4019Development 125, 4019-4032 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV3876

Muscle and tendon morphogenesis in the avian hind limb

Gabrielle Kardon

DCMB Group, Duke University, LSRC Building, Research Drive Durham, NC 27708-1000, USAPresent address: Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USAe-mail: gkardon@rascal.med.harvard.edu

Accepted 13 July; published on WWW 14 September 1998

The proper development of the musculoskeletal system in thetetrapod limb requires the coordinated development of muscle,tendon and cartilage. This paper examines the morphogenesisof muscle and tendon in the developing avian hind limb. Basedon a developmental series of embryos labeled with myosin andtenascin antibodies in whole mount, an integrative descriptionof the temporal sequence and spatial pattern of muscle andtendon morphogenesis and their relationship to cartilagethroughout the chick hind limb is presented for the first time.Anatomically distinct muscles arise by the progressivesegregation of muscle: differentiated myotubes first appear asa pair of dorsal and ventral muscle masses; these massessubdivide into dorsal and ventral thigh, shank and foot musclemasses; and finally these six masses segregate into individualmuscles. From their initial appearance, most myotubes areprecisely oriented and their pattern presages the pattern offuture, individual muscles. Anatomically distinct tendonsemerge from three tendon primordia associated with the majorjoints of the limb. Contrary to previous reports, comparison ofmuscle and tendon reveals that much of their morphogenesis

is temporally and spatially closely associated. To test whetherreciprocal muscle-tendon interactions are necessary for correctmuscle-tendon patterning or whether morphogenesis of eachof these tissues is autonomous, two sets of experiments wereconducted: (1) tendon development was examined inmuscleless limbs produced by coelomic grafting of early limbbuds and (2) muscle development was analyzed in limbs wheretendon had been surgically altered. These experimentsdemonstrate that in the avian hind limb the initialmorphogenetic events, formation of tendon primordia andinitial differentiation of myogenic precursors, occurautonomously with respect to one another. However, latermorphogenetic events, such as subdivision of muscle massesand segregation of tendon primordia into individual tendons,do require to various degrees reciprocal interactions betweenmuscle and tendon. The dependence of these latermorphogenetic events on tissue interactions differs betweendifferent proximodistal regions of the limb.

Key words: muscle, tendon, limb, chick

SUMMARY

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INTRODUCTION

In the tetrapod limb the arrangement of over forty musclesa precise pattern of attachment via tendons to the limb skelprovides an extraordinary range of motor activity. The propdevelopment of this musculoskeletal system requires coupled development of three tissues: muscle, tendon cartilage. How are muscle, tendon and cartilage morphogencoordinated and a musculoskeletal system assembled fthese tissues during development of the avian limb? In paper I examine the morphogenesis of muscle and tendontheir relation to cartilage in the developing chick hind limb.

Embryological studies of chick indicate that the limmusculoskeletal system is derived from two mesodermal populations (Chevallier et al., 1977; Christ et al., 1977; Kieand Chevallier, 1979; Ordahl and Le Douarin, 1992); musis derived from the lateral dermomyotome of somites adjacto the limb bud, while tendon and cartilage are derived direcfrom the limb bud mesoderm . Beginning at st 17 (Hamburand Hamilton, 1951), somitic myogenic precursors migrainto the hind limb bud (Hayashi and Ozawa, 1991; Jacob et

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1979) and then aggregate and differentiate into dorsal anventral muscle masses (Hayashi and Ozawa, 1991). Withithese muscle masses, segregation of individual, anatomicaldistinct muscles in a stereotyped temporal sequence and spatpattern creates the basic muscle pattern (Pautou et al., 198Romer, 1927; Schroeter and Tosney, 1991; Wortham, 1948Concurrently, cartilage cell precursors condense from the limbud mesoderm and via a process of bifurcation, segmentatioand proliferation produce the limb skeleton (Shubin andAlberch, 1986). Morphogenesis of tendon is the leasunderstood aspect of musculoskeletal development. Fromstudies of the distal-most tendons, tendon appears to first arifrom the limb mesoderm as tendon primordia whichsubsequently divide into individual, anatomically distincttendons (Hurle et al., 1989, 1990; Ros et al., 1995).

The temporal and spatial relationship between muscletendon and cartilage morphogenesis during limb developmenis unclear. Although mature myotendinous junctions do nobegin to form until st 34 (Tidball, 1989), developing muscle andtendon may be closely associated as early as st 25-26. Studof normal muscle and tendon development in the chick wing

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and leg (Sullivan, 1962; Wortham, 1948) suggest that muscand tendons individuate in contact and in tandem. Howevother studies of mouse (Milaire, 1963) suggest that muscle tendon initially develop in spatial isolation. The temporal aspatial relationship between developing tendon and cartilhave only been studied in the foot (Hurle et al., 1989, 1990; Ret al., 1995). These investigations of distal tendon formatsuggest that tendons develop separately from their cartilorigin and insertion sites and only attach relatively late (afte30). Whether this is true for more proximal tendons is unknow

What mechanisms specify the pattern and coordinate morphogenesis of muscle and tendon? Quail-chick chimstudies have established that muscle pattern is autonomously pre-specified within the somitic myogenprecursor cells (Chevallier et al., 1977; Jacob and Christ, 19but see Lance-Jones and Van Swearingen, 1995). Instmuscle appears to be patterned by the somatopleural mesoof the limb bud (Jacob and Christ, 1980). Yet it is unclear wspecific components of the somatopleure, whether undifferentiated limb mesoderm, the developing tendons andthe developing skeleton are the source of patterninformation. The role of tendon in specification of muscpattern was generally rejected because muscle and tendon thought to develop in spatial isolation and only subsequenconnect (e.g. Hauschka, 1994). Conversely, the role of muin specification of tendon pattern seemed unlikely in light experiments showing that the distal tendons of the wing anddevelop in the absence of muscle (Brand et al., 1985; Kieny Chevallier, 1979; Shellswell and Wolpert, 1977). Theobservations have lead researchers to dismiss the importanmuscle-tendon interactions in muscle and tendon pattspecification. Instead, Shellswell and Wolpert (1977) suggesthat muscle and tendon develop completely autonomously frone another. They proposed that muscle and tendon patterare ultimately coordinated because of their mutual use o‘three-dimensional system of positional informationpresumably present within the undifferentiated limb mesode

To elucidate the mechanisms that pattern and coordinmuscle and tendon development in the limb, I have taken approaches. First, I describe the temporal sequence and sppattern of muscle and tendon morphogenesis and their relato cartilage in the developing chick hind limb. Unlike previoudescriptions which relied on histological sections, thdescription is based on specimens doubly-labeled wmolecular markers for differentiated myotubes and tendon aanalyzed in whole mount with confocal microscopy. With thtechnique, an integrative description of the morphogenesismuscles and tendons throughout the entire limb is presenfor the first time. The close temporal and spatial associationmuscle and tendon morphogenesis suggests that reciprinteractions may be important for specifying and coordinatimuscle-tendon pattern. Second, I test whether muscle-teninteractions are necessary for correct muscle-tendon patteror whether morphogenesis of each of these tissuesautonomous. Using experimental manipulations, I haexamined tendon development in the absence of muscle analyzed muscle development in limbs where tendon has bsurgically altered. These experiments show that in the avhind limb the initial morphogenetic events, initiadifferentation of myogenic precursors and formation of tendprimordia, occur autonomously with respect to one anoth

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However, later morphogenetic events do require, to variodegrees, reciprocal interactions between muscle and tendInterestingly, the dependence of these later morphogeneevents on tissue interactions differs between differeproximodistal regions of the limb.

MATERIALS AND METHODS

Description of normal muscle-tendon patternTo describe the normal development of muscle-tendon pattern, chick (Gallus gallus) embryos were incubated at 37°C for 5-10 dayand killed at stages 24-35 (Hamburger and Hamilton, 1951). Embrywere exsanguinated, eviscerated, and hind limbs separated fromrest of the embryo. The length of each fresh hind limb (from thposterior body-limb junction, parallel to the proximal-distal axis, tthe distal limb tip) was measured with an optical micrometer. Limbwere doubly stained in whole mount with the monoclonal antibodF59 to adult myosin fast heavy chain isoforms and the polyclonantibody HB1 to tenascin to follow muscle and tendon developmerespectively, and analyzed on a confocal microscope (see below).

Coelomic graft surgeryTo analyze tendon development in the absence of muscle, 29 earl16 hind limb buds (donor embryos had 28-30 somites) were isolafrom migratory myogenic cells by placing them into st 16 hoscoelomic cavities. Grafted limbs were harvested at 6-10 days of todevelopment (st 27-35), stained with the antibody F59 to confirm thmuscle cells had not invaded the limbs and the antibody HB1 to follotendon development, and analyzed on a confocal microscope. Tweseven of the 29 limbs were muscleless.

Tendon removal surgeryTo assess the effect of tendon primordia on the formation of musmasses, the dorsal proximal tendon primordium was removed froright hind limbs (fresh limb length 1.0-1.5 mm) of 16 st 24-25embryos. Because of the temporal and spatial stereotypy of tenddevelopment, tendon primordia could be located by comparison wmaps of normal tendon development. The vitelline membrane wremoved and a small incision in the chorion and amnion made abothe surgery site. The tendon primordium and overlying ectoderm weremoved using a combination of suction with a 400 µm (inner)diameter glass pipette and cutting with a fine tungsten needle. Limwere harvested 2-3 days after surgery (st 28-30) by which time tectoderm had generally healed over the surgery site. To check thattendon primordium was removed, the excised primordium was stainwith HB1 to confirm that it contained high levels of tenascin. Also ia small number of cases (5), limbs with excised tendon primordia westained with HB1 directly after surgery, to confirm that the tenascipositive primordia were removed.

Two sets of control experiments were also conducted. The effeof the surgical manipulation were assessed by sham removal of dorsal proximal tendon primordium. The tendon primordium wacompletely removed as described above and then carefully replaon 18 limbs. These limbs were harvested 2 days after surgery (st 29). The effects of the ectoderm above the tendon primordium weassessed by removing just the ectoderm above the dorsal proxitendon primordium. Ectoderm was removed by local application vmouth pipette of 2% trypsin in CMF tyrodes and gentle scraping wia fine tungsten needle on 12 limbs. The limbs were harvested 2 dlater (st 27-29) by which time the ectoderm had generally healed. embryos were stained with the antibodies F59 and HB1 in whomount and analyzed on a confocal microscope.

Immunocytochemistry of whole mount and sectionedembryosA modification of Klymkowsky and Hanken’s (1991) whole-moun

4021Muscle and tendon morphogenesis

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Thigh Muscles

Iliotibialis lateralis (IL)

Iliotibialis cranialis (IC)Ambiens (AMB)Femorotibialis internus (FTI)Femorotibialis externus (FTE)

Iliofibularis (IF)Iliotrochantericus cranialis (ITCR)Iliotrochantericus medius (ITCM)Iliotrochantericus caudalis (ITC)

Iliofemoralis internus (IFI)

Iliofemoralis externus (IFE)

Extensor digitorum longus (EDL)Tibialis cranialis (TC)Fibularis longus (FL)Fibularis brevis (FB)

Extensor hallucis longus (EHL)Abductor digiti 2 (AB2)Extensor proprius 3 (EP3)Extensor brevis digiti 4 (EB4)

Obturatorius (OBT)Puboischiofemoralis (PIF)Ischiofemoralis (ISF)

Flexor cruris lateralis (FCL)Flexor cruris medialis (FCM)

Caudofemoralispars caudalis (CFC)

DORSAL VENTRAL

Shank MusclesGastrocnemius intermedius (GM)Gastrocnemius internus (GI)Plantaris (P)Flexor digitorum longus (FDL)Flexor hallucis longus (FHL)Flexor perforatus 2 (FP2)Flexor perforatus 3 (FP3)Flexor perforatus 4 (FP4)Flexor perforans et perforatus 3 (FPP3)Flexor perforans et perforatus 2 (FPP2)Gastrocnemius externus (GE)

Foot MusclesFlexor hallucis brevis (FHB)Adductor digiti 2 (AD2)Abductor digiti 4 (AB4)

pars pelvica (FCLP)pars accessoria (FCLA)

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muscles of the chicken leg. Names according to the nomina anatomica3). (left) Lateral view of a galliform bird leg (Dendragapus obscurus),59).

immunocytochemical technique was used to stain muscle and tenin whole normal and manipulated chick hind limbs. Limbs were fixeovernight at 4°C with 4% paraformaldehyde and then bleachovernight with Dent’s bleach (50% methanol, 10% DMSO, 15H2O2). Specimens were washed for 3 hours in PBS, stained overnat room temperature with primary antibodies (in 5% serum, 20DMSO), washed for 5 hours in PBS, stained overnight at rootemperature with secondary antibodies (in 5% serum, 20% DMSwashed for 5 hours in PBS, dehydrated, and cleared in Murray’s c(33% benzyl alcohol, 66% benzyl benzoate).

Muscle was stained with the monoclonal antibody F59 to admyosin fast heavy chain isoforms (provided by FE Stockdale) whilabels virtually all embryonic primary myotubes (Miller et al., 1985Miller and Stockdale, 1986). All specimens (with the exception of 34-35 limbs) contain only differentiated primary myotubes (Fredeand Landmesser, 1991). Tendon primordia and differentiated tendwere stained with the polyclonal antibody HB1 to tenascin (providby H R Erickson). The monoclonal antibody M1B4 to tenascin (frothe Developmental Studies Hybridoma Bank) gave staining patteidentical to those of HB1.

Paraffin sectionsFor a finer level of analysis of normal muscle and tendodevelopment, four st 27-28 embryos were embedded in parafsectioned (either longitudinally along dorsal-ventral planes or aloanterior-posterior planes) at 10 µm intervals and stained with F59 andHB1 antibodies. The protocol was similar to the whole-mouprotocol except that the antibodies were incubated at 4oC, secondaryantibodies were incubated for only 2 hours, and PBS washes wreduced to 1 hour.

Analysis of muscle-tendon patternMuscle-tendon pattern in normal and manipulated chick limbs wanalyzed with a Zeiss laser scanning confocal microscope. Specimwere mounted in clearing agent and optically sectioned longitudinaalong dorsal-ventral planes at 20 µm intervals. Muscle-tendon patternwas examined on individual, digitallycaptured sections using a computerprogram for compositing stacks ofsequential sections (developed on theMacintosh computer by M R Johnston).Identification of 18 thigh muscles wasaided by descriptions by Romer (1927)and Schroeter and Tosney (1991) andidentification of 15 shank and 7 footmuscles was aided by descriptions ofWortham (1948) and Pautou and co-workers (Pautou et al., 1982). Eachmuscle was identified by itscharacteristic shape, position, myotubeorientation, origin and insertion.Identification of st 28-35 muscles wasconfirmed by microdissection of clearedspecimens on a polarized lightdissection microscope.

Anatomically distinct muscles werelabeled as either ‘identifiable’ or‘segregated’. Identifiable muscles arenot separate from neighboring muscles,but are distinguished by fibers whoseangles are distinct from anddiscontinuous with neighboring fibers.Segregated muscles are separated fromneighboring muscles by at least 10 µmand are physically separable bymicrodissection.

Tendons were identified by tenascin

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staining that was significantly greater than and visibly discontinuofrom neighboring tissue. Individual, anatomically distinct tendons weidentified by their characteristic shape, position and cartilagattachment. More mature tendons were also distinguishable in whmount by high concentrations of collagen (revealed by polycloncollagen I antibodies and birefringent banding) and in paraffin sectioby condensations of compact mesenchymal cells. Continuity tenascin-labeled tendon with muscle constituted muscle-tendon contand continuity of tenascin-labeled tendon with cartilage (identifiable whole mount by tenascin-labeled perichondrium) constituted musccartilage attachment.

TerminologyThe 40 chick thigh, shank and foot muscles with their tendonexamined are listed in Fig. 1. In the thigh, femorotibialis externuincludes both femorotibialis externus and medius, obturatoriuincludes both lateral and medial parts, iliotibialis lateralis includeboth pre- and post-acetabular parts, and puboischiofemoralis incluboth medial and lateral parts. In the shank, the small popliteus mushas not been included. Throughout the text, origin and insertiomuscle heads refer to the muscle regions attaching to origin ainsertion tendons. Origin tendons are always proximal, and insertitendons distal.

RESULTS

Normal muscle and tendon developmentTenascin identifies tendon primordia and individual,anatomically distinct tendonsTenascin labeled with the polyclonal HB1 antibody (or thmonoclonal M1B4 antibody) was found to identify both the earltendon primordia and individual, anatomically distinct tendonarising from these primordia (Figs 2 and 3). This agrees wiHurle and colleagues’ (Hurle et al., 1989, 1990; Ros et al., 199

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of muscle and tendon morphogenesis in the chick hind leg. Each panel isof a stack(s) of optical dorsal-ventral sections showing only the dorsallimb. Anterior is at the top and distal is at the right of each panel. Thet MHC is shown in green; the HB1 antibody to tenascin is shown in red.easured in fixed limbs (approximately 75% of fresh limb length). Scalescle abbreviations see Fig. 1. (A) St 24. Dorsal proximal tendone in the limb before the differentiation of limb myotubes. Axiale differentiated by this stage. (B) St 25. Myotubes of the thigh musclele just proximal to the tendon primordium. (C) Late st 25. Intermediate is now visible. The thigh muscle mass appears just proximal to themordium; the shank muscle mass appears between the proximal and primordia. (D) St 26-27. Within the shank muscle mass, the origin head

tendon is beginning to form (arrowhead). Within the still unsegregated the orientation of myotubes precedes and predicts later compartments into the IL, IF and FTE muscles. (E) St 28. Thigh and shank musclegated into anatomically distinct muscles in tandem and in contact to the individual tendons of origin and insertion. Arrowhead indicates originf TC. The foot muscle mass is now distinguishable. The dorsal distal is just barely visible, superficial to the metatarsals and distal to the footost of the dorsal thigh, shank and foot muscle masses have clearly

ividual muscles in contact with their origin and insertion tendons.

finding that tenascin is the first identifiable extracellular matcomponent of chick distal tendon blastemas and developtendons. Also, reports by others (Chiquet and Fambrough, 19Swadison and Mayne, 1989) show localization of tenascin in myotendinous junctions of adult chicken tendons.

Within the developing limb, ligaments, perichondria odeveloping cartilages and satellite cells accompanyingrowing nerves also contain tenascin(Chiquet and Fambrough, 1984; Martiniand Schachner, 1991; Wehrle andChiquet, 1990; Wehrle-Haller et al., 1991)and so may potentially obscureidentification of tendon. However,centrally located ligaments andperichondria are readily distinguishedfrom the generally peripherally locatedtendons. Tenascin-labeled nerves(particularly the fibularis and tibialisnerves) migrate adjacent to tendonprimordia (Martini and Schachner, 1991;Wehrle-Haller et al., 1991), but are easilydistinguishable from tendon in embryosyounger than st 28 by examining stainedspecimens under polarized light (thecollagen ensheathing nerves isbirefringent, while immature tendon isnot). In limbs older than st 28, mostnerves are no longer tenascin positive (inagreement with Wehrle-Haller et al.,1991) and so do not complicateidentification of tendon primordia andindividuated tendons.

Three pairs of tendon primordia formin association with the major joints ofthe hind limbBetween st 24 and 27, three pairs oftendon primordia appear in a generallyproximal to distal sequence in thedeveloping hind limb (Figs 2 and 3). Onepair, here termed the proximal tendonprimordia, appears dorsally and ventrallyat the thigh-shank junction (the futureknee). The second pair, here termed theintermediate tendon primordia, appearsdorsally and ventrally at the shank-footjunction (the future intertarsal joint). Thethird pair, here termed the distal tendonprimordia, appears dorsally andventrally at the distal end of the foot atthe future metatarsal-phalangeal andinterphalangeal joints. Proximal andintermediate tendon primordia appearearliest on the dorsal side of the limb (st24-25). On the ventral side, theintermediate tendon primordium isdistinct early (by st 25), being bothtenascin rich and morphologicallyidentifiable as a pronounced swelling atthe ventral base of the foot. The ventralproximal tendon primordium is not

Fig. 2. Dorsal view a dorsal projection side of a right hind F59 antibody to fasLimb lengths are mbar, 400 µm. For muprimordium is visiblmyotubes (left) havmass are now visibtendon primordiumproximal tendon priintermediate tendonof TC with its originthigh muscle mass,that will individuatemasses have segresegregation of theirhead and tendon otendon primordiummuscle mass. (F) Msegregated into ind

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The spatial relationship between the tendon primordia anthe underlying cartilage and superficial ectoderm differbetween the three pairs of primordia. Both the dorsal anventral proximal tendon primordia extend from the ectoderma

4023Muscle and tendon morphogenesis

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f muscle and tendon morphogenesis in the chick leg. (A-F) Projectionsf the limbs shown in Fig. 2A-F. Anterior is at the top and distal is at thehe F59 antibody to fast MHC is shown in green; the HB1 antibody toimb lengths are measured in fixed limbs. Scale bar, 400 µm. For muscle

ig. 1. (A) St 24. No ventral tendon primordia are visible. (B) St 25. The tendon primordium appears before the differentiation of limb myotubes.h muscle mass differentiation begins proximal to ventral intermediate(D) St 26-27. Ventral proximal and intermediate tendon primordiatact thigh and shank muscle masses. (E) St 28. OBT muscle has thigh muscle mass. Shank and foot muscle masses have formed in proximal and intermediate tendon primordia, but the distal tendont visible. (F) St 30. Most of the ventral thigh and shank muscle masses

o individual muscles in contact with their origin and insertion tendons.mass is still unsegregated.

basement membrane to the surfaces of the femur, tibia fibula (Fig. 4A). In contrast, the dorsal and ventral intermediaand distal tendon primordia lie subjacent to the ectodermbasement membrane, but do not extend to the underlycartilages (Fig. 4A).

The three pairs of tendon primordia also differ in fine scastructure. While tenascin appears to have an amorphdistribution in the proximal tendon primordia, tenascin in thintermediate and distal tendon primordia appears to be higorganized in a meshwork of radial and concentric fibers (Fig. 4

Thigh, shank and foot muscle masses differentiate inbetween the three pairs of tendon primordiaWhile muscle cell precursors populate thelimb as the early bud forms (Goulding etal., 1994; Williams and Ordahl, 1994),they do not begin to form myotubes (andexpress F59) until just after the proximal(on the dorsal side) or intermediate (onthe ventral side) tendon primordia canbe seen (Figs 2 and 3). Myotubesdifferentiate in a roughly proximal-distalprogression with differentiation of thighmyotubes beginning at early stage 25,shank myotubes at stage 25 and footmyotubes at st 26. The proximal-distalprogression of differentiation occurs atapproximately the same rate on the dorsaland ventral sides of the limb (dorsaldifferentiation slightly precedes ventral).In all three muscle-forming regions,muscle cells differentiate adjacent to thetenascin gridwork of tendon primordia(Fig. 4A,C).

Most strikingly, the muscle massesdifferentiate in a highly stereotypedspatial pattern with reference to thetendon primordia: thigh myotubesdifferentiate just proximal to the proximaltendon primordia, shank myotubes formin between the proximal and intermediatetendon primordia, and foot myotubesform in between the intermediate anddistal tendon primordia (Figs 2 and 3).This differentiation of myotubes withinthree particular limb regions bounded bytendon primordia sets up the fundamentaldivision of muscle into thigh, shank andfoot muscles. Although bounded bytendon primordia, the thigh, shank andfoot muscle masses are initiallysomewhat continuous. Gradually theseconnections are lost and most of themuscle masses are spatially isolated fromone another by st 30.

The tendon primordia not only initiallybound the three muscle masses, butsubsequently become the anatomicalpartners of the individuated thigh, shankand foot muscles. The proximal tendonprimordia will give rise to the insertion

Fig. 3. Ventral view oof the ventral side oleft of each panel. Ttenascin, in green. Labbreviations see Fventral intermediate(C) Late St 25. Thigtendon primordium. appear with and consegregated from thebetween the ventralprimordium is not yehave segregated intVentral foot muscle

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tendons of the thigh muscles and the origin tendons of the shanmuscles, the intermediate tendon primordia will form theinsertion tendons of the shank muscles and the origin tendonof the foot muscles, and the distal tendon primordia will formthe insertion tendons of the foot muscles and part of theinsertion tendons of shank muscles inserting into the phalange

Myotubes initially differentiate aligned within the limb inthe ‘correct’ orientation which presages the fiberdirection of future individuated musclesFrom their initial appearance, most myotubes are preciselyoriented within the limb (Fig. 4D), and their orientationcorrectly predicts the fiber orientation of the future individuated

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Fig. 4.Detailed views of muscle and tendon morphogenesis in thechick hind limb. (A,C-E) The F59 antibody to fast MHC is shown ingreen; the HB1 antibody to tenascin in red. (A) The spatialrelationship between the tendon primordia and the underlyingcartilage differs between the different tendon primordia.Longitudinal optical section of st 27 limb (dorsal at top, distal atright) shows that the proximal tendon primordium extends betweethe ectoderm and the tibia but intermediate tendon lies just subjacto the ectoderm. mt, metatarsal. (B) Ventral projection (anterior attop, distal at right) of ventral distal tendon primordium. Primordiumappears as a highly organized network of radial and concentric fibSt 29 limb stained in whole mount with HB1. mt, metatarsal.(C) Longitudinal paraffin section (dorsal at top, distal at right) of st27 limb showing differentiation of shank myotubes (green) adjacebut not within ventral intermediate tendon primordium (red).(D) Prior to the segregation of thigh and shank muscle masses,individual myotubes are aligned in a distinctive orientation whichpresages the fiber direction of future individuated muscles. Ventraoptical section (anterior at top, distal at right) of st 27 limb stainedwhole mount. (E,F) Individuated tendon of IC muscle is rich intenascin and consists of condensations of flattened mesenchymacells. Longitudinal paraffin section (anterior at top, distal at right) ost 28 limb viewed with fluorescence (E) and DIC (F). Scale bars(A,B,D) 200 µm, (C) 50 µm, (E,F) 25 µm.

muscles of which the myotubes will be a part. This precalignment of myotubes is particularly obvious in the thigwhere adjacent myotubes may be oriented at 60-90o angles withrespect to one another (Fig. 4D). The dramatic differencesmyotube orientation in the thigh reflect the later, nearly radarrangement of adult thigh muscles around the femur (Fig.As a consequence of the early (st 26) distinctive array myotubes in the thigh, many individual anatomically distin

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muscles are distinguishable long before the thigh muscle mabegins segregating at st 28-29 (Fig. 5). In the shank and fomost myotubes are aligned initially along the proximal-distaaxis, reflecting the generally longitudinal arrangement of adushank and foot (Fig. 1). As a result, individual muscles in thshank and foot are not as readily identifiable before segregatof shank and foot muscle masses.

Although most myotubes are ‘correctly’ oriented, there isome imprecision in the orientation of early myotubes. A smapercentage (less than 10%) of myotubes in st 26-29 limbs, aincorrectly oriented (Fig. 2E). By st 30 incorrectly orientedmyotubes are rarely found.

Individual muscles and their origin and insertion tendonsemerge in contact and in tandemBeginning at st 26, muscle masses start to segregate iindividual muscles adjacent to and in tandem with thsegregation from tendon primordia of their individual originand insertion tendons (Figs 2, 3, 5). The first indication omuscle mass segregation is the appearance of individumuscle origin and insertion heads (discrete regions from whimyotubes appear to radiate; Fig. 2D,E). All individual musclewere found to cleave from adjacent muscle beginning at tmuscle’s origin or insertion end, in a proximal to distal or distato proximal progression (in agreement with Schroeter anTosney, 1991). No muscles were found to cleave from thcenter of the muscle outwards towards the origin and insertiends (in contrast to Pautou et al., 1982).

Individual tendons are first distinguishable as discrete regioof tenascin staining adjacent to their forming partner muscorigin or insertion heads (Fig. 2D,E). In paraffin sections, thestenascin-positive tendons are shown to correspond to regionscondensations of mesenchymal cells (Fig. 4E,F). In the casethe distal tendon primordia, individual tendons do not emergdirectly from the primordia. Instead, these primordia subdividinto four tendinous blastemas associated with the four digits. later stages, these blastemas segregate into individual tend(Figs 2E,F, 3F and 6).

With their adjacent tendons, individual muscles separafrom muscle masses between st 28 and 35 in a stereotytemporal sequence and spatial pattern (summarized in Fig. The sequence and spatial pattern of splitting events agrees wprevious studies of the thigh (Schroeter and Tosney, 1991) ashank (Pautou et al., 1982). In general, segregation of musmasses proceeds in a proximal to distal progression, aindividuation of dorsal muscles in the thigh, shank and foooccurs before individuation of comparable ventral muscles. Aexamination of splitting events within the thigh, shank and foomuscle masses reveals no obvious overall organization to thsequence. However, the segregation of the ventral shamuscles inserting into the phalanges appears to be constraiby the future topography of their distal insertion tendons (Fi6C). In the adult, the tendons of flexor digitorum longus ardeep (adjacent to bone) and extend to the distal-mophalanges; the tendons of flexor perforatus et perforans 2 a3 are intermediate and extend to the penultimate phalangand the tendons of flexor perforatus 2, 3 and 4 are superficand extend to more proximal phalanges. During cleavage of tventral shank muscle mass, flexor digitorum longuindividuates first, succeeded by flexor perforans et perforat2 and 3 and finally followed by flexor perforatus 2, 3 and 4.

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4025Muscle and tendon morphogenesis

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Distal tendons inserting into the phalanges are derivedfrom multiple sourcesThe long distal tendons of the shank and foot muscles inserinto the phalanges are derived from multiple parts (Fig. 6). Tproximal parts of these tendons are derived from the intermedand/or distal tendon primordia. The distal parts are from doand ventral tissue extensions from the metatarsal/phalangeinterphalangeal joints. These dorsal and ventral extensionstenascin-positive and appear concurrently with the formation

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Fig. 5. Summary of muscle, tendonand cartilage assembly in the chickhind limb. In all stages, identifiable,but unsegregated muscles (lightgreen) are distinguished by fiberswhose angles are distinct from anddiscontinuous with neighboringfibers; segregated muscles (darkgreen) are separated fromneighboring muscles and arephysically separable bymicrodissection. The presence ofidentifiable, but unsegregatedmuscles indicates that a prepatternof anatomical muscles is present inmuscle masses before actualmuscle mass segregation. Ingeneral, segregated musclesseparate from their muscle massesin contact and in tandem with theirorigin and insertion tendons (pink).The timing of tendon attachment tocartilage (blue) relative to muscleindividuation varies in differentregions of the limb. Attachment oftendons to the phalanges is a lateevent. Limb lengths are measuredin fixed limbs (approximately 75%of fresh limb length).

tingheiate

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their associated joints and joint capsules (Fig. 6B). For fomuscles, the insertion tendons are derived from the distal tendprimordia and the dorsal or ventral extensions from the joints. Fshank muscles inserting into the phalanges (extensor digitorlongus, flexor digitorum longus, flexor hallucis longus, flexoperforatus et perforans 2 and 3 and flexor perforatus 2, 3 andthe insertion tendons are derived from the intermediate and distendon primordia as well as the dorsal or ventral extensions frothe joints (Fig. 6B). Initially, these three segments of each tend

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4026

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muscles inserting into the phalanges are derived from multiple, disparaten of shank and foot of st 32 limb. Anterior at bottom and distal at right of) The muscles for digits 2 and 3, FPP2 and FPP3, are stained with F59nd their insertion tendons with HB1 antibody to tenascin (red). B showshows tendon (tibial cartilage is also heavily labeled). Tendons of FPP2rived from three parts (labeled 1, 2, and 3). (1) Intermediate tendonrimordium and (3) ventral extension (barely visible) at the base of the

3. At this stage parts 1 and 2 of FPP2 are just beginning to connect justile parts 1 and 2 of FPP3 are not yet connected. (C) Ventral tendons of thege has been removed; modified after Hudson et al., 1959).

are separate from one another. Gradually, the tendon segmfrom the intermediate tendon primordia join with the segmefrom the distal primordia in the region of the tibial cartilage anthe segments from the distal primordia elongate and attach todorsal and ventral extensions.

Attachment of tendons to their cartilage origin andinsertion sites differs between thigh, shank and footmusclesThe last step in the assembly of muscle and tendon is attachment of tendons to their cartilage origin and insertisites during st 26 to 35 (Fig. 5). The timing and method tendon attachment to cartilage differs between the thigh, shand foot muscles. The proximal tendon primordia that give rto the insertion tendons of thigh muscle and origin tendonsshank muscles extends to the underlying femur, tibia and fibfrom the earliest stages that they can be visualized (Fig. 4During the process of tendon splitting and muscle cleavatendon contact with cartilages is maintained and refined iattachment sites spatially appropriate to their associamuscles.

Shank muscle insertiontendons and foot muscle originand insertion tendons are notassociated with cartilageinitially. The distal tendons ofshank muscles inserting intothe tarsometatarsus (tibialiscranialis, fibularis longus andbrevis, plantaris andgastrocnemial muscles), arederived from the intermediatetendon primordia and initiallylie subjacent to the ectodermalbasement membrane. Afterindividuation of their musclepartners, they extend deeply tothe appropriate cartilages. Forshank and foot musclesinserting into the phalanges,tendon attachment is delayed inassociation with the lateappearance of distal cartilages.Attachment of these insertiontendons to cartilage resultsfrom the connection of two orthree initially separate tendonsegments, the distal-most ofwhich is attached to cartilage(Fig. 6).

Tendon development inmuscleless limbsDuring normal development,three pairs of tendon primordiaform and subdivide in closeassociation with the developingthigh, shank and footmusculature. The myoblasts ordifferentiating myotubes of themuscle masses may be critical

Fig. 6. Distal tendons of shanksources. (A,B) Ventral projectioeach panel. Scale bar, 400 µm. (Aantibody to fast MHC (green) aonly HB1 staining and so just s(pink) and FPP3 (blue), are deprimordium, (2) Distal tendon psecond phalange of digits 2 ordistal to the tibial cartilage, whadult galliform foot (tibial cartila

entsntsd the

theonofankise ofulaA).ge,ntoted

for establishing the tendon primordia and/or directing thsegregation of tendon primordia into individual tendons. Ttest these hypotheses, tendon development was examinedmuscleless limbs. These limbs were produced by graftinyoung limb buds into the coelom before myoblasts hamigrated into them.

Tendon primordia form autonomously with respect tomuscle, but aspects of their subsequent morphogenesisrequire interactions with muscleIn the absence of muscle, the three dorsal and ventral pairstendon primordia appear autonomously in a normal temporsequence and spatial pattern (Fig. 7A). The tendon primordappear in a proximal to distal sequence in association with tappropriate joints. In addition, they maintain theircharacteristic spatial relationship with the underlying cartilagand superficial ectoderm: the proximal tendon primordiextend from the ectodermal basement membrane to tcartilages of the knee, while the intermediate and distprimordia lie subjacent to the ectodermal basememembrane. On a fine scale, the arrangement of tenascin i

4027Muscle and tendon morphogenesis

lyess

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Fig. 7. Dorsal (A,D,G) and ventral (J) views of tendon development in the absence of muscle in comparison with normal dorsal (B,C,E,F andH,I) and ventral (K,L) tendon and muscle development. Each panel is a projection of a stack(s) of optical sections showing dorsal or ventralviews of limb. Anterior is at the top of all panels while distal is at the right in A-I and at the left in J-L. (A,B,D,E,G,H,J,K) show only HB1antibody to tenascin. C,F,I and L show HB1 in red and F59 antibody to fast MHC in green. Scale bar = 500 µm. (A-C) Dorsal proximal (green),intermediate (pink), and distal (blue) tendon primordia form in a normal temporal sequence and spatial pattern in both limbs with (B,C) andwithout (A) muscles. (D-F) The dorsal proximal (green), intermediate (pink), and distal tendon (blue) primordia segregate into individualtendons at st 29-30 during normal development (E,F). In the absence of muscle, dorsal proximal and intermediate tendon primordia do notindividuate, but instead degenerate (D). Possible insertion tendon of EDL muscle has developed in the absence of muscle (pink arrowhead in D,compare with pink arrowhead in E). However, the distal tendon primordium begins the process of segregation as individual tendon blastemas(blue) arise in association with each digit. (G-L) Dorsal (G) and ventral (J) distal tendons in muscleless limbs individuate from distal tendonblastemas in a normal temporal sequence and spatial pattern as compared with limbs with muscle (H,I,K,L) However, the earliest formedtendon, a dorsal tendon associated with digit 4, has already begun to degenerate proximally in the absence of muscle (blue arrowhead in G,compare with blue arrowhead in H).

meshwork of radial and concentric fibers in the distal tendprimordia is maintained in the absence of muscle (data shown).

The proximal and intermediate tendon primordia do nsegregate into individual tendons, but instead degenerate witmuscle (Fig. 7D). In 4 st 30-31 limbs, one or two distinct regioof tenascin staining are visible, which may correspond to shinsertion tendons (Fig. 7D). However, these tendons are mless robust than their normal counterparts (Fig. 7E) and thidentity as anatomically distinct tendons is uncertain. Unlikenormal development, these tendons are never found at lstages. In general, these results indicate that both maintenance and subsequent segregation of the proximal intermediate tendon primordia require muscle.

In contrast, both the dorsal and ventral distal tendon primor

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subdivide autonomously into individual tendons in a generalnormal temporal sequence and spatial pattern in musclellimbs (Fig. 7D,G and J). The distal tendon primordia initiallyform superficial to all four metatarsals (Fig. 7A). From eacprimordium, four tendinous blastemas emerge superficial to eadigit (Fig. 7D). Subsequently, individual tendons emerge fromthe tendon blastemas and connect with tendinous extensiarising from the metatarsal-phalangeal and interphalangejoints (Fig. 7G,J). However, maintenance of distal tendons dorequire interactions with muscle because in the absence muscle these tendons gradually degenerate (Fig. 7G).

Muscle development in the absence of tendonprimordiaThe differentiation of myotubes within three particular region

4028

fal

sf

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G. Kardon

l view of muscle development in limbs with the dorsal proximal tendonemoved. (A-F) Projections of a stack(s) of optical sections through with HB1 antibody to tenascin in red and F59 to fast MHC in green. re experimental limbs; B, D, and F are their contralateral control limbs.at time of surgery. Removal of the proximal tendon primordium ismatically (tendon primordia are shown as open circles, myogenic grey) and on experimental limb (A) as compared with control limb (B).ys after surgery ectopic muscle (arrow) differentiates between the thighuscle masses, superficial to the knee. This ectopic muscle appears to be aion of FTE muscle. Note that tendons, such as the distal tendon of IFed from the dorsal proximal primordium reappear, either because therdium was not completely removed or because of subsequent regulationping limb. (E-F) Ectopic muscle (arrow) between the thigh and shankes persists 3 days after surgery. (G) Comparison of ectopic muscle andls in experimental limb shown in C. Upper panel shows F59 staining andB1 staining. Ectopic muscle appears in a region of low tenascin

le bar, 250 µm (for all images).

8

of the limb bounded by tendon primordia suggests that tenmay be critical for establishing the basic division of muscinto thigh, shank and foot musculature. To test this hypothethe dorsal proximal tendon primordium was surgicalremoved from young limb buds (Fig. 8A-B). During normadevelopment, the dorsal proximal tendon primordium definthe non-muscle region between the thigh and shank mumasses and later forms the insertion tendons (primarily patellar tendon) of the thigh muscles and the origin tendonthe shank muscles.

Tendon primordia restrict the location of muscledifferentiation within the limbTwo days after removal of the tendon primordium (st 28-2the ectoderm generally healed over the surgery site, but mof the underlying mesoderm stained weakly for tenascin compared with the normally high levels of tenascin found herThree days after surgery (st 30) tendons start to appear insurgery region, either because the tendon primordium wascompletely removed or because of subsequent regulation bydeveloping limb. One day after surgery (st 27),dorsal thigh and shank muscles are often (4 of 5limbs, not shown) truncated in the region adjacentto the surgery site, but by st 28 the number,arrangement and fiber orientation of thesemuscles is normal (16 of 16 limbs; Fig. 8C,E).However, in 9 of 11 st 28-29 limbs, myotubes arealso visible in the surgery region between thethigh and shank muscles and superficial to theknee (Fig. 8C as compared with D). No myotubesare normally found in this region. Similar ectopicmuscles are present in st 30 embryos (5 of 5limbs; Fig. 8E as compared with F). Closeexamination of the distribution of ectopicmyotubes and tenascin reveals that ectopic muscleappears in regions with low concentrations oftenascin (Fig. 8G). Ectopic myotubes were notoriented randomly, but instead appeared to beeither distal extensions of the thigh femorotibialisexternus muscle (Fig. 8C) or a proximal extensionof the shank muscle, tibialis cranialis (not shown).

To control for unintended effects due to surgeryand from removal of the ectoderm overlying thetendon primordium, two sets of controlexperiments were performed. Sham removals ofthe dorsal proximal tendon primordia did not altermuscle pattern (in particular, no ectopic musclesformed) in 8 out of 13 cases. In the remaining 5cases, some ectopic myotubes were found inbetween the thigh and shank muscle masses, butthese myotubes were generally found in regionswhere small amounts of mesoderm had beenremoved accidentally during surgery. Theseobservations suggest that the formation of ectopicmuscle is not due to wounding the ectoderm andmesoderm. Removal of only the ectodermoverlying the dorsal tendon primordium also doesnot alter muscle pattern (in 10 out of 11 cases),eliminating a possible inhibitory effect of ectodermon muscle differentiation. In both controlexperiments, tendon pattern is unaffected.

Fig. 8. Dorsaprimordium rlimbs labeledA, C and E a(A-B) Limbs shown diagraprecursors in(C-D) Two daand shank mdistal extensmuscle, derivtendon primoby the develomuscle masstenascin levelower panel Hstaining. Sca

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The appearance in experimental, but not in control, limbs oectopic muscle in the knee region suggests that during normdevelopment the tenascin-rich proximal tendon primordiumlocally excludes muscle differentiation.

DISCUSSION

Formation of anatomically distinct tendonsAnalysis of whole mount tenascin-stained limbs indicatethat tendon initially appears in the limb as three pairs oprimordia located dorsally and ventrally superficial to theknee, intertarsal and metatarsal/phalangeal aninterphalangeal joints. These primordia subdivide intoindividual tendons associated with each of the jointsproximal tendon primordia give rise to the insertion tendonof thigh muscles and the origin tendons of shank muscleintermediate primordia give rise to the insertion tendons oshank muscles and the origin tendons of foot muscles andistal primordia give rise to the insertion tendons of foo

4029Muscle and tendon morphogenesis

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mplified model of muscle, tendon and cartilage assembly in the ventrald limb. Muscle masses and tendon primordia form adjacent to onest 25-29) and segregate into individual muscles and tendons in contactndem (st 29-35). While proximal tendons attach to cartilage almostely (st 29), distal tendon attachment to cartilage is delayed (st 30-35). tendons of shank muscles inserting into the phalanges are derived fromrces: intermediate and distal tendon primordia and extensions from the

angeal joints (st 29-35). In the final panel, spheres represent the junctionnt tendon sources (in the embryo these junctions are not readily). Note that not all muscles are represented, the proximal tendonm appears relatively later during development of the ventral side, andlexity of tendon attachments to the phalanges is only present on thede.

muscles and some shank muscles. From the proximal intermediate tendon primordia, individual tendons appearemerge directly as tenascin-rich condensations mesenchymal cells. The distal tendon primordium initialappears as a wide swath (probably equivalent to mesenchyme lamina of Hurle et al., 1989, 1990; Ros et 1995) of tenascin meshwork superficial to all foumetatarsals. From this swath four tendinous blastemas found (in agreement with Hurle et al., 1989, 1990; Ros et 1995) to emerge superficial to each metatarsal, asubsequently from these blastemas individual tendons app

Most individual tendons arise from only one of the threpairs of tendon primordia. However, tendons of shank and fmuscles inserting into the phalanges are derived from twothree sources. For these shank muscles, insertion tendonderived from the intermediate and distal tendon primordia,well as from dorsal or ventral tendon extensionsfrom the cartilage insertion sites. These extensionshave also been noted by Hurle and co-workers(Hurle et al., 1989, 1990; Ros et al., 1995). Theseinsertion tendons are the only tendons in the limbthat cross multiple muscles and joints. For footmuscles, insertion tendons are derived from thedistal tendon primordium and the dorsal or ventralextensions. The relatively late connection betweenthese disparate segments is the last step in tendonmorphogenesis.

Formation of anatomically distinctmusclesExamination of limbs whole mount antibodystained for differentiated myotubes and tendonreveals two events in muscle morphogenesis notpreviously recognized from histological studies:(1) in association with tendon primordia, thesubdivision of dorsal and ventral muscle massesinto thigh, shank and foot muscle masses, and (2)the early alignment of myotubes within the musclemasses.

Muscle patterning begins with the migration andaggregation of myoblasts into the dorsal and ventralregions of the limb. This early division of limbmuscle into dorsal and ventral muscle has long beenrecognized (Romer, 1922). My study reveals that thedorsal and ventral muscle masses are furthersubdivided as myotubes differentiate. Thedifferentiation of myotubes in between the threedorsal-ventral pairs of tendon primordia results inthe formation of thigh, shank and foot musclemasses. This subdivision is a dynamic process; onboth dorsal and ventral sides of the limb musclemasses are initially connected, but gradually theseconnections are lost. Through this subdivisionprocess a dorsal extensor and ventral flexor musclegroup become associated with each of the majorsegments (stylopod, zeugopod, autopod) of the limb.

From their initial appearance within the thigh,shank and foot muscle masses, most myotubes arearranged in a highly structured array and their fiberorientation correctly predicts the fiber orientationof the future individuated muscles of which the

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myotubes will be a part. As a result, many individual muscleare distinguishable long before segregation of the musmasses. Previous studies of limb morphogenesis (Pautou et1982; Romer, 1927; Schroeter and Tosney, 1991; Wortha1948) have not reported this early distinctive array of fiborientations and assumed the muscle masses whomogeneous because fiber orientations were not readapparent in the transverse histological sections used. HoweMcClearn and Noden (1988) have also found that in thdeveloping quail head myotubes differentiate in a highordered array which presages the future fiber orientation anatomically distinct muscles.

The final event in the creation of individual, anatomicalldistinct muscles is the physical segregation of muscle massThe stereotyped temporal sequence and spatial patternsegregation of thigh, shank and foot muscle masses (outline

4030

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Fig. 10. Model of muscle-tendon interactions during muscle and tendon morphogenesis (dorsal view). Distribution of myogenic precursors isshown in light green, differentiated myotubes in dark green, and tendon primordia and individuated tendons in pink. (A) Tendon primordiaform autonomously. (B) Proximal, intermediate and distal tendon primordia establish (black arrows) the boundaries within which myotubesdifferentiate and thus subdivide the dorsal and ventral (not shown) aggregates of myogenic precursors into thigh, shank and foot masses ofmyotubes. The muscle masses, in turn, are necessary (white arrows) for maintenance of the proximal and intermediate tendon primordia.(C) Segregation of proximal and intermediate tendon primordia into individual tendons and their subsequent maintenance depends oninteractions (black arrows) with muscle. Distal tendon primordium segregates into individual tendons autonomously, but their subsequentmaintenance depends on interactions (white arrows) with muscle.

Fig. 5) agrees in general with the previous findings of Schroeand Tosney (1991) and Pautou and coworkers (1982).

Muscle, tendon and skeleton assembly in the avianhind limbThis study suggests that the coordination of muscle and tenmorphogenesis generally results from the close spatial temporal association of these tissues throughout thdevelopment (summarized in Fig. 9). Tendon initially appeain the limb as three dorsal and ventral pairs of tendprimordia. Nearly concurrently, muscle masses differentiatea highly stereotyped spatial pattern in between these tenprimordia. The result is a proximal-distal sequence alternating tendon primordia and muscle masses. From thmuscle masses and adjacent tendon primordia, anatomicdistinct muscles and tendons individuate in contact andtandem. After individuation of muscles and their tendonmature, functional myotendinous junctions begin to form at34 (Tidball, 1989).

For shank and foot muscles inserting into the phalanges,coordination of muscle and tendon morphogenesis complicated by the derivation of insertion tendons from boadjacent tendon primordia and distally remote sources. Thlong insertion tendons are derived from the intermediate anddistal tendon primordia as well as from dorsal or ventral tendextensions from the cartilage insertion sites. Developmenthese muscles with their correct distal tendons not only requthat muscle and adjacent tendon primordia segregcoordinately, but that proximal tendon segments connappropriately with distal tendon segments.

These observations of the spatial and temporal relationsbetween muscle and tendon morphogenesis help resolvcontroversy in the literature. Histological studies of normmuscle and tendon in the leg and wing, by Wortham (194and Sullivan (1962), strongly suggest that muscle and ditendons individuate in contact and in tandem. HowevMilaire’s (1963) study of muscle and tendon developmentthe mouse suggests that certain (distal) tendon blastedevelop in spatial isolation from their muscle bellies. My stufinds that the proximal segment of certain distal tendo(tendons of shank muscles inserting into the phalanges) ddevelop in contact and in tandem with their muscle belliwhile the distal segments develop in spatial isolation from th

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partner muscles and only subsequently connect with thmuscles via the proximal tendon segments.

In addition to coordination of muscle and tendonmorphogenesis, tendon and cartilage development must alsocoordinated so that tendons attach to the appropriate cartilaregions. I have found that the timing and mechanism of tendoattachment to cartilage differs between different regions of thlimb. Tendons derived from the proximal tendon primordiumattach to their cartilage origin and insertion sites nearlconcurrently with their formation. This is a consequence of thearly connection between the proximal tendon primordium anthe underlying cartilages. Subsequent attachment of individutendons to particular cartilage sites results from refinement of ttendon primordia connection to the cartilages. Tendons derivefrom the intermediate and distal tendon primordia lie initiallysubjacent to the ectoderm and only later attach (in many musclvia connection to distal tendon segments) to the appropriacartilage sites, many of which form much later in developmenThis relatively late cartilage attachment of more distal tendonhas also been observed by Wortham (1948), Sullivan (1962) aHurle and coworkers (Hurle et al., 1989, 1990). How tendonattach to the correct cartilage sites is unknown.

Specification of muscle and tendon pattern in thelimbBoth analysis of normal development and experimentamanipulations of muscle and tendon provide strong evidencthat in the avian hind limb some aspects of muscle and tendpatterning are autonomous with respect to one another, whother steps require reciprocal interactions between the tissu(summarized in Fig. 10).

Beginning at st 24, the tendon primordia appeaautonomously with respect to muscle in association with thmajor joints of the limb. Unlike previous investigations (Brandet al., 1985; Kieny and Chevallier, 1979), this study finds thain muscleless limbs the three dorsal and ventral pairs of tendprimordia develop in a normal proximal-distal temporalsequence in association with the major joints of the limb. Iaddition, the fine scale structure of the tendon primordia maintained in the absence of muscle.

After migration into the dorsal and ventral sides of the limbthe subdivision of myogenic precursors into thigh, shank anfoot masses of differentiated myotubes appears to be intimate

4031Muscle and tendon morphogenesis

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linked with the tendon primordia. During normal developmenmasses of myotubes differentiate in between and are bounby the three pairs of tendon primordia. The generally excluspattern of muscle and tendon suggests that the tendon primodefine regions in which myotubes do not differentiatExperimental manipulations of the tendon primordia provifurther support for the role of tendon in regulating the formatiof three pairs of muscle masses. Removal of the dorsal proxitendon primordium results in the formation of ectopic muscin between the thigh and shank muscle masses. Removal oftendon primordium presumably allows invasive myogenprecursors to migrate in and differentiate where they normawould not. This ectopic muscle forms despite the likely removof some resident myogenic precursors. Also, contexperiments suggest that the ectopic muscle is not stimulaby tissue wounding or ectodermal removal. Differentiation ectopic muscle in regions lacking tenascin-labeled tendprimordium suggests that during normal development tendprimordia may locally exclude myotube differentiation.

It is unclear whether exclusionary signals from the tendprimordia are solely responsible for the establishment proximal and distal muscle mass boundaries. Removal of dorsal proximal tendon primordium only partially disrupts thsegregation of the thigh and shank muscle masses. It is howpossible that the surgery may have only partially removed tendon primordium. Alternatively, the surgery may havcompletely removed the tendon primordium, but because otfactors also inhibit myotube differentiation in this region onlyrelatively small number of ectopic myotubes differentiate her

Within the thigh, shank and foot muscle masses, specificaof the pattern of individual, anatomically distinct muscleappears to occur early within the muscle masses and doesdepend on tendon. Previous researchers (e.g. Pautou et al., Schroeter and Tosney, 1991; Shellswell and Wolpert, 1977) hsuggested that muscle pattern is specified as the muscle mphysically segregate into individual muscles, becausegregation of apparently homogenous muscle masses wafirst manifestation of the pattern of individual muscles. Howevthe analysis of normal muscle development suggests individual muscles are specified earlier within the forminmuscle masses. Whole-mount analysis reveals that mumasses are not homogeneous; within the masses, most myoimmediately differentiate oriented in a highly structured arrwhich prefigures the array of future individuated muscles. Timplies that the patterning information necessary to specindividual muscles is present early in the somtopleumesoderm. The specific tissue or molecular identity of tpatterning information is unknown. Tendon appears unlikelyprovide the patterning information because tenascin labelingthe mesoderm reveals no obvious prepattern and individtendons do not emerge from the tendon primordia until after initial muscle pattern is established. Potentially, the acquisitof differential hox gene expression within the muscle masmay specify muscle pattern. Recently, Yamamoto and colleag(1998) have found that hoxa-11and hoxa-13are expressedwithin specific subregions of the wing muscle masses.

What governs the final segregation of muscle masses individual, anatomically distinct muscles is unknown, but tendmay play a role. Analysis of normal development shows thmuscle masses segregate into individual muscles in contactin tandem with the segregation from tendon primordia of th

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individual origin and insertion tendons. The autonomy withrespect to muscle of the segregation of the distal tendon primordinto individual tendons and the close contact of distal tendonwith their partner foot muscle indicates that distal tendons, iparticular, may guide the segregation of the foot muscle mass

The importance of muscle interactions for the segregation otendon primordia into individual tendons differs for differentproximodistal regions of the limb. After their formation, theproximal and intermediate tendon primordia, which normallydevelop in close contact with their partner muscles, requirinteractions with muscle for their maintenance and subsequesegregation into individual tendons. In the absence of musclthese tendon primordia do not segregate into individuatendons, but instead degenerate. In contrast, the distal tendprimordia, which develops in spatial isolation from some otheir partner muscles (the shank muscles), segregatautonomously into individual tendons, but requires interactionwith muscle for subsequent maintenance of these tendons.the absence of muscle, the distal tendon primordia segregatea normal temporal sequence and spatial pattern into individutendons, but these tendons subsequently degenerate. Thfindings are in agreement with the work of Kieny andChevallier (1979) on the wing.

Implications for the neomorphic origin of tetrapoddigitsFor the past century there has been considerable debate onevolutionary origin of digits (metacarpals/metatarsals anphalanges) in tetrapod paired appendages (reviewed by Shuet al., 1997). One group of researchers has argued that antecedents of digits are present in the fins of sarcopterygifish, the sister group of tetrapods (Gregory and Raven, 194Watson, 1913). In opposition to this hypothesis, others havargued that digits are unique (neomorphic) to tetrapod(Holmgren, 1939, 1933). In recent years, the bulk odevelopmental, genetic and paleontologic data supports thypothesis that digits are neomorphic structures (Ahlberg anMilner, 1994; Shubin and Alberch, 1986; Sordino et al., 1995)

The data presented here on muscle and tendon developmin the chick hind limb lend support to the hypothesis that digitare evolutionary novelties. Significant differences exist betweethe morphogenesis of tendons in the thigh and shank and thoin the foot. Unlike the more proximal tendons, the distal tendondevelop by a two step process; the distal tendon primordsegregate into four tendon blastemas associated with each of digits and these blastemas in turn subdivide into individuatendons. In addition, unlike the more proximal tendons, distatendons develop for the most part in spatial isolation from themuscle partners. Furthermore, analysis of muscleless limbreveals that while the segregation of the proximal anintermediate tendon primordia depends on interactions witmuscle, individuation of distal tendons is autonomous withrespect to muscle. Finally, distal tendons recently have beeshown to differ from more proximal tendons in their molecularidentity; distal tendons express the transcription factors six and six 2 (Oliver et al., 1995) and the eph-related receptotyrosine kinase cek-8(Patel et al., 1996) while proximal tendonsdo not. These four observations concur that the morphogeneprocesses governing tendon development are quite differentthe foot from the rest of the limb. Potentially, this differencereflects the novel evolutionary origin of tetrapod digits.

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I thank F. E. Stockdale and H. R. Erickson for antibodies and R. Johnston for help with graphics and designing the compuprogram (Gab-o-matic Pro) for analyzing sections. H. Crenshaw,Lance-Jones, D. R. McClay, K. K. Smith, S. A. Wainwright anmembers of the D. R. McClay lab provided much advice on tresearch and the manuscript. Support for this research was provby a Duke Dissertation Fellowship and Sigma Xi grant to G. Kardand an NIH grant HD 14483 to D. R. McClay.

REFERENCES

Ahlberg, P. E. and Milner, A. R. (1994). The origin and early diversificationof tetrapods.Nature368, 507-514.

Baumel, J. J., King, A. S., Breazile, J. E., Evans, H. E. and Vanden Berge,J. C. (1993). Handbook of Avian Anatomy: Nomina Anatomica Avium, 2ndEdition, 779pp. Nuttall Ornithological Club, Cambridge.

Brand, B., Christ, B. and Jacob, H. J.(1985). An experimental analysis ofthe developmental capabilities of distal parts of avian leg buds. Am. J. A173, 321-340.

Chevallier, A., Kieny, M. and Mauger, A. (1977). Limb-somite relationship:origin of the limb musculature. J. Embryol. Exp. Morphol. 41, 245-258.

Chiquet, M. and Fambrough, D. M. (1984). Chick myotendinous antigen.1.A monoclonal antibody as a marker for tendon and muscle morphogeneJ. Cell Biol. 98, 1926-1936.

Christ, B., Jacob, H. J. and Jacob, M.(1977). Experimental analysis of theorigin of the wing musculature in avian embryos. Anat. Embryol.150, 171-186.

Fredette, B. J. and Landmesser, L. T.(1991). Relationship of primary andsecondary myogenesis to fiber type development in embryonic chmuscle.Dev. Biol.143, 1-18.

Goulding, M., Lumsden, A. and Paquette, A. J.(1994). Regulation of Pax-3 expression in the dermomyotome and its role in muscle developmDevelopment 120, 957-971.

Gregory, W. K. and Raven, H. C.(1941). Studies on the origin and earlyevolution of paired fins and limbs. Ann. N. Y. Acad. Sci.42, 273-360.

Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in thedevelopment of the chick embryo. J. Morphol.88, 49-92.

Hauschka, S. D.(1994). The embryonic origin of muscle. In Myology(ed. A.G. Engel and C. Franzini-Armstrong), pp. 3-73. McGraw-Hill, New York

Hayashi, K. and Ozawa, E. (1991). Vital labeling of somite-derivedmyogenic cells in the chicken limb bud. Roux’s Arch. Dev. Biol.200, 188-192.

Holmgren, N. (1933). On the origin of the tetrapod limb. Acta Zool.24, 185-294.

Holmgren, N. (1939). Contribution to the question of the origin of the tetrapolimb. Acta Zool. 20, 89-124.

Hudson, G. E., Lanzillotti, P. J. and Edwards, G. D.(1959). Muscles of thepelvic limb in galliform birds. Am. Midland Nat. 61, 1-67.

Hurle, H. M., Hinchliffe, J. R., Ros, M. A., Critchlow, M. A. and Genis-Galvez, J. M. (1989). The extracellular matrix architecture relating tmyotendinous pattern formation in the distal part of the developing chlimb: an ultrastructural, histochemical and immunocytochemical analysCell Diff. Dev. 27, 103-120.

Hurle, J. M., Ros, M. A., Ganan, Y., Macias, D., Critchlow, M. andHinchliffe, J. R. (1990). Experimental analysis of the role of ECM in thpatterning of the distal tendons of the developing limb bud. Cell Diff. Dev.30, 97-108.

Jacob, H. J. and Christ, B.(1980). On the formation of muscular pattern inthe chick limb. In Teratology of the Limbs(ed. H. J. Merker, H. Nau and D.Neubert), pp. 89-97. Walter de Gruyter and Co., Berlin.

Jacob, M., Christ, B. and Jacob, H. J.(1979). The migration of myogeniccells from the somites into the leg region of avian embryos. Anat. Embry157, 291-309.

Kieny, M. and Chevallier, A. (1979). Autonomy of tendon development inthe embryonic chick wing. J. Embryol. Exp. Morphol. 49, 153-165.

Klymkowsky, M. W. and Hanken, J. (1991). Whole-mount staining ofXenopusand other vertebrates. In Xenopus laevis: Practical Uses in Celland Molecular Biology(ed. B. K. Kay and H. B. Peng), pp. 413-435Academic Press, San Diego.

Lance-Jones, C. and Van Swearingen, J.(1995). Myoblasts migrating into

M.ter C.dheidedon

nat.

sis.

ick

ent.

.

d

oickis.

e

ol.

.

the limb bud at different developmental times adopt different fates in thembryonic chick hindlimb. Dev. Biol.170, 321-337.

Martini, R. and Schachner, M. (1991). Complex expression pattern oftenascin during innervation of the posterior limb buds of the developinchicken. J. Neurosci. Res.28(2), 261-279.

McClearn, D. and Noden, D. M. (1988). Ontogeny of architecturalcomplexity in embryonic quail visceral arch muscles. Am. J. Anat.183, 277-293.

Milaire, J. (1963). Etude morphologique et cytochimique du developpemedes membres chez la souris et chez la taupe. Arch. Biol. (Liege)74, 129-317.

Miller, J. B., Crow, M. T. and Stockdale, F. E.(1985). Slow and fast myosinheavy chain content defines three types of myotubes in early muscle cultures.J. Cell Biol.101, 1643-1650.

Miller, J. B. and Stockdale, F. E.(1986). Developmental regulation of themultiple myogenic cell lineages of the avian embryo. J. Cell Biol.103, 2197-2208.

Oliver, G., Wehr, R., Jenkins, N. A., Copeland, N. G., Cheyette, B. N. R.,Hartenstein, V., Zipursky, S. L. and Gruss, P.(1995). Homeobox genesand connective tissue patterning. Development 121, 693-705.

Ordahl, C. P. and Le Douarin, N. M.(1992). Two myogenic lineages withinthe developing somite.Development 114, 339-353.

Patel, K., Nittenberg, R., D’Souza, D. D., Irving, C., Burt, D., Wilkinson,D. G. and Tickle, C.(1996). Expression and regulation of Cek-8, a cell tocell signaling receptor in developing chick limbs. Development122, 1147-1155.

Pautou, M.-P., Hedayat, I. and Kieny, M.(1982). The pattern of muscledevelopment in the chick leg. Arch. Anat. Microsc. Morph. Exp. 71, 193-206.

Romer, A. S. (1922). The locomotor apparatus of certain primitive andmammal-like reptiles. Bull. Am. Mus. Nat. Hist.46, 517-606.

Romer, A. S.(1927). The development of the thigh musculature of the chicJ. Morph. Phys.43, 347-385.

Ros, M. A., Rivero, F. B., Hinchliffe, J. R. and Hurle, J. M. (1995).Immunological and ultrastructural study of the developing tendons of thavian foot. Anat. Embryol. 192, 483-496.

Schroeter, S. and Tosney, K. W.(1991). Spatial and temporal patterns ofmuscle cleavage in the chick thigh and their value as criteria for homologAm. J. Anat. 191, 325-350.

Shellswell, G. B. and Wolpert, L.(1977). The pattern of muscle and tendondevelopment in the chick wing. In Vertebrate Limb and SomiteMorphogenesis(ed. D. A. Ede, J. R. Hinchliffe and M. Balls), pp. 71-86.Cambridge University Press, Cambridge.

Shubin, N., Tabin, C. and Carroll, S.(1997). Fossils, genes and the evolutionof animal limbs. Nature 388, 639-648.

Shubin, N. H. and Alberch, P.(1986). A morphogenetic approach to theorigin and basic organization of the tetrapod limb. Evol. Biol.26, 319-387.

Sordino, P., van der Hoeven, F. and Duboule, D.(1995). Hox geneexpression in teleost fins and the origin of vertebrate digits. Nature 375, 678-681.

Sullivan, G. E. (1962). Anatomy and embryology of the wing musculature othe domestic fowl (Gallus). Aust. J. Zool. 10, 458-518.

Swadison, S. and Mayne, R.(1989). Location of the integrin complex andextracellular matrix molecules at the chicken myotendinous junction. CeTiss. Res. 257, 537-543.

Tidball, J. G. (1989). Structural changes at the myogenic cell surface durinthe formation of myotendinous junctions. Cell Tiss. Res.257, 77-84.

Watson, D. M. S.(1913). On the primitive tetrapod limb. Anatomica Anzeiger44, 24-27.

Wehrle, B. and Chiquet, M. (1990). Tenascin is accumulated alongdeveloping peripheral nerves and allows neurite outgrowth in vitroDevelopment 110, 401-415.

Wehrle-Haller, B., Koch, M., Baumgartner, S., Spring, J. and Chiquet, M.(1991). Nerve-dependant tenascin expression in the developing chick libud. Development 112, 627-637.

Williams, B. A. and Ordahl, C. P. (1994). Pax-3 expression in segmentalmesoderm marks early stages in myogenic cell specification. Development120, 785-796.

Wortham, R. A. (1948). The development of the muscles and tendons in tlower leg and foot of chick embryos. J. Morphol.83, 105-148.

Yamamoto, M., Gotoh, Y., Tamura, K., Tanaka, M., Kawakami, A., Ide,H. and Kuroiwa, A. (1998). Coordinated expression of Hoxa-11and Hoxa-13 during limb muscle patterning. Development125, 1325-1335.