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

Abstract

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.

Keywords: co-option; convergent evolution; feathers; gene family; keratin.

FIg. 1.

Comparative analysis of the type I keratin gene cluster in terrestrial vertebrates. (A) Map of the type I keratin gene clusters in representative species of the main clades of terrestrial vertebrates. Genes are symbolized by arrowheads that point in the direction of transcription. Putative orthology is indicated by equal colors. Note that KRT222 encodes a keratin-like protein with an incomplete intermediate filament domain. (B) Cysteine counts of proteins encoded by the genes shown in panel (A). Asterisks mark keratins for which only incomplete amino acid sequences were available and the real number of cysteine residues is probably higher than indicated by the respective bars. Lizard K36L1 has 46 cysteine residues which is higher than the maximum value of this graph. Species: Chicken (Gallus gallus), alligator (Alligator sinensis), turtle (Chrysemys picta), lizard (Anolis carolinensis), snake (Protobothrops mucrosquamatus), frog (Xenopus tropicalis).

FIg. 2.

Comparative analysis of the type II keratin gene cluster in terrestrial vertebrates. (A) Map of the type II keratin gene clusters in representative species of the main clades of terrestrial vertebrates. Genes are symbolized by arrowheads that point in the direction of transcription. Putative orthology is indicated by equal colors. Note that KRT18 encodes a type I keratin. (B) Cysteine counts of proteins encoded by the genes shown in panel (A). An asterisk over snake HBS2 indicates that only an incomplete amino acid sequence was available and the number of cysteine residues is probably higher than indicated. Alligator K78LT has 66 cysteine residues which is higher than the maximum value of this graph. Information about the species is provided in the legend of figure 1.

FIg. 3.

Phylogenetic analysis of type I keratins. A combined evolutionary model of maximum likelihood (ML) and Bayesian analysis of type I keratins is shown. Red circles indicate groups with bootstrap ≥75% obtained by ML analysis and posterior probability ≥0.95 obtained by Bayesian analysis. The scale bar represents substitutions/site. HAS, hard acidic sauropsid-specific keratin.

FIg. 4.

Phylogenetic analysis of type II keratins. A combined evolutionary model of maximum likelihood (ML) and Bayesian analysis of type I keratins is shown. Red circles indicate groups with bootstrap ≥75% obtained by ML analysis and posterior probability ≥0.95 obtained by Bayesian analysis. The scale bar represents substitutions/site. HBS, hard basic sauropsid-specific keratin; K78LT, K78 long tail domain.

FIg. 5.

HBS1 keratin is expressed in feathers. (A) Quantitative RT-PCR analysis of HBS1 in different chicken tissues on embryonic day 18. The expression was normalized to that of keratin 5. The relative mRNA concentration (rel. mRNA conc.) is shown in arbitrary units (a.u.). The difference between expression in feathers and other tissues from n = 3 chickens was significant. (B) Western blot analysis of HBS1 in chicken tissues on embryonic day 18. The membrane was reprobed with anti-GAPDH as a loading control. kDa, kilo-Dalton. (C) Western blot analysis of HBS1 in chicken feathers and beak collected on the first day after hatching. Ponceau staining of total protein is shown to confirm equal loading of lanes. An asterisk indicates the position of corneous beta-proteins which represent a major amount of soluble proteins of feathers. M, molecular mass marker. (D and E) Immunohistochemical staining (red) of HBS1 in wing feathers. The sections were counterstained with hematoxylin (blue). The image in panels (E) was recorded at higher magnification than the image in panel (D) where the same detail is indicated by a box. (F) Negative control (neg. ctrl.) staining in which the primary antibody was replaced by preimmune serum. Size bars: 200 µm (D), 50 µm (E and F).

FIg. 6.

Evolutionary model of the origin of cysteine-rich keratins in amniotes. The numbers and types of cysteine (Cys)-rich type I (A) and type II (B) keratins per species were mapped onto a simplified phylogenetic tree of tetrapods. The origin and loss (flash symbol) events were inferred from the presence or absence of particular keratin types in modern species and their known phylogenetic relationships. Numbers of cysteine (# Cys) per keratin are indicated. Asterisks indicate numbers of Cys residues that were determined in partial protein sequences; the actual numbers may be higher. HK, hair keratin; HAS, hard acidic sauropsid-specific keratin; HBS, hard basic sauropsid-specific keratin; My, million years.