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International audience; CLIPR-59 is a new member of the cytoplasmic linker proteins (CLIP) family mainly localized to the trans-Golgi network. We show here that Clipr-59 expression in mice is restricted to specific pools of neurons, in particular motoneurons (MNs), and progressively increases from embryonic day 12.5 (E12.5) until the first postnatal days. We generated a Clipr-59 knockout mouse model that presents perinatal lethality due to respiratory defects. Physiological experiments revealed that this altered innervation prevents the normal nerve-elicited contraction of the mutant diaphragm that is reduced both in amplitude and fatigue-resistance at E18.5, despite unaffected functional muscular contractility. Innervation of the mutant diaphragm is not altered until E15.5, but is then partially lost in the most distal parts of the muscle. Ultrastructural observations of neuromuscular junctions (NMJs) in the distal region of the diaphragm reveal a normal organization, but a lower density of nerve terminals capped by terminal Schwann cells in E18.5 mutant when compared with control embryos. Similar defects in NMJ stability, with a hierarchy of severity along the caudo-rostral axis, are also observed in other muscles innervated by facial and spinal MNs in Clipr-59 mutant mice. Clipr-59 deficiency therefore affects axon maintenance but not axon guidance toward muscle targets. Thus, CLIPR-59 is involved in the stabilization of specific motor axons at the NMJ during mouse late embryogenesis and its role is crucial for mouse perinatal development.

International audience; In vertebrates, a remarkable diversity of sex determining mechanisms is observed, ranging from environmental to genetic sex determination (ESD and GSD). In teleosts, sex determination mechanisms include both environmental and genetic regulation and are extremely diverse, changeable, and labile. In addition, many sex-determining genes have been already found in this group of vertebrates showing that SD genes can be extremely variable even between closely related organisms. Hence, teleost fish have emerged as interesting models to study the macroevolution of sex determining (SD) mechanisms within vertebrates. The cavefish Astyanax mexicanus belongs to the Characiform group. Different populations of Astyanax have been described, including pigmented river-dwelling and several depigmented blind cave populations. These populations are still inter-fertile and cave populations are known to have evolved from a surface ancestral population around 100,000 years ago. In addition these Astyanax populations can be also reared under laboratory conditions quite easily and are amendable to genetic manipulations. Altogether this makes surface and cave populations a particularly interesting evolutionary genetic model system for comparative and microevolution studies. We thus initiated studies on the microevolution of SD and SD genes in the cavefish Astyanax mexicanus, and used restriction site-associated DNA (RAD) sequencing and pool-sequencing approaches to identify sex-biased molecular markers in both cave and surface populations. Our first results led to the identification of a candidate master sex-determining gene that potentially triggers SD in both cave and surface populations. Despite this conservation of a strict GSD and the same master sex-determining gene, our results also pin point that SD in Astyanax mexicanus has already evolved between surface and cave populations and suggest a quick evolutionary transition between an old polygenic SD system in surface population towards a more recent and simple monofactorial system in cave population.

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.

International audience; Pigmentation is a diverse and ecologically relevant trait in insects. Pigment formation has been studied extensively at the genetic and biochemical levels. The temporality of pigment formation during animal development, however, is more elusive. Here, we examine this temporality, focusing on yellow, a gene involved in the formation of black melanin. We generated a protein-tagged yellow allele in the fruit fly Drosophila melanogaster, which allowed us to precisely describe Yellow expression pattern at the tissue and cellular levels throughout development. We found Yellow expressed in the pupal epidermis in patterns prefiguring black pigmentation. We also found Yellow expressed in a few central neurons from the second larval instar to adult stages, including a subset of neurons adjacent to the clock neurons marked by the gene Pdf. We then specifically examined the dynamics of Yellow expression domain and subcellular localization in relationship to pigment formation. In particular, we showed how a late step of re-internalization is regulated by the large low-density lipoprotein receptor-related protein Megalin. Finally we suggest a new function for Yellow in the establishment of sharp pigmentation pattern boundaries, whereby this protein may assume a structural role, anchoring pigment deposits or pigmentation enzymes in the cuticle.

In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass., This work was funded by Région Centre, Région Bretagne (EVOVERT grant number 049755; PEPTISAN project), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d'Orléans and Université Pierre et Marie Curie. BGG benefited from a Région Bretagne fellowship.

International audience; The brain reward circuitry plays a key role in emotional and motivational behaviors, and its dysfunction underlies neuropsychiatric disorders such as schizophrenia, depression and drug addiction. Here, we characterized the neuronal activity pattern induced by acute amphetamine administration and during drug-seeking behavior in the zebrafish, and demonstrate the existence of conserved underlying brain circuitry. Combining quantitative analyses of cfos expression with neuronal subtype-specific markers at single-cell resolution, we show that acute d-amphetamine administration leads to both increased neuronal activation and the recruitment of neurons in the medial (Dm) and the lateral (Dl) domains of the adult zebrafish pallium, which contain homologous structures to the mammalian amygdala and hippocampus, respectively. Calbindin-positive and glutamatergic neurons are recruited in Dm, and glutamatergic and γ-aminobutyric acid (GABAergic) neurons in Dl. The drug-activated neurons in Dm and Dl are born at juvenile stage rather than in the embryo or during adulthood. Furthermore, the same territory in Dm is activated during both drug-seeking approach and light avoidance behavior, while these behaviors do not elicit activation in Dl. These data identify the pallial territories involved in acute psychostimulant response and reward formation in the adult zebrafish. They further suggest an evolutionarily conserved function of amygdala-like structures in positive emotions and motivated behavior in zebrafish and mammals.

International audience; Both excessive daytime sleepiness (EDS) and rapid eye movement (REM) sleep deregulation are part of Parkinson's disease (PD) non-motor symptoms and may complicate dopamine replacement therapy. We report here that dopamine agonists act differentially on sleep architecture in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine macaque monkey. Continuous sleep and wake electroencephalographic monitoring revealed no effect of the selective dopamine D2 receptor agonist quinpirole on EDS, whereas the selective dopamine D1 receptor agonist SKF38393 efficiently alleviated EDS and restored REM sleep to baseline values. The present results question the relevance of abandoning D1 receptor agonist treatment in PD as it might actually improve sleep-related disorders.

International audience; Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

International audience; The calcium sensing receptor (CaSR) is expressed by subpopulations of neuronal and glial cells throughout the brain and is activated by extracellular calcium [Formula: see text] . During development, the CaSR regulates neuronal cell growth and migration as well as oligodendroglial maturation and function. Emerging evidence suggests that in nerve terminals, CaSR is implicated in synaptic plasticity and neurotransmission. In this review, we analyze the roles attributed to CaSR in regulating diverse brain functions, including central regulation of body fluid composition and blood pressure. We also discuss the potential relevance of Ca(2+)-sensing in brain by other family C G protein-coupled receptors. Finally, evidence that the CaSR contributes to the pathogenesis of various brain disorders raises the possibility that pharmacological modulators of the CaSR may have therapeutic benefit.

International audience; SHG angular intensity pattern (SHG-AIP) of healthy and proteolysed muscle tissues are simulated and imaged here for the first time to our knowledge. The role of the spatial distribution of second-order nonlinear emitters on SHG-AIP is highlighted. SHG-AIP with two symmetrical spots is found to be a signature of healthy muscle whereas SHG-AIP with one centered spot in pathological mdx muscle is found to be a signature of myofibrillar disorder. We also show that SHG-AIP provides information on the three-dimensional structural organization of myofibrils in physiological and proteolysed muscle. Our results open an avenue for future studies aimed at unraveling more complex physiological and pathological fibrillar tissues organization.

International audience; The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID(50)) of 37.25mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID(50) of 0.5mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC(50)) of 20.3μM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC(50) of 7.28μM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC(50) of 0.19μM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.

International audience; The open-endedness of a system is often defined as a continual production of novelty. Here we pin down this concept more fully by defining several types of novelty that a system may exhibit, classified as variation, innovation, and emergence. We then provide a meta-model for including levels of structure in a system’s model. From there, we define an architecture suitable for building simulations of open-ended novelty-generating systems and discuss how previously proposed systems fit into this framework. We discuss the design principles applicable to those systems and close with some challenges for the community.

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology., Quantitative analysis of embryonic cell dynamics from large data sets remains a major challenge in the field of developmental biology. Here the authors develop software and a workflow to reconstruct cell lineage trees from 3D time lapse imaging data sets from several developing organisms including zebrafish, tunicates and sea urchins.

International audience; In this chapter, available evidence on the toxicological profile of spirolides and other lipophilic cyclic imine toxins is reviewed, highlighting their chemical structure, the phytoplankton species involved in their production, their pharmacokinetics/toxicokinetics and experimental toxicity, and their molecular targets and mechanisms of action. These phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Their chemical structure is represented by a macrocycle, with the ring size between 14 and 27, and two conserved features that include the cyclic imine group and spiroketal ring system. The producers of spirolides, gymnodimines, and pinnatoxins have been identified as being the dinoflagellates Alexandrium ostenfeldii/peruvianum, Karenia selliformis, and Vulcanodinium rugosum. Their acute toxicity, appraised by the mouse bioassays, classifies them as “fast-acting” toxins because they induce rapid onset of neurological symptoms followed by death within a few minutes. The spirolide congeners are the most toxic after intraperitoneal injection, while there are indications that pinnatoxins are the most toxic group after oral administration. The neurotoxic effects reported for these phycotoxins are mostly due to their specific interaction with the muscle and neuronal types of nicotinic acetylcholine receptors which are the principal molecular targets of spirolides, gymnodimines, and pinnatoxins, so far studied. Hence, these phycotoxins exhibit both high affinity and broad specificity on nicotinic receptors, indicating that their sites of interaction in the receptors include amino acid residues highly conserved among animal species.

International audience; The ability to regenerate damaged cells in the retina varies tremendously among species, being restricted for most of them to specific developmental stages. Regarding vertebrates, only the newt was thought to exhibit full regenerative capacity upon retinectomy in the adulthood. The recent discovery that the anuran amphibian Xenopus can regenerate its retina after metamorphosis opened new avenues to investigate the cellular and molecular mechanisms involved in this process. In this review, we provide an historical overview of regeneration studies in Xenopus. Particular emphasis is given to the cellular sources contributing to retinal replacement, the involvement of tissue interactions and the importance of the injury paradigm. We also describe recent progress and promises in the field brought by the development of 3D tissue culture methods and transgenic Xenopus models.

The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis. Stem Cells 2012;30:2784–2795

International audience; Pinnatoxins belong to the cyclic imine (CI) group of marine toxins with a unique toxicological profile. The need for structural integrity of the aliphatic 7-membered cyclic imine for the potent bioactivity of pinnatoxins has been experimentally demonstrated. In this study, we probe interconversion of the natural cyclic imine and its open form, pinnatoxin A amino ketone (PnTX AK), under physiologically relevant aqueous conditions. Our studies demonstrate the high stability of PnTX A. The unusual stability of the imine ring in PnTX A has implications for its oral toxicity and detoxification. These studies, as well the access to PnTX amino ketone, were enabled by the total synthesis of (+)-pinnatoxin A completed previously in our laboratory.

International audience; Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

International audience; The timing of commitment and cell-cycle exit within progenitor populations during neurogenesis is a fundamental decision that impacts both the number and identity of neurons produced during development. We show here that microRNA-9 plays a key role in this process through the direct inhibition of targets with antagonistic functions. Across the ventricular zone of the developing zebrafish hindbrain, miR-9 expression occurs at a range of commitment stages. Abrogating miR-9 function transiently delays cell-cycle exit, leading to the increased generation of late-born neuronal populations. Target protection analyses in vivo identify the progenitor-promoting genes her6 and zic5 and the cell-cycle exit-promoting gene elavl3/HuC as sequential targets of miR-9 as neurogenesis proceeds. We propose that miR-9 activity generates an ambivalent progenitor state poised to respond to both progenitor maintenance and commitment cues, which may be necessary to adjust neuronal production to local extrinsic signals during late embryogenesis.

ObjectivesTissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach.MethodsThe activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice.ResultsHedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis.ConclusionsInhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.

Chantier qualité GA; International audience; Every model species requires its own developmental table. Astyanax mexicanus, a teleost fish comprising both sighted river and blind cave populations, is becoming more and more important in the field of developmental and evolutionary biology. As such, a developmental staging table is increasingly necessary, particularly since comparative analysis of early developmental events is widely employed by researchers. We collected freshly spawned embryos from surface fish and Pachón cavefish populations. Embryos were imaged every 10-12 min during the first day of development, and less frequently in the following days. The results provide an illustrated comparison of selected developmental stages from one cell to hatching of these two populations. The two morphs show an essentially synchronous development regarding major events such as epiboly, neurulation, somitogenesis, heart beating, or hatching. We also present data on particular morphological characters appearing during larval development, such as eye size, yolk regression, swim bladder, and fin development. Some details about the development of F1 Pachón cave×surface hybrids are also given. Comparisons are made with Danio rerio (zebrafish) development.

Motivation: Multi-view microscopy techniques such as Light-Sheet Fluorescence Microscopy (LSFM) are powerful tools for 3D + time studies of live embryos in developmental biology. The sample is imaged from several points of view, acquiring a set of 3D views that are then combined or fused in order to overcome their individual limitations. Views fusion is still an open problem despite recent contributions in the field. Results: We developed a wavelet-based multi-view fusion method that, due to wavelet decomposition properties, is able to combine the complementary directional information from all available views into a single volume. Our method is demonstrated on LSFM acquisitions from live sea urchin and zebrafish embryos. The fusion results show improved overall contrast and details when compared with any of the acquired volumes. The proposed method does not need knowledge of the system's point spread function (PSF) and performs better than other existing PSF independent fusion methods. Availability and Implementation: The described method was implemented in Matlab (The Mathworks, Inc., USA) and a graphic user interface was developed in Java. The software, together with two sample datasets, is available at http://www.die.upm.es/im/software/SPIMFusionGUI.zip A public release, free of charge for non-commercial use, is planned after the publication of this article. Contact: jlrubio@die.upm.es; nadine.peyrieras@inaf.cnrs-gif.fr; mledesma@die.upm.es Supplementary Information: Supplementary data are available at Bioinformatics online.

International audience; Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower 'efficacy dose/toxicity dose' ratio as compared with Dbait/PEI, coDbait was selected for clinical trials.

International audience; In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

International audience; A major prerequisite for the investigation of tissue-specific processes is the identification of cis-regulatory elements. No generally applicable technique is available to distinguish them from any other type of genomic non-coding sequence. Therefore, researchers often have to identify these elements by elaborate in vivo screens, testing individual regions until the right one is found. Here, based on many examples from the literature, we summarize how functional enhancers have been isolated from other elements in the genome and how they have been characterized in transgenic animals. Covering computational and experimental studies, we provide an overview of the global properties of cis-regulatory elements, like their specific interactions with promoters and target gene distances. We describe conserved non-coding elements (CNEs) and their internal structure, nucleotide composition, binding site clustering and overlap, with a special focus on developmental enhancers. Conflicting data and unresolved questions on the nature of these elements are highlighted. Our comprehensive overview of the experimental shortcuts that have been found in the different model organism communities and the new field of high-throughput assays should help during the preparation phase of a screen for enhancers. The review is accompanied by a list of general guidelines for such a project.

International audience; Although the simultaneous presence of tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) is considered as a phenotypic signature of dopamine (DA) neurons, it has been suggested that they are not uniformly expressed in all dopaminergic brain nuclei. Moreover, in nonmammalian vertebrates, two tyrosine hydroxylase genes (TH1 and TH2) are found, and they exhibit different expression patterns in zebrafish brains. Here we present a detailed description of the distribution of TH1, TH2, AADC, DAT, and VMAT2 transcripts, in relation to TH and DA immunoreactivity to better characterize dopaminergic nuclei in the adult zebrafish forebrain. TH2-positive cells in the hypothalamus are strongly DA immunoreactive (DAir), providing direct evidence that they are dopaminergic. DAir cells are also found in most TH1-positive or TH-immunoreactive (THir) nuclei. However, the DAir signal was weaker than THir in the olfactory bulb, telencephalon, ventral thalamus, pretectum, and some posterior tubercular and preoptic nuclei. These cell populations also exhibited low levels of VMAT2 transcripts, suggesting that low DA is due to a lower vesicular DA accumulation. In contrast, cell populations with low levels of AADC did not always have low levels of DA. DAT transcripts were abundantly expressed in most of the TH1- or TH2-positive territories. In addition, DAT and/or VMAT2 transcripts were found in some periventricular cell populations such as in the telencephalon without TH1 or TH2 expression. Thus, expression patterns of dopaminergic cell markers are not homogeneous, suggesting that the gene regulatory logic determining the dopaminergic phenotype is unexpectedly complex.

International audience; Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP(3)R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP(3)R isoforms were present in adult muscle. IP(3)R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP(3)R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.

Alzheimer’s disease (AD) is the neurodegenerative disorder provoking memory loss and cognitive dysfunction in elderly patients. It is the fourth cause of death in the developed countries (after heart disease, cancer and stroke). This disease is incurable for the moment, only palliative and long-term treatment is available having acetylcholinesterase and a7 nicotinic acetylcholine receptor as molecular targets. The key event of AD seems to be beta amyloid peptide formation and aggregation, however a reduced synthesis of neurotransmitter – acetylcholine – was also observed in the etiology of this dysfunction. Additionally, amyloid peptide Ab binds to a7 nicotinic acetylcholine receptor to induce either receptor activation or inhibition in an Ab concentration-dependent mode. Ab oligomers induce Tau phosphorylation via a7 nicotinic receptor activation as well. In our synthetic work, we took advantage of ‘‘click-chemistry’’ to couple modified acetylcholinesterase inhibitors (Cl- orNH2-tacrine and Cl-NH2- and acyl-huprine) to azide-alkyne derivatized agonists of a7 nicotinic acetylcholine receptor in order to obtain a series of multi-target directed ligands which could ideally simultaneously interact with acetylcholinesterase and with the a7 nicotinic acetylcholine receptor. The acetylcholinesterase inhibition from the synthesized two-target ligands ranged between 3 and 15 nM. The agonistic behaviour of the two-target compounds was evaluated by manual and automated two-electrode voltage clamp on Xenopus oocytes expressing human a7 nicotinic acetylcholine receptors. Among the double functionalized organic molecules, one compound showed an in vitro inhibitory activity towards human acetylcholinesterase in nanomolar scale and an agonistic activity in the micromolar range. Moreover, compound 1 showed a good ability to cross the blood–brain barrier. It is worth noticing that at high concentrations the referred compound 1 showed a pronounced antagonistic activity towards the nicotinic receptor. The synthetic precursors were as well tested on Xenopus oocytes: (i) the agonistic activity of the agonist moiety towards a7 nicotinic acetylcholine receptors in nanomolar scale was verified and (ii) the presumed nicotinic antagonistic activity of the anticholinesterase precursor group was also demonstrated by this approach explaining why at high concentrations, the partial agonist compound 1 behaves as an antagonist of a7 nicotinic acetylcholine receptors.

International audience; Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

It was first demonstrated in a teleost fish that the stimulatory control of the gonadotrope axis by GnRH can be counterbalanced by an inhibitory control exerted by dopamine (DA). Later on, this inhibitory dopaminergic control was found in various vertebrate species. However the functional importance of this regulatory pathway varies according to the species. To deepen our knowledge on this inhibitory dopaminergic system, we used the zebrafish (Danio rerio) as a model, in which numerous molecular tools are available.First we demonstrated that DA indeed plays a role in the neuroendocrine control of zebrafish reproduction. By injecting a dopamine receptor antagonist together with an agonist of the GnRH (GnRHa), we were able to stimulate LH expression in the pituitary, and to reactivate the spawning cycles in sexually regressed old females, an effect of which was not produced by the GnRHa alone.We then studied the neuroanatomical basis of this inhibitory control. After observing the expression of the D2-DA receptors subtypes in LH cells, we highlighted numerous dopaminergic terminals on- or in the vicinity of- these cells. We then localized, by DiI retrograde tracing experiments in adult zebrafish, the dopaminergic cell bodies giving rise to these projections in the most antero-Ventral part of the preoptic area. We have called these hypophysiotropic neurons the preoptico-Hypophysial (POH) DA neurons.We next studied the development of POHDA neurons. Taking advantage of early anatomical landmarks, we followed the embryonic development of these cells. We showed that the first POHDA neurons arise at around 72 hours post fertilization (hpf), more than 24 hours later that the DA neurons in the neighbor suprachiasmatic nucleus (SCDA). This late differentiation would explain why POHDA neurons have not been studied in the developing embryo so far. We showed that contrary to the number of the SCDA neurons, which is constant all along the fish life, that of POHDA neurons increases proportionally to the growth of the fish due to continuous neurogenesis. Finally, we examined the expression profiles of developmental genes related to the regionalization of the anterior forebrain. We showed that the genetic networks involved in the development of POHDA and SCDA populations are at least partly different. To summarize, this work demonstrates for the first time the existence of a dopaminergic inhibitory control of gonadotrope function in zebrafish. It describes the anatomy of the preoptico-Hypophyseal dopaminergic system supporting these DA actions and the setting up of these neurons during embryonic development. We show that these neuroendocrine population displays neurogenesis even during adulthood. Our findings also provide the genetic bases for future functional studies on the development of POHDA, a poorly studied neuroendocrine DA population.; C’est chez un téléostéen qu’il a été démontré pour la première fois que le contrôle stimulateur de l’axe gonadotrope par la GnRH peut être contrebalancé par un contrôle inhibiteur assuré par la dopamine (DA). Ce contrôle dopaminergique inhibiteur a été retrouvé par la suite chez diverses espèces de vertébrés. Cependant l’importance fonctionnelle de cette voie inhibitrice de régulation varie beaucoup d’une espèce à l’autre. Pour approfondir nos connaissances sur ce système dopaminergique inhibiteur de la reproduction, nous avons utilisé le poisson zèbre (Danio rerio), un modèle de vertébrés pour lequel de nombreux outils moléculaires sont disponibles.Nous avons d’abord démontré qu’il existait bien un contrôle dopaminergique de la fonction gonadotrope dans cette espèce: en injectant un antagoniste dopaminergique en même temps qu’un analogue de la GnRH, nous avons pu stimuler l’expression de la LH dans l’hypophyse et réactiver les cycles de ponte chez des femelles âgées sexuellement régressées, un effet qui n’est pas produit par l’agoniste de la GnRH seul. Nous avons ensuite étudié le substrat neuroanatomique de cette action inhibitrice. Après avoir observé l’expression par les cellules à LH des sous-Types de récepteurs D2 qui existent chez le poisson-Zèbre, nous avons mis en évidence de nombreuses terminaisons dopaminergiques sur- ou à proximité- de ces cellules gonadotropes. Nous avons ensuite localisé, par des expériences de traçage rétrograde chez l’adulte, les corps cellulaires des neurones dopaminergiques qui émettent ces projections, dans la partie la plus antéro-Ventrale de l'aire préoptique. Nous avons appelé ces neurones hypophysiotropes: les neurones dopaminergiques préoptico-Hypophysaires (POHDA). Nous nous sommes aussi intéressés au développement des neurones POHDA. Grâce à des repères anatomiques précoces, nous avons pu les répérer précisément au sein de l’aire préoptique et suivre leur apparition sur des embryons de plus en plus jeunes. Nous avons ainsi mis en évidence que les tout premiers neurones POHDA n’apparaissent qu’à partir de 72 heures post-Fécondation (hpf), soit plus de 24 h après les neurones dopaminergiques du noyau suprachiasmatique (SCDA) voisin. Cette différenciation tardive explique probablement pourquoi les neurones POHDA ont jusque-Là été ignorés dans toutes les études de développement. En outre, nous avons montré que contrairement au nombre des neurones SCDA qui reste constant tout au long de la vie du poisson-Zèbre, celui des neurones POHDA continue d’augmenter tant que le poisson continue à grandir de manière allométrique, grâce à une neurogenèse continue. Enfin, nous avons examiné les profils d'expression de plusieurs gènes en relation avec la régionalisation du cerveau antérieur. Cette étude a permis de montrer que les réseaux génétiques impliqués dans le développement des populations SCDA et POHDA sont au moins en partie différents.Ces travaux démontrent pour la première fois l’existence d’un contrôle dopaminergique inhibiteur de la fonction gonadotrope chez le poisson-Zèbre. Ils décrivent l’anatomie de ce système dopaminergique chez l’adulte, sa mise en place au cours du développement et ses capacités de neurogenèse continue. Ils apportent chez le poisson-Zèbre des bases génétiques sur l’identité régionale de l’aire préoptique qui vont permettre d’aborder des études fonctionnelles sur le développement de ces neurones neuroendocrines mal connus.

International audience

International audience; Macrocyclic spiroimine phycotoxins constitute a growing class of structurally related marine neurotoxins contaminating shellfish (Molgó et al., 2014). Most of them are strong antagonists of nicotinic acetylcholine receptors (nAChRs), and a few ones showed moderate activities against muscarinic acetylcholine receptors (mAChRs). During recent years this field has witnessed important developments with regard to chemical synthesis, X-ray crystallography, electrophysiology, detection methods and molecular modelling. In this study, the conformational flexibility of representative cyclic imine phycotoxins (gymnodimines, spirolides, pinnatoxins, pteriatoxins, spiro-prorocentrimine and prorocentrolides) was evaluated using long-timescale all-atom molecular dynamics simulations in OPLS-AA force field.

International audience; and the United States of America, attended this meeting organized by the French Society of Toxinology (SFET, www.sfet.asso.fr). Should a reminder be appropriate, please note that the SFET missions aim at (i) promoting research in all domains concerning the venoms and toxins from animal, vegetal or microbial origins, i.e. the chemical, immunological, environmental, pharmacological, physiological domains, along with mode of action, structure-function relationships, drug-design and applicability questionings; (ii) favouring the meeting of colleagues in the field, communication to other scientists, education and training at all levels, and dissemination to more general audiences; (iii) improving fundamental research on toxinology and enhance its relevance and applications. We warmly thank all those who made this meeting achievable, and we particularly acknowledge the cogent contribution of Professor Alan Harvey, Editor-in-chief of Toxicon, who made the publication of the RT21 proceedings possible as a Special Issue focused on Freshwater and Marine toxins. This Special Issue gathers 16 original peer-reviewed articles and reviews, and 48 abstracts, all reflecting those works and data that were presented during the SFET meeting. For decades, scientists have been working on terrestrial toxins. The main reason is that the accessibility to the producers-venomous animals, pathogenic bacteria or poisonous plants-and the related human health issues in endemic areas have motivated pharmacologists and physiologists to study the mode of action of natural toxic molecules found just outside of laboratories. Moreover, natural products have long been used in traditional medicine as drugs to treat a variety of human diseases. A well-known example is morphine, which keeps being the most widely used molecule to alleviate pain. However, the exploration of shorelines and deep oceans with modern equipment, the recreational use of seas and the emergence of eutrophication of freshwaters (a process where water bodies receive excess nutrients that stimulate excessive phytoplankton and bacteria growth) in occidental and developing countries have contributed to the molecular identification of toxic cocktails from aquatic producers. The human colonization of coastal waters and the growing activity along the rivers have increased the probability of human interactions with toxin producers. Nowadays, sea snakes, cone snails, cnidarians, toxic microalgae, parasitic bacteria are common features of the global concern in modern toxicology. Moreover, should one take into account the recent approval of marine toxins as chemical templates to design new therapeutic agents directed toward major human pathologies-i.e. pain and cancer (Prialt and Yondelis, respectively)-one would be convinced that pharmacological active molecules found underwater may represent an endless pipeline for the next generation of natural drugs.

Adult neural stem cells (aNSCs) are defined by their self-Renewal and multipotency, which allow them to generate both neurons and glial cells in the adult brain. Contrary to mammals, the zebrafish brain maintains numerous neurogenic zones in the adult, among which the most characterized is the pallial ventricular zone. It is composed of radial glial cells serving as aNSCs. Which embryonic neural progenitors are at the origin of these aNSCs is still unknown. This work aims to determine the relative contributions of two embryonic neural progenitor populations, the «proneural clusters» (involved in embryonic neurogenesis) and the « progenitor pools » (characterized by a delayed neurogenesis), to the formation of aNSCs in the zebrafish pallium. First, using genetic lineage tracing techniques, we were able to identify the embryonic neural progenitor population at the origin of a subpopulation of aNSCs located in the dorso-Medial part of the pallium. The her4:ERT2CreERT2 transgenic driver line, combined with pharmacological treatments inhibiting the Notch signalling pathway, allowed showing that neural progenitors giving rise to dorso-Medial pallial aNSCs express the « Enhancer of split » her4 gene, specifically expressed in « proneural clusters » from very early stages of development. As a second step, clonal analyses as well as spatially controlled recombinations by laser highlighted that aNSCs of the zebrafish lateral pallium do not derive from her4-Positive embryonic progenitors maintained by the Notch pathway, but from a restricted population of neuroepithelial cells located in the embryonic telencephalic roof plate. These cells display « progenitor pool » specific features, as for instance the expression of non-Canonical her genes (independent of Notch signalling) such as her6 and her9, the expression of components of signalling pathways such as Wnt, BMP, FGF, and a late neurogenesis onset. These progenitors progressively generate, from juvenile stages, the vast majority of the aNSCs of the lateral pallium. Most interestingly, a small population of these neuroepithelial cells persists in the postero-Lateral pallium at adult stage and keeps generating de novo aNSCs of this brain region. In addition to identifying the origin of pallial aNSCs in the zebrafish, this study also delivers information on the progressive maturation steps that embryonic progenitors undergo to generate aNSCs, and highlights similarities and differencies existing between the dorso-Medial and lateral progenitors. Finally, this work also permits tracing the neurons generated by stem cells at different stages. This reveals for the first time the progressive formation of the different zebrafish pallial compartements, and allows appreciating their homologies with the mouse pallial regions., Les cellules souches neurales adultes (aNSCs) sont définies par des fonctions d’auto-Renouvellement et de multipotence qui leur permettent de générer dans le cerveau adulte tant des neurones que des cellules gliales. Contrairement aux mammifères, le cerveau de poisson zèbre présente de nombreuses zones de neurogenèse adulte dont la plus caractérisée est la zone ventriculaire du pallium. Elle est composée de cellules de glies radiaires qui font office de aNSCs dans cette partie du cerveau. Quels progéniteurs neuraux embryonnaires sont sélectionnés pour être à l’origine de ces aNSCs reste mal connu. Ce travail a pour objectif de déterminer la contribution relative de deux populations de progéniteurs neuraux embryonnaires, les “clusters proneuraux” (impliqués dans la neurogenèse embryonnaire) et les “pools de progéniteurs” (caractérisés par une neurogenèse tardive), dans la formation des aNSCs du pallium de poisson zèbre. Dans un premier temps, à l’aide de techniques génétiques de lignage cellulaire, nous avons pu identifier la population de progéniteurs neuraux embryonnaires à l’origine d’une sous-Population des aNSCs située dans la partie dorso-Médiane du pallium. Des expériences de lignage utilisant la lignée de poisson zèbre her4:ERT2CreERT2 combinées à des traitements inhibiteurs de la voie de signalisation Notch nous ont permis de déterminer que les progéniteurs neuraux donnant naissance aux aNSCs du pallium dorso-Médian expriment le gène « Enhancer of split » her4, qui caractérise les “clusters proneuraux”, ce dès des stades très précoces du développement. Dans un second temps, des analyses clonales ainsi que des recombinaisons spatialement contrôlées par laser nous ont permis de mettre en évidence que les aNSCs de la partie latérale du pallium de poisson zèbre ne proviennent pas de progéniteurs embryonnaires exprimant her4 et maintenus par la voie Notch, mais d’une population restreinte de cellules neuroépitheliales situées dans la plaque du toit du télencéphale embryonnaire. Ces cellules présentent des caractéristiques spécifiques des “pool de progéniteurs”, à savoir l’expression de gènes her non-Canoniques (dont l’expression n’est pas dépendante de la voie de signalisation Notch) tels que her6 et her9, l’expression de ligands de voies de signalisation telles que Wnt, BMP et FGF, et une neurogenèse tardive. Elles génèrent progressivement, à partir du stade juvénile, une grande partie des aNSCs du pallium latéral. De plus, une partie de ces cellules neuroépitheliales persistent dans le pallium latéral postérieur chez l’adulte et continuent de former de novo des aNSCs dans cette région du cerveau. Outre la vision globale que cette étude nous a permis d’avoir sur l’origine embryonnaire de la totalité des aNSCs du pallium de poisson zèbre, elle a aussi délivré des informations sur les étapes de maturation progressive des progéniteurs embryonnaires pour former les aNSCs, et les similitudes et divergences qui existent entre la population dorso-Médiane et latérale à ce sujet. Enfin, en traçant les neurones issus des cellules souches à différents stades, cette étude a pour la première fois mis en évidence la formation progressive des compartiments neuronaux du pallium de poisson zèbre, et ainsi permis d’apprécier les homologies de ces compartiments avec les régions du pallium de souris.

International audience; Radial glial progenitors play pivotal roles in the development and patterning of the spinal cord, and their fate is controlled by Notch signaling. How Notch is shaped to regulate their crucial transition from expansion toward differentiation remains, however, unknown. miR-132 in the developing zebrafish dampens Notch signaling via a cascade involving the transcriptional corepressor Ctbp2 and the Notch suppressor Sirt1. At early embryonic stages, high Ctbp2 levels sustain Notch signaling and radial glial expansion and concomitantly induce miR-132 expression via a double-negative feedback loop involving Rest inhibition. The changing balance in miR-132 and Ctbp2 interaction gradually drives the switch in Notch output and radial glial progenitor fate as part of the larger developmental program involved in the transition from embryonic to larval spinal cord.

Summary In the adult brain, self-renewal is essential for the persistence of neural stem cells (NSCs) throughout life, but its regulation is still poorly understood. One NSC can give birth to two NSCs or one NSC and one transient progenitor. A correct balance is necessary for the maintenance of germinal areas, and understanding the molecular mechanisms underlying NSC division mode is clearly important. Here, we report a function of the Sonic Hedgehog (SHH) receptor Patched in the direct control of long-term NSC self-renewal in the subependymal zone. We show that genetic conditional activation of SHH signaling in adult NSCs leads to their expansion and the depletion of their direct progeny. These phenotypes are associated in vitro with an increase in NSC symmetric division in a process involving NOTCH signaling. Together, our results demonstrate a tight control of adult neurogenesis and NSC renewal driven by Patched., Graphical Abstract, Highlights Ptc inactivation in the adult NSCs expands the NSC pool and depletes their progeny Neurogenesis blockade is related to the increase of NSC symmetric division PTC exerts a central role via NOTCH, in the regulation of adult NSC self-renewal, In the adult brain, self-renewal is essential for the persistence of neural stem cells throughout life. Traiffort, Ruat, and colleagues report that the Sonic Hedgehog receptor Patched controls long-term self-renewal of these cells in the subependymal zone by increasing their symmetric division in a process involving NOTCH signaling.

International audience; Abstract Astyanax mexicanus, a teleost fish comprising both sighted river-dwelling and blind cave-dwelling morphs, is becoming increasingly used in the field of developmental and evolutionary biology. Thus, new experimental and technological tools are needed on this emerging fish model by the expanding scientific community. Here, we describe Astyanax husbandry and egg spawning habits, a prerequisite to the successful establishment of Astyanax transgenic lines. We then compare two different transgenesis methods on both surface and cave Astyanax. Both meganuclease (I-SceI)- and transposase (Tol2)-mediated transgenesis are equivalently efficient, resulting in ∼40% mosaic transgenic fish in F0. Furthermore, the transmission rate was analyzed in F1 in the case of the I-SceI method and was found to be 16%. Finally, the transgene was found stable up the F3 generation, demonstrating the feasibility of generating stable transgenic lines in Astyanax and opening a wide range of possibilities for this fish model.

Background Enhanced food-finding efficiency is an obvious adaptive response to cave environments. Here, we have compared the food-finding abilities of Astyanax surface fish and blind cavefish young larvae in their first month of life, in the dark. Results Our results show that enhanced prey capture skills of cavefish are already in effect in fry soon after the yolk is depleted and the young larvae must find food for themselves. Moreover, using prey capture competition assays on surface fish fry with lensectomies, we showed that eye-dependent developmental processes are not the main determinant for enhanced prey capture skills. Finally, using F2 hybrid larvae resulting from crosses between surface fish and cavefish, we found that reduced eyes do not confer a selective advantage for prey capture by fry in the dark. Conclusion We discuss these data with regards to our current developmental and genetic understanding of cavefish morphological and behavioral evolution.

International audience; Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

International audience; The facial neural crest (FNC), a pluripotent embryonic structure forming craniofacial structures, controls the activity of brain organisers and stimulates cerebrum growth. To understand how the FNC conveys its trophic effect, we have studied the role of Smad1, which encodes an intracellular transducer, to which multiple signalling pathways converge, in the regulation of Foxg1. Foxg1 is a transcription factor essential for telencephalic specification, the mutation of which leads to microcephaly and mental retardation. Smad1 silencing, based on RNA interference (RNAi), was performed in pre-migratory FNC cells. Soon after electroporation of RNAi molecules, Smad1 inactivation abolished the expression of Foxg1 in the chick telencephalon, resulting in dramatic microcephaly and partial holoprosencephaly. In addition, the depletion of Foxg1 activity altered the expression Otx2 and Foxa2 in di/mesencephalic neuroepithelium. However, when mutated forms of Smad1 mediating Fgf and Wnt signalling were transfected into FNC cells, these defects were overcome. We also show that, downstream of Smad1 activity, Dkk1, a Wnt antagonist produced by the FNC, initiated the specification of the telencephalon by regulating Foxg1 activity. Additionally, the activity of Cerberus in FNC-derived mesenchyme synergised with Dkk1 to control Foxg1 expression and maintain the balance between Otx2 and Foxa2.

International audience; We use Astyanax mexicanus, a single species with surface-dwelling forms (SF) and blind de-pigmented cave forms (CF), to study mechanisms underlying the evolution of brain and behaviour. In CF, the origin of changes in complex motivated behaviours (social, feeding, sleeping, exploratory) is unknown. Here we find a hyper-aminergic phenotype in CF brains, including high levels and neurotransmission indexes for serotonin, dopamine and noradrenaline, and low monoamine oxidase (MAO) activity. Although MAO expression is unchanged in CF, a pro106leu mutation is identified in the MAO coding sequence. This mutation is responsible for low MAO activity and high serotonin neurotransmission index in CF brains. We find the same mutated allele in several natural CF populations, some being independently evolved. Such occurrence of the same allele in several caves may suggest that low MAO activity is advantageous for cave life. These results provide a genetic basis for several aspects of the cavefish 'behavioural syndrome'.

International audience; In mammals, eupnoeic breathing is periodically interrupted by spontaneous augmented breaths (sighs) that include a larger-amplitude inspiratory effort, typically followed by a post-sigh apnoea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre-Bötzinger complex (preBötC) can generate the distinct inspiratory motor patterns for both eupnoea- and sigh-related behaviour. During mouse embryonic development, the preBötC begins to generate eupnoeic rhythmicity at embryonic day (E) 15.5, but the network's ability to also generate sigh-like activity remains unexplored at prenatal stages. Using transverse brainstem slice preparations we monitored the neuronal population activity of the preBötC at different embryonic ages. Spontaneous sigh-like rhythmicity was found to emerge progressively, being expressed in 0/32 slices at E15.5, 7/30 at E16.5, 9/22 at E17.5 and 23/26 at E18.5. Calcium imaging showed that the preBötC cell population that participates in eupnoeic-like discharge was also active during fictive sighs. However, patch-clamp recordings revealed the existence of an additional small subset of neurons that fired exclusively during sigh activity. Changes in glycinergic inhibitory synaptic signalling, either by pharmacological blockade, functional perturbation or natural maturation of the chloride co-transporters KCC2 or NKCC1 selectively, and in an age-dependent manner, altered the bi-phasic nature of sigh bursts and their coordination with eupnoeic bursting, leading to the generation of an atypical monophasic sigh-related event. Together our results demonstrate that the developmental emergence of a sigh-generating capability occurs after the onset of eupnoeic rhythmogenesis and requires the proper maturation of chloride-mediated glycinergic synaptic transmission.

International audience; The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

International audience; Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca(2+) homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2-like 10 (Nrz) is required for Ca(2+) signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca(2+) release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca(2+) transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca(2+). Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca(2+) transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca(2+) signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

Background. The majority of patients receiving the platinum-based chemotherapy drug oxaliplatin develop peripheral neurotoxicity. Because this neurotoxicity involves ROS production, we investigated the efficacy of mangafodipir, a molecule that has antioxidant properties and is approved for use as an MRI contrast enhancer. Methods. The effects of mangafodipir were examined in mice following treatment with oxaliplatin. Neurotoxicity, axon myelination, and advanced oxidized protein products (AOPPs) were monitored. In addition, we enrolled 23 cancer patients with grade ≥2 oxaliplatin-induced neuropathy in a phase II study, with 22 patients receiving i.v. mangafodipir following oxaliplatin. Neuropathic effects were monitored for up to 8 cycles of oxaliplatin and mangafodipir. Results. Mangafodipir prevented motor and sensory dysfunction and demyelinating lesion formation. In mice, serum AOPPs decreased after 4 weeks of mangafodipir treatment. In 77% of patients treated with oxaliplatin and mangafodipir, neuropathy improved or stabilized after 4 cycles. After 8 cycles, neurotoxicity was downgraded to grade ≥2 in 6 of 7 patients. Prior to enrollment, patients received an average of 880 ± 239 mg/m 2 oxaliplatin. Patients treated with mangafodipir tolerated an additional dose of 458 ± 207 mg/m 2 oxaliplatin despite preexisting neuropathy. Mangafodipir responders managed a cumulative dose of 1,426 ± 204 mg/m 2 oxaliplatin. Serum AOPPs were lower in responders compared with those in nonresponders. Conclusion. Our study suggests that mangafodipir can prevent and/or relieve oxaliplatin-induced neuropathy in cancer patients. Trial registration. Clinicaltrials.gov NCT00727922.

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience

International audience

International audience; The paper presents numerical algorithms leading to an automated cell tracking and reconstruction of the cell lineage tree during the first hours of animal embryogenesis. We present results obtained for large-scale 3D+time two-photon laser scanning microscopy images of early stages of zebrafish (Danio rerio) embryo development. Our approach consists of three basic steps – the image filtering, the cell centers detection and the cell trajectories extraction yielding the lineage tree reconstruction. In all three steps we use nonlinear partial differential equations. For the filtering the geodesic mean curvature flow in level set formulation is used, for the cell center detection the motion of level sets by a constant speed regularized by mean curvature flow is used and the solution of the eikonal equation is essential for the cell trajectories extraction. The core of our new tracking method is an original approach to cell trajectories extraction based on finding a continuous centered paths inside the spatio-temporal tree structures representing cell movement and divisions. Such paths are found by using a suitably designed distance function from cell centers detected in all time steps of the 3D+time image sequence and by a backtracking in the steepest descent direction of a potential field based on this distance function. We also present efficient and naturally parallelizable discretizations of the aforementioned nonlinear PDEs and discuss properties and results of our new tracking method on artificial and real 4D data.

The fish Astyanax mexicanus presents, within the same species, several populations of river-dwelling surface fish (SF) and blind cave-living fish (CF). In blind cavefish, the eyes first develop almost normally during embryogenesis. But 24 hours after fertilization (hpf), when the embryo hatches, the lens enters apoptosis, which triggers the progressive degeneration of the entire eye. My thesis project aimed at understanding the mechanism leading to lens apoptosis, which was so far unknown. We reasoned that the defect(s) should take place during the early stages of lens development. The lens develops from a placode, a thickening of the ectoderm at the neurula stage. All placodes, giving rise to sense organs of the head, originate from the “panplacodal” field, located at the border of the anterior neural plate at 10 hpf. We compared the patterning of the panplacodal field in the 2 morphs, using in situ hybridizations for placodal marker genes. In CF, the lens placode territory is reduced at 10 hpf, and the lens is smaller at all stages examined. Conversely, the olfactory placode is enlarged, and gives rise to a bigger olfactory epithelium in CF. The modifications in size of these two placodes could result evolutionarily from a trade-off between these two sensory components. Developmentally, the modified patterning of the panplacodal field in CF is at least partly due to the spatial and temporal differences in the expression of Shh and Fgf (and perhaps Bmp4) signaling molecules.We hypothesized that the small size of the lens could be the direct cause of its apoptosis, through a lack of community effect. We performed partial laser ablation of lens precursor cells at 12-14hpf in surface fish (thereby mimicking the CF lens size). Apoptosis in the resulting small lens of SF larvae at 60hpf was not enhanced, showing that small size is not sufficient to induce apoptosis. Lens apoptosis could also result from morphogenesis defects or from a problem in cell lineage. We are performing two-photon live imaging, from 10 to 24 hpf, of SF and CF embryos previously injected at the one cell stage with H2B-mCherry and Ras-GFP mRNAs to label nuclei and membranes. First results on surface fish show that we can back-track lens cells to the panplacodal field, and follow morphogenesis and divisions. Lens differentiation is also affected in cavefish: at least 5 crystallins, which are lens structural components, are not expressed correctly in CF, based on in situ hybridization and qPCR data. However the functional role of two of these expression modifications / losses was tested and, individually, they don’t seem to explain the apoptosis phenotype. We propose that a combination of several crystallins expression defects would explain CF lens apoptosis.Finally, and more globally, evolutionary forces that led to eye loss in Astyanax mexicanus are not yet understood. Through a transcriptome-wide molecular evolution approach, we identified fixed mutations in transcripts between SF and CF, and we could show an accumulation of mutations in “eye genes” in CF. This suggests that the selection is relaxed on these genes, that have maybe become useless in the dark. Similarly, CF crystallin sequences seem to accumulate fixed mutations at a high rate, considering their low polymorphism level., Le poisson Astyanax mexicanus présente, au sein de la même espèce, plusieurs populations de poissons de rivières (SF) et de poissons de grottes aveugles (CF). Chez les poisons cavernicoles aveugles, les yeux se développent presque normalement pendant l’embryogenèse. Mais 24 heures après la fécondation (hpf), quand l’embryon éclot, le cristallin entre en apoptose, ce qui déclenche la dégénérescence progressive de l’œil entier. Mon projet de thèse visait à comprendre le mécanisme conduisant à l’apoptose du cristallin, jusqu’alors totalement incompris, en partant du postulat selon lequel le défaut devait avoir lieu pendant les stades précoces du développement du cristallin. Le cristallin se développe à partir d’une placode, un épaississement de l’ectoderme au stade neurula. Toutes les placodes, qui donnent naissance à des organes des sens de la tête, sont issues du champ panplacodal, situé à la bordure de la plaque neurale antérieure à 10 hpf. Nous avons comparé la régionalisation de ce champ chez les deux morphes, par hybridations in situ de gènes marqueurs des différentes placodes. Chez le CF, le territoire présomptif du cristallin est réduit à 10 hpf, et le cristallin est plus petit à tous les stades étudiés. D’autre part, la placode olfactive est étendue, et donne naissance à un épithélium olfactif plus large chez le CF. Les modifications de taille de ces deux placodes pourraient être le résultat évolutif d’un « trade-off » entre ces deux composantes sensorielles. La régionalisation modifiée du champ panplacodal chez le CF est due au moins partiellement à des différences spatiales et temporelles d’expression des molécules de signalisation Shh, Fgf, et peut-être Bmp4.Nous avons pensé que la petite taille du cristallin pouvait être la cause directe de son entrée en apoptose, par un défaut d’effet de communauté. Nous avons réalisé une ablation laser partielle des cellules précurseurs du cristallin à 12-14 hpf chez l’embryon SF, mimant ainsi la taille du cristallin CF. L’apoptose dans le petit cristallin des larves SF à 60 hpf n’a pas été augmentée, ce qui montre que la petite taille n’est pas suffisante pour induire l’apoptose.L’apoptose du cristallin pourrait aussi provenir de défauts de morphogenèse ou d’un problème de lignage cellulaire. Nous utilisons donc l’imagerie biphoton in vivo sur des embryons SF et CF, de 10 à 24 hpf, préalablement injectés au stade une cellule avec des ARNm de H2B-mCherry et Ras-GFP pour marquer les noyaux et les membranes. Les premiers résultats sur les poissons de surface montrent que nous pouvons suivre à rebours les cellules du cristallin de la fin du film jusqu’au champ panplacodal, et étudier la morphogenèse et les divisions.La différenciation du cristallin est également affectée chez le CF : au moins 5 cristallines, qui sont des protéines structurales du cristallin, ne sont pas exprimées correctement chez le CF, d’après des hybridations in situ et des qPCR. Cependant, le rôle fonctionnel de deux de ces modifications d’expression a été testé, et individuellement, elles n’expliquent pas le phénotype apoptotique. Nous émettons l’hypothèse qu’une combinaison de défauts d’expression de plusieurs cristallines serait à l’origine de l’apoptose du cristallin CF. Enfin, et plus largement, les forces évolutives qui ont conduit à la perte de l’œil chez Astyanax mexicanus ne sont pas encore comprises. Par une étude d’évolution moléculaire à l’échelle du transcriptome nous avons identifié des mutations fixées entre SF et CF, et avons pu mettre en évidence une accumulation de mutations dans des « gènes d’yeux » chez les CF. Cela suggère un relâchement de la pression de sélection sur ces gènes, peut-être devenus inutiles dans l’obscurité. De même, les séquences des cristallines de CF paraissent accumuler des mutations fixées à un taux élevé vu leur bas niveau de polymorphisme.