In situ hybridisation of a large repertoire of muscle-specific transcripts in fish larvae: the new superficial slow-twitch fibres exhibit characteristics of fast-twitch differentiation

Adult fish have two main skeletal muscle types anatomically separated in the myotome: a superficial layer of slow oxidative fibres (the red muscle)covering a deeper, larger mass of fast glycolytic fibres (the white muscle). Very often, a layer of fibres called intermediate fibres, identified on the basis of their histochemical properties, separates the slow and the fast muscle fibres. It is now well established that the embryonic formation of the superficial slow muscle fibres involves the lateral migration of adaxial cells, which flank the notochord, from the deep part of the myotome toward the lateral surface (Devoto et al.,1996; Stoiber, 1998). The fast muscle fibres of the myotomal muscle arise from non-migrating lateral presomitic cells that do not contact the notochord. The specification of the different muscle cell identities depends upon distinct levels of activity of the morphogens of the hedgehog family (Du et al., 1997; Blagden et al., 1997; Wolff et al., 2003). The early differentiation of embryonic muscle fibres in fish embryos involves a complex temporal sequence of gene activations (Xu et al., 2000; Hall et al.,2003) including transient expression of fast(Chauvigné et al., 2005; Bryson-Richardson et al., 2005)as well as slow (Chauvigné et al.,2005) muscle isoforms in superficial slow fibres and deep fast fibres, respectively. Following embryonic myogenesis, an expansion of the myotome occurs in fish larvae involving the recruitment of new muscle fibres in germinal zones (Veggetti et al.,1990; Johnston et al.,1998; Galloway et al.,1999; Rowlerson and Veggetti,2001). It has been shown that the addition of new slow fibres in the borders of the myotome does occur even in zebrafish mutants that lack hedgehog signalling (Barresi et al.,2001). This indicates that the mechanisms regulating the identity of the new slow fibres produced in larvae are distinct from those specifying the slow fate of adaxial cells in early embryos. In spite of the developmental importance of the muscle expansion occurring in the larvae, very little has been reported regarding the gene activations that lead to the differentiation of new muscle fibres and the maturation of original (embryonic) muscle fibres in post-hatching embryos. In this study, we took advantage of the identification of a large repertoire of muscle ESTs for examining the developmental expression of muscle genes in the expanding myotome of fish larvae. Our major finding is that the slow fibres that are added externally to the single superficial layer of embryonic slow muscle transiently express several fast muscle isoforms. This challenges the simple view that the superficial part of the developing fish myotome is composed of a population of muscle fibres with only slow characteristics.

Eggs from rainbow trout Oncorhynchus mykiss Walbaum were collected in the experimental facilities of the INRA Drennec fish farm(Finistère, France). After artificial insemination, eggs were incubated at 10°C in recirculating dechlorinated water. Chemical water parameters were regularly monitored. Oxygen levels were always above 98% saturation. Before fixation, larvae were rapidly anaesthetised with phenoxyethanol (Sigma,Poole, UK).

Fast myosin heavy chain, slow myosin heavy chain, fast myosin light chain 1 and fast myosin light chain 3 cDNAs have been characterised previously(Gauvry and Fauconneau, 1996; Rescan et al., 2001; Thiebaud et al., 2001). Other muscle-specific cDNAs have been identified from a large-scale, rainbow trout 3′ and 5′ sequencing project (AGENAE research program). Digoxigenin-labelled antisense RNA probes were synthesised from a PCR-amplified template using T3 RNA polymerase. Single and double in situ hybridisations were performed on transverse sections of rainbow trout embryos according to the method of Gabillard et al.(2003) with minor modifications. The double in situ hybridisations were performed with fluorescent markers. The green fluorescence was obtained, once the hybridisation was performed, by incubation of sections with mouse anti-digoxigenin antibody (Roche) followed by an incubation with Alexa Fluor 488-conjugated rabbit-derived anti-mouse IgG antibodies (Molecular Probes,Leiden, The Netherlands). The red fluorescence was obtained by incubation of sections with goat anti-fluorescein antibodies (Vector, Peterborough, UK)followed by incubation with Alexa Fluor 594-conjugated donkey-derived anti-goat IgG antibodies (Molecular Probes). FITC (revealing Alexa Fluor 488)and Texas Red (revealing Alexa Fluor 594) filters were then used for confocal microscopy. The confocal microscope system used in this study was a Leica TCS NT (Milton Keynes, UK) equipped with Kr/Ar laser and mounted on a Leica DMB microscope

At prehatching stages, around 27 d.p.f. (days post-fertilisation) the genes for the fast muscle-specific proteins [myosin heavy chain (MyHC), myosin light chain (MyLC1, MyLC3), troponin C, troponin I, myosin binding protein C and tropomyosin; Table 1) were uniformly expressed in the deep fast muscle fibres(Fig. 1A–C). From hatching (35 d.p.f.) onwards, while several myosepta separating adjacent W-shaped myotomes are visible in transverse sections, we observed a progressive rarefaction of the fast muscle transcripts within the fibres of the deep domain of the myotome while they were abundant in the fibres located in dorsal, ventral and lateral domains of the fast muscle mass.(Fig. 1D–I). Double in situ hybridisation with fluorescent antisense riboprobes revealed,in post-hatched embryos, that there was not a strict overlap of the labelling of the fast muscle isoform transcripts(Fig. 2A–C). This indicates that fast muscle isoform transcripts are present in differing proportions in fast muscle fibres.

To know more about the metabolic differentiation of the muscle fibres of the fish larvae, we investigated the developmental expression of several enzymes involved in muscle metabolism. At the eyed stage (20 d.p.f.),transcripts for citrate synthase, cytochrome oxidase component IV and succinate dehydrogenase, were present throughout the whole myotome. This indicated that the energy supply in the superficial slow and the deep fast muscle fibres depends mostly on aerobic metabolism. As the myotome matures, at around 45 d.p.f., these transcripts that encode enzymes of the aerobic metabolic pathway became confined to the superficial slow fibres and, to a lesser extent, to the lateral fast fibres(Fig. 3A–C). However,intense staining for enolase β, lactate dehydrogenase A and aldolase A transcripts, which encode enzymes regulating anaerobic glycogenolysis, were found throughout the whole fast muscle mass. Taken together, these observations demonstrate the emergence, during yolk resorption, of a distinct lateral subpopulation of fast aerobic fibres.

During the larval stages, the superficial slow muscle layer thickens by the addition of new layers of muscle fibres externally to the single superficial layer of original (embryonic) slow muscle fibres(Fig. 4). This recruitment is predominant in the regions closest to the lateral line. During this myotome expansion, slow myosins (light and heavy chains), slow troponin T and slow myosin binding protein C were expressed in both deep embryonic (original) and superficial neoformed slow fibres (Fig. 5A–D). However, unexpectedly, several fast isoforms RNAs,including MyLC1, MyLC3, MyHC, as well as fast myosin binding protein C, but not fast tropomyosin nor fast troponin I and fast troponin C, were found to selectively accumulate in the superficial neoformed slow muscle. This expression was first observed in 40 d.p.f. embryos, in a monolayer of small slow fibres covering the original embryonic slow fibres(Fig. 6A–D). In later stages of myotome expansion, whereas the slow muscle had thickened by the addition of multilayers of neoformed fibres, the expression of fast isoforms was observable only within the most superficial neoformed slow fibres and was no longer detectable in the deeper layers of neoformed slow fibres(Fig. 6E–H). This shows that the expression of fast isoforms was only transient in the neoformed slow fibres. Double in situ hybridisation using a MyLC1 probe specific for the slow twitch fibres and probes corresponding to fast MyLC1, fast MyLC3,fast MyHC and fast myosin binding protein C(Fig. 6I–K)further confirmed that the external fibres of the slow muscle exhibited both slow and fast characteristics.

Although the cellular mechanisms of post-embryonic muscle growth has been described in many fish species (for reviews, see Stoiber et al., 1999; Rowlerson and Veggetti, 2001)very little is known regarding the gene transcriptions that underlie the differentiation of newly recruited muscle fibres and the maturation of embryonic muscle fibres in fish larvae. In this study, to complete our knowledge on fibre differentiation during muscle expansion in fish larvae, we carried out single and double in situ hybridisation with a large repertoire of muscle-specific riboprobes. Our results show that around the time of hatching,slow and fast muscle isoforms, such as myosin, tropomyosin and troponin, were distributed throughout the slow and fast muscle, respectively. This spatial distribution of fast and slow muscle isoform transcripts is consistent with the previous identification of two major fibre types in recently hatched zebrafish larvae as shown by means of immunohistochemistry using anti-myosin sera (Van Raamsdonk et al.,1982). However, in the course of post-hatching development, a redistribution of transcripts for fast muscle isoforms was observed in the growing myotome: these latter accumulated mostly in the borders of the fast muscle mass. Furthermore, double in situ hybridisation revealed that the fast isoform transcripts accumulated in differing proportions within individual fast fibres, revealing an unexpected fibre diversity in fast muscle. In the light of observations on the spatial distribution of different sized white fibres in fish larvae (Veggetti et al.,1990; Rowlerson and Vegetti,2001; Galloway et al.,1999) and more especially in salmonid larvae(Stickland et al., 1988), it is likely that the fibres situated in the borders of the fast muscle and that accumulate high level of fast isoform transcripts correspond to muscle fibres added during myotome expansion of the trout larvae. In keeping with this growth pattern, it has been suggested that myogenic cells originating from the perimyotomal mesenchymal tissue are a possible source of new muscle fibres(Stoiber and Sänger,1996).

Regarding the metabolic differentiation, we observed that transcripts for enzymes of the aerobic metabolic pathways were present throughout the whole myotome at the eyed stage. This observation is consistent with the general statement that energy metabolism in embryos and larvae of fish is almost entirely aerobic (Wieser,1995). During subsequent maturation of the myotome, transcripts encoding enzymes of the aerobic metabolic pathway were found to selectively accumulate in the superficial slow oxidative fibres and, to a lesser extent,in the most lateral fast muscle fibres, which were also shown to express enzymes of glycolysis. Given their location and their metabolic properties, it is probable that these lateral fast fibres correspond to the presumptive intermediate or pink muscle previously identified in older fish on the basis of their histochemical properties, such as the pH sensitivity of the mATPase and intermediate SDH activity (Sanger and Stoiber, 2001). The appearance of fast aerobic muscle fibres at the end of yolk resorption in trout coincides with that observed in zebrafish and red sea bream (van Raamsdonk et al.,1982; Matsuoka and Iwai,1984). Recently, Wolff et al.(2003) have identified a subpopulation of fast fibres (MFFs) that express the Engrailed 1 and 2 homoproteins but not slow MyHC in 24 h post-fertilisation zebrafish embryos. Given the location of these Engrailed-expressing fast muscle fibres beneath the superficial slow fibres and in the vicinity of the horizontal myoseptum,it would be of interest to determine whether this subpopulation of fast fibres prefigures the aerobic fast muscle fibres. To our knowledge, so far, no intermediate muscle fibre-specific myosin isoforms have been reported,although peptide mapping has suggested they may exist(Scapolo and Rowlerson, 1987). The generalisation of in situ hybridisations of muscle-specific EST-derived riboprobes in various fish species will help, in the near future,to determine whether a specific contractile differentiation, if any, takes place in the intermediate muscle fibres.

Our major and unexpected finding was that a subset of superficial slow fibres exhibit fast characteristics. Indeed, simple and double in situ hybridisation showed that the small diameter slow fibres that are added laterally to the embryonic slow fibres co-expressed both slow and fast-twitch muscle isoforms, in particular fast myosin heavy and light chains. The expression of fast muscle isoforms in the superficial slow muscle is consistent with, and extends, the work by Johnston et al.(1998) who reported, using peptide mapping, the presence of adult fast myosin light chain isoforms in superficial slow muscle extracts of herring yolk-sac larvae. Our observations are also consistent with previous histochemical data reporting variable myosin ATPase (mATPase) activity between deep and peripheral slow muscle fibres of gilthead seabream larvae (Ramirez-Zarzoza, 1995), and support the pioneer work by Mascarello and colleagues(1995) showing heterogeneous myosin immunostaining in slow muscle fibres of developing sea bream larvae. It is important to note, however, that in contrast to fast growing large fish species such as trout or sea bream, small fish such as zebrafish, which are largely used for developmental studies, display limited recruitment of new slow myofibres from hatching onwards. Therefore, we can not exclude that some phenotypic variations may exist in post-embryonic muscle fibres, from one fish species to another, in relation to growth patterns.

The expression of fast twitch muscle isoforms in newly recruited slow fibres is only transient, showing that the final phenotype of the neoformed superficial fibres in larvae is ultimately slow. It is important to note that such a down-regulation of fast muscle genes has also been observed early in the adaxially derived embryonic slow fibres during trout(Chauvigné et al., 2005)and zebrafish (Bryson-Richardson et al.,2005) development. The expression of fast skeletal muscle isoform in embryonic muscle fibres destined to become slow has also been described previously in chick (Sweeney et al.,1989) showing that this property is largely shared among vertebrates. Thus the transient expression of fast muscle isoforms may be necessary to build the initial myofibrillar infrastructure in nascent slow muscle fibres before the complete replacement of fast muscle isoforms by slow ones. The gradual acquisition of the slow phenotype in newly recruited superficial fibres during larval life raises a question about the mechanisms controlling the developmental programme expression of muscle genes in these fibres. It is possible, as shown in differentiating primary and secondary myofibres in chick (Lefeuvre et al.,1996), that neuronal inputs resulting from the invasion of motoneurones in myotome participates to the regulation of muscle-specific gene transcription in fish larvae. In order to investigate this, it would be of interest to use surgical and pharmacological methods to interfere with innervation of the fish larvae myotome. Also, signalling molecules that are secreted by neighbouring tissues may influence the myogenic programme of newly recruited slow muscle fibres. In this regard, it is interesting to note that Wnt11, a member of a family secreted proteins implicated in vertebrate myotome patterning, is produced by the adaxially derived embryonic slow fibres(Makita et al., 1998). This inductive influence may regulate, through the stabilisation of cytosolicβ catenin (Seidensticker and Behrens,2000) the differentiation programme of myogenic cells that form new slow fibres laterally to the embryonic slow fibres. One of the best ways to test the hypothesis of such a regulation would be to examine the phenotypic properties of nascent myofibres during the larval period in the zebrafish mutant silberblick that lacks the Wnt11 signalling pathway(Heisenberg et al., 2000). Several other mutants such as pipetail, you too and Acerebellar that lack the Wnt5, sonic hedgehog and Fgf8 signalling pathways, respectively (Rauch et al.,1997; Ingham and Kim,2005; Reifers et al.,1998) may also be useful for examining the regulation of the myogenic programme of nascent slow fibres by signalling molecules.

The cellular source of the newly recruited larval slow fibres is not yet clearly identified. In fish embryos some external cells separate the superficial slow fibres from the epidermis. These cells, which have been described in zebrafish (Waterman,1969), herring (Johnston,1993), sea bass and sea bream(Veggetti et al., 1990; Lopez-Albors et al., 1998),have been proposed to be, during the larval period, a source of myoblasts contributing to the addition of new superficial slow muscle fibres in the region close to the lateral line (Veggetti et al., 1990). This hypothesis is supported by the recent observation that external cells on the surface of the fish myotome express the myogenic determination genes Pax3 and Pax7(Groves et al., 2005; Devoto et al., 2005). However,the fact that the external cells do not exhibit feature of muscle differentiation such as the presence of myofibrils and that they are positive for collagen I led to the suggestion that they form an epithelial cell layer sharing many characteristics with amniote dermatome(Rescan et al., 2005). These two interpretations are, however, not exclusive and it is quite possible that the external cell layer produces both myogenic and dermal precursors. If so,the fish external cell layer would form a structure homologous to the dermomyotome in the amniotes (Devoto et al., 2005). Nevertheless, it is clear that only a lineage tracing analysis can definitely demonstrate that the somitic external cell layer does produce myogenic precursors contributing to the recruitment of muscle fibres during fish larval stages.

This work was supported by grants from the Institut National de la Recherche Agronomique, OFIMER, IFOP and the CIPA. We thank François Piumi for providing the EST cDNA clones and Roselyne Primault for her help in the observation of sections by confocal microscopy (Service de Microscopie Electronique, Faculté de Médecine, Université de Rennes I, France).