Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells.

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.

Female Germ Cell Development in Chickens and Humans: The Chicken Oocyte Enriched Genes Convergent and Divergent with the Human Oocyte.

The development of germ cells and other physiological events in the differentiated ovary of humans are highly conserved with several mammalian species, except for the differences in timing. However, comparative knowledge on this topic is very scarce with respect to humans and lower vertebrates, such as chickens. In chickens, female germ cells enter into meiosis around embryonic day (E) 15.5 and are arrested in meiotic prophase I as primary oocytes. The oocytes arrested in meiosis I are accumulated in germ-cell cysts; shortly after hatching, they are enclosed by flattened granulosa cells in order to form primordial follicles. In humans, the process of meiotic recombination in female germ cells begins in the 10-11th week of gestation, and primordial follicles are formed at around week 20. In this review, we comprehensively elucidate both the conservation and the species-specific differences between chickens and humans with respect to germ cell, oocyte, and follicle development. Importantly, we provide functional insights into a set of chicken oocyte enriched genes (from E16 to 1 week post-hatch) that show convergent and divergent expression patterns with respect to the human oocyte (from week 11 to 26).

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens.

BACKGROUND: Germ cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage. RESULTS: We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later. CONCLUSIONS: We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

Amplification of immunity by engineering chicken MDA5 combined with the C terminal domain (CTD) of RIG-I.

Innate immune system is triggered by pattern recognition receptors (PRRs) recognition. Retinoic acid-inducible gene 1 (RIG-I) is a major sensor that recognizes RNA ligands. However, chickens have no homologue of RIG-I; instead, they rely on melanoma differentiation-associated protein 5 (MDA5) to recognize RNA ligands, which renders chickens susceptible to infection by influenza A viruses (IAVs). Here, we engineered the cMDA5 viral RNA sensing domain (C-terminal domain, CTD) such that it functions similarly to human RIG-I (hRIG-I) by mutating histidine 925 into phenylalanine, a key residue for hRIG-I RNA binding loop function, or by swapping the CTD of cMDA5 with that of hRIG-I or duck RIG-I (dRIG-I). The engineered cMDA5 gene was expressed in cMDA5 knockout DF-1 cells, and interferon-beta (IFN-ß) activity and expression of interferon-related genes were measured after transfection of cells with RNA ligands of hRIG-I or human MDA5 (hMDA5). We found that both mutant cMDA5 and engineered cMDA5 triggered significantly stronger interferon-mediated immune responses than wild-type cMDA5. Moreover, engineered cMDA5 reduced the IAV titer by 100-fold compared with that in control cells. Collectively, engineered cMDA5/RIG-I CTD significantly enhanced interferon-mediated immune responses, making them invaluable strategies for production of IAV-resistant chickens. KEY POINTS: • Mutant chicken MDA5 with critical residue of RIG-I (phenylalanine) enhanced immunity. • Engineered chicken MDA5 with CTD of RIG-I increased IFN-mediated immune responses. • Engineered chicken MDA5 reduced influenza A virus titers by up to 100-fold.

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability.

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3-12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells.

BACKGROUND: NANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs. RESULTS: We first identified the transcription start site of cNANOG by 5'-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5' flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG. CONCLUSIONS: We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin.

BACKGROUND: The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian. RESULTS: In the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability. CONCLUSIONS: Taken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens.

The deleted in azoospermia like (DAZL) is required for germ cells development and maintenance. In chickens, the mRNA and protein of DAZL, a representative maternally inherited germ plasm factor, are detected in the germ plasm of oocyte, zygote, and all stages of the intrauterine embryos. However, it is still insufficient to explain the origin and specification process of chicken germ cells, because the stage at which the zygotic transcription of DAZL occurs and the stage at which the maternal DAZL RNA/protein clears have not yet been fully identified. Moreover, a comprehensive understanding of the expression of DAZL interacting genes during the germ cells specification and development and zygotic genome activation (ZGA) is lacking in chickens. In this study, we identified a set of DAZL interacting genes in chickens using in silico prediction method. Then, we analyzed the whole-transcriptome sequencing (WTS)-based expression of DAZL and its interacting genes in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage embryos (EGK.I to EGK.X). In the results, DAZL transcripts are increased in the zygote (onset of transcription), maintained the increased level until EGK.VI, and decreased from EGK.VIII (possible clearance of maternal RNAs). Among the DAZL interacting genes, most of them are increased either at 1st ZGA or 2nd ZGA, indicating their involvement in germ cells specification and development.

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

The innate immune system, which senses invading pathogens, plays a critical role as the first line of host defense. After recognition of foreign RNA ligands (e.g., RNA viruses), host cells generate an innate immune or antiviral response via the interferon-mediated signaling pathway. Retinoic acid-inducible gene I (RIG-1) acts as a major sensor that recognizes a broad range of RNA ligands in mammals; however, chickens lack a RIG-1 homolog, meaning that RNA ligands should be recognized by other cellular sensors such as melanoma differentiation-associated protein 5 (MDA5) and toll-like receptors (TLRs). However, it is unclear which of these cellular sensors compensates for the loss of RIG-1 to act as the major sensor for RNA ligands. Here, we show that chicken MDA5 (cMDA5), rather than chicken TLRs (cTLRs), plays a pivotal role in the recognition of RNA ligands, including poly I:C and influenza virus. First, we used a knockdown approach to show that both cMDA5 and cTLR3 play roles in inducing interferon-mediated innate immune responses against RNA ligands in chicken DF-1 cells. Furthermore, targeted knockout of cMDA5 or cTLR3 in chicken DF-1 cells revealed that loss of cMDA5 impaired the innate immune responses against RNA ligands; however, the responses against RNA ligands were retained after loss of cTLR3. In addition, double knockout of cMDA5 and cTLR3 in chicken DF-1 cells abolished the innate immune responses against RNA ligands, suggesting that cMDA5 is the major sensor whereas cTLR3 is a secondary sensor. Taken together, these findings provide an understanding of the functional role of cMDA5 in the recognition of RNA ligands in chicken DF-1 cells and may facilitate the development of an innate immune-deficient cell line or chicken model.

A novel F-box domain containing cyclin F like gene is required for maintaining the genome stability and survival of chicken primordial germ cells.

The stability and survival of germ cells are controlled by the germline-specific genes, however, such genes are less known in the avian species. Using a microarray-based the National Center for Biotechnology Information Gene Expression Omnibus dataset, we found an unigene (Gga.9721) that upregulated in the chicken primordial germ cells (PGCs). The unigene showed 97% identities with an uncharacterized chicken cyclin F like gene. The predicted chicken cyclin F like gene was further characterized through expression and regulation in the chicken PGCs. The sequence analysis revealed that the gene shows identities with cyclin F gene and contains an F-box domain. The expression of chicken cyclin F like was detected specifically in the gonads, PGCs, and germline cells. The knockdown of cyclin F like gene resulted in DNA damage and apoptosis in the PGCs. The genes related to stemness and germness were downregulated, whereas, genes related to apoptosis and DNA damage response were increased in the PGCs after the knockdown of chicken cyclin F like. We further observed that the Nanog homeobox controlled the transcriptional activity of chicken cyclin F like gene in PGCs. Collectively, the chicken cyclin F like gene, which is not reported in any other species, is required for maintaining the genome stability of germ cells.

Host-Specific Restriction of Avian Influenza Virus Caused by Differential Dynamics of ANP32 Family Members.

BACKGROUND: Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. The ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. METHODS: In this study, we targeted chicken ANP32A using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. RESULTS: We showed that chicken ANP32A only, not ANP32B and ANP32E, plays a pivotal role in supporting vPol activity of AIVs. Furthermore, we found that the human ANP32C, ANP32D, and ANP32E have suppressive effects on vPol activity in contrast to human ANP32A and ANP32B. CONCLUSIONS: Chicken and human ANP32 family members had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.

Zygotic genome activation in the chicken: a comparative review.

Maternal RNAs and proteins in the oocyte contribute to early embryonic development. After fertilization, these maternal factors are cleared and embryonic development is determined by an individual's own RNAs and proteins, in a process called the maternal-to-zygotic transition. Zygotic transcription is initially inactive, but is eventually activated by maternal transcription factors. The timing and molecular mechanisms involved in zygotic genome activation (ZGA) have been well-described in many species. Among birds, a transcriptome-based understanding of ZGA has only been explored in chickens by RNA sequencing of intrauterine embryos. RNA sequencing of chicken intrauterine embryos, including oocytes, zygotes, and Eyal-Giladi and Kochav (EGK) stages I-X has enabled the identification of differentially expressed genes between consecutive stages. These studies have revealed that there are two waves of ZGA: a minor wave at the one-cell stage (shortly after fertilization) and a major wave between EGK.III and EGK.VI (during cellularization). In the chicken, the maternal genome is activated during minor ZGA and the paternal genome is quiescent until major ZGA to avoid transcription from supernumerary sperm nuclei. In this review, we provide a detailed overview of events in intrauterine embryonic development in birds (and particularly in chickens), as well as a transcriptome-based analysis of ZGA.

Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.-Jung, K. M., Kim, Y. M., Keyte, A. L., Biegler, M. T., Rengaraj, D., Lee, H. J., Mello, C. V., Velho, T. A. F., Fedrigo, O., Haase, B., Jarvis, E. D., Han, J. Y. Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Identification and expression analysis of alpha tocopherol transfer protein in chickens fed diets containing different concentrations of alpha-tocopherol.

Among the eight forms of vitamin E, the liver preferentially releases α-tocopherol into the circulation and it is distributed to the non-liver tissues. In the hepatocytes, alpha tocopherol transfer protein (TTPA) specifically recognizes α-tocopherol with 2R-configuration and facilitates its intracellular transfer. The identification and characterization of TTPA expression have not been demonstrated in avian species. Therefore, the objectives of this study were to identify avian TTPAs, to compare the sequence conservation, phylogenetic relationship, protein interactions, and disease associations of chicken TTPA with those of human and vertebrate TTPA, and to characterize the tissue expression of the TTPA gene in chickens fed diets supplemented with different amounts of α-tocopherol. Our results suggest that the chicken TTPA was highly conserved with the human and vertebrate TTPA, and consisted of a cellular retinaldehyde binding protein and TRIO guanine exchange factor (CRAL_TRIO) domain. Feeding diets supplemented with increasing amounts of α-tocopherol (25 IU/Kg, 50 IU/Kg, or 100 IU/Kg) to broiler chickens had no effects on growth performance compared with feeding basal diets containing no supplemental α-tocopherol. The expression of TTPA gene was detected high in the liver of chickens in response to dietary α-tocopherol concentrations, whereas its expression was very low or undetectable in the non-liver tissues. In conclusion, the chicken TTPA protein sequence is highly conserved with other avian and vertebrate TTPA protein sequences. The higher expression of TTPA gene in the chicken liver in response to dietary α-tocopherol concentrations may suggest its crucial role in transporting α-tocopherol in the chicken liver.

Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways.

The activating leukocyte immunoglobulin-like receptors (LILRAs) play an important role in innate immunity. However, most of the LILRA members have not been characterized in avian species including chickens. The present study is the first attempt at cloning, structural analysis and functional characterization of two LILRAs (LILRA2 and LILRA6) in chickens. Multiple sequence alignments and construction of a phylogenetic tree of chicken LILRA2 and LILRA6 with mammalian proteins revealed high conservation between chicken LILRA2 and LILRA6 and a close relationship between the chicken and mammalian proteins. The mRNA expression of LILRA2 and LILRA6 was high in chicken HD11 macrophages and the small intestine compared to that in several other tissues and cells tested. To examine the function of LILRA2 and LILRA6 in chicken immunity, LILRA2 and LILRA6 were transfected into HD11 cells. Our findings indicated that LILRA2 and LILRA6 are associated with the phosphorylation of Src kinases and SHP2, which play a regulatory role in immune functions. Moreover, LILRA6 associated with and activated MHC class I, ß2-microglobulin and induced the expression of transporters associated with antigen processing but LILRA2 did not. Furthermore, both LILRA2 and LILRA6 activated JAK-STAT, NF-κB, PI3K/AKT and ERK1/2 MAPK signaling pathways and induced Th1-, Th2- and Th17-type cytokines and Toll-like receptors. Collectively, this study indicates that LILRA2 and LILRA6 are essential for macrophage-mediated immune responses and they have the potential to complement the innate and adaptive immune system against pathogens.

Role of Epigenetic Regulation by the REST/CoREST/HDAC Corepressor Complex of Moderate NANOG Expression in Chicken Primordial Germ Cells.

Primordial germ cells (PGCs), the precursors of gametes, have regulatory mechanisms involving transcription factors and epigenetic modifications. The transcription factor NANOG is a key regulator of germ cell and embryonic stem cell characteristics. However, the epigenetic regulation of NANOG with a histone deacetylase (HDAC) complex in PGCs has not been studied. In this study, we investigated the epigenetic regulation, and in particular the histone acetylation, of NANOG in chicken PGCs. Intriguingly, although NANOG was highly activated in chicken PGCs, the upstream region of its promoter was moderately suppressed by histone deacetylation. HDAC inhibition induced histone H3 lysine 9 acetylation (H3K9ac) and derepressed NANOG expression. Furthermore, knockdown studies revealed that HDAC complex members, such as RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3), are important epigenetic modulators of NANOG expression in chicken PGCs. We demonstrate that moderate regulation of NANOG by the REST/CoREST/HDAC complex might be crucial for maintaining the integrity of PGCs.