Research Note: Association of single nucleotide polymorphism of AKT3 with egg production traits in White Muscovy ducks (Cairina moschata).

Prior studies on transcriptomes of hypothalamus and ovary revealed that AKT3 is one of the candidate genes that might affect egg production in White Muscovy ducks. The role of AKT3 in the uterus during reproductive processes cannot be overemphasized. However, functional role of this gene in the tissues and on egg production traits of Muscovy ducks remains unknown. To identify the relationship between AKT3 and egg production traits in ducks, relative expression profile was first examined prior to identifying the variants within AKT3 that may underscore egg production traits [age at first egg (AFE), number of eggs at 300 d (N300D), and number of eggs at 59 wk (N59W)] in 549 ducks. The mRNA expression of AKT3 gene in high producing (HP) ducks was significantly higher than low producing (LP) ducks in the ovary, oviduct, and hypothalamus (P < 0.05 or 0.001). Three variants in AKT3 (C-3631A, C-3766T, and C-3953T) and high linkage block between C-3766T and C-3953T which are significantly (P < 0.05) associated with N300D and N59W were discovered. This study elucidates novel knowledge on the molecular mechanism of AKT3 that might be regulating egg production traits in Muscovy ducks.

Long noncoding RNA ZFP36L2-AS functions as a metabolic modulator to regulate muscle development.

Skeletal muscle is the largest metabolic organ in the body, and its metabolic flexibility is essential for maintaining systemic energy homeostasis. Metabolic inflexibility in muscles is a dominant cause of various metabolic disorders, impeding muscle development. In our previous study, we found lncRNA ZFP36L2-AS (for "ZFP36L2-antisense transcript") is specifically enriched in skeletal muscle. Here, we report that ZFP36L2-AS is upregulated during myogenic differentiation, and highly expressed in breast and leg muscle. In vitro, ZFP36L2-AS inhibits myoblast proliferation but promotes myoblast differentiation. In vivo, ZFP36L2-AS facilitates intramuscular fat deposition, as well as activates fast-twitch muscle phenotype and induces muscle atrophy. Mechanistically, ZFP36L2-AS interacts with acetyl-CoA carboxylase alpha (ACACA) and pyruvate carboxylase (PC) to induce ACACA dephosphorylation and damaged PC protein stability, thus modulating muscle metabolism. Meanwhile, ZFP36L2-AS can activate ACACA to reduce acetyl-CoA content, which enhances the inhibition of PC activity. Our findings present a novel model about the regulation of lncRNA on muscle metabolism.

Single-cell RNA sequencing of preadipocytes reveals the cell fate heterogeneity induced by melatonin.

Obesity is a global epidemic health disorder and associated with several diseases. Body weight-reducing effects of melatonin have been reported; however, no investigation toward examining whether the beneficial effects of melatonin are associated with preadipocyte heterogeneity has been reported. In this study, we profiled 25 071 transcriptomes of normal and melatonin-treated preadipocytes using scRNA-seq. By tSNE analysis, we present a cellular-state landscape for melatonin-treated preadipocytes that covers multiple-cell subpopulations, defined as cluster 0 to cluster 13. Cluster 0 and cluster 1 were the largest components of normal and melatonin-treated preadipocytes, respectively. G0S2, an inhibitor of adipose triglyceride lipase (ATGL), was significantly upregulated in cluster 0 and downregulated in cluster 1. We redefined cluster 0 as the G0S2-positive cluster (G0S2+ ) and cluster 1 as the G0S2-negative cluster (G0S2- ). Through pseudotime analysis, the G0S2- cluster cell differentiation trajectory was divided into three major structures, that is, the prebranch, the lipid catabolism branch, and the cell fate 2 branch. In vitro, G0S2 knockdown enhanced the expression levels of ATGL, BAT markers and fatty acid oxidation-related genes, but inhibited C/EBPα and PPARγ expression. In vivo, knockdown of G0S2 reduced the body weight gain in high-fat-fed mice. The beneficial effects of the G0S2- cell cluster in promoting lipolysis and inhibiting adipogenesis are dependent on two major aspects: first, downregulation of the G0S2 gene in the G0S2- cluster, resulting in activation of ATGL, which is responsible for the bulk of triacylglycerol hydrolase activity; and second, upregulation of FABP4 in the G0S2- cluster, resulting in inhibition of PPARγ and further reducing adipogenesis.

Long noncoding RNA SMUL suppresses SMURF2 production-mediated muscle atrophy via nonsense-mediated mRNA decay.

As the world population grows, muscle atrophy leading to muscle wasting could become a bigger risk. Long noncoding RNAs (lncRNAs) are known to play important roles in muscle growth and muscle atrophy. Meanwhile, it has recently come to light that many putative small open reading frames (sORFs) are hidden in lncRNAs; however, their translational capabilities and functions remain unclear. In this study, we uncovered 104 myogenic-associated lncRNAs translated, in at least a small peptide, by integrated transcriptome and proteomic analyses. Furthermore, an upstream ORF (uORF) regulatory network was constructed, and a novel muscle atrophy-associated lncRNA named SMUL (Smad ubiquitin regulatory factor 2 [SMURF2] upstream lncRNA) was identified. SMUL was highly expressed in skeletal muscle, and its expression level was downregulated during myoblast differentiation. SMUL promoted myoblast proliferation and suppressed differentiation in vitro. In vivo, SMUL induced skeletal muscle atrophy and promoted a switch from slow-twitch to fast-twitch fibers. In the meantime, translation of the SMUL sORF disrupted the stability of SMURF2 mRNA. Mechanistically, SMUL restrained SMURF2 production via nonsense-mediated mRNA decay (NMD), participating in the regulation of the transforming growth factor ß (TGF-ß)/SMAD pathway and further regulating myogenesis and muscle atrophy. Taken together, these results suggest that SMUL could be a novel therapeutic target for muscle atrophy.

Control of preadipocyte proliferation, apoptosis and early adipogenesis by the forkhead transcription factor FoxO6.

AIMS: Previous studies have shown that the forkhead transcription factor FoxO6 involved in memory consolidation and hepatic glucose homeostasis. Here we asked whether chicken FoxO6 may regulate preadipocyte proliferation, apoptosis and early adipogenesis. MAIN METHODS: Overexpression and knockdown of FoxO6 were performed and evaluated through cell proliferation methods, Oil-Red-O staining, and specific marker expression. Chromatin immunoprecipitation (ChIP) assay was performed to confirm cyclin G2 (CCNG2) as a direct target gene of FoxO6. KEY FINDINGS: FoxO6 is ubiquitously expressed in different chicken tissues and highly expressed in liver, abdominal fat, and preadipocytes in cultured cell. FoxO6 overexpression decreased preadipocyte proliferation by causing G1-phase cell-cycle arrest, whereas inhibition of FoxO6 showed the opposite effects. Overexpression or knockdown of FoxO6 significantly altered the mRNA and protein levels of cell-cycle related markers, such as CCNG2, cyclin dependent kinase inhibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1A (CDKN1A) and cyclin D2 (CCND2). During preadipocyte proliferation, FoxO6 targets and induces expression of CCNG2, as confirmed by ChIP assay and qPCR. In addition, FoxO6 induces preadipocyte apoptosis through increasing the protein expression levels of cleaved caspase-3 and cleaved caspase-8. Moreover, FoxO6 at the early stage of adipogenesis suppressed mRNA and protein levels of the key early regulators of adipogenesis, such as PPARγ and C/EBPα. SIGNIFICANCE: The results demonstrate that FoxO6 controls preadipocyte proliferation, apoptosis and early adipogenesis, and point to new approaches for further studies related to obesity.

A Novel DNA Methyltransferase Dnmt3a3 Splice Variant Represses Preadipocyte Proliferation and Differentiation.

Proliferation and differentiation of preadipocyte are essential for the formation of fat tissues. However, the genes that regulate the early stage of preadipocyte differentiation in chicken have remained elusive. Here we identify a novel spliced variant of the DNA methyltransferase Dnmt3a gene, named Dnmt3a3, that controls early preadipocyte differentiation. Dnmt3a3 expression is increased at the onset of preadipocyte differentiation and remains elevated during differentiation. Overexpression of Dnmt3a3 in preadipocytes markedly inhibits proliferation and cell-cycle progression, and this is accompanied by inhibition of the mRNA and protein level of cell-cycle control genes, such as p21 and p27. In addition, forced expression of Dnmt3a3 in differentiating preadipocytes represses early preadipocyte differentiation, and this was found to be accompanied by inhibition of the mRNA expression levels of early preadipocyte differentiation markers, such as GATA2, GATA3, C/EBPα, C/EBPß, AP2, and PPARγ, or the protein levels of GATA3, C/EBPß, and PPARγ. Taken together, these data demonstrate the participation of Dnmt3a3 in the proliferation and differentiation process of chicken primary preadipocyte cells.

MiR-34b-5p Mediates the Proliferation and Differentiation of Myoblasts by Targeting IGFBP2.

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.

LncIRS1 controls muscle atrophy via sponging miR-15 family to activate IGF1-PI3K/AKT pathway.

BACKGROUND: Recent studies indicate important roles for long noncoding RNAs (lncRNAs) in the regulation of gene expression by acting as competing endogenous RNAs (ceRNAs). However, the specific role of lncRNAs in skeletal muscle atrophy is still unclear. Our study aimed to identify the function of lncRNAs that control skeletal muscle myogenesis and atrophy. METHODS: RNA sequencing was performed to identify the skeletal muscle transcriptome (lncRNA and messenger RNA) between hypertrophic broilers and leaner broilers. To study the 'sponge' function of lncRNA, we constructed a lncRNA-microRNA (miRNA)-gene interaction network by integrated our previous submitted skeletal muscle miRNA sequencing data. The primary myoblast cells and animal model were used to assess the biological function of the lncIRS1 in vitro or in vivo. RESULTS: We constructed a myogenesis-associated lncRNA-miRNA-gene network and identified a novel ceRNA lncRNA named lncIRS1 that is specifically enriched in skeletal muscle. LncIRS1 could regulate myoblast proliferation and differentiation in vitro, and muscle mass and mean muscle fibre in vivo. LncIRS1 increases gradually during myogenic differentiation. Mechanistically, lncIRS1 acts as a ceRNA for miR-15a, miR-15b-5p, and miR-15c-5p to regulate IRS1 expression, which is the downstream of the IGF1 receptor. Overexpression of lncIRS1 not only increased the protein abundance of IRS1 but also promoted phosphorylation level of AKT (p-AKT) a central component of insulin-like growth factor-1 pathway. Furthermore, lncIRS1 regulates the expression of atrophy-related genes and can rescue muscle atrophy. CONCLUSIONS: The newly identified lncIRS1 acts as a sponge for miR-15 family to regulate IRS1 expression, resulting in promoting skeletal muscle myogenesis and controlling atrophy.

lncRNA-Six1 Is a Target of miR-1611 that Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis.

Emerging studies indicate important roles for non-coding RNAs (ncRNAs) as essential regulators in myogenesis, but relatively less is known about their function. In our previous study, we found that lncRNA-Six1 can regulate Six1 in cis to participate in myogenesis. Here, we studied a microRNA (miRNA) that is specifically expressed in chickens (miR-1611). Interestingly, miR-1611 was found to contain potential binding sites for both lncRNA-Six1 and Six1, and it can interact with lncRNA-Six1 to regulate Six1 expression. Overexpression of miR-1611 represses the proliferation and differentiation of myoblasts. Moreover, miR-1611 is highly expressed in slow-twitch fibers, and it drives the transformation of fast-twitch muscle fibers to slow-twitch muscle fibers. Together, these data demonstrate that miR-1611 can mediate the regulation of Six1 by lncRNA-Six1, thereby affecting proliferation and differentiation of myoblasts and transformation of muscle fiber types.

Characterization of miRNA and their target gene during chicken embryo skeletal muscle development.

MicroRNAs (miRNAs) are non-coding RNAs that regulate mRNA expression by degradation or translational inhibition. We investigated the underlying molecular mechanisms of skeletal muscle development based on differentially expressed genes and miRNAs. We compared mRNA and miRNA from chicken skeletal muscle at embryonic day E11, E16 and one day post-hatch (P1). The interaction networks were constructed, according to target prediction results and integration analysis of up-regulated genes with down regulated miRNAs or down-regulated genes with up-regulated miRNAs with |log2fold change| ≥ 1.75, P < 0.005. The miRNA-mRNA integration analysis showed high number of mRNAs regulated by a few number of miRNAs. In the E11_VS_E16, comparison group we identified biological processes including muscle maintenance, myoblast proliferation and muscle thin filament formation. The E11_VS_P1 group comparison included negative regulation of axon extension, sarcomere organization, and cell redox homeostasis and kinase inhibitor activity. The E16_VS_P1 comparison group contained genes for the negative regulation of anti-apoptosis and axon extension as well as glomerular basement membrane development. Functional in vitro assays indicated that over expression of miR-222a and miR-126-5p in DF-1 cells significantly reduced the mRNA levels of the target genes CPEB3 and FGFR3, respectively. These integrated analyses provide several candidates for future studies concerning miRNAs-target function on regulation of embryonic muscle development and growth.

MiR-16-5p targets SESN1 to regulate the p53 signaling pathway, affecting myoblast proliferation and apoptosis, and is involved in myoblast differentiation.

The proliferation, apoptosis, and differentiation of myoblasts are essential processes in skeletal muscle development. During this developmental process, microRNAs (miRNAs) play crucial roles. In our previous RNA-seq study (accession number GSE62971), we found that miR-16-5p was differentially expressed between fast and slow growth in chicken. In this study, we report that miR-16-5p could inhibit myoblast proliferation, promote myoblast apoptosis, and repress myoblast differentiation by directly binding to the 3' UTR of SESN1, which is also differentially expressed. Overexpression of SESN1 significantly promoted the proliferation, inhibited apoptosis, and induced differentiation of myoblasts. Conversely, its loss of function hampered myoblast proliferation, facilitated myoblast apoptosis, and inhibited myoblast differentiation. Interestingly, we found SESN1 could regulate p53 by a feedback mechanism, thereby participating in the regulation of p53 signaling pathway, which suggests that this feedback is indispensable for myoblast proliferation and apoptosis. Altogether, these data demonstrated that miR-16-5p directly targets SESN1 to regulate the p53 signaling pathway, and therefore affecting myoblast proliferation and apoptosis. Additionally, SESN1 targets myogenic genes to control myoblast differentiation.

Circular RNAs are abundant and dynamically expressed during embryonic muscle development in chickens.

The growth and development of skeletal muscle is regulated by proteins as well as non-coding RNAs. Circular RNAs (circRNAs) are universally expressed in various tissues and cell types, and regulate gene expression in eukaryotes. To identify the circRNAs during chicken embryonic skeletal muscle development, leg muscles of female Xinghua (XH) chicken at three developmental time points 11 embryo age (E11), 16 embryo age (E16) and 1 day post hatch (P1) were performed RNA sequencing. We identified 13,377 circRNAs with 3,036 abundantly expressed and most were derived from coding exons. A total of 462 differentially expressed circRNAs were identified (fold change > 2; q-value < 0.05). Parental genes of differentially expressed circRNAs were related to muscle biological processes. There were 946 exonic circRNAs have been found that harbored one or more miRNA-binding site for 150 known miRNAs. We validated that circRBFOX2s promoted cell proliferation through interacted with miR-206. These data collectively indicate that circRNAs are abundant and dynamically expressed during embryonic muscle development and could play key roles through sequestering miRNAs as well as other functions.

Systematic transcriptome-wide analysis of mRNA-miRNA interactions reveals the involvement of miR-142-5p and its target (FOXO3) in skeletal muscle growth in chickens.

The goal of this study was to perform a systematic transcriptome-wide analysis of mRNA-miRNA interactions and to identify candidates involved in the interplay between miRNAs and mRNAs that regulate chicken muscle growth. We used our previously published mRNA (GSE72424) and miRNA (GSE62971) deep sequencing data from two-tailed samples [i.e., the highest (h) and lowest (l) body weights] of Recessive White Rock (WRR) and Xinghua (XH) chickens to conduct integrative analyses of the miRNA-mRNA interactions involved in chicken skeletal muscle growth. A total of 162, 15, 173, and 27 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of the WRRh vs. XHh, WRRh vs. WRRl, WRRl vs. XHl, and XHh vs. XHl comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRRh vs. XHh contrast were associated with developmental disorders. Importantly, the WRRh vs. XHh contrast miRNA-mRNA network was enriched in IGF-1 signaling pathway genes, including FOXO3. A dual-luciferase reporter assay showed that FOXO3 was a target of miR-142-5p. Furthermore, miR-142-5p overexpression significantly decreased FOXO3 mRNA levels and promoted the expression of growth-related genes. These data demonstrated that miR-142-5p targets FOXO3 and promotes growth-related gene expression and regulates skeletal muscle growth in chicken. Comprehensive analysis facilitated the identification of miRNAs and target genes that might contribute to the regulation of skeletal muscle development. Our results provide new clues for understanding the molecular basis of chicken growth.

Chicken CCDC152 shares an NFYB-regulated bidirectional promoter with a growth hormone receptor antisense transcript and inhibits cells proliferation and migration.

The chicken coiled-coil domain-containing protein 152 (CCDC152) recently has been identified as a novel one implicated in cell cycle regulation, cellular proliferation and migration by us. Here we demonstrate that CCDC152 is oriented in a head-to-head configuration with the antisense transcript of growth hormone receptor (GHR) gene. Through serial luciferase reporter assays, we firstly identified a minimal 102 bp intergenic region as a core bidirectional promoter to drive basal transcription in divergent orientations. And site mutation and transient transfected assays showed that nuclear transcription factor Y subunit beta (NFYB) could bind to the CCAAT box and directly transactivate this bidirectional promoter. SiRNA-mediated NFYB depletion could significantly down-regulate the expression of both GHR-AS-I6 and CCDC152. Additionally, the expression of GHR-AS-I6 was significantly up-regulated after CCDC152 overexpression. Overexpression of CCDC152 remarkably reduced cell proliferation and migration through JAK2/STAT signaling pathway. Thus, the GHR-AS-I6-CCDC152 bidirectional transcription unit, as a novel direct target of NFYB, is possibly essential for the accelerated proliferation and motility of different cells.

Molecular characterization, expression profile of the FSHRgene and its association with egg production traits in muscovy duck.

Follicle-stimulating hormone (FSH) and its receptor play a key role in the follicular development and regulation of steroidogenesis in the ovary and spermatogenesis in the testis. The purpose of this study was to characterize themuscovy duck FSHR gene, identify SNPs and their association with egg production traits in muscovy ducks. Here, we cloned the complementary DNA (cDNA) sequence of FSHR, and examined the expression patterns of FSHR gene in adult female muscovy duck tissues. The cloned cDNA of the muscovy duck FSHR gene shared high similarity to those of pekin duck (Anas platyrhynchos) (95.7%) and chicken (93.2%). Three different muscovy duck FSHR transcripts were identified. Quantitative real-time PCR (RT-qPCR) results showed that the FSHR gene was expressed in all the 14 tested tissues, and the highest expression level was seen in the ovary. A total of 16 SNPs were identified, among which, four SNPs were located in the coding region of FSHR. The SNP C320T is significantly associated with egg production at 59 weeks of age (P < 0.05), whereas the SNP A227G is significantly associated with age at first egg stage (P < 0.05). These results suggest that the two SNPs (A227G and C320T) of FSHR gene are associated with egg production traits and could be potential markers that can be used for marker-assisted selection programmes to increase egg production in muscovy duck.

miR-16 controls myoblast proliferation and apoptosis through directly suppressing Bcl2 and FOXO1 activities.

Myogenesis mainly involves several steps including myoblast proliferation, differentiation, apoptosis and fusion. Except for muscle specific regulators, few miRNAs were proved to coordinate this complex process. Here, we reported that miR-16 inhibited myoblast proliferation and promoted myoblast apoptosis by directly targeting Bcl2 and FOXO1. The expression level of miR-16 was significantly decreased in the hypertrophic pectoral muscle compared to the normal pectoral muscle in chicken. In vitro, elevating miR-16 significantly inhibited myoblast proliferation and promoted myoblast apoptosis, resulting in about 11.2% cells arrested in G1 phase and 12.3% apoptotic cells in the early stage. Bioinformatic and biochemical analyses revealed Bcl2 and FOXO1 as direct targets of miR-16. Consist to the effect of miR-16 on myogenesis, specific inhibition of Bcl2 or FOXO1 significantly suppressed myoblast proliferation and induced myoblast apoptosis, indicating that both Bcl2 and FOXO1 contributed to miR-16 regulatory function in myogenesis. Interestingly, FOXO1, as the core target, mediated multiple growth-related pathways induced by miR-16 such as PI3K-AKT-MAPK and PI3K-AKT-mTOR. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that 234 annotated genes bound by FOXO1 in the early-differentiated myoblasts, which were significantly enriched in myogenic proliferation, death and hypotrophy. Altogether, we proposed that miR-16 acted as a coordinated mediator to suppress myogenesis in avian through the control of myoblast proliferation and apoptosis. These findings have provided a novel mechanism whereby miR-16 represses Bcl2 and FOXO1 expression to maintain myoblast growth and skeletal muscle mass.

MiR-34b-5p Suppresses Melanoma Differentiation-Associated Gene 5 (MDA5) Signaling Pathway to Promote Avian Leukosis Virus Subgroup J (ALV-J)-Infected Cells Proliferaction and ALV-J Replication.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that has a similar replication cycle to multiple viruses and therefore can be used as a model system for viral entry into host cells. However, there are few reports on the genes or microRNAs (miRNAs) that are responsible for the replication of ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of miR-34b-5p was significantly upregulated in ALV-J-infected chicken spleens compared to non-infected chicken spleens, but melanoma differentiation-associated gene 5 (MDA5) had the opposite expression pattern. In this study, a dual-luciferase reporter assay showed that MDA5 is a direct target of miR-34b-5p. In vitro, overexpression of miR-34b-5p accelerated the proliferation of ALV-J-infected cells by inducing the progression from G2 to S phase and it promoted cell migration. Ectopic expression of MDA5 inhibited ALV-J-infected cell proliferation, the cell cycle and cell migration, and knockdown of MDA5 promoted proliferation, the cell cycle and migration. In addition, during ALV-J infections, MDA5 can detect virus invasion and it triggers the MDA5 signaling pathway. MDA5 overexpression can activate the MDA5 signaling pathway, and thus it can inhibit the mRNA and protein expression of the ALV-J env gene and it can suppress virion secretion. In contrast, in response to the knockdown of MDA5 by small interfering RNA (siRNA) or an miR-34b-5p mimic, genes in the MDA5 signaling pathway were significantly downregulated (P < 0.05), but the mRNA and protein expression of ALV-J env and the sample-to-positive ratio of virion in the supernatants were increased. This indicates that miR-34b-5p is able to trigger the MDA5 signaling pathway and affect ALV-J infections. Together, these results suggest that miR-34b-5p targets MDA5 to accelerate the proliferation and migration of ALV-J-infected cells, and it promotes ALV-J replication, via the MDA5 signaling pathway.

Genome-wide association study of aggressive behaviour in chicken.

In the poultry industry, aggressive behaviour is a large animal welfare issue all over the world. To date, little is known about the underlying genetics of the aggressive behaviour. Here, we performed a genome-wide association study (GWAS) to explore the genetic mechanism associated with aggressive behaviour in chickens. The GWAS results showed that a total of 33 SNPs were associated with aggressive behaviour traits (P < 4.6E-6). rs312463697 on chromosome 4 was significantly associated with aggression (P = 2.10905E-07), and it was in the intron region of the sortilin-related VPS10 domain containing receptor 2 (SORCS2) gene. In addition, biological function analysis of the nearest 26 genes around the significant SNPs was performed with Ingenuity Pathway Analysis. An interaction network contained 17 genes was obtained and SORCS2 was involved in this network, interacted with nerve growth factor (NGF), nerve growth factor receptor (NGFR), dopa decarboxylase (L-dopa) and dopamine. After knockdown of SORCS2, the mRNA levels of NGF, L-dopa and dopamine receptor genes DRD1, DRD2, DRD3 and DRD4 were significantly decreased (P < 0.05). In summary, our data indicated that SORCS2 might play an important role in chicken aggressive behaviour through the regulation of dopaminergic pathways and NGF.

Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken.

miRNAs have been widely investigated in terms of cell proliferation and differentiation. However, little is known about their effects on bird growth. Here we characterized the promoter of miR-206 in chicken and found that the preferable promoter was located in 1200 bp upstream of pri-miR-206. In this region, many key transcription factors, including MyoD, c-Myb, CEBPα/ß, AP-4, RAP1, Brn2, GATA-1/2/3, E47, Sn, upstream stimulatory factor (USF) and CdxA, were predicted to bind and interact with miR-206 promoter. Overexpression of MyoD sharply increased miR-206 expression in both fibroblast and myoblast cells, and also the regulation in the myoblast cells was much stronger, indicating that miR-206 was regulated by MyoD combined with other muscle specific transcriptional factors. Aiming to further investigate the relationship between miR-206 mutation and transcriptional expression, total of 23 SNPs were identified in the two distinct bird lines by sequencing. Interestingly, the motif bound by MyoD was individually destroyed by G-to-C mutation located at 419 bp upstream of miR-206 precursor. Co-transfecting MyoD and miR-206 promoter in DF-1 cells, the luciferase activity of promoter containing homozygous GG types was significantly higher than CC ones (p < 0.05). Thus, this mutation caused low expression of miR-206. Consistently, eight variants including G-419C mutation exhibited a great effect on birthweight through maker-trait association analysis in F2 population (p < 0.05). Additionally, the regulation of miR-206 on embryo muscle mass mainly by increasing MyoG and muscle creatine kinase (MCK) expression (p < 0.05) with little change in MyoD, TMEM8C and myosin heavy chain (MHC). In conclusion, our findings provide a novel mutation destroying the promoter activity of miR-206 in birds and shed new light to understand the regulation mechanism of miR-206 on the embryonic muscle growth.