ej th,Og sioa n egiY Sokol Y. Sergei and Ossipova* closure Olga Itoh*, tube Keiji neural and vertebrate intercalations for cell essential and is constriction apical in functions GEF-H1 ARTICLE RESEARCH 2542 2014 March 16 Accepted 2013; December 3 Received work ` this USA. to 10029, equally NY contributed York, of authors New School *These Sinai, Icahn Mount Biology, al., at Regenerative Medicine and et Developmental Wallingford of Department 2012; al., fusion et their Suzuki and 1970; Keller, 1999; folds Schroeder, Keller, and neural 1991; Davidson 2001; bilateral Schoenwolf, of and of Poznanski convergence (Colas constriction appearance dorsal 1992a; apical and the al., intercalations proceeds, drive et cell development Keller embryonic cells, As 2011; neuroepithelial main 1997). al., the al., et et to 2004). (Gray conform plan Kuroda, body to and complex behaviors Robertis exhibit morphogenetic is (De progenitors neuroectoderm tissue organizer gastrulation, neural produced During Spemann are Initially, that the factors steps. secreted by that by distinct ectoderm process dorsal of complex in specified a number of is a development system regulate The involves nervous that understood. central poorly events vertebrate are and the cellular closure (Harris and tube models common molecular genetic neural the most mouse 2010), in the process Juriloff, been this among have in genes are 200 implicated than more defects and abnormalities, tube developmental neural Although INTRODUCTION Morphogenesis Epiboly, constriction, intercalation, Apical Cell closure, tube Neural closure. WORDS: tube KEY neural to lead cells. that ectoderm movements GEF-H1 morphogenetic deep of in functions of identify newly activity of observations our protrusive together, Taken accumulation apical apical stimulated by and marked – MLC, phosphorylated constriction apical ectopic F-actin kinase-dependent Rho-associated and induced in studies, Rab11 GEF-H1 gain-of-function overexpressed In phosphorylation, defects cells. (MLC) GEF-H1-depleted documented in chain accumulation light analysis cell II these marker myosin regulates and Molecular GEF-H1 intercalation that behaviors. cell suggesting different by abnormal constriction, at rescued apical morphants revealed were GEF-H1 stages which of developmental defects, tracing Lineage tube RNA. neural GEF-H1 severe neurulation in in resulted RhoA-specific functions a that factor GEF-H1/Lfc/ArhGEF2, in exchange for nucleotide function guanine processes a Here closure. report morphogenetic tube neural we many including development regulate vertebrate during GTPases family Rho ABSTRACT uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,24–53doi:10.1242/jcs.146811 2542–2553 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Xenopus mro.Mrhln-eitddpeino GEF-H1 of depletion Morpholino-mediated embryos. Xenopus c tblnadFatni uefca coem– ectoderm superficial in F-actin and -tubulin f,Moi I h GTPases, Rho II, Myosin Lfc, , ` ula oeet pclbslpoentreig–ytteeis stability, there junction yet cell in – as implicated targeting such protein been – apical–basal also processes movement, has cellular nuclear of II variety sliding Myosin and a to, filaments. binding actin 2009). by al., force along, contractile et (Lecuit the Vicente-Manzanares 2001). activity controls 2010; require II al., and al., Myosin involve et localization Rauzi behaviors and 2011; et II al., myosin et components Tullio cell in cytoskeletal changes 2009; constricting dynamic many al., of and et reorganization intercalatory (Rolo II main Both myosin the muscle are behaviors vertebrate cell underlying neurulation. movements these morphogenetic to together, contributors Taken 2013). ncl dein(iuoae l,20;Cage l,2008) al., 2010; (Birkenfeld et cytokinesis al., 2010), Chang et al., et (Heasman Tsapara 2006; migration 2009; al., al., cell et Nalbant 2011), et al., al., (Birukova et et (Yamahashi adhesion Ren implicated 2002; been cell al., has (Birkenfeld et GEF-H1 in Krendel cells, RhoA mammalian 1999; cultured al., for In et 1998). specific Glaven 2008; is al., et that GEF further homology-domain for underlie that essential mechanisms analysis therefore neurulation. morphogenetic In-depth is vertebrate closure. the proteins tube of neural GEF understanding in individual protein this role ArhGEF11 whether of to conserved morphogenesis as embryos, tube a question a neural has raising zebrafish in 2007), chicken al., defects In et the exhibit (Panizzi in 2012). not process PDZ-RhoGEF/ do al., morphants this only in et present, implicated at (Nishimura been GEFs, has many ArhGEF11 involve to Ha likely 1997; al., Ko invagination et tissue 1998; with (Barrett associated gastrulation al., is during that invagination et constriction pit apical (Kashef lens In the migration 2011). optic al., during crest et constriction (Plageman neural apical Rho in and The have 2009) functions movements. 2004) al., extension Trio (Tanegashima et convergent GEF (Miyakoshi ARHGEF19) in Net1 implicated mesoderm- as ubiquitous been and known The 2008) al., (also functions. et WGEF developmental specific the specific of context assigned the for 2005). in challenge al., GTPases et Rho significant (Rossman of organism a regulation whole the presents of specificities localization studies subcellular diverse expression, GTPases, and to Rho spatiotemporal vertebrate of related activators unique 70 are are which with least MCF2), that as at (GEFs) known of (also factors 2005; Dbl existence Hall, exchange and The nucleotide Jaffe 2008). (Habas 2005; guanine Hall, al., Rho neurulation 2001; et small al., vertebrate et Kinoshita II, Habas in 2003; myosin roles al., to et essential Similar play tasks. to GTPases recruited specific selectively these is II perform myosin how of understanding limited n ftecuilrgltr fnua uecouei non- is closure tube neural of regulators crucial the of One E-1LcAhE2i irtbl-idn Dbl- microtubule-binding a is GEF-H1/Lfc/ArhGEF2 been have GEFs RhoA-specific existing the of few a Only lc ta. 07.Atog etbaenuuainis neurulation vertebrate Although 2007). al., et ¨lsch Drosophila hGF sesnilfor essential is RhoGEF2 , ce n Perrimon, and ¨cker

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Fig. uedfcswr enuo sn h morpholino the inhibited MO efficiently 1A–C,F; co-injection using GEF-H1a (Fig. MO in S1A). 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Fig. marker; pan-neural-tissue (a ora fCl cec 21)17 5225 doi:10.1242/jcs.146811 2542–2553 127, (2014) Science Cell of Journal Sox2 xrsindmi Fg ADE aanot data 2A,D,E; (Fig. domain expression t ts.*** -test. Sox2 oanwsmaue ttemid- the at measured was domain P , .01 The 0.0001. nsitu in Sox2 a oebroadly more was yrdzto for hybridization n ausaeshown are values nsitu in LacZ

Journal of Cell Science erltb lsr Ospv ta. 04 Fg EF.B contrast, By 3E,F). in (Fig. implicated 2014) been al., 2007) et has (Ossipova closure Harland, that tube marker neural and endosome recycling accumulate not Lee a apical did Rab11, light cells 2009; superficial of the Additionally, II Sokol, markers 3A–D). molecular (Fig. and myosin known (Choi MO regulatory F-actin, GEF-H1 constriction and with phosphorylated injected (MLC) been express chain had to that side failed the on neural superficial cells the that plate observed We 3). (Fig. constriction apical ARTICLE RESEARCH netdwt E-1M.Temdie()i niae yteboe ie AB hshrltdmoi Ilgtcan(MC e) CD -ci,(E,F) F-actin, (C,D) red), (pMLC; chain light B II embryos. 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Journal of Cell Science EERHARTICLE RESEARCH 2546 wild-type or RNAs 2002). GEF-H1-C53R al., GEF-H1, et encoding with (Krendel GEF-H1 by (GEF-H1-C53R) GEF-H1 mutation human for C53R used active the constitutively assay used pathway gain-of-function we microtubules, the a establish of activity to analysis wanted the we initiate To embryonic in constriction ectoderm apical ectopic triggers GEF-H1 (a Xdd1 activin-A- contrast, we on of By MO Surprisingly, form 4G–J). 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(red) embryos stage a Eight-cell at epiboly. as in ectoderm green) GEF-H1 thick (mGFP; of abnormally role contains The MO) (C) (GEF-H1 legend. embryo intercalations. GEF-H1-depleted cell (B) radial (co). regulate to oligonucleotide functions GEF-H1 4. Fig. ayre l,21) ned hs fet fGFH were GEF-H1 of effects 2007; these Harland, Indeed, and 2010). are (Lee al., changes constriction granule et morphological apical Sawyer pigment of increased These characteristic by 5A,B). (Fig. accompanied the at accumulation injection, bending tissue of caused By RNAs site both embryos. gastrulation, four-cell of of onset the blastomeres animal into microinjected convergent activin-dependent in and cells. intercalations not mesodermal observations radial of but in These extension GEF-H1 constriction, shown). of role not apical selective data a 1996; support (Sokol, behavior nvivo in opeetGFH niiinb associated by inhibition GEF-H1 prevent To . 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Journal of Cell Science EERHARTICLE RESEARCH ta. 97 elre l,19a elre l,19b Keller, 1992b; al., 2550 et Keller 1992a; al., (Elul et understood Keller cell poorly 1980). both 1997; are regulate layers al., convergence mediolateral germ that et different dorsal mechanisms in Although the process drive neuroectoderm, this and to for 2008). mesoderm thought in specifically extension al., are convergent required intercalations mesodermal et is regulate Rho GEF-H1 (Tanegashima unrelated to an that functions explanation WGEF, GEF, whereas potential is extension, One convergent result MO. neural explants GEF-H1 this pole by of animal inhibited not activin-induced mesodermal was of behaviors, extension cell specificity ectodermal the convergent to regulating Attesting in the shown). 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RhoA, alternative tight al., to of et due be regulation (Birkenfeld principle, targets in in GEF-H1 might, molecular regulating of behaviors by involvement cell effect the constriction diverse upon Nevertheless, its apical view activity. and exerts observed Indeed, II the intercalation GEF-H1 myosin we GEF-H1. cell that of that both reiterating indicate target on MLC GEF-H1 II, cellular phosphorylated of major modulation in myosin a of changes is parallel of roles the II constriction pleiotropic functions myosin apical that The and known al., closure. intercalations et the tube with cell redundantly (Meiri neural in functions GEF-H1 in abnormalities protein GEFs tube this other neural that exhibit suggesting during 2012), not movements the lacking cell do mice and gene Surprisingly, shape closure. tube cell neural regulate that processes u eut sals oe o E-1i utpesignaling multiple in GEF-H1 for roles establish results Our ora fCl cec 21)17 5225 doi:10.1242/jcs.146811 2542–2553 127, (2014) Science Cell of Journal eltdclsa oprdwt hto nnetdcells uninjected 20 of bar: that GEF-H1- Scale with in (arrow). compared staining as pMLC cells of depleted lack with * panel GFP. corresponding of the localization from staining pMLC the of ectoderm ( the embryos in injected morphology causes cell and inner C) polarization disorganized (CoMO, (red) oligonucleotide pMLC morpholino with control interferes the not MO, but (GEF-H1 D) oligonucleotide morpholino GEF-H1 The (C,D) hw nB B B. in shown B inner (arrow, in cells protrusions ectoderm GEF-H1-positive the apical to leads of Myc–GEF-H1 formation of axis Overexpression apical–basal (B) The indicated. 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Journal of Cell Science olr .L,Wbr . ldi,M n oo,S Y. S. Sokol, and M. Mlodzik, U., Weber, L., G. Dollar, lsoee tte48cl tg.Lcfrs ciiywsmeasured dorsal-animal was two activity Luciferase into stage. GEF-H1-C53R 4–8-cell RNAs with co-injected the was ROCK at 2002) (Busche blastomeres al., p2MA.luc dominant-negative et reporter Posern and/or control 2008; mutated 200), al., the in Myc et or 1 500), p3DA.luc Roche, reporter 12CA5, in (clone and 1 HA 1/1000) B2, antibodies 200), (Abcam, clone in The Cruz, (Santa MLC 1 10+. GFP Roche, stage phosphorylated 9E10, at against (clone embryos were six from used prepared were lysates ARTICLE RESEARCH 2552 R. Keller, and A. M. Koehl, T., Elul, C. S. Ekker, H. Kuroda, and M. E. Robertis, De E. R. Keller, and A. L. C. Davidson, G. Y. Schoenwolf, S. and F. Sokol, J. and Colas, O. Ossipova, E., Gerstenzang, W., C. M. Chu, Y. G. S. Bokoch, Sokol, and and F. C. S. Z. Choi, Chang, J., Birkenfeld, P., Nalbant, C., Y. Chang, G. Posern, and H. Genth, S., Julien, A., Descot, S., Busche, and G. K. Birukov, M., G. Bokoch, B., Gorshkov, D., Adyshev, A., A. Birukova, K. Matter, and S. M. Balda, S., Citi, F., D’Atri, S., Aijaz, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146811/-/DC1 online available material Supplementary months. material 12 Supplementary after number release [grant for Health PMC of in Institutes Deposited National S.Y.S.]. the to by NS40972 supported was research This the Funding experiments. wrote the and performed data O. the O. analyzed and experiments, I. the K. designed manuscript. S.S. and O.O. K.I., contributions Author interests. competing no declare authors The discussion. interests for Competing laboratory We Sokol plasmids. the for of IL) members OH), Urbana-Champaign, thank Cincinnati, Illinois, also Hospital, of Children’s (University Sinai, (Cincinnati Wang Mount Yingxiao Lang at Richard Medicine NY), of Biochemistry, York, School of New (Icahn Institute Soriano Planck Phillip of (Max Germany), College Posern Martinsried, Einstein Guido (Albert NY), Marlow Bronx, Florence Medicine, Dermardirossian, Celine thank We (Fan previously Acknowledgements described as 11, 1998). stage al., reached et embryos sibling when ikned . abn,P,Yo,S .adBkc,G M. G. Bokoch, and H. S. Bokoch, Yoon, and P., M. Nalbant, K. J., Hahn, Birkenfeld, O., Pertz, P., B. Bohl, P., Nalbant, J., G., Birkenfeld, Raposo, J., Eckert, C., Flores-Maldonado, A., Punn, G., Benais-Pont, J. Settleman, and M. 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