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Genotypic and phenotypic analysis of muscles from dystrophic - normal mouse chimeras Peterson, Alan Clarke 1973

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GENOTYPIC AND PHENOTYPIC ANALYSIS OF MUSCLES FROM DYSTROPHIC—NORMAL  MOUSE CHIMERAS  by  ALAN CLARKE PETERSON B.Sc,  U n i v e r s i t y o f 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 a c c e p t t h i s t h e s i s as conforming required  THE  to the  standard  UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1973  In p r e s e n t i n g t h i s an  thesis in p a r t i a l fulfilment  of the requirements  advanced degree at the U n i v e r s i t y of B r i t i s h Columbia,  the L i b r a r y s h a l l make i t  freely  available for reference  I agree  thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s r e p r e s e n t a t i v e s . of  that  and study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s for  for  this  I t i s understood t h a t copying or p u b l i c a t i o n  t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my  written permission.  Department of  Medical  Genetics  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  A p r i l 27, 1973  ABSTRACT  1. 2J (dy  Artificial  c h i m e r i c mice, mosaic f o r muscular d y s t r o p h i c  2J /dy 2.  ) and normal (SWV +/+)  These c h i m e r i c mice demonstrated no c l i n i c a l  the d y s t r o p h i c 3.  genotypes were s u c c e s s f u l l y produced. features of  condition.  A maximum s t i m u l a t e d  t w i t c h a n a l y s i s o f the a n t e r i o r  t i b i a l i s muscles o f these chimeras a l s o r e v e a l e d no f u n c t i o n a l d e f i c i e n c e s c h a r a c t e r i s t i c o f the d y s t r o p h i c 4. was  condition.  The e l e c t r o p h o r e t i c phenotype o f Mod-1  resolved with  (malic enzyme)  a h i g h v o l t a g e m i c r o s t a r c h g e l system and a  q u a n t i t a t i v e a n a l y s i s was  developed to d e t e c t and e s t i m a t e  the  r e l a t i v e muscle f i b e r n u c l e a r m o s a i c i s m i n s i n g l e muscles from the chimeras.  The measured a c t i v i t y o f 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 p r e v i o u s l y 5. in  dy  The p r e s e n c e o f g e n e t i c a l l y d y s t r o p h i c n u c l e i was  the m a j o r i t y 2J  reported.  o f chimera muscles examined.  detected  The o v e r a l l content  of  2J /dy  n u c l e i from a l l the muscle samples examined was  be 58%.  This indicated that g e n e t i c a l l y dystrophic  normally  to muscle morphogenesis and t h a t g e n e t i c a l l y d y s t r o p h i c  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 d e g e n e r a t i o n environment.  ii  cells  estimated  to  contribute muscle  i n t h i s a r t i f i c i a l mosaic  6.  W i t h i n s i n g l e chimeras  extensively.  the c o m p o s i t i o n o f muscles v a r i e d  I n d i v i d u a l muscles of both normal and p r i m a r i l y  d y s t r o p h i c g e n e t i c c o m p o s i t i o n were d e t e c t e d . 7. muscles, to  H i s t o l o g i c a l a n a l y s i s o f samples of a n t e r i o r  prepared  determine  place.  i n 1 u t h i c k p l a s t i c s e c t i o n s , was  normal h i s t o l o g i c a l phenotype were  C o n v e r s e l y , muscles w i t h no d e t e c t a b l e d y s t r o p h i c c o m p o s i t i o n  were observed dystrophy.  phenotypes.  Examples o f muscles w i t h p r i m a r i l y d y s t r o p h i c g e n o t y p i c  c o m p o s i t i o n and remarkably revealed.  undertaken  i f any p r e c l i n i c a l muscle d e g e n e r a t i o n had taken  These muscles had e s s e n t i a l l y normal 8.  tibialis  to have f o c i o f d e g e n e r a t i o n c h a r a c t e r i s t i c o f muscular  These r e s u l t s , a l t h o u g h not e x t e n s i v e , a r e s u g g e s t i v e 2J  t h a t the muscle d e g e n e r a t i o n observed mice i s secondary fiber  i n d y s t r o p h i c (dy  2J /dy  )  t o a primary l e s i o n r e s i d i n g o u t s i d e the muscle  proper. 9.  The p o s s i b l e s i m i l a r i t i e s between these  c e l l u l a r mosaics  artificial  and a human X - l i n k e d g e n e t i c mosaic,  the heterozygous  female f o r Duchenne dystrophy, a r e p r e s e n t e d . 10. of  The r e s u l t s o f these s t u d i e s were d i s c u s s e d i n terms  the a p p l i c a t i o n of c h i m e r i c mice i n the d e l i n e a t i o n o f the  primary l e s i o n i n mouse muscular  iii  dystrophy.  TABLE OF CONTENTS  Page ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  vi  LIST OF FIGURES  v i i  LIST OF APPENDICES  ix  ACKNOWLEDGMENTS  x  Chapter I.  II.  III.  INTRODUCTION  1  Muscular d y s t r o p h y i n the mouse  2  Muscular d y s t r o p h y as a p r i m a r y myopathy  2  Muscular d y s t r o p h y as a primary neuropathy  4  Muscular d y s t r o p h y i n c h i m e r i c mice  9  GENERAL MATERIALS AND METHODS  11  1.  Mice: s o u r c e and maintenance  11  2.  P r o d u c t i o n o f chimeras  12  3.  Chimeras produced  16  MUSCLE FUNCTION OF DYSTROPHIC—NORMAL  CHIMERIC MICE  M a t e r i a l s and Methods 1.  21 22  D e t e r m i n a t i o n o f the maximum i s o m e t r i c t w i t c h o f the a n t e r i o r t i b i a l i s muscle  iv  22  R e s u l t s and D i s c u s s i o n 1.  2.  23  V i s u a l observation normal chimeras  o f the d y s t r o p h i c — 23  Maximum i s o m e t r i c t w i t c h o f a n t e r i o r t i b i a l i s muscles  IV.  23  GENETIC CONSTITUTION OF MUSCLES FROM CHIMERAS  29  Mod-1 e l e c t r o p h o r e t i c v a r i a t i o n  31  Mod-1 genotype o f chimeras  32  M a t e r i a l s and Methods  34  1.  Tissue preparation  34  2.  E l e c t r o p h o r e s i s o f Mod-1  34  3.  Q u a n t i t a t i o n o f r e l a t i v e Mod-1 band a c t i v i t y  38  R e s u l t s and D i s c u s s i o n 1.  Mod-1 e l e c t r o p h o r e t i c p a t t e r n  38  2.  C a l c u l a t i o n o f c o r r e c t i o n f a c t o r s f o r Mod-1 isoenzymes  41  M o d - l / M o d - l and M o d - l / M o d - l estimate of n u c l e a r c o n t e n t o f muscles from chimeras  42  3.  V.  a  a  b  b  HISTOLOGICAL EVALUATION OF MUSCLES FROM DYSTROPHIC— NORMAL CHIMERAS  57  M a t e r i a l s and Methods  57  R e s u l t s and D i s c u s s i o n  58  1.  Dystrophic  2.  D y s t r o p h i c — n o r m a l chimeras: a n t e r i o r t i b i a l i s muscles  3.  VI.  38  muscle: A n t e r i o r t i b i a l i s  58  58  D y s t r o p h i c — n o r m a l chimeras: s e l e c t e d muscles  62  GENERAL DISCUSSION  69  BIBLIOGRAPHY APPENDIX  72  v  77  LIST OF TABLES  Table 1.  Page Number and E x t e r n a l Sex o f C h i m e r i c Mice Produced f o r t h i s Study  19  2.  Chimera Number 7; Mod-l^ E s t i m a t e o f D y s t r o p h i c Content  45  3.  Chimera Number 8; M o d - l  E s t i m a t e o f D y s t r o p h i c Content  46  4.  Chimera Number 9: Mod-1  E s t i m a t e o f D y s t r o p h i c Content  47  5.  Chimera Number 10; M o d - l  b  E s t i m a t e o f D y s t r o p h i c Content  48  6.  Chimera Number 11; M o d - l  b  E s t i m a t e o f D y s t r o p h i c Content  49  7.  Chimera Number 16; M o d - l  b  E s t i m a t e o f D y s t r o p h i c Content  50  8.  Chimera Number 17; Mod-1  E s t i m a t e o f D y s t r o p h i c Content  51  9.  G e n e t i c C o n s t i u t i o n o f Muscles A n a l y s e d f o r Mod-l^; Summary  b  vi  52  LIST OF FIGURES  Figure  Page  1.  Hemoglobin electrophoretogram  2.  Osciloscope display of maximum isometric twitch  20  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 g e l electrophoretic apparatus  36  5.  Micro gels with sample applied p r i o r to electrophoresis  36  6.  Electrophoretogram of Mod-1 heterozygote  39  7.  Mod-1: a c t i v i t y r a t i o of heterozygote pattern from muscle supernatant Electrophoretograms of Mod-1 from i n d i v i d u a l dystrophic —normal chimera muscles with corresponding densitometer traces  43  A n t g j i o ^ t i b i a l i s muscle from 4 month o l d dystrophic (dy /dy ) mouse  59  8.  9. 10.  40  A n t e r i o r 2 t i b i a l i s muscle from 4 month old dystrophic (dy  /dy  ) mouse  59  11.  L e f t anterior t i b i a l i s of chimera 8  60  12.  L e f t 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  vii  17.  L e f t b i c e p s f e m o r i s o f chimera 10  64  18.  L e f t b i c e p s f e m o r i s o f chimera 10  64  19.  L e f t b i c e p s f e m o r i s o f chimera 16  65  20.  L e f t b i c e p s f e m o r i s of chimera 16  65  21.  L e f t b i c e p s f e m o r i s of chimera 16  66  viii  LIST OF APPENDICES  Appendix I.  FOCAL program: Mod-1 e s t i m a t e o f n u c l e a r c o n t e n t  ix  CHAPTER 1 INTRODUCTION  There a r e a l a r g e number o f e n v i r o n m e n t a l l y and g e n e t i c a l l y determined  d i s e a s e s o f v e r t e b r a t e muscle.  The term  'muscular  d y s t r o p h y ' i s r e s e r v e d 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 p r o g r e s s i v e w a s t i n g d i s e a s e s o f s k e l e t a l muscle.  Because no d i r e c t e v i d e n c e o f  abnormal i n n e r v a t i o n was o r i g i n a l l y d e s c r i b e d w i t h these muscle c o n d i t i o n s , they were c o l l e c t i v e l y thought o f as primary t h a t 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 r e s i d e s w i t h i n the muscle f i b e r s themselves  (Walton,  There has r e c e n t l y been a r e e v a l u a t i o n o f t h i s h y p o t h e s i s 1971  and McComas, S i c a , and Campbell,  evidence c o n c l u s i v e l y demonstrating l e s i o n produced  1961). (Dubowitz,  1971) w i t h emphasis now b e i n g  p l a c e d on the p o s s i b l e primary r o l e o f the nervous  initial  myopathies,  system.  However,  the s i t e and n a t u r e o f the  by the mutant genes has not been r e p o r t e d .  The p r e s e n t study d e s c r i b e s the use of a r t i f i c i a l  chimeric  mice, o f d y s t r o p h i c — n o r m a l c o m p o s i t i o n , t o d e l i n e a t e the primary of  a muscular  dystrophy mutation o f the mouse.  which supports a non-primary  lesion  Evidence i s p r e s e n t e d  myopathogenesis o f t h i s p a r t i c u l a r muscle  disease. These c h i m e r i c mice may a l s o serve a u s e f u l r o l e as a model 1  2  of  the female " c a r r i e r " o f the human X - l i n k e d Duchenne d y s t r o p h y  mutation.  Muscular d y s t r o p h y i n the mouse A r e c e s s i v e m u t a t i o n , d y s t r o p h i a m u s c u l a r i s ( gene symbol dy) c a u s i n g muscular d i s c o v e r e d i n 1951 the f i r s t  dystrophy i n the 129/Re mouse s t r a i n  was  ( M i c h e l s o n , R u s s e l l , and Harman, 1955).  I t was  g e n e t i c a l l y - d e t e r m i n e d muscle d i s e a s e , c o n s i d e r e d to be a  primary myopathy, i d e n t i f i e d i n an e x p e r i m e n t a l animal of known 2J g e n e t i c background. in  1969  of  these muscular  of  A new  i n the WK/Re s t r a i n  allele,  dy  , a t the dy_ l o c u s was d i s c o v e r e d  (Meier and Southard,  1970).  The  phenotype  d y s t r o p h i e s d i f f e r s o n l y i n the r a t e o f p r o g r e s s i o n 2J the d i s e a s e , the dy mutant h a v i n g a slower c o u r s e .  Muscular d y s t r o p h y as a_ primary myopathy D i r e c t e v i d e n c e , demonstrating muscular  d y s t r o p h y r e s i d e s w i t h i n 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 o f Duchenne muscular  Meryon i n 1852 in  t h a t the p r i m a r y l e s i o n i n  comments on the absence  the c e n t r a l nervous  system  o f any s t r u c t u r a l a b n o r m a l i t y  ( i n Dubowitz, 1971).  v a t i o n s have confirmed t h i s f i n d i n g  d y s t r o p h y by  Subsequent o b s e r -  (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 o f dy/dy mice, M i c h e l s o n , R u s s e l l  and Harman (1955) r e p o r t e d t h a t h i s t o l o g i c a l examination o f a l l l e v e l s of  the c e n t r a l nervous  system r e v e a l e d no demonstrable  pathology.  Muscle p r e p a r a t i o n s r e v e a l e d : p r o l i f e r a t i o n o f sarcolemmal  nuclei;  3. i n c r e a s e 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 i n c r e a s e i n the amount of c o n n e c t i v e and  some f a t t y replacement.  tissue  Subsequent s t u d i e s have confirmed  the  major h i s t o l o g i c a l f i n d i n g s (West and Murphy, 1960). In an experiment designed muscle pathology,  to c h a r a c t e r i z e the n a t u r e  of  the  O'Steen (1962) demonstrated d i f f e r e n c e s i n the  growth response o f normal and  dystrophic  (dy/dy) muscle.  muscle c u l t u r e s i n d i f f u s i o n chambers implanted  Dystrophic  i n dystrophic  and  normal h o s t s underwent a c h a r a c t e r i s t i c growth response ending i n fibrosis.  Normal muscle grew w e l l i n b o t h h o s t s .  These r e s u l t s were  i n t e r p r e t e d as d e m o n s t r a t i n g t h a t the p r i m a r y l e s i o n was the muscle f i b e r s and  t h a t an e n v i r o n m e n t a l e f f e c t was  In a s i m i l a r experiment w i t h mice, R o l s t o n and  d y s t r o p h i c muscle to the kidney  hosts.  He  inherent i n  very u n l i k e l y .  (1972) t r a n s p l a n t e d normal  c a p s u l e of normal and  r e p o r t e d 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  dystrophic  similar  conclusions.  These experiments can be c r i t i c i z e d on one major p o i n t . H i s t o l o g i c a l l y , muscle from d y s t r o p h i c mice p r e s e n t s of a b n o r m a l i t i e s  a wide spectrum  i n c l u d i n g an apparent i n c r e a s e i n c o n n e c t i v e  tissue.  An accompanying i n c r e a s e i n f i b r o c y t e c o n c e n t r a t i o n would t h e r e f o r e expected i n d y s t r o p h i c muscle.  T h i s c o u l d r e a d i l y e x p l a i n the  be  eventual  f i b r o s i s response of the c u l t u r e d or t r a n s p l a n t e d d y s t r o p h i c muscle. Conclusive  evidence  t h a t no c i r c u l a t i n g f a c t o r i s r e s p o n s i b l e  f o r the muscle breakdown i n mouse dystrophy  was  demonstrated by Pope  and Murphy (1960) w i t h c o n j o i n e d normal and  dystrophic  pairs.  4 Attempts w i t h r e t r o g r a d e h i s t o l o g i c a l a n a l y s e s have f a i l e d to  demonstrate  a p r e c i s e l e s i o n which c o u l d i m p l i c a t e the muscle  fiber directly. Platzer  I n an e x t e n s i v e u l t r a s t r u c t u r a l study w i t h dy/dy  (1971) demonstrated  t h a t the f i r s t  mice,  s t r u c t u r a l s i g n s of muscle  d e g e n e r a t i o n o c c u r o n l y i n the d i f f e r e n t i a t e d muscle f i b e r s . observed no a b n o r m a l i t y i n the myogenic p r o c e s s .  The f i r s t  She structural  a l t e r a t i o n was the presence o f s w o l l e n s a r c o p l a s m i c r e t i c u l u m ; b u t t h i s change was n o t noted u n t i l day 19 o f g e s t a t i o n expected date of b i r t h ) .  (day 19 o r 20 i s  She concluded t h a t t h i s a l t e r a t i o n i n the  s a r c o p l a s m i c r e t i c u l u m was a r e s u l t o f e i t h e r a f a u l t y membrane system i n t h e muscle o r a f a i l u r e o f a t r o p h i c i n f l u e n c e o f the nerve on the muscle. Attempts  to c h a r a c t e r i z e the l e s i o n b i o c h e m i c a l l y have a l s o  proven i n c o n c l u s i v e .  A l t h o u g h i n both mouse and human d y s t r o p h i e s  t h e r e a r e numerous changes i n s e v e r a l m e t a b o l i c pathways 1963  (Russell,  and Penn, C l o a k and Rowland, 1972) t h e r e has never been r e c o r d e d  a c o n f i r m e d example o f a s t r u c t u r a l change o r t o t a l absence  o f any  muscle p r o t e i n o r enzyme.  Muscular  dystrophy as a_ primary  neuropathy  A l t h o u g h the evidence f o r a primary myopathogenesis o f the muscular  d y s t r o p h i e s has been l i m i t e d , t h i s t r a d i t i o n a l t h e o r y has  been adhered  to s t r o n g l y .  Only r e c e n t l y has t h i s view been c h a l l e n g e d  and a v a r i e t y o f experiments have i n d i c a t e d a p o s s i b l e n e u r o g e n i c cause of these d e f e c t s .  5  B u l l e r , E c c l e s and E c c l e s  (1960) demonstrated t h a t f a s t and  slow muscles o f the c a t , namely f l e x o r d i g i t o r u m longus and s o l e u s muscles, r e c i p r o c a l l y changed cross innervation.  Dubowitz  their contractile properties  after  (1967a) extended these s t u d i e s to  demonstrate by h i s t o c h e m i c a l t e c h n i q u e s t h a t the b i o c h e m i c a l p r o p e r t i e s a s s o c i a t e d w i t h f a s t and slow muscles were a l s o changed experimental s i t u a t i o n .  i n this  These r e s u l t s demonstrated a s p e c t s o f the  profound i n f l u e n c e of the nerve on the muscle's c o n t r a c t i l e p r o p e r t i e s and b i o c h e m i c a l n a t u r e .  Moreover, Dubowitz  (1967b) observed  a h i s t o l o g i c a l l y myopathic appearance o f many muscles d u r i n g the e a r l y phases of r e i n n e r v a t i o n .  These o b s e r v a t i o n s h e l p e d form the  b a s i s f o r the t h e o r y t h a t the muscular d y s t r o p h i e s may r e s u l t a p r i m a r y l e s i o n w i t h i n the nervous system.  from  E v i d e n c e has accumulated  from a v a r i e t y o f s o u r c e s which g i v e s support to t h i s t h e o r y . 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 d y s t r o p h i c mice  (dy/dy).  With micro  e l e c t r o d e s they were a b l e to monitor the response o f s i n g l e muscle f i b e r s a f t e r b o t h d i r e c t and i n d i r e c t  ( v i a nerve) s t i m u l a t i o n .  A  f i b e r was c o n c l u d e d to be denervated i f i t responded to d i r e c t b u t n o t to i n d i r e c t s t i m u l a t i o n . 27% were d e n e r v a t e d .  Of 181 d y s t r o p h i c f i b e r s examined,  Of 208 f i b e r s t e s t e d i n l i t t e r - m a t e  j L . j 2 . , +/+ and dy/+ mice, o n l y 5 o r 2% were d e n e r v a t e d . p r e s e n t two p o s s i b l e e x p l a n a t i o n s f o r t h i s r e s u l t .  48 o r  controls,  The a u t h o r s  The d e n e r v a t i o n  c o u l d take p l a c e i n the v i c i n i t y o f the neuromuscular j u n c t i o n o r c o u l d r e s u l t from a n e c r o t i c segment w i t h i n the muscle f i b e r  which,  6  by b l o c k i n g e l e c t r i c a l and nervation  t r o p h i c s t i m u l i , causes f u n c t i o n a l  i n the d i s t a l segment of the Extensive  p a t i e n t s has  units.  i n d i c a t e d t h a t the muscle l o s s i s not  This r e s u l t implicates  have been r e p o r t e d by H a r r i s and mice (dy/dy).  The  generalized  C u r r i e , 1970).  smaller  the  Similar results  W i l s o n (1971) f o r muscular  s i g n i f i c a n t l y reduced i n  dystrophic muscles  dystrophic  However, the mean t w i t c h t e n s i o n of s i n g l e d y s t r o p h i c  u n i t s was  as  r e s u l t s from a l o s s of motor  number of motor u n i t s i n a n t e r i o r t i b i a l i s  c o n s i s t e n t l y e s t i m a t e d to be  mice.  dystrophic  the motor neuron as a s i t e of  primary l e s i o n (McComas, S i c a and  was  fiber.  motor u n i t a n a l y s i s i n muscular  expected w i t h a primary myopathy, but  de-  motor  than t h a t of normal u n i t s .  Evidence a g a i n s t a s i m p l e d e n e r v a t i o n  p a t h o g e n e s i s has  been  r e p o r t e d by Law  and Atwood (1972).  They demonstrated a n o n - e q u i v a l e n c e  of s u r g i c a l and  n a t u r a l denervation  i n d y s t r o p h i c mouse muscles.  The  e l e c t r i c a l " c a b l e " p r o p e r t i e s , measured w i t h i n t r a c e l l u l a r m i c r o e l e c t r o d e s , of f u n c t i o n a l l y i n n e r v a t i o n f i b e r s of d y s t r o p h i c  muscle  showed reduced r e s i s t a n c e .  function-  Dystrophic  f i b e r s which were not  a l l y innervated  had  a s i g n i f i c a n t l y higher  were i n n e r v a t e d  but  i n n e i t h e r case d i d they approach the v a l u e  normally innervated In m o d i f i e d  r e s i s t a n c e than those which for  fibers. t i s s u e c u l t u r e experiments i n which normal muscle  b i o p s i e s can d i f f e r e n t i a t e to m u l t i n u c l e a t e d stages w i t h c r o s s s t r i a t i o n s , B i s h o p , G a l l u p ,  myoblast and Skeate and  myotube 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 d y s t r o p h i c muscle.  No  s i g n i f i c a n t d i f f e r e n c e s were found i n the  l a g phase a t the b e g i n n i n g  of d i f f e r e n t i a t i o n ,  the l e n g t h s  breadths of the d i f f e r e n t i a t e d myotubes or i n the success c u l t u r e and  subsequent s u b c u l t u r e .  for this result  Amongst a number of  and of  initial  explanations  they conclude " t h a t some environmental or  neural  f a c t o r r e s p o n s i b l e f o r the d y s t r o p h i c c h a r a c t e r of the muscle i n v i v o i s l a c k i n g i n the i n v i t r o c o n d i t i o n s " . Attempts to h i s t o l o g i c a l l y i d e n t i f y d e f e c t s w i t h i n the dyst r o p h i c nervous system have p r o v i d e d f o r the neurogenic t h e o r y .  further supportive  C u r t i s , Abrams and  evidence  Harman (1961) r e p o r t e d  t h a t the motor end p l a t e s on d y s t r o p h i c muscle showed p r o g r e s s i v e l o s s of f i n e d e t a i l , apparent e l o n g a t i o n and units.  fragmentation  into  sub-  Ragab (1971) r e p o r t e d u l t r a s t r u c t u r a l changes i n the motor  end p l a t e s on h i s t o l o g i c a l l y normal muscle f i b e r s from d y s t r o p h i c mice.  He observed t h a t 40%  appeared normal w h i l e  60%  of the nerve endings of these  e x h i b i t e d v a r i o u s types of  abnormalities  i n c l u d i n g : r e d u c t i o n i n the numbers of s y n a p t i c v e s i c l e s ; amounts of n e u r o f i l a m e n t o u s complexity elongated  fibers  large  m a t e r i a l ; r e d u c t i o n i n the number  of the p o s t - s y n a p t i c  f o l d s and  and  the o c c a s i o n a l presence of  v e s i c l e s and m u l t i v e s i c u l a r b o d i e s .  H a r r i s , W a l l a c e and Wing (1972) r e p o r t e d a l a r g e r e d u c t i o n i n the t o t a l number of m y e l i n a t e d  axons i n the nerves s u p p l y i n g  a n t e r i o r t i b i a l i s muscle of d y s t r o p h i c a mean axon number of 512.5  (dy/dy) mice.  the  They observed  i n normal mice but a mean of o n l y 233.3  8 i n d y s t r o p h i c mice.  This reduction occurred  of axon diameter b u t the p a c k i n g normal s u g g e s t i n g  over the whole range  o f the axons w i t h i n the nerve was  a developmental f a i l u r e .  Attempts to t r a c e  this  l e s i o n to the s p i n a l c o r d , by d e m o n s t r a t i n g a r e d u c t i o n i n the number of h i s t o l o g i c a l l y normal a n t e r i o r horn motor neurons, have not been successful  (Josph and Netsky, 1972, Papapetropoulos and  Bradley,  1972). To  t e s t d i r e c t l y f o r a p a t h o l o g i c a l i n f l u e n c e o f the d y s t r o p h i c  nervous system, S a l a f s k y and  (1971) t r a n s p l a n t e d muscles between normal  d y s t r o p h i c mice o f the 129 s t r a i n .  the a n t e r i o r t i b i a l i s muscle regenerated  Dystrophic normally  to t h i s muscle bed of g e n e t i c a l l y normal mice.  muscle minces of when t r a n s p l a n t e d  Normal muscle d i d not  regenerate i n dystrophic hosts.  Functional innervation  occurred  w i t h i n 75 days i n the d y s t r o p h i c  t r a n s p l a n t and i n d i r e c t  gave r i s e t o a p p r o x i m a t e l y 75% o f c o n t r o l muscle t e n s i o n . evidence i s v e r y  s t r i k i n g b u t i s not c o n c l u s i v e .  stimulation This  I t was not p o s s i b l e  to demonstrate t h a t the presumed d y s t r o p h i c muscle regenerated genetically dystrophic. i n 5 dystrophic hosts  A l t h o u g h normal muscle f a i l e d  a f u n c t i o n a l muscle regenerated  yielded a perceptible contraction.  was  to regenerate i n 1 and  There a r e a number o f parameters  which d i f f e r s i g n i f i c a n t l y between normal and d y s t r o p h i c mice t h a t c o u l d account f o r the observed r e s u l t s .  The mean body weight o f  normal mice used i n S a l a f s k y ' s study was 27.88 g. whereas the dyst r o p h i c mice weighed o n l y  15.23 g.  terms o f the a v a i l a b l e b l o o d  supply  This d i f f e r e n c e , considered i n alone,  c o u l d e x p l a i n the reduced  9 response of muscle r e g e n e r a t i o n  i n the d y s t r o p h i c h o s t s .  importance of the v a s c u l a r supply documented  The  i n r e g e n e r a t i n g muscle i s w e l l  ( C a r l s o n , 1972).  Muscular dystrophy  i n c h i m e r i c mice  In 1961, Tarkowski demonstrated a unique e x p e r i m e n t a l  mani-  p u l a t i o n o f mouse embryos which y i e l d s s i n g l e mice t h a t a r e mosaics f o r 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,  c h i m e r i c , a l l o p h e n i c , and t e t r a p a r e n t a l .  This manipulation  of p r e -  i m p l a n t a t i o n embryos has l e d to s t u d i e s 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 M i n t z ,  normal and p a t h o l o g i c a l developmental phenomena.  1971), o f b o t h With the number o f  known g e n e t i c markers i n mice, i t i s p o s s i b l e w i t h many o f these chimeras to i d e n t i f y w i t h i n a p a r t i c u l a r t i s s u e o r organ the g e n e t i c o r i g i n o f the c e l l s .  Because these mice a r e e s s e n t i a l l y  d i f f e r i n g only i n t h e i r preimplantation e x c e p t i o n a l l y powerful  t o o l to study  p a t h o l o g i c a l genotypes without regenerative manipulation. One n o t a b l e e x c e p t i o n  o r i g i n , they p r o v i d e an  the i n t e r a c t i o n s o f normal and  the i n t e r v e n t i o n o f s u r g i c a l o r  (For review see N e s b i t t and G a r t i e r , 197L)  i n v o l v e s the immune system which may demon-  s t r a t e unique f e a t u r e s w i t h i n these chimeras (Wegmann, and  Hellstrbm,  "normal",  HellstrHm,  1971).  I t was p o s t u l a t e d t h a t a f u n c t i o n a l , h i s t o l o g i c a l and compos i t i o n a l a n a l y s i s o f c h i m e r i c mice d e r i v e d from the f u s i o n o f g e n e t i c a l l y d y s t r o p h i c and g e n e t i c a l l y normal embryos would p r o v i d e a unique  10  i n s i g h t i n t o the p a t h o g e n e s i s o f muscular d y s t r o p h y . a p p r o p r i a t e c e l l marker to  By u s i n g an  i n the c h i m e r i c mice i t would be p o s s i b l e  determine, i n t h 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-  t r o p h i c c e l l s d i f f e r e n t i a t e d n o r m a l l y to c o n t r i b u t e to mature I t would be of i n t e r e s t the  phenotype and  extent and type o f muscle mosaicism o f t h i s a r t i f i c i a l  mosaic s t a t e mimicked dystrophy. for  to see i f the c l i n i c a l  muscle.  cellular  the c a r r i e r o f the human X - l i n k e d Duchenne  These females a r e c o n s i d e r e d t o be g e n e t i c a l l y mosaic  t h i s X - l i n k e d gene due to random i n a c t i v a t i o n o f one X chromosome,  and they show v a r i a b l e c l i n i c a l , b i o c h e m i c a l and h i s t o p a t h o l o g i c a l f e a t u r e s o f the d i s e a s e .  T h i s v a r i a b i l i t y has been a t t r i b u t e d t o  v a r i a b l e g e n e t i c c o n s t i t u t i o n o f the muscles If  (Emery,  1964).  c h i m e r i c muscles composed p r i m a r i l y o f e i t h e r  genetically  d y s t r o p h i c o r g e n e t i c a l l y normal f i b e r n u c l e i c o u l d be found, an attempt c o u l d be made to c h a r a c t e r i z e the n a t u r e of the p r i m a r y l e s i o n .  If  g e 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 p a t h o l o g y r e s u l t s from a p r i m a r y l e s i o n w i t h i n the muscle f i b e r ,  the h i s t o l o g i c a l  phenotype o f t h e s e muscles would be expected to be normal.  Similarly,  g e n e t i c a l l y d y s t r o p h i c muscles s h o u l d demonstrate the c h a r a c t e r i s t i c features of dystrophic histopathology.  I f t h i s was n o t found, some  o t h e r f a c t o r , presumably n e u r a l , would be i m p l i c a t e d as the cause o f the  d e g e n e r a t i v e response o f the muscle.  CHAPTER I I GENERAL MATERIALS AND  1.  Mice: i.  s o u r c e and inbred  The SWV and was of  METHODS  maintenance  strains  s t r a i n of mice was  developed by Dr. J . R.  Miller  a t the F34-F36 g e n e r a t i o n of i n b r e e d i n g d u r i n g the c o u r s e  t h i s experiment.  These mice are a l b i n o .  A l l other inbred s t r a i n s  of mice were purchased from the  J a c k s o n L a b o r a t o r y (Bar Harbor, Maine).  ii.  dystrophic  mice  The d y s t r o p h i c mice used i n t h i s study were purchased from 2J the  Jackson L a b o r a t o r y .  exclusively.  The new  d y s t r o p h i c a l l e l e , dy  The homozygous d y s t r o p h i c mice were e i t h e r  i n which case they were a t the N^ - N6F1  , was used  used directly,  g e n e r a t i o n of b a c k c r o s s i n g  to C57BL/6J from the WK/Re s t r a i n , or b r e d to produce f u r t h e r homozygous d y s t r o p h i c mice.  Embryos f o r mosaic p r o d u c t i o n were o b t a i n e d 2J 2J  from matings of homozygous (dy  /dy  ) pairs.  The d y s t r o p h i c  mice  used and mice o f the C57BL/6J s t r a i n have b l a c k coat p i g m e n t a t i o n . 11  12  iii.  timed  matlngs  SWV females used f o r timed matings were caged f o u r p e r cage and the males were caged determined  singly.  i n groups o f  S i n g l e females,  to be i n p r o e s t r u s by v a g i n a l s i g n s , were p l a c e d w i t h  males p r i o r to m i d n i g h t and checked  f o r c o p u l a t i o n p l u g s the f o l l o w i n g  morning. To p r o v i d e timed matings  o f d y s t r o p h i c mice,  i t was n e c e s s a r y  to c o n t i n u o u s l y c o h a b i t a t e males and females as p a i r s . were checked  These females  every morning f o r the presence o f a c o p u l a t i o n p l u g .  The day on which a c o p u l a t i o n p l u g was observed was d e s i g n a t e d as day 0 o f pregnancy.  iv.  maintenance o f the mice  A l l mice used i n t h i s study were m a i n t a i n e d i n t h e Zoology V i v a r i u m a t the U n i v e r s i t y o f B r i t i s h Columbia. were kept on an 18 hour l i g h t p e r i o d  The animal rooms  (6 A.M. to 12 m i d n i g h t ) and a  6 hour dark p e r i o d . A l l mice were p r o v i d e d w i t h a c o n t i n u o u s water s u p p l y .  Mice  of the d y s t r o p h i c c o l o n y were f e d P u r i n a Mouse Chow; a l l o t h e r mice, P u r i n a L a b o r a t o r y Chow.  Optimal maintenance and b r e e d i n g  performance  was observed w i t h these d i e t s .  2.  P r o d u c t i o n o f chimeras The  t e c h n i q u e s employed to produce  the chimeras used i n t h i s  study were d e r i v e d from the p r o t o c o l designed by M u l l e n and Whitten  13  (1971).  A b r i e f d e s c r i p t i o n o f the procedure i s i n c l u d e d below  w i t h emphasis on a d a p t a t i o n s employed i n the p r e s e n t s t u d y .  i.  c o l l e c t i o n of embryos  With the l i g h t - d a r k c y c l e m a i n t a i n e d i n the mouse rooms, embryos were g e n e r a l l y a t the 8 - c e l l s t a g e by 12 noon of day  2.  At t h i s time they were s t i l l w i t h i n the lumen of the F a l l o p i a n 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  tube.  dislocation  and the u t e r i n e horns, w i t h the F a l l o p i a n tubes a t t a c h e d , were removed. The F a l l o p i a n tubes were f l u s h e d by i n s e r t i n g a s h o r t b e v e l 30 gauge needle, f i t t e d  to a s y r i n g e w i t h prewarmed c u l t u r e media, i n t o  j u n c t u r e of the tube and the u t e r u s .  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 (Clay Adams, No. A-1478).  the  square c a v i t y  The embryos were p i c k e d up and  slides  transferred,  w i t h a custom made b r e a k i n g p i p e t t e , to f r e s h media. A l t h o u g h the m a j o r i t y o f embryos c o l l e c t e d from SWV  female  mice were o f c o n s i s t e n t l y good q u a l i t y , a t the 8 - c e l l s t a g e w i t h no s i g n of b l a s t o m e r e death, t h i s was embryos.  not the case w i t h the d y s t r o p h i c  These embryos o f t e n were r e t a r d e d i n development  (less  8 c e l l s ) and f r e q u e n t l y showed e v i d e n c e of blastomere d e a t h . the zona p e l l u c i d a was  than  When  removed these embryos o f t e n shed b l a s t o m e r e s  which were observed as s i n g l e c e l l s l y i n g a d j a c e n t to the b l a s t o c y s t a f t e r the c u l t u r e p e r i o d .  T h i s response and the g e n e r a l l y  c o n d i t i o n o f these embryos may  r e p r e s e n t some e a r l y  poor  developmental  e f f e c t of the d y s t r o p h i c genotype but more l i k e l y r e s u l t s from the  14  g e n e r a l l y d e b i l i t a t e d c o n d i t i o n o f t h e d y s t r o p h i c females  which  produce eggs and/or p r o v i d e a p r e i m p l a n t a t i o n environment  o f poor  quality.  ii.  f u s i o n and c u l t u r i n g o f embryos  The n o n - c e l l u l a r zona p e l l u c i d a s u r r o u n d i n g t h e 8 - c e l l embryo was  removed by pronase d i g e s t i o n  ( M i n t z , 1962).  Immediately  upon  s w e l l i n g o f t h e zona, t h e embryos., were washed i n s e v e r a l changes of f r e s h c u l t u r e media and, i f n e c e s s a r y , r a p i d l y p i p e t t e d u n t i l t h e zona broke f r e e .  The z o n a - f r e e embryos were then p l a c e d s i n g l y i n  micro drops o f c u l t u r e media under p a r a f f i n  oil.  The same procedure was then c a r r i e d out w i t h a second s e t of embryos o f d i f f e r i n g genotype.  When t h e micro drops c o n t a i n e d  the two g e n e t i c a l l y d i f f e r e n t embryos, a p i p e t t e was used t o nudge the p a i r s t o g e t h e r .  T h i s procedure was r e p e a t e d two o r t h r e e times  to i n s u r e t h a t t h e embryos were a g g r e g a t i n g . D u r i n g t h i s e n t i r e p r o c e d u r e , except f o r b r i e f  interruption  f o r n e c e s s a r y m a n i p u l a t i o n s , t h e embryos were kept i n c u l t u r e media m a i n t a i n e d a t 3 7 ° C i n an atmosphere o f p r e - h u m i d i f i e d 5% CO2 i n a i r . When t h e embryos had aggregated, the c u l t u r e s were p l a c e d i n t o an a n a e r o b i c j a r , f l o o d e d w i t h 5% CC^ i n a i r and i n c u b a t e d a t 3 7 ° C.  W i t h i n 24 hours t h e m a j o r i t y of t h e aggregated embryos had  differentiated  i n t o f u l l y developed s i n g l e b l a s t o c y s t s .  15  iii. The  embryo t r a n s f e r 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 a t l e a s t one p r e v i o u s l i t t e r by normal mating.  Care was  taken  to i n s u r e t h a t these females were i n p r o e s t r u s p r i o r t o p l a c i n g them w i t h a vasectomized  male.  The presence o f a c o p u l a t i o n p l u g the  f o l l o w i n g morning was evidence  o f pseudopregnancy.  Routinely,  females i n day 2 o f pseudopregnancy were used as r e c i p i e n t s o f day 3 embryos (day 2 embryos p l u s 24 hours i n v i t r o ) . B l a s t o c y s t s were t r a n s f e r r e d s i n g l y o r i n s m a l l c l u t c h e s of 2 o r 3 to the r i g h t u t e r i n e horn of the r e c i p i e n t dams.  These  dams were a n e s t h e t i z e d w i t h Nembutal (sodium p e n t o b a r b i t a l , 50 mg/ml, Abbott L a b o r a t o r i e s , M o n t r e a l ) a d m i n i s t e r e d  a t 1.8 mg/kg.  After  p l u c k i n g the h a i r from the back, a s m a l l d o r s a l m i d l i n e i n c i s i o n was made i n the s k i n f o l l o w e d 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 u t e r i n e h o r n near the ovary.  The u t e r i n e horn was  brought t o the e x t e r i o r by p a s s i n g a loop o f t h r e a d around the o v a r i a n end o f the h o r n .  Care was taken n o t t o damage the F a l l o p i a n tube  or t o expose the ovary.  The t h r e a d a l s o served  during b l a s t o c y s t t r a n s f e r .  t o h o l d the u t e r u s  A s m a l l h o l e was made i n t h e u t e r i n e w a l l  w i t h a 26 gauge d i s p o s a b l e n e e d l e  and the t r a n s f e r p i p e t t e was i n t r o -  duced i n t o the u t e r i n e lumen i n an o v a r i a n d i r e c t i o n and the b l a s t o cysts expelled.  No a i r was a l l o w e d  to e n t e r the u t e r u s .  The t h r e a d  was removed, the u t e r u s was r e t u r n e d t o the body c a v i t y , and the d o r s a l s k i n i n c i s i o n was c l o s e d w i t h 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).  iv.  foster nursing  R o u t i n e l y , 17 days f o l l o w i n g t h e t r a n s f e r procedure (day 19 o f pseudopregnancy o f 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 s u r v i v i n g f e t u s e s were removed by C a e s a r i a n s e c t i o n .  These term f e t u s e s were  then p l a c e d w i t h newborn l i t t e r s to be f o s t e r n u r s e d .  Females o f  the SWV s t r a i n , which had g i v e n b i r t h w i t h i n 48 hours, were s u c c e s s f u l f o s t e r mothers.  3.  Chimeras  produced  N i n e t e e n mice were produced by the above t e c h n i q u e d u r i n g the c o u r s e o f t h i s study. experiments chimera  Of t h e s e , 11 form the b a s i s o f subsequent  and these mice were numbered i n o r d e r o f b i r t h  (or C) number 7 t o C number 17 ( T a b l e 1 ) .  i.  d e m o n s t r a t i o n o f mosaicism  All  chimeras used i n t h i s study demonstrated  both a l b i n o 2J  and pigmented at  from  coat c o l o r .  SWV mice a r e a l b i n o and dy  2J /dy  mice,  the N^ - Ng g e n e r a t i o n o f b a c k c r o s s i n g t o C57BL/6J were b l a c k .  No attempt  to q u a n t i t a t e o r a n a l y s e the chimera p i g e m e n t a t i o n p a t t e r n  was made.  H i s t o l o g i c a l a n a l y s i s o f the pigmented  e p i t h e l i u m of the  r e t i n a s from the chimeras a l s o r e v e a l e d both a l b i n o and pigmented cells. Hemoglobin a n a l y s i s by an e l e c t r o p h o r e t i c t e c h n i q u e ( M a r t i n  17  and P e t r a s , 1971) demonstrated and C57BL/6J) from each chimera phoretogram  ii.  b o t h Hbb-s (SWV) and Hbb-d (WK/Re tested.  The hemoglobin  electro-  i s p r e s e n t e d i n F i g u r e 1.  dystrophic—normal  chimeras 2J  A t o t a l o f A5 homozygous d y s t r o p h i c (dy  2J /dy  ) embryos were  o b t a i n e d o f which 24 were s u c c e s s f u l l y f u s e d t o SWV embryos and developed  to the b l a s t o c y s t s t a g e .  A f t e r embryo t r a n s f e r t o r e c i -  p i e n t dams, 9 l i v i n g mice were d e l i v e r e d by C a e s a r i a n s e c t i o n . These a r e r e f e r r e d Mice number 2J (dy  t o s u b s e q u e n t l y as chimeras number 8 to 16. 13 and 14 showed o n l y b l a c k p i g m e n t a t i o n  2J /dy  litter.  ) and d i e d w i t h the r e s t o f t h e i r f o s t e r mothers Presumably  these two mice were not chimeras  d i d n o t e x h i b i t the patchy pigmented  s i n c e they  and a l b i n o s k i n .  12 developed s e v e r e s k i n l e s i o n s p r o b a b l y due to a m i t e and d i e d .  Chimera number  of a mite i n f e c t i o n . it  normal  Chimera number infection  15 developed a p r o l a p s e d rectum and s i g n s  A f t e r a s i n g l e treatment w i t h a "mite d i p " ,  died. The remaining 5 chimeras  o f t h i s type were a n a l y s e d 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 muscle c o m p o s i t i o n . iii. normal—normal  o f muscular  d y s t r o p h y and f o r  chimeras  Two o t h e r chimeras, number 7 and 17, d e r i v e d from f u s i o n o f  18  g e n e t i c a l l y normal embryos, were a n a l y s e d dystrophic—normal a C57BL/10J and SWV  chimeras.  i n the same way as t h e  Chimera number 7 was produced  embryo f u s i o n and chimera number  from an unknown w i l d type embry6 (genotype +/?, from c o l o n y , mated t o G57BL/10J) f u s e d to a SWV  embryo.  17 was  from produced  the^dystrophic  TABLE 1 Number and E x t e r n a l Sex o f Chimeric Mice Produced f o r t h i s Study  Females  Type o f chimera  1. d y  2 J  /dy  2 J  ~+/+  (SWV)  Chimera number  Chimera number  10 12 16  8  (3) 2. +/+  (C57BL/10J) — +/+  Total  9 11 13 14 15  (6)  9  7  1  17  1  (SWV)  3. +/? x +/+ +/+  Males  (C57BL/10J)-  (SWV) 11 VO  20  -s  Hbb- d  o r i gin •  1  1  • • •1 t  • • ••  Figure 1.  + SWV SWV  C57BL/6J  C57BL/6J WK/Re C 8 C 9 C 10  III • II  C11  i i  C16  C15  Hemoglobin electrophoretogram. are designated as C.  Chimeras  CHAPTER I I I MUSCLE FUNCTION OF DYSTROPHIC—NORMAL  The c l i n i c a l  phenotype o f t h e d y s t r o p h i c — n o r m a l  was s t u d i e d t o determine  motor a b i l i t y .  D y s t r o p h i c mice (dy  feet, alternately flex by t h e t a i l ,  and extend  Southard,  loss  2J /dy  ) clasp t h e i r hind  t h e i r toes and l e g s when  lifted  drag one h i n d l e g o r the o t h e r when w a l k i n g , and  e x h i b i t a mild opisthotonus and  chimeras  the e x t e n t , i f any, o f f u n c t i o n a l 2J  of  CHIMERIC MICE  1970).  (arched p o s i t i o n o f the body) (Meier  I n l a t e r stages of the d i s e a s e p r o c e s s 2J 2J  (3 to 4 months), the dy  /dy  mice, m a i n t a i n e d  a t the U n i v e r s i t y  of  B r i t i s h Columbia, developed  of  the h i n d limbs and s e v e r e c o n t r a c t u r e s (permanent c o n t r a c t i o n  of  muscles). It  is difficult  marked k y p h o s i s , permanent p a r a l y s i s  to a c c u r a t e l y a s s e s s s l i g h t d e f i c i e n c y o f  motor f u n c t i o n i n mice by v i s u a l i n s p e c t i o n .  Loss o f t o t a l l y  normal muscle f u n c t i o n i n the d y s t r o p h i c — n o r m a l chimeras missed by t h i s c r i t e r i o n .  T h e r e f o r e , t o q u a n t i t a t i v e l y a s s e s s muscle  f u n c t i o n , the maximum s t i m u l a t e d t w i t c h o f the a n t e r i o r muscle was measured.  c o u l d be  tibialis  T h i s muscle was s e l e c t e d t o r e p r e s e n t muscle  f u n c t i o n i n the chimeras  because i t i s known to be a f f e 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 d y s t r o p h i c (dy/dy) mice ( S a l a f k y , 1971). T h i s muscle can be s t i m u l a t e d i n d i r e c t l y by the p e r o n e a l and  nerve  the r e s u l t i n g t w i t c h i s a f u n c t i o n of the muscle-nerve u n i t .  M a t e r i a l s and Methods 1.  D e t e r m i n a t i o n of the maximum i s o m e t r i c t w i t c h of the a n t e r i o r tibialis  muscle  A f t e r a n e s t h e s i a (sodium on the two h i n d l e g s was tibialis  c l i p p e d and  the s k i n over the a n t e r i o r  and b i c e p s f e m o r i s muscles was  a n t e r i o r t i b i a l i s muscle was  removed.  i s o l a t e d and  (Champion Serum P r o o f S i l k No. was  p e n t o b a r b i t a l , 80 mg/kg), the h a i r  6-0).  The  tendon of the  t i e d with f i n e s i l k  The a n t e r i o r t i b i a l i s  then f r e e d of c o n n e c t i v e t i s s u e by a s h a l l o w i n c i s i o n  c e e d i n g from the tendon up both s i d e s of the muscle. nerve was  The  thread  muscle  prosciatic  exposed by an i n c i s i o n p a r a l l e l to the d o r s a l edge of  the  b i c e p s f e m o r i s and c u t d i s t a l to :the p o i n t of b i f u r c a t i o n of the p e r o n e a l nerve. p o s i t i o n e d and  The mouse was  i t s tendon was  thread. and  The  i t s leg  secured to the c o r k f l o o r of a p l a s t i c chamber.  a n t e r i o r t i b i a l i s muscle was and  p l a c e d on i t s s i d e and  The  s t r e t c h e d t o i t s normal r e s t i n g l e n g t h  secured to the s t r a i n gauge w i t h the a t t a c h e d  s c i a t i c nerve was  p o s i t i o n e d on a b i p o l a r s i l v e r e l e c t r o d e  the p r o x i m a l end of the nerve was  cut.  D u r i n g the c o u r s e of the o p e r a t i o n a l l exposed t i s s u e s were c o n s t a n t l y bathed  i n 35-37° C p h y s i o l o g i c a l s a l i n e .  the p l a s t i c chamber w i t h the p r e p a r a t i o n was  P r i o r to r e c o r d i n g ,  f l o o d e d w i t h 35-37° C  23  m i n e r a l o i l to prevent c o o l i n g and d r y i n g o f the muscle and nerve. Square wave p u l s e s (15 v o l t s ) o f the 0.1 msec d u r a t i o n were a p p l i e d t o the e l e c t r o d e .  S i g n a l s from the t r a n s d u c e r (Stratham  s t r a i n gauge, Model G7B1.5-350) were l e d v i a a p o l y g r a p h p r e a m p l i f i e r ( G i l s o n M e d i c a l E l e c t r o n i c s ) t o d i s p l a y on an o s c i l o s c o p e Type 532).  (Tektronix  A l l measurements were made from photographs o f the t r a c e  taken by a kymograph camera (Grass Instrument C o . ) . Recordings from a n t e r i o r 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 d y s t r o p h i c  from d y s t r o p h i c — n o r m a l and n o r m a l — n o r m a l  (dy /dy ) 2 J  2 J  mice and  chimeras.  R e s u l t s and D i s c u s s i o n 1.  V i s u a l o b s e r v a t i o n o f the d y s t r o p h i c — n o r m a l chimeras No c l i n i c a l  f e a t u r e c h a r a c t e r i s t i c o f the d y s t r o p h i c  was observed i n the d y s t r o p h i c —  condition  normal chimeras i n d a i l y o b s e r v a t i o n s  from b i r t h t o two months o r i n weekly o b s e r v a t i o n from two months to death.  N e i t h e r l i f t i n g by the t a i l n o r d e p r e s s i n g the h i n d limbs  caused any marked l e g o r t o e f l e x i n g and never r e s u l t e d i n f o o t dragging.  The h e h a v i o r of t h e s e d y s t r o p h i c — n o r m a l chimeras was  i n d i s t i n g u i s h a b l e from t h a t o f normal  2.  mice.  Maximum i s o m e t r i c t w i t c h o f a n t e r i o r t i b i a l i s  muscles  Examples o f the o s c i l o s c o p e d i s p l a y s o b t a i n e d from the d y s t r o p h i c — normal chimeras a r e shown i n F i g u r e 2. apparatus was n o t s u f f i c i e n t  The frequency response o f the  t o permit c a l c u l a t i o n o f the c o n t r a c t i o n  24  i.  Figure  2.  O s c i l o s c o p e d i s p l a y of maximum i s o m e t r i c t w i t c h 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 o f 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 t i m e s . The maximum s t i m u l a t e d t w i t c h t e n s i o n i n grams f o r a l l t e s t e d i s p r e s e n t e d i n F i g u r e 3.  The r e s u l t s from SWV  mice  mice  (X  10.00), a l t h o u g h s l i g h t l y lower, agree f a v o r a b l y w i t h those o b t a i n e d by S a l a f s k y  (1971) f o r normal mice o f the 129 s t r a i n 2J  S i m i l a r l y , the d y s t r o p h i c  (dy  (X  11.41).  2J /dy  ) mice gave c o n t r a c t i o n s  that  were s l i g h t l y weaker than those o b t a i n e d by S a l a f s k y f o r d y s t r o p h i c (dy/dy) mice; X 1.80  was  and X 2.97  not expected because  the dy  respectively. 2J  T h i s lower response  mutant i s c o n s i d e r e d to produce  a m i l d e r form of dystrophy w i t h a l a t e r o n s e t . The n o r m a l — n o r m a l  chimeras produced a mean c o n t r a c t i o n  s l i g h t l y h i g h e r (11.30) than the i n b r e d SWV i n d i c a t i v e of h e t e r o s i s .  mice and i s p r o b a b l y  These r e s u l t s a r e c l o s e to those o b t a i n e d  f o r the h y b r i d SWV-C57BL/10J F^ mice  (X 10.81) and t h e r e f o r e do not  p r e s e n t a major problem i n d e t e c t i n g s l i g h t d e f i c i e n c e s i f p r e s e n t i n the d y s t r o p h i c — n o r m a l chimeras. When examined, the d y s t r o p h i c — n o r m a l chimeras ranged i n age from 6 to 12 months which p r o v i d e d time f o r the p r o g r e s s i v e  changes  of d y s t r o p h y to become m a n i f e s t but not to be confounded by  senile  changes a s s o c i a t e d w i t h extreme  aging.  A l t h o u g h 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 s t r o n g e r contraction  (X 13.52) than any of the c o n t r o l c l a s s e s .  The  results  from the l e f t and r i g h t l e g s of the i n d i v i d u a l chimeras were v e r y 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  result.  D y s t r o p h i c Chimeras  Control  20  N o r m a l Chimeras  18  c o "<>/ c a> I-  16  E  8  14 12  + sd  10  C3 a*.  6  o  4 2  Mouse  Fl  SWV  Muscles  8  5  10  5-8 32.7 10.0  Age  (months)  W e i g h t (grams) T w i t c h (grams)  X 36.4 X 10.8  2J  C 9 L R  C 8  C10  7  C11 L R  L R  L R  3-5  6  9  10  10  33.5  29.5  39.0  49.0  A  16.0 j 1.8  13.5  C16 L R 12  C 7  i !  L R  ;  10  L R 12  32.6  43.5  32.0 i i  0  1  7  11.3 as  F i g u r e 3.  Maximum i s o m e t r i c t w i t c h o f a n t e r i o r t i b i a l i s muscles. Mouse: F l i s SWV-C57BL/10J; C i s chimera. Muscles: numbers a r e number of muscles a n a l y z e d ; L and R i s l e f t and r i g h t ; b r o k e n l i n e s r e p r e s e n t s u b o p t i m a l p r e p a r a t i o n s and a r e not i n c l u d e d 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 .  27  A l t h o u g h the g r e a t e r mean response o f the d y s t r o p h i c — n o r m a l chimeras was unexpected, i t i s apparent t h a t these chimeras, except for  chimera number 9, demonstrated c o n t r a c t i o n s s i m i l a r t o those o f  the  normal chimeras.  i s not r e a d i l y  The much l a r g e r response of chimera number 9  explainable.  These maximum t w i t c h r e s u l t s a r e c o m p e l l i n g e v i d e n c e a g a i n s t f u n c t i o n a l d e f i c i e n c e s o f the nerve-muscle u n i t a t the maximum s t i m u l u s l e v e l i n the d y s t r o p h i c — n o r m a l chimeras. their c l i n i c a l  This supports  evaluation.  To e x p l a i n the absence o f any d e t e c t a b l e d y s t r o p h i c  phenotype  i n the d y s t r o p h i c — n o r m a l chimeras s e v e r a l p o s s i b i l i t i e s may be entertained.  1.  The muscles o f t h e s e chimeras may be composed e n t i r e l y o f gene-  t i c a l l y normal components. a.  Three s i t u a t i o n s c o u l d produce t h i s  state:  a s e l e c t i v e overgrowth o f normal myoblasts d u r i n g muscle morphogenesis.  b.  a r e p o p u l a t i o n 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 m y o b l a s t s . c.  a functionally fibers;  h e t e r o z y g o u s c o n d i t i o n i n the muscle  t h a t i s , where d y s t r o p h i c n u c l e i a r e p r e s e n t ,  normal n u c l e i occupy a p l a c e i n the same muscle  fiber  mimicking a h e t e r o z y g o u s c o n d i t i o n i n the muscle syncytium.  28  2.  The p e r i p h e r a l motor nerves may  t i c a l l y normal neurons.  Two  be composed p r i m a r i l y of gene-  obvious phenomena c o u l d produce  this  state: a.  a developmental s e l e c t i o n a g a i n s t 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 d y s t r o p h i c motor nerves accompanied  by a x o n a l s p r o u t i n g of g e n e t i c a l l y  normal motor nerves to r e i n n e r v a t e any denervated  muscle  fibers.  3.  Some o t h e r f a c t o r such as v a s c u l a r s u p p l y (Hathaway, Engel and  Z e l l w e g e r , 1970) may  be r e s p o n s i b l e f o r the muscle d e g e n e r a t i o n i n  muscular d y s t r o p h y .  In these chimeras, the t i s s u e r e s p o n s i b l e f o r  t h i s f a c t o r c o u l d be o f g e n e t i c a l l y normal  origin.  CHAPTER IV GENETIC CONSTITUTION OF MUSCLES FROM CHIMERAS  S e l e c t e d muscles from the chimeras were a n a l y s e d f o r t h e i r genetic  constitution.  genetically dystrophic  The r e s u l t s were expected to i n d i c a t e i f c e l l s s u f f e r e d any developmental f a i l u r e o r  s e l e c t i v e e l i m i n a t i o n by the p a t h o l o g i c a l p r o c e s s i n the mosaic environment. The known g e n e t i c  v a r i a n t s o f mice p r o v i d e s e v e r a l  differences  between c e l l s o f two s t r a i n s , b u t , 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 v e r y l i m i t e d . F r y e and E d i d i n rapidly intermix 1972, p e r s o n a l  (1970) found t h a t c e l l s u r f a c e  after c e l l fusion.  antigens  E d i d i n and Fambrough  (Fambrough,  communication) have shown t h a t the muscle f i b e r  membrane a l s o has f l u i d p r o p e r t i e s and t h a t a n t i g e n s on the myotube s u r f a c e a r e f r e e to move l a t e r a l l y . antigen  Labelled antibodies  to s p e c i f i c  markers would t h e r e f o r e 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 o f two types of n u c l e i o r t h e i r d i s t r i b u t i o n w i t h i n the muscle f i b e r s of the c h i m e r a s . The w e l l d e f i n e d useful i n analysing  enzyme a c t i v i t y v a r i a n t s which have been  non-synctial  29  chimera t i s s u e , e_.&.  liver,  30  (Wegmann, 1970  and  Condamine, C u s t e r and  M i n t z , 1971)  r e a d i l y a p p l i c a b l e to s k e l e t a l muscle a n a l y s i s . i n muscle t i s s u e of d i f f e r e n t f i b e r f a s t - t w i t c h red and  types,  1972)  not  normal o c c u r r e n c e  i-.e^. f a s t - t w i t c h w h i t e ,  slow-twitch intermediate,  i n the enzyme l e v e l s f o r g l y c o l y t i c and  The  are  with inherent  differences  o x i d a t i v e metabolism  (Close,  would not permit a simple a n a l y s i s . There a r e , however, s e v e r a l e l e c t r o p h o r e t i c v a r i a n t s  can be  applied  to the a n a l y s i s of whole muscle p r e p a r a t i o n s .  mouse, these f a l l i.  that  i n t o two  general  In  the  classes:  v a r i a n t s which y i e l d o n l y a s i n g l e p r o t e i n band i n  e i t h e r homozygote but b o t h p a r e n t a l bands i n the h e t e r o z y g o t e ; e_.&., hemoglobin b e t a c h a i n v a r i a n t s ii. but  (Hbb)  (Wegmann and  Gilman, 1970).  v a r i a n t s which y i e l d s i n g l e bands i n the homozygotes,  i n which the codominant e x p r e s s i o n  h e t e r o z y g o t e r e s u l t s i n more t h a t the  of the a l l e l e s i n two  parental  types.  a d d i t i o n a l h y b r i d bands, w i t h m o b i l i t i e s i n t e r m e d i a t e two  parental  types,  second c l a s s of e l e c t r o p h o r e t i c v a r i a n t s  can be used to determine the n u c l e a r  the n u c l e i must be  If only  the  subunits  in addition  In chimeras, produced from embryos d i f f e r i n g i n  analysed.  the  i s useful  arrangement w i t h i n  homozygous type of enzyme, the p a t t e r n can be  between  Ruddle, 1968).  f o r g e n o t y p i c c o n t e n t a n a l y s i s of s k e l e t a l muscle and  fibers.  These  r e s u l t from an a s s o c i a t i o n of p r o t e i n  to produce the a c t i v e enzyme (Shows and This  the  the muscle the  of the muscle e l e c t r o p h o r e t o g r a m  2 parental  p r o t e i n bands a r e  i n muscle f i b e r s composed e n t i r e l y of  resolved,  single  31 n u c l e i types.  I f any h y b r i d bands are r e s o l v e d , some or a l l of  the muscle i s composed of f i b e r s t h a t have formed from a f u s i o n of d i f f e r e n t  types of myoblasts and  types of n u c l e i (Mintz and  Mod-1  therefore contain  different  Baker, 1967).  electrophoretic variation A g e n e t i c v a r i a n t of the s u p e r n a t a n t NADP-malate dehydrogenase  (malic enzyme) from mice t h a t demonstrates h y b r i d banding r e p o r t e d by Henderson (1966). malate: NADP o x i d o r e d u c t a s e ,  T h i s enzyme i s d e s i g n a t e d decarboxylating  e l e c t r o p h o r e t i c v a r i n a t s are designated  the codominant a l l e l e s Mod-1  Mod-1  locus  (Shows, Chapman and  as  L-  1.1.1.40) and  Mod-la and  a by  (EC  was  Mod-lb c o n t r o l l e d  b and  Mod-1  Ruddle,  r e s p e c t i v e l y , at  The  forms of the enzyme found i n AKR  and  heterozygous mice ( F l AKR/C3H) c o n t a i n e d  enzyme, i n t e r m e d i a t e  between the two  r e s o l u t i o n t h a t she o b t a i n e d the h e t e r o z y g o t e c o n t a i n e d  C3H  a h y b r i d form of  p a r e n t a l bands.  d i d not a l l o w her  Shows and  system.  The  the  electrophoretic  to determine whether  t h r e e o f f i v e bands.  Ruddle (1968) who  i s a c t i v e o n l y as a t e t r a m e r .  presented  used a d i f f e r e n t e l e c t r o p h o r e t i c  They i n t e r p r e t e d t h i s p a t t e r n to r e p r e s e n t  p a r e n t a l bands was  slow  mice, r e s p e c t i v e l y .  E v i d e n c e f o r a f i v e band h e t e r o z y g o t e p a t t e r n was by  the  1970).  Henderson (1966) demonstrated the presence of f a s t and migrating  the  The  an enzyme t h a t  a c t i v i t y r a t i o of the h y b r i d  v i s u a l l y observed to be  and  1:4:6:4:1 which i s the  p r e d i c t e d phenotype f o r t e t r a m e r i c enzymes composed of 2  subunit  32  types t h a t a s s o c i a t e randomly.  The c o m p o s i t i o n of these e l e c t r o -  p h o r e t i c a l l y d i s t i n c t bands i s assumed that y i e l d s  to be the e x p a n s i o n of (a + b)  l a a a a ; 4aaab; 6aabb; 4abbb;  band t h a t m i g r a t e d f u r t h e s t  lbbbb.  4  In t h e i r system, the  (lbbbb) s t a i n e d v e r y f a i n t l y , and they  c o n s i d e r e d t h i s to r e s u l t from the pH of the e l e c t r o p h o r e t i c system. A d d i t i o n a l independent e v i d e n c e f o r a t e t r a m e r i c was r e p o r t e d by Baker and M i n t z (1969).  structure  Using a d i f f e r e n t  system, they a l s o r e p o r t e d a f i v e banded e l e c t r o p h o r e t i c a f o r Mod-1  buffer  phenotype  b /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.  T h e i r study i n c l u d e d a n a l y s i s o f t i s s u e s from ' a l l o p h e n i c ' mice d e r i v e d from M o d - l / M o d - l a  a  and Mod-l^/Mod-1^ embryo t y p e s .  From  the mosaic muscle they r e s o l v e d f i v e bands i n v a r i o u s r a t i o s f u r t h e r supported random a s s o c i a t i o n of the t e t r a m e r i c  that  subunits.  L i v e r from these a l l o p h e n i c mice y i e l d e d o n l y the homozygous Mod-la and Mod-lb p r o t e i n bands, i n d i c a t i n g t h a t b o t h c e l l in this  types c o e x i s t e d  tissue. Engle and Wolfe (1971) examined the phenotype of the  enzyme i n d e v e l o p i n g h e t e r o z y g o u s embryos  Mod-1  and demonstrated the  synchronous a c t i v a t i o n of b o t h a l l e l e s . Mod-1  genotype of chimeras In the p r e s e n t 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 ( R u s s e l l ,  1972, p e r s o n a l communication).  Because the d y s t r o p h i c mice used were o n l y a t the N, to N, F l  33  g e n e r a t i o n of b a c k c r o s s i n g t o  C57BL/6J from WK/Re, i t would  have been p o s s i b l e to use w i t h c e r t a i n t y any was  not shared  by  these  two  not  enzyme v a r i a n t t h a t  strains.  In the e l e c t r o p h o r e t i c p a t t e r n s from the chimera muscles, the presence of  'a' type s u b u n i t s would r e p r e s e n t  n u c l e i a c t i v i t y and nuclei.  the normal  SWV  'b' type s u b u n i t s , g e n e t i c a l l y d y s t r o p h i c  From s i n g l e muscles, the r a t i o o f  'a' and  'b'  subunits  c o u l d be used to a s s i g n the r e l a t i v e n u c l e a r c o n t r i b u t i o n of two  cell  the  types.  T h i s type of e l e c t r o p h o r e t i c a n a l y s i s however, i s s u b j e c t to the l i m i t a t i o n s p r e s e n t e d  f o r the h i s t o c h e m i c a l a s s a y s .  r e l a t i v e c o n c e n t r a t i o n of the enzyme employed was v a r i o u s muscle f i b e r  types,  the e l e c t r o p h o r e t o g r a m  used to g i v e a g e n e r a l e s t i m a t e muscle.  The  I f the  not e q u a l i n the could only  be  of the t o t a l n u c l e i r a t i o s i n the  c o n t r i b u t i o n from the f i b e r s demonstrating  low  c o n c e n t r a t i o n of the assay enzyme would be underestimated i f detected  at a l l .  b a s i s of wet (Czok and  M a l i c enzyme demonstrates equal a c t i v i t y , on  the  weight, i n both r e d and w h i t e f i b e r s of r a b b i t muscle  BUcher, 1960).  A l t h o u g h no s i m i l a r s t u d i e s have been  r e p o r t e d f o r mice, t h e r e i s no obvious reason  to expect  different  results. Mod-1  g e n e t i c v a r i a n t s t h e r e f o r e , were t h e o r e t i c a l l y  f o r the n u c l e a r marker r e q u i r e d to i d e n t i f y and extent and I t was  quantitate  type of mosaicism i n the chimeras used i n t h i s  on t h i s b a s i s t h a t techniques  ideal the  study.  were e s t a b l i s h e d to r e s o l v e  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 o f Mod-1  and to develop a q u a n t i t a t i v e  system t h a t c o u l d be used to e s t i m a t e c e l l or n u c l e i r a t i o s i n t h e mosaic t i s s u e s . M a t e r i a l s and Methods 1.  Tissue preparation 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  by sodium p e n t o b a r b i t a l i n j e c t i o n .  chimeras  The chimeras were d i s s e c t e d  i m m e d i a t e l y f o l l o w i n g the maximum t w i t c h experiment.  Individual  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 q u i c k frozen i n l i q u i d nitrogen. c o n t a i n e r s a t -70°  These t i s s u e s were s t o r e d i n s e a l e d  C u n t i l f u r t h e r processed.  F r o z e n 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  thick)  w i t h a r a z o r b l a d e 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' m i c r o homogenizer.  The homogenate was  centrifuged at  3° C f o r 15 minutes a t 15,000 x g and the s u p e r n a t a n t a t 90,000 x g f o r an a d d i t i o n a l 30 m i n u t e s . s u p e r n a t a n t was  recentrifuged  The r e s u l t i n g c l e a r  d i v i d e d i n t o two e q u a l p a r t s and f r e e z e d r i e d .  P r i o r t o 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 o f f r o z e n muscle s t a r t i n g w e i g h t .  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 s u p e r n a t a n t .  w e i g h i n g more than a p p r o x i m a t e l y 70 mg.  2.  E l e c t r o p h o r e s i s of Attempts  This yielded a Muscles  were a n a l y s e d s u c c e s s f u l l y .  Mod-1  t o d u p l i c a t e the t e c h n i q u e s of Shows and Ruddle (1968)  and Baker and M i n t z (1969) p r o v i d e d 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 t y p e s . a l t h o u g h comparable  The heterozygous enzyme p a t t e r n ,  to t h e i r r e s u l t s , was  an a c c u r a t e q u a n t i t a t i v e a n a l y s i s .  not r e a d i l y a d a p t a b l e to  Lack o f p r e c i s e r e s o l u t i o n and  e x c e s s i v e s t a i n i n g a c t i v i t y between the bands p r e s e n t e d the major problems. To i n c r e a s e the r e s o l u t i o n , a micro s t a r c h g e l t e c h n i q u e , designed by T s u y u k i , R o b e r t s , K e r r and Ronald for  high voltage electrophoresis.  p i c t u r e d i n F i g u r e s 4 and 5. i. ii. iii.  (1966) was  adapted  The apparatus and g e l s a r e  Advantages  of t h i s micro system a r e :  sharp d e f i n i t i o n o f the bands from h i g h v o l t a g e p r o c e d u r e s , s m a l l sample volumes can be a n a l y s e d (0.005 m l ) , t h i n g e l s t h a t can be c l e a r e d and scanned  directly  with a densitometer. a.  Buffer  system  The b u f f e r system used was  a m o d i f i c a t i o n f o r h i g h v o l t a g e of  t h a t r e p o r t e d by Baker and M i n t z (1969) . of  2000 ml double d i s t i l l e d water,  Trizma base added  to pH 8.4.  18.08  The b r i d g e b u f f e r  consisted  g c i t r i c a c i d anhydrous  The g e l b u f f e r was  and  12.5 ml b r i d g e  b u f f e r d i l u t e d to 250 ml w i t h d i s t i l l e d water and r e a d j u s t e d to pH 8.4 w i t h Trizma base. b.  P r e p a r a t i o n o f the g e l  A 12% g e l was of  Electrostarch.  and poured d i r e c t l y  produced by adding 250 ml of g e l b u f f e r to 30 g  The g e l was  cooked to E l e c t r o s t a r c h  specifications  (without d e g a s s i n g ) i n t o the g e l mould.  s e t t i n g f o r 90 minutes a t room temperature, the g e l was  After  placed  into  36  F i g u r e 5.  M i c r o g e l s w i t h samples a p p l i e d electrophoresis.  p r i o r to  37 a r e f r i g e r a t o r for an a d d i t i o n a l 30 minutes to pre cool the g e l . Gel s l i c e s , 1/16 inch thick, were prepared and the sample applied to s l o t s cut i n the g e l . c.  Running conditions  The apparatus was covered with p l a s t i c f i l m and placed into a r e f r i g e r a t o r (5° C) with a high power fan to d i s s i p a t e heat from both top and bottom surfaces of the g e l .  Electrophoresis was carried  out for 5 hours with a voltage gradient of 21 V/cm across the g e l . d.  Mod-1 enzyme assay  Enzyme a c t i v i t y was v i s u a l i z e d 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 a g i t a t i o n . A section of the g e l , including the o r i g i n and the Mod-1 zone, was placed i n a-covered P e t r i plate with 40 ml of the assay s o l u t i o n , covered and continuously agitated on a rotator enclosed i n a dark 37° C incubator.  When the gels had stained, they were removed from the  incubator and fixed i n a gel wash consisting of 5 parts water, 5 parts methanol and 1 part g l a c i a l a c e t i c a c i d . i n this s o l u t i o n .  The stained gels were stored  38 3.  Quantitation of r e l a t i v e Mod-1 a.  band a c t i v i t y  Densitometry  The fixed gels were slowly heated i n a bath of glycerine u n t i l the white starch approaced o p t i c a l c l a r i t y .  The gels were then  transferred to a clear p l a s t i c P e t r i plate and covered with a glass microscope s l i d e .  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 r e l a t i v e  areas only were required so no correction of the integration numbers to calculate r e a l area was made.  Where the peaks were not completely  i s o l a t e d , a perpendicular l i n e from the lowest point between them was projected to the base l i n e .  No correction f o r t h i s 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 i s presented i n Figure 6.  The presence of 5 bands i n the heterozygote i s consistent with  previous reports.  However, the measured a c t i v i t y r a t i o was not  consistent with the expected 1:4:6:4:1 r a t i o .  The band corresponding  to that of the homozygous Mod-1^/Mod-1^ type (i.e.. bbbb) stained faintly.  Shows and Ruddle (1968) reported that t h i s band stained  f a i n t l y i n their system and heterozygote phenotypes pictured by  39  Band Composition 1 aaaa  SWV  4 aaab 6 aabb 4 abbb 1 bbbb  C57BL/6J  origin  F i g u r e 6.  E l e c t r o p h o r e t o g r a m o f Mod-1 h e t e r o z y g o t e . Note the f a i n t s t a i n i n g o f the 1 bbbb band.  50-  40  o o  a  40-  o CD  £ o  30CO  —  +1  0)  RJ  20isd  •+-»  a.  Ui  10-  Band C o m p o s i t i o n  1a  Observed  Ratio  1.00  Expected  Ratio  Correction F i g u r e 7.  Factor(£f)  4  4a b  6a b  2.19  2.53  3  2  2  4a b  3  1b  4  .59  .10  1  4  6  4  1  -  1.83  2.37  6.78  10.00  Mod-1: a c t i v i t y r a t i o of h e t e r o z y g o t e p a t t e r n from muscle s u p e r n a t a n t ,  Baker and M i n t z Due  (1969) a l s o l a c k e d i n t e n s i v e s t a i n i n g o f t h i s band.  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 o f Mod-la and Mod-lb enzymes,  c a r e was  taken to ensure adequate  s t a i n i n g times were p r o v i d e d to  d e t e c t a l l the bands p r e s e n t .  2.  C a l c u l a t i o n o f 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 o f d e n s i t o m e t e r t r a c e s of heterozygous e l e c t r o p h o r e t o g r a m s , the a c t u a l r a t i o of the a c t i v i t y was 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  muscle  observed  observed  a c t i v i t y r a t i o o f these p r e p a r a t i o n s i s p r e s e n t e d i n F i g u r e 7.  Due  to  t h 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 t h e r e was  heterozygous  no e x p e r i m e n t a l e v i d e n c e , was  enzyme c o n c e n t r a t i o n was r a t i o , presuming  made.  assumed to be i n the t h e o r e t i c a l 1:4:6:4:1  t h a t the a c t i v i t y o f Mod-1  but the observed a c t i v i t y was  a  measure Mod-1  b  and Mod-1  a r e s u l t of d i f f e r i n g  a c t i v i t i e s o f the v a r i o u s t e t r a m e r i c p r o t e i n t y p e s . a  The  genes i s e q u a l ,  enzymatic T h e r e f o r e , to  b and Mod-1  gene p r o d u c t s , the observed a c t i v i t y  c o r r e c t e d by s u i t a b l e f a c t o r s .  The  a c t i v i t y than the 1 bbbb band and  was  1 aaaa band had r e l a t i v e l y h i g h e r  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 o r each o f the remaining 4 h e t e r o z y g o t e bands was  c a l c u l a t e d by d i v i d i n g the observed a c t i v i t y  expected a c t i v i t y which was  into  the  generated from the a s s i g n e d 1 aaaa v a l u e .  From the c o r r e c t e d 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 o f a and  b s u b u n i t s can be c a l c u l a t e d and used as an e s t i m a t e o f the Mod-1 Mod-l  a  and Mod-1°/Mod-1^ n u c l e a r c o n t e n t of mosaic  tissues.  /  A FOCAL  computer program, d e s i g n e d to p r o v i d e these c a l c u l a t i o n s i s p r e s e n t e d  42  i n Appendix 1. To t e s t t h i s approach, s u p e r n a t a n t s were determined and C h r i s t i a n ,  p r o t e i n c o n c e n t r a t i o n s o f muscle by 260/280 uv a b s o r p t i o n r a t i o s  (Warburg  1942) and a 50% aaaa and 50% bbbb m i x t u r e was  electrophoresed.  The e l e c t r o p h o r e t o g r a m and d e n s i t y t r a c e o f t h i s  m i x t u r e r e v e a l e d o n l y t h e two ' p a r e n t a l ' bands and c o r r e c t i o n o f the i n 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 o f heterozygous and M o d - l a Mod-1  P  phenotypes,  content, u s i n g the d e r i v e d c o r r e c t i o n f a c t o r s ,  c o n t e n t o f 50% ± s.d. 2%.  f a c t o r s , when a p p l i e d  t o t h e a c t i v i t y r a t i o o f Mod-1 e l e c t r o p h o r e t o g r a m s , a b  t o determine  f u r t h e r t h e degree  o f Mod-1  and Mod-1  content.  of accuracy of t h i s  procedure was made and w i t h t h e i n h e r e n t assumptions subsequent  yielded  T h e r e f o r e , the d e r i v e d c o r r e c t i o n  p r o v i d e an a c c e p t a b l y a c c u r a t e assignment No attempt  a n a l y s e d f o r Mod-1  r e q u i r e d , the  measurements a r e c o n s i d e r e d o n l y as an e s t i m a t e o f t h e  r e l a t i v e nuclear content. 3.  Si  Mod-1 /Mod-1  Si  muscles from  b b and Mod-1 /Mod-1 e s t i m a t e o f n u c l e a r c o n t e n t o f chimeras  A t o t a l o f 59 muscles from 7 chimeras  (5 d y s t r o p h i c — n o r m a l and  2 n o r m a l — n o r m a l ) were a n a l y s e d f o r Mod-1 phenotype. -normal chimeras, presence o f SWV  In the d y s t r o p h i c -  s u b u n i t s o f 'a' type c o u l d o n l y r e s u l t from t h e  (normal) n u c l e i whereas s u b u n i t s o f 'b' type c o u l d  2J 23 o n l y r e s u l t from t h e presence o f dy /dy nuclei.  A3  f  ure 8.  m  E l e c t r o p h o r e t o g r a m s of Mod-1 from i n d i v i d u a l d y s t r o p h i c — n o r m a l chimera muscles. Corresponding densitometer t r a c e s a r e p r e s e n t e d above the e l e c t r o p h o r e t o g r a m s .  44  Examples  of the e l e c t r o p h o r e t o g r a m s and the c o r r e s p o n d i n g  d e n s i t o m e t e r t r a c e s , from the d y s t r o p h i c — ^ n o r m a l chimeras a r e p r e s e n t e d i n F i g u r e 8.  The i n d i v i d u a l muscle i n t e g r a t i o n  results  from each chimera a r e t a b u l a t e d i n T a b l e s 2, 3, 4, 5, 6, 7 and V i r t u a l l y every p o s s i b l e e l e c t r o p h o r e t i c p a t t e r n was m a j o r i t y of the muscles demonstrated h y b r i d Mod-1  found.  8.  The  bands i n d i c a t i n g  t h a t they were composed, e n t i r e l y o r i n p a r t , o f h e t e r o k a r y o n muscle f i b e r s , jL.e_., muscle f i b e r s c o n t a i n i n g b o t h g e n e t i c a l l y and g e n e t i c a l l y normal n u c l e i .  No muscle was  e n t i r e l y of d y s t r o p h i c n u c l e i .  The h i g h e s t observed 'b' c o n t e n t  for  a s i n g l e muscle was  number 10.  T h i s may  found t h a t  dystrophic consisted  94% f o r the r i g h t psoas muscle from chimera  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 p o p u l a t i o n of muscles examined no  ;!b  of  normal  f  had been l a r g e r .  A n a l y s i s of some muscles  demonstrated  c o n t r i b u t i o n and t h e r e f o r e , t h e s e muscles were composed o n l y nuclei.  The range of muscle c o m p o s i t i o n found w i t h i n s i n g l e chimeras was  spectacular.  t r i c e p s was was  For example,  composed o f 94% normal n u c l e i whereas the r i g h t  composed of 94% d y s t r o p h i c n u c l e i .  a l s o found between l e f t the of  i n chimera number 10, the r i g h t  Considerable v a r i a t i o n  was  and r i g h t muscles i n s i n g l e chimeras, e_.j>.,  l e f t and r i g h t g l u t e u s maximus i n chimera number 10 was 67% and 21% d y s t r o p h i c n u c l e i  composed  respectively.  A summary o f the r e s u l t s i s p r e s e n t e d i n T a b l e 9. 'b' c o n t e n t " was  psoas  "Average  c a l c u l a t e d by summing the observed 'b' c o n t e n t f o r  TABLE 2 Chimera Number 7; Mod-1 Muscle 1.  biceps  2. 3.  Side femoris  E s t i m a t e of D y s t r o p h i c aaaa  aaab  Content aabb  abbb  bbbb  Total  % 'b'  84  121  94  left  0  0  0  37  g l u t e u s maximus  right  2  3  10  35  5  55  76  pectoralis  left right  0 15  0 20  4 37  26 10  62 5  92 87  94 60  Summary: i. ii. iii.  T o t a l 'b' s u b u n i t o f muscles examined = 88% Muscle w i t h h i g h e s t 'b' c o n t e n t = l e f t b i c e p s femoris (94%) Muscle w i t h h i g h e s t 'a' c o n t e n t = r i g h t p e c t o r a l (40%)  TABLE 3 Chimera Number 8; Mod-1 Muscle  E s t i m a t e of D y s t r o p h i c  Content  Side  aaaa  aaab  aabb  left right  28 108  43 117  121 93  13 9  abbb  bbbb  Total  % 'b  0 0  205 327  48 35  1.  biceps  2.  gastrocnemius  right  112  7  0  0  0  119  3  3.  g l u t e u s maximus  left right  73 78  32 15  9 6  6 0  0 0  120 99  29 12  4.  masseter  left right  52 83  14 5  11 5  7 2  3 0  87 95  47 16  5.  pectoralis  left right  47 106  5 5  7 7  2 6  3 18  64 142  44 63  6.  psoas  left right  13 17  3 10  5 5  2 0  1 0  24 32  51 22  7.  semimembranosis  left  4  4  20  25  7  60  75  8.  tongue  25  0  0  0  6  31  71  9.  triceps  left right  62 32  16 27  5 23  0 9  3 4  86 95  33 53  10.  vastus  right  5  3  2  0  0  10  25  11.  vastus medialis  right  12  6  2  0  0  20  19  femoris  lateralis  Summary: i. ii. iii.  T o t a l 'b' s u b u n i t o f muscles examined = 43% Muscle w i t h h i g h e s t 'b' c o n t e n t = l e f t semimembranosis (75%) Muscle w i t h h i g h e s t 'a' c o n t e n t = r i g h t gastrocnemius (97%)  TABLE 4 Chimera Number 9; Mod-l  1.  b  Estimate of Dystrophic Content  Muscle  Side  aaaa  aaab  aabb  abbb  bbbb  Total  % 'b'  psoas  left  41  22  57  98  54  272  79  TABLE 5 Chimera Number 10; Mod-1 Muscle femoris  Estimate  of D y s t r o p h i c  Content  Side  aaaa  aaab  aabb  abbb  bbbb  Total  % 'b  left  42  16  79  96  92  325  83  1.  biceps  2.  gastrocnemius  left right  75 33  26 15  5 13  0 7  0 0  106 68  13 42  3.  g l u t e u s maximus  left right  55 134  72 128  68 25  55 0  28 0  278 287  67 21  4.  masseter  left right  0 14  16 39  30 85  5.  pectoralis  left right  6.  psoas  7.  triceps  7 6  4 4  5 7  0 8  41 51  2 11  3 23  4 32  62 93  112 210  92 88  left right  157 13  59 3  50 7  7 9  41 61  314 93  63 94  left right  138 58  52 7  21 0  4 0  215 65  22 5  0 0  Summary: i. ii. iii.  T o t a l 'b' s u b u n i t o f muscles examined = 73% Muscle w i t h h i g h e s t 'b' c o n t e n t = r i g h t psoas (94%) Muscle w i t h h i g h e s t 'a' c o n t e n t = r i g h t t r i c e p s (95%)  oo  TABLE 6 Chimera Number 11; Mod-1 Muscle tibialis  E s t i m a t e of D y s t r o p h i c aaab  Content  Side  aaaa  aabb  left right  24 30  4 3  2 0  0 0  0 0  30 33  12 4  45  18  13  5  4  85  49  abbb  bbbb  Total  % 'b  1.  anterior  2.  biceps femoris  left  3.  gastrocnemius  left right  90 161  53 41  20 17  6 2  2 0  171 221  33 17  4.  g l u t e u s maximus  left right  115 135  41 93  14 72  7 23  3 3  180 326  34 41  5.  tongue  42  0  0  0  17  59  80  6.  vastus  176 33  44 11  12 2  3 0  0 0  235 46  16 13  lateralis  left right  Summary: i. ii. iii.  T o t a l 'b' s u b u n i t o f muscles examined = 35% Muscle w i t h h i g h e s t 'b' c o n t e n t = tongue (80%) Muscle w i t h h i g h e s t 'a' c o n t e n t = r i g h t a n t e r i o r t i b i a l i s (96%)  TABLE 7 Chimera Number 16; Mod-1 Muscle 1.  biceps  femoris  2.  g l u t e u s maximus  E s t i m a t e of D y s t r o p h i c aaab  Content  Side  aaaa  aabb  abbb  bbbb  Total  left right  208 151  0 0  0 0  0 0  0 0  208 151  left right  39  0 0  0 0  0 0  0 0  39  +  +  % 'b 0 0 0  3.  psoas  left right  19 93  7 0  0 0  0 0  0 0  26 93  10 0  4.  triceps  left right  88 101  0 0  0 0  0 0  0 0  88 101  0 0  * exceeded c a p a c i t y o f densitometer Summary: i. ii. iii.  T o t a l 'b' s u b u n i t o f muscles examined = 1% Muscle w i t h h i g h e s t 'b' c o n t e n t = l e f t psoas(10%) Muscle w i t h h i g h e s t 'a' c o n t e n t = a l l others(100%)  o  TABLE 8 Chimera Number 17; Mod-1^ E s t i m a t e of D y s t r o p h i c Muscle  Side  1.  biceps  femoris  2.  g l u t e u s maximus  3.  triceps  4.  vastus  5.  vastus medialis  lateralis  right  aaaa  aaab  Content  aabb  abbb  bbbb  % 'b'  1  6  11  14  20  52  86  left  14  2  0  0  0  16  5  left right  26 91  2 6  0 5  0 12  0 13  28 127  3 61  right  11  14  6  0  0  31  27  right  61  0  2  8  14  85  70  Summary: i. ii. iii.  Total  T o t a l 'b' s u b u n i t o f muscles examined = 67% Muscle w i t h h i g h e s t 'b' c o n t e n t = r i g h t b i c e p s femoris (86%) Muscle w i t h h i g h e s t 'a' c o n t e n t = l e f t t r i c e p s (97%)  TABLE 9 G e n e t i c C o n s t i t u t i o n o f Muscles Analysed Chimera  f o r Mod-1^:  Average d y s t r o p h i c content  'b'  Summary T o t a l d y s t r o p h i c content 'b'  (normal—normal) C # 7 C # 17  81 42  88 67  C # 7 and C # 17  61  82  # 8 # 9 (one muscle o n l y ) # 10 # 11 // 16  37 79 54 30 1  43 79 73 35 1  C # 8, 9, 10, 11, and 16  40  58  46  62  (dystrophic—normal) C C C C C  (combined d y s t r o p h i c and normal) C # 7, 8, 9, 10, 11, 16, and 17  53 each muscle and d i v i d i n g by the number o f muscles. values r e f l e c t  the average  These  percentage  'b' c o n t e n t p e r muscle w i t h no c o n s i d e r a t i o n  of the muscle s i z e . "Total  'b' c o n t e n t " was c a l c u l a t e d by summing the observed  i n t e g r a t i o n r e s u l t s and s o l v i n g 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 o f d y s t r o p h i c n u c l e i more a c c u r a t e l y because t h e a c t i v i t y o f the e l e c t r o p h o r e s e d enzyme was g e n e r a l l y observed  to be p r o p o r t i o n a l t o the s t a r t i n g muscle s i z e  (muscles  below 50 mg y i e l d e d j u s t p e r c e p t i b l e a c t i v i t y whereas a c t i v i t y o f l a r g e r muscles i n c r e a s e d w i t h the weight o f the f r o z e n m u s c l e ) .  If 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  t r a c e would be expected  to t h i s c a l c u l a t e d The and  total  t o s o l v e f o r a 'b' v a l u e c l o s e  percent.  'b' c o n t e n t i n n o r m a l — n o r m a l chimeras  i n d y s t r o p h i c — n o r m a l chimeras  f o r a l l the mosaics was 62%. sample would be 50% i f no c e l l h i g h e r v a l u e s observed  58%.  was 82%  The o v e r a l l *b' c o n t e n t  The expected  'b' c o n t e n t o f a l a r g e  type had a s e l e c t i v e advantage.  f o r these chimeras  The  l i k e l y r e f l e c t o n l y the  l i 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 p r e s e n t c o n c l u s i v e evidence t h a t g e n e t i c a l l y d y s t r o p h i c n u c l e i a r e p r e s e n t w i t h i n the muscle f i b e r s composing the s k e l e t a l muscle o f these chimeras. o n l y 1 chimera  In  (chimera number 16) was t h e r e a d r a m a t i c a l l y h i g h  frequency of muscles composed e n t i r e l y o f normal 'a' n u c l e i . t h i s was observed  i n o n l y 1 chimera  Because  i t i s i n t e r p r e t e d as r e p r e s e n t i n g  54  one extreme  o f 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 l o s s of the g e n e t i c a l l y d y s t r o p h i c  nuclei.  This observation confirms previous h i s t o l o g i c a l evidence that no developmental a b n o r m a l i t y e x i s t s i n d y s t r o p h i c muscle 1971).  (Platzer,  A l t h o u g h the number o f chimeras and the number o f muscles  from each was  not l a r g e , t h e r e does not appear to be any d e f i c i t of  dystrophic n u c l e i  ( d y s t r o p h i c — n o r m a l chimeras have a t o t a l  c o n t e n t of 58% amongst the muscles examined).  Furthermore,  'b' dystrophic  myoblasts a r e c a p a b l e of f u s i n g to normal myoblasts d u r i n g the m u l t i n u c l e a t i o n s t a g e as demonstrated by the l a r g e number o f muscles w i t h h y b r i d Mod-1 The t o t a l  bands. 'b' c o n t e n t v a l u e o f 58% a l s o i m p l i e s t h a t no  s e l e c t i v e death o f d y s t r o p h i c n u c l e i had taken p l a c e . were between 6 and t h e r e was place.  The  chimeras  12 months o f age when they were k i l l e d and thus  c o n s i d e r a b l e time f o r any s p e c i f i c d e g e n e r a t i o n to take  Furthermore, some muscles demonstrated  l a r g e p o p u l a t i o n s of  'homozygous' d y s t r o p h i c 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 o b s e r v a t i o n s a r e t h e r e f o r e u n l i k e l y  to be the r e s u l t of a h e t e r o k a r y o n muscle s t r u c t u r e which mimicked  the  normal g e n e t i c h e t e r o z y g o t e . The  normal myogenesis  and maintenance  of the g e n e t i c a l l y  d y s t r o p h i c n u c l e i t h a t a p p a r e n t l y o c c u r s i n these chimeras p r o v i d e s i n d i r e c t s u p p o r t i v e evidence f o r Salafsky's  (1971) t r a n s p l a n t  studies.  A l t h o u g h the chimera r e s u l t s p r o v i d e no e v i d e n c e f o r the r e g e n e r a t i o n step r e q u i r e d i n h i s experiment, they do demonstrate  that  genetically  55  d y s t r o p h i c muscle f i b e r s can form and be m a i n t a i n e d i n the mosaic environment.  I t seems l i k e l y ,  therefore, that dystrophic  muscle  f i b e r s s h o u l d a l s o be a b l e to r e g e n e r a t e and be m a i n t a i n e d i n g e n e t i c a l l y normal h o s t s . The range of g e n e t i c c o m p o s i t i o n w i t h i n 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 b o t h s i d e s , demonstrates t h a t each muscle must be c o n s i d e r e d as a unique c o m b i n a t i o n . 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 t h a t the maximum t w i t c h  results  of the a n t e r i o r t i b i a l i s muscles were a p p a r e n t l y v e r y u n i f o r m between l e f t and r i g h t muscles of the same chimera. Finally,  the e l e c t r o p h o r e t i c r e s u l t s p r o v i d e some i n s i g h t  i n t o the u s e f u l n e s s of the chimeras as a model of the female c a r r i e r of the X - l i n k e d Duchenne d y s t r o p h y (Emery,  1964).  G e a r h a r t and  M i n t z (1972) proposed t h a t the v a r i a b l e mosaicism they observed among eye muscles o f n o r m a l — n o r m a l  chimeras p r o v i d e s a model f o r  the p h e n o t y p i c v a r i a b i l i t y known to o c c u r i n muscles of women heterozygous f o r Duchenne d y s t r o p h y .  The c l i n i c a l  and  functional  phenotype of the d y s t r o p h i c — n o r m a l chimeras, which a r e known to 2J have dy  23 /dy  interpretation  n u c l e i i n t h e i r muscle f i b e r s i m p l i e s t h a t  their  i s not c o r r e c t .  A h i s t o l o g i c a l e v a l u a t i o n of the d y s t r o p h i c — n o r m a l chimeras was  undertaken to e v a l u a t e t h i s h y p o t h e s i s .  CHAPTER V HISTOLOGICAL EVALUATION OF MUSCLES FROM DYSTROPHIC—NORMAL CHIMERAS Based on both the c l i n i c a l evaluation and the maximum twitch examination (Chapter I I I ) , i t was expected that a h i s t o l o g i c a l evaluation of these chimeras would reveal only s l i g h t or no pathological lesions c h a r a c t e r i s t i c of muscular dystrophy.  If a normal h i s t o l o g i c a l  phenotype was observed i n muscles composed primarily of dystrophic n u c l e i i t would support a non-primary myopathogenesis f o r the mouse dystrophy.  S i m i l a r l y , i f muscles of genetically normal  composition were observed to have f o c i of pathological change, some factor, such as abnormal innervation, could be postulated as the primary cause of the muscle pathology. Although extensive h i s t o l o g i c a l 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 the dy  2J /dy  mutant i s a v a i l a b l e .  Meier and Southard (1970) 2J 2J  reported that the histopathological features of dy were similar to those of the dy/dy mice.  /dy  mice  They d i f f e r e d only i n  that the former demonstrated a reduction i n the extent of abnormalities  56  57  t h a t " c o n s i s t e d , i n whole o r p a r t , of l o s s of s t r i a t i o n ,  coagulation  n e c r o s i s , r e g e n e r a t i v e 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  internal  rowing of n u c l e i " .  M a t e r i a l s 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 a n t e r i o r t i b i a l i s 2J muscles of dy  2J /dy  and  c h i m e r i c mice was  done.  Muscles s e l e c t e d  a_ p r i o r i w i t h i n t e r e s t i n g g e n e t i c c o n s t i t u t i o n from the chimeras were a l s o examined.  Because the f i r s t  c l i n i c a l s i g n s of the  disease  i n d y s t r o p h i c mice a r e r e c o g n i z a b l e from h i n d limb m u s c u l a t u r e involvment,  o n l y muscles from the h i n d limbs of the dystrophic==  normal chimeras were c o n s i d e r e d  for close  examination.  To a c h i e v e 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 a c c u r a t e l y a s s e s s s l i g h t d e f i c i e n c e s , a l l muscle samples were prepared  for 1 u thick plastic sections.  A r a z o r b l a d e s e c t i o n (approximately  2 mm  t h i c k ) was  a c r o s s the b e l l y of the i n d i v i d u a l muscles immediately d i s s e c t i o n from the mouse. hours i n c o l d 2.5%  i n 0.1M  done a t approximately  d e h y d r a t i o n was  7.3  M c a c o d y l a t e b u f f e r f o r 1/2  post f i x e d f o r 1 hour i n 2% OsO^ F i x a t i o n was  after  their  These muscle samples were f i x e d f o r 4  g l u t e r a l d e h y d e , b u f f e r e d to pH  b u f f e r , r i n s e d w i t h 0.1  taken  then  c a c o d y l a t e b u f f e r , pH  7.3.  while  A f t e r dehydration  an a l c o h o l s e r i e s , f o l l o w e d by 30 minutes i n p r o p y l e n e mixture  cacodylate  hour and  5° C i n a r e f r i g e r a t o r  done a t room temperature.  muscles were p l a c e d i n t o a 1:1  with  oxide,  of L u f t ' s epon ( L u f t ,  through the 1961)  58 and p r o p y l e n e o x i d e f o r 24 h o u r s . 60° C f o r 20 to 24 hours  F i n a l embedding was  performed  at  i n epon.  The muscle t i s s u e s thus prepared were c u t i n t o  1 u  thick  s e c t i o n s on a R e i c h e r t u l t r a m i c r o t o m e u s i n g a g l a s s k n i f e . d r y i n g on a g l a s s s l i d e ,  the s e c t i o n s were s t a i n e d w i t h 1%  After toluidine  b l u e and mounted w i t h Permount.  R e s u l t s and D i s c u s s i o n 1.  D y s t r o p h i c muscle;  anterior  tibialis  C r o s s s e c t i o n s of a n t e r i o r t i b i a l i s muscle from a 4 month 2J old  dy  2J /dy  mouse a r e p r e s e n t e d i n F i g u r e s 9 and  10.  The most  s t r i k i n g f e a t u r e 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  s m a l l , presumably r e g e n e r a t i n g f i b e r s , a r e common.  Hypertrophied  f i b e r s a r e s c a t t e r e d throughout. i s common.  Evidence of c e n t r a l n u c l e a t i o n  Many f i b e r s l a c k the c h a r a c t e r i s t i c p o l y g o n a l shape o f  normal muscle f i b e r s and a r e round  instead.  Hyaline degeneration,  f i b e r s p l i t t i n g and areas o f f i b r o s i s can a l s o be 2.  Dystrophic—normal Although  o n l y 1 chimera  chimeras:  t h i s muscle was  anterior  observed.  tibialis  a n a l y s a b l e f o r n u c l e a r genotype i n  (chimera number 11), i t was  examined to see i f the  f u 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 f e a t u r e s . As expected,  t h i s a n a l y s i s r e v e a l e d v e r y few f e a t u r e s t h a t were  a t y p i c a l o f normal muscle. i n F i g u r e 11, 12,  13 and  14.  Examples of these s e c t i o n s a r e p r e s e n t e d The g e n e r a l a r c h i t e c t u r e o f the  w i t h compact p o l y g o n a l f i b e r s , was  normal.  muscles,  The most common f e a t u r e  59  F i g u r e 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  dystrophic  (dy  2J  /dy  ) mouse,  mag. x 600.  60  F i g u r e 11.  Left anterior t i b i a l i s  o f chimera 8.  F i g u r e 12.  Left anterior t i b i a l i s  o f chimera  10.  mag.  mag.  x  240,  x  240.  Figure 13.  Right anterior t i b i a l i s of chimera 9. mag. x 220  F i g u r e 14.  Right a n t e r i o r t i b i a l i s  o f chimera 9, mag. x 600  62  i n d i c a t i v e of any  d y s t r o p h i c p a t h o l o g i c a l change was  the presence  of o c c a s i o n a l c e n t r a l n u c l e a t i o n .  3.  D y s t r o p h i c — n o r m a l chimeras: Figures  15 and  s e l e c t e d muscles  16 a r e s e c t i o n s from the l e f t 2J  of chimera number 8.  The  t h i s muscle was  Apart  and  23%.  estimated  dy  g l u t e u s maximus  2J /dy  nuclear  content  of  from the r a r e example of c e n t r a l n u c l e a t i o n  possible fatty i n f i l t r a t i o n  t h i s muscle demonstrates normal  features. Figures  17 and  of chimera number 10. t h i s muscle was content  was  83%.  18 a r e s e c t i o n s from the l e f t b i c e p s 2J 2J The No  estimated  dy  /dy  nuclear  h i n d limb muscle of h i g h e r  available for histological analysis.  examples of c e n t r a l n u c l e a t i o n and  femoris  content  of  dystrophic  Although rare  p o s s i b l y degenerating  fibers  were observed, the h i s t o l o g i c a l f e a t u r e s of t h i s muscle were a l s o remarkably normal. Figures  19,  20 and  21 are s e c t i o n s of the l e f t b i c e p s  muscle from chimera number 16. 2J dy  e l e c t r o p h o r e t i c e v i d e n c e of  2J /dy  n u c l e i c o u l d be r e s o l v e d so t h i s muscle was  not c o m p l e t e l y , with  No  g e n e t i c a l l y normal.  primarily, i f  S e v e r a l s m a l l f o c i of  l e s i o n s c h a r a c t e r i s t i c of d y s t r o p h i c myopathology, are  These i n c l u d e : marked v a r i a t i o n i n f i b e r s i z e , 1.e_. , and  femoris  regenerating  frequent It  fibers; fiber  degeneration present.  hypertrophied  s p l i t t i n g ; coagulation necrosis;  examples of c e n t r a l n u c l e a t i o n . i s u n l i k e l y t h a t these o b s e r v a t i o n s  are the r e s u l t  of  and  63  64  Figure  18.  Left biceps  femoris  of chimera 10.  mag.  x  600.  65  66  F i g u r e 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— normal chimera killed at 10 months of age.  Furthermore, the unique  histological features of senile muscle, e_.£. 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 t i b i a l i s muscles i s 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 i s however, 2J only preliminary.  No muscle was found with only dy  23  /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 p r e s e n t study demonstrates  the u s e f u l n e s s o f the c h i m e r i c  mouse to d e l i n e a t e the s i t e of the primary l e s i o n i n the complex p a t h o l o g y of muscular  dystrophy.  In d y s t r o p h i c — n o r m a l chimeras, b o t h g e n e t i c a l l y d y s t r o p h i c and normal n u c l e i were d e t e c t e d i n the s k e l e t a l muscle. no apparent d e g e n e r a t i o n of g e n e t i c a l l y n u c l e i which were e s t i m a t e d to c o n t r o l muscle examined.  There  was  d y s t r o p h i c muscle f i b e r 58% o f the mature  chimera  T h i s i s e v i d e n c e t h a t the primary l e s i o n ,  resulting  i n the muscle w a s t i n g of g e n e t i c a l l y d y s t r o p h i c mice, r e s i d e s o u t s i d e the muscle f i b e r . demonstrated  to be arranged  l i k e muscle f i b e r s . myoblasts  Furthermore,  d y s t r o p h i c n u c l e i were  i n b o t h h e t e r o k a r y o n and homokaryon  These r e s u l t s imply t h a t g e n e t i c a l l y d y s t r o p h i c  c o n t r i b u t e n o r m a l l y t o muscle morphogenesis.  The d y s t r o p h i c - n o r m a l chimeras were c l i n i c a l l y normal. o b s e r v a t i o n may  indicate  This  t h a t the p h e n o t y p i c s i m i l a r i t i e s o f the  2J mouse dy  and human Duchenne d y s t r o p h y a r e i n c i d e n t a l t o primary  l e s i o n s of d i f f e r e n t natures.  Alternatively,  mouse t i s s u e to t h i s p a t h o l o g i c a l genotype,  the response o f normal  i n c h i m e r i c mice,  may  be s u f f i c i e n t to d e v e l o p m e n t a l l y o r 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 f o r unknown r e a s o n , remain u n c h a l l e n g e d i n the human genetic  mosaic.  69  70  The  muscle f u n c t i o n  evidence that was  the  analysis  of the  factor responsible  either, selected  against  chimeras p r o v i d e s  f o r the d y s t r o p h i c  i n development or was  further  phenotype  functionally  compensated f o r i n the mature a n i m a l . Histological analysis rare scattered pathology. normal and  of mature s k e l e t a l muscles demonstrated  l e s i o n s w i t h c h a r a c t e r i s t i c s of d y s t r o p h i c  muscle  These f o c i were p r e s e n t i n muscles of both g e n e t i c a l l y p r i m a r i l y dystrophic  s u p p o r t s the gives further  composition.  This  observation  i n t e r p r e t a t i o n of the muscle c o m p o s i t i o n r e s u l t s i n s i g h t i n t o the p o s s i b l e n a t u r e and  and  l o c a t i o n of  the  primary l e s i o n . Recent attempts to c h a r a c t e r i z e implicated  the p e r i p h e r a l  p r e s e n t study a r e not Genetically functional  nervous system.  inconsistent  dystrophic  (je.£. t r o p h i c  s k e l e t a l muscle.  influence)  The  s i t e of the The  l e s i o n have  r e s u l t s of  the  with this hypothesis.  motor nerves c o u l d  end-plate) d e t e r i o r a t i o n leading of the  the  undergo a p r o g r e s s i v e  or m o r p h o l o g i c a l  (e.£.  motor-  to p a r t i a l or complete d e n e r v a t i o n  simultaneous presence of  normal nerves i n the chimeras c o u l d  genetically  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  r e p r e s e n t t h i s dynamic p r o c e s s .  degeneration could motor n e r v e s .  r e p r e s e n t the  The  scattered  l o s s of s i n g l e g e n e t i c a l l y  These denervated muscle f i b e r s c o u l d  r e c e i v e normal i n n e r v a t i o n normal c o n s t i t u t i o n by  foci  from a d j a c e n t neurons of  c o l l a t e r a l axonal sprouting  of  dystrophic  subsequently genetically and  thus r e g a i n  a  71  normal h i s t o l o g i c a l appearance. This hypothesis can be tested.  Techniques are presently  available to estimate the size of i n d i v i d u a l motor u n i t s .  I f the  above hypothesis i s correct, the s i z e of the motor units i n these chimeras would be larger than motor units i n corresponding muscles of genetically normal mice.  Furthermore, the r e l a t i v e 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 i n the gene function analysis of a genetically determined developmental muscle disease of mice.  The l e t h a l muscular dysgenesis, mdg, mutant described by  Gluecksohn-Waelsch (1963) has been characterized u l t r a s t r u c t u r a l l y by Platzer and Gluecksohn-Waelsch (1972).  The action of this gene  r e s u l t s i n a t o t a l f a i l u r e of normal muscle morphogenesis with the f i r s t a l t e r a t i o n - swollen sarcoplasmic reticulum - detectable by day 14 of gestation.  Bowden-Essien (1972) demonstrated by i n v i t r o  studies that the mdg mutant may i n t e r f e r e with muscle c o n t r a c t i l i t y by a f f e c t i n g properties of the c e l l membrane.  A muscle composition  analysis of mature mdg/mdg—normal chimeras would be expected to r e f l e c t the developmental presence of the muscular dysgenic c e l l s . This could be demonstrated by the recovery of only the genetically normal n u c l e i or by recovery of the mdg/mdg n u c l e i s p a t i a l l y arranged only i n heterokaryon muscle f i b e r s .  I f the l a t t e r s i t u a t i o n was  observed, 1.e_., a rescue of the mdg/mdg n u c l e i , l i m i t s of the basic c e l l u l a r l e s i o n produced by the mutant would be defined.  BIBLIOGRAPHY  Adams, R, D., D, Denny-Brown and C. M. Pearson. 1962. D i s e a s e s of Muscle, 2nd. e d i t i o n , Harper and Row, New York. Baker, W. B. and B. M i n t z . 1969. Subunit s t r u c t u r e and gene c o n t r o l of mouse NADP-malate dehydrogenase. Biochem. Genet. 1\ 351-360. B i s h o p , A., B. G a l l u p , Y. Skeate and V. Dubowitz. 1971. 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H e r e d i t a r y mouse muscular d y s t r o p h y w i t h p a r t i c u l a r emphasis on p a t h o g e n e s i s and attempts a t t h e r a p y . Ann. N. Y. Acad. S c i . 138: 4-13. West, W. T. and E. D. Murphy. 1960. H i s t o p a t h o l o g y o f h e r e d i t a r y , p r o g r e s s i v e muscular d y s t r o p h y i n i n b r e d s t r a i n 129 mice. Anat. Rec. 137: 279-295.  APPENDIX 1 FOCAL program: Mod-1 C-8K  FOCAL  estimate o f n u c l e a r content  31969  01.01 01.05 31.10 01.15 01.20 01.25 01.30 01.35 01.40 01.45 01.50 01.55 01.60 01.65 01.70 01.75 01.80 01.85 01.90  E D T T f T T F T r T T S S T T S F T  02.10 02.1t>  F 1=1*5*5 C H = C H + ( ( T R ( I > - R t I ) > t 2 ) / R ( 1 ) 1 " C H I SQUARE"CH* ! ! ! ! ; G 1.01  04.05 04.10  S CF(1>=1*S CF(2)=l.fc3»S S C F ( ^ ) = 6 . 7 8 * 5 CFC5*=10  4*A "SAMPLE"SA»!! £5.03*" At4 4(At3*B) 6<At2*Bt2>" " 4(A*Bt3> Bf4 TOTAL"*M*"ACTIVITV ?" I=1*5;A " " T R ( I > * S 5 1=S 1 +TRC 1 > ; T " ";S Z*f ( 1 )=TR( 1 > * C F ( I ; bT*!*"PROTEIN "if I = 1*5;T O i l ) * " " J S C I = C7 + C ? t l > C T * ! " P R . R A T I O "*S P C = 1 . 2 5 i = l * 5 ; S CRC I )=C-/ ( I ) /CT; T C K C l ) * " ";S TT=TT+TRCI) !*"A C0NTE.MT " > r i = l * 5 ; S P C = P C - . 2 5 * S AG ( I ) = CR ( I > * PC 1=1*5;T A C C 1 ) * " "*S HT=AT+AC(I> A T * ! * S B T = 1 - A T ; T "B C J . M i £ \ T "* F I = 1 * 5 ; T C R C 1 ) - A C ( i ) * " " BT*i*"TH.PR.RAT«" A l = A T t 4 ; S A 2 = 4 * C A T T 3*3T>* S A3= 6* C A T t 2 « 6T* 2 ) A 4 = 4 * t A T * B T t 3 ) ; S A5 = BTt 4 Al*" "A2*" "A3*" "A4*" "A5*! " i H • R A T « A C T " i S R C 1 ) = A 1/CFC 1 ); S RC2 > = A 2 / C F ( 2 ) R l 3 >=AJ/CF (. 3 ) ; S R C 4 ) = A 4 / C F C 4 ) ; S RC5)=A5/CFC5) 1 = 1*5*3 R T = R T + R ( 1 ) * T R U ) * " RT*!»F I=1*5;S R ( I ) = R < I ) * < T T / R T )  CF(3)=2-37  

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