Transcriptional Characteristics Showed That miR-144-y/FOXO3 Participates in Embryonic Skin and Feather Follicle Development in Zhedong White Goose

Abstract

Skin and feather follicle development are essential processes for goose embryonic growth. Transcriptome and next-generation sequencing (NGS) network analyses were performed to improve the genome of Zhedong White goose and discover the critical genes, miRNAs, and pathways involved in goose skin and feather follicle morphogenesis. Sequencing output generated 6,002,591,668 to 8,675,720,319 clean reads from fifteen libraries. There were 1234, 3024, 4416, and 5326 different genes showing differential expression in four stages, E10 vs. E13, E10 vs. E18, E10 vs. E23, and E10 vs. E28, respectively. The differentially expressed genes (DEGs) were found to be implicated in multiple biological processes and pathways associated with feather growth and development, such as the Wnt signaling pathway, cell adhesion molecules, ECM-receptor interaction signaling pathways, and cell cycle and DNA replication pathways, according to functional analysis. In total, 8276 DEGs were assembled into twenty gene profiles with diverse expression patterns. The reliability of transcriptome results was verified by real-time quantitative PCR by selecting seven DEGs and five miRNAs. The localization of forkhead box O3 (FOXO3), connective tissue growth factor (CTGF), protein parched homolog1 (PTCH1), and miR-144-y by in situ hybridization showed spatial-temporal expression patterns and that FOXO3 and miR-144-y have an antagonistic targeting relationship. The correlation coefficient of FOXO3 and miR-144-y was -0.948, showing a strong negative correlation. Dual-luciferase reporter assay results demonstrated that miR-144-y could bind to the expected location to suppress the expression of FOXO3, which supports that there is a targeting relationship between them. The detections in this report will provide critical insight into the complex molecular mechanisms and breeding practices underlying the developmental characteristics of skin and feather follicles in Zhedong white geese.

Keywords: feather follicles development; gene expression; goose; skin; transcriptome sequencing.

Figure 1

Developmental pattern of skin and feather follicles in embryonic Zhedong white geese. The vertical rows show the time gradient, and the horizontal rows show the results of different stains. Magnified: 100×; Bar: 100 μm.

Figure 2

Principal Component Analysis (PCA) relationships between groups. The PC1 coordinate represents the first principal component and the percentage in brackets shows the contribution of the first principal component to the variation in the samples; the PC2 coordinate indicates the second principal component and the percentage in brackets indicates the contribution of the second principal component to the variation in the samples. The colored points in the graph indicate the individual samples.

Figure 3

GO analysis of genetic differences in skin and feather follicle development in Zhedong white geese. (A–D) shows the comparison group of E13, E18, E23, and E28 with E10. The results are summarized into three major groups: Biological process, cellular component, and molecular function. The Y-axis displays the percentage of genes, while the X-axis represents the second level term of the gene ontology.

Figure 4

KEGG analysis of genetic differences in skin and feather follicle development in Zhedong white geese. (A–D) shows the comparison group of E13, E18, E23, and E28 with E10. The abscissa represents the number of enriched genes, and the ordinates represent signal pathways.

Figure 5

Trend analysis of differential genes for skin and feather follicle development in Zhedong white geese. (A) Summary of trends in differentially expressed genes (DEGs). The colored profiles (p < 0.05) indicate significant enrichment and, conversely, non-significant enrichment. Similar color patterns are shown in profiles with the same expressive tendency. (B) The number of genes in a gene profile. (C–E) Trend plots, GO and KEGG analysis of profile 0. (F–H) Trend plots, GO and KEGG analysis of profile 16. (I–K) Trend plots, GO and KEGG analysis of profile 18. (L–N) Trend plots, GO and KEGG analysis of profile 19. Profiles C-F-E-L: Each X-axis indicates the embryonic stages (E10, E13, E18, E23, and E28); the Y-axis displays variations in expression. Four profiles (profiles C-F-E-L) represent the major transcriptional trajectories.

Figure 6

Validation of the RNA-Seq and miRNA-Seq data by RT-qPCR using 7 selected DEGs and 5 selected miRNAs. (A–G) The relative expression pattern and FPKM of seven genes (FOXO3, CTGF, PTCH1, NDRG1, FGFBP1, TPRS1, and MFAP5). (H–L) The relative expression pattern and TPM of five miRNAs (let-7-y, miR-103-y, miR-107-z, miR-181-y, and miR-183-x). As reference genes for miRNA and mRNA testing, respectively, U6, 18S rRNA, and GAPDH were used. The results are shown as the means ± SEM of three replicates.

Figure 7

In situ hybridization of selected genes and miRNA. The horizontal column shows the mRNA/miRNA expression localization of CTGF, PTCH1, FOXO3, and miR-144-y, and the vertical column shows the expression based on different embryonic stages. Magnified: 20×; Bar: 50 μm.

Figure 8

miRNA-mRNA interaction network and targeting validation. (A) Part of the interaction diagram between miRNA and target mRNA; the diamonds in blue correspond to the miRNAs and the circles in pink indicate the gene abbreviations. (B) Schematic representation of miR-144-y binding to the FOXO3-3′UTR target site. (C) Dual-luciferase reporter gene assay of miR-144-y interacting with FOXO3-3′UTR. *** p < 0.001.