Spatial molecular anatomy of germ layers in the gastrulating Cynomolgus monkey embryo-Supplemental Materials
During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of Cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation. This Supplemental Materials containing Table S1-7, Supplementary file 1-10 and Supplementary video 1. They are also available at our website: http://sop.ccla.ac.cn.
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3D reconstructions of gastrulating monkey embryos The Vaa3D (version 3.5) and Amira (version 6.0.1) were used for the 3D reconstruction process, consisting of alignment, segmentation and visualization (de Bakker Bernadette et al., 2016; Peng et al., 2010). In brief, grey scale processing was performed by Photoshop CC (Adobe Systems), and alignment of the stitched serial images was mainly implemented by manual after automatically adjustments. The aligned 3D images were segmented into epiblast/ectoderm, primitive streak, hypoblast/endoderm and mesoderm. Then, triangulated surface files were made using the SurfaceGen function, and triangle reduction (Simplify) and surface smoothing (SmoothSurface) were also applied for visualization. Besides, the Vaa3D was also applied to aligned sections to visualize the 3D structure of specific tissues. Determination of gastrulation cell number and mitotic activity The number of cells contained in epiblast/ectoderm, mesoderm and hypoblast/endoderm was determined by counting nuclei on every section of the gastrulating embryos. The total score was then adjusted by applying Abercrombie's correction formula (Abercrombie, 1946) to give an estimate of the actual cell number. In detail, the average number of cells per section (P): P=A M/(L+M) A is the crude count of number of nuclei fragments, M is the thickness of the section (M=5μm in this study), and L the average length of the nuclei (L=10μm in this work). The mean population doubling time is computed on the assumption that all cells divide and no cells are lost from the population due to cell death or exit from the population by migration. The mean population doubling time can be calculated from the equation (Snow, 1977), Ct means the cell number at t stage: T=∆t/〖C_(t+1)/C〗_t The enumeration of cell numbers was based on the findings of in vitro cultured human embryos at three timepoints (E10, E12 and E14) (Xiang et al., 2019) and cell counts in our sectioned materials across the four timepoints ( E17, E18, E19 and E20). The mean population doubling time at the pre-gastrulation stages (n=2) is about 50.8 hours and this is halved to about 23.4 hours during gastrulation (n=3).