A novel gene, BENI is required for the convergent ... · A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation Motohiro Homma a, Masafumi

03 (2007) 270–280www.elsevier.com/locate/ydbio

Developmental Biology 3

A novel gene, BENI is required for the convergent extensionduring Xenopus laevis gastrulation

Motohiro Homma a, Masafumi Inui a, Akimasa Fukui a,1, Tatsuo Michiue a,b,Koji Okabayashi c, Makoto Asashima a,b,c,⁎

a Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japanb AIST (Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi,

Tsukuba, Ibaraki 305-8566, Japanc ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan

Received for publication 29 November 2005; revised 31 October 2006; accepted 7 November 2006Available online 11 November 2006

Abstract

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-β family protein, induces mesodermal/endodermaltissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI(Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal(NLS), and two other NLSs in the C-terminal domain. BENI mRNAwas localized to the animal hemisphere at the gastrula stages and to ectodermexcept for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects andinhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealedreduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholinooligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animalcaps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, andthat this regulation is crucial for Xbra expression.© 2006 Elsevier Inc. All rights reserved.

Keywords: Xenopus laevis; Convergent extension; Activin-like signaling; BENI; Xbra; Nuclear localization signal (NLS)

Introduction

Gastrulation movements are complex movements of cellsand tissues that are regulated temporally and spatially toestablish the three germ layers. In Xenopus, gastrulationmovements are region-specific: epiboly of the animal hemi-sphere (Keller, 1980), involution of the involuting marginalzone (IMZ) (Hardin and Keller, 1988), vegetal rotation(Winklbauer and Schürfeld, 1999), and convergent extensionfollowed by involution (Keller, 1986; Keller and Danilchik,

⁎ Corresponding author. Department of Life Sciences (Biology), Graduateschool of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku,Tokyo 153-8902, Japan. Fax: +81 3 5454 4330.

E-mail address: [email protected] (M. Asashima).1 Present address: Division of Biological Sciences, Graduate School of

Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2006.11.014

1988). In convergent extension during gastrulation, severallayers of cells intercalate along the radius of the embryo (radialintercalation) to produce fewer layers and longer arrange-ment of the cells (medio-lateral intercalation) (Wilson et al.,1989; Keller and Tibbetts, 1989; Wilson and Keller, 1991;Keller et al., 2000).

Regulation of the convergent extension movement ingastrulation involves three Wnt gene family member: Wnt-4,Wnt-5a, and Wnt-11 (Du et al., 1995). Wnt-5a and Wnt-11 areexpressed maternally and during early development of X. laevisand can activate the non-canonical Wnt/JNK pathway and Wnt/Ca2+ pathway following PKC/Cdc42 activation to regulategastrulation (reviewed in Kühl, 2002; Tada et al., 2002, Shi,2003). Wnt-11 (Ku and Melton, 1993) is a direct target of Xbra(Saka et al., 2000), and when expression of Xbra was inducedby activin in vitro using animal caps, stable activation occurred

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271M. Homma et al. / Developmental Biology 303 (2007) 270–280

only within a narrow range of activin concentrations (Gurdon etal., 1994). Recently, Ninomiya et al. (2004) suggested that agraded activin-induced antero-posterior polarity is required forconvergent extension in vitro. However, the detailed molecularbasis for control mechanisms of Xbra expression in response toactivin remains unclear.

Here we report the isolation of BENI, a novel moleculeinvolved in the regulation of convergent extension in gas-trulation. BENI was identified from a microarray screen ofactivin-responsive genes (Fukui et al., unpublished). BENIhas three predicted nuclear transport signal regions andlocalizes to the nucleus. Both overexpression and depletion ofBENI cause defects of convergent extension in gastrulationvia regulation of Xbra expression. Since cell differentiationoccurs normally, the effects of BENI appear specific to cellmigration.

Materials and methods

Eggs and embryos

Xenopus eggs were obtained by injecting adult females with 300 U of humanchorionic gonadotropin (Gestron; Kawasaki-Mitaka Seiyaku, Japan); they werethen fertilized in vitro. Staging of embryos was according to Nieuwkoop andFaber (1956). The jelly coat was removed with Steinberg's solution containing4% cysteine hydrochloride (pH 7.8).

RT-PCR

Total RNA (500 ng) isolated from embryos was used to generate first-strandcDNA. Primers used in this study were as follows: BENI (forward; 5′-CGGGATCCCGAGCTGTTGATCCCTGTGC-3′ and reverse; 5′-CATTTT-TAAGATCTAGATTCTCC-3′), ornithine decarboxylase (ODC) (forward;5′-GAACAGCTTCAGCAATGACG-3′ and reverse; 5′-GGAGAGAGTCTT-CACAATGGC-3′).

Whole-mount in situ hybridization

Whole-mount in situ hybridization was carried out as described elsewhere(Harland, 1991). Digoxigenin (DIG)-labeled RNA probe was synthesizedusing T3 or SP6 polymerase and a DIG RNA Labeling Mix, 10× conc.(Roche, Germany). The probe was then digested into approximately 200-bpfragments. The collected embryos were fixed with 1× MEMFA (0.1 MMOPS, 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde, pH 7.4) for2 h. The probe was hybridized to a developmental series of albino Xenopusembryos. The color reaction was performed using BM purple (Roche,Germany).

Histological examination

For X-gal staining, embryos were fixed in MEMFA for 1 h at 4°C, washed inPBS, and incubated in X-gal buffer (1 mg/ml X-gal, 10 mMK3Fe(CN6), 10 mMK4Fe(CN6), 1 mM MgCl2, and 0.1% Tween-20 in PBS) at 37°C. Forhistological examination, embryos were fixed in Bouin's solution for 3 h. Theywere then dehydrated through a graded ethanol series, transferred to xylene forembedding in paraffin, and sectioned at 10–20 μm.

Construction of expression vector and microinjections

The open reading frame of BENI was PCR amplified from the cloneMXL1736-8950048 of Xenopus laevis IMAGE cDNA clones (Open Biosys-tems, USA) using the primers, 5′-CGGGATCCCGATGTCTCTTATGAAG-

GAACG-3′ or 5′-CGGGATCCCGAGCTGTTGATCCCTGTGC-3′ to produceBENI mRNA with or without BENI-MO target sequence, respectively, and 5′-CCATCGATGGGGCTGATACCCTGGGTCTTTTTCGAAGGG-3′. Each PCRproduct was digested with BamHI and ClaI and cloned in-frame into pCS2-GFP(pCS2-BENI-EGFP and pCS2-BENImut-EGFP). To construct NLS mutants, theprimers, 5′-CGGGATCCCGATGCCTCATTACATTCCCCGGCCACCAGG-3′ and 5′-CCATCGATGGAGTTGAGCTTTGAGATTTCCCAAGTTCC-3'were used. The IMAGE cDNA clone and the constructs were linearized byNotI and transcribed with the mMESSAGE mMACHINE SP6 Kit (Ambion,Texas, USA). Unless otherwise noted, 1 ng of mRNA was injected intoblastomeres of 4-cell stage embryos. When necessary, the mRNAwas coinjectedwith 400 pg of lacZ mRNA, as a lineage tracer.

Morpholino oligonucleotide

Morpholino oligonucleotides (MO) were designed and supplied by GeneTools, LLC (Oregon, USA). MOs used in this study were as follows: BENI-MO(5′-TTCTTGTATTTTCAGCACAGGGATC-3′), Control-MO (5′-CCTC-TTACCTCAGTTACAATTTATA-3′). TSC-22-MO was designed as describedpreviously (Hashiguchi et al., 2004).

Animal cap assay

For the animal cap assays, mRNA andMOwere injected into animal poles of 4-cell stage embryos. Animal caps were dissected at stage 8 and cultured in 0.1%BSA(Sigma) in 1× Steinberg's solution containing 25 ng/ml recombinant human activinA, which was prepared as described previously (Eto et al., 1987). The explantswere photographed at stages 18–20 and harvested for RT-PCR. The primersequences used were as previously described (Fukui et al., 2000; Myoishi etal., 2004) and as follows: Mix1, 5′-AAGGTCAGACGTCAAGGTGC-3′ and5′-AGACATTGGATGATGCCTGG-3′; Endodermin, 5′-TCGCAGATGCTCT-TACTTGG-3′ and 5′-TAGATGCTGTTCTGGCCTCC-3′.

Results

Sequence analysis of X. laevis BENI

BENI was registered in GenBank (Accession NumberBC043737) and encodes the hypothetical protein, MGC52859(Klein et al., 2002; Strausberg et al., 2002). BENI was isolatedfrom a X. laevis IMAGE cDNA clone library (Open bio-systems, USA) from stage 17–19 embryos. The ORF was2025 bp with a predicted sequence of 674 amino acids. Theonly functional motifs or domains revealed by sequenceanalysis were three predicted nuclear localization signals(NLS) (Figs. 1A, B). These NLSs comprise one bipartitesignal (Hicks and Raikhel, 1995) at amino acids 654–670 andtwo previously classified pat-4 signals (Zheng et al., 2004):one at amino acids 7–11 (RKPKK), including two overlappingpat-4 signals (RKPK and KPKK), and another at amino acids668–672 (RKRPR), including two overlapping pat-4 signals(RKRP and KRPR). Each pat-4 signal includes two overlappingpat-4 NLSs (RKPK and KPKK). A BLASTP search of theBENI amino acid sequence revealed homologues in X. tro-picalis (Protein Accession Number: AAH75584), H. sapiens(AAH16613, AAI61234%20, and BAB14718), M. musculus(BAC27080 and BAC29771), R. norvegicus (XP_220257),and T. nigroviridis (CAG01099 and CAG04344) (Fig. 1C).These homologues have more than 57% identity with ahighly conserved 58-amino acid N-terminal region in BENI(Fig. 1D).

272 M. Homma et al. / Developmental Biology 303 (2007) 270–280

Temporal and spatial distribution of BENI during development

The temporal expression patterns of BENI during Xenopusembryogenesis were analyzed by RT-PCR (Fig. 2A). The genewas expressed maternally and at low levels until stage 8.Expression of BENI then increased markedly up to stage 10,after which the level of expression remained constant.

The spatial expression pattern of BENI was examined bywhole-mount in situ hybridization. BENI mRNAwas localizedto the ectoderm from stage 11 (Figs. 2B–D) to stage 42 (Figs.2L, M). At stage 11, the mRNA was expressed in ectodermlayers, particularly around the animal pole region and modestly

Fig. 1. Amino acid sequence alignments of the both the N-terminal region (A) and C-and bipartite predicted nuclear localization signals (NLS) are boxed in red and yellowamino acid sequence (C) and in the N-terminal region (1–58 aa of BENI) (D).

around the yolk plug (Figs. 2B–D). At the neurula and tailbudstages, BENI mRNAwas localized in epidermal cells, but not inthe neural region, and expression in the cement gland was alsosignificant (Figs. 2E–K). Transverse sections along the dor-soventral axis of embryos from stage 10.5 showed a gradientof BENI mRNA expression in the epidermal layer, with highexpression in the animal pole region and low expression inthe marginal zone (Figs. 2N–Q). In the marginal zone, Xbraalso showed a gradient pattern of expression, with high expres-sion in the inner layer and low expression in the outer layer(Figs. 2R, S). This implied a reciprocal expression of Xbra andBENI in the marginal zone.

terminal region (B) of BENI with related proteins obtained from GenBank. Pat-4, respectively. Percent amino acid identities of BENI homologues in the whole


Disruption of BENI leads to abnormal gastrulation movements

To investigate the function of BENI in Xenopusdevelopment, we conducted a loss-of-function experimentusing an antisense morpholino oligonucleotide (MO). Thisapproach depends on the MO blocking translation of theBENI mRNA (Summerton and Weller, 1997; Heasman,2002). To test the specificity of the BENI-MO, weconstructed pCS2-BENI-EGFP and pCS2-BENImut-EGFP,the latter of which contains BENI mRNA without the

Fig. 2. Temporal and spatial expression pattern of BENI during Xenopus developmentby RT-PCR. RT-PCR using an ornithine decarboxylase (ODC)-specific primer was careverse transcriptase showed no contamination of genomicDNA [ODC(RT−)]. (B–Q)in situ hybridization. Embryos were hybridized with antisense (C, D, F, G, I, K, M–Pvegetal view. (E, F) Stage 17, lateral view. (G) Stage 17, anterior view. (H, I) Stage 22,Transverse section of the embryo at stage 10.5. Panels P and Q are magnified views

BENI-MO target sequence. When BENI-MO was co-injected with either BENI-EGFP or BENImut-EGFPmRNA into the animal pole of 4-cell embryos, translationof BENI was inhibited in the presence of BENI-EGFP butnot with BENImut-EGFP (Figs. 3A, B). This resultconfirmed the efficiency of BENI-MO.

BENI-MO was next injected into both dorsoanimal blas-tomeres of 4-cell embryos. The BENI-MO-injected embryosshowed a gastrulation defective phenotype (Fig. 3C), suggest-ing that BENI is required for the proper morphogenetic

. (A) Temporal expression pattern of BENI during Xenopus development detectedrried out in parallel to control the amount of input RNA [ODC]. RT-PCR withoutSpatial expression pattern of BENI during development detected bywhole-mount, Q) and sense probes (B, E, H, J, L). (B, C) Stage 11, animal view. (D) Stage 11,lateral view. (J, K) Stage 29/30, lateral view. (L, M) Stage 40, lateral view. (N–S)of panels N and O, respectively. Xbra expression (R–S). Scale bars, 100 μm.


movement of the cells from the dorsal blastomere duringgastrulation.

Gastrulation defect caused by BENI mRNA

To further examine BENI function in Xenopus develop-ment, we examined the effects of overexpression andtranslational inhibition on involution and thinning (radialintercalation). BENI mRNA (1 ng) or BENI-MO (34 ng) wasinjected into both dorsoanimal blastomeres of 4-cell embryos.Embryos showed no obvious delay at stage 10 (Fig. 4A), buta more detailed examination of the deep layers of mRNA-injected embryos revealed a delayed thinning of theinvoluting marginal zone (IMZ). The MO-injected embryosshowed no difference in dorsal IMZ from the control,uninjected embryos (Figs. 4A, B). This result suggested that

Fig. 3. Activity of BENI antisense morpholino oligonucleotide (BENI-MO). (A) BE(4 pmol=34 ng) in the animal pole of Xenopus embryos. The embryos were raisedmicroscope (A) and animal caps of the embryos were harvested for SDS-PAGE anawith anti-GFP and anti-α-tubulin (B). (C) Injection of BENI-MO (4 pmol=34 ng) at bstage 37/38. Scale bars, 1 mm.

BENI is involved in thinning during the first half ofgastrulation.

We investigated whether the effect of overexpression ofBENI is specific for the dorsal marginal zone. The BENImRNA was injected into both ventroanimal blastomeres of4-cell embryos. The mRNA-injected embryos at stage 10showed a thick ventral IMZ (Figs. 4C, D), suggesting thatBENI is involved in thinning of both the dorsal and ventralmarginal zones. Furthermore, we examined the effect ofBENI on epiboly. BENI MOs were injected into animal poleregions of 2-cell embryos and β-gal tracer was injected oneB1 blastomere of 32-cell embryos to visualize the cellmovement. The red-gal-positive cells in control embryosappeared as a narrow stream extending from the animal poleto the blastopore, whereas the cells in embryos injected withTSC-22MO, as a positive control, appeared as a broad body,

NI-EGFP (300 ng) or BENImut-EGFP (300 ng) was coinjected with BENI-MOuntil stage 9 and the EGFP was visualized and observed under a fluorescent

lysis. The animal cap lysates were subjected to SDS-PAGE and immunoblottedoth dorsal animal blastomeres of 4-cell embryos. The embryos were observed at


indicating abnormal epiboly (Fig. 4E; Hashiguchi et al.,2004). The BENI MO-injected embryos exhibited a similarphenotype to control embryos rather than to the TSC-22 MO-injected embryos (Fig. 4E). This result suggested that BENI isnot involved in involution.

Fig. 4. Gastrulation defect caused by BENI mRNA. (A) lacZ mRNA (400 pg), BENIwere injected into both dorsoanimal blastomeres of 4-cell embryos. Vegetal view of emEmbryos were histologically examined and observed under diffuse light (middle panmiddle panels (bottom panels). Double-headed arrow indicates width of dorsal IMZ. YIMZ was measured. (C) lacZ mRNA or BENI mRNA and lacZ mRNAwas injected istage 10 (top panels). Highly magnified ventral IMZ of top panels (bottom panels). Doof ventral IMZ was measured. (E) lacZ mRNA (control, upper column), lacZ mRNAcolumn) were injected into dorsoanimal blastomeres of 4-cell embryos. At stage 13,

BENI is required for proper elongation of animal caps

To examine the possible role of BENI in convergentextension during gastrulation, we next performed animal capassays. Animal caps treated with mesodermal inducer exhibited

mRNA (1 ng) and lacZ mRNA, or BENI-MO (4 pmol=34 ng) and lacZ mRNAbryos at stage 10 (top panels). There was no discernible delaying of gastrulation.els). High magnification view of dorsal involuting marginal zone (IMZ) of theellow arrowheads indicate the dorsal lip. Scale bar, 200 μm. (B) Width of dorsalnto both ventroanimal blastomeres of 4-cell embryos. Embryos were bisected atuble-headed arrow indicates width of ventral IMZ. Scale bar, 200 μm. (D) Widthand BENI MO (middle column), and lacZ mRNA and TSC-22 mRNA (lower

embryos were stained with Red-gal and the staining pattern observed.


morphogenic elongation characteristic of gastrulation, and theyexpressed various marker genes (Symes and Smith, 1987;Tamai et al., 1999). Injection of BENImRNA severely inhibitedactivin-induced elongation of the animal caps, whereasinjection of BENI-MO increased elongation compared touninjected animal caps (Fig. 5A). These results implicatedBENI in medio-lateral intercalation. To investigate whether

Fig. 5. BENI is involved in gastrulation movements and affects the expression of XbBENI mRNA inhibits elongation of animal cap explants in response to activin. Fo(4 pmol=34 ng). Animal caps from uninjected or injected embryos were left untreateround. (B) BENI regulates the expression of Xbra in vitro. Four-cell embryos were ininjected embryos were left untreated or treated with 25 ng/ml activin and culturedmarker), goosecoid, and Chordin (dorsal mesodermal marker), Xbra (pan-mesoderobserved in the absence of reverse transcriptase [RT(−)]. (D) Neither BENI mRNA nanalyzed by RT-PCR. NCAM (neural marker), Hox B9 (spinal chord marker) typeendodermin (endoderm marker) were examined. ODC served as a loading controlBENI mRNA reduced the expression of Xbra in vivo. Spatial expression patternmRNA (300 pg) or the BENI-MO (4 pmol=34 ng), together with lacZ mRNA (300 pactivity was visualized with Red-gal substrate as a tracer. Embryos were viewed fro

BENI was affecting the responsiveness of animal caps toactivin, we examined the expression of mesoderm markers. TheBENI-overexpressing animal caps showed no expression of thepan-mesodermal marker, Xbra, whereas other marker genes(Mix.1, goosecoid, and Chordin) were normally expressed(Fig. 5B). The BENI-MO-injected animal caps showed anormal gene expression pattern (Fig. 5B). In addition, we

ra both in vitro and in vivo, but does not modulate cell differentiation. (A) Theur-cell embryos were injected with the BENI mRNA (1 ng) or the BENI-MOd or treated with 25 ng/ml activin and cultured for 12 h. Control caps remainedjected with the BENI mRNA or the BENI-MO. Animal caps from uninjected orfor 3 h. The animal caps were analyzed by RT-PCR. Mix. 1 (mesoendodermalmal marker) were examined. ODC served as a loading control. No signal wasor BENI-MO altered the expression of the marker genes. The animal caps wereII collagen (notochord marker), muscle actin (m-actin) (muscle marker), and. No signal was observed in the absence of reverse transcriptase [RT(−)]. (E)of Xbra at stage 10.5 detected by whole-mount in situ hybridization. BENIg), was injected at the 4-cell stage into both dorsal blastomeres. β-galactosidasem the vegetal side.

Fig. 6. The C-terminal NLS is predominant in the subcellular localization of BENI. (A) Schematic structure of BENI and its derivatives lacking the N-terminal NLS(N-NLS) including a Pat-4 signal (red) in the conserved region (gray), and C-terminal NLS regions (C-NLS) including a Pat-4 and bipartite signal (yellow). (B) Thesubcellular localization of BENI and its derivatives. BENI-EGFP and its NLS deletion mutants mRNA (330 ng) or EGFPmRNA as control was injected in the animalpole of Xenopus embryos. The embryos were raised until stage 9 and the EGFP was visualized and observed under a fluorescent microscope. Scale bar, 100 μm. (C)Expression of BENI and its derivatives. Animal caps harvested from panel B were lysed and subjected to SDS-PAGE for immunoblotting with anti-GFP and anti-α-tubulin. (D, E) Injection of BENI-EGFP and its NLS deletion mutants mRNA at both dorsal animal blastomeres of 4-cell embryos. The embryos were observed atstage 24. Scale bar, 1 mm.



examined the expression of various molecular markers to assessany effect of BENI on cell differentiation, and found no evidentdifference in expression of these markers between the BENImRNA-injected, BENI-MO-injected, and activin-treated animalcaps (Fig. 5C). This result suggests that BENI plays no role inmesoderm induction. Taken together, these results implicate acrucial role for BENI in regulating convergent extension duringgastrulation but not in the specification of cell fate.

We next asked whether BENI is involved in the regulation ofXbra expression in vivo. To address this, we examined thespatial expression pattern of Xbra by whole-mount in situhybridization in the presence of BENI mRNA or BENI-MO. Atstage 10.5, the BENImRNA-injected embryos showed inhibitedexpression of the Xbra mRNA, which was localized around theblastopore (Fig. 5D). It should be noted that the expressionpattern of Xbra did not change in the BENI-MO-injectedembryos.

BENI is localized to nuclei

The BENI protein has three predicted nuclear localizationsignal regions: one in the N-terminal conserved region, and theothers in the C-terminal pat-4 and bipartite signal sequence. Toinvestigate the subcellular localization of BENI, we constructedseveral NLS mutants (Fig. 6A), tagged with GFP. Immunoblot-ting with anti-GFP antibody revealed that all mutants showed thesame level of expression (Fig. 6C). In the cells expressing EGFPand BENI-ΔNLS-EGFP, GFP signal was distributed over theentire cell, whereas the cells expressing BENI-EGFP showedfluorescence signal predominantly at the cell nuclei (Fig. 6B).Interestingly, while the N-terminal deletion construct, BENI-ΔN-NLS-EGFP, showed nuclear localization, the C-terminaldeletion construct, BENI-ΔC-NLS-EGFP, showed a pattern ofexpression similar to that of EGFP and BENI-ΔNLS-EGFP(Fig. 6B). This result implicated the C-terminal NLS in thenuclear localization of BENI.

We next examined the effects of the overexpression of theNLS mutants on the embryo phenotype. Overexpression ofBENI-EGFP and BENI-ΔN-NLS-EGFP caused an abnormalcurled phenotype, whereas BENI-ΔNLS-EGFP overexpressionhad no significant effect (Figs. 6D, E). This result is functionallyconsistent with the localization of BENI to the nucleus.Interestingly, BENI-ΔC-NLS-EGFP showed a slight effect onthe embryo phenotype. Therefore, the C-terminus NLS may benecessary but not sufficient for BENI localization, and the NLSmotif at the N-terminus might act as a secondary step tostrengthen the localization. The abnormal phenotype inducedby BENI-EGFP was not rescued by an increasing dose ofBENI-ΔNLS-EGFP (Fig. 6D).

Discussion

This study reports a novel activin-responsive gene, BENI,which is involved in the convergent extension during gastrula-tion in Xenopus embryos. In vivo and in vitro experimentsrevealed a role for BENI in regulating the expression of Xbraduring gastrulation.

BENI homologues in vertebrates

The highly conserved N-terminal region in BENI that sharesidentity of greater than 57% with proteins from several otherspecies suggested that these proteins have an importantfunction. However, the function of this conserved domaincould not be predicted from its sequence. A SOSUI search(Hirokawa et al., 1998) of the BENI amino acid sequencerevealed no transmembrane region and predicted BENI to be asoluble protein.

Searches in the BLAST database for protein sequences(BLASTP) revealed one Xenopus tropicalis, three human,two murine, one rat, and two green puffer nonidenticalputative proteins with significant similarity to the N-terminalregion of BENI. Alignment of the predicted amino acidsequences of these proteins (referred to below as the BENIprotein family) reveals a sequence similarity, includingseveral invariant residues among members of the family(Fig. 1). In addition, mammal homologues (H. sapiensBAB14718, M. musculus BAC27080 and BAC29771)demonstrate a broad resemblance to BENI in addition totheir sequence similarities. All BENI family members havepredicted NLS motifs, and the localization of BENI-GFP tothe nucleus implicates a novel nuclear protein family. Itshould be noted that the BLASTP search did not identify anypredicted BENI homologues in other organisms including thefruit fly or ascidians, suggesting that this family might bespecific to vertebrates.

The regulation of Xbra expression by BENI in response toactivin-like signaling

Expression of BENI mRNA was observed in the epidermallayer, mainly around the animal pole when detected at lowsensitivity, while expression was observed also in theepidermal layer of the dorsal lip region at high sensitivity.In contrast, the Xbra mRNA was expressed in the inner cellsof the dorsal lip region when detected at low sensitivity, andin the epidermal layer of the dorsal lip region at highsensitivity (Figs. 2N–Q). These two genes exhibited putativereciprocal expressions, in that neither gene was expressed inregions where the other gene was expressed strongly, and inthe epidermal layer of the dorsal lip, both genes weremoderately expressed.

The expression of Xbra, a pan-mesoderm marker gene, isinduced by activin-like signaling, FGF signaling (Smith et al.,1991), and canonical Wnt signaling (Vonica and Gumbiner,2002), and BMP signaling, although it is not clear if this is adirect effect (Re'em-Kalma et al., 1995). Furthermore, embryo-nic FGF (eFGF) and Xbra form a positive feedback regulatoryloop (Isaacs et al., 1994; Schulte-Merker and Smith, 1995; Tadaet al., 1997). Our results showed that BENI-gain-of-functioninhibits Xbra expression in vivo and in vitro. A recent studyfound that inhibition of insulin-like growth factor (IGF)signaling induces Xbra expression (Carron et al., 2005). It istherefore possible that BENI acts downstream of this signaltransduction to regulate Xbra transcription.


The role of BENI in convergent extension during gastrulation

In the present experiments, microinjection with BENI-MOsignificantly decreased the translation of BENI-GFP, and bothBENI-MO and BENI mRNA injection induced a curledphenotype in vivo. In Fig. 4, we demonstrated that ectopicexpression of BENI delayed the IMZ thinning. These resultssuggested that BENI is involved with radial intercalation duringthe first half of gastrulation. Together with the results presentedin Fig. 5A, these findings suggested that BENI function isspecific for radial and mediolateral intercalation. Indeed, wealso demonstrated that BENI is not required for epiboly,suggesting that the effect of loss of BENI function does notextend to the NIMZ (non-involuting marginal zone).

These results are reminiscent of the phenotypes induced byWnt11 or Cdc42 mutants due to the disruption of the non-canonical Wnt pathway (Tada and Smith, 2000; Penzo-Mendezet al., 2003). Furthermore, BENI mRNA inhibited Xbraexpression in vivo and in vitro, and Xbra, a direct transcriptionfactor of Wnt11, is involved in the convergent extension ofgastrulation (Conlon and Smith, 1999; Kwan and Kirschner,2003). The inhibition and activation of the elongation of activin-treated animal caps in both BENI-gain-of-function and loss-offunction experiments in this study correlate with previousreports of the inhibition of Xbra by both the dominant-negativeform of Xbra and overexpression of Xbra (Kwan and Kirschner,2003). It is probable therefore that BENI regulates Xbraexpression to induce correct convergent extension duringgastrulation.

In involution, BENI mRNA-injected embryos formed thedorsal lip and pigment line normally, suggesting that BENI isnot required for the involution that occurs as initial cellingression during gastrulation (Hardin and Keller, 1988). On theother hand, the BENI mRNA-injected embryos formed a cellmass around the blastopore lip and extensive migration on theinner surface of the ectoderm was suppressed. This phenotype isreminiscent of a previously reported case involving thetranslation inhibition of Xoom, which encodes a membraneprotein related to actin (Hasegawa et al., 1999; Hasegawa andKinoshita, 2000). Furthermore, the spatial expression pattern ofBENI at the gastrula stage is coincident with that of Xoom(Hasegawa et al., 2001). However, Hasegawa and Kinoshitareported that the inhibition of translation of Xoom does notinhibit convergent extension during gastrulation in vitro.Therefore, our results suggest a functional relationship betweenBENI and Xoom, but the mechanisms by which these twomolecules exert their effect on convergent extension ingastrulation may differ, and further experiments are needed toclarify the role of BENI in embryogenesis.

Acknowledgments

We thank Drs. M. Taira and N. Ueno for their gifts ofplasmids. This work was supported in part by a grant-in-aid forScientific Research from the Ministry of Education, Science,Sports, Culture and Technology, Japan, by a Grant-in-Aid (BioDesign Program) from the Ministry of Agriculture, Forestry and

Fisheries. M.H. is also supported by the Research Fellowshipsof the Japan Society for the promotion of Science for YoungScientists.

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A novel gene, BENI is required for the convergent ... · A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation Motohiro Homma a, Masafumi