J. Embryol. exp. Morph. 96, 79-97 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
79
Embryonic differentiation of fibre types in normal,
paralysed and aneural avian superior oblique muscle
G. S. SOHAL AND D. W. SICKLES
Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912, USA
SUMMARY
•
The influence of innervation on the initial differentiation of muscle fibre types was investigated by using the trochlear nucleus-superior oblique muscle system of duck. The adult
muscle is composed of three types of fibres (designated as type I, II, III) as identified with the
histochemical techniques for ATPase pH sensitivity. Type I fibre ATPase activity was acidstable, alkali-labile; type II alkali-stable, acid-labile; and type III both acid- and alkali-stable.
These types showed variable mitochondrial ar-glycerophosphatase dehydrogenase, nicotinamide
adenine dinucleotide tetrazolium reductase, and phosphorylase activity. Type I and IIfibresare
primarily located in the portion of the muscle adjacent to the orbit whereas the rest of the muscle
is primarily composed of type III fibres. In the normally developing muscle, type II and III fibres
are present as early as embryonic day 9; one day prior to the arrival of nervefibresin the muscle.
Type Ifibresarefirstobserved on embryonic day 17. On day 22 the percentages of type I, II and
III fibres are 29, 53 and 18, respectively. As the development progressed the percentages of type
I and II fibres decrease and after hatching 76 % of the fibres belong to type III, 17 % to type II
and only 7 % to type I. In embryos paralysed with daily application of 3 mg d-tubocurarine
(d-TC) from day 9 onwards the differentiation of type II and III fibres occurs, but type I fibres
were never observed in the paralysed muscles. These muscles also contained significantly fewer
myotubes than the normal muscle. By contrast, when the muscle was made aneural by
permanent destruction of motor neurones on embryonic day 7 all three types of fibres differentiated. When embryos with aneural muscles were also subjected to d-TC treatment the type I
fibres failed to differentiate. It is concluded that the initial differentiation of fibre types is
independent of innervation and that primary myotubes are capable of differentiating into all
three types of fibres. The absence of type I fibres in curarized muscles may be due to some
unique effect of d-TC on the muscle itself.
INTRODUCTION
Two main types of muscle fibres, twitch and tonic, can be distinguished in the
adult skeletal muscle of lower vertebrates on the basis of their morphology,
metabolism, pattern of innervation and contractile speed (Kuffler & VaughanWilliams, 1953; Couteaux, 1955; Ginsborg & Mackay, 1961; Hess, 1961, 1970;
Page, 1969). Twitch and tonic fibres can be distinguished histochemically at the
light microscopic level according to their myosin-ATPase staining patterns (Guth
& Samaha, 1969). In slow twitch muscle fibres (type I) the ATPase activity is acidstable and alkali-labile (Barnard, Lyles & Pizzey, 1982). In contrast, the fast twitch
Key words: musclefibretypes, innervation, avian muscle, adenosine triphosphate (ATP).
80
G. S. SOHAL AND D. W. SICKLES
fibres have alkali-stable and acid-labile ATPase activity (type II). In the slow tonic
(type III) fibres the ATPase activity is resistant to both acid and alkali treatment.
The role of motor nerve fibres in regulating the slow and fast muscle fibres has
been investigated in newly hatched chicken muscle (Jirmanova, Hnik & Zelena,
1971; Bennett & Pettigrew, 1974; Hnik, Jirmanova & Syrovy, 1977; Cosmos,
Butler, Allard & Mazliah, 1979). These studies indicate that the muscle fibres
become slow or fast as a result of the type of nerve fibres innervating them. This
point has been illustrated by cross reinnervation of slow tonic and fast twitch
muscles. For example, Gordon and colleagues (Gordon, Perry, Srihari & Vrbova,
1971; Gordon & Vrbova, 1975) removed one anterior latissimus dorsi (ALD) and
one posterior latissimus dorsi (PLD) muscle, minced each, and switched their
positions before placing them back. This arrangement resulted in reinnervation
of the minced (regenerating) ALD muscle by the nerve of the PLD muscle and
vice versa. After regeneration and reinnervation, the ALD muscle fibres acquired
characteristics of those normally seen in the fast fibres (PLD) and PLD became the
slow muscle. In other words, the muscle fibre types transformed as a result of the
nerve impulse activity, trophic substances or because of their new location. When
a slow twitch muscle in its normal location is innervated by a nerve from the fast
muscle it acquires properties characteristic of the fast muscle (Buller, Eccles &
Eccles, 1960; Mommaerts et al. 1977). Likewise, when the nerve to a fast muscle is
artificially stimulated at low frequency, the muscle becomes slow (Salmons &
Sreter, 1976). This type of electrical stimulation of nerve induces synthesis of slow
myosin in fast fibres (Pette & Schnez, 1977; Rubinstein et al. 1978). These
experiments suggest that the expression of slow and fast muscle fibres in the
mature muscle depends on the impulse activity of the innervating motor neurones.
The objective of the present study was to investigate whether the initial
differentiation of fibre types, as detected by myosin-ATPase staining, during the
course of embryonic development is also regulated by the motor neurones. The
superior oblique muscle in duck embryo was used as a model to study fibre typing
for several reasons. The extraocular muscles contain a mixture of fibre types
(Maier, Eldred & Edgerton, 1972) which provides a unique situation to examine
the effects of various experimental manipulations on the differentiation of all three
fibre types in the same muscle. This muscle receives its innervation solely from a
relatively small neurone pool, the trochlear nucleus. The trochlear nucleus is
located dorsally in the midbrain which provides easy access for microsurgical
destruction of innervation during development. Since eyes are usually visible
through the egg shell window, the eye movements can be used as a rough index to
monitor the effectiveness of neuromuscular transmission blockade or loss of
innervation. Finally, a good deal of information on the development of connections between the trochlear nucleus and the superior oblique muscle already
exists for the duck embryo (Sohal et al. 1985). In this report we describe the type
of muscle fibres found in the adult superior oblique muscle and compare the
differentiation of fibre types between the muscles developing normally with those
either paralysed or developing in the absence of innervation.
Musclefibretyping in development
81
MATERIALS AND METHODS
Animals and paralysis
Eggs of White Peking duck were incubated in a force-draft incubator at 37-5°C and in a
humidity of approximately 80 %. On the third day of incubation an opening in the shell of all
eggs was made with a sander to acquire access to the embryo for application of curare, for
microsurgery and for direct visual observations. Embryos were paralysed with daily application
of 3mg d-tubocurarine (d-TC, Sigma) in 0-1 ml saline from day 9 onwards. This is two days
before the neuromuscular transmission begins in the trochlear nucleus-superior oblique muscle
as determined electrophysiologically (Stoney & Sohal, 1978). d-TC solution was directly
dropped onto the vascularized chorioallantoic membrane through the egg-shell window.
Previous studies on embryonic motility have shown that this dosage of d-TC is sufficient to
abolish all visible movements of the limb and eye (Sohal & Wrenn, 1984). A total of 38 embryos
was paralysed and the results are based on 26 embryos that survived the treatment.
Destruction of motor innervation
The trochlear nucleus becomes identifiable in histological sections on embryonic day 7
(Sohal, 1976). The trochlear nerve fibres first enter the superior oblique muscle on day 10 as
determined by retrograde flow of HRP injected into the muscle on various days of embryonic
development (Sohal & Holt, 1978). The trochlear nucleus, the sole source of innervation of the
superior oblique muscle, was destroyed on embryonic day 7 as described previously (Sohal &
Wrenn, 1984). Briefly, the microsurgical procedure involved cutting the vitelline membrane in a
relatively avascular region, raising the head with a glass rod, and destruction of the dorsal
midbrain with Malis Bipolar coagulator (Codlman & Shurtleff, Inc.). The embryo was replaced
and the egg was sealed. The entire procedure took less than 1 min per embryo. The muscle was
made aneural in 85 embryos and the results are based on 22 embryos that survived the operation.
Because of the radical nature of the operation this treatment results in permanent, bilateral
destruction of the trochlear and oculomotor nuclear complexes innervating the extraocular
muscles (Sohal & Wrenn, 1984). The destruction of motor neurones was confirmed from
histological sections of the brain and the lack of muscle innervation was verified with silver
staining (see below).
Curarized aneural muscle
In addition to the normal, paralysed, and aneural preparations described above a fourth
group of embryos was also utilized. In this group the use of d-TC and motor neurone destruction
was combined. In other words embryos with aneural muscle also received d-TC in the same
amounts and for the same duration as described, above for the paralysed group. A total of 24
embryos was subjected to this dual treatment and the results are based on nine surviving cases.
Histochemical staining
The superior oblique muscle was dissected, mounted on a piece of cork with OCT compound
and rapidly frozen in liquid nitrogen. Serial transverse sections at 10 jum thickness were cut with
a cryostat and alternate sections were processed for histochemical demonstration of (i) alkalinestable actomyosin-ATPase activity, (ii) acid-stable actomyosin-ATPase activity or (iii) motor
endplate cholinesterase activity and silver staining of nerve fibres. ATPase staining was performed according to the method of Guth & Samaha (1970) as modified by Butler & Cosmos
(1981). To be more specific on critical details, alkaline preincubations were conducted for 10 min
at pH10-0 at room temperature. Acid preincubations were initially performed for 1-5 min at
pH3-7 to 4-7 at room temperature. pH lability patterns of all three muscle fibres from all ages
permitted the use of only two pHs (10-0 and 4-3) in reliable identification of various fibre types
and these pHs were used throughout this study. Adjacent sections were processed for combined
cholinesterase and silver impregnation staining according to the method outlined by Toop (1976)
to verify absence of innervation in the aneural muscle. Muscles on embryonic days 9-12,15-17,
82
G. S. S O H A L AND D . W. S I C K L E S
22 and 27 (hatching); and of 3-week post-hatching and adult ducks (minimum age 1 year) were
used for histochemical staining. The number of animals utilized varied from one to eight per age.
In order to characterize fully the fibre types in the superior oblique muscle, additional
histochemical methods for demonstration of metabolic enzymes were performed on 3-week
post-hatching and adult animals, ar-glycerophosphate dehydrogenase (ar-GPDH) activity was
demonstrated by the method of Wattenburg & Leong (1960) with a 30-min incubation.
Nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) activity was demonstrated by a 20-min incubation using the method of Scarpelli, Hess & Pearse (1958). Phosphorylase (Phos) activity was shown by the protocol outlined in Pearse (1972) with an incubation
time of 90 min including 20 % ethanol to inhibit branching enzyme activity.
Fibre counts
Quantification offibretypes for the normally developing muscle was performed on embryonic
days 9, 12, 16, 22, 27 and 3 weeks post-hatching. Quantification of fibre types in paralysed and
aneural muscles was done on embryonic days 16 and 22. Fibre type populations were calculated
from photographs (final magnification of X250 or 500) of serial sections of the muscle processed
for alkaline- and acid-stable ATPase activity. Due to the uneven distribution of muscle fibre
types across the muscle, all musclefibreswithin the muscle sections were counted. All unstained
fibres in the acid preincubation sections were type II fibres; all unstained fibres in the alkaline
preincubation sections were type I. The number of type IIIfibreswas determined by subtraction
(total number of fibres minus type I and type II = number of type III). All counts were made
blind and all fibres fell into one of the three categories. Quantification offibretypes of the adult
muscle was not attempted.
Statistics
The fibre type counts of 16- and 22-day paralysed and aneural muscles were compared to
normals with a two-way analysis of variance. A one-way AN OVA and Tukey's HSD post hoc
test was used to determine significant differences in total number and number of each fibre type
in normal, paralysed and aneural muscles. A Student's Mest was used to determine differences
between 16- and 22-day muscles.
RESULTS
Histochemical staining of the adult muscle fibres
Qualitative analysis of actomyosin-ATPase staining patterns revealed three
types of muscle fibres in the adult superior oblique muscle. Type I fibres contain an
ATPase activity that is stable to preincubation media of an acidic pH and labile
under alkaline conditions (Figs 1,2). Following exposure to acidic media of pH 3-7
for 1-5 min, the ATPase activity of type I fibres is completely inactivated. A small
amount of activity is retained after exposure to pH3-9, a moderate amount at
p H 4 1 , and all activity is retained at pH4-3 and higher. Following a 10-min
preincubation at pH 10-0, all activity is lost. Type II fibres lose their activity when
exposed to preincubation media of pH3-7 to 4-1 (Fig. 1). At pH4-3 a punctate
staining pattern is observed in some of these fibres and is likely to be due to mitochondrial Ca 2+ activated ATPase (Samaha & Yunis, 1973). At pH4-5 and above,
the ATPase activity of these fibres is moderate to high. These fibres are intensely
stained following exposure to pH10-0 preincubation (Figs 1, 2). Type III fibres
Muscle fibre typing in development
83
possess ATPase activity that is resistant to both acidic and alkaline conditions
(Figs 1, 3). These fibres are intensely stained following pH10-0 and pH4-3 to 4-7
preincubations. At pHs of 3-9 to 4-1 the ATPase activity is retained in most of the
fibres; however, some fibres show a partial lability of this activity as indicated by
their moderate staining.
Histochemical staining to demonstrate the relative activities of glycolysis
(or-GPDH), glycogenolysis (Phos) and oxidative (NADH-TR) metabolism showed
a considerable variability of activity within each fibre type (Figs 2-4). ar-GPDH
activity paralleled the alkaline-stable ATPase activity; type II and III fibres
possessed moderate to high activity while type I fibres had little activity of this
enzyme. In general, Phos activity was lowest in type I fibres. However, as may be
observed in Figs 2,3, this activity was extremely variable among the different types
of fibres. Some type I fibres contained Phos activity that was greater than some
type II and III fibres. The range of NADH-TR activity in all three fibre types was
also widespread as fibres with low, moderate, and high activity could be observed
in all three types of fibres (Figs 2, 3, 4). In addition, a small number of type III
fibres showed undetectable amounts of NADH-TR activity. In general, type I
and II fibres are located at the peripheral zone adjacent to the orbit whereas the
type III fibres are concentrated at the global (central) portion of the muscle
(Figs 2, 3, 5). Correlation between ATPase pH labilities and the activity of
metabolic enzymes in the adult muscle fibre types is summarized in Fig. 4.
Preincubation pH
3-7
3-9
4-1
4-3
4-5
4-7
10-0
7
/
Unstained
Darkly stained
Lightly stained
Lightly stained due to
mitochondrial ATPase
Moderately stained
Fig. 1. pH lability patterns of myosin-ATPase activity in the superior oblique muscle
from embryonic day 17 to hatching and in the adult. Reliable identification of fibre
types could be performed using pH 10-0 and 4-3 preincubations. Type I fibres are
unstained at pH 10-0, most type IIfibresare unstained at pH4-3, and type III fibres are
intensely stained at both pHs.
84
G. S. SOHAL AND D. W. SICKLES
Histochemical staining of the embryonic muscle fibres
Although the percentage of type I, II and III fibres in the superior oblique
muscle varied with age (see below), the same pH lability patterns as in the adult
n
w-u
Fig. 2. Muscle fibre types at the orbital margin of the adult superior oblique muscle.
(A) Alkaline-stable ATPase; (B) acid-stable ATPase; (C) NADH-TR; (D) a-GDPH;
(E) phosphorylase activity. This region of the muscle is almost exclusively composed of
type I and II musclefibres.Note the relative staining intensity of each muscle fibre type
with these histochemical techniques. Bar, 100/mi.
Muscle fibre typing in development
85
muscle were observed in the embryonic muscle. Reliable identification of type I, II
and III embryonic fibres could be made using only pH 4-3 and 10-0 preincubations.
We therefore define type I fibres as those that are stained following pH4-3
•m
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Fig. 3. Muscle fibre types in the central portion of the adult superior oblique muscle.
(A) Alkaline-stable ATPase; (B) acid-stable ATPase; (C) NADH-TR; (D) ar-GDPH;
(E) phosphorylase activity. This region is almost exclusively composed of type III
fibres. Bar, 100 [im.
86
G.
ATPase
(10-0)
s .SOHAL
ATPase
(4-3)
AND D .
w. SICKLES
aGPD H
Phos
NADH-TR
o-@ o
o® o
III
C J Unstained
Moderately stained
K | P Lightly stained
Intensely stained
(p|) Mitochondrial staining
Fig. 4. Summary of activities of myosin-ATPase and selected metabolic enzymes in
type I, II and III fibres in the adult superior oblique muscle.
preincubation and unstained at pHlO-0; type II fibres are stained at pH10-0
and unstained at pH4-3 and type III fibres are intensely stained at both pHs. The
word 'fibre' is used throughout this study purely for the sake of consistency even
though some stained cells represent myotubes, especially during earlier stages of
development.
Normal muscle
In the normally developing muscle, type II and III fibres can be identified as
early as embryonic day 9 (Fig. 7). A majority of the fibres (82 %) belong to type III
category. From days 9 to 16 the fibre types remain the same but the proportion of
type II fibres increases to 39% (366 fibres) while the proportion of type III
decreases to 61 % (571 fibres) on day 16 (Table 1). Type I fibres are first seen on
embryonic day 17 (data not shown). On day 22 the percentages of type I, II and III
fibres are 29, 53, and 18, respectively (Figs 5-7). On day 22 there is a significant
increase in the number of type I and II fibres as well as in the total number of
muscle fibres as compared to day 16 (Table 1). The small marginal and the large
central portion of the muscle becomes identifiable also on embryonic day 22
(Fig. 5). The transition between the two zones is abrupt. After day 22 the
Fig. 5. Alkaline-stable (A,C) and acid-stable (B,D) ATPase activity in the superior
oblique muscle on embryonic day 22 (A,B) and 3 weeks post-hatching (C,D). During
development, the proportion of type IIIfibresincreases until the vast majority of fibres
are type III at 3 weeks post-hatching. The marginal (arrow) and the central portion of
the muscle can be distinguished on embryonic day 22. Bar, 400 jwm.
87
Muscle fibre typing in development
•
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•
•
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0
0
107 ± 16"-c
Type I
Type II
366 ± 16
255 ± 39"
177 ± 25"
571 ± 33
179 ± 18"
109 ± 23"
Type III
Total
937 ± 40
434 ± 56"
392 ± 36"
Type II
1820 ± 129a
112 ± 15a'"
277 ± 60"'c
Type I
1018 ± 149a
0"
139 ± 18"'c
628 ± 125
256 ± 42"
73 ± 12"'c
Type III
Embryonic day 22
Total
3455 ± 370a
367 ± 53"
489 ± 69"
Data are expressed as mean ± S.E.M. a, b) and c indicate significant differences (P<0-05). a, comparison between day 16 and day 22.
b, comparison of paralysed or aneural with ilormal muscle, c, comparison between paralysed and aneural muscles. N = 4-6 animals in each
category. Data on curarized aneural muscles are not reported because of lack of significant sample size as only 1 to 2 embryos were sacrificed
daily from day 16 to 22.
Group
Normal
Paralysed
Aneural
Embryonic day 16
Table 1. Number of muscle fibres in the normal, paralysed and aneural superior oblique muscle
n
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00
oo
P
00
00
W. SiCKL
Musclefibretyping in development
89
percentage of type I and II fibres decreases whereas the type III increases. In a
3-week post-hatching muscle approximately 76 % of the fibres are type III, 17 %
type II and only 7 % type I (Fig. 7).
Paralysed muscle
The paralysed muscles were considerably smaller than normal at the corresponding ages. Histologically the muscle cells were much smaller in diameter
(Fig. 6). A total muscle fibre count indicated a significant decrease as an average
of 434 cells on embryonic day 16 and 367 cells on day 22 was noticed (Table 1).
The normal muscle at the same ages had an average of 937 and 3455 cells,
respectively (Table 1). The total fibre count in the paralysed muscle on day 16 is
not significantly different from day 22. Type II and III fibres were present in the
paralysed muscle from the onset of paralysis. On day 16, 58 % (255 fibres) were
type II and the rest belonged to the type III (179 fibres) category (Fig. 7, Table 1).
On day 22 the paralysed muscle contained an average of 112 (33 %) type II and 256
(67%) type III fibres (Table 1). Both of these counts are significantly lower as
compared to the 22-day normal. A comparison of fibre types between day 16 and
22 paralysed muscle indicated a significant decrease in the type II fibres on day 22
(Table 1). Type I fibres were never observed in the paralysed muscles (Figs 6, 7).
Thus, paralysis of the embryo prevents the differentiation of type I but not type II
and III fibres.
Aneural muscle
The aneural muscles were also much smaller than normal muscles. Histologically the aneural muscle cells resembled those in the paralysed muscle (Fig. 6).
The total number of cells was not significantly different from the paralysed muscle
as an average of 392 fibres on day 16 and 489 on embryonic day 22 was found in the
aneural muscle (Table 1). In both cases there was a severe atrophy of the muscle as
compared to the normals. In spite of this the marginal and the central zone of the
muscle could be distinguished on day 22 (Fig. 6).
Initially the aneural muscle showed type II and III fibres (data not shown). On
embryonic day 16 type I fibres were also present. The muscle on this day contained
27% (107) type I, 45% (177) type II, and 28% (109) type III fibres (Fig. 7,
Table 1). It should be pointed out that the appearance of type I fibres in the
aneural muscle is one day earlier than that seen in the normal muscle. As compared to the normal muscle on day 16, the aneural muscle contained significantly
fewer type II and III fibres but significantly more type I fibres. A comparison
between the aneural and paralysed muscle showed a significant difference in type I
but not type II and III fibres on day 16. On day 22 a majority of the fibres belongs
to the type II class, i.e. 28 % (139) type 1,57 % (277) type II and 15 % (73) type III
(Figs 6, 7, Table 1). Although the percentage of each fibre type in the aneural
muscle on day 22 resembles that of the normal, the fibre counts in each category
are significantly lower in the aneural muscle. A comparison between the paralysed
and aneural muscle on day 22 indicated a significant increase in the type I and II
90
G. S. SOHAL AND D. W. SICKLES
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91
Muscle fibre typing in development
100 -i
n = 4
n = 6
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lOO-i
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IIIIII
nun
nun
nun
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9 days
12 days
16 days
22 days
27 days
nun
3 weeks
post-hatch
Fig. 7. Percentages (ordinate) of type I, II and IIIfibresin the normal, paralysed, and
aneural superior oblique muscle during embryonic development and after hatching.
Type II and III fibres are present as early as day 9 and at all subsequent ages and type I
fibres appear on day 17 in the normal muscle. In the paralysed muscles type I fibres are
absent. In the aneural muscles all three types of fibres are present.
fibres and a significant decrease in the type III fibres in the aneural muscle.
No significant differences exist between the day-16 and day-22 aneural muscle
(Table 1). These results indicate that the initial differentiation of three fibre types
in the superior oblique muscle occurs in the absence of innervation.
Curarized aneural muscle
Under the combined experimental conditions we were interested in determining
whether or not type I muscle fibres developed. Embryos in this group showed the
most severe atrophy of muscles (Fig. 8). Muscles in all cases contained fewer than
200 muscle fibres, which is roughly half the number observed in paralysed and
aneural muscles. Over the time period examined (16-22 days), type I fibres were
not observed (Fig. 8). The other two fibre types were present in proportions
similar to those reported for the 22-day paralysed muscle in Table 1.
Fig. 6. Alkaline-stable (A,D,G) and acid-stable (B,E,H) ATPase activity and cholinesterase/silver staining (C,F,I) in the normal (A,B,C), paralysed (D,E,F), and aneural
(G,H,I) superior oblique muscle on embryonic day 22. Identification of all three fibre
types can easily be made in the normal and aneural muscles. Paralysed muscles do not
develop type I fibres, i.e. all fibres stain intensely with alkaline-stable ATPase. Silver
staining demonstrates nerve fibres in normal and paralysed muscle (small arrows in
C,F) but not in the aneural muscle. Large arrow points to the marginal portion of the
muscle. Bar for A,B,C, 200jum; bar for D-I, 100pm.
92
G. S. SOHAL AND D. W. SICKLES
8A
9- C -
B
Fig. 8. Alkaline-stable ATPase (A) and silver/cholinesterase (B) staining of curarized
aneural superior oblique muscle on embryonic day 20. The stained fibres represent
type II and type III. Type I fibres are absent. Bar, 50 fjm.
DISCUSSION
Fibre types in the adult muscle
The results of this investigation indicate that at least three types of fibres are
present in the superior oblique muscle of the adult duck. Type I fibre ATPase
activity is acid-stable, alkali-labile; type II fibres possess alkali-stable and acidlabile ATPase; and type III fibre ATPase is both acid- and alkali-stable. No
consistent pattern of staining was observed with metabolic enzyme activities.
These observations on fibre types are in general agreement with other studies on
the extraocular muscles of birds and mammals (Miller, 1967; Yellin, 1969; Maier
etal. 1972).
The distribution of type I, II and III fibres within the superior oblique muscle is
not homogeneous. Detailed quantitative studies of Maier etal. (1972) on the crosssectional area of superior oblique muscle fibres in the marginal and central
portions of adult quail, pigeon and canary have revealed significant differences in
the diameter of fibres between the two portions of the muscle. Segregation of
fibres into two distinct zones has been observed in the extraocular muscles of fish,
amphibians, birds and mammals (Kilarski & Bigaj, 1969; Yellin, 1969; Maier etal.
1972). However, not all extraocular muscles exhibit different regions of fibre
segregation. Segregation of zones is a feature unique to the superior and inferior
oblique and rectus muscles. The mechanism by which segregation of fibre types is
Musclefibretyping in development
93
brought about is currently unknown. It has been suggested by Maier et al. (1972)
that the two zones may result from differential interactions between the motor
neurone axons and the developing myotubes. Since we have shown segregation of
fibre zones in aneural muscle it is concluded that motor neurone influences are not
directly involved in this process.
Differentiation of the embryonic fibre types
In the present study type II and III fibres could be identified with ATPase
staining as early as day 9 in the normally developing muscle. This observation
indicates that differentiation of type II and III fibre occurs very early in development; even prior to the arrival of motor nerve fibres in the muscle. Our previous
studies utilizing retrograde uptake of horseradish peroxidase have indicated that
the trochlear nerve fibres first appear in the superior oblique muscle on embryonic
day 10 (Sohal & Holt, 1978). We have also shown that at this time the muscle is
primarily composed of myoblasts and myotubes (Sohal & Holt, 1980). Together,
these observations suggest that the initial differentiation of muscle cells into type
II and III is independent of direct neural influences and that young myotubes can
be stained for ATPase activity characteristic of type II and III muscle fibres.
In the present study type I, II and III fibres, in proportions similar to the normal
muscle, especially on day 22, were observed in muscle developing in the absence of
innervation. The simplest explanation for these findings is that a direct contact by
the motor neurones is not essential for the initial expression of muscle fibre types
during the course of embryonic development. This interpretation is consistent with
the recent observations of others. For example, switching of motor neurone pools
during development has no effect on fibre type differentiation (Khaskiya et al.
1980; Butler, Cosmos & Brierley, 19826; Laing & Lamb, 1983). Butler et al
(1982a) reported that in the absence of motor neurones the differentiation of
myotubes into slow and fast fibres occurs in the brachial muscles of the chick
embryo. Phillips & Bennett (1984) recently reported differentiation of type I, II
and III fibres in forelimb muscles of chick after early removal of neural tube.
Although this aspect of our study confirms the conclusions drawn by the above
authors it is difficult to compare our data with these studies since quantification of
fibre types was not reported in the above studies and owing to the fact that in our
study all three different types of muscle fibres were examined in a single muscle
under several conditions.
The results of the present study also indicate that the type I fibres do not appear
in muscles innervated but paralysed with d-TC. This observation confirms the
finding of McLennan (1983a) in the leg muscles of the chick embryo. Why type I
fibres do not appear following d-TC treatment while their differentiation is not
affected in the absence of innervation is unknown. It is not due to a delay
in differentiation and maturation as these developmental processes are more
severely affected in the aneural than in the paralysed muscle (Sohal & Holt, 1980).
It has been suggested by McLennan (1983a) that loss of twitch activity prevents
the differentiation of type I fibres. Although our results are consistent with his
94
G. S. SOHAL AND D. W. SICKLES
suggestion they also suggest another possibility. It is possible that some unique
action of d-TC on muscle, independent of innervation, is responsible for lack of
type I differentiation since type I fibres were also absent in our curarized aneural
muscles. In addition to the well-known effect of d-TC in decreasing twitch
response of muscle it has been reported that d-TC has a direct effect on the muscle
in that it increases the baseline tension in innervated and denervated adult
mammalian muscles (Mclntyre, King & Dunn, 1945; Bean & Elwell, 1951; Katz
& Eakins, 1967). Although the effects of d-TC on tension of embryonic avian
muscles are currently unknown it is possible that such muscles may also respond to
d-TC with an increase in baseline tension. This assumption would imply that
increased tension somehow prevents the appearance of type I fibres. This could
explain the absence of type I fibres in our curarized innervated and curarized
aneural muscles. An alternative explanation has been provided by Gauthier, Ono
& Hobbs (1984). These authors observed that following treatment with d-TC, a
type of myosin that is not normally present was observed in the ALD (slow) but
not in the PLD muscle of chick embryo as detected immunocytochemically. They
suggested that changes in myosin synthesis, rather than selective loss of slow
myosin, may be related to the different composition of motor neurone pools as
paralysis augments motor neurone survival by reducing the magnitude of normally
occurring cell death (Pittman & Oppenheim, 1978). This would imply that the new
myosin synthesis may be directed by the additional motor neurones. It should be
pointed out that d-TC also prevents the death of the trochlear motor neurones in
the duck embryo (Sohal, Leshner & Swift, 1983).
The results of the present study and of others indicate that motor neurones
do not specify differentiation of fibre types during the course of embryonic
development. This is contrary to the results of the cross-reinnervation experiments in the newly hatched chicken which have shown that the motor neurones
control muscle fibre types. Apparently, some time during the course of embryonic
development the motor neurones begin to exert control over the muscle. It
appears that the muscles lose their independence some time after the initial nervemuscle connections have been established. Neural control of muscle soon after
innervation during in vivo development has recently been demonstrated (Toutant,
Toutant, Renaud & Le Douarin, 1979; Renaud, Gardahaut, Rouaud & Le
Douarin, 1983). These authors reported the persistence of type I fibres, which
normally disappear during development, following artificial electrical stimulation
of the PLD muscle in the chick. The precise mechanisms by which muscle cells lose
their independence or the motor neurones exert their control over the muscle are
unknown.
Finally, a comment should be made regarding the influence of innervation on
the production of myotubes. The myotubes present at the beginning of myogenesis
are termed primary myotubes (Kelly & Zacks, 1969). The myoblasts at the periphery of the primary myotube fuse to form secondary myotubes. As myogenesis
proceeds the secondary myotubes are separated from the primary myotubes to
form distinct muscle fibres (Ontell, 1977). In the present study the number of
Muscle fibre typing in development
95
myotubes in the curarized muscle was similar to that seen in the aneural muscle
and in both cases contained significantly fewer myotubes than in the normal
muscle. This decrease is interpreted as lack of generation of secondary myotubes
since d-TC treatment (McLennan, 19836) and a lack of innervation (Harris, 1981)
are known to inhibit the production of secondary but not primary myotubes. Since
all three fibre types were seen in the aneural muscle our results suggest that
primary myotubes are capable of differentiating into type I, II and III fibres (but
see McLennan, 19836). Our results also suggest that some fibres must switch types
as the muscle matures. This is based on the fact that from day 16 to 22 there is a
significant decrease in the number of type II fibres in the curarized muscles. It is
unlikely that reduction in type II fibres is due to selective degeneration as the total
number of primary myotubes remains stable during this period. In the curarized
muscle type I fibres were absent and yet all fibres were stained. The number of
type II and type III fibres differs significantly between the paralysed and the
aneural muscle whereas there is no significant difference in the total number of
myotubes between these two groups.
We thank Teena Knox and Greg Oblak for technical assistance, Sharlene Booker for typing
the manuscript, and Harry Davis for statistics. This work was supported by grants from the
National Institutes of Health (HD17800, HD18280, OH02020).
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{Accepted 27 February 1986)