Cell Tissue Res (1992) 267:99-104
Cell&Tissue Research 9 Springer-Verlag 1992
Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes Miranda D. Grounds 1, Kerryn L. Garrett 2, May C. Lai 2 Woodring E. Wright 3, and Manfred W. Beilharz 2 1 Department of Pathologyand 2 Department of Microbiology,Queen ElizabethII Medical Centre, The Universityof Western Australia, Nedlands, WesternAustralia,Australia 6009 3 Department of Cell Biologyand Anatomy,The University of Texas SouthwesternMedical Center at Dallas, Dallas, TX 75235, USA Received February25, 1991 / AcceptedJuly 20, 1991
Summary. The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoDl and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoDl and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo. Key words: Skeletal muscle - Myogenesis - Muscle regeneration - MyoDl - Myogenin - In situ hybridisation - Mouse (Swiss SJL/J)
The recent discovery of a family of skeletal muscle specific genes involved in the early stages of determination and differentiation of muscle cells has provided a powerful approach for investigating important aspects of myogenesis. The most extensively studied of these proteins have been MyoD1 which was originally isolated from mouse myoblasts (Davis et al. 1987; reviewed Weintraub Offprint requests to: M.D. Grounds
et al. 1991), and myogenin which was isolated from rat myoblasts (Wright et al. 1989). Until recently, cardiac ~-actin was considered to be the earliest muscle specific gene expressed by striated muscle precursor cells (mpc) during differentiation (Gunning etal. 1987; Sassoon etal. 1988; Lawrence et al. 1989), but it now appears that the MyoDl and myogenin gene products represent earlier and more precise markers for skeletal mpc (Hopwood et al. 1989; Sassoon et al. 1989; Scales et al. 1990). The expression of MyoD1 and myogenin (or their homologous genes) has been studied in vivo during embryogenesis in developing muscles of mice (Sassoon et al. 1989; Buckingham et al. 1991) and other species, but to date there have been no descriptions of MyoDl or myogenin gene expression during regeneration of mature muscle in vivo. Muscle precursor cells of mature uninjured muscle are essentially quiescent and are arrested in Go (or an equivalent state): this contrasts with the situation in embryonic muscle where populations of proliferating mpc are at various stages of differentiation (Sassoon et al. 1989). When mature skeletal muscle is injured or transplanted, the quiescent mononuclear mpc are activated, proliferate and fuse to form multinucleated young muscle cells (myotubes). It is widely considered that the mpc of mature muscle are derived exclusively from satellite cells which lie between the plasmalemma and external lamina of muscle fibres (Mauro 1961; Bischoff 1979; reviewed Mazanet and Franzini-Armstrong 1980); however, the possibility also exists that mpc might be recruited in vivo from cells other than satellite cells (reviewed Grounds 1990, 1991). To date, there are no markers which can readily identify the source of these undifferentiated mpc proliferating in vivo, or which can positively distinguish early mpc from other mononuclear cells present in the regenerating tissue (reviewed in Grounds 1991). It was therefore considered that the mRNA of the MyoD1 or myogenin genes might represent very early and specific markers for the identification of skeletal mpc in vivo in regenerating mature muscle. In this paper the transcription of these genes was examined in regener-
100 a f i n g a d u l t m o u s e m u s c l e b y use o f in situ h y b r i d i s a t i o n to localise m R N A for M y o D 1 a n d m y o g e n i n o n tissue sections.
Materials and methods
Muscle injury Thirty inbred Swiss SJL/J mice were used in experiments. Animals were housed in a clean, warm animal holding facility. Muscle trauma was induced in the tibialis anterior (TA) muscles of 24 adult (6-8 week old) male SJL/J mice by a single standard crush injury inflicted with artery forceps, as described by Grounds and McGeachie (1989). All surgical procedures were carried out under halothane anaesthesia. Animals were sacrificed at varying times from 0 to 12 days after injury (see Table l) and muscle tissue examined by in situ hybridisation for transcription of MyoD1 and myogenin mRNA.
Probes Riboprobes were prepared from a 1.8 kb full length cDNA MyoD1 clone (Davis etal. 1987) and a 1.5 kb cDNA myogenin clone (Wright et al. 1989). A further 0.7 kb myogenin probe was also prepared which lacked the c-myc homology domain present in both MyoD1 and myogenin. Anfisense and sense riboprobes of MyoD1 and myogenin, and the antisensc myogenin minus c-myc probe were labelled with 35S-UTP (Amersham) to a specific activity of 0.2-1 x 10 s cpm/gg of template DNA, then hydrolysed in carbonate buffer (0.4 M NaHCO3, 0.6 M Na2CO3, pH 10.2) at 60~ C to reduce the probe size to approximately 150 nucleotides.
In situ hybridisation Muscle tissue for in situ hybridisation was fixed in 4% paraformaldehyde for about 4 h and processed for embedding in paraffin wax. Sections 5 gm thick were cut and mounted on gelatin/chrome alum-subbed slides (Gall and Pardue 1971). Following removal
TaMe 1. Distribution of cells expressing MyoD1 mRNA at various times after crush injury to the tibialis anterior muscle of mice. - , 60 cells/zone. Results were averaged from duplicate slides in the same experiment and three independent in situ hybridisation experiments Time post injury (h) 0
6 12 18 24 36 48 96 120 168 a 192 ~ 288 a
Zone 1
Zone 2
Zone 3
Zone 4
--
--
~
--
+ ++ ++ + ++ ++ ++ ++ ++ _ +
+ +++ +++ +++ +++ +++ +++ +++ ++ + +
+ +++ +++ ++++ ++++ ++++ ++ ++ + +
+ ++ +++ ++ +++ § + -_ -
a Positive cells at these time points had a markedly weaker signal. Expression of myogenin followed a similar time-course pattern
of wax and rehydration, sections were post-fixed in 4% paraformaldehyde for 5 min, then treated with the following solutions with washes of phosphate-buffered saline (pH 7.2) between each treatment: 0.2 N HC1 2 min; 0.01% Triton X-100 1.5 min; 50 pg/ mI proteinase K (Boehringer-Mannheim, Penzberg, FRG) i5 rain at 37~ C; 0.09 M triethanolamine/0.25% acetic anhydride 10 rain. Sections were then dehydrated in graded concentrations of ethanol. RNase control slides were treated with 100 gg/ml RNase A (Sigma, St_ Louis, Mo., USA) in 300 mM NaCI, 10 mM TRIS at 37~ for 30 min prior to the acetylation step. Probe was applied to sections at 5 x 10a cpm/gl in hybridisation cocktail (Sassoon et al. 1989) under sealed glass coverslips and hybridised at 50~ for 16 h. Following hybridisation, coverslips were removed and the slides washed in four changes of double-strength saline sodium citrate (SSC) (SSC: 0.15M NaC1; 0.015M trisodium citrate, pH 7.0), containing 0.1% Triton X-100, 1 mM EDTA and 5 pM dithiothreitol at 60~ C for 15 min each, then in one-tenth-strength SSC with the same additions for a further 30 rain at 60~ C. To remove nonspecifically bound probe, slides were treated with 40 pg/ml RNase A (Sigma) and 10 U/ml T1 RNase (BoehringerMannheim) for 40 min at 37~ C, followed by six changes of doublestrength SSC at 60~ C for 10 min each. Dehydrated air-dried slides were coated in NTB-2 emulsion (Eastman Kodak Co., Rochester, N.Y., USA) exposed for 6-7 days at 4~ C, developed in D19 (Kodak), fixed in Hypam (Ilford) and stained lightly with Giemsa.
Analysis of sections The distribution of mRNA-positive cells throughout the regenerated muscle was analysed in zones as shown in Fig. I and Table 1.
Results In situ h y b r i d i s a t i o n s h o w e d t h a t M y o D 1 a n d m y o g e n i n genes h a d a s i m i l a r p a t t e r n o f e x p r e s s i o n o v e r the time c o u r s e e x a m i n e d . Cells p o s i t i v e for M y o D 1 o r m y o g e n i n m R N A were i n f r e q u e n t in u n i n j u r e d a d u l t T A m u s c l e (results n o t shown), a n d in T A m u s c l e e x a m i n e d i m m e d i ately after i n j u r y (Table 1, time zero). T h e small n u m b e r o f p o s i t i v e cells seen were m o n o n u c l e a r . Increased amounts of MyoDl and myogenin transcripts were a p p a r e n t in m o n o n u c l e a r cells as e a r l y as 6 h after i n j u r y a n d the n u m b e r s o f p o s i t i v e cells inc r e a s e d m a r k e d l y b y 24 h. T h e p o s i t i v e cells were initially c o n c e n t r a t e d in an a r e a lying a b o u t 1.6-2.6 m m p r o x i m a l to the site o f injury. Fig. 1 shows M y o D ] e x p r e s s i o n in a l o n g i t u d i n a l section 24 h after crush injury, a n d illustrates the z o n a l analysis used in the l o c a l i s a t i o n o f m p c s h o w i n g p o s i t i v e p r o b e h y b r i d i s a t i o n . Z o n e s were d e f i n e d a t i n c r e a s i n g d i s t a n c e s p r o x i m a l f r o m the site o f i n j u r y : they r e l a t e d o n l y to d i s t a n c e a n d n o t to the h i s t o l o g i c a l a p p e a r a n c e o f the tissue. T h e n u m b e r s o f cells in each zone giving positive signal were c o u n t e d a n d a r e t a b u l a t e d in Table 1. R e p r e s e n t a t i v e a r e a s for M y o D 1 m R N A e x p r e s s i o n i l l u s t r a t e d in Fig. 2 A - D corr e s p o n d to the g r a d i n g system u s e d in Table 1. Essentially the s a m e d i s t r i b u t i o n o f m y o g e n i n - p o s i t i v e cells w a s seen for all time points. R N a s e t r e a t m e n t o f c o n s e c u t i v e sections (Fig. 2 E ) r e m o v e d h y b r i d i s a t i o n signal. A f t e r 48 h, n u m b e r s o f p o s i t i v e m o n o n u c l e a r cells d e c l i n e d a n d s h o w e d a n a p p a r e n t shift t o w a r d the site o f crush i n j u r y (Table l). A f t e r 8 d a y s t h e n u m b e r o f p o s i t i v e cells was similar to p r o - i n j u r y levels. Positive h y b r i d i s a -
101
Fig. 1. In situ hybridisation of a MyoD1 riboprobe to a longitudinal section of mouse tibialis anterior muscle 24 h after crush injury. A-D correspond to zones 1-4 respectively,used for analysisin Table 1. The a r r o w denotes the crush site
tion was observed only in mononuclear cells and was very weak or absent in newly formed myotubes (Fig. 3). A myogenin probe that lacked the c-myc homology domain gave very similar results (Fig. 2F) to probes for MyoDl and myogenin which both contained the homology region.
Discussion
The use of in situ hybridisation shows the rapid induction and repression of MyoD1 and myogenin genes in mononuclear mpc in regenerating muscle. The low levels of mRNA for these genes in uninjured mature skeletal muscle are consistent with data of Wright et al. (1989) and Eftimie et al. (1991) who reported 15-fold less myogenin mRNA in adult compared with foetal mouse muscle. The parallel transcription of the genes for MyoD1 and myogenin in regenerating muscle suggests co-expression of these genes within individual mpc, although the data cannot exclude the possibility that MyoD1 and myogenin genes are being expressed in different populations of mpc. If the genes are being co-expressed within the same cell this indicates either a simple co-activation, or the in vivo transcriptional cross-activation that has been observed between MyoDl and myogenin in vitro (Braun et al. 1989; Thayer et al. 1989). Transcripts of both genes appear at the same time in developing muscles of mouse embryo limbs, although myogenin is expressed before MyoD1 in myotomal cells of the somites (Sassoon et al. 1989). In regenerating mouse muscle our previous autoradiographic studies show that mpc in vivo do not start to synthesise DNA before 24 h after injury, and that replication peaks around 3 days and is essentially finished by 6 days (Grounds and McGeachie 1989). The
present study shows a large number of cells transcribing MyoD1 and myogenin by 24 h, a period too short for significant mpc replication to have occurred. It would appear that these positive cells are probably initiating replication at the same time as they are expressing MyoD1 and myogenin genes. The data suggests that these genes are expressed by activated mpc, and that most of these mpc undergo at least one cell division before fusion, since myotubes are rarely seen before 6072 h after injury. Studies in embryonic mice also indicate that the expression of MyoD1 occurs in replicating mpc of developing muscles (Sassoon et al. 1989; discussed in Choi et al. 1990). The early appearance of MyoD1 and myogenin mRNA thus does not seem to represent a commitment by mpc in vivo to imminent terminal differentiation. Tissue culture studies indicate a complex interaction between the myogenic regulatory gene products and other helix-loop-helix proteins; one possibility is that a negative regulator such as Id (Benezra et al. 1990) might form heterodimers with the MyoD1/myogenin gene products and thereby affect DNA binding during the replicative phase of mpc in vivo. The rapid activation (by 6 h) of mpc distant from the site of direct injury suggests a response to a change in the environment of the muscle fibre as a result of injury which is rapidly translated throughout the length of the fibre (e.g., altered electrical properties, ion fluxes, release of proteases), before the influx of inflammatory cells and associated changes in growth factor availability occurs. It has been demonstrated that mpc can move both longitudinally down muscle fibres towards a site of injury (Schultz et al. 1985) and between regenerating fibres across connective tissue barriers (Phillips et al. 1990). In agreement with these observations, our results suggest that with time the population of activated mpc moves towards the site of injury (Table 1), and encounters changes in growth factor levels capable of stimulating replication (reviewed: Grounds 1991).
Fig. 2A-F. In situ hybridisation on muscle sections illustrates signal representative of the five grading levels - to + + + + shown in Table 1. A + 6 h post injury in zone I with antisense M y o D l probe; B + + 36 h post injury in zone 1 with antisense M y o D l probe; C + + + 24 h post injury in zone 2 with antisense MyoD1 probe; D + + + + 24 h post injury in zone 3 with antisense MyoD1 probe; E - - RNase control 24 h post injury in zone 3 with MyoD1 probe.
A to E are all at the same magnification. The grading system assesses the numbers of labelled cells, but there is also a variation in signal intensity which is evident in these photos. In A and B, arrows indicate the labelled mpc. F 48 h post injury with antisense myogenin probe, showing a zonal trend similar to that of MyoD1 expression
103 the myogenic process in vivo where the control of events would appear to be more tightly regulated than indicated by in vitro studies.
Acknowledgements. The excellent technical assistance of Marilyn Vague, and typing of the manuscript by Min Seats within the University Department of Pathology is gratefully acknowledged. We thank Terry Partridge for early discussions in relation to this work. This research was supported by grants from the National Health and Medical Research Council of Australia (M.D.G., M.W.B.), and an Australian Postgraduate Research Award (K.L.G.).
References
Fig. 3. High-power view of MyoDl-positive cells in zone 1 at 4 days post injury. Strong hybridisation is present in some mononuclear cells but this signal is clearly very low or absent in newly formed myotubes (arrows)
In our study, strong positive hybridisation was observed only in mononuclear cells and not in newly formed multinucleated myotubes. This result suggests that in vivo the transcription of these genes has ceased or has been strongly down-regulated at the time o f the fusion event. This contrasts with tissue culture studies where both MyoD1 and myogenin m R N A have been reported to persist in myotubes (Montarras et al. 1989; Thayer et al. 1989; Wright et al. 1989). Such differences between our in vivo and the in vitro data may be accounted for entirely by the marked differences between the environment and history of myogenic cells in regenerating muscle, and those of transfected and myogenic cell lines in tissue culture. In addition, an important aspect o f fusion between myogenic cells in vivo is fusion of myoblasts or myotubes with damaged myofibres (Robertson et M. ~990) and the influence o f innervation (Eftimie et al. 1991), a situation that does not arise in tissue culture. The transcription of the MyoD1 and myogenin genes provides a powerful tool for identifying activated mpc in vivo. Assuming that all o f the mpc do express MyoD1 and myogenin, one important implication is that the majority of mpc that contribute to regeneration may come from sites distant from the actual site of injury, which itself contributes relatively few cells. The movement of mpc is particularly relevant to potential myoblast transfer therapy which is an active area of research with clinical implications (Partridge et al. 1989; Hughes and Blau 1990; Partridge 1991). Present knowledge of mpc behaviour in vivo is only rudimentary and further work is required to define the source and nature of these cells. The observation o f a rapid decrease in M y o D l and myogenin m R N A upon myotube formation during regeneration, emphasises the importance of monitoring
Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H (1990) The protein Id: a negative regulator of helix-loop-helix DNA binding proteins, Cell 61:49-59 Bischoff R (1979) Tissue culture studies on the origin of myogenic cells during muscle regeneration in the rat. In: Mauro A (ed) Muscle regeneration. Raven Press, New York, pp 13-29 Braun T, Bober E, Buschhausen-Denker G, Kohtz S, Grzeschik KH, Arnold HH (1989) Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products. EMBO J 8 : 3617-3625 Buckingham M, Lyons G, Oft M-O, Catala F (1991) Myogenesis in the mouse. J Cell Biochem t 5C: 2 Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H (1990) MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc Natl Acad Sci USA 87:7988-7992 Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000 Eftimie R, Brenner HR, Buonanno A (1991) Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc Natl Acad Sci USA 88:1349-1353 Gall JG, Pardue ML (1971) Nucleic acid hybridisation in cytological preparations. Methods Enzymol 38:470--480 Grounds MD (1990) Factors controlling skeletal muscle regeneration. In: Kakulas BA, Mastaglia FL (eds) Pathogenesis and therapy of Duchenne and Becker muscular dystrophy. Raven Press, New York, pp 177-185 Grounds MD (1991) Towards understanding skeletal muscle regeneration. Pathol Res Pract 187:1-22 Grounds MD, McGeachie JK (1989) A comparison of muscle precursor replication in crush injured skeletal muscle of Swiss and BALBc mice. Cell Tissue Res 255 : 385-391 Gunning P, Hardeman E, Wade R, Ponte P, Bains W, Blau HM, Kedes L (1987) Differential patterns of transcript accumulation during human myogenesis. Mol Cell Biol 7:4100-4114 Hopwood ND, Pluck A, Gurdon JB (1989) MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J 8:3409-3417 Hughes SM, Blau HM (1990) Migration of myoblasts across basal lamina during skeletal muscle development. Nature 345:350353 Lawrence JB, Taneja K, Singer RH (1989) Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridisation. Dev Biol 133:235 246 Mauro A (1961) Satellite cells of skeletal muscle fibres. J Biophys Biochem Cytol 9:493-495 Mazanet R, Franzini-Armstrong C (1980) The satellite cell. In: Engel AG, Banker BQ (eds) Myology. McGraw-Hill, New York, pp 285-307
104 Montarras D, Pinset C, Chelly J, Kahn A, Gros F (1989) Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells. EMBO J 8 : 2203-2207 Partridge TA (1991) Myoblast transplantation: a possible therapy for inherited myopathies. Muscle Nerve 14:197-212 Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM (1989) Conversion of mdx myofibres from dystrophin-negative to dystrophin-positive by injection of normal myoblasts. Nature 337:176-179 Phillips GD, Hoffman JR, Knighton DR (1990) Migration of myogenic cells in the rat extensor digitorum longus muscle studied with the split autograft model. Cell Tissue Res 262:81-88 Robertson TA, Papadimitriou JM, Mitchell CA, Grounds MD (1990) Fusion of myogenic cells in vivo: an ultrastructural study of regenerating murine skeletal muscle. J Struct Biol 105:170182 Sassoon DA, Garner I, Buckingham M (1988) Transcripts of c~cardiac and c~-skeletal actins are early markers for myogenesis in the mouse embryo. Development ~04:155-164
Note added in proof. In latei~experiments using digoxigenin-labelled riboprobes, some persistance of MyoD1 and myogenin mRNA in newly formed myotubes was noted.
Sassoon D, Lyons G, Wright WE, Lin V, Lassar A, Weintraub H, Buckingham M (1989) Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341:303-307 Scales JB, Olson EN, Perry M (1990) Two distinct Xenopus genes with homology to MyoDl are expressed before somite formation in early embryogenesis. Mol Cell Biol 10:1516--1524 Schultz E, Jaryszak DL, Valliere CR (1985) Response of satellite cells to focal skeletal muscle injury. Muscle Nerve 8:217-222 Thayer MJ, Tapscott SJ, Davis RL, Wright WE, Lassar AB, Weintraub H (1989) Positive autoregulation of the myogenic determination gene MyoD1. Cell 58:241-248 Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S, Shuang Y, Lassar A (1991) The MyoD gene family: nodal point during specification of the muscle cell lineage. Science 251:671-766 Wright WE, Sassoon DA, Lin VK (1989) Myogenin a factor regulating myogenesis has a domain homologous to MyoDl. Cell 56:607 617
Cell&Tissue Research 9 Springer-Verlag 1992
Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes Miranda D. Grounds 1, Kerryn L. Garrett 2, May C. Lai 2 Woodring E. Wright 3, and Manfred W. Beilharz 2 1 Department of Pathologyand 2 Department of Microbiology,Queen ElizabethII Medical Centre, The Universityof Western Australia, Nedlands, WesternAustralia,Australia 6009 3 Department of Cell Biologyand Anatomy,The University of Texas SouthwesternMedical Center at Dallas, Dallas, TX 75235, USA Received February25, 1991 / AcceptedJuly 20, 1991
Summary. The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoDl and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoDl and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo. Key words: Skeletal muscle - Myogenesis - Muscle regeneration - MyoDl - Myogenin - In situ hybridisation - Mouse (Swiss SJL/J)
The recent discovery of a family of skeletal muscle specific genes involved in the early stages of determination and differentiation of muscle cells has provided a powerful approach for investigating important aspects of myogenesis. The most extensively studied of these proteins have been MyoD1 which was originally isolated from mouse myoblasts (Davis et al. 1987; reviewed Weintraub Offprint requests to: M.D. Grounds
et al. 1991), and myogenin which was isolated from rat myoblasts (Wright et al. 1989). Until recently, cardiac ~-actin was considered to be the earliest muscle specific gene expressed by striated muscle precursor cells (mpc) during differentiation (Gunning etal. 1987; Sassoon etal. 1988; Lawrence et al. 1989), but it now appears that the MyoDl and myogenin gene products represent earlier and more precise markers for skeletal mpc (Hopwood et al. 1989; Sassoon et al. 1989; Scales et al. 1990). The expression of MyoD1 and myogenin (or their homologous genes) has been studied in vivo during embryogenesis in developing muscles of mice (Sassoon et al. 1989; Buckingham et al. 1991) and other species, but to date there have been no descriptions of MyoDl or myogenin gene expression during regeneration of mature muscle in vivo. Muscle precursor cells of mature uninjured muscle are essentially quiescent and are arrested in Go (or an equivalent state): this contrasts with the situation in embryonic muscle where populations of proliferating mpc are at various stages of differentiation (Sassoon et al. 1989). When mature skeletal muscle is injured or transplanted, the quiescent mononuclear mpc are activated, proliferate and fuse to form multinucleated young muscle cells (myotubes). It is widely considered that the mpc of mature muscle are derived exclusively from satellite cells which lie between the plasmalemma and external lamina of muscle fibres (Mauro 1961; Bischoff 1979; reviewed Mazanet and Franzini-Armstrong 1980); however, the possibility also exists that mpc might be recruited in vivo from cells other than satellite cells (reviewed Grounds 1990, 1991). To date, there are no markers which can readily identify the source of these undifferentiated mpc proliferating in vivo, or which can positively distinguish early mpc from other mononuclear cells present in the regenerating tissue (reviewed in Grounds 1991). It was therefore considered that the mRNA of the MyoD1 or myogenin genes might represent very early and specific markers for the identification of skeletal mpc in vivo in regenerating mature muscle. In this paper the transcription of these genes was examined in regener-
100 a f i n g a d u l t m o u s e m u s c l e b y use o f in situ h y b r i d i s a t i o n to localise m R N A for M y o D 1 a n d m y o g e n i n o n tissue sections.
Materials and methods
Muscle injury Thirty inbred Swiss SJL/J mice were used in experiments. Animals were housed in a clean, warm animal holding facility. Muscle trauma was induced in the tibialis anterior (TA) muscles of 24 adult (6-8 week old) male SJL/J mice by a single standard crush injury inflicted with artery forceps, as described by Grounds and McGeachie (1989). All surgical procedures were carried out under halothane anaesthesia. Animals were sacrificed at varying times from 0 to 12 days after injury (see Table l) and muscle tissue examined by in situ hybridisation for transcription of MyoD1 and myogenin mRNA.
Probes Riboprobes were prepared from a 1.8 kb full length cDNA MyoD1 clone (Davis etal. 1987) and a 1.5 kb cDNA myogenin clone (Wright et al. 1989). A further 0.7 kb myogenin probe was also prepared which lacked the c-myc homology domain present in both MyoD1 and myogenin. Anfisense and sense riboprobes of MyoD1 and myogenin, and the antisensc myogenin minus c-myc probe were labelled with 35S-UTP (Amersham) to a specific activity of 0.2-1 x 10 s cpm/gg of template DNA, then hydrolysed in carbonate buffer (0.4 M NaHCO3, 0.6 M Na2CO3, pH 10.2) at 60~ C to reduce the probe size to approximately 150 nucleotides.
In situ hybridisation Muscle tissue for in situ hybridisation was fixed in 4% paraformaldehyde for about 4 h and processed for embedding in paraffin wax. Sections 5 gm thick were cut and mounted on gelatin/chrome alum-subbed slides (Gall and Pardue 1971). Following removal
TaMe 1. Distribution of cells expressing MyoD1 mRNA at various times after crush injury to the tibialis anterior muscle of mice. - , 60 cells/zone. Results were averaged from duplicate slides in the same experiment and three independent in situ hybridisation experiments Time post injury (h) 0
6 12 18 24 36 48 96 120 168 a 192 ~ 288 a
Zone 1
Zone 2
Zone 3
Zone 4
--
--
~
--
+ ++ ++ + ++ ++ ++ ++ ++ _ +
+ +++ +++ +++ +++ +++ +++ +++ ++ + +
+ +++ +++ ++++ ++++ ++++ ++ ++ + +
+ ++ +++ ++ +++ § + -_ -
a Positive cells at these time points had a markedly weaker signal. Expression of myogenin followed a similar time-course pattern
of wax and rehydration, sections were post-fixed in 4% paraformaldehyde for 5 min, then treated with the following solutions with washes of phosphate-buffered saline (pH 7.2) between each treatment: 0.2 N HC1 2 min; 0.01% Triton X-100 1.5 min; 50 pg/ mI proteinase K (Boehringer-Mannheim, Penzberg, FRG) i5 rain at 37~ C; 0.09 M triethanolamine/0.25% acetic anhydride 10 rain. Sections were then dehydrated in graded concentrations of ethanol. RNase control slides were treated with 100 gg/ml RNase A (Sigma, St_ Louis, Mo., USA) in 300 mM NaCI, 10 mM TRIS at 37~ for 30 min prior to the acetylation step. Probe was applied to sections at 5 x 10a cpm/gl in hybridisation cocktail (Sassoon et al. 1989) under sealed glass coverslips and hybridised at 50~ for 16 h. Following hybridisation, coverslips were removed and the slides washed in four changes of double-strength saline sodium citrate (SSC) (SSC: 0.15M NaC1; 0.015M trisodium citrate, pH 7.0), containing 0.1% Triton X-100, 1 mM EDTA and 5 pM dithiothreitol at 60~ C for 15 min each, then in one-tenth-strength SSC with the same additions for a further 30 rain at 60~ C. To remove nonspecifically bound probe, slides were treated with 40 pg/ml RNase A (Sigma) and 10 U/ml T1 RNase (BoehringerMannheim) for 40 min at 37~ C, followed by six changes of doublestrength SSC at 60~ C for 10 min each. Dehydrated air-dried slides were coated in NTB-2 emulsion (Eastman Kodak Co., Rochester, N.Y., USA) exposed for 6-7 days at 4~ C, developed in D19 (Kodak), fixed in Hypam (Ilford) and stained lightly with Giemsa.
Analysis of sections The distribution of mRNA-positive cells throughout the regenerated muscle was analysed in zones as shown in Fig. I and Table 1.
Results In situ h y b r i d i s a t i o n s h o w e d t h a t M y o D 1 a n d m y o g e n i n genes h a d a s i m i l a r p a t t e r n o f e x p r e s s i o n o v e r the time c o u r s e e x a m i n e d . Cells p o s i t i v e for M y o D 1 o r m y o g e n i n m R N A were i n f r e q u e n t in u n i n j u r e d a d u l t T A m u s c l e (results n o t shown), a n d in T A m u s c l e e x a m i n e d i m m e d i ately after i n j u r y (Table 1, time zero). T h e small n u m b e r o f p o s i t i v e cells seen were m o n o n u c l e a r . Increased amounts of MyoDl and myogenin transcripts were a p p a r e n t in m o n o n u c l e a r cells as e a r l y as 6 h after i n j u r y a n d the n u m b e r s o f p o s i t i v e cells inc r e a s e d m a r k e d l y b y 24 h. T h e p o s i t i v e cells were initially c o n c e n t r a t e d in an a r e a lying a b o u t 1.6-2.6 m m p r o x i m a l to the site o f injury. Fig. 1 shows M y o D ] e x p r e s s i o n in a l o n g i t u d i n a l section 24 h after crush injury, a n d illustrates the z o n a l analysis used in the l o c a l i s a t i o n o f m p c s h o w i n g p o s i t i v e p r o b e h y b r i d i s a t i o n . Z o n e s were d e f i n e d a t i n c r e a s i n g d i s t a n c e s p r o x i m a l f r o m the site o f i n j u r y : they r e l a t e d o n l y to d i s t a n c e a n d n o t to the h i s t o l o g i c a l a p p e a r a n c e o f the tissue. T h e n u m b e r s o f cells in each zone giving positive signal were c o u n t e d a n d a r e t a b u l a t e d in Table 1. R e p r e s e n t a t i v e a r e a s for M y o D 1 m R N A e x p r e s s i o n i l l u s t r a t e d in Fig. 2 A - D corr e s p o n d to the g r a d i n g system u s e d in Table 1. Essentially the s a m e d i s t r i b u t i o n o f m y o g e n i n - p o s i t i v e cells w a s seen for all time points. R N a s e t r e a t m e n t o f c o n s e c u t i v e sections (Fig. 2 E ) r e m o v e d h y b r i d i s a t i o n signal. A f t e r 48 h, n u m b e r s o f p o s i t i v e m o n o n u c l e a r cells d e c l i n e d a n d s h o w e d a n a p p a r e n t shift t o w a r d the site o f crush i n j u r y (Table l). A f t e r 8 d a y s t h e n u m b e r o f p o s i t i v e cells was similar to p r o - i n j u r y levels. Positive h y b r i d i s a -
101
Fig. 1. In situ hybridisation of a MyoD1 riboprobe to a longitudinal section of mouse tibialis anterior muscle 24 h after crush injury. A-D correspond to zones 1-4 respectively,used for analysisin Table 1. The a r r o w denotes the crush site
tion was observed only in mononuclear cells and was very weak or absent in newly formed myotubes (Fig. 3). A myogenin probe that lacked the c-myc homology domain gave very similar results (Fig. 2F) to probes for MyoDl and myogenin which both contained the homology region.
Discussion
The use of in situ hybridisation shows the rapid induction and repression of MyoD1 and myogenin genes in mononuclear mpc in regenerating muscle. The low levels of mRNA for these genes in uninjured mature skeletal muscle are consistent with data of Wright et al. (1989) and Eftimie et al. (1991) who reported 15-fold less myogenin mRNA in adult compared with foetal mouse muscle. The parallel transcription of the genes for MyoD1 and myogenin in regenerating muscle suggests co-expression of these genes within individual mpc, although the data cannot exclude the possibility that MyoD1 and myogenin genes are being expressed in different populations of mpc. If the genes are being co-expressed within the same cell this indicates either a simple co-activation, or the in vivo transcriptional cross-activation that has been observed between MyoDl and myogenin in vitro (Braun et al. 1989; Thayer et al. 1989). Transcripts of both genes appear at the same time in developing muscles of mouse embryo limbs, although myogenin is expressed before MyoD1 in myotomal cells of the somites (Sassoon et al. 1989). In regenerating mouse muscle our previous autoradiographic studies show that mpc in vivo do not start to synthesise DNA before 24 h after injury, and that replication peaks around 3 days and is essentially finished by 6 days (Grounds and McGeachie 1989). The
present study shows a large number of cells transcribing MyoD1 and myogenin by 24 h, a period too short for significant mpc replication to have occurred. It would appear that these positive cells are probably initiating replication at the same time as they are expressing MyoD1 and myogenin genes. The data suggests that these genes are expressed by activated mpc, and that most of these mpc undergo at least one cell division before fusion, since myotubes are rarely seen before 6072 h after injury. Studies in embryonic mice also indicate that the expression of MyoD1 occurs in replicating mpc of developing muscles (Sassoon et al. 1989; discussed in Choi et al. 1990). The early appearance of MyoD1 and myogenin mRNA thus does not seem to represent a commitment by mpc in vivo to imminent terminal differentiation. Tissue culture studies indicate a complex interaction between the myogenic regulatory gene products and other helix-loop-helix proteins; one possibility is that a negative regulator such as Id (Benezra et al. 1990) might form heterodimers with the MyoD1/myogenin gene products and thereby affect DNA binding during the replicative phase of mpc in vivo. The rapid activation (by 6 h) of mpc distant from the site of direct injury suggests a response to a change in the environment of the muscle fibre as a result of injury which is rapidly translated throughout the length of the fibre (e.g., altered electrical properties, ion fluxes, release of proteases), before the influx of inflammatory cells and associated changes in growth factor availability occurs. It has been demonstrated that mpc can move both longitudinally down muscle fibres towards a site of injury (Schultz et al. 1985) and between regenerating fibres across connective tissue barriers (Phillips et al. 1990). In agreement with these observations, our results suggest that with time the population of activated mpc moves towards the site of injury (Table 1), and encounters changes in growth factor levels capable of stimulating replication (reviewed: Grounds 1991).
Fig. 2A-F. In situ hybridisation on muscle sections illustrates signal representative of the five grading levels - to + + + + shown in Table 1. A + 6 h post injury in zone I with antisense M y o D l probe; B + + 36 h post injury in zone 1 with antisense M y o D l probe; C + + + 24 h post injury in zone 2 with antisense MyoD1 probe; D + + + + 24 h post injury in zone 3 with antisense MyoD1 probe; E - - RNase control 24 h post injury in zone 3 with MyoD1 probe.
A to E are all at the same magnification. The grading system assesses the numbers of labelled cells, but there is also a variation in signal intensity which is evident in these photos. In A and B, arrows indicate the labelled mpc. F 48 h post injury with antisense myogenin probe, showing a zonal trend similar to that of MyoD1 expression
103 the myogenic process in vivo where the control of events would appear to be more tightly regulated than indicated by in vitro studies.
Acknowledgements. The excellent technical assistance of Marilyn Vague, and typing of the manuscript by Min Seats within the University Department of Pathology is gratefully acknowledged. We thank Terry Partridge for early discussions in relation to this work. This research was supported by grants from the National Health and Medical Research Council of Australia (M.D.G., M.W.B.), and an Australian Postgraduate Research Award (K.L.G.).
References
Fig. 3. High-power view of MyoDl-positive cells in zone 1 at 4 days post injury. Strong hybridisation is present in some mononuclear cells but this signal is clearly very low or absent in newly formed myotubes (arrows)
In our study, strong positive hybridisation was observed only in mononuclear cells and not in newly formed multinucleated myotubes. This result suggests that in vivo the transcription of these genes has ceased or has been strongly down-regulated at the time o f the fusion event. This contrasts with tissue culture studies where both MyoD1 and myogenin m R N A have been reported to persist in myotubes (Montarras et al. 1989; Thayer et al. 1989; Wright et al. 1989). Such differences between our in vivo and the in vitro data may be accounted for entirely by the marked differences between the environment and history of myogenic cells in regenerating muscle, and those of transfected and myogenic cell lines in tissue culture. In addition, an important aspect o f fusion between myogenic cells in vivo is fusion of myoblasts or myotubes with damaged myofibres (Robertson et M. ~990) and the influence o f innervation (Eftimie et al. 1991), a situation that does not arise in tissue culture. The transcription of the MyoD1 and myogenin genes provides a powerful tool for identifying activated mpc in vivo. Assuming that all o f the mpc do express MyoD1 and myogenin, one important implication is that the majority of mpc that contribute to regeneration may come from sites distant from the actual site of injury, which itself contributes relatively few cells. The movement of mpc is particularly relevant to potential myoblast transfer therapy which is an active area of research with clinical implications (Partridge et al. 1989; Hughes and Blau 1990; Partridge 1991). Present knowledge of mpc behaviour in vivo is only rudimentary and further work is required to define the source and nature of these cells. The observation o f a rapid decrease in M y o D l and myogenin m R N A upon myotube formation during regeneration, emphasises the importance of monitoring
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