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First published online 29 September 2004
doi: 10.1242/dev.01412

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Requirement for Par-6 and Bazooka in Drosophila border cell migration

Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA

View larger version (71K): [in a new window]   Fig. 1. Par-6 and Baz expression in the ovary. (A) Schematic of egg chambers at stages 8-10. Nurse cells (nc), and oocyte (o) are indicated. Border cells (green) are shown prior to, during and after migration. (B-M) Single confocal sections of wild-type anterior follicle cells (B-C'), and border cell clusters at the initiation of migration (E-F'), during migration (H-I'), and at the completion of migration (K-L'). Schematics of wild type stage 8 (D), early stage 9 (G), mid stage 9 (J) and stage 10 (M) egg chambers depict the position of the border cells (red) in the adjacent confocal images. Baz staining (green) is shown in panels (B,E,H,K) and double labeling with rhodamine phalloidin (red) to mark F-actin in panels (B',E',H',K'). Co-localization appears yellow. Par-6 staining (green) is shown in panels (C,F,I,L) and double labeling with phalloidin (red) in panels (C',F',I',L'). Polar cells (pc) are indicated in migrating clusters. High levels of Baz and Par-6 expression at border cell/border cell boundaries are indicated by arrows. (N-P') Three-dimensional confocal reconstruction and schematic representation of a border cell cluster in mid migration. The orientation of the border cell cluster in panel N is identical to that of the border cell cluster shown in panel H. Par-6 (green) and E-cadherin (red) are shown. Par-6 expression at the apices of the central polar cells (pc) is indicated by two straight lines.

View larger version (77K): [in a new window]   Fig. 2. Loss of Par-6 and Baz impedes border cell migration. (A-D) Single confocal section of stage 10 egg chambers stained with anti-Singed antibody (red) or GFP (green) that are wild type (wt) (A) or contain mosaic clones for a null mutation in the baz (B-C) or the par-6 (D) locus. Homozygous mutant cells are labeled by the absence of GFP. o, Oocyte. Scale bar: 50 µm. (E) slbo-Gal4 driver, expressed in the border cells (UAS-GFP) (green), but not in the polar cells, Fascilin III (red). (F-L) Confocal images of border cell clusters that are wild type (F,H,J) or that express either two copies of RNAi par-6 in a par-6 heterozygous null background (G,K) or two copies of RNAi baz in a baz heterozygous null background (I,L). RNAi transgenes were activated using slbo-Gal4. (F-I) Merged confocal z sections of border cell clusters stained with anti-Singed antibody (red) (F-I) showing either Par-6 (green) (F,G) or Baz (green) (H,I). The position of the central polar cells (pc) is indicated by two straight lines. Scale bar: 5 µm. (J-L) Single confocal sections of stage 10 egg chambers showing Singed (green) and F-actin (red). Scale bar: 50 µm. Arrows indicate border cell cluster.

View larger version (22K): [in a new window]   Fig. 3. Quantification of the delay in border cell migration caused by the depletion of Baz or Par-6 protein. RNAi lines and control line crossed to slbo-Gal4, upd-Gal4 or upd-Gal4; slbo-Gal4, driving expression of sense and antisense RNA in the border cells, polar cells or both. Two copies of the RNAi par-6 and two copies of RNAi baz transgenes were activated respectively. Quantification of border cell migration shown as the proportion of egg chambers in which the border cells migrated: 0-25% (yellow), 26-50% (blue), 51-75% (red) and 76-100% (black) of the normal distance. n, number of egg chambers examined.

View larger version (49K): [in a new window]   Fig. 4. Loss of Par-6 results in disorganized border cell clusters and a misdistribution of E-cadherin and ßps-integrin. (A-B) Confocal sections of wild-type migrating border cell clusters stained with anti-Singed antibody. (C-E) Morphology changes due to activation of two copies of the RNAi par-6 transgene in the border cells and the two central polar cells, using slbo-Gal4, expressed in the border cells, and upd-Gal4, expressed in the polar cells. Arrows show lamellipodia and long processes in cells depleted of Par-6. (F) E-cadherin protein expression in a wild-type border cell cluster. Border cell/border cell and border cell/polar cell boundaries express higher levels of E-cadherin than border cell/nurse cell junctions which are indicated by arrowheads. (G) Border cells depleted of Par-6. E-cadherin levels are high throughout the cluster. ßps-integrin expression is disrupted in Par-6 depleted (I) versus wild-type cells (H).

View larger version (103K): [in a new window]   Fig. 5. Overexpression of Par-6 and Baz in the border cells disrupts polarity. (A-D) Single confocal section of stage 10 egg chambers stained with anti-aPKC. Wild-type egg chambers are shown in A and C. Egg chambers in which Par-6 and Baz are overexpressed in centripetal and posterior follicle cells, which are indicated by the arrows, are shown in B and D. C and D show higher magnification of the cells in A and B. Scale bar: 50 µm. (E-T) Confocal images of border cell clusters, stained for different polarity markers that are wild type (E-H,M-P) or overexpress Par-6 and Baz (I-L,Q-T) using slbo-Gal4. Scale bar: 5 µm. (E-L) Three-dimensional confocal reconstruction of migrating border cells, stained for aPKC (red) and E-cadherin (green). Each panel represents a different view of the same cluster. (M-T) Confocal z sections of border cell clusters of the indicated genotypes: E-cadherin (green) and Singed (red) (M,Q); Sdt (green) and Singed (red) (N,R); ßps-integrin (green) and rhodamine phalloidine (red) (O,S); and -tubulin (green) (P,T).

View larger version (25K): [in a new window]   Fig. 6. Overexpression of Par-6 and Baz in the border cells delays border cell migration. Quantification of border cell migration defects caused by overexpression of Par-6 and Baz. Quantification of border cell migration shown as the proportion of egg chambers in which the border cells migrated: 0-25% (yellow), 26-50% (blue), 51-75% (red) and 76-100% (black) of the normal distance. n, number of egg chambers examined.

View larger version (76K): [in a new window]   Fig. 7. Par-6 and Baz localization are altered in slbo, domeless, and myosin VI mutants. Single confocal image section of a (A) wild type (wt) and (B) slbo1310/slbo1310 female sterile mutant stained for Par-6 (green), rhodamine phalloidin (red), and Fascilin III (blue). (C) Wild type and (D) dominant-negative domeless (UAS-domeCYT)-expressing border cells stained for Par-6 (green) and Singed (red). Polar cells are indicated (pc). Confocal z section of border cells from (E) wild type and (F) MyosinVI-depleted cells (Antisense MyoVI) that have been stained for Baz (green) and Singed (red).