Journal of Cell Science 106, 1291-1300 (1993) 1291 Printed in Great Britain © The Company of Biologists Limited 1993

Transient expression of the intermediate filament nestin during skeletal muscle development

Thomas Sejersen* and Urban Lendahl Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-17177 Stockholm, Sweden *Author for correspondence

SUMMARY

It has previously been established that skeletal muscle nestin, and was analysed in the human development is accompanied by changes in the compo- myogenic cell line G6 before and after in vitro differ- sition of intermediate filaments: vimentin is expressed entiation. Despite its more distant evolutionary and predominantly in myoblasts and desmin in adult structural relationship to the other two intermediate fil- myotubes. We show that the intermediate filament tran- aments, nestin formed a cytoplasmic filamentous net- sitions during muscle development are more complex, work indistinguishable from that of desmin and and involve a transient expression of the recently dis- vimentin, both in undifferentiated myoblasts and after covered intermediate filament nestin. Nestin RNA is differentiation to multinuclear myotubes. In conclusion, expressed predominantly early, in a biphasic pattern, our data suggest that nestin is an integrated component and is markedly downregulated in adult rat muscle, of the dynamic intermediate filament network during whereas desmin RNA becomes more abundant through- muscle development and that nestin copolymerizes with out development. Nestin was found up to the desmin and vimentin at stages of coexpression. postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of Key words: intermediate filaments, myogenesis, differentiation

INTRODUCTION similar, important roles in other tissues. It is thus of inter- est to characterize the spatial and temporal expression pat- The is composed of microtubuli, microfila- terns of various intermediate filaments during tissue devel- ments and intermediate filaments. While the former two opment, and to learn how the individual interact components are present in most cell types, intermediate fil- with each other in the cell. This may be particularly impor- ament expression is much more dynamic. More than 40 dif- tant for tissues that undergo complex morphogenetic ferent intermediate filaments have been characterized and changes during development. In this report we analyse the they are expressed with very distinct temporal and spatial expression and intracellular distribution of the recently dis- patterns (for review see Steinert and Liem, 1990; Stewart, covered intermediate filament nestin during the course of 1993). Based on structural criteria, intermediate filament muscle development. , including the nuclear , are currently divided Nestin comprises the class VI intermediate filaments into six classes (Lendahl et al., 1990; for review see Stein- (Lendahl et al., 1990), and belongs to the same evolution- ert and Liem, 1990), and these six classes fall into two main ary branch as neurofilaments and (Dahlstrand et evolutionary branches (Dahlstrand et al., 1992b; Dodemont al., 1992b; Dodemont et al., 1990). The expression of nestin et al., 1990; for review see Weber et al., 1991). Little is has primarily been analysed in the central nervous system still known about the specific functions of individual inter- (CNS), where it is expressed transiently in CNS stem cells, mediate filament genes, but expression of mutant versions and is later replaced by neurofilaments and glial fibrillary of a in transgenic mice resulted in severe acidic protein (GFAP) in neurons and astrocytes, respec- defects in skin organization (for review see Fuchs and tively (Lendahl et al., 1990). There are however several Coulombe, 1992). Furthermore, the genetic skin diseases lines of evidence suggesting that nestin is also expressed in epidermolysis, bullosa simplex and epidermolytic hyperk- muscle. First, nestin was originally discovered as an epi- eratosis were recently shown to be caused by mutations in tope expressed in neuroepithelial cells and myotomes. keratin genes (for review see Fuchs and Coulombe, 1992). Second, nestin mRNA is found in the developing skeletal These data show that one type of intermediate filament is muscle of rat embryos (Lendahl et al., 1990). Third, a rhab- important for organization of a specific tissue type, and it domyosarcoma tumor, which is of muscle origin, was pos- is conceivable that other intermediate filaments may play itive for nestin immunoreactivity (Dahlstrand et al., 1992a). 1292 T. Sejersen and U. Lendahl

Finally, dissection of the rat nestin promoter in transgenic MATERIALS AND METHODS mice revealed a regulatory element that directs expression in developing skeletal muscle (Zimmerman et al., 1994). Cell culture Myogenesis, the development of skeletal muscle, is a The G6 human myoblast cell line was derived from a clone orig- complex, multistep process. Somite cells become deter- inating from thigh muscle of a 73-day-old, aborted fetus (Jin et mined to form muscle precursor cells in the myotome. al., 1993). Growth medium used for myoblast proliferation was Mononucleate muscle precursor cells proliferate and Ham´s nutrient mixture F-10 with 20% fetal calf serum and 0.5% migrate to their final destinations, cease DNA replication, chicken embryo extract. Differentiation medium was Dulbecco´s and subsequently fuse into multinucleate myotubes. This modified Eagle´s medium with 5% horse serum. process is influenced by growth factors and myogenic deter- mination factors (Olson et al., 1991). The formation of RNA blot analysis myotubes is accompanied by the activation of genes encod- Purifications of total RNA, selection of poly(A)+ RNA, gel frac- ing muscle-specific proteins. Several of these genes, e.g. tionation, RNA blottings, RNA hybridizations, washing proce- and heavy and light chains, exist in embry- dures, and determinations of sizes of the transcripts were per- onic, neonatal and adult forms, and are expressed in a formed essentially as described earlier (Jin et al., 1991). sequential order during muscle development (Buckingham, 1985). DNA probes It is well established that two intermediate filaments, For northern blot hybridizations the following antisense oligonu- vimentin and desmin, are synthesized in skeletal muscle cleotide probes were used for developing rat muscle: cells (Gard and Lazarides, 1980; Osborn et al., 1982). mouse nestin 48mer, Vimentin and desmin are closely related, both belonging to (5¢-G G T C C C T G G G A A T C C T G G A T T T C T T C T G T G T C C A G- the class III intermediate filaments. They are, however, rel- ACCACTTTCTTGT-3¢) (Zimmerman et al., 1994); atively distantly related to nestin, which resides on the other hamster desmin 48mer, main evolutionary branch (see Weber et al., 1991, for (5¢-C T T C A G A A C C C C T T T G T T C A G G G C T G G T T T C T C G G- review). During early stages of avian and mammalian AAGTTGAGAGCAG-3¢) (Quax et al., 1984); embryogenesis the intermediate filament network of imma- mouse vimentin 48mer, ture muscle cells was previously reported to be made exclu- (5¢-G T C T C A T T G A T C A C C T G T C C A T C T C T G G T C T C A A C- sively of vimentin (Bennett et al., 1979; Gard and CGTCTTAATCAGG-3¢) (EMBL database). Lazarides, 1980; Zehner and Paterson, 1983). Later, For G6 cells the following probes were used: myoblasts and early myotubes express both vimentin and human desmin 48mer, desmin. In mouse and human myotubes vimentin disappears (5¢-CTTGATCATCACCGTCTTCTTGGTATGGACCTCAG- shortly after fusion, whereas in chicken myotubes coex- AACCCCTTTGCTCAGG-3¢) (Li et al., 1989), human vimentin 48mer, pression of vimentin and desmin has been reported (Ben- (5¢-CAGGAGTGTCCTTTTTGAGTGGGTATCAACCAGAG- nett et al., 1979; Gard and Lazarides, 1980; Zehner and GGAGTGAATCCAG-3¢) (Ferrari et al., 1986). Paterson, 1983). When myoblasts fuse to form myotubes As control, a second 50mer from human vimentin was used: desmin first has a diffuse longitudinal intracellular distrib- (5¢-CGTGATGCTGAGAAGTTTCGTTGATAACCTGTCCA- ution in the cytoplasm. As the myotubes mature and sar- TCTCTAGTTTCAACC-3¢). comeres are organized into visible cross-striations, desmin Mouse a -actin pAM 91 (Minty et al., 1981) and PstI fragments becomes localized to the Z bands, where it has been found of mouse fast myosin heavy chain HHC 32 (Weydert et al., 1983) to connect myofibrils to the sarcolemma and to attach actin were also used as probes. Human nestin RNA was analysed with filaments to the Z bands (Granger and Lazarides, 1979; a genomic probe containing the first of the human nestin Holtzer et al., 1982; Tokuyasu et al., 1983). However, it gene (Dahlstrand et al., 1992b). has been shown that muscle differentiation apparently pro- ceeds normally in vitro when the vimentin and desmin net- Protein blot analysis works are disrupted (Schultheiss et al., 1991). Cultured cells were scraped in PBS, lysed in SDS sample buffer, The apparent dispensability of vimentin and desmin and and boiled briefly (Laemmli, 1970). A 2 mg sample from each the fact that a more distantly related intermediate filament differentiation state was subjected to electrophoresis in a dena- is also expressed in muscle prompted us to characterize the turing polyacrylamide (12%) gel and the electrophoresed proteins temporal and spatial distribution of nestin in muscle. We were blotted onto nitrocellulose filters (Millipore) by a Bio-Rad have analysed the mRNA expression and protein distribu- electroblotter. After preadsorption with 5% non-fat dried milk and tion of nestin, and compared it with that of desmin and 20% fetal calf serum in PBS overnight at 4°C, parallel filters were vimentin during skeletal muscle development and in vitro incubated overnight at 4°C with the rabbit anti-nestin antiserum myogenic differentiation. Our data demonstrate that nestin no. 130 (Dahlstrand et al., 1992a) diluted 1:2000 in PBS, a mouse is expressed predominantly early in muscle development, monoclonal anti-desmin antibody (DE-B-5, Boehringer) diluted but can be detected in the Z bands of postnatal myofibers. 1:800 in PBS, or a mouse monoclonal anti-vimentin antibody In contrast, desmin is more abundant at later, more mature (Vim 3B4, Boehringer) diluted 1:80 in PBS, and then rinsed three times in PBS. Antibody binding was detected by use of alkaline stages of muscle development. When expressed in the same phosphatase-conjugated second antibodies (Dakopatts) or by cell nestin appears to copolymerize with both desmin and second antibodies followed by avidin and biotinylated horserad- vimentin, suggesting that during stages of coexpression net- ish peroxidase (Dakopatts), according to the manufacturer’s sug- works are produced that are mixtures of evolutionarily quite gestions. RainbowTM protein molecular mass markers (Amer- diverged intermediate filaments. sham) were used. Nestin expression in muscle development 1293

Immunohistochemistry Skeletal muscle from the thigh of a 15.5 days post-coitum (dpc) rat embryo and from the thigh of a 15-day-old postnatal rat was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 mm sections. Sections were deparaffinized for 3´ 10 minutes in xylene, rehydrated, and incubated for 5 minutes with 3% hydro- gen peroxide, followed by a 20 minute incubation with 3% bovine serum albumin in PBS. Adjacent sections were subsequently stained with the anti-nestin antiserum (diluted 1:1000 in PBS), or with the antibodies to desmin (diluted 1:200 in PBS), or to vimentin (diluted 1:50 in PBS). The immunostaining was visual- ized by use of avidin and biotinylated horseradish peroxidase (Dakopatts). Double-immune staining of postnatal muscle was performed as outlined below. To visualize the morphology of the tissue, additional sections were stained with eosin/hematoxilin.

Immunocytochemistry G6 cells grown on coverslips were fixed for 10 minutes with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS for 3 minutes, rinsed three times in PBS, incubated for 30 minutes with 3% bovine serum albumin in PBS, and subsequently incubated overnight at room temperature with the anti-nestin anti- serum (diluted 1: 500 in PBS), or with antibodies to desmin (diluted 1: 200 in PBS) or to vimentin (diluted 1: 20 in PBS). Fol- lowing three 5-minute washes in PBS, the coverslip was incubated with an appropriate second antibody (FITC goat anti-rabbit IgG, Boehringer (605210), diluted 1:100 or RTITC goat anti-mouse IgG, Boehringer (605140), diluted 1:100) for 2 hours at room tem- perature and washed 3´ 5 minutes in PBS. For double-immune staining samples were first incubated with the anti-nestin anti- serum and its appropriate second antibody, followed by either Fig. 1. RNA blot analysis of nestin, vimentin and desmin desmin or vimentin antisera and the appropriate second antibody, expression in developing skeletal muscle. A 9 mg sample of with washes in PBS for 3´ 5 minutes between each incubation. poly(A)+ RNA was analysed in each lane, from the following Control experiments where either of the first antibodies was omit- tissues and stages: whole 13.5 dpc embryo (d 13 w.e.); crude thigh ted resulted in loss of signal corresponding to the omitted anti- muscle tissue from 15.5 dpc (d 15 m.); 17.5 dpc (d 17 m.); 18.5 body, demonstrating that no cross-hybridization occurred. dpc (d 18 m.); 20.5 dpc (d 20 m.); and from newborn (d 1 m.); 7 Immunoreactive material was visualized in a fluorescence micro- days postnatal (d 7 m.); 14 days postnatal (d 14 m.); 21 days scope and photographed with Kodak 400 ASA T-max film. postnatal (d 21 m.); and from a 5-month-old rat (Adult m.). The same filter was sequentially hybridized with probes to rat nestin (Nes), vimentin (Vim), desmin (Des), and myosin heavy chain RESULTS (MHC). Sizes are denoted in kb to the right.

Analysis of mRNA levels of nestin, vimentin and Nestin and vimentin transcripts were found in early post- desmin during rat muscle development natal thigh muscle, but the levels became downregulated by To characterize the expression of nestin during muscle postnatal day 21, and were strongly reduced in the muscle development, and to compare it with the expression pat- from a 5-month-old rat. The relative levels of nestin mRNA terns of desmin and vimentin, poly(A)+ RNA from a 13.5 were transiently reduced around birth, producing a bipha- dpc embryo and from rat thigh muscle from various stages sic expression pattern, whereas vimentin mRNA levels were of development was analysed by the northern blotting tech- more uniform during the same time period. nique with probes specific to the rat nestin, vimentin and desmin genes (Fig. 1). In all three cases a single, specific Immunohistochemical analysis of intermediate hybridization signal was obtained, corresponding to the filament proteins in pre- and postnatal rat skeletal expected mRNA size for each intermediate filament gene. muscle The sizes of the nestin, vimentin and desmin mRNAs were Given the expression of nestin in both pre- and postnatal 6.7, 2.0 and 2.6 kb, respectively. muscle, we next decided to analyse the distribution of the Desmin and vimentin transcripts were easily detected in nestin protein at an early and a late stage of muscle devel- the 13.5 dpc embryos, whereas nestin transcripts were opment, and to compare it with that of vimentin and desmin. barely visible. In prenatal muscle tissue from 15.5 dpc rat Sections from fixed, embedded thigh muscle from a 15.5 thigh muscle nestin and vimentin transcripts were more dpc rat embryo and from a 15 day postnatal rat were pro- abundant, while desmin RNA was almost undetectable. At duced. Cross-sections of embryonic thigh muscle, together later stages, the expression of desmin gradually increased with surrounding mesodermal and epidermal tissue, were to reach maximal levels in the adult rat, with kinetics sim- easily identified by eosin/hematoxilin staining on such sec- ilar to the increase in myosin heavy chain expression. tions (Fig. 2). 1294 T. Sejersen and U. Lendahl

Fig. 2. Immunohistochemical analysis of intermediate filament localization in embryonic skeletal muscle. Cross-sections of thigh muscle from a 15.5 dpc rat embryo were stained with eosin/hematoxilin (HTX/eosin) or with antisera specific to nestin (Nes), vimentin (Vim), or desmin (Des). Immunoreactive material was visualized by a peroxidase reaction. Bars, 100 mm.

Adjacent sections from the 15.5 dpc muscle were stained banded structure (Fig. 3c,d). Since it has previously been with antisera specific to nestin, vimentin and desmin (Fig. established that desmin is localized at the Z bands (Granger 2). Staining with the polyclonal anti-nestin antiserum and Lazarides, 1979), we conclude that nestin in young showed immunoreactivity in developing muscle fibres, and myofibers is also located at Z bands. We frequently in endothelial cells (Fig. 2). The staining was evenly dis- observed that desmin staining (Fig. 3d) in muscle fibrils tributed in most muscle fibres in the thigh muscle. Stain- was more widespread than nestin staining (Fig 3c). This ing of endothelial cells has previously been observed during was also seen when alternate sections were stained with human brain development (Tohyama et al., 1992), and in nestin and desmin, respectively (data not shown). This is adult human brain and brain tumors (Dahlstrand et al., probably due to a broader expression pattern for desmin, as 1992a; Tohyama et al., 1992). The monoclonal antibody to suggested by the RNA data, but we can not formally vimentin produced a more widespread pattern. Developing exclude the possibility that the accessibility for the anti- skeletal muscle was stained, as well as the surrounding con- nestin antiserum was different than for the anti-desmin anti- nective tissue and dermis, but no staining was observed in body. In contrast to the nestin and desmin experiments, we endothelial cells (Fig. 2). Finally, the monoclonal anti- could not detect any immunoreactivity using the anti- desmin antibody produced a distinct and evenly distributed vimentin antibody (data not shown), in accordance with pre- staining pattern in the muscle fibres, with little or no stain- vious observations in mouse and man (Bennett et al., 1979; ing of other mesodermal or epidermal tissues (Fig. 2). Gard and Lazarides, 1980; Zehner and Paterson, 1983). In the postnatal day 15 thigh muscle, a stage when nestin mRNA levels were reduced but still detectable (Fig. 1), lon- Intermediate filament expression and intracellular gitudinal sections were analysed with the three different location during in vitro differentiation of the antibodies (Fig. 3). The anti-nestin antiserum stained a myogenic cell line G6 subset of muscle fibers in a banded pattern (Fig. 3a). The The extensive temporal overlaps in expression and the colo- desmin antibody produced a similar banded staining of the calization of nestin and desmin in Z bands prompted us to myofibers (Fig. 3b). By double immunofluorescence it was analyse the intracellular localization of the proteins in more shown that nestin and desmin antisera stain the same detail. We found the human myoblast cell line G6, derived Nestin expression in muscle development 1295 from a 73-day-old aborted fetus (Jin et al., 1993), to be par- creatine phosphokinase, a -actin and myosin heavy chain ticularly suitable for this purpose. First, G6 myoblasts dif- (Jin et al., 1993). Second, upon differentiation both typical ferentiate spontaneously under low serum conditions to elongated myotubes and very flattened myotubes are form multinucleate myotubes, and the morphological differ- formed, and the latter are particularly suitable for a detailed entiation is accompanied by changed expression patterns of morphological study of the intermediate filament network the muscle determination genes Myf3-6 and induction of in the early myotube.

Fig. 3. Immunohistochemical analysis of nestin and desmin in postnatal skeletal muscle. Longitudinal sections from postnatal day 15 rat thigh muscle were analysed with antibodies to nestin (a) and desmin (b). Immunoreactive material was visualized by a peroxidase reaction. Double immunofluorescence analysis of nestin (c) and desmin (d) in the same section. Bars, 50 mm. 1296 T. Sejersen and U. Lendahl

A B

Fig. 5. Identification of intermediate filament proteins in western blots. A 2 mg sample of protein from the G6 cell line under proliferating myoblast conditions (P), or after 8, 15, 24, 48 and 72 hours in differentiation medium was western blotted with antisera to nestin (Nes), desmin (Des) and vimentin (Vim). Migration of molecular mass markers of indicated size in kDa is shown on the left margin. Fig. 4. RNA blot analysis of intermediate filament expression during in vitro differentiation of a human myogenic cell line. A 5 mg sample of poly(A)+ RNA was analysed in each lane. (a) G6 vimentin and b-actin the filter was rehybridized with a dif- cells cultured at low density or high density in growth medium, ferent probe for vimentin. An identical pattern was obtained and G6 myoblasts cultured for 1, 2 or 3 days in differentiation with the second vimentin probe (data not shown). To con- medium (denoted Diff 1d, Diff 2d, and Diff 3d, respectively). trol for tissue specificity of intermediate filament expression (b) G11 fibroblasts (G11 Fb), G6 myoblasts in growth medium a fibroblast cell line, G11, derived from the same human (G6 Mb), G6 myotubes after 6 days in differentiation medium (G6 fetus as G6, was analysed. Expression of vimentin and b- Mt), E6 myoblasts in growth medium (E6 Mb), and E6 myotubes 6 days in differentiation medium (E6 Mt). Sizes are denoted in actin was observed, but not of nestin, desmin and a -actin, kilobases to the right. showing that nestin and desmin are not promiscuously expressed in all cell lines (Fig. 4). To prove that the different antisera did not cross-react We first analysed the G6 cells for expression of the three we performed immunoblot experiments on proteins from intermediate filaments. In northern blot experiments the G6 cell line. The anti-nestin antiserum produced a pre- mRNAs of sizes expected for nestin, vimentin and desmin dominant appoximately 200 kDa band from the various were observed in both undifferentiated and differentiated stages of in vitro differentiation, and in addition bands of cells, using probes for the three human genes (Fig. 4). slightly lower molecular masses (Fig. 5). The latter have Vimentin RNA levels were reduced during differentiation. been observed previously, and probably reflect partial As control, the same filter was rehybridized with a probe degradation (Dahlstrand et al., 1992a). The antibodies to detecting a - and b-actin. Differentiation resulted, as desmin and vimentin produced a set of prominent bands in expected, in downregulation of b-actin and upregulation of the 50-60 kDa range, and very weak bands in the 100-200 a -actin RNAs. The downregulation of vimentin and b-actin kDa range (Fig. 5). The reasons for the presence of the was more pronounced after six days in differentiation additional bands in the 50-60 kDa range observed for the medium, both in the G6 cell line and in cells from the E6 desmin, and in particular for the vimentin antisera, are not line, another myogenic clone derived from the same fetus known. They could possibly represent partial proteolysis or as G6 (Fig. 4). During the same period a -actin and myosin post-translational modifications, as previously observed heavy chain (MHC) mRNA levels were elevated, as (Granger and Lazarides, 1979), but may also be caused by expected. Interestingly, the reduction of b-actin mRNA cross-reactivity to other intermediate filament proteins in levels parallels that of vimentin in both experiments. To the same size range. In any case, the western blot experi- rule out any possibility of cross-hybridization between ment strongly argues against the possibility that the anti- Nestin expression in muscle development 1297

Fig. 6. Intracellular localization of intermediate filaments by double immunofluorescence experiments. Undifferentiated G6 cells were fixe d and stained with a rabbit polyclonal anti-nestin antiserum (a) and a mouse monoclonal anti-vimentin antibody (b); or with the anti-nestin antiserum (c) and a mouse monoclonal anti-desmin antibody (d). G6 cells after 3 days in differentiation medium were stained with the anti- nestin antiserum (e) and the anti-vimentin antibody (f); or with the anti-nestin antiserum (g) and the anti-desmin antibody (h). Note the perinuclear distribution of the intermediate filaments in the undifferentiated cells and the visualization of long fibrils in the sac-like myotubes in the differentiated cells. Nestin and vimentin or desmin networks are indistinguishable in the majority of cells. Bar, 10 mm. 1298 T. Sejersen and U. Lendahl nestin antiserum cross-reacts with lower molecular mass for PDGF b-receptor transcripts (Jin et al., 1993), and may intermediate filaments. reflect the effect of dramatic but transient hormonal changes Access to non-cross-reacting antibodies produced in dif- around the time of birth. ferent species made it possible to analyse the intermediate The expression pattern can thus be viewed as a transi- filament network in G6 cells by double-label immunofluo- tion from one type of intermediate filament to another, i.e. rescence cytochemistry for nestin and either of the other from nestin and vimentin during the early stages to desmin two intermediate filaments (Fig. 6a-h). In undifferentiated in the fully differentiated muscle. Developmental succes- cells all three antibodies revealed a typical intermediate fil- sions of intermediate filaments are also found in skin, where ament pattern, with strong perinuclear staining and fila- different keratin genes are expressed at particular stages of ments radiating out towards the periphery of the cell (Fig. keratinocyte maturation (Fuchs and Coulombe, 1992), and 6a-d). Most myoblasts displayed the three intermediate fil- in the developing CNS. Interestingly, in the early CNS aments, and the patterns of nestin and vimentin (Fig. 6a,b) nestin and vimentin are coexpressed in the mitotically or nestin and desmin (Fig. 6c,d) were indistinguishable in active neuroepithelium of the neural tube and, upon differ- the vast majority of cells, including areas with a more elab- entiation to neurons and glial cells, nestin is downregulated orate filamentous network. However, a variation in the rel- and replaced by neurofilaments and GFAP, respectively ative staining intensity between the individual intermediate (Lendahl et al., 1990). Thus, the transient expression of both filament proteins was noted in some cells. After in vitro nestin and vimentin, succeeded by the expression of another differentiation a large proportion of the myoblasts differ- intermediate filament in the differentiated cell type, is a entiated and fused into multinucleated myotubes (Fig 6e- common feature for both muscle and CNS development. It h). In some of the resulting myotubes, large flat sac-like appears, however, that the period of coexpression between structures were observed. Immunocytochemical staining nestin and the subsequent intermediate filament is more revealed the presence of nestin, vimentin and desmin in the extended in muscle than in CNS development. myotubes, including the flat structures. The filaments were Recent experiments have addressed how the regulatory much longer in the multinuclear cells, and sometimes ran regions mediating the CNS and muscle expression of nestin almost in parallel in the flat areas, while the perinuclear are organized. Various regions from the nestin gene were organization was less pronounced in multinuclear cells (Fig. fused to a reporter gene and analysed for expression in 6e-h). The filament pattern could be analysed in detail in transgenic mice. Two distinct regulatory regions were iden- the flat areas and, again, nestin appeared to colocalize with tified: the second is sufficient to reproduce the devel- vimentin (Fig. 6e,f) and desmin (Fig. 6g,h), producing oping CNS expression pattern, while the first intron directed indistinguishable patterns. lacZ expression to the myotomes of the somites in the embryo. The sequences in the first intron contain two so called E box motifs (ACACGTGG and GCAGCTGG), sug- gesting that nestin transcription may be regulated by bind- DISCUSSION ing of myogenic transcription factors of the helix-loop-helix type. Myogenic differentiation is closely associated with com- plex changes in morphology and cellular organization. It is Indistinguishable filamentous networks therefore reasonable to assume that changes in the compo- Nestin produces a cytoplasmic filamentous pattern indis- sition of the cytoskeleton are an inherent feature of this tinguishable from that of vimentin and desmin in the myo- process. To investigate this in more detail, we have genic cell line G6. Considering the evolutionary relation- analysed the expression pattern and intracellular localiza- ship between the genes this finding may not have been tion of the recently discovered intermediate filament nestin expected a priori. It has previously been shown that closely during skeletal muscle development. Our results show that related intermediate filament proteins, belonging to the nestin is expressed predominantly early, but with consider- same class, can copolymerize, whereas more distantly able temporal and spatial overlap with both vimentin and related proteins may not. Thus, the class III intermediate desmin, and that the three proteins produce cytoplasmic, fil- filament proteins vimentin and GFAP copolymerize (Sharp amentous networks that are indistinguishable by conven- et al., 1982), and vimentin and desmin are found in the tional immunocytochemistry. same filaments in cultured cells (Quinlan and Franke, 1982; Tölle et al., 1986). However, vimentin and , which Distinct expression patterns during muscle belong to different classes, form quite distinct cytoplasmic development structures (Franke et al., 1979). The class VI intermediate During muscle development the expression patterns for the filament nestin is evolutionarily less closely related to three intermediate filaments are distinct. Nestin mRNA is vimentin and desmin, and in fact belongs to the other main most abundant during early stages, and sharply downregu- branch of the cytoplasmic intermediate filament gene lated in the adult muscle, whereas desmin mRNA levels family, together with internexin and the neurofilaments increase throughout muscle development. Vimentin mRNA (Dahlstrand et al., 1992b). In the central a -helical domain, levels are also highest during the early stages, but the which is critical for polymerization of intermediate fila- expression profile is monophasic in contrast to nestin, which ments (see Stewart, 1993, for review), vimentin and desmin has a biphasic mode of expression, with reduced mRNA are 74 % conserved at the level. In contrast, levels around birth. This has previously been observed also nestin is only 28 and 30 % conserved to desmin and Nestin expression in muscle development 1299 vimentin, respectively, in this domain; yet the filament pat- is tempting to speculate that aberrant organization of nestin terns are indistinguishable. and desmin may contribute to the pathology of certain The intermediate filament network undergoes a dramatic myopathies. In support of this, several cases of myopathy reorganization from the primarily perinuclear organization have been reported to be associated with accumulation of in G6 myoblasts to the long parallel fibers in multinuclear large, dense desmin-containing sarcoplasmic bodies, not myotubes. The fact that nestin, vimentin and desmin pro- integrated into the normal intermediate filament network duce a common type of network before in vitro differen- (Edström et al., 1980; Pellissier et al., 1989; Prelle et al., tiation, and another, different form after differentiation, 1992; Rappaport et al., 1988). suggests that the reorganization takes place in a concerted Our data thus suggest that nestin is a bona fide compo- manner for the three intermediate filaments. It is reasonable nent of the dynamic intermediate filament network in skele- to assume that this is also the case in vivo, since nestin is tal muscle and, although we still know very little about its expressed in the same cells as vimentin and desmin during function, the data provide a more complete picture of the early muscle development, and since nestin and desmin are transitions in expression during muscle development. In found in the Z bands of postnatal myofibers. addition, the transient nestin expression pattern may be helpful in the diagnosis of pathogenic conditions accompa- A dynamic intermediate filament network in nied by muscle regeneration. Intermediate filaments have developing muscle been widely used in diagnosis of tumors and other patho- The described findings highlight a longstanding question: logical conditions because of their tissue-specific why are different intermediate filaments expressed at dif- expression and distinct cytoplasmic location (Osborn and ferent times during development of tissues like muscle, Weber, 1989). Transient expression of nestin in developing CNS (Lendahl et al., 1990) and skin (Fuchs and Coulombe, CNS and its reappearance in CNS tumors make nestin an 1992), when they form very closely related, if not identi- interesting CNS tumor marker (Dahlstrand et al., 1992a; cal, filamentous networks? One possibility is that the indi- Tohyama et al., 1992), and it will be interesting to learn if vidual intermediate filaments do not confer qualitative dif- nestin also reappears in myopathogenic situations. ferences on the filamentous network per se, but that the members expressed early are particularly suited to establish We thank Mrs Gabriella Dombos for excellent technical assis- the filamentous structures. This could be achieved by faster tance. This work was supported by grants from the Swedish polymerization kinetics or better de novo polymerization Cancer Society and Karolinska Institutets fonder (T.S., U.L.), the characteristics. In muscle, this may be the role of nestin and Swedish Child Cancer Fund (T.S.) and by the Swedish Medical Research Council, Margaret and Axel Ax:son Johnsons Stiftelse, vimentin, whereas desmin, found in the differented Knut and Alice Wallenbergs Stiftelse, Magn. Bergvalls Stiftelse, myotubes, may instead form a more robust and long-lived and Stiftelsen Lars Hiertas Minne (U.L.). network, better suited to be maintained in differentiated cells. It is interesting to observe that the order of interme- diate filament expression in developing CNS, i.e. transient REFERENCES nestin and vimentin expression, also fits this model, and that neurofilaments and GFAP may play special roles in the Bennett, G. S., Fellini, S. A., Toyama, Y. and Holtzer, H. (1979). differentiated CNS cells. Redistribution of intermediate filament subunits during skeletal An alternative view is that the various intermediate fil- myogenesis and maturation in vitro. J. Cell Biol.82, 577-584. Buckingham, M. E. (1985). Actin and myosin multigene families: Their aments in fact provide qualitative differences for the fil a- expression during the formation of skeletal muscle. Essays Biochem. 20, mentous structure, and that cells at different developmen- 77-109. tal stages have different requirements for their Côté, F., Collard, J.-F. and Julien, J.-P. (1993). Progressive neuronopathy cytoskeleton. At present, we cannot distinguish between in transgenic mice expressing the human neurofilament heavy gene: a these two models, and the question of detailed functions mouse model of amyotrophic lateral sclerosis. Cell 73, 35-46. Dahlstrand, J., Collins, V. P. and Lendahl, U.(1992a). Expression of the for the different intermediate filaments has been difficult class VI intermediate filament nestin in human central nervous system to approach. During myogenic differentiation in vitro tumors. Cancer Res. 52, 5334-5341. desmin and vimentin appear to be dispensable, and Dahlstrand, J., Zimmerman, L. B., McKay, R. D. G. and Lendahl, U. myotubes are formed in the presence of disrupted desmin (1992b). Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments. J. Cell Sci. 103, 589-597. and vimentin networks (Schultheiss et al., 1991), which Dodemont, H., Riemer, D. and Weber, K. (1990). Structure of an suggests that the functions may only be revealed in vivo. invertebrate gene encoding cytoplasmic intermediate filament (IF) The best evidence for intermediate filament function comes proteins: implications for the origin and the diversification of IF proteins. from the linkage between mutations in keratins and certain EMBO J. 9, 4083-4094. genetic skin diseases and experiments with mutated ker- Edström, L., Thornell, L.-E. and Eriksson, A. (1980). A new type of hereditary distal myopathy with characteristic sarcomplasmic bodies and atin genes introduced into transgenic mice (see Fuchs and intermediate (skeletin) filaments. J. Neurol. Sci. 47, 171-181. Coulombe, 1992, for review). In addition, overexpression Ferrari, S., Battini, R., Kaczmarek, L., Rittling, S., Calabretta, B., de of neurofilament genes in transgenic mice produces a Riel, J. K., Philiponis, V., Wei, J.-F. and Baserga, R. (1986). Coding motoneuron phenotype similar to that found in patients sequence and growth regulation of the human vimentin gene. Mol. Cell. Biol. 6, 3614-3620. with amyotrophic lateral sclerosis (Côté et al., 1993; Xu Franke, W. W., Schmid, E., Winter, S., Osborn, M. and Weber, K. et al., 1993). Similar experiments in transgenic mice with (1979). Widespread occurrence of intermediate-sized filaments of the nestin, vimentin and desmin may reveal more about their vimentin-type in cultured cells from diverse vertebrates. Exp. Cell Res. biological functions, interplay in vivo, and possible roles 123, 25-46. in neuromuscular disorders. Given the data on keratins it 1300 T. Sejersen and U. Lendahl

Fuchs, E. and Coulombe, P. A. (1992). Of mice and men: Genetic skin Quinlan, R. A. and Franke, W. W. (1982). Heteropolymer filaments of diseases of keratin. Cell 69, 899-902. vimentin and desmin in vascular smooth muscle tissue and cultured baby Gard, D. L. and Lazarides, E. (1980). The synthesis and distribution of hamster kidney cells demonstrated by chemical crosslinking. Proc. Nat. desmin and vimentin during myogenesis in vitro. Cell 19, 263-275. Acad. Sci. USA 79, 3452-3456. Granger, B. L. and Lazarides, E. (1979). Desmin and vimentin coexist at Rappaport, L., Contard, F. and Samuel, J. L. (1988). Storage of the periphery of the myofibril Z disc. Cell 18, 1053-1063. phosphorylated desmin in a familial myopathy. FEBS Lett. 231, 421- Holtzer, H., Bennett, G. S., Tapscott, S. J., Croop, J. M. and Toyama, Y. 425. (1982). Intermediate-size filaments: changes in synthesis and distribution Schultheiss, T., Lin, Z., Ishikawa, H., Zamir, I., Stoeckert, J. and in cells of the myogenic and neurogenic lineage. Cold Spring Harbor Holtzer, H. (1991). Desmin/vimentin intermediate filaments are Symp. Quant. Biol.46, 317-329. dispensible for many aspects of myogenesis. J. Cell Biol. 114, 953- Jin, P., Farmer, K., Ringertz, N. and Sejersen, T. (1993). Proliferation 966. and differentiation of human fetal myoblasts is regulated by PDGF-BB. Sharp, G., Osborn, M. and Weber, K. (1982). Occurrence of two different Differentiation 54, 47-54. intermediate filament proteins in the same filament in situ with a human Jin, P., Sejersen, T. and Ringertz, N. (1991). Recombinant platelet- glioma cell line. Exp. Cell Res. 141, 385-395. derived growth factor-BB stimulates growth and inhibits differentiation Steinert, P. M. and Liem, R. K. H. (1990). Intermediate filament of rat L6 myoblasts. J. Biol. Chem.266, 1245-1249. dynamics. Cell 60, 521-523. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly Stewart, M. (1993). Intermediate filament structure and assembly. Curr. of the head of the bacteriophage T4. Nature 227, 680-685. Opin. Cell Biol. 5, 3-11. Lendahl, U., Zimmerman, L. B. and McKay, R. D. G. (1990). CNS stem Tohyama, T., Lee, V. M.-Y., Rorke, L. B., Marvin, M., McKay, R. D. G. cells express a new class of intermediate filament protein. Cell 60, 585- and Trojanowski, J. (1992). Nestin expression in embryonic human 595. neuroepithelium and in human neuroepithelial tumor cells. Lab. Invest. Li, Z., Lilienbaum, A., Butler-Browne, G. and Paulin, D. (1989). Human 66, 303-313. desmin-coding gene: Complete sequence, characterization and Tokuyasu, K. T., Dutton, A. H. and Singer, S. J. (1983). Immunoelectron regulation of expression during myogenesis and development. Gene 78, microscopic studies of desmin (skeletin) localization and intermediate 243-254. filament organization in chicken skeletal muscle. J. Cell Biol. 96, 1727- Minty, A. J., Caravatti, M., Robert, B., Cohen, A., Daubas, P., Weydert, 1735. A., Gros, F. and Buckingham, M. (1981). Mouse actin messenger Tölle, H.-G., Weber, K. and Osborn, M. (1986). Microinjection of RNAs. J. Biol. Chem. 256, 1008-1014. monoclonal antibodies to vimentin, desmin, and GFA in cells which Olson, E. C., Brennan, T. J., Chakraborty, T., Cheng, T. C., Cserjesi, P., contain more than one IF type. Exp. Cell Res. 162, 462-474. Edmondson, D., James, G. and Li, L. (1991). Molecular control of Weber, K., Riemer, D. and Dodemont, H. (1991). Aspects of the evolution myogenesis: antagonism between growth and differentiation. Mol. Cell. of the /intermediate filament protein family: a current analysis of Biochem. 104, 7-13. invertebrate intermediate filament proteins. Biochem. Soc. Trans. 19, Osborn, M., Geisler, N., Shaw, G., Sharp, G. and Weber, K. (1982). 1021-1023. Intermediate filaments. Cold Spring Harbor Symp. Quant. Biol. 46, 413- Weydert, A., Daubas, P., Caravatti, M., Minty, A., Bugaisky, G., Cohen, 429. A., Robert, B. and Buckingham, M. (1983). Sequential accumulation of Osborn, M. and Weber, K. (1989). Cytoskeletal Proteins in Tumor mRNAs encoding different myosin heavy chain isoforms during skeletal Diagnosis. Cold Spring Harbor Laboratory Press, New York. muscle development in vivo detected with a recombinant Pellissier, J. F., Pouget, J., Charpin, C. and Figarella, D. (1989). identified as coding for an adult fast myosin heavy chain from mouse Myopathy associated with desmin type intermediate filaments. J. Neurol. skeletal muscle. J. Biol. Chem.258, 13867-13874. Sci. 89, 49-61. Xu, Z., Cork, L. C., Griffin, J. W. and Cleveland, D. W. (1993). Increased Prelle, A., Moggio, M., Comi, G. P., Gallanti, A., Checcarelli, N., expression of neurofilament subunit NF-L produces morphological Bresolin, N., Ciscato, P., Fortunato, F. and Scarlato, G. (1992). alterations that resemble the pathology of human motor neuron disease. Congenital myopathy associated with abnormal accumulation of desmin Cell 73, 23-33. and . Neuromusc. Disord. 2, 169-175. Zehner, Z. E. and Paterson, B. M. (1983). Vimentin Quax, W. J., van den Heuvel, R., Egberts, W. V., Quax-Jeuken, Y. E. F. during myogenesis: two functional transcripts from a single copy gene. M. and Bloemendal, H. (1984). Intermediate filament cDNAs from Nucl. Acids Res.11, 8317-8332. BHK-21 cells: Demonstration of distinct genes for desmin and vimentin Zimmerman, L., Parr, B., Lendahl, U., Gavin, B., Mann, J., in all vertebrate classes. Proc. Nat. Acad. Sci. USA 81, 5970-5974. Cunningham, M., Vassileva, G., McMahon, A. and McKay, R. (1994). Nestin expression in muscle development 1301

Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron (in press).

(Received 22 June 1993 - Accepted 23 August 1993)