A complex genomic rearrangement involving the endothelin 3 locus causes dermal hyperpigmentation in the chicken

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Neural crest progenitors of the melanocyte lineage: coat colour patterns revisited

Neural crest-derived melanoblasts are the progenitors of melanocytes, the pigment cells of the skin, hair and choroid. Previous studies of adult chimaeric mice carrying different coat colour markers have suggested that the total melanocyte population is derived from a small number of melanoblast progenitors, each of which generates a discrete unilateral transverse band of colour. This work also suggested minimal mixing of cells between clones. We have used two complementary approaches to assess the behaviour of migrating clones of melanoblasts directly in the developing embryo. First, we made aggregation chimaeras between transgenic Dct-lacZ and non-transgenic embryos, in which lacZ is a marker for melanoblasts. Second, we generated transgenic mice carrying a modified lacZ reporter construct containing a 289 base pair duplication (laacZ) under the control of the Dct promoter. The laacZ transgene is normally inactive, but reverts to wild-type lacZ at low frequency, labelling a cell and all of its progeny at random. Mosaic embryos containing labelled melanoblast clones were generated. In contrast to previous data, chimaeric and mosaic embryonic melanoblast patterns suggest that: (1) there is a large number of melanoblast progenitors; (2) there is a pool of melanoblasts in the cervical region; (3) different cell dispersion mechanisms may operate in the head and trunk regions; and (4) there is extensive axial mixing between clones.     

Smyth Chicken Melanocyte Autoantibodies: Cross-Species Recognition,In Vivo Binding,and Plasma Membrane Reactivity of the Antiserum

Smyth line (SL) chickens, which develop a depigmenting disorder similar to human vitiligo, produce circulating anti-melanocyte antibodies (Austin, L.M. et al., (1992) The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo. Clin. Immunol. Immunopathol., 64:112–120). In order to characterize these autoantibodies, we studied the reactivity of cultured chicken, mouse, and human melanocytes, as well as frozen sections of chicken feather follicles and embryonic eyes, against SL serum, employing indirect immunofluorescence. Light Brown Leghorn (LBL) serum was used as a negative control. Chicken (SL and LBL), mouse, and human melanocytes exhibited greater fluorescence with SL serum than with LBL serum (up to a 1:60,000 dilution). The fluorescent pattern was predominant along the perimeter of the cells, suggesting plasma membrane staining. Fluorescence-activated flow cytometry analysis and immunocytochemical localization at the ultrastructural level using intact chicken cells supported this hypothesis. Melanocytes were readily stained in cryosections of regenerating feather follicles and embryonic eyes incubated with SL, but not LBL, serum. In addition, amelanotic melanocytes in albino chicken feathers reacted with SL serum. SL serum also preferentially stained cells emigrating from cultured avian neural tubes and within the dermis of the proliferative germ of regenerating feather follicles suggesting that melanoblasts express the antigens. We conclude that Smyth line serum contains melanocyte autoantibodies that cross-react with mouse and human melanocytes, are able to bind to pigment cells within tissues, and recognize antigens expressed in the cytoplasm and on the surface of melanocytes and melanoblasts.     

中国地方品种鸡白细胞介素2基因的克隆及遗传进化分析(英)

鸡白细胞介素 2 (chIL 2 )是近年来新发现的一类细胞因子 .根据Sundick等发表的鸡IL 2基因序列设计一对特异性引物 ,从ConA体外激活的脾淋巴细胞中提取mRNA ,通过RT PCR方法分别扩增和克隆了我国仙居鸡、丝羽乌骨鸡两个地方品种和艾维茵商品肉鸡IL 2cDNA .仙居鸡、丝羽乌骨鸡和艾维茵商品肉鸡IL 2基因的编码区均由 42 9nt组成 ,编码一个由 143个氨基酸组成的前体蛋白 .基因 5′端含有 17个核苷酸 ,3′端含有 2 85个核苷酸组成的非编码区 ,3′ UTR中含有 5个重复的“ATTTA”序列 .编码蛋白氨基酸与来自GenBank的Kestrel、Obese和SC品系的来杭鸡比较 ,氨基酸的突变主要发生在 2 8～ 3 2位 .而这一区域仙居鸡和丝羽乌骨鸡的编码氨基酸序列与Kestrel来杭鸡相同 ,艾维茵商品肉鸡与Obese和SC来杭鸡相同 .基因系统进化树分析表明 ,仙居鸡、丝羽乌骨鸡和Kestrel来杭鸡具有很近的亲缘关系 ,艾维茵商品肉鸡与Obese和SC来杭鸡具有很近的亲缘关系 .中国的药用鸡品种———丝羽乌骨鸡在非常保守的 13 3位发生了氨基酸突变     

The Delayed Entry of Thoracic Neural Crest Cells into the Dorsolateral Path Is a Consequence of the Late Emigration of Melanogenic Neural Crest Cells from the Neural Tube

Neural crest cells migrate along two pathways in the trunk: the ventral path, between the neural tube and somite, and the dorsolateral path, between the somite and overlying ectoderm. In avian embryos, ventral migration precedes dorsolateral migration by nearly 24 h, and the onset of dorsolateral migration coincides with the cessation of ventral migration. Neural crest cells in the ventral path differentiate predominantly as neurons and glial cells of the peripheral nervous system, whereas those in the dorsolateral path give rise to the melanocytes of the skin. Thus, early- and late-migrating neural crest cells exhibit unique morphogenetic behaviors and give rise to different subsets of neural crest derivatives. Here we present evidence that these differences reflect the appearance of specified melanocyte precursors, or melanoblasts, from late- but not early-migrating neural crest cells. We demonstrate that serum from Smyth line (SL) chickens specifically immunolabels melanocyte precursors, or melanoblasts. Using SL serum as a marker, we first detect melanoblasts immediately dorsal and lateral to the neural tube beginning at stage 18, which is prior to the onset of dorsolateral migration. At later stages every neural crest cell in the dorsolateral path is SL-positive, demonstrating that only melanoblasts migrate dorsolaterally. Thus, melanoblast specification precedes dorsolateral migration, and only melanoblasts migrate dorsolaterally at the thoracic level. Together with previous work (Erickson, C. A., and Goins, T. L.,Development121, 915–924, 1995), these data argue that specification as a melanoblast is a prerequisite for dorsolateral migration. This conclusion suggested that the delay in dorsolateral migration (relative to ventral migration) may reflect a delay in the emigration of melanogenic neural crest cells from the neural tube. Several experiments support this hypothesis. There are no melanoblasts in the ventral path, as revealed by the absence of SL-positive cells in the ventral path, and neural crest cells isolated from the ventral path do not give rise to melanocytes when explanted in culture, suggesting that early, ventrally migrating neural crest cells are limited in their ability to differentiate as melanocytes. Similarly, neural crest cells that emigrate from the neural tubein vitroduring the first 6 h fail to give rise to any melanocytes or SL-positive melanoblasts, whereas neural crest cells that emigrate at progressively later times show a dramatic increase in melanogenesis under identical culture conditions. Thus, the timing of dorsolateral migration at the thoracic level is ultimately controlled by the late emigration of melanogenic neural crest cells from the neural tube.     

Hyperpigmentation Results in Aberrant Immune Development in Silky Fowl (Gallus gallus domesticus Brisson)

The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte.     

Growth of Neural Crest Cells In Vitro Is Enhanced By Extracts From Silky Fowl Embryonic Tissues

In the Silky Fowl (SF) breed of chicken, most of the internal organs are infiltrated with melanocytes. Previous studies have shown that this generalized mesodermal pigmentation is not due to a cell autonomous abnormality of the melanocytes but to environmental factors able to promote both the homing of pigment cell precursors in abnormal embryonic sites and their proliferation and differentiation. To analyse the mode of these environmental cues, we tested the effect of SF embryo extract (SFEE) on cultured quail neural crest cells as compared with that of EE from normal chickens of the JA57 strain (JA57EE). We found that SFEE enhances crest cell proliferation as judged by ³H-TdR incorporation and cell counting. In contrast, no effect of SFEE was observed either on the proportion of cultured cells that are engaged into the melanocytic differentiation pathway or on the amount of melanin produced by each differentiated pigment cell. The simple observation, however, reveals that SFEE has a significant effect on pigmentation of the cultured quail neural crest cells. This effect has therefore to be accounted for by the general increase in cell number induced by SFEE. The question is raised as to whether the in vivo SF phenotype is generated exclusively by this mechanism.     

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Phylogenetic analysis in chicken breeds inferred from complete cytochrome b gene information

Complete cytochrome b (cyt b) gene (1140 bp) nucleotide sequences were used to investigate characteristics of the genetic constitution of Chunky broiler chickens, and these were compared with the Hy-Line and WL-GM (Garber) line of White Leghorn, the GSP line of Fayoumi, the BM-C line of Black Minorca (egg-chickens), and an outgroup of wild-origin Japanese quail. A high genetic difference (five haplotypes) was observed at the cytochrome b region in the Chunky broiler in contrast to the high homologies observed among the other chicken breeds (egg-purpose). Chunky broilers can be distinguished from the other breeds (White Leghorn, Fayoumi, and Black Minorca) at positions 552 and 779. The molecular phylogenetic tree exhibited genetic differences within Chunky broilers, and between Chunky broilers and the other three chicken breeds. As a result, some chicken strains or breeds apparently different from the other egg-chickens may have contributed to the Chunky broiler formation. Artificial selection may be one of the biggest factors causing nucleotide diversity in the chicken breeds.     

Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro

Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited. To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium. The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes. The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid. About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction. Eumelanin content in p/p was extremely reduced compared to P/P. The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit. However, the reactivities in p/p were lower than in P/P. Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them. These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs.     

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.     

Single nucleotide polymorphism analysis on chicken extracelluar fatty acid binding protein gene and its associations with fattiness trait

Fattiness is an important parameter to estimate meat quality, which has high heritability. In this experiment, F2 chickens derived from Broilers crossing to Silky were used to study the effect of extracellular fatty acid binding protein (EX-FABP) gene on abdominal fat accumulation. 1.6 kb of the 5' region of the gene was amplified by six pairs of primers, and then single nucleotide polymorphisms (SNPs) were detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. There were four nucleotides variations found, A-G at -1807, C-A at -1805, T-C at -1011 and a C insertion at -1000 respectively. The result of least square analysis suggests that the birds with BB genotype defined by the second pair of primer have a higher abdominal fat weight and abdominal fat percentage than the birds with the other genotypes (AA and AB). It implied that EX-FABP gene could be a candidate locus or linked to a major gene to significantly affect abdominal fat traits in chicken.     

Genome-wide association study of body weight in chicken F2 resource population

Chicken body weight is an economically important trait and great genetic progress has been accomplished in genetic selective for body weight. To identify genes and chromosome regions associated with body weight, we performed a genome-wide association study using the chicken 60 k SNP panel in a chicken F2 resource population derived from the cross between Silky Fowl and White Plymouth Rock. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance. A chicken chromosome 4 (GGA4) region approximately 8.6 Mb in length (71.6-80.2 Mb) had a large number of significant SNP effects for late growth during weeks 7-12. The LIM domain-binding factor 2 (LDB2) gene in this region had the strongest association with body weight for weeks 7-12 and with average daily gain for weeks 6-12. This GGA4 region was previously reported to contain body weight QTL. GGA1 and GGA18 had three SNP effects on body weight with genome-wide significance. Some of the SNP effects with the significance of "suggestive linkage" overlapped with previously reported results.     

利用微卫星标记分析四川8个地方鸡品种遗传多样性

通过选用30个多态性较好的微卫星标记,检测了四川省8个地方鸡品种：峨嵋黑鸡、泸宁鸡、旧院黑鸡、米易鸡、石棉草科鸡、凉山崖鹰鸡、兴文乌骨鸡、沐川乌骨鸡的遗传多样性。利用等位基因频率计算出各群体的平均遗传杂合度（H）、多态信息含量（PIC）和群体间的DA遗传距离。结果表明：30个微卫星位点中有24个微卫星位点在8个鸡群体中的多态信息含量均为高度多态,可作为有效的遗传标记用于各鸡品种的遗传多样性和系统发生关系的分析。各鸡种的杂合度都较高,平均杂合度范围是0.629~0.681,最高的是泸宁鸡（0.681）,最低的是旧院黑鸡（0.629）。据分析可能是由于交通闭塞,形成了家禽品种的多种多型,而且杂合度的高低与PIC值的大小体现了较高的一致性。对DA遗传距离的计算表明：用UPGMA法进行聚类分析,结果8个鸡品种被聚为3类：Ⅰ类：峨嵋黑鸡、米易鸡、泸宁鸡、旧院黑鸡聚为一类。米易鸡、泸宁鸡先聚在一起,然后又与峨嵋黑鸡在较近的距离聚在一起,然后再与旧院黑鸡聚在一起;Ⅱ类：石棉草科鸡、兴文乌骨鸡、沐川乌骨鸡聚为一类。兴文乌骨鸡、沐川乌骨鸡在较近的距离聚在一起,然后又与石棉草科鸡在较近的距离聚在一起;Ⅲ类：凉山崖鹰鸡独自聚为一类。这与几个鸡品种的来源、分化、选育历史及地理分布是一致的。     

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.