Background: Elongases of very long chain fatty acids (ELOVLs), a family of first rate-limiting enzymes in the synthesis of long-chain fatty acids, play an essential role in the biosynthesis of complex lipids. Disrupting any of ELOVLs affects normal growth and development in mammals. Genetic variations in ELOVLs are associated with backfat or intramuscular fatty acid composition in livestock. However, the effects of ELOVL gene family on breeding selection and lipid deposition in different tissues are still unknown in chickens. Results: Genetic variation patterns and genetic associations analysis showed that the genetic variations of ELOVL genes were contributed to breeding selection of commercial varieties in chicken, and 14 SNPs in ELOVL2-6 were associated with body weight, carcass or fat deposition traits. Especially, one SNP rs17631638T > C in the promoter of ELOVL3 was associated with intramuscular fat content (IMF), and its allele frequency was significantly higher in native and layer breeds compared to that in commercial broiler breeds. Quantitative real-time PCR (qRT-PCR) determined that the ELOVL3 expressions in pectoralis were affected by the genotypes of rs17631638T > C. In addition, the transcription levels of ELOVL genes except ELOVL5 were regulated by estrogen in chicken liver and hypothalamus with different regulatory pathways. The expression levels of ELOVL1-6 in hypothalamus, liver, abdominal fat and pectoralis were correlated with abdominal fat weight, abdominal fat percentage, liver lipid content and IMF. Noteworthily, expression of ELOVL3 in pectoralis was highly positively correlated with IMF and glycerophospholipid molecules, including phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol and phospholipids inositol, rich in ω-3 and ω-6 long-chain unsaturated fatty acids, suggesting ELOVL3 could contribute to intramuscular fat deposition by increasing the proportion of long-chain unsaturated glycerophospholipid molecules in pectoralis. Conclusions: In summary, we demonstrated the genetic contribution of ELOVL gene family to breeding selection for specialized varieties, and revealed the expression regulation of ELOVL genes and their potential roles in regulating lipid deposition in different tissues. This study provides new insights into understanding the functions of ELOVL family on avian growth and lipid deposition in different tissues and the genetic variation in ELOVL3 may aid the marker-assisted selection of meat quality in chicken.

The insulin-like growth factor (IGF) system plays an indispensable role in embryonic and postnatal development in mammals. However, the effects of the system on growth, carcass, and egg-laying traits, and diversified selection have not been systematically studied in chickens. In the present study, firstly the composition and gene structures of the chicken IGF system were investigated using phylogenetic tree and conserved synteny analysis. Then the effects of the genetic variations in the IGF system genes on breeding of specialized varieties were explored by principal component analysis. In addition, the spatiotemporal expression properties of the genes in this system were analyzed by RT-qPCR and the functions of the genes in egg production performance and growth were explored by association study. Moreover, the effects of IGF-binding proteins 3 (IGFBP3) on skeletal muscle development in chicken were investigated by cell cycle analysis, 5-ethynyl-2'-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays. The results showed that the chicken IGF system included 13 members which could be classified into 3 groups based on their amino acid sequences: IGF binding proteins 1 to 5 and 7 (IGFBP1-5 and 7) belonged to the first group; IGF 1 and 2 (IGF1 and IGF2), and IGF 1 and 2 receptor (IGF1R and IGF2R) belonged to the second group; and IGF2 binding proteins 1-3 (IGF2BP1-3) belonged to the third group. The IGF2BP1 and 3, and IGFBP2, 3, and 7 genes likely contributed more to the formation of both the specialized meat-type and egg-type lines, whereas IGFBP1 and 5 likely contributed more to the formation of the egg-type lines. The SNPs in the IGF2BP3 and IGFBP2 and 5 genes were significantly associated with egg number, and SNPs in the IGFBP3 promoter region were significantly associated with body weight, breast muscle weight and leg muscle weight. The IGFBP3 inhibited proliferation but promoted differentiation of chicken primary myoblasts (CPMs). These results provide insights into the roles of the IGF system in the diversified selection of chickens. The SNPs associated with egg-laying performance, growth, and carcass traits could be used as genetic markers for breeding selection of chickens in the future.

Background: Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. Results: Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2 . TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. Conclusions: For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

MiRNAs have been found to be involved in the regulation of ovarian function as important post-transcriptional regulators, including regulators of follicular development, steroidogenesis, cell atresia, and even the development of ovarian cancer. In this study, we evaluated the regulatory role of gga-miR-449b-5p in follicular growth and steroid synthesis in ovarian granulosa cells (GCs) of laying hens through qRT-PCR, ELISAs, western blotting and dual-luciferase reporter assays, which have been described in our previous study. We demonstrated that gga-miR-449b-5p was widely expressed in granulosa and theca layers of the different-sized follicles, especially in the granulosa layer. The gga-miR-449b-5p had no significant effect on the proliferation of GCs, but could significantly regulate the expression of key steroidogenesis-related genes (StAR and CYP19A1) (p < 0.01) and the secretion of P4 and E2 (p < 0.01 and p < 0.05). Further research showed that gga-miR-449b-5p could target IGF2BP3 and downregulate the mRNA and protein expression of IGF2BP3 (p < 0.05). Therefore, this study suggests that gga-miR-449b-5p is a potent regulator of the synthesis of steroid hormones in GCs by targeting the expression of IGF2BP3 and may contribute to a better understanding of the role of functional miRNAs in laying hen ovarian development.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a vital secreted factor that promotes the occurrence of obesity in mammals. However, the effects of GPNMB on abdominal fat deposition is still unknown in chickens. In this study, we looked into the genetic and expression association of GPNMB gene with abdominal fat traits in chicken, and found that a genetic variation rs31126482 in GPNMB promoter was significantly associated with abdominal fat weight (AFW, P < 0.05) and abdominal fat percentage (AFP, P < 0.01). Express profile analysis of the GPNMB indicated that the gene was mainly expressed in abdominal fat tissue, and its expression level was strongly positively correlated with AFW (R-2 = 0.6356, P = 4.10E-05) and AFP (R-2 = 0.6450, P = 2.90E-05). We then investigated biological function of GPNMB on adipogenesis in chicken, and found that GPNMB could inhibit abdominal preadipocyte proliferation, but promote abdominal preadipocyte differentiation and lipid deposition. Furthermore, we explored regulatory mechanism of GPNMB gene in chicken, and detected one nonclassical estrogen regulatory element (AP1) and one peroxisome proliferator-activated receptor alpha (PPAR alpha) responsive element in the 2 kb promoter region of GPNMB gene, and demonstrated that estrogen could up-regulate GPNMB mRNA expression in adipose tissue and primary abdominal preadipocytes, while PPAR alpha could down-regulate GPNMB expression in primary preadipocytes. Taken together, this study brings new insights into understanding the function and transcriptional control of GPNMB gene, and provides genetic markers for breeding selection to improve abdominal fat traits in chicken.

Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.