(A) dGe-1 protein levels are strongly reduced in dGe-1^Δ5 GLC. Western blot of dGe-1 protein in extract of 0-2 h wt or dGe-1^Δ5 GLC embryos probed with rat anti-dGe-1 antibodies. The band just above 150 kDa, which is dramatically reduced in the mutant extract, represents dGe-1 protein. Khc protein serves as a loading control. (B-C) dGe-1 signal is absent from the nurse cells and oocyte of dGe-1^Δ5 GLC. Immunodetection of dGe-1 protein in wt (B) and dGe-1^Δ5 GLC (C) ovaries using rat anti-dGe-1 antiserum. (D) Western blot analysis of ovarian extracts from wt and dGe-1^Δ5 GLC ovaries, probed with mouse anti-Me31B and anti-Cup antibodies and rabbit anti-Khc antibody. Khc serves as a loading control. (E–K) Immunostaining of wt (E–G), dGe-1^Δ5 GLC (H–J) and dGe-1^Δ5/dGe-1^Δ5; dGe-1 Rescue (dGe-1 R) (K–M) S7 egg-chambers using a rabbit anti-Me31B antibody (E, H, K) or a mouse anti-Cup antibody (F, I, L). Overlays are shown in G, J and M. Bar, 10 µm.

(A–C) Immunodetection of dGe-1 protein in wt (A, B) and dGe-1^Δ5 GLC (C) ovaries using a rabbit anti-dGe-1 antibody. (A, B) In wt ovaries at S9 (A) or S10 (B), dGe-1 protein is enriched at the oocyte posterior pole. (C) The posterior dGe-1 signal observed in wt oocytes (B) is greatly reduced in dGe-1^Δ5 GLC oocytes. (D–F) In a wt egg-chamber at late S9, dGe-1 enriched at the posterior pole of the oocyte (green, D) and partially colocalizes with Stau protein (red, E). The overlay of the two antibody signals is shown in (F). (G–I) Colocalization of dGe-1 protein (green, G) and osk mRNA (red, H) in wt S9 oocytes expressing oskMS2 mRNA and MCP-RFP protein (red, H), to which the RNA is tethered; at S9 oskMS2 mRNA distribution reflects that of endogenous osk mRNA [33]. Overlay of dGe-1 and oskMS2 mRNA signals shown in (I). (J–K) dGe-1 protein staining in wt and stau^D3 oocytes. (J, J′) dGe-1 protein is enriched at the posterior pole in wt oocytes. (K and K′) In stau^D3 mutant oocytes, which fail to localize osk mRNA, the amount of dGe-1 at the posterior is dramatically reduced. (L–N) Double immunostaining of dGe-1 and Stau proteins in a nutritionally restricted wt egg-chamber. In starved wt oocytes, dGe-1 protein (green, L) is present in large particles, where it colocalizes with Stau protein (red, M). Overlay of dGe-1 and Stau stainings (N). (O–Q) Distribution of dGe-1 protein and osk mRNA in starved wt egg-chambers expressing oskBoxB mRNA and λN-GFP protein, to which the RNA is tethered. In nutritionally restricted wt oocytes, dGe-1 (red, O) is present in large particles, where it partially colocalizes with oskBoxB mRNA (green, P). Overlay of dGe-1 and osk mRNA signals shown in (Q). Bar, 50 µm.

(A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B, the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1^Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR (Figure S1B) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.

(A–I) Simultaneous detection of osk mRNA (red) and protein (green) by FISH coupled with immunodetection using a rabbit anti-Osk antibody. osk mRNA (A) and protein (B) colocalize in a posterior crescent at the posterior pole of wt oocytes at S10. (D–I) Examples of the aberrant distribution of osk mRNA and protein in dGe-1^Δ5 GLC egg-chambers. Overlays of osk mRNA and Osk protein signals (C, F, I). DNA stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). (J) Quantification (%) of osk mRNA localization patterns in S10 egg-chambers in different genetic backgrounds. Normal and abnormal osk mRNA localization are represented as black and grey bars, respectively. n represents the number of embryos analyzed. (K–M) Loss of maternal dGe-1 causes aberrant cuticle patterning in some embryos derived from dGe-1^Δ5 GLC. Lateral view of embryos oriented anterior to the left, ventral side down. (K) In a wt embryo, posterior structures represented by the abdominal denticle belts are clearly visible along the ventral side. In a portion of embryos derived from dGe-1^Δ5 GLC, several denticle belts are missing (L) and, in extreme cases, all are absent (M). (N) Quantification of posterior group phenotypes produced in different genetic backgrounds. Embryos lacking at least one (weak) or all (strong) abdominal denticle belts were quantified. n represents the number of embryos analyzed. Bar, 50 µm.

(A) Drosophila wt (upper panel) or dGe-1^Δ5 GLC (lower panel) ovarian extracts treated with cycloheximide and analyzed by centrifugation in a 10–45% sucrose density gradient. osk and actin mRNAs in the gradient fractions were detected by RNase protection assay. Absorption at 260 nm was measured to situate the monoribosome peak (80S) and polysomes. (B) Quantification of the RNase protection assays presented in (A). The relative levels of osk and actin mRNAs present in each fraction are indicated as a % of the total levels of both mRNAs present in wt (upper panel) and dGe-1^Δ5 GLC (lower panel) ovarian extracts. (C–D) Distribution of Stau protein in starved wt (C) or dGe-1^Δ5 GLC (D) ovaries. In nutritionally restricted wt oocytes, Stau (green) is present in large particles, which fail to form in starved dGe-1^Δ5 GLC ovaries. Bar, 50 µm.