The Trichohyalin-Like Protein Scaffoldin Is Expressed in the Multilayered Periderm during Development of Avian Beak and Egg Tooth.

Scaffoldin, an S100 fused-type protein (SFTP) with high amino acid sequence similarity to the mammalian hair follicle protein trichohyalin, has been identified in reptiles and birds, but its functions are not yet fully understood. Here, we investigated the expression pattern of scaffoldin and cornulin, a related SFTP, in the developing beaks of birds. We determined the mRNA levels of both SFTPs by reverse transcription polymerase chain reaction (RT-PCR) in the beak and other ectodermal tissues of chicken (Gallus gallus) and quail (Coturnix japonica) embryos. Immunohistochemical staining was performed to localize scaffoldin in tissues. Scaffoldin and cornulin were expressed in the beak and, at lower levels, in other embryonic tissues of both chickens and quails. Immunohistochemistry revealed scaffoldin in the peridermal compartment of the egg tooth, a transitory cornified protuberance (caruncle) on the upper beak which breaks the eggshell during hatching. Furthermore, scaffoldin marked a multilayered peridermal structure on the lower beak. The results of this study suggest that scaffoldin plays an evolutionarily conserved role in the development of the avian beak with a particular function in the morphogenesis of the egg tooth.

Methionine improves feather follicle development in chick embryos by activating Wnt/ß-catenin signaling.

This study was conducted to explore the regulatory role of methionine (Met) in feather follicle and feather development during the embryonic period of chicks. A total of 280 fertile eggs (40 eggs/group) were injected with 0, 5, 10, 20 mg of L-Met or DL-Met/per egg on embryonic day 9 (E9), and whole-body feather and skin tissues were collected on E15 and the day of hatching (DOH). The whole-body feather weight was determined to describe the feather growth, and the skin samples were subjected to hematoxylin and eosin staining and Western blotting for the evaluation of feather follicle development and the expressions of Wingless/Int (Wnt)/ß-catenin signaling pathway proteins, respectively. The results showed that L- or DL-Met did not affect the embryo weight (P > 0.05), but increased the absolute and relative whole-body feather weights. Specifically, 5 and 10 mg of L-Met and 5, 10, and 20 mg of DL-Met significantly increased the absolute feather weight at E15 (P < 0.05), and 10 mg of L-Met and 5 and 10 mg of DL-Met significantly increased the absolute and relative feather weight on the DOH (P < 0.05). Moreover, a main effect analysis suggested that changes in the embryo and feather weights were related to the Met levels (P < 0.05) but not the Met source (P > 0.05). The levels of L- and DL-Met were quadratically correlated with the absolute and relative feather weights of chicks on the DOH (P < 0.05). Correspondingly, all doses of L- and DL-Met significantly increased the diameter and density of feather follicles on the DOH (P < 0.05), as well as the activity of Wnt/ß-catenin on E15 and the DOH (P < 0.05). In conclusion, injection of either L- or DL-Met can improve feather follicle development by activating Wnt/ß-catenin signaling, and thereby promoting feather growth; furthermore, no difference in feather growth was found between L- and DL-Met treatments. Our findings might provide a nutritional intervention for regulating feather growth in poultry production.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

Exploration of key regulators driving primary feather follicle induction in goose skin.

The primary feather follicles are universal skin appendages widely distributed in the skin of feathered birds. The morphogenesis and development of the primary feather follicles in goose skin remain largely unknown. Here, the induction of primary feather follicles in goose embryonic skin (pre-induction vs induction) was investigated by de novo transcriptome analyses to reveal 409 differentially expressed genes (DEGs). The DEGs were characterized to potentially regulate the de novo formation of feather follicle primordia consisting of placode (4 genes) and dermal condensate (12 genes), and the thickening of epidermis (5 genes) and dermal fibroblasts (17 genes), respectively. Further analyses enriched DEGs into GO terms represented as cell adhesion and KEGG pathways including Wnt and Hedgehog signaling pathways that are highly correlated with cell communication and molecular regulation. Six selected Wnt pathway genes were detected by qPCR with up-regulation in goose skin during the induction of primary feather follicles. The localization of WNT16, SFRP1 and FRZB by in situ hybridization showed weak expression in the primary feather primordia, whereas FZD1, LEF1 and DKK1 were expressed initially in the inter-follicular skin and feather follicle primordia, then mainly restricted in the feather primordia. The spatial-temporal expression patterns indicate that Wnt pathway genes DKK1, FZD1 and LEF1 are the important regulators functioned in the induction of primary feather follicle in goose skin. The dynamic molecular changes and specific gene expression patterns revealed in this report provide the general knowledge of primary feather follicle and skin development in waterfowl, and contribute to further understand the diversity of hair and feather development beyond the mouse and chicken models.

Expression patterns of three JAK-STAT pathway genes in feather follicle development during chicken embryogenesis.

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK-STAT) pathway is shown to restrain the hair follicles in catagen and telogen and prevent anagen reentry in murine hair follicle cycling. The early roles of JAK-STAT pathway genes in skin development remain uncharacterized in mouse and chicken models. Here, we revealed the expression patterns of three JAK-STAT pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6-E10 stages which are key to feather follicle morphogenesis. Multiple sequence alignment of the three genes from chicken and other species all showed a closely related homology with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and followed with the focally restricted signals in the feather follicles of neck and body skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three genes displayed stronger expression in feather follicles of neck skin than that of body skin. The expression levels of JAK1 and TYK2 were much stronger than those of JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the much stronger expression in neck and body skin at later stages (E8-E10) than earlier stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange the proximal-distal axis of feather follicles, respectively, during the morphogenesis of feather follicles in embryonic chicken skin.

Feather arrays are patterned by interacting signalling and cell density waves.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation.

Getting to the root of scales, feather and hair: As deep as odontodes?

While every jawed vertebrate, or its recent ancestor, possesses teeth, skin appendages are characteristic of the living clades: skin denticles (odontodes) in chondrichthyans, dermal scales in teleosts, ducted multicellular glands in amphibians, epidermal scales in squamates, feathers in birds and hair-gland complexes in mammals, all of them showing a dense periodic patterning. While the odontode origin of teleost scales is generally accepted, the origin of both feather and hair is still debated. They appear long before mammals and birds, at least in the Jurassic in mammaliaforms and in ornithodires (pterosaurs and dinosaurs), and are contemporary to scales of early squamates. Epidermal scales might have appeared several times in evolution, and basal amniotes could not have developed a scaled dry integument, as the function of hair follicle requires its association with glands. In areas such as amnion, cornea or plantar pads, the formation of feather and hair is prevented early in embryogenesis, but can be easily reverted by playing with the Wnt/BMP/Shh pathways, which both imply the plasticity and the default competence of ectoderm. Conserved ectodermal/mesenchymal signalling pathways lead to placode formation, while later the crosstalk differs, as well as the final performing tissue(s): both epidermis and dermis for teeth and odontodes, mostly dermis for teleosts scales and only epidermis for squamate scale, feather and hair. We therefore suggest that tooth, dermal scale, epidermal scale, feather and hair evolved in parallel from a shared placode/dermal cell unit, which was present in a common ancestor, an early vertebrate gnathostome with odontodes, ca. 420 million years ago.

Embryonic morphology observation and HOXC8 gene expression in crest cushions of Chinese Crested duck.

The Chinese Crested duck (Fengtou duck) reappeared in China recently. Along with white feathers and a black bill and feet, the Fengtou duck has a high feather crest. This breed can be used for ornamental purposes or as a model organism; however, little is known about the genetic basis and development of its distinct morphological features. In this study, we observed the skull and feather crest of Fengtou duck in the embryonic stages. As a result, the protuberances of the head integument could be clearly observed at embryonic stage E9, and small perforations in the skull were first visible at E13 and were clearer at E15. Besides, intracranial fat in a small number of individuals was found starting at E15, and a small number of osteophytes was found at E18. In addition, hematoxylin-eosin (HE) and Oil Red-O staining of the crest cushion and intracranial tissue revealed fat tissue accumulation. Previous studies demonstrated that homeobox c8 (HOXC8) played a critical role in chicken crest formation. Here, we cloned the HOXC8 from Fengtou duck and determined that its transcript was highly expressed in the crest cushion; moreover, HOXC8 was detected in this tissue with a molecular weight of 38 kDa in the Fengtou duck. In conclusion, embryos of Fengtou duck have different small protuberances and perforations in the skull, including accumulation of intracranial tissue and osteophytes in some cases. Furthermore, HOXC8 may regulate the formation of the crest. These findings provide novel insight into the ontogenesis of the crest cushion in crested ducks and a basis for future studies on their evolutionary origins.

Coupling of apical-basal polarity and planar cell polarity to interpret the Wnt signaling gradient in feather development.

Sensing a global directional cue to orient cell growth is crucial in tissue morphogenesis. An anterior-posterior gradient of Wnt signaling controls the helical growth of feather branches (barbs), and thus the formation of bilateral feathers. However, it remains unclear how the keratinocytes sense this gradient and orient barb growth. Here, we show that in chicken, owing to feather branching, the global Wnt gradient is subdivided into periodic barbs. Within each barb, the anterior barbule plate cells tilt before the posterior cells. The core planar cell polarity gene Prickle1 is involved, as knockdown of its expression resulted in no cell shape change and no barb tilting. Furthermore, perturbation of the Wnt gradient leads to diffusive Prickle1 expression and loss of barb orientation. Finally, the asymmetric distribution of Wnt6/Fzd10 is coordinated by the apical-basal polarity of the barbule plate keratinocytes, which is in turn regulated by the Par3/aPKC machinery. Our data elucidate a new mechanism through which the global Wnt signaling gradient is interpreted locally to construct complex spatial forms.

TGF-ß Family Signaling in Epithelial Differentiation and Epithelial-Mesenchymal Transition.

Epithelia exist in the animal body since the onset of embryonic development; they generate tissue barriers and specify organs and glands. Through epithelial-mesenchymal transitions (EMTs), epithelia generate mesenchymal cells that form new tissues and promote healing or disease manifestation when epithelial homeostasis is challenged physiologically or pathologically. Transforming growth factor-ßs (TGF-ßs), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs) have been implicated in the regulation of epithelial differentiation. These TGF-ß family ligands are expressed and secreted at sites where the epithelium interacts with the mesenchyme and provide paracrine queues from the mesenchyme to the neighboring epithelium, helping the specification of differentiated epithelial cell types within an organ. TGF-ß ligands signal via Smads and cooperating kinase pathways and control the expression or activities of key transcription factors that promote either epithelial differentiation or mesenchymal transitions. In this review, we discuss evidence that illustrates how TGF-ß family ligands contribute to epithelial differentiation and induce mesenchymal transitions, by focusing on the embryonic ectoderm and tissues that form the external mammalian body lining.

A chemotaxis model of feather primordia pattern formation during avian development.

The orderly formation of the avian feather array is a classic example of periodic pattern formation during embryonic development. Various mathematical models have been developed to describe this process, including Turing/activator-inhibitor type reaction-diffusion systems and chemotaxis/mechanical-based models based on cell movement and tissue interactions. In this paper we formulate a mathematical model founded on experimental findings, a set of interactions between the key cellular (dermal and epidermal cell populations) and molecular (fibroblast growth factor, FGF, and bone morphogenetic protein, BMP) players and a medially progressing priming wave that acts as the trigger to initiate patterning. Linear stability analysis is used to show that FGF-mediated chemotaxis of dermal cells is the crucial driver of pattern formation, while perturbations in the form of ubiquitous high BMP expression suppress patterning, consistent with experiments. Numerical simulations demonstrate the capacity of the model to pattern the skin in a spatial-temporal manner analogous to avian feather development. Further, experimental perturbations in the form of bead-displacement experiments are recapitulated and predictions are proposed in the form of blocking mesenchymal cell proliferation.

Emergent cellular self-organization and mechanosensation initiate follicle pattern in the avian skin.

The spacing of hair in mammals and feathers in birds is one of the most apparent morphological features of the skin. This pattern arises when uniform fields of progenitor cells diversify their molecular fate while adopting higher-order structure. Using the nascent skin of the developing chicken embryo as a model system, we find that morphological and molecular symmetries are simultaneously broken by an emergent process of cellular self-organization. The key initiators of heterogeneity are dermal progenitors, which spontaneously aggregate through contractility-driven cellular pulling. Concurrently, this dermal cell aggregation triggers the mechanosensitive activation of ß-catenin in adjacent epidermal cells, initiating the follicle gene expression program. Taken together, this mechanism provides a means of integrating mechanical and molecular perspectives of organ formation.

The differential expression of MC1R regulators in dorsal and ventral quail plumages during embryogenesis: Implications for plumage pattern formation.

Melanin pigmentation patterns are ubiquitous in animals and function in crypsis, physical protection, thermoregulation and signalling. In vertebrates, pigmentation patterns formed over large body regions as well as within appendages (hair/feathers) may be due to the differential distribution of pigment producing cells (melanocytes) and/or regulation of the melanin synthesis pathway. We took advantage of the pigmentation patterns of Japanese quail embryos (pale ventrum and patterned feathers dorsally) to explore the role of genes and their transcripts in regulating the function of the melanocortin-1-receptor (MC1R) via 1. activation: pro-opiomelanocortin (POMC), endoproteases prohormone convertase 1 (PC1) and 2 (PC2), and 2. inhibition-agouti signaling and agouti-related protein (ASIP and AGRP, respectively). Melanocytes are present in all feather follicles at both 8 and 12 days post-fertilisation (E8/E12), so differential deposition of melanocytes is not responsible for pigmentation patterns in embryonic quail. POMC transcripts expressed were a subset of those found in chicken and POMC expression within feather follicles was strong. PC1 was not expressed in feather follicles. PC2 was strongly expressed in all feather follicles at E12. ASIP transcript expression was variable and we report four novel ASIP transcripts. ASIP is strongly expressed in ventral feather follicles, but not dorsally. AGRP expression within feather follicles was weak. These results demonstrate that the pale-bellied quail phenotype probably involves inhibition of MC1R, as found previously. However, quail may require MC1R activation for eumelanogenesis in dorsal feathers which may have important implications for an understanding of colour pattern formation in vertebrates.

Review: cornification, morphogenesis and evolution of feathers.

Feathers are corneous microramifications of variable complexity derived from the morphogenesis of barb ridges. Histological and ultrastructural analyses on developing and regenerating feathers clarify the three-dimensional organization of cells in barb ridges. Feather cells derive from folds of the embryonic epithelium of feather germs from which barb/barbule cells and supportive cells organize in a branching structure. The following degeneration of supportive cells allows the separation of barbule cells which are made of corneous beta-proteins and of lower amounts of intermediate filament (IF)(alpha) keratins, histidine-rich proteins, and corneous proteins of the epidermal differentiation complex. The specific protein association gives rise to a corneous material with specific biomechanic properties in barbules, rami, rachis, or calamus. During the evolution of different feather types, a large expansion of the genome coding for corneous feather beta-proteins occurred and formed 3-4-nm-thick filaments through a different mechanism from that of 8-10 nm IF keratins. In the chick, over 130 genes mainly localized in chromosomes 27 and 25 encode feather corneous beta-proteins of 10-12 kDa containing 97-105 amino acids. About 35 genes localized in chromosome 25 code for scale proteins (14-16 kDa made of 122-146 amino acids), claws and beak proteins (14-17 kDa proteins of 134-164 amino acids). Feather morphogenesis is periodically re-activated to produce replacement feathers, and multiple feather types can result from the interactions of epidermal and dermal tissues. The review shows schematic models explaining the translation of the morphogenesis of barb ridges present in the follicle into the three-dimensional shape of the main types of branched or un-branched feathers such as plumulaceous, pennaceous, filoplumes, and bristles. The temporal pattern of formation of barb ridges in different feather types and the molecular control from the dermal papilla through signaling molecules are poorly known. The evolution and diversification of the process of morphogenesis of barb ridges and patterns of their formation within feathers follicle allowed the origin and diversification of numerous types of feathers, including the asymmetric planar feathers for flight.

Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules.

The morphogenesis of feathers is a complex process that depends on a tight spatiotemporal regulation of gene expression and assembly of the protein components of mature feathers. Recent comparative genomics and gene transcription studies have indicated that genes within the epidermal differentiation complex (EDC) encode numerous structural proteins of cornifying skin cells in amniotes including birds. Here, we determined the localization of one of these proteins, termed EDMTFH (Epidermal Differentiation Protein starting with a MTF motif and rich in Histidine), which belongs to a group of EDC-encoded proteins rich in aromatic amino acid residues. We raised an antibody against an EDMTFH-specific epitope and performed immunohistochemical investigations by light microscopy and immunogold labeling by electron microscopy of chicken embryos at days 14-18 of development. EDMTFH was specifically present in the subperiderm, a transient layer of the embryonic epidermis, and in barbs and barbules of feathers. In the latter, it partially localized to bundles of so-called feather beta-keratins (corneous beta-proteins, CBPs). Cells of the embryonic periderm, the epidermis proper, and the feather sheath were immunonegative for EDMTFH. The results of this study indicate that EDMTFH may contribute to the unique mechanical properties of feathers and define EDMTFH as a common marker of the subperiderm and the feather barbules. This expression pattern of EDMTFH resembles that of epidermal differentiation cysteine-rich protein (EDCRP) and feather CBPs and is in accordance with the hypothesis that a major part of the cyclically regenerating feather follicle is topologically, developmentally and evolutionarily related to the embryonic subperiderm.

Generation of bioengineered feather buds on a reconstructed chick skin from dissociated epithelial and mesenchymal cells.

Various kinds of in vitro culture systems of tissues and organs have been developed, and applied to understand multicellular systems during embryonic organogenesis. In the research field of feather bud development, tissue recombination assays using an intact epithelial tissue and mesenchymal tissue/cells have contributed to our understanding the mechanisms of feather bud formation and development. However, there are few methods to generate a skin and its appendages from single cells of both epithelium and mesenchyme. In this study, we have developed a bioengineering method to reconstruct an embryonic dorsal skin after completely dissociating single epithelial and mesenchymal cells from chick skin. Multiple feather buds can form on the reconstructed skin in a single row in vitro. The bioengineered feather buds develop into long feather buds by transplantation onto a chorioallantoic membrane. The bioengineered bud sizes were similar to those of native embryo. The number of bioengineered buds was increased linearly with the initial contact length of epithelial and mesenchymal cell layers where the epithelial-mesenchymal interactions occur. In addition, the bioengineered bud formation was also disturbed by the inhibition of major signaling pathways including FGF (fibroblast growth factor), Wnt/ß-catenin, Notch and BMP (bone morphogenetic protein). We expect that our bioengineering technique will motivate further extensive research on multicellular developmental systems, such as the formation and sizing of cutaneous appendages, and their regulatory mechanisms.

Nuclear ß-catenin localization supports homology of feathers, avian scutate scales, and alligator scales in early development.

Feathers are an evolutionary novelty found in all extant birds. Despite recent progress investigating feather development and a revolution in dinosaur paleontology, the relationship of feathers to other amniote skin appendages, particularly reptile scales, remains unclear. Disagreement arises primarily from the observation that feathers and avian scutate scales exhibit an anatomical placode-defined as an epidermal thickening-in early development, whereas alligator and other avian scales do not. To investigate the homology of feathers and archosaur scales we examined patterns of nuclear ß-catenin localization during early development of feathers and different bird and alligator scales. In birds, nuclear ß-catenin is first localized to the feather placode, and then exhibits a dynamic pattern of localization in both epidermis and dermis of the feather bud. We found that asymmetric avian scutate scales and alligator scales share similar patterns of nuclear ß-catenin localization with feathers. This supports the hypothesis that feathers, scutate scales, and alligator scales are homologous during early developmental stages, and are derived from early developmental stages of an asymmetric scale present in the archosaur ancestor. Furthermore, given that the earliest stage of ß-catenin localization in feathers and archosaur scales is also found in placodes of several mammalian skin appendages, including hair and mammary glands, we hypothesize that a common skin appendage placode originated in the common ancestor of all amniotes. We suggest a skin placode should not be defined by anatomical features, but as a local, organized molecular signaling center from which an epidermal appendage develops.

Development, regeneration, and evolution of feathers.

The feather is a complex ectodermal organ with hierarchical branching patterns. It provides functions in endothermy, communication, and flight. Studies of feather growth, cycling, and health are of fundamental importance to avian biology and poultry science. In addition, feathers are an excellent model for morphogenesis studies because of their accessibility, and their distinct patterns can be used to assay the roles of specific molecular pathways. Here we review the progress in aspects of development, regeneration, and evolution during the past three decades. We cover the development of feather buds in chicken embryos, regenerative cycling of feather follicle stem cells, formation of barb branching patterns, emergence of intrafeather pigmentation patterns, interplay of hormones and feather growth, and the genetic identification of several feather variants. The discovery of feathered dinosaurs redefines the relationship between feathers and birds. Inspiration from biomaterials and flight research further fuels biomimetic potential of feathers as a multidisciplinary research focal point.

Immunodetection of type I acidic keratins associated to periderm granules during the transition of cornification from embryonic to definitive chick epidermis.

The change in the modality of cornification from embryonic to definitive epidermis in the chick has been studied using immunocytochemistry and electron microscopy to show that the initial soft cornification based on an acidic type I alpha-keratin transits to a definitive hard cornification based on beta-proteins in the claw, scales and feathers. The first two periderm layers contain acidic keratins associated with periderm granules and participate in a mild form of cornification before shedding of the periderm. The transition from embryonic to adult cornification is best seen in the transitional layers of the claw where numerous periderm granules merge with packets or bundles of corneous beta-proteins. This process is hardly seen in scale and feathers where periderm granules remain most in the periderm or in the feather sheath. Periderm granules disappear in corneocytes generated underneath the periderm in scales or in the transitional layer in claws and are replaced by beta-proteins associated to other types of acidic alpha-keratins. This process produces a mechanically resistant corneous material underneath the softer periderm, adapted to terrestrial demand for mechanical protection in scales and in the dorsal part of the claw, the unguis. In the ventral part of the claw, the sub-unguis, scarce or no beta-proteins are accumulated resulting in a softer corneous layer. The study indicates that specific alpha-keratins form the cytoskeletal framework of definitive corneocytes in claws, scales and feathers, and that specialized corneous beta-proteins are deposited over this framework to produce epidermal layers with higher mechanical resistance.

Developmental expression of chicken FOXN1 and putative target genes during feather development.

FOXN1 is a member of the forkhead box family of transcription factors. FOXN1 is crucial for hair outgrowth and thymus differentiation in mammals. Unlike the thymus, which is found in all amniotes, hair is an epidermal appendage that arose after the last shared common ancestor between mammals and birds, and hair and feathers differ markedly in their differentiation and gene expression. Here, we show that FOXN1 is expressed in embryonic chicken feathers, nails and thymus, demonstrating an evolutionary conservation that goes beyond obvious homology. At embryonic day (ED) 12, FOXN1 is expressed in some feather buds and at ED13 expression extends along the length of the feather filament. At ED14 FOXN1 mRNA is restricted to the proximal feather filament and is not detectable in distal feather shafts. At the base of the feather, FOXN1 is expressed in the epithelium of the feather sheath and distal barb and marginal plate, whereas in the midsection FOXN1 transcripts are mainly detected in the barb plates of the feather filament. FOXN1 is also expressed in claws; however, no expression was detected in skin or scales. Despite expression of FOXN1 in developing feathers, examination of chick homologs of five putative mammalian FOXN1 target genes shows that, while these genes are expressed in feathers, there is little similarity to the FOXN1 expression pattern, suggesting that some gene regulatory networks may have diverged during evolution of epidermal appendages.