/. Embryol. exp. Morph., Vol. 17, 2, pp. 349-358, April 1967
349
Printed in Great Britain
Somite segmentation in amphibian embryos: is
there a transmitted control mechanism?
By E. M. DEUCHAR 1 & A. M. C. BURGESS 1
From the Department of Anatomy, University College London
In all vertebrate embryos, somite segmentation begins in the occipital region
and proceeds from here caudally with remarkable regularity. The segmentation
pattern and the shape, size and number of somites formed are constant for each
species, and it appears as if some quite complex mechanism must control their
development. That this control mechanism must be versatile as well as precise
is shown, for instance, by the fact that haploid newts have a larger number of
cells per somite than the normal diploid, while polyploids have smaller numbers
of somite cells than normal, so that the somite sizes are very nearly the same in
all cases: the number of somites per embryo is also always the same as in the
diploid (Fankhauser, 1945). Further, when mesoderm is either added to or
excised from the somite region of the archenteron roof experimentally (Waddington & Deuchar, 1953), there is regulation so that the overall shape of the somites
remains nearly normal, and again, their number per embryo is unchanged from
that typical of the species.
Any attempted explanation of what may initiate and control somite formation
in the embryo has to account for two main phenomena: the tendency of the
cells to associate in segmental groups, and the regular cranio-caudal sequence,
both temporal and spatial, in which these groups form. It is clear that whatever
property determines the grouping of the cells must arise earliest in cells of the
occipital region, since segmentation begins here first. It is difficult to envisage
how the rest of the somite tissue takes its timing for somite formation from
this region, unless some influence spreads caudalwards from it. Several kinds
of mechanism have been postulated in the past, but none is very satisfactory
and all have hardly any experimental evidence to support them. Berrill (1955),
for instance, suggested that the governing factor was a wave of mitotic activity
from cranial to caudal regions. He did not, however, explain how this wave arose.
Moreover, its existence is not confirmed by observations of mitotic activity.
Again unsatisfactorily and with insufficient evidence, it has been postulated
(Deuchar, 1966) that the movements of the somite cells may be partly controlled
by the rate of synthesis by them of some myosin-like protein which mediates
1
Authors' address: Dept of Anatomy, University College, Gower Street, London,
W.C.I, U.K.
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E. M. DEUCHAR & A. M. C. BURGESS
their contraction. In addition to this, some passive vertical stretching of the
cells may take place as the neural folds close during neurulation, and since this
occurs in cranio-caudal sequence from the occipital region, it might be partly
responsible for the sequence of somite formation.
When one considers more closely the possible means by which metabolic
products, whether arising from the ATPase activity of myosin or from other
biochemical processes, could control the formation of periodic structures as
they diffuse caudalwards, a number of further problems arises. Waddington &
Deuchar (1953) pointed out that Volterra equations governing the diffusion of
chemical products are inadequate to account for regulations of size and shape
in experimental conditions. They suggested that Turing's (1952) mathematical
expressions were more suitable. Turing had set out to explain how, in a ring of
cells, random changes (e.g. in the rate of passage of metabolites from one cell
to the next) could gradually give rise to a periodicity in the concentration of
metabolites in the cells. But it is questionable whether this theory could really
be applied to the early archenteron roof of the embryo as Waddington suggests,
since this is an open-ended sheet of cells and not a closed ring. Further, MaynardSmith (1960) has pointed out that where relatively large numbers of units are
being formed (usually 32-36 somites per embryo), any random control process
would be far too open to numerical error—forming, perhaps, one somite too
many or too few. Turing's system could only satisfactorily be applied to the
development of smaller series of units, such as the digits of hand or foot, for
then a small variation in the length of the 'period' is far less likely to result in
formation of a whole unit more or less than the norm.
All of the theories of somite segmentation, varied and incomplete as they are,
have in common the assumption that some influence is passing from the cranial
end caudally, somite by somite, as segmentation proceeds. A possible exception
is the suggestion made by Waddington (1956) that somite formation may depend
on the adhesiveness of cells to one another becoming different in the anteroposterior and medio-lateral directions; he writes of the process 'spreading
backwards', but it seems likely that he was thinking of a temporal succession
of stages rather than a diffusion of an actual physical agent. However, the idea
of an actual passage of a substance or other influence would seem a very reasonable assumption to anyone who has observed the somites forming in orderly
cranio-caudal sequence. However, there is so far no experimental evidence
either for or against it in amphibians. In chick embryos, Spratt (1957) believed
that there were * somite centres' on either side of Hensen's node, which initiated
somite formation and were essential to it. Bellairs (1963) has questioned the
validity of Spratt's view, since she obtained somite formation in posterior parts
of chick embryos after transections which isolated them from any possible
influence emanating caudal from near the node.
The present series of experiments was designed to test whether, in amphibian
embryos, any essential initiating stimulus or subsequent controlling influence
Embryonic somite segmentation
351
passes from the occipital region caudalwards, or whether posterior somites can
form autonomously, in normal orientation, when the somite tissue anterior to
them is deleted or displaced.
In all, four types of operation were performed. In series 1, lengths of neural
tube and somites were excised unilaterally just caudal to the region that had
already segmented, to see if, when separated by a gap from this anterior region,
tissue behind the level of operation could still segment normally. In series 2
the posterior half of the embryo was completely severed from the anterior half,
to see if segmentation would develop normally in complete isolation from any
possible influence of the occipital region. In series 3 and 4 the degree to which
cranio-caudal orientation was determined in the somite tissue was tested. Series 3
involved antero-posterior reversal of complete transverse pieces of the neural
plate and underlying mesoderm, at neurula stages, while in the operations of
group 4, only the somite mesoderm was reversed, unilaterally, at a level just
caudal to the segmented region in tailbud stage embryos. The somites of this
reversed region and the tissues caudal to it were then compared with the
corresponding regions of the non-operated side.
MATERIALS AND METHODS
Embryos of the South African clawed toad, Xenopus laevis, the alpine newt,
Triturus alpestris and the Axolotl, Ambystoma mexicanum, were used. Xenopus
embryos were obtained by injection of the adult toads with chorionic gonadotrophin (Pregnyl: Organon Laboratories Ltd.). Newt and Axolotl embryos
were obtained during the breeding season at Wilhelmshaven. All embryos were
demembranated manually and operated on under sterile conditions in Holtfreter
saline at pH 7. Glass bridges were used to hold grafted pieces in position and
these were left for about 2 h to heal, in full-strength Holtfreter saline, before
transferring the embryos to one-tenth Holtfreter saline for further development.
They were kept until the equivalent of hatching stages, by which time they were
motile and the somite segmentation showed clearly. They were then fixed either in
Bouin's or Smith's fixatives, dehydrated, and cleared in methyl benzoate. Some of
the results could be seen in the cleared specimens, but the majority of embryos
were sectioned at 10/* and examined histologically after staining in Weigert's
haematoxylin and eosin.
RESULTS
(1) Deletions of somite mesoderm and neural tube
A piece of the somite mesoderm, together with the lateral half of the neural
tube adjacent to it, was excised from the cervical region of the late neurula of
Xenopus before segmentation had begun. A length equivalent to about 3-4
somites was removed on the right side only (Fig. la). 22 neurulae were treated
in this way. In addition, 15 embryos were operated on at the tail-bud stage
352
E. M. DEUCHAR & A. M. C. BURGESS
(stage 24-25 of Nieuwkoop & Faber, 1956) and in these the mesoderm and
neural tube were removed from near the tail, just posterior to the last somite
formed. This left a little unsegmented trunk mesoderm as well as the tail bud
intact posterior to the deletion (Fig. 1 b).
(b)
Excised
Excised
Glass rod
Yolk plug
(ft
Scfi
Fig. 1. Diagrams illustrating the operations of series 1-4. (a, b) Series 1: deletions of
somite mesoderm unilaterally at (a) late neurula and (b) tail-bud stages, (c) Division
of late gastrula into anterior and posterior halves, (d, e) Reversals of neural plate
and underlying tissues at (d) early neural and (e) late neurula stages. (/, g) Anteroposterior reversals of somite mesoderm unilaterally at (/) late neurula and (g) tail-bud
stages. (N.B. Reversals in a horizontal plane, so that dorso-ventral orientation unchanged.) a = anterior, p = posterior.
The results of both these operations showed clearly that normal segmentation
could occur caudal to the deleted tissue. Every one of the twenty-two embryos
operated at the neurula stage showed normal somites continuing from behind
the operated region. No somite tissue was present in the deletion zone; only a
few dead cells lay loosely on the surface in some instances. Similarly, all of the
embryos operated on at the tail-bud stage had absolutely normal somite tissue
caudal to the deletion. In three of these embryos, however, a little somite tissue
appeared also in the operated region, suggesting that deletion had not been
complete as it was in the other twelve.
Embryonic somite segmentation
353
(2) Isolation of the posterior tissues by halving
Since it was possible that even in the majority of the embryos of group 1
where there was definitely no somite tissue left in the deletion zone some
influence might still have passed from anterior to posterior (either via the
somites of the opposite side of or via other tissues of the embryo), it was decided
to test the effects of completely isolating the posterior from the anterior half
before segmentation had begun. This is easily achieved by bisecting the embryo
transversely with a piece of very thin glass rod (Ruud & Spemann, 1922). This
rod is laid across the embryo to compress it into two halves, then left for about
2 h for the severance to be complete and the halves to heal (Fig. 1 c). Almost
100% survival of the halves was obtained with newt and Axolotl embryos, but
only 50% with Xenopus embryos. Anterior and posterior halves were then
cultured separately until the equivalent of hatching stages.
Forty-three newt embryos were halved at the small yolk plug stage (Harrison
stage 12), and of these 41 of the posterior halves showed normal segmentation:
27 Axolotl embryos were halved at the small yolk plug stage, and of these,
25 posterior halves showed normally segmented somites; in addition, 48 were
halved at early neurula stages (Harrison stage 15) and 46 of these showed
normal segmentation in the posterior halves.
There were 28 survivors of operations on late gastrula stages of Xenopus
(Nieuwkoop & Faber stage 12); 19 out of the 28 posterior halves showed
segmented somites; the other 9 showed no differentiation of either neural tissue
or somites.
All of the above results on posterior halves were observed in histological
preparations as well as externally. Not all of the anterior halves were examined
histologically, however. Out of 51 which were examined in histological detail,
30 showed the occipital somites clearly, while the other 21 had somitic mesoderm
which did not appear to have differentiated. It was clear from these observations
that the occipital somite tissue was included in the anterior halves, so that the
somite segmentation in posterior halves could not have been initiated from
here, and they must have segmented independently.
(3) Reversals of neural plate and underlying mesoderm
The majority of these operations were carried out on Xenopus embryos.
A complete transverse sector of the neural plate and underlying tissues (i.e.
archenteron roof mesoderm, and endoderm of both right and left sides) was
excised and then replaced in reversed antero-posterior orientation (Fig. 1 d, e).
It was left with a glass bridge over it, to heal, for about 2 h, before transferring it
to a culture dish.
Out of 40 Xenopus embryos operated at the neural groove stage (Nieuwkoop
& Faber stage 12|-13), 37 developed with normal orientation of the somite
tissue both in and behind the operated region. In the other three embryos, the
354
E. M. DEUCHAR & A. M. C. BURGESS
graft had become partly detached and had developed into a tail-piece projecting
vertically upwards. The somites in this piece were therefore out of line with those
of the host. The latter effect was obtained, again owing to partial detachment of
the reversed graft, in the great majority (30/33) of another group of Xenopus
embryos that were operated on at the late neurula stage. In all cases, segmentation and orientation of the somites were normal posterior to the graft and were
clearly unaffected by its disorientation.
Reversals of the neural plate and underlying tissues were also carried out in
22 newt embryos at early neural plate stages. Apart from seven embryos that
died, all of these showed normal segmentation and orientation, both in the
graft region and posterior to it. The graft had healed successfully in all these cases
and did not tend to project upwards.
(4) Reversals of somite mesoderm
Operations were performed unilaterally, usually on the left side of the embryo.
A piece of somite mesoderm, equivalent to about 5 somites, was excised from
a region immediately behind the most posterior somite, if segmentation had
already begun, or from the cervical region at presegmentation stages (Fig. 1/, g).
It was replaced beneath the ectoderm, in reversed cranio-caudal orientation,
and left to heal for 2 h under a glass bridge if necessary.
On Xenopus embryos, 7 reversals of cervical mesoderm were carried out at
the early neurula stage; all of these showed normal segmentation afterwards.
Out of 32 operated at the late neurula stage (Nieuwkoop & Faber stage 19-20)
all were again absolutely normal. The same occurred with 17 embryos operated
at the early tail-bud stage (Nieuwkoop & Faber stage 23-25) and 27 embryos
operated at hatching stages in the extreme tail region (Nieuwkoop & Faber
stage 28-29). The remaining eight embryos of these two last series gave indeterminate results.
Thirty-one newt embryos were subjected to mesoderm reversals at the early
tail-bud stage (Harrison stage 24-26). In some of these the tail became flexed
towards the operated side, but otherwise their external appearance was normal
apart from occasional oedema near the operation site. On histological examination, it was found that 28 of the 31 operated had a reduced quantity of somite
tissue on the operated side. The other 3 embryos appeared normal. Segmentation was normal in operated regions despite the reduced tissue and it corresponded
with that on the non-operated side.
Reduction of somite tissue on the operated side was also the only abnormal
feature in twenty-three newt embryos that had been operated on at late neurula
stages (Harrison 19-20).
The results of the various operations in series 1-4 are summarized in Table 1.
Embryonic somite
355
segmentation
Table 1. Summary of experimental results
Species
Xenopus
Triturus
Axolotl
Xenopus
Xenopus
Triturus
Total
operations
Stage
Result
Series 1. Deletions of somite mesoderm and neural tube
22
Neurula
All 22 normal posterior to deletion
15
Tail bud
All 15 normal posterior to deletion; 3 with
some somite tissue in deletion area.
Series 2. Division into cranial and caudal halves
Late gastrula
43
41 posterior halves segmented normally;
2 not segmented
Late gastrula
27
25 posterior halves segmented normally;
2 died
48
Early gastrula
46 posterior halves segmented normally;
2 not segmented
28
Late gastrula
19 posterior halves segmented normally;
9 no differentiation
Series 3. Removals of neural plate and underlying tissues
37 with normal orientation and
40
Neural groove
segmentation
3 with tail-piece projecting upwards
Late neurula
3 normal; 30 with projecting
33
tail-piece
22
Early neurula
15 normal; 7 died
Series 4. Reversals of somite mesoderm
Xenopus
Triturus
7
32
21
31
31
Early neurula
Late neurula
Early tail bud
Hatching stage
Early tail bud
All 7 normal
All 32 normal
17 normal; 4 indeterminate
27 normal; 4 indeterminate
28 with less somite tissue; 3 normal.
All with normal segmentation
DISCUSSION
The outstanding feature of all these experiments is the apparent lack of effect
of any of the operations on the normal segmentation and orientation of the
somite tissue, both in and caudal to operated regions. To explain these findings
it has to be assumed that at the stages used for operations, events controlling
segmentation had not yet taken place, or were still modifiable. The halving and
deletion experiments also show clearly that segmentation takes place independently of any possible influence passing caudalwards from the occipital region
immediately beforehand. Tail regions also appear uninfluenced by the deletion
or reversal of somite tissue anterior to them.
From the foregoing, it has to be concluded that no essential stimulus or
instruction is passed caudal during the segmentation of progressively more
356
E. M. DEUCHAR & A. M. C. BURGESS
posterior regions of the somite mesoderm. Each region appears to be autonomous
with respect to its ability to segment. In amphibian embryos there is clearly
nothing equivalent to Spratt's postulated 'somite centre' of the chick. The
reversal experiments show, on the other hand, that some overall influence from
surrounding tissue governs the antero-posterior orientation of the somites, since
grafted, reversed pieces took on a new orientation to correspond with the rest of
the axis. (This orientation is evident externally, by the fact that the intersomitic
grooves are V-shaped, with the point of the V projecting anteriorly.) Clearly the
orientation is not irrevocably fixed until after the end of neurulation since it
could be altered in the grafted, reversed pieces.
In conclusion, it seems necessary to withdraw all theories on the mechanism
of somite segmentation that insist on its being governed by a continuous process
travelling cranio-caudally. There seems no justification for believing that
successive groups of somite cells affect their neighbours in any way that directly
controls their differentiation. The timing mechanism which determines that
occipital mesoderm shall segment earliest and other regions segment in craniocaudal sequence behind it must be initiated earlier on in development. It may
perhaps result from the sequence of the invagination of the mesoderm in
the gastrula. This possibility could perhaps be tested by experimental interchanges of somite mesoderm between anterior and posterior sites during
gastrulation.
SUMMARY
Operations have been carried out on Xenopus, newt and Axolotl embryos
in order to test the ability of somite mesoderm to segment and orientate normally
after deletions or reversals of somite tissue anterior to it. Unilateral pieces of
somite mesoderm were deleted in a first series of experiments. In a second
series, embryos were transected into anterior and posterior halves before the
beginning of segmentation. In a third series, pieces of neural plate together
with the underlying tissue were reversed cranio-caudally and in a fourth series,
pieces of the somite mesoderm of one side only were similarly reversed.
Except for some of the neural plate reversals, where owing to detachment of
the graft a projecting tail-piece developed and was orientated vertically, all
operations resulted in perfectly normal segmentation both in and caudal to the
operated region. It is concluded that theories which postulate the passage of
some controlling influence cranio-caudally, or some cranial initiator like
Spratt's 'somite centre' in the chick, cannot hold true for amphibian embryos.
The orientation of the somites, however, as evidenced by the direction of their
V-shaped intersomitic grooves, appears to be governed by some overall orientation in the host, since reversed pieces reorientate according to the new surroundings.
Embryonic somite segmentation
357
RESUME
Segmentation des somites chez les embryons d'Amphibiens: y a-t-il
un mecanisme de regulation transmis ?
On a realise des operations sur des embryons de Xenope, de Triton et
d'Axolotl, pour eprouver l'aptitude du mesoderme somitique a se segmenter et
a s'orienter normalement apres destruction ou inversion du tissu somitique
anterieur a lui. Des fragments de mesoderme somitique ont ete detruits unilateralement dans une premiere serie d'experiences. Dans une deuxieme serie,
des embryons ont ete coupes en deux moities anterieure et posterieure avant le
debut de la metamerisation. Dans une troisieme serie, des fragments de plaque
neurale, avec le tissu sous-jacent, ont ete inverses dans le sens cranio-caudal et
dans une quatrieme serie on a inverse de la meme maniere des fragments de
mesoderme somitique d'un seul cote.
Sauf dans le cas de quelques inversions de plaque neurale ou, le greffon
s'etant detache, un fragment de queue s'est developpe et oriente verticalement,
toutes les operations ont abouti a une metamerisation parfaitement normale a
la fois dans la region operee et dans la region plus caudale par rapport a elle.
On en conclut que les theories qui postulent le passage d'une certaine influence
regulatrice cranio-caudalement, ou l'existence d'une sorte d'initiateur cranien
tel que le 'centre somitique' de Spratt chez l'embryon de poulet, ne peuvent etre
valables pour les embryons d'Amphibiens. L'orientation des somites, neanmoins,
manifestee par la direction de leurs sillons intersomitiques en forme de V,
apparait soumise a quelque orientation generate chez l'hote, puisque des
fragments inverses se reorientent selon leur nouvelle position.
We should like to thank Mr L. G. Strang for collaboration in some of the earlier experiments,
and Miss G. Weedon and Miss S. Bevan for invaluable assistance with the histological work.
We are also most grateful to Miss J. Astafiev for drawing Fig. 1.
One of us (E.M.D.) would like to thank Professor H. Tiedemann for the opportunity to
carry out part of this work, on newt and Axolotl embryos, in his department at the MaxPlanck Institut fiir Meeresbiologie, Wilhelmshaven.
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