Figure 1.
Synthesis of GFP-Tud fusion proteins in Drosophila ovaries.
(A) Representation of the Tud protein with Tudor (1–8) and Tudor-like (1′-2′) domains depicted in purple. Fragments of Tud [33] used to design the transgenes are indicated below the map. (B) (Upper panel) Western blot analysis of GFP-Tud fusion proteins synthesized in ovaries of transgenic females using anti-GFP antibodies followed by alkaline phosphatase-conjugated antibodies. (Lower panel) The blot was then probed for ribosomal P40, as loading control.
Figure 2.
Spatial distribution of GFP-Tud fusion proteins during oogenesis.
GFP-Tud proteins were detected in fixed egg chambers. Panels of the left display previtellogenic egg chambers whereas panels on the right exhibit vitellogenic egg chambers. (A) The GFP-Tud-JOZ fusion protein accumulated in nurse cell and oocyte nuclei. In a stage 10 egg chamber no localization of this protein could be detected at the posterior pole of the oocyte. (B) The GFP-Tud-9A1 fusion protein could be found in the nuage and dispersed in punctuate structures in the cytoplasm. In stage 10 egg chamber the fusion protein accumulated at the posterior pole of the oocyte. (C) The GFP-Tud-3ZS+L fusion protein was present in both nuage and oocyte of previtellogenic egg chambers and localized in the pole plasm of a late stage 9 egg chamber. (D–E). Both GFP-Tud- 3ZS+L-N and GFP-Tud-3ZS+L-C fusion proteins corresponding to the N- and C-moieties of GFP-Tud-3ZS+L, respectively, displayed a pattern of distribution in both nuage and pole plasm similar to the original GFP-Tud- 3ZS+L fusion protein with the exception that the staining intensity was lower and that the pole plasm accumulation of the N-terminal moiety was significantly reduced.
Figure 3.
Spatial distribution of GFP-Tud fusion proteins during early embryogenesis.
GFP-Tud proteins were detected in fixed 0-2 hour embryos produced by females synthesizing the GFP-Tud-9A1 (A) or GFP-Tud-3ZS+L (B) fusion proteins. Although both fusion proteins could be detected at the posterior pole of the early embryo, the pole plasm localization of GFP-Tud-9A1 was significantly reduced compared to that of GFP-Tud-3ZS+L.
Figure 4.
A C-terminal segment encompasses the Tud function.
(A) Wild-type embryos at the syncytial blastoderm stage, or corresponding embryos derived from (B) homozygous tud1 females and tud1 females expressing (C) GFP-Tud-9A1, (D) GFP-Tud-3ZS+L, and (E) GFP-Tud-3ZS+L-C transgenes. Vas (red) and DNA (green). Only the GFP-Tud-3ZS+L transgene can restore pole cell formation in tud1 embryos.
Figure 5.
Recruitment of GFP-Tud3ZS+L by short Osk protein.
Distribution of Osk and GFP-Tud-3ZS+L in (A) wild-type and (B) UAS-osk-M1R; nosGal4 stage 9 egg chambers. Osk protein and GFP-Tud-3ZS+L co-localized only in pole plasm. Overexpression of the short form of Osk during oogenesis led to ectopic accumulation of Osk and GFP-Tud-3ZS+L (Upper panels) at the anterior pole of the oocyte and (Lower panels) in cytoplasmic particles in the nurse cells.
Figure 6.
Immunoblot detection of Tud protein during oogenesis and early embryogenesis.
Protein extracts from ovaries and early embryos were run on 6% SDS-polyacrylamide gels, transferred to an Immobilon-P membrane and probed with anti-Tud antibodies (TUD65). The relative molecular masses of the marker protein bands are indicated on the left.