Znf706 regulates germplasm formation and primordial germ cell development in zebrafish

The cell fate of primordial germ cells (PGCs) in zebrafish is pre-determined by maternally deposited germplasm, which is packaged into ribonucleoprotein complex in oocytes and, after fertilization, inherited into PGCs promoting germline fate in embryos. However, the maternal factors regulating the assembly of germplasm and PGC development remain poorly understood. In this study, we report that the maternal transcription factor Znf706 regulates the assembly of germplasm factors into a granule-like structure localized perinuclearly in PGCs during migration. Maternal and zygotic mutants of znf706 (MZznf706) exhibited deficient germplasm at the early embryonic stage, decreased PGC numbers with ectopic PGC locations during PGC migration, and lower female ratio in adulthood. Notably, the implementation of Znf706 CUT&Tag and RNA-seq on immature oocytes uncovered that Znf706 in stage I oocytes may act as a transcriptional activator and mediate mRNA binding, translational regulation, and metabolic pathways of oocytes. Hence, we propose that Znf706 is crucial for germplasm assembly and PGC development in zebrafish. Overall design: Wild-type and znf706(tsu7d/tsu7d) mutants generated by CRISPR/Cas9 were used for mRNA-seq and Znf706 CUT&Tag assaies. In the context of the current investigation, a protocol for the isolation of oocytes from juvenile female fish was developed, aimed at maximizing yield and maintaining cellular integrity. The first step involved the euthanization of the WT and znf706-/- juvenile female fish (3-month-old) by submerging them in an ice-water mixture. After euthanization, the abdominal cavity of the fish was carefully opened using surgical scissors and the ovaries were bilaterally detached from the abdomen by utilizing forceps. The excised ovaries were promptly transferred into a 6-well plate preloaded with 2 ml digestive buffer (cell dissociation buffer, PCM, PC-33689) and incubated for 2-3 hrs at a temperature of 28.5?. To enhance the efficacy of the digestive process, the 6-well plate was shaken every half hour during incubation. Once the digestion period was complete, the L-15 medium, which had been preheated to 28.5?, was added to the digestive buffer to halt the digestion process effectively. To eliminate residual impurities, the oocytes were subjected to a series of washing steps involving the addition of fresh L-15 medium, followed by gentle resuspension of the oocytes, a process repeated 2-3 times to ensure thorough cleansing. Approximately 600 stage I oocytes and 150 stage II oocytes were collected manually with a pipette, with two biological replicates each for znf706-/- and WT through the aforementioned procedure and then transferred into RNase-free 1.5 ml tubes, for quick-frozen with liquid nitrogen and preserved at -80? for short-term storage. All the liquid nitrogen-freezing samples (stages I and II oocytes) were sent to Novogene for were sent to Novogene for RNA-seq library preparation and sequencing in the Hiseq-PE150 platform. The operation from the anesthetization of adult fish to oocyte digestion is the same as that for RNA-seq. It is worth noting that CUT&Tag is a technique capturing genomic DNA, thus, it is imperative to eliminate the contamination from the granulosa cells and other somatic cells that envelop the oocytes. In our study, we performed the digestion by cell dissociation buffer (PCM, PC-33689) to get rid of somatic cells. The oocyte surface exhibits a pronounced smoothness, signifying the removal of somatic cells, which would be further validated by DAPI staining. Then, the medium containing the oocytes was drawn through a 100 µm cell strainer using a pipette, followed by a 2-minute incubation period to allow for the gravitational sedimentation of the oocytes at the bottom of the plate. To remove impurities, the sample was thoroughly washed 2-3 times with a preheated L-15 medium. Approximately 5,000 WT stage I oocytes were collected, followed by library construction using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904) and sequencing (Novogene, Hiseq-PE150 platform). The primary antibody used in the experimental group is rabbit anti-Znf706 (Aviva, arp32987-P050, 2.5µg / 50µl), while rabbit anti-IgG antibody (Cell Signaling Technology, 2729s, 2.5 µg / 50µl) in the control group. The secondary antibody was goat anti-rabbit IgG H&L (Abcam, ab6702, 1:100 dilution). The experimental group underwent two biological replicates.