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