Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3.

Factors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.

Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

The transcriptional and epigenomic foundations of ground state pluripotency.

Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as "2i" postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.

DNA Damage Signaling-Induced Cancer Cell Reprogramming as a Driver of Tumor Relapse.

Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.

Evidence implicating sequential commitment of the founder lineages in the human blastocyst by order of hypoblast gene activation.

Successful human pregnancy depends upon rapid establishment of three founder lineages: the trophectoderm, epiblast and hypoblast, which together form the blastocyst. Each plays an essential role in preparing the embryo for implantation and subsequent development. Several models have been proposed to define the lineage segregation. One suggests that all lineages specify simultaneously; another favours the differentiation of the trophectoderm before separation of the epiblast and hypoblast, either via differentiation of the hypoblast from the established epiblast, or production of both tissues from the inner cell mass precursor. To begin to resolve this discrepancy and thereby understand the sequential process for production of viable human embryos, we investigated the expression order of genes associated with emergence of hypoblast. Based upon published data and immunofluorescence analysis for candidate genes, we present a basic blueprint for human hypoblast differentiation, lending support to the proposed model of sequential segregation of the founder lineages of the human blastocyst. The first characterised marker, specific initially to the early inner cell mass, and subsequently identifying presumptive hypoblast, is PDGFRA, followed by SOX17, FOXA2 and GATA4 in sequence as the hypoblast becomes committed.

Gastruloid-derived primordial germ cell-like cells develop dynamically within integrated tissues.

Primordial germ cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here, we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of gastruloid-derived PGCLCs (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extra-embryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, owing to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments.

Publisher Correction: Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Conceptualising commercial entities in public health: beyond unhealthy commodities and transnational corporations.

Most public health research on the commercial determinants of health (CDOH) to date has focused on a narrow segment of commercial actors. These actors are generally the transnational corporations producing so-called unhealthy commodities such as tobacco, alcohol, and ultra-processed foods. Furthermore, as public health researchers, we often discuss the CDOH using sweeping terms such as private sector, industry, or business that lump together diverse entities whose only shared characteristic is their engagement in commerce. The absence of clear frameworks for differentiating among commercial entities, and for understanding how they might promote or harm health, hinders the governance of commercial interests in public health. Moving forward, it is necessary to develop a nuanced understanding of commercial entities that goes beyond this narrow focus, enabling the consideration of a fuller range of commercial entities and the features that characterise and distinguish them. In this paper, which is the second of three papers in a Series on commercial determinants of health, we develop a framework that enables meaningful distinctions among diverse commercial entities through consideration of their practices, portfolios, resources, organisation, and transparency. The framework that we develop permits fuller consideration of whether, how, and to what extent a commercial actor might influence health outcomes. We discuss possible applications for decision making about engagement; managing and mitigating conflicts of interest; investment and divestment; monitoring; and further research on the CDOH. Improved differentiation among commercial actors strengthens the capacity of practitioners, advocates, academics, regulators, and policy makers to make decisions about, to better understand, and to respond to the CDOH through research, engagement, disengagement, regulation, and strategic opposition.

Defining and conceptualising the commercial determinants of health.

Although commercial entities can contribute positively to health and society there is growing evidence that the products and practices of some commercial actors-notably the largest transnational corporations-are responsible for escalating rates of avoidable ill health, planetary damage, and social and health inequity; these problems are increasingly referred to as the commercial determinants of health. The climate emergency, the non-communicable disease epidemic, and that just four industry sectors (ie, tobacco, ultra-processed food, fossil fuel, and alcohol) already account for at least a third of global deaths illustrate the scale and huge economic cost of the problem. This paper, the first in a Series on the commercial determinants of health, explains how the shift towards market fundamentalism and increasingly powerful transnational corporations has created a pathological system in which commercial actors are increasingly enabled to cause harm and externalise the costs of doing so. Consequently, as harms to human and planetary health increase, commercial sector wealth and power increase, whereas the countervailing forces having to meet these costs (notably individuals, governments, and civil society organisations) become correspondingly impoverished and disempowered or captured by commercial interests. This power imbalance leads to policy inertia; although many policy solutions are available, they are not being implemented. Health harms are escalating, leaving health-care systems increasingly unable to cope. Governments can and must act to improve, rather than continue to threaten, the wellbeing of future generations, development, and economic growth.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

Mapping the Lobbying Footprint of Harmful Industries: 23 Years of Data From OpenSecrets.

Policy Points Our research reveals the similarities and differences among the lobbying activities of tobacco, alcohol, gambling, and ultraprocessed food industries, which are often a barrier to the implementation of public health policies. Over 23 years, we found that just six organizations dominated lobbying expenses in the tobacco and alcohol sectors, whereas the gambling sector outsourced most of their lobbying to professional firms. Databases like OpenSecrets are a useful resource to monitor the commercial determinants of health. CONTEXT: Commercial lobbying is often a barrier to the development and implementation of public health policies. Yet, little is known about the similarities and differences in the lobbying practices of different industry sectors or types of commercial actors. This study compares the lobbying practices of four industry sectors that have been the focus of much public health research and advocacy: tobacco, alcohol, gambling, and ultraprocessed foods. METHODS: Data on lobbying expenditures and lobbyist backgrounds were sourced from the OpenSecrets database, which monitors lobbying in the United States. Lobbying expenditure data were analyzed for the 1998-2020 period. We classified commercial actors as companies or trade associations. We used Power BI software to link, analyze, and visualize data sets. FINDINGS: We found that the ultraprocessed food industry spent the most on lobbying ($1.15 billion), followed by gambling ($817 million), tobacco ($755 million), and alcohol ($541 million). Overall, companies were more active than trade associations, with associations being least active in the tobacco industry. Spending was often highly concentrated, with two organizations accounting for almost 60% of tobacco spending and four organizations accounting for more than half of alcohol spending. Lobbyists that had formerly worked in government were mainly employed by third-party lobby firms. CONCLUSIONS: Our study shows how comparing the lobbying practices of different industry sectors offers a deeper appreciation of the diversity and similarities of commercial actors. Understanding these patterns can help public health actors to develop effective counterstrategies.

Nanog is the gateway to the pluripotent ground state.

Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.

Sensitivity Analysis of Upper Limb Musculoskeletal Models During Isometric and Isokinetic Tasks.

Sensitivity coefficients are used to understand how errors in subject-specific musculoskeletal model parameters influence model predictions. Previous sensitivity studies in the lower limb calculated sensitivity using perturbations that do not fully represent the diversity of the population. Hence, the present study performs sensitivity analysis in the upper limb using a large synthetic dataset to capture greater physiological diversity. The large dataset (n = 401 synthetic subjects) was created by adjusting maximum isometric force, optimal fiber length, pennation angle, and bone mass to induce atrophy, hypertrophy, osteoporosis, and osteopetrosis in two upper limb musculoskeletal models. Simulations of three isometric and two isokinetic upper limb tasks were performed using each synthetic subject to predict muscle activations. Sensitivity coefficients were calculated using three different methods (two point, linear regression, and sensitivity functions) to understand how changes in Hill-type parameters influenced predicted muscle activations. The sensitivity coefficient methods were then compared by evaluating how well the coefficients accounted for measurement uncertainty. This was done by using the sensitivity coefficients to predict the range of muscle activations given known errors in measuring musculoskeletal parameters from medical imaging. Sensitivity functions were found to best account for measurement uncertainty. Simulated muscle activations were most sensitive to optimal fiber length and maximum isometric force during upper limb tasks. Importantly, the level of sensitivity was muscle and task dependent. These findings provide a foundation for how large synthetic datasets can be applied to capture physiologically diverse populations and understand how model parameters influence predictions.