Cellular structure of dinosaur scales reveals retention of reptile-type skin during the evolutionary transition to feathers.

Fossil feathers have transformed our understanding of integumentary evolution in vertebrates. The evolution of feathers is associated with novel skin ultrastructures, but the fossil record of these changes is poor and thus the critical transition from scaled to feathered skin is poorly understood. Here we shed light on this issue using preserved skin in the non-avian feathered dinosaur Psittacosaurus. Skin in the non-feathered, scaled torso is three-dimensionally replicated in silica and preserves epidermal layers, corneocytes and melanosomes. The morphology of the preserved stratum corneum is consistent with an original composition rich in corneous beta proteins, rather than (alpha-) keratins as in the feathered skin of birds. The stratum corneum is relatively thin in the ventral torso compared to extant quadrupedal reptiles, reflecting a reduced demand for mechanical protection in an elevated bipedal stance. The distribution of the melanosomes in the fossil skin is consistent with melanin-based colouration in extant crocodilians. Collectively, the fossil evidence supports partitioning of skin development in Psittacosaurus: a reptile-type condition in non-feathered regions and an avian-like condition in feathered regions. Retention of reptile-type skin in non-feathered regions would have ensured essential skin functions during the early, experimental stages of feather evolution.

Preservation of corneous ß-proteins in Mesozoic feathers.

Fossil proteins are valuable tools in evolutionary biology. Recent technological advances and better integration of experimental methods have confirmed the feasibility of biomolecular preservation in deep time, yielding new insights into the timing of key evolutionary transitions. Keratins (formerly α-keratins) and corneous ß-proteins (CBPs, formerly ß-keratins) are of particular interest as they define tissue structures that underpin fundamental physiological and ecological strategies and have the potential to inform on the molecular evolution of the vertebrate integument. Reports of CBPs in Mesozoic fossils, however, appear to conflict with experimental evidence for CBP degradation during fossilization. Further, the recent model for molecular modification of feather chemistry during the dinosaur-bird transition does not consider the relative preservation potential of different feather proteins. Here we use controlled taphonomic experiments coupled with infrared and sulfur X-ray spectroscopy to show that the dominant ß-sheet structure of CBPs is progressively altered to α-helices with increasing temperature, suggesting that (α-)keratins and α-helices in fossil feathers are most likely artefacts of fossilization. Our analyses of fossil feathers shows that this process is independent of geological age, as even Cenozoic feathers can comprise primarily α-helices and disordered structures. Critically, our experiments show that feather CBPs can survive moderate thermal maturation. As predicted by our experiments, analyses of Mesozoic feathers confirm that evidence of feather CBPs can persist through deep time.

Insights of keratin geometry from agro-industrial wastes: A comparative computational and experimental assessment.

Understanding the structural properties of keratin is of great importance to managing their potential application in keratin-inspired biomaterials and its management of wastes. In this work, the molecular structure of chicken feather keratin 1 was characterized by AlphaFold2 and quantum chemistry calculation. The predicted IR spectrum of the N-terminal region of feather keratin 1, consisting of 28 amino acid residues, was used to assign the Raman frequencies of the extracted keratin. The MW of experimental samples were 6 & 1 kDa while the predicted MW (∼10 kDa) of ß-keratin. Experimental analysis shows the magnetic field treatment could affect the functional and surface structural properties of keratin. The particle size distribution curve illustrates the dispersion of particle size concentration, while TEM analysis demonstrates the reduction of particle diameter to 23.71 ± 1.1 nm following treatment. High-resolution XPS analysis confirmed the displacement of molecular elements from their orbital.

Valorization of feather waste in Brazil: structure, methods of extraction, and applications of feather keratin.

This systematic review presents the potential of using feather waste as a ß-keratin source, including the Brazilian scenario in the generation of this byproduct. The structure and properties of α- and ß-keratin, the methods commonly reported to extract keratin from poultry feathers, and applications of feather keratin-based materials are also covered in this review. The literature search for poultry production data in Brazil was conducted for the last 2 years, for the period 2021-2022. A broad literature search for extraction methods and applications of feather keratin was done for the period 2001-2022. The poultry industry is one of the largest sectors of the food industry, and Brazil was the third-largest world producer of chicken meat with more than six billion chickens slaughtered in 2021. Poultry feathers constitute about 7% weight of broilers; thus, it can be estimated that about one million tons of poultry feathers were generated in Brazil in 2021, and the improper disposal of this byproduct contributes to environmental problems and disease transmission. The most common method of reusing feathers is the production of feather meal. From economic and environmental points of view, it is advantageous to develop processes to add value to this byproduct, including the extraction of keratin. Among natural biodegradable polymers, keratin-based materials have revolutionized the field of biomaterials due to their biocompatibility and biodegradability, allowing their application in biomedical, pharmaceutical, chemical, and engineering areas.

Molecular Structure and Dynamics in Wet Gecko ß-Keratin.

Molecular dynamics simulations are performed to investigate the molecular picture of water sorption in gecko keratin and the influence of relative humidity (RH) on the local structure and dynamics in water-swollen keratin. At low RHs, water sorption occurs through hydrogen bonding of water with the hydrophilic groups of keratin. At high RHs (>80%), additional water molecules connect to the first "layer" of amide-connected water molecules (multimolecular sorption) through hydrogen bonds, giving rise to a sigmoidal shape of the sorption isotherm. This causes the formation of large chain-like clusters surrounding the hydrophilic groups of keratin, which upon a further increase of the RH form a percolating water network. An examination of the dynamics of water molecules sorbed in keratin demonstrates that there are two states, bound and free, for water. The dynamics of water in these states depends on the RH. At low RHs, large-scale translational motions of tightly bound water molecules to keratin are needed to remake the entire hydration shell of the keratin. At high RHs (>80%), the water molecules more quickly exchange between the two states. The center-of-mass mean-square displacement of water molecules indicates a hopping motion of water molecules in the keratin solvation shell. The hopping mechanism is more pronounced at RHs < 80%. At higher RHs, water translation through water clusters (water network) dominates. We have observed two regimes for the dependence of dynamical properties on the RH: a regime of gradual increase of the dynamics over 10% < RH < 80% and a regime of drastic dynamic acceleration at RH > 80%. The latter regime begins exactly where the water uptake and the volume swelling also increase much more and where a drastic change in the elastic properties of gecko keratin has been observed. A nearly linear relation between the relaxation times for all dynamical processes and the water content of gecko keratin is observed.

Selective IR super-resolution imaging of ß-keratins at the bulk or interface in feather detected by using a nonlinear optical process.

We developed the new IR super-resolution microscope by using a 4-wave mixing (4-wave), which is a third-order nonlinear optical process, and carried out the IR super-resolution imaging of the cross section of the rachis of an avian feather. We clearly observed strong signals in the entire region of the rachis at the amide I vibration of ß-keratin in both of the XXYY and YYXX polarization combination. These results are different from images detected by using the vibrational sum-frequency generation (VSFG) method. While the VSFG imaging detects molecules only from the interface, the 4-wave method enables us to observe the signal from the bulk area. We concluded that the four repeating units of ß-keratins in the bulk area which are suggested by X-ray diffraction studies are visualized in the 4-wave detected method. We also applied two IR super-resolution microscopies for the barb and discuss the site dependence of the orientation, distribution and concentration of ß-keratin.

Electron microscopic analysis in the gecko Lygodactylus reveals variations in micro-ornamentation and sensory organs distribution in the epidermis that indicate regional functions.

Possible pattern variations of micro-ornamentation in different areas of the skin in the gecko Lygodactylus have been analyzed by scanning and transmission electron microscopy. A map of micro-ornamentation present in various areas of the skin has been obtained. Differences in micro-ornamentation pattern and sensory organ distribution were detected. The "spinulated pattern" consists of shorter spinulae in dorsal versus ventral scales, and spinules are shorter in inner scale surface and hinge regions with respect to the outer scale surface. The spines derive from the accumulation of struts of corneous material mainly composed of corneous beta proteins (CBPs, formerly indicated as beta-keratins) that merge into pointed micro-ornamentation. The 3D-accumulation of CBPs within Oberhautchen cells can vary in some regions of different scales during Oberhautchen-beta cell differentiation, perhaps also under physical tensile forces derived from continuous scale growth. Three other main patterns of micro-ornamentation were detected and indicated as "corneous belts," "corneous dendritic ramification," and "serpentine-pit and groove." These variations from the typical spinulated pattern present in gecko epidermis are interpreted as transitional regions where the accumulation of corneous material in Oberhautchen cells that merges with underlying beta-cells gives rise to nonspinulated surfaces. Spinulated sensory organs with bristles and lenticular-shaped or knob-like tactile corpuscles are more numerous in ventral scales of the tail tip close to adhesive pads and near the digital pads. These regions are likely those most involved in the fine control of movements and response to vibrational stimuli derived from air and objects movements, including potential preys or predators.

Water uptake by gecko ß-keratin and the influence of relative humidity on its mechanical and volumetric properties.

Grand canonical ensemble molecular dynamics simulations are done to calculate the water content of gecko ß-keratin as a function of relative humidity (RH). For comparison, we experimentally measured the water uptake of scales of the skin of cobra Naja nigricollis. The calculated sigmoidal sorption isotherm is in good agreement with experiment. To examine the softening effect of water on gecko keratin, we have calculated the mechanical properties of dry and wet keratin samples, and we have established relations between the mechanical properties and the RH. We found that a higher RH causes a decrease in the Young's modulus, the yield stress, the yield strain, the stress at failure and an increase in the strain at failure of the gecko keratin. At low RHs (less than 80%), the change in the mechanical properties is small, with most of the changes occurring at higher RHs. The changes in the macroscopic properties of the keratin are explained by the action of sorbed water on the molecular scale. It causes keratin to swell, thereby increasing the distances between amino acids. This has a weakening effect on amino acid interactions and softens the keratin material. The effect is more pronounced at higher RHs.

Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Structure of Keratin.

Keratins, as a group of insoluble and filament-forming proteins, mainly exist in certain epithelial cells of vertebrates. Keratinous materials are made up of cells filled with keratins, while they are the toughest biological materials such as the human hair, wool and horns of mammals and feathers, claws, and beaks of birds and reptiles which usually used for protection, defense, hunting and as armor. They generally exhibit a sophisticated hierarchical structure ranging from nanoscale to centimeter-scale: polypeptide chain structures, intermediated filaments/matrix structures, and lamellar structures. Therefore, more and more attention has been paid to the investigation of the relationship between structure and properties of keratins, and a series of biomimetic materials based on keratin came into being. In this chapter, we mainly introduce the hierarchical structure, the secondary structure, and the molecular structure of keratins, including α- and ß-keratin, to promote the development of novel keratin-based biomimetic materials designs.

Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

Evolution of single gyroid photonic crystals in bird feathers.

Vivid, saturated structural colors are conspicuous and important features of many animals. A rich diversity of three-dimensional periodic photonic nanostructures is found in the chitinaceous exoskeletons of invertebrates. Three-dimensional photonic nanostructures have been described in bird feathers, but they are typically quasi-ordered. Here, we report bicontinuous single gyroid ß-keratin and air photonic crystal networks in the feather barbs of blue-winged leafbirds (Chloropsis cochinchinensis sensu lato), which have evolved from ancestral quasi-ordered channel-type nanostructures. Self-assembled avian photonic crystals may serve as inspiration for multifunctional applications, as they suggest efficient, alternative routes to single gyroid synthesis at optical length scales, which has been experimentally elusive.

Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids).

The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7-10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.

Immunolocalization of epidermal differentiation complex proteins reveals distinct molecular compositions of cells that control structure and mechanical properties of avian skin appendages.

The epidermal differentiation complex (EDC) is a cluster of genes that encode structural proteins of skin derivatives with variable mechanical performances, from the scales of reptiles and birds to the hard claws and beaks, and to the flexible but resistant corneous material of feathers. Corneous proteins with or without extended beta-regions are produced from avian genomes, and include the largely prevalent corneous beta proteins (CßPs, formerly indicated as beta-keratins), and minor contribution from histidine-rich proteins, trichohyalin-like proteins (scaffoldin), loricrin, and other proteins rich in cysteine or other types of amino acids. The light-microscopic and ultrastructural immunolocalization of major and minor EDC-proteins in avian skin (feather CßPs, EDKM, EDWM, EDMTFH, EDDM, and scaffoldin) suggests that each specific appendage consists of a particular mix of these proteins in addition to the main proteins containing a peculiar beta-region of 34 amino acids, indicated as feather/scale/claw/beak CßPs (fCßPs, sCßPs, cCßPs, bCßPs). This indicates that numerous proteins of the EDC are added to the variable meshwork of intermediate filament keratins to produce avian epidermis with different mechanical and functional properties. Although the specific roles for these proteins are not known they likely make an important contribution to the final material properties of the different skin appendages of birds. The highest number of sauropsid CßPs is found in birds, suggesting a relation to the evolution of feathers, and additional epidermal differentiation proteins have contributed to the evolutionary adaptations of avian skin.

Structure and topology of the linkers in the conserved lepidosaur ß-keratin chain with four 34-residue repeats support an interfilament role for the central linker.

The ß-keratin chain with four 34-residue repeats that is conserved across the lepidosaurs (lizards, snakes and tuatara) contains three linker regions as well as a short, conserved N-terminal domain and a longer, more variable C-terminal domain. Earlier modelling had shown that only six classes of structure involving the four 34-residue repeats were possible. In three of these the 34-residue repeats were confined to a single filament (Classes 1, 2 and 3) whereas in the remaining three classes the repeats lay in two, three or four filaments, with some of the linkers forming interfilament connections (Classes 4, 5 and 6). In this work the members of each class of structure (a total of 20 arrangements) have been described and a comparison has been made of the topologies of each of the linker regions. This provides new constraints on the structure of the chain as a whole. Also, analysis of the sequences of the three linker regions has revealed that the central linker (and only the central linker) contains four short regions displaying a distinctive dipeptide repeat of the form (S-X)2,3 separated by short regions containing proline and cysteine residues. By analogy with silk fibroin proteins this has the capability of forming a ß-sheet-like conformation. Using the topology and sequence data the evidence suggests that the four 34-residue repeat chain adopts a Class 4a structure with a ß-sandwich in filament 1 connected through the central linker to a ß-sandwich in filament 2.

How a Bird Gets Its Feathers: Insights from Chromatin Looping.

Mechanisms controlling skin heterogeneity are poorly understood. In this issue of Developmental Cell, Liang et al. show that in chicken, the difference in ß-keratin genes expressed in feathered and scaly skin is regulated via typical enhancers, while differential expression within individual feathers correlates with chromatin looping within the gene cluster.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

General and specific microscopic characteristics of the dorsal tail scales and the spines of the crest in the tuatara Sphenodon pucntatus (Reptilia; Rhynchocephalia; Sphenodontidae).

Dorsal crest scales and those of the tail spines of the tuatara (Sphenodon punctatus) represent different specializations involved in display and protection. Erection of the dorsal crest occurs in males during combat and courtship, but tail spines are not noticeably involved in these activities. In both scale derivatives corneous beta proteins (CBPs, formerly called beta-keratins) and intermediate filaments keratins (IFKs) were determined by immunolabelling. The dermis is dense with few sparse fibrocytes surrounded by collagen bundles, the latter rather randomly oriented in the crest scales. In the tail ridge scales banded collagen I fibrils form more regular, orthogonally aligned bundles of alternating layers with connections to the basal epidermal membrane. A conglomerate of dermal melanonophores and iridophores is present under the epidermis. The iridophores are the likely origin of the whitish colour of the crest. The epidermis shows a thicker beta-layer with serrated/indented corneocytes in the tail scales while the beta layer is reduced in the crest but contains CBPs. A relatively thick mesos layer is present in both scale derivatives, especially in the crest where its role, aside from limiting transpiration, is not known. The alpha-layer is formed by corneocytes with irregular perimeter and sparse desmosomal remnants. The high labelling intensity for CBPs in the beta-layer disappears in the mesos layer but occurs, albeit strongly reduced, in the alpha-layer as in the other body scales. The take-home message is that the dense dermis and its apical beta-layer strengthen mechanically the ridge spines while the crest is mainly supported by the firm but pliable and less dense or regular dermis.

Theoretical approaches to study the optical response of the red-legged honeycreeper's plumage (Cyanerpes cyaneus).

In this paper, we investigate the unusual color effect exhibited by the plumage of the heads of Cyanerpes cyaneus males, whose color turns from green to turquoise as the angle between the illumination and observation directions is increased. This singular color effect is characteristic of species that have quasi-ordered nanostructures of short-range order within the feather barbs. However, among species of the same family and even within feather patches of the same individual, one can find barbs with different characteristics, both macroscopic (curvature, shape, cross-sectional area) and in their internal microstructure. We apply the Korringa-Kohn-Rostoker method with the averaging technique to model the reflectance spectra for different angles of incidence and explain the dependence of the observed color with the incidence-collection angle. To investigate the influence of the disorder in the optical response of the spongy matrix, we apply the integral method for a two-dimensional cylinder system that simulates the distribution of air cavities within the $ \beta $ß-keratin medium. The experimental reflectance was interpreted as the result of multiple reflections in the internal interfaces generated by large air voids present within the spongy matrix. The application of rigorous methods to the study of natural photonic structures is of fundamental relevance for the design of efficient bioinspired artificial materials.

The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3.

Feather waste is the highest protein-containing resource in nature and is poorly reused. Bioconversion is widely accepted as a low-cost and environmentally benign process, but limited by the availability of safe and highly efficient feather degrading bacteria (FDB) for its industrial-scale fermentation. Excessive focuses on keratinase and limited knowledge of other factors have hindered complete understanding of the mechanisms employed by FDB to utilize feathers and feather cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently degrade feather to products with high amino acid content, useful as a nutrition source for animals, plants and microorganisms. Using multiple omics and other techniques, we reveal how SCUT-3 turns on its feather utilization machinery, including its colonization, reducing agent and protease secretion, peptide/amino acid importation and metabolism, oxygen consumption and iron uptake, spore formation and resuscitation, and so on. This study would shed light on the feather utilization mechanisms of FDBs.