GLIA 41185-194 (1991)

Schwann Cell Precursors and Their Development KRISTJAN R. JESSEN AND RHONA MIRSKY Department of Anatomy and Developmental Biology, University College London, London WClE 6BT, U.K.

KEY WORDS

Nerve development, Glial precursors, Myelination, Cyclic AMP

ABSTRACT

During development of peripheral nerves, an apparently homogeneous pool of embryonic Schwann cells gives rise to two morphologically and antigenically distinct mature Schwann cell types. These are the myelin-forming cells associated with axons of larger diameter and the non-myelin-forming cells associated with axons of smaller diameter. The development of these cells from precursors that can be identified in early embryonic nerves can be followed with the help of antigenic differentiation markers. This development depends on Schwann cells retaining a close association with axons. The effect of axons can be mimicked in vitro by agents that elevate CAMPlevels. This has given rise to the idea that the effects of axon-associated signals in Schwann cell development are to a significant extent mediated via elevation in Schwann cell CAMPlevels. In vitro, the CAMP induced progression of cells from a premyelination state to a myelination state depends on withdrawal from the cell cycle. It is therefore possible that in vivo, the timing of myelin formation by individual Schwann cells is determined by signals that suppress proliferation.

THE MORPHOLOGY OF NERVE DEVELOPMENT By embryo day (E) 14-15 major peripheral nerves in the rat, such as the sciatic nerve, can easily be identified as discrete anatomical structures. These nerves consist of tight bundles of axons accompanied by rapidly proliferating cells that lack basal lamina and give rise to flattened sheet-like processes. As we will discuss below, these cells have been identified as Schwann cell precursors on the basis of their antigenic properties and survival ability in vitro. In morphological terms the Schwann cell precursors and their relationship to axons have not yet been studied in detail. Ongoing proliferation gives rise to Schwann cells that start dividing the nerve into separate axon-Schwann cell units or “families,”each of which consists of a large number of axons communally enveloped by one or two Schwann cells (Gamble and Breathnach, 1965; ZiskindConhaim, 1988; Webster and Favilla, 1984). The number of axons per family falls steadily because of Schwann cell proliferation and axon death. Simultaneously, the larger axons of each family start segregat01991 Wiley-Liss, Inc

ing from their fellows and come to lie isolated in the Schwann cell cytoplasm. At this stage connective tissue spaces have developed throughout the nerve and the Schwann cells, while still ensheathing axons communually, assemble their own basal lamina (Gamble and Breathnach, 1965; Webster et al., 1973). This step is an obligatory event in Schwann cell development, since further maturation, in particular myelination, is blocked if basal lamina formation is experimentally disrupted (Bunge et al., 1986; Eldridge et al., 1987). Cell division and segregation of axons eventually leads to the attainment of a 1:l ratio between Schwann cells and the larger axons. This event is followed by cessation of cell division, which, in turn, appears to be a key event in the induction of myelination (see below). In the rat sciatic nerve, Schwann cells in a 1:l ratio with axons are common at birth and the first myelin wraps form during the first 24 h of life. In this nerve, the first mature non-myelin-forming Schwann cells appear during the Address reprint requests to K.R. Jessen, De artment o f h a t o m y and Developmental Biology, University College London, &owe, Street, London WClE 6BT, U.K.

186

JESSEN AND MIRSKY TABLE 1. The molecular phenotype of Schwann cells

Anticren" (1) CNPase Po MBP MAG P170k

PLP p2

(2) GFAP NGF receptor N-CAM L-1 A5E3 Ran-2 (3) Sl00 Vimentin Laminin The lipid antigens 04, 08, 09 Galactocerebroside Seminohid

Myelin-forming Schwann cells

Non-myelin-forming Schwann cells

+ + + + + + +b ~

+c

-

+ + + + + +

nd ~

-

nd nd -

+

+ + + + + + + + + + +

Short-term cultured cells without neurons

Precursors nd -

nd nd nd -

+

nd

+ + + -

+ +-

"The antigens in section (1) are expressed mainly by myelin-forming cells, those in section (2) by non-myelin-forming cells and those in section (3)by both. For references, see text. nd, not determined. bPresent on some myelin-forming Schwann cells only. CPresentmainly a t nodes of Ranvier, very little elsewhere.

third postnatal week (Diner, 1965). Their development is largely complete 2 to 3 weeks later, which coincides with cessation of significant levels of Schwann cell proliferation (Friede and Samorajski 1968;Jessen et al., 1985). Thus, the development of Schwann cells, like that of cells in many other systems, is characterized by an embryonic and neonatal phase of rapid proliferation, followed by cessation of division and final differentiation. Although it has been established that Schwann cell development is to a very large extent regulated by axon-derived signals, understanding of the molecules and signalling mechanisms involved in this process is still limited. The final outcome is the generation of two strikingly dissimilar Schwann cells, the myelin-forming ones which wrap around essentially all axons with a diameter larger than 1 pm (Friede, 1972) and the non-myelin-forming cells which are only about one tenth of the length of the myelin-forming cells and which accommodate axons in troughs along their surface. MYELIN- AND NON-MYELIN-FORMING CELLS CAN BE IDENTIFIED BY SEPARATE SETS OF MOLECULAR MARKERS IN VIVO Before further discussion of the Schwann cell precursor and its developmental fate, it is necessary to establish how Schwann cells can be described and distinguished using antigenic markers. The morphological difference between the two Schwann cell types is reflected in their molecular makeup (Table 1). Only the myelin-formingcells express a series of myelin proteins including Po, myelin basic protein (MBP), myelin associated glycoprotein (MAG), and P2 (group 1 in Table 1) (Hahn et al., 1987; Martini and Schachner, 1986; Mir-

sky et al., 1980; Shuman et al., 1986; Trapp et al., 1981, 1984; Trapp and Quarles, 1984). The nonmyelin-forming cells express another set of proteins exemplified by the intermediate filament protein GFAP and NGF receptors (group 2 in Table 1)(Ferrari et al., 1990; Jessen and Mirsky, 1984; Jessen et al., 1984, 1987a, 1990; Mirsky and Jessen, 1984; Mirsky et al., 1986; Reiger et al., 1986; Yen and Fields, 1981). It is noteworthy that, with the exception of NGF receptors, the proteins of non-myelin-formingSchwann cells are also expressed by other major groups of nonmyelin-forming glia, namely, astrocytes in the CNS and enteric glial cells of the myenteric ganglia (Mirsky and Jessen, 1990). None of these proteins are expressed at significant levels in cells that make myelin. The role of the myelin proteins, both within the myelin sheath and in interactions between Schwann cells and axons, is rapidly becoming clearer (Hudson, 1990). Less is known about the function of the protein markers of non-myelin-forming cells. N-CAM and L1 are probably involved in axon-Schwann cell adhesion (Seilheimer et al., 1989),but the function of GFAP and the surface proteins A5E3 and Ran-2 is obscure. The presence of NGF receptors on non-myelin-forming Schwann cells is particularly intriguing. In cultured neonatal Schwann cells most, or all, of these receptors are of the low affinity type, whereas biological actions of NGF are generally ascribed to interaction with high affinity receptors (Johnson et al., 1988). Both Schwann cell types express certain restricted molecules in common (group 3, Table 1)(Brockeset al., 1979; Eccleston et al., 1987; Jessen and Mirsky, 1985; Jessenet al., 1985,1990; Mataet al., 1990;Mirskyet al., 1990; Stefansson et al., 1982). The most surprising of these are the glycolipids galactocerebroside and the 0 4 antigen, which is probably sulphatide, since in the CNS

S C H W A " CELL DEVELOPMENT

both of these lipids are restricted to cells of the myelin lineage. Another common molecule, the Ca++-binding protein S100,is an important Schwann cell marker. It is the only Schwann cell antigen that can be used as a general Schwann cell marker in situ and in culture, since both myelin- and non-myelin-forming cells express it, it is not present in other cell types in the nerve, and its expression, once triggered, is relatively stable so that denervated cells, both in vivo and in neuron-free culture, remain SlOO positive. No other known Schwann cell antigen fulfills these criteria. SCHWANN CELL PRECURSORS We have found that the glial cells present in E14-15 rat sciatic nerve lack two fundamental properties of cells from older nerves, i.e., the ability to survive in vitro in routine serum-containing media, and expression of the Schwann cell marker SlOO (Jessen, Gavrilovic, and Mirsky, unpublished). In culture the cells can be rescued by factor(s) present in certain conditioned media (from 3T3 fibroblasts, but not from the glial line 33B). These cells have a characteristic flattened morphology in vitro and already express most of the same proteins that characterize non-myelin-forming Schwann cells in adult nerves (Table 1).The main exception is GFAP which appears later in Schwann cell development (following section). In culture these cells can be distinguished from fibroblastic cells by antibodies against the NGF receptor 1192-IgGor 217c(Ran-l)l, which has previously been established as a marker for cultured Schwann cells (Ferrari et al., 1990;Fields and Dammerman, 1985; Johnson et al., 1988). These growth factor dependent, SlOO negative cells of E14-15 nerves are precursors to the Schwann cells that appear in subsequent development, namely, SlOO positive cells that are viable in routine culture media without added growth factors. These criteria for distinguishing between rat Schwann cells and their precursors can in part be traced to the work of Holton and Weston (19821,who distinguished between quail Schwann cells and precursors by the use of S100. In the rat sciatic nerve, SlOO positive Schwann cells first appear at E15-16 and their development will be delineated in the following section. At present we do not know whether proliferation of Schwann cell precursors can be driven by the same mitogens that are active for neonatal cells, or what survival factors they require. It will also be of great interest to learn how Schwann cells are generated from precursors. Two observations indicate that the two steps involved, i.e., acquisition of survival independence and of S100, are regulated independently. First, dissociation of El6 nerves yields some NGF receptor positive cells with the morphology of Schwann cell precursors that are viable but lack S100, in addition to SlOO positive viable Schwann cells. This suggests that growth factor independence is acquired fi% and that SlOO appears later. Second, the NGF receptor positive,

187

SlOO negative cells do not complete their transit from precusors to Schwann cells in these cultures but remain SlOO negative, at least for a period of several days. There is evidence from studies on the quail suggesting that the missing signal necessary for progression to SlOO positive Schwann cells is provided by neurons (Holton and Weston, 1982) and similar observations have been made on the rat sciatic nerve precursors (Jessen, Morgan, and Mirsky, unpublished) and on rat neural crest cultures (Smith-Thomas and Fawcett, 1989; Smith-Thomas et al., 1990). If this is confirmed, the effect of the neuronal signal in question is unusual in being irreversible, since SlOO expression persists in Schwann cells removed from axonal contact, although such cells sometimes contain lower levels of the protein. In contrast, the phenotypic changes induced by neuronSchwann cell signals in subsequent Schwann cell development are rapidly reversible, as we will see in the following section. Even less is known about the earlier step in precursor development, i.e., the acquisition of survival factor independence. One possibility is that Schwann cells, but not precursors, are autocrine, and therefore self-sufficient with respect to the factor(s) in question. Alternatively, cell maturation may render the factor(s) redundant. However this may be, the apparent requirement of E14-15 precursors for extrinsic growth factor(s1raises another interesting question: which cell provides this support during normal nerve development? The outgrowing axons are an attractive candidate, since they are the most prominent cellular element present in the immediate neighbourhood of the precursor cells (see the first section). Our preliminary observations indicate that neurons would be capable of the task, since media conditioned by purified neurons, and co-culturing with neurons, both effectively rescue Schwann cell precursors in vitro. THE DEVELOPMENT OF SCHWANN CELLS As a prelude to a discussion of the underlying regulatory mechanism we will now describe how Schwann cell development from El6 can be analysed as a sequence of appearance and disappearance of known molecules or characterised antigens (Fig. 1). In the rat sciatic nerve, the first Schwann cells (S100 positive cells viable in vitro in routine media) appear at E15-16 (Jessen et al., 1989).The first step in Schwann cell development is the appearance of the surface lipid antigen 0 4 (sulphatide), which is first seen between El6 and El7 and appears exclusively on SlOO positive cells (Mirsky et al., 1990).At El8 over 95% of the cells isolated from the nerve are SlOO positive Schwann cells and about 40%of them express 04, while at E20 essentially all Schwann cells have become 0 4 positive. Galactocerebroside first appears at E18-19 and essentially all Schwann cells express this lipid bv the 5th postnatal week (Jessen et al., i985; Mirski et ai., 1980;Ranscht et al., 1982).During the first half of this period of approx-

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JESSEN AND MIRSKY

myelin proteins

r------1

\ N-M Sch I

Survival

i---_---J

I I

Sch

Precursor I I

I

I

I

Sib0

04

-

I

I

I 3rd postnatal E 15-17

ElE-P4-5

weeK

Fig. 1. Develo ment of the molecular phenotype of myelin-forming and non-myelin-FormingSchwann cells from precursor cells in the rat sciatic nerve. Sch, Schwann cell; N-M Sch in heavy frame, mature non-myelin-forming Schwann cell; N-M Sch in stippled frame, cells with the molecular phenotype similar to mature non-myelin-forming cells, that will progress to myelination; M Sch, myelin-forming Schwann cell; Gal-C, galactocerebroside. indicates molecules that are down-regulated during differentiation of the Schwann cell along the myelin pathway. All other Schwann cell molecules or properties indicated are first seen at the time points indicated. Cells acquire the ability to survive alone in culture from embryo day 15 onwards; SlOO first appears in Schwann cells between embryo days 15-16 and is

present in 95%of Schwann cells a t embryo day 18; 0 4 first a pears shortly after SlOO at embryo day 16 and is resent in 95%of Sciwann cells at embryo day 20; galactocerebroside Erst appears on cells in the m elin forming pathway at embryo day 18 and the first Po-positive c e b a, seen during the first ostnatal day. By ostnatal day5 the first myelin-formin Schwann celg have down-reguyated molecules such as GFAP, N - C d A5E3, and NGF rece tors, which are associated with non-myelin-forming Schwann cells. eells in the non-myelin-forming pathway acquire Gal-C from the third postnatal week onwards without progressing to myelination. Differentiation of these cells is complete by the fifth to sixth postnatal weeks.

imately 5 weeks, most or all of the cells that acquire galactocerebroside progress to myelin formation. This involves, first, synthesis of the myelin proteins, MAG, Po MBP, and P2 (see Table 1).This up-regulation of protein synthesis is followed by down-regulation in the expression of a series of proteins (group 2 in Table 1) including GFAP, N-CAM, and NGF receptors (Fig. 2) (Jessen et al., 1987a, 1990). These proteins are essentially absent from adult myelinated fibers. Cells that acquire galactocerebroside without progressing to myelination first appear during the 3rd postnatal week (Jessen et al., 1985). These cells do not synthesize the myelin proteins and continue to express N-CAM, NGF receptors, and other proteins of that group. They represent the first mature non-myelinforming cells. The generation of these cells continues during the next 2-3 weeks, by which time essentially all Schwann cells in the nerve express galactocerebroside and the development of the nerve is largely complete (Friede and Samorajski, 1968; Jessen et al., 1985; Webster and Favilla, 1984). When Schwann cells are withdrawn from contact or close proximity with axons, e.g., by placing them in neuron free cultures or by nerve sectioning, there is a very rapid reversal of the Schwann cell phenotype (Table 1).In myelin-forming cells, the myelin proteins are down-regulated and GFAP, N-CAM, A5E3, and NGF receptors are all re-expressed (Jessen et al., 1990). Similarly, both non-myelin-forming cells and myelin-

forming cells lose surface expression of 0 4 and galactocerebroside (Mirsky et al., 1980,1990).These and many other observations (e.g., Aguayo et al., 1976; Jessen et al., 1987b; Weinberg and Spencer, 1976) support the hypothesis that up-regulation of myelin protein expression, the formation of the myelin sheath and downregulation of GFAP, N-CAM, A5E3, and NGF receptors in the myelin pathway, and the induction of galactocerebroside and 0 4 in both pathways, depend on axonto-Schwann cell signalling, involving membrane-membrane interactions or the secretion of very short range soluble factors. The evidence indicates that these signals continue to act throughout life to maintain the two distinct Schwann cell phenotypes seen in normal nerves. In being so reliant on axon-associated signals, Schwann cell development differs strikingly from gliogenesis in the rat optic nerve (Raff, 1989). Although most of the overt phenotypic changes seen in Schwann cell development are extrinsically signalled, rather than intrinsically programmed, there is now some direct evidence that intrinsically programmed changes are also involved. This comes from the observation that GFAP, normally first seen at E18, will appear in Schwann cell cultures prepared from E15-17 nerves and maintained without neurons (Jessen et al., 19901, although unmyelinated axons probably up-regulate levels of GFAP expression on nonmyelin-forming Schwann cells in subsequent development (Mokuno et al., 1989; Neuberger and Cornbrooks,

+

,

189

S C H W A " CELL DEVELOPMENT

1989). Programmed changes might well modulate the effects of axon-associated signals. The Schwann cell response to a continuous and unchanging axonal signal could, for instance, change over time due intrinsically regulated maturation, thus generating a temporal pattern in Schwann cell development (see the following section). Although they are central to Schwann cell development, the axon-Schwann cell signalling mechanisms are still essentially unknown in terms of the identity of the putative neuron associated molecules or their receptors on Schwann cells. On the other hand, it is now clear that elevation of intracellular CAMPin Schwann cells mimics the effects of these signals remarkably faithfully. One explanation for this might be that axonSchwann cell signals act by activating the adenyl cyclase-cyclic AMP pathway in Schwann cells. These issues are discussed in the next section.

THE INVOLVEMENT OF CYCLIC AMP IN SCHWA"

NON- MY ELIN

+ + + + NEWBORN

GFAP N-CAM L1

-

AXON-DEPENDENT REVERS1BLE DOWN-REGULATION

~

~

-

Fig. 2. Axon-dependent reversible down-regulation of Schwann cell molecules that occurs in the myelin-forming pathway shortly after the onset of myelin formation.

CELL DEVELOPMENT

The current interest in CAMP as a possible developmental signal for Schwann cells derives from two earlier observations which implicated CAMPin the regulation of both proliferation and differentiation in Schwann cells (Raff et al., 1978a,b; Sobue and Pleasure, 1984). It now appears possible that the mitogenic effect of cAMP may be indirect and stems from its ability to upregulate expression of receptors for growth factors that stimulate Schwann cell proliferation, while the proliferative state, in turn, has been shown to be an important regulator of the cAMP differentiation signal. These entwined issues will here be dealt with separately at first. Cyclic AMP and Schwann Cell Proliferation Four polypeptide growth factors, glial growth factor (GGF), transforming growth factor-beta (TGFP), platelet derived growth factor (PDGF), and fibroblast growth factor (FGF), have been identified as mitogens for cultured rat Schwann cells (Davis and Stroobant, 1990; Eccleston et al., 1987,1990; Raff et al., 1978a). In serum containing media, cAMP analogues and agents that elevate intracellular cAMP levels, such as forskolin and cholera toxin, synergize with each of these factors to promote Schwann cell DNA synthesis; CAMPelevating agents alone also stimulate Schwann cell division in the presence of serum, presumably via synergy with serum derived factors. In contrast, in serum free culture media, the growth promoting effect of CAMPis essentially absent and the same holds for the polypeptide factors when applied in isolation. A combination of CAMP elevation with GGF, PDGF, or FGF in serum free medium, however, stimulates DNA synthesis (Stewart et al., 1991). Thus, for stimulation of Schwann cell DNA synthesis a co-operationbetween CAMPelevation and these polypeptide growth factors is obligatory but does not

require serum factors. At least two different explanations are possible. One is offered by the finding that, at least in serum containing media, CAMPelevation upregulates the expression of PDGF receptors on Schwann cells (Weinmaster and Lemke, 1990).Although unstimulated Schwann cells also express PDGF receptors (Eccleston et al., 19901, it is quite possible that increased receptor expression leads to increased PDGF response. If receptors for the other growth factors are regulated in the same way this mechanism alone might account for the synergy with CAMP.Alternatively, CAMPmay in some circumstances act as a direct Schwann cell mitogen requiring the simultaneous activation of pathway(s) operated by polypeptide growth factors to stimulate DNA synthesis, as envisaged in some other cell types (Rosengurt, 1986). The question of whether CAMP elevation is in all circumstances a necessary part of the events leading to stimulation of Schwann cell DNA synthesis is still open. One of the most potent Schwann cell mitogen is activity associated with heparan sulphate proteoglycan on the surface ofaxonal membranes (Ratner et al., 1985,19881, but it is still controversial whether axolemmal fractions containing this mitogen cause a significant elevation of intracellular CAMPin Schwann cells (Meador-Woodruff et al., 1984). The amount of CAMP elevation that would be significant in this context is unclear, particularly since CAMPcould rise in restricted pools or compartments within the cell without being easily detectable in whole cell measurements. For the subsequent discussion it is important to note that there are two different situations in which CAMP elevation does not lead to increased Schwann cell proliferation. As mentioned above, when CAMPis elevated in the absence of serum and other growth factors no DNA synthesis occurs. Alternatively, even when CAMP is elevated in the presence of growth factors proliferation

190

JESSEN AND MIRSKY

can be markedly suppressed by plating the cells at high density. This density dependent growth inhibition is likely to be due to the secretion of, as yet unidentified, growth inhibitorb). Cyclic AMP and Schwann Cell Differentiation In 1984 Sobue and Pleasure found that exposure to cAMP analogues induced re-expression of galactocerebroside in cultures of neonatal Schwann cells which had previously lost galactocerebroside expression due to removal from axons. In similar experiments CAMPelevation has since been shown to induce many other phenotypic changes associated with Schwann cell differentiation in vivo. This includes flattening and formation of large membraneous expansions (Sobue et al., 1986; Morgan et al., 1990),induction of expression of a 170kD protein found in myelin (Shuman et al., 1988),of the myelin specific proteins Po and MBP and corresponding mRNAs (Lemke and Chao, 1988; Morgan et al., 1990), of the SCIP gene (Monuki et al., 19891,and of the early lipid antigen 0 4 (Mirsky et al., 1990). Surface expression of the basement membrane components is also up-regulated following exposure to CAMP analogues (Baron van Evercooren, 1986). This is a disparate collection of events, since some are associated with early Schwann cell development, while others occur relatively late; some are myelination specific, while others occur in the differentiation of both Schwann cell types. Nevertheless, in addition to being induced by CAMP,they all have in common that in vivo they are regulated by axon-associated signals (see the prior section). It is therefore of particular interest that axolemmal membranes have been reported to elevate intracellular cAMP levels in Schwann cells (Ratner et al., 19851, althou& this remains to be confirmed (Meador-Woodruff et al., 1984). Furthermore, many gene regulatory proteins have CAMPresponsive elements; cAMP effects expression of POU type gene regulatory proteins in Schwann cells, and there are precedents for CAMPas the second messenger in the transduction of ligandreceptor interactions at the cell surface to changes in gene activity (Dumont et al., 1989). It is therefore possible that a crucial part of axon-Schwann cell signalling in vivo operates via elevation of Schwann cell CAMP levels. The fact that CAMP triggers a broad spectrum of developmental responses in Schwann cells opens the intriguing possibility that a single axon-associated signalling mechanism, operating via CAMP,drives much of Schwann cell development, including 0 4 expression, myelination, and galactocerebroside synthesis in both non-myelin- and myelin-forming cells. The axon-associated signal in question should be present as early as E15-16 or earlier and be associated with both myelinated and unmyelinated axons. Both of these features distinguish this scenario from an alternative possibility, viz., that a signal associated selectively with myeli-

Fig. 3. Immunoblot of Poinduction in Schwann cells. (SN)Extract of 5-day-old rat sciatic nerve, 25 Fg loaded. Lanes 1-3: extracts from dissociated Schwann cell cultures from 5-day-old rat sciatic nerve treated with agents that elevate or mimic CAMP. Lane 1: CAMP analogues; lane 2: cholera toxin, lane 3: foreskolin, lane 4: no treatment, 31 p,g of extract loaded in each lane. Note that at the P position, which is arrowed, an immunoreactive band is visible in t i e sciatic nerve lane and in each of lanes 1-3 but not in lane 4 (control).In the sciatic nerve extract a lower breakdown product of Po is also visible. (From Morgan et al., 1990 with permission.)

nated axons “the myelination signal” elevates CAMPin certain Schwann cells only, thereby inducing them to form myelin. It should be possible to distinguish experimentally between these possibilities in the near future. The idea that axon associated CAMPelevating factor(s) provide a general primary signal for the development of Schwann cells from their earliest generation in embryonic nerves takes account of more of the available data than does the suggestion that cAMP acts as a specific myelination signal. It nevertheless leaves two fundamental questions unanswered. First, why does Schwann cell development diverge, generating two distinct Schwann cell types? This problem will be discussed in the following section. Second, how is the temporal pattern in Schwann cell development generated? If we consider the development of myelin-forming cells, for instance, why would axonally induced CAMP elevation trigger 0 4 expression at E15-16 but not lead to significant Po synthesis and suppression of GFAP, N-CAM,and NGF-receptors until after birth? The effect of a constant input, e.g., one that maintains elevated cAMP levels, on gene activity or metabolism depends, of course, on a multitude of intracellular factors. If they change during the developmental period, a constant input could, in principle, trigger different events at different times. One possibility is that such a change in the factors that determine the translation of the CAMP signal occurred as a result of intrinsically programme maturation. For instance, cells in E15-16 nerves that are already able to express 0 4 in response to CAMP elevation (Mirsky et al., 1990) might be unable to respond t o the same stimulus by significant Po synthesis.

SCHWA" CELL DEVELOPMENT

191

Fig. 4. Induction of P in Schwann cells treated with cholera toxin. Po- ositive Schwann cefls cultured from 5-day-old rat sciatic nerve. Celrs were cultured on a laminin substrate and treated with cholera toxin and labelled with Po antibodies after 3 days of treatment. Cells were viewed with fluorescein optics to visualise P,. Note the flattened shape of the Schwann cells and the Poimmunofluorescence distributed throughout the whole area of each cell. Nuclei of three intensely Po-positivecells are indicated by arrows.

That response might not be acquired until in the perinatal period, a time when Po is first expressed at high levels in normal development. Our preliminary experiments do not support this type of regulation of CAMP responsiveness, and there is, as yet, no other evidence that the way in which Schwann cells respond to CAMP elevation changes according to an intrinsic programme in the period of El5 to the postnatal stage. It is therefore important that an alternative way of regulating the CAMPresponse has now been established (Morgan et al., 1990).As we will discuss below, it turns out that the response of cultured Schwann cells to agents that elevate CAMPlevels differs markedly, depending on whether they are proliferating, as they are in embryonic nerves, or quiescent, as they are from the onset of myelination. The Ability of CAMPto Induce Myelin-Related Differentiation is Inversely Related to Proliferation, While 0 4 Induction is Proliferation Independent

As discussed previously, Fig. 5 . Induction of a myelin-related phenotype in cultured Schwann Schwann cell proliferation in sparse to medium dense cells. Double label immunofluorescence usin monoclonalantibodies to Schwann cell cultures, provided the cells are simulta- P and N-CAM antibodies (A$$), or monoJona1 antibodies to Po and GkAP antibodies. Schwann cells from 5-day-old sciatic nerve were neousb' exposed to Serum factors, Serum PIUS identified treated with CAMPanalogues for 3 days under conditions where both growth factors, such as FGF or PDGF, or growth factors induced and uninduced cells were present in reasonable numbers. Cultures were viewed with (A) fluorescein o tics to visualise Po, (B) in routine serum-free media N2, rhodamine optics to visualise N-CAM, or (8) base contrast optics tenstein and %to, 1979). cyclic AMP elevation alone (arrow indicates nucleus of the Po- ositive celr). Note that the P does not stimulate DNA synthesis in se-um-free me- positive cell seen in (A) is unlabellea with antibodies to N-CAM (Bg, while surrounding PQ negative cells are intensely N-CAM positive. dium and has Only effect in very dense Note that the Po positive cell has assumed a flattenedmorphology, seen even in the presence of serum or growth factors. It is in(C).

192

JESSEN AND MIRSKY GROWTH INHIBITORS OR WITHDRAWAL OF SYNERGISTIC

G R O W H FACTORS

MYELINATION

-

cAMP ELEVATION

Fig. 6. The proliferative state may control the response to CAMP in vivo. It is envisaged that axon-dependentelevation of CAMP induces earl differentiation (04expression)under conditions in which CAMPsimultaneously synergizes with growth zctors to romote Schwann cell proliferation. Withdrawal from the cell cycle (brought about as indicated as via o&er mechanisms) triggers myelination, since in quiescent cells the ongoing CAMPelevation induces myelination.

therefore possible to test the effects of raised cAMP levels on molecular expression in Schwann cells under conditions of mitotic quiescence, on the one hand, and under conditions where the CAMP elevation simultaneously leads to rapid DNA synthesis, on the other. In this type of experiment we have found that under proliferating conditions cAMP elevation effectively triggers 0 4 expression and that 0 4 expression and DNA synthesis are compatible in individual cells. Under these conditions, however, Schwann cells do not progress from the 0 4 positive proliferative state to a myelination-related state of differentiation. Under nonproliferative conditions, however, the response of CAMP elevation is quite different. A majority of Schwann cells now assumes a molecular and morphological phenotype strikingly similar to that of myelin-forming Schwann cells in vivo. They express high levels of the major myelin protein Po,which can be detected either by SDS immunoblotting techniques (Fig. 3) or by immunohistochemistry (Fig. 41,and generally assume a flattened morphology with large membranous expansions. In addition, down-regulation of GFAP, N-CAM, A5E3, and NGF receptors, all changes which occur specifically in myelin-forming cells in vivo, is also seen in those Schwann cells in which Poinduction is strongest (Fig. 5 ) (Morgan et al., 1990). Thus, in cultured Schwann cells exposed to CAMP elevating agents, entry into the myelin pathway, from a pre-myelin state characterized by 04 expression and proliferation, depends on withdrawal from the cell cycle (Fig. 6). In vivo, also, 0 4 appears on proliferating cells between E l 6 and 20 while high levels of Po synthesis

and the formation of the myelin sheath occurs later and only in cells that have stopped dividing. It is therefore possible that the principle revealed in vitro in these experiments also applies in vivo. According to this hypothesis, CAMP elevation, presumably induced by axon-associated factors, acts as a primary signal that triggers 0 4 expression and synergizes with growth factors in driving Schwann cell proliferation in the pre-myelination period. Withdrawal from the cell cycle, first seen in Schwann cells at birth, acts as a secondary signal, which, when superimposed on CAMPelevation, induces myelination in cells associated with the larger axons. As yet very little is known about how cessation of Schwann cell division is brought about during development. In principle, at least three mechanisms could be envisaged: down-regulation of receptors for mitogenic growth factors, decrease in the availability of such factors, or the advent of active growth inhibition. With respect to the last possibility, which is increasingly being considered in other systems, recent studies have defined at least two proteins that, in the rat, are potential Schwann cell growth inhibitors, i.e., type I collagen and interferon-gamma (Eccleston et al., 1989b,c).Type I collagen inhibits Schwann cell DNA synthesis in routine cultures, while the inhibitory effect of interferongamma is seen in cells stimulated to divide by cAMP elevating agents in the presence of serum or growth factors. In this context it is interesting that some neurons show interferon-like immunoreactivity (Ljungdahl et al., 1989). Growth inhibition in Schwann cells is also caused by a protein secreted by cultured Schwann cells

SCHWA" CELL DEVELOPMENT

(Ecclestonet al., 1991; Muir et al., 1990)and byfactor(s1 associated with enteric neurons (Eccleston et al., 1989b). The hypothesis outlined above (Fig. 6) does not, as it stands, explain the generation of non-myelin-forming cells. At least two possibilities can be envisaged. First, whether a cell forms myelin or not, when withdrawn from the cell cycle, might depend on the degree of CAMP elevation. This could differ between cells associated with one large axon and cells associated with smaller axons. There is no evidence for or against this idea. Second, it can be speculated that a structural polarization of Schwann cells generated by flattening between two surfaces, i.e., that of the axon membrane and of the extracellular matrix, is a prerequisite for CAMPelevation inducing myelination, i.e., a prerequisite for galactocerebroside positive cells to progress further along the developmental path. There are several precedents for polarization affecting cell function (for discussion in the context of Schwann cell biology see Bunge et al., 1986). In Schwann cells, such polarization would be most effectively achieved in a cell associated with a single axon with a diameter which was large with respect to the size of the Schwann cell. This hypothesis is attractive in that it incorporates both recent work on CAMP and Schwann cell differentiation, and the earlier notion, based on the close correlation between myelination and axon diameter, that axon diameter has a crucial role in determining myelination (e.g., Friede, 1972). ACKNOWLEDGMENTS We would like to thank all the past and present members of our laboratory for their contribution to many of the experiments described here. The work has been supported by grants from the Medical Research Council of Great Britain, Action Research for the Crippled Child, the Wellcome Trust, and the Multiple Sclerosis Society of Great Britain and Northern Ireland. REFERENCES Aguayo, A.J., Charron, L., and Bray, G.M. (1976)Potential of Schwann cells from unmyelinated nerves to produce myelin: A quantitative ultrastructural and autoradiographic study. J . Neurocytol., 51565-573. Baron van Evercooren,A,, Gansmuller, A,, Gumgel, M.! Baumann! N.! and Klienman, H.K. (1986) Schwann cell di erentiation an uatro, extracellular matrix deposition and interaction. Deu. Neurosca., 8:182-196. Bottenstein, J.E. and Sato, G.H. (1979) Growth of rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. U.S.A., 76514-517. Brockes, J.P., Fields, K.L., and Raff, M.C. (1979) Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of eripheral nerve. Brain Res., 165:105-118. Bunge, R.P., gunge, M.B., and Eldridge, C.F. (1986) Linkage between axonal ensheathment and basal lamina production by Schwann cells.Annu. Reu. Neurosci., 9:305-328. Davis, J.B. and Stroobant, P. (1990) Platelet-derived owth factors and fibroblast growth factors are mitogenic for rat Sc Kann cells. J. Cell Biol., 110:1353-1360. Diner, 0. (1965) Les cellules de Schwann en mitose et leurs rapports avec les axones au cours du developpementdu nerf sciatique chez le rat. C.R. Acad. Sci., 261:1731-1734.

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