Download The distribution of muscle fibre types in chick embryo wings

J. Embryol. exp. Morph. 78, 67-82 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
The distribution of muscle fibre types in chick
embryo wings transplanted to the pelvic region is
normal
By N. G. LAING 1 AND A. H. LAMB 1
From the Department of Pathology, University of Western Australia
SUMMARY
Chick embryo wing buds were transplanted to the pelvic region in place of, or in addition to,
the hindlimb bud prior to innervation. The wrist muscle ulnimetacarpalis dorsalis (umd) wa$ innervated by middle-dorsal or middle-ventral motoneurons in the lumbar lateral motor column
(LMC) in a rostrocaudal position which varied with the rostrocaudal position of the wing. Despite the heterotopic innervation the subsequent development of the distributions of fast and
slow muscle fibres, as judged by ATPase staining, was normal in all muscles examined. The
pattern of innervation in the umd, as judged by acetylcholinesterase staining also developed
normally. It is probable that muscle fibre type is intrinsically, not neurogenically, determined.
INTRODUCTION
In the previous paper we observed that muscle fibre types differentiate early
in the development of the wrist muscle ulnimetacarpalis dorsalis (umd). In this
paper we set out to investigate the factors which determine muscle fibre type.
The prevailing hypothesis is that the muscle fibres have their type imposed by the
innervating nerve (Buller, Eccles & Eccles, 1960; Bennett & Pettigrew, 1974a).
The only experimental evidence for the hypothesis is from studies of cross innervation by foreign nerves in juveniles and adults (e.g. Bennett & Pettigrew,
1974a) when the nerves have already been in contact with muscle. This contact
may itself have specified the motoneurones. We therefore decided to carry out
a cross innervation study in which the nerves had not previously contacted
muscles. Wings were transplanted to the pelvic region of chick embryos at E3,
which is before axons have grown into the limbs (Oppenheim & Heaton, 1975;
Roncali, 1970). The distribution of muscle fibre types was found to be remarkably similar to that in control wings. Some of the results have previously been
reported in brief (Laing & Lamb, 1982).
METHODS
Fertile eggs from a local hatchery were incubated in a humidified forceddraught incubator at 38°C. At stages 18-19 (Hamburger & Hamilton, 1951),
1
Authors' address: Department of Pathology, University of Western Australia, Queen
Elizabeth II Medical Centre, Nedlands, 6009, Perth, Western Australia.
68
N. G. LAING AND A. H. LAMB
which were found to be the best stages for operations, the embryo was exposed
and the right wing bud removed by cutting with electrolytically sharpened tungsten needles as close as possible to the posterior cardinal vein. One of two
experimental regimes was followed. In one, the right leg bud was removed in a
similar manner to the wing bud and replaced by the wing bud. In the other, the
wing bud was pushed into a slit made between the hindlimb bud and the vein to
produce a supernumerary wing (Hamburger, 1939). The hole in the shell was
sealed with Sellotape and the egg replaced in the incubator. At 17 days of
incubation (E17) some of the embryos were re-exposed and horseradish
peroxidase (HRP) injected into the umd to label the motoneuron pool. All
embryos were fixed at E18 and processed for HRP histochemistry and/or
ATPase and acetylcholinesterase (ACh.E) histochemistry. All the methods
were described in detail in the previous paper (Laing & Lamb, 1983).
RESULTS
Several outcomes of the operation were observed. In many cases the wing was
absent. When present it was often internalized and growing in the coelomic
cavity, or it was highly abnormal. Well-formed wings were found in 71 % of
surviving embryos (59% of operated embryos died prior to E17/E18). However, even in the well-formed wings there was often a complete or partial absence
of musculature particularly distal to the elbow or wrist. The variations in the
amount of muscle may relate to variations in the innervation of such grafts as
observed by Hamburger (1939). He found grafts were best innervated when they
arose dorsally close to the vertebral column. Movements of the thumb (digit 2:
Sullivan, 1962) in ovo were a good indication of a well-muscled limb. Such a limb
stained for ACh.E is shown in Fig. 1.
Acetylcholinesterase staining
The pattern of end plates in the wrist muscle ulnimetacarpalis dorsalis {umd)
as displayed by ACh.E staining was the same in the displaced wings as on the
contralateral control side (Fig. 2). ACh.E spots were distributed throughout the
length of the slow head. The fast head usually had one or two bands of end plates.
The distributions of end plates on individual muscle fibres confirmed the differences apparent in whole mounts. In five embryos 100 ACh.E-stained muscle
fibres were teased from both the fast and slow heads of the umd of control and
displaced wings. The majority of muscle fibres teased from the slow head of both
control and operated umds had multiple end plates (76 ± 5 % and 72 ± 8 %
respectively, n = 5 in both cases). The majority of fibres in the fast heads had a
single end plate (99 ± 1 % and 98 ± 1 %, n = 5). The values for the operated,
control and normal wings are not significantly different (P > 0-1, Mann-Whitney
U-test) (see Laing & Lamb, 1983 for data on normal wings). In the slow heads
Muscle fibre types in transplanted wings
69
1
Fig. 1. Whole-mount preparation of a hind limb from an E18 embryo with a supernumerary wing stained for ACh.E. Bar = 5 mm.
there was a relationship between the length of a teased muscle fibre fragment and
the number of end plates upon it (Fig. 3). The distributions of values for three
arbitrarily chosen ranges of fragment lengths were similar in the control and
operated muscles. The slow head tended to be shorter in the displaced wings than
in the control wings (Table 1) and thus there were fewer long fragments in the
operated sample. Such variations within the subdivisions of lengths account for
the slight variations between the control and operated samples. In the control
and operated fast heads the distributions of muscle fibres with single and multiple
end plates were almost identical (Fig. 4). There was no sign of a 'half-way' state
with approximately 50 % of the muscle fibre fragments singly and 50 % multiply
innervated in either of the 'operated' umd heads.
70
N . G. LAING AND A. H. LAMB
2A
Fig. 2. Comparison of the umd muscles in normal (A) and displaced (B) wings from
E18 embryos stained for ACh.E. Bar = 0-5 mm.
There was no significant difference (P>0-05, Kolmogorov-Smirnov twosample test) in the inter end-plate distances in the slow head of the umds of
control and displaced wings (Fig. 5).
ATPase staining
Transverse sections cut through the forearm of E18 embryos and stained for
ATPase at pH 4-3 revealed the pattern of acid-labile (pale) and acid-stable (dark)
fibres within the whole forearm musculature. Most of the muscles displayed a mixture of pale and dark fibres. The distribution of the mixture was characteristic of
each muscle and was retained in the displaced wings. Sections taken through the
mid forearm in both control and displaced wings show how accurately the pattern
is conserved (Fig. 6). The supinator is closely applied to the radius and its dorsal
part has many dark fibres whereas its ventral part is composed entirely of pale
fibres. The pronator sublimis has very few dark fibres whereas its neighbour the
pronatorprofundus has many. The extensor metacarpi radialis has few dark fibres
Muscle fibre types in transplanted wings
71
control (n = 337)
operated (n = 349)
"rrrrr:
3020109
10
B 40-
3
2
"3
20-1
*
10H
11
12
13
control (n = 121)
operated (n = 137)
30-
1 2
3
4
5
6
9
10
11 12
13
- control (n = 42)
. operated (n = 14)
C 403020101
2
3
4 5 6 7 8
9 10 11 12
Number of end plates per fragment
13
Fig. 3. Comparison of the length of an E18 muscle fibre fragment and the number
of end plates upon it for the slow head of the umd in displaced and control wings. A:
fragment length less than 500[xm, B: 500-1000(im and C: greater than 1000jum.
Table 1. Length of umd muscle heads in control and displaced wings
Slow heads
A)
Embryo Control
1
2
3
4
5
Operated Percentage
(m)
(jmi)
2029
2206
2265
2324
1765
1735
1471
1706
1794
1324
Fast heads
B)
86%
67%
75%
77%
75%
Embryo Control
1
2
3
4
5
Operated Percentage
(jum)
(jum)
1029
950
857
909
971
971
900
824
771
1152
94%
95%
96%
85%
119%
72
N. G. LAING AND A. H. LAMB
A 100-
B 100501-1000 fan
^ 5 0 0 jum
90-
90-
—control (n = 490)
... operated (n = 470)
30-
30-
20-
20-
10-
control (n = 10)
operated (n = 30)
10-
0
0
Number of end plates per fragment
Fig. 4. Comparison of the length of a muscle fibre fragment and the number of end
plates upon it for the fast heads in displaced and control wings. A: fragment length
less than 500^m, (control and operated histograms were identical). B: fragment
length 500-1000jum. Data from the same E18 embryos as in Fig. 3.
2220control (n = 1144)
operated (n = 873)
1816•a 1 4 -
12-
642-
100
200
300
400
500
Distance between end plates
600
Fig. 5. Distribution of inter end plate distances on single muscle fibre fragments
teased from the slow heads of displaced and control wings. Data from the same E18
embryos as Fig. 3.
Muscle fibre types in transplanted wings
73
D
emr
6A
B
Fig. 6. Distribution of muscle fibre types as shown by ATPase staining at pH4-3:
slow fibres are dark. Transverse sections cut through the mid-forearm of E18 embryos. A: control wing, B: displaced wing, emr: extensor metacarpi radialis, FCU:
flexor carpi ulnaris, pp: pronator profundus, ps: pronator sublimis, s: supinator, r:
radius, u: ulna, D: dorsal, V: ventral. Bar = 0-5 mm.
74
N.
G. LAING AND A. H. LAMB
*
*
*
'
•
..VV".'-'••"-'-•WRi
7A
f
>•"•*-•
.'• ^i^'* /I—
B
Fig. 7. Distribution of muscle fibre types as shown by ATPase staining at pH4-3
(slow fibres are dark). Transverse sections through E18 forearms near elbow. A:
control wing. B: displaced wing, b: brachialis, r. radius, u: ulna. Bar = 0-5 mm.
Muscle fibre types in transplanted wings
pH 9-4
pH 4-3
- v
* •>
Fig. 8. ATPase staining of transverse sections of the umd. A: control, B-E various
operated umds showing degrees of loss of the fast head. F: fast head, S: slow head.
Bar = 0-5 mm.
75
76
N. G. LAING AND A. H. LAMB
Fig. 9. HRP-labelled motoneurons (arrows) in the lumbar spinal cord following
injection of the slow head of the umd in displaced wings. Dark-field illumination.
Bar = 0 1 mm.
Muscle fibre types in transplanted wings
11
and the flexor carpi ulnaris has a pattern of dark fibres at one end with very few
at the other as it twists round on itself. Sections taken nearer the elbow show the
brachialis to be composed almost exclusively of dark fibres in both control and
displaced wings (Fig. 7). In confirmation of the findings with ACh.E staining, the
fast and slow heads of the umd are easily recognizable after ATPase staining of
the displaced wings (Fig. 8). An interesting finding depicted in Fig. 8 was that the
fast head was in most cases smaller than on the control side and sometimes
entirely absent. This is presumably a mild manifestation of the process which
sometimes results in no muscle distal to the elbow. It is perhaps significant that
it is the later developing fast head (Laing & Lamb, 1983) which is affected. The
loss of the fast head was not related to the type of graft. A variable number of
pale fibres appeared in the predominantly dark slow head, the number often
being elevated compared to the control side. Also, a variable number of dark
fibres appeared in the fast head. If there were no dark fibres in the control muscle
there tended to be none in the displaced muscle and if there were a few in the
control muscle there were also a few in the displaced muscle.
N=4
n = 34
N=4
n = 15
Fig. 10. Composite diagrams showing the position of all labelled motoneurons obtained from each of the four types of displaced wings. A: wing replacing leg, B: wing
rostral to leg, C: wing dorsal to and level with leg, D: wing caudal to leg. The patterns
were consistent from embryo to embryo. The position of each labelled cell was drawn
by lining up the different sections using the central canal and the edges of the white
and grey matter as markers. N: number of embryos, n: number of cells.
78
N . G. LAING AND A. H. LAMB
A 101
8-
N =4
n = 34
64200
B 10-
10
20
30
40
50
60
70
80
90 100
8-
N=4
n = 15
64200
10
20
30
40
50
60
70
80
90 100
= 24
0
10
20
30
40
50
60
70
80
90
100
10
20
30
40
50
60
70
80
90
100
D 10-1
0
Fig. 11. Distribution of the labelled cells in the rostrocaudal axis. Different embryos
normalized as in the previous paper (Laing & Lamb, 1983). A: wing replacing leg,
B: wing rostral to leg, C: wing dorsal to and level with leg, D: wing caudal to leg. N:
number of embryos, n: number of cells.
Muscle fibre types in transplanted wings
79
Horseradish peroxidase labelling of motoneurons
The slow head of the umd was chosen for HRP injection because of its superficial position and because the motoneuron pools to the two heads are indistinguishable in normal embryos (Laing & Lamb, 1983). The position of labelled
motoneurons in the mediolateral axis of the lumbar lateral motor column (LMC)
varied considerably, but tended to be middle dorsal or middle ventral (Figs 9 and
10). These positions correspond to those of the motor pools to the extensor
hallucis brevis and extensor digitorum brevis (Hollyday, 1980). Such correlations
in the mediolateral axis are, however, of limited value because the motoneurons
surviving after grafting may not be the ones which survive normal motoneuron
death, (e.g. all the normal medial motoneurons may die after grafting so that the
most medial motoneurons seen would be lateral in a normal embryo). In the
rostrocaudal axis, the position of the labelled cells depended to a large extent on
the position of the grafted wing. With the wing replacing the leg, or lying caudal
or dorsal supernumerary to the leg, the rostral end of the motoneuron pool was
around 40 % of the length of the LMC from its rostral end (Fig. 11). In ten such
embryos none of the 152 labelled cells were found further rostrally. This position
corresponds to the position of the pools to the short extensors in the hind limb
(Hollyday, 1980). On the other hand, in each of four embryos in which the wing
was rostral to the leg, cells were found rostral to the 40% mark (Fig. 11).
Nevertheless, the fact that they were in an intermediate position in the
mediolateral axis is consistent with previous observations, that muscles derived
from the dorsal muscle mass are always innervated by intermediate or lateral
LMC motoneurons (Hollyday, 1981).
DISCUSSION
The distributions of muscle fibre types were remarkably unchanged in wings
innervated by lumbar motoneurons. There are a number of possible explanations for this. Axonal guidance within the limb may be under such precise control
that the appropriate types of axons are always led to correct regions of the limb,
corresponding to the observed patterns of fast and slow fibres. In this way it
would still be possible for fibre type to be determined by the innervating axon
(neurogenically determined). However, such an explanation places extraordinary demands on the mechanism of axon guidance. This is especially true in view
of the normal pattern even when the wing muscles received innervation from
regions of the spinal cord which do not normally innervate homologous regions
of the hind limb (e.g. umd innervated by rostral motoneurons). Neurogenic
determination is also unlikely following the recent work of Butler, Cosmos &
Brierley (1982) on the development of fibre types in aneural wings. They found
that despite the lack of innervation, the ATPase patterns of the wing were the
same as normal for similar stages of development.
80
N. G. LAING AND A. H. LAMB
The probability that muscle fibres are intrinsically determined raises some
interesting questions. How do the motoneurons become correctly matched with
the appropriate muscle fibres? This depends on whether or not the motoneurons
are themselves intrinsically determined. There is some evidence for intrinsic
determination of the spatial type of motoneurons in that axons from
motoneurons in certain regions of the spinal cord preferentially grow towards
certain regions of the limb (Lance-Jones & Landmesser, 1981) and certain
motoneurons appear to be incompatible with certain limb regions (Lamb, 1979,
1981). However, it is not known whether motoneurons are intrinsically determined for fast/slow type. If they are, then it would be easy to envisage a simple
matching process whereby motoneurons can only make or retain contact with
the appropriate fibre type. If they are not, it would be necessary for the
motoneuron to be specified by the muscle fibres it contacts. However, at this
stage of development chick embryo muscle fibres are multiply innervated
(Srihari & Vrbova, 1978; Pettigrew, Lindeman & Bennett, 1979; Pockett, 1981),
begging the question of whether single motoneurons contact more than one
muscle fibre type.
Our results are at variance in some respects with a recent study by Khaskiye
et al. (1980), who transplanted lumbar spinal cord in place of brachial cord prior
to limb innervation. They found that the resulting patterns of end-plate
distributions, as revealed by ACh.E staining, in the anterior and posterior
latissimus dorsi muscles (ALD and PLD) were altered, the main change being
a shift towards distributed innervation in the normally focally innervated PLD.
However, the ATPase patterns were close to normal. Thus, there was a dissociation of ATPase typing from the cholinesterase patterns. A similar dissociation
is seen in muscles of embryos paralysed by neuromuscular blockade (Laing,
1982a and unpublished observations) which suggests there may be a functional
reason for the discrepancy between the effects of cord and limb transplants.
Nevertheless, the fact that the ATPase patterns are unchanged in either
procedure adds further support to intrinsic muscle fibre type determination.
If motoneurons are specified for fast/slow properties, the unchanged patterns
of muscle fibre types in wings innervated by lumbar motoneurons indicate that
fast/slow properties are important in matching motoneuron and muscle fibre.
This means that the 'hierarchy of neuronal specificities' sometimes hypothesized
to account for the formation of neuromuscular connections (e.g. Hollyday, 1981)
must be extended to include fast/slow properties.
The formation of the patterns of muscle fibre types may provide an explanation for the puzzling phenomenon of motoneuron death in which approximately
50 % of embryonic motoneurons die (Prestige, 1967; Hamburger, 1975; Oppenheim & Majors-Willard, 1978; Laing, 1982/?; Lance-Jones, 1982). If
motoneurons and muscle fibres are both intrinsically specified, then motoneuron
death might be acting to correct any imbalances between the proportions of fast
and slow motoneurons innervating a muscle and the proportions of fast and slow
Muscle fibre types in transplanted wings
$1
muscle fibres. Studies of the proportions of axons initially invading each muscle
may solve this question.
Throughout these experiments we received excellent technical assistance from Jane Eccleston, Jane Diggins and Janine van Noort. The work was supported by the National Health and
Medical Research Council of Australia and the Muscular Dystrophy Research Association of
Western Australia. We would like to thank Philip Sheard for his comments on the manuscript.
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