Akira Nakamura ¹, Reiko Amikura ², Masanori Mukai ², Satoru Kobayashi ², and Paul F. Lasko ^1, *

¹ Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.
² 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@maclan.mcgill.ca

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. 

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.

Fig. 1. Distribution of Pgc RNA during oogenesis and embryogenesis. (A) Germarium through stage 6; Pgc RNA is expressed from germarium region 2B and localized in the posterior region of the oocyte. (B) Stage 8 and (C) stage 9 egg chambers showing Pgc RNA localization to the anterior, close to the oocyte-nurse cell border. (D) Stage 11 egg chamber with Pgc RNA enriched at the posterior pole plasm of the oocyte. No detectable signal in somatic follicle cells was observed at any stage of oogenesis. (E) Cleavage stage embryo in which the Pgc RNA is highly concentrated in pole plasm. (F) Cellular blastoderm embryo and (G) stage 10 embryo with Pgc RNA incorporated into pole cells. (H) Cleavage embryo hybridized with sense Pgc probe as a control. (I and J) In situ hybridization examined at the electron microscopic level reveals that Pgc RNA is localized in polar granules in (I) the pole plasm of cleavage embryos and (J) the pole cells at the syncytial blastoderm stage. The embryo in (I) was embedded, thin-sectioned, and hybridized with a double-stranded DIG-labeled Pgc DNA probe after sectioning (23); the embryo in (J) was hybridized before embedding. In both cases the Pgc probe hybridized over the entire polar granule. Bar, 200 nm; M, mitochondrion; pg, polar granule. 

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 osk ³⁰¹/osk ³⁰¹ 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), < 10 ^-4] 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. 

Fig. 2. (A) Genomic organization around Pgc. A total of 4.8 kb of genomic sequence containing Pgc has been deposited in GenBank under accession number U66411. Sequence specific to the minor 1.3-kb transcript region is delineated by a striped box. The gp150 gene (16) ends about 800 base pairs (bp) upstream from the Pgc transcription initiation site. A putative type III alcohol dehydrogenase gene [T3dh; BLAST scores 4.6 × 1013 with Bacillus methanolicus C1 methanol dehydrogenase (24); 6.9 × 10 ^-43 with a partial human cDNA clone (GenBank accession number H78978)] is transcribed from the opposite strand of sequences overlapping the Pgc intron and a portion of the exon specific to the 1.3-kb Pgc transcript (striped box). T3dh is transcribed in 12- to 24-hour embryos and larvae. (B) Nucleotide sequence of the 0.7-kb cDNA of Pgc (sequences corresponding to both the smaller and larger transcripts have been deposited in GenBank under accession numbers U66409 and U66410, respectively). A putative polyadenylation signal [AATATA, frequently used in Drosophila genes that are expressed in ovaries (25)] is indicated by double underlining. (C) Alignment of the potential translational start site for the longest ORF in the 0.7-kb Pgc transcript with a consensus sequence derived from actual translational start sites (17). Frequency refers to the percentage of actual start sites, as given in (17), that have the same nucleotide as does the Pgc sequence in the listed position. Rank refers to how frequent a particular nucleotide found in Pgc is in actual start sites; a value of 1 means the most common, a value of 4 means the least common. (D) Codon usage table for a 46-amino acid (AA) ORF (nucleotides 117 to 254) whose AUG codon is in a favorable context for translation. For all amino acids encoded by more than one codon and present in the ORF, the expected percentage (%) in Drosophila ORFs, as computed from published tables (26), is compared with the actual distribution of codons (#) in the Pgc ORF. Although some amino acids (notably Ser, Asp, Glu, and Cys) are encoded favorably, many others (such as Arg, Phe, Ala, Pro, Thr, and Gly) diverge substantially from Drosophila codon usage. The longest ORF in the minor 1.3-kb Pgc transcript extends for 92 codons; this ORF largely overlaps the T3dh coding sequence on the opposite strand and also has poor Drosophila codon usage.

Fig. 3. Distribution of Pgc RNA in embryos produced by maternal patterning mutants. Pgc RNA is not posteriorly localized in embryos from (A) osk ⁵⁴/osk ⁵⁴, (B) vas ^PD/vas ^PD, or (C) tud ^WC/tud ^WC mothers but is normally localized in embryos from (D) nos ^L7/nos ^L7 mothers. (E) Pgc RNA is mislocalized at the anterior in embryos from females carrying the P[ry ⁺, osk-bcd3'UTR] transgene (5). The maternal-effect Bic-C and Bic-D mutations induce a mirror-image duplication of the abdomen (but not pole cells) as a result of ectopic osk and nos localization to the anterior pole of embryos(4, 27). In embryos from (F) Bic-C ^AA4/CyO and (G)
Bic-D ^{71. 34}/Bic-D ^IIIE48 mothers, Pgc RNA is diffusely localized at the anterior. 

Fig. 4. Antisense Pgc expression affects germ cell migration and maintenance of pole plasm components. (A) A wild-type (w ^-) embryo and (B) an embryo from a female carrying two copies of the hsp70-AS-Pgc transgene (2×AS-Pgc embryo) were hybridized with a Pgc probe. The Pgc signal was undetectable in 2×AS-Pgcembryos. (C) w ^- and (D) 2×AS-Pgcembryos at the cleavage stage hybridized with a gcl probe. Initial localization of gcl RNA to the pole plasm is normal in 2×AS-Pgcembryos. (E) w ^- and (F) 2×AS-Pgcembryos at the cellular blastoderm stage hybridized with a gcl probe. In 2×AS-Pgcembryos, signals for gcl in the pole
cells were significantly reduced. Essentially identical results were obtained with a probe for nos (28). In (G to N), w ^- and 2×AS-Pgc embryos are stained with affinity-purified antibody to VAS (anti-VAS). At the cellular blastoderm stage, pole cells of (G) w ^- embryos and (H) 2×AS-Pgc embryos stain with equal intensity. At stage 10, VAS staining is noticeably weaker in (J) 2×AS-Pgcembryos than in (I) w ^- embryos. This difference is much more obvious at stage 12 [w ^- (K) and 2×AS-Pgc (L)]. At stage 14, pole cells are incorporated into embryonic gonads (M and N). In 2×AS-Pgc embryos few or no anti-VAS-stained pole cells were incorporated into the embryonic gonads (arrow points to gonads lacking pole cells). We frequently found clusters of pole cells outside of embryonic gonads in 2×AS-Pgc embryos(29). All 2×AS-Pgc embryos shown were embryos from females homozygous for the AS55 AS-Pgc insertion mated with w ^- males. The AS26 and AS58 AS-Pgc
 lines gave similar results, but the AS19 AS-Pgc line showed no significant effects on germ cell migration or maintenance of pole plasm components. This transgene induced only a slight decrease in ovarian Pgc RNA concentrations and had essentially no effect on subsequent fertility (Table 1). 

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-Pgcembryos, 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-Pgcembryos. 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.
^* 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).

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 Xenopusgerm 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-bcd3'UTR 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. 

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