Extended Data Fig. 6: The role of FGF and WNT signaling in the regulation of segmentation clock properties. | Nature

Extended Data Fig. 6: The role of FGF and WNT signaling in the regulation of segmentation clock properties.

From: In vitro characterization of the human segmentation clock

Extended Data Fig. 6

a, Immunofluorescence staining for dpERK on day 2 of differentiation, following 3 h of treatment with DMSO (vehicle control) or the FGFR inhibitor PD173074 (250 nM) in CL medium. n = 4 independent experiments. b, Left, qRT–PCR for the FGF ligands FGF17 and FGF8 on days 1–4 of human iPS cell differentiation. Relative expression is shown as the fold change relative to iPS cells at day 0. Mean ± s.d. n = 3 biological replicates. Right, qRT–PCR for the FGF ligand Fgf8 on days 2–6 of mouse ES cell differentiation. Relative expression is shown as the fold change relative to ES cells at day 0. Mean ± s.d. n = 3 biological replicates. c, Time-course qRT–PCR for the FGF target gene SPRY4 in PSM cells derived from human iPS cells, during the 10 h immediately after treatment with vehicle control (DMSO) or 250 nM PD03. Relative expression is shown as the fold change relative to ES cells at day 0. Mean ± s.d. n = 3 biological replicates. d, Immunofluorescence staining for dpERK in PSM cells derived from human iPS cells (top) or from mouse ES cells (bottom), treated with DMSO or PD03 (250 nM). n = 4 independent experiments. e, Immunofluorescence staining for dpERK (left), β-catenin and nuclear stain (right) in PSM cells derived from human iPS cells, treated with vehicle control (DMSO) or 2 μM XAV. n = 4 independent experiments. Scale bar, 100 μm. f, Representative examples of HES7–Achilles fluorescence intensity over the course of 45 h in a small area of interest within human cultures treated with DMSO (vehicle control), the MAPK inhibitor PD0325901 (250 nM), or the FGFR inhibitor PD173074 (250 nM) in CLFBR medium. n = 16 independent experiments. g, HES7–Achilles fluorescence intensity over the course of 45 h in a small ROI within human cultures treated with DMSO (vehicle control), 2 μM XAV or 12 μM IWR-1 in CLFBR medium. n = 3 independent experiments. h, HES7-Achilles oscillatory period of individual cells treated with vehicle control (DMSO), 2 μM XAV, 250 nM PD17, or 100 nM, 250 nM or 500 nM PD03 on day 2 of differentiation. Mean ± s.d. One-way ANOVA P values (NS, not significant): 0.9929, 0.4097, 0.9998, 0.9845 and 0.7425, from left to right on the graph. n = 27 (XAV), n = 48 (100 nM PD03), n = 57 (all others) cells. i, Average fluorescence intensity profiles for automatically tracked individual HES7-Achilles human cells treated with vehicle control (DMSO) or increasing doses of PD03 (100 nM, 250 nM and 500 nM) on day 2 of differentiation. Mean ± 95% confidence interval. n = 68 cells (control), 45 cells (100 nM), 35 cells (250 nM) or 36 cells (500 nM). j, Representative examples of HES7–Achilles fluorescence intensity profiles in a small ROI within human cultures treated with increasing doses of PD03 (100 nM, 250 nM and 500 nM) or vehicle control (DMSO). n = 8 independent experiments. k, Number of HES7–Achilles oscillations before arrest in small ROIs within cultures treated with increasing doses of PD03 (100 nM, 250 nM and 500 nM). One-way ANOVA: 100 nM versus 250 nM, P = 0.0042; 100 nM versus 500 nM, P = 2.0 × 10−5. n = 6 independent experiments. l, Average HES7–Achilles fluorescence intensity in small ROIs over the course of the oscillatory regime (before the arrest of oscillations) in cells treated with vehicle control (DMSO) or increasing doses of PD03 (100 nM, 250 nM and 500 nM). The middle hinge corresponds to the median, the lower and upper hinges correspond to the first and third quartiles, and the lower and upper whiskers correspond to the minimum and maximum. One-way ANOVA: control versus 100 nM, P = 6.7 × 10−17; control versus 250 nM, P = 6.5 × 10−21; control versus 500 nM, P = 1.9 × 10−22; 100 nM versus 250 nM, P = 1.1 × 10−17; 100 nM versus 500 nM, P = 2.5 × 10−18. n = 6 independent experiments. m, Representative HES7–Achilles fluorescence intensity profiles for PSM cells derived from mouse ES cells, treated with vehicle control (DMSO), 2 μM XAV, or 100 nM, 250 nM or 500 nM PD03. n = 12 (control, XAV and 100 nM PD03) or n = 10 (250 nM and 500 nM PD03) independent experiments. n, Histograms showing the instantaneous phase difference relative to control for individual cells treated with vehicle control (DMSO), 2 μM XAV, 250 nM PD17, or 100 nM, 250 nM or 500 nM PD03. Details are given in ‘Phase shifts’ in Methods. n was fixed at 11,000 observations. o, Quantification of the average phase difference (in degrees) for HES7–Achilles oscillations in small ROIs in cells treated with 250 nM PD17, or 100 nM, 250 nM or 500 nM PD03 relative to control (DMSO) cells. The middle hinge corresponds to the median, the lower and upper hinges correspond to the first and third quartiles, respectively, and the lower and upper whiskers correspond to the minimum and maximum, respectively. n = 13 (PD17), n = 17 (100 nM), n = 7 (250 nM) or n = 11 (500 nM) independent experiments. p, q, Time-lapse qRT–PCR for the cyclic genes HES7 (p) and LFNG (q) in PSM cells derived from human iPS cells, under control (DMSO) and 250-nM PD03 conditions. Samples were taken every 30 min immediately after treatment. Relative expression is shown as the fold change relative to ES cells at day 0. Mean ± s.d. n = 3 technical replicates. r, Outline of the experimental strategy used to assess the effect of FGF inhibition in primary mouse PSM cells carrying the LuVeLu reporter. The tail bud is dissected from E9.5 transgenic embryos, and cells are dissociated for seeding on fibronectin micropatterns. Oscillations of the LuVeLu reporter are examined in each micropattern. s, LuVeLu fluorescence intensity profiles in mouse tail-bud explant cells cultured on CYTOO micropatterns in CLFBR medium containing DMSO (vehicle control) or increasing doses of PD03 (0.4 μM, 0.65 μM and 10 μM). n = 2 independent experiments. t, Number of LuVeLu oscillations before arrest in mouse tail-bud explant cells cultured on CYTOO micropatterns treated with DMSO (vehicle control) or increasing doses of PD03 (0.4 μM, 0.65 μM and 10 μM). Mean ± s.d. One way ANOVA: 0.4 μM versus 0.65 μM, P = 0.0642; 0.4 μM versus 10 μM, P = 8.4 × 10−9; 0.65 μM versus 10 μM, P = 2.9 × 10−6. n = 10 micropatterns (0.4 μM), n = 7 micropatterns (0.65 μM and 10 μM) u, Average period of LuVeLu oscillations in mouse tail-bud explant cells cultured on CYTOO micropatterns treated with DMSO (vehicle control) or increasing doses of PD03 (0.4 μM, 0.65 μM and 10 μM). Mean ± s.d. One way ANOVA: control versus 0.4 μM, P = 0.2785; control versus 0.65 μM, P = 0.0658; control versus 10 μM, P = 2.7 × 10−6; 0.4 μM versus 0.65 μM, P = 0.831; 0.4 μM versus 10 μM, P = 3.05 × 10−4; 0.65 μM versus 10 μM, P = 4 × 10−3. n = 18 micropatterns (DMSO), n = 16 micropatterns (0.4 μM), n = 12 micropatterns (0.65 μM) and n = 6 micropatterns (10 μM).

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