Xiangping Liu

Migration and accumulation of primordial germ cells of early embryo in quail.

In this research, we have identified primordial germ cells (PGCs) in quail embryo using Quail Hemangioblastic Lineage (QH1) monoclonal antibody analysis. Quail PGCs originated from the opaca of unincubated blastodisc, and then transferred to the pellucida and the germinal crescent. At 27 hours post-incubation, a few PGCs first appeared in blood vessels of the pellucida, where many PGCs accumulated at 36 hours post-incubation. The PGCs scattered or clustered from head to omphalo mesenteric and mainly settled down in the mesenchymal blood vessels of head at 45 hours post-incubation. The size of PGCs population increased significantly (P<0.05) from stage XII (12.8±4.82 µm) to primitive streak stage (106.7±8.74 µm) and from Head process stage (95.8±19.74 µm) to tenth somite stage (199.4±19.97 µm). It is concluded that the PGCs scattered in the head area before migration to the germinal crescent and distributed randomly in both gland. The number of PGCs varied at different stages with two peaks, primitive streak stage (18 hours post-incubation) and tenth somite (36 hours post-incubation).

Developmental research on the origin and phylogeny of quails

Around the world, there are 20 types of wild and about 70 domestic quail breeds or strains, including laboratory and commercial quail. Although all domestic quails were derived from wild strains, many obvious differences are evident today. However, how these differences occurred and which wild population was the first to be domesticated, remains unclear. This paper systematically presents the history of the development of domestic quail in China from 770 B.C. to the end of the 20 century. Taking into account recent research on some structural loci of domestic and wild quail, and in the light of recent survey reports of the present general situation of these birds, particularly in respect of their ecological performance and differences between wild and domestic quail, this review puts forward a new thesis for resolving the current uncertainty about the origin of domestic quail. It is suggested that unlike those of Japanese origin, Chinese quail are probably the earlier and more direct ancestor of most kinds of the domestic quail found around the world. Moreover, the review analyzes the possible evolutionary path to domestic quail, which is mainly a result of the flow of people from Japan to China. On the assumption that more and more wild quail populations are endangered, it aims to provide a basis for renewing knowledge of wild quail resources and supporting the protection and use of these valuable worldwide stocks. This is especially important in China, the last country in the world to have so many wild quail populations. Furthermore, this new insight can promote and assist the world commercial quail industry to develop and flourish.

A mutation in the NLRC5 promoter limits NF-κB signaling after Salmonella Enteritidis infection in the spleen of young chickens.

To date, the functions of the NLRC5 in chickens remain undefined. In the current study, chicken NLRC5 was cloned and an A1017G mutation was detected in its promoter region. The relative expression levels of the NLRC5 and key NF-κB pathway genes, IKKα, IKKβ, NF-κB, IL-6, IL-1β and IFN-γ, in the spleens of wild and mutant type birds, AA and GG, were determined using FQ-PCR at 7 day post-infection (DPI) with Salmonella Enteritidis. Additionally, the bacterial burden in the caecum and various immune response parameters were measured to evaluate immune responses. All of the examined immune response parameters were significantly different between the AA chickens and the GG chickens. Specifically, the mRNA expression levels of IKKα, NF-κB, IL-6, IL-1β and IFN-γ were higher in AA chickens than those in GG chickens, while the mRNA expression levels of NLRC5 were lower in AA chickens than those in GG chickens (P<0.05). Moreover, the mRNA expression levels of TLR4 and MyD88 were not affected in either group. Collectively, considering former NLRC5 functional study in vitro, the wild genotype birds presented with better resistance to Salmonella Enteritidis through the actions of the NLRC5 and subsequent inhibition of the NF-κB pathway in chickens.

Discovery of novel long non-coding RNAs induced by subgroup J avian leukosis virus infection in chicken

Abstract Avian leukosis virus subgroup J (ALV‐J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent decades. Here, using high throughput transcriptome sequencing of HD11 and CEF cells infected with ALV‐J, a set of 4804 novel long non‐coding transcripts and numerous differentially expressed long non‐coding RNAs (lncRNAs) were identified. We also found that they share relatively shorter transcripts and fewer exon numbers compared to mRNA. Correlation analysis suggested that many lncRNAs may activate gene expression in an enhancer‐like manner other than through transcriptional regulation. Expression level analyses in vivo showed that three lncRNAs (NONGGAT001975.2, NONGGAT005832.2 and NONGGAT009792.2) may be associated with immune response regulation and could function as novel biomarkers for ALV‐J infection. Our findings provides new insight into the pathological process of ALV‐J infection and should serve as a high‐quality resource for further research on epigenetic influences on disease‐resistance breeding as well as immune system and genomic studies. HighlightsCatalog of lncRNAs during ALV‐J infection in both somatic and immune cells were provided.Functional differences in noncoding RNA regulation between them characterized for the first time.Chicken lncRNAs are shorter, have fewer exons and shorter exons than protein‐coding genes.Many lncRNAs may activate gene expression in an enhancer‐like manner other than transcriptional regulation.We found and validated three lncRNAs that could function as novel biomarkers for ALV‐J infection.

Discovery of piRNAs Pathway Associated with Early-Stage Spermatogenesis in Chicken

Piwi-interacting RNAs (piRNAs) play a key role in spermatogenesis. Here, we describe the piRNAs profiling of primordial germ cells (PGCs), spermatogonial stem cells (SSCs), and the spermatogonium (Sp) during early-stage spermatogenesis in chicken. We obtained 31,361,989 reads from PGCs, 31,757,666 reads from SSCs, and 46,448,327 reads from Sp cells. The length distribution of piRNAs in the three samples showed peaks at 33 nt. The resulting genes were subsequently annotated against the Gene Ontology (GO) database. Five genes (RPL7A, HSPA8, Pum1, CPXM2, and PRKCA) were found to be involved in cellular processes. Interactive pathway analysis (IPA) further revealed three important pathways in early-stage spermatogenesis including the FGF, Wnt, and EGF receptor signaling pathways. The gene Pum1 was found to promote germline stem cell proliferation, but it also plays a role in spermatogenesis. In conclusion, we revealed characteristics of piRNAs during early spermatogonial development in chicken and provided the basis for future research.

Study on genetic coadaptability of wild quail populations in China

Genetic coadaptability of wild Japanese quail, wild Common quail and Domestic quail populations in China was studied using 7 microsatellite DNA markers and Monte Carlo method to test genetic disequilibrium. The molecular effects of genetic coadaptability were analyzed through a new statistical model of neutral site. The results showed that genetic coadaptability dominated the genetic disequilibrium of the three quail populations, and totally 16.67%, 9.66% and 10.05% of non-allelic combinations were in the genetic disequilibrium in wild Japanese quail, wild Common quail and Domestic quail populations, respectively. Genetic coadaptability existed at almost all the tested sites. In the molecular point of view, genetic coadaptability plays an important role of keeping lots of polymorphisms in natural populations. Therefore, it is another key factor to the genetic disequilibrium in the population except for linkage. The results enrich the conceptions and connotations of genetic disequilibrium, and help us know more about genetic coadaptability and its effects, and lay a foundation of evaluation and protection of wild quail genetic resources in China.

Discovery of microRNAs during early spermatogenesis in chicken

Spermatogenesis is a complex process that involves many elements. However, until now, little is known at the molecular level about spermatogenesis in poultry. Here we investigated microRNAs and their target genes that may be involved in germ cell development and spermatogonial in chicken. We used next-generation sequencing to analyze miRNA expression profiles in three types of germline cells: primordial germ cells (PGCs), spermatogonial stem cells (SSCs), and spermatogonia (Sp) during early stage of spermatogenesis. After validated the candidate miRNAs and corresponding genes’ expression in three types of cells, we found 15 miRNAs that were enriched 21 target genes that may be involved in spermatogenesis. Among the enriched miRNAs, miR-202-5p/3p were up-regulated in the Sp library and down-regulated in the PGCs library. Through RT-qPCR and Dual-Luciferase reporter assay, we confirmed that miR-202-5p bind to LIMK2 and involved in germ cell development. Collectively, we firstly discover the novel miRNAs, like miR-202-5p, and its related genes and pathways, expressed during the early spermatogonial stage in chicken, which will provide new clues for deciphering the molecular mechanism of the miRNAs regulating germline stem cell differentiation and spermatogenesis in chicken.

DNA methylation-mediated transcription factors regulate Piwil1 expression during chicken spermatogenesis.

The P-element induced wimpy testis (Piwi) protein family is responsible for initiating spermatogenesis and maintaining the integrity of germ cells and stem cells, but little is known regarding its transcriptional regulation in poultry. Here, we characterized the methylation status of the Piwil1 promoter in five different spermatogenic cell lines using direct bisulfite pyrosequencing and determined that methylation correlates negatively with germ cell type-specific expression patterns of piwil1. We demonstrated that methylation of the −148 CpG site, which is the predicted binding site for the transcription factors TCF3 and NRF1, was differentially methylated in different spermatogenic cells. This site was completely methylated in PGCs (primordial germ cells), but was unmethylated in round spermatids. A similar result was obtained in the region from +121 to +139 CpG sites of the Piwil1 promoter CpG island, which was predicted to contain SOX2 binding sites. In addition, demethylation assays further demonstrated that DNA methylation indeed regulates Piwil1 expression during chicken spermatogenesis. Combined with transcription factor binding site prediction, we speculate that methylation influences the recruitment of corresponding transcription factors. Collectively, we show the negative correlation between promoter methylation and piwil1 expression and that the spatiotemporal expression of chicken Piwil1 from the PGC stage to the round spermatid stage is influenced by methylation-mediated transcription factor regulation.

Expression patterns of NLRC5 and key genes in the STAT1 pathway following infection with Salmonella pullorum.

NLRC5, a protein belonging to the NOD-like receptor protein family (NLRs), is highly expressed in immune tissues and cells. NLRC5 plays an important role in the immune response of humans, where its regulatory mechanism has been elucidated. However, the function and regulation of NLRC5 in chickens remains unclear. In this study, temporal expression characteristics of NLRC5 and associated genes in the STAT1 pathway in chickens following infection with Salmonella pullorum were investigated using quantitative real-time polymerase chain reaction and hierarchical cluster analyses. The role of transcription factor STAT1 in NLRC5 promoter activity was studied via point mutation of the STAT1-binding cis-element and dual-luciferase assays. Our results showed a strong correlation between NLRC5 and NF-κB. In addition, STAT1 played a crucial role in NLRC5 promoter activity, and may be activated via the interferon pathway. There was also a close relationship between CD80 and NF-κB, and CD80 may up-regulate NF-κB expression and enhance its protein activity in chickens. These findings reveal the temporal characteristics of chicken NLRC5 and STAT1 genes during S. pullorum infection, and highlight the role of STAT1 in NLRC5 promoter activity. This information aids our understanding of the regulatory mechanisms of NLRC5 and associated genes, and will help elucidate their function in the immune response of chickens.

Comprehensive Transcriptome Analysis Reveals Competing Endogenous RNA Networks During Avian Leukosis Virus, Subgroup J-Induced Tumorigenesis in Chickens

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that induces myeloid tumors and hemangiomas in chickens and causes severe economic losses with commercial layer chickens and meat-type chickens. High-throughput sequencing followed by quantitative real-time polymerase chain reaction and bioinformatics analyses were performed to advance the understanding of regulatory networks associated with differentially expressed non-coding RNAs and mRNAs that facilitate ALV-J infection. We examined the expression of mRNAs, long non-coding RNAs (lncRNAs), and miRNAs in the spleens of 20-week-old chickens infected with ALV-J and uninfected chickens. We found that 1723 mRNAs, 7,883 lncRNAs and 13 miRNAs in the spleen were differentially expressed between the uninfected and infected groups (P < 0.05). Transcriptome analysis showed that, compared to mRNA, chicken lncRNAs shared relatively fewer exon numbers and shorter transcripts. Through competing endogenous RNA and co-expression network analyses, we identified several tumor-associated or immune-related genes and lncRNAs. Along transcripts whose expression levels significantly decreased in both ALV-J infected spleen and tumor tissues, BCL11B showed the greatest change. These results suggest that BCL11B may be mechanistically involved in tumorigenesis in chicken and neoplastic diseases, may be related to immune response, and potentially be novel biomarker for ALV-J infection. Our results provide new insight into the pathology of ALV-J infection and high-quality transcriptome resource for in-depth study of epigenetic influences on disease resistance and immune system.