A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 7
Mapping changes in patterning after inhibition of Notch activity.
A: Culture method where a posterior explant of an E8.5 embryo is cultured in N2B27 media with the Notch inhibitor LY at 150 nM or an equal amount of DMSO for 12 h. B: Representative 3D renders of explants for each condition, stained for LaminB1 or SOX2, TBXT, and TBX6 indicating normal tissue morphology but a reduction in TBXT and TBX6 expression. Anterior (A), posterior (P), dorsal (D), ventral (V) axes are indicated. Scale bars indicate 100 μm. C: Analysis pipeline to evaluate the effect of Notch inhibition on patterning. After using PRINGLE to create an EpiMap, the epiblasts are folded at the midline and clustered with k-means to demark comparable ROIs. Patterning metrics are compared between DMSO and LY in these regions. One way ANOVA is conducted to identify the statistical significance of the difference of patterning metrics, regions are filled with associated colors to denote the p-value. D: Contour plots of TF normalized fluorescence intensity (NFI) projected onto the EpiMap with the difference in DMSO vs. LY conditions and associated p-values per ROI. Displaying an increase of SOX2 NFI in the PS and a posterior withdrawal of the TBXT and TBX6 domains in the Notch inhibited condition. E: Contour plots of NFI gradient steepness projected onto the EpiMap with the difference in DMSO vs. LY conditions and associated p-values per ROI. Note the overall decrease in TBXT and TBX6 gradient steepness, with a new posteriorly located nonlinear TBXT gradient. F: Contour plot of TBX6 local heterogeneity (CV) on EpiMap. Note the statistically significant decrease in the CLE and NSB like regions TBX6. Data for Fig 7 (D–F): Data file 3, https://doi.org/10.5281/zenodo.15802710.