Neuroscience Letters, 109 (1990) 18 22 Elsevier ScientificPublishers Ireland Ltd,
18
NSL 06621
Myosin heavy chain expression in developing rat intrafusal muscle fibers Jan Kucera 1 and Jon Walro 2 1Department of Neurology, School of Medicine, Boston University, Boston, MA 02118 (U.S.A) and :Department of Anatorny, College qf Medicine, Northeastern Ohio Universities, Rootstown, OH 44272 (U.S.A.;
(Received 7 August 1989: Revised version received 18 September 1989; Accepted 25 September 1989) Key wordsv Spindledevelopment: Myosin isoform; Intrafusal fiber
The immunocytochemical expression of several isoforms of myosin heavy chains (MHC) was determined in developing intrafusal and extrafusal fibers of the soleus muscle of prenatal and postnatal rats. At the onset of spindle assembly, both bag2 intrafusal myotubes and primary extrafusal myotubes bound a slow-twitch MHC antibody, whereas the bag~and chain myotubes expressed a fast-twitch MHC isoform identical to that expressed by secondary extrafusal myotubes. Subsequently, developing intrafusal fibers began to express unique myosin isoforms, and ceased to express some of the myosin isoforms present initially. The initial similarity in MHC composition of intrafusal and extrafusal fibers suggests that these two kinds of mammalian muscle cell originate from a common pool of bipotential myotubes. Differences in MHC expression by intrafusal and extrafusal fibers in adult muscles might result from the effect of sensory neurons on the developing intrafusal myotubes.
Intrafusal fibers of rat muscle spindles develop from undifferentiated myotubes when contacted by primary sensory axons during ontogeny [7, 14]. Mature intrafusal fibers express myosin isoforms that are not contained in extrafusal fibers of adult rats [5, 9 11]. However, whether intrafusal fibers express the spindle-specific myosins from the onset of their f o r m a t i o n , or whether they acquire these myofibrillar proteins in the course of their m a t u r a t i o n process has received only limited a t t e n t i o n [13]. The present study examined the patterns of expression of several isoforms of myosin heavy chains ( M H C s ) d u r i n g o n t o g e n y of intrafusal a n d extrafusal muscle fibers of the rat. A total of 12 fetal, n e o n a t a l a n d y o u n g adult offspring of 3 p r e g n a n t S p r a g u e Dawley rats were used. The day of a p p e a r a n c e o f sperm o n vaginal swabs was designated as day 0 o f gestation. G e s t a t i o n lasted 21-23 days, with 80% of rats delivering on day 22. The day o f birth was designated as p o s t n a t a l day 0. Rats were anesthetized Correspondence." J. Kucera, Division of Neurology (127), Veterans Administration Medical Center, 150 S. Huntington Ave., Boston, MA 02130, U.S.A.
0304-3940/90/$ 03.50 C(~:1990 Elsevier ScientificPublishers Ireland Ltd.
19 with sodium pentobarbital (35 mg/kg i.p.). Individual soleus muscles of postnatal rats or entire calf musculature of prenatal rats were removed, quenched in isopentane cooled to 160°C with liquid nitrogen, and cut transversely into serial sections of 8 am thickness in a cryostat. The sections were processed for either alkali-stable or acid-stable myofibrillar adenosine 5'-triphosphatase (mATPase) at pH 9.4, or were reacted with one of 4 monoclonal antibodies known to bind to different isoforms of MHCs [2, 5, 8]. Two antibodies were raised against chicken muscles, one (ALD58 or ATO) against the slow anterior latissimus dorsi [l 2] and the other (MF30 or ANT) against the mixed slow- and fast-twitch pectoralis major muscle [1]. Two antibodies were raised against mammalian muscles, one (NOQ7.5.4D or MST) against human slow-twitch muscle fibers [8] and the other (WBMHC-f or MFT) against rabbit fasttwitch muscle fibers [2]. Binding of the primary antibody was localized by an immunoperoxidase reaction utilizing the ABC (avidin-biotin-complex) method (Vectastain P4002 kit, Vector Labs., CA) and a substrate reagent containing diaminobenzidine and hydrogen peroxide [5]. The ATO and ANT antibodies bind to the slow-tonic and neonatal-twitch MHC isoforms, whereas the MST and MFT antibodies bind to the adult slow-twitch or fast-twitch MHC isoforms, respectively, in rat intrafusal and extrafusal fibers [2, 5, 8]. Spindles were recognized as encapsulations of small-diameter muscle fibers. The encapsulated fibers were classified as nuclear bag or nuclear chain intrafusal fibers according to the arrangement of equatorial nuclei, fiber size, and sequence of development. Bag fibers have more equatorial myonuclei, are thicker and longer, and develop earlier than chain fibers [7]. Bag2 fibers were discriminated from bagl fibers according to three criteria [5, 6, 7]: (i) bag2 fibers have greater cross-sectional areas and are longer; (ii) bag2 fibers display more nuclear profiles at the equator; and (iii) bag2 fibers are the first intrafusal fibers to form. Patterns of mATPase staining were also used to distinguish bagl, bag2 and chain fibers in postnatal spindles [5]. Spindles were subdivided into the central (equatorial and juxtaequatorial) and polar regions
[5]. Two specimens of the soleus muscle each obtained at days 18-21 of gestation (G) and at postnatal days (P) 0, 2, 4 and 60 were examined for binding of the MHC antibodies by extrafusal and intrafusal fibers. Developing soleus muscles contained large (primary) extrafusal myotubes surrounded by medium and/or small (secondary) extrafusal myotubes. All extrafusal myotubes of prenatal and neonatal muscles bound ANT, but not ATO. Primary myotubes also strongly bound MST, whereas the secondary myotubes bound either MFT or both MFT and MST. Extrafusal fibers of adult muscles bound either MST (type I fibers) or MFT (type IIA fibers) or both MFT and MST (type IIC fibers), but not ATO. Similar patterns of MHC expression have been reported previously in rats [8]. The earliest stage at which spindles can be identified in the rat soleus muscle is G 18 [7]. At this stage spindles consist of a single myotube, which eventually matures into the bag2 fiber [6, 7]. The G18 bag2 myotubes strongly bound MST and ANT, but not ATO or MFT, similar to the primary extrafusal myotubes. The myotubes destined to become the bagl fiber assemble on GI9 [6, 7]. These myotubes expressed
20
MFT and ANT, but not ATO or MST, similar to some secondary extrafusal myotubes. The two myotubes which eventually become chain fibers assemble between G21 and P4 [6, 7]. All chain myotubes of prenatal or neonatal spindles strongly expressed MFT and ANT, similar to nascent bagl fibers and secondary extrafusal myotubes. Some chain myotubes were also reactive to MST, analogous to some of the secondary extrafusal myotubes (Fig. 1).
Fig. 1. Reactivity of intrafusal (A D) and extrafusal (E, F) fibers to ATO, ANT, MST and MFT antibodies in a soleus muscle removed at day 22 of gestation. The extrafusal myofibers bind either a slow-twitch (s) antibody, or a fast twitch (f) antibody, or both (m). The bag1 (b0 and chain (c) intrafusal myotubes (C, D) have the same pattern of antibody binding as do some of the extrafusal myofibers (E, F). The bag2 (b2) intrafusat myotube, but none of the extrafusal myofibers binds a slow-tonic antibody (A). Bars= 10 /~m.
21
bag2 Vf.////////////////////////i.
~ / i / / / / / / / / / / / / / / / / / / .
bag, ~ffJffff~/~ 7//////~,
I
ATO
~11 ~f/ff/ff~
ANT MST MFT
I
ATO
I
:'f////////.,~
ANT
MST
r,,'/////.,'////////.,"///..;
"///v'X/////A
MFT
1st c h a i n ATO
ANT
I ,'///.¢//Z,
;~///////////////Z
MST
I
MFT
2rid chain ATO
m I ~] t-q
days
strong moderate absent
,
u
f
n
18
19
20
21
f
22 (0) 1
ANT
MST MFT
V/fZ
I i
f
2
3
,
4
adult
Fig. 2. Schematicrepresentationof the reactivityof bag~,bag2, and chain intrafusalfibers to ATO, ANT, MST and MFT antibodiesin the course of spindle prenatal and postnatal development.Key to intensity of stainingis shown at lowerleft. Timescaleis in days before (left)and after birth (right). Patterns of M H C expression changed in the course of spindle development. Strong ATO reactivity was visible in the juxtaequatorial region of bag: and bag2 fibers at birth, and spread into the polar regions of bag1 fibers with increasing maturity. The ATO reactivity appeared concurrently with the attenuation or loss of MFT and ANT reactivity in bagl fibers, and MST binding in bag2 fibers. In contrast, developing chain fibers retained the two M H C isoforms (ANT and MFT) present at the onset of their assembly. As a result of the differential patterns of MHC maturation, bag2 fibers co-expressed 3, chain fibers 2, and bagn fibers only one of the MHC isoforms in the central region of the adult spindles (Fig. 2). Whether the intrafusal myotubes originate from the same pool of myoblasts as do the extrafusal fibers, or whether they originate from precursor muscle cells of a separate lineage is controversial [6, 7, 13, 14]. Nascent bag:, bag2, and chain myotubes of prenatal spindles expressed MHCs similar to those contained in primary or secondary extrafusal myotubes of the same stage of development. The MHC isoforms specific for adult intrafusal fibers, such as ATO, were not detectable during the initial assembly of intrafusal myotubes. Rather, this isoform appeared during the subsequent stages of spindle development. The initial similarity in MHC content between intrafusal and extrafusal myotubes is consistent with the hypothesis [6] that intrafusal fibers arise from the same precursor muscle cells that give rise to the extrafusal fibers. Differences in M H C expression between intrafusal and extrafusal fibers likely arise from a specific regulatory effect of an environmental cue on the differentiating intrafusal myotubes. Differentiation of intrafusal myotubes is either arrested or altered by ablating innervation to prenatal or neonatal rat spindles [4, 14], thus the nerve supply probably regulates expression of some MHCs in spindles. Afferents rather than efferents may regulate the MHC of intrafusal fibers because neonatal
22 d e a f f e r e n t a t i o n , b u t n o t d e e f f f e r e n t a t i o n , p r e c l u d e s t h e e x p r e s s i o n o f A T ( ) in s p i n d l e s [3, 4, 14]. H o w e v e r , t h e r o l e o f o t h e r f a c t o r s , s u c h as m u s c l e a c t i v i t y o r t e n s i o n , o r i n t r a c a p s u l a r m i l i e u , in m y o s i n d i f f e r e n t i a t i o n o f i n t r a f u s a l f i b e r s r e m a i n s to b e elucidated. This study was supported by an NIH Grant NS25796 to J.K., and by the Veterans Administration. Hybridoma
ALD58
and MF30
were provided by the Developmental
B a n k in I o w a . N O Q 7 . 5 . 4 D
and WBMHC-f
Studies
w e r e gifts f r o m M . R . F i t z -
simons and W. Brown respectively, from the U.K. Technical assistance was provided b y C. H e r r e r i a .
I Bader, D., Masaki, T. and Fischman, D.A., lmmunochemical analysis of myosin heavy chains during avian myogenesis in vivo and in vitro, J. Cell Biol., 95 (1982) 763 770. 2 Ecob-Prince, M., Hill, M. and Brown, W., Immunocytochemical demonstration of myosin heavy chain expression in human muscle, J. Neurol. Sci., 91 (1989) 71 78. 3 te Kronnie, G., Donselaar, Y., Soukup, T. and Zelena, J., Development of immunohistochemical characteristics of intrafusal fibres in normal and deefferented rat muscle spindles, Histochemistry, 74 (1982) 355 366. 4 Kucera, J. and Walro, J.M., The effect of neonatal deafferentation or deefferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat, Histochemistry, 90 (1988) 151 160. 5 Kucera, J. and Walro, J.M., Postnatal expression of myosin heavy chains in muscle spindles of the rat, Anat. Embryol., 179 (1989) 369 376. 6 Kucera, J., Walro~ J.M. and Reichler~ J., lnnervation of developing intrafusal muscle fibers in the rat, Am. J. Anat., 183 (1988) 344 358. 7 Milburn, A., The early development of muscle spindles in the rat, J. Cell Sci., 12 (1973) 175- 195. 8 Narusawa, M., Fitzsimons, R.B., Izumo, S., Nadal-Ginard, B., Rubinsteim N.A. and Kelly, A.M., Slow myosin in developing rat skeletal muscle, J. Cell Biol., 104 (1987) 447 459. 9 Pierobon-Bormioli, S., Sartore, S., Vitadello, M. and Schiaffino, S., "Slow' myosins in vertebrate skeletal muscle, J. Cell Biol., 85 (1980) 672 681. 10 Rowlcrson, A., Gorza, L. and Schiaffino, S., Immunohistochemical identification of spindle fiber types in mammalian muscle using type-specific antibodies to isoforms of myosin. In I.A. Boyd and M.H. Gladden (Eds.), The Muscle Spindle, MacMillan, London, 1985, pp. 29 34. I I Rowlerson, A., Early type-differentiation of intrafusal fibers. In P. Hnik, T. Soukup, R. Vejsada, and J. Zelena (Eds.), Mechanoreceptors: development, structure and function, Plenum, New York, 1988, pp. 45 56. 12 Shafiq, S.A, Shimuzu, T. and Fischmam D.A., Heterogeneity of type I skeletal muscle fibers revealed by monoclonal antibody to slow myosin, Muscle Nerve, 7 (1984) 38(~387. 13 Thornell, L.-E., Grove, B.K., Pedrosa, F., Butler-Browne, G.S., Dhoot, G.K. and Fischman, D.A., Expression of slow tonic myosin in muscle spindle fibers early in mammalian development. In F. Stockdale (Ed.), UCLA Symposia on Cellular and Molecular Biology of Muscle Development, Vol. 93, Liss, New York, 1988, pp. 271 480. 14 Zelena, J. and Soukup, T., The differentiation of intrafusal fibers types in rat muscle spindles after motor denervation, Cell Tissue Res., 153 (1974) 115 136.
18
NSL 06621
Myosin heavy chain expression in developing rat intrafusal muscle fibers Jan Kucera 1 and Jon Walro 2 1Department of Neurology, School of Medicine, Boston University, Boston, MA 02118 (U.S.A) and :Department of Anatorny, College qf Medicine, Northeastern Ohio Universities, Rootstown, OH 44272 (U.S.A.;
(Received 7 August 1989: Revised version received 18 September 1989; Accepted 25 September 1989) Key wordsv Spindledevelopment: Myosin isoform; Intrafusal fiber
The immunocytochemical expression of several isoforms of myosin heavy chains (MHC) was determined in developing intrafusal and extrafusal fibers of the soleus muscle of prenatal and postnatal rats. At the onset of spindle assembly, both bag2 intrafusal myotubes and primary extrafusal myotubes bound a slow-twitch MHC antibody, whereas the bag~and chain myotubes expressed a fast-twitch MHC isoform identical to that expressed by secondary extrafusal myotubes. Subsequently, developing intrafusal fibers began to express unique myosin isoforms, and ceased to express some of the myosin isoforms present initially. The initial similarity in MHC composition of intrafusal and extrafusal fibers suggests that these two kinds of mammalian muscle cell originate from a common pool of bipotential myotubes. Differences in MHC expression by intrafusal and extrafusal fibers in adult muscles might result from the effect of sensory neurons on the developing intrafusal myotubes.
Intrafusal fibers of rat muscle spindles develop from undifferentiated myotubes when contacted by primary sensory axons during ontogeny [7, 14]. Mature intrafusal fibers express myosin isoforms that are not contained in extrafusal fibers of adult rats [5, 9 11]. However, whether intrafusal fibers express the spindle-specific myosins from the onset of their f o r m a t i o n , or whether they acquire these myofibrillar proteins in the course of their m a t u r a t i o n process has received only limited a t t e n t i o n [13]. The present study examined the patterns of expression of several isoforms of myosin heavy chains ( M H C s ) d u r i n g o n t o g e n y of intrafusal a n d extrafusal muscle fibers of the rat. A total of 12 fetal, n e o n a t a l a n d y o u n g adult offspring of 3 p r e g n a n t S p r a g u e Dawley rats were used. The day of a p p e a r a n c e o f sperm o n vaginal swabs was designated as day 0 o f gestation. G e s t a t i o n lasted 21-23 days, with 80% of rats delivering on day 22. The day o f birth was designated as p o s t n a t a l day 0. Rats were anesthetized Correspondence." J. Kucera, Division of Neurology (127), Veterans Administration Medical Center, 150 S. Huntington Ave., Boston, MA 02130, U.S.A.
0304-3940/90/$ 03.50 C(~:1990 Elsevier ScientificPublishers Ireland Ltd.
19 with sodium pentobarbital (35 mg/kg i.p.). Individual soleus muscles of postnatal rats or entire calf musculature of prenatal rats were removed, quenched in isopentane cooled to 160°C with liquid nitrogen, and cut transversely into serial sections of 8 am thickness in a cryostat. The sections were processed for either alkali-stable or acid-stable myofibrillar adenosine 5'-triphosphatase (mATPase) at pH 9.4, or were reacted with one of 4 monoclonal antibodies known to bind to different isoforms of MHCs [2, 5, 8]. Two antibodies were raised against chicken muscles, one (ALD58 or ATO) against the slow anterior latissimus dorsi [l 2] and the other (MF30 or ANT) against the mixed slow- and fast-twitch pectoralis major muscle [1]. Two antibodies were raised against mammalian muscles, one (NOQ7.5.4D or MST) against human slow-twitch muscle fibers [8] and the other (WBMHC-f or MFT) against rabbit fasttwitch muscle fibers [2]. Binding of the primary antibody was localized by an immunoperoxidase reaction utilizing the ABC (avidin-biotin-complex) method (Vectastain P4002 kit, Vector Labs., CA) and a substrate reagent containing diaminobenzidine and hydrogen peroxide [5]. The ATO and ANT antibodies bind to the slow-tonic and neonatal-twitch MHC isoforms, whereas the MST and MFT antibodies bind to the adult slow-twitch or fast-twitch MHC isoforms, respectively, in rat intrafusal and extrafusal fibers [2, 5, 8]. Spindles were recognized as encapsulations of small-diameter muscle fibers. The encapsulated fibers were classified as nuclear bag or nuclear chain intrafusal fibers according to the arrangement of equatorial nuclei, fiber size, and sequence of development. Bag fibers have more equatorial myonuclei, are thicker and longer, and develop earlier than chain fibers [7]. Bag2 fibers were discriminated from bagl fibers according to three criteria [5, 6, 7]: (i) bag2 fibers have greater cross-sectional areas and are longer; (ii) bag2 fibers display more nuclear profiles at the equator; and (iii) bag2 fibers are the first intrafusal fibers to form. Patterns of mATPase staining were also used to distinguish bagl, bag2 and chain fibers in postnatal spindles [5]. Spindles were subdivided into the central (equatorial and juxtaequatorial) and polar regions
[5]. Two specimens of the soleus muscle each obtained at days 18-21 of gestation (G) and at postnatal days (P) 0, 2, 4 and 60 were examined for binding of the MHC antibodies by extrafusal and intrafusal fibers. Developing soleus muscles contained large (primary) extrafusal myotubes surrounded by medium and/or small (secondary) extrafusal myotubes. All extrafusal myotubes of prenatal and neonatal muscles bound ANT, but not ATO. Primary myotubes also strongly bound MST, whereas the secondary myotubes bound either MFT or both MFT and MST. Extrafusal fibers of adult muscles bound either MST (type I fibers) or MFT (type IIA fibers) or both MFT and MST (type IIC fibers), but not ATO. Similar patterns of MHC expression have been reported previously in rats [8]. The earliest stage at which spindles can be identified in the rat soleus muscle is G 18 [7]. At this stage spindles consist of a single myotube, which eventually matures into the bag2 fiber [6, 7]. The G18 bag2 myotubes strongly bound MST and ANT, but not ATO or MFT, similar to the primary extrafusal myotubes. The myotubes destined to become the bagl fiber assemble on GI9 [6, 7]. These myotubes expressed
20
MFT and ANT, but not ATO or MST, similar to some secondary extrafusal myotubes. The two myotubes which eventually become chain fibers assemble between G21 and P4 [6, 7]. All chain myotubes of prenatal or neonatal spindles strongly expressed MFT and ANT, similar to nascent bagl fibers and secondary extrafusal myotubes. Some chain myotubes were also reactive to MST, analogous to some of the secondary extrafusal myotubes (Fig. 1).
Fig. 1. Reactivity of intrafusal (A D) and extrafusal (E, F) fibers to ATO, ANT, MST and MFT antibodies in a soleus muscle removed at day 22 of gestation. The extrafusal myofibers bind either a slow-twitch (s) antibody, or a fast twitch (f) antibody, or both (m). The bag1 (b0 and chain (c) intrafusal myotubes (C, D) have the same pattern of antibody binding as do some of the extrafusal myofibers (E, F). The bag2 (b2) intrafusat myotube, but none of the extrafusal myofibers binds a slow-tonic antibody (A). Bars= 10 /~m.
21
bag2 Vf.////////////////////////i.
~ / i / / / / / / / / / / / / / / / / / / .
bag, ~ffJffff~/~ 7//////~,
I
ATO
~11 ~f/ff/ff~
ANT MST MFT
I
ATO
I
:'f////////.,~
ANT
MST
r,,'/////.,'////////.,"///..;
"///v'X/////A
MFT
1st c h a i n ATO
ANT
I ,'///.¢//Z,
;~///////////////Z
MST
I
MFT
2rid chain ATO
m I ~] t-q
days
strong moderate absent
,
u
f
n
18
19
20
21
f
22 (0) 1
ANT
MST MFT
V/fZ
I i
f
2
3
,
4
adult
Fig. 2. Schematicrepresentationof the reactivityof bag~,bag2, and chain intrafusalfibers to ATO, ANT, MST and MFT antibodiesin the course of spindle prenatal and postnatal development.Key to intensity of stainingis shown at lowerleft. Timescaleis in days before (left)and after birth (right). Patterns of M H C expression changed in the course of spindle development. Strong ATO reactivity was visible in the juxtaequatorial region of bag: and bag2 fibers at birth, and spread into the polar regions of bag1 fibers with increasing maturity. The ATO reactivity appeared concurrently with the attenuation or loss of MFT and ANT reactivity in bagl fibers, and MST binding in bag2 fibers. In contrast, developing chain fibers retained the two M H C isoforms (ANT and MFT) present at the onset of their assembly. As a result of the differential patterns of MHC maturation, bag2 fibers co-expressed 3, chain fibers 2, and bagn fibers only one of the MHC isoforms in the central region of the adult spindles (Fig. 2). Whether the intrafusal myotubes originate from the same pool of myoblasts as do the extrafusal fibers, or whether they originate from precursor muscle cells of a separate lineage is controversial [6, 7, 13, 14]. Nascent bag:, bag2, and chain myotubes of prenatal spindles expressed MHCs similar to those contained in primary or secondary extrafusal myotubes of the same stage of development. The MHC isoforms specific for adult intrafusal fibers, such as ATO, were not detectable during the initial assembly of intrafusal myotubes. Rather, this isoform appeared during the subsequent stages of spindle development. The initial similarity in MHC content between intrafusal and extrafusal myotubes is consistent with the hypothesis [6] that intrafusal fibers arise from the same precursor muscle cells that give rise to the extrafusal fibers. Differences in M H C expression between intrafusal and extrafusal fibers likely arise from a specific regulatory effect of an environmental cue on the differentiating intrafusal myotubes. Differentiation of intrafusal myotubes is either arrested or altered by ablating innervation to prenatal or neonatal rat spindles [4, 14], thus the nerve supply probably regulates expression of some MHCs in spindles. Afferents rather than efferents may regulate the MHC of intrafusal fibers because neonatal
22 d e a f f e r e n t a t i o n , b u t n o t d e e f f f e r e n t a t i o n , p r e c l u d e s t h e e x p r e s s i o n o f A T ( ) in s p i n d l e s [3, 4, 14]. H o w e v e r , t h e r o l e o f o t h e r f a c t o r s , s u c h as m u s c l e a c t i v i t y o r t e n s i o n , o r i n t r a c a p s u l a r m i l i e u , in m y o s i n d i f f e r e n t i a t i o n o f i n t r a f u s a l f i b e r s r e m a i n s to b e elucidated. This study was supported by an NIH Grant NS25796 to J.K., and by the Veterans Administration. Hybridoma
ALD58
and MF30
were provided by the Developmental
B a n k in I o w a . N O Q 7 . 5 . 4 D
and WBMHC-f
Studies
w e r e gifts f r o m M . R . F i t z -
simons and W. Brown respectively, from the U.K. Technical assistance was provided b y C. H e r r e r i a .
I Bader, D., Masaki, T. and Fischman, D.A., lmmunochemical analysis of myosin heavy chains during avian myogenesis in vivo and in vitro, J. Cell Biol., 95 (1982) 763 770. 2 Ecob-Prince, M., Hill, M. and Brown, W., Immunocytochemical demonstration of myosin heavy chain expression in human muscle, J. Neurol. Sci., 91 (1989) 71 78. 3 te Kronnie, G., Donselaar, Y., Soukup, T. and Zelena, J., Development of immunohistochemical characteristics of intrafusal fibres in normal and deefferented rat muscle spindles, Histochemistry, 74 (1982) 355 366. 4 Kucera, J. and Walro, J.M., The effect of neonatal deafferentation or deefferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat, Histochemistry, 90 (1988) 151 160. 5 Kucera, J. and Walro, J.M., Postnatal expression of myosin heavy chains in muscle spindles of the rat, Anat. Embryol., 179 (1989) 369 376. 6 Kucera, J., Walro~ J.M. and Reichler~ J., lnnervation of developing intrafusal muscle fibers in the rat, Am. J. Anat., 183 (1988) 344 358. 7 Milburn, A., The early development of muscle spindles in the rat, J. Cell Sci., 12 (1973) 175- 195. 8 Narusawa, M., Fitzsimons, R.B., Izumo, S., Nadal-Ginard, B., Rubinsteim N.A. and Kelly, A.M., Slow myosin in developing rat skeletal muscle, J. Cell Biol., 104 (1987) 447 459. 9 Pierobon-Bormioli, S., Sartore, S., Vitadello, M. and Schiaffino, S., "Slow' myosins in vertebrate skeletal muscle, J. Cell Biol., 85 (1980) 672 681. 10 Rowlcrson, A., Gorza, L. and Schiaffino, S., Immunohistochemical identification of spindle fiber types in mammalian muscle using type-specific antibodies to isoforms of myosin. In I.A. Boyd and M.H. Gladden (Eds.), The Muscle Spindle, MacMillan, London, 1985, pp. 29 34. I I Rowlerson, A., Early type-differentiation of intrafusal fibers. In P. Hnik, T. Soukup, R. Vejsada, and J. Zelena (Eds.), Mechanoreceptors: development, structure and function, Plenum, New York, 1988, pp. 45 56. 12 Shafiq, S.A, Shimuzu, T. and Fischmam D.A., Heterogeneity of type I skeletal muscle fibers revealed by monoclonal antibody to slow myosin, Muscle Nerve, 7 (1984) 38(~387. 13 Thornell, L.-E., Grove, B.K., Pedrosa, F., Butler-Browne, G.S., Dhoot, G.K. and Fischman, D.A., Expression of slow tonic myosin in muscle spindle fibers early in mammalian development. In F. Stockdale (Ed.), UCLA Symposia on Cellular and Molecular Biology of Muscle Development, Vol. 93, Liss, New York, 1988, pp. 271 480. 14 Zelena, J. and Soukup, T., The differentiation of intrafusal fibers types in rat muscle spindles after motor denervation, Cell Tissue Res., 153 (1974) 115 136.
