Capturing avian somatic cells using feather pulp fibroblast culture as a non-invasive approach to biobanking endangered birds.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Transgenic animals have become important tools for biological research. In mammals, several methods are available to introduce foreign DNA into the mammalian genome. Gene transfer in birds is a relatively complicated process because of the unique aspects of avian reproduction, and avian transgenics has largely been limited to the use of retroviral/lentiviral vectors. However, the temporal and spatial aspects of the development of avian primordial cells (PGCs) provides easy access to the germline and several methods can be used to produce germline chimeras. Initially, the ability to make germline chimeras has given rise to efforts to establish avian embryonic stem cells (ESCs)lines. In the chick, ESCs can be cultured from the area pellucida of the unincubated embryo. These cells show several features of stem cells including the capacity to give rise to all somatic tissues. However, competency to give rise to the germline has been difficult to achieve, most likely due to the mechanisms of avian germline development. As an alternative to embryonic stem cells, the long term culture of avian PGCs hold significant promise for non-viral methods of manipulating the avian genome. Using a combination of STO feeder layers, conditioned media, fibroblast growth factor-2 and stem cell factor, stable lines of chick PGCs have been established from single embryos. These lines express several markers of germ cells and have been in continuous culture for over a year. Many of the lines retain their ability to populate the germinal ridge when injected into early embryos and can give rise to functional gametes at sexual maturity. Like that observed in mammals, under specific culture conditions, PGCs can give rise to embryonic germ (EG) cells. However, as observed with ESCs, avian EG cells only give rise to somatic cells. Hence, the long-term culture of primordial germ cells opens new applications for avian germ cell biology, avian transgenics, germplasm preservation and stem cell biology.

In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.

BackgroundAvian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.Principal FindingsWe confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.ConclusionsThe establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

Tenascin-C is a large hexameric extracellular matrix glycoprotein associated with epithelial-mesenchymal interactions, connective tissue development, and the formation of the central nervous system. Tenascin-C also lines the pathways followed by migrating avian neural crest cells, although its role in neural crest morphogenesis remains unclear. In vitro, tenascin-C interferes with cell-fibronectin interactions, and promotes the motility of many cell types including the neural crest. To determine if tenascin-C is a consistent component of matrices through which invasive embryonic cells migrate, we have investigated if tenascin-C is associated with 2 additional populations of motile, embryonic cells: primordial germ cells and hematopoietic progenitor cells. We have found that HNK-1, a monoclonal antibody used as a marker of neural crest, also stains avian primordial germ cells. Double-label immunohistochemistry reveals that tenascin-C is found in the mesenchyme adjacent to the ventral half of the dorsal aorta where the primordial germ cells penetrate the vessel wall, and both tenascin-C and fibronectin are present in the extracellular matrix through which the primordial germ cells migrate to reach the genital ridges. Unlike fibronectin, which is found throughout the splanchnic mesoderm, tenascin-C is concentrated in the proximal part of the splanchnic region where the primordial germ cells are concentrated. In embryos where the gonadal anlagen are surgically removed before the primordial germ cells leave the bloodstream, ectopic primordial germ cells were found exclusively in head and trunk mesenchyme containing tenascin-C. Like primordial germ cells, a subset of hematopoietic progenitor cells migrate through the mesenchyme ventral to the dorsal aorta where they form hematopoietic clusters. Others bud directly into the lumen of the aorta. Anti-tenascin-C stains the mesenchyme surrounding the migrating cells as well as the basal surfaces of the cells that appear to be budding into the lumen. In situ hybridization with a tenascin-C-specific cDNA probe shows that the major sources of the tenascin-C mRNA in this region are the hematopoietic progenitor cells themselves as well as the cells in the wall of the ventral aorta. mRNAs encoding 3 major splice variants of tenascin-C were identified by reverse transcriptase polymerase chain reaction (PCR) in the embryonic aorta and adjacent mesenchyme dissected from both the region of primordial germ cell and hematopoietic precursor cell migration. These experiments indicate that tenascin-C is a component of the migratory environment for many motile cells in the early embryo, where it has the potential to mediate cell-fibronectin interactions.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.

Direct introduction of cryopreserved embryonic gonadal germ cells (GGC) into a sterile chicken surrogate host to reconstitute a chicken breed has been demonstrated as a feasible approach for preserving and utilizing chicken genetic resources. This method is highly efficient using male gonads; however, a large number of frozen female embryonic gonads is needed to provide sufficient purified GGC for the generation of fertile surrogate female hosts. Applying this method to indigenous chicken breeds and other bird species is difficult due to small flock numbers and poor egg production in each egg laying cycle. Propagating germ cells from the frozen gonadal tissues may be a solution for the biobanking of these birds. Here, we describe a simplified method for culture of GGC from frozen embryonic 9.5 d gonads. At this developmental stage, the germ cells are autonomously shed into medium, yielding hundreds to thousands of mitosis-competent germ cells. The resulting cultures of GGC have over 90% purity, uniformly express SSEA-1 and DAZL antigens and can re-colonize recipient's gonads. The GGC recovery rate from frozen gonads are 42% to 100%, depending on length of cryopreservation and the breed or line of chickens. Entire chicken embryos can also be directly cryopreserved for later gonadal isolation and culture. This storage method is a supplementary approach to safeguard local indigenous chicken breeds bearing valuable genetic traits and should be applicable to the biobanking of many bird species.

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

Pronounced early differentiation underlies zebra finch gonadal germ cell development

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

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

Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC. At Days 30 and 31 of pregnancy centromeric repeats and IGF2-H19 DMD sequences acquired new methylation in male PGC, while in female PGC these sequences were completely demethylated by Day 30 and remained hypomethylated at Day 31. To characterize methylation changes that PGC undergo in culture, the methylation status of embryonic germ cells (EGCs) derived from PGC at Day 26 of pregnancy was examined. Centromeric repeats and IGF2-H19 DMD sequences were similarly methylated in both male and female EGC and hypermethylated in female EGC compared with female PGC at the same embryonic age. Our results show that, similar to murine PGC, porcine PGC undergo genome-wide DNA demethylation shortly after arrival in the genital ridges. When placed in culture porcine PGC terminate their demethylation program and may acquire new DNA methylation marks. To our knowledge, this is the first report regarding epigenetic re-programming of genital ridge PGC in the pig.

In previous studies, primordial germ cells (PGCs) of the chicken were successfully stored in liquid nitrogen, and viable progenies were obtained after transfer of the frozenthawed PGCs into recipient embryos. This study was performed in order to examine whether PGCs of the Japanese quail could be preserved under conditions similar to those for the cryopreservation of chicken PGCs. First, quail PGCs purified from embryonic blood were stored in liquid nitrogen for one to two weeks. The viability of the frozenthawed PGCs was 85% on average, indicating that the PGCs were successfully preserved. Then, gonadal PGCs enriched from the gonadal anlage of wild-type and F1 (AMRP×SBPN) quail embryos at 5 days of incubation were stored in liquid nitrogen for up to 5 months, and tranferred to quail embryos of the other strain. Upon mating with F1 (AMRP×SBPN) quail, progenies derived from the donor PGCs were obtained from two out of 12 manipulated quail. The proportion of donor derived progenies in these two germline chimeric quail was 2.4% and 2.5%. These results indicated that the quail gonadal PGCs stored in liquid nitrogen retained the ability to differentiate into functional gametes, and that the genetic resources of the quail could be conserved by cryopreserving their PGCs.

In vertebrates, the germ line stem cell population arises from extraembryonic regions and must actively migrate to the gonadal anlagen. In the avian embryo, the process of germ line stem cell migration proceeds through a series of active and passive migratory phases. The germ line stem cells or primordial germ cells (PGCs) located in the epiblast of the unincubated embryo translocate to the hypoblast between stages X-XIII (E-G&K). Subsequently, during gastrulation the PGCs are passively carried by the hypoblast to the germinal crescent at about stage 4 (H&H). As blood islands develop and the embryo becomes vascularized, the PGCs are passively carried through the blood stream between stages 13-16 (H&H). Subsequently, the PGCs leave the blood vessels and actively migrate to the gonadal anlagen. Recently, it has become possible to culture chicken germ line stem cells using PGC populations harevested from embryonic blood. We have established several cultures of the germ line stem cells. To examine the ability of the cultured germ cells to migrate, an in ovo assay was developed. The PGCs were loaded with a vital fluorescent dye (PKH-26) and injected into the stage X embryos. At stage 28-30 (H&H), the gonads were removed and examined for the presence of fluorescent PGCs indicating that the injected germ cells successfully migrated. Variability in the ability of cultured PGCs to migrate was found. Therefore, we compared the global gene expression profile between a line of germ line stem cells that exhibited good migration and a line with low migration. A chicken 44K Agilent microarray was used to interrogate the expression profiles of the two cell lines. Total RNA was obtained from 4 replicate cell cultures labeled by Cy3 or Cy5 with dye swap. Gene signal intensity was globally normalized by LOWESS and expressed on natural log scale. A mixed model including the fixed effects of dye, line, and random effects of slide and array was used to identify differentially expressed genes at P < 0.05. Of the 42,035 identified genes scanned, 5013 genes showed differential expression. As expected, several germ cell specific genes were not differentially expressed including VASA and DAZL. Of the differentially expressed genes 32 showed a greater than five-fold difference between the two lines. However, one gene showed an 18 fold difference, BU455352, Gga.19961 (alpha-3-catenin). This gene produces a beta-catenin-binding protein that mediates strong cell-cell adhesion and is associated with cell adhesion and migration in other cells. The results of this study confirm that cultured avian PGCs can retain their ability to migrate to the gonadal anlagen after long-term culture and suggests that alpha-3-catenin may be associated with avian germ cell migration.