Figure 5. Distribution of PhosphoERK1/2 in Control and Netrin-Treated Endoderm and E11.5 Lung.

ERK1/2 activity is assayed by whole mount immunohistochemistry by using antibody specific for phosphorylated forms of the protein. (A–E) endoderm cultured in Matrigel and 30 ng/ml FGF7 either without (A and D) or with 50 µg/ml Netrin-1 (B) or Netrin-4 (C and E). Note the elevated levels of phosphoERK1/2 in the secondary buds (A and D) but more uniform distribution in Netrin-treated samples (B, C, and E). The focal plane is through the lumen of the cysts in (C) and (I) and near the surface for (A, B, D–F, and H). Inset in (E) shows absence of phosphoERK1/2 in the internalized knobs in Netrin-4-treated samples. (F) Total ERK1/2 is distributed uniformly among budding and interbudding zone. (G) Western blot analysis shows that the relative level of phosphoERK1/2 is decreased in Netrin-4-treated samples. (H and I) In control samples cultured with 250 ng/ml FGF10, localized phosphoERK1/2 is restricted to the tips of elongated secondary buds (H), while Netrin-4-treated endoderm shows a uniform, low level of phosphoERK1/2 activity (I). (J–O) PhosphoERK1/2 activity in normal E11.5 lung. Note that highest ERK1/2 activity is localized to the distal tip of endodermal buds, and initiating buds shows higher phosphoERK than elongating buds (compare arrow and arrowhead in [K]). (L–O) When labeled together with TOTO-3 (blue, to visualize nuclei) and Alexa Fluor 488 phalloidin (green), a sharp boundary is seen between epithelial cells in the dilated distal tip of buds, which show high ERK1/2 activity (red), and the stalk region showing low levels. (M), (N), and (O) correspond to the blue, pink, and white squares in (L), respectively. Scale bar = 100 µm for (A)–(I), 20 µm for (J)–(L), and 10 µm for (M)–(O).