Biology subjects

Brunet, T.

Publications and source records attributed to Brunet, T..

15 recordsLinked to original sources

bioRxiv2026

Single-Cell Multimodal Profiling Reveals Maternal Diabetes-Nkx2-5 Interaction in Congenital Heart Disease in a Genetic Model Recapitulating Human Embryopathies

The etiological complexity of congenital heart disease (CHD) arises from the interaction between genetic and environmental factors. We hypothesized that environmental exposure could potentiate a genetic susceptibility, leading to an increased incidence of specific cardiac defects. To test this hypothesis, we used betaV59M+ female mice exhibiting severe hyperglycemia (>28 mmol/L) without dyslipidemia, a model we characterized that faithfully recapitulates human diabetic embryopathy. These females were crossed with males haploinsufficient for Nkx2-5, a transcription factor essential for cardiac development and implicated in human CHD. Our results show that the combination of genetic susceptibility and a hyperglycemic maternal environment (GxE) leads to a higher frequency and severity of cardiac malformations, including ventricular septal defects specifically involving the muscular septum, univentricular morphologies, and dilation with thinning of the myocardium. These structural abnormalities were associated with transcriptional changes affecting the contractile identity of the chamber myocardium. Single-nucleus multiomic profiling (snRNA-seq and snATAC-seq) of the E9.5 cardiac region revealed that the interaction between maternal diabetes and Nkx2-5 haploinsufficiency profoundly reshapes transcriptional and chromatin accessibility landscapes. These changes affect not only metabolic pathways, but also cell cycle progression in second heart field (SHF) progenitors, likely contributing to the observed structural cardiac defects. Multiomic integration further identifies a disrupted indirect Nkx2-5-Aldh1a2 regulatory axis in posterior SHF progenitors, while demonstrating that maternal diabetes selectively affects a restricted subset of Nkx2-5-dependent regulatory elements associated with cardiomyocyte maturation. Our findings establish a comprehensive single-cell multiomic resource that will facilitate the integration of human GWAS and other genomic datasets, enabling the identification and functional interpretation of disease-associated loci.

developmental biology↗

bioRxiv2026

Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.

microbiology↗

bioRxiv2026

Rapid repurposing of microvillar content drives a flagellate-to-amoeboid switch in the closest relative of animals

Animal cells extensively remodel their cytoskeleton during differentiation and can notably switch between two major motility modes: flagellum-based swimming and actin-based crawling. We previously showed that choanoflagellates, the closest living relatives of animals and classically viewed as obligate flagellated swimmers, can retract their collar complex and adopt an amoeboid form within seconds under spatial confinement, independently of regulated gene expression. Here, using live imaging, ultrastructural expansion microscopy, and cryo-electron tomography in Salpingoeca rosetta, we identify rapid, cell-wide cytoskeletal remodeling as the ultrastructural basis of this switch. Unconfined choanoflagellates lack a detectable actin cortex but display an apical flagellum and cortical microtubules, with F-actin being largely restricted to microvilli. Confinement triggers calcium release from intracellular stores, which induces microvillar retraction and absorption of microvillar material into the cell body, including actin, ezrin-radixin-moesin 1, and plasma membrane. Remodeling of the internalized F-actin and repurposing of associated proteins supports de novo actin cortex formation, which is necessary for amoeboid motility. In parallel, cortical microtubules are disassembled, and the reabsorbed microvillar plasma membrane increases the surface area of the cell body, allowing the cell to flatten under confinement. Cryo-electron tomography reveals stepwise actin reorganization from internalized microvillar bundles to a cortical contractile meshwork combining bundles and scattered filaments. This work reveals considerable ultrastructural plasticity in the cytoskeletal architecture of choanoflagellates and supports an ancestral role for microvilli as reservoirs of membrane and cytoskeleton to potentiate cell phenotypic transitions.

evolutionary biology↗

bioRxiv2026

Predicting the operon structure of the Mycococcus xanthus genome using the novel software DiscOperon

ABSTRACT Motivation: Myxococcus xanthus is a predatory soil bacterium with a large genome of 9.14 MB due to a genome duplication event. While complete genome sequences of M. xanthus are available, gene annotation remains challenging due to its size and the resulting large number of duplicated genes. Operons, so syntenic block of genes that are co-regulated in bacterial genomes, are an important resource to help predict gene function accurately. Results: In order to help improve the annotation of complex genomes such as the one from M. xanthus, we developed a novel operon prediction tool, DiscOperon, which combines gene expression data with homology searches to identify syntenic blocks: Co-expression data of neighbouring genes across the genome are first used to define gene clusters, which are then used to search for conserved syntenic blocks in fully sequenced bacterial genomes using sequence homology searches. This strategy enables DiscOperon to account for gene insertions, rearrangements and deletions, which is its most distinguishing feature. We have tested DiscOperon against ground truths gene pair information on 3 different species from ODB and RegulonDB and compared it to state-of-the-art and still available operon prediction software and we demonstrate its general usability for operon prediction of any bacterial complete genome. We have applied DiscOperon to predict the operons of M. xanthus, which we are making available for the research community. Availability and implementation: DiscOperon is lightweight, user-friendly python tool with minimal dependencies. It is freely available at https://gitlab.com/habermann_lab/discoperon for general usage. Contact: Theo Brunet (theo.brunet@univ-amu.fr); Bianca Habermann (bianca.habermann@univ-amu.fr). Supplementary information: The operon-structured and annotated M. xanthus genome is available from this manuscript, as well as from Zenodo (https://doi.org/10.5281/zenodo.21976167). We furthermore plan to submit the M. xanthus operon information to the operon database OBD.

bioinformatics↗

bioRxiv2024

MitoMAMMAL: a genome scale model of mammalian mitochondria predicts cardiac and BAT metabolism

Mitochondria perform several essential functions in order to maintain cellular homeostasis and mitochondrial metabolism is inherently flexible to allow correct function in a wide range of tissues. Dysregulated mitochondrial metabolism can therefore affect different tissues in different ways which presents experimental challenges in understanding the pathology of mitochondrial diseases. System-level metabolic modelling is therefore useful in gaining in-depth insights into tissue-specific mitochondrial metabolism, yet despite the mouse being a common model organism used in research, there is currently no mouse specific mitochondrial metabolic model available. In this work, building upon the similarity between human and mouse mitochondrial metabolism, we have created mitoMammal, a genome-scale metabolic model that contains human and mouse specific gene-product reaction rules. MitoMammal is therefore able to model mouse and human mitochondrial metabolism. To demonstrate this feature, using an adapted E-Flux2 algorithm, we first integrated proteomic data extracted from mitochondria of isolated mouse cardiomyocytes and mouse brown adipocyte tissue. We then integrated transcriptomic data from in vitro differentiated human brown adipose cells and modelled the context specific metabolism using flux balance analysis. In all three simulations, mitoMammal made mostly accurate, and some novel predictions relating to energy metabolism in the context of cardiomyocytes and brown adipocytes. This demonstrates its usefulness in research relating to cardiac disease and diabetes in both mouse and human contexts.

systems biology↗

bioRxiv2024

A fast and robust gene knockout method for Salpingoeca rosetta clarifies the genetics of choanoflagellate multicellular development

As the closest living relatives of animals, choanoflagellates offer crucial insights into the evolutionary origin of animals. Notably, certain choanoflagellate species engage in facultative multicellular development that resembles the early stages of embryogenesis. In the past few years, Salpingoeca rosetta has emerged as a tractable model for choanoflagellate cell biology and multicellular development, in particular through mutant screens and CRISPR/Cas9-mediated gene knockout (KO). However, existing KO pipelines have variable and sometimes low efficiency, frequently requiring isolation and genotyping of hundreds of clones without guarantee to obtain a KO strain. Here, we present a robust method for gene inactivation in S. rosetta that relies on insertion by CRISPR/Cas9 of a single 1.9 kb cassette encoding both a premature termination sequence and an antibiotic resistance gene. We show that this approach allows robust, fast and efficient isolation of KO clones after antibiotic selection. As a proof of principle, we first knocked out all three genes previously reported to regulate S. rosetta multicellular development in a published mutant screen (rosetteless, couscous and jumble), and confirmed that all three KOs abolished multicellular development. To showcase the potential of this method for de novo characterization of candidate developmental genes, we then inactivated three homologs of genes in the Hippo pathway: hippo, warts and yorkie, which together control cell proliferation and multicellular size in animals. Interestingly, warts KO rosettes were consistently about twice as large as their wild-type counterparts, showing our KO pipeline can reveal novel loss-of-function phenotypes of biological interest. Thus, this method has the potential to accelerate choanoflagellate functional genetics.

evolutionary biology↗

bioRxiv2024

Mixed clonal-aggregative multicellularity entrained by extreme salinity fluctuations in a close relative of animals

Multicellularity evolved multiple times independently during eukaryotic diversification1-4. Two distinct mechanisms underpin multicellularity5: clonality (serial cell division without sister-cell separation) and aggregation (whereby independent cells assemble into a multicellular entity). Clonal and aggregative multicellularity are traditionally considered mutually exclusive1,6-9, with rare exceptions10, and evolutionary hypotheses have addressed why multicellularity might diverge toward one or the other extreme3,4. Both animals and their sister group, the choanoflagellates, are currently only known to acquire multicellularity clonally4,11-13. Here, we show that the choanoflagellate Choanoeca flexa14 forms motile and contractile cell monolayers (or "sheets") through multiple mechanisms: C. flexa sheets can form purely clonally, purely aggregatively, or by a combination of both processes. We characterise the life history of C. flexa in its natural environment - ephemeral splash pools on the island of Curacao - and show that C. flexa undergoes reversible transitions between unicellularity and multicellularity during cycles of evaporation and refilling. Different splash pools house genetically distinct strains of C. flexa, between which aggregation is constrained by kin recognition15-18. We show that clonal-aggregative multicellularity serves as a versatile strategy for the robust re-establishment of multicellularity in this variable and fast-fluctuating environment. Our findings challenge former generalisations about choanoflagellates and expand the option space of choanozoan multicellularity.

evolutionary biology↗

bioRxiv2023

Swimming, Feeding and Inversion of Multicellular Choanoflagellate Sheets

The recent discovery of the striking sheet-like multicellular choanoflagellate species Choanoeca flexa that dynamically interconverts between two hemispherical forms of opposite orientation raises fundamental questions in cell and evolutionary biology, as choanoflagellates are the closest living relatives of animals. It similarly motivates questions in fluid and solid mechanics concerning the differential swimming speeds in the two states and the mechanism of curvature inversion triggered by changes in the geometry of microvilli emanating from each cell. Here we develop fluid dynamical and mechanical models to address these observations and show that they capture the main features of the swimming, feeding, and inversion of C. flexa colonies.

biophysics↗

bioRxiv2023

Sema7A and Sema4D Heterodimerization is Essential for Membrane Targeting and Neocortical Wiring

Disruption of neocortical circuitry and architecture in humans causes numerous neurodevelopmental disorders. Neocortical cytoarchitecture is orchestrated by various transcription factors such as Satb2 that control target genes during strict time windows. In humans, mutations of SATB2 cause SATB2 Associated Syndrome (SAS), a multisymptomatic syndrome involving intellectual disability, speech delay, epilepsy and craniofacial defects. We show that Satb2 controls neuronal migration and axonal outgrowth by inducing the expression of the GPI-anchored protein, Sema7A. We find that heterodimerization with Sema4D increases targeting of Sema4D to the membrane and is required for Sema7A function. Finally, we report that membrane localization and pos- translational modification of the Sema7A-Sema4D complex is disrupted by a novel de novo mutation in Sema4D (Q497P) that is associated with epilepsy in humans. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/527998v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@db698org.highwire.dtl.DTLVardef@4ee980org.highwire.dtl.DTLVardef@c3d9d6org.highwire.dtl.DTLVardef@12a135_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISema7A is a direct Satb2 target that drives neuronal migration and axon outgrowth C_LIO_LISema7A exerts its effect by heterodimerizing with Sema4D at neurites and growth cones C_LIO_LISema7A increases cell surface localization of Sema4D C_LIO_LIDe novo human Sema4D-Q497P mutation causes epilepsy, inhibits post-translational processing & surface localization C_LI eTOCSema7A is a direct target of the transcription factor Satb2. Sema7A promotes normal migration and axon outgrowth in cortical neurons by modulating reverse signaling via Sema4D. These processes are dependent on Sema7A-Sema4D heterodimerization and membrane localization; insufficient transcription of Sema7A or incomplete glycosylation of Sema4D inhibit this progression.

neuroscience↗

bioRxiv2022

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility1-3, immunity4,5, development6-9 and locomotion10,11, the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals12. In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs)13,14. We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS, sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of these, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP15. We found that treatment with NO induces sustained contractions in C. flexa by signaling through an sGC/cGMP pathway. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa - contractions resulting in a switch from feeding to swimming - resembles the effect of NO in sponges1-3 and cnidarians11,16,17, where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

evolutionary biology↗

bioRxiv2020

Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression and a severe phenotype. Contrastingly, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay and encode truncated proteins, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.

genetics↗

bioRxiv2020

A flagellate-to-amoeboid switch in the closest living relatives of animals

The evolution of different cell types was a key process of early animal evolution1-3. Two fundamental cell types, epithelial cells and amoeboid cells, are broadly distributed across the animal tree of life4,5 but their origin and early evolution are unclear. Epithelial cells are polarized, have a fixed shape and often bear an apical cilium and microvilli. These features are shared with choanoflagellates - the closest living relatives of animals - and are thought to have been inherited from their last common ancestor with animals1,6,7. The deformable amoeboid cells of animals, on the other hand, seem strikingly different from choanoflagellates and instead evoke more distantly related eukaryotes, such as diverse amoebae - but it has been unclear whether that similarity reflects common ancestry or convergence8. Here, we show that choanoflagellates subjected to spatial confinement differentiate into an amoeboid phenotype by retracting their flagella and microvilli, generating blebs, and activating myosin-based motility. Choanoflagellate cell crawling is polarized by geometrical features of the substrate and allows escape from confined microenvironments. The confinement-induced amoeboid switch is conserved across diverse choanoflagellate species and greatly expands the known phenotypic repertoire of choanoflagellates. The broad phylogenetic distribution of the amoeboid cell phenotype across animals9-14 and choanoflagellates, as well as the conserved role of myosin, suggests that myosin-mediated amoeboid motility was present in the life history of their last common ancestor. Thus, the duality between animal epithelial and crawling cells might have evolved from a temporal phenotypic switch between flagellate and amoeboid forms in their single-celled ancestors3,15,16.

evolutionary biology↗

bioRxiv2019

Light-regulated collective contractility in a multicellular choanoflagellate

Collective cell contractions that generate global tissue deformations are a signature feature of animal movement and morphogenesis. Nonetheless, the ancestry of collective contractility in animals remains mysterious. While surveying the Caribbean island of Curacao for choanoflagellates, the closest living relatives of animals, we isolated a previously undescribed species (here named Choanoeca flexa sp. nov.), that forms multicellular cup-shaped colonies. The colonies rapidly invert their curvature in response to changing light levels, which they detect through a rhodopsin-cGMP pathway. Inversion requires actomyosin-mediated apical contractility and allows alternation between feeding and swimming behavior. C. flexa thus rapidly converts sensory inputs directly into multicellular contractions. In this respect, it may inform reconstructions of hypothesized animal ancestors that existed before the evolution of specialized sensory and contractile cells.\n\nOne Sentence SummaryA newly described choanoflagellate species forms cup-shaped colonies that reversibly invert their curvature in response to light.

cell biology↗

bioRxiv2017

Whole-body single-cell sequencing of the Platynereis larva reveals a subdivision into apical versus non-apical tissues

Animal bodies comprise a diverse array of tissues and cells. To characterise cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii1-4. We identify five transcriptionally distinct groups of differentiated cells that are spatially coherent, as revealed by spatial mapping5. Besides somatic musculature, ciliary bands and midgut, we find a group of cells located at the apical tip of the animal, comprising sensory-peptidergic neurons, and another group composed of non-apical neural and epidermal cells covering the rest of the body. These data establish a basic subdivision of the larval body surface into molecularly defined apical versus non-apical tissues, and support the evolutionary conservation of the apical nervous system as a distinct part of the bilaterian brain6.

evolutionary biology↗

bioRxiv2017

The origin of animal multicellularity and cell differentiation

How animals evolved from their single-celled ancestors over 600 million years ago is poorly understood. Comparisons of genomes from animals and their closest relatives - choanoflagellates, filastereans and ichthyosporeans - have recently revealed the genomic landscape of animal origins. However, the cell and developmental biology of the first animals have been less well examined. Using principles from evolutionary cell biology, we reason that the last common ancestor of animals and choanoflagellates (the Urchoanozoan) used a collar complex - a flagellum surrounded by a microvillar collar - to capture bacterial prey. The origin of animal multicellularity likely occurred through the modification of pre-existing mechanisms for extracellular matrix synthesis and regulation of cytokinesis. The progenitors of animals likely developed clonally through serial division of flagellated cells, giving rise to sheets of cells that folded into spheres by a morphogenetic process comparable to that seen in modern choanoflagellate rosettes and calcareous sponge embryos. Finally, we infer that cell differentiation evolved in the animal stem-lineage by a combination of three mechanisms: division of labor from ancient plurifunctional cell types, conversion of temporally segregated phenotypes into spatially segregated cell types, and functional innovation.

evolutionary biology↗