A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 5
Mapping the properties of cells in relation to their local neighborhood.
A: The coefficient of variation (CV) is used as a metric for the degree of local heterogeneity in fluorescence intensity values for a cell and its direct neighbors (in 3D), defined as the standard deviation divided by the mean of fluorescence intensity values for a cell and its neighbors. Neighbors are defined as in Fig 2A. B: Violin Superplots showing CV values for SOX2, TBXT, and TBX6 within NMP-like cells as defined in Fig 3. Local heterogeneity (CV) in TBX6 is greater than TBXT, which is greater than local heterogeneity in SOX2. Dots indicate the mean for each of 19 embryos. Statistical tests performed with paired t test and Benjamini–Hochberg post-hoc correction.. *** = p < 0.001. C: Maps of local variability in TF expression across the epiblast. Contour plots showing CV values mapped onto EpiMap. Data is averaged from multiple embryos as described in Fig 4). U-shaped white lines indicate the NMP ROI as measured in Fig 3. High local variability (CV) for TBXT and TBX6 is observed within the NMP ROI. D: Heightened TBXT and TBX6 CV to the NMP region and decreases into the mesodermal PS. Lines indicate the fit of a non-parametric multiple regression curve and shading indicates 0.05 and 0.95 confidence intervals calculated as (mean ± 1.96 * (σ/(√n)). E: The neighbor ratio (NR) metric can distinguish patterning scenarios where a cell is (1) surrounded by cells with an averaged expression that is higher expression than itself, (2) the same as itself, or (3) lower expression than itself. Defined as ln(Cell TF value/ Average Neighbor TF value). F: Comparing the ‘high’ cells in the TF+ populations (upper 50th percentile) for SOX2, TBXT, and TBX6 to synthetic data assuming a normal distribution for a cell’s expression and its neighbors. This shows a decrease in NR, indicating TBX6+ high cells are surrounded by cells expressing lower TBX6 levels than expected within a random pattern. Points indicate average NR per embryo, shaded areas show confidence intervals of 0.05 and 0.95 calculated as (mean ± 1.96 * (σ/(√n)). Cells were binned into even intervals and only cells within these intervals were considered for confidence intervals and statistical summary metrics. G: Statistical analysis of the difference in observed and synthetic data NR values along pseudospace bins using (i) earth mover’s distance between normalized distributions for each embryo and (ii) p-values resulting from one way ANOVA between observed and synthetic embryo means. The difference in TBX6 NR values is statistically significant in the NMP ROI with a high difference in distributions compared to TBXT or SOX2. Shaded areas show confidence intervals of 0.05 and 0.95. Dashed line in (ii) indicates p-value = 0.05. Data for Fig 5 (B–D and F–G): Data file 1, https://doi.org/10.5281/zenodo.15802710.