Carol A. Erickson

Neural crest cell migratory pathways in the trunk of the chick embryo

Neural crest cells migrate during embryogenesis to give rise to segmented structures of the vertebrate peripheral nervous system: namely, the dorsal root ganglia and the sympathetic chain. However, neural crest cell arise from the dorsal neural tube where they are apparently unsegmented. It is generally agreed that the somites impose segmentation on migrating crest cells, but there is a disagreement about two basic questions: exactly pathways do neural crest cells use to move through or around somites, and do neural crest cells actively migrate or are they passively dispersed by the movement of somite cells? The answers to both questions are critically important to any further understanding of the mechanisms underlying the precise distribution of the neural crest cells that develop into ganglia. We have done an exhaustive study of the locations of neural crest cells in chick embryos during early stages of their movement, using antibodies to neural crest cells (HNK-1), to neural filament-associated protein in growing nerve processes (E/C8), and to the extracellular matrix molecule laminin. Our results show that Some neural crest cells invade the extracellular space between adjacent somites, but the apparent majority move into the somites themselves along the border between the dermatome/myotome (DM) and the sclerotome. Neural crest cells remain closely associated with the anterior half of the DM of developing somites as they travel, suggesting that the basal lamina of the DM may be used as a migratory substratum. Supporting this idea is our observation that the development of the DM basal lamina coincides in time and location with the onset of crest migration through the somite. The leading front of neural crest cells advance through the somite while the length of the DM pathway remains constant, suggesting active locomotion, at least in this early phase of development. Neural crest cells leave the DM at a later stage of development to associate with the dorsal aorta, where sympathetic ganglia form, and to associate with newly emerging fibers of the ventral root nerve, where they presumably give rise to neuronal supportive cells. Thus we propose that the establishment of the segmental pattern of the peripheral ganglia and nerves depends on the timely development of appropriate substrata to guide and distribute migrating neural crest cells during the early stages of embryogenesis.

The making of a melanocyte: the specification of melanoblasts from the neural crest

Melanocytes differentiate from the neural crest (NC), which is a transient population of cells that delaminates from the neural tube and migrates extensively throughout the embryo during vertebrate development. Melanoblast specification from NC precursors is a progressive process during which initially pluripotent cells become restricted to the melanogenic lineage and adopt the gene expression profile and morphology of melanocytes. This specification process is governed primarily by Wnt and BMP signaling molecules, although other signaling pathways, such as those activated by Kit and Endothelin 3, can also stimulate melanogenesis. The transcriptional repressor FoxD3 occupies a central role in melanocyte fate determination by repressing melanogenesis in premigratory NC cells and in other NC lineages.

Descriptive and experimental analysis of the dispersion of neural crest cells along the dorsolateral path and their entry into ectoderm in the chick embryo

We have characterized the dispersion of neural crest cells along the dorsolateral path in the trunk of the chicken embryo and experimentally investigated the control of neural crest cell entry into this path. The distribution of putative neural crest cells was analyzed in plastic sections of embryos that had been incubated for 24 hr in HNK-1 antibody, a procedure that we show successfully labels neural crest cells in the dorsolateral path and ectoderm. In accord with earlier observations, crest cells delay entering the dorsolateral path until a day or more after their counterparts have colonized the ventral path. However, once crest cells enter, they disperse rapidly through the path dorsal to the somite but still delay migrating dorsal to the intersegmental space. During dispersion, crest cells invade the ectoderm at sites associated with local disruptions in the basal lamina which may be caused by crest cells. Finally, deleting the dermamyotome releases an inhibition of neural crest cell migration: crest cells enter the dorsolateral path precociously. We speculate that the epithelial dermatome may transiently produce inhibitory substances and that emerging dermis may provide a long-distance, stimulatory cue.

Stripes and belly-spots – a review of pigment cell morphogenesis in vertebrates

Pigment patterns in the integument have long-attracted attention from both scientists and non-scientists alike since their natural attractiveness combines with their excellence as models for the general problem of pattern formation. Pigment cells are formed from the neural crest and must migrate to reach their final locations. In this review, we focus on our current understanding of mechanisms underlying the control of pigment cell migration and patterning in diverse vertebrates. The model systems discussed here - chick, mouse, and zebrafish - each provide unique insights into the major morphogenetic events driving pigment pattern formation. In birds and mammals, melanoblasts must be specified before they can migrate on the dorsolateral pathway. Transmembrane receptors involved in guiding them onto this route include EphB2 and Ednrb2 in chick, and Kit in mouse. Terminal migration depends, in part, upon extracellular matrix reorganization by ADAMTS20. Invasion of the ectoderm, especially into the feather germ and hair follicles, requires specific signals that are beginning to be characterized. We summarize our current understanding of the mechanisms regulating melanoblast number and organization in the epidermis. We note the apparent differences in pigment pattern formation in poikilothermic vertebrates when compared with birds and mammals. With more pigment cell types, migration pathways are more complex and largely unexplored; nevertheless, a role for Kit signaling in melanophore migration is clear and indicates that at least some patterning mechanisms may be highly conserved. We summarize the multiple factors thought to contribute to zebrafish embryonic pigment pattern formation, highlighting a recent study identifying Sdf1a as one factor crucial for regulation of melanophore positioning. Finally, we discuss the mechanisms generating a second, metamorphic pigment pattern in adult fish, emphasizing recent studies strengthening the evidence that undifferentiated progenitor cells play a major role in generating adult pigment cells.

MMP‐2 plays an essential role in producing epithelial‐mesenchymal transformations in the avian embryo

To investigate the roles that matrix‐degrading proteases may have in development of the chicken embryo, we documented the expression pattern of matrix metalloprotease‐2 (MMP‐2, 72‐kDa type IV collagenase or gelatinase A) and perturbed its function in vitro and in vivo. MMP‐2 is expressed as neural crest cells detach from the neural epithelium during an epithelial‐mesenchymal transformation (EMT) but is rapidly extinguished as they disperse. It is also expressed in the sclerotome and in the dermis at the time that the EMT is initiated, and also as these cells migrate, and is down‐regulated once motility has ceased. These patterns suggest that MMP‐2 plays a role in cell motility during the EMT and during later morphogenesis. Inhibitors of MMPs, including BB‐94 and TIMP‐2 (tissue inhibitor of metalloprotease‐2), prevent the EMT that generates neural crest cells, both in tissue culture and in vivo, but do not affect migration of the cells that have already detached from the neural tube. Similarly, knockdown of MMP‐2 expression in the dorsal neural tube using antisense morpholino oligos perturbs the EMT, but also does not affect migration of neural crest cells after they have detached from the neural tube. On the other hand, when somites in culture are treated with TIMP‐2, some mesenchymal cells are produced, suggesting that they undergo the EMT, but show greatly reduced migration through the collagen gel. MMP‐2 is also expressed in mesenchyme where tissue remodeling is in progress, such as in the developing feather germs, in the head mesenchyme, in the lateral plate mesoderm, and in the limb dermis, especially in the regions where tendons are developing. Comparisons of these expression patterns in multiple embryonic tissues suggest a probable role for MMP‐2 in the migration phase of the EMT, in addition to mesenchyme dispersion and tissue remodeling. Developmental Dynamics 229:42–53, 2004.

FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism.

The first neural crest cells to emigrate from the neural tube are specified as neurons and glial cells and are subsequently followed by melanocytes of the skin. We wished to understand how this fate switch is controlled. The transcriptional repressor FOXD3 is expressed exclusively in the neural/glial precursors and MITF is expressed only in melanoblasts. Moreover, FOXD3 represses melanogenesis. Here we show that avian MITF expression begins very early during melanoblast migration and that loss of MITF in melanoblasts causes them to transdifferentiate to a glial phenotype. Ectopic expression of FOXD3 represses MITF in cultured neural crest cells and in B16-F10 melanoma cells. We also show that FOXD3 does not bind directly to the MITF promoter, but instead interacts with the transcriptional activator PAX3 to prevent the binding of PAX3 to the MITF promoter. Overexpression of PAX3 is sufficient to rescue MITF expression from FOXD3-mediated repression. We conclude that FOXD3 controls the lineage choice between neural/glial and pigment cells by repressing MITF during the early phase of neural crest migration.

Neural crest development: the interplay between morphogenesis and cell differentiation.

The final pattern of tissues established during embryogenesis reflects the outcome of two developmental processes: differentiation and morphogenesis. Avian neural crest cells are an excellent system in which to study this interaction. In the first phase of neural crest cell migration, neural crest cells separate from the neural epithelium via an epithelial-mesenchymal transformation. We present three models to account for this process: (1) separation by asymmetric mitosis, (2) separation by generating tractional force in order to rupture cell adhesions and (3) loss of expression or function of cell-cell adhesion molecules that keep the presumptive neural crest cells tethered to the neural epithelium. Evidence is presented that the segregation of the neural crest lineage apart from the neural epithelium is caused by the epithelial-mesenchymal transformation. Once they have detached from the neural tube, neural crest cells take two pathways in the trunk of the chick embryo: (1) the ventral path between the neural tube and somite, where neural crest cells give rise to neurons and glial cells of the peripheral nervous systems, and (2) the dorsolateral path between the ectoderm and dermamyotome of the somite, where they differentiate into pigment cells of the skin. We present data to suggest that the migration and differentiation along the ventral path is controlled primarily by environmental cues, which we refer to as the environment-directed model of neural crest morphogenesis. Conversely, only melanoblasts can migrate into the dorsolateral space, and the ability to invade that path is dependent upon their early specification as melanoblasts. We call this the phenotype-directed model for neural crest cell migration and suggest that this latter model for the positioning of neural crest derivatives in the embryo may be more common than previously suspected. These observations invite a re-examination of patterning of other crest derivates, which previously were believed to be controlled by environmental cues.

Control of neural crest cell dispersion in the trunk of the avian embryo

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology and behavior of quail neural crest cells in artificial three-dimensional extracellular matrices☆

Neural crest cells migrate extensively through a complex extracellular matrix (ECM) to sites of terminal differentiation. To determine what role the various components of the ECM may play in crest morphogenesis, quail (Coturnix coturnix japonica) neural crest cells have been cultured in three-dimensional hydrated collagen lattices containing various combinations of macromolecules known to be present in the crest migratory pathways. Neural crest cells migrate readily in native collagen gels whereas the cells are unable to use denatured collagen as a migratory substratum. The speed of movement decreases linearly as the concentration of collagen in the gel increases. Speed of movement of crest cells is stimulated in gels containing 10% fetal calf serum and chick embryo extract, 33 micrograms/ml fibronectin cell-binding fragments, 3 mg/ml chondroitin sulfate, or 3 mg/ml chondroitin sulfate proteoglycan when compared to rates of movement through collagen lattices alone. Low concentrations of hyaluronate (250-500 micrograms/ml) in a 750 micrograms/ml collagen gel do not alter rates of movement over collagen alone, but higher concentrations (4 mg/ml) greatly inhibit migration. Conversely, hyaluronate (250 micrograms/ml) significantly increases speed of movement if the crest cells are cultured in high concentration collagen gels (2.5 mg/ml), suggesting that hyaluronate is expanding spaces and consequently enhancing migration. The morphology and mode of movement of neural crest cells vary with the matrix in which they are grown and can be correlated with their speed of movement. Light and scanning electron microscopy reveal rounded, blebbing cells in matrices associated with slower translocation, whereas rounded cells with branching filopodia or lamellipodia are associated with rapid translocation. Bipolar cells with long processes are observed in cultures of rapidly moving cells that appear to be adhering strongly, as well as in cultures of cells that are stationary for long periods. These data, considered with the known distribution of macromolecules in the early embryo, suggest the following: (1) Both collagen and fibronectin can act as preferred substrata for migration. (2) Chondroitin sulfate and chondroitin sulfate proteoglycan increase speed of movement, but probably do so by decreasing adhesiveness and thereby producing more frequent detachment. In the embryo, crest cells would most likely avoid regions containing high concentrations of chondroitin sulfate. (3) Hyaluronate cannot act as a substratum for migration, but in low concentrations it can open spaces in the matrix and consequently may stimulate movement. The complex interactions of combined matr

Migratory pathways of HNK-1-immunoreactive neural crest cells in the rat embryo.

In avian embryos, the precursors of the peripheral nervous system, the neural crest cells, migrate along precise pathways limited to the anterior half of each somite and the intersomitic space. This segmental migration foreshadows the development of segmented peripheral ganglia and thus may be critical to normal neuronal development. We report here that a remarkably similar pattern of migration of HNK-1-immunoreactive cells, which we believe to be neural crest cells, exists in the rat embryo, suggesting that the underlying mechanisms of neural crest guidance may be the same in avian and mammalian embryonic development.