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Several lines of evidence suggest that Wnt genes play a critical role in regulating development of the vertebrate embryo. To address the role that this family may play in the development of the chicken central nervous system (CNS), we have used a PCR based strategy to clone partial sequences for Wnt genes. At least six different Wnt genes are expressed in the developing CNS of the chick embryo. The domains of expression overlap either partially or completely, and are expressed in spatial domains that prefigure morphological subunits of the embryonic neural tube. Wnt-1 and Wnt-4 are first expressed in the open neural plate in the region of the presumptive mesencephalon. Wnt-3a expression is first observed in the rhombencephalic regions of the open neural plate. After neural tube closure, when the embryonic subdivisions of the neural tube became apparent, Wnt-1, Wnt-3a and Wnt-4 are all broadly expressed in partially overlapping domains in the mesencephalon and caudal diencephalon, as well as in the rhombencephalon and spinal cord. The mesencephalic expression patterns are subsequently modified such that Wnt-1 and Wnt-4 are expressed in a characteristic ring just rostral to the isthmus, at the mesencephalic/metencephalic junction; and Wnt-1 and Wnt-3a expression become restricted to the dorsal midline. Wnt-1, Wnt-3a, Wnt-4, Wnt-5a and Wnt-8b are expressed in one or two caudal subdivisions of the developing diencephalon, the synencephalon and posterior parencephalon, but do not extend ventral to the zona limitans interparencephalica. In contrast, Wnt-7b is expressed in the anterior parencephalon. Both Wnt-7b and Wnt-8b are expressed in telencephalic portions of the secondary prosencephalon. The timing and spatial distribution of Wnt-gene expression in the chick embryo further support the general hypothesis that Wnt genes play key roles in patterning the developing vertebrate nervous system.

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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.

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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.

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Axon navigation during vertebrate limb innervation has been shown to be associated with position-dependent changes in size and complexity of the axon growth cones, and sometimes with bifurcation of terminal growth cones and axon branching (Hollyday and Morgan-Carr, companion paper). We have further examined axon branching and asked whether it extends to the projection of collaterals to different nerves. Injections of horseradish peroxidase or Dil were made into individual peripheral nerves in the wings of chick embryos at stages 28-35, and the trajectories of solidly labeled axons were traced proximally from the injection site in tissue sections. During stages when the peripheral nerves were first forming in the shoulder region, collaterals of retrogradely labeled axons were frequently observed to project into uninjected nerves proximal to the injection site. These two-nerve collaterals were formed by a small percentage of axons in a high percentage of the embryos studied and could occur in both motor and sensory axons. Two-nerve collateral projections were observed between nerves separated along both the proximodistal and anteroposterior axes of the limb, but they were limited in spatial extent to nerves supplying adjacent limb regions and were never seen between nerves projecting to widely disparate regions of the limb. Collaterals were not seen at the plexus projecting to both dorsal and ventral pathways. The apparent frequency of two-nerve collaterals was found to decline progressively from stage 28-29 to stage 32; no two-nerve collaterals were seen in the proximal wing at stage 33 and older. The mechanism of their elimination is presently unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the limb plexus, motor axons destined for limb muscles diverge along separate pathways to innervate muscles derived from either the dorsal or ventral premuscle masses. We have examined the axonal guidance cues involved in this initial, specific pathway choice at the plexus by making dorsoventral (D/V) limb bud reversals prior to innervation. Chick/quail chimeras were used to determine the proximodistal level of the reversal in tissue sections. The specificity of the projections to dorsal or ventral nerve trunks was assessed by retrograde HRP labeling at ages prior to motoneuron death. Axons corrected for the reversal when the level of the graft was proximal to the plexus, and when the reversed limb and its gross nerve pattern were normal. If all of these conditions were not satisfied, aberrant innervation patterns were observed. Axonal trajectories were analyzed within the host tissue, at the host-graft border, and within rotated tissue to determine where along the pathway guidance cues might be located. Special attention was given to cases in which axons compensated for the reversal to project in accord with the positions of their soma in the lateral motor column. In these correcting cases, after normal D/V sorting in the spinal nerves of the host, motor axons altered their trajectories upon entering rotated graft tissue as they approached and traversed the plexus. Because corrections were within rotated tissue and not proximal to it, the D/V pathway cues are unlikely to be long-range target-derived signals, but rather appear to be closely associated with positional information in the plexus region and also more proximally in the tissue surrounding the distal spinal nerves.

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Intramuscular injections of the retrograde tracers 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and horseradish peroxidase (HRP) were used to map the motor pools innervating axial muscles in the cervical and thoracic regions of the chicken. We found that motor pool position is well correlated with the muscle's embryonic origin, and not necessarily with its position. Muscles of myotomal (exclusively somitic) origin were innervated by medially positioned motoneurons in the median motor column, and the motor pools supplying these muscles were somatotopically organized. Muscles having a dual embryonic origin, from the somites and lateral plate, were innervated by motoneurons positioned further laterally within the median motor column. The relationship between motor pool position and embryonic origin of the muscle may be a general principle of ventral horn organization.

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The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.

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Neural crest cells from brachial levels of the neural tube populate the ventral roots, spinal nerves, and peripheral nerves of the chick forelimb where they give rise to Schwann cells. The distribution of neural crest cells in the developing forelimb was examined using homotopic and heterotopic chick-quail chimeras to label neural crest cells from subsets of the brachial spinal segments. Neural crest cells from particular regions of the spinal cord populated ventral roots and spinal nerves adjacent to or immediately posterior to the graft. Crest cells also populated the brachial plexus in accord with their segmental origins. In the forelimb, neural crest cells populated muscle nerves with anterior brachial spinal segments populating nerves to anterior musculature of the forelimb and posterior brachial spinal segments populating nerves to posterior musculature. Similar patterns were seen following both homotopic and heterotopic transplantation. In both types of grafts, the distribution of neural crest cells largely matched the sensory and motor projection pattern from the same spinal segmental level. This suggests that neural crest-derived Schwann cells from a particular spinal segment may use sensory and motor fibers emerging from the same segmental level as substrates to guide their migration into the periphery.

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The role of muscle cells in the survival of embryonic motoneurons projecting to the developing wing was directly examined. Embryos lacking muscle in one of their wings were produced by surgically removing the embryonic precursors of muscle cells, the somites. The resulting limb lacked only muscle cells, with the derivatives of the other limb contributor, the lateral plate mesoderm, left intact. Counts of apparently healthy lateral motor column (LMC) motoneurons supplying normal wings between stages 28 and 36 showed little decline in motoneuron number until stage 34; approximately 24% of the motoneurons died between stages 34 and 36. In contrast, the number of LMC motoneurons supplying muscleless wings declined progressively from stages 28 to 36. This decline resulted in the loss of about 77% of the motoneurons present at stage 28. In addition, the LMCs supplying muscleless wings had fewer motoneurons at all stages examined than similarly staged controls; this difference ranged from 27% in the youngest cases to 75% in the older embryos. Motoneurons were lost equivalently from all rostrocaudal levels of the brachial LMC. From these studies we conclude that motoneurons survival depends on the presence of muscle cells in the developing wing. In the absence of muscle cells, motoneuron death was increased compared to normal embryos at stages prior to the onset of naturally occurring cell death.

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Modern neuronanatomical techniques were used to investigate the development of the avian sympathetic preganglionic cell column in the spinal cord of the chick embryo. [3H]thymidine autoradiography indicated that the majority of these preganglionic, or "Terni column" neurons are generated between stages 18 and 24 (days 2-4). This coincides with the genesis of the somatic motoneurons in the thoracic levels of the cord, and therefore differences in the time of origin cannot explain the divergent fates of these two neuronal populations. Data obtained from short-survival autoradiographic experiments indicated that many early born cells remain close to the ventral region of the ventricular epithelium until day 5 of incubation. Ventral root injections used to label retrogradely neurons projecting an axon into the ventral root (Terni cells and somatic motoneurons) have labeled neurons next to the ventricular epithelium at the same early stages. Thus, it seems likely that some Terni cells, if not all, maintain medial positions and do not migrate laterally to join a common motor column before initiating a dorsal migration. Analysis of a closely staged series of embryos, whose Terni column neurons were retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP), revealed that between days 5 and 8 of incubation, Terni column neurons migrated dorsally to attain their adult position adjacent to the central canal. These changes in position were reflected in the changing morphology of the Terni column neurons, visualized by the Golgi-like HRP labeling. The positions of the migrating Terni cells differed from those of commissural cells, indicating that these fibers are not the substrate for the dorsal migration. The dorsal migration of Terni column cells was not disrupted by the surgical removal of the sympathetic ganglia, the synaptic targets of these neurons, nor by disruption of spinal afferents. Taken together, these results suggest that the migratory behavior of Terni cells in distinctive when compared to that of somatic motoneurons, and that local and/or intrinsic cues within the spinal cord guide the dorsal migration of Terni column cells.

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Intramuscular injections of HRP were used to map the spinal cord location of motoneurons innervating wing and shoulder girdle muscles in newly hatched chicks. Motor pools are grouped in the lateral motor column in relationship to embryonic origin of the muscles: muscles derived from the ventral muscle mass are innervated by medially lying motor pools, while muscles derived from the dorsal mass are innervated by lateral pools. Motor pool position is also well correlated with nerve supply. Muscles innervated by nerves diverging from common nerve trunks are innervated by neighboring motor pools. The rostro-caudal organization of the motor pools reflects both proximo-distal and antero-posterior axes of the limb with proximal and anterior muscles innervated from rostral motor pools.

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Anterograde transport of HRP was used to map initial segmental projection patterns of motor and sensory axons to the chick embryo wing. At the earliest stages of innervation, before the peripheral nerves had formed, motor axons were distributed as two loosely organized sheets, one directed towards the dorsal muscle mass and the other to the ventral. Labeled axons emerging from a given spinal cord segment were distributed within the sheets in a pattern that reflected the origin of the axons along the rostro-caudal axis of the lateral motor column. The segmental specificity of the motor projections was analyzed again after the axonal sheets had formed recognizable nerve trunks and muscle nerves. As was observed at the younger stages, axons were distributed in a pattern that was correlated with both the relative antero-posterior (A/P) and proximo-distal (P/D) position of the forming nerve, with cranial spinal nerves projecting to anterior and proximal limb regions. Whereas most axons emerging from a given ventral root projected in accord with what would be expected from the known adult motor pool maps, segmentally unexpected projections were frequently observed. The proportion of aberrantly projecting axons appears to be quite small, and in most embryos, it was impossible to determine whether the erroneous projections originated from unbranched axons or were collateral branches of others. The findings indicate that the initial projections of motor axons to the developing wing are patterned along the A/P axis of the wing and are largely accurate; however, the guidance processes are not sufficiently precise to exclude fibers entirely from inappropriate nerves. It is likely that later developmental processes act to fine-tune the initial projection pattern.

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The role of myogenic cells in accurate pathway selection and muscle nerve formation was studied in chick embryos. Myotubes were eliminated from the forelimb of the chick embryo by extirpating the somites, which give rise to myogenic cells. Other elements of the wing tissue, connective tissue, and cartilagenous elements derived from the somatopleure, were left intact. Injections of WGA-HRP were made into either dorsal or ventral nerve trunks in the wing and the positions of retrogradely labeled motoneurons determined. The positions of the motoneurons within the brachial lateral motor column were appropriate for the injection made. Thus, the accuracy of the motoneuronal projections was unaffected by the absence of muscle cells. The absence of myotubes was not correlated with the absence of muscle nerves. Muscle nerves were consistently observed in muscleless wings until stage 36, the oldest stage examined. Muscle nerves in muscleless wings differed from those in normal wings in that they were smooth and stubby, and lacked the normal pattern of intramuscular nerve branches. From these studies we conclude that muscle cells are not necessary for accurate motor axon guidance into the periphery along the routes of major nerve trunks, nor for the formation of muscle nerves. By inference, somatopleural derivatives provide sufficient cues for selection of specific axonal pathways and for patterning of muscle nerves within the chick limb.

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The topographic organization of the motor nuclei supplying individual thoracic muscles in postnatal rats was investigated by horseradish peroxidase (HRP) labelling from the periphery. To determine whether aspects of the same organization are apparent at early developmental stages, we used HRP to label neurons contributing axons to the two primary rami of thoracic nerves in rat fetuses ranging in age from the 13th day of gestation to birth. This developmental period includes stages during which peripheral nerves and muscles are forming. The results show that motoneurons in the appropriate positions in the ventral horn have axons in each primary ramus even at stages prior to the innervation of muscle. However, in fetuses at certain developmental stages, there are also neurons located outside the motor nuclei which send axons into the peripheral nerves.

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In this paper we report investigations of the innervation of duplicated limb segments to test whether the addition of limb segments along the proximodistal axons could stimulate the growth of appropriate motoneurons into double occurrences of these muscles. Our evidence indicates that it does not. Using retrograde horseradish peroxidase nerve-tracing techniques and reconstructions of experimental limbs, we investigated the motor projection to parallel and serially duplicated legs. In all cases, host limb segments were normally innervated. In a control experiment involving a host thigh connected to a graft calf, the innervation of both segments was normal. In serially duplicated limb segments, however, we found abnormal innervation. In limbs of the type thigh-thigh-calf-foot, the innervation of the second thigh was accomplished by calf motoneurons. In limbs consisting of thigh-calf-calf-foot, the duplicated calf was served by foot motoneurons. The general pattern was that muscles were innervated as a function of their position along the proximodistal axis, irrespective of their identity. In no limb were axons found distal to the third limb segment even before the period of normal cell death. Despite the mismatched innervation with regard to the thigh/calf/foot distinction, axons retained their characteristic selectivity for either dorsally or ventrally derived muscles. The findings suggest that the projection of axons along the proximodistal axis of the limb is influenced by proximal growth cues associated with the formation of the limb plexus as well as by competitive interactions in the distal limb tissue.

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Several studies have demonstrated that motor axons can discriminate between dorsally and ventrally derived muscles. In this paper we present evidence that (1) the pathway axons take in the limb constrain their access to either dorsally or ventrally derived muscles, and therefore (2) the axon's ability to discriminate between dorsal and ventral is expressed already at the level of pathway selection into the limb. Surgically manipulated hindlimbs were produced consisting of a normal host thigh connected to a dorsoventrally rotated calf or to rotated and duplicated donor limb segments. The limb rotations were done distal to the level at which axons select a dorsally or ventrally destined pathway through the limb, such that at the level of the rotation, axons in each nerve were confronted with the opposite-from-normal set of muscles. In this situation, the relative influence of pathway availability versus dorsal/ventral muscle recognition could be assessed. The innervation of rotated limb segments was, in all cases, opposite from normal. Motoneurons which normally innervate dorsal muscles innervated ventrally derived muscles that had been rotated into a dorsal position. Likewise, normally ventrally destined axons served dorsal muscles in the rotated segments. Thus, motor axons did not alter their distal path to reach their normal set of muscles. While these results do not rule out intrinsic dorsal/ventral differences between muscles, they do demonstrate that muscle surface recognition is not necessary to account for dorsal/ventral discrimination in the innervation of normal, supernumerary, or duplicated limbs, nor is it sufficient to account for the innervation of rotated limb segments. These results also indicate that pathway guidance cues are an important influence on innervation patterns.

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