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The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function.

Tanaka-Matakatsu M, Miller J, Du W - Protein Cell (2014)

Bottom Line: These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction.These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects.Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

View Article: PubMed Central - PubMed

Affiliation: Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, 60637, USA.

ABSTRACT
Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

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Eyeless promotes context-dependent cell growth in wing discs. (A) Diagram of third instar larval wing disc shows three distinct regions. The region between outer- and inner-white circles will develop into the fly wing hinge, while inner pouch region forms fly wing blade. The bottom Notum region will develop into the future scutellum. (B–D) Ey expressing Flp-out clones (marked with GFP in green) in the three distinct regions (white arrows). Wing discs were counter stained with DAPI (blue). The median size clones are shown. The scale bars are 50 μm in (A) and 100 μm in (B–D). (E) Box-whisker plot for FLP-out ectopic clone size induced in the pouch, notum and hinge regions. Number of clones (n) are indicated in the figure for each genotype. Single heat shock at 34°C for 10 min was applied at AEL 47 ± 1 h larva and cultured additional 48 h at 25°C. The P values to the β-Gal expressing control clone are: P = 0.064 (Ey), P = 0.58 × 10−9 (Ey Sd) in pouch. The P = 5.62 × 10−5 (Ey) and P = 0.32 (Ey Sd) in notum, the P < 0.95 (Ey), P = 2.28 × 10−7 (Ey Sd) in hinge
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Fig1: Eyeless promotes context-dependent cell growth in wing discs. (A) Diagram of third instar larval wing disc shows three distinct regions. The region between outer- and inner-white circles will develop into the fly wing hinge, while inner pouch region forms fly wing blade. The bottom Notum region will develop into the future scutellum. (B–D) Ey expressing Flp-out clones (marked with GFP in green) in the three distinct regions (white arrows). Wing discs were counter stained with DAPI (blue). The median size clones are shown. The scale bars are 50 μm in (A) and 100 μm in (B–D). (E) Box-whisker plot for FLP-out ectopic clone size induced in the pouch, notum and hinge regions. Number of clones (n) are indicated in the figure for each genotype. Single heat shock at 34°C for 10 min was applied at AEL 47 ± 1 h larva and cultured additional 48 h at 25°C. The P values to the β-Gal expressing control clone are: P = 0.064 (Ey), P = 0.58 × 10−9 (Ey Sd) in pouch. The P = 5.62 × 10−5 (Ey) and P = 0.32 (Ey Sd) in notum, the P < 0.95 (Ey), P = 2.28 × 10−7 (Ey Sd) in hinge

Mentions: Recent studies indicate that Pax6 regulates multiple transcriptional networks that regulate cell proliferation as well as differentiation (Farhy et al., 2013). To characterize the effect of Eyeless on the rate of cell growth and proliferation in vivo, we used the “flip-out” GAL4 driver (Act>GAL4) approach to co-activate permanent, heritable expression of UAS regulated targets in random clones of cells (Duman-Scheel et al., 2002; Neufeld et al., 1998; Xin et al., 2002). This technique uses heat shock to induce FLP recombinase, which induces the generation of random clones of GAL4 expression cells at precisely defined time point for cell growth analysis. Specifically 47 ± 1 h larvae were heat shocked to turn on Gal4 expression, which activates UAS-target gene expression including the UAS-GFP in discrete clones in imaginal discs. After another 48 h of growth, the wing discs were dissected and the size of the clones was analyzed. Wing disc can be divided into the wing pouch, hinge, and the notum region (Fig. 1A). Analysis of the control and the Ey-expressing clones in different wing disc regions revealed that the ability of Ey to promote growth varies depending on the region in which the clone is located. While Ey expressing clones are significantly larger than the β-Gal controls located in the notum region, no significant difference were observed for the clones located in the pouch or hinge regions (Fig. 1B–E). These results show that Ey can promote cell growth in a context-dependent manner.Figure 1


The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function.

Tanaka-Matakatsu M, Miller J, Du W - Protein Cell (2014)

Eyeless promotes context-dependent cell growth in wing discs. (A) Diagram of third instar larval wing disc shows three distinct regions. The region between outer- and inner-white circles will develop into the fly wing hinge, while inner pouch region forms fly wing blade. The bottom Notum region will develop into the future scutellum. (B–D) Ey expressing Flp-out clones (marked with GFP in green) in the three distinct regions (white arrows). Wing discs were counter stained with DAPI (blue). The median size clones are shown. The scale bars are 50 μm in (A) and 100 μm in (B–D). (E) Box-whisker plot for FLP-out ectopic clone size induced in the pouch, notum and hinge regions. Number of clones (n) are indicated in the figure for each genotype. Single heat shock at 34°C for 10 min was applied at AEL 47 ± 1 h larva and cultured additional 48 h at 25°C. The P values to the β-Gal expressing control clone are: P = 0.064 (Ey), P = 0.58 × 10−9 (Ey Sd) in pouch. The P = 5.62 × 10−5 (Ey) and P = 0.32 (Ey Sd) in notum, the P < 0.95 (Ey), P = 2.28 × 10−7 (Ey Sd) in hinge
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Eyeless promotes context-dependent cell growth in wing discs. (A) Diagram of third instar larval wing disc shows three distinct regions. The region between outer- and inner-white circles will develop into the fly wing hinge, while inner pouch region forms fly wing blade. The bottom Notum region will develop into the future scutellum. (B–D) Ey expressing Flp-out clones (marked with GFP in green) in the three distinct regions (white arrows). Wing discs were counter stained with DAPI (blue). The median size clones are shown. The scale bars are 50 μm in (A) and 100 μm in (B–D). (E) Box-whisker plot for FLP-out ectopic clone size induced in the pouch, notum and hinge regions. Number of clones (n) are indicated in the figure for each genotype. Single heat shock at 34°C for 10 min was applied at AEL 47 ± 1 h larva and cultured additional 48 h at 25°C. The P values to the β-Gal expressing control clone are: P = 0.064 (Ey), P = 0.58 × 10−9 (Ey Sd) in pouch. The P = 5.62 × 10−5 (Ey) and P = 0.32 (Ey Sd) in notum, the P < 0.95 (Ey), P = 2.28 × 10−7 (Ey Sd) in hinge
Mentions: Recent studies indicate that Pax6 regulates multiple transcriptional networks that regulate cell proliferation as well as differentiation (Farhy et al., 2013). To characterize the effect of Eyeless on the rate of cell growth and proliferation in vivo, we used the “flip-out” GAL4 driver (Act>GAL4) approach to co-activate permanent, heritable expression of UAS regulated targets in random clones of cells (Duman-Scheel et al., 2002; Neufeld et al., 1998; Xin et al., 2002). This technique uses heat shock to induce FLP recombinase, which induces the generation of random clones of GAL4 expression cells at precisely defined time point for cell growth analysis. Specifically 47 ± 1 h larvae were heat shocked to turn on Gal4 expression, which activates UAS-target gene expression including the UAS-GFP in discrete clones in imaginal discs. After another 48 h of growth, the wing discs were dissected and the size of the clones was analyzed. Wing disc can be divided into the wing pouch, hinge, and the notum region (Fig. 1A). Analysis of the control and the Ey-expressing clones in different wing disc regions revealed that the ability of Ey to promote growth varies depending on the region in which the clone is located. While Ey expressing clones are significantly larger than the β-Gal controls located in the notum region, no significant difference were observed for the clones located in the pouch or hinge regions (Fig. 1B–E). These results show that Ey can promote cell growth in a context-dependent manner.Figure 1

Bottom Line: These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction.These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects.Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

View Article: PubMed Central - PubMed

Affiliation: Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, 60637, USA.

ABSTRACT
Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

Show MeSH
Related in: MedlinePlus