A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 2
3D Epiblast projection and alignment method: PRINGLE.
A: Single cell analysis pipeline: individual nuclei are identified from 3D confocal images and manually labeled as epiblast or non-epiblast using the PickCells software. Fluorescence intensity is quantified within each nucleus and the neighbors of each cell in 3D are identified. B: Summary of PRINGLE method to project the 3D nuclei centroids in 2D and align multiple embryos: The distance of nuclei centroids are calculated from the midline and notochord along principal curves in transverse and sagittal sections, respectively. These distances are then used as coordinates to project nuclei in 2D and create an unfolded epiblast. Unfolded maps generated from multiple embryos are then registered together by normalizing nuclei coordinates relative to tissue landmarks: the midline and edge of epiblast are used as landmarks for the left/right (L/R) axis, and the notochord origin and primitive streak posterior tip as landmarks for anterior/posterior (A/P) axis. However relative positions produce a distortion, therefore relative positions are normalized to the average epiblast width. This registration procedure results in an ‘average’ epiblast map in 2D. D: dorsal, V: ventral, A: Anterior, P: Posterior, M: Midline, L: Lateral edge of epiblast. This approach is explained in more detail in S1 Fig. C: A four-somite-pair epiblast showing SOX2, TBXT, and TBX6 expression (measured as outlined in (a)) mapped onto the average epiblast shape of multiple four-somite-pair epiblasts (established as outlined in (b)). Points represent nuclei centroids. Data for Fig 2 (C): Data file 1, https://doi.org/10.5281/zenodo.15802710.