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Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca

Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myotubes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulated of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets., Article, Animal Science Journal. 92(1) : e13597 (2021)

Skeletal muscle myoblasts are myogenic precursors that develop into myofibers during muscle formation. Improvement of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myocytes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myocytes, myotube formation, and myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

Article, Poultry Science. 97(2): 650-657. (2018)

Myoblasts play a central role during skeletal muscle formation and growth. Precise understanding of myoblast properties is thus indispensable for meat production. Herein, we report the cellular characteristics and gene expression profiles of primary-cultured myoblasts of layer and broiler chickens. Broiler myoblasts actively proliferated and promptly differentiated into myotubes compared to layer myoblasts, which corresponds well with the muscle phenotype of broilers. Transcriptomes of layer and broiler myoblasts during differentiation were quantified by RNA sequencing. Ontology analyses of the differentially expressed genes (DEGs) provided a series of extracellular proteins as putative markers for characterization of chicken myogenic cells. Another ontology analyses demonstrated that broiler myogenic cells are rich in cell cycle factors and muscle components. Independent of these semantic studies, principal component analysis (PCA) statistically defined two gene sets: one governing myogenic differentiation and the other segregating layers and broilers. Thirteen candidate genes were identified with a combined study of the DEGs and PCA that potentially contribute to proliferation or differentiation of chicken myoblasts. We experimentally proved that one of the candidates, enkephalin, an opioid peptide, suppresses myoblast growth. Our results present a new perspective that the opioids present in feeds may influence muscle development of domestic animals., Article

We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonial stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells (TCs) from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated TCs were 91.00% ± 2.12% and 88.00% ± 1.87% in juvenile and adult testes, respectively, and immunohistochemical evaluation indicated the expression of integrin alpha-6 (ITGA6), GDNF family receptor alpha-1 (GFRA1), and Deleted in azoospermia-like (DAZL) in specific TCs. SSCs were purified by differential plating of TCs and then subjected to quantitative reverse transcription-polymerase chain reaction, which showed differential expression of SSC-specific, and germness and stemness-related genes. Coculture of quail SSCs with mouse embryonic fibroblasts and Sertoli cells as a feeder layer resulted in the generation of stable SSC colonies and short-term cultivation, and the expression of SSC and germ cell markers was maintained during several passages of culture. Collectively, these results demonstrate the efficient isolation and characterization of quail SSCs and the suitability of Sertoli cells as a feeder layer for in vitro culture of quail SSCs. Quail SSCs will facilitate the production of germline chimeras and transgenesis.

Toll-like receptors (TLRs) are a group of sensory receptors which are capable of recognizing a microbial invasion and activating innate immune system responses, including inflammatory responses, in both immune and non-immune cells. However, TLR functions in chick myoblasts, which are myogenic precursor cells contributing to skeletal muscle development and growth, have not been studied. Here, we report the expression patterns of TLR genes as well as TLR ligand-dependent transcriptions of interleukin (IL) genes in primary-cultured chick myoblasts. Almost TLR genes were expressed both in layer and broiler myoblasts but TLR1A was detected only in embryonic layer chick myoblasts. Chick TLR1/2 ligands, Pam3CSK4 and FSL-1, induced inflammatory ILs in both layer and broiler myoblasts but a TLR4 ligand, lipopolysaccharide, scarcely promoted. This is the first report on TLR ligand-dependent inflammatory responses in chick myoblasts, which may provide useful information to chicken breeding and meat production industries.

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2., Article, Journal of Reproduction and Development.62(2):143-149(2016)

Article, International Journal of Molecular Sciences. 18(11): 2245. (2017)

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors – phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) – all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca2+ rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca2+ oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.

The ex vivo culture of avian embryos is a technique for the long-term culturing of embryos outside of their own shell and shell membrane. It allows easy access to the developing embryos and embryo manipulation. The two-step system is widely applied when the culture is performed after oviposition. Japanese quail as well as bobwhite quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been more limited than those on the Japanese quail. We have developed a more simplified ex vivo culture protocol for the two species of quail embryos from the blastoderm stage through to hatching using a single surrogate eggshell. Hatchabilities of 31% and 27% were obtained in bobwhite quail and Japanese quail embryos, respectively. The simple system described in the present study is an easy and acceptable procedure., Article, JOURNAL OF POULTRY SCIENCE. 51(2):202-205 (2014)

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP(+)/DsRed(+) hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products., Article, ANIMAL SCIENCE JOURNAL. 88(11):1880-1885 (2017)

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species.

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated. In the present studies, sexually matured males and females of the type-l were mated each other. Form these mating. chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of Muscular dystrophy. were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens Could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and Survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain. The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy., Article, JOURNAL OF POULTRY SCIENCE. 46(2): 123-126(2009)

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy. It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens., Article, JOURNAL OF POULTRY SCIENCE. 46(1): 46-51(2009)

Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the efficiency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for 0, 20, 40 or 60s before incubation. The irradiated embryos manifested delayed development at 60 h of incubation. They also showed reduced numbers of circulating PGCs at stages 14 and 15 and of gonadal PGCs at stage 30. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for 20 s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for 20 s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage 30. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the efficiency of incorporation of donor PGCs., Article, JOURNAL OF POULTRY SCIENCE. 45(4): 292-297(2008)

Recently, many results have been reported regarding the pluripotency of bone marrow cells (BMCs) with the aim of benefiting regenerative medicine for humans. Particularly, vessel formation by hematopoietic stem cells or vascular endothelial stem cells which were derived from bone marrow has received considerable interest, since the mechanism of vessel formation has been found to be involved in neoangiogenesis of serious diseases such as cancer. Most work on neoangiogenesis and regeneration has involved mammalian experimental systems, however the avian model is useful since the process of neoangiogenesis and regeneration of vessels can be observed with the whole embryo culture system. We have established a novel system using early chick embryos, where a portion of blood vessels are degenerated by UV irradiation, and vessel regeneration is then studied. Incubated embryos were partially covered with aluminum foil, from the embryonic body to the dorsal marginal vein, and irradiated with UV for 1 min. Donor BMCs were obtained from the femurs and tibias of chicks aged 10 days, fluorescently labeled with PKH26 and injected into the anterior vitelline vein of the recipients. In BMC-treated embryos the donor BMCs were observed around the UV-degenerated vessels, and regeneration of blood vessels occurred, in contrast to the untreated embryos. These results indicate that avian BMCs have the ability to participate in vessel regeneration, and the avian model used here may be a useful tool for studies of vessel neoangiogenesis and repair.

In avian species, primordial germ cells (PGC) use the vascular system as a vehicle to transport them to the future gonadal region. The aim of this study was to elucidate the details of migration system and size of the PGC population in the early chicken embryo. We analyzed whole chicken embryos during stages X and 2 to 17 by immunohistochemical staining using specific antibody raised against chicken vasa homolog. At stage X, PGC were dense in the central zone of the area pellucida. Following the formation of the primitive streak, PGC moved anteriorly to the edge of the extraembryonic region. The size of the PGC population increased gradually during stages X (130.4 +/- 31.9) to 10 (439.3 +/- 93.6). At stage 10, PGC began to accumulate in the region anterior to the head, and then we could observe that PGC invaded into the vascular system in this region. At stage 11, the number of PGC decreased in the region anterior to the head (129.8 +/- 42.5 to 46.7 +/- 4.2) and increased in the blood vessels (194.0 +/- 41.6 to 285.0 +/- 7.5). No PGC could be recognized in the intermediate mesoderm, the future gonadal region, until stage 14, but they first appeared there at stage 15. The number of PGC recognized in the intermediate mesoderm increased from stage 15 to 17. Interestingly, the number of PGC between the left and right sides of this region was consistently and significantly different (P < 0.05) in females and males. The present study mainly clarified that chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively left the blood vessels and migrated to the intermediate mesoderm from stage 15.

A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.

Our previous study demonstrated that elongated spermatids and sperm carrying the female-specific W-chromosome of the sex-reversed domestic fowl can activate the mouse oocyte, but whether they can fertilize the avian oocyte and lead to a developing zygote remains undetermined. A single sperm isolated from the semen and testis of normal rooster and from a testis of sex-reversed hen was microinjected into a quail oocyte and cultured for 20 to 24 h. Blastoderms were fixed, cleaved, nuclei stained by 4',6'-diamidino-2-phenylin-dole, and developmental stages were assessed. In the normal rooster group, ejaculated and testicular sperm induced blastodermal development in 22.6 and 20% of the quail oocytes, respectively. The developmental stages ranged from IV to VII. In the sex-reversal group, 20% of injected testicular sperm induced blastodermal development. The blastodermal stages varied from stage III to VI. Blastoderms after 4',6'-diamidino-2-phenylindole staining were assayed by PCR to identify the W chromosome of either chicken sperm or quail oocyte. The PCR assay results showed that 2 out of 9 developed blastoderms microinjected with sperm of sex-reversed hen were identified containing the female-specific W chromosome derived from sex-reversed hen. From these results, it is concluded that chicken sperm bearing the W chromosome possess fertilizing ability and can function to stimulate blastoderm development similar to that of normal chicken sperm carrying the Z chromosome.

The timing and biological events associated with germ cell specification in chickens have not been determined yet. In this study, we report the origin of primordial germ cells (PGCs) and germ plasm dynamics through investigation of the expression of the chicken homolog of deleted in azoospermia-like (cDAZL) gene during germ cell specification. Asymmetric localization of germ plasm in the center of oocytes from preovulatory follicle stages leads to PGCs being formed in the center. During cleavage stages, DAZL expression pattern changes from a subcellular localization to a diffuse form before and after zygotic genome activation. Meanwhile, PGCs exhibit transcriptional active status during their specification. In addition, knockdown studies of cDAZL, which result in reduced proliferation, aberrant gene expression profiles, and PGC apoptosis in vitro, suggest its possible roles for PGC formation in chicken. In conclusion, DAZL expression reveals formation and initial positioning of PGCs in chickens.