Transcriptional regulation of Nplp1 in adult Tv1 neurons.

(A–F) coldsRNAi downregulated Nplp1 but not ap, eya, or dimm in adult Tv1. (A,B) Expression of Nplp1 (red), apGal4,UAS-nEGFP (green) and Col (blue) in adult Tv4 neurons at A10 at 29°C. Col expression is lost and Nplp1 is downregulated in coldsRNAi (B) compared to w1118 control (A). (C) Quantification of Nplp1 immunoreactivity in w1118 (n = 29) and coldsRNAi (n = 20) at A10 at 29°C. (D–F) coldsRNAi did not affect Dimm immunoreactivity (D), apGal4,UAS-nEGFP fluorescence (E) or Eya immunoreactivity (F) compared to w1118 at A10 at 29°C. coldsRNAi (D n = 24; E n = 22; F n = 21). w1118 control (D n = 25; E n = 27; F n = 24). (G–I) apdsRNAi significantly reduced Nplp1 (G) and Dimm (H) immunoreactivity compared to w1118 at A20 at 29°C. (G) * p<0.0001 apdsRNAi (n = 30) compared to w1118 control (n = 36). (H) * p<0.001 apdsRNAi (n = 13) compared to w1118 control (n = 22) (I) Dimm restoration (UAS-dimm) in apdsRNAi background only partially rescued Nplp1 downregulation at A15 at 29°C. * p<0.0001 apdsRNAi (n = 22) compared to w1118 control (n = 19). ** p<0.0001 apdsRNAi;UAS-dimm (n = 23) compared to apdsRNAi and w1118 controls. (J–L) eyadsRNAi significantly reduced Nplp1 (J) and Dimm (K) immunoreactivity compared to w1118 at A10 at 29°C. (L) Dimm restoration (UAS-dimm) in eyadsRNAi background failed to rescue Nplp1 immunoreactivity at A15 at 29°C. * p<0.0001 eyadsRNAi and eyadsRNAi; UAS-dimm (n = 19) compared to w1118 control. NSD, no significant difference between eyadsRNAi and eyadsRNAi;UAS-dimm. eyadsRNAi (J n = 23; K n = 23; L n = 22). w1118 control (J n = 30; K n = 25; L n = 26). (M–O) dimmdsRNAi downregulated Nplp1 and PHM in adult Tv1. (M,N) Tv1 neurons expressing Nplp1 (red), apGal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. Nplp1 is downregulated and PHM is lost in dimmdsRNAi (n = 18) (M) compared to w1118 control (n = 15) (N). (O) dimmdsRNAi significantly reduced Nplp1 immunoreactivity compared to w1118 at A10 at 29°C. * p<0.0001 dimmdsRNAi (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/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); coldsRNAi (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/UAS-coldsRNAi); apdsRNAi (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi); apdsRNAi; UAS-dimm (UAS-dicer2/+; apGal4/UAS-dimm; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi); eyadsRNAi (UAS-dicer2/UAS-eyadsRNAi; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi; UAS-dimm (UAS-dicer2/UAS-eyadsRNAi; apGal4/UAS-dimm; tub-Gal80TS, UAS-nEGFP/+); dimmdsRNAi (UAS-dicer2/+; apGal4/UAS-eyadsRNAi; tub-Gal80TS, UAS-nEGFP/+); rev4,dimmdsRNAi (UAS-dicer2/+; apGal4/rev4, UAS-dimmdsRNAi; tub-Gal80TS, UAS-nEGFP/+).

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