Project description:Gastrulation is the highly coordinated process by which the early embryo breaks symmetry, establishes germ layers and a body plan, and sets the stage for organogenesis. As early mammalian development is challenging to study in vivo, stem cell-derived models have emerged as powerful surrogates, e.g. human and mouse gastruloids. However, although single cell RNA-seq (scRNA-seq) and high-resolution imaging have been extensively applied to characterize such in vitro embryo models, a paucity of measurements of protein dynamics and regulation leaves a major gap in our understanding. Here we sought to address this by applying quantitative proteomics to human and mouse gastruloids at four key stages of their differentiation (naïve ESCs, primed ESCs, early gastruloids, late gastruloids). To the resulting data, we perform network analysis to map the dynamics of expression of macromolecular protein complexes and biochemical pathways, including identifying cooperative proteins that associate with them. With matched RNA-seq and phosphosite data from these same stages, we investigate pathway-, stage- and species-specific aspects of translational and post-translational regulation, e.g. finding peri-gastrulation stages of human vs. mice to be discordant with respect to the mitochondrial transcriptome vs. proteome, and nominating novel kinase-substrate relationships based on phosphosite dynamics. Finally, we leverage correlated dynamics to identify conserved protein networks centered around congenital disease genes. Altogether, our study showcases the potential of intersecting proteomics and in vitro embryo models to advance our understanding of early mammalian development in ways that may not be possible through transcriptomics alone.
Project description:Time-course analysis of cell type specification in a 3-dimensional gastruloid differentiation system initiated from mouse embryonic stem (ES) cells and biased towards hemato-endothelial cell production.
Project description:Gastruloids are highly scalable, three-dimensional assemblies generated from pluripotent stem cells that recapitulate fundamental principles of embryonic pattern formation in vitro. Using single cell RNA and multiome sequencing we provide a comprehensive resource mapping cellular states and cell types found during gastruloid development and compare them to the in vivo embryo. We further develop a high throughput gastruloid handling and imaging pipeline to spatially monitor cell type emergence and unfolding of symmetry breaking during gastruloid development. We report spatial variability of pluripotency states in early gastruloids that determines a binary cell response to Wnt activation. While cells situated in the core of the gastruloid revert to an ectopic pluripotent state, peripheral cells differentiate to a primitive streak like state. These two populations then cause gastruloids to break radial symmetry, allowing axial elongation and commitment to the three embryonic germ layers. Finally by performing a phenotypic compound screen, we perturb thousands of gastruloids at relevant developmental time points deriving a phenotypic landscape and inferring molecular regulator networks underlying gastruloid development. Employing this resource, we improve the formation of anterior structures in the existing gastruloid model, using a dual Wnt modulation approach to differentiate an anterior ectopic pluripotent core to anterior ecto- and endodermal structures. This work gives is a resource to understand how gastruloids develop and, more generally, how homogenous cell populations can generate complex patterns in vitro.
Project description:We use single cell RNA sequencing to describe the transcriptional changes during gastruloid development from 24 to 84 hours with 12 hours intervals.