Cell lineage analysis of the avian neural crest

Neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. A major unanswered question concerning the neural crest is when and how the neural crest cells become determined to adopt a particular fate. We have explored the developmental potential of trunk neural crest cells in avian embryos by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells within the dorsal neural tube. We find that premigratory and emigrating neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. These 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 emigration from the neural tube either during their migration or at their sites of localization. To determine whether neural crest cells become restricted during their migration, we have microinjected individual trunk neural crest cells with dye shortly after they leave the neural tube or as they migrate through the somite. We find that a majority of the clones derived from migrating neural crest cells appear to be multipotent; individual migrating neural crest cells gave rise to both sensory and sympathetic neurons, as well as cells with the morphological characteristics of Schwann cells, and other nonneuronal cells. Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. These data demonstrate that migrating trunk neural crest cells, like their premigratory progenitors, can be multipotent. They give rise to cells in multiple neural crest derivatives and contribute to both neuronal and non-neuronal elements within a given derivative. Thus, restriction of neural crest cell fate must occur relatively late in migration or at the final destinations

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. Thus, restriction of neural crest cell fate must occur relatively late in migration or at the final sites of neural crest cell localization

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway

Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling

Analysis of neural crest cell migration in the mouse has been difficult due to the lack of reliable cell markers. Recently, we found that injection of DiI into the chick neural tube marks premigratory neural crest cells whose endfeet are in contact with the lumen of the neural tube (Serbedzija et al. Development 106, 809–819 (1989)). In the present study, this technique was applied to study neural crest cell migratory pathways in the trunk of the mouse embryo. Embryos were removed from the mother between the 8th and the 10th days of development and DiI was injected into the lumen of the neural tube. The embryos were then cultured for 12 to 24 h, and analyzed at the level of the forelimb. We observed two predominant pathways of neural crest cell migration: (1) a ventral pathway through the rostral portion of the somite and (2) a dorsolateral pathway between the dermamyotome and the epidermis. Neural crest cells were observed along the dorsolateral pathway throughout the period of migration. The distribution of labelled cells along the ventral pathway suggested that there were two overlapping phases of migration. An early ventrolateral phase began before E9 and ended by E9.5; this pathway consisted of a stream of cells within the rostral sclerotome, adjacent to the dermamyotome, that extended ventrally to the region of the sympathetic ganglia and the dorsal aorta

Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent

Segmental migration of trunk neural crest: time-lapse analysis reveals a role for PNA-binding molecules

Trunk neural crest cells migrate through the somites in a striking segmental fashion, entering the rostral but not caudal sclerotome, via cues intrinsic to the somites. Attempts to define the molecular bases of these cues have been hampered by the lack of an accessible assay system. To examine trunk neural crest migration over time and to perturb candidate guiding molecules, we have developed a novel explant preparation. Here, we demonstrate that trunk regions of the chicken embryo, placed in explant culture, continue to develop apparently normally for 2 days. Neural crest cells, recognized by prelabeling with DiI or by poststaining with the HNK-1 antibody, migrate in the somites of the explants in their typical segmental pattern. Furthermore, this paradigm allows us to follow trunk neural crest migration in situ for the first time using low-light-level videomicroscopy. The trajectories of individual neural crest cells were often complex, with cells migrating in an episodic mode encompassing forward, backward and lateral movements. Frequently, neural crest cells migrated in close-knit groups of 2–4 cells, moving at mean rates of migration of 10–14 µm/hour. Treatment of the explants with the lectin peanut agglutinin (PNA) both slowed the rate and altered the pattern of neural crest migration. Neural crest cells entered both the rostral and caudal halves of the sclerotome with mean rates of migration ranging from 6 to 13 µm/hour. These results suggest that peanut agglutinin-binding molecules are required for the segmental patterning of trunk neural crest migration. Because this approach permits neural crest migration to be both observed and perturbed, it offers the promise of more direct assays of the factors that influence neural crest development

Developmental potential of trunk neural crest cells in the mouse

The availability of naturally occurring and engineered mutations in mice which affect the neural crest makes the mouse embryo an important experimental system for studying neural crest cell differentiation. Here, we determine the normal developmental potential of neural crest cells by performing in situ cell lineage analysis in the mouse by microinjecting lysinated rhodamine dextran (LRD) into individual dorsal neural tube cells in the trunk. Labeled progeny derived from single cells were found in the neural tube, dorsal root ganglia, sympathoadrenal derivatives, presumptive Schwann cells and/or pigment cells. Most embryos contained labeled cells both in the neural tube and at least one neural crest derivative, and numerous clones contributed to multiple neural crest derivatives. The time of injection influenced the derivatives populated by the labeled cells. Injections at early stages of migration yielded labeled progeny in both dorsal and ventral neural crest derivatives, whereas those performed at later stages had labeled cells only in more dorsal neural crest derivatives, such as dorsal root ganglion and presumptive pigment cells. The results suggest that in the mouse embryo: (1) there is a common precursor for neural crest and neural tube cells; (2) some neural crest cells are multipotent; and (3) the timing of emigration influences the range of possible neural crest derivatives

Order and coherence in the fate map of the zebrafish nervous system

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns

Violation of cell lineage restriction compartments in the chick hindbrain

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development