Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development

Abstract

The segregation and maintenance of a dedicated germline in multicellular organisms is essential for species propagation in the sexually reproducing metazoan kingdom. The germline is distinct from somatic cells in that it is ultimately dedicated to acquiring the "totipotency" and to regenerating the offspring after fertilization. The most striking feature of germ cells lies in the presence of characteristic membraneless germ granules that have recently proven to behave like liquid droplets resulting from liquid-liquid phase separation (LLPS). Vasa/Ddx4, a faithful DEAD-box family germline marker highly conserved across metazoan species, harbors canonical DEAD-box motifs and typical intrinsically disordered sequences at both the N-terminus and C-terminus. This feature enables it to serve as a primary driving force behind germ granule formation and helicase-mediated RNA metabolism (e.g., piRNA biogenesis). Genetic ablation of Vasa/Ddx4 or the catalytic-dead mutations abolishing its helicase activity led to sexually dimorphic germline defects resulting in either male or female sterility among diverse species. While recent efforts have discovered pivotal functions of Vasa/Ddx4 in somatic cells, especially in multipotent stem cells, we herein summarize the helicase-dependent and -independent functions of Vasa/Ddx4 in the germline, and discuss recent findings of Vasa/Ddx4-mediated phase separation, germ granule formation and piRNA-dependent retrotransposon control essential for germline development.

Keywords: DEAD-box helicase; Germline; Phase separation; Spermatogenesis; Spermiogenesis; Transposable elements (TE); piRNA.

Fig. 1

Vasa orthologs are evolutionarily conserved and harbor intrinsically disordered regions at both the N-terminus and C-terminus. A The phylogenetic tree of Vasa orthologs from 12 animal species with schematic domain composition illustrated on the right side. Estimated divergence times are obtained from the TimeTree database [102]. B In silico prediction of protein disorder tendency for sequences of Vasa orthologs from mouse (blue), human (red), Drosophila melanogaster (black), and Zebrafish (yellow). Residues above the dotted line are predicted to be disordered by IUPred2A [103]. Conservative regions (DEAD-box helicase domain) are indicated by colored rectangles below. C Multi-sequence alignment of amino acids for Vasa orthologs across 12 metazoan species using Jalview [104]. Amino acids highlighted in red color represent conserved motifs in DEAD-box helicase family (Motifs I–VI) with names labeled, respectively, below. The green color highlights the divergent acidic C-terminal sequence across 12 species

Fig. 2

Schematic diagrams of germline development and dynamic distribution of nuage in Drosophila and mice. A Anatomy of the ovaries in a single ovariole illustrating nuage formation during Drosophila oogenesis. The Drosophila ovaries comprise 16–20 tubular structures, termed ovarioles, which are arranged as a production line-like assembly of differentiating egg chambers to produce mature eggs (oogenesis). The functional unit of the Drosophila ovary is termed the ovarian follicle, or egg chamber, originating from the germarium that is localized to the anterior tip in each ovariole. Germline stem cells (GSCs) (red) undergo asymmetric cell division, giving rise to one daughter stem cell that remains in contact with the Cap cells, and the other daughter cell destined to differentiate into Cystoblast (CB). Following four rounds of successively synchronous mitotic cell divisions, 16 interconnected cystocytes (light red) are produced, and subsequently bud off the germarium once they are surrounded by somatic follicle cells. During the development of each egg chamber, one of the 16 cystocytes is committed to meiosis and divides into the oocyte at the posterior end, whereas the remaining cells develop into polyploid nurse cells, which are interconnected through the intercellular bridges. Vasa-enriching nuage in nurse cells and Vasa-containing polar granules in oocyte are highlighted in green color. S, stage; B schematic diagram of nuage distribution (green color) in the male mouse germline. Primordial germ cells (PGCs) are induced in a subpopulation of the epiblast in the early embryos starting on embryonic day 6.5 (E6.5). Ddx4 is detectable in PGCs from E10 onwards. Pi-bodies and piP-bodies are present in prospermatogonia in the perinatal testis. Intermitochondrial cement (IMC) is present in the spermatocytes. The chromatoid body (CB) is assembled in haploid spermatids. All granules (green) are localized around the nuclear membrane within the cytoplasm