/ . Embryol. exp. Morph. Vol. 64, pp. 295-304,1981 2 9 5Printed in Great Britain © Company of Biologists Limited 1981

The formation of somites andearly myotomal myogenesis in Xenopus laevis,

Bombina variegata and Pelobates fuscus*

ByLEOKADIA KIELB6WNA1

From the Department of General Zoology, Zoological Institute,Wroclaw University, Poland

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

The structure of the nonsegmented mesoderm and of the newly formed myotomes inPelobates 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 mononuclearmuscle cells was not observed in the light microscope.

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

INTRODUCTION

Somite formation in Xenopus laevis differs from that of other Amphibia.Before segmentation the cells of the unsegmented paraxial mesoderm lie at rightangles to the embryo's long axis and radially relative to the premyocoel. Duringsegmentation they rotate by 90°. The myotomes seem to be only one cell long(Hamilton, 1969; Cooke & Zeeman, 1976). Striated myonbrils are visible withthe light microscope in the mononuclear myoblasts (Kietbowna, 1966, 1980;Muntz, 1975). Thin and thick myofilaments were observed in the electronmicroscope in cells of a newly-forming somite. The progression from disorgan-ized myofilaments to a fully organized sarcomere with a complete sarcotubularsystem can be found within a single cell (Blackshaw & Warner, 1976). Multi-nuclear myotubes appear in X. laevis at a more advanced developmental stage(stage 45) (Kielbowna, 1966, 1980; Muntz, 1975).

* This research was supported by Nencki Institute of Experimental Biology, PolishAcademy of Sciences, Warsaw, Poland.

1 Author's address: Department of General Zoology, Zoological Institute of the Universityof Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland.

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In Bombina variegata the myotomal muscle fibres differentiate in the sameway as in X. laevis. Striated myofibrils are visible with the light microscope, inthe mononuclear myoblasts, and lie parallel to the embryo's long axis, coveringwhole length of the myotome (Kielbowna & Koscielski, 1979). In the mono-nuclear cells at a more advanced stage of differentiation (stage 43) maturemyofibrils and a sarcotubular system were found in electron microscope(Kielbowna, in preparation). Multinuclear myotubes are formed in B. variegatain the postembryonal phase (stage 45) (Kielbowna & Koscielski, 1979).

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

MATERIAL AND METHODS

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

The embryos and tadpoles of Bombina variegata were derived from a labora-tory stock. Their developmental stages were determined by comparison withthe 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 whichhad been standing for 24 h at 20-22 °C. The developmental stages of P. fuscuswere determined on the basis of external features. The stage of early and lateneurula, 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 tadpoleswere fixed in formalin (pH 6-9 buffered according to Lille) and in Smith'sfixative, then dehydrated, embedded in paraffin and cut in 7 /*m sections. Inorder to examine the spatial arrangement of cells the embryos were sectionedtransversely, sagitally and horizontally. The sections were stained with

Somitogenesis and myogenesis in amphibia 297

Delafield's haematoxylin and eosin, safranin and fast green according to Selman& Pawsey (1965). To detect the presence of myofibrils the sections were stainedwith iron haematoxylin. Additionally, the formation of the myotomes inB. variegata was studied using semi-thin sections. The latter were preparedfrom the material fixed for 1-2 h in 0-1 M-phosphate-buffered 3 % glutaralde-hyde, pH 7-2 at 4 °C, rinsed in phosphate buffer (pH 7-2), dehydrated andembedded in Epon.

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

RESULTS

(a) Xenopus laevis (Daudiri). During the early neurula stage the nonsegmentedparaxial mesoderm, as seen in transverse section, consists of two layers ofcolumnar cells, connected with one another medially (Fig. 1, 13). The cavitybetween the layers is called the premyocoel (Hamilton, 1969). Somitogenesisbegins in the late neurala. The somites are formed consecutively in an antero-posterior direction.

Somite formation in X. laevis involves a rotation of the premyotomal cells.Its consecutive stages can be observed on horizontal and sagittal sections of theseries of somites in consecutive positions (stage 20-21). In horizontal sectionsthe cells of the newly formed somite are arranged perpendicularly relative tothe long axis of the body. In the two next older somites, the cells change theirangle of inclination, and in the fourth somite they come to lie parallel to thelong axis of the embryo (Fig. 16). During this movement the medial tip of thecell in the unsegmented mesoderm comes to lie at the anterior face of the somiteand the premyocoel tip moves posteriorly. The change in orientation of all thesomite cells is similar. The successive phases of the rotation of the whole cellblock can be seen in sagittal sections (Fig. 2). During the rotation the premyo-coel 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 hatchingphase (stage 32), nonsegmented mesoderm remains only in the tail. At stage 37the whole paraxial mesoderm is segmented. A size increase of the myotomalmyoblasts follows somitogenesis. The myoblasts of the oldest myotomes at thestage 37 are longer than the youngest ones, as they begin their developmentearlier. In the myoblast cytoplasm striated myofibrils appear.

Until stage 36 the myotomes contain only the differentiating mononuclearmyoblasts. From stage 37 on, mesenchymal cells proliferate into the intermyo-tomal 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 donot undergo mitosis.

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

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

During the hatching phase (stage 32) the nonsegmented mesoderm occupiesthe tail. In the trunk, different stages of myotome formation are observed. Inthe 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 towardsthe head.

At stage 36 the whole paraxial mesoderm is segmented. The population ofmyotome cells is homogeneous. At stage 37 and in subsequent stages prolifer-ating mesenchymal cells invade the intermyotomal spaces and move into themyotomes, between the myoblasts (Figs. 9, 20.

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

(c) Pelobates fuscus (Laurenti). In transverse sections of the neural plate andneural groove stages the paraxial mesoderm forms cell blocks (Fig. 15). Seg-mentation of the paraxial mesoderm, studied in horizontal and sagittal sections,begins in the cephalic region at the stage of neural tube closure, and proceedsanteroposteriorly. The cell arrangement in the newly formed somites resemblesthat of the nonsegmented mesoderm cells.

FIGURES 1-2 Xenopus lae visFig. 1. Nonsegmented paraxial mesoderm (mes) with premycoel (pm). Transversesection. Stage 17. Delafield's haematoxylin, eosin.Fig. 2. Rotation of myotomes (my). Sagittal section. Stage 20. Safranin, fast green.

FIGURES 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 thecaudal 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.

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During the later developmental stages, somitogenesis proceeds caudally. Atthe 10-somite stage, the myotomal cells in the four oldest somites becomeorganized: three to five cells assume a position along the myotomal long axisand 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 havemultinuclear myotubes. The myotubes result from the fusion of three to fivemyoblasts. In the younger myotomes various stages of myotube formation arevisible. Only the tail bud is composed of nonsegmented mesoderm.

At the young tadpole stage (6 mm long) myotubes are present in all themyotomes, but the myotubes of the older myotomes are longer and their myo-fibrils 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).

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

Mitotic figures were found in the nonsegmented mesoderm cells, in pre-fusion myoblasts and in mesenchymal cells. In myotubes no mitoses wereobserved.

DISCUSSION

The nonsegmented paraxial mesoderm in X. laevis consists of two cell layerswith the premyocoel between them. As a result of the rotation of the whole cellblock, the premyotomal cells change their position from perpendicular toparallel relative to the embryo long axis. Thus the myoblast length equals themyotome length. The results obtained are consistent with those of Hamilton(1969) and Cooke & Zeeman (1976). In two other species, i.e. in B. variegata andP. fuscus, the nonsegmented paraxial mesoderm is a compact cell block withoutany premycoel. In B. variegata the myotomal cells elongate parallel to the embryolong axis, each one extending finally over the whole length of a myotome. In

FIGURES 7-9 Bombina variegataFig. 7. Transverse section of the trunk myotomes (my). Stage 32. Delafield'shaematoxylin, 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) Stage39. Delafield's haematoxylin, eosin.

FIGURES 10-12 Pelobates fuscusFig. 10. Multinuclear myotubes (mt). Embryo 6 mm long. Sagittal section. Dela-field's haematoxylin, eosin.Fig. 11. Myofibrils (mf) in multinuclear myotubes (mt). Embryo 6 mm long. Ironhaematoxylin.Fig. 12. Mesenchymal cells (me) between the myotubes (mt). Embryo 10 mm long.Delafield's haematoxylin, eosin. Scale line = 100/tm.

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P.fuscus the mononuclear myotomal cells do not individually extend over thewhole length of the myotome but fuse to form numerous myotubes orientedparallel 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 myo-blasts (Kielbowna, 1966, 1980; Muntz, 1975; Kordylewski, 1978; Kielbowna &Koscielski, 1979). The myoblasts in X. laevis attain morphological and functionalmaturity when mononuclear (Muntz, 1975; Blackshaw & Warner, 1976).Electromicroscopic studies show that both mature myofibrils and completesarcotubular system arise in the mononuclear cells (Blakshaw & Warner, 1976).In the mononuclear cells in B. variegata, mature myofibrils and a sarcotubularsystem develop as in X. laevis (Kielbowna, in preparation).

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

In P.fuscus, myogenesis, studied in the light microscope, begins with myo-blast 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 ofthe nuclear DNA level to 8C (Kielbowna, 1966). In B. variegata the differ-entiating myoblasts contain tetraploid quantities of DNA (Kietbowna &Koscielski, 1979). In P.fuscus, however, the myotube growth occurs in thepresence of several nuclei, probably diploid.

Mesenchymal cells appear in the myotomes of X. laevis, B. variegata andP.fuscus in the postembryonic phase. In X. laevis the cells originate from thesclerotome (Ryke, 1953). The mesenchymal cells in X. laevis exhibit fibroblasticand probably myoblastic potential (Kielbowna, 1980). Accumulations of smallernuclei (like satellite cells) at the ends of the myotome cells appear to be the firststage in the development of multinucleation. The 'satellite cells' probably fusewith the myotome cell whose original large nucleus becomes smaller, as in themultinuclear myotome cell all the nuclei are equal-sized and small (Muntz, 1975)The myoblastic role of mesenchymal cells has been demonstrated in B. variegata

FIGURES 13-21 Xenopus laevis, Bombina variegata and Pelobates fuscusFig. 13-15. Nonsegmented paraxial mesoderm in Xenopus laevis (Fig. 13), Bombinavariegata (Fig. 14) and Pelobates fuscus (Fig. 15). Transverse sections, (mes), non-segmented paraxial mesoderm; (pm), premyocoel.Fig. 16-18. Formation of somites in Xenopus laevis (Fig. 16), Bombina variegata(Fig. 17) and Pelobates fuscus (Fig.-18). 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 musclecells; (mt), myotubes; (me), mesenchymal cells.

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myotomes. Multinuclear muscle fibres in B. variegata arise as a result of thefusion of myotomal myoblasts (primary myoblasts) with myoblasts of mesen-chymal origin (secondary myoblasts). The nuclei of the multinucleate cells varyin size and DNA content (nuclear dimorphism). The larger nuclei of the primarymyoblasts retain 4C DNA, whereas the smaller nuclei of the secondary myo-blasts are diploid (Kielbowna & Koscielski, 1979). In P.fuscus mesenchymalmyoblasts fuse with the multinuclear myotube, resulting in its conspicuouselongation (Kielbowna, in preparation).

These comparisons of X. laevis, B. variegata and P. fuscus show that myo-tomal myogenesis can occur as the sequel to any one of a variety of differentmodes of myotome formation.

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