Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and mRNAs during feather and scale development and has produced a highly diverse, but manageable list of miRNA-mRNA duplexes for future validation experiments.

Keywords: ALDH1A3 and FGF20; Chicken; Evolution; Feather; Genome; MicroRNA; Scale; Tenascin C; β-Keratin.

Fig. 1

(a) Graphically displays the number of differentially expressed miRNA genes for all 14 comparison groups. (b) Illustrates a hierarchical clustering dendrogram of the biological replicates grouped by their respective microarray sample and based on the normalized intensity values of mRNA genes using the Euclidean similarity measure.

Fig. 2

Displays the probable birth of chicken miRNA genes across the tetrapod phylogeny (Jarvis et al., 2014; Crawford et al., 2015) based on the presence or absence of the miRNA chicken genes in genome searches (see also Supplementary file 2 for detailed searched results). The numbers for each broad taxonomic classification (Tetrapoda, Amniota, Sauria etc.) are the accumulative number of chicken miRNA genes found in the most recent common ancestor. The red number next to the clade names are the number of miRNA genes that are not found in that clade but are found in the hierarchical most recent common ancestor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

(a) Illustrates the nucleotide alignment of Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3) sequences from 45 tetrapod species of which 37 are avian species. The dashed black rectangle highlights the miRNA target site conserved in birds to the exclusion of other tetrapods. (b) Illustrates the nucleotide alignment of three chicken Tenascin C (TNC) alternative splice variants. The dashed black rectangle highlights the miRNA target site that is conserved in two of the three splice variants. (c) Illustrates the nucleotide alignment of 13 chicken claw β-keratins from the two claw β-keratin clades (Greenwold et al., 2014; Zhang et al., 2014). The dashed black rectanglehighlights the miRNA target site conserved in the clade 2 claw β-keratins.