J. Embryol. exp. Morph. 78, 53-66 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
53
Development and motor innervation of a distal
pair of fast and slow wing muscles in the chick
embryo
ByN. G. LAING 1 AND A. H. LAMB 1
From the Department of Pathology, University of Western Australia
SUMMARY
The chick wrist muscle ulnimetacarpalis dorsalis (umd) has two heads. Using myosin
ATPase and acetylcholinesterase (ACh.E) staining it was shown that one of the heads is
composed almost entirely of acid-stable muscle fibres with multiple end plates (slow muscle
fibres) and the other of acid-labile fibres with single end plates (fast muscle fibres). The
development of the muscle was traced from E7 (Stage 32-33) when it is a relatively
homogeneous mass, to E18. The two heads of the muscle are first distinguishable, by ATPase
staining, at E8 (Stage 33-34) prior to their cleaving. Both heads of the muscle are innervated
by motoneurons positioned laterally in the lateral motor column in spinal segments 15 and 16.
There is no observable difference in the positions of the motoneuron pools to the two heads.
At E18 the motoneurons innervating the fast head tend to be slightly larger than those innervating the slow head.
INTRODUCTION
There is considerable interest in the differentiation of fast and slow muscles in
the embryo and the role of the innervating motoneurons. The anterior and
posterior latissimus dorsi muscles (ALD and PLD) have been particularly
popular as subjects of study since their description by Ginsborg & Mackay (1960,
1961) because they are composed almost exclusively of slow and fast muscle
fibres respectively. Unfortunately, their origin from the vertebral column
precludes their use in experiments based on manipulation of the limb bud as a
means of testing the effect of foreign innervation on the fast and slow properties.
Another pair of fast and slow muscles was therefore sought in more distal regions
of the limb buds. The ulnimetacarpalis dorsalis muscle (umd), which lies in the
distal part of the wing (Hudson & Lanzilloti, 1964), was found to be an ideal
alternative. This paper describes the development of the muscle and some
features of its motor innervation.
1
Authors' address: Hospital and University Pathology Services, Sir Charles Gairdner Hospital and University of Western Australia, The Queen Elizabeth II Medical Centre, Nedlands
6009, Western Australia.
54
N. G. LAING AND A. H. LAMB
METHODS
Muscle histochemistry
Chick embryos of various ages from day 7 of incubation (E7), st 32-33 (Hamburger & Hamilton, 1951), to day 18 of incubation (E18) (st44) were killed by
decapitation.
In some cases the wings, from elbow to digits, were dissected free and frozen
in foil tubes of freezing compound (Lab-Tek OCT) by partial immersion in
isopentane (Fluka) cooled to -160 °C by liquid nitrogen. The wings were serially
sectioned (16/mi) in a cryostat and the sections stained for myosin ATPase
(Dubowitz & Brooke, 1973). In other cases the whole wing was stained for
acetylcholinesterase (ACh.E) (Koelle & Friedenwald, 1949).
In order to visualize the end plates on teased muscle fibres, the umd was
prefixed in situ for l h in formol calcium, dissected free, then stained with the
Karnovsky & Roots (1964) ACh.E incubation medium for 1 h while being continuously agitated. The muscles were post-fixed in formol calcium for several
days and cleared in glycerol. Individual muscle fibres were then teased from the
muscle using electrolytically sharpened tungsten needles held in micromanipulators (Narishige).
The Koelle & Friedenwald (1949) method was used for whole mounts because
it gave greater penetration into the muscles. The Karnovsky & Roots (1964)
technique was used for single fibres because it gave better definition of individual
end plates.
Horseradish peroxidase (HRP) labelling of neurons
One or other of the heads of the umd was injected at E17 with a 20 % solution
of HRP (Sigma Type VI) dissolved in pH 7-0 phosphate buffer. The solution was
injected using a glass microelectrode broken back to a tip diameter of
approximately 20 /im and held in a micromanipulator. The wing was kept stationary by a fine wire Sellotaped to the shell of the egg. The next day, the embryos
were killed by decapitation and the brachial region of the spinal cord was fixed
for 4h in 2-5% glutaraldehyde in pH7-0 phosphate buffer containing 5%
sucrose. The cords were then washed until they sank in pH 7 0 phosphate buffer
containing 20 % sucrose and frozen in the same way as the wings. Serial sections
(20/im) were cut in a cryostat. When dry, the sections were immersed in 1 %
cobalt chloride in 0-1 M-Tris buffer (pH7-4) for 15 min, washed in Tris buffer for
5 min, then in two 5-min changes of pH 7-4 phosphate buffer. The sections were
immersed in a 0-05 % solution of diaminobenzidine (Sigma) in pH 7-4 phosphate
buffer for 15 min after which H2O2 was added to a concentration of 0-01 % and
incubation continued for a further 15 min. The sections were then rinsed,
dehydrated, cleared and mounted. Labelled motoneurones whose perimeters
could be clearly distinguished were drawn at a magnification of x 900 with the aid
An avian fast/slow muscle pair
55
r
1A
B
Fig. 1. A: low and B: high-magnification photographs of the wrist region containing
the ulnimetacarpalis dorsalis stained for acetylcholinesterase. The superficial head
has many choJinesterase deposits throughout its length. The deep head has, in this
case, two bands of cholinesterase deposits, E18: st44. s: superficial head, d: deep
head. Bar = 1 mm in A, Bar = 0-5mm in B.
56
N. G. LAING AND A. H. LAMB
of a camera lucida and the area of the cells calculated using a MOP-1 image
analyser (Carl Zeiss).
RESULTS
Muscle histochemistry
The umd was first noticed as a likely fast/slow muscle pair in the whole-mount
ACh.E preparation at E18: st44 (Fig. 1). The superficial head of the muscle had
many end plates throughout its length whereas the deep head had a small number
of bands of larger end plates. Similar staining patterns are found in the slow ALD
and fast PLD respectively (Ginsborg & Mackay, 1961). At E l l (st37-38) the
pattern of end plates was the same as at E18 (st44). At E10 (st36) no end-plate
staining could be obtained in the fast head though the slow head showed a similar
pattern to that at E18.
Dissection of the muscle revealed that the deep head was larger than it appeared because the main bulk of the muscle was deep to the superficial head. This
anatomical relationship may clearly be seen in the transverse sections through
the umd of E18 embryos stained for ATPase (Fig. 21, J). The great majority of
the muscle fibres of the superficial head stained darkly both after acid (pH4-3)
and alkali (pH9-4) preincubation, but the muscle fibres of the deep head stain
darkly only after alkali preincubation. The muscle fibres of the superficial head
are thus acid stable and those of the deep head acid labile. The acid-stable and
acid-labile characteristics correspond respectively with the properties of the
embryonic ALD and PLD (Toutant, Toutant, Renaud & LeDouarin, 1979).
Thus the superficial head of the umd is a slow muscle and the deep head a fast
muscle as judged by ATPase staining. A finding of unknown significance was
that, in most cases (e.g. Fig. 2J), a few muscle fibres of opposite type from the
majority were found in each head in a distribution that varied from one embryo
to another.
The fast and slow nature of the two heads was confirmed by the end-plate
distributions on teased muscle fibres. One-hundred muscle fibres with one or
more end plates were teased from each head of the muscle in five E18 embryos.
Of the 500 fragments from the deep heads 488 (97-6 %) had only one end plate,
11 (2-2%) had two, and one had seven (0-2%), On the other hand only 101
(20-2 %) of the fragments from the superficial head had one end plate, 131
Fig. 2. ATPase staining of transverse sections through the umd at various ages.
Serial sections were cut, the sections depicted at each age are close to each other, but
not necessarily adjacent. Acid-stable fibres are dark at pH4-3 and acid-labile fibres
are pale. In all cases where a fast head is present the sections are from the point at
which the fast head appeared largest. A, B E7 (st32-33); C, D E8 (st33-34); E, F
E9 (st35); G, H E12 (st39) and I, J E18 (st44). In all cases scale bar = 0-2 mm. Left
figure (A, C, E, G & I): pH9-4, right figure (B, D, F, H & J): pH4-3. s: slow head,
f: fast head.
An avian fast/slow muscle pair
pH4-3
pH 9-4
2A
57
B
D
p
V^4^--
\
58
N. G. LAING AND A. H.
LAMB
(26-2 %) had two, and 268 (53-6 %) had more than two. Thus the majority of the
fragments from the superficial head were multiply innervated (80 ± 9 % for the
five embryos) while the majority of the fragments from the deep head were singly
innervated (98 ± 2 %, n = 5).
The longest single fragments obtained from the slow head were over 2 mm long
and thus approaching the total length of the muscle. There was a positive correlation between the length of a muscle fragment and the number of end plates upon
it (Fig. 3) up to the maximum number of end plates seen on one fragment which
was nineteen. The mean distance between the end plates on the fragments from
the slow head was 110 ± 57 fxm (S.D., range 14-490^m, n = 1325) (Fig. 4). The
muscle fibres of the fast head were smaller in diameter than those of the slow
100 i
<500jum
fast (n = 464)
slow (n = 340)
10
1001
15
^
^
500-1000 /xm
fast (n = 36)
slow (n = 125)
5
10
15
Number of end plates per fragment
20
Fig. 3. Comparison of the length of a muscle fibre fragment and the number of end
plates upon it for both fast and slow heads of the umd at E18. (A) fragment length
<500,um; (B) 500-1000 jum; (C) 1000-1500 fim and (D) >1500Jum.
An avian fast/slow muscle pair
59
head at all later stages (Fig. 2) and were therefore more difficult to tease apart.
The longest fragments obtained from the fast head were nevertheless over half
the length of the muscle at around 1 mm long.
ATPase staining at various ages revealed the development of the two heads of
the muscle (Fig. 2). At E7 (st32-33) the muscle was one homogeneous mass with
acid-stable fibres present (Fig. 2A, B). At E8 (st33-34) there were the first
indications of two heads (Fig. 2C, D), and at E9 (st35) the presence of two heads
was clear (Fig. 2E, F). At these early stages the fast (acid-labile) head was
proportionately much smaller than at more mature stages but its appearance was
approaching that found in E18 embryos by E12: st39 (Fig. 2G, H). The fast head
was shorter than the slow head at all stages and at the earliest stages was present
in very few sections.
HRP labelling of motoneurons
The fast and slow heads of the umd could easily be distinguished at E17 in ovo
with the aid of the dissecting microscope. The division into the two heads visible
in Fig. IB was clear following HRP injection since the brown discolouration
total n = 1325
100
200
300
400
500
Distance between end plates
Fig. 4. Distribution of inter end-plate distances on single muscle fibre fragments
teased from the slow head of the umd at E18.
60
N. G. LAING AND A. H. LAMB
Fig. 5. HRP-labelled cells (arrows) in the brachial spinal cord after injection of the
slow (A) and fast (B) heads of the umd in E18 embryos, w: white matter, g: grey
matter, *: examples of red-blood corpuscles. Bar = 20jUm.
An avian fast/slow muscle pair
61
caused by the HRP would reach, but not cross, the divide, leaving one muscle
head brown and the other white.
Labelled motoneurons were found in the lateral part of the lateral motor
column of spinal segments 15 and 16 after injection of both heads (Fig. 5). The
positions of all labelled motoneurons found were plotted with a camera lucida.
There was no observable difference in the mediolateral positions of the
motoneuron pools to the slow and fast heads (Fig. 6), nor could any difference
be distinguished between the pools in the rostrocaudal axis (Fig. 7). There was
a tendency for segment 15 to contain more labelled motoneurons after injections
into the fast head 65-7 % ± 14-5 % (s.n.) (n = 7), than after injections into the
slow head, 51-8% ±28-8% (n = 4), but the difference was not significant
(Mann-Whitney U-test). This tendency, similar to that reported by De Santis,
Hoekman & Limwongse (1977), arose in our study through variations in the
position of the boundary between segments 15 and 16. The boundary was defined
as the section midway between the caudal-most section containing ventral root
15 and the rostral-most section containing ventral root 16 (Hamburger, 1958).
The boundary was about two thirds of the way from the rostral end of the lateral
motor column, the point where most labelled cells were found. Thus, a slight
variation in the position of the boundary markedly altered the ratio of
motoneurons in the two segments.
The mean areas of the somas of the 'slow' motoneurons was 294-5 //m2 (n = 87,
S.D. 97-4jum2) and the area of the 'fast' motoneurons 376-6/im2 (n = 84, S.D.
Fast
Slow
S15
S16
Fig. 6. Composite diagram showing the positions of all the labelled motoneurons
pooled from all embryos. The position of each labelled cell was drawn by lining up
the sections from different embryos using the central canal and the edge of the white
matter as markers (Landmesser, 1978). A: Motoneurons to the slow head (102
motoneurons in four embryos). B: Motoneurons to the fast head (103 motoneurons
in seven embryos). S15: segment 15, S16: segment 16.
EMB78
62
N. G. LAING AND A. H . LAMB
A
slow head
10
8
js>
6
1 4
2 2
20
10
30
40
50
60
70
90
100
B
fast head
Fl n
10
rostral
20
30
40
50
60
Position in LMC
70
80
90 100
caudal
Fig. 7. Distributions of the HRP-labelled cells in Fig. 6 in the rostrocaudal axis.
Results were pooled from all embryos. The distributions in the rostrocaudal axis
were normalized by dividing the lateral motor column (LMC) into 100 equal bins.
The section before the most rostral section containing LMC cells was taken as zero
and the caudalmost section containing LMC cells as 100. A: Motoneurons to the slow
head. B: Motoneurons to the fast head.
20-
10-
/ .......
'f
//////
7/ i
.low head motoneurons
I"ast head motoneurons
/ /
' i i
-
7A
77Wj77!
'//
—i
100
200
300
400
500
600
Motoneuron soma area (jum2)
700
>
800
Fig. 8. Distribution of soma sizes for motoneurons to slow and fast heads of the
ulnimetacarpalis dorsalis. The motoneuron sizes were grouped in bins of 50 jum2 and
the number in each bin expressed as a percentage of the total number of
motoneurons labelled from that head of the muscle.
117-9/im2). The difference was statistically significant, (P< 0-001, t-test). Plotting the sizes of the cells (Fig. 8) revealed little sign of a bimodal distribution
amongst the motoneurons innervating the slow head and only slight indications
of a bimodal distribution for those innervating the fast head.
An avian fast/slow muscle pair
63
DISCUSSION
The superficial and deep heads of the ulnimetacarpalis dorsalis have been
shown to be slow and fast respectively by the histological criteria of myosin
ATPase staining and of gross and individual fibre ACh.E staining. It is interesting that a difference could be seen between the two heads with ATPase staining
at E8, prior to the completion of their cleavage which, according to Sullivan
(1962), occurs between E10 and E12. In fact the differential staining is the first
evidence of there being two heads. Toutant etal. (1979) noted differential staining of ALD and PLD as early as E8 and Butler & Cosmos (1981) noted that the
ALD and PLD were distinguishable with ATPase staining at E6 (st29) which is
prior to the completion of their cleavage. This very early differentiation of fast
and slow muscles by ATPase staining may be contrasted with the fact that differences in muscle contraction speed do not arise until some time between E15
and E18 (Gordon & Vrbova, 1975). The same may be said for the kitten where
hindlimb muscle fibres may be differentiated by ATPase at birth (Nystrom,
1968), but not by electrophysiology (Buller, Eccles & Eccles, 1960). The larger
size of the acid-stable fibres of the superficial head corresponds with observations
on the ALD and PLD (Gordon, Perry, Tuffery & Vrbova, 1974; Toutant et al.
1979).
The results obtained with ACh.E staining are similar to findings in other
muscles. End-plate staining can be obtained in calf muscles from E12-E13
(Drachman, 1963) and in the ALD and PLD from E l l onwards (Bennett &
Pettigrew, 1974). The mean distance between end plates in the slow head is, at
110±57/im (S.D.), somewhat less than figures published for the ALD after
various procedures. Burden (1977), using radioactive obungarotoxin found an
inter end-plate distance of 223 ± 60/^m (S.D.) at E19. Using ACh.E, Bennett &
Pettigrew (1974) found an inter end-plate distance of 164±8/um (S.E.M.) at
E l l - 1 3 , Pittman & Oppenheim (1979) 158 ± 74[xm (S.D.) at E17, 163 ± 78/mi
at E18, Gordon etal. (1974) 133 ± 4 fim (S.E.M.) at E21 and Khaskiye etal. (1980)
128±4/im (S.E.M.) at E16. The distance between end plates on slow fibres
increases with the age of the embryo (Bennett & Pettigrew, 1974). Thus the
difference in results between umd and ALD may reflect differences in technique
or that umd development lags behind ALD development (distal wing muscles
complete cleavage later than proximal muscles, Sullivan, 1962), or a real difference between the muscle fibres of umd and ALD.
The motoneuron projection patterns to the umd are consistent with earlier
studies. The innervation of umd by spinal segments 15 and 16 corroborates the
report by Bennett, Lindeman & Pettigrew (1979) that the forearm muscles,
flexor carpi ulnaris and flexor digitorum profundus are innervated by those segments. The umd is derived from the dorsal muscle mass (Sullivan, 1962) and its
innervation by motoneurons lying laterally in the motor column is consistent with
other reports of topographical correspondence between the mediolateral axis of
64
N. G. LAING AND A. H. LAMB
the cord and the ventrodorsal axis of the limb (Lamb, 1976; Landmesser, 1978).
Considering the close approximation of the fast and slow heads of umd it is not
surprising that their motor pools overlap almost completely. De Santis et al.
(1977) noted only a slight difference in the positions of the ALD and PLD
motoneuron pools and they attributed this to the separate origins of the two
muscles.
The tendency for motoneurons to the fast head of umd to be slightly larger
than those to the slow head is similar to that reported in the cat (Burke et al.
1977). However, in our study we did not find such a marked bimodal distribution,
perhaps because our measurements were taken from embryos before adult size
distributions were established (Hollyday & Hamburger, 1976). Another
possibility is that some of the intrafusal fibres of avians are innervated by axon
collaterals of large motoneurons (Barker, 1968; Dorward, 1970), thus reducing
the number of small motoneurons present in the avian spinal cord.
Our results show that the fast and slow heads of the umd do not have mature
proportions until E12. This confirms the findings of other workers (Romer, 1927;
Wortham, 1948; Sullivan, 1962), that the development of distal wing muscles
lags behind muscle development in other regions and it may help to explain the
relatively prolonged time course of naturally occurring motoneuron death in the
brachial cord (Oppenheim & Majors-Willard, 1978; Laing, 1982).
In the following paper we examine the effect of transplanting the wing bud to
the lumbar region on the development of muscle fibre types in the umd and
forearm muscles.
Throughout these experiments we received excellent technical assistance from Jane Eccleston and Jane Diggins. The work was supported by the Australian National Health and Medical
Research Council and the Muscular Dystrophy Research Association of W.A. We would like
to thank Mr Philip Sheard for criticizing the manuscript.
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