Biology subjects

Sutcliffe, C.

Publications and source records attributed to Sutcliffe, C..

8 recordsLinked to original sources

bioRxiv2025

Characterisation of the dual roles of senescent-like T cells that arise during healthy and unhealthy ageing

Ageing is accompanied by progressive remodelling of the immune system, but chronic metabolic disease may accelerate this process and drive qualitatively distinct forms of immune dysfunction. Here, we used type 2 diabetes (T2D) as a model of unhealthy immune ageing to identify a distinct population of CD8 TEMRA cells that accumulates in older individuals with T2D. These cells were highly differentiated, oligoclonally expanded and had shorter telomeres, consistent with an increased replicative history and premature senescence-like state. Unlike conventional TEMRA cells, which can preserve cytotoxic function through acquisition of NK-cell receptors, T2D-associated TEMRA cells showed reduced surface expression of KLRG1, NKG2D and NKG2A together with defective receptor recycling. TGF{beta}1 was elevated in T2D and reproduced several features of this phenotype in vitro, including increased TEMRA differentiation, reduced NK-receptor expression, altered receptor trafficking and induction of p21. Functionally, these cells displayed reduced TCR-triggered degranulation, correlating with impaired cytotoxicity and altered tissue distribution in individuals with T2D. Rather than providing effective immune surveillance, the accumulation of these highly differentiated TEMRA cells with diminished effector capacity may compromise immune function. Together, these findings identify a distinct senescent-like CD8 TEMRA state linking metabolic inflammation to dysregulated T cell differentiation.

immunology↗

bioRxiv2025

Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

The regulation of mRNA decay is important for numerous cellular and developmental processes. Here, we use the patterning gene even-skipped (eve) in the early Drosophila embryo to investigate the contribution of mRNA decay to shaping the mature striped expression pattern. Using mathematical models to analyse live and fixed imaging data, we show that for eve stripes 2 and 4, spatially regulated degradation rates outperform models with constant or mRNA age-dependent decay. Uniform mRNA decay rates perturb definition of eve stripes 2 and 4 in silico, and we show that altering eve mRNA numbers in vivo by changing the UTR results in dysregulation of the anterior-posterior pair-rule network and embryo segmentation defects. Overall, these data demonstrate how eve mRNA instability can function with transcriptional regulation to define sharp expression domain borders. We suggest that spatially regulated mRNA stability may be widely used to sculpt expression patterns during development.

developmental biology↗

bioRxiv2025

Exploiting the SunTag system to study the developmental regulation of mRNA translation

The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila. Repression of the maternal hunchback (hb) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantitate translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky as [~]5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different UTRs. We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantitate translation of the short gastrulation (sog) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.

developmental biology↗

bioRxiv2024

Ovarian germline stem cell dedifferentiation is cytoneme-dependent

Progenitor cell dedifferentiation is important for stem cell maintenance during tissue repair and age-related stem cell decline. Here, we use the Drosophila ovary as a model to study the role of cytonemes in BMP signalling-directed germline stem cell (GSC) maintenance and dedifferentiation of germ cells to GSCs. We provide evidence that differentiating germ cell cysts extend longer cytonemes that are more polarised towards the niche during dedifferentiation to reactivate BMP signalling. The presence of additional somatic cells in the niche is associated with a failure of germ cell dedifferentiation, consistent with the formation of a physical barrier to cytoneme-niche contact and outcompetition of germ cells for BMP. Using BMP beads in vitro, we show that these are sufficient to induce cytoneme-dependent contacts in Drosophila tissue culture cells. We demonstrate that the Enabled (Ena) actin polymerase is localised to the tips of germ cell cytonemes and is necessary for robust cytoneme formation, as its mislocalisation reduces the frequency, length and directionality of cytonemes. During homeostasis, specifically perturbing cytoneme function through Ena mislocalisation impairs GSC fitness by reducing GSC BMP signalling and niche occupancy. Disrupting cytonemes by targeting Ena during dedifferentiation reduces germ cell BMP responsiveness and the ability of differentiating cysts to dedifferentiate. Overall, our results provide evidence that cytonemes play a fundamental role in establishing polarised signalling and niche occupancy during stem cell maintenance and dedifferentiation. Significance StatementFertility depends not only on germline stem cell (GSC) maintenance during homeostasis, but also on the ability of germ cells to reverse their developmental programme and dedifferentiate during ageing- or stress-induced GSC loss. Here, using the Drosophila ovary as a model, we show that cytonemes are required for GSC asymmetric signalling, niche occupancy and fitness during homeostasis. During dedifferentiation, we demonstrate that cytonemes are necessary for differentiating germ cells to re-access the self-renewal signal and establish polarised signalling, recolonise an empty niche and dedifferentiate to germline stem cells. Our data identify cytonemes as a novel target for strategies aimed at improving stem cell fitness or dedifferentiation, which has huge therapeutic potential for the regeneration of damaged tissues.

developmental biology↗

bioRxiv2024

A simple MiMIC based approach for tagging endogenous genes to visualise live transcription in vivo

Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem loops into endogenous genes requires laborious CRISPR genome editing. Here we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging and live imaging transcription of endogenous genes and the long non-coding RNA, roX1, in the embryo. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.

developmental biology↗

bioRxiv2022

Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila

A BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. In this study, we combine genome editing, structural and developmental approaches to study Sog function in Drosophila. We generate a sog knockout fly stock, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we test the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the SogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, we use experimental negative stain EM imaging and hydrodynamic data to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. We discuss how this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation. Summary statementThe authors model the structure of Sog and establish a new sog knockout fly stock that they validate for the testing of specific sog mutations.

developmental biology↗

bioRxiv2021

Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo

The Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. Here we adapt the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using singlemolecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

developmental biology↗

bioRxiv2019

Modulation of promoter occupancy dictates the transcriptional response to graded BMP signalling levels in the Drosophila embryo

SUMMARYMorphogen gradients specify cell fates during development, with a classic example being the BMP gradient’s conserved role in embryonic dorsal-ventral axis patterning. Here we elucidate how the BMP gradient is interpreted in the Drosophila embryo, by combining live imaging with computational modelling to infer transcriptional burst parameters at single cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, we show that BMP signaling controls burst frequency by regulating the promoter activation rate. We provide evidence that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. We suggest that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell fate decisions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗