zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis

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Developmental Biology 229, 102–118 (2001)doi:10.1006/dbio.2000.9967, available online at http://www.idealibrary.com on

Zebrafish mdk2, a Novel Secreted Midkine,Participates in Posterior Neurogenesis

Christoph Winkler*,† and Randall T. Moon**Howard Hughes Medical Institute, Department of Pharmacology, and Center forDevelopmental Biology, University of Washington School of Medicine, Seattle,Washington 98195-7750; and †Department of Physiological Chemistry I,Biocenter, University of Wuerzburg, 97074 Wuerzburg, Germany

Patterning the neural plate in vertebrates depends on complex interactions between a variety of secreted growth factors.Here we describe a novel secreted factor in zebrafish, named mdk2, related to the midkine family of heparin-binding growthfactors that is involved in posterior neural development. mdk2 is expressed shortly after the onset of gastrulation in thepresumptive neural plate cells of the epiblast, and this expression is enhanced by exogenous retinoic acid. Ectopic expressionof mdk2 enhances neural crest cell fates at the lateral edges of the caudal neural plate, concomitant with a repression ofnterior structures and mesendodermal and ectodermal markers. Reciprocally, ectopic expression of a dominant negativedk2 results in severe deficiencies of structures posterior to the midbrain–hindbrain boundary, with negligible effects on

nterior structures. In these embryos, the expression of hindbrain and neural crest markers is strongly reduced, and theormation of posterior primary moto- and sensory neurons is blocked. Analyses in mutant zebrafish embryos shows thatxpression of mdk2 is independent of FGF8 and nodal-related-1 signaling, but is under negative control of BMP signaling.hese data support the hypothesis that mdk2 participates in posterior neural development in zebrafish. © 2001 Academic Press

Key Words: neural induction; neural patterning; midkines; neural crest; posteriorization; planar signaling; heparin-binding growth factors; expression cloning.

INTRODUCTION

Based on transplantation experiments in amphibian em-bryos Nieuwkoop postulated a two-step model for neuralinduction and patterning (Toivonen and Saxen, 1968; Nieuw-koop, 1985). In a first “activation” step signals from thedorsal lip or “Spemann organizer” of the gastrula instructoverlaying ectoderm to become neuroectoderm, whichoriginally shows anterior characteristics. In a second“transformation” step this anterior neural tissue is progres-sively shifted toward more posterior fates by signals thatoriginate either from the organizer-derived axial mesodermunderlying the neuroectoderm (vertical signaling) or fromsignals traveling through the plane of the ectoderm (planarinduction).

The first neural inducer identified in frogs, noggin, wasisolated in an activity screen for axis-rescuing molecules(Smith and Harland, 1992). Noggin and chordin (Sasai et al.,994) are secreted from the Spemann organizer and bindith high affinity to members of the BMP family (Zimmer-
an et al., 1996; Piccolo et al., 1996). By blocking the
102

ability of BMP to induce epidermal cell fates, the BMPantagonists allow neural induction to occur as a defaultpathway (reviewed by Hemmati-Brivanlou and Melton,1997). In zebrafish, mutants lacking the chordin gene showa greatly decreased neural plate (Hammerschmidt et al.,1996). Thus, BMP antagonists are expressed in the rightplace and time, with the appropriate activities, to partici-pate in Nieuwkoop’s activation step of neural induction.

Signaling factors that may promote posterior neural cellfates and thus function in the transformation step aremembers of both the Wnt (McGrew et al., 1995, 1999) andthe FGF (Cox and Hemmati-Brivanlou, 1995; Lamb andHarland, 1995) families. They have been shown to workcooperatively (McGrew et al., 1997) in the presence of BMPantagonists to induce the expression of posterior neuralmarkers in Xenopus animal cap explants. Recent worksuggests that Wnt signaling, in addition to its caudalizingactivity, is involved in the earliest phase of neural induc-tion by antagonizing BMP4 and sensitizing ectoderm tobecome neural (Baker et al., 1999). Wnts and FGFs are
expressed in posterior dorsal mesoderm during gastrulation
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103Neural Patterning by Zebrafish mdk2

and are therefore candidates for vertical inducers or modi-fiers. A third posteriorizing molecule known to be involvedin pattern formation and neurogenesis in vertebrates isretinoic acid (see Muhr et al., 1999; Pierani et al., 1999).

Molecular models of neural induction and patterning alsoneed to consider the likely involvement of additional fac-tors (reviewed by Streit and Stern, 1999). Transplantationand cell ablation experiments in zebrafish (Woo and Fraser,1997; Houart et al., 1998) suggest that signals other thanorganizer-derived BMP antagonists may be responsible forthe induction and patterning of the early nervous system.Transplantation studies also suggest that planar rather thanvertical signals play an important role in neural inductionand patterning in zebrafish (Woo and Fraser, 1998). This issupported by analysis of cyclops/squint double-mutant em-ryos that lack axial mesoderm and endoderm but developnervous system with a strikingly normal pattern along thenteroposterior axis (Feldman et al., 1998). The molecular

nature of these signals that induce and pattern the earlynervous system in fish are unknown. We therefore designeda cross-species strategy to identify novel factors in zebrafishthat possess the activity to induce neural gene expressionusing Xenopus animal cap explants. We report the isolationof a novel secreted factor, mdk2, that is expressed duringgastrulation and we present evidence suggesting that it isrequired for posterior neural development of zebrafish.

MATERIALS AND METHODS

Isolation of Zebrafish mdk2 by Expression CloningUsing Xenopus Animal Caps

An adult zebrafish brain cDNA library (a gift from J. Ngai,University of California at Berkeley) cloned into l-ZipLox (GibcoBRL) was used to screen for factors with neural-inducing activities.DNA from phage pools was either obtained directly by standardPEG purification or after excision of phage inserts into pZL1plasmids (Gibco BRL) and RNAs were transcribed from DNA poolsusing the Ambion SP6 mMessage mMachine Kit (McGrew et al.,1997). Between 1 and 10 ng of synthetic RNA was injected intoanimal poles of both blastomeres of a two-cell stage Xenopusembryo. Twenty embryos per analyzed pool were raised to stage 8and animal caps were dissected and cultured in 13 MBS (Gurdonand Wickens, 1983) until control siblings reached stage 21. Thenthe induction of neural marker expression was determined byRT-PCR as described (McGrew et al., 1997). In the first round ofscreening pools with 50,000 independent clones by RT-PCR, weidentified one pool that induced expression of the panneuralmarker NCAM. After four rounds of sib selection and activityscreening three independent subpools were isolated, with 200clones each, that induced expression of neural markers (data notshown). Interestingly, all three subpools induced different patternsof neural markers, suggesting that different neural-inducing activi-ties were present and that the initially observed NCAM inductionin the original pool likely resulted from an interplay of differentfactors. One subpool of 200 independent clones was further sibselected by activity screening of pools of 23 clones and, finally,single clones, resulting in the isolation of independent clones with
neural-inducing activity. As the pool of 23 clones showed stronger
Copyright © 2001 by Academic Press. All right

neural-inducing activity than each single factor alone, we concludethat both factors act synergistically, with each other or withadditional factors in the pool.

RNA Injections into Zebrafish Embryos

The regions containing the coding sequence for full-lengthmdk2 (aa 1–147) and C-terminally truncated versions mdkD38 (aa1–73) and mdkD35 (aa 1–98) were amplified from the pZL plasmidcarrying the excised phage insert using the following primerscontaining a consensus Kozak site, in-frame stop codon, andupstream BamHI and downstream XhoI restriction sites: mk00359-GGGGATCCCCACCATGCGGAGTTTGTTCTC-39) as up-

stream primer and mk004 (59-GGCTCGAGCAAGTTAGTTT-TCCTTCCC-39), mk008 (59-GGGCTCGAGCTAGACTTTGCA-CTTGGTTTTC-39), and mk005 (59-GGGCTCGAGCTAAGTA-GTGTCACATTCGGC-39) as downstream primers, respectively.For construction of a MYC-epitope tagged mdk2 version, prim-ers mk003 and mk014 (59-GGGCTCGAGGTTTTCCTT-CCCTTTTCC-39) were used. The amplified fragments werecloned into the BamHI and XhoI sites of pCS21 (a gift fromD. Turner and R. Rupp). The C-terminal MYC-epitope wasinserted into the XhoI site by direct ligation of annealed epitopeprimers containing terminal XhoI sites (forward 59-TCG-AGGAACAGAAGCTGATTAGCGAAGAAGATCTGC-39, reverse59-TCGAGCAGATCTTCTTCGCTAATCAGCTTCTGTTCC-39). All plasmids were linearized with NotI for synthesis of cappedRNAs.

About 200 pg of RNA was injected into one cell of two- tofour-cell stage zebrafish embryos. In the majority of embryos thisleads to a unilateral distribution of RNA as described earlier (Bladeret al., 1997), which is the cause of the unilateral effects of ectopicexpression observed at 12 h postfertilization (hpf). For rescueexperiments (Table 1) constant amounts of mdk2 RNA wereco-injected with control RNA encoding prolactin and increasingamounts of mdkD38 RNA. Injection of control prolactin RNA has noffect on development in frogs and fish (McGrew et al., 1995; dataot shown).

Retinoic Acid Treatment

All-trans-retinoic acid (RA; Sigma) was diluted as 1 mM stock inDMSO and stored at 220°C in the dark. Serial dilutions (1026 to0210 M) were made in 0.33 Danieau’s solution (17.4 mM NaCl,.21 mM KCl, 0.12 mM MgSO4, 0.18 mM Ca(NO3)2, 1.5 mMepes, pH 7.2) just prior to use. Zebrafish embryos were collected

nd raised in 0.33 Danieau’s. At the dome stage (4.3 hpf), thencubation medium was replaced with RA-containing medium andhe embryos were kept in the dark until 80% epiboly (8.5 hpf).mbryos were fixed in 4% paraformaldehyde and prepared for initu hybridization or used directly for RT-PCR. Control embryosere incubated in 0.1% DMSO in 0.33 Danieau’s solution.

In Situ Hybridization and Immunostaining

For analysis of mdk2 expression an antisense riboprobe wastranscribed with T7 from the full-length cDNA in the originalpZL1 vector digested with SalI (sense: NotI and T3). In situhybridizations of this and published clones were performed accord-ing to Hauptmann and Gerster (1994) and embryos were mountedin glycerol for photography. Immunostaining for MYC-mdk2 ex-
pression on previously in situ-stained embryos was done according
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104 Winkler and Moon

to standard protocols using the anti-MYC antibody clone 9E10(Santa Cruz Biotechnology, Inc.) and a horseradish peroxidase-coupled anti-mouse secondary antibody.

RESULTS

Cross-Species Expression Cloning of mdk2

We have used the established Xenopus animal cap explant

FIG. 1. Protein sequence and structure of mdk2. (A) Sequence co142 aa; M mk, mouse midkine, 140 aa; H mk, human midkine, 14as “.”, conserved cysteine residues as “*”. Residues conserved betSchematic structure of mdk2. Intradomain disulfide bridges of cysty brackets. I, signal peptide; III, hinge region separating N- (II) fromalues represent levels of identity between mdk2 and its closest kectors used for injection of RNA encoding dominant-negative mdk

mdkD35 includes the hinge region and parts of domain IV.

assay to screen for neural-inducing activities in pools derived i


rom an adult zebrafish brain library. Sib selection and activitycreening resulted in the isolation of a positive subpool of 23lones that contained two independent neural-inducing fac-ors. One of these clones, encoding mdk2, showed weakeural-inducing activity in the frog explants and inducedxpression of the panneural marker nrp1 and the cement glandarker XAG after injection of high doses (2 ng) of RNA (data

ot shown). In this report, we describe the expression andctivities of mdk2 (GenBank Accession No. AY008836) dur-

ison of mdk2 to known vertebrate midkines (X mk, frog midkineAmino acid residues identical to mdk2 are indicated by “-”, gapsXenopus and mouse, but different in mdk2, are labeled “1”. (B)

conserved between mammalian midkines and mdk2 are indicatedrminal domain; V, VII, conserved heparin binding sites. Percentagen relative, frog midkine/pleiotrophic factor a. Bottom: Expressiono zebrafish embryos. mdkD38 contains the N-terminal domain only,

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The isolated mdk2 cDNA clone contains an open readingrame of 147 amino acids with an N-terminal stretch of 22ydrophobic residues that likely functions as a signal se-uence. mdk2 secretion was confirmed by cell cultureransfections in which MYC-tagged mdk2 protein wasetected both in cell lysates and in the cell culture super-atant (data not shown). A database search revealed anverall identity of mdk2 to its closest relative, the frogidkine/pleiotrophic factor a (Tsujimura et al., 1995), of

56.2% on the amino acid level. This value is lower than thehomology between frog and human (Tsutsui et al., 1991)midkine (63.6%) and lower than the homology between frogmidkine and pleiotrophic factor b, another member of the

idkine family with distinct expression pattern and func-ion during embryogenesis (Tsujimura et al., 1995). In total,0 amino acids that are conserved between frog and mousere changed between frog and zebrafish mdk2 (Fig. 1A). Inhe C-terminal domain we found three stretches of aminocids that are 100% conserved between mdk2 and frog,ouse, or human midkine (Fig. 1B). Two of these encode

utative heparin-binding sites (Asai et al., 1997). The thirdtretch encodes the so-called hinge region separating the N-nd C-terminal domain as demonstrated by NMR solution-tructure analysis (Iwasaki et al., 1997). The sequencesetween these stretches show 43 and 44% amino aciddentity. There are three glutamine residues present atonserved positions (two in the N-terminal domain, Q65,67; one in the C-terminal domain, Q 118; Fig. 1B). These

lutamine residues are known to serve as substrates forissue type II transglutaminase that is responsible forimerization of murine midkines (Mahoney et al., 1996;ojima et al., 1997). Based on the low sequence similaritynd differences in the expression patterns as outlined be-ow, we propose that mdk2 encodes a novel member of the

idkine/pleiotrophic factor family.

FIG. 2. Expression of mdk2 during zebrafish embryogenesis. (A)ontrol. (B–N) RNA whole-mount in situ hybridization using full-lnset of mdk2 expression in the epiblast in a region overlaying thrrow indicates anterior border of mdk2 expression overlaying the

hroughout the presumptive neural plate. Arrow indicates leadinpiboly at the level of shield region (dorsal to the right). Dorsoventrf shield region (as boxed in E). mdk2 transcripts are excluded fromo the left. Regionalization of mdk2 expression in the early neuid–hindbrain boundary (MHB). Arrows demarcate expression at

abeling with eng2 (in red) demarcating future MHB (arrowheads).late. (I) Higher magnification of MHB region as in H showing oveiew. Arrowhead indicates future MHB, arrows denote expressionrrows mark the developing hindbrain exhibiting a gradient of

ndicates strong mdk2 expression at the MHB. (L) 12-h embryo, dort the MHB (arrowhead), and in a gradient in the hindbrain, but exf MHB region. Double labeling with eng2 (red). eng2 is expressedf the metencephalon (arrow). (N) 14-h embryo, lateral view. mdkrrows), and in dissociating prechordal plate cells underneath the fndicate dissociating prechordal plate. (P) Late mdk2 expression in fo
ube (arrows).

Expression of mdk2 RNA during EarlyEmbryogenesis

We analyzed the temporal expression of mdk2 by RT-PCR (Fig. 2A) and detected no maternal mdk2 transcriptsbefore midblastula transition. The first zygotic transcriptswere detectable at shield stage, then expression increased at80% epiboly, when the presumptive neural plate is formed.Expression was maintained through hatching (Fig. 2A). OurRT-PCR analyses do not rule out the possibility that mdk2exists in more than one splice variant.

Using whole-mount in situ hybridization, mdk2 expres-sion was first detected at 60% epiboly (late shield stage) inthe epiblast cells that overlay the earliest involuting pro-spective mesendodermal cells, the hypoblast (Fig. 2B).Double-staining analysis revealed that mdk2 starts to beexpressed slightly later than the panneural marker fkd3(data not shown). As gastrulation proceeds, mdk2 expres-sion colocalizes with fkd3 throughout the forming pre-sumptive neural-plate. Its anterior boundary overlies theleading edge of the extending axial hypoblast (Figs. 2C and2D; see also dorsal view of embryo double labeled with gscin Fig. 6C). In optical cross sections, we observed a dorsal-to-ventral gradient of mdk2 expression in the epiblast (Fig.2E). Cells located directly above the forming notochordshowed reduced expression (Fig. 2F). At 80% epiboly thefirst signs of regionalization of mdk2 transcripts becomeapparent (Fig. 2G). Double labeling with eng2 showed thatmdk2 expression is highest at the level of the futuremid–hindbrain boundary (MHB), where eng2 and mdk2expression overlaps (Figs. 2H and 2I). In more posteriorregions, mdk2 is expressed in two lateral stripes at thedges of the converging neural plate. As convergence pro-eeds at 90% epiboly, expression is strongest at the futureHB and in the anterior hindbrain. The two lateral do-

CR of different embryonic stages. b-catenin was used as loadingmdk2 as probe. (B) 60% epiboly (lateral view, dorsal to the right).

voluting hypoblast (arrows). (C) Close-up of shield region as in B.rior edge of involuting mesendoderm. (D) 70% epiboly. Expressione of axial hypoblast. (E) Optical section through embryo at 70%dient of mdk2 expression in the epiblast. (F) Higher magnificationinvoluting mesendoderm. (G) 80% epiboly, dorsal view, anterior

late. Arrowhead indicates elevated expression at the prospectivel edges of the neural plate. (H) 90% epiboly, dorsal view. Doublews indicate mdk2 expression at the edges of the posterior neuraling expression of mdk2 (blue) and eng2 (red). (J) Bud stage, dorsal

e edges of the forming neural keel. (K) Lateral view of embryo in J.expression with its maximum at the anterior end. Arrowhead

iew of head region. mdk2 is expressed in the diencephalon (arrow),d from the eye fields and midbrain. (M) 12-h embryo, dorsal views the MHB (bracket), whereas mdk2 marks the anteriormost edgeression at the MHB (arrowhead), in the dorsal neural tube (smallain (large arrows). (O) Frontal view of head region as in N. Arrowsnd dorsal midbrain, MHB (arrowhead), hindbrain, and dorsal neural

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FIG. 3. Ectopic expression of mdk2 promotes posterior cell fates and blocks anterior development. (A, B) 80% epiboly embryos, lateralview. Expression of flh (also known as Znot; arrowhead) is unaffected in uninjected (A) and mdk2-injected embryos (B), but gsc (arrow) isepressed in B. (C–F) 12-h embryos, dorsal view, anterior to the top. (C) isl1 expression (arrows) in an uninjected embryo. (D) Repression ofsl1 expression in the polster region of a mdk2 injected embryo (arrows). Trigeminal ganglion cells appear normal (arrowheads in C and D).E) pax2 and krox20 expression in an uninjected embryo. (F) Reduction of pax2 expression in the MHB (arrows) in mdk2-injected embryo.
Expression of krox20 in rhombomeres 3 and 5 (arrowheads) is unaffected. (G, H) 80% epiboly embryos, dorsal view, anterior to the top. (G)

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mains of mdk2 expression in the posterior part of theembryo converge until they end up in the dorsal neural keel(Fig. 2J).

Expression in the differentiating hindbrain is highly dy-namic. Whereas in earlier stages expression is highest in theanterior half of the developing hindbrain (Fig. 2K), theexpression later shifts posteriorly (Fig. 2L). At these stages,mdk2 is expressed at high levels in the diencephalon (Figs.2K–2P), a region where pax6 and otx2 are also expressed.

ouble labeling with eng2 indicates that mdk2 expressionn addition marks the anteriormost edge of the metencepha-on (Fig. 2M). At 14 hpf we found a transient expression inlusters of cells originating from the dissociating prechordallate that at this stage undergo a process of epithelial–esenchymal transformation and end up in the hatching

land (Figs. 2N and 2O). At later stages mdk2 is expressed inhe diencephalon, in the dorsal roof of the mesencephalon,t the MHB, in the rhombencephalon, and in the dorsaleural tube. It is excluded from the telencephalon and theye fields, as well as from all mesendodermal derivativesFig. 2P).

Ectopic mdk2 Suppresses Head Formation andEnhances Premigratory Neural Crest

The activity of mdk2 in the Xenopus animal cap assaytogether with its pattern of expression is consistent with apossible function as a planar secreted factor during neuralinduction and early patterning. To further test for thisactivity, RNA encoding full-length mdk2 was injected intoearly zebrafish embryos and the effect on development wasanalyzed by in situ hybridization (Fig. 3).

Ectopic expression of mdk2 had a strong posteriorizingeffect on development. Although expression of gsc wasnitiated normally during shield formation at 50% epibolydata not shown), the maintenance of expression in therechordal plate was completely blocked in 51% of thembryos analyzed (n 5 93; Fig. 3B relative to control 3A),ith reduced expression of gsc in the remaining embryos.

otx2 expression in the anterior neural plate of an uninjected emReduction of otx2 expression in a mdk2-injected embryo. Border eand P, lateral view). (I) Diamond-shaped expression of otx2 in the coin an uninjected embryo. (J) Ectopic mdk2 expression results in a c(anterior to the arrowhead) and a reduction at the posterior edgesuninjected embryo. (L) Expansion of fkd6-positive cells by ectopic exfkd6 domains (arrows) and a substantial increase in the number of exembryo. (N) Anterior fusion of fkd6 domains (arrows). krox20 exprLateral views (O, P) show mdk2-induced repression of anterior heaembryos. tb, tail bud. (Q, R) Higher magnification dorsal view of fembryo. Arrows indicate the width of neural crest precursor popuepiboly embryos, dorsal view. (S) Local repression of gsc expressioin the hypoblast (brown staining, arrow). (T) Higher magnificatiomdk2-MYC-positive cell (brown, arrowhead). All embryos were inje
embryo, except in S and T in which 20 pg DNA encoding MYC-tagged

he effect of ectopic mdk2 expression on mesoderm devel-pment was restricted to anterior domains, as expression ofnot (flh) in the posterior mesoderm was unaffected (Figs.A and 3B). Also, expression of the anterior mesodermalarker isl1 in the prechordal plate-derived polster regionas strongly reduced at the one-somite stage in 74% of

nalyzed embryos (n 5 76; arrow, Fig. 3D, relative toontrol, Fig. 3C).In parallel to the reduction of marker expression in the

nterior mesoderm, we found severe patterning abnormali-ies in the overlaying anterior neural plate. Both pax6 (dataot shown) and otx2 expression were reduced in 74 (n 5 19)nd 81% (n 5 32) of injected embryos, respectively. otx2xpression showed generally reduced levels in injectedmbryos at early gastrulation (arrows, Fig. 3H, relative toontrol, Fig. 3G). In addition, embryos injected with mdk2id not display strong otx2 expression at the caudal bordersepresenting the future MHB. As convergence progressed atail-bud stage, the otx2 domain showed an abnormal shapeFig. 3J relative to control, Fig. 3I). Expression at the caudalorder was reduced, and the anterior half of the diamond-haped otx2 domain was missing.Supporting the idea that mdk2 interferes with anterior

ell fates, only expression of anterior markers was reducedn embryos expressing ectopic mdk2. Expression of pax2 athe MHB was only slightly reduced in response to ectopicdk2 (Fig. 3F, arrow, compared to control, Fig. 3E). Expres-

ion of the hindbrain marker krox20 in rhombomeres r3 and5 appeared normal (arrowheads, Fig. 3F, compared to con-rol, Fig. 3E). The number of isl1-positive primary motoneu-ons and sensory neurons was not significantly altered byctopic mdk2 expression (Fig. 4Q, relative to control, Fig.P). However, we observed a strong enhancement of premi-ratory neural crest as assayed by forkhead-6 (fkd6) expres-ion (Figs. 3K–3R). At 12 hpf of normal development premi-ratory neural crest arises at the edges of the neural plateith its anterior border appearing as two well-separatedopulations of cells (arrows, control embryos, Figs. 3K andM). There is no fkd6 expression in the anterior head-

Note pronounced expression at the caudal borders (arrows). (H)sion is lacking (arrows). (I–R) 12-h embryos, dorsal view (except Oging anterior neural plate with pronounced caudal borders (arrows)d shape of otx2 expression with repression in the anterior domainws). (K) fkd6 expression in premigratory neural crest cells of an

sion of mdk2. This leads to a complete anterior fusion of the lateralsing cells. (M) Double labeling of fkd6 with krox20 in an uninjectedn shows that anteroposterior register is not significantly changed.ctures (arrow-brackets) in mdk2-injected (P) versus uninjected (O)xpression in neural crest in uninjected (Q) and mdk2-injected (R)s, arrowheads show isolated population of crest cells. (S, T) 80%DNA-injected embryo around single mdk2-MYC-expressing cell

a different embryo with repression of gsc in cells surrounding awith 200 pg RNA encoding mdk2 into 1 cell of the 2- to 4-cell stage

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forming region (control embryo, Fig. 3O). In embryos ex-pressing ectopic mdk2, the anterior fkd6 domains fuse andstart at the anteriormost edge of the embryo, as anteriorneural and mesodermal structures are repressed (arrows,Figs. 3L, 3N, and 3P). Costaining of fkd6 with krox20 as apositional reference indicates that tissue anterior to fkd6-positive cells (arrows in Fig. 3M) is absent in injectedembryos (Fig. 3N), leading to anterior fusion and ectopicfkd6 expression at the anterior edge of the shortened neuralplate. Also, the number of fkd6-positive cells increases.Whereas the two lateral stripes of neural crest usually are3–5 cells wide, in mdk2-injected embryos this is increasedto 10–15 cell diameters (Fig. 3R compared to control, Fig.3Q).

To analyze the distance of action by which mdk2 re-presses anterior fates, we produced single-cell clones ex-pressing a MYC-epitope-tagged mdk2 protein. At 80% epi-boly, expression of mdk2 in cells located in the prospectiveneural plate had no effect on gsc expression in the underly-ing hypoblast (data not shown). However, when mdk2 wasectopically expressed in single cells located in the axialhypoblast, it was able to repress gsc expression in theneighboring one or two rows of cells (Figs. 3S and 3T; mdk2protein in brown, gsc in purple).

To further test the hypothesis that mdk2 encodes a signalwith posteriorizing activity, we analyzed expression of theanterior marker emx1 in the dorsal telencephalon (Moritaet al., 1995) and the posterior marker hoxC10 in the caudalCNS of the tail bud (Prince et al., 1998) in injected embryos(Fig. 5). Eighty-four percent of the embryos injected withmdk2 RNA (n 5 32) showed a truncation of the head regionwith 56% of the embryos exhibiting an anteriorly reducedemx1 domain (Figs. 5B, 5F, and 5J relative to controls, Figs.5A, 5E, and 5I) and 28% of the embryos completely lackingemx1-expressing cells (Figs. 5C, 5G, and 5K). On the otherhand, we found a significant upregulation of posteriorhoxC1 expression in the tail bud of 34% of embryosinjected with mdk2 RNA (Figs. 5C and 5O compared tocontrols, Fig. 5A and 5M).

In summary, these overexpression studies demonstratethat ectopic mdk2 activity results in the repression ofanterior marker expression (gsc, isl1, emx1, pax2, pax6,otx2) and the enhancement of more posterior expressiondomains, such as fkd6 in premigratory neural crest cells andhoxC10 in the caudalmost CNS of the tail bud.

mdk2 Is Required for Posterior Neurogenesis

It has been shown that both N- and C-terminal domainsin human midkine are able to form covalently linkeddimers (Kojima et al., 1997). The C-terminal domainsexhibit full biological activity, e.g., heparin binding andpromotion of neurite outgrowth in vitro, whereas theN-terminal domain is not required for this function (Mura-matsu et al., 1994). Based on these findings, we designedtwo C-terminal-truncated variants by deleting parts
(mdkD35) or all of the C-terminal domain (mdkD38) (Fig. 1B).

Since ectopic expression of both forms gave the samephenotypic results, we concentrated on mdkD38 for furtherfunctional analyses. We performed rescue experiments byco-injecting a constant amount of wild-type mdk2 RNAwith increasing amounts of RNA encoding mdkD38 (Table 1and Figs. 4A–4D) to show that this truncated version actsspecifically in a dominant-interfering manner. Whereas75% of embryos injected with mdk2 developed no headstructures, co-injection of a threefold greater amount of thedominant-negative form decreased this to 30%. This showsthat mdkD38 acts as a dominant-negative mutant and can beused to interfere with mdk2 function. To analyze thedominant-negative phenotypes, mdkD38 RNA was injectednto early zebrafish embryos and the resulting phenotypesere compared to those of embryos injected with wild-typedk2 RNA. Whereas 72% of embryos injected with mdk2NA (n 5 32) show severe anterior deficiencies (Fig. 4E,

eyes absent, cyclopia, no forebrain), only 13% of themdkD38-injected embryos (n 5 54) exhibited any deficien-cies in head development (Fig. 4F). With both forms ofmdk2, embryos showed posterior deficiencies, but the mal-formations observed in mdk2-injected embryos were dis-tinct and less severe than the truncations in mdkD38-injected embryos (compare mdk2 in Fig. 4E with mdkD38 inig. 4F). The clear difference in anterior phenotypes dem-nstrates that ectopic expression of mdkD38 does not result

in a gain of mdk2 function.At a molecular level, early expression of the posterior

mesodermal marker flh (100%, n 5 72, data not shown) andthe anterior mesodermal marker gsc (Fig. 4H compared tocontrol, Fig. 4G) and isl1 (data not shown) appeared normalin 94 and 87% of mdkD38-injected embryos (n 5 49 and 40),espectively. Also, expression of anterior neural markersike pax2 (arrowheads, Fig. 4J compared to control, Fig. 4I;ormal expression in 96% of injected embryos; n 5 55) andax6 (100%, n 5 14; data not shown) appeared unalteredompared to control embryos, in contrast to the effects seenn mdk2-injected embryos (Figs. 3A–3F). The only anterior

arker that we found affected by dominant-negativedkD38 was expression of emx1 in the dorsal telencephalon.

ifty-nine percent of injected embryos (n 5 44) showed anpregulation and caudal expansion of the emx1 domainoward the diencephalon (Figs. 5D, 5H, and 5L relative toontrols, Figs. 5A, 5E, and 5I), a region where endogenousdk2 is strongly expressed at later stages (see Figs. 2L–2P).his indicates that mdk2, in addition to its early posterior-

zing function, might play a distinct role in the anterioread at later stages of development.Other deficiencies observed caudal to the MHB in em-

ryos injected unilaterally with mdkD38 were at the level ofthe rhombomeres. Thirty-eight percent of embryos exam-ined (n 5 45) showed a clear reduction of krox20 markerexpression in r3 and r5 (arrows, Fig. 4J, compared to control,Fig. 4I). Furthermore, 15 of 35 analyzed embryos showed asevere reduction in fkd6 expression in premigratory neuralcrest cells (arrow, Fig. 4M, compared to control, Fig. 4K, and
mdk2-injected embryo, Fig. 4L). Examination of embryos

h1

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FIG. 4. Repression of posterior neural fates by the dominant interfering mdk2 variant mdkD38. (A to D) Coexpression of mdkD38 rescuesead deficiencies induced by ectopic mdk2 expression. (A) Embryos exhibiting a strong posteriorized phenotype at 48 h after injection with10 pg mdk2 and 330 pg prolactin RNAs. Note that ectopic mdk2 leads to specific repression of head and eye structures and to some curved

tails. (B) Partial rescue of head deficiencies by co-injection of 110 pg mdk2, 220 pg mkD38, and 110 pg prolactin RNAs. (C) Greater rescueof head deficiencies by co-injection of 110 pg mdk2 and 330 pg mkD38. The majority of head structures appear indistinguishable from thosef uninjected control embryos (D). The posterior defects observed are likely due to mdkD38 interfering with endogenous mdk2 function. (E,) 24-h embryos injected with 200 pg wild-type mdk2 (E) and mdkD38 (F). Note anterior truncation resulting from gain of function (E,

arrowheads) and posterior deficiencies resulting from loss of function (F, arrowheads). (G) Lateral view of gsc expression in an uninjectedembryo at 80% epiboly. (H) gsc expression (arrow) is unaffected in mdkD38-injected embryos. (I–O) 12-h embryos, dorsal view, anterior tothe top. (I) pax2 (arrowhead) and krox20 (arrows) expression in an uninjected embryo. (J) Repression of krox20 expression in rhombomeres

and 5 of an embryo unilaterally injected with mdkD38. Expression of pax2 at MHB appears normal compared to I. (K) fkd6 staining (red)in an uninjected embryo. (L) Expansion of the fkd6-positive neural crest domain in mdk2-injected embryo. (M) Repression of fkd6 by mdkD38

in one half of a unilaterally injected embryo (arrow). (N–R) Ectopic mdkD38 expression blocks formation of posterior moto- and sensoryneurons. (N) isl1 expression in Rohon–Beard sensory neurons (rb) and motoneurons (mn) of an uninjected embryo at 12 hpf. (O) Unilateralack of both neural fates in an embryo unilaterally injected with mdkD38. (P–R) Dorsal view of 10-h embryos. (P) isl1 expression in an

uninjected embryo. (Q) Primary neurons appear unaffected in a mdk2-injected embryo. (R) Note unilateral absence of sensory neurons
(arrowhead) in mdkD38-injected embryo.

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for the formation of primary moto- and sensory neuronsrevealed a complete lack of these neurons (arrows, Fig. 4O,unilaterally injected on the left side, compared to control,

FIG. 5. mdk2 activity affects emx1 expression pattern in the anterviews of emx1 expression (arrow) in the telencephalon and hoxc1embryos injected with 150 pg RNA encoding mdk2 (B, C) and with(B) or complete repression (C) of emx1 expression (arrows) and expanhand, emx1 expression (arrow) appears caudally expanded in mdkD3

ateral views, I–L dorsal views, anterior to the left). The emx1 eompletely missing (G, K) in mdk2-injected embryos compared t

embryos (H, L). (M–P) Dorsal views of hoxC10 expression in the taicontrol embryo in M), but is not significantly affected in mdkD38-ilightly earlier stage (12 hpf compared to 14 hpf for control and mdktaining.

Fig. 4N). Both isl1 subtypes (50%, n 5 60 analyzed em- w


ryos) and huC-positive subtypes (63%, n 5 8; data nothown) were affected. As the interference with mdk2 func-ion leads to a repression of development of neural crest, as

rebrain and hoxC10 expression in the posterior CNS. (A–D) Lateralpression (arrowhead) in the tail bud of a control embryo (A) andpg RNA encoding dominant-negative mdkD38 (D). Note reductionof hoxC10 (C, arrowhead) in mdk2-injected embryos. On the othercted embryos (D). (E–L) Higher magnification of head regions (E–Hsion domain is shortened (F, J) and dislocates ventrally (F) or istrol embryos (E, I), while it extends caudally in mdkD38-injected. hoxC10 is upregulated in mdk2-injected embryos (O, compare toed embryos (P). Note that the mdkD38-injected embryo in P is at aected embryos), which accounts for the apparently weaker hoxC10

ior fo0 ex500

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ell as posterior moto- and sensory Rohon–Beard neurons,


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this suggests a mdk2 function in the earliest steps of neuralpecification. However, ectopic expression of mdkD38 did

not affect early panneural fkd3 expression (normal expres-sion in n 5 67 injected embryos; data not shown). Thissuggests that endogenous mdk2 function is not required forthe first phase of neural induction (the activation step), butis essential for the specification of posterior neural cellfates. Interestingly, ectopic mdkD38 expression did not sig-nificantly reduce hoxC10 expression in the posterior CNSof the tail bud. This suggests that mdk2 is not required forthe earliest steps in formation of the posterior neural plate,that other factors take over the function of mdk2 duringthis process, or that ectopic mdkD38 does not persist atsufficient levels to modulate hoxC10 in later stage em-bryos.

Expression of mdk2 in Mutant Zebrafish Embryos

Zebrafish mutants offer the possibility of analyzing epi-genetic relationships of mdk2 with other known signalingfactors involved in neural induction and patterning. Inacerebellar (ace) mutants, the MHB and cerebellum arelacking as a consequence of missing FGF8 function (Reiferset al., 1998). As mdk2 is strongly expressed in the prospec-tive MHB region, and its ectopic activity interferes withpax2 expression, we tested whether mdk2 acts downstreamof FGF8. In situ hybridization of ace mutant embryos at 24

pf showed that mdk2 expression is missing in the cerebel-um due to the lack of this structure in ace embryos

(arrows, Fig. 6B, compared to control, Fig. 6A). Otherwise,mdk2 expression seemed normal in all other tissues, in-cluding diencephalon, hindbrain, and spinal cord. Further-more, we did not observe any changes in mdk2 expressionn a cluster of mutant ace embryos at 80% epiboly (data not

TABLE 1Rescue of mdk2-Induced Head Defects by Coexpression of the Do

Injected RNA n

mdk2c 110 pg 1181 prolactin 330 pg

mdk2 110 pg 401 mdkD38 220 pg1 prolactin 110 pg

mdk2 110 pg 961 mdkD38 330 pg

a The observed head defects include cyclopia and complete ladevelopment.

b Posterior deficiencies include shortened trunks and tails, kinkemdkD38 cause posterior deficiencies, but with different phenotypes,espectively (also compare Figs. 5E and 5F).

c The amount of injected mdk2 RNA is approx threefold higherd The observed anterior deficiencies were significantly less sev

cyclopia vs missing eyes and brain structures.

hown). This suggests that neither induction nor mainte-


ance of mdk2 expression in regions outside the cerebellums dependent on FGF8 function. We, however, cannot ex-lude the possibility that mdk2 expression is under FGF8ontrol in the cerebellum.Mutations in the cyclops (cyc) gene nodal-related 1 (znr1)

ead to deficiencies in dorsal mesendoderm, resulting ineural defects and cyclopia (Sampath et al., 1998). In cycutant embryos, gsc expression is repressed; however, both

the anterior expansion and the level of mdk2 expression arenot affected (Figs. 6C and 6D). This suggests that mdk2expression is not dependent on znr1 signaling from the axialmesoderm, but could be affected by signaling from theectoderm.

BMP signaling in the ectoderm inhibits neural develop-ment and promotes epidermal differentiation. To testwhether mdk2 is affected by BMP signaling, we analyzed itsexpression in chordino (chd) mutants during early gastru-lation. chd mutant embryos are ventralized (Schulte-Merker et al., 1997) and show an expansion of ventral eve1expression (arrowheads, Fig. 6F, relative to control, Fig. 6E).In chd mutant embryos, the onset of mdk2 expression isblocked (arrow, Fig. 6F, compared to control, Fig. 6E),suggesting that initiation of mdk2 expression is undernegative control of BMP signaling. This was confirmed byanalyzing dorsalized snailhouse (snh, bmp7; Mullins et al.,1996) and swirl (swr, bmp2b; Nguyen et al., 1998) mutantembryos. In both mutants, identified by reduced eve1expression (arrowheads, Figs. 6H and 6J, compared to con-trols, Figs. 6G and 6I), we found strong ventral expansion ofthe mdk2 domain (arrows, Figs. 6H and 6J, compared tocontrols, Figs. 6G and 6I). This confirms that BMP signalingnegatively regulates expression of mdk2 during gastrula-tion.

As murine midkines were originally identified as RA-

nt-Interfering Variant mdkD38

Head defectsa Posterior deficienciesb

89 (75%) 78 (66%)

19 (48%)d 29 (73%)

29 (30%)d 54 (56%)

eyes and fore- and midbrain structures, scored after 2 days of

ils, or malformed somites. It should be noted that both mdk2 andably reflecting posteriorization and lack of posterior development,

necessary to induce a mdk2-specific phenotype.han defects observed when mdk2 was injected alone, i.e., weak

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cells (Tomomura et al., 1990), we asked whether mdk2xpression is also inducible by RA. Treatment of zebrafishmbryos with high doses of RA (1026 or 1027 M) between

dome stage and 80% epiboly resulted in a complete repres-sion or significant reduction of endogenous mdk2 expres-sion as assayed by in situ hybridization (Figs. 6K–6P) orRT-PCR (data not shown). However, we observed an in-crease in mdk2 expression when lower doses (1028 or 1029

M) were used, showing that expression of mdk2 is depen-ent on the dose of RA applied.Taken together, this suggests that in the gastrulating

mbryo mdk2 expression is induced and modulated by aradient of RA activity; whereas independent of znr1 and

FGF8 signaling, initiation of mdk2 expression is undernegative control of BMP signaling.

DISCUSSION

mdk2 Encodes a Novel Member of the MidkineFamily That Is Expressed in the PresumptiveNeural Plate

The mdk2 sequence shows homology with the midkinefamily of secreted, heparin-binding growth factors. Severalsequence features are conserved between known mamma-lian midkines and mdk2. For example, two highly con-erved consensus sites for putative heparin-binding motifsere found. Heparin binding has been shown to enhanceimerization of human midkine and is required for itseurite outgrowth-promoting activity (Kojima et al., 1997).

Although the overall structure identifies mdk2 as a mem-er of the midkine family there are significant differences inhe amino acid sequence. In addition, we found clearifferences in the embryonic expression patterns that sug-est that mdk2 is an orthologue of this family, but withovel and distinct functions during embryogenesis com-ared to the corresponding frog and mammalian midkines.nterestingly, an additional zebrafish midkine-related se-uence has been published in GenBank (Accession No.W281401) that is slightly closer related to frog midkine

han mdk2.Neural induction is believed to start at the onset of

astrulation when cells of the involuting mesoderm secreteignals that instruct ectoderm to adopt a neural fate (re-iewed in Hemmati-Brivanlou and Melton, 1997). Theemporal and spatial expression pattern of mdk2 is consis-ent with a function during neural induction and earlyatterning. However, unlike known neural inducers thatre expressed in the cells of the dorsal lip and are known tontagonize BMPs, mdk2 is expressed in the cells of theresumptive neural plate overlaying the involuting mesen-oderm. Therefore, secreted mdk2 could serve as a planar
ignal instructing the forming neural plate. a

The Activity of mdk2 during Neurogenesis

We isolated mdk2 based on its neural-inducing activity inenopus animal cap explants. However, this activity wasery low compared to known neural inducers and requiredigh doses of injected RNA (2 ng per injected embryo). Thenly markers induced were panneural nrp-1 and the ante-ior ectodermal marker XAG. Neither NCAM nor otherorebrain or posterior markers were induced. The findinghat no NCAM, but only the more sensitive marker nrp-1,as induced shows that mdk2 has only limited neural-

nducing activity. The observed activity in animal capxplants is very similar to the activity of the relatedenopus midkine XMK in this assay (Yokota et al., 1998).his could be explained by the high degree of similarityetween both proteins. However, the difference in sequencend expression pattern suggests that mdk2 is a member of

this gene family with novel function that is specific forteleost neural development and different from XMK func-tion in frogs. It has been shown that ectopic expression ofXMK in frogs antagonizes mesoderm induction and pro-motes neural development (Yokota et al., 1998). Injection ofXMK RNA into frog embryos led to the development ofhypertrophic heads. Ectopic expression of mdk2 expressionin zebrafish embryos had a strikingly different effect.Rather than enlarged heads, the injected fish embryosdeveloped without a head. In situ hybridization showedhat from midgastrula stages onward anterior mesodermalgsc, isl1) and neural (emx1, pax2, pax6, otx2) markers wereeduced and the phenotypic defects observed included se-ere cyclopia, lacking eyes, or complete lack of head struc-ures up to the level of the MHB.

The downregulation of gsc expression by mdk2 suggestshat one early function of mdk2 is to keep anterior ectoder-al genes from being inappropriately expressed in posterior

egions of the neural plate (Fig. 7). In zebrafish, gsc isxpressed in both anterior mesendoderm and ventral neu-oectoderm (Thisse et al., 1994). At the moment, it isnclear whether endogenous mdk2 acts exclusively on oner both of these gsc expression domains. It is possible thatndogenous mdk2 regulates only ectodermal gsc expressionnd that the effects on anterior mesoderm (like reduction ofsl1 in the prechordal plate) observed after injection areaused by ectopic mdk2 expression in the anterior meso-erm. Our finding that expression of dominant-negativedkD38 had no significant effects on anterior mesodermal

evelopment, and in particular did not lead to an expansionf gsc expression, is in agreement with the idea thatndogenous mdk2 function is restricted to neuroectoderm.owever, the possibility remains that endogenous mdk2rotein secreted from the neural plate directly affects ex-ression of anterior mesodermal genes.Using DNA injections into late embryos that resulted in

ingle cells or small cell clones expressing high levels ofpitope-tagged mdk2, we found that the observed repres-ion of gsc is a direct and specific consequence of mdk2
ctivity acting at short range. Specifically, cells directly

omeg(msE7


FIG. 6. mdk2 expression is independent of FGF8 and nodal-related1 signaling, inhibited by BMP signaling, and upregulated by low dosesf exogenously applied RA. (A, B) Lateral views of mdk2 expression in 24-hpf wild-type (wt, A) and acerebellar (ace, B) mutant embryos.dk2 expression at the MHB (arrow) is absent due to lack of cerebellar structure. Otherwise expression appears unaffected (arrowheads).

y, eye. (C, D) Dorsal views of mdk2 (arrowhead) and gsc (arrow) expression in wt (C) and cyclops (cyc, D) mutant embryos at 90% epiboly.sc is repressed in cyc embryos, but mdk2 expression appears unaffected. (E–P) Lateral views, dorsal to right. (E, F) Expression of eve1arrowhead) and mdk2 (arrow) in wt and chordino (chd) mutant embryos at 70% epiboly. eve1 expression is expanded in chd embryos, but

dk2 is repressed (arrow in F compared to E). (G–J) Expression of eve1 (arrowhead) and mdk2 (arrow) in wt (G, I), snailhouse (snh, H), andwirl (swr, J) mutant embryos at 80% epiboly. While eve1 is repressed in snh and swr, mdk2 expression is ventrally expanded. (K–P)xpression levels of mdk2 (arrowhead) are dependent on the dose of exogenously applied RA. (K–N) Dose response of mdk2 expression in0% epiboly embryos. RA applied at 1027 M reduces mdk2 expression (L), whereas lower doses (M, N) increase expression when compared

to untreated embryos (K). (O) Expression of mdk2 in an untreated embryo at 70% epiboly. (P) Complete repression of mdk2 expression by
treatment with RA at a dose of 1026 M.

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adjacent to mdk2-positive cells showed reduced gsc expres-sion. Interestingly, single cell clones that expressed mdk2in the ectoderm had no effect on underlying gsc expressionin the mesoderm, thus providing further evidence thatendogenous mdk2 function is restricted to the ectoderm.These findings together with the fact that endogenousmdk2 is expressed throughout the entire early posteriorneural plate suggest that mdk2 protein presumably acts atshort range also in the neuroectoderm.

Widespread ectopic expression of mdk2 after RNA injec-tion resulted in a widening of the posterior neural plate andan enhancement of neural crest. It has been shown thatneural crest formation in frogs requires BMP antagonists incombination with Wnt signaling (LaBonne and Bronner-Fraser, 1998). Our observation of enhanced neural crestformation after ectopic mdk2 expression opens the possi-bility that mdk2 protein interferes with BMP signaling.This hypothesis could also explain the weak neural-inducing activity of mdk2 in Xenopus animal cap explants.

Recently, it has been shown that a gradient of BMPactivity mediated by organizer-derived, diffusible antago-nists is responsible for setting up the pattern in the earlyneural plate in zebrafish (Barth et al., 1999). Our data alsosuggest that mdk2 activity in the ectoderm is involved inneural patterning. It seems possible that secreted mdk2interacts with BMP signaling in the early ectoderm and thatthis interaction leads to the generation of positional infor-mation that is required for the formation of neural crest andprimary neuronal fates in the caudal neural plate.

To further analyze the prospective function of mdk2

FIG. 7. Simplified model of mdk2 function in patterning theneural plate of gastrulating zebrafish embryos (anterior to the top).Expression of mdk2 in the plane of the presumptive neural plate isinduced and regulated by a gradient of RA activity. Initially, mdk2transcription is under negative control of BMP signaling anddirectly follows the onset of expression of the panneural markerfkd3. Secreted mdk2 then acts by suppressing genes expressed inthe anterior neuroectoderm, like gsc (Thisse et al., 1994), otx2 (Li etal., 1994), and possibly mesendodermal genes. It further permitsspecification of posterior neural fates, i.e., primary moto- andsensory neurons. Subsequent enhancement of premigratory neuralcrest (hatched areas) at the edges of the caudal neural plate might beachieved by interactions of secreted mdk2 with a gradient of BMPactivity in the ectoderm (as described by LaBonne and Bronner-Fraser, 1998; Nguyen et al., 1998; Barth et al., 1999).

during posterior neural development we used a dominant- a


interfering approach. Ectopic expression of dominant-negative mdkD38 caused posterior truncations and blockedthe formation of premigratory neural crest and primaryneurons. This strongly suggests that mdk2 or related mid-kines are required for the development and differentiationof posterior neural derivatives. Significantly, the structuresthat were affected by the dominant-negative mdk2 were thesame structures that normally express mdk2.

mdk2 is expressed slightly later than panneural fkd3, andmdk2 transcription is under negative control of BMP sig-naling and dependent on chordin function. This suggeststhat mdk2 is involved in processes that follow the firstactivation phase of neural induction according to Nieuw-koop’s model. This is also supported by the observation thatpanneural fkd3 expression and formation of anterior tri-geminal ganglia are not blocked by dominant-negativemdkD38, suggesting that anterior neural induction is notffected. However, formation of both caudal neural crestnd primary neurons is dependent on mdk2 function.herefore, mdk2 could play a role in the transformation of

nitially induced anterior neural tissue to more posteriorates. We did not observe expansion of early anterior neural

arkers at the expense of posterior fates after interferingith mdk2 function with dominant-negative mdkD38. Thisight be explained by the presence of additional factors in

he embryo that limit anterior neural development in thebsence of mdk2 function.Consistent with the idea that mdk2 is involved in poste-

ior transformation, we found an enhancement of hoxC10xpression in the posterior CNS after ectopic mdk2 expres-ion. We did not, however, observe any significant reduc-ion of hoxC10 expression in embryos injected withominant-negative mdkD38. This again suggests either thatdk2 is not required for the first steps of posterior trans-

ormation or that here also other posteriorizing factorsompensate for the lack of mdk2 activity in the injectedmbryos.During gastrulation mdk2 is expressed throughout the

resumptive neural plate with its anterior boundary over-apping the caudal gsc domain (Fig. 6C). Therefore, it wasot surprising that we did not observe any reduction of gsc,

sl1, pax2, and pax6 marker expression in the head region ofdkD38-injected embryos. On the other hand, more poste-

ior fates were strongly affected, thus reflecting a possiblearly function of mdk2 in posterior neurogenesis. Lateruring development, however, mdk2 is also expressed in

the anterior CNS. The only anterior neural marker wefound affected by high doses of mdkD38 was emx1, which isnormally restricted to the dorsal telencephalon. Loss ofmdk2 function resulted in a caudal expansion of emx1toward the diencephalon. Endogenous mdk2 is stronglyexpressed in the diencephalic region. This therefore indi-cates a possible second function of mdk2 at later stages ofdevelopment, which is to set the caudal limit of emx1expression in the telencephalon and prevent it from beingexpressed in the diencephalon. As emx1, but not other
nterior markers, was affected, we suggest that the interac-

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tion between mdk2 and emx1 is rather direct. Futureanalyses will be necessary to test whether and how theexpanded emx1 expression affects normal head develop-ment.

Ectopic expression of wild-type mdk2 is sufficient toblock anterior structures and to enhance posterior develop-ment. Similar effects have been described for RA in frogs(Ruiz i Altaba and Jessell, 1991). In zebrafish, exogenous RAblocks the differentiation of specific brain regions (Holderand Hill, 1991). Here we have shown that expression ofmdk2 can be modulated by exogenously applied RA. Thedifferential response to increasing doses of RA suggests thatendogenous mdk2 transcription is regulated by a RA gradi-ent in the gastrulating embryo. The presence of suchgradients confined to posterior regions of the gastrulatingembryo has been suggested from studies using RA-responsive transgenes (Rossant et al., 1991; Joore et al.,1997). Therefore, the pattern of mdk2 expression is consis-tent with the pattern of retinoid activity in zebrafish inboth a spatial and a temporal manner. During early stages,highest RA activity is observed at the involuting marginwhere repression of mdk2 occurs. Lower RA activity in thepresumptive neural plate coincides with the induction ofmdk2 expression. During later gastrulation, high RA activ-ity is mainly observed in a posterior dorsal domain, whereasmdk2 expression is abundant at the lateral edges of theneural plate. We therefore suggest that RA is a directinducer of mdk2 and regulates its expression during gastru-lation. mdk2 is the first secreted factor known to beregulated by RA during gastrulation.

Other factors are also likely to regulate expression andactivity of mdk2. We analyzed mdk2 expression in variouszebrafish mutants and found that the onset of mdk2 tran-scription is under negative control of ectodermal BMPsignaling. It appears to act downstream of BMP7 andBMP2b. This again suggests that mdk2 does not functionduring early neural induction in a pathway alternative toBMP antagonists, but that it acts during the second, i.e.,transforming phase of neural induction.

mdk2 as a Planar Signal in Neural Patterning

The neural-inducing activity of mdk2 in Xenopus animalcap explants was low compared to known neural inducerslike noggin and chordin. These are in part “vertical” induc-ers that are secreted by the organizer and axial mesodermand therefore signal toward the overlaying ectoderm. El-egant transplantation experiments have shown that in earlygastrulae of zebrafish nonaxial mesendodermal cells fromthe germ ring provide the source for secreted signals thattransform initially neuralized ectoderm into more posteriorfates (Woo and Fraser, 1997). These signals of unknownnature apparently travel through the plane of ectoderm toinstruct caudal neural progenitors. mdk2 on the other handis expressed in the presumptive neural plate and probablyalso signals in a planar fashion. It is possible that mdk2
transcription is induced by signals emerging from the germ

ing; however, based on our findings, we favor the idea thatdk2 is induced by RA and cooperates with the caudalizing

ignals derived from nonaxial mesendoderm.The significant role of planar induction in the neural

atterning process in zebrafish was recently confirmed bydditional transplantation experiments (Woo and Fraser,998). Transplantation of cells from the prospective hind-rain region at 80% epiboly into ventral locations showedhat these cells are completely committed to become hind-rain cells, although at the corresponding stage there is noirect contact between the respective ectodermal regionnd involuting mesoderm. Therefore, vertical contact withxial mesoderm is not required for cells to adopt a hindbraindentity (Woo and Fraser, 1998). The source for the planaratterning signal originates from outside the shield region,s ablation of the shield at 60% epiboly does not preventindbrain formation (Shih and Fraser, 1996). mdk2 is ex-ressed before 80% epiboly at high levels throughout therospective posterior neural plate outside the shield region,nd interfering with its function abolishes hindbrain differ-ntiation. Therefore, mdk2 is a strong candidate for being alanar signal postulated by these grafting experiments.The receptors for midkines and in particular mdk2 are

ot yet known. It will be interesting to see in the futurehether mdk2 functions through its own receptors and

ignaling pathways or by antagonizing BMP activity in theastrulating embryo.

ACKNOWLEDGMENTS

We thank J. Ngai for providing the zebrafish brain cDNA library;M. Schartl for discussions and support; C. Thorpe, J. Altschmied,and R. Dorsky for comments on the manuscript; and D. Goetz forassistance with the explant assays. C.W. was supported by along-term fellowship from the Human Frontier Science ProgramOrganization (HFSPO), and R.T.M. is an Investigator of the HowardHughes Medical Institute.

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Received for publication April 19, 2000Revised September 20, 2000

Accepted September 20, 2000
Published online November 28, 2000

zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis

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