SEARCH

SEARCH BY CITATION

Keywords:

  • myogenesis;
  • muscle growth;
  • dermomyotome;
  • Pax7;
  • MEF2;
  • fish muscle;
  • fiber type

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow-growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast-growing salmonid with exceptionally large embryos. Results confirm the three-phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second- and third-phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies. Developmental Dynamics 236:1106–1114, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The rate of somatic growth in developing fish is species-specific and can display great intraspecific variation in response to external factors such as temperature and food supply. Individual growth is determined and limited by the growth of the predominant tissue, the trunk muscle. In all vertebrates, muscle growth during development occurs by a combination of muscle fiber recruitment (hyperplasia) and the subsequent enlargement of these fibers (hypertrophy). Data from juvenile and adult fish indicate that the intensity and the time pattern in which a teleost species activates these two fundamental growth mechanisms are closely associated with the species' speed of growth and ultimate size. In large and/or fast-growing fish, hyperplasia and hypertrophy persist to a large body size (Weatherley et al.,1988; Steinbacher et al.,2006b), whereas small and slow-growing fish largely rely on hypertrophy and the rate of hyperplasia is rather low (Weatherley et al.,1988; Veggetti et al.,1993). However, knowledge is still poor as to how these interspecific differences in the utilization of the hyperplastic growth mode commence during the earliest stages of life.

It is well established in fish and other vertebrate groups that hyperplastic muscle growth during early ontogeny occurs in phases. The first phase is primary myotome formation from the paraxial mesoderm in the embryo. In teleost fish, this phase is traditionally subdivided into two parts: (1) Initially, before somite formation, myogenic precursors are determined adjacent to the notochord (adaxial cells) and develop during mediolateral migration to form the superficial slow fiber layer. (2) Subsequently, lateral paraxial cells within the newly formed somite give rise to fast fibers after they have been passed by the migrating slow fiber precursors (Devoto et al.,1996; Stoiber et al.,1998). This completed, a second phase of muscle fiber recruitment (“stratified growth”) begins to add new muscle cells to the myotome surface, at first creating growth zones at the dorsal and ventral extremes and at the lateral boundary of the fast muscle domain (Rowlerson and Veggetti,2001). A third phase involves muscle fiber recruitment between the fibers already existing at positions scattered throughout the myotome, thus giving a mosaic-like appearance to muscle cross-sections (“mosaic growth”). The intensity of mosaic growth and the time of its first appearance in development vary between teleost species, to the extreme of a complete lack of this growth mode in species that remain small in their ultimate size, such as the guppy Poecilia reticulata (Weatherley et al.,1988; Veggetti et al.,1993). In most species, mosaic growth is likely to begin only after the fish have entered the larval period (Rowlerson and Veggetti,2001).

Recent evidence from pearlfish, a large but slow-growing cyprinid, suggests that stratified hyperplasia during second-phase myogenesis depends substantially upon myogenic cells from the dermomyotome (DM; Steinbacher et al.,2006b; for characterization of fish DM, see Devoto et al.,2006). Myogenic cell allocation during mosaic hyperplasia is less well understood. Previous attempts of explanation include activation of residual satellite cell-like cells (Koumans and Akster,1995) and immigration of myogenic cell from the myotome surface (Stoiber and Sänger,1996). It remains unclear whether DM cells ultimately also drive mosaic growth, which is plausible and has been shown for “third-phase” myotome colonization by satellite cell precursors in the amniotes (Gros et al.,2005; Relaix et al.,2005).

Salmonid fish are known to develop to a large size during embryogenesis. They hatch at an advanced stage of development compared with many other fish and have the potential of a very fast posthatching growth (Blaxter,1988). In the present study, we combine molecular techniques and analysis of morphology (light microscopy, transmission electron microscopy [TEM]) to investigate myogenic cell activation during the three phases of myogenesis in embryos and larvae of brown trout, a fast-growing freshwater salmonid of large adult size (50–70 cm). Specifically, we examine MyoD and Myogenin gene expression in this species using in situ hybridization (ISH). These genes are both members of the myogenic regulatory factor (MRF) family of basic helix–loop–helix—containing transcription factors. MyoD is an essential regulator of muscle cell determination, whereas Myogenin is commonly regarded as a regulator of muscle cell differentiation. ISH for MyHCs and MyHCf is used for fiber type characterization of newly recruited muscle cells. This examination is supplemented by immunostaining for MEF2, another marker of myogenic cells, and for Pax7, a known marker of DM-related myogenic progenitor cells in amniotes.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The results presented are organized according to developmental stages, initially based on somite counts and later related to probable thresholds for muscle development (hatching, onset of free swimming, first exogenous feeding). Note that the correlation of somite numbers with developmental events (e.g., epiboly) is not directly comparable to that of the well-known zebrafish because of the difference in the final number of somites (brown trout about 62 rather than only 30–34 in zebrafish).

MyoD and Myogenin Expression During the Three Phases of Muscle Formation

For first-phase myogenesis, the present study has confirmed (results not shown) that the sequence and timing of MyoD and Myogenin expression in brown trout embryos occurs in a manner similar to that known for many other teleost species (zebrafish: Devoto et al.,1996, Weinberg et al.,1996; rainbow trout: Delalande and Rescan,1999; pearlfish: Steinbacher et al.,2006b). A secondary expansion of the myotome by recruitment of new muscle cells becomes evident when the fish approach the 40-somite stage. By this time, semithin sections of somites in which the adaxial cells are in the middle of their lateral migration consist of a medially located myotome and a lateral portion of cuboidal to columnar cells, which most likely include the dermomyotome (DM) (Fig. 1A,B). A more detailed examination reveals that the myotome is composed of dorsoventrally flattened myofibril-containing cells (most probably developing slow fibers) interdigitated with polygonal myofibril-free cells (most probably fast fiber precursors; Fig. 1C). Immunostaining using anti-myosin sera and ISH for MyHCs confirms that the somite centers already contain slow fibers with initial myofibril strands (Fig. 1D,E). In contrast to the flattened cells of the myotome centers, the dorsal extreme of the myotome exhibits a transition from flattened cells proximally to rounded or polygonal cells distally, which join the DM layer at the edge of the somite (Fig. 1A). This finding is the first indication of the formation of superficial growth zones (stratified hyperplasia, second-phase myogenesis) generated by DM-derived myogenic cells. The myogenic fate of the cells at the DM edges is confirmed by double-immunolabeling for Pax7 and MEF2 (albeit from a slightly more advanced state of somite differentiation, Fig. 1F). In somites that have not yet completed adaxial cell migration, it is unclear whether the DM is confined to a superficial monolayer (as in later stages), or whether it also includes the morphologically similar cells immediately underneath the superficial layer (Fig. 1B). Unfortunately, anti-Pax7 immunostaining fails to clarify this situation as Pax7 is not yet present at this time, whereas ISH for MyoD and Myogenin is in support of a myogenic nature of these cells (compare Fig. 1D,E,G,H). By contrast, ISH for MyoD and Myogenin fails to provide information on hyperplasia elsewhere in the myotome at this stage because the myotomes are entirely positive for these markers without further discrimination between old somitic cells and possible newly recruited cells (Fig. 1G,H).

thumbnail image

Figure 1. Embryos with 40–45 somites. A: Semithin section showing that the myotome is covered by a dermomyotome (DM) consisting of cuboidal to columnar cells. Inset: Transmission electron microscopy (TEM) photomicrograph of the dorsal extreme of the somite. There is a ventral to dorsal transition (asterisk) from flattened myotomal cells toward rounded or polygonal cells resembling the DM. B,C: Electron micrographs from the somite center: the DM is underlaid by one to two layers of DM-like cells (B); flattened myofibril-containing slow fibers are alternately stacked with myofibril-free fast fiber precursors (C). Arrows indicate myofibrils. D: The F59 antibody stains slow fiber myofibrils at high intensities, myofibril fragments in fast fibers at medium intensities. E: In situ hybridization (ISH) staining with MyHCs probe is confined to slow fibers. F: Double-immunolabeling for Pax7 (green) and MEF2 (red). The cells of the DM layer and some cells within the myotome are Pax7+. At the dorsal extreme, some of these cells also stain for MEF2 and Hoechst 33258 (blue) resulting in a white appearance (arrows). G:MyoD transcript is present within the entire myotome; the DM is unstained. H:Myogenin is expressed throughout the myotome. dm, dermomyotome; ep, epidermis; FF, fast fibers; myo, myotome; nc, notochord; SF, slow fibres; spc, spinal cord. Dorsal is at the top. Scale bars = 50 μm in A,D–H, 10 μm in inset in A, 20 μm in B, 2 μm in C.

Download figure to PowerPoint

A similar failure of MyoD and Myogenin to detect hyperplasia is evident in embryos with 50 and 60 somites in which the myotomes are still entirely stained by these probes (Fig. 2A,B). Semithin sections of these animals reveal a considerable expansion of the myotome in both the dorsoventral and the mediolateral direction (Fig. 2C). Within each myotome, the deep bulks of developing fast muscle are covered laterally by a monolayer of slow fibers (Fig. 2D). The fast muscle appears as an “unorderly” mixture of myofibril-containing fibers at different states of differentiation, interspersed with numerous cells that appear darker than the fibers by their more condensed cytoplasm and may be regarded as undifferentiated due to their lack of myofibrils and the occasional occurrence of mitotic division (Fig. 2C). The insertion of such undifferentiated cells between the pre-existing fibers is likely to be the first indication of mosaic hyperplasia (third-phase myogenesis). Lateral to the slow fibers, the myotome is still entirely covered by the DM. The lateral region of the DM layer is now thinner and consists of approximately isodiametric cells. The terminal regions still comprise columnar cells and bend over the myotome extremes where the DM cells appear to intermingle with small undifferentiated myotomal cells (Fig. 2C).

thumbnail image

Figure 2. Embryos with 60 somites. A,B:MyoD (A) and Myogenin (B) are expressed in all myotome cells (dermomyotome [DM] unstained). C: Semithin section of dorsal quadrant. A monolayer of radially oriented flattened slow fiber profiles constitutes the myotome surface. Fast muscle domains are interspersed with less differentiated cells that may still undergo mitotic division (inset). The lateral layer of cuboidal DM cells seems to merge into the myotome at the dorsal extreme (arrow). D: The 4/96-3c antibody stains slow fiber myofibrils at high intensities, fast fiber myofibrils at medium intensities. dm, dermomyotome; ep, epidermis; FF, fast fibers; myo, myotome; nc, notochord; SF, slow fibers; spc, spinal cord. Dorsal is at the top. Scale bars = 50 μm.

Download figure to PowerPoint

In prehatching trout embryos that have acquired their final number of somites, expression of MyoD and Myogenin begins to decrease in the fibers of the myotome centers but persists at the dorsal and ventral extremes and in some small cells in the centers and along the lateral border of the fast muscle domains (Fig. 3A,B). At the latter sites, transcript concentrations of Myogenin are usually much higher than those of MyoD (compare Fig. 3A with B). When hatching is imminent, further cells containing the MyoD and Myogenin transcript arise at the lateral surface of the slow muscle insertion at the horizontal septum (next to the lateral line nerve; Fig. 3C). These cells are also positive for MyHCs (see Fig. 4E), thus indicating the onset of formation of the multilayered slow muscle wedge that is found at this site in more advanced stages (see below). The detection of myogenic cells by ISH is confirmed by immunostaining for MEF2 and examination of semithin sections. The latter show small (presumably new) cells at the myotome extremes, and at the lateral boundary and also scattered over the interior of the fast muscle (Fig. 3D). Combined immunolabeling for MEF2 and the DM-related muscle progenitor marker Pax7 reveals that some double-labeled cells also occur at the extremes of the myotome and in the myotome centers (Fig. 3E). A continuous DM cover of the myotome is maintained and is also Pax7+ (Fig. 3D,E). The lateral DM cells now begin to take a squamous shape, and those at the still inward-curved dorsal and ventral terminations are cuboidal and appear to merge into the underlying myotome.

thumbnail image

Figure 3. A–K: Animals shortly before or at hatching (A–E), at onset of free swimming (F–H), and at first feeding (I–K). Stratified and mosaic hyperplasia shown on cross-sections hybridized with MyoD (A,F,I) and Myogenin (B,C,G,J) probe, immunolabeled with Pax7 (red) and MEF2 (green), and counterstained with Hoechst 33258 (blue, E), and on semithin sections (D,H,K). Inset in H: Transmission electron microscopy (TEM) photomicrograph of a flattened cell on the lateral surface of a slow fiber, which is most probably a residual DM cell. Arrows indicate growth zone at the myotome extremes, open arrows indicate lateral fast muscle growth zone, arrowheads indicate mosaic growth within the fast muscle centers, open arrowheads indicate lateral slow muscle growth zone. Dorsal is at the top. dm, dermomyotome; FF, fast fibers; hs, horizontal septum; ln, lateral line nerve; ms, myoseptum; SF, slow fibers; spc, spinal cord. Scale bars = 100 μm in A–G,I,J, 50 μm in H,K.

Download figure to PowerPoint

thumbnail image

Figure 4. Fiber type determination of myogenic cells recruited during stratified and mosaic muscle growth using MyHC probes. A,B: The 60-somite embryos. A: Cytoplasmic domains of slow fibers stain for MyHCs; slow fiber myofibrils and fast fibers are unstained. B:MyHCf transcript is present within all fast fibers and within the mid-lateral slow fibers. C: Swim-up stage. MyHCs+ cells constitute the dorsal termination of the slow fiber layer (arrow) and are inserted between the slow fibers next to a myoseptum. D:MyHCf probe stains all fast muscle. E: First feeding stage. Spots reactive to MyHCs probe arise at the surface and within the zone of newly formed slow fibers next to the horizontal septum (open arrowheads). F: First feeding stage. Detail showing MyHCf expression within fast fibers and within newly formed slow fibers next to the horizontal septum (open arrowheads). Dorsal is at the top. FF, fast fibers; hs, horizontal septum; ms, myoseptum; nc, notochord; SF, slow fibers; spc, spinal cord. Scale bars = 100 μm.

Download figure to PowerPoint

In animals of the swim-up stage and at the onset of exogenous feeding, patterns of MyoD and Myogenin expression are similar to those described for the prehatching/hatching stage. Thus, transcript-containing cells are clustered at the myotome extremes and along the lateral surface of the slow fiber layer next to the horizontal septum and are inserted between the fibers of the myotome centers (Fig. 3F,G,I,J). Semithin sections reveal considerable further expansion of the fast muscle area, which has again improved its irregular mosaic cytoarchitecture. This finding is particularly true for the distal dorsal and ventral portions of the myotomes. Here, differentiated muscle fibers of various sizes (including very small) intermingle with less differentiated (myofibril-free) small cells that are inserted between them (Fig. 3H). Thin sheets of similarly undifferentiated, mainly flattened cells delimit the myotomes at their extreme dorsal and ventral curvatures. These curvatures are overlaid by loosely aligned squamous cells that may correspond to the previously much thicker terminal regions of the DM. There is no evidence of further existence of a continuous lateral DM layer but TEM analysis reveals that individual elongated cells that may be DM residues that have persisted at the slow muscle surface (inset in Fig. 3H). By the time of first feeding, the slow muscle insertion at the horizontal septum has clearly expanded by the addition of two or three layers of small new fibers (Fig. 3K).

MyHC Gene Expression as a Means of Characterizing Fiber Types of Newly Recruited Muscle Cells

MyHC isoforms are the most important molecular determinants of muscle fiber type (Wigmore and Evans,2002). Thus, ISH for MyHCs and MyHCf was used here to determine the fiber type of the muscle cells recruited during the three phases of myogenesis in the trout myotome at a very early state of fiber differentiation.

At the 50- and 60-somite stage, the MyHCs transcript is present within the cytoplasm of all slow fibers in the anal area myotomes examined (Fig. 4A). MyHCf expression occurs within all fast muscle (Fig. 4B). Additionally, MyHCf is detected in many slow fibers, especially in the mid-lateral area (next to the horizontal septum).

From the completion of somitogenesis in the prehatching stage onward, MyHCs expression ceases throughout most of the slow muscle layer but persists in a few cells at the dorsal and ventral terminations and next to the myosepta (Fig. 4C). By the time of hatching, small MyHCs+ spots also arise along the lateral surfaces of the slow muscle insertions at the horizontal septa. Such spots are more numerous by the time of first feeding when the insertions have added two to three layers of small new fibers (Fig. 4E). ISH for the detection of MyHCf in the more advanced stages from prehatching to first feeding reveals a continued uniform presence of the transcript in all fast muscle areas (Fig. 4D,F), including the growth zones at the dorsal and ventral extremes and at the lateral surface, which also stain for MyoD and Myogenin (cf. Fig. 3A–C,E, F,I,J), and at slightly reduced levels also within many slow fibers. In most slow fibers, expression of MyHCf has ceased by the time of first feeding, this with the exception of the small new fibers at the mid-lateral slow muscle insertion (Fig. 4F).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The present results using brown trout provide a comprehensive characterization of MyoD and Myogenin expression over all three of the phases of myogenesis. This finding supplements our previous study of myogenic cell recruitment during axial muscle formation in pearlfish using MEF2D expression (Steinbacher et al.,2006b). The information gained from the present study is also new and important because it refers to a fast-growing teleost fish of relatively large final size. Thus, our knowledge of developmental patterns is extended beyond that documented for slower growing and/or smaller species such as zebrafish and pearlfish (Barresi et al.,2001; Steinbacher et al.,2006b). The importance of this difference in growth rates for developmental patterns is only now becoming apparent. Linked to this, we have shown that the onset of the second and third myogenic phases is simultaneous, which is different to other previously known teleost patterns that exhibit progression of consecutive phases. Furthermore, we suggest that Pax7 is expressed in the myogenic cells that account for second- and third-phase myogenesis. In the remainder of this discussion, we address these results in more detail as they appear within each of the three myogenic phases, and finally, we consider some of the implications of the rapid somatic growth of salmonid fish for interpreting teleost growth strategies.

First-Phase Myogenesis: The Initial Formation of the Myotome

The present results reveal that first-phase myogenesis in brown trout occurs in a manner similar to that known for other teleost species (Devoto et al.,1996; Weinberg et al.,1996; Delalande and Rescan,1999; Steinbacher et al.,2006b). The similarities relate to the sequence and timing of muscle-specific gene expression and to the general patterns of slow and fast muscle formation within the somite. However, examination of the cellular patterns of slow muscle formation in brown trout provide a new, plausible explanation as to how slow and fast muscle cells change position during the mediolateral migration of the slow muscle cells. This examination is enabled by an open lattice arrangement of the laterally advancing slow muscle cells, which allows for a free passage of the fast muscle precursors to the medial side of the slow muscle cells. TEM analysis also demonstrates that the slow fiber front is riddled with gaps so that myofibril-containing slow fibers alternate with less developed myofibril-free fast fiber precursors (Fig. 1B,C). Recently, a similar but more regular slow fiber open lattice has been found in sturgeon embryos (Steinbacher et al.,2006a). We suggest that the trout may reflect the true situation of as to how the fast fibers gain their final position relative to the slow fibers in fish. This suggestion is in contrast to the apparently closed front of migrating slow muscle cells found in zebrafish and other cyprinids (Devoto et al.,1996; Stoiber et al.,1998), which may indeed be rather permeable.

Second-Phase Myogenesis (Stratified Hyperplasia)

The present study demonstrates that, after the first phase of myogenesis, MyoD and Myogenin expression is maintained in those areas of the myotome that are known to be stratified growth zones because of the presence of small (presumably new) cells detectable on semithin sections. These zones are at the myotome extremes, at the lateral boundary of the fast muscle, and at the lateral boundary of the slow muscle next to the horizontal septum (Fig. 3A–C,F,G,I,J). This finding suggests that MyoD and Myogenin transcripts are largely restricted to myoblasts and/or young myofibers that arise in these growth zones, thus making MyoD and Myogenin useful markers of myogenic cell recruitment during second-phase myogenesis.

The present study further shows that myotome expansion by stratified growth of the second-phase myogenesis in the brown trout begins at an early point in myotome development (similar to the pearlfish) but is much more vigorous than previously described in the pearlfish (Steinbacher et al.,2006b). This finding means that the appearance of small (new) muscle cells at the myotome extremes begins when the slow fibers have not yet finished their lateral migration (Fig. 1A–H). The early occurrence of stratified hyperplasia in the prehatching stage has been also found in other salmonid species (Atlantic salmon: Stickland et al.,1988; rainbow trout: Stoiber and Sänger,1996; Xie et al.,2001). The beginning of stratified growth at a point as early as in the brown trout has been recently detected in a slow-growing cyprinid fish, the pearlfish (Steinbacher et al.,2006b). This finding is in conflict with previous work on a variety of other teleost species suggesting a delay of stratified growth until the time of hatching or even until after first feeding (review Rowlerson and Veggetti,2001). However, in the light of the present findings, it may be speculated that a precocious onset of stratified hyperplasia as in brown trout and pearlfish is more common among teleosts than previously thought but has been overlooked through methodological limitations to (nonmolecular) techniques, which have little capacity to detect very small numbers of myogenic cells.

MyoD and Myogenin expression patterns do not provide any information as to the possible origins of the newly recruited muscle cells at the stratified growth zones. However, semithin sections suggest that small cells from the DM edges merge into the growth zones at the myotome extremes (Figs. 1A, 2C, 3D,H). Similarly, double-immunolabeling reveals that these sites contain cells in which Pax7 (a factor that occurs throughout the DM) is colocalized with MEF2 (a marker of myogenic cells; Figs. 1F, 3E). Thus, it may be hypothesized that at least some of the newly recruited muscle cells at these growth sites derive from the DM, as also suggested for the pearlfish (Steinbacher et al.,2006b). A continued supply of myogenic cells to second-phase hyperplasia by this mechanism would then benefit from the sustained existence of a columnar/cuboidal DM epithelium in the trout embryo. This finding would allow a much higher concentration of (mitotically competent) precursor cells per unit surface area than the quickly flattening DM of other teleost species, such as the pearlfish (Devoto et al.,2006; Steinbacher et al.,2006b).

Regarding fiber type classification of the newly recruited muscle cells, results are in accordance with the findings of other authors investigating various teleost species (e.g., Veggetti et al.,1990; Rowlerson et al.,1995; Galloway et al.,1999; Steinbacher et al.,2006b). ISH with MyHC probes confirms that the growth zones at the myotome extremes mostly generate fast fibers (Fig. 4D), whereas only a few slow fibers arise at the terminations of the superficial slow muscle layer (Fig. 4C). This arrangement drives the rapid and highly allometric dorsoventral growth of the slow and fast muscle types in this species resulting in the well-known recession of the slow muscle layer from the myotome extremes. The new muscle cells that appear at the lateral boundary of the fast muscle just beneath the superficial slow fiber layer are exclusively fast type (Fig. 4D,F). The new fibers at the lateral surface of the slow fiber monolayer next to the horizontal septum are most probably slow type, although they are positive for both MyHCs and MyHCf (Fig. 4E,F). They provide the means for the rapid establishment of the slow muscle wedge that exists at this site in adult teleosts. The finding that slow fibers of brown trout also initially bear features of the fast fiber phenotype is consistent with previous studies in zebrafish (Xu et al.,2000; Bryson-Richardson et al.,2005), rainbow trout (Rescan et al.,2001; Chauvigné et al.,2006), and pearlfish (Steinbacher et al.,2006b).

Third-Phase Myogenesis (Mosaic Hyperplasia)

Similar to the second-phase myogenesis, the present study demonstrates that MyoD- and Myogenin-expressing cells in the fast muscle centers are most likely myogenic and account for mosaic growth. This is again underpinned by morphological analysis (presence of small mosaic cells at various states of differentiation between the pre-existing fast fibers) and the immunostaining results (presence of MEF2+ cells at the same sites).

One of the most surprising results of the present study is that fast muscle mosaic hyperplasia in the brown trout begins exceptionally early and increases, just as with stratified hyperplasia, from the time immediately after completion of adaxial cell migration. By this time, semithin section analysis reveals that the fast muscle areas already represent a mixture of cells at varying states of myogenic differentiation including very small cells (Fig. 2C). This finding infers that, in this species, first-phase myogenesis is followed, without interruption, by the onset of second-phase and third-phase myogenesis simultaneously. Most other teleost species that have so far been analyzed initiate fast muscle stratified and mosaic hyperplasia consecutively. Usually, the onset of mosaic growth occurs in the advanced larval or early juvenile stages (review Rowlerson and Veggetti,2001). Some small teleosts such as the guppy (P. reticulata) appear to be entirely devoid of mosaic fast muscle hyperplasia (Veggetti et al.,1993). However, an early occurrence of mosaic growth before or at hatching has been reported for the rainbow trout (Nag and Nursall,1972; Stoiber and Sänger,1996) and the Atlantic salmon (Johnston and McLay,1997). This finding suggests an intrinsically “salmonid” feature of premature mosaic fast fiber recruitment that does not exist in other teleost groups.

The origin of the myogenic cells that account for mosaic hyperplasia in teleosts remains unclear. Here, we have been able to demonstrate that mosaic fast muscle in brown trout contains Pax7+ myogenic progenitors (Fig. 3E). Double-labeling with MEF2 indicates that most of the these cells are indeed myogenic. In amniotes, it has been shown that the Pax7+ cells that account for the third phase of myogenesis originate from the DM (Gros et al.,2005; Relaix et al.,2005). Thus, it is tempting to speculate that a similar situation may be present in fish. The precursor cells would then detach from the fish DM and migrate deeper into the fast muscle bulk before they differentiate. Such a process of precursor cell immigration from the myotome surface (and by means of the myoseptal gaps) has been proposed for the rainbow trout, albeit without reference to the DM (Stoiber and Sänger,1996). However, further research using lineage analysis techniques will be required to clarify this issue.

Some Implications for Teleost Growth Strategies

The present study demonstrates that myogenesis in the trout embryo is a multistage process occurring in three phases. This finding is in agreement with the situation in other teleosts (Rowlerson and Veggetti,2001; Steinbacher et al.,2006b) and also similar to multistage myogenesis in amphibians (Xenopus) and amniotes (chick, mouse; e.g., Venters et al.,1999; Grimaldi et al.,2004; Cinnamon et al.,2006). However, in trout, there is the important difference of a precocious activation of third-phase myogenesis simultaneously with the second phase, occurring immediately after the first phase (see above). Third-phase mosaic hyperplasia is known to be responsible for the strong increase of muscle mass in teleost larvae and juveniles (Rowlerson and Veggetti,2001) and for sustained growth of the adults until fish have attained approximately 44% of their maximum size (Weatherley et al.,1988). Thus, mosaic growth occurs over a much longer period in large fish during their development than in smaller fish. The present study now provides evidence that this growth mode is also a key determinant of somatic growth during embryonic life. If activated early in myogenesis, as in the brown trout, the increase of embryonic body mass is rather high. If activated only after a considerable delay, embryonic body mass increase is low to moderate, even in species that attain large adult size, as the pearlfish (Steinbacher et al.,2006b). The coincidental and vigorous activation of the second- and third-phase myogenesis early in the embryonic period may provide a more efficient strategy to establish a large potential for posthatching (juvenile) body growth than the successive and more delayed activation of these phases in the embryos of other fish (e.g., cyprinids). In salmonids, the capability of intense embryonic body growth may be an intrinsic evolutionary prerequisite to allow for individual survival at later life stages, particularly in the cannibalistic fry of these fish. This strategy would then supplement maternal traits such as egg size and timing of reproduction that have been identified in studies of other salmonid species as key factors that influence the offspring's competitive ability, dispersal, foraging, vulnerability to predation, and sensitivity to climatic conditions in the larval period (e.g., Einum and Fleming,2000; Sundström et al.,2005).

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

All brown trout (Salmo trutta lacustris [Salmonidae]) used in the study were laboratory reared from artificially inseminated eggs. Parent animals were caught in the Mondsee lake near Salzburg, Austria. Rearing temperature increased from 6.5°C at the time of fertilization to 8°C at the embryo/larva transition (± 0.5°C) as a result of natural seasonal changes. Rate of water flow was kept constant throughout the experiment. Larvae were offered commercial fish food of appropriate particle size. Samples (n ≥ 6) of successive developmental stages were taken within the period from 30% epiboly in the embryo to the larvae just after onset of exogenous feeding. All animals were overanesthetised with MS-222 (Sigma) and, before fixation, larger individuals were cut into smaller pieces to allow sufficient fixative penetration.

Specimens intended for whole-mount ISH were fixed in phosphate (PBS) buffered 4% paraformaldehyde (8 hr; 4°C), rinsed twice in PBS, and subjected to a graded transfer into 98% methanol in which they were stored at −20°C until required for further processing. Procedures applied were after Wilkinson (1992), with minor modifications (Steinbacher et al.,2006b). Plasmids with cDNA encoding for rainbow trout MyoD1 isoform, Myogenin, MyHCf, and MyHCs were provided by P.-Y. Rescan, INRA Rennes, France. Nucleotide sequences have been described by Gauvry and Fauconneau (1996), Rescan et al. (1995), and Rescan et al. (2001). Plasmid linearization and synthesis of digoxigenin-labeled antisense and sense riboprobes followed the protocol of Sive et al. (2000). Stained specimens were photographed through a Leica Wild M10 stereomicroscope.

For ISH and immunostaining of sections, freshly killed animals were cryofixed by plunging into 2-methyl-butane cooled to near its freezing point (−158°C). Serial 10 μm transverse and horizontal sections were cut on a Leitz 1720 cryostat and collected on silane-coated slides, dried for 1 hr at room temperature and stored under liquid nitrogen until required for further processing. Details of the ISH protocol are given in Steinbacher et al. (2006b).

Primary antisera used for immunostaining were the following: murine monoclonal IgG1 against chicken fast myosin (F59, provided by F. Stockdale, Stanford University, Stanford, California), diluted 1:10; rabbit polyclonal anti-teleost slow myosin (4/96-3c, provided by A. Rowlerson, King's College London, UK), diluted 1:500; murine monoclonal IgG1 anti-chicken Pax7 (DSHB), diluted 1:20; and rabbit polyclonal anti-human MEF2 (Santa Cruz), diluted 1:100. Secondary antibodies applied were Cy3-labeled rabbit anti-mouse IgG (1:100; Jackson), Alexa 568- or Alexa 488-conjugated goat anti-rabbit (1:800; Molecular Probes), and Alexa 488- or Alexa 546-conjugated goat anti-mouse IgG1 (1:800; Molecular Probes). The detailed protocol is provided in Steinbacher et al. (2006b).

Specimens for semithin section histology and TEM were processed as previously described by Stoiber et al. (1998,2002). Transverse sections were cut using a Reichert Ultracut S microtome. Semithin sections (1.5 μm) were mounted on glass slides and, after de-resination with sodium methylate, stained with a combination of azure II–methylene blue and basic fuchsin (Stoiber et al.,1998). Results were photographed through a Reichert Polyvar microscope. Ultrathin sections (60–80 nm) were mounted on Formvar-coated 75-mesh copper grids, contrasted with aqueous solutions of uranyl acetate and lead citrate and viewed with a Zeiss EM 910 electron microscope at 80 kV. Images were photographed digitally using Gatan Image Acquisition software. From the 40-somite stage onward, all sectioning performed in this study was confined to positions from mid-trunk to just posterior of the anus.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Synnoeve Tholo, Adda Maenhardt, and Andreas Zankl (all University of Salzburg, Austria) for their excellent technical support. Brown trout were provided by Hans-Peter Gollmann, BAW Scharfling, Mondsee, Austria.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Barresi MJF, D'Angelo JA, Hernandez LP, Devoto SH. 2001. Distinct mechanisms regulate slow-muscle development. Curr Biol 11: 14321438.
  • Blaxter JHS. 1988. Pattern and variety in development. In: HoarWS, RandallDJ, editors. Fish physiology. Vol. 11. The physiology of developing fish. New York: Academic Press. p 158.
  • Bryson-Richardson RJ, Daggett DF, Cortes F, Neyt C, Keenan DG, Currie PD. 2005. Myosin heavy chain expression in zebrafish and slow muscle composition. Dev Dyn 233: 10181022.
  • Chauvigné F, Ralliére C, Cauty C, Rescan PY. 2006. 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. J Exp Biol 209: 372379.
  • Cinnamon Y, Ben-Yair R, Kalcheim C. 2006. Differential effects of N-cadherin-mediated adhesion on the development of myotomal waves. Development 133: 11011112.
  • Delalande JM, Rescan PY. 1999. Differential expression of two nonallelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss). Dev Genes Evol 209: 432437.
  • Devoto SH, Melancon E, Eisen JS, Westerfield M. 1996. Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development 122: 33713380.
  • Devoto SH, Stoiber W, Hammond CL, Steinbacher P, Haslett JR, Barresi MJF, Patterson SE, Adiarte EG, Hughes SM. 2006. Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish. Evol Dev 8: 101110.
  • Einum S, Fleming IA. 2000. Selection against late emergence and small offspring in Atlantic salmon (Salmo salar). Evolution Int J Org Evolution 54: 628639.
  • Galloway TF, Kjorsvik E, Kryvi H. 1999. Muscle growth in yolk-sac larvae of the Atlantic halibut as influenced by temperature in the egg and yolk-sac stage. J Fish Biol 55: 2643.
  • Gauvry L, Fauconneau B. 1996. Cloning of a trout fast skeletal myosin heavy chain expressed both in embryo and adult muscle and in myotubes neoformed in vitro. Comp Biochem Physiol 115B: 183190.
  • Grimaldi A, Tettamanti G, Martin BL, Gaffield W, Pownall ME, Hughes SM. 2004. Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis. Development 131: 32493262.
  • Gros J, Manceau M, Thome V, Marcelle C. 2005. A common somitic origin for embryonic muscle progenitors and satellite cells. Nature 435: 954958.
  • Johnston IA, McLay HA. 1997. Temperature and family effects on muscle cellularity at hatch and first feeding in Atlantic salmon (Salmo salar L.). Can J Zool 75: 6474.
  • Koumans JTM, Akster HA. 1995. Myogenic cells in development and growth of fish. Comp Biochem Physiol 110A: 320.
  • Nag AC, Nursall JR. 1972. Histogenesis of white and red muscle fibres of trunk muscles of a fish Salmo gairdneri. Cytobios 6: 227246.
  • Relaix F, Rocancourt D, Mansouri A, Buckingham M. 2005. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435: 948953.
  • Rescan PY, Gauvry L, Paboeuf G. 1995. A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes. FEBS Lett 362: 8992.
  • Rescan PY, Collet B, Ralliere C, Cauty C, Delalande JM, Goldspink G, Fauconneau B. 2001. Red and white muscle development in the trout (Oncorhynchus mykiss) as shown by in situ hybridisation of fast and slow myosin heavy chain transcripts. J Exp Biol 204: 20972101.
  • Rowlerson AM, Veggetti A. 2001. Cellular mechanisms of post-embryonic muscle growth in aquaculture species. In: JohnstonIA, editor. Fish physiology. Vol. 18. Muscle development and growth. San Diego: Academic Press. p 103140.
  • Rowlerson A, Mascarello F, Radaelli G, Veggetti A. 1995. Differentiation and growth of muscle in the fish Sparus aurata (L): II. Hyperplastic and hypertrophic growth of lateral muscle from hatching to adult. J Muscle Res Cell Motil 16: 223236.
  • Sive HL, Grainger RM, Harland RM. 2000. Whole-mount in situ hybridization. In: SiveHL, GraingerRM, HarlandRM, editors. Early development of Xenopus laevis. A laboratory manual. New York: Cold Spring Habor Laboratory Press. p 249274.
  • Steinbacher P, Haslett JR, Sänger AM, Stoiber W. 2006a. Evolution of myogenesis in fish: a sturgeon view of the mechanisms of muscle development. Anat Embryol (Berl) 211: 311322.
  • Steinbacher P, Haslett JR, Six M, Gollmann HP, Sänger AM, Stoiber W. 2006b. Phases of myogenic cell activation and possible role of dermomyotome cells in teleost muscle formation. Dev Dyn 235: 31323143.
  • Stickland NC, White RN, Mescall PE, Crook AR, Thorpe JE. 1988. The effect of temperature on myogenesis in embryonic development of the Atlantic salmon (Salmo salar L.). Anat Embryol (Berl) 178: 253257.
  • Stoiber W, Sänger AM. 1996. An electron microscopic investigation into the possible source of new muscle fibres in teleost fish. Anat Embryol (Berl) 194: 569579.
  • Stoiber W, Haslett JR, Goldschmid A, Sänger AM. 1998. Patterns of superficial fibre formation in the European pearlfish (Rutilus frisii meidingeri) provide a general template for slow muscle development in teleost fish. Anat Embryol (Berl) 197: 485496.
  • Stoiber W, Haslett JR, Steinbacher P, Freimüller M, Sänger AM. 2002. Tonic fibres in axial muscle of cyprinid fish larvae: their definition, possible origins and functional importance. Anat Embryol (Berl) 205: 113124.
  • Sundström LF, Lohmus M, Devlin RH. 2005. Selection on increased intrinsic growth rates in coho salmon, Oncorhynchus kisutch. Evolution Int J Org Evolution 59: 15601569.
  • Veggetti A, Mascarello F, Scapolo PA, Rowlerson A. 1990. Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.): an ultrastructural and morphometric study. Anat Embryol (Berl) 182: 110.
  • Veggetti A, Mascarello F, Scapolo PA, Rowlerson A, Candia Carnevali MD. 1993. Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study. Anat Embryol (Berl) 187: 353361.
  • Venters SJ, Thorsteinsdottir S, Duxson MJ. 1999. Early development of the myotome in the mouse. Dev Dyn 216: 219232.
  • Weatherley AH, Gill HS, Lobo AF. 1988. Recruitment and maximal diameter of axial muscle fibres in teleosts and their relationship to somatic growth and ultimate size. J Fish Biol 33: 851859.
  • Weinberg ES, Allende ML, Kelly CS, Abdelhamid A, Murakami T, Andermann P, Doerre OG, Grunwald DJ, Riggleman B. 1996. Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 122: 271280.
  • Wigmore PM, Evans DJR. 2002. Molecular and cellular mechanisms involved in the generation of fiber diversity during myogenesis. Int Rev Cytol 216: 175232.
  • Wilkinson DG. 1992. Whole mount in situ hybridization of vertebrate embryos. In: WilkinsonDG, editor. In situ hybridization: a practical approach. Oxford: Oxford University Press. p 7583.
  • Xie SQ, Mason PS, Wilkes D, Goldspink G, Fauconneau B, Stickland NC. 2001. Lower environmental temperature delays and prolongs myogenic regulatory factor expression and muscle differentiation in rainbow trout (Oncorhynchus mykiss) embryos. Differentiation 68: 106114.
  • Xu Y, He J, Wang X, Lim TM, Gong Z. 2000. Asynchronous activation of 10 muscle-specific protein (MSP) genes during zebrafish somitogenesis. Dev Dyn 219: 201215.