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Myogenic defect in muscular dystrophy of the chicken
DEVELOPMENTAL
BIOLOGY
Myogenic
48,
Defect
T. A. LINKHART,’ Department
(1976)
447-457
of Avian
in Muscular
G. W. YEE, Sciences,
Dystrophy
P. S. NIEBERG,
University
Accepted
of Californm,
September
of the Chicken AND B. W. WILSON Davis,
California
95616
25,1975
Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at 3’12 days of incubation (stage 19-20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves. INTRODUCTION
Several inherited neuromuscular abnormalities such as the muscular dystrophies have been proposed to arise from defects in trophic regulation (7, 19, 35, 451, the complex group of interactions between nerve and muscle that establish and maintain many properties of skeletal muscles (10, 15, 24, 26). Recently, there has been much interest in whether the primary defects in neuromuscular disorders are expressed in the muscle fibers themselves, in the nerves that innervate them or in other cells affecting nerve and muscle function (18, 35, 46). Inherited muscular dystrophy of the chicken is a neuromuscular disorder thought to arise from abnormal trophic regulation. Expression of the codominant gene (am) affects the development of several properties of fast twitch glycolytic skeletal muscle fibers, a process which is known to be regulated by trophic nervemuscle interactions (3, 8, 11, 31, 51, 52). In addition, there are alterations in biochemical and physiological properties of my’ Present University
address: Department of Washington, Seattle,
of Biochemistry, Wash. 98105. 447
Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
oneural transmission (1, 30, 32). The present investigation was undertaken to determine whether such characteristics of inherited muscular dystrophy of the chicken are due to a failure of nerves to maintain and regulate the muscles they innervate or whether the muscles themselves fail to respond to trophic influences of their nerves. Limb buds were transplanted at stages 19-20 (25) between normal and dystrophic chick embryos. Muscles of the transplanted limbs developed and became innervated by nerves and subject to the systemic regulation of the hosts. Donor and host wing muscles were analyzed several weeks after hatching for several properties characteristic of dystrophic muscles. Several distinctive electrophysiological, histochemical and enzymatic differences between normal and dystrophic muscles were selected as markers of the phenotypic expression of the dystrophic gene: From a few weeks after hatching, dystrophic muscles exhibit abnormal electromyographic (EMG) patterns, particularly repetitive electrical discharges in response to mechanical stimulation similar to those found in myotonia of human muscle (29)
448
DEVELOPMENT
BIOLOGY
and easily distinguishable from brief “insertion potentials” characteristic of normal muscles. Affected muscles of dystrophic chickens maintain high activity, extrajunctional localization and low molecular weight isozymes of acetylcholinesterase (AChE) characteristic of embryonic chick muscles (51). Dystrophic muscles also have high nonspecific cholinesterase (BChE) activity (52). In addition, lactic dehydrogenase (LDH) activity is low (8, 311, succinic dehydrogenase (SDH) activity is high (3, 111, and average fiber diameter is increased in muscles with dystrophy. We briefly reported in a previous article (34) that average fiber diameter and AChE properties were associated with the genotype of the donor limb in a series of transplantation experiments. In this report, AChE levels and localizations are studied in more detail and the EMG patterns and LDH and SDH activities of donor and host muscles are examined. The results suggest that subsequent to stages 19-20, expression of all abnormalities of dystrophic muscle examined in the study is a property of limb tissue, and that nerves of dystrophic chickens are capable of maintaining and regulating genetically normal muscle. METHODS
Lines of Birds
Used
Homozygous dystrophic embryos were from Line 413 maintained by the Department of Avian Sciences, University of California, Davis, California. This line is primarily of New Hampshire background and was recently derived from homozygous dystrophic Line 304 which had been selected for several years for early onset of the disorder and is characterized by pronounced muscle fiber hypertrophy (36). Birds of Line 413 also exhibit hypertrophy of muscles affected by the dystrophy. The normal embryos were from a commercial White Leghorn flock (Donsing Hatcheries, Rio Linda, California). Limb
Bud Transplantation
Embryos
were
incubated
for 3l/2 days
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48, 1976
(Jamesway 252B incubator, 37&C, 58% humidity), candled, laid on their sides, and cooled to room temperature. Approximately 1.5 ml of albumen was removed with a syringe and curved 13 gauge (Horsely Venoclysis) needle inserted through a small hole in the pointed end of each egg, which was then sealed with paraffin. A l-cm2 hole was cut in the side of each egg and the embryo was staged (25). Only stages 19-20 were used. The vitelline membrane was peeled back, and the chorionic and amnionic membranes were opened with watchmaker’s forceps (at the seroamniotic connection) in the region of the right wing limb bud. The wing bud was severed as closely as possible to the body with fine black glass needles and exchanged with one from another embryo. The donor wing bud was held in place on the host embryo by two or three fine black glass tacks, with normal position and orientation maintained. Eggs were placed under a warming lamp for 2 to 3 hr while healing of the limb to the host occurred. The tacks were removed, the chorion and amnion pinched closed, and 0.1-0.2 ml of amphotericin B (12.5 pg/ml) and penicillin-streptomycin (250 units/ml each) in Tyrode’s solution were added. The eggs were sealed with parafilm and incubated on their sides on an egg flat which was rocked daily by elevating one side of the flat. Four types of operations were performed: normal autotransplants were made by removing and reattaching the limb buds of normal embryos; normal limb buds were transplanted onto normal embryos and onto dystrophic embryos; dystrophic limb buds were transplanted onto normal embryos. Analyses Monopolar clinical electromyographic (EMG) electrodes (hand-held) coupled to a Beckman EMG preamplifier and Dynagraph recorder were used to examine muscles of host and donor wings. Electromyographic analysis was performed with light pentabarbital (IV) anesthesia 1 day
LINKHART
et
al.,
Muscular
Dystrophy
of the Chicken
449
between donor and host muscles were perbefore each bird was sacrificed. Electrical formed using Student’s t test (2). discharges elicited by insertion or slight Frozen cross sections (12 pm) were cut in movement (1 mm) of the electrode needle a cryostat at - 18°C within 1 day of sacrificwere monitored on an oscilloscope, reing the animal. Sections air-dried on glass corded on magnetic tape (TEAC Data Casslides were incubated for succinic dehydrosette Recorder), and photographed later genase activity (41) or for cholinesterase from playback of the tape record. activity (52) or were fixed (4% buffered Biceps muscles were usually used for formalin) and stained with hematoxylin analyses. The superficial pectoralis and and eosin. the extensor carpi radialis were also analyzed in two birds of each set. The birds RESULTS were sacrificed by exsanguination at 6 to General Appearance 25 weeks after hatching. Limb muscles were immediately removed and placed in Approximately 10% of the operated emice-cold phosphate-buffered physiological bryos hatched, dystrophic embryos having saline (pH 7.4). A sample from the center a lower survival rate than normal emof the muscle was frozen in iso-pentanebryos. Most of the embryos died within 48 liquid nitrogen for histochemistry and his- hr of surgery. Donor limbs were morphotology. The remainder of each muscle was logically normal in most of the embryos stripped of connective tissue and homoge- which died after 5 days of incubation. Alnized (Teflon pestle tissue grinder, A. H. most all of the operated chicks that Thomas, Philadelphia, Pa.) in cold phos- hatched had functional transplanted limbs phate-buffered saline (pH 7.4; l/4, wet of normal size and shape. From 2 to 3 weeks after hatching, genetically dysw/v). Homogenates were centrifuged trophic wings, whether of host or donor (5OOg, 20 min) and the superantants were origin, began to exhibit functional impairused for subsequent enzyme assays. ment characteristic of muscular dystrophy Cholinesterase activities of samples frozen less than one week and thawed once, of the chicken. The muscles of these limbs, were assayed by the spectrophotometric including the breast muscles near the method of Ellman et al., (17). Specific ace- shoulder of dystrophic limbs, were grossly tylcholinesterase (AChE) and nonspecific hypertrophied and appeared rigid from pseudocholinesterase (BChE) activities chronic contraction. Genetically normal wings retained normal functions. Dyswere distinguished in spectrophotometric assays, cryostat sections, and acrylamide trophic hosts, unable to flex their own ungels by the use of specific substrates and operated left wings, were able to flex their inhibitors. AChE activity is defined as ace- transplanted normal right wings. tylthiocholine hydrolysis in the presence of In about half the hatched birds, the wing derived from a donor embryo aplop4 M iso-OMPA (tetraisopropyl-pyrophosphoramide, Sigma, St. Louis, MO.) an peared to be rejected 4 weeks or longer inhibitor of BChE. BChE activity is de- after hatching. These wings became swollen and exudative, similar to the situation fined as butyrylthiocholine hydrolysis. AChE enzymes of the chicken hydrolyze described by Eastlick and Chehak (16) for acetylthiocholine much more rapidly than hatched chicks possessing transplanted butyrylthiocholine; BChE enzymes hydrolegs. The swelling and exudation occurred lyze both substrates at approximately the in both normal and dystrophic hosts and may have involved immunological reacsame rate (53). Lactic dehydrogenase activity of fresh samples was determined spec- tions of the hosts since none of eight autotrophotometrically as described by Carditransplants showed any indications of it 25 net et al. (8). Paired statistical analyses weeks after hatching. Only birds with mor-
450
DEVELOPMENT
BIOLOGY
phologically normal wings showing no signs of rejection were used for analysis. All birds analyzed had transplanted wings which appeared anatomically normal. Biceps muscles had normal points of origin and insertion and normal position of the blood vessels and nerves supplying them. Analysis of biceps muscles are reported below. Analysis of the extensor carpi radialis in the forewing and the pectoralis near its insertion on the humerus performed for two birds of each set of transplants gave similar results. Electromyographic
Potentials
Genetically dystrophic biceps, whether of donor (Fig. la) or host (Fig. Id) exhibited repetitive insertion potentials characteristic of dystrophic chick muscle. Potentials initially repeat at high frequencies and gradually subside. These often lasted as long as 5 sec. “Insertion activity” of normal muscle (whether of donor or host) consisted of short bursts of activity which abated rapidly (usually within 0.2 set; Figs. lb and 1~). Cholinesterase
Activities
Both AChE (reported previously, 34) and BChE activities were low in normal host biceps muscles, in autotransplanted normal biceps muscles, and in normal donor biceps muscles whether on normal or dystrophic hosts. Activities were significantly higher (p < 0.001) in dystrophic host biceps and dystrophic donor biceps on normal hosts (Table 1). High AChE activity in homogenates of genetically dystrophic chick muscles was accompanied by the presence of embryonic AChE isozymes and extrajunctional AChE localization. Genetically normal muscle fibers, whether innervated by nerves of normal or dystrophic chickens (Figs. 2A, B, D, and E) had AChE activity only at myoneural junctions and in the sarcoplasm near junctions. Many genetically dystrophic muscle fibers (Figs. 2C and F) had sarcoplasmic AChE activity not associated
VOLUME
48, 1976
with myoneural junctions. Figure 2 also illustrates the fiber hypertrophy characteristic of the dystrophic chickens used in the study (all sections in Fig. 2 are same magnification). Average fiber diameter of genetically dystrophic muscles was significantly greater than contralateral normal muscles regardless of which was donor or host (34). Lactic Dehydrogenase
Activity
LDH activities (Table 1) of genetically normal biceps whether of host or donor origin were significantly higher (p < 0.001) than contralateral dystrophic biceps while there were no differences between host and donor muscles in normal to normal transplants or normal autotransplants. Succinic
Dehydrogenase
Activity
Cross sections of host and donor biceps (Fig. 3) had low SDH activity in normal host (Figs. 3b and 3d) and donor (Figs. 3a and 3e) fibers (less than 1% of the fibers had positive SDH activity). Dystrophic host (Fig. 30 and donor (Fig. 3c) muscles had many fibers which were high in SDH activity. DISCUSSION
In this and in a brief previous report (34), analyses of the several properties characteristic of dystrophic muscle gave consistent results; genetically dystrophic muscle was phenotypically dystrophic, regardless of the genotype of the innervating nerves. Nerves of genetically dystrophic chickens supported development and maintenance of genetically normal muscle. The results demonstrate that major characteristics of inherited muscular dystrophy of the chicken are properties of the limb and its muscles and not of the host and its nerves after stages 19-20, the time when the transplants were performed. It is probable that the abnormality originates in the muscle fibers themselves, although the experiments reported cannot
LINKHART
et al..
Muscular
Dystrophy
of the Chicken
FIG. 1. Electromyography of donor and host biceps muscles. EGM recordings were made with two monopolar needle electrodes placed 2-3 mm apart in the muscle; a ground electrode was placed near the muscle. Slight movement of one of the needle electrodes is followed by a brief burst of electrical activity in normal muscle, by a prolonged repetitive discharge in dystrophic muscle. Potentials were recorded on magnetic tape; photographs were made from display of the tape record on an oscilloscope; the horizontal sweep was triggered by the onset of the electrical discharge. (Al, dystrophic donor in normal host: t B). contralateral normal host; CC), normal donor in dystrophic host; ID), contralateral dystrophic host. Note that the time base in A and D is five times longer than in (B) and (Cl.
rule out a primary or associated abnormality in other limb tissues, such as blood vessels and connective tissue cells, as has been proposed for Duchenne muscular dystrophy in humans (9, 27, 28). Abnormali-
ties in vascular and connective ments of the limbs of dystrophic have been described (4, 51, but on nerve and muscle functions Considering past evidence that
tissue elechickens their effect is unclear. dystrophy
452
DEVELOPMENTAL
ENZYME Transplant
ACTIVITIES
type
Age (weeks)
BIOLOGY
OF BICEPS
VOLUME
TABLE
1
MUSCLES
FROM
Number of birds
DONOR
48, 1976
AND
HOST
Biceps IllUSCk
WINGS”
A/r&/g
wet weight
AChE
BChE
LDH
l-25
8
Donor (N) Host (N)
1.1 + 0.8 1.4 + 0.7
0.39 + 0.16 0.35 L 0.23
3310 5 650 3120 c 560
6-10
4
Donor (N) Host (N)
1.5 * 1.0 1.0 + 0.3
0.59 + 0.17 0.55 + 0.10
2350 5 350 2580 -c 550
Dystrophic limb transplanted onto normal host
6-14
6
Donor (D) Host (N)
16.6 + 13.1* 1.2 f 0.3
8.6 k 4.8’ 0.7 f 0.4
970 2 390’ 2190 -t 520
Normal limb transplanted dystrophic host
6-8
5
Donor (N) Host (D)
2.1 + 0.7’ 13.1 + 4.7
1.0 + 0.8’ 7.6 A 2.8
3020 f 380’ 1820 t 480
Normal
autotransplant
Normal limb transplanted normal host
onto
onto
n Enzyme activities are averages + standard deviations of homogenates of biceps muscles from transplanted donor and contralateral host limbs. Fiber diameters are averages + standard deviations of 100 fibers from each biceps of two birds at 6 weeks of age. Statistically significant differences are indicated by *, P < 0.001 by Student’s t-distribution of paired variates.
of the chicken involves a defect in neurally mediated muscle maturation, the data support the idea that genetically dystrophic muscle in the chicken lacks the ability to respond to trophic influence of its innervating nerve. However, this study does not rule out the presence of abnormalities in the nerves that are not associated with the parameters we measured. For example, Albuquerque and Warnick (1) have reported presynaptic alterations in dystrophic chickens. Whether these arise from direct expression of dystrophy in the motor nerves or as secondary effects of the abnormal muscles which they innervate is not known, and may be resolved by electrophysiological studies of normal and dystrophic nerve and muscle combinations produced by limb bud transplantation (Michael Latinsky, personal communication). Whether neural influence before stages 19-20 affects subsequent phenotypic expression of the limb was not dealt with in our investigation. In a recent study, Vetrano and his colleagues (13, 47) transplanted neural tube sections from dystrophic to normal chick embryos at stage 14 (50 hr of incubation) and found that muscles of l&day normal host embryos had high thymidine kinase activity characteristic of dystrophic embryos (50). However, reciprocal transplants of normal neural tube to dystrophic hosts were not done and it was unclear whether the dys-
trophic neural transplants might have been contaminated with dystrophic myogenic cells. Regardless, our results show that nerves play no role after stages 19-20 in expression of several important properties of dystrophic chick muscle. “Preliminary results” of Cosmos and Butler (12) suggested that inherited muscular dystrophy of the chicken is myogenic. They found that normal muscle minces regenerated in dystrophic hosts and maintained normal histological appearance, lipid distribution, and succinic dehydrogenase activity. However, the report did not include results of normal to normal controls, host muscle “buds” regenerated together with donor muscle, and there was a “progressive loss of the transplanted tissue” when dystrophic muscle was transplanted into normal hosts. These problems were avoided in the present study. Limbs were transplanted at a stage in development preceding muscle differentiation (6, 28, 45) and nerve outgrowth and innervation (20,21, 22,23,44). Development of the transplanted limb occurred normally and the nerve-muscle combinations were allowed to mature to an age at which several different properties of dystrophic muscle were unambiguously expressed. Contamination of hosts with nerves of donor origin was not possible because the nerve cell bodies were located in the spinal cord. Contamination of donor limb buds with my-
F ‘IG. 2. Cytochemical localization of AChE activity in donor and host biceps muscles. Cross secti inc ubated with acetylthiocholine and lo-” M iso-OMPA. AChE appears as dark stain at motor endplates ext rajunctional activity (e) appears in the sarcoplasm of many fibers in genetically dystrophic muscle. nor ,mal donor in normal host; (B), contralateral normal host; (CI, dystrophic donor in normal host; con .tralateral normal host; (E), normal donor in dystrophic host; (F), contralateral dystrophic host. were taken with identical exposures under identical conditions. x 105. phc rtographs 453
I ml; I Ai, ( D:, All
FIG. 3. Histochemical localization of succinic dehydrogenase activity in donor and host biceps muscles. Cross sections were incubated for SDH activity with Na succinate and nitro blue tetrazolium. Activity appears as dark granules. SDH “positive” fibers (arrows) have more granules distributed throughout the sarcoplasm. (A), normal donor in normal host; (B), contralateral normal host; (0, dystrophic donor in normal host; CD), contralateral normal host; (E), normal donor in dystrophic host; (F), contralateral dystrophic host. All photographs were taken with identical exposures under identical conditions. x 230. 454
LINKHART
et al.,
Muscular
ogenic cells of the hosts was possible but the fact that the phenotypes of donor and host muscles were significantly different in transplants between normal and dystrophic embryos strongly suggests that such contamination was negligible. Whether inherited muscular dystrophies of humans and other mammals are myogenic or neurogenic in origin is unclear. Electrophysiological studies of motor neurons and neuromuscular transmission, transplantation of muscle minces or whole muscles, culture of normal and dystrophic nerve and muscle cells, and production of allophenic mosaics of normal and dystrophic mouse embryos have resulted in equivocal and often conflicting results (14, 35, 40, 42). There is little reason to expect that all muscular dystrophies have either a common molecular basis or a common cellular origin. Indeed, the dichotomy “nerve or muscle” may prove conceptually inadequate to deal with the complicated cell interactions that occur during development and maturation of normal and abnormal muscles. Although our investigations (32, 33, 39, 48, 49, 51, 531 strongly suggest that the lesion in regulation of dystrophic chick muscle lies in the ability of the muscles to respond to normal neural influence, the mechanism of regulation which is disrupted has not yet been identified. “Trophic factors” transferred from nerve to muscle, acetylcholine released by the nerve, and contractile activity itself have been proposed as mechanisms of trophic regulation (15,26,43). Recent evidence suggests that AChE activity and localization of major biochemical abnormalities in dystrophic chick muscle, are regulated in part by contractile activity (33, 39, 48, 49). Perhaps inherited muscular dystrophy of the chicken is caused by a failure of the muscle to respond to its own neurally mediated contractions. Supported in part by a grant from the Muscular Dystrophy Associations of America, Inc., and PHS Grant NS10957. Dr. T. A. Linkhart was a National
Dystrophy
of
the (‘hIcken
455
Science Foundation Predoctoral Trainee in Physiology. We gratefully acknowledge Dr. LJ. K. Abbott for her help and encouragement in development of the transplantation technique, Mr. F. Lantz and Mr. J. Adams for maintaining the experimental animals, Dr. it. G. Taylor and Dr. R. E. Burger for providing help, facilities, and equipment for the electromyographic analysis, Mr. J. Schenkel for his initial work in the study, and Dr. M. Rathbone and Mr. F. Vetrano for providing us with their unpublished results. REFERENCES 1. ALBUQUERQUE, E. X., and WARNICK, J. E. (1971). Electrophysiological observations in normal and dystrophic chicken muscles. Science 172, 1260-1263. 2. ALDER, H. L., and ROESSLER, E. B. (1964). “Introduction to Probability and Statistics,” 3rd ed. Freeman and Co., San Francisco, Calif. 3. ASHMORE, C. R., and DOERR, L. (1971). Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick. Exp. Nurology 30, 431-446. 4. ASHMORE, C. R., DOERR, L., and SOMES, R. G., JR. (1968). Microcirculation: Loss of an enzyme activity in chickens with hereditary muscular dystrophy. Science 160, 319-320. 5. ASMUNDSON, V. S., KRATZER, F. H., and JULIAN, L. M. (1966). Inherited myopathy in the chicken. Ann. N.Y. Acad. Sci. 138, 1-17. 6. BONNER, P. H., and HAUSCHKA, S. D. (19741. Clonal analysis of vertebrate myogenesis: I. Early developmental events in the chick limb. Develop. Biol. 37, 317-328. 7. BRADLEY, W. G. (1971). Nerve, muscle, and muscular dystrophy. Dev. Med. Child Neural. 13. 5288531. 8. CARDINET, G. H., FREEDLAND, R. A., TYLER, W. S., and JULIAN, L. M. (1972). Morphologic, histochemical, and quantitative enzyme study of hereditary avian muscular dystrophy. Amer. J. Vet. Res. 33, 1671-1684. 9. CAZZATO, G. (1968). Considerations about a possible role played by connective tissue proliferation and vascular disturbances in the pathogenesis of progressive muscular dystrophy. Europ. Neural. 1, 158-179. 10. CLOSE, D. R. (1972). Dynamic properties of mammalian skeletal muscle. Physiol. Rev. 52. 129-197. 11. COSMOS, E., and BUTLER, J. (1967). Differentiation of fiber types in muscle of normal and jystrophic chickens. In “Exploratory Conceit: in Muscular Dystrophy and Related Disorders” (A. T. Milhorat, ed.), pp. 197-204. Excerpta Medica, Amsterdam, The Netherlands.
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26. HARRIS, A. J. (19741. Inductive functions of the nervous system. Ann. Rev. Physiol. 36, 251305. 27. HATHAWAY, P. W., ENGEL, W. K., and ZELI~ WEGER, H. (19701. Experimental myopathy after microarterial embolization. Comparison with childhood X-linked pseudohypertrophic muscular dystrophy. Arch. Neurol. 22, 365378. 28. HILFER, S. R., SEARLS, R. L., and FONTE, V. G. (1973). An ultrastructural study of early myogenesis in the chick wing bud. Develop. Biol. 30, 374-391. 29. HOLLIDAY, T. A., VAN METER, J. R., JULIAN, L. M., and ASMUNDSON, V. S. (19651. Electromyography of chickens with inherited muscular dystrophy. Amer. J. Physiol. 209,871-876. 30. JEDRZEJCZYK, J., WIECKOWSKI, J., RYMASZEWSKA, T., and BARNARD, E. A. (1973). Dystrophic chicken muscle: Altered synaptic acetylcholinesterase. Science 180, 406-408. 31. KAPLAN, N. O., and CAHN, R. D. (1962). Lactic dehydrogenases and muscular dystrophy of the chick. Proc. Nat. Acad. Sci. USA 48, 2123-2130. 32. LEBEDA, F. J., WARNICK, J. E., and ALBUQUERQUE, E. X. (1974). Electrical and chemosensitive properties of normal and dystrophic chicken muscles. Erp. Neurol. 43, 21-37. 33. LINKHART, T. A., and WILSON, B. W. (1975). Role of muscle contraction in trophic regulation of chick muscle acetylcholinesterase activity. Exp. Neurol. 48, 557-568. 34. LINKHART, T. A., YEE, G. W., and WILSON, B. W. (19751. Myogenic defect in acetylcholinesterase regulation in muscular dystrophy of the chicken. Science 187, 549-551. 35. MCCOMAS, A. J., SICA, R. E. P., UFTON, A. R. M., and PETITO, F. (1974). Sick motoneurons and muscle disease. Ann. N.Y. Acad. Sci. 228, 261-279. 36. MCMURTY, S. L., JULIAN, L. M., and ASMUND SON, V. S. (1972). Hereditary muscular dystrophy of the chicken. Quantitative histopathological findings of the Pectoralis at 6 weeks of age. Arch. Path. 94, 217-224. 37. MENDELL, J. R., ENGEL, W. K., and DERRER, E. C. (19711. Duchenne muscular dystrophy: functional &hernia reproduces its characteristic lesions. Science 172, 1143-1145. 38. MENDELL, J. R., ENGEL, W. K., and DERRER, E. C. (1972). Increased plasma enzyme concentrations in rats with functional ischaemia of muscle provide a possible model of Duchenne muscular dystrophy. Nature (London) 239, 522524. 39. PATTERSON, G. T., and WILSON, B. W. (1975). Distribution of extrajunctional acetylcholinnesterase in muscle. I. Normal and dystrophic
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of the Chicken
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47. VETRANO, F. (19741. The role of the spinal chord in the pathogenesis of hereditary muscular dystrophy in the domestic fowl. Masters Thesis, McMaster University, Hamilton, Ontario, Canada. 48. WALKER, C. R., and WILSON, B. W. (19741. Regulation of AChE in muscle cultures with electrical stimulation. J. Cell Biol. 63, 362a. 49. WALKER, C. R., and WILSON, B. W. (19751. Control of acetylcholinesterase by activity of cultured muscle cells. Nature (London) 256,215216. 50. WEINSTOCK, I. M., and DJU, M. Y. (19671. Phosphorylation of thymidine in developing breast muscle of the normal and dystrophic chicken. Life Sci. 6, 797-802. 51. WILSON, B. W., KAPLAN, M. A., MERHOFF, W. C., and MORI, S. S. (19701. Innervation and the regulation of acetylcholinesterase activity during development of normal and dystrophic muscle. J. Exp. Zool. 174, 39-54. 52. WILSON, B. W., LINKHART, S. G., and NIEBERG, P. A. (1973a). Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. J. Exp. Zool. 186, 187-192. 53. WILSON, B. W., LINKHART, T. A., WALKER, C. R., and NIEBERG, P. A. (1973bl. Tissue cholinesterase in plasma of chick embryos and dystrophic chickens. J. Neural. Sci. 18. 333-350.
BIOLOGY
Myogenic
48,
Defect
T. A. LINKHART,’ Department
(1976)
447-457
of Avian
in Muscular
G. W. YEE, Sciences,
Dystrophy
P. S. NIEBERG,
University
Accepted
of Californm,
September
of the Chicken AND B. W. WILSON Davis,
California
95616
25,1975
Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at 3’12 days of incubation (stage 19-20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves. INTRODUCTION
Several inherited neuromuscular abnormalities such as the muscular dystrophies have been proposed to arise from defects in trophic regulation (7, 19, 35, 451, the complex group of interactions between nerve and muscle that establish and maintain many properties of skeletal muscles (10, 15, 24, 26). Recently, there has been much interest in whether the primary defects in neuromuscular disorders are expressed in the muscle fibers themselves, in the nerves that innervate them or in other cells affecting nerve and muscle function (18, 35, 46). Inherited muscular dystrophy of the chicken is a neuromuscular disorder thought to arise from abnormal trophic regulation. Expression of the codominant gene (am) affects the development of several properties of fast twitch glycolytic skeletal muscle fibers, a process which is known to be regulated by trophic nervemuscle interactions (3, 8, 11, 31, 51, 52). In addition, there are alterations in biochemical and physiological properties of my’ Present University
address: Department of Washington, Seattle,
of Biochemistry, Wash. 98105. 447
Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
oneural transmission (1, 30, 32). The present investigation was undertaken to determine whether such characteristics of inherited muscular dystrophy of the chicken are due to a failure of nerves to maintain and regulate the muscles they innervate or whether the muscles themselves fail to respond to trophic influences of their nerves. Limb buds were transplanted at stages 19-20 (25) between normal and dystrophic chick embryos. Muscles of the transplanted limbs developed and became innervated by nerves and subject to the systemic regulation of the hosts. Donor and host wing muscles were analyzed several weeks after hatching for several properties characteristic of dystrophic muscles. Several distinctive electrophysiological, histochemical and enzymatic differences between normal and dystrophic muscles were selected as markers of the phenotypic expression of the dystrophic gene: From a few weeks after hatching, dystrophic muscles exhibit abnormal electromyographic (EMG) patterns, particularly repetitive electrical discharges in response to mechanical stimulation similar to those found in myotonia of human muscle (29)
448
DEVELOPMENT
BIOLOGY
and easily distinguishable from brief “insertion potentials” characteristic of normal muscles. Affected muscles of dystrophic chickens maintain high activity, extrajunctional localization and low molecular weight isozymes of acetylcholinesterase (AChE) characteristic of embryonic chick muscles (51). Dystrophic muscles also have high nonspecific cholinesterase (BChE) activity (52). In addition, lactic dehydrogenase (LDH) activity is low (8, 311, succinic dehydrogenase (SDH) activity is high (3, 111, and average fiber diameter is increased in muscles with dystrophy. We briefly reported in a previous article (34) that average fiber diameter and AChE properties were associated with the genotype of the donor limb in a series of transplantation experiments. In this report, AChE levels and localizations are studied in more detail and the EMG patterns and LDH and SDH activities of donor and host muscles are examined. The results suggest that subsequent to stages 19-20, expression of all abnormalities of dystrophic muscle examined in the study is a property of limb tissue, and that nerves of dystrophic chickens are capable of maintaining and regulating genetically normal muscle. METHODS
Lines of Birds
Used
Homozygous dystrophic embryos were from Line 413 maintained by the Department of Avian Sciences, University of California, Davis, California. This line is primarily of New Hampshire background and was recently derived from homozygous dystrophic Line 304 which had been selected for several years for early onset of the disorder and is characterized by pronounced muscle fiber hypertrophy (36). Birds of Line 413 also exhibit hypertrophy of muscles affected by the dystrophy. The normal embryos were from a commercial White Leghorn flock (Donsing Hatcheries, Rio Linda, California). Limb
Bud Transplantation
Embryos
were
incubated
for 3l/2 days
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48, 1976
(Jamesway 252B incubator, 37&C, 58% humidity), candled, laid on their sides, and cooled to room temperature. Approximately 1.5 ml of albumen was removed with a syringe and curved 13 gauge (Horsely Venoclysis) needle inserted through a small hole in the pointed end of each egg, which was then sealed with paraffin. A l-cm2 hole was cut in the side of each egg and the embryo was staged (25). Only stages 19-20 were used. The vitelline membrane was peeled back, and the chorionic and amnionic membranes were opened with watchmaker’s forceps (at the seroamniotic connection) in the region of the right wing limb bud. The wing bud was severed as closely as possible to the body with fine black glass needles and exchanged with one from another embryo. The donor wing bud was held in place on the host embryo by two or three fine black glass tacks, with normal position and orientation maintained. Eggs were placed under a warming lamp for 2 to 3 hr while healing of the limb to the host occurred. The tacks were removed, the chorion and amnion pinched closed, and 0.1-0.2 ml of amphotericin B (12.5 pg/ml) and penicillin-streptomycin (250 units/ml each) in Tyrode’s solution were added. The eggs were sealed with parafilm and incubated on their sides on an egg flat which was rocked daily by elevating one side of the flat. Four types of operations were performed: normal autotransplants were made by removing and reattaching the limb buds of normal embryos; normal limb buds were transplanted onto normal embryos and onto dystrophic embryos; dystrophic limb buds were transplanted onto normal embryos. Analyses Monopolar clinical electromyographic (EMG) electrodes (hand-held) coupled to a Beckman EMG preamplifier and Dynagraph recorder were used to examine muscles of host and donor wings. Electromyographic analysis was performed with light pentabarbital (IV) anesthesia 1 day
LINKHART
et
al.,
Muscular
Dystrophy
of the Chicken
449
between donor and host muscles were perbefore each bird was sacrificed. Electrical formed using Student’s t test (2). discharges elicited by insertion or slight Frozen cross sections (12 pm) were cut in movement (1 mm) of the electrode needle a cryostat at - 18°C within 1 day of sacrificwere monitored on an oscilloscope, reing the animal. Sections air-dried on glass corded on magnetic tape (TEAC Data Casslides were incubated for succinic dehydrosette Recorder), and photographed later genase activity (41) or for cholinesterase from playback of the tape record. activity (52) or were fixed (4% buffered Biceps muscles were usually used for formalin) and stained with hematoxylin analyses. The superficial pectoralis and and eosin. the extensor carpi radialis were also analyzed in two birds of each set. The birds RESULTS were sacrificed by exsanguination at 6 to General Appearance 25 weeks after hatching. Limb muscles were immediately removed and placed in Approximately 10% of the operated emice-cold phosphate-buffered physiological bryos hatched, dystrophic embryos having saline (pH 7.4). A sample from the center a lower survival rate than normal emof the muscle was frozen in iso-pentanebryos. Most of the embryos died within 48 liquid nitrogen for histochemistry and his- hr of surgery. Donor limbs were morphotology. The remainder of each muscle was logically normal in most of the embryos stripped of connective tissue and homoge- which died after 5 days of incubation. Alnized (Teflon pestle tissue grinder, A. H. most all of the operated chicks that Thomas, Philadelphia, Pa.) in cold phos- hatched had functional transplanted limbs phate-buffered saline (pH 7.4; l/4, wet of normal size and shape. From 2 to 3 weeks after hatching, genetically dysw/v). Homogenates were centrifuged trophic wings, whether of host or donor (5OOg, 20 min) and the superantants were origin, began to exhibit functional impairused for subsequent enzyme assays. ment characteristic of muscular dystrophy Cholinesterase activities of samples frozen less than one week and thawed once, of the chicken. The muscles of these limbs, were assayed by the spectrophotometric including the breast muscles near the method of Ellman et al., (17). Specific ace- shoulder of dystrophic limbs, were grossly tylcholinesterase (AChE) and nonspecific hypertrophied and appeared rigid from pseudocholinesterase (BChE) activities chronic contraction. Genetically normal wings retained normal functions. Dyswere distinguished in spectrophotometric assays, cryostat sections, and acrylamide trophic hosts, unable to flex their own ungels by the use of specific substrates and operated left wings, were able to flex their inhibitors. AChE activity is defined as ace- transplanted normal right wings. tylthiocholine hydrolysis in the presence of In about half the hatched birds, the wing derived from a donor embryo aplop4 M iso-OMPA (tetraisopropyl-pyrophosphoramide, Sigma, St. Louis, MO.) an peared to be rejected 4 weeks or longer inhibitor of BChE. BChE activity is de- after hatching. These wings became swollen and exudative, similar to the situation fined as butyrylthiocholine hydrolysis. AChE enzymes of the chicken hydrolyze described by Eastlick and Chehak (16) for acetylthiocholine much more rapidly than hatched chicks possessing transplanted butyrylthiocholine; BChE enzymes hydrolegs. The swelling and exudation occurred lyze both substrates at approximately the in both normal and dystrophic hosts and may have involved immunological reacsame rate (53). Lactic dehydrogenase activity of fresh samples was determined spec- tions of the hosts since none of eight autotrophotometrically as described by Carditransplants showed any indications of it 25 net et al. (8). Paired statistical analyses weeks after hatching. Only birds with mor-
450
DEVELOPMENT
BIOLOGY
phologically normal wings showing no signs of rejection were used for analysis. All birds analyzed had transplanted wings which appeared anatomically normal. Biceps muscles had normal points of origin and insertion and normal position of the blood vessels and nerves supplying them. Analysis of biceps muscles are reported below. Analysis of the extensor carpi radialis in the forewing and the pectoralis near its insertion on the humerus performed for two birds of each set of transplants gave similar results. Electromyographic
Potentials
Genetically dystrophic biceps, whether of donor (Fig. la) or host (Fig. Id) exhibited repetitive insertion potentials characteristic of dystrophic chick muscle. Potentials initially repeat at high frequencies and gradually subside. These often lasted as long as 5 sec. “Insertion activity” of normal muscle (whether of donor or host) consisted of short bursts of activity which abated rapidly (usually within 0.2 set; Figs. lb and 1~). Cholinesterase
Activities
Both AChE (reported previously, 34) and BChE activities were low in normal host biceps muscles, in autotransplanted normal biceps muscles, and in normal donor biceps muscles whether on normal or dystrophic hosts. Activities were significantly higher (p < 0.001) in dystrophic host biceps and dystrophic donor biceps on normal hosts (Table 1). High AChE activity in homogenates of genetically dystrophic chick muscles was accompanied by the presence of embryonic AChE isozymes and extrajunctional AChE localization. Genetically normal muscle fibers, whether innervated by nerves of normal or dystrophic chickens (Figs. 2A, B, D, and E) had AChE activity only at myoneural junctions and in the sarcoplasm near junctions. Many genetically dystrophic muscle fibers (Figs. 2C and F) had sarcoplasmic AChE activity not associated
VOLUME
48, 1976
with myoneural junctions. Figure 2 also illustrates the fiber hypertrophy characteristic of the dystrophic chickens used in the study (all sections in Fig. 2 are same magnification). Average fiber diameter of genetically dystrophic muscles was significantly greater than contralateral normal muscles regardless of which was donor or host (34). Lactic Dehydrogenase
Activity
LDH activities (Table 1) of genetically normal biceps whether of host or donor origin were significantly higher (p < 0.001) than contralateral dystrophic biceps while there were no differences between host and donor muscles in normal to normal transplants or normal autotransplants. Succinic
Dehydrogenase
Activity
Cross sections of host and donor biceps (Fig. 3) had low SDH activity in normal host (Figs. 3b and 3d) and donor (Figs. 3a and 3e) fibers (less than 1% of the fibers had positive SDH activity). Dystrophic host (Fig. 30 and donor (Fig. 3c) muscles had many fibers which were high in SDH activity. DISCUSSION
In this and in a brief previous report (34), analyses of the several properties characteristic of dystrophic muscle gave consistent results; genetically dystrophic muscle was phenotypically dystrophic, regardless of the genotype of the innervating nerves. Nerves of genetically dystrophic chickens supported development and maintenance of genetically normal muscle. The results demonstrate that major characteristics of inherited muscular dystrophy of the chicken are properties of the limb and its muscles and not of the host and its nerves after stages 19-20, the time when the transplants were performed. It is probable that the abnormality originates in the muscle fibers themselves, although the experiments reported cannot
LINKHART
et al..
Muscular
Dystrophy
of the Chicken
FIG. 1. Electromyography of donor and host biceps muscles. EGM recordings were made with two monopolar needle electrodes placed 2-3 mm apart in the muscle; a ground electrode was placed near the muscle. Slight movement of one of the needle electrodes is followed by a brief burst of electrical activity in normal muscle, by a prolonged repetitive discharge in dystrophic muscle. Potentials were recorded on magnetic tape; photographs were made from display of the tape record on an oscilloscope; the horizontal sweep was triggered by the onset of the electrical discharge. (Al, dystrophic donor in normal host: t B). contralateral normal host; CC), normal donor in dystrophic host; ID), contralateral dystrophic host. Note that the time base in A and D is five times longer than in (B) and (Cl.
rule out a primary or associated abnormality in other limb tissues, such as blood vessels and connective tissue cells, as has been proposed for Duchenne muscular dystrophy in humans (9, 27, 28). Abnormali-
ties in vascular and connective ments of the limbs of dystrophic have been described (4, 51, but on nerve and muscle functions Considering past evidence that
tissue elechickens their effect is unclear. dystrophy
452
DEVELOPMENTAL
ENZYME Transplant
ACTIVITIES
type
Age (weeks)
BIOLOGY
OF BICEPS
VOLUME
TABLE
1
MUSCLES
FROM
Number of birds
DONOR
48, 1976
AND
HOST
Biceps IllUSCk
WINGS”
A/r&/g
wet weight
AChE
BChE
LDH
l-25
8
Donor (N) Host (N)
1.1 + 0.8 1.4 + 0.7
0.39 + 0.16 0.35 L 0.23
3310 5 650 3120 c 560
6-10
4
Donor (N) Host (N)
1.5 * 1.0 1.0 + 0.3
0.59 + 0.17 0.55 + 0.10
2350 5 350 2580 -c 550
Dystrophic limb transplanted onto normal host
6-14
6
Donor (D) Host (N)
16.6 + 13.1* 1.2 f 0.3
8.6 k 4.8’ 0.7 f 0.4
970 2 390’ 2190 -t 520
Normal limb transplanted dystrophic host
6-8
5
Donor (N) Host (D)
2.1 + 0.7’ 13.1 + 4.7
1.0 + 0.8’ 7.6 A 2.8
3020 f 380’ 1820 t 480
Normal
autotransplant
Normal limb transplanted normal host
onto
onto
n Enzyme activities are averages + standard deviations of homogenates of biceps muscles from transplanted donor and contralateral host limbs. Fiber diameters are averages + standard deviations of 100 fibers from each biceps of two birds at 6 weeks of age. Statistically significant differences are indicated by *, P < 0.001 by Student’s t-distribution of paired variates.
of the chicken involves a defect in neurally mediated muscle maturation, the data support the idea that genetically dystrophic muscle in the chicken lacks the ability to respond to trophic influence of its innervating nerve. However, this study does not rule out the presence of abnormalities in the nerves that are not associated with the parameters we measured. For example, Albuquerque and Warnick (1) have reported presynaptic alterations in dystrophic chickens. Whether these arise from direct expression of dystrophy in the motor nerves or as secondary effects of the abnormal muscles which they innervate is not known, and may be resolved by electrophysiological studies of normal and dystrophic nerve and muscle combinations produced by limb bud transplantation (Michael Latinsky, personal communication). Whether neural influence before stages 19-20 affects subsequent phenotypic expression of the limb was not dealt with in our investigation. In a recent study, Vetrano and his colleagues (13, 47) transplanted neural tube sections from dystrophic to normal chick embryos at stage 14 (50 hr of incubation) and found that muscles of l&day normal host embryos had high thymidine kinase activity characteristic of dystrophic embryos (50). However, reciprocal transplants of normal neural tube to dystrophic hosts were not done and it was unclear whether the dys-
trophic neural transplants might have been contaminated with dystrophic myogenic cells. Regardless, our results show that nerves play no role after stages 19-20 in expression of several important properties of dystrophic chick muscle. “Preliminary results” of Cosmos and Butler (12) suggested that inherited muscular dystrophy of the chicken is myogenic. They found that normal muscle minces regenerated in dystrophic hosts and maintained normal histological appearance, lipid distribution, and succinic dehydrogenase activity. However, the report did not include results of normal to normal controls, host muscle “buds” regenerated together with donor muscle, and there was a “progressive loss of the transplanted tissue” when dystrophic muscle was transplanted into normal hosts. These problems were avoided in the present study. Limbs were transplanted at a stage in development preceding muscle differentiation (6, 28, 45) and nerve outgrowth and innervation (20,21, 22,23,44). Development of the transplanted limb occurred normally and the nerve-muscle combinations were allowed to mature to an age at which several different properties of dystrophic muscle were unambiguously expressed. Contamination of hosts with nerves of donor origin was not possible because the nerve cell bodies were located in the spinal cord. Contamination of donor limb buds with my-
F ‘IG. 2. Cytochemical localization of AChE activity in donor and host biceps muscles. Cross secti inc ubated with acetylthiocholine and lo-” M iso-OMPA. AChE appears as dark stain at motor endplates ext rajunctional activity (e) appears in the sarcoplasm of many fibers in genetically dystrophic muscle. nor ,mal donor in normal host; (B), contralateral normal host; (CI, dystrophic donor in normal host; con .tralateral normal host; (E), normal donor in dystrophic host; (F), contralateral dystrophic host. were taken with identical exposures under identical conditions. x 105. phc rtographs 453
I ml; I Ai, ( D:, All
FIG. 3. Histochemical localization of succinic dehydrogenase activity in donor and host biceps muscles. Cross sections were incubated for SDH activity with Na succinate and nitro blue tetrazolium. Activity appears as dark granules. SDH “positive” fibers (arrows) have more granules distributed throughout the sarcoplasm. (A), normal donor in normal host; (B), contralateral normal host; (0, dystrophic donor in normal host; CD), contralateral normal host; (E), normal donor in dystrophic host; (F), contralateral dystrophic host. All photographs were taken with identical exposures under identical conditions. x 230. 454
LINKHART
et al.,
Muscular
ogenic cells of the hosts was possible but the fact that the phenotypes of donor and host muscles were significantly different in transplants between normal and dystrophic embryos strongly suggests that such contamination was negligible. Whether inherited muscular dystrophies of humans and other mammals are myogenic or neurogenic in origin is unclear. Electrophysiological studies of motor neurons and neuromuscular transmission, transplantation of muscle minces or whole muscles, culture of normal and dystrophic nerve and muscle cells, and production of allophenic mosaics of normal and dystrophic mouse embryos have resulted in equivocal and often conflicting results (14, 35, 40, 42). There is little reason to expect that all muscular dystrophies have either a common molecular basis or a common cellular origin. Indeed, the dichotomy “nerve or muscle” may prove conceptually inadequate to deal with the complicated cell interactions that occur during development and maturation of normal and abnormal muscles. Although our investigations (32, 33, 39, 48, 49, 51, 531 strongly suggest that the lesion in regulation of dystrophic chick muscle lies in the ability of the muscles to respond to normal neural influence, the mechanism of regulation which is disrupted has not yet been identified. “Trophic factors” transferred from nerve to muscle, acetylcholine released by the nerve, and contractile activity itself have been proposed as mechanisms of trophic regulation (15,26,43). Recent evidence suggests that AChE activity and localization of major biochemical abnormalities in dystrophic chick muscle, are regulated in part by contractile activity (33, 39, 48, 49). Perhaps inherited muscular dystrophy of the chicken is caused by a failure of the muscle to respond to its own neurally mediated contractions. Supported in part by a grant from the Muscular Dystrophy Associations of America, Inc., and PHS Grant NS10957. Dr. T. A. Linkhart was a National
Dystrophy
of
the (‘hIcken
455
Science Foundation Predoctoral Trainee in Physiology. We gratefully acknowledge Dr. LJ. K. Abbott for her help and encouragement in development of the transplantation technique, Mr. F. Lantz and Mr. J. Adams for maintaining the experimental animals, Dr. it. G. Taylor and Dr. R. E. Burger for providing help, facilities, and equipment for the electromyographic analysis, Mr. J. Schenkel for his initial work in the study, and Dr. M. Rathbone and Mr. F. Vetrano for providing us with their unpublished results. REFERENCES 1. ALBUQUERQUE, E. X., and WARNICK, J. E. (1971). Electrophysiological observations in normal and dystrophic chicken muscles. Science 172, 1260-1263. 2. ALDER, H. L., and ROESSLER, E. B. (1964). “Introduction to Probability and Statistics,” 3rd ed. Freeman and Co., San Francisco, Calif. 3. ASHMORE, C. R., and DOERR, L. (1971). Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick. Exp. Nurology 30, 431-446. 4. ASHMORE, C. R., DOERR, L., and SOMES, R. G., JR. (1968). Microcirculation: Loss of an enzyme activity in chickens with hereditary muscular dystrophy. Science 160, 319-320. 5. ASMUNDSON, V. S., KRATZER, F. H., and JULIAN, L. M. (1966). Inherited myopathy in the chicken. Ann. N.Y. Acad. Sci. 138, 1-17. 6. BONNER, P. H., and HAUSCHKA, S. D. (19741. Clonal analysis of vertebrate myogenesis: I. Early developmental events in the chick limb. Develop. Biol. 37, 317-328. 7. BRADLEY, W. G. (1971). Nerve, muscle, and muscular dystrophy. Dev. Med. Child Neural. 13. 5288531. 8. CARDINET, G. H., FREEDLAND, R. A., TYLER, W. S., and JULIAN, L. M. (1972). Morphologic, histochemical, and quantitative enzyme study of hereditary avian muscular dystrophy. Amer. J. Vet. Res. 33, 1671-1684. 9. CAZZATO, G. (1968). Considerations about a possible role played by connective tissue proliferation and vascular disturbances in the pathogenesis of progressive muscular dystrophy. Europ. Neural. 1, 158-179. 10. CLOSE, D. R. (1972). Dynamic properties of mammalian skeletal muscle. Physiol. Rev. 52. 129-197. 11. COSMOS, E., and BUTLER, J. (1967). Differentiation of fiber types in muscle of normal and jystrophic chickens. In “Exploratory Conceit: in Muscular Dystrophy and Related Disorders” (A. T. Milhorat, ed.), pp. 197-204. Excerpta Medica, Amsterdam, The Netherlands.
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12. COSMOS, E., and BUTLER, J. (1972). Differentiation of muscle transplanted between normal and dystrophic chickens, In “Research in Muscle Development and the Muscle Spindle” (B. Q. Banker, R. J. Przybylski, J. P. Van Der Meulen, and M. Victor, eds.), pp. 149-162. Excerpta Medica, Amsterdam, The Netherlands. 13. RATHBONE, M. P., STEWART, P. A., and VETRANO, F. (1975). Dystrophic spinal cord transplants induce abnormal thymidine kinase activity in normal muscle. Science 189, 1106-1107. 14. DOUGLAS, W. B., and COSMOS, E. (1975). Histochemical responses of murine dystrophic muscles cross-innervated by sciatic nerves of normal mice. In “Exploratory Concepts in Muscle (ID” (A. T. Milhorat, ed.1. Excerpta Medica, Amsterdam, The Netherlands. 15. DRACHMAN, D. B. (1974). The role of acetylcholine as a neurotrophic transmitter. Ann. N.Y. Acad. Sci. 228, 160-176. 16. EASTLICK, H. L., and CHEHAK, J. D. (1953). Studies on transplanted embryonic limbs of the chicken: The pathology in post-hatch stages. Growth 17, 45-65. 17. ELLMAN, G. L., COURTNEY, K. D., ANDRES, V., and FEATHERSTONE, R. M. (1961). A new and rapid calorimetric determination of acetylcholine&erase activity. Biochem. Pharm. 1, 8895. 18. EMERY, A. E. H., and GOSDEN, C. (1974). A neurogenic component in muscular dystrophy. J. Med. Genetics 11, 76-79. 19. ENGEL, W. (19701. Selective and non-selective susceptibility of muscle fiber types, a new approach to human neuromuscular diseases. Arch. Neural. 22, 97-116. 20. FILOGAMO, G., and GABELLA, G. (1967). The development of neuromuscular correlations in vertebrates. Arch. Biol. (Liege) 78, S-60. 21. GIACOBINI, G. (1972). Embryonic and postnatal development of choline acetyltransferase activity in muscles and sciatic nerve of the chick. J. Neurochem. 19, 1401-1403. 22. GIACOBINI, G., FILOGAMO, G., WEBER, M., Be QUET, P., and CHANGEUX, J. P. (19731. Effects of a snake a-neurotoxin on the development of innervated skeletal muscles in chick embryo. Proc. Nat. Acad. Sci. USA 70, 1708-1712. 23. GRIM, M. (1970). Differentiation of myoblasts and the relationship between somites and the wing bud of the chick embryo. 2. Anat. Entwickl. Gesch. 132, 260-271. 24. GUTH, L. (19681. “Trophic” influences of nerve on muscle. Physiol. Rev. 48, 645-687. 25. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morph. 88,49-92.
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26. HARRIS, A. J. (19741. Inductive functions of the nervous system. Ann. Rev. Physiol. 36, 251305. 27. HATHAWAY, P. W., ENGEL, W. K., and ZELI~ WEGER, H. (19701. Experimental myopathy after microarterial embolization. Comparison with childhood X-linked pseudohypertrophic muscular dystrophy. Arch. Neurol. 22, 365378. 28. HILFER, S. R., SEARLS, R. L., and FONTE, V. G. (1973). An ultrastructural study of early myogenesis in the chick wing bud. Develop. Biol. 30, 374-391. 29. HOLLIDAY, T. A., VAN METER, J. R., JULIAN, L. M., and ASMUNDSON, V. S. (19651. Electromyography of chickens with inherited muscular dystrophy. Amer. J. Physiol. 209,871-876. 30. JEDRZEJCZYK, J., WIECKOWSKI, J., RYMASZEWSKA, T., and BARNARD, E. A. (1973). Dystrophic chicken muscle: Altered synaptic acetylcholinesterase. Science 180, 406-408. 31. KAPLAN, N. O., and CAHN, R. D. (1962). Lactic dehydrogenases and muscular dystrophy of the chick. Proc. Nat. Acad. Sci. USA 48, 2123-2130. 32. LEBEDA, F. J., WARNICK, J. E., and ALBUQUERQUE, E. X. (1974). Electrical and chemosensitive properties of normal and dystrophic chicken muscles. Erp. Neurol. 43, 21-37. 33. LINKHART, T. A., and WILSON, B. W. (1975). Role of muscle contraction in trophic regulation of chick muscle acetylcholinesterase activity. Exp. Neurol. 48, 557-568. 34. LINKHART, T. A., YEE, G. W., and WILSON, B. W. (19751. Myogenic defect in acetylcholinesterase regulation in muscular dystrophy of the chicken. Science 187, 549-551. 35. MCCOMAS, A. J., SICA, R. E. P., UFTON, A. R. M., and PETITO, F. (1974). Sick motoneurons and muscle disease. Ann. N.Y. Acad. Sci. 228, 261-279. 36. MCMURTY, S. L., JULIAN, L. M., and ASMUND SON, V. S. (1972). Hereditary muscular dystrophy of the chicken. Quantitative histopathological findings of the Pectoralis at 6 weeks of age. Arch. Path. 94, 217-224. 37. MENDELL, J. R., ENGEL, W. K., and DERRER, E. C. (19711. Duchenne muscular dystrophy: functional &hernia reproduces its characteristic lesions. Science 172, 1143-1145. 38. MENDELL, J. R., ENGEL, W. K., and DERRER, E. C. (1972). Increased plasma enzyme concentrations in rats with functional ischaemia of muscle provide a possible model of Duchenne muscular dystrophy. Nature (London) 239, 522524. 39. PATTERSON, G. T., and WILSON, B. W. (1975). Distribution of extrajunctional acetylcholinnesterase in muscle. I. Normal and dystrophic
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