Appendages, such as wings of a fly or limbs of a vertebrate,are excellent models to study the principles of patterning andmorphogenesis. In the adult these structures are used for avariety of behaviors, including locomotion. Although supportstructures of the adult vertebrate limb are generated within thelimb bud, its dynamic elements are derived from the somiticmesoderm and neural tube. Recent studies show that regionalpatterns set up in the mesenchyme-filled limb bud guidemuscle precursors and developing motor axons to their properlocation within the limb. Subsequent development of theneuromuscular system is regulated by cell surface interactionsbetween pre-specified muscle fibers and motor axons.
Addresses*Department of Neurobiology, Pharmacology and Physiology,University of Chicago, Chicago, Illinois 60637, USA†Gene Expression Laboratory, The Salk Institute for Biological Studies,La Jolla, California 92037, USACorrespondence: Kamal Sharma; e-mail: [email protected]
AbbreviationsPSA polysialic acid Shh sonic hedgehog
IntroductionIn the adult limb, properties of individual muscles arecomplemented by the distinct physiology of the motorneurons that innervate them. Limb movements resultfrom the coordinated activation of sets of musclesanchored to the skeleton so as to produce flexion or exten-sion at a joint. Given the functional interdependence ofneural, muscular and skeletal elements, it is likely that thedevelopment of these structures is tightly coordinated. Aswe learn more about the patterning of the limb bud anddevelopment of the motor neurons and muscles, there isincreasing evidence that cell–cell interactions play animportant role. The following discussion reviews recentstudies that have identified molecular mechanisms of cel-lular interactions in the development of the limbneuromuscular system.
Limb muscles and motor neurons that innervate them areclosely related to other muscles and motor neurons. Twotypes of muscles, epaxial and hypaxial, develop at most rostral–caudal levels, and sets of complementary motorneurons are generated within the spinal cord. At the limblevels some hypaxial muscle precursors migrate into thelimb bud, proliferate and generate limb muscles. Similarly,at the corresponding levels of the spinal cord, motor neurons that innervate limb muscles are generated in addition to the normal complement of epaxial and hypaxial motor neurons.
Development of the limb bud, muscles and motor neuronshas been reviewed extensively. Our aim in this review is toidentify a few, critical features of the neuromuscular sys-tem in the vertebrate limb and to discuss how thesefeatures are acquired during embryonic development.
Limb motor neuronsMotor neurons develop in the ventral neural tube. In theadult spinal cord, motor neurons that innervate muscles ofsimilar embryonic origin are arranged in columns. Withineach column, motor neurons that innervate one muscle areaggregated together as a motor pool. Motor neurons thatinnervate epaxial and hypaxial muscles are generated inthe spinal cord at most axial levels. At the limb levels,motor neurons that innervate dorsal and ventral limb mus-cles are arranged in two additional columns (Figures 1a,b).Typically, the position of motor neurons within thesecolumns corresponds to the location of muscles that theiraxons innervate. For example, anterior and proximal limbmuscles are innervated by motor neurons in the anteriorand medial part of the corresponding limb motor column.This precise topography may develop through the genera-tion of different motor neuron subtypes during embryonicdevelopment. The types of motor neurons that will differ-entiate at any spinal cord level are determined before anyovert signs of motor neuron differentiation are evident[1,2]. A number of cell-extrinsic and -intrinsic signals havebeen identified that act sequentially [3] to determine cellfate in the motor neuron lineage.
Induction of motor neurons requires sonic hedgehog (Shh) tobe secreted from the underlying notochord and floor plate.Shh diffuses from the source and acts as a ventralizing mor-phogen to set up genetically distinct domains of neuralprecursors along the dorsal–ventral axis in a concentration-dependent manner [4,5]. Shh-mediated signaling is requiredfor the generation of all motor neuron subtypes [6–8]. Motorneurons at the thoracic and limb levels are generated fromprecursors that express the paired-domain homeodomaintranscription factor Pax6 ([9]; Figure 1c). Postmitotic motorneurons at these levels express the LIM (Lin II, Islet 1, Mec3)homeodomain factors Isl1/2 and the homeodomain factorHB9/mnr1/2 ([10,11,12••,13,14]; Figure 1c). In addition,motor neurons that will innervate epaxial, hypaxial and limbmuscles express unique combinations of LIM-homeo-domain factors ([10,12••]; Figure 1c). These transcriptionfactors regulate translocation of motor neuron cell bodies tospecific motor columns and target their axons to appropriatemuscle groups [12••,15•,16••]. Expression of LIM-home-odomain genes in motor neurons is very dynamic. Initially allcell types express Lhx3/4 [12••]. Early function of these twogenes is necessary for the development of epaxial, hypaxialand limb motor neuron subtypes [12••]. Soon after exitingthe cell cycle, as motor neurons begin to express Isl1, Lhx3/4
Development of the limb neuromuscular systemKamal Sharma* and Juan Carlos Izpisúa Belmonte†
Development of the limb neuromuscular system Sharma and Belmonte 205
expression is turned off in the hypaxial and limb motor neu-rons. Epaxial motor neurons continue to express Lhx3/4 astheir axons innervate the appropriate muscle targets
(Figure 1c). Dynamic regulation of Lhx3/4 expressionappears to be sufficient for the divergence of epaxial versushypaxial/limb motor neuron lineage [15•].
Figure 1
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Motor neuron and muscle lineage in vertebrates. (a) A schematicrepresentation of motor neuron subtypes and muscles that develop atthe limb and non-limb (thoraic) levels of the trunk. Note that epaxial(blue) and hypaxial (green) motor neurons and muscles are generatedthroughout the trunk region. At the limb levels two additional motorneuron subtypes are generated (red and yellow) to innervate the dorsal(red) and ventral (yellow) limb muscles. (b) Motor neurons (red) thatinnervate epaxial (blue), dorsal (red) and ventral (yellow) limb and bodywall muscles (green) are organized in discrete columns within thespinal cord. (c) Neuronal precursors (gray) are specified to a motorneuron fate through the action of Shh that induces Pax6 expression ina dividing precursor population. Generation of different motor neuron
subtypes (see [b] for the color code) requires a combination ofdifferent transcription factors. Development of the dorsal limb motorneurons (red) requires a retinoid (RA) signal. Note that RALDH2 isexpressed in neurons that appear to be the ventral limb motor neurons(yellow). (d) Pax3 is expressed in the epaxial as well as hypaxial muscleprecursors. Epaxial muscles are generated from precursors in themedial part of the dermomyotome that express low levels of Pax3 (lightgray), whereas hypaxial muscles (green) are generated from precursorslocated at the lateral lip of the dermomyotome (dark gray) that expressc-met and higher levels of Pax3. A subset of these lateral precursorsexpresses Lbx1 and migrates into the developing limb bud to form thedorsal (red) and the ventral (yellow) limb muscles.
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Like hypaxial motor neurons, limb motor neurons expressIsl1/2 and HB9. At limb levels, a subset of hypaxial motorneurons expresses retinaldehyde dehydrogenase 2(RALDH2, an enzyme in the retinoic acid synthesis path-way). These early generated cells might represent motorneurons that will innervate ventral limb muscles(Figure 1c). Expression of RALDH2 results in the genera-tion of an active zone for retinoid signaling in the ventralspinal cord [17–22]. Retinoid signaling through nuclearhormone receptors activates proliferation [23•] of motorneuron precursors, perhaps resulting in increased numbersof motor neurons at limb levels. Moreover, retinoid signal-ing through the RXR receptors activates expression ofLim1 in the later born subset of motor neurons that inner-vates dorsal limb muscles ([23•]; Figure 1c). Thus, thelimb motor neurons develop as a specialized derivative ofhypaxial motor neurons just as limb muscles are derivedfrom hypaxial muscle precursors (see below).
Generation of limb musclesDevelopment of skeletal muscles begins in the somiticmesoderm. As is the case with neural tube, dorsal–ventralpatterning of the somitic mesoderm requires morphogenssecreted by the ventrally located notochord and the dor-sal ectoderm (see [24,25] for review). These morphogens(for example, Shh and Wnt1) specify the ventral somiticcells to a sclerotome fate and the dorsal cells to a dermomyotome fate.
Muscle precursors are generated throughout the medial–lateral extent of the dermomyotome. Cells in the dermomy-otome express Pax3, a paired domain transcription factor([26]; Figure 1d). Epaxial muscles are generated from Pax3-positive precursors located in the medial dermomyotome,whereas hypaxial muscle precursors are present in the later-al dermomyotome [27–29]. In response to inductive signalsfrom the nearby neural tube and ectoderm, the medial pre-cursors activate myf5 expression and differentiate in situ [30].The hypaxial precursors are farther from the neural tube andexpress c-met (Figure 1d), a cell surface receptor that isrequired for the migration of muscle precursors into the limb[31,32]. Despite the expression of c-met in all hypaxial mus-cle precursors, the function of c-met and its ligand (SF/HGF)is necessary only for the development of limb muscles[31,32]. Hypaxial muscles of the body wall develop normallyin the absence of c-met function; however, limb muscles arenot generated, as in the SF/HGF knockout [32]. Theseobservations suggest that limb precursors are different fromhypaxial precursors. A subpopulation of lateral precursors canselectively respond to signals from the limb level lateral platemesoderm that induces these precursors to migrate into thelimb. Consistent with these findings, Lbx1 is expressed inthe lateral dermomyotomal lip at limb levels ([33];Figure 1d). In Lbx1-deficient mice most limb muscle pre-cursors fail to migrate into the limb, and limb muscledevelopment is compromised [34]. Together, these observa-tions suggest that limb muscle precursors are specializedderivatives of hypaxial muscle precursors.
Muscles in the limb have distinct morphological and phys-iological properties, and a few studies have beenconducted to ask whether muscle precursors that migrateinto the limb are already specified to generate specifictypes of muscles. In the chick, early migrating limb muscleprecursors are biased towards a slow phenotype, and thelate migrating precursors form predominately fast musclefibers ([35] and see below).
Building a functional limbMuscles and their motor innervation determine kineticproperties of the limb. Structural elements (for example,bones and tendons) provide the static infrastructure rela-tive to which muscles contract to generate movement.Building a functional limb requires generation of thesekinetic and static elements from motor neurons in thespinal cord, muscle precursors in the somitic mesodermand mesenchyme in the limb bud. Development of physi-ologically specific and structurally appropriate motor andmuscle components of the limb proceeds in a well definedtemporal sequence. Each step in this sequence establishescritical functional characteristics of the neuromuscular sys-tem within the limb. The discussion below is focused onthree characteristics; first, coordination of extensor andflexor muscle function; second, motor control of individualmuscles; and third, matching of muscle fiber physiologywith motor innervation.
Coordination of extensor and flexor muscle functionRhythmic movement across a joint requires alternate con-traction of the flexor and extensor muscles. Muscles thatfunction as flexors and extensors develop at stereotypiclocations within the limb and anchor onto specific bonesthrough tendons [36,37]. Motor neurons that innervatethese muscles have distinct electrical properties and arewired to generate alternating motor outputs [38]. For theflexor and extensor muscles to produce appropriate bend-ing at a joint, coordinated development of theneuromuscular system and mesenchyme-derived bonesand tendons is necessary.
Specification of motor neurons and muscle precursorstakes place in the neural tube and somitic mesoderm,respectively. Immediately following these events, motoraxons and muscle precursors invade the limb bud. Motoraxons choose a dorsal or ventral nerve trunk (for example,[16••]) and muscle precursors aggregate into dorsal andventral muscle masses (for example, [37]). This simplebinary decision sets the stage for development of an impor-tant functional specificity of the limb neuromuscularsystem. In the thigh, shank and ankle, extensor musclesare primarily derived from the dorsal muscle masses,whereas flexor muscles develop mostly from the ventralmuscle masses. Similarly, motor neurons that choose thedorsal and ventral nerve trunk are primarily extensors andflexors, respectively. This raises the questions of howthese decisions made — are motor neurons and muscleprecursors specified to a dorsal (extensor) versus ventral
Development of the limb neuromuscular system Sharma and Belmonte 207
(flexor) fate prior to arrival at the limb bud; and how do theextensor and flexor muscles and motor axons target to theappropriate dorsal and ventral location within the limbbud. On both counts, we understand more about howmotor axons make these binary decisions. Not surprisingly,motor axons actively interact with the environment in thelimb bud in order to target the appropriate location cor-rectly [16••]. Limb motor neurons, like limb muscleprecursors, express c-met, and SF/HGF promotes thegrowth of limb motor axons [39].
Before motor axons and muscle precursors arrive, mor-phogens secreted by three patterning centers have alreadydetermined a spatial map within the limb bud. A number ofstudies have implicated the ZPA (zone of polarizing activity),ectoderm and AER (apical ectodermal ridge) in determina-tion of the anterior–posterior, dorsal–ventral andproximal–distal axes of the limb bud, respectively. Along thedorsal–ventral axis mesenchymal cells show restricted geneexpression. Motor axons enter the limb at the boundarybetween the dorsal Lmx1b-positive and ventral Lmx1b-neg-ative mesenchymal cells at the base of the limb bud. Asdiscussed above, two molecularly distinct motor neuronsinnervate limb muscles. Axons from Lim1-positive andLim1-negative limb motor neurons are intermixed untilarrival at the base of the limb bud. Upon encountering thelimb mesenchyme, Lim1-positive motor neurons selectivelygrow into the dorsal (Lmx1B+) compartment (Figure 1b–d).These observations have led to the suggestions that expres-sion of Lim1 by the motor neurons and Lmx1B by themesenchymal cells dower motor axons with a mechanism fortargeting appropriate dorsal, flexor muscles [16••].
Motor control of individual musclesSoon after the aggregation of muscle precursors into ven-tral and dorsal masses, a series of morphogenetic eventsleads to the cleavage of each mass into three subsets thatwill generate the dorsal and ventral muscles of the thigh,shank and ankle [37]. These events are tightly coordinatedboth spatially and temporally with the formation of ten-dons from the limb mesenchyme [37]. Within each musclemass, groups of myotubes are generated in distinct orien-tations presaging the orientation of the differentiatedmuscles and thereby determining its action at a joint [37].Although these morphogentic events have been well doc-umented, we do not understand the molecular basis ofmuscle mass cleavage and how muscles are generatedwithin each compartment.
As muscle formation progresses, motor axons that haveselectively targeted the dorsal and the ventral musclemasses undergo a series of defasciculations leading to theformation of individual nerves for each muscle. As thenerve trunk grows to a specific region within the limb, a setof motor axons defasciculates from the other axons and tar-gets a specific region that will develop into one specificmuscle. How do motor axons make this decision? Itappears that genetic determination of the motor neuron
subtypes and epigentic mechanisms based on interactionsbetween motor axons and targets are required.
Motor neurons are specified to innervate particular mus-cles early in development [1,2]. Although we understandthe genetic mechanisms motor neurons use to target axonsto specific nerve trunks [12••,15•,16••], molecular differ-ences between pools of motor neurons that innervate onemuscle remain unidentified (see [40,41]). It is likely that
Figure 2
Specificity of the motor–muscle connections. (a) Motor neurons thatinnervate the same muscle are located in discrete groups termedmotor pools. Within a muscle, the site of termination of individual motoraxons corresponds to the relative position of the motor neuron cellbody within the motor pool. This specificity is controlled by gradedexpression of cell surface receptors and ligands present on the motoraxons and muscle fibers (see text for details). (b) Muscles with fastmyosin heavy chain (MyHC) are innervated by the fast motor neurons(Fmn) in the presence of high levels of polysialic acid (PSA). Nervebranching patterns of the fast motor neurons are strikingly differentfrom the branching pattern of the slow motor neurons (Smn).
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such differences exist. Subsets of motor pools selectivelyexpress transcription factors of the ETS class [40,41].Expression of these factors in select motor pools and cor-responding sensory neurons is dependent on interactionswith the peripheral muscle targets. As the search continuesfor factors that mediate motor pool to muscle matching, itis important to state that such factors are probably inducedbefore motor neurons contact their target and indepen-dently of contact with the muscle target.
Motor neurons within a motor pool innervate muscle fibersin a topographically organized fashion. Motor neurons in arostral position within a motor pool innervate rostral fibersin a muscle (Figure 2a). Two of the five known ephrin-Agenes (ephrin A1 and A5) are expressed at higher concen-tration in the rostral muscles compared to the caudal.Motor neurons express three Eph A receptors (EphA3, 4and 5). The growth of caudal motor neuron axons is moresensitive to inhibition by ephrin A5 than the rostral motoraxons. This differential expression of ephrins and Ephreceptors on the motor axons and the muscles is partlyresponsible for topographic specificity of the motor–muscle connections [42].
Matching of muscle fiber physiology withmotor innervationDuring normal neuromuscular function, muscles rarely actas a simple ON/OFF switch. Graded response of muscles tomotor commands is the key to smooth, elegant muscle func-tion. This property of the neuromuscular system isdependent on the development of specific characteristics inmuscle and motor components. First, muscle fibers are het-erogeneous and express different myosin subtypes. Musclesfibers that generate force during muscle contraction (extra-fusal fibers) are either ‘slow’ and express MyHC I(β) or ‘fast’and express MyHC IIa, IIb or IIx(d) [43]. Second, motorneurons that innervate slow muscles have different electri-cal and morphological properties from those innervating fastmuscles. Third, fewer slow fibers are innervated by thesame motor neuron compared with fast fibers.
Determination of the slow versus fast muscle type appearsto start at the muscle precursor stage. In the chick, earlymigrating muscle precursors are more likely to generateslow muscles, compared to the late migrating precursors[35]. However, at the epithelial somite stage, muscle typeis not yet specified. In the craniofacial skeletal muscles,muscle fiber type is determined by interactions betweenthe migrating muscle precursors and the neural-crest-derived connective tissue precursors [44,45].
As motor axons reach a differentiating muscle, a series ofbranches are formed at the axon terminal. Slow and fastprimary muscle regions have characteristic patterns ofnerve branching. In the slow muscles nerves run parallel tothe muscle fibers, axons extend throughout the muscleand the innervation of myofibers is made by axon collater-als. In the fast muscles, nerves run perpendicular to the
muscle fibers and axons show reductive branching ([46];Figure 2b). When motor neurons are forced to innervatenovel muscle targets, the fast and slow motor neuronsselectively innervate novel fast and slow muscle targets,suggesting that molecular differences exist betweenfast/slow muscles and motor neurons [46]. Either the sameor related molecular differences in the motor axons allowfast/slow motor axons to fasciculate with each other ontheir way to the appropriate muscle target [47]. Recently,Landmesser and colleagues [48••] have identified the mol-ecular nature of one such cue. In chick, the plantarismuscle and motor neurons that innervate it are of the slowvariety. In quail, this muscle and its motor neurons are fasttype. In chick–quail hindlimb chimeras, slow chick motorneurons make slow type motor projections onto the fastquail plantaris muscle. This pattern switches to the fasttype when polysialic acid (PSA) is removed enzymatically,suggesting that the slow pattern in this mismatch situationis dependent on PSA in the muscle and nerve (Figure 2b).However, removing PSA in the normal chick has no effecton the slow innervation pattern [48••]. Together, thesestudies suggest that the fast and slow motor neurons andmuscle fibers share cell surface properties that determinethe specificity of the neuromuscular system.
ConclusionsWe now understand some of the important steps in devel-opment of an integrated neuromuscular system in thevertebrate limbs. Transcription factors and signaling mole-cules that regulate successive steps in the specification ofindividual neuronal and muscle cell types have been iden-tified. How developing motor axons communicate withmuscles and mesenchyme derived connective tissue is notunderstood. Further studies are needed to identify cellsurface receptors and ligands that allow motor axons andmuscle precursors to read positional information within thelimb bud. Understanding how these receptor–ligand inter-actions regulate the neuron and muscle cell physiology willbe the next challenge for the molecular cell physiologists.
AcknowledgementsWe are thankful to Cynthia Lance-Jones and Michael O’Donovan fordiscussion and Manuel Utset for critical reading of the manuscript. KS issupported by startup funds from the University of Chicago and research inJCIB laboratory is supported by grant from the National Science Foundation.
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In the adult, fast and slow muscles of the limb are innervated by motorneurons with different electrical properties. In a series of three studies,Landmesser and colleagues have traced this motor–muscle specificityback to early embryonic development. They have used surgical [46], ret-rograde axon tracing [47] and biochemical manipulation [48•• ] tech-niques on early stage chicken embryos to determine that the motor axonsof the fast and slow varieties share molecular differences before makingcontact with the limb muscles. They show that PSA is one such molecule[48•• ]. Knowing that the fast and slow motor neuron axons express dif-ferent surface molecules at early stages of development should helpfocus molecular studies on a narrow window of time during embryogene-sis. For example, are these molecular differences direct targets of thetranscription factors that specify motor neuron identity, and what mecha-nisms restrict the expression of such molecules to either fast or slowmotor neurons?
