Transcriptional heterogeneity and cell cycle regulation as central determinants of primitive endoderm priming
- University of Copenhagen, Denmark
Abstract
During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNA-seq in the contexts of self-renewal, priming and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESCs and PrE differentiation, we find it is not just responsible for ESCs heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tight relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.
Data availability
The sc-RNAseq data used in this study has been deposited in the Gene Expression Omnibus and are available under the accession number GSE200534. Previously published Nowotschin et al., 2019 data that were used here are available under accession number GSE123046.
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Transcriptional Heterogeneity and Cell Cycle Regulation as Central Determinants of Primitive Endoderm PrimingNCBI Gene Expression Omnibus, GSE200534.
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The Emergent Landscape of the Mouse Gut Endoderm at Single-Cell ResolutionNCBI Gene Expression Omnibus, GSE123046.
Article and author information
Author details
Funding
Lundbeckfonden (R198-2015-412)
- Joshua M Brickman
Danish Agency for Science and Higher Education (DFF- 8020-00100B)
- Joshua M Brickman
Danish National Research Foundation (DNRF116)
- Joshua M Brickman
Lundbeckfonden (R286-2018-1534)
- Marta Perera
Lundbeckfonden (R303-2018-2939)
- Rita Soares Monteiro
Danish National Research Foundation (DNRF116)
- Ala Trusina
Novo Nordisk Fonden (NNF21CC0073729)
- Joshua M Brickman
Novo Nordisk Fonden (NNF17CC002785)
- Joshua M Brickman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Perera et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Transcriptional heterogeneity and cell cycle regulation as central determinants of primitive endoderm priming
eLife 11:e78967.
https://doi.org/10.7554/eLife.78967
Further reading
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- Developmental Biology
During the trunk to tail transition the mammalian embryo builds the outlets for the intestinal and urogenital tracts, lays down the primordia for the hindlimb and external genitalia, and switches from the epiblast/primitive streak (PS) to the tail bud as the driver of axial extension. Genetic and molecular data indicate that Tgfbr1 is a key regulator of the trunk to tail transition. Tgfbr1 has been shown to control the switch of the neuromesodermal competent cells from the epiblast to the chordoneural hinge to generate the tail bud. We now show that in mouse embryos Tgfbr1 signaling also controls the remodeling of the lateral plate mesoderm (LPM) and of the embryonic endoderm associated with the trunk to tail transition. In the absence of Tgfbr1, the two LPM layers do not converge at the end of the trunk, extending instead as separate layers until the caudal embryonic extremity, and failing to activate markers of primordia for the hindlimb and external genitalia. The vascular remodeling involving the dorsal aorta and the umbilical artery leading to the connection between embryonic and extraembryonic circulation was also affected in the Tgfbr1 mutant embryos. Similar alterations in the LPM and vascular system were also observed in Isl1 null mutants, indicating that this factor acts in the regulatory cascade downstream of Tgfbr1 in LPM-derived tissues. In addition, in the absence of Tgfbr1 the embryonic endoderm fails to expand to form the endodermal cloaca and to extend posteriorly to generate the tail gut. We present evidence suggesting that the remodeling activity of Tgfbr1 in the LPM and endoderm results from the control of the posterior PS fate after its regression during the trunk to tail transition. Our data, together with previously reported observations, place Tgfbr1 at the top of the regulatory processes controlling the trunk to tail transition.
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- Developmental Biology
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Mutations in Sonic Hedgehog (SHH) signaling pathway genes, for example, Suppressor of Fused (SUFU), drive granule neuron precursors (GNP) to form medulloblastomas (MBSHH). However, how different molecular lesions in the Shh pathway drive transformation is frequently unclear, and SUFU mutations in the cerebellum seem distinct. In this study, we show that fibroblast growth factor 5 (FGF5) signaling is integral for many infantile MBSHH cases and that FGF5 expression is uniquely upregulated in infantile MBSHH tumors. Similarly, mice lacking SUFU (Sufu-cKO) ectopically express Fgf5 specifically along the secondary fissure where GNPs harbor preneoplastic lesions and show that FGFR signaling is also ectopically activated in this region. Treatment with an FGFR antagonist rescues the severe GNP hyperplasia and restores cerebellar architecture. Thus, direct inhibition of FGF signaling may be a promising and novel therapeutic candidate for infantile MBSHH.
