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Development 138 (9): 1697-1703

Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

Yan Yan, Natalie Denef, Charm Tang, and Trudi Schüpbach*

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA


Figure 1
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Fig. 1. Mutations in PI4KIIIalpha lead to oocyte polarity defects. (A) CG10260, the Drosophila PI4KIIIalpha homolog. Alleles PI4KIIIalphaFQ88, PI4KIIIalphaGJ86, PI4KIIIalphaFY24 and PI4KIIIalphaGS27 contain early stop codons at Gln78, Trp1242, Lys1751 and Gln1778, respectively. Protein domains are annotated with CDD search (Marchler-Bauer et al., 2009Go). PI4Ka, phosphoinositide 4-kinase (PI4K) accessory domain; PI4Kc, PI4K, type III, alpha isoform, catalytic domain. (B-E') Wild-type egg chambers (B,D) and egg chambers containing PI4KIIIalphaGS27 (C) or PI4KIIIalphaFQ88 (E) mutant posterior follicle cells (PFCs), marked by the absence of GFP (green) and stained for DNA (blue, B,C) or Grk (red, D,E). The DNA (B',C') and Grk (D',E') channels are also shown separately. The oocyte nucleus is located in the dorsal-anterior corner in a wild-type egg chamber (B', arrow). In mutant PFCs, the oocyte nucleus remains at the posterior of the oocyte (C', arrow). Similarly, Grk localization is disrupted in the mutant egg chamber (E). (F-G') A wild-type egg chamber (F) and an egg chamber containing PI4KIIIalphaGS27 mutant PFCs (G) expressing Kin-lacZ and stained for β-gal (red). The β-gal channel is shown separately in F',G'. In the wild-type egg chamber, Kin-β-gal localizes to the posterior of the oocyte (F). In the egg chamber containing a large PI4KIIIalphaGS27 PFC clone, Kin-β-gal is mislocalized to the center of the oocyte (G). (H,H') Egg chambers stained for Staufen (red), which forms a tight crescent at the posterior of a wild-type egg chamber (H', red arrow), but is mislocalized to the center of the oocyte in an egg chamber containing a PI4KIIIalphaFQ88 PFC clone (H', white arrow). Scale bars: 10 µm.

 

Figure 2
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Fig. 2. Effects of PI4KIIIalpha mutations on different signaling pathways. Mutant cells are marked by the absence of GFP (green), except in B where we generated unmarked clones in order to visualize the 10xSTAT-GFP reporter and PI4KIIIalpha mutant cells were identified by the loss of their monolayered epithelial structure outlined by actin staining. (A,A') A Drosophila egg chamber containing PI4KIIIalpha mutant PFCs, expressing the EGFR signaling reporter kekkon-lacZ and stained for β-gal (red in A, gray in A'). (B,B') An egg chamber containing PI4KIIIalpha mutant PFCs, expressing the JAK/STAT signaling reporter 10xSTAT-GFP (green). Both EGFR and JAK/STAT signaling pathways were correctly activated and transduced in the PI4KIIIalpha mutant PFCs, as indicated by the normal levels of β-gal staining (A,A') and GFP signal (B,B'). (C-F') Egg chambers containing PI4KIIIalpha mutant PFCs stained for phosphorylated Histone H3 (PH3, red, C), Actin (red, D), DNA (blue, C,D), Cut (red, E) and Hnt (red, F); the PH3, Cut and Hnt channels are also shown separately (C',E',F'); the boxed region in D is shown at higher magnification in D' and D'' (DNA channel only). PI4KIIIalpha mutant PFCs remained in the mitotic cycle after stage 6, as indicated by the presence of PH3-positive cells (C', arrow) and multilayered cells with smaller nuclei (D'', arrow). PI4KIIIalpha mutant PFCs failed to downregulate Cut (E) and to upregulate Hnt (F) after stage 6. These results indicate that Notch signaling is compromised in PI4KIIIalpha mutant PFCs. (D,E) Note that PI4KIIIalpha mutant cells at the lateral side of the egg chambers show normal epithelial structure (D) and correctly downregulated Cut expression (E), as in the wild-type cells. (G-H') Egg chambers containing PI4KIIIalpha mutant FCs, expressing the Hippo signaling reporters ex-lacZ (G) and diap1-lacZ (H), stained for β-gal (red in G,H; gray in G',H'). Upregulation of both reporters indicates that Hippo signaling is disrupted in PI4KIIIalpha mutant FCs. Scale bars: 10 µm.

 

Figure 3
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Fig. 3. Merlin mislocalization from the apical membrane in PI4KIIIalpha mutant follicle cells and eye disc cells. Clone boundaries are marked by dotted white lines. Mutant cells are marked by the absence of GFP (green), except in G where we generated unmarked clones in order to visualize the Ubi-PH-PLC{delta}-GFP reporter. PI4KIIIalpha mutant cells were identified by the abnormal actin structure on the apical domain. FCs and eye disc cells are orientated with the apical side up. (A-B') The follicular epithelium (A) and the imaginal eye disc epithelium (B) containing PI4KIIIalpha mutant cells stained for Merlin (red in A,B; gray in A',B'). (C-F') Egg chambers containing PI4KIIIalpha (C,E) or sav (D,F) mutant FCs stained for Expanded (red in C,D; gray in C',D') and Kibra (red in E,F; gray in E',F'). Merlin, Expanded and Kibra localization in wild-type cells is indicated with red arrows. Merlin disappears from the apical and junctional region of the mutant cells (A',B', white arrow), whereas Expanded (C', white arrow) and Kibra (E', white arrow) are upregulated but remain localized. Upregulation of Expanded (D', white arrow) and Kibra (F', white arrow) is similarly observed in sav mutant FCs. (G-J') Egg chambers containing PI4KIIIalpha mutant cells, expressing the PI(4,5)P2 reporter Ubi-PH-PLC{delta}-GFP (G), stained for GFP (green in G; gray in G'), actin (red in G-J; gray in H'), Cad99C (blue in I; gray in I') and phospho-ERM proteins (blue in J; gray in J'). The PI(4,5)P2 reporter is absent from the apical membrane of the mutant cells (G', white arrow), in contrast to the wild-type cells (G', red arrow). PI4KIIIalpha mutant cells exhibit abnormal actin spikes on the apical side (H', white arrow) as marked by the microvillus marker Cad99C (I', white arrow). The apical microvilli region of PI4KIIIalpha mutant cells shows depletion of phospho-ERM proteins (J', white arrow). Scale bars: 10 µm.

 


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