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First published online 13 September 2006
doi: 10.1242/dev.02565

Development 133, 4005-4013 (2006)
Published by The Company of Biologists 2006
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Interaction between Polo and BicD proteins links oocyte determination and meiosis control in Drosophila

Vincent Mirouse1, Etienne Formstecher2 and Jean-Louis Couderc1,*

1 Inserm, U384, Clermont-Ferrand, F-63001 France; Univ Clermont 1, UFR Médecine, 28, place Henri Dunant, Clermont-Ferrand, F-63001 France.
2 Hybrigenics SA, 3-5 Impasse Reille, 75014 Paris, France.


Figure 1
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  		Fig. 1. Interaction between BicD and Polo in two-hybrid and in vivo. (A) Yeast two-hybrid interaction between Polo and the N-terminal part of BicD (amino acids 120-350). The whole Polo-Box domain [Polo-Boxes 1 and 2 (red) and a short helix represented in gray] but not the kinase domain (green) are required for the interaction. (B) Immunoprecipitation using anti-GFP antibodies (IP-GFP) from ovary extracts from wild-type (lane 1), GFP-Polo (lane 2) and Ubi-NlsGFP (lane 3) flies, showing BicD specifically coprecipitates with GFP-Polo (lane 2, IP-GFP).

 

Figure 2
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  		Fig. 2. Polo localization during the early steps of oogenesis. (A) Schematic representation of a Drosophila germarium focusing on oocyte determination and meiosis progression. (B) General view of GFP-Polo expression (green) in a germarium and a stage 2 follicle. Arrowheads indicate Polo spots in region 2 and 3. (C-E) Enlargements of (B). (C) In region 1, Polo is found in many dots in each interphase germline cell, but is not colocalized with centrosomes (CP309, red). (D) In region 2a, Polo dots are progressively restricted to the pro-oocytes but do not show colocalization with BicD (blue) or centrosomes (red). (E) In region 3, Polo is found at the anterior or lateral part of the oocyte whereas BicD and centrosomes are in the posterior region. (F,G) A germarium expressing GFP-Polo (green) stained for SCs [C(3)G, red] and fusome and follicular cells (Hts, blue). Polo concentrates in meiotic cells from region 2a (F) to region 3 / stage 1 follicle (G). (H) Single confocal section of cysts in region 2 showing that GFP-Polo (green) is not localized on the remnant fusome (red). (I,J) Endogenous Polo protein (green) co-stained with SCs (red) shows a similar dynamic localization to GFP-Polo in regions 1 (I), 2 and 3 (J) of the germarium. (K) polo mRNA in situ hybridization. Strong expression of polo is detected in the germline in region 1 of germarium (arrow) and in follicle cells in region 2a (arrowhead).

 

Figure 3
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  		Fig. 3. Polo localization is dependent on BicD, egl and the Dynein complex. (A) BicDr5 germline clones marked by the absence of NlsGFP. (B) BicDPA66 homozygous germarium. (C) eglwu50/RC12 ovariole and (D) dmnK16109 germline clones marked by the absence of GFP. In A to D, the first images show SCs (red, white in the second picture), GFP-Polo (green dots, white in the third picture) plus NlsGFP (green nuclei, white nuclei in the third picture) and Hts (blue) in A and D. Polo localization is not restricted in any of these different genotypes, and the meiosis is not maintained in any cell of the cyst (at least 50 mutant cysts were scored for each genotype). In BicD (A) and egl (C) null mutants, meiosis starts in all the cells in region 2a whereas in BicDPA66 (B) and dmn (D) mutants normal initiation of meiosis in two to four cells is observed.

 

Figure 4
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  		Fig. 4. Loss of function of Polo affects meiotic progression and BicD polarized transport. (A) A wild-type germarium stained for SCs (red, white in A') and BicD (green, white in A''). SC formation is initiated in two to four cells in region 2a, and is then restricted to one cell in region 2b. BicD accumulation in meiotic cells begins in region 2a. Through region 2b, BicD redistributes to the posterior of the oocyte. (B) A polo1/polo9 germarium. Meiosis does not initiate properly in region 2a. Cysts often contain abnormal SCs, and meiosis restriction to one cell is delayed. BicD starts to accumulate in pro-oocytes only in region 2b. In region 3, BicD is still at the anterior of the oocyte. (C) A mosaic polo9 follicle at stage 7 with a single mutant cell (arrow) marked by the absence of nuclear GFP (green, white in C'). In this nurse cell, the amount of BicD protein (red, white in C'') is higher than in the neighboring wild-type nurse cells, but lower than in the oocyte (arrowhead). DNA is shown in blue.

 

Figure 5
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  		Fig. 5. Polo overexpression affects meiosis progression and oocyte differentiation. (A) UAS-Polo overexpression with a germline-specific driver leads to the formation of SCs (white) in more than four cells per cyst in region 2a, and to a delay in meiotic restriction in region 2b. (B) Overexpression of GFP-Polo. C(3)G (red, white in B'), BicD (blue, white in B''), and GFP-Polo (green, white in B'''). (B') Meiosis can initiate in more than four cells, and cysts in region 3 still contain four cells in meiosis, thus indicating a strong delay in meiosis restriction to one cell. (B'') Polo overexpression leads to a delay in BicD accumulation in the oocyte and localization to the posterior of the oocyte. (B''') Cysts from region 2a to region 3 contain an unusually high number of Polo dots that are not restricted to one cell. There is a correlation between cells containing the strongest Polo speckles and meiotic cells in the cyst in region 3.

 

Figure 6
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  		Fig. 6. Polo overexpression restores BicDPA66 protein function during early oogenesis. (A,B) C(3)G is in red and BicD in green. (A) BicDPA66 ovariole with no cell in meiosis from stage 2b of the germarium, and diffuse localization of BicD. (B) BicDPA66 ovariole overexpressing Polo. Each follicle contains an oocyte in meiosis, which accumulates BicD. (C) DNA staining of later stage follicles in a BicDPA66 ovariole overexpressing Polo. Stage 4 and stage 6 follicles contain an oocyte marked by the presence of the condensed DNA (arrowheads). The follicle at stage 9 is degenerating. (D) Model of a positive feedback loop between Polo and BicD proteins during meiosis restriction and oocyte differentiation. The Polo kinase is required to trigger meiosis and to activate BicD-dependent transport. In turn, BicD is required for the transport of oocyte determinants and for Polo localization.

 




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