Myogenesis in Xenopus laevis and in Bombina variegata is similar despite differences in the structure of the nonsegmented mesoderm and in the formation of the myotomes. In X. laevis the nonsegmented mesoderm consists of two cell layers with the premyocoel between them. During somitogenesis the premyoblasts rotate covering subsequently the whole myotome length. In B. variegata the premyocoel is absent. The myotomal cells change their shape and elongate, attaining ultimately the whole myotome length. The morphologically mature mononuclear muscle cells in both species result from myogenesis beginning in similarly arranged myoblasts. The multinuclear myotubes arise in the swimming tadpole (stage 45).

The structure of the nonsegmented mesoderm and of the newly formed myotomes in Pelobates fuscus is similar to that of B. variegata, while the process of myogenesis is different. It begins in the multinuclear myotubes. The stage of morphologically mature mononuclear muscle cells was not observed in the light microscope.

The results suggest that myotomal myogenesis is related neither to any particular type of nonsegmented mesoderm structure nor to any specific mode of myotome formation.

Somite formation in Xenopus laevis differs from that of other Amphibia. Before segmentation the cells of the unsegmented paraxial mesoderm lie at right angles to the embryo’s long axis and radially relative to the premyocoel. During segmentation they rotate by 90°. The myotomes seem to be only one cell long (Hamilton, 1969; Cooke & Zeeman, 1976). Striated myofibrils are visible with the light microscope in the mononuclear myoblasts (Kiełbówna, 1966, 1980; Muntz, 1975). Thin and thick myofilaments were observed in the electron microscope in cells of a newly-forming somite. The progression from disorganized myofilaments to a fully organized sarcomere with a complete sarcotubular system can be found within a single cell (Blackshaw & Warner, 1976). Multinuclear myotubes appear in X. laevis at a more advanced developmental stage (stage 45) (Kiełbówna, 1966, 1980; Muntz, 1975).

In Bombina variegata the myotomal muscle fibres differentiate in the same way as in X. laevis. Striated myofibrils are visible with the light microscope, in the mononuclear myoblasts, and lie parallel to the embryo’s long axis, covering whole length of the myotome (Kiełbówna & Koscielski, 1979). In the mononuclear cells at a more advanced stage of differentiation (stage 43) mature myofibrils and a sarcotubular system were found in electron microscope (Kiełbówna, in preparation). Multinuclear myotubes are formed in B. variegata in the postembryonal phase (stage 45) (Kiełbówna & Koscielski, 1979).

The aim of the study was to establish whether or not myogenesis in the myotomes of B. variegata is preceded by a rotation of the premyotomal cells similar to that which occurs in X. laevis. X. laevis and B. variegata belong to closely related families: Pipidae and Discoglossidae. X. laevis is wholly aquatic while B. variegata prefers the aquatic environment. Myotome formation and myotomal myogenesis in these two species were compared with the analogous processes in Pelobates fuscus (Pelobatidae), which is mainly land-dwelling. Relations between the structure of the nonsegmented mesoderm and the formation of myotomes and myotomal myogenesis were not studied.

In order to present the differences in somite formation between Xenopus laevis (Daudin), Bombina variegata (L.) and Pelobates fuscus (Laurenti) the author repeated the investigations of Hamilton (1969) and Cooke & Zeeman (1976). Mature specimens of X. laevis were induced with gonadotrophic hormone to spawn. 500 i.u. of the hormone (Biogonadyl) in 0·5 cc. of distilled water were injected into the dorsal lymphatic sacs of each animal. The developmental stages were determined according to the Normal Table for Xenopus laevis (Nieuwkoop & Faber, 1956). For the experiments, embryos and tadpoles at a series of developmental stages from 14 to 40 were used.

The embryos and tadpoles of Bombina variegata were derived from a laboratory stock. Their developmental stages were determined by comparison with the stages of X. laevis. Embryos and tadpoles at the stages 14– 40 were used.

The spawn of Pelobates fuscus was collected in natural ponds near Wroclaw. The developing spawn of all the species studied was kept in tap water which had been standing for 24 h at 20–22°C. The developmental stages of P. fuscus were determined on the basis of external features. The stage of early and late neurula, 10 somites, tail-bud stage (14–15 somites) and tadpole stages (4·5– 10 mm long) were studied.

The membranes of the embryos were removed. The embryos and tadpoles were fixed in formalin (pH 6·9 buffered according to Lille) and in Smith’s fixative, then dehydrated, embedded in paraffin and cut in 7 μm sections. In order to examine the spatial arrangement of cells the embryos were sectioned transversely, sagitally and horizontally. The sections were stained with

Delafield’s haematoxylin and eosin, safranin and fast green according to Selman & Pawsey (1965). To detect the presence of myofibrils the sections were stained with iron haematoxylin. Additionally, the formation of the myotomes in B. variegata was studied using semi-thin sections. The latter were prepared from the material fixed for 1–2 h in 0±1 M-phosphate-buffered 3% glutaraldehyde, pH 7±2 at 4°C, rinsed in phosphate buffer (pH 7·2), dehydrated and embedded in Epon.

The studies on the somite development were confined to myotome development, disregarding dermatome and sclerotome.

(a) Xenopus laevis (Daudin)

During the early neurula stage the nonsegmented paraxial mesoderm, as seen in transverse section, consists of two layers of columnar cells, connected with one another medially (Fig. 1, 13). The cavity between the layers is called the premyocoel (Hamilton, 1969). Somitogenesis begins in the late neurala. The somites are formed consecutively in an anteroposterior direction.

Fig. 1-2

Xenopus laevisFig. 1. Nonsegmented paraxial mesoderm (mes) with premycoel (pm). Transverse section. Stage 17. Delafield’s haematoxylin, eosin. Fig. 2. Rotation of myotomes (my). Sagittal section. Stage 20. Safranin, fast green.

Fig. 1-2

Xenopus laevisFig. 1. Nonsegmented paraxial mesoderm (mes) with premycoel (pm). Transverse section. Stage 17. Delafield’s haematoxylin, eosin. Fig. 2. Rotation of myotomes (my). Sagittal section. Stage 20. Safranin, fast green.

Somite formation in X. laevis involves a rotation of the premyotomal cells. Its consecutive stages can be observed on horizontal and sagittal sections of the series of somites in consecutive positions (stage 20-21). In horizontal sections the cells of the newly formed somite are arranged perpendicularly relative to the long axis of the body. In the two next older somites, the cells change their angle of inclination, and in the fourth somite they come to lie parallel to the long axis of the embryo (Fig. 16). During this movement the medial tip of the cell in the unsegmented mesoderm comes to lie at the anterior face of the somite and the premyocoel tip moves posteriorly. The change in orientation of all the somite cells is similar. The successive phases of the rotation of the whole cell block can be seen in sagittal sections (Fig. 2). During the rotation the premyocoel disappears automatically. The results wholly confirm the data of Hamilton (1969) and Cooke & Zeeman (1976).

At the tail-bud stage somitogenesis proceeds caudally. During the hatching phase (stage 32), nonsegmented mesoderm remains only in the tail. At stage 37 the whole paraxial mesoderm is segmented. A size increase of the myotomal myoblasts follows somitogenesis. The myoblasts of the oldest myotomes at the stage 37 are longer than the youngest ones, as they begin their development earlier. In the myoblast cytoplasm striated myofibrils appear.

Until stage 36 the myotomes contain only the differentiating mononuclear myoblasts. From stage 37 on, mesenchymal cells proliferate into the intermyotomal spaces and then move into the myotomes, between the myoblasts (Fig. 19).

Mitotic figures were observed in the mesoderm cells before somite formation, and in the mesenchymal cells. The differentiating mononuclear myoblasts do not undergo mitosis.

(b) Bombina variegata (L.)

In transverse sections of the neural-plate stage (stage 15) and the neural-fold stage (stage 17), blocks of the paraxial mesoderm cells without any premyocoel are visible (Figs. 3, 14). The first somites arise in the cephalic region. At the stage of neural-tube closure (stage 19), 6–7 somites are visible in sagittal and horizontal sections, the somite cells being arranged in a disorderly manner as in non-segmented mesoderm. In the later stages, somitogenesis progresses anteroposteriorly.

Fig. 3-6

Bombina variegataFig. 3. Nonsegmented paraxial mesoderm (mes). Transversal section. Stage 20. Delafield’s haematoxylin, eosin. Fig. 4. Premyoblasts undergoing an oriented growth (pm). Sagittal section of the caudal part of the embryo. Stage 32. Delafield’s haematoxylin, eosin. Fig. 5. As in Fig. 4. Semi-thin section. Methyl blue. Fig. 6. Mononuclear myoblasts (m). Sagittal section of the trunk myotomes. Stage 32. Delafield’s haematoxylin, eosin.

Fig. 3-6

Bombina variegataFig. 3. Nonsegmented paraxial mesoderm (mes). Transversal section. Stage 20. Delafield’s haematoxylin, eosin. Fig. 4. Premyoblasts undergoing an oriented growth (pm). Sagittal section of the caudal part of the embryo. Stage 32. Delafield’s haematoxylin, eosin. Fig. 5. As in Fig. 4. Semi-thin section. Methyl blue. Fig. 6. Mononuclear myoblasts (m). Sagittal section of the trunk myotomes. Stage 32. Delafield’s haematoxylin, eosin.

At stage 23 in the four oldest somites the round cells become lenticular and then spindle-shaped. The elongation of the myoblasts is directed towards the proximal and distal myotome borders. The myoblast elongation takes place in each myotome consecutively. In the myoblasts covering the whole myotome length myofibrils appear.

During the hatching phase (stage 32) the nonsegmented mesoderm occupies the tail. In the trunk, different stages of myotome formation are observed. In the few youngest myotomes the myoblasts are in the process of elongating (Figs. 4, 5, 17). In the remaining ones the myoblasts are already elongated (Figs. 6, 7, 8). The myoblast length (i.e. the myotome length) increases towards the head.

Fig. 7-9

Bombina variegateFig. 7. Transverse section of the trunk myotomes (my). Stage 32. Delafield’s haematoxylin, eosin. Fig. 8. Myofibrils (mf) in mononuclear myoblasts of the trunk myotomes. Stage 32. Iron haematoxylin. Fig. 9. Mesenchymal cells (me) between the mononuclear muscle cells (mm) Stage 39. Delafield’s haematoxylin, eosin.

Fig. 7-9

Bombina variegateFig. 7. Transverse section of the trunk myotomes (my). Stage 32. Delafield’s haematoxylin, eosin. Fig. 8. Myofibrils (mf) in mononuclear myoblasts of the trunk myotomes. Stage 32. Iron haematoxylin. Fig. 9. Mesenchymal cells (me) between the mononuclear muscle cells (mm) Stage 39. Delafield’s haematoxylin, eosin.

At stage 36 the whole paraxial mesoderm is segmented. The population of myotome cells is homogeneous. At stage 37 and in subsequent stages proliferating mesenchymal cells invade the intermyotomal spaces and move into the myotomes, between the myoblasts (Figs. 9, 20.

Mitotic figures were occasionally found in the nonsegmented mesoderm and mesenchymal cells. No mitosis was observed in differentiating myoblasts.

(c) Pelobates fuscus (Laurenti)

In transverse sections of the neural plate and neural groove stages the paraxial mesoderm forms cell blocks (Fig. 15). Segmentation of the paraxial mesoderm, studied in horizontal and sagittal sections, begins in the cephalic region at the stage of neural tube closure, and proceeds anteroposteriorly. The cell arrangement in the newly formed somites resembles that of the nonsegmented mesoderm cells.

During the later developmental stages, somitogenesis proceeds caudally. At the 10-somite stage, the myotomal cells in the four oldest somites become organized: three to five cells assume a position along the myotomal long axis and a few parallel rows of cells form in the myotome (Fig. 18).

At the tail-bud stage (14–15 somites), the four or five oldest myotomes have multinuclear myotubes. The myotubes result from the fusion of three to five myoblasts. In the younger myotomes various stages of myotube formation are visible. Only the tail bud is composed of nonsegmented mesoderm.

At the young tadpole stage (6 mm long) myotubes are present in all the myotomes, but the myotubes of the older myotomes are longer and their myofibrils are more numerous than those of the younger myotomes (Fig. 10, 11). The number of nuclei in the myotubes of all myotomes is equal (3–5).

Fig. 10-12

Pelobates fuscusFig. 10. Multinuclear myotubes (mt). Embryo 6 mm long. Sagittal section. Delafield’s haematoxylin, eosin. Fig. 11. Myofibrils (mf) in multinuclear myotubes (mt). Embryo 6 mm long. Iron haematoxylin. Fig. 12. Mesenchymal cells (me) between the myotubes (mt). Embryo 10 mm long. Delafield’s haematoxylin, eosin. Scale line = 100 μm.

Fig. 10-12

Pelobates fuscusFig. 10. Multinuclear myotubes (mt). Embryo 6 mm long. Sagittal section. Delafield’s haematoxylin, eosin. Fig. 11. Myofibrils (mf) in multinuclear myotubes (mt). Embryo 6 mm long. Iron haematoxylin. Fig. 12. Mesenchymal cells (me) between the myotubes (mt). Embryo 10 mm long. Delafield’s haematoxylin, eosin. Scale line = 100 μm.

Fig. 13-21

Xenopus laevis, Bombina variegata and Pelobates fuscusFig. 13-15. Nonsegmented paraxial mesoderm in Xenopus laevis (Fig. 13), Bombina variegata (Fig. 14) and Pelobates fuscus (Fig. 15). Transverse sections, (mes), nonsegmented paraxial mesoderm; (pm), premyocoel. Fig. 16-18. Formation of somites in Xenopus laevis (Fig. 16), Bombina variegata (Fig. 17) and Pelobates fuscus (Fig. l8). Horizontal sections, (my), myotomes; (m), myoblasts; (mt), myotubes. Fig. 19-21. Mononuclear muscle cells in Xenopus laevis (Fig. 19), Bombina variegata (Fig. 40) and myotubes in Pelobates fuscus (Fig. 21). (mm), mononuclear muscle cells; (mt), myotubes; (me), mesenchymal cells.

Fig. 13-21

Xenopus laevis, Bombina variegata and Pelobates fuscusFig. 13-15. Nonsegmented paraxial mesoderm in Xenopus laevis (Fig. 13), Bombina variegata (Fig. 14) and Pelobates fuscus (Fig. 15). Transverse sections, (mes), nonsegmented paraxial mesoderm; (pm), premyocoel. Fig. 16-18. Formation of somites in Xenopus laevis (Fig. 16), Bombina variegata (Fig. 17) and Pelobates fuscus (Fig. l8). Horizontal sections, (my), myotomes; (m), myoblasts; (mt), myotubes. Fig. 19-21. Mononuclear muscle cells in Xenopus laevis (Fig. 19), Bombina variegata (Fig. 40) and myotubes in Pelobates fuscus (Fig. 21). (mm), mononuclear muscle cells; (mt), myotubes; (me), mesenchymal cells.

In older tadpoles (10 mm long) the mesenchymal cells proliferate into the intermyotomal spaces and move into the myotomes between the myotubes (Fig. 12, 21).

Mitotic figures were found in the nonsegmented mesoderm cells, in prefusion myoblasts and in mesenchymal cells. In myotubes no mitoses were observed.

The nonsegmented paraxial mesoderm in X. laevis consists of two cell layers with the premyocoel between them. As a result of the rotation of the whole cell block, the premyotomal cells change their position from perpendicular to parallel relative to the embryo long axis. Thus the myoblast length equals the myotome length. The results obtained are consistent with those of Hamilton (1969) and Cooke & Zeeman (1976). In two other species, i.e. in B. variegata and P. fuscus, the nonsegmented paraxial mesoderm is a compact cell block without any premycoel. In B. variegata the myotomal cells elongate parallel to the embryo long axis, each one extending finally over the whole length of a myotome. In P.fuscus the mononuclear myotomal cells do not individually extend over the whole length of the myotome but fuse to form numerous myotubes oriented parallel to the myotome long axis.

The similar orientation of the myotomal cells in X. laevis and B. variegata, though due to different morphogenetic movements, results in similar myogenesis. The differentiation of the myotomal muscle fibres begins in mononuclear myoblasts (Kiełbówna, 1966, 1980; Muntz, 1975; Kordylewski, 1978; Kiełbówna & Koscielski, 1979). The myoblasts in X. laevis attain morphological and functional maturity when mononuclear (Muntz, 1975; Blackshaw & Warner, 1976). Electromicroscopic studies show that both mature myofibrils and complete sarcotubular system arise in the mononuclear cells (Blakshaw & Warner, 1976). In the mononuclear cells in B. variegata, mature myofibrils and a sarcotubular system develop as in X. laevis (Kiełbówna, in preparation).

The multinuclear myotubes in X. laevis and B. variegata form at the young tadpole stage (stage 45) (Kiełbówna, 1966, 1980; Muntz, 1975; Kiełbówna & Koscielski, 1979).

In P.fuscus, myogenesis, studied in the light microscope, begins with myoblast fusion, the stage of mature mononuclear muscle cells being omitted. Myofibrils first appear in multinuclear myotubes.

The growth of mononuclear muscle cells in X. laevis involves an increase of the nuclear DNA level to 8C (Kiełbówna, 1966). In B. variegata the differentiating myoblasts contain tetraploid quantities of DNA (Kiełbówna & Koscielski, 1979). In P.fuscus, however, the myotube growth occurs in the presence of several nuclei, probably diploid.

Mesenchymal cells appear in the myotomes of X. laevis, B. variegata and P.fuscus in the postembryonic phase. In X. laevis the cells originate from the sclerotome (Ryke, 1953). The mesenchymal cells in X. laevis exhibit fibroblastic and probably myoblastic potential (Kiełbówna, 1980). Accumulations of smaller nuclei (like satellite cells) at the ends of the myotome cells appear to be the first stage in the development of multinucleation. The ‘satellite cells’ probably fuse with the myotome cell whose original large nucleus becomes smaller, as in the multinuclear myotome cell all the nuclei are equal-sized and small (Muntz, 1975) The myoblastic role of mesenchymal cells has been demonstrated in B. variegate myotomes. Multinuclear muscle fibres in B. variegata arise as a result of the fusion of myotomal myoblasts (primary myoblasts) with myoblasts of mesenchymal origin (secondary myoblasts). The nuclei of the multinucleate cells vary in size and DNA content (nuclear dimorphism). The larger nuclei of the primary myoblasts retain 4C DNA, whereas the smaller nuclei of the secondary myoblasts are diploid (Kiełbówna & Koscielski, 1979). In P. fuscus mesenchymal myoblasts fuse with the multinuclear myotube, resulting in its conspicuous elongation (Kiełbówna, in preparation).

These comparisons of X. laevis, B. variegata and P. fuscus show that myotomal myogenesis can occur as the sequel to any one of a variety of different modes of myotome formation.

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