ULTRASTRUCTURE OF DEVELOPING HUMAN MUSCLE:
T H E PROBLEM OF MULTINUCLEATION
OF STRIATED MUSCLE C E L L S
GUILBERTO MINGUETTI * W. G. P. MAIR **
S t r i a t e d muscle cells of v e r t e b r a t e s are of mesodermal origin and particularly of the p a r a x i a l mesoderm by w a y of the segmentally a r r a n g e d somites. Centain p a r t s of the v o l u n t a r y musculature, e.g. those of the limbs, t o n g u e and orbit a p p e a r to arise independently of the somites, a n d differentiate in situ in the mesenchyme l. T h e p a r a x i a l mesoderm consists of longitudinal m a s s e s on either side of the n o t o c h o r d and a r i s e s from the intra-embryonic mesoderm. In man, by the twentyfirst d a y of development, the p a r a x i a l m e -soderm on either side of the n o t o c h o r d a l process and, later, of the definitive notochord, begins to segment into paired cubical m a s s e s called somites. D u r i n g the period of somite formation, which in man e x t e n d s a p p r o x i m a t e l y from the twenty-first to thirtieth day of development, the e m b r y o is said to be in the somite s t a g e . Altogether 42-44 p a i r s of somites are formed in the h u m a n embryo, a n d in e m b r y o s of 32 d a y s (5mm C.R. l e n g t h ) each somite b e g i n s to differentiate into t h r e e p o r t i o n s : a myotome which gives origin to muscle, a dermotome which is concerned with the i n t e g u m e n t a r y tissues and a sclerotome which is related to the development of the axial skeleton. T h e cells of the myotome are embryonic muscle cells or m y o b l a s t s which begin to u n d e r g o elongation and from condensed g r o u p s of cells with their long axis parallel1 1
. T h e y each contain a single nucleus and proliferate by mitosis.
Different muscles differ in the time at wich the first m y o b l a s t s a r e r e c o g -nizable and in their subsequent differentiation. Muscle fibres in the t o n g u e differentiate well a h e a d of those in the limbs and trunk. In t h e t o n g u e cross s t r i a t e d fibrils can be found a s early as six w e e k s 10
. In the limbs, however, only after the 8th week of embryonic life m a y the muscle cells be identified a s much more complex units called m y o t u b e s . T h e s e cells a r e larger, have parallel sides and an axial r o w of multiple nuclei lying in a pale core of cytoplasm. C r o s s s t r i a t e d fibrils lie at t h e p e r i p h e r y along the long axis of the cell. From
Trabalho realizado no Instituto de N e u r o l o g i a da Universidade de L o n d r e s : * P H . D . , Professor Adjunto do Departamento de Clínica Médica (Neurologia) da Universidade Federal do Paraná (Curitiba, Paraná B r a s i l ) ; ** M.D., F.R. C.Pth., Consultant N e u -ropathologist, I n s t i t u t e of Neurology. Queen Square, London WC1N 3B6G, England.
Acknowledgement — The authors are indebted to Mr. B. Young for his excelent technical
the 8th w e e k o n w a r d s new m y o b l a s t s continue to differentiate from the m e s e n -chyme. Later, d u r i n g the 5th m o n t h of development the nuclei of m a t u r e m y o t u b e s begin to move to their p e r m a n e n t surface position u n d e r n e a t h the p l a s m a m e m b r a n e , the cells r e a c h i n g the s t a g e of myofibres. Now, v e r y n u m e -r o u s and well o -r g a n i s e d myofib-rils occupy the cent-ral p a -r t of the fib-re. It is the general belief t h a t from this s t a g e o n w a r d s v o l u n t a r y muscle continues to g r o w in two w a y s : by recruitment of new m y o b l a s t s from adjacent mesen-chyme, and by p r o g r e s s i v e e n l a r g e m e n t of the muscle fibres, the last becoming relatively more and more i m p o r t a n t the older the foetus 6 , 7 , 9 , 1 0 , 2 7
.
T h e mechanism of how muscle fibres become multinucleated d u r i n g embryonic m y o g e n e s i s and r e g e n e r a t i o n in vivo, or d u r i n g the process of diffe-rentiation in tissue cultures in vitro h a s been the subject of continuous dis-cussion. No definite solution h a s yet been found. T h r e e e x p l a n a t i o n s exist as to how the l a r g e , multinucleated skeletal muscle fibres arise from m y o b l a s t s —
a) Each muscle fibre is a syncytium resulting from the fusion of m a n y s e p a r a t e
cells. b) E a c h m y o b l a s t g r o w s m a r k e d l y in length but the rapid multiplication of the nuclei by amitosis is not accompanied by division of the cytoplasm. Hence a multinucleated cell is produced. c) Both p r o c e s s e s of development m a y occur. T h e aim of the p r e s e n t investigation is to b r i n g some contribution for better knowledge of this matter.
MATERIAL, A N D METHODS
T h e material used in this investigation i s derived from t w e n t y seven human foetuses r a n g i n g from nine w e e k s to nine m o n t h s development. T h e y have been studied in
detail in longitudinal and transverse sections b y electron microscopy; s o m e 300 grids were examined. F o e t u s e s of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 24, 28, 32, 36 and
40 w e e k s were studied. The age of minute foetuses w a s established by the obstetricians w h o supplied the specimens and w a s calculated according to the crown-rump length
(C.R. l e n g t h ) . T h e a g e s of foetuses of 5, 7, 8 and 9 months development were based on the menstrual age. They were newborn prematures w h o died w i t h i n 24 hours of
birth. Specimens w e r e removed soon after death in these cases to avoid the occurrence of post mortem changes in the muscle. The specimens were obtained from various
gynaecological centres in England and Brazil. The age of the earlier specimens, 9-18 weeks, w a s based on the crown-rump measurement while that of the older specimens,
20-40 weeks, w a s determined b y the menstrual age. In both instances this information w a s provided by the gynaecologist who attended the patients.
The muscle w a s usually taken from the thigh, even muscle from the minute foetuses,
and laid immediately on a piece of card and k e p t s l i g h t l y stretched b y means of pins applied to either end of the specimen. The specimens w e r e then immersed in cold
3% glutaraldehyde in S^rensen's phosphate buffer at pH 7.4. After 2 hours of fixation the muscle held b y the o i n s w a s released and cut in small pieces of about 1-2 mm
thick. These were washed in t w o changes of S I r e n s e n ' s buffer at pH 7.4. for 15 minutes each. P o s t fixation w a s carried out for 2 hours at room temperature in cold 1% osmium
propylene oxide and in a mixture of equal parts of propylene oxide and Epon 812. F i n a l l y they were embedded in fresh Epon mixture. Sections, 1-2^ thick were cut on an L K B Ultratome and stained w i t h toluidine blue by the method of Trump et al. ( 2 6 ) and examined b y l i g h t microscopy. Thin sections of appropriate regions were collected on copper grids, stained by uranyl acetate and lead citrate acc. to R e y n o l d s (22) and
examined in Siemens Elmiskop I.
R E S U L T S
In the present s t u d y some observations m a y s u g g e s t a manner by w h i c h
multi-nucleation is achieved. Myoblasts u n d e r g o i n g m i t o s i s are prominent b e t w e e n nine and eighteen w e e k s development. Some of these cells u n d e r g o i n g m i t o s i s are intimately
interposed between m y o t u b e s running longitudinally and all the cells are contained
in the same, basement membrane tube. ( F i g s . 1, 2, 3, 4). W h i l e the plasma membranes
of t h e cells in mitosis were a l w a y s clearly defined those of m y o b l a s t s in t h e interphase in a similar situation in relation to m y o t u b e s are not a l w a y s clearly defined ( F i g s . 5, 6).
If this i s due to degeneration of the membranes it m a y explain h o w additional nuclei
may be incorporated into the m y o t u b e s in the later s t a g e s of their maturation.
Degeneration of adjacent plasma m e m b r a n e s of m y o t u b e s enclosed in the same
and m y o b l a s t s similarly disposed. F u r t h e r the nuclei of m y o t u b e s lie in consecutive r o w s along the l e n g h t of the cells and two nuclei in parallel were never found in the
same m y o t u b e to s u g g e s t that degeneration of t h e plasma membrane of cells in parallel may have occured.
Evidence of the occurrence of mitosis or amitosis of m y o t u b e s w a s not observed. However some m y o b l a s t s w e r e seen w h i c h it may be thought show evidence of amitosis (Fig. 7). These m y o b l a s t s were a l w a y s in the neighbourhood of myotubes. However
even if these m y o b l a s t s did divide b y amitosis this observation adds no support to our knowledge of how multinucleation of m y o t u b e s may occur since the division of the
nucleus w a s accompanied b y division of the cytoplasm so that two cells result from the division and not a multinucleated muscle cell. Had this observation been made by
light microscopy the erroneous impression would have been obtained that tha nucleus was dividing amitotically within the m y o t u b e since by light microscopy neither the
plasma nor the basement membranes of the closely adjacent muscle cells can be identified.
Yet in the present study, within the same basement membrane tube cells were observed at different s t a g e s of development w h i c h showed deep finger-like cytoplasmic
indentations: the cytoplasm of the more mature cell w a s indented by that of the less
mature cell ( F i g s . 8, 9, 10). The indentations were bounded by the plasma membranes
COMMENTS
S c h w a n n e x p r e s s e d the opinion t h a t , in the pig, the muscle fibres a r o s e by the alignment of the primitive cells into parallel r o w s in which these cells
w e r e a r r a n g e d in an e n d - t o - e n d f a s h i o n2 4
. He considered t h a t linear coalescent of these cells p r o d u c e d the e l o n g a t e d multinucleated muscle fibres. T h i s is called the polygenistic or polycellular t h e o r y . T h i s polygenistic view w a s soon contested by R e m a k w h o believed t h a t each multinucleated muscle fibre a r o s e by g r o w t h and n u c l e a r division of a single embryonic cell, avoiding the process of c y t o d i e r e s i s2 1
. R e m a k ' s view w a s t e r m e d the monogenistic or unicellular theory. T h e e m p h a s i s placed on amitotic events a t the end of t h e nineteenth c e n t u r y reinforced the a c c e p t a n c e of the unicellular mechanism of multinuclea-tion of muscle fibres, b u t b r o u g h t m o r e confusion to the m a t t e r . M a n y recent r e p o r t s r e - e m p h a s i z e the thesis t h a t amitotic division of m y o b l a s t a n d m y o t u b e nuclei w i t h o u t cytoplasmic division l e a d s to multinucleation 3,18,20.
N u m e r o u s other w o r k s have been c a r r i e d out to reinforce both t h e poly-genistic a n d the monopoly-genistic theories. T h i s literature is confusing a n d there h a s been much conflict of opinion a s to how the m y o b l a s t becomes converted into the multinucleated a n d highly specialized muscle fibre. A modification of the polygenistic t h e o r y s t i p u l a t e s t h a t multinucleation is achieved by the fusion of mitotically dividing mononucleated m y o b l a s t s and or with a m y o t u b e con-taining non-dividing nuclei.
Direct o b s e r v a t i o n s in tissue cultures on the fusion of m y o b l a s t s into
multinucleated m y o t u b e s w e r e r e p o r t e d by Rinaldini ( 1 9 5 7 )2 3
, Holtzer ( 1 9 5 8 )1 2
, Cooper and K o n i g s b e r g (1959)5, K o n i g s b e r g (1964 and 1973)13,14 a n d O k a z a k i
and Holtzer ( 1 9 6 6 )1 9
. Muscle syncytium a r i s i n g a s a result of cell fusion h a s also been d e m o n s t r a t e d by several other techniques which will now be discussed. T h e r e is no evidence of D N A s y n t h e s i s either by the m i c r o s p e c t r o p h o t o m e t r i c
d e t e r m i n a t i o n of F e u l g e n - D N A per nucleus 2,8,15( 0r by r a d i o a u t o g r a p h i c
tech-niques to detect the i n c o r p o r a t i o n of isotopically labelled D N A p r e c u r s o r s . T h e syncytial nuclei do not i n c o r p o r a t e a n y of the labelled p r e c u r s o r s a l t h o u g h mononucleated cells do. Later, some of t h e s e m o n o n u c l e a t e d cells a p p e a r within the syncytium 4.25.28. T h e occurrence of fusion of m y o b l a s t s in vivo w a s
d e m o n s t r a t e d in e x p e r i m e n t s by Mintz a n d Baker ( 1 9 6 7 )1
? .
of cells t h a t a r e usually fusiform w i t h r a t h e r r e g u l a r m e m b r a n e s and which, after c e a s i n g to divide, begin to fuse to form a syncytium. T h e w o r k of K o n i g s b e r g a n d his c o l l a b o r a t o r s , however, a r e b a s e d on e x p e r i m e n t s with e m b r y o s of chickens, mice and r a t s . H u m a n m y o b l a s t s are flatter cells t h a n those of birds, mice and r a t s a n d the only convincing time for t h e s e a u t h o r s to distinguish clonal colonies of h u m a n m y o b l a s t s is w h e n these cells have a l r e a d y shown some differentiation into m y o t u b e s .
In a p r e v i o u s r e p o r t the a u t h o r s o b s e r v e d t h a t m y o b l a s t s , the cells from which muscle is derived, a r e mononucleated cells which d u r i n g the period from nine to eighteen w e e k s development s h o w the g r e a t e s t d e g r e e of mitotic a c t i v i t y1 6
. D u r i n g this period the m y o b l a s t s m a y occur in isolation or in g r o u p s and also in association w i t h more m a t u r e muscle cells, the m y o t u b e s . T h e m y o b l a s t s have a l a r g e single nucleus a n d a relatively small a m o u n t of cytoplasm. T h e plasma m e m b r a n e of the m y o b l a s t is a well defined electron dense s t r u c t u r e . B a s e m e n t m e m b r a n e d o e s not occur a r o u n d isolated m y o b l a s t s but is seen a r o u n d m y o b l a s t s lying in apposition to m y o t u b e s (Fig. 11), p a r -ticularly d u r i n g mitotic division. M y o b l a s t s which a p p e a r to have u n d e r g o n e amitotic division also occur but two d a u g h t e r cells arise from this division and not a multinucleated cell. M y o t u b e s a r e also p r e s e n t at nine w e e k s development a n d a r e p r e v a l e n t in the muscle up to eighteen w e e k s development. T h e y a r e multinucleated cells w h o s e nuclei lie in a r o w in the centre of the cell and w h o s e myofibrils lie peripherally. T h e striated p a t t e r n of the myofibril is evident even a t nine w e e k s development. Some m y o t u b e s exhibit a well defined p l a s m a m e m b r a n e s u r r o u n d e d by a distinct b a s e m e n t m e m b r a n e but g r o u p s of m y o t u b e s also occur which s h a r e a common b a s e m e n t m e m b r a n e tube.
In the p r e s e n t investigation it w a s possible to observe t h a t disintegration of the plasma m e m b r a n e s of adjacent m y o b l a s t s and m y o t u b e s a p p e a r s to occur in longitudinally disposed cells of those c a t e g o r i e s which s h a r e a common b a s e m e n t m e m b r a n e tube and this m a y help to explain h o w further nuclei may be i n c o r p o r a t e d into well developed m y o t u b e s . Deep indentations of the cyto-plasm and the p l a s m a m e m b r a n e s of adjacent m y o t u b e s a t different s t a g e s of development a r e occasionally seen. T h i s gives rise to finger-like p r o c e s s e s a l o n g the surfaces of contact of both cells. T h e significance of such p r o c e s s e s is a subject for speculation. After eighteen w e e k s development the muscle is formed almost exclusively of muscle fibres, the nuclei of which lie at the p e r i p h e r y of the cell and the g r e a t e r p a r t of the cell contains well o r g a n i s e d myofibrils. From a b o u t t w e n t y w e e k s development the muscle d o e s not contain m y o b l a s t s which are isolated from the other muscle cells but t h e r e occur beneath the b a s e m e n t m e m b r a n e of the muscle fibres mononucleated cells similar in a p p e a r a n c e to cells found in the s a m e situation a s in adult muscle. T h e s e latter cells a r e satellite cells or m y o b l a s t s .
SUMMARY
observe t h a t disintegration of the p l a s m a m e m b r a n e s of adjacent m y o b l a s t s and m y o t u b e s which s h a r e a common b a s e m e n t m e m b r a n e t u b e a p p e a r s to occur in longitudinally disposed cells of those c a t e g o r i e s . T h i s m a y help to explain h o w further nuclei m a y be i n c o r p o r a t e d into well developed m y o t u b e s a n d how the s t r i a t e d muscle cells become multinucleated during embryonic myogenesis and r e g e n e r a t i o n in vivo.
RESUMO
Ultraestrutura do músculo humano em desenvolvimento: o problema da multinucleação das células musculares estriadas.
As células m u s c u l a r e s e s t r i a d a s dos v e r t e b r a d o s s ã o de origem mesodérmica, particularmente do m e s o d e r m a p a r a - a x i a l localizado nos somitos. No e m b r i ã o humano de 32 dias c a d a somito inicia um processo de diferenciação em t r ê s p o r ç õ e s : o miótomo que dá origem ao músculo, o d e r m á t o m o e o esclerótomo. No miótomo proliferam a s células m u s c u l a r e s e m b r i o n á r i a s m o n o n u c l e a d a s , os mioblastos. A p a r t i r da oitava s e m a n a de vida e m b r i o n á r i a j á s ã o o b s e r v a d a s células m u s c u l a r e s m a i s diferenciadas, os m i o t u b o s : s ã o células com m a i s de um núcleo de localização central. Ao r e d o r d o s núcleos, são o b s e r v a d a s a s miofibrilas. A partir do quinto mês de vida intra-interina, os núcleos m i g r a m p a r a a periferia do citoplasma, p a r a a s p o r ç õ e s sub-sarcolêmicas, d a n d o origem à s fibras m u s c u l a r e s p r o p r i a m e n t e ditas. H á três t e o r i a s s o b r e o mecanismo de multinucleação d a s células m u s c u l a r e s : a) c a d a célula muscular multinu¬ cleada é um sincício resultante da fusão de v á r i a s células m u s c u l a r e s m o n o -n u c l e a d a s ; b) divisão do -núcleo, sem divisão do citoplasma ( a m i t o s e ) ; c) ocor-rência de a m b o s os p r o c e s s o s . No p r e s e n t e t r a b a l h o , foram e s t u d a d a s por microscopia eletrônica células m u s c u l a r e s provenientes de 27 fetos h u m a n o s v a r i a n d o de 9 s e m a n a s de vida e m b r i o n á r i a até 9 meses. Foi possível se o b s e r v a r que mioblastos em mitose são c o m u n s entre os miotubos e que os mesmos localizam-se entre a m e m b r a n a p l a s m á t i c a e a m e m b r a n a basal dos miotubos. Foi possível a i n d a se o b s e r v a r em v á r i o s c a m p o s e x a m i n a d o s a p r e s e n ç a de mio-b l a s t o s s i t u a d o s n a s e x t r e m i d a d e s de m i o t u mio-b o s ou entre dois miotumio-bos longi-tudinalmente dispostos cujas m e m b r a n a s p l a s m á t i c a s j u s t a p o s t a s e n c o n t r a v a m - s e em d e g e n e r a ç ã o s u g e r i n d o u m a possível a n e x a ç ã o d a s células mais jovens
( m i o b l a s t o s ) pelas células mais diferenciadas ( m i o t u b o s ) . Se isso realmente ocorre, o fato poderia em p a r t e explicar o fenômeno d a multinucleação d a s célu-las m u s c u l a r e s e s t r i a d a s .
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