SEARCH

SEARCH BY CITATION

Keywords:

  • gut;
  • skin;
  • melanocyte;
  • neural crest;
  • development

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The development of melanocytes from neural crest-derived precursors that migrate along the dorsolateral pathway has been attributed to the selection of this route by cells that are fate-restricted to the melanocyte lineage. Alternatively, melanocytes could arise from nonspecified cells that develop in response to signals encountered while these cells migrate, or at their final destinations. In most animals, the bowel, which is colonized by crest-derived cells that migrate through the caudal branchial arches, contains no melanocytes; however, the enteric microenvironment does not prevent melanocytes from developing from crest-derived precursors placed experimentally into the bowel wall. To test the hypothesis that the branchial arches remove the melanogenic potential from the crest-derived population that colonizes the gut, the Silky fowl (in which the viscera are pigmented) was studied. Sources of crest included Silky fowl and quail vagal and truncal neural folds/tubes, which were cultured or explanted to chorioallantoic membranes alone or together with branchial arches or limb buds from Silky fowl, White Leghorn, or quail embryos. Crest and mesenchyme-derived cells were distinguished by using the quail nuclear marker. Melanocytes developed from Silky fowl and quail crest-derived cells. Melanocyte development from both sources was inhibited by quail and White Leghorn branchial arches (and limb buds), but melanocyte development was unaffected by branchial arch (and limb buds) from Silky fowl. These observations suggest that a factor(s) that is normally expressed in the branchial arches, and is lacking in animals with the Silky mutation, prevents cells with a melanogenic potential from colonizing the bowel. Anat Rec 268:16–26, 2002. © 2002 Wiley-Liss, Inc.

The neural crest of vertebrate embryos is a transient structure that is comprised of precursor cells that ultimately give rise to a plethora of terminally differentiated cell types (Le Douarin, 1982; Anderson, 1989; Weston, 1991). The neural crest disappears because its cells dissipate by migrating along defined pathways that lead them to a wide variety of final destinations. Previous studies have suggested that at least some of the cells of the premigratory crest are multipotent (Bronner-Fraser and Fraser, 1988, 1989), continue to be so after migration has begun (Fraser and Bronner-Fraser, 1991), are still multipotent as they migrate through branchial arches (Ito and Sieber-Blum, 1993), and remain multipotent after they arrive at their target organs (Rothman et al., 1990, 1993; Duff et al., 1991; Sextier-Sainte-Claire Deville et al., 1994; Lo and Anderson, 1995). The conclusion that crest-derived precursors are multipotent has been supported by analyzing the progeny of clones of these cells in vitro (Sieber-Blum and Cohen, 1980; Baroffio et al., 1988; Dupin et al., 1990; Ito and Sieber-Blum, 1991, 1993; Sextier-Sainte-Claire Deville et al., 1992; Stemple and Anderson, 1992, 1993). Although it is clear that some premigratory and early migrating crest-derived cells are multipotent (Sieber-Blum et al., 1993), many cells of the same population appear to be specified and fate restricted (Henion and Weston, 1997). Since the premigratory crest population is heterogeneous, it is possible that subsets of specified crest-derived cells choose to migrate along lineage-specific pathways, while subsets of nonspecified crest-derived cells migrate along pathways chosen independently of lineage. The microenvironment that nonspecified crest-derived cells encounter along their migratory pathway or in their target organs may contribute to specifying their lineage and restricting their fate. A model has been presented that accounts for the patterning of crest derivatives by attributing it to the differential selection of appropriate migratory pathways by precursors with cell-autonomous properties that have become fate-restricted at the time they segregate from the neural tube (Erickson and Goins, 1995; Reedy et al., 1998a, b; Faraco et al., 2001). Evidence suggests that the leading edge of the laterally migrating neural crest population contains pluripotent precursors (Sieber-Blum et al., 1993), whereas the trailing edge consists primarily of cells that have been specified to develop as melanocytes (Artinger and Bronner-Fraser, 1992).

The possibility that crest-derived cells become fate-specified before they migrate, and then select the migratory pathways they ultimately follow on the basis of that predetermined specification, has most strongly been supported by studies of cell migration from the truncal level of the neural crest (Erickson and Goins, 1995; Reedy et al., 1998a, b; Faraco et al., 2001). A substantial proportion of the subpopulations of cells in the truncal neural crest have been demonstrated by in vitro clonal studies to be lineage-restricted (Sieber-Blum and Cohen, 1980; Ito and Sieber-Blum, 1991, 1993; Henion and Weston, 1997). At this axial level, crest-derived cells that are specified to develop as melanocytes preferentially depart from the neural tube after neurogenic precursors have done so; moreover, the choice made by these late-migrating precursors to migrate along the dorsolateral pathway is apparently related to their having been specified as melanocytes (Erickson and Goins, 1995; Reedy et al., 1998a, b; Faraco et al., 2001). For example, following back-transplantation into younger host embryos, crest-derived cells that have been specified as melanocytes immediately and preferentially exploit the dorsolateral migration pathway. In contrast, similarly back-transplanted crest-derived cells that have not been specified, or which have been specified to develop as neurons, are excluded from the dorsolateral pathway. More recently, the winged-helix transcription factor, FoxD3, was shown to be expressed by all crest-derived lineages except the late-emigrating melanoblasts (Kos et al., 2001). FoxD3 represses melanogenesis, and by doing so it may allow other neural crest lineages to develop. FoxD3 expression has also been postulated to play a role in the segregation of the neural crest lineage from the neuroepithelium.

The vagal level of the crest develops earlier than that of the trunk and has been shown to give rise to three (rather than two, as in the trunk) waves of precursor emigration (Reedy et al., 1998b). It is therefore possible that the proportion of nonspecified cells in the vagal crest is higher than in the trunk, particularly in the early wave of vagal crest-derived precursors that colonize the branchial arches. The early vagal crest-derived cells initially express FoxD3 (Kos et al., 2001). In vitro, vagal crest-derived cells, sampled before their entry into the posterior branchial arches, have the capacity to give rise to melanocytes (Ito and Sieber-Blum, 1991). Vagal crest-derived cells in the branchial arches, however, are not melanoblasts and do not form melanocytes when explanted in vitro (Ciment, 1983; Reedy et al., 1998b); moreover, clones obtained from crest-derived cells isolated from within the branchial arches also do not give rise to melanocytes (Ito and Sieber-Blum, 1993). Vagal crest-derived cells migrate through the caudal branchial arches to reach targets such as the gut and the heart, within which they do not form melanocytes (Payette et al., 1984; Tucker et al., 1986; Kirby, 1987; Phillips et al., 1987; Pomeranz and Gershon, 1990; Burns and Le Douarin, 2001). On the other hand, vagal and other crest-derived cells give rise to large numbers of melanocytes within the bowel if they are permitted to migrate into explants of gut without passing through the branchial arches (Le Douarin and Teillet, 1974).

The failure of cells that enter or pass through the branchial arches to display a melanocytic potential may be due to the absence of such cells from the population of vagal crest-derived cells that colonizes the branchial arches. Conceivably, as in the trunk, fate specification may direct the choice of migratory routes taken by melanogenic precursors at the vagal level of the neuraxis. Crest-derived cells able to give rise to melanocytes may emigrate from the vagal neural crest later than the subpopulation that colonizes the branchial arches and migrate along a different dorsolateral pathway. Alternatively, multipotent crest-derived precursors could actually enter the branchial arches and receive signals from the microenvironment of the branchial arches that bias them against melanogenesis. If so, then the microenvironment rather than prior determination would be responsible for the nonmelanocytic fate of this population of crest-derived cells. The patterning of neural crest derivatives may thus be the result of both determined progenitors following migratory pathways chosen according to their specified fates, and of undetermined precursors migrating along pathways chosen independently of lineage, which encounter developmental cues on their route. It is thus important to determine whether the microenvironment of the branchial arches can suppress the ability of multipotent crest-derived cells to give rise to melanocytes.

Ectopic development of melanocytes occurs naturally in mutant Silky fowl (Dunn and Jull, 1927; Hallet and Ferrand, 1984). The viscera of Silky animals are pigmented because they contain ectopic crest-derived melanocytes. Within the Silky gut, the melanocytes are found in the region of the enteric plexuses, suggesting that they arise from crest-derived cells that follow the normal crest migration pathway in the bowel, but develop abnormally. The melanocytic defect in the Silky animals is not neural crest autonomous (Hallet and Ferrand, 1984). When quail neural tubes are grafted into Silky embryos, quail crest-derived cells form melanocytes within the viscera of the resulting chimeric embryos. In contrast, when Silky neural tubes are grafted into White Leghorn chick embryos, the viscera remain unpigmented. Quail crest cells will even colonize the bowel and other organs of Silky fowl embryos that are grafted to the coelomic cavity of quail host embryos (Ferrand and L'Hermite, 1985). These experiments with Silky fowl suggest that subsets of crest-derived cells that migrate ventrally have the capacity to develop as melanocytes and are normally prevented from doing so, either by the microenvironment (which is abnormal in Silky fowl) they encounter along their migratory pathway or by the microenvironment in the organs they colonize.

Although crest-derived cells from the trunk that are specified to develop as melanocytes preferentially migrate dorsolaterally (Erickson and Goins, 1995), the observation that quail crest-derived cells migrating in Silky hosts give rise to melanocytes in the gut and other viscera (Hallet and Ferrand, 1984; Ferrand and L'Hermite, 1985), makes it clear that cells with a melanogenic potential (at least in these birds) can also migrate ventrally. When quail neural rudiments are implanted directly into the primordial bowel of White Leghorn chick hosts and grown as chorioallantoic membrane (CAM) grafts, crest-derived cells form melanocytes in the enteric plexuses of the host gut (Le Douarin and Teillet, 1974). The White Leghorn bowel thus does not prevent the expression of the melanocyte phenotype by quail crest cells. Therefore, the failure of the quail crest-derived cells that migrate to the bowel in chimeric quail Leghorn embryos to give rise to melanocytes is likely due to an effect exerted on migrating crest-derived cells before they reach the gut. The current experiments were carried out to test the hypothesis that the branchial arches, through which crest-derived cells migrate en route to the bowel, act as a barrier that prevents the colonization of internal organs by cells with a melanogenic potential. Conceivably, the branchial arches could exclude crest-derived cells that have been specified as melanocytes. If Silky fowl branchial arches lack this ability, specified melanogenic cells could migrate through them to reach their ectopic destinations. Alternatively, the microenvironment of the branchial arches might suppress the melanogenic potential of crest-derived cells, specified or not.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

Fertilized quail eggs were obtained from Karasoulas Farms (Lake Elsinore, CA). Silky fowl eggs were obtained from Karasoulas Farms, Stromberg Farms (MI), Duane Urch (Owatonna, MN), and Dr. Ursula Abbott (University of California–Davis). White Leghorn chicken eggs were purchased from Truslow Farms (MD). All eggs were incubated at 38°C and 60–70% relative humidity in a forced-draft incubator. Chick embryos were staged according to Hamburger and Hamilton (1951) (H-H stages), while quail embryos were staged according to Zacchei (1961) (Z stages). White Leghorn animals have a cell autonomous pigmentation defect; however, this mutation involves the late degeneration of melanocytes and does not affect their differentiation from crest-derived precursors (Jimbow et al., 1974; Reedy et al., 1998b).

Tissue Culture

Neural folds were removed with sharpened tungsten needles from the vagal levels of the neuraxis of quail (Z stages 5–7), Silky fowl, or White Leghorn embryos (H-H stages 7–9). Branchial arches 3 and 4, and hind limb buds were removed from chick and Silky fowl embryos at H-H stages 19–21, and from quail embryos at Z stages 15 and 16. Epithelium and blood vessels were dissected away from the arches and only the remaining mesenchyme was retained. The apical ectodermal ridge and epithelium were removed from the limb buds and, again, only the mesenchyme was retained. The neural folds, branchial arches, and limb buds were placed alone or together on polycarbonate filters (0.6 μm pore size, Nuclepore™, Costar; Scientific Corp., Cambridge, MA). For co-cultures of the neural folds with either branchial arch or limb bud mesenchyme, the neural folds were plated first, and then covered by the co-cultured mesenchyme. In additional experiments, the truncal neural tube was removed from quail (Z stages 9–11) and Silky fowl (H-H stages 11–13) embryos (12–18 somites). The neural tubes were briefly incubated in Mg2+/Ca2+-free phosphate-buffered saline (PBS) containing 0.25% Dispase™ (Worthington, Lakeland, NJ) and cleaned of adherent tissue. The neural tubes were then placed on filters as described above, either by themselves or in combination with branchial arch or limb bud mesenchyme. The filters carrying the tissues were suspended on wire grids in 35-mm organ culture dishes over a medium consisting of Eagle's minimum essential medium (MEM) supplemented with 15% heat-inactivated horse serum and 10% chick embryo extract (E11; CEE) (Howard and Gershon, 1993). Tissues were incubated at 37°C in an atmosphere of 5% CO2 for 7–10 days.

CAM Grafts

Vagal neural folds, branchial arch mesenchyme, and truncal neural tubes were explanted from quail, Silky fowl, and White Leghorn embryos as described above. Vagal neural tubes (levels of somites 2–6), obtained from quail embryos of 10 somites (Z stage 8), were also used in these experiments. The neural tubes, both truncal and vagal, were cleaned of adherent tissue as described above. Tissues were placed on Nuclepore™ filters or polycarbonate track-etched filters (Poretics Corp., Livermore, CA, and Millipore Corp., Bedford, MA), which were then inverted and placed on the CAMs of E6 or E7 chick embryos so that the tissue was sandwiched between the filters and the CAMs. The grafts were maintained on the CAMs for 9–13 days. The neural folds, tubes, and branchial arch mesenchyme were grown on the CAMs by themselves or, alternatively, the neural structures were co-grafted with the branchial arch mesenchyme. Following maintenance on the CAM, the grafts were dissected free from the membranes in PBS for processing.

Histology

Cultures and grafts were fixed for 1–2 hr in Carnoy's solution in the dark at 4°C. The fixed tissues were dehydrated through a graded series of ethanols, cleared in xylene, embedded in paraffin, and sectioned at 8–10 μm. Sections were stained by the method of Feulgen and Rossenbeck (1924) to demonstrate the quail nuclear marker DNA (Le Douarin, 1973). In some experiments, crest-derived cells in the cultures and CAM grafts were identified specifically with NC-1 monoclonal antibodies (Vincent and Thiery, 1984). Bound antibodies were located with goat anti-mouse secondary antibodies coupled to alkaline phosphatase and visualized with a blue reaction product (#5300; Vector Laboratories Inc., Burlingame, CA). The stained sections were examined microscopically using both brightfield and Nomarski interference contrast optics. Quail cells were identified by their characteristic Feulgen-stained nucleolar-associated heterochromatin. Melanocytes were identified by their content of brown-black pigment granules. Individual cells were recognized as separate by using interference-contrast optics. Every third section was analyzed quantitatively. Cell numbers were determined by counting nuclei. Images were digitized with a microcomputer-imaging device (MCID IV; Imaging Research Inc., St. Catherines, Canada). The total area of the tissue was measured by computer-assisted morphometry. The proportions of the total area occupied by chick cells, quail cells, and pigment cells of quail or chick origin were then determined. Data was recorded both as the number of cells of each type per unit area, and as the percent of total area occupied by each type of cell.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Individual Tissues

Melanocytes were observed in all cultures of quail (5/5; Fig. 1A) and Silky (7/7; Fig. 1B) vagal neural folds. They were also observed in all cultures of quail and Silky truncal neural tube, which included the presumptive neural crest (not illustrated). In contrast, no melanocytes were observed in cultures of quail (0/17; Fig. 1C), Silky (0/13; Fig. 1D), or White Leghorn (0/14) branchial arch, or in quail (0/11; Fig. 1E), Silky (0/9; Fig. 1F), or White Leghorn (0/4) limb bud mesenchyme. These observations (Table 1A) established that the quail and Silky neural folds and tubes were, as expected, good sources of melanogenic precursor cells. The experiments also established that there were no such cells in the branchial arch and limb bud mesenchyme prepared for the current experiments. Therefore, the branchial arch and limb bud mesenchymal explants were not contaminated with melanocytes from the skin, and any endogenous crest-derived cells they may have contained were unable to give rise to melanocytes under the conditions of these experiments.

thumbnail image

Figure 1. Melanocytes develop in cultures of neural folds, but not in those of branchial arches or limb buds. Tissues were removed from embryos at the specified ages and cultured on Nuclepore™ filters. A: Quail neural fold (Qnf) explanted at Z stage 6. The presence of melanocytes is evident in the gross pigmentation of the culture. B: Silky fowl neural fold (Snf) explanted at H-H stage 8. Again the presence of melanocytes is evident in the gross pigmentation of the culture. C: Quail branchial arch (Qba) explanted at Z stage 15. There are no melanocytes. D: Silky fowl branchial arch (Sba) explanted at H-H stage 20. There are no melanocytes. E: Quail limb bud (Qlb) explanted at Z stage 16. There are no melanocytes. F. Silky fowl limb bud (Slb) explanted at H-H stage 20. There are no melanocytes. Marker = 1.0 mm.

Download figure to PowerPoint

Table 1A. Pigmentation of cultures/CAM grafts*
SourceNeural fold/tubeBranchial archLimb bud
  • *

    Pigmented cultures were defined as those in which melanocytes with a dendritic morphology were present within the body of the culture.

Silky fowl7/70/130/9
Quail5/50/170/11
White leghornND0/140/4

Co-cultures

The vagal neural folds from quail and Silky fowl were combined with the branchial arch and posterior limb bud mesenchyme of quail, Silky fowl, and White Leghorn origin to determine the effects of these tissues on the migration of crest-derived cells and their expression of a melanocytic phenotype (Table 1B). Crest-derived cells from both the quail and Silky fowl neural folds were found to invade the branchial arch and the limb bud mesenchyme, regardless of whether it was of quail, Silky fowl, or White Leghorn origin (Fig. 2). No significant differences were found between quail and Silky crest-derived cells in their ability to invade the mesenchyme of either the branchial arches or the limb buds. Crest-derived cells from the Silky or White Leghorn neural folds that invaded quail branchial arch or limb bud mesenchyme were readily identified, because the branchial arch (but not the crest-derived cells) exhibited the quail nuclear marker, which is retained in these organotypic tissue cultures (Fig. 2A). Similarly, quail crest-derived cells could be recognized when they invaded the Silky or Leghorn branchial arch or limb bud mesenchyme, because the invading crest-derived cells exhibited the quail marker while the branchial arch cells did not (Fig. 2B). Silky neural folds were not combined with White Leghorn mesenchyme, because it would be impossible, in such a combination, to identify the source of cells in the mesenchyme. When Silky neural folds were co-cultured with quail branchial arch (Fig. 2A, C, and E) or limb bud mesenchyme (Fig. 3A), there were few or no melanocytes in the cultures. The rare melanocytes that did develop (in 11/26 co-cultures) were of Silky fowl origin, but they were limited to a very small area on the periphery of the cultures, rather than in the mesenchyme. The Silky pigment cells in the co-cultures were exceptionally small, did not acquire dendrites, and assumed an unusual round configuration, suggesting that their contact with the branchial arches caused them to develop abnormally. These rare melanocytes were thus not considered to have integrated into the cultures, and the cultures in which they were found were not classified as pigmented (Table 1B). Nonpigmented crest-derived cells of Silky origin, however, did enter and distribute themselves throughout the quail branchial arch or limb bud mesenchyme. In contrast, when quail crest-derived cells invaded the Silky branchial arches (Fig. 2B, D, and F) or the Silky limb bud mesenchyme (Fig. 3B), the invading cells gave rise to melanocytes (Table 1B). These co-cultures of neural fold and mesenchyme always contained many melanocytes; the melanocytes were all of quail origin, and they were very well distributed throughout the Silky mesenchyme. When quail neural folds were co-cultured with White Leghorn branchial arch or limb bud mesenchyme, there were almost no melanocytes in the cultures, and those that were observed were not located inside the mesenchyme (not illustrated).

Table 1B. Pigmentation of co-cultures/CAM grafts*
Neural fold/tubeBranchial archLimb bud
Silky fowlQuailWhite leghornSilky fowlQuailWhite leghorn
  • *

    Pigmented cultures were defined as those in which melanocytes with a dendritic morphology were present within the body of the culture.

Silky fowl1/10/18ND1/10/8ND
Quail21/210/20/78/80/20/8
thumbnail image

Figure 2. Crest-derived cells invade co-cultured branchial arches but give rise to melanocytes only when the branchial arches are of Silky fowl origin. The recipient branchial arches and limb buds were sectioned and stained to demonstrate DNA to visualize the distinct pattern of nucleolar-associated heterochromatin (nuclear marker) that identifies quail cells. The cultures were serially sectioned. A: Co-culture of Silky fowl neural fold and quail branchial arch. Neither quail cells (arrows) nor any of the crest-derived cells of Silky fowl origin (arrowheads) contain melanin. B: Co-culture of quail neural fold and Silky fowl branchial arch. Many pigmented melanocytes are present. Both pigmented and nonpigmented quail cells (arrows) can be distinguished; however, Silky fowl cells (arrowheads) are all nonpigmented. C: Co-culture of Silky fowl neural fold and quail branchial arch as in A, but viewed as a whole mount. There are no melanocytes. D: Co-culture of quail neural fold and Silky fowl branchial arch as in B, but viewed as a whole mount. Many melanocytes are evident. E: Co-culture of Silky fowl neural fold and quail branchial arch. Almost no melanocytes can be seen (compare to F). F: Co-culture of quail neural fold and Silky fowl branchial arch. Note that melanocytes have thoroughly invaded the branchial arch mesenchyme. Markers: A and B = 25 μm; C and D = 1.0 mm; E and F = 100 μm.

Download figure to PowerPoint

thumbnail image

Figure 3. Crest-derived cells invade co-cultured limb buds but give rise to melanocytes only when the limb buds are of Silky fowl origin. A: Co-culture of Silky fowl neural fold and quail limb bud viewed as a whole mount. There are no melanocytes. B: Co-culture of quail neural fold and Silky fowl limb bud viewed as a whole mount. Many melanocytes are evident. Markers = 1.0 mm.

Download figure to PowerPoint

In control experiments, Silky and quail neural folds were cultured with branchial arch and limb bud mesenchyme of the same species. In these control studies, it was impossible to identify specifically the cells from the co-cultured neural folds that invaded the branchial arch or limb bud mesenchyme. Nevertheless, when present, melanocytes could be recognized in these types of culture. When the neural folds, branchial arches, and limb bud mesenchyme were all of Silky origin, melanocytes were present in the branchial arch (Fig. 4A) and limb bud mesenchyme (Fig. 4B). The melanocytes in these cultures, moreover, were well distributed throughout the mesenchyme. In contrast, when quail neural folds were co-cultured with quail branchial arch (Fig. 4C) or limb bud mesenchyme (Fig. 4D), the melanocytes were usually absent or, when present, were quite rare and confined to small regions at the edges of the cultures. The rare pigmented cells in the all-quail co-cultures were small, nondendritic, rounded cells that appeared as little black dots at the periphery of the cultures.

thumbnail image

Figure 4. Control co-cultures. Silky and quail neural folds were co-cultured with branchial arches or limb buds of the same species. A: Co-culture of Silky fowl neural fold and branchial arch. Many melanocytes are present. B: Co-culture of Silky fowl neural fold and limb bud. The area within the rectangle is shown at a higher magnification in the inset. Many melanocytes are present. C: Co-culture of quail neural fold and branchial arch. No melanocytes can be detected. D: Co-culture of quail neural fold and limb bud. No melanocytes can be detected. Markers = 25 μm.

Download figure to PowerPoint

CAM Grafts

Although experiments with organotypic tissue cultures suggested that quail and White Leghorn branchial arch and limb bud mesenchyme are able to inhibit the migration and/or development of cells with a melanogenic potential, in vitro conditions do not exactly mimic the in situ situation. We therefore sought to analyze the effects of branchial arches on migration and/or development of cells with a melanogenic potential by using a different method. The interactions of crest-derived cells with branchial arch mesenchyme were studied in CAM grafts. In CAM grafts, the branchial arches and the migrating neural crest-derived cells are isolated from their natural locations in the embryo as they are in culture, but the milieu in which they interact is different.

Vagal neural folds and truncal neural tubes of quails were transplanted to the CAMs of E6-7 chick embryos together with Silky fowl or White Leghorn branchial arch mesenchyme. When co-grafted with Silky fowl branchial arch, the quail neural folds and neural tubes produced crest-derived cells that invaded the branchial arch mesenchyme and gave rise to many melanocytes in the Silky branchial arch tissue (Fig. 5A–C). The melanogenic precursors from the truncal neural tube appeared to be more invasive of the branchial arches than were their equivalents from the vagal neural folds or tubes (not illustrated). In contrast, quail crest-derived cells invaded the White Leghorn branchial arches, but the cells that did so gave rise to almost no melanocytes (Fig. 5D and E).

thumbnail image

Figure 5. CAM grafts. Crest-derived cells invade co-grafted branchial arches but give rise to melanocytes only when the branchial arches are of Silky fowl origin. A–C: Co-graft of quail neural fold and Silky Fowl branchial arch. Many melanocytes are evident and surround the developing cartilage. At higher magnification (B, inset) the melanocytes can be seen to express the quail nuclear marker (arrow) and to be well infiltrated into the mesenchyme of the branchial arch (C). D and E (inset): Co-graft of quail neural fold and White Leghorn branchial arch. The branchial arch mesenchyme has been infiltrated by many quail cells (arrowheads) but none are pigmented. Markers: A = 100 μm; B = 25 μm; C = 100 μm; D = 100 μm; E = 25 μm.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The current experiments were carried out to test the hypothesis that normal branchial arches have the ability to suppress the melanogenic potential of crest-derived cells. This idea does not conflict with previous proposals that those crest-derived cells that have been specified as melanogenic prior to migration will preferentially migrate along the dorsolateral pathway (Erickson and Goins, 1995; Henion and Weston, 1997). Instead, this hypothesis considers that the crest-derived cell population that migrates ventrally contains nonspecified cells that will develop as melanocytes unless they are prevented from doing so by factor(s) they encounter within the migratory pathway. FoxD3, which inhibits melanogenesis, has been reported to be expressed by ventrally-migrating crest-derived cells (Kos et al., 2001), but they may not all do so, and FoxD3 expression may not be sustained in other cells. For example, FoxD3 expression is lost from the majority of crest-derived cells in the branchial arches. The crest-derived cell population is heterogeneous and contains both nonspecified and fate-restricted cells (Henion and Weston, 1997); therefore, the fact that cells of the population that has been specified as melanocytes preferentially migrate dorsolaterally does not prevent melanocytes from also arising from nonspecified cells that migrate ventrally. A signal encountered after migration has begun, for example, may initiate expression of FoxD3 in a subset of crest-derived cells that do not yet express it and/or maintain FoxD3 expression in a subset of ventrally migrating cells that would otherwise lose it. The target viscera that ventrally migrating crest-derived cells ultimately colonize do not contain melanocytes, yet they do not themselves prevent melanocytes from developing from melanogenic precursors that are made to colonize them (Le Douarin and Teillet, 1974). Therefore, if the population of crest-derived cells that enters the branchial arches contains cells that have not been fate-restricted and/or have a melanogenic potential, then cells capable of giving rise to melanocytes must be removed from the population that migrates through the branchial arches. This removal could be accomplished by regulating expression of FoxD3. Alternatively, the branchial arches may prevent the passage of crest-derived cells that are competent to form melanocytes.

Silky fowl were used to examine the putative role of the branchial arches in removing cells with a melanogenic potential from the crest-derived population that migrates through them because the viscera of these birds contain neural crest-derived melanocytes (Dunn and Jull, 1927; Hallet and Ferrand, 1984); moreover, the ectopic presence of pigment cells in the Silky viscera is not neural crest autonomous (Hallet and Ferrand, 1984; Ferrand and L'Hermite, 1985) and is thus not due to a FoxD3-dependent selection by crest cells of migration pathways to follow (Kos et al., 2001). The ability of the branchial arches to prevent the colonization of the gut by melanogenic precursors may thus be abnormal in Silky fowl. The migration of vagal crest-derived cells was studied because the crest at this level contains a mixture of committed and pluripotent cells (Ito and Sieber-Blum, 1991, 1993) and is the source of cells that migrate through the caudal branchial arches to reach the gut (Payette et al., 1984; Tucker et al., 1986; Kirby, 1987; Phillips et al., 1987; Pomeranz and Gershon, 1990; Burns and Le Douarin, 2001), which itself has been demonstrated to lack the capacity to inhibit the development of melanocytes (Le Douarin and Teillet, 1974). Experiments were carried out with both organotypic tissue cultures and explants grown as CAM grafts. The experimental design allowed crest-derived cells (the origin of which could be identified) to migrate into branchial arch or limb bud mesenchyme in the cultures or CAM grafts, and determined the effect of that migration on the development of melanocytes.

Vagal neural folds and truncal neural tubes from both quail and Silky fowl were each found to be good sources of melanogenic crest-derived cells. In contrast, no melanocytes were found to develop from any crest-derived cells that might have been present in explants of branchial arch or limb bud mesenchyme from quail, Silky, or White Leghorn chick embryos. In the co-cultures, therefore, any melanocytes found in the recipient branchial arch or limb bud mesenchyme were derived from the donor tissue. This conclusion was supported by observations of the origin of melanocytes in co-cultures. Quail crest-derived cells (identified by their nuclear markers) were found to give rise to melanocytes in co-cultured Silky branchial arches and Silky limb bud mesenchyme. In contrast, Silky crest-derived cells did not produce significant numbers of melanocytes in quail branchial arches or limb bud mesenchyme (the cells of which displayed the quail nuclear marker). Similarly, no melanogenesis was seen when quail crest-derived cells colonized branchial arches or limb bud mesenchyme of White Leghorn origin. The difficulty of forming melanocytes within the quail and White Leghorn mesenchyme was not due to a difference between the donors in the invasiveness of crest-derived precursors, because recipient tissues were well colonized by donor cells in each case. Moreover, the difference did not appear to be due to a defect in the ability of the donor crest to colonize recipient mesenchyme from another species, because in controls, in which the donor crest was co-cultured with branchial arches or limb bud mesenchyme of the same species, quail and White Leghorn crest-derived cells still failed to give rise to melanocytes in the branchial arches or limb bud mesenchyme. Significant numbers of melanocytes were again formed only when the recipient tissues were of Silky origin. These observations are consistent with the idea that the mesenchyme of Silky branchial arches and limb buds is permissive for melanogenesis, while that of quails and White Leghorn chicks is not.

Results obtained with co-cultures of crest-derived precursors and recipient mesenchyme were essentially reproduced in experiments carried out with CAM grafts. Explants of Silky fowl, quail, and White Leghorn branchial arches all lacked melanocytes when grown alone as CAM grafts. Once more, however, crest-derived cells migrating from quail neural tubes (trunk) and neural folds (vagal) invaded Silky fowl branchial arches in co-grafts and gave rise to abundant melanocytes in the Silky tissue, and the melanocytes were all of quail origin. Crest-derived cells from quail neural tubes and folds were equally able to invade White Leghorn branchial arches; however, in doing so they gave rise to almost no melanocytes. The formation of melanocytes was thus dependent on the origin of the recipient tissue, not the source of the donor's crest-derived cells. Melanocytes formed within Silky but not White Leghorn branchial arches.

It is interesting that neither in cultures nor in CAM grafts were melanocytes found in Silky fowl branchial arches (0/13) when these were grown by themselves. The arches contain endogenous crest-derived cells at the time of their explantation (H-H stages 19–21) (Le Douarin and Kalcheim, 1999). Since Silky Fowl branchial arches do not prevent melanogenesis when crest-derived cells that are competent to form melanocytes invade them, the failure of melanocytes to develop in these explants suggests that the crest-derived cells that they do contain lack a melanogenic potential. This observation thus supports the idea that the earliest population of crest-derived cells to migrate away from the neuraxis are not competent to form melanocytes (Reedy et al., 1998a, b). The melanocytes that arise in the Silky fowl branchial arches when they are co-cultured or grafted with neural folds or tubes must thus be derived entirely from the neural folds or tubes. It follows that the absence of melanocytes in cultures and CAM grafts of quail and White Leghorn branchial arches grown without a source of melanogenic precursors may also be due to the absence of melanogenic cells in the early-migrating population of crest-derived cells that have colonized these structures. On the other hand, in contrast to Silky fowl, quail and White Leghorn branchial arches prevent the development of melanocytes when the melanogenesis-competent population of crest-derived cells from neural folds or tubes colonizes them. Clearly, Silky fowl branchial arches permit crest-derived cells to form melanocytes, while quail and White Leghorn branchial arches do not.

The development of melanocytes in Silky fowl has previously been investigated using Smyth line serum (which detects a tyrosinase-related protein) as a marker for melanoblasts, and HNK-1 antibodies as a marker for cells of neural crest origin (Reedy et al., 1998b; Faraco et al., 2001). The data from those experiments have been interpreted to support a theory, called “the phenotype-directed model,” that crest-derived cells are specified before they migrate and that specification determines the migratory pathway that crest-derived cells select. The early dorsolaterally migrating cells that colonize the branchial arches at the vagal level are thought to lack a melanogenic potential and, as noted above, our data support this conclusion. The failure of branchial arch crest-derived cells to give rise to melanocytes is thus attributed entirely to the absence of melanogenic precursors, not to an ability of the branchial arch environment to prevent crest cells from giving rise to melanocytes. Precursors with the ability to form melanocytes are postulated to emanate from the vagal neural crest at a later time and to not enter the branchial arches. On the other hand, Silky fowl crest-derived cells do traverse the branchial arches and migrate all the way to the bowel, where they form melanocytes. These visceral melanocytes must thus be derived, not from the early-migrating vagal crest-derived cells that initially populate the caudal branchial arches, but from the later-migrating population, which has a melanogenic potential and continues migrating through the Silky fowl branchial arches to reach the gut. Since there are no visceral melanocytes in the bowel in quail or White Leghorn embryos, the branchial arches of these birds must prevent the late-migrating cells that are able to form melanocytes from migrating to their gut.

The current experiments suggest that vagal crest-derived cells are indeed influenced by the microenvironment of the branchial arches. Vagal crest-derived cells that enter the branchial arches, in co-culture experiments or in CAM grafts, do not develop normally as melanocytes except when the branchial arches are derived from Silky fowl. All three waves of crest-derived precursors that have been detected at the vagal level should be represented in explants of neural folds (Reedy et al., 1998b); moreover, the culture and CAM graft conditions provide adequate time for crest-derived cells to differentiate. The fact that crest-derived cells are able to form melanocytes in the Silky but not in the quail or White Leghorn mesenchyme suggests that the microenvironment of the Silky branchial arch (and limb bud mesenchyme) is permissive for the development of melanocytes while that of quail and White Leghorn embryos is not. The defect in Silky fowl is clearly not limited to the branchial arches, which were not found to differ with respect to their effect on melanogenesis from the limb buds. However, by reason of their location, the branchial arches are in a position to affect the migration of melanogenic cells to the viscera, while the limb buds are not.

An extensive abnormal ventral migration of melanogenic crest-derived precursors has been found to occur in Silky fowl embryos that does not occur in their White Leghorn counterparts (Faraco et al., 2001). This migration, which appears to confirm the idea that the Silky phenotype is brought about by an environmental rather than a neural crest-specific defect, is associated with an absence of the peanut agglutinin-binding proteins of barrier tissues that normally restrict melanoblasts to the dorsolateral pathway of crest-derived cell migration. The ventral migration of crest-derived cells in Silky fowl has been interpreted to suggest that the loss of barrier molecules that restrict the migration of specified crest-derived precursors to the dorsolateral pathway accounts for the abnormal distribution of melanocytes in the Silky fowl. This explanation, however, cannot solely explain the current observations. In both co-cultures and CAM grafts, donor crest-derived cells from vagal and truncal sources from all species tested entered the recipient mesenchyme equally well no matter what its origin. If the mesenchyme of the quail and White Leghorn branchial arches is a barrier to the migration of crest-derived cells, therefore, its function must be restricted to a melanogenesis-competent subset of the late-migrating vagal crest-derived cells. The presence of melanocytes in Silky fowl branchial arches could thus be explained by their inability to cause multipotent crest-derived cells to lose their melanogenic potential, or to prevent a subset of crest-derived cells that are specified to be melanocytes from migrating into them.

The possibility that the branchial arches cause the loss of the potential of crest-derived cells to give rise to melanocytes is consistent with data derived from experiments carried out with phorbol esters. Phorbol esters appear to favor the development of pigment cells from unspecified crest-derived precursors in vitro, apparently by preferentially directing such cells to the melanogenic pathway of differentiation (Sieber-Blum and Sieber, 1981). Phorbol esters also reverse the developmental restriction of crest-derived cells, permitting cells that have migrated ventrally, such as the presumptive Schwann cells in the dorsal root ganglia of older embryos, to regain their ability to develop as melanocytes (Ciment et al., 1986). Crest-derived cells that have been treated with a phorbol ester reach ectopic locations when transplanted into host embryos (Sears and Ciment, 1988). Some of these ectopic cells develop as melanocytes, even though they do not migrate along the dorsolateral pathway. These data suggest that environmental factors influence the fates of at least some crest-derived cells, and that cells with the ability to give rise to melanocytes can be found in targets reached by crest-derived cells that have not followed the migratory pathway normally traversed by melanocytes. Fate restriction may thus not be finally accomplished even for the late-migrating crest-derived cells at the time they segregate from the neural tube, and may not be the only determinant of the pathway these cells choose to follow when they migrate. The ability of the branchial arches to interfere with the realization of the melanogenic potential of nonspecified crest-derived cells may allow the branchial arches to remove the melanogenic potential from the crest-derived cell population that passes through them. Upon entering the normal branchial arches, therefore, the crest-derived cell population migrating to the gut and the heart may initially contain crest-derived precursors that can develop as melanocytes. In all but Silky fowl, in which the microenvironment of the branchial arches lacks the melanocyte-suppressing factor(s), this potential is removed from the crest-derived cell population as it traverses the branchial arches, accounting for the lack of melanocytes in the bowel and heart, and the inability of crest-derived cells from these organs to form melanocytes (Ciment and Weston, 1983). In Silky fowl, however, the removal of cells with a melanogenic potential does not occur, allowing crest-derived cells to pass through and pigment the viscera. Conceivably, the branchial arches may deliver a signal (absent in Silky fowl) to crest-derived cells that initiates and/or maintains the expression in subsets of crest-derived cells of FoxD3. The current observations thus support the idea that the microenvironment of structures, such as the branchial arches, through which precursors migrate, plays a role in the specification of cells derived from the neural crest. Fate specification need not precede the migration of all crest-derived cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Sitara Kommareddi, Andrew Francella, and Juhayna Kassem for technical assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED