud 4 (black arrowhead) is bigger than the hair follicles. (C) Lef1 expression on Fgf10^–/– embryo. (D) Mammary bud 4 is still present at E13.5. (E) Transillumination picture of dissected skin corresponding to wild-type female embryo at E18.5. Note that five pairs of nipples are clearly visible. (F) Carmin Red staining of normal mammary gland 4 showing a ramified structure with numerous end buds (white arrowheads). (G) Transillumination picture of dissected skin corresponding to Fgf10^–/– female embryos at E18.5. Note that only one pair of nipples is visible. (H) Carmin Red staining of mutant mammary gland 4 showing a single elongated sprout (white arrowhead). (I) Carmin Red staining of cleared fat pads transplanted with wild-type mammary gland 4 and cultured for 4 weeks. The epithelium of wild-type mammary gland 4 (black arrow) has extensively ramified into the cleared fat pad in both directions. (J) High magnification of the transplanted mammary gland (asterisk) showing a nicely ramified epithelium emerging from it. (K) The epithelium of mutant mammary gland 4 (black arrow) has also ramified into the cleared fat pad in both directions. Note the presence of numerous end buds (small black arrows). (L) High magnification of the transplanted mammary gland (asterisk) showing a ramified epithelium emerging from it and hairs (dark stripes) at the graft side. B4, mammary bud 4; fp, fat pad; G1-5, mammary glands; ln, lymph node. Scale bar, A, 800 µm; B, 309 µm; C, 800 µm; D, 309 µm; E, 1800 µm; F, 80 µm; G, 1800 µm; H, 80 µm; I, 2600 µm; J, 315 µm; K, 2100 µm; L, 270 µm.