Akira Nakamura, Reiko Amikura, Masanori Mukai, Satoru Kobayashi, Paul F. Lasko ^*

In Drosophila embryos, germ cell formation is induced by specialized cytoplasm at the posterior of the egg, the pole plasm. Pole plasm contains polar granules, organelles in which maternally produced molecules required for germ cell formation are assembled. An untranslatable RNA, called Polar granule component (Pgc), was identified and found to be localized in polar granules. Most pole cells in embryos produced by transgenic females expressing antisense Pgc RNA failed to complete migration and to populate the embryonic gonads, and females that developed from these embryos often had agametic ovaries. These results support an essential role for Pgc RNA in germline development.

A. Nakamura and P. F. Lasko, Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.
R. Amikura, M. Mukai, S. Kobayashi, Institute of Biological Sciences, Gene Experiment Center, and Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
^*   To whom correspondence should be addressed. E-mail: paul_lasko{at}maclan.mcgill.ca

Genetic screens have identified several maternally acting Drosophila genes with functions that are required for the formation of both abdomen and pole cells (4). Three of these genes, oskar (osk), vasa (vas), and tudor (tud), are central to pole plasm assembly. Mislocalization of high concentrations of osk RNA to the anterior pole induces functional pole plasm at the anterior (5). The activities of vas and tud are both required downstream of osk for ectopic pole cell formation, and OSK, VAS, and TUD proteins are all components of polar granules (6, 7). Polar granule assembly is completed later with the localization of numerous other RNAs and proteins to the posterior cytoplasm. In contrast to osk, vas, and tud, which are essential both for abdomen formation and for pole cell formation, the RNAs localized later are only required for some aspects of pole plasm function. For example, nanos (nos) RNA localized in pole plasm is required for abdomen formation and for correct pole cell migration into the embryonic gonads, but not for pole cell formation per se (8). Two other late-localizing RNAs, mitochondrial large ribosomal RNA (mtlrRNA) and germ cell-less (gcl), are involved specifically in pole cell formation (9, 10). However, because neither gcl nor mtlrRNA alone can induce pole cells at ectopic sites (10, 11), it is likely that unidentified additional pole plasm components operate cooperatively with gcl and mtlrRNA in pole cell formation.

To identify such molecules, we used mRNA differential display to screen for RNA species that are present in wild-type embryos but absent or rare in mutant embryos that fail to form pole cells (12). From this screening process, we isolated a cDNA whose transcript is localized in polar granules and we named the gene Polar granule component (Pgc). Pgc RNA is first detectable in germarium region 2B of ovaries when it is localized in the oocyte, and it continues to be concentrated at the posterior of the oocyte until stage 7 (Fig. 1A). In stage 8, the RNA no longer accumulates in the posterior of the oocyte but instead accumulates at the anterior of the oocyte close to the oocyte-nurse cell border (Fig. 1B). Through stages 9 and 10 the RNA spreads posteriorly along the oocyte cortex (Fig. 1C), and a posterior concentration becomes detectable at stage 11 (Fig. 1D). In cleavage embryos, Pgc RNA is highly concentrated in pole plasm (Fig. 1E). Later, Pgc RNA is incorporated into pole cells, and the small amount of unlocalized Pgc RNA is rapidly degraded from the somatic region of the embryo (Fig. 1F). Pgc RNA remains detectable in pole cells until stage 10 of embryogenesis, when they pass through the posterior midgut primordium (Fig. 1G). Ultrastructural analysis revealed that Pgc RNA is localized in polar granules, both in the pole plasm and in the pole cells of syncytial blastoderm embryos (Fig. 1, I and J). Within the polar granules the distribution of Pgc RNA differs from that of mtlrRNA. mtlrRNA is concentrated on the surface of polar granules, frequently at the boundaries between polar granules and mitochondria of early-cleavage embryos; after pole cell formation, mtlrRNA signal is undetectable on polar granules (13). In contrast, a Pgc probe hybridized throughout the entire polar granule, and signals were detected even on polar granules in pole cells.

We cloned more than 30 Pgc cDNAs (14) that hybridize to a major transcript of 0.7 kb and a minor transcript of 1.3 kb; the expression level of the larger transcript was less than 1% of that of the smaller. Pgc is expressed only in female germ cells. Both transcripts were detected in RNA prepared from fertile adult females, ovaries, and early-stage embryos; however, the transcripts were undetectable in RNA prepared from late-stage embryos, larvae, and pupae and from adult males and sterile females from osk³⁰¹/osk³⁰¹ mothers, which produce embryos that fail to form pole cells at 25°C (15). Sequence analysis of the cDNAs and corresponding genomic DNA indicates that both transcripts are derived from the same gene (Fig. 2A). Pgc maps to a gene-rich area of chromosome region 58D, with the 3 end of the gp150 gene (16) less than 1 kb proximal to the 5 end of Pgc. A putative type III alcohol dehydrogenase gene (T3dh), transcribed from the opposite strand, is nested in the Pgc intron and overlaps a portion of the exon specific to the minor 1.3-kb Pgc transcript (Fig. 2A). For the following reasons we conclude that Pgc encodes an untranslatable RNA. In the major 0.7-kb transcript, the longest open reading frame (ORF) (nucleotides 480 to 692; Fig. 2B) would encode a polypeptide of 71 amino acids, but its AUG codon is in an extremely poor context for translation initiation (17) (Fig. 2C). A shorter 46-codon ORF (nucleotides 117 to 254) begins with an AUG in a good translation initiation context, but it has poor Drosophila codon usage (Fig. 2D). No highly homologous [probability of a chance match, P(N), < 10⁴] sequences were obtained in BLAST searches of the nonredundant nucleic acid sequence database when any ORFs or the nucleotide sequences of either Pgc transcript were analyzed.

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We examined embryos produced by mothers homozygous for various posterior-group mutations to determine the effects of such mutations on Pgc RNA localization. Embryos from osk, vas, and tud homozygous females failed to localize Pgc RNA in pole plasm (Fig. 3, A to C), and Pgc RNA is undetectable at the cellular blastoderm stage in these embryos. In contrast, nos embryos localized Pgc RNA normally and incorporated it into pole cells (Fig. 3D). Ectopic Pgc RNA localization to the anterior was observed (Fig. 3E) in embryos from females carrying the osk-bcd3UTR transgene (5). In embryos from either Bicaudal-C or Bicaudal-D females, Pgc RNA was mislocalized to the anterior in a diffuse manner (Fig. 3, F and G), as has been reported for other pole plasm RNAs (4).

osk-bcd3UTR

To produce flies with reduced Pgc function, we made transgenic lines carrying a hybrid gene in which antisense Pgc is expressed under the control of the hsp70 promoter (18). To eliminate nonspecific deleterious effects on subsequent embryonic development, which we observed when even wild-type flies were heat shocked during mid-to-late oogenesis, in subsequent experiments we analyzed the effect of antisense Pgc expression on pole cell development by comparing embryos from females carrying two copies of the hsp70-AS-Pgc transgene (2×AS-Pgc embryos) cultured at constant temperature (25°C) without heat shocking. As judged by in situ hybridization with a strand-specific Pgc probe, the amount of localized Pgc RNA was greatly reduced in 2×AS-Pgc embryos (Fig. 4, A and B). Although Pgc is expressed in female germ cells throughout oogenesis, we did not observe any defect in oogenesis in females expressing antisense Pgc.

We analyzed the spatial distributions of several RNAs and proteins that are localized in pole plasm in 2×AS-Pgc embryos. The posterior concentration of all pole plasm components analyzed appeared to be essentially normal in these embryos at the cleavage stage (Fig. 4, C and D); however, in postblastodermal development, localized nos, gcl, and VAS signals were reduced in intensity (Fig. 4, E to K). Furthermore, we observed defects in pole cell migration in the 2×AS-Pgc embryos. In wild-type embryos, an average of 28 pole cells complete migration and associate with mesodermal tissue during stage 14 to form the two embryonic gonads (6) (Fig. 4, I, K, and M). In 2×AS-Pgc embryos, the ability of pole cells to complete migration and colonize the gonad is dramatically impaired (Fig. 4, J, L, and N). Three of four 2×AS-Pgc lines, with substantially reduced Pgc RNA concentrations, showed a slight reduction from 34 to between 25 and 27 in the number of VAS-positive migrating pole cells at stage 12 (Table 1). In subsequent development, many pole cells died or failed to migrate into the embryonic gonads; at stage 14 the median pole cell number was four to five per gonad in the three 2×AS-Pgc lines (Table 1). To confirm these effects on adult fertility, we examined the gonads of adult females that developed from 2×AS-Pgc embryos. Most embryos from these lines hatched and completed development, but, consistent with the failure of pole cells to colonize the embryonic gonads, up to 53% of adult ovaries were agametic (Table 1). These defects in germ cell proliferation correlate with a specific decrease in the amount of Pgc RNA (Table 1).

Table 1. Correlation between Pgc RNA amount and numbers of functional pole cells in progeny from females carrying two copies of the hsp70-AS-Pgc transgene. Relative Pgc RNA amount was determined by densitometric quantitation of Northern (RNA) hybridizations of a strand-specific probe to polyadenylated RNA from ovaries of the indicated lines. The filter was rehybridized with a probe for the ribosomal protein gene RpS15a (30) for loading control. Hatch rate, pole cell numbers, and ovary phenotype were scored for progeny from females of the indicated lines mated with w males. Agametic ovaries were frequently observed in w progeny from females carrying one copy of hsp70-AS-Pgc mated with w males, indicating that the agametic ovary phenotype was caused by maternally supplied antisense Pgc RNA. Line PgcRNA amount (%)* Hatching rate percent (n) Number of pole cells/stage 12 embryo (n) Distribution of pole cell number in gonads of stage 14 embryos Adult ovaries  6 5 4 3 2 1 0 With eggs Agametic w 100 95.8 (409) 34.2  ±  5.2  (44) 231 1 0 0 0 0 0 310 0 AS19 63 76.5 (562) 32.8  ±  5.5  (36) 175 3 2 1 1 0 0 586 12 AS26 2 94.9 (196) 27.4  ±  6.2  (55) 94 39 35 31 26 39 30 266 296 AS55 13 93.4 (455) 25.7  ±  6.5  (45) 103 35 27 30 24 18 19 275 133 AS58 40 82.9 (316) 25.0  ±  6.3  (25) 99 20 18 23 20 32 30 410 112 * Pgc RNA amounts normalized to RpS15a RNA amounts and presented relative to the w control. Numbers of cells that stained with affinity-purified anti-VAS. Wild-type stage 14 gonads have an average of 14 pole cells (6).

Our results suggest that the untranslatable Pgc RNA has an essential role in the differentiation of pole cells into functional, proliferative germ cells. In contrast to gcl, which is thought to be primarily required for pole cell formation (9, 11), reduction of the Pgc RNA concentration has only a modest effect on initial pole cell formation. However, between stages 12 and 14, pole cells in 2×AS-Pgc embryos are severely compromised in their ability to migrate into the gonads and develop into functional germline stem cells. We believe that the effects we observed of antisense Pgc expression on germ cell establishment result from a specific interference with endogenous Pgc function for the following reasons: bicoid and osk RNAs were normally localized in cleavage embryos from all of the hsp70-AS-Pgc lines (19), and 2×AS-Pgc eggs hatched at high efficiency and developed into viable morphologically normal adults (Table 1). We hypothesize that reduction of the Pgc RNA concentration in the antisense lines leads to reduced stability of polar granules after their initial formation because Pgc RNA is an integral component of polar granules and the concentrations of various pole plasm components are reduced in postblastodermal pole cells of 2×AS-Pgc embryos. No abdominal defects were observed in 2×AS-Pgc embryos; however, because our results are based on a reduction of localized Pgc RNA concentrations, we cannot exclude a role for Pgc in abdominal specification. Null mutations may reveal additional functions for Pgc.

In both Drosophila and Xenopus, germ plasm can induce germ cell fate (1, 20). In addition, specific components of germ plasm appear to be conserved between these two evolutionary diverse animals (8, 21). A group of untranslatable RNAs, called Xlsirts, are localized in Xenopus germ plasm and are required for anchoring of Vg1 RNA to the vegetal cortex of the oocyte (22). Although the exact role, if any, of Xlsirts in germ cell establishment is unclear, our results suggest that, like the Xlsirts, Pgc RNA functions in the maintenance of germ plasm integrity. Further analysis of the composition and role of Drosophila polar granules will be of relevance to understanding the molecular basis of germ cell determination in both invertebrates and vertebrates.

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31. We thank A. Ephrussi for providing us the osk cDNA clone and for the osk-bcd3UTR lines, H. Foley for secretarial assistance, and C. Lévesque for fly food preparation. Supported by research grants from Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada (NCIC), with funds from the Canadian Cancer Society. A.N. is a Japan Society for the Promotion of Science postdoctoral fellow. P.L. is a Research Scientist of the NCIC.

21 August 1996; accepted 23 October 1996