Journal of Cell Science eta evu system for nervous cells the central glial of in development required postembryonic are normal Inx2 and Ogre Innexins Article Research necnetdb w xrclua op n single The to space a C-termini. intercellular the in and and dock hemichannels membranes hemichannels; cell N- neighbouring loops multimeric in intracellular into extracellular assemble and domains proteins transmembrane loop, two four intracellular by topology: membrane interconnected have the connexin connexins same and the where Innexins of 1999). the 2007), members Paul, and from Saier, (White formed and family are (Cx) Yen junctions gap 2003; of al., al., et bulk (Panchin et humans Bruzzone including chordates, 2000; present (Phelan, in are numbers organisms (Panx), small pannexins in prechordate termed innexins, in Divergent 2005). genes gap-junction known elegans originally Caenorhabditis (Inx), diseases Innexins assembled families. in are gene identified human junctions two gap of (Wei products level, the molecular cataract several from the and At gap-junctional 2004). in deafness al., et neuropathies, Defective implicated peripheral function. including is and normal communication for development essential are that tissue coupling metabolic fundamental electrical differentiation, and in and homeostasis proliferation involved cell and as is kingdom such processes animal channels the gap-junction in ubiquitous through communication Intercellular Introduction words: Key channels the functional In of channels. formation induces homotypic (Inx2) Inx2 Innexin2 from with paired Ogre distinct signalling in of properties the co-expression paracrine expression two with however, regulates releasing heterologous channels, channels; that of by by gap-junction pore independently intercellular each homotypic demonstrate, aqueous act from form we central form also one a Here can proteins hemichannels, with neurogenesis. Hemichannels channel two of postembryonic molecules. complete of signalling a families composite small form a and two to molecules. is ions space are these extracellular of channel Connexins the exchange intercellular pannexins. of in cell–cell termed Each dock Members sequences directly. junctions. Hemichannels communicate divergent chordates. gap cells. to as apposed constitute systems chordates to multicellular which in in exclusive numbers of cells small assemblies neighbouring proteins in permit persist analogous to and evolved prechordates functionally have in that found families are gene Innexins two of one are Innexins Summary 10.1242/jcs.117994 doi: 3823–3834 126, ß Science Cell of Journal 2013 June 4 Accepted ( correspondence for *Author 2 1 Holcroft E. Catherine agtdepeso fRAitreec rngns ed oasgiiatrdcini h ieo h avlnrossse and system nervous larval postembryonic the normal of for cells size glial the in in required crucially reduction are system. significant innexins nervous these a either central that to conclude the of We leads of Downregulation adults. development transgenes, cells. surviving glial in interference defects in RNA behavioural and of neuroepithelia expression proliferative targeted in Ogre with colocalises aln Phelan Pauline aut fLf cecs nvriyo acetr acetrM39T UK 9PT, M13 Manchester Manchester, UK of 7NJ, University CT2 Sciences, Canterbury Life Kent, of of Faculty University Biosciences, of School 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. pi aginrdcd(ogre) reduced ganglion optic nei,Cnei,Gpjunction, Gap Connexin, Innexin, rspiamelanogaster Drosophila 1, * [email protected] Lnemne l,19) r h only the are 1999), al., et (Landesman 1 ila .Jackson D. William , ) Pea ta. 98 and 1998) al., et (Phelan Drosophila samme fthe of member a is evu ytm Glia system, Nervous , 1 e-sagLin Wei-Hsiang , Drosophila Drosophila eisi al avewt h eciaino quiescent adult- embryonic of re-modelled new reactivation with produce neurogenesis together the to which, of with divide neurons wave These 1985; specific larvae This neuroblasts. early Kankel, embryonic 1992). in and Kankel, begins was and (Lipshitz architecture in neural Watanabe defects neurogenesis to the attributed and was postembryonic phenotype flies This nervous smaller wild-type disorganized. small significantly highly of were had lobes those flies optic than development the Mutant particular, neural 1985). in systems; in Kankel, implicated and (Lipshitz and screening mutagenesis melanogaster Drosophila eihne()cnans i ftoo oedifferent more or two both, of or mix one, a which proteins. in contain(s) as channels to hemichannel(s) referred heteromeric (hereafter homotypic), is and species innexin hemichannel as heterotypic) different each a to which of in referred composed channels (hereafter heterotypic These identical composition homomeric pore. are respectively. hemichannels subunit two aqueous species, homomeric the in which types: protein in central channel channels one, intercellular homotypic possible a than of three composed more assemble with heteromeric, or or channel homomeric one, be complete can Hemichannels a form pi aginreduced ganglion optic 2 nei aiy rgnlyietfe sagn seta for essential gene a as identified originally family, innexin alihBassiri Kayleigh , avlcnrlnrossse,w idta n2partially Inx2 that find we system, nervous central larval Xenopus twsdsoee yconventional by discovered was It . ( ogre 1 ihr .Baines A. Richard , ogre oye,ta geaoede not does alone Ogre that oocytes, soeo ih nei ee in genes innexin eight of one is ) or inx2 Drosophila eetvl ngi,by glia, in selectively 2 and 3823 Journal of Cell Science el Fg AG,mmrn urnsrcre in recorded paired the currents in membrane expressed 1A–G), those (Fig. to equivalent cells ng), (5 amounts RNA n otg-eedn anrfor manner voltage-dependent and otg estvt Fg EG rage) ntetaeshown trace no the or weak In very triangles). showed 1E), 1E,G, which Inx2 (Fig. (Fig. in and sensitivity pairs Ogre cell voltage expressed in induced cells Channels both squares). 1G, Fig. plot, a through voltage cell transjunctional ( depolarising one and Stepping hyperpolarising clamp channels. of Ogre voltage functional series expressed form dual cells both to by which failed in activity pairs channel Cell gap-junction electrophysiology. recorded form and to paired protein in oocytes RNA Ogre corresponding the of expressed we ability channels, the investigate To of Expression Results expression. innexin explore of of and consequences downregulation CNS the cell-specific expression larval investigating early by the significance the functional in describe their innexins two We cell homotypic these same coupling. of form the pattern in intercellular not Inx2 regulate does with in interact innexin to can expression this but channels that intercellular heterologous show We by oocytes. channels, the junction of of development. neural role member postembryonic the early of a in analyses innexins further other as or no the this been identification on have work there its initial family, innexin pre-dated the Since which al., 2008). gene, al., et et (Younossi-Hartenstein Awasaki supporting 2003; neurons in part compartmentalizing important an cells and Glial play (CNS). and system simultaneously nervous develop central adult the form neurons, egbu Fg B al ) ucinlcnutne( conductance Junctional 1). Table 1B; (Fig. neighbour 3824 n2hmtpccanl.Telte yesoe te voltage steep ( current showed junctional type state from latter steady The significantly sensitivity; The 1). differed channels. (Table channels homotypic pairs these Inx2 Inx2/Inx2 of of properties that voltage than higher statistically, estvt bevd nsvrlcl ar hr a oeiec of evidence as no response was voltage there a pairs cell several in observed; sensitivity eln thigher at decline eenteetial ope Fg D;teaverage the 1D); Inx2 other (Fig. the coupled and electrically Ogre expressed not cell were one which from in recordings configurations. pairs different show Oocyte in 1D,E Inx2 with Fig. Ogre 1C. co-expressing 2000) pairs voltage- Fig. cell al., in form confirmed et is to (Stebbings this oocytes shown and in previously channels was proteins functional homotypic Inx2 sensitive these potential study). encoding this for 2002; no homotypic transcripts candidate with in form as co-expressed good are tested frequently not Ogre were a does with ng is interactions Ogre 20 1F; Inx2 to Thus, (Fig. up channels. coupling. oocytes of of control amounts evidence water-injected RNA in 1). Table that equated cells hnes(i.1)wt naverage functional of an formation with the in 1E) resulted (Fig. however, not channels pair, water- do a both of proteins of in Inx2 cells the that and Ogre that of from Co-expression demonstrating channels. heterotypic different form 1), significantly (Table pairs not injected was pairs Inx2 eercre sn w-lcrd otg lmig The clamping. voltage two-electrode that oocytes ( using single current/voltage in recorded expressed were were RNAs the hemichannels, V j nti td,w osdrtecmeec fOr ofr gap- form to Ogre of competency the consider we study, this In odtriewehrOr n n2fr non-junctional form Inx2 and Ogre whether determine To tp Fg A i o lctjntoa urnsi t paired its in currents junctional elicit not did 1A) (Fig. steps ) ora fCl cec 2 (17) 126 Science Cell of Journal I j r ierat linear are s ogre I m V / V j .Ti stemxmmlvlo voltage of level maximum the is This s. m I and j eainhp r hw nFg H At 1H. Fig. in shown are relationships ) eelna tall at linear were s inx2 V j f1–0m n hwaslight a show and mV 10–40 of s Drosophila in V Xenopus j § 0m Fg Cand 1C (Fig. mV 10 g j ise Sebnse al., et (Stebbings tissues V lgty lhuhnot although slightly, I j j s. elndi time- a in declined ) oocytes ogre g g j j nthese in ) -injected fOgre/ of Xenopus Xenopus G ogre j / V j ae2 ramxueof mixture a or 2) lane aiu urnsaesoni i.1H; Fig. in shown innexins. are both expressing currents cells By in Maximum greater was circles). which of open magnitude 1H, (Fig. and with controls injected cells water-injected from contrast, different significantly in not were those circles) filled 1H, (Fig. cells n n2epesn el.Atog ihaonsof amounts high Although Ogre- cells. in than Inx2-expressing Inx2-, and in marked more was which sensitivity, voltage r-nuaino h nioiswt necs fthe of excess an with antibodies the by of eliminated were which pre-incubation In bands, single microscopy. recognised anti-Inx2 C- and immunofluorescence of and unique characterised blots were western blotting 2A) to (Fig. western Inx2 raised and by Ogre antibodies of peptides polyclonal terminal purified Affinity antibodies innexin of Characterisation dsrbdi ealblw eosrtsta ncdw of knockdown 6 that inx2 demonstrates Fig. below) panels). right detail 3D, in the in (described (Fig. antibody peptide left corresponding with the incubated (lower of were presence 3–5) tissues anti-Inx2 Figs the detail, when and in abolished labelling (described panel) was systems of nervous left larval pattern early (upper in characteristic panel) the anti-Ogre that innexins with shows of seen 2D knockdown Fig. 2D) 6). (RNAi)-mediated (Fig. (Fig. competition interference peptide RNA by and demonstrated was tissues legend). mass whole 2 Fig. molecular (see oligomers Higher innexin be non-specific. could and are bands RNA-injected to antibody cells common this Bands that water-injected protein. confirming Inx2 4), for lane specific panel, is right expressing 2C, cells (Fig. in cells band corresponding a label ogre not did but 3) is band lane a antibody detected this anti-Inx2 Similarly, that protein. were at demonstrating Ogre 4) lane for anti-Ogre, panel, specific left with 2C, (Fig. probed cells of control water-injected mix or equal an or when detectable 1) lane panel, fractions inx2 left panel). 2C, membrane (right protein. (Fig. anti-Inx2 oocyte RNA translated a and of detected panel) detect (left Anti-Ogre blots to anti-Ogre western with antibodies labelled shows the 2C in with Fig. RNAs cells corresponding the western these in expressing antibodies by the Xenopus proteins examined of Inx2 specificity was and the these blot Ogre size, of Since identical SDS-PAGE nature 2013). of 2008) hydrophobic are al., Deber, the of in et and by Phelan (Rath reports for 1998; innexins proteins accounted previous al., be et might with of Phelan and 2000; consistent al., migration is et (Stebbings proteins this anomalous kDa; The 42 slightly 2B). of (Fig. mass peptides to migrated unlabelled corresponding hs xrsigIx n geaeidctv fafunctional a of indicative and are proteins. alone these Ogre between Inx2 and interaction expressing Inx2 cells expressing in those hemichannels and induce channels did recordings shown). cell not single (data unreliably in the albeit ng) raising currents, 10 non-junctional cells, (to paired in injected coupling amount intercellular induce to failed aiaino h nioisfrimnfursec aeln of labelling immunofluorescence for antibodies the of Validation h bevddfeecsi h rpriso intercellular of properties the in differences observed The , inx2 7kai el expressing cells in kDa 37 Fg C ih ae,ln )o nwtrijce control water-injected in or 1) lane panel, right 2C, (Fig. Ns(i.2,lf ae,ln ) hsbn a not was band This 3). lane panel, left 2C, (Fig. RNAs ngilclsb Nidaaial eue anti-Ogre reduced dramatically RNAi by cells glial in Fg H rage)ehbtdnnjntoa urns the currents, non-junctional exhibited triangles) 1H, (Fig. oye,wihlc noeosinxn,adprobing and innexins, endogenous lack which oocytes, , 7ka lgtyblwterddcdmolecular deduced their below slightly kDa, 37 inx2 Drosophila epesn el Fg C etpnl ae2) lane panel, left 2C, (Fig. cells -expressing inx2 , 7kabn nclsexpressing cells in band kDa 37 and inx2 avlnrossses anti-Ogre systems, nervous larval ogre inx2 Fg H qae)o both or squares) 1H, (Fig. Ns(i.2,rgtpanel, right 2C, (Fig. RNAs N Fg C ih panel, right 2C, (Fig. RNA I m xiie modest exhibited ogre ogre ogre RNA ogre ogre and or Journal of Cell Science hnes C xrsino n2i ohclso aridcstefraino hnesta r togysniiet otg;sed state steady voltage; to sensitive strongly are that channels of formation the induces pair a of cells both in Inx2 of Expression (C) channels. ( ooyi rhtrtpccniuainadrcre yda elvlaecapn.Bt el fapi eecapda odn oeta ( potential holding a at clamped were pair a of cells Both clamping. voltage cell dual by recorded and configuration heterotypic or homotypic otie ihntesmos ( symbols. the within contained nwihbt oye xrs geadIx omcanl ihn,o eywa,vlaesensitivity; voltage weak, very or no, with channels form Inx2 and Ogre express oocytes both which in ( voltage transjunctional generate to duration) seconds (5 steps mV 10 in hyperpolarised or depolarised then was 1) (cell cell eesepdtruhasre fhproaiigaddplrsn ebaeptnil ( potentials membrane depolarising and hyperpolarising of series a through stepped were n otg-eedn anrfor manner voltage-dependent and urn ( current oepisa higher at pairs some n2hmtpccanl surs n hnesi ge n n2epesn elpis(rage) o each For (triangles). pairs cell Inx2-expressing and Ogre- in channels and (squares) channels homotypic Inx2 ucsiesteps). successive nwtrijce otos(pncrls and circles) (open controls water-injected in Lpht n akl 95,w eeitrse oetbihthe establish to interested were we 1985), of Kankel, and role (Lipshitz known the the in Inx2 Given and Ogre of Localisation target their recognise antibodies proteins. the that demonstrating these further in respectively, labelling, cells, 6E,F) (Fig. anti-Inx2 or 6A,B) (Fig. step. the of end the to close made measurements in conductance non-junctional and junctional of properties Differential 1. Fig. netdwith injected combination. A–F ersnaietae rmpie elrcrig.()Vlaese rtcl B ar nwihbt el xrs gefi ofr ucinlint functional form to fail Ogre express cells both which in Pairs (B) protocol. step Voltage (A) recordings. cell paired from traces Representative ) I 2 A eurdt ananteohrcl cl )a h odn oeta a eodd ucinlcnutne( conductance Junctional recorded. was potential holding the at 2) (cell cell other the maintain to required nA) ; inx2 Xenopus I m surs or (squares) / V V j m .()Wtrijce otosaenteetial ope.( coupled. electrically not are controls Water-injected (F) s. oye eemconetdwith microinjected were oocytes eainhp r lt fiiilcret eodda h einn ftese,vru otg,atrsbrcino odn aus Currents values. holding of subtraction after voltage, versus step, the of beginning the at recorded current, initial of plots are relationships ogre H ogre w-lcrd otg lm eodnsfo igeocts hl-elmmrn urns( currents membrane Whole-cell oocytes. single from recordings clamp voltage Two-electrode ) V j and steps npsebyncneurogenesis postembryonic in inx2 § ogre 0m.()Cl ar nwihoecl xrse geadteohrIx ontfr ucinlcanl.()Pairs (E) channels. functional form not do Inx2 other the and Ogre expresses cell one which in pairs Cell (D) mV. 10 (triangles). ijce el fle ice)aeidsigihbe urnssgiiatygetrta oto r rsn ncells in present are control than greater significantly Currents indistinguishable. are circles) (filled cells -injected g Drosophila j ,nraie otervle at values their to normalized s, ogre I m ausaemean are values 5ng), (5 CNS inx2 5n)o i of mix a or ng) (5 6 ...for s.e.m. G V eainhpbtenjntoa odcac n rnjntoa otg for voltage transjunctional and conductance junctional between Relationship ) aiaaddsa eul,wihaetetreso retinal of targets the are which adult medulla, the are of distal neurons precursors to and outer rise The neural gives lamina (opc) neuroepithelia. lobe centre proliferative proliferation optic two optic At in (ALH). development, dividing hatching larval of actively larval in stage after proteins two hours this therefore, 24–40 the We, of systems, process. this distribution nervous in relative Ogre the with act examined to Inx2 of potential j 5 Xenopus 0m,aepotda mean as plotted are mV, 10 V m n 0m ussapidfr5scnswt 0scn a between gap second 20 a with seconds 5 for applied pulses mV 20 ; 5 ogre oye xrsigIx n ge niiulyo in or individually Ogre, and Inx2 expressing oocytes – el;sm ro asaecnandwti h symbols. the within contained are bars error some cells; 5–7 and inx2 g la eurmn o neis3825 innexins for requirement Glial j scntn at constant is 5n fec)RA.FrAGclswr ardin paired were cells A–G For RNAs. each) of ng (5 V j tp steady-state step, 6 ...for s.e.m. V g j j , pt 0m n elnssihl in slightly declines and mV 40 to up s V m j )i ucinlcret( current junctional is S) 5 V n 5 1 2 I – ar.Sm ro asare bars error Some pairs. 4–5 m V g eercre stecells the as recorded were ) h j V.Smlaeul,the Simultaneously, mV). ; a acltdfrom calculated was g V j erae natime- a in decreases h )of 2 0m.One mV. 40 I j 52 ercellular I2)/ I j V j . Journal of Cell Science itr of mixture with injected lane), per equivalent ( cell absence (one at the oocytes in band panel) single (right a anti-Inx2 or detect panel) (left anti-Ogre (B) with left. probed the ( on proteins. Inx2 indicated and are Ogre of sequences C-terminal unique osdrdculdif coupled considered nioyfrbnigt el ftebanlbsadvnrlgnlo seFg –) cl a:50 bar: Scale 3–5). th Figs th that (see with fact compete ganglion effectively the ventral (lower antigens with and anti-Inx2 Peptide consistent or respectively. lobes peptide, is panels) brain Inx2 (upper panels) the or ( anti-Ogre right Ogre of RNA. with and of cells labelled single excess (left to an were a 3 of systems binding with lane panels) Nervous for (right injected in z-series. antibody presence cells band confocal or the a kDa panels) migh (left from than 37 2) absence images lane RNAs the the panel, single individual and right are the bands 1; Images of these lane systems. panel, of levels nervous (left intensity lower bands lower received mass The molecular cells higher respectively. injected The Inx2, non-specific. and are Ogre cells, water-injected of including lanes, all to common with injected oocytes a detects Ogre antibodies. anti-innexin of Characterisation 2. Fig. and (Hofbauer brain that demonstrated central Kankel and and Watanabe which lobes 1990). complex, Campos-Ortega, produces optic lobula the (ipc) the and medulla interconnect centre proximal the proliferation of neurons inner The photoreceptors. 3826 H geIx/geIx 53 6% 4.49 (64%) 25/39 Ogre+Inx2/Ogre+Inx2 geIx /7(% 0.16 (0%) 0/17 Ogre/Inx2 n2Ix 72 6% 3.41 (65%) 17/26 Inx2/Inx2 geOr /3(% 0.17 (0%) 0/23 Ogre/Ogre el1cl ar ope/oa % Mean (%) coupled/total Pairs 2 1/cell Cell ucinlconductance, Junctional 2 O/H 2 /5(% 0.11 (0%) 0/45 O ogre ora fCl cec 2 (17) 126 Science Cell of Journal , and 7kapoeni el netdwith injected cells in protein kDa 37 inx2 inx2 g , j . 7kai h bec fcmeigppie obnsaevsbei h rsneo etd.()Mmrn rtisfrom proteins Membrane (C) peptide. of presence the in visible are bands no peptide; competing of absence the in kDa 37 5n ah ae3,btnti oye netdwith injected oocytes in not but 3), lane each, ng (5 0.5 N ln )o ae ln ) ih ae,at-n2dtcsa detects anti-Inx2 panel, Right 4). (lane water or 2) (lane RNA g al .Jntoa opigin coupling Junctional 1. Table j Drosophila ma for (mean m S. n avlnrossses h aesaenihorn ae 8CSeuvlnsprln)ctfo h aebo and blot same the from cut lane) per equivalents CNS (8 lanes neighbouring are panels The systems. nervous larval elpis,wscluae sdsrbdi h eedt i.1a 0m rnjntoa otg tp ar were Pairs step. voltage transjunctional mV 10 a at 1 Fig. to legend the in described as calculated was pairs), cell innexin ogre ( A rnmmrn T)tplg finxn.Atbde eerie osotppie orsodn to corresponding peptides short to raised were Antibodies innexins. of topology (TM) Transmembrane ) B , N 1 g ae1 ramxueof mixture a or 1) lane ng, (10 RNA C Nso ae,wr rbdwt niOr lf ae)adat-n2(ih ae) etpnl anti- panel, Left panel). (right anti-Inx2 and panel) (left anti-Ogre with probed were water, or RNAs etr lt aeldwt niinxnatbde.Pstoso oeua asmres(nkDa) (in markers mass molecular of Positions antibodies. anti-innexin with labelled blots Western ) Xenopus 2 rpeec + fa xeso geo n2ppie epciey ohantibodies Both respectively. peptide, Inx2 or Ogre of excess an of (+) presence or ) ogre oyepisijce ihinxnRNAs innexin with injected pairs oocyte hpdsrcuewt eta oetruhwihteoptic the which dome- through characteristic hole a central ipc a forms the with in centre structure findings. weakly proliferation these shaped and confirm outer opc data Our the The 1992). in Kankel, and strongly (Watanabe expressed is Ogre N ln )o ae ln ) h ekrbnsjs eo h innexins the below just bands weaker The 4). (lane water or 1) (lane RNA , 7kapoeni oye netdwith injected oocytes in protein kDa 37 ogre m m. and g j D inx2 ( muoloecnelbligof labelling Immunofluorescence ) m 6 6 6 6 6 S; .739 1.27 .217 0.02 .626 1.16 .323 0.03 .245 0.02 Ns( gec,ln ) hc sntpeetin present not is which 3), lane each, ng (5 RNAs 6 s.e.m.) inx2 N 1 g ae2 ra or 2) lane ng, (10 RNA Drosophila corresponding e eoligomers be t n aes in panels) Xenopus larval co- e Journal of Cell Science ssgetv fgilcls ahrta ern cnimdin (confirmed neurons than rather 5). cells, expression 4, glial and of Figs pattern of expressed The 3G–L) suggestive 3M–O). (Fig. widely (Fig. ipc is lobes ganglion brain also ventral the merged of the and were cells and the other Inx2 in opc as colocalised in and partially overlap the and, Ogre significant the showed arrows) expression. to in demonstrate, 3C,F,I) 3E,H, predominantly (Fig. (Fig. expression images localised membrane of innexins plasma centres Both ipc proliferation levels the 3B,E,H). the in (Fig. in similar expression found weaker also clearly with with was was opc, Inx2 expression the 3A,D,G). Ogre of (Fig. 3A–I). cells (Fig. centre in opc proliferation apparent the inner to ribbon-like medial The lies brain. the enters stalk ein hc nlddusandcls a uniid hr was There quantified. whole was cells, the unstained as included underestimates Values which be region, could colocalisation. centres but partial proliferation for intermediate the value reliable for were the of (0.53) and lobes indicative (0.39), brain ipc nonetheless the the (0.52), of for opc glia that the presumptive than for greater value suggestive PCC was S1. The 0.87), which colocalisation. ventral PCC, Table perfect the arrowheads; near along material of 3N, cells (Fig. supplementary the midline for in ganglion observed was given correlation one are Highest with consistent and were (ICQ) another and quotient (PCC) correlation coefficient intensity correlation the Pearson’s of performed. was values analysis Calculated colocalisation images, labelled double in oass ute h xeto vra fOr n n2signals Inx2 and Ogre of overlap of extent the further assess To xrsigcls cl as –,40 A–C, innexin- the bars: of 4 Scale some Fig. of cells. See identity expressing M). of glial (arrows, the row of midline a confirmation the in for of found side is either midline on alone the puncta Ogre along whereas cells N) in (arrowheads, colocalise ganglion, Inx2 ventral the and In Ogre which (M–O) of arrowheads. some by in lobes, indicated expressed are the extensively throughout brain cells are the glial Inx2 between presumptive and situated Ogre gland, the (G–I). ring in lobes the evident Plasma of also E). is cells proteins (arrow, large the D–F of in localisation centres seen membrane proliferation as the H) of arrows, cells localise (G–I, of two Inx2 membrane in and plasma Ogre shown the J–L). A–C, at and in (G–I centres. planes depicted both focal as in different lobe, similar the right is in The staining weakly (G–L) more Inx2 and (ipc). (opc) centre strongly centre inner is outer the Ogre partially in centres. Inx2 expressed proliferation and focusing the Ogre A–C, in centres. in colocalise proliferation depicted optic as ganglion the lobe, ventral on left and The (D–L) (D–F) lobes magnification (M–O). brain Higher the (D–O) of anteriorly. views gland attached ring is the ganglion; B) ventral (rg, and system lobes nervous brain the developing showing the of view Low magnification (A–C) orange/yellow). appears colocalisation ( rjcin famxmmo ieiae,fo confocal a from 1 images, at five taken of or z-series maximum images, a single of are anti-Ogre projections Shown with antibodies. labelled anti-Inx2 double and ALH) hours the (24–40 in larvae colocalise partially Inx2 Drosophila and Ogre 3. Fig. itiuin ( distribution, C , F la eurmn o neis3827 innexins for requirement Glial , I , L , O h vra fOr rd n n2(green; Inx2 and (red) Ogre of overlay the ) avlCNS. larval B , E , H m , tp.( steps. m K , N n2dsrbto and distribution Inx2 ) evu ytm of systems Nervous A , D , G , J m , M ;DO 20 D–O, m; )Ogre Drosophila m m. Journal of Cell Science A ytmwsue omr pcfcclswt F.UAS- GFP. with cells specific mark to GAL4– used the mCD8::GFP was neurons, not system and glia, UAS indeed were centres merged proliferation the S1). of Table of material regions inspection unstained (supplementary of CNS those visual Ogre- the to similar the with were coefficients in consistent images; arrows; 3M, staining 0.06) (Fig. Inx2 ganglion ventral PCC the and in puncta Ogre bilateral positive between correlation no 3828 aeldwt nei n F niois(is4 ) Repo 5). mesectoderm-derived ne of the subset 4, a being and (Figs glia exceptions midline ventral cells, antibodies glial GFP particular most double marks were is and flies which innexin transgenic expression, these with from labelled systems nervous Larval e of control the under expressed was lav ocnimta h nei-xrsigclsotieo the of outside cells innexin-expressing the that confirm To GL,wihdie expressio drives which -GAL4, ora fCl cec 2 (17) 126 Science Cell of Journal F uinta localis that fusion GFP a , ywl hrceie nembryos, in characterised well ly repo v otgi Hle ta. 1995; al., et (Halter glia root rve nalpsmttcneurons. postmitotic all in n GL,t ae la el,or cells, glial label to -GAL4, st h lsamembrane, plasma the to es ;Fg M ros;ti osbyrpeet tiigi h tracheal the in staining represents in possibly arrows this 4D–F, arrows); 3M, (Fig. ganglion Fig. E; ventral the a in in puncta not found Repo-negative channel was has alone dorsoventral Ogre the identity markers. line their that glia the although (surface) are of these 4F,L); channel midline Fig. the possibly in arrowheads along 4D–F,J–L, cells (Fig. were ganglion proteins Repo-positive ventral Both prominent labelled. were in 2003) al., present et Beckervordersandforth Dumstrei 1995; 2008; al., neuropile al., et and et Ito cortex 1998; surface, al., of et categories (Hartenstein broad glia the of each in cells with co-localised Inx2 and 4A–F) significantly (Fig. Ogre Both larvae. 4G–L) that early to (Fig. of relation CNS in the expression in innexin Repo shows of 4 Fig. 2012). al., et Stork la el.Tedsrbto finxnsann ntebanlobes brain the in staining gangli innexin ventral and of of 4A–C,G–I) membrane distribution (Fig. plasma The the cells. at accumulate glial proteins the that indicating i.4 geadIx r xrse ngilclsi the in cells glial in expressed are Inx2 and Ogre 4. Fig. xrs ge(opr n ) cl as 25 bars: Scale K). and that E the neuropile (compare in ventral Ogre expressed the express not of is cells lobes It tracheal brain (J–L). presumptive the ganglion of ventral similar cells and a Repo-positive (G–I) in in expressed Ogre is to Inx2 pattern anti-GFP. and anti-Inx2 with lobes, brain the In (A–C) anti-GFP. a of 1 projections at taken are z-series Images confocal with antibodies. labelled GFP double and were innexin ALH) hours (24–40 systems mCD8::GFP CNS. larval r ato h rcelsse.( system. possibly tracheal and the negative of Repo part the are are of E) side (arrows, midline either midline the on ventral along puncta and Ogre-labelled neuropile F). the (arrowheads, and ganglion the of expression Ogre of ganglion, that ventral overlaps Ogre the express In also which (D–F) of (B,C). many (A), cells glial in expressed orange/ appears innexin (colocalisation ( and panel yellow). (green) right the GFP the in of in labelling overlay (red) innexin the panel, and left panel the middle in shown is labelling A – F la el eemre yepeso fUAS- of expression by marked were cells Glial ne h oto of control the under rprtoslble ihat-geand anti-Ogre with labelled Preparations ) repo GPi la el tteltrlmargins lateral the at cells glial in -GFP encnimduigspecific using confirmed been n(i.4D–F,J– (Fig. on iaeal ymtia o of row symmetrical bilaterally m tp.I ahrow, each In steps. m G repo – L repo rprtoslabelled Preparations ) repo GL.Nervous -GAL4. GPi widely is -GFP )sget that suggests L) die GFP, -driven m repo m. -GFP Journal of Cell Science N Fg )sget httepoen r eurdi hs cells these in required are larval proteins the of the cells that glial suggests in 4) Inx2 (Fig. and CNS Ogre of ganglion expression strong The the exiting of Downregulation neurons K–L). and of E–F in axons arrowheads glia in 5D–F,J–L; the Inx2 (Fig. and 3 Ogre with Figs of motor in expression associated 5 seen shows and Fig. innexins additionally and of arrowheads). arrowheads) 4 localisation 5D,J, and glial 5G, (Fig. the of (Fig. ganglion some ventral confirms lobes the the the on of body neurons in neurons mushroom arrowheads), deep 5A, cortical (Fig. neurons lobes including brain the of neurons, surface Elav-positive in Ogre express to 1992). known 4C) Kankel, are (Fig. that and lobes neuroblasts (Watanabe scattered brain be the to likely in are visible cells innexin-positive negative, w contact in develops which system, ete ge(i.5–)nrIx Fg GL a expressed was 5G–L) (Fig. Inx2 nor 5A–F) (Fig. Ogre Neither ogre and inx2 t la el.Cutr fRepo- of Clusters cells. glial ith nteCNS the in ciiy hrceie yiaiiyt l,dfiut akn and walking difficulty fly, to inability by characterised activity, Movie a material and supplementary 1). 6C,D) (see (Fig. the defects adults in of behavioural surviving of size protein and variety the 6A,B) Ogre in CNS- (Fig. of reduction larvae the a of loss CNS with The and associated rg). was arrow) flies 6B, transgenic 6B, (Fig. (Fig. gland the ring of centres of associated proliferation reduction including lobes midline cells, optic brain non-targeted the CNS; in and the persisted ganglion near protein developing the The 6A,B). cells in (Fig. the elsewhere in than of marked ganglion protein ventral less cells attenuated was glial significantly expression knockdown in Ogre levels cells. glial ncdw h xrsino h rtisi cell-specific to a RNAi in of UAS- with consequences proteins of the the expression coupled shows 6 of was Fig. expression manner. system the GAL4–UAS knockdown the is the this if establish case, To development. postembryonic normal for ogre ncdw dl le xiie mardlocomotor impaired exhibited flies adult knockdown rjcin facnoa -eistkna 1 at taken z-series are confocal Images a antibodies. of GFP projections double and were innexin ALH) with hours labelled (24–40 systems Nervous GAL4. UAS- of differentiated expression in expressed not are neurons. Inx2 larval and Ogre 5. Fig. as 25 Scale cells bars: K,L). glial (arrowheads, processes in neuronal found the ventral Elav-positive is surrounding the in but In expressed J) (J–L) not (arrowheads, 4. is neurons 3, Ogre, Figs like in Inx2, shown ganglion, as glial lobes and centre the proliferation of optic (H, cells the Inx2 in express expressed is not Inx2 the do I). of G) neurons (arrowheads, Elav-positive bodies lobes, mushroom optic the In (G–I) E,F). (arrowheads, axons neuronal ( the glial with Ogre-labelled associate D). cells ventral (arrowheads, the processes In (D–F) and 4. bodies 3, Figs in shown ganglion, as glial is in here expression seen optic Ogre cells the B). of (arrows, latter cells centre optic The Ogre-labelled proliferation the (B,C). by of Ogre surrounded insertion express are of not group site do the A) at (arrowheads, and stalk lobe anterior the the in of neurons region Elav-positive lobe, optic the In (A–C) ahrow, each gen n nei rd ntergtpanel. right the GFP in ( of (red) overlay innexin the and and panel (green) middle the in labelling innexin G–L A – F la eurmn o neis3829 innexins for requirement Glial rprtoslble ihat-geadanti-GFP. and anti-Ogre with labelled Preparations ) rprtoslble ihat-n2adanti-GFP. and anti-Inx2 with labelled Preparations ) ogreRNAi m elav m. elav GP u o ge sepesdi erlcell neural in expressed is Ogre, not but -GFP, GPlbligi hw ntelf panel, left the in shown is labelling -GFP otioi ern eemre by marked were neurons Postmitotic mCD8::GFP rUAS- or inx2RNAi ne h oto of control the under repo -GAL4-directed pcfclyto specifically m tp.In steps. m elav - Journal of Cell Science UAS- el n eutdi rmtcrdcini h ieo the of size the in reduction dramatic these a in in specifically resulted protein Inx2 and with of seen cells levels that reduced than, effectively severe more albeit of ogre days to, few similar a of remarkably within died consequence rapidly generally The and flies continuously eclosion. These back its legs. was on its fly lay moving and the uncoordinated, itself cases, right extreme was to In unable defects. behaviour sensorimotor of Grooming indicative behaviour. circling g F). ventral and The E images. control (compare in CNS saturation larval the developing hence through the preparations, taken knockdown of z-series 50 confocal and size of bars: control the projections for Scale in are used F. E,F) in reduction were and in (arrow) dramatic settings (A,B outlined centres preparations acquisition a immunolabelled proliferation image with of same optic correlates Images The the adulthood. expression systems. to in Inx2 survive persists of not do Expression loss flies (F) The knockdown controls. cells. in glial centres in proliferation reduced optic the in and development. CNS impairs and levels protein reduces significantly cells glial in expression innexin of innexinRNAi downregulation RNAi-mediated 6. 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(rg). optic nervous ( gland Adult (cbr), ring (C,D) brain B). the central gangl and in the ventral A of and and (compare (br) ganglion (arrow) lobes ventral centres brain and proliferation lobes the in brain optic of reduced cells the considerably glial is in in expression persists expressed cell Expression strongly Glial is (B) Ogre centres. proliferation controls, optic In the (A) antibodies. anti-Ogre with immunofluorescence by E , F ) ogreRNAi inx2 ncdw.Tree xrsinof expression Targeted knockdown. ncdw.Nrossses(6husAH aeldb muoloecnewt niIx niois E n2i togyepesdi la cells glial in expressed strongly is Inx2 (E) antibodies. anti-Inx2 with immunofluorescence by labelled ALH) hours (36 systems Nervous knockdown. ora fCl cec 2 (17) 126 Science Cell of Journal osrcswr xrse ngilclsudrtecnrlof control the under cells glial in expressed were constructs / n repo 5 –.* 3–7. GL ( -GAL4 P 5 .05 ** 0.0015, ogre m ABEF;75 (A,B,E,F); m H eaiedfeecsi evu ytmsz,etmtdb esrn h ufc rao w-iesoa mgs uha those as such images, two-dimensional of area surface the measuring by estimated size, system nervous in differences Relative ) ncdw) UAS- knockdown); inx2 P # .05 nardStudent’s unpaired 0.0005, ncdw Fg EF was 6E,F) (Fig. knockdown m CD.( (C,D). m inx2RNAi inx2RNAi G loecneitniyi muoaeldnrossses uniidb esrn integrated measuring by quantified systems, nervous immunolabelled in intensity Fluorescence ) ogilcells glial to / TM6B,Tb t ts.Fygntps UAS- genotypes: Fly -test. repo ( inx2 GL.( -GAL4. oto) UAS- control); ogre soitdwt eutoso 7 n 3 ntesz fthe of size the in 33% and 27% of flies reductions in with intensity associated in the decrease and 41% control expressing a from revealed systems larvae fluorescence nervous F). knockdown of immunolabelled Quantification and in eclose. 6B intensity to Fig. failed (compare expression stage Inx2 same early the Unlike the at Typically, flies knockdown 6E,F). (Fig. of CNS system larval nervous developing ncdws oepoeni tl eetbei el ttevnrlmidline. ventral the at cells in detectable still is protein some knockdowns; A–D ) ogre ogre inx2RNAi ogreRNAi ncdw le,tasei le ihrdcdglial reduced with flies transgenic flies, knockdown ncdw.(,)Nrossses(6husAH labelled ALH) hours (36 systems Nervous (A,B) knockdown. inx2 ogre ogreRNAi / ncdw le a mle hnta of that than smaller was flies knockdown repo / ncdwsi oal mle hnta fcontrols. of that than smaller notably is knockdowns TM6B,Tb GL ( -GAL4 inx2 rUAS- or rngn Fg G.Ti was This 6G). (Fig. transgene inx2 ncdwsbti considerably is but knockdowns knockdown). ogreRNAi / TM3,Sb o v)adin and (vg) ion ( ogre h nervous the ro bars Error . developing nlo is anglion inx2 UAS- control); sisting ogre Journal of Cell Science neisaewdl n yaial xrse during expressed have dynamically studies previous electrical and and mediate 2002) other al., the 2011) widely et and (Stebbings al., These development are circuits. et neural (Wu adult innexins defined Inx7 in Murphey, and and signalling Trimarchi 2008; Inx6 al., et ShakB 1997), Phelan complete. 1996; from al., far et is genes (Phelan individual expression the of of patterns functions temporal and and spatial precise of Knowledge inx6 of phenotype in similarity and the 6C,D) (Fig. by flies supported knockdown is related on the target constructs of RNAi the Specificity of proteins. effects off-target to attributed and, be glia in These the required critically observations). that not importantly, unpublished are was proteins (our these this CNS that suggest Inx3, larval data the of in case Inx2 the and Ogre In of that overlaps shown). partially expression its not as surprising somewhat (data in glia observed in was phenotype transgenes No expressing 2002). al., flies et and (Tazuke 2008) specific al., cell et (Ostrowski CNS embryonic of exception the with Lpht n akl 95 seDiscussion). (see 1985) Kankel, and (Lipshitz UAS- of expression drive to rncit rmsvno which, of seven from transcripts of genome The cells. these in proteins Discussion the is of innexin will function studies the one Further address of another. to of loss required presence be the by the by whereby for for redundancy, roles compensated accounted genetic subtle obvious be having by proteins an could the or yield knockdown, This not of shown). levels did inadequate not cells these (data to phenotype are constructs RNAi innexins target driver both in GAL4 adult studies the preliminary although the centres, which proliferation of Interestingly, optic the morphology in normal. expressed gross and was size CNS the and development, UAS- UAS- expressing or flies transgenic neurons, in expressed ope iha5%rdcini avlbansz Fg 6H). (Fig. of size downregulation was brain upon larval observed 6G) phenotype in (Fig. inx2 severe reduction more intensity 51% The a immunofluorescence of with 6H, coupled measurements (Fig. in expressing CNS by flies adult reduction indicated In and as area. panel) respectively, surface upper panel), 6H, lower (Fig. brain larval r htOr n n2aerqie ngi o normal concert. in and/or other. for the independently of act glia presence could expression the proteins and, by in the influenced Their overlap Thus, is required protein partially CNS. one of cells are activity the these Inx2 in of patterns and development (Ostrowski Ogre Inx7 study postembryonic this and of that findings 2002) key The al., Curtin are development. neural et 1985; in (Curtin 2008) Kankel, al., ShakB et and 2002), (Lipshitz al., Ogre et for roles highlighted Kankel, and (Lipshitz that larvae fact as 1985). die the protein, by functional The part, all stages. lacking in larval supported, early at is Ogre former than Inx2 for requirement greater ossetwt h idn htOr n n2aenot are Inx2 and Ogre that finding the with Consistent nadto okokn down knocking to addition In and hnof than inx2RNAi inx7 r on tsm tg ntenrossystem. nervous the in stage some at found are , ogre shakBRNAi Drosophila ne h oto of control the under ih elc h ee fkokono a or knockdown of level the reflect might ogre inx7 hc srqie nsbeso lai the in glia of subsets in required is which , c855a and , RNAi ogreRNAi inx2 inx3RNAi a ih nei-noigloci, innexin-encoding eight has ogre Egre l,20)wsue to used was 2007) al., et (Egger osrcst l te innexins other all to constructs shakB ncdw hntpscannot phenotypes knockdown inx2RNAi ogre and rngn o t intended its for transgene , inx2 , oso-ucinmutants loss-of-function inx5RNAi ogre elav ,weused inx4 rngns 68% a transgenes, ogre GL completed -GAL4 , nvitro in inx2 ,whichisgerm- ulmutants, null or , repo inx3 ogreRNAi channel , inx6RNAi -GAL4 , inx5 ogre , oye,mgiueo eihne urnsvre with varies currents hemichannel single in of expressed Ogre+Inx2 When magnitude mean conductance. the a affecting oocytes, intercellular of without cells of sensitivity, both cells. voltage level, in the Inx2 Inx2-expressing reduces with pair Ogre in of co-expression conductance Specifically, form intercellular not also does co-expressed Inx3, modifies when but like independently, al., Ogre, these channels et that intercellular itself (Stebbings demonstrate of channels which we intercellular channels. Here Inx3, form properties 2000). of to homotypic competent co-expression the not by forms is that influenced Inx2 are demonstrated gap-junction channels system, form studies to this proteins Previous of In ability the channels. examine to system co-operatively Paired act Inx2 and Ogre aiu erldgnrto rnua iigmtns Circling mutants. adult in wiring seen of neural those failure of or supplementary the reminiscent degeneration are to neural and (see due various develop are to phenotypes circuitry presumably neural 1) behavioural sensorimotor Movie and material The locomotor defective to activity. of leads small Loss a adults in CNS. despite surviving expression, the abnormal briefly Ogre Inx2, survive morphologically glial of and and case reduced eclosion the with normal to In Flies develop CNS. contrast, larvae. for larval as the marked cells die a of flies to size glial leads the glia in in in the reduction specifically of protein either required Downregulation of CNS. expression postembryonic critically Ogre the of that are development knockdown, Inx2 cell-specific by and demonstrated, have postembryonic We for cells glial development in neural required are act Inx2 they and the Ogre do action? types in of cell mechanism which types the required in is both so, what cell and If Are development? different questions. normal several for in in prompts proteins brain Inx CNS patterns larval two developing the of overlapping overlapping both in presence The largely cells highly contrast, ganglion. glial a ventral of By and in populations lobes neurons. Neither several expressed, ipc. in postmitotic the extensively pattern, overlap in in are extent significant lesser found proteins a show is to innexins and innexin opc two in the expressed The in also is particularly anlage. Inx2 1992). that optic find Kankel, we the and and here, (Watanabe confirmed is centres This larval proliferation the in lobe expressed is optic Ogre that demonstrated studies and Previous precursors neural in cells expressed glial are Inx2 and Ogre neclua hneswt rprisdsic rmInx2 form from to distinct dock properties that with channels hemichannels preferentially might intercellular Inx2 heteromeric populations and distinct Ogre and two assemble Alternatively, of hemichannels presence channels. the of homomeric reflect then membrane, form would co-expressing the cells from might at Recordings channels. once to intercellular Inx2, Inx2; homotypic Ogre by like of aided Trafficking be Ogre, might findings. membrane these the explain to mechanisms uhamcaima n2adIx aebe hw oform to shown been have 2006). Inx3 al., and et Inx2 (Lehmann for did as hetero-oligomers precedent mechanism injected some also a RNA is such of There currents. amount non-junctional the induce high increasing at although even RNA channels because latter of the levels favour We channels. homotypic Xenopus . Inx2 oye aebe sdwdl saheterologous a as widely used been have oocytes . ogre la eurmn o neis3831 innexins for requirement Glial ge hr r ubro possible of number a are There Ogre. i o nuehmtpcintercellular homotypic induce not did nvitro in ogre ucinin function Journal of Cell Science la el agl erdcsthe reproduces largely cells glial hog rvso fsgaln oeue including molecules and Caldwell 1993; signalling al., et of (Ebens hypothesis Perlecan development and attractive provision neuronal Anachronism regulate an to known is through are former cells to cells glial The these because from signals neurons. of role hemichannel transfer in primary developing be the might Alternatively, innexins glial neurons. of network, and/or supports glial turn, extensive in the not crucial which, of both be maintenance Intercellular and are might hypothesize development cells for glial which neighbouring to exclusive. among mechanisms, communication possible non-junctional is mutually and it junctional regulate 2003). development might al., Inx2 et as and neuronal Starich acting Ogre glial-expressed 2007; to how al., 2010) al., considering et al., et In (Elias et Huang molecules Samuels ATP 2006; adhesion 2008; al., cell as al., et et such Spray Thompson 2003; 2007; functional molecules Paul, of signalling and formation (Goodenough release the that from have hemichannels also ranging proteins functions these that additional evidence is intercellular coupled there between Increasingly, molecules forming of cells. transfer by direct permit act that channels proteins gap-junction Classically, CNS? the postembryonic of regulate development proteins gap-junction do How postembryonic neuronal regulate of stages to that subsequent cells conclusion development. possibly, glial quite the in and, to act neurogenesis lead innexins two data these accumulated The phenotype. of data Our phenotype Downregulation 1985). neural Kankel, of the downregulation and that the of demonstrating (Lipshitz in provided latter non-autonomy in lobes the support optic analysis cellular gene the of mosaic the within was evidence for genetic 1989), some requirement al., Interestingly, et a Singh precursors. in 1985; with neuroblasts system with Kankel, nervous consistent coupled the and scattered of study), (Lipshitz size this the mutants and in (also reduction 1992) profound centres the and Kankel, wild- (Lipshitz the proliferation and in therein (Watanabe protein types optic the cell of expression specific type strong to null The not 1985). putative but Kankel, of of CNS site the CNS The flies pupae. to these or The and larvae hypomorphs system, late analysis. of as that visual died of than smaller the even focus highly was in mutants the mutation. with least was the at but of which architecture, adulthood severity neuronal to a the abnormal survived was the with lobes, mutants defect optic correlated the Hypomorphic major which particularly CNS, of The the the of extent 1985). that size at the Kankel, Kankel in mutations reduction and and various (Lipshitz Lipshitz of locus of phenotype the studies characterised early from 2004)] neurogenesis al., et development. Bauer neural 2002; in al., et (Bauer morphogenesis epithelial 2007). phenotype al., supine et early (Connell-Crowley a at themselves) exhibit patterning right adults to axon as (inability in and defects stages have developmental cyclin-dependent p35, the in activator, mutations with kinase Flies mutations synaptogenesis 1993). and al., with growth et flies axon (Phillis in in involved seen molecules is various (Eberl grooming in lobes Abnormal optic 1997). the al., particularly et brain, the of degeneration with in described first was behaviour 3832 hsi h is td oimplicate to study first the is This ora fCl cec 2 (17) 126 Science Cell of Journal inx2 ogre a nw oei postembryonic in role known a has rdcsarmral similar remarkably a produces pirouette inx2 ogre ogre uat n associated and mutants wihi novdin involved is [which ciiywsmapped was activity uatphenotype. mutant ogre ogre ogre in itnusigteeaekyqetosfrftr studies. future for questions and key possible, are are scenarios these both distinguishing However, 2011). al., et (Awasaki transcribed 0mnt ahi . ali B-,btenpiayadsecondary and primary between PBT-W, (Amersham). in ECL-Plus with NaCl development M to prior 0.5 and antibodies in wash 20-minute at,19;Pr ta. 01 og ta. 02,E-cadherin 2002), al., TGF- et and Voigt 2003) 2001; al., al., et et (Dumstrei Park 1998; Datta, curdadaaye nplm otae(esos90o 00 Axon 10.0; or 9.0 (versions software pClamp in or were Data 1320A analysed PC. Digidata a to and a USA) an through Devices, or acquired Instruments/Molecular interfaced cells) results. (Axon recording same (paired cells) digitizer cell the amplifiers 1322A (single Paired yielded GeneClamp500 polarity amplifier 1. two pulse Fig. using Axoclamp-2B of to made reversal were legend involve and Recordings the not clamp. hyperpolarising did in voltage sequential that electrode provided applied protocols two are 2008) which al., pulses, protocols, et recording depolarising Phelan the 1998; of al., et Details Phelan 1981; al., et neto,uigte‘urs-uho’mto Cla,1984). 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Ltd, (1:500). Laboratories 488 (Vector Fluor Vectashield Alexa in anti-mouse and (1:500) secondary 633 of Fluor application Insight Alexa simultaneous Cruz, by anti-rabbit (Santa followed anti-GFP 1:100) mouse anti- UK; innexin and Ltd, rabbit 1:1000) with Biotechnology with anti-Inx2, labelling simultaneously or 1:10 double incubated post- (anti-Ogre, For were Inx preparations were paraformaldehyde. antibodies, 4% preparations with GFP in signal Zenon-labelled and minutes same 20 labelling. the for conventional, achieve fixed to with required as was Zenon, concentration anti-Inx2 in increase wsk,T,Hag . ’onr .B n e,T. Lee, and B. M. O’Connor, Y., Huang, T., Awasaki, T. Lee, and K. Ito, S.-L., Lai, T., Awasaki, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.117994/-/DC1 online available material Supplementary BB/J005002/1 Biological number W.H.L.]. [grant and and grant R.A.B. project Biotechnology to a 00/A1/ and number the C.E.H.] [grant to studentship by G/06183 PhD a supported Council: Research was Sciences work This preparation. Funding manuscript and project, microscopy, the phenotypic of and K.B.: supervision immunolabelling and P.P.: W.D.J., design flies; transgenic P.P., of recordings; analysis hemichannel R.A.B.: N. C.E.H.: support, and contributions Meyel microscopy Author Van D. for discussion. Barrett, helpful Brown A. for Blackburn, Peco I. E. E. generation Allen, the antibodies, M. on collaborating Kirkwood, for innexin Davies J. and of Bacon J. thank We Acknowledgements respectively. London), Research, Medical for Institute (National Salecker repo UAS- Vienna Bloomington the UAS- chance. by (randomized) arise Drosophila scrambled not multiple did %Robs colocalisation against cases, of all tested measures values In positive images with that (Rrand). 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D. larval Paul, early and embryonic late the in cord. cells nerve glial Drosophila of development buds. taste mouse in communication cell D. S. Roper, R473-R474. ifrnito fteotclbsi rspiamelanogaster. Drosophila in lobes 198 optic the of differentiation cells. glial of Pattern II. brain. Drosophila melanogaster. Drosophila in response USA auditory an disrupt that utpso la el nteDoohl mroi eta ev oda eae to related as cord nerve ventral embryonic Drosophila expression. the gene and in lineage cells glial of Subtypes ncrns ou:agyorti ertdb laihbt erbatproliferation. neuroblast inhibits glia by secreted Cell glycoprotein a locus: anachronism aneac fgi ucini h mroi evu ytmo Drosophila of system nervous embryonic the in function glia melanogaster. of maintenance M. 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