Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.     

Quail melanoblast migration in two breeds of fowl and in their hybrids: evidence for a dominant genic control of the mesodermal pigment cell pattern through the tissue environment

In the Silkie fowl large numbers of melanocytes invade most internal tissues and organs. The factors involved in this internal pigment cell pattern were studied by grafting quail neural tube segments into White Leghorn, White Silkie, and F1 hybrids (White Silkie male X White Leghorn female). Sections of quail neural tube five somites long, excised at the level of the last formed somites, were grafted isotopically and ischoronically. Various tissues and organs (mesenteries, muscles, testis, ovary, mesonephros, metanephros, and adrenals) excised from the internal region corresponding to the peripheral transverse strip of quail melanocytes, were studied after staining by the Feulgen-Rossenbeck technique. Despite some variations in pigment cell density, Silkie and hybrid grafted embryos exhibited an extensive quail internal pigmentation similar to the melanocyte distribution in the Silkie breed. In white Leghorn host embryos, the internal pigmentation remained limited. These results show the part played by tissular factors in the expression of the Silkie pigment phenotype and that this genetic tissular character is dominant. On the contrary, White Leghorn embryos, grafted with Silkie neural tube segments, never exhibited any internal pigmentation; the melanocytes deriving from the grafted Silkie neural tube were only localized at the dermoepidermal level. Thus, the migrating and/or differentiating capabilities of the Silkie premelanoblasts are different from those of quail premelanoblasts. The sex-linked inhibitor of the White Leghorn tissue interferes at the level of the pigment cells of chickens but not of quails.     

Bh (black at hatch) gene appears to be expressed in melanocytes of feather germs in the Japanese quail (Coturnix coturnix japonica)

The Bh (black at hatch) gene was examined to determine whether it is expressed in plumage melanocytes by analyzing pigmentation patterns of Bh melanocytes placed in the micro-environment of the feather germs of quail embryos with pink eyes. These host quails genetically lack a large part of plumage melanin. The Bh locus in these almost white quails is wild-type. When Bh neural crest cells were transplanted orthotopically into the host embryos, wild-type and Bh /+ melanocytes, which differentiated from the transplanted neural crest cells, formed plumage pigmentation patterns characteristic of each genotype in the micro-environment of the host feather germs. Brown plumage pigmentation, which was very similar to that of 10-day Bh / Bh embryos, was also observed in the feather germs of host embryos that received Bh neural crest cells, although the genotype of the donors could not be determined. These donors died before pigmentation of their feather germs occurred. The results demonstrate that pigmentation patterns of Bh menalocytes are not altered in the micro-environment of the host germs, suggesting that the Bh gene is autonomous in Bh melanocytes and is expressed in melanocytes of both Bh and the host feather germs, and that it causes the normal pigmentation pattern to be altered.     

Avian spinal cord chimeras. I. Hatching ability and posthatching survival in homo- and heterospecific chimeras

Quail-chick spinal cord chimeras were constructed by grafting isotopically, at the brachial level, the neural tube of a quail embryo into a chick of the same developmental stage. The chimeras were allowed to hatch and their behavior and survival after birth were observed. We found that if white Leghorns of the rapid-feathering strain were taken as hosts, the ability of the operated embryos to hatch was higher than in the slow-feathering wild-type chickens. The important point arising from this study is that the establishment of the neuronal circuits and of the connexions of the grafted neurons to their peripheral and central targets occurs between cells of two different species in such a way that normal behavior of the chimera is ensured. These animals can stand, walk, and fly as normal chickens do. Moreover, the size reached by the fragment of quail spinal cord implanted into the chick axial structures is larger than it would have been in the donor at the same age. This results in perfectly normal morphogenesis of the vertebrae which develop from the chick somites at the level of the graft. The pigment pattern of the chick feathers colonized by quail melanoblasts of graft origin is very close to that of the quail, albeit somewhat different, probably due to the different size of the feathers in the two species. Normality of the chimeras is only transient. During the second month of their life they develop a neurological syndrome characterized first by the paralysis of the wings and later by their inability to stand. In strong contrast, spinal cord chimeras constructed between two histoincompatible chickens, remain healthy and seem to develop a complete tolerance to the graft. What seems to be the development of an immune rejection of the grafted neural tube in the quail-chick spinal cord chimeras is now under investigation.     

Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9)

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.     

Developmental integration of feather growth and pigmentation and its implications for the evolution of diet-derived coloration

Variation in avian coloration is produced by coordinated pigmentation of thousands of growing feathers that vary in shape and size. Although the functional consequences of avian coloration are frequently studied, little is known about its developmental basis, and, specifically, the rules that link feather growth to pigment uptake and synthesis. Here, we combine biochemical, modeling, and morphometric techniques to examine the developmental basis of feather pigmentation in house finches (Carpodacus mexicanus)--a species with extensive variation in both growth dynamics of ornamental feathers and their carotenoid pigmentation. We found that the rate of carotenoid uptake was constant across a wide range of feather sizes and shapes, and the relative pigmented area of feathers was independent of the total amount of deposited carotenoids. Analysis of the developmental linkage of feather growth and pigment uptake showed that the mechanisms behind partitioning the feather into pigmented and nonpigmented parts and the mechanisms regulating carotenoid uptake into growing feathers are partially independent. Carotenoid uptake strongly covaried with early elements of feather differentiation (the barb addition rate and diameter), whereas the pigmented area was most closely associated with the rate of feather growth. We suggest that strong effects of carotenoid uptake on genetically integrated mechanisms of feather growth and differentiation provide a likely route for genetic assimilation of diet-dependent coloration.     

Die Ausbreitung der Melanoblasten bei den Embryonen verschiedenfarbiger Hühner

Summary The first melanoblasts are found in the embryos of the black Rheinländer and Plymouth Rock chickens at the fifth day of development in the occipital and lumbal region. The further spread of the pigment cells is equal in both races. But at the eight day the pigmented area is much more extended in the Plymouth Rocks. These embryos possess at this time more than twice the amount of melanoblasts which are more pigmented than in Rheinländer embryos of the same age. During the further development this differences are equalized.In both races the colour pattern of the chickens are different and have no similarities with the pattern of adult animals. The different patterns of chickens may be explained by time-dependent, genetic differences during the differentiation of the melanoblasts. The colouration in the adult fowls must be due to different genetic factors, which become effective during the development of the juvenile feathers.     

Insights Into Pigmentary Phenomena Provided by Grafting and Chimera Formation in the Axolotl

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, and grafts of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

Plumage pigmentation and expression of its regulatory genes during quail development--histochemical analysis using Bh (black at hatch) mutants

The plumage on the dorsal trunk of normal quail embryos exhibits longitudinal black and brown stripes of pigments produced by melanocytes. However, this pigmentation pattern disappeared in Bh (black at hatch) heterozygous and homozygous embryos because of overall black and brown pigmentation of plumages, respectively. To investigate the mechanisms of the pigment pattern formation of plumage and clarify the roles of the Bh locus in the pattern formation, we examined the expression pattern of genes relating to melanocyte development (Mitf, MelEM antigen, Kitl, Kit and EdnrB2) and melanin pigment production (Dct, Tyrp1, Tyr and Mmp115) in Bh mutant and wild-type embryos throughout development. As a result, we found that MelEM antigen was expressed in melanoblasts committed to produce black pigment before apparent melanogenic gene expression, and that Bh heterozygotes and homozygotes showed abnormal expression patterns of the MelEM antigen. These results indicate that MelEM antigen is a good marker for melanoblasts committed to produce black pigment, and suggests that the Bh locus directs melanocytes to produce eumelanin in proper positions.     

A comparative study of myogenic cell invasion of the avian wing and leg bud.

The borders of myogenic cell invasion of avian wing and leg buds were determined using the interspecific grafting technique between quail and chick embryos. Distal parts of quail limb buds were grafted ectopically into the coelomic cavity of chick embryos. The presence or absence of skeletal muscle was investigated in histological sections of the reincubated grafts. A comparison between the borders of myogenic cell invasion of the wing and leg buds showed that the differences in the position of the distal most muscles in the adult avian limbs could be a consequence of the cranio-caudal sequence of development.     

TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor.

We have used a probe derived from TRP-2/DT to detect migratory melanoblasts shortly after they emerge from the neural crest, as early as 10 days post coitum (dpc). TRP-2/DT expression is otherwise restricted to the presumptive pigmented retinal epithelium, the developing telencephalon and the endolymphatic duct. The pattern of steel and c-kit hybridisation in the developing brain differed from that of TRP-2. TRP-1 and tyrosinase probes also detected melanoblasts but were both expressed later in development than TRP-2. We used the TRP-2/DT probe to investigate the way that the Steel-dickie (Sld) mutation interferes with melanocyte development, and found that the membrane-bound steel growth factor which is missing in Sld/Sld mutants is necessary for the survival of melanoblasts but not for their early migration and initial differentiation.     

Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development

The endothelin receptor B gene (Ednrb) encodes a G-protein-coupled receptor that is expressed in a variety of cell types and is specifically required for the development of neural crest-derived melanocytes and enteric ganglia. In humans, mutations in this gene are associated with Waardenburg-Shah syndrome, a disorder characterized by pigmentation defects, deafness and megacolon. To address the question of whether melanocyte development depends entirely on a cell-autonomous action of Ednrb, we performed a series of tissue recombination experiments in vitro, using neural crest cell cultures from mouse embryos carrying a novel Ednrb-null allele characterized by the insertion of a lacZ marker gene. The results show that Ednrb is not required for the generation of early neural crest-derived melanoblasts but is required for the expression of the differentiation marker tyrosinase. Tyrosinase expression can be rescued, however, by the addition of Ednrb wild-type neural tubes. These Ednrb wild-type neural tubes need not be capable of generating melanocytes themselves, but must be capable of providing KIT ligand, the cognate ligand for the tyrosine kinase receptor KIT. In fact, soluble KIT ligand is sufficient to induce tyrosinase expression in Ednrb-deficient cultures. Nevertheless, these tyrosinase-expressing, Ednrb-deficient cells do not develop to terminally differentiated, pigmented melanocytes. Pigmentation can be induced, however, by treatment with tetradecanoyl phorbol acetate, which mimics EDNRB signaling, but not by treatment with endothelin 1, which stimulates the paralogous receptor EDNRA. The results suggest that Ednrb plays a significant role during melanocyte differentiation and effects melanocyte development by both cell non-autonomous and cell-autonomous signaling mechanisms.     

Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis

Summary Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (> 400 K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.     

Dominant white plumage-color somatic mosaic in the domestic fowl

An unusual feather-color mosaic of the variegated type is described in the fowl. The bird, a female, has the e+/e+ wild-type plumage pattern with light tan or buffish colored feathers intermingled over her sides and dorsal surface. This condition is bilateral and symmetrical with whole feathers being diluted in some areas and parts of feathers in others. Breeding tests with e+/e+ males showed that the bird was heterozygous for dominant white (l/i+) and homozygous for e+. All her e+/e+, l/i+ offspring were of the red-pyle pattern as would be expected from that genotype. The possible cause of this mosaic condition may be due either to the loss of the l-bearing locus or to a reverse mutation to i+ in neural crest cells destined to become the primordial melanoblasts.