Regulation of genes involved in Schwann cell development and differentiation

B. Cast&ma

L6pez and M. Nieto-Sampedro (Eds.)

Prqress in Brain Research,Vol. 132 0 2001 Elsevier Science B.V. AU rights reserved

CHARTER 1

Regulation of genes involved in Schwann cell development and differentiation R. M irslq ‘3*, D.B. Parkinson ‘, Z. Dong 2, C. Meier 3, E. Calle ‘, A. Brennan ‘, P. Topilko4, B.S. Harris ‘, H.J.S. Stewart5 and K.R. Jessen ’ ’ Department of Anatomy and Developmental Biology, University College London, Gower Street, WClE 6BT London, UK 2 Reneuron Ltd, London, UK ’ Ruhr-Universitaet, Bochum, Germany 4 Ecole Normal Superieure, Paris, France 5 Sussex University, Brighton UK

The Schwann cell lineage and its origin in the neural crest Most Schwann cells develop from the neural crest (Le Douarin et al., 1991). They exist in mature nerves in two distinctly different forms, non-myelinating and myelinating Schwann cells, which are present in comparable numbers throughout the peripheral nervous system (PNS). The two main intermediate cells involved in the generation of these cells from neural crest cells are the Schwann cell precursor, typically found in rat nerves at embryo day (E) 14 and 15 (mouse El2 and 13), and the immature Schwann cell, present from El7 (mouse E15) to around birth. Subsequently the immature Schwann cells start to differentiate, first along the myelin pathway, with mature non-myelinating cells appearing later. The lineage therefore involves three main transition points, i.e. the transition of crest cells to precursors, of precursors to immature Schwann cells and lastly the final, and largely reversible, formation

*Corresponding author: Rhona Mirsky, Department of Anatomy and Developmental Biology, University College London, Gower Street, WCIE 6BT London, UK. Tel.: +44-20-7679-3380; Fax: +44-17-1380-7349; E-mail: [email protected]

of the two mature Schwann cell types (Mirsky and Jessen, 1996; Jessen and Mirsky, 1997, 1998). It is still not clear how the neural crest, a group of cells that also gives rise to other lineages, including neurons and melanocytes, gives rise to cells of the glial lineage. At least in birds it appears that many crest cells have entered distinct lineages at the onset of crest migration (Henion and Weston, 1997), and it is likely that instructive signals play an important role in regulating the appearance of the different cell types. For example, in rat neural crest cells cultured under clonal conditions, application of i3-neuregulins biases crest differentiation towards glia by blocking entry to the neuronal lineage (Shah et al., 1994). Using comparable conditions, transforming growth factor B (TGFB) promotes the generation of smooth muscle cells while bone morphogenetic protein 2 (BMP2) stimulates entry to the autonomic neuronal lineage (Shah et al., 1996). These experiments indicate that b-neuregulin may act instructively to promote glial differentiation, while other growth factors bias the crest cells towards other lineages. They remain, however, to be reconciled with evidence obtained from mice lacking ErbB3 protein, a major receptor for b-neuregulin in crest cells and early glia (see below) (Meyer and Birchmeier, 1995; Riethmacher et al., 1997). These studies suggest that at least in the cephalic neural crest, h-neuregulin is re-

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quired for neurogenesisin vivo and show that in the ErbB33- m ice, the number of dorsal root ganglion (DRG) neurons is initially normal, rather than excessive, as would be expected if h-neuregulinswere acting instructively to promote glial differentiation in vivo. Previously, the lack of early glial differentiation markers made studies on early glial developmentdifficult, although the SMP protein proved useful in the chick/quail system (Cameron-Curry et al., 1993). It turns out, however, that in rat and chick, some m igrating crest cells express the gene for the major peripheral myelin protein Pa (Bhattacharyya et al., 1991; Zhang et al., 1995; Lee et al., 1997). In normal adult rat nerves, Pa gene expression is restricted to myelinating Schwann cells, although during development, it is expressedat much lower basal levels in immature Schwann cells (Cheng and Mudge, 1996; Lee et al., 1997). The striking axonally induced increase in POsynthesis in myelinating Schwann cells is therefore an up-regulation of pre-existing basal levels rather than novel gene expression. This is m irrored by axonally driven suppressionof basal Pa levels in mature non-myelinating Schwanncells (Lee et al., 1997). There is unambiguousPa expressionin Schwann cell precursors and, significantly, in a subpopulation of late m igrating crest cells in the trunk region of rats from El1 onwards (Bhattacharyya et al., 1991; Lee et al., 1997). Clusters of Pa-positive crest cells appear to be preferentially located near b3-tubulin-positive axons which are projecting from the neural tube and at sites of condensing DRGs.

Since there are no published reports of Pa expression in cells other than Schwann cells or satellite cells, it is likely, although not proven, that POexpression in crest cells marks those cells that have just entered the glial lineage, although they, and cells which have progressed even further along the lineage in vivo, may still retain the potential to give rise to other lineages when placed in an appropriateenvironment (Hagedom et al., 1999; Morrison et al., 1999). These authors have an alternative explanation of their data, suggesting that Pa expression occurs in multipotent neural crest cells before entry into the glial lineage. Schwann cell precursors and the generation of Schwann cells Schwann cell precursors are present in peripheral nerves of rat embryos at El4/15 (mouse El2/13) (Jessenand M irsky, 1997). Since these cells express low, basal Pa levels, we can trace the lineage from its origin in the neural crest to immature Schwann cells in perinatal nerves. Some of the distinctive phenotypic properties of the Schwann cell precursors are listed in Table 1. While almost all the cells in El4 and 15 rat nerves are precursors,by El7 (mouse El5), nearly all the cells are Schwann cells. The generation of Schwann cells from precursors therefore takes place over a relatively short time span, although the differences between these two cells extend to a number of diverse and apparentlyunrelated phenotypic features. At present, there is no clear evidence for a precursor population in mature nerves.

TABLE 1 Some of the main differences between Schwann cell precursors and Schwann cells in the sciatic nerve of rat and mouse Precursors

Schwann cells

Die by apoptosis when removed from axons and plated in vitro; no autocrine loops SlOO (cytoplasmic)- a Do not divide in response to FGFsb Flattened with extensive cell-cell contacts in vitro occ Motility+++

Full survival under some conditions; due to autocrine loops S 100 (cytoplasmic)+ Divide in response to FGFs Bi- or tri-polar in vitro 04+ c Motility+

aPrecursors are SlOO-negative (and Schwann cells are SlOO-positive) when observed with our routine immunolabeling methods. Low levels of SlOO immunoreactivity are detectable, however, in many precursors in the mouse, when the sensitivity of the immunolabeling method is increased. bThis applies to rat only. In the mouse, FGF is a mitogen under identical conditions. ‘This has only been tested thoroughly in the mouse.

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A remarkable feature of the Schwann cell precursor is its acute dependenceon axonal survival signals. There is now extensive evidence, obtained first in vitro (Jessenet al., 1994; Dong et al., 1995) and subsequently from gene knockout animals (Meyer and Birchmeier, 1995; Riethmacher et al., 1997, Morris et al., 1999), that the axonal signal that regulates precursor survival is p-neuregulin, signaling through ErbB3 and ErbB2 receptors (reviewed in Jessenand M irsky, 1997; M irsky and Jessen, 1999). fi-Neuregulin also supportsthe conversionof isolated Schwann cell precursors to Schwann cells in vitro with a time course that is similar to that of Schwann cell generation from precursors in vivo (Dong et al., 1995) and acts as an axon-associatedm itogen and survival factor for perinatal Schwann cells (Morrissey et al., 1995; Grinspan et al., 1996; Syroid et al., 1996; Trachtenberg and Thompson, 1996). It is therefore a crucial regulator of the early Schwann cell lineage. Recent experiments using the Cre-lox system to conditionally knockout ErbB2 receptors in Schwann cells, further suggestthat neuregulins may be involved in some unknown way in the early stages of myelination, since these m ice have hypomyelinated nerves (Garratt et al., 2000). The postnatal formation of myelinating and non-myelinating cells Early in rodent postnatal life, immature Schwann cells diverge, generating the myelinating cells that ensheath the large diameter axons and the nonmyelinating cells that accommodatesmall diameter axons in invaginations in their surface membranes. All evidence suggeststhat this process is driven by axonal signals, although the molecules involved in this aspectof axon-Schwann cell communication are still unknown. While biochemical and morphological changesoccur in both types of Schwann cell differentiation, they are much more extensive in myelinating cells (Jessen and M irsky, 1992). These cells carry out a large amount of membranesynthesis and wrapping neededto form the myelin sheath,and undergo extensive changes in gene expression including up-regulation of one set of proteins, the myelin proteins including PO, myelin basic protein (MBP) and PMP22, followed by down-regulation of another set of proteins, exemplified by neural cell adhesion

molecule (N-CAM), p75NGF (nerve growth factor) receptor and glial fibrillary acidic protein (GFAP), that are expressedby immature Schwann cells and mature non-myelinating cells (Jessen et al., 1990). Remarkably, these axon-inducedchangesare largely reversible. If mature Schwanncells lose contact with axons, either following nerve transection or on dissociation and culture, they promptly undergo radical changesin morphology and gene expressionleading to developmentalregression of individual Schwann cells and myelin breakdown. These processes are accompaniedby Schwanncell proliferation (Fawcett and Keynes, 1990).The eventualoutcome is the generation of a single population of cells that are comparable, although not identical, to immature Schwann cells in neonatal nerves (Gould et al., 1992; Jessen and M irsky, 1992). The relatively high expression of neurotrophic factors and cell adhesion molecules by these cells provides an environment conducive to axonal re-growth (Scherer and Salzer, 1996). Thus, the dramatic regression response of Schwann cells to loss of axon contact in damagednerves, together with the autocrine mechanismsthat allow Schwann cells to survive in the absenceof axonal contact (see below) forms the basis for nerve regeneration and repair in the PNS (Jessenand M irsky, 1999). Transcription factors in Schwann cell differentiation Three transcription factors are known to have dramatic effects on Schwann cell development,including myelination. These are Sox-10 a member of the SRY-like high mobility group (HMG) domain class of transcription factors (Topilko et al., 1997; Kuhlbrodt et al., 1998a;Southard-Smithet al., 1998), the POU domain factor Ott-6 (SCIP, tst-1) and the zinc finger protein Krox-20. Creation of transgenic m ice null for Ott-6 or Krox-20, results in a delay (Ott-6) or arrest (Krox-20) of Schwann cell myelination (Topilko et al., 1994; Bermingham et al., 1996; Jaegle et al., 1996). The myelination arrest in Ott-6-/- m ice is only temporary, suggestingthat there may be other POU domain factors in Schwann cells that can replace Ott-6 during myelination (Jaegle and Meijer, 1998). Inactivation of Ott-6 or Krox-20 in m ice blocks Schwann cell development at the pro-myelination

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stage. At this point, Schwann cells due to myelinate envelop large diameter axons, making up to one and a half turns round the axon, but fail to form concentric compactedmyelin rings. The strong myelination-associatedup-regulation of the proteins POand MBP does not take place, although mRNA levels are reportedto be normal (Topilko et al., 1994; Bermingham et al., 1996; Jaegle et al., 1996). There is some evidencethat a number of other unrelatedways of interfering with Schwanncell developmentalso blocks myelination at a similar stage. Pro-myelin arrest is, for instance, seen in neuron-Schwann cell co-cultures where myelination is prevented by using antibodies to galactocerebroside(Ranscht et al., 1987). Similarly, m ice in which Pa protein is m ildly overexpressedor knocked out, show reduced myelination with many cells appearingto stall at the pro-myelin stage (Martini and Carenini, 1998; Wrabetz et al., 2000; Yin et al., 2000). Nevertheless,the identification of mutations in Egr-1 (Krox-20) in two patients with Charcot-Marie-Tooth disease points to a direct role for this transcription factor in myelination (Warner et al., 1998, 1999; Timmerman et al., 1999). Recent evidence suggests that Sox-10 is important not only at early stages of differentiation of the neural crest, but also at later stagesin Schwann cell development.It is expressedin neural crest cells, in cells of the Schwann cell lineage and in oligodendrocytes (Kuhlbrodt et al., 1998a,b; Southard-Smith et al., 1998). It activates expression of the PO gene in peripheral glial cells both in vivo and in culture, and the promoter contains multiple binding sites for Sox proteins (Peirano et al., 2000). Furthermore, a human patient with a mutation in Sox-10, not only had Waardenburg-Hirschsprung syndrome, the normal consequenceof disruption of the Sox-10 gene in human heterozygotes,but also had peripheral and central dysmyelinating neuropathy. This is likely to be becausethis particular mutation in Sox-10 protein causesan extension of the normal protein, leading it to act in a dominant-negativefashion to disrupt both central and peripheral myelination programmes (Inoue et al., 1999). Other transcription factors that may be important in this system include the zinc finger protein Krox-24 (Topilko et al., 1997; Kuhlbrodt et al., 1998b; Southard-Smithet al., 1998). This factor is expressedstrongly at early stagesof development of the Schwann cell lineage, and is strongly up-reg-

ulated after nerve transection or crush. Our recent results, comparing axonal regeneration after nerve crush in normal and Krox-24-/- m ice, suggest that lack of IQ-ox-24 has little effect on the speed of axonal regeneration,but other parametersremain to be explored. It will also be interesting to see whether the ongoing search for cell type specific B class basic helix-loop-helix (bHLH) factors in Schwanncells proves successful, given the recent identification of B-class HLH proteins in the oligodendrocytelineage (Lu et al., 2000; Zhou et al., 2000). The promoters of the myelin genes POand MBP, and the p75NGF receptor all contain the activating E box sequence to which bHLH dimers bind. This, together with the fact that Schwann cell precursors and Schwanncells have recently been shown to express two groups of proteins, A class bHLH proteins and Id factors, thought to regulate specific B bHLH activity (Stewart et al., 1997), makes it likely that important B class geneswill be identified in Schwanncells. Recent studies in our laboratory indicate that transcription factors of the Ets family are involved in Schwann cell survival responses, while activation of c-jun promotes cell death (see below). A more comprehensivelist of transcription factors found in the Schwann cell lineage and a discussion of their possible functions can be found in Stewart et al. (1996). Survival and death pathways in Schwann cell precursors and Schwann cells We and others have provided strong evidence that the survival of Schwanncell precursorsis acutely dependenton fl-neuregulin derived from axons (above). The survival of Schwanncells, however, is regulated in a different way. In the distal stump of transected nerves, Schwann cells survive for several months in the absenceof axons, although their number gradually declines and they lose some of their responsiveness to extrinsic signals (Grinspan et al., 1996; Trachtenbergand Thompson, 1996; Li et al., 1998). Likewise, neonatalSchwann cells survive well without neurons in vitro when plated at moderatedensity in serum-containingor serum-free medium. In contrast, Schwann cell precursors die rapidly in vitro, even when plated at very high density (Jessen et al., 1994). These observations show that there is a

7 change in survival regulation during Schwann cell development. While the survival of Schwann cell precursors depends on axonal signals, Schwann cells can survive in the absenceof axons. This ability to survive in the absence of axons is critical in the context of nerve regeneration. Schwann cells are left without axons in the nerve segment distal to an injury. For successful repair, the axons must enter the distal stump and regrow into their target tissues. This axon re-growth depends on the presence of living Schwann cells in the distal nerve segment, probably because the axons require interactions with Schwann cell associated adhesion molecules and trophic factors (Fawcett and Keynes, 1990; Nadim et al., 1990). Nerve regeneration, therefore, depends on the mechanism that allows Schwann cells to survive in the absenceof axons. We have recently shown that Schwann cells acquire the ability to survive without axons by establishing an autocrine survival loop (Meier et al., 1999). We have demonstrated that these survival loops exist in Schwann cells from El8 and postnatal nerves, but do not exist in Schwann cell precursors. Others have shown that if nerves of newborn rats are transected, considerable cell death results in the distal stump, indicating that axons still provide detectable survival input mediated by l%neuregulinsto the Schwann cell population, while this has become insignificant 1-2 weeks later (Grinspan et al., 1996; Syroid et al., 1996; Trachtenberg and Thompson, 1996). This indicates that the switch from axondependent survival to an axon-independent autocrine survival regulation occurs gradually as Schwann cells develop from precursors and mature in early postnatal nerves. We have investigated the molecular identity of the Schwann cell derived survival signal. It is not mitogenic for Schwann cells and this observation, plus the fact that it does not promote precursor survival, render it unlikely that the Schwann cell signal is simply b-neuregulin, although it does not exclude the possibility that very low levels of b-neuregulin might be a component of the signal. Our experiments indicate that the autocrine survival activity resides in a cocktail of growth factors - including insulinlike growth factor 2 (IGF-2), platelet-derived growth factor-BB (PDGF-BB) and neurotrophin-3 (NT3) that acts synergistically to block Schwann cell death

(Meier et al., 1999). In addition to the autocrine function of this signal, the secretion of these factors by Schwann cells could have more general significance since they are all known to regulate survival and differentiation of other cells, including neurons. It has also been reported that leukemia inhibitory factor (LIF) can provide survival signals to neonatal Schwann cells in the presence of other growth factors (Dowsing et al., 1999). To investigate the intracellular pathways used for neuregulin- and autocrine-induced survival, we used retroviral infection to express DNA binding domains of two members of the Ets transcription factor family, Ets- 1 and PU- I, in cultured neonatal Schwann cells. A member of the Ets family, GABPcl, regulates B-neuregulin-induced transcription of acetylcholine receptor 6 subunit at the neuromuscular junction (Fromm and Burden, 1998) so we reasoned by analogy that members of this family might be involved in NRG signaling in Schwann cells. Another member of the family, Erm, has recently been reported to distinguish satellite cells in DRG from Schwann cells, and is also regulated by fi-neuregulin (Hagedorn et al., 2000). The DNA binding domains function as truns-acting dominant-negative molecules by preventing binding of the same or highly related Ets transcription family members to their cellular target genes. In other cell systems, Ets factors activate genes involved in growth control, transformation and development, and we found that expression of dominant-negative Ets- 1 prevented Schwann cell survival in response to both l$neuregulin and the IGF-2, PDGF, NT3 autocrine cocktail, but did not affect survival signals activated by lysophosphatidic acid. Dominant-negative PU-1 had little effect in any of the three systems tested. Using RT-PCR, we identified several Ets family members present in mouse nerves at E12.5 and postnatal day zero. These had much stronger homology to Ets- 1 than PU-1, confirming the functional significance of the data obtained using the dominant-negative molecules, and included GABPcl and NEts, both of which were localized to nuclei of cultured Schwann cells (Parkinson et al., 1999; Parkinson, Langner, Jessen and Mirsky, unpublished observations). In addition to positive survival factors, factors that actively cause apoptosis may also play a role in Schwann cell death after injury or infection. In the

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nervous system, nerve growth factor (NGF), acting on the p75-neurotrophin receptor, may function in this way both in retinal developmentand in Schwann cells (Frade et al., 1996; Frade and Barde, 1999; Soilu-Hanninen et al., 1999). We have found that TGFbs have similar effects on neonatal Schwann cells both in vivo and in vitro, causing apoptosis under a variety of conditions. The apoptotic effect is blocked by the combined presence of p-neuregulins and autocrine signals, and application of TGFfi kills Schwann cells in the distal stump of cut neonatal nerves, but not in normal nerves. TGFI3 induces apoptosis by activating c-jun and API dependent transcription in Schwann cells, while infection with a dominant-negative form of c-jun, v-jun, is sufficient to cause apoptosis in the presence of applied TGFb. As Schwann cells mature, they no longer undergo apoptosis in response to TGFb, and this is correlated with a failure of the applied TGFb to induce high levels of phosphorylation of c-jun on serine-63. Application of caspase inhibitors blocks the TGFB-induced cell death, but not cell death induced by deprivation of l3-neuregulinor autocrine factors, indicating that these are downstream targets of the TGF@-inducedc-jun response(Parkinson, Dong, Bunting, Meier, Marie, Mirsky and Jessen, under revision). TGFfis have a variety of other effects on Schwann cells including inhibitory effects on myelination, proliferative and phenotypic effects (for reviews see Mirsky and Jessen, 1996; Scherer and Salzer, 1996). Both Schwann cells and neurons express TGFb isoforms, so TGFB could potentially act in an autocrine or paracrine fashion either during normal development or alternatively in adult nerves in responseto inflammation or injury. Conclusions The work reviewed here shows that Schwann cells and their precursors respond to both neuronal and autocrine signals during development and in adult life. Other evidence suggests that they in turn act as a source of diverse developmental signals that influence both neurons and the mesenchymal cells that form the perineurial sheath (Jessenand Mirsky, 1999; Parmantier et al., 1999). These signals influence both differentiation and survival in early nerves. The transcription factors Ott-6, Krox-20 and

Sox-10 are crucial to peripheral nerve development and Schwann cell myelination. During early development of the Schwann cell lineage, Schwann cell precursors depend strictly on a paracrine mode of survival support from neurons, while Schwann cells respond to both paracrine and autocrine survival signals. In both precursors and Schwann cells, the paracrine signal is fl-neuregulin, while the principal components of the autocrine survival signal are IGF-2, PDGF-BB and NT3, acting in conjunction with laminin. In addition, TGFbs can promote active Schwann cell death in culture and in transected neonatalnerves. Transcription factors of the Ets family mediate Schwann cell survival in response to b-neuregulins and autocrine growth signals (IGF-2, PDGF-BB and NT3), while active Schwann cell death in response to TGFbs is mediated through phosphorylation of the transcription factor c-jun. Abbreviations bHLH BMP2 DRG E GFAP HMG IGF-2 LIF MBP N-CAM NGF NT3 p75NGF PDGF-BB PNS TGFb

basic helix loop helix bone morphogenic protein 2 dorsal root ganglion embryonic day glial fibrillary acidic protein high mobility group insulin-like growth factor 2 leukemia inhibitory factor myelin basic protein neural cell adhesionmolecule nerve growth factor neurotrophin-3 P75 nerve growth factor receptor platelet derived growth factor-BB peripheral nervous system transforming growth factor b

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