Neural crest cells and motor axons in avians: Common and distinct migratory molecules

It has long been thought that the same molecules guide both trunk neural crest cells and motor axons as these cell types grow and extend to their target regions in developing embryos. There are common territories that are navigated by these cell types: both cells grow through the rostral portion of the somitic sclerotomes and avoid the caudal half of the sclerotomes. However, these cell types seem to use different molecules to guide them to their target regions. In this Review, I will discuss the common and distinct methods of migration taken by trunk neural crest cells and motor axons as they grow and populate their target regions through chick embryos at the level of the trunk.Key words: migration, axon, motor neuron, trunk neural crest cells, chick     

The differing effects of occipital and trunk somites on neural development in the chick embryo

In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.     

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.     

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.     

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.     

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.     

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.     

Effects of mesodermal tissues on avian neural crest cell migration

We have used microsurgical techniques to investigate the effects of embryonic mesodermal tissues on the pattern of chick neural crest cell migration in the trunk. Segmental plate or lateral plate mesenchyme was transplanted into regions encountered by neural crest cells. We found that neural crest cells are able to migrate through lateral plate mesenchyme but not through segmental plate tissue until this tissue differentiates into a sclerotome. After this stage, segmental migration is controlled by the subdivision of the sclerotome into a rostral and a caudal half; when the rostrocaudal orientation of the sclerotomes is reversed by rotating the segmental plate 180 degrees about its rostrocaudal axis, neural crest cells migrate through the portion of the sclerotome that was originally rostral.     

Anterior/Posterior Influences on Neural Crest-Derived Pigment Cell Differentiation

The neural crest of vertebrate embryos has been used to elucidate steps involved in early embryonic cellular processes such as differentiation and migration. Neural crest cells form a ridge along the dorsal midline and subsequently they migrate throughout the embryo and differentiate into a wide variety of cell types. Intrinsic factors and environmental cues distributed along the neural tube, along the migratory pathways, and/or at the location of arrest influence the fate of neural crest cells. Although premigratory cells of the cranial and trunk neural crest exhibit differences in their differentiation potentials, premigratory trunk neural crest cells are generally assumed to have equivalent developmental potentials. Axolotl neural crest cells from different regions of origin, different stages of development, and challenged with different culture media have been analyzed for differentiation preferences pertaining to the pigment cell lineages. We report region-dependent differentiation of chromatophores from trunk neural crest at two developmental stages. Also, dosage with guanosine produces region-specific influences on the production of xanthophores from wild-type embryos. Our results support the hypothesis that spatial and temporal differences among premigratory trunk neural crest cells found along the anteroposterior axis influence developmental potentials and diminish the equivalency of axolotl neural crest cells.     

J1/tenascin-related molecules are not responsible for the segmented pattern of neural crest cells or motor axons in the chick embryo

It has been suggested that substrate adhesion molecules of the tenascin family may be responsible for the segmented outgrowth of motor axons and neural crest cells during formation of the peripheral nervous system. We have used two monoclonal antibodies (M1B4 and 578) and an antiserum [KAF9(1)] to study the expression of J1/tenascin-related molecules within the somites of the chick embryo. Neural crest cells were identified with monoclonal antibodies HNK-1 and 20B4. Young somites are surrounded by J1/tenascin immunoreactive material, while old sclerotomes are immunoreactive predominantly in their rostral halves, as described by other authors (Tan et al. 1987--Proc. natn. Acad. Sci. U.S.A. 84, 7977; Mackie et al. 1988--Development 102, 237). At intermediate stages of development, however, immunoreactivity is found mainly in the caudal half of each sclerotome. After ablation of the neural crest, the pattern of immunoreactivity is no longer localised to the rostral halves of the older, neural-crest-free sclerotomes. SDS-polyacrylamide gel electrophoresis of affinity-purified somite tissue, extracted using M1B4 antibody, shows a characteristic set of bands, including one of about 230 x 10(3), as described for cytotactin, J1-200/220 and the monomeric form of tenascin. Affinity-purified somite material obtained from neural-crest-ablated somites reveals some of the bands seen in older control embryos, but the high molecular weight components (120-230 x 10(3] are missing. Young epithelial somites also lack the higher molecular mass components. The neural crest may therefore participate in the expression of J1/tenascin-related molecules in the chick embryo. These results suggest that these molecules are not directly responsible for the segmented outgrowth of precursors of the peripheral nervous system.     

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.     

Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.     

Sequential activation of butyrylcholinesterase in rostral half somites and acetylcholinesterase in motoneurones and myotomes preceding growth of motor axons

By applying double-staining procedures that combine cholinesterase histochemistry (acetyl- and butyrylcholinesterase, respectively) as indicators of neuronal and myotomal tissue differentiation on longitudinal sections, together with detection of motor axons with antibodies to G4 antigen, we here describe the spatiotemporal expression of all components of the segmental motor units along the trunk of chicken embryos between stages 16-20. In particular, BChE expression is spatially elevated on the rostral part of the differentiating somite. About 2-3 somites more rostrally (and thus developmentally later), AChE is expressed almost simultaneously in a nonsegmented fashion in neuronal cell bodies of the ventral horn and in the corresponding dermomyotomes. There it is first detectable in a rostromedial sector. With a delay (4-6 somites compared with AChE in motoneurones), motor axons begin to grow exclusively through the BChE-rich sclerotomal space towards the AChE-activated myotome anlage. On motor axons, AChE detection is significantly retarded. We conclude that the rostrocaudal segmental asymmetry is not restricted to the sclerotomes (which other authors have described before by using different markers), but it extends into the dermomyotome, in which cholinesterases introduce an early subdivision. Hence, the entire process of first myotome differentiation, motor axon growth and establishment of first target contacts are taking place within the rostral half somite. We suggest that both cholinesterases might be involved in processes of motor unit differentiation and fibre guidance.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

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.     

Migrating neural crest cells in the trunk of the avian embryo are multipotent

Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural tube either during their migration or at their sites of localization. Here, we test the developmental potential of migrating trunk neural crest cells by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells as they migrate through the somite. By two days after injection, the LRD-labelled clones contained from 2 to 67 cells, which were distributed unilaterally in all embryos. Most clones were confined to a single segment, though a few contributed to sympathetic ganglia over two segments. A majority of the clones gave rise to cells in multiple neural crest derivatives. Individual migrating neural crest cells gave rise to both sensory and sympathetic neurons (neurofilament-positive), as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells (both neurofilament-negative). Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. Our data demonstrate that migrating trunk neural crest cells can be multipotent, giving rise to cells in multiple neural crest derivatives, and contributing to both neuronal and non-neuronal elements within a given derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Molecular differences between the rostral and caudal halves of the sclerotome in the chick embryo

It is known that both neural crest cell migration and motor axon outgrowth in most vertebrate embryos are segmented because of restrictions imposed upon their distribution by the neighbouring sclerotomes, each of which is divided into a rostral and a caudal half. The caudal half does not allow crest migration or axon outgrowth, while the rostral half does. In this paper, we investigate the expression of proteins and glycoproteins in the two halves of the sclerotome of the chick embryo at stages between 20 and 32 pairs of somites by two-dimensional SDS-polyacrylamide gel electrophoresis. We find that the patterns of expression are complex, and that polypeptides and glycoproteins vary both spatially and temporally: of those that are expressed differentially by the sclerotome, some differ quantitatively and others qualitatively. Some macromolecules change their spatial distribution with developmental age, and some appear or disappear as the embryos become older.     

Cholinesterases preceding major tracts in vertebrate neurogenesis

The role of acetylcholinesterase (AChE) in neurotransmission is well known. But long before synapses are formed in vertebrates, AChE is expressed in young postmitotic neuroblasts that are about to extend the first long tracts. AChE histochemistry can thus be used to map primary steps of brain differentiation. Preceding and possibly inducing AChE in avian brains, the closely related butyrylcholinesterase (BChE) spatially foreshadows AChE-positive cell areas and the course of their axons. In particular, before spinal motor axons grow, their corresponding rostral sclerotomes and myotomes express BChE, and both their neuronal source and myotomal target cells express AChE. Since axon growth has been found inhibited by acetylcholine, it is postulated that both cholinesterases can attract neurite growth cones by neutralizing the inhibitor. Thus, the early expression of both cholinesterases that is at least partially independent from classical cholinergic synaptogenesis, sheds new light on the developmental and medical significance of these enzymes.