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

Abstract

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.

Fig. 1.

Comparative analysis of the keratin type I and type II gene clusters in fully aquatic and terrestrial mammals. Comparison of the type I and II keratin gene clusters of human, cattle, dolphin, orca, baiji, sperm whale, minke whale, manatee, and elephant indicate a loss of several keratin genes in fully aquatic mammals, including epidermal differentiation-associated type 1 keratins K10 and K9 and type 2 keratins K1, K2, and K77 (red arrows). The direction of arrows indicates the orientation of gene transcription. Only genes considered to be intact (encoding a functional protein) are shown. The position of a cluster of genes encoding keratin-associated proteins (KAPs) is indicated within the type I keratin gene cluster. Heterodimerization interactions between keratins of particular interest for the present study are indicated by double-headed arrows and the main expression sites are shown above and below the depiction of the gene clusters. Note that K222 has a unique structure and the orthology relationships of the K6 isoforms and of K81 and K86-like proteins are uncertain in cetaceans.

Fig. 2.

Analysis of transcriptomes of dolphin skin shows high abundance of hyperproliferation-associated keratins K6 and K17. (A) Published transcriptome data of bottlenose dolphin skin samples (n = 116; Neely et al. 2018) were analyzed for the expression levels of keratin genes. The number of RNA-seq reads were normalized to the expression level of ALAS1 as a reference gene. a.u., arbitrary units. (B–D) Schematic comparison of epidermal thickness and keratin expression in normal (B) and psoriatic (C) human epidermis and cetacean (D) epidermis. Skin sections stained with hematoxylin and eosin are shown at equal magnifications to facilitate comparison of epidermal thickness. Keratins expressed in human (according to literature cited in the main text) and dolphin skin (this study) are indicated. Keratins K6 and K17 are induced when keratinocyte hyperproliferation during wound healing or in psoriasis leads to thickening of the epidermis. Scale bars: 500 μm.

Fig. 3.

Keratin K23 is expressed in differentiated epidermal keratinocytes. (A) Expression levels of KRT23 in human tissues were obtained from the GTEx database. Bars indicate means and error bars indicate standard deviations. *, P < 0.05 (t-test); n.s., not significant; sal., salivary; RPKM, reads per kilobase of transcript per million mapped reads. (B) Expression of KRT23 during differentiation of primary human keratinocytes in vitro. Human epidermal keratinocytes were cultured in monolayer culture under subconfluent proliferation (prol)-enhancing conditions, under postconfluent differentiation (diff)-enhancing conditions for 3 and 7 days, and in skin equivalent (SE) cultures. KRT23 expression was determined by quantitative real-time PCR using ALAS1 as a reference gene for normalization. (C) Immunohistochemical analysis of K23 (red) demonstrated expression in the most differentiated living layers of human epidermis. (D) Replacing the primary antibody by immunoglobulin from nonimmunized guinea pigs abolished the staining and confirmed its specificity. sc, stratum corneum, derm, dermis, epid, epidermis. Scale bars: 50 µm. (E) Western blot analysis of K23 in human keratinocytes differentiating in vitro. Protein lysates from keratinocytes were consecutively analyzed with primary antibodies against K23, K2, K5, and GAPDH. The positions of molecular weight markers are shown on the right. kDa, kilo-Dalton. (F) Schematic summary of K23 evolution in vertebrates, inferred from the presence or absence of Krt23 genes in modern species mapped onto a phylogenetic tree (Zhou et al. 2011).

Fig. 4.

Two isoforms of K10 are generated by alternative splicing in terrestrial mammals. (A) Schematic depiction of the exon–intron organization of Krt10 and splicing leading to K10 and K10x1 isoforms. (B) Alignment of nucleotide sequences around the two alternative splice donor sites of the Krt10 gene and the two mRNA variants. Amino acid sequences encoded by the mRNAs are shown. *, end of protein. (C) Alignment of carboxy-terminal amino acid sequences of K10 variants from different species. (D) Schematic model of the evolution of K10. IF, intermediate filament domain, CTM, carboxy-terminal motif. (E) Alignment of carboxy-terminal amino acid sequences of human intermediate filament proteins. Residues belonging to the evolutionarily ancestral CTM are indicated by yellow shading. Note that the amino acid sequences begin with residue 579 of human K10 and K10x1 in panels B and C, and with residue 584 of human K10 and residue 590 of human K10x1 in panel E. (F) Nucleotide sequence alignment of Krt10 exon 7 (indicated by green and blue lines) and adjacent introns of manatee and elephant. GT/AG splice site motifs are underlined. Substitutions of splice sites are indicated by grey shading. Also note sequence deletions (indicated by dashes) in the manatee. (G) The evolution of the Krt10 gene is indicated on a phylogenetic tree (Zhou et al. 2011). Asterisks indicate the origin of new traits and bolt symbols indicate the loss of Krt10. ***, triplication of Krt10 in the opossum.

Fig. 5.

Schematic overview of epidermal keratin evolution. Phylogenetic relationships of mammalian species and the presence or absence of intact epidermal keratin genes (+ and – symbols in the table) are indicated. Gene inactivations that can be inferred from the species distribution of genes are indicated by bolt symbols on the phylogenetic tree (Zhou et al. 2011). The tasmanian devil is shown as a representative of marsupials. Blue lines and fonts indicate fully aquatic life.