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First published online 23 April 2008
doi: 10.1242/dev.017103

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The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis

¹ Department of Biochemistry, Chang Gung University, Tao-Yuan, 333, Taiwan.
² Department of Anatomy, Chang Gung University, Tao-Yuan, 333, Taiwan.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Subcellular localization of Gurken in follicle cells. Anterior of the egg chamber is towards the left and dorsal side faces upwards. (A-D) The localization of Gurken in the posterior follicle cells during early oogenesis stages was dependent on shibire. Gurken was labeled with antibody (green). The oocyte and follicle cell cortex were visualized by phalloidin staining (red). Gurken was easily detected in the posterior follicle cells around the oocyte at permissive temperature in EQ1::shits egg chambers (A,B). These punctate signals were significantly reduced at a non-permissive temperature (C,D). (E-H) Gurken (red) colocalized with early endosomes (labeled with GFP-Rab5, E,F), and late endosomes (labeled with GFP-Rab7, G,H) in posterior follicle cells. Arrows indicate colocalization in the merged images (F',H'). (I-L) During middle oogenesis when Gurken (green) shows an asymmetrical localization in the oocyte, little Gurken could be detected in dorsal follicle cells. At stage 8 (I,J) and 9 (K,L), Gurken signals in dorsal follicle cells were detected in about 17% of the egg chambers. Scale bars: 10 µm in A-I; 20 µm in J,K; 40 µm in L.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Expression pattern of the HRP-Grk fusion protein. (A) The schematic of the HRP-Grk fusion protein. SP, signal peptide; EGF, EGF repeat motif; TM, transmembrane domain; Cyt, cytoplasmic domain. (B-M) The localization of HRP-Grk was detected by the anti-HRP antibody (green), and the egg chamber organization was identified by phalloidin staining (red). The wild-type egg chambers served as a negative control (B). HRP staining in the oocyte at stage 7 (C), stage 8 (D) and stage 9 (E) showed similar patterns to endogenous Gurken staining. The punctate HRP signal was detected not only in the dorsal (arrows in F and G) follicle cells but also in the ventral follicle cells (arrows in H,I). These punctate signals were markedly reduced at a non-permissive temperature in shibire mutant background (J-M). (N-Q) Some HRP signal (red) in follicle cells colocalized with an early endosomal marker, GFP-Rab5, in the dorsal (N,N',O) and ventral (P,P',Q) follicle cells (arrows indicate punctate signals that overlapped in merged images). Anterior of the egg chamber is towards the left and dorsal side faces upwards. Scale bars: 20 µm in B-E; 10 µm in F-Q.

View larger version (119K): [in this window] [in a new window]   Fig. 3. The Grk-Egfr complex is internalized in follicle cells through Rab5-Rab7 endocytic pathways. (A-C) Clonal analysis of mosaic egg chambers shows that the anti-Egfr antibody staining (red) is specific as it is absent from the mutant cells. (D-F') HRP-Grk (red) was colocalized with Egfr (green) in dorsal follicle cells. (G-I) Punctate HRP signals (red) were much less abundant in the top/Egfr mutant follicle cells (star indicates non-GFP cells) compared with adjacent normal cells (arrows indicate punctate HRP signals in wild-type cells). (J-O) Internalized Egfr (red) was colocalized with Rab5 (J-L) and Rab7 (M-O) in the follicle cells. Oo, oocyte; arrows indicate punctate signals that overlapped in merged images (D-F,J-L,M-O); the bracket area was enlarged in F',L',O'. Scale bars: 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 4. The degradation of HRP-Grk in follicle cells. (A) Schematic representation of our sectioning strategy. The data shown below were all taken from cross-section profiles of stage 9 egg chambers that included oocyte nucleus. (B) Micrograph of a 1 µm cryosection. N, oocyte nucleus; Oo, oocyte; FC, follicle cells. (C-G) Electron micrographs of stage 9 oocytes immunolabeled with anti-HRP (10 nm gold particles). The labeling density of HRP-Grk at TGNs (brackets) is higher on the dorsal side (C) than on the ventral side (D) of the egg. (E) Quantitation of HRP-Grk labeling per TGN unit in the oocyte. n=310. Among the organelles, the single-membrane delimited MVB/lysosome (arrows) exhibited the most concentrated labeling, both in the dorsal (F) and the ventral (G) follicular cells. M, mitochondria. (H-J) The MVB/lysosomes in follicle cells of late stage 9 OreR egg chamber. Electron micrographs of MVB/lysosomes in the dorsal (I) and lateral (J) follicular cells. (H) Semi-quantification of the volume density of MVB/lysosomes in follicular cells. The average density ratio (bar) in dorsal, and lateral follicle cells is 0.0060 and 0.0109, respectively. The difference between the dorsal and lateral follicle cells is meaningful in the nonparametric Wilcoxon test (two-tailed, P=0.007). (K) Semi-quantification of the volume density of MVB/lysosomes in follicle cells of late stage 9 grkHF/grkHF egg chambers. The average density ratios (bar) for dorsal and lateral are 0.0062 and 0.0065, respectively. Scale bars: 20 µm in B; 100 nm in C,D,F,G,I,J.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Overexpression of CblL altered Gurken distribution. Anterior of the egg chamber is towards the left and dorsal side faces upwards. (A-C) Gurken expression pattern (green) at late stage 9 egg chambers was altered when Cbl was overexpressed. In wild-type egg chambers, Gurken accumulated in the dorsal anterior corner (A). Slightly reduced expression of Gurken was detected in egg chambers with overexpression of CblS driven by GR1-Gal4 (B). Significantly reduced expression of total Gurken in egg chamber in which CblL was overexpressed (C). (D) The percentage of late stage 9 egg chambers that had normal Gurken protein expression. *P>0.05; **P<0.01. (E-H) The increased HRP-Grk signal (green) in dorsal follicle cells (E) was colocalized with CblL, which was ectopically expressed by EQ-Gal4 and stained with 8C4 antibody (F, red). (H) The composite image of several confocal sections of an egg chamber with an HRP-grk transgene and CblL overexpression. (I-K) Some of these HRP-Grk signals were also colocalized with Rab5 (I), Rab7 (J) and Egfr (K). Arrows indicate punctate signals that overlapped in merged images. The bracket area is enlarged in E',F',G',I',J',K'. Scale bars: 40 µm in A-C; 20 µm in E-J; 10 µm in K.

View larger version (47K): [in this window] [in a new window]   Fig. 6. The Gurken internalization was blocked in Cbl mutant follicle cells. Anterior of the egg chamber is towards the left and dorsal side faces upwards. Wild-type cells were marked by anti-Myc antibody (red). Mosaic egg chamber containing Cbl mutant clones and an HRP-grk transgene were obtained from females of the genotype e22cFLP/grkHFHRP-grk; CblF165FRT80B/MFRT80B. (A,B) Gurken expression (green) pattern was not markedly altered in egg chambers containing Cbl mutant follicle cell clones. (C,D,F-H) Punctate HRP-Grk signal was significantly reduced in the posterior (C,D), dorsal (F,G) and ventral (H) follicle cells, in which Cbl was homozygous mutant (areas indicated by brackets). When a large Cbl mutant clone was present in the dorsal region, the punctate HRP-Grk signal could be detected in follicle cells situated more posteriorly (F, arrows indicate punctate HRP signals; G, open stars indicate the 10th follicle cells) than in wild-type egg chambers (E). The 1st main body follicle cell was the one overlying the border between nurse cells and the oocyte (indicated by a star). Scale bars: 40 µm in A,B; 20 µm in C,D; 10 µm in E-H.

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