Origin of feathers: Feather beta (beta) keratins are expressed in discrete epidermal cell populations of embryonic scutate scales

The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.     

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.     

Dinosaur protofeathers: pushing back the origin of feathers into the Middle Triassic?

Reports of primordial feathers (protofeathers) in dinosaurs have received widespread interest. Recently, it was proposed that a novel protofeather in the theropod dinosaur Beipiaosaurus completes the transitional series in the evolution of the feather and provides the first evidence of filamentous feathers as display in nonavian theropods. A more far-reaching evolutionary ramification is the claim that these structures push the origin of monofilamentous integumentary structures into the Middle Triassic or earlier. I discuss problems with the analyses within the broader context of studies concerning the hypothesis of protofeathers, and show that affinity between the integumentary structures in Beipiaosaurus and feathers is improbable. The scientific methodology is questioned by its failure to make phenomena perceivable by objective means, by questionable rationalizations in critical issues, and by lack of consideration of exceptions to the postulated thesis. The notion that primordial feathers occurred in a clade more inclusive than the Coelurosauria and that it is supported by the presence of integumental structures in Psittacosaurus is analyzed and rejected.     

The colour of fossil feathers

Feathers are complex integumentary appendages of birds and some other theropod dinosaurs. They are frequently coloured and function in camouflage and display. Previous investigations have concluded that fossil feathers are preserved as carbonized traces composed of feather-degrading bacteria. Here, an investigation of a colour-banded feather from the Lower Cretaceous Crato Formation of Brazil revealed that the dark bands are preserved as elongate, oblate carbonaceous bodies 1-2mum long, whereas the light bands retain only relief traces on the rock matrix. Energy dispersive X-ray analysis showed that the dark bands preserve a substantial amount of carbon, whereas the light bands show no carbon residue. Comparison of these oblate fossil bodies with the structure of black feathers from a living bird indicates that they are the eumelanin-containing melanosomes. We conclude that most fossil feathers are preserved as melanosomes, and that the distribution of these structures in fossil feathers can preserve the colour pattern in the original feather. The discovery of preserved melanosomes opens up the possibility of interpreting the colour of extinct birds and other dinosaurs.     

The evolutionary origin and diversification of feathers

Progress on the evolutionary origin and diversification of feathers has been hampered by conceptual problems and by the lack of plesiomorphic feather fossils. Recently, both of these limitations have been overcome by the proposal of the developmental theory of the origin of feathers, and the discovery of primitive feather fossils on nonavian theropod dinosaurs. The conceptual problems of previous theories of the origin of feathers are reviewed, and the alternative developmental theory is presented and discussed. The developmental theory proposes that feathers evolved through a series of evolutionary novelties in developmental mechanisms of the follicle and feather germ. The discovery of primitive and derived fossil feathers on a diversity of coelurosaurian theropod dinosaurs documents that feathers evolved and diversified in nonavian theropods before the origin of birds and before the origin of flight. The morphologies of these primitive feathers are congruent with the predictions of the developmental theory. Alternatives to the theropod origin of feathers are critique and rejected. Hypotheses for the initial function of feathers are reviewed. The aerodynamic theory of feather origins is falsified, but many other functions remain developmentally and phylogenetically plausible. Whatever their function, feathers evolved by selection for a follicle that would grow an emergent tubular appendage. Feathers are inherently tubular structures. The homology of feathers and scales is weakly supported. Feathers are composed of a suite of evolutionary novelties that evolved by the duplication, hierarchical organization, interaction, dissociation, and differentiation of morphological modules. The unique capacity for modular subdivision of the tubular feather follicle and germ has fostered the evolution of numerous innovations that characterize feathers. The evolution of feather keratin and the molecular basis of feather development are also discussed.     

Origin of archosaurian integumentary appendages: the bristles of the wild turkey beard express feather-type beta keratins

The discovery that structurally unique "filamentous integumentary appendages" are associated with several different non-avian dinosaurs continues to stimulate the development of models to explain the evolutionary origin of feathers. Taking the phylogenetic relationships of the non-avian dinosaurs into consideration, some models propose that the "filamentous integumentary appendages" represent intermediate stages in the sequential evolution of feathers. Here we present observations on a unique integumentary structure, the bristle of the wild turkey beard, and suggest that this non-feather appendage provides another explanation for some of the "filamentous integumentary appendages." Unlike feathers, beard bristles grow continuously from finger-like outgrows of the integument lacking follicles. We find that these beard bristles, which show simple branching, are hollow, distally, and express the feather-type beta keratins. The significance of these observations to explanations for the evolution of archosaurian integumentary appendages is discussed.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.     

Avian scale development. IX. Scale formation by scaleless (sc/sc) epidermis under the influence of normal scale dermis

Unlike normal scutate scales whose outer and inner epidermal surfaces elaborate β (β-keratins) and α (α-keratins) strata, respectively, the scaleless mutant's anterior metatarsal epidermis remains flat and elaborates only an α stratum. Reciprocal epidermal-dermal recombinations of presumptive scale tissues from normal and mutant embryos have demonstrated that the scaleless defect is expressed only by the epidermis. In fact, the scaleless anterior metatarsal epidermis is unable to undergo placode formation. More recently, it has been determined that the absence of epidermal placode morphogenesis into a definitive scale ridge actually results in the establishment of a scale dermis which is incapable of inducing the outer and inner epidermal surfaces of scutate scales. Can the initial genetic defect in the scaleless anterior metatarsal epidermis be overcome by replacing the defective dermis with a normal scutate scale dermis, i.e., a dermis with scale ridges already present? Or, are the genes involved in the production of a β stratum regulated by events directly associated with morphogenesis of the epidermal placode? In the present study, we combined scaleless anterior metatarsal epidermis (stages 36 to 42) with normal scutate scale dermis (stage 40, 41, or 42) old enough to have acquired its scutate scale-inducing ability. After 7 days of growth as chorioallantoic membrane grafts, we observed grossly and histologically, typical scutate scales in these recombinant grafts. Electron microscopic and electrophoretic analyses have verified that these recombinant scales are true scutate scales. The scaleless mutation, known to be expressed initially by the anterior metatarsal epidermis, can be overcome by exposing this epidermis to appropriate inductive cues, i.e., cues that direct the differentiation of the outer and inner epidermal surfaces of the scutate scales and the production of specific structural proteins. We have determined that the time between stages 38 and 39 is the critical period during which the normal scutate scale dermis acquires these inductive abilities.     

The Integumentary Morphology of Modern Birds--An Overview

Avian integument is thin, elastic, and loosely attached to thebody, giving birds the freedom of movement needed for flight.Its epidermis is both keratinized and lipogenic, and the skinas a whole acts as a sebaceous secretory organ. The skin iscovered by feathers over most of the body, but many birds showcolored bare skin or integumentary outgrowths on the head andneck. Heavily cornified epidermis covers the beak, claws, spurs,and the scales on the legs and feet. These structures (exceptthe back of the leg and underside of the foot) contain beta-keratinlike that in reptilian scales. Most birds have sebaceous secretoryglands at the base of the tail and in the ear canals. Feathersare the most numerous, elaborate, and diverse of avian integumentaryderivatives. Their diversity is due to the possibilities inherentin their basic plan of a shaft with two orders of branches andthe use of modified beta-keratin as a strong, light, and plasticbuilding material. The evolution of feathers in birds has beenaccompanied by the development of complex systems for producingcolors and patterns, the innovations of feather arrangementand follicles with their musculature and innervation, and theprocess and control of molting.     

Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering

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.     

Epidermal-dermal tissue interactions between mutant and normal embryonic back skin: site of mutant gene activity determining abnormal feathering is in the epidermis.

The site of the scaleless gene's activity in the development of abnormal feathers was determined by reciprocally recombining epidermis and dermis between normal and scaleless chick embryos and culturing the recombinants for seven days on the chorioallantoic membrane. When recombined with a common dermal source, feather development is enhanced by scaleless high line as compared to scaleless low line epidermis. Against a common responding tissue, 7-day normal back epidermis, significant differences were not found in feather inducing ability between normal, scaleless high line and scaleless low line dermis. It was concluded that, in relation to abnormal feathering, these tissue interactions reveal that the site of the scaleless gene's activity is the epidermis. A model of tissue interaction in the development of normal and abnormal feathers is presented. According to the model, the focus of the scaleless mutation and the genes accumulated by selection for high or low feather numbers is the epidermis, the effect being that the reactivity of the epidermis to dermal stimuli is altered. Subsequently, the epidermis controls the morphogenetic organization of the dermis. The scaleless dermis is presumed to contain normal positional information for the determination of feather structure and pattern.     

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.     

Avian scale development: XI. Immunoelectron microscopic localization of alpha and beta keratins in the scutate scale

Epithelial-mesenchymal interactions play important roles in morphogenesis, histogenesis, and keratinization of the vertebrate integument. In the anterior metatarsal region of the chicken, morphogenesis results in the formation of distinct overlapping scutate scales. Recent studies have shown that the dermis of scutate scales is involved in the expression of the beta keratin gene products, which characterize terminal differentiation of the epidermis on the outer scale surface (Sawyer et al.: Dev. Biol. 101:8-18, '84; Shames and Sawyer: Dev. Biol. 116:15-22, '86; Shames and Sawyer: In A.A. Moscona and A. Monroy (eds), R.H. Sawyer (Vol. ed): Current Topics in Developmental Biology. Vol. 22: The Molecular and Developmental Biology of Keratins. New York: Academic Press, pp. 235-253, '87). Since alpha and beta keratins are both found in the scutate scale and are members of two different multigene families, it is important to know the precise location of these distinct keratins within the epidermis. In the present study, we have used protein A-gold immunoelectron microscopy with antisera made against avian alpha and beta keratins to specifically localize these keratins during development of the scutate scale to better understand the relationship between dermal cues and terminal differentiation. We find that the bundles of 3-nm filaments, characteristic of tissues known to produce beta keratins, react specifically with antiserum which recognizes beta keratin polypeptides and are found in the embryonic subperiderm that covers the entire scutate scale and in the stratum intermedium and stratum corneum making up the platelike beta stratum of the outer scale surface. Secondly, we find that 8-10-nm tonofilaments react specifically with antiserum that recognizes alpha keratin polypeptides and are located in the germinative basal cells and the lowermost cells of the stratum intermedium of the outer scale surface, as well as in the embryonic alpha stratum, which is lost from the outer surface of the scale at hatching. The alpha keratins are found throughout the epidermis of the inner surface of the scale and the hinge region. Thus, the present study further supports the hypothesis that the tissue interactions responsible for the formation of the beta stratum of scutate scales do not directly activate the synthesis of beta keratins in the germinative cells but influence these cells so that they or their progeny will activate specific beta keratin genes at the appropriate time and place.     

Evo-Devo of amniote integuments and appendages

Integuments form the boundary between an organism and the environment. The evolution of novel developmental mechanisms in integuments and appendages allows animals to live in diverse ecological environments. Here we focus on amniotes. The major achievement for reptile skin is an adaptation to the land with the formation of a successful barrier. The stratum corneum enables this barrier to prevent water loss from the skin and allowed amphibian / reptile ancestors to go onto the land. Overlapping scales and production of beta-keratins provide strong protection. Epidermal invagination led to the formation of avian feather and mammalian hair follicles in the dermis. Both adopted a proximal - distal growth mode which maintains endothermy. Feathers form hierarchical branches which produce the vane that makes flight possible. Recent discoveries of feathered dinosaurs in China inspire new thinking on the origin of feathers. In the laboratory, epithelial - mesenchymal recombinations and molecular mis-expressions were carried out to test the plasticity of epithelial organ formation. We review the work on the transformation of scales into feathers, conversion between barbs and rachis and the production of "chicken teeth". In mammals, tilting the balance of the BMP pathway in K14 noggin transgenic mice alters the number, size and phenotypes of different ectodermal organs, making investigators rethink the distinction between morpho-regulation and pathological changes. Models on the evolution of feathers and hairs from reptile integuments are discussed. A hypothetical Evo-Devo space where diverse integument appendages can be placed according to complex phenotypes and novel developmental mechanisms is presented.     

Bristles before down: A new perspective on the functional origin of feathers

Over the course of the last two decades, the understanding of the early evolution of feathers in nonavian dinosaurs has been revolutionized. It is now recognized that early feathers had a simple form comparable in general structure to the hairs of mammals. Insight into the prevalence of simple feathers throughout the dinosaur family tree has gradually arisen in tandem with the growing evidence for endothermic dinosaur metabolisms. This has led to the generally accepted opinion that the early feather coats of dinosaurs functioned as thermo insulation. However, thermo insulation is often erroneously stated to be a likely functional explanation for the origin of feathers. The problem with this explanation is that, like mammalian hair, simple feathers could serve as insulation only when present in sufficiently high concentrations. The theory therefore necessitates the origination of feathers en masse. We advocate for a novel origin theory of feathers as bristles. Bristles are facial feathers common among modern birds that function like mammalian tactile whiskers, and are frequently simple and hair‐like in form. Bristles serve their role in low concentrations, and therefore offer a feasible first stage in feather evolution.     

Avian Scale Development XIV: A Study of Cell Proliferation in the Epidermis of the Scaleless, sc/sc, Mutant

Embryonic induction has been demonstrated in numerous studies, yet the molecular basis for induction still eludes investigators. Components of the extracellular matrix (ECM), cell adhesion molecules (CAM), diffusable factors, as well as direct cell-cell contact, have been implicated in the early induction of avian feathers and scales. Although feathers and scales differ in many aspects, they are similar in that they appear initially as discrete and orderly arranged epidermal placodes. In the case of scutate scales, the cells of the epidermal placode are nonproliferative, while the cells of the interplacode regions are highly proliferative. In this study, I compare the proliferative activity of normal scale cells with that of the epidermal cells from embryos of the scaleless (sc/sc) mutant chicken which does not undergo epidermal placode formation and therefore lacks scutate scales. These results show that prior to the time that placodes would normally form, the proliferative activity of the scaleless epidermal cells is similar to that seen in normal epidermal cells. Likewise, the cessation of cell proliferation seen in normal placodes occurs in the epidermal basal cells of the sc/sc shank. It is the high rate of proliferation seen for the epidermal basal cells of the normal interplacode region and the outer surface of the scale ridge that never develops in the sc/sc epidermal cells.     

Integument structure in ornithischian dinosaurs (Hypsilophodontia,Ornithopoda) from the Late Jurassic of Transbaikalia

Integumentary structures of ornithischain dinosaurs of the taxon Hypsilophodontia (Ornithopoda) from the Ukureiskaya Formation (Upper Jurassic) of the Kulinda locality (Transbaikal Region, Russia) are described in detail. It is shown that members of this group had so-called bristle scales, integumentary appendages previously unknown in ornithischian dinosaurs. These are relatively small horn plates embedded in the skin, the distal margin of which has several long, flat, and probably constantly growing bristles. The monobristle variant of bristle scale is probably homologous to the protofeather of theropods; if this is the case, it is possible to reconstruct the protofeather as an elongated and constantly growing scale.     

Evolution of dinosaur epidermal structures

Spectacularly preserved non-avian dinosaurs with integumentary filaments/feathers have revolutionized dinosaur studies and fostered the suggestion that the dinosaur common ancestor possessed complex integumentary structures homologous to feathers. This hypothesis has major implications for interpreting dinosaur biology, but has not been tested rigorously. Using a comprehensive database of dinosaur skin traces, we apply maximum-likelihood methods to reconstruct the phylogenetic distribution of epidermal structures and interpret their evolutionary history. Most of these analyses find no compelling evidence for the appearance of protofeathers in the dinosaur common ancestor and scales are usually recovered as the plesiomorphic state, but results are sensitive to the outgroup condition in pterosaurs. Rare occurrences of ornithischian filamentous integument might represent independent acquisitions of novel epidermal structures that are not homologous with theropod feathers.     

The Fossil Record of Feather Evolution in the Mesozoic

The oldest known feathers from the Late Jurassic are alreadymodern in form and microscopic detail. Because these oldestexamples are assignable to an extinct branch (Sauriurae) ofthe basal avian dichotomy, their features must have been establishedat a significantly earlier date. The skin of a wide varietyof dinosaurs is now known and is unlikely to represent a predecessorto a feather bearing integument. Examples of feathered dinosaursresult from erroneous identification of internal structuresas part of the skin covering, and from the confusion of flightlessbirds from the Early Cretaceous of China with dinosaurs.