ORIGINAL ARTICLE

Trichohyalin-Like Have Evolutionarily Conserved Roles in the Morphogenesis of Skin Appendages Veronika Mlitz1, Bettina Strasser1, Karin Jaeger1, Marcela Hermann2, Minoo Ghannadan1, Maria Buchberger1, Lorenzo Alibardi3, Erwin Tschachler1 and Leopold Eckhart1

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to . In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner of . The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin. Journal of Investigative Dermatology (2014) 134, 2685–2692; doi:10.1038/jid.2014.204; published online 5 June 2014

INTRODUCTION et al., 2012; Kypriotou et al., 2012). This apparent similarity of S100 fused-type proteins (SFTPs) are a family of epidermal domains has led to the hypothesis that SFTP proteins, which comprises seven members in human and have arisen by the fusion of a S100 with a flanking gene mouse, namely cornulin (encoded by the gene, Crnn), within the epidermal differentiation complex (Markova et al., filaggrin (Flg), filaggrin 2 (Flg2), hornerin (Hrnr), (Rptn), 1993; Krieg et al., 1997; Contzler et al., 2005). trichohyalin (Tchh), and trichohyalin-like 1 (Tchhl1) (Henry Mammalian SFTPs have distinct expression patterns and et al., 2012; Kypriotou et al., 2012). The SFTP genes are functions, which are best characterized for filaggrin, tricho- located in the epidermal differentiation complex on human hyalin, and cornulin (Henry et al., 2012; Kypriotou et al., 1q21 and mouse chromosome 3q. SFTPs consist 2012). Filaggrin is expressed in differentiating keratinocytes of of an amino-terminal S100 domain that is followed by a the epidermis where it supposedly aggregates segment of 200 to more than 4,000 amino acid residues of intermediate filaments (Sandilands et al., 2009). In addition, highly diverse sequences among the various SFTPs. Many filaggrin is a major source of histidine that is converted to an SFTPs contain repetitive sequences with a high abundance of endogenous protectant against ultraviolet radiation, urocanic arginine, glutamic acid, , glycine, serine, and/or acid (Barresi et al., 2011). Mutations in the FLG gene are histidine residues (Henry et al., 2012). In this regard, the SFTPs associated with vulgaris and atopic dermatitis resemble proteins that are encoded by epidermal differ- (Sandilands et al., 2009; Brown and McLean, 2012). entiation complex genes in close vicinity to SFTPs (Henry Trichohyalin is expressed in the of the hair and other sites of hard cornification (Rothnagel and Rogers, 1986; O’Keefe et al., 1993; Rogers, 2004). It has a very high 1Department of Dermatology, Medical University of Vienna, Vienna, Austria; 2Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical content of glutamic acid and arginine residues, and many of University of Vienna, Vienna, Austria and 3Dipartimento di Scienze the latter are deiminated to (Tarcsa et al., 1996). Biologiche, Geologiche ed Ambientali (BiGeA), Universita` di Bologna, Polymorphisms in the TCHH gene determine the shape of Bologna, Italy human hair (Medland et al., 2009). Both filaggrin and Correspondence: Leopold Eckhart, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. trichohyalin accumulate in the form of characteristic intra- E-mail: [email protected] cellular granules known as and trichohyalin Abbreviations: SFTP, S100 fused-type protein; mRNA, messenger RNA granules, respectively. Cornulin is predominantly expressed Received 3 January 2014; revised 28 March 2014; accepted 11 April 2014; not only in the epithelium of the esophagus but also in accepted article preview online 29 April 2014; published online 5 June 2014 the inner root sheath of the hair follicles and in the

& 2014 The Society for Investigative Dermatology www.jidonline.org 2685 V Mlitz et al. Conserved Roles of S100 Fused-Type Proteins

upper epidermis (Xu et al., 2000; Henry et al., 2012). Cornulin a S100-fused type genes

has been implicated in esophageal squamous cell carcinoma LCE5A Crnn Flg2 Flg Hrnr Rptn Tchh Tchhl1 S100A11 (Chen et al., 2013) and atopic eczema (Liede´n et al., 2009). Human (H.s.) EDC1 Scfn Recent advances in genome sequencing have facilitated the Alligator (A.m.) identification of homologs of human skin proteins in non- Chicken (G.g.) model species, leading to new concepts regarding the evolu- Lizard (A.c.) tion of hair and other skin appendages (Eckhart et al., 2008). b Central domain Here, we apply comparative genomics in the investigation of S100 C-terminal motif SFTPs and define the evolutionarily conserved roles of SFTPs. H.s. Crnn 495 aa We identify SFTPs in sauropsids, i.e. reptiles and birds, and A.m. Crnn 446 aa G.g. Crnn 384 aa suggest an origin of SFTPs in early amniotes. Comparative H.s. Tchh 1,943 aa gene expression analyses in chicken and human tissues point A.m. Scfn 2,678 aa to conserved principles of morphogenesis of skin appendages. G.g. Scfn ~3,200 aa An improved understanding of the evolution of SFTPs will A.c. Scfn >688 aa

provide a framework for further investigations of the biological c 100% functions and dermatological relevance of proteins such as 90% trichohyalin and filaggrin. 80% 70% RESULTS 60% Identification of nonmammalian S100 fused-type proteins 50% 40% Publicly available genome sequences of nonmammalian 30%

vertebrates were screened for the presence or absence of % Of total amino acids 20% SFTP genes. Indeed, two genes encoding proteins with an 10% amino-terminal S100 domain, a long domain of low sequence 0% Tchh Scfn Scfn Scfn Crnn Crnn Crnn Tchhl1 Rptn Flg Flg2 Hrnr complexity, and conserved sequence elements at the carboxy H.s. A.m. G.g. A.c. A.m G.g. H.s. H.s. H.s. H.s. H.s. H.s. terminus were identified at reptilian and avian genome loci Arg Glu Gln Ser Gly His Other homologous to those of the human SFTP gene cluster (Figure 1a and b; Supplementary Figure S1 online). Similar Figure 1. Identification and phylogenetic analysis of nonmammalian genes to their human homologs, the SFTP genes of sauropsids were encoding S100 fused-type proteins (SFTPs). (a) The chromosomal loci containing the SFTP genes of different species are depicted. Genes are arranged in tandem and with the same orientiation as the next represented by arrows pointing from the 50 to the 30 end. The genes flanking the 0 gene downstream, S100A11 (Figure 1a). On the 5 side, the SFTPs on the left side have not been assigned a name yet, with the exception of sauropsidian SFTP genes were flanked by genes apparently LCE5A (late cornified envelope 5A); they are provisionally denoted here as similar to cornified envelope genes of the epidermal differ- EDC1 (epidermal differentiation complex gene 1). (b) Scheme of protein entiation complex (Figure 1a; Supplementary Figure S2 domains and sizes. Protein segments containing sequence repeats are indicated online). By contrast, the genome of an amphibian, the clawed in dark gray. Lengths are indicated by numbers of amino acid residues (aa). The frog, Xenopus tropicalis, contained an S100A11 gene but no proteins are not drawn to scale. (c) Amino acid composition of the central domain of SFTPs. Differently colored portions of each column indicate the genes encoding SFTPs. Similarly, no homologs of SFTPs were percent of total amino acid residues present in the central domain. A.c., Anolis found in fish genomes. Thus, SFTP genes appear to be present carolinensis; A.m., Alligator mississippiensis; G.g., Gallus gallus; H.s., Homo in amniotes (mammals and sauropsids) but not in other sapiens. vertebrates. A phylogenetic analysis showed that the SFTPs of different sauropsidian species represented orthologs of two proteins, i.e. a homolog of mammalian cornulin and a newly identified (filaggrin, filaggrin 2, and hornerin) and trichohyalin-type SFTP that we tentatively named scaffoldin (proposed gene proteins (trichohyalin, trichohyalin like 1, and repetin) con- name Scfn; see below). Genes encoding cornulin (Crnn) were tained only mammalian proteins. identified in chickens, budgerigars, alligators, and pythons but Similar to their mammalian counterparts, the sauropsidian not in the green anole lizard (Anolis carolinensis) (Figure 1a; SFTP genes consisted of three exons with the coding sequence Supplementary Table S1 online; Supplementary Figure S1a being located in exons 2 and 3 (Supplementary Table S1 online). Amplification and sequencing of the corresponding online). Sequence alignment showed conservation of putative chromosomal locus of the lizard confirmed the absence of a binding sites for transcription factors AP-1 (Jang et al., 1996) Crnn gene (not shown). Scfn genes were identified in all the and KLF4 (Patel et al., 2006) in the proximal promoters above-mentioned species including the lizard (Figure 1a; of several sauropsidian and mammalian SFTP genes Supplementary Table S1 online; Supplementary Figure S1b (Supplementary Figure S4 online). Most likely owing to the online). Mammalian and nonmammalian SFTPs formed four enormous length of up to 10 kbp and the presence of highly phylogenetic clades (Supplementary Figure S3 online), of repetitive sequence elements (see below), the third exon of the which only the cornulin clade comprises both mammalian Scfn gene of several species was not completely covered by and sauropsidian homologs. By contrast, the scaffoldin clade the available genome sequences. However, the complete was sauropsid-specific and the clades of filaggrin-type proteins coding sequence (8037 nucleotides), with the exception of

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seven unidentified nucleotides, was available for the alligator Scaffoldin and cornulin are expressed in the periderm and skin (Supplementary Figure S1 online). Moreover, we sequenced appendages of chicken B2500 nucleotides on both ends of the Scfn gene of the Next, we determined the expression patterns of SFTP chicken strain, Tetra SL, that we used for further investigations genes in chicken and in the green anole lizard. Reverse of SFTPs (accession number KC700629). There were several transcription–PCR showed that cornulin was expressed in differences in the nucleotide sequence of Scfn in Tetra SL the esophagus of chicken, thus resembling its human ortholog chicken and in the red jungle fowl genome sequence (Xu et al., 2000), whereas scaffoldin was not present in this deposited in the GenBank (International Chicken Genome tissue (Supplementary Figure S7a online). Both cornulin and Sequencing Consortium, 2004), most likely reflecting scaffoldin were expressed in the tongue and, at lower levels, differences in the chicken strains and errors in the assembly in the skin and in the claw-forming part of the toes. By of the whole-genome sequence. Moreover, we performed contrast, tissues that did not contain keratinocytes lacked long-range PCR on chicken genomic DNA and determined the expression of Crnn and Scfn (Supplementary Figure S7a size of the product by agarose gel electrophoresis in online). In the lizard, scaffoldin messenger RNA (mRNA) comparison with DNA length standards. We estimate the was detected only in the tips of the toes, which mostly contain length of the coding sequence of the Scfn gene to be B9,600 the claw-forming epidermis (Supplementary Figure S7b nucleotides. Accordingly, the protein encoded by the chicken online). Scfn gene has B3,200 amino acids and a molecular weight of To localize the expression of SFTP genes in individual cells 385 kDa. The lengths of scaffoldins of the chicken and within tissues, we subjected a set of epithelial tissues of alligator species (2,678 aa) are comparable to that of human chicken to mRNA in situ hybridization with antisense trichohyalin (1,943 aa), whereas all cornulin proteins of the sequence probes specific to the 30-untranslated regions of same three species were in the size range of 380–500 amino Crnn and Scfn. Cornulin was expressed in the suprabasal acids (Figure 1b). epithelial layers of the esophagus (Figure 2a), whereas scaf- foldin mRNA was not detected there (Figure 2b). Suprabasal SFTPs of sauropsids are most similar to mammalian cornulin and epidermal keratinocytes of chicken skin did not express SFTPs; trichohyalin however, both cornulin and scaffoldin mRNAs were detected The protein domains of sauropsid SFTPs contained several in the embryonic periderm (Figure 2c and d). Moreover, the features that are also present in mammalian SFTPs, indicating mRNAs of both SFTPs were present in the differentiating inheritance from common ancestral molecules. In the S100 epidermal cells of the ventral part of the claw apparatus, the domains of sauropsid cornulin and scaffoldin proteins, the subunguis (Figure 2e and f). In the dorsal part of the claw, the amino acid residues implicated in calcium binding (Zimmer unguis, SFTP expression was confined to the periderm, et al., 2013) as well as many residues at other positions were whereas cells giving rise to the mature claw did not express conserved (Supplementary Figure S5 online). Similarly, cornulin nor scaffoldin (Figure 2e and f, and data not shown). sequence elements at the carboxy terminus of SFTPs were The corresponding controls with sense probes did not yield conserved (Supplementary Figure S6 online). In particular, the signals, confirming the specificity of the method (Supple- motif LYxY(L/I/V)QxQ(K/R) was present in diverse, although mentary Figure S8 online). not in all, SFTPs. Between the S100 domain and the conserved carboxy terminus, several sauropsidian scaffoldin proteins con- Scaffoldin is expressed in epithelial scaffoldings of hard skin tained sequence repeats (Supplementary Figure S1c online). appendages Alligator scaffoldin contained 40 moderately conserved To determine the expression pattern of chicken scaffoldin at sequence repeats of 45 amino acids, and a sequence unit of the protein level, we generated a specific antiserum and 37–39 amino acids was repeated B65 times in chicken performed immunochemical analyses (Figure 3). Western blot scaffoldin (Supplementary Figure S1c online). The amino acid analysis revealed a band compatible with the predicted size of composition of the central segment of sauropsidian SFTPs, 385 kDa in embryonic beak, toe, skin, and tongue (Figure 3a), irrespective of the presence or absence of repeats, showed a whereas adult skin as well as muscle were negative (data not clear bias to the presence of a small subset of amino acid shown). In a preparation of the beak of E18 and, to a lesser residues that are also enriched in the corresponding region of extent, also in other samples, at least one additional band with mammalian SFTPs (Figure 1c) (Henry et al., 2012). This domain an apparently higher molecular weight was detected, a feature of scaffoldin proteins was particularly rich in arginine (17% of that is reminiscent of the immunoblot pattern reported for amino acid residues in alligator, 18% in chicken) and glutamic mammalian trichohyalin (Me´chin et al., 2005). Consistently, acid (17% in alligator, 19% in chicken), being most similar to low-molecular-weight proteins, indicative of filaggrin-type trichohyalin among human SFTPs (Figure 1c). The alligator, proteolytic processing (Sandilands et al., 2009), were not chicken, and human cornulin proteins were rich in glutamine detected (Figure 3a). Pre-adsorption of the antiserum with (14%) (Figure 1c). For comparison, the proteins encoded by the peptide used for immunization abolished the high-mole- the entire contain, on average, 5.8% arginine, cular-weight signals, confirming the specificity of the method 6.8% glutamic acid, and 4.6% glutamine (Scherer, 2011). (not shown). Notably, high contents of serine, glycine, and histidine The in situ distribution of the scaffoldin protein was residues were found only in mammalian SFTPs, i.e. filaggrin, determined by immunohistochemical analysis of chicken filaggrin 2, and hornerin. embryonic tissues. Scaffoldin was expressed at high amounts

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Cornulin Scaffoldin

P P SP SP E E

* *

Figure 2. In situ hybridization of cornulin and scaffoldin mRNAs in chicken. The expression of mRNAs of cornulin (a, c, e) and scaffoldin (b, d, f) was analyzed by in situ hybridization of sections through the esophagus of adult chicken (a, b), skin including the periderm of chicken embryos at embryonic day E18 (c, d), and the claw unit of chicken embryos at embryonic day E14 (e, f). Bars ¼ 20 mm(a–d), 200 mm(e, f). E, epidermis; P, periderm; SP, subperiderm. Folded areas of the section in e are indicated by asterisks. The position of the dermo-epidermal junction is marked by a line. The broken lines indicate the borders of the subperiderm. Control stainings with sense probes were negative (Supplementary Figure S8 online).

in the peridermal compartment of the egg tooth on embryonic from the periderm but expression in the upper layers of the day E10 (Figure 3b), in the periderm of embryonic epidermis epidermis of both embryonic and adult mouse skin (Figure3c),inthefeathersheathatE18(Figure3d),atthe (Supplementary Figure S10 online and data not shown). borders of the lingual papillae (Figure 3e), and in the epithelial Cornulin was also expressed in the hyponychium of the cells below the forming tip of the claw (Figure 3f). In the unit, which is the mammalian homolog of the sauropsidian periderm above the egg tooth (Figure 3b, inset), in the feather subunguis (data not shown). Trichohyalin was previously sheath (Figure 3d), and in the subunguis of the claw (Figure 3f, reported to be absent from the periderm but was expressed inset), scaffoldin accumulated in distinct granules, most likely transiently in the suprabasal embryonic epidermis (Lee et al., identical to the so-called periderm granules (Sawyer et al., 1999). Immunohistochemical analysis of the human nail unit 1974; Kuraitis and Bowers, 1978). The specificity of the using an antibody against the granular form of trichohyalin immunoreaction was confirmed by pre-adsorption of the (O’Guin et al., 1992) demonstrated that trichohyalin was antibody with the immunogenic peptide that abolished strictly confined to epithelial cells in a segment of the hypony- staining, whereas pre-adsorption with an unrelated peptide chium, beneath the forming nail tip (Figure 4). In contrast to a had no effect (Supplementary Figure S9 online). previous report (O’Keefe et al., 1993), no expression was detected in the nail matrix and in the epidermis. These results The human nail unit contains trichohyalin at a site homologous were consistently obtained in the nail samples from four to the expression site of chicken scaffoldin different donors. Thus, trichohyalin is specifically expressed As the investigation of sauropsidian SFTPs revealed expression in the epithelium beneath the tip of the nail but not in the in the periderm and in the epithelium flanking the claw epithelial cells that form the nail itself. The conserved tips on the ventral side, we wondered whether the correspond- expression patterns of scaffoldin and trichohyalin in claws, ing sites in mammals also contained SFTPs. Immunohisto- nails, feathers, and hair, as well as in filiform papillae of the chemical investigations suggested the absence of cornulin tongue of chicken and humans, suggest evolutionary ancient

2688 Journal of Investigative Dermatology (2014), Volume 134 V Mlitz et al. Conserved Roles of S100 Fused-Type Proteins Beak Toe Skin Tongue Beak Toe Skin Tongue P kDa 460 238 163 E 71 55 41 31

WB: Scfn Ponceau

SP FS E P

F

CI

Sub

Figure 3. Immunochemical analysis of scaffoldin expression in chicken. Western blot analysis of scaffoldin and Ponceau staining of proteins blotted onto the membrane (a). (b–f) Immunohistochemical staining of scaffoldin (red), indicated by arrows, in the periderm of the egg tooth at E10 (b, inset: periderm granules), in the periderm of leg skin at E18 (c), in the feather sheath at E18 (d), in the tongue of an adult chicken (e), and in the subunguis of the claw apparatus of a 3-day-old chick (f). The position of the dermo-epidermal junction is marked by a line. Broken lines indicate the borders of the subperiderm. Bars ¼ 40 mm (b-d), 300 mm(e, f). Cl, claw; E, epidermis; F, feather; FS, feather sheath; P, periderm; SP, subperiderm; Sub, subunguis; WB, western blot.

and important roles of scaffoldin and trichohyalin in epithelial Besides the S100 domain and the sequence motif in the scaffoldings of growing skin appendages. carboxy-terminal domain, a high content of glutamine in the central domain has apparently been inherited from an DISCUSSION ancestral SFTP. Glutamine is the target of transglutamination The results of this study uncover SFTPs in sauropsids and, during keratinocyte cornification. The content of other amino thereby, imply an evolutionary origin of SFTPs before the acids has greatly diversified during the evolution of SFTPs divergence of mammals and sauropsids B310 million years resulting in glutamic acid– and arginine-rich proteins such ago (Donoghue and Benton, 2007). The comparison of as trichohyalin and scaffoldin on one end and glycine- and nucleotide sequences of SFTP gene promoters (Supplemen- serine-rich proteins such as filaggrin on the other end. Most tary Figure S4 online) and the comparison of amino acid likely, the primordial structure of SFTPs was similar to that of sequences of SFTPs from sauropsids and mammals cornulin, which has an intermediate content of glutamic acid, (Supplementary Figures S5 and S6 online) revealed sequence arginine, glycine, and serine in its central domain (Figure 1c). elements that have been conserved up to the present. The According to this hypothesis, cornulin appears to have conserved features of human SFTP genes and proteins are retained many features of the ancestral SFTP from which other likely to be relevant in skin biology and dermatology, and SFTPs evolved by gene duplication and more pronounced therefore should be further investigated in future studies. sequence modification. Notably, cornulin is expressed in

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a np Gene duplications leading to SFTPs diversification of SFTPs Human nb Crnn, Tchh, Flg, four others Orgin of Crnn Loss of Crnn Mouse hyn Crnn, Tchh, Flg, four others Lizard np , Scfn Python Crnn, Scfn

nm nb hyn Alligator Crnn, Scfn Chicken Crnn, Scfn np bc Frog Fish

nb hyn –400 –300 –200 –100 Million years ago * Figure 5. Schematic overview of S100 fused-type protein (SFTP) evolution. The distribution of SFTPs among extant vertebrates and the inferred gene Figure 4. Immunohistochemical detection of trichohyalin in the human origin and loss events are shown on a phylogenetic tree. The divergence times nail unit. Longitudinal sections of the human nail unit were immunostained of evolutionary lineages are indicated on the timescale at the bottom. with an antibody against human trichohyalin. The immunostaining (red) was Arrows with dotted lines indicate possible origins of the second SFTP gene restricted to distinct cells of the hyponychium (a, b). A schematic overview of either before or after the evolutionary divergence of sauropsids and the nail unit is depicted in the inset of panel (a). The red box in the inset mammals. Additional SFTP gene duplications have occurred in the mammalian corresponds to the area shown in panel a.(b) Shows a detail at higher lineage. magnification. A negative control reaction with an antibody isotype yielded no signal (c). An asterisk marks an artifactual discontinuity caused by the sectioning of the nail. The growth direction of the nail is indicated by arrows (a, c). Bars ¼ 200 mm(a, c), 20 mm(b). hyn, hyponychium; nb, nail bed; nm, gene duplications with subsequent sequence modifications. nail matrix; np, nail plate. However, the origin of an ancestral filaggrin-type gene in stem amniotes (before the separation of the mammalian and sauropsidian lineages) and the subsequent loss of this gene a wider variety of human and murine epithelia than other in sauropsids cannot be excluded. The hypothesis of SFTP loss SFTPs, including the esophagus, the interfollicular epidermis, is exemplified by the presence of cornulin in both mammals the tongue, the inner root sheath of the hair, and the nail and many sauropsids, indicative of an origin in stem amniotes, apparatus (Contzler et al., 2005; Supplementary Figure S10 and absence of cornulin in the lizard (Figure 1a). Further online; Supplementary Figure S11 online, and our unpub- phylogenetic analyses, including molecular dating using SFTP lished data). Similarly, the ancestral cornulin protein may have genes from a large panel of species, may help to define the had a common role in epithelia of several body sites, and times of origin of individual SFTP genes. additional SFTPs with specialized functions and restricted The expression patterns of cornulin and scaffoldin in expression patterns in the skin and its appendages have chicken share important features with those of cornulin and evolved later. trichohyalin in mammals, respectively. Our in situ hybridiza- Scaffoldin is a newly identified sauropsidian SFTP that has tions of chicken cornulin and scaffoldin as well as the an amino acid composition similar to that of mammalian immunostaining of chicken scaffoldin and human trichohyalin trichohyalin. A phyologenetic analysis separated the scaffoldin demonstrate that these SFTPs are strongly expressed in the proteins of the sauropsidian species from the clade of epithelium beneath the forming tip of the claw/nail. This mammalian trichohyalin-type proteins (trichohyalin, tricho- epithelium is known as subunguis in chicken and appears to hyalin-like 1, repetin) as well as from other SFTPs (Supple- be homologous to the proximal hyponychium or nail isthmus mentary Figure S3 online). As the amino acid sequences of the in humans (Perrin, 2007). In the tongue, mammalian tricho- central domains of SFTPs are highly divergent (Supplementary hyalin and cornulin are confined to the anterior side of the Figure S1 online) and carboxy-terminal sequences are incom- filiform papillae, which in many species (including chicken pletely conserved (Supplementary Figure S6 online), the and mouse) form a nail-like tip. Interestingly, scaffoldin is also phylogenetic analysis had to be restricted to the S100 domains expressed in the feather sheath, whereas the mammalian of SFTPs. Considering this limitation, it remains uncertain SFTPs, cornulin, and trichohyalin are expressed in the inner whether the Scfn and Tchh genes have originated by inde- root sheath of the hair (Rothnagel and Rogers, 1986; Contzler pendent duplications of the primordial SFTP gene, or whether et al., 2005). Feathers and hair are structurally different skin there was a single gene duplication that yielded a gene from appendages, which, however, have in common the require- which Scfn and Tchh evolved. The origin of the ancestral Scfn ment for mechanical stabilization or scaffolding by an gene may have occurred either before or after the divergence ensheathing epithelium (Alibardi, 2003; Wu et al., 2004). of the evolutionary lineages that have led to the formation of The comprehensive characterization of the possible roles of sauropsidians and mammals and, accordingly, the most recent scaffoldin in sculpting the complex structures of feathers ancestor of sauropsids and mammals may have had one or (Chang et al., 2004) will require further studies. Interestingly, two SFTP genes (Figure 5). The confinement of filaggrin-type cornulin and scaffoldin are expressed in the periderm, which SFTPs to mammals is likely to be caused by -specific transiently covers the embryonic epidermis of chicken

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(Alibardi, 2009), with particularly high levels of scaffoldin (Accession number KC700626) as well as the adult toe of the lizard being present above the forming egg tooth (Figure 3b), (accession number KC700627) were submitted to the GenBank. whereas SFTPs have not been detected yet in the periderm of mammals. Taken together, SFTPs of sauropsids and some mRNA in situ hybridization SFTPs of mammals are expressed in diverse epithelial struc- The primers Crnn-s1 (50-ACCAGGAGGAACCACAGACA-30), Crnn-a1 tures that support the formation of hard skin appendages such (50-GGCAGAGGCAGCCTAATACA-30), Scfn-s1 (50-CCACTGCAATA as the egg tooth, the filiform papillae of the tongue, nails, TTTAGTCACTTTCC-30), and Scfn-a1 (50-TGAAGAAGAAACCCCAA feathers, and hair. We propose that these SFTP-expressing TGC-30) were used for PCR amplifications of 30-untranslated cells function as scaffolds during the outgrowth of appendages regions of Crnn and Scfn from chicken genomic DNA. The amplicons from the skin. Comparative investigations of SFTPs in phylo- were cloned in sense and antisense orientation into pCR2.1- genetically diverse species may help to further define the TOPO plasmids (Life Technologies, Paisley, UK) and transcribed mechanistic roles of cornulin, scaffoldin, and trichohyalin in in vitro using the DIG RNA labeling kit (Roche Applied Science), epithelial scaffoldings and to identify the ancestral skin which introduces digoxigenin (DIG)-UTP into RNA probes. appendage from which filiform papillae, nails, feathers, and Agarose gel electrophoreses and dot blot assays validated antisense hair have evolved. and sense probes. Hybridization experiments were performed accord- ing to a previously published protocol (Weninger et al.,1996) MATERIALS AND METHODS omitting the RNA digestion step. Antisense and sense probes were Ethics statement applied at concentrations of 5 and 10 ng ml 1,respectively, All animal procedures were approved by the Animal Care and Use at hybridization temperatures of 55 1C(cornulin)and501C Committee of the Medical University of Vienna and conducted (scaffoldin). Only those stainings obtained with antisense probes for according to the guidelines established by the Animal Care and which the corresponding sense probes did not yield to staining were Use Committee of the Medical University of Vienna. Human toe considered specific. samples were obtained from cadavers of individuals who had died of diseases not associated with dystrophies of the nails, as described Generation of antibodies and immunochemical assays previously (Ja¨ger et al., 2007). The study was approved by the Ethics The oligopeptide RYERTREDIAAEAE, corresponding to 14 amino Committee of the Medical University of Vienna and complied with acid residues of the consensus sequence of the repeat unit in the the principles of the Declaration of Helsinki. central domain of chicken scaffoldin, was synthesized by Genecust Europe, Dudelange, Luxembourg and coupled to keyhole limpet Sequence queries, alignments, and phylogenetic analyses hemocyanin. Mice received six injections of 100 mg of the oligopep- Mammalian SFTP sequences were used as queries in tBLASTn tide to generate antisera. Western blot and immunohistochemistry searches in all vertebrate genome sequences available in the with antiserum dilutions of 1:2,000 gave specific signals that were not GenBank (http://www.ncbi.nlm.nih.gov/). Amino acid sequences of obtained with pre-immune sera. The specific immunoreactivity was S100 domains were aligned using CLUSTALW, and a phylogenetic blocked by pre-incubation with antigenic peptide (4 mg peptide per tree was built with PHYLIP using the Jones–Taylor–Thornton matrix as 1 ml antiserum). distance algorithm and the neighbor-joining method for clustering. For western blot experiments, proteins were extracted from Trees were displayed using the TreeView program (Page, 1996). tissues with the Precellys homogenizer (Peqlab) using the NuPAGE LDS Sample buffer (Life Technologies) and electrophoresed Nucleic acid preparation, PCR, and sequence analysis through ExcelGel SDS Gradient 8–18 (GE Healthcare Life Genomic DNA was prepared from chicken and anole lizard samples Sciences, Buckinghamshire, UK). For immunohistochemical ana- according to a standard protocol (Strauss, 2001). A region spanning lyses, tissue samples were fixed with 7.5% formaldehyde and the repeat-rich region of exon 3 of the chicken Scfn gene was embedded in paraffin. Antigen retrieval was performed by incuba- amplified using Expand Long Range enzyme mix (Roche Applied tion in citrate buffer (pH 6.0) for all antigens investigated. Science, Basel, Switzerland) using the primers 50-AGGAAGGCA In addition, some nail sections were treated with protease K CAATCAACCAC-30 and 50-CACGACAAACCTCTGCTTCA-30.PCRs (20 mgml 1)at371C for 20 min before trichohyalin immunohis- of shorter gene segments were performed using DreamTaq DNA tochemistry, which yielded essentially the same results as the polymerase (Thermo Scientific, Waltham, MA). The amplicons were protocol involving antigen retrieval with citrate buffer. The sections purified, sequenced, and the resulting sequences were submitted to were incubated with mouse anti-scaffoldin antibody (1:2,000), the GenBank (accession numbers: KC700629 for chicken scaffoldin mouse anti-trichohyalin antibody clone AE15 (1:500; Santa Cruz and KC700628 for anole lizard scaffoldin). Biotechnology, Dallas, Texas), rabbit anti-cornulin antibody RNA was extracted from homogenized tissues with peqGOLD (1:100; Abcam, Cambridge, UK) or rabbit anti-keratin K6 antibody TriFast (Peqlab, Erlangen, Germany) and reverse transcribed using (1:500; Covance, Princeton, NJ). Biotinylated sheep anti-mouse Ig iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to (1:200; GE, Chalfont, UK) and goat anti-rabbit Ig (1:100; Vector the manufacturers’ instructions. Reverse transcription–PCR was per- Laboratories, Burlingame, CA) were used as secondary antibodies. formed using DreamTaq DNA polymerase (Thermo Scientific) with Sheep or goat serum (10%) was added to the secondary antibody to the primers listed in Supplementary Table S2 online. The sequence of prevent unspecific binding. The sections were then incubated with a cornulin cDNA derived from the embryonic tongue of chicken the streptavidin–biotin–horseradish peroxidase complex and (accession numbers GQ149127 and KC700625) and the sequence of 3-amino-9-ethylcarbazole (DakoCytomation, Glostrup, Denmark), scaffoldin cDNA derived from the embryonic skin of chicken and counterstained with hematoxylin.

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CONFLICT OF INTEREST Kypriotou M, Huber M, Hohl D (2012) The human epidermal differentiation The authors state no conflict of interest. complex:cornified envelope precursors, S100 proteins and the ‘fused genes’ family. Exp Dermatol 21:643–9 ACKNOWLEDGMENTS Lee SC, Lee JB, Kook JP et al. (1999) Expression of differentiation markers We thank Michael Mildner, Heinz Fischer, Wolfgang Sipos, and Lutz Langbein during fetal skin development in humans: immunohistochemical studies for helpful discussions and Susanne Karner for excellent technical support. The on the precursor proteins forming the cornified cell envelope. J Investig authors acknowledge all providers of public genome sequences that were used Dermatol Symp Proc 112:882–6 in the present study. This work was supported by the Austrian Science Fund (FWF): P23801 grant to LE, by the European Cooperation in Science and Liede´n A, Ekelund E, Kuo IC et al. (2009) Cornulin, a marker of late epidermal Technology (COST) Action BM0903 (Skin Barrier in Atopic Diseases, Skin- differentiation, is down-regulated in eczema. Allergy 64:304–11 BAD), and by a research grant from CE.R.I.E.S., Neuilly, France. Markova NG, Marekov LN, Chipev CC et al. (1993) Profilaggrin is a major epidermal calcium-binding protein. Mol Cell Biol 13:613–25 Medland SE, Nyholt DR, Painter JN et al. (2009) Common variants in the SUPPLEMENTARY MATERIAL trichohyalin gene are associated with straight hair in Europeans. Am J Supplementary material is linked to the online version of the paper at http:// Hum Genet 85:750–5 www.nature.com/jid Me´chin MC, Enji M, Nachat R et al. (2005) The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and REFERENCES subcellular locations. 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