Fig. 3. Wnt5a inhibits lateral branching in vivo and HGF-induced branching morphogenesis in vitro. (A-C) Wnt5a inhibits lateral branching in vivo. Carmine staining of murine mammary glands treated with slow-release pellets containing BSA (A) or Wnt5a in the contralateral gland (B). The pellet is marked. (C) The average number of secondary branches per 0.5 cm of the primary branch was calculated from three separate fields for each gland. Error bars represent the standard deviation of the average from the three fields. A paired Student's t-test indicated that the differences were significant. (D-G) Wnt5a inhibits branching and growth in mammary organoids in culture. Phase contrast images showing primary mammary organoids untreated or treated with HGF alone or in combination with Wnt5a (D). Quantification of primary branch number demonstrates a statistically significant reduction in branching in cultures treated with Wnt5a compared to HGF alone, determined by a Student's t-test (E; P<0.05). Quantification of primary branch length shows that Wnt5a inhibited the extent of branching from the organoids, (F; P<0.05). Organoids treated with Wnt5a showed a statistically significant reduction in proliferation as determined by BrdU incorporation (G; P<0.05), illustrated to the right.