Download Comparative analysis of amphibian somite

/ . Embryol. exp. Morph. Vol. 66, pp. 1-26, 1981
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Comparative analysis of
amphibian somite morphogenesis: cell
rearrangement patterns during rosette
formation and myoblast fusion
By B. WOO YOUN 1 AND GEORGE M. MALACINSKP
Program in Cellular, Molecular, and Developmental Biology, Department of
Biology, Indiana University
SUMMARY
Detailed SEM observations of the changes in cellular morphology, arrangements, and
contacts that occur during the process of somite formation were made in two species of urodele
amphibians, Ambystoma mexicanum and Pleurodeles wait Hi, and one species of anuran
amphibian, Rana sphenocephala. After fixation, embryos were fractured transversely, horizontally, and parasagittally, and the intrasomitic cellular arrangement pattern was examined
with the SEM. It was found that Ambystoma and Pleurodeles embryos followed exactly the
same development sequence in rosette formation and myoblast fusion. Rana somites did not,
however, appear to form rosettes. Those myotomal cells underwent fusion immediately after
a few segmentations occurred.
Patterns of cellular rearrangement were also described during urodele rosette formation at
the time of somite segmentation and during myoblast fusion. Extensive changes in cell shape
and orientation appeared to occur during those processes. When cells changed their orientation, they often exhibited a triangular configuration. Probable roles of these triangular-shaped
cells in rosette formation and myoblast fusion are discussed.
During the initial period of myoblast or myotomal cell fusion, cellsfirstsend out specialized
cell processes and then establish their cell-cell contacts. The establishment of such contacts
eventually leads to tight membrane appositions and fusion. Since myoblast fusion appeared
to occur between two cells which were tandemly arranged in a rosette, the origin of multinuclearity in the fused cells is discussed.
Finally, comparative analyses of the pattern of somite formation and subsequent muscle
development were made between different species of amphibians. The possibility is discussed that patterns of somitogenesis may provide useful indicators for determining how
different families of amphibians evolved.
1
Author''s present address: Department of Biology, Princeton University, Princeton, N.J.
08540, U.S.A.
2
Author's address (for all correspondence): Department of Biology, Indiana University,
Bloomington, Indiana 47401, U.S.A.
2
B. W. YOUN AND G. M. MALACINSKI
INTRODUCTION
Somite formation is a major morphogenetic event of axial structure formation
during amphibian embryogenesis. Consequently, various aspects of somitogenesis have recently been studied. These include control mechanisms which
regulate both the number and size of somites (Hamilton, 1969; Cooke, 1975;
Elsdale, Pearson & Whitehead, 1976; Cooke, 1978; Pearson & Elsdale, 1979;
Elsdale & Pearson, 1979); the kinematic nature of somite segmentation (Deuchar
& Burgess, 1967; Pearson & Elsdale, 1979); comparative analyses of somite
formation among different amphibian species (Hamilton, 1969); and the nature
of myoblast fusion in various amphibian species (Loeffler, 1968, 1970; Muntz,
1975). It has emerged from those analyses that in most amphibian species somites
form in a manner which is comparable to somitogenesis in many other vertebrates (e.g. chick). That is, each somite forms initially as a cluster of cells
organized in a 'rosette' configuration. Initially, individual somites form by the
pinching off of a group of cells from the paraxial mesoderm. Later, the majority
of the somite cells, which are myotomal in character and mononucleate,
elongate anteroposteriorly and fuse end to end with other cells of the same
somite to give multinucleate muscle fibres. Somite formation in the anuran,
Xenopus laevis does not, however, follow this typical vertebrate pattern. In
Xenopus the presumptive somite cells elongate in the mediolateral direction
prior to segmentation. Somite segmentation then occurs as transverse fissures
isolate successive blocks of spindle-shaped cells. Bundles of the spindle-shaped
cells in newly segmented somites then rotate through 90°. The cells come to lie
parallel to the notochord and stretch from end to end of the length of the somite
(Youn & Malacinski, 1981). Several hours later, these myotomal cells differentiate as uninucleate muscle fibres.
With recent advances in preparative techniques for scanning electron microscopy, it is now possible to observe the details of cellular morphology, arrangements, and contacts during the process of somite formation in various vertebrate
embryos. In the chick embryo, for example, the surface mesoderm which lies on
either side of the anteriormost end of the primitive streak (just posterior to the
Hensen's node) has been examined in stereo with the scanning electron microscope (SEM). Meier (1979) has found that this region is organized into tandemly
aligned, repeating circular domains (about 180/tm in diameter). As these
structures ('somitomeres') are added to the embryonic axis during neurulation,
they condense considerably and undergo morphogenesis to become mature
somites. Chick somites appear, therefore, to emerge from pre-existing somitomeres. That observation suggests that the paraxial mesoderm is programmed
relatively early during axial structure development. The pattern of rosette
formation at the time of somite segmentation has been studied by Bellairs
(1979) with 'fractured' chick embryos. Those studies have led to the proposal
that two factors may be responsible for changes in cell shape during somite
Amphibian somite morphogenesis
3
segmentation: (1) collagen fibrils; and (2) a change in cell-to-cell adhesiveness.
The arrangement of the cells in more highly differentiated somites has also been
examined by that author. The results have led to the suggestion that the chick
resembles Xenopus in that the myotomal cells undergo rotation and become
oriented in an anteroposterior direction.
Detailed SEM investigations of amphibian somite segmentation have been
restricted almost exclusively to the anuran amphibian, Xenopus laevis. In
previous studies changes in cellular morphology, arrangement, and contacts
among the paraxial mesodermal cells of Xenopus have been examined (Youn,
Keller & Malacinski, 1980). The role individual cell shape changes play during
the 90° rotation of Xenopus myotomal cells has also been studied (Youn &
Malacinski, 1981). In a few other anuran and urodele amphibian species the
dorsal surface of the somite and mesoderm and the temporal sequence of somite
segmentation have also recently been described (Youn et al. 1980). No detailed
SEM observation of the changes in cellular morphology, arrangement and
contacts that occur during the process of somite formation has, however, been
made in the various species of amphibians which display the typical vertebrate
rosette pattern. The present paper employs two species of the urodele amphibians,
Ambystoma mexicanum and Pleurodeles wait Hi, and one species of the anuran
amphibian, Rana sphenocephala for a comparative study. The developmental
timing and sequence of rosette formation and myoblast fusion were observed.
The patterns of cellular rearrangements were described during rosette formation
at the time of somite segmentation and during myoblast fusion. The results of
these studies provided no evidence for the presence of the chick type of somitomere-like structures in the paraxial mesoderm. It was also observed that the
pattern of rosette formation and myoblast fusion in Rana is quite different from
the urodele species. Special attention was, therefore, devoted to the comparative
aspects of somite formation in these various species of amphibia.
MATERIALS AND METHODS
Ambystoma mexicanum and Pleurodeles waltlii embryos were obtained from
natural spawnings. Rana sphenocephala eggs were artificially inseminated according to the method outlined by Rugh (1962). Jelly layers were removed with fine
watchmaker's forceps in dechlorinated tap water. The stagings of the urodele
embryos followed Bordzilovskaya & Dettlaff (1979) for Ambystoma, and
Gallien & Durocher (1957) for Pleurodeles. Embryos of Rana sphenocephala
were staged according to Shumway (1940) for Rana pipiens. Embryos at
appropriate stages were fixed overnight at 4 °C in a cacodylate-buffered (0-1 M,
pH 7-2) 2-5 % glutaraldehyde solution. The fixed embryos were rinsed in 0-1 M
cacodylate buffer. The epidermis of each embryo was then peeled off with a fine
steel knife and watchmaker's forceps. The neural tube, and sometimes also the
notochord, were removed from the 'peeled' embryos. Dissected embryos were
4
B. W. YOUN AND G. M. MALACINSKI
then post-fixed in cacodylate-buffered (0-1 M, pH 7-5) osmium tetroxide for
3 h at 4 °C. In order to expose the cells which reside in the interior of the
somitic mesoderm, a horizontal or parasagittal fracture was made through the
somite file and the unsegmented mesoderm at (or above) the level of the notochord. Occasionally, a transverse fracture was made through the unsegmented
plate to reveal a cross-sectional view of the morphology, arrangement, and contacts of the cells. Fine tungsten needles and forceps were employed for preparing
those fractures.
Following dehydration in a graded alcohol series, samples were critical-point
dried from liquid CO 2 in a Pelco critical-point drier (Model H, Ted Pella Co.).
Specimens were then mounted on aluminium stubs with conductive silver paint
and .coated with gold-palladium (60:40) in a sputter coater E5100 (Polaron
Equipment Ltd). These were examined at 20 kV in an Etec U-1.scanning electron
microscope and photographed on Polaroid type 55 positive-negative film. More
than 20 embryos of each stage were examined under the SEM. Five developmental stages were chosen to describe the timing and sequence of rosette formation and myoblast fusion.
RESULTS
1. Developmental timing and sequence of rosette formation and myoblast fusion
Figures 1 and 2 display a stage series of horizontally or parasagittally fractured
embryos of Ambystoma and Rana. In the case of Ambystoma (Fig. 1), each
somite is formed by the pinching off of a group of cells from the paraxial mesoderm. Those cells then become organized into a rosette configuration. Details of
how the myotomal cells rearrange themselves into a rosette will be described in
the next section of this report. Each of the segmented somites remain as rosettes
until stage 27 (12-13 somites, see Fig. lc). This observation confirms an earlier
report by Loeffler (1968). Analyses of histological preparations had suggested
that the critical period for myoblast fusion occurs between stages 28 and 34. Our
SEM observations indicate that that is indeed the case. The embryo shown in
Fig. Id has formed about 15 somites (stages 28-29), and the myoblasts in the
anterior six somites have either completed or are in the process of undergoing
fusion. Later, at about stage 32 (20 somites, see Fig. 1 e), the anterior twelve
somites show signs of myoblast fusion. The fusion of myoblasts, therefore, takes
place in an anteroposterior direction. It also appears that the fusion process
proceeds in a mediolateral direction. This can be observed in the sixth somite of a
stage-28 to -29 embryo (Fig. 1 d), and the twelfth somite of a stage-32 embryo
(Fig. 1 e). In those examples, fusion of myoblasts is just about to begin in those
particular planes of the horizontal fracture. Only a group of cells in the medial
side can be seen to be stretched from end to end of the somites. Myoblast fusion
does not, therefore, occur synchronously throughout individual somites.
It is difficult to estimate accurately how long it takes for the cells of a single
somite to complete the process of fusion by simple examination of the mor-
*. .*'
Fig. 1. An atlas of somite development in Ambystoma mexicanum. Embryos were fractured longitudinally except in {b) where
the embryo was parasagittally fractured in order to reveal the pattern of intrasomitic cellular arrangement. Whole embryo
views are shown on the left, and higher-magnification pictures of the fractured somite mesoderm are on the right side, (a) Stages
19-20; (b) stages 21-22; (c) stages 27-28; id) stages 28-29; (e) stage 32. Bars in the lower-magnification pictures represent
0-5 mm. Bar at the upper left corner of the higher-magnification micrograph in (a) represents 01 mm for all the highermagnification micrographs in Fig. 1. Ant, anterior; Post, posterior, for all the embryos shown here.
5*
IS-
B. W. YOUN AND G. M. MALACINSKI
Amphibian somite morphogenesis
1
phology of cells in these particular fracture planes. The fractures were made at
slightly different dorsoventral levels in different embryos. Nevertheless, a rough
approximation of the time required for rosette formation and myoblast fusion
can be made. For example, the 6th somite at about stage 20 (approximately
70 h after fertilization at 20°, Fig. 1 a) is a newly formed somite which is beginning to assume the rosette form. It remains rosette-shaped until stages 27-28
(about 90 h, Fig. 1 c). At about stages 28-29 (approximately 95 h, Fig. 1 d), the
cells of the 6th somite start to fuse from the medial side. Consequently, the
rosette configuration lasts for approximately 25 h at 20°. At stages 32 (about
113 h, Fig. 1 e), the 6th somite appears to have completed the process of myoblast
fusion. Thus it takes about 18 h at 20° for completion of the fusion process.
In Pleurodeles, also a urodele amphibian, the pattern of rosette formation is
exactly the same as that in Ambystoma. Moreover, the process of myoblast
fusion follows exactly the same sequence as seen in Ambystoma embryos
(results not shown). When the Pleurodeles embryo reaches the 20-somite stage,
the anterior eleven somites appear either to be undergoing fusion or to have
already completed the fusion process. It should be pointed out here that unlike
the rosettes of the chick embryo, those in the urodele amphibians are much
more closely defined, with long cells radiating from the centres (see Discussion).
Scanning electron micrographs in Fig. 2 indicate that the pattern of rosette
formation and myoblast fusion in Rana is completely different from that of the
urodele species studied here. A group of cells first becomes separated from the
paraxial mesoderm. The cells do not then exhibit any detectable sign of the
formation of the urodele-type rosettes (e.g. Fig. 2). In some cases, cells appear to
be arranged radially around much of the circumference of several somites (e.g.
see somite no. 5 in Fig. 2b and somite no. 14 in Fig. 2e). The apparent difference
in the mode of somite cell organization between Rana and the urodeles may
derive in part from the shorter length and more rounded shape of Rana cells
compared with urodele cglls (see below, Figs 3 and 4). However, differences in
the degree of cell elongation between species may only give a superficial and
variable indication of the functional polarization of the cells caused by the
cytoskeletal organization or the arrangement of organelles. In a few somites
Fig. 2. An atlas of somite development in Rana sphenocephala. Embryos were
fractured longitudinally to show the dorsal aspects of the intrasomitic cellular
arrangement pattern. Whole embryo views are seen on the left side, and highermagnification micrographs taken from the fractured somite mesoderm are on the
right side, (a) Stage 15; (b) stage 15+; (c) stage 16; (d) stage 18; (e) stage 20. Marked
areas in (c), (d) and (e) indicate a few posteriormost somites (about six) in which
the myotomal cells are undergoing fusion. Cells in the somites anterior to those
seem to have completed the fusion process at these particular fraction planes. Bars
in the lower-magnification micrographs represent 0-3 mm. Bar at the upper left
corner of the higher-magnification micrograph in (a) represents 01 mm for all the
higher-magnification pictures in Fig. 2. Ant. anterior; Post, posterior, for all the
embryos pictured here.
B. W. YOUN AND G. M. MALACINSKI
Fig. 3. (a) Dorsal view of the longitudinally fractured, unsegmented mesoderm of
Ambystoma embryo, right caudal to the last segmented somite. Cells of various sizes
are seen to be arranged in various directions. No specialized structure such as somitomeres, can be detected in this figure. This micrograph was taken from the embryo
shown in Fig. \c. Med, medial; Lat, lateral; Ant, anterior; Post, posterior. Bar
represents 25 /on.
(b) Dorsal view of the longitudinally fractured, unsegmented mesoderm of
Rana embryo, showing that cells appear to be oriented somewhat mediolaterally.
The width of the paraxial mesoderm at this particular fracture plane appears to consist of approximately four cells. The number of cells does not seem to change even
after somite segmentation (see arrow for the segmentation line). No somitomerelike structure can be observed in this micrograph. This picture was taken from the
embryo shown in Fig. 2c. Med, medial; Lat, lateral; Ant, anterior; Post, posterior.
Bar represents 25 /im.
anterior to the just separated paraxial mesoderm cells, fusion is already under
way (Figs. 2 c, d, and e). It is interesting to note here that the cells of approximately six caudal somites are always observed in the process of fusion (see
indicated areas in Figs. 2c, d, and e), whereas the rest of the anterior somites
appear to have completed the process. As in the case of Ambystoma, the fusion
process proceeds in the mediolateral direction. The cellular rearrangement
pattern at the time of somite segmentation and during myotomal cell fusions
will be dealt with later in this paper.
Amphibian somite
morphogenesis
ft
Fig. 4. Transverse view of the unsegmented mesoderm of Ambystoma (a) and Rana
(Jb) embryos. Arrow indicates approximate parasagittal plane of fractures which
will be shown in Fig. 5. Med. medial; Lat, lateral. Bars represent 01 mm.
An accurate estimation of the developmental timing of myotomal cell fusions
could not be made in this case, since a normal staging series of Rana sphenocephala has not yet been established. The developmental rate at room temperature
is, however, known to be about 20 % as fast again as Rana pipiens (J. Frost,
personal communication).
In order to approximate the timing of fusion, the 5th somite was chosen. It is
newly formed at the neural-fold stage, and the cells within it remain unfused
until the neural folds begin to fuse. Once the folds have fused, cells in the medial
side of the somite begin to fuse. By the muscular movement stage the cells in
the 5th somite appear to be completely fused. Substantially less than 24 h is
required, therefore, for completion of the fusion process.
2. Cellular rearrangement during rosette formation
In the chick embryo, somites have been shown to develop from pre-existing
somitomeres (Meier, 1979). That observation suggests an early programming of
the paraxial mesoderm which later develops into somites. In the case of amphibian
embryos, examination of the surface paraxial mesoderm has, however, failed to
provide any evidence for such somitomere-like structures (Youn et ah 1980).
In this study a further attempt was made to search for the presence of such
structures in the rosette-forming species of amphibians (urodeles). The pattern
of intrasomitic cellular arrangement was examined in the unsegmented mesoderm
which resides immediately caudal to a newly segmented somite. First, Ambystoma embryos were horizontally fractured through the unsegmented plate.
10
B. W. YOUN AND G. M. MALACINSKI
Dorsal aspects of the pattern of cells which lay deep (ventrally) in the plate were
examined with the SEM (Fig. 3d). No specialized structure which resembled the
somitomere of the chick embryo could be found. Cells in the medial side appeared,
however, to be elongated mediolaterally (see pointers in Fig. 3d).
Second, the cellular arrangement pattern of the paraxial mesoderm was
examined in transverse sections. The photograph in Fig. 4 a demonstrates that
cells which lie on the lateral side are arranged in a columnar manner, but cells
of the medial region are less well organized. Some of those in the medial region
near the notochord and the neural tube appear to be elongated in the mediolateral direction. This apparently explains why the cells of the medial side shown
in Fig. 3a were seen to be oriented in a mediolateral fashion. Fig. 4a also shows
that the paraxial mesoderm consists of two layers of cells in the lateral side and
approximately three layers in the region where the mesoderm is thickest.
These cells are, no doubt, elongated dorsoventrally. Therefore, the cellular
arrangement pattern displayed in Fig. 3 a is obtained when a fracture is made
between those cell layers. Only the upper (dorsal) portions of those dorsoventrally oriented cells which reside in the lower layer were exposed in the
horizontal section (Fig. 3d).
In Rana embryos, no somitomere-like cellular arrangement could be observed
in horizontal sections (Fig. 3b). The paraxial mesoderm consists of about four
cell layers in width at this particular fracture plane. The number of layers does
not appear to change even after the last somite is formed. As in the case of
Ambystoma embryos, the medial cells of the paraxial mesoderm are mediolaterally oriented. When viewed in transverse section (Fig. 4b), it can be
observed that the paraxial mesoderm consists of many cell layers dorsoventrally.
The highest number is found in the most medial side near the notochord and
decreases further laterally.
In order to gain insight into the cellular mechanisms involved in rosette
formation in Ambystoma and Pleurodeles embryos, it is essential to understand
the three-dimensional aspects of the cellular arrangements in the paraxial
mesoderm. The horizontal section shown in Fig. 3a reveals the morphology of
mediolaterally elongated cells but fails to display any structural details of dorsoventrally oriented cells. The transverse section in Fig. 4a provides a substantial
amount of information regarding the cell morphology and arrangement pattern
in the paraxial mesoderm. Yet that view fails to reveal any temporospatial
sequence of cell shape changes that occur in the posteroanterior direction as
the myotomal cells are progressively organized into a rosette. Nevertheless,
transverse sections can provide a general indication of how the cells in the
paraxial mesoderm might behave at the time of somite segmentation. The mediolaterally elongated cells seen in the transverse section (Fig. 4 a) and the horizontal
section (Fig. 3d) could provide the mediolaterally arrayed cells of a prospective
rosette. Cells in the middle layer of the three-layered region of the paraxial
mesoderm might serve as a source for anteroposteriorly arranged cells in either
Amphibian somite morphogenesis
11
Fig. 5. Mediolateral view of the parasagittally fractured Ambystoma (c) and
Pleurodeles (a and b) embryos, showing how changes in cell shape occur during
rosette formation. Short, thick arrows indicate the caudal segmentation line. Short
arrows point to conically shaped cells which appear to be undergoing rotation. In
these cases, rotation takes place in such a way that dorsoventrally elongated cells
become oriented anteroposteriorly. Triangular-shaped cells (long arrows) are
often observed in the anterior or the posterior corners of rosettes. Ant, anterior;
Post, posterior, for all the micrographs in Fig. 5. Bars represent 50 /«n. Same scale
of bar in (a) can be used in (b).
the anterior or posterior half of a rosette. In that case they would have to
undergo several degrees of rotation, since they appear to be elongated dorsoventrally. In the lateral region of the paraxial mesoderm, the cells are arranged
in two layers. Those cells must also rotate in order to provide the lateromedially
arranged cells of a prospective rosette.
12
B. W. YOUN AND G. M. MALACINSKI
Amphibian somite morphogenesis
13
Both the pattern of cellular arrangements in the unsegmented mesoderm and
the changes which accompany rosette formation in a newly formed somite can
be best displayed in parasagittal sections. Parasagittal fractures were made with
Pleurodeles (Figs. 5 a, b) and Ambystoma (Fig. 5 c) embryos at various stages.
The approximate location of the fracture plane is indicated by the arrow in
Fig. 4a. Since the fractures were made somewhat laterally, two layers of dorsoventrally elongated cells were usually observed in the paraxial mesoderm. It was
predicted that cells in the prospective cranial and caudal segmentation lines
should undergo extensive rearrangement in order to provide the anteroposteriorly oriented cells in the complete rosette. In Figs 5 a and c, in which the
posterior segmentation line is shown to be fully established (thick arrows), cells
in the most anterior portion of the as yet unsegmented mesoderm assume an
oblique orientation (short arrows in Figs. 5 a and c). Generally, those cells are
conical. The apex of the cells at the outside margin of the prospective rosette is
wide. The other ends are sharply pointed toward the prospective centre of the
rosette. Where segmentation is still in progress (thick arrow, Fig. 5 b), however,
the prospective anterior cells of the emerging somite do not show any detectable
sign of rotation. In such cases it was observed that in the caudal part of the
newly segmented somite, cells are often triangular shaped (long arrow, Fig. 5 b).
Such triangular-shaped cells are also frequently observed in other parts of rosettes
(long arrows, Fig. 5 a). It can be speculated that those cells are actively engaged
in rotating 90°. Perhaps they are triangular because one of their apices actively
moves towards the prospective centre of the rosette (see Discussion). As rosette
formation continues, the other apices probably retract toward the thickest
Fig. 6. (a) Dorsal view of the longitudinally fractured, unsegmented mesoderm of
Ambystoma, showing thin, thread-like projections between the cells. Bar represents
20 fim.
(b) Dorsal view of the longitudinally fractured, newly formed somite of Abmystoma, showing that cells appear to move towards the prospective centre of the
rosette (circled area) by sending out long projections (arrow). The caudal line of
segmentation is indicated by pointers. Bar represents 20 fim.
(c) Higher-magnification micrograph taken from the region near the long
projection indicated by arrow in Fig. 6 b. The long projection is seen to have tiny,
finger-like processes around its margin. Cells appear to make contacts with each
other by thin but broad projections which, in turn, send out a few finger-like
processes. Bar represents 20/tm.
id) Lateral view of the parasagittally fractured mature somite of Ambystoma,
showing thin, broad cell processes around the centre of the rosette. Bar represents
20 fim.
{e) Dorsal view of the longitudinally fractured mature somite of Ambystoma,
showing the myocoel. The lateromedial direction of the somite is indicated by long
arrow. Bar represents 20 fim.
(/) Higher-magnification micrograph of the myocoel taken from Fig. 6e,
showing specialized endings of myoblasts in the myocoel. They seem to be interconnected by a network of fibrils. Bar represents 20 fim.
14
B. W. YOUN AND G. M. MALACINSKI
Amphibian somite morphogenesis
15
portions of the cells. In this fashion dorsoventrally arranged cells could change
to an anteroposterior orientation.
Details of the changes in cell-to-cell contacts which accompany the formation
of a new somite were also examined. When observed in horizontal sections,
cells of the paraxial mesoderm appear to make contacts with each other by
many thin, thread-like projections (Fig. 6 a). In the newly segmented somite
(Fig. 6b), cells appear to move toward the prospective centre of the rosette
(small circle) by sending out long, thin, and sharply pointed projections (arrow).
When viewed under higher magnification (Fig. 6c), these cell processes appear
to bifurcate into short, finger-like projections which connect with adjacent
cells (arrows). Moreover, it was observed that cells in the newly formed somite
are attached to each other by thin, broad processes which, in turn, send out a
few finger-like projections (pointers, Fig. 6 c). Even after rosette formation is
completed, cells near the centre of the rosette continue to display specialized
cell processes (arrows, Fig. 6 d). Those processes are thin and broad and have
relatively smooth edges.
It appears that myocoel formation and the emergence of rosette centres are
separate phenomena. The micrographs in Fig. 1 display the centres of the
rosettes in each somite but not the myocoels. That may be due to the fact that
horizontal fractures were not made deep enough to uncover them. The myocoel
can be observed when a fracture is made more ventrally through the somite file.
Figure 6e shows both the centre of a rosette and the myocoel. Cells in the medial
Fig. 7. (a) Dorsal view of the longitudinally fractured somite of Ambystoma, showing
that the myoblasts start to fuse from the medial side. Short arrows indicate the
region of probably the retraction trails of two cells in the most medial side. They
begin to show an initial sign of fusion. One of them is seen to send out a long
process (pointer) on to another. Long arrows point to the prospective dermatomal
cell, which appears to have rather smooth lateral surfaces. Bar represents 20 /*m.
(6) Dorsal view of the Ambystoma myoblasts in the lateral region of the somite.
Cells in the most lateral side seem to lose their triangular or conical configuration
and become oriented anteroposteriorly. Bar represents 20 pm.
(c) Dorsal view of the longitudinally fractured somite of Ambystoma which
is in a more advanced stage of fusion than the somite shown in Fig. la. Fracture
was made more deeply (ventrally) in the lateral side than in the medial side. Arrow
indicates sclerotomal cell mass. Bar represents 20/*m.
(d) Higher-magnification micrograph taken from the lateral region of the
somite shown in Fig. 7 c. Arrow indicates thin, broad cell processes which appear
to cover the end portions of neighbouring cell surfaces which are to be fused.
Bar represents 20/tm.
(e) Dorsal view of the longitudinally fractured somite of Ambystoma in the
posterior portions. Cells in the anterior half of the somite are shown to develop
narrow and long processes (arrows), and begin to surround the shorter-looking
cells. Bar represents 20/*m.
(/) Dorsal view of the longitudinally fractured somite of Ambystoma, in which
cells appear to have already completed the fusion process. Pointers indicate dermatomal cells. The mediolateral direction of all the somites shown in Fig. 7 is indicated
by a long arrow here. Bar represents 20 /im.
16
^ T E A , * """*
B. W. YOUN AND G. M. MALACINSKI
Amphibian somite morphogenesis
17
region which are adjacent to the myocoel (opposite to the direction of the arrow
in Fig. 6e) appear to be shorter than those in the more lateral portions of the
somite. Therefore, the presence of the myocoel may not be required for cellular
rearrangements during rosette formation. Actually, the myocoel exists prior to
the rosette formation (Hamilton, 1969). Within the myocoel itself, cells appear
to be interconnected by a network of fibrils (Fig. 6/). Unlike chick embryos
(Bellairs, 1979), no mesenchymal cells were detected within the myocoel.
3. Cellular rearrangement during myoblast fusion
The process of myoblast fusion was examined in horizontally fractured
embryos of Ambystoma (Fig. 7) and Rana (Fig. 8). In the mature rosette configuration cells appear to radiate from the centre of the rosette in all directions.
In the medial and lateral sides, cells which are radiating from the centre of the
rosette are conically or triangularly shaped. In the middle region, however, cells
are somewhat rod-like or spindle-shaped, and are arranged directly in the
anteroposterior direction. Before they begin to fuse, two main events apparently
occur. First, cells in the medial side undergo extensive changes in shape. Figure
la shows that they are in the process of retracting their conical side away from
the centre and towards the medial region of the rosette (short arrows). While
that retraction process is under way, it appears that cells begin to establish endto-end contacts with each other (pointer). Those kinds of contact may be the
first indication that myoblast fusion is about to begin. It was speculated in the
previous section of this report that cell retraction is required for elongated cells
to change direction of orientation. As was shown in Fig. 5, dorsoventrally
elongated cells became arranged anteroposteriorly. Likewise, mediolaterally
arranged cells become oriented in the anteroposterior direction. Cell retraction
may, therefore, be a common mechanism used for changing direction of cell
orientation during rosette formation and myoblast fusion (see Discussion).
Second, extensive changes in cellular rearrangement occur in the lateral side.
Conically or triangularly shaped cells seem to rearrange themselves to become
elongated directly in the anteroposterior direction (Fig. 1b). Cell retraction may
also be involved in this process. As a result, radially oriented cells in the lateral
Fig. 8. Dorsal views of the longitudinally fractured somites of Rana embryos,
(a), (b), (c) and (d) are the higher-magnification micrographs taken from the 8th,
7th, 5th and 4th somites of the stage-16 embryo shown in Fig. 2 c, respectively.
Long arrows in (a) and (b) indicate probable fusion lines along which the myotomal
cells are aligned and fused later. In (a), arrow indicates long projection which may
play an active role in the fusion process. In (b), arrows indicate the cells which
appear to be elongated along the prospective fusion line. Arrow in (c) points to
widely spread cell process. In (d), most of the myotomal cells at this particular
fracture plane have completed fusion. Bar in (a) represents 20 fim for (b), (c), id)
and (/). Arrow in (e) also indicates long projections which are usually observed during
the initial period of fusion. Bar in (e) represents 20 /im. In (/), arrow points to the
cell which is near completion of fusion. The mediolateral direction of all the somite
is shown by long arrow in(/).
18
B. W. YOUN AND G. M. MALACINSKI
side change their orientation. Some of those cells which are located on the extreme
lateral side of the somite remain unfused and, at later stages, become dermatomal cells.
As mentioned before, the fusion of myoblasts proceeds in the mediolateral
direction. The observations included in Figs. 1c and Vindicate that the establishment of end-to-end contacts between cells in the anterior and posterior halves of
the rosette may lead to fusion. The higher-magnification micrograph in Fig. Id
shows that cells send out wide and broad processes (e.g. see arrow) surrounding
the partner cell with which they will fuse. Moreover, cells in the anterior half
of the rosette may play a more active role in the fusion process than those in
the posterior half. Those in the anterior half possess long and narrow processes
stretching all the way to the caudal end of the somite (see arrows in Fig. le).
Conversely, cells in the posterior half are somewhat shorter in length in this
particular fracture plane. Later, these processes may actually broaden and begin
to surround the shorter cells. Membrane fusion probably then follows.
The same kind of cell processes seen in Ambystoma at the time of myoblast
fusion can also be observed during myotomal cell fusion in Rana. Cells appear to
send out long processes toward neighbouring cells (arrows in Figs. 8 a, b). It
is also observed that cells frequently develop broad, wide processes, probably at
the end of the fusion process (arrow, Fig. 8 c). Figure 8e shows an example of
cells which are probably toward the completion of fusion (arrow). The cells are
almost completely fused, except in the posterior region where a smaller cell was
detached during the fracture leaving behind an artifactual hole. We suspect that
the fused cell was actively surrounding the detached cell underneath it in order
to complete fusion and to stretch from one end of the somite to the other. With
the aid of SEM alone, however, it is difficult to interpret cell morphologies and
contacts in terms of the fusion process itself. In order to determine precisely
where and when cell fusion is occurring, further evidence provided by the transmission electron microscope is needed.
As mentioned before, the myotomal cells in Rana undergo fusion immediately
after a few segmentations have occurred. During the fusion period they position
themselves in lines which are not exactly parallel to the long axis of the embryo.
In fact, it appears that the fusion lines are approximately 45° off the axis (long
arrows in Figs. 8 a and b). Cells in that formation which are preparing for fusion
(short arrows, Fig. &b) appear to be somewhat elongated in the direction of the
long arrow. However, Figs. 8 c and d reveal that cells which have already fused,
or are in the process of fusion, become arranged parallel to the notochord. This
observation indicates that Rana somites rotate in a fashion which resembles
somitogenesis in Xenopus (Hamilton, 1969; Youn & Malacinski, 1981). The
degree of rotation, however, is approximately 45°. That amount of rotation is
substantially less than the 90° rotation known to occur in Xenopus somites.
Like Ambystoma, the fusion process proceeds in the mediolateral direction.
It is also interesting to note here that the sclerotome does not seem to form
19
Amphibian somite morphogenesis
Step II
Parasagittal
section
(lateral view)
Step III
Shown only
on the
lateral side
Fig. 9. Schematic diagram of the changes in cell shape accompanying rosette
formation at the time of somite segmentation. Only two.layers of cells are considered. See text for detailed explanation of the cell shape changes in each step.
by partitioning from the main somite mass. Figure 1c shows the mass of sclerotomal cells lying deep in the medial wall of the somite (arrow). Those cells are
probably not of neural crest origin because neural crest cell migration has not
begun at this stage (approximately stage 32). Figure 7/clearly shows the dermatomal cells in the lateral side of the somite (pointers). These cells can be characterized by their smaller size and the many finger-like projections on their
lateral surfaces. For comparison, the lateral side of the somite shown in Fig. la
displays only cells which are larger in size and have fewer projections (long
arrows).
DISCUSSION
1. Cellular basis of rosette formation in urodele species
The scanning electron microscopic observations of the interior of the paraxial
mesoderm and somites have provided new insights into the cellular mechanisms
which direct rosette formation and myoblast fusion in Ambystoma and
Pleurodeles embryos. A variety of cellular mechanisms such as shape changes,
rearrangements, and individual cell movements are probably involved in such
processes. Our observations reveal that during somite morphogenesis some cells
appear to change orientation several times. The first change in cell arrangement
occurs during rosette formation at the time of appearance of new somites. The
parasagittal sections in Fig. 5 indicate the manner in which the double-layered
cells, which are elongated in the dorsoventral direction, become organized into
a rosette form. A particularly interesting feature of somite segmentation is the
appearance of triangular-shaped cells at the corners of rosettes. These cells may
turn through as much as 90°. The cellular arrangement pattern seen in Fig. 5 is
diagrammatically illustrated in Fig. 9. To simplify the issue, only two layers of
cells elongated dorsoventrally in parasagittal section are considered. During the
establishment of the segmentation line cells in the bottom portion of the upper
layer and top portion of the lower layer begin to send out long processes toward
the prospective centre of the rosette (see Figs. 6b, c). In order to become
organized into a rosette, cells in the anterior- and posterior-most regions of the
somite must move a longer distance towards the centre of the rosette than those
in the middle. In the process they become triangular shaped. It appears that such
20
B. W. YOUN AND G. M. MALACINSKI
cellular reshaping, followed by active cell movements, takes place only after the
segmentation lines appear. Since segmentation lines are established first in the
anterior region, cells in that region are the first to rearrange themselves into
triangular configurations. Shortly afterwards, while the caudal line of segmentation is being established, cells in the posterior region of a newly formed somite
begin to change shape.
In Step II (Fig. 9), those triangular-shaped cells undergo another change in
shape in order to complete the process of rosette formation. In more matured
somites, cells are radially arranged so that those in the anterior and the posterior
halves of the rosette become oriented more or less parallel to the long axis of the
embryo (Step III). In order to become arranged in that fashion, the apices of
the triangular-shaped cells around the outside margin of the somite appear to
move towards the future anteroposterior axis (see the direction of arrows in
Step II). This phenomenon provides one definitive example of a cell rotation
which occurs during somite morphogenesis in amphibian embryos. Another
example can be found in the rotation through 90° of myotomal cells of Xenopus
laevis somites (Hamilton, 1969). Our SEM studies have demonstrated that rotating myotomal cells of Xenopus often exhibit bent configurations (Youn &
Malacinski, 1981). That shape change may indicate that they have an intrinsic
capacity for bending. Perhaps specially oriented or polarized microtubules
(reviewed by Karfunkel, 1974; Trinkaus, 1976) participate in the process. In the
case of rosette formation, however, cell motility mechanisms might be somewhat
different. Cells retract, probably because they have a tendency to compensate
for previous migratory movements towards the centre of the rosette (see Step I).
In contrast to Xenopus somitogenesis, therefore, active cytoplasmic flow (in the
direction of arrows in step II) accompanied by membrane turnover may be
directly involved in the rotational reorientation of myotomal cells in Ambystoma
and Pleurodeles embryos (Harris, 1973). Transformation of microfilaments from
a network formation to bundles, or vice versa, during tail elongation and retraction as seen in fibroblasts in culture (Chen, 1981) could account for myotomal
cell retraction during rosette formation.
A question then arises concerning the motive force that drives cells to the
prospective centre of the rosette. Morphogenetic movements of cell groups may
be mediated by cell-cell interactions such as adhesion, contact inhibition, or
changes in the shapes of firmly affixed individual cells (Phillips, Steinberg &
Lipton, 1977). Alterations in the adhesive interactions between cells have been
thought to be active factors in morphogenetic processes (Townes & Holtfreter,
1955; Gustafson & Wolpert, 1967). Bellairs, Curtis & Sanders (1978), Bellairs,
Sanders & Portch (1980) have shown that when chick somites segment and the
somite cells subsequently differentiate, the rate of cell-cell aggregation increases
and the cells exhibit characteristic behaviour patterns. It was proposed from
those observations that changes in cellular adhesiveness play an important role
in somite formation. That proposal may imply that the surface of each cell is not
Amphibian somite morphogenesis
21
uniformly adhesive during rosette formation. Rather, it is localized largely at
one end near the prospective centre of the rosettes. Subsequently, clusters of cells
in a newly formed somite aggregate together in the area of high adhesiveness.
That presumably leads to the organization of cells into rosettes.
There is much evidence that the molecules which determine adhesive specificity may reside at or external to the cell surface. For example, research on
aggregation factors has suggested that specific glycoproteins presumably located
at the cell surface are responsible for mediating cell-cell adhesion in embryonic
chick neural tissue (Thiery, Brackenbury, Rutishauser & Edelman, 1977) and
cellular slime moulds (Gerisch, 1976). It is very likely that somitic cell rearrangements are also initiated to a significant degree by modifications in
cell-surface adhesion determinants. In order to understand the mechanisms and
forces which mediate and direct morphogenetic cell rearrangements accompanying rosette formation, it would be essential to seek further evidence for changes
in the adhesion/recognition properties of the somitic cells and to relate these
properties to specific cell-surface biochemical determinants.
Our SEM studies have revealed several major differences between somitogenesis in amphibian and in chick embryos. In chick embryos, the presence of
mesenchymal cells in the mycoel, the appearance of somitomere-like structures
in the paraxial mesoderm, the mode of sclerotome formation, and the loosely
packed nature of the somitic cells all differ from the amphibian embryos studied
here. Such variant features lead to the suggestion that amphibian and chick
somites may employ different mechanisms for rosette formation. Extensive
discussion of comparisons between amphibian and chick somitogenesis may
ultimately be, therefore, misleading.
2. Cellular basis of myoblast fusion
Extensive changes in cellular arrangement were shown to occur during the
initial period of fusion (Fig. la). Radially arranged cells in the medial side of the
rosette appeared to retract their apices from the centre of the rosette and become
arranged anteroposteriorly. Those in the lateral side also began to undergo
rotational reorientation at about the same developmental stage (Figs. 7 b, c).
Medial cells of the rosette become elongated anteroposteriorly before the lateral
ones do. This mediolateral directionality can also be observed during amphibian
somite morphogenesis in the following two instances: (1) myoblast fusion, and
(2) formation of dermatome in the lateral side and of sclerotome in the medial
side. It would be interesting to determine when and how information on the
directional morphogenesis is given to the somitic and/or presomitic cells during
development. Somite reversal experiments (Deuchar & Burgess, 1967) are
currently being undertaken to obtain such information.
While the fusion process is under way in the medial side, somites are composed
of two layers of spindle-shaped myoblasts tandemly arranged in the anterior
and the posterior halves of the rosette. Our SEM observations indicate that
22
B. W. YOUN AND G. M. MALACINSKI
Table 1. Comparative aspects of amphibian somite morphogenesis
Anurans
A.
Character
Xenopus
Rana
{Ambystoma, Pleurodeles)
Origin of mesoderm
Mode of the establishment
Presence of myocoel
Deep layer*
Diagonal
Obliterated after
segmentation
Presence of rosette-shaped
somites
Cell rotation
Absent
?
Superficial layerf
Transverse
Diagonal
Not present Present both in unsegmented mesoderm
and somites
Present
Absent
90°
45°
Cell fusion
Absent
Multinuclearity in myoblasts
or myotomal cells
Multinuclearity in myotubes
Mono
Involves
several cells
Multi
?
Irrelevant
Multi
As much as 90° in some
cells during rosette
formation and myoblast
fusion
One-to-one fusion
Multi
* According to Keller (1976).
t According to Vogt (1929).
fusions take place between two cells from each layer. Are the resulting fused
cells dinucleate? According to Holtfreter (1965), most of the myotubes of
Ambystoma maculatum somites contain five to seven nuclei. Muchmore (1965)
has examined mitoses in developing myotomes of Ambystoma maculatum. He
found that the nuclei of myoblasts divide rapidly until the mid-tailbud stage,
when mitosis ceases and the cells fuse to form the characteristic multinucleate
muscle fibres. Even prior to fusion, therefore, nuclear division in the absence of
cytoplasmic partitioning has probably occurred in the myoblasts. The multiple
fusions of mononucleate myoblasts suggested by Loeffier (1965) do not appear
to take place. Our SEM results (e.g. Fig. 7) failed to reveal signs of multiple
fusions between myoblasts.
When the cells undergo fusion, they elaborate cell processes which may play an
important role in the fusion process itself. Similar types of cell processes were
observed in each of the amphibian species included in this study. In the present
studies cells were observed to send out either long processes (see arrows in
Figs. 7e, 8 a and 2>e) or thin, broad processes (see arrows in Figs. Id and 8 c).
It can be speculated that the appearance of such long processes is characteristic
of cells which are in the initial phase of fusion. Conversely, the presence of
thin, broad processes may indicate that cells are near the completion of fusion.
Close apposition of membranes by means of such cell processes appears to
characterize the early events of membrane fusion in many experimental systems.
A direct morphogenetic role of surface cell processes produced during the process
Amphibian somite morphogenesis
23
of cell fusion has been suggested for neural fold fusion (Bancroft & Bellairs,
1975; Mak, 1976; Waterman, 1975) and for fusion in cultured muscle cells
(Fischman, 1970; Shimada, 1971,1972). Ultrastructural alteration of membranes
accompanying this process has also been the subject of several recent reports
(Shimada, 1971; Lipton & Konigsberg, 1972; Rash & Fambrough, 1973).
However, the detailed cellular mechanisms of fusion still remain obscure.
3. Comparative aspects of amphibian somite morphogenesis
Table 1 summarizes different aspects of somite formation in four species of
amphibia. Embryos of the urodele species, Ambystoma and Pleurodeles, exhibit
exactly the same pattern of somite morphogenesis. A few differences exist among
the anuran species, Xenopus and Rana. However, there are many differences in
somite formation between the anuran and urodele species. No comprehensive
comparisons will be made for each aspect listed in the table, since many of the
details have already been mentioned. Further discussion concerning the establishment of segmentation lines and the ploidy of myoblasts is, however, warranted.
First, the shape of newly formed somites in Xenopus and Rana appears to be
arrowhead-like. Youn & Malacinski (1981) have suggested that the formation
of 'arrowhead-like' somites may be caused by extensive differential movements
that occur between the prospective neural area, epidermis, and prospective
mesodermal mantle during neurulation. As a result, the segmentation lines are
made diagnonally. In the case of the urodele species, the somites appear to be
formed by transverse fissures, giving the somites a 'stump-like' shape. This
may be due to the fact that differential movements are not as prominent in
Ambystoma and Pleurodeles as in Xenopus and Rana. The question of how
different shaping mechanisms might affect the intrasomitic cellular arrangement
pattern remains obscure.
Second, with regard to the ploidy of myoblasts and myotubes, it is known that
Xenopus rotated myotomal cells remain uninucleate until about stage 45
(Kielbowna, 1975). Nuclear division then follows, and mutinucleate myotubes
are formed. Conversely, in Ambystoma and Pleurodeles embryos, multinucleate
myotubes remain in an interkinetic stage until the beginning of metamorphosis.
It was, therefore, concluded that the myotomal cells of Xenopus were unusual in
that they developed to a fully functional state and relatively large size while
remaining uninucleate (Muntz, 1975). Multinucleation and the establishment of
contractibility are obviously not causally related.
The pronounced differences in early embryonic development of the anuran
and urodelean amphibians have recently been summarized (Chung & Malacinski,
1980). Nieuwkoop & Sutasurya (1976) have also indicated that variations are
exemplified in many ways, including differences in mesoderm formation. But,
perhaps most importantly, fundamental differences in primordial germ cell
(PGC) formation exist between the two groups. Comparative analyses of the
origin of the PGCs have led to speculation that the two groups of amphibians
24
B. W. YOUN AND G. M. MALACINSKI
are only remotely related and could have originated polyphyletically from
different ancestral fishes. Table 1 also displays striking differences between the
two groups in the mode of somite formation and subsequent muscle development. However, it is perhaps not appropriate to speculate further on the origin
of the modern Amphibia with those data. Rather, we would like to suggest that
Rana may be intermediate between Xenopus and the urodele species, and that
more diversity can be found among the anuran species than the urodele species.
The pattern of somitogenesis may provide a useful indicator for determining
how different families of amphibians evolved. Further clues as to the origin of
modern Amphibia may be obtained by examining and comparing various aspects
of somitogenesis among teleosts, reptiles, and other amphibians.
We wish to express our gratitude to Dr Ray Keller for his valuable advice and continuous
encouragement during the course of this investigation. We wish to thank Dr John Frost, Dr
Sally Frost and Ms Fran Briggs (I.U. Axolotl Colony) for providing Rana, Pleurodeles and
Ambystoma embryos, respectively. We also thank Ms Diane Malacinski for assisting in
the preparation of the manuscript. This work was initiated with support from NSF PCM
77-04457 and completed with NSF PCM 80-06343.
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