FGF4 stimulation increases variability in mESCs [scRNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1077953 | ENA |
REPOSITORIES: ENA
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Project description:FGF4 is essential for Epiblast and Primitive Endoderm formation in the mouse embryo and promotes differentiation of mouse embryonic stem cells. However, different FGF-concentrations regulate gene expression quantitatively remained an open question. We used single-cell RNA sequencing to quantify transcriptional variability and dynamics upon FGF4 stimulation of Fgf4-mutant mESCs.
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Project description:Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. FGF, Wnt, and mTOR signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mESCs. Focusing on the Mediator subunit Med12 as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage, but pluripotency transitions are slowed down, and the transcriptional separation between epiblast and primitive endoderm identities is enhanced in Med12-mutant cells. These cellular phenotypes correlate with reduced biological noise upon loss of Med12. These findings suggest that Med12 regulates cellular plasticity through the priming of transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.
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Project description:Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. FGF, Wnt, and mTOR signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mESCs. Focusing on the Mediator subunit Med12 as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage, but pluripotency transitions are slowed down, and the transcriptional separation between epiblast and primitive endoderm identities is enhanced in Med12-mutant cells. These cellular phenotypes correlate with reduced biological noise upon loss of Med12. These findings suggest that Med12 regulates cellular plasticity through the priming of transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.
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Project description:Protein kinase signalling is a major mechanism by which embryonic stem cell pluripotency and differentiation is controlled. However, the pathways and components that regulate embryonic stem cell identity have not been systematically defined. Here, we employ FGF4 signalling as a model system to investigate phosphoproteome dynamics in differentiating mouse embryonic stem cells. We report identification and quantitation of more than 10,000 phosphopeptides, of which hundreds of phosphophoylation sites are regulated more than 2-fold by acute FGF4 stimulation. We hypothesise that phosphorylation sites in this dataset are relevant for regulating the transition of mouse embryonic stem cells from pluripotency towards lineage specific differentiation.
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