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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 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 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 replicatedin 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.

Mouse embryonic stem cells (mESCs) represent are the primary in vitro model for many aspects of early embryonic development. They are the descendants of the cells of the cells inner cell mass (ICM) of early blastocyst and whilst they maintain the potency of their forerunner cells, they acquire the ability to self-renew during ex vivo culture.Over the past decades considerable work has focused in understanding the molecular basis of this ex vivo self-renewal and how it relates to cellular potency. The dominant model is that the combined activities of a discrete network of transcription factors (TFs), referred to as the pluripotency network, act cooperatively to maintain their own expression and promote proliferation whilst simultaneously blocking differentiation. However useful, this model has yet to adequately explain why such a complex, self-sustaining and robust self-renewal network would have evolved in a population that exhibits negligible self-renewal in vivo. In the context of Waddingtonian lineage transitions, as a cell traverses developmental landscapes, the gene regulatory network (GRN) that establishes each discernible cell state must also define the range of lineage trajectories available to it, incorporating various inputs like signalling and positional information to choose one trajectory over the other. Here we present data that argues that the canonical pluripotency network can be divided into at least two distinct subgroups; those that promote lineage and those that balance lineage decisions, with both classes of TFs functioning in creating a state of lineage competency, similar to what is seen in the ICM. Specifically, we see that the combined activities of ESRRB and NANOG promote a gene expression signature similar to the ICM when expressed at a near 1:1 ratio. However if ESRRB expression persists in the absence of NANOG it is no longer tethered to epiblast associated loci and acts to drive the expression of primitive endoderm (PrE) associated genes. We formalise the kinetics of this interaction and show that oscillation of ERK signalling result in asynchronous fluctuations in NANOG and ESRRB ratios and a concomitant stepwise increase in PrE determinants, similar to what is seen in the ICM. From this we propose that the ex vivo culture environment selects for ratios of TFs that, whilst maintaining the expression of ICM-like genes, and as such potency, prevent cells from progressing further in development.

Mouse embryonic stem cells (mESCs) represent are the primary in vitro model for many aspects of early embryonic development. They are the descendants of the cells of the cells inner cell mass (ICM) of early blastocyst and whilst they maintain the potency of their forerunner cells, they acquire the ability to self-renew during ex vivo culture.Over the past decades considerable work has focused in understanding the molecular basis of this ex vivo self-renewal and how it relates to cellular potency. The dominant model is that the combined activities of a discrete network of transcription factors (TFs), referred to as the pluripotency network, act cooperatively to maintain their own expression and promote proliferation whilst simultaneously blocking differentiation. However useful, this model has yet to adequately explain why such a complex, self-sustaining and robust self-renewal network would have evolved in a population that exhibits negligible self-renewal in vivo. In the context of Waddingtonian lineage transitions, as a cell traverses developmental landscapes, the gene regulatory network (GRN) that establishes each discernible cell state must also define the range of lineage trajectories available to it, incorporating various inputs like signalling and positional information to choose one trajectory over the other. Here we present data that argues that the canonical pluripotency network can be divided into at least two distinct subgroups; those that promote lineage and those that balance lineage decisions, with both classes of TFs functioning in creating a state of lineage competency, similar to what is seen in the ICM. Specifically, we see that the combined activities of ESRRB and NANOG promote a gene expression signature similar to the ICM when expressed at a near 1:1 ratio. However if ESRRB expression persists in the absence of NANOG it is no longer tethered to epiblast associated loci and acts to drive the expression of primitive endoderm (PrE) associated genes. We formalise the kinetics of this interaction and show that oscillation of ERK signalling result in asynchronous fluctuations in NANOG and ESRRB ratios and a concomitant stepwise increase in PrE determinants, similar to what is seen in the ICM. From this we propose that the ex vivo culture environment selects for ratios of TFs that, whilst maintaining the expression of ICM-like genes, and as such potency, prevent cells from progressing further in development.

Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan. European Research Council (AdG MiniEmbryoBlueprint 834580)