Mapping the Fine-Scale Organization and Plasticity of the Brain Vasculature.

The cerebral vasculature is a dense network of arteries, capillaries, and veins. Quantifying variations of the vascular organization across individuals, brain regions, or disease models is challenging. We used immunolabeling and tissue clearing to image the vascular network of adult mouse brains and developed a pipeline to segment terabyte-sized multichannel images from light sheet microscopy, enabling the construction, analysis, and visualization of vascular graphs composed of over 100 million vessel segments. We generated datasets from over 20 mouse brains, with labeled arteries, veins, and capillaries according to their anatomical regions. We characterized the organization of the vascular network across brain regions, highlighting local adaptations and functional correlates. We propose a classification of cortical regions based on the vascular topology. Finally, we analysed brain-wide rearrangements of the vasculature in animal models of congenital deafness and ischemic stroke, revealing that vascular plasticity and remodeling adopt diverging rules in different models.

Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin.

Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.

Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve.

Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20-/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.

Müller glia-derived PRSS56 is required to sustain ocular axial growth and prevent refractive error.

A mismatch between optical power and ocular axial length results in refractive errors. Uncorrected refractive errors constitute the most common cause of vision loss and second leading cause of blindness worldwide. Although the retina is known to play a critical role in regulating ocular growth and refractive development, the precise factors and mechanisms involved are poorly defined. We have previously identified a role for the secreted serine protease PRSS56 in ocular size determination and PRSS56 variants have been implicated in the etiology of both hyperopia and myopia, highlighting its importance in refractive development. Here, we use a combination of genetic mouse models to demonstrate that Prss56 mutations leading to reduced ocular size and hyperopia act via a loss of function mechanism. Using a conditional gene targeting strategy, we show that PRSS56 derived from Müller glia contributes to ocular growth, implicating a new retinal cell type in ocular size determination. Importantly, we demonstrate that persistent activity of PRSS56 is required during distinct developmental stages spanning the pre- and post-eye opening periods to ensure optimal ocular growth. Thus, our mouse data provide evidence for the existence of a molecule contributing to both the prenatal and postnatal stages of human ocular growth. Finally, we demonstrate that genetic inactivation of Prss56 rescues axial elongation in a mouse model of myopia caused by a null mutation in Egr1. Overall, our findings identify PRSS56 as a potential therapeutic target for modulating ocular growth aimed at preventing or slowing down myopia, which is reaching epidemic proportions.

Tuning Physicochemical Properties of a Macroporous Polysaccharide-Based Scaffold for 3D Neuronal Culture.

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 µg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

Schwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behavior ex vivo and in vivo after exogenous transplantation in the demyelinated spinal cord. The data highlight for the first time that SC migrate preferentially along blood vessels in perivascular extracellular matrix (ECM), avoiding CNS myelin. We demonstrate in vitro and in vivo that this migration route occurs by virtue of a dual mode of action of Eph/ephrin signaling. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinß1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair.

Boundary cap cells are peripheral nervous system stem cells that can be redirected into central nervous system lineages.

Boundary cap cells (BC), which express the transcription factor Krox20, participate in the formation of the boundary between the central nervous system and the peripheral nervous system. To study BC stemness, we developed a method to purify and amplify BC in vitro from Krox20(Cre/+), R26R(YFP/+) mouse embryos. We show that BC progeny are EGF/FGF2-responsive, form spheres, and express neural crest markers. Upon growth factor withdrawal, BC progeny gave rise to multiple neural crest and CNS lineages. Transplanted into the developing murine forebrain, they successfully survived, migrated, and integrated within the host environment. Surprisingly, BC progeny generated exclusively CNS cells, including neurons, astrocytes, and myelin-forming oligodendrocytes. In vitro experiments indicated that a sequential combination of ventralizing morphogens and glial growth factors was necessary to reprogram BC into oligodendrocytes. Thus, BC progeny are endowed with differentiation plasticity beyond the peripheral nervous system. The demonstration that CNS developmental cues can reprogram neural crest-derived stem cells into CNS derivatives suggests that BC could serve as a source of cell type-specific lineages, including oligodendrocytes, for cell-based therapies to treat CNS disorders.

Glia at Transition Zones.

Neural cells are segregated into their distinct central nervous system (CNS) and peripheral nervous system (PNS) domains. However, at specialized regions of the nervous system known as transition zones (TZs), glial cells from both the CNS and PNS are uniquely present with other specialized TZ cells. Herein we review the current understanding of vertebrate TZ cells. The article discusses the distinct cells at vertebrate TZs with a focus on cells that are located on the peripheral side of the spinal cord TZs. In addition to the developmental origin and differentiation of these TZ cells, the functional importance and the role of TZ cells in disease are highlighted. This article also reviews the common and unique features of vertebrate TZs from zebrafish to mice. We propose challenges and open questions in the field that could lead to exciting insights in the field of glial biology.

Motor innervation directs the correct development of the mouse sympathetic nervous system.

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the "fight-or-flight response". Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells.

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Homozygous deletion of an EGR2 enhancer in congenital amyelinating neuropathy.

The transcription factor EGR2 is expressed in Schwann cells, where it controls peripheral nerve myelination. Mutations of EGR2 have been found in patients with congenital hypomyelinating neuropathy or Charcot-Marie-Tooth disease type 1D. In a patient with congenital amyelinating neuropathy, we observed pathological abnormalities recapitulating the peripheral nervous system phenotype of homozygous Egr2-null mice. This patient, born from consanguineous parents, showed no EGR2 immunoreactivity in Schwann cells and harbored a homozygous 10.7-kilobase-long deletion encompassing a myelin-specific enhancer of EGR2. This regulatory mutation is the first genetic abnormality associated with congenital amyelinating neuropathy in humans.

Neural tube-associated boundary caps are a major source of mural cells in the skin.

In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.

Topical delivery of mitogen-activated protein kinase inhibitor binimetinib prevents the development of cutaneous neurofibromas in neurofibromatosis type 1 mutant mice.

Cutaneous neurofibromas (cNFs) are a hallmark of patients with the neurofibromatosis type 1 (NF1) genetic disorder. These benign nerve sheath tumors, which can amount to thousands, develop from puberty onward, often cause pain and are considered by patients to be the primary burden of the disease. Mutations of NF1, encoding a negative regulator of the RAS signaling pathway, in the Schwann cell (SCs) lineage are considered to be at the origin of cNFs. The mechanisms governing cNFs development are poorly understood, and therapeutics to reduce cNFs are missing, mainly due to the lack of appropriate animal models. To address this, we designed the Nf1-KO mouse model that develops cNFs. Using this model, we found that cNFs development is a singular event and goes through 3 successive stages: initiation, progression, and stabilization characterized by changes in the proliferative and MAPK activities of tumor SCs. We found that skin trauma accelerated the development of cNFs and further used this model to explore the efficacy of the MEK inhibitor binimetinib to cure these tumors. We showed that while topically delivered binimetinib has a selective and minor effect on mature cNFs, the same drug prevents their development over long periods.

Existing and Developing Preclinical Models for Neurofibromatosis Type 1-Related Cutaneous Neurofibromas.

Neurofibromatosis type 1 (NF1) is caused by a nonfunctional copy of the NF1 tumor suppressor gene that predisposes patients to the development of cutaneous neurofibromas (cNFs), the skin tumor that is the hallmark of this condition. Innumerable benign cNFs, each appearing by an independent somatic inactivation of the remaining functional NF1 allele, form in nearly all patients with NF1. One of the limitations in developing a treatment for cNFs is an incomplete understanding of the underlying pathophysiology and limitations in experimental modeling. Recent advances in preclinical in vitro and in vivo modeling have substantially enhanced our understanding of cNF biology and created unprecedented opportunities for therapeutic discovery. We discuss the current state of cNF preclinical in vitro and in vivo model systems, including two- and three-dimensional cell cultures, organoids, genetically engineered mice, patient-derived xenografts, and porcine models. We highlight the models' relationship to human cNFs and how they can be used to gain insight into cNF development and therapeutic discovery.

Myelinating Schwann cells and Netrin-1 control intra-nervous vascularization of the developing mouse sciatic nerve.

Peripheral nerves are vascularized by a dense network of blood vessels to guarantee their complex function. Despite the crucial role of vascularization to ensure nerve homeostasis and regeneration, the mechanisms governing nerve invasion by blood vessels remain poorly understood. We found, in mice, that the sciatic nerve invasion by blood vessels begins around embryonic day 16 and continues until birth. Interestingly, intra-nervous blood vessel density significantly decreases during post-natal period, starting from P10. We show that, while the axon guidance molecule Netrin-1 promotes nerve invasion by blood vessels via the endothelial receptor UNC5B during embryogenesis, myelinated Schwann cells negatively control intra-nervous vascularization during post-natal period.

CNS/PNS boundary transgression by central glia in the absence of Schwann cells or Krox20/Egr2 function.

CNS/PNS interfaces constitute cell boundaries, because they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that depletion of Schwann cells and boundary cap cells or inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether, these data suggest that maintenance of the CNS/PNS boundary requires a Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.

[Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia]. / Système nerveux central, système nerveux pèriphérique: comment maintenir la frontière?

CNS/PNS interfaces constitute cell boundaries, defining territories with different neuronal and glial contents. Despite their potential implications for regenerative medicine, the mechanisms that restrict oligodendrocytes and astrocytes to the CNS and Schwann cells to the PNS are not known in mammals. To investigate the involvement of peripheral glia and myelin in CNS/PNS boundary maintenance, we first studied mutant mice. We found that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, resulted in CNS/PNS boundary transgression by astrocytes and oligodendrocytes, and in myelination of nerve root axons by oligodendrocytes. In contrast, no such migration was observed in mice with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. These findings suggest that CNS/PNS boundary maintenance requires a new Krox20 function independent of myelination control. We also examined a patient with congenital amyelinating neuropathy, whose Schwann cells lack KROX20 protein. Interestingly, the patient's nerve roots were also invaded by oligodendrocytes and astrocytes, indicating that CNS/PNS boundary transgression by central glia can occur in human pathological situations and that the underlying mechanisms are the same as in mutant mice.

Novel features of boundary cap cells revealed by the analysis of newly identified molecular markers.

Neural crest (NC) cells are a multipotent, highly migratory cell population that generates most of the components of the peripheral nervous system (PNS), including the glial Schwann cells (SC) and boundary cap (BC) cells. These latter cells are located at the interface between the central nervous system and PNS, at the exit/entry points of ventral motor/dorsal sensory axons and give rise to all SC in the nerve roots and to a subset of nociceptive neurons and satellite cells in the dorsal root ganglia. In the present study we have compared BC cells with two closely related cell types, NC and Schwann cell precursors (SCpr), by RNA profiling. This led to the definition of a set of 10 genes that show specific expression in BC cells and/or in their derivatives along the nerve roots. Analysis of the expression of these genes during mouse development revealed novel features, of those most important are: (i) dorsal and ventral nerve root BC cell derivatives express different sets of genes, suggesting that they have distinct properties; (ii) these cells undergo major modifications in their gene expression pattern between embryonic days 14.5 and 17.5, possibly linked to the SCpr-immature Schwann cell transition; (iii) nerve roots SC differ from more distal SC not only in their origins and locations, but also in their gene expression patterns. In conclusion, the identification of these novel makers opens the way for a detailed characterization of BC cells in both mouse and man.

Cellular Origin, Tumor Progression, and Pathogenic Mechanisms of Cutaneous Neurofibromas Revealed by Mice with Nf1 Knockout in Boundary Cap Cells.

Patients carrying an inactive NF1 allele develop tumors of Schwann cell origin called neurofibromas (NF). Genetically engineered mouse models have significantly enriched our understanding of plexiform forms of NFs (pNF). However, this has not been the case for cutaneous neurofibromas (cNF), observed in all NF1 patients, as no previous model recapitulates their development. Here, we show that conditional Nf1 inactivation in Prss56-positive boundary cap cells leads to bona fide pNFs and cNFs. This work identifies subepidermal glia as a likely candidate for the cellular origin of cNFs and provides insights on disease mechanisms, revealing a long, multistep pathologic process in which inflammation-related signals play a pivotal role. This new mouse model is an important asset for future clinical and therapeutic investigations of NF1-associated neurofibromas. SIGNIFICANCE: Patients affected by NF1 develop numerous cNFs. We present a mouse model that faithfully recapitulates cNFs, identify a candidate cell type at their origin, analyze the steps involved in their formation, and show that their development is dramatically accelerated by skin injury. These findings have important clinical/therapeutic implications.This article is highlighted in the In This Issue feature, p. 1.

Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points.

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.