Core Genes Affecting Chicken Muscle Development And Their Responses To Changes In Different Level Of Dietary Nutrition

The growth and development of chicken skeletal muscle is a complex economic trait controlled by a multi-gene coordinated regulatory molecular network.Although long-term in-depth research has made great progress,its mechanism is still unclear.In this study,we used fast-growing Arbor Acres(AA)broilers and slow-growing indigenous Lushi(LS)chickens as an ideal model for systematic comparative studies on transcriptional dynamic characteristics during muscle development in chickens.Firstly,we constructed a systematically single-cell transcriptional dataset across two key initial stages of myogenesis among two breeds,and clarified the differences in the expression of early embryonic myogenic genes between the two breeds.Secondly,we performed comparative systematic analysis of genome-wide gene expression in breast muscles during embryonic and post-hatching stages.A subset of genes involving myogenesis were identified as the main contributors to the difference of skeletal muscle development between two breeds.Thirdly,using the quantitative analysis of AS at the transcript level,we identified the dominant transcripts at each breast muscle sample.Then,we clarified the dominant transcripts switched events between two breeds.On these basis,a model of promoting skeletal muscle growth and development by feeding LS chickens with high-energy and high-protein diets was constructed,and key genes that responded to high-energy and high-protein to promote skeletal muscle development were identified.Meanwhile,this study analyzed the effect of high-energy and high-protein diets on the dominant transcript switched events of genes related to breast muscledevelopment,and clarified the molecular mechanism of switched dominant transcript affecting muscle development.The specific results are as follows:1.Analysis of the regulatory mechanism of early embryonic myogenesis among individuals with differences in skeletal muscle developmentLimited studies have looked into the effects of genetic complexities on initiation of myogenesis during somitogenesis.In this study,we performed single-cell nucleus RNA sequencing of somitics from the stages of initial myogenesis in Hamburger &Hamilton(HH)9 and primary myofiber formation in HH18 in the represented fast-growing AA broiler and slow-growing YF chicken.We reconstructed the differentiation trajectory from muscle progenitor cells to myoblasts and Mscs,and identified a novel type of cell subpopulation before myogenic differentiation.After differentially expressed analysis for muscle-related subpopulations between two breeds,we found that HOX and HB family members were significantly up-regulated in LS chickens,while TAF6,APOA1 and MEF2 C were significantly up-regulated in AA broilers,which reflect differences in the effects of early embryonic myogenesis on muscle development in AA broilers and LS chickens.These results provide novel insights into the regulation of skeletal muscle growth by early embryonic myogenesis.2.Construction of transcriptional regulatory network related to skeletal muscle developmentMyofiber growth and hypertrophy are the main factors affecting the skeletal muscle development,the molecular regulatory mechanisms responsible for such differences are still elusive.In this study,we compared the histological and transcriptomic characteristics of breast muscle across 7 key developmental stages between AA broilers and LS chickens.The key developmental periods leading to the muscle fiber morphology differences between the two breeds were from week 1 to week 3.The analysis of time series gene expression revealed that MYOG,HEY1,SOX8,and PAX7 might serve as crucial TFs required for chicken breast muscle development.Combining principal component-based developmental axis analysis,differentially expressed transcripts between breeds,and gene-weighted co-expression analysis,we identified 44 genes,including MUSTN1,RBP7,HOPX,AK1,TNFRSF6 B,ACTC1,RBP7,FABP4,THRSP,THBS1,and PPF1BP1,enriched in protein synthesis and skeletal muscle development might be the main contributors to the differences in the breast muscle mass between the two breed.These findings provide new insights into the mechanisms regulating chicken skeletal muscle growth and development.3.Analysis of dominant transcript switched events related to skeletal muscle developmentAS can regulate gene function.Different transcript switching events frequently occur during muscle growth and development,but the effect of these switching events on muscle development is still unclear.Based on the AS analysis via the transcriptomes of the above seven key developmental stages,it was found that exon skipping was the most frequent AS event,and the mutually exclusive exons were the lowest rate.Further investigation found that the 85 genes underwent switch events among the dominant isoforms between two breeds.The protein structure analysis found that 52% of the genes exhibted different domain,signal peptide,ORF or IDRs between switched dominant transcripts.Changes in the domain position between transcripts are the main differences in protein structure annotation.These results indicated that not only existed the dominant transcripts during the development of breast muscle in chicken,but the dominant transcripts were switched among different breeds,and might play different biological functions.4.Analysis of key genes and their biological functions in response to changes in dietary energy and proteinNutrient supply,especially the change of energy and protein,is the most important way to improve the growth and development of chicken skeletal muscle.But whether it regulates skeletal muscle development by changing the expression characteristics of core genes of skeletal muscle development is not clear.In this study,a model of promoting skeletal muscle development by feeding LS chickens with high-energy and high-protein diets was established,and LS chickens fed with normal laying hen diets were used as the control group for the experiment.Compared with the control group,the cross-sectional area of muscle fibers in the experimental group was significantly higher,but the number of muscle fibers was significantly lower.Meat quality-related phenotypes,such as IMF,had no significant difference between the control and the experimental group.Analysis of gene expression in breast muscle showed that six genes related to muscle growth and development,including MUSTN1,RBP7,METTL21 C,RPS29,COL6A3 and SAE1,were key genes responding to high-energy and high-protein diets.These results suggest that changes in dietary energy and protein affect skeletal muscle development by modulating the expression of genes central to skeletal muscle development.5.Effects of different nutrition on major transcripts of core genes of skeletal muscle developmentNutrient supply effects AS events,but its effect on the switched dominant transcripts of core genes related to muscle development between two breeds remains unclear.In this study,through integrated the core genes of muscle development and the genes exhibited switched dominant trasncripts between breeds,we found 4 core genes related to muscle development,named TNFRSF6 B,ASNS,SNRPG and ENSGALG00000033780(ENS33780),underwent the dominant transcript switch during muscle development.High-energy and high-protein diets change the expression levels of different spliced isoforms of TNFRSF6 B and ENS33780.The TNFRSF6 B gene mediated cell proliferation has two isoforms,TNFRSF6B-X1 and TNFRSF6B-X2.The expression of TNFRSF6B-X1 in the breast muscle of normal group was significantly higher than that of the transcript TNFRSF6B-X2,but the expression of the two isoforms was not significantly different in the breast muscle of experimental group.Further analysis of the expression characteristics of the two isoforms of TNFRSF6 B found that The TNFRSF6B-X1 was the dominant transcript in both AA broilers and LS chickens at post-hatching day 1.However,the transcript of TNFRSF6B-X2 became the dominant transcript in AA broilers,whereas TNFRSF6B-X1 remained the dominant transcript in LS chickens at post-hatching week 1.A switching event of the dominant isoform occurred in AA broilers at 1 week old,resulting in TNFRSF6B-X2 being the dominant isoform in AA broilers,while TNFRSF6B-X1 remained the dominant isoform in LS chickens.Gain-of-function assays demonstrated that both of them could promote the proliferation of chicken primary myoblasts,but only TNFRSF6B-X2 could augment myogenesis.These results suggest that high-energy and high-protein diets effect the dominant transcripts switched in breast muscle between different breeds,resulting in changes in the biological functions of genes regulating myoblasts,which may have an effect on skeletal muscle development in chicken.In summary,this study enriches the regulated theory of chicken skeletal muscle growth and development.It provides an important reference for systematically elucidated core genes and transcripts that regulate skeletal muscle growth and development under various nutrient supply conditions,rapid assessment of the effects of various nutritional conditions on chicken skeletal muscle development,and precise design of for dietary ingredients to improve skeletal muscle development.And this study has important guiding significance for improving skeletal muscle development and meat production of local chickens.