Gap junctions in Turing-type periodic feather pattern formation.

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogeneous 2D field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating nonprotein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found 7 of the 12 investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation. Interestingly, inhibition of gap junctional intercellular communication (GJIC) in the ex vivo skin explant culture allowed the sequential emergence of new feather buds at specific spatial locations relative to the existing primary buds. The results suggest that GJIC may facilitate the propagation of long-distance inhibitory signals. Thus, inhibition of GJs may stimulate Turing-type periodic feather pattern formation during chick skin development, and the removal of GJ activity would enable the emergence of new feather buds if the local environment were competent and the threshold to form buds was reached. We further propose Turing-based computational simulations that can predict the sequential appearance of these ectopic buds. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Characterization of circRNA expression profiles associated with non-Mendelian inheritance in feather growth of chickens.

1. Non-coding RNAs, such as miRNAs, play a crucial role in chicken feather growth rate. However, circular RNA (circRNA) expression profiles in fast- and slow-feathering chickens that follow and do not follow Mendelian inheritance are unclear.2. The circRNA expression profiles was analysed by RNA sequencing of hair follicles of slow-feathering chickens that follow genetic rules and fast-feathering chickens that did not follow genetic rules. Differentially expressed circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was then constructed and the key factors and regulation mechanisms controlling feather growth rate were identified.3. The results revealed that 67 circRNAs were significantly differentially expressed in hens, including 22 up-regulated and 45 down-regulated circRNAs in non-Mendelian inheritance-mediated fast-feathering hens compared with Mendelian inheritance-mediated slow-feathering hens. In addition, 16 significantly differentially expressed circRNAs were identified in cockerels, including nine up-regulated and seven down-regulated circRNAs in non-Mendelian inheritance-mediated fast- compared with Mendelian inheritance-mediated slow-feathering cocks. Moreover, circRNA-mediated ceRNA regulation of hair follicle formation was particularly abundant in the Jak-STAT, Wnt and Toll-like receptor signalling pathways. Furthermore, circABI3BP was seen to be a crucial circRNA in regulating feather growth rate, by binding with gga-miR-1649-5p to regulate SSTR2 expression.4. In conclusion, this study analysed circRNA expression profiles in fast- and slow-feathering chickens that follow and do not follow Mendelian inheritance, which laid the foundation for understanding the role of circRNA in chicken feather growth rate.

Transcriptome analysis of sexual dimorphism in dorsal down coloration in goslings.

BACKGROUND: In day-old Hungarian white goose goslings, there is a noticeable difference in dorsal down coloration between males and females, with females having darker dorsal plumage and males having lighter plumage. The ability to autosex day-old goslings based on their dorsal down coloration is important for managing them efficiently and planning their nutrition in the poultry industry. The aim of this study was to determine the biological and genetic factors underlying this difference in dorsal down colorationthrough histological analysis, biochemical assays, transcriptomic profiling, and q‒PCR analysis. RESULTS: Tissue analysis and biochemical assays revealed that compared with males, 17-day-old embryos and day-old goslings of female geese exhibited a greater density of melanin-containing feather follicles and a greater melanin concentration in these follicles during development. Both female and male goslings had lower melanin concentrations in their dorsal skin compared to 17-day-old embryos. Transcriptome analysis identified a set of differentially expressed genes (DEGs) (MC1R, TYR, TYRP1, DCT and MITF) associated with melanogenesis pathways that were downregulated or silenced specifically in the dorsal skin of day-old goslings compared to 17-day-old embryos, affecting melanin synthesis in feather follicles. Additionally, two key genes (MC1R and MITF) associated with feather coloration showed differences between males and females, with females having higher expression levels correlated with increased melanin synthesis and darker plumage. CONCLUSION: The expression of multiple melanogenesis genes determines melanin synthesis in goose feather follicles. The dorsal down coloration of day-old Hungarian white goose goslings shows sexual dimorphism, likely due to differences in the expression of the MC1R and MITF genes between males and females. These results could help us better understand why male and female goslings exhibit different plumage patterns.

Effect of a novel alkaline protease from Bacillus licheniformis on growth performance, carcass characteristics, meat quality, antioxidant capacity, and intestinal morphology of white feather broilers.

BACKGROUND: The present study was conducted to investigate the effects of dietary novel alkaline protease from Bacillus licheniformis on the growth performance, meat quality, antioxidant status and intestinal morphology of broilers. In total, 4000 broilers were randomly assigned into five groups and treated with normal control, normal control + 100 mg kg-1 protease, normal control + 200 mg kg-1 protease, normal control + 300 mg kg-1 protease and normal control + 400 mg kg-1 protease. RESULTS: Supplementing protease impacted final body weight (linear, P = 0.003; quadratic, P = 0.006) and decreased feed conversion rate (linear, P = 0.036) in broilers. Moreover, dietary protease significantly increased breast muscle rate (linear, P = 0.005; quadratic, P = 0.021) and decreased drip loss (linear, P < 0.001; quadratic, P < 0.001). In addition, dietary protease notably increased protein digestibility (linear, P = 0.001; quadratic, P = 0.006) and trypsin activity (linear, P = 0.002; quadratic, P = 0.009) in jejunum. Light microscopy revealed that the jejunum villi in the 300 mg kg-1 and 400 mg kg-1 groups exhibited greater height and a denser arrangement compared to those in the control group. The addition of protease decreased malondialdehyde content (linear, P < 0.001; quadratic, P < 0.001) and increased total antioxidant capacity (linear, P = 0.001; quadratic, P < 0.001) in pectoral muscles. CONCLUSION: The results of the present study suggest that dietary novel alkaline protease from B. licheniformis improved growth performance by affecting trypsin activity, protein digestibility, antioxidant capacity and intestinal health. © 2024 Society of Chemical Industry.

Morphogens enable interacting supracellular phases that generate organ architecture.

During vertebrate organogenesis, increases in morphological complexity are tightly coupled to morphogen expression. In this work, we studied how morphogens influence self-organizing processes at the collective or "supra"-cellular scale in avian skin. We made physical measurements across length scales, which revealed morphogen-enabled material property differences that were amplified at supracellular scales in comparison to cellular scales. At the supracellular scale, we found that fibroblast growth factor (FGF) promoted "solidification" of tissues, whereas bone morphogenetic protein (BMP) promoted fluidity and enhanced mechanical activity. Together, these effects created basement membrane-less compartments within mesenchymal tissue that were mechanically primed to drive avian skin tissue budding. Understanding this multiscale process requires the ability to distinguish between proximal effects of morphogens that occur at the cellular scale and their functional effects, which emerge at the supracellular scale.

Mechanical properties pattern the skin.

Morphogens induce variations in tissue mechanics to promote feather budding.

Deletion in KRT75L4 linked to frizzle feather in Xiushui Yellow Chickens.

Bird feathers are the product of interactions between natural and artificial selection. Feather-related traits are important for chicken selection and breeding. Frizzle feather is characterized by the abnormally development of feathers in chickens. In the current study, frizzle feather characteristics were observed in a local breed called Xiushui Yellow Chicken in Jiangxi, China. To determine the molecular mechanisms that underlie frizzle feather in Xiushui Yellow Chicken, four populations of three breeds (Xiushui Yellow Chicken with frizzle feathers, Xiushui Yellow Chicken with normal feathers, Guangfeng White-Ear Yellow Chicken, and Ningdu Yellow Chicken) were selected for whole-genome resequencing. Using a comparative genome strategy and genome-wide association study, a missense mutation (g.5281494A>G) and a 15-bp deletion (g.5285437-5285451delGATGCCGGCAGGACG) in KRT75L4 were identified as candidate mutations associated with frizzle feather in Xiushui Yellow Chicken. Based on genotyping performed in a large Xiushui Yellow Chicken population, the g.5285437-5285451delGATGCCGGCAGGACG mutation in KRT75L4 was confirmed as the putative causative mutation of frizzle feather. These results deepen the understanding of the molecular mechanisms responsible for frizzle feather, as well as facilitating the molecular detection and selection of the feather phenotype in Xiushui Yellow Chickens.

Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

A quantitative image-based protocol for morphological characterization of cellular solids in feather shafts.

During morphogenesis, cellular sheets undergo dynamic folding to build functional forms. Here, we develop an image-based quantitative morphology field (QMorF) protocol that quantifies the morphological features of cellular structures and associated distributions. Using feather shafts with different rigidities as examples, QMorF performs coarse-graining statistical measurements of the fitted cellular objects over a micro-image stack, revealing underlying mechanical coupling and developmental clues. These images give intuitive representations of mechanical forces and should be useful for analyzing tissue images showing clear cellular features. For complete details on the use and execution of this protocol, please refer to Chang et al. (2019).

Immunostaining of telomerase in embryonic and juvenile feather follicle of the chick labels proliferating cells for feather formation.

Feathers regenerate through proliferation of cells derived from follicle stem cells. Immunoloblotting for telomerase in chick embryonic and juvenile feathers shows immunopositive bands around 100 kDa, 75 and 60 kDa only in embryonic feathers, indicating fragmentation of the protein due to physiological processing or artifacts derived from protein extraction. Immunolabeling for telomerase is present in the cytoplasm and nuclei of cells of the collar epithelium and bulge located in the follicle, and in sparse cells of the dermal papilla. PCNA-immunolabeling indicates that the collar and dermal papilla contain numerous proliferating cells, including the ramogenic zone where barb ridges are formed. Ultrastructural labeling indicates that a telomerase-like protein or its fragment is localized in nucleoli and in sparse nuclear clumps, likely representing Cajal bodies. The cytoplasm shows sparse immune-gold particles, also associated to mitochondria and sparse keratin filaments. An intense labeling is present in some areas of condensing chromosomes in dividing cells. Since telomerase positive cells are also seen in suprabasal layers of the collar epithelium and in the ramogenic zone, it is suggested that they represent dividing cells, most likely transit amplifying cells that give rise to the corneocytes of feathers. The significance of telomerase localization in chromatin is unknown.

The genetic basis and robustness of naked neck mutation in chicken.

Chicken is a homeothermic animal; consequently, regardless of fluctuation in weather conditions, it maintains constant body temperature. However, in hot regions and seasons, chickens suffer from heat stress. To dissipate excess heat, besides modifying the environment, which is costly, however, chickens with efficient heat dissipation capacity might be utilized. Naked neck chickens have a higher capacity for heat loss attributable to reduced feather mass. The naked neck mutation (Na) was originated from a large insertion (~ 180 bp) integrated ~ 260-kb downstream of a protein-coding gene-GDF7 (Growth Differentiation Factor 7). Na possesses a cis-regulatory function and upregulates the expression of GDF7-a gene that exhibits a tissue-specific effect by the sensitizing action of retinoic acid. Na suppresses the development of feathers in the neck and vent. Na shows autosomal incomplete dominance and regulates several developmental processes. Na usually segregates at low frequency, which might be attributed to limited socio-cultural preferences. Specifically, in hot and humid regions, although to a varying extent, Na enhances performance, immunocompetence, and resilience to disease both in the homozygous and heterozygous state. Occasionally, naked neck chickens (especially the homozygous ones) lose comparative advantage in cool environments. Homozygous Na also results in high embryo death and reduced hatchability and diminishes floating and flying capacity. Nevertheless, selective breeding of naked neck chickens for fertility traits enhances the performance and welfare of chickens in hot and humid regions. The comparative advantage of Na needs to be studied not only from a temperature perspective and under controlled experiment but also from humidity, body weight, feed intake (absolute and relative to body weight), age, agroecology insights, and under field condition. Due to the incomplete dominant expression pattern of Na, studies need to separately report their findings for homozygous and heterozygous naked neck chicken.

Birds of a feather moult together: Differences in moulting distribution of four species of storm-petrels.

The non-breeding period of pelagic seabirds, and particularly the moulting stage, is an important, but understudied part of their annual cycle as they are hardly accessible outside of the breeding period. Knowledge about the moulting ecology of seabirds is important to understand the challenges they face outside and within the breeding season. Here, we combined stable carbon (δ13C) and oxygen (δ18O) signatures of rectrices grown during the non-breeding period of two pairs of storm-petrel species breeding in the northern (European storm-petrel, Hydrobates pelagicus, ESP; Leach's storm-petrel, Hydrobates leucorhous, LSP) and southern (black-bellied storm-petrel, Fregetta tropica, BBSP; Wilson's storm-petrel, Oceanites oceanicus, WSP) hemispheres to determine differences in moulting ranges within and between species. To understand clustering patterns in δ13C and δ18O moulting signatures, we examined various variables: species, sexes, years, morphologies (feather growth rate, body mass, tarsus length, wing length) and δ15N. We found that different factors could explain the differences within and between the four species. We additionally employed a geographical distribution prediction model based on oceanic δ13C and δ18O isoscapes, combined with chlorophyll-a concentrations and observational data to predict potential moulting areas of the sampled feather type. The northern species were predicted to moult in temperate and tropical Atlantic zones. BBSP was predicted to moult on the southern hemisphere north of the Southern Ocean, while WSP was predicted to moult further North, including in the Arctic and northern Pacific. While moulting distribution can only be estimated on large geographical scales using δ13C and δ18O, validating predictive outcomes with food availability proxies and observational data may provide valuable insights into important moulting grounds. Establishing those, in turn, is important for conservation management of elusive pelagic seabirds.

Understanding Transcriptomic and Serological Differences between Forced Molting and Natural Molting in Laying Hens.

Molting is natural adaptation to climate change in all birds, including chickens. Forced molting (FM) can rejuvenate and reactivate the reproductive potential of aged hens, but the effect of natural molting (NM) on older chickens is not clear. To explore why FM has a dramatically different effect on chickens compared with NM, the transcriptome analyses of the hypothalamus and ovary in forced molted and natural molted hens at two periods with feathers fallen and regrown were performed. Additionally, each experimental chicken was tested for serological indices. The results of serological indices showed that growth hormone, thyroid stimulating hormone, and thyroxine levels were significantly higher (p < 0.05) in forced molted hens than in natural molted hens, and calcitonin concentrations were lower in the forced molted than in the natural molted hens. Furthermore, the transcriptomic analysis revealed a large number of genes related to disease resistance and anti-aging in the two different FM and NM periods. These regulatory genes and serological indices promote reproductive function during FM. This study systematically revealed the transcriptomic and serological differences between FM and NM, which could broaden our understanding of aging, rejuvenation, egg production, and welfare issues related to FM in chickens.

The effect of dietary energy and protein level on feather, skin and nodule growth of the ostrich (Struthio camelus).

Accurate diet formulations are required to fulfil the nutrient requirements of birds in order to achieve optimal production. Knowing how the skin, nodule and feather production characteristics vary with diets of different nutrient densities will help in least-cost modelling. Feather growth and nodule development are factors that were previously neglected in ostrich diet formulation, both of which are essential for the development of a predictive production model. In this trial, 120 birds were placed in 15 pens. Varying energy regimes (high, medium and low) and accompanying protein and amino acid profile levels (level 1-5) were assigned ad libitum to each pen. A randomly selected bird from each pen was slaughtered at 1, 35, 63, 103, 159, 168 and 244 days of age. During the slaughter, each bird was weighed, stunned, exsanguinated, defeathered and eviscerated. Feathers from four regions of the skin were plucked and weighed. The shaft diameter of the wing feathers was measured. The nodule size of the tanned skin was measured for each slaughter age. The data were transformed to natural logarithms and regressed against the total feather weight and the total featherless empty body protein weight to set up allometric growth equations. A prediction equation to determine nodule size of the live bird was proposed. Feed cost optimisation is paramount, and results from this study will aid in setting up least-cost optimisation (simulation) formulation models.

Variations of Mesozoic feathers: Insights from the morphogenesis of extant feather rachises.

The rachises of extant feathers, composed of dense cortex and spongy internal medulla, are flexible and light, yet stiff enough to withstand the load required for flight, among other functions. Incomplete knowledge of early feathers prevents a full understanding of how cylindrical rachises have evolved. Bizarre feathers with unusually wide and flattened rachises, known as "rachis-dominated feathers" (RDFs), have been observed in fossil nonavian and avian theropods. Newly discovered RDFs embedded in early Late Cretaceous Burmese ambers (about 99 million year ago) suggest the unusually wide and flattened rachises mainly consist of a dorsal cortex, lacking a medulla and a ventral cortex. Coupled with findings on extant feather morphogenesis, known fossil RDFs were categorized into three morphotypes based on their rachidial configurations. For each morphotype, potential developmental scenarios were depicted by referring to the rachidial development in chickens, and relative stiffness of each morphotype was estimated through functional simulations. The results suggest rachises of RDFs are developmentally equivalent to a variety of immature stages of cylindrical rachises. Similar rachidial morphotypes documented in extant penguins suggest that the RDFs are not unique to Mesozoic theropods, although they are likely to have evolved independently in extant penguins.

The Wnt/ß-catenin signaling pathway is involved in regulating feather growth of embryonic chicks.

Avian feathers have robust growth and regeneration capability and serve as a useful model for decoding hair morphogenesis and other developmental studies. However, the molecular signaling involved in regulating the development of feather follicles is unclear. The purpose of this study was to investigate the role of the Wnt/ß-catenin pathway in regulating feather morphogenesis in embryonic chicks through in ovo injection of different doses of Dickkopf-1 (DKK1, a specific inhibitor of the target of the Wnt/ß-catenin pathway). A total of 120 fertilized embryo eggs were randomly divided into 4 treatments, including a noninjection group (control group) and groups injected with 100 µL of phosphate-buffered saline (PBS)/egg (PBS control group), 100 µL of PBS/egg containing 600-ng DKK1/egg (600-ng DKK1 group), and 100-µL PBS/egg containing 1,200-ng DKK1/egg (1,200-ng DKK1 group). Feathers and skin tissues were sampled on embryonic (E) day 15 and the day of hatching to examine the feather mass, diameter and density of feather follicles, and the protein expression of the Wnt/ß-catenin pathway. The results showed that, compared with CON and PBS treatment, the injection of DKK1 into the yolk sac of chick embryos had no significant effect on the hatching rate and embryo weight (P > 0.05), while it significantly decreased the relative mass of feathers in the whole body (P < 0.05). The high dose of DKK1 (1,200-ng DKK1/egg) decreased the relative mass of feathers on the back, chest, belly, neck, wings, head, and legs, which was more obvious than that in the 600-ng DKK1 group, which presented a dose-dependent effect. In addition, DKK1 injection significantly downregulated the protein expression levels of ß-catenin, transcription factor 4, Cyclin D1, and c-Myc (P < 0.05). The immunofluorescence result of ß-catenin was consistent with the Western blotting assay results. Altogether, these observations suggested that the Wnt/ß-catenin signaling pathway is involved in regulating feather follicle development and feather growth during the embryonic development of chicks.

The role of CTNNB1 and LEF1 in feather follicles development of Anser cygnoides and Anser anser.

BACKGROUND: Wingless-types/beta-catenin (Wnt/ß-catenin) signaling pathway is one of the most extensively studied transcriptional cascades involved in various types of organogenesis including embryonic and postnatal development. Downy feather quantity is primarily affected by follicular development and gene regulations. OBJECTIVE: This research was aimed to investigate the role of catenin beta-1(CTNNB1) and lymphoid enhancerbinding factor-1 (LEF1) on feather follicles development at different developmental stages. METHODS: Fluorescence quantitative PCR, Western-blot and immunohistochemical methods were used in Anser cygnoides and Anser anser embryos (E12, E13 E18, and E28) and after birth gosling stages (G18, G48, G88) for gene expression analysis. RESULTS: CTNNB1 and LEF1 genes were expressed in Anser cygnoides and Anser anser at different embryonic and after-birth gosling developmental stages and the expression levels were significantly different in different stages (p < 0.05). The mRNA expression of CTNNB1 and LEF1 genes reached the highest level at D88 in Anser cygnoides, while the highest expression levels were at D18 and D88 in Anser anser, and the expression levels of CTNNB1 genes at D88 in all embryonic stages were significantly lower than after-birth stages. CTNNB1 and LEF1 protein expression were the highest at E12 and E28 for Anser cygnoides feather follicles development. While at a similar stage for Anser anser, the expression of CTNNB1 and LEF1 protein was the highest at D48 and D18. Protein expression at embryonic stages was in the epidermis (E) and the hair basal plate (P), the expression site for after-birth stages was in the dermal papilla (DP). CONCLUSION: Our study illustrated that CTNNB1 and LEF1 has an impact on Anser cygnoides and Anser anser feather follicles growth and development.

Adaptation of the master antioxidant response connects metabolism, lifespan and feather development pathways in birds.

Birds (Aves) display high metabolic rates and oxygen consumption relative to mammals, increasing reactive oxygen species (ROS) formation. Although excess ROS reduces lifespan by causing extensive cellular dysfunction and damage, birds are remarkably long-lived. We address this paradox by identifying the constitutive activation of the NRF2 master antioxidant response in Neoaves (~95% of bird species), providing an adaptive mechanism capable of counterbalancing high ROS levels. We demonstrate that a KEAP1 mutation in the Neoavian ancestor disrupted the repression of NRF2 by KEAP1, leading to constitutive NRF2 activity and decreased oxidative stress in wild Neoaves tissues and cells. Our evidence suggests this ancient mutation induced a compensatory program in NRF2-target genes with functions beyond redox regulation-including feather development-while enabling significant metabolic rate increases that avoid trade-offs with lifespan. The strategy of NRF2 activation sought by intense clinical investigation therefore appears to have also unlocked a massively successful evolutionary trajectory.

Parallel Genetic Origin of Foot Feathering in Birds.

Understanding the genetic basis of similar phenotypes shared between lineages is a long-lasting research interest. Even though animal evolution offers many examples of parallelism, for many phenotypes little is known about the underlying genes and mutations. We here use a combination of whole-genome sequencing, expression analyses, and comparative genomics to study the parallel genetic origin of ptilopody (Pti) in chicken. Ptilopody (or foot feathering) is a polygenic trait that can be observed in domesticated and wild avian species and is characterized by the partial or complete development of feathers on the ankle and feet. In domesticated birds, ptilopody is easily selected to fixation, though extensive variation in the type and level of feather development is often observed. By means of a genome-wide association analysis, we identified two genomic regions associated with ptilopody. At one of the loci, we identified a 17-kb deletion affecting PITX1 expression, a gene known to encode a transcription regulator of hindlimb identity and development. Similarly to pigeon, at the second loci, we observed ectopic expression of TBX5, a gene involved in forelimb identity and a key determinant of foot feather development. We also observed that the trait evolved only once as foot-feathered birds share the same haplotype upstream TBX5. Our findings indicate that in chicken and pigeon ptilopody is determined by the same set of genes that affect similar molecular pathways. Our study confirms that ptilopody has evolved through parallel evolution in chicken and pigeon.