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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-3β inhibitor, which activates Wnt/β-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/β-catenin signaling may contribute to the formation of epithelial and mesenchymal components in the bioengineered skin., (© 2019 Japanese Society of Developmental Biologists.)

Amniote skin appendages such as feathers, hairs and scales, provide thermoregulation, physical protection and display different color patterns to attract a mate or frighten an adversary. A long-standing question is whether "reptile scale" and "avian leg scales" are of the same origin. Understanding the relation between avian feathers, avian scales and reptilian scales will enhance our understanding of skin appendage evolution. We compared the molecular and cellular profiles in chicken feather, chicken scales and alligator scales and found that chicken scutate scales are similar to chicken feathers in morphogenesis at the early placode stage. When we compared the expression of the recently identified feather-specific genes and scale-specific genes in these skin appendages, we found that at the molecular level alligator scales are significantly different from both chicken feathers and chicken scales. Furthermore, we identified a similarly diffuse putative stem cell niche in morphologically similar chicken and alligator scales. These putative stem cells participate in alligator scale regeneration. In contrast, avian feathers have a more condensed stem cell niche, which may be responsible for cycling. Thus, our results suggest that chicken and alligator scales formed independently through convergent evolution.

Background: Early feathering and late feathering in chickens are sex-linked phenotypes, which have commercial application in the poultry industry for sexing chicks at hatch and have important impacts on performance traits. However, the genetic mechanism controlling feather development and feathering patterns is unclear. Here, miRNA and mRNA expression profiles in chicken wing skin tissues were analysed through high-throughput transcriptomic sequencing, aiming to understand the biological process of follicle development and the formation of different feathering phenotypes., Results: Compared to the N1 group with no primary feathers extending out, 2893 genes and 31 miRNAs displayed significantly different expression in the F1 group with primary feathers longer than primary-covert feathers, and 1802 genes and 11 miRNAs in the L2 group displayed primary feathers shorter than primary-covert feathers. Only 201 altered genes and 3 altered miRNAs were identified between the N1 and L2 groups (fold change > 2, q value < 0.01). Both sequencing and qPCR tests revealed that PRLR was significantly decreased in the F1 and L2 groups compared to the N1 group, whereas SPEF2 was significantly decreased in the F1 group compared to the N1 or L2 group. Functional analysis revealed that the altered genes or targets of altered miRNAs were involved in multiple biological processes and pathways related to feather growth and development, such as the Wnt signalling pathway, the TGF-beta signalling pathway, the MAPK signalling pathway, epithelial cell differentiation, and limb development. Integrated analysis of miRNA and mRNA showed that 14 pairs of miRNA-mRNA negatively interacted in the process of feather formation., Conclusions: Transcriptomic sequencing of wing skin tissues revealed large changes in F1 vs. N1 and L2 vs. N1, but few changes in F1 vs. L2 for both miRNA and mRNA expression. PRLR might only contribute to follicle development, while SPEF2 was highly related to the growth rate of primary feathers or primary-covert feathers and could be responsible for early and late feather formation. Interactions between miR-1574-5p/NR2F, miR-365-5p/JAK3 and miR-365-5p/CDK6 played important roles in hair or feather formation. In all, our results provide novel evidence to understand the molecular regulation of follicle development and feathering phenotype.

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., Competing Interests: The authors have declared that no competing interests exist.

Morphogenesis of hair follicles, teeth, and mammary glands depends on inductive epithelial-mesenchymal interactions mediated by a conserved set of signalling molecules. The early development of different skin appendages is remarkably similar. Initiation of organogenesis is marked by the appearance of a local epithelial thickening, a placode, which subsequently invaginates to produce a bud. These early developmental stages require many of the same genes and signalling circuits and consequently alterations in them often cause similar phenotypes in several skin appendages. After the bud stage, these organs adopt diverse patterns of epithelial growth, reflected in the usage of more divergent genes in each.

The present ultrastructural study on regenerating feathers emphasizes the role of supportive cells in determining the branching pattern of barbs. Supportive cells are localized among developing barb and barbule cells, in marginal plates, and underneath the feather sheath, and their differentiative fate, in general, is a form of lipid degeneration. The Latter process determines the carving out of barb branching in both downfeathers and pennaceous feathers. In the latter feathers, some supportive cells (barb vane cells and cylindrical cells of marginal plates) degenerate within each barb ridge leaving separate barbules. Other supportive cells, here termed wedge cells, form columns of cornified material that merge into elongated corneous scaffolds localized among barbs and the rachis. This previously undescribed form of cornification of supportive cells derives from the aggregation of periderm and dense granules present in wedge cells. The latter cells give origin to a corneous material different from feather keratin that may initially sustain the early and soft barbules. After barbules are cornified the supportive cells scaffolds are eventually sloughed as the sheath breaks allowing the new feather to open up and form a planar vane. The corneous material of wedge cells may also contribute to molding of the overlapped nodes of barbule cells that form lateral spines or hooklets in mature barbules. Eventually, the disappearance of wedge cell scaffolding determines the regular spacing of barbs attached to the rachis in order to form a close vane.

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.

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.

To analyse the morphogenic events during skin appendage formation, it is important to have an animal model that offers distinct patterns at various stages of development and is accessible to analysis using state of the art technology. The avian integument is such a model. Combining experimental embryologic approaches, organ cultures, and gene transduction technology, we are now able to begin to address the molecular basis of pattern formation, primordium initiation, anterior-posterior axis formation, proximo-distal axis formation, phenotypic determination, and others. Parallel mechanisms are usually found in feathers and hairs, and the avian integument model has matured to be a major source of new findings in the study of skin appendage morphogenesis. More information on the avian integument model can be found at website http://www.hsc.usc.educmchuong.

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.

We have previously shown that feather formation in chick embryonic skin depends on accumulation of sulphated glycosaminoglycans in the underlying mesenchyme, and that addition of spermidine to chick embryo fibroblasts increases the extracellular sulphated glycosaminoglycans. In the present work, using histological, histochemical and biochemical procedures, we have investigated the effects on glycosaminoglycan accumulation and on epithelial differentiation of adding spermidine and bis-cyclohexylammonium sulphate, a spermidine inhibitor, to embryonic chick skin cultures. Our results demonstrate that spermidine induces an accumulation of sulphated glycosaminoglycan and an increase in feather formation, suggesting that the morphogenetic effect of spermidine may be dependent on specific glycosaminoglycan accumulation.

Classical histological studies have demonstrated the cellular organization of skin appendages and helped us appreciate the intricate structures and function of skin appendages. At this juncture, questions can be directed to determine how these cellular organizations are achieved. How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? What are the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, many new molecules expressed in a spatial and temporal specific manner during the formation of skin appendages were identified by molecular biological approaches. In this review, novel molecular techniques that are useful in skin appendage research are discussed. The distribution of exemplary molecules from different categories including growth factors, intracellular signaling molecules, homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in a diagram using feather and hair as models. We hope that these results will serve as the ground work for completing the molecular mapping of skin appendages which will refine and re-define our understanding of the developmental process beyond relying on morphological criteria. We also hope that the listed protocols will help those who are interested in this venture. This new molecular histology of skin appendages is the foundation for forming new hypotheses on how molecules are mechanistically involved in skin appendage development and for designing experiments to test them. This may also lead to the modulation of healing and regeneration processes in future treatment modalities.

Figure 9 summarizes the morphogenetic process of feather and hair. Hair of feathers are formed from a layer of homogeneously distributed mesenchymal cells. The mesenchymal cells start to condense to form foci in response to some unidentified induction signal (Fig. 9B). Several adhesion molecules, including L-CAM, N-CAM, integrin, tenascin, as well as proteoglycan, are involved. These adhesion molecules appear to have different roles in this process, because perturbation with specific antibodies leads to different aborted patterns. Hair or feather follicles then form following cell proliferation and epithelial invagination (Fig. 9C). The dermal papilla is enriched with N-CAM and tenascin, whereas the feather collar (equivalent of hair matrix) is enriched with L-CAM and PDGF receptor. Epithelial cells in the feather collar receive a signal from the dermal papilla and are able to continue to divide. Several growth factors, such as PDGF and EGF, may be involved. As epithelial cells are pushed upwards, they differentiate and keratinize in a cylindrical structure into hair. In feather, another morphogenetic event takes place to form the branched structure. The epithelial cylinder of the feather shaft invaginates to form rows of cells that die to become space and create the secondary branch or barbs (Fig. 9D). N-CAM is enriched in the cells destined to die and appears to form the border of cell groups within which the "death signal" is transmitted. In some, but not all, feathers the same process is repeated, in a way analogous to fractal formation, to form the tertiary branches or the barbules (Fig. 9E). Thus, in each step of the morphogenesis of feather and hair, different adhesion molecules are expressed and are involved in different functions: induction, mesenchymal condensation, epithelial folding, and cell death, depending on different scenarios. We have just begun to elucidate these molecular events.

Several reports have suggested that mesenchymal glycosaminoglycans (GAG) may be involved in the regulatory role of epithelial differentiation. Some researchers have pointed out that exogenous GAG affects extracellular GAG accumulation. We have therefore examined the effect of added GAG on two typical processes of avian skin differentiation: keratinization and feather formation. Glycosaminoglycans, either obtained from fibroblasts cultures (conditioned media) or purified commercially available GAG were administered to 5/6-day chick embryo back skin explants. Control cultures were supported with 199 synthetic medium, chick embryo extract or calf serum. Explants have been examined by histological and histochemical procedures. Skin explants maintained in vitro for 7 days exhibited an epithelial differentiation and a dermal histochemical reactivity which were related to the composition of the culture medium. In conditioned media from dermal fibroblasts, but not from heart or lung fibroblasts, explants always exhibited keratinization. In purified-GAG-containing media, keratinization was observed with condroitinsulphates and not with hyaluronic acid. Keratinization was always related to prevalent accumulation of hyaluronic acid in the underlying mesenchyme whereas feather formation was in relation to deposits of condroitinsulphates in dermis pulp. The above findings demonstrate that exogenous GAG is able to modulate avian skin differentiation and that this regulation is linked to an influence on the mesenchymal GAG pattern.