Biology subjects

Bernard, F.

Publications and source records attributed to Bernard, F..

8 recordsLinked to original sources

bioRxiv2026

Quantitative Description of C. elegans mRNA Landscapes From High Coverage Single Cell Transcriptomes

Single-cell RNA sequencing technology dramatically changed the way we investigate transcriptomes. However, the amount and complexity of data generated by such methods poses new challenges for biologists who are trying to extract detailed insights into the genetic programs that drive cellular functions and differentiation. To provide a more intuitive understanding of cell specific gene expression programs, we developed a novel approach for exploiting scRNA-seq data that detects individual gene expression levels in each cell, by avoiding dimensional reduction methods. This was achieved by focusing our analysis on individual cells with a high sequencing coverage (above 15000 Unique Molecular Identifiers (UMIs)). Such High Coverage Cells (HCC), were found in all five C. elegans scRNA-seq datasets we investigated and constitute direct quantitative experimental observations of the mRNA content of individual cells. Clustering the complete gene expression matrix for these cells, we identified gene sets specific for most C. elegans tissues. Among each set we found genes that are dominating cell specific transcriptomes as well as genes that are restricted to particular cell types but are a thousand fold less expressed. For each cell type or subtype we characterized, we identified a set of genes with expression restricted to those cells that were not previously associated with the corresponding tissue. Our results demonstrate that by focusing on HCCs, we can provide high-resolution quantitative descriptions of cellular expression landscapes that are immediately exploitable for researchers to generate new biological hypotheses. Overall, we demonstrate that HCCs represent a powerful and largely unexplored source of biological insights and suggest that future scRNA-seq experiments could benefit from focusing on HCC enrichment to capture and exploit the full complexity of cellular transcriptomes.

systems biology↗

bioRxiv2024

AURA: Automated Universal RNAscope Analysis for high-throughput applications

In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up to 14 mRNA probes per image. We describe procedures for storing raw data, quality control images and the use of a Python app to summarize all the results in one spreadsheet detailing the number of single or co-positive cells.

cell biology↗

bioRxiv2024

Hearing in categories aids speech streaming at the "cocktail party"

Our perceptual system bins elements of the speech signal into categories to make speech perception manageable. Here, we aimed to test whether hearing speech in categories (as opposed to a continuous/gradient fashion) affords yet another benefit to speech recognition: parsing noisy speech at the "cocktail party." We measured speech recognition in a simulated 3D cocktail party environment. We manipulated task difficulty by varying the number of additional maskers presented at other spatial locations in the horizontal soundfield (1-4 talkers) and via forward vs. time-reversed maskers, promoting more and less informational masking (IM), respectively. In separate tasks, we measured isolated phoneme categorization using two-alternative forced choice (2AFC) and visual analog scaling (VAS) tasks designed to promote more/less categorical hearing and thus test putative links between categorization and real-world speech-in-noise skills. We first show that listeners can only monitor up to [~]3 talkers despite up to 5 in the soundscape and streaming is not related to extended high-frequency hearing thresholds (though QuickSIN scores are). We then confirm speech streaming accuracy and speed decline with additional competing talkers and amidst forward compared to reverse maskers with added IM. Dividing listeners into "discrete" vs. "continuous" categorizers based on their VAS labeling (i.e., whether responses were binary or continuous judgments), we then show the degree of IM experienced at the cocktail party is predicted by their degree of categoricity in phoneme labeling; more discrete listeners are less susceptible to IM than their gradient responding peers. Our results establish a link between speech categorization skills and cocktail party processing, with a categorical (rather than gradient) listening strategy benefiting degraded speech perception. These findings imply figure-ground deficits common in many disorders might arise through a surprisingly simple mechanism: a failure to properly bin sounds into categories.

neuroscience↗

bioRxiv2022

Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs

Nematode mRNA processing involves a trans-splicing step through which a 21nt sequence from a snRNP replaces the original 5 end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods. In this study, we used Oxford Nanopores long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrated that spliced leader (SL) sequences presence at the 5 end of the messengers affected library preparation and generated sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we found evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These messengers all share the capacity to generate a 5 terminal hairpin structure mimicking the SL structure providing a mechanistic explanation for their non conformity. Altogether, our data provides the most comprehensive quantitative analysis of SL usage to date in C. elegans.

genomics↗

bioRxiv2022

Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte

Accurate positioning of the nucleus is essential. Microtubules and their associated motors are important players in this process. Although nuclear migration in Drosophila oocytes is controlled by microtubule, a role for microtubule-associated molecular motors in nuclear positioning has yet to be reported. We characterize novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, we report that, prior to migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In absence of Kinesin-1, SPD2 an essential component of the pericentriolar material is increased at the centrosomes, suggesting that Kinesin-1 associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. Our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity. Summary statementIn this study, we identified a crucial role of Kinesin-1 in centrosome clustering required for nuclear positioning and migration in the Drosophila oocyte.

developmental biology↗

bioRxiv2021

Osteoarthritis Early-, Mid- and Late-Stage Progression in the Rat Medial Meniscus Transection Model

Osteoarthritis is a degenerative disease of synovial joints affecting all tissues, including the articular cartilage and underlying subchondral bone. Osteoarthritis animal models can recapitulate aspects of human disease progression and are commonly used to test the development of drugs, biomaterials, and cell therapies for treatment. The rat medial meniscus transection (MMT) model is a surgically induced post-traumatic osteoarthritis model and is one of the most commonly used models for therapeutic development; however, it is typically used to evaluate the efficacy of therapies to prevent disease development rather than testing the treatment of disease progression in already established disease. We describe herein, the qualitative and quantitative changes to articular cartilage, subchondral bone, and formation of osteophytes in rats at early-(3-weeks post-surgery), mid-(6-weeks post-surgery) and late-(12-weeks post-surgery) stages of osteoarthritis progression. Tibiae of MMT-operated animals showed loss of proteoglycan and fibrillation formation on articular cartilage surfaces as early as 3-weeks post-surgery. Using a contrast-enhanced CT technique, quantitative, 3-dimensional analysis of the tibiae showed that the articular cartilage initially thickened at 3- and 6-weeks post-surgery and then decreased at 12-weeks post-surgery. This decrease in cartilage thickness corresponded with increased lesions in the articular cartilage, including fully degraded surfaces down to the subchondral bone layer. In this rat MMT model, subchondral bone thickening was significant at 6-weeks post-surgery and seem to follow cartilage damage. Osteophytes were found at 3-weeks post-surgery, which coincided with articular cartilage degradation. Cartilaginous osteophytes preceded mineralization suggesting that these marginal tissue growths most likely occurred through endochondral ossification. The use of the rat MMT model has predominantly been used out to 3-weeks, and most studies determine the effect of therapies to delay or prevent the onset of osteoarthritis. We provide evidence that an extension of the rat MMT model out to 6 and 12 weeks resembled more severe phenotypes of human osteoarthritis. The mid- to late-stages of rat MMT model can be used to evaluate the therapeutic efficacy of novel treatments to treat the progression of established disease -- since patients typically present in the clinic when the disease is established and becomes symptomatic, thus evaluating the efficacy of new treatments at the late stage will be important for eventual clinical translation.

pathology↗

bioRxiv2020

Phospho-regulated auto-inhibition of Cnn controls microtubule nucleation during cell division

{gamma}-tubulin ring complexes ({gamma}-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within {gamma}-TuRC-tethering proteins, including Drosophila Cnn. Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here we identify an extreme N-terminal "CM1 auto-inhibition" (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits {gamma}-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phospho-mimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to {gamma}-TuRCs is isoform-specific and that mis-regulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is auto-inhibited from binding {gamma}-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to {gamma}-TuRCs is regulated. Summary (for the online JCB table of contents and alerts)We show that auto-inhibition regulates the binding between microtubule nucleating complexes and proteins that tether them to sites of microtubule nucleation. Failure to properly regulate this binding can lead to ectopic cytosolic microtubule nucleation and major defects during cell division.

cell biology↗

bioRxiv2019

The Somatic Golgi nucleates microtubules that are directed by Kinesin-2 to maintain microtubule polarity within neurons

Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that {gamma}-tubulin localises asymmetrically to the somatic Golgi within Drosophila neurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. We propose that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites.

cell biology↗