Journal: Developmental biology

Article Title: Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut.

doi: 10.1016/j.ydbio.2004.04.039

Figure Lengend Snippet: Fig. 4. Whole mount ISH of Fgfr2b and Fgf10 mRNA in vivo and in foregut cultures. (A) Fgfr2b mRNA is expressed throughout the foregut endoderm including the respiratory tract in E9.5 embryos and in (B) control cultures. (B, C) Fgfr2b expression is not disrupted by BMS treatment. (D) At E8.5, Fgf10 is expressed in the mesoderm of thyroid; subsequently (E9.5), signals appear in lung and thyroid domains. (E) In control cultures, these domains are preserved. (F) BMS treatment of explants younger than the 15-somite stage, but not older than that (G), prevents Fgf10 mRNA expression in the presumptive respiratory mesoderm (*). RA (106 M) has no obvious effects on Fgf10 expression. Area between dotted lines (in yellow) corresponds to the region of disrupted Fgf10 expression. Scale bars in A and E represent 85 and 150 Am, respectively.

Article Snippet: In some experiments, heparin beads soaked in human recombinant FGF10 (100 ng/Al, R&D Systems) or PBS (buffer) were grafted onto foreguts after 48 h culture.

Techniques: In Vivo, Control, Expressing

Journal: Developmental biology

Article Title: Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut.

doi: 10.1016/j.ydbio.2004.04.039

Figure Lengend Snippet: Fig. 6. Exogenous FGF10 rescues lung budding and epithelial differentiation in BMS-treated cultures. PCNA staining in sagittal sections (A, D) and whole mount ISH of Ttf1 (B, E) and Sp-C (C, F) in 6-day cultures engrafted with heparin beads (human recombinant FGF10, PBS buffer). (A) FGF10 beads induced lung bud formation, local PCNA labeling and expression of Ttf1 (B) and Sp-C mRNA (C) in BMS-treated explants. PBS beads failed to induce a local increase in proliferation (D) and had no detectable Ttf1 (E) or Sp-C (F) signals in the respiratory region of foregut. Note the preserved Ttf1 staining in thyroid (B, E). (G–J) Quantitative analysis of PCNA- and TUNEL-stained sections from BMS-treated explants in the lung field. (H) Engraftment of an FGF10 bead results in a significant increase in the relative number of PCNA-labeled cells in the adjacent endoderm (G) as compared to a PBS bead. Mesodermal PCNA (G) or TUNEL labeling (I) in endoderm or mesoderm is not altered by FGF10. Graphs (H, J) represent mean and standard error; *P < 0.05. Lu, lung; Th, thyroid. Scale bar in E represents 240 Am.

Article Snippet: In some experiments, heparin beads soaked in human recombinant FGF10 (100 ng/Al, R&D Systems) or PBS (buffer) were grafted onto foreguts after 48 h culture.

Techniques: Staining, Recombinant, Labeling, Expressing, TUNEL Assay

Journal: Developmental biology

Article Title: Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut.

doi: 10.1016/j.ydbio.2004.04.039

Figure Lengend Snippet: Fig. 7. Lung agenesis and disrupted expression of Ttf1 protein and Fgf10 mRNA in the respiratory region of foregut of vitamin A-deficient rats (A–D). H&E and Ttf1 immunostaining of transverse sections of control RAS foregut demonstrates (A) ongoing tracheoesophageal separation with Ttf1 expression restricted to the ventrally located tracheal epithelium and, more caudally, (B) primary lung bud epithelium intensely labeled with Ttf1. (C) In RAD foregut, the tube is irregularly shaped (*) and shows low levels of Ttf1 expression in the ventral endoderm at the prospective respiratory region that failed to bud. (D) Strong Ttf1 signals, however, are present in the thyroid bud. (E, F) Isotopic ISH of Fgf10 mRNA in transverse sections at the level of the lung buds in control RAS demonstrates localized signal in the mesoderm of the lung (F, enlarged), limb and urogenital tract (ug). In RAD (G, H), Fgf10 expression is preserved in the limb and urogenital tract but is disrupted in the presumptive lung field of the foregut (H, high magnification). Es, esophagus; fl, forelimb; hl, hindlimb; Lu, lung; Th, thyroid; Tr, trachea; RAD, RA deficient; RAS, RA sufficient; ug, urogenital ridge. Dashed boxes in E and G delineate close-up views in F and H, respectively. Arrowheads represent signals; asterisks indicate areas that failed to bud or to express Fgf10. Scale bars in B and G represent 80 and 480 Am, respectively.

Article Snippet: In some experiments, heparin beads soaked in human recombinant FGF10 (100 ng/Al, R&D Systems) or PBS (buffer) were grafted onto foreguts after 48 h culture.

Techniques: Expressing, Immunostaining, Control, Labeling

Journal: Developmental biology

Article Title: Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut.

doi: 10.1016/j.ydbio.2004.04.039

Figure Lengend Snippet: Fig. 8. Proposed model for the role of RA in early lung morphogenesis. Early in mouse foregut organogenesis, RA signaling is active in all layers. (A) By the 15-somite stage, RA has induced proliferation of a critical mass of mesodermal cells in the lung field, while signaling in endoderm maintains identity in lung progenitor cells (in red). (B) At around 25 somites, the condensing mesodermal cells begin to locally express Fgf10 (in green), which then activates the Fgf pathway in the endoderm to expand the population of lung progenitor cells into a primary endodermal bud. (C) Once secondary buds form and branching morphogenesis starts, RA signaling is downregulated in the lung and Fgf10 expression becomes independent of RA.

Article Snippet: In some experiments, heparin beads soaked in human recombinant FGF10 (100 ng/Al, R&D Systems) or PBS (buffer) were grafted onto foreguts after 48 h culture.

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: FGF10/FGFR2 signal induces cell migration and invasion in pancreatic cancer

doi: 10.1038/sj.bjc.6604473

Figure Lengend Snippet: Expression patterns of FGFR2 and FGF10 in normal pancreas and pancreatic cancer. The magnification is shown in the right bottom corner of each figure. ( A and B ) Immunostaining of FGFR2 ( A ) and FGF10 ( B ) in normal pancreas, showing that FGFR2 is expressed weakly in ductal cells ( A , arrow) and acinar cells ( A , arrow head), and that no obvious FGF10 staining is found in normal pancreatic tissue, including ductal cells ( B , arrows). ( C and D ) Immunostaining of FGFR2 ( C ) and FGF10 ( D ) in pancreatic cancer tissues, showing that FGFR2 is expressed in cancer cells ( C ), whereas FGF10 is expressed in scattered cells in the stroma surrounding cancer cells ( D , arrows). ( E and F ) Immunostaining of FGFR2 in pancreatic cancer cells. ( E ) Representative result from the FGFR2 high expression group, indicating higher FGFR2 expression in cancer cells (arrows) compared with islets (arrow head). ( F ) Representative result from the FGFR2 low expression group, showing lower FGFR2 expression in cancer cells (arrows) compared with islet (arrow heads). ( G and H ) Immunostaining of FGF10 ( G ) and CD3 ( H ), marker for T cell. Fibroblast growth factor 10 and CD3 are both expressed in scattered cells with similar cell shape in the stroma surrounding cancer cells (arrows).

Article Snippet: They were then rinsed and blocked in 10% H 2 O 2 solution with methanol for 10 min. Next, the sections were incubated with goat anti-human FGF10 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) at 1 : 20 dilution, mouse anti-human FGFR2 monoclonal antibody (R&D Systems) at 1 : 10 dilution, or rabbit anti-human CD3 monoclonal antibody (ready-to-use without dilution) (Thermo Fisher Scientific Anatomical Pathology, Fremont, CA, USA) overnight at 4°C.

Techniques: Expressing, Immunostaining, Staining, Marker

Journal: British Journal of Cancer

Article Title: FGF10/FGFR2 signal induces cell migration and invasion in pancreatic cancer

doi: 10.1038/sj.bjc.6604473

Figure Lengend Snippet: Fibroblast growth factor 10 induces cell migration and invasion in pancreatic cell lines with FGFR2-IIIb expression. ( A ) RT–PCR analysis of FGF10 and FGFR2-IIIb in four pancreatic cell lines and cDNA obtained from normal lung tissue as a positive control. None of the cell lines express FGF10. MIA PaCa-2 and PanC-1 cells do not express FGFR2-IIIb, but CFPAC-1 and AsPC-1 do express this gene. ( B ) Representative results of cell migration (upper panels) and invasion (lower panels) for CFPAC-1 cells. Representative migrated and invaded cells are indicated with arrows. ( C and D ) Cell migration ( C ) and invasion ( D ) assay of all four cell lines cultured without (white column) or with (black column) FGF10 (100 ng ml −1 ). FGF10-induced cell migration and invasion in CFPAC-1 and AsPC-1 cells, but not in MIAPaCa-2 and PanC-1 cells. The numbers of migrated or invaded cells cultured with FGF10 are shown relative to a value of 100% for cell migration without ligand. ( E and F ) Inhibition of FGFR2-IIIb signalling by an FGFR2-IIIb/IgG chimera in CFPAC-1 cells. Migration ( E ) and invasion ( F ) assay. The numbers of migrated or invaded cells are shown relative to a value of 100% for cells cultured without FGF10 or chimera (control; white column). FGF10-induced migration and invasion in CFPAC-1 cells (black column). Addition of the FGFR2-IIIb/IgG chimera completely eliminated the effects of FGF10 (grey column), whereas the chimera itself did not affect cell migration and invasion of CFPAC-1 cells (striped column). * P <0.05.

Article Snippet: They were then rinsed and blocked in 10% H 2 O 2 solution with methanol for 10 min. Next, the sections were incubated with goat anti-human FGF10 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) at 1 : 20 dilution, mouse anti-human FGFR2 monoclonal antibody (R&D Systems) at 1 : 10 dilution, or rabbit anti-human CD3 monoclonal antibody (ready-to-use without dilution) (Thermo Fisher Scientific Anatomical Pathology, Fremont, CA, USA) overnight at 4°C.

Techniques: Migration, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, Inhibition, Control

Journal: British Journal of Cancer

Article Title: FGF10/FGFR2 signal induces cell migration and invasion in pancreatic cancer

doi: 10.1038/sj.bjc.6604473

Figure Lengend Snippet: Fibroblast growth factor 10 induces expression of mRNA for MT1-MMP and TGF- β 1 in CFPAC-1 cells. The figure shows the relative copy numbers of MT1-MMP ( A ) and TGF- β 1 ( B ) mRNA in CFPAC-1 cells cultured with 100 ng ml −1 of FGF10 for the indicated times. The MT1-MMP and TGF β 1 mRNA/GAPDH mRNA copy number ratios are shown relative to those of cells without FGF10 stimulation (0 h). The concentration of TGF- β 1 protein in medium also increased in time-dependent manner in CFPAC-1 cells 48 h after addition of FGF10 (100 ng ml −1 ) ( C ). FGF receptor-2-IIIb/IgG chimera (500 ng ml −1 ) inhibited this TGF- β 1 secretion by FGF10, whereas chimera alone did not affect TGF- β 1 secretion by itself ( D ). * P <0.05; NS=not significant.

Article Snippet: They were then rinsed and blocked in 10% H 2 O 2 solution with methanol for 10 min. Next, the sections were incubated with goat anti-human FGF10 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) at 1 : 20 dilution, mouse anti-human FGFR2 monoclonal antibody (R&D Systems) at 1 : 10 dilution, or rabbit anti-human CD3 monoclonal antibody (ready-to-use without dilution) (Thermo Fisher Scientific Anatomical Pathology, Fremont, CA, USA) overnight at 4°C.

Techniques: Expressing, Cell Culture, Concentration Assay

Journal: Development (Cambridge, England)

Article Title: Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis

doi: 10.1242/dev.080226

Figure Lengend Snippet: Chick hypothalamic axons project to the NH. ( A , E ) Scanning electron micrographs (S.e.m.), ventral (A) and side (E) views, showing prospective (A) and definitive (E) ventral NH (nh) and the median eminence (me) region of the dorsal NH. Dorsal NH derives from collar cells (blue shaded region). Anterior is to the left. ( B-D , F-H ) Fgf10 and Fgf3 expression in the forming NH, shown in lateral whole-mount views (B,F; anterior to left) or transverse sections. nh points to prospective/definitive ventral NH. Expression of Fgf3 (H) distinguishes collar/dorsal NH from ventral NH (nh). White bars indicate approximate boundary between collar/dorsal NH and ventral NH. Yellow dots outline adenohypophysis. ( I-N ) Transverse sections show that hypothalamic pioneers project to the dorsal NH, but do not initially project into the ventral NH. At E4.5, TUJ1 + pioneers (arrowhead in I) project towards the Tbx2 + midline above the adenohypophysis (outlined by yellow dots). By E6, TUJ1 + pioneers project through the collar region (arrowheads in J) but turn (arrowheads in K), rather than project into/beneath the prospective ventral NH. (L) TH + axons project to the collar region (arrowheads) at E6. Boxed area in J indicates area shown in K. (M,N) Serial adjacent transverse sections at E7 at the level of the me (M) or 50 μm more posterior (N). Arrowheads point to axons that have projected through the collar, but have not penetrated the ventral NH. ( O ) Transverse section of E10 NH. TUJ1 + axons (arrows) project beyond the Sox3 + collar region into/beneath the ventral NH. ( P ) Retrograde DiI labelling (position of DiI injection indicated by red arrowhead in E) reveals that many axons project into the NH by E12. ah, adenohypophysis/anterior pituitary; me, median eminence; nh, ventral NH. Scale bars: 100 μm in A-E,I-L; 50 μm in G,H,M-P; 150 μm in F.

Article Snippet: Fgf10 and Fgf3 blocking antibodies (Santa Cruz Biotechnology; 50 ng/μl) were added to medium or pre-incubated with tissue for 6-8 hours.

Techniques: Expressing, Injection

Journal: Development (Cambridge, England)

Article Title: Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis

doi: 10.1242/dev.080226

Figure Lengend Snippet: Forming NH directs H-NH axons in chick. ( A ) Schematic depicting hypothalamus (green, mammillary pouch; blue, prosp-NH; grey, retrochiasmatic area; yellow dots, Shh + region) and indicating regions of hypothalamic and [L] dissections (green and black boxes, respectively). Schematic insets in B-E show cultures. ( B ) Co-culture of ventral hypothalamus (hyp) and [L] explants. Axons project to prosp-NH (white arrow); right-hand panel shows high-power view of boxed region. Dotted line indicates ventral edge of hypothalamic explant. ( C ) No outgrowth is detected from [L] explant cultured alone. ( D-F ) Axons extend from [L] explant co-cultured with Fgf10 + prosp-NH explant; right-hand panel in (E) shows high-powered view of boxed region. Significantly more ( *** P <0.0001) axons extend from proximal (p) versus distal (d) face of [L] explants. Error bars represent s.e.m. ( G-J ) [L] explant from E4 Roslin GFP-chick, grafted homotypically to wild-type hypothalamus (shown schematically in G). After 2 days, axons extend from the graft towards the nascent NH (H,I; shown schematically in right-hand panel of G), re-orienting their growth as they approach NH (outlined by white dots). (I) High-powered view of boxed region in H. Axons extend towards the NH (dotted outline), but do not project deeply into it (asterisk in H). (J) Explant shown in H after fixation and in situ hybridisation with Fgf10 . mp, mammillary pouch. Scale bars: 100 μm.

Article Snippet: Fgf10 and Fgf3 blocking antibodies (Santa Cruz Biotechnology; 50 ng/μl) were added to medium or pre-incubated with tissue for 6-8 hours.

Techniques: Co-Culture Assay, Cell Culture, In Situ, Hybridization

Journal: Development (Cambridge, England)

Article Title: Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis

doi: 10.1242/dev.080226

Figure Lengend Snippet: FGFs orient H-NH axons. ( A , A′,D , D ′) Chick [L] explants cultured with protein-soaked beads shown in whole-mount view (A,D). Significantly more ( *** P <0.0001) axons extend from the proximal (p) versus distal (d) face of [L] explants (A′,D′). ( B , C , E , F ) High-powered views of boxed regions in A,D, after immunolabelling. Axons, including those expressing Vasopressin and TH (arrowheads) project from [L] explants towards Fgf10- (B,C) or Fgf3- (E,F) soaked beads. (Beads outlined by red dots.) ( G-G ″) Treatment with SU5402 abolishes FGF-stimulated outgrowth. ( H-L ′) [L] explants cultured with prosp-NH explants alone (H,H′) or with inhibitors (I-L′). ( M ) Local chemorepulsion (arrow) in proximity to an Fgf10-soaked bead. ( N , N ′) [L] explants cultured with high concentrations of FGF. In high powered views (N) white arrow points to short axons that emerge from proximal face, yellow arrows point to axons emerging from distal face. Error bars represent s.e.m. *** P <0.005. d, distal; p, proximal. Scale bars: 100 μm in A,D,G,H-L,M,N; 50 μm in B,C,E,F.

Article Snippet: Fgf10 and Fgf3 blocking antibodies (Santa Cruz Biotechnology; 50 ng/μl) were added to medium or pre-incubated with tissue for 6-8 hours.

Techniques: Cell Culture, Expressing

Journal: Development (Cambridge, England)

Article Title: Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis

doi: 10.1242/dev.080226

Figure Lengend Snippet: Prosp-NH-derived FGFs stimulate outgrowth of endothelial processes. ( A-G ) Transverse (B-F) and sagittal (G) sections through chick NH at planes indicated in A. D,E show serial adjacent sections. Arrowheads and arrows point to endothelial processes/axons outside (arrowheads) or within/beneath (arrow) ventral NH. ( H-I ) [CAMv] isolation from wild-type or Roslin-GFP embryos. ( J-K ) Short radial projections emerge from [CAMv] explants cultured alone for 48 hours. Whole-mount immunolabelling shows that these are Tie1 + (J′). Dotted circles in J′ outline explant and ring of short processes. ( L-L ″) [CAMv]-E4 prosp-NH co-culture after 24 (L) and 40 hours (L′). Arrows indicate long endothelial processes. ( L ′″) Quantitative analysis of long processes from proximal versus distal faces of [CAMv] explants. Error bars represent s.e.m. *** P <0.001. d, distal; p, proximal. ( M , M ′) High-power views, after sectioning; extending processes are DAPI + VE-cadherin + Tie1 + . ( N-R ′) GFP [CAMv] explants cultured with PBS- (N), Fgf10- (O,P) or Fgf3- (Q) soaked beads for 42 hours. FGF-beads promote significant numbers of long projections from proximal faces of [CAMv] explants. Analyses of wild-type co-cultures reveals that endothelial projections are DAPI + VE-cadherin + Tie1 + (R,R′). ( S , S ′) Wild-type [CAMv] explant cultured with Fgf10 for 40 hours. Whole-mount immunohistochemical analyses show many long (>90 μm) Tie1 + projections. Dotted circles show explant outline and reference for control process lengths (see J′). ( T , U ) Transverse sections through [CAMv] explants cultured with/without Fgf10. No significance difference is detected in DAPI + or pH3 + cells. ( V , V ′) Transverse sections through prosp-NH explants pre-soaked in anti-Fgf3 and anti-Fgf10 antibodies. Fgf10 and Tbx2 are expressed normally (compare with ). ( W ) Prosp-NH explant pre-soaked in anti-Fgf3 and anti-Fgf10 fails to provoke long process outgrowth from GFP [CAMv] explants, which appear similar to controls (compare with K). Righthand panel shows quantitative analysis. Scale bars: 70 μm in A-E,G-L″,N-Q,S,S′,V,W; 45 μm in T,U; 25 μm in M,M′,R,R′; 20 μm in F.

Article Snippet: Fgf10 and Fgf3 blocking antibodies (Santa Cruz Biotechnology; 50 ng/μl) were added to medium or pre-incubated with tissue for 6-8 hours.

Techniques: Derivative Assay, Isolation, Cell Culture, Co-Culture Assay, Immunohistochemical staining, Control