"Medicine, Faculty of"@en . "Medical Genetics, Department of"@en . "DSpace"@en . "UBCV"@en . "Peterson, Alan Clarke"@en . "2011-03-09T01:09:00Z"@en . "1973"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "1. Artificial chimeric mice, mosaic for muscular dystrophic (dy[sup 2J] /dy[sup 2J]) and normal (SWV +/+) genotypes were successfully produced.\r\n2. These chimeric mice demonstrated no clinical features of the dystrophic condition.\r\n3. A maximum stimulated twitch analysis of the anterior tibialis muscles of these chimeras also revealed no functional deficiences characteristic of the dystrophic condition.\r\n4. The electrophoretic phenotype of Mod-1 (malic enzyme) was resolved with a high voltage micro starch gel system and a quantitative analysis was developed to detect and estimate the relative muscle fiber nuclear mosaicism in single muscles from the chimeras. The measured activity of the 5 bands of the Mod-1 hetero-zygote phenotype was observed to be different from the expected ratio of 1:4:6:4:1 previously reported.\r\n5. The presence of genetically dystrophic nuclei was detected\r\nin the majority of chimera muscles examined. The overall content of (dy[sup 2J] /dy[sup 2J]) nuclei from all the muscle samples examined was estimated to be 58%. This indicated that genetically dystrophic cells contribute normally to muscle morphogenesis and that genetically dystrophic muscle fiber nuclei suffer no specific degeneration in this artificial mosaic environment. \r\n\r\n6. Within single chimeras the composition of muscles varied extensively. Individual muscles of both normal and primarily dystrophic genetic composition were detected.\r\n7. Histological analysis of samples of anterior tibialis muscles, prepared in 1 u thick plastic sections, was undertaken to determine if any preclinical muscle degeneration had taken place. These muscles had essentially normal phenotypes.\r\n8. Examples of muscles with primarily dystrophic genotypic\r\ncomposition and remarkably normal histological phenotype were\r\nrevealed. Conversely, muscles with no detectable dystrophic composition\r\nwere observed to have foci of degeneration characteristic of muscular\r\ndystrophy. These results, although not extensive, are suggestive\r\nthat the muscle degeneration observed in dystrophic (dy[sup 2J] /dy[sup 2J]) mice is secondary to a primary lesion residing outside the muscle fiber proper.\r\n9. The possible similarities between these artificial cellular mosaics and a human X-linked genetic mosaic, the heterozygous female for Duchenne dystrophy, are presented.\r\n10. The results of these studies were discussed in terms of the application of chimeric mice in the delineation of the primary lesion in mouse muscular dystrophy."@en . "https://circle.library.ubc.ca/rest/handle/2429/32218?expand=metadata"@en . "GENOTYPIC AND PHENOTYPIC ANALYSIS OF MUSCLES FROM DYSTROPHIC\u00E2\u0080\u0094NORMAL MOUSE CHIMERAS by ALAN CLARKE PETERSON B.Sc, U n i v e r s i t y of V i c t o r i a , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the F i e l d of Genetics We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1973 In presenting th i s thes is i n p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary s h a l l make i t f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of t h i s thes is for s cho lar ly purposes may be granted by the Head of my Department or by h i s representat ives . It i s understood that copying or p u b l i c a t i o n of t h i s thes is for f i n a n c i a l gain s h a l l not be allowed without my wri t ten permiss ion. Department of Medical Genetics The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date A p r i l 27, 1973 ABSTRACT 1. A r t i f i c i a l chimeric mice, mosaic for muscular dystrophic 2J 2J (dy /dy ) and normal (SWV +/+) genotypes were s u c c e s s f u l l y produced. 2. These chimeric mice demonstrated no c l i n i c a l features of the dystrophic condition. 3. A maximum stimulated twitch analysis of the anterior t i b i a l i s muscles of these chimeras also revealed no f u n c t i o n a l deficiences c h a r a c t e r i s t i c of the dystrophic condition. 4. The electrophoretic phenotype of Mod-1 (malic enzyme) was resolved with a high voltage micro starch gel system and a quantitative analysis was developed to detect and estimate the r e l a t i v e muscle f i b e r nuclear mosaicism i n s i n g l e muscles from the chimeras. The measured a c t i v i t y of the 5 bands of the Mod-1 hetero-zygote phenotype was observed to be d i f f e r e n t from the expected r a t i o of 1:4:6:4:1 previously reported. 5. The presence of g e n e t i c a l l y dystrophic n u c l e i was detected i n the majority of chimera muscles examined. The o v e r a l l content of 2J 2J dy /dy n u c l e i from a l l the muscle samples examined was estimated to be 58%. This indicated that g e n e t i c a l l y dystrophic c e l l s contribute normally to muscle morphogenesis and that g e n e t i c a l l y dystrophic muscle f i b e r n u c l e i s u f f e r no s p e c i f i c degeneration i n t h i s a r t i f i c i a l mosaic environment. i i 6. Within s i n g l e chimeras the composition of muscles varied extensively. Individual muscles of both normal and pr i m a r i l y dystrophic genetic composition were detected. 7. H i s t o l o g i c a l a nalysis of samples of anterior t i b i a l i s muscles, prepared i n 1 u thick p l a s t i c sections, was undertaken to determine i f any p r e c l i n i c a l muscle degeneration had taken place. These muscles had e s s e n t i a l l y normal phenotypes. 8. Examples of muscles with p r i m a r i l y dystrophic genotypic composition and remarkably normal h i s t o l o g i c a l phenotype were revealed. Conversely, muscles with no detectable dystrophic composition were observed to have f o c i of degeneration c h a r a c t e r i s t i c of muscular dystrophy. These r e s u l t s , although not extensive, are suggestive 2J 2J that the muscle degeneration observed i n dystrophic (dy /dy ) mice i s secondary to a primary l e s i o n r e s i d i n g outside the muscle f i b e r proper. 9. The possible s i m i l a r i t i e s between these a r t i f i c i a l c e l l u l a r mosaics and a human X-linked genetic mosaic, the heterozygous female f o r Duchenne dystrophy, are presented. 10. The r e s u l t s of these studies were discussed i n terms of the a p p l i c a t i o n of chimeric mice i n the de l i n e a t i o n of the primary l e s i o n i n mouse muscular dystrophy. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i LIST OF APPENDICES i x ACKNOWLEDGMENTS x Chapter I. INTRODUCTION 1 Muscular dystrophy i n the mouse 2 Muscular dystrophy as a primary myopathy 2 Muscular dystrophy as a primary neuropathy 4 Muscular dystrophy i n chimeric mice 9 I I . GENERAL MATERIALS AND METHODS 11 1. Mice: source and maintenance 11 2. Production of chimeras 12 3. Chimeras produced 16 I I I . MUSCLE FUNCTION OF DYSTROPHIC\u00E2\u0080\u0094NORMAL CHIMERIC MICE 21 Materials and Methods 22 1. Determination of the maximum isometric twitch of the an t e r i o r t i b i a l i s muscle 22 i v Results and Discussion 23 1. V i s u a l observation of the d y s t r o p h i c \u00E2\u0080\u0094 normal chimeras 23 2. Maximum isometric twitch of anterior t i b i a l i s muscles 23 IV. GENETIC CONSTITUTION OF MUSCLES FROM CHIMERAS 29 Mod-1 electrophoretic v a r i a t i o n 31 Mod-1 genotype of chimeras 32 Materials and Methods 34 1. Tissue preparation 34 2. Electrophoresis of Mod-1 34 3. Quantitation of r e l a t i v e Mod-1 band a c t i v i t y 38 Results and Discussion 38 1. Mod-1 electrophoretic pattern 38 2. C a l c u l a t i o n of c o r r e c t i o n f a c t o r s f o r Mod-1 isoenzymes 41 3. Mod-l a/Mod-l a and Mod-l b/Mod-l b estimate of nuclear content of muscles from chimeras 42 V. HISTOLOGICAL EVALUATION OF MUSCLES FROM DYSTROPHIC\u00E2\u0080\u0094 NORMAL CHIMERAS 57 Materials and Methods 57 Results and Discussion 58 1. Dystrophic muscle: Anterior t i b i a l i s 58 2. Dystrophic\u00E2\u0080\u0094normal chimeras: a n t e r i o r t i b i a l i s muscles 58 3. Dystrophic\u00E2\u0080\u0094normal chimeras: selected muscles 62 VI. GENERAL DISCUSSION 69 BIBLIOGRAPHY 72 APPENDIX 77 v LIST OF TABLES Table Page 1. Number and External Sex of Chimeric Mice Produced f o r t h i s Study 19 2. Chimera Number 7; Mod-l^ Estimate of Dystrophic Content 45 3. Chimera Number 8; Mod-l b Estimate of Dystrophic Content 46 4. Chimera Number 9: Mod-1 Estimate of Dystrophic Content 47 5. Chimera Number 10; Mod-l b Estimate of Dystrophic Content 48 6. Chimera Number 11; Mod-l b Estimate of Dystrophic Content 49 7. Chimera Number 16; Mod-l b Estimate of Dystrophic Content 50 8. Chimera Number 17; Mod-1 Estimate of Dystrophic Content 51 9. Genetic Constiution of Muscles Analysed f or Mod-l^; Summary 52 v i LIST OF FIGURES Figure Page 1. Hemoglobin electrophoretogram 20 2. Osciloscope display of maximum isometric twitch of chimera anterior t i b i a l i s muscles 24 3. Maximum isometric twitch of anter ior t i b i a l i s muscles 26 4. Micro starch gel electrophoretic apparatus 36 5. Micro gels with sample applied prior to electrophoresis 36 6. Electrophoretogram of Mod-1 heterozygote 39 7. Mod-1: activity ratio of heterozygote pattern from muscle supernatant 40 8. Electrophoretograms of Mod-1 from individual dystrophic \u00E2\u0080\u0094normal chimera muscles with corresponding densitometer traces 43 9. A n t g j i o ^ t i b i a l i s muscle from 4 month old dystrophic (dy /dy ) mouse 59 10. Anterior2tibialis muscle from 4 month old dystrophic (dy /dy ) mouse 59 11. Left anterior t i b i a l i s of chimera 8 60 12. Left anterior t i b i a l i s of chimera 10 60 13. Right anterior t i b i a l i s of chimera 9 61 14. Right anterior t i b i a l i s of chimera 9 61 15. Left gluteus maximus of chimera 8 63 16. Left gluteus maximus of chimera 8 63 v i i 17. L e f t biceps femoris of chimera 10 64 18. L e f t biceps femoris of chimera 10 64 19. L e f t biceps femoris of chimera 16 65 20. L e f t biceps femoris of chimera 16 65 21. L e f t biceps femoris of chimera 16 66 v i i i LIST OF APPENDICES Appendix I. FOCAL program: Mod-1 estimate of nuclear content i x CHAPTER 1 INTRODUCTION There are a large number of environmentally and g e n e t i c a l l y determined diseases of vertebrate muscle. The term 'muscular dystrophy' i s reserved f o r a p a r t i c u l a r c l a s s of i n h e r i t e d progressive wasting diseases of s k e l e t a l muscle. Because no d i r e c t evidence of abnormal innervation was o r i g i n a l l y described with these muscle conditions, they were c o l l e c t i v e l y thought of as primary myopathies, that i s , g e n e t i c a l l y determined myopathies i n which the primary l e s i o n resides w i t h i n the muscle f i b e r s themselves (Walton, 1961). There has recently been a reevaluation of t h i s hypothesis (Dubowitz, 1971 and McComas, Sica, and Campbell, 1971) with emphasis now being placed on the possible primary r o l e of the nervous system. However, evidence co n c l u s i v e l y demonstrating the s i t e and nature of the i n i t i a l l e s i o n produced by the mutant genes has not been reported. The present study describes the use of a r t i f i c i a l chimeric mice, of dystrophic\u00E2\u0080\u0094normal composition, to delineate the primary l e s i o n of a muscular dystrophy mutation of the mouse. Evidence i s presented which supports a non-primary myopathogenesis of t h i s p a r t i c u l a r muscle disease. These chimeric mice may also serve a useful r o l e as a model 1 2 of the female \" c a r r i e r \" of the human X-linked Duchenne dystrophy mutation. Muscular dystrophy i n the mouse A recessive mutation, dystrophia muscularis ( gene symbol dy) causing muscular dystrophy i n the 129/Re mouse s t r a i n was discovered i n 1951 (Michelson, R u s s e l l , and Harman, 1955). I t was the f i r s t genetically-determined muscle disease, considered to be a primary myopathy, i d e n t i f i e d i n an experimental animal of known 2J genetic background. A new a l l e l e , dy , at the dy_ locus was discovered i n 1969 i n the WK/Re s t r a i n (Meier and Southard, 1970). The phenotype of these muscular dystrophies d i f f e r s only i n the rate of progression 2J of the disease, the dy mutant having a slower course. Muscular dystrophy as a_ primary myopathy Dire c t evidence, demonstrating that the primary l e s i o n i n muscular dystrophy resides within the muscle f i b e r , has been l i m i t e d . The f i r s t d e t a i l e d d e s c r i p t i o n of Duchenne muscular dystrophy by Meryon i n 1852 comments on the absence of any s t r u c t u r a l abnormality i n the c e n t r a l nervous system ( i n Dubowitz, 1971). Subsequent obser-vations have confirmed t h i s f i n d i n g (Adams, Denny-Brown and Pearson, 1962). In the o r i g i n a l d e s c r i p t i o n of dy/dy mice, Michelson, Ru s s e l l and Harman (1955) reported that h i s t o l o g i c a l examination of a l l l e v e l s of the c e n t r a l nervous system revealed no demonstrable pathology. Muscle preparations revealed: p r o l i f e r a t i o n of sarcolemmal n u c l e i ; 3. increase i n the amount of i n t e r s t i t i a l t i s s u e ; rounded and occasional s p l i t t i n g f i b e r s ; marked increase i n the amount of connective t i s s u e and some f a t t y replacement. Subsequent studies have confirmed the major h i s t o l o g i c a l findings (West and Murphy, 1960). In an experiment designed to characterize the nature of the muscle pathology, O'Steen (1962) demonstrated differences i n the growth response of normal and dystrophic (dy/dy) muscle. Dystrophic muscle cultures i n d i f f u s i o n chambers implanted i n dystrophic and normal hosts underwent a c h a r a c t e r i s t i c growth response ending i n f i b r o s i s . Normal muscle grew w e l l i n both hosts. These r e s u l t s were interpreted as demonstrating that the primary l e s i o n was inherent i n the muscle f i b e r s and that an environmental e f f e c t was very u n l i k e l y . In a s i m i l a r experiment with mice, Rolston (1972) transplanted normal and dystrophic muscle to the kidney capsule of normal and dystrophic hosts. He reported e s s e n t i a l l y i d e n t i c a l r e s u l t s and s i m i l a r conclusions. These experiments can be c r i t i c i z e d on one major point. H i s t o l o g i c a l l y , muscle from dystrophic mice presents a wide spectrum of abnormalities including an apparent increase i n connective t i s s u e . An accompanying increase i n f i b r o c y t e concentration would therefore be expected i n dystrophic muscle. This could r e a d i l y explain the eventual f i b r o s i s response of the cultured or transplanted dystrophic muscle. Conclusive evidence that no c i r c u l a t i n g f actor i s responsible for the muscle breakdown i n mouse dystrophy was demonstrated by Pope and Murphy (1960) with conjoined normal and dystrophic p a i r s . 4 Attempts with retrograde h i s t o l o g i c a l analyses have f a i l e d to demonstrate a precise l e s i o n which could implicate the muscle f i b e r d i r e c t l y . In an extensive u l t r a s t r u c t u r a l study with dy/dy mice, Pl a t z e r (1971) demonstrated that the f i r s t s t r u c t u r a l signs of muscle degeneration occur only i n the d i f f e r e n t i a t e d muscle f i b e r s . She observed no abnormality i n the myogenic process. The f i r s t s t r u c t u r a l a l t e r a t i o n was the presence of swollen sarcoplasmic reticulum; but th i s change was not noted u n t i l day 19 of gestation (day 19 or 20 i s expected date of b i r t h ) . She concluded that t h i s a l t e r a t i o n i n the sarcoplasmic reticulum was a r e s u l t of eit h e r a f a u l t y membrane system i n the muscle or a f a i l u r e of a trophic influence of the nerve on the muscle. Attempts to characterize the l e s i o n biochemically have also proven inconclusive. Although i n both mouse and human dystrophies there are numerous changes i n several metabolic pathways (Russell, 1963 and Penn, Cloak and Rowland, 1972) there has never been recorded a confirmed example of a s t r u c t u r a l change or t o t a l absence of any muscle protein or enzyme. Muscular dystrophy as a_ primary neuropathy Although the evidence f o r a primary myopathogenesis of the muscular dystrophies has been l i m i t e d , t h i s t r a d i t i o n a l theory has been adhered to strongly. Only recently has t h i s view been challenged and a v a r i e t y of experiments have indicated a possible neurogenic cause of these defects. 5 Bu l l e r , Eccles and Eccles (1960) demonstrated that f a s t and slow muscles of the cat, namely f l e x o r digitorum longus and soleus muscles, r e c i p r o c a l l y changed t h e i r c o n t r a c t i l e properties a f t e r cross innervation. Dubowitz (1967a) extended these studies to demonstrate by histochemical techniques that the biochemical proper-t i e s associated with f a s t and slow muscles were also changed i n t h i s experimental s i t u a t i o n . These r e s u l t s demonstrated aspects of the profound influence of the nerve on the muscle's c o n t r a c t i l e proper-t i e s and biochemical nature. Moreover, Dubowitz (1967b) observed a h i s t o l o g i c a l l y myopathic appearance of many muscles during the early phases of reinnervation. These observations helped form the basis f o r the theory that the muscular dystrophies may r e s u l t from a primary l e s i o n within the nervous system. Evidence has accumulated from a v a r i e t y of sources which gives support to t h i s theory. McComas and Mrozek (1967) c l e a r l y demonstrated the presence of denervated muscle f i b e r s i n dystrophic mice (dy/dy). With micro electrodes they were able to monitor the response of s i n g l e muscle f i b e r s a f t e r both d i r e c t and i n d i r e c t (via nerve) stimulation. A f i b e r was concluded to be denervated i f i t responded to d i r e c t but not to i n d i r e c t stimulation. Of 181 dystrophic f i b e r s examined, 48 or 27% were denervated. Of 208 f i b e r s tested i n l i t t e r - m a t e c o n t r o l s , j L .j2., +/+ and dy/+ mice, only 5 or 2% were denervated. The authors present two possible explanations f o r t h i s r e s u l t . The denervation could take place i n the v i c i n i t y of the neuromuscular junction or could r e s u l t from a necrotic segment wit h i n the muscle f i b e r which, 6 by blocking e l e c t r i c a l and trophic s t i m u l i , causes f u n c t i o n a l de-nervation i n the d i s t a l segment of the f i b e r . Extensive motor u n i t analysis i n muscular dystrophic patients has indicated that the muscle loss i s not generalized as expected with a primary myopathy, but r e s u l t s from a loss of motor un i t s . This r e s u l t implicates the motor neuron as a s i t e of the primary l e s i o n (McComas, Sica and Currie, 1970). Similar r e s u l t s have been reported by Harris and Wilson (1971) f o r muscular dystrophic mice (dy/dy). The number of motor units i n a n t e r i o r t i b i a l i s muscles was c o n s i s t e n t l y estimated to be s i g n i f i c a n t l y reduced i n dystrophic mice. However, the mean twitch tension of s i n g l e dystrophic motor units was smaller than that of normal u n i t s . Evidence against a simple denervation pathogenesis has been reported by Law and Atwood (1972). They demonstrated a non-equivalence of s u r g i c a l and natural denervation i n dystrophic mouse muscles. The e l e c t r i c a l \"cable\" properties, measured with i n t r a c e l l u l a r micro-electrodes, of f u n c t i o n a l l y innervation f i b e r s of dystrophic muscle showed reduced resistance. Dystrophic f i b e r s which were not function-a l l y innervated had a s i g n i f i c a n t l y higher resistance than those which were innervated but i n neither case did they approach the value for normally innervated f i b e r s . In modified t i s s u e culture experiments i n which normal muscle biopsies can d i f f e r e n t i a t e to multinucleated myoblast and myotube stages with cross s t r i a t i o n s , Bishop, Gallup, Skeate and Dubowitz (1971) 7 observed no d i f f e r e n c e i n the performance of normal and g e n e t i c a l l y dystrophic muscle. No s i g n i f i c a n t differences were found i n the lag phase at the beginning of d i f f e r e n t i a t i o n , the lengths and breadths of the d i f f e r e n t i a t e d myotubes or i n the success of i n i t i a l culture and subsequent subculture. Amongst a number of explanations for t h i s r e s u l t they conclude \"that some environmental or neural factor responsible for the dystrophic character of the muscle i n vivo i s lacking i n the i n v i t r o conditions\". Attempts to h i s t o l o g i c a l l y i d e n t i f y defects within the dys-trophic nervous system have provided further supportive evidence for the neurogenic theory. C u r t i s , Abrams and Harman (1961) reported that the motor end plates on dystrophic muscle showed progressive loss of f i n e d e t a i l , apparent elongation and fragmentation into sub-u n i t s . Ragab (1971) reported u l t r a s t r u c t u r a l changes i n the motor end plates on h i s t o l o g i c a l l y normal muscle f i b e r s from dystrophic mice. He observed that 40% of the nerve endings of these f i b e r s appeared normal while 60% exhibited various types of abnormalities including: reduction i n the numbers of synaptic v e s i c l e s ; large amounts of neurofilamentous material; reduction i n the number and complexity of the post-synaptic folds and the occasional presence of elongated v e s i c l e s and m u l t i v e s i c u l a r bodies. H a r r i s , Wallace and Wing (1972) reported a large reduction i n the t o t a l number of myelinated axons i n the nerves supplying the anterior t i b i a l i s muscle of dystrophic (dy/dy) mice. They observed a mean axon number of 512.5 i n normal mice but a mean of only 233.3 8 i n dystrophic mice. This reduction occurred over the whole range of axon diameter but the packing of the axons within the nerve was normal suggesting a developmental f a i l u r e . Attempts to trace t h i s l e s i o n to the s p i n a l cord, by demonstrating a reduction i n the number of h i s t o l o g i c a l l y normal anterior horn motor neurons, have not been successful (Josph and Netsky, 1972, Papapetropoulos and Bradley, 1972). To test d i r e c t l y f o r a pathological influence of the dystrophic nervous system, Salafsky (1971) transplanted muscles between normal and dystrophic mice of the 129 s t r a i n . Dystrophic muscle minces of the anterior t i b i a l i s muscle regenerated normally when transplanted to t h i s muscle bed of g e n e t i c a l l y normal mice. Normal muscle did not regenerate i n dystrophic hosts. Functional innervation occurred within 75 days i n the dystrophic transplant and i n d i r e c t stimulation gave r i s e to approximately 75% of control muscle tension. This evidence i s very s t r i k i n g but i s not conclusive. I t was not possible to demonstrate that the presumed dystrophic muscle regenerated was g e n e t i c a l l y dystrophic. Although normal muscle f a i l e d to regenerate i n 5 dystrophic hosts a f u n c t i o n a l muscle regenerated i n 1 and yielded a perceptible contraction. There are a number of parameters which d i f f e r s i g n i f i c a n t l y between normal and dystrophic mice that could account f o r the observed r e s u l t s . The mean body weight of normal mice used i n Salafsky's study was 27.88 g. whereas the dys-trophic mice weighed only 15.23 g. This d i f f e r e n c e , considered i n terms of the a v a i l a b l e blood supply alone, could explain the reduced 9 response of muscle regeneration i n the dystrophic hosts. The importance of the vascular supply i n regenerating muscle i s well documented (Carlson, 1972). Muscular dystrophy i n chimeric mice In 1961, Tarkowski demonstrated a unique experimental mani-pulation of mouse embryos which y i e l d s s i n g l e mice that are mosaics for two c e l l types. These mice have been c a l l e d a r t i f i c i a l mosaic, chimeric, allophenic, and tetr a p a r e n t a l . This manipulation of pre-implantation embryos has led to studies i n numerous l a b o r a t o r i e s , e s p e c i a l l y that of Mintz (Mintz, 1969 and Mintz, 1971), of both normal and pathological developmental phenomena. With the number of known genetic markers i n mice, i t i s possible with many of these chimeras to i d e n t i f y within a p a r t i c u l a r t i s s u e or organ the genetic o r i g i n of the c e l l s . Because these mice are e s s e n t i a l l y \"normal\", d i f f e r i n g only i n t h e i r preimplantation o r i g i n , they provide an exceptionally powerful t o o l to study the in t e r a c t i o n s of normal and pathological genotypes without the intervention of s u r g i c a l or regenerative manipulation. (For review see Nesbitt and Gar t i e r , 197L) One notable exception involves the immune system which may demon-strate unique features within these chimeras (Wegmann, HellstrHm, and Hellstrbm, 1971). It was postulated that a f u n c t i o n a l , h i s t o l o g i c a l and compo-s i t i o n a l a n alysis of chimeric mice derived from the fusion of geneti-c a l l y dystrophic and g e n e t i c a l l y normal embryos would provide a unique 10 in s i g h t into the pathogenesis of muscular dystrophy. By using an appropriate c e l l marker i n the chimeric mice i t would be possible to determine, i n th i s i n t e r a c t i o n system, whether g e n e t i c a l l y dys-trophic c e l l s d i f f e r e n t i a t e d normally to contribute to mature muscle. It would be of i n t e r e s t to see i f the c l i n i c a l phenotype and the extent and type of muscle mosaicism of th i s a r t i f i c i a l c e l l u l a r mosaic state mimicked the c a r r i e r of the human X-linked Duchenne dystrophy. These females are considered to be g e n e t i c a l l y mosaic for t h i s X-linked gene due to random i n a c t i v a t i o n of one X chromosome, and they show v a r i a b l e c l i n i c a l , biochemical and h i s t o p a t h o l o g i c a l features of the disease. This v a r i a b i l i t y has been a t t r i b u t e d to v a r i a b l e genetic c o n s t i t u t i o n of the muscles (Emery, 1964). If chimeric muscles composed p r i m a r i l y of eit h e r g e n e t i c a l l y dystrophic or g e n e t i c a l l y normal f i b e r n u c l e i could be found, an attempt could be made to characterize the nature of the primary l e s i o n . I f ge n e t i c a l l y normal muscles were i d e n t i f i e d and the muscle pathology re s u l t s from a primary l e s i o n within the muscle f i b e r , the h i s t o l o g i c a l phenotype of these muscles would be expected to be normal. S i m i l a r l y , g e n e t i c a l l y dystrophic muscles should demonstrate the c h a r a c t e r i s t i c features of dystrophic histopathology. If t h i s was not found, some other f a c t o r , presumably neural, would be implicated as the cause of the degenerative response of the muscle. CHAPTER II GENERAL MATERIALS AND METHODS 1. Mice: source and maintenance i . inbred s t r a i n s The SWV s t r a i n of mice was developed by Dr. J . R. M i l l e r and was at the F34-F36 generation of inbreeding during the course of t h i s experiment. These mice are albino. A l l other inbred s t r a i n s of mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). i i . dystrophic mice The dystrophic mice used i n t h i s study were purchased from 2J the Jackson Laboratory. The new dystrophic a l l e l e , dy , was used ex c l u s i v e l y . The homozygous dystrophic mice were eit h e r used d i r e c t l y , i n which case they were at the N^ - N6F1 generation of backcrossing to C57BL/6J from the WK/Re s t r a i n , or bred to produce further homo-zygous dystrophic mice. Embryos f o r mosaic production were obtained 2J 2J from matings of homozygous (dy /dy ) p a i r s . The dystrophic mice used and mice of the C57BL/6J s t r a i n have black coat pigmentation. 11 12 i i i . timed matlngs SWV females used f o r timed matings were caged i n groups of four per cage and the males were caged s i n g l y . Single females, determined to be i n proestrus by vaginal signs, were placed with males p r i o r to midnight and checked f o r copulation plugs the following morning. To provide timed matings of dystrophic mice, i t was necessary to continuously cohabitate males and females as p a i r s . These females were checked every morning f o r the presence of a copulation plug. The day on which a copulation plug was observed was designated as day 0 of pregnancy. i v . maintenance of the mice A l l mice used i n t h i s study were maintained i n the Zoology Vivarium at the University of B r i t i s h Columbia. The animal rooms were kept on an 18 hour l i g h t period (6 A.M. to 12 midnight) and a 6 hour dark period. A l l mice were provided with a continuous water supply. Mice of the dystrophic colony were fed Purina Mouse Chow; a l l other mice, Purina Laboratory Chow. Optimal maintenance and breeding performance was observed with these d i e t s . 2. Production of chimeras The techniques employed to produce the chimeras used i n t h i s study were derived from the protocol designed by Mullen and Whitten 13 (1971). A b r i e f d e s c r i p t i o n of the procedure i s included below with emphasis on adaptations employed i n the present study. i . c o l l e c t i o n of embryos With the lig h t - d a r k cycle maintained i n the mouse rooms, embryos were generally at the 8 - c e l l stage by 12 noon of day 2. At t h i s time they were s t i l l within the lumen of the F a l l o p i a n tube. To c o l l e c t the embryos the female was k i l l e d by c e r v i c a l d i s l o c a t i o n and the uterine horns, with the F a l l o p i a n tubes attached, were removed. The F a l l o p i a n tubes were flushed by i n s e r t i n g a short bevel 30 gauge needle, f i t t e d to a syringe with prewarmed culture media, into the juncture of the tube and the uterus. The embryos were washed out the f i m b r i a l end of the tube and c o l l e c t e d i n 4 cm square c a v i t y s l i d e s (Clay Adams, No. A-1478). The embryos were picked up and transferred, with a custom made breaking pipette, to fresh media. Although the majority of embryos c o l l e c t e d from SWV female mice were of c o n s i s t e n t l y good q u a l i t y , at the 8 - c e l l stage with no sign of blastomere death, t h i s was not the case with the dystrophic embryos. These embryos often were retarded i n development (less than 8 c e l l s ) and frequently showed evidence of blastomere death. When the zona p e l l u c i d a was removed these embryos often shed blastomeres which were observed as s i n g l e c e l l s l y i n g adjacent to the blastocyst a f t e r the culture period. This response and the generally poor condition of these embryos may represent some early developmental e f f e c t of the dystrophic genotype but more l i k e l y r e s u l t s from the 14 generally d e b i l i t a t e d condition of the dystrophic females which produce eggs and/or provide a preimplantation environment of poor q u a l i t y . i i . f usion and c u l t u r i n g of embryos The no n - c e l l u l a r zona p e l l u c i d a surrounding the 8 - c e l l embryo was removed by pronase d i g e s t i o n (Mintz, 1962). Immediately upon swelling of the zona, the embryos., were washed i n several changes of fresh culture media and, i f necessary, r a p i d l y pipetted u n t i l the zona broke f r e e . The zona-free embryos were then placed s i n g l y i n micro drops of cult u r e media under p a r a f f i n o i l . The same procedure was then c a r r i e d out with a second set of embryos of d i f f e r i n g genotype. When the micro drops contained the two g e n e t i c a l l y d i f f e r e n t embryos, a pipette was used to nudge the pa i r s together. This procedure was repeated two or three times to insure that the embryos were aggregating. During t h i s e n t i r e procedure, except for b r i e f i n t e r r u p t i o n for necessary manipulations, the embryos were kept i n cult u r e media maintained at 3 7 \u00C2\u00B0 C i n an atmosphere of pre-humidified 5% CO2 i n a i r . When the embryos had aggregated, the cultures were placed into an anaerobic j a r , flooded with 5% CC^ i n a i r and incubated at 3 7 \u00C2\u00B0 C. Within 24 hours the majority of the aggregated embryos had d i f f e r e n t i a t e d into f u l l y developed s i n g l e b l a s t o c y s t s . 15 i i i . embryo transfer The r e c i p i e n t dams used i n t h i s study were a l l SWV mice which had at l e a s t one previous l i t t e r by normal mating. Care was taken to insure that these females were i n proestrus p r i o r to placing them with a vasectomized male. The presence of a copulation plug the following morning was evidence of pseudopregnancy. Routinely, females i n day 2 of pseudopregnancy were used as r e c i p i e n t s of day 3 embryos (day 2 embryos plus 24 hours i n v i t r o ) . Blastocysts were transferred s i n g l y or i n small clutches of 2 or 3 to the r i g h t uterine horn of the r e c i p i e n t dams. These dams were anesthetized with Nembutal (sodium pentobarbital, 50 mg/ml, Abbott Laboratories, Montreal) administered at 1.8 mg/kg. Aft e r plucking the h a i r from the back, a small dorsal midline i n c i s i o n was made i n the ski n followed by a l a t e r a l i n c i s i o n i n the abdominal w a l l above the r i g h t uterine horn near the ovary. The uterine horn was brought to the ex t e r i o r by passing a loop of thread around the ovarian end of the horn. Care was taken not to damage the F a l l o p i a n tube or to expose the ovary. The thread also served to hold the uterus during blastocyst t r a n s f e r . A small hole was made i n the uterine w a l l with a 26 gauge disposable needle and the transfer pipette was i n t r o -duced into the uterine lumen i n an ovarian d i r e c t i o n and the b l a s t o -cysts expelled. No a i r was allowed to enter the uterus. The thread was removed, the uterus was returned to the body c a v i t y , and the dorsal skin i n c i s i o n was closed with a s i n g l e wound c l i p (Clay Adams, 16 18 mm a u t o c l i p , No. B-2365). i v . f o s t e r nursing Routinely, 17 days following the transfer procedure (day 19 of pseudopregnancy of the r e c i p i e n t dam, day 20 of the embryos), the r e c i p i e n t dams were k i l l e d by c e r v i c a l d i s l o c a t i o n and surviving fetuses were removed by Caesarian section. These term fetuses were then placed with newborn l i t t e r s to be fos t e r nursed. Females of the SWV s t r a i n , which had given b i r t h within 48 hours, were successful f o s t e r mothers. 3. Chimeras produced Nineteen mice were produced by the above technique during the course of t h i s study. Of these, 11 form the basis of subsequent experiments and these mice were numbered i n order of b i r t h from chimera (or C) number 7 to C number 17 (Table 1). i . demonstration of mosaicism A l l chimeras used i n t h i s study demonstrated both albino 2J 2J and pigmented coat c o l o r . SWV mice are albino and dy /dy mice, at the N^ - Ng generation of backcrossing to C57BL/6J were black. No attempt to quantitate or analyse the chimera pigementation pattern was made. H i s t o l o g i c a l a nalysis of the pigmented epithelium of the retinas from the chimeras also revealed both albino and pigmented c e l l s . Hemoglobin analysis by an electrophoretic technique (Martin 17 and Petras, 1971) demonstrated both Hbb-s (SWV) and Hbb-d (WK/Re and C57BL/6J) from each chimera tested. The hemoglobin e l e c t r o -phoretogram i s presented i n Figure 1. i i . dystrophic\u00E2\u0080\u0094normal chimeras 2J 2J A t o t a l of A5 homozygous dystrophic (dy /dy ) embryos were obtained of which 24 were s u c c e s s f u l l y fused to SWV embryos and developed to the blastocyst stage. Af t e r embryo transfer to r e c i -pient dams, 9 l i v i n g mice were delivered by Caesarian section. These are referred to subsequently as chimeras number 8 to 16. Mice number 13 and 14 showed only black pigmentation 2J 2J (dy /dy ) and died with the re s t of t h e i r f o s t e r mothers normal l i t t e r . Presumably these two mice were not chimeras since they did not exhibit the patchy pigmented and albino skin. Chimera number 12 developed severe s k i n l e s i o n s probably due to a mite i n f e c t i o n and died. Chimera number 15 developed a prolapsed rectum and signs of a mite i n f e c t i o n . A f t e r a s i n g l e treatment with a \"mite dip\", i t died. The remaining 5 chimeras of t h i s type were analysed f o r c l i n i c a l and h i s t o l o g i c a l parameters of muscular dystrophy and f o r muscle composition. i i i . normal\u00E2\u0080\u0094normal chimeras Two other chimeras, number 7 and 17, derived from fusion of 18 ge n e t i c a l l y normal embryos, were analysed i n the same way as the dystrophic\u00E2\u0080\u0094normal chimeras. Chimera number 7 was produced from a C57BL/10J and SWV embryo fusion and chimera number 17 was produced from an unknown wild type embry6 (genotype +/?, from the^dystrophic colony, mated to G57BL/10J) fused to a SWV embryo. TABLE 1 Number and External Sex of Chimeric Mice Produced f o r this Study Type of chimera 1. dy 2 J/dy 2 J~+/+ (SWV) 2. +/+ (C57BL/10J) \u00E2\u0080\u0094 +/+ (SWV) 3. +/? x +/+ (C57BL/10J)-+/+ (SWV) Females Chimera number 10 12 16 (3) 7 17 Males Chimera number 8 9 11 13 14 15 (6) Total 9 1 1 11 VO 20 - s Hbb- d o r i gin \u00E2\u0080\u00A2 + 1 SWV t 1 SWV C57BL/6J \u00E2\u0080\u00A2 C57BL/6J \u00E2\u0080\u00A2 WK/Re \u00E2\u0080\u00A2 1 C 8 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 C 9 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 C 10 I I I C11 \u00E2\u0080\u00A2 II C15 i i C16 Figure 1. Hemoglobin electrophoretogram. Chimeras are designated as C. CHAPTER I I I MUSCLE FUNCTION OF DYSTROPHIC\u00E2\u0080\u0094NORMAL CHIMERIC MICE The c l i n i c a l phenotype of the dystrophic\u00E2\u0080\u0094normal chimeras was studied to determine the extent, i f any, of fu n c t i o n a l loss 2J 2J of motor a b i l i t y . Dystrophic mice (dy /dy ) clasp t h e i r hind feet, a l t e r n a t e l y f l e x and extend t h e i r toes and legs when l i f t e d by the t a i l , drag one hind l e g or the other when walking, and exhibit a mild opisthotonus (arched p o s i t i o n of the body) (Meier and Southard, 1970). In l a t e r stages of the disease process 2J 2J (3 to 4 months), the dy /dy mice, maintained at the Univ e r s i t y of B r i t i s h Columbia, developed marked kyphosis, permanent p a r a l y s i s of the hind limbs and severe contractures (permanent contraction of muscles). It i s d i f f i c u l t to accurately assess s l i g h t d e f i c i e n c y of motor function i n mice by v i s u a l inspection. Loss of t o t a l l y normal muscle function i n the dystrophic\u00E2\u0080\u0094normal chimeras could be missed by t h i s c r i t e r i o n . Therefore, to q u a n t i t a t i v e l y assess muscle function, the maximum stimulated twitch of the an t e r i o r t i b i a l i s muscle was measured. This muscle was selected to represent muscle function i n the chimeras because i t i s known to be affe c t e d by 21 22 f u n c t i o n a l c r i t e r i a i n dystrophic (dy/dy) mice (Salafky, 1971). This muscle can be stimulated i n d i r e c t l y by the peroneal nerve and the r e s u l t i n g twitch i s a function of the muscle-nerve u n i t . Materials and Methods 1. Determination of the maximum isometric twitch of the anterior t i b i a l i s muscle A f t e r anesthesia (sodium pentobarbital, 80 mg/kg), the h a i r on the two hind legs was clipped and the skin over the anterior t i b i a l i s and biceps femoris muscles was removed. The tendon of the anterior t i b i a l i s muscle was i s o l a t e d and t i e d with f i n e s i l k thread (Champion Serum Proof S i l k No. 6-0). The anterior t i b i a l i s muscle was then freed of connective ti s s u e by a shallow i n c i s i o n pro-ceeding from the tendon up both sides of the muscle. The s c i a t i c nerve was exposed by an i n c i s i o n p a r a l l e l to the dorsal edge of the biceps femoris and cut d i s t a l to :the point of b i f u r c a t i o n of the peroneal nerve. The mouse was placed on i t s side and i t s leg positioned and secured to the cork f l o o r of a p l a s t i c chamber. The anterior t i b i a l i s muscle was stretched to i t s normal r e s t i n g length and i t s tendon was secured to the s t r a i n gauge with the attached thread. The s c i a t i c nerve was positioned on a b i p o l a r s i l v e r electrode and the proximal end of the nerve was cut. During the course of the operation a l l exposed tissues were constantly bathed i n 35-37\u00C2\u00B0 C p h y s i o l o g i c a l s a l i n e . P r i o r to recording, the p l a s t i c chamber with the preparation was flooded with 35-37\u00C2\u00B0 C 23 mineral o i l to prevent cooling and drying of the muscle and nerve. Square wave pulses (15 v o l t s ) of the 0.1 msec duration were applied to the electrode. Signals from the transducer (Stratham s t r a i n gauge, Model G7B1.5-350) were led v i a a polygraph preamplifier (Gilson Medical E l e c t r o n i c s ) to display on an osciloscope (Tektronix Type 532). A l l measurements were made from photographs of the trace taken by a kymograph camera (Grass Instrument Co.). Recordings from anterior t i b i a l i s muscles were made i n s i t u from SWV, SWV-C57BL/10J F l , and dystrophic ( d y 2 J / d y 2 J ) mice and from dystrophic\u00E2\u0080\u0094normal and normal\u00E2\u0080\u0094normal chimeras. Results and Discussion 1. V i s u a l observation of the dystrophic\u00E2\u0080\u0094normal chimeras No c l i n i c a l feature c h a r a c t e r i s t i c of the dystrophic condition was observed i n the dystrophic \u00E2\u0080\u0094 normal chimeras i n d a i l y observations from b i r t h to two months or i n weekly observation from two months to death. Neither l i f t i n g by the t a i l nor depressing the hind limbs caused any marked leg or toe f l e x i n g and never resulted i n foot dragging. The hehavior of these dystrophic\u00E2\u0080\u0094normal chimeras was ind i s t i n g u i s h a b l e from that of normal mice. 2. Maximum isometric twitch of anterior t i b i a l i s muscles Examples of the osciloscope displays obtained from the d y s t r o p h i c \u00E2\u0080\u0094 normal chimeras are shown i n Figure 2. The frequency response of the apparatus was not s u f f i c i e n t to permit c a l c u l a t i o n of the contraction 24 i. Figure 2. Osciloscope d i s p l a y of maximum isometric twitch of chimera a n t e r i o r t i b i a l i s muscles. 1 and 2; l e f t and r i g h t muscles of chimera 8. 3 and 4; l e f t and r i g h t muscles of chimera 9. 5 and 6; l e f t and r i g h t muscles of chimera 10. 25 and r e l a x a t i o n times. The maximum stimulated twitch tension i n grams f o r a l l mice tested i s presented i n Figure 3. The r e s u l t s from SWV mice (X 10.00), although s l i g h t l y lower, agree favorably with those obtained by Salafsky (1971) for normal mice of the 129 s t r a i n (X 11.41). 2J 2J S i m i l a r l y , the dystrophic (dy /dy ) mice gave contractions that were s l i g h t l y weaker than those obtained by Salafsky for dystrophic (dy/dy) mice; X 1.80 and X 2.97 r e s p e c t i v e l y . This lower response 2J was not expected because the dy mutant i s considered to produce a milder form of dystrophy with a l a t e r onset. The normal\u00E2\u0080\u0094normal chimeras produced a mean contraction s l i g h t l y higher (11.30) than the inbred SWV mice and i s probably i n d i c a t i v e of he t e r o s i s . These r e s u l t s are close to those obtained for the hybrid SWV-C57BL/10J F^ mice (X 10.81) and therefore do not present a major problem i n detecting s l i g h t deficiences i f present i n the dystrophic\u00E2\u0080\u0094normal chimeras. When examined, the dystrophic\u00E2\u0080\u0094normal chimeras ranged i n age from 6 to 12 months which provided time for the progressive changes of dystrophy to become manifest but not to be confounded by s e n i l e changes associated with extreme aging. Although the r e s u l t s from these chimeras were v a r i a b l e , they produced a s u r p r i s i n g l y stronger contraction (X 13.52) than any of the co n t r o l c l a s s e s . The r e s u l t s from the l e f t and r i g h t legs of the i n d i v i d u a l chimeras were very s i m i l a r i n d i c a t i n g a b i l a t e r a l uniformity of t h i s r e s u l t . c o \" c a> I-E C3 a*. o 20 18 16 14 12 10 8 6 4 2 Contro l + sd Dy s t roph i c Chimeras Mouse Fl S W V A 2J C11 C 9 C 8 C10 C16 i 0 1 7 C 7 Musc le s 8 5 7 L R L R L R L R L R ! L R L R Age (months) 10 5-8 3-5 6 9 10 10 12 ; 10 12 Weight (grams) X 36.4 32.7 16.0 j 33.5 29.5 39.0 49.0 32.0 i 32.6 43.5 T w i t c h (grams) X 10.8 10.0 1.8 13.5 i 11.3 Normal Chimeras Figure 3. Maximum isometric twitch of anterior t i b i a l i s muscles. Mouse: F l i s SWV-C57BL/10J; C i s chimera. Muscles: numbers are number of muscles analyzed; L and R i s l e f t and r i g h t ; broken l i n e s represent suboptimal preparations and are not included i n means; C 7 L e f t was a t e c h n i c a l f a i l u r e . as 27 Although the greater mean response of the dystrophic\u00E2\u0080\u0094normal chimeras was unexpected, i t i s apparent that these chimeras, except for chimera number 9, demonstrated contractions s i m i l a r to those of the normal chimeras. The much larger response of chimera number 9 i s not r e a d i l y explainable. These maximum twitch r e s u l t s are compelling evidence against f u n c t i o n a l deficiences of the nerve-muscle unit at the maximum stimulus l e v e l i n the dystrophic\u00E2\u0080\u0094normal chimeras. This supports t h e i r c l i n i c a l evaluation. To explain the absence of any detectable dystrophic phenotype i n the dystrophic\u00E2\u0080\u0094normal chimeras several p o s s i b i l i t i e s may be entertained. 1. The muscles of these chimeras may be composed e n t i r e l y of gene-t i c a l l y normal components. Three s i t u a t i o n s could produce t h i s state: a. a s e l e c t i v e overgrowth of normal myoblasts during muscle morphogenesis. b. a repopulation of degenerated g e n e t i c a l l y dystrophic muscle f i b e r s by g e n e t i c a l l y normal myoblasts. c. a f u n c t i o n a l l y heterozygous condition i n the muscle f i b e r s ; that i s , where dystrophic n u c l e i are present, normal n u c l e i occupy a place i n the same muscle f i b e r mimicking a heterozygous condition i n the muscle syncytium. 28 2. The peripheral motor nerves may be composed p r i m a r i l y of gene-t i c a l l y normal neurons. Two obvious phenomena could produce t h i s state: a. a developmental s e l e c t i o n against g e n e t i c a l l y dystrophic neuroblasts. b. f u n c t i o n a l death of mature g e n e t i c a l l y dystrophic motor nerves accompanied by axonal sprouting of g e n e t i c a l l y normal motor nerves to reinnervate any denervated muscle f i b e r s . 3. Some other f a c t o r such as vascular supply (Hathaway, Engel and Zellweger, 1970) may be responsible f o r the muscle degeneration i n muscular dystrophy. In these chimeras, the tis s u e responsible f o r t h i s f a c t o r could be of g e n e t i c a l l y normal o r i g i n . CHAPTER IV GENETIC CONSTITUTION OF MUSCLES FROM CHIMERAS Selected muscles from the chimeras were analysed f o r t h e i r genetic c o n s t i t u t i o n . The r e s u l t s were expected to i n d i c a t e i f g e n e t i c a l l y dystrophic c e l l s suffered any developmental f a i l u r e or s e l e c t i v e elimination by the pathological process i n the mosaic environment. The known genetic variants of mice provide several differences between c e l l s of two s t r a i n s , but, t h e i r a p p l i c a t i o n as u s e f u l markers i n s k e l e t a l muscle i s very l i m i t e d . Frye and Edidin (1970) found that c e l l surface antigens r a p i d l y intermix a f t e r c e l l f u s i o n . Edidin and Fambrough (Fambrough, 1972, personal communication) have shown that the muscle f i b e r membrane also has f l u i d properties and that antigens on the myotube surface are free to move l a t e r a l l y . Labelled antibodies to s p e c i f i c antigen markers would therefore not be u s e f u l to d i s t i n g u i s h e i t h e r t o t a l r a t i o s of two types of n u c l e i or t h e i r d i s t r i b u t i o n within the muscle f i b e r s of the chimeras. The w e l l defined enzyme a c t i v i t y v a r i a n t s which have been useful i n analysing non-synctial chimera t i s s u e , e_.&. l i v e r , 29 30 (Wegmann, 1970 and Condamine, Custer and Mintz, 1971) are not r e a d i l y applicable to s k e l e t a l muscle a n a l y s i s . The normal occurrence i n muscle tis s u e of d i f f e r e n t f i b e r types, i-.e^. f a s t - t w i t c h white, fa s t - t w i t c h red and slow-twitch intermediate, with inherent differences i n the enzyme l e v e l s for g l y c o l y t i c and oxidative metabolism (Close, 1972) would not permit a simple a n a l y s i s . There are, however, several electrophoretic variants that can be applied to the analysis of whole muscle preparations. In the mouse, these f a l l into two general c l a s s e s : i . v a r i a n t s which y i e l d only a s i n g l e protein band i n e i t h e r homozygote but both parental bands i n the heterozygote; e_.&., hemoglobin beta chain variants (Hbb) (Wegmann and Gilman, 1970). i i . v a riants which y i e l d s i n g l e bands i n the homozygotes, but i n which the codominant expression of the a l l e l e s i n the heterozygote r e s u l t s i n more that the two parental types. These a d d i t i o n a l hybrid bands, with m o b i l i t i e s intermediate between the two parental types, r e s u l t from an a s s o c i a t i o n of protein subunits to produce the a c t i v e enzyme (Shows and Ruddle, 1968). This second class of electrophoretic variants i s useful f o r genotypic content analysis of s k e l e t a l muscle and i n a d d i t i o n can be used to determine the nuclear arrangement within the muscle f i b e r s . In chimeras, produced from embryos d i f f e r i n g i n the homozygous type of enzyme, the pattern of the muscle electrophoretogram can be analysed. If only the 2 parental protein bands are resolved, the n u c l e i must be i n muscle f i b e r s composed e n t i r e l y of s i n g l e 31 n u c l e i types. If any hybrid bands are resolved, some or a l l of the muscle i s composed of f i b e r s that have formed from a fusion of d i f f e r e n t types of myoblasts and therefore contain d i f f e r e n t types of n u c l e i (Mintz and Baker, 1967). Mod-1 electrophoretic v a r i a t i o n A genetic v a r i a n t of the supernatant NADP-malate dehydrogenase (malic enzyme) from mice that demonstrates hybrid banding was reported by Henderson (1966). This enzyme i s designated as L-malate: NADP oxidoreductase, decarboxylating (EC 1.1.1.40) and the electrophoretic varinats are designated Mod-la and Mod-lb c o n t r o l l e d a b by the codominant a l l e l e s Mod-1 and Mod-1 r e s p e c t i v e l y , at the Mod-1 locus (Shows, Chapman and Ruddle, 1970). Henderson (1966) demonstrated the presence of f a s t and slow migrating forms of the enzyme found i n AKR and C3H mice, r e s p e c t i v e l y . The heterozygous mice ( F l AKR/C3H) contained a hybrid form of the enzyme, intermediate between the two parental bands. The electrophoretic r e s o l u t i o n that she obtained did not allow her to determine whether the heterozygote contained three of f i v e bands. Evidence for a f i v e band heterozygote pattern was presented by Shows and Ruddle (1968) who used a d i f f e r e n t electrophoretic system. They interpreted t h i s pattern to represent an enzyme that i s a c t i v e only as a tetramer. The a c t i v i t y r a t i o of the hybrid and parental bands was v i s u a l l y observed to be 1:4:6:4:1 which i s the predicted phenotype for tetrameric enzymes composed of 2 subunit 32 types that associate randomly. The composition of these e l e c t r o -4 p h o r e t i c a l l y d i s t i n c t bands i s assumed to be the expansion of (a + b) that y i e l d s laaaa; 4aaab; 6aabb; 4abbb; lbbbb. In t h e i r system, the band that migrated furthest (lbbbb) stained very f a i n t l y , and they considered t h i s to r e s u l t from the pH of the electrophoretic system. A d d i t i o n a l independent evidence for a tetrameric structure was reported by Baker and Mintz (1969). Using a d i f f e r e n t buffer system, they also reported a f i v e banded electrophoretic phenotype a b fo r Mod-1 /Mod-1 mice i n the r a t i o of 1:4:6:4:1 by v i s u a l observation. Their study included analysis of tissues from 'allophenic' mice derived from Mod-l a/Mod-l a and Mod-l^/Mod-1^ embryo types. From the mosaic muscle they resolved f i v e bands i n various r a t i o s that further supported random as s o c i a t i o n of the tetrameric subunits. L i v e r from these allophenic mice yielded only the homozygous Mod-la and Mod-lb protein bands, i n d i c a t i n g that both c e l l types coexisted i n t h i s t i s s u e . Engle and Wolfe (1971) examined the phenotype of the Mod-1 enzyme i n developing heterozygous embryos and demonstrated the synchronous a c t i v a t i o n of both a l l e l e s . Mod-1 genotype of chimeras In the present study, mice of the SWV s t r a i n were determined to have the Mod-la enzyme and C57BL/6J, C57BL/10J and WK/Re mice were known to be Mod-lb type (Russell, 1972, personal communication). Because the dystrophic mice used were only at the N, to N, F l 33 generation of backcrossingto C57BL/6J from WK/Re, i t would not have been possible to use with c e r t a i n t y any enzyme varia n t that was not shared by these two s t r a i n s . In the electrophoretic patterns from the chimera muscles, the presence of 'a' type subunits would represent the normal SWV n u c l e i a c t i v i t y and 'b' type subunits, g e n e t i c a l l y dystrophic n u c l e i . From s i n g l e muscles, the r a t i o of 'a' and 'b' subunits could be used to assign the r e l a t i v e nuclear c o n t r i b u t i o n of the two c e l l types. This type of electrophoretic analysis however, i s subject to the l i m i t a t i o n s presented f o r the histochemical assays. If the r e l a t i v e concentration of the enzyme employed was not equal i n the various muscle f i b e r types, the electrophoretogram could only be used to give a general estimate of the t o t a l n u c l e i r a t i o s i n the muscle. The c o n t r i b u t i o n from the f i b e r s demonstrating low concentration of the assay enzyme would be underestimated i f detected at a l l . Malic enzyme demonstrates equal a c t i v i t y , on the basis of wet weight, i n both red and white f i b e r s of rabbit muscle (Czok and BUcher, 1960). Although no s i m i l a r studies have been reported f o r mice, there i s no obvious reason to expect d i f f e r e n t r e s u l t s . Mod-1 genetic variants therefore, were t h e o r e t i c a l l y i d e a l for the nuclear marker required to i d e n t i f y and quantitate the extent and type of mosaicism i n the chimeras used i n t h i s study. It was on t h i s basis that techniques were established to resolve the 34 e l e c t r o p h o r e t i c v a r i a n t s of Mod-1 and to develop a q u a n t i t a t i v e system that could be used to estimate c e l l or n u c l e i r a t i o s i n the mosaic t i s s u e s . M a t e r i a l s and Methods 1. Tissue p r e p a r a t i o n C o n t r o l mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and chimeras by sodium p e n t o b a r b i t a l i n j e c t i o n . The chimeras were d i s s e c t e d immediately f o l l o w i n g the maximum t w i t c h experiment. I n d i v i d u a l muscles and organs were i d e n t i f i e d , removed by d i s s e c t i o n and quick f r o z e n i n l i q u i d n i t r o g e n . These t i s s u e s were stored i n sealed c o n t a i n e r s at -70\u00C2\u00B0 C u n t i l f u r t h e r processed. Frozen muscles were weighed, s l i c e d i n t o t h i n s e c t i o n s (1mm t h i c k ) w i t h a r a z o r blade and homogenized i n 1 ml of d i s t i l l e d water i n a V e r t i s '45' micro homogenizer. The homogenate was c e n t r i f u g e d at 3\u00C2\u00B0 C f o r 15 minutes at 15,000 x g and the supernatant r e c e n t r i f u g e d at 90,000 x g f o r an a d d i t i o n a l 30 minutes. The r e s u l t i n g c l e a r supernatant was d i v i d e d i n t o two equal p a r t s and f r e e z e d r i e d . P r i o r to e l e c t r o p h o r e s i s , the samples were r e c o n s t i t u t e d by 0.003 ml water to 0.01 g of frozen muscle s t a r t i n g weight. This y i e l d e d a 6 to 7 f o l d c o n c e n t r a t i o n of the c e n t r i f u g e d supernatant. Muscles weighing more than approximately 70 mg. were analysed s u c c e s s f u l l y . 2. E l e c t r o p h o r e s i s of Mod-1 Attempts to d u p l i c a t e the techniques of Shows and Ruddle (1968) and Baker and Mintz (1969) provided s u c c e s s f u l r e s o l u t i o n of the 35 two homozygous enzyme types. The heterozygous enzyme pattern, although comparable to t h e i r r e s u l t s , was not r e a d i l y adaptable to an accurate quantitative a n a l y s i s . Lack of precise r e s o l u t i o n and excessive s t a i n i n g a c t i v i t y between the bands presented the major problems. To increase the r e s o l u t i o n , a micro starch gel technique, designed by Tsuyuki, Roberts, Kerr and Ronald (1966) was adapted for high voltage electrophoresis. The apparatus and gels are pictured i n Figures 4 and 5. Advantages of t h i s micro system are: i . sharp d e f i n i t i o n of the bands from high voltage procedures, i i . small sample volumes can be analysed (0.005 ml), i i i . t h i n gels that can be cleared and scanned d i r e c t l y with a densitometer. a. Buffer system The buffer system used was a modification f or high voltage of that reported by Baker and Mintz (1969) . The bridge bu f f e r consisted of 2000 ml double d i s t i l l e d water, 18.08 g c i t r i c acid anhydrous and Trizma base added to pH 8.4. The gel bu f f e r was 12.5 ml bridge buffer d i l u t e d to 250 ml with d i s t i l l e d water and readjusted to pH 8.4 with Trizma base. b. Preparation of the g e l A 12% gel was produced by adding 250 ml of gel bu f f e r to 30 g of E l e c t r o s t a r c h . The gel was cooked to E l e c t r o s t a r c h s p e c i f i c a t i o n s and poured d i r e c t l y (without degassing) into the gel mould. A f t e r s e t t i n g f o r 90 minutes at room temperature, the gel was placed into 36 Figure 5. Micro gels with samples applied p r i o r to el e c t r o p h o r e s i s . 37 a refrigerator for an additional 30 minutes to pre cool the gel. Gel slices, 1/16 inch thick, were prepared and the sample applied to slots cut in the gel. c. Running conditions The apparatus was covered with plastic film and placed into a refrigerator (5\u00C2\u00B0 C) with a high power fan to dissipate heat from both top and bottom surfaces of the gel. Electrophoresis was carried out for 5 hours with a voltage gradient of 21 V/cm across the gel. d. Mod-1 enzyme assay Enzyme activity was visualized by a modification of the assay method of Henderson (1966). 5 ml of 1.0 M Tris-HCL buffer, pH 8.0 was added to 95 ml of d i s t i l l e d water. To a 30 ml aliquot of this buffer, 390 mg of L-malic acid was added and adjusted to pH 8.0 with concentrated NaOH. This aliquot was then returned to the buffer. 3.7 mg MnC^, 20 mg triphosphopyridine nucleotide (Sigma), 2.4 mg phenazine methosulfate (Sigma) and 20 mg Nitro B Tetrazolium (Dejac Laboratories) were then added and thoroughly dissolved by agitation. A section of the gel, including the origin and the Mod-1 zone, was placed in a-covered Petri plate with 40 ml of the assay solution, covered and continuously agitated on a rotator enclosed in a dark 37\u00C2\u00B0 C incubator. When the gels had stained, they were removed from the incubator and fixed in a gel wash consisting of 5 parts water, 5 parts methanol and 1 part glacial acetic acid. The stained gels were stored in this solution. 38 3. Quantitation of relative Mod-1 band activity a. Densitometry The fixed gels were slowly heated in a bath of glycerine unt i l the white starch approaced optical c l a r i t y . The gels were then transferred to a clear plastic Petri plate and covered with a glass microscope slide. A Joyce, Loebl recording microdensitometer (wedge D 3, aperature 5 mm, s l i t 30-60) was used to produce a density trace of the electrophoretogram. b. Integration The areas under the peaks of the density trace were integrated manually with a Kosumi planimeter (bar setting 14.00). The relative areas only were required so no correction of the integration numbers to calculate real area was made. Where the peaks were not completely isolated, a perpendicular line from the lowest point between them was projected to the base line. No correction for this t a i l i n g was made. Results and Discussion 1. Mod-1 electrophoretic pattern The electrophoretogram of the Mod-lab phenotype produced by the micro high voltage electrophoretic system is presented in Figure 6. The presence of 5 bands in the heterozygote is consistent with previous reports. However, the measured activity ratio was not consistent with the expected 1:4:6:4:1 ratio. The band corresponding to that of the homozygous Mod-1^/Mod-1^ type (i.e.. bbbb) stained faintly. Shows and Ruddle (1968) reported that this band stained faintly in their system and heterozygote phenotypes pictured by 39 Band Composition 1 aaaa SWV 4 aaab 6 aabb 4 abbb 1 bbbb C57BL/6J origin Figure 6. Electrophoretogram of Mod-1 heterozygote. Note the f a i n t s t a i n i n g of the 1 bbbb band. o o a o CD \u00C2\u00A3 o CO \u00E2\u0080\u0094 +1 0) RJ \u00E2\u0080\u00A2+-\u00C2\u00BB a. Ui 50-40-30-20-10-i s d 40 Band Compo s i t i o n 1 a 4 4 a 3 b 6 a 2 b 2 4a b 3 1 b 4 O b s e r v e d Rat io 1.00 2.19 2.53 .59 .10 E x p e c t e d Rat io 1 4 6 4 1 C o r r e c t i o n Factor (\u00C2\u00A3f ) - 1.83 2.37 6.78 10.00 Figure 7. Mod-1: a c t i v i t y r a t i o of heterozygote pattern from muscle supernatant, Baker and Mintz (1969) also lacked intensive stainingof t h i s band. Due to t h i s d i f f e r e n c e i n d e t e c t a b i l i t y of Mod-la and Mod-lb enzymes, care was taken to ensure adequate s t a i n i n g times were provided to detect a l l the bands present. 2. C a l c u l a t i o n of c o r r e c t i o n f a c t o r s f o r Mod-1 isoenzymes From a s e r i e s of densitometer traces of heterozygous muscle electrophoretograms, the actual r a t i o of the a c t i v i t y was observed to be s u b s t a n t i a l l y d i f f e r e n t from the expected. The observed a c t i v i t y r a t i o of these preparations i s presented i n Figure 7. Due to th i s observed s h i f t from the t h e o r e t i c a l r a t i o , an important assumption, f o r which there was no experimental evidence, was made. The heterozygous enzyme concentration was assumed to be i n the t h e o r e t i c a l 1:4:6:4:1 a b r a t i o , presuming that the a c t i v i t y of Mod-1 and Mod-1 genes i s equal, but the observed a c t i v i t y was a r e s u l t of d i f f e r i n g enzymatic a c t i v i t i e s of the various tetrameric p r o t e i n types. Therefore, to a b measure Mod-1 and Mod-1 gene products, the observed a c t i v i t y was corrected by s u i t a b l e f a c t o r s . The 1 aaaa band had r e l a t i v e l y higher a c t i v i t y than the 1 bbbb band and i t s a c t i v i t y was set at unity. A unique c o r r e c t i o n f a c t o r f or each of the remaining 4 heterozygote bands was calculated by d i v i d i n g the observed a c t i v i t y into the expected a c t i v i t y which was generated from the assigned 1 aaaa value. From the corrected r a t i o , the r e l a t i v e c o n t r i b u t i o n of a and b subunits can be calculated and used as an estimate of the Mod-1 / Mod-l a and Mod-1\u00C2\u00B0/Mod-1^ nuclear content of mosaic t i s s u e s . A FOCAL computer program, designed to provide these c a l c u l a t i o n s i s presented 42 i n Appendix 1. To te s t t h i s approach, p r o t e i n concentrations of muscle supernatants were determined by 260/280 uv absorption r a t i o s (Warburg and C h r i s t i a n , 1942) and a 50% aaaa and 50% bbbb mixture was electrophoresed. The electrophoretogram and density trace of t h i s mixture revealed only the two 'parental' bands and c o r r e c t i o n of the in t e g r a t i o n r e s u l t s , _i.e_., 10.00 x bbbb a c t i v i t y , y i e l d e d 48% Mod-la and 52% Mod-lb. Si A s e r i e s of heterozygous phenotypes, analysed f o r Mod-1 and Mod-l P content, using the derived c o r r e c t i o n f a c t o r s , y i e l d e d a Mod-1 content of 50% \u00C2\u00B1 s.d. 2%. Therefore, the derived c o r r e c t i o n f a c t o r s , when applied to the a c t i v i t y r a t i o of Mod-1 electrophoretograms, a b provide an acceptably accurate assignment of Mod-1 and Mod-1 content. No attempt to determine further the degree of accuracy of t h i s procedure was made and with the inherent assumptions required, the subsequent measurements are considered only as an estimate of the r e l a t i v e nuclear content. Si Si b b 3. Mod-1 /Mod-1 and Mod-1 /Mod-1 estimate of nuclear content of muscles from chimeras A t o t a l of 59 muscles from 7 chimeras (5 dystrophic\u00E2\u0080\u0094normal and 2 normal\u00E2\u0080\u0094normal) were analysed for Mod-1 phenotype. In the dystrophic--normal chimeras, subunits of 'a' type could only r e s u l t from the presence of SWV (normal) n u c l e i whereas subunits of 'b' type could 2J 23 only r e s u l t from the presence of dy /dy n u c l e i . A3 f m ure 8. Electrophoretograms of Mod-1 from i n d i v i d u a l dystrophic \u00E2\u0080\u0094 n o r mal chimera muscles. Corresponding densitometer traces are presented above the electrophoretograms. 44 Examples of the electrophoretograms and the corresponding densitometer traces, from the dystrophic\u00E2\u0080\u0094^normal chimeras are presented i n Figure 8. The i n d i v i d u a l muscle i n t e g r a t i o n r e s u l t s from each chimera are tabulated i n Tables 2, 3, 4, 5, 6, 7 and 8. V i r t u a l l y every possible electrophoretic pattern was found. The majority of the muscles demonstrated hybrid Mod-1 bands i n d i c a t i n g that they were composed, e n t i r e l y or i n part, of heterokaryon muscle f i b e r s , jL.e_., muscle f i b e r s containing both g e n e t i c a l l y dystrophic and g e n e t i c a l l y normal n u c l e i . No muscle was found that consisted e n t i r e l y of dystrophic n u c l e i . The highest observed 'b' content fo r a s i n g l e muscle was 94% f o r the r i g h t psoas muscle from chimera number 10. This may be of l i m i t e d s i g n i f i c a n c e and t o t a l l y dystrophic muscles might have been observed i f the population of muscles examined had been l a r g e r . Analysis of some muscles demonstrated no ;!bf c o n t r i b u t i o n and therefore, these muscles were composed only of normal n u c l e i . The range of muscle composition found within s i n g l e chimeras was spectacular. For example, i n chimera number 10, the r i g h t t r i c e p s was composed of 94% normal n u c l e i whereas the r i g h t psoas was composed of 94% dystrophic n u c l e i . Considerable v a r i a t i o n was also found between l e f t and r i g h t muscles i n s i n g l e chimeras, e_.j>., the l e f t and r i g h t gluteus maximus i n chimera number 10 was composed of 67% and 21% dystrophic n u c l e i r e s p e c t i v e l y . A summary of the r e s u l t s i s presented i n Table 9. \"Average 'b' content\" was calculated by summing the observed 'b' content f o r TABLE 2 Chimera Number 7; Mod-1 Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b' 1. biceps femoris l e f t 0 0 0 37 84 121 94 2. gluteus maximus r i g h t 2 3 10 35 5 55 76 3. p e c t o r a l i s l e f t 0 0 4 26 62 92 94 r i g h t 15 20 37 10 5 87 60 Summary: i . T otal 'b' subunit of muscles examined = 88% i i . Muscle with highest 'b' content = l e f t biceps femoris (94%) i i i . Muscle with highest 'a' content = r i g h t pectoral (40%) TABLE 3 Chimera Number 8; Mod-1 Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b 1. biceps femoris l e f t 28 43 121 13 0 205 48 r i g h t 108 117 93 9 0 327 35 2. gastrocnemius r i g h t 112 7 0 0 0 119 3 3. gluteus maximus l e f t 73 32 9 6 0 120 29 r i g h t 78 15 6 0 0 99 12 4. masseter l e f t 52 14 11 7 3 87 47 r i g h t 83 5 5 2 0 95 16 5. p e c t o r a l i s l e f t 47 5 7 2 3 64 44 r i g h t 106 5 7 6 18 142 63 6. psoas l e f t 13 3 5 2 1 24 51 r i g h t 17 10 5 0 0 32 22 7. semimembranosis l e f t 4 4 20 25 7 60 75 8. tongue 25 0 0 0 6 31 71 9. tri c e p s l e f t 62 16 5 0 3 86 33 r i g h t 32 27 23 9 4 95 53 10. vastus l a t e r a l i s r i g h t 5 3 2 0 0 10 25 11. vastus medialis r i g h t 12 6 2 0 0 20 19 Summary: i . T o t a l 'b' subunit of muscles examined = 43% i i . Muscle with highest 'b' content = l e f t semimembranosis (75%) i i i . Muscle with highest 'a' content = r i g h t gastrocnemius (97%) TABLE 4 Chimera Number 9; Mod-l b Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b' 1. psoas l e f t 41 22 57 98 54 272 79 TABLE 5 Chimera Number 10; Mod-1 Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b 1. biceps femoris l e f t 42 16 79 96 92 325 83 2. gastrocnemius l e f t 75 26 5 0 0 106 13 r i g h t 33 15 13 7 0 68 42 3. gluteus maximus l e f t 55 72 68 55 28 278 67 r i g h t 134 128 25 0 0 287 21 4. masseter l e f t 7 4 5 0 0 16 30 r i g h t 6 4 7 8 14 39 85 5. p e c t o r a l i s l e f t 41 2 3 4 62 112 92 r i g h t 51 11 23 32 93 210 88 6. psoas l e f t 157 59 50 7 41 314 63 r i g h t 13 3 7 9 61 93 94 7. tr i c e p s l e f t 138 52 21 4 0 215 22 r i g h t 58 7 0 0 0 65 5 Summary: i . T otal 'b' subunit of muscles examined = 73% i i . Muscle with highest 'b' content = r i g h t psoas (94%) i i i . Muscle with highest 'a' content = r i g h t t r i c e p s (95%) oo TABLE 6 Chimera Number 11; Mod-1 Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b 1. anterior t i b i a l i s l e f t 24 4 2 0 0 30 12 r i g h t 30 3 0 0 0 33 4 2. biceps femoris l e f t 45 18 13 5 4 85 49 3. gastrocnemius l e f t 90 53 20 6 2 171 33 r i g h t 161 41 17 2 0 221 17 4. gluteus maximus l e f t 115 41 14 7 3 180 34 r i g h t 135 93 72 23 3 326 41 5. tongue 42 0 0 0 17 59 80 6. vastus l a t e r a l i s l e f t 176 44 12 3 0 235 16 r i g h t 33 11 2 0 0 46 13 Summary: i . T otal 'b' subunit of muscles examined = 35% i i . Muscle with highest 'b' content = tongue (80%) i i i . Muscle with highest 'a' content = r i g h t anterior t i b i a l i s (96%) TABLE 7 Chimera Number 16; Mod-1 Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b 1. biceps femoris l e f t 208 0 0 0 0 208 0 r i g h t 151 0 0 0 0 151 0 2. gluteus maximus l e f t 39 0 0 0 0 39 r i g h t + 0 0 0 0 + 0 3. psoas l e f t 19 7 0 0 0 26 10 r i g h t 93 0 0 0 0 93 0 4. tric e p s l e f t 88 0 0 0 0 88 0 r i g h t 101 0 0 0 0 101 0 * exceeded capacity of densitometer Summary: i . T o t a l 'b' subunit of muscles examined = 1% i i . Muscle with highest 'b' content = l e f t psoas(10%) i i i . Muscle with highest 'a' content = a l l others(100%) o TABLE 8 Chimera Number 17; Mod-1^ Estimate of Dystrophic Content Muscle Side aaaa aaab aabb abbb bbbb Total % 'b' 1. biceps femoris r i g h t 1 6 11 14 20 52 86 2. gluteus maximus l e f t 14 2 0 0 0 16 5 3. tr i c e p s l e f t 26 2 0 0 0 28 3 r i g h t 91 6 5 12 13 127 61 4. vastus l a t e r a l i s r i g h t 11 14 6 0 0 31 27 5. vastus medialis r i g h t 61 0 2 8 14 85 70 Summary: i . T o t a l 'b' subunit of muscles examined = 67% i i . Muscle with highest 'b' content = r i g h t biceps femoris (86%) i i i . Muscle with highest 'a' content = l e f t t r i c e p s (97%) TABLE 9 Genetic C o n s t i t u t i o n of Muscles Analysed for Mod-1^: Summary Chimera Average dystrophic content 'b' Total dystrophic content 'b' (normal\u00E2\u0080\u0094normal) C # 7 81 88 C # 17 42 67 C # 7 and C # 17 61 82 (dystrophic\u00E2\u0080\u0094normal) C # 8 37 43 C # 9 (one muscle only) 79 79 C # 10 54 73 C # 11 30 35 C // 16 1 1 C # 8, 9, 10, 11, and 16 40 58 (combined dystrophic and normal) C # 7, 8, 9, 10, 11, 16, and 17 46 62 53 each muscle and d i v i d i n g by the number of muscles. These percentage values r e f l e c t the average 'b' content per muscle with no consideration of the muscle s i z e . \" T otal 'b' content\" was calculated by summing the observed i n t e g r a t i o n r e s u l t s and solving f o r 'b'. This value probably r e f l e c t s the t o t a l c o n t r i b u t i o n of dystrophic n u c l e i more accurately because the a c t i v i t y of the electrophoresed enzyme was generally observed to be proportional to the s t a r t i n g muscle s i z e (muscles below 50 mg yielded j u s t perceptible a c t i v i t y whereas a c t i v i t y of larger muscles increased with the weight of the frozen muscle). I f a l l the muscles had been prepared as a s i n g l e sample, the r e s u l t i n g densitometer trace would be expected to solve f o r a 'b' value close to t h i s c a l c ulated percent. The t o t a l 'b' content i n normal\u00E2\u0080\u0094normal chimeras was 82% and i n dystrophic\u00E2\u0080\u0094normal chimeras 58%. The o v e r a l l *b' content f o r a l l the mosaics was 62%. The expected 'b' content of a large sample would be 50% i f no c e l l type had a s e l e c t i v e advantage. The higher values observed f o r these chimeras l i k e l y r e f l e c t only the li m i t e d sample s i z e examined. These r e s u l t s f o r the Mod-l^ marker present conclusive evidence that g e n e t i c a l l y dystrophic n u c l e i are present within the muscle f i b e r s composing the s k e l e t a l muscle of these chimeras. In only 1 chimera (chimera number 16) was there a dramatically high frequency of muscles composed e n t i r e l y of normal 'a' n u c l e i . Because t h i s was observed i n only 1 chimera i t i s interpreted as representing 54 one extreme of the v a r i a t i o n expected i n chimeras and not as a s p e c i f i c loss of the g e n e t i c a l l y dystrophic n u c l e i . This observation confirms previous h i s t o l o g i c a l evidence that no developmental abnormality e x i s t s i n dystrophic muscle (Platzer, 1971). Although the number of chimeras and the number of muscles from each was not large, there does not appear to be any d e f i c i t of dystrophic n u c l e i (dystrophic\u00E2\u0080\u0094normal chimeras have a t o t a l 'b' content of 58% amongst the muscles examined). Furthermore, dystrophic myoblasts are capable of fusing to normal myoblasts during the multinucleation stage as demonstrated by the large number of muscles with hybrid Mod-1 bands. The t o t a l 'b' content value of 58% also implies that no s e l e c t i v e death of dystrophic n u c l e i had taken place. The chimeras were between 6 and 12 months of age when they were k i l l e d and thus there was considerable time for any s p e c i f i c degeneration to take place. Furthermore, some muscles demonstrated large populations of 'homozygous' dystrophic muscle f i b e r s , e_.g_., l e f t p e c t o r a l i s of chimera number 10 (Table 5). These observations are therefore u n l i k e l y to be the r e s u l t of a heterokaryon muscle structure which mimicked the normal genetic heterozygote. The normal myogenesis and maintenance of the g e n e t i c a l l y dystrophic n u c l e i that apparently occurs i n these chimeras provides i n d i r e c t supportive evidence f o r Salafsky's (1971) transplant studies. Although the chimera r e s u l t s provide no evidence f o r the regeneration step required i n h i s experiment, they do demonstrate that g e n e t i c a l l y 55 dystrophic muscle f i b e r s can form and be maintained i n the mosaic environment. I t seems l i k e l y , therefore, that dystrophic muscle f i b e r s should also be able to regenerate and be maintained i n g e n e t i c a l l y normal hosts. The range of genetic composition within s i n g l e chimeras, both f o r d i f f e r e n t muscles and f o r the same muscle on both sides, demonstrates that each muscle must be considered as a unique combination. In t h i s l i g h t , i t i s most i n t e r e s t i n g that the maximum twitch r e s u l t s of the anterior t i b i a l i s muscles were apparently very uniform between l e f t and r i g h t muscles of the same chimera. F i n a l l y , the electrophoretic r e s u l t s provide some i n s i g h t into the usefulness of the chimeras as a model of the female c a r r i e r of the X-linked Duchenne dystrophy (Emery, 1964). Gearhart and Mintz (1972) proposed that the v a r i a b l e mosaicism they observed among eye muscles of normal\u00E2\u0080\u0094normal chimeras provides a model for the phenotypic v a r i a b i l i t y known to occur i n muscles of women heterozygous for Duchenne dystrophy. The c l i n i c a l and f u n c t i o n a l phenotype of the dystrophic\u00E2\u0080\u0094normal chimeras, which are known to 2J 23 have dy /dy n u c l e i i n t h e i r muscle f i b e r s implies that t h e i r i n t e r p r e t a t i o n i s not co r r e c t . A h i s t o l o g i c a l evaluation of the dystrophic\u00E2\u0080\u0094normal chimeras was undertaken to evaluate t h i s hypothesis. CHAPTER V HISTOLOGICAL EVALUATION OF MUSCLES FROM DYSTROPHIC\u00E2\u0080\u0094NORMAL CHIMERAS Based on both the c l i n i c a l evaluation and the maximum twitch examination (Chapter III), i t was expected that a histological evaluation of these chimeras would reveal only slight or no pathological lesions characteristic of muscular dystrophy. If a normal histological phenotype was observed in muscles composed primarily of dystrophic nuclei i t would support a non-primary myopathogenesis for the mouse dystrophy. Similarly, i f muscles of genetically normal composition were observed to have foci of pathological change, some factor, such as abnormal innervation, could be postulated as the primary cause of the muscle pathology. Although extensive histological characterization of dystrophic muscle from dy/dy mice has been reported (Michelson e^ t a l . , 1955; Ross, Pappas and Harman, 1960; West, Meier and Hoag, 1966; and West and Murphy, 1960), only limited analysis of the histopathology of 2J 2J the dy /dy mutant is available. Meier and Southard (1970) 2J 2J reported that the histopathological features of dy /dy mice were similar to those of the dy/dy mice. They differed only in that the former demonstrated a reduction in the extent of abnormalities 56 57 that \"consisted, i n whole or part, of loss of s t r i a t i o n , coagulation necrosis, regenerative a c t i v i t y , v a r i a t i o n i n f i b e r s i z e and i n t e r n a l rowing of n u c l e i \" . Materials and Methods A l i m i t e d h i s t o l g i c a l a p p r a i s a l of the anterior t i b i a l i s 2J 2J muscles of dy /dy and chimeric mice was done. Muscles selected a_ p r i o r i with i n t e r e s t i n g genetic c o n s t i t u t i o n from the chimeras were also examined. Because the f i r s t c l i n i c a l signs of the disease i n dystrophic mice are recognizable from hind limb musculature involvment, only muscles from the hind limbs of the dystrophic== normal chimeras were considered f o r close examination. To achieve c l e a r h i s t o l o g i c a l r e s o l u t i o n , adequate to accurately assess s l i g h t d e f i c i e n c e s , a l l muscle samples were prepared f o r 1 u thick p l a s t i c sections. A razor blade section (approximately 2 mm thick) was taken across the b e l l y of the i n d i v i d u a l muscles immediately a f t e r t h e i r d i s s e c t i o n from the mouse. These muscle samples were f i x e d f o r 4 hours i n cold 2.5% gluteraldehyde, buffered to pH 7.3 with cacodylate buffer, rinsed with 0.1 M cacodylate buffer for 1/2 hour and then post f i x e d f o r 1 hour i n 2% OsO^ i n 0.1M cacodylate buffer, pH 7.3. F i x a t i o n was done at approximately 5\u00C2\u00B0 C i n a r e f r i g e r a t o r while dehydration was done at room temperature. A f t e r dehydration through an alcohol s e r i e s , followed by 30 minutes i n propylene oxide, the muscles were placed into a 1:1 mixture of Luft's epon (Luft, 1961) 58 and propylene oxide for 24 hours. F i n a l embedding was performed at 60\u00C2\u00B0 C f o r 20 to 24 hours i n epon. The muscle tissues thus prepared were cut into 1 u thick sections on a Reichert ultramicrotome using a glass k n i f e . A f t e r drying on a glass s l i d e , the sections were stained with 1% t o l u i d i n e blue and mounted with Permount. Results and Discussion 1. Dystrophic muscle; a n t e r i o r t i b i a l i s Cross sections of a n t e r i o r t i b i a l i s muscle from a 4 month 2J 2J old dy /dy mouse are presented i n Figures 9 and 10. The most s t r i k i n g feature i s marked v a r i a t i o n i n f i b e r s i z e . Examples of small, presumably regenerating f i b e r s , are common. Hypertrophied f i b e r s are scattered throughout. Evidence of c e n t r a l nucleation i s common. Many f i b e r s lack the c h a r a c t e r i s t i c polygonal shape of normal muscle f i b e r s and are round instead. Hyaline degeneration, f i b e r s p l i t t i n g and areas of f i b r o s i s can also be observed. 2. Dystrophic\u00E2\u0080\u0094normal chimeras: anterior t i b i a l i s Although t h i s muscle was analysable f o r nuclear genotype i n only 1 chimera (chimera number 11), i t was examined to see i f the fu n c t i o n a l r e s u l t s were r e f l e c t e d i n normal h i s t o l o g i c a l features. As expected, t h i s a nalysis revealed very few features that were a t y p i c a l of normal muscle. Examples of these sections are presented i n Figure 11, 12, 13 and 14. The general a r c h i t e c t u r e of the muscles, with compact polygonal f i b e r s , was normal. The most common feature 59 Figure 10. A n t e r i o r t i b i a l i s muscle from 4 month o l d 2J 2J d y s t r o p h i c (dy /dy ) mouse, mag. x 600. 60 Figure 11. L e f t a n t e r i o r t i b i a l i s of chimera 8. mag. x 240, Figure 12. L e f t a n t e r i o r t i b i a l i s of chimera 10. mag. x 240. Figure 13. Right anterior t i b i a l i s of chimera 9. mag. x 220 Figure 14. Right a n t e r i o r t i b i a l i s of chimera 9, mag. x 600 62 i n d i c a t i v e of any dystrophic pathological change was the presence of occasional c e n t r a l nucleation. 3. Dystrophic\u00E2\u0080\u0094normal chimeras: selected muscles Figures 15 and 16 are sections from the l e f t gluteus maximus 2J 2J of chimera number 8. The estimated dy /dy nuclear content of t h i s muscle was 23%. Apart from the rare example of c e n t r a l nucleation and possible f a t t y i n f i l t r a t i o n t h i s muscle demonstrates normal features. Figures 17 and 18 are sections from the l e f t biceps femoris 2J 2J of chimera number 10. The estimated dy /dy nuclear content of t h i s muscle was 83%. No hind limb muscle of higher dystrophic content was a v a i l a b l e for h i s t o l o g i c a l a n a l y s i s . Although rare examples of c e n t r a l nucleation and possibly degenerating f i b e r s were observed, the h i s t o l o g i c a l features of t h i s muscle were also remarkably normal. Figures 19, 20 and 21 are sections of the l e f t biceps femoris muscle from chimera number 16. No electrophoretic evidence of 2J 2J dy /dy n u c l e i could be resolved so t h i s muscle was p r i m a r i l y , i f not completely, g e n e t i c a l l y normal. Several small f o c i of degeneration with l e s i o n s c h a r a c t e r i s t i c of dystrophic myopathology, are present. These include: marked v a r i a t i o n i n f i b e r s i z e , 1.e_. , hypertrophied and regenerating f i b e r s ; f i b e r s p l i t t i n g ; coagulation necrosis; and frequent examples of c e n t r a l nucleation. It i s u n l i k e l y that these observations are the r e s u l t of 63 64 Figure 18. L e f t biceps femoris of chimera 10. mag. x 600. 65 66 Figure 21. L e f t biceps femoris of chimera 16. mag. x 750. 67 normal changes due to aging. No abnormal histological features were observed in sections of muscle from chimera number 7, a normal\u00E2\u0080\u0094 normal chimera killed at 10 months of age. Furthermore, the unique histological features of senile muscle, e_.\u00C2\u00A3. Ringbindin, observed by Rowe (1969) in muscles from 25 month old mice, were not observed in the muscles examined in this study. Although this analysis, by design, was neither extensive nor done \"blind\" the results are striking. The minimal patholgical involvement of the anterior tibialis muscles is consistent with the functional measurements. The implications of the histological evaluation of muscles selected for genetic composition are obvious. They support previous evidence, including the muscle composition results of this study, that the primary cause of muscle degeneration, in the dystrophic mouse, is not the result of the muscle genotype. The interpretation of these histological results is however, 2J 23 only preliminary. No muscle was found with only dy /dy nuclei. It can be argued that the minimal pathological involvement of muscles with genetically dystrophic nuclei could result from the presence of the normal nuclei. Conversely, muscles determined to be composed of only genetically normal nuclei could contain a small proportion of genetically dystrophic nuclei that was not detectable by the electrophoretic system employed. These interpretations are unlikely however, because the apparent extent of degeneration was most severe in the genetically normal muscle. These observations suggest that variable mosaicism in some 68 other tissue, possibly the motor neurons, should be considered. Furthermore, the model proposed by Gearhart and Mintz (1972) to explain the variable phenotype of muscles from women heterozygous for Duchenne dystrophy should be reevaluated. CHAPTER VI GENERAL DISCUSSION The present study demonstrates the usefulness of the chimeric mouse to delineate the s i t e of the primary l e s i o n i n the complex pathology of muscular dystrophy. In dystrophic\u00E2\u0080\u0094normal chimeras, both g e n e t i c a l l y dystrophic and normal n u c l e i were detected i n the s k e l e t a l muscle. There was no apparent degeneration of g e n e t i c a l l y dystrophic muscle f i b e r n u c l e i which were estimated to co n t r o l 58% of the mature chimera muscle examined. This i s evidence that the primary l e s i o n , r e s u l t i n g i n the muscle wasting of g e n e t i c a l l y dystrophic mice, resides outside the muscle f i b e r . Furthermore, dystrophic n u c l e i were demonstrated to be arranged i n both heterokaryon and homokaryon l i k e muscle f i b e r s . These r e s u l t s imply that g e n e t i c a l l y dystrophic myoblasts contribute normally to muscle morphogenesis. The dystrophic-normal chimeras were c l i n i c a l l y normal. This observation may i n d i c a t e that the phenotypic s i m i l a r i t i e s of the 2J mouse dy and human Duchenne dystrophy are i n c i d e n t a l to primary l e s i o n s of d i f f e r e n t natures. A l t e r n a t i v e l y , the response of normal mouse ti s s u e to t h i s p a t h o l o g i c a l genotype, i n chimeric mice, may be s u f f i c i e n t to developmentally or f u n c t i o n a l l y compensate f o r d e f i c i t s , which for unknown reason, remain unchallenged i n the human genetic mosaic. 69 70 The muscle function analysis of the chimeras provides further evidence that the f a c t o r responsible f o r the dystrophic phenotype was e i t h e r , selected against i n development or was f u n c t i o n a l l y compensated for i n the mature animal. H i s t o l o g i c a l analysis of mature s k e l e t a l muscles demonstrated rare scattered l e s i o n s with c h a r a c t e r i s t i c s of dystrophic muscle pathology. These f o c i were present i n muscles of both g e n e t i c a l l y normal and p r i m a r i l y dystrophic composition. This observation supports the i n t e r p r e t a t i o n of the muscle composition r e s u l t s and gives further i n s i g h t into the possible nature and l o c a t i o n of the primary l e s i o n . Recent attempts to characterize the s i t e of the l e s i o n have implicated the peripheral nervous system. The r e s u l t s of the present study are not inconsistent with t h i s hypothesis. Genetically dystrophic motor nerves could undergo a progressive f u n c t i o n a l (je.\u00C2\u00A3. trophic influence) or morphological (e.\u00C2\u00A3. motor-end-plate) d e t e r i o r a t i o n leading to p a r t i a l or complete denervation of the s k e l e t a l muscle. The simultaneous presence of g e n e t i c a l l y normal nerves i n the chimeras could compensate f o r t h i s l o s s . The type of h i s t o l o g i c a l l e s i o n s observed i n the mature chimera s k e l e t a l muscle may represent t h i s dynamic process. The scattered f o c i of degeneration could represent the l o s s of s i n g l e g e n e t i c a l l y dystrophic motor nerves. These denervated muscle f i b e r s could subsequently receive normal innervation from adjacent neurons of g e n e t i c a l l y normal c o n s t i t u t i o n by c o l l a t e r a l axonal sprouting and thus regain a 71 normal histological appearance. This hypothesis can be tested. Techniques are presently available to estimate the size of individual motor units. If the above hypothesis i s correct, the size of the motor units in these chimeras would be larger than motor units in corresponding muscles of genetically normal mice. Furthermore, the relative motor unit size would be expected to increase with age as the number of genetically dystrophic neurons, s t i l l functioning, approached zero. The chimera technique also may be useful in the gene function analysis of a genetically determined developmental muscle disease of mice. The lethal muscular dysgenesis, mdg, mutant described by Gluecksohn-Waelsch (1963) has been characterized ultrastructurally by Platzer and Gluecksohn-Waelsch (1972). The action of this gene results in a total failure of normal muscle morphogenesis with the f i r s t alteration - swollen sarcoplasmic reticulum - detectable by day 14 of gestation. Bowden-Essien (1972) demonstrated by in vitro studies that the mdg mutant may interfere with muscle contractility by affecting properties of the c e l l membrane. A muscle composition analysis of mature mdg/mdg\u00E2\u0080\u0094normal chimeras would be expected to reflect the developmental presence of the muscular dysgenic c e l l s . This could be demonstrated by the recovery of only the genetically normal nuclei or by recovery of the mdg/mdg nuclei spatially arranged only in heterokaryon muscle fibers. 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Hereditary mouse muscular dystrophy with p a r t i c u l a r emphasis on pathogenesis and attempts at therapy. Ann. N. Y. Acad. S c i . 138: 4-13. West, W. T. and E. D. Murphy. 1960. Histopathology of hereditary, progressive muscular dystrophy i n inbred s t r a i n 129 mice. Anat. Rec. 137: 279-295. APPENDIX 1 FOCAL program: Mod-1 estimate of nuclear content C-8K FOCAL 31969 01.01 E 01.05 D 4*A \" S A M P L E \" S A \u00C2\u00BB ! ! 31.10 T \u00C2\u00A3 5 . 0 3 * \" A t 4 4 ( A t 3 * B ) 6\" 01.15 T \" 4(A*Bt3> B f 4 T O T A L \" * M * \" A C T I V I T V ?\" 01.20 f I=1*5;A \" \"TR(I>*S 5 1=S 1 +TRC 1 > ; T \" \";S Z*f ( 1 )=TR( 1 > *CF( I ; 01.25 T bT*!*\"PROTEIN \"if I = 1*5;T O i l ) * \" \" J S C I = C7 +C?tl> 01.30 T CT*!\"PR. RATIO \"*S PC=1.25 01.35 F i = l * 5 ; S CRC I )=C-/ ( I ) /CT; T C K C l ) * \" \";S TT=TT+TRCI) 01.40 T !*\"A C0NTE.MT \" > r i = l * 5 ; S PC=PC-.25*S AG ( I ) = CR ( I > * PC 01.45 r 1=1*5;T A C C 1 ) * \" \" * S H T = A T + A C ( I > 01.50 T AT*!*S BT=1-AT;T \"B CJ.Mi\u00C2\u00A3\T \"* F I = 1 * 5;T C R C 1 ) - A C ( i ) * \" \" 01.55 T B T * i * \" T H . P R . R A T \u00C2\u00AB \" 01.60 S A l = A T t 4 ; S A 2 = 4 * C A T T 3*3T>* S A3= 6* C A T t 2 \u00C2\u00AB 6T* 2 ) 01.65 S A 4 = 4 * t A T * B T t 3 ) ; S A5 = BTt 4 01.70 T A l * \" \"A2*\" \"A3*\" \"A4*\" \"A5*! 01.75 T \" i H\u00E2\u0080\u00A2RAT\u00C2\u00AB ACT\" iS RC1)=A 1/CFC 1 ); S RC2 > =A2/CF(2) 01.80 S R l 3 >=AJ/CF (. 3 ) ; S RC4)=A4/CFC4);S RC5)=A5/CFC5) 01.85 F 1 = 1*5*3 RT=RT+R(1)*T R U ) * \" 01.90 T R T * ! \u00C2\u00BB F I=1*5;S R ( I ) = R < I ) * < T T / R T ) 02.10 F 1=1*5*5 C H = C H + ( ( T R ( I > - R t I ) > t 2 ) / R ( 1 ) 02.1t> 1 \"CHI SQUARE\"CH* ! ! ! ! ; G 1.01 04.05 S CF(1>=1*S CF(2)=l.fc3\u00C2\u00BBS CF(3)=2-37 04.10 S C F ( ^ ) = 6 . 7 8 * 5 CFC5*=10 "@en . "Thesis/Dissertation"@en . "10.14288/1.0101027"@en . "eng"@en . "Genetics"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Genotypic and phenotypic analysis of muscles from dystrophic - normal mouse chimeras"@en . "Text"@en . "http://hdl.handle.net/2429/32218"@en .