Extended Data Fig. 5: Analysis of the human segmentation clock at the single-cell level. | Nature

Extended Data Fig. 5: Analysis of the human segmentation clock at the single-cell level.

From: In vitro characterization of the human segmentation clock

Extended Data Fig. 5

a, Scheme showing the insertion of a constitutively expressed pCAG-H2B-mCherry nuclear label in the safe harbour AAVS1 locus in a HES7-Achilles human-iPS-cell background. b, Diffusion (square micrometres per minute) for individual human HES7-Achilles cells automatically tracked over a period of 24 h. The middle hinge corresponds to median, lower and upper hinges correspond to first and third quartiles, respectively, and the lower and upper whiskers correspond to the minimum and maximum, respectively. n = 76 cells. c, Distribution of pairwise instantaneous phase shifts between individual oscillating human HES7-Achilles cells, binned by instantaneous distance between pairs of cells. P values for the pairwise Kolmogorov–Smirnov test are as follows: <160 μm versus 160–265 μm: 0.6407, <160 μm versus 265–530 μm: 0.1811, <160 μm versus >530 μm: 0.1340, 160–265 μm versus 265–530 μm: 0.1428, 160–265 μm versus >530 μm: 0.6784, and 265–530 μm versus >530 μm: 0.8171. n = 1,000 observations. d, Distribution of phases along the unit circle at early, middle and late time points. Each dot represents one cell. n = 144 cells. e, Illustration of phase determination. Representative raw HES7–Achilles fluorescence profile for an automatically tracked cell (left) and corresponding processed signal along with the inferred phase from Hilbert transform (right). f, Heat map of HES7–Achilles fluorescence intensity over time in automatically tracked cells. Each line represents one cell. n = 144 cells. g, Histogram of the time (hours since onset of imaging) at cell division for manually tracked human HES7-Achilles cells. n = 67 cells. h, Left, immunofluorescence staining for histone H3 phosphorylated at Ser10, in PSM cells derived from human iPS cells, treated with vehicle control (DMSO) or 5 μM aphidicolin for 24 or 48 h, starting on day 2 of differentiation. n = 5 independent experiments. Scale bar, 100 μm. Right, quantification of phosphorylated histone H3 (at Ser10) nuclei as a percentage of total nuclei. The middle hinge corresponds to 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. i, Scatter plot showing the cell-cycle time in PSM cells derived from human iPS cells, cultured in CLFBR medium. Mean ± s.d. n = 26 cells. j, Histogram of the HES7–Achilles oscillatory phase at the time of cell division in human iPSC-derived PSM cells cultures in CLFBR medium. Distribution is not significantly different from the uniform distribution: Kolmogorov-Smirnov test p=0.225. n=55 cell divisions. k, Normalized HES7–Achilles fluorescence intensity profiles for 3 individual PSM cells derived from human iPS cells, pre-treated with 5 μM Aahidicolin for 24 h. n = 6 independent experiments. l, Kuramoto order parameter over 20 h on day 3 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 5 μM aphidicolin for 24 h. The synchronization threshold is shown as the mean ± s.d. of the Kuramoto order parameter for same dataset, but with randomized phases. n = 45 cells (control) or 48 cells (aphidicolin). m, Comparison of the Kuramoto order parameter for oscillating HES7-Achilles cells treated with vehicle control (DMSO) or 5 μM aphidicolin. 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. Paired two-sided t-test, P = 0.348. n = 45 cells (control) or 48 cells (aphidicolin). n, qRT–PCR for Notch target genes HES7, NRARP and LFNG in PSM cells derived from human iPS cells, treated with vehicle control (DMSO) or 25 μM DAPT on day 2 of differentiation. Mean ± s.d. n = 3 biological replicates. o, Example of HES7–Achilles fluorescence intensity in a small ROI over a period of 45 h in cells treated with DMSO (vehicle control) or the γ-secretase inhibitor DAPT (25 μM) in CLFBR medium. n = 16 independent experiments. p, Representative example of HES7–Achilles fluorescence intensity profiles for PSM cells derived from mouse ES cells, treated with vehicle control (DMSO) or 25 μM DAPT. n = 13 independent experiments. q, Kuramoto order parameter over 20 h on day 2 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 25 μM DAPT. The synchronization threshold is shown as the mean ± s.d. of the Kuramoto order parameter for same dataset, but with randomized phases. n = 131 cells (control) or 110 cells (DAPT). r, Representative immunofluorescence staining for YAP, F-actin (phalloidin) and DAPI nuclear stain in isolated human PSM-like cells treated with DMSO or latrunculin A (350 nM). Scale bar, 50 μm. n = 4 independent experiments. s, ChIP–qPCR fold enrichment of the LFNG and HES7 promoters in chromatin pulled down with an antibody against NOTCH1, relative to isotype IgG controls. Mean ± s.d. iPS-cell control, n = 4; all other conditions, n = 3 biological replicates. t, Mean HES7–Achilles fluorescence intensity for isolated human cells cultured with 350 nM latrunculin A alone, or in combination with 25 μM DAPT. 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 = 18 cells. u, Scatter plot showing the HES7–Achilles oscillatory period for isolated human cells cultured with 350 nM latrunculin A alone, or in combination with 25 μM DAPT. Mean ± s.d. n = 47 (latrunculin A) or 22 (latrunculin A + DAPT) cells. v, Kuramoto order parameter over 18 h on day 2 of differentiation for human HES7-Achilles cells treated with DMSO, latrunculin A alone or latrunculin A in combination with DAPT. The synchronization threshold is shown as the mean ± s.d. of the Kuramoto order parameter for the same dataset, but with randomized phases. n = 53 cells (control), 18 cells (latrunculin A) or 18 cells (latrunculin A + DAPT). w, Comparison of the Kuramoto order parameter in confluent HES7-Achilles cells versus isolated cells treated with 350 nM latrunculin A alone, or in combination with 25 μM DAPT. 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. Paired one-way ANOVA with Bonferroni correction: confluent control versus LatA, P = 1.16 × 10−6; confluent control versus latrunculin A + DAPT, P = 6.8 × 10−13; latrunculin A versus latrunculin A + DAPT, P = 0.304. n = 53 cells (control), 18 cells (latrunculin A) or 18 cells (latrunculin A + DAPT).

Source data

Back to article page