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.

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.

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.

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.

A novel angiogenesis model for screening anti-angiogenic compounds: the chorioallantoic membrane/feather bud assay.

Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies. Anti-angiogenic therapeutic approaches have recently been shown to be effective for the treatment of certain cancers. Endothelial cells migrating to tumors provide them with new blood vessels that are critical for their growth and survival. We have developed a novel and rapid method to evaluate the anti-angiogenic activity of new agents consisting of a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. Unlike previous assays, this new assay assesses the effects of drugs on the ability of tissues to attract and develop their own blood supply. The CAM already has a well-developed vascular network that is capable of providing blood vessels to the non-vascularized FB, allowing for this tissue to develop feathers. As a result, the exposure of the FB to drugs for 2 days followed by attachment to the CAM for 4 days allows evaluation of the compound's ability to impact blood vessel and feather formation within the CAM-attached FB tissue. Feather formation is determined as well as expression of endothelial cell genes and proteins analyzed. Using agents with known anti-angiogenic activity including fumagillin, minocycline, zoledronic acid, doxorubicin and agents lacking anti-angiogenic activity such as melphalan, we have shown that the CAM/FB assay can accurately and rapidly assess the ability of agents to prevent blood vessel and feather development within non-vascularized tissues.

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.

Cornification of the pulp epithelium and formation of pulp cups in downfeathers and regenerating feathers.

During most of feather growth (anagen), the dermal papilla stimulates the collar epithelium to give rise to feather keratins accumulating cells that form most of the corneous material of barbs and the rachis. Aside from the induction of differentiated cells of the feather, the distal part of the papilla forms a loose connective tissue that nourishes the growing feather, termed the pulp. In the last stages of feather growth, the pulp undergoes a process of re-absorption and leaves empty cavities indicated as pulp cups surrounded by keratinized cells inside the calamus. The process of cornification of pulp cups in different species of birds has been described here by using electron microscopy and immunocytochemistry for keratins. Pulp cells accumulate bundles of soft (alpha)-keratin, but do not synthesise feather keratins as in the surrounding calamus cells. Cells of the pulp epithelium accumulate large amounts of lipids and form a softer keratinized epithelium surrounding the pulp. This type of keratinization resembles the formation of soft epidermis in apteric and interfollicular regions. The role of the cornified pulp epithelium is to limit water loss and to form a microbe barrier before the mature feather is moulted.

Abortive placode formation in the feather tract of the scaleless chicken embryo.

The featherless phenotype of the scaleless mutant provides a model for delineating the process of feather follicle formation. Initial studies established that the mutation affects the epidermis and suggested that epidermis is unable to respond to signals from underlying dermis, or propagate a reciprocal signal. The work presented here demonstrates that scaleless epidermis does indeed respond to the initial inductive signals from dermis, as indicated by the localization of nuclear beta-catenin and transient focal expression of genes expressed in the placode of wild-type feather rudiments. In the sporadic "escaper" feathers that form in scaleless, expression of many genes associated with the progression of feather development is comparable to that in wild-type embryos. An exception is the ectodysplasin receptor gene Edar, which is expressed at lower levels in mutant feather buds. These observations suggest that the scaleless mutation impairs the locally augmented expression of Edar required to stabilize the placodal fate and sustain feather development.

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.

Transcriptional control of Shh/Ptc1 signaling in embryonic development.

In vivo profiling of signal-directed gene expression patterns is a major bottleneck in studying developmental biology. A signal molecule initiates its specific gene expression pattern through the activation of certain transcription factor (TF); however, tissue heterogeneity often masks this pattern due to intercellular complexity of other signal transduction pathways. To decipher the synergistic regulation of signal-directed gene expression in the tissue level, we report here a unique transcriptional responsive element (TRE) existing in the 5'-upstream promoter regions (5'-UPR) of the genes responding to the Shh/Ptc1 signal transduction pathway during feather placode development in chicken embryos. By locating the TRE homologue and its interactive TF, we were able to reveal the gene expression pattern of the Shh/Ptc1 signaling. We firstly demonstrated that homology profiling of the 5'-UPR of the genes, Gli1, TGF-beta2 and Msx2, responding to the Shh/Ptc1 signaling showed a more than 70% conserved region. Computer alignment of the consensus sequences in the conserved region revealed a 37-nucleotide TRE sequence, containing two regulatory elements homologous to human and mouse Gli-binding sites. Activation of this newly identified Shh/Ptc1-responsive TRE by active Smo signaling in chicken hepatoepithelial carcinoma cells elicited a strong synergistic expression of the Shh/Ptc1-downstream genes. Based on previous bioinformatics and the present experimental findings, we successfully established an in vivo signaling model for the Shh/Ptc1-directed embryonic feather morphogenesis.

Involvement of Hex in the initiation of feather morphogenesis.

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.

Expression of Hex during feather bud development.

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.

Ventral vs. dorsal chick dermal progenitor specification.

The dorsal and the ventral trunk integuments of the chick differ in their dermal cell lineage (originating from the somatic and somatopleural mesoderm respectively) and in the distribution of their feather fields. The dorsal macropattern has a large spinal pteryla surrounded by semi-apteria, whereas the ventral skin has a true medial apterium surrounded by the ventral pterylae. Comparison of the results of heterotopic transplantations of distal somatopleure in place of somatic mesoderm (Mauger 1972) or in place of proximal somatopleure (our data), leads to two conclusions. These are that the fate of the midventral apterium is not committed at day 2 of incubation and that the signals from the environment which specify the ventral and dorsal featherforming dermal progenitors are different. Effectively, Shh, but not Wnt -1 signalling can induce the formation of feather forming dermis from the embryonic somatopleure. Shh is not able, however, to trigger the formation of a feather forming dermis from the extra embryonic somatopleure. This brief report constitutes the first attempt, by comparing old and new preliminary results, to understand whether dermal progenitors at different sites are specified by different signalling pathways.

Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development.

The pattern of feather buds in a tract is thought to result from the relative ratios between activator and inhibitor signals through a lateral inhibition process. We analyse the role of Drm/Gremlin, a BMPs antagonist expressed during feather pattern formation, in the dermal precursor, the dense dermis, the interbud dermis and in the posterior dermal condensation. We have altered the activity of Drm in embryonic chick skin using retroviral vectors expressing drm/ gremlin and bmps. We show that expression of endogenous drm is under the control of a feedback loop induced by the BMP pathway, and that overexpression of drm results in fusion between adjacent feather buds. We propose that endogenous BMP proteins induce drm expression in the interbud dermis. In turn, the Drm/Gremlin protein limits the inhibitory effect of BMPs, allowing the adjacent row of feathers to form. Thus, the balance between BMPs and its antagonist Drm would regulate the size and spacing of the buds.

FGF signaling is required for initiation of feather placode development.

Morphogenesis of hairs and feathers is initiated by an as yet unknown dermal signal that induces placode formation in the overlying ectoderm. To determine whether FGF signals are required for this process we over-expressed soluble versions of FGFR1 or FGFR2 in the skin of chicken embryos. This produced a complete failure of feather formation prior to any morphological or molecular signs of placode development. We further show that Fgf10 is expressed in the dermis of nascent feather primordia, and that anti-FGF10 antibodies block feather placode development in skin explants. In addition we show that FGF10 can induce expression of positive and negative regulators of feather development and can induce its own expression under conditions of low BMP signaling. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and implicate FGF10 as an early dermal signal involved in this process.

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers.

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.

Spatial and temporal pattern of Wnt-6 expression during chick development.

The WNT family of proteins is composed of several members. In the present study we isolated the full length chick Wnt-6 cDNA and analyzed its expression pattern by in situ hybridization during chick development. Wnt-6 expression is observed in the ectoderm from HH-stage 4 onwards. At HH-stages, 7-16 expression can be seen in the ectoderm overlying the segmental plate and the epithelial somite, while the ectoderm overlying the compartmentalized somite is Wnt-6 negative. Expression is also observed at the heart outflow tract and in the ectoderm overlying the pharyngeal arches. From HH-stages 17 to 27, expression is also observed at limb level, both in the dorsal and ventral ectoderm and a stronger expression in the dorsoventral boundary. Furthermore, expression in the ectoderm delimiting the somitic boundaries in the anteroposterior and mediolateral axis at limb level was observed, as well as in the ventral body wall. Expression becomes evident in the inner ear. From HH-stage 30 onwards, expression is restricted to the feather buds and to the gastrointestinal tract.

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.