Figure 2

Low concentrations of exogenous TGF-β1 induce morphogenesis of branching tubules. (A) J3B1A cells grown in a collagen gel in defined medium for a total of 10 days. (B) Parallel culture in which J3B1A cells were grown in a collagen gel for 6 days to allow cyst formation and were subsequently treated with 50 pg/ml TGF-β1 for an additional 4 days. TGF-β1 has induced the outgrowth of tube-like structures from the wall of existing cysts. (C) Treatment with 2 ng/ml TGF-β1 has resulted in the formation of numerous thin cell cords extending out into the surrounding collagen matrix. Notably, at this relatively high concentration, TGF-β1 also disrupts the organization of preformed cysts, resulting in lumen obliteration. (D) Higher magnification view of a multicellular structure formed in a culture treated with 20 pg/ml TGF-β1 for 4 days. The outgrowths enclose a patent lumen, which at least in some tubes is continuous with the cavity of the cyst. (E) Semi-thin section of a collagen gel culture of J3B1A cells treated with 50 pg/ml TGF-β1 for 4 days. Bars (A-E), 200 μm. (F) Quantitative analysis of TGF-β1-induced tubulogenesis. J3B1A cells were grown in a collagen gel for 6 days to allow cyst formation and were subsequently treated with different concentrations of TGF-β1. Tube formation was evaluated as described in Materials and Methods after 4 days of treatment. Data were expressed as mean number of outgrowths per colony ± s.e.m. from three separate experiments. p < 0.0005 for values of 20 pg/ml TGF-β1 at 2 days compared with control at 2 days, as well as for values of 100 pg/ml TGF-β1 at 2 days compared with 50 pg/ml TGF-β1 at 2 days; p < 0.0025 for values of 50 pg/ml TGF-β1 at 4 days compared with 2 days; p < 0.01 for values of 50 pg/ml TGF-β1 at 4 days compared with 20 pg/ml TGF-β1 at 4 days; p < 0.025 for values of 50 pg/ml TGF-β1 at 2 days compared with 20 pg/ml TGF-β1 at 2 days, as well as for values of 20 pg/ml TGF-β1 at 4 days compared with 2 days.