Myonuclear birthdates distinguish the origins of primary...

Development 107, 771-784 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

771

Myonuclear birthdates distinguish the origins of primary and secondary

myotubes in embryonic mammalian skeletal muscles

A. J. HARRIS1, M. J. DUXSON2, R. B. FTTZSIMONS3 and F. RIEGER4

The Neuroscience Centre and Departments of 'Physiology and 2Anatomy, University of Otago Medical School, PO Box 913, Dunedin,New Zealand.3Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK4INSERM U-J53, 17 rue du Fer-d-Moulin, 75005 Paris, France

Summary

Myotubes were isolated from enzymically disaggregatedembryonic muscles and examined with light microscopy.Primary myotubes were seen as classic myotubes withchains of central nuclei within a tube of myofilaments,whereas secondary myotubes had a smaller diameterand more widely spaced nuclei. Primary myotubes couldalso be distinguished from secondary myotubes by theirspecific reaction with two monoclonal antibodies (MAbs)against adult slow myosin heavy chain (MHC). Myonuc-lei were birthdated with [3H]thymidine autoradiographyor with 2-bromo-5'-deoxyuridine (BrdU) detected with acommercial monoclonal antibody. After a single pulse oflabel during the 1-2 day period when primary myotubeswere forming, some primary myotubes had many myo-nuclei labelled, usually in adjacent groups, while inothers no nuclei were labelled. If a pulse of label wasadministered after this time labelled myonuclei ap-peared in most secondary myotubes, while primary

myotubes received few new nuclei. Labelled and un-labelled myonuclei were not grouped in the secondarymyotubes, but were randomly interspersed. We con-clude that primary myotubes form by a nearly synchron-ous fusion of myoblasts with similar birthdates. Incontrast, secondary myotubes form in a progressivefashion, myoblasts with asynchronous birthdates fusinglaterally with secondary myotubes at random positionsalong their length. These later-differentiating myoblastsdo not fuse with primary myotubes, despite being closelyapposed to their surface. Furthermore, they do notgenerally fuse with each other, as secondary myotubeformation is initiated only in the region of the primarymyotube endplate.

Key words: cell lineage, myoblasts, myotubes, skeletalmuscle, nuclear birthdating, 2-bromo-5'-deoxyuridine,[3H]thymidine, muscle fibre types.

Introduction

Skeletal muscle fibres constitute a spectrum of cell typessharing a common theme of a sarcomeric arrangementof sliding filaments and actin-myosin interactions. Thevaried proportions of muscle fibre types within differentmuscles allow the matching of individual physiologicaldemands for endurance, strength and speed of contrac-tion. Within particular muscles, different fibre typeshave an ordered spatial distribution with an adaptiverelation to factors such as the frequency of use ofparticular motor units or uneven restrictions in intra-muscular blood flow when the muscle contracts. Thesepatterns arise early in muscle development (McLennan,1983; Butler etal. 1982) so that they reflect developmen-tal patterning, rather than adaptation to use. Musclefibres form by fusion of mononucleate myoblasts intomultinucleate myotubes (Schwann, 1847), and theemergence of different muscle fibre types may dependon their origins from different populations of myoblasts(reviewed by Stockdale and Miller, 1987). There are

two major generations of myotubes: primary myotubesform first, and are followed after a delay by secondarymyotubes, which will form the majority of muscle fibresin the adult tissue (Couteaux, 1937, 1941; Kelly andZacks, 1969; Harris, 1981a; Ross et al. 1987a). Thesetwo generations have characteristic differences in theirsequence of expression of MHC isoforms, as discussedin the preceding paper (Harris et al. 1989).

Specificity of fusion within individual classes of myo-blast in a mixed population has been shown in vitro, andthere is indirect evidence to suggest that it may occur invivo (reviewed by Stockdale and Miller, 1987). Wedescribe here experiments designed to test whetherdistinct populations of myoblasts can be detected dur-ing normal muscle development in vivo. Our resultsindicate that the nerve-dependent population of 'late'myoblasts that generates secondary myotubes do notfuse with primary myotubes, at least in the early stagesof the foetal period of muscle development. Foetal ratand mouse muscles were disaggregated into their com-ponent myotubes which could then be identified as

772 A. J. Harris and others

primary or secondary myotubes by criteria of mor-phology, time of appearance, and MHC isoform com-position. Nuclear birthdating was used to determine thetimes of origin of individual nuclei within a myotube tosee if we could distinguish different populations ofmyoblasts contributing to the cell lineages of primaryand secondary myotubes.

If the hypothesis that the two classes of myotube havedifferent cellular origins is correct, one might predictthat myoblasts present during the time of formation ofsecondary myotubes would specifically fuse with sec-ondary myotubes alone. If, on the other hand, myo-blasts fused randomly with other cells, some definitepredictions on their distribution can be made. We havealready excluded the possibility of random homophilicfusion between secondary myoblasts, as new secondarymyotubes form only in association with primary myo-tube endplates, and not randomly through the muscledespite uniform distribution of myoblasts throughoutthe tissue and equal access of myoblasts to both primaryand secondary myotubes (Duxson et al. 1989). In theearly stages of secondary myotube formation, duringE16-17 in rat diaphragm muscle (Harris, 1981a), myo-blasts would have a much greater probability of encoun-tering primary rather than secondary myotubes, andfew labelled nuclei should be found in secondarymyotubes. Only by E18, when the number of secondarymyotubes exceeds primary myotubes by more than 2:1and many secondary myotubes extend the full length ofthe muscle, would random fusion be expected to giverise to similar numbers of labelled nuclei entering bothsecondary and primary myotubes.

Duxson et al. (1989) showed also, in confirmation ofearlier work (Kelly and Zacks, 1969, Ontell and Dunn,1978), that secondary myotubes grow longitudinallyalong the surface of a single primary myotube, thusdistinguishing their pattern of growth from that ofprimary myotubes which reach from tendon to tendonfrom an early stage. We now ask whether this pattern ofgrowth is due to myoblasts fusing only with the growingends of the secondary myotube, or whether myotubesare receptive to fusion along their whole length.

The recent commercial production of monoclonalantibodies to 2-bromo-5'-deoxyuridine (BrdU) makespossible rapid birthdating of cell nuclei using immuno-logical rather than autoradiographic marking oflabelled nuclei (Gratzner, 1982). BrdU is incorporatedinto DNA in place of thymidine, and the labelled DNAcan be replicated so that the label is diluted oversuccessive cell generations. Our interpretation of thenuclear birthdating experiments is based on the theory(Bischoff and Holtzer, 1969) that myoblasts becomefusion-competent immediately after a mitosis, so thatafter incorporating label a myoblast will divide and thenone or both daughter cells may promptly fuse, or mayreenter the cell cycle; heavily labelled cells, whichremained dormant in the developing muscle withouteither dividing or fusing, would be defined as satellitecells, not myoblasts.

Incorporation of BrdU into chick myoblasts in tissueculture early in S-phase has been shown to reduce their

capacity for fusion (Lough and Bischoff, 1976), so thatthey must undergo one further mitotic division beforethey become fusion-competent, indicating that caremust be taken in interpreting results obtained with thislabel. Accordingly, all experimental results were veri-fied with parallel applications of [3H]thymidine fol-lowed by autoradiography.

Materials and methods

Experiments were done on both rats and mice. Rats (whiteWistar) were mated by coupling overnight in wire-bottomedcages, and pregnancies dated by the presence of a copulationplug at 9:00h the following morning (EO). Mice (strain 129/J)were coupled between 12:00 and 16:00h, and pregnanciesconfirmed by the presence of a copulation plug at 16:00h(EO). Thus, when comparing the timing of events betweenmice and rats, mouse embryos at a given 'embryonic day'were about 12 h younger than rats.

Frozen sectionsDiaphragm muscles were dissected from pithed embryos andpinned out on Sylgard in a Petri dish. They were fixed in 4 %paraformaldehyde in 0.1 M-phosphate buffer, pH7.4, for30min and then washed in phosphate-buffered saline (PBS).Just before freezing they were rinsed for 2min in distilledwater, and then placed horizontally on the cryotome chuck ona flat base cut from frozen Tissue-Tek, so that longitudinalsections could be made (Harris, 1981ft). Sections were col-lected on subbed slides, and processed to reveal mitoses orBrdU labelling.

Preparation of muscle disaggregatesDisaggregates were prepared from a variety of musclesincluding diaphragm, sterno-mastoid, intercostals, soleus andextensor digitorum longus, from both rats and mice. Themajority of experiments were done on diaphragm muscle.Embryos were chilled on ice for anaesthesia and their musclesdissected and pinned, well stretched, to incubate in azide-Ringers (140mM-NaCl, 5mM-KCl, 2mM-CaCl2, 4mM-Hepes,0.05% sodium azide) for 30min at room temperature. Themuscles were fixed in 30 mM-dimethylsuberimidate dihydro-chloride (DMS) (Sigma) in lOOmM-NaCl, 50mM-Tris, pH7.4,(TBS) at 37°C for 30min. Next they were incubated incollagenase-neutral protease (Bischoff, 1986) in 140 mM-NaCl, 5 mM-KCl, 2 mM-CaCl2 at 37 °C for 1-2 h, at which timemyotubes could be gently separated from their tendons andany unwanted tissue fragments removed. The remainingtissue was incubated for 30-60 min more, until gentle pipet-ting with a fire-polished Pasteur pipette sufficed to disaggre-gate the cells. In early experiments, myotubes were separatedfrom mononucleate cells by 1G sedimentation (Bischoff,1986), but it was later found more useful to retain bothmononucleate and multinucleate cells. The disaggregationprocedure released a variable number of free nuclei whichcaused some contamination by adhering to the surfaces of thedisaggregated cells. The muscle disaggregates were postfixedin 4% paraformaldehyde in 100 mM-phosphate buffer,pH7.4; centrifuged, and resuspended in distilled water. Theywere then spread on subbed slides and air dried at 4°C.

ImmunohistochemistryAfter being dried onto slides, disaggregated muscle cells wereprepared for immunohistochemistry by a wash in 0.1M-glycine in 0.1 M-NaCl, 20mM-phosphate buffer, pH7.4 (PBS),

Myonuclear birthdates and skeletal muscle fibre origins 773

rinsed in PBS, and given a mild digestion in protease K(Merck) 2/igml"1 in O.lM-Tris buffer, 5mM-EDTA, pH7.4,for 5min at room temperature, followed by 5min washes in20mM-Tris, 0.1 M-NaCl (TBS) plus 4 mM-CaCl2 to stop actionof the protease, and in PBS.

All primary antibodies were aliquotted, quickfrozen inliquid nitrogen, and stored at -80° C. After thawing for use,hybridoma supernatants were diluted with an equal quantityof 120mM-phosphate buffer, pH7.4, and further diluted, asrequired, in PBS containing 0.2% Triton X-100 and 0.5%BSA (ID). All the results presented in this paper are with themonoclonal antibodies NOQ7.5.4D or NOQ7.1.1A, twoclones raised against adult human muscle slow myosin heavychain (MHC) (Draeger etal. 1987) which bind a MHC isoformpresent in all newly formed rat primary myotubes (Narasuwaet al. 1987; Harris et al. 1989). Second antibodies includedbiotin anti-mouse (DAKO or Amersham) followed by strep-tavidin-HRP or streptavidin-biotin-HRP complex (Amer-sham); or FITC or RHO anti-mouse (DAKO). HRP activitywas revealed using diaminobenzidine as chromagen.

Location of mitotic figuresPregnant rats were anaesthetized with ether and the uterusexposed by laparotomy. Taxol (gift from Dr J. Douros) waskept frozen at -80°C as a 2.5 mM stock solution in DMSO,and diluted 1:1000 in sterile saline just before use. Hoechst33258 dye was stored at 4°C in the dark as a 1:1000 solution indistilled water, and further diluted 1:500 in PBS just beforeuse. Individual embryos were injected intraperitoneally (i.p.)with 4/il of taxol to arrest mitoses, and examined 6h later.Frozen sections were incubated in rhodamine-labelled alpha-bungarotoxin (RHO-a^BTX, gift from Dr R. Bloch) to locateendplates, and incubated for 4min in Hoechst 33258 to revealchromosomes. Sections were examined with a 40x oil immer-sion objective in a Zeiss fluorescence microscope.

Birthdating with 5-bromo-2' -deoxyuridineThe drug (purchased from Sigma) was stored desiccated at-20°C and just before use was freshly dissolved in sterilesaline. The standard procedure was i.p. injection of 5 mg intoa pregnant rat (12-25 mgkg"1) or 1 mg into a pregnant mouse(20-40 mg kg ). In experiments comparing [3H]thymidineautoradiography with BrdU immunohistochemistry, foetusesin one uterine horn were individually injected with 50 fig ofBrdU and those in the other horn with 5 fid of [3H]thymi-dine, as described below.

Slides of BrdU-treated myotube preparations were im-mersed in IM-HCI at 60 °C for 8min, or 2M-HC1 at 37 °C for30min. Time and temperature were carefully controlled, assome nonspecific nuclear labelling occurred in tissues pro-cessed in 1 M-HCI at higher temperatures or for longer times.The slides were then rinsed in 0.1 M-phosphate buffer fol-lowed by two changes of PBS. They were then incubatedovernight with anti-BrdU (purchased from Becton-Dickin-son), 1:50 dilution in ID, washed in two changes of PBS, andthe anti-BrdU revealed with a fluorescent second antibody(FITC or RHO-antimouse, DAKO, diluted 1:50 in ID). Ourroutine preparation for birthdating analysis was to distinguishmyotube types with anti-slow MHC Ab marked with the HRPsandwich technique, and then to reveal nuclei containingBrdU with a fluorescent second antibody.

[3H]thymidine birthdating, and autoradiography[6-3H]thymidine (Amersham, 5mCimmole"', lmCiml"1)was stored at 4°C as a sterile solution in distilled water. Datedpregnant rats were anaesthetized with ether and the uterusexposed with a laparotomy. Individual foetuses were visual-ized through the uterine wall and injected intraperitoneallywith 5 fxCi of the sterile solution.

Disaggregated myotube preparations containing [3H]thy-midine-labelled nuclei were incubated with anti-slow MHCantibody which was then marked with HRP by the biotin-streptavidin sandwich technique, and the HRP revealed usingdiaminobenzidine as a chromogen. Slides were rinsed in PBSand then brominated in freshly prepared IO1TIM-H2SO4,20 mM-KBr, 10 mM-H2O2 for 20 min to prevent chemography.They were coated with Kodak NTB2 emulsion, exposed for2-3 weeks and developed for 6 min in half-strength D19developer. The slides were then stained for 20 min withHarris' haematoxylin to mark nuclei, and permanentlymounted.

Criteria for scoring labelled nucleiFITC- or RHO-antiBrdU-labelled nuclei were distinguishedunder the microscope by the subjective criterion of beingobviously fluorescent. The technique used for labelling is verysensitive, and the results were comparable to those using[3H]thymidine labelling (Table 1). The total number of nucleiin a myotube segment was counted using phase-contrastillumination, and then fluorescent nuclei counted under epi-fluorescence illumination. Because of bleaching it was usualto examine only one or two segments in a field of view.

Table 1. Percentage of nuclei in primary and secondary myotubes labelled with birthdating markers[fHJthymidine or BrdURU

labelled(Eday)

% of nuclei labelledassayed(Eday) primary myotubes secondary myotubes assay

14141414

151516

161717

15161616

161617

191819

26(38*)29 (52*)34 (45*)26 (52*)

61*50 (52*)39 (39*)

5n.c.1069

None presentFew or none presentFew or none presentFew or none present

Few or none presentFew or none present

4747372233

3H (i.p.)BrdU

3H3H

BrdU3H (i.p.)3H(i.p.)

3HBrdU

3H3H3H

'Nuclei in fibre segments with 1 or more labelled nucleus, not counting segments without labelled nuclei.


Photographs were made for illustration only, and not used forquantitative analysis.

With [3H]thymidine autoradiography, most labelled nucleiwere heavily labelled under the conditions of labelling andexposure used; too heavily, for example, to be useful foranalysis of timing of the cell cycle. The threshold criterion forlabelling was 5 or more grains per nucleus; the averagebackground density was always equivalent to much less than 1grain per nucleus.

Fluorescence microscopySlides were examined using Leitz Labolux, Zeiss Universal orVANOX epifluorescence microscopes with 200W mercuryilluminators, or a Zeiss Standard epifluorescence microscopewith a 75 W xenon illuminator. Unlabelled nuclei wereidentified with phase contrast. Colour photography was withEktachrome (ASA 200) or Fuji (ASA 1600) colour negativefilm; most records were made on Ilford HP5 black and whitefilm.

Results

Location of mltotlc figures In muscleIndividual foetal rats were injected in utero with theantimitotic drug taxol, and frozen sections incubated inthe DNA label Hoechst 33258. The incidence of mitoticfigures was plotted along the length of the muscles withrespect to the location of the endplates, which werelocated with RHO-a-BTX. Diaphragm muscles fromE18 and E20 foetuses were examined: in both cases, thedistribution was uniform, with no detectable excess inthe endplate region of the muscle. We could notdistinguish between mitoses in myoblasts and mitoses infibroblasts. In preliminary experiments, we also lookedat BrdU-labelled nuclei in longitudinal frozen sectionsof foetal diaphragm muscles. These typically containedlarge numbers of labelled nuclei and it was impossibleto tell whether they were in mononucleate or multi-nucleate cells. We found no significant inhomogeneityin their longitudinal distribution along the musclesections.

Preparation of isolated myotubesWe used the technique of Bischoff (1986) to prepareisolated myotubes. Cells isolated from disaggregatedliving embryonic muscles generally were supercon-tracted with their nuclei compacted together. Next, wetried a 'natural' form of fixation, inducing rigor by roomtemperature incubation in Mg2+-free saline containing0.02% sodium azide before exposing tissues to thedisaggregating enzymes. This worked quite well withmuscles from older foetuses, but new-formed myotubesstill supercontracted. We finally used the hetero-bifunc-tional reagent dimethylsuberimidate dihydrochloride(DMS) (Expert-Bezancon et al. 1977), which providedfixation adequate to prevent contraction, while leavingthe tissue susceptible to the enzymes used for disaggre-gation of the cells. This reagent has the further advan-tage of minimally masking or denaturating antigenicdeterminants.

Bischoffs technique, applied to adult muscle, isintended to separate muscle fibres with satellite cells

still attached to their surface beneath the basal lamina.Foetal muscles contain patchy and diffuse basal lamina(Duxson et al. 1989), and we hoped to obtain myotubesfree of mononucleate cells. Most results presented herecome from myotubes with central nuclei, so there islittle risk of confusion with nuclei in tightly attachedmononucleate cells.

Timing of BrdU injectionsMuscle disaggregates prepared 3 h and 5.5 h after BrdUinjection contained large numbers of heavily labellednuclei, but none of these were in multinucleate cells.Seven hours after injection, a few labelled myonucleiwere found (mouse injected at E14; 70% of myotubeshad no labelled nuclei; 23 % had a single labellednucleus. Labelled nuclei in myotubes were more faintlylabelled than most mononucleate cell nuclei). Inmuscles disaggregated 14 h after injection, there wassubstantial labelling of nuclei in multinucleate cells.Most experiments allowed 24 h between injection andexamination of muscles. Control experiments showedno labelling when antibodies were applied to tissuesthat had not been exposed to BrdU, and no labellingwhen the second antibody was applied alone, withoutprevious exposure to anti-BrdU.

Ordered appearance of muscle fibre types: fibre sizeMyotube preparations from rat diaphragms disaggre-gated on E15 or E16, or mouse diaphragms disaggre-gated on E14 or E15, contained a uniform population oflarge diameter (15-20/an) myotubes (Fig. 1). One daylater, on E17 (rat) or E16 (mouse), the populationsbecame bimodal with the appearance of a new, smaller(5-10 /m\) diameter class of myotube (Figs 2, 3). Thesmall diameter myotubes initially had a beaded appear-ance with rounded nuclei separated by narrow threadsof cytoplasm, fitting Couteaux' (1941) description ofyoung secondary myotubes as 'like a pearl necklace'(Figs 2B, 3). With time, e.g. by E18 in rat diaphragm orsternomastoid muscles, older secondary myotubes hadbecome elongated and uniform in diameter with centralnuclei, but still remained smaller in diameter than theprimary myotubes.

Antibody stainingThe early appearing larger-diameter myotubes werestained by the two anti-slow MHC MAbs NOQ7.5.4Dor NOQ7.1.1A (Fig. 1). Mononucleate cells present atthe same time did not stain with these MAbs, or withthe muscle-specific antibody anti-desmin. In ratmuscles, a small number of myotubes which did notstain with anti-slow MHC (but did stain with anti-desmin) appeared on E16 (Fig. 3), and were morenumerous on E17. These were smaller-diameter fibres,and were still consistently of the smaller-diameter classon E18 (Fig. 4). By E19, some approached the dimen-sions of primary myotubes. The timing of appearance ofthese two classes of myotube correlates well withstructural descriptions of the time of origin of primaryand secondary myotubes, respectively, and we will usethis nomenclature from here on. We conclude that


Fig. 1. Primary myotubes isolated from embryonic muscles. (A) Fibres from an E16 rat diaphragm muscle stained with anti-slow MHC. All fibres in the disaggregate were stained, but mononucleate cells were not. Fluorescence micrographs,rhodamine-labelled second antibody. (B) Primary myotube isolated from an E16 rat diaphragm following injection of BrdUon E15 (-25 h). All nuclei in this segment are marked by anti-BrdU (FITC-labeLled second antibody). (C) Primary myotubeisolated from an E15 mouse diaphragm, following injection of [3H]thymidine on E14 (—24h). Autoradiograph viewed withNomarski optics. Labelled nuclei are marked by arrows. Calibration bars, 20 /OTI.

primary myotubes, but not secondary myotubes,reacted with the anti-slow MHC MAbs, confirming theconclusions reached in the accompanying paper (Harriset al. 1989) from experiments with frozen sections.

Nuclear birthdatingWhen BrdU or [3H]thymidine was injected into apregnant mouse or rat, or directly into the peritonealcavity of an individual foetus in utero, many nuclei inmyotubes disaggregated from muscles of the embryoscould be marked with anti-BrdU or autoradiography,respectively.

Mouse E13/14 or rat E14/15Label injected into mice on E13 or E14, or into rats onE14 or E15, was incorporated into nuclei in the largediameter myotubes stained by anti-slow MHC(Table 1). Following a single injection of label at thesetimes disaggregated diaphragm muscle myotubes exam-ined 24-48 h later included some myotube segments in

which all the nuclei were labelled (Fig. IB), and othersin which no nuclei were labelled. In partially labelledmyotube segments, the labelled nuclei often werepresent as a coherent group, rather than being mixedwith nonlabelled nuclei. The timing of the injection wascritical: for example, injecting a pregnant rat late onE15 and examining the myotubes 24 h later might resultin few labelled nuclei in primary myotubes, while mostmononucleated cells were labelled, and a very smallnumber of secondary myotubes was present, all with alarge proportion of nuclei labelled (Fig. 3).

Mouse E15 or E16, rat E16 or E17Injection of either birthdating reagent at these latertimes labelled a much smaller proportion of nuclei inprimary myotubes, but labelled nuclei were present inthe new-formed small-diameter slow MHC-negativemyotubes (secondary myotubes) (Fig. 4, Table 1).Nearly all secondary myotube segments in diaphragmor sternomastoid muscle disaggregates prepared 1-3


Fig. 2. Secondary myotubes isolated from maturing rat and mouse muscles. (A) Phase-contrast photomicrograph of fibresisolated from an E18 mouse diaphragm illustrating a primary (larger diameter) myotube and a secondary (smaller diameter)myotube. (B) Secondary myotube from an E17 rat diaphragm: BrdU was injected at 2h intervals from — 24 h to —12 hpreviously, in order to cumulatively label all nuclei undergoing mitosis in that time interval, in contrast to the pulse labellingused in the experiments whose results are summarized in Table 1. Although all secondary myotube nuclei were labelled bythis procedure, few labelled nuclei appeared in primary myotubes in this disaggregate. Fluorescence micrograph.(C) Primary and secondary myotubes from an E18 rat diaphragm, labelled with a single injection of [3H]thymidine on E17.A single labelled nucleus is present in the primary myotube, while labelled nuclei appear frequently in the secondarymyotube. (D) Primary and secondary mytotubes from an E15.5 mouse, labelled with [3H]thymidine on E14.5. Labellednuclei are seen only in secondary myotubes (arrows). (C and D): Nomarski optics. Calibration bars, 20\im.

days after injecting BrdU at these times containedlabelled nuclei. Timing of the injection was not critical:for example, in two experiments, diaphragm myotube

preparations were made from rat foetuses injected withBrdU on E16 and examined 24 h later on E17. 75 % and90 %, respectively, of primary myotube segments (de-

Myonudear birthdates and skeletal muscle fibre origins 111

Fig. 3. The earliest secondary myotubes in a disaggregate of an embryonic rat diaphragm muscle. A primary and a new-formed secondary myotube from an E16 rat embryo injected with BrdU on E15 (—21 h). A bright-field view of cells stainedwith anti-slow MHC, revealed with an HRP-linked biotin-streptavidin system. Every primary myotube reacted with theantibody, but few primary myotube nuclei were labelled by anti-BrdU. The very small number of new-formed secondarymyotubes present were not stained by anti-slow MHC and the majority of their nuclei were marked by anti-BrdU (inset,fluorescence micrograph showing FITC-linked second antibody), showing they had formed from myoblasts which haddivided and fused since E15. Calibration bar, 20 jan.

fined by reaction with anti-slow MHC) had no labellednuclei and the others typically had no more than one,whereas 85 % and 88 %, respectively, of secondarymyotube segments (slow MHC -ve) had labelled nucleiand typically these were in the majority. Similarly, in 2examples of rat diaphragm myotubes labelled on E17and examined 24 h later on E18, 82% and 93%,respectively, of primary myotube segments had nolabelled nuclei, and 75% and 100%, respectively, ofthe secondary myotube segments were labelled. Thelabelled nuclei in secondary myotubes were randomlyscattered amongst unlabelled nuclei (Fig. 5).

Only about 5 % of the nuclei in primary myotubeswere labelled on E16, E17, or E18 (Figs 2C, 6) com-pared to 30-50 % if label was injected on E14 or E15(Table 1). In one experiment, [3H]thymidine wasinjected into rat embryos on E16 and the myotubesexamined on E19. 10% of the nuclei in primarymyotubes were labelled, as compared to 37% of thenuclei in secondary myotubes. This time allowed forconsiderable dilution of label (i.e. by 2 or more mitosesoccurring after labelling and before cell fusion). How-ever, nuclei from myoblasts that had fused followingtheir first mitosis after labelling could be identified bytheir high density of autoradiographic grains. Heavilylabelled nuclei were identified under dark-field mi-croscopy, and then their location in a primary or asecondary myotube determined with bright-field mi-

croscopy. In this case, 14% of the heavily labellednuclei were in primary myotubes. If it can be assumedthat the disaggregated fibre preparation preserves pri-mary and secondary myotubes in the same proportionsas in the muscle, then this experiment indicates that onE16, 86% of the differentiated myoblasts fused intosecondary myotubes, and not with primary myotubes.As secondary myotubes are just beginning to form atthis time, no model of random fusion with any availablemyotube could account for this observation.

Patterns of fusion of myoblasts with primary andsecondary myotubesIt has long been accepted that the 'primary generation'of myotubes (Tello, 1917) forms during a brief intervalof time (Kelly and Zacks, 1969, Ontell and Kozecka,1984; Ross et al. 1981a), while the appearance of thesecondary generation is protracted (Couteaux, 1941;Betz et al. 1980; Harris, 1981a). The classical descrip-tion of primary myotube formation (reviewed by Swat-land, 1984) is that myoblasts assemble in rows, and thenfuse more or less simultaneously to form the myotubes.Secondary myotubes, on the other hand, grow longi-tudinally along the surface of their parent primarymyotube (Duxson et al. 1989). Because myoblasts areuniformly distributed throughout the muscle (Duxson etal. 1989), this pattern of growth could be explained bythe extending tip of the myotube being particularly


Fig. 4. Young secondary myotubes are not stained by anti-slow MHC antibody. All illustrations are from disaggregates ofE18 rat diaphragm muscles following injection of BrdU or [3H]thymidine on E17. The anti-slow MHC MAb is revealed witha biotin-streptavidin-HRP sandwich technique, and anti-BrdU marked with a rhodamine-labelled second antibody.(A) Bright-field and fluorescence pair of views, showing a primary myotube marked by HRP (top) and secondary myotubescontaining nuclei marked by anti-BrdU (bottom). (B) More extended view of a secondary myotube, showing scatteredlabelled and unlabelled nuclei. (C, D and E) HRP-stained primary myotubes, and unstained secondary myotubes.(C) Autoradiograph viewed with Nomarski optics, (D) and (E) bright-field microscopy. Calibration bars, 20 j/m.


* • • • * • • * * ,

• * # •

Fig. 5. Random positions of insertion of new nuclei into secondary myotubes. (A) E16 (evening) mouse diaphragmsecondary myotubes following injection of BrdU 13 h earlier. Top: a single labelled nucleus (arrow) amongst unlabellednuclei. Bottom: scattered brightly and faintly labelled nuclei in a segment of a secondary myotube. (B) Secondary myotubesfrom an E18 mouse diaphragm, BrdU injected on E16 (-50h). There is a random scattering of brightly labelled, faintlylabelled and unlabelled nuclei. Anti-BrdU revealed with FITC-labelled second antibody. (C and D) Autoradiographs ofsecondary myotubes from E18 rat diaphragm muscles, [3H]thymidine injected on E17 (-24h). (C) Bright-field;(D) Nomarski optics. Calibration bars, 20/an.

receptive to receiving a fusing myoblast. This hypoth-esis predicts that birthdating label should mark nucleinear the ends of secondary myotubes, and a pulse oflabel should mark a group of adjacent nuclei.

At all ages studied, the opposite was true; labellednuclei appeared in secondary myotubes at random

points (Figs 5 ,7) . This was regardless of whether thesecondary myotubes were new-formed and small, orwhether they were nearing maturity. Fusion patternswere analyzed (Fig. 8) by measuring the probability of alabelled secondary myotube nucleus being next to 1, 2or 3 other labelled nuclei. The probabilities were those


1 •$•*

Fig. 6. Labelled nuclei in an autoradiograph of a primary myotube (stained with anti-slow MHC) from an E18 ratdiaphragm, labelled with [3H]thymidine on E17. This myotube segment contained 52 nuclei, of which 2 were labelled. Notealso the free nuclei released from cells damaged during the disaggregation procedure, adhering to the side of the myotube.Calibration bar, 20 jun.

expected by random chance. In new-formed primarymyotubes (labelled on E14), by contrast, a labellednucleus was more likely to be next to 2 or 3 otherlabelled nuclei than can be accounted for by chance,indicating that groups of myoblasts with similar birth-dates had fused together. Labelling on E15 produced amore nearly random pattern, indicating that primarymyotubes, once formed, grew by addition of furthermyoblasts at random points.

Labelling secondary myotube precursorsSecondary myotubes also were labelled in musclesdisaggregated from E16 (mice) or E17 (rats) onwards,following injection of BrdU at times that labelledprimary myotubes. The labelling was less intense thanin the primary myotubes, and than in many of themononucleate cells (Fig. 9). These results indicate thatthe precursors of myoblasts destined to fuse intosecondary myotubes also were mitotically active at thetime that myoblasts destined to fuse into primarymyotubes were undergoing their last S-phase, with thedifference that secondary myoblast precursors reen-tered the cell cycle and their label was diluted. Thebrightly labelled mononucleate cells might include dif-ferentiating fibroblasts, and satellite cell precursors thathad withdrawn from the cell cycle.

Discussion

Recent tissue culture studies of the origins of differentmuscle fibre types indicate that these result fromhomophilic fusion of distinct groups of myoblasts withina heterogeneous population of myogenic stem cells(reviewed by Stockdale and Miller, 1987). The birthdat-ing experiments in embryonic and foetal mice and rats,which we present here, confirm that at least twomyogenic cell populations can be distinguished in vivo.These include myoblasts that fuse with each other toform primary myotubes and myoblasts that fuse withsecondary but not primary myotubes during the pro-longed progressive phase of secondary myotubegrowth.

Does BrdU inhibit differentiation?Incorporation of BrdU into chick myoblasts in tissueculture early in S-phase has been shown to reduce their

capacity for fusion (Lough and Bischoff, 1976), so thatthey must undergo one further mitotic division beforethey become fusion-competent, indicating that caremust be taken in interpreting results obtained with thislabel. We made a direct comparison between foetusesinjected with [3H]thymidine and BrdU by injectingthose in one uterine horn with one reagent, and those inthe other horn with the other. Groups of foetusesinjected on E16 and examined on E17, that is during thepeak period of production of secondary myotubes indiaphragm muscle (Harris, 1981a), were identical inmean body weight (P>0.5). Preparations of disaggre-gated myotubes from each group of animals showed noqualitative differences. Both comprised a mixture ofprimary and secondary myotubes, and the incidence oflabelled nuclei in secondary myotubes was similar(Table 1). Our results are drawn from nuclei formed inmyoblasts which had differentiated and expressed theircapacity to fuse. Thus, even if the BrdU had slightlyreduced the numbers of fusion-competent myoblasts,this should not affect our conclusions on the question ofspecificity of fusion between myoblasts and primary orsecondary myotubes.

Each technique had its own advantages: speed ofanalysis in the case of BrdU, and suitability for quanti-tative analysis in the case of [3H]thymidine autoradi-ography where both labelled and unlabelled nucleicould be distinguished, and the intensity of labellingmeasured by grain counting.

Nuclear labellingThe major results in this study concern labelled myo-tube nuclei, observed 24h after pulse labelling. Afterfinishing DNA replication, a labelled myoblast musthave undergone mitosis, and one or both daughter cellsfused with a myotube. We observed that a minimumperiod of 7h was required before any labelled nucleiwere seen in myotubes; these early fusing cells presum-ably were labelled late in S-phase. Within 24 h oflabelling, few myoblasts would have had time to reenterthe mitotic cycle and replicate a second time beforefusion. In the case of myotubes left 48 h before analysis,it was possible for nuclear label to have been diluted by2 or possibly 3 divisions subsequent to the initiallabelling. This gives rise to the possibility of overesti-mating the number of myotube nuclei whose parents


Fig. 7. Schematic illustration of the pattern of labelling ofsecondary myotube nuclei. Nuclei labelled with[3H]thymidine are represented by filled circles.(A) Examples of E17 secondary myotube segments, labelledon E16. (B) Examples of E18 secondary myotube segments,labelled on E17.

were in S-phase at the time of injecting the label, bycounting nuclei from myoblast daughter cells thatreentered the mitotic cycle before finally fusing. As weare concerned with the relative numbers of labellednuclei entering primary and secondary myotubes, re-spectively, this possibility does not disturb the in-terpretation of our results, i.e. that separate popu-lations of myoblasts contributed nuclei to primary andsecondary myotubes. A further check, in myotubesexamined 3 days after labelling, was to locate densegrain clusters under dark-field illumination (to identifyheavily labelled nuclei) and then to identify the type ofmyotube with bright-field illumination; these nucleimust have entered the myotubes within 24 h of label-ling.

Formation of primary myotubesThere was a 2 day time window (E13-14, mice; E14-15,rats) during which injection of a pulse of birthdatinglabel marked a substantial proportion of nuclei in someprimary myotubes (Fig. 1, Table 1). At the same time,other primary myotubes might have no labelled nuclei,or might contain a discrete group of neighbouringnuclei all of which were labelled (Fig. 8). For severaldays after this time, injection of BrdU or [3H]thymidineresulted in few labelled nuclei appearing in primarymyotubes. As primary myotubes increased in bothlength and diameter during this time their growth mustprimarily have been due to synthesis rather than ac-cretion of new cytoplasm and nuclei by cell fusion.

In disaggregates of embryonic muscles, primary myo-tubes appeared as classic myotubes (Schwann, 1847;Couteaux, 1941) with nuclei lying within a tube ofcontractile filaments (Figs 1, 2, 4, 6). Very shortly aftertheir formation primary myotubes reacted strongly withthe anti-slow MHC MAbs NOQ7.5.4D andNOQ7.1.1A. A quantitative study of the formation ofthe rat IVth lumbrical muscle has previously shown thatprimary myotubes all appeared within 48 h, and couldpossibly have been generated within a 24 h interval(Ross etal. 1987a).

Initiation of secondary myotube formationOur previous work has shown that formation of second-ary myotubes is initiated only in the vicinity of anendplate, although there is no absolute requirement fora nerve terminal to be physicaUy present at the precisemoment when a secondary myotube forms (see Dis-cussion in Duxson et al. 1989).

The irreversible reduction in secondary myotubenumber that follows maternal undemutrition (Wilson etal. 1988) suggests there is a critical period in develop-ment, preceding the actual appearance of new second-ary myotubes, when their potential maximum numberis determined. We cannot say whether new myotubesare initiated from a special cell type present in develop-mentally controlled numbers, or whether an inductivesignal from nerve or extracellular matrix is required totrigger the rare myoblast-myoblast fusion event thatmarks formation of a new secondary myotube.

Secondary myotube growth differed from that of


80-

60-

%40-

20"

(0

11

14-15 14-16

15-16

Pi115-16

> l

16-17 17-18

Fig. 8. Grouping of labelled nuclei in primary and secondary myotubes from rat embryos and foetuses. Each histogramrepresents one experiment, and is labelled with the day of injection of label and the day of examination. The numbers oflabelled nuclei with 0, 1, 2 or 3 labelled nuclei adjacent to them were counted (dark bars), and the distributions comparedwith those expected by chance (slashed bars). Nuclei in primary myotubes labelled on E14 were more likely to be in groupsthan expected from random chance. Nuclei in primary myotubes labelled on E15, and in secondary myotubes labelled onE16 or E17 were distributed as expected by random chance, indicating that myoblasts fused randomly at any point along thesurface of the growing myotube.

primaries in relying on the progressive addition of newcytoplasm and nuclei as myoblasts fused laterally atrandom positions along the length of the myotube. Thesecondary myotubes also maintained a characteristi-cally smaller diameter until near the time of birth, evenafter they had elongated sufficiently to connect with themuscle tendons and split laterally from their primary.

Mechanisms of primary and secondary myotubeformationOur results indicate that primary and secondary myo-tubes differ not only in their times of development,their structural relations with one another, the presenceor absence of plasticity in cell numbers, and the types ofmuscle fibre into which they will differentiate in theadult (Rubinstein and Kelly, 1981; Harris et al. 1989)but also in the manner in which their precursor myo-blasts fuse together. Primary myotubes form in conse-quence of a well-synchronised mitosis and then fusionof their myoblasts. We have not studied this processstructurally, but there are many reports in the lightmicroscope literature (reviewed by Swatland, 1984) ofalignment of myoblasts in rows followed by their fusingtogether.

Secondary myotubes form initially as binucleate cells,and then progressively elongate and increase theirnumber of nuclei, over several days. Our birthdatingexperiments show that some nuclei in a secondarymyotube are labelled regardless of when the label isinjected, at least up until the time of birth in the rat.These nuclei are inserted at random points along thelength of the myotube, so that extension of the extremi-

ties of secondary myotubes is due to growth rather thanto an accretion of new myoblasts at their ends. Againthis contrasts with primary myotubes, which display aperiod of several days after their formation when theyreceive few new nuclei.

Specificity in myogenic cell fusionThere already exists much evidence for specificity in cellfusion within developing muscles. Primary myotubenumbers are tightly regulated (Harris, 1981a; Ross etal.1987b; Wilson et al. 1988), showing that fusion betweenmyoblasts is directed rather than random. The ac-companying ultrastructural study (Duxson et al. 1989)shows that secondary myoblasts do not fuse with eachother (except in a directed fashion in proximity toprimary myotube endplates), but only with existingsecondary myotubes. Furthermore, despite their inti-mate cell-cell contacts, primary and secondary myo-tubes do not fuse with each other. The current studyadds to this the observation that there is a period inmuscle development when myoblasts selectively fusewith secondary, and not primary myotubes.

The abrupt change in nuclear labelling patterns infoetal rat muscles between E15 and E16 can only beexplained by specificity in fusion. Myoblasts withinmuscle bundles frequently contact primary and second-ary myotubes simultaneously (Duxson et al. 1989) and,early in the course of secondary myotube formation,myoblasts away from the endplate region could onlyhave contacted the primary myotube. If fusion was notdirected, but a random process with committed myo-blasts fusing with any adjacent myotube, then the onset


Fig. 9. Secondary myotube nuclei faintly stained following injection of BrdU to mark primary myotube nuclei. Primary andsecondary myotubes from an E17 rat embryo diaphragm muscle following injection of BrdU on E14. (A) Primary myotubewith brightly marked nuclei. (B) Secondary myotube with faintly marked nuclei together with brightly stained nuclei in manymononucleate cells. (C) Primary (marked by sets of double asterisks) and secondary myotubes in the same field of view toillustrate contrast in nuclear marking. The secondary myotube nuclei, faintly labelled and unlabelled, are marked by arrows.Calibration bar, 20 /im.

of secondary myotube formation would be gradual,rather than the observed abrupt phenomenon. Also,the proportion of nuclei labelled in primary myotubesshould decline slowly with time, rather than the ob-served abrupt decline.

In a freeze-fracture study of myoblast fusion in tissueculture, Kalderon and Gilula (1979) showed that myo-blasts initially were metabolically coupled via gapjunctions but the incidence of coupling was reduced to<50 % at the time fusion began. Fusion was initiated bythe appearance of regions of membrane without intra-membranous particles, and no gap junctions were seenin the fusion regions. Their results suggest that there isan intercellular recognition event which precedesfusion, and that recognition and fusion may rely onseparate mechanisms.

Specificity in fusion of myogenic cells may most

simply be explained by assuming the existence onmyoblasts of fusion proteins analogous to those de-scribed for some forms of virus-cell fusion. A viralenvelope protein binds to specific receptors on its targetcell (Dalgleish et al. 1984) with subsequent exposure ofa hydrophobic fusion protein on the virus which con-tacts the host cell membrane and initiates virus-hostfusion. This model would require different types ofmyotube to express different fusion receptors, specificfor each myoblast binding protein type; the hydro-phobic fusion proteins themselves need not possessspecificity, but would be exposed only after the specificinteraction between myoblast agonist and myotubefusion receptor.

This work was supported by the New Zealand MedicalResearch Council, the Muscular Dystrophy Association of


America (grants to A.J.H. and F.R.), the Vernon WilleyFoundation, INSERM and CNRS. A.J.H. thanks ProfessorD. Paulin and the University Paris 7 for a visiting professor-ship and M. Fardeau for hospitality in his laboratory. Supportfor the preparation of monoclonal antibodies NOQ7.5.4Dand NOQ7.7.1A by Dr Fitzsimons came from the Postgradu-ate Committee in Medicine of the University of Sydney.

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(Accepted 31 August 1989)

Myonuclear birthdates distinguish the origins of primary...

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