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. 2026 Feb 9;27(5):1146–1160. doi: 10.1038/s44319-026-00710-x

Somatic gene repression ensures physical segregation of germline and soma in Drosophila embryos

Miho Asaoka 1,✉,#, Mizuki Kayama 2,#, Tomoki Kawagoe 3,#, Makoto Hayashi 1,4, Shumpei Morita 1,2,5, Satoru Kobayashi 1,2,3,✉
PMCID: PMC12979565  PMID: 41663761

Abstract

In many animals, primordial germ cells are transiently segregated outside the somatic-cell cluster that forms the embryo’s body during early embryogenesis. This physical segregation of the germline from the soma has long been believed to be crucial for germline development, but the mechanisms controlling this segregation and its developmental significance remain unclear. Here, in Drosophila, we show that somatic gene silencing in the germline is essential for maintaining this segregation. Primordial germ cells (pole cells) lacking the Nanos- and Polar granule component (Pgc)-dependent dual repression mechanism misexpress widespread somatic genes. They form abnormal cellular protrusions, invade adjacent somatic epithelium, and intermingle with somatic cells. These mislocalized pole cells ultimately undergo cell death, whereas properly segregated cells survive. Notably, knockdown of miranda (mira), one of the somatic genes ectopically expressed, rescues these phenotypes. Our findings uncover a previously unrecognized mechanism whereby somatic gene silencing safeguards the physical boundary between the germline and the somatic cells forming the embryo’s body, highlighting its potential role in ensuring germline viability during early development.

Keywords: Germ Cell, Germ-soma Segregation, Apoptosis, Nanos, Drosophila

Subject terms: Chromatin, Transcription & Genomics; Development

Synopsis

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Drosophila primordial germ cells are spatially segregated from the soma forming the embryo body during early embryogenesis. Nanos/Pgc-dependent repression suppresses somatic genes in the germline, thereby maintaining this spatial segregation and preventing germline apoptosis.

  • In pgc- impα2OE embryos, primordial germ cells (pole cells) fail to repress expression of numerous somatic genes.

  • pgc- impα2OE pole cells form abnormal protrusions, invade underneath somatic epithelia, and eventually undergo apoptosis.

  • These phenotypes are rescued by knockdown of the somatic gene mira, ectopically expressed in pgc- impα2OE pole cells.

Drosophila primordial germ cells are spatially segregated from the soma forming the embryo body during early embryogenesis. Nanos/Pgc-dependent repression suppresses somatic genes in the germline, thereby maintaining this spatial segregation and preventing germline apoptosis.

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Introduction

The physical segregation of primordial germ cells from the soma is widely observed in many animal species (Dixon, 1994). For example, in some hymenopterans and higher dipterans, including Drosophila, primordial germ cells (pole cells) are formed at the blastoderm stage by inheriting a specific ooplasm called germ plasm, which contains maternal factors required for germline development. They are formed outside the somatic layer at this stage and remain outside the somatic cells until mid-embryogenesis. They reside inside the lumen of the developing midgut primordium, which is derived from the outside of the somatic layer at the blastoderm stage. Pole cells then pass through the midgut epithelium into the hemocoel and migrate into the embryonic gonads, where they differentiate into oocytes or sperms (Campos-Ortega and Hartenstein, 1985; Nakao et al, 2006; Nieuwkoop and Sutasurya, 1981; Tanaka, 1985). A similar spatial segregation is observed in some vertebrate species (Golkar-Narenji et al, 2023; Nieuwkoop and Sutasurya, 1979). In some reptiles and primates, primordial germ cells first appear in the extra-embryonic region, separated from the somatic-cell cluster forming an embryo (Bergmann et al, 2022; Golkar-Narenji et al, 2023; Sasaki et al, 2016). In chicks and mice, primordial germ cells are initially observed inside the somatic-cell cluster but then migrate into the extra-embryonic region and stay there until mid-embryogenesis (Matsui and Okamura, 2005; Swift, 1914; Tsunekawa et al, 2000). From these observations, the physical segregation of the germline from the soma has been recognized as a common feature across species and is thought to play an important role in germline development (Dixon, 1994). However, its underlying mechanisms and developmental significance remain poorly understood. This is due to the lack of an experimental model that disrupts this segregation.

Besides the physical segregation, somatic gene silencing in the germline has also been reported in many animal species. Expression of the genes required for somatic tissue development are specifically suppressed in primordial germ cells during early embryogenesis (Asaoka et al, 2019; Kojima et al, 2017; Lai et al, 2012; Leatherman et al, 2002; Lee et al, 2017; Martinho et al, 2004; Ohinata et al, 2005; Seydoux et al, 1996; Tomioka et al, 2002; Yamaji et al, 2008). Recently, in Drosophila, two maternal proteins, Nanos and Polar granule component (Pgc), have been identified as factors required to repress somatic gene expression in pole cells (Asaoka et al, 2019; Deshpande et al, 1999; Martinho et al, 2004). Nanos, a translational repressor, suppresses the translation of importin-α2/pendulin (imp-α2) mRNA, which encodes an adapter protein for nuclear import receptors. This results in the inhibition of Imp-α2-dependent nuclear import of transcriptional activators in pole cells (Asaoka et al, 2019). Pgc suppresses RNA polymerase II-dependent transcription by inhibiting the function of positive transcriptional elongation factor B (P-TEFb) (Hanyu-Nakamura et al, 2008). Nanos-dependent inhibition of the nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing are both required to suppress the expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and Sex-lethal (Sxl) in pole cells. Thus, Nanos- and Pgc-dependent repression acts as a “double-lock” mechanism to ensure tight inhibition of somatic gene expression in pole cells (Asaoka et al, 2019).

Here, we describe the critical role of Nanos/Pgc-dependent somatic gene repression in the spatial segregation of the germline from the soma. We also present data suggesting that the loss of this mechanism reduces the number of germline progenitors.

Results and discussion

Transcriptome of pole cells lacking the Nanos/Pgc-dependent double-lock mechanism

To comprehensively identify genes suppressed by the Nanos/Pgc-dependent ‘double-lock’ mechanism in pole cells, we first compared the transcriptomes of pole cells lacking the double-lock mechanism with those of normal pole cells. A previous study has shown that the double-lock mechanism is disrupted by overexpression of the Imp-α2 protein in pole cells mutant for maternal pgc (pgc− impα2OE pole cells) (Asaoka et al, 2019). Using fluorescence-activated cell sorting (FACS), we isolated pole cells from pgc− impα2OE and normal (w) embryos at stages 4–6 and processed these cells for RNA-sequencing (RNA-seq). We found that, compared to normal pole cells, 878 and 389 transcripts were upregulated and downregulated in pgc− impα2OE pole cells, respectively [False discovery rate (FDR) < 0.01] (Fig. 1A). ftz and Sxl, of which expression is reported to be suppressed by the double-lock mechanism in our previous paper (Asaoka et al, 2019), were observed in the “upregulated transcripts”. Gene ontology (GO) terms related to somatic development, such as “embryonic morphogenesis” and “central nervous system development”, were enriched in the upregulated transcripts (genes) (Fig. 1B; Dataset EV1). Furthermore, 389 transcripts were upregulated specifically in pgc− impα2OE pole cells, but not in pgc− or impα2OE pole cells (Fig. 1C; Dataset EV2), and the GO terms related to somatic development were also enriched in them (Fig. 1D; Dataset EV2). The above results indicate that widespread misexpression of “somatic genes” occurs in pgc− impα2OE pole cells.

Figure 1. Transcriptomic profile of pole cells lacking the “double-lock mechanism”.

Figure 1

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(A) Number of transcripts upregulated and downregulated in pgc− impα2OE pole cells compared with w pole cells (FDR < 0.01). (B) The top 15 significantly enriched GO terms for the 878 upregulated transcripts in (A), and all significantly enriched GO terms for the 389 downregulated transcripts (q < 0.01, Benjamini–Hochberg procedure). GO terms related to somatic tissue development are marked with red dots. (C) Comparison of upregulated transcripts (FDR < 0.01, versus w) between pgc−, impα2OE, and pgc− impα2OE pole cells. The number of transcripts in each category is shown. (D) All significantly enriched GO terms for the 389 transcripts specifically upregulated in pgc− impα2OE pole cells (q < 0.01, Benjamini–Hochberg procedure). GO terms related to somatic tissue development are marked with red dots. RNA sequencing was performed and analyzed using three biological replicates per genotype (n = 3). Exact FDR values for each transcript are provided in Datasets EV1–EV3. Source data are available online for this figure.

The transcripts downregulated in pgc− impα2OE pole cells were enriched for GO terms unrelated to germline development (Fig. 1B; Dataset EV3). Furthermore, we noticed that the expression of transcripts encoding germ-plasm components, such as oskar (osk), germ cell-less (gcl), nanos, P-element induced wimpy testis (piwi), Peroxiredoxin 2 (Prx2), Trapped in endoderm 1 (Tre1), and wunen-2 (wun2), was not downregulated in pgc− impα2OE pole cells (Appendix Table S1), suggesting that maternal transcripts encoding germ-plasm components were normally retained in pgc− impα2OE pole cells.

Morphological alterations of pgc−impα2OE pole cells

In normal embryos, pole cells are formed outside the somatic-cell layer at the blastoderm stage (stage 4), and they remain in the lumen of the posterior midgut primordium until the end of gastrulation (stage 9) (Fig. 2A). During these stages, the pole cells exhibit a spherical morphology (Campos-Ortega and Hartenstein, 1985) (Fig. 2B). In contrast, pgc− impα2OE pole cells formed cellular protrusions at the onset of gastrulation at stage 6. A single protrusion was formed per cell and penetrated between the epithelial cells (Figs. 2B and EV1A). The percentage of pgc− impα2OE pole cells forming protrusions was significantly higher than normal, impα2OE, or pgc− pole cells (Fig. 2C). At stages 7–9, pgc− impα2OE pole cells were located within the epithelial layer of the midgut primordium, whereas normal pole cells were in the lumen of the midgut primordium (Figs. 2A,D and EV1B). Previous studies have reported that in normal development, pole cells located in the lumen of the midgut primordium occasionally fall into the yolk region (hemocoel) during gastrulation (Turner and Mahowald, 1976). However, in pgc− impα2OE embryos, the frequency of these pole cells did not increase compared to that in normal embryos (Fig. EV1C). This suggests that pgc− impα2OE pole cells remain within the epithelial layer of the midgut primordium rather than entering the hemocoel. The percentage of pgc− impα2OE pole cells within the epithelial layer was significantly higher than that of normal, impα2OE, or pgc− pole cells (Fig. 2E). Our observations strongly suggest that pgc− impα2OE pole cells with protrusions invade and stay within the epithelial layer of the midgut rudiment, resulting in intermingling with somatic cells.

Figure 2. Pole cells lacking the double-lock mechanism form cellular protrusions and invade the somatic-cell layer.

Figure 2

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(A) Illustration of pole cell development in the Drosophila embryo. Somatic cells that give rise to the midgut (pink), somatic gonadal precursors (orange), yolk (brown). Anterior up, posterior down, ventral left, dorsal right. (B) Immunofluorescence images of posterior pole regions of normal (y w) and pgc− impα2OE embryos stained for the germline marker Vasa at stage 6. DIC images merged with the Vasa signal are shown. Arrows indicate cellular protrusions. White dotted lines outline the somatic-cell layer. Scale bar, 20 µm. (C) Beeswarm plots showing the percentage of pole cells with protrusions at stage 6 in y w, impα2OE, pgc− and pgc− impα2OE embryos. (D) Immunofluorescence images of the midgut primordium of y w and pgc− impα2OE embryos stained for Vasa at stage 7. DIC images merged with the Vasa signal are shown. White dotted lines outline the epithelium of the midgut primordium. L lumen of the midgut primordium, E epithelium of the midgut primordium. Scale bar, 20 µm. (E) Beeswarm plots showing the percentage of pole cells within the epithelium of the midgut primordium at stages 7–9 in y w, impα2OE, pgc− and pgc− impα2OE embryos. (F) Representative sequential time-lapse confocal images of the posterior pole region of pgc− impα2OE embryos with nanos-moesin-GFP transgene, which marks germ cell membranes (Sano et al, 2005) (n = 6 embryos, biological replicates). Images span from late-stage 6 to early stage 7. Thick and thin arrows indicate pole cells with and without protrusion at time 0’0”, respectively. Arrowhead indicates cellular protrusion. The upper left corner panel shows a magnified view of the boxed region at 0’00”; the lower right corner panel shows a brightened and magnified image of the boxed region at 2’40”. Note that the pole cell that has completed invasion (thick arrow) stays within the epithelium of the midgut primordium but shows reduced expression of nanos-moesin-GFP compared with pole cells outside (thin arrow). Scale bars, 20 µm. Data information: In (C, E), black dots represent individual embryos (biological replicates). Red lines represent the medians, and the boxes indicate the interquartile range (IQR; 25th–75th percentiles). All data points are shown; therefore, minima and maxima correspond to the lowest and highest dots, and whiskers are not displayed. “n” indicates the number of embryos examined per genotype. Statistical significance was assessed using Steel’s multiple comparison test versus pgc- impα2OE. *P < 0.01. Exact P values are provided in Source Data. Source data are available online for this figure.

Figure EV1. Phenotypes of pgc-impα2OE pole cells.

Figure EV1

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(A) Representative confocal images of pole cell protrusions (arrows) in pgc− impα2OE embryos double-stained with anti-Vasa antibody and DAPI at stage 6 (n = 14 embryos, biological replicates). A DIC image merged with the Vasa signal is shown on the left. The main body of the pole cell with the nucleus (arrowheads) remained on the outside, but its protrusion penetrated the somatic-cell layer (arrows). The pole cell protrusion (arrows) never reached beyond the somatic-cell layer into the yolk. (B) Representative confocal images of posterior-dorsal region of gastrulating pgc- impα2OE embryos (stage 6/7) triple-stained with anti-Vasa antibody, PY20 antibody, and DAPI (n = 22 embryos, biological replicates). A DIC image merged with Vasa is shown on the left. Arrows indicate pole cells within the epithelium of the midgut primordium. They were intermingled with the epithelial cells in the midgut primordium. In (A, B) Anterior up, posterior down, ventral left, dorsal right. Scale bars, 10 µm. (C) Beeswarm plots showing the percentage of pole cells within the hemocoel in y w and pgc− impα2OE embryos at stages 7–9. Black dots represent individual embryos (biological replicates). Red lines represent the medians, and the boxes indicate the interquartile range (IQR; 25th–75th percentiles). All data points are shown; therefore, minima and maxima correspond to the lowest and highest dots, and whiskers are not displayed. “n” indicates the number of embryos examined per genotype. Statistical significance was assessed using Mann–Whitney U test. *P < 0.01 (P = 0.00098).

This idea is supported by our live imaging. To visualize the cell membrane of pole cells in living embryos, we expressed the Moesin-GFP fusion protein only in pole cells (Sano et al, 2005). pgc− impα2OE pole cells forming protrusions at stage 6 invaded the epithelial layer of the midgut primordium during gastrulation, whereas pgc− impα2OE pole cells with spherical morphology did not invade, as far as we observed (we examined six embryos for 20 min) (Fig. 2F).

When pgc− impα2OE pole cells were transplanted and allowed to develop in wild-type hosts, the transplanted pole cells were able to invade the epithelium of the midgut primordium (Table EV1). These results clearly suggest that the Nanos/Pgc-dependent double-lock mechanism is required in the germline.

Developmental fate of pgc−impα2OE pole cells

In our live imaging, Moesin-GFP expression was maintained in spherical pole cells (thin arrows in Fig. 2F), whereas it gradually disappeared in pole cells invading the midgut primordium (thick arrows in Fig. 2F). Furthermore, in fixed samples, we often observed cell debris lacking DNA staining in the midgut primordium of pgc− impα2OE embryos. These cell debris were positive for the germline marker Vasa, indicating that they were derived from pole cells (Fig. 3A). These observations led us to hypothesize that pgc− impα2OE pole cells invading the midgut primordium undergo cell death. To test this, we examined the expression of a cell death marker, the cleaved form of the Drosophila effector caspase, Death caspase-1 (Dcp-1). In normal embryos, cleaved Dcp-1 was never detected in pole cells during gastrulation [the number of embryos examined (n) = 17]. In contrast, cleaved Dcp-1-positive pole cells were observed in 56.3% of pgc− impα2OE embryos (P vs. normal < 0.01, Fisher’s exact test, n = 16) (Fig. 3B). The percentage of pgc− impα2OE pole cells expressing cleaved Dcp-1 was significantly higher than normal pole cells (Fig. 3C). Thus, pgc− impα2OE pole cells undergo cell death during gastrulation (stages 7–9). Notably, pgc− pole cells show apoptotic phenotype only after stage 10 (Hanyu-Nakamura et al, 2019) and apoptosis is never detected in impα2OE pole cells (Asaoka et al, 2019), suggesting that the early-stage cell death of pgc− impα2OE pole cells is driven by mechanisms distinct from those underlying late-stage apoptosis in pgc− pole cells. The pgc− impα2OE pole cells with cleaved Dcp-1 signal were observed in the epithelial layer of the midgut primordium rather than in the lumen (Fig. 3D). The above observations strongly suggest that pgc− impα2OE pole cells invading the epithelial layer of the midgut primordium undergo cell death.

Figure 3. Pole cells lacking the double-lock mechanism express cleaved Dcp-1 in the midgut primordium epithelium.

Figure 3

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(A) Representative confocal images of Vasa-positive cell debris (arrows) in pgc− impα2OE embryos, visualized by triple-staining with anti-Vasa and PY20 (anti-phosphotyrosine) antibodies and DAPI (nuclear DNA) at stage 9 (n = 20 embryos, biological replicates). PY20 antibodies were used to outline all the cells (Pflanz et al, 2015). DIC image merged with the Vasa signal is shown on the left. Insets: Magnified images of the debris indicated by arrows. (B) Cleaved Dcp-1 expression in pole cells of y w and pgc− impα2OE embryos at stage 7. DIC images merged with cleaved Dcp-1 and Vasa are shown. Arrows indicate cleaved Dcp-1-positive pole cell. E epithelium of the midgut primordium, L lumen of the midgut primordium. Magnified views of the yellow square area are shown on the right. (A, B) Anterior up, posterior down, ventral left, dorsal right. White dotted lines outline the epithelium of the midgut primordium. Scale bars, 20 µm. (C, D) Beeswarm plots showing the percentage of pole cells expressing cleaved Dcp-1 in y w and pgc− impα2OE embryos at stages 7–9 (C), and in the lumen and in the epithelium of the midgut primordium in the pgc− impα2OE embryos (D). Black dots represent individual embryos (biological replicates). Red lines represent the medians, and the boxes indicate the IQR (25th–75th percentiles). All data points are shown; therefore, minima and maxima correspond to the lowest and highest dots, and whiskers are not displayed. “n” indicates the number of embryos examined per genotype or group. Statistical significance was assessed using Mann–Whitney U test in (C) and two-sided exact Wilcoxon signed-rank test in (D). *P < 0.01. Exact P values are provided in Source Data. Source data are available online for this figure.

Abnormalities in the morphology and developmental fate of pgc−impα2OE pole cells are rescued by suppressing the somatic gene miranda (mira)

Next, we searched for the gene(s) responsible for the phenotypes of pgc− impα2OE pole cells. We assumed that the misexpression of the somatic gene(s) was responsible for the phenotypes. Of the 878 transcripts upregulated in pgc− impα2OE pole cells, we first selected 190 transcripts with the GO term “Embryonic morphogenesis” (GO 0048598) (Fig. 1B; Dataset EV1). Among them, we selected 19 transcripts with GO terms related to neuroblasts [“Neuroblast differentiation” (GO 0014016) and “Central nervous system development” (GO 007417)] (Dataset EV1), because a similar cellular protrusion was reported in Drosophila quiescent neuroblasts (Tsuji et al, 2008). Among them, the expression of transcripts from achaete (ac), mira, scute (sc), and tailless (tll) was upregulated in pgc− impα2OE pole cells at a higher level [log2 fold change >2.0, average transcripts per million (TPM) > 40.0], and their expression levels were higher than those in impα2OE or pgc− pole cells (Dataset EV1). We finally focused on mira because the percentage of pgc− impα2OE pole cells expressing mira was similar to that of pole cells forming protrusions (Fig. 4A).

Figure 4. mira is one gene responsible for the phenotypes of pgc−impα2OE embryos.

Figure 4

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(A) Beeswarm plots showing the percentage of pole cells expressing ac, mira, sc, or tll mRNA in y w and pgc− impα2OE embryos at stage 6. (B) Confocal images showing mira mRNA expression in y w and pgc− impα2OE embryos triple-stained for mira mRNA, Vasa protein, and nuclear DNA (DAPI) at stage 6. Merged DIC/Vasa/DAPI images and the mira mRNA channel alone are shown. White lines outline pole cells. Scale bar, 5 µm. (C) Beeswarm plots showing the percentage of pole cells expressing mira mRNA in spherical pole cells and protrusion-forming pole cells of pgc− impα2OE embryos at stage 6. (D–I) Phenotypes of protrusion formation, invasion, and cleaved Dcp-1 expression in pole cells of pgc− impα2OE embryos with (pgc− impα2OE matGal4 > mira RNAi) or without knocking down mira (pgc− impα2OE matGal4 >). Embryos were stained for Vasa (D–G) or double-stained for Vasa and cleaved Dcp-1 (H, I). (D) Beeswarm plots showing the percentage of pole cells with protrusions at stage 6. (E) DIC images merged with the Vasa signal at stage 6. Arrow points to a protrusion. White dotted lines outline the somatic-cell layer. Scale bar, 10 µm. (F) Beeswarm plots showing the percentage of pole cells within the epithelium of the midgut primordium at stages 7–9. (G) DIC images merged with the Vasa signal at stage 7. Arrows indicate pole cells invading the epithelium of the midgut primordium. White dotted lines outline the epithelium of the midgut primordium. Scale bar, 20 µm. (H) Beeswarm plots showing the percentage of pole cells expressing cleaved Dcp-1 at stages 7–9. (I) DIC images merged with cleaved Dcp-1 and Vasa signals, along with the cleaved Dcp-1 channel alone. Arrows indicate cleaved Dcp-1-positive cells. White dotted lines outline the epithelium of the midgut primordium. The image of pgc- impα2OE matGal4 > embryo is the merged image of Vasa- and Dcp1-signals of the same embryo shown in (G). Scale bar, 20 µm. Data information: In all beeswarm plots, black dots represent individual embryos (biological replicates). Red lines represent the medians, and the boxes indicate the IQR (25th–75th percentiles). All data points are shown; therefore, minima and maxima correspond to the lowest and highest dots, and whiskers are not displayed. “n” indicates the number of embryos examined per genotype or group. Statistical significance was assessed using Mann–Whitney U test in (A, D, F, H) and two-sided exact Wilcoxon signed-rank test in (C). *P < 0.01; ns, not significant. Exact P values are provided in Source Data. Source data are available online for this figure.

In normal embryos at stages 5–6, mira mRNA was detected in all somatic cells but not in pole cells (Fig. 4A,B) (Fisher et al, 2012; Frise et al, 2010; Tomancak et al, 2002; Weiszmann et al, 2009). Ectopic mira expression was observed in pgc− impα2OE pole cells forming protrusions, but less frequently in spherical pole cells (Fig. 4B,C). Thus, we wondered whether such ectopic mira expression in pgc− impα2OE pole cells caused defects in morphology and developmental fate. We knocked down mira in pgc− impα2OE pole cells by supplying double-stranded RNA (dsRNA) against mira using the maternal-Gal4 (matGal4) driver. The frequency of pgc− impα2OE pole cells with protrusions and those invading the epithelial layer of the midgut primordium was significantly reduced when mira dsRNA was expressed (Fig. 4D–G). Furthermore, Dcp-1 expression in pgc− impα2OE pole cells was reduced by mira dsRNA (Fig. 4H,I). The above data showed that ectopic mira expression caused defects in pgc− impα2OE pole cells.

Ectopic mira expression is observed in a fraction of pgc−impα2OE pole cells

In the above experiments, we noticed ectopic mira expression in a fraction of pgc− impα2OE pole cells (Fig. 4A). To examine the cause of the differences among these pole cells, we performed single-cell RNA-seq (scRNA-seq) using pgc− impα2OE pole cells. Clustering analysis revealed that pgc- impα2OE pole cells were divided into three clusters (clusters A, B, and C) (Fig. 5A). Among them, pgc- impα2OE pole cells in cluster C expressed mira at a higher level than those in clusters A or B (Fig. 5B). In contrast, the expression of maternal transcripts localized in the germ plasm in cluster C was lower than that observed in clusters A or B (Fig. 5C). These results suggest that mira is ectopically expressed in pgc− impα2OE pole cells that inherit fewer germ-plasm components than the other pole cells. This is further supported by the observation that expression levels of the germ-plasm component mRNAs (gcl, nanos, osk, Prx2, and piwi) showed significant negative correlations with that of mira (Pearson correlation coefficient = −0.66, −0.46, −0.57, −0.58, −0.70, respectively; P < 0.01, two-sided Student’s t test).

Figure 5. Heterogeneity in pgc−impα2OE pole cells.

Figure 5

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(A) UMAP representation of three clusters identified by scRNA-seq of pole cells from pgc− impα2OE embryos at 2–3 h AEL (from the end of stage 4 to stage 6). (B, C) Boxplots showing the distribution of expression levels of mira (B) and eight major maternal germ-plasm mRNAs (C) in pole cells belonging to clusters A, B, or C. n = 6 cells (cluster A), 18 cells (cluster B), 12 cells (cluster C). The “mRNA level” represents the normalized expression values (see “Methods” and Appendix Table S2). The central lines represent the medians, and the boxes indicate the IQR (25th–75th percentiles), whiskers extend to the most extreme data points within 1.5× the IQR, and outliers are plotted. (D) Schematic drawing of central (Cent.) and peripheral (Peri.) pole cells of a stage-6 embryo. Ventral is to the left, and dorsal is to the right. The pole cells were divided into two groups: peripheral (on the edge of the pole cell cluster) and central (the remaining cells) (Slaidina and Lehmann, 2017). (E, F) Beeswarm plots showing the percentage of pole cells expressing mira mRNA (E) and the percentage of pole cells with protrusions (F) in central and peripheral pole cells of pgc− impα2OE embryos at stage 6. Black dots represent individual embryos (biological replicates). Red lines represent the medians, and the boxes indicate the IQR (25th–75th percentiles). All data points are shown; therefore, minima and maxima correspond to the lowest and highest dots, and whiskers are not displayed. “n” indicates the number of embryos examined per group. Statistical significance was assessed using Steel’s multiple comparison test versus cluster C in (B, C) (*P < 0.05; ns, not significant) and two-sided exact Wilcoxon signed-rank test in (E, F) (*P < 0.01). Exact P values are provided in Source Data. Source data are available online for this figure.

The pole cells located at the “central” region of the pole cell cluster inherit a higher amount of germ plasm than those located in the “peripheral” region (Slaidina and Lehmann, 2017) (Fig. 5D). For example, nanos mRNA levels are higher in central pole cells than in peripheral pole cells (Slaidina and Lehmann, 2017). Therefore, we speculate that “peripheral” pgc− impα2OE pole cells tend to misexpress mira. Figure 5E shows that the percentage of pgc− impα2OE pole cells expressing mira was significantly higher in the “peripheral” than in “central” region (Fig. 5E; Appendix Fig. S1A). Furthermore, the percentage of pgc− impα2OE pole cells forming protrusions was also higher in the “peripheral” region than in “central” region (Fig. 5F; Appendix Fig. S1B). Taken together, these results indicated that “peripheral” pgc− impα2OE pole cells tended to misexpress mira and formed cellular protrusions. Thus, we speculated that the peripheral pole cells invaded the midgut epithelium and eventually died until stage 9. These abnormalities of pgc− impα2OE pole cells are distinct from the ones caused by the absence of pgc. It has been reported that pgc mutation causes nanos degradation in pole cells and consequently induces abnormalities in mitosis, apoptosis, and migration of pole cells after stage 10 onward (Hanyu-Nakamura et al, 2019). Combining these early (stages 5–9) and late (stage 10 onward) abnormalities results in lower fertility of pgc− impα2OE embryos than that of pgc− mutants or impα2OE embryos (Asaoka et al, 2019).

It remains elusive how ectopic mira expression is induced in peripheral pole cells in pgc− impα2OE embryos. The simplest explanation is that maternal factor(s) within germ plasm, other than Nanos and Pgc, also contribute to mira repression. “Central” pole cells may contain this factor at a concentration above the threshold sufficient to repress mira. The “peripheral” pole cells may contain it below the threshold, making Nanos and Pgc critical for mira repression. It is also possible that Nanos and Pgc repress mira only in the peripheral pole cells. However, this scenario seems unlikely because Nanos and Pgc levels are higher in the central pole cells than in the peripheral ones (Fig. 5C). In any case, this mechanism helps the peripheral pole cells to execute germline development. Consequently, this may increase progeny production.

Nanos- and Pgc-dependent repression mechanisms act independently and redundantly to suppress somatic gene expression (Asaoka et al, 2019). Misexpression of mira, as well as protrusion formation and failure in germline/soma segregation, was enhanced in pgc− impα2OE pole cells compared to impα2OE or pgc− pole cells (Dataset EV1). mira may be expressed in the germline later in normal development and its premature expression causes the phenotypes observed in pgc− impα2OE pole cells. However, mira transcript(s) and their protein product(s) are never detected in the germline throughout normal development (Fisher et al, 2012; Shen et al, 1997). Thus, it is unlikely that premature expression of mira causes the phenotypes observed in pgc− impα2OE pole cells. In somatic cells, mira transcript(s) and protein product(s) are expressed ubiquitously at the blastoderm stage, and their expression is gradually restricted to neuroblasts in the ectoderm and stem-like cells (interstitial cell precursors: ICPs) in the midgut primordium (Matsuzaki et al, 1998; Shen et al, 1997). These cells are reported to form protrusions, the morphology of which is similar to that of pgc− impα2OE pole cells (Tepass and Hartenstein, 1995; Tsuji et al, 2008). Although it remains unknown whether protrusion formation requires mira expression in these somatic cells, it is possible that mira can induce cellular protrusions of pole cells. In neuroblasts, the Mira protein binds to the cargo proteins to transport them asymmetrically within the cells (Ikeshima-Kataoka et al, 1997; Irion et al, 2006). In pole cells, Mira misexpression may induce the asymmetric localization of protein(s) required for the formation of cellular protrusions. Mira acts together with three other proteins, Prospero (Pros), Staufen (Stau), and Brain tumor (Brat), in neuroblast (Knoblich, 2010). Interestingly, in pgc− impα2OE pole cells, the expression of pros and brat mRNAs was upregulated compared to that in normal pole cells (Dataset EV1), and stau mRNA was also detected at a significant level [Total stau expression (sum of three annotated isoforms): 31.6 ± 14.7 (TPM ± SEM, three biological replicates)]. It is necessary to investigate whether these three proteins act together with Mira for protrusion formation of pgc− impα2OE pole cells.

Our results showed that pgc− impα2OE pole cells detected in the epithelial layer of the midgut primordium expressed cleaved Dcp-1, which eventually led to cell death. It is reasonable to speculate that the cell death of pgc− impα2OE pole cells is induced directly by mira misexpression, although no evidence suggests that this gene is required for cell death. Alternatively, it is also possible that the cell death is caused by mira through mingling of pole cells with inappropriate cells, or somatic cells. For example, unfit somatic cells straying into ectopic locations are selectively eliminated by autophagy through interaction(s) with the surrounding cells (Nagata et al, 2019). In unfit Drosophila cells, autophagy upregulates the pro-apoptotic gene head involution defective (hid), which induces cleaved Dcp-1 expression (Nagata et al, 2019). The autophagy regulators Autophagy-related 1 (Atg1) and hid are activated in pgc− impα2OE pole cells (Dataset EV1), whereas their expression is undetectable in normal pole cells until gastrulation is complete (Fisher et al, 2012; Sato et al, 2007).

The expression of Nanos homologs and global transcriptional silencing are widely observed in the early primordial germ cells of numerous animals (Kurimoto et al, 2008; Mosquera et al, 1993; Nakamura and Seydoux, 2008; Sasaki et al, 2016; Subramaniam and Seydoux, 1999). In several insects, reptiles, chicks, mice, rabbits, and primates, their spatial segregation from the soma forming embryo’s body has also been reported. Primordial germ cells are transiently located in the extra-embryonic region or gut lumen. (Dixon, 1994; Golkar-Narenji et al, 2023; Matsui and Okamura, 2005; Nieuwkoop and Sutasurya, 1979, 1981; Sasaki et al, 2016). Our findings in Drosophila demonstrate that the Nanos/Pgc-dependent double-lock mechanism suppresses the somatic gene mira in the primordial germ cells, thereby maintaining this spatial separation from the soma and preventing germline apoptosis. Although no orthologs of mira have been reported outside insects (FlyBase, https://flybase.org/), other organisms may achieve a similar outcome by repressing different somatic genes through Nanos and global transcriptional silencing mechanisms. By contrast, in C. elegans, zebrafish, and Xenopus, spatial separation has not been reported. In these animals, primordial germ cells are formed and remain within the soma forming embryo’s body throughout development. Notwithstanding, primordial germ cells lacking Nanos homologs undergo apoptosis, intermingling with inappropriate somatic cells (Koprunner et al, 2001; Lai et al, 2012; Subramaniam and Seydoux, 1999). In C. elegans, Nanos homologs repress the expression of the somatic transcription factor LIN-15B to prevent germline apoptosis (Lee et al, 2017). These observations imply that somatic gene repression is a fundamental mechanism to protect primordial germ cells from a harmful somatic environment. This protection may be achieved either through physical segregation from the soma forming embryo’s body, as seen in Drosophila, or through alternative Nanos-dependent regulatory mechanisms in C. elegans. Further studies will help to clarify these mechanisms underlying germline apoptosis.

Methods

Reagents and tools table

Reagent/resource Reference or source Identifier or catalog number
Experimental models
Drosophila melanogaster strains
y w
impα2-nos3’UTR Asaoka et al, 2019
EGFP-vasa Sano et al, 2002; KYOTO Stock Center 109172
pgc Δ1 Hanyu-Nakamura et al, 2008
Df(2 R)X58-7 Bloomington Drosophila Stock Center BL-283
nanos-moesin-GFP Sano et al, 2005
maternal-Gal4 Bloomington Drosophila Stock Center BL-7063
UAS-mira RNAi Bloomington Drosophila Stock Center BL-32356
Antibodies
Chick anti-Vasa Hayashi et al, 2022
Mouse anti-Phosphotyrosine antibody PY20 BD Transduction Laboratories 610000
Rabbit anti-cleaved Drosophila Dcp1 (Asp215) Cell Signaling Technology 9578
Rabbit anti-GFP Invitrogen A-11122
Anti-Digoxigenin-POD Roche 11633716001
Anti-chicken IgY Alexa 488 ThermoFisher Scientific A-11039
Anti-chicken IgY Cy3 Jackson ImmunoResearch Laboratories 703-165-155
Anti-rabbit Cy3 Jackson ImmunoResearch Laboratories 711-165-152
Anti-mouse Cy3 Jackson ImmunoResearch Laboratories 715-165-151
Anti-mouse Alexa 633 ThermoFisher Scientific A-21050
Anti-rabbit Alexa 488 ThermoFisher Scientific A-11034
Oligonucleotides and other sequence-based reagents
ac forward primer: GGA GCA TCG TCA CAC AAT A This study
ac reverse primer: GCT GAG GTA ATA CTT GTT GGC C This study
sc forward primer: TCA TCG AGT GTG CTG TCC AC This study
sc reverse primer: TAG CTG AAG TTG GGA GTG CG This study
tll forward primer: GGA GAT CCC GGC AGT ATG TG This study
tll reverse primer: GAG GGA TGG GTC CTC TGG AT This study
mira cDNA clone BDGP Gold cDNA Collection LD02989
T7 primer This study N/A
SP6 primer This study N/A
Chemicals, enzymes, and other reagents
DAPI Sigma-Aldrich D9542
Bovine serum albumin (BSA) Sigma-Aldrich A7906
Normal Goat Serum ThermoFisher/Gibco 16210064
VECTASHIELD with DAPI Mounting Medium Vector Laboratories H-1200
pGEM-T Easy Promega A1360
T7 RNA polymerase Merck 10881767001
SP6 RNA polymerase Merck 10810274001
TSA Plus fluorescein kit PerkinElmer Life Sciences NEL741001KT
glass-bottomed dish MATSUNAMI D11130H
double-sided sticky tape 3 M Scotch w-12
silicone oil Shin-Etsu Chemical FL-100-450CS
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing Clontech 634890
SMART-Seq HT Kit Clontech 634437
TruePrep DNA Library Prep Kit V2 for Illumina Vazyme TD502
Software
Fiji Schindelin et al, 2012 https://imagej.net/software/fiji/
Imaris Oxford Instruments https://imaris.oxinst.jp/
Trimmomatic Bolger et al, 2014 http://www.usadellab.org/cms/?page=trimmomatic
FlyBase Larkin et al, 2021 https://flybase.org/
Kallisto Bray et al, 2016 https://pachterlab.github.io/kallisto/
R The R Foundation https://www.r-project.org/
edgeR Robinson et al, 2010 https://bioconductor.org/packages/release/bioc/html/edgeR.html
Seurat Hao et al, 2024 https://satijalab.org/seurat/
Metascape Zhou et al, 2019 http://metascape.org
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Fly strains

Flies were maintained on a standard Drosophila medium at 25 °C. The following fly stocks were used: y w, impα2-nos3’UTR (Asaoka et al, 2019), EGFP-vasa (Sano et al, 2002), pgcΔ1/CyO (Hanyu-Nakamura et al, 2008), Df(2 R)X58-7/CyO [Bloomington Drosophila Stock Center (BDSC) No. 283], nanos-Moe.EGFP.nanos 3’UTR (nanos-moesin-GFP) (Sano et al, 2005), w*; P{matα4-GAL-VP16}V37 (maternal-Gal4, BDSC No. 7063), y v; P{TRiP.HMS00347}attP2 (UAS-miraRNAi, BDSC No. 32356). Embryos produced from impα2-nos3’UTR/impα2-nos3’UTR, pgcΔ1/Df(2 R)X58-7, pgcΔ1 impα2-nos3’UTR/pgcΔ1 impα2-nos3’UTR females mated with y w were referred to as impα2OE, pgc−, and pgc− impα2OE embryos, respectively.

Immunostaining

Antibody staining of embryos was performed as previously described (Asaoka et al, 2019). Briefly, embryos were dechorionated in a sodium hypochlorite solution and fixed in a 1:1 mixture of heptane and fixative [4% paraformaldehyde in PBS (130 mM NaCl, 7 mM Na2HPO4, and 3 mM NaH2PO4)] for 20 min. Vitelline membranes of the fixed embryos were removed by vigorous shaking in 1:1 heptane:methanol. The following antibodies were used: chick anti-Vasa antibody (1:2000, lab stock) (Hayashi et al, 2022), mouse anti-Phosphotyrosine antibody PY20 (1:50, BD Transduction Laboratories), and rabbit anti-cleaved Drosophila Dcp1 (Asp215) antibody (1:50, Cell Signaling Technology). Signals were detected using Alexa Fluor 488-conjugated anti-chicken IgY antibody (1:400, ThermoFisher Scientific), Cy3-conjugated anti-mouse IgG antibody (1:500, Jackson ImmunoResearch Laboratories), Cy3-conjugated anti-rabbit IgG antibody (1:500, Jackson ImmunoResearch Laboratories), or Alexa Fluor 633-conjugated anti-mouse IgG antibody (1:400, ThermoFisher Scientific), as appropriate. Nuclei were visualized in embryos by staining with DAPI (2 μg/ml, Sigma-Aldrich) for 30 min after antibody staining. Stained embryos were washed three times with PBT [PBS containing 0.2% Bovine serum albumin (BSA, Sigma-Aldrich) and 0.1% Triton X-100] and mounted in VECTASHIELD with DAPI Mounting Medium (Vector Laboratories).

Z-stack confocal images were taken using a Leica TCS-SP8 confocal microscope (Leica) and analyzed using Fiji software. For analysis of cellular protrusion formation of pole cells, z-stack images were taken with the following settings: ×63 objective lens, ×2.5 digital zoom, 1024 × 1024 dpi, 0.36 μm z-step interval. The acquired confocal serial images were reconstructed into 3D structures and analyzed using Imaris software (ver. 7.6) (Oxford Instruments). The number of pole cells with protrusions was counted using the Fiji software.

In situ hybridization

cDNA fragments corresponding to ac, sc and tll were amplified from an imaginal disc cDNA library and an embryonic cDNA library (Brown and Kafatos, 1988) using the following primers: 5′-GGA GCA TCG TCA CAC AAT A-3′ and 5′-GCT GAG GTA ATA CTT GTT GGC C-3′ for ac, 5′-TCA TCG AGT GTG CTG TCC AC -3′ and 5′-TAG CTG AAG TTG GGA GTG CG -3′ for sc, and 5′-GGA GAT CCC GGC AGT ATG TG -3′ and 5′-GAG GGA TGG GTC CTC TGG AT -3′ for tll. Amplified cDNAs were cloned into pGEM-T Easy (Promega). Templates for RNA probes were amplified from these plasmids and a full-length mira cDNA clone (BDGP Gold cDNA Collection, LD02989) using T7 and SP6 primers, and digoxigenin (DIG)-labeled RNA probes were synthesized from the fragments using T7 and SP6 RNA polymerase (Merck), respectively.

The whole mount in situ hybridization of embryos was performed as previously described (Asaoka et al, 2019). Triple staining for in situ hybridization, immunostaining with anti-Vasa antibody, and DAPI staining, as shown in Figs. 4B,C, and 5E, were performed as follows. Embryos were dechorionated, fixed, and devitellinized as described in the “Immunostaining” section. All the following steps were performed using RNase-free reagents until the hybridization step was completed. Devitellinized embryos were immediately rinsed with methanol twice, dehydrated by incubating in ethanol four times (10 min each), incubated in 1:1 xylene:ethanol for 30 min, and rinsed with ethanol five times and with methanol twice (30 s each). The embryos were re-fixed in a 1:1 mixture of methanol and fixative (4% paraformaldehyde, 0.1% Tween 20 in PBS) for 5 min and washed twice with PBTw (PBS containing 0.1% Tween 20). The embryos were digested with 4 μg/mL Proteinase K in PBTw for 7 min at 23 °C. The reaction was stopped by immediately rinsing the samples once (30 s) and washing four times (2 min each) with PBTw. The embryos were post-fixed in fixative for 20 min, washed with PBTw five times (2 min each), incubated in a 1:1 mixture of PBTw and hybridization solution [HS; 50% formamide, 5× SSC (750 mM NaCl and 75 mM sodium-citrate, pH 5.0), 100 μg/mL heparin, 100 μg/mL yeast tRNA, and 0.1% Tween 20] for 10 min, incubated in HS for 10 min, and then pre-hybridized in HS for 1.5 h and hybridized in HS containing 0.5 ng/μL RNA probe for 16 h at 56 °C. After hybridization, the embryos were washed four times with the washing solution (50% formamide, 5× SSC, and 0.1% Tween 20) for 30 min each at 56 °C, with PBTw containing 75%, 50%, and 25% washing solution, and five times with PBTw for 5 min each at room temperature. The embryos were then blocked for 1 h in PBTB (PBS containing 0.5% Blocking Reagent [PerkinElmer, NEL700001KT] and 0.1% Tween 20), incubated in PBTB containing chick anti-Vasa antibody (1:1000) and anti-Digoxigenin-POD antibody (1:500, Roche, 11633716001) overnight at 4 °C, and washed with PBTw once for 30 s and five times for 10 min each. The anti-digoxigenin-POD signal was detected using the TSA Plus fluorescein kit (PerkinElmer Life Sciences, Inc.), and the embryos were washed with PBT (PBS containing 0.2% BSA and 0.1% Triton X-100) three times (15 min each), blocked for 30 min in blocking solution (PBS containing 0.2% BSA, 0.1% Triton X-100, and 5% normal goat serum), and incubated with Cy3-conjugated anti-chicken IgY antibody (1:250, Jackson ImmunoResearch Laboratories) and DAPI for 2 h. After washing with PBT twice (30 s each) and three times (10 min each), the samples were mounted in VECTASHIELD with DAPI Mounting Medium. Z-stack confocal images were taken using a Leica TCS-SP8 confocal microscope (Leica). Using Fiji software with the ROI manager function, we measured the average fluorescence intensities (intensity/pixel) of mira mRNA signals within the area occupied by individual pole cells, as defined by the outline of the Vasa signal in the Vasa-signal channel. Measurements were taken from sections through the median plane of pole cells and only from pole cells located within 15 μm of the top section of the confocal z-stack. In each section, the cells at both ends of the pole cell population were designated as peripheral pole cells, and the remaining cells were designated as central pole cells.

Live imaging of embryos

Embryos produced from w; pgcΔ1 impα2OE; nanos-moesin-GFP females mated with y w were collected at 2.5–3.5 h after egg laying (AEL), dechorionated in a sodium hypochlorite solution, and washed several times with distilled water (DW). Using forceps, embryos were aligned in a drop of PBS on a glass-bottomed dish (MATSUNAMI) and covered with a piece of thin agarose gel (1% agarose in PBS, 1 mm thick). Time-lapse images were acquired using a Leica TCS-SP8 confocal microscope (Leica).

Pole cell transplantation

Pole cell transplantation was performed as described previously, with the following modification (Asaoka et al, 2021; Nishimura et al, 2023). Donor and host embryos were collected at 40-min intervals and were allowed to develop until 80–120 min AEL. Collected embryos were dechorionated and aligned on double-sided sticky tape. In silicone oil (Shin-Etsu Chemical), pole cells were collected into one glass needle from 7 to 10 donor embryos. The collected pole cells were quickly deposited and mixed thoroughly onto the surface of an embryo and then reloaded into the needle. Approximately 10 pole cells were transplanted to the posterior pole of each host embryo. After transplantation, the host embryos were allowed to develop until stages 7–9 and then triple-stained with anti-GFP (1:500, Invitrogen), anti-Vasa, and PY3 antibodies, followed by DAPI staining as described above. Serial confocal images (z-step: 1.0 μm) were obtained using a Leica TCS-SP8 confocal microscope (Leica) and analyzed using Fiji software to determine whether GFP-positive donor pole cells were located within the epithelium of the midgut primordium or not.

mira knockdown

Embryos produced from pgcΔ1 impα2-nos3’UTR/pgcΔ1 impα2-nos3’UTR; maternal-Gal4/UAS-miraRNAi and pgcΔ1 impα2-nos3’UTR/pgcΔ1 impα2-nos3’UTR; maternal-Gal4/+ females mated with y w were used as pgc- impα2OE embryos with and without mira knockdown, respectively.

Bulk RNA-seq analysis

Embryos produced from w; +; EGFP-vasa, w; pgcΔ1 impα2-nos3’UTR/impα2-nos3’UTR; EGFP-vasa/+, and w; pgcΔ1 impα2-nos3’UTR; EGFP-vasa/+ females mated with y w were used as normal (w), impα2OE, and pgc− impα2OE embryos, respectively. One hundred pole cells were isolated from 2–3 h AEL embryos by FACS, and cDNAs were synthesized using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), as described previously (Morita et al, 2020; Shigenobu et al, 2006). Nextera XT library creation and RNA-seq were performed at the University of Minnesota Genomics Center using the HiSeq 2500 platform (Illumina), and approximately 20 million reads per sample (50-bp paired-end reads) were obtained. For RNA-seq analysis of pgc− pole cells, previously deposited raw read count data (Data ref: Morita et al, 2019; Morita et al, 2020) were retrieved and re-analyzed in the same manner as for other genotypes.

Raw reads were processed using Trimmomatic (ver. 0.36) (Bolger et al, 2014) and aligned with the transcript model of Drosophila melanogaster (Flybase; dmel-all-transcriptr-r6.17. fasta) using the Kallisto software (ver. 0.43.1) with default settings (Bray et al, 2016). Differential expression analysis was performed in R (ver. 4.4.1) using the edgeR package (ver. 4.2.1) (Robinson et al, 2010). For quality control of our dataset, the transcripts showing CPM (count-per-million) >0.5 in at least three samples were filtered. The resulting 16009 transcripts (8305 genes) were used for the following analyses and normalized by the trimmed-mean method before differential expression analysis. Differentially expressed transcripts were identified by comparing the normal (w) and experimental groups, considering batch effects to ensure accurate identification. Transcripts with an FDR (Benjamini–Hochberg procedure) of <0.01 were determined to be differentially expressed transcripts.

Gene enrichment analysis

GO functional enrichment analysis was performed using Metascape (ver. 3.5.20250701) (http://metascape.org) (Zhou et al, 2019) with default settings. The filtered transcript set [16009 transcripts (8305 genes), see “Bulk RNA-seq analysis” section in “Methods”] was used as the background gene set. Upregulated and downregulated transcripts were analyzed separately. q-values are calculated using the Benjamini–Hochberg procedure.

ScRNA-seq analysis

Embryos produced from w; pgcΔ1 impα2OE; EGFP-vasa/+ females mated with y w were used. Pole cells were isolated from 2–3 h AEL embryos and collected individually into different tubes via FACS, as described (Shigenobu et al, 2006). cDNAs were synthesized using the SMART-Seq HT Kit (Clontech), and libraries were prepared with 5 ng of cDNA input using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme). The samples were sequenced using the HiSeq 10 platform (Illumina), and approximately 5 million reads per sample (150-bp paired-end reads) were obtained. Both library preparation and sequencing were performed at the Azenta Life Sciences.

Raw reads were processed and aligned to the transcript model of Drosophila melanogaster, as described in the “Bulk RNA-seq analysis” section. The “est_count” output generated by Kallisto was used as the raw read count for further processing in R (ver. 4.4.1) using Seurat (ver. 5.1.0) (Hao et al, 2024) packages. For quality control of our dataset, transcripts showing a read count of >0 in at least one sample were filtered and used. The feature counts were log-normalized and scaled using the default options. To remove dead cells, cells in which over 7.5% of the reads were from mitochondrial genes were filtered out. The final dataset of 36 high-quality cells and 21811 features was used for downstream analysis. To assemble cells into transcriptomic clusters using meaningful features, we first performed principal component analysis (PCA) using 2000 highly variable transcripts as input. Based on the Elbow method, the first eight principal component (PC) vectors were selected as significant PCs and used as input for Uniform Manifold Approximation and Projection (UMAP) clustering, with a resolution parameter of 1.0.

Calculation of the expression level of mRNAs of mira and components of the germ plasm

To calculate the total number of transcripts corresponding to mira and germ-plasm components, normalized and scaled count data were used. For each gene, the counts of the transcripts shown in Appendix Table S2 were summed and used as the “mRNA level” in Fig. 5B,C.

Statistics and reproducibility

The statistical tests used for each experiment are indicated in the figure legends. The experiments for all figures and tables were repeated more than twice, except for the scRNA-seq analysis shown in Fig. 5.

Supplementary information

Table EV1 (10.8KB, xlsx)
Appendix (4.1MB, pdf)
Peer Review File (694.9KB, pdf)
Dataset EV1 (133.3KB, xlsx)
Dataset EV2 (37.6KB, xlsx)
Dataset EV3 (50.6KB, xlsx)
Source data Fig. 1 (19.8KB, zip)
Source data Fig. 2 (926.5KB, zip)
Source data Fig. 3 (1.3MB, zip)
Source data Fig. 4 (4.2MB, zip)
Source data Fig. 5 (17.6KB, zip)
Expanded View Figures (342.9KB, pdf)

Acknowledgements

We thank R Lehmann, A Nakamura, E Gavis, BM Mechler, the Bloomington Drosophila Stock Center, and the Drosophila Genomics Resource Center for fly stocks and materials, and Editage (www.editage.jp) for English language editing. We also thank Y Kozono for valuable discussion. This work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (23K05778 to MA, and 24H02030 to SK).

Expanded view

Author contributions

Miho Asaoka: Conceptualization; Data curation; Supervision; Funding acquisition; Investigation; Methodology; Writing—original draft; Writing—review and editing. Mizuki Kayama: Investigation. Tomoki Kawagoe: Investigation; Methodology. Makoto Hayashi: Methodology; Writing—review and editing. Shumpei Morita: Methodology. Satoru Kobayashi: Supervision; Funding acquisition; Project administration; Writing—review and editing.

Source data underlying figure panels in this paper may have individual authorship assigned. Where available, figure panel/source data authorship is listed in the following database record: biostudies:S-SCDT-10_1038-S44319-026-00710-x.

Data availability

Bulk and single-cell RNA-seq data produced in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers DRR669259–DRR669267 (https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB20673) and DRR752151–DRR752194 (https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB20688), respectively.

The source data of this paper are collected in the following database record: biostudies:S-SCDT-10_1038-S44319-026-00710-x.

Disclosure and competing interests statement

The authors declare no competing interests.

Footnotes

These authors contributed equally: Miho Asaoka, Mizuki Kayama, Tomoki Kawagoe.

Contributor Information

Miho Asaoka, Email: masaoka@tara.tsukuba.ac.jp.

Satoru Kobayashi, Email: skob@tara.tsukuba.ac.jp.

Supplementary information

Expanded view data, supplementary information, appendices are available for this paper at 10.1038/s44319-026-00710-x.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table EV1 (10.8KB, xlsx)
Appendix (4.1MB, pdf)
Peer Review File (694.9KB, pdf)
Dataset EV1 (133.3KB, xlsx)
Dataset EV2 (37.6KB, xlsx)
Dataset EV3 (50.6KB, xlsx)
Source data Fig. 1 (19.8KB, zip)
Source data Fig. 2 (926.5KB, zip)
Source data Fig. 3 (1.3MB, zip)
Source data Fig. 4 (4.2MB, zip)
Source data Fig. 5 (17.6KB, zip)
Expanded View Figures (342.9KB, pdf)

Data Availability Statement

Bulk and single-cell RNA-seq data produced in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers DRR669259–DRR669267 (https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB20673) and DRR752151–DRR752194 (https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB20688), respectively.

The source data of this paper are collected in the following database record: biostudies:S-SCDT-10_1038-S44319-026-00710-x.

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