Morphogenesis of Prechordal Plate and Notochord Requires Intact Eph/Ephrin B Signaling

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Developmental Biology 234, 470–482 (2001)doi:10.1006/dbio.2001.0281, available online at http://www.idealibrary.com on

Morphogenesis of Prechordal Plate and NotochordRequires Intact Eph/Ephrin B Signaling

Joanne Chan,*,1 John D. Mably,† Fabrizio C. Serluca,† Jau-Nian Chen,†athaniel B. Goldstein,* Matthew C. Thomas,* Jennifer A. Cleary,*aroline Brennan,‡ Mark C. Fishman,† and Thomas M. Roberts*

*Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Pathology,Harvard Medical School, †Cardiovascular Research Center, Massachusetts General Hospital,and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115;and ‡Department of Genetics, School of Biological Sciences, Queen Mary College,University of London, Mile End Road, London E1 4NS, United Kingdom

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migrationprocesses during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrinsthat represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrinB sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously toseveral Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands areconfined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins areexpressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord.Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology ofthe notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does notcompletely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesisof the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggestEph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation. © 2001 Academic Press

Key Words: Eph; ephrin B; zebrafish; gastrulation; prechordal plate; notochord; morphogenesis; cell movement.

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INTRODUCTION

Eph receptor tyrosine kinases and their ligands, theephrins, are cell surface molecules known to regulate cell-to-cell signaling. Roles for this family in developmentalprocesses include axon guidance, somitogenesis, angiogen-esis, and neural crest cell migration (reviewed in Holder andKlein, 1999). Although members of this family have alsobeen implicated in cell adhesion and other attraction-related responses (reviewed in Mellitzer et al., 2000), Eph/ephrin interactions have been most closely associated withrepulsion-mediated cell movements. Thus, binding andactivation of Eph receptors and ephrin ligands have been

1 To whom correspondence should be addressed at Departmentof Cancer Biology, Smith-970, Dana-Farber Cancer Institute, 1Jimmy Fund Way, Boston, MA 02115. Fax: 617-632-4770. E-mail:

rjochan@mbcrr.harvard.edu.

470

hown to generate a mutual exclusion effect on adjacentells allowing for the formation of rhombomere and somiteoundaries (Mellitzer et al., 1999; Xu et al., 1995; Durbin etl., 1998). In addition, retinal axons expressing an Epheceptor avoid areas of high ephrin expression in the tectumCheng et al., 1995; Nakamoto et al., 1996).

Eph receptors are divided into two major classes. Ineneral, EphA receptors bind A-ephrins that are anchored tohe plasma membrane by a glycosyl phosphatidyl-inositolGPI) linkage, and EphB receptors can bind B-ephrins thatave transmembrane and short, highly conserved cytoplas-ic domains. The EphA4 receptor is the only exception

ince it can bind B-ephrins with high affinity (Brambrilla etl., 1995; Gale et al., 1996a; reviewed in Flanagan andanderhaeghen, 1998). Membrane attachment of ephrins is

hought to promote clustering of the receptors to stimulateross-phosphorylation and initiation of a signal in the

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471Eph/Ephrin B Signaling in Morphogenesis

or the B-ephrins to their receptors also induces signalpropagation in the ligand-bearing cell (Davy and Robbins,2000; Davy et al., 1999; Holland et al., 1996; Bruckner et al.,1997). Thus, interaction of Ephs and ephrins leads tobidirectional signaling with each component acting as bothligand and receptor. Soluble ephrins can act as receptorantagonists since they can bind Eph receptors but do notinduce receptor activation (Davis et al., 1994; Winslow etal., 1995). In addition, soluble ephrins also block signalinitiation in the ligand-bearing cell by occupying Eph recep-tors, thus, inhibiting both components of Eph/ephrin sig-naling.

As the Eph/ephrin system has been implicated in activat-ing cytoskeletal changes and regulating cell movementevents in a number of developing systems (reviewed in Kaloand Pasquale, 1999; Holder and Klein, 1999), we werecurious to see if Eph/ephrin B signaling might play anearlier role in mediating morphogenetic movements duringgastrulation. Here we report the cloning and expressionpatterns of four members of the zebrafish ephrin B ligandfamily. Each ephrin B ligand displays a dynamic expressionpattern from as early as 30% epiboly to later development.To examine the functional role of Eph/ephrin B signaling,we perturbed their interaction by microinjection of RNAencoding dominant-negative soluble forms of B-ephrinsinto the zebrafish embryo. Disruption of Eph/ephrin Bsignaling generated apparent morphological defects in thenotochord and prechordal plate consistent with incorrectconvergent extension movements. Using several tissue-specific in situ markers, we did not observe any changes inell fate specification by the end of gastrulation, at theailbud stage. Our data support a role for Eph/ephrin Bs inell movement regulation during gastrulation.

MATERIALS AND METHODS

Maintenance of Zebrafish

Breeding fish were maintained at 28.5°C on a 14-h light/10-hdark cycle. Embryos were collected by natural spawning, raised in10% Hank’s saline, and staged up to 24 h (30 somites) according toKimmel et al. (1995); beyond this point, embryo stage is given ashours postfertilization.

Isolation and Characterization of cDNA Clones

The C-terminal cytoplasmic regions of mouse ephrin B1 andephrin B2 cDNAs (generously provided by John Flanagan) were

sed as probes at low stringency to screen an 18-h zebrafishmbryonic cDNA library. After three rounds of screening, fourndependent full-length cDNA clones were isolated encoding fourredicted proteins of the zebrafish ephrin B family: ephrin B1,phrin B2a, ephrin B2b, ephrin B3 (GenBank Accession Nos.F375224, AF375225, AF375226, AF375227, respectively). Nucle-tide sequences were determined using the dideoxy method by the

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RNA in Situ Hybridization

Embryos were staged according to Kimmel et al. (1995). Afterfixation overnight in 4% paraformaldehyde in PBS, embryos weretransferred and stored in methanol at 220°C. Whole-mount in situhybridization using digoxigenin-labeled antisense RNA probes wasperformed essentially as described by Xu et al. (1994). RNA probescontaining digoxigenin-11-UTP and/or fluorescein-12-UTP (bothfrom Boehringer Mannheim) were synthesized from linearizedplasmid DNA. For two-color in situ labeling, both the digoxigenin-labeled and fluorescein-labeled RNA probes were added together inthe hybridization step. First, the digoxigenin probe was visualizedusing the BM Purple alkaline phosphatase substrate (BoehringerMannheim) to give a blue/purple color. Subsequently, the fluores-ceinated probe was detected with antifluorescein antibody (Boeh-ringer Mannheim) and developed in the presence of BCIP/INT(Boehringer Mannheim), for a red/orange stain. Embryos werecleared and flat-mounted in 70% glycerol for photography.

JB-4 Plastic Sections

Zebrafish whole-mount in situ stained embryos were dehydratedin ethanol and infiltrated in JB-4 infiltration resin (JB-4 embeddingsolution A containing 1g/100 ml JB-4 catalyst; Poly-sciences Inc.).After embedding the specimen in JB-4 infiltration solution [JB-4Hardener (25:1) and hardening of blocks overnight at room tem-perature], specimens were sectioned at 5 mm using a Jung Supercut2065 microtome.

Microinjection of mRNA

Zebrafish ephrin B cDNAs encoding the extracellular domainswere subcloned into the pCS21 vector or the pCSMT vector(Turner and Weintraub, 1994) and capped RNAs were in vitrotranscribed using the Message Machine Kit (Ambion, Inc.), follow-ing the manufacturer’s instructions. A volume of 0.2 nl of mRNAat 100 ng/ml encoding soluble ephrin B2b (sol-ephrin-B2b), or aframe-shifted version of soluble ephrin B2b (shift-ephrin-B2b), or amyc-tagged version of the soluble ephrin B2b (sol-ephrin-B2b-myc),or the control nuclear GFP (GFP) was microinjected into one-cellstage zebrafish embryos using a gas driven microinjector (MedicalSystems Corp.).

RESULTS

Sequence Analysis of Predicted ZebrafishEphrin B Proteins

Using mouse ephrin B1 and ephrin B2 cDNAs as probes,we screened a zebrafish 18-h cDNA library at low strin-gency and isolated full-length cDNA clones encoding fourmembers of the ephrin B ligand family (Fig. 1A). Sequenceanalysis of these predicted proteins revealed structuralhomology to mammalian ephrin B1, B2, and B3; therefore,they have been named according to the degree of sequencehomology (as assigned by the Eph nomenclature commit-tee, Fig. 1B). All four zebrafish B-ephrin cDNAs encodetransmembrane proteins, each with a predicted signal se-quence, a membrane spanning region, and a cytoplasmic

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FIG. 1. Sequence alignment of predicted zebrafish class B ephrins. Predicted protein sequences for zebrafish ephrin B1, ephrin B2a, ephrinB2b, ephrin B3 were aligned and their phylogenetic relationships determined using the Clustal method with the DNA analysis programsfrom Lasergene software, DNA Star. (A) Amino acid sequence alignment of zebrafish B-ephrins. Identical residues are boxed in black;similar residues are boxed in gray; putative signal sequences are double-overlined in blue; the boxed green residues represent the predictedfirst amino acid of zebrafish B-ephrin proteins; conserved cysteine residues are boxed in red with an asterisk; a solid overline in greenconforms to the consensus sequence for metalloprotease cleavage; probable transmembrane domains are underlined in red. The blue arrowdenotes the beginning of the intracellular domain. The full-length cDNAs encoding zebrafish B-ephrins have been submitted to GenBank:ephrin B1 (Accession No. AF375224), ephrin B2a (Accession No. AF375225), ephrin B2b (Accession No. AF375226), ephrin B3 (Accession

o. AF375227). (B) Phylogenetic tree of mammalian are denoted “ephrin,” and zebrafish are denoted “Zephrin” B-ephrins. The mammalian

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473Eph/Ephrin B Signaling in Morphogenesis

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between each human/zebrafish ephrin B homolog pair is inthe range of 52 to 65% over the entire protein. Theintracellular domains exhibit even higher identity, in therange of 75 to 84%. There are two cDNAs that predictzebrafish homologs of mammalian ephrin B2; thus, they arenamed ephrin B2a (the same sequence as ephrin B2, Durbint al., 1998) and ephrin B2b (Fig. 1). The predicted zebrafishphrin B2a protein is more closely related to the mamma-ian ephrin B2 than zebrafish ephrin B2b, at 65 and 55%verall amino acid identities, respectively.

Expression Patterns of Ephrin Bsin Zebrafish Development

We examined the expression patterns of all four class Bephrins in the zebrafish by in situ RNA hybridization,hown in a blue stain in Figs. 2–5. The following stagesere used in the expression pattern analysis: 30% epiboly

o tailbud stage, early somitogenesis, 18 somites, 24 h, 36 h,nd 48 h. A standard set of well-characterized zebrafish initu markers has been used to help define the expressionomains for each B-ephrin. The RNA marker probes usedere goosecoid, gsc, an early dorsal marker; no tail, ntl, aotochord marker; pax2.1, expressed in the midbrain–indbrain boundary; Krox-20, krx-20, a marker for hind-rain rhombomeres 3 and 5; myoD, myoD, for demarcationf somitic borders; and the hatching gland-specific marker,gg1, which labels the anterior-most region of the axialesendoderm from late gastrula to tailbud stages (Thisse et

l., 1994; Schulte-Merker et al., 1992; Krauss et al., 1991;

ventricular zone. Scale bars for A, G 5 100 mm.

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erster, 1997). These markers are developed in a red/orangetain (Figs. 2–5).

Zebrafish Ephrin B1 Is Expressedin the Neurectoderm

Ephrin B1 mRNA is expressed diffusely by 50% epibolyand becomes more distinctive by 85% epiboly in the lateralregions of the embryo (Fig. 2). Ephrin B1 staining is coinci-dent with the lateral parts of pax2.1 staining, at the futuremidbrain–hindbrain boundary (Fig. 2A). Diffuse staining isalso seen around the anterior edge of the neural plate, in thepresumptive forebrain from 85% epiboly to the 1-somitestages (Figs. 2A and 2B). At the 1-somite stage, ephrin B1expression is prominent in the lateral regions of the pro-spective midbrain and hindbrain, as well as in a stripedpattern in the presomitic mesoderm (Fig. 2B). At later stagesof development, ephrin B1 is expressed in the posterior halfof forming somites, in adaxial cells, and in the developingnervous system (Figs. 2C–2G; Chan et al., in prep., also seefigure legends).

Ephrin B2b Is Expressed at the FutureDorsal Side of the Embryo

Ephrin B2b expression is first detected at 30% epiboly atthe future dorsal side of the embryo, confirmed by coinci-dent staining with the dorsal marker gsc (Figs. 3A, 3B, anddata not shown). Ephrin B2b expression spreads throughoutthe germ ring as the embryo develops from 40 to 60%epiboly, where it is also expressed in the embryonic shield

FIG. 2. Expression of ephrin B1 by in situ antisense RNA hybridization (A–G). Ephrin B1 staining was visualized with a blue stain, in everyanel. Antisense marker RNA probes were krx-20, myoD, ntl, and pax2.1, visualized with a red/orange stain. Dorsal views (except G, lateral

view); anterior is to the left. (A) 85% epiboly, pax2.1. Ephrin B1 is expressed in the lateral regions of the neurectoderm where it is coincidentwith the distal portion of the pax2.1 expression domain at the future midbrain–hindbrain boundary. (B) 1-somite. Ephrin B1 staining is stillprominent in the lateral regions of the future midbrain and hindbrain, and in stripes in the presomitic mesoderm. (C) Three somites,krx-20/myoD/pax2.1. Ephrin B1, staining is detected in the prospective forebrain, midbrain, and hindbrain, and a striping pattern in formingsomites. (D) 11 somites, myoD. Ephrin B1 staining in the presomitic mesoderm persists, just posterior to the last formed somite. Withinhe forming somites, its expression is more intense in the posterior half-somite. (E, F) 18 somites, krx-20/pax2.1 in E. Ephrin B1 expression

is detected in the presumptive telencephalon, diencephalon, and at the posterior tectum, just rostral to the midbrain–hindbrain boundaryas indicated by pax2.1 staining, and in all the hindbrain rhombomeres. Ephrin B1 staining becomes prominent in adaxial cells from the8-somite stage (see F). (G) 24 h, eyes removed. Expression of ephrin B1 persists in the telencephalon, the dorsal diencephalon, the posteriorectum, and the rhombomeres. Abbreviations: n, notochord; ad, adaxial cells; r3, rhombomere 3; r5, rhombomere 5; t, telencephalon, tec,ectum. Scale bars for A, D, F 5 100 mm.

FIG. 3. Expression of ephrin B2b by in situ antisense RNA hybridization (A–I). Ephrin B2b staining was visualized with a blue stain, invery panel. Antisense marker RNA probes were krx-20, myoD, and pax2.1, visualized with a red/orange stain. Animal pole view withorsal to the right (B). Dorsal views (except A, C, I, lateral views); anterior is to the left. (A, B) 30% epiboly. Ephrin B2b is asymmetricallyxpressed at the dorsal side of the embryo. (C) 75% epiboly. Ephrin B2b expression is detected in the hypoblastic layer. (D) Tail-bud stage,at-mounted. Ephrin B2b expression is most prominent in the presumptive forebrain and the presomitic mesoderm. (E, F) Three and eightomites, respectively, krx-20/myoD/pax2.1. Expression is seen in the forebrain, most intensely in the future diencephalon, in the rostral

spinal cord, in the presomitic mesoderm, and in the tailbud. (G) Ten somites, myoD, higher magnification. Ephrin B2b expression is seenin two stripes (arrows) in the presomitic mesoderm, just caudal to formed somites. Somite boundaries are indicated by vertical lines. (H)24 h. Expression is seen in the dorsal diencephalon, ventral to the epiphysis, cerebellum, and in the proliferating ventricular zone of thehindbrain rhombomeres. (I) 36 h. Ephrin B2b expression is maintained in the dorsal diencephalon and in the hindbrain rhombomeres.Abbreviations: pcp, prechordal plate; pn, presumptive notochord; r3, rhombomere 3; r5, rhombomere 5; e, epiphysis; tec, tectum; vz,

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475Eph/Ephrin B Signaling in Morphogenesis

B2b is detected in cells within the involuting hypoblast andalong the epibolizing margin (Fig. 3C). Ephrin B2b is ex-pressed in the prechordal plate and presumptive notochord.Although strong staining is detected in the anterior neuralplate, ephrin B2b is also expressed just caudal to the

idbrain–hindbrain boundary and throughout the entireresomitic mesoderm at the tailbud stage (Fig. 3D). At latertages of development, ephrin B2b expression persists in a

distinctive pattern throughout the segmentation andpharyngula periods (Figs. 3E–3I, also see figure legend).

Zebrafish Ephrin B2a Is also Expressedduring Early Development

The ephrin B2a expression pattern, especially in relationto somitogenesis, has been carefully described by Durbin etal. (1998). We independently isolated this cDNA clone fromour cDNA library screen. Ephrin B2a is also expressed early,from 30 to 50% epiboly in the germ ring (Durbin et al.,1998). During early- to midgastrula stages, ephrin B2aexpression mirrors that of ephrin B2b, described above(Figs. 3C, 4A, and 4B). At later stages, ephrin B2a isexpressed in the developing eye, in the dorsal artery, and inthe central nervous system (Figs. 4C–4H, also see figurelegend).

Zebrafish Ephrin B3 Is Expressed in the PrechordalPlate and Notochord

Localized ephrin B3 expression is first noted at 80%epiboly in the prechordal plate and presumptive notochord(Figs. 5A, 5B, and 5C). Ephrin B3 expression is also promi-

tectum. Scale bars for A, F 5 100 mm.

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B3 staining along the axial mesendoderm is hour-glassshaped, with highest expression at the rostral and caudalends. At 90% epiboly, ephrin B3 expression is also seen inthe overlaying neurectoderm, as well as in the anterior-most,pillow or polster region and in the prechordal plate (Fig. 5D).At later stages of development, ephrin B3 is expressed in theforebrain, in the midbrain tectum, and in hindbrain rhom-bomeres 3 and 5 (Figs. 5E–5I, also see figure legend).

The early expression patterns of zebrafish B-ephrins areconsistent with a role for Eph/ephrin B signaling duringgastrulation. This idea is also supported by the expressionpatterns of zebrafish Eph receptors at gastrula stages (Figs.2–5; Durbin et al., 1998; Xu et al., 1994; Cooke et al., 1997).

Overexpression of Soluble Ephrin B2bPerturbs Gastrulation

To investigate the role of Eph/ephrin B interactionsduring early development, we perturbed Eph/ephrin signal-ing by injection of RNA encoding dominant-interfering,soluble forms of ephrin B ligands into the one-cell stagezebrafish embryo. Soluble ephrin proteins can bind Ephreceptors but are unable to cluster receptors and induceintracellular tyrosine phosphorylation, thus inhibitingdownstream signaling in the receptor-bearing cell (Davis etal., 1994; Winslow et al., 1995). In addition, soluble ephrinslso block signaling in the ligand-bearing cell by occupyingph receptors and preventing endogenous ephrins fromnteracting with their cognate receptors. Overexpression ofoluble forms of each of the four ligands generated similarhenotypes (data not shown), presumably due to promiscu-

FIG. 4. Expression of ephrin B2a by in situ antisense RNA hybridization (A–H). Ephrin B2a staining was visualized with a blue stain, invery panel. Antisense marker RNA probes were krx-20 and pax2.1, visualized with a red/orange stain. Dorsal views (except B, E, F, Hateral views; and transverse section in G), anterior is to the left. (A, B) 70% epiboly. Ephrin B2a is expressed in the germ ring and shieldn A. Expression is seen in the hypoblastic layer. (C) Eight somites, krx-20/pax2.1. Ephrin B2a expression is detected in the presumptiveorebrain, diffusely in the eye field, and just caudal to the midbrain–hindbrain boundary as well as in prospective rhombomeres 1, 4, 7, andn the neural crest at the level of rhombomeres 2, 3, 4. (D) Eighteen somites, krx-20/pax2.1. Ephrin B2a expression in the eye field formsstriking ventral-to-dorsal stripe. (E) 24 h, pax2.1. Eye expression of ephrin B2a is confined to the lens primordium and developing retina

n the dorsal–temporal quadrant. (F, G) 24 h, transverse section in G. Ephrin B2a expression is seen in the endothelial cells of the dorsalrtery and is morphologically confirmed by location of the staining ventral to the notochord. (H) 36 h, eyes removed. Expression is seen inhe ventral hypothalamus and in the dorsal midline of the tectum. Expression is seen in the telencephalon. Abbreviations: pn, presumptiveotochord; r3, rhombomere 3; r5, rhombomere 5; e, epiphysis; t, telencephalon; n, notochord; da, dorsal artery; tec, tectum; pcp, prechordallate. Scale bars for A, E, F, G 5 100 mm.IG. 5. Expression of ephrin B3 by in situ antisense RNA hybridization (A–I). Ephrin B3 staining was visualized with a blue stain, in everyanel. Antisense marker RNA probes were gsc, hgg1, krx-20, myoD, and pax2.1, visualized with a red/orange stain. Dorsal views (except–D, F, H, lateral views), anterior is to the left. (A, B, C) 80% epiboly, double stained with gsc in C. Ephrin B3 is expressed in the axialesendoderm, with higher expression in the rostral and caudal ends in the hypoblastic layer (prechordal plate and presumptive notochord),here its anterior end is coincident with the gsc expression domain. (D) 90% epiboly, hgg1. Expression is seen in the overlayingeurectoderm, anterior-most pillow region, and the prechordal plate. (E) Nine somites, pax2.1/krx-20/myoD. Expression is seen in the

prospective forebrain, the midbrain, and the hindbrain, and in the rostral half of the anterior formed somites. (F, G) 24 h, eyes removed inF, double stained with pax2.1/krx-20 in G. Expression is seen in the lens primordium, ventral and dorsal diencephalon, tectum, cerebellum,telencephalon, and ventral hypothalamus. (H) 36 h, eyes removed. Ephrin B3 staining is detected in the epiphysis and maintained in therostral telencephalon and ventral hypothalamus, as well as in rhombomere 4. (I) 48 h. Expression is seen in the proliferating zone of thetectum. Abbreviations: pcp, prechordal plate; pn, presumptive notochord; r3, r4, r5, rhombomeres 3, 4, 5; e, epiphysis; t, telencephalon; tec,

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1995; Gale et al., 1996a). Since ephrin B2b expression isspecifically detected at the future dorsal side of the 30%epiboly stage embryo (Figs. 3A and 3B), we used a solubleform of the ephrin B2b cDNA construct, sol-ephrin B2b, inll subsequent experiments. To ensure that the sol-ephrin2b proteins were expressed in the embryo, a myc-taggedersion of the same construct was used in microinjection

FIG. 6. Effect of sol-ephrin B2b overexpression by the end of gastrut the tailbud stage (A–L). Sol-ephrin B2b mRNA or control GFP m

the tailbud stage. Embryos were hybridized with digoxigenin-labelmyoD, ntl, opl, papc, pax2.1, for analysis of defects in embryo morH, J, L, sol-ephrin B2b-injected embryos. (A, B) hgg1, pax2.1 in blue;domains appear widened compared with control. (C, D) gsc in reshortened and broadened. The midbrain–hindbrain boundary is ainjected embryo appears more spherical in shape. (E, F) hlx in bconsistently shorter and wider prechordal plate domain as shownstaining also demonstrate lateral expansion of both neural plate aembryo, F, hlx-positive cells are detected in the region of the futurmyoD, in blue; ntl, in red. Injected embryos in F and H represent awith the myoD staining of adaxial cells in the paraxial mesodermirregular in H, compared with control, G. (I–J) hlx, papc in blue; ntlanterior staining is consistent with C and D, with shortening andextension of the midbrain–hindbrain boundary. Ntl staining of thmesoderm. The notochord/paraxial mesoderm boundary is indiAbbreviations: pcp, prechordal plate; n, notochord; r3, r5, rhombom

xperiments to confirm protein expression in zebrafish p

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mbryo lysates by detection with an anti-myc antibodydata not shown).

Overexpression of sol-ephrin-B2b proteins resulted in aose-dependent defect in gastrula morphology evident byisual analysis using a dissecting microscope and confirmedy in situ RNA analysis with marker genes (see Table 1 andig. 6). More severe defects were observed in a larger

n. Analysis of morphological defects by of Eph/ephrin B disruptionA were injected into one-cell stage zebrafish embryos and fixed atfluorescein-labeled antisense RNA marker probes: gsc, hgg1, hlx,

ogy. (A–L) Dorsal views; A, C, E, G, I, K, control embryos; B, D, F,red. In the injected embryo, B, the hgg1, pax2.1, and ntl expression

x2.1 in blue. The gsc expression in the prechordal plate appearsaterally expanded as indicated by the pax2.1 staining in D. Theopl, pax2.1 in red. Higher magnification of embryos indicate a

the hlx staining. The opl staining of the neural plate and pax2.1e midbrain–hindbrain boundary. On the left side of the injected

dbrain–hindbrain boundary toward the neural plate (arrow). (G–H)severe phenotype. The ntl staining of the notochord is juxtaposed

e notochord/paraxial mesoderm border in the injected embryo is2.1 in red. Higher magnification of the same embryos in K, L. Theening of the prechordal plate and notochord domains and lateraltochord staining is juxtaposed with papc staining of the paraxial

by the arrowheads in control (K) versus injected embryo (L).s 3, 5; tec, tectum; mhb, midbrain–hindbrain boundary.

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mRNA, whereas very mild defects were obtained when weinjected the sol-ephrin-B2b mRNA at 4 pg. At 20 pg ofol-ephrin B2b mRNA, we could easily detect visible prob-ems with gastrula morphology in about 30% of injectedmbryos; therefore, this concentration was used in allubsequent experiments (see Table 1 and Fig. 6). Controlnjections of RNA for the green fluorescent protein, GFP, orframe-shifted version of sol-ephrin-B2b, shift-ephrin-B2b,ad no effect on embryo morphology (Table 1).In sol-ephrin B2b-injected embryos the distance between

he anterior limit of the prechordal plate and the margin ofhe gastrulating embryo was reduced from as early as0–80% epiboly by visual analysis. By the tailbud stage,njected embryos were significantly shorter along the ante-ioposterior axis and showed a more spherical shape whenompared to wild-type embryos (Figs. 6C, 6D, 6I, and 6J).he notochords in these embryos were found to be muchider (Figs. 6B, 6H, 6J, and 6L) than in controls (Fig. 6A, 6G,I, and 6K). At later stages of development, up to andncluding the 18-somite stage, injected embryos were stillound to be shorter along the anterioposterior axis and toave widened notochords (data not shown). The malforma-ions obtained by disruption of Eph/ephrin B signaling arehenotypically similar to some of the zebrafish gastrulationutants with defects in convergent extension movements

Solnica-Krezel et al., 1996; Hammerschmidt et al., 1996).

Eph/Ephrin B Disruption Alters Cell Movementbut Does Not Affect Cell Specificationby the End of Gastrulation

To investigate further the altered gastrula morphologycaused by our Eph/ephrin B interference experiment, weexamined the expression domains of a number of markergenes for the prechordal plate and notochord, the anteriorneural plate, as well as the paraxial mesoderm at the tailbudstage (Fig. 6). These include markers used previously, inaddition to the odd-paired-like gene, opl, which defines theanterior neural plate (Grinblat et al., 1998), and the pro-

TABLE 1Frequency of Gastrula Defects as Judged by In Situ Staining Using

Type of mRNAinjected

Quantity ofmRNA injected

(pg)

Total number ofinjected embryos

(N)b

sol-ephrin-B2b 20 170shift-ephrin-B2b 20 182GFP 20 185

a Embryos were injected at the one-cell stage with the mRNAs lwith in situ markers, then grouped and counted for phenotypic di

b N, total number of embryos injected in this experiment.c n, number of injected embryos scored for phenotypically norma

plate marker hlx and notochord marker ntl. These numbers are al

ocadherin papc gene which demarcates presomitic meso-

Copyright © 2001 by Academic Press. All right

erm (Yamamoto et al., 1998). In the sol-ephrin B2b in-ected embryo, the neural plate, prechordal plate, andresumptive midbrain–hindbrain domains were widened asndicated by opl, hgg1, gsc, hlx, and pax2.1 staining (Figs.A–6F, 6I, and 6J). In addition, some hlx-positive prechordallate cells are inappropriately located in the lateral region ofhe prospective forebrain–midbrain boundary on the leftide of the embryo (Fig. 6F, arrow). To examine theotochord/presomitic mesoderm border, ntl and myoDrobes were used as markers for notochord precursor cellsnd adaxial cells (Figs. 6G and 6H), respectively; while theapc probe was used to define the presomitic mesodermFigs. 6I–6L). The sol-ephrin B2b mRNA-injected embryohowed broadening of notochordal domain with an irregularorder between the cells in the presomitic mesoderm andhose in the notochord (Figs. 6G, 6H, 6K, and 6L). In a moreevere sol-ephrin B2b overexpression phenotype (Figs. 6Gnd 6H), notochord widening is accompanied by misloca-ion of some myoD-positive cells in the paraxial mesodermFig. 6H, arrowhead). Thus, Eph/ephrin B interference likelyenerated cell movement defects during gastrulation, lead-ng to the improper location of these marker positive cells.

The altered morphology of various tissues examined isonsistent with a defect in convergent extension move-ents affecting both axial and paraxial tissues. However,

ince all tissues examined are present at the tailbud stage,ur Eph/ephrin B disruption does not appear to alter cellate determination by the end of gastrulation.

DISCUSSION

Molecular Cloning and Implications of theZebrafish Ephrin B Sequences

We have isolated four full-length cDNAs encoding mem-bers of the zebrafish ephrin B family that represent the threeknown mammalian B-ephrins (Fig. 1; Gale et al., 1996a,b).In the zebrafish, some genes have been duplicated (Postleth-wait et al., 1998); therefore, it is likely that ephrin B2a and

hordal Plate and Notochord Markersa

notypicallynormal(n, %)c

Severe gastruladefect(n, %)

Mild gastruladefect(n, %)

Death(n, %)

61, 36% 59, 35% 43, 25% 7, 4%178, 98% 0, 0% 0, 0% 4, 2%181, 98% 0, 0% 0, 0% 4, 2%

above, then allowed to develop to tailbud stage, fixed and stainedces.

vere, or mild gastrula defects after in situ staining with prechordalpressed as a percentage (%) of the total number injected, N.

Prec

Phe

istedfferen

l, se

ephrin B2b are both paralogs of mammalian ephrin B2. The

s of reproduction in any form reserved.

ms2mge1sam1dadczPt

ac1wieetEsec(sig

ihlpiswmmtteenobEsasqcoss

479Eph/Ephrin B Signaling in Morphogenesis

preservation and expression of both ephrin B2 homologs inthe zebrafish suggest that they each developed an importantrole during zebrafish embryogenesis (Postlethwait et al.,1998; Force et al., 1999). Their expression patterns indistinct compartments would allow them to interact withdifferent Eph receptors on adjacent cells (see Figs. 3 and 4).The subtle changes in their predicted amino acid sequencesmay also allow the two proteins to exhibit differentialreceptor-binding affinities and to initiate alternative signal-ing cascades.

Although the extracellular domain is most divergentamong mammalian and zebrafish B-ephrins, the homologyis still quite high, at ;58% identity. Thus, zebrafish andmammalian B-ephrins likely display similar protein foldingand receptor-binding characteristics. This notion is furtherbolstered by the absolute conservation of four invariantcysteines in the extracellular domain (Fig. 1; reviewed inPandey et al., 1995). In addition, the zebrafish ephrin Bectodomain contains a motif related to the ephrin A2metalloprotease cleavage site (8 out of 10 residues conformto the consensus; Hattori et al., 2000). This motif isvirtually identical in all four zebrafish ephrin Bs (aminoacids 106–115 of zebrafish ephrin B1, Fig. 1A) and is alsoconserved in the mammalian B-ephrins (Gale et al., 1996b;Bergemann et al., 1998). Thus, like the A-ephrins, B-ephrins

ay also be cleaved after receptor engagement to mediateignal termination and repulsive movements (Hattori et al.,000). The intracellular domain among zebrafish and mam-alian B-ephrins exhibits an even greater homology, sug-

esting interactions with similar downstream effector mol-cules to initiate bidirectional signaling (Holland et al.,996; Bruckner et al., 1997). In addition to the five con-erved tyrosines, the last seven amino acids, NIYYKV, form

PDZ domain-binding site and are identical among allammalian and zebrafish B-ephrins (Fig. 1; Torres et al.,

998). In mammalian B-ephrins, this site binds PDZomain-containing proteins (Lin et al., 1999; Bruckner etl., 1999; Torres et al., 1998), a protein–protein interactionomain found in many cell surface proteins, neuronalhannels, and transporters (reviewed in Sheng, 1996). Thus,ebrafish B-ephrins may also be able to interact withDZ-domain proteins to allow formation and colocaliza-ion of large clusters of signaling molecules.

Eph/Ephrin B Signaling Regulates Cell MovementsUnderlying Gastrulation

During zebrafish gastrulation, coordinated morphoge-netic movements such as epiboly, convergent extension,and involution or ingression are involved in the formationof the three germ layers (Warga and Kimmel, 1990; re-viewed in Solnica-Krezel et al., 1995). During convergentextension, cells converge toward the dorsal midline of theembryo and mediolateral cell intercalations occur to furtherextend the anterioposterior body axis (Warga and Kimmel,1990; Kimmel et al., 1994; Gont et al., 1993; Davidson and

Copyright © 2001 by Academic Press. All right

re expressed during gastrulation, suggesting a role inell–cell recognition (Figs. 2–5; Xu et al., 1994; Cooke et al.,997; Durbin et al., 1998). Of the four zebrafish B-ephrins,e decided to use the sol-ephrin-B2b construct for micro-

njection into zebrafish embryos since ephrin B2b displaysarly localized expression at the future dorsal side of a 30%piboly stage embryo (Figs. 3A and 3B). This suggests thathe ephrin B2b protein is required early on to interact withph receptors in vivo, thus, playing a role in cell–cellignaling before the onset of gastrulation. Previous studiesstablished roles for Eph/ephrin signaling in the control ofell migrations in a number of developmental systemsreviewed in Holder and Klein, 1999). These observationsuggest that Eph/ephrin B interactions may also be involvedn the control of morphogenetic cell movements underlyingastrulation.As both Eph receptors and ephrin Bs are capable of

nteracting with PDZ domain-scaffolding molecules andave been implicated in signaling cytoskeletal changes, it isikely that both ligand- and receptor-bearing cells partici-ate in cell movement events (Torres et al., 1998; reviewedn Kalo and Pasquale, 1999). Prevention of Eph/ephrin Bignaling caused defects in embryo morphology consistentith incorrect convergent extension. Both the convergentovement of cells toward the dorsal midline and theediolateral cell intercalations were affected as evident in

he shorter and wider notochord and irregular formation ofhe notochord/paraxial mesoderm boundary (Fig. 6). How-ver, disruption of Eph/ephrin signaling had no significantffect on the relative size of notochord, prechordal plate,eural plate, or paraxial mesoderm. Thus, the specificationf these domains, at least at a gross level, appears unaffectedy the end of gastrulation. Previous experiments withph/ephrin B disruption generated a loss of myoD expres-ion within the mesoderm of injected embryos (Durbin etl., 1998). It is possible that at later stages, the defects inomite boundary formation could have resulted as a conse-uence of the gastrulation effect and/or as a result of theontinued presence of the dominant-interfering B-ephrin. Inur hands, we also saw a reduction or disruption of myoDtaining at somitogenesis stages, suggesting an alteration inomite patterning (data not shown; Durbin et al., 1998).

Molecular Mechanisms Driving ConvergentExtension Movements

Our Eph/ephrin B interference phenotype resembles thezebrafish mutants defective in gastrulation cell movementsuch as silberblick (slb), trilobite (tri), and knypek (kny)(Heisenberg et al., 2000; Heisenberg and Nusslein-Volhard,1997; Marlow et al., 1998). These mutants also display ashortened embryo axis without an apparent reduction inthe size of various expression domains. The gene respon-sible for the slb mutation was recently identified as thewnt11 homolog in the zebrafish (Heisenberg et al., 2000).Studies on the function of slb/wnt11 in zebrafish and

s of reproduction in any form reserved.

zWcc

B

B

B

C

C

D

D

D

D

D

480 Chan et al.

regulates cell movements rather than cell fate determina-tion (Heisenberg et al., 2000; Tada and Smith, 2000). Inebrafish and Xenopus experiments, loss of normal Slb/nt11 function resulted in abnormal morphogenesis that

an be rescued by a truncated form of disheveled thatannot activate GSK-3 and b-catenin. Thus, Slb/Wnt11 is

thought to specifically regulate morphogenetic movementsthrough a noncanonical wnt pathway (Heisenberg et al.,2000; Tada and Smith, 2000).

Another molecule that appears to directly influence con-vergent extension is the paraxial protocadherin, PAPC(Yamamoto et al., 1998; Kim et al., 1998). PAPC has potenthomotypic cell adhesion activity demonstrated in cell dis-sociation and reaggregation assays. It also plays a role inmorphogenesis as dominant-negative forms of PAPC inhib-its convergent extension (Yamamoto et al., 1998; Kim et al.,1998). Although no direct link has been made between theEph/ephrin Bs, Slb/Wnt11 and PAPC, these molecules sharesome common structural and signaling features and maywork to coordinate morphogenetic events essential forproper gastrulation. Both PAPC and Slb/Wnt11 are nor-mally expressed in paraxial tissues during late gastrulation(Yamamoto et al., 1998; Heisenberg et al., 2000). Geneticand experimental evidence show that Slb/Wnt11 partici-pates in the mediolateral cell intercalation events that drivethe convergent extension movements (Heisenberg et al.,2000). The early expression of ephrin B2b and papc at thefuture dorsal side by 30% epiboly suggests a requirementfor these proteins before gastrulation begins (Figs. 3A and3B; Yamamoto et al., 1998). The zebrafish EphB receptor,rtk3 (Xu et al., 1994), as well as ephrin B2b (Fig. 3D), areexpressed in the paraxial mesoderm during late gastrulastages and may regulate the proper alignment of cells at thedorsal midline. Thus, Eph/ephrin Bs likely contribute to thecoordination of morphogenetic movements along with se-creted factors and cell-adhesion molecules such as Slb/Wnt11 and PAPC.

Our expression and Eph/ephrin B interruption data sup-port a role for these molecules in convergent extensionduring gastrulation. Disruption of Eph/ephrin B signalingresulted in the abnormal cell movements that led to mor-phogenetic defects in the prechordal plate and notochord, aswell as in paraxial tissues. The availability of these fourephrin B cDNAs should provide useful reagents to dissectfurther the role of Eph/ephrin B signaling during zebrafishdevelopment.

ACKNOWLEDGMENTS

We dedicate this paper to the memory of our friend Nigel Holder.Our thanks to John Flanagan for providing mouse cDNA clones forephrin B1 and ephrin B2. We are indebted to George Serbedzija fordiscussions and technical advice. We express our gratitude to SteveWilson, Ajay Chitnis, Jeremy Green, and Chuck Stiles for helpfuldiscussions during the course of this study. The help of HenryRoehl, Andreas Vogel, Yevgenya Grinblat, and Hazel Sive in

Copyright © 2001 by Academic Press. All right

thank Dana Witten for help with the digital camera setup. Finally,we are grateful to George Serbedzija, Andres Collazo, JeremyGreen, Chuck Stiles, and Ole Gjoerup for critical reading of themanuscript. This work was supported by NIH grants to T.M.R.(HD24926) and M.C.F. (HL49579). In compliance with HarvardMedical School guidelines on possible conflict of interest, wedisclose that one of the authors (T.M.R.) has consulting relation-ships with Upstate Biotechnology and Novartis Pharmaceuticals,Inc.

REFERENCES

Anonymous. (1997). Unified nomenclature for Eph family recep-tors and their ligands, the ephrins. Eph Nomenclature Commit-tee [letter]. Cell 90, 403–404.

Bergemann, A. D., Zhang, L., Chiang, M. K., Brambilla, R., Klein,R., and Flanagan, J. G. (1998). Ephrin-B3, a ligand for the receptorEphB3, expressed at the midline of the developing neural tube.Oncogene 16, 471–480.

rambilla, R., Schnapp, A., Casagranda, F., Labrador, J. P., Berge-mann, A. D., Flanagan, J. G., Pasquale, E. B., and Klein, R. (1995).Membrane-bound LERK2 ligand can signal through three differ-ent Eph-related receptor tyrosine kinases. EMBO J. 14, 3116–3126.

ruckner, K., Pablo Labrador, J., Scheiffele, P., Herb, A., Seeburg,P. H., and Klein, R. (1999). Ephrin-B ligands recruit GRIP familyPDZ adaptor proteins into raft membrane microdomains. Neu-ron 22, 511–524.

ruckner, K., Pasquale, E. B., and Klein, R. (1997). Tyrosinephosphorylation of transmembrane ligands for Eph receptors.Science 275, 1640–1643.heng, H. J., Nakamoto, M., Bergemann, A. D., and Flanagan, J. G.(1995). Complementary gradients in expression and binding ofELF-1 and Mek4 in development of the topographic retinotectalprojection map. Cell 82, 371–381.ooke, J. E., Xu, Q., Wilson, S. W., and Holder, N. (1997). Charac-terisation of five novel zebrafish Eph-related receptor tyrosinekinases suggests roles in patterning the neural plate. Dev. GenesEvol. 206, 515–531.avidson, L. A., and Keller, R. E. (1999). Neural tube closure inXenopus laevis involves medial migration, directed protrusiveactivity, cell intercalation and convergent extension. Develop-ment 126, 4547–4556.avis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak,V., Pawson, T., Goldfarb, M., and Yancopoulos, G. D. (1994).Ligands for EPH-related receptor tyrosine kinases that requiremembrane attachment or clustering for activity. Science 266,816–819.avy, A., Gale, N. W., Murray, E. W., Klinghoffer, R. A., Soriano,P., Feuerstein, C., and Robbins, S. M. (1999). Compartmentalizedsignaling by GPI-anchored ephrin-A5 requires the Fyn tyrosinekinase to regulate cellular adhesion. Genes Dev. 13, 3125–3135.avy, A., and Robbins, S. M. (2000). Ephrin-A5 modulates celladhesion and morphology in an integrin-dependent manner.EMBO J. 19, 5396–5405.urbin, L., Brennan, C., Shiomi, K., Cooke, J., Barrios, A., Shan-mugalingam, S., Guthrie, B., Lindberg, R., and Holder, N. (1998).Eph signaling is required for segmentation and differentiation of

s of reproduction in any form reserved.

F

G

G

G

G

H

H

H

H

H

H

K

K

K

K

K

L

M

M

M

N

O

P

P

S

S

S

S

T

T

T

481Eph/Ephrin B Signaling in Morphogenesis

Flanagan, J. G., and Vanderhaeghen, P. (1998). The ephrins and Ephreceptors in neural development. Annu. Rev. Neurosci. 21,309–345.

orce, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L., andPostlethwait, J. (1999). Preservation of duplicate genes bycomplementary, degenerative mutations. Genetics 151, 1531–1545.ale, N. W., Flenniken, A., Compton, D. C., Jenkins, N., Copeland,N. G., Gilbert, D. J., Davis, S., Wilkinson, D. G., and Yancopou-los, G. D. (1996a). Elk-L3, a novel transmembrane ligand for theEph family of receptor tyrosine kinases, expressed in embryonicfloor plate, roof plate and hindbrain segments. Oncogene 13,1343–1352.ale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan,L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H.,Wilkinson, D. G., Pawson, T., Davis, S., and Yancopoulos, G. D.(1996b). Eph receptors and ligands comprise two major specific-ity subclasses and are reciprocally compartmentalized duringembryogenesis. Neuron 17, 9–19.ont, L. K., Steinbeisser, H., Blumberg, B., and de Robertis, E. M.(1993). Tail formation as a continuation of gastrulation: Themultiple cell populations of the Xenopus tailbud derive from thelate blastopore lip. Development 119, 991–1004.rinblat, Y., Gamse, J., Patel, M., and Sive, H. (1998). Determina-tion of the zebrafish forebrain: Induction and patterning. Devel-opment 125, 4403–4416.ammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A.,Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M.,Haffter, P., Heisenberg, C. P., Jiang, Y. J., Kelsh, R. N., Odenthal,J., Warga, R. M., and Nusslein-Volhard, C. (1996). Mutationsaffecting morphogenesis during gastrulation and tail formationin the zebrafish, Danio rerio. Development 123, 143–151.attori, M., Osterfield, M., and Flanagan, J. G. (2000). Regulatedcleavage of a contact-mediated axon repellent. Science 289,1360–1365.eisenberg, C. P., and Nusslein-Volhard, C. (1997). The function ofsilberblick in the positioning of the eye anlage in the zebrafishembryo. Dev. Biol. (Orlando) 184, 85–94.eisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L.,Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000).Silberblick/Wnt11 mediates convergent extension movementsduring zebrafish gastrulation. Nature 405, 76–81.older, N., and Klein, R. (1999). Eph receptors and ephrins:Effectors of morphogenesis. Development 126, 2033–2044.olland, S. J., Gale, N. W., Mbamalu, G., Yancopoulos, G. D.,Henkemeyer, M., and Pawson, T. (1996). Bidirectional signallingthrough the EPH-family receptor Nuk and its transmembraneligands. Nature 383, 722–725.alo, M. S., and Pasquale, E. B. (1999). Signal transfer by Ephreceptors. Cell Tissue Res. 298, 1–9.

im, S. H., Yamamoto, A., Bouwmeester, T., Agius, E., andRobertis, E. M. (1998). The role of paraxial protocadherin inselective adhesion and cell movements of the mesoderm duringXenopus gastrulation. Development 125, 4681–4690.

immel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., andSchilling, T. F. (1995). Stages of embryonic development of thezebrafish. Dev. Dyn. 203, 253–310.immel, C. B., Warga, R. M., and Kane, D. A. (1994). Cell cyclesand clonal strings during formation of the zebrafish central

Copyright © 2001 by Academic Press. All right

rauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U., andFjose, A. (1991). Zebrafish pax[zf-a]: A paired box-containing geneexpressed in the neural tube. EMBO J. 10, 3609–3619.

in, D., Gish, G. D., Songyang, Z., and Pawson, T. (1999). Thecarboxyl terminus of B class ephrins constitutes a PDZ domainbinding motif. J. Biol. Chem. 274, 3726–3733.arlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S. C., Abbas, L.,Weaver, M., Driever, W., and Solnica-Krezel, L. (1998). Func-tional interactions of genes mediating convergent extension,knypek and trilobite, during the partitioning of the eye primor-dium in zebrafish. Dev. Biol. (Orlando) 203, 382–399.ellitzer, G., Xu, Q., and Wilkinson, D. G. (1999). Eph receptorsand ephrins restrict cell intermingling and communication.Nature 400, 77–81.ellitzer, G., Xu, Q., and Wilkinson, D. G. (2000). Control of cellbehaviour by signalling through Eph receptors and ephrins. Curr.Opin. Neurobiol. 10, 400–408.akamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T.,Hansen, M. J., Yoon, C. H., O’Leary, D. D., and Flanagan, J. G.(1996). Topographically specific effects of ELF-1 on retinal axonguidance in vitro and retinal axon mapping i. Cell 86, 755–766.xtoby, E., and Jowett, T. (1993). Cloning of the zebrafish krox-20gene (krx-20) and its expression during hindbrain development.Nucleic Acids Res. 21, 1087–1095.

andey, A., Lindberg, R. A., and Dixit, V. M. (1995). Cell signalling.Receptor orphans find a family. Curr. Biol. 5, 986–989.

ostlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A.,Brownlie, A., Donovan, A., Egan, E. S., Force, A., Gong, Z.,Goutel, C., Fritz, A., Kelsh, R., Knapik, E., Liao, E., Paw, B.,Ransom, D., Singer, A., Thomson, M., Abduljabbar, T. S., Yelick,P., Beier, D., Joly, J. S., Larhammar, D., Rosa, F., et al. (1998).Vertebrate genome evolution and the zebrafish gene map. Nat.Genet. 18, 345–349.

chulte-Merker, S., Ho, R. K., Herrmann, B. G., and Nusslein-Volhard, C. (1992). The protein product of the zebrafish homo-logue of the mouse T gene is expressed in nuclei of the germ ringand the notochord of the early embryo. Development 116,1021–1032.

heng, M. (1996). PDZs and receptor/channel clustering: Roundingup the latest suspects. Neuron 17, 575–578.

olnica-Krezel, L., Bailey, J., Gruer, D. P., Price, J. M., Dove, W. F.,Dee, J., and Anderson, R. W. (1995). Characterization of npfmutants identifying developmental genes in Physarum. Micro-biology 141, 799–816.

olnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E., Rangini,Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D. Y.,Zwartkruis, F., Abdelilah, S., and Driever, W. (1996). Mutationsaffecting cell fates and cellular rearrangements during gastrula-tion in zebrafish. Development 123, 67–80.

ada, M., and Smith, J. C. (2000). Xwnt11 is a target of XenopusBrachyury: Regulation of gastrulation movements via Dishev-elled, but not through the canonical Wnt pathway. Development127, 2227–2238.hisse, C., Thisse, B., Halpern, M. E., and Postlethwait, J. H. (1994).Goosecoid expression in neurectoderm and mesendoderm isdisrupted in zebrafish cyclops gastrulas. Dev. Biol. (Orlando)164, 420–429.orres, R., Firestein, B. L., Dong, H., Staudinger, J., Olson, E. N.,Huganir, R. L., Bredt, D. S., Gale, N. W., and Yancopoulos, G. D.(1998). PDZ proteins bind, cluster, and synaptically colocalizewith Eph receptors and their ephrin ligands [see comments].

s of reproduction in any form reserved.

X

Y

482 Chan et al.

Turner, D. L., and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cellsto a neural fate. Genes Dev. 8, 1434–1447.

Vogel, A. M., and Gerster, T. (1997). Expression of a zebrafishCathepsin L gene in anterior mesendoderm and hatching gland.Dev. Genes Evol. 206, 477–479.

Warga, R. M., and Kimmel, C. B. (1990). Cell movements duringepiboly and gastrulation in zebrafish. Development 108, 569–580.

Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A.,Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J.,and Riggleman, B. (1996). Developmental regulation of zebrafishMyoD in wild-type, no tail and spadetail embryos. Development122, 271–280.

Winslow, J. W., Moran, P., Valverde, J., Shih, A., Yuan, J. Q., Wong,S. C., Tsai, S. P., Goddard, A., Henzel, W. J., Hefti, F. et al. (1995).

Copyright © 2001 by Academic Press. All right

receptor involved in axon bundle formation. Neuron 14, 973–981.

Xu, Q., Alldus, G., Holder, N., and Wilkinson, D. G. (1995).Expression of truncated Sek-1 receptor tyrosine kinase disruptsthe segmental restriction of gene expression in the Xenopus andzebrafish hindbrain. Development 121, 4005–4016.

u, Q., Holder, N., Patient, R., and Wilson, S. W. (1994). Spatiallyregulated expression of three receptor tyrosine kinase genesduring gastrulation in the zebrafish. Development 120, 287–299.amamoto, A., Amacher, S. L., Kim, S. H., Geissert, D., Kimmel,C. B., and De Robertis, E. M. (1998). Zebrafish paraxial protocad-herin is a downstream target of spadetail involved in morpho-genesis of gastrula mesoderm. Development 125, 3389–3397.

Received for publication October 18, 2001Revised March 28, 2001

Accepted March 28, 2001

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