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J. Embryol. exp. Morph. 83, 213-223 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
213
Motor innervation of proximally rotated chick
embryo wings
By N. G. LAING
Department of Pathology, University of Western Australia, Queen Elizabeth II
Medical Centre, Nedlands, 6009, Western Australia
SUMMARY
Chick embryo wing buds were rotated close to the lateral edge of the somites at stage 19,
prior to limb innervation. Despite the abnormal orientation of the resulting limb, the motor
pools to biceps and triceps were largely normal, as judged by electrical stimulation and
horseradish peroxidase labelling just prior to hatching. The only abnormalities were a few
caudal motoneurons innervating biceps and a few rostral motoneurons innervating triceps.
This distribution is similar to that seen normally in young embryos before the completion of
motoneuron death and it is suggested that the rotation may be keeping alive motoneurons
which otherwise would die. The morphology of the brachial plexus supplying rotated wings
was abnormal. It is concluded that axons growing into the limb bud from the spinal cord can
compensate for reversal of both the limb axes and selectively innervate appropriate muscles.
The result is consistent with others in which proximal reversal of one limb axis alone produced
normal innervation.
INTRODUCTION
Stirling & Summerbell (1979) reported that the innervation of chick embryo
wings which had been rotated prior to nerve entry, so that both the dorsoventral
(D-V) and anteroposterior (A-P) axes were reversed, was abnormal. For example, the biceps muscle was innervated by the spinal nerves which normally innervate the triceps and vice versa. From this, Stirling & Summerbell (1979) concluded that there was no selective innervation of the limb by ingrowing axons.
Since then, however, conflicting results have been obtained in a number of
studies. These have involved reversal of one limb axis only, either the D-V axis
or the A-P axis. Whether the resulting innervation is normal or abnormal apparently depends on the proximodistal level of the manipulation. D-V reversal
of the wing between the shoulder and the elbow (Summerbell & Stirling, 1981,
1982) and D-V reversal of the leg at the knee (Whitelaw & Hollyday, 1983)
produce mostly abnormal innervation, as in the original study, whereas D-V
reversal of the hind limb (Ferguson, 1983; abstract, 1978) and A-P reversal of the
wing at the body wall or the shoulder (Stirling & Summerbell, 1983) produce
mostly normal innervation. Proximal reversal of one axis can therefore result in
normal innervation and this demonstrates that outgrowing axons can selectively
innervate their targets in the limb.
214
N. G. LAING
The effect of proximal rotations with reversal of both D-V and A-P axes has
not yet been reported. In this study, dual axis rotation has been achieved using
the classical techniques of Hamburger (1939) in which the manipulated limb bud
is inserted into a slit in the body wall. Both muscles studied (biceps, triceps) were
found to receive mostly normal innervation, through an abnormal brachial
plexus. Axons growing into the embryonic limb bud can thus compensate for
reversal of both the limb axes and innervate appropriate muscles.
MATERIALS AND METHODS
Fertile eggs were 'windowed' at three days of incubation. The right wing bud
was rotated through 180° at Stage 19 (Hamburger & Hamilton, 1951). Stage 19
was chosen in order to parallel the conditions of the experiments of Stirling &
Summerbell (1979) as closely as possible apart from the level of rotation. At this
stage axons growing out from the spinal cord have not progressed further than
the ventrolateral tip of the somite and have not entered the limb (Roncali, 1970;
Bennett, Davey & Uebel, 1980; Swanson & Lewis, 1982; Hollyday, 1983). Using
techniques similar to those of Hamburger (1939), cuts were made with electrolytically sharpened tungsten needles rostral and caudal to the wing bud and
between the wing bud and the somites as close as possible to the lateral border
of the posterior cardinal vein, leaving the wing bud attached ventrally to the body
of the embryo. This attachment was then severed by gently pulling the loosened
bud away from the body while cutting through the body wall close to the ventral
surface of the wing bud. The cuts produced a pocket in the body wall into which
the rotated wing bud could be introduced. In other (sham-operated) embryos,
the wing bud was replaced in the slit without rotation. The embryos were allowed
to mature until the seventeenth day of incubation (E17) when motoneuron death
is largely completed in the brachial region of the chick embryo (Oppenheim &
Majors-Willard, 1978; Laing, 1982a). The biceps or triceps muscle was then
injected with a 20 % w/v solution of HRP (Sigma Type VI) dissolved in pH7-4
TRIS buffer containing 0-01 % poly-1-ornithine (Hadley & Trachtenberg, 1978).
The eggs were then returned to the incubator until the next day when the embryos were killed by decapitation. After dorsal laminectomy, the brachial region
of the spinal cord was dissected out and frozen in isopentane cooled in liquid
nitrogen. Serial frozen sections were cut (20jum) and stained for HRP reaction
product using a modification (Laing & Lamb, 1983) of the cobalt-intensified
diamino benzidine (DAB) method of Adams (1977). The wings were skinned at
least as far as the elbow and then, still attached to the body, placed in a bath
containing an oxygenated Ringer's solution of composition: NaCl, 137 HIM; KC1,
5 min; CaCl, 5 min; MgCl, 1 mM; phosphate buffer pH 7-4,1 HIM; HEPES pH 7-4,
5mM and glucose, 11 mM. The rostral-most and caudal-most large spinal nerves
entering the brachial plexus were stimulated using close-fitting suction electrodes
by single pulses (0-5 msec, 2-10 V) or trains of pulses (40 Hz) passed through
Innervation of rotated wings
215
constant current stimulus isolators (Neurolog). Brachial plexus morphology was
recorded in each case in hand-drawn sketches. Some of the normal and operated
wings were processed for routine histology and stained with haematoxylin and
eosin.
RESULTS
Anatomy
The right wing bud was rotated in 280 cases. Of these, 124 died prior to E17.
The wing was absent in 11 embryos and highly abnormal in 45. When present,
the rotated wings were often bent over the back with the ventral surface of the
wing apposed to the dorsal surface of the back as described by Summerbell &
Stirling (1981) for dorsoventral reversals. The musculature of the rotated wing
varied from being well developed to being totally absent. Embryos whose
operated limbs contained no muscle (21 embryos) were discarded. Any rotated
wings which contained good biceps and triceps muscles were used for HRP
and/or electrophysiology. Sometimes, the muscles of the upper arm were well
formed when those of the forearm were much reduced. Sections cut through the
limbs revealed that reduction in the musculature was due to a general reduction
of the size of the muscles and/or the total absence of individual muscles. The
muscles most frequently affected were extensor metacarpi radialis, extensor
digitorum communis, extensor metacarpi ulnaris and anconeus which are all
derived from the dorsal muscle mass (Sullivan, 1962). But, the pronator profundus and pronator sublimis, which are derived from the ventral muscle mass, were
affected in one of the seven cases examined. Sham-operated wings showed
similar results, in that the wing was sometimes missing or abnormal, but the
musculature was complete in all six cases examined and reduced compared with
the control side in only one case.
The morphology of the brachial plexus supplying the rotated wings was usually
abnormal. The plexus in normal embryos is formed mainly from spinal nerves 14,
15 and 16 (S.14, S.15 and S.16) with varying contributions from S.13 and/or
S. 17. S. 14 and S.15 fuse proximally, with S.16 merging further distally (Fig. 1 A).
In the plexus supplying rotated wings S.16 merged with S.15 more proximally
th an S. 14 in 24 out of the 26 embryos examined. In all cases, the plexus was much
closer to the vertebral column and a single or double nerve trunk entered the
wing (Fig. 1B,C). The plexus was normal in sham-operated embryos.
Electrophysiology
Electrophysiological stimulation was carried out on eighteen experimental
embryos, three normal embryos and ten sham-operated embryos. In many cases
the specimens required a period of recuperation after the trauma of dissection
before good responses to nerve stimulation were obtained and in some cases
216
N. G. LAING
17 -
13
A
Normal
1J
N
\
x
X
14-O
14 W
15;—'
is;
74
16.
/
17'
B
Rotated
Fig. 1. Commonest morphology of the brachial plexus in normal embryos (A) and
embryos with a rotated wing (B and C). Spinal nerves 13 and 17 are depicted by
dashed lines to indicate that they do not always contribute to the plexus.
there was no response at all (9 out of 25 control wings, 2 out of 10 sham-operated
wings and 8 out of 18 rotated wings). The responses, when obtained, were
remarkably similar in normal, sham-operated and rotated wings. Stimulation of
the rostral spinal nerves, S. 13 or S. 14, with single pulses always produced visible
twitches in biceps. Single pulses applied to the caudal nerves, S.16 or S.17,
produced twitches in triceps. Tetanic stimulation (40 Hz) produced elbow flexion
when applied to the rostral nerves and elbow extension when applied to the
caudal nerves (Fig. 2). However, in two out of the ten cases in which muscle
contraction was obtained in rotated wings, rostral nerve stimulation produced
weak triceps activation as well as the main biceps effect. With single pulses the
unusual effect appeared as weak twitches of the triceps and tetanic stimulation
produced weak contractions whose effectiveness was masked by the contraction
of the biceps.
HRP
HRP was injected into 42 of the embryos with well-muscled rotated wings.
Sixteen died following the injection, but in 20 of the remaining 26 embryos
labelled motoneurons were found and the histology was good enough throughout
Innervation of rotated wings
217
Fig. 2. Effect of tetanic stimulation (40 Hz) of brachial spinal nerves supplying
rotated (A,B,C) and normal wings (A',B',C). (B,B') resting position; (A,A') stimulation of S.14; (C,C) stimulation of S.16. Viewed ventrally, the host axes are the
same, rostral at the top, for both rotated and normal wings. The suction electrode
can be seen to be more caudal in (C) and (C) than in (A) and (A'). In (B) and (B')
the suction electrode is not near the plexus.
the brachial lateral motor column (LMC) to allow reconstruction of the
motoneuron pool of the injected muscle.
In normal embryos, the biceps motor pool was located predominantly in the
rostral half of the brachial LMC (Figs 3, 4). This was also the case in embryos
with rotated wings, although in three out of eleven operated embryos there were
some labelled motoneurons far caudally in the LMC. In one of these, the entire
motoneuron pool, as seen by HRP, (seven labelled cells) lay caudally, but it
showed normal electrophysiology which indicated innervation of biceps by segment 14. The results with triceps injection were similar. The triceps motor pool
was predominantly in the caudal half of the LMC in normal and operated embryos (Figs 5,6), though rostral labelled motoneurons were found in seven out
of nine experimental embryos. The mediolateral distribution of the labelled cells
was in most cases similar in normal and operated embryos (Figs 4, 6). The
dissimilarities occurred with the 'tail' motoneurons. Caudal motoneurons innervating biceps tended to be further lateral than the rostral motoneurons innervating biceps and rostral motoneurons innervating triceps tended to be more
medial than their caudal counterparts. Thus, both biceps and triceps
motoneuron pools lay from rostromedial to caudolateral in the brachial LMC
when tails were present. This is a similar pattern to that seen in stage-35 normal
218
N . G. LAING
Rotated wing biceps
4-
2-
* - . , . L . p . . . J . . .,
10
20
30
40
50
60
70
80
90
100
80
90
100
Normal biceps
I
4-
2-
0
0
10
20
Rostral
30
40
50
60
70
Brachial LMC
Caudal
Fig. 3
Normal
Rotated
Rostral
Fig. 4
Innervation of rotated wings
219
4 -. Rotated wing triceps
2-
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
100
0
Rostral
Brachial LMC
100
Caudal
Fig. 5. Rostrocaudal position of triceps motor pool in normal embryos and embryos
with rotated wings. Normal: 6 embryos, 441 labelled motoneurons; operated: 9
embryos, 728 labelled motoneurons.
embryos by Summerbell & Stirling (1981, 1982). 'Tails' were present in both
embryos which showed abnormal electrophysiological responses but also in
other embryos which showed normal electrophysiology.
In sham-operated embryos the positions of the biceps and triceps motor pools
were similar to those in normal embryos: there was no caudal 'tail' of
motoneurons following biceps injection or rostral 'tail' following triceps injection.
It can be seen in Figs 4 and 6 that the LMC on the operated side was usually
(13 out of 20 cases) reduced when compared with the control side. A similar
effect was described by Stirling & Summerbell (1983). No attempt was made to
count motoneurons in the thick frozen sections.
Fig. 3. Rostrocaudal position of biceps motor pool in normal embryos and embryos
with rotated wings. Positions of labelled neurons were normalized using a percentage
scale in which the section before the rostral-most section containing LMC cells was
taken as zero and the caudal-most section containing LMC cells was taken as 100.
Normal: 7 embryos, total of 539 labelled motoneurons; operated: 11 embryos, 478
labelled motoneurons.
Fig. 4. Mediolateral position of biceps motor pool to representative normal and
rotated wings. Camera-lucida drawings for each quarter of the length of the LMC
show all the labelled motoneurons found in that quarter. Individual sections were
aligned using the central canal and the edge of the grey matter as markers. The righthand group of drawings show one of the most abnormal cases following rotation.
220
N. G. LAING
Normal
Rotated
3
/4
4
/4
Caudal
Fig. 6. Mediolateral distribution of triceps motor pool in representative normal and
operated embryos. Right-hand set of drawings show one of the most abnormal motor
pools.
Labelled dorsal root ganglion cells were noticed in ganglia 14,15 and 16 after
injection of either biceps or triceps in normal and experimental embryos. This
indicates that the somatotopic projection of dorsal root ganglion cells is more
diffuse than that of motoneurons: a finding in agreement with that of Honig
(1982).
DISCUSSION
The results indicate that when the chick wing bud is rotated using the
technique described here, the innervation of the biceps and triceps muscles is
largely by motoneurons in normal positions. Thus, normal innervation can result
even when the wing is rotated in both axes, provided the rotation is performed
proximally enough. In compensating for the reversal, axons growing out from
the spinal cord to the limb form an abnormal plexus and then selectively innervate their appropriate muscles. This consolidates previous findings of selective
innervation following proximal reversal of one axis: the D-V axis in the hind limb
(Ferguson, 1978, 1983) and the A-P axis in the wing (Stirling & Summerbell,
1983). Stirling & Summerbell (1983) maintain that after D-V reversal of the
wing, selective innervation does not occur and that outgrowing axons art
Innervation of rotated wings
221
passively routed to inappropriate muscles. The present study demonstrates that,
as with the hind limb, selective innervation of the wing can occur following D-V
reversal. It is debatable whether proximal A-P reversal of the hind limb, with its
two nerve plexuses, would produce normal innervation, since long rostrocaudal
(R-C) reversals of the lumbar spinal cord produce abnormal innervation (LanceJones & Landmesser, 1981).
The abnormal plexus found here in rotated wings with normal motor pools
should be emphasized. It is unlikely to be an artefact of the operative technique
since the plexus was normal in sham-operated embryos. Stirling & Summerbell
(1983) mention changes in trajectory taken by labelled axons following their
proximal A-P reversals but do not discuss the general morphology of the plexus.
Ferguson (1983) notes variations in individual plexuses but does not discuss
systematic alterations which might be expected to be less dramatic in her reversals of the D-V axis than in experiments involving A-P reversal. The abnormal
plexus found in the present study would indicate that a considerable degree of
reorganization of axon outgrowth is occurring there, as the axons encounter
rotated limb tissue and respond to new positional information. The formation of
the abnormal plexus is thus an important step in the production of selective
innervation.
The reduction in muscle bulk in the rotated wings and in the LMC supplying
them is also interesting. The reduced muscle bulk could result in a reduced LMC
(eg. Laing, 19826) or a reduced LMC could lead to a reduced muscle bulk
(Harris, 1981). Since there was not such a marked reduction in muscle in the
sham-operated limbs, the operative technique itself is unlikely to be responsible
and it is therefore unlikely that the reduced LMC was a consequence of reduced
muscle bulk unless the muscle bulk is affected by rotation but not by normal
orientation of the grafted wing bud. It is more likely that the reduced muscle is
a consequence of the small LMC. The small LMC possibly results from a greater
disorder in the innervation of the rotated wing by ingrowing axons and
subsequent refinement into normal motor pools by increased motoneuron death
(Stirling & Summerbell, 1983). It is worth noting, however, that a reduced LMC
did not correlate in these present experiments with the presence of normal motor
pools without 'tails'.
Pettigrew, Lindeman & Bennett (1979) found that at early stages all segments
contributing to the brachial plexus innervate biceps, but that this alters during
the period of normal motoneuron death, until by stage 32, only segments 14 and
15 innervate biceps. Using the sensitive benzidinedihydrochloride method for
HRP staining, Summerbell & Stirling (1981) (and Stirling & Summerbell, 1983)
found biceps innervation by segment 16 and triceps innervation by segment 14
as late as stage 35 in normal embryos. The rostrocaudal and mediolateral
distributions they found at stage 35 are very similar to those found in the present
study for rotated wings at E18 (Stage 44). Since motoneuron death continues
after stage 35 in the brachial region (Oppenheim & Majors-Willard, 1978; Laing,
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N. G. LAING
1982a), the caudal 'tail' of motoneurons innervating biceps in the rotated wings,
which would usually be considered 'errors', might be motoneurons which in
normal embryos innervate the biceps at early stages but which later die. A similar
argument could be employed for the rostral motoneurons innervating triceps.
The wing rotation may thus be allowing some motoneurons which normally die
to survive. The survival of these 'tail' motoneurons following manipulation
(appropriate segment and inappropriate segment motoneurons co-existing in a
muscle: see also Lance-Jones & Landmesser (1981)) may offer a clue to the
process of normal motoneuron death by indicating a change in optimal connectivity patterns (Lamb, 1984).
The plexus has been described as 'the critical choice point' in the innervation
of the limb (Hollyday, 1981). Following D-V limb reversal (Summerbell &
Stirling, 1982) or long rostrocaudal spinal cord reversal (Lance-Jones & Landmesser, 1981) some axons can take aberrant routes in distal limb regions to reach
appropriate muscles, indicating that axons can make navigational adjustments
more distally and override the 'choice' made at the plexus. Another factor which
overrides the decision at the plexus is whether or not the motoneuron survives
the normal motoneuron death period. At early, but not late stages, all the spinal
nerves contributing to the plexus may innervate both biceps and triceps, (see
above). Similar observations have been made in Xenopus (Lamb, 1976).
Motoneuron death, which depends upon unknown interactions between the
axons and the muscle (Lamb, 1984), is responsible for the production of the adult
pattern of innervation at least in Xenopus (Lamb, 1977), and is also, therefore,
important in deciding which motoneurons innervate which muscles.
This work was supported by the National Health and Medical Research Council of Australia
and by the Muscular Dystrophy Research Association of Western Australia. Throughout the
experiments I received excellent technical assistance from Jane Eccleston and Janine van
Noort. I would like to thank Alan Lamb, Phil Sheard and Vicky Stirling for improving the
manuscript and Min Seats for her skill on the word processor.
REFERENCES
J. C. (1977). Technical considerations in the use of horseradish peroxidase as a
marker. Neuroscience 2, 141-145.
BENNETT, M. R., DAVEY, D. & UEBEL, K. (1980). The growth of segmental nerves from the
brachial myotomes into the proximal muscles of the chick forelimb during development. /.
comp. Neurol. 189, 335-357.
FERGUSON, B. (1978). Effect of dorso-ventral limb rotations on the development of motor
connections. Soc. Neurosci. Abst. 4, 111.
FERGUSON , B. (1983). Development of motor innervation of the chick following dorsal-ventral
limb bud rotations. /. Neurosci. 3,1760-1772.
HADLEY, R. T. & TRACHTENBERG, M. C. (1978). Poly-1-ornithine enhances the uptake of
horseradish peroxidase. Brain Res. 158, 1—14.
HAMBURGER, V. (1939). The development and innervation of transplanted limb primordia of
chick embryos. /. exp. Zool. 80, 347-389.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
ADAMS,
Innervation of rotated wings
223
A. J. (1981). Embryonic growth and innervation of rat skeletal muscles. I. Neural
regulation of muscle fibre numbers. Phil. Trans. R. Soc. B. 293, 257-277.
HOLLYDAY, M. (1981). Rules of motor innervation in chick embryos with supernumerary
limbs. /. comp. Neurol. 202, 439-465.
HOLLYDAY, M. (1983). Development of motor innervation of chick limbs. Prog. clin. Biol.
Res. 110A, 183-193.
HONIG, M. G. (1982). The development of sensory projection patterns in embryonic chick
hind limb. /. Physiol. 330, 175-202.
LAING, N. G. (1982a). Timing of motoneuron death in the brachial and lumbar regions of the
chick embryo. Devi Brain Res. 5, 181-186.
LAING, N. G. (19826). Motor projection patterns to the hind limb of normal and paralysed
chick embryos. J. Embryol. exp. Morph. 72, 269-286.
LAING, N. G. & LAMB, A. H. (1983). Development and motor innervation of a distal pair of
fast and slow wing muscles in the chick embryo. /. Embryol. exp. Morph. 78, 53-66.
LAMB, A. H. (1976). The projection patterns of the ventral horn to the hind limb during
development. Devi Biol. 54, 82-99.
LAMB, A. H. (1977). Neuronal death in the development of the somatotopic projections of the
ventral horn in Xenopus. Brain. Res. 134, 145-150.
LAMB, A. H. (1984). Motoneuron death in the embryo. Critical Reviews in Neurobiology, (ed.
A. Roses), Boca Raton, Florida: CRC press (in press).
LANCE-JONES, C. & LANDMESSER, L. (1981). Pathway selection by embryonic chick
motoneurons in an experimentally altered environment. Proc. R. Soc. B 214, 19-52.
OPPENHEIM, R. W. & MAJORS-WILLARD, C. (1978). Neuronal cell death in the brachial spinal
cord is unrelated to the loss of polyneuronal innervation in wing muscle. Brain Res. 154,
148-152.
PETTIGREW, A. G., LINDEMAN, R. & BENNETT, M. R. (1979). Development of the segmental
innervation of the chick forelimb. /. Embryol. exp. Morph. 49, 115-137.
RONCALI, L. (1970). The brachial plexus and the wing nerve pattern during early developmental phases in chicken embryos. Monitore zool. ital. 4, 81-98.
STIRLING, R. V. & SUMMERBELL, D. (1979). The segmentation-of axons from the segmental
nerve roots to the chick wing. Nature Lond. 278, 640-642.
STIRLING, R. V. & SUMMERBELL, D. (1983). Familiarity breeds contempt: the behaviour of
axons in foreign and familiar environments. In Limb Development and Regeneration (ed.
J. F. Fallon & A. I. Caplan), Part A, pp. 217-226. New York: Alan R. Liss, Inc.
SULLIVAN, G. E. (1962). Anatomy and embryology of the wing musculature of the domestic
fowl (Gallus). Aust. J. Zool. 10, 458-518.
SUMMERBELL, D. & STIRLING, R. V. (1981). The innervation of dorsoventrally reversed chick
wings: evidence that motor axons do not actively seek out their appropriate targets. J.
Embryol. exp. Morph. 61, 233-247.
SUMMERBELL, D. & STIRLING, R. V. (1982). Development of the pattern of innervation of the
chick limb. Am. Zool. 22, 173-184.
SWANSON, G. J. & LEWIS, J. (1982). The timetable of innervation and its control in the chick
wing bud. /. Embryol. exp. Morph. 71, 121-137.
WHITELAW, V. & HOLLYDAY, M. (1983). Neural pathway constraints in the motor innervation
of the chick hindlimb following dorso-ventral rotations of distal limb segments. /. Neurosci.
3, 1226-1233.
HARRIS,
(Accepted 11 May 1984)