A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome.

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

pathogenesis of the henny feathering trait in the Sebright bantam chicken. Increased conversion of androgen to estrogen in skin.

In female chickens of all breeds development of female feathering pattern is mediated by estrogens, whereas normal males and castrated chickens of both sexes develop male feathering. Male chickens carrying the henny feathering trait (such as the Sebright bantam and golden Campine) develop a female feathering pattern but otherwise virilize normally. To examine the possibility that the henny feathering trait is the result of increased conversion of androgen to estrogen in skin, estrogen formation from [1,2,6,7-3H]testosterone was measured in tissue slices from control breeds and chickens with the henny feathering trait. Rates of estrogen formation were undetectable or low in all control tissues other than ovary, whereas rates were high in skin and skin appendages and detectable in many tissues from Sebright and Campine birds. The increased rate of estrogen formation in skin was demonstrable in Sebright chicks and in all areas of skin biopsied in the mature bird. Furthermore, plasma levels of 17 beta-estradiol were higher in Sebright and Campine than in control male cocks. Thus, increased formation of estrogen from androgen in the peripheral tissues probably explains the henny feathering trait.

Pinching off syndrome in free-ranging white-tailed sea eagles (Haliaeetus albicilla) in Europe: frequency and geographic distribution of a generalized feather abnormality.

Generalized feather abnormalities are rarely documented in free-living birds. Pinching off syndrome (POS) is a feather abnormality in which all remiges and retrices become malformed and are lost during the nestling stage, rendering the bird unable to fly. To determine the frequency of occurrence and geographic distribution of this syndrome in white-tailed sea eagles (Haliaeetus albicilla) in Europe, we sent questionnaires to ornithologists in 19 countries within the European range of this species. We also searched for reported cases of sea eagles with feather abnormalities that met the criteria of POS. Overall, 32 nestlings or fledglings with POS were identified between 1975 and 2006. The geographic distribution of cases was primarily restricted to 4 European countries: Germany (17 cases), Poland (11 cases), the Czech Republic (3 cases), and Great Britain (1 case). Eleven eagles from Germany and 2 eagles from the Czech Republic were examined clinically. In 15 birds in which sex was determined, 8 were female and 7 were male. From 2000 to 2005, the 5-year incidence of POS in white-tailed sea eagles in Germany was 3.5 cases per 1000 birds. Although the etiology of this syndrome in wild sea eagles is unknown, our results support a possible genetic cause.

Morbidity of free-ranging white-tailed sea eagles (Haliaeetus albicilla) in Germany.

Between September 1998 and December 2006, 87 injured or sick free-ranging white-tailed sea eagles (Haliaeetus albicilla) from Germany were admitted to the Small Animal Clinic, Free University of Berlin. Most birds were adults (43%) followed by nestlings/fledglings (26%), immature birds (18%), and juveniles (13%). In all age groups more females than males were presented. Birds with trauma-associated injuries were most often admitted, followed by lead toxicosis and feather anomalies in fledglings. Collision with anthropogenic structures was the most common cause of injuries in all age classes. Lesions caused by intraspecific aggressions were mainly found in adults, whereas a generalized feather abnormality (pinching off syndrome) was diagnosed only in fledglings. Of all birds, 25% were released back to the wild, 21% died, and 54% were not releasable or were euthanatized.

A novel sex-linked mutant affecting tail formation in Hongshan chicken.

The Hongshan chicken is a Chinese indigenous breed that has two distinctly different tail types. Some chickens have stunted tails as compared to the normal phenotype, and they are termed rumpless. Rumplessness in other chicken breeds was caused by a reduction in the number of coccygeal vertebrae. However, X-ray examination showed that rumpless Hongshan chickens possess the normal number of coccygeal vertebrae. Our analyses of the main tail feathers and tissue sections led us to speculate that their stunted tail appearance may be the result of abnormal feather development. To investigate the genetic mechanism underlying rumplessness in Hongshan chickens, we analyzed the results of various crosses. The results indicated that rumplessness is a Z-linked dominant character. In addition, we chose some normal and rumpless individuals for pool-sequencing. Nucleotide diversity and Fst were calculated, and a selective sweep was detected on the Z chromosome. These analyses allowed us to reduce the search area to 71.8-72 Mb on the Z chromosome (galGal5.0). A pseudogene LOC431648 located in this region appeared a strong candidate involving in Wnt/ß-catenin signaling pathway to regulate feather development in chickens.

BMPs induce dermal markers and ectopic feather tracts.

Bone morphogenetic protein (BMP) signaling is known to be involved in multiple inductive events during embryogenesis including the development of amniote skin. Here, we demonstrate that early application of BMP-2 to the lateral trunk of chick embryos induces the formation of dense dermis, which is competent to participate in feather development. We show that BMPs induce the dermis markers Msx-1 and cDermo-1 and lead to dermal proliferation, to expression of beta-catenin, and eventually to the formation of ectopic feather tracts in originally featherless regions of chick skin. Moreover, we present a detailed analysis of cDermo-1 expression during early feather development. The data implicate that cDermo-1 is located downstream of BMP in a signaling pathway that leads to condensation of dermal cells. The roles of BMP and cDermo-1 during development of dermis and feather primordia are discussed.

Zinc deficiency in pheasant chicks fed practical diets.

A disease syndrome characterized by abnormal feathering and varus deviation of the tarsometatarsus spontaneously occurred in pheasant chicks fed a practical commercially prepared diet. The addition of supplemental Zn to the commercial diet was effective in alleviating the clinical signs, even though the commercial mix had Zn levels that met the published pheasant chick requirement. Supplemental arginine was ineffective in reducing the severity of the clinical signs. Also, when the diet was mixed in a laboratory setting, pheasant chicks did not develop abnormal feathering and leg bones. Clinical signs caused by feeding the commercial mix were not reproduced by altering dietary calcium and phosphorus levels in the laboratory mix. Neither adding a pellet binder nor removing supplemental Zn from the lab mix produced as severe leg and feather deformities as the commercial mix. Thus, unidentified dietary factors are capable of causing a Zn-deficiency syndrome in pheasant chicks fed practical diets with analyzed Zn levels meeting the required levels.

Short barb: a feather structure mutation in Japanese quail.

A type of feather structure abnormality in Japanese quail resulting in shortened barbs on contour feathers was found to be controlled by a single autosomal recessive gene, sh (short barb). The mutation was first identified in a full-sib family from the University of British Columbia wild type line. Unlike other feather structure mutations in Japanese quail reported previously in literature, the short barb mutation is not associated with poor reproduction.

"Porcupine": a feather structure mutation in Japanese quail.

A type of feather structure abnormality in Japanese quail resulting from the failure of barbs to uncoil was found to be controlled by a single autosomal recessive gene, pc (porcupine). The mutation was first identified in two birds from a population homozygous for white plumage color. Porcupine quail have poor egg production, lower fertility, and higher embryonic and chick mortality compared to wildtype or heterozygotes.

Abnormal feathering of chicks caused by scirpenol mycotoxins differing in degree of acetylation.

Graded levels of the Fusarium mycotoxins, scirpentriol (STO), 15-monoacetoxyscirpenol (15-MAS), 4,15-diacetoxyscirpenol (4,15-DAS), and 3,4,15-triacetoxyscirpenol (TAS), were fed to chicks until 3 wk of age. The primary wing feathers, which were scored visually on a scale of 1 to 5 using a newly created scoring scheme, were altered in a dose-related fashion by 15-MAS [minimum effective dose (MED) = .5 microgram/g diet], 4,15-DAS (MED = 2 micrograms/g), and STO (MED = 4 micrograms/g) but not by TAS (MED greater than 8 micrograms/g). The minimum growth inhibitory doses were 2 micrograms/g for STO, 15-MAS, and 4,15-DAS and 8 micrograms/g for TAS. The main alteration of the feathers was a frayed and missing web on the medial side of the distal half of the feather. The shafts of the feathers tended to have an accentuated medial curve. These results imply that the feather alterations associated with corn and feed infested with Fusarium spp. might be caused by trichothecene mycotoxins such as the scirpenols.

Ectopic feather follicles in a chicken teratoma or malformation.

At necropsy of an 8-week-old pullet a 0.75 X 5.0 cm. yellowish, moderately firm cigar-shaped object was seen in the greater abdominal air sac lying parallel to the border of the right hepatic lobe. A capsule of 5 tissue layers (from inside outward-stratified squamous epithelium fibrous tissue, longitudinal muslce, fibrous tissue and circular muscle) enclosed featherlike structures which had developed from the stratified squamous epithelial layer. This combination of tissue components constituted ectopic follicles and feathers in a malformation or teratoma.

A feather abnormality in chicks fed diets deficient in certain amino acids.

A feather abnormality was observed in chicks during bio-assay investigations with eleven essential amino acids. As dietary valine, leucine, isoleucine, glycine or phenylalanine and tyrosine levels decreased, a similar progressive feather abnormality became apparent. The outstanding feature of the feather abnormality was the concave structure of the feathers as they bent upward from the body. This structure gave the feathers a ragged appearance. The feather abnormality is described and illustrated. The percentage of dietary amino acids used were: valine, 0.40, 0.50, 0.60, and 0.70; leucine, 0.60, 0.68, 0.76, and 0.82; isoleucine, 0.32, 0.38, 0.44, and 0.50; glycine, 0.30, 0.35, 0.40, and 0.45; or phenylalanine and tyrosine, 0.65, 0.70, 0.75, and 0.80. Chicks fed the highest level of valine (0.50%), histidine (0.15%) or methionine to cystine ratio of 1:1 (0.30%) had normal feathers. In contrast, chicks fed the highest levels of either leucine, isoleucine, glycine or phenylalanine and tyrosine had abnormal feathers.

Valine deficiency. 2. The effect of feeding a valine-deficient diet during the starter period on performance and leg abnormality of male broiler chicks.

Using force-feeding and pair-feeding techniques, two experiments were conducted to investigate the problem of leg abnormality associated with feeding a Val-deficient diet as compared with a diet deficient in all the branched chain amino acids (BCAA) or a Val-supplemented diet. The BCAA-deficient diet contained .96, .52, and .63% of Leu, Ile, and Val, respectively. The Val-deficient diet contained 1.37 Leu, .82 Ile, and .63% Val. The Val-supplemented diet contained 1.37 Leu, .82 Ile, and .83% Val. In both studies birds fed the Val-supplemented gained more weight than birds in the other two groups. Although birds on Val-deficient and BCAA-deficient diets had similar weight gain, the former were lethargic and showed feather and leg abnormalities. Bone ash and bone calcium for Val-deficient birds were the lowest among the three groups studied (P < .05). The levels of the BCAA in plasma reflected those of the treatment diets. Plasma hydroxyproline was lowest in birds fed the Val-deficient diet (P < .05), indicating a reduction in bone collagen breakdown. Kidney function measurements were the same for birds on the Val-supplemented and Val-deficient diets. Fractional excretion of calcium in Val-deficient birds (.13%), however, was three times higher than that of Val-supplemented birds (P < .05). The results indicated that Val deficiency per se increased calcium excretion in urine and induced leg abnormality in young chickens.

Assessing the effects of the naked neck gene on chronic heat stress resistance in two genetic populations.

The effect of the naked neck (Na) gene on resistance to chronic heat stress was studied in the F2 generation of two populations of chickens genetically differing in growth. The Na gene was introduced into the Athens-Canadian randombred (ACRB), a small BW population, and into a large BW commercial broiler population. Naked neck and normally feathered birds were maintained in either a chronic heat stress (32 C) environment or a control (21 C) environment from 4 to 8 wk of age. Body weight, BW gain, feed consumption, and feed efficiency were calculated at 4, 6, and 8 wk. The 32 C environment significantly reduced gain and feed consumption at all age intervals in both populations. Feed conversion ratio (FCR) was lower in the 32 C environment in the ACRB population, but in the broiler population FCR either was the same or increased when compared with the 21 C environment. In the F2 generation of both populations the naked neck birds were significantly larger than the normally feathered birds although the two types segregated from the same parents. In the ACRB population the relative growth response was not significantly different between naked neck and normal feathered birds, indicating that the Na gene did not confer resistance to chronic heat stress in the small BW population. In the broiler population, an assessment of the relative growth response in the 32 C environment showed that the naked neck birds had a smaller reduction in BW gain and better feed efficiency than the normally feathered birds, indicating that in the large BW broiler population the Na gene did confer resistance to chronic heat stress.