Trichohyalin-like proteins have evolutionarily conserved roles in the morphogenesis of skin appendages

Abstract

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid 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 mammals. 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 epithelium 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 root sheath of hair. 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.

Fig. 1. Identification and phylogenetic analysis non-mammalian genes encoding S100 fused-type proteins (SFTPs)

(a) The chromosomal loci containing the SFTP genes of different species are depicted. Genes are represented by arrows pointing from the 5′ to the 3′-end. The genes flanking the SFTPs on the left side have not been assigned a name yet with the exception of LCE5A (late cornified envelope 5A); they are provisionally denoted here as EDC1 (epidermal differentiation complex gene 1). (b) Scheme of protein domains and sizes. Protein segments containing sequence repeats are indicated in dark grey. Lengths are indicated by numbers of amino acid residues (aa). The proteins are not drawn to scale. (c) Amino acid composition of the central domain of SFTPs. Differently colored portions of each column indicate the percent of total amino acid residues present in the central domain. A.c., Anolis carolinensis; A.m., Alligator mississippiensis; G.g., Gallus gallus; H.s., Homo sapiens.

Fig. 2. In situ hybridization of cornulin and scaffoldin mRNAs in the 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: 20 μm (a-d), 200 μm (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 (Suppl. Figure S8).

Fig. 3. Immunochemical analysis of scaffoldin expression in the 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: 40 μm (b-d). 300 μm (e and f). Cl, claw; E, epidermis; F, feather; FS, feather sheath; kDa, kilodalton; P, periderm; SP, subperiderm; Sub, subunguis; WB, Western blot.

Fig. 4. Immunohistochemical detection of trichohyalin in the human nail unit

Longitudinal sections of the human nail unit were immunostained with an antibody against human trichohyalin. The immunostaining (red) was restricted to distinct cells of the hyponychium (a, b). A schematic overview of the nail unit is depicted in the inset of panel (a). The red box in the inset corresponds to the area shown in panel (a). Panel (b) shows a detail at higher magnification. A negative control reaction with an antibody isotype yielded no signal (c). An asterisk marks an artefactual discontinuity caused by the sectioning of the nail. The growth direction of the nail is indicated by arrows (a and c). Bars: 200 μm (a, c), 20 μm (b). hyn, hyponychium; nb, nail bed; nm, nail matrix; np, nail plate.

Fig. 5. Schematic overview of S100 fused-type protein (SFTP) evolution

The distribution of SFTPs among extant vertebrates and the inferred gene origin and loss events are shown on a phylogenetic tree. The divergence times of evolutionary lineages are indicated on the timescale at the bottom. Arrows with dotted lines indicate possible origins of the second SFTP gene either before or after the evolutionary divergence of sauropsids and mammals. Additional SFTP gene duplications have occurred in the mammalian lineage.