Morphogen gradients are used repeatedly during development to pattern embryonic tissues. Absolute concentrations, duration or even temporal derivative of morphogen concentration have all been proposed to carry positional information. However, establishing the causal relationship between the spatio-temporal profile of the gradient and the resulting cellular diversity and tissue patterning is difficult to address in live embryos because of lack of tools to take control of those variables. Here, we developed microfluidics devices able to apply spatiotemporally well-defined morphogen landscapes on the basal side of human embryonic stem cells colonies, thus mimicking how BMP4 is delivered to the pluripotent epiblast during mouse gastrulation. Combining live imaging and theoretical modelling, we show that in this configuration the absolute concentration of BMP4 provides positional information and the cell identities emerging during differentiation can vary according to the shape of the gradient. Our toolbox provides a powerful means to dissect the logic of patterning and to engineer tissues precisely. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-15-CE13-0007, ANR-23-CE13-0018 International Human Frontier Science Program Organization, CDA00063/2015-C Centre National de la Recherche Scientifique, https://ror.org/02feahw73, emergence INC2023

Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions.

Organoids generating major cortical cell types in distinct compartments are used to study cortical development, evolution and disorders. However, the lack of morphogen gradients imparting cortical positional information and topography in current systems hinders the investigation of complex phenotypes. Here, we engineer human cortical assembloids by fusing an organizer-like structure expressing fibroblast growth factor 8 (FGF8) with an elongated organoid to enable the controlled modulation of FGF8 signaling along the longitudinal organoid axis. These polarized cortical assembloids mount a position-dependent transcriptional program that in part matches the in vivo rostrocaudal gene expression patterns and that is lost upon mutation in the FGFR3 gene associated with temporal lobe malformations and intellectual disability. By producing spatially oriented cell populations with signatures related to frontal and temporal area identity within individual assembloids, this model recapitulates in part the early transcriptional divergence embedded in the protomap and enables the study of cortical area-relevant alterations underlying human disorders. Cortical development is influenced by morphogen gradients. To mimic patterning events during brain development, polarized cortical assembloids are generated with the help of a localized FGF8 source.

The primitive streak (PS) is the embryonic structure from which mesoderm and definitive endoderm cells emerge to form organs and fetal tissues. The identities of the cells leaving the PS depend on both the time and the position at which they exit. Three signaling molecules, BMP4, WNT3 and NODAL, play key roles in determining these identities, but it has been difficult to disentangle the contribution of each to this process in the mouse embryo. Using 2D gastruloids that recapitulate early aspects of mouse gastrulation in vitro, we have previously shown that WNT3 and NODAL promote distal PS fates on their own, and proximal PS fates in the presence of BMP4. We now detail further the impact of BMP4 on the cell identities produced in gastruloids. Single cell RNA-seq analysis shows that prolonged exposure to BMP4 results in the presence of all the cell types, including primordial germ cells, which normally form from the most proximal part of the pluripotent epiblast. We then show that reducing the duration or intensity of gastruloid exposure to BMP4 can allow the emergence of more distal cell identities, such as paraxial mesoderm, alongside anterior PS derivatives. These results, which are consistent with embryological studies, provide key elements for understanding how the varied output of the PS is controlled. ### Competing Interest Statement The authors have declared no competing interest.

Microbial communities are shaped by complex metabolic interactions such as cooperation and competition for resources. Methods to control such interactions could lead to major advances in our ability to better engineer microbial consortia for synthetic biology applications. Here, we use optogenetics to control SUC2 invertase production in yeast, thereby shaping spatial assortment of cooperator and cheater cells. Yeast cells behave as cooperators ( i.e ., transform sucrose into hexose, a public good) upon blue light illumination or cheaters ( i.e ., consume hexose produced by cooperators to grow) in the dark. We show that cooperators benefit best from the hexoses they produce when their domain size is constrained between two cut-off length-scales. From an engineering point of view, the system behaves as a bandpass filter. The lower limit is the trace of cheaters’ competition for hexoses, while the upper limit is defined by cooperators’ competition for sucrose. Cooperation mostly occurs at the frontiers with cheater cells, which not only compete for hexoses but also cooperate passively by letting sucrose reach cooperators. We anticipate that this optogenetic method could be applied to shape metabolic interactions in a variety of microbial ecosystems.

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.

Timelapse fluorescence microscopy imaging is routinely used in quantitative cell biology. However, microscopes could become much more powerful investigation systems if they were endowed with simple unsupervised decision-making algorithms to transform them into fully responsive and automated measurement devices. Here, we report CyberSco.Py, Python software for advanced automated timelapse experiments. We provide proof-of-principle of a user-friendly framework that increases the tunability and flexibility when setting up and running fluorescence timelapse microscopy experiments. Importantly, CyberSco.Py combines real-time image analysis with automation capability, which allows users to create conditional, event-based experiments in which the imaging acquisition parameters and the status of various devices can be changed automatically based on the image analysis. We exemplify the relevance of CyberSco.Py to cell biology using several use case experiments with budding yeast. We anticipate that CyberSco.Py could be used to address the growing need for smart microscopy systems to implement more informative quantitative cell biology experiments.

The TGF-β pathway is known to behave as a classical morphogen, meaning that it can dictate cell fate decisions in a dose-dependent manner. Recent observations however showed that in addition to the absolute value of morphogen concentration, cells could also extract information from its temporal variations. In the present article we describe how to use automated microfluidics cell culture to stimulate cells with precisely defined temporal profiles of morphogens and how to engineer mouse embryonic stem cells with fluorescent reporters of pathway activity to record in real time their response to the applied stimulations. The combination of automated cell culture and of live cell reporter provides a complete toolbox to study how cells encode the information carried by time-varying TGF-β signals.

Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the for-mation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.

During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. Progress on these issues is slowed by the difficulty to record or perturb the patterning events in real time, especially in mammalian embryos that develop in utero. Because they permit easy observation and manipulation, in vitro model systems offer an exciting opportunity to dissect the rules governing the organization of the mammalian gastrula. For instance, it is sufficient to cultivate human embryonic stem cells on micropatterned substrates to reveal their self-organization potential. We present here a method to obtain micropatterned mouse Epiblast Like Cells colonies, providing a convenient way to compare spatial organization of mouse and human pluripotent stem cells and to complement the characterization of mutant embryos in a controlled environment.

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.

The TGFβ signaling pathway is a critical regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts both as an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling.

Genetics and biochemistry have defined the components and wiring of the signaling pathways that pattern the embryo. Among them, the transforming growth factor β (TGF-β) pathway has the potential to behave as a morphogen: in vitro experiments established that it can dictate cell fate in a concentration-dependent manner. How morphogens convey positional information in a developing embryo, when signal levels change with time, is less understood. Using integrated microfluidic cell culture and time-lapse microscopy, we demonstrate here that the speed of ligand presentation has a key and previously unexpected influence on TGF-β signaling outcomes. The response to a TGF-β concentration step is transient and adaptive: slowly increasing the ligand concentration diminishes the response, and well-spaced pulses of ligand combine additively, resulting in greater pathway output than with constant stimulation. Our results suggest that in an embryonic context, the speed of change of ligand concentration is an instructive signal for patterning.

Current description of biomembrane mechanics originates for a large part from W. Helfrich's model. Based on his continuum theory, many experiments have been performed in the past four decades on simplified membranes in order to characterize the mechanical properties of lipid membranes and the contribution of polymers or proteins. The long-term goal was to develop a better understanding of the mechanical properties of cell membranes. In this paper, we will review representative experimental approaches that were developed during this period and the main results that were obtained.

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer Layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

Membrane transport between intracellular compartments, entry or exit out of the cell, imply similar sequential events: membrane deformation and lipid/protein sorting during the formation of the transport intermediate (vesicle or tube), fission from the donor compartment, transport and eventually fusion with the acceptor membrane. The mechanisms behind these biological processes of membrane transformation are actively studied both in the cell biology and the biophysics contexts. Membrane nanotubes with a controlled diameter (15-500 nm) pulled out of Giant Unilamellar Vesicles (GUV) are very convenient tools to address the role of curvature in trafficking events and to measure mechanical effects due to protein binding using optical tweezers. As an example of this type of approach combining in vitro experiments and theoretical modeling, I will present our results on two proteins implied in clathrin-mediated endocytosis. Amphiphysin 1 contains a N-BAR membrane-binding domain. We have shown that at low protein density on the GUV, the distribution of proteins and the mechanical effects induced are well described by a model based on spontaneous curvature induction. At high densities, the radius and force are independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. For the entire density range, protein was found to be enriched on the tube as compared to the GUV, showing a concomitant curvature-sensing ability. I will compare this behavior with that of another protein, the dynamin, induced the scission of the clathrin-coated vesicle in cells. In contrast, there is a threshold for the tube radius above which no binding occurs, but below which dynamin forms a scaffold constricting the tube.

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density.

In recent years, the interest for proteins that exert key functions in vesicular trafficking through their ability to sense or induce positive membrane curvature has expanded. In this chapter, we first present simple protocols to determine whether a protein targets positively curved membranes with liposomes of well-defined size. Next we describe more sophisticated approaches based on the controlled deformation of giant liposomes. These approaches allow visualization and quantification of protein binding to membrane regions of high curvature by real-time fluorescence microscopy. Last we describe several functional assays to measure how membrane curvature controls the activation state of Arf1 via ArfGAP1 or the asymmetric tethering between flat and curved membranes via the golgin GMAP-210.

The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated R-Smads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.

It has often been suggested that the high curvature of transport intermediates in cells may be a sufficient means to segregate different lipid populations based on the relative energy costs of forming bent membranes. In this review, we present in vitro experiments that highlight the essential physics of lipid sorting at thermal equilibrium: It is driven by a trade-off between bending energy, mixing entropy, and interactions between species. We collect evidence that lipid sorting depends strongly on lipid-lipid and protein-lipid interactions, and hence on the underlying composition of the membrane and on the presence of bound proteins.