A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 4
Mapping the shape and steepness of gradients of transcription factor expression in 3D.
A: Method to map the shape of gradients across 3D space (i) First scale the unit vectors between a nuclei and its neighbors to the change in smoothed TF values. (ii) Then sum the vectors to obtain a single directional vector, with magnitude representing the steepness of the gradient. B: (i) Contour plots of average binned normalized fluorescence intensity (NFI) measurements for SOX2, TBXT, and TBX6 in the normalized epiblast shape using PRINGLE, as described in Fig 2. Embryos are grouped per somite pair stage during E8.5, n numbers shown per group. White lines indicate the NMP region of interest (ROI) for each individual embryo as determined using the approach shown in Fig 3. (ii) Contour plots mapping TF gradient steepness map, calculated as shown in A. Averaged data from multiple embryos is shown, as explained in (c). Gradients of TBXT and TBX6 appear to be steepest in regions corresponding to NMP-like cells (NMP ROI) and gradients of SOX2 appear to be steepest in the node streak border. C: (i) Averages of single cell NFI measurements, as measured in Fig 2A, and measurement of gradient steepness, as measured in b (ii), per embryo and somite pair stage plotted in relation to pseudo space values, corresponding to cell identities based on integration of SOX2, TBX6, and TBXT expression as determined in Fig 3. This confirms that gradients of TBX6 and TBXT gradient are steepest within the NMP ROI and decreases into the PS, with TBX6 spatially lagging behind TBXT. Solid lines indicate the fit of a non-parametric multiple regression curve and shading indicates 0.05–0.95 confidence intervals calculated as (mean ± 1.96 * (σ/(√n)). Vertical dashed lines indicate gating from fitting TF pseudospace to the N-M-bi-fated regions in Fig 3. Data for Fig 4 (B-C): Data file 1, https://doi.org/10.5281/zenodo.15802710.