CORRIGENDUM

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

MID1 and MID2 are required for Xenopus neural tube closure through the regulation of organization Makoto 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 is shown below.

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

The authors apologise to readers for this mistake. DEVELOPMENT RESEARCH ARTICLE 2329

Development 137, 2329-2339 (2010) doi:10.1242/dev.048769 © 2010. Published by The Company of Biologists Ltd MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization Makoto Suzuki1,2, Yusuke Hara1,2, Chiyo Takagi1, Takamasa S. Yamamoto1 and Naoto Ueno1,2,*

SUMMARY Closure of the neural tube requires both the change and maintenance of cell shape. The change occurs mainly through two coordinated morphogenetic events: cell elongation and apical constriction. How cytoskeletal elements, including , are regulated in this process in vivo is largely unknown. Here, we show that neural tube closure in Xenopus depends on orthologs of two : MID1, which is responsible for Opitz G/BBB syndrome in humans, and its paralog MID2. Depletion of the Xenopus MIDs (xMIDs) by morpholino-mediated knockdown disrupted epithelial morphology in the neural plate, leading to neural tube defects. In the xMID-depleted neural plate, the normal epithelial organization was perturbed without affecting neural fate. Furthermore, the xMID knockdown destabilized and caused the disorganization of microtubules, which are normally apicobasally polarized, accounting for the abnormal phenotypes. We also found that the xMIDs and their interacting Mig12 were coordinately required for microtubule stabilization during remodeling of the neural plate. Finally, we showed that the xMIDs are required for the formation of multiple epithelial organs. We propose that similar MID-governed mechanisms underlie the normal morphogenesis of epithelial tissues and organs, including the tissues affected in patients with Opitz G/BBB syndrome.

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

INTRODUCTION It is widely accepted that regulation of the neuroepithelial In vertebrates, the neural tube is the primary luminal structure of cytoskeleton is fundamental to cellular morphogenesis during early development. It is the anlage of the central nervous system neural tube closure (Colas and Schoenwolf, 2001; Copp et al., and forms from a flat neuroepithelial sheet called the neural plate. 2003; Pilot and Lecuit, 2005; Quintin et al., 2008). In particular, The lateral edges of the neural plate form ridges, called neural regulation of the actin cytoskeleton has been extensively studied, folds, along the dorsal surface, parallel to the anterior-posterior and analyses in mice, chick and Xenopus show that actin-binding axis. The neural folds continue to rise and eventually meet at the proteins and their regulators, including Shroom3, MARCKS, Rap1, dorsal midline, where they fuse to form the luminal structure of the ROCKs, p190 RhoGAP and RhoA, positively regulate apical neural tube (Colas and Schoenwolf, 2001; Copp et al., 2003; constriction via myosin activity (Copp et al., 2003; Haigo et al., Davidson and Keller, 1999). Failure of neural tube closure causes 2003; Hildebrand, 2005; Kinoshita et al., 2008; Nishimura and congenital malformations, collectively called neural tube defects Takeichi, 2008). In addition, cell adhesion molecules such as N- (NTDs), including anencephaly and spina bifida (Colas and cadherin and Nectin contribute to apical constriction by regulating Schoenwolf, 2001; Copp et al., 2003). cortical actin assembly (Morita et al., 2010; Nandadasa et al., During neural tube closure, the neuroepithelial cells undergo 2009). dynamic changes in shape, including apicobasal elongation and By contrast, the roles and regulatory mechanisms of apical constriction, which cause the tissue to bend to form the microtubules in neural tube closure have been elusive. During neural tube (Colas and Schoenwolf, 2001; Davidson and Keller, cell elongation, microtubules polymerize and assemble along the 1999). The apicobasal elongation changes the cuboidal apicobasal axis (Burnside, 1973; Handel and Roth, 1971; neuroepithelial cells into columnar cells (Burnside, 1973). Apical Karfunkel, 1971). In chick and Xenopus, microtubule constriction minimizes the apical surface of selected cells in the polymerization inhibitors induce aberrant cell morphologies and neural plate (located at hinge points), causing them to adopt wedge- defects in neural tube closure (Handel and Roth, 1971; like rather than columnar shapes (Schoenwolf and Franks, 1984). Karfunkel, 1971). In addition, non-centrosomal -tubulin, To achieve the complex morphological changes required for tube indirectly recruited to the apical side by Shroom3, participates formation, these cellular changes must be tightly controlled in time in the assembly of microtubule arrays and apicobasal cell and space. elongation (Lee et al., 2007). Thus, microtubules appear to be important in the cellular morphogenesis required for neural tube closure. Here, we show that the Xenopus orthologs of human MID1 1Division of Morphogenesis, Department of Developmental Biology, National (also known as FXY, RNF59, TRIM18) and of MID2, an MID1 Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi, paralog (also known as FXY2, RNF60, TRIM1) are crucial for Japan. 2Department of Basic Biology, School of Life Science, the Graduate University for Advanced Studies (SOKENDAI), Nishigonaka 38, Myodaiji, Okazaki 444-8585, epithelial remodeling in neural tube closure. In humans, MID1 is Aichi, Japan. responsible for X-linked Opitz G/BBB syndrome (OS), listed as OMIM 30000 (Buchner et al., 1999; Quaderi et al., 1997; Robin *Author for correspondence ([email protected]) et al., 1995). OS is characterized by midline malformations,

Accepted 17 May 2010 including hypertelorism, hypospadias, cleft lip/palate, DEVELOPMENT 2330 RESEARCH ARTICLE Development 137 (14) laryngotracheoesophageal abnormalities, imperforate anus, cardiac Embryo manipulation and microinjection defects and brain abnormalities (Fontanella et al., 2008; So et al., Capped mRNAs or Mos were injected into the appropriate region of two- 2005). or four-cell embryos. The injected embryos were cultured in 3% ϫ MID1 and MID2 encode conserved proteins associated with Ficoll/0.1 Steinberg’s Solution to stage 9, then washed and cultured in ϫ microtubules belonging to the RBCC/TRIM (N-terminal RING 0.3 Marc’s Modified Ringer’s (MMR) until the appropriate stage (Nieuwkoop and Faber, 1967). Morphogenetic defects in the morphants finger-B box-coiled coil/tripartite motif) superfamily (Buchner were analyzed at stage 16-17 unless otherwise stated. In animal cap et al., 1999; Cainarca et al., 1999; Schweiger et al., 1999; Short elongation assays, 0.5 pg activin mRNA was injected into the animal pole and Cox, 2006). MID1 and MID2 are known to be expressed of two-cell embryos. The animal cap was dissected at stage 9 and cultured during development in human, mouse and chick (Buchner et al., in Steinberg’s Solution until the sibling embryos reached stage 17. 1999; Dal Zotto et al., 1998; Granata et al., 2005; Pinson et al., 2004; Quaderi et al., 1997; Richman et al., 2002). However, RT-PCR and in situ hybridization RT-PCR and in situ hybridization were performed as described (Goda et although biochemical and in vitro cell biological studies have al., 2009). For RT-PCR with dissected tissues, the neural plate and ventral yielded some information, the physiological and developmental epidermis at stage 14 were separated from the underlying mesoderm in functions of the MID proteins are still unclear, as is the Danilchik’s For Amy Medium (DFA) (Sater et al., 1993). Ten explants pathological role of the MID1 mutant in OS. We report here that were used for each experiment. The following primers were used: xMID1, Xenopus MID1 and MID2 (xMID1 and xMID2) are essential for 5Ј-GTTGTCTTCTCTGTTGAATAA-3Ј and 5Ј-TGTATAATGGT TCT - neural tube closure through their stabilization of microtubules, GTTTGAT-3Ј; xMID2, 5Ј-GTCATGAAGTTAAGAAAACTTGCTC-3Ј which is required for cell elongation and apical constriction. We and 5Ј-ACCTTCAAGCAATTTCTTCTCTCTG-3Ј; NCAM, 5Ј-GCCTG - Ј Ј Ј propose that microtubule regulation by the MIDs is crucial for TAGAATTACAATGCTG-3 and 5 -AGCATCTTGGCTGCTGGCATT-3 ; Sox2, 5Ј-GAGGATGGACACTTATGCCCAC-3Ј and 5Ј-GGACATGC - a variety of epithelial remodeling processes during the TGTAGGTAGGCGA-3Ј; Epidermal keratin I, 5Ј-CGGTTGAAGG - development of many vertebrate species. TAACCTGA-3Ј and 5Ј-CAACCTTCCCATCAACCA-3Ј; ODC, 5Ј-CAG- CTAGCTGTGGTGTGG-3Ј and 5Ј-CAACATGGAA ACTCACACC-3Ј. MATERIALS AND METHODS The following plasmids were used for probe synthesis: xMID1 and xMID2 Xenopus MID1 MID2 Cloning of and (constructed for this study); Sox2 (XL039o24, XDB3); NCAM (Kintner and Xenopus laevis MID1 was identified as a cDNA clone, XL082d10, in Melton, 1987); N-cadherin (XL289n05ex, XDB3); Epidermal keratin I our EST database (XDB3, http://xenopus.nibb.ac.jp). Since this clone (XL056e18, XDB3); Shh (Yakushiji et al., 2007); Ptc2 (Yakushiji et al., contains a 103 bp internal non-coding sequence, we isolated the entire 2007); Gli1 (Takabatake et al., 2000); Gli3 (Takabatake et al., 2000); coding region from neurula cDNA by PCR using UTR sequence-specific HNF3 (FoxA2a) (XL016l12, XDB3); Pintallavis (FoxA4a) (XL047n03, primers (5Ј-ggaattcGCACGAGGCTGGATTTTGCTTAC-3Ј and 5Ј- XDB3); N-tubulin (Takabatake et al., 2002); Shroom3 (Haigo et al., 2003); TGTGCATTGCAATGGATTCCCAATGGC-3Ј), and cloned it into and Pax3 (XL014p10, XDB3). pCS2p+. Similarly, a partial cDNA of Xenopus laevis MID2 was obtained by PCR from neurula cDNA using primers based on the Western blotting and immunoprecipitation genomic sequence of Xenopus tropicalis (5Ј-GAATGGAA- For western blotting to test the specificity of xMID-Mo, 20 embryos at CAGCCCTGTCTCATTCT-3Ј and 5Ј-ACCTTCAAGCAATTTCT- stage 14 were lysed in 400 l lysis buffer [50 mM Tris-HCl (pH 7.5), 150 TCTCTCTG-3Ј) and cloned into pBluescript SK+. This clone contained mM NaCl, 5 mM EDTA, 0.5% NP40, 50 mM NaF, protease inhibitors]. the 5ЈUTR and 1.1 kb of the coding region, and the last 89 bp exhibited For immunoprecipitation of EGFP-tubulin, 30 embryos at the late neurula high homology (98.9%) with the 5Ј region of another cDNA clone, stage were lysed in 600 l lysis buffer. Immunoprecipitation was xlk74e03ex (deposited in NBRP Xenopus, http://www.shigen.nig.ac.jp/ performed as described (Ohkawara et al., 2003). Antibodies to GFP (598, xenopus/top.jsp), indicating that the entire coding region of xMID2 was MBL) and acetylated tubulin (TT6793, Sigma) were used. spanned by these two cDNA clones. We then isolated the entire coding Immunohistochemistry region from tailbud cDNA by PCR using UTR sequence-specific Ј Ј Ј Embryos were fixed in Dent’s Fixative (for -catenin, C-cadherin, ZO-1), primers (5 -GAATGGAACAGCCCTGTCTCATTCT-3 and 5 -GAT- low-FGT Fixative (for -tubulin and cytoplasmic acetylated tubulin) Ј  TTCCCATCCAAGTCCTTTGCTG-3 ) and cloned it into pCS2p+. (Becker and Gard, 2006) or MEMFA. Published procedures were used to Phylogenetic analysis was performed using MEGA4 software (Tamura prepare (Fagotto and Gumbiner, 1994) and stain (Suzuki et al., 2007) fish et al., 2007). GenBank accession numbers are GU362929 (xMID1) and gelatin cryosections, or thick sections (Becker and Gard, 2006), with minor GU362930 (xMID2). modifications. Antibodies to the following proteins were used: Sox2 Morpholinos, plasmids and mRNA preparation (AB5603, Chemicon); phospho-histone H3 (06-570, Upstate); active Antisense morpholino oligonucleotides (Mo) were obtained from caspase 3 (559565, BD Pharmingen); laminin (L9393, Sigma); Xen1 (DSHB); MZ15 (DSHB); -catenin (C7082, Sigma); C-cadherin (6B6, Tools. The Mo sequences were as follows: xMID-Mo, 5Ј-CAGTTCAG-  DSHB); ZO-1 (AB01003, Sanko Junyaku); -tubulin (T9026, Sigma); ACTCCAGTGTTTCCATC-3Ј; 5mis-xMID-Mo, 5Ј-CACTTGAGACT -  acetylated tubulin (T6793, Sigma); Flag (F3165 and F7425, Sigma); GFP ACAGTCTTTCGATC-3Ј; standard control-Mo, 5Ј-CCTCTTA CCT C - (598, MBL); and RFP (PM005, MBL). The secondary antibodies were AGTTACAATTTATA-3Ј. The Mig12-Mo was reported previously (Hayes anti-mouse HRP (170-6516, Bio-Rad); anti-mouse Alexa 488 (A11017, et al., 2007). Each Mo was injected at 13-17 ng per blastomere unless Molecular probes); anti-mouse Alexa 555 (A21425, Molecular probes); otherwise stated. Because neither the 5mis-xMID-Mo nor the standard anti-rabbit Alexa 488 (A11070, Molecular probes); anti-rabbit Alexa 555 control-Mo affected normal Xenopus development, we describe these Mos (A21430, Molecular probes); and anti-rabbit Cy5 (111-176-047, Jackson). as ‘control-Mo’ in this study. When necessary, sections were counterstained with Alexa 546-phalloidin Full-length or truncated forms of xMID1, xMID2, Mig12 (Hayes et al., (A22283, Molecular probes) or TO-PRO-3 (T3605, Molecular probes) to 2007), human -tubulin and human tau (MAPT) (Lu and Kosik, 2001) label F-actin or nuclei, respectively. were subcloned into pCS2p+ with or without Venus or EGFP. For rescue constructs, silent mutations were introduced into the Mo recognition sites Imaging and image analysis of the xMIDs. Mig12-GFP (Hayes et al., 2007) and Flag--globin For rescue experiments, we co-injected 0.25% Rhodamine-Dextran (D1817, (Hemmati-Brivanlou et al., 1994; Ohkawara et al., 2003) were reported Molecular Probes) with the Mo, selected embryos with appropriate previously. Capped mRNAs were synthesized with the mMESSAGE fluorescence in the neural plate, and fixed them in MEMFA when the control

mMACHINE Kit (Ambion) and purified on a NICK column (Pharmacia). embryos reached the neural-fold stage. For quantitative analysis, the distance DEVELOPMENT Microtubule and xMIDs in neurulation RESEARCH ARTICLE 2331

Fig. 1. Expression patterns of xMID1 and xMID2 through early embryogenesis. (A,B)RT-PCR analysis. (A)xMID1 and xMID2 mRNAs are expressed maternally and zygotically. Ornithine decarboxylase (ODC) was used as an internal control. The number above each lane is the embryonic stage. –RT, control experiment without reverse transcriptase. (B)Ectodermal expression of xMIDs in the early neurula (stage 14) Xenopus embryo. In the neural plate, xMID1 and xMID2 were expressed at similar levels to in ventral epidermis (Epidermis) and whole embryo (W.E.). NCAM and Sox2 are pan-neural markers. Epidermal keratin I (Epi. keratin) is an epidermal marker. (C-I)Expression pattern of xMID1 in embryos at the stages indicated (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 to the 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)xMID2 expression. 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.

between the neural folds marked by pigmented margins in the anterior spinal RESULTS cord anlage was measured. To quantify the cellular characteristics, images of Identification of the Xenopus homologs of MID1 stained sections were obtained with a Zeiss LSM510 META confocal and MID2 microscope equipped with a 63ϫ, NA 1.4, oil-immersion or a 40ϫ, NA 1.2, water-immersion objective lens. The cell height, apical width, basal width, To identify candidate molecules for regulating neural tube closure, perimeter of the apical side, and pixel intensities were all determined using we focused on MIDs that were implicated in epithelial ImageJ (NIH) software (see Fig. S4E in the supplementary material). To morphogenesis through microtubule regulation. We isolated two quantify the cell morphologies, we selected cells with easily detected tracer cDNAs that encode proteins of 668 and 687 amino acids and fluorescence and a visible nucleus. The apical width and perimeter were exhibit 92% identity to human MID1 and 83% to human MID2, defined as the distance and outline length between apical cell-cell junctions, respectively (see Fig. S1A in the supplementary material). A respectively. The cell height was defined as the maximal length along the phylogenetic analysis confirmed that the two were closest to axis perpendicular to the apical width. Similarly, the basal width was defined human MID1 and MID2 (see Fig. S1B in supplementary material) as the distance between the basal cell-cell junction with the neighboring cell and we therefore named them xMID1 and xMID2. and the line perpendicular to the apical width in contact with the opposite By reverse transcription PCR (RT-PCR), we found that both basal junction. Cell width was defined as the larger of the apical and basal widths (see Fig. S4E in the supplementary material). xMIDs were expressed throughout embryogenesis (Fig. 1A). In To quantify molecular markers, labeled areas in the apical cell junctions addition, both genes were expressed in the neural plate of the early (ZO-1, C-cadherin, -catenin) or the basal lamina (laminin) were neurula (Fig. 1B). Next, we analyzed the spatial expression of the manually selected to exclude background staining. For apical cell xMIDs in detail by in situ hybridization. Before neurulation, neither junctions, we selected the region with higher fluorescence intensity than gene was detectable (Fig. 1C; data not shown). During early to mid- detected more basally, and excluded almost all of the cell membrane with neurulation, xMID1 was upregulated uniformly in the embryo (Fig. baseline-level staining and the cytoplasm, because we could not clearly 1D), and by late neurulation its transcripts were detected in the distinguish between low-level specific signals and non-specific epithelial organs, including the neural tube, optic and otic vesicles, background labeling. Then, the total pixel intensity in each selected area cement gland and newly epithelialized somites (Fig. 1E,F). At the was measured. In the case of laminin, we drew a thin (one-pixel-wide) line tailbud stages, additional tissues expressed xMID1, including the along the basal end of the cell, at the level of the basal lamina, where the midbrain, hindbrain, pronephros, pharyngeal pouch, heart tube and laminin fluorescence was brightest, and measured the fluorescence intensity on the line. Since the basal width varied greatly among cells, we scattered epidermal cells (Fig. 1H,I). By contrast, expression of normalized the data by dividing the total intensity by the length of the line. xMID2 was undetectable at neurula stages (data not shown), Data were analyzed by Student’s t-test, and are presented as the mean ± whereas weak expression was observed in the pineal gland, otic

s.e.m. vesicle and heart tube at the tailbud stages (Fig. 1J). DEVELOPMENT 2332 RESEARCH ARTICLE Development 137 (14)

Fig. 2. Depletion of xMIDs causes neural tube defects. (A)The complementary sequence of the xMID-Mo compared with xMID1 and xMID2. The ATG codons for the first methionine are underlined. (B)Western blot analysis of the N-terminal domain of the xMID proteins, tagged with Venus at the C-terminus (xMID1-Vns, xMID2-Vns, 250 pg), probed with an anti-GFP antibody. The xMID-Mo (17 ng) did not block translation of the mRNA for Venus (Vns, 100 pg) or the mRNA for xMID1-Venus with six silent mutations in the Mo recognition site (mut-xMID1-Vns, 250 pg). (C,D)Dorsal views of the unilaterally injected 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 the distance between the neural folds in a rescue experiment. (E)Dorsal view of bilaterally injected xMID morphants at stage 20; anterior is to the top. (EЈ)Higher magnification view of the area marked by the bracket in E. (F,G)Transverse sections through the neural plate of Xenopus embryos with unilateral injection of control-Mo (F) or xMID- Mo (G). Dashed lines indicate the outlines of neural tissues, and brackets show the distance between the neural folds in a rescue experiment. (H)Average distance between the neural folds of unilaterally injected xMID morphants were dose-dependently reduced by 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. The number of embryos examined is indicated above each bar.

cells at the anterior neural plate [96% (n73 embryos) compared with 0.02% (n95 embryos) in control morphants] (Fig. 2EЈ). Co- injection of the xMID-Mo with the mRNAs of rescue constructs with silent mutations in the Mo recognition site of xMID1, xMID2, or xMID1+2 dose-dependently reversed the Mo-induced distance between the neural folds (Fig. 2H). Therefore, the knockdown of the xMIDs by the xMID-Mo specifically caused the NTDs.

Knockdown of xMIDs does not affect gastrulation movements, cell viability, neural development or primary ciliogenesis To investigate whether the effects of xMID-Mo were specific to epithelial remodeling, we examined gastrulation movement, cell viability and neural specification and patterning. xMID-Mo did not affect gastrulation movement, activin-induced animal cap elongation or cell proliferation and viability as revealed by staining for phospho-histone H3 and the apoptosis marker active caspase 3 (see Fig. S2 in the supplementary material). Knockdown of xMIDs causes neural tube defects Since MID1 and MID2 repress Shh in Hensen’s node in chicken To deplete the endogenous xMID proteins and elucidate their in vivo (Granata and Quaderi, 2003; Granata et al., 2005), and a deficiency role, we designed a specific antisense morpholino oligonucleotide in Shh activity inhibits neural plate bending (Ybot-Gonzalez et al., (xMID-Mo) that efficiently blocked the translation of not only 5Ј 2002), we examined neural specification, dorsoventral patterning UTR-xMID1-Venus, but also 5Ј UTR-xMID2-Venus, owing to the and the Shh pathway in the xMID morphants. We detected no similarity of the Mo recognition site (Fig. 2A,B). The xMID-Mo did apparent change in the markers tested, except that the delayed not reduce the protein level of a 5Ј UTR-xMID1-Venus with six silent neural tube closure resulted in expression domains that were wider mutations in the target sequence (mut-xMID1-Vns). than normal at these stages (see Fig. S2E and Fig. S3 in the The xMID-Mo, injected into one dorsal blastomere of four-cell supplementary material). From these results, we concluded that the embryos, caused a marked delay in neural tube closure (Fig. 2D,G), loss of xMID function did not affect gastrulation, cell viability or whereas the control-Mo had no effect on embryonic development neural specification and patterning. (Fig. 2C,F). In the xMID morphants, the distance between neural Recent studies have shown that mutations in ciliary genes that folds was significantly greater (by ~3- to 5-fold) than in control result in agenesis of the primary cilium, a microtubule-based sibling embryos (Fig. 2H). Furthermore, the bilateral injection of the organelle, cause NTDs, indicating some linkage between primary xMID-Mo caused severe defects in which the neural tube remained cilium formation and neural tube closure (Bisgrove and Yost, 2006). open even at the late neurula stage (Fig. 2E). In addition, xMID Hence, we analyzed the genesis of the primary cilia in the neural

morphant cells were consistently found as a dissociated clump of tube. Knockdown of the xMIDs did not obviously affect the length DEVELOPMENT Microtubule and xMIDs in neurulation RESEARCH ARTICLE 2333

Fig. 3. xMIDs regulate epithelial morphology and organization. (A-AЉ) Transverse section through the neural plate of unilaterally injected xMID morphants stained with phalloidin. (A)Apical actin assembly in the morphant cells (white arrowheads) was attenuated compared with that on the control side (open arrowheads). (AЈ)EGFP mRNA (50 pg) was co-injected as a tracer. Dashed line indicates Mo-injected cells. (AЉ)Schematic illustration of A,AЈ showing the non-Mo-containing cells outlined in blue and morphant cells in pink. (B-G)Quantification of cell morphological features in the superficial layer of the neural plate. Control-Mo (Cont.- Mo), n53 cells (8 embryos); xMID-Mo, n59 cells (7 embryos); xMID-Mo + xMID1, n56 cells (13 embryos); xMID-Mo + xMID2, n54 cells (9 embryos); xMID-Mo + xMID1&2, n42 cells (10 embryos). *P<0.05, **P<0.01, ***P<0.001, compared with xMID-Mo.

or number of primary cilia (see Fig. S2F in the supplementary an increased height-to-width ratio (Fig. 3E). Therefore, the material). Therefore, the NTDs of the xMID morphants are not exogenous xMIDs rescued the cell morphology from a rounded to attributable to a defect in primary cilium formation. a columnar shape. These data strongly suggest that the NTDs of the xMID morphants were due to defects in cellular morphogenesis in Knockdown of xMIDs induces aberrant cell the neural plate. morphology in the neural plate Next, we analyzed the epithelial cell morphology in the xMID Defects in cell-cell and cell-extracellular matrix morphants by phalloidin staining. On the control side, the contacts in the neural plate neuroepithelial cells showed normal apicobasal elongation and To dissect the cellular phenotype of the xMID morphants, we apical constriction (Fig. 3A,AЉ, blue outlines). In striking contrast, examined the localization of proteins involved in cell-cell and cell- the xMID morphant cells did not elongate, but remained rounded, extracellular matrix (ECM) contacts. In the control cells, ZO-1 and their apical constriction was perturbed (Fig. 3A-AЉ, pink (also known as TJP1), a tight junction marker (Itoh et al., 1993), outlines; see also Fig. S4A-D in the supplementary material). and C-cadherin and -catenin, the major components of the Consistent with this, the cortical actin, the assembly of which is a cadherin complex (Brieher and Gumbiner, 1994; Levine et al., prominent feature of apical constriction, was clearly attenuated 1994), were concentrated at the apical junction (Fig. 4A,C,E). By (Fig. 3A, arrowheads). contrast, at the apical junction in the xMID morphant cells, the ZO- To quantify the morphological defects in the xMID morphants 1 signal was obscure (Fig. 4B,I) and the levels of C-cadherin and (see Fig. S4E in the supplementary material), we analyzed the -catenin were severely reduced (Fig. 4D,F,J,K), although the neuroepithelial cells in the superficial and deep layers separately transcription and translation of these molecules were unaffected (Schroeder, 1970). In the xMID-depleted superficial layer, the cell (data not shown). We also examined laminin, a major component height and apical width were significantly decreased and increased, of the basal lamina (Miner et al., 1998), and found that its respectively, compared with the control (Fig. 3B-D). Consequently, localization in the basal lamina was attenuated in the xMID the ratios of cell height to width and apical width to basal width morphants (Fig. 4G,H,HЈ,L). Thus, the knockdown of xMIDs were altered (Fig. 3E,F). We observed similar defects in the deep resulted in the aberrant organization of cell-adhesive machineries layer, except that the basal width of the deep morphant cells was and the polarized distribution of the ECM. significantly increased (see Fig. S4F-J in the supplementary material). In addition, by assessing the ratio of the apical perimeter Defective microtubule organization and to the cell width, we found that the normally flat apical surface of stabilization in xMID morphants the cell tended to protrude in the xMID morphants (Fig. 3G). Thus, We next analyzed the subcellular localization of EGFP-tagged the knockdown of the xMIDs caused cellular defects in both the xMID1. Interestingly, EGFP-xMID1 colocalized with bundles of superficial and deep layers of the neural plate. non-centrosomal microtubules stained with anti--tubulin Consistent with the rescue data described above (Fig. 2H), the antibody (Fig. 5A), which are readily observed in apicobasally xMID1 and xMID2 mRNAs partially, but convincingly, rescued the elongated epithelial cells (Bacallao et al., 1989; Bartolini and apical width (Fig. 3C,F) and the ratio of the apical perimeter to the Gundersen, 2006; Lee et al., 2007). In the control columnar cell width (Fig. 3G). Although co-expression of the xMIDs did not epithelial cells, the apicobasal arrays of microtubules were also rescue the cell height, it significantly decreased the basal width readily apparent (100%, n11 cells, three embryos) (Fig. 5B). By

compared with that of control cells (Fig. 3B,D), which resulted in contrast, in the xMID morphant cells, the arrays of microtubules DEVELOPMENT 2334 RESEARCH ARTICLE Development 137 (14)

Fig. 4. xMIDs regulate the localization of adhesive molecules. (A-HЈ) 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 laminin accumulation basal to the xMID-Mo-injected cells. (HЈ)Higher-magnification view of the boxed area in H. Scale bars: 50m. (I-L)Quantification of marker intensities in the morphants at stage 15.5. For ZO-1 intensity: control-Mo, n27 sites (3 embryos); xMID-Mo, n44 sites (6 embryos). For C- cadherin: control-Mo, n16 sites (4 embryos); xMID-Mo, n20 sites (5 embryos). For -catenin: control-Mo, n7 sites (3 embryos); xMID-Mo, n15 sites (4 embryos). For laminin: control-Mo, n19 sites (4 embryos); xMID-Mo, n19 sites (3 embryos). *P<0.05, **P<0.001. (M,N)Schematic illustrations showing the control (M) and xMID (N) morphants. Rectangles indicate the regions analyzed in this study. so, somite; nt, notochord. were not polarized, and the cells were more rounded (85%, n13 tau-injected xMID morphant cells (see Fig. S5E,F in the cells, four embryos) (Fig. 5D). To assess the stability of the supplementary material). These findings suggest that xMIDs are polymerized microtubules, we analyzed their acetylation status required for the stabilization of microtubules. (Creppe et al., 2009; Verhey and Gaertig, 2007). In the control cells, filamentous and continuous staining was detected, xMIDs functionally interact with Mig12 in neural particularly in the apical region (86%, n7 cells, two embryos) tube closure (Fig. 5C). By contrast, in the rounded xMID morphant cells the Mig12 (also known as G12-like and MID1IP1), which encodes a acetylated -tubulin staining was punctate (92%, n13 cells, four MID1-interacting molecule, is expressed in the ventral midline of embryos) (Fig. 5E). Furthermore, the acetylation of the the neural plate (Berti et al., 2004; Conway, 1995; Hayes et al., overexpressed EGFP-tubulin was markedly decreased in the 2007). The Mo-mediated knockdown of Mig12 causes NTDs (Fig. xMID morphants (40±4.5%, P<0.01, n3) (Fig. 5F,G). Thus, the 6A-C) (Hayes et al., 2007) and defects in epidermal ciliogenesis microtubules of the xMID morphant cells were disorganized and (Hayes et al., 2007). To investigate the functional interaction of destabilized. xMIDs and Mig12, we performed individual injections or co- We next examined the relationship between the microtubule injections of xMID-Mo and Mig12-Mo. In morphants that received destabilization and the NTDs in the morphant embryos by co- either Mo alone at a low dose, only a slight delay in neural tube injecting the xMID-Mo with the mRNA for an unrelated closure was induced (Fig. 6D-F,H). By contrast, the co-injection of microtubule-stabilizing factor, tau (also known as MAPT), which xMID-Mo and Mig12-Mo at the same low dose induced severe is a classical microtubule-associated protein (Kanai et al., 1992; NTDs (Fig. 6G,H), suggesting that xMIDs and Mig12 interact Lu and Kosik, 2001). The forced expression of tau not only functionally. We then performed rescue experiments of xMID rescued the disrupted neural cell morphologies (see Fig. morphants with Mig12 and vice versa. The NTDs of the Mig12 S5C,D,G-L in the supplementary material), but also partially morphants were rescued by xMID1 mRNA in a dose-dependent rescued the NTDs of the xMID morphants (see Fig. S5A,B in manner (Fig. 6I). However, the NTDs in the xMID morphants were the supplementary material). Furthermore, the apicobasal not rescued by Mig12 mRNA (Fig. 6J), indicating that Mig12

polarization of the microtubule arrays was restored in the requires the xMIDs to function in neural tube closure. DEVELOPMENT Microtubule and xMIDs in neurulation RESEARCH ARTICLE 2335

morphants (Fig. 6K-M, bottom). These results highlight the functional relationship between the xMIDs and Mig12 in regulating microtubule organization and cellular morphogenesis. In vitro, when co-expressed with MID1, Mig12 colocalizes with microtubules and stabilizes them (Berti et al., 2004). To investigate the subcellular localization of Mig12, we expressed Mig12-GFP with xMID1 in animal caps and the neural plate (see Fig. S6 in the supplementary material). When expressed alone, Mig12 was distributed throughout the cells of the animal cap and neural plate (see Fig. S6A,C,D in the supplementary material). However, when co-expressed with xMID1, Mig12-GFP colocalized with the microtubules in the animal cap cells, although such colocalization was not evident in neuroepithelial cells (see Fig. S6B,E in the supplementary material). Thus, the functional interaction of the xMIDs and Mig12 appears to be highly dynamic and context- dependent, and in the neuroepithelial cells controlled physical and functional interactions allow highly organized microtubule remodeling.

xMIDs contribute to the development of other epithelial organs To further investigate the role of xMIDs in epithelial morphogenesis, we performed targeted Mo injections into the presumptive head region of embryos and found that xMID-Mo caused developmental defects of the eye (data not shown). Characterization using a neural marker, Xen1, and a notochord marker, MZ15, revealed hypoplasia of the anterior central nervous system in the xMID morphants, although the notochord formed normally (Fig. 7A,B, insets; data not shown). We also analyzed neuroepithelial cell morphologies and laminin localization in the basal lamina at the tailbud stage. The normally multi-layered structures of the brain and optic vesicles (Fig. 7A,C) were disorganized, and the neuroepithelial cells had not elongated (Fig. 7B,D). Furthermore, neuroepithelial cells had dissociated from the apical surface of the epithelial sheet, which lacked actin filaments (Fig. 7L; data not shown). The neuroepithelial cells found in the ventricle were positive for active caspase 3 (Fig. 7K,L, dashed line), suggesting anoikis, a form of apoptosis caused by 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 cause Fig. 5. xMIDs associate with and regulate microtubules. (A)Transverse section through the neural plate of a Xenopus embryo the catastrophic collapse of the central nervous system. injected with EGFP-xMID1 mRNA (50 pg) at stage 15.5, and stained with Similar defects in cell morphogenesis were found in the cement anti-GFP and anti--tubulin antibodies. Scale bar: 20m. (B-E)Transverse gland (Fig. 7E,F), where xMID1 is strongly expressed (Fig. 1F,H,I). sections through the neural plate of embryos unilaterally injected with Furthermore, in the pronephros, the epithelial cells failed to adopt a control-Mo (B,C) or xMID-Mo (D,E), and stained for -tubulin (B,D) or columnar shape or exhibit apical actin assembly, and no tubular acetylated tubulin (Ac.-Tubulin) (C,E) antibodies. Flag--globin mRNA structure was formed (Fig. 7G,H). The area in which the pronephros (250 pg) was co-injected as a tracer, and stained with an anti-Flag normally forms was filled with disorganized cell aggregates (Fig. antibody (middle panels). Bottom panels show traced drawings of cells 7H). In the foregut, derivatives of which are affected in OS patients stained by -tubulin and anti-acetylated tubulin antibodies. (F)Western (Fontanella et al., 2008; So et al., 2005), continuous apical actin blots of immunoprecipitates (IP) or lysates from embryos expressing EGFP- tubulin (EGFP-tub.) mRNA (1 ng), detected with anti-GFP and anti- failed to assemble in the endodermal cells, and the cells protruded acetylated tubulin (Acetylated-tub.) antibodies. Immunoprecipitation was apically, which caused a deformity of the luminal structure (Fig. performed using the anti-GFP antibody. (G)Quantification of 7I,J). Taken together, our findings indicate that xMIDs play a immunoprecipitation assay, showing the signal intensities of acetylated fundamental role in the remodeling of multiple epithelial tissues. tubulin from three independent experiments, normalized to those of EGFP-tubulin. Error bars indicate s.e.m. *P<0.01. DISCUSSION Role of microtubule regulation by xMIDs in cell shape changes and maintenance during neural The neuroepithelial cells of the Mig12 morphants and tube closure xMID/Mig12 double morphants resembled those of the xMID Here, we demonstrated that the xMIDs are required for normal morphants (Fig. 6K-M). Consistent with this, the assembly of neural tube closure, a multi-step event that involves neural

apicobasally polarized microtubules was decreased in the double specification, cell proliferation and morphogenetic movements DEVELOPMENT 2336 RESEARCH ARTICLE Development 137 (14)

Fig. 6. Mig12 functions with xMIDs in neural tube closure. (A,B)Dorsal views of Xenopus embryos given bilateral injections of control-Mo (A) or Mig12-Mo (B); anterior is to the top. (C)Average distance between the neural folds of unilaterally injected Mig12 morphants was reduced by the Mo-insensitive Mig12 mRNA (25, 50 pg). (D-H)Synergistic effects of Mig12-Mo and xMID-Mo on neural tube closure. Dorsal views, anterior to the top. Injection of low doses (8.4 ng) of Mig12- Mo (E) or xMID-Mo (F) caused slight delays in neural tube closure. Co-injection of Mig12- Mo and xMID-Mo (8.4 ng each) induced severe NTDs (G,H). (I)Average distance between the neural folds of unilaterally injected Mig-12 morphants was reduced dose-dependently by xMID1 mRNA (50, 100, 250, 500, 1000 pg). (J)Average distance between the neural folds of unilaterally injected xMID morphants was not reduced by Mig12 mRNA (25, 50, 100, 250, 500 pg). The number of embryos examined in C,H,I,J is indicated above each bar. (K-M)Transverse sections through the neural plate of embryos given unilateral injections of control-Mo (K, 8.4 ng), Mig12-Mo (L, 17 ng), or Mig12-Mo and xMID-Mo (M, 8.4 ng each), stained with -tubulin (top) and anti-GFP (middle) antibodies. mRNA (125 pg) encoding membrane-bound EGFP was co-injected as a tracer. Bottom panels show traced drawings of cells stained by anti--tubulin.

(Copp et al., 2003). In particular, a collective cell movement, which primary cilium formation, the impact of xMID knockdown on is based on the morphogenesis of cells in the neural plate, serves microtubules might be limited, affecting only their rearrangement as the major driving force for its invagination (Colas and and assembly along the apicobasal axis. Schoenwolf, 2001; Pilot and Lecuit, 2005; Quintin et al., 2008). In Since the cellular and molecular mechanisms of neural tube xMID morphants, the neuroepithelial cells remained rounded, and closure in amphibians are closely related to those in amniotes the localization of adhesive molecules was perturbed, indicating (Davidson and Keller, 1999), MIDs are probably required for that the epithelial organization was not maintained. The neural tube closure in amniotes, including humans. However, rearrangement and assembly of microtubules was also impaired in NTDs, such as anencephaly and spina bifida, have not been the xMID morphants. The prevention of NTDs in xMID morphants reported in OS patients, although the expression of human MID1 by the expression of another microtubule-associated protein in the developing neural tube has been reported (Pinson et al., suggests that the primary function of xMIDs is to stabilize 2004), and abnormalities of the brain, including agenesis or microtubules. From these data, we propose that the xMIDs regulate hypoplasia of the and corpus callosum, are seen cellular morphogenesis and epithelial organization during neural in OS (Fontanella et al., 2008; So et al., 2005). The overlapping tube closure through the assembly and stabilization of expression of MID1 and MID2 in developing neural tissues microtubules. Since the knockdown of the xMIDs did not cause (Buchner et al., 1999; Granata et al., 2005; Dal Zotto et al., 1998)

any obvious defects in classical microtubule function in mitosis or and the finding that MID1 and MID2 have redundant activities in DEVELOPMENT Microtubule and xMIDs in neurulation RESEARCH ARTICLE 2337

Fig. 7. xMIDs function in the developing brain and other epithelial organogeneses. (A-J)Transverse sections through the brain (A,B), optic vesicle (C,D), cement gland (E,F), pronephros (G,H) and foregut (I,J) of Xenopus embryos injected with control-Mo (A,C,E,G,I) or xMID-Mo (B,D,F,H,J), and stained with phalloidin (green), anti-laminin antibody (magenta), or anti-Flag antibody (gray). (A,B)Insets show stage 40 embryos stained for the pan-neural marker Xen1. Lateral views: anterior to the left, dorsal to the top. Arrowheads indicate head defects in the xMID morphant. (B,D)Arrowheads indicate non- continuous and attenuated laminin staining in the basal lamina. (E,F)Brackets indicate the lengths of Flag-positive cells. (G)Asterisks indicate tubular structures of the pronephros. (I,J)Asterisks indicate luminal structures of the foregut. Arrowheads indicate the lack of apical actin assembly in the xMID morphant cells. (K,L)Transverse sections through the brain of control-Mo-injected (K) and xMID-Mo- injected (L) embryos, stained with anti-active caspase 3 (left) and anti-Flag (right) antibodies. Dashed line delimits the ventricle.

chick left-right determination (Granata et al., 2005), suggest that cytoplasmic localization of Mig12-GFP was not changed by the MID1 and MID2 have redundant functions in neural tube closure gain or loss of xMID function. These data all suggest that, at least such that their role in this process is not unveiled by the in the neuroepithelial cells, the xMIDs are the main players in the knockdown or mutation of only one of them. xMID-Mig12 complex, and Mig12 might be recruited in a limited amount to finely modulate the xMID activities. Furthermore, the xMID-Mig12 collaboration is required for the functional interaction of these proteins might be dynamic and rearrangement and stabilization of microtubules tightly regulated in time and space to avoid overstabilization of in vivo the microtubules, which might lead to defects in cellular Mig12 was identified as a gene encoding a 152 amino acid protein morphogenesis. Since a regulatory subunit of protein phosphatase that is expressed in gastrula-stage zebrafish (Conway, 1995). In 2A (PP2A) binds MID1 and MID2 (Liu et al., 2001; Short et al., Cos7 cells, Mig12 colocalizes with MID1 and stabilizes 2002; Trockenbacher et al., 2001), it is possible that the PP2A microtubules, suggesting that Mig12 might function cooperatively complex is involved in this mechanism. with the xMIDs. In this study, we showed that Mig12 cooperates functionally with the xMIDs in regulating microtubule organization Molecular link between microtubules and apical during neural tube closure. However, all our data support the idea constriction that the xMIDs function as the dominant regulators of neural tube The molecular mechanisms governing cellular morphogenesis in closure. The strongest evidence for this is that the NTDs of Mig12 epithelia are well documented, especially with regard to apical morphants were rescued by xMID expression, whereas those of constriction in Drosophila (Dawes-Hoang et al., 2005; Kolsch et

xMID morphants were not rescued by Mig12. Furthermore, the al., 2007; Nikolaidou and Barrett, 2004; Pilot and Lecuit, 2005; DEVELOPMENT 2338 RESEARCH ARTICLE Development 137 (14)

Quintin et al., 2008). In vertebrates, Shroom3-mediated activation Supplementary material of ROCKs and myosin II plays a crucial role in driving apical Supplementary material for this article is available at constriction (Haigo et al., 2003; Hildebrand, 2005; Nishimura and http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.048769/-/DC1 Takeichi, 2008; Rolo et al., 2009). Our study of the xMIDs raises References the important question of how microtubules control apical Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E. H. and Simons, K. constriction in neuroepithelial cells. In Drosophila, RhoGEF2 (1989). The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J. Cell Biol. 109, 2817-2832. associates with microtubule plus ends in an EB1-dependent manner Bartolini, F. and Gundersen, G. G. (2006). Generation of noncentrosomal (Rogers et al., 2004), and inhibition of microtubule polymerization microtubule arrays. J. Cell Sci. 119, 4155-4163. prevents apical actin assembly and Myosin light chain Becker, B. E. and Gard, D. L. (2006). Visualization of the cytoskeleton in Xenopus phosphorylation, thus blocking apical constriction (Corrigall et al., oocytes and eggs by confocal immunofluorescence microscopy. Methods Mol. Biol. 322, 69-86. 2007). In addition, the enhancement of cadherin-based cell Berti, C., Fontanella, B., Ferrentino, R. and Meroni, G. (2004). Mig12, a novel adhesion is dependent on microtubules in human epithelial cells Opitz syndrome gene product partner, is expressed in the embryonic ventral (Meng et al., 2008). These data suggest that the molecular link midline and co-operates with Mid1 to bundle and stabilize microtubules. BMC Cell Biol. 5, 9. between microtubules and apical constriction is mediated by the Bisgrove, B. W. and Yost, H. J. (2006). The roles of cilia in developmental transport of key regulators of actin polymerization, myosin II disorders and disease. Development 133, 4131-4143. activation, and/or cell-cell contacts. It will therefore be intriguing Brieher, W. M. and Gumbiner, B. M. (1994). Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps. J. Cell Biol. 126, to examine whether intracellular transport is affected in xMID 519-527. morphants. Buchner, G., Montini, E., Andolfi, G., Quaderi, N., Cainarca, S., Messali, S., Bassi, M. T., Ballabio, A., Meroni, G. and Franco, B. (1999). MID2, a Insights into the molecular and pathological homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development. Hum. Mol. mechanisms of Opitz G/BBB syndrome Genet. 8, 1397-1407. We demonstrated that xMID1 is required for the morphogenesis of Burnside, B. (1973). Microtubules and microfilaments in amphibian neurulation. epithelial organs, such as the cement gland, pronephros and Am. Zool. 13, 989-1006. Cainarca, S., Messali, S., Ballabio, A. and Meroni, G. (1999). Functional foregut. Furthermore, in the xMID morphants, the epithelial cell characterization of the Opitz syndrome gene product (midin): evidence for morphology and organization were severely affected, and the homodimerization and association with microtubules throughout the cell cycle. distribution of laminin in the basal lamina was compromised. Hum. Mol. Genet. 8, 1387-1396. These data indicate that the morphogenetic defects in xMID Colas, J. F. and Schoenwolf, G. C. (2001). Towards a cellular and molecular understanding of neurulation. Dev. Dyn. 221, 117-145. morphants are due to the loss of epithelial integrity. Conway, G. (1995). A novel gene expressed during zebrafish gastrulation In OS patients, various developmental abnormalities, including identified by differential RNA display. Mech. Dev. 52, 383-391. craniofacial, urogenital, gastrointestinal and cardiovascular defects Copp, A. J., Greene, N. D. and Murdoch, J. N. (2003). The genetic basis of mammalian neurulation. Nat. Rev. Genet. 4, 784-793. are observed, although the pathological mechanisms have not been Corrigall, D., Walther, R. F., Rodriguez, L., Fichelson, P. and Pichaud, F. (2007). identified (Fontanella et al., 2008; So et al., 2005). Importantly, a Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in recent analysis of the MID1 expression pattern in the human Drosophila epithelia. Dev. Cell 13, 730-742. embryo revealed it to be expressed in various epithelial tissues, Creppe, C., Malinouskaya, L., Volvert, M. L., Gillard, M., Close, P., Malaise, O., Laguesse, S., Cornez, I., Rahmouni, S., Ormenese, S. et al. (2009). including the central nervous system, kidney primordia, and the Elongator controls the migration and differentiation of cortical neurons through pharyngeal, respiratory and gastrointestinal epithelia (Pinson et al., acetylation of alpha-tubulin. Cell 136, 551-564. 2004). In addition, MID1 is expressed in the anal folds and genital Dal Zotto, L., Quaderi, N. A., Elliott, R., Lingerfelter, P. A., Carrel, L., Valsecchi, V., Montini, E., Yen, C. H., Chapman, V., Kalcheva, I. et al. tubercle (Pinson et al., 2004). These expression patterns indicate a (1998). The mouse Mid1 gene: implications for the pathogenesis of Opitz strong correlation between epithelial MID1 expression and the syndrome and the evolution of the mammalian pseudoautosomal region. Hum. development of organs affected by OS. Although no extensive Mol. Genet. 7, 489-499. histological characterizations of tissues from OS patients have been Davidson, L. A. and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and reported, there are notable similarities in the pathological features convergent extension. Development 126, 4547-4556. of OS patients and the epithelial defects of xMID morphants. In Dawes-Hoang, R. E., Parmar, K. M., Christiansen, A. E., Phelps, C. B., Brand, addition, Mid1 shows similar epithelial expression patterns in A. H. and Wieschaus, E. F. (2005). folded gastrulation, cell shape change and the control of myosin localization. Development 132, 4165-4178. mouse (Dal Zotto et al., 1998) and chick (Richman et al., 2002). Fagotto, F. and Gumbiner, B. M. (1994). Beta-catenin localization during We propose that common mechanisms underlie the normal Xenopus embryogenesis: accumulation at tissue and somite boundaries. morphogenesis of the organs affected in OS patients and in the Development 120, 3667-3679. Xenopus embryos in this study. Taken altogether, our findings Fontanella, B., Russolillo, G. and Meroni, G. (2008). MID1 mutations in patients with X-linked Opitz G/BBB syndrome. Hum. Mutat. 29, 584-594. demonstrate the general importance of microtubule regulation by Frisch, S. M. and Screaton, R. A. (2001). Anoikis mechanisms. Curr. Opin. Cell MID1 and MID2 in cell shape change and maintenance in Biol. 13, 555-562. epithelial morphogenesis during vertebrate embryogenesis. Goda, T., Takagi, C. and Ueno, N. (2009). Xenopus Rnd1 and Rnd3 GTP-binding proteins are expressed under the control of segmentation clock and required for somite formation. Dev. Dyn. 238, 2867-2876. Acknowledgements Granata, A. and Quaderi, N. A. (2003). The Opitz syndrome gene MID1 is We thank T. C. Cox, K. S. Kosik, K. Nakayama, T. Okubo, T. Takabatake, K. essential for establishing asymmetric in Hensen’s node. Dev. Tamura and J. B. Wallingford for plasmids and reagents; and members of the Biol. 258, 397-405. N.U. laboratory and S. Nonaka laboratory for valuable discussions, comments Granata, A., Savery, D., Hazan, J., Cheung, B. M., Lumsden, A. and Quaderi, and technical assistance. This work was supported by KAKENHI (07J05064, N. A. (2005). Evidence of functional redundancy between MID proteins: 21870043 to M.S.; 17207015, 21370102 to N.U.) from the Japan Society for implications for the presentation of Opitz syndrome. Dev. Biol. 277, 417-424. the Promotion of Science (JSPS). M.S. and Y.H. were supported by JSPS Haigo, S. L., Hildebrand, J. D., Harland, R. M. and Wallingford, J. B. (2003). Research Fellowships for Young Scientists. Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr. Biol. 13, 2125-2137. Competing interests statement Handel, M. A. and Roth, L. E. (1971). Cell shape and morphology of the neural

The authors declare no competing financial interests. tube: implications for microtubule function. Dev. Biol. 25, 78-95. DEVELOPMENT Microtubule and xMIDs in neurulation RESEARCH ARTICLE 2339

Hayes, J. M., Kim, S. K., Abitua, P. B., Park, T. J., Herrington, E. R., Kitayama, Quaderi, N. A., Schweiger, S., Gaudenz, K., Franco, B., Rugarli, E. I., Berger, A., Grow, M. W., Ueno, N. and Wallingford, J. B. (2007). Identification of W., Feldman, G. J., Volta, M., Andolfi, G., Gilgenkrantz, S. et al. (1997). novel ciliogenesis factors using a new in vivo model for mucociliary epithelial Opitz G/BBB syndrome, a defect of midline development, is due to mutations in development. Dev. Biol. 312, 115-130. a new RING finger gene on Xp22. Nat. Genet. 17, 285-291. Hemmati-Brivanlou, A., Kelly, O. G. and Melton, D. A. (1994). Follistatin, an Quintin, S., Gally, C. and Labouesse, M. (2008). Epithelial morphogenesis in antagonist of activin, is expressed in the Spemann organizer and displays direct embryos: asymmetries, motors and brakes. Trends Genet. 24, 221-230. neuralizing activity. Cell 77, 283-295. Richman, J. M., Fu, K. K., Cox, L. L., Sibbons, J. P. and Cox, T. C. (2002). Hildebrand, J. D. (2005). Shroom regulates epithelial cell shape via the apical Isolation and characterisation of the chick orthologue of the Opitz syndrome positioning of an actomyosin network. J. Cell Sci. 118, 5191-5203. gene, Mid1, supports a conserved role in vertebrate development. Int. J. Dev. Itoh, M., Nagafuchi, A., Yonemura, S., Kitani-Yasuda, T. and Tsukita, S. Biol. 46, 441-448. (1993). The 220-kD protein colocalizing with cadherins in non-epithelial cells is Robin, N. H., Feldman, G. J., Aronson, A. L., Mitchell, H. F., Weksberg, R., identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA Leonard, C. O., Burton, B. K., Josephson, K. D., Laxova, R., Aleck, K. A. et cloning and immunoelectron microscopy. J. Cell Biol. 121, 491-502. al. (1995). Opitz syndrome is genetically heterogeneous, with one locus on Kanai, Y., Chen, J. and Hirokawa, N. (1992). Microtubule bundling by tau Xp22, and a second locus on 22q11.2. Nat. Genet. 11, 459-461. proteins in vivo: analysis of functional domains. EMBO J. 11, 3953-3961. Rogers, S. L., Wiedemann, U., Hacker, U., Turck, C. and Vale, R. D. (2004). Karfunkel, P. (1971). The role of microtubules and microfilaments in neurulation Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent in Xenopus. Dev. Biol. 25, 30-56. manner. Curr. Biol. 14, 1827-1833. Kinoshita, N., Sasai, N., Misaki, K. and Yonemura, S. (2008). Apical Rolo, A., Skoglund, P. and Keller, R. (2009). Morphogenetic movements driving accumulation of Rho in the neural plate is important for neural plate cell shape neural tube closure in Xenopus require myosin IIB. Dev. Biol. 327, 327-338. change and neural tube formation. Mol. Biol. Cell 19, 2289-2299. Sater, A. K., Steinhardt, R. A. and Keller, R. (1993). Induction of neuronal Kintner, C. R. and Melton, D. A. (1987). Expression of Xenopus N-CAM RNA in differentiation by planar signals in Xenopus embryos. Dev. Dyn. 197, 268-280. ectoderm is an early response to neural induction. Development 99, 311-325. Schoenwolf, G. C. and Franks, M. V. (1984). Quantitative analyses of changes in Kolsch, V., Seher, T., Fernandez-Ballester, G. J., Serrano, L. and Leptin, M. cell shapes during bending of the avian neural plate. Dev. Biol. 105, 257-272. (2007). Control of Drosophila gastrulation by apical localization of adherens Schroeder, T. E. (1970). Neurulation in Xenopus laevis. An analysis and model junctions and RhoGEF2. Science 315, 384-386. based upon light and electron microscopy. J. Embryol. Exp. Morphol. 23, 427- Lee, C., Scherr, H. M. and Wallingford, J. B. (2007). Shroom family proteins 462. regulate gamma-tubulin distribution and microtubule architecture during Schweiger, S., Foerster, J., Lehmann, T., Suckow, V., Muller, Y. A., Walter, G., epithelial cell shape change. Development 134, 1431-1441. Davies, T., Porter, H., van Bokhoven, H., Lunt, P. W. et al. (1999). The Opitz Levine, E., Lee, C. H., Kintner, C. and Gumbiner, B. M. (1994). Selective syndrome gene product, MID1, associates with microtubules. Proc. Natl. Acad. disruption of E-cadherin function in early Xenopus embryos by a dominant Sci. USA 96, 2794-2799. negative mutant. Development 120, 901-909. Short, K. M. and Cox, T. C. (2006). Subclassification of the RBCC/TRIM Liu, J., Prickett, T. D., Elliott, E., Meroni, G. and Brautigan, D. L. (2001). superfamily reveals a novel motif necessary for microtubule binding. J. Biol. Phosphorylation and microtubule association of the Opitz syndrome protein mid- Chem. 281, 8970-8980. 1 is regulated by protein phosphatase 2A via binding to the regulatory subunit Short, K. M., Hopwood, B., Yi, Z. and Cox, T. C. (2002). MID1 and MID2 homo- alpha 4. Proc. Natl. Acad. Sci. USA 98, 6650-6655. and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Lu, M. and Kosik, K. S. (2001). Competition for microtubule-binding with dual alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz expression of tau missense and splice isoforms. Mol. Biol. Cell 12, 171-184. GBBB syndrome and other developmental disorders. BMC Cell Biol. 3, 1. Meng, W., Mushika, Y., Ichii, T. and Takeichi, M. (2008). Anchorage of So, J., Suckow, V., Kijas, Z., Kalscheuer, V., Moser, B., Winter, J., Baars, M., microtubule minus ends to adherens junctions regulates epithelial cell-cell Firth, H., Lunt, P., Hamel, B. et al. (2005). Mild phenotypes in a series of contacts. Cell 135, 948-959. patients with Opitz GBBB syndrome with MID1 mutations. Am. J. Med. Genet. A Miner, J. H., Cunningham, J. and Sanes, J. R. (1998). Roles for laminin in 132A, 1-7. embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the Suzuki, M., Satoh, A., Ide, H. and Tamura, K. (2007). Transgenic Xenopus with laminin alpha5 chain. J. Cell Biol. 143, 1713-1723. prx1 limb enhancer reveals crucial contribution of MEK/ERK and PI3K/AKT Morita, H., Nandadasa, S., Yamamoto, T. S., Terasaka-Iioka, C., Wylie, C. and pathways in blastema formation during limb regeneration. Dev. Biol. 304, 675- Ueno, N. (2010). Nectin-2 and N-cadherin interact through extracellular 686. domains and induce apical accumulation of F-actin in apical constriction of Takabatake, T., Takahashi, T. C., Takabatake, Y., Yamada, K., Ogawa, M. and Xenopus neural tube morphogenesis. Development 137, 1315-1325. Takeshima, K. (2000). Distinct expression of two types of Xenopus Patched Nandadasa, S., Tao, Q., Menon, N. R., Heasman, J. and Wylie, C. (2009). N- genes during early embryogenesis and hindlimb development. Mech. Dev. 98, and E-cadherins in Xenopus are specifically required in the neural and non- 99-104. neural ectoderm, respectively, for F-actin assembly and morphogenetic Takabatake, Y., Takabatake, T., Sasagawa, S. and Takeshima, K. (2002). movements. Development 136, 1327-1338. Conserved expression control and shared activity between cognate T-box genes Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis (Daudin). Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus Amsterdam: North-Holland Publishing Company. eye development. Dev. Growth Differ. 44, 257-271. Nikolaidou, K. K. and Barrett, K. (2004). A Rho GTPase signaling pathway is Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007). MEGA4: Molecular used reiteratively in epithelial folding and potentially selects the outcome of Rho Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, activation. Curr. Biol. 14, 1822-1826. 1596-1599. Nishimura, T. and Takeichi, M. (2008). Shroom3-mediated recruitment of Rho Trockenbacher, A., Suckow, V., Foerster, J., Winter, J., Krauss, S., Ropers, H. kinases to the apical cell junctions regulates epithelial and neuroepithelial planar H., Schneider, R. and Schweiger, S. (2001). MID1, mutated in Opitz remodeling. Development 135, 1493-1502. syndrome, encodes an ligase that targets phosphatase 2A for Ohkawara, B., Yamamoto, T. S., Tada, M. and Ueno, N. (2003). Role of degradation. Nat. Genet. 29, 287-294. glypican 4 in the regulation of convergent extension movements during Verhey, K. J. and Gaertig, J. (2007). The tubulin code. Cell Cycle 6, 2152-2160. gastrulation in Xenopus laevis. Development 130, 2129-2138. Yakushiji, N., Suzuki, M., Satoh, A., Sagai, T., Shiroishi, T., Kobayashi, H., Pilot, F. and Lecuit, T. (2005). Compartmentalized morphogenesis in epithelia: Sasaki, H., Ide, H. and Tamura, K. (2007). Correlation between Shh expression from cell to tissue shape. Dev. Dyn. 232, 685-694. and DNA methylation status of the limb-specific Shh enhancer region during Pinson, L., Auge, J., Audollent, S., Mattei, G., Etchevers, H., Gigarel, N., limb regeneration in amphibians. Dev. Biol. 312, 171-182. Razavi, F., Lacombe, D., Odent, S., Le Merrer, M. et al. (2004). Embryonic Ybot-Gonzalez, P., Cogram, P., Gerrelli, D. and Copp, A. J. (2002). Sonic expression of the human MID1 gene and its mutations in Opitz syndrome. J. hedgehog and the molecular regulation of mouse neural tube closure. Med. Genet. 41, 381-386. Development 129, 2507-2517. DEVELOPMENT