Journal: Biology of reproduction

Article Title: Differential endometrial gene expression in pregnant and nonpregnant sows.

doi: 10.1095/biolreprod.109.082321

Figure Lengend Snippet: FIG. 3. Immunohistochemical localization of IL6R (a and b), LIFR (d and e), IL11RA (g and h), MUC4 (j and k), ERBB3 (m and n), FGF9 (p and q), FGFR3 (s and t), in endometrial tissue from nonpregnant (a, d, g, j, m, p, and s) and pregnant (b, e, h, k, n, q, and t) sows. Corresponding negative controls: IL6R preadsorption control (c), LIFR preadsorption control (f), IL11RA preadsorption control (i), murine IgG2a Isotype control (l), ERBB3 preadsorption control (o), murine IgG3 Isotype control (r), and murine IgG2a Isotype control (u). DG, deep glands; LE, luminal epithelium; S, stroma; SG, superficial glands. Bar ¼ 200 lm (a–f and j–u) and 500 lm (g–i).

Article Snippet: Primary antibody Manufacturer Product code Source Clonality Isotype Concentration (lg/ml) HIERa Primary antibody incubationb Anti-human LIFR R&D Systems (Minneapolis, MN) AF-249-NA Goat Polyclonal IgG 0.50 Ph 9 2 h RT Anti-human IL6R Santa Cruz (Santa Cruz, CA) sc-661 Rabbit Polyclonal IgG 0.10 Ph 6 2 h RT Anti-human IL11RA Santa Cruz sc-993 Rabbit Polyclonal IgG 8.00 Ph 9 2 h RT Anti-human FGF9 Santa Cruz sc-8413 Mouse Monoclonal IgG3 20.00 Ph 9 2 h RT Anti-human FGFR3 Santa Cruz sc-13121 Mouse Monoclonal IgG2a 8.00 Ph 9 2 h RT Anti-human MUC4 Abnova (Taipei City, Taiwan) H00004585-M07 Mouse Monoclonal IgG2a 0.20 Ph 6 2 h RT Anti-human ERBB3 Santa Cruz sc-285-G Goat Polyclonal IgG 0.67 Ph 9 o/n 48C a HIER, heat induced epitope retrieval. b RT, room temperature; o/n, over night.

Techniques: Immunohistochemical staining, Control

Journal: Developmental biology

Article Title: Loss of Fgfr2 leads to partial XY sex reversal.

doi: 10.1016/j.ydbio.2007.11.010

Figure Lengend Snippet: Fig. 7. FGF9 does not bind to the coelomic epithelium of Fgfr2 mutant XY gonads. E11.5 gonad sections were incubated with human FGF9, which was detected with an antibody specific to the human FGF9 protein (green). Human FGF9 bound to the coelomic epithelium in E11.5 XY gonad sections, but not in E11.5 XX and XY Fgfr2 mutant gonad sections. Arrowheads denote the surface of the coelomic epithelium.

Article Snippet: E11.5 frozen sections (12 μm) of XY control, XX control and XY Fgfr2 mutant gonads were treated with human FGF9 protein (0.7 μg/ ml) (R&D systems; AF-273-NA), which was detected by an antibody specific to human FGF9 (R&D systems; 273-F9).

Techniques: Mutagenesis, Incubation

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Missense mutation in the Fgf9 gene of Eks mice. ( a ) Nucleotide sequence of the Fgf9 cDNA derived from +/+ and Eks/Eks mice. Ek s mutants have an A to C substitution at position 428, which results in the replacement of Asn143 with Thr. The Eks missense mutation is indicated by the asterisk and the corresponding amino acid is shown in purple. ( b ) Structure-based sequence alignment of human FGFs. The amino acid sequence surrounding the N143T mutation in FGF9 Eks and that of its corresponding domain in other human FGF family proteins are aligned based on sequence identity. The Asn143 residue in FGF9 is highly conserved among most FGF proteins (purple box). The asterisk denotes the site of Eks mutation.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Sequencing, Derivative Assay, Residue

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Fgf9 Eks/Eks mice phenocopy Fgfr s gain-of-function mutants. ( a–l ) Defects in early specification of prospective elbow joints in Fgf9 Eks/Eks embryos. Hematoxylin and eosin staining ( a, b, g, h ) and in situ detection of Gdf5 ( c, d, i, j ) and Col2a1 ( e, f, k, l ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. In Fgf9 +/+ embryos there was Gdf5 expression at the prospective elbow joint ( i ), which was demarcated as the gap of Col2a1 expression ( k , arrow), at E11.5. In Fgf9 Eks/Eks embryos there was not Gdf5 expression at the prospective elbow joint ( j ). Scale bars, 100 µm. ( m–b’ ) Ectopic osteogenesis at the coronal sutures in Fgf9 Eks/Eks fetuses. Hematoxylin and eosin staining ( m, n, u, v ) and von Kossa staining ( o, p, w, x ) and in situ detection of Spp1 ( q, r, y, z ) and Runx2 ( s, t, a’, b’ ) in the coronal suture of Fgf9 +/+ and Fgf9 Eks/Eks fetuses at E15.5 and E16.5. Note the ectopic ossification in the suture of Fgf9 Eks/Eks at E16.5 ( v, x, z, b’ ). fb, frontal bone; pb, parietal bone. Scale bars, 100 µm.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Staining, In Situ, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects dimerization of FGF9. ( a, b ) Sedimentation equilibrium analysis of FGF9 WT and FGF9 Eks . Ln A 280nm versus radius 2 during sedimentation equilibrium at 16,000 rpm at 20°C is indicated using 0.4 mg/ml of FGF9 WT ( a ) and FGF9 Eks ( b ). The residuals are shown in the upper panels. ( c, d ) Sedimentation velocity analysis of FGF9 WT and FGF9 Eks . The sedimentation coefficient distribution for FGF9 WT ( c ) and FGF9 Eks ( d ) at the concentrations of 0.2, 0.3 and 0.4 mg/ml are shown. ( e ) Analytical gel filtration chromatography of FGF9 WT and FGF9 Eks . FGF9 WT or FGF9 Eks applied separately to Superdex 75 10/300 GL columns. Eluted FGF9 WT and FGF9 Eks were identified by absorbance at 280 nm. Arrows indicate the position of the size standards: 67 kDa, albumin; 43 kDa, ovalbumin; 25 kDa, chymotrypsinogen; 13.7 kDa, ribonuclease A.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Sedimentation, Filtration, Chromatography

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects the mitogenic activity of FGF9. ( a–g ) Dose dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing exogenous FGFR1b, 1c, 2b, 2c, 3b, 3c or 4 were treated with increasing concentrations of FGF9 WT or FGF9 Eks in the presence of 1 µg/ml heparin. Cell proliferation was determined by [ 3 H]thymidine incorporation after 36 hours in culture. ( h–n ) Heparin-dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing the respective FGFR were treated with increasing concentrations of heparin in the presence of 0.2 nM FGF9 WT or FGF9 Eks . Cell proliferation was determined as above. Data are represented as mean ± s.e.m. of triplicate assays. These results are representative of at least two independent experiments.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Activity Assay, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation reduces FGF9 affinity for heparin by impairing its dimerization. ( a ) Chromatographic analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. FGF9 WT or FGF9 Eks were loaded onto a heparin-conjugated agarose column and eluted with a linear gradient of NaCl from 120 mM to 2.0 M (black line). Elution profiles of FGF9 WT and FGF9 Eks were determined by monitoring absorbance at 280 nm. ( b, c ) Surface plasmon resonance analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. Sensorgrams indicating the interaction of FGF9 WT ( b ) and FGF9 Eks ( c ) with immobilized heparin were determined at different concentrations. The biosensor chip response is indicated on the y-axis (RU) as a function of time (x-axis) at room temperature. ( d–g ) The most probable solution structures of dimeric FGF9 WT -heparin ( d ), dimeric FGF9 Eks -heparin ( e ), monomeric FGF9 WT -heparin ( f ) and monomeric FGF9 Eks -heparin ( g ) complexes deduced by MD simulations. Heparin and protein residues that form important hydrogen bonds are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each complex is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, SPR Assay, Residue, Binding Assay

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 Eks can inhibit joint and suture development as well as FGF9 WT . ( a–g ) Inhibition of knee joint development induced by ectopic expression of Fgf9 Eks as well as Fgf9 WT . Hindlimb buds of HH stage 17 chickens were infected with RCAS- Fgf9 WT , RCAS- Fgf9 Eks , or empty RCAS virus. ( a, b ) Fgf9 expression was examined by in situ hybridization 2 days after infection. ( c–g ) Respective knee joints (arrows) were examined by Alcian blue staining ( c, d, f ) and hematoxylin and eosin staining of sections through the knee joint ( e, g ) 5 days after infection. f, femur; t, tibia. ( h–k ) Inhibition of coronal suture development by the ectopic presence of FGF9 Eks well as FGF9 WT . FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and the Spp1 expression was examined by in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( h ) and FGF9 Eks ( j ) bead implants, there was overlap of Spp1 expression in the frontal and parietal bones, which was not seen on the unoperated sides ( i, k ). fb, frontal bone; pb, parietal bone.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Inhibition, Expressing, Infection, Virus, In Situ Hybridization, Staining

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Ectopic FGF9 Eks signaling due to its hyper diffusibility. ( a–e ) Increased diffusibility of FGF9 Eks in the skull bone anlagen. FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and Spp1 expression was examined by whole-mount in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( a ) and FGF9 Eks ( c ) bead implants, we observed well-defined intense signals in the frontal and parietal bone anlagen around the implants, which were not seen on the unoperated side ( b, d ). This domain with intense Spp1 signals reflects diffusibility of exogenous FGF9 proteins. We therefore compared diffusibility of FGF9 WT and FGF9 Eks based on the induced expression domain of Spp1 ( e ). The diffusion areas (%) in the frontal and parietal bone anlagen area were estimated from the area ratio of the intense Spp1 expression against the frontal and parietal bone anlagen. Data are represented as mean ± s.e.m. of six operations. FGF9 Eks is more diffusible than FGF9 WT . Significance was determined by two-tailed Student’s t -test. ( f–h ) Increased diffusibility of FGF9 Eks in the forelimb bud. FGF9 WT or FGF9 Eks beads were implanted into forelimb buds of Fgf9 −/− embryos of E10.5 mice. Diffusion of exogenous FGF9 WT ( f ) and FGF9 Eks ( g ) after 2 hours was immunodetected using a FGF9 antibody. ( h ) The diffusion area of FGF9 Eks and FGF9 WT was measured at the level of the equator of the beads. Data are represented as mean (FGF9 WT =100%) ± s.e.m. of four (FGF9 WT ) or five (FGF9 Eks ) operations. FGF9 Eks is also more diffusible than FGF9 WT in limb buds. Significance was determined by one-tailed Student’s t -test. ( i–x ) The downstream target genes of FGF signaling, Etv4 and Etv5 , are expressed ectopically in the prospective elbow joint in Fgf9 Eks/Eks mice. Counterstaining with Giemsa ( i, j, q, r ) and in situ detection of Col2a1 ( k, l, s, t ), Etv4 ( m, n, u, v ) and Etv5 ( o, p, w, x ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. There was ectopic expression of Etv4 ( n, v ) and Etv5 ( p, x ), in the cartilaginous condensation including the prospective elbow joint position, which was demarcated as the gap of Col2a1 expression ( s , arrow), in Fgf9 Eks/Eks , which were not seen in Fgf9 +/+ mice ( m, o, u, w ). Scale bars, 100 µm. ( y ) A model for the pathogenic mechanism underlying elbow joint synosotsis in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the prospective elbow joint may inhibit the initiation of joint development. ( z ) A model for the pathogenic mechanism underlying premature fusion of the coronal suture in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the coronal suture may promote ectopic osteogenesis and subsequently induce premature fusion of the suture.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Expressing, In Situ Hybridization, Diffusion-based Assay, Two Tailed Test, One-tailed Test, In Situ

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 WT modulates FGF9 Eks action by forming FGF9 WT /FGF9 Eks heterodimers. ( a–c ) Proposed solution structures for FGF9 WT homodimer ( a ), FGF9 WT/ Eks heterodimer ( b ) and FGF9 Eks homodimer ( c ). Amino acid residues contributing to hydrogen bond formation involved in dimerization are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each dimer is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory. ( d ) FGF9 WT is capable of forming dimers with FGF9 Eks . The expression of FGF9 WT homodimers, FGF9 WT/ Eks heterodimers and FGF9 Eks homodimers was analyzed using IP/Western blots. The expression vectors for FLAG- or Myc-tagged FGF9 WT and FGF9 Eks were transfected into COS7 cells and culture supernatants were subjected to IP/Western analysis. ( e–h ) Less severe elbow joint synostosis in Fgf9 Eks/+ than Fgf9 Eks/− . Forelimbs from Fgf9 +/− , Fgf9 Eks/+ , Fgf9 Eks/− and Fgf9 Eks/Eks embryos at E17.5 were stained with Alcian blue and Alizarin red. Synostotic change is restricted to the cartilaginous component in Fgf9 Eks/+ embryos, whereas it is extended to the bony component in Fgf9 Eks/− , and Fgf9 Eks/Eks embryos. h, humerus; r, radius; u, ulna.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Residue, Binding Assay, Expressing, Western Blot, Transfection, Staining

Journal: Cancer Research

Article Title: Fibroblast Growth Factor 9 Has Oncogenic Activity and Is a Downstream Target of Wnt Signaling in Ovarian Endometrioid Adenocarcinomas

doi: 10.1158/0008-5472.can-05-3694

Figure Lengend Snippet: Figure 1. FGF9 mRNA and protein levels are up-regulated in OEAs with Wnt pathway defects. A, FGF9 expression in OEAs with Wnt pathway defects (shaded columns) was compared with OEAs with intact signaling (open columns). Relative expression based on the Affymetrix U133A microarray data is shown in arbitrary units. B, quantitative RT-PCR analysis confirms FGF9 up-regulation in Wnt pathway–defective OEAs. Columns, mean fold expression of FGF9 (normalized to HPRT) in OEAs with (shaded) and without (open) Wnt pathway deregulation; bars, SE. C, immunoblot analysis of lysates from selected primary OEAs shows readily detectable expression of FGF9 protein. Normal proliferative endometrium [NE-18(PC)] was used as a positive control.

Article Snippet: Western blots were done according to standard methods using a murine monoclonal anti-FGF9 primary antibody (R&D Systems, Minneapolis, MN) at 1:125 dilution or a murine monoclonal anti-c-myc antibody (9E10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1,000, and horseradish peroxidase–conjugated goat anti-mouse secondary antibody (Pierce) at 1:20,000 dilution.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Positive Control

Journal: Cancer Research

Article Title: Fibroblast Growth Factor 9 Has Oncogenic Activity and Is a Downstream Target of Wnt Signaling in Ovarian Endometrioid Adenocarcinomas

doi: 10.1158/0008-5472.can-05-3694

Figure Lengend Snippet: Figure 3. Activation of FGF9 is dependent on new protein synthesis. A, HEC-1A/S33Y-ER cells were either mock treated (diamonds) or treated with 4-OH-tamoxifen (4-OH; n), 1 Ag/mL cycloheximide alone (CHX; E), or both (.). RNA harvested from cells at the indicated time points was analyzed for FGF9 expression by quantitative RT-PCR as above. B, quantitative RT-PCR analysis of AXIN2 in HEC-1A/S33Y-ER cells. These cells were either mock treated (y) or treated with 4-OH-tamoxifen (n), 1 Ag/mL cycloheximide alone (E), or both (.). C, OVCA420 cells were treated with LiCl in the absence or presence of cycloheximide over a short time course to investigate the possibility that FGF9 induction is dependent on short-lived cofactors. Cells were treated with 30 mmol/L LiCl (n), 30 mmol/L LiCl and 1 Ag/mL cycloheximide (.), or 30 mmol/L NaCl plus 1 Ag/mL cycloheximide (y). In both experiments, RNA was extracted from cells harvested at the indicated time points and analyzed by quantitative RT-PCR. Relative FGF9 expression normalized to HPRT.

Article Snippet: Western blots were done according to standard methods using a murine monoclonal anti-FGF9 primary antibody (R&D Systems, Minneapolis, MN) at 1:125 dilution or a murine monoclonal anti-c-myc antibody (9E10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1,000, and horseradish peroxidase–conjugated goat anti-mouse secondary antibody (Pierce) at 1:20,000 dilution.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 1. Gene expression of FGF ligands in early OVs and FBNs derived from hiPSCs. (A) Schematic showing the timeline of early hiPSC differentiation to OVs and FBNs. At day 0 (D0), pluripotent hiPSCs are subjected to a well-established, fully defined differentiation protocol that generates anterior neuroepithelium/eye field cells by D10, followed 10 days later by the appearance of OVs and FBNs.74,75,78 (B) hiPSC-OVs and hiPSC-FBNs can be easily distinguished in live cultures by their light microscopic ap- pearances, manually picked, and cultured separately. (C) Comparative microarray analysis showing the relative expression (see accompanying heat map legend) of FGF ligands in wild-type hiPSC-OVs versus hiPSC-FBNs isolated from the same cultures at D20.74 (D) qRT-PCR analysis at D30 revealed a significantly sustained increase in the expression of FGF9 and FGF19 in hiPSC-OVs relative to hiPSC-FBNs, but not FGF8 (**P < .01).

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Gene Expression, Derivative Assay, Cell Culture, Microarray, Expressing, Isolation, Quantitative RT-PCR

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 2. Comparative gene expression of FGF ligands in early wild-type control versus (R200Q)VSX2 hiPSC-OVs. (A) No dif- ference in light microscopic appearance at day 20 (D20) was observed between wild-type control and (R200Q)VSX2 hiPSC-OVs, as expected since VSX2 is first expressed around this time. (B) Comparative RNAseq analysis showing the relative expression (see accompanying heat map legend) of FGF ligands in D30 (R200Q)VSX2 hiPSC-OVs relative to parallel D30 cultures of wild-type con- trol hiPSC-OVs. (C) Confirmatory qRT-PCR analysis of selected FGF ligands revealed significantly decreased expression of FGF9 and FGF19, but not FGF3 or FGF8, in (R200Q)VSX2 hiPSC-OV cultures relative to wild-type control hiPSC-OVs (**P < .01, ***P < .001). (D, E) qRT-PCR analyses of wild-type and (R200Q)VSX2 hiPSC-OVs showing FGF9 (D) and FGF19 (E) expres- sion levels at multiple differentiation time points between D0 and D30 (relative to D0 wild-type hiPSC-OVs; note the logarithmic y axis scale). FGF9 demonstrated a biphasic rise in expression levels between D10 and D14 and again between D20 and D30, whereas FGF19 expression increased between D16 and D30.

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Gene Expression, Control, Expressing, Quantitative RT-PCR

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 3. Treatment with exogenous FGF9, but not FGF19, substantially reduced production of pigmented RPE in (R200Q) VSX2 hiPSC-OV cultures. Culture wells containing equal amounts of adherent (R200Q)VSX2 hiPSC-OVs were treated with or without 100 ng/ml FGF9 or FGF19 beginning at day 20 (D20) of differentiation. Wells were photographed on D35 and again on D55 to qualitatively assess the relative production of pigmented RPE. Above each well is a corresponding photograph of 3 cell pellets harvested from parallel treated (or untreated) culture wells for each condition.

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques:

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 5. Early and prolonged exposure to FGF9 also led to upregulation of NR genes in (R200Q)VSX2 hiPSC-OV cultures. (A) qRT-PCR showing expression levels of selected NR genes relative to GAPDH in adherent cultures of (R200Q)VSX2 hiPSC-OVs treated with FGF9 for the same time periods investigated in Figure 4 (*P < .05 or otherwise indicated). (B) Western blot of RCVRN protein expression in untreated adherent (R200Q)VSX2 hiPSC-OV cultures (1st and 3rd lanes) and the same cultures treated with FGF9 from D20-D55 (2nd lane), D20-D90 (4th lane), or D55-90 (5th lane). (C) Western blot of VSX2 protein expression in un- treated adherent (R200Q)VSX2 hiPSC-OV cultures (1st and 3rd lanes) and the same cultures treated with FGF9 from D20-D55 (2nd lane) or D30-D55 (4th lane). Expression of ACTN protein was used as a control in panels (B) and (C).

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 6. FGF9 treatment increased ERK phosphorylation and cell proliferation in (R200Q)VSX2 hiPSC-OV cultures. (A) Western blot showing the temporal effects of FGF9 treatment on levels of phosphorylated ERK (p-ERK) in (R200Q)VSX2 hiPSC-OV cultures. Expression of unphosphorylated ERK and Actin (ACTN) is also shown. (B) Immunocytochemical analysis on fixed cryosections showing increased nuclear expression of the cell proliferation marker Ki67 in an FGF9-treated vs. untreated (R200Q)VSX2 hiPSC-OV (scale bar [ 50 mm). (C, D) qRT-PCR (C) and western blot (D) analyses showing increased gene and protein expression of the pro-proliferative marker CCND1/CCND1 (C, D) and decreased expression of the cell cycle inhibitor P27 (C) at day 55 in (R200Q)VSX2 hiPSC-OVs treated with or without FGF9 beginning at day 20.

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Phospho-proteomics, Western Blot, Expressing, Marker, Quantitative RT-PCR

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 7. Inhibition of FGF9 activity in wild-type hiPSC-OV cultures did not alter RPE or NR gene expression, ERK phosphor- ylation, or cellular proliferation. (A) Photographs taken at day 90 of culture wells containing equal amounts of adherent wild-type hiPSC-OVs treated with or without 500 ng/ml FGF9 neutralizing antibody (anti-FGF9) beginning at day 20. No qualitative differ- ence in the production of pigmented RPE was observed. (B) Western blots showing similar protein expression levels of the RPE marker RPE65 and the NR (and photoreceptor) marker RCVRN with or without treatment with FGF9-neutralizing antibody from day 20 to day 90. ACTN expression was used as a control. (C) Immunocytochemical analysis also showed no difference in nu- clear Ki67 expression in hiPSC-OV cultures with or without treatment with FGF9-neutralizing antibody from day 20 to day 90. (D) Western blot demonstrating a reduction in the level of phosphorylated ERK (p-ERK) protein in wild-type hiPSC-OVs 5 minutes after treatment with or without FGF9-neutralizing antibody (demonstrating activity of the anti-FGF9 antibody). Expression levels of ACTN and unphosphorylated ERK are also shown.

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Inhibition, Activity Assay, Gene Expression, Western Blot, Expressing, Marker, Control

Journal: American journal of ophthalmology

Article Title: The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

doi: 10.1016/j.ajo.2019.04.033

Figure Lengend Snippet: FIGURE 8. Model generated by our study series depicting the interrelated effects of VSX2, MITF, FGF9, and Wnt signaling on RPE and NR production in hPSCs. (A) Top panel: In normal hiPSC-OVs, VSX2 binds directly to and inhibits MITF and Wnt pathways genes, leading to the generation of NRPCs over RPE72,92 in the early optic vesicle. Middle panel: In the absence of functional VSX2, inhibition of pro-RPE genes is lifted and RPE production is favored over NRPCs.33 Lower panel: Application of exogenous FGF9 can tip the balance back toward NRPC production and at least partially override the phenotypic consequences of the functional loss of VSX2 activity in hiPSC-OVs (present study). (B) Simplified schematic showing the relative impacts of VSX2, MITF, FGF9, and Wnt signaling on RPE and NRPC production in hPSCs. Note that FGF9 works in parallel with VSX2 but is not strictly required for NR production and maintenance in wild-type cultures, likely due to the redundant activity of other pro-NR factors. The asterisk denotes the existence of additional pro-NR influences from factors other than FGF9. Circled numbers demarcate the following individual studies and the aspect(s) of RPE and NR production on which they focused: 1 [ Phillips et al (2014)33; 2 [ Capowski et al (2014)92; 3 [ Capowski et al (2016)72; 4 [ Gamm et al (present study).

Article Snippet: Similarly, wild-type control hiPSC retinal cultures were cultured in 500 ml RDM alone or 500 ml RDM plus neutralizing antibody against FGF9 (anti-FGF9; 500 ng/ml; R&D Systems).

Techniques: Generated, Functional Assay, Inhibition, Activity Assay

Journal: Disease Models & Mechanisms

Article Title: Deletion of Yy1 in mouse lung epithelium unveils molecular mechanisms governing pleuropulmonary blastoma pathogenesis

doi: 10.1242/dmm.045989

Figure Lengend Snippet: Molecular distinctions between CPAM and PPB lung biopsies. qRT-PCR expression analyses of DICER1 , YY1 , FGF9 , FGF10 , BMP4 , SPRY2 , ETV4 , ETV5 , ELF5 , SHH , IRX2 and IRX5 genes in human lung biopsies from male (blue) and female (pink) control, CPAM (unknown type, Type I and Type II) and PPB (unknown type, Type I, Type II and Type III) patients, indicating a specific PPB molecular signature. Means±s.e.m. are shown. * P adj<0.05, ** P adj<0.01, *** P adj<0.001 (Kruskall–Wallis, Dunn's post hoc with Bonferroni correction). CTL, control ( n =14-16); CPAM, congenital pulmonary airway malformation ( n =7); PPB, pleuropulmonary blastoma ( n =7-12).

Article Snippet: The following primary antibodies were used: anti-E-cadherin (clone 36/E-cadherin 610181, BD Biosciences; 1/5000 for human sections, 1/200 for mouse sections), anti-FGF9 (ab71395, Abcam; 1/50), anti-Ki67 (also known as Mki67; NCL-L-Ki67-MM1, Leica Biosystems; 1/200), anti-YY1 (ab109237, Abcam; 1/1000) and anti-ZEB1 (ab203829, Abcam; 1/50).

Techniques: Quantitative RT-PCR, Expressing

Journal: Disease Models & Mechanisms

Article Title: Deletion of Yy1 in mouse lung epithelium unveils molecular mechanisms governing pleuropulmonary blastoma pathogenesis

doi: 10.1242/dmm.045989

Figure Lengend Snippet: FGF9 lung epithelial expression is augmented in Yy1 flox/flox ; Tg +/Nkx2.1Cre mice. (A-E‴) Representative micrographs showing co-immunostaining of E-cadherin (green) and FGF9 (red) in control and Yy1 flox/flox ; Tg +/Nkx2.1Cre mice at postnatal day (P)21 (A-C‴) and P55 (D-E‴). FGF9 epithelial expression (arrows) was increased in the cystic areas (asterisks) of the lungs of mutants. Tissue disorganization (arrowheads) in cystic walls was observed. Boxed areas are shown at higher magnification in the corresponding images below or right. Scale bars: 100 µm.

Article Snippet: The following primary antibodies were used: anti-E-cadherin (clone 36/E-cadherin 610181, BD Biosciences; 1/5000 for human sections, 1/200 for mouse sections), anti-FGF9 (ab71395, Abcam; 1/50), anti-Ki67 (also known as Mki67; NCL-L-Ki67-MM1, Leica Biosystems; 1/200), anti-YY1 (ab109237, Abcam; 1/1000) and anti-ZEB1 (ab203829, Abcam; 1/50).

Techniques: Expressing, Immunostaining

Journal: Disease Models & Mechanisms

Article Title: Deletion of Yy1 in mouse lung epithelium unveils molecular mechanisms governing pleuropulmonary blastoma pathogenesis

doi: 10.1242/dmm.045989

Figure Lengend Snippet: Model for the involvement of YY1 in PPB pathogenesis. In the PPB context (text and arrows in red), the somatic missense mutation (asterisk) in the RNaseIIIb domain of the DICER1 protein modifies the miRNA profile, causing a bias toward the generation of miRNAs-3p over miRNAs-5p. Higher miR-125a-3p levels are found in PPB, and they bind to YY1 3′UTR to inhibit YY1 protein expression. Reduced YY1 expression correlates with altered expression levels of several lung epithelial transcription factors – ETV4 , ETV5 , ELF5 , IRX2 and IRX5 – as previously observed in Yy1 flox/flox ; Shh +/Cre mutant mice. Accordingly, SHH , a direct transcriptional target of both YY1 and ETV5, shows reduced expression in PPB lung tissues. Decreased expression of epithelium-specific genes, like CDH1 , TJP1 and the FGFR2-IIIB isoform, is also observed in PPB samples. In parallel, expression of mesenchyme-specific markers – such as SNAI1 , SNAI2 , ZEB1 , CDH2 and the FGFR2-IIIC isoform – is increased. The upregulation of ZEB1 may be explained by the reduced expression of miR-200a in PPB. High ZEB1 expression levels are known to inhibit ESRP1. Together, the data suggest that EMT is promoted in PPB and may be involved in the progression of PPB epithelial cysts toward sarcoma. Expression of the splicing regulatory factors ESRP1 and ESRP2 is reduced, likely contributing to the FGFR2 isoform switch from IIIB to IIIC observed in PPB patients. The FGFR2-IIIC receptor preferentially binds the FGF9 ligand, which is highly expressed in PPB samples and Yy1 mutant mice. This suggests that YY1 participates in the regulation of FGF9 expression. There is no clear evidence that miR-140-5p, a previously identified post-transcriptional regulator of FGF9, is associated with increased FGF9 expression in PPB samples. No change in FGF10 expression levels is seen in PPB lung samples in contrast to the dramatic increase previously observed in the Yy1 flox/flox ; Shh +/Cre mouse model. In human lung development, the exact role of FGF10 remains undefined. However, one possible explanation for the unchanged FGF10 levels in PPB is that FGF9, known to inhibit FGF10 expression in human lung, could act to maintain stable FGF10 levels. Thus, YY1 occupies a central position in the mechanism of PPB pathogenesis, as a downstream target of the abnormal epithelial DICER1-cleaved miRNA profile as well as a transcriptional regulator of key molecules of lung development. The exact role of YY1 in the observed changes of epithelial gene expression in PPB remains to be determined.

Article Snippet: The following primary antibodies were used: anti-E-cadherin (clone 36/E-cadherin 610181, BD Biosciences; 1/5000 for human sections, 1/200 for mouse sections), anti-FGF9 (ab71395, Abcam; 1/50), anti-Ki67 (also known as Mki67; NCL-L-Ki67-MM1, Leica Biosystems; 1/200), anti-YY1 (ab109237, Abcam; 1/1000) and anti-ZEB1 (ab203829, Abcam; 1/50).

Techniques: Mutagenesis, Expressing

Journal: Acta Histochemica et Cytochemica

Article Title: Immunocytochemical Colocalization of Fibroblast Growth Factor-1 with Neurotrophin-3 in Mouse Alveolar Macrophages

doi: 10.1267/ahc.11055

Figure Lengend Snippet: Expression and colocalization of FGF-1 with neurotrophins in mouse alveolar macrophages. ( A ) Double immunocytochemical staining of macrophages with FGF-1 and NGF (upper panels), with FGF-1 and BDNF (middle panel), and with FGF-1 and NT-3 (lower panels). Panels in the first column show differential interference contrast (DIC) images. Panels in the fourth column represent merged images of the second and third columns. Bars=10 µm. ( B ) Expression ratio of FGF-1, NGF, BDNF, and NT-3 in alveolar macrophages. Mean percentages of the number of macrophages immunoreactive for FGF-1, NGF, BDNF, and NT-3 to the number of total macrophages are shown. ( C ) Ratio of colocalization of FGF-1 with neurotrophins in alveolar macrophages. The percentages of double-immunoreactive macrophages to FGF-1-immunoreactive macrophages are shown. ( B, C ) Error bars represent standard error of the mean. (+), the presence of immunoreactivity; Mϕ, macrophages; N, number of microscopic fields examined (total cell numbers are indicated below); P, probability of significance.

Article Snippet: Cells were incubated for 1 hr at room temperature with the primary antibody, goat anti-human FGF-1 antibody (4 μg/ml, reacts with mouse, rat, and human FGF-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse NGF antibody (1:500, Serotec, Oxford, UK), chicken anti-mouse NGF antibody (2 μg/ml, Chemicon International, Temecula, CA, USA), rabbit anti-mouse BDNF antibody (1:1000, Chemicon International), chicken anti-human BDNF antibody (2 μg/ml, reacts with human and mouse BDNF, General Laboratories, Madison, WI, USA), sheep anti-mouse NT-3 antibody (10 μg/ml, Chemicon International), or chicken anti-human NT-3 antibody (1:100, reacts with human and mouse NT-3, Abcam, Tokyo).

Techniques: Expressing, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Immunocytochemical Colocalization of Fibroblast Growth Factor-1 with Neurotrophin-3 in Mouse Alveolar Macrophages

doi: 10.1267/ahc.11055

Figure Lengend Snippet: Colocalization of FGF-1, NGF, BDNF, and NT-3 with LAMP-2 in vesicles of mouse alveolar macrophages. ( A ) Confocal images showing colocalization of FGF-1, NGF, BDNF, and NT-3 with LAMP-2, a late endosomal and lysosomal marker. Panels in the first column show DIC images. Panels in the fourth column represent merged images of the second and third columns. Bars=5 µm. ( B ) Ratio of colocalization of FGF-1, NGF, BDNF, and NT-3 with LAMP-2 in the vesicles of alveolar macrophages. The percentages of double-immunoreactive vesicles to the vesicles immunoreactive for FGF-1, NGF, BDNF, or NT-3 are shown. (+), the presence of immunoreactivity; P, probability of significance.

Article Snippet: Cells were incubated for 1 hr at room temperature with the primary antibody, goat anti-human FGF-1 antibody (4 μg/ml, reacts with mouse, rat, and human FGF-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse NGF antibody (1:500, Serotec, Oxford, UK), chicken anti-mouse NGF antibody (2 μg/ml, Chemicon International, Temecula, CA, USA), rabbit anti-mouse BDNF antibody (1:1000, Chemicon International), chicken anti-human BDNF antibody (2 μg/ml, reacts with human and mouse BDNF, General Laboratories, Madison, WI, USA), sheep anti-mouse NT-3 antibody (10 μg/ml, Chemicon International), or chicken anti-human NT-3 antibody (1:100, reacts with human and mouse NT-3, Abcam, Tokyo).

Techniques: Marker

Journal: Acta Histochemica et Cytochemica

Article Title: Immunocytochemical Colocalization of Fibroblast Growth Factor-1 with Neurotrophin-3 in Mouse Alveolar Macrophages

doi: 10.1267/ahc.11055

Figure Lengend Snippet: Colocalization of FGF-1, NGF, BDNF, and NT-3 with EEA1 in vesicles of mouse alveolar macrophages. ( A ) Confocal images showing colocalization of FGF-1, NGF, BDNF, and NT-3 with EEA1, a early endosomal marker. Panels in the first column show DIC images. Panels in the fourth column represent merged images of the second and third columns. Bars=5 µm. ( B ) Ratio of colocalization of FGF-1, NGF, BDNF, and NT-3 with EEA1 in the vesicles of alveolar macrophages. The percentages of double-immunoreactive macrophages to the vesicles immunoreactive for FGF-1 NGF, BDNF, or NT-3 are shown. (+), the presence of immunoreactivity; P, probability of significance.

Article Snippet: Cells were incubated for 1 hr at room temperature with the primary antibody, goat anti-human FGF-1 antibody (4 μg/ml, reacts with mouse, rat, and human FGF-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse NGF antibody (1:500, Serotec, Oxford, UK), chicken anti-mouse NGF antibody (2 μg/ml, Chemicon International, Temecula, CA, USA), rabbit anti-mouse BDNF antibody (1:1000, Chemicon International), chicken anti-human BDNF antibody (2 μg/ml, reacts with human and mouse BDNF, General Laboratories, Madison, WI, USA), sheep anti-mouse NT-3 antibody (10 μg/ml, Chemicon International), or chicken anti-human NT-3 antibody (1:100, reacts with human and mouse NT-3, Abcam, Tokyo).

Techniques: Marker

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Primer sequences .

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Amplification

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Antibody dilutions, retrieval buffer and details .

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Positive Control

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Expression of FGF9, FGFR2 and fetal cell lineage markers in human fetal gonads. ( A ) Transcriptional expression of FGF9 , FGFR2 , OCT4 (POU5F1) , NR2F2 , SOX9 and CYP17A1 in ovary and testis samples from the first trimester. Gene expression is normalized to RPS29 and expressed relative to testis samples from 7–9 GWs, which were set to a value of 1. Values represent mean ± SEM and for each age group n = 6–8. Significant difference compared to fetal testes 7–9 GWs, * P < 0.05, ** P < 0.01, *** P < 0.001. GW: gestational week. ( B ) Immunohistochemical staining of FGF9, OCT4 (fetal germ cell marker), SOX9 (Sertoli cell marker, in fetal testis sample only), FOXL2 (granulosa cell marker, in fetal ovary sample only) and COUP-TFII (interstitial cell marker) in human ovary and testis samples from 10 GWs. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Marker

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on proliferation and apoptosis in human fetal gonads. Effects on expression of markers of proliferation and apoptosis following FGF9 (25 ng/ml) and SU5402 (10 μM) treatment for 14 days in ex vivo cultures of fetal ( A ) ovaries and ( B ) testes. Immunohistochemical staining for the proliferation marker BrdU (which was added to culture media for the last 6 h of culture), and the apoptosis markers cleaved PARP (cPARP) was examined. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm. ( C ) Percentage of proliferating (BrdU + ) germ cells after 14 days of culture in fetal ovaries and testes. Values represent mean ± SEM, with n = 10 (vehicle, ovaries), n = 7 (FGF9, ovaries), n = 6 (SU5402, ovaries), n = 9 (vehicle, testes), n = 8 (FGF9, testes), n = 7 (SU5402, testes). Significant difference compared to vehicle controls, * P < 0.05, *** P < 0.001. ( D ) Percentage of apoptotic (cPARP + ) germ cells after 14 days of culture in fetal ovaries and testes. Values represent mean ± SEM, with n = 10 (vehicle, ovaries), n = 8 (FGF9, ovaries), n = 6 (SU5402, ovaries), n = 10 (vehicle, testes), n = 8 (FGF9, testes), n = 7 (SU5402, testes). Significant difference compared vehicle controls, ** P < 0.001.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Ex Vivo, Immunohistochemical staining, Staining, Marker

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on the expression of germ cell markers in human fetal ovaries. ( A ) Expression pattern of germ cell markers OCT4 (oogonia), MAGE-A4 (subpopulation of oogonia transitioning into oocytes) and γH2AX (meiotic germ cells) in fetal ovary samples treated with FGF9 (25 ng/ml) and SU5402 (10 μM) for 14 days in ex vivo culture. Arrows indicate MAGE-A4 + cells. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm. ( B ) Quantification of germ cells determined as the number of OCT4 + gonocytes per mm 2 . Values represent mean ± SEM, with n = 14 (vehicle), n = 7 (FGF9) and n = 6 (SU5402). Significant difference compared to vehicle controls, *** P < 0.001. ( C ) Quantification of meiotic germ cells determined as the number of γH2AX + germ cells per mm 2 . Values represent mean ± SEM, with n = 12 (vehicle), n = 7 (FGF9) and n = 6 (SU5402). Significant difference compared to vehicle controls, * P < 0.05, ** P < 0.01.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Ex Vivo

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on expression of somatic cell markers in human fetal ovaries. Expression pattern of somatic cell markers COUP-TFII (interstitial cells), FOXL2 (granulosa cells) and WNT4 (primarily in granulosa cells) in fetal ovary samples treated with FGF9 (25 ng/ml) and SU5402 (10 μM) for 14 days in ex vivo culture. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Ex Vivo

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on expression of germ cell and somatic cell markers in human fetal testes. ( A ) Expression pattern of germ cell markers OCT4 (gonocytes), MAGE-A4 (pre-spermatogonia) and somatic cell markers SOX9 (Sertoli cells), AMH (Sertoli cells) and COUP-TFII (interstitial cells) in fetal testis samples treated with FGF9 (25 ng/ml) and SU5402 (10 μM) for 14 days in ex vivo culture. Arrows indicate MAGE-A4 + cells. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm. ( B ) Quantification of germ cells determined as the number of OCT4 + gonocytes per mm 2 . Values represent mean ± SEM, with n = 12 (vehicle), n = 8 (FGF9) and n = 7 (SU5402). Significant difference compared to vehicle controls, * P < 0.05. ( C ) Quantification of Sertoli cells determined as the number of SOX9 + cells per mm 2 . Values represent mean ± SEM, with n = 10 (vehicle), n = 8 (FGF9) and n = 7 (SU5402). Significant difference compared to vehicle controls, * P < 0.05.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Ex Vivo

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on expression of Leydig cell markers in human fetal testes. Expression pattern of fetal Leydig cell markers CYP11A1, CYP17A1 and 3β-HSD in fetal testis samples treated with FGF9 (25 ng/ml) and SU5402 (10 μM) for 14 days in ex vivo culture. Counterstaining with Mayer’s haematoxylin, scale bar corresponds to 50 μm.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Ex Vivo

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Effects of manipulating FGF signalling on steroidogenesis in human fetal testes. Quantification of steroid hormone metabolites produced in ex vivo cultures of fetal testis tissue and secreted to the media droplets. Medium was collected every 48 h throughout the 14 days culture period and was pooled for each individual tissue piece. Androgens were measured by LC-MS/MS and are shown as ratios compared to corresponding vehicle controls. ( A ) Effect of FGF9 (25 ng/ml) treatment for 14 days in ex vivo culture. Values represent mean ± SEM, with n = 16 (vehicle) and n = 16 (FGF9). Significant difference compared to vehicle control, * P < 0.05. ( B ) Effect of SU5402 (10 μM) treatment for 14 days in ex vivo culture. Values represent mean ± SEM, with n = 12 (vehicle) and n = 12 (SU5402). Significant difference compared to vehicle control, * P < 0.05.

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Produced, Ex Vivo, Liquid Chromatography with Mass Spectroscopy, Control

Journal: Human Reproduction (Oxford, England)

Article Title: Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

doi: 10.1093/humrep/dez191

Figure Lengend Snippet: Proposed involvement of FGF9/FGFR2 signalling in human fetal gonad development. A model illustrating the interaction between key factors involved in early gonad development. SRY upregulates SOX9 in Sertoli cells of the developing testes resulting in the self-promoting expression of SOX9, which is followed by expression of additional testis-promoting factors, including FGF9. FGF9 signals through FGFR2 to inhibit the pro-ovarian factor WNT4 (and the RSPO1/β-catenin pathway). The present study confirmed the FGF9-mediated inhibition of meiotic entry in female germ cells previously demonstrated in human fetal ovaries ( Frydman et al., 2017 ). Additionally, this study suggests the involvement of FGF9/FGFR2 in the expression of SOX9 in human fetal testes. Finally, the present study suggests involvement of FGF9/FGFR2 signalling in supporting germ cell survival in both sexes. 1 Reviewed in Rotgers et al., 2018 .

Article Snippet: The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200.

Techniques: Expressing, Inhibition