No abstract

Pigment patterns in the integument have long-attracted attention from both scientists and nonscientists 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.

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo . We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields . These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction . The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater . These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10-fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range .

SummaryMelanocytes 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.

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.

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.

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.

An epithelial-mesenchymal transition (EMT) is the process whereby epithelial cells become mesenchymal cells, and is typified by the generation of neural crest cells from the neuroepithelium of the dorsal neural tube. To investigate the neural crest EMT, we performed live cell confocal time-lapse imaging to determine the sequence of cellular events and the role of cell division in the EMT. It was observed that in most EMTs, the apical cell tail is retracted cleanly from the lumen of the neuroepithelium, followed by movement of the cell body out of the neural tube. However, exceptions to this sequence include the rupture of the neural crest cell tail during retraction (junctional complexes not completely downregulated), or translocation of the cell body away from the apical surface while morphologically rounded up in M phase (no cell tail retraction event). We also noted that cell tail retraction can occur either before or after the redistribution of apical-basolateral epithelial polarity markers. Surprisingly, we discovered that when an EMT was preceded by a mitotic event, the plane of cytokinesis does not predict neural crest cell fate. Moreover, when daughter cells are separated from the adherens junctions by a parallel mitotic cleavage furrow, most re-establish contact with the apical surface. The diversity of cellular mechanisms by which neural crest cells can separate from the neural tube suggests that the EMT program is a complex network of non-linear mechanisms that can occur in multiple orders and combinations to allow neural crest cells to escape from the neuroepithelium.