Fig. 2. Accelerated ductal extension, excessive side-branching and precocious lobulo-alveolar development in mutant mammary glands. (A) Whole-mount preparations of control and mutant mammary glands at different developmental stages. Carmine Red-stained whole mounts of inguinal mammary glands from control (a, b, e-g, k and l) and mutant (c, d, h-j, m and n) mice at 6 (a-d), 8 (e-j), 14 (k-n) weeks of age. Images in b, d, f, g, i, j, l, and n show higher magnifications of the areas bracketed in a, c, e, h, k and m. Note the lobulo-alveolar development on mutant glands (arrows in d, j, and n). Scaled arrows in a and c indicate the distances between the centers of the lymph node and the end of the TEBs. Box in a indicates the area where the number of branches was counted. (B) Quantitative representation of distances from the center of the lymph node to the far end of TEBs (top), and the number of branching points within the comparable boxed area (see Aa. 8 mm²) at different stages analyzed (n=45). (C) Northern blot analysis of ß-casein mRNA levels in control (C) and mutant (M) mammary glands. RNA was extracted from virgin mammary glands at 6 weeks (6W) and 10 weeks (10W). Northern blots were probed with cDNAs encoding – and ß-casein. One representative of two independent experiments is shown. The amount of ribosomal RNAs was used as loading controls. (D) Enlarged lobulo-alveolar in mutant mammary gland compared with control at 13 days after pregnancy.