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- 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 470 All rights of reproduction in any form reserved. Eph/Ephrin B Signaling in Morphogenesis 471 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 strin- gency 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