Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules

RESEARCH ARTICLE Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules a11111 Lorenzo Alibardi1, Karin Brigit Holthaus1,2, Supawadee Sukseree2, Marcela Hermann3, Erwin Tschachler2, Leopold Eckhart2* 1 Comparative Histolab and Dipartimento di Scienze Biologiche, Geologiche ed Ambientali (BiGeA), University of Bologna, Bologna, Italy, 2 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 3 Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria * OPEN ACCESS Citation: Alibardi L, Holthaus KB, Sukseree S, Hermann M, Tschachler E, Eckhart L (2016) Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules. PLoS ONE 11(12): e0167789. doi:10.1371/journal.pone.0167789 Editor: Michel Simon, INSERM, FRANCE Received: September 17, 2016 Accepted: November 21, 2016 Published: December 9, 2016 Copyright: © 2016 Alibardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The study was supported by the Austrian Science Fund (FWF): P23801 (LE) and Austrian Science Fund (FWF): P28004 (LE) (https://www. fwf.ac.at/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The morphogenesis of feathers is a complex process that depends on a tight spatiotemporal regulation of gene expression and assembly of the protein components of mature feathers. Recent comparative genomics and gene transcription studies have indicated that genes within the epidermal differentiation complex (EDC) encode numerous structural proteins of cornifying skin cells in amniotes including birds. Here, we determined the localization of one of these proteins, termed EDMTFH (Epidermal Differentiation Protein starting with a MTF motif and rich in Histidine), which belongs to a group of EDC-encoded proteins rich in aromatic amino acid residues. We raised an antibody against an EDMTFH-specific epitope and performed immunohistochemical investigations by light microscopy and immunogold labeling by electron microscopy of chicken embryos at days 14–18 of development. EDMTFH was specifically present in the subperiderm, a transient layer of the embryonic epidermis, and in barbs and barbules of feathers. In the latter, it partially localized to bundles of socalled feather beta-keratins (corneous beta-proteins, CBPs). Cells of the embryonic periderm, the epidermis proper, and the feather sheath were immunonegative for EDMTFH. The results of this study indicate that EDMTFH may contribute to the unique mechanical properties of feathers and define EDMTFH as a common marker of the subperiderm and the feather barbules. This expression pattern of EDMTFH resembles that of epidermal differentiation cysteine-rich protein (EDCRP) and feather CBPs and is in accordance with the hypothesis that a major part of the cyclically regenerating feather follicle is topologically, developmentally and evolutionarily related to the embryonic subperiderm. PLOS ONE | DOI:10.1371/journal.pone.0167789 December 9, 2016 1 / 16 An Epidermal Differentiation Protein of Subperiderm and Feathers Competing Interests: The authors have declared that no competing interests exist. Introduction The cornified skin barrier of amniotes and cornified skin appendages such as claws, hair, and feathers are formed by epidermal keratinocytes that differentiate by inducing the expression of specific sets of genes [1–4]. The stratification of the epidermis begins in the embryo and involves the establishment of the periderm as the superficial layer in all amniotes and the formation of a subperiderm in archosaurs [5–10]. Periderm and subperiderm are shed during late development when a mature cornified layer (stratum corneum) has been established by the definitive epidermis. During adult life the cornified epidermis provides the essential protection against water loss and mechanical stress whereas cornified skin appendages serve various functions including, but not limited to, grasping (claws), thermoinsulation (hair, feathers), and facilitating flight (feathers). Many of the structural components of cornifying keratinocytes are encoded in a gene cluster termed the Epidermal Differentiation Complex (EDC). In humans and other mammals, the EDC comprises genes for proteins that interact with each other to form, via transglutamination, a cornified cell envelope or with the keratin intermediate filaments during compaction of the cytoskeleton [11, 12]. Recent studies have shown that non-mammalian amniotes also have an EDC in which both orthologs of human EDC genes, such as loricrin and cornulin, and clade-specific genes which, for example, code for proteins of the scutes of turtle or the feathers of birds are located [13–15]. Proteins traditionally termed beta-keratins [16–18] but now identified as Corneous Beta Proteins (CBPs) represent a major sub-cluster of EDC genes in sauropsids while they are absent in mammals [8, 19]. These proteins of a molecular mass typically in the range of 10–18 kDa possess a characteristic central region (with a most highly conserved stretch of 34 amino acid residues) that folds into an anti-parallel beta-sheet and facilitates the formation of CBP filaments of 3–4 nm thickness [20, 21]. The intra- and intermolecular interactions of sub-domains and sequence motifs in other avian EDC proteins have remained unknown so far. Conserved sequence motifs at the amino- and carboxy-terminus of EDC proteins are likely sites of transglutamination whereas an extremely high cysteine content of epidermal differentation cysteine-rich protein (EDCRP) has been proposed to form multiple disulfide bonds that may contribute to the mechanical strengthening of feathers [22]. Labeling with tritiated histidine and autoradiography suggested that histidine-containing proteins are present in the cytoplasm and in corneous bundles of barbules, however, the identity of these protein(s) was not determined in that study [23]. The morphogenesis and maturation of feathers depends on a complex spatio-temporal cell differentiation program in which EDC-encoded and non-EDC-encoded proteins form the body of the feather whereas other proteins regulate the scaffolding function and programmed cell death of intermediate cells [14, 24–27]. Similarities in the topology and gene expression profil (...truncated)