Transient Expression of the Intermediate Filament Nestin During

Transient Expression of the Intermediate Filament Nestin During

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, vimentin and desmin 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 protein 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 cytoskeleton 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 proteins 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. genes, including the nuclear lamins, 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 internexin (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 keratin gene 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- actin and myosin 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 exon 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

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