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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., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

A strategy to mitigate the negative effects of stress on animals is to enhance their ability to beneficially respond to stressful conditions. This study aimed to assess whether prenatal ambient temperature influences the response of Japanese quail (Coturnix coturnix japonica) chicks to environmental challenges during growth. The experiment was conducted in a 2 × 2 factorial arrangement: two temperature conditions for the mothers (thermoneutral and heat stress by continuous exposure to 32 °C) and two offspring ambient temperature conditions (thermoneutral and heat stress by intermittent exposure to 34 °C for 6 h/day from 15 to 35 days of age). Heat stress in mothers led to lower laying rate, egg mass, expression of methionine sulfoxide reductase A (MSRA) gene, and antioxidant capacity as well as higher chick mortality rate (1-15 days of age). Maternal heat stress led to lower weight gain and total antioxidant capacity and higher feed conversion ratio. Maternal temperature × Offspring temperature interaction effects were observed on carbonylated protein content and HSP70, GSS, and MSRA gene expression. It was observed that, for chicks hatched from heat-stressed mothers, exposure to heat stress led to higher carbonylated protein content and HSP70 expression than exposure to thermoneutral conditions. Maternal heat stress was also responsible for increasing GSS expression in chicks grown under thermoneutral conditions. Chicks hatched from non-stressed mothers and subjected to heat stress had higher MSRA expression compared to chicks maintained in a thermoneutral environment. Our results show that, although maternal heat stress had no negative effects on performance or oxidative metabolism of offspring grown under thermoneutral conditions, it was associated with lower performance and higher protein oxidation in offspring exposed to heat stress during growth. These results could be due in part to alterations in the expression of genes related to antioxidant capacity.

Myopia, or short-sightedness, is a highly prevalent refractive disorder in which the eye's focal length is too short for its axial dimension in its relaxed state. High myopia is associated with increased risks of blinding ocular complications and abnormal eye shape. In addition to consistent findings on posterior segment anomalies in high myopia (e.g., scleral remodeling), more recent biometric and biomechanical data in myopic humans and animal models also indicate anterior segment anomalies (e.g., corneal biomechanical properties). Because the cornea is the anterior-most ocular tissue, providing essential refractive power and physiological stability, it is important to understand the biochemical signaling pathway during myopia development. This study first aimed to establish the entire chicken corneal proteome. Then, using the classical form deprivation paradigm to induce high myopia in chicks, state-of-the-art bioinformatics technologies were applied to identify eight differentially expressed proteins in the highly myopic cornea. These results provide strong foundation for future corneal research, especially those using chicken as an animal model for myopia development.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies., Competing Interests: The authors have declared that no competing interests exist.

Krüppel-like factor 7 (KLF7) has been reported to inhibit adipogenesis and regulate the development of the nervous system. However, transcription regulation of KLF7 remains poorly understood. In the current study, a 2196-bp-long 5'-flanking sequence of chicken KLF7 (-2286 bp to -91 bp, upstream of the translation start site) was studied for promoter activity, and there was a remarkable promoter activity in this sequence (P<0.05). The 5'-truncated mutation analysis showed that a minimal promoter was on the sequence from -241 bp to -91 bp. In addition, GATA2 overexpression facilitated the promoter activity of pGL3-KLF7(-2286/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91), and GATA3 overexpression inhibited the promoter activity of pGL3-KLF7(-1845/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91) in chicken preadipocytes (P<0.05). Knockdown of GATA2 expression inhibited the promoter activity of pGL3-KLF7(-1215/-91) and pGL3-KLF7(-241/-91), and knockdown of GATA3 expression facilitated the promoter activity of pGL3-KLF7(-521/-91) and pGL3-KLF7(-241/-91) (P<0.05). Additionally, overexpression and knockdown analyses showed that GATA3 inhibited KLF7 mRNA expression (P<0.05), and both overexpression and knockdown of GATA2 resulted in the downregulation of KLF7 mRNA expression in chicken preadipocytes (P<0.05). Western blot analysis in chicken preadipocytes showed that GATA2 facilitated KLF7 expression and GATA3 inhibited KLF7 expression. Mutation analysis showed that the motif of 'GGATCTATCA' (-107 bp/-98 bp) might be a cis-regulation element, which is involved in the KLF7 expression regulation by GATA3 in chicken preadipocytes. These results provided some details of KLF7 transcription regulation in chicken adipose tissue., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail., Competing Interests: The authors have declared that no competing interests exist.

Background: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling., Results: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis., Conclusions: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown., (© 2020 Wiley Periodicals LLC.)

The evolutionarily conserved lethal-7 ( let-7 ) microRNAs (miRNAs) are well-known activators of proliferative quiescence and terminal differentiation. However, in the murine auditory organ, let-7g overexpression delays the differentiation of mechano-sensory hair cells (HCs). To address whether the role of let-7 in auditory-sensory differentiation is conserved among vertebrates, we manipulated let-7 levels within the chicken auditory organ: the basilar papilla. Using a let-7 sponge construct to sequester let-7 miRNAs, we found that endogenous let-7 miRNAs are essential for limiting the self-renewal of HC progenitor cells. Furthermore, let-7b overexpression experiments revealed that, similar to mice, higher than normal let-7 levels slow/delay HC differentiation. Finally, we identify CHD7, a chromatin remodeler, as a candidate for mediating the repressive function of let-7 in HC differentiation and inner ear morphogenesis. Our analysis uncovered an evolutionarily conserved let-7-5p -binding site within the chicken Chd7 gene and its human and murine homologs, and we show that let-7g overexpression in mice limits CHD7 expression in the developing inner ear, retina and brain. Haploinsufficiency of CHD7 in humans causes CHARGE syndrome and attenuation of let-7 function may be an effective method for treating CHD7 deficiency., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFβ pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Fluorescence-activated cell sorting (FACS) in combination with yeast surface display has emerged as a vital tool for the isolation and engineering of antibodies and antibody-derived fragments from synthetic, naïve, and immune libraries. However, the generation of antibodies against certain human antigens from immunized animals, e.g., mice, can remain challenging due to the homology to the murine counterpart. Due to the phylogenetic distance from humans, avian immunization can be a powerful technique for the generation of antibodies with high specificity against human antigens. Additionally, the peculiar Ig gene diversification in chickens enables the amplification of heavy and light chain genes utilizing single primer pairs, resulting in a convenient library generation. Herein, we describe the protocol for the construction of a single chain fragment variable (scFv) library derived from chickens after immunization with epidermal growth factor receptor (EGFR) for subsequent yeast surface display as well as the screening process utilizing FACS for the isolation of high-affinity antibodies.

This study was conducted to explore the regulatory role of the target of rapamycin complex 1 (TORC1) signaling pathway in crop milk synthesis in breeding pigeons (Columba livia). Three groups of breeding pigeons in the lactation period (n = 30 pairs/group) were respectively injected with rapamycin (RAPA, a specific inhibitor of the target of rapamycin complex) at doses of 0 (vehicle, control), 0.6, or 1.2 mg/kg body weight (BW)/day via the wing vein for 7 days. The average daily feed intake (ADFI) and BW of the breeding pigeons and the BW of young squabs were respectively recorded throughout the experimental period. The breeding pigeons were sacrificed to collect their crop tissues, crop milk, and serum on the eighth day of the experiment. The results showed that neither 0.6 nor 1.2 mg/kg BW RAPA injection affected BW loss or ADFI in breeding pigeons (P > 0.05), while crop thickness and crop relative weight were significantly decreased (P < 0.05) in the 1.2 mg/kg BW rapamycin-injected group. Simultaneously, RAPA (especially at 1.2 mg/kg BW) decreased the crude protein, α s1 -casein, α s2 -casein, β-casein, and amino acid contents (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Lys, His, Arg, and Pro) of crop milk (P < 0.05) and the concentrations of albumin, total protein, and uric acid in the serum of breeding pigeons (P < 0.05). Additionally, the expression of TORC1 pathway-related proteins (TORC1, S6K1, S6, 4EBP1, and eIF4E) was downregulated in the crop tissues of breeding pigeons by 0.6 or 1.2 mg/kg BW/day RAPA injection (P < 0.05). Accordingly, the average daily gain (ADG) of young squabs declined, and the mortality rate increased significantly (P < 0.05). Together, the results showed that RAPA reduced protein and amino acid levels in the crop milk of breeding pigeons and retarded young squab growth, suggesting a crucial role of TORC1 in crop milk synthesis in breeding pigeons., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The impact of physiological stress on lipid metabolism, the metabolome, and systemic responses was examined in chickens. To incite a stress response, birds were continuously administered corticosterone (CORT) in their drinking water at three doses (0 mg/L, 10 mg/L, and 30 mg/L), and they were sampled 1, 5, and 12 days after commencement of CORT administration. Corticosterone administration to birds differentially regulated lipogenesis genes (i.e. FAS, ACC, ME, and SREBF1), and histopathological examination indicated lipid deposition in hepatocytes. In addition, CORT affected water-soluble metabolite profiles in the liver, as well as in kidney tissue and breast muscle; thirteen unique metabolites were distinguished in CORT-treated birds and this was consistent with the dysregulation of lipid metabolism due to physiological stress. Acute phase responses (APRs) were also altered by CORT, and in particular, expression of SAA1 was decreased and expression of CP was increased. Furthermore, CORT administration caused lymphoid depletion in the bursa of Fabricius and elevated IL6 and TGFβ2 mRNA expression after 5 and 12 days of CORT administration. Collectively, incitement of physiological stress via administration of CORT in chickens modulated host metabolism and systemic responses, which indicated that energy potentials are diverted from muscle anabolism during periods of stress.

In mammalian skeletal muscles, protein synthesis rates vary according to fiber types. We herein demonstrated differences in the regulatory mechanism underlying the protein synthesis in the pectoralis major (a glycolytic twitch muscle), adductor superficialis (an oxidative twitch muscle), and adductor profound (a tonic muscle) muscles of 14-day-old chickens. Under ad libitum feeding conditions, protein synthesis is significantly higher in the adductor superficialis muscle than in the pectoralis major muscle, suggesting that protein synthesis is upregulated in oxidative muscles in chickens, similar to that in mammals. In the pectoralis major muscle, fasting significantly inhibited the Akt/S6 pathway and protein synthesis with a corresponding decrease in plasma insulin concentration. Conversely, the insulin like growth factor-1 (IGF-1) mRNA levels significantly increased. These findings suggest that the insulin/Akt/S6 pathway plays an important role in the regulation of protein synthesis in the pectoralis major muscle. Interestingly, protein synthesis in the adductor superficialis muscle appears to be regulated in an Akt-independent manner, because fasting significantly decreased S6 phosphorylation and protein synthesis without affecting Akt phosphorylation. In the adductor profound muscle, IGF-1 expression, phosphorylation of Akt and S6, and protein synthesis were decreased by fasting, suggesting that insulin and/or skeletal IGF-1 appear contribute to protein synthesis via the Akt/S6 pathway. These findings revealed the differential regulation of protein synthesis depending on skeletal muscle types in chickens., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Developmental conditions can impact the adult phenotype via epigenetic changes that modulate gene expression. In mammals, methylation of the glucocorticoid receptor gene Nr3c1 has been implicated as mediator of long-term effects of developmental conditions, but this evidence is limited to humans and rodents, and few studies have simultaneously tested for associations between DNA methylation, gene expression and phenotype. Adverse environmental conditions during early life (large natal brood size) or adulthood (high foraging costs) exert multiple long-term phenotypic effects in zebra finches, and we here test for effects of these manipulations on DNA methylation and expression of the Nr3c1 gene in blood. Having been reared in a large brood induced higher DNA methylation of the Nr3c1 regulatory region in adulthood, and this effect persisted over years. Nr3c1 expression was negatively correlated with methylation at 2 out of 8 CpG sites, and was lower in hard foraging conditions, despite foraging conditions having no effect on Nr3c1 methylation at our target region. Nr3c1 expression also correlated with glucocorticoid traits: higher expression level was associated with lower plasma baseline corticosterone concentrations and enhanced corticosterone reactivity. Our results suggest that methylation of the Nr3c1 regulatory region can contribute to the mechanisms underlying the emergence of long-term effects of developmental conditions in birds, but in our system current adversity dominated over early life experiences with respect to receptor expression.

Increased exposure to light pollution perturbs physiological processes through misalignment of daily rhythms at the cellular and tissue levels. Effects of artificial light-at-night (ALAN) on diel properties of immunity are currently unknown. We therefore tested the effects of ALAN on diel patterns of cytokine gene expression, as well as key hormones involved with the regulation of immunity, in zebra finches (Taeniopygia guttata). Circulating melatonin and corticosterone, and mRNA expression levels of pro- (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines were measured at six time points across 24-h day in brain (nidopallium, hippocampus, and hypothalamus) and peripheral tissues (liver, spleen, and fat) of zebra finches exposed to 12 h light:12 h darkness (LD), dim light-at-night (DLAN) or constant bright light (LLbright). Melatonin and corticosterone concentrations were significantly rhythmic under LD, but not under LLbright and DLAN. Genes coding for cytokines showed tissue-specific diurnal rhythms under LD and were lost with exposure to LLbright, except IL-6 in hypothalamus and liver. In comparison to LLbright, effects of DLAN were less adverse with persistence of some diurnal rhythms, albeit with significant waveform alterations. These results underscore the circadian regulation of biosynthesis of immune effectors and imply the susceptibility of daily immune and endocrine patterns to ALAN.

Leucine (Leu) plays a critical regulatory role in protein synthesis, however, the effects and molecular mechanisms of Leu on crop milk protein in the domestic pigeons (Columba livia) are still unknown. Therefore, the study aimed to investigate the effects of dietary Leu supplementation on crop milk protein synthesis and the growth performance of squabs and the possible underlying mechanism. A total of 240 pairs of breeding pigeons (1102.3 ± 9.5 g/pair) were randomly assigned to 1 of 5 treatments, including a positive control (PC) diet that had adequate crude protein (crude protein, CP = 18%; Leu = 1.30%), a negative control (NC) diet that was low in CP (CP = 16%, Leu = 1.30%), and NC diets supplemented with Leu at 0.15%, 0.45%, or 1.05%. Compared with the NC diet, 0.15 to 0.45% Leu supplementation decreased BW loss and increased relative crop weight, crop thickness, and protein levels in the crop tissue and milk of breeding pigeons. However, dietary supplementation with 1.05% Leu inhibited ADFI in breeding pigeons. Dietary supplementation with 0.15 to 0.45% Leu decreased the mortality rate and increased the BW, eviscerated yield, and breast muscle yield of young squabs. The protein expression levels of the target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1), ribosomal protein S6 kinase (S6), eukaryotic initiation factor 4E binding protein 1 (4EBP1), and eukaryotic translation initiation factor 4E (eIF4E) were upregulated in the crop tissue of breeding pigeons in PC, 0.15% and 0.45% Leu-supplemented groups. Collectively, these results indicated that 0.15 to 0.45% Leu supplementation could decrease BW loss, increase milk protein synthesis in the crop of breeding pigeons, and enhance the survival rate and growth performance of young squabs through the TOR signaling pathway., (© 2019 Poultry Science Association Inc.)

Tree swallows (Tachycineta bicolor) are one of the most commonly studied wild birds in North America. They have advanced numerous research areas, including life history, physiology, and organismal responses to global change; however, transcriptomic resources are scarce. To further advance the utility of this system for biologists across disciplines, we generated a transcriptome for the tree swallow using six tissues (brain, blood, ovary, spleen, liver, and muscle) collected from breeding females. We de novo assembled 207,739 transcripts, which we aligned to 14,717 high confidence protein-coding genes. We then characterized each tissue with regard to its unique genes and processes and applied this transcriptome to two fundamental questions in evolutionary biology and endocrinology. First, we analyzed 3,015 single-copy orthologs and identified 46 genes under positive selection in the tree swallow lineage, including those with putative links to adaptations in this species. Second, we analyzed tissue-specific expression patterns of genes involved in sex steroidogenesis and processing. Enzymes capable of synthesizing these behaviorally relevant hormones were largely limited to the ovary, whereas steroid binding genes were found in nearly all other tissues, highlighting the potential for local regulation of sex steroid-mediated traits. These analyses provide new insights into potential sources of phenotypic variation in a free-living female bird and advance our understanding of fundamental questions in evolutionary and organismal biology.

Bitterness is an important taste sensation for chickens, which provides useful sensory information for acquisition and selection of diet, and warns them against ingestion of potentially harmful and noxious substances in nature. Bitter taste receptors (T2Rs) mediate the recognition of bitter compounds belonging to a family of proteins known as G-protein coupled receptors. The aim of this study was to identify and evaluate the expression of T2R7 in chicken tongue tissue and construct cT2R7-1 and cT2R7-2- expressing HEK-293T cells to access the expression of PLCβ2 and ITPR3 after exposure with different concentrations of the bitter compounds. Using real-time PCR, we show that the relative expression level of T2R7 mRNA in 5, 1, 0.1, and 10 -3  mM of camphor and erythromycin solutions and 5 mM of chlorpheniramine maleate solutions was significantly higher than that in 50 mM KCL solutions. We confirmed that the bitter taste receptor T2R7 and downstream signaling effectors are sensitive to different concentrations of bitter compounds. Moreover, T2R7-1 (corresponding to the unique haplotype of the Tibetan chicken) had higher sensitivity to bitter compounds compared with that of T2R7-2 (corresponding to the unique haplotype of the Jiuyuan black-chicken). These results provide great significance of taste response on dietary intake to improve chicken feeding efficiency in poultry production and have certain reference value for future taste research in other bird species., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Yuan Su et al.)

Ariadne homolog 2 (ARIH2), as an E3 ubiquitin ligase, is one of the important factors involved in regulating biological functions, such as inflammation and skeletal muscle degeneration. In the present study, the full-length coding sequence of Arih2 gene was cloned from Hy-Line Brown chicken. The tissue transcriptional profiles of Arih2 gene at different developmental stages were detected using quantitative real-time PCR (qRT-PCR), and the Arih2 functional characteristics in immune response were analyzed. The results showed that the full-length coding sequence of Arih2 gene was 1473 bp, encoding 490 amino acids, and conservative between different species. The Arih2 gene was transcribed in various tissues at different developmental stages, and its transcriptional activities varied significantly between multiple tissues. With the development of chicken, Arih2 gene was basically up-regulated in heart, liver, kidney, skeletal muscle and glandular stomach, but fluctuated significantly in large intestine. In immune response, the transcriptional activities of Arih2 gene exhibited significant changes in the bursa, thymus and blood (P<0.05). The results showed that Arih2 might be a multifunctional gene involved in tissue development and immune response in chicken, and have a potential possible application as diagnostic marker for identifying immune response., (© 2019 The Author(s).)

We investigated the molecular basis of seasonal testicular states in black-headed buntings (Emberiza melanocephala), in which seasonal migrations limit reproduction to a narrow time-window during the year. We examined testicular gene expressions in buntings during short day-induced photosensitive nonreproductive state (SDSE), and during long day-induced early (LDES) and late (LDLS) gonadal maturation and regressed photorefractory (LDRF) seasonal states. Using Kallisto, we pseudoaligned the RNA-Seq transcripts and calculated the transcript abundance. We found 1,799 differentially expressed genes (DEGs) between the four photoperiod-induced seasonal states. Further pairwise comparison with SDSE revealed 1,187, 1,224, and 1,238 DEGs in LDES, LDLS, and LDRF, respectively; 852 genes were common to three comparisons. We then identified genetic pathways putatively involved in seasonal testicular cycle. DEGs that enriched calcium ion binding were highly expressed in testicular maturation states. Similarly, DEGs that enriched glycolytic pathway were highly expressed in LDES and LDRF states. More specifically, quantitative polymerase chain reaction showed significant differences between the photoperiod-induced states in testicular expression of genes (HOOK1, RGS2, PRDX4, BCL6, CYFIP2, PDCD4, P2RX4, and GABRA1) involved in gametogenesis and associated pathways. Overall, these results show significant transcriptional differences, and provide insights into the molecular basis of seasonal changes in the reproductive life-history states in a photoperiodic species., (© 2019 Wiley-Liss, Inc., A Wiley Company.)

Intramuscular fat (IMF) traits are important factors that influence meat quality. However, the molecular regulatory mechanisms that underlie this trait in chickens are still poorly understood at the gene coexpression level. Here, we performed a weighted gene coexpression network analysis between IMF traits and transcriptome profile in breast muscle in the Chinese domestic Gushi chicken breed at 6, 14, 22, and 30 weeks. A total of 26 coexpressed gene modules were identified. Six modules, which included the dark gray, purple, cyan, pink, light cyan, and blue modules, showed a significant positive correlation (P < 0.05) with IMF traits. The strongest correlation was observed between the dark gray module and IMF content (r = 0.85; P = 4e-04) and between the blue module and different fatty acid content (r = 0.87~0.91; P = 5e-05~2e-04). Enrichment analysis showed that the enrichment of biological processes, such as fatty acid metabolic process, fat cell differentiation, acylglycerol metabolic process, and glycerolipid metabolism were significantly different in the six modules. In addition, the 32, 24, 4, 7, 6, and 25 hub genes were identified from the blue, pink, light cyan, cyan, dark gray, and purple modules, respectively. These hub genes are involved in multiple links to fatty acid metabolism, phospholipid metabolism, cholesterol metabolism, diverse cellular behaviors, and cell events. These results provide novel insights into the molecular regulatory mechanisms for IMF-related traits in chicken and may also help to uncover the formation mechanism for excellent meat quality traits in local breeds of Chinese chicken., (© 2019 Wiley Periodicals, Inc.)

Much attention has been paid to the broodiness of the Muscovy duck, but the molecular mechanism of broodiness remains largely unknown. In this study, the ovary tissues of Muscovy ducks during the broody and laying periods were used to investigate differentially expressed proteins (DEPs) by the iTRAQ-based proteomics approach. A total of 335 DEPs were identified, including 139 up-regulated and 196 down-regulated proteins. Six proteins (APOV1, GAL, SAA, GNB5, VLDLR and CDK1) with higher changes in expression were selected, and these proteins are mainly involved in the pathways related to reproductive performance, such as Oocyte meiosis, and PI3K-Akt signaling pathway. Steroid biosynthesis was the most significantly enriched pathway by KEGG pathway enriched analysis. The qRT-PCR analysis was applied to verify the proteomic analysis. Meanwhile, metabolomics analysis found that several important differentially expressed metabolites (DEMs) (7-dehydrodesmosterol, 25-Hydroxyvitamin D3, 7-Dehydrocholesterol, Pregnanolone, Allopregnanolone and estrogen) that were also mainly involved in Steroid biosynthesis, Steroid hormone biosynthesis and Metabolic pathways. Crucially, the changes in the abundance of these metabolites are closely related to the changes in the protein abundance of proteins identified in the same pathway, and it is always the upstream key enzymes that influence the production of downstream metabolites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The goose is one of the most important waterfowl, having lowing laying rate. Previous studies have shown the SNPs in the introns of MAGI-1 (Record-106975) and ACSF2 (Record-106582) significantly associated with egg production in geese. However, the mechanism of those SNPs influencing egg production remains unclear. In this study, the three goose breeds (Yangzhou geese, Zhedong white geese, and Carlos geese) with obviously different egg production were selected, and the allele frequency distribution and functions of those SNPs were investigated. The results suggested that the allele frequency distribution of ACSF2 was significantly different among the three goose breeds (χc 2  = 92.377, Pc = 2.29 × 10 -22 ), with the C allele appearing at frequencies of 0.29 in the Yangzhou geese and 0.94 in the Carlos geese. In contrast, the allele frequencies of MAGI-1 were not significantly different among the different goose breeds. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression of MAGI-1 with the AG genotype individuals was significantly higher than those of the AA and GG genotype. For ACSF2, the CC genotype had significantly higher expression than both the AC genotype and the AA genotype. The luciferase reporter analysis revealed that the site-directed mutation ACSF2 (A>C) significantly drove the expression activity. Further analysis suggested that the mutation altered the binding site of the transcription factor BARHL2. Binding of BARHL2 to the ACSF2 intron was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the A>C mutation of ACSF2 (Record-106582) could promote the expression by regulating the binding of BARHL2, resulting in differences in egg performance, which provided molecular insights into the effect of the polymorphism in ACSF2 on egg performance in geese., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Avian pathogenic Escherichia coli (APEC) causes great economic loss to the poultry industry worldwide. Chicken type II pneumocytes (CP II cells) secrete surfactants and modulate lung immunity to decrease the infection of the invading pathogen. Nevertheless, the pathogenesis of CP II cells to APEC infection remains poorly understood. Therefore, we conducted global gene expression profiling of CP II cells after APEC-O78 infection to explore the host-pathogen interaction. The differentially expressed genes of CP II cells to APEC infection were characterized by RNA-seq with EB-seq algorithm. In consequence, the mRNA of 18996 genes was identified, and CP II cells responded to APEC infection with marked changes in the expression of 1390 genes. Among them, there are 803 down-regulated mRNAs and 587 up-regulated mRNAs. The KEGG prediction and Gene Ontology terms analysis revealed that the major enriched pathways were related to NF-κB signaling pathway, apoptosis pathway, tight junction, and cytokine-cytokine receptor interaction and other pathways. We adopted qRT-PCR to verify the validity of the selected gene expression. The fold induction of qPCR was similar to the RNA-seq results. These results provide a better understanding of the pathogenesis of APEC, especially apoptosis pathway involved in APEC infection., Competing Interests: The authors have declared that no competing interests exist.

Background: Green light penetrates the skull and has directly affected on the secretion of melatonin in plasma, which regulates the endocrine activities to influence the muscle growth, satellite cell mitotic activity and quality properties of meat from the embryonic period to posthatch in chick. Pituitary adenylate cyclase-activating polypeptide 6-38 (PACAP6-38) could inhibit the synthesis and secretion of pineal melatonin. Finding a new way for exploring the mechanism of light-regulated muscle growth in ovo is essential for promoting the productive performance in poultry., Methods: Chick embryos were exposed to darkness (D-group) and green light (G-group) throughout the embryonic period, and injected with PACAP6-38 or saline at embryonic day 8. Plasma hormone, skeletal muscle fiber areas, satellite cell proliferation activity, paired domain homeobox transcription factor 7 and myogenic regulatory factors were observed., Results: By saline treatment, the percentage of proliferating cell nuclear antigen immunoreactive cells and mitotic activity of satellite cells in skeletal muscle were higher in G-group than those of in D-group at post-hatching day 0. With the increase of plasma melatonin, green light promoted the secretion of growth hormone (GH) and insulin like factor 1 (IGF-1) in plasma, the satellite cell proliferation, the size of muscle fiber, as well as the mRNA expressions of Pax7, myogenic regulatory factors and IGF-1R. After PACAP6-38 treatment to inhibit the secretion of melatonin in ovo, aforementioned parameters were remarkably decreased and the difference of these parameters was disappeared between D-group and G-group., Conclusion: These data indicated that stimulation with monochromatic green light during incubation enhanced the secretion of melatonin and up-regulation of GH-IGF-1 axis to activate the satellite cells proliferation and myofiber formation, involving the expression of Pax7 and myogenic regulatory factors., Competing Interests: The authors have declared that no competing interests exist.

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.

Exposure to dioxin-like compounds is consistently associated with concentration-dependent induction of cytochrome P4501A (CYP1A) enzymes in primary cultures of avian hepatocytes. We have previously demonstrated that the median effective concentration (EC50) for induction of this response is predictive of in vivo sensitivity to dioxin-like compounds in birds. We investigated sources of interindividual variation in the CYP1A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild herring gulls and considered how this variation may complicate dioxin sensitivity estimates based on the CYP1A bioassay. Concentration-dependent effects of TCDD on CYP1A mRNA expression were characterized in 55 hepatocyte cultures prepared from individual herring gull embryos. A large degree of variability was observed among the hepatocyte culture preparations. For example, 1) basal CYP1A4 and CYP1A5 mRNA expression varied by 20- and 126-fold, respectively, among individuals, and 2) exposure to TCDD induced CYP1A4 mRNA expression by 57-fold in the most responsive sample but did not significantly induce CYP1A4 mRNA expression above baseline values in 42% of hepatocyte culture preparations. Environmental and genetic factors contributing to the observed variability are discussed. Despite the large amount of interindividual variation, we conclude that reproducible EC50-based estimates of species sensitivity can be obtained from the CYP1A cell culture bioassay when samples are collected from relatively uncontaminated colonies. Environ Toxicol Chem 2019;38:660-670. © 2019 SETAC., (© 2019 SETAC.)

Melatonin is a crucial neurohormone synthesized in the pineal gland that influences the physiology of animals. The molecular mechanism of norepinephrine control of the synthesis of melatonin is well documented; however, few reports have described the effects of epinephrine on the synthesis of melatonin. In this study, the effect of epinephrine on melatonin synthesis was investigated by adding different concentrations of epinephrine or norepinephrine to broiler pineal glands cultured in vitro. In addition, we investigated how epinephrine regulates the synthesis of melatonin and the transcription of the key melatonin synthesis enzyme AANAT. We determined the abundance of melatonin, norepinephrine, and epinephrine in broiler serum and the mRNA expression levels of key enzymes under different light conditions. The minimum concentrations of epinephrine and norepinephrine required to recover the melatonin synthesis rhythm in pineal cells were 10 -13 and 10 -11  mol/L, respectively. Under various light durations, epinephrine reached maximum levels two hours earlier than melatonin. These results demonstrate for the first time that epinephrine can increase the synthesis of melatonin by increasing the transcription of AANAT.

High-throughput sequencing and proteomics technologies are markedly increasing the amount of RNA and peptide data that are available to researchers, which are typically made publicly available via data repositories such as the NCBI Sequence Read Archive and proteome archives, respectively. These data sets contain valuable information about when and where gene products are expressed, but this information is not readily obtainable from archived data sets. Here we report Chickspress (http://geneatlas.arl.arizona.edu), the first publicly available gene expression resource for chicken tissues. Since there is no single source of chicken gene models, Chickspress incorporates both NCBI and Ensembl gene models and links these gene sets with experimental gene expression data and QTL information. By linking gene models from both NCBI and Ensembl gene prediction pipelines, researchers can, for the first time, easily compare gene models from each of these prediction workflows to available experimental data for these products. We use Chickspress data to show the differences between these gene annotation pipelines. Chickspress also provides rapid search, visualization and download capacity for chicken gene sets based upon tissue type, developmental stage and experiment type. This first Chickspress release contains 161 gene expression data sets, including expression of mRNAs, miRNAs, proteins and peptides. We provide several examples demonstrating how researchers may use this resource., (© The Author(s) 2019. Published by Oxford University Press.)

CCAAT/enhancer binding protein α, β, δ (C/EBP α, β, δ) are essential transcriptional factors in regulating adipose development. However, information about their sequence characteristics and functions during adipocyte development still remains scarce in birds. In present study, we found that duck C/EBP α, β, δ differed in their phosphorylation sites and low complexity regions (LCRs) among their orthologs and paralogs. Phylogenetic analysis showed that C/EBP α, β, δ had different evolutionary patterns, and each of duck C/EBP α, β, δ was strikingly diverged from orthologs of other Aves. Results of quantitative real-time PCR exhibited that C/EBP α, β, δ were all highly expressed in duck adipose tissues. Indeed, investigations of changes in both their mRNA levels and lipid droplet content during duck adipocytes differentiation showed that their expression profiles were closely related to cellular lipid accumulation. Furthermore, hierarchical clustering analysis of the C/EBPs and lipid metabolism-related genes expression profiles showed that C/EBP α was clustered with genes related to lipolysis, lipogenesis and fatty acid desaturation, whereas C/EBP β, δ were clustered with genes related to de novo lipogenesis and fatty acid elongation, which were different from mammals. In summary, C/EBP α, β, δ of duck differ from other species in their structures and have different effects on lipid metabolism during adipocytes differentiation. This research serve as a foundation for further investigations about avian C/EBP α, β, δ in adipocytes differentiation and adipose development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the chicken, sex determination relies on a ZZ (male)/ZW (female) chromosomal system, but underlying mechanisms are still not fully understood. The Z-dosage and the dominant W-chromosome hypotheses have been proposed to underlie primary sex determination. We present a modelling approach, which assembles the current knowledge and permits exploration of the regulation of this process in chickens. Relying on published experimental data, we assembled a gene network, which led to a logical model that integrates both the Z-dosage and dominant W hypotheses. This model showed that the sexual fate of chicken gonads results from the resolution of the mutual inhibition between DMRT1 and FOXL2, where the initial amount of DMRT1 product determines the development of the gonads. In this respect, at the initiation step, a W-factor would function as a secondary device, by reducing the amount of DMRT1 in ZW gonads when the sexual fate of the gonad is settled, that is when the SOX9 functional level is established. Developmental constraints that are instrumental in this resolution were identified. These constraints establish qualitative restrictions regarding the relative transcription rates of the genes DMRT1, FOXL2 and HEMGN. Our model further clarified the role of OESTROGEN in maintaining FOXL2 function during ovary development., (© 2018 The Authors.)

Tetramethylpyrazine (TMP) is a biologically active ingredient, which is isolated from a popularChinese medicinal plant. It has been used effectively to treat ischemic heart problems, cerebrovascular and thrombotic vascular diseases. This study was designed to evaluate the effect of TMP on calciumsensing receptors in pulmonary artery smooth muscle in chickens. For this purpose forty day-old chicks were distributed into five groups: the control group, the hypoxia group (kept under low Oxygen treatment), and TMP groups (kept under low Oxygen treatment along with treatment of different concentrations of TMP). The pulmonary artery smooth muscle cells were also cultured on 6-well plates in high glucose culture medium and divided into the same five groups. We used in vivo and in vitro study models by applying immunohistochemistry, RT-qPCR assay and Western blotting analysis. Our results showed that pre-incubation with hypoxia markedly stimulated the activation of calcium-sensing receptor (CaSR) in pulmonary artery smooth muscle cells (PASMCs). The TMP decreased the mRNA and protein levels of CaSR. Treatment with TMP clearly inhibited the activation of all CaSR in a dose-dependent manner. Our data demonstrated that TMP can down-regulate the expression of CaSR. Therefore, these findings provide a new target to treat pulmonary arterial hypertension (PAH) under hypoxic conditions.

Background: MC1R, a G-protein coupled receptor with high affinity for alpha-melanocyte stimulating hormone (αMSH), modulates pigment production in melanocytes from many species and is associated with human melanoma risk. MC1R mutations affecting human skin and hair color also have pleiotropic effects on the immune response and analgesia. Variants affecting human pigmentation in utero alter the congenital phenotype of both oculocutaneous albinism and congenital melanocytic naevi, and have a possible effect on birthweight., Methods and Results: By in situ hybridization, RT-PCR and immunohistochemistry, we show that MC1R is widely expressed during human, chick and mouse embryonic and fetal stages in many somatic tissues, particularly in the musculoskeletal and nervous systems, and conserved across evolution in these three amniotes. Its dynamic pattern differs from that of TUBB3, a gene overlapping the same locus in humans and encoding class III β-tubulin. The αMSH peptide and the transcript for its precursor, pro-opiomelanocortin (POMC), are similarly present in numerous extra-cutaneous tissues. MC1R genotyping of variants p.(V60M) and p.(R151C) was undertaken for 867 healthy children from the Avon Longitudinal Study of Parent and Children (ALSPAC) cohort, and birthweight modeled using multiple logistic regression analysis. A significant positive association initially found between R151C and birth weight, independent of known birth weight modifiers, was not reproduced when combined with data from an independent genome-wide association study of 6,459 additional members of the same cohort., Conclusions: These data clearly show a new and hitherto unsuspected role for MC1R in noncutaneous solid tissues before birth., (© 2018 Wiley Periodicals, Inc.)

The light-dependent magnetic compass of birds provides orientation information about the spatial alignment of the geomagnetic field. It is proposed to be located in the avian retina, and be mediated by a light-induced, biochemical radical-pair mechanism involving cryptochromes as putative receptor molecules. At the same time, cryptochromes are known for their role in the negative feedback loop in the circadian clock. We measured gene expression of Cry1, Cry2 and Cry4 in the retina, muscle and brain of zebra finches over the circadian day to assess whether they showed any circadian rhythmicity. We hypothesized that retinal cryptochromes involved in magnetoreception should be expressed at a constant level over the circadian day, because birds use a light-dependent magnetic compass for orientation not only during migration, but also for spatial orientation tasks in their daily life. Cryptochromes serving in circadian tasks, on the other hand, are expected to be expressed in a rhythmic (circadian) pattern. Cry1 and Cry2 displayed a daily variation in the retina as expected for circadian clock genes, while Cry4 expressed at constant levels over time. We conclude that Cry4 is the most likely candidate magnetoreceptor of the light-dependent magnetic compass in birds., (© 2018 The Author(s).)

Neuromedin U (NMU) and its structurally-related peptide, neuromedin S (NMS), are reported to regulate many physiological processes and their actions are mediated by two NMU receptors (NMUR1, NMUR2) in mammals. However, the information regarding NMU, NMS, and their receptors is limited in birds. In this study, we examined the structure, functionality, and expression of NMS, NMU, NMUR1 and NMUR2 in chickens. The results showed that: 1) chicken (c-) NMU cDNA encodes a 181-amino acid precursor, which may generate two forms of NMU peptide with 9 (cNMU-9) and 25 amino acids (cNMU-25), respectively. 2) Interestingly, two cNMS transcripts encoding two cNMS precursors of different lengths were identified from chicken pituitary, and both cNMS precursors may produce a mature cNMS peptide of 9 amino acids (cNMS-9). 3) cNMU-9, cNMU-25 and cNMS-9 could activate cNMUR1 expressed in HEK293 cells potently, as monitored by three cell-based luciferase reporter systems, indicating that cNMUR1 can act as a receptor common for cNMU and cNMS peptides, whereas cNMUR2 could be potently activated by cNMS-9, but not by cNMU-9/cNMU-25. 4) cNMU and cNMUR1 are widely expressed in chicken tissues with abundant expression noted in the gastrointestinal tract, as detected by quantitative real-time PCR, whereas cNMUR2 expression is mainly restricted to the brain and anterior pituitary, and cNMS is widely expressed in chicken tissues. Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of the avian lumbosacral spinal cord. Histological evidence indicates that neuron-like cells gather in the ALs, and behavioral evidence suggests that the ALs act as a sensory organ of equilibrium during bipedal walking. Recently, using an electrophysiological method, we reported that cells showing Na + currents and action potentials exist among cells that were dissociated from the ALs. However, it was unclear which isoforms of the voltage-gated sodium channel (VGSC) are expressed in the ALs and whether cells having neuronal morphology in the ALs express VGSCs. To elucidate these points, RT-PCR and immunohistochemical experiments were performed. In RT-PCR analysis, PCR products for Na v 1.1-1.7 were detected in the ALs. The signal intensities of the Na v 1.1 and 1.6 isoforms were stronger than those of the other isoforms. We confirmed that an antibody raised against an epitope peptide of the rat VGSC had cross-reactivity to chick tissues by Western blotting, and we performed immunofluorescence staining using the antibody. The AL contained cells having neuron-like morphology and VGSC-like immunoreactivity at their cytoplasm and/or cell membranes. Filament-like structures showing GFAP-like immunoreactivity infilled intercellular spaces. The VGSC- and GFAP-like immunoreactivities did not overlap. These results indicate that the neuronal isoforms of the VGSC are mainly expressed in the AL and that the neuron-like cells in the ALs express VGSCs. Our findings indicate that AL neurons generate action potentials and send sensory information to the motor systems on the contralateral side of the spinal segment.

In palmipeds, overfeeding leads to hepatic steatosis, also called "foie gras" which is the result of many metabolic mechanisms. In order to understand these mechanisms, we decided to measure the expression of genes implicated in lipid metabolism during 12 hours (h) following the last meal of the overfeeding period. We have shown that there is a precocious expression (within 2 h) of fatty acid synthase and acyl CoA synthetase long-chain 1 in liver and muscle of mule ducks in addition with a later peak. Furthermore, di-acyl glycerol acyl transferase presents the highest induction of expression in liver and it is overexpressed quite a long time, positioning this enzyme as a key factor in hepatic steatosis. These observations are quite similar in muscle. Lipoprotein secretion is upregulated later in postprandial period, with an upregulation of apolipoprotein and microsomal triglycerides transfer protein beginning at 5 h in liver or muscle. Regarding hepatic re-uptake of lipid, lesser variations are observed, suggesting that fatty acid binding protein and very low-density lipoprotein receptor (VLDLR) are already at their maximum expression specifically in these tissues. In muscle, VLDLR and LDLR upregulation is observed 5 h after the meal, associated with an overexpression in the adipose tissue of lipase maturation factor 1 involved in the maturation of lipoprotein lipase. These findings will allow us to better understand the kinetic treatment in lipid metabolism after a meal in overfed ducks. This first report on kinetic expression will allow researcher to better target their sampling time knowing the optimal point of expression of each gene.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1 + MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1 + MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1 + MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1 + MNs committed to LMC neurons, depending on the Hox expression pattern., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

We investigated the effects of co-enzyme Q10 (Q10) and acetyl salicylic acid (ASA) on expression of Hsp70 in the protection of primary chicken myocardial cells during heat stress. Western blot analysis showed that Q10 and ASA accelerated the induction of Hsp70 when chicken myocardial cells were exposed to hyperthermia. In the absence of heat stress, however, neither Q10 nor ASA are able to upregulate Hsp70 expression. Analysis of enzymes that respond to cellular damage and pathological examination revealed that ectopic expression of ASA and Q10 alleviate cellular damage during heat stress. Quantification of heat shock factors (HSF) indicated that treatment of ASA increased the expression of HSF-1 and HSF-3 during heat stress. Treatment with Q10 resulted in the elevation of HSF-1 expression. Expression of HSF-2 and HSF-4 was not affected by ASA or Q10. Subcellular distribution analysis of HSF-1 and HSF-3 showed that in response to heat stress ASA promoted nuclear translocation of HSF-1 and HSF-3, while Q10 promoted only HSF-1 nuclear translocation. Chromatin immunoprecipitation (ChIP) analysis indicated that HSF-1 occupies the Hsp70 promoter in chicken primary myocardial cells during heat stress and under normal conditions, while HSF-3 occupies the Hsp70 promoter only during heat stress. Real-time PCR analysis revealed that ASA induces HSF-1 and HSF-3 binding to Hsp70 HSE, while Q10 only induces HSF1 binding to Hsp70 HSE, in agreement with the impact of HSF1 and HSF3 silencing on Hsp70 expression. These data demonstrate that ASA and Q10 both induce the expression of Hsp70 to protect chicken primary myocardial cells during heat stress, but through distinct pathways.

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na 2 SeO 3 , 2 mg/kg), Cd (CdCl 2 , 150 mg/kg), or Se + Cd (150 mg/kg of CdCl 2 and 2 mg/kg of Na 2 SeO 3 ) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.

To determine the negative effects of cadmium (Cd) exposure and the protective role of selenium (Se) on Cd-spiked neutrophils of chicken, forty-eight 28-day-old Isa Brown male chickens were divided randomly into four groups. Group I (control group) was fed with the basic diet containing 0.2 mg/kg Se. Group II (Se-treated group) was fed with the basic diet supplemented with Na 2 SeO 3 , and the total Se content was 2 mg/kg. Group III (Se/Cd-treated group) was fed with the basic diet supplemented with Na 2 SeO 3 ; the total Se content was 2 mg/kg and supplemented with 150 mg/kg CdCl 2 . Group IV (Cd-treated group) was fed with the basic diet supplemented with 150 mg/kg CdCl 2 . Analyses of inflammatory factors, cytokines, and heat shock protein (Hsp) messenger RNA (mRNA) expression were detected by real-time PCR (RT-PCR). Additionally, we evaluated the phagocytic rate of neutrophils in peripheral blood. First, we observed that Cd significantly induced the mRNA expression levels of inflammatory factors NF-κB, iNOS, COX-2, and TNF-α, while Se/Cd treatment reduced their mRNA expression, although these expression levels remained higher than that of the control group. In addition, the mRNA expression levels of cytokines (IL-2, IL-4, and IL-10) for the Se-treated group exhibited significant differences between the Se/Cd-treated group and the Cd-treated group. Furthermore, the mRNA expression levels of Hsps demonstrated that the Se/Cd-treated group and the Cd-treated group were significantly higher (P < 0.05) than the control group and the Se-treated group. These results demonstrated that Se presented partial protection on Cd-spiked neutrophils of chicken with Hsps being involved in the process of the Cd-spiked toxic effects in chicken peripheral blood neutrophils.

The present study investigated the dynamic expression and regulatory mechanism of transforming growth factor β (TGF-β) signaling involved in embryonic stem cells (ESCs) differentiation into male germ cells. Candidate genes involved in TGF-β signaling pathway were screened from RNA-sequencing (RNA-seq), which were further validated by quantitative real-time PCR (qRT-PCR). Bone morphogenetic protein 4 (BMP4) was used to induce differentiation of ESCs in vitro Inhibition of TGF-β signaling pathway was reflected by Western blot of SMAD2 and SMAD5 expression. Differentiating efficiency of germ cells was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS). Germ cell marker genes were assessed by qRT-PCR in the differentiation process, with activation or inhibition of TGF-β signaling pathway. In the process of in vitro induction, SMAD2 and SMAD5 were found to significantly up-regulated in BMP4 group versus the control and inhibition groups after 4 and 14 days. Expression of CKIT, CVH, DAZL, STRA8 , and INTEGRIN α6 were significantly increased in the BMP4 group compared with the control group, while down-regulated in the inhibition groups. The proportion of germ cell-like cells was decreased from 17.9% to 2.2% after 4 days induction, and further decreased from 14.1% to 2.1% after 14 days induction. Correspondingly, expression of marker genes in germ cells was significantly lower. In vivo inhibition of TGF-β signaling pathway reduced germ cells formation from 5.5% to 1.6%, and down-regulated the expression of CKIT, CVH, DAZL, STRA8 , and INTEGRIN α6 In conclusion, our study reveals the mechanism regulating spermatogonial stem cells (SSCs) and lays the basis for further understanding of the regulatory network., (© 2017 The Author(s).)

Ornithine decarboxylase antizyme 1 (OAZ1) regulates both polyamine biosynthesis and transport and acts as a tumor suppressor because of its functions in the regulation of cell proliferation, apoptosis and growth. However, the roles of OAZ1 in the regulation of polyamine metabolism, cell proliferation, apoptosis and hormone receptor expression in granulosa cells (GCs) from geese have not been determined. To define the roles of OAZ1 in primary GCs, we constructed and transfected an Oaz1 overexpression vector with a thymidine deletion into primary GCs. Oaz1 overexpression induced 65 and 30% decreases in the putrescine and spermidine contents, respectively, 225% increase in the spermine content and up-regulated the expression levels of genes associated with polyamine metabolism (but Oaz2). The Ccnd1, Pcna, Bax and Caspase 3 mRNA expression levels in GCs overexpressing Oaz1 were significantly higher than the expression levels in the vehicle group (p < 0.05). Additionally, a slight increase in the E2 concentration in the culture medium and enhanced Er, Fshr and Lhr expression were observed in the GCs 24 h after Oaz1 overexpression (p < 0.05). These data suggested that Oaz1 up-regulated the expression of genes related to polyamine metabolic enzymes and reproductive hormone receptors, disturbed intracellular polyamine homeostasis, and affected the transcription of genes associated to cell proliferation and apoptosis in goose primary GCs. Taken together, our results indicate that modifying polyamine metabolism by Oaz1 may interfere with the expression of genes involved in cell proliferation, apoptosis and responsiveness of ovary toward hormones in goose GCs.

The sterile α-motif and HD domain containing protein 1 (SAMHD1) family is a newly identified protein family, involved in innate immunity restriction. This family possesses a broad-spectrum of antiviral activity. The SAMHD1 family in chicken has not been clearly documented. Here, we expressed chicken SAMHD1 (101-614) fused with a SUMO tag in an Escherichia coli (E. coli) system. For the first time, chicken SAMHD1 (101-614) was found to possess dNTPase cleavage activities in vitro. This suggests that chicken SAMHD1 may be a potential antiviral factor against avian viruses. Through a unique purification method, the purity of the protein as estimated by SDS-PAGE was >95% after a double Ni affinity chromatography and gel filtration purification. Using a sitting-drop vapor-diffusion method, protein crystals were obtained. This study provides some essential method and information for further structure and function determinations of chicken SAMHD1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To investigate the potential protective effect of heat shock protein 70 (Hsp70) during heat stress (HS) in chicken primary myocardial cells (CPMC), a cellular model of low expression of Hsp70 was established using 200 µM quercetin, a specific inhibitor of Hsp70. Comparative analyses were done among a HS group, Hsp70 low expression (HS+Quercetin) group and quercetin treated only group (Quercetin) during different durations of HS (0, 1, 2, 3 and 5 h). Inhibition of Hsp70 expression in quercetin treatment groups was detected, and suggested that Hsp70 expression was inhibited significantly. Levels of enzymes associated with cardiac damage were measured. In the Hsp70 low expression group, levels of these enzymes were elevated significantly compared with HS group, quercetin alone didn't elevate the level of these enzymes, The Hsp70 low expression group had twofold greater apoptosis compared with the HS group after 5 h of HS which was consistent with the results of Cleaved caspase‑3 protein, no obvious apoptosis was detected in quercetin group. Levels of caspase-3 and -9 activities were significantly higher in the Hsp70 low expression group, no differences of apoptosis inducing factor (AIF) in cell nucleus were observed between two groups suggested that inhibition of Hsp70 in CPMC increased the percentage of apoptosis may involve a mitochondrial pathway but AIF was not included. Expression of Bax with Bcl‑2 and their downstream cytochrome c in two groups confirmed our hypothesis. Our findings suggest that in CPMC, Hsp70 may have a cytoprotective role during HS that may act via a mitochondrial pathway.

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

As interferon-stimulated genes (ISGs), interferon-inducible transmembrane proteins 1 and 3 (IFITM1 and IFITM3) can effectively inhibit the replication of multiple viruses. Here, goose IFITM1 and IFITM3 were cloned and identified for the first time. The two proteins share the same topological structure and several important sites critical for the antiviral functions in other species are conserved in the goose. Goose IFITM1 and IFITM3 are most closely related to their respective orthologs in ducks; these proteins exhibited high mRNA transcript levels in immune-related tissues, including the thymus, bursa of Fabricius, and Harderian gland, compared to other tissues. Moreover, goose IFITM1 was highly constitutively expressed in gastrointestinal tract tissues, while goose IFITM3 was expressed in respiratory organs. Furthermore, goose IFITM3 was activated in goose peripheral blood mononuclear cells (PBMCs) infected with Tembusu virus (TMUV) or treated with Toll-like receptors (TLRs) agonists, while only the R848 and Poly (I:C) agonists induced significant upregulation of goose IFITM1. Furthermore, goose IFITM1 and IFITM3 were upregulated in the sampled tissues, to some extent, after TMUV infection. Notably, significant upregulation of goose IFITM1 and IFITM3 was detected in the cecum and cecal tonsil, where TMUV was primarily distributed. These data provide new insights into the immune effectors in geese and promote our understanding of the role of IFITM1 and IFITM3 in the defense against TMUV., Competing Interests: The authors disclose that there are no financial and personal relationships with other people or organizations that could inappropriately influence this work.

Myozenin3 ( Myoz3 ) has been reported to bind multiple Z-disc proteins and hence play a key role in signal transduction and muscle fiber type differentiation. The purpose of current study is to better understand the basic characteristics of Myoz3 . Firstly, we cloned the ORF (open reading frame) of the Myoz3 gene. AA (amino acid) sequence analysis revealed that the Myoz3 gene encodes a 26 kDa protein which have 97% identities with that of turkey. Expression profiling showed that Myoz3 mRNA is mainly expressed in leg muscle and breast muscle. Furthermore, we investigated Myoz3 gene polymorphisms in two broiler breeds, the Yellow Bantam (YB) and the Avian. Five SNPs (single nucleotide polymorphisms) were identified in the YB breed and 3 were identified in the Avian breed. Genotypes and haplotype were constructed and their associations with carcass traits were analyzed. In the YB breed, c.516 C>T had a strong effect on both shank bone length and the [Formula: see text] value of breast muscle, and the H1H3 diplotype had the highest FC compared to other diplotypes. The markers identified in this study may serve as useful targets for the marker-assisted selection (MAS) of growth and meat quality traits in chickens.