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. 2015 Dec 8;112(49):E6770-9.
doi: 10.1073/pnas.1520566112. Epub 2015 Nov 23.

Topographical mapping of α- and β-keratins on developing chicken skin integuments: Functional interaction and evolutionary perspectives

Ping Wu  1 , Chen Siang Ng  2 , Jie Yan  3 , Yung-Chih Lai  4 , Chih-Kuan Chen  5 , Yu-Ting Lai  2 , Siao-Man Wu  2 , Jiun-Jie Chen  2 , Weiqi Luo  1 , Randall B Widelitz  1 , Wen-Hsiung Li  6 , Cheng-Ming Chuong  7
Affiliations

Topographical mapping of α- and β-keratins on developing chicken skin integuments: Functional interaction and evolutionary perspectives

Ping Wu et al. Proc Natl Acad Sci U S A. .

Abstract

Avian integumentary organs include feathers, scales, claws, and beaks. They cover the body surface and play various functions to help adapt birds to diverse environments. These keratinized structures are mainly composed of corneous materials made of α-keratins, which exist in all vertebrates, and β-keratins, which only exist in birds and reptiles. Here, members of the keratin gene families were used to study how gene family evolution contributes to novelty and adaptation, focusing on tissue morphogenesis. Using chicken as a model, we applied RNA-seq and in situ hybridization to map α- and β-keratin genes in various skin appendages at embryonic developmental stages. The data demonstrate that temporal and spatial α- and β-keratin expression is involved in establishing the diversity of skin appendage phenotypes. Embryonic feathers express a higher proportion of β-keratin genes than other skin regions. In feather filament morphogenesis, β-keratins show intricate complexity in diverse substructures of feather branches. To explore functional interactions, we used a retrovirus transgenic system to ectopically express mutant α- or antisense β-keratin forms. α- and β-keratins show mutual dependence and mutations in either keratin type results in disrupted keratin networks and failure to form proper feather branches. Our data suggest that combinations of α- and β-keratin genes contribute to the morphological and structural diversity of different avian skin appendages, with feather-β-keratins conferring more possible composites in building intrafeather architecture complexity, setting up a platform of morphological evolution of functional forms in feathers.

Keywords: Evo-Devo; beak; claw; feather; scale; skin appendage.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of avian skin appendages and RNA-seq analysis. (A–E) Schematic drawing (Left) and common β-keratin in situ hybridization (Right) of E16 embryonic skin appendages. (A) Beak. (B) Feather. (C) Scutate scale. (D) Reticulate scale. (E) Claw. Red arrow in A indicates expression of β-keratin in the oral epidermis. Green dotted line in B indicates the barb ridge. (F and G) Multidimensional scaling (MDS) plot of RNA-seq samples for α-keratin genes (F) and β-keratin genes (G). Similarities of gene expression patterns were calculated and mapped for E14 beaks, E14 feathers, E14 scutate scales, E14 reticulate scales, E14 claws, E16 scutate scales, and E16 reticulate scales. (H) Hierarchical clustering of β-keratin gene expression profile inferred from RNA-seq data; Bottom shows enriched subfamilies in each cluster. AP, axial plate; Bc, barb cortex; Bm, barb medulla; BP, barb plate; BR, barb ridge; Et, egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB; lower beak; MP, marginal plate; OS, outer surface; PD, periderm; PI, Phalange I; PM, periderm above the egg tooth; PP, pulp; RM, ramus; S, surface; Su, subunguis; UB, upper beak; Ug, unguis.
Fig. S1.
Fig. S1.
The structure of different chicken skin appendages. (A) Bright field view of skin appendages at E16. (B–F) Schematic drawing of embryonic skin appendages and the layers that comprise them. (B) Beak. (C) Feather. (D) Scutate scale. (E) Reticulate scale. (F) Claw. AP, axial plate; Bc, barb cortex; Bm, barb medulla; BP, barb plate; BR, barb ridge; Et, egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB; lower beak; MP, marginal plate; OS, outer surface; PD, periderm; PI, Phalange I; PM, periderm above the egg tooth; PP, pulp; RM, ramus; S, surface; SB, stratum basal; SC, stratum corneum; SI, stratum intermedium; SP, subperiderm; Su, subunguis; UB, upper beak; Ug, unguis.
Fig. S2.
Fig. S2.
The expression of common β-keratin transcripts in E12–E16 skin appendages. H&E (Left) and β-keratin in situ hybridization (Middle and Right, Right has higher magnification). E12, Upper; E14, Middle; E16, Lower. (A) Beak. (B) Feather. (C) Scutate scale. (D) Reticulate scale. (E) Claw. Black dotted line in A and C–E indicate the basement membrane. Green dotted line in B indicates the barb ridge. Black arrows in B indicate the barb plate. Red arrow in A indicates the oral epidermis. Red dotted circle in B indicates the ramus forming region. BP, barb plate; BR, barb ridge; Et, egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB; lower beak; OS, outer surface; PP, pulp; RM, ramus; S, surface; Su, subunguis; UB, upper beak; Ug, unguis.
Fig. S3.
Fig. S3.
RNA-seq analysis of chicken skin in different regions and developmental time. (A) PCA plot of RNA-seq samples for all genes. Similarities of gene expression patterns were calculated and mapped for E14 beaks, E14 feathers, E14 scutate scales, E14 reticulate scales, E14 claws, E16 scutate scales, and E16 reticulate scales. (B) Hierarchical clustering of α-keratin gene expression profile inferred from RNA-seq data.
Fig. 2.
Fig. 2.
In situ hybridization of α-keratin transcripts in different skin appendages at E16. Type I α-keratin genes KRT13A (A) and KRT14 (B). Type II α-keratin genes KRT75A (C) and KRT5 (D). Insets in A–D are higher magnification views of the indicated area. Black dotted line indicates the basement membrane. Green dotted line indicates the barb ridge. (E) Summary of expression of four α-keratin genes in different skin appendages. In the same feather cross-section, we used three barb ridges to present the expression of each α-keratin. To demonstrate the spatial difference in other skin appendages, we distinguished between the scutate scale outer surface and the inner surface, between the reticulate scale surface and hinge, and between the claw unguis and subunguis. Colored blocks indicate the positive epidermis layer. Light blue, KRT13; blue, KRT14; green, KRT75A; light green, KRT5. Et; egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB, lower beak, OS, outer surface; PD, periderm; S, surface; SB, stratum basal; SC, stratum corneum; SI, stratum intermedium; SP, subperiderm; Su, subunguis; UB; upper beak, Ug, unguis.
Fig. S4.
Fig. S4.
In situ hybridization of four representative α-keratin transcripts in different skin appendages at E14 and E16. Type I α-keratin, KRT13A (A); KRT14 (B). Type II α-keratin, KRT75A (C); KRT5 (D). (Upper) E14. (Lower) E16. Insets are higher magnification views of the indicated area. Black dotted line indicates the basement membrane. Et; egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB, lower beak, OS, outer surface; S, surface; Su, subunguis; UB; upper beak, Ug, unguis.
Fig. 3.
Fig. 3.
In situ hybridization of five β-keratin transcripts encoded on Chr25 and Chr27 in different skin appendages at E16. (A) Chr25-Claw9. (B) Chr25-FK12. (C) Chr25-Scale18. (D) Chr25-Ktn13. Chr27-FK12 (E). Insets in A–E are higher magnification views of the indicated area. Arrows in C indicate the expression of the Scale18 gene in the lower beak inner-oral epidermis and subunguis. Black dotted line indicates the basement membrane. Green dotted line indicates the barb ridge. (F) Summary of the expression patterns of five β-keratin genes in different skin appendages. In the same feather cross-section, we used three barb ridges to present the expression of each β-keratin gene. To demonstrate the spatial difference in other skin appendages, we distinguished between the scutate scale outer surface and inner surface, between the reticulate scale surface and hinge, and between the claw unguis and subunguis. Colored blocks indicate the positive epidermis layer. Yellow, Chr25-Claw9; pink, Chr25-FK12; red, Chr25-Scale18; orange, Chr25-Ktn13; brown, Chr27-Fk12. Et; egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB, lower beak, OS, outer surface; PD, periderm; S, surface; SB, stratum basal; SC, stratum corneum; SI, stratum intermedium; SP, subperiderm; Su, subunguis; UB; upper beak, Ug, unguis.
Fig. S5.
Fig. S5.
In situ hybridization of five representative β-keratin transcripts encoded on Chr25 and Chr27 among different skin appendages at E14 and E16. (A) Chr25-Claw9. (B) Chr25-FK12. (C) Chr25-Scale18. (D) Chr25-F. (E) Chr27-FK12. Arrows in C indicate the expression of Scale18 in lower beak inner-oral epidermis and subunguis. (Upper) E14. (Lower) E16. Insets are higher magnification views of the indicated area. Black dotted line indicates the basement membrane. Et; egg tooth; FS, feather sheath; Hg, hinge; IS, inner surface; LB, lower beak, OS, outer surface; S, surface; Su, subunguis; UB; upper beak, Ug, unguis.
Fig. 4.
Fig. 4.
Functional study shown by overexpressing α-keratins KRT5 mutant and feather β-keratin antisense form in embryonic feather development. The interdependence of α- and β-keratins in forming proper keratin network is shown by functional perturbation experiment during feather regeneration. (A–C) Bright field view of feather filament at E15. Black arrow indicates abnormal development. (D–F) H&E staining of cross-sections. Red arrow indicates the enlarged ramus. Blue arrow indicates the abnormal barb ridge without clear ramus. (G–L) SISH. (G–I) Common β-keratin staining. Green arrow indicates the β-keratin expression domain surrounding the enlarged ramus. Yellow arrow indicates the smaller β-keratin negative domain. (J–L) Common type-II α-keratin staining. Purple arrow indicates the expression of type II α-keratin, which surrounds the enlarged ramus. Black arrow indicates the abnormal expression of type II α-keratin. Dotted green lines indicate a barb ridge. (M–O) Confocal microscopy of double staining for β-keratin (green) and KRT75 (red). (A, D, G, J, and M) RCAS-GFP control. (B, E, H, K, and N) KRT5 mutant form 3. (C, F, I, L, and O) Feather keratin 8 antisense form. BP, barb plate; RM, ramus.
Fig. 5.
Fig. 5.
Summary of topographic expression patterns of α- and β-keratin genes in different skin appendages. (A) Regional differences among different skin appendages. Each line indicates the expression of keratin genes in certain appendages. The missing line indicates the negative or undetectable RNA expression. (B) Intraappendage differences of keratin expression. We only show the differentially expressed keratin genes in B. Colors represent the indicated keratin genes. (C) β-keratin gene arrangements on Chr25 and Chr27. The marked genes are used in β-keratin in situ hybridization. Claw, claw keratin; FK, feather keratin; FL, feather-like keratin; Ktn, keratinocyte keratin; Scale, scale keratin.
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