EBRs are required in vivo for regulation of tissue growth. (A) EBR deletion does not affect Ft levels. Immunoblot of trans-heterozygous EBR allele L3 wing discs. Compared to cntrl (ft::FLAG), there is no change in Ft levels in the EBR alleles. Proteins were analyzed by immunoblotting with the indicated antibodies. Actin was used as a loading control. Ft presents as multiple bands due to proteolytic processing (Feng and Irvine, 2009; Sopko et al., 2009). (B–D′) EBR deletion does not affect Ft levels or localization. XY confocal micrographs third instar wing imaginal discs containing of ft^EBR1 (B and B′), ft^ΔE (C and C′), or ft^EBR1/2 (D and D′) mutant clones (marked by absence of RFP shown in red) with Ft staining (shown in gray). XY images are orientated as dorsal up. Clonal boundaries are marked by yellow dotted lines. Scale bars are 10 µm. (E–I) Effect of trans-heterozygous EBR deletion on adult wing phenotypes. Phenotype of control (cntrl)—ft::FLAG (E), ft^EBR1 (F), ft^ΔE (G), bulk ft^EBR1/2 (class 1; H), or ft^EBR1/2 (class 2; I) wings. Scale bars are 500 µm. (J) Quantification of pupal lethality in trans-heterozygous EBR animals. Compared to cntrl (ft::FLAG) wings, ft^ΔE and ft^EBR1/2 cause a significant increase in pupal lethality. Data points indicate one vial of ∼30 pupae, with number of vials ≥10 per genotype, with mean and SD represented. ***P < 0.005 using one-way ANOVA with a Dunnett’s post-hoc test compared to the ft::FLAG control. ns denotes non-significant. (K) Quantification of adult wing size in trans-heterozygous EBR flies. Data are normalized against the mean of the cntrl (ft::FLAG). ft^EBR1 causes significant overgrowth. ft^EBR1/2 class 2 flies are isolated from the bulk ft^EBR1/2 to indicate the significant overgrowth. Data points indicate an individual wing (n ≥ 38 per genotype, class 2 flies n = 9), with mean and SD represented. ****P < 0.0001 using one-way ANOVA with a Dunnet’s post-hoc test compared to cntrl. ns denotes non-significant. (L) Quantification of adult wing roundness in trans-heterozygous EBR flies. Shape was determined by the ratio of wing length verses width. Data are normalized against the mean of the cntrl (ft::FLAG). ft^EBR1 and ft^ΔE are significantly rounder than cntrl. ft^EBR1/2 class 2 flies are isolated from the bulk ft^EBR1/2 to indicate the significant increase in roundness. Data points indicate an individual wing (n ≥ 38 per genotype, class 2 flies n = 9), with mean and SD represented. ***P = 0.0004 and ****P < 0.0001 using one-way ANOVA with a Dunnet’s post-hoc test compared to cntrl. ns denotes non-significant. WB, Western blot. Source data are available for this figure: SourceData FS4.