Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division

Abstract

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. Here we report the function of Ago1 in developing egg chambers. We find that Ago1 protein is enriched in the oocytes and is also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte which is phenocopied in dicer-1, pasha and drosha mutants. Our results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila.

Fig. 1. Ago1 distribution in Drosophila egg chambers

(A & A’) Ago1::GFP protein trap line and (B & B’) Ago1 antibody staining in the Drosophila egg chambers showing the distribution of Ago1 protein. Ago1 is distributed in the cytoplasm but it is enriched in the developing oocyte (arrow) and clearly seen in egg chamber stage 7. GFP / Ago1 are shown in green, DNA in magenta and F-actin in red (C–E) Ago1 colocalizes with Oskar at the posterior cortex of a stage-10 oocyte. Ago1 is shown red, Oskar in green, and DNA in blue Scale bars, 20 µm.

Fig. 2. Ago1 staining is reduced in ago1 mutants and increases upon overexpression

(A–F) Mutant clones (GFP −ve) show lower expression of Ago1 compared to wild-type (GFP +ve) cells. (A–C) Ago1 expression in follicle cell clones of the ago1^k08121 mutant. (D–F) Ago1 expression in germline clones. Ago1 enrichment in the oocyte was also dramatically reduced in the ago1^k08121 mutant clones compared to positive control (arrow). (G–I) Overexpression using an inducible GAL4 driving UAS-Ago1 shows overexpressing clones (GFP +ve) have brighter Ago1 staining compared to wild-type (GFP −ve) cells. Scale bars, 20 µm.

Fig. 3. Loss of Ago1 disrupts oocyte formation and germline cell division

(A) A heterozygous GFP +ve ovariole. (B) Ovariole comprised of germline cell clones of ago1^k08121. Follicle cells in late stage egg chambers seem to be degrading. (C–F) Two neighbouring ago1 mutant egg chambers, one with an oocyte and one without (orb staining marks the oocyte). (G–J) ago1^k08121 clone with only 8 nurse cells and no oocyte formation. Scale bars, 20 µm.

Fig. 4. ago1 phenotype manifests in the germarium

(A–H) Germarium with GFP +ve wild-type clones (A–D) showing normal branching of the fusomes (arrow) compared to GFP −ve ago1 mutant clones (E–H) showing less brancing of the fusomes (arrow). In merge pictures, GFP - green, HTS - red and Vasa - yellow. (I–N) Germarium with GFP +ve wild type clones (I–K) showing normal size germarium compared to the smaller GFP −ve ago1 mutant clones (L–N) germarium. In merge pictures, GFP – green and Orb – red. R1, R2 and R3 denotes to region 1, region 2 and region 3 in the germarium.

Fig. 5. Orb and Cup staining in the pseudo-oocyte

(A–D) Germarium with GFP −ve ago1 mutant clones stained with anti-Orb and anti-Cup. Orb protein is present in region 2B while Cup can only be seen in stage 1 egg chamber. (E–H) Stage 1 egg chamber with 8 nurse cells stained with anti-Orb showing the Orb protein accumulates at the pseudo-oocyte. Scale bars, 20 µm.

Fig. 6. Identification and characterization of new dicer-1 and pasha alleles

(A) Schematic representations of of Dicer-1 and Pasha proteins showing important domains and locations of the mutations. (B–G) Adult eyes with eyFLP-generated clones of the indicated mutation in the background of GMR-w-miR. Loss of miRNA activity results in more white gene product and patches of darker pigmentation.

Fig. 7. dicer-1, pasha and drosha null mutants exhibit the same phenotype as Ago-1 mutants

(A) Positive control (GFP +ve) showing a normal egg chamber. (B–G) Selected examples demonstrating the ~30–60% of germline clones for the indicated genotypes that exhibit the oocyte-less 8 nurse cell egg chamber phenotype. Six independent dicer-1 mutant alleles showing selected egg chamber exhibiting the same germline cell division and oocyte formation defect as in an ago1 mutant. (H–I) Germline clones of two independent pasha alleles. pasha^LL03360 does not show the same germline cell division or oocyte formation defects as in ago1, while pasha^36B2 phenocopies the ago1 and dicer-1 mutant phenotype. (J) drosha mutant showing the 8 nurse cells phenotype. DNA is shown in margenta. Scale bars, 20 µm.