Extended Data Figure 5.

a, Scheme showing the insertion of a constitutively expressed pCAG-H2B-mCherry nuclear label in the safe harbor AAVS1 locus in a HES7-Achilles human iPSC background. b, Diffusion (μm²/min) for individual human HES7-Achilles cells automatically tracked over a period of 24 hours. Middle hinge corresponds to median, lower and upper hinges correspond to 1^st and 3^rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=76 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 pair-wise Kolmogorov-Smirnov test are as follows: 0.6407, 0.1811, 0.1340, 0.1428, 0.6784, 0.8171. n=1000. d, Distribution of phases along the unit circle at early, middle, and late timepoints. 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, Heatmap 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 the onset of imaging) at cell division for manually tracked human HES7-Achilles cells. n=67 h, Left: Immunofluorescence staining for phosphorylated histone H3 (Ser10) in human iPSC-derived PSM cells treated with either vehicle control (DMSO) or 5μM Aphidicolin for 24 or 48 hours, starting on day 2 of differentiation. n=5. Scale bar = 100μm. Right: Quantification of phosphorylated histone H3 (Ser10) nuclei as a percent of total nuclei. Middle hinge corresponds to median, lower and upper hinges correspond to 1^st and 3^rd quartiles, lower and upper whiskers correspond to minimum and maximum. i, Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. j, Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. k, Normalized HES7-Achilles fluorescence intensity profiles for 3 individual human iPSC-derived PSM cells pre-treated with 5μM Aphidicolin for 24 hours. n=6 independent experiments. l, Kuramoto order parameter over 20 hours on day 3 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 5μM Aphidicolin for 24 hours. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=45 cells (Control), 48 cells (Aphi). m, Comparison of the Kuramoto order parameter for oscillating HES7-Achilles treated with vehicle control (DMSO) or 5μM Aphidicolin. Middle hinge corresponds to median, lower and upper hinges correspond to 1^st and 3^rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired two-sided t-test p=0.348. n=45 cells (Control), 48 cells (Aphi). n, qRT-PCR for Notch target genes HES7, NRARP and LFNG in human iPSC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT on day 2 of differentiation. Mean±SD. n=3. o, Example of HES7-Achilles fluorescence intensity in a small region of interest (ROI) over a period of 45 hours in cells treated with DMSO (vehicle control) or the γ-secretase inhibitor DAPT (25μM) in CLFBR medium. n=16 independent experiments p, Representative example of Hes7-Achilles fluorescence intensity profiles for mESC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT. n=13 independent experiments q, Kuramoto order parameter over 20 hours on day 2 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 25μM DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=131 cells (Control), 110 cells (DAPT). r, Representative immunofluorescence staining for YAP, F-actin (phalloidin) and DAPI nuclear stain in isolated human PSM-like cells treated with either DMSO or Latrunculin A (350nM). 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 ±SD. iPSC control n=4, all other conditions n=3. t, Mean HES7-Achilles fluorescence intensity for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1^st and 3^rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=18 cells. u, Scatter plot showing the HES7-Achilles oscillatory period for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Mean ±SD. n= 47 (LatA), 22 (LatA + DAPT) v, Kuramoto order parameter over 18 hours on day 2 of differentiation for human HES7-Achilles cells treated with DMSO, LatA alone or LatA in combination with DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT). w, Comparison of the Kuramoto order parameter in confluent HES7-Achilles cells vs. isolated cells treated with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1^st and 3^rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired one-way ANOVA with Bonferroni correction: Confluent control vs. LatA p=1.16e⁻⁶, Confluent control vs. LatA + DAPT p=6.8e⁻¹³, LatA vs. LatA + DAPT p=0.304. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT).