Visualization of Gurken distribution in Follicle cells

Transcription

1 Visualization of Gurken distribution in Follicle cells Wei-Ling Chang,Hsiao-Chun Pen, Yu-Wei Chang, He-Yen Chou, Willisa Liou, Li-Mei Pai Institute of Basic Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan

2 Abstract The asymmetrically localized Gurken - a TGF- homolog, acts as a morphogen to activate the Egfr in follicle cells for the axes establishment in eggs and embryos. During early stages of oogenesis, Gurken could be easily detected in the posterior follicle cells. However, Gurken is hardly detected in follicle cells during middle stages of oogenesis. Here, we have generated a HRP-Gurken fusion protein which facilitated the examination of Gurken in the endocytic route inside follicle cells. The HRP-Gurken fusion protein is functional as it rescues the phenotype of gurken null mutant. During the middle stages of oogenesis, the HRP-Gurken fusion protein is detected not only in the dorsal but also in the ventral side of follicle cells using confocal microscopy and transmission electronic microscopy. The HRP-Gurken fusion protein is internalized into follicle cells mediated by Shibire and through Rab5-associated endocytic pathways. Furthermore, the HRP-Gurken fusion protein was detected in the endocytic vesicles in follicle cells under transmission electronic microscopy. These results indicate that Gurken-Egfr complex are likely internalized into follicle cells and processed through endocytic pathways to terminate Egfr signaling. Moreover, Egfr signaling activated by Gurken was, to our knowledge, the first time visualized in the ventral follicle cells, which is consistent with hypothetic and genetic evidence. It has been well known that Cbl acts as an endocytic adaptor to mediate internalization of Egfr. Here, we document that D-Cbl facilitates the internalization of Gurken-Egfr complex into follicle cells since no HRP-Gurken was detected in D-cbl mutant cells and yet an accumulation of HRP-Gurken- positive vesicles are observed in the adjacent normal follicle cells. Taken together, these results suggest that the gradient of Gurken morphogen is regulated by the internalization of Gurken-Egfr complex through D-cbl.

3 Introduction The asymmetrically localized Gurken a TGF-a homolog, acts as a morphogen to activate the Egfr in follicle cells for the axes establishment in eggs and embryos. During early stages of oogenesis when Gurken defines the posterior follicle cell fate, Gurken could be easily detected in the posterior follicle cells. However, Gurken is hardly detected in follicle cells during middle stages of oogenesis. In order to visualize Gurken in follicle cells when Gurken determines the dorsoventral axis, we used HRP as a tracer to study the endocytic trafficking of Gurken because HRP is relatively stable within the destructive environment of the endosome and lysosomal compartments. Using immunocytochemistry by fluorescence microscopy and by electron microscopy, we study the distribution and the signal process of Gurken protein in egg chambers. It has been well known that Cbl acts as an endocytic adaptor to mediate internalization of Egfr. Over-expression of D-CblL resulted in phenotypes characteristic of reduction of Egfr signaling. According to early report, the EGFR pathway primary downstream target gene kekkon was ectopically expressed in the ventral side of egg chambers in Cbl mutant cells. These results suggest that ventral follicle cells are exposed to some Gurken protein which leads to activate EGFR pathway in Cbl mutant region. Here we are interested in whither Gurken express in the ventral side of the oocyte and how Cbl regulate EGFR signaling pathway during Drosophila oogenesis.

4 Result Figure 1. The subcellular localization of Gurken in follicle cells The subcellular localization of Gurken in posterior follicle cells during early oogenesis stages were showed from A-C. (A) It is easily to detect the punctate distribution of Gurken in the apical side of posterior follicle cells. (B) These punctate signals were significantly reduced when Shibire mutant. (C) Gurken was colocalized with early endosomes, labeled with GFP-Rab5, in the posterior follicle cells. These data indicate that Gurken is internalized into posterior follicle cells mediated by Shibire and through Rab5 associated endocytic pathway during the stages when Gurken defines the posterior follicle cell fate. (D and E) The asymmetrically localization of Gurken during middle oogenesis stages at stage 8 and 9 respectively. (D and E ) The big magnification from D and E shown only small Grk signals were detected in the dorsal follicle cells. Bars, 10 m (A-C, and D ); 20 m (D and E ); 40 m (E). Figure 2. The Gurken-Egfr complex is detected in the endocytic pathway of not only dorsal but also ventral follicle cells (A) The HRP-Gurken fusion protein. HRP was inserted into the gurken genomic DNA sequence, just downstream of the signal peptide domain and upstream of the EGF repeat domain. (B) The wild type egg chambers labeled with HRP antibody as a negative control. (C-E) The egg chambers with HRP-Grk fusion protein labeled with HRP antibody at different oogenesis stages. All egg chambers have the same patterns compared with Gurken staining. (F-G) With the HRP staining, HRP-Grk could be easily detected not only in the dorsal follicle cells but also in ventral follicle cells. (H-I) These punctate signals were significantly reduced when shibire mutant both in

5 the dorsal and ventral follicle cells. (J-J ) These HRP signals in follicle cells were colocalized with early endosomes, labeled with GFP-Rab5, in the dorsal and ventral follicle cells (arrows). (K-K ) In the Egfr mutant cells marked with no GFP signal, no HRP signal in follicle cells was detected (arrow hand). (L-M) Egf receptor also internalized into follicle cells through Rab5 and Rab7 endocytic pathway. These results suggest that the HRP signal in follicle cells is dependent on Egfr, indicating that Gurken binds to Egfr and the ligand-receptor complex enter into follicle cells mediated by Dynamin and through Rab5 associated endocytic pathway during the stages when Gurken defines the dorsoventral cell fate. Bars, 20 m (B-E); 10 m (F-K). Table 1. The biological function of HRP-Gurken in rescuing the phenotypes of grk mutants The ventralized egg shalls laid by gurken mutant could be rescued by the HRP-Gurken transgene. One copy of HRP-Gurken almost completely reverted heterozygous mutant phenotype to the wild type (99%) and two copies of this transgene rescued to a degree of that 60% eggs were normal. It indicated that HRP-Gurken fusion protein can active Egfr signaling as endogenous Gurken does, and also displays dosage dependent effects. Figure 3. The HRP-Gurken protein is degraded in the lysosome compartment (A-B) To obtain ultrathin sections that included oocyte nucleus, 0.5 micron semithin sections were first cut with long axis (used for C-F) and short (used for G-H) cross section respectively. (C-D) The HRP immuno-labeling result is the same with previous Gurken immuno-labeling, which shown that the HRP-Gurken positive ter-golgi units is significantly concentrated on the D/A corner of the oocyte and their

6 labeling density in ventral side of oocyte is much weaker. (E-F) The HRP-positive MVB or lysosome could be detected both at the dorsal and ventral follicle cells in early stage 9 egg chambers. These data indicated that after the ligand of Gurken bound to its receptors, they internalized into follicle cells and go through lysosome-degraded pathway. (G-H) The area of MVB/ lysosome is bigger in the lateral follicle cells than in the dorsal follicle cells. This result indicated that the degraded activity is higher in the lateral follicle cells. Bars, 100nm (C-F); 200nm (G-H). Figure 4. The role of D-Cbl in regulation of Gurken distribution In wild type, Grk has an accumulate pattern along the anterior-poster axis in late stage 9 egg chambers, about 89%. (B) This pattern was slightly reduced (78%) in the egg chambers with overexpression of D-CblSA1 and (C) significantly reduced (53%) in the egg chambers with overexpression of D-CblLA18. (D-F) gurken RNA expression pattern has no different between wild type egg chamber and D-CblS & L overexpression egg chambers, indicating that the reduce was accrued in protein level. (G)The punctate HRP signals were dramatically increased in follicle cells with expression of D-CblL. These results suggest the promotion of Gurken-Egfr internalization by D-CblL can change the distribution of Gurken morphogen. (H) On the other hand, a sever reduction of internalized Gurken-Egfr complex in D-cbl mutant follicle cells (myc-negative) was observed, indicating that the internalization of Gurken-Egfr complex requires the function of D-Cbl. Bars, 40 m (A-F); 20 m (G); 10 m (H).

7 Conclusion Egfr signaling activated by Gurken was, to our knowledge, the first time visualized in the ventral follicle cells.the Gurken-Egfr complex is internalized into follicle cells mediated by Shibire and through Rab5 associated endocytic pathways. The density of Gurken in the dorsal and ventral follicle cells is the same in the early stage 9 egg chambers. D-Cbl facilitates the internalization of Gurken-Egfr complex into follicle cells. The gradient of Gurken morphogen is regulated by the internalization of Gurken-Egfr complex through D-cbl.

8 Figure Legends Figure 1. The subcellular localization of Gurken in follicle cells Figure 2. The Gurken-Egfr complex is detected in the endocytic pathway of not only dorsal but also ventral follicle cells

9 Genotype OreR grkhf/+ grkhf/grkhf grkhfhrp-grk-5/+ grkhfhrp-grk-5/ grkhf grkhfhrp-grk-5/grkhfhrp-grk-5 N 100% - 87% - 54% 1% 13% 38% 100% 46% 99% 62% Table 1. The biological function of HRP-Gurken in rescuing the phenotypes of grk mutants Figure 3. The HRP-Gurken protein is degraded in the lysosome compartment

10 Figure 4. The role of D-Cbl in regulation of Gurken distribution