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: 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: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Establishment of a transgenic mouse model with the conditional deletion of Fgf9 in osteoblasts. A) Schematic showing the transgenic mouse model used in this study. B). The Cre recombinase is demonstrated to be expressed in osteoblasts (OBs) and osteocytes (OCTs) in adult Coll (2.3)-Cre; td/Tomato Red mice. C) Reverse transcription end point PCR demonstrated a successful excision of Fgf9 fragment in the RNA extract from the long bones of the Coll (2.3); Fgf9 fl/fl (Fgf9 OB−/−) mice. D) Real time PCR assessment of the level of Fgf9 mRNA (relative to GAPDH) in the long bones of 3 months old Fgf9 OB−/− mice. *p < 0.05 vs. littermate controls (Fgf9 fl/fl).

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Transgenic Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of cancellous bone mass at distal femur in the 3 month old male and female Fgf9 OB−/− mice. A). Representative μCT 3D reconstruction images of the distal femurs. B and C). Histomorphometry of cancellous bone at the distal femurs. BV, bone volume; TV, tissue volume; Tb.Th, trabecular thickness, Tb.Sp, trabecular separation; MS, mineralizing surface; MAR, mineral apposition rate, BFR, bone formation rate. ** p < 0.01, *p < 0.05 vs. the sex-matched littermate controls. ### p < 0.001, ## p < 0.01 vs. age-match Fgf9 fl/fl mice with the same genotype.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: μCT assessment of distal femur and tibiofibular junction in 3 month of Fgf9 OB−/− and littermate Fgf9 fl/fl mice.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of cortical bone at tibio-fibular junction (TFJ) in the 3 month old male and female Fgf9 OB−/− mice. A). Representative 3D reconstruction images of μCT scan at the TFJ. B and C). Histomorphometry of cortical bone at the TFJ. Ps.Pm, periosteal perimeter; Ec.Pm, endosteal perimeter; Ct.Th, cortical thickness; Med.Ar, bone marrow area; Ps.BFR, periosteal bone formation rate, Ec.BFR, endosteal bone formation rate. ** p < 0.01, *p < 0.05 vs. the sex-matched littermate controls.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Determination of mRNA levels of osteogenic marker genes in the long bones of the 3 month old Fgf9 OB−/− mice. *p < 0.05 vs. the sex-matched littermate controls.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Marker

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of bone resorption in the 3 months old Fgf9 OB−/− mice. N. OC, number of osteoclasts; BS, the length of bone surface. PYD, pyridinoline.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Effects of exogenous FGF9 on proliferation of the cultured bone marrow stromal cells (BMSCs). The BMSCs from wild type mouse were treated with exogenous FGF (5 ng/ml) for 24 h. A and B) Assessment of BMSC proliferation in the absence or presence of Akt inhibitor, MK-2206 (1 μM). Cell proliferation was determined by BrdU incorporation assay. C) Determination of Akt1 activation in BMSCs by FGF9 (5 ng/ml) using Western blots.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Cell Culture, BrdU Incorporation Assay, Activation Assay, Western Blot