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/. Embryo!, exp. Morph. Vol. 41, pp. 269-277, 1977
Printed in Great Britain © Company of Biologists Limited 1977
269
The formation of discrete muscles
from the chick wing dorsal and ventral muscle
masses in the absence of nerves
By G. B. SHELLSWELL 1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London
SUMMARY
It is well known that the different muscles in the vertebrate limb develop by a series of
splittings and subdivisions of dorsal and ventral muscle masses. The mechanism for this
process is not clear, and the suggestion from previous studies that tension exerted by the
growing limb stimulates these splittings is now thought unlikely. It has also been proposed
that nerves play an important part in the separations by physically pushing the muscle mass
apart. There is also the possibility that nerves could stimulate differential contraction of parts
of the muscle mass, leading to a shearing effect and resulting in separation.
In this study, peripheral nerves are removed by the administration of the nicotinamide
analogue 3-acetylpyridine before the start of muscle mass division. The resulting pattern of
muscle is normal although nerves are completely absent. This clearly rules out any major role
of nerves in the formation of the muscle pattern, although many authors have shown that
innervation is important for the maintenance and later development of the separated muscles.
While the mechanism of the process of division of the muscle masses remains unknown, it is
suggested that there are changes in cellular behaviour at areas corresponding to the future
spaces between the muscles. These changes may be specified by some aspect of positional
information within the limb. They might involve muscle cells stopping or reversing their
differentiation as muscle in the areas forming the future spaces, or perhaps change the adhesion
properties of the muscle cells to cause them to' sort out' and separate. Alternatively, a localized
invasion of non-muscle mesenchymal cells at the future spaces could lead to separation of
discrete muscles. These possibilities are at present under investigation.
INTRODUCTION
It is now well established that the discrete muscles in the vertebrate limb
develop from the dorsal and ventral muscle masses by way of a series of splittings
and subdivisions. This sequence of cleavages has been described by Romer (1927)
and Wortham (1948) in the development of leg muscles in the chick, and by
Sullivan (1962) in the chick wing. We have recently investigated this process of
muscle mass division in relation to its role in the formation of the pattern of
muscles in the chick wing. (Shellswell & Wolpert, 1977.)
Author's address: The A.R.C. Meat Research Institute, Langford, Bristol BS18 7DY,
U.K.
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EMB 41
270
G. B. SHELLSWELL
In that study we discussed the mechanisms for the division process which had
been proposed in earlier work. Carey (1921) and Sullivan (1962) suggested that
tension exerted by the longitudinal growth of the limb acts as a stimulus for the
splittings. There is no doubt that there is considerable lengthening of the limb
during this time; Summerbell (1976) described a doubling in the length of the
forearm region of the chick wing over the period when most of the splittings in
the muscle masses occur (stages 28-32). Although many authors have shown
that tension is important for later muscle growth and repair after injury
(reviewed by Goldspink, 1974) there have been no recent studies implicating it
in early events in myogenesis, although Hunt (1932) using a chorioallantoic
grafting technique showed that it was possible to obtain well formed muscle
groups with markedly subnormal elongation of the grafted limb.
We have suggested that splitting is an autonomous process, independent of
external forces such as tension. We proposed alterations of cellular properties
or behaviour at the areas corresponding to the future gaps between the muscles,
areas which are specified by some aspect of positional information across the
anteroposterior axis of the limb. A possible mechanism might be distance from
a reference region, similar to that proposed for cartilage elements in this axis
by Tickle, Summerbell & Wolpert (1975). These changes could affect presumptive muscle cells in the future gap areas by stopping or even reversing their
differentiation as muscle, or by changing their adhesive properties to give two
separate masses (Steinberg, 1970), analogous to compartment formation in
insects (Crick & Lawrence, 1975). Alternatively there might be some localized
invasion of non-muscle mesenchymal cells (the presumptive epi-, peri- and
endo-mysium) to form the spaces.
In this discussion we omitted to examine the role, if any, of the nerves.
Wortham (1948) suggested that the division of the leg muscle masses was
caused by migration of nerves into the future spaces, separating and pushing the
masses apart. In addition to this purely physical role of the nerves there is the
possibility that some selective nervous stimulation of different parts of the
muscle mass could produce a shearing effect, leading to physical separation.
Indeed, Landmesser & Morris (1975) found that they could cause contraction
of different parts of the undivided hindlimb muscle masses by differential nerve
stimulation. Finally, Hunt (1932) showed that although muscle would differentiate in limb-buds grafted to the chorioallantoic membrane, it was only
divided into distinct bundles with well developed associated connective tissue
if the grafts included an abundant nerve supply.
The experiments described here assess the importance of nerves in the division
of the muscle masses in the chick wing. They make use of the recent findings of
McLachlan, Bateman & Wolpert (1976) that the nicotinamide analogue
3-acetylpyridine (3-AP) has a deleterious effect on various cell types, and at
certain dosages can be particularly effective against peripheral nerves which
are almost totally destroyed after only 24 h. Peripheral nerves have been removed
Formation of muscles in the absence of nerves
271
by administration of 3-AP before and during muscle mass division, when
Roncali (1970) described their invasion as progressing towards the wrist (before
muscle mass division) or having just reached it (during muscle mass division).
Although McLachlan et al. (1976) found that the muscle blocks in treated limbs
formed the normal pattern, muscle mass division had already started when their
3-AP treatment commenced (6 days).
MATERIALS AND METHODS
Fifteen White Leghorn eggs were windowed after 3 days of incubation at
37 °C. In each case the vitelline and amniotic membranes were torn above the
embryo to prevent it from sinking down into the yolk, a few drops of balanced
salt solution containing antibiotics were added to prevent infection and the
eggs were resealed with Sellotape. Their development was closely monitored and
the hole in the membranes prevented from healing. On reaching stage 25/26
(Hamburger & Hamilton, 1951) one embryo was fixed as a control and another
eight embryos were treated with a low teratogenic dose of 0-7 mg of 3-AP
(Sigma) in 50 jn\ of distilled water. Two embryos were treated with the same dose
of 3-AP at stage 29, again with a control fixed at this stage. All treated eggs and
remaining controls were reincubated under similar conditions, and removed at
various times for staging and fixation in half strength Karnovsky fixative. In
the group treated at stage 25/26, three embryos werefixedafter 24 h reincubation,
three after 48 h and two after 72 h. In the group treated at stage 29, one was
fixed after 24 h reincubation and the other after 62 h. The remaining embryos
were fixed as controls for the different periods of reincubation used.
After fixation, both wings from all embryos were dehydrated and embedded
in Araldite. Four or five serial semi-thin (1-5 jum) transverse sections were cut
at least two proximo-distal levels in the limb, at the levels of the digits and
approximately mid-radius/ulna region. Longitudinal sections were cut in two
treated and two-control limbs to check the appearance of developing myotubes.
Sections were stained with toluidine blue, viewed and photographed in a Leitz
Orthomat photomicroscope.
RESULTS
Our earlier work (Shellswell & Wolpert, 1977) described the splitting of the
muscle masses in the radius/ulna region of the wing. The first division is in the
ventral mass at late stage 27 and then the dorsal mass divides into two masses
which subdivide again by stage 29/30. There is further binary subdivision both
dorsally and ventrally to give, by stage 36, the essentially adult pattern of six
main dorsal extensors and four ventral flexors at the mid radius/ulna proximodistal level.
Figure 1 shows the sequence of splitting in the mid radius/ulna region 24 h,
48 h and 72 h after 3-AP treatment at the pre-division stage 26. It can be seen
18-2
272
G. B. SHELLSWELL
Formation of muscles in the absence of nerves
273
that the pattern of muscles is normal, but closer examination (Fig. 2) shows the
complete absence of nerves, in agreement with McLachlan et al. (1976). The
muscle pattern was also normal in limbs where 3-AP treatment had started after
initial muscle mass division.
At the digit level the pattern of muscles was also normal (Fig. 3 (A)) and
again there was no sign of any nerves. It is interesting that the tendons at this
level appeared normal 32 h after treatment (Fig. 3 (B)) in contradiction to the
absence of tendons described by McLachlan et al (1976). This is probably due
to the different dose and longer period of reincubation after treatment used by
these authors.
Longitudinal sections of early and late stages after treatment show the normal
appearance of elongated myotubes which often show striations (Fig. 4).
Surrounding non-muscle cells which will eventually form endo-, peri- and
epi-mysium layers of muscle connective tissue were also normal both in numbers
and arrangement (e.g. flattened cells surrounding separate muscle blocks epimysium - and small cells with many processes, around and between the
groups of myotubes - peri- and endo-mysium).
DISCUSSION
The results clearly show that the continued presence of nerves is not essential
for division of the muscle mass in the chick wing. The pattern of muscles following removal of peripheral nerves by 3-AP treatment before division was normal
in all cases. This rules out the suggestion of Wortham (1948) that separation of
the distinct limb muscles is caused by a physical migration of the nerves and it
also seems that any shearing effect caused by differential nerve stimulation
cannot be involved. The normal appearance of the separating muscles also
confirms the observation of Hunt (1932) that early differentiation events in
myogenesis in vivo (myoblast fusion, myofibril formation in myotubes) are also
not dependent on nerves.
There is the possibility that although the continued presence of nerves is not
essential for the formation of the muscle pattern, the initial cues for muscle
Fig. 1. The development of the pattern of muscles after 3-AP treatment, transverse
sections at mid radius/ulna level. (A) Undivided dorsal and ventral muscles masses
(MM) at start of treatment, stage 26. (B) 24 h after treatment, stage 28 showing clear
division of the ventral muscle mass while dorsally the division is not complete.
(C) 48 h after treatment, stage 29/30. Separation of two dorsal blocks complete,
further subdivisions clear anteriorly, not complete posteriorly. (D) 72 h after treatment, stage 33. Four separate dorsal muscle blocks. There are also indications
of further division ventrally at the arrrows. (E) 72 h after treatment, slightly more
advanced stage than (D). Further subdivision has occurred dorsally giving five
blocks while the ventral blocks are showing signs of further separation. (Scale
bar = 200 /*m in each case. All limbs dorsal surface to the top, anterior surface to the
right.)
Fig. 2. Central region of mid radius/ulna level in (A) 48 hours after 3-AP treatment, stage 30. (B) Normal control stage
29/30. Nerve absent in treated limb while muscle block is unaffected. BV: blood vessel, M: muscle, iV: nerve, JR/iradius,
U: ulna.
50 urn
1
to
B
50 nm
Fig. 3. (A) T.S. digit level of limb 72 h after 3-AP treatment. (B) Higher magnification of the area
indicated showing two tendons dorsal to digit 3.
A
200//m
to
I
276
G. B. SHELLSWELL
10 nm
Fig. 4. L.S. muscle region 48 h after 3-AP treatment, stage 30, showing
striated myotubes.
division might be mediated by them. In the experiments described here nerves
in the undivided forearm muscle masses might have given these cues either before
the administration of 3-AP at stage 26 or during the 24-h period of degeneration
following treatment (i.e. up to late stage 27, about the time of the first division
of the forearm ventral muscle mass). The normal pattern of muscles in themanus
from embryos treated with 3-AP at stage 26 shows that this type of early nervemuscle interaction is not involved in the splitting of the muscle masses. Although
there is relatively little muscle in this region, what there is develops from dorsal
and ventral muscle masses by a sequence of cleavages similar to that described
for the forearm. Nerves do not reach this part of the limb until after stage 28
(Roncali, 1970). Indeed, nerves were not found more distal than the wrist in
control limbs of this stage. Even allowing for the 24-h degeneration period it is
clear that the pattern of muscles in the hand can develop normally even though
nerves never reach the muscle masses. This contrasts with the later influence
'fast' or 'slow' nerves have on muscle fibre differentiation, where information
(in this case the nature of the stimulation) passes from nerve to muscle and
determines the type of contractility shown by the muscle fibre.
How then do the individual muscles separate? There is increasing evidence
that external forces are not involved because at the time of splitting in the forearm there is no development of distal tendons which could exert tension on
the muscle mass. Indeed, removal of all regions distal to the radius and ulna
has little effect on the division process (Shellswell & Wolpett, 1977). In this
Formation of muscles in the absence of nerves
277
connection, in the shortened limbs produced with the teratogen 6-aminonicotinamide (6-AN) which McLachlan et al. (1976) suggest affects cartilage matrix
secretion, the muscle pattern is normal although the muscles themselves are
shorter (McLachlan & Shellswell, unpublished observations).
It seems then that the division of the limb muscle masses is an autonomous
process depending on some change in the behaviour of muscle or non-muscle
cells in the region of the future spaces. We do not know the exact nature of these
changes although we have suggested some possibilities as described above. We
are at present investigating these possibilities at the cellular level using the
electron microscope and the results will be published elsewhere (Shellswell, Day
& Wolpert - in preparation).
Thanks are due to A. Day for his technical help and advice, to J. McLachlan for the 3-AP
solution and his helpful comments, to Dr C. Tickle for reading and commenting on the
manuscript, and to Professor L. Wolpert for inspiration and discussion.
This work was supported by the Medical Research Council with a Research Studentship.
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