Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail.

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.