Publications by authors named "Alain Chédotal"

121 Publications

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

Elife 2021 Apr 19;10. Epub 2021 Apr 19.

The University of Queensland, Queensland Brain Institute, Brisbane, Australia.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in and knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.
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http://dx.doi.org/10.7554/eLife.61769DOI Listing
April 2021

Editorial overview: Developmental neuroscience.

Curr Opin Neurobiol 2021 Feb;66:iii-v

Institut de la Vision, INSERM, Sorbonne Université, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.conb.2021.03.006DOI Listing
February 2021

Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.

Science 2021 04;372(6538):150-156

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 Rue Moreau, 75012 Paris, France.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.
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http://dx.doi.org/10.1126/science.abe7790DOI Listing
April 2021

Loss of floor plate Netrin-1 impairs midline crossing of corticospinal axons and leads to mirror movements.

Cell Rep 2021 Jan;34(3):108654

Sorbonne Université, CNRS, INSERM, Institut de Biologie Paris-Seine (IBPS), Neuroscience Paris-Seine (NPS), 75005 Paris, France. Electronic address:

In humans, execution of unimanual movements requires lateralized activation of the primary motor cortex, which then transmits the motor command to the contralateral hand through the crossed corticospinal tract (CST). Mutations in NTN1 alter motor control lateralization, leading to congenital mirror movements. To address the role of midline Netrin-1 on CST development and subsequent motor control, we analyze the morphological and functional consequences of floor plate Netrin-1 depletion in conditional knockout mice. We show that depletion of floor plate Netrin-1 in the brainstem critically disrupts CST midline crossing, whereas the other commissural systems are preserved. The only associated defect is an abnormal entry of CST axons within the inferior olive. Alteration of CST midline crossing results in functional ipsilateral projections and is associated with abnormal symmetric movements. Our study reveals the role of Netrin-1 in CST development and describes a mouse model recapitulating the characteristics of human congenital mirror movements.
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http://dx.doi.org/10.1016/j.celrep.2020.108654DOI Listing
January 2021

Phox2a Defines a Developmental Origin of the Anterolateral System in Mice and Humans.

Cell Rep 2020 11;33(8):108425

Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada; Integrated Program in Neuroscience, McGill University, Montréal, QC H3A 2B4, Canada; Department of Anatomy and Cell Biology, McGill University, Montréal, QC H3A 0C7, Canada; Division of Experimental Medicine, McGill University, Montréal, QC H3A 2B2, Canada. Electronic address:

Anterolateral system neurons relay pain, itch, and temperature information from the spinal cord to pain-related brain regions, but the differentiation of these neurons and their specific contribution to pain perception remain poorly defined. Here, we show that most mouse spinal neurons that embryonically express the autonomic-system-associated Paired-like homeobox 2A (Phox2a) transcription factor innervate nociceptive brain targets, including the parabrachial nucleus and the thalamus. We define the Phox2a anterolateral system neuron birth order, migration, and differentiation and uncover an essential role for Phox2a in the development of relay of nociceptive signals from the spinal cord to the brain. Finally, we also demonstrate that the molecular identity of Phox2a neurons is conserved in the human fetal spinal cord, arguing that the developmental expression of Phox2a is a prominent feature of anterolateral system neurons.
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http://dx.doi.org/10.1016/j.celrep.2020.108425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713706PMC
November 2020

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Front Cell Dev Biol 2020 27;8:585675. Epub 2020 Oct 27.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.
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http://dx.doi.org/10.3389/fcell.2020.585675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652757PMC
October 2020

[The human segmentation clock rings every five hours].

Authors:
Alain Chédotal

C R Biol 2020 06 5;343(1):7-8. Epub 2020 Jun 5.

Institut de la vision, Paris, France, Membre de l'Académie des sciences.

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http://dx.doi.org/10.5802/crbiol.11DOI Listing
June 2020

[A novel method for micrometric 3D mapping of the brain vasculature].

Authors:
Alain Chédotal

C R Biol 2020 06 5;343(1):5-6. Epub 2020 Jun 5.

Institut de la vision, Paris, France, Membre de l'Académie des sciences.

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http://dx.doi.org/10.5802/crbiol.12DOI Listing
June 2020

Neural Stem Cells Direct Axon Guidance via Their Radial Fiber Scaffold.

Neuron 2020 09 23;107(6):1197-1211.e9. Epub 2020 Jul 23.

Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA; Departments of Genetics, Psychiatry, and Comparative Medicine, Kavli Institute for Neuroscience, Program in Cellular Neuroscience, Neurodegeneration, and Repair, and Yale Child Study Center, Yale University School of Medicine, New Haven, CT 06510, USA. Electronic address:

Neural stem cells directly or indirectly generate all neurons and macroglial cells and guide migrating neurons by using a palisade-like scaffold made of their radial fibers. Here, we describe an unexpected role for the radial fiber scaffold in directing corticospinal and other axons at the junction between the striatum and globus pallidus. The maintenance of this scaffold, and consequently axon pathfinding, is dependent on the expression of an atypical RHO-GTPase, RND3/RHOE, together with its binding partner ARHGAP35/P190A, a RHO GTPase-activating protein, in the radial glia-like neural stem cells within the ventricular zone of the medial ganglionic eminence. This role is independent of RND3 and ARHGAP35 expression in corticospinal neurons, where they regulate dendritic spine formation, axon elongation, and pontine midline crossing in a FEZF2-dependent manner. The prevalence of neural stem cell scaffolds and their expression of RND3 and ARHGAP35 suggests that these observations might be broadly relevant for axon guidance and neural circuit formation.
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http://dx.doi.org/10.1016/j.neuron.2020.06.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529949PMC
September 2020

Remotely Produced and Axon-Derived Netrin-1 Instructs GABAergic Neuron Migration and Dopaminergic Substantia Nigra Development.

Neuron 2020 08 19;107(4):684-702.e9. Epub 2020 Jun 19.

Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands. Electronic address:

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviors and show select disease vulnerability, including in Parkinson's disease. Despite progress in identifying mDA neuron subtypes, how these neuronal subsets develop and organize into functional brain structures remains poorly understood. Here we generate and use an intersectional genetic platform, Pitx3-ITC, to dissect the mechanisms of substantia nigra (SN) development and implicate the guidance molecule Netrin-1 in the migration and positioning of mDA neuron subtypes in the SN. Unexpectedly, we show that Netrin-1, produced in the forebrain and provided to the midbrain through axon projections, instructs the migration of GABAergic neurons into the ventral SN. This migration is required to confine mDA neurons to the dorsal SN. These data demonstrate that neuron migration can be controlled by remotely produced and axon-derived secreted guidance cues, a principle that is likely to apply more generally.
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http://dx.doi.org/10.1016/j.neuron.2020.05.037DOI Listing
August 2020

Clearing method for 3-dimensional immunofluorescence of osteoarthritic subchondral human bone reveals peripheral cholinergic nerves.

Sci Rep 2020 06 1;10(1):8852. Epub 2020 Jun 1.

Sorbonne Université, Paris, France.

The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. Osteoarthritis (OA) is associated with local low-grade inflammation, but the regulatory mechanisms are unclear. Local Ach release could have anti-inflammatory activity since articular cells express Ach receptors involved in inflammatory responses. Using the 3DISCO clearing protocol that allows whole-sample 3-dimensional (3D) analysis, we cleared human OA cartilage-subchondral bone samples to search for cholinergic nerve fibres able to produce Ach locally. We analysed 3 plugs of knee cartilage and subchondral bone from 3 OA patients undergoing arthroplasty. We found no nerves in the superficial and intermediate articular cartilage layers, as evidenced by the lack of Peripherin staining (a peripheral nerves marker). Conversely, peripheral nerves were found in the deepest layer of cartilage and in subchondral bone. Some nerves in the subchondral bone samples were cholinergic because they coexpressed peripherin and choline acetyltransferase (ChAT), a specific marker of cholinergic nerves. However, no cholinergic nerves were found in the cartilage layers. It is therefore feasible to clear human bone to perform 3D immunofluorescence. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release.
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http://dx.doi.org/10.1038/s41598-020-65873-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264130PMC
June 2020

Corneal stromal stem cells restore transparency after N injury in mice.

Stem Cells Transl Med 2020 08 7;9(8):917-935. Epub 2020 May 7.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N ) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N -injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen.
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http://dx.doi.org/10.1002/sctm.19-0306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381812PMC
August 2020

Publisher Correction: Tissue clearing and its applications in neuroscience.

Nat Rev Neurosci 2020 05;21(5):298

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41583-020-0291-5DOI Listing
May 2020

Revisiting the role of Dcc in visual system development with a novel eye clearing method.

Elife 2020 02 25;9. Epub 2020 Feb 25.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as knockouts die at birth. To circumvent this drawback, we generated an eye-specific mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of , some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, -deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.
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http://dx.doi.org/10.7554/eLife.51275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062470PMC
February 2020

Tissue clearing and its applications in neuroscience.

Nat Rev Neurosci 2020 02;21(2):61-79

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.
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http://dx.doi.org/10.1038/s41583-019-0250-1DOI Listing
February 2020

Construction and reconstruction of brain circuits: normal and pathological axon guidance.

J Neurochem 2020 04 21;153(1):10-32. Epub 2019 Nov 21.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.
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http://dx.doi.org/10.1111/jnc.14900DOI Listing
April 2020

Decoding human fetal liver haematopoiesis.

Nature 2019 10 9;574(7778):365-371. Epub 2019 Oct 9.

Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
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http://dx.doi.org/10.1038/s41586-019-1652-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861135PMC
October 2019

Roles of axon guidance molecules in neuronal wiring in the developing spinal cord.

Authors:
Alain Chédotal

Nat Rev Neurosci 2019 07;20(7):380-396

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

The spinal cord receives, relays and processes sensory information from the periphery and integrates this information with descending inputs from supraspinal centres to elicit precise and appropriate behavioural responses and orchestrate body movements. Understanding how the spinal cord circuits that achieve this integration are wired during development is the focus of much research interest. Several families of proteins have well-established roles in guiding developing spinal cord axons, and recent findings have identified new axon guidance molecules. Nevertheless, an integrated view of spinal cord network development is lacking, and many current models have neglected the cellular and functional diversity of spinal cord circuits. Recent advances challenge the existing spinal cord axon guidance dogmas and have provided a more complex, but more faithful, picture of the ontogenesis of vertebrate spinal cord circuits.
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http://dx.doi.org/10.1038/s41583-019-0168-7DOI Listing
July 2019

Shared and differential features of Robo3 expression pattern in amniotes.

J Comp Neurol 2019 08 4;527(12):2009-2029. Epub 2019 Mar 4.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

In Bilaterians, commissural neurons project their axons across the midline of the nervous system to target neurons on the opposite side. In mammals, midline crossing at the level of the hindbrain and spinal cord requires the Robo3 receptor which is transiently expressed by all commissural neurons. Unlike other Robo receptors, mammalian Robo3 receptors do not bind Slit ligands and promote midline crossing. Surprisingly, not much is known about Robo3 distribution and mechanism of action in other vertebrate species. Here, we have used whole-mount immunostaining, tissue clearing and light-sheet fluorescent microscopy to study Robo3 expression pattern in embryonic tissue from diverse representatives of amniotes at distinct stages, including squamate (African house snake), birds (chicken, duck, pigeon, ostrich, emu and zebra finch), early postnatal marsupial mammals (fat-tailed dunnart), and eutherian mammals (mouse and human). The analysis of this rich and unique repertoire of amniote specimens reveals conserved features of Robo3 expression in midbrain, hindbrain and spinal cord commissural circuits, which together with subtle but meaningful modifications could account for species-specific evolution of sensory-motor and cognitive capacities. Our results also highlight important differences of precerebellar nuclei development across amniotes.
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http://dx.doi.org/10.1002/cne.24648DOI Listing
August 2019

Synergistic Activity of Floor-Plate- and Ventricular-Zone-Derived Netrin-1 in Spinal Cord Commissural Axon Guidance.

Neuron 2019 02 17;101(4):625-634.e3. Epub 2019 Jan 17.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 Rue Moreau, 75012 Paris, France. Electronic address:

In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain.
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http://dx.doi.org/10.1016/j.neuron.2018.12.024DOI Listing
February 2019

Long-Range Guidance of Spinal Commissural Axons by Netrin1 and Sonic Hedgehog from Midline Floor Plate Cells.

Neuron 2019 02 17;101(4):635-647.e4. Epub 2019 Jan 17.

Laboratory of Brain Development and Repair, The Rockefeller University, New York, NY 10065, USA; Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

An important model for axon pathfinding is provided by guidance of embryonic commissural axons from dorsal spinal cord to ventral midline floor plate (FP). FP cells produce a chemoattractive activity, comprised largely of netrin1 (FP-netrin1) and Sonic hedgehog (Shh), that can attract the axons at a distance in vitro. netrin1 is also produced by ventricular zone (VZ) progenitors along the axons' route (VZ-netrin1). Recent studies using region-specific netrin1 deletion suggested that FP-netrin1 is dispensable and VZ-netrin1 sufficient for netrin guidance activity in vivo. We show that removing FP-netrin1 actually causes guidance defects in spinal cord consistent with long-range action (i.e., over hundreds of micrometers), and double mutant analysis supports that FP-netrin1 and Shh collaborate to attract at long range. We further provide evidence that netrin1 may guide via chemotaxis or haptotaxis. These results support the model that netrin1 signals at both short and long range to guide commissural axons in spinal cord.
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http://dx.doi.org/10.1016/j.neuron.2018.12.025DOI Listing
February 2019

Genetic Analysis of the Organization, Development, and Plasticity of Corneal Innervation in Mice.

J Neurosci 2019 02 26;39(7):1150-1168. Epub 2018 Dec 26.

Institut de la Vision, Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, F-75012 Paris, France,

The cornea has the densest sensory innervation of the body, originating primarily from neurons in the trigeminal ganglion. The basic principles of cornea nerve patterning have been established many years ago using classic neuroanatomical methods, such as immunocytochemistry and electrophysiology. Our understanding of the morphology and distribution of the sensory nerves in the skin has considerably progressed over the past few years through the generation and analysis of a variety of genetically modified mouse lines. Surprisingly, these lines were not used to study corneal axons. Here, we have screened a collection of transgenic and knockin mice (of both sexes) to select lines allowing the visualization and genetic manipulation of corneal nerves. We identified multiple lines, including some in which different types of corneal axons can be simultaneously observed with fluorescent proteins expressed in a combinatorial manner. We also provide the first description of the morphology and arborization of single corneal axons and identify three main types of branching pattern. We applied this genetic strategy to the analysis of corneal nerve development and plasticity. We provide direct evidence for a progressive reduction of the density of corneal innervation during aging. We also show that the semaphorin receptor neuropilin-1 acts cell-autonomously to control the development of corneal axons and that early axon guidance defects have long-term consequences on corneal innervation. We have screened a collection of transgenic and knockin mice and identify lines allowing the visualization and genetic manipulation of corneal nerves. We provide the first description of the arborization pattern of single corneal axons. We also present applications of this genetic strategy to the analysis of corneal nerve development and remodeling during aging.
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http://dx.doi.org/10.1523/JNEUROSCI.1401-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381234PMC
February 2019

Commissural neurons transgress the CNS/PNS boundary in absence of ventricular zone-derived netrin 1.

Development 2018 01 17;145(2). Epub 2018 Jan 17.

Sorbonne Universités, UPMC Université Paris 06, INSERM, CNRS, Institut de la Vision, 75012 Paris, France

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in hypomorphic embryos and knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in and mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.
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http://dx.doi.org/10.1242/dev.159400DOI Listing
January 2018

Non-cell autonomous control of precerebellar neuron migration by Slit and Robo proteins.

Development 2018 01 17;145(2). Epub 2018 Jan 17.

Sorbonne Universités, UPMC Paris 06, INSERM, CNRS, Institut de la Vision 75012, Paris, France

During development, precerebellar neurons migrate tangentially from the dorsal hindbrain to the floor plate. Their axons cross it but their cell bodies stop their ventral migration upon reaching the midline. It has previously been shown that Slit chemorepellents and their receptors, Robo1 and Robo2, might control the migration of precerebellar neurons in a repulsive manner. Here, we have used a conditional knockout strategy in mice to test this hypothesis. We show that the targeted inactivation of the expression of and receptors in precerebellar neurons does not perturb their migration and that they still stop at the midline. The selective ablation of the expression of all three Slit proteins in floor-plate cells has no effect on pontine neurons and only induces the migration of a small subset of inferior olivary neurons across the floor plate. Likewise, we show that the expression of Slit proteins in the facial nucleus is dispensable for pontine neuron migration. Together, these results show that Robo1 and Robo2 receptors act non-cell autonomously in migrating precerebellar neurons and that floor-plate signals, other than Slit proteins, must exist to prevent midline crossing.
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http://dx.doi.org/10.1242/dev.150375DOI Listing
January 2018

Mutations in the netrin-1 gene cause congenital mirror movements.

J Clin Invest 2017 Nov 25;127(11):3923-3936. Epub 2017 Sep 25.

Pathology Department, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.

Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.
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http://dx.doi.org/10.1172/JCI95442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663368PMC
November 2017

Neuroscience in the third dimension: shedding new light on the brain with tissue clearing.

Mol Brain 2017 07 20;10(1):33. Epub 2017 Jul 20.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 Rue Moreau, 75012, Paris, France.

For centuries analyses of tissues have depended on sectioning methods. Recent developments of tissue clearing techniques have now opened a segway from studying tissues in 2 dimensions to 3 dimensions. This particular advantage echoes heavily in the field of neuroscience, where in the last several years there has been an active shift towards understanding the complex orchestration of neural circuits. In the past five years, many tissue-clearing protocols have spawned. This is due to varying strength of each clearing protocol to specific applications. However, two main protocols have shown their applicability to a vast number of applications and thus are exponentially being used by a growing number of laboratories. In this review, we focus specifically on two major tissue-clearing method families, derived from the 3DISCO and the CLARITY clearing protocols. Moreover, we provide a "hands-on" description of each tissue clearing protocol and the steps to look out for when deciding to choose a specific tissue clearing protocol. Lastly, we provide perspectives for the development of tissue clearing protocols into the research community in the fields of embryology and cancer.
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http://dx.doi.org/10.1186/s13041-017-0314-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520295PMC
July 2017

Behavioral Consequences of a Bifacial Map in the Mouse Somatosensory Cortex.

J Neurosci 2017 07 29;37(30):7209-7218. Epub 2017 Jun 29.

Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and

The whisker system is an important sensory organ with extensive neural representations in the brain of the mouse. Patterned neural modules (barrelettes) in the ipsilateral principal sensory nucleus of the trigeminal nerve (PrV) correspond to the whiskers. Axons of the PrV barrelette neurons cross the midline and confer the whisker-related patterning to the contralateral ventroposteromedial nucleus of the thalamus, and subsequently to the cortex. In this way, specific neural modules called barreloids and barrels in the contralateral thalamus and cortex represent each whisker. Partial midline crossing of the PrV axons, in a conditional Robo3 mutant () mouse line, leads to the formation of bilateral whisker maps in the ventroposteromedial, as well as the barrel cortex. We used voltage-sensitive dye optical imaging and somatosensory and motor behavioral tests to characterize the consequences of bifacial maps in the thalamocortical system. Voltage-sensitive dye optical imaging verified functional, bilateral whisker representation in the barrel cortex and activation of distinct cortical loci following ipsilateral and contralateral stimulation of the specific whiskers. The mutant animals were comparable with the control animals in sensorimotor tests. However, they showed noticeable deficits in all of the whisker-dependent or -related tests, including Y-maze exploration, horizontal surface approach, bridge crossing, gap crossing, texture discrimination, floating in water, and whisking laterality. Our results indicate that bifacial maps along the thalamocortical system do not offer a functional advantage. Instead, they lead to impairments, possibly due to the smaller size of the whisker-related modules and interference between the ipsilateral and contralateral whisker representations in the same thalamus and cortex. The whisker sensory system plays a quintessentially important role in exploratory behavior of mice and other nocturnal rodents. Here, we studied a novel mutant mouse line, in which the projections from the brainstem to the thalamus are disrupted. This led to formation of bilateral whisker maps in both the thalamus and the cortex. The two whisker maps crowd in a space normally devoted to the contralateral map alone and in a nonoverlapping fashion. Stimulation of the whiskers on either side activates the corresponding region of the map. Mice with bilateral whisker maps perform well in general sensorimotor tasks but show poor performance in specific tests that require whisker-dependent tactile discrimination. These observations indicate that contralateral, instead of bilateral, representation of the sensory space plays a critical role in acuity and fine discrimination during somesthesis.
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http://dx.doi.org/10.1523/JNEUROSCI.0598-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546400PMC
July 2017

Introduction to the special volume on axonal development and disorders.

Dev Neurobiol 2017 07 22;77(7):807-809. Epub 2017 May 22.

Sorbonne Universités, UPMC Univ. Paris 06, INSERM, CNRS, Institut de la Vision, Paris, 75012, France.

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http://dx.doi.org/10.1002/dneu.22504DOI Listing
July 2017

Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.

Nature 2017 05 26;545(7654):350-354. Epub 2017 Apr 26.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 Rue Moreau, 75012 Paris, France.

Netrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.
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http://dx.doi.org/10.1038/nature22331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438598PMC
May 2017

A mutant with bilateral whisker to barrel inputs unveils somatosensory mapping rules in the cerebral cortex.

Elife 2017 03 28;6. Epub 2017 Mar 28.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

In mammals, tactile information is mapped topographically onto the contralateral side of the brain in the primary somatosensory cortex (S1). In this study, we describe Robo3 mouse mutants in which a sizeable fraction of the trigemino-thalamic inputs project ipsilaterally rather than contralaterally. The resulting mixture of crossed and uncrossed sensory inputs creates bilateral whisker maps in the thalamus and cortex. Surprisingly, these maps are segregated resulting in duplication of whisker representations and doubling of the number of barrels without changes in the size of S1. Sensory deprivation shows competitive interactions between the ipsi/contralateral whisker maps. This study reveals that the somatosensory system can form a somatotopic map to integrate bilateral sensory inputs, but organizes the maps in a different way from that in the visual or auditory systems. Therefore, while molecular pre-patterning constrains their orientation and position, preservation of the continuity of inputs defines the layout of the somatosensory maps.
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http://dx.doi.org/10.7554/eLife.23494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404921PMC
March 2017