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.

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.

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.

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.

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.

Shaping organs by a wingless-int/Notch/nonmuscle myosin module which orients feather bud elongation.

How organs are shaped to specific forms is a fundamental issue in developmental biology. To address this question, we used the repetitive, periodic pattern of feather morphogenesis on chicken skin as a model. Avian feathers within a single tract extend from dome-shaped primordia to thin conical structures with a common axis of orientation. From a systems biology perspective, the process is precise and robust. Using tissue transplantation assays, we demonstrate that a "zone of polarizing activity," localized in the posterior feather bud, is necessary and sufficient to mediate the directional elongation. This region contains a spatially well-defined nuclear ß-catenin zone, which is induced by wingless-int (Wnt)7a protein diffusing in from posterior bud epithelium. Misexpressing nuclear ß-catenin randomizes feather polarity. This dermal nuclear ß-catenin zone, surrounded by Notch1 positive dermal cells, induces Jagged1. Inhibition of Notch signaling disrupts the spatial configuration of the nuclear ß-catenin zone and leads to randomized feather polarity. Mathematical modeling predicts that lateral inhibition, mediated by Notch signaling, functions to reduce Wnt7a gradient variations and fluctuations to form the sharp boundary observed for the dermal ß-catenin zone. This zone is also enriched for nonmuscle myosin IIB. Suppressing nonmuscle myosin IIB disrupts directional cell rearrangements and abolishes feather bud elongation. These data suggest that a unique molecular module involving chemical-mechanical coupling converts a pliable chemical gradient to a precise domain, ready for subsequent mechanical action, thus defining the position, boundary, and duration of localized morphogenetic activity that molds the shape of growing organs.

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.

Expression patterns of prdm1 during chicken embryonic and germline development.

PRDM1 (PR domain containing 1) is a transcriptional repressor that has been identified in various species and is crucial for cell growth, differentiation and development. However, the expression pattern and role of PRDM1 in development has not been sufficiently established in birds. We therefore investigate the spatio-temporal expression of PRDM1 in various tissues, especially in the germline, during chicken development, providing the basis for functional study. Our results show that prdm1 mRNA was expressed in blastodermal cells (BCs) at stage X and in various tissues including the liver, skin, lung, kidney, eye, bursa of fabricius, spleen, proventriculus, gizzard, intestine, testis, ovary, tongue, feathers and thymus but was not or was only sparcely present in the heart, brain and skeletal muscle. The level of prdm1 mRNA was highest in the BCs among all tissues tested and significantly changed during development in many tissues, such as the blastoderm, bursa of fabricius, spleen, feathers and germline. Furthermore, the expression of the PRDM1 protein generally paralleled the mRNA results, except for in the gizzard. Immunohistochemistry also revealed that PRDM1 was localized in the smooth muscle. In addition, during germline development, PRDM1 was found to be continuously expressed in the presumptive primordial germ cells (PGCs) at stage X, the circulating PGCs in blood and the germ cells in the gonads from embryonic day 6 to adult in both males and females. The expression pattern of PRDM1 in chicken thus suggests that this protein plays an important role during chicken development, such as in BC differentiation, feather formation and germ cell specification.

Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering.

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body.

Feather regeneration as a model for organogenesis.

In the process of organogenesis, different cell types form organized tissues and tissues are integrated into an organ. Most organs form in the developmental stage, but new organs can also form in physiological states or following injuries during adulthood. Feathers are a good model to study post-natal organogenesis because they regenerate episodically under physiological conditions and in response to injuries such as plucking. Epidermal stem cells in the collar can respond to activation signals. Dermal papilla located at the follicle base controls the regenerative process. Adhesion molecules (e.g., neural cell adhesion molecule (NCAM), tenascin), morphogens (e.g., Wnt3a, sprouty, fibroblast growth factor [FGF]10), and differentiation markers (e.g., keratins) are expressed dynamically in initiation, growth and resting phases of the feather cycle. Epidermal cells are shaped into different feather morphologies based on the molecular micro-environment at the moment of morphogenesis. Chicken feather variants provide a rich resource for us to identify genetic determinants involved in feather regeneration and morphogenesis. An example of using genome-wide single nucleotide polymorphism (SNP) analysis to identify alpha keratin 75 as the mutation in frizzled chickens is demonstrated. Due to its accessibility to experimental manipulation and observation, results of regeneration can be analyzed in a comprehensive way. The layout of time dimension along the distal (formed earlier) to proximal (formed later) feather axis makes the morphological analyses easier. Therefore feather regeneration can be a unique model for understanding organogenesis: from activation of stem cells under various physiological conditions to serving as the Rosetta stone for deciphering the language of morphogenesis.

Molecular signaling in feather morphogenesis.

The development and regeneration of feathers have gained much attention recently because of progress in the following areas. First, pattern formation. The exquisite spatial arrangement provides a simple model for decoding the rules of morphogenesis. Second, stem cell biology. In every molting, a few stem cells have to rebuild the entire epithelial organ, providing much to learn on how to regenerate an organ physiologically. Third, evolution and development ('Evo-Devo'). The discovery of feathered dinosaur fossils in China prompted enthusiastic inquiries about the origin and evolution of feathers. Progress has been made in elucidating feather morphogenesis in five successive phases: macro-patterning, micro-patterning, intra-bud morphogenesis, follicle morphogenesis and regenerative cycling.

Effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation in chick embryonic cultured dorsal skin.

BACKGROUND: Retinoic acid, an active metabolite of retinol, is known to regulate cell proliferation, differentiation, and morphogenesis during normal development of many tissues. Using chick embryonic tarsometatarsal skin, we showed previously that the expression of Gbx1, a divergent homeobox gene, is increased in the epidermis through interaction with retinol-pretreated dermal fibroblasts followed by epidermal transdifferentiation to mucous epithelium. This present study was performed to elucidate the effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation. RESULTS: We showed that Gbx1 was expressed in the chick embryonic dorsal epidermis as early as at placode stage (Hamburger and Hamilton stage 31) and increased in amount during feather-bud formation. Treatment with 1 µM retinoic acid for 24 hr inhibited feather-bud formation and induced the transdifferentiation of the epidermis to a mucosal epithelium with a concomitant increase in Gbx1 mRNA expression in the epithelium. Furthermore, transient transfection of the epidermis with Gbx1 cDNA by electroporation induced elongation of the feather bud, but did not result in transdifferentiation. CONCLUSIONS: These results indicate that Gbx1 was involved in the feather-bud formation and was one of target genes of retinoic acid and that other signals in addition to Gbx1 were required for epidermal mucous transdifferentiation.

The relationship of plumage colours with MC1R (Melanocortin 1 Receptor) and ASIP (Agouti Signaling Protein) in Japanese quail (Coturnix coturnix japonica).

1. The relationship of polymorphisms in the Melanocortin 1 Receptor (MC1R) and Agouti Signalling Protein (ASIP) genes with plumage colour in Japanese quail was investigated by cloning and sequencing the entire coding regions from black, white and maroon Japanese quail embryos at 12 d of incubation. 2. Three SNPs were identified in the MC1R coding region by multiple alignment of sequences from individuals with different plumage colours. A missense C/T mutation located at 169 bp within the Open Reading Frame caused a Ile57Val mutation in the amino acid sequence, and had a significant relationship with the black colour. 3. The expression of MC1R was higher in black plumage quails than that in maroon plumage quails, whereas the expression of ASIP was higher in maroon plumage quails than that in black plumage quails. 4. It is concluded that the black plumage colour in Japanese quails may be caused by either increased production of MC1R or decreased production of ASIP.

Follicle characteristics and follicle developmental related Wnt6 polymorphism in Chinese indigenous Wanxi-white goose.

In birds, downy feather quantity mainly affected by the follicles. Wnt6, a secreted cysteine-rich protein, plays a key role in follicular development as an intercellular signaling molecule. The present study was to investigate the follicle development and Wnt6 polymorphism in Wanxi-white geese, a Chinese indigenous breed. In total, 300 fertilized eggs were hatched. At embryonic stage and on early birth goslings, the diameter and density of follicles from different sites were examined after sectioning and staining. The results showed that the diameter of primary feather follicles in thorax, venter, dorsum and flank had no difference at embryonic stage. In contrast, after birth, thorax and ventral feather follicles had greater diameter than those on dorsum and flank. Similarly, the primary feather follicle density was higher in thorax and venter than in dorsum and flank at embryonic stage. The secondary feather follicle diameter in flank was greater than that in other sites examined. The secondary follicle showed lush growth in E27 with thickest in ventral and thorax. Overall, follicle formed consistently in dorsal and flank, and follicle in thorax and ventral formed in another consistent way. The polymorphism study showed 2 single nucleotide polymorphisms of Wnt6 and 3 genotypes identified. Sequencing revealed two nucleotide transitions, T451C and A466G, which were synonymous mutations causing codons for aspartate and lysine to change from GAU to GAC and from AAA to AAG, respectively. This information about follicle development and Wnt6 polymorphisms would provide potential utilization in marker-assisted selection program for down feather selection.

From buds to follicles: matrix metalloproteinases in developmental tissue remodeling during feather morphogenesis.

Organogenesis involves a series of dynamic morphogenesis and remodeling processes. Since feathers exhibit complex forms, we have been using the feather as a model to analyze how molecular pathways and cellular events are used. While several major molecular pathways have been studied, the roles of matrix degrading proteases and inhibitors in feather morphogenesis are unknown. Here we addressed this knowledge gap by studying the temporal and spatial expression of proteases and inhibitors in developing feathers using mammalian antibodies that cross react with chicken proteins. We also investigated the effect of protease inhibitors on feather development employing an in vitro feather bud culture system. The results show that antibodies specific for mammalian MMP2 and TIMP2 stained positive in both feather epithelium and mesenchyme. The staining co-localized in structures of E10-E13 developing feathers. Interestingly, MMP2 and TIMP2 exhibited a complementary staining pattern in developing E15 and E20 feathers and in maturing feather filaments. Although they exhibited a slight delay in feather bud development, similar patterns of MMP2 and TIMP2 staining were observed in in vitro culture explants. The broad spectrum pharmacological inhibitors AG3340 and BB103 (MMP inhibitors) but not Aprotinin (a plasmin inhibitor) showed a reversible effect on epithelium invagination and feather bud elongation. TIMP2, a physiological inhibitor to MMPs, exhibited a similar effect. Markers of feather morphogenesis showed that MMP activity was required for both epithelium invagination and mesenchymal cell proliferation. Inhibition of MMP activity led to an overall delay in the expression of molecules that regulate either early feather bud growth and/or differentiation and thereby produced abnormal buds with incomplete follicle formation. This work demonstrates that MMPs and their inhibitors are not only important in injury repair, but also in development tissue remodeling as demonstrated here for the formation of feather follicles.

Differential regional expression of multiple ADAMs during feather bud formation.

The expression of seven members of the ADAM family was investigated by in situ hybridization in the developing feather buds of chicken. The expression profiles of the ADAMs in the cells and tissues of the feather buds differ from each other. ADAM9, ADAM10, and ADAM17 are expressed in the epidermis of the feather bud, whereas ADAM23 expression is restricted to the bud crest, with a distribution similar to that of sonic hedgehog. ADAM13 is not only expressed in the epidermis, but also in restricted regions of the dermis. Both ADAM12 and ADAM22 are expressed in the dermis of the feather bud, with an opposite mediolateral and anteroposterior polarity. Furthermore, the mRNAs of all investigated ADAMs show regional differences in their expression, for example, in the neck and in the roots of the leg and wing. These results suggest that ADAMs play a variety of roles during avian feather bud formation.

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.

Spots and stripes: pleomorphic patterning of stem cells via p-ERK-dependent cell chemotaxis shown by feather morphogenesis and mathematical simulation.

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction-diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large.

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.