Genomic Organization, Transcriptomic Analysis, and Functional Characterization of Avian α- and β-Keratins in Diverse Feather Forms

Chen Siang Ng 2 y Ping Wu 1 y Wen-Lang Fan 2 Jie Yan 0 1 Chih-Kuan Chen 2 6 Yu-Ting Lai 2 Siao-Man Wu 2 Chi-Tang Mao 2 4 5 Jun-Jie Chen 2 Mei-Yeh Jade Lu 2 Meng-Ru Ho 2 Randall B. Widelitz 1 Chih-Feng Chen 3 8 Cheng-Ming Chuong 1 8 Wen-Hsiung Li 2 7 0 Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University , China 1 Department of Pathology, Keck School of Medicine, University of Southern California 2 Biodiversity Research Center , Academia Sinica, Taipei, Taiwan 3 Department of Animal Science, National Chung Hsing University , Taichung, Taiwan 4 Graduate Institute of Biotechnology, National Chung Hsing University , Taichung, Taiwan 5 Molecular Biology of Agricultural Sciences, Taiwan International Graduate Program , Academia Sinica, Taipei, Taiwan 6 Institute of Ecology and Evolutionary Biology, National Taiwan University , Taipei, Taiwan 7 Department of Ecology and Evolution, University of Chicago 8 Center for the Integrative and Evolutionary Galliformes Genomics (iEGG Center), National Chung Hsing University , Taichung, Taiwan Feathers are hallmark avian integument appendages, although they were also present on theropods. They are composed of flexible corneous materials made of a- and b-keratins, but their genomic organization and their functional roles in feathers have not been well studied. First, we made an exhaustive search of a- and b-keratin genes in the new chicken genome assembly (Galgal4). Then, using transcriptomic analysis, we studied a- and b-keratin gene expression patterns in five types of feather epidermis. The expression patterns of b-keratin genes were different in different feather types, whereas those of a-keratin genes were less variable. In addition, we obtained extensive a- and b-keratin mRNA in situ hybridization data, showing that a-keratins and b-keratins are preferentially expressed in different parts of the feather components. Together, our data suggest that feather morphological and structural diversity can largely be attributed to differential combinations of a- and b-keratin genes in different intrafeather regions and/or feather types from different body parts. The expression profiles provide new insights into the evolutionary origin and diversification of feathers. Finally, functional analysis using mutant chicken keratin forms based on those found in the human a-keratin mutation database led to abnormal phenotypes. This demonstrates that the chicken can be a convenient model for studying the molecular biology of human keratin-based diseases. Introduction For birds, feathers play a crucial role in heat retention, mate attraction, protection, flight, etc. Feathers can have such diverse functions because they form different structures to adapt to functional needs in different body parts or at The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. different times of their life (Chuong et al. 2012). There are specific feather types in different body regions, and there are different branching morphologies in different parts of the same feather (Lin et al. 2013). The feather is a unique morphological innovation which might have originated from modifications of reptilian scales (Greenwold and Sawyer 2010) and evolved in nonavian dinosaurs and basal birds (Prum and Brush 2002; Wu et al. 2004; Xu et al. 2010). The successful diversification of feather forms presumably has contributed significantly to the rapid and extensive radiation of birds to become the dominant terrestrial vertebrate. The major components of feathers are a- and b-keratins, which are encoded by multigene families (Alibardi and Toni 2008). The emergence of novel, lineage-specific morphological features can be attributed to expansion of these gene families (Conant and Wolfe 2008). This has been proposed as a critical evolutionary mechanism that drives molecular diversity (Ohno 1970). For instance, the independent origin of hair and nails in mammals and baleen in whales might have been led by the expansion of a-keratin genes (Vandebergh and Bossuyt 2012). Large-scale expansions of b-keratin genes in birds and turtles were proposed to be associated with the innovation of the feather and turtle shell (Greenwold and Sawyer 2010; Li et al. 2013). In birds, five b-keratin gene subfamilies (claw, feather, feather-like, keratinocyte, and scale) have been classified by sequence heterogeneity and tissue-specific expression (Presland et al. 1989; Presland, Whitbread, et al. 1989; Whitbread et al. 1991; Greenwold and Sawyer 2010). Previous genome-wide comparative analyses in zebra finch and chicken identified several clusters of b-keratin genes; the largest two are on chromosomes 25 (Chr25) and 27 (Chr27) (Greenwold and Sawyer 2010). The acquisition of new b-keratin genes in birds was most likely correlated with functional diversification of these genes. New b-keratin genes in the expanded b-keratin multigene family might have been selected for novel functions in evolved skin appendages such as the feather of birds and the plastron and carapace of turtles. However, mapping the keratin genes within the avian genome has been extremely challenging due to the high similarity between duplicated genes. Although the expansion and radiation of the avian b-keratin genes could have contributed to the evolution of feathers and the diversification of birds, little work has been carried out to characterize their expression profiles in different feather parts and types. Coordinated expression of the acidic and basic keratins, which are encoded by the Type I and Type II a-keratin gene clusters, is also essential for skin appendage development. Characterization of the genomic organization is helpful for understanding the evolution and regulation of a- and bkeratin genes. Knowledge of the timing and tissue expression of copious a- and b-keratin genes would allow us to associate feather shape with the specific keratins produced to form the ramus, barbules, rachis, and calamus in various feather types. The availability of transcriptomic analysis tools and avian whole-genome sequences provides an excellent opportunity to study evolutionary processes and gene expression patterns that potentially account for morphological variations. In this study, we aim to identify a- and b-keratin genes involved in the formation of different types of feathers at different developmental stages. We search for and annotate the a- and b-keratin sequences in the new chicken genome assembly, and analyze the expression profiles of the a- and b-keratins during the development of different feather types by RNA-seq and by in situ hybri (...truncated)