Transcriptional regulation of Nplp1 in adult Tv1 neurons.

(A–F) col^dsRNAi downregulated Nplp1 but not ap, eya, or dimm in adult Tv1. (A,B) Expression of Nplp1 (red), ap^Gal4,UAS-nEGFP (green) and Col (blue) in adult Tv4 neurons at A10 at 29°C. Col expression is lost and Nplp1 is downregulated in col^dsRNAi (B) compared to w¹¹¹⁸ control (A). (C) Quantification of Nplp1 immunoreactivity in w¹¹¹⁸ (n = 29) and col^dsRNAi (n = 20) at A10 at 29°C. (D–F) col^dsRNAi did not affect Dimm immunoreactivity (D), ap^Gal4,UAS-nEGFP fluorescence (E) or Eya immunoreactivity (F) compared to w¹¹¹⁸ at A10 at 29°C. col^dsRNAi (D n = 24; E n = 22; F n = 21). w¹¹¹⁸ control (D n = 25; E n = 27; F n = 24). (G–I) ap^dsRNAi significantly reduced Nplp1 (G) and Dimm (H) immunoreactivity compared to w¹¹¹⁸ at A20 at 29°C. (G) * p<0.0001 ap^dsRNAi (n = 30) compared to w¹¹¹⁸ control (n = 36). (H) * p<0.001 ap^dsRNAi (n = 13) compared to w¹¹¹⁸ control (n = 22) (I) Dimm restoration (UAS-dimm) in ap^dsRNAi background only partially rescued Nplp1 downregulation at A15 at 29°C. * p<0.0001 ap^dsRNAi (n = 22) compared to w¹¹¹⁸ control (n = 19). ** p<0.0001 ap^dsRNAi;UAS-dimm (n = 23) compared to ap^dsRNAi and w¹¹¹⁸ controls. (J–L) eya^dsRNAi significantly reduced Nplp1 (J) and Dimm (K) immunoreactivity compared to w¹¹¹⁸ at A10 at 29°C. (L) Dimm restoration (UAS-dimm) in eya^dsRNAi background failed to rescue Nplp1 immunoreactivity at A15 at 29°C. * p<0.0001 eya^dsRNAi and eya^dsRNAi; UAS-dimm (n = 19) compared to w¹¹¹⁸ control. NSD, no significant difference between eya^dsRNAi and eya^dsRNAi;UAS-dimm. eya^dsRNAi (J n = 23; K n = 23; L n = 22). w¹¹¹⁸ control (J n = 30; K n = 25; L n = 26). (M–O) dimm^dsRNAi downregulated Nplp1 and PHM in adult Tv1. (M,N) Tv1 neurons expressing Nplp1 (red), ap^Gal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. Nplp1 is downregulated and PHM is lost in dimm^dsRNAi (n = 18) (M) compared to w¹¹¹⁸ control (n = 15) (N). (O) dimm^dsRNAi significantly reduced Nplp1 immunoreactivity compared to w¹¹¹⁸ at A10 at 29°C. * p<0.0001 dimm^dsRNAi (n = 19) compared to w¹¹¹⁸ control (n = 18). (P) Model depicting regulation of Nplp1 and PHM and the configuration changes between ap, eya, dimm and col from embryonic to adult Tv1. Notably, col no longer regulates dimm, eya or ap expression (denoted by X). Genotypes: w¹¹¹⁸ (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+); col^dsRNAi (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-col^dsRNAi); ap^dsRNAi (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi); ap^dsRNAi; UAS-dimm (UAS-dicer2/+; ap^Gal4/UAS-dimm; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi); eya^dsRNAi (UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+); eya^dsRNAi; UAS-dimm (UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/UAS-dimm; tub-Gal80^TS, UAS-nEGFP/+); dimm^dsRNAi (UAS-dicer2/+; ap^Gal4/UAS-eya^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+); rev4,dimm^dsRNAi (UAS-dicer2/+; ap^Gal4/rev4, UAS-dimm^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+).