Ferritin heavy chain protects the developing wing from reactive oxygen species and ferroptosis

Abstract

The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.

Fig 1. Knockdown of ferritin subunits causes growth defects and partially supresses overgrowth induced upon hippo knockdown.

(a-f) Representative male wings of indicated genotypes. (b) NubGal4 > VDRC 49536, (c) NubGal4 > VDRC 14491 (g) Quantification of wing areas. Minimum 8 wings were measured for each genotype and the average wing areas were plotted with standard error. The differences are significant between all pairs (student’s t-test, p < 0.05). Also see the related S2 Fig. All images are shown at the same scale and the scale bar in (a) corresponds to 100 microns.

Fig 2. Knockdown of the ferritin heavy chain in the pouch influences proliferation and induces excessive cell death.

Representative third instar wing imaginal discs with Nub-Gal4 driven overexpression of GFP (a and f), the weak Fer1HCH–RNAi (49536 (b and g)), the strong Fer1HCH -RNAi (12925 (c-d, h-i)) and Fer2LCH -RNAi (14491 (e and j)), in the pouch, at indicated time points. (a’-e’) shows the TUNEL staining in gray, and (f-j) shows EdU staining in gray. Wingless (Wg) staining (red in c-e) surrounds the pouch and marks the D/V boundary. All images are shown at the same scale and the scale bar in (a) corresponds to 50 microns.

Fig 3. Cells mutant for the ferritin heavy chain cannot compete against Minute cells.

Mosaic analysis of Fer1HCH⁴⁵¹, Fer2LCH³⁵ and Df(3R)Fer mutant chromosomes in wild-type (a-e, k) and Minute backgrounds (f-j and l-p). The disc size quantifications shown are from day 5 mosaic discs in a wild-type (k) and Minute backgrounds (l). Statistical significance is indicated as ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤ 0.0001. (g’) shows the GFP channel alone in gray. All discs are shown at the same scale except for the insets (i’) and (o’); they are magnified (2x zoom) and show only the GFP channel in gray. Scale bar in (o) is 100 microns. Genotypes are: a) y ubx-flp; FRT82B ubiGFP / FRT82B b) y ubx-flp; FRT82B ubiGFP / FRT82B Fer1HCH⁴⁵¹ c) y ubx-flp; FRT82B ubiGFP / FRT82B Fer2LCH³⁵ d) y ubx-flp; FRT82B ubiGFP / FRT82B Df(3R)Fer f) y ubx-flp; FRT82B M(3) ubiGFP / FRT82B g-m) y ubx-flp; FRT82B M(3) ubiGFP / FRT82B Fer1HCH⁴⁵¹ h-n) y ubx-flp; FRT82B M(3) ubiGFP / FRT82B Fer2LCH³⁵ i-o) y ubx-flp; FRT82B M(3) ubiGFP / FRT82B Df(3R)Fer.

Fig 4. Ferritin knockdown leads to drastic mitochondrial defects, reminiscent of ferroptosis.

Representative TEM images of mitochondria from (a) control discs and discs with ptc-gal4 driven (b) Fer1HCH knockdown with a strong RNAi line (102406), (c) the weaker RNAi-line (49536), (d) Fer2LCH knockdown (14491), (e) Fer1HCH overexpression and (f) Fer2LCH overexpression. (g) Quantification of mitochondria based on morphology. Intact mitochondria with their cristae visible were considered normal. Also see S6E–S6F Fig for examples for normal and abnormal mitochondria. We scored all mitochondria in at least two composites from different discs (min 74, max 2288 mitochondria per genotype). Error bars represent standard error. All images are shown at the same magnification. Scale bar in (a) is 500nm.

Fig 5. Low levels of the Ferritin heavy chain cause ROS accumulation.

(a-e) Representative wing discs of indicated genotypes. Dorsal is up, anterior is to the left. hh-Gal is expressed in the posterior compartment and nub-Gal4 is expressed in the pouch (used in panel b). (d’) shows the GstD1-GFP signal alone of the disc shown in panel (d). All discs are from day 5 larvae with the exception of panel (b), which shows a day 8 disc. (f-i) Representative wings of indicated genotypes and (j) quantification of their areas normalized to control wings (minimum 10 wings per genotype) are shown. Error bars represent standard error. P-values for t-tests are: hh-Gal4 vs Fer1HCH-i-w (p≤ 0.0001); hh-Gal4 vs Fer1HCH-i-w + SOD:Cat (p≤ 0.01); Fer1HCH-i-w vs Fer1HCH-i-w + SOD:Cat (p≤ 0.001). All discs and all wings are shown at the same scale. Scale bars in (a) and (f) are 50 and 100 microns respectively.

Fig 6. Excess Ferritin in warts discs protects against ROS formation.

(a-d’) Representative wing discs of indicated genotypes. The weaker Fer1HCH-RNAi line (49536) was used in these experiments. Dorsal is up, anterior is to the left. hh-Gal is expressed in the posterior half of the discs. Ptc (in red) marks the A/P boundary. GstD1-GFP reporter indicates ROS accumulation. DAPI labels nuclei. Scale bar in (a’) is 50 microns.