DEVELOPMENTAL DYNAMICS 213:309–321 (1998) Expression of Myogenic Regulatory Factors During Muscle Development of Xenopus: Myogenin mRNA Accumulation Is Limited Strictly to Secondary Myogenesis NATHALIE NICOLAS, CLAUDE-LOUIS GALLIEN, AND CHRISTOPHE CHANOINE* Laboratoire de Biologie du Développement, Université René Descartes, Paris, France ABSTRACT To clarify the acquisition of the adult muscle pattern in Xenopus laevis, in situ hybridization and reverse transcriptase-polymerase chain reaction were used to correlate the time course of gene expression for myogenic regulatory factors (Myf-5, MyoD, and myogenin) with the expression of contractile protein (myosin heavy chain; MHC) genes during hindlimb formation compared with their expression in dorsal body muscles. After the precocious expression of Myf-5 and MyoD mRNA in limb bud (stage 50), myogenin mRNA strongly accumulated later at paddle stages (stages 52/53) concomitantly with the accumulation of both the larval and the adult MHC mRNAs. In dorsal body muscles, as early as stage 52, myogenin transcripts accumulated in a few small, secondary myofibers expressing the adult MHC mRNA that were located along the dorsomedial edge, but they were never detected in the large, primary myofibers of the body expressing the larval MHC mRNA. During metamorphosis, the areas expressing both the adult MHC and the myogenin transcripts gradually expanded from the dorsomedial edge to the ventral side of the dorsal body muscles, accounting for the progression of the secondary ‘‘adult’’ myogenesis described previously (Nishikawa and Hayashi  Dev. Biol. 165:86–94). This work shows that, in Xenopus, the accumulation of myogenin mRNA is restricted to secondary myogenesis, including the formation of new muscles in developing limbs as well as in dorsal muscles during body remodeling. This shows that myogenin is not required for primary myogenesis, and it suggests a crucial role for myogenin in the terminal differentiation program, including myoblast fusion and the activation of adulttype muscle genes. Dev. Dyn. 1998;213:309–321. r 1998 Wiley-Liss, Inc. Key words: myogenesis; myogenic regulatory factors; myogenin; myosin heavy chains; Xenopus INTRODUCTION During the development of most vertebrate skeletal muscles, multinucleate myotubes are formed by fusion of many myoblasts (Youn and Malacinski, 1981a). Howr 1998 WILEY-LISS, INC. ever, this does not occur during the myogenesis of the primary myotome muscle of Xenopus laevis (Muntz, 1975; Kielbowna, 1980; Youn and Malacinski, 1981b), in which myoblasts are seen to differentiate into uninucleate myotubes. Later, at the onset of metamorphosis, these develop into multinucleate muscle fibers. For a long time, the fate of the primary myotomal myofibers and the origin of the multinucleated secondary myotomal myofibers have been controversial (Kielbowna, 1966, 1980; Muntz, 1975). In 1987, Boudjelida and Muntz showed that multinucleated primary myotomal fibers in Xenopus arise from amitotic division of the primary nuclei and, thus, without cell fusion. More recently, Nishikawa and Hayashi (1995) showed that these primary myotomal myofibers die. In this scenario, secondary, multinucleated myofibers arise from fusion of recently migrated adult-type myoblasts during metamorphosis (Nishikawa and Hayashi, 1994). Likewise, limb muscles, which do not appear until the middle of metamorphosis, are characterized by a classic scheme of myogenesis, including cell fusion (Muntz, 1975; Dhanarajan and Atkinson, 1981). In amphibians, metamorphosis, during which many new muscles are formed, is also a phase of terminal muscle differentiation when some muscle structural genes, such as adult myosin heavy chains (MHCs), begin to be expressed (Chanoine et al., 1987; Radice and Malacinski, 1989) and when the full range of adult fiber types arises (Kay et al., 1988; Schwartz and Kay, 1988; for review, see Radice et al., 1989). The discovery of a family of skeletal muscle specific genes, the MyoD family, which is involved in the early stages of determination and differentiation of muscle cells, provided a powerful approach for investigating important aspects of myogenesis (Sassoon, 1993). Members of the MyoD gene family, including MyoD itself (Davis et al., 1987), myogenin (Wright et al., 1989), myogenic regulatory factor 4 (MRF4; Rhodes and Konieczny, 1989; Braun et al., 1990; Miner and Wold, 1990), and Myf-5 (Braun et al., 1989), encode basic helix-loop-helix (bHLH) proteins (DNA binding tran- Grant sponsor: Association Française Contre les Myopathies. *Correspondence to: Pr. C. Chanoine, Laboratoire de Biologie du Développement, Centre Universitaire des Saints-Pères, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France. E-mail: email@example.com Received 2 June 1998; Accepted 4 August 1998 310 NICOLAS ET AL. scription factors) that are able to convert nonmuscle cells to a muscle phenotype in culture (Choi et al., 1990; Schäfer et al., 1990) and that can promote the transcription of a number of muscle-specific genes (Weintraub et al., 1991). In mammals, Myf-5 and MyoD seem to intervene at the level of the precursor muscle-cell population, whereas myogenin and probably MRF4 intervene at the level of muscle differentiation (for review, see Buckingham, 1994). Xenopus homologs of MyoD (Hopwood et al., 1989; Scales et al., 1990), Myf-5 (Hopwood et al., 1991), myogenin, and MRF4 (Jennings, 1992) have been identified, and their temporal and spatial expression has been analyzed during early embryogenesis (for review, see Gurdon et al., 1992; Jennings, 1992). Surprisingly, no myogenin transcript has been detected during early muscle development in Xenopus, and it has been suggested that myogenin could be expressed later, during multinucleation of muscle fibers at the time of metamorphosis (Jennings, 1992). Nothing is known about the expression of the bHLH regulatory factors during multinucleation of the muscle fibers in developing Xenopus. For this reason, in this work, by using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR), we analyzed the expression of Myf-5, MyoD, and myogenin in relation to the expression of muscle structural genes in developing hindlimb and compared it with their expression in dorsal body muscles. Because it has been shown that myogenin is essential for in vivo myogenesis (Venuti et al., 1995), it was of particular interest in this study to analyze the expression of the myogenin gene during the late events of primary myogenesis and during the secondary myogenesis, which takes place during metamorphosis (Nishikawa and Hayashi, 1994). This work also offers a characterization of the distinct myogenic cell lineages in Xenopus. RESULTS Developing Hindlimb In Xenopus, hindlimb buds become visible at stage 48. From this stage to stage 52, there was no detectable hybridization with probes for any of the mRNAs for the myogenic regulatory factors (MRFs), nor was there any hybridization with probes for the larval and adult MHCs in the hindlimbs (not shown). Nevertheless, by using RT-PCR, transcripts for Myf-5 and MyoD began to be detectable from stage 50, whereas myogenin mRNA was first expressed later, at stage 52, a stage that is characterized by extensive growth of the limb (Fig. 1). The analyzed transcripts were coreverse transcribed and coamplified in the same reaction with the ubiquitously expressed elongation factor 1␣ gene (EF1␣; Krieg et al., 1989) serving as an internal control for the amount of RNA tested and for the RT-PCR reproductibility. Additional controls (see Experimental Procedures) indicated clearly that the generation of PCR products is strictly dependent on the synthesis of cDNA during the RT reaction and on the presence of both Fig. 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the myogenic regulatory factors and the normalizing expressed elongation factor 1␣ (EF-1␣) mRNA during hindlimb development. Stages are those of Nieuwkoop and Faber (1967). Myf-5 and MyoD, myogenin regulatory factors (MRFs). forward and reverse primers during subsequent PCR amplification. From stage 53, there was a dramatic change in the expression of the genes for the MRFs and the MHCs in the Xenopus hindlimb (Figs. 1, 2). Transcripts for the three myogenic regulatory factors, Myf-5, MyoD, and myogenin, were detected in the proximal parts of the paddle by using in situ hybridization. The intensity of hybridization was such that the signal for myogenin ⬎ MyoD ⬎ Myf-5. Simultaneously, the transcripts for larval and adult MHCs were expressed. This paddle stage corresponds to the time of the first appearance of multinucleated myotubes in the hindlimb (Muntz, 1975). At stage 54, when the forming digits began to be observed, all of the transcripts also accumulated in the distal parts of the hindlimb (Fig. 3). In the following stage 55, there was a dramatic decrease in the levels of Myf-5 mRNA, which were now only slightly detectable in the distal parts of the hindlimb (Fig. 4E). At this stage, a clear proximal-distal gradient in the expression of larval MHC mRNA was also evident. The level of signal with the probe for larval MHC was much less in proximal parts than that in distal parts of the hindlimb (Fig. 4A). From this stage, the intensity of hybridization was such that the signal for MyoD ⬎ myogenin ⬎ Myf-5. Dorsal Body Muscles From stage 48 to stage 62, the larval MHC mRNA accumulates in all the large myofibers of the body, whereas no hybridization signal for the adult MHC and for myogenin are observed (Figs. 2A, 3B, 5C, 6B). From stage 52, the adult MHC mRNA begins to accumulate in a few small myofibers located along the dorsomedial MUSCLE DEVELOPMENT AND MRFs IN XENOPUS 311 Fig. 2. A–F: Hindlimb bud at stage 53 reacted with cRNA probes as indicated. D is a brightfield photomicrograph of the same section that is shown in darkfield in E. Sections shown A–F are in serial order. All probes are specific for the transcript indicated. Autoradiographs were exposed to emulsion for 6 days prior to development for the myosin heavy chains (MHC) probes [(E3), larval MHC; (A7), adult MHC] and 12 days prior to development for the MRF probes (Myf-5, MyoD, and myogenin). Arrowhead in A indicates that body muscles strongly react with the larval MHC (E3) probe. Arrow in C indicates nonspecific background due to the pigment. edge (Fig. 5A,B) and the periphery of the body (Fig. 6A). These small myofibers do not express the larval MHC mRNA (Figs. 5C, 6B). From stage 48, by using RT-PCR, transcripts for MyoD, Myf-5, and myogenin were detected in dorsal body muscles (Fig.7). Because, in a previous work (Jennings, 1992), no myogenin transcript was detected either in embryos or in adult frogs, we carried out in situ hybridization analysis to determine the spatial localization of the myogenin mRNA. At stage 53 (Fig. 5E,F) and at stage 54 (Fig. 3 A,C), whereas Myf-5 and MyoD transcripts were detected in all the parts of the dorsal muscles, a more discrete hybridization signal was observed for myogenin mRNA. Myogenin transcripts were never detected in the large ‘‘larval’’ myofibers (Figs. 3D, 5D) or in the small ‘‘adult’’ myofibers at the periphery of the body (Fig. 6D). From stage 48, at high magnification, some isolated mononucleate cells located at the periphery of the large myofibers (expressing the larval MHC) were seen to express myogenin mRNA (Fig. 8). From stage 52, myogenin mRNA was detected in the small muscle cells expressing the adult MHC that were located along the dorsomedial edge (Fig. 5D). No particular localization of Myf-5 or MyoD transcripts was noted in these myogenin-expressing muscle cells compared with the ‘‘larval’’ fibers (Fig. 5E,F). At stage 61/62, during anatomical metamorphosis, the areas expressing the adult MHC transcripts gradually expanded from the dorsome- dial edge to the ventral side of the dorsal body muscle (Fig. 9B): The new-forming muscle mass strongly expressed the adult MHC mRNA. Myogenin mRNA was expressed only in these ‘‘adult-type’’ muscle cells (Fig. 9D). Some areas of necrosis were observed in the ‘‘larval’’ parts of the dorsal body muscles, as noted previously by Nishikawa and Hayashi (1995; see Fig. 9A). All of these results are summarized in Figure 10. DISCUSSION This paper provides a detailed spatial and temporal analysis of Myf-5, MyoD, myogenin, and MHC gene expression in developing hindlimb of Xenopus compared with their accumulation in dorsal body muscles during the same period. Differential Expression of MHC Genes in Hindlimb and Dorsal Body Muscles In Xenopus, it is known that muscle development is characterized by a transition from larval to adult MHCs at the time of metamorphosis (Kordylewsky et al., 1989; Radice and Malacincki 1989). The interesting work of Nishikawa and Hayashi (1994) greatly contributed to a better understanding of Xenopus myogenesis, because, by using immunohistochemical analysis, it showed that, before metamorphosis (stage 53), only a small number of muscle fibers at the dorsal part of dorsal muscle expressed adult-type muscle proteins 312 NICOLAS ET AL. Fig. 3. A–G: Sections of hindlimb at stage 54 (early digits) and attached myotomal muscles reacted with probes as indicated. E is a brightfield photomicrograph of the same section that is shown in darkfield in D. Sections in A–D are in serial order. G is a brightfield photomicrograph of the same section that is shown in darkfield in F. F is 100 µm away from D. Note that the intensity of the signal for Myf-5 is greater in distal parts than in proximal parts of the hindlimb. Arrowheads in D indicate nonspecific background due to pigment. (MHC and ␤-tropomyosin). Later, during metamorphosis, the adult-type area gradually expanded from the dorsal side to the ventral side in correlation with the degeneration of preexisting larval-type muscle fibers (Nishikawa and Hayashi, 1995; present study). In accordance with those authors, we show that, during pre- and prometamorphosis, the small myofibers located along the dorsomedial edge and the periphery of the body express adult MHC mRNA. Furthermore, we show that they do not express the larval MHC transcripts, which are detected only in the large myofibers of the body. This confirms the hypothesis proposed by Nishikawa and Hayashi (1994) that the myosin isoform transition, which occurs in Xenopus myotome at the onset of metamorphosis, is achieved by new proliferation of adult-type myoblasts and not by a change in gene expression within the same cell. In contrast, in the hindlimb, multinucleated myofibers coexpress larval and adult MHC mRNA, showing a more classic scheme of myogenesis, like that observed in mammals, which is characterized by a change in gene expression in the same myofiber. Myf-5 and MyoD mRNAs Are Expressed Precociously During Hindlimb Development This work shows that MyoD and Myf-5 mRNAs accumulate precociously during hindlimb development at stage 50, before the appearance of multinucleated myotubes. The initial appearance of myogenin mRNA occurs later, and it strongly accumulates at stage 53 concomitantly with the expression of muscle structural genes (i.e., MHC genes). The different timing of expression for the individual myogenic factors suggests that each factor has a distinct role during Xenopus myogenesis. Nevertheless, it appears that, during three types of myogenesis in Xenopus, including somitogenesis, regeneration, and limb development (Hopwood et al., 1989, 1991; Rupp and Weintraub, 1991; Nicolas et al., MUSCLE DEVELOPMENT AND MRFs IN XENOPUS 313 Fig. 4. A–F: Sections of hindlimb at stage 55 reacted with probes as indicated. D is a brightfield photomicrograph of the same section that is shown in darkfield in C. Sections in A–F are in serial order. There is a proximal-distal gradient for the accumulation of Myf-5 and larval MHC (E3) mRNA. Myf-5 mRNA is still detected only in the distal parts, whereas the intensity of the signal for the larval MHC is greater in distal parts than in proximal parts of the hindlimb. 1996, 1998, present study), MyoD and Myf-5 are the first myogenic factors that are expressed, suggesting their involvement in muscle determination or/and early muscle differentiation. Previous functional studies using ectopic expression of these myogenic factors supported this hypothesis (Hopwood and Gurdon, 1990; Hopwood et al., 1991; Ludolph et al., 1994). scripts were not detected either in embryos (from stage 1 to stage 47) or in adult frog muscle. From stage 48 to stage 61/62, corresponding to late larval development of the myotome, we never detected myogenin mRNA in the primary myofibers expressing the larval MHC. To our knowledge, this represents the only case of in vivo myogenesis without myogenin expression. The work of Boudjelida and Muntz (1987) stongly supported the idea that, during this period, the majority of primary myofibers become multinucleated by amitosis, the nuclei of the uninucleate myotomal myotubes being polyploid up to octaploid in Xenopus (Kielbowna, 1966). In a recent work, Myer et al. (1997), by using chimeric mice Myogenin mRNA Accumulates During Secondary Myogenesis but Not During Primary Pyogenesis In a previous report (Jennings, 1992), myogenin mRNA was never detected during early embryogenesis of Xenopus. Jennings indicated that myogenin tran- 314 NICOLAS ET AL. Fig. 5. A–F: Transverse sections of the body at stage 53 reacted with probes as indicated. The region of the dorsomedial edge at the level of hindlimb is shown. A is a brightfield photomicrograph of the same section that is shown in darkfield in B. Sections A–F are in serial order. Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA probes. Arrows indicate nonspecific background due to pigment. containing mixtures of myogenin null and wild type cells, showed that myogenin is required in vivo to establish an extracellular environment compatible with myoblast fusion. The fact that myogenin is not expressed during primary myogenesis of Xenopus could account for this specific type of myogenesis without cell fusion, which is characterized by the death of larval fibers (Nishikawa and Hayashi, 1994, 1995) without ‘‘terminal differentiation’’ (i.e., without expression of adult structural protein gene). Based on the fact that MUSCLE DEVELOPMENT AND MRFs IN XENOPUS 315 Fig. 6. A–D: Transverse sections of the body at stage 53 reacted with probes as indicated. Detail of the periphery of the body is shown. C is a brightfield photomicrograph of the same section that is shown in darkfield in A. Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA probes. Arrows indicate nonspecific background due to pigment. bHLH myogenic regulatory factors, to at least some degree, have functionally redundant roles in myogenesis (Weintraub, 1993), it is probable that at least one of the other bHLH MRFs could play the role of myogenin during primary myogenesis in Xenopus. In contrast to the expression observed during primary myogenesis, we showed that myogenin mRNA strongly accumulates during secondary myogenesis, including the formation of new muscles in developing limbs and the new myogenesis process involving the 316 NICOLAS ET AL. Fig. 7. Reverse transcriptase-polymerase chain reaction analysis of the myogenic regulatory factors and standardization-control EF-1␣ mRNA in dorsal body muscles at stages 48 and 55. Note that the level of myogenin mRNA is higher at stage 55 than at stage 48. construction of adult-type fibers during body remodeling. In homozygous null mutant mice with a myogenin (⫺/⫺) gene, the skeletal muscle displayed a marked reduction in myofibers (Hasty et al., 1993; Nabeshima et al., 1993). However, normal numbers of myoblasts were present, indicating an essential in vivo role for myogenin in the terminal differentiation of myoblasts into myotubes. Venuti et al. (1995) reported that, in mice, the absence of myogenin may affect secondary myogenesis more severely than primary myogenesis and suggested that secondary myofibers may require myogenin-dependent activation of a specific set of genes that cannot be activated by other MRFs. In Xenopus, the restricted localization of myogenin mRNA in secondary myofibers, expressing adult contractile protein genes (i.e., adult MHC), strongly suggests a crucial role for myogenin in the acquiring of the adult muscle pattern. The fact that the expression of myogenin and expression of the adult fast MHC are associated raises the question of the involvement of this particular myogenic regulatory factor, myogenin, in the activation of this MHC gene. In this regard, it should be noted that, during muscle regeneration of Xenopus, myogenin and adult fast MHC mRNA begin to be detected at the same time, during the myoblast-to-myotube conversion (Nicolas et al., 1996). In Xenopus regenerating muscles, myogenin mRNA is expressed transiently (Nicolas et al., 1996), which is also the case during development, because myogenin transcripts were not detected in adult limb and axial muscles (Jennings, 1992; unpublished observations). The fact that the small adult fibers located at the periphery of the body were detected precociously during premetamorphosis, before the anteroposterior progression of the adult type areas (during metamorphosis; see above) initiated from the dorsomedial edge, supports the hypothesis that the myogenin gene could be expressed transiently before premetamorphosis in the periphery of the body. Fig. 8. Photomicrographs at high magnification showing longitudinal sections of the body at stage 48 (A) and at stage 54 (B,C) reacted with myogenin cRNA probes. Arrowheads indicate some isolated cells expressing myogenin mRNA. What Is the Biological Significance of the Isolated Mononucleate Cells Expressing Myogenin mRNA in Dorsal Body Muscles? The presence of some isolated mononucleate cells expressing myogenin mRNA in the body gives rise to several different hypotheses. First, they could repre- MUSCLE DEVELOPMENT AND MRFs IN XENOPUS 317 Fig. 9. A–D:Transverse sections of the body at stage 62 reacted with probes as indicated. A is a brightfield photomicrograph of the same section that is shown in darkfield in B. Sections A–D are in serial order. Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA probes. Stars in A indicate areas of necrosis. sent migrating myoblasts involved in the construction of adult muscles. However, in all of the species analyzed to date, proliferating myoblasts do not express myogenin mRNA, which is associated with muscle differentiation during myotube formation (Buckingham, 1994). On the other hand, they could represent some activated satellite fusing with primary ‘‘larval’’ myofibers and contributing to the multinucleation process. Indeed, it has been established that, in most skeletal muscle, the satellite cells fuse with their associated muscle fiber to produce multinucleate cells (Moss and Leblond, 1970). In Xenopus myotome, Boudjelida and Muntz (1987) showed that satellite cells are visible first after the majority of the primary muscle fibers have become multinucleate. They appear to be in a dormant state, and their numbers increases up to stage 59. In the same work, electron microscopic investigation did not support the hypothesis that multinucleation in Xenopus myotome could result from fusion of the satellite cells with the fiber. We have shown previously that the quiescent satellite cells in adult muscle do not express bHLH myogenic regulatory factor mRNA (Nicolas et al., 1996), whereas myogenin mRNA was detected transiently in activated, fusing satellite cells following muscle injury. For this reason, we cannot exclude the fact that some satellite cells could take part in the multinucleation process. This hypothesis is not in opposition to the fact that division of the primary myonuclei by amitosis is responsible mainly for the multinucleation of the majority of the primary ‘‘larval’’ fibers. Evidence of Distinct Myogenic Programs in Xenopus This work showed that myogenesis in Xenopus is characterized by different myogenic programs in terms of expression of both myogenic regulatory factors and contractile protein genes (see Fig. 10). Myogenin is not expressed during the ‘‘early’’ differentiation, which leads to primary myofibers expressing the larval MHC. At the onset of metamorphosis, secondary myogenesis takes place, giving rise to adult-type fibers expressing myogenin and the ‘‘adult-type’’ muscle genes (Nishikawa and Hayashi, 1994; this study). The fact that primary and secondary myogenesis are characterized by both 318 NICOLAS ET AL. Fig. 10. Expression of MRFs (MyoD, Myf-5, and myogenin) and MHC genes in developing hindlimb of Xenopus compared with their expression in dorsal body muscles. Hatched areas represent transcripts that were detected during primary myogenesis. Open areas represent transcripts that were detected during secondary myogenesis. Stages are those of Nieuwkoop and Faber (1967; N&F). distinct morphological events (i.e., with or without myoblast fusion) and distinct expression of contractile protein genes should be correlated to the absence or the presence of myogenin expression, respectively. This probably accounts for different intrinsic properties of embryonic and adult-type myoblasts. On the other hand, the differences between primary and secondary myogenesis in terms of the expression of both myogenic regulatory factors and contractile protein genes, in part, may reflect environmental differences. In particular, it is known that thyroid hormone, the levels of which increase during metamorphosis, up-regulates the adult fast MHC mRNA (Sachs et al., 1996) and can modulate the expression of some myogenic regulatory factors (Hughes et al., 1993). Even if the origin of the myoblasts involved during secondary myogenesis is not clear in Xenopus (for review, see Radice et al., 1989), the fact that the myofibers coexpressed larval and adult fast MHC during limb formation, whereas the adult myofibers only expressed the adult fast MHC during body remodeling, supports the existence of two distinct myogenic cell lineages. raised in tap water. Embryos and larvae were staged according to Nieuwkoop and Faber (1967). EXPERIMENTAL PROCEDURES Animals Adult male and female Xenopus laevis were maintained at 22°C in tap water and fed once a week. Breeding pairs were injected twice at 8-hr intervals with 100 units or 500 units (for females) and with 50 units or 150 units (for males) of human chorionic gonadotropin (Paines and Byrne Ltd., London, United Kingdom). Fertilized eggs, embryos, and tadpoles were Preparation and Prehybridization of Tissue Sections The procedure for fixing, embedding, and sectioning tissues was the same as that used for mouse embryos and was essentially the same as described by Wilkinson et al. (1987) with some modifications (Ontell et al., 1993). Briefly, tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated, and infiltrated with paraffin. Then, 6-µm-thick serial sections were mounted on TESPA coated RNase-free glass slides (Sigma, St. Louis, MO). Sections were deparaffinized in xylene, rehydrated, digested with proteinase K, postfixed, treated with dithiothreitol/ iodoacetamine/N-ethylmaleimide (to reduce nonspecific 35S-binding; Zeller and Rogers, 1989), treated with triethanolamine/acetic anhydride, washed, and dehydrated. Probe Preparation The following probes were used to generate antisense cRNAs: 1) XMyoD template is a 598-nucleotide (nt), 38 fragment (BamHI-EcoRI; position 872–1,469) of XmyoD2–24 (Hopwood et al., 1989) subcloned in pGEM 4Z (Promega Biotec, Madison, WI), cut with BamHI, and transcribed with SP6 RNA polymerase. 2) Xmyogenin template is a 522-nt fragment corresponding to a DNA sequence subcloned into pGEM7Zf(⫹), plamid pCJMG2 (Jennings, 1992), linearized with SphI, and transcribed by using SP6 RNA polymerase. 3) pSP73- MUSCLE DEVELOPMENT AND MRFs IN XENOPUS XMyf-5–2 template (Hopwood et al., 1991) was linearized by using PstI and was transcribed by using SP6 RNA polymerase. The RNA probe was a 534-nt, 38 fragment (PstI-EcoRI, position 601–1,134). 4) Adult MHC template is the A7 cDNA clone (Radice and Malacinski, 1989) cut with ApaI and transcribed with SP6 polymerase. The RNA probe was a 330-nt, 38 fragment (PstI-EcoRI). A7 transcripts are expressed in fast-type fibers (Nicolas et al., 1996). 5) Embryonic MHC template is a 330-nt, 38 fragment (PstI-EcoRI) of the original E3 cDNA clone (Radice and Malacinski, 1989) subcloned into pGEM4Z (Promega Biotec), cut with HindIII, and transcribed with SP6 RNA polymerase. E3 transcripts are detected during embryonic as well as larval development (Radice and Malacinski, 1989). For this reason, in the text, we have named it larval myosin heavy chain transcript (larval MHC). cRNA probes were made by in vitro transcription in the presence of 50 µCi [35S]UTP at 1,200 Ci/mmol (NEN research product, Boston, MA), according to the manufacturer’s instructions (Promega Biotec). However, unlabeled UTP was omitted from the reaction medium in order to achieve synthesis of RNA probes with a specific activity of 109 cpm/µg. Probes were hydrolyzed to an average of 100 nucleotides by limited alkaline hydrolysis, according to Cox et al. (1984), for efficient hybridization, and used at 50,000 cpm/µl hybridization solution. Thirty microliters of hybridization solution were loaded per section. Hybridization and Washing Procedures High-stringency conditions for hybridization and posthybridization were followed. Sections were hybridized overnight at 53°C with posthybridization washing in 2 ⫻ standard saline citrate, 50% formamide, and 50 mM dithiothrietol at 65°C for 30 min. Autoradiography was carried out with Kodak NTB-2 track emulsion (Eastman-Kodak, Rochester, NY), developed in Kodak D19 developer, and stained lightly with Giemsa. Evaluation of Hybridization Signal To compare the intensity of hybridization signal with a given cRNA probe over time, sections at each stage were hybridized with the same probe preparation, washed, dipped into emulsion, exposed, and developed together. Changes in the level of hybridization signal with a given cRNA probe over time were evaluated by taking darkfield photomicrographs with a constant light intensity and a constant time of exposure of the film to the light source. When it was desirable to compare the relative intensity of hybridization signals of different probes, at a given stage, serial sections of the hind limb muscles were reacted with the different probes, processed for autoradiography, and developed at the same time. A similar protocol using darkfield photomicrographs (as described above) with a constant light intensity and a constant time of exposure of the film to the light source was used to compare the intensity of signal for these probes at a given develop- 319 mental stage. Hybridization signals over the muscles with sense (control) probes were not above the background due to pigment. Histology For the histological results presented, transverse frozen sections, 8 µm thick, were used. They were stained with hematoxylin and eosin. RNA Extraction Total RNA was purified by using the method of Auffray and Rougeon (1980) and was checked by agarose gel electrophoresis and ethidium bromide staining. RT-PCR RT-PCR was performed in one single tube, according to Goblet and Whalen (1995) and Lin-Jones and Hauschka (1996). First-Strand cDNA Synthesis One microgram of total RNA was used for RT-PCR for all primer pairs. The RNA was denatured briefly with 25 ng of random primer (Gibco BRL, Grand Island, NY) at 65°C for 5 min. Then, 1 ⫻ RT buffer (ATGC, Noisy-le-Grand, France), 1 mM each dNTP, 10 U of RNasin (Promega), and 200 units of AMV Reverse Transcriptase (ATGC) were added to a final reaction volume of 20 µl. RT reactions were incubated for 10 min at room temperature, for 60 min at 37°C, and for 5 min at 95°C. PCR Amplification The whole RT reaction was diluted to 100 µl final volume with 1 ⫻ PCR buffer (ATGC). Multiplex PCR reaction was performed with an MRF-specific set of primers, with an EF-1␣-specific sets of primers (see below), and with 2 units of Taq polymerase (ATGC). Samples were overlaid with paraffin oil (80 µl) and amplified in a thermocycler (Appligène, Illkirch, France). The cycling parameters were as follows: The initial cycle consisted of a 95°C denaturation for 5 min, 1 min at a 55°C annealing temperature, and 1 min at a 72°C extension temperature. The remaining cycles were for 30 sec at 95°C, 1 min at 55°C, and 1 min at 72°C, with the final cycle having a 10 min extension at 72°C. Due to the different abundance of EF-1␣ and MRF transcripts, we performed nine PCR cycles with MyoD or myogenin primer pairs or 12 PCR cycles with Myf-5 primer pairs, before adding the EF-1␣ primers and continuing amplification for 19 additional cycles. Onefifth of the PCR sample was electrophoresed on 6% polyacrylamide gels. DNA was transferred onto Hybond-C super membrane (Amersham, Buckinghamshire, United Kingdom). Southern blots were hybridized with MRF- and EF-1␣-radioactive probes. The amount of EF-1␣ PCR product was quantified by using a Bio-imaging analyzer and NIH Image software (Bethesda, MD). This calibration allowed us to adjust 320 NICOLAS ET AL. the volumes of the PCR reactions loaded on the polyacrylamide gel. PCR Primers XMyf-5. The primer pair 58-ACTACTACAGTCTCCCAGGACAGAG-38 (F) and 58-AGAGTCTGGAATAGGGAGGGAGCAT-38 (R) (positions 524–774; Hopwood et al., 1991) produced a fragment of 250 base pairs (bp) from cDNA. XMyoD. The primer pair 58-AACTGCTCCGATGGCATGATGGATTA-38 (F) and 58-ATTGCTGGGAGAAGGGATGGTGATTA-38 (R) (positions 662–952; Hopwood et al., 1989) produced a fragment of 289 bp from cDNA. Xmyogenin. The primer pair 58-CCAGCCCTTATTTCTTTTCAGACCA-38 (F) and 58-AATCCCTGAGCCCTGTAATAAAACC-38 (R) (positions 35–183; Jennings, 1992) produced a fragment of 147 bp from cDNA. EF-1␣. The primer pair 58-CCTGAATCACCCAGGCCAGATTGGTG-38 (F) and 58-GAGGGTAGTCTGAGAAGCTCTCCACG-38 (R) (positions 1088–1311; Krieg et al., 1989) produced a fragment of 222 bp from cDNA. Negative Controls Negative controls were performed with samples in which the reverse transcriptase or RNA or one of the primers was omitted to detect eventual DNA contamination. All of these controls remained constantly negative. ACKNOWLEDGMENTS We thank Drs. J.B. 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