Fig. 2. Mammary placode development is asynchronous. Whole-mount in situ hybridization on embryos at E11.75 with dig-labeled antisense riboprobe for Lef1. (A) Lateral view of the embryo showing that placode 3 and placode 4 have formed and are seen as dot-like structures. (B) High magnification of the hindlimb area showing placode 4 as a dot- like structure and placode 5 as a line. (C) High magnification of the forelimb area showing placode 3 as a dot-like structure, placode 1 as a line and placode 2 as a diffuse line. (D-F) Scanning electron microscopy of the flank of a E12.5 wild-type embryo showing 5 mammary buds localized at precise points along the anteroposterior axis (3 thoracic and 2 inguinal). (E) High magnification of the box shown in D. Note that mammary bud 2, the last one to form, is the bud that is the most elevated above the surface of the epidermis. (F) High magnification of the mammary bud 2 showing individual cells at the surface of the knob-like structure. B1-5, mammary buds; P1-5, mammary placodes. Scale bar, A, 950 µm; B, 630 µm; C, 470 µm; D, 440 µm; E, 180 µm; F, 30 µm.