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/. Embryol. exp. Morph. Vol. 64, pp. 23-43, 1981
Printed in Great Britain © Company of Biologists Limited 1981
23
Somitogenesis in the amphibian
Xenopus laevis: scanning electron microscopic
analysis of intrasomitic cellular arrangements
during somite rotation
By B. WOO YOUN 1 AND GEORGE M. MALACINSKI 2
From the Program in Cellular, Molecular, and Developmental
Biology, Department of Biology, Indiana University
SUMMARY
The intrasomitic changes in cell arrangement which accompany somite rotation during
somitogenesis in Xenopus laevis were analysed with the scanning electron microscope (SEM).
Longitudinal, horizontal fractures of whole embryos were examined at various dorsoventral
levels of stage-22 to -24 embryos. Observations of the gross morphological features of
somitogenesis, and the cellular changes which accompany somite segmentation and somite
rotation were made. Several of these observations lead to modifications of previous models
for the cellular basis of somitogenesis in Xenopus. Individual cellular rearrangements, rather
than simultaneous block rotation of a whole somite, appear to be responsible for the 90°
rotation of myotomal cells within a single somite.
Cellular arrangments in fused somites were also examined. Some ultraviolet-irradiated
embryos displayed a complete lack of a notochord. The somites in those embryos were
fused across the midline beneath the neural tube. The dorsal and ventral arms of the somites
are not fused. Normal rotation occurs only in the dorsal and ventral arms while, in the
majority of cases, cells in the fused region fail to rotate normally. In some cases, individual
cells in the fused region undergo partial rearrangement. Those observations support the
notion that individual cellular rearrangements account for the rotation of the whole somite.
INTRODUCTION
Analysis of somite formation during amphibian embryogenesis provides
various opportunities for gaining insights into both the cellular basis of morphogenesis (Deuchar & Burgess, 1967) and the theoretical aspects of pattern
formation (Cooke & Zeeman, 1976). For example, attempts at understanding
the manner in which the paraxial mesoderm becomes segmented into somites
has been the object of numerous investigations. This issue has been approached
in various ways (reviewed by Bellairs, 1979), one of which is the study of the
morphological changes which occur during somite segmentation. From obser1
Authors' present address: Department of Biology, Princeton University, Princeton,
NJ 08540, U.S.A.
2
Authors' address for all correspondence: Department of Biology, Indiana University,
Jordan Hall 138 Bloomington, Tndiana 47405, U.S.A.
24
B. W. YOUN AND G. M. MALACINSKI
vations made with the light microscope, Hamilton (1969) developed a model to
describe the process of somite segmentation in Xenopus laevis. The paraxial
mesodermal cells of Xenopus embryos first elongate perpendicularly to the
notochord. The process of somite segmentation then occurs as transverse
fissures isolate successive blocks of spindle-shaped cells. Segmentation progresses
in a cranial to caudal direction. Each somite then proceeds to rotate through
90 ° with its medial edge moving forward. Individual somites thus become
single bundles of spindle-shaped cells (myoblasts) that lie parallel to the notochord. In other amphibia, however, the pattern of somite formation is quite
different. In urodeles, for example, the somites form before cell elongation, in a
manner comparable to many other vertebrates (e.g. chick, see Bellairs, 1979;
Meier, 1979) where each somite forms initially as a rosette. During subsequent
differentiation, the myotome cells alter their orientation, become elongated
anteroposteriorly, and fuse to form multinucleate muscle fibres (Loeffier, 1968).
In the case of Xenopus, several questions remain concerning the mechanical
details of somite rotation: Does a whole somite block rotate simultaneously,
or do the cells within a somite rotate as individuals? Does a centre of rotation
exist around which the other parts of the somite rotate in a manner analagous
to an axle and wheel? Do the neural tube, notochord, lateral mesoderm, or
dermatome facilitate myotome rotation? What, if any, locomotory processes
are involved as individual myotomal cells change position?
In a recent study (Youn, Keller & Malacinski, 1980), we provided scanning
electron microscopic descriptions of somitogenesis in Xenopus. Those data
demonstrated that distinctive changes in cellular morphology and arrangement
occur among the paraxial mesoderm cells, as predicted by the earlier descriptions of Hamilton (1969). The present studies were undertaken in order to
provide further insights into the mechanical basis of somite rotation. We have
examined the cellular arrangement of myotomal cells in the rotating somite
itself. When frontal fractures were made through a rotating somite at various
dorsoventral levels, groups of myotomal cells were observed to exhibit a
gradient of direction in cellular orientation. Moreover, we have extended our
discovery that ultraviolet irradiation (U.V.) of the vegetal hemisphere of the
fertilized, uncleaved Xenopus egg produces embryos which lack a notochord
and display fusion of somites along the midline beneath the neural tube (Youn
& Malacinski, 1980a). According to the model for somite morphogenesis
proposed by Hamilton (1969), fusion of somites across the midline of the
embryo should prevent the rotation of individual somites. Our results, however
demonstrate that myotomal cells in both the dorsal and ventral portions of the
somite orient in the same direction as those of normal, unfused somites. Those
in the fused region do not rotate normally. These observations contradict the
idea that simultaneous block rotations occur along the dorsoventral length of
the whole somite. Rather, individual cellular rearrangements accompanied by
the active cell movements within the somite appear to occur.
Amphibian somitogenesis
25
MATERIALS AND METHODS
Xenopus laevis embryos were obtained by either gonadotrophic hormoneinduced mating of adults (Gurdon, 1967), or artificial insemination (Wolf &
Hendrick, 1971; Youn & Malacinski, 19806). Embryos collected from natural
spawnings were chemically dejellied in a 2 % cysteine-HCL solution (pH 7-4
with Tris buffer), allowed to develop in dechlorinated tap water (DTW), and
fixed in 2 % glutaraldehyde (pH 7-6 with 0-1 M-cacodylate buffer) at Nieuwkoop
& Faber (1967) stage 22-24. Artificially inseminated eggs were U.V.-irradiated
according to the method outlined by Malacinski, Brothers & Chung (1977).
Irradiated eggs were kept in DTW until the embryos were classified at stage
22-24 according to their defects in neural structures (Malacinski, Benford &
Ching, 1977; Youn & Malacinski, 1980#). Embryos which displayed extents of
neural defects in the 0- to +2 range ( 0 - , near normal neural morphology;
+ 2, extreme microcephaly; see Youn & Malacinski, 1980#) were selected and
fixed in the 2 % glutaraldehyde solution.
Fixed embryos were washed in 0-1 M-cacodylate buffer (pH 7-6). Then the
epidermis and neural tube (sometimes together with the notochord) were
removed using a fine steel knife and forceps. In order to expose the intrasomitic
cellular morphology and arrangement, longitudinal, horizontal fractures were
made to each embryo at various dorsoventral levels (see Results for details of
the fracture methods). The fractured embryos were dehydrated in increasing
concentrations of ethanol and critical-point dried using liquid CO2. All the
specimens were mounted on aluminium stubs with conducting silver paint and
coated with gold-palladium in a sputter coater E5100 (Polaron Equipment, Ltd).
These were observed with an Etec Autoscan U-l SEM and photographed on
Polaroid Type 55 positive-negative film.
RESULTS
I. Morphological features of somitogenesis in Xenopus laevis
The cellular arrangement in the paraxial mesoderm is shown diagrammatically in Fig. 1. SEM observations of transverse sections of the paraxial mesoderm
at the early neurulation period have been made in the previous paper (Youn
et al. 1980). Such observations are summarized here in order to provide some
basic information on how changes in cellular morphology and arrangement
occur among the paraxial mesodermal cells before somite segmentation begins.
At approximately stage 12 f , the paraxial mesoderm consists of two layers of
cells. These cells are polyhedral-shaped and are perhaps slightly greater in
height than width (Fig. \a). At about stage 13 f the paraxial mesoderm continues to maintain a double-layered configuration, but the cells become more
elongated dorsoventrally (Fig. 1 b). Cells near the notochord, where the upper
and lower layers meet, have a rather irregular shape but are oriented medio-
26
B. W. YOUN AND G. M. MALACINSKI
(a)
W
(O
N
Fig. 1. Diagrammatic representation of cellular arrangements (mid-transverse view)
in the paraxial mesoderm of Xenopus laevis at about stage 12+ (a), stage 13+ (/?), and
stage 18 (c). Pointers and arrow in (c) indicate the prospective dermatomal cells
and the pre-myocoel, respectively. N, notochord. (Redrawn from Youn etal., 1980).
laterally. When the neural folds rise, changes in arrangement among the paraxial
mesoderm cells occur. By stage 18, cells in the upper layer come to lie mediolaterally whereas those in the lower layer lie dorsoventrally (Fig. lc). Cells
near the notochord assume a radial arrangment and become elongated further
mediolaterally. This arrangement is also observed in the unsegmented posterior
portion of stage-22 to -24 embryos. All the prospective myotomal cells become
more spindle-shaped than at earlier stages. The prospective dermatomal
cells (see pointers in Fig. lc) form a sheet over the lateral surface of the
prospective somite. A small pre-myocoel is visible in the unsegmented mesoderm
(see arrow in Fig. 1 c).
By about stage 18, three to four somites have formed (Youn et ah 1980).
As the elevated neural folds fuse and the whole embryo increases in length,
more somites are added posteriorly. Figure 2a displays a dissection from a
stage-22 to -24 embryo which shows the mediolateral view of the right
side of the somite files and the rest of the unsegmented mesoderm. The somite
mesoderm can be divided into three regions: (1) Region A, to which the neural
tube attaches; (2) region B, where the notochord resides; and (3) region C,
where the mesoderm contacts the endodermal roof of the gut. Cells in regions
A, B, and C originate at earlier stages from the upper, middle, and lower layers
of the paraxial mesoderm, respectively. Those three regions are sharply (pointers
in Fig. Id) delineated in the posterior portion of the unsegmented mesoderm.
That sculpturing, however, becomes less distinctive in the anterior portion of the
unsegmented plate. In the segmented mesoderm, almost no sculpturing is
observed. This is probably due to the fact that regions A and C become stretched
dorsoventrally in the more anterior unsegmented mesoderm.
It can also be seen in Fig. 2a that somite segmentation in the most anterior
part of the stage-22 to -24 embryo occurred by the formation of transverse
fissures. As a result, the shape of the anterior-most somites is 'stump-like'.
More posteriorly, somites are 'bow-shaped'. Further posteriorly - in the last
few segmented somites-they display an 'arrowhead-like' shape. The manner
in the which the various shapes are acquired at different developmental times
is not known. It is probable, however, that differential movements between the
neural ectoderm (or epidermis) and the trunk mesoderm during the periods of
Fig. 2 (a) Mediolateral view of the right side of the somitefilesand the unsegmented mesoderm of a Xenopus embryo at stage 22-24,
showing how the somite mesoderm was divided into three regions, A, B, and C. The three regions are most sharply sculptured in
the caudal end of the unsegmented plate (pointers). Ant, anterior; Post, posterior. Bar represents 01 mm.
(b) Posteriodorsal view of stage-22 to -24 Xenopus embryo. Longitudinal fractures were made in a few somites and in the most
anterior portion of the unsegmented mesoderm at or above the level of the notochord (N), in order to reveal cellular airangement
pattern. Ant, anterior; Post, posterior.
(c) Higher magnification micrograph of the most posterior two somites of the embryo shown in (b). The upper region of the
somites appears to be twisted backward in the mediolateral direction. Arrow points to the parallel arrangement of the myotomal
cells in the unsegmented mesoderm. Bar represents 50 fim.
(d) Posterotransverse view of the most posterior somite of Xenopus at late neurula stage. NF, neural fold; S, somite; N, notochord;
DM, dermatome; EP, epidermal ectoderm; AR, endodermal roof of archenteron. Bar represents 50/*m.
t^- Post *
Ptost
to
1-
28
B. W. YOUN AND G. M. MALACINSKI
neurulation and post-neurulation may, in part, contribute to the different shapes
of the somites (see Discussion).
Viewed dorsally, the upper regions of the arrowhead-like somites appear to
be twisted backwards (Fig. 2b-d). Therefore, the initial process of somite
segmentation in the upper region of the arrowhead-like somites occurs by the
formation of diagonal rather than transverse fissures. Since cells in the unsegmented mesoderm are oriented perpendicularly to the notochord (arrow in
Fig. 2 c), the development of transverse fissures should be mechanically more
simple. Questions concerning (1) the manner in which the embryo makes such
diagonal fissures which are directed against this cellular arrangement, and (2)
the role of the somite segmentation pattern in the overall process of somite
rotation are, however, beyond the scope of this study.
It should be noted here that the dermatome also segments together with the
somites (Figs. 2b, c). Kordylewski (1978) previously reported similar observations in Xenopus. These observations, however contradict the earlier description
made by Hamilton (1969) that the dermatome remains as a curtain during
somite segmentation. The fact that the dermatome also segments raises an
important question concerning the problem of somite rotation in Xenopus: Does
the dermatome also rotate? Since the medial edge of the rotating somite always
goes forward the dermatome in the lateral side moves backward to be positioned
in the intersomitic space. This process does not, however, occur in vivo. The
prospective myotomal cells should be able to rotate freely and independently
of the segmented dermatome (see below).
IT. Intrasomitic cellular arrangment in the rotating somite
Neither mediolateral nor transverse views (Figs. 2a, d) yielded information
about intrasomitic cellular arrangements in the rotating somite. Longitudinal
fractures along the whole length of the somite file at three different dorsoventral
levels were, therefore, made. An embryo in which a fracture was made through
region A is shown in Fig. 3a; through region B in Fig. 3b; and through region
C in Fig. 3 c). In all those micrographs, cells in the unsegment?d mesoderm lie
perpendicular to the notochord. In the most posterior somite, cells shown in
Fig. 3 a as well as in Fig. 3 c are oriented at about 45° to the notochord. This
observation confirms the earlier findings of Hamilton (1969). When a fracture is
made through region B, the oblique orientation of the myotomal cells in the
most posterior somite is rarely seen. As shown in Fig. 3 b, a portion of the cells
appear to have completed their rotation. Those cells located in the medial edge
are, however, actually in the process of completing rotation. In the more anterior
somites the myotomal cells seen in Fig. 3 b are oriented parallel to the long axis
of the embryo.
Two main conclusions can be drawn from observing the intrasomitic cellular
arrangements shown in Fig. 3. Firstly, there exists a dorsoventral gradient in
the myotomal cell orientation of the rotating somite. Cells in the upper (A) and
Amphibian somitogenesis
;/\
-
29
ivn
Fig. 3. Dorsal view of longitudinally fractured somite mesoderm olXenopus at stage
22-24 showing intrasomitic cellular arrangements. Fractures were made through
regions A(o), B(b), and C(c). Arrows indicate the last segmented somites. Ant,
anterior; Post, posterior. Bars represent 01 mm.
EMB 64
30
B. W. YOUN AND G. M. MALACINSKI
lower (C) regions of the rotating somite are at the same stage of cellular orientation - i.e. approximately 45° to the notochord. Most of the cells which
occupy the middle (B) region have completed rotation through 90° and lie
parallel to the notochord. This observation indicates that the cells within an
individual somite do not rotate simultaneously along the entire dorsal/ventral
length of the somite. Since cells in region B rotate first, there may actually be an
intrasomitic sequential wave of a rotational signal. That signal might arise in
the middle region first and then be propagated toward both ends of the somite.
Secondly, a gradient in the myotomal cell orientation exists within a single
fracture plane of a rotating somite (see Fig. 3 b). Observations of these various
cell orientations suggest that individual cellular 'rearrangements' rather than
the simultaneous block rotation of a whole somite is responsible for the
rotation of the myotomal cells within an individual somite by 90°.
Further evidence to support the first conclusion (re. dorsal/ventral gradient)
was obtained from observations of the type shown in Fig. 4. The two most
posterior somites were photographed ventrally or mediolaterally at various tilt
angles. In the most posterior somite (viewed ventrally), myotomal cells located
in the most ventral region are orientated almost perpendicularly to the notochord (long arrow in Fig. 4 a). However, cells which lie more dorsally approach
an orientation which is parallel to the notochord (short arrow in Fig. 4a). These
differences in cellular arrangement in the ventrodorsal direction can be observed
more clearly at higher tilt angles which reveal both the ventral and the mediolateral aspects of the rotating somite (long and short arrows, in Figs 4b, c).
When viewed mediolaterally (Fig. 4d), both the upper and lower parts of the
somite are twisted backward in the mediolateral direction. This twisted configuration of the most posterior somite in the upper region can also be seen in
Fig. 2 c. A dorsal view of the twisted ventral portion of the somite is shown in
Fig. 5.
The second conclusion (re. intrasomitic cell patterns) was further strengthened
by the results of additional examinations of myotomal cellular arrangements of
the type displayed in Fig. 6. In these cases the longitudinal fracture was made
between regions A and B (see Fig. 6 a for the lateral view of the fractured whole
embryo). A higher magnification dorsal view of the myotomal cellular arrangement is shown in Fig. 6b. As mentioned previously, cells in the unsegmented
plate are oriented perpendicularly to the long axis of the embryo (Fig. 3). Cells
in the most anterolateral portion of the unsegmented mesoderm, however,
assume an oblique orientation (short arrows in Fig. 66). A similar cellular
arrangement can also be observed in the comparable region of the unsegmented
mesoderm displayed in Fig. 3 b. This indicates that those cells are already in the
process of rotating prior to segmentation. The cellular arrangement in the
fracture plane of the posterior-most somite is particularly interesting. Cells in
the lateral and medial edges are oriented parallel to the notochord (long arrows
in Fig. 6 b). Conversely, those cells in the centre portion maintain the parallel
Amphibian somitogenesis
Fig. 4. Scanning electron micrographs of the two posterior-most (right side) somites
of Xenopus at stage 22-24. A fracture was made through region C, and the somite
file was inserted to reveal the ventral aspect of cellular arrangement pattern. These
somites were then photographed ventrally or mediolaterally at various tilt angles:
(a), 0°; (b), 20°; (c), 40°; (d), 60°. Long arrow points to the group of cells which lie
perpendicular to the long axisof the embryo. Short arrow points to the group of cells
which lie more dorsally than those indicated by the long arrow and which also appear
to assume an oblique orientation at higher tilt angles. Ant, anterior; Post,
posterior. Bar in (d) represents 50/tm for all the figures shown in Fig. 4.
31
32
B. W. YOUN AND G. M. MALACINSKI
Fig. 5. Dorsal view of the posterior-most somite of Xenopus at stage 22-24 showing
the parallel cellular arrangement and twisted configuration in the most ventral portion of the somite. A fracture was made through region C. Bar represents 20 /*m.
orientation up to a certain point (pointer in Fig. 6b) where they turn sharply
toward the notochord.
These observations support the second conclusion that individual cellular
rearrangements, rather than block rotation of a whole somite, is involved in
the turning of the myotomal cells by 90°. These observations also yield another
important conclusion - that the rotating myotomal cells have elastic properties.
That is, cells bend while in the rearrangement process. Moreover, cells in the
medial edge of the most posterior somite which lay parallel to the notochord
appear to reside deeper (more ventral) than those other regions of the somite
(thick arrow in Fig. 6 a). This parallel arrangement of the myotomal cells can
also be observed if a fracture is made more ventrally through this region (Fig.
3 b). This observation also supports the conclusion that the turning of the cells
in the middle region (B) occurs prior to rotation of cells in regions A and C.
It is also worthwhile to examine the myoblast cellular morphology and the
cell-to-cell contacts in both the unsegmented mesoderm and the segmented
somites. Most cells in the caudal portion of the unsegmented mesoderm are
'rod-like' rather than'spindle-like'. They are in contact with each other through
small protrusions (Fig. 6c). In this region the lateral ends of cells are round
and smooth, and display only a few protrusions. Unfortunately, it is difficult to
analyse the detailed cellular morphology and contacts in the medial side. The
presence of large amounts of extracellular fibrillar materials obscure those
contacts. Occasionally it was observed that many cells send out small protrusions which, in turn, appear to connect to the notochord by a network of
extracellular fibrils (short arrow in Fig. 6 c). In the most anterior region where the
myotomal cells are beginning to change their orientation, cells also show changes
Amphibian somitogenesis
33
in morphology - especially at their ends. Their anterior or medial ends become
flat and broad (pointers in Fig. 6c), whereas their lateral ends become narrow
and spindle-shaped (long arrows in Fig. 6 c). There appears to be less extracellular material in this medial space compared with the posterior portion. This
is perhaps mainly due to the fact that the fracture was made at slightly different
dorsoventral levels (arrow in Fig. 6a). In the most posterior somite, parallel
and oriented cells display broadened ends at the posterior side (pointers in
Fig. 6d). Cells which are in the process of rotating and bending are narrow and
sharply pointed at both ends (long arrows in Fig. 6d). These observations imply
that the extensive changes in cellular morphology occur during myotomal
cellular rearrangement and rotation (see Discussion). In the more anteriorly
located somites cells become spindle-shaped. Among those a few cells may
maintain blunt ends at the posterior side (short arrows in Fig. 6d). In this region
extracellular fibrillar materials are abundant only in the region between the
notochord and the intersomitic space.
III. Intrasomitic cellular arrangement in fused somites
Previous studies by Malacinski et al. (1975, 1977) have demonstrated that
ultraviolet irradiation (254 nm) of the vegetal hemisphere of the fertilized,
uncleaved egg results in abnormal axial structure development. The range of
defects in the formation of the axial structures is very broad. Recently, Youn
& Malacinski (1980a) employed the SEM to examine various types of defective
embryos. In that study the following notations were employed to categorize
the types of abnormal embryos employed: 0, no apparent irradiation-induced
damage to axial structures; 0~, slightly microcephalic; + 1 , microcephalic; +2,
extremely microcephalic; + 3 , poor head morphology and shortened axial
structures. In the present study U.V.-irradiated embryos which displayed those
neural defects were examined after the epidermis was peeled was observed. It
was discovered, for example, that 0~ to +3 embryos lacked a notochord. One
result of notochordlessness is the fusion of somites across the midline underneath the neural tube (Fig. 7). The process of somite segmentation in irradiated
embryos appeared, however, to occur normally. It was observed that somite
counts of 0 to + 2 embryos were approximately the same as those of normal,
unirradiated control embryos.
If each block of segmented somites on either side must rotate through 90°
with its medial edge moving forward, the process of rotation should be prevented
by the fusion of somites which occurs in irradiated embryos. Several observations on irradiated embryos were made. Embryos showing 0~ to +2 U. V. defects
were selected at stage 22-24. Longitudinal fractures were made through the
upper (A), middle (B), and lower (C) regions as shown in Fig. 7. The intrasomitic cellular arrangement was then examined. In Fig. 8a a typical embryo
in which a fracture was made through region A is shown. Examination of the
intrasomitic cellular arrangement reveals that the prospective myotomal cells
34
—
B. W. YOUN AND G. M. MALACINSKI
<aj (b)
N
i
1
Fig. 6. Dynamic changes in myotomal cell shape during rotation. Somite rotation
is a dynamic process which produces strikingly different cell shapes at different
developmental times and at various dorsoventral levels. The process of rotation
must occur very rapidly at the time of somite segmentation. Therefore, the probability of obtaining a fracture/section in which cells in the last segmenting somite show
a bent configuration and different morphologies at their ends during the process
of rotation is very low (we estimate that probability to be less than 1 %).
Fig. 6. (a) Lateral view of the posterior half of Xenopus embryo at stage 22-24
showing how the fracture was made. Arrow points to a deeply fractured region in
the unsegmented plate. Ant, anterior; Post, posterior. Bar represents 0-1 fim.
(b) Higher magnification of dorsal view of the longitudinally fractured somite
mesoderm of the embryo shown in (a) displaying myotomal cellular arrangement of
the last five somites and the unsegmented plate. The oblique orientation of the cells
in the most anterior portion of the unsegmented plate is indicated by the short
arrows. Long arrows point to cells arranged in parallel in the lateral and medial
edges of the most posterior somite. A cell with a bent configuration is indicated by
the pointer. N. notochord. Bar represents 0-1 mm.
(c) Higher magnification micrograph of the unsegmented mesoderm (dorsal view)
taken from the region indicated in (b). Additional information on the cellular morphology arrangement, and contacts can be obtained from this micrograph. Short arrow
points to an example of a cell process which might have been connected to the notochord through a network of extracellular fibrils. Long arrows indicate sharply
pointed lateral (prospective caudal) ends of the turning myotomal cells. Pointers
35
Amphibian somitogenesis
Fracture
levels
Fig. 7. Schematic diagram of the cross-sectional view of the fused somite. The
three regions A, B, and C (also see Fig. Id) are indicated. Approximate levels of
fractures made in each region for observation are indicated by solid lines.
undergo the same rotation processes seen in normal embryos (Fig. 3 and 6).
]n the unsegmented plate, cells lie perpendicularly to the long axis of the
embryo. Cells in the most posterior somites rotate, while those in the rest of the
somites become oriented parallel to the long embryonic axis. In Fig. 8Z>, a
fracture through region A is displayed. A few mistakes in the process of somite
segmentation can be observed. The somites on either side are not matched
symmetrically (the figure legend contains a detailed description of this abnormal,
asymmetric segmentation). The same cellular arrangement can, however, be seen
along the long axis of the embryo.
When a fracture was made through region C (Fig. 8 c), a similar rotation
process was also observed. The perpendicular arrangement of cells in the
unsegmented mesoderm, the oblique orientation of the turning cells in the
most posterior somite, and the parallel arrangement in the more anteriorly
located somites were all observed. The turning myotomal cells in the most
posterior somite reside, however, in a reverse orientation compared with those
in Figs. 8#, b. This is because in Fig. 8c the fractured embryo was inverted to
reveal the ventral aspects of the intrasomitic cellular arrangement.
The intracellular arrangement in the middle (fused) region is complex (Fig. 9).
Cells in this region are oriented in several directions. Cells in the unsegmented
plate are elongated in the mediolateral direction. The unsegmented plate is a
indicate broad, flattened protrusions at the prospective anterior ends of the cell
which are about to turn.
(d) Higher magnification micrograph of the last three somites taken from the
region indicated in (b). In the most caudal somite some cells are already arrayed
parallel to the notochord with their posterior ends being broad (pointers), while
others still show a perpendicular orientation. Cells which are in the process of
rotating and bending appear to have narrow and sharply pointed ends (long arrows).
Short arrows indicate cells with blunt ends at the posterior side. Bar represents
20/<m for (c) and (cl).
36
B. W. YOUN AND G. M. MALACINSKI
Fig. 8. (a) Dorsal views of the myotomal cellular arrangement in the upper (A)
region of the fused somites in a U.V.-irradiated Xenopus embryo a tstage 22-24.
Arrow indicates the most posterior somite. Ant, anterior; Post, posterior for all the
embryos shown in Fig. 8. Bar represents 01 mm.
(6) Dorsal view of myotomal cellular arrangement in the upper (A) region of
the fused somites. U.V. irradiation sometimes causes mismatched, asymmetric
fusion. An extra somite which forms as a result of the mismatch is indicated by
thin, long arrow. However, the same perpendicular orientation in the unsegmented
mesoderm and parallel arrangement in the somites are obtained in this abnormal
embryo. Thick, short arrow points to the most posterior somite which contains
the cells with oblique orientation. Bar represents 01 mm.
(c) Ventral view of myotomal cellular arrangement in the lower region (C) of the
fused somites. After a fracture was made, the embryo was inverted to reveal the
ventral aspect of the cellular arrangement pattern. Arrow indicates the most posterior
somite. Bar represents 01 mm.
Amphibian somitogenesis
lb)
Fig. 9. Dorsal views of the myotomal cellular arrangement in the middle (B) fused
region of the fused somites in U.V.-irradiated Xenopus embryos. Approximately
60 notochordless embryos from thtee separate irradiations were examined. Each
of the three irradiations represented spawnings of different females. Three typical
examples are shown in this figure. Pointers in (a) and (c) indicate cells which exhibit
oblique orientations. Thin, short arrows in (a), (b), and (c) point to a group of
cells in the lateral side of somites which assume parallel orientation. Sometimes
fractures were made through the level slightly higher (more dorsally) than other
regions of the same embryo, such that all the myotomal cells indicated by long
arrows in (/?) are perfectly arranged parallel to the long axis of the embryo. Thick
short arrows indicate the most posterior somites. Ant, anterior; Post, posterior.
Bars represent 01 mm.
37
38
B. W. YOUN AND G. M. MALACINSKI
few cells wide rather than just one cell wide. In some of the posterior-most
somites, cells either do not display any definite orientation (Fig. 9 a), show the
correct parallel arrangement pattern (Fig. 9 b), or display the perpendicular
orientation (Fig. 9 c). The parallel arrangement shown in Fig. 9 b (long arrows)
may be due to the fact that the fracture level is slightly higher (more dorsal)
than other regions of the same embryo, or the same, comparable regions of
other embryos shown in Figs. 9 a, c. Therefore, the parallel-cell arrangement
appears to reflect the cell orientation pattern only in the upper region (Figs. 9 a,
9b). Further anteriorly, cells become more spindle-shaped and tend to maintain
the perpendicular arrangement. Again, the parallel arrangement of the cells
seen frequently in the lateral side (short arrows in Figs 9a-c) can be regarded
as a continuation of the cell orientation pattern from the upper region. The
oblique orientation of the cells (pointers in Figs. 9 a, c) can be observed if they
are in a transitory zone, changing from the parallel to the perpendicular
arrangement. It is possible that this represents independent rotation of cells
originally derived from the left and right sides of the embryos.
The turning of prospective myotomal cells by 90° in the upper and lower
regions (Fig. 8) even under the condition of somite fusion provides additional
support for the conclusion drawn in the previous section of this report: Individual cellular rearrangement, rather than simultaneous block rotation of a
whole somite, plays an important role in the rotation process. In the previous
section of this report it was also suggested that since the myotomal cells in the
middle region turn first, the motive force for the cells in the upper and lower
regions to turn might be exerted from the middle region of the somite and
propagated towards both ends. That suggestion probably does not pertain to
irradiated embryos since cells in the middle region tend to be oriented perpendicularly to the long axis of the embryo (Fig. 9). These observations imply
that the prospective myotomal cells in each dorsoventral level have an intrinsic
capacity for cellular rearrangement and, therefore, turn independently of each
other. The fusion of somites somehow prevents the cells in the middle region
from behaving in the normal fashion (see Discussion).
DISCUSSION
The first point for discussion is the relative roles that morphogenetic movements of the mesodermal mantle, the superficial epidermis, and the prospective
neural area play in the shaping of Xenopus somites. It was shown in Fig. 2a
that in a mediolateral view the shape of the somites displays a progressive change
in the anteroposterior direction from a stump-like to an arrowhead-like morphology. The somite reversal experiments of Deuchar and Burgess (1967) have
also shown that grafted, reversed pieces take on a new orientation to correspond
with the rest of the host axis. They suggested from those results that some overall
influence from surrounding tissues governed the anteroposterior orientation of
Amphibian somitogenesis
39
the somites, and that the orientation was not irrevocably fixed until after the
end of the neurulation. The manner in which the surrounding tissue might
affect the overall shaping of the somites at different developmental times could
be explained in part by the results of vital-dye mapping experiments.
Keller (1976) placed dye marks through the double-layered prospective neural
area and the underlying prospective dorsal mesoderm at stage 13 and studied
the relative movements of these layers during neurulation. Examination of the
locations of the dye marks in cross sections at either stage 18 or 22 revealed
that relative anteroposterior movements occur between the dorsal epidermis
and the lateral somite mesoderm (i.e. Fig. 2a, region C) and also between the
neural plate and the somite mesoderm (i.e. Fig. 2a, region A) further medially.
The neural and dorsal epidermal ectoderm moves posteriorly relative to the
somite mesoderm. This movement increases in the more posterior regions.
The medial register of the somite mesoderm and the overlying neural or
epidermal ectoderm (i.e. Fig. 2a, region B), is however, essentially preserved
throughout neurulation. In the regions where the relative anteroposterior
movement takes place, the upper (A) and lower (C) portions of the somites
come to lie further posteriorly with respect to the middle portion (B). The extent
of the contribution of relative movements varies in different regions of the
embryo. Somites which form in the posterior region of the embryo acquire
an arrowhead-like shape, since most of the movement occurs in this region.
Those which develop more anteriorly lose the arrowhead-like shape and
assume a bow-like configuration. The shape of the first few somites located in
the most anterior region of the embryo is stump-like, because they are least
affected by that relative anteroposterior movement.
The second point for discussion concerns the rotation of somites. Is somite
rotation a property of the individual myotomal cells or the whole somite?
Cooke (1977) observed with light microscopic examination of horizontal sections through whole embryos that cells of each somite block maintain a close
contact, yet move partly as individuals. Our SEM findings support that observation. Individual cellular rearrangements, rather than the simultaneous block
rotation of a whole somite, appear to be responsible for the rotation of myotomal cells by 90° (Figs. 4 and 6). Individual cell rearrangements do not,
however, alone account for the orientation pattern of somite cells in U.V.irradiated embryos. Cells in the fused region tend to be oriented perpendicularly
to the long axis of the embryo (see Fig. 9).
There are at least two possible explanations for the abnormal behaviour of
cells in the fused somite region of U.V.-irradiated embryos. Firstly, cells in that
region may not be truly myotomal, in the sense that they could have originated
from the group of cells which would normally have become the notochord.
Those cells may still have some of the characteristics of notochordal cells at these
relatively early stages (stage 22-24). Such cells conceivably could be unable to
exhibit the rotational movements of authentic myotomal cells. Further detailed
40
B. W. YOUN AND G. M. MALACINSKI
ultrastructural studies using transmission electron microscopy are needed in
order to test this possibility. Secondly, if the cells in the fused region are indeed
true myotomal cells, then the bilateral separation of somites caused by the
presence of the notochord must be considered essential for the rotation of
myotomal cells by 90°. In U.V.-irradiated embryos cells in the upper and lower
(unfused) portions of the somite were shown to be arranged in a normal,
parallel orientation (Fig. 8). The manner in which the fusion of somites hinders
the rotational behaviour of the myotomal cells remains obscure. Furthermore,
the possibility that the neural tube, notochord, lateral mesoderm, or dermatome
might aid in myotome rotation was not tested in the present study. Such structures are known to be involved in the proper shaping of chick somites by exerting packing-pressure on them (Packard & Jacobson, 1979).
The third and last point for discussion concerns the mechanisms of motile
and adhesive control through which the myotomal cells as individuals might
execute such rearrangements during somite rotation. Cell shape changes have
been implicated in many developmental processes involving tissue folding,
bending, invagination, etc. For example, cell shape changes are a primary cause
of neural-fold formation (reviewed by Karfunkel, 1974) "and mesodermal
migration following involution (Keller & Schoenwolf, 1977). Various mechanisms of motile and adhesive control are postulated to be involved in those
processes (Trinkaus, 1976). In chick somitogenesis, Meier (1979) has reported
that somite cell rearrangements precede the process of somite segmentation.
Changes in cell contacts appear to be responsible for the formation of the
'somitomere-like structure', the segmentation process, and rosette formation
(Bellairs, 1979; Bellairs, Curtis & Sanders, 1978). These changes may be due to
changes in cell adhesion.
The scanning electron micrographs in Fig. 6 also reveal that extensive
changes in cellular morphology occur during the rotation of Xenopus myotomal
cells by 90°. The turning process itself can be divided into several distinct
steps in which the cells exhibit typical changes in morphology and orientation
(Fig. 10).
(a) Step I. During this initial period of rotation, myotomal cells in the most
anterior region of the unsegmented plate begin to change their arrangements
and assume oblique orientations. Meanwhile, those cells also display changes
in morphology, especially at their ends. Their medial or prospective anterior
ends are flat and broad, whereas their lateral or prospective posterior ends
become narrow and spindle-shaped. Thus, the cells become polarized. The
broad, flattened protrusions at the prospective anterior ends resemble the
lamellipodia of cells which are propagated in culture. They are attached to
neighbouring cells by short filiform protrusions. Such lamelliform protrusions
may actually function in locomotion and, thus, affect the rearrangement of
myotomal cells. At present, however, there is no direct evidence to support this
speculation.
Amphibian somitogenesis
Unsegmented
mesoderm
Last segmented
somite
41
Anterior somites
Fig. 10. Schematic diagram of the myotomal cells in the somite mesoderm showing
how changes in cell shape and the presence of cellular processes and contacts might
be related to the rotation process. Cells exhibiting typical morphologies were selected
from Figs 6 c and 6d and redrawn.
(b) Step II. As the process of rotating and bending continues, cells become
narrow and sharply pointed at both ends. It is important to determine whether
component cells of the myotome retain their original neighbour relationships
during rotation. Cell slippage and neighbour exchanges could cause relaxation
of the tensions that produce cell bending. Our SEM observations (e.g., Fig. 6)
show that individual myotomal cells are in contact with many of their neighbouring cells along their sides by many short finger-like protrusions. Each cell
appears to remain firmly affixed to its neighbours during rotation. In such cases,
bent cells are, most likely, under considerable tension. In order to endure such
tension, the cells may need strong cell-to-cell adhesive interactions at both ends.
The changes in shape that take place in fibroblasts and similar cells when they
are migrating in culture (reviewed by Trinkaus, 1976) could provide insight
into the mechanics of somite rotation. When these cells are attached to the
substrate by both ends, they are put under tension. As mentioned above, both
ends of the bent cells appear to be narrow and sharply pointed. Bent cells are
bipolar and usually have an elastic property. Polarity and elasticity may depend
on contractile changes in the cytoskeletal network of the cell. Contractile
proteins are known to be active in generating cell shape changes (reviewed in
Amer. Zool., vol. 13 (1973), 'Factors controlling cell shape during development';
see also Burnside, 1978). Those types of proteins are also involved in cell
movements (reviewed in Ciba Symp., vol.14 (1973), 'Locomotion of tissue cells').
It is not known, however, how relationships between neighbouring cells
affect cell shape changes which accompany the rotation of a whole somite.
Since the myotomal cells in the middle region (B) start to rotate first, environmental and/or intrinsic clues may initially be given to those cells. Since the
cells are thought to remain firmly affixed to their neighbours (see above), cell
shape changes in those cells could act together to produce tensions which result
in the sequential rotation of other cells in the upper (A) and the lower (C)
regions. Thus, a whole somite can be regarded as a deformable solid.
(c) Steps III and IV. In Step 111 the caudal ends of cells become round while
42
B. W. YOUN AND G. M. MALACINSKI
the anterior ends continue to maintain a sharply pointed configuration. At the
same time the cells become oriented parallel to one another. The caudal end
becomes round in shape, perhaps because the cell has been released from the
tension at that end. Finally, in Step IV, the anterior end acquires a round shape.
In these later stages it seems that changes in cell shape and orientation could
be related to the changes of tension applied to various portions of the myotomal
cells during rotation. Conversely, the initial process of rotation (Steps I and II)
may require both cellular motile activities and the generation of tension.
Although the present SEM observations on the myotomal cellular morphology
and the distribution of cell processes and contacts suggest that individual
cellular rearrangement accounts for the process of somite rotation, it is still
difficult to reconstruct the process with certainty from the static images obtainable with SEM. In order to understand how the turning of the myotomal cells
by 90° occurs in vivo, it would be necessary to examine behaviour properties
of the cells in vitro under various conditions. In vitro studies with different cell
types have contributed to an understanding of the machinery used in cell shape
changes and motile behaviour. For example, Bellairs et al. (1978, 1980) have
shown that unsegmented mesoderm, newly segmented mesoderm, and differentiating somites exhibit striking differences in behaviour when explanted in vitro.
Analysis of the dense cultures of human lung fibroblasts by Elsdale & Wasoff
(1976) has revealed that cells in closely packed arrays do not behave independently but are influenced in both their shape and orientation by global constraints arising out of their interactions. These studies may serve as a guide
to what factors might affect the behavioural pattern of the myotomal cells of
Xenopus in vivo. Thus, the use of both cell culture and organ culture techniques
probably represent the next logical step in any attempts to establish the casual
relationships which actively promote amphibian somitogenesis.
We wish to thank Dr Ray Keller for this advice and encouragement during the course of
this investigation. This work was initiated with financial support from NSF PCM 77-04457,
and completed with NSF PCM 80-06343 and NASA NAGW-60 support.
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(Received 3 October 1980, revised 10 February 1981)