Abstract
Skeletal muscle development at the embryonic stage is critical for muscle growth in poultry, during which microRNAs (miRNAs) play a vital role. Geese are one of the main poultry species, but how miRNAs regulate skeletal muscle development in geese during the embryonic stage remains poorly understood. This study characterized histological features and miRNA expression profiles during leg muscle development in Daozhou Grey goose (a prized Chinese breed) embryos at three stages (E14, E21, and E28). The objective was to identify differentially expressed miRNAs (DEMs) associated with muscle development in Daozhou Grey goose and utilize bioinformatics to predict the potential biological functions of these DEMs. Histological observations revealed stage-specific muscle development, marked by myoblast proliferation at E14, differentiation at E21 and muscle fiber maturation at E28. A total of 340 known and 270 novel miRNAs were identified by miRNA sequencing, with miR-148a-3p, miR-1a-3p, miR-100-5p, miR-206, and miR-92-3p being the most abundant. Furthermore, differential expression analysis identified 105, 107 and 70 DEMs across E14 vs E21, E14 vs E28, and E21 vs E28 comparisons, respectively. The expression patterns of the twelve randomly selected DEMs (9 known and 3 novel miRNAs) were validated by Quantitative real-time polymerase chain reaction (qRT-PCR), confirming the sequencing results. Functional enrichment analysis of predicted miRNA target genes revealed involvement of the MAPK, TGF-β and Notch signaling pathways in embryonic myogenesis. The miRNA-mRNA interaction network analysis of the top 10 most DEMs and their target genes revealed key regulatory nodes such as let-7k-5p-MAP3K1, miR-133a-3p-FZD7, miR-133c-3p-STAT3, miR-187-3p-ZEB2, and miR-205b-SETD3, with inverse expression patterns validated by qRT-PCR. It was hypothesized that the above pathways as well as these miRNA-mRNA target relationships play an important role in the regulating embryonic muscle growth and development of the Daozhou Grey goose. These findings provide the first comprehensive miRNAs profile of embryonic leg muscle in vivo in geese, offering a basic reference and theoretical framework for further research on molecular regulatory mechanisms of goose muscle development.