Journal: Nature protocols

Article Title: Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells

doi: 10.1038/nprot.2016.170

Figure Lengend Snippet: Troubleshooting.

Article Snippet: We obtained the permission to use hPSCs from our Institutional Review Board (IRB) and institutional Embryonic Stem Cell Research Oversight (ESCRO) committee. human FGF2 (Peprotech, 100-18B) human FGF9 (R&D systems, 273-F9-025/CF) human Noggin (Peprotech, 120-10C) L-GlutaMAX (Life Technologies, 35050-061) O.C.T compound (Fisher Scientific, 23-730-571) Paraformaldehyde 16% (PFA, Electron Microscopy Sciences, RT15710) !

Techniques: Concentration Assay, Microscopy

Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: ( A ) Quantification of scratch wound closure of E13.5 growth-arrested primary dermal fibroblasts treated with FGF9 (200 ng/ml) or FGF9 and SU5402 (20 µM). At 24 hr, FGF9 treatment resulted in greater wound-closure at 8 hr (36.17 ± 5.04%, p=0.0490) and at 24 hr (97.11 ± 3.09%, p=0.0133) compared to DMSO control (all treatments n = 5 experiments, each performed with freshly extracted primary dermal fibroblast cell population). Wound closure was not altered when cells were treated with both FGF9 and SU5402 inhibitor (p=0.2563). ( B ) Quantification of transwell migration assay of E13.5 primary dermal fibroblasts. Migration was significantly increased when FGF9 (200 ng/ml) was added to lower or both upper and lower chambers (p=0.0003 and p=0.0010, respectively; for both n = 6 experiments, each performed with freshly extracted primary dermal fibroblast cell population). No statistical difference was observed between FGF9 treatments (p=0.4033). ( C ) Quantification of nuclei density in E13.5 dermis explants treated for 3 hr with beads loaded with FGF9 (100 µg/ml) or 0.1% BSA vehicle control. Density measured from a single optical slice at mid-bead. FGF9 bead induces an increase in density within 15 µm radius from the bead relative to BSA control (p=0.033, n = 7 explants), but not between 15 and 30 µm radius (p=0.236, n = 7 explants). ( D ) Whole-mount RNA in situ hybridization of dermal samples treated with FGF9 beads for 3, 8, and 16 hr. Induction of Spry4 and Dusp6 , but not Sox2 expression (purple) was observed around the bead at all time points ( n indicates induction/total samples, induction was tested in two independent experiments with skin samples derived from at least two different litters.). ( E ) 2 hr EdU incorporation into dermis organ cultures after overnight incubation with BSA (left), FGF20 (center), FGF9 (right) loaded beads. Note the increased number of proliferating cells around the FGF9 bead ( n = 5 explants). Error bars represent SD. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Scale bar = 30 µm. See also and . 10.7554/eLife.36468.027 Figure 6—figure supplement 1—source data 1. Values used to quantify FGF9-induced cellular changes. Values used to quantify fibroblast wound closure in the presence of DMSO, SU5402, FGF9 +DMSO, or FGF9 +SU5402 . Values used to quantify E13.5 primary fibroblast transwell migration in control, FGF9 in lower chamber, and FGF9 in seeding and lower chambers . Values used to quantify fibroblast density in response to BSA or FGF9-loaded beads at 0–15 µm and 15–30 µm distance from the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Control, Transwell Migration Assay, Migration, RNA In Situ Hybridization, Expressing, Derivative Assay, Incubation

Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: 3 hr incubation of BSA-, FGF20- or FGF9-loaded beads with E13.5 wildtype ( A, B, C ) or Fucci mKO ( D ) dermises. Cdkn1a expression was assayed in these samples using ( A ) whole-mount RNA in situ hybridization of dermal samples. Cdkn1a expression (purple) was not observed around the bead (0/20 each condition, in four independent experiments with skin samples derived from four different litters.) ( B ) section radioactive in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters), and ( C ) section in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters). ( D ) Cell cycle exit was assessed using Fucci mKO reporter allele (representing G 0 /G 1 cell cycle phase) in the 30 µm surrounding the center of the bead (0/10 each condition in two independent experiments with skin samples from two different litters). ( E ) Quantification of percent total cells surrounding the bead positive for Fucci-mKO . No significant difference was observed between any of the groups. Error bars represent SD. Scale bar = 50 µm. See also and . 10.7554/eLife.36468.029 Figure 6—figure supplement 2—source data 1. Values used to quantify FGF20 or FGF9 induced Fucci-mKO expression. Values used to quantify the percent of total cells expressing Fucci-mKO 30 µm surrounding the center of the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Incubation, Expressing, RNA In Situ Hybridization, Derivative Assay, In Situ Hybridization

Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Treatment with rFgf10 (200 n /ml) of wild type explant lung culture for 48 hr. qRT-PCR shows that Nolz-1 and Wnt2 are not changed in the rFgf10 treated group compared to the vehicle control. Student’s t -test, P > 0.05, n = 4. Scale bar, 500 μm. (B) Treatment with rFgf9 (200 ng/ml) results in enlarged epithelia in wild-type explant lungs cultured for 48 hr. The qRT-PCR shows that Nolz-1 , Wnt2 and Lef1 mRNAs are increased in rFgf9 treated group in wild type lungs. Student’s t -test, * P < 0.05, ** P < 0.01, n = 4. Western blotting showed that rFgf9 treatment increases Nolz-1 protein by 99% in wild type lung culture compared to the vehicle-treated group. Student’s t -test, * P < 0.05, n = 3. (C) Working hypothesis. Nolz-1 controls the proliferation of mesenchymal cells and the growth of epithelial branches through the regulation of Wnt2 signaling in the early stages of the development of the lungs. In the late stages of development, Nolz-1 acts non-cell autonomously to regulate the development of epithelial cells through Wnt2 signaling. Fgf9 acts upstream to regulate Nolz-1 expression in developing lungs.

Article Snippet: The lung explants were cultivated with recombinant mouse Fgf9 protein (200 ng/ml, R & D Systems, Inc.) or Fgf10 protein (200 ng/ml, R & D Systems, Inc.).

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

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 1 FGF9 increases total neuronal length in Q7 and Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 24 and 48 h and then fixed for immunofluorescent staining using a βIII tubulin antibody. Immunofluorescent images show neuronal morphology in Q7 (a) and Q111 (b) cells at 24 and 48 h after FGF9 treatment. βIIItubulin: green color. Hoechst 33342: blue color. Quantitation results in the immunofluorescent images provide a comparison of total outgrowth in the Q7 (c) and Q111 (d) cells. Double asterisks represent p < 0.01, triple asterisks represent p < 0.001, N = 45–103 cells from three different batches

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Cell Culture, Staining, Quantitation Assay, Comparison

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 2 FGF9 increases expression levels of neuronal morphology–related proteins in Q7 and Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 48 h, and then subjected to western blotting. Western blotting was performed in Q7 (a) and Q111 (c) cells using βIII tubulin,

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 3 FGF9 increases expression levels of neuronal synaptic markers in Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 48 h and then subjected to western blotting. Western blotting was performed in the Q7 (a) and Q111 (c) cells using synaptophysin and PSD-95 antibodies. γ-tubulin was used as an internal control. Quantitation results after western blotting pro- vide a comparison of these markers in the Q7 (b) and Q111 (d) cells. Triple asterisks represent p < 0.001. N = 6

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Expressing, Cell Culture, Western Blot, Control, Quantitation Assay, Comparison

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Schematic illustrating the 2 general theoretical models explaining the emergence of repeating patterns: Turing reaction–diffusion-driven systems and mesenchymal self-organisation. (B) Schematic of the process of hair follicle formation depicting acquisition of epidermal foci of Dkk4 in placodes coupled with the underlying mesenchymal cell accumulation. (C) Pattern responses of skin to stimulation and repression of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and wingless-related integration site (WNT) pathways. Detection of the epidermal placode marker Dkk4 and cell arrangement through green fluorescent protein (GFP) signal in the TCF/Lef::H2B-GFP line in E13.5 mouse dorsal skin explant cultures treated for 27 h with BMP4 (500 ng/ml), LDN193189 (BMP receptor inhibitor) (10 μM), FGF9 (1 μg/ml), SU5402 (FGFR inhibitor) (25 μM), CHIR99021 (GSK3 inhibitor to stimulate WNT/β-catenin signalling) (10 μM), or IWR-1 (WNT/β-catenin signalling inhibitor) (50 μM). Scale bars: 250 μm. (D) Heatmap depicting quantitative reverse transcription polymerase chain reaction (qRT-PCR) fold changes of candidate genes in response to 6 h stimulation or inhibition of the BMP, FGF, or WNT signalling pathways. Pie charts show the fraction of dermal (yellow) to epidermal (purple) expression of each gene detected in unstimulated skin (see ). Statistical significance from control skins was calculated using a Student t test (* p < 0.05 and > ± 1.8-fold change). The raw numerical data for the heatmap can be found in . (E) Gene regulatory network derived from the transcriptional responses to BMP, FGF, or WNT pathway stimulation shown in (D). (F) Dispersion curves calculated from matrix of interactions show that Turing instability (curve breaking the x-axis) can be achieved by a regulatory network with this structure when components of each pathway can diffuse (see for details).

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Diffusion-based Assay, Marker, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Inhibition, Expressing, Control, Derivative Assay, Dispersion

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Single frames from time-lapse sequences of E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explant culture captured by confocal microscopy. Dashed circles indicate ultimate condensate location. Scale bar: 50 μm. (B) Analysis of tracked cells showing the probability of joining the dermal condensate based upon initial location relative to its centre. Two hundred and forty individual cells were tracked across 8 condensates from 4 independent skins. (C) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue). Tracking was halted on cell entry. (D) Plots showing the mean Euclidean angle (top) and mean level of persistence (bottom) of condensate-entering and intercondensate cells for 6-h windows relative to time of entry into the condensate. Error bars represent SEM (condensate cells n = 9, 14, and 20 and intercondensate n = 263, 245, and 197 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test ( p < 0.0001 and p < 0.001 for angle and persistence, respectively) followed by Mann–Whitney U tests with Bonferroni’s correction (** p < 0.01). The raw numerical tracking data (for B, C, and D) can be found in . (E) Detection of a molecular prepattern prior to dermal condensate formation. TCF/Lef::H2B-GFP skin explants were fixed at intermediate stages of pattern formation, imaged to detect GFP, and Dkk4 expression determined in the same skin sample. Asterisk represents an area where Dkk4 -positive foci are present but corresponding dermal condensates are absent. Scale bar: 500 μm. (F) Time-lapse images of E12.75 TCF/Lef::H2B-GFP dorsal skin explants cultured with recombinant fibroblast growth factor (FGF) 9- or bovine serum albumin (BSA)-loaded beads. Cells accumulate around FGF9-loaded beads. Scale bar: 250 μm.

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Confocal Microscopy, MANN-WHITNEY, Expressing, Cell Culture, Recombinant

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Detection of green fluorescent protein (GFP) signal, Dkk4 expression, and histological appearance of TCF/Lef::H2B-GFP skin explants cultured with fibroblast growth factor (FGF) 9 (1 μg/ml), LDN193189 (LDN) (10 μM), or both agents. Large, periodically spaced dermal condensates form in the absence of epidermal placodes when FGF and LDN are administered. Scale bars: H2B-GFP & Dkk4 : 250 μm, haemotoxylin and eosin (HE): 50 μm. (B) Frozen sections of TCF/Lef::H2B-GFP dorsal skin explants treated as indicated and stained for neural cell adhesion molecule (NCAM) expression. Arrows indicate epidermal placode. Scale bar: 20 μm. (C) Expression of epidermal placode ( Shh , Edar , Bmp2 ) and dermal condensate ( Bmp4 , Sox2 ) marker genes in dorsal skin explants cultured with FGF and LDN. Scale bar: 250 μm. (D) Epidermis and dermis isolated from E13.5 TCF/Lef::H2B-GFP skin explants cultured with FGF9 and LDN, counterstained with propidium iodide (PI) and imaged using confocal microscopy. Epidermis is unpatterned while the large dermal condensates are accompanied by cell depletion from the intervening spaces. Scale bar: 100 μm.

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Expressing, Cell Culture, Staining, Marker, Isolation, Confocal Microscopy

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of E13.5 or E13.75 (with condensates) skins treated with transforming growth factor (TGF) β2, fibroblast growth factor (FGF) 9, or bone morphogenetic protein (BMP) 4 for 8 or 24 h, respectively, followed by assessment of transcript abundance. TGFβ2 upregulates expression of genes associated with cell movement and the extracellular matrix. Statistical significance from control was calculated using a Student t test (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent SEM from at least 3 independent experiments. (C) Cell aggregation at FGF9 beads in E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants. TGFβ2 (100 ng/ml) or LY2109761 (25 μM) is present in the culture medium as indicated. TGFβ2 enhances aggregation at FGF9 beads, while LY2109761 suppresses cell accumulation. (D) FGF9 presence in culture medium does not detectably increase cell recruitment to TGFβ2 beads. (E, F) Quantification of areas of high cell density around FGF9- or TGFβ2-coated beads under conditions as indicated. Statistical significance was calculated using Student t tests (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent SEM of at least 3 independent experiments. Scale bars: 250 μm. The raw numerical values (for A, B, E, and F) can be found in .

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control

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