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1 DOI: 1.138/nc3497 In th formt provi y th uthors n unit. -4 min -2 min min 8 min RFP::CAAX -15 μm μm RFP::CAAX High Low R F/B of D/V clls 1.5 picl 48 sc 6 sc 66 sc 7 sc 78 sc 9 sc 126 sc sl for intrcltion uring intrcltion Bsl (-15μm) Apicl (μm) c R F/B of of pk3 RBD mong D/V clls uring rostt progrssion 1.5 * * for intrcltion uring intrcltion rostt rsolv rostt Grmn xtnsion (%) 8 **** 6 4 DN-Rc.N17 D gm D cph Apicl ( μm) Control mryo n of stg 6 ( min) lry srg 8 (15 min) Bsl (-15 μm) RBD pk3 ::GFP High Low Control mryo n of stg 6 ( min) rly stg 8 (15 min) Apicl ( μm) f Bsl (-15 μm) GFP::Rc1 High Low Supplmntry Figur 1 Chrctristics of rostt formtion n GBE in wil-typ n DN-Rc1 T17N mryos. () Forc-color confocl tim-lps imgs of mryo xprssing RFP::CAAX uring rostt formtion. Brightr colors not high fluorscnt intnsity. D/V n A/P clls r inict y fill n opn ots, rspctivly. min rprsnts th tim of rostt pttrn formtion. ( ) Quntifiction of R F/B of D/V clls in mryos xprssing RFP::CAAX for n uring rostt formtion t th picl n sl sction (n=1 D/V clls from 2 mryos). () Two-photon tim-lps imgs of mryos xprssing Utrophin::GFP uring rostt formtion t th picl (μm) n th sl (-15μm) sction. Color ots inict clls in rostt. R rrows inict th migrtion irction of intrclting D/V clls. sc rprsnts th ginning of imging. (c) Tim progrssion of front-to-ck rtio (R F/B ) of pk3 RBD ::GFP intnsity msur uring th rostt formtion t sl sction (n = 6 rostts, 2 mryos). () Tissu xtnsion of wil-typ (n=19) n tissu-lvl xprssion of DN-Rc TN17 mryos (n=44). Avrg vlus r shown s thick horizontl lins. ( ) Schmtic illustrtion of th tissu xtnsion msurmnt. Th lngth twn th postrior n of grmn n th postrior n of th mryo (D gm, lck rrow) is ivi y th lngth twn th cphlic furrow n th n of mryo (D cph, r rrow). (-f) Forc-color two-photon imgs of pk3 RBD ::GFP () n GF- P::Rc1 (f) uring fst GBE stg of vntroltrl pithlium in mryos. Dt shown rprsnt mn ± SEM in, n c. Sttisticl tsts wr on y stunt s t-tst. * n *** not P < 5 n P < 1, rspctivly. : not significnt. Scl rs, 1 μm Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
2 Apicl ( μm) Bsl (-15 μm) (μm) - min -8 min min 23 min zip (MHC) RNAi - 24 min min 8 min 3 min Prcntg fo rostt rsoluction (%) simultnous sl first picl first no sl rostt sl fil picl fil sl norml 75 5 c Clustring cofficint (n = 5) Apicl Bsl pr-rostt rostt rsolv Stgs sqh (MRLC) RNAi - 6 min min 3 min min Apicl ( μm) Apicl ( μm) j f Bsl (-15 μm) Protrusion prcntg pr D/V cll pir (%) n = 66 n = 46 n = 38 g (μm) D 9 A 18 P - 9 V Protrusion orinttion g Bsl (-15 μm) D 9 V cll D cll A 18 P - 9 V Protrusion orinttion Pr-rostt Rostt Rsolv h Sp of D/V clls t sl sction (μm/min) 1.5 l * **** i Num. of rsolv sl rostts / 1 4 μm 2 / 3 min n = 146 * * n = 39 n = 44 n = 83 n = 82 Y27632 zip RNAi k Topologicl stgs of 6-cll rostt No 4 No 2 No 6 No 1 No 5 No 3 No 2 No 4 No 1 No 6 No 3 No 5 No 2 No 1 No 4 No 3 No 6 No 5 m Protrusion; min (Numr of circls) = 4 (6); min (Dimtr rtio) = 1.6 (2.1). n No Protrusion; min (Numr of circls) = 4 (2); min (Dimtr rtio) = 1.6 (1.5). Multipl innr ntwork connctions form twn clls of pr-rostt pttrn No innr ntwork connctions mong clls of th rostt pttrn Multipl innr ntwork connctions rstor twn clls of rsolv rostt pttrn Clustring cofficint Clustring cofficint = Clustring cofficint Supplmntry Figur 2 Quntifictions of H-1152, Y injctions n sqh, zip RNAi xprimnts n nlysis of rostt topology trnsition n sl protrusions. () Rostt formtion with injction. () Frction of rostts with trt (56 rostts, 3 mryos) n (118 rostts, 6 mryos) mryos. (c) Clustring cofficint of rostt progrssion in trtmnt (n=5 rostts from 3 mryos). (, ) Rostt formtion in zip RNAi () n mryos (). In, -, fill n opn ots inict D/V n A/P clls, rspctivly. In -, th t r rprsnttiv of 5 rostts pr conition. R opn circls not th timing of rostt formtion (min). (f) Frction of D/V clls with protrusions in control, injct, n mryos. (g, g ) Distriution of th orinttion of sl protrusion in injct (n=46), n (n=36) mryos. A, P, D, n V not ntrior, postrior, orsl n vntrl irction, rspctivly. (h) Sp of D/V cll t th sl sction (-15μm, n=7,6,5 rostts in wil-typ, injct n mryos, rspctivly). (i) Numr of rostts ppring in wil-typ, Y injct, injct, zip RNAi, n mryos. (j) Grphicl illustrtion of th topologicl stgs of 6-cll rostt. Thr topologiclly istinct stgs r pr-rostt, rostt, n rsolv stgs. R lins inict th connctions twn clls mong th rostt. (k) Grphicl rprsnttions of cll connctions mong th rostt (r lins). Cntrs of ch cll r rprsnt y numr nos. (l) Tl of clustring cofficint n prmtrs msur y Cytoscp. (m-n) Exmpls of th moifi kissing-circl lgorithm to tct protrusions. (m) A cll ws consir s protruing cll. Thr r 6 circls within th cll, n th imtr of ch circl is lss thn 4% of th lrgr circl. (n) A cll ws consir s non-protruing cll. Thr r only 2 circls within th cll, n th imtr of smllr circl is 5% of th lrgst circl. Dt shown rprsnt mn ± SEM in c, h n i. Sttisticl tsts wr on y stunt s t-tst in h. * n *** not P<5 n P<1, rspctivly. Scl rs, 1μm Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
3 Apicl ( μm) Stg 6 Stg 7 Src42A srna injction -22 min -8 min min 12 min Apicl ( μm) Apicl ( μm) Bsl (-15 μm) Src42A srna injction -9 min -4 min min Src42A srna injction -17 min -1 min min 14 min Apicl ( μm) Bsl (-15 μm) DN-Src42A -39 min -16 min min 17 min (-15 μm) Apicl ( μm) cbsl Bsl (-15 μm) pk3 RBD :: GFP Bsl (-15 μm) GFP::Rc1 f Exprssion rltiv to Rpl Src64B RNAi Src42A Src64B Src42A srna injction g Num. of picl rostts /1 4 μm 2 / 3 min h Prcntg of rostt formtion (%) srna injction simultnous sl-first picl-first 75 5 Src64B RNAi 23% 68% 9% % 28% 59% 28% 44% 1% % 23% 4% + + i Grmn xtnsion (%) * OrgonR Src64B RNAi Supplmntry Figur 3 Anlyss of Src42A n Src64B knockowns. () Exmpl of svr pithlium fcts in strong mryos t stg 6 n stg 7. R rrowhs inict th gps twn pithli clls. Yllow ots inict th clls prcociously lmint from pithlium uring cllulriztion. (-c) Two-photon imgs of rostt progrssion in Src42A srna injct () or ominnt-ngtiv DN-Src42A inuc mryos (c). () Forc-color tim-lps imgs of pk3 RBD ::GFP () n GFP::Rc1 () uring rostt formtion in Src42A srna injct mryos. In -, colour ots ll clls mong rostt, min rprsnts th tim whn picl rostt forms. (f) Fol chng of Src42A n Src64B mrna in, Src64B RNAi (lft n mil rs, inuc y GAL4-UAS), n Src42A srna injct mryos rltiv to th control (UAS-Src42A, UAS-Src64B corrsponing to ch group, n Orgon-R s control for injctions, n=3 inpnnt iologicl rplicts for ch group). Pls rfr to Supplmntry Tl 4 for tils. (g) Numr of picl-rostts ppring in wil-typ (n=146 rostts from 7 mryos) n (n=167 rostts from 7 mryos) mryos. (h) Frction of iffrnt rostt phnotyps in wil-typ (146 rostts, from 7 mryos), Src42A srna injct (57 rostts from 6 mryos), Src64B srna injct (29 rostts from 4 mryos), n co-injction of Src42A n Src64B srna mryos (35 rostts from 6 co-injct mryos). Th frction of sl-first n picl-first rostts in Src64B injct mryos ws similr to tht of wil-typ. Morovr, th frction in th mryos with oul supprssion of Src42A n Src64B i not xcrt th sl fcts compr with singl supprssion of Src42A. (i) Extnsion of grmn in wil-typ (n=19) n Src64B RNAi (n=) mryos. Th gr of GBE ly in Src64B RNAi ws to lssr xtnt compr to tht of (Fig. 5k). Ths t inict tht Src64B i not ply mjor rol in sl rostt formtion n GBE. Dt shown rprsnt mn ± SEM in f n g. Sttisticl tsts wr on y stunt s t-tst in g n i. * nots P < 5. Scl rs, 1 μm Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
4 Phlloiin Mrg Apicl Phlloiin Phlloiin Bsl Phlloiin c OrgonR c Phlloiin Phlloiin OrgonR DN-Src42A Apicl ( μm) Bsl (-12 μm) Phlloiin Supplmntry Figur 4 n ctin locliztion n th msorm morphology in wil-typ n Src42A knockown mryos. (-) n phlloiin t th picl () n soltrl sction () of wil-typ mryo. (, ) High mgnifiction of th ox rgion in n. ( ) nrich t th sl rostt n co-locliz with ctin. (c-c ) Ltrl viw of n ctin t GBE stg 7-8 vntrl pithlium from wil-typ, Orgon-R (c) n knockown (c ) mryos. (- ) Cross-sctionl viw of msorm t stg 7-8 gstrultion mryos. Th tissu intgrity of invgint msorm mintins in knockown ( ) similr to th wil-typ () mryo. () Distriution of n phlloiin of sl fctiv rostt in DN-Src42A mryo. In -, -, c-c, - imgs r rprsnttiv of 4-5 mryos for ch pnl. In, img is rprsnttiv of mor thn 5 rostts. Scl rs, 1 μm Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
5 Prcntg of T1 trnsition (%) simultnous sl first picl first 14% % 1% wiltyp Apicl Bsl (-18μm) min 2 min 5 min 8 min Bsl (-18μm) Apicl pgfp::utr c for intrcltion uring intrcltion R F/B of D/V clls uring T1 trnsition 1.5 ** picl sl picl sl pk::gfp GFP::Rc1 ** Num. of rostts / 1 4 μm 2 / 3 min _picl _sl toll268_picl toll268_sl v_picl v_sl Supplmntry Figur 5 Chrctristics of sl intrcltion uring T1 trnsition, n nlyss of rostt fcts in v, n Toll-2, 6, 8 knockowns. () Frctions of T1 trnsitions with picl-first (whit), sl-first (light-gry), simultnous (rk-gry) phnotyps in wil-typ mryos (n=45 T1 trnsitions from 3 mryos). () Confocl tim-lps imgs of mryo xprssing pgfp::utr uring T1 trnsition. Only smll numr of clls wr photoctivt. D/V n A/P clls r inict y fill n opn ots, rspctivly. min rprsnts th tim of th formtion of sl nw junction. Whit rrowhs inict th irction of two intrclting D/V clls uring T1 trnsition. Whit ott lins rprsnt th shp of clls with wkr fluorscnc. (c) Quntifictions of front-to-ck rtio (R F/B ) of D/V clls in mryos xprssing pk3 RBD ::GFP (n=1 D/V clls) n GFP::Rc1 (n=16 D/V clls) intnsity for n uring T1 trnsition t th picl n sl sction. () Avrg numr of rostts count t th picl sction (μm) n sl sction (-15μm) in wil-typ (n=4 mryos), v RNAi (n=9 mryos) n tripl-knockown of Toll-2,6,8 mryos (n=6 mryos). Scl r, 1 μm Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
6 Supplmntry Tl Lgns Supplmntry Tl 1 Brightfil imging of GBE fcts from DN-Rc n Src knockown scrning. () GBE fcts of mtrnl lo ominnt-ngtiv knockown of Rc1 scrning. () GBE fcts of mtrnl lo RNAi n ominnt-ngtiv knockown of Src42A n Src64B scrning. (c) GBE fcts from scrning of Src42A zygotic mutnts. W foun tht rmoving th zygotic contriution of Src42A cus littl or no ffcts on GBE ut l to othr morphognsis fcts in ltr vlopmnt, suggsting tht mtrnl, ut not zygotic, contriution of Src42A is criticl for GBE. Supplmntry Tl 2 RNAi scrning to intify possil rgultors of soltrl-rostt formtion. Supplmntry Tl 3 Squncs of primr us to synthsiz srnas. Th squncs of th forwr n rvrs primrs us to synthsiz srnas ginst Src42A, Src64B, vn-skipp, Toll-2, Toll-6, trnscripts r list. Annling squncs r highlight in ol. Th rmining squnc is T7 promotr. Supplmntry Tl 4 Sttistics sourc t. Supplmntry Vio Lgns Supplmntry Vio 1 Rostt formtion t iffrnt picosl sctions in wil-typ mryo from two-photon liv imging of Utr-GFP. Supplmntry Vio 2 3D rconstruction of rostt formtion from two-photon liv imging of Utr-GFP. Supplmntry Vio 3 Two-photon liv imging of Pk3 RBD -GFP t th sl (-15 μm) pithlium in wil-typ GBE mryo. Supplmntry Vio 4 Two-photon liv imging of GFP-Rc1 t th sl (-15 μm) pithlium in wil-typ GBE mryo. Supplmntry Vio 5 Brightfil movi of GBE fct of n mryo xprssing oth zip RNAi n. Th mryo is rprsnttiv from ovr 9 fctiv mryos. Supplmntry Vio 6 Wtr-injct mryo xprssing Utr-GFP uring GBE y two-photon imging of picl n sl sction. Supplmntry Vio 7 v srna injct mryo xprssing Utr-GFP uring GBE y two-photon imging of picl n sl sction. Supplmntry Vio 8 Toll-2,6,8 tripl knockown mryo xprssing Utr-GFP uring GBE y two-photon imging of picl n sl sction Mcmilln Pulishrs Limit, prt of Springr Ntur. All rights rsrv.
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