Soft Epidermis Research Articles

Immunolabeling for filaggrin and acidic keratins in the granular layer of mammalian epidermis indicates that an acidic-basic interaction is involved in cornification

The soft epidermis of mammals derives from the accumulation of keratohyaline granules in the granular layer, before maturing into corneocytes. Main proteins accumulated in the granular layer are pro-filaggrin and filaggrin that determine keratin clumping and later moisturization of the stratum corneum that remains flexible. This soft epidermis allows the high sensitivity of mammalian skin. Presence and thickness of the stratum granulosum varies among different species of mammals and even between different body regions of the same animal, from discontinuous to multilayered. These variations are evident using antibodies for filaggrin, a large protein that share common epitopes among placentals. Here we have utilized filaggrin antibodies (8959 and 466) and an acidic keratin antibody (AK2) for labeling placental, marsupial and monotreme epidermis. AK2 labeling appears mainly to detect K24 keratin, and less likely other acidic keratins. Immunoreactivity for filaggrin is absent in platypus, discontinuous in Echidna and in the tested marsupials. In placentals, it is inconstantly or hardly detected in the thin epidermis of bat, rodents, and lagomorphs with a narrow, mono-stratified and/or discontinuous granular layer. In contrast, where the granular layer is continuous or even stratified, both filaggrin and AK2 antibodies decorate granular cells. The ultrastructural analysis using the AK2 antibody on human epidermis reveals that a weak labeling is associated with keratohyalin granules and filamentous keratins of transitional keratinocytes and corneocytes. This observation suggests that basophilic filaggrin interacts with acidic keratins like K24 and determines keratin condensation into corneocytes of the stratum corneum.

Tracing the entry process of submicrometre plastics in soybean sprouts by leaf-derived fluorescent carbon dots

As a global emerging contaminant, microplastics (MPs) in water or soil can accumulate in vegetables, making them easily ingested through the diet. With excellent and tunable optical properties, carbon dots (CDs) are highly advantageous for tracing the entry process of MPs. Originally, long-wavelength CDs were synthesized from leaf-derived extracts, and fluorescent submicrometer plastics (CDs-MPs) with clean surfaces and concentrated particle sizes were obtained by soap-free microemulsion polymerization. The concentration of CDs-MPs exhibits a significant linear relationship with long-wavelength fluorescence intensity (λEx/λEm: 415/676 nm). Soybean sprouts (SBS), as an important type of food, are susceptible to contamination of MPs due to their soft epidermis and rapidly growing biomass. The results showed that CDs-MPs could be embedded into the cortex of SBS and enter the plant with cell division and elongation, leading to an increase in pore size on the cell wall surface. After entering the root system, CDs-MPs will pass through the Casparian strip and migrate in the vessels. Then, CDs-MPs enter the leaves through vascular bundles, and the distribution and size of epicuticular wax on leaves have changed. Furthermore, SBS showed resistant growth and increased levels of oxidative response when exposed to MPs/CDs-MPs. It is the first study to demonstrate the application of leaf-derived CDs in the prevention of MPs pollution by revealing the migration behavior of submicrometre plastics in SBS.

Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

Loading of microplastics by two related macroalgae in a sea area where gold and green tides occur simultaneously

Macroalgae are important components of offshore ecosystems and can also cause algal blooms. Microplastics (MPs) have been found in macroalgae and exhibit interactions during algal blooms. Ulva prolifera and Sargassum horneri are common algae in the Yellow Sea in China and are also the major macroalgae that cause green and gold tides. However, the loading of MPs by S. horneri along the coast of China has not been investigated, and the loading characteristics of MPs by two related macroalgae in a sea area where gold and green tides occur simultaneously have not been reported. Because U. prolifera and S. horneri occur simultaneously in the Yellow Sea, we investigated the characteristics of MPs in U. prolifera and S. horneri at 9 stations in the Yellow Sea located along the migration paths of green and gold tides. It was found that U. prolifera exhibited a higher abundance of MPs (0.065 ± 0.047 items/g−1 fresh weight) and ratio of the MP abundance in macroalgae to that in seawater (Rm/Rs = 97.33) than S. horneri (0.016 ± 0.016 items/g−1 fresh weight, Rm/Rs = 24.56). Moreover, the MPs in U. prolifera showed increased diversity in shape, color and material than those in S. horneri, and the particle sizes in the former were also smaller. Fast-growing biomass, slender branches, hollow air sacs and soft epidermis may allow U. prolifera to load more MPs. However, these mechanisms have not been fully established and merit further study. The relationship between severe algal blooms and MP pollution should be considered.

Anoxia can increase the rate of decay for cnidarian tissue: Using Actinia equina to understand the early fossil record.

An experimental decay methodology is developed for a cnidarian model organism to serve as a comparison to the many previous such studies on bilaterians. This allows an examination of inherent bias against the fossilisation of cnidarian tissue and their diagnostic characters, under what conditions these occur, and in what way. The decay sequence of Actinia equina was examined under a series of controlled conditions. These experiments show that cnidarian decay begins with an initial rupturing of the epidermis, followed by rapid loss of recognisable internal morphological characters. This suggests thatbacteria work quicker on the epidermis than autolysis does on the internal anatomy. The data also showthat diploblastic tissue is not universally decayed more slowly under anoxic orreducing conditions than under oxic conditions. Indeed, some cnidarian characters decay more rapidly under anoxic conditions than they do under oxic conditions. This suggests the decay pathways acting may be different to those affecting soft bilaterian tissue such as soft epidermis and internal organs. What is most important in the decay of soft polyp anatomy is the microbial community, which can be dominated by oxic or anoxic bacteria. Different Lagerstätte, even of the same type, will inevitably have subtle difference in their bacterial communities, which among other factors, could be a control on soft polyp preservation leading to either an absence of compelling soft anthozoans (Burgess Shale) or an astonishing abundance (Qingjiang biota).

Immunolocalization of beta‐proteins and alpha‐keratin in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material

AbstractImmunolocalization of beta‐proteins in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material, Acta Zoologica, Stockholm. The corneous layer of soft‐shelled turtles derives from the accumulation of higher ratio of alpha‐keratins versus beta‐proteins as indicated by gene expression, microscopic, immunocytochemical and Western blotting analysis. Type I and II beta‐proteins of 14–16 kDa, indicated as Tu2 and Tu17, accumulate in the thick and hard corneous layer of the hard‐shelled turtle, but only type II is present in the thinner corneous layer of the soft‐shelled turtle. The presence of proline–proline and proline–cysteine–hinge dipeptides in the beta‐sheet region of all type II beta‐proteins so far isolated from the epidermis of soft‐shelled turtles might impede the formation of beta‐filaments and of the hard corneous material. Western blot analysis suggests that beta‐proteins are low to absent in the corneous layer. The ultrastructural immunolocalization of Tu2 and Tu17 beta‐proteins shows indeed that a diffuse labelling is seen among the numerous alpha‐keratin filaments present in the precorneous and corneous layers of the soft epidermis and that no dense corneous material is formed. Double‐labelling experiments confirm that alpha‐keratin prevails on beta‐proteins. The present observations support the hypothesis that the soft material detected in soft‐shelled turtles derives from the prevalent activation of genes producing type II beta‐proteins and high levels of alpha‐keratins.

Ultrastructural pattern of loricrin localization in the epidermis of the red kangaroo Macropus rufus (Marsupialia, Mammalia) in relation to the formation of stratum corneum in mammalian epidermis

The ultrastructural immunolocalization of loricrin, a major protein of the cell corneous envelope is known for the epidermis of monotremes and placental mammals, but not in the soft epidermis of marsupials. This information would allow a definitive generalization on the role of loricrin in the process of keratinization in mammalian epidermis. The present study on the hairy epidermis of the red kangaroo has shown that the immunolabelling for this protein is diffuse in the upper spinosus layer and is peripherally associated with keratohyalin granules in the granular layer. Immunolabelling, however, is poor or absent in the darker part of keratohyalin granules which likely contains pro-filaggrin and filaggrin. In transitional and corneous cells, loricrin concentrates along the forming cell corneous envelope as in the hairy epidermis of other mammals. However, in the softer part of the corneous layer, rich in lipids, the labelling tends to disappear. Loricrin therefore appears to be connected with the strengthening of the cell corneous envelope of mature corneocytes, but becomes poorly detected in soft corneocytes. Based on the present and previous information, the distribution of loricrin in the epidermis of different mammals and in the epidermis of birds and reptiles is discussed.

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.

X-ray diffraction patterns and anatomical properties of claw tissues of beef and dairy cattle

SUMMARYMedial claws from the right hind feet were obtained post mortem from four 19–20-month-old beef heifers and from four 28-month-old first-calving dairy heifers 3–4 days postpartum. X-ray diffraction (XRD) studies were undertaken on samples of soft and hard (cornified) integumental tissues of dorsal wall, sole and heel (bulb) for varying sites and planes of exposure. The measurements were interpreted as defining diffraction patterns and intermolecular spacings of cytoskeletal and extracellular fibrous structural proteins. The orientation of these proteins was examined in relation to physical characteristics and function including bearing of body weight by these tissues.Physical measurements taken included impression hardness which showed typically greater values for wall than sole and variable differences between horn of dairy and beef origin and husbandry systems. Claws from dairy heifers had significantly smaller values for toe (dorsal wall) angle, claw height and heel height and thickness of solear horn and heel soft tissue. Although few were studied, the results reflected typical husbandry origins and indicated the susceptibility to the lesion formation well recognized in postpartum dairy cattle.Typical XRD patterns for horn samples showed defined arcs of reflectance on the equatorial axis consistent with findings for the presence of α-helices in fibrils reported to occur in other hard-keratin-containing integumental tissues. However, reflectance on the meridional axis also reported for these other tissues was not detected. A similar defined pattern was obtained for less than 0·10 of samples of internal soft pre-cornified epidermal and attached dermal tissue although the values for intermolecular ‘d’ spacing for these were consistent with those reported for type I collagen. Diffuse reflection patterns were thus evident for the majority of samples of soft tissue epidermis and dermis and also for adipose tissue of the digital cushion.The formation of defined arcs of reflectance allowed the determination of fibril alignment in wall and solear horn. For the orientated samples of dorsal wall horn tissue, the outer layer showed a longitudinal angle of orientation essentially maintained proximal to distal. This pattern was maintained throughout the depth of horn at the proximal site. In contrast, layers in mid-wall and towards the distal edge showed a greater circumferential (horizontal) orientation in sections collected anterior to posterior towards the inner corial, including laminar, tissues. The orientation of fibrils in inner wall horn appears to relate to the direction of load-bearing forces in connecting horn to the distal phalanx. Horizontal alignment of fibrils was observed in the sole. In presenting the long axis of cells to the ground surface this orientation may facilitate erosive forces and contribute to the thinning of cornified sole horn under adverse underfoot conditions.

Soft epidermis of a scaleless snake lacks beta-keratin

Beta-keratins are responsible for the mechanical resistance of scales in reptiles. In a scaleless crotalus snake (Crotalus atrox), large areas of the skin are completely devoid of scales, and the skin appears delicate and wrinkled. The epidermis of this snake has been assessed for the presence of beta-keratin by immunocytochemistry and immunoblotting using an antibody against chicken scale beta-keratin. This antibody recognizes beta-keratins in normal snake scales with molecular weights of 15-18 kDa and isoelectric points at 6.8, 7.5, 8.3 and 9.4. This indicates that beta-keratins of the stratum corneum are mainly basic proteins, so may interact with cytokeratins of the epidermis, most of which appear acidic (isoelectric points 4.5-5.5). A beta-layer and beta-keratin immunoreactivity are completely absent in moults of the scaleless mutant, and the corneous layer comprises a multi-layered alpha-layer covered by a flat oberhautchen. In conclusion, the present study shows that a lack of beta-keratins is correlated with the loss of scales and mechanical protection in the skin of this mutant snake.

Pigmentation, impression hardness and the presence of melanosomes in bovine claw tissue

The physical properties of hardness and dry matter and the histological characteristics of dermal and epidermal tissues were investigated in lateral and medial claws obtained post mortem from mature crossbred female cattle. Claws containing pigmented (n=22) or non-pigmented (n=22) coronary wall horn were studied. Impression hardness, measured on the dorsal border of claws, proximo-distal at intervals of 5 mm from the lower perioplic line was shown to increase to the 20 mm lower site. Similarly, in comparison with values for pigmented horn, pooled measurements for non-pigmented horn showed greater hardness from the lower perioplic line to the 40 mm lower site but were not different for subsequent measurements towards the distal edge. Differences in values recorded for sole and heel and for coronary wall horn measured under the distal edge were not different for claws varying in coronary horn pigmentation. The absence of differences in dry matter at measurement sites suggested that differences in impression hardness values were not caused by variation in water content of horn. Histological examination of pigmented claw wall horn and underlying soft tissue showed pigment-containing cells in the coronary epidermis but not coronary dermis or in dermis or epidermis of laminae. Pigmentation appeared concentrated in melanosomes in cells along the basement membrane and was present uniformly throughout the epidermis and in cells lining horn tubules. There was no pigmentation detected in the soft tissue epidermis of claws selected visually for non-pigmented wall horn. The presence of melanosomes in epidermal cells from the pigmented coronary region was demonstrated by electron microscopy. The results suggest that pigmentation in the coronary region is associated with reduced initial development of hardness. The possible antagonism between antioxidant properties of melanins and requirement for an oxidative environment for disulphide bond formation in the cytoskeleton of horn cells is discussed.

Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales

The characteristics of scaled skin of reptiles is one of their main features that distinguish them from the other amniotes, birds and mammals. The different scale patterns observed in extant reptiles result from a long evolutive history that allowed each species to adapt to its specific environment. The present review deals with comparative aspects of epidermal keratinization in reptiles, chelonians (turtles and tortoises), lepidosaurian (lizards, snakes, sphenodontids), archosaurians (crocodilians). Initially the morphology and cytology of reptilian scales is outlined to show the diversity in the epidermis among different groups. The structural proteins (alpha-keratins and associated proteins), and enzymes utilized to form the corneous layer of the epidermis are presented. Aside cytokeratins (alpha-keratins), used for making the cytoskeleton, reptilian alpha-keratinocytes produce interkeratin (matrix) and corneous cell envelope proteins. Keratin bundles and degraded cell organelles constitute most of the corneous material of alpha-keratinocytes. Matrix, histidine-rich and sulfur-rich proteins are produced in the soft epidermis and accumulated in the cornified cell envelope. Main emphasis is given to the composition and to the evolution of the hard keratins (beta-keratins). Beta-keratins constitute the hard corneous material of scales. These small proteins are synthesized in beta-keratinocytes and are accumulated into small packets that rapidly merge into a compact corneous material and form densely cornified layers. Beta-keratins are smaller proteins (8–20 kDa) in comparison to alpha-keratins (40–70 kDa), and this size may determine their dense packing in corneocytes. Both glycine–sulfur-rich and glycine–proline-rich proteins have been so far sequenced in the corneous material of scales in few reptilian species. The latter keratins possess C- and N-amino terminal amino acid regions with sequence homology with those of mammalian hard keratins. Also, reptilian beta-keratins possess a central core with homology with avian scale/feather keratins. Multiple genes code for these proteins and their discovery and sequentiation is presently an active field of research. These initial findings however suggest that ancient reptiles already possessed some common genes that have later diversified to produce the specific keratin-associated proteins in their descendants: extant reptiles, birds and mammals. The evolution of these small proteins in lepidosaurians, chelonians and archosaurians represent the next step to understand the evolution of cornification in reptiles and derived amniotes (birds and mammals).

Immunolocalization and characterization of beta-keratins in growing epidermis of chelonians

Beta-keratins constitute most of the corneous material of carapace and plastron of turtles. The production of beta-keratin in the epidermis of a turtle and tortoise (criptodirians) and of a species of pleurodiran turtle was studied after injection of tritiated proline during the growth of carapace, plastron and claws. Growth mainly occurs near hinge regions along the margins of scutes and along most of the claws (growing regions). Proline incorporation occurs mainly in the growing centers, and is more specifically associated with beta-keratin synthesis. Proline-labeled bands of protein at 12–14 kDa and 25–27 kDa, and 37 kDa, in the molecular weight range of beta-keratins, were isolated from the soft epidermis of turtles 3 h after injection of the labeled amino acid. After extraction of epidermal proteins, an antibody directed against a chicken beta-keratin was used for immunoblotting. Bands of beta-keratin at 15–17 kDa, 22–24 kDa, and 36–38 kDa appear in all species. Beta-keratin is present in the growing and compact stratum corneum of the hard (shell) and soft (limbs, neck and tail) epidermis. This was confirmed using a specific antibody against a turtle beta-keratin band of 15–16 kDa. The latter antibody recognized epidermal protein bands in the range of 15–16 kDa and 29–33 kDa, and labels beta-keratin filaments. This result indicates that different forms of beta-keratins are produced from low molecular weight precursors or that larger aggregate form during protein preparation. The present study shows that beta-keratin is abundant in the scaled epidermis of tortoise but also in the soft epidermis of pleurodiran and cryptodiran turtles, indicating that this form of hard keratin is constitutively expressed in the epidermis of chelonians.

Proliferation in the epidermis of chelonians and growth of the horny scutes

The proliferation of the epidermis in soft skin, claws, and scutes of the carapace and plastron in the tortoise (Testudo hermanni) and the turtle (Chrysemys picta) were studied using autoradiographic and immunocytochemical methods. During the growing season, a basal keratinocyte in the epidermis of soft skin and claws takes 5-9 days to migrate into the corneous layer. In the tortoise, during fall/winter (resting season) a few alpha-keratin cells are produced in soft epidermis and hinge regions among scutes and occasional beta-keratin cells in the outer scute surface. When growth is resumed in spring (growing season), cell proliferation is intense, mainly around hinge regions and tips of marginal scutes. No scute shedding occurs and numerous beta-keratin cells are produced around the hinge regions, while alpha-keratin cells disappear. Beta-cells form a new thick corneous layer around the hinge regions, which constitute the growing rings of scutes. Beta-keratin cells produced in more central parts of scutes maintain a homogeneous thickness of the corneous layer along the whole scute surface. In the turtle, a more complicated process of scute growth occurs than in the tortoise. At the end of the growing season (late fall) the last keratinocytes formed beneath the old stratum corneum of the outer scale surface and hinge regions produce more alpha- than beta-keratin. These thin alpha-keratin cells form a scission layer below the old stratum corneum, which extends from the hinge regions toward the center of scutes and the tip of marginal scutes. In the resting season (fall/winter) most cells remain within the germinative layer of the carapace and plastron and a few alpha-cells move in 7-9 days into the corneous layer above hinge regions. In the following spring/summer (growing season) a new generation of beta-keratin cells is produced beneath the scission layer from the hinge region and more central part of the scutes. The epidermis of the inner surface of scutes and hinge regions contains most of the cells incorporating thymidine and histidine, while the remaining outer scute surface is less active. It takes 5-9 days for a newly produced beta-cell to migrate into the corneous layer. These cells form a new corneous layer that extends the whole scute surface underneath the maturing scission layer. The latter contains lipids and eventually flakes off, determining shedding of the above outer corneous layer in late spring or summer.

Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.

On the origin of feathers

AbstractThe appearance of feathers defines the appearance of birds. A number of changes defined, preceded or accompanied the event. The changes were hierarchical in nature and included revolutions in genomic organization (i.e., HOX and the feather keratin genes), protein sequence and shape, the large scale organization of proteins into filaments, and in the geometry of the cells and their roles in the follicle. Changes at each of these levels differ or produced different products than found in its analog in reptiles. They are essentially unique to birds and produced an evolutionary novelty. I used analysis of extant structure and information on development to reconstruct key events in the evolution of feathers. The ancestral reptilian epidermal structure, while probably a scale or tubercles, is still unidentified. The structural genes of feather proteins (φ‐keratin) are tandem repeats probably assembled from pre‐existing exons. They are unlike the alpha‐keratin of vertebrate soft epidermis. Amino‐acid composition, shape, and behavior of feather keratins are unique among vertebrates. The 3‐dimensional organization of the follicle and the developmental processes are also unique.Although we lack a complete understanding of the appearance and early role of feathers, they are clearly the results of novel events.