Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

Abstract

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.

Single‐cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway‐related gene expression profiles during mitotic arrest

“…Libraries were sequenced with a 2 × 100 bp paired‐end protocol on a Novaseq‐6000 platform (Illumina, San Diego, CA, USA). Detailed information is shown in our previous studies [ 5 , 13 ].…”

“…In chickens, during the differentiation of primordial gem cells (PGCs) into sex‐specific germ cells, female germ cells enter meiosis and maintain meiotic arrest in G2/M phase until hatching, whereas male germ cells asynchronously enter mitotic arrest on embryonic day 14 (E14) and then enter into the resting phase (mitotic quiescence state). At 10 weeks after hatching, they re‐enter the cell cycle and begin to differentiate [ 3 , 4 , 5 ]. The mitotic arrest in germ cells is regulated by multiple signaling pathways in diverse species [ 6 , 7 , 8 , 9 , 10 ].…”

“…Recently, we developed germ‐cell tracing chicken models expressing germ‐cell‐specific fluorescent markers and were able to perform scRNA‐seq of germ cells at each stage of embryonic development using single‐cell transcriptomic approach [ 13 ]. Using this model, we identified the mitotic‐arrested prospermatogonia cluster at three time points (E12, E16, and hatch) and found an epigenetic reprogramming schedule of mitotic‐arrested chicken prospermatogonia [ 5 ]. As a follow‐up study, here we investigate signaling pathways that regulate male embryonic germ cells during mitotic arrest for the first time using a single‐cell transcriptomic approach.…”

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

“…Furthermore, it was reported that the genital ridge cells secrete a chemoattractant stromal cell-derived factor 1 (SDF1), which is received by PGC G protein-coupled receptor C-X-C motif chemokine receptor 4 (CXCR4) during the migration of PGCs toward the genital ridge [ 19 ]. Additionally, the migrating PGCs undergo genome-wide DNA demethylation, and de novo DNA methylation is then established in PGCs entering the gonads [ 2 , 4 ].…”

“…The specification of PGCs was reported to take place via either an epigenesis mode (particularly in Mammalia and Caudata) or an inherited mode (in Aves, Anura, Teleostei, and Insecta) in animals [ 1 ]. Irrespective of these specification modes, the specified PGCs migrate to the gonads and simultaneously undergo epigenetic reprogramming, including genome-wide DNA demethylation and dynamic changes in histone modifications [ 2 , 3 , 4 ]. Fertility functions, including germ cell development in females and males, are essential for producing healthy offspring [ 5 ].…”