Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.     

A new membrane antigen revealed by monoclonal antibodies is associated with motoneuron axonal pathways

Chick embryonic motoneurons selectively grow out from the spinal cord as the first step of their selective axonal growth. In order to detect the molecules responsible for motoneuron outgrowth from the cord, we produced and immunohistochemically screened many monoclonal antibodies (MAbs) against cord and somite. We found that two of them, called M7412 and M7902, selectively bound to the cell surface of the anterior half of the sclerotome, where motoneurons selectively extend their axons. Immunohistochemistry and immunoblot results were identical for these antibodies and the antigen was called M7412 antigen. Although neural crest cells also migrate into the anterior half of the sclerotome, the expression of M7412 antigen by sclerotome cells was independent of the neural crest, because neural crest removal did not affect the appearance of the antigen. Furthermore, MAb M7412 bound to the mesenchymal cells along presumptive major nerve trunks in the limb and to the structures surrounding myotubes in muscles during the formation of intramuscular nerve branches. These results suggest that M7412 antigen might be a substrate for general, but not specific, growth of motoneuron axons. If this is the case, we must also infer that some molecule inhibitory for motoneuron growth is localized in the posterior half of sclerotome, because at upper cervical levels the M7412 antigen was also expressed intensely in the posterior sclerotome, whereas motoneurons still grew only into the anterior half. The M7412 antigen was transiently expressed in such various tissues as somite; muscles; blood vessels; spinal cord cells, especially motoneurons innervating the limb; and dorsal root and other peripheral ganglion cells. The M7412 antigenic molecule was extractable with NP40 from a membrane fraction of whole chick embryos and its molecular weight was estimated to be 70 kDa from immunoblot analysis. Thus, our monoclonal antibodies have revealed a new membrane-associated molecule which is likely to play a role in cell-cell interactions during development of motoneurons.     

Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.     

Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19–20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.     

Spinal nerve segmentation in the chick embryo: analysis of distinct axon-repulsive systems

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules.     

Somite polarity and segmental patterning of the peripheral nervous system

The analysis of the outgrowth pattern of spinal axons in the chick embryo has shown that somites are polarized into anterior and posterior halves. This polarity dictates the segmental development of the peripheral nervous system: migrating neural crest cells and outgrowing spinal axons traverse exclusively the anterior halves of the somite-derived sclerotomes, ensuring a proper register between spinal axons, their ganglia and the segmented vertebral column. Much progress has been made recently in understanding the molecular basis for somite polarization, and its linkage with Notch/Delta, Wnt and Fgf signalling. Contact-repulsive molecules expressed by posterior half-sclerotome cells provide critical guidance cues for axons and neural crest cells along the anterior-posterior axis. Diffusible repellents from surrounding tissues, particularly the dermomyotome and notochord, orient outgrowing spinal axons in the dorso-ventral axis ('surround repulsion'). Repulsive forces therefore guide axons in three dimensions. Although several molecular systems have been identified that may guide neural crest cells and axons in the sclerotome, it remains unclear whether these operate together with considerable overall redundancy, or whether any one system predominates in vivo.     

Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: partial and complete deletion of the somite

The development of patterned axon outgrowth and dorsal root ganglion (DRG) formation was examined after partially or totally removing chick somitic mesoderm. Since the dermamyotome is not essential and a full complement of limb muscles developed, alterations in neural patterns could be ascribed to deletion of sclerotome. When somitic tissue was completely removed, axons extended and DRG formed, but in an unsegmented pattern. Therefore the somite does not elicit outgrowth of axons or migration of DRG precursors, it is not a manditory substratum and it is not required for DRG condensation. These results suggest that posterior sclerotome is relatively inhibitory to invasion, an inhibition that is released when sclerotome is absent. When somites were partially deleted, axonal segmentation was not lost proportionally with the amount of sclerotome removed, suggesting that properties that may vary with sclerotome volume (such as diffusible cues) do not play a primary role. Instead, spinal nerves lost segmentation only when ventral sclerotome was deleted, regardless of whether dorsal sclerotome was or was not removed. This strongly suggests that axonal segmentation is imposed by direct interactions between growth cones and extracellular matrices or surfaces sclerotome cells. While DRG tended to be normally segmented when ventral sclerotome was deleted and to lose segmentation when dorsomedial sclerotome was absent, a coordinate loss of DRG segmentation with sclerotome volume could not be ruled out. However it is clear that axonal and DRG segmentation are independent. Observations on a subset of embryos in which the notochord was displaced relative to the spinal cord suggest that the ventromedial sclerotome surrounding the notochord inhibits axon advance. Posterior and ventromedial sclerotome are hypothesized to act as barriers to axon outgrowth due to some feature of their common cartilaginous development. Specific innervation patterns were also examined. When the notochord was displaced toward the control limb, axons on this side made and corrected projection errors, suggesting that the notochord can influence the precision of axonal pathway selection. In contrast, motor axons that entered the limb on all operated sides innervated muscle with their normal precision despite the absence of the somite and axonal segmentation. Therefore, the somite and the process of spinal nerve segmentation are largely irrelevant to the specificity of motoneuron projection.     

The distribution of neural crest-derived Schwann cells from subsets of brachial spinal segments into the peripheral nerves innervating the chick forelimb.

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.     

T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo.

Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity.     

The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

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.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

The neural tube origin of ventral root sheath cells in the chick embryo

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.     

Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral half-sclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite. It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed. Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.     

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.     

Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish.

The HNK-1 glycoepitope, carried by many cell recognition molecules, is present in the developing posterior lateral line nerve and on other primary axons of zebrafish. To elucidate the function of HNK-1 in vivo, the antibody 412 to HNK-1 was injected into zebrafish embryos at 16 h post fertilization (hpf). The injected antibody bound specifically to axons carrying HNK-1. This treatment selectively affected the growth of either one or both posterior lateral line nerves in 39% of the experimental cases (13 of 33 animals), which was significantly more (P<0.0002) than in uninjected, vehicle injected, and non-immune IgG injected controls (1.2% of the animals; one of 85 animals), as assessed at 27 or 33 hpf. Other HNK-1 immunoreactive nerves, such as the ventral motor nerves were unaffected, indicating that antibody binding per se did not interfere with axon growth. The primordium of the posterior lateral line was not affected in its caudal migration and in depositing differentiating neuromasts along the trunk, showing that injections did not retard development and that initial formation of lateral line organs is probably independent of contact with nerve fibers. We suggest that the HNK-1 glycoepitope is an important modulator of embryonic nerve growth.     

Distribution of motor and sensory fibers in the intercostal nerves. Significance in reconstructive surgery.

If the distribution of the types of nerve fibers in the various intercostal nerves is taken into consideration, an intercostal nerve segment can be an acceptable donor nerve graft for sensory and/or motor nerve replacements. We describe the distribution of motor and sensory axons in various segments of the upper and lower intercostal nerves.     

Evolution of the arthropod neuromuscular system. 2. Inhibitory innervation of the walking legs of a scorpion: Vaejovis spinigerus (Wood, 1863), Vaejovidae, Scorpiones, Arachnida

Inhibitory motoneurons which supply the leg musculature are identified and characterized in the scorpion, Vaejovis spinigerus (Wood, 1863) (Vaejovidae, Scorpiones, Arachnida). (1) Successive intracellular muscle fiber recordings from antagonists, and correlation of the monitored inhibitory postsynaptic potentials with spikes in motor nerves, suggest supply of the scorpion leg musculature by common inhibitory motoneurons. (2) Anti-GABA immunohistochemistry is combined with transmission electron microscopy to estimate the number of inhibitory motor axons present in the main leg nerve. The number of immunoreactive axons decreases toward more distal leg segments, from 14 to 18 in the basis to 6-8 in the tibia. No immunoreactive axons are detected beyond the tibia. (3) The distribution of putative inhibitory neurons in the subesophageal ganglion mass is determined by anti-GABA immunohistochemistry, revealing notable similarities to the situation in pterygote insects. This provides a framework for the characterization of the inhibitory motoneurons. (4) Backfills from leg nerves are combined with anti-GABA immunocytochemistry to identify inhibitory motoneurons in the central nervous system. Putative inhibitory motoneurons occur in three clusters per hemi-segment. Two clusters are located near the posterior edge of the neuromere, one lateral, the other more medial, and both contain ca. 8-10 cell bodies. The third cluster consists of two somata located contralaterally, just off the ganglion midline.