Comparative genomics of sirenians reveals evolution of filaggrin and caspase-14 upon adaptation of the epidermis to aquatic life.

The mammalian epidermis has evolved to protect the body in a dry environment. Genes of the epidermal differentiation complex (EDC), such as FLG (filaggrin), are implicated in the barrier function of the epidermis. Here, we investigated the molecular evolution of the EDC in sirenians (manatees and dugong), which have adapted to fully aquatic life, in comparison to the EDC of terrestrial mammals and aquatic mammals of the clade Cetacea (whales and dolphins). We show that the main subtypes of EDC genes are conserved or even duplicated, like late cornified envelope (LCE) genes of the dugong, whereas specific EDC genes have undergone inactivating mutations in sirenians. FLG contains premature stop codons in the dugong, and the ortholog of human CASP14 (caspase-14), which proteolytically processes filaggrin, is pseudogenized in the same species. As FLG and CASP14 have also been lost in whales, these mutations represent convergent evolution of skin barrier genes in different lineages of aquatic mammals. In contrast to the dugong, the manatee has retained functional FLG and CASP14 genes. FLG2 (filaggrin 2) is truncated in both species of sirenians investigated. We conclude that the land-to-water transition of sirenians was associated with modifications of the epidermal barrier at the molecular level.

Evolutionary origin of Hoxc13-dependent skin appendages in amphibians.

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.

Comparative genomics of monotremes provides insights into the early evolution of mammalian epidermal differentiation genes.

The function of the skin as a barrier against the environment depends on the differentiation of epidermal keratinocytes into highly resilient corneocytes that form the outermost skin layer. Many genes encoding structural components of corneocytes are clustered in the epidermal differentiation complex (EDC), which has been described in placental and marsupial mammals as well as non-mammalian tetrapods. Here, we analyzed the genomes of the platypus (Ornithorhynchus anatinus) and the echidna (Tachyglossus aculeatus) to determine the gene composition of the EDC in the basal clade of mammals, the monotremes. We report that mammal-specific subfamilies of EDC genes encoding small proline-rich proteins (SPRRs) and late cornified envelope proteins as well as single-copy EDC genes such as involucrin are conserved in monotremes, suggesting that they have originated in stem mammals. Monotremes have at least one gene homologous to the group of filaggrin (FLG), FLG2 and hornerin (HRNR) in placental mammals, but no clear one-to-one pairwise ortholog of either FLG, FLG2 or HRNR. Caspase-14, a keratinocyte differentiation-associated protease implicated in the processing of filaggrin, is encoded by at least 3 gene copies in the echidna. Our results reveal evolutionarily conserved and clade-specific features of the genetic regulation of epidermal differentiation in monotremes.

Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1.

Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1.

Heme Oxygenase-1 Is Upregulated during Differentiation of Keratinocytes but Its Expression Is Dispensable for Cornification of Murine Epidermis.

The epidermal barrier of mammals is initially formed during embryonic development and continuously regenerated by the differentiation and cornification of keratinocytes in postnatal life. Cornification is associated with the breakdown of organelles and other cell components by mechanisms which are only incompletely understood. Here, we investigated whether heme oxygenase 1 (HO-1), which converts heme into biliverdin, ferrous iron and carbon monoxide, is required for normal cornification of epidermal keratinocytes. We show that HO-1 is transcriptionally upregulated during the terminal differentiation of human keratinocytes in vitro and in vivo. Immunohistochemistry demonstrated expression of HO-1 in the granular layer of the epidermis where keratinocytes undergo cornification. Next, we deleted the Hmox1 gene, which encodes HO-1, by crossing Hmox1-floxed and K14-Cre mice. The epidermis and isolated keratinocytes of the resulting Hmox1f/f K14-Cre mice lacked HO-1 expression. The genetic inactivation of HO-1 did not impair the expression of keratinocyte differentiation markers, loricrin and filaggrin. Likewise, the transglutaminase activity and formation of the stratum corneum were not altered in Hmox1f/f K14-Cre mice, suggesting that HO-1 is dispensable for epidermal cornification. The genetically modified mice generated in this study may be useful for future investigations of the potential roles of epidermal HO-1 in iron metabolism and responses to oxidative stress.

Transglutaminase Activity Is Conserved in Stratified Epithelia and Skin Appendages of Mammals and Birds.

The cross-linking of structural proteins is critical for establishing the mechanical stability of the epithelial compartments of the skin and skin appendages. The introduction of isopeptide bonds between glutamine and lysine residues depends on catalysis by transglutaminases and represents the main protein cross-linking mechanism besides the formation of disulfide bonds. Here, we used a fluorescent labeling protocol to localize the activity of transglutaminases on thin sections of the integument and its appendages in mammals and birds. In human tissues, transglutaminase activity was detected in the granular layer of the epidermis, suprabasal layers of the gingival epithelium, the duct of sweat glands, hair follicles and the nail matrix. In the skin appendages of chickens, transglutaminase activity was present in the claw matrix, the feather follicle sheath, the feather sheath and in differentiating keratinocytes of feather barb ridges. During chicken embryogenesis, active transglutaminase was found in the cornifying epidermis, the periderm and the subperiderm. Transglutaminase activity was also detected in the filiform papillae on the tongue of mice and in conical papillae on the tongue of chickens. In summary, our study reveals that transglutaminase activities are widely distributed in integumentary structures and suggests that transglutamination contributes to the cornification of hard skin appendages such as nails and feathers.

Evolutionary diversification of epidermal barrier genes in amphibians.

The epidermal differentiation complex (EDC) is a cluster of genes encoding components of the skin barrier in terrestrial vertebrates. EDC genes can be categorized as S100 fused-type protein (SFTP) genes such as filaggrin, which contain two coding exons, and single-coding-exon EDC (SEDC) genes such as loricrin. SFTPs are known to be present in amniotes (mammals, reptiles and birds) and amphibians, whereas SEDCs have not yet been reported in amphibians. Here, we show that caecilians (Amphibia: Gymnophiona) have both SFTP and SEDC genes. Two to four SEDC genes were identified in the genomes of Rhinatrema bivittatum, Microcaecilia unicolor and Geotrypetes seraphini. Comparative analysis of tissue transcriptomes indicated predominant expression of SEDC genes in the skin of caecilians. The proteins encoded by caecilian SEDC genes resemble human SEDC proteins, such as involucrin and small proline-rich proteins, with regard to low sequence complexity and high contents of proline, glutamine and lysine. Our data reveal diversification of EDC genes in amphibians and suggest that SEDC-type skin barrier genes have originated either in a common ancestor of tetrapods followed by loss in Batrachia (frogs and salamanders) or, by convergent evolution, in caecilians and amniotes.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus.

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.