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First published online 27 July 2004
doi: 10.1242/dev.01286

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Interaction with eIF5B is essential for Vasa function during development

Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montréal, Québec H3A 1B1, Canada

View larger version (20K): [in a new window]   Fig. 1. The Vas C terminus is required for eIF5B interaction. (A) Schematic of Vas, showing motifs characteristic of DEAD-box proteins. (B) Deletions in Vas were tested against eIF5B for direct interaction in the yeast two-hybrid system, using a plate-based assay for ß-galactosidase activity. Four replicates were tested for each sample, and were compared to a positive control (full-length Vas, 1-661) and a negative control (Vas 1-310). Interaction with eIF5B is indicated by +, and absence of interaction by -. (C) Further deletions in Vas, ending between residues 595 and 621, were tested against eIF5B in the same system. (D) The region surrounding residues V616, P617 and D618 of Vas (indicated by a box), was compared with homologous proteins in other species, and with more distantly related Drosophila DEAD-box proteins (eIF4A, Ddx1 and Dhh1). Residues that are identical between homologous proteins are shown in red.

View larger version (16K): [in a new window]   Fig. 2. Deletions within residues 616-618 of Vas impair eIF5B-interaction. (A) Specific Vas deletions, which were transformed into yeast strain EGY48, were tested for their level of protein expression in yeast on a western blot probed with anti-Vas. As a loading control, the blot was also probed with anti-Actin (ICN Biomedicals). (B) Vas deletions were tested for interaction with eIF5B in the yeast two-hybrid system using a liquid assay for ß-galactosidase activity. The positive control for interaction is the full-length Vas protein (1-661). The value shown for each sample represents the average of three replicates, and the error bars represent s.d. (C) Vas deletions were also tested for interaction with Osk, using the same system.

View larger version (42K): [in a new window]   Fig. 3. Localization and expression of transgenic GFP-fusion proteins. For each transgenic genotype, localization of GFP is demonstrated in early and late stages of oogenesis: (A,B) vas+; (D,E) vas616-618; (G,H) vas616; (J,K) vas617; (M,N) vas618. Protein expression in ovaries of transgenic flies is demonstrated on western blots probed with anti-Vas. (C) Vas+; (F) Vas616-618; (I) Vas616; (L) Vas617; (O) Vas618. Flies contained two copies of each transgene in the wild-type background. For each sample, the faster migrating band represents endogenous Vas and the slower migrating band represents the transgenic GFP-Vas.

View larger version (59K): [in a new window]   Fig. 4. Vas-eIF5B interaction is required for the progression of oogenesis. (A) Western blot of ovarian extracts from wild-type (OreR), vasPH165, vasPH165;P{vas+} and vasPH165;P{vas617} females was probed with anti-Vas. The same blot was probed with anti-4E-BP as a loading control. (B) Ovaries from vas PH165 females produce few mature stage 14 eggs owing to a developmental arrest during oogenesis. (C) Expression of a wild-type vas+ transgene rescues this defect of the vasPH165 mutation, allowing the production of abundant stage14 eggs. (D) Ovaries from vasPH165;P{vas617} females resemble those of vasPH165, exhibiting a similar developmental arrest during oogenesis and producing few mature eggs.

View larger version (69K): [in a new window]   Fig. 5. Vas-eIF5B interaction is important for the establishment of dorsoventral polarity in the egg, and for grk regulation. Dark-field photographs of dorsal appendage phenotypes (A-F). Eighty-two percent of vasPH165;P{vas+} eggs have two dorsal appendages (A). Dorsal appendages in vasPH165;P{vas617} eggs reveal a range of phenotypes: 22% have two dorsal appendages (B), 67% exhibit one semi-fused or fully fused dorsal appendage (C-E) and 11% have no dorsal appendages (F). (G-L) grk RNA and Grk protein were visualized in the early stages of oogenesis, through in situ hybridization and immunostaining. (J-L) Grk protein is shown in red; DAPI staining of DNA is shown in blue. (G,J) In vasPH165;P{vas+} ovaries, both RNA and protein are enriched in the developing oocyte. (H-L) In both vasPH165 and vasPH165;P{vas617} ovaries, grk RNA is enriched in the oocyte (H,I); however, Grk protein is barely detectable (K,L). Grk protein was examined in single confocal sections using the same laser settings for each genotype.

View larger version (32K): [in a new window]   Fig. 6. Pole plasm assembly in the presence of reduced Vas-eIF5B interaction. (A) Western blot of ovarian extracts from wild-type (OreR), vasPD, vasPD;P{vas+} and vasPD;P{vas617} females was probed with anti-Vas. The same blot was probed with anti--Tubulin as a loading control. (B-M) Stage 10 ovaries from OreR, vasPD, vasPD;P{vas+} and vasPD;P{vas617} females were labeled for Osk (B-E), Vas (F-I) and Tud (J-M) by immunostaining.

View larger version (71K): [in a new window]   Fig. 7. Vas-eIF5B interaction is vital for pole cell formation. Pole cells were visualized at the cellular blastoderm stage using either GFP-Vas or immunostaining for Nos protein. The pictures shown are representative for each genotype. (A,B) Using GFP-Vas as a marker in live embryos, pole cells could be detected at the posterior of the majority of vasPD;P{vas+} embryos (A), but in fewer than 1% of vasPD;P{vas617} embryos (B). (C) Using immunostaining for Nos as a marker for pole cells, Nos-positive cells could be detected at the posterior in 58% of vasPD;P{vas+} embryos. (D,E) In 81% of vasPD;P{vas617} embryos, no Nos-positive cells were apparent at the same stage (D), whereas in 5%, one to three Nos-positive cells could be detected at the posterior (E). (F) In 14% of vasPD;P{vas617} embryos, several Nos-positive cells were detected but were positioned elsewhere within the embryo.

View larger version (78K): [in a new window]   Fig. 8. Reduced Vas-eIF5B binding does not abrogate somatic patterning or Nos deployment. RNA in situ hybridization for ftz was used as an indicator of somatic segmentation at the cellular blastoderm stage. (A) Fifty percent of vasPD;P{vas617} embryos revealed a normal ftz distribution of seven transverse stripes. (B,C) In 20% of vasPD;P{vas617} embryos, stripes 4-6 were weaker and less defined than the others (B), and in 30%, more severe defects such as deletions and fusions of these segments were apparent (C). (D) Nos protein, visualized through immunostaining in 0- to 2-hour-old embryos, was present at the posterior of 95% of vasPD;P{vas+} embryos. (E,F) Posterior Nos was detectable in 55% of the vasPD;P{vas617} embryos (E), and undetectable in the remaining 45% (F).