vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development

Sylvia Styhler 1 Akira Nakamura 0 1 Andrew Swan 1 Beat Suter 1 Paul Lasko ) 1 0 Present address: Institute of Biological Sciences, University of Tsukuba , Tsukuba, Ibaraki 305 , Japan 1 Department of Biology, McGill University , 1205 Avenue Docteur Penfield, Montreal, Quebec , Canada H3A 1B1 SUMMARY The Drosophila gene vasa is required for pole plasm assembly and function, and also for completion of oogenesis. To investigate the role of vasa in oocyte development, we generated a new null mutation of vasa, which deletes the entire coding region. Analysis of vasa-null ovaries revealed that the gene is involved in the growth of germline cysts. In vasa-null ovaries, germaria are atrophied, and contain far fewer developing cysts than do wild-type germaria; a phenotype similar to, but less severe than, that of a null nanos allele. The null mutant also revealed roles for vasa in oocyte differentiation, anteriorposterior egg chamber patterning, and dorsal-ventral follicle patterning, in addition to its better-characterized Segregation of the germline from the soma is a central feature of animal development. In Drosophila, the germline is determined through the activities of maternally expressed RNAs and proteins which colocalize in the pole plasm at the posterior pole of the egg (reviewed by Rongo and Lehmann, 1996). Pole cells, the progenitors of the germline, form very early in embryogenesis, then, beginning at gastrulation, they migrate into the interior of the embryo and ultimately associate with the gonadal mesoderm to form the embryonic gonads (reviewed by Williamson and Lehmann, 1996). Beginning in larval development, germ cells proliferate and differentiate in order to carry out spermatogenesis and oogenesis; among the structures assembled during oogenesis is new pole plasm, which specifies the germline for the subsequent generation of individuals. Genetic and molecular studies have identified numerous genes which are required for pole plasm assembly and subsequent posterior segment specification and germ cell formation; many of these genes are expressed during oogenesis and produce mRNAs and/or proteins which localize in pole plasm or in polar granules, specialized organelles contained within the pole plasm (reviewed by Rongo and Lehmann, 1996). Analysis of the expression of these genes supports an early hypothesis (Mahowald, 1968) that translational control is a major mechanism regulating Drosophila germline functions in posterior embryonic patterning and pole cell specification. The anterior-posterior and dorsal-ventral patterning phenotypes resemble those observed in gurken mutants. vasa-null oocytes fail to efficiently accumulate many localized RNAs, such as Bicaudal-D, orb, oskar, and nanos, but still accumulate gurken RNA. However, GRK accumulation in the oocyte is severely reduced in the absence of vasa function, suggesting a function for VASA in activating gurken translation in wild-type ovaries. development. The product of the vasa (vas) gene, a DEADbox-family protein which is localized in polar granules and which shares the enzymatic functions of the translation initiation factor eIF4A (Hay et al., 1988; Lasko and Ashburner, 1988; Liang et al., 1994), is a candidate germline-specific translational regulator. For instance, levels of the short isoform of OSKAR protein (OSK), a molecule central to pole plasm assembly (Ephrussi et al., 1991; Kim-Ha et al., 1991; Ephrussi and Lehmann, 1992), are greatly reduced in vas mutant ovaries (Markussen et al., 1995; Rongo et al., 1995). Another pole plasm mRNA whose translation may be activated by VAS is nanos (nos), as nos RNA carrying an intact translational regulation element in its 3 UTR is completely inactive in embryos derived from vas mutant ovaries (Gavis et al., 1996; Dahanukar and Wharton, 1996). While the activities of pole plasm components such as VAS have been most thoroughly studied with respect to their function in pole cell formation and specification of the posterior soma, clearly some genes involved in pole plasm assembly also function in other stages of germline development. For instance, females homozygous for either of two strong nos alleles exhibit defects in germ cell proliferation (Lehmann and Nsslein-Volhard, 1991; Wang et al., 1994). Furthermore, pole cells lacking maternal nos function fail to complete migration and do not associate with the embryonic gonadal mesoderm (Kobayashi et al., 1996), indicating a role for nos in the transition from pole cell to functional germ cell. Similarly, various vas alleles have defects in oogenesis and lay few or no eggs (Lasko and Ashburner, 1988, 1990; Lehmann and Nsslein-Volhard, 1991; Schpbach and Wieschaus, 1991). Females trans-heterozygous for Df(2L)A267 and Df(2L)TE116-GW18, two large deletion mutations which both include vas, were reported to be blocked in early vitellogenic stages of oogenesis (Lasko and Ashburner, 1988). Analysis of whether this phenotype was caused solely by loss of vas function has been confounded by the fact that these transheterozygous deficiency lines are haploid for a large number of genes, but that, aside from large deficiencies, a clearly null allele of vas did not exist. Four EMS-induced alleles of vas, vasD1, vasQ6, vasQ7 and vasD5, also lead to greatly reduced fertility, with many egg chambers blocked as for the transheterozygous deficiency females (Lehmann and NssleinVolhard, 1991). The few eggs produced by females homozygous for these alleles often lack dorsal appendages and have the micropyle, a specialized vitelline membrane structure normally found only at the anterior of the egg, duplicated at the posterior (Lehmann and Nsslein-Volhard, 1991). Again, whether these phenotypes represent the results of a complete loss of vas function is unknown. vasQ6 and vasD5 are missense mutations which alter single amino acids of VAS and both alleles produce substantial amounts of mutant protein (Liang et al., 1994), so neither of these mutations is likely to be null. For vasD1 and vasQ7 the molecular nature of the mutation is unknown, but the vas coding region is unaffected in these mutant alleles. In this paper, we have used a new vas null allele, vasPH165, a small deletion which we generated by imprecise P-element excision, to investigate in detail the role of vas in events of oogenesis prior to pole plasm assembly. We found that abrogation of vas function results in defects in many aspects of oogenesis including control of cystocyte divisions, oocyte differentiation, and specification of posterior and dorsal follicle cell-derived structures. Furthermore, vasPH165 oocytes only weakly concentrate many oocyte-localized RNAs, although some oocyte-specific molecules, including gurken (grk; Schpbach, 1987; Neuman-Silberberg and Schpbach, 1993) RNA, remain concentrated in the oocyte in vas mutant ovaries. However, in the case of grk, translation is severely reduced in the absence of vas function. This provides evidence that VAS is (...truncated)