Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

Abstract

Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2B and WNT16), BMP (BMP3, BMP4, and BMP7), EDAR (EDAR and EDARADD), and FGF (FGFR2 and FGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-β, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

Keywords: expression pattern; induction; long non-coding RNA; skin development; wool follicle.

FIGURE 1

Identification of long non-coding RNAs (lncRNAs) and mRNAs involved in primary wool follicle induction stages in carpet wool sheep fetal skins. (A,B) The morphology of skin and wool follicles at stages 0 and 1 of primary wool follicle development were observed by hematoxylin and eosin (H&E) staining (Left) are 200×, scale bars represent 100 μm. (Right) are 400×, scale bars represent 50 μm, which is the magnification of the segment in the red frame). Epi, epidermis, Der, dermis, Pc, placode, Dc, dermal condensation. At stage 1 (B), the hair placode and dermal condensation were clearly detected compared to the homogeneous epidermal layers at stage (A). (C) Coding potentiality filter using Coding Potential Calculator, Pfam, Phylogenetic codon substitution frequency, and Coding-Non-Coding Index. (D) Exon number distribution of lncRNAs and mRNAs. (E) Transcript lengths distribution of lncRNAs and mRNAs. (F) Open reading frame length distribution of lncRNAs and mRNAs. (G) Conservation of the sequence in lncRNAs and mRNAs was analyzed using Phast (v1.3) software. (H) Differentially expressed transcripts in sheep skin between the two stages of primary wool follicle development. Red, green, and blue dots in the graph represent transcripts that were significantly upregulated, significantly downregulated, and not significantly changed between these two stages, respectively.

FIGURE 2

Gene Ontology (GO) analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 enrichment biological processes for differentially expressed lncRNA targets and mRNAs are listed. (C) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in wool follicle and skin development. (D) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in key signaling pathways in wool follicle development. The orange and blue circles represent the number of differentially expressed lncRNA targets and mRNAs, respectively. Their intersections represent the number of differentially expressed targets of differentially expressed lncRNAs.

FIGURE 3

KEGG analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q-value of each pathway.

FIGURE 4

Interaction network in primary wool follicle induction. (A) mRNA–mRNA interaction network involved in hair follicle development was constructed and is presented. (B) lncRNA–mRNA interaction network related to hair follicle development is displayed. Red and green represent upregulated and downregulated, respectively. Circles and squares represent mRNAs and lncRNAs, respectively.

FIGURE 5

Expression levels of differently expressed mRNAs and lncRNAs involved in primary wool follicle induction were validated by qRT-PCR. Data are presented as mean ± SEM (n = 6). ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t-test).

FIGURE 6

Expression pattern of XLOC_297809 and XLOC_764219 at stage 1 of primary wool follicle induction. (A,B) qRT-PCR and in situ hybridization of XLOC_297809. XLOC_297809 was expressed in placodes and dermal condensations. (C,D) qRT-PCR and in situ hybridization of XLOC_764219. XLOC_764219 was expressed in epidermis, placodes, and dermal condensations. Data are presented as the mean ± SEM (n = 6). ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t-test). Skin sections at stage 1 of primary wool follicle development in sheep fetus were displayed at 400× (scale bars represent 20 μm). Epi, epidermis, Der, dermis, Pc, placode, Dc, dermal condensation.