Smyth Line Research Articles

Immune Activities in Choroids of Visually Impaired Smyth Chickens With Autoimmune Vitiligo.

Vitiligo is a common dermatological disorder affecting 1–2% of the world’s population. It is characterized by postnatal, autoimmune destructions of melanocytes in the skin, resulting in patches of depigmentation. Autoimmunity in vitiligo may also affect melanocytes in non-integumental tissues, including the eyes where choroidal melanocytes are the target of the autoimmune response. The Smyth line (SL) of chicken is the only animal model that spontaneously and predictably develops all clinical and biological manifestations of autoimmune vitiligo. In SL vitiligo (SLV), destruction of epidermal melanocytes in growing feathers (GFs) involves a melanocyte-specific, Th1-mediated cellular immune response. Smyth chickens may also exhibit uveitis and vision impairment. Previous studies established a strong association between SLV and vision impairment, including similar pathology in affected eyes and GFs. To determine the presence, types, and activities of choroid infiltrating mononuclear cells, we collected eyes before, near onset, and during active SLV from sighted, partially blind, and blind SL chickens. All SL chickens with vision impairment had SLV. Immunohistochemistry and quantitative reverse transcriptase-PCR analyses revealed mononuclear cell and cytokine expression profiles in the autoimmune destruction of melanocytes in choroids that are identical to those described in GF, demonstrating the systemic nature of autoimmunity against melanocytes in SLV. In addition, we observed aberrant melanogenesis in SL eyes. The immunopathogenesis in SL vision impairment resembles human vitiligo-associated ocular diseases, especially Vogt–Koyanagi–Harada syndrome and sympathetic ophthalmia. Hence, the Smyth chicken autoimmune vitiligo model provides the opportunity to expand our understanding of spontaneous autoimmune pigmentation disorders and to develop effective treatment strategies.

Mardivirus Infection and Persistence in Feathers of a Chicken Model Harboring a Local Autoimmune Response

Herpesvirus of turkey (HVT) is commonly used as a vaccine to protect chickens against Marek’s disease. Following vaccination, HVT infects feathers where it can be detected in all chicken lines examined. Unlike the parental Brown line (BL), Smyth line (SL) chickens develop vitiligo, due to autoimmune destruction of melanocytes in feathers. Previous reports showed a strong inflammatory response in Smyth chickens’ feathers at vitiligo onset, that subsided once melanocytes were destroyed, and depigmentation was complete. Here, we questioned whether the local autoimmune response in the Smyth model influences HVT infection and persistence in feathers. For this, one-day-old SL and BL chickens were vaccinated with Newcastle disease (rHVT-ND). Vitiligo was scored and HVT loads in pigmented and non-pigmented growing feathers were quantified regularly over 20 weeks. Chickens of both lines showed moderate HVT loads in feathers. At the onset of active vitiligo, the HVT load was significantly higher in SL compared to BL feathers. However, no difference in HVT loads was noticed between pigmented and non-pigmented feathers from SL chickens. Therefore, surprisingly, the inflammatory response in feathers of SL chickens did not inhibit HVT infection and persistence, but on the contrary, temporarily promoted HVT infection in feathers.

Ex vivo killing of primary melanocytes by syngeneic mononuclear cells from active, autoimmune vitiligo lesions in Smyth chickens (BA7P.152)

Abstract In humans autoimmune vitiligo is characterized by the loss of pigment-producing melanocytes in the skin. The vitiligo-prone Smyth line (SL) chicken together with the MHC-matched parental Brown line (BL) and vitiligo-resistant Light Brown Leghorn line of chickens constitute a spontaneous, non-transgenic animal model for this disorder. Cell-mediated immunity (CMI) is known to contribute to the etiopathology of vitiligo. Furthermore, CMI was demonstrated in vivo in vitiliginous SL chickens based on the delayed wattle response (DWR) to melanocyte lysates. Immunohistochemical analysis of DWR-positive tissue revealed extensive T cell infiltration indicative of a CMI response. In order to examine CMI in SL vitiligo (SLV) ex vivo we developed a co-culture assay using primary effector and target cells from growing feathers (the target tissue). Mononuclear cells (MNCs) were harvested from feathers of chickens with active SLV and BL controls. Target cells (melanocytes) were isolated from pigmented feathers prior to development of SLV. To detect melanocyte-specific death a modified flow cytometric method of Lee-MacAry et al (2001) was used. Regardless of whether melanocytes were isolated from chickens that did or did not eventually develop SLV, co-culture with MNCs from SLV chickens resulted in greater melanocyte death compared to controls. Use of this ex vivo system with sorted MNCs from active, autoimmune SLV lesions will help identify roles of individual subsets in melanocyte death.

Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

BackgroundThe Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.ResultsIllumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP).ConclusionsVarious potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-707) contains supplementary material, which is available to authorized users.

Characterization of melanocyte-specific autoantibodies in vitiligo-prone Smyth line chickens. (BA2P.128)

Abstract Autoimmune vitiligo is characterized by the loss of pigment-producing melanocytes (MC) in the skin. The autoimmune vitiligo-prone Smyth line (SL) of chicken together with the parental Brown line (BL) and vitiligo-resistant Light Brown Leghorn (LBL) chickens constitutes an excellent animal model for this disorder. Although cell-mediated immunity has long been known to contribute to the etiopathology of SL vitiligo (SLV) the role of humoral immunity is less clear. We recently completed a time-course study examining levels of IgG specific to MC lysate in SL and control plasma. Results showed elevated (P<0.05) MC-lysate specific IgG levels in SL chickens 1-2 weeks prior to and throughout SLV development. To evaluate the binding characteristics of MC-specific IgG, primary MC isolated from growing feathers were incubated with IgG purified from the serum of SLV (birds with SLV) and control chickens. IgG binding was evaluated by indirect immunofluorescent staining and flow cytometry. Regardless of the source of MC (SL, BL, LBL), significant binding was only observed with SLV-IgG (mean fluorescence intensity increased approximately 100%). This effect was not observed when MC were incubated with IgG purified from egg yolk of SLV hens. Further characterization of autoantibodies from SLV chickens may provide insight into their role in the loss of MC in SLV. Studies evaluating such mechanisms as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity are underway.

Understanding mechanisms of vitiligo development in Smyth line of chickens by transcriptomic microarray analysis of evolving autoimmune lesions

BackgroundThe Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather) of SL chickens at different vitiligo (SLV) states.ResultsCompared to the reference sample, which was from Brown line chickens (the parental control), 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE) (P ≤ 0.05) in feather samples collected from SL chickens that never developed SLV (NV), from SLV chickens prior to SLV onset (EV), during active loss of pigmentation (AV), and after complete loss of melanocytes (CV). Comparisons of gene expression levels within SL samples (NV, EV, AV and CV) revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death.ConclusionsThe microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune response plays a predominant role in melanocyte loss.

IFN-γ, IL-21, and IL-10 Co-Expression in Evolving Autoimmune Vitiligo Lesions of Smyth Line Chickens

The Smyth line (SL) of chicken is an excellent animal model for human autoimmune vitiligo. In SL vitiligo (SLV) post-natal loss of melanocytes in feathers appears to be due to cell-mediated immunity. In this study, leukocyte-infiltration and associated expression (RNA) of immune function-related cytokines in growing feathers were investigated throughout SLV-development and -progression. Both leukocyte infiltration and cytokine expression levels started to increase near visible SLV onset (early SLV), reached peak levels during active SLV, and decreased to near pre-vitiligo levels after complete loss of melanocytes. Specifically, significant increases were noticed in relative proportions of T cells, B cells, and MHC II-expressing cells during active SLV. Levels of T cell infiltration were higher than those of B cells, with more CD8+ than CD4+ cells throughout SLV. Elevated leukocyte infiltration in early and active SLV was accompanied by increased levels of cytokine expression, especially in interferon-gamma, interleukin (IL)-10 and IL-21. Low expression of IL-4 and IL-17 did not suggest important roles of Th2 and Th17 cells in SLV pathogenesis. Taken together, SLV appears to be a Th1 polarized autoimmune disease, whereby interferon-gamma expression is strongly associated with parallel increases in IL-10 and IL-21, particularly during early and active stages of SLV.

Impact of the hypomethylating agent 5-azacytidine on dendritic cells function

Recent evidence suggested that 5-azacytidine (5-aza) can impact important immune functions via epigenetic modifications, making it an attractive candidate for pharmacologic manipulation of the immune system. The aim of this work was to study the effects of 5-aza on human dendritic cells (DC) generated from peripheral blood monocytes, and to test the type of immune response induced in patients treated with 5-aza. On the phenotypic level, CD40 and CD86 expression was significantly increased on mature DC exposed to 5-aza (5-aza-DC), compared with control untreated DC. Mature control DC and mature 5-aza-DC secreted comparable amounts of interleukin (IL)-6, IL-12p70, IL-23, and tumor necrosis factor-α. However, mature 5-aza-DC secreted significantly lower levels of IL-10 and IL-27 compared to mature control DC (p = 0.04 and p = 0.005, respectively). In the peripheral blood of 14 patients (7 males and 7 females; age range, 53-81 years) with advanced myeloid malignancies (8 acute myeloid leukemia and 6 myelodysplastic syndrome) treated with 5-aza, there was a significant decrease of IL-4-secreting CD4(+) T cells (p = 0.001), and a significant increase of IL-17A- and IL-21-secreting CD4(+) T cells (p = 0.003 and p = 0.01, respectively, compared to 5 healthy donors) suggesting a Th17 response pattern in the blood of patients receiving 5-aza. In all, these data suggest potentially novel mechanisms of action of epigenetic therapies, such as 5-aza, which may have broader implications for immunotherapeutic strategies.

Abnormal morphology of melanosomes in the autoimmune vitiligo-prone Smyth line chicken does not appear to be due to alteration in lipid composition (83.16)

Abstract The Smyth line (SL) chicken is an important animal model for autoimmune vitiligo. SL vitiligo is multifactorial in nature including genetic susceptibility, immune system components and environmental triggers. Genetic susceptibility to SL vitiligo appears to be manifested in part by an inherent melanocyte defect. SL melanosomes (pigment-carrying organelles) have irregular-shaped membranes with pigmented extensions that may be due to abnormal lipid composition. Lipids can affect protein topology and trafficking (and hence protein degradation and presentation), membrane stability and oxidative stress resistance. To compare melanosomal lipids from SL and control lines of chickens, melanosomes were isolated from growing feathers of 4-week-old SL, Brown line (parental control) and Light Brown Leghorn (vitiligo-resistant) chickens and lipids were extracted. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with thin-layer chromatography (TLC) was used to determine expression differences in different classes of lipids by using TLC-MALDI imaging technique. Total fatty acid composition was examined by gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters. Statistical analysis of preliminary data shows no significant differences in total fatty acid and phospholipid composition in melanosomes from SL and control chickens. Hence, lipid composition may not explain the melanosomal abnormalities observed in SL melanocytes.

Pigmented growing feathers from autoimmune vitiligo-prone Smyth line chickens have heightened sensitivity to 4-tertiary butyl phenol (83.19)

Abstract The Smyth line (SL) chicken model for vitiligo exhibits spontaneous, autoimmune loss of melanocytes in growing feathers. Onset of SL vitiligo (SLV) can be observed between 6 and 20 wks of age and affects 85-95% of SL chickens. Multiple factors contribute to the melanocytes’ cell death, including genetic susceptibility to vitiligo as well as environmental factors that trigger autoimmune loss of SL melanocytes (e.g., live turkey herpesvirus (HVT) administration at hatch). In humans, phenolic derivatives, such as 4-tertiary butyl phenol (4-TBP), have been shown to trigger vitiligo in susceptible individuals. 4-TBP is structurally similar to tyrosine, the initial substrate of tyrosinase in melanin synthesis. In addition to interfering with melanin production, 4-TBP exposure results in oxidative stress. To examine the effect of 4-TBP in SL and control chickens, pigmented growing feathers were collected from 1, 4 and 7 week-old SL, Brown Line (BL, parental control, <1% vitiligo) and Light Brown Leghorn (LBL, vitiligo resistant) chickens that did or did not receive HVT. The newest three mm growth (the melanocyte-containing, active pigmentation site) of the feathers was cultured with 4-TPB (200 μM) and the production of oxidative radicals was monitored in vitro. Independent of HVT treatment, 4-TBP induced more oxidative stress (P < 0.05) in feathers from SL than BL and LBL chickens, suggesting heightened sensitivity of melanocytes to 4-TBP in vitiligo-prone SL chickens.

Global transcriptome analysis of feathers from Smyth line chickens revealed different gene-expression profiles at various stages of autoimmune vitiligo (83.17)

Abstract The Smyth line (SL) chicken is an animal model for autoimmune vitiligo (SLV). SL chickens develop spontaneous, post-hatch, autoimmune melanocyte loss in growing feathers (GF). With repeatable access to GF and the high incidence (85-95%) of SLV, SLV development can be examined in the same bird. A microarray study was conducted to investigate the global transcriptomic profile in GF from SL and Brown line (BL, parental control) chickens. RNA pools were made from GF of BL chickens, SL chickens that never developed SLV (S0), and SL chickens before (S1), during (S2) and at complete (S3) SLV. Complementary RNA probes were generated from BL and SL (S0~S3) RNA, labeled with Cy3 and Cy5, respectively and hybridized on a chicken 4x44 K Agilent microarray. The slide was scanned and normalized signal intensities were analyzed to identify differentially expressed (DE, >2x) genes between BL and SL samples. There were 504, 518, 1124, and 1151 DE genes containing 262, 226, 658, and 680 unique DE genes in S0, S1, S2 and S3, respectively. S0 and S1 had similar proportions of up-and down-regulated DE genes while up-regulated DE genes predominated in S2 and S3. The highest expressed DE genes were related to immune activity and cell death. Ingenuity Pathway Analysis software was used to determine biological functions and pathways of DE genes. The study provides comprehensive information from global gene expression to functionalities and biological networks of DE genes in the process of SLV.

Using the chicken feather as a window into cell-mediated tissue responses (99.12)

Abstract Cell-mediated immune (CMI) responses are typically tested by injecting the skin and monitoring the skin reaction. In chickens, various integumental tissues are suitable to measure the CMI reaction, including the wattle, wing web, toe web and foot pad. Using the Smyth line chicken vitiligo model to study the spontaneous autoimmune destruction of pigment cells in feathers, we have found that one growing feather-tip (3 mm diameter by 8 mm length) yields generous amounts of material for down-stream gene-expression-, histological-, and cell population-analyses. Moreover, growing feathers are easily accessible for microinjection, removal of a feather is minimally invasive, and feathers are very suitable for repeated sampling during an immune response. To investigate the potential of the feather as a window into tissue immune responses, time-course studies were conducted comparing leukocyte infiltration in wattles, wing webs and growing feathers in response to injection of vehicle, PHA or LPS, and during the recall response to Mycobacterium butyricum. Based on histology and immunohistochemical staining, leukocyte infiltration profiles over a 72 hour period were similar in all three tissues tested. Hence, the feather provides excellent opportunity to monitor and assess CMI responses and immunomodulation strategies in situ and via down-stream analyses. Support: Arkansas Experiment Station Animal Health Grant

Oxidative stress and antioxidant levels in Feather-derived and Embryo-derived Melanocytes from Vitiligo-Prone smyth line and normally pigmented chickens.

Oxidative stress and antioxidant levels in Feather-derived and Embryo-derived Melanocytes from Vitiligo-Prone smyth line and normally pigmented chickens.

Antioxidant capacity and Oxidative stress in vitiliginous smyth line chickens and controls.

Antioxidant capacity and Oxidative stress in vitiliginous smyth line chickens and controls.

Apoptosis in feathers of Smyth line chickens with autoimmune vitiligo

Vitiligo is an acquired dermatological disorder characterized by a loss of epidermal melanocytes resulting in depigmentation of the skin. Mechanisms underlying the destruction of melanocytes in vitiligo remain unclear. An animal model to study spontaneously occurring autoimmune vitiligo is the mutant Smyth line (SL) of chickens. This investigation was designed to determine whether the pathogenesis of depigmentation in Smyth line chicken vitiligo (SLV) involves an apoptotic mechanism. Terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) was used to detect in situ cell apoptosis in cryostat sections of 2-week-old regenerating feathers. Two-week-old regenerating feathers were obtained from SL chickens and their normally pigmented controls including the parental Brown line (BL) and Light Brown Leghorn (LBL) chickens at 6, 8, 10 and 12 weeks of age. In feathers from vitiliginous SL chickens, the number of TUNEL+ cells was significantly ( P≤0.05) higher than that in the feathers of non-vitiliginous SL, BL or LBL chickens. These TUNEL+ cells were primarily located in the epithelial barb ridge where melanocyte cell bodies are located. The extent of this apoptosis in the feathers of SLV chickens varied with the severity of depigmentation of the feathers (i.e., highest in active depigmentation), suggesting a close association between apoptosis and the disappearance of melanocytes. In addition to TUNEL staining, most sections were double-stained with monoclonal antibodies specific to either CD8 or MHC class II molecules to further explore the relationship between CD8+ feather-infiltrating lymphocytes and this increase in apoptotic cells. Compared to normally pigmented controls, the number of CD8+ and MHC class II+ cells in the feather pulp and the barb ridge increased 2–4 weeks before the visible onset of SLV, and was directly related to the changes in the number of TUNEL+ cells prior to, at onset and during depigmentation. Moreover, some of these infiltrating CD8+ cells were localized next to or near the TUNEL+ cells. These observations suggest that enhanced apoptosis in the feather of SLV chickens is a pathogenic mechanism involved in the death of melanocytes and appears to be induced by infiltrating cytotoxic T lymphocytes (CD8+).

Melanocyte-specific cell mediated immune response in vitiliginous Smyth line chickens

Smyth line chickens (SL) are an animal model to study human autoimmune vitiligo. Vitiligo in SL chickens is characterized by a post-hatch loss of feather melanocytes (MC) resulting in feather depigmentation. The etiopathology of this disease remains unclear, however, a hypothesis that cell-mediated immunity (CMI) plays an important role in the destruction of melanocytes has been proposed based on previous studies. To support this hypothesis, the delayed wattle response (DWR) to MC lysates was examined in SL chickens and normally pigmented, MHC-matched controls (Brown line, BL; Light Brown Leghorn, LBL) to determine whether vitiliginous SL chickens have MC-specific CMI. Chickens were challenged in one wattle with a lysate of either embryo- or feather-derived cultured MC. Lysates of chick-embryo-fibroblasts (CEF) associated turkey herpesvirus (HVT) vaccine and syngeneic CEF were used as positive and negative control, respectively. Vitiliginous SL chickens exhibited a significantly greater DWR to feather-MC but not to embryo-MC and CEF than nonvitiliginous SL chickens and normally pigmented controls. The DWR to feather-MC peaked at 3 to 5 days post feather-MC lysate injection. Immunohistochemical staining of DWR-positive wattle tissue revealed extensive lymphocyte infiltration at the site of feather-MC injection consisting primarily of T cells (TCR2+, CD4+ or CD8+ T cells). These observations support the presence of MC-specific CMI in SL chickens with vitiligo. The anti-MC CMI activity appears to be specific to MC derived from feathers. SDS–PAGE analysis of different sources of MC lysates revealed a 43 kDa missing band in the lysate of feather-MC established from SL chickens. No other obvious protein pattern differences that could account for the feather MC-specific CMI response in vitiliginous SL chickens were observed among the MC lysates.

Herpesvirus Connection in the Expression of Autoimmune Vitiligo in Smyth Line Chickens

The Smyth line (SL) chicken is an animal model for human vitiligo, a common acquired depigmentary disorder affecting about 1-2% of people worldwide. The vitiligo-like depigmentation in SL chickens typically develops when the birds are between 6 and 14 weeks of age and may affect 70-95% of hatch mates. The development of SL vitiligo is considered to depend on two interacting components, namely an inherent melanocyte defect and an autoimmune reaction to melanocytes. Recently, a role for an environmental factor in the expression of vitiligo was suggested by the observation that only 10% of SL chicks imported from the University of Massachusetts (UM) and reared in isolation at biosecurity level 2 (BSL 2) at the University of Arkansas (UA) exhibited vitiligo. Following further assessment of environmental differences between UA and UM SL chickens, three environmental factors that may have influenced the expression of SL vitiligo were identified. Included were housing condition, status of Mycoplasma synoviae infection, and turkey herpesvirus (HVT) vaccination status. Studies were subsequently conducted at UA and UM to assess the role of these environmental factors in the expression of SL vitiligo. M. synoviae infection was not found necessary for vitiligo expression in SL chickens. However, HVT emerged as a strong candidate for an important environmental factor in SL vitiligo. The connection between HVT and SL vitiligo was confirmed for both BSL 2 and conventional housing. Therefore, the observations reported here suggest a strong causative link between HVT infection and SL vitiligo.

Molecular characterization of the Smyth chicken sublines and their parental controls by RFLP and DNA fingerprint analysis

The Smyth line (SL) chicken, a model for autoimmune human vitiligo, is characterized by a spontaneous posthatch epidermal pigment loss (vitiligo). Even though the immunological and morphological changes accompanying the vitiligo process have been well studied, the genetics of this phenomenon remains elusive. The SL lines have been maintained by nonpedigreed matings since their inception, and therefore, the inbreeding status is unknown. The present study was designed to provide an estimate of the inbreeding coefficients and the molecular genetic profiles of the SL sublines, each homozygous for a different MHC haplotype and their MHC-matched parental control (BL) sublines. The DNA fingerprint analysis revealed that there is a moderate level of inbreeding within the SL and BL parental sublines. Of the two SL sublines studied, SL101 had the highest level of inbreeding (0.948). Similarly, its parental control line (BL101) was more inbred than the parental subline of SL102 (BL102). The very high level of similarity between the SL sublines and their respective parental control lines is shown further by the similarity index (SI) estimates (SI between SL101 and BL101 was 0.949 and that between SL102 and BL102 was 0.932). Restriction fragment length polymorphism (RFLP) analysis of the endogenous viral genes (avian leukosis virus subgroup E, ALVE) showed that five ALVE-related BamH1 fragments were present in the SL101 and four in SL102 sublines, whereas the parental BL101 and BL102 sublines had five and six fragments, respectively. SL101 and SL102 shared two fragments, but the frequencies were different. Similarly, BL101 and BL102 shared two fragments. SL101 and BL101 shared three fragments, and SL102 and BL102 also shared three fragments.

Hyperpigmentation in the Silkie fowl correlates with abnormal migration of fate-restricted melanoblasts and loss of environmental barrier molecules.

In most homeothermic vertebrates, pigment cells are confined to the skin. Recent studies show that the fate-restricted melanoblast (pigment cell precursor) is the only neural crest lineage that can exploit the dorsolateral path between the ectoderm and somite into the dermis, thereby excluding neurons and glial cells from the skin. This does not explain why melanoblasts do not generally migrate ventrally into the region where neurons and glial cell derivatives of the neural crest differentiate, or why melanoblasts do not escape from the dorsolateral path once they have arrived at this destination. To answer these questions we have studied melanogenesis in the Silkie fowl, which is a naturally occurring chicken mutant in which pigment cells occupy most connective tissues, thereby giving them a dramatic blue-black cast. By using markers for neural crest cells (HNK-1) and melanoblasts (Smyth line serum), we have documented the development of the Silkie pigment pattern. The initial dispersal of melanoblasts is the same in the Silkie fowl as in Lightbrown Leghorn (LBL), White Leghorn (WLH), and quail embryos. However, by stage 22, when all ventral neural crest cell migration has ceased in the WLH, melanoblasts in the Silkie embryo continue to migrate between the neural tube and somites to occupy the sclerotome. This late ventral migration was confirmed by filling the lumen of the neural tube with DiI at stage 19 and observing the embryos at stage 26. No DiI-labeled cells were observed in the sclerotome of LBL embryos, whereas in the Silkie embryos DiI-filled cells were found as far ventral as the mesentery. In addition to this extensive ventral migration, we also observed considerable migration of melanoblasts from the distal end of the dorsolateral space into the somatic mesoderm (the future parietal peritoneum), and into the more medioventral regions where they accumulated around the dorsal aorta and the kidney. The ability of melanoblasts in the Silkie embryos to migrate ventrally along the neural tube and medially from the dorsolateral space is correlated with a lack of peanut agglutinin (PNA) -binding barrier tissues, which are present in the LBL embryo. The abnormal pattern of melanoblast migration in the Silkie embryo is further exaggerated by the fact that the melanoblasts continue to divide, as evidenced by BrdU incorporation (but the rate of incorporation is not greater than seen in the LBL). Results from heterospecific grafting studies and cell cultures of WLH and Silkie neural crest cells support the notion that the Silkie phenotype is brought about by an environmental difference rather than a neural crest-specific defect. We conclude that melanoblasts are normally constrained to migrate only in the dorsolateral path, and once in that path they generally do not escape it. We further conclude that the barriers that normally restrain melanoblast migration in the chicken are not present in the Silkie fowl. We are now actively investigating the nature of this barrier molecule to complete our understanding of melanoblast migration and patterning.

Analysis of the Effect of Endogenous Viral Genes in the Smyth Line Chicken Model for Autoimmune Vitiligo

The Smyth line (SL) chicken, an animal model for autoimmune human vitiligo, is characterized by a spontaneous posthatch pigment loss, determined to be the result of an autoimmune phenomenon. Because endogenous virus (EV) genes have been reported to be associated with a number of autoimmune diseases of human and animal models, we designed this experiment to investigate the role of EV in the SL vitiligo by using the complete sequence of Rous-associated virus-2 as a probe for EV. An F(2) resource population was developed by the matings of SL and parental control (BL) chickens. Linkage disequilibrium between vitiligo and EV was apparent (16.2-kb SacI fragment, P </= 0.05 and a 19-kb HindIII fragment, P </= 0.03). Methylation analyses revealed that the EV and endogenous avian retroviral (EAV) genes were methylated in both the SL and BL sublines of chickens; therefore, methylation does not appear to be responsible for the differences in the expression of vitiligo between SL and BL sublines. Expression of the EV genes correlated with the disease in vitiliginous SL101 birds and also in 5-Azacytidine-induced vitiliginous BL101 parental control chickens. Only one EV locus was detected in the unrelated Light Brown Leghorn control chickens (1q14) by in situ hybridization, whereas 3 EV loci were identified in SL101 and BL101 chickens (1p25, 2q26, and an unidentifiable microchromosome). Our observations indicate that EV genes may play a role in the induction of autoimmune vitiligo in the SL chicken model.