Biology subjects

Novault, S.

Publications and source records attributed to Novault, S..

6 recordsLinked to original sources

bioRxiv2026

Single-cell spatially resolved transcriptomic characterization of the developing mouse cochlea

The cochlea, the sensory organ of hearing, functions as a frequency analyzer, analogous to a musical instrument. During development, while the medio-lateral axis supports differentiation of sensory cells and their surrounding supporting cells, the longitudinal axis underlies frequency-dependent properties of the cochlea. The combination of these two gene expression gradients defines unique physiological attributes of each cell intimately linked to its position within the cochlea. To determine which cochlear cell-types have a transcriptomic signature sensitive to these two gradients and identify the underlying gene regulatory networks, we took advantage of the advent of spatial single cell transcriptomics methodologies. We therefore generated a spatial transcriptomic atlas reaching single cell resolution based on the Visium HD technique, a sequencing-based technology that employ arrays of spatially barcoded probes to capture RNA molecules unbiasedly from histological tissue sections. Spatial transcriptional changes during embryonic stages, E14 and E16, as well as during postnatal development, P1 and P8, were investigated. Based on this dataset, not only cell-type assignment in single cell RNA-seq experiments could be validated, but the classification for some cell-types could be refined. Gradients of gene expression along the medio-lateral and longitudinal axes in multiple cell-types together with their temporal dynamics across development were also uncovered. Altogether, this atlas paves the way for deciphering gene regulatory networks controlling gene expression as a function of position in the cochlear cell types, providing a valuable resource for the design of efficient, robust and safe gene therapy strategies.

cell biology↗

bioRxiv2023

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

cell biology↗

bioRxiv2023

Extracellular vesicles production regulates fluconazole resistance in Cryptococcus neoformans

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCEExtracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

microbiology↗

bioRxiv2022

TATTOO-seq delineates spatial and cell type-specific regulatory programs during limb patterning

The coordinated differentiation of progenitor cells into specialized cell types and their spatial organization into distinct domains is central to embryogenesis. Here, we applied a new unbiased spatially resolved single-cell transcriptomics method to identify the genetic programs that underlie the emergence of specialized cell types during limb development and their integration in space. We uncovered combinations of transcription factors whose expression patterns are predominantly associated with cell type specification or spatial position, enabling the deconvolution of cell fate and position identity. We demonstrate that the embryonic limb undergoes a complex multi-scale re-organization upon perturbation of one of its spatial organizing centers, including the loss of specific cell populations, specific alterations in the molecular identities of other pre-existing cell states and changes in their relative spatial distribution. Altogether, our study shows how multi-dimensional single-cell and spatially resolved molecular atlases could reveal the interconnected genetic networks that regulate the intricacies of organogenesis and its reorganization upon genetic alterations.

systems biology↗

bioRxiv2021

Identification of fetal liver stromal subsets in spectral cytometry using the parameter autofluorescence

The fetal liver is the main hematopoietic organ during embryonic development. The fetal liver is also the unique anatomical site where hematopoietic stem cells expand before colonizing the bone marrow, where they ensure life-long blood cell production and become mostly resting. The identification of the different cell types that comprise the hematopoietic stroma in the fetal liver is essential to understand the signals required for the expansion and differentiation of the hematopoietic stem cells. We used a panel of monoclonal antibodies to identify fetal liver stromal cells in a 5-laser equipped spectral flow cytometry analyzer. The "Autofluorescence Finder" of SONY ID7000 software identified two distinct autofluorescence emission spectra. Using autofluorescence as a fluorescence parameter we could assign the two autofluorescent signals to three distinct cell types and identified surface markers that characterize these populations. We found that one autofluorescent population corresponds to hepatoblasts and cholangiocytes whereas the other expresses mesenchymal transcripts and was identified as stellate cells. Importantly, after birth, autofluorescence becomes the unique identifying property of hepatoblasts because mature cholangiocytes are no longer autofluorescent. These results show that autofluorescence used as a parameter in spectral flow cytometry is a useful tool to identify new cell subsets that are difficult to analyze in conventional flow cytometry.

developmental biology↗

bioRxiv2020

New insights into Cryptococcus extracellular vesicles suggest a new structural model and an antifungal vaccine strategy

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

microbiology↗