Biology subjects

Ford, M. J.

Publications and source records attributed to Ford, M. J..

9 recordsLinked to original sources

bioRxiv2026

FluoroFate: A generalisable platform for time-resolved single-cell analysis of cell fate enables quantification of cell death dynamics

Fundamental cellular decisions of life and death are governed by intricate and tightly regulated intracellular signalling pathways that determine whether cells proliferate, enter quiescence, or undergo programmed cell death (apoptosis). Live-cell fluorescence imaging enables these processes to be observed in real time at single-cell resolution, but two problems limit their study. First, existing biosensors do not allow apoptotic status and cell cycle progression to be resolved in tandem within the same cell. Second, interpreting live-cell imaging data is challenging even where multiplex reporters exist, as the biological meaning of fluorescent signals depends on their temporal ordering, and large-scale imaging experiments generate complex, multidimensional data that are difficult to analyse systematically and at scale. Here we address both problems. We present FluoroFate, a generalisable and user-friendly graphical interface-driven tool for time-resolved single-cell analysis of multiplex live-cell imaging datasets, which integrates existing, robust deep learning-based segmentation, cell tracking, and temporal classification methods to quantify fluorescent reporter dynamics in individual cells across time without the need for specialist computational expertise. Alongside FluoroFate, we develop tricistronic Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci) and apoptosis biosensors, enabling simultaneous monitoring of cell cycle progression and caspase activation within the same cell. Applying FluoroFate, we resolve apoptotic and non-apoptotic cell death at the single-cell level based on the temporal ordering of Annexin V and propidium iodide signals, identifying distinct kinetic and phenotypic cell death profiles in response to pharmacological perturbation. We highlight divergent temporal dynamics and modes of cell death between birinapant and cycloheximide treatment, reflecting differences in how TNF/TNFR1 signalling is disrupted by these agents. At the single-cell level, we uncover parallel, independently regulated death programmes, demonstrating that loss of RIPK1 selectively impairs apoptotic cell death whilst leaving non-apoptotic death largely unaffected. We then use FluoroFate to analyse timelapse images of our combined Fucci-apoptosis reporters, resolving cell cycle progression and caspase activation within the same cell over time. Together, FluoroFate and our new cell cycle and apoptosis biosensors represent a broadly applicable platform for extracting mechanistic insight from live-cell imaging data.

cell biology↗

bioRxiv2024

Aging-associated vacuolation of multi-ciliated cells in the distal mouse oviduct reflects unique cell identity and luminal microenvironment

The female reproductive organs present with the earliest aging characteristics, such as a decline in fertility and estrous cyclicity. While age-related changes in the ovary are well-documented, it is unclear if any age-associated changes occur in the other female reproductive organs, such as the oviduct/fallopian tube. The recent recognition of the distal end of the fallopian tube as the tissue of origin of high-grade serous tubal ovarian carcinomas (HGSCs), and that its patient demographic is strongly biased to postmenopausal women, motivated us to investigate age-associated changes in this organ. At the distal end of aged oviducts in mice, we found vacuolated multi-ciliated cells (MCCs) with a severely apically displaced and deformed nucleus. This phenotype was unique to the distal oviduct epithelium -infundibulum (INF) and ampulla (AMP). Ovariectomy did not affect the timeline of MCC vacuolation, suggesting little involvement of ovulation and hormonal regulation. MCC vacuolation was induced in hypoxia or hydroxyurea treatments in in vitro organotypic culture of all oviduct regions, not limited to the INF/AMP epithelium. This suggests high oxygen demand in MCCs, compared to other cell types, and a uniquely stressed INF/AMP epithelial microenvironment in vivo. We found that the blood circulation of INF/AMP depended on the ovarian artery, different from the rest of the oviduct epithelium and its circulation declined along with ovarian activities. We conclude that a decline in local blood circulation and distinct cellular identity of the INF/AMP epithelium caused age-associated MCC vacuolation, reflecting its mild, chronically stressed microenvironment.

physiology↗

bioRxiv2021

Modeling High-grade serous ovarian carcinoma using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing

Ovarian cancer remains the most lethal gynecological cancer today. High-grade serous ovarian carcinoma (HGSC) is the most common and lethal type of ovarian cancer and is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing. We mutated tumor suppressor genes associated with HGSC in two different combinations; Brca1, Tp53, Pten with/without Lkb1 and successfully generated HGSC, however, with different latencies and pathophysiology. By utilizing Cre lineage tracing in our system, we visualized peritoneal micrometastases in an immune-competent environment. Because our strategy is flexible in selecting mutation combinations and targeting areas, it would be useful for generating ovarian cancer mouse models. Significance StatementHigh grade serous ovarian cancer (HGSC) is the most common ovarian malignancy but our knowledge of early tumorigenesis is still quite limited due to late diagnosis of patients. We developed a new strategy of generating mouse ovarian cancer models using a combination of in vivo electroporation and CRISPR-mediated genome editing. We demonstrated that a combination of three tumor suppressor gene mutations (Brca1, Tp53 and Pten) is sufficient to develop HGSCs. Interesting, an additional mutation in Lkb1 drastically changed tumor latency, penetrance and pathophysiology through changing its cell-of-origin. Our strategy is highly flexible in selection of mutation combinations and targeting areas in immune competent mice and useful to study early tumorigenesis of ovarian cancer.

cancer biology↗

bioRxiv2020

Anatomical and cellular heterogeneity in the mouse oviduct-- its potential roles in reproduction and preimplantation development

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive tissue that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are basic coiling patterns and turning points in the coiled mouse oviduct can serve as reliable landmarks for luminal morphological regionalities. We identified previously unrecognized anatomical structures in the isthmus and uterotubal junction (UTJ) that likely play important roles in reproduction. Interestingly, during ovulation, the isthmus was transiently plugged by a thick mucus, keeping the oocytes within the ampulla. Preimplantation embryos travelled along the oviduct and formed a queue within small compartments of the UTJ before uterine entry. Taken together, the oviduct luminal epithelium had highly diverse luminal structures with distinct cell populations reflecting its complex functions in reproduction.

physiology↗

bioRxiv2020

Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis

Owing to technical advances in single cell biology, the appreciation of cellular heterogeneity has increased, which has aided our understanding of organ function, homeostasis and disease progression. The oviduct (also known as the fallopian tube in humans) is the distal-most portion of the female reproductive tract. It is essential for reproduction and the proposed origin of high grade serous ovarian carcinoma (HGSOC). In mammals, the oviduct is morphologically segmented along the ovary-uterus axis into four evolutionally conserved regions. It is unknown however if there is a diversification of epithelial cell characteristics between these regions. In this study, we identified transcriptionally distinct populations of secretory and multiciliated cells restricted to the distal and proximal regions of the oviduct. We demonstrated that these distal and proximal populations are distinct lineages specified early in Mullerian duct development and are maintained separately. These results aid our understanding of epithelial development, homeostasis and initiation of disease from the oviduct.

developmental biology↗

bioRxiv2020

Prom1 expression does not mark a stem/progenitor population in the mouse oviduct epithelium

The oviduct or fallopian tube is the site of fertilization and preimplantation embryonic development. The epithelium lining the oviduct consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis still remains poorly understood. CD133/Prom1 has been used to identify adult stem cell populations in various organs and often associated with cancer stem cell property. Using a Cre-recombinase based lineage tracing strategy, we found that CD133/Prom1 expression was not associated with a stem/progenitor population in the oviduct but marked a sub population of multiciliated and secretory cells which did not propagate. Interestingly, Prom1 expressing secretory cells rapidly transition to multiciliated cells and progressively migrate to the tips of epithelial folds in the ampulla. Our results show that CD133/Prom1 expression cannot be used as a progenitor/stem cell marker in the mouse oviduct.

developmental biology↗

bioRxiv2020

A mobile sex-determining region, male-specific haplotypes, and rearing environment influence age at maturity in Chinook salmon

Variation in age at maturity is an important contributor to life history and demographic variation within and among species. The optimal age at maturity can vary by sex, and the ability of each sex to evolve towards its fitness optimum depends on the genetic architecture of maturation. Using GWAS of RAD sequencing data, we show that age at maturity in Chinook salmon exhibits sex-specific genetic architecture, with age at maturity in males governed by large (up to 20Mb) male-specific haplotypes. These regions showed no such effect in females. We also provide evidence for translocation of the sex-determining gene between two different chromosomes. This has important implications for sexually antagonistic selection, particularly that sex-linkage of adaptive genes may differ within and among populations based on chromosomal location of the sex-determining gene. Our findings will facilitate research into the genetic causes of shifting demography in Chinook salmon as well as a better understanding of sex-determination in this species and Pacific salmon in general.

evolutionary biology↗

bioRxiv2018

Functionally Coherent Transcription Factor Target Networks Illuminate Control of Epithelial Remodelling and Oncogenic Notch

BackgroundCell identity is governed by gene expression, regulated by Transcription Factor (TF) binding at cis-regulatory modules. Decoding the relationship TF binding patterns and the regulation of cognate target genes is nontrivial, remaining a fundamental limitation in understanding cell decision-making mechanisms. Identification of TF physical binding that is biologically neutral is a current challenge. We studied cell identity in the context of Epithelial to Mesenchymal Transition (EMT), a cell programme fundamental for normal embryonic development that contributes to tumour progression and fibrosis.\n\nResultsWe developed the NetNC software for discovery of functionally coherent TF targets. NetNC was applied to analyse gene regulation by the EMT TFs Snail, Twist in early embryogenesis and also to modENCODE HOT regions. Predicted neutral binding accounted for 50% to [≥]80% of candidate target genes assigned from significant binding peaks. Novel gene functions and network modules were identified, including regulation of chromatin organisation and crosstalk with notch signalling. Orthologues of predicted TF targets discriminated breast cancer molecular subtypes and NetNC analysis predicted new gene functions; for example, evidencing networks that reshape Waddingtons landscape during EMT-like phenotype switching. Predicted invasion roles for SNX29, ATG3, UNK and IRX4 were validated using a tractable cell model.\n\nConclusionsWe found extensive neutral TF binding across the nine datasets examined and showed that NetNC performs well in identifying functionally coherent targets. HOT regions had comparatively high functional coherence. Our results illuminate conserved molecular networks that regulate epithelial remodelling in development and disease, with potential implications for precision medicine.

systems biology↗

bioRxiv2017

A Multi-Stage Representation Of Cell Proliferation As A Markov Process

The stochastic simulation algorithm commonly known as Gillespies algorithm (originally derived for modelling well-mixed systems of chemical reactions) is now used ubiquitously in the modelling of biological processes in which stochastic effects play an important role. In well mixed scenarios at the sub-cellular level it is often reasonable to assume that times between successive reaction/interaction events are exponentially distributed and can be appropriately modelled as a Markov process and hence simulated by the Gillespie algorithm. However, Gillespies algorithm is routinely applied to model biological systems for which it was never intended. In particular, processes in which cell proliferation is important (e.g. embryonic development, cancer formation) should not be simulated naively using the Gillespie algorithm since the history-dependent nature of the cell cycle breaks the Markov process. The variance in experimentally measured cell cycle times is far less than in an exponential cell cycle time distribution with the same mean.\n\nHere we suggest a method of modelling the cell cycle that restores the memoryless property to the system and is therefore consistent with simulation via the Gillespie algorithm. By breaking the cell cycle into a number of independent exponentially distributed stages we can restore the Markov property at the same time as more accurately approximating the appropriate cell cycle time distributions. The consequences of our revised mathematical model are explored analytically as far as possible. We demonstrate the importance of employing the correct cell cycle time distribution by recapitulating the results from two models incorporating cellular proliferation (one spatial and one non-spatial) and demonstrating that changing the cell cycle time distribution makes quantitative and qualitative differences to the outcome of the models. Our adaptation will allow modellers and experimentalists alike to appropriately represent cellular proliferation - vital to the accurate modelling of many biological processes - whilst still being able to take advantage of the power and efficiency of the popular Gillespie algorithm.

cell biology↗