Content-aware frame interpolation (CAFI): deep learning-based temporal super-resolution for fast bioimaging.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.

Collective signalling drives rapid jumping between cell states.

Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.

Clearing the slate: RNA turnover to enable cell state switching?

The distribution of mRNA in tissue is determined by the balance between transcription and decay. Understanding the control of RNA decay during development has been somewhat neglected compared with transcriptional control. Here, we explore the potential for mRNA decay to trigger rapid cell state transitions during development, comparing a bistable switch model of cell state conversion with experimental evidence from different developmental systems. We also consider another potential role for large-scale RNA decay that has emerged from studies of stress-induced cell state transitions, in which removal of mRNA unblocks the translation machinery to prioritise the synthesis of proteins that establish the new cell state.

Controlling periodic long-range signalling to drive a morphogenetic transition.

Cells use signal relay to transmit information across tissue scales. However, the production of information carried by signal relay remains poorly characterised. To determine how the coding features of signal relay are generated, we used the classic system for long-range signalling: the periodic cAMP waves that drive Dictyostelium collective migration. Combining imaging and optogenetic perturbation of cell signalling states, we find that migration is triggered by an increase in wave frequency generated at the signalling centre. Wave frequency is regulated by cAMP wave circulation, which organises the long-range signal. To determine the mechanisms modulating wave circulation, we combined mathematical modelling, the general theory of excitable media, and mechanical perturbations to test competing models. Models in which cell density and spatial patterning modulate the wave frequency cannot explain the temporal evolution of signalling waves. Instead, our evidence leads to a model where wave circulation increases the ability for cells to relay the signal, causing further increase in the circulation rate. This positive feedback between cell state and signalling pattern regulates the long-range signal coding that drives morphogenesis.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Distributed and centralized control during differentiation.

A persistent view of cell fate choices during development entails centralized control by so-called master regulators. A recent single-cell study of the large-scale fate specification during mammalian gastrulation (Mittnenzweig et al., 2021) implies the prevalence of more distributed forms of control.

Cell and molecular transitions during efficient dedifferentiation.

Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing Dictyostelium cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.

What Is a Transcriptional Burst?

The idea that gene activity can be discontinuous will not surprise many biologists - many genes are restricted in when and where they can be expressed. Yet during the past 15 years, a collection of observations compiled under the umbrella term 'transcriptional bursting' has received considerable interest. Direct visualization of the dynamics of discontinuous transcription has expanded our understanding of basic transcriptional mechanisms and their regulation and provides a real-time readout of gene activity during the life of a cell. In this review, we try to reconcile the different views of the transcriptional process emerging from studies of bursting, and how this work contextualizes the relative importance of different regulatory inputs to normal dynamic ranges of gene activity.

Live imaging of ERK signalling dynamics in differentiating mouse embryonic stem cells.

Stimulation of the ERK/MAPK pathway is required for the exit from pluripotency and onset of differentiation in mouse embryonic stem cells (ESCs). The dynamic behaviour of ERK activity in individual cells during this transition is unclear. Using a FRET-based biosensor, we monitored ERK signalling dynamics of single mouse ESCs during differentiation. ERK activity was highly heterogeneous, with considerable variability in ERK signalling between single cells within ESC colonies. Different triggers of differentiation induced distinct ERK activity profiles. Surprisingly, the dynamic features of ERK signalling were not strongly coupled to loss of pluripotency marker expression, regardless of the differentiation stimulus, suggesting the normal dynamic range of ERK signalling is not rate-limiting in single cells during differentiation. ERK signalling dynamics were sensitive to the degree of cell crowding and were similar in neighbouring cells. Sister cells from a mitotic division also showed more similar ERK activity, an effect that was apparent whether cells remained adjacent or moved apart after division. These data suggest a combination of cell lineage and niche contributes to the absolute level of ERK signalling in mouse ESCs.

The fate of cells undergoing spontaneous DNA damage during development.

Embryonic development involves extensive and often rapid cell proliferation. An unavoidable side effect of cell proliferation is DNA damage. The consequences of spontaneous DNA damage during development are not clear. Here, we define an approach to determine the effects of DNA damage on cell fate choice. Using single cell transcriptomics, we identified a subpopulation of Dictyostelium cells experiencing spontaneous DNA damage. Damaged cells displayed high expression of rad51, with the gene induced by multiple types of genotoxic stress. Using live imaging, we tracked high Rad51 cells from differentiation onset until cell fate assignment. High Rad51 cells were shed from multicellular structures, excluding damaged cells from the spore population. Cell shedding resulted from impaired cell motility and defective cell-cell adhesion, with damaged cells additionally defective in activation of spore gene expression. These data indicate DNA damage is not insulated from other aspects of cell physiology during development and multiple features of damaged cells prevent propagation of genetic error. Our approach is generally applicable for monitoring rare subpopulations during development, and permits analysis of developmental perturbations occurring within a physiological dynamic range.

Transition state dynamics during a stochastic fate choice.

The generation of multiple fates from a uniform cell population via self-organisation is a recurring feature in development and regeneration. However, for most self-organising systems, we have little understanding of the processes that allow cells to become different. One of the clearest examples of developmental self-organisation is shown by Dictyostelium, with cells segregating into two major fates, stalk and spore, within multicellular aggregates. To characterise the gene expression decisions that underlie this cell fate bifurcation, we carried out single cell transcriptomics on Dictyostelium aggregates. Our data show the transition of progenitors into prespore and prestalk cells occurs via distinct developmental intermediates. Few cells were captured switching between states, with minimal overlap in fate marker expression between cell types, suggesting states are discrete and transitions rapid. Surprisingly, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

The Atypical MAP Kinase ErkB Transmits Distinct Chemotactic Signals through a Core Signaling Module.

Signaling from chemoattractant receptors activates the cytoskeleton of crawling cells for chemotaxis. We show using phosphoproteomics that different chemoattractants cause phosphorylation of the same core set of around 80 proteins in Dictyostelium cells. Strikingly, the majority of these are phosphorylated at an [S/T]PR motif by the atypical MAP kinase ErkB. Unlike most chemotactic responses, ErkB phosphorylations are persistent and do not adapt to sustained stimulation with chemoattractant. ErkB integrates dynamic autophosphorylation with chemotactic signaling through G-protein-coupled receptors. Downstream, our phosphoproteomics data define a broad panel of regulators of chemotaxis. Surprisingly, targets are almost exclusively other signaling proteins, rather than cytoskeletal components, revealing ErkB as a regulator of regulators rather than acting directly on the motility machinery. ErkB null cells migrate slowly and orientate poorly over broad dynamic ranges of chemoattractant. Our data indicate a central role for ErkB and its substrates in directing chemotaxis.

Promoter-mediated diversification of transcriptional bursting dynamics following gene duplication.

During the evolution of gene families, functional diversification of proteins often follows gene duplication. However, many gene families expand while preserving protein sequence. Why do cells maintain multiple copies of the same gene? Here we have addressed this question for an actin family with 17 genes encoding an identical protein. The genes have divergent flanking regions and are scattered throughout the genome. Surprisingly, almost the entire family showed similar developmental expression profiles, with their expression also strongly coupled in single cells. Using live cell imaging, we show that differences in gene expression were apparent over shorter timescales, with family members displaying different transcriptional bursting dynamics. Strong "bursty" behaviors contrasted steady, more continuous activity, indicating different regulatory inputs to individual actin genes. To determine the sources of these different dynamic behaviors, we reciprocally exchanged the upstream regulatory regions of gene family members. This revealed that dynamic transcriptional behavior is directly instructed by upstream sequence, rather than features specific to genomic context. A residual minor contribution of genomic context modulates the gene OFF rate. Our data suggest promoter diversification following gene duplication could expand the range of stimuli that regulate the expression of essential genes. These observations contextualize the significance of transcriptional bursting.

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.

Symmetry breaking in development and stochastic gene expression.

The prevailing emphasis in developmental biology since the expansion of the molecular biology age has been that developmental decisions are instructive. A cell differentiates to become a specific cell type because it receives a signal, whereas its neighbor that does not receive the signal adopts a different fate. This emphasis has been generally accepted, largely because of the success of this view in tractable invertebrate model organisms, and the widespread similarities in molecular regulation to the development of more complex species. An alternative emphasis, that cells make their own decisions, has until the past decade been conspicuously silent. Here I trace the re-emergence of our appreciation of single cell decision-making in development, and how widespread this phenomenon is likely to be. I will focus the discussion on the potential role of stochastic gene expression in generating differences between cells in the absence of simple instructive signals and highlight the complexity of systems proposed to involve this type of regulation. Finally, I will discuss the approaches required to fully test hypotheses that noisy gene regulation can be extrapolated through developmental time to accurately specify cell fate. WIREs Dev Biol 2017, 6:e284. doi: 10.1002/wdev.284 For further resources related to this article, please visit the WIREs website.

Generation of Single-Cell Transcript Variability by Repression.

Gene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation.

A continuum model of transcriptional bursting.

Transcription occurs in stochastic bursts. Early models based upon RNA hybridisation studies suggest bursting dynamics arise from alternating inactive and permissive states. Here we investigate bursting mechanism in live cells by quantitative imaging of actin gene transcription, combined with molecular genetics, stochastic simulation and probabilistic modelling. In contrast to early models, our data indicate a continuum of transcriptional states, with a slowly fluctuating initiation rate converting the gene between different levels of activity, interspersed with extended periods of inactivity. We place an upper limit of 40 s on the lifetime of fluctuations in elongation rate, with initiation rate variations persisting an order of magnitude longer. TATA mutations reduce the accessibility of high activity states, leaving the lifetime of on- and off-states unchanged. A continuum or spectrum of gene states potentially enables a wide dynamic range for cell responses to stimuli.

Gene Regulation: Stable Noise.

Transcriptional regulation is noisy, yet despite this variability, embryonic development reproducibly generates form and function. Recent work demonstrates that patterns of transcriptional activity in embryos are stably inherited through mitosis. These observations have implications for how accuracy arises in development.