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. 2017 Aug 30;6:e25301. doi: 10.7554/eLife.25301

Figure 4. Babo, through dSmad2 and Tkv, through Mad, regulate Wts and Melt.

Graph Legend: Red is the percentage of R8 photoreceptors that expressed Rh6, Blue is the percentage that expressed Rh5 and Purple is Co-expression of Rh5 +Rh6. Error bars report standard error of the mean. Paired t-tests were performed against percentages of Rh5 expression and significance is denoted where applicable (***=p ≤ 0.01, **=p ≤ 0.05, ns = not significant). Where significance (or non-significance) is noted, follow the horizontal bar above the significance value to its termination in a vertical tick to find the comparative genotype. Percentage values for each genotype are listed as (Rh5/(Rh5 +Rh6)/Rh6). (A) Expressing UAS-Dcr2 and UAS-dSmad2RNAi with lGMR-Gal4 reduced pale subtypes (lGMR-Gal4 >UAS-Dcr2; UAS-dSmad2RNAi) (15/0/85) (n = 4) and this reduction was maintained even when UAS-Babo* was co-expressed (lGMR-Gal4 >UAS-Dcr2; UAS-dSmad2RNAi; UAS-Babo*) (23/0/77) (n = 7) (B). (C) Expressing UAS-Dcr2 and UAS-madRNAi with lGMR-Gal4 also reduced pale subtypes (lGMR-Gal4 >UAS-Dcr2; UAS-MadRNAi) (4/0/96) (n = 7). (D) Expressing UAS-Tkv* could not rescue MadRNAi-induced loss of pale subtypes (lGMR-Gal4 >UAS-Dcr2; UAS-MadRNAi, UAS-Tkv*) (6/0/94) (n = 9). (E) Quantification of A-D. (F) Wild-type retinas express Melt (red) and Wts (green) in mutually exclusive subsets of R8 cells; Melt (red) with Rh5 (blue) in the pale subset and Wts (green) with Rh6 (not shown) in the alternate, yellow R8 subset. (G) Removing wts (lGMR-Gal4 > UAS-Dcr2; UAS-WtsRNAi) (100/0/0) (n = 8) led to total loss of yellow subtype while removing melt (lGMR-Gal4 > UAS-Dcr2; UAS-MeltRNAi) (2/8/90) (n = 5) resulted in the opposite, loss of pale subtypes (H). (I) Expressing Babo* increased pale subtypes (blue), which was concomitant with a loss of Wts expression (green). (J) Removing melt in retinas that overexpress Babo* (lGMR-Gal4 > UAS-Dcr2; UAS-Babo*; UAS-MeltRNAi) (0/0/100) (n = 11) prevented Babo*-induced pale subtypes. (K) Overexpression of Tkv* increased the number of ommatidia expressing Rh5 (blue) in R8. In this genotype, increased Rh5 expression was often co-expressed with Rh6 (magenta). Increased Rh5 expression was coincident with loss of Wts, even in some instances where Rh6 was still present (white arrows); however, Wts remained in other R8 cells that co-expressed Rh5 + Rh6 (yellow arrowheads). (L) Removing melt from Tkv* retinas (lGMR-Gal4 > UAS-Dcr2; UAS-Tkv*; UAS-MeltRNAi) (7/3/90) (n = 46) resulted in a loss of Tkv*-induced Rh5 expression. (M) While significantly different from wtsRNAi controls (due to extremely low variance), removing wts prevented increased yellow subtypes seen in dSmad2RNAi (lGMR-Gal4 > UAS-Dcr2; UAS-dSmad2RNAi; UAS-WtsRNAi) (95/0/5) (n = 6) and MadRNAi (lGMR-Gal4 > UAS-Dcr2; UAS-MadRNAi; UAS-WtsRNAi) (97/0/3) (n = 12) (N) retinas. (O) lGMR-Gal4 > UAS Yki retinas exhibited 100% pale subtypes (100/0/0) (n = 7). (P) Expressing UAS-Yki overcame the high incident of yellow subtypes induced by expressing BaboRNAi (lGMR-Gal4 > UAS-Dcr2; UAS-BaboRNAi; UAS-Yki) (100/0/0) (n = 6), TkvRNAi (lGMR-Gal4 > UAS-Dcr2; UAS-TkvRNAi; UAS-Yki) (100/0/0) (n = 6) (Q), MadRNAi (lGMR-Gal4 >UAS-Dcr2; UAS-MadRNAi; UAS-Yki) (100/0/0) (n = 6) (R) and dSmad2RNAi (lGMR-Gal4 >UAS-Dcr2; UAS-dSmad2RNAi; UAS-Yki) (100/0/0) (n = 6) (S). (T) Quantification of G-H, J, L-S. Letters (P, Q, O…) have been added to applicable bars to help identify corresponding image panels.

Figure 4.

Figure 4—figure supplement 1. Med, Babo, and Tkv are required for R8 subtypes.

Figure 4—figure supplement 1.

Graph Legend: Red is the percentage of R8 photoreceptors that expressed Rh6, Blue is the percentage that expressed Rh5 and Purple is Co-expression of Rh5 + Rh6. Error bars report standard error of the mean. Paired t-tests were performed against percentages of Rh5 expression and significance is denoted where applicable (**=p≤0.05). N values are listed on the graph. Percentage values for each genotype are listed as (Rh5/(Rh5 + Rh6)/Rh6). (A) Removing the co-SMAD med resulted in decreased pale subtypes (15/1/83) similar to removing its binding partners mad or dSmad2 – compare to Dcr2; lGMR control (32/6/62). (B) Overexpressing Babo* using lGMR-Gal4 lead to expression of Melt (green) and Rh5 (blue). (C) Overexpression of Tkv* using lGMR-Gal4 also lead to expression of Melt (green) and Rh5 (blue), although in some cases where Rh5 and Rh6 become co-expressed (magenta), Melt expression remained absent (yellow arrowheads). These are likely the same ommatidia that retain wts expression (see Figure 4K).