Biology subjects

Knight, R. D.

Publications and source records attributed to Knight, R. D..

7 recordsLinked to original sources

bioRxiv2025

Open Raman Microscopy (ORM): A Modular Hardware and Software Framework for Accessible Raman Imaging

Raman microscopy is a label-free, non-destructive imaging tool for spatially resolved chemical fingerprinting. Its powerful ability to reveal molecular information has driven rapid growth in applications across fields as varied as materials science, environmental analysis, and biomedical research. Despite its versatility, the accessibility of Raman microscopy is limited by expensive commercial setups and the technical barriers faced by researchers attempting to build custom systems. Here, we introduce an Open Raman Microscopy (ORM) framework based on a readily accessible modular microscopy platform. The ORM platform provides configurations for both high-throughput imaging and confocal imaging. We developed a dedicated python-based control and acquisition software, the ORM-Integrated Raman and Imaging Software (ORM-IRIS) designed to accommodate modular integration and control of components, including the laser source, spectrometer, and translational stages. Implemented across three institutions we demonstrate the ORM platform for high-throughput imaging of articular cartilage tissue, confocal three-dimensional imaging of a zebrafish embryo, and imaging of gold colloid decorated surfaces for surface enhanced Raman spectroscopy. Together, this open-source hardware and software framework enhances the accessibility of Raman microscopy across an expanding range of scientific applications.

biophysics↗

bioRxiv2025

Muscle regeneration can be rescued in a telomerase deficient zebrafish model of ageing by MMP inhibition

Ageing progressively impairs skeletal muscle regeneration, contributing to reduced mobility and quality of life in the ageing population. Whilst the molecular changes underlying muscle ageing have been well characterised, their impact on muscle stem cell (muSC) behaviour during regeneration remains poorly understood. Here, we leverage the telomerase-deficient tert mutant zebrafish larvae as an in vivo model of accelerated ageing to perform real-time analysis of muSC dynamics following muscle injury. We demonstrate that the ageing-like inflammatory environment in tert mutant disrupts muSC migration, impairs activation and proliferation, and compromises regenerative capacity. We further show that sustained inflammation, mediated by persistent macrophage presence and elevated matrix metalloproteinase (MMP) activity directly limits muSC recruitment and migration efficiency. Pharmacological inhibition of MMP9/13 activity and genetic depletion of macrophages partially restore muSC migratory behaviour and regenerative outcomes. Notably, we demonstrate that muSC migration dynamics correlate with regenerative success, providing a functional readout for therapeutic screening. Our findings reveal zebrafish tert mutants offer a tractable system for dissecting ageassociated changes to cell behaviour and for identifying rejuvenation interventions.

cell biology↗

bioRxiv2021

Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.

developmental biology↗

bioRxiv2021

Systems analysis reveals ageing-related perturbations in retinoids and sex hormones in Alzheimer's and Parkinson's diseases

Neurodegenerative diseases (NDDs), including Alzheimers (AD) and Parkinsons diseases (PD), are complex heterogeneous diseases with highly variable patient responses to treatment. Due to the growing evidence for ageing-related clinical and pathological commonalities between AD and PD, these diseases have recently been studied in tandem. In this study, we analyse transcriptomic data from AD and PD patients, and stratify these patients into three subclasses with distinct gene expression and metabolic profiles. Through integrating transcriptomic data with a genome-scale metabolic model and validating our findings by network exploration and co-analysis using a zebrafish ageing model, we identify retinoids as a key ageing-related feature in all subclasses of AD and PD. We also demonstrate that the dysregulation of androgen metabolism by three different independent mechanisms is a source of heterogeneity in AD and PD. Taken together, our work highlights the need for stratification of AD/PD patients and development of personalised and precision medicine approaches based on the detailed characterisation of these subclasses.

systems biology↗

bioRxiv2020

Myofiber stretch induces tensile and shear deformation of muscle stem cells in their native niche

BackgroundMuscle stem cells (MuSCs) are requisite for skeletal muscle regeneration and homeostasis. Proper functioning of MuSCs, including activation, proliferation, and fate decision, is determined by an orchestrated series of events and communication between MuSCs and their niche consisting of the host myofiber and neighbouring cells. A multitude of biochemical stimuli are known to regulate fate and function of MuSCs. However, in addition to biochemical factors, it is conceivable that MuSCs residing between basal lamina and sarcolemma of myofibers are subjected to mechanical forces during muscle stretch-shortening cycles due to myofascial connections between MuSCs and myofibers. MuSCs have been shown to respond to mechanical forces in vitro but it remains to be proven whether physical forces are also exerted on MuSCs in their native niche and whether they contribute to the functioning and fate of MuSCs. MethodsMuSCs deformation in their native niche resulting from mechanical loading of ex vivo myofiber bundles were visualized utilizing mT/mG double-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 hour pulsating fluid shear stress with a peak shear stress rate of 8.8 Pa/s. After treatment, nitric oxide and mRNA expression levels of genes involved in regulation of MuSC proliferation and differentiation were determined. ResultsEx vivo stretching of extensor digitorum longus and soleus myofiber bundles caused compression as well as tensile and shear deformation of MuSCs in their niche. MuSCs responded to pulsating fluid shear stress in vitro with increased nitric oxide production and an upward trend in iNOS mRNA levels, while nNOS expression was unaltered. Pulsating fluid shear stress enhanced gene expression of c-Fos, Cdk4, and IL-6, while expression of Wnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, and MGF remained unchanged. ConclusionsWe conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive as evident by pulsating fluid shear stress-mediated expression of factors by MuSCs known to promote proliferation.

cell biology↗

bioRxiv2019

Cell Tracking Profiler: a user-driven analysis framework for evaluating 4D live cell imaging data

Accurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo. Using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm we have established a ground truth for muSC cell behaviour. This revealed that variability in segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) operates through the freely available Icy platform, and allows user-managed cell tracking from 3D time-lapsed datasets to provide measures of cell shape and movement. Using dimensionality reduction methods, multiple correlation and regression analyses we identify myosin II-dependent parameters of muSC behaviour during regeneration. CTP and the associated statistical tools we have developed thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets. SummaryAnalysis of cell shape and movement from 3D time-lapsed datasets is currently very challenging. We therefore designed Cell Tracking Profiler for analysing cell behaviour from complex datasets and demonstrate its effectiveness by analysing stem cell behaviour during muscle regeneration in zebrafish.

cell biology↗

bioRxiv2016

Myotome adaptability confers developmental robustness to somitic myogenesis in response to fibre number alteration.

Summary StatementHomeostatic interactions between muscle stem cells and fibres during myogenesis ensure the correct muscle size is formed independent of fibre number in zebrafish\n\nAbstractBalancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how muscle stem/precursor cell (MPC) numbers are tightly regulated during zebrafish somitic muscle development. MPCs expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myodfh261 mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7+ MPC population in the DM. Subsequently, myodfh261 mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myodfh261 mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myodfh261 mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size.

developmental biology↗