Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila

Abstract

Regulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.

Fig. 1. Knockdown of Top2 causes reduced wing growth.

A, B Knockdown of Top2 reduces wing size in females. The A/P boundary is indicated by a dashed line. PCV and ACV are posterior and anterior crossvein, respectively. L2-L5 are longitudinal veins. (A) en > GFP/+ (n = 12, x̅ ± s = 1 ± 0.039), (B’) en > GFP > Top2 RNAi^JF01300 (n = 12, x̅ ± s = 0.945 ± 0.031). Scale bars are 300 µm. (B′) An enlarged view of (B). Knockdown of Top2 shows defects in the region between L3 and L5 veins in the female wing. (C, D) Knockdown of Top2 reduces wing in male. (C) en > GFP/+ (n = 12, x̅ ± s = 1 ± 0.039), (D) en > GFP > Top2 RNAi^JF01300 (n = 13, x̅ ± s = 0.850 ± 0.092). (D′) An enlarged view of (D). Knockdown of Top2 shows loss of wing area including L4 in males. The distal region of L4 and 5 veins are thicker than normal. (E) Quantification of relative wing sizes in (A, B). (F) Quantification of relative wing sizes in (C, D). Statistical analysis in E, F by unpaired two-tailed student t-test, ***P < 0.001, ****P < 0.0001. Error bars in (E, F) are SD. Scale bars are 300 μm (A, C) and 100 μm (B′, D′).

Fig. 2. Top2 RNAi increases cell death.

A–B’’’ en > GFP/+ shows no change in the DAPI and Dcp-1 staining in the posterior region at 29 °C. A–A’’’ Apical section. B-B’’’ Basal section. Staining for GFP, DAPI, and Dcp-1 is as indicated in each panel. Yellow dotted lines in (A’–D’’) indicate the A/P boundary. Scale bars in (A–D) are 50 µm. a anterior, p posterior. C–D’’’ en > GFP > Top2 RNAi^JF01300 shows increased Dcp-1 staining in the posterior region at 29 °C (3/3 discs; 100%). C–C’’’ Apical section. Dcp-1 staining is weakly increased (White arrow) (C”). D–D’’’ Basal section. Dcp-1 staining is strongly increased (White arrow in D"). DAPI staining is decreased (White arrow) in the posterior apical region (C’) while it is accumulated (White arrow) in the posterior basal region (D’).

Fig. 3. Knockdown of Top2 causes defects in eye and head development.

A–D Knockdown of Top2 in females by three strong RNAi lines using ey-GAL4 causes pupal lethality at 25 °C. A ey/+ control. B–D Phenotypes in the head regions from dead pupae. B ey > Top2 RNAi^GD4570. 100% late pupal lethality (N = 52). C ey > Top2 RNAi^VSH330177. 100% late pupal lethality (N = 46). (D) ey > Top2 RNAi^10223R-2. 94.2% late pupal lethality (N = 52) (All three escapers are females). Scale bars in (A) and (E) are 150 µm. Note that depletion of Top2 in male eye discs by four different RNAi lines (B–D and Top2 RNAi^JF01300) causes pupal lethality with similar defects in the head. F–I Variable eye phenotypes of ey > Top2 RNAi^JF01300 adult females. E ey/+ control. F no eye (8%, N = 50), (G) Strong reduction (4%), (H) Intermediate reduction (26%), (I) Mild reduction (62%). N number of animals.

Fig. 4. Top2 shows genetic interaction with Tctp in organ growth.

A–D Top2^Suo1/+ enhances Tctp RNAi eye phenotype in females. A ey/+ (n = 10, x̅ ± s = 1 ± 0.05), (B) Top2^Suo1/+ (n = 10, x̅ ± s = 0.933 ± 0.034), (C) ey > Tctp RNAi/+ (n = 10, x̅ ± s = 0.687 ± 0.06), (D) ey > Tctp RNAi/Top2^Suo1 (n = 8, x̅ ± s = 0.569 ± 0.069). Scale bar in (A) is150 µm. E Quantification of relative eye sizes in (A–D). Statistical analysis in E by unpaired two-tailed student t-test, **P < 0.01 and ****P < 0.0001. Error bars in (E) are SD. F–M Wing phenotypes of Tctp RNAi and Top2^Suo1/+ in males (F–I) and females (J–M). F, J nub/+ control, (G, K) Top2^Suo1/+, H, L Intermediate phenotype showing wing size reduction, (I, M) Strong phenotype showing size reduction with severe folding. Scale bars in (F), (J) are 300 µm. N, O Genetic interaction between Tctp RNAi and Top2^Suo1/+ in males (N) and females (O). Top2^Suo1/+ weakly enhances the intermediate phenotype to the strong phenotype in males (N) while strongly enhancing it in females (O). N number of animals.

Fig. 5. Tctp RNAi reduces Top2 protein levels.

A–A’’’ en > GFP/+ shows no significant change in the Top2 level in the posterior domain at 25 °C. A GFP, (A’) DAPI, (A’’) Top2, (A’’’) Merge. Scale bars in (A–D) are 50 µm. B-B’’’ en > GFP > Tctp i shows a decrease in Top2 level at 25 °C. (11/14 discs; 78.6%) (B) GFP, (B’) DAPI, (B’’) Top2, (B’’’) Merge. The white arrow shows an area of decreased Top2 in the posterior region. C–C’’’ en > GFP > Diap1 shows little change in the Top2 level at 25 °C. (C) GFP, (C’) DAPI, (C’’) Top2, (C’’’) Merge. D–D’’’ en > GFP > Tctp i > Diap1 shows rescue of Top2 level at 25 °C (8/12 discs; 75%). D GFP, (D’) DAPI, (D’’) Top2, (D’’’) Merge. E, F Western blots stained for Top2, Tctp, and β-Tubulin. (E) Top2 protein level is decreased in Tctp-depleted S2 cells (representative of three independent experiments). F Tctp protein level is decreased in Top2-depleted S2 cells (representative of three independent experiments).

Fig. 6. Overexpression of Top2 can partially suppress the Tctp RNAi phenotype.

A, B Genetic interaction between Tctp RNAi and Top2 overexpression by UAS-Top2 lines (UAS-Top2-1 to UAS-Top2-5). A Females. Control progeny (nub > Tctp i/+) from a cross between nub > Tctp RNAi and w¹¹¹⁸ flies shows 100% wing defects with no suppression. Tctp RNAi phenotypes are suppressed by Top2 overexpression from three out of five UAS-Top2 lines (lines 2, 3, and 5) (B) Males. Control progeny (nub > Tctp i/+) shows 100% wing defects with no suppression. Tctp RNAi phenotypes are suppressed by Top2 overexpression from all five UAS-Top2 lines. N number of animals. Numbers in the bars indicate % adults showing the suppression.