Journal of Cell Science namme fabslmtza hlm h ndra Our Cnidaria. the phylum, metazoan basal a of member a in unknown. is systems nervous animals’ other in Nanos al., for et in role (Haraguchi A unidentified mouse, 2003). is done function its the through but Work system, 2008) In nervous central Gavis, studied. mechanism. and well the Brechbiel unclear 2004; in an least al., Nanos et the of (Ye function morphogenesis far the by Lastly, 2011). Drosophila is al., system et Juliano 2010; Wu nervous functional al., 2010; of et al., absence (Ewen-Campen the et organisms in non-model even in well practice studies is common and been Using 2010) species. has Xie, animal marker of and variety Shen a evolutionarily in 2000; be documented to al., seems et contrast, in (Mochizuki 2012). cells, conserved germ al., in et Nanos of (Schmitt-Engel role The insects in experimentally functions. demonstrated these with consistent 2008). are Gavis, animals various and Finally, in Brechbiel patterns 2004; 2004). of al., system al., et nervous the (Ye et in functions fate some (Hayashi somatic fulfils suppresses Nanos differentiation by and stem premature gonads, germ patterning the of and to posterior self-renewal migration mediates their fly it cells, for where development, for necessary cell roles is main it three inhibiting established first, studies Nanos: subsequent and 1991) innovation. arthropod or in and insect cells an germ is in patterning Nanos of posterior neurons functions in and the role nematocytes that its propose between but We imbalance indirectly. conserved, numerical only evolutionarily that patterning are axial suggesting development affecting and neural defects, maturation. intact, these nematocyte remained caused preventing have polarity but might anterior-posterior two neuroblasts, between basic of switch expense the a the of that as at Downregulation acts nematoblasts Nanos2 neurons. of Hence, of numbers Nanos2 nematoblasts. the numbers post-mitotic increasing the committed, pathways, of decreased differentiation maturation but blocked progenitors, Nanos2 Furthermore, cell effect. stinging embryonic of numbers rnlto ftertre ee.Nnswsfrtdsrbda a as repress described its first proteins was with Nanos in concert these genes. marker in posterior target Together, their act of a Pumilio. to translation as known acting partner is protein canonical binding It RNA repressor. domain, translational Zn-finger a is Nanos Introduction words: Key cnidarian the Using animals. other in experimentally demonstrated posterior been in acts not also have Nanos flies, functions In maintenance. these and development but cell Hydractinia germ development, for important neural marker, and germline pan-metazoan a is Nanos Summary 10.1242/jcs.127233 doi: 3192–3203 126, ß Science Cell Ireland of Galway, Journal 2013 Ireland, April of 15 University Accepted National (REMEDI), Institute ( Medicine correspondence Regenerative for and *Author Sciences Natural of Frank* School Uri cnidarian and determination Kanska a Justyna fate in cell studies neural by in revealed Nanos for roles New 3192 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. eamda tdigterl fNnsi erldevelopment neural in Nanos of role the studying at aimed We been only has patterning, posterior Nanos, of role identified first The coi xrsinas asdptenn eet,btteewr o soitdwt eeuaino n inln,showing signaling, Wnt of deregulation with associated not were these but defects, patterning caused also expression ectopic hunchback yrza netbae ifrnito,Se el,Nuoeei,Nematogenesis Neurogenesis, cells, Stem Differentiation, Invertebrate, Hydrozoa, a hw htNnsi eurdfrdendrite for required is Nanos that shown has ehv noee oe oe o ao nnua elft eemnto.Etpcepeso of expression Ectopic determination. fate cell neural in Nanos for roles novel uncovered have we Drosophila [email protected] rnlto.Scn,i scuilfrgerm for crucial is it Second, translation. LhanadNu and (Lehmann Nanos1 ) Nanos sepesdi the in expressed is Nanos ¨sslein-Volhard, sagermline a as Drosophila expression eo okalit ryadpeao tissues. mechanical/ predator delivering and prey and upon into tubule (cnida). cocktail barbed discharge venom a a nematocyst extruding explosively stimulation, a called chemical can include capsule, cells Nematocysts known post-Golgi (also chemo/mechanosensory sensory/ nematocyte specific These or specialized a cell, cnidocyte). is stinging 2009). the as cnidarians al., type, of et cell in feature (Galliot effector least defining animals at main higher are, with The neurons conserved specify well because that part, embryonic and animals mechanisms adult molecular the accessible in the renewed being and continuously is it short and analysis a expression gene has to amenable manipulation. basis, is and daily development, a all on lineages. to cell progenitors interstitial germ provides called and continuously cells, somatic short, al., stem for the of i-cells et Throughout or population cells, a Atlantic. Plickert cycle, organisms North 2010; life European animal’s model al., the in et as representative Chapman established 2007; include 2012). well al., that a et quite Some types (Putnam display cells. cell are stem group and few neural cnidarians this with gland/secretory, plan muscle, of epithelial, body Members simple hydroids. relatively and jellyfishes Nanos. of role the ancient into insight and provide evolution could cellular Metazoa early the a of a base the at at of cnidarians development role neural in the resolution gene cnidarian investigating important first, potentially enables twofold: was simplicity study morphological this conducting for rationale h ndra evu ytmi fpriua neetbecause interest particular of is system nervous cnidarian The corals, anemones, sea includes Cnidaria phylum The yrcii echinata Hydractinia nvivo in eod h ioa hlgntcpsto of position phylogenetic pivotal the Second, . Fg )i omncnidarian common a is 1) (Fig. Hydractinia Nanos2 Nanos2 eerhArticle Research erdcssexually reproduces a h opposite the had nrae the increased Journal of Cell Science rmtedatasml fthe of assembly draft Hydractinia the S1). from Fig. material the (supplementary also al. et including Extavour of analysis, data this repeated Hydractinia have We that suggesting duplication. 2005), cnidarian al., two et the (Extavour has phylogeny previously Nanos A generated others. been by cnidarians other in identified also eiiilyperformed initially animals We normal in patterns Expression Pumilio echinata hereafter an named The PCR. RACE were (and by obtained genes bilaterian were 2010) sequences and coding with cnidarian complete al., fragments The to cDNA similarity et two sequence identified high we (Soza-Ried library) 454 databases unpublished EST sequences Searching gene analyzing and Obtaining Results maturation. to proliferation through specification from Hydractinia xrsinpten of patterns expression genomic genomes cnidarians cnidarian 2 other that encode suggesting in databases, nor transcriptomic draft, and genome or database EST Pumilio single A sn anadls ffnto xeiet eso that show we experiments function of loss and gain Using Nanos1 eune eenihrietfe nthe in identified neither were sequences Nanos eune,adorpyoeyi ossetwt the with consistent is phylogeny our and sequences, yrcii Pumilio Hydractinia ao2cnrl eaoeei tdfeetstages different at nematogenesis controls Nanos2 eoeCnotu)adnamed and Consortium) Genome n 1 and and hereafter , Nanos yrcii ciaaNanos1 echinata Hydractinia Nanos2 Pumilio nsitu in ao2 Nanos1 Nanos2, ee r euto ieg specific lineage a of result a are genes epciey Two respectively. , yrdzto osuytenormal the study to hybridization Pumilio genes. eesqec a obtained was sequence gene Hydractinia Additional . and Nanos Pumilio eoe(The genome Nanos and Hydractinia Hydractinia ee were genes Nanos Zygotic . Nanos2 genes. or , ue n ne ellyr as nw seiemsand epidermis as known origin. (also embryonic like layers their hydroids in of In cell that regardless Note respectively, inner the 2C). gastrodermis), to (Fig. and refer ectoderm usually ‘endoderm’ outer the in and in nematocytes ‘ectoderm’ literature maturing and cnidarian in endoderm second, the mass; both cell endodermal the nematoblasts dividing in of clusters in first, differentiation: hpf), nematocyte (48 larva planula cytoplasm. their developing in the capsule by maturing identified nematocyst early be can (Lindgens or and nematoblasts migratory clusters are postmitotic becoming nematocytes or second, after 2004); nests proliferate al., characteristic et nematoblasts form as they first, committed ways: two morphologically identified by be can nematoblasts, progenitors, and nematocytes their Hydrozoan 2C). Fig. 1). (nematocytes; cells Fig. stinging head; as gastrulation to referred (also polyp polar oral the adult of the pole into into with develops following end develops However, posterior 2006). larval pole al., the species metamorphosis this et (Plickert on end posterior in Later larval the 2003). al., formed et (Wikramanayake (Freeman, is oocytes where cnidarian pole of the beenblastopore pole also is this has This at 1981). expression off given similar are hydrozoan bodies A the the in 2A,B). early of (Fig. of observed nuclei pole the stages around oral deposition cleavage Nuage-like prospective a in the and embryo in deposited 2006). al., asymmetrically et (Plickert stage in transcription ntegsrl,a 4husps etlzto hf n nthe in and (hpf) fertilization post hours 24 at gastrula, the In gastrulation, Following ao nciainnmtgnss3193 nematogenesis cnidarian in Nanos Hydractinia Hydractinia i.1 h iecceadcln tutr of structure colony and cycle Hydractinia life The 1. Fig. Nanos2 Nanos2 Clytia o xml,icls ern and neurons i-cells, example, for , omne ttemorula/gastrula the at commences Nanos2 . a xrse ntosae of stages two in expressed was b a xrse ndeveloping in expressed was (Lecle ctnni tblzdadthe and stabilized is -catenin a aenltranscript, maternal a was ` ee l,21) Polar 2012). al., et re Journal of Cell Science Nanos2 Nanos2 Fg D upeetr aeilFg 2) ul mature devoid Fully were cells, battery S2C). and in mounted of tubule being coiled position, Fig. apical with their capsule by material their by tentacles recognized supplementary the nematocytes, in nematocytes 2D; column, maturing body (Fig. positioned, the basally of in part polyp, in upper the and the of in base nematocytes the migrating at single clusters nematoblasts mitotic in animals and development embryonic late cell metamorphosis. in outer epidermis) the to or migrate (‘ectoderm’ and layer origin endodermal adult, embryonic the of of are ‘ectoderm’ but the in found predominantly are nematocytes of pattern Expression 2. Fig. 3194 noemlcl as(oe ne)adi auignmtctsi h coem(ihrist roha) ahdln ersnstemsge.Tel The mesoglea. the represents line Dashed arrowhead). inset, (higher focus, ectoderm of the out in surrounding, nematocytes the hpf). maturing from (24 in results Preplanula and background (C) inset) (arrowheads). (lower perinuclear mass and cell pole endodermal oral prospective the in expressed eaoye ntetnals(pe ne) EF eulpolyps. Sexual (E,F) inset). (upper tentacles the in nematocytes polyp. feeding oy.Istsoshg anfcto fpttv nifrnitdicl.( i-cell. undifferentiated putative of magnification high shows Inset polyp. fa4 p mro I 2hflra J rmr oy iwdfo below. from viewed polyp Primary (J) larva. hpf 72 (K) (I) column. embryo. hpf 48 a of nml r retdwt h rlpl p n noem c coem o auigoct;S,mtrn pr;t etce;s tln.Saebars: Scale stolons. s, tentacles; t, sperm; maturing Sp, oocyte; maturing Oo, ectoderm; ec, endoderm; en, 50 up. F), pole oral the with oriented are animals Nanos2 m () 200 m(C), splmnaymtra i.SC.I eulplp,the polyps, sexual In S2C). Fig. material (supplementary + rb akderydvlpn emclsi ohml and male both in cells germ developing early marked probe ora fCl cec 2 (14) 126 Science Cell of Journal Ncol1+ rncit;ol aal oiindnmtctswere nematocytes positioned basally only transcripts; Nanos2 m Nanos2 D,150 (D), m eaolssi auefeigplp (L) polyp. feeding mature a in nematoblasts sepesdi eaolsscutr ntelwrpr lwrist,i niiulmgaignmtbat mdl ne) n nmaturing in and inset), (middle nematoblasts migrating individual in inset), (lower part lower the in clusters nematoblasts in expressed is a loepesdi metamorphosed in expressed also was Nanos2 m G,10 (G), m , Ncol1 m Gist ) 180 H), inset, (G m and Rfamide Nanos2+ nnra animals. normal in Nanos2 Rfamide+ mauenmtctsi h es nesiilsae fteetdr.()Epeso nmature in Expression (D) ectoderm. the of spaces interstitial dense the in nematocytes immature m I,300 (I), m sepesdi eae()adml F al emcls G xrsinof Expression (G) cells. germ early (F) male and (E) female in expressed is H-K ern namtr edn oy.Istsoshg anfcto ftrenuos All neurons. three of magnification high shows Inset polyp. feeding mature a in neurons Ncol1+ ) Ncol1 m ( .J,400 m.(J), A-G nmls utemr,ismN eesi l te eltypes cell other all cnidarian, in related by levels undetectable mRNA observed its were never Furthermore, but males. S2E), in Fig. material (supplementary gametes to contrast el eebigucmitdicls ae ntervr large Fig. very material supplementary their 2G; No on (Fig. S2A,B). cytoplasm based little in and i-cells, expressed nucleus also uncommitted was resembling it but cells 2G), (Fig. In nematocytes maturing S2D). and and Fig. developing material late strong supplementary stolons, the in 2E,F; (Fig. undetectable gametes was mature but individuals, female eaolssaeol iil ntesoosbtnti h on oy’ body polyp’s young the in not but stolons the in visible only are nematoblasts xrsin H ihrmgiiainof magnification Higher (H) expression. ) Nanos2 Nanos1 m .(K). m. Nanos2 xrsin w-el()adfu-el()saeembryos. stage (B) four-cell and (A) Two-cell expression. Nanos2 a togyepesdi al eeoigfemale developing early in expressed strongly was Nanos2 sepesdi rlfrtn eaols lsesi the in clusters nematoblast proliferating in expressed is ltahemisphaerica Clytia Nanos2 in xrsinwsosre neihla el,in cells, epithelial in observed was expression Hydra nsitu in xrsinwsosre nnematoblasts in observed was expression Mciuie l,2000). al., et (Mochizuki yrdzto.Ntwrh,i a in Noteworthy, hybridization. Ncol1+ , CheNanos1 eaolssi h endoderm the in nematoblasts Nanos2 100 a similar a has m (A–G,E, m gtblue ight nprimary in Nanos2 is Journal of Cell Science Drosophila Nanos2 mammalian resembles system specific. female xrsinptena mammalian mammalian as pattern 2000). expression al., et (Mochizuki Nanos1 as expression RAi eetbeol nnmtgnssbtnti mature Minicollagen in type. not cell but nematogenesis other in any only nematocytes. by detectable capsule expressed is nematocyst the mRNA not of part are form proteins and These (Kurz 1991). markers al., differentiation et nematocyte cnidarian the classical of are pattern in expression marker studied lineage the nematocyte studied known also we nematogenesis, Hence, morphologies. cells. somatic some nematoblast in least at had with but overlapped definitely, cells expression identified the be type not cell the of could The at latter S2G). clusters Fig. the nematoblasts material in (supplementary dividing polyps in of and base S2F) Fig. material to similar system, nervous the in ige eaolssi h noemlcl aso 4hpf 24 of of clusters mass in never almost cell and 2H,I), endodermal (Fig. larvae the in and in embryos nematoblasts single, Because Pumilio mammalian , sepesdi emclsadi utptn tmcells stem multipotent in and cells germ in expressed is Hydractinia rncit eefudi emcls(supplementary cells germ in found were transcripts single Nanos2 yrcii Ncol1 Hydractinia Nanos2 CheNanos2 yrcii Nanos2 Hydractinia nanos Nanos1 sml specific, male is previously. eei xrse nbt emclsand cells germ both in expressed is gene xrsinwsascae with associated was expression Nanos2 yrcii Nanos1 Hydractinia (Lecle sntepesdi emcls The cells. germ in expressed not is Nanos1 yrcii Nanos2 Hydractinia RAwsfrtepesdin expressed first was mRNA ` Ncol1 ee l,21) In 2012). al., et re ngr el u nypartly only but cells germ in Ncol1 Nanos2-3 xrsini h nervous the in expression e,unlike Yet, . yrcii Nanos1 Hydractinia n te minicollagens other and hc a o been not has which , oee,while However, . a similar a has Hydractinia . Pumilio Hydra is , Gese l,19)adi ern Hykw ta. 2004). al., et (Hayakawa neurons in and 1995) gene al., proneural et The (Grens neurites. the stain Ash they cells quantify because pan-neuronal to counting difficult is by A it antibodies not 2L). tubulin anti-acetylated (Fig. of is type and identify: in cell neurons to cnidarians, difficult other is in any marker marker in neuronal expressed used commonly eaolsscutr n nmtrn eaoye,we nematocytes, maturing in and clusters nematoblasts efre w-oo double two-color performed ern Ktuuae l,2003), al., et (Katsukura definitiveneurons a the sought chosen therefore (hereafter have We and 2009). marker al., neuronal et (Marlow system progenitors, polyps. their developing i-cells, to but stolons versa, the from from vice nematocytes migrate or mature generally stolons of the migration to for polyps evidence no stolons. the in is in occurs and There polyps nematogenesis the in that locations: anatomical however, different two proliferating Note, in 2K). rarely (Fig. only clusters and nematoblasts post-mitotic had single, animals polyps, metamorphosed Young Ncol1 nematoblasts. dividing ndsic oan ln h eaoyedfeetainpathway differentiation nematocyte the along domains distinct in that found We 3). (Fig. Nanos2 ie h xrsinptenof pattern expression the Given eaoye r huh obln oteciainnervous cnidarian the to belong to thought are Nematocytes konin (known el rmrl ntersoos(i.2) nmature In 2J). (Fig. stolons their in primarily cells + Ncol1 obte nesadgn xrsinaognematogenesis along expression gene understand better to Rfamide Nematostella ao nciainnmtgnss3195 nematogenesis cnidarian in Nanos a xrse nabl-iefsin rmrl in primarily fashion, belt-like a in expressed was Hydra vrapn xrsinpten.Saebr:200 bars: Scale patterns. expression overlapping coexpressing of ( magnification nematoblasts. higher shows Inset below. 100 express to started ( have cells some where a of magnification genes; two and the red of stained expression the in overlap partial Nanos2 i.3 Double 3. Fig. B rmr oy rwn nagassie iwdfrom viewed slide, glass a on growing polyp Primary ) .Atog o xrse nall in expressed not Although ). m (C). m as . ( Ncol1 A Cnash Nknsie l,21) n ihteuse the with and 2012), al., et (Nakanishi vriwo edn oy hwn only showing polyp feeding a of Overview ) C Nanos2 nsitu in nsitu in lnl av hwn partially showing larva Planula ) and sepesdi ohnematoblasts both in expressed is ) Elav Nanos2 RFamide Nanos2 yrdzto for hybridization upe ne hw higher shows Inset purple. sas o xrse nall in expressed not also is yrdzto of hybridization epesn eaols cluster nematoblast -expressing Fmd precursor RFamide Nanos2 sadfntv n most and definitive a is eelreyexpressed largely were nproliferating in Ncol1 Ncol1 Hydractinia Ncol1 . m and (A,B), m Ncol1 gene and is Journal of Cell Science opat;Fg B n eaopoe oys(0 decrease, (50% polyps metamorphosed larvae and 4B) both Fig. in morphants; nematocytes mature significantly decrease, and (37.5% 4A) a Fig. had morphants; 12 embryos control; to morphant comparing that of number same found reduced block the Table We material at translation (supplementary morpholino S1). control as scrambled a embryos. used A was with in concentration mM. 0.25 microinjected levels at protein were morpholino Nanos2 eggs lower Fertilized antisense morpholino to a in used required we oligonucleotide be this, might test pattern, Nanos2 To expression nematogenesis. that early its thought on we Based above, well Nanos2. described of as decided not function we the cells), study is sensory to are role (which nematoblasts whereas proliferating neuronal cells, germ its in expressed However, Because in mammals. established understood. well in is cells germ and in Nanos of role The of Downregulation nematocytes. mature from absent maturation, nematocyte late as resumes after dissipates mRNA its soon but clusters, Hence, nematoblast 3A). mitotic in (Fig. expressed interface the at overlap little only with 3196 nrae neurogenesis increases ora fCl cec 2 (14) 126 Science Cell of Journal Ncol1 Ncol1 P rncito essadi ananduntil maintained is and ceases transcription Ncol1 xrsincommences. expression , Nanos2 .5 9cnrlaiascutdad49 and counted animals control 49 0.05; P yrcii Nanos1 Hydractinia 5 oiienmtbat 3.%decrease (32.5% nematoblasts positive .1 0cnrlaiascutdand counted animals control 10 0.01; eue eaoeei and nematogenesis reduces Nanos2 a xrse loin also expressed was Nanos2 a exclusively was Drosophila expression Nanos2 is xrsinof expression of decrease in a increase with metamorphosis. associated an post also was numbers but It tentacle neurons. nematocytes, of and number the nematoblasts of number otherwise. normal appeared but morphants, in 4C,D) Fig. h oto opoio etr ltuigat-i-a antibody anti-His-tag using blot Western morpholino. control the ( polyp per tentacles tentacle 5.9 of reduced average significantly an had from they numbers, polyps; animals. sibling metamorphosed of batch we single of effect a this on number experiment minimize each To the performed individuals. with between animals work varies we heterogeneous neurons because control that genetically also, with Note wild-type, 4E,F). injected Fig. morphants; animals 16 to increase, (36% compared of morpholino numbers higher neurons had morphants positive that found We probe. by done was This neurons. 4C). Fig. P aecnann h rdc ftecnrlmrhln u o in not but morpholino the the control with in the one kDa) of the (37 product size the expected containing approximate lane the at band a detected otos o14tnalsprplp( polyp per tentacles 1.4 to controls, , h pcfct ftemrhln a eosrtdby demonstrated was morpholino the of specificity The decreased in resulted Nanos2 of downregulation Hence, ncigdw fNns loipce h opooyof morphology the impacted also Nanos2 of down Knocking of effect the analyzed then We .01 9cnrlaiasad2 opat counted; morphants 29 and animals control 29 0.0001; Nanos2 Nanos2 Hstgwt the with -His-tag opoio(i.4G). (Fig. morpholino nsitu in P etce ,soo.Saebr:150 bars: Scale stolon. t, s, morpholino. tentacle; Nanos2 morpholino with control not of but presence the in tag showing rmr oys(F control polyps and primary (F) morphant in neurons oto lnle(E and planulae (E) control morphant in (arrowheads) neurons oto (D control ( (C polyps control primary and metamorphosed (C) morphant in capsules lnl ave(B control larvae and planula (B) morphant in capsules ( oto opoioijce ave(A larvae injected morpholino control in nematoblasts ncdw fNanos2. of knockdown Morpholino-mediated 4. Fig. , D B oiidDP tiigo nematocyst of staining DAPI Modified ) etcenmeso opat()and (D) morphant of numbers Tentacle ) .3 oto avecutdand counted larvae control 7 0.03; n yrdzto sn an using hybridization 5 5 7%decrease, (77% 25) nvitro in Nanos2 9 rmr oys ( polyps. primary ) Nanos2 Nanos2 yteie Nanos2-His- synthesized 9 9 .( ). .( ). 9 oneuainon downregulation .( ). C G Nematocyst ) F opoioand morpholino etr blot Western ) ) opat()and (A) morphant ( A Rfamide+ NCol1+ ) E P ) n , Rfamide+ Rfamide Rfamide 5 nvitro in 0.0001; 9 m 2 in 32) ). m. 9 ). Journal of Cell Science n splmnaymtra i.S) The S3). 3 Fig. the material at (eGFP) (supplementary protein the fluorescent end green of enhanced with consisting expressing in-frame construct animals a transgenic Actin1 used generated We we ectopically. this, excessive of For effect the study to on went then We Ectopic 02.Hne sn hscntutealdu osuynot study in to naturally gene do that us the cells express epithelial in not enabled effect long-term its also construct of but embryogenesis, consequences this the using only Hence, of types 2012). cell other (Ku all to animal in switches the quiescent being activity larval specific, its epithelial the metamorphosis become in following and embryogenesis However, in stage. ubiquitously active is promoter tbeGPepeso a vdn 44 or later hours 24–48 evident was expression GFP Stable etlzdeg eemconetdwt h osrc solution. construct the with microinjected were eggs Fertilized rmtrusra ftefull the of upstream promoter Nanos2 ne ta. 00 iln ta. 01 uf tal., et Duffy 2011; al., et Millane 2010; al., et ¨nzel expression Nanos2 Hydractinia Nanos2 bqiosoeepeso in overexpression ubiquitous oigsqec,fused sequence, coding . yrcii Actin1 Hydractinia Nanos2 Hydractinia expression. Nanos2 9 Fg A vrg nraeo 110%; of increase which significantly average were 5A; embryos, nematoblasts (Fig. of transgenic in numbers higher the in that expected, nematoblasts overexpressed counted we only driving shown). their stage of promoter (not level expression the same larval of GFP the independent normal the of appeared expression consisting GFP injected reaching construct animals Nanos2– Control a before normal. fewer with appeared with died cells Those expressing S4B). GFP them Fig. defects, material of developmental (supplementary most severe having and (supplementary distorted, expression Nanos2–GFP mosaics looked high the a mostly therefore and with Animals of process were S4A). stochastic Fig. material integration embryos a is injected The genome the embryo’s S4A). the into Fig. construct material (supplementary eas ao2dwrglto opoie nematogenesis, compromised downregulation Nanos2 Because Nanos2 ao nciainnmtgnss3197 nematogenesis cnidarian in Nanos Nanos2 rngnclra oprn owl-yelarvae wild-type to comparing larvae transgenic niNo1atbde hwd as showed, antibodies Anti-NCol1 . l nml r retdwt hi rlpoles 200 oral bars: their Scale with up. oriented are animals polyp. transgenic All in number tentacle excessive (G wild-type and (F (G) larva transgenic wild-type and (F) transgenic el ntasei E n wild-type and (E (E) transgenic in cells (D yeaias rngnc()adwild-type and (C (C) Transgenic wild- animals. and type transgenic in capsules nematocyst (asterisk). mouth ( the to relative tentacle lowest (B wild-type (A and (A) Transgenic nematoblasts. ( cellular of and consequences expression Gene 5. Fig. C A 9 9 9 9 9 , 9 , ave ( larvae. ) D B av.Tasei D n wild-type and (D) Transgenic larva. ) rmr oy.Dse ie niaethe indicate lines Dashed polyp. primary ) eaopoe rmr oys Note polyps. primary metamorphosed ) etce fpiayplp.( polyps. primary of tentacles ) av.Tasei B n wild-type and (B) Transgenic larva. ) Cl nioysann of staining antibody NCol1 ) oiidDP tiigt visualize to staining DAPI Modified ) F , G ) Nanos2 P Rfamide , m m. .0;3 wild-type 33 0.005; coi expression. ectopic ern in neurons + E BrdU+ ) 9 ,adin and ), Journal of Cell Science vrg nrae u a olne ttsial significant statistically longer no was 25% 5B; (Fig. but ( NCol1+ polyps primary increase) of hours numbers 24 and average in the elevated cells still nematogenesis, epithelial was affect cells to longer shifted no expression metamorphosis, could transgene’s Following the ones). transgenic when 34 counted, larvae nta,w esrdteraeaedniyadfudno found and density numbers. average excessive their their measured to in we than due Instead, higher in animals counted was directly not nematocytes post-metamorphic were in mature Nematocytes expected. animals of as wild-types transgenic number post-metamorphic the in which reversed wild- 11 was significantly analyzed; 45%; trend animals we of contained transgenic 5 decrease and larvae larvae (average type nematocytes and nematoblasts, transgenic mature the of embryos fewer numbers in counted transgenic elevated we the nematocytes these despite when mature that, Surprisingly, discovered of 5B). (Fig. numbers line tentacle 3198 yeaiasi hc Cl+nmtbat eealways never In and were column line. body nematoblasts tentacle polyp the the NCol1+ of wild- part reached lower which to the compared to in restricted as animals polyps primary type transgenic in broadened otat eosre NCol1 observed we contrast, P 5 .) h xrsindmi fNo1wsas still also was NCol1 of domain expression The 0.1). ora fCl cec 2 (14) 126 Science Cell of Journal + Nanos2 eaolsswl bv the above well nematoblasts P rngncaias in animals, transgenic , .3 Fg C.This 5C). (Fig. 0.03) nrae infcnl rma vrg f93 etce per tentacles numbers 9.32 of Their ( average polyp 6A,B). an (Fig. wild-type from significantly tentacles abnormal but increased positioned, supernumerary, displayed was phenotypes correctly also mildest The animals morphologies. metamorphosed 25 polyps numbers, counted, wild-types metamorphosed 22 larvae, and in animals; larvae decrease transgenic 83% embryos, of (average transgenic in of numbers ones). wild- transgenic 18 than increase; counted; cells 90.5% embryos 5E; positive (Fig. BrdU animals of type numbers higher significantly efudta rngncaiasoverexpressing animals labeling. BrdU transgenic by that embryos found wild-type We versus transgenic in cells Nanos2 proportionately were numbers animals. below), transgenic nematocyte (see in tentacles higher that more concluded had polyps we polyps transgenic As wild-type 5D). and (Fig. transgenic between differences significant nadto otecagsi eaolssadneurons and nematoblasts in changes the to addition In the that found We neurons. of numbers the at looked then We osuytesuc fteecsienmtbatnmesin numbers nematoblast excessive the of source the study To bv.t etce;H,hprlsi tln p rmr oy.Saebars: Scale from polyp. photographed primary was Pp, which stolon, 200 F, hyperplastic in HS, except tentacles; are t, up animals above. pole All oral morphology. the normal display with but oriented cells epithelial its in are GFP ( Stolons focus. numbers. of tentacle out secondary, normal shown all have but polyps tentacles, identical supernumerary genetically displays still polyp primary ( structure. ( polyp. polyps. metamorphosed in stolons Hyperplastic ( (B) mouth. normally the positioned around tentacles supernumerary with polyps metamorphosed of Consequences 6. Fig. C , D m uenmrr n coi etce nyugmetamorphosed young in tentacles ectopic and Supernumerary ) A,300 (A), m rngnclra,w oprdtenmeso S-phase of numbers the compared we larvae, transgenic E rmr oy opiigabl ihtnalsadn other no and tentacles with ball a comprising polyp Primary ) Rfamide F rngnccln eea ek otmtmrhss The metamorphosis. post weeks several colony Transgenic ) m n B,180 (B), m 5 P G oiienuoswr infcnl reduced significantly were neurons positive , 4 o1.8i rngncoe ( ones transgenic in 14.28 to 54) rngncscnayplpepesn Nanos2- expressing polyp secondary Transgenic ) .01 i.5F,G). Fig. 0.0001; Nanos2 m C,250 (C), m overexpression. P m , D,350 (D), m .01 9wild-type 19 0.0001; ( A , B Young ) m Nanos2 (E). m n 5 had 34) Journal of Cell Science omlmrhlge,cnandnmru tnigcells stinging had numerous tentacles ectopic contained All ectopically 6C,D). morphologies, them (Fig. grew column not normal post had body also A which the but develop. immediately polyps down tentacles to in supernumerary week observed evident was one only phenotype least not severe at animals; more required was transgenic but metamorphosis, in phenotype 53% abnormal of (increase ln ae hthdnra etcenumbers. tentacle identical normal genetically had first its that epithelia to the mates excessive comparing its clone later, 6F), to their due (Fig. weeks recognizable number still tentacle several in was colonies to these in expression up polyp Nevertheless, Nanos2–GFP 6F,G). (Fig. strong normal the completely despite appeared ( newly metamorphosis) of polyp all during primary emerges and phenotypes the that weeks after the next polyps clonal the Concomitantly, budded over dissipated down. transgenic gradually other shut animals in had expression and cells tissue, epithelial to 6E). restricted (Fig. structure excessive allogeneic further any a on without as by tentacles developed damage animals of three ball Finally, direct inflict swollen a colonies. Mu to other exposed to with 1992; not contact were are al., used animals transgenic et However, are tissues. Lange, stolons that 1989; al., nematocytes Hyperplastic McFadden et encounters 1972; Lange 2002). 1984; allogeneic (Ivker, as al., form animals during et organs histoincompatible stolons these between animals, modified hyperplastic normal shrimp developed In brine temporarily kill also 6B,D). were (Fig. to animals and ability stolons transgenic their cells, Many by battery attested nauplii. as epithelial functional in fully mounted (nematocytes) olwn eaopoi,tetaseesepeso became expression transgene’s the metamorphosis, Following P i.e. , .01.This 0.0001). h is polyp first the ¨ller, h coi etcefrainin that formation and signaling, tentacle Wnt no canonical ectopic affect and the not polyps does wild-type Nanos2 i that in else shows as anywhere end detected oral was mRNA very the to of restricted ubiquitous expression more the depolarized, then However, a Wnt, have also of if should that upstream thought We were 2010). al., et Nanos2 Duffy 2006; al., et (Plickert ubiquitous ao sacasclgr elmre nvrulyalanimals all virtually inhibition the in is action marker of mode cell general Its germ al.). et classical (Ewen-Campen a is Nanos Discussion signaling. 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Duffy the 2006; shown Wnt of al., reminiscent et canonical 2010), Plickert al., 2006; the al., et et of (Lee tentacles canonical activation ectopic by induces Ectopic controlled signaling. is polarity Wnt anterior–posterior cnidarians, In Wnt is development independent tentacle excessive mediated Nanos2 Nanos2 Mu 2004; ovrey coi n ciainb ramn with treatment by activation Wnt ectopic Conversely, Hydractinia etce ,soo.Saebr:150 bars: Scale stolon. s, tentacle; canonical activate ( ectopically signaling. to Wnt azakenpaullone with treated polyp xrsinpten ( pattern. ( expression (arrowhead). tip oral very ( between Relation 7. Fig. A xrsin(i.7,) ugsigthat suggesting 7C,D), (Fig. expression le ta. 04;Toe l,20) rv ubiquitous drove 2006), al., et Teo 2004b; al., et ¨ller ) Nanos2 ao nciainnmtgnss3199 nematogenesis cnidarian in Nanos hogotislf yl n sakontre fits of target known a is and cycle life its throughout rngncpiayplpexpressing polyp primary transgenic D ) Nanos2 C Ubiquitous ) Wnt3 Nanos2 Nanos2 xrsini oto,utetdaia.t, animal. untreated control, in expression B omlplpwt similar with polyp Normal ) Nanos2 ntasei nml remained animals transgenic in se xlsvl nteoa pole oral the in exclusively essed h nml(i.7,) This 7A,B). (Fig. animal the n m Nanos2 n aoia n signaling. Wnt canonical and eitdecsietentacles excessive mediated A,200 (A), m Nanos2 rngncaiaswas animals transgenic xrsini primary in expression rngncanimals transgenic Wnt3 Wnt3 m Wnt3 (C). m Nanos2 edrn it rendering , expression. nya the at only Wnt3 Wnt3 Wnt3 sa is in Journal of Cell Science te nml ssac,adbsdecuieyo oaie Nanos polarized on in exclusively function based similar and a scarce, for is evidence animals 2003), other al., et (Lall insects many results been The only has 2007). cell affecting al., a of et progenitor, such (Miljkovic-Licina of previously neural existence proposed The nematocyte neurons. and common nematocytes a Alternatively, other too. of on affected numbers a be that would exclude not types in cannot have cell but We result types neurons. cell as other such would their counted derivatives, i-cell promotes This to other in also them commitment. decrease and promoting following i-cells, nematogenesis proliferation on to acting committed and be become nematocytes may between Nanos2 neurons. balance Therefore, which the case. nematocytes, case, determining the to the not were similar this was ectopic metamorphosis, If post by nematoblasts. of produced of inhibited numbers case maturation the were increased in their numbers as neuroblasts merely Nanos2, for that marker and unlikely definite is increased no it have neuroblasts, we early Although stages. metamorphic eaesll pteil rngncaiashd ncontrast, expression in had, transgene’s of animals numbers the Transgenic lower when epithelial. solely metamorphosis became after until eaoye ept aiga xesof excess an having despite nematocytes upeetr aeilFg 2–) n xliswhy explains and S2A–D), 2C,D; (Fig. Fig. cells these Hydractinia material in pattern expression supplementary they its where with tentacles consistent the in destination until final lose stages their differentiation maturation reach last cells An nematocyte their the prevent maturation. nematoblast late may promoting Nanos2 while in nematocyte Hence, migration, migration. to Nanos2 inhibiting related be that of may in suggests role This acts stages. additional metamorphosis also post increasing early Nanos2 by and nematocytes mature pre nematoblasts, of in numbers of decreased but numbers proliferation, their increased determination. in fate resulted cell neural in Nanos functions. Nanos studying in informative been more of mammalian system be have nervous may the simpler might far of by complexity system The system. the nervous nervous to mammalian perhaps owing the al., nervous role overlooked, visible the et in concert, Siemen mammalian in Nanos 2011; act no usually of al., the Pumilio et and canonical Marrero Nanos causes in Because 2010; 2011). al., Nanos’ function absence et (Vessey does contrast, system complete Pumilio, By its partner, is abnormality. as (Nanos1) developmental homologues however, unknown, the which, Nanos of In function three is The system. 2007). of nervous the al., out in expressed one et 2008), only (Kadyrova Gavis, mouse, composition and subunit Brechbiel glutamate receptor postsynaptic with 2004; and growth, terminal al., presynaptic with et (Ye morphogenesis In context. this in similarly function they that both assume Because mice. documented, well and is flies Nanos cells on (Kadyrova germ done in work Nanos enzymes on of based role deadenylating The 2007). mRNA al., mediate recruiting et also can by it but mRNAs, degradation target its of translation of 3200 lhuhNnssest eivle npseirptenn in patterning posterior in involved be to seems Nanos Although ned u eut on oanvladuepce oefor role unexpected and novel a to point results our Indeed, system. nervous the in Nanos of role the is understood Less Nanos2 Drosophila Nanos2 oooswr xrse ngr el ti esnbeto reasonable is it cells germ in expressed were homologs ora fCl cec 2 (14) 126 Science Cell of Journal xrsinmnplto r umrzdi i.8A. Fig. in summarized are manipulation expression aveoverexpressing larvae xrsin tpmgaigadflymtr.Ti is This mature. fully and migrating stop expression, ao a enascae ihdendrite with associated been has Nanos , Rfamide Rfamide ern nbt r-adpost- and pre- both in neurons + Nanos2 ern hudhv been have should neurons + Nanos2 xrsinwskyin key was expression Nanos2 a nyfwmature few only had Ncol1+ Nanos2 overexpression Hydractinia progenitors, Hydractinia Rfamide+ ol act could ubr fmtr eaoye Fg ,) ncnrs,ectopic contrast, In in 5,6). activation increasing (Figs nematocytes Wnt the mature with of numbers concomitant post-metamorphosis, evident led ttepelrasae(lcete l,20) different 2006), 25), al., of (out et cases (Plickert three stage ectopic pre-larva from the at already only the Second, ( 2C). (Fig. defects polarity stages polarized larval for not and evidence gastrulae the was although in of pole 2A), expression First, oral (Fig. prospective embryo hypothesis: the early at this lines concentrated six were dispute However, transcripts 2002). evidence (Meinhardt, pole of to posterior corresponds as head larval cnidarians, cnidarian the in the and role structures metamorphic similar oral post are a tentacles support of could tentacles animals ectopic transgenic the of presence Mciuie l,20;Tra ta. 04 xaore l,2005; al., et Gonza Extavour 2004; and al., et Torras Torras 2000; al., et and (Mochizuki (Pilon leeches as 1997), such Weisblat, animals various in patterns expression ietrl nciainai atrig oeta ih ean be might that no role has a it innovation. a patterning, arthropod suggest add axis or and insect lineage, cnidarian data neural in a Our self-renewal, role in direct differentiation. maintenance, Nanos for of cell role similar prevention stem and germ of migration, in role independent conserved development. acts nematocytes of Nanos phase later that the in suggest Pumilio final may destination. their nematoblasts, final prevents promotes their but reach Pumilio Nanos2 they nematocytes, until development maturing maturation early nematocyte of later in migration In overlap mature. patterns by partner, cycle, Ncol1 replaced cell its and expression silenced the with is exit transcription concert nematoblasts their in as Later, act nematoblasts. to as likely is Pumilio, During Nanos2 fate. i-cells phase, neuronal promotes represses it this and first, nematocytes the to In commitment nematogenesis. that of phases suggest distinct studies of Analysis functional 8B. Fig. in presented formation. tentacle cnidarian normal in role polyps, a eventually have and (Mu tentacles, resorbed in being results i-cells of elimination emsriigtasei nml a omlmorphologies normal had animals long- Fourth, Fifth, transgenic 4). 6). surviving (Fig. (Fig. numbers term tentacles tentacle and extra nematocyte lowering than other Nanos2 defects polarity a eeeaetnals(acmadCmbl,1978; regeneration Campbell, tentacle) that (hence, showed head normal and studies of for recent requited but (Marcum is 1978), neurogenesis Fujisawa, tentacles and Sugiyama regenerate free nematocyte) therefore that can (and alternative i-cell the hypothesis note, exclude defects, Of cannot hypothesis. polarity but the formation, than tentacle favor rather extra tentacles drove nematocytes, we of ectopic numbers Hence, mediated excessive Nanos2 Wnt-independent. in that 6F,G). are unaltered (Fig. showing 7A,B), cells was (Fig. epithelial polarity, their of axis in expression Nanos2 Finally, ectopic despite ae oehr ao a hw rvosyt aea have to previously shown was Nanos together, Taken is nematogenesis in function Nanos2 summarizing model A phase, oneuainhdn feto xsplrt xetof except polarity axis on effect no had downregulation xrsinpten,oelpigol nproliferating in only overlapping patterns, expression Hydra Nanos2 Nanos2 MlkvcLcn ta. 07.In 2007). al., et (Miljkovic-Licina ´ Hydractinia e-rso 05 Lecle 2005; lez-Crespo, le ta. 04) ec,nmtctsmay nematocytes Hence, 2004a). al., et ¨ller Hydractinia xrsin hr,wt h xeto of exception the with Third, expression. xrsinrsmsutlsign cells stinging until resumes expression Nanos2 Wnt3 ao euao fcnidarian of regulator major a , ti td)adohrcnidarians other and study) (this coi xrsinddntcause not did expression ectopic i.e. mro rlzsteanimals the oralizes embryos Nanos2 Nanos2 Nanos2 coi etce)became tentacles) ectopic Ncol1 xrsinptenand pattern expression ` sepesdi two in expressed is ee l,21) The 2012). al., et re rngncanimals transgenic tteedo the of end the At . Hydractinia Nanos2 Nanos2 Nanos2 Nanos2 Nanos Hydra and , Journal of Cell Science eiuoyeLst ytm(rmg,ctn.L61.W de 1 was added We protein L4611). cat.no. Nanos2-His-tag (Promega, System purpose, Lysate Reticulocyte this the same using For the synthesized oligonucleotide. at at injected morpholino pl were 100 morpholinos with scrambled microinjected were control synthesized (see embryos a and concentration. stage As designed oligonucleotides cell were mM. One-two 0.25–0.5 sequence) morpholino Tools. for Gene S1 block by Table translation material supplementary conjugated Fluorescein downregulation Morpholino-mediated 4–5 of concentration a at plasmid the (Ku microinjected of were described volume embryos pl stage 200 cell One-two with S3). the Fig. material of (supplementary consisting of vector expression a prepared We animals transgenic of Generation (Fermentas). protocol 50 manufacturer’s at performed the was RNA to T7 hybridization and SP6 according using synthesized were polymerases (Roche) probes RNA labeled Fluorescein situ In (Mu situ In CsCl mM 100 in treatment pulse spawning. a light-induced 1973). following by Buchal, once hour fish induced an ground was about by collected supplemented Metamorphosis week were a They Embryos times seawater. week. in five a cultured nauplii and shrimp SCUBA, brine by fed Bay were Galway in collected were Animals culture Animal Methods and Materials Nanos2 olwdtepooo yTldn ta.(oeaoe l,2012). al., et (Toledano al. et Toledano by protocol the followed rncito-rnlto ecinwspromdwt h o-oigscrambled non-coding the with performed was reaction transcription-translation etr ltigwspromdt eiyteseiiiyo h rnlto block translation the of specificity the verify to performed was Blotting Western yrdzto a efre sdsrbd(aesie l,19) I-and DIG- 1996). al., et (Gajewski described as performed was hybridization hybridization opoiot 50 to morpholino ne ta. 00 uf ta. 02 iln ta. 2011). al., et Millane 2012; al., et Duffy 2010; al., et ¨nzel Nanos2 nvitro in ue nfaewt nacdgenfursetprotein fluorescent green enhanced with frame in fused m rncito-rnlto ecin scnrl a control, As reaction. transcription-translation l rncito-rnlto ytmTNT system transcription-translation ˚ .Frfluorescent For C. yrcii Actin1 Hydractinia nsitu in m g/ rmtr driving promoter, yrdzto we hybridization m spreviously as l H m 7Coupled T7 lof1mM le and ¨ller nsitu In nfl.W hrfr one l Cl+clsi ige neirpseirconfocal anterior-posterior single, a in counted cells NCol1+ be all to counted high therefore too We were full. nematoblasts anti in O of Suat polyclonal numbers Dr The from rabbit Germany). gift Heidelberg, kind the of a 2010), using al., et visualized (Adamczyk antibody were NCol1 progenitors cell Stinging cells Nematoblasts glycerol. secondary of in counting A11059). of mounted and no. and Labeling dilution PBS cat. Triton 1:500 (Invitrogen, 0.25% pre-absorbed with antibody washed anti-mouse in PBS. were 488 they hour Triton Finally, 1 dark Fluor 0.25% of the for Alexa in dilution in incubated 1:500 antibody, temperature Unspecific and in BSA PBS temperature PBS. room Triton 1% room 0.25% Triton at at with with washed hour 0.25% then 1 blocked they with (Roche), for anti-BrdU Next was incubated washed pH7.2. then antibodies then glycine were Samples HCl, M the 0.4 M of with 2 binding then with PBS, treated in paraformaldehyde. then with washed were and fixed were hour were they 1 specimens later for The minutes BrdU Forty mM seawater. 200 with containing washed seawater in incubated 90% were Animals in mounted staining were BrdU Animals temperature. GS/BSA/PBS room in at 1:500 diluted hour 1 were glycerol/PBS. for Invitrogen) room IgG, incubated A-11008, anti-rabbit at above. The and and goat as hour 488 A-11012 (GS/BSA/PBS). 1 and re-blocking no. 594 and then for BSA/PBS Fluor cat. BSA/PBS (Alexa incubated (BSA/PBS), in with antibodies and PBS secondary washes serum Pre-adsorbed GS/BSA/PBS three in goat by in BSA followed were 5% 1:500 temperature, 2% Samples then diluted in 100%). in and was minutes and minutes antibody minutes 30 75 30 60 50, for for for (25, blocked blocked PBS steps in and four through paraformaldehyde rehydrated ethanol 4% in in dehydrated fixed were Animals Immunohistochemistry a niois(:00dlto,SnaCu,ctn.S-0) olwdb HRP- and by 656120) followed no. cat. SC-803), substrate. Invitrogen, no. chemiluminescent dilution, cat (1:1000 a IgG Cruz, anti-rabbit Santa Goat anti-His- dilution, conjugated rabbit (1:1000 using antibodies detected were tag proteins His-tagged Synthesized, morpholino. ersnsdvlpn eaoytcpueadterdellipsoid capsule. red nematocyst the mature circle and represents Red capsule nuclei. nematocyst represent developing ellipsoids represents or circles Blue mature. is hs,i cso -el,pooignmtctsadrepressing and nematocytes promoting i-cells, on acts it phase, first Nanos2 auainutlte ec hi ia etnto,mutdin mounted destination, where final cells, their battery final reach their they prevents until but maturation nematocytes, maturing of migration by phase, replaced is cycle, and cell stops the transcription committed exit of nematoblasts proliferation As promotes nematoblasts. then It fate. neuronal *** i.8 umr fteefcsof effects in the role of Summary 8. Fig. P ao nciainnmtgnss3201 nematogenesis cnidarian in Nanos , Nanos2 .01 ( 0.0001. Hydractinia sepesdi w itntpae fnmtgnss nthe In nematogenesis. of phases distinct two in expressed is xrsinrsms ao2te cst promote to acts then Nanos2 resumes. expression B oe o h oeo ao2i nematogenesis. in Nanos2 of role the for Model ) . Nanos2 ( A umr fefcs ** effects; of Summary ) RAdsiae n h el fully cells the and dissipates mRNA Nanos2 Ncol1 tteedo the of end the At . iepeso n its and misexpression P Nanos2 , ¨ bk(University zbek 0.05, Ncol1 Hydra Journal of Cell Science uf,D . lcet . une,T,Tlan .adFak U. Frank, and W. Tilmann, T., Kuenzel, G., Plickert, J., D. Duffy, uf,D . iln,R .adFak U. Frank, and C. R. Millane, J., D. T., Duffy, Mitros, E., S. Hampson, O., Simakov, F., E. Kirkness, A., J. Chapman, rcbe,J .adGvs .R. E. Gavis, and L. J. Brechbiel, dmzk . ekr,C,Blsbaain .G,Ymd,S,Mrksi S., Murakoshi, S., Yamada, G., P. Balasubramanian, C., Zenkert, P., Adamczyk, References at PhD http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.127233/-/DC1 online her available material of Supplementary support in award Fund Hayes recipient Investigator a Crawford Principal dissertation. was Thomas (SFI) J.K. a 11/PI/1020]. Ireland of and Foundation 07/IN.1/B943 numbers Science the [grant a performed is U.F. J.K. experiments, Funding paper. the the wrote U.F. designed and J.K. U.F. and experiments, and J.K. contributions O Author Suat from gift technical kind for Heidelberg). a acknowledged of was continuous (University kindly antibody John their are Anti-NCol1 MacLoughlin, for Callanan assistance. Eoin Terry laboratory Lawless, and Frank Albert Galvin the discussions. and of support members thank We Acknowledgements maximum prepared acid the and amino using method, option. of software, NJ/BioNJ (NNI) model 5.10 the Nearest-Neighbor-Interchange (JTT) using MEGA the Jones-Taylor-Thornton the and phylogenetic the by substitution replicates) with inferred (100 method was (http://www.ebi.ac.uk/Tools/msa/ likelihood Bootstrapped Nanos manually. ClustalW2 of tree adjusted conserved previously using of a and Alignment prepared on and clustalw2) 2005). based al., GenBank was established et from domains (Extavour were taken analysis domains were phylogenetic conserved animals described their various of from boundaries sequences protein Nanos analyses microscopy. Phylogenetic brightfield using animals the in cells the all expressing Neurons animal. the counted neurons choosing each Rfamide+ in layer, cells confocal of single number for highest counted the were with animal one per cells BrdU+ All in cells increase nematocysts. S-phase (9% of animal number them total different the between for proxy difference the a significant as among no counted animals distribution found transgenic we 80 alternative, but and distal As most tentacles stolons). numbers the and in column high nematocysts animals. body entire difficult their (tentacles, is the compartments metamorphosis of post to these week z-stacks a confocal Under than due full older microscope. animals in in in Nematocysts confocal counted nematocysts visible. Counting were a were larvae nuclei, and of and not 2002) channel and embryos al., capsules, FITC et nematocyst only the (Szczepanek conditions described under poly- as them DAPI the viewing with staining capsules by nematocyst counted their were nematocytes Mature Nematocysts animal. counted each in cells software. of Minitab Student’s two-sample the number the highest by performed the were analyses with Statistical one the choosing plane, 3202 inln rmtsoa u upessaoa tutrsin structures aboral suppresses but oral promotes signaling n regeneration. and al. et D. Busam, of J., genome Borman, G., dynamic P. The Balasubramanian, (2010). T., Rattei, T., Weinmaier, 271-281. inln rstl uigailptenn n tmcl proliferation. cell stem and patterning axial during crosstalk signaling morphogenesis. dendrite in function o-uftdcodotnsaiie ebaetblto nciainorganelles. cnidarian in tubulation Chem. membrane O Biol. and stabilizes J. W. T. Holstein, chondroitin T., Gojobori, non-sulfated S., J. Hwang, K., Sugahara, ora fCl cec 2 (14) 126 Science Cell of Journal 285 P Development Rfamide 25613-25623. , 5 .1) eteeoeto h ubro etce napolyp a in tentacles of number the took therefore We 0.119). eevsaie by visualized were 137 20) pta euaino ao srqie o its for required is nanos of regulation Spatial (2008). Hydra 3057-3066. , m f2 idtp n 9 and wild-type 24 of m ur Biol. Curr. . Nature 21) etsokpoenadWnt and protein shock heat A (2012). 18 464 nsitu in 745-750. , 592-596. , yrdzto.W counted We hybridization. Hydractinia c guaaecnet of contents -glutamate Nanos2 t ¨ ts ehdusing method -test bk S. zbek, e.Biol. Dev. metamorphosis 21) Wnt (2010). transgenic 21) A (2010). ¨ zbek 362 , aauh,S,Tua . iaia . aak,Y,Nmr-iaaahd A., Nomura-Kitabayashid, Y., Sasaoka, S., Kitajima, M., Tsuda, S., Haraguchi, emn,R n Nu and R. Lehmann, ohzk,K,Sn,H,Kbysi . ihmy-uiaa .adFujisawa, and C. Nishimiya-Fujisawa, S., Kobayashi, H., Sano, K., Mochizuki, B. Galliot, and L. Ghila, S., Chera, M., Miljkovic-Licina, H. Meinhardt, aah,Y,Hysi .adKbysi S. Kobayashi, and M. Hayashi, Y., Hayashi, T. Fujisawa, and C. Fujisawa, E., Hayakawa, e,P . ag . au,D .adMridl,M Q. M. Martindale, and Q. D. Matus, K., Pang, N., P. Lee, Lecle iln,R . asa . uf,D . eih,C,Cniga,S,Plickert, S., Cunningham, C., Seoighe, J., D. Duffy, J., Kanska, C., R. Millane, L. W. R. L. Rotundo, Buss, and and J. M. P. McFarland, S., Tsoulfas, C. McFadden, A., Darr, G., S. Rossi, E., Marrero, rn,A,Msn . as,J .adBd,H R. H. Bode, and L. J. Marsh, E., Mason, A., Grens, Q. M. Martindale, and Q. D. Matus, K., Pang, G., C. Extavour, ag,R . ik .H n Mu and H. M. Dick, G., R. Lange, A. W. Mueller, and G. Plickert, R., Lange, H. N. Patel, and Z. M. Ludwig, S., Lall, alw .Q,Siatv,M,Mts .Q,Rksr .S n atnae .Q. M. Martindale, and S. D. Rokhsar, Q., D. Matus, M., Srivastava, Q., H. Marlow, alo,B,Qiun,M,Gia . eRs,R,MlkvcLcn,M n Chera, and M. Miljkovic-Licina, R., Rosa, de L., Ghila, M., Quiquand, B., G. Galliot, Plickert, and J. Schlossherr, T., Leitz, M., Gajewski, G. Freeman, G. C. Extavour, and E. E. Schwager, B., Ewen-Campen, uz .M,Hlti,T . er,B . ne,J n ai,C N. C. David, and J. Engel, M., B. Petri, W., T. Holstein, M., E. Kurz, T. Lemcke, and L. Meijer, Ku M., Leost, K., Lauenroth, T. C., Sugiyama, and Kunick, J. C. Grimmelikhuijzen, N., C. David, Y., Katsukura, acm .A n apel .D. R. Campbell, and A. B. Marcum, ayoa .Y,Hbr,Y,Le .H n hro,R P. R. Wharton, and H. M. T. G. Lee, Y., Wessel, Habara, and Y., L. Z. Kadyrova, S. Swartz, E., C. Juliano, ides . osen .W n eha,U. Technau, and W. T. Holstein, D., Lindgens, ve,F B. F. Ivker, ne,T,Hiran . rn,U,Mu U., Frank, R., Heiermann, T., ¨nzel, eta evu ytmi ipnal o omldevelopment. normal for dispensable is system nervous Y. central Saga, and K. Kurokawa, e.GnsEvol. Genes Dev. T. 67. eeCAHi h ifrnito aha fsnoynuosi h tentacles. the in neurons sensory of pathway Evol. differentiation Genes the in CnASH gene 731. in activity oei otro atr omto fthe of formation pattern posterior in role cnidarians. in polarity and signaling 17 Wnt of evolution come: xrsino O oantasrpinfactor. transcription domain POU a of U. expression Frank, and G. brain. wild-type young in a became body old an plan: body in ontogeny Colony I. hydroids. aei rspiagr line. germ Drosophila in fate the gene: specification fate cell a of elfraini h ndra Clytia. cnidarian the in formation cell E. Houliston, hydroid the in historecognition invertebrate, ttenuouclrsynapse. Pumilio- neuromuscular protein the RNA-binding at the 2 by acetylcholinesterase of regulation Translational vectensis, Nematostella of system nervous cnidarian. the anthozoan of an development and Anatomy (2009). S. cell germ bilaterian of evolution the and anemone sea mechanisms. a specification in patterns expression nanos specification. line germ of machinery Zool. Diptera. the of outside patterning in collagens G. Plickert, chimeras. and cnidarian and the S. animals in R. cells transgenic stem Schwarz, of potential M., differentiation Helling, A., Nonn, nesiilcells. interstitial ndracnttt oe aiyo erppiewt opoeei ativity. morphogenetic with Biol. neuropeptide Dev. of Arch. family novel a constitute Cnidaria larva. oto fmtra ylnBmN yNnsi h rspiagermline. Drosophila the in Nanos by mRNA B Cyclin maternal Development of control zknaloei eetv niio fgyoe ytaekns- beta. kinase-3 of synthase glycogen larvae of Lett. 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