[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our understanding of the location-specific and breed-specific genes and pathways but also provides some candidate molecular targets for increasing muscle products and meat quality by genetic control.
[Show abstract][Hide abstract] ABSTRACT: Incubation temperature has an immediate and long-term influence on the embryonic development in birds. DNA methylation as
an important environment-induced mechanism could serve as a potential link between embryos’ phenotypic variability and temperature
variation, which reprogrammed by DNA (cytosine-5)-methyltransferases (DNMTS) and Methyl-CpG binding domain proteins (MBPS) 3&5 (MBD3&5). Five genes in DNMTS and MBPS gene families were selected as target genes, given their important role in epigenetic modification. In this study, we aimed
to test whether raising incubation temperature from 37.8°C to 38.8°C between embryonic days (ED) 1–10, ED10–20 and ED20–27
have effect on DNA methylation and whether DNMTS, MBPS play roles in thermal epigenetic regulation of early development in duck. Real-time quantitative PCR analysis showed that
increased incubation temperature by 1°C has remarkably dynamic effect on gene expression levels of DNMTS and MBPS. Slight changes in incubation temperature significantly increased mRNA levels of target genes in breast muscle tissue during
ED1–10, especially for DNMT1, DNMT3A and MBD5. In addition, higher temperature significantly increased enzyme activities of DNMT1 in leg muscle during ED10–20, liver tissue
during ED1–10, ED20–27 and DNMT3A in leg muscle and breast muscle tissue during ED10–20. These results suggest that incubation
temperature has an extended effect on gene expression levels and enzyme activities of DNMTS and MBPS, which provides evidence that incubation temperature may influence DNA methylation in duck during early developmental stages.
Our data indicated that DNMTS and MBPS may involved in thermal epigenetice regulation of embryos during the early development in duck. The potential links between
embryonic temperature and epigenetic modification need further investigation
[Show abstract][Hide abstract] ABSTRACT: In this study, we aimed to use duck breast muscle and leg muscle, the 2 main productive muscle organs, as a model to elucidate
the molecular mechanism controlling how the 2 muscles have different deposition capabilities, and to analyze the mechanisms
facilitating duck muscle development posthatching. Peking duck breast muscle and leg muscle were collected 3, 7, and 16 wk
posthatching. The morphology of the myofibers was observed by paraffin sectioning the muscles. The expression of genes involved
in protein metabolism [mammalian target of rapamycin (mTOR), RPS6-p70-protein kinase (S6K), forkhead box O1 (FoxO1), muscle RING finger 1 (MuRF1), and atrogin-1 (MAFbx)] was detected using real-time quantitative PCR and Western blot assays, and the results indicated that breast muscle had
a stronger capacity for both protein synthesis and protein degradation compared with leg muscle. Satellite cell frequency
declined during muscle development in both tissues, and the expression of Pax3/7, satellite cell marker genes, was not significantly different between breast muscle and leg muscle. No notable apoptosis
was observed in either tissue. The results of this study suggest that protein metabolism signaling is the main reason promoting
duck skeletal muscle mass gain.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstrated by the development of lipid droplets and the expression of key marker genes including peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (CEBP/α) and adipocyte fatty acid-binding protein (FABP4). Our results showed that duck primary preadipocytes began to adhere 12 h after seeding as short spindle shapes or litter triangles, which grew quickly 3 days post attachment and maintained stable after day 7. After 8 days the preadipocytes were induced to differentiate into mature adipocytes, which were stained red by oil red O. Additionally, it showed that during preadipocyte differentiation PPARγ mRNA was highly expressed at day 3, while CEBP/α and FABP4 mRNA peaked at day 5 and 8, respectively. These results indicate that we have successfully isolated and cultured Peking duck preadipocytes and successfully induced them to differentiate into mature adipocytes. This work could lay a foundation for further research into waterfowl adipogenesis.
[Show abstract][Hide abstract] ABSTRACT: The Pax3 gene has been proven to play a crucial role in determining myogenic progenitor cell fate during embryonic myogenesis; however, the molecular role of Pax3 in myoblast development during later stages of myogenesis is unknown. We hypothesized that Pax3 would function in myoblast proliferation and differentiation; therefore, we employed three short hairpin RNAs (shRNAs) (shRNA1, shRNA2, and shRNA3) that target Pax3 to characterize the function of Pax3 in duck myoblast development. The mRNA and protein expression levels of Pax3 in duck myoblasts were detected using real-time PCR and Western blotting. Cell proliferation was assessed using the MTT and BrdU assays, while cell differentiation was assayed using immunofluorescence labeling with a MyoG antibody. Additionally, folic acid (FA), which is a rescue tool, was added into the medium of duck myoblasts to indirectly examine the function of Pax3 on duck myoblast proliferation and differentiation. The results revealed that one of the shRNA vectors, shRNA1, could significantly and stably reduce the expression of Pax3 (P < 0.05). Silencing Pax3 by shRNA1 significantly reduced the proliferation and differentiation of duck myoblasts (P < 0.05) due to downregulated expression of myogenic regulator factors. These trends could be rescued by adding FA; and Pax7, a paralog gene of Pax3, was involved in those processes. Overall, Pax3 had a positive function in duck myoblast proliferation and differentiation by modulating the expression of myogenic regulation factors, and shRNA targeting of Pax3 might be a new approach for understanding the function of Pax3 in the development of diverse tissues.
No preview · Article · Oct 2013 · Molecular and Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Myogenic enhancer transcription factor 2c (MEF2c), one of the members of the MEF2 family of transcription factors, plays an important role in mammalian muscle development. However, the role of MEF2c in avian muscle development still remains unclear. To understand the function of MEF2c in avian muscle development, we first cloned the duck MEF2c coding domain sequence (CDS) and analyzed MEF2c expression in duck muscle tissues of embryos from 10 days of incubation to 1 week after birth using real-time PCR technology. The results showed that the duck MEF2c CDS consists of 1,398 nucleotides that encode 465 amino acids. The MEF2c duck protein contains a MADS domain, a MEF2 domain and a HJURP_C domain with high homology to related proteins in other organisms. Different expression levels of MEF2c were found in skeletal, smooth and cardiac muscle. Therefore, these results indicated that duck MEF2c has two conserved domains (a MADS and a MEF2 domain), is an indispensable regulator of muscle development, and plays an important role in the development of duck muscle.
[Show abstract][Hide abstract] ABSTRACT: IGF-1 is one of important factors which can make an effect on differentiation and development of tissues and organs. The present study was to observe the effects of IGF-1 to duck's embryo, liver and heart's development, by in ovo feeding IGF-1 to hatching duck eggs. Firstly, 0.5mL IGF-1 diluted in saline at different concentrations including 0, 80, 100 and 120ng/mL were injected into duck eggs and the results showed that 100ng/mL was the adjust concentration which had significant effects on duck embryo, liver and heart's development in later periods of hatching stages comparing with the control group. Then, the adjusted concentration of IGF-1 was used to investigate the developmental roles of IGF-1 on duck embryo and organ's development. The results showed that IGF-1 had a positive effect on duck embryonic development, especially had an obvious effect on liver. It was concluded that IGF-1 may play an important role in the process of duck liver development during the hatching stages.
No preview · Article · Dec 2012 · International Journal of Poultry Science
[Show abstract][Hide abstract] ABSTRACT: Paired box (Pax) proteins 3 and 7, which are two members of the PAX gene family, are important to animal skeletal muscle development acting their roles by mediating the expression of MyoD. These three genes expressed in several of tissues and organs in vertebrates. In order to investigate whether or not the relationships between Pax3, Pax7 and MyoD in other tissues are still similar with that in skeletal muscle, we detected the expression of Pax3, Pax7 and MyoD in duck different tissues and organs using real-time PCR technical. Results showed that these three genes not only expressed in skeletal muscle tissues, but also expressed in other tissues like heart, brain, spleen, fat and so on. The Pax3 and Pax7 also have coordinate roles in heart, spleen and brain, which are similar with that in skeletal muscle. Results indicate that the MyoD, Pax3 and Pax7 may involve in other tissues development besides skeletal muscle in duck and may also act on a similar mechanism between Pax3/7 and MyoD in skeletal muscle.
No preview · Article · Dec 2012 · Journal of Applied Animal Research
[Show abstract][Hide abstract] ABSTRACT: POU1F1 is an essential factor that regulates the development and reproduction of animal. The objective of the current research was to screen for polymorphism, expression of POU1F1 and their association with carcass quality traits. A total of 126 Erlang mountainous chickens from two strains (SD02 and SD03) were employed for testing. Seventeen single nucleotide polymorphisms (SNPs) were detected, but only two SNPs (g.96217999 T > C and g.96219442 C > T) were associated with carcass quality traits. In SD03 chicken, g.96217999 T > C genotypes were significantly associated with body weight (BW), carcass weight (CW), eviscerated weight (EW), and semi-eviscerated weight (SEW; P < 0.05), and was highly significantly associated with breast muscle weight (BMW) and abdominal fat weight (AW; P < 0.01). g.96219442 C > T was significantly associated with BW, EW, SEW (P < 0.05). However, these two SNPs were not significantly associated with any carcass traits in SD02 chicken. Diplotypes showed that in SD03 chicken, the haplotype [C: C] was the most favorable haplotype because it was associated with higher BW, CW, SEW, EW, BMW, and AW (P < 0.05). On the contrary, haplotype [T: T] was associated with lower carcass quality traits (P < 0.01). In addition, qRT-PCR revealed that at 13 weeks, the POU1F1 mRNA expression was significantly higher in breast muscle of cock compared to that of hens (P < 0.05), whereas there was no significant correlation between POU1F1 expression and carcass traits. These results suggested that POU1F1 could be a potential candidate gene for carcass traits in chicken.
No preview · Article · Jun 2012 · Molecular Biology Reports
[Show abstract][Hide abstract] ABSTRACT: Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.
No preview · Article · Feb 2012 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: 1. The objective of the research was to investigate the molecular evolutionary relationships between the duck myogenic determination factors (MYOD) gene family members and their roles in muscle development. 2. The four members of the duck MYOD gene family were cloned using RT-PCR, and their relative mRNA expression during duck muscle development was measured using qRT-PCR. 3. The results showed that MyoD and Myf5 clustered together, as did MyoG and MRF4 based on their complete amino acid sequence and the basic helix-loop-helix domain. Results of the evolutionary level analysis were consistent with that of the differential expression patterns during duck breast muscle development. As determined by qRT-PCR, MyoD and Myf5 were highly expressed in 22-day embryos, while MyoG and MRF4 expression was high in 14-day embryos. 4. We conclude that the entire MYOD gene family in the duck originated from a common ancestral gene and evolved after two duplication events. The roles of the MYOD gene family members in duck muscle development are similar to those in mammals.
Preview · Article · Aug 2011 · British Poultry Science
[Show abstract][Hide abstract] ABSTRACT: Myoblasts isolated from duck embryonic muscle were purified and in vitro cultured. External characteristics were observed by using the immunofluorescence technique, and growth curve of duck embryonic myoblasts was established after measuring with the MTT method. Moreover, mRNA expression of three marker genes, the Desmin, the muscle creatine kinase (Mck) and the troponin C (Tnnc), which could reflect the development status of myofibers, were detected each 24 h for cultured cells by using the qPCR technique. Results showed that the in vitro cultured duck myoblasts went through a series of developmental stages, including the proliferation of myoblasts, the differentiation of multi-nuclei myotubes, and the formation of myofiber. The cultured duck embryonic myoblasts entered into a logarithmic stage approximately on the fourth day after seeding. Accompanying with its progressive growth before entering into the logarithmic phase, the myoblasts also showed some differentiation phenomena, reflected by a low expression level of Desmin and high expression level of the Mck and Tnnc genes. During the rapid growth of the logarithmic phase, there was a high expression of the Desmin gene, and a low expression level of the Mck gene and the Tnnc gene in the cultured myoblasts. The expression profiles of the three marker genes for muscle development could be used for distinguishing the different developmental stages of in vitro cultured myoblasts at the molecular level, which would be more accurate and more feasible than observing the external characteristics of the cultured cells.
[Show abstract][Hide abstract] ABSTRACT: Myostatin (MSTN) plays a key role in the negative regulation of muscle growth and development during embryogenesis. The MSTN genes have different genetic characteristics in vertebrates: sole gene in mammals, gene duplication in fish, and alternative splicing in birds. To investigate the alternative splicing sites and developmental expression patterns of the duck MSTN genes, the mRNA and genome sequences were cloned, and the expression patterns were detected during breast muscle and leg muscle development by real-time PCR. In our study, four alternatively spliced forms of MSTN mRNA were found in the developing skeletal muscle of Peking duck, including two novel alternatively spliced transcripts, MSTN-c and MSTN-d. As a result of alternative splicing at the common GT-AG processing sites, MSTN-b and MSTN-c retained only the N-terminal TGFβ-propeptide superfamily domains. However, MSTN-d was not missing these domains, in contrast to MSTN-a. The real-time PCR results showed that there was no significant difference between breast muscle and leg muscle in MSTN-a mRNA expression, also in MSTN-b and MSTN-c. MSTN-a and MSTN-b have significant higher expressions than MSTN-c and MSTN-d, suggesting that they play the major role during embryo muscle development.
No preview · Article · Apr 2011 · Comparative Biochemistry and Physiology Part D Genomics and Proteomics
[Show abstract][Hide abstract] ABSTRACT: The EST sequence of goose (Anser cygnoides) Stearoyl-CoA desaturase 1(SCD-1) was obtained from a subtractive cDNA library. To further investigate the role of SCD-1 in lipid metabolism in geese, 5'-RACE and 3'-RACE were carried out in this study to obtain the complete cDNA sequence of goose SCD-1, which contained a 29-bp 5' UTR, a 1074-bp open reading frame (ORF) encoding 357 amino acids, and a 125-bp 3' UTR. The expression of SCD-1 was measured in several tissues, and the effects of overfeeding on the expression of SCD-1 were studied. The results of real time RT-PCR demonstrated that, compared to the brain, goose SCD-1 mRNA was more abundant in the liver. Overfeeding markedly increased the mRNA expression of SCD-1 in the liver of Sichuan White and Landes geese, and gene expression was markedly higher in the Sichuan White goose than in the landes goose. The mRNA abundance of SCD-1 in the liver had significant positive correlations with triacylglycerol (TG) content in liver lipids and in the levels of plasma insulin and very low-density lipoproteins (VLDL) levels in Sichuan white geese. However, the mRNA abundance of SCD-1 in the livers of Landes geese had only significant positive correlations with the TG content in liver lipids. In conclusion, SCD-1 is not only critical for hepatic steatosis in geese but is also important for the difference in lipid deposition in the livers of the two breeds.
No preview · Article · Nov 2010 · Molecular Biology Reports
[Show abstract][Hide abstract] ABSTRACT: In order to investigate the developmental differences between the duck breast muscle and leg muscle tissues during the embryonic stage to neonatal stages, as well as the expression profile of MyoD between the two muscle tissues, the morphologic characteristics in the two muscle tissues during duck embryo stages at E14, E18, E22, E27 and D7 were compared through the muscle paraffin sections. The coding domain sequence of duck MyoD gene was cloned, and then the expression of MyoD in duck leg muscle and breast muscle during embryo stage on E10, E14, E18, E22, E27 and D7 was detected using qRT-PCR method. Results showed that the developmental status of the duck breast muscle in embryonic phrases lag behind that of leg muscle. The CDS of duck MyoD gene consists of 894 nucleotides, and showed relatively high similarity with the gene of other species. The MyoD mRNA expressed in both kinds of muscle tissues and the expression profile had a similar trend, although the expression level of MyoD in the breast muscle was significantly higher than that in the leg muscle at each developmental stages (p<0.05). Results suggested that MyoD might have potential functions in controlling muscle fiber phenotype during the secondary myogenesis of muscle development. These fundamental works may provide some valuable clues for knowing the roles of MyoD in the myogenesis and the muscle fiber type differentiation in birds.
[Show abstract][Hide abstract] ABSTRACT: Liu, H., Wang, J., Han, C, Jia, J., Si, J., Huang, K. and Li, L. 2010. Cloning of MRF4 gene CDS and its mRNA expression in heart tissues during duck embyronic development. J. Appl. Anim. Res., 37: 185–189.In order to investigate the effect of MRF4 in heart muscle, complete coding region of duck MRF4 gene was cloned. It consisted of 723 nucleotides and codes for 240 AA, including a bHLH domain. Relative mRNA developmental expression of MRF4 in heart tissues at different embryo stages and newborn birds (lw) was investigated. Duck MRF4 mRNA was expressed in each stage and the expression level of the day 18 stage was higher than that of other stages (P
No preview · Article · Jun 2010 · Journal of Applied Animal Research