Maternal Nanos inhibits Importin-α2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. Here, we show that Nos represses somatic gene expression by inhibiting translation of maternal importin-α2 (impα2) mRNA. Mis-expression of Impα2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, we propose that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a ‘double-lock’ mechanism to inhibit somatic gene expression in germline progenitors.


Introduction
How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification [1]. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development [2][3][4][5][6][7]. In other species, germline progenitors are specified by inductive signals from surrounding tissues [8,9]. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors [10][11][12][13][14][15][16], implying that this phenomenon is critical for separation of the germline from the soma.
In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage [3][4]. In pole cells of blastoderm embryos (stage [4][5], the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos) [10,15,17]. Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting positive transcriptional elongation factor b (P-TEFb) function [17]. By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals [18]. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate [19]. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells [15]. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown.
Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3´UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide [20][21][22]. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3´UTR and recruiting Nos to the RNA/protein complex [23,24]. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation [25,26]. Furthermore, deletion of NREs causes hb translation in pole cells [25,26], suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-likesequence-dependent manner [27]. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) [26]. These findings led us to speculate that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3´UTRs.
Here, we report that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3Ú TR [28]. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins [28][29][30][31]. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on our observations, we propose that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells.

Nos, along with Pum, represses production of Impα2 in pole cells
Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells [28] (Fig 1A). However, we found that expression of Impα2 protein was at background levels in pole cells [28] (Fig 1D  and 1G). Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRElike sequence) in its 3´UTR [25,28] (Fig 2A), we assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, we first monitored the expression of the Impα2 protein in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos (Fig 1D-1F and 1I and S1 Fig). Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells (Fig 1B  and 1C), these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells.
We next investigated whether this repression is mediated by the NRE-like sequence in the impα2 3´UTR. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively) (Fig 2A), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells (Fig 2B, 2F and 2G). By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells (Fig 2C, 2F and 2G). Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied (Fig 2E, 2F and 2G), as well as when impα2 ΔNRE mRNA was supplied (Fig 2D, 2F and 2G). Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos (Fig 2F and 2G), these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells.
The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides (Fig 2A). The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5´-HWWD UGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex (Fig 7 in the article [22]). Hence, we asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, we performed electrophoretic mobility shift assay (EMSA) using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro [22]. We found that Nos and Pum  F'), and y w females with (impα2-nos3´UTR) (H and H') or without two copies of impα2-nos3´UTR (y w) (G and G'). Stage-5 embryos were stained with anti-Impα2 23aa antibody (green, D-H), which recognizes only Impα2 protein among the Importin-α family of proteins [48]. DIC images (D'-H') are also shown. Arrows and arrowheads point to pole cells with and without Impα2 expression, respectively. Scale bars, 20 μm (C) and 10 μm (H'). (I) Fluorescence intensities of Impα2 protein signals in pole cells of embryos derived from nos/+, nos, pum, y w, and impα2-nos3´UTR females. Embryos together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT) (Fig 2H and S2 Fig), whereas alteration of the NRE-like sequence (mut) (S2 Fig) abolished this interaction (Fig 2H). These results demonstrate that Nos and Pum are able to interact with the impα2 3´UTR in an NRE-like sequence-dependent manner. The observations described above led us to conclude that Nos, along with Pum, directly represses impα2 translation in pole cells.

Nos suppresses nuclear import of a transcription factor, Ftz-F1, by repressing Impα2
Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS) [28][29][30][31]. We predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, we focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells [32][33][34]. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells (Fig 3A, 3B, 3E, 3F, 3J and 3K). In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos (Fig 3C, 3G, 3H and 3J). Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells (Fig 3G, 3H and 3K). To determine whether this aberrant concentration of Ftz-F1 was caused by misexpression of Impα2, we expressed Impα2 ectopically in pole cells of normal embryos (Fig 1H  and 1I). To this end, impα2 mRNA in which the 3´UTR was replaced with the nos 3´UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3´UTR [35,36]. The percentage of these embryos (impα2-nos3´UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/ cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells (Fig 3D, 3I, 3J and 3K). These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1.

Mis-expression of Impα2 in the absence of Pgc function results in ectopic expression of somatic genes in pole cells
Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells [15]. Because Ftz-F1 is required for proper expression of ftz in the soma [37][38][39][40][41], we asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells [42], but never expressed in pole cells [percentage of embryos expressing ftz in pole cells (pe) = 0%; number of embryos examined (n) = 283] (Fig 4A and 4G). By contrast, in impα2-nos3´UTR embryos, ftz mRNA was rarely detectable in pole cells (pe = 8.9%, n = 45) (Fig 4B, 4C and 4G). We assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, we expressed Impα2 in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3´UTR embryos), and found that the frequency of ftz expression was drastically increased (pe = 51.4%, n = 74) (Fig 4F and 4G), compared to those of impα2-nos3´UTR embryos (P < 0.01) and the embryos lacking Pgc (pgc embryos) (pe = 34.9%, n = 109, from late stage 4 to stage 6 were stained with anti-Impα2 23aa antibody, and fluorescence intensities of Impα2 signals were measured (see Materials and Methods). Mean values of fluorescence intensities (± SE) are shown. For each genotype, 7-17 embryos were examined. The numbers of pole cells measured are shown in parentheses. Significance was calculated using paired t-test ( � : P < 0.01, n.s.: P > 0. 05). https://doi.org/10.1371/journal.pgen.1008090.g001 Nos inhibits Importin-α2-dependent nuclear import  (Table 1). Therefore, we conclude that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc.
In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3 UTR embryos (S3 Fig). Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3´UTR pole cells than pgc or impα2-nos3´UTR pole cells (S3 Fig). We next examined expression of the sex-determination gene Sxl in early pole cells, because Sxl is also repressed by nos in both male and female pole cells [15]. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3´UTR, pgc, and pgc impα2-nos3´UTR embryos (P > 0.1, vs. y w) (S4 Fig). By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3´UTR embryos than in impα2-nos3´UTR, and pgc embryos (S4 Fig). These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/ or Sxl in pole cells.
During the course of the experiments described above, we happened to observe that impα2-nos3´UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis (Fig 7). Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, as reported previously [17], we examined the effect of impα2-nos3´UTR in pgc/+ background (Fig 7A). The percentage of dysgenic ovaries in pgc/+ impα2-nos3´UTR females derived from pgc/+ impα2-nos3´UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3´UTR females (Fig 7A). In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present (S5 Fig). Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3´UTR males derived from pgc impα2-nos3´UTR mothers mated with y w males were higher than those in pgc and impα2-nos3´UTR males (Fig 7B). In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic) (S5 Fig). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses (Fig 7), our data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, we cannot test whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed [19].

Mechanism of repression of somatic gene expression in pole cells by Nos and Pgc
Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes [30,[48][49][50][51][52][53][54]. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3 [28,49,55]. impα1/Kap-α1/ CG8548 mRNA is not detectable in pole cells during early embryogenesis [56,57], and its protein product is ubiquitously expressed at a very low level throughout embryogenesis [48]. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation [58,59], and production of Impα3 protein is upregulated during the blastoderm stage [55,58] (S6 Fig). Because Impα3 production was independent of maternal nos activity (S6 Fig), it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become Nos inhibits Importin-α2-dependent nuclear import transcriptionally active during gastrulation [60][61][62][63][64], when Impα2 is undetectable in these pole cells [28]. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation [10,17]. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood [28]. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes. We found that depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone (Figs 4G and 5H), probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, we propose that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation [17]; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators.
Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species [18]. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells [65]. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein [16]. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres [66]. Here, we demonstrate that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, we propose that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi [17,[67][68][69]. Therefore, we propose that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development. positive (bottom) for PH3 in stage-8 embryos. DIC images merged with PH3-and Vasa-signals are also shown. Right: percentages of embryos carrying 0 (white), 1 (gray), 2 (pale green), or �3 (green) pole cells with PH3 signal are shown. The numbers of embryos examined are shown in parentheses. Significance was calculated using Fisher's exact test ( � : P < 0.05, n.s.: P > 0.5). (B) Expression of an apoptotic marker, cleaved Caspase-3, was examined in pole cells of embryos derived from y w, impα2-nos3'UTR, pgc/pgc (pgc), and pgc impα2-nos3'UTR females mated with y w males. Stage-10-16 embryos were double-stained with anti-cleaved Caspase-3 (green) and anti-Vasa (magenta) antibodies. Left: representative images of pole cells negative (top) and positive (bottom) for cleaved Caspase-3 in stage-12 embryos. DIC images merged with cleaved Caspase-3-and Vasa-signals are also shown. Right: percentages of pole cells expressing cleaved Caspase-3 are shown. The numbers of pole cells examined are shown in parentheses. For each genotype, 10-26 embryos were examined. Significance was calculated using Fisher's exact test ( � : P < 0.01, n.s.: P > 0.1). (C) Stage-14-16 embryos derived from y w, impα2-nos3'UTR, pgc/Df (pgc), and pgc impα2-nos3'UTR females mated with y w males were stained with anti-Vasa antibody (magenta). Left: representative images of pole cells within (top) and outside of (bottom) gonads in stage-14 embryos. DIC images merged with Vasa signal are also shown. Dotted line shows contour of embryonic gonads. Right: percentages of pole cells incorporated in the embryonic gonads. Total numbers of pole cells examined are shown in parentheses. For each genotype, 10-20 embryos were examined. Significance was calculated using Fisher's exact test ( � : P < 0.01, n.s.: P > 0.1). Scale bars, 10 μm (A-C). https://doi.org/10.1371/journal.pgen.1008090.g006

UASp-impα2 WT and
The nucleotide sequences of the above constructs were confirmed by sequencing, and then the constructs were transformed into flies. To establish impα2-nos3´UTR flies, germline transformation was performed as described previously [75] using y w embryos as recipients. Two independent w + transformants for each transgene were mated with y w females to establish homozygous stocks. Data shown in figures were obtained from one of the two independent transformant lines, as we found no significant difference between the two lines. To establish UASp-impα2 WT and UASp-impα2 ΔNRE flies, germline transformation was performed using embryos derived from y vas-φ-zh2A w; ZH-attP-58A females [72], and a single transformant line was established for each transgene, as described previously [76].

Staging of embryos
Developmental stages of Drosophila embryos were determined according to Campos-Ortega and Hartenstein [77]. In this study, stage-4 embryos that had finished the 13th somatic nuclear division and retained round nuclei before cellularization were referred to as "late stage-4 embryos".

Immunostaining
Antibody staining of embryos was performed as described previously [43]. For anti-Impα2 staining, embryos were fixed in 2 ml of 1:1 mixture of heptane and fixative I [3.7% formalin in PBS (130 mM NaCl, 7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 )] for 10 min with vigorous shaking. Two different antibodies were used, anti-Impα2 23aa and anti-Impα2 2/3 (gifts from B. M. Mechler), which were raised against the 23-amino acid residues of the C-terminal region and two-thirds of Impα2 protein, respectively [28,48]. For the experiments shown in Fig 1,  We observed no significant difference in the results obtained using these two antibodies, except that anti-Impα2 23aa antibody often caused a non-specific signal on the embryo surface.
For anti-Eve staining, embryos were fixed in 2 ml of 1:1 mixture of heptane and fixative II for 5 min with vigorous shaking. Guinea pig anti-Eve antibody 634 [1:200 dilution, Asian Distribution Center for Segmentation Antibodies at National Institute of Genetics (NIG), Japan] [78] and Cy3-conjugated anti-guinea pig IgG antibody (1:500 dilution, Jackson ImmunoResearch) were used.
All embryos, ovaries, and testes stained with fluorochrome-conjugated secondaries were mounted in Vectashield (Vector Laboratories) or ProLong Diamond (Molecular Probes). Zstack confocal images were taken from each embryo using a Zeiss LSM 5 Pascal (Zeiss), Zeiss LSM 510 Meta (Zeiss), Leica TCS-NT (Leica), or Leica TCS-SP8 (Leica) confocal microscope. Optical slices were analyzed using Zeiss LSM 5 Image Browser (Zeiss), ImageJ, or Fiji software. In Figs 2F, 3J, 6A and S1, the numbers of signal-positive pole cells located from the top to median plane of embryos were counted in confocal serial images.

In situ hybridization
Digoxigenin (DIG)-labeled RNA probes were synthesized with SP6, T7, or T3 RNA polymerase in the presence of DIG-labeled uridine triphosphate (UTP) (Boehringer-Mannheim), using fulllength impα2 cDNA clone K9, full-length 1817-bp ftz cDNA (a gift from H. Ueda), a 985-bp eve cDNA fragment (ntd 231-1215 of GenBank accession no. BT029151), or an 848-bp Sxl cDNA fragment (ntd 572-1419 of GenBank accession no. NM167112) as the template. Whole-mount in situ hybridization of embryos was performed essentially according to the methods reported by Tautz and Pfeifle [80], with several modifications [81]. For staining with impα2 probe, fixed embryos were treated at 23˚C for 3 min with PBT (130 mM NaCl, 7 mM Na 2 HPO 4 , 3mM NaH 2 PO 4 , 0.1% Tween-20) containing 50 μg/ml Proteinase K, and the reaction was immediately stopped by treating twice for 30 sec each with PBT containing 2 mg/ml glycine. Hybridization was performed for 16 hr at 60˚C in hybridization solution (50% formamide, 5 x SSC, 0.1% Tween 20, 0.05 mg/ml heparin, 0.1 mg/ml yeast tRNA) containing 0.6 μg/ml impα2 RNA probe. Post-hybridization washing was performed six times (30 min each) at 60˚C in a solution containing 50% formamide, 5× SSC, and 0.1% Tween-20. Embryos were incubated for 30 min with Fab fragments of anti-DIG antibody conjugated with HRP (600 U/l, Boehringer-Mannheim), then the signal was enhanced using the TSA-Biotin System (PerkinElmer Life Sciences, Inc.) and Streptavidin-Cy3 conjugate (1:2000 dilution, Jackson ImmunoResearch). For staining with ftz, eve or Sxl probe, the fixed embryos were treated at room temperature for 15 min with PBT containing 7 μg/ml Proteinase K, and then the reaction was stopped as described above. Hybridization was performed for 16 hr at 56˚C in hybridization solution containing 0.5 μg/ml of ftz, eve or Sxl RNA probe. The embryos were washed five times (30 min each) at 56˚C in hybridization solution, and then rinsed in PBT containing 75%, 50%, and 25% hybridization solution for 5 min each, and in PBT five times for 5 min each. The embryos were incubated with HRP-conjugated anti-DIG antibody (300 U/l) for 16 hr at 4˚C, and the signal was enhanced using TSA-Plus Fluorescein System (PerkinElmer Life Sciences, Inc.). Embryos were mounted in Vectashield (Vector Laboratories) or ProLong Diamond (Molecular Probes). Z-stack confocal images were taken from each embryo using a Zeiss LSM 5 Pascal (Zeiss), Leica TCS-NT (Leica), or Leica TCS-SP8 (Leica) confocal microscope. Optical slices were analyzed using Zeiss LSM 5 Image Browser (Zeiss), or Fiji software. In Figs 4G, 5H, S3 and S4, the numbers of signal-positive pole cells located from the top to median plane of embryos were counted in confocal serial images.

Quantification of Impα2 and Impα3 signals and nuclear localization of Ftz-F1
Embryos from late stage 4 to stage 6 were stained with anti-Impα2 23aa antibody. Serial optical sections (1.3 μm thick, 4-5 sections per pole cell) were obtained using a confocal microscope (LSM 510 Meta, Zeiss). Embryos from late stage 4 to stage 5 were stained with anti-Impα3 antibody, and serial optical sections (1.0 μm thick, 6-8 sections per pole cell) were obtained using a TCS-SP8 confocal microscope (Leica). Fluorescence intensities from the area occupied by individual pole cells (judged by the outline of the cell in the DIC image) were determined in sections through the median plane of pole cells. Fluorescence intensities were measured in all pole cells located within 15 μm of the top section of confocal serial images. Average fluorescence intensities (intensity/pixel) were calculated.
Embryos from late stage 4 to stage 5 were double-stained with anti-Ftz-F1 antibody and propidium iodide or DAPI as described above. Under a confocal microscope (Zeiss LSM 510 Meta, Zeiss), serial optical sections (1.3 μm thick, 4-5 sections per pole cell) were obtained. We examined all pole cells located within 18.2 μm of the top section of confocal serial images. To quantify Ftz-F1 distribution in the nucleus of a single pole cell, fluorescence intensities from the area occupied by the nucleus were determined for each section using ImageJ, and then summed. The nuclear area was judged as the propidium iodide-or DAPI-positive area. To quantify Ftz-F1 distribution in the cytosol of pole cells, we measured fluorescence intensity from the whole area of a pole cell (judged by the outline of the cell in the DIC image), and then fluorescence intensity of the cytosolic area was calculated by subtracting the nuclear intensity from the whole-cell intensity. Average fluorescence intensities (intensity/pixel) were calculated for both nuclear and cytoplasmic areas, and the ratio of nuclear to cytoplasmic intensity was calculated.

Injection of double-stranded RNA (dsRNA) against impα2 mRNA
Template DNA was amplified from impa2 cDNA clone K9 by PCR using forward primer 5´-G CGCGAATTAACCCTCACTAAAGGGCTCCCGAACAGATCGTCG-3´(ntd 1483-1500 of GenBank accession no. BT003258) and reverse primer 5´-GCGCGAATTAACCCTCACTA AAGGGAATCATTCAAACAATTCATTATTTATTGACAACTTTG-3´(complementary to ntd 2447-2483 of GenBank accession no. BT003258), both of which contain the promoter sequences for T3 RNA polymerase (shown in bold) at their 5´-ends. dsRNA was transcribed in vitro from the amplified DNA with T3 RNA polymerase (MEGAscript T3 kit, Ambion). dsRNA (0.1 nl of a 1.7 μg/μl solution) was injected into the posterior pole of pgc nos embryos at early stage 2. Because knockout of maternal impα2 mRNA results in developmental arrest at early cleavage stage [82], we performed partial knockdown of impα2 mRNA by precisely regulating the injection volume using a thin glass needle (hole diameter = 3 μm). Injected embryos were fixed in a 1:1 mixture of heptane and fixative II for 20 min, and the vitelline membrane was removed in PBS using a tungsten needle. Fixed embryos were processed for in situ hybridization with an antisense ftz RNA probe, as described above. The pole cells located within 30 μm of median section of confocal serial images were counted.

Nos and Pum protein purification
Recombinant Nos and Pum proteins were expressed in KRX E. coli cells (Promega) as described previously [22] using the Nos expression plasmid pFN18K NosZC (aa 289-401) (a gift from A. C. Goldstrohm) and the Pum expression plasmid pFN18K Pum RNA-binding domain (aa 1091-1426) (a gift from A. C. Goldstrohm). For Nos expression, cells were cultured in 2×YT medium with 25 μg/ml kanamycin and 2 mM MgSO 4 at 37˚C to an OD 600 of 0.7-0.9, and then protein expression was induced with 0.1% (w/v) rhamnose for 3 hr. For Pum expression, cells were cultured at 37˚C in the same medium to an OD 600 of 0.6, and then at 16˚C to an OD 600 of 0.7-0.9. Protein expression was induced with 0.1% rhamnose for 14-16 hr at 16˚C. Nos and Pum proteins were purified essentially as described by Weidmann et al. [22], with the following modifications. Nos and Pum proteins with Halo tag were purified by incubating with Magne HaloTag beads (Promega) overnight at 4˚C. Beads were washed three times with Wash Buffer (50 mM Tris-HCl pH 8.0, 2 mM MgCl 2 , 1 M NaCl, 1 mM DTT, 0.5% [v/v] NP-40), and three times with Elution Buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM DTT, 20% [v/v] glycerol). Then, the beads were resuspended in Elution Buffer containing AcTEV protease (Invitrogen) and incubated for 24 hr at 4˚C to cleave Nos or Pum protein from the Magne HaloTag beads. The beads were then removed using a MagneSphere magnetic separation stand (Promega).

Electrophoretic mobility shift assay (EMSA)
Synthetic Cy5-labeled impα2 RNA fragment (IDT, Tokyo), shown in S2 Fig, were 2 μM), and Nos (1.2 μM) were incubated in RNA binding buffer for 3 hr at 4˚C. Native polyacrylamide TBE mini-PROTEAN gel (5%, Bio-Rad) was pre-run for 2.5 hr at 50 V, and then 10 μl of each sample was loaded and the gel was run at 50 V for 2 hr 10 min at 4˚C. A Typhoon FLA 7000 laser scanner (GE Healthcare) was used to image EMSA.
Supporting information S1 Fig. Nos and Pum repress mis-expression of Impα2 in pole cells. Expression of Impα2 was examined in pole cells of embryos derived from nos/+, nos/nos (nos), pum Msc /TM3 (pum Msc /+), pum FC8 /TM3 (pum FC8 /+), and pum Msc /pum FC8 (pum) females. Embryos from late stage 4 to stage 6 were stained with anti-Impα2 2/3 antibodies [28]. Percentages of embryos containing 0 (white), 1-3 (pale orange), 4-6 (orange), and �7 (red) pole cells with Impα2 signal are shown. The numbers of embryos examined are shown in parentheses. Significance was calculated using Fisher's exact test ( � : P < 0.01). (A) Expression of eve mRNA was examined in pole cells of embryos from late stage 4 to stage 5. Embryos were derived from y w females with (impα2-nos3'UTR) or without two copies of impα2-nos3'UTR (y w), and nos/nos (nos), pgc/Df (pgc), and pgc/pgc; impα2-nos3'UTR/ impα2-nos3'UTR (pgc impα2-nos3'UTR) females mated with y w males. Percentages of embryos carrying 0 (white), 1 (gray), 2-4 (pale green), or �5 (green) pole cells with eve mRNA signal are shown. The numbers of embryos examined are shown in parentheses. Significance was calculated using Fisher's exact test ( � : P < 0.05, n.s.: P > 0.5). (B) Expression of Eve protein was examined in pole cells of embryos from late stage 4 to stage 5. Embryos were derived from y w, nos, impα2-nos3'UTR, pgc and pgc impα2-nos3'UTR females mated with y w males, as described above. Percentages of embryos carrying 0 (white), 1-3 (pale orange), 4-6 (orange), or �7 (red) pole cells with Eve signal are shown. The numbers of embryos examined are shown in parentheses. Significance was calculated using Fisher's exact test ( � : P < 0.01, n.s.: Fluorescence intensities of Impα3 protein signals in pole cells of embryos derived from y w and nos females. Embryos from late stage 4 to stage 5 were stained with anti-Impα3 antibody, and fluorescence intensities of Impα3 signals were measured (see Materials and Methods). Mean values of fluorescence intensities (± SE) are shown. The numbers of pole cells measured are shown in parentheses. 12 and 10 embryos were examined for y w and nos, respectively. Significance was calculated using paired t-test (n.s.: P > 0.1). (B, C) Stage-5 embryos derived from y w (A) and nos (B) females were stained for Impα3 protein. In pole cells, as well as in somatic cells, Impα3 was mainly detected on the nuclear envelope and in the nuclei [58]. Arrows and arrowheads point to pole cells expressing Impα3 on the nuclear envelope, with or without signal in their nuclei, respectively. Scale bar, 10 μm. (TIF) with injections; Drs. R. Lehmann, B. M. Mechler, A. C. Goldstrohm, H. Ueda, E. Gavis, and C. S. Parker for materials; Drs. R. Niwayama and A. Kimura for their technical assistance in quantification of nuclear localization of Ftz-F1; Drs. A. C. Goldstrohm and F. Suto for technical suggestions for protein purification; and Dr. Y. Hiromi for helpful discussion. We also thank the Bloomington Drosophila Stock Center for providing us with fly strains, and the Developmental Studies Hybridoma Bank and Asian Distribution Center for Segmentation Antibodies for providing antibodies.