DAZL plays a crucial role throughout the entire process of germ cell development, starting from the early stage of PGCs to the final formation of gametes. During the early stages of embryo development, DAZL is responsible for specifying the germ cell lineage and promoting the differentiation of pluripotent cells into germ cells by activating the expression of the DAZL gene [1]. Defects or abnormalities in DAZL can disrupt the development of germline stem cells, leading to reproductive disorders [2,3]. Interestingly, studies have shown that introducing the DAZL gene can partially rescue the impaired germ cell development in animals with DAZL-knockout mutations [4].

Chicken PGCs are a type of germline stem cell that exhibits a consistent and significant expression of DAZL. These specialized cells can be derived from embryos and cultured in vitro for extended periods without losing their ability to transmit genetic information [5]. Consequently, chicken PGCs have emerged as invaluable tools for investigating the functional aspects of genes in germline stem cells. Similar to mammals, the presence of DAZL is closely associated with the origin and central formation of PGCs in chickens [6]. Following their specification, chicken PGCs migrate from the central area pellucida of the blastoderm to the germinal crescent. Eventually, they enter the vascular system and settle in the genital ridge where they develop early gonads alongside surrounding somatic cells. Notably, throughout these intricate biological processes, DAZL exhibits robust expression [7], underscoring its vital role in facilitating the development of chicken PGCs. Despite its importance in chicken PGCs development, the mechanisms underlying how DAZL regulates gene expression and the gene network or regulatory pathways associated with the DAZL gene in chicken PGCs remain poorly understood.

In mammalian germ cells, DAZL plays a pivotal role in regulating essential biological processes by binding to RNAs. Extensive research has documented that DAZL specifically interacts with the 3′ UTR regions of genes, thereby exerting control over germ cell specialization and development [1,2], promoting germ cell survival [8], facilitating gametogenesis [9,10], influencing oocyte maturation [11], and orchestrating the transition from oocyte to zygote [12]. Moreover, DAZL's impact on germ cell development extends beyond RNA interactions as it also engages in protein-to-protein interactions. Protein immunoprecipitation techniques had been employed to reveal DAZL's regulatory role in pluripotency, differentiation, and apoptosis during the development of PGCs [13]. To gain further insights into the function of DAZL in chicken PGCs, exploring protein-to-RNA and protein-to-protein analyses holds immense promise.

In this study, we delved into the intricate functionality of DAZL in chicken PGCs and elucidated the intricate gene network orchestrated by DAZL-regulating RNAs and proteins. To investigate the impact of DAZL, we employed cutting-edge CRISPR/Cas9 technology to silence its expression in PGCs, allowing us to meticulously examine cell survival and proliferation. Subsequently, we conducted rigorous RIP-Seq and Co-IP-MS/MS analyses to unravel the transcriptomic and proteomic networks influenced directly by DAZL. Our groundbreaking findings shed light on how DAZL directly governs key genes associated with germline development, pluripotency, and cell proliferation in chicken PGCs. Notably, we discovered a collaborative partnership between DAZL and DDX4 in orchestrating the intricate gene regulatory network within chicken PGCs. These compelling results serve as a solid foundation for further exploring the functional role of DAZL in chicken germline cells.