Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 4. (A) Glands inhibited with rFGFR2b were rescued with FGF7 and FGF10, which increased the number of end buds. Glands were treated with 1.6 µg/ml of rFGFR2b, and increasing doses of FGFs were added. Glands cultured with rFGFR2b were rescued only by FGF7 (500 ng/ml) and FGF10 (1000 ng/ml), and not by FGF1 (500 ng/ml) and FGF3 (1000 ng/ml), by other ligands for FGFR2b, or by FGF2 (200 ng/ml) or BMP7 (200 ng/ml). (B) A combination of neutralizing antibodies to FGF1, FGF7 and FGF10 was required to significantly inhibit branching morphogenesis (ANOVA, **P<0.01). Individual antibodies to FGFs (25 µg/ml), or combinations of either two or three antibodies or similar concentrations of either mouse or goat IgGs, were incubated with E12 SMGs for 48 hours. Individual anti-FGF antibodies or combinations of two anti-FGF antibodies (not shown) did not significantly inhibit branching. The number of buds was expressed as a ratio of the number of buds at 48 hours/number of buds at 2 hours (T48/T2).

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Cell Culture, Incubation

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 6. FGF-induced morphogenesis is a result of localized proliferation. (A) Individual FGFs, FGF1 (200 ng/ml), FGF2 (100 ng/ml), FGF4 (100 ng/ml), FGF7 (100 ng/ml) and FGF10 (500 ng/ml), stimulate proliferation of epithelial buds by 8 hours, before any morphogenesis has occurred. BMP4 (100 ng/ml) and BMP7 (100 ng/ml) did not stimulate epithelial proliferation above control levels. Scale bar: 50 µm. BrdU was detected with a Cy3-labeled antibody, the nuclei were stained with SYBR-green, and fluorescence was quantitated using MetaMorph software and expressed as the ratio of BrdU:SYBR-green pixels. At least five glands/condition were used for quantitation, and the experiments were repeated twice with similar results. (B) FGFs induce localized proliferation resulting in distinct morphologies by 44 hours. FGF1 and FGF10 stimulate proliferation at the tips of the ducts. FGF2 and FGF7 stimulate proliferation over the entire buds, and FGF7 also induces duct proliferation. BrdU was detected with a Cy3-labeled antibody, and nuclei were stained with SYBR-green. Scale bar: 100 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Control, Labeling, Staining, SYBR Green Assay, Software, Quantitation Assay

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 5. (A) Individual FGFs and BMPs have distinct morphological effects on isolated epithelium cultured in growth factor-reduced Matrigel for 44 hours. FGF1-, FGF4- and FGF10-treated epithelium form duct-like structures, whereas FGF2 and FGF7 promote bud formation. Epithelium treated with FGF8, BMP4 or BMP7 alone do not grow. Numbers indicate concentrations in ng/ml. (B) Combinations of FGFs resulted in phenotypes with more complex morphology than any growth factor alone. FGF4/FGF1 resulted in larger glands with longer ducts. FGF10/FGF2 and FGF7/FGF1 both resulted in elongated stalks and enlarged buds. Combinations of FGF10 or FGF7 with either BMP4 (shown) or BMP7 (similar result, not shown) did not grow, suggesting BMPs antagonize FGF7 and FGF10. FGF2/FGF7/FGF10 resulted in multiple stalks and buds. FGF1/FGF2/FGF10 resulted in the most complex morphology with elongated ducts, multiple branch points and enlarged terminal epithelial buds. Scale bars: 200 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Isolation, Cell Culture

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 7. FGFR1 and FGFR2 are expressed throughout the epithelium. (A) FGFR1 staining (red) is increased around the edge of the bud with FGF7 treatment, and near the tip of the duct with FGF10 treatment. E- cadherin (green) defines the cell junctions of the epithelium, and SYBR-green stains the nucleus (pseudocolored blue). (B) FGFR2 staining (red) is localized throughout the epithelium in a punctate pattern and shows less cell membrane localization with FGF7 than with FGF10 treatment. E-cadherin (green) defines the cell junctions of the epithelium and SYBR- green stains the nucleus (pseudocolored blue). Scale bars: 20 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Staining, SYBR Green Assay, Membrane

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 8. FGF7- and FGF10-mediated morphogenesis is ERK1/2 dependent. FGF7-mediated morphogenesis is also PI3K dependent. FGF7- (200 ng/ml) and FGF10- (1500 ng/ml) mediated morphogenesis is inhibited by the FGFR inhibitor SU5402 (SU, 2.5 µM), the MEK1/2 inhibitor UO126 (UO, 10 µM), and by rFGFR2b (R2b, 5 µg/ml), but not by the broad PKC inhibitor GO6983 (GO, 1.5 µM), or by rFGFR1b (R1b, 10 µg/ml). FGF7-mediated morphogenesis is also inhibited by the PI3K inhibitor LY294002 (LY, 10 µM). Star indicates an inhibited phenotype. The inhibitors were added to the media for 44 hours, and the number of buds (for FGF7) and the length of ducts (for FGF10) were measured using MetaMorph software and expressed as a ratio at 44 hours/time 0. *, P<0.5; **, P<0.01. See Movie 2 in supplementary material, which shows FGF7- and FGF10-treated epithelium cultured for 36 hours.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Software, Cell Culture

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: 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. α6 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.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Produced, Membrane, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Activation Assay, Zymography, Control

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 10. Working model of how FGF7 and FGF10 signaling through FGFR2b regulates morphogenesis. The model summarizes our findings, and the dotted lines show other potential mechanisms: MMP2 may regulate FGFR1 cleavage; FGF1 expression may stimulate both FGFR1 and FGFR2; cofactors or co-receptors may specify the localization of FGF binding and, therefore, where proliferation occurs.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Expressing, Binding Assay