Convergent Evolution of Cysteine-Rich Keratins in Hard Skin Appendages of Terrestrial Vertebrates.

Terrestrial vertebrates have evolved hard skin appendages, such as scales, claws, feathers, and hair that play crucial roles in defense, predation, locomotion, and thermal insulation. The mechanical properties of these skin appendages are largely determined by cornified epithelial components. So-called "hair keratins," cysteine-rich intermediate filament proteins that undergo covalent cross-linking via disulfide bonds, are the crucial structural proteins of hair and claws in mammals and hair keratin orthologs are also present in lizard claws, indicating an evolutionary origin in a hairless common ancestor of amniotes. Here, we show that reptiles and birds have also other cysteine-rich keratins which lack cysteine-rich orthologs in mammals. In addition to hard acidic (type I) sauropsid-specific (HAS) keratins, we identified hard basic (type II) sauropsid-specific (HBS) keratins which are conserved in lepidosaurs, turtles, crocodilians, and birds. Immunohistochemical analysis with a newly made antibody revealed expression of chicken HBS1 keratin in the cornifying epithelial cells of feathers. Molecular phylogenetics suggested that the high cysteine contents of HAS and HBS keratins evolved independently from the cysteine-rich sequences of hair keratin orthologs, thus representing products of convergent evolution. In conclusion, we propose an evolutionary model in which HAS and HBS keratins evolved as structural proteins in epithelial cornification of reptiles and at least one HBS keratin was co-opted as a component of feathers after the evolutionary divergence of birds from reptiles. Thus, cytoskeletal proteins of hair and feathers are products of convergent evolution and evolutionary co-option to similar biomechanical functions in clade-specific hard skin appendages.

Differential Evolution of the Epidermal Keratin Cytoskeleton in Terrestrial and Aquatic Mammals.

Keratins are the main intermediate filament proteins of epithelial cells. In keratinocytes of the mammalian epidermis they form a cytoskeleton that resists mechanical stress and thereby are essential for the function of the skin as a barrier against the environment. Here, we performed a comparative genomics study of epidermal keratin genes in terrestrial and fully aquatic mammals to determine adaptations of the epidermal keratin cytoskeleton to different environments. We show that keratins K5 and K14 of the innermost (basal), proliferation-competent layer of the epidermis are conserved in all mammals investigated. In contrast, K1 and K10, which form the main part of the cytoskeleton in the outer (suprabasal) layers of the epidermis of terrestrial mammals, have been lost in whales and dolphins (cetaceans) and in the manatee. Whereas in terrestrial mammalian epidermis K6 and K17 are expressed only upon stress-induced epidermal thickening, high levels of K6 and K17 are consistently present in dolphin skin, indicating constitutive expression and substitution of K1 and K10. K2 and K9, which are expressed in a body site-restricted manner in human and mouse suprabasal epidermis, have been lost not only in cetaceans and manatee but also in some terrestrial mammals. The evolution of alternative splicing of K10 and differentiation-dependent upregulation of K23 have increased the complexity of keratin expression in the epidermis of terrestrial mammals. Taken together, these results reveal evolutionary diversification of the epidermal cytoskeleton in mammals and suggest a complete replacement of the quantitatively predominant epidermal proteins of terrestrial mammals by originally stress-inducible keratins in cetaceans.

Evolution of Trichocyte Keratins.

The evolution of keratins was closely linked to the evolution of epithelia and epithelial appendages such as hair. The characterization of keratins in model species and recent comparative genomics studies have led to a comprehensive scenario for the evolution of keratins including the following key events. The primordial keratin gene originated as a member of the ancient gene family encoding intermediate filament proteins. Gene duplication and changes in the exon-intron structure led to the origin of type I and type II keratins which evolved further by nucleotide sequence modifications that affected both the amino acid sequences of the encoded proteins and the gene expression patterns. The diversification of keratins facilitated the emergence of new and epithelium type-specific properties of the cytoskeleton. In a common ancestor of reptiles, birds, and mammals, a rise in the number of cysteine residues facilitated extensive disulfide bond-mediated cross-linking of keratins in claws. Subsequently, these cysteine-rich keratins were co-opted for an additional function in epidermal follicular structures that evolved into hair, one of the key events in the evolution of mammals. Further diversification of keratins occurred during the evolution of the complex multi-layered organisation of hair follicles. Thus, together with the evolution of other structural proteins, epithelial patterning mechanisms, and development programmes, the evolution of keratins underlied the evolution of the mammalian integument.

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.

An In Vitro Model of Avian Skin Reveals Evolutionarily Conserved Transcriptional Regulation of Epidermal Barrier Formation.

The function of the skin as a barrier against a dry environment evolved in a common ancestor of terrestrial vertebrates such as mammals and birds. However, it is unknown which elements of the genetic program of skin barrier formation are evolutionarily ancient and conserved. In this study, we determined the transcriptomes of chicken keratinocytes (KCs) grown in monolayer culture and in an organotypic model of avian skin. The differentiation-associated changes in global gene expression were compared with previously published transcriptome changes of human KCs cultured under equivalent conditions. We found that specific keratins and genes of the epidermal differentiation complex were upregulated during the differentiation of both chicken and human KCs. Likewise, the transcriptional upregulation of genes that control the synthesis and transport of lipids, anti-inflammatory cytokines of the IL-1 family, protease inhibitors, and other regulators of tissue homeostasis was conserved in the KCs of both species. However, some avian KC differentiation-associated transcripts lack homologs in mammals and vice versa, indicating a genetic basis for taxon-specific skin features. The results of this study reveal an evolutionarily ancient program in which dynamic gene transcription controls the metabolism and transport of lipids as well as other core processes during terrestrial skin barrier formation.

A Stress Response Program at the Origin of Evolutionary Innovation in the Skin.

The skin epithelium, ie, the epidermis, of dolphins and whales (cetaceans) is up to 50 times thicker than that of humans and other mammals living on land. Recently, comparative genomics revealed further striking differences in the cytoskeleton of the outer layers of the epidermis in aquatic and terrestrial mammals. Cetaceans lack the cytoskeletal keratins, which make up more than half of the total protein mass in the cornified epidermal layer of terrestrial mammals under homeostatic conditions. By contrast, orthologs of stress-inducible epithelial keratins are conserved in cetaceans and these keratins are constitutively expressed in their skin. Thus, the epidermal stress response program of a terrestrial common ancestor of modern mammals has become the default program of epidermal differentiation and a central component of the unique cutaneous organization of cetaceans. We propose that phenotypic plasticity during stress responses plays important roles in the evolution of the skin.

Comparative genomics suggests loss of keratin K24 in three evolutionary lineages of mammals.

Keratins are the main cytoskeletal proteins of epithelial cells and changes in the expression of keratins have contributed to the evolutionary adaptation of epithelia to different environments. Keratin K24 was proposed to be a differentiation marker of epidermal keratinocytes but the significance of K24 expression in the epidermis versus other tissues has remained elusive. Here, we show by RT-PCR, western blot, and immunofluorescence analyses that K24 is highly expressed in the epithelium of the cornea whereas its expression levels are significantly lower in other stratified epithelia including in the epidermis. To investigate the evolutionary history of K24, we screened the genome sequences of vertebrates for orthologs of the human KRT24 gene. The results of this comparative genomics study suggested that KRT24 originated in a common ancestor of amniotes and that it was lost independently in three clades of mammals, i.e. camels, cetaceans, and a subclade of pinnipeds comprising eared seals and the walrus. Together, the results of this study identify K24 as component of the cytoskeleton in the human corneal epithelium and reveal previously unknown differences of keratin gene content among mammalian species.

Immunolocalization and phylogenetic profiling of the feather protein with the highest cysteine content.

Feathers are the most complex skin appendages of vertebrates. Mature feathers consist of interconnected dead keratinocytes that are filled with heavily cross-linked proteins. Although the molecular architecture determines essential functions of feathers, only few feather proteins have been characterized with regard to their amino acid sequences and evolution. Here, we identify Epidermal Differentiation protein containing DPCC Motifs (EDDM) as a cysteine-rich protein that has co-evolved with other feather proteins. The EDDM gene is located within the avian epidermal differentiation complex (EDC), a cluster of genes that has originated and diversified in amniotes. EDDM shares the exon-intron organization with EDC genes of other amniotes, including humans, and a gene encoding an EDDM-like protein is present in crocodilians, suggesting that avian EDDM arose by sequence modification of an epidermal differentiation gene present in a common ancestor of archosaurs. The EDDM protein contains multiple sequence repeats and a higher number of cysteine residues than any other protein encoded in the EDC. Immunohistochemical analysis of chicken skin and skin appendages showed expression of EDDM in barb and barbules of feathers as well as in the subperiderm on embryonic scutate scales. These results suggest that the diversification and differential expression of EDDM, besides other EDC genes, was instrumental in facilitating the evolution of the most complex molecular architecture of feathers.