Biology subjects

Saunders, T. E.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Condensation of the Drosophila Nerve Cord is Oscillatory and depends on Coordinated Mechanical Interactions

During development, organs must form with precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we combine in toto live-imaging, biophysical and genetic perturbations, and atomic force microscopy to characterize the condensation of the Drosophila ventral nerve cord (VNC) during embryonic development at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process, but instead occurs through oscillatory contractions alternating from anterior and posterior ends. The VNC mechanical properties spatially and temporally vary during its condensation, and forces along its longitudinal axis are spatially heterogeneous, with larger ones exerted between neuromeres. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. Finally, we show that these outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays due to the different time scales on which the mechanical processes act, and effective frictional interactions. In summary, we have defined the complex and progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

developmental biology↗

bioRxiv2020

Scaling of Internal Organs during Drosophila Embryonic Development

Many species show a diverse range of sizes; for example, domestic dogs have large variation in body mass. Yet, the internal structure of the organism remains similar, i.e. the system scales to organism size. Drosophila melanogaster has been a powerful model system for exploring scaling mechanisms. In the early embryo, gene expression boundaries scale very precisely to embryo length. Later in development, the adult wings grow with remarkable symmetry and scale well with animal size. Yet, our knowledge of whether internal organs initially scale to embryo size remains largely unknown. Here, we utilise artificially small Drosophila embryos to explore how three critical internal organs - the heart, hindgut and ventral nerve cord (VNC) - adapt to changes in embryo morphology. We find that the heart scales precisely with embryo length. Intriguingly, reduction in cardiac cell length, rather than number, appears to be important in controlling heart length. The hindgut - which is the first chiral organ to form - displays scaling with embryo size under large-scale changes in the artificially smaller embryos but shows few hallmarks of scaling within wild-type size variation. Finally, the VNC only displays weak scaling behaviour; even large changes in embryo geometry result in only small shifts in VNC length. This suggests that the VNC may have an intrinsic minimal length, which is largely independent of embryo length. Overall, our work shows that internal organs can adapt to embryo size changes in Drosophila. but the extent to which they scale varies significantly between organs.

developmental biology↗

bioRxiv2020

Spatiotemporal sensitivity of embryonic heart specification to FGFR signaling in Drosophila

Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl signaling during embryogenesis.

developmental biology↗

bioRxiv2020

Aster repulsion drives local ordering in an active system

Biological systems are a form of active matter, which often undergo rapid changes in their material state, e.g. liquid to solid transitions. Yet, such systems often also display remarkably ordered structures. It remains an open question as to how local ordering occurs within active systems. Here, we utilise the rapid early development of Drosophila melanogaster embryos to uncover the mechanisms driving short-ranged order. During syncytial stage, nuclei synchronously divide (within a single cell defined by the ellipsoidal eggshell) for nine cycles after which most of the nuclei reach the cell cortex. Despite the rapid nuclear division and repositioning, the spatial pattern of nuclei at the cortex is highly regular. Such precision is important for subsequent cellularisation and morphological transformations. We utilise ex vivo explants and mutant embryos to reveal that microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. For large networks of nuclei, such as in the embryo, we predict - and experimentally verify - the formation of force chains. The ex vivo extracts enabled us to deduce the force potential between single asters. We use this to predict how the nuclear division axis orientation in small ex vivo systems depend on aster number. Finally, we demonstrate that, upon nucleus removal from the cortex, microtubule force potentials can reorient subsequent nuclear divisions to minimise the size of pattern defects. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters can drive ordering within an active system.

biophysics↗

bioRxiv2020

A molecular approach to studying Hymenoptera diets using polistine wasps

O_LIThe study of animal diets has benefited from the rise of high-throughput DNA sequencing applied to stomach content or faecal samples. The latter can be fresh samples used to describe recent meals, or older samples, which can inform about past feeding activities. For most invertebrates, however, it is difficult to access historical samples, due to the small size of the animals and the absence of permanent defecation sites. Therefore, sampling must be repeated to account for seasonal variation and to capture the overall diet of a species. C_LIO_LIThis study develops a method to describe the overall diet of nest-building Hymenoptera based on a single sampling event, by analysing prey DNA from faeces accumulated in brood cells. We collected 48 nests from two species of introduced paper wasps (Polistes chinensis, and P. humilis) in the urban and peri-urban areas of Auckland, New Zealand, and selected two samples per nest. One from brood cells in the outer layer of the nest to represent the most recent diet, and one from brood cells in an inner layer to represent older diet. C_LIO_LIDiet differed between species, although both fed mainly on Thysanoptera, Lepidoptera and Acariformes. Prey taxa identified to species level included both agricultural pests and native species. Prey communities consumed were significantly different between inner and outer nest samples suggesting seasonal variation in prey availability and/or a diversification of the wasps diet as the colony grows. We also show for the first time potential predation of marine organisms by Polistes wasps. C_LIO_LIOur study provides field evidence that polistine wasps feed on agricultural pests, supporting the hypothesis that some social wasp species could have a suppressing effect on agricultural pests. The proposed methodology is readily applicable to other nest-building Hymenoptera and has the potential to provide comprehensive knowledge about their diet with minimum sampling effort. Such knowledge is Essential to measure the ecological impact of invasive Vespidae and support the conservation of native invertebrate biodiversity. C_LI

ecology↗

bioRxiv2019

Independent evolution of lateral inhibition mechanisms in different lineages of land plants: MpFEW RHIZOIDS1 miRNA-mediated lateral inhibition controls rhizoid cell patterning in Marchantia polymorpha

Lateral inhibition patterns differentiated cells during development in bacteria, metazoans and land plants. Tip-growing rhizoid cells develop among flat epidermal cells in the epidermis of the early diverging land plant Marchantia polymorpha. We show that the majority of rhizoid cells develop individually but some develop in linear, one-dimensional clusters of between two and seven rhizoid cells in wild type plants. The distribution of rhizoid cells can be accounted for within a simple model of lateral inhibition. The model also predicted that, in the absence of lateral inhibition, rhizoid cell clusters would be two-dimensional with larger clusters than those formed with lateral inhibition. Rhizoid differentiation in Marchantia polymorpha is positively regulated by the ROOT HAIR DEFECTIVE SIX-LIKE1 (MpRSL1) basic Helix Loop Helix (bHLH) transcription factor which is directly repressed by the FEW RHIZOIDS1 (MpFRH1) miRNA. To test if MpFRH1 miRNA acts during lateral inhibition we generated loss-of-function mutants that did not produce the MpFRH1 miRNA. Two-dimensional clusters of rhizoids develop in Mpfrh1loss-of-function (lof) mutants as predicted by the model for plants that lack lateral inhibition. Furthermore, clusters of up to nine rhizoid cells developed in the Mpfrh1lof mutants compared to a maximum number of seven observed in wild type. The higher steady state levels of MpRSL1 mRNA in Mpfrh1lof mutants indicate that MpFRH1-mediated lateral inhibition involves the repression of MpRSL1 activity. Together the modelling and genetic data indicate that the pattern of cell differentiation in the M. polymorpha epidermis is consistent with a lateral inhibition process in which MpFRH1 miRNA represses MpRSL1. This discovery suggests that novel mechanisms of lateral inhibition may operate in different lineages of land plants, unlike metazoans where the conserved Delta-Notch signaling system controls lateral inhibition in diverse metazoan lineages.

developmental biology↗

bioRxiv2019

Stochastic activation and bistability in a Rab GTPase regulatory network.

Rab GTPases are the central regulators of intracellular traffic. Their function relies on a conformational change triggered by nucleotide exchange and hydrolysis. While this switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate a biochemical signal in space and time is unclear. Here, we combine in vitro reconstitution experiments with theoretical modeling to study a minimal Rab5 activation network. We find that positive feedback in this network gives rise to bistable switching of Rab5 activation and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Together, our findings reveal new insights into the non-equilibrium properties and general principles of biochemical signaling networks underlying the spatiotemporal organization of the cell.

systems biology↗

bioRxiv2019

Interfacial energy constraints are sufficient to align cells over large distances

During development and wound healing, cells need to form long-ranged ordered structures to ensure precise formation of organs and repair damage. This requires cells to locate specific partner cells to which to adhere. How such cell matching reliably happens is an open problem, particularly in the presence of biological variability. Here, we use an equilibrium energy model to simulate how cell matching can occur with subcellular precision. A single parameter - encapsulating the competition between selective cell adhesion and cell compressibility - can reproduce experimental observations of cell alignment in the Drosophila embryonic heart. This demonstrates that adhesive differences between cells (in the case of the heart, mediated by filopodia interactions) are sufficient to drive cell matching without requiring cell rearrangements. The biophysical model can explain observed matching defects in mutant conditions and when there is significant biological variability. We also demonstrate that a dynamic vertex model gives results consistent with the equilibrium energy model. Overall, this work shows that equilibrium energy considerations are consistent with observed cell matching in cardioblasts, and has potential application to other systems, such as neuron connections and wound repair. Statement of SignificanceCells often need to identify specific neighboring cells, such as during wound repair and forming neural connections. Here, we develop a biophysical model of such cell-cell interactions within the context of the developing heart. We demonstrate that precise cell matching can occur by minimizing the energy costs of interfacial interactions. This model can explain a breadth of experimental observations despite it being a steady-state approximation of a dynamic system. This opens the possibility that such approaches may be applicable to other systems, providing a powerful yet simple framework for understanding cell matching.

systems biology↗

bioRxiv2019

Shh induces symmetry breaking in the presomitic mesoderm by inducing tissue shear and orientated cell rearrangements

Future boundaries of skeletal muscle segments are determined in the presomitic mesoderm (PSM). Within the PSM, future somitic cells undergo significant changes in both morphology and position. How such large-scale cellular changes are coordinated and the effect on the future border formation is unknown. We find that cellular rearrangements differ between cell populations within the PSM. In contrast to lateral somitic cells, which display less organized rearrangement, the adaxial cell layer undergoes significant tissue shearing with dorsal and ventral cells sliding posteriorly. This shear is generated by orientated intercalations of dorsally and ventrally located adaxial cells, which induces a chevron-like pattern. We find Shh signaling is required for the tissue shear and morphogenesis of adaxial cells. In particular, we observe Shh-dependent polarized recruitment of non-muscle myosin IIA drives apical constrictions, and thus the intercalations and shear. This reveals a novel role for Shh in regulating cell mechanics in the PSM.

developmental biology↗

bioRxiv2019

Embryonic geometry underlies phenotypic variation in decanalized conditions

During development, many mutations cause increased variation in phenotypic outcomes, a phenomenon termed decanalization. Such variations can often be attributed to genetic and environmental perturbations. However, phenotypic discordance remains even in isogenic model organisms raised in homogeneous environments. To understand the mechanisms underlying phenotypic variation, we used as a model the highly precise anterior-posterior (AP) patterning of the early Drosophila embryo. We decanalized the system by depleting the maternal bcd product and found that in contrast to the highly scaled patterning in the wild-type, the segmentation gene boundaries shift away from the scaled positions according to the total embryonic length. Embryonic geometry is hence a key factor predetermining patterning outcomes in such decanalized conditions. Embryonic geometry was also found to predict individual patterning outcomes under bcd overexpression, another decanalizing condition. Further analysis of the gene regulatory network acting downstream of the morphogen identified vulnerable points in the networks due to limitations in the available physical space.

developmental biology↗

bioRxiv2018

Shaping the zebrafish myotome by differential friction and active stress

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During fish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to play a role in swimming. The final myotome shape can be altered by perturbing muscle cell differentiation or by altering the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after its segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the myotome and the surrounding tissues is spatially varying, resulting in spatially heterogeneous friction. Using a vertex model, we show that the interplay of differential spreading and friction is sufficient to drive the initial phase of myotome shape formation. However, we find that active stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the myotome observed in wildtype embryos. A final ingredient for formation and maintenance of the chevron shape is tissue plasticity, which is mediated by orientated cellular rearrangements. Our work sheds a new light on how a spatio-temporal sequence of local cellular events can have a non-local and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

biophysics↗

bioRxiv2018

MicroRNAs buffer genetic variation at specific temperatures during embryonic development

Successful embryogenesis requires the coordination of developmental events. Perturbations, such as environmental changes, must be buffered to ensure robust development. However, how such buffering occurs is currently unknown in most developmental systems. Here, we demonstrate that seven miRNAs are differentially expressed during Drosophila embryogenesis at varying temperatures within natural physiological ranges. Lack of miR-3-309, -31a, -310c, -980 or -984c causes developmental delays specifically at a given temperature. Detailed analysis on miR-310c and -984c shows that their targets are typically mis-expressed in mutant backgrounds, with phenotypes more pronounced at temperatures where miRNAs show highest expression in wild-type embryos. Our results show that phenotypes may arise at specific temperatures while remaining silent at others, even within typical temperature ranges. Our work uncovers that miRNAs mask genetic variation at specific temperatures to increase embryonic robustness, highlighting another layer of complexity in miRNA expression.

developmental biology↗

bioRxiv2018

The mirtron miR-1010 functions in concert with its host gene SKIP to maintain synaptic homeostasis

Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that larvae lacking the mirtron miR-1010 are unable to grow properly and pupariate. We show that miR-1010 downregulates nAcR{beta}2. Increase of cortical nAcR{beta}2 mediated by neural activity elevates the level of intracellular Ca2+, which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcR{beta}2 mRNA levels (negative feedback loop). Our results demonstrate how a mirtron, in coordination with its host gene, contributes to maintaining homeostasis.

developmental biology↗

bioRxiv2018

Temporal development of Drosophila embryos is highly robust across a wide temperature range

Development is a precisely coordinated process in both space and time. Spatial precision has been quantified in a number of developmental systems, and, for example, such data has contributed significantly to our understanding of morphogen gradient interpretation. However, comparatively little quantitative analysis has been performed on timing and temporal coordination during development. Here, we use Drosophila to explore the temporal robustness of embryonic development within physiologically normal temperatures. We find that development is temporally very precise across a wide range of temperatures in all three Drosophila species investigated. However, we find temperature dependence in the heterochronicity. A simple model incorporating history-dependence can explain the developmental temporal trajectories. Interestingly, the history-dependence is temperature specific with either effective negative or positive feedback at different temperatures. We also find that embryos are surprisingly robust to shifting temperatures during embryogenesis. We further identify differences between tropical and temperate species that are suggestive of different potential mechanisms regulating temporal development depending on the local environment. Overall, our data shows that Drosophila embryonic development is temporally robust across a wide range of temperatures but there are species specific differences.

developmental biology↗

bioRxiv2018

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here we introduce morphogen-age measurements as a novel approach to retrieve key parameters in morphogen dynamics. Using a tandem fluorescent timer (tFT) as a protein-age sensor we find a gradient of increasing age of Bicoid (Bcd) along the anterior-posterior (AP) axis in the early Drosophila embryo. Quantitative analysis retrieves parameter that are most consistent with the synthesis-diffusion-degradation (SDD) model underlying Bcd-gradient formation, and rule out some other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bcd gradient formation, and demonstrate how morphogen age-information can complement knowledge about movement, abundance and distribution, which should be widely applicable for other systems.

developmental biology↗

bioRxiv2018

Pcdh18a-positive tip cells instruct notochord formation in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not known how these processes act together in a coordinated way during notochord formation. Analysing the tissue flow, we determined the displacement of the axial mesoderm and identified, relative to the ectoderm, an actively-migrating notochord tip cell population and a group of trailing notochordal plate cells. Molecularly, these tip cells express Protocadherin18a, a member of the cadherin superfamily. We show that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms these tip cells into a cohesive and fast migrating cell group. In turn, these tip cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework, and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation of the posterior, axial cells. Indeed, grafting experiments validated the predictions and induced ectopic notochord-like rods. Our findings indicate that the tip cells influence the trailing mesodermal cell sheet by inducing the formation of the notochord.

developmental biology↗

bioRxiv2017

Disrupting different Distal-less exons leads to ectopic and missing eyespots accurately modeled by reaction-diffusion mechanisms

Eyespots on the wings of nymphalid butterflies represent colorful examples of the process of pattern formation, yet the developmental origins and the mechanisms behind eyespot differentiation are still not fully understood. Here we re-examine the function of Distal-less (Dll) in eyespot development, which is still unclear. We show that CRISPR-Cas9 induced exon 2 mutations in Bicyclus anynana leads to exon skipping and ectopic eyespots on the wing. Exon 3 mutations, however, lead to null/missense transcripts, missing eyespots, lighter wing coloration, loss of scales, and a variety of other phenotypes implicating Dll in the process of eyespot differentiation. Reaction-diffusion modeling enabled exploration of the function of Dll in eyespot formation, and accurately replicated a wide-range of mutant phenotypes. These results confirm that Dll is a required activator of eyespot development, scale growth and melanization and point to a new mechanism of alternative splicing to achieve Dll over-expression phenotypes.

developmental biology↗

bioRxiv2017

Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila {beta}-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of {beta}-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

developmental biology↗