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First published online 25 July 2007
doi: 10.1242/dev.010447

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The planar polarity pathway promotes coordinated cell migration during Drosophila oogenesis

Rebecca Bastock^* and David Strutt

Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Core planar polarity gene function is required in the border cells. Anterior is to the left and border cells are migrating towards the right, in this and subsequent figures. Statistical significances are indicated on charts as ***P<0.001 and **P<0.01; all P values and numbers of clusters examined are shown in Table S1 in the supplementary material for UAS/GAL4 experiments and Table S2 in the supplementary material for mosaic cluster analysis. (A) Schematic of border cell migration and outer follicle cell rearrangement. Anterior polar follicle cells (red) recruit adjacent outer follicle cells (light green) to form the border cells (dark green). The border cell cluster delaminates from the follicular epithelium and begins to migrate posteriorly at the beginning of stage 9, normally completing its journey by the end of this stage. Concomitantly, the outer follicle cells rearrange so that they no longer cover the nurse cells. In wild-type chambers, the border cell cluster migrates at such a rate that it approximately keeps up with the posterior movement of the outer follicle cells. (B,C) Charts showing the extent of border cell migration relative to outer follicle cell rearrangement for clusters in which either fz, dsh or stbm transcripts have been either knocked-down by UAS-RNAi constructs at 29°C (B) or overexpressed using UAS constructs at 25°C (C) under the control of the border cell-specific slbo-GAL4 driver (Rørth et al., 1998). Coloured bars indicate the proportion of clusters found ahead of the outer follicle cells, in approximately the same position (`normal') or lagging behind, for either sibling controls or experimental conditions. `Ahead' or `behind' are defined as being more than the diameter of a nurse cell nucleus away from the trailing edge of the rearranging outer follicle cells. Either knockdown of fz, dsh or stbm or overexpression of fz or stbm causes a significant increase in the number of clusters `behind' and an accompanying decrease in the number of clusters showing a `normal' rate of migration. (D) Chart showing the proportions of genetically mosaic clusters recovered for the strong alleles fz15 and stbm6 with both polar follicle cells retaining gene function, but with either wild-type border cells leading (pink bars) or mutant border cells leading (blue bars). In both genotypes, there is a statistically significant (P=0.003) preponderance for wild-type border cells to be found at the leading edge of the migrating cluster. Mutant cells in the cartoons are represented by grey shading, with leading cells to the right and lagging cells to the left.

View larger version (104K): [in this window] [in a new window]   Fig. 2. Border cell clusters lacking core planar polarity gene function show normal expression of slow border cells, DE-Cadherin and Stat92E. (A-D) slow border cells expression as revealed by the slbo-lacZ reporter (Montell et al., 1992); ß-gal immunolabelling (red) and DE-Cadherin (DE-Cad) expression (green) in migrating border cell clusters from wild-type (A), fz21/fz15 (B), stbm6 (C) and dsh1 (D) individuals. High levels of nuclear-localised lacZ gene product in border cells is indicated by arrowheads. We observed that, in wild-type clusters, ß-gal levels were lower at early stage 9 than at the end of stage 9, whereas, in mutant clusters, ß-gal levels were generally higher throughout migration. We assume that ß-gal accumulates progressively within the border cells after the onset of gene expression, and that the delayed migration seen in the mutant backgrounds results in higher accumulation at equivalent stages of migration. (E-H') DE-Cad (green/white) and actin (red) distribution in migrating border cell clusters from wild-type (E,E'), fz21 (F,F'), stbm6 (G,G') and dsh1 (H,H') individuals. Border cells are marked by red dots and polar follicle cells by white asterisks. (I-K) Stat92E (green) and Armadillo (Arm, red) distribution in migrating border cell clusters from wild-type (I), fz21 (J) and stbm6 (K) individuals. High levels of nuclear-localised Stat92E in border cells is indicated by arrowheads.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Core planar polarity genes regulate the border cell actin cytoskeleton. Polar follicle cells are marked with white asterisks and border cells are marked with red dots. (A-G) Migrating border cell clusters, fixed to enhance preservation of the actin structures (see Materials and methods). In wild-type clusters (A) large actin-rich protrusions can be seen (arrowheads). In fz15/fz23 (B), dsh1 (C), stbm6 (D) and slbo-GAL4/UAS-fz- RNAi (E) mutants, the cytoskeleton appears fuzzy and large protrusions are rarely seen. Overexpression of fz (F) and stbm (G) under the control of slbo-GAL4 also disrupts the formation of large actin-rich protrusions. GAL4 experiments were carried out at 29°C. (H-L') Migrating border cell clusters stained for actin (red/white), expressing slbo-Gal4, UAS-GFP (green) at 25°C. Expression of dominant-negative RhoN19 (H), RhoN19 and fz- RNAi (I), fz-RNAi (J), dominant-active RhoV14 (K), and RhoV14 and fz-RNAi (L). The UAS-fz-RNAi insertion used was chosen because it gives weaker phenotypes than the insertion used for other experiments (e.g. panels E and N), with some actin-rich protrusions still being visible (J). Expressing dominant-negative RhoN19 results in border cells becoming long, thin and not migrating effectively (H), and co-expressing fz-RNAi has no effect on this phenotype (I). Cells expressing dominant-active RhoV14 become very round with an even cytoskeleton (K), and co-expressing fz-RNAi (L) ameliorates this phenotype, with the cells appearing less round and producing actin-rich protrusions (arrowheads). (M-N') Migrating border cell clusters, stained for actin (red), expressing GFP-RhoA (green/white). In wild-type clusters, GFP-RhoA colocalises with actin-rich protrusions at the cell surface (M), which are lost in cells expressing fz-RNAi under the control of slbo-GAL4 at 25°C (N), resulting in a partial redistribution of GFP-RhoA to the cytoplasm. Border cell clusters expressing fz-RNAi under the control of slbo-GAL4 showed an average cytoplasmic level of GFP-RhoA of 24.0% of peak membrane levels (n=10), compared with 15.4% for control clusters lacking the slbo-GAL4 driver (n=9), these results being statistically significant at the P<10-5 level (t-test).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Core polarity gene function in the polar follicle cells affects border cell migration. (A) Chart showing the extent of border cell migration for clusters in which either fz, dsh or stbm transcripts have been knocked-down by UAS-RNAi constructs under the control of the polar follicle cell-specific upd-GAL4 driver at 29°C (Tsai and Sun, 2004). Knockdown of fz transcripts causes a significant increase in the number of clusters `behind' (see Fig. 1), whereas knockdown of dsh causes no delay in migration. Knockdown of stbm in flies carrying two copies of the endogenous stbm locus causes a mild delay in border cell migration, which is greatly enhanced by the removal of one copy of the endogenous locus. (B) Chart showing the proportions of genetically mosaic clusters recovered for the strong alleles fz15 and stbm6 with both polar follicle cells lacking gene function, and either wild-type border cells leading (pink bars) or mutant border cells leading (blue bars). Mutant cells in the cartoons are represented by grey shading, with leading cells to the right and lagging cells to the left. In the small number of fz mosaic clusters recovered (n=6), we saw no clusters with a wild-type border cell leading, which only deviates from the null hypothesis that border cell position is random at a significance level of P=0.034. In the stbm mosaic clusters recovered (n=10), both wild-type and mutant border cells are seen leading, and the result fits the null hypothesis that border cell position is random (P=0.5). (C) Chart showing the proportions of genetically mosaic clusters recovered for the strong fz15 allele with only one polar follicle cell lacking gene function. Two classes of clusters were recovered (n=15); both had the non-mutant polar follicle cell touching the leading border cell, with the genotype of this leading border cell approximately equally distributed between wild type and mutant. The leading position of the polar follicle cells strongly deviates from the null hypothesis that polar cell position is random (P=0.0003), whereas the position of the border cells fits the hypothesis that this is random with respect to the genotype of the border cell (P=0.71). The data suggest that border cell position is determined by the genotype of the polar follicle cell with which they make junctional contact, regardless of the genotype of the border cell. (D) Chart showing the proportions of genetically mosaic clusters recovered for the strong stbm6 allele with only one polar follicle cell lacking gene function. Two classes of clusters were recovered (n=9); both had non-mutant border cells leading the cluster, with the genotype of the polar follicle cell touching the leading border cell being either mutant or non-mutant. The leading position of wild-type border cells does not fit the null hypothesis that position is random (P=0.018). The position of the wild-type polar cells fits the hypothesis that this is randomly determined (P=0.51).

View larger version (64K): [in this window] [in a new window]   Fig. 5. Fz and Stbm proteins are localised within the border cell cluster. Illustrations (top) show border cells (white), polar follicle cells (grey) and Fz (red); the direction of migration is towards the right (grey arrows). (A-D) Border cell clusters stained for Fz (red) and actin (green). Fz is localised in the adherens junctions of the polar follicle cells (A) and apical regions of the border cells (B) prior to migration. During migration, Fz localisation is retained in the junctional region that the polar follicle cells share with the border cells (C) and is within the migratory regions of the border cells (D). This pattern of localisation is lost in egg chambers mutant for fz, consistent with the immunolabelling being specific (data not shown). (E,G) Egg chambers stained for Armadillo or actin (red) and Stbm (green). Stbm is localised to the polar follicle cell adherens junctions (arrow). This pattern of localisation is lost in egg chambers mutant for stbm (data not shown). (F,H) Egg chambers stained for Armadillo or actin (red) and Dsh-GFP (green). Dsh-GFP is seen in a punctate pattern in polar follicle cell and border cell cytoplasm, and also partially overlaps the adherens junction region (arrow).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Model of Fz and Stbm interactions in the pupal wing epithelium and border cell cluster. In the wing, Fz and Stbm mutually reinforce the localisation of each other in opposing junctions of neighbouring cells (rounded black arrows) and inhibit the localisation of each other in adjacent regions of the same cell (grey bars). Distally localised Fz within the same cell promotes the production of a single distal actin-rich trichome, via Dsh and RhoA function (Axelrod, 2001; Strutt, 2001; Strutt et al., 1997). In addition, proximally localised Stbm is thought to promote trichome formation at the opposite end by an uncharacterised mechanism (Adler et al., 2004). In the border cell cluster, Fz and Stbm are localised to the junctional regions in which the epithelial polar follicle cells and the partly epithelial border cells make contact. Because Fz-expressing polar follicle cells promote the migration of Stbm-expressing border cells in a contact-dependent manner, we infer that Fz in the junctions of polar cells promotes the localisation of Stbm to the junctions of border cells. In turn, this would lead to Fz localisation to the non-junctional (mesenchymal) migratory regions of the border cells. Fz in border cells locally modulates the formation of appropriate actin structures, probably via Dsh and RhoA, as in the wing. In addition, Stbm in the junctions of border cells promotes the formation of actin structures at a distance in the migratory region. In this way, Stbm localised to junctions and Fz in the migratory region both independently promote migration. Consistent with our mosaic analysis, this scheme predicts that (i) contact with an Fz-expressing polar cell promotes border cell migration (Fig. 4B,C), (ii) Fz-expressing polar cells are only able to promote the migration of border cells that express Stbm (Fig. 1D, Fig. 4D), and (iii) Stbm is required in the polar cells for efficient border cell migration (Fig. 4B), but the border cells do not need to touch the Stbm-expressing polar cell (Fig. 4D).