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Highlights • • A novel Vcan mouse allele, VcanAA, has ADAMTS protease-resistant versican. • • VcanAA/AA mice are viable and develop soft tissue-syndactyly (STS) • • VcanAA/AA STS is rendered more severe in combination with Adamts20Bt/Bt. • • Mice lacking the versican GAGβ domain, but not the GAGα domain, also have STS. • • The versican GAGβ proteolytic fragment versikine is necessary for web regression., Two inherent challenges in the mechanistic interpretation of protease-deficient phenotypes are defining the specific substrate cleavages whose reduction generates the phenotypes and determining whether the phenotypes result from loss of substrate function, substrate accumulation, or loss of a function(s) embodied in the substrate fragments. Hence, recapitulation of a protease-deficient phenotype by a cleavage-resistant substrate would stringently validate the importance of a proteolytic event and clarify the underlying mechanisms. Versican is a large proteoglycan required for development of the circulatory system and proper limb development, and is cleaved by ADAMTS proteases at the Glu441-Ala442 peptide bond located in its alternatively spliced GAGβ domain. Specific ADAMTS protease mutants have impaired interdigit web regression leading to soft tissue syndactyly that is associated with reduced versican proteolysis. Versikine, the N-terminal proteolytic fragment generated by this cleavage, restores interdigit apoptosis in ADAMTS mutant webs. Here, we report a new mouse transgene, VcanAA, with validated mutations in the GAGβ domain that specifically abolish this proteolytic event. VcanAA/AA mice have partially penetrant hindlimb soft tissue syndactyly. However, Adamts20 inactivation in VcanAA/AA mice leads to fully penetrant, more severe syndactyly affecting all limbs, suggesting that ADAMTS20 cleavage of versican at other sites or of other substrates is an additional requirement for web regression. Indeed, immunostaining with a neoepitope antibody against a cleavage site in the versican GAGα domain demonstrated reduced staining in the absence of ADAMTS20. Significantly, mice with deletion of Vcan exon 8, encoding the GAGβ domain, consistently developed soft tissue syndactyly, whereas mice unable to include exon 7, encoding the GAGα domain in Vcan transcripts, consistently had fully separated digits. These findings suggest that versican is cleaved within each GAG-bearing domain during web regression, and affirms that proteolysis in the GAGβ domain, via generation of versikine, has an essential role in interdigital web regression.

Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond.

Primary sensory afferents of the dorsal root ganglion (DRG) that innervate the skin detect a wide range of stimuli, such as touch, temperature, pain, and itch. Different functional classes of nociceptors project their axons to distinct target zones within the developing skin, but the molecular mechanisms that regulate target innervation are less clear. Here we report that the Nogo66 receptor homolog NgR2 is essential for proper cutaneous innervation. NgR2−/−mice display increased density of nonpeptidergic nociceptors in the footpad and exhibit enhanced sensitivity to mechanical force and innocuous cold temperatures. These sensory deficits are not associated with any abnormality in morphology or density of DRG neurons. However, deletion of NgR2 renders nociceptive nonpeptidergic sensory neurons insensitive to the outgrowth repulsive activity of skin-derived Versican. Biochemical evidence shows that NgR2 specifically interacts with the G3 domain of Versican. The data suggest that Versican/NgR2 signaling at the dermo-epidermal junction actsin vivoas a local suppressor of axonal plasticity to control proper density of epidermal sensory fiber innervation. Our findings not only reveal the existence of a novel and unsuspected mechanism regulating epidermal target innervation, but also provide the first evidence for a physiological role of NgR2 in the peripheral nervous system.

Patients with the very rare eye pathology Wagner disease (OMIM #143200) present with an abnormal (empty) vitreous, retinal detachment and altered electroretinogram (ERG). The disease is progressive and can eventually lead to blindness. No therapy can be offered to date. The genetic basis is the presence of mutations in the VCAN gene, encoding the large extracellular matrix molecule versican, which is a component of the vitreous. All identified mutations map to the canonical splice sites flanking exon 8, resulting in low number of aberrant splice products and a severe increase in two (V2, V3) of the four naturally occurring splice variants. The pathomechanism of Wagner's disease is poorly understood and a mouse model may afford further insight. The hdf -/- mice, named for their initial phenotype of heart defects, carry a null allele for Vcan that leads to embryonic lethality when homozygous, but heterozygote animals are viable. Here we investigated a possible eye phenotype in the heterozygous animals. While the overall morphology of retina and ciliary body appears to be normal, older (17 months) mutant animals show a decrease in ERG signaling profiles affecting the a-, b- and c-waves. This aspect of altered ERG profile demonstrates similarities to the human disease manifestation and underlines the suitability of heterozygous hdf+/- mice as a model for Wagner disease.

Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human skin.

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate. The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.

Versican is a member of the family of large aggregating chondroitin sulfate proteoglycans which are one of the major constituents of brain extracellular matrix (ECM). We examined the expression of versican splice variants at mRNA and protein levels in normal human brain and in gliomas, medulloblastomas, schwannomas, neurofibromas, and meningiomas. RT-PCR revealed transcripts for V0 and V1 in all tissues. V2 mRNA was restricted to gliomas and normal brain, while V3 mRNA was detected in all tissues except for medulloblastomas. Immunohistochemistry using antibodies to the glycosaminoglycan (GAG)-alpha attachment domain of versican (present in V0 and V2) revealed decreased staining of most glioma ECMs compared to normal neuropil, while some abnormal tumor vessels, but not normal cerebral vessels, were GAG-alpha-positive. Expression of the GAG-beta attachment domain (present in V0 and V1) was faint in normal neuropil and cerebral vessels, but increased in tumor vessels and was absent in most glioma ECMs. Both GAG-alpha and GAG-beta were expressed in connective tissue of all nonglial tumors. Our data suggest that V2 is the major versican isoform of normal neuropil and glioma ECM. Furthermore, increased expression in tumor vessels and decreased expression in glioma ECM of the anti-adhesive versican may be related to marked local invasivity and rarity of extracranial metastasis of gliomas.

The complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of these isoforms is indispensable for life, a constitutive and complete inactivation may even preclude any in vivo studies of later expressed splice-variants in mice. To explore function of the tissue-restricted versican V2 isoform during central nervous system maturation, for instance, we had to circumvent the early embryonic lethality of the complete knockout by employing a novel splice-variant-specific gene ablation approach. For this purpose, we introduced a preterm translational stop codon preceded by an ER-retention signal (KDEL) into the alternatively spliced exon 7 of the VCAN gene. This way the synthesis of the V2- and the V0-forms of the proteoglycan was entirely abolished in the mutant mice, most likely mediated by a KDEL-promoted intracellular degradation of the mutant fragment and by a nonsense-mediated decay mechanism. The expression of the vitally important V1-isoform and the smallest V3-variant remained, however, unaffected. Here we provide the details of our targeting strategy, the screening procedure, the generation of isoform-specific antibodies, and the transcript analysis and we supply the experimental protocols for the biochemical and immunohistological examinations of the mutant mouse strain Vcan(tm1.1Dzim).

We have cloned an alternatively spliced glycosaminoglycan attachment domain (GAG-alpha) of human versican from cDNA libraries derived from U251MG glioma cells. Inserted carboxyl-terminal of the hyaluronan-binding region, this domain adds another 987 amino acids to the original versican (V1) core protein giving rise to the large V0 isoform with 3396 amino acids and 17-23 putative glycosaminoglycan attachment sites. The GAG-alpha domain is encoded by exon 7 of the human versican gene (Naso et al., J. Biol. Chem., 32999-33008). Sequence comparisons revealed a slight similarity to the alternative splice domain of PG-M, further supporting the notion that PG-M is the chicken homologue of versican. On immunoblots of a proteoglycan preparation from U251MG culture medium, anti-GAG-alpha antibodies reacted exclusively with the larger of two versican core proteins recognized by antibodies against the original GAG-beta domain. Using reverse transcription-polymerase chain reaction, we detected both the V0 and V1 isoforms in the cerebral cortex, aorta, intervertebral disc, liver, myometrium, and prostate, whereas keratinocytes exclusively expressed versican V1. In brain tissue, we identified a short versican variant (V2) including only the GAG-alpha domain. By expressing particular splice forms of versican, cells may control the hydration properties of their pericellular hyaluronan coat and thus could modulate interactions with the extracellular matrix or neighboring cells.

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity-purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down-regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.

The complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of these isoforms is indispensable for life, a constitutive and complete inactivation may even preclude any in vivo studies of later expressed splice-variants in mice. To explore function of the tissue-restricted versican V2 isoform during central nervous system maturation, for instance, we had to circumvent the early embryonic lethality of the complete knockout by employing a novel splice-variant-specific gene ablation approach. For this purpose, we introduced a preterm translational stop codon preceded by an ER-retention signal (KDEL) into the alternatively spliced exon 7 of the VCAN gene. This way the synthesis of the V2- and the V0-forms of the proteoglycan was entirely abolished in the mutant mice, most likely mediated by a KDEL-promoted intracellular degradation of the mutant fragment and by a nonsense-mediated decay mechanism. The expression of the vitally important V1-isoform and the smallest V3-variant remained, however, unaffected. Here we provide the details of our targeting strategy, the screening procedure, the generation of isoform-specific antibodies, and the transcript analysis and we supply the experimental protocols for the biochemical and immunohistological examinations of the mutant mouse strain Vcan(tm1.1Dzim).

Peanut agglutinin-binding disaccharides and chondroitin sulfate mark transient mesenchymal barriers to advancing motor and sensory axons innervating the hindlimbs during chick development. Here we show that the vast majority of these carbohydrates are at the critical stage and location attached to the versican splice variants V0 and V1. We reveal that the isolated isoforms of this extracellular matrix proteoglycan suppress axon extension at low concentrations and induce growth cone collapse and rapid retraction at higher levels. Moreover, we demonstrate that versican V0 and/or V1, recombinantly expressed in collagen-I gels or ectopically deposited in the hindlimbs of chicken embryosin ovo, cause untimely defasciculation and axon stalling. Consequently, severe disturbances of nerve patterning are observed in the versican-treated embryos. Our experiments emphasize the inhibitory capacity of versicans V0 and V1 in axonal growth and evidence for their function as basic guidance cues during development of the peripheral nervous system.

Oligodendrocyte myelin glycoprotein (OMgp) is expressed by both neurons and oligodendrocytes in the central nervous system (CNS). It has been implicated in growth cone collapse and neurite outgrowth inhibition by signaling through the Nogo receptor and paired immunoglobulin-like receptor B (PirB). OMgp was also reported to be an extracellular matrix (ECM) protein surrounding CNS nodes of Ranvier and proposed to function as 1) an inhibitor of nodal collateral sprouting and 2) an important contributor to proper nodal and paranodal architecture. However, we show here that the anti-OMgp antiserum used in previous studies to define the functions of OMgp at nodes is not specific. Among all reported nodal ECM components, the antiserum exhibited strong cross-reactivity against versican V2 isoform, a chondroitin sulfate proteoglycan. Furthermore, the OMgp antiserum labeled OMgp-null nodes, but not nodes from versican V2-deficient mice, and pre-adsorption of the OMgp antiserum with recombinant versican V2 blocked nodal labeling. Analysis of CNS nodes in OMgp-null mice failed to reveal any nodal or paranodal defects, or increased nodal collateral sprouting, indicating that OMgp does not participate in CNS node of Ranvier assembly or maintenance. We successfully identified a highly specific anti-OMgp antibody and observed OMgp staining in white matter only after initiation of myelination. OMgp immunoreactivity decorated the surface of mature myelinated axons, but was excluded from compact myelin and nodes. Together, our results strongly argue against the nodal localization of OMgp and its proposed functions at nodes, and reveal OMgp’s authentic localization relative to nodes and myelin.

The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix surrounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile; however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.

The basic concept, that specialized extracellular matrices rich in hyaluronan, chondroitin sulfate proteoglycans (aggrecan, versican, neurocan, brevican, phosphacan), link proteins and tenascins (Tn-R, Tn-C) can regulate cellular migration and axonal growth and thus, actively participate in the development and maturation of the nervous system, has in recent years gained rapidly expanding experimental support. The swift assembly and remodeling of these matrices have been associated with axonal guidance functions in the periphery and with the structural stabilization of myelinated fiber tracts and synaptic contacts in the maturating central nervous system. Particular interest has been focused on the putative role of chondroitin sulfate proteoglycans in suppressing central nervous system regeneration after lesions. The axon growth inhibitory properties of several of these chondroitin sulfate proteoglycans in vitro, and the partial recovery of structural plasticity in lesioned animals treated with chondroitin sulfate degrading enzymes in vivo have significantly contributed to the increased awareness of this long time neglected structure.

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin ( http://www.provitro.de ). Supplementary data are online available at http://pathologie-ccm.charite.de (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes.

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell–like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest–derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

We have isolated and characterized the proteoglycan isoforms of versican from bovine brain extracts. Our approach included (i) cDNA cloning and sequencing of the entire open reading frame encoding the bovine versican splice variants; (ii) preparation of antibodies against bovine versican using recombinant core protein fragments and synthetic peptides; (iii) isolation of versican isoforms by ammonium sulfate precipitation followed by anion exchange and hyaluronan affinity chromatography; and (iv) characterization by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining or immunoblotting. Our results demonstrate that versican V2 is, together with brevican, a major component of the mature brain extracellular matrix. Versicans V0 and V1 are only present in relatively small amounts. Versican V2 migrates after chondroitinase ABC digestion with an apparent molecular mass of about 400 kDa, whereas it barely enters a 4-15% polyacrylamide gel without the enzyme treatment. The 400-kDa product is recognized by antibodies against the glycosaminoglycan-alpha domain and against synthetic NH2- and COOH-terminal peptides. Our preparations contain no major proteolytic products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.

We studied the distribution of the large hyaluronan-binding proteoglycan versican (also known as PG-M) in human adult tissues using affinity-purified polyclonal antibodies that recognize the core protein of the prominent versican splice variants VO and V1. Versican was present in the loose connective tissues of various organs and was often associated with the elastic fiber network. Furthermore, it was localized in most smooth muscle tissues and in fibrous and elastic cartilage. Versican staining was also noted in the central and peripheral nervous system, in the basal layer of the epidermis, and on the luminal surface of some glandular epithelia. In blood vessels, versican was present in all three wall layers of veins and elastic arteries. In muscular arteries the immunoreactivity was normally restricted to the tunica adventitia. However, it appeared in the media and the split elastica interna of atherosclerotically transformed vessel walls. Our survey of the distribution of versican in normal human tissues now forms the basis for extended studies of potentially aberrant versican expression during pathogenic processes.

SummaryRapid action potential propagation in myelinated axons requires Na+ channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na+ channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na+ channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na+ channel clustering.