Transcriptional regulation of Nplp1 in adult Tv1 neurons.

<p>(A–F) <i>col<sup>dsRNAi</sup></i> downregulated Nplp1 but not <i>ap</i>, <i>eya</i>, or <i>dimm</i> in adult Tv1. (A,B) Expression of Nplp1 (red), <i>ap<sup>Gal4</sup>,UAS-nEGFP</i> (green) and Col (blue) in adult Tv4 neurons at A10 at 29°C. Col expression is lost and Nplp1 is downregulated in <i>col<sup>dsRNAi</sup></i> (B) compared to <i>w<sup>1118</sup></i> control (A). (C) Quantification of Nplp1 immunoreactivity in <i>w<sup>1118</sup></i> (n = 29) and <i>col<sup>dsRNAi</sup></i> (n = 20) at A10 at 29°C. (D–F) <i>col<sup>dsRNAi</sup></i> did not affect Dimm immunoreactivity (D), <i>ap<sup>Gal4</sup>,UAS-nEGFP</i> fluorescence (E) or Eya immunoreactivity (F) compared to <i>w<sup>1118</sup></i> at A10 at 29°C. <i>col<sup>dsRNAi</sup></i> (D n = 24; E n = 22; F n = 21). <i>w<sup>1118</sup></i> control (D n = 25; E n = 27; F n = 24). (G–I) <i>ap<sup>dsRNAi</sup></i> significantly reduced Nplp1 (G) and Dimm (H) immunoreactivity compared to <i>w<sup>1118</sup></i> at A20 at 29°C. (G) * p<0.0001 <i>ap<sup>dsRNAi</sup></i> (n = 30) compared to <i>w<sup>1118</sup></i> control (n = 36). (H) * p<0.001 <i>ap<sup>dsRNAi</sup></i> (n = 13) compared to <i>w<sup>1118</sup></i> control (n = 22) (I) Dimm restoration <i>(UAS-dimm)</i> in <i>ap<sup>dsRNAi</sup></i> background only partially rescued Nplp1 downregulation at A15 at 29°C. * p<0.0001 <i>ap<sup>dsRNAi</sup></i> (n = 22) compared to <i>w<sup>1118</sup></i> control (n = 19). ** p<0.0001 <i>ap<sup>dsRNAi</sup>;UAS-dimm</i> (n = 23) compared to <i>ap<sup>dsRNAi</sup></i> and <i>w<sup>1118</sup></i> controls. (J–L) <i>eya<sup>dsRNAi</sup></i> significantly reduced Nplp1 (J) and Dimm (K) immunoreactivity compared to <i>w<sup>1118</sup></i> at A10 at 29°C. (L) Dimm restoration <i>(UAS-dimm)</i> in <i>eya<sup>dsRNAi</sup></i> background failed to rescue Nplp1 immunoreactivity at A15 at 29°C. * p<0.0001 <i>eya<sup>dsRNAi</sup></i> and <i>eya<sup>dsRNAi</sup></i>; <i>UAS-dimm</i> (n = 19) compared to <i>w<sup>1118</sup></i> control. NSD, no significant difference between <i>eya<sup>dsRNAi</sup></i> and <i>eya<sup>dsRNAi</sup></i>;<i>UAS-dimm. eya<sup>dsRNAi</sup></i> (J n = 23; K n = 23; L n = 22). <i>w<sup>1118</sup></i> control (J n = 30; K n = 25; L n = 26). (M–O) <i>dimm<sup>dsRNAi</sup></i> downregulated Nplp1 and PHM in adult Tv1. (M,N) Tv1 neurons expressing Nplp1 (red), <i>ap<sup>Gal4</sup>,UAS-nlsEGFP</i> (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. Nplp1 is downregulated and PHM is lost in <i>dimm<sup>dsRNAi</sup></i> (n = 18) (M) compared to <i>w<sup>1118</sup></i> control (n = 15) (N). (O) <i>dimm<sup>dsRNAi</sup></i> significantly reduced Nplp1 immunoreactivity compared to <i>w<sup>1118</sup></i> at A10 at 29°C. * p<0.0001 <i>dimm<sup>dsRNAi</sup></i> (n = 19) compared to <i>w<sup>1118</sup></i> control (n = 18). (P) Model depicting regulation of Nplp1 and PHM and the configuration changes between <i>ap</i>, <i>eya</i>, <i>dimm</i> and <i>col</i> from embryonic to adult Tv1. Notably, <i>col</i> no longer regulates <i>dimm</i>, <i>eya</i> or <i>ap</i> expression (denoted by X). <i>Genotypes: w<sup>1118</sup></i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/+; tub-Gal80<sup>TS</sup>, UAS-nEGFP/+</i>); <i>col<sup>dsRNAi</sup></i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/+; tub-Gal80<sup>TS</sup>, UAS-nEGFP/UAS-col<sup>dsRNAi</sup></i>); <i>ap<sup>dsRNAi</sup></i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/+; tub-Gal80<sup>TS</sup>, UAS-nEGFP/UAS-ap<sup>dsRNAi</sup></i>); <i>ap<sup>dsRNAi</sup>; UAS-dimm</i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/UAS-dimm; tub-Gal80<sup>TS</sup>, UAS-nEGFP/UAS-ap<sup>dsRNAi</sup></i>); <i>eya<sup>dsRNAi</sup></i> (<i>UAS-dicer2/UAS-eya<sup>dsRNAi</sup>; ap<sup>Gal4</sup>/+; tub-Gal80<sup>TS</sup>, UAS-nEGFP/+</i>); <i>eya<sup>dsRNAi</sup></i>; <i>UAS-dimm</i> (<i>UAS-dicer2/UAS-eya<sup>dsRNAi</sup>; ap<sup>Gal4</sup>/UAS-dimm; tub-Gal80<sup>TS</sup>, UAS-nEGFP/+</i>); <i>dimm<sup>dsRNAi</sup></i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/UAS-eya<sup>dsRNAi</sup>; tub-Gal80<sup>TS</sup>, UAS-nEGFP/+</i>); <i>rev4,dimm<sup>dsRNAi</sup></i> (<i>UAS-dicer2/+; ap<sup>Gal4</sup>/rev4, UAS-dimm<sup>dsRNAi</sup>; tub-Gal80<sup>TS</sup>, UAS-nEGFP/+</i>).</p>