Abstract
A large resource of epitope-tagged and Cre/CreERT2-expressing mouse models are available for studying germ granules and germline development. Germ granules are proteinaceous, membraneless organelles (MLO) involved in germ cell differentiation and maturation; however, their protein and RNA transcript constituents, as well as their functional mechanisms remain incompletely understood. Herein, we generated a versatile germline mouse model through combinatorially tagging DDX4 to enable simultaneous expression of three cistronic coding products (C-terminally tagged DDX4 - DDX45HA, EGFP, and CreERT2) under the control of the endogenous Ddx4 promoter. By leveraging the high-affinity HA tag, we optimized an efficient workflow to purify germ granules (Chromatoid body, CB) from spermatids, and characterized their protein and RNA transcript composition. Moreover, we explored and ascertained that DDX4-mediated, phase-separation dependent CB integrity is functionally important for recruiting distinctive long RNA transcripts and for the biogenesis of pachytene- and TE-derived piRNAs. Together, our study generated a versatile germline mouse model with a multiplicity of applications for germline study, and provided mechanistic insights into germline development as dictated by germ granules.