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First published online July 12, 2005
doi: 10.1242/10.1242/dev.01911

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Function of the ETS transcription factor Yan in border cell migration

¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
² Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
³ University of Chicago, 5801 South Ellis Avenue, Chicago, IL 60637, USA
⁴ The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (68K): [in a new window]   Fig. 1. Yan is dynamically expressed in migrating border cells. (A) BCs are specified at stage 8 of oogenesis at the anterior pole of the ovarian follicle cell epithelium. They exit from the epithelium at stage 9 to migrate in between the NCs to reach the NC-oocyte (OOC) boundary at stage 10 of oogenesis. (B-D) Expression of yanP(lacZ) in egg chambers. Arrows indicate BCs and arrowheads centripetal cells. (B) yanP(lacZ) is expressed in anterior and posterior terminal cells at early stages of oogenesis, and in BCs that exit the epithelium. (C) At stage 9, yanP(lacZ) is strongly expressed in outer BCs, while it is weakly detectable in the main body follicle cells. (D) At stage 10, yanP(lacZ) expression broadens and shows the highest levels in BCs and centripetal cells. (E-G) Staining of egg chambers with anti-Yan antibodies (green) and Alexa-568-phalloidin (red). Arrows mark BCs. Magnified boxes show the progressive decrease in Yan protein (gray) levels during migration. (E) Yan is strongly expressed in BCs, whereas it is hardly detectable in polar cells (yellow arrowhead). (F). Yan expression becomes weaker after BCs exit from the epithelium and progressively decreases thereafter. (G) Yan is hardly detectable in BCs that have reached the NC-oocyte boundary at stage 10 of oogenesis.

View larger version (80K): [in a new window]   Fig. 2. Yan is required for the invasive migration of border cells. (A) Comparison of BC migration in stage 10 egg chambers between wild type (WT) and three different yan mutant alleles. Ninety-five percent of wild-type BCs reach the NC-oocyte boundary. Seventy-nine percent of completely mutant yan443 or yan884 mutant BC clusters are detectable at the anteriormost pole of the follicular epithelium and do not show any signs of migration, while approximately 15% of yan443 or yan884 mutant BCs migrate less than 25% of the way towards the oocyte. Only 2% of yan443 or yan884 mutant BCs reach the NC-oocyte boundary on time. BCs of the weaker yan1 allele exit from the epithelium but are delayed in their migration to the oocyte. Sixty-seven percent of mutant BCs migrate less then 25% of the way towards the oocyte, while 25% reach the NC-oocyte boundary. (B,C) Stage 10 egg chambers stained with Alexa-568-phalloidin (red) and DAPI (blue). Wild-type cells are marked by expression of GFP and yan mutant cells by lack of GFP expression. (B) Wild-type BC clusters reach the NC-oocyte boundary at stage 10 of oogenesis. (C) yan443 mutant BC clusters remain part of the anterior follicle cell epithelium. (C') High magnification image of a yan443 mutant BC cluster. Arrows indicate the boundary between BCs and squamous follicle cells.

View larger version (106K): [in a new window]   Fig. 3. Accumulation of DE-Cad in yan mutant border cells. The expression of Fas3 and DE-Cad was compared between yan/+ (WT) and yan mutant clones (see Materials and methods for details). (A-D') Actin is visualized by Alexa-568-phalloidin (red). (A-D) Expression of GFP (blue) marks non-mutant cells in stage 9 mosaic mutant egg chambers. (A'-D') yan443 mutant follicle cell clones are marked by the absence of GFP (blue). (A,A') Slbo is expressed both in yan mutant and non-mutant BCs. (B,B') The polar cell marker Fas3 is restricted to polar cells, even when outer BCs lack Yan expression. (C,C') DE-Cad accumulates in yan443 mutant BCs at the boundaries between outer BCs and polar cells (asterisk), as well as at the interface between BCs and squamous follicle cells (arrow). (D,D') Mutant BCs accumulate high levels of DE-Cad on their surface (arrows) and connect non-mutant BCs and the follicular epithelium. Asterisks mark polar cells.

View larger version (47K): [in a new window]   Fig. 4. Yan regulates DE-Cad expression levels. (A-C) Egg chambers are stained for actin (with Alexa-568-phalloidin, red), anti-DE-Cad (green) and DAPI (blue). (A'-C') Higher magnification views of A-C; grayscale images of DE-Cad allow the visualization of DE-Cad expression levels. Yellow arrows point towards polar cells, white arrowheads point at outer BCs, white arrows point at the interface between BCs and squamous follicle cells. (A,A') In wild type, high levels of DE-Cad are found at the boundary between polar cells and outer BCs, as well as between outer BCs. DE-Cad is also strongly detectable at the edge of outer BCs. (B,B') DE-Cad surface expression is reduced in outer BCs where UAS-yanACT is ectopically expressed using slbo-Gal4. (C,C') Expression of UAS-DE-Cad5,9 in BCs by slbo-Gal4; arrow in C indicates arrested BCs. (D) Functional interaction between DE-Cad and Yan to regulate BC migration. In wild type, 95% of BCs reach the NC-oocyte boundary. Ectopic expression of UAS-yanACT induces strong BC migration defects (60% of BCs migrate less than 25%, 26% migrate 25-50%, 12% migrate 50-75% and 2% complete their migration). Weak expression of UAS-DE-Cad partially suppresses UAS-yanACT migration defects (37% migrate less than 25%, 29% migrate 25-50%, 29% migrate 50-75% and 5% reach the nurse cell-oocyte boundary). Forced expression of UAS-DE-Cad5,9 delays BC migration (20% migrate less than 25%, 30% migrate 25-50%, 32% migrate 50-75% and 18% complete their migration by stage 10 of oogenesis). n>100.

View larger version (57K): [in a new window]   Fig. 5. Yan modulates endocytosis. (A-C) Live FM1-43 uptake in Drosophila SL2 cells. FM1-43 membrane incorporation in 20 minutes after yanRNAi (A), overexpression of yanACT (B) and gfpRNAi control (C). (D) Kinetics of FM1-43 uptake is accelerated in cells overexpressing yanACT (red) and decreased in yanRNAi cells (blue), when compared with a gfpRNAi control (orange). (E) Twenty-minute FM1-43 uptake after yanRNAi, gfpRNAi and MT-YanACT in triplicate. (F,G) Stage 10 egg chamber, stained with anti-DE-Cad (green), Alexa-568-phalloidin (red) and DAPI (blue). slbo-Gal4, UAS-Rab5(S43N)-expressing BCs (G) show impaired ability to migrate and elevated levels of cortical DE-Cad expression when compared with control egg chambers (F). (H) Overexpression of dominant-negative Rab5 in BCs delays their migration (46% migrate less than 25%, 25% migrate between 25-50%, 15% migrate 50-75% and 14% complete their migration by stage 10 of oogenesis).

View larger version (54K): [in a new window]   Fig. 6. Activated Yan enhances slbo mutant border cell migration defects. (A,B) slbo1310 mutant egg chamber stained with Alexa-568-phalloidin (red) and anti-Yan (green). Arrows indicate BCs. (A) Yan is normally expressed in slbo1310 mutant egg chambers at stage 9. (B) Yan is strongly expressed in border cells of slbo1310 mutant egg chambers that fail to migrate towards the oocyte at stage 10. (C) Statistical representation of BC migration defects. Overexpression of wild-type Yan in BCs has only minor effects on BC migration. By contrast, expression of YanACT specifically in BCs delays their migration; 38% completely failed to migrate, 22% migrated less than 25% and 26% of BC clusters migrate less than 50% towards the NC-oocyte boundary. Interestingly, this phenotype can be enhanced by removing one copy of slbo1310 resulting in 72% of BCs that completely failed to migrate, 17% that migrated less than 25% and 11% that migrated less than 50% towards the oocyte. Expression of Yan and YanACT enhances the BC migration defects of slbo mutant egg chambers. These BC migration defects are significantly stronger than the defects observed in homozygous slbo1310 mutant egg chambers, where 56% of mutant BCs show no migration and 42% migrate less than 25% of the way towards the oocyte.

View larger version (83K): [in a new window]   Fig. 7. Yan expression depends on JAK/STAT and Notch pathways. (A) JAK/STAT and Notch signaling around the anterior and posterior polar cells (green) specify terminal cell populations during oogenesis. (B,C) ß-galactosidase (ß-gal) staining of a Su(H)-lacZ Notch activity reporter. Arrows indicate BCs. (B) ß-Gal is dynamically expressed during early oogenesis, strongly in BCs of stage 9 egg chambers. (C) ß-Gal is strongly expressed in BCs of stage 10 egg chambers. (D-K') Egg chambers are stained with Alexa-568-phalloidin (red) to visualize actin. Arrows indicate BCs and arrowheads mark squamous cells. (D) BCs migrate normally in Nts egg chambers at the permissive temperature. (E) BCs have migration defects in Nts egg chambers at the restrictive temperature. (F-H) Slbo expression is shown in green; (F'-K') Yan is shown in green. (F,F') Wild-type control BC cluster expressing Slbo and Yan. (G,G') Nts BCs have reduced Slbo and Yan expression. (H,H') Slbo and Yan are normally expressed in BCs where Serrate (Ser) is ectopically expressed. (I,J) stat92E mutant follicle cells are positively marked by GFP expression (green, arrows), whereas wild-type cells do not express GFP (arrowhead). (I',J') stat92E mutant BCs do not express Yan (arrows), whereas Yan expression (green) is normal in wild-type cells (arrowhead). (K,K') tai61G1 mutant BC clones are negatively marked by lack of GFP expression. (K) tai mutant BCs do not express GFP (green). (K') The expression of Yan in tai mutant BCs and squamous follicle cells was similar to that observed in wild-type control egg chambers (F').

View larger version (69K): [in a new window]   Fig. 8. RTKs signaling downregulates Yan. (A) Yan (blue) is strongly expressed in BCs that delaminate from the follicular epithelium, but expression gradually decreases as BCs migrate along the increasing gradient of Pvf1 (orange). The inverse expression levels of Yan and Pvf1 suggest that PVR activity counteracts Yan expression in migrating BCs. (B-E') Egg chambers are stained with Alexa-568-phalloidin (red) to visualize actin. Arrows indicate BCs and arrowheads the squamous follicle cells. (B-E) Anti-Slbo (green) and actin (red) staining; (B'-E') anti-Yan (green) and actin (red) staining. (B,B') Slbo and Yan are strongly expressed in wild-type BCs that delaminate from the follicular epithelium. (C) Slbo is normally expressed in BCs that ectopically express activated PVR. (C') By contrast, Yan is undetectable in BCs but is expressed normally in squamous follicle cells where slbo-Gal4 is inactive. Similarly, expression of either activated EGFR (D,D') or activated Raf (E,E') in BCs downregulates Yan but not Slbo. All experiments and stainings were done in parallel, and images were taken with the same settings, together with experiments in Fig. 7F-H. The expression levels of Yan in squamous follicle cells serve as an internal control. (F,G) ß-Gal staining indicates yan expression in yanP(lacZ) (F) and yanP(lacZ)/slbo-Gal4; UAS-PVR (G) stage 10 egg chambers. Arrows indicate BCs; insets show yan mRNA expression by in situ hybridization. (H,I) Stage 10 egg chamber stained with anti-Slbo antibody (red), anti-ß-Gal antibody indicating pnt-lacZ expression (green), DAPI (blue) and Alexa-568-phalloidin (gray). (H) Pnt-lacZ is expressed in posterior cells and is absent in wild-type BCs. (I) Expression of activated PVR in BCs arrests their migration. These BCs express Slbo but do not express pnt-lacZ.

View larger version (19K): [in a new window]   Fig. 9. Integrative model of Yan regulation and function during border cell migration. (A) The JAK/STAT and Notch pathways specify the group of anterior terminal cells (a-TC) around the pair of polar cells (PC, green). Cells that are directly adjacent to the anterior polar cells are specified as BCs expressing Slbo. With the exception of the polar cells, Yan (blue) is expressed in all a-TCs, and becomes upregulated immediately prior to their transit from a static, epithelial state to a migratory state (dark blue). Posterior terminal cells (red) are specified by Gurken (EGF) signaling. Pvf1 (orange), secreted from the oocyte, guides BCs towards the oocyte. As BCs face increasing Pvf1 levels from anterior to posterior, Yan expression levels decrease (light blue circles). (B) Regulatory relationships between signal transduction pathways that control BC migration. JAK/STAT and Notch signaling pathways regulate the expression of slbo and yan, whereas PVR and EGFR induction lead to Yan phosphorylation and its inactivation. The transient upregulation of Yan at the initiation of BC migration facilitates DE-Cad turnover at the plasma membrane to enable BCs to make and break adhesive contacts, and to promote detachment from the epithelium and cell movement. Coordinated, dynamic changes in cell adhesion and cytoskeletal organization enable BCs to migrate in a stereotypic fashion.