Extended Data Fig. 7: Intestinal zinc stainings.

a, Validation of the zinc-sensitive dye, FluoZin-3AM, in adult and larval Malpighian tubules. The tubules of w¹¹¹⁸ adults have less zinc than those of wild-type (OrR) adults; levels can be increased by supplementing their adult diet with 1 mM ZnCl₂ for 3 days (left three panels). A more modest reduction in zinc levels is observed in larval tubules of second-instar w¹¹¹⁸ larvae relative to wild-type OrR larvae (right two panels). b, FluoZin-3AM staining in the middle midgut of second-instar wild-type larvae (OrR, which harbour a wild-type w gene), w mutant larvae (w¹¹¹⁸), w mutant larvae with a mini-w transgene (UAS-Rheb/+) and hodor mutant larvae (which are mutant for w but carry mini-w transgenes). # denotes the copper cell region and * denotes the iron cell region. The panels to the right show higher-magnification images of the copper cell region. Zinc levels are higher in the copper cell region of wild-type larvae relative to the other genotypes, which have comparable zinc levels. The bottom panel shows FluoZin-3AM staining of a wild-type (OrR) adult midgut. There is no apparent zinc enrichment in the copper cell region (#). c, Quantification of intestinal zinc in the copper cell region. In both c and d, larvae were raised on a low-yeast diet. d, Wild-type OrR larvae reach the pupal stage significantly faster than w¹¹¹⁸ in low-yeast conditions, whereas hodor^−/− still causes a significant developmental delay in either a genetic background with an intact w gene (w⁺;hodor^−/−) or when backcrossed eight times into a w mutant background lacking the w gene (w;hodor^−/−). e, Heterozygous lines carrying mini-w develop faster than w¹¹¹⁸ larvae in low-yeast conditions. Scale bars; 50 μm (a); 500 μm (b); 50 μm (inset). See Supplementary Information for sample sizes and full genotypes. For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. For cases in which more than two groups were compared, an ordinary one-way ANOVA was performed with a Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Box plots: line, median; box, 75th–25th percentiles; whiskers, minimum to maximum.