Spatio-temporal dynamics of early somite segmentation in the chicken embryo.

During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 (hes4) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.

Birds of a feather flock together: a dataset for Clock and Adcyap1 genes from migration genetics studies.

Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1 mRNAs, in the chick inner ear.

Prosaposin is a glycoprotein that is widely conserved in vertebrates. It serves as a precursor for saposins A, B, C, and D, which are necessary activators of lysosomal sphingolipid hydrolases. It can also act as a neurotrophic factor. Prosaposin plays a crucial role in the mammalian vestibuloauditory system because it prevents progressive deafness and severe vestibular dysfunction. Prosaposin can exhibit a neurotrophic effect through the G protein-coupled receptor (GPR), and GPR37 and GPR37L1 are its candidate receptors. In this study, we examined the expression patterns of prosaposin, GPR37, and GPR37L1 mRNAs in postnatal day 0 chick vestibuloauditory organs by in situ hybridization. Prosaposin mRNA expression was observed in all vestibular end organs, the vestibular and spiral ganglions, whereas no hybridization signal was observed in the auditory organ, namely basilar papilla. While GPR37L1 mRNA expression was observed in the oligodendrocytes/Schwann cells in the vestibular ganglion, GPR37 mRNA expression was observed in the crista ampullaris base region. These findings suggest that prosaposin expression in the auditory hair cells is acquired uniquely in mammals partly due to the loss of regeneration upon maturation and improved autophagic activity in mammalian auditory hair cells. In addition, as GPR37L1 expression in the chick glial cells differed from GPR37 expression in mammalian glial cells, the roles of GPR37 and GPR37L1 for prosaposin may differ between birds and mammals.

Residues L55 and W69 of Tva Mediate Entry of Subgroup A Avian Leukosis Virus.

The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Effects of a FCBP gene polymorphism, location, and sex on Young's modulus of the tenth primary feather in racing pigeons.

Young's modulus (E) is a measure for stiffness of a material and a higher E means a higher stiffness. The respective polymorphism of the feather corneous beta-protein gene causes the replacement of glycine by cysteine. We looked for possible effects of the three FCBP genotypes on E in the 10th primaries of racing pigeons. However, we did not find a statistically significant difference of E between the genotypes, even within the sexes and/or within different locations under our test conditions. Our findings do not preclude the possibility that under other conditions (temperature, moisture) an influence of the glycine/cysteine polymorphism on E may exist. Compared to the more proximal locations of the rachis (base and middle) we observed lower values for E in the distal region (tip). The 10th primary constitutes the leading edge of the pigeon wing and this special function may require higher stiffness in the proximal parts of the shaft. We observed significantly higher values of E in females than in males, which result only from statistically significantly higher values in the middle region. The higher stiffness of female primaries may also contribute to the better results of hens compared to cocks in pigeon races.

Dynamic transcriptome and chromatin architecture in granulosa cells during chicken folliculogenesis.

Folliculogenesis is a complex biological process involving a central oocyte and its surrounding somatic cells. Three-dimensional chromatin architecture is an important transcription regulator; however, little is known about its dynamics and role in transcriptional regulation of granulosa cells during chicken folliculogenesis. We investigate the transcriptomic dynamics of chicken granulosa cells over ten follicular stages and assess the chromatin architecture dynamics and how it influences gene expression in granulosa cells at three key stages: the prehierarchical small white follicles, the first largest preovulatory follicles, and the postovulatory follicles. Our results demonstrate the consistency between the global reprogramming of chromatin architecture and the transcriptomic divergence during folliculogenesis, providing ample evidence for compartmentalization rearrangement, variable organization of topologically associating domains, and rewiring of the long-range interaction between promoter and enhancers. These results provide key insights into avian reproductive biology and provide a foundational dataset for the future in-depth functional characterization of granulosa cells.

Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales.

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

Expression atlas of avian neural crest proteins: Neurulation to migration.

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

Correlation between small-scale methylation changes and gene expression during the development of myopia.

Visually induced changes in the expression of early growth response-1 (EGR1), FBJ osteosarcoma oncogene (FOS), and NGFI-A binding protein-2 (NAB2) appear to form a part of a retinal network fundamental to ocular growth regulation, and thus, the development of myopia (short-sightedness). However, it is unclear how environmental (visual) cues are translated into these molecular changes. One possibility is through epigenetic modifications such as DNA methylation, a known regulator of such processes. By sequencing bisulfite-converted DNA amplicons, this study examined whether changes in DNA methylation occur within specific regulatory and promoter regions of EGR1, FOS, and NAB2 during the periods of increased and decreased ocular growth in chicks. Visually induced changes in ocular growth rates were associated with single-point, but not large-scale, shifts in methylation levels within the investigated regions. Analysis of methylation pattern variability (entropy) demonstrated that the observed methylation changes are occurring within small subpopulations of retinal cells. This concurs with previous observations that EGR1 and FOS are differentially regulated at the peptide level within specific retinal cell types. Together, the findings of this study support a potential role for DNA methylation in the translation of external visual cues into molecular changes critical for ocular growth regulation and myopia development.

The double-edged sword role of TGF-ß signaling pathway between intrauterine inflammation and cranial neural crest development.

Intrauterine infection would harm a developing embryo/fetus, thereby increasing the risk of developmental malformation. But, whether or not the infection-induced inflammation affects neural crest development still remains obscure. In this study, we employed meta-analysis to demonstrate the potential correlation between infection-induced inflammation and craniofacial anomalies, which was usually derived from the problems in neural crest cell development. The correlation was further verified by inflammatory cytokine release and the activation of nuclear factor kappa-light-chain enhancer of activated B cells signaling in lipopolysaccharide-treated HH10 chicken embryos. In such an inflammatory condition, AP-2α- and Pax7-labeled pre-migratory and migratory neural crest cells in HH10 chicken embryos were significantly less than the ones in control. The bioinformatics analysis of RNA-seq data demonstrated that the principal differential gene expression occurred in transforming growth factor-beta (TGF-ß) signaling pathway, which was confirmed by the subsequent experimental results of quantitative PCR and immunofluorescent staining. Under this inflammatory circumstance, whole-mount in situ hybridization, immunofluorescence, and quantitative PCR showed the gene expression changes of key EMT-related transcription factors including upregulated Msx1, downregulated Slug, and FoxD3, as well as adhesion molecules and extracellular matrix protein including upregulated Cadherrin6B, E-cadherin, N-cadherin, and Laminin at the dorsal portion of neural tube of HH10 chicken embryos. Meanwhile, the bioinformatics analysis of RNA-seq data also manifested the differential gene expressions relevant to cell proliferation, which was confirmed by proliferating cell nuclear antigen Western blot data and co-immunofluorescence staining of human natural killer-1 and phosphorylated histone H3. In brief, this study revealed for the first time that the double-edged sword role of TGF-ß signaling pathway between intrauterine inflammation (protective role) and cranial neural crest development (harmful role).

Genome-wide survey and functional analysis reveal TCF21 promotes chicken preadipocyte differentiation by directly upregulating HTR2A.

BACKGROUND/AIM: Previously, we showed that transcription factor 21 (TCF21) promotes chicken preadipocyte differentiation. However, the genome-wide TCF21 binding sites and its downstream target genes in chicken adipogenesis were unknown. METHODS: ChIP-Seq and RNA-Seq were used to screen candidate targets of TCF21. qPCR and luciferase reporter assay were applied to verify the sequencing results. Western blotting, oil red-O staining and pharmacological treatments were performed to investigate the function of 5-hydroxytryptamine receptor 2A (HTR2A), one of the bonafide direct downstream binding targets of TCF21. RESULTS: A total of 94 candidate target genes of TCF21 were identified. ChIP-qPCR, RT-qPCR, and luciferase reporter assay demonstrated that HTR2A is one of the bonafide direct downstream binding targets of TCF21. HTR2A expression in adipose tissue was upregulated in fat line broilers. Also, the abundance of HTR2A gradually increased during the adipogenesis process. Interestingly, pharmacological enhancement or inhibition of HTR2A promoted or attenuated the differentiation of preadipocytes, respectively. Furthermore, HTR2A inhibition impaired the TCF21 promoted adipogenesis. CONCLUSIONS: We profiled the genome-wide TCF21 binding sites in chicken differentiated preadipocytes revealing HTR2A as the direct downstream target of TCF21 in adipogenesis.

Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

New insights into genetics underlying of plumage color.

Plumage color can be considered as a social signal in chickens and a breeding identification tool among breeders. The relationship between plumage color and trait groups of immunity, growth and fertility is still a controversial issue. This research aimed to determine the genome-wide additive and epistatic variants affecting plumage color variation in chickens using the chicken Illumina 60k high-density SNP array. Two scenarios of genome-wide additive association studies using all SNPs and independent SNPs were carried out. To perform epistatic association analysis, the LD pruning approach was used to reduce the complexity of the analysis. We detected seven novel significant loci using all of the SNPs in the model and 14 SNPs using the LD pruning approach associated with plumage color. Moreover, 89 significantly associated SNP-SNP interactions (P-value <10-6 ) distributed in 25 chromosomes were identified, indicating that all of the signals together putatively influence the quantitative variation of plumage color. By annotating genes relevant to top SNPs, we have distinguished 18 potential candidate genes comprising HNF4beta, CKMT1B, TBC1D22A, RPL8, CACNA2D1, FZD4, SGMS1, IRF8, OPTN, LOC420362, TRABD, OvoDA1, DAD1, USP6, RBM12B, MIR1772, MIR1709 and MIR6696 and also 89 putative gene-gene combinations responsible for plumage color variation in chickens. Furthermore, several KEGG pathways including metabolic pathway, cytokine-cytokine receptor interaction, focal adhesion, melanogenesis, glycosaminoglycan biosynthesis-keratan sulfate and sphingolipid metabolism were enriched in the gene-set analysis. The results indicated that plumage color is a highly polygenic trait which, in turn, can be affected by multiple coding genes, regulatory genes and gene-gene epistasis interactions. In addition to genes with additive effects, epistatic genes with tiny individual effect sizes but significant effects in a pair have the potential to control plumage coloration in chickens.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Transcriptomic Analysis of Laying Hens Revealed the Role of Aging-Related Genes during Forced Molting.

Molting in birds provides us with an ideal genetic model for understanding aging and rejuvenation since birds present younger characteristics for reproduction and appearance after molting. Forced molting (FM) by fasting in chickens causes aging of their reproductive system and then promotes cell redevelopment by providing water and feed again. To reveal the genetic mechanism of rejuvenation, we detected blood hormone indexes and gene expression levels in the hypothalamus and ovary of hens from five different periods during FM. Three hormones were identified as participating in FM. Furthermore, the variation trends of gene expression levels in the hypothalamus and ovary at five different stages were found to be basically similar using transcriptome analysis. Among them, 45 genes were found to regulate cell aging during fasting stress and 12 genes were found to promote cell development during the recovery period in the hypothalamus. In addition, five hub genes (INO80D, HELZ, AGO4, ROCK2, and RFX7) were identified by WGCNA. FM can restart the reproductive function of aged hens by regulating expression levels of genes associated with aging and development. Our study not only enriches the theoretical basis of FM but also provides insights for the study of antiaging in humans and the conception mechanism in elderly women.

Integrated Analysis Reveals a lncRNA-miRNA-mRNA Network Associated with Pigeon Skeletal Muscle Development.

Growing evidence has demonstrated the emerging role of long non-coding RNA as competitive endogenous RNA (ceRNA) in regulating skeletal muscle development. However, the mechanism of ceRNA regulated by lncRNA in pigeon skeletal muscle development remains unclear. To reveal the function and regulatory mechanisms of lncRNA, we first analyzed the expression profiles of lncRNA, microRNA (miRNA), and mRNA during the development of pigeon skeletal muscle using high-throughput sequencing. We then constructed a lncRNA-miRNA-mRNA ceRNA network based on differentially expressed (DE) lncRNAs, miRNAs, and mRNAs according to the ceRNA hypothesis. Functional enrichment and short time-series expression miner (STEM) analysis were performed to explore the function of the ceRNA network. Hub lncRNA-miRNA-mRNA interactions were identified by connectivity degree and validated using dual-luciferase activity assay. The results showed that a total of 1625 DE lncRNAs, 11,311 DE mRNAs, and 573 DE miRNAs were identified. A ceRNA network containing 9120 lncRNA-miRNA-mRNA interactions was constructed. STEM analysis indicated that the function of the lncRNA-associated ceRNA network might be developmental specific. Functional enrichment analysis identified potential pathways regulating pigeon skeletal muscle development, such as cell cycle and MAPK signaling. Based on the connectivity degree, lncRNAs TCONS_00066712, TCONS_00026594, TCONS_00001557, TCONS_00001553, and TCONS_00003307 were identified as hub genes in the ceRNA network. lncRNA TCONS_00026594 might regulate the FSHD region gene 1 (FRG1)/ SRC proto-oncogene, non-receptor tyrosine kinase (SRC) by sponge adsorption of cli-miR-1a-3p to affect the development of pigeon skeletal muscle. Our findings provide a data basis for in-depth elucidation of the lncRNA-associated ceRNA mechanism underlying pigeon skeletal muscle development.