Biology subjects

Renaud, Y.

Publications and source records attributed to Renaud, Y..

13 recordsLinked to original sources

bioRxiv2025

Enhanced Multiplexed Single-Cell RNA-Sequencing for Accurate Detection of Treatment Effects Without Batch Correction in the Avian Embryonic Model

Single-cell RNA sequencing has revolutionized our ability to explore cellular heterogeneity and is a powerful tool to study the impact of environmental perturbations on multiple cell states. However, environmental perturbations can be subtle, and the associated biological effects could be masked by experimental noise and bioinformatic processing, especially when the samples are generated separately. Multiplexing strategies have been developed to label each sample and process them together to reduce experimental noise, but existing multiplexing methods often fall short for non-human and non-mouse models in established single-cell RNA sequencing protocols like the BD Rhapsody technology. To address this gap, we combined Lipid Modified Oligonucleotide (LMO) cell tagging with the BD Rhapsody platform to achieve efficient and scalable multiplexing of early embryonic chick cells under different environmental conditions. This species-agnostic LMO approach overcomes limitations of antibody-based multiplexing methods that are often restricted to human and mouse systems, and the only multiplexing option available with the BD Rhapsody system. In our study, we successfully compartmentalized and multiplexed chick embryonic cells under different treatment conditions, analyzing up to 40,000 viable cells per experiment. This strategy minimized experimental noise, eliminating the need for bioinformatics-based batch correction. As a result, we were able to achieve high-quality transcriptomic profiling with minimal loss of critical biological information and identified subtle biological differences that were masked when using data integration pipelines. Our workflow provides an adaptable, robust solution for LMO-tagged single-cell analyses of complex non-human models with the BD Rhapsody technology and opens new avenues for developmental biology research by accurately capturing treatment-induced effects in embryonic tissues.

developmental biology↗

bioRxiv2024

A dual histone code specifies the binding of heterochromatin protein Rhino to a subset of piRNA source loci

Animal germ cells deploy a specialized small RNA-based silencing system, called the PIWI-interacting RNA (piRNA) pathway, to prevent unwanted expression of transposable elements and maintain genome integrity. In Drosophila germ cells, the majority of piRNA populations originate from dual-strand piRNA clusters, genomic regions highly enriched in transposable element (TE) fragments, via an elaborate protein machinery centred on the heterochromatin protein 1 homolog, Rhino. Although Rhino binds to peptides carrying trimethylated H3K9 in vitro, it is not fully understood why in vivo only a fraction of H3K9me3-decorated heterochromatin is occupied by Rhino. Recent work revealed that Rhino is recruited to a subset of piRNA clusters by the zinc finger protein Kipferl. Here we identify a Kipferl-independent mode of Rhino targeting that, in addition to the previously established role of H3K9me3, also depends on the histone H3 lysine 27 methyltransferase Enhancer of Zeste. At Kipferl-independent sites, we find that Rhino, through its chromodomain, specifically binds to loci marked by both H3K9me3 and H3K27me3. Although the exact mechanism of how Rhino binding is influenced by dual histone modifications remains unclear from a structural and biochemical perspective, our work suggests that combinatorial modifications can play a crucial role in influencing the specificity of chromatin-binding protein interactions. These findings provide an enhanced understanding of the multifaceted mechanisms by which Rhino targets piRNA source loci highlighting the sophisticated epigenetic landscape governing TE silencing in Drosophila germ cells. Our work further reveals a role for dual histone modifications defining the binding specificity of a key chromatin protein.

molecular biology↗

bioRxiv2023

Amalgam plays a dual role in controlling the number of leg muscle progenitors and regulating their interactions with developing tendon

Formation of functional organs requires cell-cell communication between different cell lineages, and failure in this communication can result in severe developmental defects. Hundreds of possible interacting pairs of proteins are known, but identifying the interacting partners that ensure a specific interaction between two given cell types remains challenging. Here, we use the Drosophila leg model and our cell type-specific transcriptomic datasets to uncover the molecular mediators of cell-cell communication between tendon and muscle precursors. Through the analysis of gene expression signatures of appendicular muscle and tendon precursor cells, we identify two candidates for early interactions between these two cell populations: Amalgam (Ama) encoding a secreted protein and Neurotactin (Nrt) known to encode a membrane-bound protein. Developmental expression and function analyses reveal that: i) Ama is expressed in the leg myoblasts, whereas Nrt is expressed in adjacent tendon precursors, and ii) in Ama and Nrt mutants, myoblast-tendon cell-cell association is lost, leading to tendon developmental defects. Furthermore, we demonstrate that Ama acts downstream of the FGFR pathway to maintain the myoblast population by promoting cell survival and proliferation in a Nrt-independent manner. Together, our data pinpoint Ama and Nrt as molecular actors ensuring early reciprocal communication between leg muscle and tendon precursors, a prerequisite for the coordinated development of the appendicular musculoskeletal system.

developmental biology↗

bioRxiv2023

Adenine methylation is very scarce in the drosophila genome and not erased by the Ten Eleven Translocation dioxygenase.

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in drosophila, for which the erasure of this epigenetic mark has been ascribed to the Ten Eleven Translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.

molecular biology↗

bioRxiv2023

Gap junction-dependent amino acid transfer promotes germ cell growth

Gap junctions allow the exchange of small molecules between cells. How this function could be used to promote cell growth is not yet fully understood. During Drosophila ovarian follicle development, germ cells, which are surrounded by epithelial somatic cells, undergo massive growth. We found that this growth depends on gap junctions between these cell populations, with a requirement for Innexin4 and Innexin2, in the germ cells and the somatic cells, respectively. Translatomic analyses revealed that somatic cells express enzymes and transporters involved in amino acid metabolism that are absent in germ cells. Among them, we identified an amino acid transporter required for germline growth. Its ectopic expression in the germline can compensate for its absence in somatic cells. Moreover, affecting either gap junctions or amino-acid import in somatic cells induces P-bodies in the germ cells, a feature associated with an arrest of translation. Finally, in somatic cells, innexin2 expression and gap junction assembly are regulated by the insulin receptor/PI3K kinase pathway. Overall, these results support the view that metabolic transfer through gap junction promotes cell growth and illustrate how such a mechanism can be integrated into a developmental programme, coupling growth control by extrinsic systemic signals with the intrinsic coordination between cell populations.

developmental biology↗

bioRxiv2022

Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy. It is caused by the excessive expansion of non-coding CTG repeat, which when transcribed affect functions of RNA-binding factors. Specifically, MBNL1 is sequestered in nuclear foci while CELF1 is stabilised, with adverse effects on alternative splicing, processing and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, has been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis was unknown. Here, we used Drosophila DM1 models to explore miR-1 involvement in cardiac dysfunction in DM1. We found that miR-1 down-regulation in the heart led to dilated cardiomyopathy (DCM), a DM1-associated phenotype. We then combined in silico screening for miR-1 targets with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. We identified Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp and its human ortholog Col15A1 were both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. Importantly, when overexpressed in the heart, Mp induced DCM, whereas its attenuation ameliorated the DCM phenotype in aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 are thus critical in DM1-associated DCM.

genetics↗

bioRxiv2022

Sexually dimorphic activation of innate antitumour immunity prevents adrenocortical carcinoma development

In contrast with most cancers, adrenocortical carcinomas (ACC) are more frequent in women than men, but the underlying mechanisms of this sexual dimorphism remain elusive. Homozygous deletion of the negative WNT pathway regulator ZNRF3 is the most frequent alteration in ACC patients. Here, we show that Cre-mediated inactivation of Znrf3 in steroidogenic cells of the mouse adrenal cortex is associated with sexually dimorphic tumour progression. Indeed, although most knockout female mice develop metastatic carcinomas over an 18 month-time course, adrenal hyperplasia gradually regresses in male knockout mice. This male-specific regression is associated with induction of senescence and recruitment of macrophages, which differentiate as active phagocytes that clear-out senescent preneoplastic cells. Macrophage recruitment is also observed in female mice. However, it is delayed and dampened compared to males, which allows for tumour progression. Interestingly, testosterone treatment of female knockouts is sufficient to induce senescence, recruitment of phagocytic macrophages and regression of hyperplasia. We further show that although macrophages are present within adrenal tumours at 18 months, MERTKhigh active phagocytes are mostly found in indolent lesions in males but not in aggressive tumours in females. Consistent with our observations in mice, analysis of RNA sequencing data from the TCGA cohort of ACC shows that phagocytic macrophages are more prominent in men than women and associated with better prognosis. Altogether, these data establish that phagocytic macrophages prevent aggressive ACC development in male mice and suggest that they may play a key role in the unusual sexual dimorphism of ACC in patients.

cancer biology↗

bioRxiv2021

Characterization of the Drosophila adult hematopoietic system reveals a rare cell population with differentiation and proliferation potential.

While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, we show that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, our transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many gene expressed in embryonic blood cells. Interestingly, we identify a small set of blood cells that do not express differentiation markers but maintain the progenitor marker domeMeso. Yet, we show that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, we find that some of them can differentiate and proliferate in response to bacterial infection. In sum, our results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood.

developmental biology↗

bioRxiv2020

Differential activation of JAK-STAT signaling in blood cell progenitors reveals functional compartmentalization of the Drosophila lymph gland.

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on progenitors located in lymph gland anterior lobes (AP), ignoring the progenitors from the posterior lobes (PP). Using in situ expression mapping and transcriptome analysis we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated. Stat92E knockdown in PP or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable. HighlightsWe provide an in situ and transcriptome map of larval blood progenitors Posterior lymph gland progenitors are refractory to immune challenge STAT activation after wasp parasitism maintains posterior progenitors

developmental biology↗

bioRxiv2020

m6A RNA methylation regulates promoter proximal pausing of RNA Polymerase II

RNA Polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing. The m6A methyltransferase complex (MTC), with the nuclear reader Ythdc1, are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body, and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

molecular biology↗

bioRxiv2019

New muscle identity code components identified by TRAP control shape and size of muscle subsets in Drosophila

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

developmental biology↗

bioRxiv2019

A Nanog-dependent gene cluster initiates the specification of the pluripotent epiblast

The epiblast (Epi) is the source of embryonic stem (ES) cells and all embryonic tissues. It differentiates alongside the primitive endoderm (PrE) in a randomly distributed \"salt and pepper\" pattern from the inner cell mass (ICM) during preimplantation of the mammalian embryo. NANOG and GATA6 are key regulators of this binary differentiation event, which is further modulated by heterogeneous FGF signalling. When and how Epi and PrE lineage specification is initiated within the developing embryo is still unclear. Here we generated NANOG and GATA6 double KO (DKO) mouse embryos and performed single-cell expression analyses. We found that the ICM was unable to differentiate in the DKO mice, allowing us to characterize the ICM precursor state. The normally heterogeneous expression of Fgf4 between cells was significantly reduced in DKO ICMs, impairing FGF signalling. In contrast, several pluripotency markers did still display cell-to-cell expression variability in DKO ICMs. This revealed a primary heterogeneity independent of NANOG, GATA6 and FGF signalling that may also be conserved in humans. We found that NANOG is key in the initiation of epiblast specification already between the 16- and 32-cell stages, enabling the cell-clustered expression of many pluripotency genes. Our data uncover previously unknown biology in the early mouse embryo with potential implications for the field of pluripotent stem cells in human and other mammals.

developmental biology↗

bioRxiv2018

Straightjacket/α 2δ 3 deregulation is associated with cardiac conduction defects in Myotonic Dystrophy type 1

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a complex toxic CTG repeat disorder involving misbalance between two RNA- binding factors, MBNL1 and CELF1. How this pathogenic DM1 condition translates into cardiac conduction disorders remains poorly understood. Here, we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and identified associated gene deregulations using TU-tagging based transcriptional profiling of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels and among them increased expression of straightjacket/2{delta}3 that encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heart beating, a hallmark of affected conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular 2{delta}3 expression is low in healthy mice and humans but significantly elevated in ventricular muscles from DM1 patients with conduction defects. Taken together, this suggests that reducing the straightjacket/2{delta}3 transcript levels in ventricular cardiomyocytes could represent a strategy to prevent conduction defects and in particular intraventricular conduction delay associated with DM1 pathology.

genomics↗