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The first synthetic genetic oscillator or ‘repressilator’ is simplified using insights from stochastic theory, thus achieving remarkably precise and robust oscillations and informing current debates about the next generation of synthetic circuits and their potential applications in cell-based therapies. The repressilator reborn The first genetic oscillator, known as the 'repressilator', was arguably a leader in the field of synthetic biology at the beginning of this century. It is, however, notoriously unreliable. Now Johan Paulsson and colleagues have succeeded in reducing error propagation and information losses in the original design, not by adding control loops, but by removing existing features, achieving remarkably precise and robust oscillations. The fact that such precise behaviour can be achieved in a relatively simple circuit, despite interaction with a noisy organism, should instruct current debates about cell-based therapies and the reliability of synthetic approaches in complex and variable environments. Synthetically engineered genetic circuits can perform a wide variety of tasks but are generally less accurate than natural systems. Here we revisit the first synthetic genetic oscillator, the repressilator 1 , and modify it using principles from stochastic chemistry in single cells. Specifically, we sought to reduce error propagation and information losses, not by adding control loops, but by simply removing existing features. We show that this modification created highly regular and robust oscillations. Furthermore, some streamlined circuits kept 14 generation periods over a range of growth conditions and kept phase for hundreds of generations in single cells, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. Our results suggest that even the simplest synthetic genetic networks can achieve a precision that rivals natural systems, and emphasize the importance of noise analyses for circuit design in synthetic biology.

Cells rely on the precise action of proteins that detect and repair DNA damage. However, gene expression noise causes fluctuations in protein abundances that may compromise repair. For the Ada protein in Escherichia coli, which induces its own expression upon repairing DNA alkylation damage, we found that undamaged cells on average produce one Ada molecule per generation. Because production is stochastic, many cells have no Ada molecules and cannot induce the damage response until the first expression event occurs, which sometimes delays the response for generations. This creates a subpopulation of cells with increased mutation rates. Nongenetic variation in protein abundances thus leads to genetic heterogeneity in the population. Our results further suggest that cells balance reliable repair against toxic side effects of abundant DNA repair proteins.

Cell fate decision circuits must be variable enough for genetically identical cells to adopt a multitude of fates, yet ensure that these states are distinct, stably maintained, and coordinated with neighboring cells. A long-standing view is that this is achieved by regulatory networks involving self-stabilizing feedback loops that convert small differences into long-lived cell types. We combined regulatory mutants and in vivo reconstitution with theory for stochastic processes to show that the marquee features of a cell fate switch in Bacillus subtilis—discrete states, multigenerational inheritance, and timing of commitments—can instead be explained by simple stochastic competition between two constitutively produced proteins that form an inactive complex. Such antagonistic interactions are commonplace in cells and could provide powerful mechanisms for cell fate determination more broadly.

Embryos must communicate instructions to their constituent cells over long distances. These instructions are often encoded in the concentration of signals called morphogens. In the textbook view, morphogen molecules diffuse from a localized source to form a concentration gradient, and target cells adopt fates by measuring the local morphogen concentration. However, natural patterning systems often incorporate numerous co-factors and extensive signaling feedback, suggesting that embryos require additional mechanisms to generate signaling patterns. Here, we examine the mechanisms of signaling pattern formation for the mesendoderm inducer Nodal during zebrafish embryogenesis. We find that Nodal signaling activity spans a normal range in the absence of signaling feedback and relay, suggesting that diffusion is sufficient for Nodal gradient formation. We further show that the range of endogenous Nodal ligands is set by the EGF-CFC co-receptor Oep: in the absence of Oep, Nodal activity spreads to form a nearly uniform distribution throughout the embryo. In turn, increasing Oep levels sensitizes cells to Nodal ligands. We recapitulate these experimental results with a computational model in which Oep regulates the diffusive spread of Nodal ligands by setting the rate of capture by target cells. This model predicts, and we confirm in vivo, the surprising observation that a failure to replenish Oep transforms the Nodal signaling gradient into a travelling wave. These results reveal that patterns of Nodal morphogen signaling are shaped by co-receptor-mediated restriction of ligand spread and sensitization of responding cells.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile. During animal development, a single fertilized cell gives rise to different tissues and organs. This ‘patterning’ process depends on signaling molecules that instruct cells in different positions in the embryo to acquire different identities. To avoid mistakes during patterning, each cell must receive the correct amount of signal at the appropriate time. In a process called ‘inhibitory feedback’, a signaling molecule instructs cells to produce molecules that block its own signaling. Although inhibitory feedback is widely used during patterning in organisms ranging from sea urchins to mammals, its exact purpose is often not clear. In part this is because feedback is challenging to experimentally manipulate. Removing the inhibitor disrupts feedback, but also increases signaling. Since the effects of broken feedback and increased signaling are intertwined, any resulting developmental defects do not provide information about what feedback specifically does. In order to examine the role of feedback, it is therefore necessary to disconnect the production of the inhibitor from the signaling process. In developing embryos, a well-known signaling molecule called Nodal instructs cells to become specific types – for example, a heart or gut cell. Nodal also promotes the production of its inhibitor, Lefty. To understand how this feedback system works, Rogers, Lord et al. first removed Lefty from zebrafish embryos. These embryos had excessive levels of Nodal signaling, did not develop correctly, and could not survive. Bathing the embryos in a drug that inhibits Nodal reduced excess signaling and allowed them to develop successfully. In these drug-treated embryos, inhibitor production is disconnected from the signaling process, allowing the role of feedback to be examined. Drug-treated embryos were less able to tolerate fluctuations in Nodal signaling than normal zebrafish embryos, which could compensate for such disturbances by adjusting Lefty levels. Overall, it appears that inhibitory feedback in this patterning system is important to compensate for alterations in Nodal signaling, but is not essential for development. Understanding the role of inhibitory feedback will be useful for efforts to grow tissues and organs in the laboratory for clinical use. The results presented by Rogers, Lord et al. also suggest the possibility that drug treatments could be developed to help correct birth defects in the womb.

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis . We show that the motile state is ‘memoryless’, exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend. This study shows that Bacillus subtilis switches from a solitary, motile lifestyle to a multicellular, sessile state in a random, memoryless fashion, but that the underlying gene network is buffered against its own stochastic variation to tightly time the reverse transition; thus bacteria keep track of time to force their progeny to cooperate during the earliest stage of multicellular growth. Cell fate determined by the click of a clock Genetically identical cells can make different cell-fate decisions in response to explicit extracellular triggers but also as a reaction to apparently stochastic or randomly generated stimuli arising within the cell. Is the underlying noise in gene expression a 'bug' or a key part of the cellular program? This study, a joint project from the labs of Richard Losick and Johan Paulsson, shows that chance has a role, at least for Bacillus subtilis bacteria. B. subtilis cells face a dramatic cell-fate decision, the transition between the solitary, motile state and the multicellular, chained state. The authors find that the bacterium switches from solitary to multicellular states according to a random, memoryless molecular mechanism, but that the underlying gene network is buffered against its own stochastic variations for the reverse transition. Thus bacteria keep track of time in order to force their progeny to cooperate with each other — a process that may inspire similarly quantitative research on development and cancer.

Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The ‘specification model’ postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the ‘migration model’ posits that Toddler signaling regulates mesendodermal cell migration downstream of Nodal signaling. Here, we test key predictions of both models. We find that in toddler mutants Nodal signaling is initially normal and increasing endoderm specification does not rescue mesendodermal cell migration. Mesodermal cell migration defects in toddler mutants result from a decrease in animal pole-directed migration and are independent of endoderm. Conversely, endodermal cell migration defects are dependent on a Cxcr4a-regulated tether of the endoderm to mesoderm. These results suggest that Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling and indirectly affects endodermal cell migration via Cxcr4a-signaling.

The advent of spatial omics has revolutionized our understanding of tissue biology; however, these technologies remain largely descriptive and do not capture how changes in gene regulation propagate across spatial neighborhoods. While perturbation methods and foundation models aim to model the impact of genetic perturbations, these methods are limited to single-cell approaches that lack spatial resolution. Other studies can delineate morphological domains based on transcriptional similarity, but not spatial functional microniches. We address this major unmet need by developing SpaceTravLR (Spatially perturbing Transcription factors, Ligands and Receptors), a novel interpretable machine learning approach that generalizes across tissues and species, uncovering spatial features linked to functional outcomes, thereby capturing functional microniches with spatial resolution. SpaceTravLR infers how single or combinatorial genetic perturbations rewire signals across the tissue neighborhood, by propagating effects through underlying spatially resolved molecular networks, thereby modeling how perturbations can reshape both the targeted cell and its surrounding neighborhood. SpaceTravLR defines novel spatial microniches across a range of tissues at different scales of organization (niches, neighborhoods and tissues), disease and developmental contexts. SpaceTravLR's perturbation predictions are made solely from spatial omics data and closely align with experimental validation or known outcomes based on mechanistic studies. Critically, our approach enables the generation of mechanistic hypotheses underlying identified niches. We show SpaceTravLR discovered a novel mechanism for that drives the spatial location of a pathogenic population of allergen-specific T helper 2 (Th2) cells as they develop in the lymph node, which was experimentally validated in a murine model. Overall, SpaceTravLR provides a novel interpretable and experimentally validated framework for uncovering how genes act individually and combinatorially through cell-intrinsic and cell-extrinsic circuits to shape spatial tissue organization and function.

Recent evidence demonstrates that eukaryotic genomes encode thousands of evolutionarily novel proteins that originate from non-coding DNA and can contribute to species-specific adaptations. Yet, it remains unclear how these incipient proteins-whose sequences are entirely new to nature-navigate the cellular environment to bring about phenotypic change. Here, we conduct a systematic investigation of yeast proteins with enhanced growth phenotypes, revealing the early stages of cellular integration. We find that these proteins are strongly enriched at the endoplasmic reticulum (ER) relative to conserved proteins, and that they integrate into cellular systems through conserved membrane targeting, trafficking, and degradation pathways. Despite having unrelated sequences, ER-localized proteins share a common molecular signature: a C-terminal transmembrane domain that likely enables recognition by conserved post-translational ER insertion pathways. After insertion, ER-localized proteins traffic from the ER and their homeostasis is regulated by conserved proteasomal and vacuolar degradation pathways. Our findings demonstrate that ancient targeting and degradation pathways can accommodate young proteins sharing a convergent molecular signature. These pathways may act as selective filters, biasing which young proteins persist.

A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creaHng designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light-sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.