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First published online 3 November 2004
doi: 10.1242/dev.01460

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Temporal complexity within a translational control element in the nanos mRNA

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (13K): [in a new window]   Fig. 1. Tagged nos and nos-tub3'UTR transgenes. (A) The gfp-nos and gfp-nos-tub3'UTR transgenes contain GFP sequences (dark shading) inserted at the N terminus of the genomic nos coding region (light shading). Both transgenes include the nos promoter and 5' regulatory sequences (Pnos), 5'UTR (left black bar) and polyadenylation signal. Gfp-nos (top) bears the intact nos 3'UTR (right black bar), whereas these sequences have been replaced by -tubulin 3'UTR sequences (tub) in gfp-nos-tub3'UTR (bottom). (B) The hemagglutinin (HA) epitope tagged nos-tub3'UTR transgene is identical to gfp-nos-tub3'UTR, except that an N-terminal HA tag replaces GFP. The nos-tub:TCE and TCE mutant transgenes carry insertions of wild-type and mutant TCE sequences (shown in C), respectively (hatched box). Transgenes in A and B are drawn to scale, except for introns. (C) Nucleotide changes associated with the SRE– (circles), TCEIIA (squares) and TCEIIIA (hexagons) mutations are indicated on the TCE secondary structure. The following mutations are not shown: TCEIIIA/U^C72, a compensatory mutation to TCEIIIA that restores base pairing with three A to U substitutions; and TCEIIIGC/GC, which changes the alternating U-A and A-U base pairs in the distal region of stem-loop III to alternating G-C and G-C base pairs (Crucs et al., 2000).

View larger version (110K): [in a new window]   Fig. 2. GFP-Nos distribution during early to middle stages of oogenesis. (A-C) Fixed 6 x gfp-nos egg chambers, with GFP-Nos visualized directly and the actin cytoskeleton labeled red with rhodamine-phalloidin. (A) GFP-Nos (green) expression is strong in regions 1 and 2 of germarium (g, inset) and in the nurse cells during stages 5-8. In addition to forming cytoplasmic particles, GFP-Nos is enriched at the periphery of the nurse cell nuclei (arrows). GFP-Nos can also be detected in the oocyte during midoogenesis (arrowhead). (B) Stage 9 egg chamber with high levels of GFP-Nos in the nurse cells (nc) and perinuclear enrichment. Lower levels of GFP-Nos are present in the oocyte (oo). (C) Stage 10 egg chamber with loss of perinuclear GFP-Nos localization as nurse cells prepare for dumping. (D) High-magnification image of nurse cell nuclei from a stage 5 6 x gfp-nos egg chamber showing (i) DNA stained with Hoescht's dye; (ii) GFP-Nos; (iii) anti-Vas immunofluorescence; (iv) merge of i-iii. GFP-Nos colocalizes with Vas at the nuclear periphery. A similar distribution of GFP-Nos is observed in gfp-nos:tub3'UTR egg chambers at the stages shown in A-C.

View larger version (104K): [in a new window]   Fig. 3. GFP-Nos distribution at late stages of oogenesis. (A-C) Lower power images of the anterior region of fixed 6 x gfp-nos egg chambers used for staging. (D-F) Higher power images of the posterior region of egg chambers shown in A-C. (A,D) Stage 11 egg chamber, with transfer of GFP-Nos produced in the nurse cells to the oocyte during nurse cell dumping. (B,E) Stage 12 oocyte, with GFP-Nos distributed uniformly throughout the ooplasm. (C,F) Stage 13 oocyte, in which GFP-Nos has disappeared from the anterior and a gradient of GFP-Nos is observed emanating from the posterior pole. This gradient is also detected in 2 x gfp-nos oocytes (see Fig. S1 in the supplementary material). The actin cytoskeleton is labeled with rhodamine-phalloidin (red) in all panels.

View larger version (51K): [in a new window]   Fig. 4. Restriction of GFP-Nos to the posterior requires the nos 3'UTR. All images are confocal projections of fixed oocytes. The muscular sheath that encases the ovary is still attached in these preparations as revealed by staining with rhodamine-phalloidin (red). (A) Control oocyte lacking GFP-Nos. (B) Stage 13 6 x gfp-nos oocyte with GFP-Nos gradient emanating from the posterior pole. (C) Stage 13 gfp-nos:tub3'UTR oocyte displaying a uniform distribution of GFP-Nos.

View larger version (34K): [in a new window]   Fig. 5. Differential effect of TCE mutations on translational repression during oogenesis and embryogenesis. (A) Northern analysis of total RNA from nos-tub3'UTR (tub), nos-tub:TCE (TCE) or nos-tub:TCE mutant (TCEIIA, TCE[SRE–], TCEIIIA) ovaries. Transgene RNAs, detected with a nos probe, were normalized to the rp49 control to determine their relative abundance, indicated below. (B) Immunoblot analysis of HA-Nos protein in extracts of stage 14 egg chambers from wild-type (WT), nos-tub3'UTR (tub), nos-tub:TCE (TCE) or nos-tub:TCE mutant derivatives (TCEIIA, TCE[SRE–], TCEIIIA) using an anti-HA antibody. The antibody crossreacts with a protein that co-migrates with HA-Nos, as well as a more rapidly migrating protein in all samples. Snf protein was monitored as a loading control. In stage 14 oocytes, the wild-type TCE and stem-loop II mutants (TCEIIA, TCE[SRE–]) prevent accumulation of HA-Nos, whereas stem-loop III mutants (TCEIIIA and others shown in Fig. S2) do not. Analysis of total ovarian extract [TCE(total)] confirms that nos-tub:TCE RNA is expressed and translated at earlier stages of oogenesis. (C) Immunoblot analysis of HA-Nos protein in extracts of stage 10 (st10) egg chambers and stage 14 (st14) oocytes dissected from nos-tub:TCE (TCE) or wild-type ovaries. (D) Immunoblot analysis of HA-Nos protein in extracts of total ovary or stage 14 (st14) oocytes from nos-tub:TCE females mutant for smg [smg1/Df(ScfR6)]. (E) Immunoblot analysis of HA-Nos protein in extracts of embryos from the same transgenic and wild-type lines shown in B. HA-Nos protein is detected in early embryos for all of the TCE mutants.

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