Biology subjects

Ashe, H. L.

Publications and source records attributed to Ashe, H. L..

11 recordsLinked to original sources

bioRxiv2025

Genomic foundation models reveal RNA-binding proteins regulating translation and mRNA degradation during Drosophila development

BackgroundThe regulation of mRNA decay and translation is crucial for cellular function and development; however, the complex interplay of RNA-binding proteins (RBPs) regulating these processes remains incompletely understood. Recent advances in genomic foundation models present new opportunities for decoding the regulatory grammar embedded within mRNA untranslated regions (UTRs). Here, we leverage explainable artificial intelligence to systematically identify RBP motifs that influence translation and mRNA decay during Drosophila melanogaster development. ResultsWe extended the training of GENA-LM Fly, a genomic foundation model, on all 5 and 3 UTR pairs from the D. melanogaster genome. We separately fine-tuned it using ribosome density (RD) and mRNA decay (half-life) data from the embryonic maternal-to-zygotic transition (MZT). Using SHapley Additive exPlanations (SHAP) analysis, we identified the sequence regions most influential for prediction and performed motif enrichment analysis to discover associated RBP binding sites. We identified 42 unique RBPs associated with increasing (n=23; e.g., Rnp4f, Mxt) or decreasing (n=19; e.g., Aret/Bruno, Rox8, Sxl, Orb2) RD and 18 unique RBPs associated with increasing (n=6) and decreasing (n=12; e.g., Cnot4, Rbp9, Rox8) mRNA half-life. Using publicly available PAR-CLIP data, we validated our Orb2 signal in a Drosophila cell line. Furthermore, feature ablation and shuffling experiments revealed the contributions of different sequence components to model performance. Our approach significantly outperformed naive high-versus-low RD comparisons, demonstrating the power of model explainability in biological discovery. ConclusionsThis study demonstrates that genomic foundation models, when combined with explainability methods, can discover meaningful biology even without drastically improving the underlying prediction accuracy. The identified RBP motifs provide new insights into post-transcriptional regulatory elements that govern RD and decay during early development.

bioinformatics↗

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↗

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↗

bioRxiv2024

An avidity-driven mechanism of extracellular BMP regulation by Twisted gastrulation

During dorsoventral patterning of bilaterian embryos, the conserved regulator Twisted gastrulation (Tsg) modulates BMP signalling by binding Chordin/Short gastrulation (Sog). Here we elucidate the mechanism by which Tsg interacts with Sog/Chordin to promote formation of the inhibitory Tsg-Sog/Chordin-BMP complex and regulate BMP signalling extracellularly. We identify and validate in vitro a hydrophobic interface in the Tsg C-terminal domain that binds Chordin. Mutation of this epitope in Drosophila Tsg (TsgL100A) results in an unexpectedly mild perturbation to embryonic BMP gradient formation. We show that a protosome-specific Tsg C-terminal extension also binds Sog, and the presence of this second binding site allows partial rescue of Sog interaction with TsgL100A in the presence of BMP. Consistent with this, a truncated Tsg protein lacking both Sog binding regions is unable to support BMP gradient formation in vivo. As our data show that disruption of either Sog binding site in Tsg, but not both, can be overcome by Tsg-BMP and Sog-BMP interactions, we present a new avidity-driven mechanism of BMP gradient formation that will be relevant to a broad range of developmental contexts. Summary statementIdentification and mutational analysis of the binding epitopes that mediate Twisted gastrulation interaction with Short gastrulation/Chordin reveals an avidity-based model of embryonic BMP gradient formation.

developmental biology↗

bioRxiv2023

Modulation of transcription burst amplitude underpins dosage compensation in the Drosophila embryo

Dosage compensation, the balancing of X linked gene expression between sexes and to the autosomes, is critical to an organisms fitness and survival. In Drosophila, dosage compensation involves hypertranscription of the male X chromosome. Here we use quantitative live imaging and modelling at single-cell resolution to determine the mechanism underlying X chromosome dosage compensation in Drosophila. We show that the four X chromosome genes studied undergo transcriptional bursting in male and female embryos. Mechanistically our data reveal that transcriptional upregulation of male X chromosome genes is primarily mediated by a higher RNA polymerase II initiation rate and burst amplitude across the expression domain. In contrast, burst frequency is spatially modulated in nuclei within the expression domain in response to different transcription factor concentrations to tune the transcriptional response. Together, these data show how the local and global regulation of distinct burst parameters establish the complex transcriptional outputs underpinning developmental patterning.

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↗

bioRxiv2022

Modelling global mRNA dynamics during Drosophila embryogenesis reveals a relationship between mRNA degradation and P-bodies

Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single molecule imaging we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3 ends in P-bodies in the early embryo, consistent with 5 to 3 degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.

developmental biology↗

bioRxiv2020

Scalable inference of transcriptional kinetic parameters from MS2 time series data

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSMotivationC_ST_ABSThe MS2-McP (MS2 coat protein) live imaging system allows for visualisation of transcription dynamics through the introduction of hairpin stem-loop sequences into a gene. A fluorescent signal at the site of nascent transcription in the nucleus quantifies mRNA production. computational modelling can be used to infer the promoter states along with the kinetic parameters governing transcription, such as promoter switching frequency and polymerase loading rate. However, modelling of the fluorescent trace presents a challenge due its persistence; the observed fluorescence at a given time point depends on both current and previous promoter states. A memory-adjusted Hidden Markov Model (mHMM) was recently introduced to allow inference of promoter activity from MS2-McP data. However, the computational time for inference scales exponentially with gene length and the mHMM is therefore not currently practical for application to many eukaryotic genes. ResultsWe present a scalable implementation of the mHMM for fast inference of promoter activity and transcriptional kinetic parameters. This new method can model genes of arbitrary length through the use of a time-adaptive truncated compound state space. The truncated state space provides a good approximation to the full state space by retaining the most likely set of states at each time during the forward pass of the algorithm. Testing on MS2-MCP fluorescent data collected from early Drosophila melanogaster embryos indicates that the method provides accurate inference of kinetic parameters within a computationally feasible timeframe. The inferred promoter traces generated by the model can also be used to infer single-cell transcriptional parameters. AvailabilityPython implementation available at https://github.com/ManchesterBioinference/burstInfer, along with code to reproduce the examples presented here.

bioinformatics↗

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↗

bioRxiv2018

Drosophila ovarian germline stem cell cytocensor projections dynamically receive and attenuate BMP signalling

In the Drosophila ovarian germline, BMP signals released by niche cells promote germline stem cell (GSC) maintenance. Although BMP signalling is known to repress expression of a key differentiation factor, it remains unclear whether BMP-responsive transcription also contributes positively to GSC identity. Here, we identify the GSC transcriptome using RNA-seq, including the BMP-induced transcriptional network. Based on these data, we provide evidence that GSCs form two types of cellular projections. Genetic manipulation and live ex vivo imaging reveal that both classes of projection allow GSCs to access a reservoir of Dpp held away from the GSC-niche interface. Moreover, the microtubule-rich projections, termed cytocensors, formed downstream of BMP have additional functionality, which is to attenuate BMP signalling, allowing dynamic modulation of signal transduction to facilitate differentiation following GSC division. We propose that cytocensors will be synthesised by other types of cells in diverse contexts to calibrate signalling responses.

developmental biology↗