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First published online 18 April 2007
doi: 10.1242/dev.02832

Development 134, 1955-1965 (2007)
Published by The Company of Biologists 2007
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A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte

Alejandra Clark*, Carine Meignin and Ilan Davis{dagger}

The Wellcome Trust Centre for Cell Biology, Michael Swann building, The University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.


Figure 1
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  		Fig. 1. Injected grk RNA moves from the nurse cells into the oocyte and localizes specifically, in the same way as the endogenous transcript. (A,C,E,G) grk RNA (A,C,E) and bcd RNA (G) localization at 15 minutes after injection; (A',C',E',G') at 60 minutes after injection. (B,D,F,H) In situ hybridization showing endogenous grk mRNA (B,D,F) and bcd mRNA (H) in equivalent-stage egg chambers (RNA in red, DNA in cyan). (A) Stage 7 (also see Movie 1 in the supplementary material); (C) young stage 8; (E,G) stage 9 (also see Movie 2 in the supplementary material) egg chambers. grk and bcd RNA injected into nurse cells is transported into the oocyte and localizes in the same way as the endogenous mRNAs. (A',E',G) Arrows indicate sites of apparent ring canal accumulation. (I) lacZ RNA (pink) was injected into the nurse cell, as shown by the outline of the egg chamber (grey). (I',I'') At 15 (I') and 60 (I'') minutes after injection. Injected lacZ RNA accumulates in the nurse cells, with little movement into the oocyte and no localization. In all panels, anterior is to the left and dorsal is to the top. Scale bars: 40 µm.

 

Figure 2
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  		Fig. 2. grk RNA particles specifically accumulate at apparent ring canals. (A,B) Trails of grk RNA (also see Movie 3 in the supplementary material) and lacZ RNA injections into wild-type nurse cells. Schematics on the left represent the site of injection (red outline) and show the average speed±standard error of the mean (S.E.), the number of egg chambers injected and the total number of particles tracked for analysis (n). Trails at 1 second intervals were superimposed (1 minute duration). Red arrows indicate the direction of RNA particle movement; broken lines show those particles which seem to follow nurse cell cytoplasmic streaming, and were thus not tracked for analysis. Anterior is to the left and dorsal is to the top. Scale bar: 5 µm.

 

Figure 3
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  		Fig. 3. grk RNA particles move in directed paths towards ring canals. (A,B) Injected and subsequently fixed egg chambers show that grk RNA (A) but not lacZ RNA (B) accumulates at the ring canal (arrows). (B) Arrowhead points to a ring canal itself. (C) Frames from a time-lapse movie of a nurse cell injected with grk RNA (red) in an Actin-GFP (green) egg chamber (also see Movie 4 in the supplementary material). Panels correspond to successive time points, at 5 second intervals. Arrows indicate two grk RNA particles moving on the same path towards a ring canal that is connected to the oocyte. (D) grk RNA (red) accumulation at the ring canal at approximately 3 minutes after injection. (E) grk RNA particle (red) moving through a ring canal shown as a trail, successive time-points (at 5 second intervals) being superimposed on each other. Notice the difference in distances between RNA particles moving through the ring canal (E) and those moving towards ring canals (C). At the bottom of the figure is a table showing the corresponding grk RNA particle statistics. In all panels, the oocyte is to the right. (C-E) Red is grk RNA; green is Actin-GFP. Scale bar: 5 µm.

 

Figure 4
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  		Fig. 4. Microtubules are required for grk RNA movement towards the ring canals and for transport into the oocyte. (A) Tau-GFP egg chambers show an enrichment of microtubules (MTs) at the ring canals (arrows). (A') Higher-magnification of ring canal in A. (B-E) Tau-GFP (green) living egg chambers injected with grk RNA (red) at 15 (B,D) and 60 (C,E) minutes after injection. (B,C) grk control; (D,E) grk-colcemid co-injection. (B',C',D',E') Only injected RNA is shown, at the corresponding time points. (F,G) Higher-magnification of boxed regions shown in B and D. Control (F) and Colcemid-treated (G) egg chambers at 30 minutes after injection with grk RNA. Notice how, in the absence of MTs, grk RNA particles fail to accumulate at the ring canals as they do in controls (F, arrowhead). In all panels, anterior is to the left and dorsal is to the top. Scale bar: 5 µm.

 

Figure 5
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  		Fig. 5. Dynein is required for the efficient transport of grk RNA into the oocyte and for its localization. (A-D) Wild type (A,B; wt) and dhc mutant (C,D; dhc) stage-8 egg chambers injected with grk RNA (pink) in the nurse cells. Notice the decrease in grk RNA transport and localization in the oocyte in dhc mutants (compare B' with D') (also see Movie 5 in the supplementary material). (A-D) Grey is brightfield illumination showing the outline of the nuclei and oocyte. Arrowheads indicate the position of the oocyte nucleus. (A',B',C',D') Injected grk RNA only. (E-L) grk (E-H) and osk (I-L) mRNA in situ hybridization in wild-type (E,F and I,J; wt) and dhc mutant (G,H and K,L; dhc) egg chambers. (E,G,I,K) Stage 3-4 egg chambers; (F,H) stage 6 egg chambers; (J,L) stage 11A egg chambers. Notice the reduction in grk and osk mRNA signal and localization in the oocyte of dhc mutants (arrowheads; compare E' with G' and F' with H' for grk mRNA, and I' with K' for osk mRNA). (L) In late-stage egg chambers, cytoplasmic foci of osk mRNA on the periphery of dhc mutant egg chambers is observed (arrows). (E-L) Cyan is DNA; red is mRNA; black and white panels show mRNA only. In all panels, anterior is to the left and dorsal is to the top.

 

Figure 6
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  		Fig. 6. Dynein is required for the efficient transport of grk RNA particles towards ring canals. (A,B) Trails of grk RNA injected into wild-type (A; wt) and dhc mutant (B) nurse cells. Schematics on the left represent the site of injection (red outline) and show the average speed±standard error of the mean, the number of egg chambers injected and the total number of particles tracked for analysis. Trails at 1 second intervals were superimposed (0.5 minute duration). Red arrows indicate the direction of RNA-particle movement; broken lines show those particles that seem to follow nurse cell cytoplasmic streaming, and were thus not tracked for analysis. A decrease in the speed of grk RNA particles in dhc mutants is observed by the differences in distance travelled (*) (also see Movie 6 in the supplementary material). Anterior is to the left and dorsal is to the top. Scale bar: 5 µm in B for A,B.

 

Figure 7
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  		Fig. 7. BicD protein is recruited by grk and bcd RNA and is necessary for grk RNA accumulation at ring canals. (A-D) Stage 8-9 egg chambers injected with RNA (red); BicD protein (green) and DNA (blue) are also shown (acquired at 20x). Outlined region represents the area shown (acquired at 100x) in the panels to the right (RNA, BicD and merged). (A-A''') Uninjected egg chamber showing the BicD localization pattern around the oocyte nucleus. (B-C''') Injected grk RNA (B') and bcd RNA (C') colocalize (arrows) with BicD (B'' and C'', respectively). (D-D''') Injected lacZ RNA forms particles that do not accumulate (D') and that do not colocalize with BicD (D''). (E,H) Immunofluorescence showing BicD localization in wild-type controls (E) and BicDmom (H) egg chambers. Notice the absence of BicD protein in BicDmom egg chambers (H). (F,G,I,J) Wild-type control (F,G) and BicDmom (I,J) egg chambers injected with grk RNA at 15 (F,I) and 60 (G,J) minutes after injection. In the absence (H-J) of BicD protein, grk RNA ring canal accumulation (arrow) and localization in the oocyte is strongly reduced. In all panels, anterior is to the left and dorsal is to the top (except D, dorsal side where the oocyte nucleus is).

 




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