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SummaryThe contractile properties of adult myofibers are shaped by their Myosin heavy chain (MYH) isoform content. We identify by snATAC-seq a 42kb super-enhancer (SE) at the locus regrouping the fast Myh (fMyh) genes. By 4C-seq we show that active fMyh promoters interact with the SE by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the SE recapitulates the endogenous spatio-temporal expression of adult fMyh genes. In situ deletion of the SE by CRISPR/Cas9 editing demonstrates its major role in the control of associated fMyh genes, and deletion of two fMyh genes at the locus reveals an active competition of the promoters for the shared SE. Last, by disrupting the organization of fMyh, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Summary Single-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)., Graphical abstract, Highlights • snRNA-seq captures essentially all cell populations present in the skeletal muscle tissue • Can identify rare myonuclei populations, like neuromuscular and myotendinous myonuclei • snRNA-seq circumvents cell-dissociation-induced modifications on gene expression • In contrast to single-cell RNA-seq, snRNA-seq allows us to process snap frozen biopsies, Single-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.

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium., Whether skeletal muscle fibre gene expression is coordinated as a whole in different nuclei in the fibre is unclear. Here, the authors use single nucleus RNAseq and ATACseq to show the transcriptome heterogeneity of muscle nuclei in the adult mouse fibre, with correlations between the two datasets.

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. By snRNA-seq and snATAC-seq, we showed that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we showed that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a unique mechanism of coordination of gene expression in a syncytium.

Notice à reprendre en chantier qualité avec la version finale de l’Editeur; International audience; We 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.

International audience; Trio, 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.

Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons.SIGNIFICANCE STATEMENTChanges in the expression of neuronal chloride transporters, such as KCC2, occur during postnatal development and are induced in a variety of neurological and psychiatric conditions. Such changes are expected to primarily impact GABA signaling because GABAA receptors are predominantly permeable to chloride ions. However, the KCC2 transporter forms clusters near glutamatergic synapses and interacts with several actin-related proteins. We show that KCC2 is strictly required for LTP expression at hippocampal excitatory synapses. This effect is due to KCC2 interaction with the Rac1/PAK signaling pathway that controls actin polymerization. Suppressing this interaction promotes actin polymerization thereby hindering AMPA receptor traffic upon KCC2 suppression. Alterations of KCC2 expression therefore impact not only GABAergic signaling but also glutamatergic synaptic function and long term plasticity.

The precerebellar system provides an interesting model to study tangential migrations. All precerebellar neurons (PCN) are generated in the most alar part of the hindbrain in a region called rhombic lip. PCN first emit a leading process and then translocate their nuclei inside it, a mechanism called nucleokinesis. In the past few years, molecular cues that could affect those processes have been investigated, with a special care on: (i) the identification of extrinsic factors directing cell migration and axon elongation as well as neuronal survival during development; (ii) intracellular reorganizations of the cytoskeleton during nucleokinesis in response to chemotropic factors. The signaling cascades, including regulators of actin and microtubule cytoskeleton, in response to diffusible guidance factors have raised an increasing attention. We will here review the role of guidance cues involved in PCN migration in particular netrin-1, Slit and Nr-CAM. We will also consider Rho-GTPases that have been proposed to mediate axon outgrowth and neuronal migration, especially in response to netrin-1, and which may act as a relay between extracellular signals and intracellular remodeling. Recent findings from in vitro pharmacological inhibition of various Rho-GTPases and over-expression of effectors bring molecular cues that, in accordance with anatomical data, fit the idea that nucleokinesis and axon outgrowth are not strictly coupled events during PCN migration.

Recent human genetics approaches identified the Aristaless-related homeobox (ARX) gene as the causative gene in X-linked infantile spasms, Partington syndrome, and non-syndromic mental retardation as well as in forms of lissencephaly with abnormal genitalia. The ARX predicted protein belongs to a large family of homeoproteins and is characterised by a C-terminal Aristaless domain and an octapeptide domain near the N-terminus. In order to learn more about ARX function, we have studied in detail Arx expression in the central nervous system during mouse embryonic development as well as in the adult. During early stages of development, Arx is expressed in a significant proportion of neurons in the cortex, the striatum, the ganglionic eminences and also in the spinal cord. In the adult, expression of Arx is still present and restricted to regions that are known to be rich in GABAergic neurons such as the amygdala and the olfactory bulb. A possible role for Arx in this type of neurons is further reinforced by the expression of Arx in a subset of GABAergic interneurons in young and mature primary cultures of cortical neuronal cells as well as in vivo. Moreover, these data could explain the occurrence of seizures in the great majority of patients with an ARX mutation, due to mislocalisation or dysfunction of GABAergic neurons. We also performed ARX wild-type and mutant over-expression experiments and found that the different ARX mutations tested did not modify the morphology of the cells. Moreover, no abnormal cell death or protein aggregation was observed, hence suggesting that more subtle pathogenic mechanisms are involved.

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.

The precerebellar nuclei (PCN) originate from the rhombic lip, a germinal neuroepithelium adjacent to the roof plate of the fourth ventricle. We first report here that, in chicken, the Brn3a-expressing postmitotic medullary cells that produce the inferior olive (ION, the source of cerebellar climbing fibres) originate from a dorso-ventral domain roughly coinciding with the hindbrain vestibular column. Whereas Foxd3 expression labels the whole mature ION but is only detected in a subpopulation of ION neuroblasts initiating their migration, we report that Brn3a allows the visualization of the whole population of ION neurons from the very beginning of their migration. We show that Brn3a-positive neurons migrate tangentially ventralwards through a characteristic dorso-ventral double submarginal stream. Cath1 expressing progenitors lying just dorsal to the ION origin correlated dorso-ventral topography with the prospective cochlear column (caudal to it) and generate precerebellar nuclei emitting mossy-fiber cerebellar afferents. We used the chick–quail chimaera technique with homotopic grafts at HH10 to determine the precise fate map of ION precursors across the caudal cryptorhombomeric subdivisions of the medullary hindbrain (r8–r11). We demonstrate that each crypto-rhombomere contributes to two lamellae of the ION, while each ION sub-nucleus originates from at least two contiguous crypto-rhombomeres. We then questioned how rhombomere identity is related to the plasticity of cell type specification in the dorsal hindbrain. The potential plasticity of ectopically HH10 grafted ION progenitors to change their original fate in alternative rostrocaudal environments was examined. Heterotopic grafts from the presumptive ION territory to the pontine region (r4–r5) caused a change of fate, since the migrated derivatives adopted a pontine phenotype. The reverse experiment caused pontine progenitors to produce derivatives appropriately integrated into the ION complex. Grafts of ION progenitor domains to myelomeres (my) 2–3 also showed complete fate regulation, reproducing spinal cord-like structures, whereas the reverse experiment revealed the inability of my2–3 to generate ION cell types. This was not the case with more caudal, relatively less specified myelomeres (my5–6). Interestingly, when heterotopically grafted cells are integrated dorsally, they do not change their phenotype. Our results support the hypothesis that positional information present in the hindbrain and spinal cord at early neural tube stages controls the specific fates of ventrally migrating PCN precursors.

International audience; Netrin-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.

International audience; BACKGROUND: 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.

During the embryonic development of the hindbrain, movements of neuronal clusters allow the formation of mature “pools”, in particular for inferior olivary (ION) and facial motor (fMN) nuclei. The cellular mechanisms of neuron clustering remain uncharacterized. We report that the absence of the Rho–guanine exchange factor Trio, which can activate both RhoG and Rac1in vivo, prevents the proper formation of ION and fMN subnuclei. Rac1, but not RhoG, appears to be a downstream actor in Trio-induced lamellation. In addition, we report thatCadherin-11is expressed by a subset of neurons through the overall period of ION and fMN parcellations, and defects observed intriomutant mice are located specifically in Cadherin-11-expressing regions. Moreover, endogenous Cadherin-11 is found in a complex with Trio when lamellation occurs. Altogether, those results establish a link between Trio activity, the subsequent Rac1 activation, and neuronal clusters organization, as well as a possible recruitment of the Cadherin-11 adhesive receptor to form a complex with Trio.

L’empreinte parentale est un phenomene epigenetique complexe qui regule l’expression monoallelique de certains genes en fonction de l’origine parentale de l’allele. Les genes soumis a empreinte (GSE) participent au developpement placentaire et fœtal. Des defauts d’empreinte de certains GSE entrainent des syndromes cliniques associant une pathologie placentaire a l’origine notamment d’un defaut de croissance fœtale, et des manifestations neuro-developpementales. Environ 70 GSE ont ete identifies a ce jour chez l’homme. Nous avons developpe une strategie de criblage haut debit pour identifier de nouveaux GSE dans le placenta humain. Une premiere serie de validation a permis de confirmer le statut d’empreinte d’une dizaine de nouveaux genes [1] . Recemment, nous avons revele le statut de GSE pour le gene DSCAM (Down Syndrome Cellular Adhesion Molecule). Ce gene, qui se trouve dans la region critique du syndrome de Down, est ainsi le premier GSE identifie sur le chromosome 21. Notre etude montre que c’est l’allele paternel qui est exprime dans le placenta. Des resultats preliminaires nous laissent penser que l’empreinte de ce gene pourrait etre regulee par une region differentiellement methylee en amont du gene. L’etude par immunohistochimie de coupes placentaires montre une expression dans les cellules endotheliales et le syncytiotrophoblaste pouvant suggerer un role dans l’angiogenese placentaire via son ligand Netrin. Une etude est en cours a partir d’un modele cellulaire de trophoblaste avant et apres syncytialisation pour analyser son role dans ce type cellulaire. Par ailleurs, DSCAM est connu pour son role dans la mise en place des connections inter-neurones [2] . Une etude est en cours sur des modeles murins afin d’analyser le statut d’empreinte de ce gene dans le tissu nerveux et les consequences d’un defaut d’expression de son ligand Netrin sur le developpement placentaire.

During embryonic development, tangentially migrating precerebellar neurons emit a leading process and then translocate their nuclei inside it(nucleokinesis). Netrin 1 (also known as netrin-1) acts as a chemoattractant factor for neurophilic migration of precerebellar neurons (PCN) both in vivo and in vitro. In the present work, we analyzed Rho GTPases that could direct axon outgrowth and/or nuclear migration. We show that the expression pattern of Rho GTPases in developing PCN is consistent with their involvement in the migration of PCN from the rhombic lips. We report that pharmacological inhibition of Rho enhances axon outgrowth of PCN and prevents nuclei migration toward a netrin 1 source, whereas inhibition of Rac and Cdc42 sub-families impair neurite outgrowth of PCN without affecting migration. We show, through pharmacological inhibition, that Rho signaling directs neurophilic migration through Rock activation. Altogether, our results indicate that Rho/Rock acts on signaling pathways favoring nuclear translocation during tangential migration of PCN. Thus, axon extension and nuclear migration of PCN in response to netrin 1 are not strictly dependent processes because: (1)distinct small GTPases are involved; (2) axon extension can occur when migration is blocked; and (3) migration can occur when axon outgrowth is impaired.