Muscle development, growth, and regeneration occur throughout the life cycle of vertebrates. Myogenesis occurs in four consecutive, time-distinct but overlapping stages in amniotes, including embryo, fetus, neonate, and adult[18]. Fetal and neonatal myogenesis is key for muscle growth and maturation. Adult myogenesis is necessary for postpartum growth and repairing damaged muscles[19]. Primary and secondary fibers are produced during poultry embryonic and fetal development; after that, the number of myofibers remains stable[20] except during damage repair.
The small RNA sequence detection range is 18-30nt endogenous RNA, including miRNA, siRNA, and piRNA. However, the main objective of this study was to detect miRNAs related to muscle development of the chicken embryo to promote the genetic improvement of Chengkou mountain chickens. Therefore, 2047 miRNAs were detected by small RNA sequencing of the embryonic leg muscle of Chengkou Mountain chickens at different developmental time points. The differential analysis identified 196 differentially expressed miRNAs, indicating the significance of the 196 miRNAs in the muscle development of the chicken embryo.
A WGCNA systematic biological method described the patterns of gene association among the different samples and identified highly covariant gene sets, candidate biomarker genes, or therapeutic targets for use in medical and biological fields. WGCNA revealed the TERThigh-specific miR-17-92 cluster can targets biological processes enriched in the TERTlow cancer in a pan-cancer analysis[21]. Elsewhere, RNA sequencing and WGCNA of cord blood samples from fetal growth-restricted cases and controls combined with maternal peripheral blood quantification revealed miR-42-5p and miR-1306-3p as potential fetal growth restrictors[22]. In calves, WGCNA identified bta-miR-145 and bta-miR-199a-3p as important hub miRNAs regulating rumen development, immune system, and protein digestion[23]. This technique also established that DYNLL2 and its target miR-148-3p are important regulators of chicken myogenesis[24]. In this study, the WGCNA technique revealed key modules and hub genes related to leg muscles of embryos at four different development stages. Thus, many interesting genes for muscle development-related biological process and signaling pathways were identified through GO and KEGG hub gene target analysis. The WGCNA analysis identified 537 miRNAs in the greenyellow module, highly correlated (r = 0.92) with E12, 78 miRNAs in the magenta module for E16, 78 miRNAs in the purple module for E19, and 49 miRNAs in the cyan module for E21. These results account for a quarter of the total muscle miRNA, indicating that the progression of muscle development in the embryo is extremely important during this period. The results are consistent with an earlier study of E12 to E21, the period of rapid maturity of muscle fiber during the embryonic development of Chengkou mountain chicken[17].
Previous studies have shown that miRNAs are involved in muscle development; miR-222a and miR-126-5p significantly reduced the CPEB3 and FGFR3 mRNA levels in chicken embryo fibroblasts[25], emphasizing the significance of miRNA-target interactions in embryonic muscle regulation. A luciferase reporter gene assay showed that miR-1 targets chicken ACVR2B UTR directly, but network analysis predicted that ACVR2B targets gga-miR-101, gga-miR-1a and gga-miR-499 [26]. Although gga-miRNA-454-3p does not affect primary myoblast differentiation, it inhibits differentiation by targeting the myotube-associated protein SBF2[27]. A study focused on the chicken skeletal muscle indicated that miR-29b-1-5p inhibits the proliferation of chicken primary myoblasts and promotes the differentiation of myoblasts via an effective target gene, ANKRD9[28]. Meanwhile, miR-29b-1-5p and miR-133a-5p are sponges for circFGFR2 in skeletal muscle proliferation and differentiation[29]. MiR-133 was earlier established as a miRNA specifically expressed in skeletal muscles[30]. High-throughput sequencing revealed novel_miR_158, novel_miR_144, novel_miR_291, and miR-205a as crucial miRNAs for skeletal muscle development in Bian chicken, suggesting their vital function in chicken growth[31].
The proliferation and differentiation of skeletal muscle satellite cells are crucial in skeletal muscle development, especially during repair after muscle injury. MiR-21-5p targets KLF3 and regulates skeletal muscle satellite cell proliferation and differentiation[32]. MicroR-27b-3p regulates the proliferation and differentiation of chicken primary myoblasts by targeting MSTN[33]. Moreover, miRNA-214 regulates chicken myoblast proliferation and differentiation by targeting TRMT61A[34]. The miRNA-gene pairs gga-miR-499-5p/SOX6 and gga-miR-196-5p/CALM1 might affect muscle fiber performance using a miRNA-mRNA integrated analysis[35]. All these miRNAs, except novel_miRNA (naming rules were different), were detected in this sequencing data, and gga-miR-499-5p was the candidate hub miRNA within the purple module for E19, suggesting its role in chicken muscle development.
Besides, gga-miR-130b-5p, gga-miR-1643-5p, gga-miR-12218-5p, and gga-miR-132b-5p were critical for muscle development at E12, E16, E19, and E21 embryo stages in Chengkou mountain chickens. Multiple genes related to muscle development were identified by predicting the targets of these miRNAs. MEF2C, a member of the myocyte enhancer factor 2 family of MADS (MCM1, agamous, deficiens, serum response factor), is an important regulator of cardiac myogenesis and right ventricular development. This gene is mainly expressed in cardiac precursor cells before linear cardiac tube formation in mice, and MEF2C mutation prevents right ventricle formation in mice[36].
Meanwhile, MEF2C probably synergizes with MyoD through amplification to establish skeletal muscle commitment during cardiac and skeletal myogenesis[37]. Activation of satellite cells regulates the repair of injured human skeletal muscles. However, after knocking out the MEF2A, MEF2C, and MEF2D genes, satellite cells only proliferated but failed to differentiate, showing the dependence of skeletal muscle regeneration on MEF2[38]. ERBB4 (Erb-B2 receptor tyrosine kinase 4), a Tyr protein kinase family member and the epidermal growth factor receptor subfamily, regulates muscle differentiation[39]. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade has also been identified as a key factor in myogenesis. Nonetheless, the STAT3 isoform is critical for satellite cell migration and myogenic differentiation because it mediates the expression of muscle-specific myogenic factors[40]. The SOCS (Suppressor of Cytokine Signaling) family of proteins down-regulates STAT activation[41]. Other target genes such as BRAF[42], DAAM1[43], FZD4[44, 45], and NF1[46, 47] affect muscle development in the same or different ways.
GO, and KEGG enrichment results showed that some muscle development entries were significantly enriched, including the Wnt, ErbB, MAPK, and Notch signaling pathways. As previously predicted, DAAM1, FZD4, and WNT16 constitute the Wnt signaling pathway that regulates the critical ability of muscles to break down and reorganize fibers during development. Wnt signaling is involved in muscle remodeling[48]. Consequently, correct activation of the Wnt signaling pathway is essential during the various steps of muscle formation[49]. Therefore, deficiency of Wnt signaling effectors during pregnancy leads to marked tissue damage and muscle dysplasia[50]. ERBB4 represents the ErbB signaling pathway, whose inhibition leads to non-denervated skeletal muscle growth in mice, but activation causes an opposite outcome[51].
In this study, the predicted members of the MAPK signaling pathway were PPM1A, NF1, MEF2C, and TRAF2. Ras-MAPK signaling promotes neuroactivity-dependent differentiation of slow muscle fibers in vivo[52]. Besides, the p38 MAPK is activated during myoblast differentiation, and it also affects the activity of the MEF2 family of transcription factors, suggesting that p38 may be involved in the myogenic program[53]. Early morphogenesis of skeletal muscles during chicken embryo development requires transient activation of the Notch signaling pathway to drive terminal differentiation of muscle progenitors[54]. Notch and NRG signaling antagonistically regulate the synthesis and degradation of the cardiomyocyte extracellular matrix in a mouse trabecular model, which is critical for the individualization and rearrangement growth of trabecular units[55]. Generally, the growth and development of organisms is a complex process, often regulated by several signaling pathways. Studies have shown that myogenic progenitor cell differentiation transitions from Notch to Wnt signaling. The temporal balance between Notch and Wnt signaling coordinates the precise progression of muscle precursor cells along the myogenic lineage pathway[56].