Gene expression in the two breeds at various developmental stages
Of the five prenatal stages, the numbers of DE genes in the two sheep breeds were highest at 70 and 120 d, indicating that rapid myofiber proliferation included numerous genes during the myogenic process. However, two surges of myofiber proliferation appeared at 85 and 120 d in Texel fetuses, probably because the expression of more genes associated with myoblast proliferation and differentiation at 70 d prepared for the myofiber hyperplasia that followed at 85 d, and more genes related to the immune system were differentially expressed between T70 and U70 (Figure 6), which led to larger total counts of DE genes. The difference between the two sheep breeds in the timing of myogenesis in terms of transcriptomic levels was similar to that found between pig breeds . Cagnazzo et al. showed that myogenesis-related gene expression is greater in early Duroc (a breed with more intramuscular fat) embryos than in early Pietrain (a highly muscled breed) embryos at 14-49 d of gestation, whereas the opposite was found in late embryos (63-91 d of gestation) . The present findings suggest that highly muscled breeds have a longer myogenic process during prenatal stages and that myogenesis is more intense in late-stage fetuses, which was validated by Pax7-cell staining in longissimus muscle cross sections (Figure 3). However, whether this myogenic process is the same in sheep as in pigs at earlier embryonic stages remains to be investigated.
We found that the immune and hematological systems' development and function were most overrepresented of all physiological systems and functions, accompanied by muscle system development, which was consistent with a microarray developmental analysis in cattle fetuses . Recent discoveries have revealed complex interactions between skeletal muscle and the immune system that regulate muscle regeneration and myogenesis, and many immune molecules, such as tumor necrosis factor-alpha (TNF-α), NF-kB, interleukin (IL)-4, IL-6, IL-10, and leukemia inhibitory factor are involved in muscle cell proliferation and differentiation [33–38]. In most cases, whether perturbation in the same signaling pathways related to myogenesis occurs will determine normal myogenic development or muscle disorders. Therefore, the pathways associated with immune and muscle disorders in this work are valuable for human myopathy at the prenatal stage. The hematological system is also implicated in myogenesis [39–43], which probably reflects the systemic requirement for muscle function during fetal development.
Nervous system development was overrepresented in the last two development stages in the two sheep breeds. Neurons are indispensable for maintaining normal muscle physiological function and also parallel the skeletal muscle in auxology. Comprehensive and close interactions occur between muscles and the nervous system during development [44–46]. Muscle development is regulated by the central nervous system in Drosophila and pigs [47, 48], and muscle fiber type is dependent on the pattern of innervation of a muscle established due to differential projection patterns between fast and slow motoneurons [49, 50]. Animals with different muscle phenotypes undergo diverse innervation patterns during fetal development. Double-muscled cattle have an additional 13-26% increased branching in terminal axons compared to that in normal cattle caused by a real increase in the number of myofibers .
We identified several valuable canonical pathways directly associated with muscle development and function, myogenesis, myoblast proliferation, and the cell cycle in muscle at various developmental stages, such as calcium , CXCR4 [53–55], and VEGF signaling . Furthermore, other pathways involved in adipose and muscle metabolism, such as inositol metabolism, steroid biosynthesis, EIF2 signaling, glycerolipid metabolism, the glycolysis/gluconeogenesis pathway, and N-glycan biosynthesis, are significantly affected during development. Steelman et al. suggested that Wnt signaling is a potential downstream target of myostatin for postnatal skeletal muscle growth and hypertrophy in mice . However, we focused on the transcriptome during prenatal stages rather than postnatal stages and found that various canonical pathways are predominant in skeletal muscle development at different stages due to a myostatin mutation in sheep.
DE genes involved in muscle and adipose development
STAT3, a member of the STAT family and a cooperator in the Janus kinase pathway, plays a dual role in the regulation of myoblast proliferation and differentiation, but is dependent on interactions with various cofactors [58–62]. We found that STAT3 expression was higher in U70 than T70, which suggests that STAT3 is most probably associated with the differentiation rather than proliferation of myoblasts. The GO analysis of DE genes between T70 and U70 confirmed our presumption, and muscle fiber hyperplasia was more intense in T70 (Figure 2) as well.
ALDOA is responsible for significant activation during the differentiation of chicken primary myoblasts and plays an important role in muscle gene transcription [63, 64]. In our study, a higher ALDOA expression level in U70 indicated that more myoblasts exited the cell cycle and entered differentiation in U70 than those in T70. The leptin receptor (LEPR), a protein secreted from adipocytes, is responsible for fat mass regulation via leptin in the hypothalamus [65, 66]. LEPR expression was higher in U70 than in T70. Whether the higher LEPR expression at prenatal stages is associated with high postnatal adipose deposition remains to be investigated.
Both hyperplasia and hypertrophy continued in skeletal muscle at 85 d. FBP2, which encodes a gluconeogenesis regulatory enzyme that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate, was expressed higher in T85 than in U85. A previous study suggested that FBPase participates in some nuclear processes during the development and regeneration of skeletal muscle [67, 68]. Additionally, PRG3 expression was also higher in T85. Previous studies have shown that PRG3 plays a role in some early aspects of skeletal myogenesis [69–72].
The expression levels of PIK3R1, TSHR, and PON1 were higher in U85 and U100 than that in T85 or T100. Previous studies have demonstrated that these genes are involved in fat metabolism and adipocyte development [73–76].
PDK4, a gene that was upregulated in T100, is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation . In addition, PDK4 is significantly associated with intramuscular fat and muscle water content , indicating that PDK4 is involved in meat quality.
TXNRD1 is a variant of thioredoxin reductase, an important selenoprotein that maintains cellular redox balance and regulates several redox-dependent processes during apoptosis, cell proliferation, and differentiation [79–81]. LEF1 is a transcription factor involved in the regulation of myogenesis. Loss of Lef1-mediated repression results in an increased number of cells expressing Pax-7 and Pax-3, suggesting that Wnt signaling via Lef1 acts to regulate the number of premyogenic cells in somites . In our study, LEF1 expression was higher in U100 than that in T100, which is consistent with the rapid proliferation of myofibers in U100. Although transcription factor activity is predominantly manifested at the protein level, the difference in LEF1 expression level indicated a potential difference of LEF1 between breeds.
OVAR-DQB1 (sheep MHC class II) was expressed much higher in T100 and T120, particularly in T120 (Table 7), compared with U 100 and U120. Results from Karpati et al. indicated that the class I molecule may be involved in the fusion of myogenic cells during muscle regeneration . Honda and Rostami demonstrated that the expression of class I antigens on muscle cells is not only immunologically modulated but also developmentally regulated, and that these antigens may play a role in cell recognition and interaction during the myogenic fusion process. The presence of the antigens, however, was transitory, and they disappeared as myoblasts fused and differentiated into multinucleate myotubes .
CEBPB, an important transcription factor, acts as a indispensable regulator of adipocyte differentiation during adipogenesis [85–87]. We found that CEBPB expression was higher in skeletal muscle in T120 than in U120, indicating a key difference in adipogenesis between Texel and Ujumqin sheep. However, which breeds contain more adipose mass than the other at this developmental stage remains to be investigated.
TRRAP, which was downregulated in T120, in contrast to that in U120, is vital for embryonic survival and control of the mitotic checkpoint [88–91]. Deletion of TRRAP leads to a reduced level of beta-catenin ubiquitination, a lower degradation rate, and accumulation of the beta-catenin protein, whereas TRRAP knockdown results in abnormal retention of beta-catenin at chromatin and concomitant hyperactivation of the canonical Wnt pathway . However, the canonical Wnt pathway is downregulated in the absence of myostatin through beta-catenin, whereas the Wnt/calcium pathway is upregulated , suggesting that TRRAP may negatively regulate skeletal muscle development between the canonical Wnt and the Wnt/calcium pathways through beta-catenin at 120 d. Further efforts are warranted to test the effect of TRRAP on skeletal muscle development.
Other than the genes discussed above, we found that MSTN expression was lower in Texel than in Ujumqin sheep through the five development stages (Table 8). This result indicated that MSTN downregulation contributed to the hyperplasia and hypertrophy of myofibers in Texel sheep through the second half of gestation. Moreover, the genes discussed above are potential myostatin targets for further investigation.