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.

Intermittent mild cold acclimation ameliorates intestinal inflammation and immune dysfunction in acute cold-stressed broilers by regulating the TLR4/MyD88/NF-κB pathway.

To investigate the potential protective effect of prior cold stimulation on broiler intestine induced by acute cold stress (ACS). A total of 384 one-day-old broilers were divided into control (CON), ACS, cold stimulation Ⅰ (CS3+ACS), and cold stimulation Ⅱ (CS9+ACS) groups. Broilers in CON and ACS groups were reared normally, and birds in CS3+ACS and CS9+ACS groups were reared at 3℃ and 9℃ below CON group for 5 h, respectively, on alternate days from d 15 to 35. Broilers in ACS, CS3+ACS, and CS9+ACS groups were subjected to 10℃ for 24 h on d 43. Eventually, small intestine tissues were collected for histopathological observation and indexes detection. The results showed that intestinal tissues in all ACS-broilers exhibited inflammatory cell infiltrates, microvilli disruption, reduced villus length in jejunum and increased crypt depth in jejunum and ileum. Whereas these phenomena were relatively light in CS3+ACS group. Compared to CON group, mRNA expression of the TLR4/MyD88/NF-κB pathway-related genes (TLR4, MyD88, NF-κBp65, COX-2, iNOS, PTGEs, TNF-α), Th1/Th17-derived cytokines (IL-1ß, IL-2, IL-8, IL-12, IFN-γ, IL-17), and HSPs (HSP40, HSP60, HSP70, HSP90) was upregulated (P < 0.05), and that of Th2-deviated cytokines (IL-4, IL-6, IL-10, IL-13) and IκBα was downregulated (P < 0.05) in small intestine in almost all ACS-broilers. Compared to ACS group, mRNA expression of most of the TLR4/MyD88/NF-κB pathway-related genes, Th1/Th17-derived cytokines, and HSPs was downregulated and that of Th2-derived cytokines was upregulated in CS3+ACS group (P < 0.05). Protein expression levels of TLR4, MyD88, p-p65/p65, p-IκBα/IκBα, IKK, TNF-α, IL-1ß, IL-10, and HSPs were similar to their mRNA expression. The concentration of sIgA and activities of CAT, SOD, and GSH-px were decreased and MDA and H2O2 were increased in ACS and CS9+ACS groups compared to CON group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to gut immune dysfunction; while mild cold stimulation at 3℃ below normal rearing temperature alleviated cold stress-induced intestinal injure and dysfunction by modulating the TLR4/MyD88/NF-κB pathway in broilers.

Effects of immunosuppression-associated gga-miR-146a-5p on immune regulation in chicken macrophages by targeting the IRKA2 gene.

Stress-induced immunosuppression (SIIS) is one of the common problems in intensive poultry production, which brings enormous economic losses to the poultry industry. Accumulating evidence has shown that microRNAs (miRNAs) were important regulators of gene expression in the immune system. However, the miRNA-mediated molecular mechanisms underlying SIIS in chickens are still poorly understood. This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken SIIS. A stress-induced immunosuppression model was successfully established via daily injection of dexamethasone and analyzed miRNA expression in spleen. Seventy-four differentially expressed miRNAs (DEMs) was identified, and 229 target genes of the DEMs were predicted. Functional enrichment analysis the target genes revealed pathways related to immunity, such as MAPK signaling pathway and FoxO signaling pathway. The candidate miRNA, gga-miR-146a-5p, was found to be significantly downregulated in the Dex-induced chicken spleen, and we found that Dex stimulation significantly inhibited the expression of gga-miR-146a-5p in Chicken macrophages (HD11). Flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8) and other assays indicated that gga-miR-146a-5p can promote the proliferation and inhibit apoptosis of HD11 cells. A dual-luciferase reporter assay suggested that the Interleukin 1 receptor associated kinase 2 (IRAK2) gene, which encoded a transcriptional factor, was a direct target of gga-miR-146a-5p, gga-miR-146a-5p suppressed the post-transcriptional activity of IRAK2. These findings not only improve our understanding of the specific functions of miRNAs in avian stress but also provide potential targets for genetic improvement of stress resistance in poultry.

Integrated transcriptomic analysis on chicken ovary reveals CYP21A1 affects follicle granulosa cell development and steroid hormone synthesis.

Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.

Pigeon novel-miR-741 targets OTUD1 to inhibit proliferation and promote apoptosis of crop fibroblasts.

Trichomonas gallinae (T. gallinae) is a globally distributed protozoan parasite and could cause serious damage to the pigeon industry. MiRNAs have important roles in regulating parasite infection, but its impacts on T. gallinae resistance have rarely been reported. In the present study, we identified a new miRNA (novel-miR-741) and its predicted target OTU deubiquitinase 1 (OTUD1) that might be associated with immunity to T. gallinae in pigeon. Novel-miR-741 and OTUD1 over-expression vectors and interference vectors were constructed. Results from dual luciferase activity assay demonstrated that OTUD1 was a downstream target of novel-miR-741. The Cell Counting Kit-8 and apoptosis assays showed that novel-miR-741 inhibited the proliferation and promoted apoptosis of pigeon crop fibroblasts. Meanwhile, mRNA levels of OTUD1 were significantly reduced in novel-miR-741 mimic-transfected fibroblasts, while mRNA levels of OTUD1 were significantly increased in the novel-miR-741 inhibitor-transfected fibroblasts. The regulatory roles of si-OTUD1 on fibroblasts proliferation, apoptosis, and migration were similar to novel-miR-741 mimic. Our findings demonstrated that novel-miR-741 inhibited the proliferation, and migration of crop fibroblasts, while OTUD1 promoted the proliferation and migration of crop fibroblasts. Therefore, the regulation of OTUD1 by novel-miR-741 was proposed as a potential therapeutic strategy for T. gallinae.

DNA methylation promotes the expression of PPARγ transcript 1 at least in part by preventing NRF1 binding to the promoter P1 of chicken PPARγ gene.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis. Our previous study revealed that chicken PPARγ has 3 alternative promoters named as P1, P2, and P3, and the DNA methylation of promoter P3 was negatively associated with PPARγ mRNA expression in abdominal adipose tissue (AAT). However, the methylation status of promoters P1 and P2 is unclear. Here we assessed promoter P1 methylation status in AAT of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The results showed that promoter P1 methylation differed in AAT between the lean and fat lines of NEAUHLF at 7 wk of age (p < 0.05), and AAT expression of PPARγ transcript 1 (PPARγ1), which was derived from the promoter P1, was greatly higher in fat line than in lean line at 2 and 7 wk of age. The results of the correlation analysis showed that P1 methylation was positively correlated with PPARγ1 expression at 7 wk of age (Pearson's r = 0.356, p = 0.0242), suggesting P1 methylation promotes PPARγ1 expression. To explore the underlying molecular mechanism of P1 methylation on PPARγ1 expression, bioinformatics analysis, dual-luciferase reporter assay, pyrosequencing, and electrophoresis mobility shift assay (EMSA) were performed. The results showed that transcription factor NRF1 repressed the promoter activity of the unmethylated P1, but not the methylated P1. Of all the 4 CpGs (CpG48, CpG49, CpG50, and CpG51), which reside within or nearby the NRF1 binding sites of the P1, only CpG49 methylation in AAT was remarkably higher in the fat line than in lean line at 7 wk of age (3.18 to 0.57, p < 0.05), and CpG49 methylation was positively correlated with PPARγ1 expression (Pearson's r = 0.3716, p = 0.0432). Furthermore, EMSA showed that CpG49 methylation reduced the binding of NRF1 to the P1. Taken together, our findings illustrate that P1 methylation promotes PPARγ1 expression at least in part by preventing NRF1 from binding to the promoter P1.

Effects of L-Leu-L-Leu peptide on growth, proliferation, and apoptosis in broiler intestinal epithelial cells.

Small peptides are nutrients and bioactive molecules that have dual regulatory effects on nutrition and physiology. They are of great significance for maintaining the intestinal health and production performance of broilers. We here cultured the primary small intestinal epithelial cells (IEC) of chicken in a medium containing L-Leu (Leu) and L-Leu-L-Leu (Leu-Leu) for 24 h. The untreated cells were considered as the control group. The growth, proliferation, and apoptosis of IEC were examined. By combining RNA-seq and label-free sequencing technology, candidate genes, proteins, and pathways related to the growth, proliferation, and apoptosis of IEC were screened. Immunofluorescence detection revealed that the purity of the isolated primary IEC was >90%. The Leu-Leu group significantly promoted IEC growth and proliferation and significantly inhibited IEC apoptosis, and the effect was better than those of the Leu and control groups. Using transcriptome sequencing, four candidate genes, CCL20, IL8L1, IL8, and IL6, were screened in the Leu group, and one candidate gene, IL8, was screened in the Leu-Leu group. Two candidate genes, IL6 and RGN, were screened in the Leu-Leu group compared with the Leu group. Nonquantitative proteomic marker sequencing results revealed that through the screening of candidate proteins and pathways, found one growth-related candidate protein PGM3 and three proliferation-related candidate proteins RPS17, RPS11, and RPL23, and two apoptosis-related candidate proteins GPX4 and PDPK1 were found in the Leu-Leu group compared with Leu group. In short, Leu-Leu could promote IEC growth and proliferation and inhibit IEC apoptosis. On combining transcriptome and proteome sequencing technologies, multiple immune- and energy-related regulatory signal pathways were found to be related to IEC growth, proliferation, and apoptosis. Three candidate genes of IL8, IL6, and RGN were identified, and six candidate proteins of PGM3, RPS17, RPS11, RPL23, GPX4, and PDPK1 were involved in IEC growth, proliferation, and apoptosis. The results provide valuable data for preliminarily elucidating small peptide-mediated IEC regulation pathways, improving the small peptide nutrition theoretical system, and establishing small peptide nutrition regulation technology.

Genome-wide association studies of anserine and carnosine contents in the breast meat of Korean native chickens.

Histidine-containing dipeptides (HCDs), such as anserine and carnosine, are enormously beneficial to human health and contribute to the meat flavor in chickens. Meat quality traits, including flavor, are polygenic traits with medium to high heritability. Polygenic traits can be improved through a better understanding of their genetic mechanisms. Genome-wide association studies (GWAS) constitute an effective genomic tool to identify the significant single-nucleotide polymorphisms (SNPs) and potential candidate genes related to various traits of interest in chickens. This study identified potential candidate genes influencing the anserine and carnosine contents in chicken meat through GWAS. We performed GWAS of anserine and carnosine using the Illumina chicken 60K SNP chip (Illumina Inc., San Diego, CA) in 637 Korean native chicken-red-brown line (KNC-R) birds consisting of 228 males and 409 females. The contents of anserine and carnosine in breast meat of KNC-R chickens were investigated. The mean value of the anserine and carnosine are 29.12 mM/g and 10.69 mM/g respectively. The genomic heritabilities were moderate (0.24) for anserine and high (0.43) for carnosine contents. Four and nine SNPs were significantly (P < 0.05) associated with anserine and carnosine, respectively. Based on the GWAS result, the 30.6 to 31.9 Mb region on chicken chromosome 7 was commonly associated with both anserine and carnosine. Through the functional annotation analysis, we identified HNMT and HNMT-like genes as potential candidate genes associated with both anserine and carnosine. The results presented here will contribute to the ongoing improvement of meat quality to satisfy current consumer demands, which are based on healthier, better-flavored, and higher-quality chicken meat.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens.

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

NR5A1 and NR5A2 regulate follicle development in chicken (Gallus gallus) by altering proliferation, apoptosis, and steroid hormone synthesis of granulosa cells.

Chicken ovarian follicle development is regulated by complex and dynamic gene expression. Nuclear receptor 5A1 and 5A2 (NR5A1 and NR5A2, respectively) are key genes that regulate steroid hormone production and gonadal development in mammals; however, studies on follicular development in the chicken ovary are scarce. In this study, we investigated the functions of NR5A1 and NR5A2 on follicle development in chickens. The results showed that the expression of NR5A1 and NR5A2 was significantly higher in small yellow follicles and F5. Furthermore, the expression of NR5A1 and NR5A2 was significantly higher in follicular tissues of peak-laying hens (30 wk) than in follicular tissues of late-laying hens (60 wk), with high expression abundance in granulosa cells (GC). The overexpression of NR5A1 and NR5A2 significantly promoted proliferation and inhibited apoptosis of cultured GC; upregulated STAR, CYP11A1, and CYP19A1 expression and estradiol (E2) and progesterone (P4) synthesis in GC from preovulatory follicles (po-GC); and increased STAR, CYP11A1, and CYP19A1 promoter activities. In addition, follicle-stimulating hormone treatment significantly upregulated NR5A1 and NR5A2 expression in po-GC and significantly promoted FSHR, CYP11A1, and HSD3B1 expression in GC from pre-hierarchical follicles and po-GC. The core promoter region of NR5A1 was identified at the -1,095- to -483-bp and -2,054- to -1,536-bp regions from the translation start site (+1), and the core promoter region of NR5A2 was at -998 to -489 bp. Two single nucleotide polymorphisms (SNP) were identified in the core promoter region of the NR5A1 gene, which differed between high- and low-yielding chicken groups. Our study suggested that NR5A1 and NR5A2 promoted chicken follicle development by promoting GC proliferation and E2 and P4 hormone synthesis and inhibiting apoptosis. Moreover, we identified the promoter core region or functional site that regulates NR5A1 and NR5A2 expression.

Molybdenum and cadmium co-induce apoptosis and ferroptosis through inhibiting Nrf2 signaling pathway in duck (Anas platyrhyncha) testes.

Cadmium (Cd) and high molybdenum (Mo) are injurious to the body. Previous research has substantiated that Cd and Mo exposure caused testicular injury of ducks, but concrete mechanism is not fully clarified. To further survey the toxicity of co-exposure to Cd and Mo in testis, 40 healthy 8-day-old Shaoxing ducks (Anas platyrhyncha) were stochasticly distributed to 4 groups and raised with basic diet embracing Cd (4 mg/kg Cd) or Mo (100 mg/kg Mo) or both. At the 16th wk, testis tissues were gathered. The characteristic ultrastructural changes related to apoptosis and ferroptosis were observed in Mo or Cd or both groups. Besides, Mo or Cd or both repressed nuclear factor erythroid 2-related factor 2 (Nrf2) pathway via decreasing Nrf2, Heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), Glutamate-cysteine ligase catalytic subunit (GCLC) and Glutamate-cysteine ligase modifier subunit (GCLM) mRNA expression of and Nrf2 protein expression, then stimulated apoptosis by elevating Bcl-2 antagonist/killer-1 (Bak-1), Bcl-2-associated X-protein (Bax), Cytochrome complex (Cyt-C), caspase-3 mRNA expression, cleaved-caspase-3 protein expression and apoptosis rate, as well as reducing B-cell lymphoma-2 (Bcl-2) mRNA expression and ratio of Bcl-2 to Bax, and triggered ferroptosis by upregulating Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), transferrin receptor (TFR1) and Prostaglandin-Endoperoxide Synthase 2 (PTGS2) expression levels, and downregulating ferritin heavy chain 1 (FTH1), ferritin light chain 1 (FTL1), ferroportin 1 (FPN1), solute carrier family 7 member 11 (SCL7A11) and glutathione peroxidase 4 (GPX4) expression levels. The most obvious changes of these indexes were observed in co-treated group. Altogether, the results announced that Mo or Cd or both evoked apoptosis and ferroptosis by inhibiting Nrf2 pathway in the testis of ducks, and co-exposure to Mo and Cd exacerbated these variations.

PI3K/AKT pathway modulation and cold acclimation alleviation concerning apoptosis and necroptosis in broiler thymus.

Moderate cold stimulation regulates the thymus's growth and function and facilitates cold acclimatization in broilers. However, the underlying mechanism remains unknown. To explore the possible mechanism of the thymus in cold-acclimated broilers against cold stress, 240 one-day-old Arbor Acres (AA) broilers were assigned to 2 groups randomly. The control group (C) was housed at conventional temperatures. The temperature during the first week was 33°C to 34°C. Between the ages of 8 and 32 d, the temperature was lowered by 1°C every 2 d, i.e., gradually from 32°C to 20°C, and then maintained at 20°C until 42 d of age. The cold-acclimated group (C-3) was housed at the same temperature as C from 1 to 7 d after birth. Between 8 and 42 d, the temperature of C-3 was 3°C colder than C. After 24 h exposure to acute cold stress (ACS) at 42 d, C and C-3 were named as S and S-3. The results showed that ACS was able to induce oxidation stress, modulate PI3K/AKT signal, and cause necroptosis and apoptosis in broiler thymus. By contrast, cold acclimation could alleviate apoptosis and necroptosis induced by cold stress via alleviating oxidative stress, efficiently activating the PI3K/AKT signal, as well as decreasing apoptotic and necrotic genes' levels. This study offers a novel theoretical basis for cold acclimation to improve the body's cold tolerance.

Curcumin alleviates cecal oxidative injury in diquat-induced broilers by regulating the Nrf2/ARE pathway and microflora.

This study evaluated the alleviative effect of curcumin (CUR) on the diquat (DQ)-induced cecal injury in broilers. A total of 320 one-day-old Cobb broilers were selected and randomly divided into 4 treatments, namely control, DQ, CUR 100, and CUR150 groups. The control and DQ groups were fed a basal diet, while the CUR 100 and CUR150 groups were fed the basal diet supplemented with 100 and 150 mg/kg CUR, respectively. Each group had 8 replicates, with 10 broilers per replicate. On day 21 of the experiment, 1 broiler was selected from each replicate and intraperitoneally injected 20 mg/kg body weight of DQ for DQ, CUR 100, and CUR 150 groups. Broilers in control group received equivalent volume of saline. Broilers were euthanized 48h postinjection for tissue sampling. The results showed that DQ injection could cause oxidative stress and inflammatory reactions in the cecum, affecting the fatty acid production and flora structure, thus leading to cecum damage. Compared with the DQ group, the activity of superoxide dismutase, the level of interleukin 10, acetic acid, and total volatile fatty, and the abundance of nuclear factor E2-related factor 2, copper and zinc superoxide dismutase and catalase mRNA in the cecal mucosa of broilers in the CUR group increased significantly (P < 0.05). However, the levels of malondialdehyd, reactive oxygen species, tumor necrosis factor-alpha, and the expression of cysteine-aspartic acid protease-3 and tumor necrosis factor-alpha decreased significantly (P < 0.05) in the CUR group. In addition, CUR treatment alleviated the damage to the cecum and restored the flora structure, and Lactobacillus and Lactobacillaceae promoted the alleviative effect of CUR on DQ. In summary, CUR could alleviate the cecal injury caused by DQ-induced oxidative damage and inflammatory reactions by regulating the Nrf2-ARE signaling pathway and intestinal flora, thus protecting the cecum.

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.