Anat Embryol (1992) 185:143-153
Anatomy and Embryology 9 Springer-Verlag1992
Myosin isoform transitions during development of extra-ocular and masticatory muscles in the fetal rat F. Mascarello 1 and A.M. Rowlerson 2
1 Istituto di Anatomia degli AnimaliDomesticicon Istologiaed Embriologia,Universitfidi Milano, via Celoria 10, 1-20133 Milano, Italy 2 Department of Physiology,The MedicalSchool, Bristol BS8 1TD, and Department of Physiology,U.M.D.S., St. Thomas's Hospital, Campus, London SEI 7EH, U.K. Accepted August5, 1991 Summary. The late fetal development of rat extra-ocular
and masticatory muscles was examined by myosin immunohistochemistry. The pattern of slow and neonatal myosin isoform expression in primary and secondary myotubes in these muscles was generally similar to that seen by others in limb muscles. We observed a consistent difference between the Sprague-Dawley and Wistar rats in the degree of maturity reached by all muscles studied at a particular age. In both strains, extra-ocular muscles were also about one day in advance of the masticatory muscles. Thus, secondary myotubes were first seen at E17 in Wistar extraocular muscles, at E18 in SpragueDawley extra-ocular muscles and Wistar masticatory muscles, and at E19 in Sprague-Dawley masticatory muscles. There was a strikingly early and complete type differentiation of primary myotubes in extraocular muscles, and tonic myosin first appeared before birth in presumptive extrafusal tonic fibres in the orbital layer of the oculorotatory muscles. Throughout the late fetal period, retractor bulbi was composed of fast myotubes only, but these myotubes were not arranged in classical clusters. In the masticatory muscles at E17/EI8 some slow primary myotubes started to express tonic myosin, and these presumptive spindle bag2 fibres were located only in regions of the muscles known to contain spindles in the adult. Presumptive bag1 fibres appeared about a day later (initially without tonic myosin), and in the region of the spindle cluster in anterior deep masseter extrafusal secondary myotube production appeared to be suppressed. Key words: Muscle development - Extra-ocular muscles - Masticatory muscles - Rat fetus
Introduction
Muscle fibres are produced in a two-stage process (Kelly and Zacks 1969; Ontell and Kozeka 1984). First, early Offprint requests to: A.M. Rowlerson(London address)
(embryonic) myoblasts fuse to form immature fibres (primary myotubes) which have a highly distinctive morphology and account for about 10-20% of the final muscle fibre number. Then, after a delay of 2 or more days, late (fetal) myoblasts fuse in a nerve-dependent process to form the so-called secondary myotube population which gives rise to the rest of the muscle fibres (Harris 1981). In mammalian muscle all primary myotubes pass through an early stage in which they contain the 'slow' isoform of myosin characteristic of adult slow-twitch fibres (Narusawa et al. 1987; Vivarelli et al. 1988; Condon et al. 1990a). They soon differentiate in a nervedependent manner (Harris et al. 1989; Condon et al. 1990b) into those which continue to express slow myosin, and are presumed to become the slow fibres in the adult, and those which lose the slow myosin and become fast fibres (probably type IIA) in the adult (Hoh et al. 1988; Hoh and Hughes 1989). Secondary myotubes do not initially contain slow myosin. Most become fast fibres in the adult (Narusawa et al. 1987; Hoh et al. 1988) and in their case the embryonic myosin is replaced first by neonatal and then by adult myosin (Whalen et al. 1981; Harris et al. 1989). In future slow muscles, many of the secondaries start to express slow myosin and become slow fibres in the adult (Narusawa et al. 1987; Hoh et al. 1988; Hoh and Hughes 1989; Harris et al. 1989). In the rat, fast fibres in limb muscles generally reach their adult myosin composition at about a month after birth, when all the remaining neonatal myosin has been replaced by the appropriate (e.g. IIA, IIB) fast myosin (Whalen et al. 1981 ; Butler Browne et al. 1982; d'Albis et al, 1991). Slow twitch fibre development has been well studied (Narusawa et al. 1987; Harris et al. 1989; Condon et al. 1990a, b), but the development of slow-tonic fibres has received very little attention, probably because they are rare in mammalian muscles (see Morgan and Proske 1984). We have examined the development of masticatory and extra-ocular muscles in the rat, where these muscles contain a few slow fibres of the slow twitch type and
144 b o t h varieties o f m a m m a l i a n slow-tonic fibres, i.e. extrafusal t o n i c fibres in the e x t r a - o c u l a r muscles a n d i n t r a f u sal (spindle) b a g fibres in the m a s t i c a t o r y muscles. T h e bag fibres o f muscle spindles are a p a r t i c u l a r l y interesting case d e v e l o p m e n t a l l y , because their characteristic expression o f t o n i c m y o s i n ( P i e r o b o n - B o r m i o l i et al. 1980; R o w l e r s o n et al. 1985) is d e p e n d e n t u p o n sensory ( n o t m o t o r ) i n n e r v a t i o n . P r e s u m p t i v e spindle b a g fibres c a n be recognised very early i n muscle d e v e l o p m e n t b y their tonic m y o s i n ( R o w l e r s o n 1988; T h o r n e l l et al. 1988; K u cera a n d W a l r o 1990; A n t o n i o u et al. 1990). This t o n i c m y o s i n first a p p e a r s a b o u t I d a y after c o n t a c t with the sensory nerve t e r m i n a l is established, a n d is seen in fibres o f b o t h p r i m a r y m y o t u b e origin (the spindle bag2 fibre) a n d early s e c o n d a r y m y o t u b e origin (the b a g l fibre) ( K u c e r a a n d W a l r o 1990). I n the rat masseter, m a n y o f the muscle spindles p r e s e n t are c o n c e n t r a t e d together in a c o m p l e x cluster a r r a n g e m e n t ( K a r l s e n 1965; M a i e r 1979; R o w l e r s o n et al. 1988), which is very striking, b u t its f u n c t i o n a n d / o r origin is u n e x p l a i n e d . O u r observations o f the spindle cluster in fetal rats suggest developm e n t a l events are a n i m p o r t a n t factor in g e n e r a t i n g this structure. We were also interested to see if the early developm e n t o f m a s t i c a t o r y a n d e x t r a - o c u l a r muscles was comp a r a b l e to t h a t in l i m b muscles, as p o s t n a t a l r e p l a c e m e n t o f n e o n a t a l m y o s i n b y a d u l t f o r m s is slower a n d / o r inc o m p l e t e in some fibres in these muscles (Wieczorek et al. 1985; d ' A l b i s et al. 1986, 1991). I n c i d e n t a l l y , this choice o f muscle g r o u p s also offered the o p p o r t u n i t y to e x a m i n e fibre type d e v e l o p m e n t i n a muscle which is exclusively fast in the a d u l t (retractor bulbi), u n l i k e the m u c h - s t u d i e d h i n d - l i m b fast muscles which c o n t a i n a few slow fibres.
Materials and methods
Muscle samples for immunohistochemistry. Late fetal (gestational ages E16 E21) and neonatal (day of birth and 7 days postnatal) rats were killed by decapitation. The rats used were obtained from two different sources: Wistar strain bred 'in house' at the Medical School of the Universiy of Bristol, and Sprague-Dawley supplied by Charles River, in Milan. The convention used in this paper for calculating gestational age is that the day on which the copulation plug was found is called E0. The Wistar rats normally give birth on E21 and the Sprague-Dawley rats normally give birth on E20 or E21. Comparing the degree of muscle development reached in these two strains on any given day, our impression was that it seemed about a day 'younger' overall in the SpragueDawley strain than in the Wistar rats, and that development was more advanced in extraocular than in masticatory muscles. For the sake of clarity, results from the two strains are therefore presented together according to the stage of muscle development reached, rather than simply by gestational age. Except in the oldest animals, no attempt was made to dissect out the masticatory or extra-ocular muscle. Instead, the roof of the skull and the brain were removed from the head, and in some cases skeletal muscle from an adult rat packed in the space created. The whole head was then quickfrozen in melting isopentane. Subsequently, 10-~tmcryostat sections were cut from these blocks either skip-serially (every third or fourth section collected) or as a complete series. Separate series of sections were cut in the frontal and
transverse planes, and at some ages yet another series was taken perpendicular to the optic nerve, specifically for examination of the extraocular muscles. Occasional sections were stained for mATPase activity or haematoxylin and eosin, but most were stained in alternating order with polyclonal antibodies specific for slow myosins (anti-I), tonic myosin only (anti-tonic) and neonatal/ embryonic myosins (anti-NE). Antibody binding was visualised by the indirect immunoperoxidase method. The adult muscle incorporated in the muscle blocks, and hence present in the sections, acted as an internal reference or ' control' for the staining reactions of the fetal and neonatal muscle. The specificity of the three primary antibodies used resides in their reaction with the appropriate myosin heavy chains and is summarised in Table 1. Anti-I has been used widely previously on muscle sections, and reacts with both slow-twitch and slow-tonic fibres (Mascarello etal. 1982; Rowlerson et al. 1983, 1985, 1988) although on immunoblotting the reaction with tonic myosin is clearly weaker (Fig. i). It gives no significant reaction with embryonic or neonatal myosins. Antitonic and anti-NE were raised recently; the preparation and testing of anti-NE has been described fully elsewhere (Scapolo et al. 1991), and anti-tonic is described below. Myotubes were identified as primary or secondary on morphological grounds, including their arrangement in clusters, diameter and profile in transverse section. Immature fibres or myotubes reacting strongly with anti-I and weakly with anti-NE were regarded as 'slow', and those giving the inverse reaction as 'fast'.
Preparation and testing of Anti-tonic antibody. The tonic fibre isoform of myosin was obtained by brief high salt extraction of myofibrils made from adult chicken anterior latissimus dorsi (ALD) muscle. The extract was first clarified by centrifugation, and the myosin then precipitated by dialysis against a very low ionic strength solution. Myosin heavy chains were extracted from this crude myosin sample by SDS-PAGE on 8% gels. The myosin heavy chain bands were revealed by potassium acetate staining and then cut out. These gel pieces were then ground to a sIurry in 0.1 M Na2 CO3 (pH 9.3) and stored in this solution for 48 h. The resulting supernatant containing extracted heavy chains was then adjusted to pH 7.4 and used to immunise New Zealand white rabbits after a sample had been examined by SDS-PAGE followed by silver staining to check that there was no contamination by other proteins. Briefly, the immunisation schedule consisted of three sub-cutaneous injections (at multiple sites) of myosin (
Anatomy and Embryology 9 Springer-Verlag1992
Myosin isoform transitions during development of extra-ocular and masticatory muscles in the fetal rat F. Mascarello 1 and A.M. Rowlerson 2
1 Istituto di Anatomia degli AnimaliDomesticicon Istologiaed Embriologia,Universitfidi Milano, via Celoria 10, 1-20133 Milano, Italy 2 Department of Physiology,The MedicalSchool, Bristol BS8 1TD, and Department of Physiology,U.M.D.S., St. Thomas's Hospital, Campus, London SEI 7EH, U.K. Accepted August5, 1991 Summary. The late fetal development of rat extra-ocular
and masticatory muscles was examined by myosin immunohistochemistry. The pattern of slow and neonatal myosin isoform expression in primary and secondary myotubes in these muscles was generally similar to that seen by others in limb muscles. We observed a consistent difference between the Sprague-Dawley and Wistar rats in the degree of maturity reached by all muscles studied at a particular age. In both strains, extra-ocular muscles were also about one day in advance of the masticatory muscles. Thus, secondary myotubes were first seen at E17 in Wistar extraocular muscles, at E18 in SpragueDawley extra-ocular muscles and Wistar masticatory muscles, and at E19 in Sprague-Dawley masticatory muscles. There was a strikingly early and complete type differentiation of primary myotubes in extraocular muscles, and tonic myosin first appeared before birth in presumptive extrafusal tonic fibres in the orbital layer of the oculorotatory muscles. Throughout the late fetal period, retractor bulbi was composed of fast myotubes only, but these myotubes were not arranged in classical clusters. In the masticatory muscles at E17/EI8 some slow primary myotubes started to express tonic myosin, and these presumptive spindle bag2 fibres were located only in regions of the muscles known to contain spindles in the adult. Presumptive bag1 fibres appeared about a day later (initially without tonic myosin), and in the region of the spindle cluster in anterior deep masseter extrafusal secondary myotube production appeared to be suppressed. Key words: Muscle development - Extra-ocular muscles - Masticatory muscles - Rat fetus
Introduction
Muscle fibres are produced in a two-stage process (Kelly and Zacks 1969; Ontell and Kozeka 1984). First, early Offprint requests to: A.M. Rowlerson(London address)
(embryonic) myoblasts fuse to form immature fibres (primary myotubes) which have a highly distinctive morphology and account for about 10-20% of the final muscle fibre number. Then, after a delay of 2 or more days, late (fetal) myoblasts fuse in a nerve-dependent process to form the so-called secondary myotube population which gives rise to the rest of the muscle fibres (Harris 1981). In mammalian muscle all primary myotubes pass through an early stage in which they contain the 'slow' isoform of myosin characteristic of adult slow-twitch fibres (Narusawa et al. 1987; Vivarelli et al. 1988; Condon et al. 1990a). They soon differentiate in a nervedependent manner (Harris et al. 1989; Condon et al. 1990b) into those which continue to express slow myosin, and are presumed to become the slow fibres in the adult, and those which lose the slow myosin and become fast fibres (probably type IIA) in the adult (Hoh et al. 1988; Hoh and Hughes 1989). Secondary myotubes do not initially contain slow myosin. Most become fast fibres in the adult (Narusawa et al. 1987; Hoh et al. 1988) and in their case the embryonic myosin is replaced first by neonatal and then by adult myosin (Whalen et al. 1981; Harris et al. 1989). In future slow muscles, many of the secondaries start to express slow myosin and become slow fibres in the adult (Narusawa et al. 1987; Hoh et al. 1988; Hoh and Hughes 1989; Harris et al. 1989). In the rat, fast fibres in limb muscles generally reach their adult myosin composition at about a month after birth, when all the remaining neonatal myosin has been replaced by the appropriate (e.g. IIA, IIB) fast myosin (Whalen et al. 1981 ; Butler Browne et al. 1982; d'Albis et al, 1991). Slow twitch fibre development has been well studied (Narusawa et al. 1987; Harris et al. 1989; Condon et al. 1990a, b), but the development of slow-tonic fibres has received very little attention, probably because they are rare in mammalian muscles (see Morgan and Proske 1984). We have examined the development of masticatory and extra-ocular muscles in the rat, where these muscles contain a few slow fibres of the slow twitch type and
144 b o t h varieties o f m a m m a l i a n slow-tonic fibres, i.e. extrafusal t o n i c fibres in the e x t r a - o c u l a r muscles a n d i n t r a f u sal (spindle) b a g fibres in the m a s t i c a t o r y muscles. T h e bag fibres o f muscle spindles are a p a r t i c u l a r l y interesting case d e v e l o p m e n t a l l y , because their characteristic expression o f t o n i c m y o s i n ( P i e r o b o n - B o r m i o l i et al. 1980; R o w l e r s o n et al. 1985) is d e p e n d e n t u p o n sensory ( n o t m o t o r ) i n n e r v a t i o n . P r e s u m p t i v e spindle b a g fibres c a n be recognised very early i n muscle d e v e l o p m e n t b y their tonic m y o s i n ( R o w l e r s o n 1988; T h o r n e l l et al. 1988; K u cera a n d W a l r o 1990; A n t o n i o u et al. 1990). This t o n i c m y o s i n first a p p e a r s a b o u t I d a y after c o n t a c t with the sensory nerve t e r m i n a l is established, a n d is seen in fibres o f b o t h p r i m a r y m y o t u b e origin (the spindle bag2 fibre) a n d early s e c o n d a r y m y o t u b e origin (the b a g l fibre) ( K u c e r a a n d W a l r o 1990). I n the rat masseter, m a n y o f the muscle spindles p r e s e n t are c o n c e n t r a t e d together in a c o m p l e x cluster a r r a n g e m e n t ( K a r l s e n 1965; M a i e r 1979; R o w l e r s o n et al. 1988), which is very striking, b u t its f u n c t i o n a n d / o r origin is u n e x p l a i n e d . O u r observations o f the spindle cluster in fetal rats suggest developm e n t a l events are a n i m p o r t a n t factor in g e n e r a t i n g this structure. We were also interested to see if the early developm e n t o f m a s t i c a t o r y a n d e x t r a - o c u l a r muscles was comp a r a b l e to t h a t in l i m b muscles, as p o s t n a t a l r e p l a c e m e n t o f n e o n a t a l m y o s i n b y a d u l t f o r m s is slower a n d / o r inc o m p l e t e in some fibres in these muscles (Wieczorek et al. 1985; d ' A l b i s et al. 1986, 1991). I n c i d e n t a l l y , this choice o f muscle g r o u p s also offered the o p p o r t u n i t y to e x a m i n e fibre type d e v e l o p m e n t i n a muscle which is exclusively fast in the a d u l t (retractor bulbi), u n l i k e the m u c h - s t u d i e d h i n d - l i m b fast muscles which c o n t a i n a few slow fibres.
Materials and methods
Muscle samples for immunohistochemistry. Late fetal (gestational ages E16 E21) and neonatal (day of birth and 7 days postnatal) rats were killed by decapitation. The rats used were obtained from two different sources: Wistar strain bred 'in house' at the Medical School of the Universiy of Bristol, and Sprague-Dawley supplied by Charles River, in Milan. The convention used in this paper for calculating gestational age is that the day on which the copulation plug was found is called E0. The Wistar rats normally give birth on E21 and the Sprague-Dawley rats normally give birth on E20 or E21. Comparing the degree of muscle development reached in these two strains on any given day, our impression was that it seemed about a day 'younger' overall in the SpragueDawley strain than in the Wistar rats, and that development was more advanced in extraocular than in masticatory muscles. For the sake of clarity, results from the two strains are therefore presented together according to the stage of muscle development reached, rather than simply by gestational age. Except in the oldest animals, no attempt was made to dissect out the masticatory or extra-ocular muscle. Instead, the roof of the skull and the brain were removed from the head, and in some cases skeletal muscle from an adult rat packed in the space created. The whole head was then quickfrozen in melting isopentane. Subsequently, 10-~tmcryostat sections were cut from these blocks either skip-serially (every third or fourth section collected) or as a complete series. Separate series of sections were cut in the frontal and
transverse planes, and at some ages yet another series was taken perpendicular to the optic nerve, specifically for examination of the extraocular muscles. Occasional sections were stained for mATPase activity or haematoxylin and eosin, but most were stained in alternating order with polyclonal antibodies specific for slow myosins (anti-I), tonic myosin only (anti-tonic) and neonatal/ embryonic myosins (anti-NE). Antibody binding was visualised by the indirect immunoperoxidase method. The adult muscle incorporated in the muscle blocks, and hence present in the sections, acted as an internal reference or ' control' for the staining reactions of the fetal and neonatal muscle. The specificity of the three primary antibodies used resides in their reaction with the appropriate myosin heavy chains and is summarised in Table 1. Anti-I has been used widely previously on muscle sections, and reacts with both slow-twitch and slow-tonic fibres (Mascarello etal. 1982; Rowlerson et al. 1983, 1985, 1988) although on immunoblotting the reaction with tonic myosin is clearly weaker (Fig. i). It gives no significant reaction with embryonic or neonatal myosins. Antitonic and anti-NE were raised recently; the preparation and testing of anti-NE has been described fully elsewhere (Scapolo et al. 1991), and anti-tonic is described below. Myotubes were identified as primary or secondary on morphological grounds, including their arrangement in clusters, diameter and profile in transverse section. Immature fibres or myotubes reacting strongly with anti-I and weakly with anti-NE were regarded as 'slow', and those giving the inverse reaction as 'fast'.
Preparation and testing of Anti-tonic antibody. The tonic fibre isoform of myosin was obtained by brief high salt extraction of myofibrils made from adult chicken anterior latissimus dorsi (ALD) muscle. The extract was first clarified by centrifugation, and the myosin then precipitated by dialysis against a very low ionic strength solution. Myosin heavy chains were extracted from this crude myosin sample by SDS-PAGE on 8% gels. The myosin heavy chain bands were revealed by potassium acetate staining and then cut out. These gel pieces were then ground to a sIurry in 0.1 M Na2 CO3 (pH 9.3) and stored in this solution for 48 h. The resulting supernatant containing extracted heavy chains was then adjusted to pH 7.4 and used to immunise New Zealand white rabbits after a sample had been examined by SDS-PAGE followed by silver staining to check that there was no contamination by other proteins. Briefly, the immunisation schedule consisted of three sub-cutaneous injections (at multiple sites) of myosin (
