Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary

Abstract

In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. Here we present data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam.

Figure 1. Germline cells within germaria co-express Mei-P26, Bgcn, Sxl and Bam.

(A) A wild-type germarium stained for Mei-P26 (red), GFP-Bgcn (green) and DNA (blue). (B) A wild-type germarium stained for Mei-P26 (red), Sxl (green) and DNA (blue). (C) A wild-type germarium stained for Mei-P26 (red), Bam (green) and DNA (blue). Scale bars represent 10 µm.

Figure 2. Mei-P26 physically interacts with Bgcn.

(A) V5-tagged Mei-P26 co-immunoprecipitates Myc tagged Bgcn from S2 cell extracts. GFP-Bgcn immunoprecipitates with Mei-P26 in (B) wild-type whole ovary extracts and (C) bam^▵86 mutant ovary extracts. (D) Various combinations of Bam, Bgcn, Mei-P26, Ago1 bait and prey constructs were tested in a LexA based yeast-2-hybrid β-galactosidase assay.

Figure 3. Sxl and Bam associate with Mei-P26.

(A) Sxl immunoprecipitates with Mei-P26 in ovarian extracts from newly eclosed females. (B) Heat-shock induced Bam::HA and Sxl co-immunoprecipitate with Mei-P26. The levels of Sxl that associate with Mei-P26 do not appear to change in the presence of Bam::HA. (C) Extracts from whole ovaries were fractionated and the resulting eluents were probed for the presence of various proteins using western blot analysis. Mei-P26, GFP-tagged Bgcn, Sxl and HA-Bam were observed over a range of fractions but co-peaked in fraction 30. The control proteins Ter94 and Actin peaked in fractions 34 and 37 respectively.

Figure 4. Loss of snf and Mei-P26 results in mis-regulation of Nanos expression during cyst development.

(A) Control, (B) snf¹⁴⁸ homozygous and (C) mei-P26^mfs1 homozygous germaria stained for Nanos (green), Bam (red) and DNA (blue). Overlapping Nanos and Bam expression is readily observed in both mei-P26 and snf mutant ovaries. (D) A mei-P26^mfs1 clonal germarium stained for GFP (green) and Bam::HA (red). Both heterozygous cells (magenta arrow) and homozogous mei-P26 mutant clones (white arrow) appear to express similar levels of Bam::HA. Scale bars represent 10 µm.

Figure 5. Mei-P26 and Sxl associate with nanos mRNA.

(A) Immunoprecipitation of Sxl and Mei-P26 enriches for nanos mRNA but not actin mRNA. (B) Graph showing the ratio of mRNA pulled down in an Sxl IP versus a control IP and a Mei-P26 IP versus a control IP as measured by real time qualitative RT-PCR.