Straznicky 1967.pdf

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Acla Bioi. Hung. 18(4), 437 -448 (1967)THE DEVELOPMENT OF THE INNERVATION AND THEMUSCULATURE OF WINGS INNERVATED BY THORACIC

NERVESK STRAZNICKY

I NSTITl'TE OF ANATOMY. HISTOLOGY AND EMBRYOLOGY, MEDICAL UNIVERSITY, pf:cs (HEAD. n. FLERKOj(Receiwd Octoher 7. 19(6)

SynopsisThe. brachial segruents of the spinal cord of chicken emhryos "ere repJacecl bytboracic segment6. and the development of the wing musculature aod innp,rvation as

studied in 8- to 16-day-old embryos aud in 4-week-old cbickens. Wing muscles witbthoracic innervation developed normally till tbe 10th rlay of incubation. From that timeon tbe muscle. gradually deteriorated. and by the 16th day the ruu,c1e lissue hrcnroereplaced hy nclipose nnd connective lissue tllr


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tained well-differentiated muscle tissue even at the time of hatching. In Con-Ira:>t 1 the latter finding, SZEKELY and SZENTAGOTHAI [17] found no musclesaftt'r hatching in extra limhs which were grafted into the somites at the trunklevel. Thes(' ('xtra limbs wcre highly sensitive to mechanical stimuli. Althoughthese authors [171 did not gin- a dC'lailed account of the type of inl1f'rvation,whether only sensory_ or both sensory and motor nen'es contributed to theinnervation of th(' graft it could IJp assumed that in those large nerve trunkswhich entered the grafts, motor nerves might have he('n prescnt as well. Fromthis short and by no means complete sun'cy on the lit ('rat urI', it seems thatmuscle tissue can dev(']op undl'r nervell' conditions, or with inappropriateinnervation; however a specific nerve supply which arises from the limh segments of the cord is nf'cessary to complete the differentiation and to maintainthe muscle. This specificity, nevertheles, may be explaincd in a simple quantitative sense, assuming that thoracic segments give origin to lIIany fewermotor fibers than brachial segments do aud tbal hecause of its unfavourableposition I he graftpd limh is l'('llt'lratpd only hy such a small fraction of the motor fibers that the innt'rvation is not sufficient for the maintt'nance of the muscles.

To tudy thi prohlt'rn, in the present work the brachial cord of chickenembryos was removed and replaced hy the thoracic cord, takt'n from otheremhryos of the same age. This experimental arrangement gives the n e r v e ~ fromthe grafted thoracic eord segments a much better chance to invade thc host'sown wings, which are left in the normal positions. The developmenl of the inuerYlItion and that of the lIIusculature of such wings was studied in diITf'rf'ntstages of incubation and after hatl'hiug.

Material and methodThe present experiments were performed on chicken embryos. On the 3rd day of iucu

bation. al stages IS - 16 HAMBURGER and HAMILTON IlJj the hracbial segments of the spinaleord were rcmoved (segments 13 - 16). and a part of tbe thoracic cord of equal length takenfrom auother emhryo was transplanted to fill the gap accurately. The method of tram.plantationwas fully descrihed in an eurlier paper 1141 Out of a total of 375 operated embryos 60 success'fully operated ones were killed between the Bth and 16tb day of incubation. wbile a few of themwere brougbt up to the time of batching. The sacrificed embryos were fixed either in 10 percent formalin or Susa solution for bistological investigatioo. Serial sections were made fr()Inthe yertebral column. inclnding the transplanted part of the spinal cord. Unly those cases werefurther investigated in which normallv developed spinal cord was found. In the successfullyoperated embryos the development of the llluscle tisSllc was investigated histologicallv. Sectionsprepared from of each emhryo were. stained with haematoxylin-eosin.those from tbe other ,dngwere either impregnated witb tbe Abraham- Bielsehowsky II) silver lechnilJue. or the cholincstcra (eh F. reaction wa.< studied according to LERllER and O R N T I ~ [ I I I In hatchedchickens the peripberal digtribut ion pa llerH of nen co was examined in macroscopical di . .ertion


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[ ) ~ H l . P M ~ : I 1 OF THE I N ~ t , f l , H I 0 7 < 439Ohservations

Between thc 8th and 10th days of incubation embryos wpre sacrificedd[lily anf th .. ir willAs submitted to histological iuvestigatioll,Five embryos we're studipd [It ag .. of 8 - 9 days. In hacmatoxylin-t'(,sin

~ t a i D ( d s e c t i o n ~ IJOtl1 homogeneous and striated mnscl(' fiLers were found. Thetriattod fihers wpre starting to IWCOJl\( ' organized illto lIluscle hUlldltos. Spactos

Ipft hetween the fiLers weI''' fillfd \\ ith embryonic mcsenchyme c('lIs. In controlcmhryos of th .. ~ a m e age the d i f f t r e n t i a t i l l ~ muscle fibcrs showed l,out thpsaUle picture. The fibers wert', probahly, somewhat thicker. ltoaving less roomfor inttorfihrillar spaces [lnd rtlativf'ly few undifferentiated mesenchynle cellswere present. The innenation of "'in/ : muscles. as revt'aled in silvcr-illll'regnat..d sp('cimens, consistpeJ of richly sprouting plexiform ucrve hundles, fromwhich occasionally thin hran('hes emcq:ed and got lost among thl:" musclefibers. In this respt'ct 110 difff'rencf' was found bctwrcn 1I0rmai allrl operatedembryos, except that nen'es could more p[lsi'y If' shown ill the normal wings.The histochf'mical loc[llization of ChE activity showed again a simil[lr pictuftin the wing IHusclf's both in normal and operated embryos: it wa found inl'int> brown staining particles scalt('fcd evenly t hroughollt the llluscle fibers.

Thirtt'f'n opcrated e l I l h r y o ~ \\ f'rr examined on the 10th day of incubation.The first sign wllidl distinguished them from normal embryos W[lS Ihat thcir\\ ings 'vere somewhat smaller in size. Among the devl'l0l'ing muscle fibers largl'giant cells appeared, 20 30 u ill diameter (Fig. lb). Their cytoplasm show .. ,lthe same pink t ~ o l o U I as did the young musde fibers stained with hapmatoxylin-posin. Inside the cytoplasm one or two nuclei wert.' fonnd. The form and shapeof thest' cells rt'srrnblcd Vf'ry mlll'll the giant cells known to he present illneoplast ic tissue of I I I uscle origin. \l { I regarded them [IS drtcriorat illg musclefillt'rs and their presence was th .. first sign of mllselt> dp.structiou. The interfibrillar spacf's were larger, [lnd mort' mesf'nchyme cells were prcsent than innormal embryos (Fig. la , in which ouly a few mcsenchyme cells and a largenumb .. r of yuung muscle-forming cl'lIs. with occasional miloses, \\erl' found.The cross-striation of normal muscle fih .. rs was morc distinct and the stainingof nuclei lighter than ill operated embryos. _ The innervation of the musclesdid not chang too much as compared to the previous stage. In normal embryosthe plexiform nen'e bundles gavc origin to s vera] fine fibers, which run paralIf' to the muscle fibers (Fig,. h . The fine branches occasionally tf'rrninatt>rl illsmall oval t'nlal'gelllents (fig. Id). Bt>causl" of the absence of ChE activity onIhl' surface of muscle fibers at this agl:", wr could not definitely decide whethrrthese enlargements effl:"ct('d neuromlls('ular connections or they were growthcuncs commonly fnund on the tip of growing nerve fibl:"rs in tissue culturt> andrve I"egellPratioll experilllt'uts. Thf' innl'fvation I)f thr' UlUH.:h> in opf'rat('ot>mbryos did not difff'r much from that of the normal ones. The plexifnrm


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44{) K. STRAZNICKY

Fig Irs Muscle t i ue from the 'ing of a 10-dav-old normal emhryo. Haematoxylin-eMin< 600'Fig lb Muscle ti 'nc from the wing of an operated embryo of the same age. The Rrrow pointsto a giallt cell. Hacmatoxylin-eosill, x 600Fig Ie Fine nerve fibers emcrginl : from a plexiform nerve bnndle in the wing muscle of aJO-day-old normal embryo. Ahraham- Aielsebow.;k,y's silver technique. Some of the finefiLer.s arc slightly retouched. Y 600Fig ld An oval n l ~ r m n t on the tip of a growing lIerve fiber from the same embryo.AhraL:iIll- Bielschowsky's silver techniquc. > 900Fig Ie. ChE activity in the form of fine and coarse granules within the muscle fibers of a 10

day-olu normal embryo. Lchrer-- Ornstein tcchni'lue, /600Fig If Mnscle tiS'ne from the wing of a 12-da),-0Id normal embryo. Haematoxylin-eosin,\00Fig 19 The salllc from a 12-day-old operated embryo. Note the phagocytes and a giant cellamong the muscle cells. Haemato ylin-eo,in. ;600

- (.ta BiotoSi('(J ACfI l mlo imlUlrum Hungaricru 18 }1.J67


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UEVELOI'\lENT OF THE 1J\I'>EnVATIlJi\ 441

nerve hundles were slllaller in size and nUllloer, and we never could find enlargements on the tip of the finl' fihers. In histochemical specimens the salllefine granulation indicative of high ChE activity was shown in tile muscle fibcrsboth of the normal (Fig. 1rvo. Abraham -- Bit'lscllo"sky's silvt'T technique, " 900F'f _ I i . ChE actiVIty in the wiug musclt' or a 12-Ja)ul" normal clIIlor>u. Arruw, puint \0 the

primor


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44 K. STIlAlNICKY

Fig 2a Muscle tissue from the wiug of a I ~ d a y o l d normal embryo. Haelllatoxylin-eosio.v 600Fig 2b The same from an operated embryo of the same age. A few muscle fibers. connective

tissue cells anrl a multinucl"atcd giant c cll can he scen. HaemlJtoxylin-eosin. > 600Fig 2c Arrows point to. primitive eod-plates in the wing muscle of a I ~ d a y o l d normalembryo. Abraham- Rielschowsky's silver technique, y 900Fig 2d ChE activity in the wing muscle of the same embryo. The end-plates primordia arcarranged in irrcgular rows. Lehrer Ornstein technique, x 300 .Fig 2. Long nerve fibers in the wiog of a }.l-day-old operated embryo. Ahrahiim- Hielschowsky's silver t"elmi,!u", / YFig f Muscle tissue from the "'jng of a 16-day-old normal embryo. Hacmatoxylineosill.,600Acla Biologica AcnJemlC1t SrirnHurum Hungarua L , 1967


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insidl' thl: muscle' fillers (Fig. Ik), against a dark-stainl'd hackground of diffu!:'l'I'nzymc activity.

Sixtl:en opl'rated emhryos werl: a\ ailahle for histological invl'stigation atthe age of 13-14 days. In hapmatoxylin-eosin-stained sections a further de structioll of lIIuscle was confirmed. Thc muscle fiber hunrllt's w,rt' hreaking up(Fig. 2b). In sevPfal instancl's solitary fihers and llluitinucleatpd ~ i n t Cl'lIs.or fragmcnts of them, occurrl'd thickly con'r( 'd with phaf!ocytes. Because oftill' t':\tpnsi\ e destruction of the sarcopla


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444 K STRAZNICK Y

ed the picture of the matured striated muscle tissue (Fig. 2j). In impregnated sections of normal wiugs the primitive end-plates showed further signsof differentiation. Imtead of t hI: dichotomous branching seen in the earlierstage, the terminal fibers broke up in tuft-like endings covering about one halfof the width of musclc fibers whcll vicwed from above. Sections in differentplanes r ..vealed differpnt forms of the enrl-plates: they might appear in elongated club-shaped or in circular forms (Fig. 2h . Sites of high enzyme activitywere increased both in number and size. They occupied oval or round fidds onthe surface of muscle filwrs, soml'times as large as a half of the width of a fiber.The size and shape of these fields was very similar to that of the tuft-like endings found in imprcgnated sections (Fig. 2g . The diffuse sarcoplasmic activityvirtually ( l i s a p p l a r f ~ d . Several nervf' 11II1\(lIe5 were seen running bl't\\ccn fatlobules and among cells of t he scar tissue in winf:s of operated embryos. t wasdifficult to show the nerves since the silver was taken up mostly by the nucleiof connective tissue cells.

Five operated l'mbryos hatched and these were kept alive for a periollof 5 wceks. As was described carlicr (13) t he wings innervated by thoracicspinal cord were motionless aud e,-ery joint 1H l ame ankylotic. The macroscopic appearance of the spinal cord and the hrachial plexus. as rl'ported in dptail earlier (14), gave a normal picture. The brachial plexus, as in normal chickens, was o m p o s ~ of [ollr ~ p j l l a l nl'rves, Itut lhi' branches were markedly thinner. Dissection of t hl winp:s revealed a normal peripheral arborization patlprnof the main ncrve trunks; both t he median and t he radial nerve followed theirnorlllal course. The musculaturp was rl pIac-l d It) fat tissue and conneeti e tis"ue save for a tiny hundle at the site of the triceps muscle. This continued in along tendon and attaehl'd to the olecranon. All shoulder muscles were present,although mud.l slIIalipr in sizl than thuSt, uf normal emhryos.

In 3 hatched chickens ventral roots of the transplanted, as well as ofthe host's own, thoracic segments and those of normal brachial cord sf>g:mcntsWl re slIhjf ctf>d to hi;;tulugical inve,;tigation (Table I). The ventral root fibers

IThe nlimber oJ venlra roor Jlbers rn 1I0rm,, brachi,,/ _


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f)EVELOPMEJ\T OF TilE I N N E n v ~ T 1 i 445

of the transplautf'd spinal I:ord were considerably increased in Humber wben Icumpared to the number of motor fih"rs of normal thoracic segments. This : .increase was most marked in the 14th and 15th segments, somewhat less intht' 13th and 16th sef(ments. In tIlt' grafted thoracic segments tbprt' were abouttwice as many motor fibers as in normal thoraci,' segments. but only one fifthI,f them were coming from normal brachial spinal cord ;cgn\l?nts.

The fibers diameter spectrum showed that the larger fibers founu inventral roots of the host's own thoracic cord Wf'rt' 6.:; II in diameter, and therange of fiber diameters had a peak at 311. In the ventral roots of llraftcd seg-ments the largest fibers were found to he 5 in diameter and the peak appl'ur-ed at 2.:; fl.

In two chickens at ages of 3 and 5 weeks after hatching, the veutral rootemerging from the trausplanted thoracic cord segment that replaced tht' 2ndhrachial Sf'grnt'Jlt was snered. Five days later degeneratt'd ncrve fibers werefound in ahundauee throughout the length of the radial nerve (Fig. 2j , whichoriginated mainly from the 2nd brachial sl'gment. WI' also observed degenerat-ed fihers in tht' scar tissue which [('placed the wing U1l1sculaturf'. A failure toshow degenerated fibf'rs in the dorsal root excluded the possibility that sensoryfibers from the spinal gauglion which ~ l t t hav(' been injured d u r i n ~ operation

~ i g n i f i a D t l y contrihuted to the degeneration.

DiscussionFTl m the rrsults pr(,sf'nted the question raispd in the introduction can be

answerrd in the affirrnativf', namely lhat the musculature of wings needs aspecific lH'rve supply for the normal differentiatioll alld maintenance of itsstructure. The apparl'1I1 contradiction bf'tween this and EASTLlr.KS [5.19] results suggesting that llormal muscle tissue develops in coelomic leI graftswith thoracie innervation, can probahly he explaint'd hy the fact that a coelo- ,.mic graft, madf' aftt'r 2 to 3 days of ineuhation, mainly attachl's to Ihe cranial iiiI ,part of the body wall and rpcl'ivI's wing llerves as w?11. In fact. K\STLICK [5] ,also clescriht'S musclr dest ruet ion in t hp presence of nt'rve fihrr5. These mighthavt' emerged f rom' he thoracic segments of the cord. whilf' tllllst' arround iiwhich a normal mU5culatur(' dt'vt'loped might have lH'en of hrachial o r i ~ i n . I,In t'arlit:'r f'xperimt'nts [15 ]we have succeeded iu showing that hracllial i n n . ~ r -vat ion is an atlf''1uatp.nrrn' supply for leg llluscles, thereforf' it sl'f:'cificity of Int'r\'e supply dops not exist ill tt'rms of wing or I('g. , IThe df'V'e1opmental history of muscles with thoracic innervation can he , Isummarized a,; follows. Embryonic myohla ;ts, apparently hy their inhrrcnt. 01potentiality. arc eapahlt' of diff('rt'ntiating into striated muscle fibers without Itill' I'rt'spner of any nent 's [:: O], or with tllOraril'. illnervatioll. Th" main difft'r-('nee betwf'f'u the furlher d t e l o p m ~ n t of muscles with normal or i lh thorae-

'C'(1 BW({)i;I tU ',nd ml'I "'n""'("lflllll Hllnl; . lrrrcJf ' 1\, }Q );


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fJ h. T H ~ Z N I \

ic innervation, is that in th(' lalle rcase only a vcry small numher of embryonic IIH'sl'uch) OIl ' cells call transform into l I l u ~ c 1 e - f o r m i n g cells, aud tht' majoritygivf' " rise to diffprent connective tissllP cells, which destroy and then replacl'thf' muscl,'. Appareutly thl' multiplication of the muscle by the division ofdifferentiatinl fibers occurs also milch less frt'qllently than in normal musell'.From 10th day 011 no mort' Ill musclt'-foTluing cdls are visible. the developf'dfibers stop d i f f l n n l i a t i l l ~ and arl' gradually rl'plact'd hy connectivp tissupwithin 6 days.

There is a 51 riking difft'rt'llce also ill the development of normal and t horacic innervation of \\ in/ : 111115(,11'''. Sinct' dt'finitive nPllromllsclllar cnd-platt'sappear at ahollt the second week artf'r hatching. \\ e could ohserve only till'vpry first phase of their development. Tht' study of this narrow section of endplate deveh'l'mellt shows lliut so-called" Pl,'xijorm Z1('ischenstadien as described by TELLO [I8] and BOECKE in mammalian emhryos, a diffuse typeof innervation, prceedes the individual type 0'( muscle innenation in the chicken as w('lI. On thl' 10th {lay fint terminal filwrs It'ave the pleXiform ncrvf'bundle and terminate with small oval enlargements on individual muscle fibers. One day later the high ChE activity in small oval areas on the sarcoplusrn is suggpslivp or all {,rfedive ueuromuscular contact estahlished by tht"terminal fibers. o r d i n ~ to the shape and location of these t'nzYllle-activf'areas, they can he regarded as primordia of definitive end-plates. which continuously grow in size during d,,\elopment. In the muscles of operated cmbryosthese end.plate primordia do not de\ e1op, and those fine braucuiug nerve fiberswhich made neuromuscular connections in normal musclt", run a long coursl'among the df'geueruting Illuscle fibers and eounective tissue cells. Not bcingable to develop a reliablt' techniqut', we could uot decide if thest" loose Ut'uromuscular connect ions were ever functional. From a few observations Illade before killing the t'mhryos, we obtained thc vaguc impression that movempntscould be elicited in tht' shoulder joint hy llIef'hanical stimulation, anti to amuch less extent in the elbow and in the wrist. We again t'annot tell whetherthese were neurogcnic 01' myogenie movements. After the 12th day of incubation, we did not succeed in e v o k i n ~ any movements.

The 10th day seemS to be a critical pcriod in th development of thelIIuscle. 'fhi,,; is the tilllt' \l'hf'll all intimate neuromuscular conuection bt'gins todevelop, and also when muscle r1pstruction ),pgins ill thl' utypir'ully iUllPrvuterlmuscle. t would be, ho,':t'ver. misleadinl ; to conclude that the Illuscle dt'stl'Uction occurs on account of the lack of elld-plate formatiou seen first on the thday of incubation. Tht' tramformation of mesenchyme cells into phagocytesinstead of into muscle-forming cells as early as the 9th day, may suggest a neuromuscular conuection whieh develops in normal embryos earlier than couldbe shown by histologif'al mf'thorls. and which is ahsent in lhe case of tllllrUl'il'innervation.

Acta Bio(ogl( n Ar dt'mIQI' .-'Cll nllarum Hun..annu 18. 191 7


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Ul:\ ':1.0PMEi'T OF THE I:'ONEll' " IUi ' 447

Although the total numher of themotorfiht'rs leaving the thoraciC' segmentswas It'ss than oul'fifth nf!henumherofmotorfibersfrom a normal hrachial cord, the diminution of fibers cannot accoullt for th .. extensive wuscll'i ..strul'lion. I f it were so, we could have found smaller or larger hUUIJ l .. Saround the few motnr fib!'rs


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448 K STRAZNICKY

19. WORTnAM, R. A., EASTLICK H. L. (1960) Studies on transplanted embryonic limbs of thechick. VI. The development of muscles and tendons in nerveless and weakly innervatedchick limb grafts. J. orp 106, 131146.20. ZELENA, J. (1962) The effect of denervation on muscle development. In GUTMANN E.The denervoted muscle. Publishing House of the Czechoslovak Academy of Sciences,Prague.

OlE ENTWICKLUNG DEI:t I ~ N R V T I O N UND MUSKULATUR DER V O ~ THORA KALEN NERVEN INNERVIERTE"i FLOCELN

In Hiihneremhryoncn wurden statt brachialen Segmenten des Riickenmarkes thorakaleSep;mente transplantiert. Ole Ent\\icklung der Fliigelmuskulatur unn dcren Innervatiou wurdeim Alter 8 his 16 Tagen uon S Woehcn nach der Bebriitung untersucht. Die Entwirklung dcrvon den thorakalen Segmenten innervierten FJiigehnuskulatur verlief his zum 10. Tage derInkubation norulaJ. l\"ach clem 10. Tage hCl';ann rine graduelle Degeneration der Muskdu. so doGam 16. Tap;r in den FliigeJn statt Muskeln nur Fett und Bindegewehe zu finden war. Es konutemit Hilfe der SilberteclUlik und Cholinesterase Mcthode festgestellt werden. daO die Entwicklungder ersten neurolUuskuHiren Endpbitteben in n o r m ~ n Fliigeln zeitlic!> mit clem Anfong derDcgeneration der Fliigelrnuskulatur der operierten EmQJ;,.vonen zusa millen trift. In I1uhnern miteinem tborakalen Riickenmarktransplantal konuten narh dcr Lasion d",r "wei teo braehialenVornerwurzel degenerierte :" ervenfasern ill den Fliigrln beobaehter ,,crnen. Die Ergebnissespreehen dafiir. daG die Fliigelmllskcln mit thorakaler Illoervatioll trotz dcs VorLalldenseinsder motorisehen !'lervenfasero. eben so zngrunde geheo, als die Mnskelo des von anderen Authoren beschriebeneo neneolosen Gliedertransplantates. Znr norma leu Enlwicklung der Musk"]nis! eine spezifische Innervation von der Gliederhohe des Riiekenmarkes notig.

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Straznicky 1967.pdf