Developmental Transcriptional Networks Are Required to Maintain Neuronal Subtype Identity in the Mature Nervous System

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply “locked-in,” but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain

Changes in Tv4 network configuration for maintenance.

<p>(A–E) <i>eya</i> regulates FMRFa independently of BMP signaling. (A,B) Nuclear pMad accumulation (red) in Tv4 (arrowhead) was downregulated in <i>eya^dsRNAi</i> flies (B) compared to <i>w¹¹¹⁸</i> control (A). (C) Expression of <i>eya^dsRNAi</i> in the presence of constitutively-activated Thickveins and Saxophone BMP type I receptors (<i>TSA</i>) activated pMad accumulation (red) in all Tv neurons (including Tv4; arrow) but failed to rescue FMRFa (blue) compared to <i>w¹¹¹⁸</i> control. (D) Quantification of pMad immunoreactivity in Tv4 nucleus in <i>w¹¹¹⁸</i> and <i>eya^dsRNAi</i> flies. * p<0.0001 eya<i>^dsRNAi</i> (n = 30) compared to <i>w¹¹¹⁸</i> control (n = 30). (E) Quantification of FMRFa immunoreactivity in Tv4 in <i>w¹¹¹⁸</i>, <i>eya^dsRNAi</i> and <i>eya^dsRNAi</i>; <i>TSA</i>. * p<0.0001 eya<i>^dsRNAi</i> (n = 38) and <i>eya^dsRNAi</i>; <i>TSA</i> (n = 42) compared to <i>w¹¹¹⁸</i> control (n = 34). NSD, no significant difference between eya<i>^dsRNAi</i> and <i>eya^dsRNAi</i>; <i>TSA</i>. (F–H) <i>eya</i> does not regulate <i>dimm</i> in adult Tv4. <i>dimm</i> (red) in Tv neurons (blue) is maintained in <i>eya^dsRNAi</i> (G) compared to <i>w¹¹¹⁸</i> control (F). (H) Quantification of Dimm immunoreactivity in Tv4. There is no significant difference between <i>eya^dsRNAi</i> (n = 24) and <i>w¹¹¹⁸</i> (n = 25) controls (p = 0.67). (I–K) <i>ap</i> regulates <i>dimm</i> in adult Tv4. <i>dimm</i> (red) in Tv neurons (blue) is downregulated in <i>ap^dsRNAi</i> (J) compared to <i>w¹¹¹⁸</i> control (I). (K) Quantification of Dimm immunoreactivity in Tv4. * p<0.0001 <i>ap^dsRNAi</i> (n = 24) compared to <i>w¹¹¹⁸</i> (n = 35). (L) Model depicting regulatory configuration of <i>ap</i>, <i>eya</i>, <i>dimm</i> and BMP signaling from embryonic to adult Tv4. The dependence of <i>dimm</i> on <i>eya</i> expression is not maintained (X). <i>Genotypes</i>: <i>w¹¹¹⁸</i> (A,D, F,H, I,J) (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>eya^dsRNAi</i> flies (B,D,E, G,H) (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>eya^dsRNAi; TSA</i> flies (C,E) (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/UAS-tkv^A, UAS-sax^A; tub-Gal80^TS, UAS-nEGFP/+</i>). <i>ap^dsRNAi</i> flies (J),K (<i>UAS-dicer2/+; ap^Gal4; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi</i>).</p

<i>dimm</i> maintains peptidergic phenotype and <i>dac</i> has an enhanced maintenance function.

<p>(A,B) Representative images of adult Tv4 neurons expressing FMRFa peptide (red), <i>ap^Gal4,UAS-nlsEGFP</i> (green) and Dimm (blue) at A10 at 29°C. FMRFa is downregulated and Dimm is lost in <i>rev4,dimm^dsRNAi</i> (B) compared to <i>w¹¹¹⁸</i> control (A). (C,D) Quantification of FMRFa peptide at A10 (C) and FMRFa transcript at A20 (D) in individual adult Tv4 neurons at 29°C. (C)* p<0.0001 <i>rev4,dimm^dsRNAi</i> (n = 19) compared to <i>w¹¹¹⁸</i> control (n = 42). (D)* p<0.0001 <i>rev4,dimm^dsRNAi</i> (n = 30) compared to <i>w¹¹¹⁸</i> control (n = 58). (E,F) Representative images of Tv4 neurons expressing mature FMRFa peptide (red), <i>ap^Gal4,UAS-nlsEGFP</i> (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. PHM is lost in <i>rev4,dimm^dsRNAi</i> (n = 26) (F) compared to <i>w¹¹¹⁸</i> control (n = 30) (E). (G,H) Images of adult Tv4 neurons expressing FMRFa peptide (red), <i>ap^Gal4,UAS-nEGFP</i> (green) and Dac (blue) at A10 at 29°C. FMRFa is downregulated and Dac immunoreactivity is lost in <i>dac^dsRNAi</i> (H) compared to <i>w¹¹¹⁸</i> control (G). (I,J) Quantification of FMRFa peptide in individual adult Tv4 neurons at A10 at 29°C (I), and in L1 larval Tv4 neurons in <i>dac</i> null mutants. (J) * p<0.0001 <i>dac^dsRNAi</i> (n = 26) compared to <i>w¹¹¹⁸</i> control (n = 47). (J) ** P = 0.02 <i>dac^−/−</i> (n = 31) compared to <i>w¹¹¹⁸</i> control (n = 22). <i>Genotypes</i>: <i>w¹¹¹⁸</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>rev4,dimm^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/rev4, UAS-dimm^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>dac^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/UAS-dac^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>dac^−/−</i> (<i>dac³/dac^{Df(3L)EXEL 7066}</i>).</p

Transcription factors <i>ap</i>, <i>eya</i>, <i>sqz</i> are required for persistent FMRFa expression in the adult Tv4 neuron.

<p>(A) Cartoon illustrating adult induction of dsRNAi constructs in adult Tv neurons using the TARGET system. (B,C, E,F, H,I) Images of adult Tv4 neurons expressing FMRFa transcript (red), <i>ap^Gal4,UAS-nlsEGFP</i> (green) and Eya (blue) after A15 (B–G) or A20 (H–J) of <i>dsRNAi</i> expression at 29°C. (B,C) Eya immunoreactivity is eliminated and FMRFa is downregulated in <i>eya^dsRNAi</i> (C) compared to <i>w¹¹¹⁸</i> control (B). (E,F) FMRFa is downregulated in <i>sqz^IE</i>,<i>sqz^dsRNAi</i> (F) compared to <i>w¹¹¹⁸</i> control (E). (H,I) FMRFa is downregulated in <i>ap^P44;ap^dsRNAi</i> (I) compared to <i>w¹¹¹⁸</i> control (H). (D,G,J) Quantification of FMRFa transcript in individual adult Tv4 neurons at A15 (D,G) or A20 (J) at 29°C. (D) * p<0.0001 eya<i>^dsRNAi</i> (n = 15) and <i>eya^dsRNAi; eya^CliIID</i> (n = 13) compared to <i>w¹¹¹⁸</i> (n = 35) and <i>eya^CliIID</i> controls. ** p<0.0001 compared to eya<i>^dsRNAi</i> alone. (G) * p<0.0001 <i>sqz^IE; sqz^dsRNAi</i> (n = 39) compared to <i>w¹¹¹⁸</i> (n = 40), <i>sqz^IE</i> (n = 21), and <i>sqz^dsRNAi</i> (n = 25) controls. (J) * p<0.0001 <i>ap^P44; ap^dsRNAi</i> (n = 25) compared to <i>w¹¹¹⁸</i> (n = 47) and <i>ap^P44</i> (n = 25) controls. *** p<0.005 compared to ap<i>^dsRNAi</i> (n = 32) alone. <i>Genotypes</i>: <i>w¹¹¹⁸</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP</i>) (A–C) <i>eya^CliIID</i> (<i>UAS-dicer2/+; ap^Gal4/eya^CliIID; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>eya^dsRNAi</i> (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>eya^dsRNAi; eya^CliIID</i> (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/eya^CliIID; tub-Gal80^TS, UAS-nEGFP/+</i>). (E–G) <i>sqz^IE</i> (<i>UAS-dicer2/+; ap^Gal4/sqz^IE; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>sqz^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-sqz^dsRNAi</i>); <i>sqz^IE; sqz^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/sqz^IE; tub-Gal80^TS, UAS-nEGFP/UAS-sqz^dsRNAi</i>). (H–I) <i>ap^P44</i> (<i>UAS-dicer2/+; ap^Gal4/ap^P44; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>ap^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi</i>); <i>ap^P44; ap^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/ap^P44; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi</i>).</p

Adult Tv1 and Tv4 neurons maintain Nplp1 and FMRFa and a subset of embryonic transcription factors.

<p>(A–B) Outline of transcription factor network configuration during specification and differentiation of Tv1 (A) and Tv4 (B) neurons at embryonic stages. Tv1 expresses Nplp1 (blue). Tv4 expresses FMRFa (red). Tv1 and Tv4 neurons both express PHM. Arrows represent known regulatory relationships. (C–J) Representative images from Tv clusters in adults (green) in thoracic hemisegments 1 and 3. Tv4 neurons (arrows) express FMRFa (white). Tv1 neurons (arrowheads) express Nplp1 (blue). Tv1 and Tv4 retain expression of transcription factors <i>ap</i> (C–I, green), <i>eya</i> (C red, J green) and <i>dimm</i> (D, red). Tv1 neurons also retains <i>col</i> (F, red). Tv4 neurons also retain <i>dac</i> (E, red) and <i>sqz</i> (J, red). Tv1 and Tv4 do not express transcription factors <i>grh</i> (G, red), <i>cas</i> (H red) or <i>nab</i> (I, red) in adults. Genotype: (C–I) <i>FMRFa-LacZ,ap^Gal4/+; UAS-nEGFP/+</i>. (J) <i>UAS-nEGFP/+</i>; sqz<i>^Gal4/+</i>. (K,L) Outline of transcription factors present in fully differentiated adult Tv1 (K) and Tv4 (L) neurons. Grey arrows indicate known developmental interactions between transcription factors in embryonic Tv cluster neurons that, herein, we test in the adult.</p

Transcriptional regulation of Nplp1 in adult Tv1 neurons.

<p>(A–F) <i>col^dsRNAi</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^Gal4,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^dsRNAi</i> (B) compared to <i>w¹¹¹⁸</i> control (A). (C) Quantification of Nplp1 immunoreactivity in <i>w¹¹¹⁸</i> (n = 29) and <i>col^dsRNAi</i> (n = 20) at A10 at 29°C. (D–F) <i>col^dsRNAi</i> did not affect Dimm immunoreactivity (D), <i>ap^Gal4,UAS-nEGFP</i> fluorescence (E) or Eya immunoreactivity (F) compared to <i>w¹¹¹⁸</i> at A10 at 29°C. <i>col^dsRNAi</i> (D n = 24; E n = 22; F n = 21). <i>w¹¹¹⁸</i> control (D n = 25; E n = 27; F n = 24). (G–I) <i>ap^dsRNAi</i> significantly reduced Nplp1 (G) and Dimm (H) immunoreactivity compared to <i>w¹¹¹⁸</i> at A20 at 29°C. (G) * p<0.0001 <i>ap^dsRNAi</i> (n = 30) compared to <i>w¹¹¹⁸</i> control (n = 36). (H) * p<0.001 <i>ap^dsRNAi</i> (n = 13) compared to <i>w¹¹¹⁸</i> control (n = 22) (I) Dimm restoration <i>(UAS-dimm)</i> in <i>ap^dsRNAi</i> background only partially rescued Nplp1 downregulation at A15 at 29°C. * p<0.0001 <i>ap^dsRNAi</i> (n = 22) compared to <i>w¹¹¹⁸</i> control (n = 19). ** p<0.0001 <i>ap^dsRNAi;UAS-dimm</i> (n = 23) compared to <i>ap^dsRNAi</i> and <i>w¹¹¹⁸</i> controls. (J–L) <i>eya^dsRNAi</i> significantly reduced Nplp1 (J) and Dimm (K) immunoreactivity compared to <i>w¹¹¹⁸</i> at A10 at 29°C. (L) Dimm restoration <i>(UAS-dimm)</i> in <i>eya^dsRNAi</i> background failed to rescue Nplp1 immunoreactivity at A15 at 29°C. * p<0.0001 <i>eya^dsRNAi</i> and <i>eya^dsRNAi</i>; <i>UAS-dimm</i> (n = 19) compared to <i>w¹¹¹⁸</i> control. NSD, no significant difference between <i>eya^dsRNAi</i> and <i>eya^dsRNAi</i>;<i>UAS-dimm. eya^dsRNAi</i> (J n = 23; K n = 23; L n = 22). <i>w¹¹¹⁸</i> control (J n = 30; K n = 25; L n = 26). (M–O) <i>dimm^dsRNAi</i> downregulated Nplp1 and PHM in adult Tv1. (M,N) Tv1 neurons expressing Nplp1 (red), <i>ap^Gal4,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^dsRNAi</i> (n = 18) (M) compared to <i>w¹¹¹⁸</i> control (n = 15) (N). (O) <i>dimm^dsRNAi</i> significantly reduced Nplp1 immunoreactivity compared to <i>w¹¹¹⁸</i> at A10 at 29°C. * p<0.0001 <i>dimm^dsRNAi</i> (n = 19) compared to <i>w¹¹¹⁸</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: w¹¹¹⁸</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>col^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-col^dsRNAi</i>); <i>ap^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi</i>); <i>ap^dsRNAi; UAS-dimm</i> (<i>UAS-dicer2/+; ap^Gal4/UAS-dimm; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi</i>); <i>eya^dsRNAi</i> (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>eya^dsRNAi</i>; <i>UAS-dimm</i> (<i>UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/UAS-dimm; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>dimm^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/UAS-eya^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+</i>); <i>rev4,dimm^dsRNAi</i> (<i>UAS-dicer2/+; ap^Gal4/rev4, UAS-dimm^dsRNAi; tub-Gal80^TS, UAS-nEGFP/+</i>).</p