Identification of reptilian genes encoding hair keratin-like proteins suggests a new scenario for the evolutionary origin of hair

Abstract

The appearance of hair is one of the main evolutionary innovations in the amniote lineage leading to mammals. The main components of mammalian hair are cysteine-rich type I and type II keratins, also known as hard alpha-keratins or "hair keratins." To determine the evolutionary history of these important structural proteins, we compared the genomic loci of the human hair keratin genes with the homologous loci of the chicken and of the green anole lizard Anolis carolinenis. The genome of the chicken contained one type II hair keratin-like gene, and the lizard genome contained two type I and four type II hair keratin-like genes. Orthology of the latter genes and mammalian hair keratins was supported by gene locus synteny, conserved exon-intron organization, and amino acid sequence similarity of the encoded proteins. The lizard hair keratin-like genes were expressed most strongly in the digits, indicating a role in claw formation. In addition, we identified a novel group of reptilian cysteine-rich type I keratins that lack homologues in mammals. Our data show that cysteine-rich alpha-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.

Fig. 1.

Comparison of the α-keratin gene family loci in amniotes. The chromosomal loci containing the type I (A) and type II (B) keratin genes of humans, anole, and chicken are schematically depicted. Genes are represented by arrows pointing in the 5′ to 3′ end direction. Black arrows represent homologues of hair keratins. Note that only 1 of a series of human keratin-associated protein (KAP) genes and 4 of 11 hair keratin genes at the human type I keratin gene locus are shown.

Fig. 2.

(A and B) Numbers of cysteine residues in various keratins. Cysteine residues were counted in all new anole and chicken keratins and in all human hair keratins. For mammalian non-hair keratins, only representative examples are shown. The black portion of each column indicates the number of cysteines in the central domain of the keratin protein. Note that the cysteine counts of aHAS1, aHAS2, and aHAS4 do not contain the as-yet uncharacterized tail domains.

Fig. 3.

Phylogenetic analysis of sauropsid and mammalian keratins. Phylogenetic trees of selected type I (A) and type II (B) keratins were built using the neighbor-joining method. The branch lengths in the tree are proportional to the number of substitutions per site (scale: 0.1 substitutions per site). a, anole; c, chicken; h, human; m, mouse.

Fig. 4.

RT-PCR analysis of anole hard keratin expression. RNA was prepared from various organs of A. carolinensis and analyzed by RT-PCR with keratin-specific primers. The ubiquitously expressed protease caspase-3 was amplified as a control (Con.) for the quality of the RNA preparations. PCRs without cDNA template were performed to control for purity of reagents (lane “control”).

Fig. 5.

Immunohistochemical localization of anole hard keratins aHA1 and aHB1. Cryosections of A. carolinensis digits were analyzed by immunohistochemistry using antisera specific for aHA1 (A and B) and aHB1 (C and D). Red staining marks expression of these keratins. Preincubation of the antisera with peptides corresponding to the respective immunization epitope (B and D) blocked the staining and confirmed the specificity of the antisera. Preincubation of the anti-aHA1 antiserum with the aHB1 peptide and vice versa did not block the reaction. In B, white asterisks indicate artefacts caused by folding of tissue fragments over the sectioned claw. (Insets) Larger magnifications of the boxed areas. cl, claw; m, matrix; sc, scale. [White scale bar, 100 μm; black bar, 20 μm.]

Fig. 6.

Schematic overview of hard keratin gene evolution. The presence of hard keratins in various vertebrate species was mapped onto a phylogenetic tree of vertebrates. The number of functional type I and type II keratins is shown on the right. The number of sauropsid-specific cysteine-rich type I keratins (aHAS1–4 and cHAS1–2) is indicated separately (+4 and + 2, respectively). Stars indicate gene innovation events and a “strike” symbol indicates the loss of the type I hair keratin-like gene(s) in the lineage leading to chicken.