Specific guidance of motor axons to duplicated muscles in the developing amniote limb

The effect of alteration of limb pattern upon motor axon guidance has been investigated in chick embryos. Following grafting of the zone of polarizing activity (ZPA) into the anterior margin of the early limb bud, limbs develop with forearms duplicated about the anteroposterior axis. The position of motoneurones innervating the duplicated posterior forearm extensor EMU was mapped by retrograde transport of horse radish peroxidase (HRP). The motor pool labelled from injection into the anteriorly duplicated EMU muscle is consistently similar to that supplying the posterior EMU muscle on the unoperated side of the embryo. In those cases where the axons are well filled, their trajectories from the injection site are observed to change position within the radial nerve to specifically innervate the duplicated muscle. The axons modify their trajectories proximal to the level of limb duplication in a region where there is no change in the pattern of overt differentiation of the limb cells. This suggests that axons may use a cell's positional value to navigate and provides significant support for the theory of positional information.     

Muscle founder cells regulate defasciculation and targeting of motor axons in the Drosophila embryo.

During Drosophila embryogenesis, motor axons leave the central nervous system (CNS) as two separate bundles, the segmental nerve (SN) and intersegmental nerve (ISN). From these, axons separate (defasciculate) progressively in a characteristic pattern, initially as nerve branches and then as individual axons, to innervate target muscles [1] [2]. This pattern of branching resembles the outgrowth and defasciculation of motor axons from the neural tube of vertebrate embryos. The factors that trigger nerve branching are unknown. In vertebrate limbs, the branched innervation may depend on mesodermal cues, in particular on the connective tissues that organise the muscle pattern [3]. In Drosophila, the muscle pattern is organised by specific mesodermal cells, the founder myoblasts, which initiate the development of individual muscles [4][5][6]. Founder myoblasts fuse with neighbouring non-founder myoblasts and entrain these to a specific muscle programme, which also determines their innervation [4] [7]. In the absence of mesoderm, ISN and SN can form, but motor axons fail to defasciculate from these bundles [7]. The cue(s) for nerve branching therefore lie within the mesoderm, most likely in the muscles and/or in the precursor cells of the adult musculature [8]. Here, we show that founder myoblasts are the source of the cue(s) that are required to trigger defasciculation and targeted growth of motor axons. Moreover, we found that a single founder myoblast can trigger the defasciculation of an entire nerve branch. This suggests that the muscle field is structured into sets of muscles, each expressing a common defasciculation cue for a particular nerve branch.     

The projection patterns of the ventral horn to the hind limb during development.

Horseradish peroxidase is injected into specified regions of the hind limb bud of Xenopus laevis tadpoles at serial stages of development. Ventral horn cells projecting to the injection sites become labelled by the retrograde axonal transport of the enzyme. By mapping the labelled cells the developing pattern of projection of the ventral horn to the hind limb is charted. The earliest patterns of projection suggest that the first motor axons to invade the limb grow to the mesenchyme nearest to their point of entry. Thereafter, however, the projection patterns begin to resemble the adult patterns and indicate that subsequently invading axons are guided to limb regions related to the location of their cell bodies in the ventral horn. Further abrupt changes of the projection patterns leading to the final adult patterns are seen at the time of onset of ventral horn cell degeneration and just prior to the onset of limb movements.     

Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral pathfinding of limb motor axons

The Eph family of tyrosine kinase receptors has recently been implicated in various processes involving the detection of environmental cues such as axonal guidance, targeted cell migration and boundary formation. We have inactivated the mouse EphA4 gene to investigate its functions during development. Homozygous EphA4 mutant animals show peroneal muscular atrophy correlating with the absence of the peroneal nerve, the main dorsal nerve of the hindlimb. This phenotype is also observed, although with a lower penetrance, in heterozygotes. During normal hindlimb innervation, motor axons converge towards the sciatic plexus region at the base of the limb bud, where they must choose between dorsal and ventral trajectories within the limb. Among the axons emerging from the sciatic plexus, dorsal projections show higher levels of EphA4 protein than ventral axons. In EphA4 mutant mice, presumptive dorsal motor axons fail to enter the dorsal compartment of the limb and join the ventral nerve. Our data therefore suggest that the level of EphA4 protein in growing limb motor axons is involved in the selection of dorsal versus ventral trajectories, thus contributing to the topographic organisation of motor projections.     

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.     

Reformation of specific neuromuscular connections during axolotl limb regeneration: evidence that the first contacts are correct

Retrograde neuronal tracing with horseradish peroxidase was used to determine the position in the spinal cord of the motor neurone pools of a proximal (biceps) and a distal (extensor digitorum) limb muscle at various times during axolotl limb regeneration. It was found that from the earliest stages of muscle redifferentiation (as judged by light and electron microscopic analysis) the vast majority of axons innervating the regenerating muscles came from cells within the bounds of the normal motor neurone pool for each muscle. A few incorrect projections were noted in that the regenerating proximal muscle was sometimes innervated by some cells caudal to its normal motor neurone pool. The results are discussed in terms of mechanisms that may be operating in the regenerating limb to ensure that specific neuromuscular connections are made.     

Eph:ephrin-B1 forward signaling controls fasciculation of sensory and motor axons

Axon fasciculation is one of the processes controlling topographic innervation during embryonic development. While axon guidance steers extending axons in the accurate direction, axon fasciculation allows sets of co-extending axons to grow in tight bundles. The Eph:ephrin family has been involved both in axon guidance and fasciculation, yet it remains unclear how these two distinct types of responses are elicited. Herein we have characterized the role of ephrin-B1, a member of the ephrinB family in sensory and motor innervation of the limb. We show that ephrin-B1 is expressed in sensory axons and in the limb bud mesenchyme while EphB2 is expressed in motor and sensory axons. Loss of ephrin-B1 had no impact on the accurate dorso-ventral innervation of the limb by motor axons, yet EfnB1 mutants exhibited decreased fasciculation of peripheral motor and sensory nerves. Using tissue-specific excision of EfnB1 and in vitro experiments, we demonstrate that ephrin-B1 controls fasciculation of axons via a surround repulsion mechanism involving growth cone collapse of EphB2-expressing axons. Altogether, our results highlight the complex role of Eph:ephrin signaling in the development of the sensory-motor circuit innervating the limb.     

Extrinsic influences on limb muscle organisation

Skeletal muscle within the vertebrate limb originates from the somite. Much work has focussed upon the role of secreted signalling molecules of the Hedgehog, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt families plus their associated antagonists in establishing somitic cell types, yet there is no consensus on how these signals combine to influence muscle patterning. When somitic cells migrate into the limb bud, they become subject to a new set of guidance and patterning cues. Here we discuss the possible roles played by signalling proteins, particularly Hedgehogs, in guiding the cells of the limb musculature to their fate.     

Development of the Pattern of Innervation of the Chick Limb

SYNOPSIS. There is a consistent repeatable pattern of innervationof the vertebrate limb by sensory and motor axons from the spinalcord. We have been looking at the factors which control thegeneration of this pattern in the developing chick limb. Thegross pattern of nerves in the limb is controlled by the limbtissue and arises as a result of the axon's response to itslocal environment. Each segmental root innervates a characteristic territory inthe limb. Each muscle is innervated by a characteristicallylocated motoneurone pool in the cord. Three models explaining the generation of these patterns arepresented: active selection, selective cell death, and passivedeployment. A review of recent experiments leads us to concludethat there is evidence for both passive deployment and local,short range, active selection.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Neuroanatomical clues to peripheral locomotor control in small crustaceans (Artemia salina).

Brine shrimp (Artemia salina) were prepared for light and electron microscopy at several stages. Immersion-fixed, rapid Golgi impregnations demonstrated two distinct neuronal types in thoracic appendages of mature, freely swimming Artemia. Isolated motor neurons had large cell somas and thick, radiating dendrites at the body wall-limb junction. A long, elaborate axon extended into the limb. Groups of a second type of neuron with smaller somas and very thin, radiating processes occurred in the distal limb near presumably tactile bristles. Thick axons from motor neurons were traced to terminals associated with limb muscle. Both muscle and axon were best seen with Nomarski optics. Motor axons possessed elongate, irregularly shaped boutons en passant and morphologically variable boutons terminaux; the latter included huge endings with knobbed projectiles arising from thick collaterals, or smaller, round boutons from thin collaterals. In addition, a thick unidentified axon coursed longitudinally within the central body wall, sending short collaterals peripherally. The elaborate peripheral neurons described in this Golgi study may be anatomical correlates for the extraordinary coordination of mature brine shrimp. Because Artemia movements resemble those of leech and decapods, which have been studied extensively electrophysiologically, the possibility of similarly elaborate peripheral structures supplementing central control of locomotion in those invertebrates should be considered.     

GDNF acts as a chemoattractant to support ephrinA-induced repulsion of limb motor axons

Despite the abundance of guidance cues in vertebrate nervous systems, little is known about cooperation between them. Motor axons of the lateral motor column (LMC(L)) require two ligand/receptor systems, ephrinA/EphA4 and glial cell line-derived neurotrophic factor (GDNF)/Ret, to project to the dorsal limb. Deletion of either EphA4 or Ret in mice leads to rerouting of a portion of LMC(L) axons to the ventral limb, a phenotype enhanced in EphA4;Ret double mutants. The guidance errors in EphA4 knockouts were attributed to the lack of repulsion from ephrinAs in the ventral mesenchyme. However, it has remained unclear how GDNF, expressed dorsally next to the choice point, acts on motor axons and cooperates with ephrinAs. Here we show that GDNF induces attractive turning of LMC(L) axons. When presented in countergradients, GDNF and ephrinAs cooperate in axon turning, indicating that the receptors Ret and EphA4 invoke opposite effects within the same growth cone. GDNF also acts in a permissive manner by reducing ephrinA-induced collapse and keeping the axons in a growth-competent state. This is the first example of two opposing cues promoting the same trajectory choice at an intermediate target.     

SF/HGF is a mediator between limb patterning and muscle development.

Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.     

Lineage and pattern in the developing vertebrate limb

Skeletal development in the vertebrate limb occurs independently of that of associated muscles and nerves. Patterning of muscles and nerves within the vertebrate limb depends on cues provided by the developing skeleton. Recent work suggests that skeletal pattern formation depends on spatially periodic prepatterns of extracellular matrix, the biosynthesis of which may be stimulated by diffusible growth factors. In concert with the regulation of limb bud size and shape by endogenous retinoids and other substances, this mechanism could explain how characteristic limb asymmetries are generated.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

Induced neuroma formation and target muscle perturbation in the giant fiber pathway of the Drosophila temperature-sensitive mutant shibire

Summary The temperature-sensitive mutation shibire (shi) in Drosophila melanogaster is thought to disrupt membrane recycling processes, including endocytotic vesicle pinch-off. This mutation can perturb the development of nerves and muscles of the adult escape response. After exposure to a heat pulse (6 h at 30° C) at 20 h of pupal development, adults have abnormal flight muscles. Wing depressor muscles (DLM) are reduced in number from the normal six to one or two fibers, and are composed of enlarged fibers that appear to represent fiber fusion; large spaces devoid of muscle fibers suggested fiber deletion. The normal five motor axons are present in the peripheral nerve PDMN near the ganglion. However, while some motor axons pass dorsally to the extant fibers, other motor axons lacking end targets pass into an abnormal posterior branch and terminate in a neuroma, i.e., a tangle of axons and glia without muscle target tissue. Hemisynapses are common in axons of the proximal PDMN and within the neuroma, but they are rarely seen in control (no heat pulse) shi or wild-type flies. All surviving muscle fibers are innervated; no muscle tissue exists without innervation. Fibrillar fine structure and neuromuscular synapses appear normal. Fused fibers have dual innervation, suggesting correct and specific matching of target tissue and motor axons. Motor axons lacking target fibers do not innervate erroneous targets but instead terminate in the neuroma. These results suggest developmental constraints and rules, which may contribute to the orderly, stereotyped development in the normal flight system. The nature of the anomalies inducible in the flight motor system in shi flies implies that membrane recycling events at about 20 h of pupal development are critical to the formation of the normal adult nerve-muscle pattern for DLM flight muscles.     

The pattern of innervation in serially duplicated axolotl limbs: further evidence for the existence of local pathway cues?

The innervation of the biceps muscle was examined in regenerated and vitamin A-induced serially duplicated axolotl forelimbs using retrograde transport of horseradish peroxidase. The regenerated biceps muscle becomes innervated by motor neurones in the same position in the spinal cord as the normal biceps motor pool. In previous experiments in which the innervation of a second copy of a proximal limb muscle was examined in serially duplicated limbs (Stephens, Holder & Maden, 1985), the duplicate muscle was found to become innervated by motor neurones that would normally have innervated distal muscles. In the present study, the innervation of the second copy of biceps was examined under conditions designed to encourage nerve sprouting from 'correct' biceps axons. Following either partial limb denervation or denervation coupled with removal of the proximal biceps, the second copy of the muscle was still innervated by inappropriate motor neurones, which again would normally innervate distal limb muscles. These results are interpreted as evidence for the necessity for an appropriate local environment for axonal growth to allow reformation of a correct pattern of motor innervation in the regenerated limb.     

Genetic and molecular analyses of motoneuron development

Motoneurons have distinct identities and muscle targets. Recent classical and molecular genetic studies in flies and vertebrates have begun to elucidate how motoneuron identities and target specificities are established. Many of the same molecules participate in the guidance of both vertebrate and fly motor axons. It is less clear, however, whether the same molecular mechanisms establish vertebrate and fly motoneuron identities.