Angélique Desoeuvre

Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain.

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.

Efficient In Vivo Electroporation of the Postnatal Rodent Forebrain

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

Diversity and spatiotemporal dynamics of bacterial communities: physicochemical and other drivers along an acid mine drainage

Deciphering the biotic and abiotic factors that control microbial community structure over time and along an environmental gradient is a pivotal question in microbial ecology. Carnoulès mine (France), which is characterized by acid waters and very high concentrations of arsenic, iron, and sulfate, provides an excellent opportunity to study these factors along the pollution gradient of Reigous Creek. To this end, biodiversity and spatiotemporal distribution of bacterial communities were characterized using T-RFLP fingerprinting and high-throughput sequencing. Patterns of spatial and temporal variations in bacterial community composition linked to changes in the physicochemical conditions suggested that species-sorting processes were at work in the acid mine drainage. Arsenic, temperature, and sulfate appeared to be the most important factors that drove the composition of bacterial communities along this continuum. Time series investigation along the pollution gradient also highlighted habitat specialization for some major members of the community (Acidithiobacillus and Thiomonas), dispersal for Acidithiobacillus, and evidence of extinction/re-thriving processes for Gallionella. Finally, pyrosequencing revealed a broader phylogenetic range of taxa than previous clone library-based diversity. Overall, our findings suggest that in addition to environmental filtering processes, additional forces (dispersal, birth/death events) could operate in AMD community.

Diversity and Distribution of Arsenic-Related Genes Along a Pollution Gradient in a River Affected by Acid Mine Drainage.

Some microorganisms have the capacity to interact with arsenic through resistance or metabolic processes. Their activities contribute to the fate of arsenic in contaminated ecosystems. To investigate the genetic potential involved in these interactions in a zone of confluence between a pristine river and an arsenic-rich acid mine drainage, we explored the diversity of marker genes for arsenic resistance (arsB, acr3.1, acr3.2), methylation (arsM), and respiration (arrA) in waters characterized by contrasted concentrations of metallic elements (including arsenic) and pH. While arsB-carrying bacteria were representative of pristine waters, Acr3 proteins may confer to generalist bacteria the capacity to cope with an increase of contamination. arsM showed an unexpected wide distribution, suggesting biomethylation may impact arsenic fate in contaminated aquatic ecosystems. arrA gene survey suggested that only specialist microorganisms (adapted to moderately or extremely contaminated environments) have the capacity to respire arsenate. Their distribution, modulated by water chemistry, attested the specialist nature of the arsenate respirers. This is the first report of the impact of an acid mine drainage on the diversity and distribution of arsenic (As)-related genes in river waters. The fate of arsenic in this ecosystem is probably under the influence of the abundance and activity of specific microbial populations involved in different As biotransformations.

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.

Release of arsenite, arsenate and methyl-arsenic species from streambed sediment affected by acid mine drainage: a microcosm study

Environmental context Arsenic-rich waters generated from the oxidation of mining wastes are responsible for the severe contamination of river waters and sediments located downstream from mining sites. Under certain environmental conditions, the affected riverbed sediments may represent a reservoir for arsenic from which this toxic element may be released into water, mainly as a consequence of microbial activity. Abstract The (bio-)geochemical processes driving As mobilisation from streambed sediments affected by acid mine drainage (AMD) were investigated, and the structure of the bacterial community associated with the sediments was characterised. Microcosm experiments were set up to determine the effect of oxygen, temperature (4 and 20°C) and microbial activity on As mobilisation from contrasting sediments collected during high- (November 2011) and low- (March 2012) flow conditions in the Amous River, that received AMD. Distinct bacterial communities thrived in the two sediments, dominated by Rhodobacter spp., Polaromonas spp. and Sphingomonads. These communities included only few bacteria known for their capacity to interact directly with As, whereas biogeochemical processes appeared to control As cycling. Major As mobilisation occurred in the AsIII form at 20°C in anoxic conditions, from both November and March sediments, as the result of successive biotic reductive dissolution of Mn- and Fe-oxyhydroxides. The later process may be driven by Mn- and Fe-reducing bacteria such as Geobacter spp. and possibly occurred in combination with microbially mediated AsV reduction. The involvement of other bacteria in these redox processes is not excluded. Biomethylation occurred only with the sediments collected at low-flow during oxic and anoxic conditions, although no bacteria characterised so far for its ability to methylate As was identified. Finally, sorption equilibrium of AsV onto the sediment appeared to be the main process controlling AsV concentration in oxic conditions. Comparison with field data shows that the later process, besides biomethylation, may be of relevance to the As fate in AMD-affected streams.

Agrin-Signaling Is Necessary for the Integration of Newly Generated Neurons in the Adult Olfactory Bulb

In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na+K+-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na+K+-ATPase is of functional importance in this system.

Spatial Distribution of Eukaryotic Communities Using High-Throughput Sequencing Along a Pollution Gradient in the Arsenic-Rich Creek Sediments of Carnoulès Mine, France

Microscopic eukaryotes play a key role in ecosystem functioning, but their diversity remains largely unexplored in most environments. To advance our knowledge of eukaryotic microorganisms and the factors that structure their communities, high-throughput sequencing was used to characterize their diversity and spatial distribution along the pollution gradient of the acid mine drainage at Carnoulès (France). A total of 16,510 reads were retrieved leading to the identification of 323 OTUs after normalization. Phylogenetic analysis revealed a quite diverse eukaryotic community characterized by a total of eight high-level lineages including 37 classes. The majority of sequences were clustered in four main groups: Fungi, Stramenopiles, Alveolata and Viridiplantae. The Reigous sediments formed a succession of distinct ecosystems hosting contrasted eukaryotic communities whose structure appeared to be at least partially correlated with sediment mineralogy. The concentration of arsenic in the sediment was shown to be a significant factor driving the eukaryotic community structure along this continuum.

Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage

The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ.