Reduction of Myc expression during regeneration also disrupts margin fate specification (Related to Fig 5).

(A) Margin defects detected in adult wings from undamaged control (w¹¹¹⁸) and UAS-Myc/+ discs. rnGAL4, GAL80^ts/TM6B females were crossed to w¹¹¹⁸ or UAS-Myc males and taken through the protocol shown in Fig 1A. (B) Frequency of margin defects in adult wings after disc regeneration for control (w¹¹¹⁸) (n = 103), brat¹/+ (n = 203), dm⁴/+ (n = 94) and dm⁴/+; brat¹/+ (n = 94) wings, from three independent experiments. (C) Quantification of Myc fluorescence intensity in undamaged control (w¹¹¹⁸) (n = 12) and dm⁴/+ (n = 11) discs. w¹¹¹⁸ males were crossed to w¹¹¹⁸ or dm⁴/FM7i, ActGFP females and dissected when the animals were third instar. Area for fluorescence intensity measurement was defined by wing pouch morphology and the elevated Myc expression domain in the wing pouch. (D) Quantification of Myc fluorescence intensity in R0 control (w¹¹¹⁸) (n = 13), R0 dm⁴/+ (n = 10), R24 control (w¹¹¹⁸) (n = 13), and R24 dm⁴/+ (n = 10) discs. Area for fluorescence intensity measurement was defined by the elevated Myc expression domain in the wing pouch. (E) Quantification of Myc fluorescence intensity in undamaged control (VDRC genetic background line, called control) (n = 14), MycRNAi#1/+ (n = 12), and MycRNAi#2/+ (n = 13) discs. rnGAL4, GAL80^ts/TM6B females were crossed to the control, MycRNAi#1, or MycRNAi#2 males. The animals were shifted to 30°C during early third instar and kept there for 28 hours then dissected. MycRNAi#1/+ *** p < 0.000007, MycRNAi#2/+ *** p < 0.00002. Area for fluorescence intensity measurement was defined by wing pouch morphology. (F) Quantification of Myc fluorescence intensity in R0 control (n = 13), R0 MycRNAi#1/+ (n = 15), R0 MycRNAi#2/+ (n = 13), R24 control (n = 13), R24 MycRNAi#1/+ (n = 13), and R24 MycRNAi#2/+ (n = 13) discs. Fluorescence intensity was measured in the area marked by Anti-Nubbin immunostaining. *** p < 0.00007. (G,H) Anti-Myc immunostaining in undamaged (G) and regenerating R24 (H) Myc^P0/Y imaginal wing discs. Dashed line outlines the wing pouch defined by anti-Nubbin immunostaining. (I) Margin tissue lost as a percentage of total wing perimeter for adult wings after disc damage and regeneration in the noted genotypes. (J, K) Immunostaining for Cut in control (J) and Myc^P0/Y (K) regenerated (R72) discs showing margin disruption in the mutants.(L) Pupariation rates after disc regeneration for control (w¹¹¹⁸) (n = 216), brat¹/+ (n = 114) and UAS-Myc/+ (n = 209) animals, from three independent experiments. Error bars represent SEM. Student’s T-test used for statistical analyses.

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