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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01321

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Germ cell-autonomous Wunen2 is required for germline development in Drosophila embryos

¹ Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
² Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

View larger version (125K): [in a new window]   Fig. 1. Maternal N14 mutant embryos have a defect in pole cell maintenance. Wild-type embryos (A,C,E,G) and N14m– embryos from N14/Df(2R)w45-19g females (B,D,F,H) were stained for Vasa to visualize pole cells. Anterior is towards the left in all panels. (A-F) Lateral views. (G,H) Top views. In wild-type embryos, pole cells were formed at the posterior pole of the blastoderm-stage embryo (A), and carried into the posterior midgut primordium. At stage 10, pole cells moved through the midgut epithelium and migrated dorsally along its basal surface (C). Next, pole cells moved into the mesoderm at stage 11 (E), and were finally incorporated into the embryonic gonad at stage 16 (G). In N14m– embryos, a normal number of pole cells was formed (B). These pole cells migrated normally through the midgut epithelium and moved dorsally along its surface (D). However, the number of Vasa-positive pole cells was rapidly reduced at stage 11 (F), and few or no pole cells were incorporated into the gonad (H).

View larger version (87K): [in a new window]   Fig. 2. Pole cells in N14m– embryos die after exiting the midgut. Pole cells in N14m– embryos were labeled with a photoactivatable lineage tracer, caged fluorescein (green), and an antibody against Vasa (red). Anterior is towards the left and dorsal is upwards. (A-C) At stage10, uncaged-fluorescein/Vasa double-positive pole cells were observed on the surface of the midgut. (D-F) From stage 11 onwards, the fluorescein-marked cells disappeared. The remaining fluorescein-positive cells were left on the dorsal side of the midgut surface. (G-I) At stage 12, few fluorescein-marked cells were observed. Scale bar: 40 µm.

View larger version (30K): [in a new window]   Fig. 3. Pole cell death in N14m– embryos might occur via a caspase 3-independent pathway. Pole cells in N14m– embryos were directly labeled using a caged fluorescein (A, green), and were stained for cleaved caspase 3 (B, red) and Vasa (C, blue). (D) Merged image of A-C. Cleaved caspase 3 was never detected in the dying pole cells on the surface of the midgut, in which the fluorescein signal remained but the Vasa signal was reduced (arrowheads). Arrows indicate the active caspase 3-positive mis-migrated pole cells, which are usually seen even in wild-type embryos. Scale bar: 40 µm.

View larger version (129K): [in a new window]   Fig. 4. N14 mutation does not affect the distribution of pole plasm components. Wild-type (A-D) and N14m– (E-H) embryos at the cellular blastoderm stage. Embryos were stained for nanos RNA (A,E), gcl RNA (B,F), pgc RNA (C,G) or Nanos protein (D,H). These RNAs and protein were incorporated normally into pole cells in N14m– embryos (E-H) as in wild-type embryos (A-D).

View larger version (56K): [in a new window]   Fig. 5. N14 mutation does not affect zygotic gene expression in pole cells. (A,B) Embryos at the syncytial blastoderm stage were stained with the antibody H5 (green) and an antibody against Vasa (red). The H5 antibody labeled somatic nuclei, but not pole cell nuclei in N14m– embryos (B) as well as in wild-type embryos (A). Scale bar: 20 µm. (C-E) Expression of ß-galactosidase in pole cells was examined in embryos from nanos-Gal4-VP16 females crossed with UAS-lacZ males. (C,D) Lateral views of stage 10 embryos. In both N14m– (D) and wild-type (C) embryos, ß-galactosidase was expressed in pole cells. (E) The stage-dependent expression of ß-galactosidase was examined in N14m– background (black squares) and wild-type background (white squares). The percentage of embryos with ß-galactosidase-positive pole cells was plotted against the embryonic stage. In both N14m– and wild-type background, the expression of zygotic ß-galactosidase was detected from stage 9. After stage 12, the number of embryos expressing ß-galactosidase was diminished in N14m– embryos, owing to pole cell loss.

View larger version (16K): [in a new window]   Fig. 6. N14 is an allele of wun2. (A) Gene organization of the N14 locus. The N14 mutant phenotype was not complemented by Df(2R)w45-19g or Df(2R)w73-1, but was by Df(2R)Np1. These deficiencies defined the N14 locus within 100 kb of a genomic region that contains nine identified and predicted genes. A series of transgenic constructs that was used to identify the responsible gene for the N14 mutant phenotype is shown below. Only the constructs in which wun2 was intact (white boxes), rescued the N14 mutant phenotype. (B) An 8 kb HincII fragment (Pwun2-8k) rescued the N14 mutant phenotype, but the Pwun2-8k fragment, which had a partial deletion in the wun2 RNA-coding region, did not. The N14 chromosome had a nonsense mutation at the 111th Trp codon of the wun2-coding region. Mobilization of a P-element insertion, EP2650, generated a wun2 deletion (wun2). Embryos from N14/wun2 females showed the N14 mutant phenotype, confirming that wun2 is the responsible gene for the N14 mutation (Table 1).

View larger version (66K): [in a new window]   Fig. 7. Distribution of wun2 RNA. Wild-type embryos (A-C) and embryos from wun2/Df(2R)w45-19g females (wun2 embryos; D) were stained for wun2 RNA. (A) wun2 RNA was detected throughout the stage-2 embryo. (B,B') At stage 4, wun2 RNA remained in pole cells, while the signal in somatic cells disappeared. (B') Highlighting of the posterior pole of the embryo shown in B. (C) At stage 5, wun2 RNA was detected in pole cells and in a posterior stripe. (D) Stage 5 wun2 embryo showing the absence of wun2 RNA in pole cells but its presence in a posterior stripe.

View larger version (76K): [in a new window]   Fig. 8. Pole cell-specific expression of wun2 rescues the maternal wun2 mutant phenotype. Embryos were stained for Vasa to visualize pole cells. (A-C) Embryos from a cross of wun2N14/Df(2R)w45-19g; nanos-Gal4-VP16/+ females with Oregon-R males. (D-F) Embryos from a cross of wun2N14/Df(2R)w45-19g; nanos-Gal4-VP16/+ females with EP2650 males. (G-I) Embryos from a cross of nanos-Gal4-VP16 females with EP2650 males. (D-F) Expression of wun2 in pole cells fully rescued the maternal wun2 mutant defect. These pole cells were moderately dispersed at the late stages of embryogenesis (F), as in embryos overexpressing wun2 in pole cells (I) (Starz-Gaiano et al., 2001). The maternal wun2 mutant phenotype was not rescued with nanos-Gal4-VP16 driver alone (A-C) or with no driver (data not shown).

View larger version (70K): [in a new window]   Fig. 9. Wun2 activity in pole cells competes with somatic Wun and Wun2 activities for pole cell survival. Embryos were stained for Vasa to visualize pole cells. (A) A wun2m– embryo showing the drastic pole cell loss phenotype. (B) An embryo lacking both maternal Wun2 and zygotic Wun and Wun2 showing partial suppression of the pole cell death phenotype of wun2m– embryos. (C) Overexpression of wun2 in the mesoderm leads to pole cell death. (D) An embryo overexpressing wun2 both in pole cells and the mesoderm shows a partially suppressed pole cell death phenotype caused by the overexpression of wun2 in the mesoderm alone (C).