LIN28 is essential for the maintenance of chicken primordial germ cells.

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

Intra-amniotic administration of l-glutamine promotes intestinal maturation and enteroendocrine stimulation in chick embryos.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling.

Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation.

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging 'neural plate border' as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast.

Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells.

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.

Dissociation of chick embryonic tissue for FACS and preparation of isolated cells for genome-wide downstream assays.

In order to process samples by fluorescence-activated cell sorting (FACS), it is essential to obtain a single-cell suspension of dissociated cells. Numerous protocols and commercial reagents are available; however, each requires optimization for specific tissue types. Here, we describe an optimized protocol for dissociating dissected chick embryos across a broad span of developmental stages. We also provide protocols for processing targeted cell populations isolated using FACS for ATAC-seq, RNA-seq, and chromatin immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Ling and Sauka-Spengler (2019) and Williams et al. (2019).

A cell atlas of the chick retina based on single-cell transcriptomics.

Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates - photoreceptor, horizontal, bipolar, amacrine, retinal ganglion, and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral, and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell, and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse, and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.

The evolutionary relationships of organisms and of genes have long been studied in various ways, including genome sequencing. More recently, the evolutionary relationships among the different types of cells that perform distinct roles in an organism, have become a subject of inquiry. High throughput single-cell RNA sequencing is a technique that allows scientists to determine what genes are switched on in single cells. This technique makes it possible to catalogue the cell types that make up a tissue and generate an atlas of the tissue based on what genes are switched on in each cell. The atlases can then be compared among species. The retina is a light-sensitive tissue that animals with a backbone, called vertebrates, use to see. The basic plan of the retina is very similar in vertebrates: five classes of neurons ­ the cells that make up the nervous system ­ are arranged into three layers. The chicken is a highly visual animal and it has frequently been used to study the development of the retina, from understanding how unspecialized embryonic cells become neurons to examining how circuits of neurons form. The structure and role of the retina have been studied in many vertebrates, but detailed descriptions of this tissue at the molecular level have been largely limited to mammals. To bridge this gap, Yamagata, Yan and Sanes generated the first cell atlas of the chicken retina. Additionally, they developed a gene editing-based technique based on CRISPR technology called eCHIKIN to label different cell types based on genes each type switched on selectively, providing a means of matching their shape and location to their molecular identity. Using these methods, it was possible to subdivide each of the five classes of neurons in the retina into multiple distinct types for a total of 136. The atlas provided a foundation for evolutionary analysis of how retinas evolve to serve the very different visual needs of different species. The chicken cell types could be compared to types previously identified in similar studies of mouse and primate retinas. Comparing the relationships among retinal cells in chickens, mice and primates revealed strong similarities in the overall cell classes represented. However, the results also showed big differences among species in the specific types within each class, and the genes that were switched on within each cell type. These findings may provide a foundation to study the anatomy, physiology, evolution, and development of the avian visual system. Until now, neural development of the chicken retina was being studied without comprehensive knowledge of its cell types or the developmentally important genes they express. The system developed by Yamagata, Yan and Sanes may be used in the future to learn more about vision and to investigate how neural cell types evolve to match the repertoire of each species to its environment.

Chicken Interspecies Chimerism Unveils Human Pluripotency.

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.

A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method.

Canine distemper virus (CDV) is the causative agent of canine distemper (CD), which is one of the most important infectious diseases affecting wild and domestic carnivores. Vaccination represents an effective approach to prevent CDV infection among domestic carnivores. Canarypox-vectored recombinant CD vaccines (such as Recombitek CDV, PureVax Ferret Distemper, and Merial) with the CDV hemagglutinin (H) and fusion (F) genes can induce a potent immune response in dogs and ferrets. However, the vaccine's effectiveness varies with the species. In the current study, we developed a highly efficient recombinant canarypox virus termed as "ALVAC-CDV-M-F-H/C5-" that contained CDV virus-like particles (VLPs) by using the CRISPR/Cas9 gene editing method, which enabled concurrent expression of the matrix (M), H, and F genes. The recombinant strain provided faster seroconversion than the parent strain among minks as well as provided higher rates of antibody positivity than the parent strain among foxes and minks even before the administration of a second booster vaccination. We demonstrated, for the first time, that the CRISPR/Cas9 system can be applied for the rapid and efficient modification of the ALVAC-CDV-F-H genome and also that a high-dose new recombinant strain that produces CDV VLPs may present good outcomes in the prevention of CD among foxes and minks.

Deletion of LORF9 but not LORF10 attenuates Marek's disease virus pathogenesis.

Marek's disease virus (MDV) genome contains a number of uncharacterized long open reading frames (LORF) and their role in viral pathogenesis has not been fully investigated. Among them, LORF9 (MDV069) and LORF10 (MDV071) are locate at the right terminus of the MDV genome unique long region (UL). To investigate their role in MDV pathogenesis, we generated LORF9 or LORF10 deletion and revertant viruses. In vitro growth kinetics results show that both LORF9 and LORF10 are not essential for virus growth in cell culture. However, LORF9, but not LORF10, is involved in MDV early cytolytic replication in vivo, as evidenced by limited viral antigen expression in lymphoid organs of LORF9 deletion virus inoculated chickens. MDV genome copy number data further confirmed that LORF9 is important for MDV replication in spleen during early cytolytic phase. Deletion of LORF9 also partially impairs the replication of MDV in feather follicle epithelium (FFE); however, it can still establish latency and transformation. In addition, pathogenesis studies show that deletion of LORF9, but not LORF10, result in significant reduction of MDV induced mortality and slightly reduce MDV associated tumors of inoculated chickens. Importantly, we confirmed these results with the generation of LORF9 and LORF10 revertant viruses that fully restore the phenotypes of parental MDV. In conclusion, our results show that deletion of LORF9, but not LORF10, significantly impair viral replication in lymphoid organs during early cytolytic phase and attenuate Marek's disease virus pathogenesis.

Multiscale cardiac imaging spanning the whole heart and its internal cellular architecture in a small animal model.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

The heart is our hardest-working organ and beats around 100,000 times a day, pumping blood through a vast system of vessels to all areas of the body. Specialized heart cells make the heart contract rhythmically, enabling it to work efficiently. Contractile molecules inside these cells, called myofibrils, align within the heart cells, and heart cells align to each other, so that the heart tissue contracts effectively. However, when the heart has defects or is diseased this organization can be lost, and the heart may no longer pump blood efficiently, leading to sometimes life-threatening complications. For example, around one in a hundred newborn babies suffer from congenital heart defects, and despite medical advances, these conditions remain the main cause of non-infectious mortality in children. Many cases of congenital heart disease are diagnosed before a baby is born during an ultrasound scan. However, these scans, as well as subsequent diagnostic tools, lack the precision to detect problems within the heart cells. Now, Rykiel et al. used two complementary imaging techniques known as micro-computed tomography and scanning electron microscopy to acquire pictures of the whole heart as well as of the organization inside the heart cells. This made it possible to capture the structure of the heart tissue at both micrometer (the whole heart) and nanometer resolution (the inside of the cells), and to study what happens within the heart and its cells when the heart has a defect. Rykiel et al. tested the imaging technology on the hearts of chicken embryos, at stages equivalent to a five to six-month-old human fetus, and compared a healthy heart with a heart with a defect called tetralogy of Fallot. They found that the tissues in the heart with a defect had a sponge-like appearance, with increased space in between cells. Moreover, the myofibrils of the heart with a defect were aligned differently compared to those in the normal heart. More research is needed to fully understand what happens when the heart has a defect. However, the imaging technology used in this study offers the possibility of examining the heart at an unprecedented level of detail. This will deepen our understanding of how structural heart defects arise and how they affect the pumping of the heart, and will give us clues to design better treatments for patients with heart defects and other heart anomalies.

Effects of gonadotropins on testis cell subpopulations of newly hatched chicks treated during embryonic development.

The aim of the present study was to evaluate histological and stereological changes, as well as the variations in the number and size of cells from diverse cell subpopulations in testes of newly hatched chicks treated with follicle stimulating hormone (FSH) and luteinizing hormone (LH) during embryonic development. Stereological results indicated that in FSH-treated chicks total volume of the tubular compartment constitutes most of the testis. In contrast, the total volume of interstitial tissue constitutes most of the testis of LH-treated chicks. Results indicate that the number of germ and Sertoli cells increases as a result of FSH and LH treatment, but in FSH-treated testis, Sertoli cells were the most numerous cell type in seminiferous tubules; whereas germ cells were the most numerous cell type in testis of LH-treated chicks. Results also indicate there was a larger total volume of Leydig cells in the testes of FSH- and LH-treated chicks. The larger volume of Leydig cells in FSH-treated chicks is due to a larger cellular volume of these cells, and not due to the number, which remains constant. In contrast, in testes of LH-treated chicks, there is a larger number and volume of Leydig cells. These results indicate the testes of chick embryos respond to FSH and LH treatment, with there being modifications in the seminiferous tubules and interstitial tissue, but these changes differ markedly, indicating that FSH and LH have differential effects on chick testes.

Isolation and Propagation of Coronaviruses in Embryonated Eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

Dynamic morphoskeletons in development.

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes.

Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro.

Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.

Testosterone Promotes the Proliferation of Chicken Embryonic Myoblasts Via Androgen Receptor Mediated PI3K/Akt Signaling Pathway.

Testosterone (T) is essential for muscle fiber formation and growth. However, the specific mechanism by which T regulates skeletal muscle development in chicken embryos remains unclear. In this study, the role of T in myoblast proliferation both in vivo and in vitro was investigated. Results showed that the T administration significantly increased the ratio of breast muscle and leg muscle. T induced a significant increase in the cross-sectional area (CSA) and density of myofiber and the ratio of PAX7-positive cells in the skeletal muscle. Exogenous T also induced the upregulation of myogenic regulatory factors (MRFs) and cyclin-dependent kinases (CDK2)/Cyclin D1 (CCND1) and protein levels of androgen receptor (AR), p-Akt and PAX7. Furthermore, T treatment significantly promoted myoblasts cultured in vitro entering a new cell cycle and increased PAX7-positive cells. The mRNA and protein expression of AR and PAX7 were upregulated when treated with T compared to that of the control. The addition of T induced proliferation accompanied by increasing AR level as well as PI3K (Phosphoinositide 3-kinase)/Akt activation. However, T-induced proliferation was attenuated by AR, PI3K, and Akt-specific inhibitors. These data indicated that the pro-proliferative effect of T was regulated though AR in response to the activation of PI3K/Akt signalling pathway.

Visualizing mesoderm and neural crest cell dynamics during chick head morphogenesis.

Vertebrate head morphogenesis involves carefully-orchestrated tissue growth and cell movements of the mesoderm and neural crest to form the distinct craniofacial pattern. To better understand structural birth defects, it is important that we characterize the dynamics of these processes and learn how they rely on each other. Here we examine this question during chick head morphogenesis using time-lapse imaging, computational modeling, and experiments. We find that head mesodermal cells in culture move in random directions as individuals and move faster in the presence of neural crest cells. In vivo, mesodermal cells migrate in a directed manner and maintain neighbor relationships; neural crest cells travel through the mesoderm at a faster speed. The mesoderm grows with a non-uniform spatio-temporal profile determined by BrdU labeling during the period of faster and more-directed neural crest collective migration through this domain. We use computer simulations to probe the robustness of neural crest stream formation by varying the spatio-temporal growth profile of the mesoderm. We follow this with experimental manipulations that either stop mesoderm growth or prevent neural crest migration and observe changes in the non-manipulated cell population, implying a dynamic feedback between tissue growth and neural crest cell signaling to confer robustness to the system. Overall, we present a novel descriptive analysis of mesoderm and neural crest cell dynamics that reveals the coordination and co-dependence of these two cell populations during head morphogenesis.

A reviewed chronology of primordial germ cells migration in the chick embryo.

INTRODUCTION: Primordial Germ Cells (PGCs) are present in all sexually reproducing animals. They differentiate into spermatozoa or oocytes and are therefore responsible for the transmission of genetic and epigenetic information across generations. In birds, PGCs are first observed in the center of the blastodisc at stage Eyal-Giladi X. With the formation of the primitive streak, germ cells are translocated anteriorly to the germinal crescent. At stage Hamburger- Hamilton 10-12, they enter the vasculature before migrating through the dorsal mesentery towards the genital ridges. MATERIAL AND METHODS: Embryos from stages Hamburger-Hamilton (HH) 16 to 22 were collected. Blood samples were taken from the dorsal aorta and from the heart in order to perform blood smears and PAS staining. Embryos were dissected and fixed in Serra's medium. Sections were placed on slides for PAS staining. A sample of each embryo was collected for DNA extraction and PCR in order to determine the sex of the embryos. RESULTS: PGCs were observed in blood circulation until stage HH 20 on blood smears and until stage HH 19 on histological sections. The first PGCs arrived in the genital ridges were observed from stage HH 17. A few germ cells were still migrating in the dorsal mesentery at stage HH 22. The aim of this study was to review the chronology of the migration of PGCs in chick embryos.

An endogenous retroviral element exerts an antiviral innate immune function via the derived lncRNA lnc-ALVE1-AS1.

Endogenous retroviruses (ERVs) constitute an important component of animal and human genomes and are usually silenced by epigenetic mechanisms in adult cells. Although ERVs were recently reported to be linked to early development, tumorigenesis and autoimmune disease, their impacts on antiviral innate immunity and the underlying mechanisms have not been elucidated. Here, we provide the first direct evidence of an endogenous retroviral element affecting antiviral innate immunity via its derived antisense long non-coding RNA (lncRNA). We found that an antisense lncRNA, which is called lnc-ALVE1-AS1 and is transcribed from the endogenous avian leukosis virus in chromosome 1 (ALVE1), distinctly inhibited the entry and replication of exogenous retroviruses in chicken embryonic fibroblasts (CEFs). This behaviour is at least in part attributed to the induction of an antiviral innate immune pathway by ALVE1 activation, suggesting that an activated endogenous retroviral element may induce antiviral defence responses via its derived antisense lncRNA. We also found that lnc-ALVE1-AS1 mediated these effects by activating the TLR3 signalling in the cytoplasm. Our results provide novel insights into the antiviral innate immune function of ERVs, suggesting that ERVs may play an important role in antiviral defences and provide new strategies for the development of new vaccines.

gga-miR-146c Activates TLR6/MyD88/NF-κB Pathway through Targeting MMP16 to Prevent Mycoplasma Gallisepticum (HS Strain) Infection in Chickens.

Mycoplasma gallisepticum (MG), a pathogen that infects chickens and some other birds, triggers chronic respiratory disease (CRD) in chickens, which is characterized by inflammation. The investigation of microbial pathogenesis would contribute to the deep understanding of infection control. Since microribonucleic acids (miRNAs) play a key role in this process, gga-mir-146c, an upregulated miRNA upon MG infection, was selected according to our previous RNA-sequencing data. In this paper, we predicted and validated that MMP16 is one of gga-miR-146c target genes. Results show that MMP16 is the target of gga-miR-146c and gga-miR-146c can downregulate MMP16 expression within limits. gga-miR-146c upregulation significantly increased the expression of TLR6, NF-κB p65, MyD88, and TNF-α, whereas the gga-miR-146c inhibitor led to an opposite result. gga-miR-146c upregulation effectively decreased apoptosis and stimulated DF-1 cells proliferation upon MG infection. On the contrary, gga-miR-146c inhibitor promoted apoptosis and repressed the proliferation. Collectively, our results suggest that gga-miR-146c upregulation upon MG infection represses MMP16 expression, activating TLR6/MyD88/NF-κB pathway, promoting cell proliferation by inhibiting cell apoptosis, and, finally, enhancing cell cycle progression to defend against host MG infection.

Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.

Primordial germ cells (PGCs) are precursors of germline cells that can generate sperm and eggs in adults, making them promising tools for transgenic animal preparation and germplasm preservation, especially in avians. In this study, we purified the PGCs from circulating embryonic blood of Chinese Meiling chickens using Nycodenz density centrifugation, and characterized them by alkaline phosphatase (AKP) staining, periodic acid-Schiff (PAS) staining and stage-specific embryonic antigen-1 (SSEA-1) immunostaining and PGC-specific gene amplification. The purified PGCs were also labeled with PKH26 and transferred into donor chicken embryos at the Hamburger-Hamilton (HH) stage 14 to 16, and cells with red fluorescence were observed in the gonads of 8-d-old embryos. When using about 200 PGCs isolated from Chinese Meiling chickens, microinjection into the dorsal aortas of recipient chickens with white feathers at stage HH14 to 16 resulted in germline chimeras that hatched and attained sexual maturity. The frequency of donor-derived yellow-feathered offspring from germline chimeric chickens was 12.6 ± 2.6% after mating with the white-feathered chickens. These results demonstrate that we had successfully purified the PGCs from the Chinese Meiling chicken. These germline cells could be used to preserve Chinese Meiling chickens.