Biology subjects

Saunders, T. E.

Publications and source records attributed to Saunders, T. E..

At least 19 recordsLinked to original sources

bioRxiv2025

Identification of optimal fluorophores for use in the Drosophila embryo

The use of fluorescent proteins has become ubiquitous throughout the life sciences as a key method for understanding molecular, cellular and tissue scale processes. Genetically encodable fluorophores have enabled stable genetic lines to be generated in a large array of organisms. There are now suites of fluorophores available, particularly in the green and red spectra. Yet, which fluorophore works best in vivo can depend on a range of factors, both extrinsic (e.g. pH, temperature) and intrinsic (e.g. photobleaching, brightness). While fluorophores have been well characterised in cell culture, such measures within in vivo systems are more limited. Here, we present a quantitative screen of nine green and eight red fluorophore lines in Drosophila, with the fluorescent protein expressed from the same genomic location and imaged under identical conditions. We analyse the expression of the fluorophores in both early and late Drosophila embryos. We provide a quantitative analysis of the bleaching and folding rates. We find amongst the green fluorophores that the suitable choice - e.g., mEGFP, mNeonGreen, mStayGold - depends on timing and imaging requirements. For the red fluorophores, mScarlet-I performed consistently well, though no particular fluorophore stood out as ideal under all conditions. These results provide a powerful database for selecting optimal fluorophores for imaging in the Drosophila embryo in green and red channels.

developmental biology↗

bioRxiv2025

A structural transition ensures robust formation of skeletal muscle

During organ development cells undergo significant morphological and positional changes. Yet, by the end of organogenesis, internal organ structure is typically robustly defined, with cells tightly packed. It remains an open question as to how the three-dimensional (3D) internal structure of an organ emerges reliably, particularly when there are multiple cell types interacting and dynamic boundary constraints. Here, we utilise quantitative live imaging and 3D morphological measures of the developing zebrafish myotome to unravel how early muscle organisation emerges. Contrary to the textbook view of muscle fibres as cylindrical, myocytes undergo an ordered chiral twist, the direction and magnitude of which depends on their position within the myotome. Further, cells skew and rearrange, seemingly to facilitate close packing of neighbouring muscle fibres. Cell movement undergoes a rapid decline in speed once the cells span the myotome segment. We find that cell packing is altered in mutants that disrupt cell fate or cell fusion, even though the final muscle segments remain largely confluent. Biophysical perturbation reveals that the cells are mechanically plastic, able to adjust to changes in the local cellular environment and boundary constraints. Taking these results together, we propose that the early myotome undergoes a structural transition, from a fluid-like state into a frozen state, resembling glass-like behaviour. Cellular plasticity in response to varying boundary constraints may be a general mechanism for ensuring robust organ morphogenesis in dense 3D tissues.

developmental biology↗

bioRxiv2025

Dynamic expression and differential requirement of the myocyte fusogen Myomixer during distinct myogenic episodes in the zebrafish

Skeletal muscle formation involves the fusion of myocytes into precisely aligned, multinucleated myofibres. These fibres continue to grow through reiterative rounds of myocyte fusion, incorporating new myonuclei and supporting muscle growth, repair and regeneration over organismal life span. The vertebrate-specific myocyte fusogens, Myomaker (Mymk) and Myomixer (Mymx), are crucial for generating multinucleated skeletal muscles. While the role of the transmembrane protein Mymk is well established, expression dynamics of mymx and the function of the Mymx micropeptide is less well understood. Here, using quantitative imaging and a mymx knockout strain, we explored the impact on myogenesis at different life stages of the zebrafish. We demonstrate that during the initial phase of muscle formation, mymx has a spatiotemporally varied expression across all axes of the developing myotome. On Mymx loss, myotome morphogenesis is disrupted, with both cell and tissue structure impacted. Moreover, we could show differential effects of Mymk versus Mymx loss on myocyte fusion and muscle growth. Finally, we report that perturbation to adult muscle multinucleation and size impacted bone development, again with different phenotypic severities among the two fusogen mutants. Together, our work provides key insights into the interplay between myocyte fusion, myotome morphogenesis and acquisition of final adult form.

developmental biology↗

bioRxiv2025

Boundary constraints can determine pattern emergence

The robust patterning of cell fates during embryonic development requires precise coordination of signalling gradients within defined spatial constraints. Using a geometrically confined in vitro system derived from human embryonic stem cells, we demonstrate that patterning of neuromesodermal progenitors (NMPs) during axial elongation is driven by boundary-dependent mechanisms. Despite extensive work on radial fate patterning in confined 2D systems, the quantitative role of boundary conditions in shaping spatiotemporal dynamics remains unclear. Here, we show that a minimal reaction-diffusion model coupled with a simplified gene regulatory network accurately predicts spatial patterns across diverse geometries. Guided by its predictions, we identify Wnt signalling as a key component of the activator signal. Inhibition of Wnt secretion preserved initiation of patterning but disrupted subsequent morphogenesis, indicating distinct mechanisms govern pattern establishment versus maintenance. Our findings reveal how geometry encodes positional information that directs molecular patterning, providing insight into how spatial constraints and signalling dynamics guide robust tissue self-organisation during development.

systems biology↗

bioRxiv2025

Stress anisotropy in 3D active curved structures

Layers composed of lateral connections prevail in biological systems from subcellular membranes to epithelial sheets. This work presents a continuum framework to describe the effects of mechanical forces within a 3D curved layer with supporting lateral mesh. We provide detailed discussion on the emergence of stress anisotropy as a function of depth in different curvature settings, building on Lou et al. Phys. Rev. Lett. 130, 108401 (2023). We principally consider an epithelial monolayer to explain how the interplay between layer curvature and cell mechanics determines the stress anisotropy. We show that this can lead to irregular cellular shapes in 3D, including scutoid-like geometries. Our framework is general, and can be extended to a diverse set of biologically relevant systems.

biophysics↗

bioRxiv2024

Regulation of bicoid mRNA throughout oogenesis and early embryogenesis impacts protein gradient formation

The transcription factor Bicoid (Bcd) protein guides early Drosophila patterning and is the best-characterised morphogen. The source of the morphogen, bcd mRNA, is maternally deposited during oogenesis and localised to the anterior pole of the mature oocyte. While the spatiotemporal interpretation of the Bcd morphogen gradient has been intensely studied, when and where Bcd protein is produced and how this protein gradient is dynamically shaped remains contentious. Here, we use the SunTag reporter system to quantitatively examine the spatiotemporal profile of bcd mRNA translation in vivo. We show that association with Processing bodies (P bodies) in mature oocytes prevent premature bcd mRNA translation. Following egg activation, bcd mRNA dissociates from P bodies and translation is observed at the anterior pole. Translation remains restricted to the anterior domain throughout early development, even after nuclear migration in the syncytial blastoderm. At cellularisation, translation ceases and the remaining bcd mRNA associates with reformed P bodies, which appear to block any further translation. We use these observations to create a new modified source-diffusion-degradation model of Bcd gradient formation that has spatiotemporally varying production. Overall, our study reveals that bcd mRNA translation is tightly controlled in space and time during oogenesis and early embryogenesis.

developmental biology↗

bioRxiv2024

CellMet: Extracting 3D shape metrics from cells and tissues

During development and tissue repair, cells reshape and reconfigure to ensure organs take specific shapes. This process is inherently three-dimensional (3D). Yet, in part due to limitations in imaging and data analysis, cell shape analysis within tissues have been studied as a two-dimensional (2D) approximation, e.g., the Drosophila wing disc. With recent advances in imaging and machine learning, there has been significant progress in our understanding of 3D cell and tissue shape in vivo. However, even after gaining 3D segmentation of cells, it remains challenging to extract cell shape metrics beyond volume and surface area for cells within densely packed tissues. In order to extract 3D shape metrics, we have developed CellMet. This user-friendly tool enables extraction of quantitative shape information from 3D cell and tissue segmentation. It is developed for extracting cell scale information from densely packed tissues, such as cell face properties, cell twist, and cell rearrangements. Our method will improve the analysis of 3D cell shape and the understanding of cell organisation within tissues. Our tool is open source, available at https://github.com/TimSaundersLab/CellMet.

developmental biology↗

bioRxiv2024

Receptor binding and tissue architecture explain the morphogen local-to-global mobility transition

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control. SIGNIFICANCEMeasurements of morphogen diffusivity vary significantly depending on experimental approach. Such differences have been used to argue against diffusion as a viable mechanism of morphogen gradient formation. Here, we demonstrate that accounting for the local tissue architecture in concert with including receptor binding is sufficient to explain a range of biological observations. This demonstrates that (hindered) diffusion-driven transport is a viable mechanism of gradient formation.

biophysics↗

bioRxiv2023

The positioning mechanics of microtubule asters in Drosophila embryo explants

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modelling, we demonstrate that these asters likely generate an effective pushing force over short distances. Significance StatementUsing cytosolic explants from Drosophila syncytial embryos combined with quantitative microscopy and perturbations, de-Carvalho et al., reveal the mechanical forces separating Drosophila microtubule asters. Aster separation drives precise nuclear positioning in multinucleated embryo cells, a vital process for tissue formation and gene expression during subsequent embryo development.

biophysics↗

bioRxiv2022

Optogenetic control of YAP can enhance the rate of wound healing

BackgroundTissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display limited ability to repair injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the YAP, a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury. MethodsWe express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay and anchorage-independent colony formation assay. ResultsUtilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing. ConclusionsThis study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.

cell biology↗

bioRxiv2022

Long-ranged formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution. Yet, how morphogen gradients form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. We utilise fluorescence correlation spectroscopy (FCS) and perturbations to Bcd, to dissect Bcd dynamics at multiple spatial and temporal locations. In both the cytoplasm and nucleus, we find two dynamic modes for Bicoid diffusion dynamics, consisting of fast and slow populations of Bcd. Surprisingly, there are spatial differences in Bcd diffusivity along the AP-axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in the Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. We subsequently investigated which binding elements of Bcd are playing a role in its dynamics. In the nucleus, we demonstrate that the slower mode of Bcd transport is due to Bcd DNA binding. Addition of the Bcd homeodomain to eGFP::NLS can qualitatively replicate the observed Bcd concentration profile, suggesting this domain is the primary region regulating Bcd dynamics. This study provides a detailed analysis of morphogen dynamics at different spatial and temporal locations, revealing multiple modes of transport. These results explain how a long-ranged gradient can form while retaining a steep profile through much of its range.

developmental biology↗

bioRxiv2022

Curvature-induced cell rearrangements in biological tissues

On a curved surface, epithelial cells can adapt to geometric constraints by tilting and by exchanging their neighbors from apical to basal sides, known as an apicobasal T1 (AB-T1) transition. The relationship between cell tilt, AB-T1 transitions, and tissue curvature still lacks a unified understanding. Here, we propose a general framework for cell packing in curved environments and explain the formation of AB-T1 transitions under different conditions. We find that steep curvature gradients can lead to cell tilting and induce AB-T1 transitions. Conversely, large curvature anisotropy can drive AB-T1 transitions by hydrostatic pressure. The two mechanisms compete to determine the impact of tissue geometry and mechanics on optimized cell rearrangements in 3D.

biophysics↗

bioRxiv2022

Protective geometry and reproductive anatomy as candidate determinants of clutch size variation in pentatomid bugs

Many animals lay their eggs in clusters. Eggs on the periphery of clusters can be at higher risk of mortality. We asked whether the most commonly occurring clutch sizes in pentatomid bugs could result from geometrical arrangements that maximize the proportion of eggs in the clusters interior. Although the most common clutch sizes do not correspond with geometric optimality, stink bugs do tend to lay clusters of eggs in shapes that protect increasing proportions of their offspring as clutch sizes increase. We also considered whether ovariole number, an aspect of reproductive anatomy that may be a fixed trait across many pentatomids, could explain observed distributions of clutch sizes. The most common clutch sizes across many species correspond with multiples of ovariole number. However, there are species with the same number of ovarioles that lay clutches of widely varying size, among which multiples of ovariole number are not over-represented. In pentatomid bugs, reproductive anatomy appears to be more important than egg mass geometry in determining clutch size uniformity. In addition, within this group of animals that has lost most of its variation in ovariole number, clutches with a broad range of shapes and sizes may still be laid.

ecology↗

bioRxiv2022

Quantitative analysis of three-dimensional cell organisation and concentration profiles within curved epithelial tissues

Organogenesis involves folding of flat epithelial tissues into three-dimensional (3D) shapes. Quantitative analysis in 3D of the concentration gradients of biochemical and mechano-chemical cues that shape the tissue has remained a challenge, due to the complex tissue geometries. Towards addressing this, we present a methodology that transforms the cartesian images acquired by high-resolution confocal microscopy of curved tissues into a laminar organisation from the outer-most tissue surface. We further detail a data-based intensity correction method to account for the intensity variations that arise as a consequence of the global geometry. Applying our approach to the dome-shaped Drosophila wing disc, we quantitatively estimate the concentration profiles of different biochemical signals, including the Wingless morphogen gradient. The laminar data-organisation method also enabled visualisation of apical and basal layers facilitating an accurate 3D reconstruction of cell shapes within the pseudostratified epithelium. We find that the columnar disc proper cells have irregular 3D shapes and undergo frequent apico-basal cell intercalations, concomitantly leading to cell neighbour exchanges along the apico-basal axis. The workflow of image processing described here may be employed to quantify 3D concentration gradients and 3D cellular organisation in any layered curved tissue, provided a marker describing the reference surface manifold is available.

developmental biology↗

bioRxiv2022

Echolocation-like model of directed cell migrationwithin growing tissues

During development and regeneration, cells migrate to specific locations within growing tissues. These cells can respond to both biochemical signals and mechanical cues, resulting in directed migration. Such migration is often highly stereotypic. Yet, how cells respond to migratory signals in a robust manner within a growing domain remains an open problem. Here, we propose a model of directed migration in growing tissues motivated by echolocation. The migrating cells generate a signaling gradient that induces a response signal from the moving system boundary. This response signal mediates cellular adhesion to the surrounding matrix and hence modulates the cell migration. We find that such a mechanism can align a series of cells at stable positions within growing systems and can effectively scale to system size. Finally, we discuss the relevance of such a model to fibroblast migration and location within the developing zebrafish caudal fin, which may be regulated by opposing signaling gradients of Slit-Robo pathway components. Significance StatementHow do cells reliably migrate within growing environments? Here, we show that cells can take advantage of an echolocation-like process, whereby they induce a response from the tissue boundary. As they approach the boundary, the response signal strengthens and brings the cell to a fixed position from the boundary. This simple system may be applicable to fibroblast migration in the fin.

biophysics↗

bioRxiv2021

Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

cell biology↗

bioRxiv2021

DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells

In a previous study, it was reported that Yap1 and Wwtr1 in zebrafish regulates the morphogenesis of the posterior body and epidermal fin fold (Kimelman, D., et al. 2017). We report here that DNA damage induces apoptosis of epidermal basal cells (EBCs) in zebrafish yap1-/-;wwtr1-/- embryos. Specifically, these mutant EBCs exhibit active Caspase-3, Caspase-8 and {gamma}H2AX, consistent with DNA damage serving as a stimulus of the extrinsic apoptotic pathway in epidermal cells. Live imaging of zebrafish epidermal cells reveals a steady growth of basal cell size in the developing embryo, but this growth is inhibited in mutant basal cells followed by apoptosis, leading to the hypothesis that factors underscoring cell size play a role in this DNA damage-induced apoptosis phenotype. We tested two of these factors using cell stretching and substrate stiffness assays, and found that HaCaT cells cultured on stiff substrates exhibit more numerous {gamma}H2AX foci compared to ones cultured on soft substrates. Thus, we propose that substrate rigidity modulates genomic stress in the developing epidermal cell, and that Yap1 and Wwtr1 are required for its survival.

developmental biology↗

bioRxiv2021

Optogenetic manipulation of YAP cellular localisation and function

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attached a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression, cell proliferation, and anchorage-independent growth. Similarly, we can utilise optoYAP in zebrafish embryos to modulate target genes. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

cell biology↗