Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation.

Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation.

Transcriptional heterogeneity and cell cycle regulation as central determinants of Primitive Endoderm priming.

During embryonic development cells acquire identity as they proliferate, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to 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 (PrE) lineage. Since ESCs are derived from the inner cell mass (ICM) of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing dynamically interconverting subfractions primed for either of the two ICM lineages, Epiblast and PrE. Here, we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture promotes Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, this change is accompanied by a counter-intuitive increase in G1 length, also observed in vivo. While fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signalling is a key regulator of ESC differentiation and PrE specification, we find it is not just responsible for ESCs heterogeneity, but also the inheritance of similar cell cycles between sisters and cousins. Taken together, our results indicate a tight relationship between transcriptional heterogeneity and cell cycle regulation in lineage specification, 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.

Dynamic lineage priming is driven via direct enhancer regulation by ERK.

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.

Naïve human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable naïve extra-embryonic endoderm.

Embryonic stem cells (ESCs) exist in at least two states that transcriptionally resemble different stages of embryonic development. Naïve ESCs resemble peri-implantation stages and primed ESCs the pre-gastrulation epiblast. In mouse, primed ESCs give rise to definitive endoderm in response to the pathways downstream of Nodal and Wnt signalling. However, when these pathways are activated in naïve ESCs, they differentiate to a cell type resembling early primitive endoderm (PrE), the blastocyst-stage progenitor of the extra-embryonic endoderm. Here, we apply this context dependency to human ESCs, showing that activation of Nodal and Wnt signalling drives the differentiation of naïve pluripotent cells toward extra-embryonic PrE, or hypoblast, and these can be expanded as an in vitro model for naïve extra-embryonic endoderm (nEnd). Consistent with observations made in mouse, human PrE differentiation is dependent on FGF signalling in vitro, and we show that, by inhibiting FGF receptor signalling, we can simplify naïve pluripotent culture conditions, such that the inhibitor requirements closer resemble those used in mouse. The expandable nEnd cultures reported here represent stable extra-embryonic endoderm, or human hypoblast, cell lines.This article has an associated 'The people behind the papers' interview.

Transcriptomics of dorso-ventral axis determination in Xenopus tropicalis.

Amphibian embryos provide a powerful system to study early cell fate determination because their eggs are externally fertilised, large, and easy to manipulate. Ultraviolet (UV) or lithium chloride (LiCl) treatment are classic embryonic manipulations frequently used to perturb specification of the dorso-ventral (DV) axis by affecting the stability of the maternal Wnt mediator ß-catenin. Such treatments result in the formation of so-called ventralised or dorsalised embryos. Although these phenotypes have been well described with respect to their morphology and some aspects of gene expression, their whole transcriptomes have never been systematically characterised and compared. Here we show that at the early gastrula stage UV-treated embryos are transcriptionally more closely related to untreated embryos than to LiCl-treated embryos. Transcriptional comparisons with dissected ventral and dorsal regions of unperturbed gastrula embryos indicate that UV and LiCl treatments indeed enrich for ventral and dorsal cells, respectively. However, these treatments also affect the balance of neural induction in the ectodermal germ layer, with LiCl stimulating pro-neural BMP inhibition and UV preferentially generating epidermis because of elevated BMP levels. Thus the transcriptomes of UV- and LiCl-treated embryos can best be described as ventro-epidermalised and dorso-neuralised. These descriptions notwithstanding, our profiling reveals several hitherto uncharacterized genes with differential expression along the DV axis. At least one of these genes, a RNF220-like ubiquitin ligase, is activated dorsally by ß-catenin. Our analysis of UV/LiCl-mediated axis perturbation will enhance the mechanistic understanding of DV axis determination in vertebrates.

Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus.

Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.