Reduction of brat expression does not cause enhanced growth or margin defects during normal development (Related to Fig 1).

(A) Adult wing area measured using ImageJ after mounting and imaging wings, for undamaged control (w¹¹¹⁸) (n = 63 female and 70 male) and brat¹/+ (n = 38 female and 48 male) wings. rnGAL4, GAL80^ts/TM6B females were crossed to w¹¹¹⁸ or brat¹/SM6-TM6B males and taken through the protocol shown in Fig 1A. Differences in size are extremely small though statistically significant (p = 0.01 for females and 2.15x10^-14 for males). (B) Adult wing sizes after disc regeneration for control (w¹¹¹⁸) (n = 599), brat¹/+ (n = 199), brat¹⁹²/+ (n = 237), brat¹⁵⁰/+ (n = 235) and brat¹¹/+ (n = 188) wings, from three independent experiments. (C) Margin defects detected in adult wings from undamaged control (w¹¹¹⁸) and brat¹/+ discs. rnGAL4, GAL80^ts/TM6B females were crossed to w¹¹¹⁸ or brat¹/SM6-TM6B males and taken through the protocol shown in Fig 1A. Margin defects detected in the undamaged wings were never as severe as the ones seen in brat¹/+ wings after disc regeneration. A representative wing with margin defects is shown. (D) Anti-Brat immunostaining in undamaged control (attP2) and bratRNAi/+ discs. rnGAL4, GAL80^ts/TM6B females were crossed to attP2 or bratRNAi males. Larvae were kept at 18°C and shifted to 30°C on day 7 AEL. Discs were dissected 24 hours after the shift to 30°C. Quantification of Brat fluorescence intensity in undamaged control (attP2) (n = 15) and bratRNAi/+ (n = 15) discs. Area for fluorescence intensity measurement was defined by wing pouch morphology and Anti-Myc co-immunostaining. * p = 0.02. (E) Margin defects detected in adult wings from undamaged control (attP2) and bratRNAi dics. rnGAL4, GAL80^ts/TM6B females were crossed to attP2 or bratRNAi males. Larvae were kept at 18°C and shifted to 30°C on day 7 AEL and kept there until eclosion. (F) Frequency of margin defects seen in adult wings after disc regeneration for control (w¹¹¹⁸) (n = 240), brat¹/+ (n = 191), brat¹⁹²/+ (n = 196), brat¹⁵⁰/+ (n = 213) and brat¹¹/+ (n = 152) wings. Wings in (F) are from the same experiments as (B). (G) Quantification of fluorescence intensity of anti-Brat immunostaining at R0 in control (n = 10) and UAS-bratRNAi (n = 5) discs showing no reduction of protein levels. Error bars represent SEM. Student’s T-test used for statistical analyses. Scale bars are 100 μm. Scale bars for adult wings are 0.5 mm.

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