Biology subjects

Schmutz, S.

Publications and source records attributed to Schmutz, S..

9 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↗

bioRxiv2026

Bacterial targeting of host paraspeckles uncovers a new SFPQ-based regulation

Nuclear functions are key in protecting cells against infections, yet intracellular pathogens like Legionella pneumophila can exploit these mechanisms to survive. We characterized a L. pneumophila protein, LpDot1, which shares sequence similarity with the eukaryotic catalytic domain of the histone methyltransferase DOT1. Structure determination, together with biochemical and biophysical analyses, revealed that LpDot1 methylates non-histone nuclear proteins, notably the splicing factor proline-glutamine rich protein (SFPQ). Importantly, LpDot1 targets the previously uncharacterized K518, located on an important structural motif of SFPQ, therefore impairing its dimerization in vitro. During infection, L. pneumophila modulates SFPQ abundance and activities in a LpDot1-dependent manner, thereby hijacking paraspeckle organization and the host cell splicing machinery, leading to alternative splice variants of infection related genes such as NF-kB2 and CD45. To our knowledge, this is the first report of a bacterial effector directly modifying paraspeckle dynamics, providing new insight into previously uncharacterized eukaryotic regulatory pathways.

microbiology↗

bioRxiv2024

Single-cell and in situ spatial analyses reveal the diversity of newly born hematopoietic stem cells and of their niches

Hematopoietic stem cells (HSCs) and more committed progenitors (collectively referred to as HSPCs) emerge from vessels during development, via Endothelial-to-Hematopoietic Transition (EHT). Recently, using the zebrafish embryo, we showed that two EHT cell types emerge from the aorta, raising the question of their subsequent fate. To address this issue, we established a complex pipeline based on single-cell photoconversion and transgenic lines to characterize the transcriptomic profiles of single EHT cell type progenies. We obtained, at unprecedented resolution in the early larva, a cartography of HSPCs and highly diversified differentiated populations, notably NK-like cell types, innate lymphoid cells and early eosinophils. We show that the two EHT cell types previously characterized indeed lead to differentially fated cells, with significant differences in thymus colonization and T-lymphoid lineage commitment. Using HSPC signatures retrieved from our datasets - namely gata2b and cd34/podocalyxin -, and to address niches, we performed in situ gene expression analyses via RNAscope. Unexpectedly, we unveil a niche contacting the supra-intestinal artery. Finally, integration with previous datasets reveal that our populations contain potential developmental HSCs bearing signatures highly similar with adult HSCs. Summary StatementSingle cell photoconversion of emerging hematopoietic precursor cells and transcriptomics unravel the diversity of hematopoietic stem and progenitor cell populations and homing in developmental niches in toto, during zebrafish development.

developmental 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↗

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

Distinct Phenotypes of SARS-CoV-2 Isolates Reveal Viral Traits Critical for Replication in Primary Human Respiratory Cells

Since entering the human population, SARS-CoV-2 (the causative agent of COVID-19) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from non-human Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally-occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E, and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin-cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific virus replication, and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants-of-concern.

microbiology↗

bioRxiv2020

The Flemmingsome reveals an ESCRT-to-membrane coupling required for completion of cytokinesis

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal novel actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among those proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins were highly recruited first at the midbody then at the abscission site, and their depletion delayed abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 were essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, revealing novel common requirements in cytokinesis, exosome formation and HIV budding.

cell biology↗

bioRxiv2017

Cnidarian cell type diversity revealed by whole-organism single-cell RNA-seq analysis

A hallmark of animal evolution is the emergence and diversification of cell type-specific transcriptional states. But systematic and unbiased characterization of differentiated gene regulatory programs was so far limited to specific tissues in a few model species. Here, we perform whole-organism single cell transcriptomics to map cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal that display complex tissue-level bodyplan organization. We uncover high diversity of transcriptional states in Nematostella, demonstrating cell type-specific expression for 35% of the genes and 51% of the transcription factors (TFs) detected. We identify eight broad cell clusters corresponding to cell classes such as neurons, muscles, cnidocytes, or digestive cells. These clusters comprise multiple cell modules expressing diverse and specific markers, uncovering in particular a rich repertoire of cells associated with neuronal markers. TF expression and sequence analysis defines the combinatorial code that underlies this cell-specific expression. It also reveals the existence of a complex regulatory lexicon of TF binding motifs encoded at both enhancer and promoters of Nematostella tissue-specific genes. Whole organism single cell RNA-seq is thereby established as a tool for comprehensive study of genome regulation and cell type evolution.

genomics↗