Biology subjects

Meunier, A.

Publications and source records attributed to Meunier, A..

12 recordsLinked to original sources

bioRxiv2026

Objective method to estimate the duration of the DNAreplication cycle without perturbation in Escherichia coli.

All cells, including unicellular organisms such as Escherichia coli, depend on the robustness of their cell cycle for proliferation. The cell cycle is central to the organisms highly resilient proliferation program and is thus a key focus in many areas of cell biology. However, studying the DNA replication cycle in bacteria remains challenging due to technical limitations. The durations of the replicative and post-replicative phases (known as the C and D periods, respectively) are inferred from the analysis of cellular DNA content distributions, obtained through microscopy or flow cytometry. This analysis typically requires pharmacological treatments to conduct replication run-off experiments, which help distinguish between cells that have or have not yet initiated DNA replication. Four decades ago, Skarstad, Steen and Boye showed that flow cytometry profiles measuring DNA amount per cell from exponentially growing cells could provide sufficient information to infer the durations of cell cycle periods at low growth rates. In this study, we propose an objective and automated approach that implements this idea to estimate cell cycle parameters for any growth conditions. Specifically, we validate a Nested Sampling method to estimate cell cycle parameters directly from flow cytometry data, eliminating the dependence on bacterial strain sensitivity to drugs. This tool, available as a Python package, allows for the accurate and minimally biased estimation of the C+D period under any biologically relevant conditions. Given its independence from pharmacological treatments, we anticipate broad adoption of this tool, especially as we show that most natural isolates of E. coli are not amenable to the state of the art replication run-off experiments.

microbiology↗

bioRxiv2025

Unraveling Enteroendocrine Cell lineage dynamics and associated gene regulatory networks during intestinal development

Enteroendocrine cells (EECs) are rare intestinal epithelial cells producing multiple hormones that regulate essential aspects of digestion and energy. EEC subtypes, their hormone repertoire and differentiation mechanisms from intestinal stem cells have been characterized in the adult intestine. However, although EECs need to be functional from birth, as their absence causes severe intestinal malabsorption in newborns, the mechanisms underlying EEC subtype specification during development are largely unknown. We used mouse embryos, human pluripotent stem cell derived intestinal organoid models and single-cell transcriptomics to characterize EEC lineages and dynamics during development. Our findings demonstrate that in both mice and humans, the majority of EECs are specified during development through similar differentiation trajectories as observed in the adult intestine. This suggests that EEC subtypes specification occurs independently of fully organized crypt-villus structures and stimulation by diet or microbiota. However, the emergence of certain EEC subtypes depends on tissue maturation. Finally, our integrative approach infers lineage-specific regulators dynamically, identifying new candidates controlling EEC differentiation in the developing human gut. HIGHLIGHTSO_LIMost enteroendocrine cells (EECs) are specified during development. C_LIO_LICrypt-villus organization, diet or microbiota are not essential for EEC formation. C_LIO_LIIn pluripotent stem cell derived human intestinal organoids (HIOs), EECs differentiate along 3 main trajectories, one driven by maturation processes. C_LIO_LINovel Gene Regulatory Networks are identified governing human EEC differentiation in HIOs. C_LI

developmental biology↗

bioRxiv2024

Identification of a new cell cycle variant during multiciliated cell differentiation

2A complex and conserved regulatory network drives the cell cycle. Individual components of this network are sometimes used in differentiated cells, i.e. to control organelle destruction in mammalian lens cells or light response in land plants. Some differentiated cells co-opt cell-cycle regulators more largely, to increase their ploidy using a cell cycle variant named endoreplication. Using single-cell RNA-seq profiling and functional assays in differentiating multiciliated cells, we identified a novel type of cell cycle variant that supports cytoplasmic organelle, rather than nuclear content amplification. This variant operates in post-mitotic, centriole-amplifying differentiating multiciliated cells and is characterized by (i) a circular trajectory of the transcriptome, (ii) sequential expression of more than 70% of the genes involved in S, G2 and M-like progression along this trajectory, and (iii) successive waves of cyclins. This cell cycle variant is tailored by the expression of the non-canonical cyclins O and A1 - which replace the transcriptionally silent cyclins E2 and A2 - and by the silencing of the APC/C inhibitor Emi1, two switches also detected in male meiosis, another variant of the canonical cell cycle where centriole and DNA replications are uncoupled. Re-expressing Cyclin E2, cyclin A2 or Emi1 is sufficient to induce partial replication and mitosis, suggesting that change in the regulation of expression of a few cell cycle key players drives a qualitative and quantitative tuning of Cdk activity, allowing the diversion of the cell cycle in the multiciliation variant. We also propose that this new cell cycle variant relies on the existence of a cytoplasmic - or centriolar - Cdk threshold, lower than the S-phase threshold, which affects only the cytoplasmic reorganization. One-Sentence SummaryMCC progenitors undergo a final, tailored iteration of the cell cycle during differentiation, to drive centriole amplification without DNA replication or mitosis.

cell biology↗

bioRxiv2024

Cyclin O controls entry into the cell-cycle variant required for multiciliated cell differentiation

Multiciliated cells (MCC) ensure proper fluid circulation in various organs in metazoans. Their differentiation is marked by the massive ampliication of cilia-nucleating centrioles and is known to be controlled by various cell cycle components. Tn a companion study, we show that the differentiation of MCC is driven by a genuine cell-cycle variant characterized by sequential and wave-like expression of canonical and non-canonical cyclins such as Cyclin O (CCNO). Patients with CCNO mutations exhibit a subtype of Primary Ciliary Dyskinesia (PCD) designated as Reduced Generation of Multiple Motile Cilia (RGMC), yet the role of CCNO during MCC differentiation remains unclear. Here, using mice and human cellular models, single cell transcriptomics and functional studies, we show that Cena is activated during a strategic temporal window at the crossroads between the onset of MCC differentiation, the entry into the MCC cell cycle variant, and the activation of the centriole biogenesis program. We ind that the absence of Cena leads to a block of MCC progenitor differentiation at the G1/S-like transition, just before the beginning of centriole formation. This leads to a complete lack of centrioles and cilia in mouse brain and human airway MCC. Altogether, our study identifies CCNO as a core regulator of entry into the MCC cell cycle variant and shows that the coupling of centriole biogenesis to an S-like phase, maintained in MCC, is dependent on CCNO. One sentence summaryCyclin O is necessary for multiciliated cells to enter their differentiation cell cycle variant and allows the massive amplification of centrioles, which serve as basal bodies for cilia nucleation.

cell biology↗

bioRxiv2024

Microtubule-dependent orchestration of centriole amplification in brain multiciliated cells

Multiciliated cell (MCC) differentiation is a calibrated version of the canonical cell cycle. The MCC cell cycle variant sustains amplification of centrioles for the nucleation of dozens of motile cilia, while avoiding cell division. In this study, we show that the MCC cell cycle variant is an accelerated version of the canonical cell cycle, which superposes two cycles of centriole biogenesis, in order to obtain multiple mature centrioles within a single -instead of double- cell cycle iteration. We further show that the precocious maturation of amplified procentrioles is even determinant for their spatial self-organization, disengagement and apical migration for cilia nucleation. Our findings collectively suggest that the decomposition of centriole biogenesis over two cycle iterations in dividing cells may have been adopted to ensure the growth of a solitary primary cilium, and exemplify how minimal deviations of the canonical cell cycle allow MCC progenitors to both amplify, and accelerate, centriole biogenesis for vital motile ciliogenesis.

cell biology↗

bioRxiv2023

Neuro-Cognitive Multilevel Causal Modeling: A Framework that Bridges the Explanatory Gap between Neuronal Activity and Cognition

Explaining how neuronal activity gives rise to cognition arguably remains the most significant challenge in cognitive neuroscience. We introduce neuro-cognitive multilevel causal modeling (NC-MCM), a framework that bridges the explanatory gap between neuronal activity and cognition by construing cognitive states as (behaviorally and dynamically) causally consistent abstractions of neuronal states. Multilevel causal modeling allows us to interchangeably reason about the neuronal- and cognitive causes of behavior while maintaining a physicalist (in contrast to a strong dualist) position. We introduce an algorithm for learning cognitive-level causal models from neuronal activation patterns and demonstrate its ability to learn cognitive states of the nematode C. elegans from calcium imaging data. We show that the cognitive-level model of the NC-MCM framework provides a concise representation of the neuronal manifold of C. elegans and its relation to behavior as a graph, which, in contrast to other neuronal manifold learning algorithms, supports causal reasoning. We conclude the article by arguing that the ability of the NC-MCM framework to learn causally interpretable abstractions of neuronal dynamics and their relation to behavior in a purely data-driven fashion is essential for understanding more biological systems whose complexity prohibits the development of hand-crafted computational models.

neuroscience↗

bioRxiv2023

Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation.

Ependymal cells (ECs) are multi-ciliated cells in the brain that contribute to cerebrospinal fluid flow. ECs are specified at embryonic stages but differentiate later in development. Differentiation depends on genes such as GEMC1 and MCIDAS with E2F4/5, but also on cell cycle related factors. In the mouse brain, we observe that nuclear deformation accompanies EC differentiation. Tampering with these deformations either by decreasing F-actin levels or by severing the link between the nucleus and the actin cytoskeleton blocks differentiation. Conversely, increasing F-actin by knocking out the Arp2/3 complex inhibitor Arpin or artificially deforming the nucleus activates differentiation. These data are consistent with actin polymerization triggering nuclear deformation and jump-starting the signaling that produces ECs. A player in this process is the retinoblastoma1 (RB1) protein whose phosphorylation prompts MCIDAS activation. Overall, this study reveals an important role for actin-based mechanical inputs to the nucleus as controlling factors in cell differentiation.

developmental biology↗

bioRxiv2021

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

ObjectiveMice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin secreting beta cells, causing diabetes. In human, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. MethodsWe generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. In addition, we searched whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. ResultsCUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1268 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements frequently overlapping within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA, RFX or PBX transcription factors. Moreover, we found that 22% of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 targets include transcription factors known to have important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself. Remarkably, we uncovered that NEUROG3 binds transcriptional regulator genes with enriched expression in human fetal pancreatic alpha (e.g., IRX1, IRX2), beta (e.g., NKX6-1, SMAD9, ISX, TFCP2L1) and delta cells (ERBB4) suggesting that NEUROG3 could control islets subtype programs. Moreover, NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8 and PCSK1). In addition, we unveiled a panel of ncRNA potentially regulated by NEUROG3. Lastly, we identified several T2DM risk SNPs within NEUROG3 peaks suggesting a possible developmental role of NEUROG3 in T2DM susceptibility. ConclusionMapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation. HighlightsO_LINEUROG3 CUT&RUN analysis revealed 1268 target genes in human pancreatic endocrine progenitors (PEPs) C_LIO_LINEUROG3 binding sites overlap with active chromatin regions in PEPs. C_LIO_LI1/5 of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3. C_LIO_LINEUROG3 targets islet specific TFs and regulators of insulin secretion. C_LIO_LISeveral T2DM risk allelles lie within NEUROG3 bound regions. C_LI

genomics↗

bioRxiv2020

The rise and the fall of a Pseudomonas aeruginosa epidemic lineage in a hospital

The biological features that allow a pathogen to survive in the hospital environment are mostly unknown. The extinction of bacterial epidemics in hospitals is mostly attributed to changes in medical practice, including infection control, but the role of bacterial adaptation has never been documented. We analyzed a collection of Pseudomonas aeruginosa isolates belonging to the Besancon Epidemic Strain (BES), responsible for a 12-year nosocomial outbreak, using a genotype-to-phenotype approach. Bayesian analysis estimated the emergence of the clone in the hospital five years before its opening, during the creation of its water distribution network made of copper. BES survived better than the reference strains PAO1 and PA14 in a copper solution due to a genomic island containing 13 metal-resistance genes and was specifically able to proliferate in the ubiquitous amoeba Vermamoeba vermiformis. Mutations affecting amino-acid metabolism, antibiotic resistance, lipopolysaccharide biosynthesis, and regulation were enriched during the spread of BES. Seven distinct regulatory mutations attenuated the overexpression of the genes encoding the efflux pump MexAB-OprM over time. The fitness of BES decreased over time in correlation with its genome size. Overall, the resistance to inhibitors and predators presumably aided the proliferation and propagation of BES in the plumbing system of the hospital. The pathogen further spread among patients via multiple routes of contamination. The decreased prevalence of patients infected by BES mirrored the parallel and convergent genomic evolution and reduction that affected bacterial fitness. Along with infection control measures, this may have participated in the extinction of BES in the hospital setting. ImportanceBacterial pathogens are responsible for nosocomial outbreaks, but the sources of contamination of the hospitals are mostly unclear and the role of bacterial evolution in the extinction of outbreaks has never been considered. Here, we found that an epidemic strain of the pathogen Pseudomonas aeruginosa contaminated the drinking water network of a hospital due to its tolerance to copper and predatory amoeba, both present in the water pipes. The extinction of the outbreak occurred concomitantly with parallel and convergent genome evolution and a reduction in the size of the bacterial genome that correlated with the fitness of the pathogen. Our data suggest that pathogen evolution participated in the extinction of an outbreak in a hospital setting.

microbiology↗

bioRxiv2019

Gravity-based microfiltration reveals unexpected prevalence of circulating tumor cell clusters in ovarian cancer

Circulating tumor cells (CTCs) are rare (few cells per milliliter of blood) and mostly isolated as single cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate. Here, we introduce gravity-based microfiltration (G{micro}F) for facile isolation of cCTCs while minimizing unwanted cluster disaggregation, with [~]85% capture efficiency. G{micro}F from orthotopic ovarian cancer mouse models, from 17 epithelial ovarian cancer (EOC) with either localized or metastatic disease, and from 13 metastatic colorectal cancer liver metastasis (CRCLM) patients uncovered cCTCs in every case, with between 2-100+ cells. cCTCs represented between 5-30% of all CTC capture events, and 10-80% of CTCs were clustered; remarkably, in 10 patients, most CTCs were circulating not as scCTCs, but as cCTCs. G{micro}F uncovered the unexpected prevalence and frequency of cCTCs including sometimes very large ones in EOC patients, and motivates additional studies to uncover their properties and role in disease progression.

bioengineering↗

bioRxiv2019

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production

ObjectiveEnteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function, however, the role of Rfx6 in EECs differentiation remained to be elucidated.\n\nMethodsWe examined the molecular, cellular and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes.\n\nResultsRfx6 is expressed in the gut endoderm; later it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, whose differentiation programs are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1- or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine.\n\nConclusionThese studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

developmental biology↗

bioRxiv2018

Dynamics of centriole amplification in centrosome-depleted brain multiciliated progenitors

Centrioles are essential microtubule-based organelles organizing cilia and centrosomes. Their mode of biogenesis is semi-conservative: each pre-existing centriole scaffolds the formation of a new one, a process coordinated with the cell cycle. By contrast, multiciliated progenitors with two centrosomal centrioles massively amplify centrioles to support the nucleation of hundred of motile cilia and transport vital fluids. This occurs through cell type-specific organelles called deuterosomes, composed of centrosome-related elements, and is regulated by the cell cycle machinery. Deuterosome-dependent centriole amplification was proposed for decades to occur de novo, i.e. independently from pre-existing centrioles. Challenging this hypothesis, we recently reported an accumulation of procentriole and deuterosome precursors at the centrosomal daughter centriole during centriole amplification in brain multiciliated cells. Here we further investigate the relationship between the centrosome and the dynamic of centriole amplification by (i) characterizing the centrosome behavior during the centriole amplification dynamics and (ii) assessing the dynamics of amplification in centrosome-depleted cells. Surprisingly, although our data strengthen the centrosomal origin of amplified centrioles, we show limited consequences in deuterosome/centriole number when we deplete centrosomal centrioles. Interestingly, in absence of centrosomal centrioles, procentrioles are still amplified sequentially from a single focal region, characterized by microtubule convergence and pericentriolar material (PCM) self-assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.

cell biology↗