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.     

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.     

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.     

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.     

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.     

Neuregulin-1 type III determines the ensheathment fate of axons

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.     

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.     

Specific functions for ERK/MAPK signaling during PNS development

We have established functions of the stimulus-dependent MAPKs, ERK1/2 and ERK5, in DRG, motor neuron, and Schwann cell development. Surprisingly, many aspects of early DRG and motor neuron development were found to be ERK1/2 independent, and Erk5 deletion had no obvious effect on embryonic PNS. In contrast, Erk1/2 deletion in developing neural crest resulted in peripheral nerves that were devoid of Schwann cell progenitors, and deletion of Erk1/2 in Schwann cell precursors caused disrupted differentiation and marked hypomyelination of axons. The Schwann cell phenotypes are similar to those reported in neuregulin-1 and ErbB mutant mice, and neuregulin effects could not be elicited in glial precursors lacking Erk1/2. ERK/MAPK regulation of myelination was specific to Schwann cells, as deletion in oligodendrocyte precursors did not impair myelin formation, but reduced precursor proliferation. Our data suggest a tight linkage between developmental functions of ERK/MAPK signaling and biological actions of specific RTK-activating factors.     

erbb3 and erbb2 are essential for schwann cell migration and myelination in zebrafish

BACKGROUND: Myelin is critical for efficient axonal conduction in the vertebrate nervous system. Neuregulin (Nrg) ligands and their ErbB receptors are required for the development of Schwann cells, the glial cells that form myelin in the peripheral nervous system. Previous studies have not determined whether Nrg-ErbB signaling is essential in vivo for Schwann cell fate specification, proliferation, survival, migration, or the onset of myelination. RESULTS: In genetic screens for mutants with disruptions in myelinated nerves, we identified mutations in erbb3 and erbb2, which together encode a heteromeric tyrosine kinase receptor for Neuregulin ligands. Phenotypic analysis shows that both genes are essential for development of Schwann cells. BrdU-incorporation studies and time-lapse analysis reveal that Schwann cell proliferation and migration, but not survival, are disrupted in erbb3 mutants. We show that Schwann cells can migrate in the absence of DNA replication. This uncoupling of proliferation and migration indicates that erbb gene function is required independently for these two processes. Pharmacological inhibition of ErbB signaling at different stages reveals a continuing requirement for ErbB function during migration and also provides evidence that ErbB signaling is required after migration for proliferation and the terminal differentiation of myelinating Schwann cells. CONCLUSIONS: These results provide in vivo evidence that Neuregulin-ErbB signaling is essential for directed Schwann cell migration and demonstrate that this pathway is also required for the onset of myelination in postmigratory Schwann cells.     

Vimentin regulates peripheral nerve myelination

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.     

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.     

Neuregulin 1 type III/ErbB signaling is crucial for Schwann cell colonization of sympathetic axons

Analysis of Schwann cell (SC) development has been hampered by the lack of growing axons in many commonly used in vitro assays. As a consequence, the molecular signals and cellular dynamics of SC development along peripheral axons are still only poorly understood. Here we use a superior cervical ganglion (SCG) explant assay, in which axons elongate after treatment with nerve growth factor (NGF). Migration as well as proliferation and apoptosis of endogenous SCG-derived SCs along sympathetic axons were studied in these cultures using pharmacological interference and time-lapse imaging. Inhibition of ErbB receptor tyrosine kinases leads to reduced SC proliferation, increased apoptosis and thereby severely interfered with SC migration to distal axonal sections and colonization of axons. Furthermore we demonstrate that SC colonization of axons is also strongly impaired in a specific null mutant of an ErbB receptor ligand, Neuregulin 1 (NRG1) type III. Taken together, using a novel SC development assay, we demonstrate that NRG1 type III serves as a critical axonal signal for glial ErbB receptors that drives SC development along sympathetic axons.     

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.     

Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.     

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.     

Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.