Single-cell RNA sequencing reveals surface markers of primordial germ cells in chicken and zebra finch.

Primordial germ cells (PGCs) in avian species exhibit unique developmental features, including the ability to migrate through the bloodstream and colonize the gonads, allowing their isolation at various developmental stages. Several methods have been developed for the isolation of avian PGCs, including density gradient centrifugation, size-dependent separation, and magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) using a stage-specific embryonic antigen-1 (SSEA-1) antibody. However, these methods present limitations in terms of efficiency and applicability across development stages. In particular, the specificity of SSEA-1 decreases in later developmental stages. Furthermore, surface markers that can be utilized for isolating or utilizing PGCs are lacking for wild birds, including zebra finches, and endangered avian species. To address this, we used single-cell RNA sequencing (scRNA-seq) to uncover novel PGC-specific surface markers in chicken and zebra finch. We screened for genes that were primarily expressed in the PGC population within the gonadal cells. Analyses of gene expression patterns and levels based on scRNA-seq, coupled with validation by RT-PCR, identified NEGR1 and SLC34A2 as novel PGC-specific surface markers in chickens and ESYT3 in zebra finches. Notably, these newly identified genes exhibited sustained expression not only during later developmental stages but also in reproductive tissues.

Prognostic factors of first-onset optic neuritis based on diagnostic criteria and antibody status: a multicentre analysis of 427 eyes.

BACKGROUND: Optic neuritis (ON) prognosis is influenced by various factors including attack severity, underlying aetiologies, treatments and consequences of previous episodes. This study, conducted on a large cohort of first ON episodes, aimed to identify unique prognostic factors for each ON subtype, while excluding any potential influence from pre-existing sequelae. METHODS: Patients experiencing their first ON episodes, with complete aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) antibody testing, and clinical data for applying multiple sclerosis (MS) diagnostic criteria, were enrolled. 427 eyes from 355 patients from 10 hospitals were categorised into four subgroups: neuromyelitis optica with AQP4 IgG (NMOSD-ON), MOG antibody-associated disease (MOGAD-ON), ON in MS (MS-ON) or idiopathic ON (ION). Prognostic factors linked to complete recovery (regaining 20/20 visual acuity (VA)) or moderate recovery (regaining 20/40 VA) were assessed through multivariable Cox regression analysis. RESULTS: VA at nadir emerged as a robust prognostic factor for both complete and moderate recovery, spanning all ON subtypes. Early intravenous methylprednisolone (IVMP) was associated with enhanced complete recovery in NMOSD-ON and MOGAD-ON, but not in MS-ON or ION. Interestingly, in NMOSD-ON, even a slight IVMP delay in IVMP by >3 days had a significant negative impact, whereas a moderate delay up to 7-9 days was permissible in MOGAD-ON. Female sex predicted poor recovery in MOGAD-ON, while older age hindered moderate recovery in NMOSD-ON and ION. CONCLUSION: This comprehensive multicentre analysis on first-onset ON unveils subtype-specific prognostic factors. These insights will assist tailored treatment strategies and patient counselling for ON.

Comparative single-cell transcriptomic analysis reveals differences in signaling pathways in gonadal primordial germ cells between chicken (Gallus gallus) and zebra finch (Taeniopygia guttata).

Primordial germ cells (PGCs) have been used in avian genetic resource conservation and transgenic animal production. Despite their potential applications to numerous avian taxa facing extinction due to habitat loss and degradation, research has largely focused on poultry, such as chickens, in part owing to the difficulty in obtaining intact PGCs from other species. Recently, phenotypic differences between PGCs of chicken and zebra finch, a wild bird with vocal learning, in early embryonic development have been reported. In this study, we used advanced single-cell RNA sequencing (scRNA-seq) technology to evaluate zebra finch and chicken PGCs and surrounding cells, and to identify species-specific characteristics. We constructed single-cell transcriptome landscapes of chicken gonadal PGCs for a comparison with previously reported scRNA-seq data for zebra finch. We identified interspecific differences in several signaling pathways in gonadal PGCs and somatic cells. In particular, NODAL and insulin signaling pathway activity levels were higher in zebra finch than in chickens, whereas activity levels of the downstream FGF signaling pathway, involved in the proliferation of chicken PGCs, were higher in chickens. This study is the first cross-species single-cell transcriptomic analysis targeting birds, revealing differences in germ cell development between phylogenetically distant Galliformes and Passeriformes. Our results provide a basis for understanding the reproductive physiology of avian germ cells and for utilizing PGCs in the restoration of endangered birds and the production of transgenic birds.

Transplantation and enrichment of busulfan-resistant primordial germ cells into adult testes for efficient production of germline chimeras in songbirds†.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.

Efficacy of part-time patching in preventing recurrence after bilateral lateral rectus recession in children with intermittent exotropia.

BACKGROUND: This study evaluate the efficacy of part-time patching in preventing recurrence after bilateral lateral rectus recession (BLR) in patients with intermittent exotropia (IXT). METHODS: A total of 190 children aged 3-13 years who experienced recurrence after BLR for IXT and received part-time patching were retrospectively reviewed. The patching was prescribed for 2 h per day for more than 6 months. Patients who had a recurrence of 18 PD or more underwent reoperation. Changes in exodeviation and reoperation ratio after part-time patching were analyzed. RESULTS: A total of 34 patients (17.9%) received reoperation after part-time patching, and the reoperation ratio after 2 years was 20.3% as per the Kaplan-Meier survival analysis. Patients with a recurrence of 7 to 10 PD showed a significantly better effect compared to those with a recurrence of more than 10 PD (p < 0.001), and the reoperation ratio was also lower in the survival analysis (p = 0.004). The factor associated with reoperation in patients with part-time patching was the duration between the operation and the initiation of part-time patching (hazard ratio [HR] = 1.006, p = 0.002). CONCLUSIONS: Part-time patching was effective in maintaining the efficacy of surgery and delaying the need of reoperation after BLR. This effect was better in patients with a recurrence of ≤ 10 PD.

Efficient gene delivery into the embryonic chicken brain using neuron-specific promoters and in ovo electroporation.

BACKGROUND: The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging. METHODS: In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters. RESULTS: Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4. CONCLUSION: We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.

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

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

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

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

Generation and characterization of genome-modified chondrocyte-like cells from the zebra finch cell line immortalized by c-MYC expression.

BACKGROUND: Due to their cost effectiveness, ease of use, and unlimited supply, immortalized cell lines are used in place of primary cells for a wide range of research purposes, including gene function studies, CRISPR-based gene editing, drug metabolism tests, and vaccine or therapeutic protein production. Although immortalized cell lines have been established for a range of animal species, there is still a need to develop such cell lines for wild species. The zebra finch, which is used widely as a model species to study the neurobiological basis of human speech disorders, has been employed in several functional studies involving gene knockdown or the introduction of exogenous transgenes in vivo; however, the lack of an immortalized zebra finch cell line has hampered precise genome editing studies. RESULTS: Here, we established an immortalized cell line by a single genetic event, expression of the c-MYC oncogene, in zebra finch embryonic fibroblasts and examined its potential suitability for gene targeting investigations. Retroviral vector-mediated transduction of c-MYC was used to immortalize zebra finch primary fibroblasts; the transformed cells proliferated stably over several passages, resulting in the expression of chondrocyte-specific genes. The transfection efficiency of the immortalized cells was much higher than that of the primary cells. Targeted knockout of the SOX9 gene, which plays a role in the differentiation of mesenchymal progenitor cells into chondrocytes, was conducted in vitro and both apoptosis and decreased expression levels of chondrogenic marker genes were observed in edited cells. CONCLUSIONS: The c-MYC induced immortalized chondrocyte-like cell line described here broadens the available options for establishing zebra finch cell lines, paves the way for in-depth biological researches, and provides convenient approaches for biotechnology studies, particularly genomic modification research.

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

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

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

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

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

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

The transcriptome of early chicken embryos reveals signaling pathways governing rapid asymmetric cellularization and lineage segregation.

The phylogenomics and comparative functional genomics of avian species were investigated in the Bird 10,000 Genomes (B10K) project because of the important evolutionary position of birds and their value as a research model. However, the systematic profiling of transcriptional changes prior to oviposition has not been investigated in avian species because of the practical difficulties in obtaining pre-oviposited eggs. In this study, a total of 137 pre-oviposited embryos were collected from hen ovaries and oviducts and subjected to RNA-sequencing analyses. Two waves of chicken zygotic genome activation (ZGA) were observed. Functionally distinct developmental programs involving Notch, MAPK, Wnt and TGFß signaling were separately detected during cleavage and area pellucida formation. Furthermore, the early stages of chicken development were compared with the human and mouse counterparts, highlighting chicken-specific signaling pathways and gradually analogous gene expression via ZGA. These findings provide a genome-wide understanding of avian embryogenesis and comparisons among amniotes.

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

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

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Zygotic genome activation in the chicken: a comparative review.

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

Pachydrusen, choroidal vascular hyperpermeability, and punctate hyperfluorescent spots.

PURPOSE: To investigate the relationship between pachydrusen and features of choroidal vascular hyperpermeability (CVH) and punctate hyperfluorescent spots (PHS) on serial imaging in patients with polypoidal choroidal vasculopathy (PCV) or pachychoroid neovasculopathy (PNV). METHODS: Patients diagnosed between January 2007 and June 2016 at 2 high-volume, tertiary hospitals were retrospectively reviewed with serial multimodal imaging assessment. The primary outcome was the association between drusen subtypes (hard/soft drusen, subretinal drusenoid droplets, or pachydrusen) with CVH and PHS, previously described in central serous chorioretinopathy. RESULTS: Among the 105 eyes (105 patients; mean age, 67.0 years), 87 (82.9%) were diagnosed with PCV and 18 (17.1%) with PNV. Pachydrusen was the most frequently identified subtype (54 eyes, 51.4%). CVH (72.2% vs 41.4%, P = 0.021) and PHS (72.2% vs 44.8%, P = 0.041) were observed with greater frequency in PNV eyes. Significant correlations were found between CVH and PHS (phi coefficient φ 0.30, P = 0.003), and PHS with pachydrusen (φ 0.20, P = 0.040). Over a mean follow-up of 74.8 months, new drusen co-localizing to PHS were noted in 22 (21.0%) eyes (φ 0.54, P < 0.001). CONCLUSION: We observed a trend of pachydrusen appearing in conjunction with PHS in PCV or PNV. Frequent localization of new drusen to these choroidal lesions was observed over long-term follow-up. PHS may be a form of late-staining "forme fruste" drusen, possibly associated with micro-ischemic changes to the choriocapillaris.

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

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

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

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

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

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