Convergent evolution of cysteine-rich proteins in feathers and hair

Abstract

Background: Feathers and hair consist of cornified epidermal keratinocytes in which proteins are crosslinked via disulfide bonds between cysteine residues of structural proteins to establish mechanical resilience. Cysteine-rich keratin-associated proteins (KRTAPs) are important components of hair whereas the molecular components of feathers have remained incompletely known. Recently, we have identified a chicken gene, named epidermal differentiation cysteine-rich protein (EDCRP), that encodes a protein with a cysteine content of 36%. Here we have investigated the putative role of EDCRP in the molecular architecture and evolution of feathers.

Results: Comparative genomics showed that the presence of an EDCRP gene and the high cysteine content of the encoded proteins are conserved among birds. Avian EDCRPs contain a species-specific number of sequence repeats with the consensus sequence CCDPCQ(K/Q)(S/P)V, thus resembling mammalian cysteine-rich KRTAPs which also contain sequence repeats of similar sequence. However, differences in gene loci and exon-intron structures suggest that EDCRP and KRTAPs have not evolved from a common gene ancestor but represent the products of convergent sequence evolution. mRNA in situ hybridization demonstrated that chicken EDCRP is expressed in the subperiderm layer of the embryonic epidermis and in the barbule cells of growing feathers. This expression pattern supports the hypothesis that feathers are evolutionarily derived from the subperiderm.

Conclusions: The results of this study suggest that convergent sequence evolution of avian EDCRP and mammalian KRTAPs has contributed to independent evolution of feathers and hair, respectively.

Figure 1

EDCRP is expressed in the skin containing feather follicles and in late embryonic skin of the chicken. (A) Schematic depiction of the chicken EDCRP gene. White boxes indicate exons, and grey shading marks the coding region. Arrows indicate the position primer annealing sites for the amplification of cDNAs. (B) Skin from the wings, containing feather follicles (+), and skin from the lower part of the legs, lacking feather follicles (−), was prepared from embryonic (days E10, E14 and E18) and adult chicken. RNA was extracted and reverse-transcribed to cDNA, which was subjected to PCRs specific for EDCRP and a control gene (caspase-3). PCR without template cDNA was performed as negative control reaction.

Figure 2

EDCRP is expressed in the subperiderm and the feathers of the chicken. Leg skin on embryonic day E18 (A, B) and growing feathers (E18) in longitudinal sections (C, D) and cross-sections (E-H) were subjected to in situ hybridization with either anti-sense probes specific for EDCRP mRNA (A, C, E, G) or sense probes as negative controls (B, D, F, H). bl, barbules; der, dermis; epi, epidermis; me, medulla of a barb ridge; per, periderm; sh, feather sheath; sub, subperiderm; pu, pulp. Size bars, 40 μm (A, B, E, F, G, H), 20 μm (C, D).

Figure 3

Embryonic epidermal stratification and expression of EDCRP are maintained in feather follicles. (A) The skin layers during embryonic development and within a feather follicle are shown schematically. The topology of the layers is maintained. The permanent structures of feathers are made by cells that correspond to the embryonic subperiderm whereas other cells of the feather follicle degenerate during the feather morphogenesis process. The development of feathers within the feather follicle is repeated in the adult animal. Note that the cross-section through the tubular feather follicle shows feather elements (red squares) that are connected by barbs (indicated by light red color) located outside of the plane of section. B) Three-dimensional depiction of the topology of epithelial layers in the feather follicle. The colors show equivalence of the feather follicle layers to the embryonic skin layers in panel A.

Figure 4

Avian EDCRPs contain conserved sequences at the amino-terminus and the carboxy-terminus as well as a variable number of conserved sequence repeats in the central segment. Amino acid sequences of epidermal differentiation cysteine-rich protein (EDCRP) from various bird species were aligned. Vertical lines separate the amino-terminus, the repeats of the central region and the carboxy-terminus. Hyphens were introduced to optimize the alignment. Color shading highlights the amino acid residues C, K, P and Q, which are assumed to be important for the function of the protein (see main text). x, amino acid residue missing because of gaps in genome sequences. Species: Adélie penguin (Pygoscelis adeliae), canary (Serinus canaria), chicken (Gallus gallus), duck (Anas platyrhynchos), egret (Egretta garzetta), emperor penguin (Aptenodytes forsteri), falcon (Falco cherrug), flycatcher (Ficedula albicollis), loon (Gavia stellata), mesite (Mesitornis unicolor), pigeon (Columba livia), ostrich (Struthio camelus australis), tinamou (Tinamus guttatus), zebra finch (Taeniopygia guttata).

Figure 5

Amino acid sequence alignment of avian EDCRPs, lizard EDCRP and representative human cysteine-rich KRTAPs. Sequence similarity is indicated by the following symbols: “*”, conserved in 5/5 proteins; “:”, conserved in 4/5 proteins; “.”, conserved in 3/5 proteins. Color shading highlights the amino acid residues C, K, P and Q, which are assumed to be important for the function of the protein (see main text). Species: chicken (Gallus gallus), human (Homo sapiens), lizard (Anolis carolinensis), pigeon (Columba livia). KRTAP, keratin-associated protein.

Figure 6

Differences in exon-intron structures and gene loci argue against common ancestry of EDCRP and KRTAPs. The exon-intron structures of EDCRP and KRTAP genes as well as their locations in the EDC and in the type 1 keratin gene cluster, respectively, are depicted schematically. Grey shading marks the coding region within exons. The inferred gene composition of ancestral genomes is shown below the schematics of the modern genomes. It remains uncertain whether the ancestral gene of avian and lizard EDCRPs was already cysteine (C)-rich.

Figure 7

Scenario for the evolution of cysteine-rich proteins, feathers and hair. The putative origins of the epidermal differentiation complex (EDC), the subperiderm, avian and lizard EDCRPs as well as KRTAPs are indicated by vertical arrows on a schematic phylogenetic tree of vertebrates.