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Background Germ cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage. Results We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later. Conclusions We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

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.