A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 8
Using PRINGLE to examine scaling of axial patterning between chick and mouse embryos.
A: Diagram of pre-somitic Hamburger-Hamilton stage 6 embryo, approximate field of view used in confocal imaging shown by red box (the developing head and notochord are not indicated). Confocal image stacks of HH6 embryo labeled with hybridization chain reaction (HCR) of SOX2, TBXT, and MSGN1 transcripts represented with a max projection, a single-z plane and 3D render, highlighting the difficulty in representing raw chick imaging data due to undulating nature of epithelium. B: outline of pipeline. C: Cytoplasm inference method for HCR quantification by peri-nuclear segmentation of DAPI signal. Nuclear segmentation performed with cellpose. D: Output of semi-supervised machine-learning classifier, with post-processing, to predict whether a nucleus is epiblast or non-epiblast, showing high accuracy. E: Size comparison of chick HH6 and 4-somite pair (SP) mouse epiblasts shown by PRINGLE projections to normalized A-P and L-R positions. Average SOX2/TBXT cytoplasmic signal for chick and SOX2/TBXT nuclear protein for mouse represented by colored contours (average of four embryos). White lines represent midline (vertical line) and position of node-streak border (intersection with horizontal line). F: The absolute and relative positions of peak paraxial mesoderm to the node streak border (NSB) and PS end show scaling of the focal point to ~30% of primitive streak length despite a large difference in absolute length. Peak paraxial mesoderm is defined as nuclei in the top 90th percentile for paraxial mesoderm marker expression, MSGN1 in chick and TBX6 in mouse. Error bars show standard deviation. G: Comparison of relative global gradient maps for mouse and chick epiblasts show similar “M” shape for SOX2, TBXT “T” shape, and elliptical paraxial mesoderm pattern by TBX6 in mouse and MSGN1 in chick. H: Density plot showing the pairwise expression of SOX2 and TBXT, manual thresholds shown as black dashed lines outlining SOX2+/TBXT+ population. I: Average density distribution ((no. cells per bin)/(total no. cells)) of SOX2+/TBXT+ cells forms a u-shape around the PS, similar to mouse NMP region (Fig 3). White dashed line indicates the position of the node-streak border. Data for Fig 8 (D–I): Data file 4, https://doi.org/10.5281/zenodo.15802710.