Analyse The Regulatory Mechanism Involved In The Difference Of Skeletal Muscle Development In Heiwu And Peking Duck

Myogenesis in avian has a similar development character with other vertebrates, the myoblast number is almostly fixed during the embryonic stage. After post-hatching, protein deposition is main contributor for muscle mass gain, a process displayed as the lengthen and thicken of myofibers. The priviously studies have domenstranted that muscle deposition capability exites differences among breeds and locations. To date, the resarch about the difference of muscle development between species or locations is mainly focus on phenotypic index or some single genes, but the detail molecular mechanism related to these differences is still unknown. Herein, this study employed the leg muslce and pectoral muslce of two phenotypic difference duck species as research materials, acrooding to compare the character of duck skeletal muscle development at different time point from embryo stage and post-hatch stage, to clear Heiwu and Peking duck skeletal development displayed species-specific and locations-specific. The RNA-Seq was used to identify differentially expressed genes related to leg muscle and pectoral muscle myogenesis in Heiwu and Peking ducks. In vitro, the function of Pax3 in on duck myoblast roliferation and differentiation was identified.The main results as following:(1) During duck embryo stage, compared to Heiwu duck. Peking duck has a quickly muscle growth ability, and leg muscle weight is significantly different between two duck species. Meanwhile, leg muscle has a higher development ability than pectoral muscle at the same duck species. (P<0.05).(2) Some genes(IGF-1,PI3K,AKT,Pax3 and Pax7) involved in muscle development have a different expression pattern among four skeletal muscles, suggested the difference of muscle development between locations and species is already exist at embryo stage.(3) During Peking duck post-hatching, compared to leg muscle fibers, pectoral muscle fibers have a higher density of myofiber and myonuclei. Verse vice, the diameter and cross-section area of myofiber have a higher number in leg muscle fiber than pectoral muscle fiber (P<0.05).(4) Both protein synthesis genes (mTOR and S6K) and protein degradation genes (FoxO1, MuRF1 and MAFbx) have a significantly different expression pattern between leg muscle and pectoral muscle, and pectoral muscle has a higher expression level than leg muscle (P<0.05), indicated pectoral muscle has a higher capability in protein metabolism than leg muscle.(5) With the week increase, satellite cell frequency (Pax3 and Pax7) decreased at both pectoral muscle and leg muscle, but it has no difference between two muscles (P>0.05). Based on the Hoechst-stained and the detection of caspase-3 activity, no obvious apoptosis is observed in both muscles (P>0.05).(6) In this study, all four skeletal muscle libraries obtain over 1.2G clean reads by using RNA-Seq. All data from this study demonstrated that obvious differences in gene expression between leg muscle and pectoral muscle (338 genes) and between Heiwu duck and Peking duck (88 genes). Intriguingly, more location-specific genes (246 for Heiwu duck and 258 for Peking duck) were identified compared with breed-specific gene (20 for leg muscle and 54 for pectoral muscle).(7) The KEGG results revealed that focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction are the three main pathways responsible for muscle development differences between leg and pectoral muscles, and only ribosomes is mainly involved in muscle development differences between Heiwu and Peking duck.(8) One shRNA is successfully constructed to target and silence the expression of duck Pax3(>70%).(9) After silencing Pax3, both the proliferation and differentiation of duck myoblast are significantly inhibited, by reducing the expression of myogenic genes (MyoD, Myf5 and MyoG) (P<0.05).(10) Adding folic acid to the medium of duck myoblast find that folic acid can significantly increase the expression of myogenic genes (MyoD and MyoG) and Pax7 (P<0.05), to rescue the duck myoblast proliferation and differentiation decreased by shRNA silencing Pax3.In all, this study proved that skeletal muscle development is different between species and locations during duck embryo, and protein metabolism signaling maybe is the main reason for muscle deposition difference between leg muscle and pectoral muscle, focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction are the three main pathways responsible for muscle development differences between leg and pectoral muscles, and ribosomes is thereason for muscle development differences between Heiwu and Peking duck. Pax3 has a positive effect on duck myoblast proliferation and differentiation. All these results are helpful for understand the molecular basis of skeletal muscle development difference between species and locations.