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.

A source of exogenous oxidative stress improves oxidative status and favors pheomelanin synthesis in zebra finches.

Some organisms can modulate gene expression to trigger physiological responses that help adapt to environmental stress. The synthesis of the pigment pheomelanin in melanocytes seems to be one of these responses, as it may contribute to cellular homeostasis. We experimentally induced environmental oxidative stress in male zebra finches Taeniopygia guttata by the administration of the herbicide diquat dibromide during feather growth to test if the expression of genes involved in pheomelanin synthesis shows epigenetic lability. As pheomelanin synthesis implies decreasing the availability of the main cellular antioxidant (glutathione), it is expected to cause oxidative stress unless a protective mechanism limits pheomelanin synthesis and thus favors the antioxidant capacity. However, diquat exposure did not only improve the antioxidant capacity of birds, but also upregulated the expression of a gene (AGRP) that promotes pheomelanin synthesis in feather melanocytes, leading to the development of darker plumage coloration. No changes in the expression of other genes involved in pheomelanin synthesis (Slc7a11, Slc45a2, MC1R, ASIP and CTNS) were detected. DNA methylation levels only changed in MC1R, suggesting that epigenetic modifications other than changes in methylation may regulate AGRP expression lability. Our results suggest that exogenous oxidative stress induced a hormetic response that enhanced the oxidative status of birds and, consequently, promoted pheomelanin-based pigmentation, supporting the idea that birds adjust pheomelanin synthesis to their oxidative stress conditions.

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.

Involvement of selective epithelial cell death in the formation of feather buds on a bioengineered skin.

Selective cell death by apoptosis plays important roles in organogenesis. Apoptotic cells are observed in the developmental and homeostatic processes of several ectodermal organs, such as hairs, feathers, and mammary glands. In chick feather development, apoptotic events have been observed during feather morphogenesis, but have not been investigated during early feather bud formation. Previously, we have reported a method for generating feather buds on a bioengineered skin from dissociated skin epithelial and mesenchymal cells in three-dimensional culture. During the development of the bioengineered skin, epithelial cavity formation by apoptosis was observed in the epithelial tissue. In this study, we examined the selective epithelial cell death during the bioengineered skin development. Histological analyses suggest that the selective epithelial cell death in the bioengineered skin was induced by caspase-3-related apoptosis. The formation of feather buds of the bioengineered skin was disturbed by the treatment with a pan-caspase inhibitor. The pan-caspase inhibitor treatment suppressed the rearrangement of the epithelial layer and the formation of dermal condensation, which are thought to be essential step to form feather buds. The suppression of the formation of feather buds on the pan-caspase inhibitor-treated skin was partially compensated by the addition of a GSK-3ß inhibitor, which activates Wnt/ß-catenin signaling. These results suggest that the epithelial cell death is involved in the formation of feather buds of the bioengineered skin. These observations also suggest that caspase activities and Wnt/ß-catenin signaling may contribute to the formation of epithelial and mesenchymal components in the bioengineered skin.

Technical note: Induction of pluripotent stem cell-like cells from chicken feather follicle cells.

Pluripotent stem cells including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are regarded as representative tools for conservation of animal genetic resources. Although ESC have been established from chicken, it is very difficult to obtain enough embryos for isolation of stem cells for avian conservation in most wild birds. Therefore, the high feasibility of obtaining the pluripotent cell is most important in avian conservation studies. In this study, we generated induced pluripotent stem cell-like cells (iPSLC) from avian Feather Follicular cells (FFC). Avian FFC are one of the most easily accessible cell sources in most avian species, and their reprogramming into pluripotent stem cells can be an alternative system for preservation of avian species. Intriguingly, FFC had mesenchymal stromal cells (MSC)-like characteristics with regard to gene expression, protein expression, and adipocyte differentiation. Subsequently, we attempted to generate iPSLC from FFC using retroviral vectors. The FFC-iPSLC can proliferate with the stem pluripotent property and differentiate into several types of cells in vitro. Our results suggest that chicken FFC are an alternative cell source for avian cell reprogramming into pluripotent stem cells. This experimental strategy should be useful for conservation and restoration of endangered or high-value avian species without sacrificing embryos.

Pheomelanin molecular vibration is associated with mitochondrial ROS production in melanocytes and systemic oxidative stress and damage.

Vibrations in covalent bonds affect electron delocalization within molecules, as reported in polymers. If synthesized by living cells, the electron delocalization of polymers affects the stabilization of cellular free radicals, but biomolecular vibration has never been considered a potential source of cytotoxicity. Here we show that the vibrational characteristics of natural pheomelanin and eumelanin contribute to feather color expression in four poultry breeds with different melanin-based pigmentation patterns, but only the vibrational characteristics of pheomelanin are related to the production of reactive oxygen species (ROS) in the mitochondria of melanocytes and to systemic levels of cellular oxidative stress and damage. This association may be explained by the close physical contact existing between mitochondria and melanosomes, and reveals an unprecedented factor affecting the viability of organisms through their pigmentation. These findings open a new avenue for understanding the mechanism linking pheomelanin synthesis to human melanoma risk.

Feather on the Cap of Medicine.

Through cyclic regeneration, feather stem cells are molded into different shapes under different physiological states. With its distinct morphology, context-dependent growth, and experimental manipulability, the feather provides a rich model to study growth control, regeneration, and morphogenesis in vivo. Recent examples include novel insights revealed by transient perturbation with chemotherapeutic reagents and irradiation during feather growth.

Testing chemotherapeutic agents in the feather follicle identifies a selective blockade of cell proliferation and a key role for sonic hedgehog signaling in chemotherapy-induced tissue damage.

Chemotherapeutic agents induce complex tissue responses in vivo and damage normal organ functions. Here we use the feather follicle to investigate details of this damage response. We show that cyclophosphamide treatment, which causes chemotherapy-induced alopecia in mice and man, induces distinct defects in feather formation: feather branching is transiently and reversibly disrupted, thus leaving a morphological record of the impact of chemotherapeutic agents, whereas the rachis (feather axis) remains unperturbed. Similar defects are observed in feathers treated with 5-fluorouracil or taxol but not with doxorubicin or arabinofuranosyl cytidine (Ara-C). Selective blockade of cell proliferation was seen in the feather branching area, along with a downregulation of sonic hedgehog (Shh) transcription, but not in the equally proliferative rachis. Local delivery of the Shh inhibitor, cyclopamine, or Shh silencing both recapitulated this effect. In mouse hair follicles, those chemotherapeutic agents that disrupted feather formation also downregulated Shh gene expression and induced hair loss, whereas doxorubicin or Ara-C did not. Our results reveal a mechanism through which chemotherapeutic agents damage rapidly proliferating epithelial tissue, namely via the cell population-specific, Shh-dependent inhibition of proliferation. This mechanism may be targeted by future strategies to manage chemotherapy-induced tissue damage.

Ultrastructural characteristics of 5BrdU labeling retention cells including stem cells of regenerating feathers in chicken.

Feathers regenerate from stem cells localized in a region of the follicle indicated as the bulge of the collar. Stem cells are slow cycling cells and some of these cells can be identified after labeling experiments using 5-bromo-deoxyuridine to detect label retaining cells (5BrdU LRCs). The present electron microscopic analysis of 5BrdU LRCs has described the ultrastructural characteristics of small cells present in the bulge region of the follicle in regenerating feathers of chickens that include stem cells. Labeled feather stem cells are smaller than 10 lm in average diameter, possess large nuclei with high nuclear/cytoplasmic ratio, and contain evenly distributed ribosomes, sparse bundles of intermediate filaments, scarce or no endoplasmic reticulum, and few mitochondria. The nuclei are mainly euchromatic with a variable amount of heterochromatin clumps and the nucleoli show developed granular and fibrillar components. These features indicate that feather stem cells are transcriptionally active cells for ribosomal and proteins synthesis. The cell surface of feather stem cells often shows small and irregular folds resembling microvilli in contact with the surrounding cells. The latter characteristics may be related to the exchange of molecules and/or with the migration of stem cells among other epithelial cells of the collar epithelium.

Identification of a feather ß-keratin gene exclusively expressed in pennaceous barbule cells of contour feathers in chicken.

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of ß-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BlSK1). The BlSK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BlSK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BlSK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken.

Clusters of iron-rich cells in the upper beak of pigeons are macrophages not magnetosensitive neurons.

Understanding the molecular and cellular mechanisms that mediate magnetosensation in vertebrates is a formidable scientific problem. One hypothesis is that magnetic information is transduced into neuronal impulses by using a magnetite-based magnetoreceptor. Previous studies claim to have identified a magnetic sense system in the pigeon, common to avian species, which consists of magnetite-containing trigeminal afferents located at six specific loci in the rostral subepidermis of the beak. These studies have been widely accepted in the field and heavily relied upon by both behavioural biologists and physicists. Here we show that clusters of iron-rich cells in the rostro-medial upper beak of the pigeon Columbia livia are macrophages, not magnetosensitive neurons. Our systematic characterization of the pigeon upper beak identified iron-rich cells in the stratum laxum of the subepidermis, the basal region of the respiratory epithelium and the apex of feather follicles. Using a three-dimensional blueprint of the pigeon beak created by magnetic resonance imaging and computed tomography, we mapped the location of iron-rich cells, revealing unexpected variation in their distribution and number--an observation that is inconsistent with a role in magnetic sensation. Ultrastructure analysis of these cells, which are not unique to the beak, showed that their subcellular architecture includes ferritin-like granules, siderosomes, haemosiderin and filopodia, characteristics of iron-rich macrophages. Our conclusion that these cells are macrophages and not magnetosensitive neurons is supported by immunohistological studies showing co-localization with the antigen-presenting molecule major histocompatibility complex class II. Our work necessitates a renewed search for the true magnetite-dependent magnetoreceptor in birds.

EROD activity and stable isotopes in seabirds to disentangle marine food web contamination after the Prestige oil spill.

In this study, we measured via surgical sampling hepatic EROD activity in yellow-legged gulls from oiled and unoiled colonies, 17 months after the Prestige oil spill. We also analyzed stable isotope composition in feathers of the biopsied gulls, in an attempt to monitor oil incorporation into marine food web. We found that yellow-legged gulls in oiled colonies were being exposed to remnant oil as shown by hepatic EROD activity levels. EROD activity was related to feeding habits of individual gulls with apparent consequences on delayed lethality. Capture-recapture analysis of biopsied gulls suggests that the surgery technique did not affect gull survival, giving support to this technique as a monitoring tool for oil exposure assessment. Our study highlights the combination of different veterinary, toxicological and ecological methodologies as a useful approach for the monitoring of exposure to remnant oil after a large oil spill.

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.

Prevalence and molecular characterization of meq in feather follicular epithelial cells of Korean broiler chickens.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens. Meq is the relevant oncogene and four isoforms, long (L)-meq, meq, short (S)-meq and very short (VS)-meq, have been identified. Although MD is important in the poultry industry, the prevalence and molecular properties of Korean MD virus (MDV) among broiler chickens remain unclear. Therefore, we characterized meq in pooled feather tips sampled at 3- and 5-week-old chickens from 21 unvaccinated and 22 vaccinated broiler farms via nested-PCR and nucleotide sequence analysis. Multiple bands consisting of L-meq, meq, and S-meq amplicons were observed in a commercial vaccine (CVI988 + HVT), 1 (4.8%) and 5 samples (22.7%) from unvaccinated and vaccinated farms, respectively. A strong meq amplicon was observed in a MD-related tumor tissue, 6 (28.6%) and 1 (4.5%) samples from unvaccinated and vaccinated farms, respectively. Six and one amplicons from unvaccinated (28.6%) and vaccinated farms (4.5%), respectively, were differentiated from CVI988 by nucleotide sequence analysis. Therefore, the relatively high rate of meq in the unvaccinated broiler farms constitutes support for vaccination. However, the existence of CVI988-related meq in unvaccinated chickens necessitates further study regarding the origins and pathoimmunological effects of the viruses on chickens.

Cytological Aspects of the Differentiation of Barb Cells During the Formation of the Ramus of Feathers / Aspectos Otológicos de la Diferenciación de Células Barba Durante la Formación de las Ramas de las Plumas

The present ultrastructural study on developing and regenerating feathers of chick and zebrafinch describes the ultrastructural changes that occur during the differentiation of barb cells that leads to the formation of the ramus of barbs. Differently from barbule and barb cortical cells that accumulate feather keratin, barb medullary cells undergo to lipid degeneration. Eventually, lipids disappear and medullary cells become empty cavities in the central part of the ramus. In barb medullary cells feather keratin is accumulated in few peripheral bundles that merge with those of cortical cells to fom the wall of the ramus. The latter is joined with branching barbules. The process that controls the transition from keratin-synthesizing to lipid-producing barb cells remains unknown. The accumulation of lipids among keratin bundles confirms the capability of beta-keratin cells to undergo an intense lipidogenesis under specific conditions.

La presente investigación ultra estructural sobre el desarrollo y regeneración de plumas en polluelos y gorrión cebra (Taeniopygia guttata castanotis) describe los cambios ultraestructurales que pueden ocurrir durante la diferenciación de células barbas que lleva a la formación de las ramas de las barbas. Diferente a las barbas pequeñas y a las células barbas corticales que acumulan queratina en las plumas, las células barbas medulares se convierten en cavidades vacías en la parte central de la rama. En células barbas medulares la queratina de la pluma es acumulada en algunos fascículos periféricos que se unen con aquellos de las células corticales para formar la pared de la rama. Este último se une luego a pequeñas barbas en ramas. Aún es desconocido el proceso que controla la transición de la síntesis de queratina a células barbas produciendo lípidos. La acumulación de lípidos entre los acumulos de queratina confirma la capacidad de las células beta-queratina a someterse a una lípido génesis intensa bajo condiciones específicas.

Mapping stem cell activities in the feather follicle.

It is important to know how different organs 'manage' their stem cells. Both hair and feather follicles show robust regenerative powers that episodically renew the epithelial organ. However, the evolution of feathers (from reptiles to birds) and hairs (from reptiles to mammals) are independent events and their follicular structures result from convergent evolution. Because feathers do not have the anatomical equivalent of a hair follicle bulge, we are interested in determining where their stem cells are localized. By applying long-term label retention, transplantation and DiI tracing to map stem cell activities, here we show that feather follicles contain slow-cycling long-term label-retaining cells (LRCs), transient amplifying cells and differentiating keratinocytes. Each population, located in anatomically distinct regions, undergoes dynamic homeostasis during the feather cycle. In the growing follicle, LRCs are enriched in a 'collar bulge' niche. In the moulting follicle, LRCs shift to populate a papillar ectoderm niche near the dermal papilla. On transplantation, LRCs show multipotentiality. In a three-dimensional view, LRCs are configured as a ring that is horizontally placed in radially symmetric feathers but tilted in bilaterally symmetric feathers. The changing topology of stem cell activities may contribute to the construction of complex feather forms.

EGF signaling patterns the feather array by promoting the interbud fate.

Feather buds form sequentially in a hexagonal array. Bone morphogenetic protein (BMP) signaling from the feather bud inhibits bud formation in the adjacent interbud tissue, but whether interbud fate and patterning is actively promoted by BMP or other factors is unclear. We show that epidermal growth factor (EGF) signaling acts positively to establish interbud identity. EGF and the active EGF receptor (EGFR) are expressed in the interbud regions. Exogenous EGF stimulates epidermal proliferation and expands interbud gene expression, with a concurrent loss of feather bud gene expression and morphology. Conversely, EGFR inhibitors result in the loss of interbud fate and increased acquisition of feather bud fate. EGF signaling acts directly on the epidermis and is independent of BMP signaling. The timing of competence to interpret interbud-promoting signals occurs at an earlier developmental stage than previously anticipated. These data demonstrate that EGFR signaling actively promotes interbud identity.

Establishment of feather follicle stem cells as potential vehicles for delivering exogenous genes in birds.

The present study was performed to develop a culture system for feather keratinocyte stem cells to enable the genetic manipulation of endangered avian species. The feather follicle cells were isolated from growing feathers of adult White Leghorn chicken. Leukemia inhibitory factor (LIF) was used to maintain the characterization of the keratinocyte colony-forming cells (KCFCs). The EGFPN1 plasmid DNA retroviral vector was used to deliver Green Fluorescent Protein (GFP) gene, which was introduced to the KCFCs by lipofection. After removal of the fibroblast-like cells, the feather KCFCs attached to the substrate within 24 h of seeding. The cells continued to proliferate for at least 30 days in the presence of LIF. The cell-adhesion molecules such as integrin beta1 and CD49c were immunocytochemically positive in the cells. The KCFCs differentiated into barbular cells and pennaceous feather vane in the LIF-free medium. The GFP gene-transfected KCFCs stably expressed GFP. The present results indicate that the KCFCs derived from feather follicles are closely related to multipotent stem cells. In addition, gene manipulation of such stem cells may be useful for the production of chimera in avian species.

Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.

Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution.

Epithelial appendages are derivatives of epithelia that elaborate to form specialized structures and functions. The appendage can protrude out, such as in teeth and feathers, or invaginate in, such as in glands. The epithelia can be ectodermal, such as in hairs, or endodermal, such as in livers. Using feather as a prototype of epithelial appendage, we study the molecular signals involved in the successive stages of epithelial-mesenchymal interactions during morphogenesis. We propose that these form the basics of gene networks, which can be integrated to gene supernetwork and totinetwork. Because the unit of development is molecular pathway rather than single molecule, and the unit of morphogenesis is cell group rather than single cell, we make the analogy between genes/developmental pathways and words/sentences. The study of developmental pathways in epithelial appendage organogenesis will help us to understand the grammar of genes and the basic rules in constructing regulated new growth. This knowledge may contribute to the study of cancer biology (deregulated new growth) and organ regeneration.