/ . Embryo/, exp. Morph. Vol. 55, pp. 93-108, 1980
Printed in Great Brtiain © Company of Biologists Limited 1980
93
An experimental analysis of somite segmentation
in the chick embryo
By RUTH BELLAIRS 1 AND MARIANNE VEIN! 2
Department of Anatomy and Embryology, University College London,
and Zoological Laboratory of the University of Athens, Greece
SUMMARY
In a previous paper it was suggested that collagen fibrils play an important role in the
process of somite segmentation. This paper was designed mainly to test that concept. In one
series of experiments, embryos were treated with either a,a'-dipyridyl or L-azetidine-2carboxylic acid, which are analogues that interfere with the formation of normal collagen.
The reagents led to a reduction in the numbers of somites that formed, as well as to the
production of other anomalies such as overall diminution in size and retardation. The older
the embryo at the time of treatment, the further posteriorly were the major anomalies located.
It is concluded that these results lend some support to the concept.
In a second series of experiments an incision was made along one side of the neural tube
and notochord to separate it from the segmental plate on one side. The result was that many
more somites formed on the unoperated (control) side of the embryo than on the operated
side. It is concluded that these results also lend support to the concept; but that they are of
interest also in relation to the mechanisms involved in the control of somite numbers. In a
third group of experiments, attempts were made to obtain somites in the absence of endoderm.
Although this was not possible using surgery, it was achieved by treating the young embryos
with u.v. irradiation. It was concluded that the presence of endoderm is not essential for the
segmentation of mesoderm.
INTRODUCTION
There have been a number of theories which attempt to explain how the somites
form from unsegmented mesoderm (see reviews by Bellairs, 1963,1979; Bellairs &
Portch, 1977; Lanot, 1971; Nicolet, 1971). Early ideas of a simple inductive
influence from neighbouring tissues have been replaced by the more recent
concept of a multistep process. The first stage appears to be the cleaving of the
original mass of presumptive somitic mesoderm into right and left sides (Lipton
& Jacobson, 19746); this is normally brought about by the regression of Hensen's
node, and the concomitant laying down of the notochord, but it can be stimulated by an incision alongside (Lipton & Jacobson, 19746) or through (Menkes
& Sandor, 1977) the primitive streak. Each mass of presumptive somite mesoderm
then becomes condensed into a band of mesenchymal cells, the segmental plate,
1
Author's address: Department of Anatomy and Embryology, University College, Gower
Street, London, WC1 6BT, U.K.
2
Author's address: Zoological Laboratory of the University of Athens, Athens, Greece.
7
EMB55
94
R BELLAIRS AND M. VEINI
and these lie one on either side of the notochord. Eventually, these bands of
segmental plate become reformed into somites.
The events involved in this final step whereby cells are rearranged to form a
somite, have been investigated recently by experimental and morphological
techniques. Bellairs, Curtis & Sanders (1978), using the Couette viscometer
technique of Curtis (1969), showed that cell to cell adhesiveness increases at the
time of segmentation. It was suggested that this increase did not take place
uniformly over the surface of each cell but was localized at one end, the result
being that clusters of cells aggregated at their regions of high adhesiveness and
so formed a clump of cells, a somite. This initial adhesion was then supplemented
by the development of tight junctions.
At the same time as this aggregation occurs however, the cells become elongated
and fibroblast-like in shape. This is probably essential from the mechanical point
of view to permit the orderly assemblage of sufficient number of cells in each
somite, since if the cells were rounded only three or four could attach to each
other at the regions of high adhesiveness. This change in cell shape was studied
with the scanning electron microscope by Bellairs (1979), who showed that at
about the time of segmentation, the distal end of each somite cell becomes
anchored by collagen fibrils to an adjacent epithelium (i.e. neural tube, notochord, ectoderm, or endoderm). It had already been suggested by Lipton &
Jacobson (1974&) that those fibrils which ran from the somite to the neural tube
and notochord helped to 'stabilize' the somite once it had formed. Bellairs
(1979), however, suggested that these same fibrils also played an essential role in
the segmentation process itself, by acting like guy ropes and exerting a tension on
the cells. This tension not only enabled each cell to become elongated but also
permitted the somite as a whole to accommodate its shape to the space available
between the adjacent tissues.
Most of the experiments described below were performed in order to test this
hypothesis. They were not designed to answer the question of how an embryo
controls the number of somites that form. That problem will be considered in
a later paper.
MATERIALS AND METHODS
Hens' eggs were incubated for about 18, 33 or 44 h, so that they were at about
stage 3+ , 9, or 12 respectively, in the table of Hamburger & Hamilton (1951).
They were then explanted on to watch glasses, using the technique of New (1955),
and the number of somites was recorded. The experimental interference was
carried out immediately after explantation, and each embryo was then incubated
for a further 12-24 h before fixation; some were inspected after only 3-5 h had
elapsed to check that the somites present at the start of the experiment had not
dispersed. In addition, the last formed somites were sometimes marked with a
spot of carmine to establish their identity. A series of normal embryos was
Experiments on somite segmentation
95
cultured concurrently to act as a general control although an additional and
special control was incorporated in each group of experiments.
(I) Chemical reagents
Specimens were treated with a,a'-dipryridyl (Sigma) or with L-azetidine-2carboxylic acid (Sigma) applied in 1 ml Pannett and Compton's saline (Pannett &
Compton, 1924) to the ventral surface of the embryo immediately after explantation. Suitable concentrations of the reagents were found to be 0-5-1-0 mM. The
purpose of these experiments was to interfere with the normal development of
collagen fibrils. (See Discussion for rationale.) Special control experiments
consisted of treating the embryos with 1 ml of saline alone, or with a solution of
ferrous sulphate to counteract the effect of a,a'-dipyridyl (which is an iron
chelator).
(II) Surgical experiments
(a) Incisions. An incision was made along one side of the neural tube and
notochord, the other side being left as a special control. This operation separated
the segmental plate from the neural tube and notochord at one side, but not at
the other (Figs. 6, 7). The tissues on either side of the hole were then pulled apart
slightly to prevent re-adhesion and healing. The purpose of this experiment was
to test the idea that the neural tube and notochord played some role in initiating
segmentation by acting as anchorage sites for the collagen fibrils.
(b) Removal of endoderm. Pieces of endoderm were stripped from beneath
the segmental plate at one side of the embryo. The other, unoperated side acted
as the special control. The purpose of this experiment was to test the idea that
attachment to the endoderm was important for segmentation.
(III) Ultraviolet irradiation
These experiments were carried out on blastoderms explanted at stage 4-5 in
the usual way, with the ventral surface lying uppermost. The ventral side of the
area pellucida was exposed to a dosage of 518 erg/mm2 for periods of 1 min
from a u.v. lamp stationed 15 mm above it. The purpose of this experiment was
to prevent the endoderm from forming. Control experiments consisted of
irradiating specimens which were covered with a glass plate.
RESULTS
(I) Chemical reagents
(a) Fifty-two embryos were treated at stages 9-12 (7-16 pairs of somites) with
a,a'-dipyridyl at a concentration of either 0-5 mM (23 specimens) or 1 mM
(29 specimens) in 1 ml. Pannett and Compton's saline. A further 26 embryos
treated with weaker doses (0-25 mM) appeared to develop normally. Twenty-four
control embryos were treated with 1 ml Pannett and Compton saline. The precise
7-2
96
R. BELLAIRS AND M. VEINI
Experiments on somite segmentation
97
concentration of a,a'-dipyridyl did not seem to be critical provided it was above
a minimal level since the same range of results was obtained with both concentrations. This was probably because the chemical became diluted further by the
thin layer of saline lying over the embryo in the culture dish.
Five of the treated embryos and two of the controls were dead after 18 h. The
remainder of the controls had reached stage 13 or 14 but none of the treated
embryos had developed so far. Only five had formed additional somites whilst in
the remainder certain changes had taken place in the original somites so that in
relatively mild cases they were about twice as wide as long, whilst in the severer
ones the original somites had dispersed. At the posterior end of the trunk,
the tissues were frequently necrotic and fragile (Fig. 2). Sections across this
region showed that the segmental plate mesoderm had largely disappeared
and a wide space lay on either side of the neural tube (Fig. 5). By contrast with
the trunk region, the anterior part of the same embryo was usually in a relatively
healthy condition with all its organs intact, and frequently possessed a beating
heart (Fig. 3). The younger the embryo at the time of treatment however, the
more likely were the anterior regions to be affected.
It was typical of all specimens to be shorter in length than the control embryos.
This appeared to be the result of a contraction of the embryo as a whole, since
in even the most mildly affected specimens, the neural tube was no longer straight
but thrown into a series of pleats (Fig. 4). The size of the area pellucida also was
less than that of the control specimens. In addition, most of the treated embryos
FIGURES
1-5
Fig. 1. Embryo fixed 28 h after being treated with a solution of 0-5 mM of Lazetidine. The embryo, which possessed 10 pairs of somites at the time of treatment,
now possesses none, and is abnormally short in length. Haematoxylin and eosin.
xl6-5.
Fig. 2. Embryo fixed 24 h after being treated with a solution of 0-5 mM a-a'dipyridyl. The embryo, which possessed 16 pairs of somites at the time of treatment
has developed no more, and many of the original ones have dispersed. The embryo
is abnormally short in length and has begun to disintegrate in the posterior trunk
region. Haematoxylin and eosin. x 10.
Figs. 3, 5. Transverse sections across an embryo treated with a solution of 0-5 mM
a-a'-dipyridyl and exhibiting symptoms comparable to those shown in Fig. 2. The
thoracic region (Fig. 3) has developed normally and a well formed heart is present,
but the somites have disappeared from the trunk region (Fig. 5). n.p., Neural plate;
l.p., lateral plate. Haematoxylin and eosin. x 100 and 135, respectively.
Fig. 4. Posterior end of an embryo fixed 24 h after treatment with a solution of
0-5 mM of a-a'-dipyridyl. The embryo which was at stage 14 (22 pairs of somites)
has failed to develop any further somites. A line of carmine particles (c) which was
placed at the level of the last somite at the time of treatment can be seen as a dark
line and no additional somites lie posterior to it. Note also that the neural tube
(arrows) is thrown into a series of pleats posterior to the carmine, x 68.
98
R. BELLAIRS AND M. VEINI
exhibited certain oedematous bulges, especially in the pro-amniotic areas on
either side of the head.
When ten embryos were treated with 0-5 mM of a,a'-dipyridyl at stages 4-5,
similar results were obtained. In comparison with four control specimens, the
experimentally treated ones were retarded and short in length. By the time that
the control embryos had reached stage 10, and possessed about 10 pairs of
somites, none of the treated embryos had developed beyond about stage 8,
though because they possessed anomalies of the head region, it was difficult to
be confident as to their stage; however, none possessed somites.
A further special control experiment was performed by treating three embryos
with 1 mM of a,a'-dipyridyl in the usual manner, and then flushing it away with
saline after a period of 2 h. It was, however, found that the development of these
specimens did not differ significantly from companion embryos in which the
a,a'-dipyridyl was not flushed away. Similarly, the addition of ferrous sulphate
in concentrations below the toxic level failed to abolish all the effects of the
a,a'-dipyridyl. Thus when ferrous sulphate was added alone at a concentration
of 00015 mg/1-00 ml of Pannett & Compton's saline, it led to distortion and
death in three out of seven specimens, although the rest were normal. When
added simultaneously with an equal volume of a,a'-dipyridyl to 11 embryos, all
but one showed the characteristic appearance of a,a'-dipyridyl-treated embryos
whilst the remaining one was normal.
(b) Fifteen embryos were treated at stages 9—11 with L-azetidine-2-carboxylic
acid at a concentration of 0-5 mM in 1 ml Pannett and Compton saline. Twentyfour control embryos were treated with Pannett and Compton's saline (as already
reported in para, la, above).
Six of the embryos treated with L-azetidine appeared to be normal and reached
about the same stage as the control specimens after 24 h. The anomalies found in
the remainder paralleled those found after treatment with a,a'-dipyridyl. Seven
specimens added no more somites but retained their original ones although these
became short and wide relative to the long axis of the embryo; three of the
embryos possessed well-formed and beating hearts.
In the remaining two embryos the original somites disappeared completely
(Fig. 1). Embryos treated with L-azetidine were often stunted and frequently
oedematous.
(II) Surgical experiments
(a) Incisions. This operation was performed on 34 embryos. Two died after
about 5 h but each of the surviving 32 specimens formed more somites on the
control side than on the operated side. A typical example is shown in Figs. 6-8.
At the time of the operation the embryo was at about stage 8 and possessed
five pairs of somites (Fig. 6). An incision was made to the left of the neural tube,
just posterior to the last somite and extending into the area opaca; the two sides
of the embryo were then pulled apart (Fig. 7). After 20 h the space between the
Experiments on somite segmentation
99
two regions had widened, probably due to the centrifugal pull exerted by the
area opaca, and the two areas of somitic mesoderm were widely separated. It was
found that there were now 16 somites on the unoperated (right) side and only
eight on the operated (left) side. (Fig. 8 shows the dorsal side as opposed to
Figs. 6 and 7, which show the ventral side.) Moreover, the segmental plate of the
unoperated side was more than twice the length of that of the operated side
(Fig. 8).
in seven specimens the number of somites on each side of the incision were
counted at 3-4 h after the operation, and in every case additional somites had
formed on the unoperated (control) side but not on the experimental side.
A similar result was obtained in specimens where the slit was less extensive and
where the two regions were not so widely separated. If the slit was even greater,
the embryo became highly distorted and reduced to a narrow rim of tissue lying
just within the glass ring of the culture dish; specimens of this extreme type have
not been included in the results.
In eight embryos no new somites formed at all on the operated side, but since
each of these developed only about three or four extra somites on the unoperated
side, it seemed likely that their development as a whole was retarded.
In a further series of control experiments (six specimens) a longitudinal
incision was made through the neural fold to one side of the midline, so that
about three quarters of the tube lay to one side of the slit and one quarter to the
other. After 20 h it was found that an equal number of somites formed on each
side of the slit (Figs. 9-11).
(b) Removal of pieces of endoderm from beneath segmental plate. In 16 embryos
at stages 8-11 a piece of endoderm was removed from beneath the entire length
of the segmental plate at one side. After 24 h further incubation five specimens
were discarded because no further somites had formed, even on the unoperated
(control) side.
In nine embryos the endoderm appeared to have either regenerated or repaired
over the denuded area, and the same number of somites was present on the
control and operated sides, though occasionally they appeared to be rather short
and wide. In the remaining two specimens there were fewer somites on the
operated than on the control side.
(c) Operations (a) and (b) combined. In a series of ten embryos operations (a)
and (b) were carried out simultaneously on the same embryo. All of these
developed in the same way as the embryos in group (a) and all formed more
somites on the unoperated than on the operated side.
(d) Removal of endoderm from area pellucida at stages 4-5. In ten embryos
the endoderm was removed from the area pellucida. At this stage it adheres firmly
to the upper layers, especially in the vicinity of the anterior end of the primitive
streak, so that it was not always possible to be confident of having made a clean
dissection. After 24 h further incubation one specimen was found to be retarded
and was discarded. Four were normal apart from having diplocardia and the
100
R. BELLAIRS AND M. VEINI
Experiments on somite segmentation
Fig. 9. Control embryo explanted into New culture at about stage 9 possessing
eight pairs of somites. A longitudinal cut has been made through the neural tube
just to the left of the midline. Photographed alive from the ventral surface, x 12.
Figs. 10,11. Same embryo as shown in Fig. 9 after 18 h of incubation. Photographed
alive from the dorsal side. The embryo is now at about stage 13+ and possesses
20 pairs of somites. The neural tissue divides unequally around the split with a
thicker region lying to the left of the split; there is no difference in the numbers or
pattern of the somites on the two sides of the split, x 14 and 35, respectively.
FIGURES
6-8
Fig. 6. Embryo explanted into New culture at stage 8 possessing four to five pairs of
somites. Viewed from the ventral surface and photographed alive. C = spot of
carmine, x 12-5.
Fig. 7. Same embryo as shown in Fig. 6, after a slit has been made between the
neural tube (n.t.) and the segmental plate (s.p.) on the left side. The two sides of
the embryo have been pulled apart, x 12-5.
Fig. 8. Same embryo as shown in Figs. 6 and 7, after 20 h of incubation and seen
from the dorsal surface. Note the difference in the numbers of somites on the two
sides, x 14.
101
102
R. BELLAIRS AND M. VEINI
12
13
Fig. 12. Transverse section through an embryo which lacks endoderm but possesses
well-formed somites (s). This embryo was irradiated with u.v. at stage 5 and fixed
24 h later. Haematoxylin and eosin. x 135.
Fig. 13. Transverse section through a control embryo which possesses endoderm (e)
and well formed somites (s). Haematoxylin and eosin. x 135.
Fig. 14. Longitudinal section through an embryo lacking endoderm but possessing
somites. This embryo was irradiated with u.v. at stage 5 and fixed 24 h later. Note
the incomplete intersegmental furrows (arrows). Haematoxylin and eosin. x 135.
Experiments on somite segmentation
103
remainder appeared completely normal. Each had developed to about stage 8-10
and histological sections showed that a new endoderm had formed.
(Ill) Ultraviolet irradiation
Irradiation was performed on a total of 93 embryos, in 74 of which the
endoderm had been removed prior to treatment and in 19 of which it had been
left intact. Similar results were obtained in the two series which will therefore be
pooled. After 24 h further incubation 16 embryos were dead; 24 appeared to be
normal though retarded in comparison with the control specimens and many of
them possessed no somites at all; 35 possessed wide or doubled heads, sometimes
accompanied by diplocardia, and in four of these a single central row of somites
was present; 18 developed a normal embryonic axis and were at a stage comparable to the control embryos and possessed about six pairs of somites. However, in eight of these specimens the endoderm appeared to be absent in the region
of the somites, a fact which was confirmed by serial sections. Figure 12 is a
transverse section across an embryo irradiated at stage 5 and fixed 24 h later.
The endoderm is totally absent beneath the notochord and somites, though some
extra-cellular material lies beneath them. Figure 13 is a transverse section across
the somite region of a control embryo, in which the endoderm is clearly visible.
Another endodermless embryo is shown in longitudinal section in Fig. 14. It will
be seen that although a clear row of somites has formed, some of the intersegmental furrows are incomplete. This anomaly occurred symmetrically in the
two rows of somites.
DISCUSSION
The main objective of this paper has been to test the hypothesis of Bellairs
(1979) that collagen plays an important, and probably essential, role in enabling
the segmental plate to become formed into somites. Subsidiary aims have been
to investigate the significance of the neural tube, notochord and also the endoderm, as anchorage sites for the collagen fibrils.
The importance of the collagen has been tested by treating the embryos with
analogues which interfere with the formation of normal collagen. The action of
a,a'-dipyridyl has been studied extensively on chick tendon cells in tissue culture
(see discussion by Prockop, Berg, Kiririkko & Uitto, 1976). a,a'-dipyridyl is an
iron chelator which inhibits the hydroxylation of proline by the enzymes prolyl
and lysyl hydroxylases, so that protocollagen is synthesized instead of procollagen.
This substance collects in the cells which are unable to secrete it in any appreciable
amount, unless the cultures are treated with a ferrous rich medium after about
2 h, which will remove the remainder of the a,a'-dipyridyl.
L-azetidine similarly affects the rate at which procollagen is secreted, though in
a different way.
In our experiments we have shown that if collagen synthesis is inhibited with
either of these two reagents, the segmentation of somites is also interrupted. It is
104
R. BELLAIRS AND M. VEINI
relevant that the regions of greatest vulnerability seemed to be located the further
posteriorly, the later the embryos. Thus, embryos possessed primarily head
anomalies if treated at stages 4-5, mid-trunk anomalies at stages 9-10, and more
posterior trunk anomalies at stages 11-12.
This may reflect the morphological regions in which collagen synthesis is most
active at these particular times. It is already known (Cohen & Hay, 1971; Hay,
1973) that the neural tube of the chick secretes collagen at least from stage 12, so
it seems likely that it begins to do so soon after it has itself developed. Similarly,
Bancroft & Bellairs (1976) have shown that collagen fibrils are present around
the notochord from about stage 9. As the effects of the chemical reagents increase,
either because of a higher dosage or because of a longer period of treatment, they
often start to include the already formed somites which begin to change shape
and ultimately to dissociate. They probably have an effect also on collagen
throughout the embryo as a whole, since the tension of the entire blastoderm
seems to be disturbed, the body becoming shorter and the neural tube becoming
pleated.
It seems likely that in addition to their inhibition of collagen synthesis, these
reagents may have had other effects on the embryo. For example oedema is
common in the pro-amniotic region of the treated embryos and this is probably
caused by the swelling of hyaluronate, which has been shown to be the predominant glycosaminoglycan of the head region at this stage (Fisher & Solursh,
1977). There is also some general retardation of differentiation and growth of the
treated embryos in comparison with the saline-treated controls. In assessing the
present results, moreover, it is important to remember that somites are relatively
easily inhibited from developing by treating the embryo with a variety of different
chemicals, e.g. BudR (Lee, Deshpande & Kalmus, 1974) and LSD (Hart, 1975)
many of which may reflect merely a generalized toxicity or a lowering of the rate
of protein synthesis. Certainly, in view of the smaller size of the treated embryos
in the present experiments it appears that there has been some reduction in the
overall rate of protein synthesis. However, this retardation is unlikely to be the
major cause of the failure to form additional somites since the treated embryos
have often differentiated at their anterior ends to a stage when a considerable
number of extra somites should have formed in the trunk. Moreover, the fact
that the two analogues produce the same effect, even though they interfere with
different parts of the collagen-synthesizing programme lends support to the
conclusion that, whatever else they may be doing, they are primarily affecting
somite segmentation by interfering with collagen synthesis.
The idea that somites are normally induced by neural tissue or by notochord
has been put forward by several groups of workers, mainly as a result of experiments in which grafts of Hensen's node have been implanted into lateral parts
of the area opaca, though these concepts lost much of their impact when it was
shown that somites could form in the absence of neural tissue and of notochord
(Bellairs, 1963; Christ, 1970; Menkes & Sandor, 1977). Recently, however,
Experiments on somite segmentation
105
Lipton & Jacobson (19746) suggested that induction of the presumptive somite
mesoderm might occur at about the time that the neural plate develops, and that
once the segmental plate has formed, it has become 'imprinted' with a 'prepattern of segmentation'. They state:' The neural plate appears to be the principal
inductor of somites.' Experiments carried out by Lipton & Jacobson (19746) to
test this idea of induction by the neural plate have been criticized eleswhere
(Bellairs & Portch, 1977, p. 450) on the grounds that their grafts probably
included some presumptive somite mesoderm. Indeed, this criticism has now
become even more plausible as a result of the experiments of Hornbruch,
Summerbell & Wolpert (1979), who have shown that when Hensen's node is
grafted at the edge of the area pellucida, the secondary axis that forms possesses
somites derived entirely from the graft. Nevertheless, many authors have pointed
out there is indeed a close morphological relationship between the presumptive
somite mesoderm and the neural plate; it is important to remember however
that the exact nature of the relationship of the two tissues in these early stages
has yet to be elucidated. The whole question of induction of somites will shortly
be discussed in a further publication.
More evidence is available about the role of the neural tissue and notochord
in the later stages of somite development. Lipton & Jacobson (19746) have
suggested that they act as a substrate to which the somites, once they are formed,
become anchored by collagen fibrils, and there is evidence that in their absence
the somites dissociate (Lipton & Jacobson, 19746; Packard & Jacobson, 1976;
Sandor & Amels, 1970). This emphasis on the importance of the collagen fibrils
has been extended further by Bellairs (1979), who has pointed out that the presumptive somite cells become subjected to tension as they aggregate into somites,
and that this tension is supplied by the attachment of the distal ends of the cells
by collagen fibrils to the adjacent epithelia. The most important of these sites
are the neural tube and notochord, which is probably related to the fact that these
are the most active synthesisers of collagen at this time, but similar attachments
pass to the ectoderm, the endoderm, and the aorta, and it appears that under
experimental conditions, these tissues alone may be sufficient to promote some
segmentation (Christ, Jacob & Jacob, 1972). This appears to be the case in the
incision experiments reported in this paper. However, it is of significance that
although somites do form from segmental plate not in contact with neural plate
or notochord, there are fewer of them than on the control side.
One possible explanation, that some of the somites on the experimental side
of the embryo have dissociated, seems unlikely, for the following reasons. First,
in those embryos that were inspected at 3-4 h after the operation, there was
evidence that the numbers of somites was always greater on the control side.
Second, the segmental plate (i.e. the as yet unsegmented mesoderm) is usually
clearly visible on the experimental side of the embryo, and is frequently longer
than that on the control side (see Fig. 8). It might be argued that the stretching
and distortion around the expanded slit have inhibited somite information, but
106
R. BELLAIRS AND M. VEINI
in those control specimens where a similar expansion occurred, equal numbers of
somites formed on each side of the embryo.
It is concluded therefore that in the absence of the neural tissue and notochord,
segmentation takes place more slowly, and it is suggested that this is at least
partly because of the reduction in the amount of collagen. Another factor,
however, to be considered is that Hensen's node and the remains of the primitive
streak are present on the control side but absent on the experimental side. This
is likely to be important since Lipton & Jacobson (1974&) have shown that as
Hensen's node regresses the mesoderm on either side of it becomes converted
into segmental plate. It is likely therefore that additional segmental plate has
formed on the control side, but not on the experimental side. If that is so, however,
it implies that some relationship exists between the events at the anterior and
posterior ends of the segmental plate, i.e. the speed at which segmental plate
adds to itself at the posterior end may be related to the speed at which it becomes
transformed into somites at its anterior end. Some evidence to support this
relationship comes from the fact that between about stages 9 and 13, the segmental
plate contains material for a standard number of somites, usually about 12 (Packard
& Jacobson, 1976). Further analysis of the control of somite numbers will be
presented in a later paper.
The importance of the endoderm in promoting segmentation has been tested
previously by Sandor & Amels (1971) by dissecting the endoderm from beneath
the segmental plate, but as Packard & Jacobson (1976) have already pointed out,
these experiments were probably carried out after the time when any inductive
effect from the endoderm might take place. Our attempts, reported in section lib
of the Results, to obtain embryos which lacked endoderm beneath the segmental
plate by dissecting it from younger embryos, were also unsuccessful because the
tissue regenerated. However, by treatment with the u.v. we were able to produce
embryos in which the endoderm was missing beneath an extensive region of the
embryonic axis. These clearly demonstrate that the segmental plate can form
somites in the absence of endoderm, other epithelia being present.
Interestingly, though most of the somites were normal, certain anomalies in
segmentation occurred which resemble those found in amphibian embryos as a
result of heat shock (Cooke, 1975, 1978; Elsdale, Pearson & Whitehead, 1976;
Pearson & Elsdale, 1979c, b).
It is possible that in the present experiments they were a direct result of the
u.v. treatment, although similar anomalies may be brought about by heat shock
treatment in the chick. These will be discussed in a later paper.
We wish to thank the University of Kuwait for financial support in carrying out this
research. Dr Veini also acknowledges grants for travel and maintenance from EMBO and
NATO.
We are most grateful to Miss Doreen Bailey for her skilful technical assistance, and to
Professor R. D. Harkness and Dr R. Harwood for advice on the choice of analogues.
Experiments on somite segmentation
107
REFERENCES
BANCROFT, M. & BELLAIRS, R. (1976). The development of the notochord in the chick embryo,
studied by scanning and transmission electron microscopy. /. Embryol. exp. Morph. 35,
383-401.
BELLAIRS, R. (1963). The development of somites in the chick embryo. J. Embryol. exp.
Morph. 11, 697-714.
BELLAIRS, R. (1979). The mechanism of somite segmentation in the chick embryo. /. Embryol.
exp. Morph. 51, 227-243.
BELLAIRS, R., CURTIS, A. S. G. & SANDERS, E. J. (1978). Cell adhesiveness and embryonic
differentiation. J. Embryol. exp. Morph. 46, 207-213.
BELLAIRS, R. & PORTCH, P. A. (1977). Somite formation in the chick embryo. In Vertebrate
Limb and Somite Morphogenesis, British Society of Developmental Biology Symposium 3
(ed. D. A. Ede, J. R. Hinchliffe & M. Balls). Cambridge University Press.
CHRIST, B. (1970). Experimente zur Lageentwicklung der Somiten. Verh. Anat. Ges. Ergantz.
2, 126 B, 555-564.
CHRIST, B., JACOB, H. J. & JACOB, M. (1972). Experimentelle Untersuchungen zur Somitenetstehung beim Huhnerembryo. Z. Anat. EntGesch. 138, 82-97.
COHEN, A. M. & HAY, E. D. (1971). Secretion of collagen by embryonic neuroepithelium at
the time of spinal cord-somite interaction. Devi Biol. 26, 578-605.
COOKE, J. (1975). The control by somite number during morphogenesis of a vertebrate,
Xenopus laevis. Nature, Lond. 254, 196-199.
COOKE, J. (1978). Somite abnormalities caused by short heat shocks to pre-neurula stages of
Xenopus laevis. J. Embryol. exp. Morph. 45, 283-294.
CURTIS, A. S. G. (1969). The measurement of cell adhesiveness by an absolute method.
/ . Embryol. exp. Morph. 22, 305-325.
ELSDALE, T., PEARSON, M. & WHITEHEAD, M. (1976). Abnormalities in somite segmentation
following heat shock to Xenopus embryos. /. Embryol. exp. Morph. 35, 625-635.
FISHER, M. & SOLURSH, M. (1977). Glycosamino-glycan localization and role in maintenance
of tissue spaces in the early chick embryo. /. Embryol. exp. Morph. 48, 195-207.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HART, N. A. (1975). The effect of LSD on somite number in explanted chick embryo.
Experientia 91, 97-99.
HAY, E. D. (1973). Origin and role of collagen in the embryo. Am. Zool. 19, 1085-1107.
HORNBRUCH, A., SUMMERBELL, D. & WOLPERT, L. (1979). Somite formation in the early
chick embryo following grafts of Hensen's node. /. Embryol exp. Morph. 51, 51-62.
LANOT, R. (1971). La formation des somites mesodermiques des Vertebres. Anns de Biologic
10, 193-226.
LEE, H.-Y., DESHPANDE, A. K. & KALMUS, G. W. (1974). The effects of 5-bromodeoxyuridine (BrdU) on morphogenesis of the early chick embryo. Wilhelm Roux Arch. EntwMech. Org. 174, 102-106.
LIPTON, B. H. & JACOBSON, A. G. (1947a). Analysis of normal somite development. Devi
Biol. 38, 73-90.
LIPTON, B. H. & JACOBSON, A. G. (19746). Experimental analysis of the mechanisms of
somite morphogenesis. Devi Biol. 38, 91-103.
MENKES, B. & SANDOR, S. (1977). Somitogenesis; regulation potencies, sequence determination and primordial interactions. In Vertebrate Limb and Somite Morphogenesis. British
Society of Developmental Biology Symposium, no. 3 (ed. D. A. Ede, J. R. Hinchliffe
& M. Balls). Cambridge University Press.
NEW, D. A. T. (1955). A new technique for the cultivation of chick embryos in vitro. J.
Embryol. exp. Morph. 3, 320-331.
NICOLET, G. (1971). Avian gastrulation. Adv. Morphogen. 9, 231-262.
PACKARD, D. S. & JACOBSON, A. G. (1976). The influence of axial structures on chick somite
formation. Devi Biol. 59, 36-48.
108
R. BELLAIRS AND M. VEINI
D. S. & JACOBSON, A. G. (1979). Analysis of the physical forces that influence the
shape of chick somites. /. exp. Zool. 207, 81-92.
PANNETT, C. A. & COMPTON, A. (1924). The cultivation of tissues in saline embryonic juice.
Lancet, 206, 381.
PEARSON, M. & ELSDALE, T. (1979 a). Somitogenesis in amphibian embryos. I. Experimental
evidence for an interaction between two temporal factors in the specification of somite
pattern. /. Embryol. exp. Morph. 51, 27-50.
PEARSON, M. & ELSDALE, T. (19796). Somitogenesis in amphibia. II. Origins in early
embryogenesis of two factors involved in somite specification. /. Embryol. exp. Morph.
53, 245-267.
PROCKOP, D. J., BERG, R. A., KIVIRIKKO, K. J. & UITTO, J. (1976). Intracellular steps in the
biosynthesis of collagen. In Biochemistry of Collagen (ed. G. N. Ramachandran & A. H.
Reddi). New York: Plenum Press.
SANDOR, S. & AMELS, D. (1970). Researches on the development of axial organs. VI. The
influence of the neural tube on somitogenesis. Rev. roumaine Embryol. Cytol. (Serie
Embryol.) 1, 49-57.
SANDOR, S. & AMELS, D. (1971). Researches on the development of axial organs in the chick
embryo. VII. Rev. roumaine Embryol. Cytol. (Serie Embryol.) 8, 37-42.
PACKARD,
(Received 8 August 1979, revised 27 September 1979)