QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vanzo, N. F. Articles by Ephrussi, A. Search for Related Content PubMed PubMed Citation Articles by Vanzo, N. F. Articles by Ephrussi, A. Social Bookmarking                         What's this?

Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1 – 69117 Heidelberg, Germany

View larger version (83K): [in a new window]   Fig. 1. osk mRNA maintenance at the posterior pole of the oocyte requires Long Osk. (A) osk mRNA in situ hybridization to whole-mount ovaries (left panels) and freshly laid eggs (right panels; anterior is towards the left and dorsal towards the top) of osk RNA-null females oskA87/Df(3R)p-XT103, expressing either a wild-type osk transgene (upper panels), oskM1R encoding Short Osk (middle panels) or oskM139L encoding Long Osk (lower panels). The arrow indicates residual osk mRNA in embryos produced by females expressing Short Osk alone. (B) Comparison of the delocalization pattern of osk mRNA in stage 10 oocytes of the osk nonsense mutant osk84/Df(3R)p-XT10 (upper panel) and of the RNA-null mutant oskA87/Df(3R)p-XT103 expressing oskM1R. (C) Western blot analysis of Osk isoforms produced in ovaries shown in A, and of -Tubulin (an internal loading control). Oregon R is the wild-type reference strain.

View larger version (57K): [in a new window]   Fig. 2. Long Osk, but not Short Osk, can anchor at the posterior pole of the oocyte. (A-E) Confocal images of Osk isoforms detected by immunostaining of osk RNA-null ovaries oskA87/Df(3R)p-XT103, expressing either a wild-type osk transgene (upper panels), oskM1R encoding Short Osk (middle panels) or oskM139L encoding Long Osk (lower panels). The rabbit anti-Osk polyclonal serum used in these experiments detects both Osk isoforms. During late stage 10, Long Osk remains tightly anchored at the posterior pole in a crescent-shaped pattern (bottom panels), as observed when both isoforms are expressed (top panels). At the same stage, Short Osk shows either a ‘dotty’ localization close to the posterior cortex (3%) or is strongly detached in aggregated particles that disperse in the bulk ooplasm (97%) (middle panels). (B) Magnification of the posterior tip of the oldest egg chamber shown in A. (C-E) Co-localization of pole plasm components at the posterior tip of stage 10 egg chambers. (C) Merged confocal images of osk mRNA (green) and Osk protein (red) simultaneously detected by in situ hybridization and antibody staining. Merged confocal images of immunohistochemically detected Staufen (red) and Osk (green) (D), and of Oskar (green) and Vasa (red) (E).

View larger version (33K): [in a new window]   Fig. 3. Long Osk anchors both osk RNA and Short Osk at the posterior pole of the oocyte. (A,B) osk mRNA and (C,D) Osk isoforms in ovaries of osk RNA-null females oskA87/Df(3R)p-XT103, expressing only oskM1R (A,C) or both oskM1R and oskM139L (B,D). Co-expression of Short Osk and Long Osk (B,D) suppresses the granular detachment of both osk mRNA and Short Osk observed when Short Osk is expressed alone (A,C). Note that mRNA detection in A,B is colorimetric, rather than fluorescence based, as shown in Fig. 1A. (E) Long Osk expression promotes Short Osk accumulation during oogenesis. Quantification of Short Osk in ovary extracts from two independent lines (noted 1 and 2) expressing oskM1R alone or together with oskM139L. A significantly greater amount of Short Osk, both phosphorylated and unphosphorylated, accumulates in the presence of Long Osk.

View larger version (65K): [in a new window]   Fig. 4. Proper patterning requires the combinatorial activity of Long Osk and Short Osk. (A) Hatching rate of embryos and cuticle analysis of unhatchers with patterning defects produced by osk RNA-null females oskA87/Df(3R)p-XT103, expressing the indicated transgenes. Number of eggs scored to calculate hatch rates are osk wild type (n=90); oskM1R [n=125 (line 1) and 49 (line 2)]; oskM1R+oskM139L [n=561 (line 1) and 147 (line 2)]; oskM139L (n>300). [(1) and (2) refer to the lines analyzed by western blotting in Fig. 3E.] *The remaining 12% of unhatchers develop cuticles without obvious patterning defects. (B) Posterior defects (left panel), anterior defects (middle panel), and both anterior and posterior defects (right panel) observed in progeny of females expressing only oskM1R. None of these defects is detected when oskM1R and oskM139L are co-expressed (C). Anterior is towards the top and ventral is towards the left.

View larger version (41K): [in a new window]   Fig. 5. Long and Short Osk are required for efficient pole cell formation. (A-C) Vasa immunostaining reveals pole cells in embryos produced by oskA87/Df(3R)p-XT103 females expressing a wild-type osk transgene (A), the two transgenes oskM1R and oskM139L (B) or only oskM1R (C). Compared with embryos produced by females expressing both Osk isoforms (A,B), a significant reduction in pole cell number is observed in embryos produced by females expressing only Short Osk (C). (D) Fertility of the adult female progeny was evaluated by ovary dissection. Note that the two independent lines expressing oskM1R or both oskM1R and oskM139L were subjected to Osk western blotting analysis [denoted as (1) and (2) in Fig. 3E]. The number of ovaries dissected in each case was oskWT (n=41), oskM1R [n=54 (line 1) and 51 (line 2)], oskM1R and oskM139L [n=52 (1) and 61 (2)].

View larger version (38K): [in a new window]   Fig. 6. Ectopic anchoring of Osk to the entire oocyte cortex requires Long Osk. Ectopic overexpression of the two Osk isoforms (A) and Short Osk alone (B) from the UAS-osk-K10 3'UTR and UAS-oskM1R-K10 3'UTR transgenes, respectively, in wild-type ovaries. When both isoforms are expressed (A), Osk is readily detected along the entire cortex of oocytes of stages 7/8 to 10. When only Short Osk is expressed (B), no cortical staining is observed other than at the posterior pole, where endogenous Osk (Long and Short) is expressed and localized. In both cases, the brightness of the immunostaining in the subcortical region of the nurse cells reveals the massive overproduction of Osk isoforms. (C) Analysis of overexpressed Osk isoforms produced from the transgenic ovaries by western blotting.

View larger version (49K): [in a new window]   Fig. 7. The N-terminal extension of Long Osk is not sufficient for posterior anchoring. ß-Galactosidase staining of wild-type (A,B) and osk84/Df(3R)pXT103 ovaries (C,D) expressing the m1414lacwt transgene, which encodes a translational fusion of the first 139 amino acids of Long Osk to ß-galactosidase (Gunkel et al., 1998). The chimeric protein is detected at the posterior pole of wild-type oocytes during stage 10 onwards (A,B), whereas in the absence of endogenous Osk activity, the fusion delocalizes during stage 10 (C,D).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter