Leucine-Rich Repeat Transmembrane Proteins Instruct Discrete Dendrite Targeting in an Olfactory Map

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12 hrs APF N/n82 Hon t l, 2009 Luin-Rih Rpt Trnsmmrn Protins

1 Howr Huhs Mil Institut n prtmnt o Bioloy, Stnor Univrsity, Stnor,

CA 91125, USA; 3 Struturl Bioloy Cntr, Ntionl Institut o Gntis, n

Jpn. Supplmntry Fiur 1 nti-cps ps-gl4 16 hrs APF 18 hrs APF 24 hrs

Tim ours nlysis o Cps xprssion urin vlopmnt.

inst Cps (ntrl pnls) n ps-gl4-rivn mc8-gfp (riht pnls).

1 12 hrs APF N/n82 Hon t l, 2009 Luin-Rih Rpt Trnsmmrn Protins Instrut isrt nrit Trtin in n Oltory Mp Wizh Hon 1, Hito Zhu 1, Christophr J. Pottr 1, Grill Brsh 1, Mitsuhiko Kurusu 2,3, Ki Zinn 2, Liqun Luo 1 1 Howr Huhs Mil Institut n prtmnt o Bioloy, Stnor Univrsity, Stnor, CA 94305, USA; 2 ivision o Bioloy, Cliorni Institut o Thnoloy, Psn, CA 91125, USA; 3 Struturl Bioloy Cntr, Ntionl Institut o Gntis, n prtmnt o Gntis, Th Grut Univrsity or Avn Stuis, Mishim , Jpn. Supplmntry Fiur 1 nti-cps ps-gl4 16 hrs APF 18 hrs APF 24 hrs APF 30 hrs APF 36 hrs APF 48 hrs APF Supplmntry Fiur 1. Tim ours nlysis o Cps xprssion urin vlopmnt. (-) Th vlopin ntnnl los wr stin rom 12 h APF to 48 h APF y ntiois inst Cps (ntrl pnls) n ps-gl4-rivn mc8-gfp (riht pnls). Nuropil is visuliz in th lt pnls y N-hrin (-, rom 12 h APF to 36 h APF) or n82 (, 48 h APF). Cps protin is prsnt in th vlopin ntnn lo throuhout th prio whn PN nrits r mkin thir trt isions, n it is not istriut vnly s ompr to th nuropil mrkrs. Sl rs rprsnt 10 µm. All ims r sinl onol stions. Ntur Nurosin: oi: /nn.2442

2 Supplmntry Fiur 2 Hon t l, C155-Gl4 GH146-Flp 5 UAS>stop>mC8GFP 25% 10 ps-gl4 GH146-Flp 5 UAS>stop>mC8GFP 25% A2 A6 L2v L2 A4 L5 M4 C2 M5 M1 M6 A1 A2 A4 C1 C2 C3 L3 P1m A1 A3 A7l L1 M2 A3 P1l C3m M4 M6 GH146-Flp + GH146-Flp! A2 A6 L2v L2 A4 L5 M4 C2 M5 M1 M6 A1 A2 A4 C1 C2 C3 L3 P1m A1 A3 A7l L1 M2 A3 P1l C3m M4 M6 Cps + 10 ps-gl4 y-flp 5 UAS>stop>mC8GFP 25% A2 A6 L2v L2 A4 L5 M4 C2 M5 M1 M6 A1 A2 A4 C1 C2 C3 L3 P1m A1 A3 A7l L1 M2 A3 P1l C3m M4 M6 Cps! N Supplmntry Fiur 2. Intrstionl xprssion pttrns rom irnt Flp n Gl4 lins. Exprssion o Flp-out GFP rportr UAS>stop>mC8-GFP () t th intrstion o pn-nurl C155- Gl4 n PN-spii GH146-Flp in ult, whih trmins th PNs tht r positiv or GH146-Flp, () t th intrstion o ps-gl4 n PN-spii GH146-Flp in ult, whih trmins th Cps-positivs PNs mon GH146-positiv PNs, n () t th intrstion o ps-gl4 n ORN-spii y-flp, whih trmins th Cps-positiv ORNs. W sor 46 o ~50 lomruli (x xs) tht r onsistntly intiil. Th rst o th lomruli r mor vril in siz n morpholoy, lot in th postrior ntnnl lo, n r GH146-ntiv. Th y xs rprsnt th prnt o ntnnl los in whih prtiulr lomrulus is innrvt y GFP-positiv nurons. Th lowr prnt o th intrstionl mrkr xprssion in ps-gl4 ll PNs () ompr to C155-Gl4 ll PNs () my u to lowr xprssion lvl o ps-gl4 in ult PNs ompr with C155-Gl4. Th lowr prnt o th intrstionl mrkr xprssion in iniviul PNs () ompr with ORNs () likly rsult rom two tors. First, Flp-mit romintion my not omplt, thror not ll lls t th intrstion r ll. Thr r ~20 ol mor ORNs thn PNs innrvtin sinl lomrulus on vr, thror it is mor likly or lomrulus to ll y t lst on ORN xon thn t lst on PN nrit. Son, y-flp is turn on rly n xprss in ll ORN prursors, whrs GH146-Flp is turn on rltivly lt n only xprss in post-mitoti PNs, whih urthr rss th hn o rorin ps-gl4 xprssion. In orr to ompnst or th stohsti ntur n trmin ll Cps-positiv PN lsss, w quntii th xprssion pttrn in 18 inpnnt ntnnl los, n sint ny lomruli tht r ll in t lst 2 inpnnt ntnnl los to trts o Cps-positiv PNs. (n=24, 18, 8 or -). Bs on th omprison o () n (), th orrltion twn Cps xprssion in ORNs n PNs is not sttistilly siniint (X 2, p>0.3). In ition, PNs riv rom th ltrl nurolst (ll orn in lrvl st) r prrntilly Cps-positiv (9 out o 11). PNs riv rom th ntroorsl lin n sprt into two suroups: 6 out o 7 PNs orn in mryos r Cps-positiv n 3 out o 11 PNs orn in lrv r Cps-positiv. In summry, thr is no lr-ut rltionship twn Cps xprssion n lin/irth orr. Ntur Nurosin: oi: /nn.2442

3 Supplmntry Fiur 3 Hon t l, 2009 NB LOF mistrtin % Norml innrvtion Loss-o-innrvtion Etopi innrvtion Cps + Cps! PN xprssion ORN xprssion LOF Mistrtin A1 A2 L3 M1 C2 A4 M5 Siniint? A4 C2 C1 C3 P1m A1 A3 A7l L1 M2 L2 L2v L5 M4 M6 A2 A6 A3 P1l C3m M4 M6 Ys, p<0.001 No, p>0.7 Siniint? Ys, p<0.001 No, p>0.2 PN xprssion ORN xprssion C2/A4/M1 LOF Mistrtin PN xprssion ORN xprssion ME Mistrtin PN xprssion ORN xprssion A2 C2 C1 P1m A1 A4 C3 L3 A1 A3 A7l L1 M2 L2 L2v L5 M1 M4 M5 M6 A2 A4 A6 C2 A3 P1l C3m M4 M6 A2 C2 C1 P1m A1 A4 C3 L3 A1 A3 A7l L1 M2 L2 L2v L5 M4 M5 M6 A2 A6 A3 P1l C3m M4 M6 L2 L2v C2 A4 M1 A2 A6 M6 L5 M4 M5 P1m A1 A2 A4 C1 C2 C3 L3 A1 A3 A7l L1 M2 A3 P1l C3m M4 M6 Siniint? Ys, p<0.01 No, p>0.3 Siniint? Ys, p<0.01 No, p>0.3 Siniint? Ys, p<0.001 No, p>0.1 Supplmntry Fiur 3. Sttistis o mistrtin iss with rr to Cps xprssion in PNs or ORNs. Quntiition o lomrulr innrvtion pttrn in ps!/! nurolst lons (), ps!/! sinl ll lons (-) n Cps misxprssion in sinl ll lons () wr tkn rom Fiur 2, 3t, 3v n 4j. W Cps xprssion inormtion low iniviul lsss o PNs n ORNs (orn, Cpspositiv; lu, Cps-ntiv). W trmin whthr th prrn o mistrtin vnts tht our in lomrulr trts o Cps-positiv or Cps-ntiv PNs (or ORNs) is sttistilly siniint, n show th orrsponin p-vlus to th riht o Cps xprssion pttrn. In h s, thr is siniint orrltion twn whr mistrtin ours n Cps xprssion pttrn in PNs, ut no siniint orrltion twn whr mistrtin ours n Cps xprssion pttrn in ORNs. In ition, thr is no lr-ut rltionship twn topi trts n th lin/irth orr o orrsponin PNs. Ntur Nurosin: oi: /nn.2442

Hon t l, 2009 Supplmntry Fiur 4 WT WT ps

4 Hon t l, 2009 Supplmntry Fiur 4 WT WT ps / ps / ME Rsu Supplmntry Fiur 4. Norml xon trtin o PNs in ps mutnt n misxprssion. Axon trtin to th mushroom oy lyx n ltrl horn (outlin) o sinl ll lons (-) n lons (-) r shown or thr notyps s init. Cps misxprssion in Cps-ntiv PNs or Cps loss in Cps-positiv PNs i not hn th lssspii ltrl horn roriztion pttrns. S Mthos or lss intiition. wil-typ, n=10; ps!/!, n=10; misxprssion, n=10; wil-typ, n=10; ps!/!, n=7; rsu, n=6. Ntur Nurosin: oi: /nn.2442

Hon t l, 2009 Supplmntry Fiur 5 n82 GH146::mC8GFP Or47-rC2 mr Norml h Shit Supplmntry Fiur 5. Loss o Cps in PNs os not isrupt th propr trtin o ORN xons.

In th ntrior stion o th ntnnl lo, PN nrits trtin to A1 (soli outlin) n (sh outlin) n istinuish ov th kroun o th lss ns nrits o th pn-ntnnl-lo-projtin PN (top pnl, son rom lt).

vntrl nurolst lons, w oun orsomil shit o nrits trtin to th lomrulus, suh tht it xtnsivly orrs th A1 lomrulus (n xmpl is shown in th ottom pnl).

2), n oun tht in ll 4 ss Or47 xons shit orrsponinly, suh tht th synpti mthin twn vntrl PN n Or47 xon is not isrupt, spit th loss o Cps in vntrl PNs.

5 Hon t l, 2009 Supplmntry Fiur 5 n82 GH146::mC8GFP Or47-rC2 mr Norml h Shit Supplmntry Fiur 5. Loss o Cps in PNs os not isrupt th propr trtin o ORN xons. Norml GH146-Gl4 ll vntrl nurolst MARCM lons onsist o unilomrulr PNs tht trt nrits to th A1,, n L1 lomruli, n pn-ntnnl-loprojtin PN 1. In th ntrior stion o th ntnnl lo, PN nrits trtin to A1 (soli outlin) n (sh outlin) n istinuish ov th kroun o th lss ns nrits o th pn-ntnnl-lo-projtin PN (top pnl, son rom lt). A1 n r sprt y th A1 lomrulus. Howvr, in 4 out o 13 ps!/! vntrl nurolst lons, w oun orsomil shit o nrits trtin to th lomrulus, suh tht it xtnsivly orrs th A1 lomrulus (n xmpl is shown in th ottom pnl). W itionlly ll ORNs tht normlly trt to th lomrulus with trnsn Or47-rC2 (r. 2), n oun tht in ll 4 ss Or47 xons shit orrsponinly, suh tht th synpti mthin twn vntrl PN n Or47 xon is not isrupt, spit th loss o Cps in vntrl PNs. Pnls rom lt to riht: n82 stinin tht lls ll lomruli; MARCM llin o vntrl nurolst lons y mc8-gfp; Or47-rC2 llin o Or47 ORN xons; mr o th thr hnnls. Gnotyp: hsflp 122 UAS-mC8-GFP; GH146-Gl4 UAS-mC8-GFP / Or47-rC2; ps 28s FRT2A / G80 FRT2A. Rrns: 1. Mrin, E.C., Jris, G.S., Komiym, T., Zhu, H. & Luo, L. Rprsnttion o th lomrulr oltory mp in th rosophil rin. Cll 109, (2002). 2. Brnik,., Chihr, T., Couto, A. & Luo, L. Wirin stility o th ult rosophil oltory iruit tr lsion. J. Nurosi. 26, (2006). Ntur Nurosin: oi: /nn.2442

Hon t l, 2009 Supplmntry Fiur 6 24 hrs APF 36 hrs APF

lip-out toxin RiinA in th intrstionl rion o Pl-Gl4 n

This strty lts lmost ll ORNs or thir xons ntr th

Compr with norml ORNs (-), most o th lt ORNs i or il

Antnnl los n ORN ntry unls r outlin y ott lins.

6 Hon t l, 2009 Supplmntry Fiur 6 24 hrs APF 36 hrs APF 48 hrs APF Ault Wil typ h ORN ltion Supplmntry Fiur 6. Eiy o pn-orn ltion urin vlopmnt. (-) Pl- Gl4 is xprss in ll ORNs urin n tr ORN xons rriv t th ntnnl lo. (-h) Pl-Gl4 n y-flp r utiliz to riv th xprssion o lip-out toxin RiinA in th intrstionl rion o Pl-Gl4 n y-flp. This strty lts lmost ll ORNs or thir xons ntr th vlopin ntnnl lo. Compr with norml ORNs (-), most o th lt ORNs i or il to sn xons to th ntnnl lo n w ORN xons rriv t th o th ntnnl lo t 24 h APF (). Ths rminin xons o not ntr th ntnnl lo n vntully i (-h). Antnnl los n ORN ntry unls r outlin y ott lins. Pl-Gl4 lso lls som ntrl nurons, notly in th suosophl nli (vntrl to th ntnnl los), whih r not lt us ths nurons o not xprss y- Flp. All ims r sinl onol stions. Gnotyp: Pl-Gl4, y-flp; UAS-mC8-GFP; UAS>stop>RiinA / UAS-ps. Ntur Nurosin: oi: /nn.2442

Trn-ovrxprssin PNs in nurolst or sinl-ll

7 Hon t l, 2009 Supplmntry Fiur 7 PN lpn ps-gl4, GH146-Flp UAS>stop>mC8GFP trn-lz WT ps + OE trn + Mr Supplmntry Fiur 7. Trn ovrxprssion phnotyps n ovrlppin xprssion with Cps in PNs. (-) nrit trtin o wil-typ n Trn-ovrxprssin PNs in nurolst or sinl-ll MARCM lons. Ovrxprssion o Trn (P{GS6}10885) in nurolst lons (,) or sinl ll lons () uss stron mistrtin o nrits to othr lomruli ompr to wil-typ ontrols (,,). () Exprssion o trn-lz (r) tothr with ps-gl4, GH146-Flp, UAS>stop>mC8-GFP (rn). A mnii viw o th ll ois is shown in th lowr pnls. Ntur Nurosin: oi: /nn.2442

Present state Next state Q + M N

Present state Next state Q + M N Qustion 1. An M-N lip-lop works s ollows: I MN=00, th nxt stt o th lip lop is 0. I MN=01, th nxt stt o th lip-lop is th sm s th prsnt stt I MN=10, th nxt stt o th lip-lop is th omplmnt o th prsnt stt I