Fig. 9. FGF-mediated morphogenesis is MMP-dependent. (A, part a,b) MMP2 is localized in the mesenchyme and in the epithelium at the periphery of the growing buds, while MMP9 is localized only in the mesenchyme. Perlecan, produced by the mesenchyme, stains the basement membrane. LSCM sections of E13 cultured glands. Scale bar: 100 µm. (A, part c,d) MMP2 is localized at the periphery of the epithelium and in residual mesenchyme cells (dotted outline) in both (c) FGF7- and (d) FGF10-treated epithelium. (A, part e,f) MMP9 is localized only in the residual mesenchyme cells (dotted outline) in both FGF7- and FGF10-treated epithelium. 6 integrin and peanut lectin (PNA) are epithelial cell markers. Scale bars: 20 µm. (B) The relative levels of gene expression of MMPs, FGFs and FGFRs were compared by real-time PCR in salivary epithelium treated with either FGF7 (200 ng/ml) or FGF10 (1500 ng/ml) for 44 hours. MMP2 gene expression was increased 4-fold, FGF1 expression was increased 2.5-fold and FGFR1b expression was increased 2-fold in FGF7-treated epithelial rudiments, when compared with FGF10-treated epithelial rudiments. (C) FGF7 increases MMP2 production and activation after 44 hours. The culture media from epithelium treated with FGF7 or FGF10, as well as media from a dish with laminin-1 alone, was assayed by gelatin zymography after 24 and 44 hours of FGF treatment. (D) FGF7- and FGF10-mediated morphogenesis is inhibited by GM6001 (5 µM), a broad MMP inhibitor, and by a neutralizing antibody to FGF1 (25 µg/ml). Neither a DMSO carrier control (shown) nor an IgG control (not shown) inhibited morphogenesis.