MID1 and MID2 are required for Xenopus neural tube closure through the regulation ofmicrotubule organizationMakoto Suzuki, Yusuke Hara, Chiyo Takagi, Takamasa S. Yamamoto and Naoto Ueno

There was an error published in Development 137, 2329-2339.

In the RT-PCR and in situ hybridization section on p. 2330, the primer pair for xMID1 was incorrect. The correct primer pair isshown below.

5¢-AGTGTGGTTTCCTATGAGCTA-3¢ and 5¢-TGTATAATGGTTCTGTTTGAT-3¢

The authors apologise to readers for this mistake.

Development 138, 385 (2011) doi:10.1242/dev.062976© 2011. Published by The Company of Biologists Ltd

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INTRODUCTIONIn vertebrates, the neural tube is the primary luminal structure ofearly development. It is the anlage of the central nervous systemand forms from a flat neuroepithelial sheet called the neural plate.The lateral edges of the neural plate form ridges, called neuralfolds, along the dorsal surface, parallel to the anterior-posterioraxis. The neural folds continue to rise and eventually meet at thedorsal midline, where they fuse to form the luminal structure of theneural tube (Colas and Schoenwolf, 2001; Copp et al., 2003;Davidson and Keller, 1999). Failure of neural tube closure causescongenital malformations, collectively called neural tube defects(NTDs), including anencephaly and spina bifida (Colas andSchoenwolf, 2001; Copp et al., 2003).

During neural tube closure, the neuroepithelial cells undergodynamic changes in shape, including apicobasal elongation andapical constriction, which cause the tissue to bend to form theneural tube (Colas and Schoenwolf, 2001; Davidson and Keller,1999). The apicobasal elongation changes the cuboidalneuroepithelial cells into columnar cells (Burnside, 1973). Apicalconstriction minimizes the apical surface of selected cells in theneural plate (located at hinge points), causing them to adopt wedge-like rather than columnar shapes (Schoenwolf and Franks, 1984).To achieve the complex morphological changes required for tubeformation, these cellular changes must be tightly controlled in timeand space.

It is widely accepted that regulation of the neuroepithelialcytoskeleton is fundamental to cellular morphogenesis duringneural tube closure (Colas and Schoenwolf, 2001; Copp et al.,2003; Pilot and Lecuit, 2005; Quintin et al., 2008). In particular,regulation of the actin cytoskeleton has been extensively studied,and analyses in mice, chick and Xenopus show that actin-bindingproteins and their regulators, including Shroom3, MARCKS, Rap1,ROCKs, p190 RhoGAP and RhoA, positively regulate apicalconstriction via myosin activity (Copp et al., 2003; Haigo et al.,2003; Hildebrand, 2005; Kinoshita et al., 2008; Nishimura andTakeichi, 2008). In addition, cell adhesion molecules such as N-cadherin and Nectin contribute to apical constriction by regulatingcortical actin assembly (Morita et al., 2010; Nandadasa et al.,2009).

By contrast, the roles and regulatory mechanisms ofmicrotubules in neural tube closure have been elusive. Duringcell elongation, microtubules polymerize and assemble along theapicobasal axis (Burnside, 1973; Handel and Roth, 1971;Karfunkel, 1971). In chick and Xenopus, microtubulepolymerization inhibitors induce aberrant cell morphologies anddefects in neural tube closure (Handel and Roth, 1971;Karfunkel, 1971). In addition, non-centrosomal -tubulin,indirectly recruited to the apical side by Shroom3, participatesin the assembly of microtubule arrays and apicobasal cellelongation (Lee et al., 2007). Thus, microtubules appear to beimportant in the cellular morphogenesis required for neural tubeclosure.

Here, we show that the Xenopus orthologs of human MID1(also known as FXY, RNF59, TRIM18) and of MID2, an MID1paralog (also known as FXY2, RNF60, TRIM1) are crucial forepithelial remodeling in neural tube closure. In humans, MID1 isresponsible for X-linked Opitz G/BBB syndrome (OS), listed asOMIM 30000 (Buchner et al., 1999; Quaderi et al., 1997; Robinet al., 1995). OS is characterized by midline malformations,including hypertelorism, hypospadias, cleft lip/palate,

Development 137, 2329-2339 (2010) doi:10.1242/dev.048769© 2010. Published by The Company of Biologists Ltd

1Division of Morphogenesis, Department of Developmental Biology, NationalInstitute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi,Japan. 2Department of Basic Biology, School of Life Science, the Graduate Universityfor Advanced Studies (SOKENDAI), Nishigonaka 38, Myodaiji, Okazaki 444-8585,Aichi, Japan.

*Author for correspondence (nueno@nibb.ac.jp)

Accepted 17 May 2010

SUMMARYClosure of the neural tube requires both the change and maintenance of cell shape. The change occurs mainly through twocoordinated morphogenetic events: cell elongation and apical constriction. How cytoskeletal elements, including microtubules,are regulated in this process in vivo is largely unknown. Here, we show that neural tube closure in Xenopus depends onorthologs of two proteins: MID1, which is responsible for Opitz G/BBB syndrome in humans, and its paralog MID2. Depletion ofthe Xenopus MIDs (xMIDs) by morpholino-mediated knockdown disrupted epithelial morphology in the neural plate, leading toneural tube defects. In the xMID-depleted neural plate, the normal epithelial organization was perturbed without affectingneural fate. Furthermore, the xMID knockdown destabilized and caused the disorganization of microtubules, which are normallyapicobasally polarized, accounting for the abnormal phenotypes. We also found that the xMIDs and their interacting proteinMig12 were coordinately required for microtubule stabilization during remodeling of the neural plate. Finally, we showed thatthe xMIDs are required for the formation of multiple epithelial organs. We propose that similar MID-governed mechanismsunderlie the normal morphogenesis of epithelial tissues and organs, including the tissues affected in patients with Opitz G/BBBsyndrome.

KEY WORDS: Neural tube closure, Microtubule, MID1, MID2, Opitz syndrome, Epithelial remodeling, Xenopus

MID1 and MID2 are required for Xenopus neural tubeclosure through the regulation of microtubule organizationMakoto Suzuki1,2, Yusuke Hara1,2, Chiyo Takagi1, Takamasa S. Yamamoto1 and Naoto Ueno1,2,*

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laryngotracheoesophageal abnormalities, imperforate anus, cardiacdefects and brain abnormalities (Fontanella et al., 2008; So et al.,2005).

MID1 and MID2 encode conserved proteins associated withmicrotubules belonging to the RBCC/TRIM (N-terminal RINGfinger-B box-coiled coil/tripartite motif) superfamily (Buchneret al., 1999; Cainarca et al., 1999; Schweiger et al., 1999; Shortand Cox, 2006). MID1 and MID2 are known to be expressedduring development in human, mouse and chick (Buchner et al.,1999; Dal Zotto et al., 1998; Granata et al., 2005; Pinson et al.,2004; Quaderi et al., 1997; Richman et al., 2002). However,although biochemical and in vitro cell biological studies haveyielded some information, the physiological and developmentalfunctions of the MID proteins are still unclear, as is thepathological role of the MID1 mutant in OS. We report here thatXenopus MID1 and MID2 (xMID1 and xMID2) are essential forneural tube closure through their stabilization of microtubules,which is required for cell elongation and apical constriction. Wepropose that microtubule regulation by the MIDs is crucial fora variety of epithelial remodeling processes during thedevelopment of many vertebrate species.

MATERIALS AND METHODSCloning of Xenopus MID1 and MID2Xenopus laevis MID1 was identified as a cDNA clone, XL082d10, inour EST database (XDB3, http://xenopus.nibb.ac.jp). Since this clonecontains a 103 bp internal non-coding sequence, we isolated the entirecoding region from neurula cDNA by PCR using UTR sequence-specificprimers (5�-ggaattcGCACGAGGCTGGATTTTGCTTAC-3� and 5�-TGTGCATTGCAATGGATTCCCAATGGC-3�), and cloned it intopCS2p+. Similarly, a partial cDNA of Xenopus laevis MID2 wasobtained by PCR from neurula cDNA using primers based on thegenomic sequence of Xenopus tropicalis (5�-GAATGGAA -CAGCCCTGTCTCATTCT-3� and 5�-ACCTTCAAGCAATTTCT -TCTCTCTG-3�) and cloned into pBluescript SK+. This clone containedthe 5�UTR and 1.1 kb of the coding region, and the last 89 bp exhibitedhigh homology (98.9%) with the 5� region of another cDNA clone,xlk74e03ex (deposited in NBRP Xenopus, http://www.shigen.nig.ac.jp/xenopus/top.jsp), indicating that the entire coding region of xMID2 wasspanned by these two cDNA clones. We then isolated the entire codingregion from tailbud cDNA by PCR using UTR sequence-specificprimers (5�-GAATGGAACAGCCCTGTCTCATTCT-3� and 5�-GAT -TTCCCATCCAAGTCCTTTGCTG-3�) and cloned it into pCS2p+.Phylogenetic analysis was performed using MEGA4 software (Tamuraet al., 2007). GenBank accession numbers are GU362929 (xMID1) andGU362930 (xMID2).

Morpholinos, plasmids and mRNA preparationAntisense morpholino oligonucleotides (Mo) were obtained from GeneTools. The Mo sequences were as follows: xMID-Mo, 5�-CAGTTCAG -ACTCCAGTGTTTCCATC-3�; 5mis-xMID-Mo, 5�-CACTTGAGACT -ACAGTCTTTCGATC-3�; standard control-Mo, 5�-CCTCTTA CCT C -AGTTACAATTTATA-3�. The Mig12-Mo was reported previously (Hayeset al., 2007). Each Mo was injected at 13-17 ng per blastomere unlessotherwise stated. Because neither the 5mis-xMID-Mo nor the standardcontrol-Mo affected normal Xenopus development, we describe these Mosas ‘control-Mo’ in this study.

Full-length or truncated forms of xMID1, xMID2, Mig12 (Hayes et al.,2007), human -tubulin and human tau (MAPT) (Lu and Kosik, 2001)were subcloned into pCS2p+ with or without Venus or EGFP. For rescueconstructs, silent mutations were introduced into the Mo recognition sitesof the xMIDs. Mig12-GFP (Hayes et al., 2007) and Flag--globin(Hemmati-Brivanlou et al., 1994; Ohkawara et al., 2003) were reportedpreviously. Capped mRNAs were synthesized with the mMESSAGEmMACHINE Kit (Ambion) and purified on a NICK column (Pharmacia).

Embryo manipulation and microinjectionCapped mRNAs or Mos were injected into the appropriate region of two-or four-cell embryos. The injected embryos were cultured in 3%Ficoll/0.1� Steinberg’s Solution to stage 9, then washed and cultured in0.3� Marc’s Modified Ringer’s (MMR) until the appropriate stage(Nieuwkoop and Faber, 1967). Morphogenetic defects in the morphantswere analyzed at stage 16-17 unless otherwise stated. In animal capelongation assays, 0.5 pg activin mRNA was injected into the animal poleof two-cell embryos. The animal cap was dissected at stage 9 and culturedin Steinberg’s Solution until the sibling embryos reached stage 17.

RT-PCR and in situ hybridizationRT-PCR and in situ hybridization were performed as described (Goda etal., 2009). For RT-PCR with dissected tissues, the neural plate and ventralepidermis at stage 14 were separated from the underlying mesoderm inDanilchik’s For Amy Medium (DFA) (Sater et al., 1993). Ten explantswere used for each experiment. The following primers were used: xMID1,5�-GTTGTCTTCTCTGTTGAATAA-3� and 5�-TGTATAATGGT TCT -GTTTGAT-3�; xMID2, 5�-GTCATGAAGTTAAGAAAACTTGCTC-3�and 5�-ACCTTCAAGCAATTTCTTCTCTCTG-3�; NCAM, 5�-GCCT G -TAGAATTACAATGCTG-3� and 5�-AGCATCTTGGCT GCTGGCATT-3�;Sox2, 5�-GAGGATGGACACTTATGCCCAC-3� and 5�-GGACATGC -TGTAGGTAGGCGA-3�; Epidermal keratin I, 5�-CGGTTGAAGG -TAACCTGA-3� and 5�-CAACCTTCCCATCAACCA-3�; ODC, 5�-CAG -CTAGCTGTGGTGTGG-3� and 5�-CAACATGGAA ACTCACACC-3�.The following plasmids were used for probe synthesis: xMID1 and xMID2(constructed for this study); Sox2 (XL039o24, XDB3); NCAM (Kintner andMelton, 1987); N-cadherin (XL289n05ex, XDB3); Epidermal keratin I(XL056e18, XDB3); Shh (Yakushiji et al., 2007); Ptc2 (Yakushiji et al.,2007); Gli1 (Takabatake et al., 2000); Gli3 (Takabatake et al., 2000);HNF3 (FoxA2a) (XL016l12, XDB3); Pintallavis (FoxA4a) (XL047n03,XDB3); N-tubulin (Takabatake et al., 2002); Shroom3 (Haigo et al., 2003);and Pax3 (XL014p10, XDB3).

Western blotting and immunoprecipitationFor western blotting to test the specificity of xMID-Mo, 20 embryos atstage 14 were lysed in 400 l lysis buffer [50 mM Tris-HCl (pH 7.5), 150mM NaCl, 5 mM EDTA, 0.5% NP40, 50 mM NaF, protease inhibitors].For immunoprecipitation of EGFP-tubulin, 30 embryos at the late neurulastage were lysed in 600 l lysis buffer. Immunoprecipitation wasperformed as described (Ohkawara et al., 2003). Antibodies to GFP (598,MBL) and acetylated tubulin (TT6793, Sigma) were used.

ImmunohistochemistryEmbryos were fixed in Dent’s Fixative (for -catenin, C-cadherin, ZO-1),low-FGT Fixative (for -tubulin and cytoplasmic acetylated tubulin)(Becker and Gard, 2006) or MEMFA. Published procedures were used toprepare (Fagotto and Gumbiner, 1994) and stain (Suzuki et al., 2007) fishgelatin cryosections, or thick sections (Becker and Gard, 2006), with minormodifications. Antibodies to the following proteins were used: Sox2(AB5603, Chemicon); phospho-histone H3 (06-570, Upstate); activecaspase 3 (559565, BD Pharmingen); laminin (L9393, Sigma); Xen1(DSHB); MZ15 (DSHB); -catenin (C7082, Sigma); C-cadherin (6B6,DSHB); ZO-1 (AB01003, Sanko Junyaku); -tubulin (T9026, Sigma);acetylated tubulin (T6793, Sigma); Flag (F3165 and F7425, Sigma); GFP(598, MBL); and RFP (PM005, MBL). The secondary antibodies wereanti-mouse HRP (170-6516, Bio-Rad); anti-mouse Alexa 488 (A11017,Molecular probes); anti-mouse Alexa 555 (A21425, Molecular probes);anti-rabbit Alexa 488 (A11070, Molecular probes); anti-rabbit Alexa 555(A21430, Molecular probes); and anti-rabbit Cy5 (111-176-047, Jackson).When necessary, sections were counterstained with Alexa 546-phalloidin(A22283, Molecular probes) or TO-PRO-3 (T3605, Molecular probes) tolabel F-actin or nuclei, respectively.

Imaging and image analysisFor rescue experiments, we co-injected 0.25% Rhodamine-Dextran (D1817,Molecular Probes) with the Mo, selected embryos with appropriatefluorescence in the neural plate, and fixed them in MEMFA when the controlembryos reached the neural-fold stage. For quantitative analysis, the distance

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between the neural folds marked by pigmented margins in the anterior spinalcord anlage was measured. To quantify the cellular characteristics, images ofstained sections were obtained with a Zeiss LSM510 META confocalmicroscope equipped with a 63�, NA 1.4, oil-immersion or a 40�, NA 1.2,water-immersion objective lens. The cell height, apical width, basal width,perimeter of the apical side, and pixel intensities were all determined usingImageJ (NIH) software (see Fig. S4E in the supplementary material). Toquantify the cell morphologies, we selected cells with easily detected tracerfluorescence and a visible nucleus. The apical width and perimeter weredefined as the distance and outline length between apical cell-cell junctions,respectively. The cell height was defined as the maximal length along theaxis perpendicular to the apical width. Similarly, the basal width was definedas the distance between the basal cell-cell junction with the neighboring celland the line perpendicular to the apical width in contact with the oppositebasal junction. Cell width was defined as the larger of the apical and basalwidths (see Fig. S4E in the supplementary material).

To quantify molecular markers, labeled areas in the apical cell junctions(ZO-1, C-cadherin, -catenin) or the basal lamina (laminin) weremanually selected to exclude background staining. For apical celljunctions, we selected the region with higher fluorescence intensity thandetected more basally, and excluded almost all of the cell membrane withbaseline-level staining and the cytoplasm, because we could not clearlydistinguish between low-level specific signals and non-specificbackground labeling. Then, the total pixel intensity in each selected areawas measured. In the case of laminin, we drew a thin (one-pixel-wide) linealong the basal end of the cell, at the level of the basal lamina, where thelaminin fluorescence was brightest, and measured the fluorescenceintensity on the line. Since the basal width varied greatly among cells, wenormalized the data by dividing the total intensity by the length of the line.Data were analyzed by Student’s t-test, and are presented as the mean ±s.e.m.

RESULTSIdentification of the Xenopus homologs of MID1and MID2To identify candidate molecules for regulating neural tube closure,we focused on MIDs that were implicated in epithelialmorphogenesis through microtubule regulation. We isolated twocDNAs that encode proteins of 668 and 687 amino acids andexhibit 92% identity to human MID1 and 83% to human MID2,respectively (see Fig. S1A in the supplementary material). Aphylogenetic analysis confirmed that the two genes were closest tohuman MID1 and MID2 (see Fig. S1B in supplementary material)and we therefore named them xMID1 and xMID2.

By reverse transcription PCR (RT-PCR), we found that bothxMIDs were expressed throughout embryogenesis (Fig. 1A). Inaddition, both genes were expressed in the neural plate of the earlyneurula (Fig. 1B). Next, we analyzed the spatial expression of thexMIDs in detail by in situ hybridization. Before neurulation, neithergene was detectable (Fig. 1C; data not shown). During early to mid-neurulation, xMID1 was upregulated uniformly in the embryo (Fig.1D), and by late neurulation its transcripts were detected in theepithelial organs, including the neural tube, optic and otic vesicles,cement gland and newly epithelialized somites (Fig. 1E,F). At thetailbud stages, additional tissues expressed xMID1, including themidbrain, hindbrain, pronephros, pharyngeal pouch, heart tube andscattered epidermal cells (Fig. 1H,I). By contrast, expression ofxMID2 was undetectable at neurula stages (data not shown),whereas weak expression was observed in the pineal gland, oticvesicle and heart tube at the tailbud stages (Fig. 1J).

2331RESEARCH ARTICLEMicrotubule and xMIDs in neurulation

Fig. 1. Expression patterns of xMID1 and xMID2 through early embryogenesis. (A,B)RT-PCR analysis. (A)xMID1 and xMID2 mRNAs areexpressed maternally and zygotically. Ornithine decarboxylase (ODC) was used as an internal control. The number above each lane is the embryonicstage. –RT, control experiment without reverse transcriptase. (B)Ectodermal expression of xMIDs in the early neurula (stage 14) Xenopus embryo. Inthe neural plate, xMID1 and xMID2 were expressed at similar levels to in ventral epidermis (Epidermis) and whole embryo (W.E.). NCAM and Sox2are pan-neural markers. Epidermal keratin I (Epi. keratin) is an epidermal marker. (C-I)Expression pattern of xMID1 in embryos at the stagesindicated (lower right in each panel). (C)Dorso-posterior view, dorsal to the top. (D,E)Dorsal view, anterior to the top. (F)Anterior view, dorsal tothe top. (G)Dorsal view, labeled with sense probe (S), anterior to the top. (H,I)Lateral view, anterior to the left, dorsal to the top. (J,K)xMID2expression. Lateral view, anterior to the left, dorsal to the top. (K)Sense probe (S). cg, cement gland; hb, hindbrain; ht, heart tube; mb, midbrain;nt, neural tube; op, optic vesicle; ot, otic vesicle; pg, pineal gland; pn, pronephros; pp, pharyngeal pouch; pr, proctodeum; so, somite.

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Knockdown of xMIDs causes neural tube defectsTo deplete the endogenous xMID proteins and elucidate their in vivorole, we designed a specific antisense morpholino oligonucleotide(xMID-Mo) that efficiently blocked the translation of not only 5�UTR-xMID1-Venus, but also 5� UTR-xMID2-Venus, owing to thesimilarity of the Mo recognition site (Fig. 2A,B). The xMID-Mo didnot reduce the protein level of a 5� UTR-xMID1-Venus with six silentmutations in the target sequence (mut-xMID1-Vns).

The xMID-Mo, injected into one dorsal blastomere of four-cellembryos, caused a marked delay in neural tube closure (Fig. 2D,G),whereas the control-Mo had no effect on embryonic development(Fig. 2C,F). In the xMID morphants, the distance between neuralfolds was significantly greater (by ~3- to 5-fold) than in controlsibling embryos (Fig. 2H). Furthermore, the bilateral injection of thexMID-Mo caused severe defects in which the neural tube remainedopen even at the late neurula stage (Fig. 2E). In addition, xMIDmorphant cells were consistently found as a dissociated clump of

cells at the anterior neural plate [96% (n73 embryos) comparedwith 0.02% (n95 embryos) in control morphants] (Fig. 2E�). Co-injection of the xMID-Mo with the mRNAs of rescue constructs withsilent mutations in the Mo recognition site of xMID1, xMID2, orxMID1+2 dose-dependently reversed the Mo-induced distancebetween the neural folds (Fig. 2H). Therefore, the knockdown of thexMIDs by the xMID-Mo specifically caused the NTDs.

Knockdown of xMIDs does not affect gastrulationmovements, cell viability, neural development orprimary ciliogenesisTo investigate whether the effects of xMID-Mo were specific toepithelial remodeling, we examined gastrulation movement, cellviability and neural specification and patterning. xMID-Mo did notaffect gastrulation movement, activin-induced animal capelongation or cell proliferation and viability as revealed by stainingfor phospho-histone H3 and the apoptosis marker active caspase 3(see Fig. S2 in the supplementary material).

Since MID1 and MID2 repress Shh in Hensen’s node in chicken(Granata and Quaderi, 2003; Granata et al., 2005), and a deficiencyin Shh activity inhibits neural plate bending (Ybot-Gonzalez et al.,2002), we examined neural specification, dorsoventral patterningand the Shh pathway in the xMID morphants. We detected noapparent change in the markers tested, except that the delayedneural tube closure resulted in expression domains that were widerthan normal at these stages (see Fig. S2E and Fig. S3 in thesupplementary material). From these results, we concluded that theloss of xMID function did not affect gastrulation, cell viability orneural specification and patterning.

Recent studies have shown that mutations in ciliary genes thatresult in agenesis of the primary cilium, a microtubule-basedorganelle, cause NTDs, indicating some linkage between primarycilium formation and neural tube closure (Bisgrove and Yost, 2006).Hence, we analyzed the genesis of the primary cilia in the neuraltube. Knockdown of the xMIDs did not obviously affect the length

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Fig. 2. Depletion of xMIDs causes neural tube defects. (A)Thecomplementary sequence of the xMID-Mo compared with xMID1 andxMID2. The ATG codons for the first methionine are underlined.(B)Western blot analysis of the N-terminal domain of the xMIDproteins, tagged with Venus at the C-terminus (xMID1-Vns, xMID2-Vns,250 pg), probed with an anti-GFP antibody. The xMID-Mo (17 ng) didnot block translation of the mRNA for Venus (Vns, 100 pg) or themRNA for xMID1-Venus with six silent mutations in the Mo recognitionsite (mut-xMID1-Vns, 250 pg). (C,D)Dorsal views of the unilaterallyinjected morphants; anterior is to the top. Control-Mo (C) or xMID-Mo(D) was injected into the right dorsal blastomere at the four-cell stage.Dashed lines indicate the boundaries between the neural and non-neural ectoderm. The black bracket marked by an asterisk indicates thedistance between the neural folds in a rescue experiment. (E)Dorsalview of bilaterally injected xMID morphants at stage 20; anterior is tothe top. (E�)Higher magnification view of the area marked by thebracket in E. (F,G)Transverse sections through the neural plate ofXenopus embryos with unilateral injection of control-Mo (F) or xMID-Mo (G). Dashed lines indicate the outlines of neural tissues, andbrackets show the distance between the neural folds in a rescueexperiment. (H)Average distance between the neural folds ofunilaterally injected xMID morphants were dose-dependently reducedby the Mo-insensitive xMID1 (50, 100, 200, 500 pg), xMID2 (50, 100,200, 500 pg), or xMID1+2 (25, 50, 100, 250 pg each) mRNAs. Thenumber of embryos examined is indicated above each bar.

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or number of primary cilia (see Fig. S2F in the supplementarymaterial). Therefore, the NTDs of the xMID morphants are notattributable to a defect in primary cilium formation.

Knockdown of xMIDs induces aberrant cellmorphology in the neural plateNext, we analyzed the epithelial cell morphology in the xMIDmorphants by phalloidin staining. On the control side, theneuroepithelial cells showed normal apicobasal elongation andapical constriction (Fig. 3A,A�, blue outlines). In striking contrast,the xMID morphant cells did not elongate, but remained rounded,and their apical constriction was perturbed (Fig. 3A-A�, pinkoutlines; see also Fig. S4A-D in the supplementary material).Consistent with this, the cortical actin, the assembly of which is aprominent feature of apical constriction, was clearly attenuated(Fig. 3A, arrowheads).

To quantify the morphological defects in the xMID morphants(see Fig. S4E in the supplementary material), we analyzed theneuroepithelial cells in the superficial and deep layers separately(Schroeder, 1970). In the xMID-depleted superficial layer, the cellheight and apical width were significantly decreased and increased,respectively, compared with the control (Fig. 3B-D). Consequently,the ratios of cell height to width and apical width to basal widthwere altered (Fig. 3E,F). We observed similar defects in the deeplayer, except that the basal width of the deep morphant cells wassignificantly increased (see Fig. S4F-J in the supplementarymaterial). In addition, by assessing the ratio of the apical perimeterto the cell width, we found that the normally flat apical surface ofthe cell tended to protrude in the xMID morphants (Fig. 3G). Thus,the knockdown of the xMIDs caused cellular defects in both thesuperficial and deep layers of the neural plate.

Consistent with the rescue data described above (Fig. 2H), thexMID1 and xMID2 mRNAs partially, but convincingly, rescued theapical width (Fig. 3C,F) and the ratio of the apical perimeter to thecell width (Fig. 3G). Although co-expression of the xMIDs did notrescue the cell height, it significantly decreased the basal widthcompared with that of control cells (Fig. 3B,D), which resulted in

an increased height-to-width ratio (Fig. 3E). Therefore, theexogenous xMIDs rescued the cell morphology from a rounded toa columnar shape. These data strongly suggest that the NTDs of thexMID morphants were due to defects in cellular morphogenesis inthe neural plate.

Defects in cell-cell and cell-extracellular matrixcontacts in the neural plateTo dissect the cellular phenotype of the xMID morphants, weexamined the localization of proteins involved in cell-cell and cell-extracellular matrix (ECM) contacts. In the control cells, ZO-1(also known as TJP1), a tight junction marker (Itoh et al., 1993),and C-cadherin and -catenin, the major components of thecadherin complex (Brieher and Gumbiner, 1994; Levine et al.,1994), were concentrated at the apical junction (Fig. 4A,C,E). Bycontrast, at the apical junction in the xMID morphant cells, the ZO-1 signal was obscure (Fig. 4B,I) and the levels of C-cadherin and-catenin were severely reduced (Fig. 4D,F,J,K), although thetranscription and translation of these molecules were unaffected(data not shown). We also examined laminin, a major componentof the basal lamina (Miner et al., 1998), and found that itslocalization in the basal lamina was attenuated in the xMIDmorphants (Fig. 4G,H,H�,L). Thus, the knockdown of xMIDsresulted in the aberrant organization of cell-adhesive machineriesand the polarized distribution of the ECM.

Defective microtubule organization andstabilization in xMID morphantsWe next analyzed the subcellular localization of EGFP-taggedxMID1. Interestingly, EGFP-xMID1 colocalized with bundles ofnon-centrosomal microtubules stained with anti--tubulinantibody (Fig. 5A), which are readily observed in apicobasallyelongated epithelial cells (Bacallao et al., 1989; Bartolini andGundersen, 2006; Lee et al., 2007). In the control columnarepithelial cells, the apicobasal arrays of microtubules were alsoreadily apparent (100%, n11 cells, three embryos) (Fig. 5B). Bycontrast, in the xMID morphant cells, the arrays of microtubules

2333RESEARCH ARTICLEMicrotubule and xMIDs in neurulation

Fig. 3. xMIDs regulate epithelialmorphology and organization.(A-A�) Transverse section through the neuralplate of unilaterally injected xMID morphantsstained with phalloidin. (A)Apical actinassembly in the morphant cells (whitearrowheads) was attenuated compared withthat on the control side (open arrowheads).(A�)EGFP mRNA (50 pg) was co-injected as atracer. Dashed line indicates Mo-injected cells.(A�)Schematic illustration of A,A� showing thenon-Mo-containing cells outlined in blue andmorphant cells in pink. (B-G)Quantification ofcell morphological features in the superficiallayer of the neural plate. Control-Mo (Cont.-Mo), n53 cells (8 embryos); xMID-Mo, n59cells (7 embryos); xMID-Mo + xMID1, n56cells (13 embryos); xMID-Mo + xMID2, n54cells (9 embryos); xMID-Mo + xMID1&2, n42cells (10 embryos). *P<0.05, **P<0.01,***P<0.001, compared with xMID-Mo.

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were not polarized, and the cells were more rounded (85%, n13cells, four embryos) (Fig. 5D). To assess the stability of thepolymerized microtubules, we analyzed their acetylation status(Creppe et al., 2009; Verhey and Gaertig, 2007). In the controlcells, filamentous and continuous staining was detected,particularly in the apical region (86%, n7 cells, two embryos)(Fig. 5C). By contrast, in the rounded xMID morphant cells theacetylated -tubulin staining was punctate (92%, n13 cells, fourembryos) (Fig. 5E). Furthermore, the acetylation of theoverexpressed EGFP-tubulin was markedly decreased in thexMID morphants (40±4.5%, P<0.01, n3) (Fig. 5F,G). Thus, themicrotubules of the xMID morphant cells were disorganized anddestabilized.

We next examined the relationship between the microtubuledestabilization and the NTDs in the morphant embryos by co-injecting the xMID-Mo with the mRNA for an unrelatedmicrotubule-stabilizing factor, tau (also known as MAPT), whichis a classical microtubule-associated protein (Kanai et al., 1992;Lu and Kosik, 2001). The forced expression of tau not onlyrescued the disrupted neural cell morphologies (see Fig.S5C,D,G-L in the supplementary material), but also partiallyrescued the NTDs of the xMID morphants (see Fig. S5A,B inthe supplementary material). Furthermore, the apicobasalpolarization of the microtubule arrays was restored in the

tau-injected xMID morphant cells (see Fig. S5E,F in thesupplementary material). These findings suggest that xMIDs arerequired for the stabilization of microtubules.

xMIDs functionally interact with Mig12 in neuraltube closureMig12 (also known as G12-like and MID1IP1), which encodes aMID1-interacting molecule, is expressed in the ventral midline ofthe neural plate (Berti et al., 2004; Conway, 1995; Hayes et al.,2007). The Mo-mediated knockdown of Mig12 causes NTDs (Fig.6A-C) (Hayes et al., 2007) and defects in epidermal ciliogenesis(Hayes et al., 2007). To investigate the functional interaction ofxMIDs and Mig12, we performed individual injections or co-injections of xMID-Mo and Mig12-Mo. In morphants that receivedeither Mo alone at a low dose, only a slight delay in neural tubeclosure was induced (Fig. 6D-F,H). By contrast, the co-injection ofxMID-Mo and Mig12-Mo at the same low dose induced severeNTDs (Fig. 6G,H), suggesting that xMIDs and Mig12 interactfunctionally. We then performed rescue experiments of xMIDmorphants with Mig12 and vice versa. The NTDs of the Mig12morphants were rescued by xMID1 mRNA in a dose-dependentmanner (Fig. 6I). However, the NTDs in the xMID morphants werenot rescued by Mig12 mRNA (Fig. 6J), indicating that Mig12requires the xMIDs to function in neural tube closure.

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Fig. 4. xMIDs regulate the localization of adhesive molecules. (A-H�) Transverse sections through the neural plate of unilaterally control-Mo-injected (Cont.-Mo) (A,C,E,G) and xMID-Mo-injected (B,D,F,H) Xenopus embryos at stage 15.5, stained with antibodies against ZO-1 (A,B), C-cadherin (C-cad.) (C,D), -catenin (-cat.) (E,F), or laminin (G,H). Flag--globin mRNA (250 pg) was co-injected as a tracer, and stained with an anti-Flag antibody (magenta). (A-F)Dashed lines indicate Flag-positive Mo-injected cells. (H)Arrowheads indicate the attenuation of lamininaccumulation basal to the xMID-Mo-injected cells. (H�)Higher-magnification view of the boxed area in H. Scale bars: 50m. (I-L)Quantification ofmarker intensities in the morphants at stage 15.5. For ZO-1 intensity: control-Mo, n27 sites (3 embryos); xMID-Mo, n44 sites (6 embryos). For C-cadherin: control-Mo, n16 sites (4 embryos); xMID-Mo, n20 sites (5 embryos). For -catenin: control-Mo, n7 sites (3 embryos); xMID-Mo, n15sites (4 embryos). For laminin: control-Mo, n19 sites (4 embryos); xMID-Mo, n19 sites (3 embryos). *P<0.05, **P<0.001. (M,N)Schematicillustrations showing the control (M) and xMID (N) morphants. Rectangles indicate the regions analyzed in this study. so, somite; nt, notochord.

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The neuroepithelial cells of the Mig12 morphants andxMID/Mig12 double morphants resembled those of the xMIDmorphants (Fig. 6K-M). Consistent with this, the assembly ofapicobasally polarized microtubules was decreased in the double

morphants (Fig. 6K-M, bottom). These results highlight thefunctional relationship between the xMIDs and Mig12 in regulatingmicrotubule organization and cellular morphogenesis.

In vitro, when co-expressed with MID1, Mig12 colocalizes withmicrotubules and stabilizes them (Berti et al., 2004). To investigatethe subcellular localization of Mig12, we expressed Mig12-GFPwith xMID1 in animal caps and the neural plate (see Fig. S6 in thesupplementary material). When expressed alone, Mig12 wasdistributed throughout the cells of the animal cap and neural plate(see Fig. S6A,C,D in the supplementary material). However, whenco-expressed with xMID1, Mig12-GFP colocalized with themicrotubules in the animal cap cells, although such colocalizationwas not evident in neuroepithelial cells (see Fig. S6B,E in thesupplementary material). Thus, the functional interaction of thexMIDs and Mig12 appears to be highly dynamic and context-dependent, and in the neuroepithelial cells controlled physical andfunctional interactions allow highly organized microtubuleremodeling.

xMIDs contribute to the development of otherepithelial organsTo further investigate the role of xMIDs in epithelial morphogenesis,we performed targeted Mo injections into the presumptive headregion of embryos and found that xMID-Mo caused developmentaldefects of the eye (data not shown). Characterization using a neuralmarker, Xen1, and a notochord marker, MZ15, revealed hypoplasiaof the anterior central nervous system in the xMID morphants,although the notochord formed normally (Fig. 7A,B, insets; data notshown). We also analyzed neuroepithelial cell morphologies andlaminin localization in the basal lamina at the tailbud stage. Thenormally multi-layered structures of the brain and optic vesicles (Fig.7A,C) were disorganized, and the neuroepithelial cells had notelongated (Fig. 7B,D). Furthermore, neuroepithelial cells haddissociated from the apical surface of the epithelial sheet, whichlacked actin filaments (Fig. 7L; data not shown). The neuroepithelialcells found in the ventricle were positive for active caspase 3 (Fig.7K,L, dashed line), suggesting anoikis, a form of apoptosis causedby the loss of cell adhesion (Frisch and Screaton, 2001). Moreover,a continuous basal ECM failed to form, as indicated by the non-continuous and attenuated laminin staining (Fig. 7B,D, arrowheads).Thus, the NTDs that develop in xMID morphants ultimately causethe catastrophic collapse of the central nervous system.

Similar defects in cell morphogenesis were found in the cementgland (Fig. 7E,F), where xMID1 is strongly expressed (Fig. 1F,H,I).Furthermore, in the pronephros, the epithelial cells failed to adopt acolumnar shape or exhibit apical actin assembly, and no tubularstructure was formed (Fig. 7G,H). The area in which the pronephrosnormally forms was filled with disorganized cell aggregates (Fig.7H). In the foregut, derivatives of which are affected in OS patients(Fontanella et al., 2008; So et al., 2005), continuous apical actinfailed to assemble in the endodermal cells, and the cells protrudedapically, which caused a deformity of the luminal structure (Fig.7I,J). Taken together, our findings indicate that xMIDs play afundamental role in the remodeling of multiple epithelial tissues.

DISCUSSIONRole of microtubule regulation by xMIDs in cellshape changes and maintenance during neuraltube closureHere, we demonstrated that the xMIDs are required for normalneural tube closure, a multi-step event that involves neuralspecification, cell proliferation and morphogenetic movements

2335RESEARCH ARTICLEMicrotubule and xMIDs in neurulation

Fig. 5. xMIDs associate with and regulate microtubules.(A)Transverse section through the neural plate of a Xenopus embryoinjected with EGFP-xMID1 mRNA (50 pg) at stage 15.5, and stained withanti-GFP and anti--tubulin antibodies. Scale bar: 20m. (B-E)Transversesections through the neural plate of embryos unilaterally injected withcontrol-Mo (B,C) or xMID-Mo (D,E), and stained for -tubulin (B,D) oracetylated tubulin (Ac.-Tubulin) (C,E) antibodies. Flag--globin mRNA(250 pg) was co-injected as a tracer, and stained with an anti-Flagantibody (middle panels). Bottom panels show traced drawings of cellsstained by -tubulin and anti-acetylated tubulin antibodies. (F)Westernblots of immunoprecipitates (IP) or lysates from embryos expressing EGFP-tubulin (EGFP-tub.) mRNA (1 ng), detected with anti-GFP and anti-acetylated tubulin (Acetylated-tub.) antibodies. Immunoprecipitation wasperformed using the anti-GFP antibody. (G)Quantification ofimmunoprecipitation assay, showing the signal intensities of acetylatedtubulin from three independent experiments, normalized to those ofEGFP-tubulin. Error bars indicate s.e.m. *P<0.01.

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(Copp et al., 2003). In particular, a collective cell movement, whichis based on the morphogenesis of cells in the neural plate, servesas the major driving force for its invagination (Colas andSchoenwolf, 2001; Pilot and Lecuit, 2005; Quintin et al., 2008). InxMID morphants, the neuroepithelial cells remained rounded, andthe localization of adhesive molecules was perturbed, indicatingthat the epithelial organization was not maintained. Therearrangement and assembly of microtubules was also impaired inthe xMID morphants. The prevention of NTDs in xMID morphantsby the expression of another microtubule-associated proteinsuggests that the primary function of xMIDs is to stabilizemicrotubules. From these data, we propose that the xMIDs regulatecellular morphogenesis and epithelial organization during neuraltube closure through the assembly and stabilization ofmicrotubules. Since the knockdown of the xMIDs did not causeany obvious defects in classical microtubule function in mitosis or

primary cilium formation, the impact of xMID knockdown onmicrotubules might be limited, affecting only their rearrangementand assembly along the apicobasal axis.

Since the cellular and molecular mechanisms of neural tubeclosure in amphibians are closely related to those in amniotes(Davidson and Keller, 1999), MIDs are probably required forneural tube closure in amniotes, including humans. However,NTDs, such as anencephaly and spina bifida, have not beenreported in OS patients, although the expression of human MID1in the developing neural tube has been reported (Pinson et al.,2004), and abnormalities of the brain, including agenesis orhypoplasia of the cerebellar vermis and corpus callosum, are seenin OS (Fontanella et al., 2008; So et al., 2005). The overlappingexpression of MID1 and MID2 in developing neural tissues(Buchner et al., 1999; Granata et al., 2005; Dal Zotto et al., 1998)and the finding that MID1 and MID2 have redundant activities in

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Fig. 6. Mig12 functions with xMIDs inneural tube closure. (A,B)Dorsal views ofXenopus embryos given bilateral injections ofcontrol-Mo (A) or Mig12-Mo (B); anterior is tothe top. (C)Average distance between theneural folds of unilaterally injected Mig12morphants was reduced by the Mo-insensitiveMig12 mRNA (25, 50 pg). (D-H)Synergisticeffects of Mig12-Mo and xMID-Mo on neuraltube closure. Dorsal views, anterior to thetop. Injection of low doses (8.4 ng) of Mig12-Mo (E) or xMID-Mo (F) caused slight delays inneural tube closure. Co-injection of Mig12-Mo and xMID-Mo (8.4 ng each) inducedsevere NTDs (G,H). (I)Average distancebetween the neural folds of unilaterallyinjected Mig-12 morphants was reduceddose-dependently by xMID1 mRNA (50, 100,250, 500, 1000 pg). (J)Average distancebetween the neural folds of unilaterallyinjected xMID morphants was not reduced byMig12 mRNA (25, 50, 100, 250, 500 pg). Thenumber of embryos examined in C,H,I,J isindicated above each bar. (K-M)Transversesections through the neural plate of embryosgiven unilateral injections of control-Mo (K,8.4 ng), Mig12-Mo (L, 17 ng), or Mig12-Moand xMID-Mo (M, 8.4 ng each), stained with-tubulin (top) and anti-GFP (middle)antibodies. mRNA (125 pg) encodingmembrane-bound EGFP was co-injected as atracer. Bottom panels show traced drawingsof cells stained by anti--tubulin.

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chick left-right determination (Granata et al., 2005), suggest thatMID1 and MID2 have redundant functions in neural tube closuresuch that their role in this process is not unveiled by theknockdown or mutation of only one of them.

xMID-Mig12 collaboration is required for therearrangement and stabilization of microtubulesin vivoMig12 was identified as a gene encoding a 152 amino acid proteinthat is expressed in gastrula-stage zebrafish (Conway, 1995). InCos7 cells, Mig12 colocalizes with MID1 and stabilizesmicrotubules, suggesting that Mig12 might function cooperativelywith the xMIDs. In this study, we showed that Mig12 cooperatesfunctionally with the xMIDs in regulating microtubule organizationduring neural tube closure. However, all our data support the ideathat the xMIDs function as the dominant regulators of neural tubeclosure. The strongest evidence for this is that the NTDs of Mig12morphants were rescued by xMID expression, whereas those ofxMID morphants were not rescued by Mig12. Furthermore, the

cytoplasmic localization of Mig12-GFP was not changed by thegain or loss of xMID function. These data all suggest that, at leastin the neuroepithelial cells, the xMIDs are the main players in thexMID-Mig12 complex, and Mig12 might be recruited in a limitedamount to finely modulate the xMID activities. Furthermore, thefunctional interaction of these proteins might be dynamic andtightly regulated in time and space to avoid overstabilization ofthe microtubules, which might lead to defects in cellularmorphogenesis. Since a regulatory subunit of protein phosphatase2A (PP2A) binds MID1 and MID2 (Liu et al., 2001; Short et al.,2002; Trockenbacher et al., 2001), it is possible that the PP2Acomplex is involved in this mechanism.

Molecular link between microtubules and apicalconstrictionThe molecular mechanisms governing cellular morphogenesis inepithelia are well documented, especially with regard to apicalconstriction in Drosophila (Dawes-Hoang et al., 2005; Kolsch etal., 2007; Nikolaidou and Barrett, 2004; Pilot and Lecuit, 2005;

2337RESEARCH ARTICLEMicrotubule and xMIDs in neurulation

Fig. 7. xMIDs function in the developing brainand other epithelial organogeneses.(A-J)Transverse sections through the brain (A,B), opticvesicle (C,D), cement gland (E,F), pronephros (G,H)and foregut (I,J) of Xenopus embryos injected withcontrol-Mo (A,C,E,G,I) or xMID-Mo (B,D,F,H,J), andstained with phalloidin (green), anti-laminin antibody(magenta), or anti-Flag antibody (gray). (A,B)Insetsshow stage 40 embryos stained for the pan-neuralmarker Xen1. Lateral views: anterior to the left, dorsalto the top. Arrowheads indicate head defects in thexMID morphant. (B,D)Arrowheads indicate non-continuous and attenuated laminin staining in thebasal lamina. (E,F)Brackets indicate the lengths ofFlag-positive cells. (G)Asterisks indicate tubularstructures of the pronephros. (I,J)Asterisks indicateluminal structures of the foregut. Arrowheads indicatethe lack of apical actin assembly in the xMIDmorphant cells. (K,L)Transverse sections through thebrain of control-Mo-injected (K) and xMID-Mo-injected (L) embryos, stained with anti-active caspase3 (left) and anti-Flag (right) antibodies. Dashed linedelimits the ventricle.

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Quintin et al., 2008). In vertebrates, Shroom3-mediated activationof ROCKs and myosin II plays a crucial role in driving apicalconstriction (Haigo et al., 2003; Hildebrand, 2005; Nishimura andTakeichi, 2008; Rolo et al., 2009). Our study of the xMIDs raisesthe important question of how microtubules control apicalconstriction in neuroepithelial cells. In Drosophila, RhoGEF2associates with microtubule plus ends in an EB1-dependent manner(Rogers et al., 2004), and inhibition of microtubule polymerizationprevents apical actin assembly and Myosin light chainphosphorylation, thus blocking apical constriction (Corrigall et al.,2007). In addition, the enhancement of cadherin-based celladhesion is dependent on microtubules in human epithelial cells(Meng et al., 2008). These data suggest that the molecular linkbetween microtubules and apical constriction is mediated by thetransport of key regulators of actin polymerization, myosin IIactivation, and/or cell-cell contacts. It will therefore be intriguingto examine whether intracellular transport is affected in xMIDmorphants.

Insights into the molecular and pathologicalmechanisms of Opitz G/BBB syndromeWe demonstrated that xMID1 is required for the morphogenesis ofepithelial organs, such as the cement gland, pronephros andforegut. Furthermore, in the xMID morphants, the epithelial cellmorphology and organization were severely affected, and thedistribution of laminin in the basal lamina was compromised.These data indicate that the morphogenetic defects in xMIDmorphants are due to the loss of epithelial integrity.

In OS patients, various developmental abnormalities, includingcraniofacial, urogenital, gastrointestinal and cardiovascular defectsare observed, although the pathological mechanisms have not beenidentified (Fontanella et al., 2008; So et al., 2005). Importantly, arecent analysis of the MID1 expression pattern in the humanembryo revealed it to be expressed in various epithelial tissues,including the central nervous system, kidney primordia, and thepharyngeal, respiratory and gastrointestinal epithelia (Pinson et al.,2004). In addition, MID1 is expressed in the anal folds and genitaltubercle (Pinson et al., 2004). These expression patterns indicate astrong correlation between epithelial MID1 expression and thedevelopment of organs affected by OS. Although no extensivehistological characterizations of tissues from OS patients have beenreported, there are notable similarities in the pathological featuresof OS patients and the epithelial defects of xMID morphants. Inaddition, Mid1 shows similar epithelial expression patterns inmouse (Dal Zotto et al., 1998) and chick (Richman et al., 2002).We propose that common mechanisms underlie the normalmorphogenesis of the organs affected in OS patients and in theXenopus embryos in this study. Taken altogether, our findingsdemonstrate the general importance of microtubule regulation byMID1 and MID2 in cell shape change and maintenance inepithelial morphogenesis during vertebrate embryogenesis.

AcknowledgementsWe thank T. C. Cox, K. S. Kosik, K. Nakayama, T. Okubo, T. Takabatake, K.Tamura and J. B. Wallingford for plasmids and reagents; and members of theN.U. laboratory and S. Nonaka laboratory for valuable discussions, commentsand technical assistance. This work was supported by KAKENHI (07J05064,21870043 to M.S.; 17207015, 21370102 to N.U.) from the Japan Society forthe Promotion of Science (JSPS). M.S. and Y.H. were supported by JSPSResearch Fellowships for Young Scientists.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.048769/-/DC1

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2339RESEARCH ARTICLEMicrotubule and xMIDs in neurulation

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