Figure 3(a) Schematic of extracting kymograph information from tissue data by averaging Venus::HES5 intensities observed in E10.5 heterozygous spinal cord slices to generate one intensity profile in the dorsal-ventral axis per timepoint (see Materials and Methods). (b) Representative kymograph data showing spatiotemporal Venus::HES5 expression profile along ventral-dorsal direction in a 15μm wide apical region and observed over 14h; local bands of 20μm width in D-V; region of interest markers indicate: *low to high, **high to low and ***re-occurring high/low activity in the same area. (c) Hierarchical clustering of apical Venus::HE5 expression from one representative experiment showing behaviour in the same area over time; columns represent fluctuations in Venus::HES5 intensity in small local areas (bands) obtained by dividing the spatial signal into non-overlapping 20μm regions and normalizing to the mean and standard deviation of each region over time (z-scoring); data has been subject to a Gaussian blur pre-processing step (see Appendix Figure S2b and Methods). (d) Persistence of Venus::HES5 in 20μm regions expressed as continuous time intervals when signal in the band is high or low compared to its mean (see Methods); individual datapoints (gray) indicate quantification of high and low persistence time obtained from over 300 thin bands collected from multiple tissues with 2 z-stacks per tissue and 2 repeats (left and right of ventricle) per z-stack; dots indicate paired medians of 5 independent experiments; statistical test is paired t test of median per experiment with two tail significance and p=0.7171. (e) Persistence of Venus::HES5 levels in high and low states taken from 60 tracked single cells collected from 3 independent experiments; paired t test not significant p=0.0533. (f) Relative distance between cell pairs computed from relative 3D Euclidean distance between nuclei over 12-15h; dots indicate median distance over tracking period, horizontal lines show mean and SD of 14 cell pairs from 3 experiments. (g) Spearman correlation coefficients computed in the same cell pairs from Venus::HES5 and H2B::mCherry (control) nuclear intensity timeseries; markers in each condition indicate pairs; black dots indicate median correlation coefficients per experiment (4 pairs, 3 pairs and 7 pairs), lines show median of 14 pairs from 3 experiments; paired t test with significance p=0.0058. (h) Representative example timeseries of raw Venus::HES5 raw intensity in cells pairs identified as remaining in close proximity; insert shows Venus::HES5 intensity relative to the tissue mean intensity; r-values indicate Spearman correlation coefficients between time traces over all co-existing timepoints. (i) Detrended Venus::HES5 fluorescent intensity timeseries (after z-scoring) corresponding to examples in (h); red arrows indicate in-phase peaks. (j) Density phase plots from instantaneous Hilbert phase reconstruction at multiple timepoints over a 12-14h period; dots indicate the phase angle in Cell 1 and Cell 2 from 14 pairs collected from 3 experiments; colormap indicates probability density showing accumulation of phase values predominantly along the (0,0) and (2π, 2π) diagonal; light colours indicate most frequent. (k) Graphic representation of a neuroepithelial tissue with nuclei colour-coded to indicate clusters of high or low HES5 expression. The tissue is illustrated at three different time points to depict how clusters of cells can dynamically switch from high to low or low to high while the periodic spatial pattern is maintained. In the example time traces (corresponding to the three grey and one red highlighted nuclei), synchronised ultradian oscillations are shown as being overlayed on the slow-varying higher-amplitude switching dynamics.