/. Embryol. exp. Morph. Vol. 64, pp. 295-304,1981
Printed in Great Britain © Company of Biologists Limited 1981
295
The formation of somites and
early myotomal myogenesis in Xenopus laevis,
Bombina variegata and Pelobates fuscus*
ByLEOKADIA KIELB6WNA1
From the Department of General Zoology, Zoological Institute,
Wroclaw University, Poland
SUMMARY
Myogenesis in Xenopus laevis and in Bombina variegata is similar despite differences in the
structure of the nonsegmented mesoderm and in the formation of themyotomes. 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.
INTRODUCTION
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 myonbrils are visible with
the light microscope in the mononuclear myoblasts (Kietbowna, 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) (Kielbowna, 1966, 1980; Muntz, 1975).
* This research was supported by Nencki Institute of Experimental Biology, Polish
Academy of Sciences, Warsaw, Poland.
1
Author's address: Department of General Zoology, Zoological Institute of the University
of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland.
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L. KIELB6WNA
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 (Kielbowna & 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
(Kielbowna, in preparation). Multinuclear myotubes are formed in B. variegata
in the postembryonal phase (stage 45) (Kielbowna & 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.
MATERIAL AND METHODS
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 (4-510 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 /*m sections. In
order to examine the spatial arrangement of cells the embryos were sectioned
transversely, 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 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.
RESULTS
(a) Xenopus laevis (Daudiri). 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.
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.
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Somitogenesis and myogenesis in amphibia
299
(b) Bombina variegata (L.). In transverse sections of the neural-plate stage
(stage 15) and the neural-fold stage (stage 17), blocks oftheparaxialmesoderm
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.
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.
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.
FIGURES 1-2 Xenopus lae vis
Fig. 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.
F I G U R E S 3-6 Bombina variegata
Fig. 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.
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Somitogenesis and myogenesis in amphibia
301
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).
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.
DISCUSSION
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
7-9 Bombina variegata
Fig. 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.
FIGURES
10-12 Pelobates fuscus
Fig. 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/tm.
FIGURES
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Somitogenesis and myogenesis in amphibia
303
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 (Kielbowna, 1966, 1980; Muntz, 1975; Kordylewski, 1978; Kielbowna &
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 (Kielbowna, in preparation).
The multinuclear myotubes in X. laevis and B. variegata form at the young
tadpole stage (stage 45) (Kielbowna, 1966, 1980; Muntz, 1975; Kielbowna &
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 (Kielbowna, 1966). In B. variegata the differentiating myoblasts contain tetraploid quantities of DNA (Kietbowna &
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 (Kielbowna, 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. variegata
F I G U R E S 13-21 Xenopus laevis, Bombina variegata and Pelobates fuscus
Fig. 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.-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 muscle
cells; (mt), myotubes; (me), mesenchymal cells.
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L. KIELBOWNA
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 (Kielbowna & Koscielski, 1979). In P.fuscus mesenchymal
myoblasts fuse with the multinuclear myotube, resulting in its conspicuous
elongation (Kielbowna, 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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