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In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl− cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1β2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology., GABAA receptors mediate most inhibitory synaptic transmission in the brain. Here authors used concatemeric α1β2γ2 GABAA receptors to introduce gain-of-desensitization mutations one subunit at a time, revealing non-concerted rearrangements with a key contribution of the γ2 subunit during desensitization.

International audience; The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

Background and ObjectivesTo test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models.MethodsThe effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting–sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein–GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin.ResultsIn cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination.DiscussionTF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.

International audience; In larval xenopus, locomotor-induced oculomotor behavior produces gaze-stabilizing eye movements to counteract the disruptive effects of tail undulation during swimming. While neuronal circuitries responsible for feed-forward intrinsic spino-extraocular signaling have recently been described, the resulting oculomotor behavior remains poorly understood. Conveying locomotor CPG efference copy, the spino-extraocular motor command coordinates the multi-segmental rostrocaudal spinal rhythmic activity with the extraocular motor activity. By recording sequences of xenopus tadpole free swimming, we quantified the temporal calibration of conjugate eye movements originating from spino-extraocular motor coupled activity during pre-metamorphic tail-based undulatory swimming. Our results show that eye movements are produced only during robust propulsive forward swimming activity and increase with the amplitude of tail movements. The use of larval isolated in vitro and semi-intact fixed head preparations revealed that spinal locomotor networks driving the rostral portion of the tail set the precise timing of the spino-extraocular motor coupling by adjusting the phase relationship between spinal segment and extraocular rhythmic activity with the swimming frequency. The resulting spinal-evoked oculomotor behavior produced conjugated eye movements that were in phase opposition with the mid-caudal part of the tail. This time adjustment is independent of locomotor activity in the more caudal spinal parts of the tail. Altogether our findings demonstrate that locomotor feed-forward spino-extraocular signaling produce conjugate eye movements that compensate specifically the undulation of the mid-caudal tail during active swimming. Finally, this study constitutes the first extensive behavioral quantification of spino-extraocular motor coupling, which sets the basis for understanding the mechanisms of locomotor-induced oculomotor behavior in larval frog.

International audience; Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

International audience; G-protein-coupled receptors (GPCR) are the most widely used system of communication used by cells. They sense external signals and translate them into intracellular signals. The information is carried mechanically across the cell membrane, without perturbing its integrity. Agonist binding on the extracellular side causes a change in receptor conformation which propagates to the intracellular side and causes release of activated G-proteins, the first messengers of a variety of signaling cascades.Permitting access to powerful electrophysiological techniques, ion channels can be employed to monitor precisely the most proximal steps of GPCR signaling, receptor conformational changes, and G-protein release. The former is achieved by physical attachment of a potassium channel to the GPCR to create an Ion-Channel Coupled Receptor (ICCR). The latter is based on the use of G-protein-regulated potassium channels (GIRK). We describe here how these two systems may be used in the Xenopus oocyte heterologous system with a robotic system for increased throughput.

International audience; Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.

International audience; Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.

International audience; Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) enzymes are bi-functional enzymes encoded by four different genes (pfkfb1, pfkfb2, pfkfb3, pfkfb4) in vertebrates. They are involved in the regulation of glycolysis: they catalyze the synthesis and the degradation of F-2,6-BP (fructose-2,6-bisphosphate), the most potent allosteric activator of phosphofructokinase 1 (Pfk1), a key glycolytic enzyme. By producing F-2,6-BP, Pfkfb enzymes allow glycolysis to proceed, while by degrading F-2,6-BP they block glycolysis. As major regulators of glycolysis, Pfkfb enzymes are involved in cancer: tumor cells have a higher glycolytic rate compared to normal cells, even in the presence of adequate oxygen levels (Warburg effect) and several cancer cell lines express elevated levels of Pfkfb enzymes. Glycolysis is also important for energy and metabolite production in proliferating cells. In embryos, however, the role of glycolysis and the expression of glycolysis regulators remain to be explored. Here, we provide a phylogenetic analysis of Pfkfb enzymes in vertebrates, and we detail the expression pattern of pfk1, pfkfb1, pfkfb2, pfkfb3, and pfkfb4 genes in Xenopus laevis embryos. We show that pfkfb transcripts expression is overlapping at blastula and gastrula stages and that from neurulation to tadpole stages, they display tissue-specific, complementary and dynamic expression patterns.

Excitability differs among muscle fibers and undergoes continuous changes during development and growth, yet the neuromuscular synapse maintains a remarkable fidelity of execution. Here we show in two evolutionarily distant vertebrates (Xenopus laevis cell culture and mouse nerve-muscle ex-vivo) that the skeletal muscle cell constantly senses, through two identified calcium signals, synaptic events and their efficacy in eliciting spikes. These sensors trigger retrograde signal(s) that control presynaptic neurotransmitter release, resulting in synaptic potentiation or depression. In the absence of spikes, synaptic events trigger potentiation. Once the synapse is sufficiently strong to initiate spiking, the occurrence of these spikes activates a negative retrograde feedback. These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability. DOI: http://dx.doi.org/10.7554/eLife.12190.001, eLife digest Nerve cells communicate with each other, and with targets such as muscle cells, at junctions called synapses. The nerve cell before the synapses releases a chemical called a neurotransmitter, which binds to receptors on the cell after the synapses. However, the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner. For this, it requires feedback from the second cell. This feedback is particularly important at synapses between nerve cells and muscle cells, which are known as neuromuscular junctions. The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise. Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes. Ouanounou et al. have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter. Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals. The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is. The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell. In response to the first calcium signal, the muscle cell sends positive feedback to the neuron, telling it to increase neurotransmitter release further. In response to the second signal, the muscle cell sends negative feedback to reduce neurotransmitter release. Thus, when neurotransmitter release is not enough to activate the muscle, positive feedback dominates and neurotransmitter release increases. However, when the muscle is activated, the two types of feedback act in balance to maintain efficient communication across the synapse. The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals, including the specific molecule (or molecules) that regulate neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.12190.002

During swimming in the amphibian Xenopus laevis, efference copies of rhythmic locomotor commands produced by the spinal central pattern generator (CPG) can drive extraocular motor output appropriate for producing image-stabilizing eye movements to offset the disruptive effects of self-motion. During metamorphosis, X. laevis remodels its locomotor strategy from larval tail-based undulatory movements to bilaterally synchronous hindlimb kicking in the adult. This change in propulsive mode results in head/body motion with entirely different dynamics, necessitating a concomitant switch in compensatory ocular movements from conjugate left–right rotations to non-conjugate convergence during the linear forward acceleration produced during each kick cycle. Here, using semi-intact or isolated brainstem/spinal cord preparations at intermediate metamorphic stages, we monitored bilateral eye motion along with extraocular, spinal axial and limb motor nerve activity during episodes of spontaneous fictive swimming. Our results show a progressive transition in spinal efference copy control of extraocular motor output that remains adapted to offsetting visual disturbances during the combinatorial expression of bimodal propulsion when functional larval and adult locomotor systems co-exist within the same animal. In stages at metamorphic climax, spino-extraocular motor coupling, which previously derived from axial locomotor circuitry alone, can originate from both axial and de novo hindlimb CPGs, although the latter's influence becomes progressively more dominant and eventually exclusive as metamorphosis terminates with tail resorption. Thus, adaptive interactions between locomotor and extraocular motor circuitry allows CPG-driven efference copy signaling to continuously match the changing spatio-temporal requirements for visual image stabilization throughout the transitional period when one propulsive mechanism emerges and replaces another.

International audience; DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.

International audience; Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

International audience; Microinjection is the most flexible transfection method in terms of choice of reagents to inject into cells. But this method lacks the high throughput to compete with less flexible methods like chemical- or viral-based approaches. Various approaches have been pursued to increase the throughput by automating the microinjection process. However, these approaches focused solely on the microinjection itself and disregarded the tasks before and after the injection, which also belong to the critical time path of the whole process, that is, sorting out viable cells from a cell suspension, placing the cell for injection, and collecting the cell after the injection. In the approach with our XenoFactor, we demonstrate a system capable of running the whole process automatically. By optimizing the XenoFactor for Xenopus laevis oocytes, we could demonstrate the successful automated injection. Starting from a suspension with a mixture of defolliculated oocytes at different stages and quality levels, the manual approach requires 1 day in total for the preparation of 400 microinjected oocytes. The XenoFactor takes only 4h for the same amount and delivers injected oocytes of reproducible quality and without the fatigue symptoms experienced during the manual approach.

Beyond the Greatwall Protein phosphorylation and dephosphorylation provide a central mechanism that controls the eukaryotic cell division cycle and entry of cells into mitosis. A form of protein phosphatase 2A (PP2A) has an important role inhibiting phosphorylation-dependent activation of cyclin-dependent kinase 1 (CDK1) itself and also dephosphorylating substrates of the active CDK1 that promote mitosis. PP2A activity is inhibited when another protein kinase, known as Greatwall, is activated (see the Perspective by Virshup and Kaldis ). Mochida et al. (p. 1670 ) and Gharbi-Ayachi et al. (p. 1673 ) searched for substrates of Greatwall that might participate in the cell cycle regulatory machinery. When phosphorylated by Greatwall, a pair of small related proteins, Arpp19 and α-endosulfine, inhibited activity of PP2A. These effects were critical for regulation of mitosis in Xenopus egg extracts and in human cancer cells. Greatwall itself is phosphorylated and activated by CDK1—thus, apparently contributing to a feed-forward loop that contributes to the switchlike commitment of cells to mitosis.

International audience; Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide ("target protector") that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This "target protector and rescue assay" demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein.

International audience; In Xenopus embryos, maternal cyclins drive the first 12 cell divisions after which several cyclins are terminally degraded, including cyclin B2. Cyclin B2 disappearance is due to transcription-mediated mRNA deadenylation at the midblastula transition, when transcription initiates and the cell cycle lengthens. To further define the mechanism, we characterized proteins capable of binding cyclin B2 3'UTR. We show that ElrA and AUF1 compete for binding to regions containing cytoplasmic polyadenylation elements (CPEs), with AUF1 binding increasing at the midblastula transition. Deletion of both CPEs abrogates polyadenylation but has no effect on deadenylation or binding of ElrA or AUF1. Overexpression of ElrA or AUF1 does not alter cyclin B2 mRNA stability. These results show that ElrA and AUF1 bind to cyclin B2 mRNA independent of CPEs and function by binding other elements.

International audience; The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel.

International audience; BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.

SummaryNMDA receptors (NMDARs) are heteromeric assemblies of NR1 and NR2(A–D) subunits with properties heavily influenced by the type of NR2 subunit incorporated. While NMDARs with only one type of NR2 subunit have been extensively characterized, little is known about receptors containing two different NR2 subunits, despite compelling evidence that such triheteromeric receptors exist in vivo. We used a point-mutation approach that allows isolation of recombinant triheteromeric NMDARs possessing two different NR2 N-terminal domains (NTDs). We show that in receptors associating the NR2A-NTD (sensing nanomolar Zn) and the NR2B-NTD (sensing ifenprodil), each NTD binding site retains selective high affinity for its ligand. However, each ligand produces only partial inhibition, and maximal inhibition requires occupancy of both NR2-NTDs by their respective ligands. Similarly, NR1/2A/2C receptors are inhibited by zinc with high potency but low efficacy. Therefore, interactions between homologous N-terminal domains determine the unique pharmacological properties of triheteromeric NMDARs.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. The SLC7A7 gene, mutated in LPI, encodes the y(+)LAT-1 protein, which is the light subunit of the heterodimeric CAA transporter in which 4F2hc is the heavy chain subunit. Co-expression of 4F2hc and y(+)LAT-1 induces the y(+)L activity. This activity is also exerted by another complex composed of 4F2hc and y(+)LAT-2, the latter encoded by the SLC7A6 gene and more ubiquitously expressed than SLC7A7. On the basis of both the pattern of expression and the transport activity, y(+)LAT-2 might compensate for CAA transport when y(+)LAT-1 is defective. By expression in Xenopus laevis oocytes and mammalian cells, we functionally analysed two SLC7A7 mutants, E36del and F152L, respectively, the former displaying a partial dominant-negative effect. The results of the present study provide further insight into the molecular pathogenesis of LPI: a putative multiheteromeric structure of both [4F2hc/y(+)LAT-1] and [4F2hc/y(+)LAT-2], and the interference between y(+)LAT-1 and y(+)LAT-2 proteins. This interference can explain why the compensatory mechanism, that is, an increased expression of SLC7A6 as seen in lymphoblasts from LPI patients, may not be sufficient to restore the y(+)L system activity.

International audience; Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.

International audience; EDEN-BP (embryo deadenylation element-binding protein) binds specifically to the EDEN motif in the 3'-untranslated regions of maternal mRNAs and targets these mRNAs for deadenylation and translational repression in Xenopus laevis embryos. EDEN-BP contains three RNA recognition motifs (RRMs) and is related to the elav family of RNA-binding proteins. In the present study we show that the two N-terminal RRMs of EDEN-BP are necessary for the interaction with EDEN as well as a part of the linker region (between RRM2 and RRM3). Using a band shift assay we show that two different complexes are formed according to the size and, therefore, the functional nature of the EDEN motif. Finally, we show that EDEN-BP can form a dimer in a two-hybrid assay. Accordingly, we suggest that the functional configuration of EDEN-BP is a dimer.

International audience; From recent findings about the indirect effect of metformin (MET) targeted on the respiratory chain complex I, we reconsidered this question and tried to determine the causality of any alteration at this enzymatic level using Xenopus laevis oocytes. Addition of MET (50 microM) reduced by 40% the rotenone-sensitive activity of complex I only in incubating intact oocytes but not in mitochondria isolated by differential centrifugation. The drug prior injected inside these cells had also no measurable effect. In spite of this and the weak binding of MET to the mitochondrial fraction, there was a fairly good correlation between the marked inhibitory action of MET on complex I and its progressive appearance within the oocyte cytoplasm. The intriguing observation that MET as a liposomal form was again able to exert its role when added directly to isolated mitochondria is in accordance with a membrane-mediated uptake and vesicular routing of MET. Furthermore, a temperature-dependent effect was clearly shown. At 4 degrees, oocytes failed to take up efficiently MET and accordingly its subsequent action on respiration was therefore lost. Likewise, MET transport was hindered and inhibition of complex I totally disappeared when a structural analog, asymmetrical dimethylarginine (ADMA), was placed together with MET either at an identical concentration or in excess. These data strongly support the view that MET may recognise some specific membranous sites, likely belonging to effector systems, before penetrating the cell in a bound state via an obscure endocytotic event which still has to be identified.

Producción Científica, The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions., Ministerio de Economía, Industria y Competitividad (Project BFU2014-53469P), Instituto de Saludo Carlos III (grant RD16/0011/0003)

The regulation of Plasmodium falciparum protein phosphatase type 1 (PfPP1) activity remains to be deciphered. Data from homologous eukaryotic type 1 protein phosphatases (PP1) suggest that several protein regulators should be involved in this essential process. One such regulator, named PfI2 based on its primary sequence homology with eukaryotic inhibitor 2 (I2), was recently shown to be able to interact with PfPP1 and to inhibit its phosphatase activity, mainly through the canonical ‘RVxF’ binding motif. The details of the structural and functional characteristics of this interaction are investigated here. Using NMR spectroscopy, a second site of interaction is suggested to reside between residues D94 and T117 and contains the ‘FxxR/KxR/K’ binding motif present in other I2 proteins. This site seems to play in concert/synergy with the ‘RVxF’ motif to bind PP1, because only mutations in both motifs were able to abolish this interaction completely. However, regarding the structure/function relationship, mutation of either the ‘RVxF’ or ‘FxxR/KxR/K’ motif is more drastic, because each mutation prevents the capacity of PfI2 to trigger germinal vesicle breakdown in microinjected Xenopus oocytes. This indicates that the tight association of the PfI2 regulator to PP1, mediated by a two-site interaction, is necessary to exert its function. Based on these results, the use of a peptide derived from the ‘FxxR/KxR/K’ PfI2 motif was investigated for its potential effect on Plasmodium growth. This peptide, fused at its N-terminus to a penetrating sequence, was shown to accumulate specifically in infected erythrocytes and to have an antiplasmodial effect. Structured digital abstract PfPP1 and PfI2 bind by nuclear magnetic resonance (1, 2) PP1 physically interacts with PfPP1 by anti tag coimmunoprecipitation (View interaction) PfI2 binds to PfPP1 by enzyme linked immunosorbent assay (View interaction) PfI2 binds to PfPP1 by surface plasmon resonance (1, 2, 3, 4)

International audience; ClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50 %. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome.

International audience; ClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50 %. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome.

International audience; The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

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.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain., This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the Fondation Pierre Gilles de Gennes (FPGG0039), the “Association pour la Recherche sur le Cancer” (ARC 4972 and ARC 5115 to M.W.; FRCDOC20120605233 and LABEX Memolife to H.J.G.).

International audience; We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

PurposeThe chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ-aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABAA receptor (GABAAR) mediated transmission, with a particular focus on the action of CX3CL1 on the use-dependent decrease (rundown) of the GABA-evoked currents (IGABA), a feature underlying the reduction of GABAergic function in epileptic tissue.MethodsPatch-clamp recordings were obtained from cortical pyramidal neurons in slices from six MTLE patients after surgery. Alternatively, the cell membranes from epileptic brain tissues of 17 MTLE patients or from surgical samples and autopsies of nonepileptic patients were microtransplanted into Xenopus oocytes, and IGABA were recorded using the standard two-microelectrode voltage-clamp technique. Immunohistochemical staining and double-labeling studies were carried out on the same brain tissues to analyze CX3CR1 expression.Key FindingsIn native pyramidal neurons from cortical slices of patients with MTLE, CX3CL1 reduced IGABA rundown and affected the recovery of IGABA amplitude from rundown. These same effects were confirmed in oocytes injected with cortical and hippocampal MTLE membranes, whereas CX3CL1 did not influence IGABA in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with MTLE, CX3CR1 immunoreactivity was higher in MTLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells.SignificanceThese findings indicate a role for CX3CL1 in MTLE, supporting recent evidence on the relevance of brain inflammation in human epilepsies. Our data demonstrate that in MTLE tissues the reduced GABAergic function can be modulated by CX3CL1. The increased CX3CR1 expression in microglia and the modulation by CX3CL1 of GABAergic currents in human epileptic brain suggests new therapeutic approaches for drug-resistant epilepsies based on the evidence that the propagation of seizures can be influenced by inflammatory processes.

International audience; Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (XLB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (XBL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1 was present in low concentration. No peptides were identified in secretions of either XLB or XBL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis×X. muelleri F1 hybrids and no novel peptide expression.

International audience; The neural crest is a population of highly migratory and multipotent cells, which arises from the border of the neural plate in vertebrate embryos. In the last few years, the molecular actors of neural crest early development have been intensively studied, notably by using the frog embryo, as a prime model for the analysis of the earliest embryonic inductions. In addition, tremendous progress has been made in understanding the molecular and cellular basis of Xenopus cranial neural crest migration, by combining in vitro and in vivo analysis. In this review, we examine how the action of previously known neural crest-inducing signals [bone morphogenetic protein (BMP), wingless-int (Wnt), fibroblast growth factor (FGF)] is controlled by newly discovered modulators during early neural plate border patterning and neural crest specification. This regulation controls the induction of key transcription factors that cooperate to pattern the premigratory neural crest progenitors. These data are discussed in the perspective of the gene regulatory network that controls neural and neural crest patterning. We then address recent findings on noncanonical Wnt signaling regulation, cell polarization, and collective cell migration which highlight how cranial neural crest cells populate their target tissue, the branchial arches, in vivo. More than ever, the neural crest stands as a powerful and attractive model to decipher complex vertebrate regulatory circuits in vivo.

International audience; Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

International audience; Phosphorylation of DNA ligase I has been analyzed during Xenopus laevis early development. The enzyme, which is involved in DNA replication and DNA repair events, is accumulated during oogenesis to reach a maximum in the stage VI oocyte, and remains at a constant level during maturation. When maturation of the oocyte is induced (in vivo or in vitro), this leads to a post-translational modification of the protein. In stage VI oocytes, a DNA ligase I of apparent molecular mass 180 kDa is detected immunologically whereas a 190-kDa form is found in unfertilized eggs and persists until the tadpole stage. This modification is due to phosphorylation performed by a protein kinase that is turned on 3-4 h after induction of the maturation. Activation of the kinase requires protein synthesis, and appearance of phosphorylated DNA ligase coincides with activation of histone H1 kinase activity. Induction of DNA ligase I modification and maturation are induced in the absence of protein synthesis following injection of maturation promoting factor into oocytes. Immunoprecipitated oocyte DNA ligase I is phosphorylated and its molecular mass modified by purified cyclin B/p34cdc2 in vitro. DNA ligase I phosphorylation is not induced in oocyte extract where only mitogen-activated-protein kinase is induced. Phosphorylation of DNA ligase I induced by cdc2 kinase occurs at the time new DNA replication and recombination activities appear in eggs.

Summary Background Self-generated body movements require compensatory eye and head adjustments in order to avoid perturbation of visual information processing. Retinal image stabilization is traditionally ascribed to the transformation of visuovestibular signals into appropriate extraocular motor commands for compensatory ocular movements. During locomotion, however, intrinsic "efference copies" of the motor commands deriving from spinal central pattern generator (CPG) activity potentially offer a reliable and rapid mechanism for image stabilization, in addition to the slower contribution of movement-encoding sensory inputs. Results Using a variety of in vitro and in vivo preparations of Xenopus tadpoles, we demonstrate that spinal locomotor CPG-derived efference copies do indeed produce effective conjugate eye movements that counteract oppositely directed horizontal head displacements during undulatory tail-based locomotion. The efference copy transmission, by which the extraocular motor system becomes functionally appropriated to the spinal cord, is mediated by direct ascending pathways. Although the impact of the CPG feedforward commands matches the spatiotemporal specificity of classical vestibulo-ocular responses, the two fundamentally different signals do not contribute collectively to image stabilization during swimming. Instead, when the CPG is active, horizontal vestibulo-ocular reflexes resulting from head movements are selectively suppressed. Conclusions These results therefore challenge our traditional understanding of how animals offset the disruptive effects of propulsive body movements on visual processing. Specifically, our finding that predictive efference copies of intrinsic, rhythmic neural signals produced by the locomotory CPG supersede, rather than supplement, reactive vestibulo-ocular reflexes in order to drive image-stabilizing eye adjustments during larval frog swimming, represents a hitherto unreported mechanism for vertebrate ocular motor control.

International audience; Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

International audience; Cell growth, proliferation, differentiation and survival are influenced by the availability of oxygen. The effect of hypoxia on embryonic cells and the underlying molecular mechanisms to maintain cellular viability are still poorly understood. In this study, we show that hypoxia during Xenopus embryogenesis rapidly leads to a significant developmental delay and to cell apoptosis after prolonged exposure. We provide strong evidence that hypoxia does not affect somitogenesis but affects the number of mitotic cells and muscle-specific protein accumulation in somites, without interfering with the expression of MyoD and MRF4 transcription factors. We also demonstrate that hypoxia reversibly decreases Akt phosphorylation and increases the total amount of the translational repressor 4E-BP, in combination with an increase of the 4E-BP associated with eIF4E. Interestingly, the inhibition of PI3-kinase or mTOR, with LY29002 or rapamycin, respectively, triggers the 4E-BP accumulation in Xenopus embryos. Finally, the overexpression of the non-phosphorylatable 4E-BP protein induces, similar to hypoxia, a decrease in mitotic cells and a decrease in muscle-specific protein accumulation in somites. Taken together, our studies suggest that 4E-BP plays a central role under hypoxia in promoting the cap-independent translation at the expense of cap-dependent translation and triggers specific defects in muscle development.

International audience; Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

International audience; In contrast to mammals, the brain of adult non-mammalian vertebrates exhibits a higher proliferative and/or neurogenic activity. To provide new models on this issue, we have examined origin, distribution and fate of proliferating cells in the entire brain of juvenile and adult Xenopus laevis. Using immunohistochemistry for the Proliferation Cell Nuclear Antigen (PCNA), and/or the thymidine analog, 5-Bromo-2' deoxyUridine (BrdU), the labeled cells are located in ventricular zones of the olfactory bulbs, cerebral hemispheres, preoptic region, ventral hypothalamus and cerebellum. Qualitatively, the highest level of proliferative cells was found in the telencephalic ventricles. By using in situ hybridization/immunocytochemistry double-labeling techniques, we demonstrate for the first time in post-metamorphic frog brain that the proliferative cells are localized in very close vivinity to the radial glial cells, progenitor cells that we have also identified in the ventricular layer using classical molecular markers (BLBP, Vimentin). In addition, after long post-BrdU administration survival times ranging between 14 and 28days, BrdU labeling combined with immunohistochemistry for markers of cell migration (DoubleCortin) or radial glial cells (BLBP), reveals that the proliferative cells are able to migrate from the ventricular zone into the brain parenchyma, most likely by migrating along the radial processes. Finally, at survival time of 28days and by using a combination of BrdU labeling and in situ hybridization for markers of differentiation states (Neuro-β-tubulin, Proteolipid Protein), we demonstrate that newborn cells can differentiate in large portion into either neurons or oligodendrocytes.

International audience; PTK7 is a tyrosine kinase receptor implicated in planar cell polarity, a process with multiple implications at the cellular and organism levels. Loss of function of PTK7 leads to profound morphogenetic defects in the mouse, such as neural tube defects, misorientation of stereocilia in the inner ear, and impaired polarized cell movements. The planar cell polarity pathway is classically assigned to a non-canonical Wnt pathway, which does not rely on b-catenin transcriptional activity. We recently revealed that PTK7 is implicated in b-catenin-dependent developmental processes in mammalian and Xenopus systems. Based on data recently obtained by our group as well as others, we discuss how PTK7 could be involved in canonical and non-canonical Wnt pathways, and which implications are expected from these data in physiology and physiopathology.

International audience; To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.

International audience; The neural crest (NC) is a stem cell-like population that arises at the border of neural and non-neural ectoderm. During development, NC undergoes an epithelio-mesenchymal transition (EMT), i.e. loss of epithelial junctions and acquisition of pro-migratory properties, invades the entire embryo and differentiates into a wide diversity of terminal tissues. We have studied the implication of Rho pathways in NC development and previously showed that RhoV is required for cranial neural crest (CNC) cell specification. We show here that the non-canonical Wnt response rhoU/wrch1 gene, closely related to rhoV, is also expressed in CNC cells but at later stages. Using both gain- and loss-of-function experiments, we demonstrate that the level of RhoU expression is critical for CNC cell migration and subsequent differentiation into craniofacial cartilages. In in vitro cultures, RhoU activates pathways that cooperate with PAK1 and Rac1 in epithelial adhesion, cell spreading and directional cell migration. These data support the conclusion that RhoU is an essential regulator of CNC cell migration.

Little is known about the respective contributions of cell proliferation and cell death to the control of vertebrate forebrain growth. The homeodomain protein barhl2 is expressed in the diencephalons of Xenopus , zebrafish, and mouse embryos, and we previously showed that Barhl2 overexpression in Xenopus neuroepithelial cells induces Caspase3-dependent apoptosis. Here, barhl2 is shown to act as a brake on diencephalic proliferation through an unconventional function of Caspase3. Depletion of Barhl2 or Caspase3 causes an increase in diencephalic cell number, a disruption of the neuroepithelium architecture, and an increase in Wnt activity. Surprisingly, these changes are not caused by decreased apoptosis but instead, are because of an increase in the amount and activation of β-catenin, which stimulates excessive neuroepithelial cell proliferation and induces defects in β-catenin intracellular localization and an up-regulation of axin2 and cyclinD1 , two downstream targets of β-catenin/T-cell factor/lymphoïd enhancer factor signaling. Using two different sets of complementation experiments, we showed that, in the developing diencephalon, Caspase3 acts downstream of Barhl2 in limiting neuroepithelial cell proliferation by inhibiting β-catenin activation. Our data argue that Bar homeodomain proteins share a conserved function as cell type-specific regulators of Caspase3 activities.

The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3 ; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.