Biology subjects

Fletcher, A. G.

Publications and source records attributed to Fletcher, A. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Widespread coupling of promoters and terminators of transcription in bacteria

Transcription underpins the expression of genetic information and is viewed as a series of independent initiation and termination events. In bacteria, promoters and terminators of transcription are therefore considered distinct regulatory elements. Here, we challenge this view. Genome-scale analyses reveal that promoters and terminators are extensively coupled. Thus, initiation and termination sites, for adjacent transcription units, frequently overlap. This conserved organisation arises because the DNA sequences, which direct termination, also contribute to promoter function. This couples neighbouring transcription units and generates regulatory interference between RNA polymerases. These findings define a universal mechanism for coordinating transcription across bacterial genomes.

microbiology↗

bioRxiv2026

Notch signalling governs human enteric nervous system progenitor dynamics

The enteric nervous system (ENS) is the main branch of the peripheral nervous system that innervates the gastrointestinal tract controlling vital functions. It arises during embryogenesis via migration, proliferation and differentiation of neural crest-derived ENS progenitors. Perturbation of these processes, caused by mutations in key signalling pathway components and transcription factors, prevents progenitor colonisation of the distal gut causing aganglionic phenotypes and enteric neuropathies such as Hirschsprung (HSCR) disease. While animal models implicate Notch signalling in ENS specification, its role in human ENS progenitor cell fate decisions remains unclear. Here, we employ a human pluripotent stem cell-based model to show that Notch signalling is a key regulator of human ENS progenitor dynamics. Quantitative modelling of our in vitro data indicates that Notch inhibition accelerates progenitor differentiation without substantially altering neuronal versus glial lineage bias. Furthermore, we establish that Notch signalling controls human ENS progenitor migration by influencing migration velocity and directionality. Together, these findings provide mechanistic insights into how Notch signalling disruption may contribute to the pathogenesis of human intestinal aganglionosis.

developmental biology↗

bioRxiv2026

Quantification and statistical comparison of cell-state transition kinetics using a parametric failure-based model

Successful development of multicellular organisms requires cells to transition between states with precise timing. Distinct cell states are often understood as being maintained by stabilizing regulatory networks, such that a complete cell-state transition requires network rewiring through partial dismantling of the current state and concurrent reconfiguration into a new one. Empirically, these transitions are often investigated by quantifying the gain or loss of expression of a small number of state-specific markers, frequently a single proxy. A general quantitative framework for comparing the kinetics of such transitions across experimental conditions is lacking. Here, we show that the delayed Weibull distribution provides a natural description of cell-state transition kinetics when transition is viewed as the cumulative consequence of many molecular events, whose timing may vary between cells and conditions, analogous to system failure in reliability theory. This formulation yields a compact model with interpretable parameters describing the delay before transition onset, the characteristic timescale of transition, the temporal form of the transition hazard, and the fraction of cells competent to respond. Together, this framework provides a practical and interpretable approach for quantifying the kinetics of cell-state transitions and how they are altered by perturbation.

systems biology↗

bioRxiv2026

GPU acceleration of cell-based simulations in Chaste using FLAME GPU 2

Chaste is an open-source C++ library providing a general-purpose framework for cell-based simulations of biological tissues. It has been applied to a wider range of biological processes, including morphogenesis, carcinogenesis, and wound healing. Such simulations often involve numerous mechanical interactions between neighbouring cells, making them computationally demanding. Graphical Processing Units (GPUs), with their highly parallel architectures, offer a powerful means to accelerate these computations, enabling larger, more detailed simulations and improving research productivity. FLAME GPU 2 is a GPU-accelerated simulator for domain-independent complex systems that maps formal agent descriptions written in scripting language to optimized CUDA code. In this work, FLAME GPU 2 is integrated with Chaste to accelerate force calculations in a class of cell-based simulations, demonstrating the feasibility of GPU acceleration within existing CPU-based frameworks. The GPU-accelerated implementation is validated against the original CPU version, achieving up to 93.6x speedup in force calculations and 3.72x speedup for full simulations across various cell population sizes. Moreover, the approach enables smaller mechanics time steps, without incurring significant data transfer overhead, thereby improving the accuracy of mechanical modelling. This enhancement increases the fidelity of cell position calculations in non-equilibrium simulations and improves dynamic accuracy as cells approach equilibrium.

cell biology↗

bioRxiv2025

Beyond pulling: microtubule pushing forces contribute to robust spindle orientation in regular and irregular cell shapes

Oriented cell division is fundamental to development and tissue organization, requiring precise control of both spindle positioning and orientation. While cortical pulling forces mediated by dynein motor proteins are well-established drivers of spindle dynamics, the contribution of microtubule polymerization-based pushing forces remains unclear. We developed a generalizable computational biophysical model that integrates both pulling and pushing mechanisms to investigate spindle behavior across diverse cell types and geometries. This model successfully recapitulates experimental observations in three well-studied models: Drosophila follicular epithelial cells, Drosophila neuroblasts, and the early C. elegans embryo. Systematic analysis reveals that while pulling forces are the primary drivers of directed spindle orientation, pushing forces play crucial supporting roles by preventing spindle stalling and promoting alignment dynamics, particularly at high initial misalignment angles. We further applied our model to irregularly shaped zebrafish endothelial cells, which present unique challenges due to their non-spherical morphology and dynamic shape changes during mitosis. Our results demonstrate that asymmetric cortical force generator distributions, potentially localized at cell-cell junctions, can account for the observed off-center spindle positioning in these cells. This work provides a unified framework for understanding how the interplay between cell geometry, molecular polarity cues, and competing physical forces determines spindle dynamics, offering new insights into both canonical and non-canonical division orientations across cell types.

biophysics↗

bioRxiv2025

Open Benchmarking for Cell-Based Multiscale Models: Lessons from a Community Initiative

The emergence of virtual human twins (VHT) in biomedical research has sparked interest in multiscale in silico modelling frameworks, particularly in their application bridging cellular to tissue levels. Among the diverse array of multiscale modelling tools, off-lattice center-based agent-based models (CBM) offer a promising approach due to their depiction of cells in 3D space, closely resembling biological reality. Despite the proliferation of CBM tools addressing various biomedical challenges, a comprehensive and systematic comparison among them has been elusive. This paper presents a community-driven benchmark initiative aimed at evaluating and comparing CBM for biomedical applications, akin to successful efforts in other scientific domains such as the Critical Assessment of Protein Structure Prediction (CASP). Enlisting developers from leading tools like BioDynaMo, Chaste, PhysiCell,TiSim, and CompuTiX, we devised a benchmark scope, defined metrics, and established reference datasets to ensure a meaningful and equitable evaluation. Unit tests targeting different solvers within these tools were designed, ranging from diffusion and mechanics to cell cycle simulations and growth scenarios. Results from these tests demonstrate varying tool implementations in handling diffusion, mechanics, and cell cycle equations, emphasising the need for standardised benchmarks and interoperability. Discussions among the community underscore the necessity for defining gold standards, fostering interoperability, and drawing lessons from analogous benchmarking experiences. The outcomes, disseminated through a public platform in collaboration with OpenEBench, aim to catalyse advancements in computational biology, offering a comprehensive resource for tool evaluation and guiding future developments in cell-level simulations. This initiative endeavours to strengthen and expand the computational biology simulation community through continued dissemination and performance-oriented benchmarking efforts to enable the use of VHT in biomedicine.

systems biology↗

bioRxiv2024

Donor embryonic stem cells impede host epiblast specification in 8-cell stage chimeras by crowding and FGF4 signalling

During mouse embryo compaction, outer cells become trophectoderm, while inner cells form the inner cell mass (ICM), later differentiating into primitive endoderm and epiblast - source of embryonic stem cells (ESCs) - during blastocyst formation. Trophectoderm specification is driven by position-governed polarisation, while primitive endoderm specification is positively regulated by FGF4 signalling from the ICM and epiblast. When injected into an 8-cell stage morula, ESCs can exclude host cells from the epiblast, leading to mice derived entirely from these cells. While evidence suggests roles for ESC-produced FGF4 and physical crowding in host cell displacement from the ICM, the interplay between these possible mechanisms has yet to be dissected, in part due to the lack of studies using Fgf4-/- ESCs. Here, we combine chimaera titration assays with mathematical modelling to study these mechanisms of host cell displacement. Both Fgf4+/+ and Fgf4-/- ESCs displaced host cells from the epiblast, while only Fgf4-/- injected embryos reduced primitive endoderm and increased trophectoderm, indicating sequential exclusion by displacement crowding followed by FGF4 signalling. SUMMARY STATEMENTIn mouse blastocyst chimeras, donor cells displace host cells from the epiblast by a combination of crowding and FGF4 signalling.

developmental biology↗

bioRxiv2023

The Mechanical Influence of Densification on Initial Epithelial Architecture

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayer epithelium - whether protective, secretory, absorptive, or filtrative -relies on regular tissue architecture with respect to the apical-basal axis. Using an unbiased 3D analysis pipeline developed in our lab, we previously showed that epithelial tissue architectures in culture can be divided into distinct developmental categories, and that these are intimately connected to cell density: at sparse densities, cultured epithelial cell layers have a squamous morphology (Immature); at intermediate densities, these layers develop lateral cell-cell borders and rounded cell apices (Intermediate); cells at the highest densities reach their full height and demonstrate flattened apices (Mature). These observations prompted us to ask whether epithelial architecture emerges from the mechanical constraints of densification, and to what extent a hallmark feature of epithelial cells, namely cell-cell adhesion, contributes. In other words, to what extent is the shape of cells in an epithelial layer a simple matter of sticky, deformable objects squeezing together? We addressed this problem using a combination of computational modeling and experimental manipulations. Our results show that the first morphological transition, from Immature to Intermediate, can be explained simply by cell crowding. Additionally, we identify a new division (and thus transition) within the Intermediate category, and find that this second morphology relies on cell-cell adhesion.

cell biology↗

bioRxiv2023

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins

During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.

cell biology↗

bioRxiv2022

In-silico and in-vitro morphometric analysis of intestinal organoids

Organoids offer a powerful model to study cellular self-organisation, the growth of specific tissue morphologies in-vitro, and to assess potential medical therapies. However, the intrinsic mechanisms of these systems are not entirely understood yet, which can result in variability of organoids due to differences in culture conditions and basement membrane extracts used. Improving the standardisation of organoid cultures is essential for their implementation in clinical protocols. Developing tools to assess and predict the behaviour of these systems may produce a more robust and standardised biological model to perform accurate clinical studies. Here we developed an algorithm to automate crypt-like structure counting on intestinal organoids in both in-vitro and in-silico images. In addition, we modified an existing two-dimensional agent-based mathematical model of intestinal organoids to better describe the system physiology, and evaluated its ability to replicate budding structures compared to new experimental data we generated. The crypt-counting algorithm proved useful in approximating the average budding structures found in our in-vitro intestinal organoid culture images on days 3 and 7 after seeding. Our changes to the in-silico model maintain the potential to produce simulations that replicate the number of budding structures found on days 5 and 7 of in-vitro data. The present study aims to aid in quantifying key morphological structures and provide a method to compare both in-vitro and in-silico experiments. Our results could be extended later to 3D in-silico models.

systems biology↗

bioRxiv2022

Bespoke data augmentation and network construction enable image classification on small microscopy datasets

Recent work has indicated a need for increased temporal resolution for studies of the early chick brain. Over a 10-hour period, the developmental potential of progenitor cells in the HH10 brain changes, and concomitantly, the brain undergoes subtle changes in morphology. We asked if we could train a deep convolutional neural network to sub-stage HH10 brains from a small dataset (<200 images). By augmenting our images with a combination of biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (<250) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data. SUMMARY STATEMENTWe train a deep convolutional network that can be generally applied to accurately classify chick embryos from images. Saliency analyses show that classification is based on biologically relevant features.

developmental biology↗

bioRxiv2021

insideOutside: an accessible algorithm for classifying interior and exterior points, with applications in embryology

A crucial aspect of embryology is relating the position of individual cells to the broader geometry of the embryo. A classic example of this is the first cell-fate decision of the mouse embryo, where interior cells become inner cell mass and exterior cells become trophectoderm. Fluorescent labelling, imaging, and quantification of tissue-specific proteins have advanced our understanding of this dynamic process. However instances arise where these markers are either not available, or not reliable, and we are left only with the cells spatial locations. Therefore, a simple, robust method for classifying interior and exterior cells of an embryo using spatial information is required. Here, we describe a simple mathematical framework and an unsupervised machine learning approach, termed insideOutside, for classifying interior and exterior points of a three-dimensional point-cloud, a common output from imaged cells within the early mouse embryo. We benchmark our method against other published methods to demonstrate that it yields greater accuracy in classification of nuclei from the pre-implantation mouse embryos and greater accuracy when challenged with local surface concavities. We have made MATLAB and Python implementations of the method freely available. This method should prove useful for embryology, with broader applications to similar data arising in the life sciences.

developmental biology↗

bioRxiv2021

ChemChaste: Simulating spatially inhomogenous biochemical reaction-diffusion systems for modelling cell-environment feedbacks

MotivationSpatial organisation plays an important role in the function of many biological systems, from cell fate specification in animal development to multi-step metabolic conversions in microbial communities. The study of such systems benefits from the use of spatially explicit computational models that combine a discrete description of cells with a continuum description of one or more chemicals diffusing within a surrounding bulk medium. These models allow the in silico testing and refinement of mechanistic hypotheses. However, most existing models of this type do not account for concurrent bulk and intracellular biochemical reactions and their possible coupling. ResultsHere, we describe ChemChaste, an extension for the open-source C++ computational biology library Chaste. ChemChaste enables the spatial simulation of both multicellular and bulk biochemistry by expanding on Chastes existing capabilities. In particular, ChemChaste enables: (i) simulation of an arbitrary number of spatially diffusing chemicals; (ii) spatially heterogeneous chemical diffusion coefficients; and (iii) inclusion of both bulk and intracellular biochemical reactions and their coupling. ChemChaste also introduces a file-based interface that allows users to define the parameters relating to these functional features without the need to interact directly with Chastes core C++ code. We describe ChemChaste and demonstrate its functionality using a selection of chemical and biochemical exemplars, with a focus on demonstrating increased ability in modelling bulk chemical reactions and their coupling with intracellular reactions. Availability and implementationChemChaste is a free, open-source C++ library, available via GitHub at https://github.com/OSS-Lab/ChemChaste under the BSD license. ContactO.Soyer@warwick.ac.uk or a.g.fletcher@sheffield.ac.uk

systems biology↗

bioRxiv2021

Competition Between Cell-Cell and Cell-Substrate Adhesion Determines Epithelial Monolayer Architecture in Culture

Organ surfaces are lined by epithelial monolayers - sheets of cells that are one-cell thick. This architecture underlies tissue function, and its loss is associated with disease, including cancer. Studies of in-plane epithelial cell behaviors show that a developing epithelium behaves as a fluid in respect to the tissue plane, and can therefore readily adapt to varying mechanical influences during morphogenesis. We asked the question of how monolayer architecture is achieved, and whether it demonstrates the same fluid behavior. To address this problem, we cultured MDCK (Madin-Darby Canine Kidney) cell layers at different densities and timepoints and analyzed their architectures using a novel tool, Automated Layer Analysis (ALAn), which we introduce here. Our experimental and theoretical results lead us to propose that epithelial monolayer architecture is governed by a balance of counteracting forces due to cell-cell and cell-substrate adhesion, and that this balance is influenced by cell density. MDCK cells do not undergo obvious rearrangement along the apical-basal axis; instead, cells that do not contact the substrate aggregate on top of the monolayer. Our findings therefore imply that monolayered architecture is under more rigid control than planar tissue shape in epithelia.

cell biology↗

bioRxiv2021

Adhesion dynamics regulate cell intercalation behaviour in an active tissue

Cell intercalation is a key cell behaviour of morphogenesis and wound healing, where local cell neighbour exchanges can cause dramatic tissue deformations such as body axis extension. Here, we develop a mechanical model to understand active cell intercalation behaviours in the context of an epithelial tissue. Extending existing descriptions, such as vertex models, the junctional actomyosin cortex of every cell is modelled as a continuum morphoelastic rod, explicitly representing cortices facing each other at bicellular junctions. Cells are described directly in terms of the key subcellular constituents that drive dynamics, with localised stresses from the contractile actomyosin cortex and adhesion molecules coupling apposed cortices. This multi-scale apposed-cortex formulation reveals key behaviours that drive tissue dynamics, such as cell-cell shearing and flow of junctional material past cell vertices. We show that cell neighbour exchanges can be driven by purely junctional mechanisms. Active contractility and viscous turnover in a single bicellular junction are sufficient to shrink and remove a junction. Next, the 4-way vertex is resolved and a new, orthogonal junction extends passively. The adhesion timescale defines a frictional viscosity that is an important regulator of these dynamics, modulating tension transmission in the tissue as well as the speeds of junction shrinkage and growth. The model additionally predicts that rosettes, which form when a vertex becomes common to many cells, are likely to occur in active tissues with high adhesive friction. SIGNIFICANCECell intercalation, or neighbour exchange, is a crucial behaviour that can drive tissue deformations, dissipate stress and facilitate wound healing. Substantial experimental work has identified the key molecular players facilitating intercalation, but there remains a lack of consensus and understanding of their physical roles. Existing biophysical models that represent cell-cell contacts with single edges cannot study the continuous dynamics of intercalation, involving shear between coupled cell cortices. Deriving a continuum description of the cell cortex, explicitly coupling neighbouring cortices with adhesions, we define the biophysical conditions required for successful neighbour exchanges. Furthermore, we show how the turnover of adhesion molecules specifies a viscous friction that regulates active tissue dynamics.

biophysics↗

bioRxiv2019

The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less knowledge is available. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions. In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of adherens junctions. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular adherens junctions, the first of its kind. Clonal analysis suggests that pair-wise homophilic adhesion is necessary and sufficient for Sdk tricellular vertex localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where adherens junctions are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension, using quantitative methods. We find that apical cell shapes are strikingly abnormal in sdk mutants. Moreover, adhesion at apical vertices is compromised in rearranging cells, with holes forming and persisting throughout axis extension. Finally, we show that polarized cell intercalation is decreased and abnormal in sdk mutants. Mathematical modeling of the cell behaviours supports the conclusion that the T1 transitions of polarized cell intercalation are delayed in sdk mutants. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the striking cell shape phenotype of sdk mutant embryos.

developmental biology↗

bioRxiv2018

Cross-talk between Hippo and Wnt signalling pathways in intestinal crypts: insightsfrom an agent-based model

Intestinal crypts are responsible for the total cell renewal of the lining of the intestines, occurring every 4-7 days in humans. This rapid turnover is governed by the complex interplay between signalling pathways and the cell cycle within individual cells in the crypts. The role of Wnt signalling in governing cell proliferation and differentiation in the intestinal crypt has been extensively studied, with increased signalling found towards the lower regions of the crypt. Recent studies have shown that the Wnt signalling gradient found within the crypt may arise as a result of division-based spreading from a Wnt reservoir at the crypt base. The discovery of the Hippo pathways involvement in maintaining crypt homeostasis is more recent; a mechanistic understanding of Hippo pathway dynamics, and its possible cross-talk with the Wnt pathway, remains lacking. To explore how the interplay between these pathways may control crypt homeostasis and dysplasia we developed and analysed a mathematical model coupling an ordinary differential equation description of Wnt and Hippo signalling with a cell-based description of cell movement, proliferation and contact inhibition. Furthermore, we compared an imposed Wnt gradient with a division-based Wnt gradient model. Our results suggest that Hippo signalling affects the Wnt pathway by reducing the presence of free cytoplasmic {beta}-catenin, causing cell cycle arrest. This process slows the rate of crypt turnover, previously attributed only to mutations in Wnt pathway components commonly observed in colorectal cancers. We also show that a division-based spreading of Wnt can form a Wnt gradient, resulting in proliferative dynamics comparable to imposed-gradient models. This suggests that imposed-gradient models are a reasonable approximation of experimentally observed Wnt gradients, however they provide little insight into the source of Wnt. Finally, a simulated APC double mutant, with misregulated Wnt and Hippo signalling activity, is predicted to be capable of completing a more rapid monoclonal conversion of the crypt than a Wnt-only mutant.\n\nAuthor summaryColorectal cancer is the fourth leading cause of cancer death worldwide. It is generally established that colorectal cancer occurs upon disruption of the dynamics of crypts, tubular indentations lining the walls of the gastrointestinal tract that are responsible for renewal of the intestinal epithelium. Thus, understanding the mechanisms that regulate intestinal crypt dynamics is instrumental to understand how colorectal cancer can emerge. We used mathematical modelling to explore how crypt dynamics emerge from the interactions across several biological scales, including mutations that cause an abnormal cell response to changes at the cell or tissue level, cell proliferation and motility within the crypt. Our model describes, in each cell within the crypt, levels and dynamics of genes involved in two signalling pathways - the canonical Wnt and Hippo pathways - relevant for cell proliferation and adhesion. In simulations, we predict how the crosstalk between these pathways affects crypt dynamics in both healthy and dysplastic cases. Furthermore, our simulations match recent biological evidence of a novel mechanism, based on cell division, causing heterogeneous and graded localisation of different cell types (dividing or not) within the crypt. Our results suggest the possibility of combined disease treatment, targeting both the Wnt and Hippo pathways.

systems biology↗

bioRxiv2018

Inferring Tumour Proliferative Organisation from Phylogenetic Tree Measures in a Computational Model

We use a computational modelling approach to explore whether it is possible to infer a tumours cell proliferative hierarchy, under the assumptions of the cancer stem cell hypothesis and neutral evolution. We focus on inferring the symmetric division probability for cancer stem cells in our model, as this is believed to be a key driving parameter of tumour progression and therapeutic response. Given the advent of multi-region sampling, and the opportunities offered by them to understand tumour evolutionary history, we focus on a suite of statistical measures of the phylogenetic trees resulting from the tumours evolution in different regions of parameter space and through time. We find strikingly different patterns in these measures for changing symmetric division probability which hinge on the inclusion of spatial constraints. These results give us a starting point to begin stratifying tumours by this biological parameter and also generate a number of actionable clinical and biological hypotheses including changes during therapy, and through tumour evolution.

cancer biology↗