DEV02832 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables 1-2 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Injected grk, K10 and I factor RNAs accumulate at the ring canals and are transported into the oocyte where they localise. (A-C) Tau-GFP (green) egg chambers injected with (A) grk, (B) K10, (C) I factor RNAs. Black and white panels show RNA at corresponding time points. All three RNAs accumulate at the ring canals (A′′-C′′) and localise in the oocyte (A′′′-C′′′).

• Supplemental Figure S2 -

  Fig. S2. The microtubule distribution in dhc mutants is indistinguishable from that of wild-type controls. (A,B) Panels showing the distribution of microtubules (Tau-GFP) at the ring canals in (A) wild type and (B) dhc mutants. Red dotted line separates nurse cell cytoplasm (left) from oocyte (oo, right). Scale bar: 5 μm.

• Supplemental Figure S3 -

  Fig. S3. Dynein is required for the efficient transport of bcd RNA into the oocyte and localisation. (A,B) bcd mRNA in situ hybridisation in wild-type (A) and dhc mutant (B) stage 10 egg chambers. (A′,B′) bcd mRNA only. Note that like in the case of osk, bcd mRNA accumulates in the nurse cell cytoplasm (not in the nuclei) of dhc mutant egg chambers.

• Supplemental Figure S4 -

  Fig. S4. grk RNA-particle movements are distinct from the general flow of cytoplasm from nurse cell to oocyte. (A,B) DIC images of stage 8 egg chamber showing cytoplasmic movement near the ring canal (A). (B) Trails of time lapse movie showing the direction of cytoplasmic movement as indicated by red arrows. Note the absence of bulk cytoplasmic flow towards the ring canal connecting to the oocyte (oocyte is to the right).

• Supplemental Figure S5 -

  Fig. S5. Model describing a four-step mechanism of delivery of grk mRNA from the nurse cell cytoplasm to its final destination in the dorso-anterior corner of the oocyte. Dynein- and BicD-dependent transport of grk RNA particles along a specific network of microtubules leads to a rapid accumulation of grk mRNA in front of the ring canal. Transport through the ring canal is rate limiting and may involve a different mechanisms. Once in the oocyte, grk RNA is transported by Dynein in two further steps to the dorso-anterior corner (as previously shown), where it is anchored.

• Movie 1 -

  Movie 1. Injected grk RNA is transported into the oocyte and localizes like the endogenous transcript. Stage 7 wild-type egg chamber whose nurse cell has been injected with grk RNA (20×). Movie starts 10 minutes after injection and lasts for 1 hour with a time-lapse interval of 1 minute. grk RNA moves toward the ring canal into the oocyte, localizing first anteriorly in the oocyte and then posteriorly, forming a crescent by the oocyte nucleus.

• Movie 2 -

  Movie 2. Injected grk RNA is transported into the oocyte and localizes like the endogenous transcript. Stage 9 wild-type egg chamber whose nurse cell has been injected with grk RNA (20×). Movie starts 10 minutes after injection and lasts for 1 hour with a time-lapse interval of 1 minute. grk RNA moves towards the ring canal into the oocyte and localizes anteriorly by the oocyte nucleus (dorsal side is down).

• Movie 3 -

  Movie 3. grk RNA particles move towards and accumulate near the ring canals. Stage 8 wild-type egg chamber whose nurse cell has been injected with grk RNA (100×). Movie starts approximately 3 minutes after injection and lasts for 3 minutes with a time-lapse interval of 1 second. (Oocyte is facing up.) grk RNA particles move in directed paths towards the ring canals, accumulating in front of the ring canals. Some RNA particles move into the oocyte. Note the difference in speed of RNA particles moving towards and through the ring canal. Upon entry into the oocyte, some RNA particles remain static, whereas most move into other focal planes and cannot be followed.

• Movie 4 -

  Movie 4. grk RNA particles move towards and through the ring canal. Stage 8 Act-GFP (green) egg chamber injected in the nurse cell with grk RNA (red) (100×). Movie starts approximately 2 minutes after injection and lasts for 3 minutes, with a time-lapse interval of 5 seconds. (The oocyte is pointing up.) grk RNA particles move towards the ring canals and accumulate in front of the ring canals. A few RNA particles move into the oocyte.

• Movie 5 -

  Movie 5. grk RNA transport into the oocyte (stage 7) requires Dynein. Stage 7 dhc mutant egg chamber whose nurse cell has been injected with grk RNA (20×). Movie starts 10 minutes after injection and lasts for 1 hour with a time-lapse interval of 1 minute. grk RNA movement into the oocyte is inefficient in dhc mutant egg chambers (compare with Movie 1).

• Movie 6 -

  Movie 6. grk RNA transport into the oocyte (stage 9) requires Dynein. Stage 9 dhc mutant egg chamber whose nurse cell has been injected with grk RNA (100×). Movie starts 3 minutes after injection and lasts for 3 minutes, with a time-lapse interval of 1 second. (The oocyte is pointing up.) grk RNA-particle movement is impaired in dhc mutants; no ring canal accumulation is evident within the imaging period (compare with Movie 3).