Supplementary Figure1: ClustalW comparison between Tll, Dsf and NR2E1.

Transcription

1 P-Box Dsf MG-TAG--DRLLD-IPCKVCGDRSSGKHYGIYSCDGCSGFFKR 39 NR2E MSKPAGSTSRILD-IPCKVCGDRSSGKHYGVYACDGCSGFFKR 42 Tll MQSSEGSPDMMDQKYNSVRLSPAASSRILYHVPCKVCRDHSSGKHYGIYACDGCAGFFKR :.:***** *:*******:*:****:***** D-Box T/A-Box Dsf SIHRNRIYTCKATGDLKGRCPVDKTHRNQCRACRLAKCFQSAMNKDAVQHERGPRKPKLH 99 NR2E1 SIRRNRTYVCKSG--NQGGCPVDKTHRNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR 100 Tll SIRRSRQYVCKSQ--KQGLCVVDKTHRNQCRACRLRKCFEVGMNKDAVQHERGPRNSTLR 118 **:*.* *.**: :* * ************** **:: *************...:: Dsf PQLHHHHHHAAAAAAAAHHAAAAHHHHHHHHHAHAAAAHHAAVAAAAASGLHHHHHAMPV 159 NR2E1 KQVALYFR GHKEENGAAAHFPSAALPAP Tll RHMAMYKDAMMGAG EMPQIPAEILMNTAALTGFPGVPMPMP :: :.* :..... Dsf SLVTNVSASFNYTQHISTHPPAPAAPPSGFHLTASGAQQGPAPPAGHLHHGGAGHQHATA 219 NR2E AFFTAVTQLEPH Tll GLPQRAGHHPAHMAAFQPPPS :.* Dsf FHHPGHGHALPAPHGGVVSNPGGNSSAISGSGPGSTLPFPSHLLHHNLIAEAASKLPGIT 279 NR2E Tll Dsf ATAVAAVVSSTSTPYASAAQTSSPSSNNHNYSSPSPSNSIQSISSIGSRSGGGEEGLSLG 339 NR2E GLELA 145 Tll AAAVLDLSVP 190.*.: Dsf SESPRVNVETETPSPSNSPPLSAGSISPAPTLTTSSGSPQHRQMSRHSLSEATTPPSHAS 399 NR2E1 AVS TTP Tll RVP HHP * Dsf LMICASNNNNNNNNNNNNGEHKQSSYTSGSPTPTTPTPPPPRSGVGSTCNTASSSSGFLE 459 NR2E ERQTLVSLAQPTPKYPH E 169 Tll VHQGHHGFFSPTAAYMNALATR A 219 :*.**. Dsf LLLSPDKCQELIQYQVQHNTLLFPQQLLDSRLLSWEMLQETTARLLFMAVRWVKCLMPFQ 519 NR2E1 VNGTP-----MYLYEVAT ESVCESAARLLFMSIKWAKSVPAFS 207 Tll LPPTP------PLMAA EHIKETAAEHLFKNVNWIKSVRAFT 254 : :*. * : *::*. ** :.* *.:.* Dsf TLSKNDQHLLLQESWKELFLLNLAQWTIPLDLTPILESPLIRERVLQDEAT---QTEMKT 576 NR2E1 TLSLQDQLMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQA 267 Tll ELPMPDQLLLLEESWKEFFILAMAQYLMPMNFAQLLFVYES--ENANREIMGMVTREVHA 312 *. ** :**:::*:*:*:* :**: :*:: :* : : *::: Dsf IQEILCRFRQITPDGSEVGCMKAIALF--APETAG NR2E1 LQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGS EL 305 Tll FQEVLNQLCHLNIDSTEYECLRAISLFRKSPPSASSTEDLANSSILTGSGSPNSSASAES 372 :**:: :: :: *.:* *::.* * * :. Dsf --LCDVQPVEMLQDQAQCILSDHVRLRYPRQATRFGRLLLLLPSLRTIRAATIEALFFKE 667 NR2E1 RSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKK 365 Tll RGLLESGKVAAMHNDARSALHNYIQRTHPSQPMRFQTLLGVVQLMHKVSSFTIEELFFRK 432 : : : ::::*: *.::: :* *. ** ** :: ::.:. *** :**:: Dsf TIGNVPIARLLRDMYTMEPAQVDK 691 NR2E1 TIGNVPITRLLSDMYKSSDI Tll TIGDITIVRLISDMYSQRKI ***::.*.**: ***. Supplementary Figure1: ClustalW comparison between Tll, Dsf and NR2E1. The degree of amino acid identity and similarity between Tll, Dsf and Nr2e1 is equivalent. Tll shows 81 and 41% identity with NR2E1 in the DNA binding domain and ligand binding domain (LBD) (purple residues) respectively 20,24. Dsf shows slightly higher amino acid identity in its LBD (44%) 36. Tll and Dsf are only 32% identical in their LBD. Dsf is identical to NR2E1 in the P-Box (red residues) while Tll

2 differs from NR2E1 in one residue in this domain. On the other hand, compared to Dsf, Tll has a higher degree of amino acid identity to NR2E1 in the D-Box (green residues) and T/A-Boxes (blue residues). Supplementary Figure 2: Pan-neuronal knock-down of tll strongly reduces Tll protein levels. Western blot to detect Tll levels in elav>tll RNAi heads compared to control flies (elav/y and tll RNAi /+) using rabbit anti-nr2e1 antibody. A non-specific band, denoted by the asterisk, shows roughly equal loading levels in the different samples. Tll levels in the pan-neuronal knock-down flies are strongly reduced compared to controls. MW standard is shown next to the blot.

3 Supplementary Figure 3: Adult knock-down is sufficient to cause increased fighting frequencies. Fighting frequencies of elav-gal4>uas-tll RNAi in a TubP-GAL80 ts background and control lines containing only one component of the binary expression system (elav- GAL4/+ and TubP-GAL80 ts /+; UAS-tll RNAi /+). All flies were reared at 18 C and on the day of eclosion were either moved to 25 C (grey bars) or maintained at 18 C (open bars) for 7 days. Only elav-gal4>tll RNAi ; TubP-GA80 ts flies that were shifted to 25 C after eclosion (i.e. in which tll was knocked down in the adult) showed significantly increased fighting frequencies (Kruskal-Wallis ANOVA, * denotes P<0.05, n = minimum of 40 pairs for each genotype). Bar graphs represent means +/- s.e.m.

4 Supplementary Figure 4: Tll protein localization in the PI stained with NR2E1 in wild-type and in dsf deletion flies. a, Immunofluorescence image of a wild-type adult brain stained with anti-nr2e1 antibody detecting Tll protein. b, Immunofluorescence image of a brain from a Df(2L)clot7/dsf 3 fly stained with anti-nr2e1 (red) and anti-dlg (green). Arrowheads denote the pars intercerebralis. Scale bar = 100 m.

5 Supplementary Figure 5: Fighting frequencies of males with PI specific knockdown of a second RNAi line against tll, tll RNAi2, are significantly higher than controls. Fighting frequencies of males with PI specific knock-down of UAS-tll RNAi2 [51] compared to control lines containing only one component of the binary expression system (50Y/+, c929/+, dilp2/+ and UAS-tll RNAi2 /+). Asterisks denote statistically significantly higher medians in the males in which tll RNAi2 is knocked down compared to males expressing only one component of the binary system (Kruskal-Wallis ANOVA,* denotes P<0.05, ** denotes P<0.01 and *** denotes P<0.001, n = minimum of 70 pairs for each genotype). Bar graphs represent means +/- s.e.m.

6 Supplementary Figure 6: Effect of PI specific knock-down of tll on other behaviours Courtship index (a), and latency (b) are not significantly different between males in which tll is knocked down (50Y>tll RNAi ) compared to the single binary factor controls (50Y/+ and UAS-tll RNAi ). Mating duration (c) and latency (d) and total amount of sleep (e) also show no significant differences between the different genotypes. However, activity levels (f) in tll knock-down males (50Y>tllR NAi ) were significantly lower compared to control males (50Y/+ and tll RNAi /+) (ANOVA,** denotes P<0.01). Courtship analysis, n = 10; mating assays, n = 30; sleep and activity assays, n = 32. Bar graphs represent means +/- s.e.m.

7 Supplementary Figure 7: Pan-neuronal knock-down of Atro decreases Atro protein levels. Western blot to detect Atro levels in elav>atro IR heads compared to control flies (elav/y and Atro IR /+) using rabbit anti-atrophin. Anti-Dlg was used as a loading control. Atro levels are significantly reduced in the pan-neuronal knock-down flies compared to controls. MW standard is shown next to the blot.

8 Supplementary Figure 8: Atro staining in the adult fly brain. a, Immunofluorescence image of a wild-type adult brain stained with anti-atrophin (red) and anti-dlg (green). A subset of confocal sections was composited to create this image. b, Immunofluoresence image of the same brain as in (a) without the green channel and a larger number of sections composited to create the final image. Arrowheads denote the pars intercerebralis. Scale bar = 100 m.

9 Supplementary Figure 9: Bimolecular fluorescent complementation between Tll and Atro shows direct interaction in vitro. a-d, NYFP-MYC-tagged Atro transfected into S2 cells does not show YFP signal in green, but can be detected with antibodies against the Myc tag (red channel). e-h, CYFP-HA-tagged Tll transfected into S2 cells does not show green YFP signal, but can be detected with antibodies against the HA tag (red channel). i-l, NYFP-tagged Atro and CYFP-tagged Tll transfected together into S2 cells show a strong green YFP signal in the nucleus. The interaction can also be detected with antibodies against Tll (red channel). m-p, NYFP-MYC-tagged Atro and CYFP-HA-tagged Tll transfected together into S2 cells show a clear YFP signal in the nucleus. The interaction can also be detected with antibodies against Atro (red channel). Scale bar = 20 m.

10 Supplementary Figure 10: Bimolecular fluorescent complementation between Tll and Atro shows direct interaction in vivo. a-d, Bimolecular fluorescent complementation in vivo in adult brains shows no YFP signal when only one of the two BiFC components is expressed in the PI. a, b, When only one component is expressed, no YFP signal is detected but the single component can be detected with an antibody against its tag (HA in Tll-HA-CYFP in b). c, d When the other component is expressed, again no YFP signal is detected but the single component can be detected with an antibody against its tag (MYC in Atro- MYC-CYFP in d). Scale bar = 100 m.

11 Supplementary Figure 11: Activation of dilp2 expressing neurons with the bacterial sodium channel, UAS-NaChBac, significantly increases fighting frequencies and is fully suppressed by blocking neuropeptide processing. Fighting frequencies in dilp2>nachbac males are significantly higher than the single binary factor component controls (Kruskal-Wallis ANOVA, *** denotes P<0.001, n = 70 pairs for each genotype). dilp2 GAL4 is expressed almost exclusively in a subset of PI neurons (Fig. 3C). The effect of genetic activation of dilp2 expressing neurons is completely suppressed when amon RNAi is co-expressed (black bar), showing that the aggression inducing effect of PI activation also depends on a neuropeptide-based mechanism. Bar graphs represent the means +/- s.e.m.

12 Supplementary Figure 12: Quantitative RT-PCR of pan-neuronal amon knockdown. Quantitative RT-PCR on RNA extracted from brains from elav/y, UAS-amon RNAi /+ and elav>amon RNAi males using primers against amon showed an almost complete loss of amon expression in the pan-neuronal knock-down animals. Results are the average from three biological replicates. Bar graphs represent the means +/- s.e.m.

13 tailless cdna sequence ttaaaacgccggcggtcctcacagcagacaacacaacccatcgtgatctcagcgagtccacatcggagt aaccaaggatatatcgaatatatcacacaatccgcaataccgccgtccacccaaaccgttaaaacaaaa atccaaaacgactcaaagatacaccagtgccaagtgaaattcaatttgtgcaagcgtttctacaaaaat cgccaaaattacgccccacatcggtatgcagtcgtcggagggttcaccagacatgatggatcagaaata CAACAGCGTGCGTCTTTCGCCAGCGGCATCGAGTCGCATTCTATACCATGTGCCCTGCAAAGTCTGCAG AGATCACAGCTCCGGCAAGCATTACGGCATCTACGCCTGTGATGGCTGCGCCGGATTCTTCAAGAGGAG CATTCGGAGATCCCGGCAGTATGTGTGCAAGTCGCAGAAGCAGGGACTCTGTGTGGTGGACAAGACGCA CAGGAACCAATGTAGGGCTTGCCGACTGAGGAAGTGCTTTGAGGTCGGAATGAACAAGGATGCAGTGCA GCACGAGCGGGGACCGCGGAACTCCACTCTGCGTCGCCACATGGCCATGTACAAGGATGCCATGATGGG CGCCGGCGAGATGCCACAAATACCCGCCGAAATTCTGATGAACACGGCTGCCTTGACCGGCTTTCCTGG AGTACCGATGCCCATGCCTGGCCTGCCCCAGAGGGCTGGTCATCATCCTGCTCACATGGCTGCCTTCCA GCCGCCACCATCGGCTGCCGCTGTCTTGGACTTATCCGTGCCACGAGTGCCCCATCACCCGGTGCACCA AGGACACCACGGTTTCTTCTCGCCCACCGCCGCCTACATGAATGCCCTGGCCACTCGGGCCCTGCCCCC CACTCCTCCGCTGATGGCAGCTGAGCACATCAAGGAAACCGCGGCGGAACACCTATTCAAGAACGTCAA CTGGATCAAGAGCGTACGGGCCTTCACCGAACTGCCCATGCCGGATCAGCTGCTCCTGCTGGAGGAGTC CTGGAAGGAGTTCTTCATCCTGGCCATGGCCCAGTACCTAATGCCCATGAATTTCGCCCAGCTGCTGTT CGTCTACGAGTCCGAGAATGCCAACCGGGAGATCATGGGCATGGTGACCCGCGAGGTGCACGCCTTCCA GGAGGTGCTGAACCAACTGTGCCATCTGAACATTGACAGCACCGAGTACGAGTGTCTGAGGGCTATTTC GCTCTTCCGTAAGTCACCACCGTCGGCAAGTTCTACCGAGGATTTAGCCAACAGCTCAATCCTGACAGG AAGCGGCAGCCCGAACTCCTCGGCCTCTGCTGAATCCAGGGGTCTTCTGGAGTCGGGAAAAGTGGCGGC CATGCACAACGATGCCCGGAGTGCGCTGCACAACTACATCCAGAGGACCCATCCCTCGCAGCCCATGCG ATTCCAGACGCTCTTGGGCGTGGTGCAGCTGATGCACAAGGTCTCAAGCTTCACCATCGAGGAGCTGTT CTTCCGAAAGACCATCGGCGACATCACCATTGTGCGCCTCATCTCCGACATGTACAGTCAGCGCAAGAT CTGAaaagtatgtagagcctagactaatcgccgcactcgaagtgccttccaagtgctgggaactgtgat aatctcggaagaagcgctttggacaatactcgatcagtgaaatcaacgatttctcatatccaggagtcg agccttaaaatacgtacacaacactcaccttaataccttacctaaacagaactcgaagtaatcttagct aaagtctctcagaccatccagatgtgtttcaaattgcattcgcaaaagtttcaactttgcctgttaaat acgtcaatcgtagttttaaacactttagttttaagcgcatattattagctttaggatttggaaaaataa ttattc Atrophin cdna sequence ccccccccccgcagtttttgaaagggtgaccgacggatggctgtgtttgtgcaaattaaccgaaatata catttccggctactcgctgcctcctcgctgagcaacaactacaaaaacaacaacaccaataacaacagc aggaacaacaatatcaagaacaacaggagcaaagcaacaagaaccaccacgaccgccgcccaccccctt tgaaaatccatcgacacagcaccacatcaccatcgtcgtcatcatcatcatcatcctcagcaacttgga gtctggaatctggagacggagaccgtagaccggagttactttgccgcctgctgctggagagaggagcat cttcgtgcgggacgcctcgacgatacacaaccccgactacggattcatgttgaattttcatatcgtaga gggcataaaaaggcttaacactggaccggaagcgtgtttttaaagggtgatggcggcctccactcaagg AGAAATTCGAGTGGGTCCCGGCCACCAGGTAAACGATGTCTATGCAAAACTGCCCGATTATAATCCAAT CTCAAGCTTCCCCATCGACAAGGAAACCGATGAACGTGAACTAGAGGAATCAAGATGGAGTCCAGGCGT TGTGGCCGATGGCGACTTGTTAATGTTCTTGCGTGCGGCTCGATCCATGGCTGCATTTCAAGGAATGTG TGATGGTGGTTTAGAAGACGGTTGTTTGGCTGCTAGTCGCGACGACACTACAATAAACGCACTCGACGT GCTCCACGATTCTGGCTACGATCCAGGCAAAGCTCTACAAGCGCTCGTAAAGTGCCCCGTTTCGAAGGG CATCGACAAGAAGTGGACCGAGGACGAAACAAAGAAATTCATCAAGGGTCTGCGTCAGTTTGGGAAGAA CTTCTTCCGCATCCATAAGGACTTGCTGCCGCACAAGGACACGCCGGAGCTGGTCGAGTTCTACTATCT GTGGAAGAAGACGCCCGGCGCGAACAATAATCGGCCACACAGGCGACGCCGCCAAAGCGCCCTGCGACG CAACCGTGTCACGCGGGCCAACAACAGCAACAGCAACACTCCTCCGAAGAAGGAGGACACTCCAGAACC ACAAACTGCGACGACGGCGACGGCGGCGGCAACCGCGGCGTCCGAGACGGCGAGTCGCTCCTCGCCCGC TGTCTCCAAGGAGGAGAACAGCTCGCTCACCGAGGACGACGCCAGCGAGTGCGACAGTGATTCGAGTCT GACCCACAAAAGGGATGAATCACCCTCAAGGATGAGGACGCGTAACAAGCAACAGAACAACAACAGCAG CACCAGCAGCGGTAACAACACGGCCGGCAACGGTGGCGGTAACGCCACATCCATAAGCAGCGGATCAAC CGGCGGCGGTGCCGCTGGCGGCAATAGTTCGTCTAAGGATCAATCAGCCAACGCCGTGGCTAATGGCAA GCGACCCAAGAGGGGCTCCGAAACACCGGACGTTTCCGGCGGAGCCTCGGTCGATAGTCCCAAGACACC GACGAAGGCTGTGGCCGAGAGTTCAGCCAATAAGCGCAAGGGTGGCAAGCAGGATACGCCCAACAAGAA GAAGCGAACGGAGCAGGAGTCCAACGAGCCAAGCGCCCATGAGGAGAATGCCATCAAGGAGAAGCGCAA GCGACCGGACAGCCCGGTTGAGAGTATGAACTCGGATAGCAGACCGGATTCAGTGCTCGACGATGGGGA ATCGAATACCACGGACACCACCACCGCCGAGCAGCAGTCCACAAAGGACAGCAAGGAGACGGTCAGCTG CAAGGAGGAGCGCGAAATGGTCACCAACGATCTGGAGGCCAAGGCCGAGGAAAAGGCCATCAAGGCAGA

14 GGCTTTGGCCGAAGACAGCAAGGATAGCGCCATCAAGAACATGGACGAGGAGACAAACATCCAGGCGCC TAGCAGTGCAGACACTAGTTTGGTGGATGGTCCTAATCCCAATGCCCTGCCCAGTCCGGTAGCCGCACC AATCACAATGAAGGTACCAACAATTGCCACCGTTGAGGCGCTGAACGCGTCCGTCGACCGCAAGGAGGC CATCGAGAAGATGGAGTCGTGCGACAGCGATCCGGAGATGCTTAAAAAACTGGCAACCATTAAGCAGGA AGTATCTCCGCAGCAGCAACAGCACATGCAACAGCAATCACAGCAGCAGATGCAGCAGCAACTCGCTCC AGTTGGCATACCGCAACCTCCGTCTTGCCCGCCATCGGAATCAGTCTATATCAAAAAAGAGCCCATGGA GGACTCGATGGACGCCACCTGCAATCAGAACAGCAACGAACCGCAGGACCTGAAGGTGAAGATCGAGAT TAAAAACGAGGATGCATTGAAGCATAGTGCTGGAGGTCTGCCGCCTTCAGGTCCGTGTGCACCGCCTTC AGCTCTACATCCGCTTTCCGGAGCTCCGGTAGAGAGCGGCCAGGAGCCACTGCACCTGCAACACATGCC TCATGGGCAAGTAACGACGCAACCGCCCCCTGGCTACCTAATTGATGGCCAGCTAAAGTATGGACCATC GGGACAAGGCGTGCCTCCACAGCCACCACAACTGCACAGCGATGCGGCTGGAGGAGTCAGCGGAGCACC ACCTGGAGCGCCAACCACGCCCCAGAAGTATCCGCCCGAGATGGAGATGAAGTTCGCTCCTCAGGATCT CAAGTATCCCCCACCGCCGCCTCTAGATGCACTCAAGTACAGCCAGGAGATGCAAGCTGCGGCGGCGGC AGCGGCTGCTGCTGGCAAATACGATATGAAGTATATGATGGAACAGCAGGGCAAGTACAATGTGGAGTT GTCAGCTGCCCATCAGCCGCCAAGCAAGCCAGGCTACCAGGACTCGCTTAAGATACCCGATATTAAGCC CGGTTTCGGCCACCTGCCGCACAACGTGGGCTCTCCGCTGGACGCCGCCCATAAATACGGACCGCCTCC GACGTCGCAAGAGTCCCAGCAACAGCAACCCCAGCCGCCGGCACATCAGGTACCGCCGGGAGCAACTCC ACCACCTGGTATCGCCATGCCCAAGCCGCACTATCAACACGATGTGCAAACACCACCGTTGGGACGGCC CTTCGAGCCGACCGGACTTATGCTCAAGTATGGCGATCCATTGGCAGCCAAATACGGCCCGCCCCAGGA TCTCAAGTACCCGATGCCTCCGGTCTCTCAGGCGGGACCAGCGGACGTAAAGCCCTATGGCGGCGAGAA TCTGATCAAGTCCTCACCGTACGGACCGCCGCCGGAGAGTCCTATCGATGCCTCTGCGCGCTCTACACC TGGTCAGGACAGCCAGGGCAGCAATAGCAATTCACAGCCGCCCTCAATGCCCCCGCAACCGCAGCAGTT CCAGTCGCCGCATCCCTCGCCGCATATGCCTTCGCCAGCAGGAGGTGGTCTACCACCGGGAATGCATCC GCAAAATCTCATCCACGGCCCGCCACCAGGTGCAGCGGGTGGCAGTGGTCCCCAGCCACCTCCACCGCC CACATCGCTACACCAGCCAACGCCCACGTCTGCAGGTCCACCCAGTCTGCAACATGGACTACATCCTGG CCATCAACACTCACAGCTGTCTGTGGCATCATCGATACCGCCGAGCTCGATTGGAATTCCTCCCACGCT CTCGACTATGGCGCCCTCGCACATGCACCCGCACCTTCATCCACATGCGCATCTGCAGGGTCTCCATCG GCCGCACGATCTGCCGCCCAGTATGCATCCACATGCACCCATGCCGCTGTCGTTGCAGGGACATCCGCA GCACGGACATGGATTGCCGCCCTCGCACACTTCTCAGCAACAGCAGCAGCAGCAACAACAACAACAGCC CGGCGGACCAGCTGGTACGGTGCGAACTCCGTCACCAGCCCAGCAGCCGCCGAGATCCATGCACGATCC GCAATCGTCTCGAGAGCCGCCCACATCGCAGCCTTCGACCACTATGGCAGGATCGAGTGGTCCGGGTGG ACCACCGCCGCAACAGTCGCCGCATGCGCATCGCACATCACCGTTGCCAGGGCTCGCGGGTAGTGGTCC TCCACCCCCGGGACTCATCGGTCATCCGATGGCCATACACCCGCACCTAGCCCACTTGCCGCCCGGACA TCCCGCTCACGCAGCGCTGGCCCATCCTGGACACCATCTGCTGTCGCACTCGATAGCGGGCTTGGGTCC TGGTGGTGGACCCATCGCGTTGCTGGCCGGTCCCGGCGGTCTTGGTGGTATTCCAGAGTCCGCTCTAAG TCGCCGCACCCCGCCCTCACATCTGCCACACTCGCATGCCTCTTCGGCCCCACTGACGGCTCACTCGGT GGCCAGTATGACGTCTACCAGTATGTCGCTGACCACCAGCACGGTGCCATCGTCCGCCTTTAGCCGCGC CAGTCCCAGCGTACAGATCTCGAGCAGTGGAGGAGGACCTTCAGGGCCCGGAAGCGTTGGACCTGGAGG AATGCCAAACTCGTCGGCAGCAGCGGCTGCTGCGGCAGCTGCTCATCGGGCAGCCTCACCGGCATCCAG CGTAAGCAGCCTAAGTCGGCAGAGTCCGCTGCATCCGGTGCCGCAGTCGCCGCTCAGCCATCATCCCTC GTCCTCTGCGTTATCCGCCGCAGCAGCTGCTGTGGCGGAGCGGGATCGACATGCGCTGATGCGTCAGCA ATCGCCACACATGACTCCACCCCCGGTGTCCAACGCCTCTTTAATGGCGAGTCCTCTGAGCAAGATGTA CGCTCCTCAACCGGGTCAGAGAGGCTTGGGAACATCACCGCCACCGCATTTGCGGCCAGGAGCATCGCC GCCGGTCATTCGTCACCCGCAGATGCCTCTACCGTTGCCACTGATTGCGCCTGGCGGAGGAATCCCGCA GATTGGAGTGCATCCGGGTCAGTCACCGTATCCGCACCCGCTTCTGCATCCCTCGGTATTCTACTCGCC GCATCACCATCCATTTAATTCGCCATATGGCTATGCGCCCTATGGTCCTGGATTCCCGGCATACATGAA GCCGCCACCACAGCCGGGACAGCTCGATCCGGCAGCCGTGATGGCGGCCCACCATGCCGGATTGCAAGG ACCGCCGCCCCAGCAGATGCGCCAGGACGAGCAGAATGCAGCGGCCGCCGCTGCACAAGCAGCTGCTGA GAAACAACACCAAGCGGCTGCAGCAGCGGCAGCCCAGCAGCACAAGGCGCCGCAACAACAACAGCCTGG CGGAATGCCACCCAACAAACCGCCGACGCCAAAGACGCCACAGGGTCCGGGCGGTGGGATGCCCCCAGG AATGGGTGGACCGGGAACACCGACGGGACTGCCGCCAGGTGCCTATCCTGGCAGCCATATGCCGGGATA TCCACAAGGGCCGCCTCATGGGTCACCCTTTGCGCCACAAGATGGTCAGCCTCACGGCTTGAAGCCCAC ATCGCACATGGACGCCCTGCGAGCGCATGCACACTCAGCCAACTCGGCGGGTATGGGCGGTGGACACCA TCCGACGGAGCCATTGCCCATTGATATTGAACCGGATCCAGAGCCAGAGATTCCCAGTCCAACGCACAA TATACCACGTGGTCCCAGTCCCGAAGCAAAACCGGACGACACCGAATGCCATCGCTCTCAGTCTGCCAT ATTTGTGCGCCACATCGATCGTGGGGATTACAATTCGTGCACGAGAACAGATTTGATCTTCAAGCCGGT GGCCGACTCAAAGTTGGCCCGCAAGCGTGAAGAACGCGACCGCAAGCTGGCCGAAAAGGAGCGTGAGCG GCGACAGCAGCAGCAGCAACAACAACAGCAGCAGCAACAACAGCAAGCAGCGGCCGCGCAACAGGCGGC ACAGCAAGCCAAGATGAAGGCTGAGCTGAAGCCACCGTATGCGGATACGCCGGCACTGCGTCAACTGTC CGAGTACGCTCGTCCCCACGTCGCCTTCAGTCCTGTTGAACAGATGGTGCCATATCATCATCCAATGGG CCCCATGTACAGAGAGAGGGAACTGGAGGAGATTAAAAACGCACAAGCTGCTGCGGCGAGTCAATCCCG

15 ACTAGATCCGCACTGGATGGAATACTATCGACGCGGCATCCACCCCTCGCAGTTCCCGCTGTATGCGAA TCCGGCGATATCGCAGATGGAGAGGGAGCGTCTGGGAATTCCACCTCCGCACCATGTGGGGTTGGACCC GGGCGAGCACATGGTGCGTATGCCGCAACCACCGGAGGCCGGTTTCCAACTGCCACCGAATGTTGGCCA GTATCCGCGGCCAAATATGCTTATACCTAGGGAGCCGCACTCGGATGTCCTGCTGCGCATGTCCTATGC CGACCAACTACAGTATTTACAGGCCGCCGAGTTTCAGCGACAGTCCCTGCATGATCAGTACTTTAGACA ACGGCCCAGATAAgtggacagacagcaattgtagagagcaagcaactgaacaaagacaatactcgggcc gcggccgtgaggaactgctttgtgtgatttttgtgttcaagcgtttacattttgttttccacacgcaca accacccaacatactcacaaaacaaacatcaaaacatcaacatcatctgcagagacgacgtcagcgtat ggctcaagaatccagaaaccagaaaaccaagcgactttaagttgcgcaacgccttgtcacgtttttatg taaaaagaatcagctgcgttaatcgggagtggttcgtcgattgttccggctgcatacagcacacttcac accagacactcactgaccaaggcatccagaatcaaaaagcagaaaccaactacaaaactaaactaaaca aaacaggacacgatggtcgcaatatcttaagtatcctcttcatagtgtatagtgttaaattcttaaacg taagcgagatatggagcatgttaaatacggtttagacttattaaggcaatggaacaaaatcgaacttgg cacacaaatttttatggaggttttcaaacagcttatatattatatgtatatgaatgtgctacacttcga caaaaactatctagtaagcttcacaaatcaattgatttagacacgatgataactaagcaaaaccaaaac acaaactcaccagcacttgctgcaggaggcaaggtacataaattagtatagacaaacgattaaggatag acgaatcgagcgctcatatatatattcgagagtattgtacaaatggattcggagatagttttcttttta caacacagtttggcactctgcgcattagagatacgcctaatattatttaaatgtatttacttccagcca caattgaactatataaggtttatatatgcttgctgctttcgactttttagctaaaataaaatggtaaac gaatcacgccaatgcctagatttcgctttttgtatgtagtccttgtttgtgttagcctagccagcagct ttgcctaccaactaactgctactcattatttattattttgctatgactagagtattataggggttggga tatgaaagttttgtaaaaagtgcataaaaaataacgaaataattatatgggtattatgtgaatgatttg atgctgctctcaaacccaaaagcacagcacggctttcgatgcttgtgacgtgccagcatttgaagcagc tgcaattttgagtgcttaatttatattagtaatttatacaattattatacgctaaaaaatgcattgaat tcacacaatatttcaaaagatttagtatgtaaggccaacaaaccgctaaggtggatggattcaatggta aacaaaagtgtgaattatactaaacaaaatgagaatccacaaagtaaaaatatttttaaatacggtttt ttatttcacattttgaatatacacattgatataaatatattgtagctatatactaattatgaaagttta tcgtaggagtctcgcaagcatattcaatggcacacaaacgacaaaaaacaaaaaaaataatacaagatt tggcgtagttaacatttaagaagtgtaaagtaaagatccatacatatttatatatatatacaaaaaaaa acaaaacaaatgcagctatattattgcagctatattactaaataaattataaaacaaaaagatcatact gaaaagataaattattaaaacctgtaaaaactaagtaaaacaaaataaaattagttac attagttaccaaatccc Supplementary Figure 13: Sequences targeted by RNAi constructs in tll, dsf, Sbb, Atro, and amon. In both the tll and Atrophin cdna sequence, the ORF is marked by capitalized letters, while the UTRs are marked by small caps. The yellow highlighted region in tll (bp ) corresponds to the region of the mrna sequence that is targeted by UAStll RNAi1 (UAS-tll JF02537 from ref. 39) and the blue highlighted region (bp and bp ) corresponds to the mrna sequences targeted by UAS-tll RNAi2 (from ref. 50). The yellow highlighted region in Atrophin (bp ) corresponds to the mrna sequence that is targeted by UAS-Atro IR (from ref. 52) and the purple highlighted region (bp ) corresponds to the mrna sequence targeted by UAS-Atro RNAi (from ref. 39)

16 Supplementary Figure 14: Full-length image of immunoblot of pan-neuronal tll knock-down. Full-length western blot showing Tll levels in males in which tll is knocked down in all neurons (elav-gal4>tll RNAi ) compared to control males (elav-gal4/y and UAStll RNAi ). Rabbit anti-nr2e1 antibody recognizes several non-specific bands. Tll migrates slightly below 50kDa.

17 Supplementary Figure 15: Full-length images of immunoblots of pan-neuronal Atro knock-down. Full-length western blot showing Atro levels in males in which atro is knocked down in all neurons (elav-gal4>atro IR ) compared to controls (elav-gal4/y and UAS- Atro IR ). The same blot was probed with Atro antibody (left) and with Dlg antibody (right). The MW standard is shown between the two blots. The rabbit anti-atro antibody recognizes several non-specific bands. Atro migrates around 250 kda, Dlg migrates slightly above 100kDa.

18 Supplementary Table 1: Similarity of Tll and Dsf to NR2E1. Identical residues Similar residues Weakly similar residues Gaps Tll Dsf Approximately the same number of amino acid residues are identical, similar and weakly similar between both fly paralogs and the mouse gene. Tll has slightly fewer gaps (indels) than Dsf.