Fig. 3.

Biological responses of isolated LG and SMG epithelial explants to FGF7, FGF10, and FGF10-HS mutants in an ECM-diffusion assay. Growth responses of epithelial explants of LG (A) or SMG (B). (C) The morphology of the LG explant (the ratio between bud width and length) correlated with the difference in HS affinity and gradient formation of the FGF. The ratio of the width to the length of epithelial buds was determined in four independent experiments (5 to 7 explants of each kind); the Student's t test was used for statistical analysis. (D) BrdU labeling (red) of SMG explants grown in the presence of FGFs. An antibody specific for syndecan-1 (green) was used to label all of the epithelial cells. Explants exposed to the sharp gradients produced by the restricted diffusion of FGF10 and FGF10^K195E showed proliferating cells only at the bud tips (which are near the interface between the gel and the medium). In contrast, FGF10^R193K and FGF10^T197K induced cell proliferation at the tips and also more distally in the explants. Both FGF7 and FGF10^R187V, which could diffuse freely throughout the gel, induced cell proliferation throughout the whole explant. (E) Different FGFs permeating into the gel are differentially captured by HS (indicated schematically in red) and are thus likely to form different gradients that elicit different cellular responses. Quantification of BrdU labeling in SMG (F) and LG (G) explants exposed to FGF ligands. Explants exposed to FGF10^R187V showed the same labeling profile as those that were exposed to FGF7. BrdU quantification for LG and SMG explants was performed in four independent experiments (6 to 8 explants of each kind).