Ventral Muscle Mass Research Articles

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B.lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B.lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B.lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

Burn-Induced Microglia Activation is Associated With Motor Neuron Degeneration and Muscle Wasting in Mice

Burn injury (BI) leads to both systemic and neuro-inflammation and is associated with muscle wasting and weakness, which increase morbidity and mortality. Disuse atrophy is concomitantly present in BI patients. Most studies have focused on muscle with little attention to role of central nervous system (CNS) in the neuromuscular changes. We tested the hypothesis that BI-induced muscle wasting stems from CNS microglia activation and cytokines and chemokine release, which is associated with spinal ventral horn motor neuron degeneration. Body surface (35%) BI, immobilization alone (Immob), BI with immobilization (BI + Immob), or Sham BI were administered to mice. Spinal cord (L3-L4 segments) and skeletal muscle tissues were harvested on days 7 and 14 after perturbations to examine microglia, motor neuron, and skeletal muscle changes. BI and BI + Immob significantly (P < 0.05) activated microglia, evidenced by its increased density around motor neurons, upregulated neuroinflammation-marker, translocator protein 18 kDa expression and inflammatory cytokines (interleukin-1β, tumor necrosis factor-α) and/or chemokines (CXCL2) expression at days 7 and 14. Ventral horn motor neurons apoptosis and downregulation were observed at both periods after BI and was significantly magnified by concomitant BI + Immob. BI and more prominently BI + Immob disintegrated and fragmented the pretzel-shaped synapse and was associated with significantly decreased gastrocnemius, tibialis, and soleus muscle masses. BI induces microglia proliferation and activation (cytokine and chemokine release), degeneration of ventral horn motor neurons and muscle mass loss, all of which were accentuated by concomitant immobilization. The mechanisms connecting microglia activation and motor neuron degeneration to muscle mass loss require further delineation.

Reconstructing pectoral appendicular muscle anatomy in fossil fish and tetrapods over the fins-to-limbs transition.

The question of how tetrapod limbs evolved from fins is one of the great puzzles of evolutionary biology. While palaeontologists, developmental biologists, and geneticists have made great strides in explaining the origin and early evolution of limb skeletal structures, that of the muscles remains largely unknown. The main reason is the lack of consensus about appendicular muscle homology between the closest living relatives of early tetrapods: lobe-finned fish and crown tetrapods. In the light of a recent study of these homologies, we re-examined osteological correlates of muscle attachment in the pectoral girdle, humerus, radius, and ulna of early tetrapods and their close relatives. Twenty-nine extinct and six extant sarcopterygians were included in a meta-analysis using information from the literature and from original specimens, when possible. We analysed these osteological correlates using parsimony-based character optimization in order to reconstruct muscle anatomy in ancestral lobe-finned fish, tetrapodomorph fish, stem tetrapods, and crown tetrapods. Our synthesis revealed that many tetrapod shoulder muscles probably were already present in tetrapodomorph fish, while most of the more-distal appendicular muscles either arose later from largely undifferentiated dorsal and ventral muscle masses or did not leave clear correlates of attachment in these taxa. Based on this review and meta-analysis, we postulate a stepwise sequence of specific appendicular muscle acquisitions, splits, and fusions that led from the ancestral sarcopterygian pectoral fin to the ancestral tetrapod forelimb. This sequence largely agrees with previous hypotheses based on palaeontological and comparative work, but it is much more comprehensive in terms of both muscles and taxa. Combined with existing information about the skeletal system, our new synthesis helps to illuminate the genetic, developmental, morphological, functional, and ecological changes that were key components of the fins-to-limbs transition.

Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development.

Initiation of primary myogenesis in amniote limb muscles

Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are "seeded" by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects. We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3(+ve) myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7(+ve) cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis. Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects.

Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing

We have mapped the motor neurons (MNs) supplying the major hindlimb muscles of transgenic (C57/BL6J-ChAT-EGFP) and wild-type (C57/BL6J) mice. The fluorescent retrograde tracer Fluoro-Gold was injected into 19 hindlimb muscles. Consecutive transverse spinal cord sections were harvested, the MNs counted, and the MN columns reconstructed in 3D. Three longitudinal MN columns were identified. The dorsolateral column extends from L4 to L6 and consists of MNs innervating the crural muscles and the foot. The ventrolateral column extends from L1 to L6 and accommodates MNs supplying the iliopsoas, gluteal, and quadriceps femoris muscles. The middle part of the ventral horn hosts the central MN column, which extends between L2 and L6 and consists of MNs for the thigh adductor, hamstring, and quadratus femoris muscles. Within these longitudinal columns, the arrangement of the different MN groups reflects their somatotopic organization. MNs innervating muscles developing from the dorsal (e.g., quadriceps) and ventral muscle mass (e.g., hamstring) are situated in the lateral and medial part of the ventral gray, respectively. MN pools belonging to proximal muscles (e.g., quadratus femoris and iliopsoas) are situated ventral to those supplying more distal ones (e.g., plantar muscles). Finally, MNs innervating flexors (e.g., posterior crural muscles) are more medial than those belonging to extensors of the same joint (e.g., anterior crural muscles). These data extend and modify the MN maps in the recently published atlas of the mouse spinal cord and may help when assessing neuronal loss associated with MN diseases.

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.

Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis

The basic helix-loop-helix transcription factor MyoD is a central actor that triggers the skeletal myogenic program. Cell-autonomous and non-cell-autonomous regulatory pathways must tightly control MyoD expression to ensure correct initiation of the muscle program at different places in the embryo and at different developmental times. In the present study, we have addressed the involvement of Sim2 (single-minded 2) in limb embryonic myogenesis. Sim2 is a bHLH-PAS transcription factor that inhibits transcription by active repression and displays enhanced expression in ventral limb muscle masses during chick and mouse embryonic myogenesis. We have demonstrated that Sim2 is expressed in muscle progenitors that have not entered the myogenic program, in different experimental conditions. MyoD expression is transiently upregulated in limb muscle masses of Sim2(-/-) mice. Conversely, Sim2 gain-of-function experiments in chick and Xenopus embryos showed that Sim2 represses MyoD expression. In addition, we show that Sim2 represses the activity of the mouse MyoD promoter in primary myoblasts and is recruited to the MyoD core enhancer in embryonic mouse limbs. Sim2 expression is non-autonomously and negatively regulated by the dorsalising factor Lmx1b. We propose that Sim2 represses MyoD transcription in limb muscle masses, through Sim2 recruitment to the MyoD core enhancer, in order to prevent premature entry into the myogenic program. This MyoD repression is predominant in ventral limb regions and is likely to contribute to the differential increase of the global mass of ventral muscles versus dorsal muscles.

Sim1 and Sim2 expression during chick and mouse limb development

The Drosophila Single minded (Sim) transcription factor is a master regulator of cell fate during midline development. The homolog mouse Sim1 and Sim2 genes are important for central nervous system development. Loss of mSim1 activity leads to an absence of specific neuroendocrine lineages within the hypothalamus, while overexpression of mSim2 leads to behavioural defects. We now provide evidence that vertebrate Sim genes might be important for limb muscle formation. We have examined by in situ hybridisation the expression of the Sim1 and Sim2 genes during limb development in chick and mouse embryos. The expression of both Sim genes is mainly associated with limb muscle formation. We found that each Sim gene has a similar temporal and spatial expression pattern in chick and mouse embryonic limbs, although with some differences for the Sim2 gene between species. In chick or mouse embryonic limbs, Sim1 and Sim2 display non-overlapping expression domains, suggesting an involvement for Sim1 and Sim2 proteins at different steps of limb muscle formation. Sim1 gene expression is associated with the early step of muscle progenitor cell migration in chick and mouse, while the Sim2 gene is expressed just after the migration process. In addition, chick and mouse Sim2 gene expression is enhanced in limb ventral muscle masses versus dorsal ventral muscle masses. Our results provide a basis for further functional analysis of the Sim genes in limb muscle formation.

Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling

Vertebrate muscle morphogenesis is a complex developmental process, which remains quite yet unexplored at cellular and molecular level. In this work, we have found that sculpturing programmed cell death is a key morphogenetic process responsible for the formation of individual foot muscles in the developing avian limb. Muscle fibers are produced in excess in the precursor dorsal and ventral muscle masses of the limb bud and myofibers lacking junctions with digital tendons are eliminated via apoptosis. Microsurgical experiments to isolate the developing muscles from their specific tendons are consistent with a role for tendons in regulating survival of myogenic cells. Analysis of the expression of Raldh2 and local treatments with retinoic acid indicate that this signaling pathway mediates apoptosis in myogenic cells, appearing also involved in tendon maturation. Retinoic acid inhibition experiments led to defects in muscle belly segmentation and myotendinous junction formation. It is proposed that heterogeneous local distribution of retinoids controlled through Raldh2 and Cyp26A1 is responsible for matching the fleshy and the tendinous components of each muscle belly.

Expression of avian Groucho-related genes ( Grgs) during embryonic development

Groucho-related genes ( Grgs) encode transcriptional co-repressors of Lef/Tcf and Hes proteins, which are mediators of Wnt and Notch signalling, respectively. Thus, they are important players in the developmental processes controlled by Wnt and Notch signalling, including lateral inhibition, segmentation and dorso-ventral patterning. We have cloned the avian homologues of Grg genes and examined their expression pattern by whole-mount in situ hybridisation between Hamburger–Hamilton (HH) stages 3 and 24. At HH stage 3, Grg gene expression is detected in the primitive streak and Hensen's node. Later, Grg genes are expressed at high levels in the developing head fold and by HH stage 11, throughout the anterior CNS and in the ventricular zone of the neural tube. In addition, Grg2, Grg4 and Grg5 are expressed in the notochord. In the paraxial mesoderm, Grg genes are activated as soon as somites form. As somites mature, Grg1 and Grg5/AES are expressed predominantly in the medial myotome and dermomyotome, whereas Grg2, Grg3 and Grg4 are expressed throughout the myotome. In HH stage 20 limbs, Grg1, Grg3 and Grg4 transcripts are more abundant in the posterior limb bud, whereas Grg2 and Grg5/AES are expressed throughout. By HH stage 24, Grg1, Grg2 and Grg3 become localized to the dorsal and ventral limb muscle masses, whereas Grg4 and Grg5/AES occupy a more central and ventro-proximal domain, respectively. Overall, our expression data are consistent with a role for Grg genes in Lef/Tcf and Wnt signalling during somitogenesis and with a role in Hes and Notch signalling in neurogenesis.

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.

Regulation of myogenic differentiation in the developing limb bud.

The limb myogenic precursors arise by delamination from the lateral dermomyotome in response to signals from the lateral plate mesoderm. They subsequently migrate into the developing limb bud where they switch on the expression of the myogenic regulatory factors, MyoD and Myf5, and coalese to form the dorsal and ventral muscle masses. The myogenic cells subsequently undergo terminal differentiation into slow or fast fibres which have distinct contractile properties determining how a muscle will function. In general, fast fibres contract rapidly with high force and are characterized by the expression of fast myosin heavy chains (MyHC). These fibres are needed for movement. In contrast, slow fibres express slow MyHC, contract slowly and are required for maintenance of posture. This review focuses on the molecular signals that control limb myogenic development from the initial delamination and migration of the premyogenic cells to the ultimate formation of the complex muscle pattern and differentiation of slow and fast fibres.

Developmental expression of the POU transcription factor qBrn-2 during somitic myogenesis in quail.

We prepared a specific antiserum to the qBrn-2 protein and examined the developmental distribution of this protein during quail somitic myogenesis. In contrast to its mammalian homolog N-Oct-3, qBrn-2 exhibited an impressive spatio-temporal profile in somitic myogenesis, in addition to the orthodox expression observed in the developing neural tube. In somites, qBrn-2 was expressed in the outer epithelial cells, but not in the core cells. During the somite differentiation, qBrn-2 expression was enhanced and restricted to myotome. The location of qBrn-2 expression seemed to overlap with that of myf5 and myoD in myotome. However, in cells that just began to express myf5 or myoD, qBrn-2 expression was not obvious. As embryonic development proceeded, qBrn-2 positive cells in myotome migrated dorsally and ventrally, and qBrn-2 expression was still observed at dorsal and ventral muscle masses in the forelimb. On the basis of our observations, it seems that qBrn-2 may play important roles in the determination, differentiation and migration of muscle precursor cells, in addition to its known roles in neurogenesis.

Early specification of limb muscle precursor cells by the homeobox gene Lbx1h.

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met ( ref. 6) and those encoded by homeobox-containing genes, including c-Met (ref. 6), Tbx1 (ref. 7), Mox2 (ref. 8), Six1 and Six2 (ref. 9), Pitx2, Pax3 (refs 10,11) and Lbx1h (refs 12,13), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevented an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Here we show that Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, led to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect was caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. Our results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles.

Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud.

Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud.

Muscle and tendon morphogenesis in the avian hind limb.

The proper development of the musculoskeletal system in the tetrapod limb requires the coordinated development of muscle, tendon and cartilage. This paper examines the morphogenesis of muscle and tendon in the developing avian hind limb. Based on a developmental series of embryos labeled with myosin and tenascin antibodies in whole mount, an integrative description of the temporal sequence and spatial pattern of muscle and tendon morphogenesis and their relationship to cartilage throughout the chick hind limb is presented for the first time. Anatomically distinct muscles arise by the progressive segregation of muscle: differentiated myotubes first appear as a pair of dorsal and ventral muscle masses; these masses subdivide into dorsal and ventral thigh, shank and foot muscle masses; and finally these six masses segregate into individual muscles. From their initial appearance, most myotubes are precisely oriented and their pattern presages the pattern of future, individual muscles. Anatomically distinct tendons emerge from three tendon primordia associated with the major joints of the limb. Contrary to previous reports, comparison of muscle and tendon reveals that much of their morphogenesis is temporally and spatially closely associated. To test whether reciprocal muscle-tendon interactions are necessary for correct muscle-tendon patterning or whether morphogenesis of each of these tissues is autonomous, two sets of experiments were conducted: (1) tendon development was examined in muscleless limbs produced by coelomic grafting of early limb buds and (2) muscle development was analyzed in limbs where tendon had been surgically altered. These experiments demonstrate that in the avian hind limb the initial morphogenetic events, formation of tendon primordia and initial differentiation of myogenic precursors, occur autonomously with respect to one another. However, later morphogenetic events, such as subdivision of muscle masses and segregation of tendon primordia into individual tendons, do require to various degrees reciprocal interactions between muscle and tendon. The dependence of these later morphogenetic events on tissue interactions differs between different proximodistal regions of the limb.

Coordinated expression of Hoxa-11 and Hoxa-13 during limb muscle patterning

The limb muscle precursor cells migrate from the somites and congregate into the dorsal and ventral muscle masses in the limb bud. Complex muscle patterns are formed by successive splitting of the muscle masses and subsequent growth and differentiation in a region-specific manner. Hox genes, known as key regulator genes of cartilage pattern formation in the limb bud, were found to be expressed in the limb muscle precursor cells. We found that HOXA-11 protein was expressed in the premyoblasts in the limb bud, but not in the somitic cells or migrating premyogenic cells in the trunk at stage 18. By stage 24, HOXA-11 expression began to decrease from the posterior halves of the muscle masses. HOXA-13 was expressed strongly in the myoblasts of the posterior part in the dorsal/ventral muscle masses and weakly in a few myoblasts of the anterior part of the dorsal muscle mass. Transplantation of the lateral plate of the presumptive wing bud to the flank induced migration of premyoblasts from somites to the graft. Under these conditions, HOXA-11 expression was induced in the migrating premyoblasts in the ectopic limb buds. Application of retinoic acid at the anterior margin of the limb bud causes duplication of the autopodal cartilage and transformation of the radius to the ulna, and at the same time induces duplication of the muscle pattern along the anteroposterior axis. Under these conditions, HOXA-13 was also induced in the anterior region of the ventral muscles in the zeugopod. These results suggest that Hoxa-11 and Hoxa-13 expression in the migrating premyoblasts is under the control of the limb mesenchyme and the polarizing signal(s). In addition, these results indicate that these Hox genes are involved in muscle patterning in the limb buds.

Chick wing innervation. II. Morphology of motor and sensory axons and their growth cones during early development

The development and distribution of neuronal projections to the developing chick wing was studied using anterograde transport of horseradish peroxidase (HRP). Small injections of HRP were made into motor or sensory neuronal populations in order to visualize individual axons and their associated growth cones. Motor growth cones were observed in different regions of the embryo at different stages, in a proximal-to-distal pattern of distribution which paralleled the process of axon outgrowth and nerve formation. Different growth cone morphologies were associated with differing regions of the developing projection. In the spinal nerves, axons destined for the limb were unbranched and terminated in simply shaped growth cones. As axons approached the developing limb and entered the plexus region, their growth cones became more complex and larger primarily because of widening, and they sometimes branched, producing processes which could extend tens of microns from a tricorne branch point on the parent axon. Both motor and sensory fibers showed similar morphological changes in the plexus region. A distinctively shaped growth cone expanded on its leading edge was observed, sequentially apparent in the distal spinal nerves, in the plexus region, in the loosely organized axonal sheets projecting to the uncleaved dorsal or ventral muscle masses, and where muscle nerves diverged from nerve trunks and within muscle nerves. It is likely that some of these are transitional growth cones preparing to branch, because complex and branched growth cones were also observed in these regions. Branched axons oriented along the anteroposterior axis were similarly observed in the plexus region and distal to the plexus when axons first projected to the limb bud. At somewhat older stages when the basic peripheral nerve branching pattern had formed, motor growth cones were observed in common nerve trunks and in individual muscle nerves, but they were no longer found in the plexus region. Branched axons were likewise restricted to these peripheral locations. Taken together, these observations suggest that one of the ways in which axons navigate is by exploration in the form of growth cone widening, and in some cases terminal bifurcation which may produce axon branches. Selection of the most appropriately directed growth cone process and/or precocious axonal branches may be one of the ways in which axons respond to specific growth cues which guide axons into the limb bud. Alternatively, this precocious branching may be an early neurotrophic response to developing muscle and play no significant role in axon navigation.

Chick wing innervation. I. Time course of innervation and early differentiation of the peripheral nerve pattern.

Anterograde transport of horseradish peroxidase was used to map the initial projection patterns of motor and sensory axons innervating the wing of the chick embryo. Injections which resulted in labeling large numbers of motor and sensory axons, separately or in combination, were used to define the time course of innervation and to visualize the progressive morphogenesis of the peripheral nerve pattern. Motor axons emerged from the spinal cord and accumulated near the ventromedial border of the myotome where they remained for up to 16 hours before growing into the plexus region and limb bud. Despite the known later time of sensory neuron production, the first sensory axons projected to the wing at the same time as motor axons. When axons first entered the wing bud, they were distributed in two loosely organized sheets of axon fascicles, one projecting to dorsal muscle mass, the other to ventral muscle mass. The width of the sheets was between one-third to one-half the width of the wing bud, and this distance was more than twice the diameter of the proximal nerve trunks measured at stage 28. In the proximal limb the basic pattern of peripheral nerves emerged gradually from stages 26 to 28. During these stages, the loosely organized sheets of axonal fascicles seen at younger stages were progressively transformed into several coherent nerve trunks and muscle nerves extended from common nerve trunks. The implication of these observations is that many outgrowing axons appear not to follow preformed pathways corresponding to the mature peripheral nerve branching pattern. This pattern may instead result from axonal recognition of cues within a largely undifferentiated limb bud, and from the subsequent bundling together of loosely organized axon fascicles. These events occur concurrently with limb growth and differentiation.