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

Home , Goosecoid protein, Rhombomere

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 Notochord Requires Intact Eph/Ephrin B Signaling

Joanne Chan,*,1 John D. Mably,† Fabrizio C. Serluca,† Jau-Nian Chen,† Nathaniel B. Goldstein,* Matthew C. Thomas,* Jennifer A. Cleary,* Caroline 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 migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed 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 of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/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.

INTRODUCTION shown to generate a mutual exclusion effect on adjacent cells allowing for the formation of rhombomere and somite Eph receptor tyrosine kinases and their ligands, the boundaries (Mellitzer et al., 1999; Xu et al., 1995; Durbin et ephrins, are cell surface molecules known to regulate cell- al., 1998). In addition, retinal axons expressing an Eph to-cell signaling. Roles for this family in developmental receptor avoid areas of high ephrin expression in the tectum processes include axon guidance, somitogenesis, angiogen- (Cheng et al., 1995; Nakamoto et al., 1996). esis, and neural crest cell migration (reviewed in Holder and Eph receptors are divided into two major classes. In Klein, 1999). Although members of this family have also general, EphA receptors bind A-ephrins that are anchored to been implicated in cell adhesion and other attraction- the plasma membrane by a glycosyl phosphatidyl-inositol related responses (reviewed in Mellitzer et al., 2000), Eph/ (GPI) linkage, and EphB receptors can bind B-ephrins that ephrin interactions have been most closely associated with have transmembrane and short, highly conserved cytoplas- repulsion-mediated cell movements. Thus, binding and mic domains. The EphA4 receptor is the only exception activation of Eph receptors and ephrin ligands have been since it can bind B-ephrins with high afﬁnity (Brambrilla et al., 1995; Gale et al., 1996a; reviewed in Flanagan and 1 To whom correspondence should be addressed at Department Vanderhaeghen, 1998). Membrane attachment of ephrins is of Cancer Biology, Smith-970, Dana-Farber Cancer Institute, 1 thought to promote clustering of the receptors to stimulate Jimmy Fund Way, Boston, MA 02115. Fax: 617-632-4770. E-mail: cross-phosphorylation and initiation of a signal in the [email protected]. receptor-bearing cell. Furthermore, binding of either the A-

or the B-ephrins to their receptors also induces signal RNA in Situ Hybridization propagation in the ligand-bearing cell (Davy and Robbins, Embryos were staged according to Kimmel et al. (1995). After 2000; Davy et al., 1999; Holland et al., 1996; Bruckner et al., fixation overnight in 4% paraformaldehyde in PBS, embryos were 1997). Thus, interaction of Ephs and ephrins leads to transferred and stored in methanol at Ϫ20°C. Whole-mount in situ bidirectional signaling with each component acting as both hybridization using digoxigenin-labeled antisense RNA probes was ligand and receptor. Soluble ephrins can act as receptor performed essentially as described by Xu et al. (1994). RNA probes antagonists since they can bind Eph receptors but do not containing digoxigenin-11-UTP and/or fluorescein-12-UTP (both induce receptor activation (Davis et al., 1994; Winslow et from Boehringer Mannheim) were synthesized from linearized al., 1995). In addition, soluble ephrins also block signal plasmid DNA. For two-color in situ labeling, both the digoxigenin- initiation in the ligand-bearing cell by occupying Eph recep- labeled and fluorescein-labeled RNA probes were added together in tors, thus, inhibiting both components of Eph/ephrin sig- the hybridization step. First, the digoxigenin probe was visualized naling. using the BM Purple alkaline phosphatase substrate (Boehringer Mannheim) to give a blue/purple color. Subsequently, the fluores- As the Eph/ephrin system has been implicated in activat- ceinated probe was detected with antifluorescein antibody (Boeh- ing cytoskeletal changes and regulating cell movement ringer Mannheim) and developed in the presence of BCIP/INT events in a number of developing systems (reviewed in Kalo (Boehringer Mannheim), for a red/orange stain. Embryos were and Pasquale, 1999; Holder and Klein, 1999), we were cleared and flat-mounted in 70% glycerol for photography. curious to see if Eph/ephrin B signaling might play an earlier role in mediating morphogenetic movements during gastrulation. Here we report the cloning and expression JB-4 Plastic Sections patterns of four members of the zebrafish ephrin B ligand Zebrafish whole-mount in situ stained embryos were dehydrated family. Each ephrin B ligand displays a dynamic expression in ethanol and infiltrated in JB-4 infiltration resin (JB-4 embedding pattern from as early as 30% epiboly to later development. solution A containing 1g/100 ml JB-4 catalyst; Poly-sciences Inc.). To examine the functional role of Eph/ephrin B signaling, After embedding the specimen in JB-4 infiltration solution [JB-4 we perturbed their interaction by microinjection of RNA Hardener (25:1) and hardening of blocks overnight at room tem- ␮ encoding dominant-negative soluble forms of B-ephrins perature], specimens were sectioned at 5 m using a Jung Supercut into the zebrafish embryo. Disruption of Eph/ephrin B 2065 microtome. signaling generated apparent morphological defects in the notochord and prechordal plate consistent with incorrect Microinjection of mRNA convergent extension movements. Using several tissue- Zebrafish ephrin B cDNAs encoding the extracellular domains specific in situ markers, we did not observe any changes in were subcloned into the pCS2ϩ vector or the pCSMT vector cell fate specification by the end of gastrulation, at the (Turner and Weintraub, 1994) and capped RNAs were in vitro tailbud stage. Our data support a role for Eph/ephrin Bs in transcribed using the Message Machine Kit (Ambion, Inc.), follow- cell movement regulation during gastrulation. ing the manufacturer’s instructions. A volume of 0.2 nl of mRNA at 100 ng/␮l encoding soluble ephrin B2b (sol-ephrin-B2b), or a frame-shifted version of soluble ephrin B2b (shift-ephrin-B2b), or a myc-tagged version of the soluble ephrin B2b (sol-ephrin-B2b-myc), MATERIALS AND METHODS or the control nuclear GFP (GFP) was microinjected into one-cell stage zebrafish embryos using a gas driven microinjector (Medical Maintenance of Zebrafish Systems Corp.).

Breeding fish were maintained at 28.5°C on a 14-h light/10-h dark cycle. Embryos were collected by natural spawning, raised in RESULTS 10% Hank’s saline, and staged up to 24 h (30 somites) according to Kimmel et al. (1995); beyond this point, embryo stage is given as Sequence Analysis of Predicted Zebrafish hours postfertilization. Ephrin B Proteins Using mouse ephrin B1 and ephrin B2 cDNAs as probes, Isolation and Characterization of cDNA Clones we screened a zebrafish 18-h cDNA library at low stringency and isolated full-length cDNA clones encoding four The C-terminal cytoplasmic regions of mouse ephrin B1 and members of the ephrin B ligand family (Fig. 1A). Sequence ephrin B2 cDNAs (generously provided by John Flanagan) were analysis of these predicted proteins revealed structural used as probes at low stringency to screen an 18-h zebrafish homology to mammalian ephrin B1, B2, and B3; therefore, embryonic cDNA library. After three rounds of screening, four they have been named according to the degree of sequence independent full-length cDNA clones were isolated encoding four predicted proteins of the zebrafish ephrin B family: ephrin B1, homology (as assigned by the Eph nomenclature commit- ephrin B2a, ephrin B2b, ephrin B3 (GenBank Accession Nos. tee, Fig. 1B). All four zebrafish B-ephrin cDNAs encode AF375224, AF375225, AF375226, AF375227, respectively). Nucle- transmembrane proteins, each with a predicted signal se- otide sequences were determined using the dideoxy method by the quence, a membrane spanning region, and a cytoplasmic Dana-Farber Cancer Institute Molecular Biology Core Facility. domain (Figs. 1A and 1B). The amino acid sequence identity

FIG. 1. Sequence alignment of predicted zebrafish class B ephrins. Predicted protein sequences for zebrafish ephrin B1, ephrin B2a, ephrin B2b, ephrin B3 were aligned and their phylogenetic relationships determined using the Clustal method with the DNA analysis programs from 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 predicted first amino acid of zebrafish B-ephrin proteins; conserved cysteine residues are boxed in red with an asterisk; a solid overline in green conforms to the consensus sequence for metalloprotease cleavage; probable transmembrane domains are underlined in red. The blue arrow denotes 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 No. AF375227). (B) Phylogenetic tree of mammalian are denoted “ephrin,” and zebrafish are denoted “Zephrin” B-ephrins. The mammalian sequences were taken from human ephrin B1, B2, and B3 from GenBank.

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 474 Chan et al. between each human/zebrafish ephrin B homolog pair is in Gerster, 1997). These markers are developed in a red/orange the range of 52 to 65% over the entire protein. The stain (Figs. 2–5). intracellular domains exhibit even higher identity, in the range of 75 to 84%. There are two cDNAs that predict Zebrafish Ephrin B1 Is Expressed zebrafish homologs of mammalian ephrin B2; thus, they are in the Neurectoderm named ephrin B2a (the same sequence as ephrin B2, Durbin et al., 1998) and ephrin B2b (Fig. 1). The predicted zebrafish Ephrin B1 mRNA is expressed diffusely by 50% epiboly ephrin B2a protein is more closely related to the mamma- and becomes more distinctive by 85% epiboly in the lateral lian ephrin B2 than zebrafish ephrin B2b, at 65 and 55% regions of the embryo (Fig. 2). Ephrin B1 staining is coinci- overall amino acid identities, respectively. dent with the lateral parts of pax2.1 staining, at the future midbrain–hindbrain boundary (Fig. 2A). Diffuse staining is also seen around the anterior edge of the neural plate, in the Expression Patterns of Ephrin Bs presumptive forebrain from 85% epiboly to the 1-somite in Zebrafish Development stages (Figs. 2A and 2B). At the 1-somite stage, ephrin B1 expression is prominent in the lateral regions of the pro- We examined the expression patterns of all four class B spective midbrain and hindbrain, as well as in a striped ephrins in the zebrafish by in situ RNA hybridization, pattern in the presomitic mesoderm (Fig. 2B). At later stages shown in a blue stain in Figs. 2–5. The following stages of development, ephrin B1 is expressed in the posterior half were used in the expression pattern analysis: 30% epiboly of forming somites, in adaxial cells, and in the developing to tailbud stage, early somitogenesis, 18 somites, 24 h, 36 h, nervous system (Figs. 2C–2G; Chan et al., in prep., also see and 48 h. A standard set of well-characterized zebrafish in figure legends). situ markers has been used to help define the expression domains for each B-ephrin. The RNA marker probes used were goosecoid, gsc, an early dorsal marker; no tail, ntl,a Ephrin B2b Is Expressed at the Future notochord marker; pax2.1, expressed in the midbrain– Dorsal Side of the Embryo hindbrain boundary; Krox-20, krx-20, a marker for hind- Ephrin B2b expression is first detected at 30% epiboly at brain rhombomeres 3 and 5; myoD, myoD, for demarcation the future dorsal side of the embryo, confirmed by coinci- of somitic borders; and the hatching gland-specific marker, dent staining with the dorsal marker gsc (Figs. 3A, 3B, and hgg1, which labels the anterior-most region of the axial data not shown). Ephrin B2b expression spreads throughout mesendoderm from late gastrula to tailbud stages (Thisse et the germ ring as the embryo develops from 40 to 60% al., 1994; Schulte-Merker et al., 1992; Krauss et al., 1991; epiboly, where it is also expressed in the embryonic shield Oxtoby and Jowett, 1993; Weinberg et al., 1996; Vogel and (data not shown). From 75% epiboly, expression of ephrin

FIG. 2. Expression of ephrin B1 by in situ antisense RNA hybridization (A–G). Ephrin B1 staining was visualized with a blue stain, in every panel. 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 coincident with the distal portion of the pax2.1 expression domain at the future midbrain–hindbrain boundary. (B) 1-somite. Ephrin B1 staining is still prominent 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 forming somites. (D) 11 somites, myoD. Ephrin B1 staining in the presomitic mesoderm persists, just posterior to the last formed somite. Within the 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 boundary as indicated by pax2.1 staining, and in all the hindbrain rhombomeres. Ephrin B1 staining becomes prominent in adaxial cells from the 18-somite stage (see F). (G) 24 h, eyes removed. Expression of ephrin B1 persists in the telencephalon, the dorsal diencephalon, the posterior tectum, and the rhombomeres. Abbreviations: n, notochord; ad, adaxial cells; r3, rhombomere 3; r5, rhombomere 5; t, telencephalon, tec, tectum. Scale bars for A, D, F ϭ 100 ␮m. FIG. 3. Expression of ephrin B2b by in situ antisense RNA hybridization (A–I). Ephrin B2b staining was visualized with a blue stain, in every panel. Antisense marker RNA probes were krx-20, myoD, and pax2.1, visualized with a red/orange stain. Animal pole view with dorsal to the right (B). Dorsal views (except A, C, I, lateral views); anterior is to the left. (A, B) 30% epiboly. Ephrin B2b is asymmetrically expressed at the dorsal side of the embryo. (C) 75% epiboly. Ephrin B2b expression is detected in the hypoblastic layer. (D) Tail-bud stage, ﬂat-mounted. Ephrin B2b expression is most prominent in the presumptive forebrain and the presomitic mesoderm. (E, F) Three and eight somites, 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 seen in 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 the hindbrain 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, ventricular zone. Scale bars for A, G ϭ 100 ␮m.

B2b is detected in cells within the involuting hypoblast and B3 staining along the axial mesendoderm is hour-glass along the epibolizing margin (Fig. 3C). Ephrin B2b is ex- shaped, with highest expression at the rostral and caudal pressed in the prechordal plate and presumptive notochord. ends. At 90% epiboly, ephrin B3 expression is also seen in Although strong staining is detected in the anterior neural the overlaying neurectoderm, as well as in the anterior-most, plate, ephrin B2b is also expressed just caudal to the pillow or polster region and in the prechordal plate (Fig. 5D). midbrain–hindbrain boundary and throughout the entire At later stages of development, ephrin B3 is expressed in the presomitic mesoderm at the tailbud stage (Fig. 3D). At later forebrain, in the midbrain tectum, and in hindbrain rhom- stages of development, ephrin B2b expression persists in a bomeres 3 and 5 (Figs. 5E–5I, also see figure legend). distinctive pattern throughout the segmentation and The early expression patterns of zebrafish B-ephrins are pharyngula periods (Figs. 3E–3I, also see figure legend). consistent with a role for Eph/ephrin B signaling during gastrulation. This idea is also supported by the expression Zebrafish Ephrin B2a Is also Expressed patterns of zebrafish Eph receptors at gastrula stages (Figs. during Early Development 2–5; Durbin et al., 1998; Xu et al., 1994; Cooke et al., 1997). The ephrin B2a expression pattern, especially in relation to somitogenesis, has been carefully described by Durbin et Overexpression of Soluble Ephrin B2b al. (1998). We independently isolated this cDNA clone from Perturbs Gastrulation our cDNA library screen. Ephrin B2a is also expressed early, from 30 to 50% epiboly in the germ ring (Durbin et al., To investigate the role of Eph/ephrin B interactions 1998). During early- to midgastrula stages, ephrin B2a during early development, we perturbed Eph/ephrin signal- expression mirrors that of ephrin B2b, described above ing by injection of RNA encoding dominant-interfering, (Figs. 3C, 4A, and 4B). At later stages, ephrin B2a is soluble forms of ephrin B ligands into the one-cell stage expressed in the developing eye, in the dorsal artery, and in zebrafish embryo. Soluble ephrin proteins can bind Eph the central nervous system (Figs. 4C–4H, also see figure receptors but are unable to cluster receptors and induce legend). intracellular tyrosine phosphorylation, thus inhibiting downstream signaling in the receptor-bearing cell (Davis et al., 1994; Winslow et al., 1995). In addition, soluble ephrins Zebrafish Ephrin B3 Is Expressed in the Prechordal also block signaling in the ligand-bearing cell by occupying Plate and Notochord Eph receptors and preventing endogenous ephrins from Localized ephrin B3 expression is first noted at 80% interacting with their cognate receptors. Overexpression of epiboly in the prechordal plate and presumptive notochord soluble forms of each of the four ligands generated similar (Figs. 5A, 5B, and 5C). Ephrin B3 expression is also promi- phenotypes (data not shown), presumably due to promiscu- nent in cells of the hypoblast (Figs. 5B and 5C). The ephrin ous binding with several Eph receptors (Brambrilla et al.,

FIG. 4. Expression of ephrin B2a by in situ antisense RNA hybridization (A–H). Ephrin B2a staining was visualized with a blue stain, in every panel. Antisense marker RNA probes were krx-20 and pax2.1, visualized with a red/orange stain. Dorsal views (except B, E, F, H lateral views; and transverse section in G), anterior is to the left. (A, B) 70% epiboly. Ephrin B2a is expressed in the germ ring and shield in A. Expression is seen in the hypoblastic layer. (C) Eight somites, krx-20/pax2.1. Ephrin B2a expression is detected in the presumptive forebrain, diffusely in the eye field, and just caudal to the midbrain–hindbrain boundary as well as in prospective rhombomeres 1, 4, 7, and in 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 forms a striking ventral-to-dorsal stripe. (E) 24 h, pax2.1. Eye expression of ephrin B2a is confined to the lens primordium and developing retina in the dorsal–temporal quadrant. (F, G) 24 h, transverse section in G. Ephrin B2a expression is seen in the endothelial cells of the dorsal artery and is morphologically confirmed by location of the staining ventral to the notochord. (H) 36 h, eyes removed. Expression is seen in the ventral hypothalamus and in the dorsal midline of the tectum. Expression is seen in the telencephalon. Abbreviations: pn, presumptive notochord; r3, rhombomere 3; r5, rhombomere 5; e, epiphysis; t, telencephalon; n, notochord; da, dorsal artery; tec, tectum; pcp, prechordal plate. Scale bars for A, E, F, G ϭ 100 ␮m. FIG. 5. Expression of ephrin B3 by in situ antisense RNA hybridization (A–I). Ephrin B3 staining was visualized with a blue stain, in every panel. Antisense marker RNA probes were gsc, hgg1, krx-20, myoD, and pax2.1, visualized with a red/orange stain. Dorsal views (except B–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 axial mesendoderm, with higher expression in the rostral and caudal ends in the hypoblastic layer (prechordal plate and presumptive notochord), where its anterior end is coincident with the gsc expression domain. (D) 90% epiboly, hgg1. Expression is seen in the overlaying neurectoderm, 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 in F, 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 the rostral telencephalon and ventral hypothalamus, as well as in rhombomere 4. (I) 48 h. Expression is seen in the proliferating zone of the tectum. Abbreviations: pcp, prechordal plate; pn, presumptive notochord; r3, r4, r5, rhombomeres 3, 4, 5; e, epiphysis; t, telencephalon; tec, tectum. Scale bars for A, F ϭ 100 ␮m.

FIG. 6. Effect of sol-ephrin B2b overexpression by the end of gastrulation. Analysis of morphological defects by of Eph/ephrin B disruption at the tailbud stage (A–L). Sol-ephrin B2b mRNA or control GFP mRNA were injected into one-cell stage zebrafish embryos and fixed at the tailbud stage. Embryos were hybridized with digoxigenin-labeled or fluorescein-labeled antisense RNA marker probes: gsc, hgg1, hlx, myoD, ntl, opl, papc, pax2.1, for analysis of defects in embryo morphology. (A–L) Dorsal views; A, C, E, G, I, K, control embryos; B, D, F, H, J, L, sol-ephrin B2b-injected embryos. (A, B) hgg1, pax2.1 in blue; ntl in red. In the injected embryo, B, the hgg1, pax2.1, and ntl expression domains appear widened compared with control. (C, D) gsc in red; pax2.1 in blue. The gsc expression in the prechordal plate appears shortened and broadened. The midbrain–hindbrain boundary is also laterally expanded as indicated by the pax2.1 staining in D. The injected embryo appears more spherical in shape. (E, F) hlx in blue; opl, pax2.1 in red. Higher magnification of embryos indicate a consistently shorter and wider prechordal plate domain as shown by the hlx staining. The opl staining of the neural plate and pax2.1 staining also demonstrate lateral expansion of both neural plate and the midbrain–hindbrain boundary. On the left side of the injected embryo, F, hlx-positive cells are detected in the region of the future midbrain–hindbrain boundary toward the neural plate (arrow). (G–H) myoD, in blue; ntl, in red. Injected embryos in F and H represent a more severe phenotype. The ntl staining of the notochord is juxtaposed with the myoD staining of adaxial cells in the paraxial mesoderm. The notochord/paraxial mesoderm border in the injected embryo is irregular in H, compared with control, G. (I–J) hlx, papc in blue; ntl, pax2.1 in red. Higher magnification of the same embryos in K, L. The anterior staining is consistent with C and D, with shortening and widening of the prechordal plate and notochord domains and lateral extension of the midbrain–hindbrain boundary. Ntl staining of the notochord staining is juxtaposed with papc staining of the paraxial mesoderm. The notochord/paraxial mesoderm boundary is indicated by the arrowheads in control (K) versus injected embryo (L). Abbreviations: pcp, prechordal plate; n, notochord; r3, r5, rhombomeres 3, 5; tec, tectum; mhb, midbrain–hindbrain boundary.

1995; Gale et al., 1996a). Since ephrin B2b expression is embryo lysates by detection with an anti-myc antibody specifically detected at the future dorsal side of the 30% (data not shown). epiboly stage embryo (Figs. 3A and 3B), we used a soluble Overexpression of sol-ephrin-B2b proteins resulted in a form of the ephrin B2b cDNA construct, sol-ephrin B2b,in dose-dependent defect in gastrula morphology evident by all subsequent experiments. To ensure that the sol-ephrin visual analysis using a dissecting microscope and confirmed B2b proteins were expressed in the embryo, a myc-tagged by in situ RNA analysis with marker genes (see Table 1 and version of the same construct was used in microinjection Fig. 6). More severe defects were observed in a larger experiments to confirm protein expression in zebrafish proportion of embryos injected with 40 pg of sol-ephrin-B2b

TABLE 1 Frequency of Gastrula Defects as Judged by In Situ Staining Using Prechordal Plate and Notochord Markersa

Quantity of Total number of Phenotypically Severe gastrula Mild gastrula Type of mRNA mRNA injected injected embryos normal defect defect Death injected (pg) (N) b (n,%)c (n,%) (n,%) (n,%) sol-ephrin-B2b 20 170 61, 36% 59, 35% 43, 25% 7, 4% shift-ephrin-B2b 20 182 178, 98% 0, 0% 0, 0% 4, 2% GFP 20 185 181, 98% 0, 0% 0, 0% 4, 2%

a Embryos were injected at the one-cell stage with the mRNAs listed above, then allowed to develop to tailbud stage, ﬁxed and stained with in situ markers, then grouped and counted for phenotypic differences. b N, total number of embryos injected in this experiment. c n, number of injected embryos scored for phenotypically normal, severe, or mild gastrula defects after in situ staining with prechordal plate marker hlx and notochord marker ntl. These numbers are also expressed as a percentage (%) of the total number injected, N.

mRNA, whereas very mild defects were obtained when we derm (Yamamoto et al., 1998). In the sol-ephrin B2b in- injected the sol-ephrin-B2b mRNA at 4 pg. At 20 pg of jected embryo, the neural plate, prechordal plate, and sol-ephrin B2b mRNA, we could easily detect visible prob- presumptive midbrain–hindbrain domains were widened as lems with gastrula morphology in about 30% of injected indicated by opl, hgg1, gsc, hlx, and pax2.1 staining (Figs. embryos; therefore, this concentration was used in all 6A–6F, 6I, and 6J). In addition, some hlx-positive prechordal subsequent experiments (see Table 1 and Fig. 6). Control plate cells are inappropriately located in the lateral region of injections of RNA for the green fluorescent protein, GFP,or the prospective forebrain–midbrain boundary on the left a frame-shifted version of sol-ephrin-B2b, shift-ephrin-B2b, side of the embryo (Fig. 6F, arrow). To examine the had no effect on embryo morphology (Table 1). notochord/presomitic mesoderm border, ntl and myoD In sol-ephrin B2b-injected embryos the distance between probes were used as markers for notochord precursor cells the anterior limit of the prechordal plate and the margin of and adaxial cells (Figs. 6G and 6H), respectively; while the the gastrulating embryo was reduced from as early as papc probe was used to define the presomitic mesoderm 70–80% epiboly by visual analysis. By the tailbud stage, (Figs. 6I–6L). The sol-ephrin B2b mRNA-injected embryo injected embryos were significantly shorter along the ante- showed broadening of notochordal domain with an irregular rioposterior axis and showed a more spherical shape when border between the cells in the presomitic mesoderm and compared to wild-type embryos (Figs. 6C, 6D, 6I, and 6J). those in the notochord (Figs. 6G, 6H, 6K, and 6L). In a more The notochords in these embryos were found to be much severe sol-ephrin B2b overexpression phenotype (Figs. 6G wider (Figs. 6B, 6H, 6J, and 6L) than in controls (Fig. 6A, 6G, and 6H), notochord widening is accompanied by misloca- 6I, and 6K). At later stages of development, up to and tion of some myoD-positive cells in the paraxial mesoderm including the 18-somite stage, injected embryos were still (Fig. 6H, arrowhead). Thus, Eph/ephrin B interference likely found to be shorter along the anterioposterior axis and to generated cell movement defects during gastrulation, lead- have widened notochords (data not shown). The malforma- ing to the improper location of these marker positive cells. tions obtained by disruption of Eph/ephrin B signaling are The altered morphology of various tissues examined is phenotypically similar to some of the zebrafish gastrulation consistent with a defect in convergent extension move- mutants with defects in convergent extension movements ments affecting both axial and paraxial tissues. However, (Solnica-Krezel et al., 1996; Hammerschmidt et al., 1996). since all tissues examined are present at the tailbud stage, our Eph/ephrin B disruption does not appear to alter cell fate determination by the end of gastrulation. Eph/Ephrin B Disruption Alters Cell Movement but Does Not Affect Cell Specification by the End of Gastrulation DISCUSSION To investigate further the altered gastrula morphology caused by our Eph/ephrin B interference experiment, we Molecular Cloning and Implications of the examined the expression domains of a number of marker Zebrafish Ephrin B Sequences genes for the prechordal plate and notochord, the anterior We have isolated four full-length cDNAs encoding mem- neural plate, as well as the paraxial mesoderm at the tailbud bers of the zebrafish ephrin B family that represent the three stage (Fig. 6). These include markers used previously, in known mammalian B-ephrins (Fig. 1; Gale et al., 1996a,b). addition to the odd-paired-like gene, opl, which defines the In the zebrafish, some genes have been duplicated (Postleth- anterior neural plate (Grinblat et al., 1998), and the pro- wait et al., 1998); therefore, it is likely that ephrin B2a and tocadherin papc gene which demarcates presomitic meso- ephrin B2b are both paralogs of mammalian ephrin B2. The

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Eph/Ephrin B Signaling in Morphogenesis 479 preservation and expression of both ephrin B2 homologs in are expressed during gastrulation, suggesting a role in the zebrafish suggest that they each developed an important cell–cell recognition (Figs. 2–5; Xu et al., 1994; Cooke et al., role during zebrafish embryogenesis (Postlethwait et al., 1997; Durbin et al., 1998). Of the four zebrafish B-ephrins, 1998; Force et al., 1999). Their expression patterns in we decided to use the sol-ephrin-B2b construct for micro- distinct compartments would allow them to interact with injection into zebrafish embryos since ephrin B2b displays different Eph receptors on adjacent cells (see Figs. 3 and 4). early localized expression at the future dorsal side of a 30% The subtle changes in their predicted amino acid sequences epiboly stage embryo (Figs. 3A and 3B). This suggests that may also allow the two proteins to exhibit differential the ephrin B2b protein is required early on to interact with receptor-binding affinities and to initiate alternative signal- Eph receptors in vivo, thus, playing a role in cell–cell ing cascades. signaling before the onset of gastrulation. Previous studies Although the extracellular domain is most divergent established roles for Eph/ephrin signaling in the control of among mammalian and zebrafish B-ephrins, the homology cell migrations in a number of developmental systems is still quite high, at ϳ58% identity. Thus, zebrafish and (reviewed in Holder and Klein, 1999). These observations mammalian B-ephrins likely display similar protein folding suggest that Eph/ephrin B interactions may also be involved and receptor-binding characteristics. This notion is further in the control of morphogenetic cell movements underlying bolstered by the absolute conservation of four invariant gastrulation. cysteines in the extracellular domain (Fig. 1; reviewed in As both Eph receptors and ephrin Bs are capable of Pandey et al., 1995). In addition, the zebrafish ephrin B interacting with PDZ domain-scaffolding molecules and ectodomain contains a motif related to the ephrin A2 have been implicated in signaling cytoskeletal changes, it is metalloprotease cleavage site (8 out of 10 residues conform likely that both ligand- and receptor-bearing cells partici- to the consensus; Hattori et al., 2000). This motif is pate in cell movement events (Torres et al., 1998; reviewed virtually identical in all four zebrafish ephrin Bs (amino in Kalo and Pasquale, 1999). Prevention of Eph/ephrin B acids 106–115 of zebrafish ephrin B1, Fig. 1A) and is also signaling caused defects in embryo morphology consistent conserved in the mammalian B-ephrins (Gale et al., 1996b; with incorrect convergent extension. Both the convergent Bergemann et al., 1998). Thus, like the A-ephrins, B-ephrins movement of cells toward the dorsal midline and the may also be cleaved after receptor engagement to mediate mediolateral cell intercalations were affected as evident in signal termination and repulsive movements (Hattori et al., the shorter and wider notochord and irregular formation of 2000). The intracellular domain among zebrafish and mam- the notochord/paraxial mesoderm boundary (Fig. 6). How- malian B-ephrins exhibits an even greater homology, sug- ever, disruption of Eph/ephrin signaling had no significant gesting interactions with similar downstream effector mol- effect on the relative size of notochord, prechordal plate, ecules to initiate bidirectional signaling (Holland et al., neural plate, or paraxial mesoderm. Thus, the specification 1996; Bruckner et al., 1997). In addition to the five con- of these domains, at least at a gross level, appears unaffected served tyrosines, the last seven amino acids, NIYYKV, form by the end of gastrulation. Previous experiments with a PDZ domain-binding site and are identical among all Eph/ephrin B disruption generated a loss of myoD expres- mammalian and zebrafish B-ephrins (Fig. 1; Torres et al., sion within the mesoderm of injected embryos (Durbin et 1998). In mammalian B-ephrins, this site binds PDZ al., 1998). It is possible that at later stages, the defects in domain-containing proteins (Lin et al., 1999; Bruckner et somite boundary formation could have resulted as a conse- al., 1999; Torres et al., 1998), a protein–protein interaction quence of the gastrulation effect and/or as a result of the domain found in many cell surface proteins, neuronal continued presence of the dominant-interfering B-ephrin. In channels, and transporters (reviewed in Sheng, 1996). Thus, our hands, we also saw a reduction or disruption of myoD zebrafish B-ephrins may also be able to interact with staining at somitogenesis stages, suggesting an alteration in PDZ-domain proteins to allow formation and colocaliza- somite patterning (data not shown; Durbin et al., 1998). tion of large clusters of signaling molecules. Molecular Mechanisms Driving Convergent Eph/Ephrin B Signaling Regulates Cell Movements Extension Movements Underlying Gastrulation Our Eph/ephrin B interference phenotype resembles the During zebrafish gastrulation, coordinated morphoge- zebrafish mutants defective in gastrulation cell movement netic movements such as epiboly, convergent extension, such as silberblick (slb), trilobite (tri), and knypek (kny) and involution or ingression are involved in the formation (Heisenberg et al., 2000; Heisenberg and Nusslein-Volhard, of the three germ layers (Warga and Kimmel, 1990; re- 1997; Marlow et al., 1998). These mutants also display a viewed in Solnica-Krezel et al., 1995). During convergent shortened embryo axis without an apparent reduction in extension, cells converge toward the dorsal midline of the the size of various expression domains. The gene respon- embryo and mediolateral cell intercalations occur to further sible for the slb mutation was recently identified as the extend the anterioposterior body axis (Warga and Kimmel, wnt11 homolog in the zebrafish (Heisenberg et al., 2000). 1990; Kimmel et al., 1994; Gont et al., 1993; Davidson and Studies on the function of slb/wnt11 in zebrafish and Keller, 1999). Zebrafish B-ephrins and several Eph receptors Xenopus suggest that this member of the wnt family

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 480 Chan et al. regulates cell movements rather than cell fate determina- thank Dana Witten for help with the digital camera setup. Finally, tion (Heisenberg et al., 2000; Tada and Smith, 2000). In we are grateful to George Serbedzija, Andres Collazo, Jeremy zebrafish and Xenopus experiments, loss of normal Slb/ Green, Chuck Stiles, and Ole Gjoerup for critical reading of the Wnt11 function resulted in abnormal morphogenesis that manuscript. This work was supported by NIH grants to T.M.R. can be rescued by a truncated form of disheveled that (HD24926) and M.C.F. (HL49579). In compliance with Harvard Medical School guidelines on possible conflict of interest, we cannot activate GSK-3 and ␤-catenin. Thus, Slb/Wnt11 is disclose that one of the authors (T.M.R.) has consulting relation- thought to specifically regulate morphogenetic movements ships with Upstate Biotechnology and Novartis Pharmaceuticals, through a noncanonical wnt pathway (Heisenberg et al., Inc. 2000; Tada and Smith, 2000). Another molecule that appears to directly influence convergent extension is the paraxial protocadherin, PAPC (Yamamoto et al., 1998; Kim et al., 1998). PAPC has potent REFERENCES homotypic cell adhesion activity demonstrated in cell dis- sociation and reaggregation assays. It also plays a role in morphogenesis as dominant-negative forms of PAPC inhib- Anonymous. (1997). Unified nomenclature for Eph family recep- its convergent extension (Yamamoto et al., 1998; Kim et al., tors and their ligands, the ephrins. Eph Nomenclature Commit- tee [letter]. Cell 90, 403–404. 1998). Although no direct link has been made between the Bergemann, A. D., Zhang, L., Chiang, M. K., Brambilla, R., Klein, Eph/ephrin Bs, Slb/Wnt11 and PAPC, these molecules share R., and Flanagan, J. G. (1998). Ephrin-B3, a ligand for the receptor some common structural and signaling features and may EphB3, expressed at the midline of the developing neural tube. work to coordinate morphogenetic events essential for Oncogene 16, 471–480. proper gastrulation. Both PAPC and Slb/Wnt11 are nor- Brambilla, R., Schnapp, A., Casagranda, F., Labrador, J. P., Berge- mally expressed in paraxial tissues during late gastrulation mann, A. D., Flanagan, J. G., Pasquale, E. B., and Klein, R. (1995). (Yamamoto et al., 1998; Heisenberg et al., 2000). Genetic Membrane-bound LERK2 ligand can signal through three differ- and experimental evidence show that Slb/Wnt11 partici- ent Eph-related receptor tyrosine kinases. EMBO J. 14, 3116– pates in the mediolateral cell intercalation events that drive 3126. the convergent extension movements (Heisenberg et al., Bruckner, K., Pablo Labrador, J., Scheiffele, P., Herb, A., Seeburg, 2000). The early expression of ephrin B2b and papc at the P. H., and Klein, R. (1999). Ephrin-B ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neu- future dorsal side by 30% epiboly suggests a requirement ron 22, 511–524. for these proteins before gastrulation begins (Figs. 3A and Bruckner, K., Pasquale, E. B., and Klein, R. (1997). Tyrosine 3B; Yamamoto et al., 1998). The zebrafish EphB receptor, phosphorylation of transmembrane ligands for Eph receptors. rtk3 (Xu et al., 1994), as well as ephrin B2b (Fig. 3D), are Science 275, 1640–1643. expressed in the paraxial mesoderm during late gastrula Cheng, H. J., Nakamoto, M., Bergemann, A. D., and Flanagan, J. G. stages and may regulate the proper alignment of cells at the (1995). Complementary gradients in expression and binding of dorsal midline. Thus, Eph/ephrin Bs likely contribute to the ELF-1 and Mek4 in development of the topographic retinotectal coordination of morphogenetic movements along with se- projection map. Cell 82, 371–381. creted factors and cell-adhesion molecules such as Slb/ Cooke, J. E., Xu, Q., Wilson, S. W., and Holder, N. (1997). Charac- Wnt11 and PAPC. terisation of five novel zebrafish Eph-related receptor tyrosine Our expression and Eph/ephrin B interruption data sup- kinases suggests roles in patterning the neural plate. Dev. Genes port a role for these molecules in convergent extension Evol. 206, 515–531. Davidson, L. A., and Keller, R. E. (1999). Neural tube closure in during gastrulation. Disruption of Eph/ephrin B signaling Xenopus laevis involves medial migration, directed protrusive resulted in the abnormal cell movements that led to mor- activity, cell intercalation and convergent extension. Develop- phogenetic defects in the prechordal plate and notochord, as ment 126, 4547–4556. well as in paraxial tissues. The availability of these four Davis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak, ephrin B cDNAs should provide useful reagents to dissect V., Pawson, T., Goldfarb, M., and Yancopoulos, G. D. (1994). further the role of Eph/ephrin B signaling during zebrafish Ligands for EPH-related receptor tyrosine kinases that require development. membrane attachment or clustering for activity. Science 266, 816–819. Davy, A., Gale, N. W., Murray, E. W., Klinghoffer, R. A., Soriano, ACKNOWLEDGMENTS P., Feuerstein, C., and Robbins, S. M. (1999). Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine We dedicate this paper to the memory of our friend Nigel Holder. kinase to regulate cellular adhesion. Genes Dev. 13, 3125–3135. Our thanks to John Flanagan for providing mouse cDNA clones for Davy, A., and Robbins, S. M. (2000). Ephrin-A5 modulates cell ephrin B1 and ephrin B2. We are indebted to George Serbedzija for adhesion and morphology in an integrin-dependent manner. discussions and technical advice. We express our gratitude to Steve EMBO J. 19, 5396–5405. Wilson, Ajay Chitnis, Jeremy Green, and Chuck Stiles for helpful Durbin, L., Brennan, C., Shiomi, K., Cooke, J., Barrios, A., Shan- discussions during the course of this study. The help of Henry mugalingam, S., Guthrie, B., Lindberg, R., and Holder, N. (1998). Roehl, Andreas Vogel, Yevgenya Grinblat, and Hazel Sive in Eph signaling is required for segmentation and differentiation of providing in situ marker cDNAs is also much appreciated. We the somites. Genes Dev. 12, 3096–3109.

Flanagan, J. G., and Vanderhaeghen, P. (1998). The ephrins and Eph Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U., and receptors in neural development. Annu. Rev. Neurosci. 21, Fjose, A. (1991). Zebrafish pax[zf-a]: A paired box-containing gene 309–345. expressed in the neural tube. EMBO J. 10, 3609–3619. Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L., and Lin, D., Gish, G. D., Songyang, Z., and Pawson, T. (1999). The Postlethwait, J. (1999). Preservation of duplicate genes by carboxyl terminus of B class ephrins constitutes a PDZ domain complementary, degenerative mutations. Genetics 151, 1531– binding motif. J. Biol. Chem. 274, 3726–3733. 1545. Marlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S. C., Abbas, L., Gale, N. W., Flenniken, A., Compton, D. C., Jenkins, N., Copeland, Weaver, M., Driever, W., and Solnica-Krezel, L. (1998). Func- N. G., Gilbert, D. J., Davis, S., Wilkinson, D. G., and Yancopou- tional interactions of genes mediating convergent extension, los, G. D. (1996a). Elk-L3, a novel transmembrane ligand for the knypek and trilobite, during the partitioning of the eye primor- Eph family of receptor tyrosine kinases, expressed in embryonic dium in zebrafish. Dev. Biol. (Orlando) 203, 382–399. floor plate, roof plate and hindbrain segments. Oncogene 13, Mellitzer, G., Xu, Q., and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. 1343–1352. Nature 400, 77–81. Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, Mellitzer, G., Xu, Q., and Wilkinson, D. G. (2000). Control of cell L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., behaviour by signalling through Eph receptors and ephrins. Curr. Wilkinson, D. G., Pawson, T., Davis, S., and Yancopoulos, G. D. Opin. Neurobiol. 10, 400–408. (1996b). Eph receptors and ligands comprise two major specific- Nakamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T., ity subclasses and are reciprocally compartmentalized during Hansen, M. J., Yoon, C. H., O’Leary, D. D., and Flanagan, J. G. Neuron embryogenesis. 17, 9–19. (1996). Topographically specific effects of ELF-1 on retinal axon Gont, L. K., Steinbeisser, H., Blumberg, B., and de Robertis, E. M. guidance in vitro and retinal axon mapping i. Cell 86, 755–766. (1993). Tail formation as a continuation of gastrulation: The Oxtoby, E., and Jowett, T. (1993). Cloning of the zebrafish krox-20 multiple cell populations of the Xenopus tailbud derive from the gene (krx-20) and its expression during hindbrain development. late blastopore lip. Development 119, 991–1004. Nucleic Acids Res. 21, 1087–1095. Grinblat, Y., Gamse, J., Patel, M., and Sive, H. (1998). Determina- Pandey, A., Lindberg, R. A., and Dixit, V. M. (1995). Cell signalling. tion of the zebrafish forebrain: Induction and patterning. Devel- Receptor orphans find a family. Curr. Biol. 5, 986–989. opment 125, 4403–4416. Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A., Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., Brownlie, A., Donovan, A., Egan, E. S., Force, A., Gong, Z., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Goutel, C., Fritz, A., Kelsh, R., Knapik, E., Liao, E., Paw, B., Haffter, P., Heisenberg, C. P., Jiang, Y. J., Kelsh, R. N., Odenthal, Ransom, D., Singer, A., Thomson, M., Abduljabbar, T. S., Yelick, J., Warga, R. M., and Nusslein-Volhard, C. (1996). Mutations P., Beier, D., Joly, J. S., Larhammar, D., Rosa, F., et al. (1998). affecting morphogenesis during gastrulation and tail formation Vertebrate genome evolution and the zebrafish gene map. Nat. in the zebrafish, Danio rerio. Development 123, 143–151. Genet. 18, 345–349. Hattori, M., Osterfield, M., and Flanagan, J. G. (2000). Regulated Schulte-Merker, S., Ho, R. K., Herrmann, B. G., and Nusslein- cleavage of a contact-mediated axon repellent. Science 289, Volhard, C. (1992). The protein product of the zebrafish homo- 1360–1365. logue of the mouse T gene is expressed in nuclei of the germ ring Heisenberg, C. P., and Nusslein-Volhard, C. (1997). The function of and the notochord of the early embryo. Development 116, silberblick in the positioning of the eye anlage in the zebrafish 1021–1032. embryo. Dev. Biol. (Orlando) 184, 85–94. Sheng, M. (1996). PDZs and receptor/channel clustering: Rounding Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., up the latest suspects. Neuron 17, 575–578. Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000). Solnica-Krezel, L., Bailey, J., Gruer, D. P., Price, J. M., Dove, W. F., Silberblick/Wnt11 mediates convergent extension movements Dee, J., and Anderson, R. W. (1995). Characterization of npf mutants identifying developmental genes in Physarum. Micro- during zebrafish gastrulation. Nature 405, 76–81. biology 141, 799–816. Holder, N., and Klein, R. (1999). Eph receptors and ephrins: Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E., Rangini, Effectors of morphogenesis. Development 126, 2033–2044. Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D. Y., Holland, S. J., Gale, N. W., Mbamalu, G., Yancopoulos, G. D., Zwartkruis, F., Abdelilah, S., and Driever, W. (1996). Mutations Henkemeyer, M., and Pawson, T. (1996). Bidirectional signalling affecting cell fates and cellular rearrangements during gastrula- through the EPH-family receptor Nuk and its transmembrane tion in zebrafish. Development 123, 67–80. ligands. Nature 383, 722–725. Tada, M., and Smith, J. C. (2000). Xwnt11 is a target of Xenopus Kalo, M. S., and Pasquale, E. B. (1999). Signal transfer by Eph Brachyury: Regulation of gastrulation movements via Dishev- receptors. Cell Tissue Res. 298, 1–9. elled, but not through the canonical Wnt pathway. Development Kim, S. H., Yamamoto, A., Bouwmeester, T., Agius, E., and 127, 2227–2238. Robertis, E. M. (1998). The role of paraxial protocadherin in Thisse, C., Thisse, B., Halpern, M. E., and Postlethwait, J. H. (1994). selective adhesion and cell movements of the mesoderm during Goosecoid expression in neurectoderm and mesendoderm is Xenopus gastrulation. Development 125, 4681–4690. disrupted in zebrafish cyclops gastrulas. Dev. Biol. (Orlando) Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and 164, 420–429. Schilling, T. F. (1995). Stages of embryonic development of the Torres, R., Firestein, B. L., Dong, H., Staudinger, J., Olson, E. N., zebrafish. Dev. Dyn. 203, 253–310. Huganir, R. L., Bredt, D. S., Gale, N. W., and Yancopoulos, G. D. Kimmel, C. B., Warga, R. M., and Kane, D. A. (1994). Cell cycles (1998). PDZ proteins bind, cluster, and synaptically colocalize and clonal strings during formation of the zebrafish central with Eph receptors and their ephrin ligands [see comments]. nervous system. Development 120, 265–276. Neuron 21, 1453–1463.

Turner, D. L., and Weintraub, H. (1994). Expression of achaete- receptor involved in axon bundle formation. Neuron 14, 973– scute homolog 3 in Xenopus embryos converts ectodermal cells 981. to a neural fate. Genes Dev. 8, 1434–1447. Xu, Q., Alldus, G., Holder, N., and Wilkinson, D. G. (1995). Vogel, A. M., and Gerster, T. (1997). Expression of a zebrafish Expression of truncated Sek-1 receptor tyrosine kinase disrupts Cathepsin L gene in anterior mesendoderm and hatching gland. the segmental restriction of gene expression in the Xenopus and Dev. Genes Evol. 206, 477–479. zebrafish hindbrain. Development 121, 4005–4016. Warga, R. M., and Kimmel, C. B. (1990). Cell movements during Xu, Q., Holder, N., Patient, R., and Wilson, S. W. (1994). Spatially epiboly and gastrulation in zebrafish. Development 108, 569– regulated expression of three receptor tyrosine kinase genes during gastrulation in the zebrafish. Development 120, 287–299. 580. Yamamoto, A., Amacher, S. L., Kim, S. H., Geissert, D., Kimmel, Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A., C. B., and De Robertis, E. M. (1998). Zebrafish paraxial protocad- Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J., herin is a downstream target of spadetail involved in morpho- and Riggleman, B. (1996). Developmental regulation of zebrafish genesis of gastrula mesoderm. Development 125, 3389–3397. MyoD in wild-type, no tail and spadetail embryos. Development 122, 271–280. Received for publication October 18, 2001 Winslow, J. W., Moran, P., Valverde, J., Shih, A., Yuan, J. Q., Wong, Revised March 28, 2001 S. C., Tsai, S. P., Goddard, A., Henzel, W. J., Hefti, F. et al. (1995). Accepted March 28, 2001 Cloning of AL-1, a ligand for an Eph-related tyrosine kinase Published online May 11, 2001