Peripheral glia: Schwann cells in motion

Neuregulin signaling through ErbB receptors is known to play an essential role in Schwann cell proliferation, survival and myelination. Recent studies in zebrafish provide a peek at living Schwann cells migrating along axons in vivo and suggest that ErbB signaling, while not required for cell movement per se, is required to maintain the directed migration of these cells.     

Neuronal Neuregulin 1 type III directs Schwann cell migration

During peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves.     

ErbB receptors and the development of the nervous system

Tyrosine kinase receptors and their ligands allow communication between cells in the developing and adult organism. An extensive line of research has revealed that ‘neuregulins’, a family of EGF-like factors that signal via ErbB receptors, are used frequently for cell communication during nervous system development, and control a spectacular spectrum of developmental processes. For instance, during development of the peripheral nervous system, Schwann cells require neuronally-produced neuregulin (Nrg1) for growth, migration and myelination, neural crest cells rely on mesenchymally-generated Nrg1 signals for migration, while muscle requires neuronally-produced Nrg1 for the differentiation of a muscle spindle. In the central nervous system, neuregulin signals allow cells to act as guideposts or as barriers for axons during pathfinding. Neuregulin signals are also important in other organs, but the nervous system functions have received recently considerable attention due to the finding that particular haplotypes of Nrg1 and ErbB4 predispose to schizophrenia. Understanding the neuregulin signaling system can thus contribute to define causes of this devastating mental disorder.     

FAK is required for axonal sorting by Schwann cells

Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.     

Insights on the role of adhesion related molecules on stem cells

The development of the peripheral nervous system (PNS) is a highly dynamic process, during which motor and sensory axons innervate distal targets, such as skeletal muscles and skin. Axonal function depends critically on support from Schwann cells, the main glial cell type in the PNS. Schwann cells originate from the neural crest, migrate along outgrowing axons and associate with axons along their entire length prior to ensheathment or myelination. How axonal growth and the migration of Schwann cells is coordinated at the level of reciprocal axon-glial signaling is the fascinating subject of ongoing research. Neuregulin-1 (NRG1) type III, an axonal membrane-bound ligand for receptor tyrosine kinases of the ErbB family, acts as a “master regulator” of peripheral myelination. In addition, NRG1-ErbB signaling directs the development of the Schwann cell lineage and regulates the proliferation and survival of Schwann cells. Studies in zebrafish have identified a direct role of NRG1 type III in Schwann cell migration, but to what extend NRG1 serves a similar function in the mammalian PNS is not clear. We have employed a mouse superior cervical ganglion explant culture system, in which the migration of endogenous Schwann cells along outgrowing axons can be visualized by time-lapse imaging. Using this approach, we found that NRG1 type III-ErbB signaling regulates the colonization of distal axonal segments by Schwann cells. However, our data suggest an indirect effect of NRG1 type III-ErbB signaling via the support of Schwann cell survival in proximal axonal regions rather than a direct effect on Schwann cell motility.     

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.     

Neuregulin, a factor with many functions in the life of a schwann cell

The signalling system comprising the ligand Neuregulin-1, and its receptors, ErbB2 and ErbB3, plays multiple and important roles in glial development. These include functions in early development of neural crest cells, in expansion of the Schwann cell precursor pool and in myelination. Neuregulin is one of the crucial axon-derived signals that influence development of Schwann cells. These are specialized cells that ensheath peripheral axons and provide electrical insulation. Schwann cells have also long been implicated in providing more than a simple ensheathing function. Compelling evidence for this has emerged from the analysis of mice lacking these cells, resulting from a non-functional or compromised Neuregulin signalling system. They serve as a model to study glia-nerve interactions in vivo and indicate that Schwann cells provide important neurotrophic signals, and also cues that regulate perineurium development and nerve fasciculation.     

Axonal regulation of myelination by neuregulin 1

Neuregulins comprise a family of epidermal growth factor-like ligands that interact with ErbB receptor tyrosine kinases to control many aspects of neural development. One of the most dramatic effects of neuregulin-1 is on glial cell differentiation. The membrane-bound neuregulin-1 type III isoform is an axonal ligand for glial ErbB receptors that regulates the early Schwann cell lineage, including the generation of precursors. Recent studies have shown that the amount of neuregulin-1 type III expressed on axons also dictates the glial phenotype, with a threshold level triggering Schwann cell myelination. Remarkably, neuregulin-1 type III also regulates Schwann cell membrane growth to adjust myelin sheath thickness to match axon caliber precisely. Whether this signaling system operates in central nervous system myelination remains an open question of major importance for human demyelinating diseases.     

ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.     

Nrg1/ErbB signaling networks in Schwann cell development and myelination

Neuregulin-1 (Nrg1) provides a key axonal signal that regulates Schwann cell proliferation, migration and myelination through binding to ErbB2/3 receptors. The analysis of a number of genetic models has unmasked fundamental mechanisms underlying the specificity of the Nrg1/ErbB signaling axis. Differential expression of Nrg1 isoforms, Nrg1 processing, and ErbB receptor localization and trafficking represent important regulatory themes in the control of Nrg1/ErbB function. Nrg1 binding to ErbB2/3 receptors results in the activation of intracellular signal transduction pathways that initiate changes in Schwann cell behavior. Here, we review data that has defined the role of key Nrg1/ErbB signaling components like Shp2, ERK1/2, FAK, Rac1/Cdc42 and calcineurin in development of the Schwann cell lineage in vivo. Many of these regulators receive converging signals from other cues that are provided by Notch, integrin or G-protein coupled receptors. Signaling by multiple extracellular factors may act as key modifiers and allow Schwann cells at different developmental stages to respond in distinct manners to the Nrg1/ErbB signal.     

Dysregulation of ErbB Receptor Trafficking and Signaling in Demyelinating Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with the majority of cases involving demyelination of peripheral nerves. The pathogenic mechanisms of demyelinating CMT remain unclear, and no effective therapy currently exists for this disease. The discovery that mutations in different genes can cause a similar phenotype of demyelinating peripheral neuropathy raises the possibility that there may be convergent mechanisms leading to demyelinating CMT pathogenesis. Increasing evidence indicates that ErbB receptor-mediated signaling plays a major role in the control of Schwann cell-axon communication and myelination in the peripheral nervous system. Recent studies reveal that several demyelinating CMT-linked proteins are novel regulators of endocytic trafficking and/or phosphoinositide metabolism that may affect ErbB receptor signaling. Emerging data have begun to suggest that dysregulation of ErbB receptor trafficking and signaling in Schwann cells may represent a common pathogenic mechanism in multiple subtypes of demyelinating CMT. In this review, we focus on the roles of ErbB receptor trafficking and signaling in regulation of peripheral nerve myelination and discuss the emerging evidence supporting the potential involvement of altered ErbB receptor trafficking and signaling in demyelinating CMT pathogenesis and the possibility of modulating these trafficking and signaling processes for treating demyelinating peripheral neuropathy.     

Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development

During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific conditional gene targeting to show that members of the Rho GTPases, cdc42 and rac1, have different and essential roles in axon sorting by Schwann cells. Our results indicate that although cdc42 is required for normal Schwann cell proliferation, rac1 regulates Schwann cell process extension and stabilization, allowing efficient radial sorting of axon bundles.     

Neuregulin1 and ErbB expression in the uninjured and regenerating olfactory mucosa

Neuregulin1, a protein involved in signaling through the ErbB receptors, is required for the proper development of multiple organ systems. A complete understanding of the expression profile of Neuregulin1 is complicated by the presence of multiple isoform variants that result from extensive alternative splicing. Remarkably, these numerous protein products display a wide range of divergent functional roles, making the characterization of tissue-specific isoforms critical to understanding signaling. Recent evidence suggests an important role for Neuregulin1 signaling during olfactory epithelium development and regeneration. In order to understand the physiological consequences of this signaling, we sought to identify the isoform-specific and cell type-specific expression pattern of Neuregulin1 in the adult olfactory mucosa using a combination of RT-qPCR, FACS, and immunohistochemistry. To complement this information, we also analyzed the cell-type specific expression patterns of the ErbB receptors using immunohistochemistry. We found that multiple Neuregulin1 isoforms, containing predominantly the Type I and Type III N-termini, are expressed in the uninjured olfactory mucosa. Specifically, we found that Type III Neuregulin1 is highly expressed in mature olfactory sensory neurons and Type I Neuregulin1 is highly expressed in duct gland cells. Surprisingly, the divergent localization of these Neuregulin isoforms and their corresponding ErbB receptors does not support a role for active signaling during normal turnover and maintenance of the olfactory mucosa. Conversely, we found that injury to the olfactory epithelium specifically upregulates the Neuregulin1 Type I isoform bringing the expression pattern adjacent to cells expressing both ErbB2 and ErbB3 which is compatible with active signaling, supporting a functional role for Neuregulin1 specifically during regeneration.     

Signaling through Arf6 guanine-nucleotide exchange factor cytohesin-1 regulates migration in Schwann cells

During development of the peripheral nervous system (PNS), Schwann cells migrate along neuronal axons before initiating myelination of the axons. While intercellular signals controlling migration, between Schwann cells and peripheral neurons, are established, how their intracellular transduction of the signals into Schwann cells still remains to be clarified. Here, we show that cytohesin-1, a guanine-nucleotide exchange factor (GEF), and the effector Arf6 are required for migration of primary Schwann cells. Knockdown of cytohesin-1 or Arf6 in Schwann cells, as well as treatment with the chemical cytohesin inhibitor SecinH3 or knockout of cytohesin-1, inhibits peripheral neuronal conditioned medium-mediated migration. Similar effects are also observed following stimulation with each of growth factors contained in a conditioned medium, suggesting that cytohesin-1 plays a role in transducing soluble ligand signals from neurons. Reintroduction of small interfering (si)RNA-resistant cytohesin-1 into Schwann cells reverses blunted migration in the siRNA-transfected Schwann cells, illustrating the importance of cytohesin-1 in migration. On the other hand, introduction of cytohesin-1 that harbors the Tyr-382 mutation, which is an amino acid that is important for its activation, failed to reverse the reduction in primary Schwann cell migration. These results suggest that signaling through cytohesin-1 is required for Schwann cell migration, revealing a novel mechanism for Schwann cell migration.     

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.     

A dual role for ErbB2 signaling in cardiac trabeculation

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.     

Neuregulin-mediated ErbB3 signaling is required for formation of zebrafish dorsal root ganglion neurons

Dorsal root ganglia (DRGs) arise from trunk neural crest cells that emerge from the dorsal neuroepithelium and coalesce into segmental streams that migrate ventrally along the developing somites. Proper formation of DRGs involves not only normal trunk neural crest migration, but also the ability of DRG progenitors to pause at a particular target location where they can receive DRG-promoting signals. In mammalian embryos, a receptor tyrosine kinase proto-oncogene, ErbB3, is required for proper trunk neural crest migration. Here, we show that in zebrafish mutants lacking ErbB3 function, neural crest cells do not pause at the location where DRGs normally form and DRG neurons are not generated. We also show that these mutants lack trunk neural crest-derived sympathetic neurons, but that cranial neural crest-derived enteric neurons appear normal. We isolated three genes encoding neuregulins, ErbB3 ligands, and show that two neuregulins function together in zebrafish trunk neural crest cell migration and in DRG formation. Together, our results suggest that ErbB3 signaling is required for normal migration of trunk, but not cranial, neural crest cells.