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First published online March 1, 2004
doi: 10.1242/10.1242/dev.01034

Development 131, 1401-1410 (2004)
Published by The Company of Biologists 2004
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PKA-R1 spatially restricts Oskar expression for Drosophila embryonic patterning

Shoko Yoshida1,*, H-Arno J. Müller2, Andreas Wodarz2 and Anne Ephrussi1,{dagger}

1 European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
2 Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany



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  		Fig. 1. The 18304 locus is required for the control of oskar activity in anteroposterior patterning of the embryo. (A-C) Cuticle preparations of a wild-type embryo (A) and embryos derived from 18304 germline clones (B,C), which show patterning defects ranging from deletion of the head (B), to complete mirror-image duplication of posterior structures (abdominal segments and filzkörper material; C). (D) Embryo derived from 18304, oskar double-mutant mother. Such embryos completely lack posterior structures and show the oskar single-mutant phenotype.

 


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  		Fig. 2. Oskar protein is ectopically expressed in the 18304 mutant. (A,E) oskar RNA localization in stage 10 wild-type and 18304 hemizygote mutant egg chambers revealed by in situ hybridization. oskar RNA localizes correctly at the posterior pole in wild-type (A) and 18304 mutant (E) oocytes. (B-D,F-H) Anti-Oskar immunostaining of wild-type and 18304 hemizygous egg chambers and embryos. (B,F) Stage 6 egg chambers of wild-type (B) and 18304 hemizygous (F) females. In the wild type, Oskar protein is not expressed before posterior localization of the RNA and is not detected at this stage (B), whereas it accumulates prematurely (arrow) in 18304 hemizygous oocytes (F). (C,G) Stage 10 egg chambers of wild type (C) and 18304 hemizygotes (G). In the wild type, Oskar protein is detected as a tight crescent at the oocyte posterior pole (C), whereas, in the 18304 mutant, it is observed all around the cortex of nurse cells and the oocyte (arrows), as well as at the posterior pole (G). The signal observed in the nuclei of wild-type and 18304 follicle cells, and in nurse cells is also observed in oskar protein-null egg-chambers (data not shown), indicating that it is background. (D,H) 0-2 hour-old embryo of wild type (D) and 18304 hemizygotes (H). In wild-type embryos, Oskar protein is maintained at the posterior pole as a tight crescent (D). In 18304 mutant embryos, Oskar protein is mislocalized all around the cortex, with a higher accumulation at the posterior pole (H). (I) Northern blot analysis of wild-type and 18304 hemizygote ovarian poly(A)+ RNA, probed with an oskar cDNA probe (top panel). No difference is detected in oskar RNA expression levels between wild type and the 18304 mutant. The blot was re-probed with rp49 cDNA as a loading control (bottom panel). (J) Western blot analysis of protein extracts of wild-type and 18304 mutant ovaries. Probing with anti-Oskar antiserum reveals that both isoforms of Oskar protein are expressed at higher levels in 18304 than in wild type (top and middle panels). The blot was re-probed with an anti-{alpha}-Tubulin antibody as a loading control (bottom panel).

 


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  		Fig. 3. The 18304 locus encodes Drosophila Pka-R1. (A) Diagram of the genomic region defined by the overlap of two deficiencies that fail to complement the 18304 maternal-effect phenotype. The position of the proximal breakpoint of Df(3L)ri-XT106 and the distal breakpoint of Df(3L)ME107 (shown in brackets) were identified by RFLPs specific for the 18304 chromosome (see Materials and methods). Six gene candidates for the 18304 locus map to this region: Pka-R1 (four alternative transcripts are shown), CG3288, CG13255, CSN3, CG11456 and CG32432 (Berkeley Drosophila Genome Project). (B) Domain organization of Drosophila PKA regulatory subunit type 1. One isoform (RA) comprises a dimerization domain at the N terminus, followed by an inhibitory domain and two cAMP-binding domains. The other two isoforms (RB and RD) lack the dimerization domain. (C) Western blot analysis using human PKA-R1ß antibody to reveal the expression of PKA-R1 in the Drosophila ovary. A single band of 50 kD is detected in ovarian extracts and corresponds to the size of the RA isoform. (D) Alignment of D. melanogaster, H. sapiens, M. musculus and C. elegans PKA-R1. In 18304, a conserved arginine residue in the inhibitory domain is mutated to glutamine and, in E1, a conserved glycine residue is mutated to aspartic acid. (E-G) Subcellular localization of PKA-R1 protein during oogenesis revealed by anti-PKA-R1 immunostaining (E). PKA-R1 is detected in the cytoplasm, with an accumulation at the cell membrane. (F) Rhodamine-conjugated phalloidin reveals the subcortical actin cytoskeleton in the oocyte, nurse cells and follicle cells. (G) Merged image of E and F showing co-localization of PKA-R1 and actin at the cell cortex.

 


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  		Fig. 4. PKA catalytic activity is increased in Pka-R118304. PKA activity in extracts of Pka-R118304/Df(3L)ri-XT106 adult flies was measured in the absence or in the presence of 5 µM cAMP. In the absence of exogenous cAMP, Pka-R118304 mutant extracts show an approximately 1.2-fold increase in PKA activity over wild type (left panel). In the presence of saturating concentrations of cAMP, mutant extracts show a 1.5- to 2-fold elevation in PKA activity over the wild type, revealing an excess of PKA catalytic subunit activity in the Pka-R118304 mutant (right panel). This is a summary of assays performed independently three times; bars indicate s.e.m. The differences in PKA activities between wild type and mutant in the presence or absence of exogenous cAMP are statistically significant as determined by a two-sample t-test (P<0.001).

 


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  		Fig. 5. Mutations in the Drosophila PKA catalytic subunit DC0 suppress the Pka-R118304 maternal-effect bicaudal phenotype. The patterning defects of embryos derived from CyO/+; Pka-R118304/Pka-R1E1 (Pka-R1 single mutant) and DC0E95/+; Pka-R118304/Pka-R1E1 (Pka-R118304 mutant lacking one wild-type copy of DC0) mothers were evaluated by cuticle analysis. In the Pka-R1 single mutant, only 18% of the embryos appear wild type, and 65% of the embryos display a phenotype reflecting ectopic posterior patterning activity (29% head deletion, 36% bicaudal). Removal of one wild-type copy of DC0 completely suppresses this phenotype, and 88% percent of the embryos display a wild-type cuticle pattern. A similar suppression was observed when the DCOH2 allele was tested.

 


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  		Fig. 6. Overexpression of the RA isoform of PKA-R1 causes a reduction in PKA activity and Oskar protein accumulation. (A) Wild-type adult wing. (B) An ectopic wing outgrowth, which is observed when the RA isoform is overexpressed ubiquitously using an actinGAL4 driver. The penetrance of this phenotype is about 30%. (C,D) oskar RNA localization in stage 10 wild-type egg-chambers and in egg chambers in which RA was overexpressed using the actinGAL4 driver, revealed by in situ hybridization. oskar RNA localizes correctly at the posterior pole in wild type (C) and in RA-overexpressing oocytes (D). (E) Northern blot analysis of wild-type and 18304 hemizygote ovarian poly(A)+ RNA probed with an oskar cDNA probe (top panel). The blot was re-probed with rp49 cDNA as a loading control (bottom panel), revealing that there was no difference in oskar RNA expression levels between wild-type and RA-overexpressing ovaries. (F) Western blot analysis of protein extracts of wild-type and RA-overexpressing ovaries (actinGAL4 and nanosGAL4VP16), probed with anti-Oskar antiserum. Both isoforms of Oskar protein are expressed at significantly lower levels in the extracts of ovaries in which RA is overexpressed (top). The blot was re-probed with anti-{alpha}-Tubulin antibody as a loading control (bottom).

 

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© The Company of Biologists Ltd 2004