Biology subjects

Fouillade, C.

Publications and source records attributed to Fouillade, C..

7 recordsLinked to original sources

bioRxiv2024

autoFISH - a modular toolbox for sequential smFISH experiments

Fluorescence in situ hybridization (FISH) allows for spatial and quantitative profiling of gene expression by visualizing individual RNA molecules. Here, we introduce automated FISH (autoFISH), a comprehensive toolbox to conduct automated single molecule FISH (smFISH) experiments that is both cost-effective and versatile. This includes detailed plans for constructing the necessary equipment, open-source software for control, reliable experimental protocols, and analysis workflows based on our FISH-quant analysis package. Validation experiments with both cell lines and tissue samples confirmed the systems robustness. We demonstrate standard and amplified smFISH, along with a modified protocol for tissue clearing that enhances nuclear retention while preserving background reduction efficiency.

molecular biology↗

bioRxiv2024

Radiotherapy triggers pro-angiogenic signaling in human lung

Radiotherapy is one of the main therapeutic options for the treatment of lung cancer. Although highly efficient, radiation cause severe damages to normal tissue and radio-induced toxicities vary from mild pneumonitis to pulmonary fibrosis. The mechanism leading to these toxicities remain unclear. To investigate the molecular responses of human lung to radiotherapy, we analyzed, by single cell RNAseq, lung tissue resected in the vicinity of the tumor (i.e. treated with radiation) and compared the transcriptional profiles of the distinct lung populations from the same patient removed at distance from the tumor (i.e. non-treated with radiation). Analysis of six lung samples from patients suffering from Pancoast tumor, a rare lung malignancy that requires neo-adjuvant radiotherapy before surgery, revealed a strong induction of VEGF signaling after radiotherapy. Expression of VEGFA, one of the canonical pro-angiogenic ligands, was found upregulated in multiple cell populations in lung exposed to high doses of radiation. Irradiated capillaries, particularly gCap cells, expressing KDR/VEGFR2, present transcriptional profile similar to tip cells, characterized by sprouting and motility capacities. In addition, we identified a sub-population of alveolar macrophages expressing FLT1/VEGFR1, a receptor for VEGFA, in lung tissues treated by radiotherapy. Cell-Cell communication analysis revealed that FLT1/VEGFR1 positive macrophages interact with tip cells after radiotherapy through IL1B-IL1R signaling. Lastly, analysis of mouse single cell dataset confirmed the increase in the proportion of gCap cells presenting a tip-like phenotype after radiation injury. Altogether, this study describes, at the single cell level, the pro-angiogenic responses of human lung after radiotherapy. These results will lead to a better understanding of the physiopathology of lung radiation injury and may pave the way to optimize treatments to improve patients quality of life.

cancer biology↗

bioRxiv2024

Conserved signals orchestrate self-organization and symmetry breaking of bi-layered epithelia during development and regeneration

Organ development relies on complex molecular mechanisms that guide initially homogeneous populations of stem cells to differentiate into specialized cell types within defined spatial patterns. While stable during homeostasis, the proper spatial organization of cell types must be re-established in case of tissue injury for successful regeneration of organ shape and function. How cells commit to a differentiation path is a central question in stem cell research; however, the coordination between tissue geometry and cell fate specification remains enigmatic. To elucidate the molecular mechanisms instructing self-organization and symmetry breaking of epithelial stem cells, we developed a multi-faceted approach combining in vitro organoids, ex vivo embryonic tissue explants, and single-cell quantitative imaging to investigate the dynamic acquisition of cell fate in four bi-layered epithelia, during embryonic development but also in regeneration. Our findings indicate that tissue architecture is the primary determinant of cell fate decisions in these tissues. Upon the initial cell internalization event, the homogeneous population of stem cell break symmetry. Through genetic and pharmacological perturbations, we have demonstrated that a tightly coordinated interplay between Hippo/YAP and Notch signaling is essential for conveying information from tissue architecture to functional cell differentiation and stem cell potency restriction. Globally, this study uncovers the inherent capacity of stem cells to self-organize into multicellular structures, where the precise position of each differentiated cell is critical to instruct their differentiation choices during embryonic development and regeneration.

developmental biology↗

bioRxiv2024

A DIY guide for image-based spatial transcriptomic: TLS as a case example

Spatial RNA profiling methods provide insight to the cellular heterogeneity and spatial architecture of complex, multi-cellular systems. Combining molecular and spatial information provides important clues to study tissue architecture in development and disease. Here, we present a comprehensive do-it-yourself guide to perform such experiments at reduced costs leveraging open-source approaches. This guide spans the entire life cycle of a project, from its initial definition to experimental choices, wet lab approaches, instrumentation and analysis. As a concrete example, we focus on Tertiary lymphoid structures (TLS), which we use to develop typical questions that can be addressed by these approaches.

bioinformatics↗

bioRxiv2023

A point cloud segmentation framework for image-based spatial transcriptomics

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It hence presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is thus applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and cell type calling. On experimental data, our method also demonstrates proficiency in estimating RNA profiles that align with established scRNA-seq datasets. Importantly, ComSeg exhibits a particular efficiency in handling complex tissue, positioning it as a valuable tool for the community.

bioinformatics↗

bioRxiv2022

Induction of telomerase in p21-positive cells counteracts capillaries rarefaction in aging mice lung

Telomerase is required for long-term cell proliferation and linked to stem cells. This is evident in the lung where short telomeres are associated with lung dysfunction. We constructed a mouse model in which the telomerase (Tert) is expressed from the p21Cdkn1a promoter. We found that this peculiar Tert expression curb age-related emphysema and pulmonary perivascular fibrosis in old mice. In old mice lungs, such Tert expression preferentially occurs in endothelial cells where it reduces the number of senescent endothelial cells. Remarkably, we report that Tert counteracts the age-related decline in capillary density. This was associated with an increased number of Cd34+ cells identified as a subclass of capillary cells with proliferative capacity. Expression of catalytically inactive Tert neither prevents the decline of capillary density in old mice nor protects against age-related emphysema and fibrosis. These findings reveal that telomerase decreases age-decline of pulmonary functions by sustaining microvasculature regeneration and outgrowth.

molecular biology↗

bioRxiv2022

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages

Aging is characterized by gradual immune dysfunction and increased risk for many diseases, including respiratory infections. Genomic instability is thought to play a central role in the aging process but the mechanisms that damage nuclear DNA in aging are insufficiently defined. Cells that migrate or reside within confined environments experience forces applied to their nucleus, leading to transient nuclear envelope (NE) ruptures. NE ruptures are associated with DNA damage, and Lamin A/C is required to limit these events. Here, we show that Lamin A/C protects lung alveolar macrophages from NE rupture and hallmarks of aging. Lamin A/C ablation in immune cells results in a selective depletion of lung alveolar macrophages (AM) and a heightened susceptibility to influenza infection. Lamin A/C-deficient AM that persist display constitutive nuclear envelope rupture marks, DNA damage and p53-dependent senescence. In wild-type mice, we found that AM migrate within constricted spaces in vivo, at heights that induce NE rupture and DNA damage. AM from aged wild-type mice and from Lamin A/C-deficient mice share an upregulated lysosomal signature with CD63 expression, and we find that CD63 is required to clear damaged DNA in macrophages. We propose that induction of genomic instability by NE disruption represents a mechanism of aging in alveolar macrophages.

immunology↗