Tubulin Tyrosination Is Required for the Proper Organization and Pathfinding of the Growth Cone

International audienceBACKGROUND: During development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood. METHODOLOGY/FINDINGS: Here, we have dissected the role of a post-translational modification of the last amino acid of the alpha-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL(-/-)) through in vivo, ex vivo and in vitro analyses. TTL(-/-) neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL(-/-) growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL(-/-) neurons can rescue Myosin IIB localization. CONCLUSIONS/SIGNIFICANCE: In the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development

Genome-wide association study identifies six new loci influencing pulse pressure and mean arterial pressure.

Numerous genetic loci have been associated with systolic blood pressure (SBP) and diastolic blood pressure (DBP) in Europeans. We now report genome-wide association studies of pulse pressure (PP) and mean arterial pressure (MAP). In discovery (N = 74,064) and follow-up studies (N = 48,607), we identified at genome-wide significance (P = 2.7 × 10(-8) to P = 2.3 × 10(-13)) four new PP loci (at 4q12 near CHIC2, 7q22.3 near PIK3CG, 8q24.12 in NOV and 11q24.3 near ADAMTS8), two new MAP loci (3p21.31 in MAP4 and 10q25.3 near ADRB1) and one locus associated with both of these traits (2q24.3 near FIGN) that has also recently been associated with SBP in east Asians. For three of the new PP loci, the estimated effect for SBP was opposite of that for DBP, in contrast to the majority of common SBP- and DBP-associated variants, which show concordant effects on both traits. These findings suggest new genetic pathways underlying blood pressure variation, some of which may differentially influence SBP and DBP

Differential roles of Netrin-1 and its receptor DCC in inferior olivary neuron migration.

International audienceNetrin-1 was previously shown to be required for the tangential migration and survival of neurons that will form the inferior olivary nucleus (ION). Surprisingly, the compared analysis of mutant mice lacking either Netrin-1 or its major receptor DCC reveals striking phenotypic differences besides common features. Although ectopic stops of ION cell bodies occur in the same positions along the migratory stream in both mutants, the ION neurons' number is not affected by the lack of DCC whereas it is reduced in Netrin-1 mutant mice. Thus, cell death results from the absence of Netrin-1 and not from neuron mis-routing, arguing for a role of Netrin-1 as a survival factor in vivo. The secretion of Netrin-1 by the floor plate (FP) is strictly required - whereas DCC is not - to avoid ION axons' repulsion by the FP and allows them to cross it. Leading processes of neurons of other caudal precerebellar nuclei (PCN) cannot cross the FP in either mutant mouse, suggesting differential sensitivity or mechanism of action of Netrin-1 for leading processes of ION and other PCN neurons

Trio GEF mediates RhoA activation downstream of Slit2 and coordinates telencephalic wiring

International audienceTrio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in T rio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders

Extraction and sequencing of single nuclei from murine skeletal muscles

International audienceSingle-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021)

Distinct roles of Rac1/Cdc42 and Rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1

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CLIPR-59: a protein essential for neuromuscular junction stability during mouse late embryonic development.

International audienceCLIPR-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

The Belgian food consumption survey: aims, design and methods

A national food consumption survey was organised for the first time in Belgium in 2004. A total of 3,200 individuals were interviewed. The target population contained all residents of Belgium who are 15 years or older. The individuals were selected by a multistage procedure from the National Register. Information on food intake was collected using two non-consecutive 24h recalls in combination with a self-administered food frequency questionnaire. Additional data on sociodemographic and lifestyle characteristics were obtained with a face-to-face questionnaire. Moreover information regarding food safety on household level was recorded. The fieldwork was spread over one year to anticipate seasonal effects and was carried out by trained dieticians. In this paper a general description of the aims, the design, the sampling procedure and the questionnaires of the Belgian food consumption survey are given.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A genome-wide search for new imprinted genes in the human placenta identifies DSCAM as the first imprinted gene on chromosome 21

Notice à reprendre en chantier qualité avec la version finale de l’EditeurInternational audienceWe identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications