(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 ap (C–I, green), eya (C red, J green) and dimm (D, red). Tv1 neurons also retains col (F, red). Tv4 neurons also retain dac (E, red) and sqz (J, red). Tv1 and Tv4 do not express transcription factors grh (G, red), cas (H red) or nab (I, red) in adults. Genotype: (C–I) FMRFa-LacZ,ap^Gal4/+; UAS-nEGFP/+. (J) UAS-nEGFP/+; sqz^Gal4/+. (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.

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

(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/+).

Following terminal differentiation, Tv1 and Tv4 maintain expression of terminal differentiation genes (green text). Tv1 (PHM and Nplp1). Tv4 (PHM and FMRFa). The expression of transcription factors in grey text is lost by larval stages, all transcription factors in black text are retained in adult Tv1/4 neurons. Notably, all black transcription factors have been implicated in differentiation of subtype-specific neuropeptide expression in postmitotic Tv1/4 neurons. In contrast, all grey transcription factors have been implicated in the specification of Tv neuron subtype fates, and not in direct differentiation of subtype neuropeptide gene expression. All remaining transcription factors are required for maintained Nplp1 and FMRFa expression (black arrows), but their cross-regulatory relationships are mostly lost in adults (grey arrows). The only transcription factors that maintain their embryonic configuration are ap and eya's requirement for dimm in Tv1, and eya's continued regulation of active BMP signaling in Tv4. Dac plays an enhanced role in FMRFa expression in adult Tv4 neurons (red arrow).

(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), ap^Gal4,UAS-nlsEGFP (green) and Eya (blue) after A15 (B–G) or A20 (H–J) of dsRNAi expression at 29°C. (B,C) Eya immunoreactivity is eliminated and FMRFa is downregulated in eya^dsRNAi (C) compared to w¹¹¹⁸ control (B). (E,F) FMRFa is downregulated in sqz^IE,sqz^dsRNAi (F) compared to w¹¹¹⁸ control (E). (H,I) FMRFa is downregulated in ap^P44;ap^dsRNAi (I) compared to w¹¹¹⁸ 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^dsRNAi (n = 15) and eya^dsRNAi; eya^CliIID (n = 13) compared to w¹¹¹⁸ (n = 35) and eya^CliIID controls. ** p<0.0001 compared to eya^dsRNAi alone. (G) * p<0.0001 sqz^IE; sqz^dsRNAi (n = 39) compared to w¹¹¹⁸ (n = 40), sqz^IE (n = 21), and sqz^dsRNAi (n = 25) controls. (J) * p<0.0001 ap^P44; ap^dsRNAi (n = 25) compared to w¹¹¹⁸ (n = 47) and ap^P44 (n = 25) controls. *** p<0.005 compared to ap^dsRNAi (n = 32) alone. Genotypes: w¹¹¹⁸ (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP) (A–C) eya^CliIID (UAS-dicer2/+; ap^Gal4/eya^CliIID; tub-Gal80^TS, UAS-nEGFP/+); eya^dsRNAi (UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+); eya^dsRNAi; eya^CliIID (UAS-dicer2/UAS-eya^dsRNAi; ap^Gal4/eya^CliIID; tub-Gal80^TS, UAS-nEGFP/+). (E–G) sqz^IE (UAS-dicer2/+; ap^Gal4/sqz^IE; tub-Gal80^TS, UAS-nEGFP/+); sqz^dsRNAi (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-sqz^dsRNAi); sqz^IE; sqz^dsRNAi (UAS-dicer2/+; ap^Gal4/sqz^IE; tub-Gal80^TS, UAS-nEGFP/UAS-sqz^dsRNAi). (H–I) ap^P44 (UAS-dicer2/+; ap^Gal4/ap^P44; tub-Gal80^TS, UAS-nEGFP/+); ap^dsRNAi (UAS-dicer2/+; ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi); ap^P44; ap^dsRNAi (UAS-dicer2/+; ap^Gal4/ap^P44; tub-Gal80^TS, UAS-nEGFP/UAS-ap^dsRNAi).

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

(A–F) Representative images of Tv4 neurons (arrows) and Tv1 neurons (arrowheads) at larval stage 1 (A–C) and adults (D–F). (B,E) Induction of ectopic FMRFa expression (arrowhead, red) in Tv1 (identified with Nplp1, blue) after misexpression of dac and constitutively-activated Thickveins and Saxophone BMP receptors (TSA) in embryos (B n = 33) and for 5 days in adults (E n = 31). (C) Misexpression of col in embryonic Tv neurons using ap^Gal4 initiates ectopic Nplp1 expression (arrow, blue) in Tv4 neurons (identified with FMRFa, red) (C n = 24). (F) Misexpression of col and upregulation of dimm for 5 days in adults using ap^Gal4 initiates ectopic Nplp1 expression (arrow, blue) in Tv4 neurons (identified with FMRFa, red) (F n = 42). Genotypes: (A–C) Larval stage 1 w¹¹¹⁸ (ap^Gal4/+; UAS-nEGFP/+); TSA;dac (ap^Gal4/UAS-tkv^A, UAS-sax^A; UAS-nEGFP/UAS-dac); col (ap^Gal4/UAS-col; UAS-nEGFP/+). (D-F) Adult w¹¹¹⁸ (ap^Gal4/+; tub-Gal80^TS, UAS-nEGFP/+); TSA;dac (ap^Gal4/UAS-tkv^A, UAS-sax^A; tub-Gal80^TS, UAS-nEGFP/UAS-dac); dimm;col (ap^Gal4/UAS-col; tub-Gal80^TS, UAS-nEGFP/UAS-dimm).