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High-resolution maps of embryonic development suggest that acquisition of cell identity is not limited to canonical germ layers but proceeds via alternative routes. Despite evidence that visceral organs are formed via embryonic and extra-embryonic trajectories, the production of organ-specific cell types in vitro focuses on the embryonic one. Here we resolve these differentiation routes using massively parallel single-cell RNA sequencing to generate datasets from FOXA2

Infected cells communicate through secreted signaling molecules like cytokines, which carry information about pathogens. How differences in cytokine secretion affect inflammatory signaling over space and how responding cells decode information from propagating cytokines are not understood. By computationally and experimentally studying NF-κB dynamics in cocultures of signal-sending cells (macrophages) and signal-receiving cells (fibroblasts), we find that cytokine signals are transmitted by wave-like propagation of NF-κB activity and create well-defined activation zones in responding cells. NF-κB dynamics in responding cells can simultaneously encode information about cytokine dose, duration, and distance to the cytokine source. Spatially resolved transcriptional analysis reveals that responding cells transmit local cytokine information to distance-specific proinflammatory gene expression patterns, creating "gene expression zones." Despite single-cell variability, the size and duration of the signaling zone are tightly controlled by the macrophage secretion profile. Our results highlight how macrophages tune cytokine secretion to control signal transmission distance and how inflammatory signaling interprets these signals in space and time., Science Advances, 8 (35), ISSN:2375-2548

Significance How repressive heterochromatic states propagate along chromosomes and are subsequently maintained for many cell divisions remain important unanswered questions in biology. Combining mathematical modeling and single-cell measurements, we find that heterochromatin does not propagate in a purely linear manner as often assumed. Rather, sudden transitions can affect large domains globally at once. We suggest this is due to long-range interactions that bring distant nucleosomes close to each other to facilitate their modification. This supports the notion that the compartmentalization of chromatin components within the nucleus and the folding of chromatin into loops dynamically control heterochromatin propagation in three dimensions by bringing nucleosomes and their modifiers into close proximity., Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation.

The differentiation of ICM cells into epiblast (EPI) and primitive endoderm (PE) is central in embryonic development. It is known that FGF4 signaling is important in this process, but it remains unclear how cells can be correctly partitioned. Here we model the NANOG-GATA6-FGF4 network, and test all 64 logical regulatory combinations for their ability to partition a group of cells. We found that nearly all the logic combinations allowed for correct partitioning, including a minimal network where self-activation of NANOG and GATA6 was inactivated. However such self-activation increased the robustness of the system. Furthermore, the model also captured the reported changes in cell proportions in response to FGF perturbations. This constrains the possible regulatory logic and predicts the presence of an “OR” gate in cell-cell communication. We repeatedly found that FGF4 coordinated the decision in two phases: A convergence and a bifurcation phase. First FGF4 negative feedback drives the cells to a balanced “battle” state where most cells have intermediate levels of both regulators, thus being double positive. Subsequent bifurcation happens at constant FGF4 level. Together our results suggest that the frequently observed state of multipotency during differentiation may be an emergent phenomenon resulting from inter-cellular negative feedbacks.

Spatiotemporal balancing of cellular proliferation and differentiation is crucial for postnatal tissue homoeostasis and organogenesis. During embryonic development, pancreatic progenitors simultaneously proliferate and differentiate into the endocrine, ductal and acinar lineages. Using in vivo clonal analysis in the founder population of the pancreas here we reveal highly heterogeneous contribution of single progenitors to organ formation. While some progenitors are bona fide multipotent and contribute progeny to all major pancreatic cell lineages, we also identify numerous unipotent endocrine and ducto-endocrine bipotent clones. Single-cell transcriptional profiling at E9.5 reveals that endocrine-committed cells are molecularly distinct, whereas multipotent and bipotent progenitors do not exhibit different expression profiles. Clone size and composition support a probabilistic model of cell fate allocation and in silico simulations predict a transient wave of acinar differentiation around E11.5, while endocrine differentiation is proportionally decreased. Increased proliferative capacity of outer progenitors is further proposed to impact clonal expansion., The pancreas arises from a small population of cells but how individual cells contribute to organ formation is unclear. Here, the authors deconstruct pancreas organogenesis into clonal units, showing that single progenitors give rise to heterogeneous multi-lineage and endocrinogenic single-lineage clones.

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress”, we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress agents cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen inS. cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and co-chaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER-protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.SIGNIFICANCEApproximately one third of eukaryotic proteins are synthesized on ribosomes attached to the endoplasmic reticulum (ER) membrane. Many of these polypeptides co- or post-translationally translocate into the ER, wherein they fold and mature. An ER quality-control system proofreads these proteins by facilitating their folding and modification, while eliminating misfolded proteins through ER-associated degradation (ERAD). Yet, the fate of many secretory proteins during ER stress is not completely understood. Here, we uncovered an ER-stress induced “protein reflux” system that delivers intact, folded ER luminal proteins back to the cytosol without degrading them. We found that ER protein reflux works in parallel to ERAD and requires distinct ER-resident and cytosolic chaperones and co-chaperones.

Phase separation of soluble proteins into insoluble deposits is associated with numerous diseases. However, protein deposits can also function as membrane-less compartments for many cellular processes. What are the fitness costs and benefits of forming such deposits in different conditions? Using a model protein that phase separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast. The environmental condition and the cellular demand for the protein function emerge as key determinants of fitness. Protein deposit formation can lead to cell-to-cell differences in free protein abundance between individuals. This results in variable manifestation of protein function and a continuous range of phenotypes in a cell population, favoring survival of some individuals in certain environments. Thus, protein deposit formation by phase separation might be a mechanism to sense protein concentration in cells and to generate phenotypic variability. The selectable phenotypic variability, previously described for prions, could be a general property of proteins that can form phase separated assemblies and may influence cell fitness.Stand-first textUsing a model protein that phase separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast.Bullet pointsThe presented approach identifies and quantifies different fitness effects associated with protein deposit formation due to phase separationThe environmental condition and the cellular demand for the protein function emerge as key determinants of fitness upon protein deposit formationVariability in protein deposit formation can lead to cell-to-cell differences in free protein abundance between individualsProtein phase separation can generate a continuous range of phenotypes in a cell population

Outstanding questions in the chromatin field bear on how large heterochromatin domains are formed in space and time. Positive feedback, where histone-modifying enzymes are attracted to chromosomal regions displaying the modification they catalyse, is believed to drive the formation of these domains; however, few quantitative studies are available to assess this hypothesis. Here we quantified the de novo establishment of a naturally occurring ∼20-kb heterochromatin domain in fission yeast through single-cell analyses, measuring the kinetics of heterochromatin nucleation in a region targeted by RNAi and its subsequent expansion. We found that nucleation of heterochromatin is stochastic and can take from one to ten cell generations. Further silencing of the full region takes another one to ten generations. Quantitative modelling of the observed kinetics emphasizes the importance of local feedback, where a nucleosome-bound enzyme modifies adjacent nucleosomes, combined with a feedback where recruited enzymes can act at a distance., Chromosomes contain large heterochromatin domains. Here, the authors measure the kinetics of heterochromatin formation in fission yeast and show both global and local feedbacks by nucleosome-bound enzymes are important for formation and stability of the large heterochromatin domains.

Phase separation of soluble proteins into insoluble deposits is associated with numerous diseases. However, protein deposits can also function as membrane-less compartments for many cellular processes. What are the fitness costs and benefits of forming such deposits in different conditions? Using a model protein that phase-separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast. The environmental condition and the cellular demand for the protein function emerge as key determinants of fitness. Protein deposit formation can influence cell-to-cell variation in free protein abundance between individuals of a cell population (i.e., gene expression noise). This results in variable manifestation of protein function and a continuous range of phenotypes in a cell population, favoring survival of some individuals in certain environments. Thus, protein deposit formation by phase separation might be a mechanism to sense protein concentration in cells and to generate phenotypic variability. The selectable phenotypic variability, previously described for prions, could be a general property of proteins that can form phase-separated assemblies and may influence cell fitness. This work was supported by the Medical Research Council (MC_U105185859; M.M.B., M.T., C.R., and N.S.G.), Marie Curie Actions (FP7-PEOPLE-2012- IEF-330352, to M.T.; and FP7-PEOPLE-2011-IEF299105, to N.S.G.), FEBS LongTerm Fellowships (N.S.G.), Beatriu de Pinos fellowships (M.T.), and the Ministerio de Economía y Competitividad (SAF2017-82158-R, SAF2015-72518-EXP, and RYC-2012-09999; M.T.). N.S.G. is a recipient of the MRC Centenary Award. M.M.B. is a Lister Institute Research Prize Fellow. We thank Gian Gaetano for supporting N.S.G. and Sean Munro for the gift of strain Y03157

Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs in the absence of maternal information and in the presence of transcriptional stochasticity. How does the preimplantation embryo ensure robust, reproducible development in this context? It utilizes a versatile toolbox that includes complex intracellular networks coupled to cell—cell communication, segregation by differential adhesion, and apoptosis. Here, we ask whether a minimal set of developmental rules based on this toolbox is sufficient for successful blastocyst development, and to what extent these rules can explain mutant and experimental phenotypes. We implemented experimentally reported mechanisms for polarity, cell—cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells’ competence of fibroblast growth factor (FGF)/extracellular signal—regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network., Author summary The first 4.5 days of mammalian embryo development proceeds without maternal information and is remarkably robust to perturbations. For example, if an early embryo is cut in half, it produces 2 perfectly patterned, smaller embryos. Where does the information guiding this development come from? Here, we explore this issue and ask whether a model composed of a simple set of rules governing cell behavior and cell—cell interactions produces in silico embryos. This agent-based computational model demonstrates that 4 rules, in which a cell makes decisions based on its neighbors to adopt polarity, make lineage choices, alter its adhesion, or die, can recapitulate blastocyst development in silico. By manipulating these rules, we could also recapitulate specific phenotypes at similar frequencies to those observed in vivo. One interesting prediction of our model is that the duration of cell—cell communication through fibroblast growth factor (FGF) signaling controls scaling of a region of the blastocyst, and we confirmed this experimentally. Taken together, our model specifies a set of rules that provide a framework for self-organization, and it is this self-organizing embryogenesis that may be an enabler of stochastic variation in evolution.

All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor–ligand pair can evolve through network-altering mutations independently of receptor–ligand binding, and suggest a potential role for such mutations in disease., Co-evolution of a new receptor-ligand pair will affect the downstream signal transduction network. Here, the authors use experimental evolution of yeast mating receptor Ste2 to show the effect of enhanced binding affinity and weakened interactions with the network's negative regulators on protein evolution.

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated tocause programmed cell death. We discovered thatthioredoxin-interacting protein (TXNIP) isa critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small moleculeIRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.

Asymmetric damage segregation (ADS) is a mechanism for increasing population fitness through non-random, asymmetric partitioning of damaged macromolecules at cell division. ADS has been reported across multiple organisms, though the measured effects on fitness of individuals are often small. Here, we introduce a cell-lineage-based framework that quantifies the population-wide effects of ADS and then verify our results experimentally in E. coli under heat and antibiotic stress. Using an experimentally validated mathematical model, we find that the beneficial effect of ADS increases with stress. In effect, low-damage subpopulations divide faster and amplify within the population acting like a positive feedback loop whose strength scales with stress. Analysis of protein aggregates shows that the degree of asymmetric inheritance is damage dependent in single cells. Together our results indicate that, despite small effects in single cell, ADS exerts a strong beneficial effect on the population level and arises from the redistribution of damage within a population, through both single-cell and population-level feedback.

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality., Author Summary The state of chromatin, heterochromatin or euchromatin, affects homologous recombination in eukaryotes. We study mating-type switching in fission yeast to learn how recombination is regulated in heterochromatin. Fission yeast exists as two mating-types, P or M, determined by the allele present at the expressed mat1 locus. Genetic information for the P and M mating-types is stored in two silent heterochromatic cassettes, mat2-P and mat3-M. Cells can switch mating-type by a replication-coupled recombination event where one of the silent cassettes is used as donor to convert mat1. Mating-type switching occurs in a directional manner where mat2-P is a preferred donor in M cells and mat3-M is preferred in P cells. In this study, we investigated factors responsible for these directed recombination events. We found that two portable recombination enhancers within the heterochromatic region compete with each other and direct recombination in a cell-type specific manner. We also found that heterochromatin plays an important role in directionality by biasing competitions between the two enhancers. Our findings suggest a new model for directed recombination in a heterochromatic domain and open the field for further studies of recombination regulation in other chromatin contexts.

Heat shock response is a stress response to temperature changes and a consecutive increase in amounts of unfolded proteins. To restore homeostasis, cells upregulate chaperones facilitating protein folding by means of transcription factors (TF). We here investigate two heat shock systems: one characteristic to gram negative bacteria, mediated by transcriptional activator sigma32 in E. coli, and another characteristic to gram positive bacteria, mediated by transcriptional repressor HrcA in L. lactis. We construct simple mathematical model of the two systems focusing on the negative feedbacks, where free chaperons suppress sigma32 activation in the former, while they activate HrcA repression in the latter. We demonstrate that both systems, in spite of the difference at the TF regulation level, are capable of showing very similar heat shock dynamics. We find that differences in regulation impose distinct constrains on chaperone-TF binding affinities: the binding constant of free sigma32 to chaperon DnaK, known to be in 100 nM range, set the lower limit of amount of free chaperon that the system can sense the change at the heat shock, while the binding affinity of HrcA to chaperon GroE set the upper limit and have to be rather large extending into the micromolar range., 17 pages, 5 figures

Background Waves propagating in "excitable media" is a reliable way to transmit signals in space. A fascinating example where living cells comprise such a medium is Dictyostelium D. which propagates waves of chemoattractant to attract distant cells. While neutrophils chemotax in a similar fashion as Dictyostelium D., it is unclear if chemoattractant waves exist in mammalian tissues and what mechanisms could propagate them. Results We propose that chemoattractant cytokine waves may naturally develop as a result of NF-κ B response. Using a heuristic mathematical model of NF-κ B-like circuits coupled in space we show that the known characteristics of NF-κ B response favor cytokine waves. Conclusions While the propagating wave of cytokines is generally beneficial for inflammation resolution, our model predicts that there exist special conditions that can cause chronic inflammation and re-occurrence of acute inflammatory response.

Unfolded protein response (UPR) is a stress response to increased levels of unfolded proteins in the endoplasmic reticulum (ER). To deal with this stress, all eukaryotic cells share a well-conserved strategy--the upregulation of chaperons and proteases to facilitate protein folding and to degrade the misfolded proteins. For metazoans, however, an additional and seemingly redundant strategy has been evolved--translation attenuation (TA) of proteins targeted to the ER via the protein kinase PERK pathway. PERK is essential in secretory cells, such as the pancreatic β-cells, but not in non-secretory cell types. We have recently developed a mathematical model of UPR, focusing on the interplay and synergy between the TA arm and the conserved Ire1 arm of the UPR. The model showed that the TA mechanism is beneficial in highly fluctuating environment, for example, in the case where the ER stress changes frequently. Under highly variable levels of ER stress, tight regulation of the ER load by TA avoids excess amount of chaperons and proteases being produced. The model also showed that TA is of greater importance when there is a large flux of proteins through the ER. In this study, we further expand our model to investigate different types of ER stress and different temporal profiles of the stress. We found that TA is more desirable in dealing with the translation stress, for example, prolonged stimulation of proinsulin biosynthesis, than the chemical stress.

Endoplasmic Reticulum is a cellular organelle where membrane and extracellular proteins are folded with the help of chaperons. Insulin is one example of such extracellular proteins. Unfolded Protein Response (UPR) is a cell response to an increased level of unfolded proteins in ER. In pancreatic β-cells failure in UPR leads to accumulation of unfolded insulin in Endoplasmic reticulum and eventual cell death. This is thought to be one of the causes of type two diabetes.

Increased levels of unfolded proteins in the endoplasmic reticulum (ER) of all eukaryotes trigger the unfolded protein response (UPR). Lower eukaryotes solely use an ancient UPR mechanism, whereby they up-regulate ER-resident chaperones and other enzymatic activities to augment protein folding and enhance degradation of misfolded proteins. Metazoans have evolved an additional mechanism through which they attenuate translation of secretory pathway proteins by activating the ER protein kinase PERK. In mammalian professional secretory cells such as insulin-producing pancreatic β-cells, PERK is highly abundant and crucial for proper functioning of the secretory pathway. Through a modeling approach, we propose explanations for why a translation attenuation (TA) mechanism may be critical for β-cells, but is less important in nonsecretory cells and unnecessary in lower eukaryotes such as yeast. We compared the performance of a model UPR, both with and without a TA mechanism, by monitoring 2 variables: ( i ) the maximal increase in ER unfolded proteins during a response, and ( ii ) the accumulation of chaperones between 2 consecutive pulses of stress. We found that a TA mechanism is important for minimizing these 2 variables when the ER is repeatedly subjected to transient unfolded protein stresses and when it sustains a large flux of secretory pathway proteins which are both conditions encountered physiologically by pancreatic β-cells. Low expression of PERK in nonsecretory cells, and its absence in yeast, can be rationalized by lower trafficking of secretory proteins through their ERs.

SummaryDisruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing “ER stress” whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems.

Background Mutations accumulate as a result of DNA damage and imperfect DNA repair machinery. In higher eukaryotes the accumulation and spread of mutations is limited in two primary ways: through p53-mediated programmed cell death and cellular senescence mediated by telomeres. Telomeres shorten at every cell division and cell stops dividing once the shortest telomere reaches a critical length. It has been shown that the rate of telomere attrition is accelerated when cells are exposed to DNA damaging agents. However the implications of this mechanism are not fully understood. Results With the help of in silico model we investigate the effect of genotoxic stress on telomere attrition and apoptosis in a population of non-identical replicating cells. When comparing the populations of cells with constant vs. stress-induced rate of telomere shortening we find that stress induced telomere shortening (SITS) increases longevity while reducing mutation rate. Interestingly, however, the effect takes place only when genotoxic stresses (e.g. reactive oxygen species due to metabolic activity) are distributed non-equally among cells. Conclusions Our results for the first time show how non-equal distribution of metabolic load (and associated genotoxic stresses) combined with stress induced telomere shortening can delay aging and minimize mutations.