Cytochemical and molecular characteristics of the process of cornification during feather morphogenesis

Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as “feather keratin”. This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.     

Review: cornification,morphogenesis and evolution of feathers

Feathers are corneous microramifications of variable complexity derived from the morphogenesis of barb ridges. Histological and ultrastructural analyses on developing and regenerating feathers clarify the three-dimensional organization of cells in barb ridges. Feather cells derive from folds of the embryonic epithelium of feather germs from which barb/barbule cells and supportive cells organize in a branching structure. The following degeneration of supportive cells allows the separation of barbule cells which are made of corneous beta-proteins and of lower amounts of intermediate filament (IF)(alpha) keratins, histidine-rich proteins, and corneous proteins of the epidermal differentiation complex. The specific protein association gives rise to a corneous material with specific biomechanic properties in barbules, rami, rachis, or calamus. During the evolution of different feather types, a large expansion of the genome coding for corneous feather beta-proteins occurred and formed 3–4-nm-thick filaments through a different mechanism from that of 8–10 nm IF keratins. In the chick, over 130 genes mainly localized in chromosomes 27 and 25 encode feather corneous beta-proteins of 10–12 kDa containing 97–105 amino acids. About 35 genes localized in chromosome 25 code for scale proteins (14–16 kDa made of 122–146 amino acids), claws and beak proteins (14–17 kDa proteins of 134–164 amino acids). Feather morphogenesis is periodically re-activated to produce replacement feathers, and multiple feather types can result from the interactions of epidermal and dermal tissues. The review shows schematic models explaining the translation of the morphogenesis of barb ridges present in the follicle into the three-dimensional shape of the main types of branched or un-branched feathers such as plumulaceous, pennaceous, filoplumes, and bristles. The temporal pattern of formation of barb ridges in different feather types and the molecular control from the dermal papilla through signaling molecules are poorly known. The evolution and diversification of the process of morphogenesis of barb ridges and patterns of their formation within feathers follicle allowed the origin and diversification of numerous types of feathers, including the asymmetric planar feathers for flight.     

The Evolution of Feathers*

Existing hypotheses on the evolution of feathers are reviewed with the assumptions that feather evolved from reptilian scales and that pennaceous feathers evolved before downy feathers. Observations with a scanning electron microscope demonstrate that basic to the structure of pennaceous feathers is the lamelliform structure of barbules, the planes of which are oriented at right angles to the plane of the feather vane. Thus the structure of the vane is more open than generally realized. The airtight vane of flight feathers is assumed a later specialization. Most of the existing hypotheses assume that the feather acts as a relatively solid barrier between the skin of the bird and the exterior and they are therefore not in agreement with the actual structure of feathers. A hypothesis is needed which explains the adaptive value of a pennaceous feather being porous. The hypothesis is put foward that feathers evolved due to selection for a water-repellent integument. For purely physical reasons a porous surface repels water drops more strongly than does a solid surface of the same material. Physicists have pointed out that the structure of feathers conforms closely with the theoretical requirements for water-repellency. Possibly feathers started to evolve on reptiles living at the seashore, where the main advantage of increased water-repellency was to reduce cooling from evaporation of water off a wet integument.     

Is the denser contour feather structure in pale grey than in pheomelanic brown tawny owls Strix aluco an adaptation to cold environments?

In colour polymorphic species morphs are considered to be adaptations to different environments, where they have evolved and are maintained because of their differential sensitivity to the environment. In cold environments the plumage insulation capacity is essential for survival and it has been proposed that plumage colour is associated with feather structure and thereby the insulation capacity of the plumage. We studied the structure of contour feathers in the colour polymorphic tawny owl Strix aluco. A previous study of tawny owls in the same population has found strong selection against the brown morph in cold and snowy winters whereas this selection pressure is absent in mild winters. We predicted that grey morphs have a denser and more insulative plumage, enabling them to survive better in cold climate compared to brown ones. The insulative plumulaceous part of the dorsal contour feathers was larger and the fine structure of the plumulaceous part of the feather was denser in grey tawny owls than in brown ones. In the ventral contour feathers the plumulaceous part of the feather was denser in females than in males and in older birds without any differences between morphs. Our study suggests that insulative microscopical feather structures differ between colour morphs and we propose that feather structure may be a trait associated with morph‐specific survival in cold environments.     

Shh-Bmp2 signaling module and the evolutionary origin and diversification of feathers

To examine the role of development in the origin of evolutionary novelties, we investigated the developmental mechanisms involved in the formation of a complex morphological novelty-branched feathers. We demonstrate that the anterior-posterior expression polarity of Sonic hedgehog (Shh) and Bone morphogenetic protein 2 (Bmp2) in the primordia of feathers, avian scales, and alligator scales is conserved and phylogenetically primitive to archosaurian integumentary appendages. In feather development, derived patterns of Shh-Bmp2 signaling are associated with the development of evolutionarily novel feather structures. Longitudinal Shh-Bmp2 expression domains in the marginal plate epithelium between barb ridges provide a prepattern of the barbs and rachis. Thus, control of Shh-Bmp2 signaling is a fundamental component of the mechanism determining feather form (i.e., plumulaceous vs. pennaceous structure). We show that Shh signaling is necessary for the formation and proper differentiation of a barb ridge and that it is mediated by Bmp signaling. BMP signaling is necessary and sufficient to negatively regulate Shh expression within forming feather germs and this epistatic relationship is conserved in scale morphogenesis. Ectopic SHH and BMP2 signaling leads to opposing effects on proliferation and differentiation within the feather germ, suggesting that the integrative signaling between Shh and Bmp2 is a means to regulate controlled growth and differentiation of forming skin appendages. We conclude that Shh and Bmp signaling is necessary for the formation of barb ridges in feathers and that Shh and Bmp2 signaling constitutes a functionally conserved developmental signaling module in archosaur epidermal appendage development. We propose a model in which branched feather form evolved by repeated, evolutionary re-utilization of a Shh-Bmp2 signaling module in new developmental contexts. Feather animation Quicktime movies can be viewed at http://fallon.anatomy.wisc.edu/feather.html.     

Molecular and microstructural inventory of an isolated fossil bird feather from the Eocene Fur Formation of Denmark

An isolated, yet virtually intact contour feather (FUM‐1980) from the lower Eocene Fur Formation of Denmark was analysed using multiple imaging and molecular techniques, including field emission gun scanning electron microscopy (FEG‐SEM), X‐ray absorption spectroscopy and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). Additionally, synchrotron radiation X‐ray tomographic microscopy (SRXTM) was employed in order to produce a digital reconstruction of the fossil. Under FEG‐SEM, the proximal, plumulaceous part of the feather revealed masses of ovoid microstructures, about 1.7 μm long and 0.5 μm wide. Microbodies in the distal, pennaceous portion were substantially smaller (averaging 0.9 × 0.2 μm), highly elongate, and more densely packed. Generally, the microbodies in both the plumulaceous and pennaceous segments were aligned along the barbs and located within shallow depressions on the exposed surfaces. Biomarkers consistent with animal eumelanins were co‐localized with the microstructures, to suggest that they represent remnant eumelanosomes (i.e. eumelanin‐housing cellular organelles). Additionally, ToF‐SIMS analysis revealed the presence of sulfur‐containing organics – potentially indicative of pheomelanins – associated with eumelanin‐like compounds. However, since there was no correlation between melanosome morphology and sulfur content, we conclude these molecular structures derive from diagenetically incorporated sulfur rather than pheomelanin. Melanosomes corresponding roughly in both size and morphology with those in the proximal part of FUM‐1980 are known from contour feathers of extant parrots (Psittaciformes), an avian clade that has previously been reported from the Fur Formation.     

Infrared analysis of contour feathers: The conservation of body heat radiation in birds

1. We examined infrared absorption spectra in contour feathers of 10 species of birds to compare infrared features of the downy and pennaceous regions with the mechanisms of conservation of body heat radiation.     

Structural coloration in a fossil feather

Investigation of feathers from the famous Middle Eocene Messel Oil Shale near Darmstadt, Germany shows that they are preserved as arrays of fossilized melanosomes, the surrounding beta-keratin having degraded. The majority of feathers are preserved as aligned rod-shaped eumelanosomes. In some, however, the barbules of the open pennaceous, distal portion of the feather vane are preserved as a continuous external layer of closely packed melanosomes enclosing loosely aligned melanosomes. This arrangement is similar to the single thin-film nanostructure that generates an iridescent, structurally coloured sheen on the surface of black feathers in many lineages of living birds. This is, to our knowledge, the first evidence of preservation of a colour-producing nanostructure in a fossil feather and confirms the potential for determining colour differences in ancient birds and other dinosaurs.     

Linking the molecular evolution of avian beta (β) keratins to the evolution of feathers

Feathers of today's birds are constructed of beta (β)-keratins, structural proteins of the epidermis that are found solely in reptiles and birds. Discoveries of "feathered dinosaurs" continue to stimulate interest in the evolutionary origin of feathers, but few studies have attempted to link the molecular evolution of their major structural proteins (β-keratins) to the appearance of feathers in the fossil record. Using molecular dating methods, we show that before the appearance of Anchiornis (～155 Million years ago (Ma)) the basal β-keratins of birds began diverging from their archosaurian ancestor ～216?Ma. However, the subfamily of feather β-keratins, as found in living birds, did not begin diverging until ～143?Ma. Thus, the pennaceous feathers on Anchiornis, while being constructed of avian β-keratins, most likely did not contain the feather β-keratins found in the feathers of modern birds. Our results demonstrate that the evolutionary origin of feathers does not coincide with the molecular evolution of the feather β-keratins found in modern birds. More likely, during the Late Jurassic, the epidermal structures that appeared on organisms in the lineage leading to birds, including early forms of feathers, were constructed of avian β-keratins other than those found in the feathers of modern birds. Recent biophysical studies of the β-keratins in feathers support the view that the appearance of the subfamily of feather β-keratins altered the biophysical nature of the feather establishing its role in powered flight.     

Development and evolutionary origin of feathers

Avian feathers are a complex evolutionary novelty characterized by structural diversity and hierarchical development. Here, I propose a functionally neutral model of the origin and evolutionary diversification of bird feathers based on the hierarchical details of feather development. I propose that feathers originated with the evolution of the first feather follicle-a cylindrical epidermal invagination around the base of a dermal papilla. A transition series of follicle and feather morphologies is hypothesized to have evolved through a series of stages of increasing complexity in follicle structure and follicular developmental mechanisms. Follicular evolution proceeded with the origin of the undifferentiated collar (stage I), barb ridges (stage II), helical displacement of barb ridges, barbule plates, and the new barb locus (stage III), differentiation of pennulae of distal and proximal barbules (stage IV), and diversification of barbule structure and the new barb locus position (stage V). The model predicts that the first feather was an undifferentiated cylinder (stage I), which was followed by a tuft of unbranched barbs (stage II). Subsequently, with the origin of the rachis and barbules, the bipinnate feather evolved (stage III), followed then by the pennaceous feather with a closed vane (stage IV) and other structural diversity (stages Va-f). The model is used to evaluate the developmental plausibility of proposed functional theories of the origin of feathers. Early feathers (stages I, II) could have functioned in communication, defense, thermal insulation, or water repellency. Feathers could not have had an aerodynamic function until after bipinnate, closed pennaceous feathers (stage IV) had evolved. The morphology of the integumental structures of the coelurisaurian theropod dinosaurs Sinosauropteryx and Beipiaosaurus are congruent with the model's predictions of the form of early feathers (stage I or II). Additional research is required to examine whether these fossil integumental structures developed from follicles and are homologous with avian feathers. J. Exp. Zool. (Mol. Dev. Evol.) 285:291-306, 1999.Copyright 1999 Wiley-Liss, Inc.     

Population differences in the structure and coloration of great tit contour feathers

Contour feathers cover most of the avian body and play critical roles in insulation, social communication, aerodynamics, and water repellency. Feather production is costly and the development of the optimum characteristics for each function may be constrained by limited resources or time, and possibly also lead to trade‐offs among the different characteristics. Populations exposed to different environmental conditions may face different selective pressures, resulting in differences in feather structure and coloration, particularly in species with large geographical distributions. Three resident populations of great tit Parus major L. from different latitudes differed in feather structure and coloration. Individuals from the central population exhibited less dense and longer contour feathers, with a higher proportion of plumulaceous barbs than either northern or southern birds, which did not differ in their feather structure. Ultraviolet reflectance and brightness of the yellow of the contour feathers of the breast was higher for the southern than for the northern population. Birds with greener plumage (higher hue) had less dense but longer feathers, independently of the population of origin. Differences in feather structure across populations appear to be unrelated to the contour feather colour characteristics except for hue. Nutritional and time constraints during molt might explain the pattern of feather structure, whereas varying sexual selection pressure might underlie the coloration patterns observed. Our results suggest that different selective pressures or constraints shape contour feather traits in populations exposed to varying environmental conditions. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 82–91.     

The evolutionary origin of feathers.

Previous theories relating the origin of feathers to flight or to heat conservation are considered to be inadequate. There is need for a model of feather evolution that gives attention to the function and adaptive advantage of intermediate structures. The present model attempts to reveal and to deal with, the spectrum of complex questions that must be considered. In several genera of modern lizards, scales are elongated in warm climates. It is argued that these scales act as small shields to solar radiation. Experiments are reported that tend to confirm this. Using lizards as a conceptual model, it is argued that feathers likewise arose as adaptations to intense solar radiation. Elongated scales are assumed to have subdivided into finely branched structures that produced a heat-shield, flexible as well as long and broad. Associated muscles had the function of allowing the organism fine control over rates of heat gain and loss: the specialized scales or early feathers could be moved to allow basking in cool weather or protection in hot weather. Subdivision of the scales also allowed a close fit between the elements of the insulative integument. There would have been mechanical and thermal advantages to having branches that interlocked into a pennaceous structure early in evolution, so the first feathers may have been pennaceous. A versatile insulation of movable, branched scales would have been a preadaptation for endothermy. As birds took to the air they faced cooling problems despite their insulative covering because of high convective heat loss. Short glides may have initially been advantageous in cooling an animal under heat stress, but at some point the problem may have shifted from one of heat exclusion to one of heat retention. Endothermy probably evolved in conjunction with flight. If so, it is an unnecessary assumption to postulate that the climate cooled and made endothermy advantageous. The development of feathers is complex and a model is proposed that gives attention to the fundamental problems of deriving a branched structure with a cylindrical base from an elongated scale.     

Interpopulation variation in contour feather structure is environmentally determined in great tits

Background

The plumage of birds is important for flying, insulation and social communication. Contour feathers cover most of the avian body and among other functions they provide a critical insulation layer against heat loss. Feather structure and composition are known to vary among individuals, which in turn determines variation in the insulation properties of the feather. However, the extent and the proximate mechanisms underlying this variation remain unexplored.

Methodology/Principal Findings

We analyzed contour feather structure from two different great tit populations adapted to different winter regimes, one northern population in Oulu (Finland) and one southern population in Lund (Sweden). Great tits from the two populations differed significantly in feather structure. Birds from the northern population had a denser plumage but consisting of shorter feathers with a smaller proportion containing plumulaceous barbs, compared with conspecifics from the southern population. However, differences disappeared when birds originating from the two populations were raised and moulted in identical conditions in a common-garden experiment located in Oulu, under ad libitum nutritional conditions. All birds raised in the aviaries, including adult foster parents moulting in the same captive conditions, developed a similar feather structure. These feathers were different from that of wild birds in Oulu but similar to wild birds in Lund, the latter moulting in more benign conditions than those of Oulu.

Conclusions/Significance

Wild populations exposed to different conditions develop contour feather differences either due to plastic responses or constraints. Environmental conditions, such as nutrient availability during feather growth play a crucial role in determining such differences in plumage structure among populations.     

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation.

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.     

A New Species of Pengornithidae (Aves: Enantiornithes) from the Lower Cretaceous of China Suggests a Specialized Scansorial Habitat Previously Unknown in Early Birds

We describe a new enantiornithine bird, Parapengornis eurycaudatus gen. et sp. nov. from the Lower Cretaceous Jiufotang Formation of Liaoning, China. Although morphologically similar to previously described pengornithids Pengornis houi, Pengornis IVPP V18632, and Eopengornis martini, morphological differences indicate it represents a new taxon of the Pengornithidae. Based on new information from this specimen we reassign IVPP V18632 to Parapengornis sp. The well preserved pygostyle of the new specimen elucidates the morphology of this element for the clade, which is unique in pengornithids among Mesozoic birds. Similarities with modern scansores such as woodpeckers may indicate a specialized vertical climbing and clinging behavior that has not previously been inferred for early birds. The new specimen preserves a pair of fully pennaceous rachis-dominated feathers like those in the holotype of Eopengornis martini; together with the unique morphology of the pygostyle, this discovery lends evidence to early hypotheses that rachis-dominated feathers may have had a functional significance. This discovery adds to the diversity of ecological niches occupied by enantiornithines and if correct reveals are remarkable amount of locomotive differentiation among Enantiornithes.     

Convergent evolution of cysteine-rich proteins in feathers and hair

Background

Feathers and hair consist of cornified epidermal keratinocytes in which proteins are crosslinked via disulfide bonds between cysteine residues of structural proteins to establish mechanical resilience. Cysteine-rich keratin-associated proteins (KRTAPs) are important components of hair whereas the molecular components of feathers have remained incompletely known. Recently, we have identified a chicken gene, named epidermal differentiation cysteine-rich protein (EDCRP), that encodes a protein with a cysteine content of 36%. Here we have investigated the putative role of EDCRP in the molecular architecture and evolution of feathers.

Results

Comparative genomics showed that the presence of an EDCRP gene and the high cysteine content of the encoded proteins are conserved among birds. Avian EDCRPs contain a species-specific number of sequence repeats with the consensus sequence CCDPCQ(K/Q)(S/P)V, thus resembling mammalian cysteine-rich KRTAPs which also contain sequence repeats of similar sequence. However, differences in gene loci and exon-intron structures suggest that EDCRP and KRTAPs have not evolved from a common gene ancestor but represent the products of convergent sequence evolution. mRNA in situ hybridization demonstrated that chicken EDCRP is expressed in the subperiderm layer of the embryonic epidermis and in the barbule cells of growing feathers. This expression pattern supports the hypothesis that feathers are evolutionarily derived from the subperiderm.

Conclusions

The results of this study suggest that convergent sequence evolution of avian EDCRP and mammalian KRTAPs has contributed to independent evolution of feathers and hair, respectively.

     

Feather melanin and microstructure variation in dark‐eyed junco Junco hyemalis across an elevational gradient in the Selkirk Mountains

Variation in feather melanism and microstructure can arise through sexual selection and ecological functional drivers. Melanin‐based plumage traits are associated with sexual dichromatism and the intensity of sexual selection in many avian species, but also have several ecological benefits such as protection against ultra‐violet (UV) radiation, camouflage, and feather strength. Additionally, feather microstructure influences thermoregulation. Plumage variation across species is well documented; however, the relative role of sexual selection and ecological drivers in intra‐specific and within‐population variation is less established. We investigated UV reflectance, melanism, and feather microstructure in a population of Oregon dark‐eyed juncos Junco hyemalis oreganus between high (1900–2200 m a.s.l.) and low (450–800 m a.s.l.) elevations in the Selkirk Mountains to evaluate potential sexual selection and ecological drivers of variation. We found no difference in UV reflectance or lightness (melanism) of head feathers between elevations, but individuals at high elevation had lighter (less melanism) and less brown (less pheomelanin) body contour feathers than at low elevations. High elevation individuals also had longer contour feathers with more pronounced plumulaceous regions. Sexual dichromatism did not vary between elevations, leading us to reject sexual selection in favour of ecological functional drivers of plumage variation in this system. To our knowledge, this is the first study to identify within‐population differences in feather melanism and microstructure between different elevations.     

On the identification of feather structures in stem-line representatives of birds: evidence from fossils and actuopalaeontology

Dinosaurs with fossilized filamentous integument structures are usually preserved in a highly flattened state. Several different feather types have been described on this basis, but the two-dimensional preservation of specimens during fossilization makes the identification of single feather structures difficult due to overlapping feather structures in vivo. Morphological comparison with the diversity of recent feather types is therefore absolutely vital to avoid misinterpretation. To simulate the preservation process, a cadaver of recent Carduelis spinus (European siskin) was flattened in a printing press. Afterwards, the structure of the plumage was compared with the morphology of a single body feather from the same specimen. In comparison with the single feather, the body plumage of the flattened bird looked rather filamentous. It was almost impossible to identify single structures, and in their place, various artefacts were produced. The investigation of plumage in a specimen of the Mesozoic bird Confuciusornis sanctus reveals similar structures. This indicates that flattening of specimens during fossilization amplifies the effect of overlapping among feathers and also causes a loss of morphological detail which can lead to misinterpretations. The results are discussed in connection with some dubious feather morphologies in recently described theropods and basal birds. Based on recent feather morphology, the structure of so-called proximal ribbon-like pennaceous feathers (PRPFs) found in many basal birds is reinterpreted. Furthermore, the morphology of a very similar-looking feather type found in the forelimb and tail of an early juvenile oviraptorosaur is discussed and diagnosed as the first feather generation growing out of the feather sheath. Thus, the whole plumage of this theropod might represent neoptile plumage.