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First published online November 11, 2004
doi: 10.1242/10.1242/dev.01515

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Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia

Veit Riechmann^* and Anne Ephrussi

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (35K): [in a new window]   Fig. 1. Par-1 phosphorylates Exu in vitro and in vivo. (A) In vitro translated and 35S radiolabelled Exu protein subjected to SDS-PAGE and autoradiography. Exu protein incubated in kinase buffer alone migrates as three bands consisting of a major band at 60 kDa and two weaker protein bands that migrate more slowly (lane 2). As phosphorylation usually reduces the electrophoretic mobility of a protein, the presence of the two weak bands of Exu suggests that the protein is phosphorylated by kinases present in the in vitro translation extract. The absence of the slowly migrating protein bands after 2 hours treatment with lambda phosphatase shows that Exu is phosphorylated (lane 3). Phosphorylation is dramatically increased after incubation with Par-1 kinase, as revealed by the absence of the 60 kDa fast migrating band and the appearance of a slowly migrating protein doublet at 66 kDa (lane 1). (B) Western blot of ovarian extracts of females of the indicated genotypes probed with an antibody against Exu (upper panel) or Tubulin (lower panel) as a loading control. par-1W3 is a null allele, and par-19A and par-1574 are hypomorphic alleles. par-1 overexpression in ovaries was achieved by placing par-1 under the control of the Gal4 UAS system and expressing a UAS-par-1 transgene in the germline using the nanosGal4:VP16 driver.

View larger version (35K): [in a new window]   Fig. 4. Mapping and mutation of Exu phosphorylation sites. (A) Position of the two potential 14-3-3 binding sites (red) in Exu protein (grey bar). Sequences of site A (amino acids 434-440) and B (amino acids 453-459) and of the corresponding mutations (A1-A6 and B1-B3) are shown below. (B-D) Autoradiographic images of SDS polyacrylamide gels showing in vitro translated and radiolabelled wild-type or mutant Exu proteins after incubation with Par-1. Mutations in site A and B are indicated above the lane. (E) Western blot of ovarian extracts probed with an antibody against Exu (upper panel) or Tubulin (lower panel) as a loading control. Exu protein was expressed from transgenes using the endogenous exu promoter in an exu-null background (exuXL/exuVL). Transgenes encode either wild-type or mutant Exu protein. The nature of the mutation is indicated above the corresponding lane. The first lane shows extracts from exu null mutant ovaries expressing Exu-GFP protein, in which all Par-1 phosphorylation sites are mutated (GFP A6B3). This mutant protein runs in a single band, indicating absence of phosphorylation. The second lane shows extracts from wild-type ovaries expressing Exu-GFP. Exu-GFP is phosphorylated and migrates as a triplet around 100 kDa while untagged Exu migrates around 66 kDa.

View larger version (42K): [in a new window]   Fig. 2. exu and par-1 are required for bicoid mRNA localisation. (A-C) In situ hybridisation showing bicoid mRNA localisation in entire egg chambers, by projection of eight confocal sections. (D-I) Single confocal sections in the middle of the oocyte showing bicoid mRNA (red) and DNA (green) at stage 9 (D-F) and 10b (G-I) of oogenesis. Anterior is leftwards, and posterior is rightwards. Genotypes are indicated. All females were grown at 18°C.

View larger version (124K): [in a new window]   Fig. 3. Par-1, Exu-GFP and bicoid mRNA localise to patches in the nurse cells. (A-C) Confocal section in the apical region of the nurse cells of egg chambers expressing Exu-GFP. (A) Par-1 protein is visualised with an anti-Par-1 antibody (red) and Exu-GFP with an antibody against GFP in the wild type. Both proteins accumulate in patches and colocalise almost throughout the whole nurse cell cytoplasm. Par-1 is also detected at the membranes of the nurse cells. (B) Double staining for Exu-GFP (green) detected with antibody against GFP and bicoid mRNA (red) in the wild type. (C) exu null mutants (exuXL/exuVL) expressing Exu-GFP protein in which all phosphorylation sites are mutated (Exu-GFPA+B) double stained for GFP (green) and bicoid mRNA (red). (D,E) Confocal sections through the middle of a wild type egg chamber expressing wild-type Exu-GFP (D), and an exu null mutant egg chamber expressing Exu-GFPA+B (E). Exu-GFPs are visualised with fluorescence of the GFP.

View larger version (58K): [in a new window]   Fig. 5. Exu phosphorylation is required for proper formation of the Bicoid protein gradient. (A-C) Confocal sections through the middle of early cleavage stage embryos hybridised with a bicoid RNA probe. (A) In all wild-type embryos (n=20), bicoid mRNA was localised anteriorly. (B) In embryos from females expressing the exuA+B transgene in an exu-null mutant background, only 70% of the embryos (n=32) show anteriorly localised bicoid mRNA, while the other 30% show no anterior signal. (C) In embryos from exu-null mutants (exuXL/exuVL) all embryos (n=19) lack anterior bicoid mRNA. (D-F) Blastoderm embryos stained for Bicoid protein (green) and DNA (red). Right panel shows Bicoid protein alone. All embryos were fixed and stained in parallel, and pictures were taken with the same setup with a digital camera and a light microscope. (D) In all wild-type embryos (n=27), a clear Bicoid gradient with similar extension was observed. (E) In embryos from females expressing the exuA+B transgene in an exu null mutant background, no Bicoid was detected in 27% of the embryos, while in all of the remaining embryos the extension of the gradient is reduced compared with wild-type embryos (n=30). (F) In all exu-null mutants examined (n=34) no Bicoid protein was detectable.

View larger version (90K): [in a new window]   Fig. 6. Exu phosphorylation is required for bicoid mRNA localisation and embryonic patterning. (A-E) In situ hybridisation showing bicoid mRNA (red) in confocal sections of stage 9 (left) and stage 10b (right) egg chambers. DNA is shown in green. Wild-type (A) or mutant (B-E) Exu protein was expressed from transgenes in an exu-null mutant background (exuXL/exuVL). Sequences of site A and B are indicated, and mutations are shown in red. All mutations affect bicoid mRNA localisation at stage 9 (B-E). bicoid mRNA localisation defects recover at stage 10b when only site A or B is mutant (B,C), and partially recovers when serines 438, 440 and 457 are mutant (D). No recovery is observed when all relevant serines in site A and B are simultaneously mutated. (A'-E') Embryos in the right panel are derived from females of the same genotype as egg chambers in the left panel. Embryos were stained with anti-Eve antibody to visualise segmentation. Ten embryos from each genotype were randomly chosen for analysis with the light microscope. Those embryos that show maximal anterior extension of the first Eve stripe are shown. The average position of the first Eve stripe of the ten analysed embryos is indicated as percentage of egg length. The average position of the first Eve stripe in exu null mutants (exuXL/exuVL) is at 26.9% egg length. Black line indicates the position of first Eve stripe in embryos expressing wild type Exu. The extension of the anterior shift of the first Eve stripe in the mutants (B'-E') corresponds to the severity of the bicoid mRNA localisation defects during oogenesis (B-E). All females were grown at 18°C. Anterior is leftwards; posterior is rightwards.

View larger version (71K): [in a new window]   Fig. 7. bicoid mRNA localisation during late oogenesis. In situ hybridisation showing bicoid mRNA localisation in confocal sections of stage 12 egg chambers. (A) In the wild-type bicoid mRNA accumulates at the anterior of the oocyte. (B) Although bicoid mRNA is detectable at the anterior of exu null mutants most of the mRNA appears to be unlocalised at the cortex or in the ooplasm. (C) In mutants in which Exu phosphorylation is abolished, bicoid mRNA is enriched at the anterior indicating partial recovery of the complete loss of bicoid mRNA localisation during mid-oogenesis. Genotypes are indicated. All females were grown at 18°C. Anterior is leftwards; posterior is rightwards.

View larger version (96K): [in a new window]   Fig. 8. exu independent functions of par-1 in bicoid mRNA localisation. In situ hybridisation showing bicoid mRNA localisation in confocal sections of stage 9 egg chambers, in which in the wild-type mRNA is localised as a ring around the anterior margin of the oocyte. (A,B) exu-null mutant egg chambers either expressing no transgene (A) or a transgene encoding the activated ExuSer457Glu (B). Both egg chambers are from siblings from the same cross and have been stained in parallel. The ExuSer457Glu encoding transgene rescues bicoid mRNA localisation completely, as revealed by the restriction of bicoid mRNA to the anterior corners of the oocyte. The anterior bicoid mRNA ring appears as two discrete dots in the confocal section. (C,D) Egg chambers from par-1 mutant siblings either expressing no transgene (C) or the ExuSer457Glu encoding transgene (D). Both egg chambers have been stained in parallel. The ExuSer457Glu protein is unable to fully rescue the cortical localisation of bicoid mRNA as the mRNA is no longer tightly restricted to the anterior corners but spreads along the lateral and anterior cortex of the oocyte (D). (E,F) For clarity only the oocyte of the egg chamber is shown. bicoid mRNA is restricted to the anterior corners in the wild type (E) and is dispersed throughout the ooplasm in homozygous exuPJ mutants (F). In homozygous par-19A exuPJ double mutants (G), bicoid mRNA is cortically localised resembling the situation in par-1 single mutants.

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