Abstract
During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.
Highlights
BMP4 is expressed by the extraembryonic ectoderm and later (E7.5-E8.0) in the allantois, amnion and chorion. (B,D,E) Representative confocal images of immunostained gastrulating embryos showing BMP signaling activity based on nuclear localization of pSMAD1/5/8
Primitive streak (PS) and Mesoderm 1 and 2 (Meso1/2) were manually selected on confocal images of transverse cryosections using ImageJ software as shown in right-hand panel
(D,F) High magnification confocal optical sections of a region of the colony edge after 72 hr of differentiation
Summary
Middle panels show quantification of SOX2, BRACHYURY and CDX2 immunostaining voxel fluorescence intensity, in arbitrary units (a.u.), from colony center (0) to edge (500). Quantification of immunostaining voxel fluorescence intensity of differentiated TGFP/+ and Sox17GFP/+ cell lines, in arbitrary units (a.u.), from colony center (0) to edge (500).
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