Schwann Cell Development☆

Schwann Cell Development☆ R Mirsky and KR Jessen, University College London, London, United Kingdom ã 2015 Elsevier Inc. All rights reserved.

Introduction The Schwann Cell Lineage Markers of Schwann Cell Development Gliogenesis from the Neural Crest The Role of SOX10 in PNS Gliogenesis ß-Neuregulin-1 and the Neural Crest Schwann Cell Precursors ß-Neuregulin-1 and Schwann Cell Precursors Notch and Schwann Cell Precursors Immature Schwann Cells Boundary cap Cells Give Rise to Schwann Cells in Spinal Roots Functions of Schwann Cell Precursors and Schwann Cells Trophic Support of Neuronal Survival Morphogenesis: Maintaining the Normal Structure of the Spinal Cord, Nerve Trunks and Neuromasts The Generation of Immature Schwann Cells, Endoneurial Fibroblasts and Melanocytes The Transition from Immature Schwann Cells to Myelination Radial Sorting Control of Schwann Cell Numbers The Onset of Myelination The Role of ß-Neuregulin-1 in Myelination The Role of Cyclic AMP in Promoting Myelination Myelin-Related Transcription Factors Negative Regulation of Myelination

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Introduction Schwann cells, found in association with axons in peripheral nerves, are the main glial cell type in the peripheral nervous system (PNS). They exist in two quite distinct forms, either making myelin sheaths round large axons to speed impulse conduction or alternatively enclosing groups of smaller axons, holding them together in tight bundles. A mature peripheral nerve therefore consists of a mixture of myelinated and unmyelinated axon-Schwann cell units, usually referred to as nerve fibers. This article provides an overview of the development of Schwann cells from their origins in the neural crest and briefly of their transformation after nerve injury. The generation of PNS glia parallels the formation of glial cells in the central nervous system (CNS). Nevertheless, many of the key molecules that control gliogenesis differ between the two systems.

The Schwann Cell Lineage The neural crest is a transient group of cells that segregates from the dorsal neural tube and gives rise to a variety of derivatives, including autonomic and sensory neurons, chromaffin cells, glial cells including Schwann cells and satellite cells of the autonomic and sensory ganglia, fibroblastic cells, smooth muscle cells and melanocytes (Figure 1 here). The majority of Schwann cells are derived from the group of neural crest cells that migrate ventrally through the anterior part of the somites, although Schwann cells in the dorsal and ventral roots are derived from a specialized group of cells called boundary cap cells (below) that also gives rise to some neurons and satellite glial cells in dorsal root sensory ganglia (DRG). Mature myelinating and non-myelinating Schwann cells arise from the neural crest via two intermediate stages, the Schwann cell precursor and the immature Schwann cell (Figure 2 here). Schwann cell precursors are the glial cells found in embryonic day (E) mouse E12-13 nerves (rat E14-15). Immature Schwann cells are generated from Schwann cell precursors and populate mouse nerves from E15-16 (rat E17-18) to around the time of birth when myelination starts. At the immature Schwann cell stage, Schwann cells that by chance are associated with large axons receive axon-associated signals that instruct them to make myelin ☆

Change History: September 2014. R Mirsky and KR Jessen introduced small edits in the text of the article including citations, added the sections ‘Applications’ and ‘References’, and added Figures.

Reference Module in Biomedical Research

http://dx.doi.org/10.1016/B978-0-12-801238-3.04757-7

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Primitive streak Neural plate

Neural fold Neural groove

Neural crest cell

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3 Neural tube Notochord Figure 1 The neural crest. In a process known as neurulation, the neural plate, which is found along the dorsal surface of an embryo, gradually folds in on itself to form the neural groove. As the neural folds fuse to form the neural tube, the neural crest cells segregate from the tips of the folds. After taking up an initial position at the dorsal surface of the tube, the crest cells in the trunk region soon migrate along one of two major streams: in a lateral direction (1) to give rise to epaxial (dorsal) melanocytes in the skin, and in a ventral direction (2,3) to give rise to neurons in dorsal root sensory ganglia (DRG) and glia (2), or glia, hypaxial (ventral) melanocytes, autonomic neurons and chromaffin cells (3). Neural crest cells in the most anterior part of the trunk, the cardiac crest, also generate fibroblasts and smooth muscle cells, and the cephalic crest in the head region also forms the cells of cartilage and bone. The mechanisms that allow the apparently homogenous population of crest cells to generate such diversity have been intensively studied. It is considered likely that some neural crest cells are already committed to certain fates, whereas others are multipotent. Although some cells may enter lineages in a stochastic and undirected manner, a combination of positive and negative instructive signals probably plays an important part in directing neural crest cell differentiation. How migrating neural crest cells, which initially move through immature connective tissue on each side of the neural tube, end up as Schwann cell precursors in tight association with axons in early embryonic nerves which in turn give rise to immature Schwann cells, endoneurial fibroblasts and ventral melanocytes is not clear, either in terms of their detailed migratory route or the signals that cause these cells to adopt an early glial phenotype. From Jessen, K. R. and Mirsky, R. (2005). Nature Reviews Neuroscience 6, 671–682, with permission.

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Figure 2 The Schwann cell lineage. Schematic illustration showing both developmental and injury induced transitions. Black arrows: normal development. Red arrows: the Schwann cell injury response. Stippled arrows: post-repair formation of myelin and Remak cells. Embryonic dates E refer to mouse development. The embryonic phase of Schwann cell development involves three transient cell populations. First, migrating neural crest cells, which are discussed further in Figure 1. Second, Schwann cell precursors. These cells express various differentiation markers that are not found in neural crest cells, including brain fatty acid binding protein (BFABP), protein zero (P0) and desert hedgehog (Dhh) (Figure 3). At any one time, a rapidly developing population of cells – such as the glia of embryonic nerves – will contain some cells that are rather more advanced than others. Third, immature Schwann cells. All immature Schwann cells are considered to have the same developmental potential, and their fate is determined by axons with which they associate. Myelination occurs only in Schwann cells that by chance envelop large diameter axons – Schwann cells that ensheath small diameter axons progress to become mature non-myelinating cells. After nerve injury Schwann cells in adult nerves undergo transdifferentiation to generate a repair cell type (Bungner cell) that promotes axonal regeneration and functional recovery. Modified from Jessen, K. R. and Mirsky, R. (2005). Nature Reviews Neuroscience 6, 671–682, with permission.

sheaths, and adopt the specific gene expression profile appropriate for myelination. Mature non-myelinating Schwann cells appear about 2 weeks after myelination starts. These cells also express genes that differentiate them from myelinating cells and typically hold several small diameter axons individually in troughs that run along the cell surface, forming unmyelinated (Remak) fibers. Three major developmental steps therefore define the lineage. That is, the transition from migrating neural crest cells to axonassociated Schwann cell precursors, the transition from precursors to immature Schwann cells, and finally the divergence of this population to form the two mature Schwann cell types found in adult nerves. At all stages there is a continuous close association between these cells and axons. It is a striking feature of all the cell types that they are highly dependent on survival factors, mitogens and differentiation signals from axons. Another notable feature is plasticity. For example, mature myelinating and non-myelinating Schwann cells respond to nerve injury by converting to a repair Schwann cell phenotype that is tailored to support neuronal survival and regeneration and Schwann cell precursors can be diverted, to other neural crest derivatives (below). In vivo, rapid proliferation is a characteristic of all early stages of the lineage, namely, neural crest cells, Schwann cell precursors and immature Schwann cells. In contrast, the onset of myelination is clearly linked with cell cycle exit, and both myelinating and non-myelinating cells are quiescent in normal adult nerves. The cells retain, however, the potential to proliferate because they re-enter the cell cycle when they transdifferentiate to repair cells in response to nerve injury. Apoptotic cell death is also a feature of developing cells in early nerves whereas myelinating and mature non-myelinating cells are normally resistant to apoptosis.

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Figure 3 Changes in phenotypic profile as cells progress through the embryonic Schwann cell lineage. Each stage involves characteristic relationships with surrounding tissues and distinctive signaling properties (indicated in the panels below the lineage drawing. Also shown are some of the molecular markers of the lineage. They fall into four groups: (i) Markers that show no significant change between the three stages. (ii) Markers that are upregulated during development. Some of these are upregulated at the crest to Schwann cell precursor transition; another group is upregulated at the Schwann cell precursor to immature Schwann cell transition; (iii) Markers that are down-regulated at the Schwann cell precursor to immature Schwann cell transition. Shared profiles are indicated by distinct colors. The gene expression shown here is based on observations of endogenous genes rather than on observations of reporter genes in transgenic animals. Note that Cadherin 19 (Cad 19) is exclusively expressed in Schwann cell precursors. Glial fibrillary acidic protein (GFAP) is a late marker of Schwann cell generation, as significant expression is not seen until about the time of birth. GFAP is reversibly suppressed in myelinating cells. Schwann cell precursors have been shown to be S100 calcium-binding protein (S100)-negative and Schwann cells S100-positive using routine immunohistochemical methods – however, low levels of S100 are detectable in many mouse Schwann cell precursors when the sensitivity of the assay is significantly increased. a4 integrin; AP2a, activator protein 2a; BFABP, brain fatty acid binding protein; Dhh, desert hedgehog; ErbB3, neuregulin receptor; GAP43, growth-associated protein 43; L1, L1 adhesion molecule; N-cad, N-cadherin; OCT6, octamer-binding transcription factor 6; 04, lipid antigen; PLP, proteolipid protein; PMP22, peripheral myelin protein 22kDa; P0, protein zero; p75NTR, p75 neurotrophin receptor; SOX10, SRY box 10. Modified from Jessen, K. R. and Mirsky, R. (2005). Nature Reviews Neuroscience 6, 671–682, with permission.

Markers of Schwann Cell Development Each cell stage in the embryonic phase of the lineage can be defined by a distinct combination of differentiation markers (Figure 3 here). Additional criteria such as morphology, relationships to other cells and tissues and response to extrinsic signals can also be used to define the cell types and to study the signals that are important in controlling progression from one stage to the next.

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The markers can be divided into five different categories depending on expression patterns (Figure 3). The first category, exemplified by the transcription factor SRY (sex determining region Y) box 10 (SOX10), includes molecules that are present at all developmental stages; the second, exemplified by the transcription factor activator protein 2a (AP2a), includes genes/proteins present at high levels in neural crest cells and Schwann cell precursors but which are down-regulated in immature Schwann cells; the third category, of which cadherin 19 is at present the best known example, represents genes expressed only in Schwann cell precursors; the fourth category represents genes/proteins expressed by Schwann cell precursors and immature Schwann cells but not by neural crest cells, such as brain fatty acid binding protein (BFABP), myelin protein zero (P0) and connexin 29. The last category represents molecules such as S100 or glial fibrillary acidic protein (GFAP) that are present at high levels on immature Schwann cells but are low or absent from neural crest cells or Schwann cell precursors. It should be emphasized that the use of technologies that allow comparative analysis across the genome reveal larger sets of different genes, but which of these prove most useful in study of mechanisms that control development of the lineage remains to be determined. Several additional criteria provide crucial information in analyzing Schwann cell development. Schwann cell precursors and immature Schwann cells, but not neural crest cells, share a feature characteristic of glial cells in both the PNS and CNS, namely their close physical apposition with axons (neurons). Schwann cell precursors also differ from migrating crest cells in their response to survival factors and in being relatively insensitive to the neurogenic actions of bone morphogenetic protein (BMP) 2. They are also more sensitive to the actions of Notch (below) and are strongly biased towards the generation of Schwann cells rather than other neural crest derivatives. A striking difference between Schwann cells and Schwann cell precursors is the ability of Schwann cells to support their own survival in the absence of axons, using autocrine survival circuits. The cytoarchitecture of the nerves at the stage when they contain Schwann cell precursors (E12-13) is also distinctly different from that of nerves containing immature Schwann cells (from E15-16) (below).

Gliogenesis from the Neural Crest The generation of more differentiated cells from stem cells or cells that share some stem cell properties, including the neural crest, is a topic that continues to arouse widespread interest. At present the consensus is that cell specification involves interplay between cell-autonomous intracellular signaling and extracellular cues arising from the niche in which the stem cell resides. The best evidence is that generation of Schwann cells and other glia from the neural crest involves both of these mechanisms.

The Role of SOX10 in PNS Gliogenesis In the generation of glial cells from the neural crest, the transcription factor SOX10 is the most important player known so far, because it is the only gene known to be essential for this process. Initially expressed by all migrating neural crest cells, its expression persists in the developing satellite glial cells of the DRG, in Schwann cell precursors and in Schwann cells of peripheral nerves. SOX10 is, however, down-regulated very early in neurogenesis. In line with this, neurons are initially generated in normal numbers in mice in which SOX10 has been inactivated. In contrast, satellite cells and Schwann cells fail to develop in these animals. In place of satellite cells (i.e. glia) the DRG contain a population of neural crest-like cells and nerve trunks also contain a few neural crestlike cells but no Schwann cell precursors, showing that in the absence of SOX10 glial specification is blocked. Experiments also show that SOX10 plays a role in specifying and maintaining the glial phenotype. It may act in part by up-regulating levels of the neuregulin receptor ErbB3, thus increasing the responsiveness of neural crest cells to the growth factor ß-neuregulin-1 (below). Later in development, there is evidence that it acts in conjunction with other transcription factors such as KROX20 (EGR2) to regulate the promoters of genes that are crucial in myelination such as P0 and connexin 32 (below).

ß-Neuregulin-1 and the Neural Crest ß-neuregulin-1, signaling through its receptors ErbB2 and ErbB3, is a growth factor that has multiple important functions throughout Schwann cell development and maturity. In neural crest cell cultures it inhibits the development of neurons, a function that might lead indirectly to increased development of glia. Over-production of neurons has not, however, been noted in mutants that lack components of the neuregulin signaling pathway so it is not clear that ß-neureglin-1 regulates neuronal numbers in vivo. When ß-neuregulin-1 is added to migrating neural crest cell cultures it increases the proportion of Schwann cells generated. Nevertheless these cultures generate Schwann cells even in the absence of added neuregulin, and the same is true when the appearance of Schwann cell precursors from neural crest cell cultures is monitored. Furthermore, in mouse mutants in which neuregulin signaling has been abrogated in vivo, satellite glia in the DRG are generated although Schwann cell precursors are severely depleted (below). In these mutants most neural crest cells fail to migrate ventrally below the level of the DRG to the sites where sympathetic ganglia are formed, resulting in underdeveloped (hypoplastic) ganglia. In sum these data indicate that in principle ß-neuregulin-1 is not required for the generation of glia from the neural crest, although it modulates this process by suppressing neurogenesis, and by promoting migration of neural crest cells or early crest derivatives, in particular those that will form sympathetic ganglia. There is also evidence that ß-neuregulin-1 accelerates the transition of Schwann cell precursors to Schwann cells (below).

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Schwann Cell Precursors In peripheral nerve trunks, Schwann cell precursors represent the first clearly defined stage of glial differentiation and are the cells from which immature Schwann cells are derived. They represent the large majority of cells in the limb nerves of E12/13 mice (E14/15 rats). They are initially seen at the edge of early embryonic nerves but are later seen inside them as well. They communally surround large groups of axons with their sheet-like processes and divide the nerves into territories. At this stage, the nerves are compact structures that contain essentially no connective tissue and are not vascularized (Figure 4 here).

ß-Neuregulin-1 and Schwann Cell Precursors A vital role for ß-neuregulin-1 in the Schwann cell lineage is indicated by the observation, mentioned earlier, that Schwann cell precursors, and later Schwann cells, are absent or severely depleted in the peripheral nerves of mouse mutants that lack neuregulin signaling. This probably reflects the major role of ß-neuregulin-1 as an essential survival factor and mitogen for Schwann cell precursors, although impaired migration might also play a part (above). Axonally-derived ß-neuregulin-1 is an essential survival factor and mitogen for Schwann cell precursors in vitro and it has been shown that the most important isoform expressed by axons is the membrane-bound ß-neuregulin-1 type III (Figure 5 here). It is the major isoform present in DRG and motor neurons in vivo,

Figure 4 Schwann cell precursors (SCP) and immature Schwann cells (iSch) in embryonic nerves. Upper panel (SCP): transverse section of E14 rat sciatic nerve. Schwann cell precursors branch among axons (downward large arrow) and are found in close apposition to axons at the surface of the nerve (upward large arrow). A dividing Schwann cells precursor can be seen (small arrow). Connective tissue is not found within the nerve. Lower panel (iSch). Extracellular connective tissue space (turquoise), which contains mesenchymal cells, surrounds the nerve but is essentially absent from the nerve itself. These nerves are also free of blood vessels and the axons are of smaller and more uniform diameter than those seen in mature nerves. Magnification, x2000. Transverse section of E18 rat sciatic nerve at the same magnification. One or a few immature Schwann cells together surround several axons of varying sizes, forming compact groups (families; asterisk). A dividing Schwann cell is present (double arrows). Connective tissue (turquoise) containing blood vessels (large arrow) is present throughout the nerve surrounding individual families. Bracket indicates the developing perineurium. Adapted from Jessen, K. R. and Mirsky, R. (2005). Nature Reviews Neuroscience 6, 671–682, with permission.

Schwann Cell Development

Type I

Type II

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Type III

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C

N C

C Immunoglobulin (lg)-like domain Domain rich in potential glycosylation sites

C Hydrophobic sequence with similarities to internal signal sequence

EGF-like domain Transmembrane domain

Cysteine-rich domain

Figure 5 Neuregulin-1. ß-neuregulin-1 seems to have exceptionally numerous and varied functions in Schwann cell biology. It is involved in neural crest migration, and has been implicated in the specification of neural crest stem cells and shown to be essential for the survival of Schwann cell precursors. It is also involved in Schwann cell generation, proliferation and survival. In postnatal nerves, ß-neuregulin-1 is a positive regulator of myelination and myelin sheath thickness but paradoxically, at least in experiments it appears to drive the dedifferentiation of Schwann cells in injured nerve fibers. No other molecule has been proposed to be so comprehensively involved in the control of Schwann cell development. There are a surprising number (>15) of neuregulin-1 a and ß protein isoforms. The schematic structures of the main isoforms found in the nervous system are shown above. Although splice variants without the transmembrane domain exist for all these isoforms, transmembrane isoforms (as shown here together with the products of a proteolytic cleavage in the juxta-membrane area) predominate in the nervous system. The epidermal growth factor (EGF) domain is found in all bioactive forms of neuregulin-1 and is sufficient for activation of ErbB receptor kinase. The type III ß-neuregulin-1 isoform is expressed in axons and is the main regulator of survival of Schwann cell precursors and myelin sheath thickness. It is thought to have two membranespanning domains and to undergo proteolytic cleavage that generates a membrane-attached protein carrying the EGF domain. Further processing is controlled positively by BACE1 and negatively by ADAM17 (Tace). ß-neuregulin-1 shows high affinity binding to two receptors, ErbB3 and ErbB4, whereas a related protein, ErbB2, acts as a co-receptor in ErbB3-ErbB2 and ErbB4-ErbB2 complexes. The former is the main receptor in peripheral glial cells. The action of axonal ß-neuregulin-1 type III on ErbB3-ErbB2 in developing Schwann cells is probably the best established molecular signaling pathway betweens neurons and glia in the PNS. From Jessen, K. R. and Mirsky, R. (2005). Nature Reviews Neuroscience 6, 671–682, with permission.

and in mice in which this isoform has been selectively eliminated, although peripheral nerves are initially populated by Schwann cell precursors, few remain after E14. In vivo, Schwann cell precursors die after nerve injury and this can be prevented by externally applied ß-neuregulin-1. Taken together, this indicates that axonal ß-neuregulin-1 type III is a key survival signal in embryonic Schwann cell development, being an essential survival factor for Schwann cell precursors. In vitro experiments show that in addition to promoting survival, ß-neuregulin-1 accelerates the transition from Schwann cell precursors to immature Schwann cells. This transition is also controlled by another signaling system present in embryonic nerves, that is endothelin. In contrast to neuregulin, endothelins negatively regulate Schwann cell generation, shown for instance by the observation that Schwann cells are generated prematurely in rats in which the endothelin B receptor is inactivated.

Notch and Schwann Cell Precursors Notch transmembrane receptors and their ligands delta, jagged and serrate are known to influence glial cell fate choices in the developing nervous system. In the CNS, the classical view of Notch signaling is that it acts to maintain neural stem cells in an undifferentiated state, but recent evidence suggests that it can promote the appearance of radial glia, astrocytes and Muller cells. It also stimulates the formation of oligodendrocyte precursors while inhibiting their progression to myelinating cells. The question of whether Notch instructively promotes gliogenesis from neural crest cells is still controversial. Notch activation in neural crest cells inhibits the generation of neurons in vivo and in vitro, while in cultures derived from a subpopulation of cells from E14 rat sciatic nerves it increases the number of GFAP-positive Schwann cells and the rate at which they are generated. There is also evidence that Notch, like ß-neuregulin-1, acts on early glial cells, stimulating Schwann cell precursors to generate Schwann cells. Notch also promotes the proliferation of immature Schwann cells in vitro and in vivo. Although it is not settled whether the only actions of Notch are to accelerate lineage progression and stimulate proliferation, or whether Notch also directs crest cells to the glial lineage, there are strong parallels between the actions of Notch and ß-neuregulin-1 in this system. Both signals suppress neurogenesis in crest cells, and stimulate Schwann cell generation from precursors and Schwann cell proliferation.

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Immature Schwann Cells The switch from Schwann cell precursor to immature Schwann cell between E13 and E15 in mouse (rat E15-17) involves a coordinated change in molecular expression, and in the response of the cells to survival signals and mitogens. This is accompanied by architectural reorganization that involves the appearance of blood vessels and connective tissue in the nerve, and the appearance of basal laminae on the surface of individual Schwann cells. Distinct layers of perineurium also appear at the nerve surface (Figure 4). The mechanisms of cell survival also change with this transition. Schwann cell precursors are acutely dependent on ß-neuregulin-1 for survival (above) whereas immature Schwann cells acquire autocrine survival mechanisms. This enables Schwann cells to support their own survival in the absence of axons, a mechanism that is likely to be important for axonal regeneration after injury. The autocrine survival factors that have been identified include a cocktail of insulin growth factor (IGF)-2, platelet-derived growth factor (PDGF)-BB and neurotrophin (NT)-3, potentiated by laminin, lysophosphatidic acid (LPA) and leukemia inhibitory factor (LIF) and it has been shown that Schwann cells can express neuregulins after nerve injury, which may also contribute to their survival. This switch in survival strategy makes biological sense. Precursor survival is completely dependent on neuronally-derived ß-neuregulin-1, a property that may serve to match axon and Schwann cell precursor numbers and to keep precursors confined to developing nerves as they grow through body tissues to reach their targets (below). In contrast, the fact that Schwann cells can survive in the absence of axons ensures that if axonal injury occurs the surviving Schwann cells can provide essential factors to support axonal regrowth. Little is known about the transcription factors that control the precursor to Schwann cell transition. Levels of AP2a are high in precursors and drop sharply in immature Schwann cells and enforced expression of AP2a in vitro retards the transition. Endothelin also regulates the transition in a negative fashion, as mentioned above. As the cells transit from Schwann cell precursors to Schwann cells, developmental options narrow. Dedifferentiation of immature Schwann cells leading to the emergence of Schwann cell precursors has not so far been observed definitively, and Schwann cells in culture are resistant to signals such as BMPs and fibroblast growth factor 2 that can induce the generation of other neural crest derivatives from Schwann cell precursors.

Boundary cap Cells Give Rise to Schwann Cells in Spinal Roots Schwann cells in spinal roots have a different origin from the majority of Schwann cells in peripheral nerves. They are derived from boundary cap cells, a specialized set of cells that originate from the neural crest. These cells are found during embryonic development clustered in groups where dorsal and ventral roots enter and exit the spinal cord. They can be identified by the expression of the transcription factor KROX20 (EGR2) (and other distinctive genes) long before this gene appears in myelinating Schwann cells (below). Lineage tracing studies of the fate of these cells in vivo have revealed that they give rise not only to the Schwann cells of the dorsal and ventral roots but also to a small subset of nociceptive neurons and some satellite cells within the DRG. Few boundary cap cell-derived glia were detected in spinal nerves. Therefore these findings do not affect the classical view that the majority of Schwann cells of limb nerves originate from migrating neural crest cells.

Functions of Schwann Cell Precursors and Schwann Cells Trophic Support of Neuronal Survival A major function of glial cells is to provide trophic support for neurons both during development and in adult life. In the case of Schwann cell precursors and immature Schwann cells, persuasive support for this view comes from experiments in which these cells have been deleted from peripheral nerves. In mouse mutants that lack SOX10, ß-neuregulin-1 isoform III or the neuregulin receptors ErbB2 or ErbB3, Schwann cell precursors and Schwann cells are depleted or absent owing to the importance of these molecules in gliogenesis and glial survival (above). Importantly, in these mutants most of the DRG and motor neurons that project into limb nerves die by E14 and E18 respectively although they are initially generated in normal numbers. This suggests that an important function of precursors and immature Schwann cells is to provide essential survival signals for developing neurons. Impaired axon-target contacts may also contribute to sensory and motor death in the ß-neuregulin-1 isoform III mutants. Taken together with the evidence that axons provide essential ß-neuregulin-1-mediated survival support for Schwann cell precursors that was discussed earlier, these studies indicate that early in nerve development neurons and glia depend on each other for survival, a dependence that persists throughout life.

Morphogenesis: Maintaining the Normal Structure of the Spinal Cord, Nerve Trunks and Neuromasts An important morphogenetic role for the early glia of ventral roots has been revealed by studies using several mouse mutants, all of which have in common the absence of glial cells from nerve roots. In these mice the cell bodies of motor neurons are displaced into the ventral roots. Comparable observations have been made in the chick. This indicates that an important function of boundary cap

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cells, or the Schwann cell precursors that are derived from them, is to maintain the normal location of motor neurons within the spinal cord. Another morphogenetic function for immature Schwann cells and their precursors is that of holding peripheral nerve trunks together in a unified structure, since in mouse mutants that lack these cells, large nerves separate into a number of smaller bundles. Remarkably, however, even without accompanying glial cells the nerves initially grow out into limbs more or less correctly and find their way to their target areas. A morphogenetic function for neural crest derived glia is also seen in the lateral line nerve of zebrafish, where developing glial cells (immature Schwann cells or their precursors) control nerve fasciculation and the formation of secondary neuromasts, organs that are specialized to detect water movements.

The Generation of Immature Schwann Cells, Endoneurial Fibroblasts and Melanocytes Lineage tracing studies have revealed two unexpected roles for Schwann cell precursors. They show that the relatively small population of fibroblasts found within the nerve at birth (5-10% of total cells) arises from cells in the nerve that express desert hedgehog and p75 neurotrophin receptor (p75NTR) and are therefore presumably Schwann cell precursors. This accords with the observation that not only Schwann cells but also fibroblasts appear in the nerve at the precursor-Schwann cell transition. Earlier experiments demonstrated that PNS glia from rodents and bird nerves have the potential to generate melanocytes. Recent experiments have confirmed and expanded these earlier findings, showing that many melanocytes are generated from Schwann cell precursors that migrate away from developing nerves to skin. Direct lineage tracing reveals that neural crest and Schwann cell progenitor-derived melanocytes are differentially restricted to the epaxial and hypaxial body domains, respectively. The generation of melanocytes requires down-regulation of the transcription factor FoxD3, and cross-regulatory interactions between the transcription factors Sox2 and Mitf. An additional example of the unexpected developmental potential of early PNS glia was mentioned earlier. Boundary cap cells are tightly associated with axons and express glial-associated genes such as P0 and KROX20 and can therefore be regarded as having the phenotype of early glial cells. Nevertheless these cells give rise not only to glia but also to some DRG neurons during normal development. This is reminiscent of the observation that early CNS glia, namely radial glia, generate both astrocytic glial cells and neurons and is in accord with the emerging concept that early glia can act as multipotent progenitors in both the developing PNS and CNS.

The Transition from Immature Schwann Cells to Myelination In rodents, the transition from Schwann cell precursors to immature Schwann cells is essentially complete by E18, and at that time nerves have also acquired the basic tissue architecture known from postnatal nerves, since they now contain relatively large extracellular spaces, connective tissue, fibroblasts and blood vessels. At this stage the Schwann cells, which have started to assemble a basal lamina, still ensheath relatively large groups of axons communally (Figure 4). Myelination starts around birth. This requires radial sorting, a process of radical change in the configuration of Schwann cells and axons that allows the larger diameter axons that will become myelinated to acquire a 1:1 relationship with individual Schwann cells, a configuration that is a prerequisite for myelin formation. At the same time as radial sorting is taking place, Schwann cell numbers are adjusted to the number of axons by control of proliferation and survival. During this period a number of signaling systems probably act together to prevent premature myelination.

Radial Sorting This process, which allows individual Schwann cells to become associated with single large diameter axons, is rapidly being dissected at the molecular level, although many aspects remain unclear. Defects in radial sorting are seen in the absence of laminin isoforms, in the absence of the laminin receptor ß1 integrin, in the absence of focal adhesion kinase (FAK) and in downstream targets of these molecules such as cdc42, Rac1 and ILK. Radial sorting also fails in the clawpaw mutant mouse, which has a mutation in Lgi4, secreted by Schwann cells. It binds to a disintegrin and metalloprotease (ADAM)22 on axons. Lack or mutation in either Lgi4 or ADAM22 causes sorting defects and severe hypomyelination. In the absence of laminin, or the laminin receptor dystroglycan, Schwann cell proliferation is also impaired and apoptosis increased. Interestingly, these functions are less affected by the absence of ß1 integrin. Cell movement is a crucial component of radial sorting and it is possible that factors that control Schwann cell migration in culture could also affect radial sorting. They include the growth factors ß-neuregulin-1, IGFs, and NT-3, all of which promote migration, and brain derived neurotrophic factor (BDNF), which inhibits it. Levels of activation of the small GTPase Rac also regulate Schwann cell-axon interactions and in other cell types low levels of rac activation promote directionally persistent cell migration, whereas high levels promote random migration. There is also evidence that the p38 mitogen-activated protein kinase pathway is required just prior to myelination, perhaps for the correct alignment of axons and Schwann cells.

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Schwann Cell Development

Control of Schwann Cell Numbers During the process of sorting and myelination that occurs around birth it becomes important to match the numbers of Schwann cells and axons. By then, the period of neuronal cell death is largely over. This process is therefore controlled by a balance between rates of Schwann cell proliferation and death. Experiments using cultured cells suggest that axons are the major stimulators of Schwann cell proliferation, an idea supported by the observation that in vivo Schwann cell proliferation decreases as Schwann cells lose contact with axons in transected nerves of newborn animals. In co-cultures of Schwann cells and DRG neurons, ß-neuregulin-1 acts as a major axonal mitogen, although this has not yet been confirmed in vivo. Transforming growth factor (TGF) ßs are also potential mitogens that are present in developing nerves. Mouse Schwann cells that lack TGFß type II receptor proliferate more slowly in vivo than normal Schwann cells, demonstrating that TGFß is normally involved in regulating Schwann cell proliferation during nerve development. Schwann cell survival in developing nerves is likely to be controlled by a balance between factors that support survival and those that promote cell death. Axonally-derived ß-neuregulin-1 and autocrine circuits provide survival support (above), together with laminin acting through dystroglycan and integrin receptors. Two signals that promote cell death have been identified in vivo. TGFß is one of these. Deletion of TGFß type II receptors suppresses developmental death in E18 to newborn mouse nerves and cell death after newborn nerve transaction is also lower in these mutant nerves. Signals acting through the p75NTR, possibly nerve growth factor, are required for the cell death that occurs after neonatal nerve transection but not for normally occurring developmental death.

The Onset of Myelination Schwann cell myelination is a remarkable example of cell-cell interaction in which the association of an immature Schwann cell with a large diameter axon induces a radical change in morphological and molecular phenotype. This leads to the formation of the myelin sheath and reciprocal changes in axonal membrane proteins, ion channels and cytoskeleton that enable saltatory conduction along large nerves. The role of the basal lamina in promoting myelination is well-established and the molecular mechanisms involved are beginning to be revealed.

The Role of ß-Neuregulin-1 in Myelination ß-neuregulin-1 type III is one of the crucial axonal signals involved in controlling Schwann cell myelination. Mutant mice heterozygous for this isoform have thinner myelin sheaths, while over-expression results in thicker myelin sheaths and induces myelination of axons that would not normally be myelinated. ß-neuregulin-1 activates signaling pathways that positively regulate myelination, including the phosphatidyl inositol 3 kinase, ERK and protein kinase A pathways.

The Role of Cyclic AMP in Promoting Myelination Experiments in zebrafish and mice have revealed and important role for the G-protein coupled receptor (GPR)126 and protein kinase A in promoting Schwann cell myelination. Mice lacking GPR126 in Schwann cells fail to myelinate. The mechanism involves elevation of intracellular cyclic AMP levels, stimulation of protein kinase A activity and transcription factors of the CREB family, confirming long standing experiments in vitro that showed that elevation of intracellular cAMP levels in Schwann cells promoted a myelin-related phenotype.

Myelin-Related Transcription Factors The most important transcription factor involved in myelination is Krox20 (Egr2), which acts together with its partners, the NGF1A-binding proteins 1 and 2 (NAB1/2) to up-regulate a large number of myelin genes and proteins. Absence of either Krox20 or alternatively the combined absence of NAB1/2, both of which are upregulated by Krox20, results in Schwann cell arrest at the 1:1 pro-myelin stage and failure to myelinate, indicating the central importance of the Krox20/NAB1/2 complex for gene activation and myelination. In humans Krox20 (Egr2) mutations are associated with hereditary sensory and motor neuropathies. The octamer-binding transcription factor 6 (Oct6), and to a lesser extent brain 2 class III POU-domain protein (Brn2), control the timing of myelination. In mice that lack Oct6, myelination is severely delayed, probably because Oct6 is required for up-regulation of Krox20 at the appropriate time in development, whilst Brn2 can partially compensate for loss of Oct6. In vitro the transcription factor NFkB and the Sloan-Kettering Institute proto-oncogene (SKI) are expressed prior to myelination and in Schwann cell-neuron co-cultures lack of NFkB or SKI in Schwann cells results in failure to myelinate. Similarly transcription factors of the NFAT family are likely to be involved at least in the timing of myelination since deletion of the NFAT activator calcineurin B in neural crest cells results in delayed radial sorting and myelination.

Schwann Cell Development

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Negative Regulation of Myelination While the onset of myelination is characterized by activation of pro-myelin pathways, recent studies indicate that signaling pathways that inhibit myelination also play a role in the timing of myelination and in transdifferentiation of Schwann cells after injury. One of these inhibitory signals is the c-Jun amino (N)-terminal kinase (JNK) pathway, and in particular the transcription factor c-Jun, recently shown to play a crucial role in transdifferentiation of Schwann cells in regenerating nerves after injury. c-Jun is active in immature Schwann cells, where it is required for ß-neuregulin-1 and TGFß-induced signaling, at least in cultured cells. The pathway is inactivated as individual cells start to myelinate using a mechanism that depends in part on Krox20. In mice in which Krox20 has been inactivated, the JNK pathway is still active, proliferation remains high and the cells arrest at the pro-myelin stage of development. When expression of JNK pathway constituents, and in particular c-Jun is enforced in cultured Schwann cells, myelination in neuron-Schwann cell cultures is blocked and myelin gene expression that would normally result from pro-myelin signals such as Krox20 is also blocked. Nevertheless in mice with specific inactivation of c-Jun in Schwann cells myelination proceeds normally whereas nerve regeneration after nerve injury is severely compromised with failure of axon growth and widespread death of sensory neurons and facial motor neurons, indicating a crucial role for c-Jun in the generation of the repair Schwann cell phenotype. The transcription factor SOX2 also promotes proliferation, inhibits myelination when over-expressed in Schwann cells in neuron-Schwann cell co-cultures, remains high in mutant mice expressing low levels of Krox20 (Egr2) and is re-expressed in Schwann cells after nerve injury. Similarly, Notch signaling, which, as mentioned above, promotes proliferation in immature Schwann cells, is down-regulated as cells start to myelinate in vivo, while enforced expression of Notch in Schwann cells delays the onset of myelination in vivo, and removal of the Notch pathway transcription factor RBPJ accelerates it. This pathway is also active after nerve injury and mice lacking RBPJ in Schwann cells show delayed myelin breakdown. At present it is unclear how these diverse inhibitory pathways are interlinked and the details of how they interact with pro-myelin signals are just starting to be revealed.

Further Reading Adameyko I, Lallemend F, Aquino JB, Pereira JA, Topilko P, Mu¨ller T, Fritz N, Beljajeva A, Mochii M, Liste I, Usoskin D, Suter U, Birchmeier C, and Ernfors P (2009) Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin. Cell 139: 366–379. Arthur-Farraj PJ, Latouche M, Wilton DK, Quintes S, Chabrol E, Banerjee A, Woodhoo A, Jenkins B, Rahman M, Turmaine M, Wicher GK, Mitter R, Greensmith L, Behrens A, Raivich G, Mirsky R, and Jessen KR (2012) c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron 75: 633–647. Birchmeier C and Nave K-A (2008) Neuregulin-1, a key axonal signal that drives Schwann cell growth and differentiation. Glia 56: 1491–1497. Cornell RA and Eisen JS (2005) Notch in the pathway: the roles of Notch signaling in neural crest development. Seminars in Cell & Developmental Biology 16: 663–672. Dong Z, Brennan A, Liu N, et al. (1995) NDF is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors. Neuron 15: 585–596. Feltri ML and Wrabetz LJ (2005) Laminins and their receptors in Schwann cells and hereditary neuropathies. Journal of the Peripheral Nervous System 10: 128–143. Garratt AN, Britsch S, and Birchmeier C (2000) Neuregulin, a factor with many functions in the life of a Schwann cell. BioEssays 22: 987–996. Fleck D, Garratt AN, Haass C, and Willem M (2012) BACE1 dependent neuregulin processing: review. Current Alzheimer Research 9: 178–183. Guo L, Lee AA, Rizvi TA, Ratner N, and Kirschner LS (2013) The protein kinase A regulatory subunit R1A (Prkar1a) plays critical roles in peripheral nerve development. The Journal of Neuroscience 33: 17967–17975. Jaegle M, Ghazvini M, Mandemakers W, et al. (2003) The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development. Genes & Development 17: 1380–1391. Jessen KR and Mirsky R (2005) The origin and development of glial cells in peripheral nerves. Nature Reviews. Neuroscience 6: 671–682. Jessen KR and Mirsky R (2008) Negative regulation of myelination: relevance for development, injury, and demyelinating disease. Glia 56: 1552–1565. Jessen KR and Mirsky R (2013) The Schwann cell lineage: cellular transitions during development and after injury. In: Kettenmann H and Ransom B (eds.) Neuroglia, 3rd ed, pp. 159–171. Oxford, UK: Oxford University Press. Jessen KR, Mirsky R, and Lloyd AC (2014) Schwann cells: development and role in nerve repair. In: Barres B, Freeman M, and Stevens B (eds.) Glia, Cold Spring Harbor Perspectives in Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, In press. Joseph NM (2004) Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells. Development 131: 5599–5612. Kaucka´ M and Adameyko I (2014) Non-canonical functions of the peripheral nerve. Experimental Cell Research 321: 17–24. Kubu CJ, Orimoto K, Morrison SJ, et al. (2002) Developmental changes in Notch1 and numb expression mediated by local cell-cell interactions underlie progressively increasing delta sensitivity in neural crest stem cells. Developmental Biology 244: 199–214. Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, and Yan R (2006) Bace1 modulates myelination in the central and peripheral nervous system. Nature Neuroscience 9: 1520–1525. La Marca R, Cerri F, Horiuchi K, Bachi A, Feltri ML, Wrabetz L, Blobel CP, Quattrini A, Salzer JL, and Taveggia C (2011) TACE (ADAM17) inhibits Schwann cell myelination. Nature Neuroscience 14: 857–865. Mogha A, Benesh AE, Patra C, Engel FB, Scho¨neberg T, Liebscher I, and Monk KR (2013) Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation. The Journal of Neuroscience 33: 17976–17985. Monk KR, Naylor SG, Glenn TD, Mercurio S, Perlin JR, Dominguez C, Moens CB, and Talbot WS (2009) A G protein-coupled receptor is essential for Schwann cells to initiate myelination. Science 325: 1402–1405. Nagarajan R, Svaren J, Le N, et al. (2001) EGR2 mutations in inherited neuropathies dominant-negatively inhibit myelin gene expression. Neuron 30: 355–368. Le N, Nagarajan R, Wang JY, et al. (2005) Analysis of congenital hypomyelinating Egr2Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination. Proceedings of the National Academy of Sciences of the United States of America 102: 2596–2601. Lobsiger CS, Taylor V, and Suter U (2002) The early life of a Schwann cell. Biological Chemistry 383: 245–253. Maro GS, Vermeren M, Voiculescu O, et al. (2004) Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nature Neuroscience 7: 930–938. Michailov GV, Sereda MW, Brinkmann BG, et al. (2004) Axonal neuregulin-1 regulates myelin sheath thickness. Science 304: 688–689. Mirsky R and Jessen KR (2005) Molecular signaling in Schwann cell development. In: Dyck PJ and Thomas PK (eds.) Peripheral neuropathy, 4th ed, pp. 341–376. Philadelphia, USA: Elsevier Saunders.

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