Biology subjects

Gilmour, D.

Publications and source records attributed to Gilmour, D..

9 recordsLinked to original sources

bioRxiv2026

A chemigenetic fluorescence lifetime-based biosensor for imaging of ERK activity

Extracellular signal-regulated kinase (ERK) governs many cellular processes, and its spatiotemporal activity dynamics are important for downstream signaling specificity. Existing fluorescent ERK activity biosensors, however, fall short in enabling spectrally multiplexed, spatially resolved, or deep tissue measurements. Here, we introduce a chemigenetic, single-fluorophore biosensor, WHaloEKAR, that reports ERK activities through changes in fluorescence lifetime. WHaloEKAR combines large lifetime changes (-0.81 ns) with the spectral versatility of the self-labeling protein HaloTag giving access to orange, red, and far-red ERK biosensors. The latter enabled the investigation of heterogeneous ERK activity at distinct subcellular locations in cultured cell lines and primary cortical rat neurons via fluorescence lifetime imaging microscopy (FLIM). Moreover, multiplexed FLIM measurements allowed to study ERK activity in parallel to the second messengers calcium and cyclic adenosine monophosphate. Finally, WHaloEKAR enabled the investigation of ERK activity in 3D samples as well as in live animals.

cell biology↗

bioRxiv2026

Shield-4i: A Whole-mount Multiplexed Imaging Platform for Studying Multiscale Information Flow in 3D Multicellular Systems

Self-organization in multicellular systems emerges from reciprocal interactions across spatiotemporal scales. Understanding how subcellular organization, tissue remodeling and developmental outcome are coordinated, thus requires simultaneous profiling of biological processes spanning orders of magnitudes in space and time. Yet, a unified experimental and computational framework for capturing these multiscale properties across in vivo and stem cell-derived systems has been lacking. Here, we introduce Shield-4i, a high-throughput, versatile, and accessible method for automated in toto iterative immunofluorescence imaging of whole-mount structures at subcellular resolution. Through polyepoxide-mediated inter- and intramolecular crosslinking, Shield-4i preserves sample integrity during repeated SDS-based elution cycles. We benchmark this method in gastrulating zebrafish and post-implantation mouse embryos and demonstrate its applicability to stem cell-derived 3D gastruloids, achieving up to 30-plex measurements of proteins and their post-translational modifications across hundreds of samples. To enable scalable analysis, we developed a dedicated 3D workflow supporting OME-Zarr-based and FAIR-compliant data storage, standardized processing, and multiscale feature extraction. Applying this framework to investigate gastruloid self-organization, we quantify how cellular physicochemical state and signaling properties encode cell position along embryonic axes and connect molecular patterning and fate decisions to morphological symmetry breaking at the multicellular scale. Together, Shield-4i provides a high-content in toto spatial proteomics platform for dissecting multiscale information flow and self-organization in multicellular systems.

systems biology↗

bioRxiv2025

Multiplexed embryo profiling links cellular state to zygotic genome activation in single cells

Multicellular self-organization relies on complex interactions across multiple length scales, shaping the transcriptional activity and molecular phenotype of individual cells. Despite advances in spatial omics technologies, mapping protein state composition at high spatial resolution to capture the molecular phenotype across whole-mount structures has remained challenging. Here, we introduce high-throughput 3D iterative indirect immunofluorescence imaging (3D-4i) for the simultaneous detection of multiple proteins and protein states across the entire embryo, and a 3D-dedicated computer vision pipeline for quantifying morphological and molecular properties across subcellular, single-cell, and whole-embryo scales in hundreds of samples. Applying this pipeline to early zebrafish embryos undergoing mid-blastula transition, we determine the cell cycle phase for each cell across the embryo, allowing us to uncover the spatiotemporal dynamics by which global meta-synchronous mitotic waves transition to cell cycle desynchronization. Using statistical analysis, we find that cell cycle phase is the major source of variability in transcription within a division cycle, and combining this with analysis of key transcription factors and chromatin modifier state enables accurate prediction of transcriptional output during zygotic genome activation in individual cells. Collectively, our results demonstrate the strength of 3D-4i in quantifying multimodal effects across spatiotemporal scales and show how it can be utilized to unravel the complex contribution of causal factors that collectively drive multicellular self-organization.

systems biology↗

bioRxiv2025

A scalable human-zebrafish xenotransplantation model reveals gastrosome-mediated processing of dying neurons by human microglia

Microglia engulf dying neurons through efferocytosis, a critical function in both development and disease. How microglia process the engulfed neuronal material--especially lipids--remains poorly understood, despite its central role in neurodegeneration. Thus, we developed HuZIBRA, a scalable in vivo xenotransplantation model in which human iPSC-derived microglia-like cells (iMGLs) are introduced into the developing zebrafish brain (zf-hiMG), a system characterized by high levels of neuronal cell death and amenable to precise genetic and pharmacological manipulation. We show that human microglia-like cells recognize and engulf apoptotic zebrafish neurons, indicating conserved efferocytic mechanisms. In these cells, engulfed neuronal material accumulates into a distinct, lipid-rich intracellular compartment, the gastrosome, which we also observed in iMGLs placed in a human brain-like environment. The size of the human gastrosome dynamically reflects neuronal cell death levels and is regulated by key genes, including TREM2 and SLC37A2. Pharmacological inhibition of the cholesterol transporter NPC1 induces gastrosome expansion and lipid accumulation, recapitulating pathological features of Niemann-Pick disease type C. Thus, HuZIBRA provides a powerful in vivo platform to uncover cell-autonomous adaptive responses of human microglia to apoptotic and metabolic stress, with the gastrosome emerging as a key integrator of neuronal debris processing and disease-relevant lipid metabolism.

cell biology↗

bioRxiv2025

Nuclear rerouting of paracrine Fgf3 in source cells represses target genes to pattern morphogen responses

Morphogen gradients direct tissue patterning by inducing dose-dependent transcriptional responses, yet ligand-producing cells often respond differently from their neighbors. Using the zebrafish lateral line organogenesis model, we uncover a cell-autonomous role for the paracrine ligand Fgf3. Transcriptomic profiling and quantitative single-molecule imaging identify target genes, including the chemokine scavenger cxcr7b, whose expression decreases both when FGF receptor signaling is inhibited and when Fgf3 is overexpressed. High-resolution live imaging reveals nuclear accumulation of Fgf3 in producing cells, whereas neighbors receive only extracellular ligand, a feature also observed in other embryonic tissues. Mosaic gain- of-function and nanobody-mediated degradation demonstrate that the nuclear pool of Fgf3 autonomously represses specific targets without impairing canonical receptor signaling. Structure-guided comparative assays indicate nuclear targeting as a latent property of several paracrine FGFs. Dual secreted-nuclear functionality of FGF ligands may represent an intrinsic symmetry-breaking mechanism during organogenesis.

developmental biology↗

bioRxiv2024

In toto analysis of multicellular arrangement reduces embryonic tissue diversity to two archetypes that require specific cadherin expression

Breakthroughs in transcriptomics are providing exciting new ways to characterise cellular diversity within organisms1-3. Yet, organisms are more than the sum of their differentiated cells, as the biological function of most cell types only emerges when they are organised into tissues with characteristic architectures4. The mechanisms that drive architectural diversification at the tissue level remain poorly understood, in part due to a general lack of methods for directly comparing different organisational patterns. Here we establish nuQLOUD, an efficient imaging and computational framework that reduces complex tissues to clouds of nuclear positions, enabling the extraction of cell-type agnostic architectural features. Applying nuQLOUD to whole developing zebrafish embryos reveals that global tissue diversity can be efficiently reduced to two primary archetypes, termed amorphous and crystalline. We investigate the molecular drivers of tissue archetypes by focussing on cadherin cell adhesion molecules and demonstrate that the expression domains of major cadherins segregate along tissue-archetypal lines. Further spatiotemporal analysis and targeted perturbation of cadherin expression in different organs identifies N-Cadherin as a general driver of the amorphous archetype. Thus, this systematic investigation of architectural diversity provides a new way to conceptualise embryonic organisation and understand drivers of tissue diversification within a standardised framework.

developmental biology↗

bioRxiv2022

A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterised by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarised vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterised by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.

cell biology↗

bioRxiv2021

Conserved exchange of paralog proteins during neuronal differentiation

Gene duplication enables the emergence of new functions by lowering the general evolutionary pressure. Previous studies have highlighted the role of specific paralog genes during cell differentiation, e.g., in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. While ~80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B subunits of the COPII complex. Altering the ratio between these two proteins via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

systems biology↗

bioRxiv2020

An Image-Based Data-Driven Analysis of Cellular Architecture in a Developing Tissue

Quantitative microscopy is becoming increasingly crucial in efforts to disentangle the complexity of organogenesis, yet adoption of the potent and ever-growing toolbox provided by modern data science has been slow, primarily because data-driven tools focus on "big data" whereas microscopy produces "rich data". We tackle this issue using a newly developed algorithm for point cloud-based morphometry to unpack the rich information encoded in high-resolution 3D image data into a straightforward numerical representation. This allowed us to employ machine learning for multi-modal data integration of cell morphology, intracellular organization, gene expression and annotated contextual knowledge that facilitates data exploration. We apply these techniques to construct and explore a quantitative atlas of cellular architecture for the zebrafish posterior lateral line primordium, an experimentally tractable model of complex self-organized organogenesis. In doing so, we are able to retrieve both previously established and novel biologically relevant patterns, demonstrating the potential of our data-driven approach.

developmental biology↗