/. Embryol exp. Morph. Vol. 45, pp. 215-227, 1978
Printed in Great Britain © Company of Biologists Limited 1978
215
Scanning electron microscopic
observations of the development of the somites and
their innervation in anuran larvae
ByLESZEK KORDYLEWSKI1
From the Department of Comparative Anatomy, Jagiellonian University,
Cracow
SUMMARY
The development of the paraxial mesoderm in tadpoles of Xenopus, Bufo and Rana was
observed with a scanning electron microscope. In addition to examination of the differentiation of the surface and the interior of the somites, some attention was also paid to the
transformation of the material of the neural crests and to the innervation of the developing
myotome.
INTRODUCTION
Amphibian larvae are suitable material for the examination of myogenesis.
This is because in a relatively large embryo one can observe the development of
muscle which precedes the differentiation of nerve tissue. Thus motor innervation approaches the trunk muscle tissue when the latter is already functionally
differentiated (Muntz, 1975). What is more, particularly in Xenopus, the myotubes are mononuclear and multinucleation occurs at metamorphosis in a rather
synchronous way (Kieibowna, 1966; Muntz, 1975; Blackshaw & Warner,
1976o).
The development of the locomotor system in amphibian larvae has been
usually studied from sections in the light and electron microscopes (Hamilton,
1969; Muntz, 1975; Blackshaw & Warner, 1976a). These observations were
supported by functional experiments (Blackshaw & Warner, ]976a,b). The
present paper represents an attempt to visualize directly the three-dimensional
organization of the early muscle and nerve systems in anuran larvae by use of
the scanning electron microscope (SEM).
The SEM examination of vertebrate embryos done hitherto has given
excellent results (Waterman, 1972; England & Wakely, 1977; Wakely &
England, 1977). However, in Amphibia, external surfaces or other epithelia
have been mainly studied (Kessel, Beams & Shih, 1974; Lofberg, 1974a;
1
Author's address: Department of Comparative Anatomy, Institute of Zoology,
Jagiellonian University, Krupnicza 50, 30-060 Krakow, Poland.
216
L. KORDYLEWSKI
Dierickx & Waele, 1975). It seems that, after careful fracturing of amphibian
embryos, the interior of the animal, seen by SEM, also reveals many interesting
details. Penetration by this method deep into the tissues of a developing organism is easier than in the case of an adult individual.
MATERIALS AND METHODS
Developing spawn of Rana temporaria and Bufo bufo was collected from its
natural localities in spring. Spawn of Xenopus was obtained by injection of
gonadotropin (Biogonadyl, Pol fa) into mature pairs of toads. The developmental stages were determined according to Michniewska-Predygier & Pigori
(1957) and Nieuwkoop & Faber (1967). During fixation with buffered glutaraldehyde (pH 7-4) the tadpoles were fractured and ectoderm and some or all
of the somites were removed. In some specimens gut, neural tube and notochord
were also removed. With this procedure it was possible in several individuals to
visualize the lateral, proximal, ventral, caudal or cranial surfaces of the somites,
myomeres and also the neural tube. In addition the structures present in the
cavities, namely neural crests, nerves and ganglia were also exposed. In some
cases, when the somites were damaged, their interior was also visualized. The
prepared fragments of the embryos were fixed for 2 h in glutaraldehyde, then
after rinsing they were postfixed in 1-2 % buffered osmium tetroxide for 1 h.
After dehydration the specimens were dried at the critical point with CO2,
mounted on holders and coated with carbon and gold in an evaporator. They
were observed with the scanning electron microscope JSM-35.
RESULTS
Early stages
Segmentation of the mesodermal cell masses leads to the appearance of
crescent-like somites convex cranially (Fig. 1). The somites have the same width
over their whole height, but they are thickest at the level of the notochord. The
tops of the somites overlap the neural tube, where they touch the material of
the neural crests. These last are built of loose, stellate cells (Fig. 2). The ventral
ends of the somites overlap the entodermal gut: they approach the nephrotome.
The somites are compact, built of oval cells (diameter ca. 15-20/mi), which
protrude out of the somite surface (Figs. 3-6). The surface of the cells is not
FIGURES 1 AND 2
Fig. 1. Tadpole of R. temporaria, st. 17-18. Part of the ectodermal layer (ect) removed. Mesoderm forms 14 somites (s), unsegmented in the tail bud (tb). Neural
crests (nc), entoderm (ent.) 50 x .
Fig. 2. R. temporaria, st. 19. Somites (s) partially removed. Neural crest cells (nc)
visible between ectoderm (ect) and neural tube (nt). 250 x .
SEM observations of inner vat ion of somites of anuran larvae 211
218
L. KORDYLEWSKI
SEM observations of innervation of somites of anuran larvae 219
smooth, but rather covered by a network of multidirectional fibrils (Fig. 6).
The only elements of the internal cell structure that are marked on the cell
surfaces are the yolk platelets (Figs. 5, 12). The cells are tightly packed with
them, so that through the cell surface the yolk platelets manifest themselves as
subtle granulations. In the case of cell damage they spill out profusely (Figs. 4, 5,
single arrows).
The number of somites increases during development by the gradual appearance of furrows in the non-metamerized mesoderm (Figs. 4-6). This process
starts cranially and extends caudally. In Figs. 1 and 4 are shown the last somites
not fully segmented from the mesodermal material of the tail bud. New, caudal
somites are less differentiated than the cranial ones. Thus, in the same individual,
independently of its developmental stage (about 25-35 in Xenopus and 17-20 in
Rana and Bufo) one can find various developmental stages of the somites along
the craniocaudal extent of the animal. Therefore the degree of the differentiation
of the muscle elements is not specific for the particular developmental stage of
the tadpole. However, the development of the muscle elements in a particular
somite, at least in Xenopus, seems to be remarkably synchronous.
Further development
The majority of cells that build the somite undergo transformation into
embryonic muscle fibres. At stage 26 in Xenopus there are elongated, spindleshaped cells in the interior of the somite. They lie parallel to the long axis of the
embryo. They extend from the cranial to the caudal edge of the somite, touching
in this way both the preceding somite and the following one. These cells are
still rich in yolk platelets which can be seen through the cell surface (Fig. 11).
The somites in Xenopus are covered laterally with a single cell layer of flat
oval cells. These cells are also rich in yolk. They adjoin each other by tiny
branches. Partial removal of this layer (Figs. 10, 11), or its splitting (Fig. 3,
arrows) exposes the elongated myoblasts that lie underneath.
The proximal side of the somite (Fig. 9) is not covered by a flat cell layer. The
polygonal outlines of the cells on this side of the somite are due to the radial
FIGURES 3-6
Surfaces of the somite after removal of the ectoderm.
Fig. 3. X. laevis, st. 26. Under an external layer of flat cells elongated myoblasts are
visible (arrows). 250 x .
Fig. 4. R. temporaria, st. 18. Yolk platelets visible in the damaged somite (single
arrow). Somite partially segmented from the caudal mesoderm (double arrow).
180 x .
Fig. 5. B. bufo, st. 17. Yolk platelets at the site of damage (arrow). Two inter-somite
furrows are visible. 460 x .
Fig. 6. /?. temporaria, st. 18. Enlarged fragment of Fig. 4 in the region of an intersomite furrow. Cells connected by fibrous material. 680 x .
220
L. KORDYLEWSKI
SEM observations of innervation of somites of anuran larvae 221
distribution of the myoblasts, which are directed perpendicularly to this surface.
When the cranial surface of the somite was shown by the removal of the preceding somite (Fig. 7), the ends of the elongated myocytes that lie parallel to
the long axis of the tadpole were also visible (Fig. 8). No specialized structures
in the region of inter-somite junction were noticed with SEM. It seems that the
surface fibrils are more abundant at these ends of the myocytes than at the ones
at the proximal surface (Fig. 8). It is worth mentioning that in this region the
ends of the developing muscle fibres show acetylcholinesterase activity (unpublished data).
On the lateral layer of flat cells thin (diameter about 0-8 /mi), branched,
anastomosing fibres are present (Figs. 10, 12, 13). They were not found on the
proximal surface of the somite. Similar fibrous structures appear between the
neural tube and the proximal surface of the somite in its upper part, but they
are considerably thicker (Fig. 7, arrow head).
In the older larva (e.g. Xenopus st. 46) the metamerization of the myomeres is
maintained as the continuation of the former segmentation of the mesoderm
into somites. The muscle fibres observed in the tail (Fig. 16) are more cylindrical
than spindle-like. They lie parallel to the long axis of the tadpole. They also
extend from the cranial to the caudal edge of the myomere. The dorsal and the
ventral fibres are shifted caudally in relation to the intermediate fibres; this is
related to the bent shape of the myomeres. The shape of the cell surfaces does
not indicate the presence of yolk in the cytoplasm. On the other hand clear
transverse striation is present, which indicates that the contractile apparatus
has already developed. When the cell surface is damaged, in the cell interior
numerous thin myofibrils oriented parallel to the long axis of the cell are visible
(Fig. 16, double arrow). At the ends of the muscle fibres (i.e. at the myotome
limits) single oval cells are sporadically present (Fig. 16, arrow).
In the vicinity of the muscle connective tissue elements are present: stellate
fibroblasts, also capillaries and effused erythrocytes (Fig. 16). In the region of
the inter-somite junctions long fibres (diameter about 3/tm) approach the
FIGURES 7-9
Fig. 7. X. laevis, st. 26. Ectoderm (ect) and entoderm (ent) partially removed. Larva
broken transversely, cranial part removed. Notochord («), neural tube (nt), left and
right somites (Is, rs). Fibrous structures (arrow) come out from the neural tube to the
region of the removed left somite. Lateral, cranial and ventroproximal surfaces of
the somites are visible. 300 x .
Figs. 8, 9. Enlarged fragments of Fig. 7.
Fig. 8. Cranial surface of the somite after removal of the preceding somite; the ends
of the spindle-shaped myoblasts are visible. Arrows show the edge of the somite.
On its right side a layer of flat cells covering the lateral side of the somite is present
(dermatome). Notochord (n). 710 x .
Fig. 9. Ventro-proximal surface of the somite after removal of entoderm. Three kinds
of cells are visible: large, smaller round, and elongated - spindle-shaped (myoblasts).
480 x .
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L. KORDYLEWSKI
SEM observations of innervation of somites of anuran larvae 223
muscle. They originate in the region of the neural tube and are directed laterocaudally (Figs. 17, 18).
After removal of somites in Bufo bufo (st. 19-20) conical aggregations of cells
were found, which lie on the notochord, but emerge from the neural tube. They
occur metamerically and are directed lateroventrally (Fig. 14).
DISCUSSION
The descriptions of the organization and development of the amphibian
somite presented hitherto were based on histological investigations (Kitchin,
1949; Muchmore, 1951; Waddington & Deuchar, 1953; Hamilton, 1969;
Loeffler, 1969; Kiefbowna, 1975; Muntz, 1975), electron microscopic observations (Nakao, 1976; Blackshaw & Warner, 1976a) or attempted to correlate the
examined structures with their functions (Muntz, 1975; Cooke, 1975; Blackshaw
& Warner, 19766). After successive removal of various layers of the embryo,
the external surfaces of the somites, the cells contained in them and the structures located in the spaces between somites, ectoderm, notochord, neural tube
and gut, were visualized with the SEM. It was also possible to observe some of
their developmental changes. The early somites that are separated caudally
from the mass of isometric mesodermal cells by furrowing, are crescent like.
This shape of somite is maintained later, up to the advanced myotome, but
dramatic changes occur in the shape of the cells enclosed in it. These became
elongated, spindle-shaped; due to rotation they orient parallel to the long
axis of the embryo (Hamilton, 1969). The majority of cells extend through the
whole length of the somite. The cells are rich in yolk, which results in a characteristic 'rolling landscape' topography of their surfaces (Lofberg, 19746). These
large cells in Xenopus are mononuclear myoblasts (Muntz, 1975) that each
contain a single polyploid nucleus (Kierbowna, 1966).
The single layer of flat cells that covers the lateral surfaces of the somites
(Figs. 8, 10, 11, 12) might represent the dermatome. The description of the
experiment by Blackshaw & Warner (1976 a) indicates that these authors also
removed only the ectoderm, while the somites remained covered by the derma-
FIGURES
10-13
Fig. 10. Isolated somites of X. laevis, st. 26. Lateral layer of flat cells (dermatome)
partially removed from the cranial somites. 150 x.
Figs. 11-13. Enlarged fragments of Fig. 10.
Fig. 11. Spindle-shaped myoblasts after removal of dermatome. They extend from
end to end of the somite and are full of yolk platelets. 490 x .
Fig. 12. Network of fibres covering the lateral layer of flat cells (dermatome). 1030 x .
Fig. 13. Branched ending interdigitating with the fibrous projections of the cells.
Through the surfaces of the lateral layer cells yolk platelets in the cytoplasm are
visible. 2530 x.
15-2
224
17
L. KORDYLEWSKI
SEM observations of innervation of somites of anuran larvae 225
tome. However, according to Hamilton (1969), the dermatome in Xenopus does
not show the metameric segmentation which was observed in the present study.
The differences in the formation of the dermatome described by Hamilton (1969)
among different species were not noticed in the present study.
Other details of the somite cell surface, namely the fine fibrous protrusions
pointing in all directions, seem to be composed of an extracellular material rather
than to represent cell body projections. Lofberg (1974 c) has compared similar
mesh works of fine fibrils covering the neural crest cells in axolotl, when seen
with the SEM and the transmission electron microscope. He came to the conclusion that these fibrils are made of a fuzzy extracellular substance that can
stabilize and coordinate the movements of the migrating cells.
The branched anastomosing fibres present on the dermatome could perhaps
be mesenchymatic connective tissue elements. However, it seems more probable
that they are elements of the nervous system, namely the projections of the
Rohon-Beard sensory cells (Muntz, 1975) that are extending on the surface of
the dermatome. Nerve tissue elements are undoubtedly present in the vicinity
of the neural tube. Neural crest cells in the early stages (after neurulation) were
observed with the SEM by Lofberg (1974c). In the present study they appear
as loose, stellate, multibranched cells (Fig. 2) that occur in the cavity between
the neural tube and the top parts of the somites (Fig. 1). Later on they probably
give rise to the conical structures seen in Bufo (Figs. 14, 15) on the neural tube.
These cell aggregations probably represent the ganglia or motor root anlagen,
which contain Rohon-Beard fibres (Muntz, 1975). In the older larvae some
fibres are visible running from the vicinity of the neural tube caudolaterally to
the myotomes. They reach the latter in the region of myosepta (Figs. 17, 18).
FIGURES
14-18
Fig. 14. B. bufo, st. 19-20. Ectoderm and left somites removed. Visible neural tube
{nt), notochord (n) and entoderm (ent). Metameric conical structures come out
laterally from the neural tube, some of t iem removed. 70 x .
Fig. 15. Enlarged fragment of Fig. 14. Cones are formed by cells wrapping the
elongated ones. Notochord (n), neural tube (nt). 390 x .
Fig. 16. X. laevis, st. 46. Skin removed. Part of the trunk muscle visible, divided
into myomeres. Every muscle fibre extends from end to end of the myomere. Single
oval cell present at the ends of the fibres (arrow). Striation of the fibres prominent.
In the injured fibres myofibrils visible (double arrow). Capillaries (c), effused red
cells on the fibres. 510 x .
Fig. 17. X. laevis, st. 46. Notochord, spinal cord and upper part of the muscle
removed. Longfibreapproaches from the region of spinal cord to the furrow between
myomeres. Metameric marks of the similar structures removed are visible in the
respective regions. 270 x .
Fig. 18. X. laevis, st. 46. Skin, notochord and the left muscle removed. Nerve
approaches from under the spinal cord (sc) ventrocaudally to the right muscle visible
in the background. It enters the muscle in the region of myoseptum (arrow).
Broken fragments of other similar structures visible in respective metameric places.
260 x .
226
L. KORDYLEWSKI
They probably represent the motor branches that originate from the ventral
roots (Kappers, Huber & Crosby, 1960). Similar innervation, which gradually
develops in the caudal direction, was described by Blackshaw & Warner (19766).
The observation of Kietbowna (1975), that depletion of yolk occurs in the
older larvae, was confirmed in the present paper. In muscle fibres this process is
synchronous with the development of the contractile apparatus, which becomes
manifested by external striation and the presence of myofibrils in the cell
interior (Fig. 16).
The small oval cells located at the ends of the elongated muscle cells (Fig. 16)
probably represent non-differentiated elements of the myogenic cell line. They
might be the carriers of the diploid nuclei (Kielbowna, 1975), which after
mitoses enrich the muscle fibres with new nuclei. Such nuclei, similarly located,
were observed with the light microscope by Muntz (1975).
The findings of other authors (Hamilton, 1969; Muntz, 1975; Blackshaw &
Warner, 1976), that the differentiation of the muscle elements precedes complete
innervation of the somite, have been fully confirmed in the present study. The
stimulation of the non-innervated somites is due to the existence of an electric
coupling of the somites, as has been shown by Blackshaw & Warner (1976a).
These authors also assume that the gap junctions between the neighbouring
somites represent the morphological equivalents responsible for the low resistance pathways of the impulse. Our observations of the somite-to-somite surfaces
with SEM did not show any specialized structures of this sort. However, the
abundance of the extracellular fibrils on this surface may indicate that their
very close contact is much greater than between the other somite surfaces and
the other tissues surrounding them, where electrical coupling has not been
shown (Blackshaw & Warner, 1976a). It is also worth mentioning that early
acetylcholinesterase activity in the tadpoles is indicated in the region of the intersomite furrow (Kordylewski, unpublished data). This is where the coupling
occurs, and also where the early neuromuscular junction will develop later on.
I wish to express my cordial thanks to Professor Dr W. Kilarski for his kind permission to use
his SEM Laboratory, as well as for his comments. I am grateful to Professor Dr H. Szarski
for reading the manuscript. I also thank very much Mr P. J. D. Fletcher who helped me in
the final draft of this paper.
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(Received 8 November 1977, revised 6 January 1978)