Universal calcium fluctuations inHydramorphogenesis.

Understanding the collective physical processes that drive robust morphological transitions in animal development necessitates the characterization of the relevant fields involved in morphogenesis. Calcium (Ca2+) is recognized as one such field. In this study, we demonstrate that the spatial fluctuations of Ca2+duringHydraregeneration exhibit universal characteristics. To investigate this phenomenon, we employ two distinct controls, an external electric field andheptanol, a gap junction-blocking drug. Both lead to the modulation of the Ca2+activity and a reversible halting of the regeneration process. The application of an electric field enhances Ca2+activity in theHydra's tissue and increases its spatial correlations, while the administration ofheptanolinhibits its activity and diminishes the spatial correlations. Remarkably, the statistical characteristics of Ca2+spatial fluctuations, including the coefficient of variation and skewness, manifest universal shape distributions across tissue samples and conditions. We introduce a field-theoretic model, describing fluctuations in a tilted double-well potential, which successfully captures these universal properties. Moreover, our analysis reveals that the Ca2+activity is spatially localized, and theHydra's tissue operates near the onset of bistability, where the local Ca2+activity fluctuates between low and high excited states in distinct regions. These findings highlight the prominent role of the Ca2+field inHydramorphogenesis and provide insights into the underlying mechanisms governing robust morphological transitions.

Metabolic cost of rapid adaptation of single yeast cells.

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.

Hydra Regeneration: Closing the Loop with Mechanical Processes in Morphogenesis.

The convergence of morphogenesis into viable organisms under variable conditions suggests closed-loop dynamics involving multiscale functional feedback. We develop the idea that morphogenesis is based on synergy between mechanical and bio-signaling processes, spanning all levels of organization: molecular, cellular, tissue, up to the whole organism. This synergy provides feedback within and between all levels of organization, to close the loop between the dynamics of the morphogenesis process and its robust functional outcome. Hydra offer a powerful platform to explore this direction, thanks to their simple body plan, extraordinary regeneration capabilities, and the accessibility and flexibility of their tissues. Our recent experiments show that structural inheritance of the actomyosin organization directs body-axis formation during Hydra regeneration. Morphogenesis is then stabilized through dynamic cytoskeletal reorganization induced by the inherited structure. The observed cytoskeletal stability and reorganization capabilities suggest that mechanical feedback integrates with biochemical processes to establish viable patterns and ensure canalization.

Electric-Induced Reversal of Morphogenesis in Hydra.

Morphogenesis involves the dynamic interplay of biochemical, mechanical, and electrical processes. Here, we ask to what extent can the course of morphogenesis be modulated and controlled by an external electric field? We show that at a critical amplitude, an external electric field can halt morphogenesis in Hydra regeneration. Moreover, above this critical amplitude, the electric field can lead to reversal dynamics: a fully developed Hydra folds back into its incipient spheroid morphology. The potential to renew morphogenesis is reexposed when the field is reduced back to amplitudes below criticality. These dynamics are accompanied by modulations of the Wnt3 activity, a central component of the head organizer in Hydra. The controlled backward-forward cycle of morphogenesis can be repeated several times, showing that the reversal trajectory maintains the integrity of the tissue and its regeneration capability. Each cycle of morphogenesis leads to a newly emerged body plan in the redeveloped folded tissue, which is not necessarily similar to the one before the reversal process. Reversal of morphogenesis is shown to be triggered by enhanced electrical excitations in the Hydra tissue, leading to intensified calcium activity. Folding back of the body-plan morphology together with the decay of a central biosignaling system, indicate that electrical processes are tightly integrated with biochemical and mechanical-structural processes in morphogenesis and play an instructive role to a level that can direct developmental trajectories. Reversal of morphogenesis by external fields calls for extending its framework beyond program-like, forward-driven, hierarchical processes based on reaction diffusion and positional information.

The unforeseen challenge: from genotype-to-phenotype in cell populations.

Biological cells present a paradox, in that they show simultaneous stability and flexibility, allowing them to adapt to new environments and to evolve over time. The emergence of stable cell states depends on genotype-to-phenotype associations, which essentially reflect the organization of gene regulatory modes. The view taken here is that cell-state organization is a dynamical process in which the molecular disorder manifests itself in a macroscopic order. The genome does not determine the ordered cell state; rather, it participates in this process by providing a set of constraints on the spectrum of regulatory modes, analogous to boundary conditions in physical dynamical systems. We have developed an experimental framework, in which cell populations are exposed to unforeseen challenges; novel perturbations they had not encountered before along their evolutionary history. This approach allows an unbiased view of cell dynamics, uncovering the potential of cells to evolve and develop adapted stable states. In the last decade, our experiments have revealed a coherent set of observations within this framework, painting a picture of the living cell that in many ways is not aligned with the conventional one. Of particular importance here, is our finding that adaptation of cell-state organization is essentially an efficient exploratory dynamical process rather than one founded on random mutations. Based on our framework, a set of concepts underlying cell-state organization-exploration evolving by global, non-specific, dynamics of gene activity-is presented here. These concepts have significant consequences for our understanding of the emergence and stabilization of a cell phenotype in diverse biological contexts. Their implications are discussed for three major areas of biological inquiry: evolution, cell differentiation and cancer. There is currently no unified theoretical framework encompassing the emergence of order, a stable state, in the living cell. Hopefully, the integrated picture described here will provide a modest contribution towards a physics theory of the cell.

Single-cell protein dynamics reproduce universal fluctuations in cell populations.

Protein variability in single cells has been studied extensively in populations, but little is known about temporal protein fluctuations in a single cell over extended times. We present here traces of protein copy number measured in individual bacteria over multiple generations and investigate their statistical properties, comparing them to previously measured population snapshots. We find that temporal fluctuations in individual cells exhibit the same properties as those previously observed in populations. Scaled fluctuations around the mean of each trace exhibit the universal distribution shape measured in populations under a wide range of conditions and in two distinct microorganisms; the mean and variance of the traces over time obey the same quadratic relation. Analyzing the individual protein traces reveals that within a cell cycle protein content increases exponentially, with a rate that varies from cycle to cycle. This leads to a compact description of the trace as a 3-variable stochastic process -exponential rate, cell cycle duration and value at the cycle start- sampled once a cycle. This description is sufficient to reproduce both universal statistical properties of the protein fluctuations. Our results show that the protein distribution shape is insensitive to sub-cycle intracellular microscopic details and reflects global cellular properties that fluctuate between generations.

Multiple genomic changes associated with reorganization of gene regulation and adaptation in yeast.

Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.

Dynamics of the cell-cycle network under genome-rewiring perturbations.

The cell-cycle progression is regulated by a specific network enabling its ordered dynamics. Recent experiments supported by computational models have shown that a core of genes ensures this robust cycle dynamics. However, much less is known about the direct interaction of the cell-cycle regulators with genes outside of the cell-cycle network, in particular those of the metabolic system. Following our recent experimental work, we present here a model focusing on the dynamics of the cell-cycle core network under rewiring perturbations. Rewiring is achieved by placing an essential metabolic gene exclusively under the regulation of a cell-cycle's promoter, forcing the cell-cycle network to function under a multitasking challenging condition; operating in parallel the cell-cycle progression and a metabolic essential gene. Our model relies on simple rate equations that capture the dynamics of the relevant protein-DNA and protein-protein interactions, while making a clear distinction between these two different types of processes. In particular, we treat the cell-cycle transcription factors as limited 'resources' and focus on the redistribution of resources in the network during its dynamics. This elucidates the sensitivity of its various nodes to rewiring interactions. The basic model produces the correct cycle dynamics for a wide range of parameters. The simplicity of the model enables us to study the interface between the cell-cycle regulation and other cellular processes. Rewiring a promoter of the network to regulate a foreign gene, forces a multitasking regulatory load. The higher the load on the promoter, the longer is the cell-cycle period. Moreover, in agreement with our experimental results, the model shows that different nodes of the network exhibit variable susceptibilities to the rewiring perturbations. Our model suggests that the topology of the cell-cycle core network ensures its plasticity and flexible interface with other cellular processes, without a need for an optimal setting of the kinetic parameters.

Universal protein fluctuations in populations of microorganisms.

The copy number of any protein fluctuates among cells in a population; characterizing and understanding these fluctuations is a fundamental problem in biophysics. We show here that protein distributions measured under a broad range of biological realizations collapse to a single non-gaussian curve under scaling by the first two moments. Moreover, in all experiments the variance is found to depend quadratically on the mean, showing that a single degree of freedom determines the entire distribution. Our results imply that protein fluctuations do not reflect any specific molecular or cellular mechanism, and suggest that some buffering process masks these details and induces universality.

Canalized Morphogenesis Driven by Inherited Tissue Asymmetries in Hydra Regeneration.

The emergence and stabilization of a body axis is a major step in animal morphogenesis, determining the symmetry of the body plan as well as its polarity. To advance our understanding of the emergence of body axis polarity, we study regenerating Hydra. Axis polarity is strongly memorized in Hydra regeneration even in small tissue segments. What type of processes confer this memory? To gain insight into the emerging polarity, we utilize frustrating initial conditions by studying regenerating tissue strips which fold into hollow spheroids by adhering their distal ends of opposite original polarities. Despite the convoluted folding process and the tissue rearrangements during regeneration, these tissue strips develop in a reproducible manner, preserving the original polarity and yielding an ordered body plan. These observations suggest that the integration of mechanical and biochemical processes supported by their mutual feedback attracts the tissue dynamics towards a well-defined developmental trajectory biased by weak inherited cues from the parent animal. Hydra thus provide an example of dynamic canalization in which the dynamic rules are instilled, but, in contrast to the classical picture, the detailed developmental trajectory does not unfold in a programmatic manner.

Plasticity of body axis polarity in Hydra regeneration under constraints.

One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to explore the plasticity of polarity determination during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the polarity and original positions of the rings along the body axes of the parent animals. Under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. Importantly, the site of head regeneration exhibits a strong bias toward the edges of the tissue, even when this involves polarity reversal. In particular, we observe head formation at an originally aboral tissue edge, which is not compatible with models of Hydra regeneration based only on preexisting morphogen gradients or an injury response. The site of the new head invariably contains an aster-like defect in the organization of the supra-cellular ectodermal actin fibers. While a defect is neither required nor sufficient for head formation, we show that the defect at the new head site can arise via different routes, either appearing directly following excision as the tissue seals at its edge or through de novo defect formation at the fusion site. Altogether, our results show that the emergence of a polar body axis depends on the original polarity and position of the excised tissues as well as structural factors, suggesting that axis determination is an integrated process that arises from the dynamic interplay of multiple biochemical and mechanical processes.

Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge.

Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be 'pre-designed' to lead to an adaptive metabolic state on physiological timescales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3, from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge to which they adapted over approximately 10 generations. Using genome-wide expression arrays, we show here that a global transcriptional reprogramming (>1200 genes) underlies the adaptation. A large fraction of the responding genes is nonreproducible in repeated experiments. These results show that a nonspecific transcriptional response reflecting the natural plasticity of the regulatory network supports adaptation of cells to novel challenges.

Synthetic gene recruitment reveals adaptive reprogramming of gene regulation in yeast.

The recruitment of a gene to a foreign regulatory system is a major evolutionary event that can lead to novel phenotypes. However, the evolvability potential of cells depends on their ability to cope with challenges presented by gene recruitment. To study this ability, we combined synthetic gene recruitment with continuous culture and online measurements of the metabolic and regulatory dynamics over long timescales. The gene HIS3 from the histidine synthesis pathway was recruited to the GAL system, responsible for galactose utilization in the yeast S. cerevisiae. Following a switch from galactose to glucose--from induced to repressed conditions of the GAL system--in histidine-lacking chemostats (where the recruited HIS3 is essential), the regulatory system reprogrammed to adaptively tune HIS3 expression, allowing the cells to grow competitively in pure glucose. The adapted state was maintained for hundreds of generations in various environments. The timescales involved and the reproducibility of separate experiments render spontaneous mutations an unlikely underlying mechanism. Essentially all cells could adapt, excluding selection over a genetically variable population. The results reveal heritable adaptation induced by the exposure to glucose. They demonstrate that genetic regulatory networks have the potential to support highly demanding events of gene recruitment.

Structural Inheritance of the Actin Cytoskeletal Organization Determines the Body Axis in Regenerating Hydra.

Understanding how mechanics complement bio-signaling in defining patterns during morphogenesis is an outstanding challenge. Here, we utilize the multicellular polyp Hydra to investigate the role of the actomyosin cytoskeleton in morphogenesis. We find that the supra-cellular actin fiber organization is inherited from the parent Hydra and determines the body axis in regenerating tissue segments. This form of structural inheritance is non-trivial because of the tissue folding and dynamic actin reorganization involved. We further show that the emergence of multiple body axes can be traced to discrepancies in actin fiber alignment at early stages of the regeneration process. Mechanical constraints induced by anchoring regenerating Hydra on stiff wires suppressed the emergence of multiple body axes, highlighting the importance of mechanical feedbacks in defining and stabilizing the body axis. Together, these results constitute an important step toward the development of an integrated view of morphogenesis that incorporates mechanics.

Dynamics of protein distributions in cell populations.

A population of cells exhibits wide phenotypic variation even if it is genetically homogeneous. In particular, individual cells differ from one another in the amount of protein they express under a given regulatory system under fixed conditions. Here we study how protein distributions in a population of the yeast S. cerevisiae are shaped by a balance of processes: protein production--an intracellular process--and protein dilution due to cell division--a population process. We measure protein distributions by employing reporter green fluorescence protein (gfp) under the regulation of the yeast GAL system under conditions where it is metabolically essential. Cell populations are grown in chemostats, thus allowing control of the environment and stable measurements of distribution dynamics over many generations. Despite the essential functional role of the GAL system in a pure galactose medium, steady-state distributions are found to be universally broad, with exponential tails and a large standard-deviation-to-mean ratio. Under several different perturbations the dynamics of the distribution is observed to be asymmetric, with a much longer time to build a wide expression distribution from below compared with a fast relaxation of the distribution toward steady state from above. These results show that the main features of the protein distributions are largely determined by population effects and are less sensitive to the intracellular biochemical noise.

Universality, complexity and the praxis of biology: Two case studies.

The phenomenon of biology provides a prime example for a naturally occurring complex system. The approach to this complexity reflects the tension between a reductionist, reverse-engineering stance, and more abstract, systemic ones. Both of us are reductionists, but our observations challenge reductionism, at least the naive version of it. Here we describe the challenge, focusing on two universal characteristics of biological complexity: two-way microscopic-macroscopic degeneracy, and lack of time scale separation within and between levels of organization. These two features and their consequences for the praxis of experimental biology, reflect inherent difficulties in separating the dynamics of any given level of organization from the coupled dynamics of all other levels, including the environment within which the system is embedded. Where these difficulties are not deeply acknowledged, the impacts of fallacies that are inherent to naive reductionism are significant. In an era where technology enables experimental high-resolution access to numerous observables, the challenge faced by the mature reductionist-identification of relevant microscopic variables-becomes more demanding than ever. The demonstrations provided here are taken from two very different biological realizations: populations of microorganisms and populations of neurons, thus making the lesson potentially general.

Induced mutations in yeast cell populations adapting to an unforeseen challenge.

The modern evolutionary synthesis assumes that mutations occur at random, independently of the environment in which they confer an advantage. However, there are indications that cells facing challenging conditions can adapt rapidly, utilizing processes beyond selection of pre-existing genetic variation. Here, we show that a strong regulatory challenge can induce mutations in many independent yeast cells, in the absence of general mutagenesis. Whole genome sequencing of cell lineages reveals a repertoire of independent mutations within a single lineage that arose only after the cells were exposed to the challenging environment, while other cells in the same lineage adapted without any mutation in their genomes. Thus, our experiments uncovered multiple alternative routes for heritable adaptation that were all induced in the same lineage during a short time period. Our results demonstrate the existence of adaptation mechanisms beyond random mutation, suggesting a tight connection between physiological and genetic processes.

Population dynamics of metastable growth-rate phenotypes.

Neo-Darwinian evolution has presented a paradigm for population dynamics built on random mutations and selection with a clear separation of time-scales between single-cell mutation rates and the rate of reproduction. Laboratory experiments on evolving populations until now have concentrated on the fixation of beneficial mutations. Following the Darwinian paradigm, these experiments probed populations at low temporal resolution dictated by the rate of rare mutations, ignoring the intermediate evolving phenotypes. Selection however, works on phenotypes rather than genotypes. Research in recent years has uncovered the complexity of genotype-to-phenotype transformation and a wealth of intracellular processes including epigenetic inheritance, which operate on a wide range of time-scales. Here, by studying the adaptation dynamics of genetically rewired yeast cells, we show a novel type of population dynamics in which the intracellular processes intervene in shaping the population structure. Under constant environmental conditions, we measure a wide distribution of growth rates that coexist in the population for very long durations (>100 generations). Remarkably, the fastest growing cells do not take over the population on the time-scale dictated by the width of the growth-rate distributions and simple selection. Additionally, we measure significant fluctuations in the population distribution of various phenotypes: the fraction of exponentially-growing cells, the distributions of single-cell growth-rates and protein content. The observed fluctuations relax on time-scales of many generations and thus do not reflect noisy processes. Rather, our data show that the phenotypic state of the cells, including the growth-rate, for large populations in a constant environment is metastable and varies on time-scales that reflect the importance of long-term intracellular processes in shaping the population structure. This lack of time-scale separation between the intracellular and population processes calls for a new framework for population dynamics which is likely to be significant in a wide range of biological contexts, from evolution to cancer.

Cellular plasticity enables adaptation to unforeseen cell-cycle rewiring challenges.

The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is the emergence of a yet-unknown general, non-specific mechanism allowing fast inherited adaptation to unforeseen challenges.

Epigenetically heritable alteration of fly development in response to toxic challenge.

Developing organisms have evolved a wide range of mechanisms for coping with recurrent environmental challenges. How they cope with rare or unforeseen challenges is, however, unclear as are the implications to their unchallenged offspring. Here, we investigate these questions by confronting the development of the fly, D. melanogaster, with artificial tissue distributions of toxic stress that are not expected to occur during fly development. We show that under a wide range of toxic scenarios, this challenge can lead to modified development that may coincide with increased tolerance to an otherwise lethal condition. Part of this response was mediated by suppression of Polycomb group genes, which in turn leads to derepression of developmental regulators and their expression in new domains. Importantly, some of the developmental alterations were epigenetically inherited by subsequent generations of unchallenged offspring. These results show that the environment can induce alternative patterns of development that are stable across multiple generations.