Primordial germ cell migration in zebrafish...Primordial germ cell migration in zebraﬁsh 5297...

INTRODUCTION

While the specification mechanisms of primordial germ cells(PGCs) and the position where these cells are specified candiffer, a common theme for many species is that the germcells are formed in regions distinct from the site where thegonad will form. Hence, the PGCs have to migrate towardsthe future gonad, a process that has been studied in Xenopusand particularly in Drosophila and mouse where moderngenetic approaches have been applied (reviewed in Howard,1998; Rongo et al., 1997; Wylie, 1999). The generalconclusion from these studies is that the movements of thePGCs towards the gonadal region, where they associate withcells of mesodermal origin, rely on directional cues providedby the somatic environment (e.g., Anderson et al., 1999;Jaglarz and Howard, 1994; Matsui et al., 1990; Moore et al.,1998; Zhang et al., 1996). A classical example from themouse is that of the c-kit receptor, which is expressed in thePGCs, and its ligand Steel, which is expressed by somatic

cells along the migratory route. Receptor-ligand interactionis required to support migration and survival of the PGCs(Bernex et al., 1996; Matsui et al., 1990). Mouse PGCs havealso been shown to migrate towards explants of target tissue,suggesting that they are attracted towards the genital ridge bylong-range signaling (Godin et al., 1990). Finally, a role forextracellular matrix (ECM) molecules in the PGC migrationprocess has been suggested by the finding of specificinteractions between ECM molecules and PGCs and by thephenotype of PGCs lacking specific receptors for componentsof the ECM (e.g., Anderson et al., 1999). In Drosophila, adetailed description of the migration process coupled withgenetic analysis allowed the identification of tissues andmolecules required for PGC migration (reviewed in Howard,1998; Rongo et al., 1997; Williamson and Lehmann, 1996;Wylie, 1999). Drosophila PGCs are formed at the posteriorpole of the early embryo and then together with somatic cellsparticipate in the morphogenetic movement forming theposterior midgut (PMG). Subsequently, the PGCs actively

5295Development 126, 5295-5307 (1999)Printed in Great Britain © The Company of Biologists Limited 1999DEV3057

In many organisms, the primordial germ cells have tomigrate from the position where they are specified towardsthe developing gonad where they generate gametes.Extensive studies of the migration of primordial germ cellsin Drosophila, mouse, chick and Xenopus have identifiedsomatic tissues important for this process anddemonstrated a role for specific molecules in directing thecells towards their target. In zebrafish, a unique situationis found in that the primordial germ cells, as marked byexpression of vasa mRNA, are specified in random positionsrelative to the future embryonic axis. Hence, the migratingcells have to navigate towards their destination fromvarious starting positions that differ among individualembryos. Here, we present a detailed description of themigration of the primordial germ cells during the first 24hours of wild-type zebrafish embryonic development. Wedefine six distinct steps of migration bringing theprimordial germ cells from their random positions beforegastrulation to form two cell clusters on either side of themidline by the end of the first day of development. To

obtain information on the origin of the positional cuesprovided to the germ cells by somatic tissues during theirmigration, we analyzed the migration pattern in mutants,including spadetail, swirl, chordino, floating head, cloche,knypek and no isthmus. In mutants with defects in axialstructures, paraxial mesoderm or dorsoventral patterning,we find that certain steps of the migration process arespecifically affected. We show that the paraxial mesodermis important for providing proper anteroposteriorinformation to the migrating primordial germ cells andthat these cells can respond to changes in the globaldorsoventral coordinates. In certain mutants, we observeaccumulation of ectopic cells in different regions of theembryo. These ectopic cells can retain both morphologicaland molecular characteristics of primordial germ cells,suggesting that, in zebrafish at the early stages tested, thevasa-expressing cells are committed to the germ celllineage.

Key words: Zebrafish, Primordial germ cell, Cell migration, vasa

SUMMARY

Identification of tissues and patterning events required for distinct steps in

early migration of zebrafish primordial germ cells

Gilbert Weidinger‡, Uta Wolke‡, Marion Köprunner, Michael Klinger* and Erez Raz¶

Department of Developmental Biology, Institute of Biology I, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany*Present address: Faculty of Biology, University of Konstanz, Universitätsstrasse 10, D-78434 Konstanz, Germany‡These authors contributed equally to this work¶Author for correspondence (e-mail: [email protected])

Accepted 21 September; published on WWW 9 November 1999

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migrate through the midgut epithelium towards the gonadalmesoderm with which they align and coalesce. Two genesspecifically affecting this migration are expressed in somaticcells (Moore et al., 1998; van Doren et al., 1998; Zhang etal., 1996, 1997): the wunen gene is required for repulsion ofPGCs, thereby bringing them closer to the mesodermal targetwhereas columbus functions in attracting them towards thegonadal mesoderm.

Until recently, studying the migration of PGCs in fish hasrelied on their identification by morphological criteria atrelatively late stages, usually around the beginning ofsomitogenesis (e.g., Gevers et al., 1992). The ability to followPGC migration in fish was revolutionized by the cloning of thezebrafish vasa gene homolog, which is exclusively expressedin PGCs (Olsen et al., 1997; Yoon et al., 1997). vasa wasoriginally identified in Drosophila as a maternal effect generequired for the formation of the abdominal segments and forgerm cell specification (Hay et al., 1988; Lasko and Ashburner,1988; Schüpbach and Wieschaus, 1986). In situ hybridizationof 4-cell-stage zebrafish embryos showed specific localizationof the vasa transcript in four stripes at the edges of the first twocleavage planes, suggesting that specification of germ cells isachieved by localization of cytoplasmic components as inother organisms such as Drosophila, C. elegans and Xenopus(Wylie, 1999; Yoon et al., 1997). At the 32-cell stage, thetranscript is detected in four cells that subsequently divide togive rise to four cell clusters located close to the blastodermmargin at late blastula stages. During subsequent developmentthese vasa-expressing cells migrate and at 24 hourspostfertilization (hpf) end up in two bilateral rows around theanterior end of the yolk extension. These cells that maintainvasa expression at least up to late larval stages are primordialgerm cells based on their morphology and position (Yoon etal., 1997).

Here we provide a detailed analysis of the early stages ofPGC migration in zebrafish. We find that this process can bedivided into six distinct steps, some of which appear to beshared with somatic tissues, whereas others seem to reflectactive migration of PGCs relative to their somatic neighbors.Analysis of the migration process in mutant embryos showsthat some of these steps can be specifically affected. Thisallows us to propose which structures or processes areinvolved in directing the migration of the PGCs. As a resultof abnormal migration, in some mutants we observeaccumulation of cells in ectopic regions. As judged bymorphological and molecular criteria, these ectopic PGCs canmaintain their predetermined fate in a foreign somaticenvironment at the early stages tested.

MATERIALS AND METHODS

Zebrafish maintenance and mutant strainsZebrafish (Danio rerio) were maintained as described previously(Westerfield, 1995). Mutant strains used: chordino, dintm84

(Hammerschmidt et al., 1996); cloche, clom39 (Stainier et al., 1995);floating-head, flhtk241 (Odenthal et al., 1996); knypek, knym818 (A.Chitnis, K. Artinger and W. Driever, personal communication;Solnica-Krezel et al., 1996); no isthmus, noitu29a (Brand et al., 1996);spadetail, sptb104 (Kimmel et al., 1989); swirl, swrta72a (Mullins et al.,1996).

Whole-mount in situ hybridization and histologyTwo-color in situ hybridization was performed as described by Jowettand Lettice (1994) with modifications according to Hauptmann andGerster (1994). In some cases, light-blue color was obtained using aβ-galactosidase-conjugated anti-digoxigenin antibody and X-Gal forsubsequent color reaction (Hauptmann, 1999). The following probeswere used for whole-mount in situ mRNA hybridization: din (Miller-Bertoglio et al., 1997), gata2 (Detrich et al., 1995), hoxa-2 (Prince etal., 1998), myoD (Weinberg et al., 1996), papc (Yamamoto et al.,1998), pax2.1 (formerly paxb, (Krauss et al., 1991)), pax8 (Pfeffer etal., 1998) and vasa (Olsen et al., 1997; Yoon et al., 1997).

Methacrylate sections were performed using the JB-4 Plus resin(Polyscience Inc.) according to the manufacturer’s protocol.

Determination of PGC numbervasa-positive cells were counted using a stereomicroscope at ×150magnification. The number of vasa-positive PGCs varies amongembryos of the same clutch. Importantly, the number of PGCs mayalso be influenced by the genetic background. For example, mutantchordino and wild-type siblings in the TL genetic background haveabout 25 PGCs at 24 hpf (e.g., Table 1), while embryos with ABgenetic background typically have as many as 50 PGCs at 24 hpf (e.g.,Table 2). For these reasons, description of mutant phenotypes was doneby comparing the PGC number or behavior to wild-type siblings.Additionally, to minimize the variability, analysis of the migrationprocess in wild-type and in mutant embryos was usually performed byfixing embryos of the same clutch at different times of development.

vasa cDNA amplification and injection of mRNA Full-length vasa cDNA (as published by Yoon et al. (1997)) wasamplified from ovary cDNA using the following primers: 5′ primer(TCA GGC TCT TCA CGC GTG TCC ACC TGC TAC), 3′ primer(TTT TGT CAC CAG TAT CCG TCT TTA TTT TGA) (italic sequenceis homologous to vasa). Except for a few amino acid changes that weattribute to polymorphisms between strains (identical changes weredetected in independent PCR reactions), the sequence of the clonedvasa cDNA is identical to that published by Yoon et al. (1997).Capped vasa mRNA was synthesized using the Ambion MessageMachine kit and zebrafish embryos were injected at the 1- to 2-cellstage with 100-200 pg of vasa mRNA. In situ hybridization was

G. Weidinger and others

Table 1. PGC phenotype of chordino (din) mutant embryosAverage PGC Embryos with PGCs Proportion of PGCs

Embryos analyzed number per embryo* ventral-posterior of main clusters ventral-posterior of main clusters

Stage wt siblings din wt siblings din wt siblings din wt siblings din

60% epiboly 13 15 23.1±5.7 22.0±4.6 100% 100% 60.0%±11.4% 63.4%±13.7%80% epiboly 14 14 21.9±4.8 21.6±4.3 100% 100% 58.9%±13.7% 56.0%±13.1%2 somite 24 9 24.6±7.3 23.3±6.9 92% 100% 19.4%±13.1% 34.7%±19.3%8 somites 19 14 33.7±8.6 34.4±8.4 100% 100% 21.8%±9.6% 41.8%±13.6%24 hpf 52 55 26.4±7.4 27.4±8.9 15% 95% 1.5%±4.2% 27.4%±14.4%

* in TL genetic background (see Materials and Methods).

5297Primordial germ cell migration in zebrafish

performed on the injected embryos at different stages. Judged fromthe signal intensity at early gastrulation, most cells in the injectedembryos received higher levels of vasa mRNA than the endogenouslevel of vasa expressed by the PGCs.

RESULTS

Migration of primordial germ cells in wild-typeembryosTo identify possible sources for signals and define tissues thatmay participate in directing PGCs towards their target, weanalyzed the migration of PGCs relative to forming somaticstructures in the first 24 hours of development. As discussedby Yoon et al. (1997), based on their morphology and positionat larval stages, the vasa-expressing cells are PGCs. It isformally possible that some cells do not maintain expressionof vasa or that not all of the vasa-positive cells at a certainstage were expressing it at earlier stages. Our results (seebelow) do not support these options, but rather are consistentwith the notion that, during the first 48 hpf, the vasa mRNA isa reliable, stable marker for PGCs and that cells expressing itare related by lineage. Throughout this work then, the analysisof the migration of the vasa-expressing cells during the first 24hours of development was performed by observing the positionof these cells at different stages and deducing movements thatwould connect the different arrangements observed at differenttime points.

Observing the migration of the vasa-expressing PGCs iscomplicated by the fact that the position of the four PGCclusters at blastula stages seems to be determined by theorientation of the early cleavage planes (Yoon et al., 1997),which is random with respect to the future dorsoventral axis(Abdelilah et al., 1994; Helde et al., 1994). To prove that theinitial relation of the four PGC clusters relative to the dorsalaspect of the embryo is indeed random, we analyzed embryosbefore gastrulation. At dome stage (late blastula, 4.5 hpf), thefour PGC clusters, each containing about four cells, are foundclose to the blastoderm margin, equidistant from each other ina square-like arrangement (Fig. 1A-C, upper panel). Wedetermined the orientation of this square relative to the dorsalpart of the embryo (visualized by chordino expression) in 30

embryos, proving that the arrangement of the clusters israndom relative to the dorsal aspect of the embryo (data notshown). Three examples for initial cluster arrangements areshown in Fig. 1. The dorsal side of the embryo can be flankedby two PGC clusters (starting position A, Fig. 1A), in anintermediate arrangement one of the clusters is located off themiddle of the chordino expression domain either on the left orthe right side (starting position B, Fig. 1B), or one of theclusters lies exactly at or very close to the dorsal side (startingposition C, Fig. 1C). Therefore, in contrast to other organisms,within each zebrafish embryo, the PGCs start their migrationfrom different dorsoventral positions and these starting pointsdiffer between individual embryos. Below we describe themovements that take place during the next 20 hours ofdevelopment bringing the PGCs to their position at the 1-daystage. These movements can be divided into six steps, some ofwhich are temporally distinct, while others occursimultaneously.

Step I, convergence towards the dorsalAt the 60% epiboly stage (6.5 hpf, early gastrulation), we stillfind three classes of PGC arrangements. The positions of PGCclusters and the frequencies with which each arrangement isfound (data not shown) imply that they derive from the threearrangements described for the dome stage; however, at 60%epiboly, the PGC clusters are located more dorsally (Fig. 1A-C, middle panel). This movement starts relatively early, beforethe onset of gastrulation, since at shield stage (6 hpf, onset ofgastrulation), the square formed by the PGC clusters is alreadylost in most embryos (Fig. 2A). This first step of PGCmigration appears to be shared with somatic cells that at thisstage undergo compaction (Warga and Nüsslein-Volhard,1998) and convergence movements (Solnica-Krezel et al.,1995) towards the dorsal.

Step II, exclusion from the dorsal midlineAs the embryonic shield (the zebrafish organizer) is formed,PGC clusters located very close to the dorsal (starting positionC) are excluded from the midline to occupy a slightly morelateral position while more lateral clusters continue to convergedorsally. In general, no germ cells are observed in the extremedorsal region by 60% epiboly (Fig. 1C, middle panel).

Table 2. PGC phenotype of spadetail (spt) mutant embryosAverage PGC Embryos with PGCs Proportion of PGCs

Embryos analyzed number per embryo* anterior of main clusters anterior of main clusters

Stage wt siblings spt wt siblings spt wt siblings spt wt siblings spt

1 somite 13 10 50.4±9.7 56.2±10.8 46% 100% 2.4%±3.3% 35.9%±15.0%5 somites 29 25 39.1±6.4 39.7±12.6 48% 93% 3.7%±5.0% 21.3%±13.2%24 hpf 85 53 45.6±9.6 47.4±8.4 17% 96% 1.0%±2.6% 16.5%±10.6%48 hpf 8 10 n.d. 49.8±12.1 0% 100% 0% 26.9%±10.3%

Embryos with PGCs Proportion of PGCsposterior of main clusters posterior of main clusters

Stage wt siblings spt wt siblings spt

1 somite 100% 100% 17.7%±10.0% 21.6%±8.1%5 somites 97% 100% 22.3%±13.8% 18.6%±12.2%24 hpf 20% 72% 1.1%±3.0% 8.7%±9.7%48 hpf 0% 75% 0% 9.1%±9.8%

n.d., not done* in AB genetic background (see Materials and Methods).

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Step IIIa, alignment along the anterior border of thetrunk mesodermPGC clusters initially positioned close to the dorsal side alignalong the boundary between trunk and head paraxial mesodermmarked by the anterior border of the paraxial protocadherin(papc) (Yamamoto et al., 1998) expression domain. Thealignment can be first observed at the 60% epiboly stage and

is shown in Fig. 1D for the 90% epiboly stage. As developmentproceeds, this pattern becomes progressively more defined asthe PGCs form a 1- to 3-cell-wide line on either side of theaxis at the anteroposterior level of the first somite (Fig. 1A-C,lower panel). Cross sections of this region at the 1- and 4-somite stage reveal that the aligned PGCs are always in closecontact with the yolk syncytial layer (YSL) and the overlaying


Fig. 1. Migration of PGCs in wild-typeembryos. The PGCs are labeled using the vasamRNA probe (dark blue) and other structuresare labeled in red or in light blue with theprobes indicated. Embryos at somitogenesisstages were deyolked and flattened. (A-C) Thethree basic initial PGC arrangements relative tothe dorsal aspect of the embryo are shown atthe dome stage (upper panel), where dorsal ismarked by the chordino expression domain. At60% epiboly (middle panel) and the 2-somitestage (lower panel) also three classes ofarrangements are found at frequencies expectedfrom the three initial arrangements at the domestage. The 60% epiboly stages are stained withchordino (PGC clusters are marked witharrowheads), the 2-somite stages with myoDmarking the adaxial cells, papc expressed inthe segmental plate and the forming somites,and pax8 staining the otic placodes. Dome and60% epiboly stages are shown in animal viewwith dorsal up. (D,E) PGCs align along theborder of the trunk mesoderm that expressespapc both on the dorsal (D) and the ventral (E)side at the 90% epiboly stage. Note that noPGCs are found on the notochord, which isdevoid of papc staining in D. (F) A 1-somite-stage embryo showing the alignment ofposterior PGCs (arrowheads) at the lateralborder of the broad expression domain of pax8in the pronephric anlage. Pax8 also stains theotic placodes and myoD the adaxial cells (darkblue). (G,H) Cross-sections of 4-somite-stageembryos after whole-mount in situhybridization with vasa. (G) At the level ofsomite 1, two medially located PGCs are seenin contact with the YSL and the overlyingparaxial mesoderm (arrowheads), whereas twolaterally located PGCs have lost contact withthe YSL and extend up to the ectoderm(arrows). (H) A posterior trailing PGC (arrow)at the level of somite 7 found at the lateralmargin of the mesoderm in contact with theYSL and the ectoderm. (I) 8-somite-stageembryo stained with myoD (adaxial cells andsomites) and pax2.1 (pronephros, otic placodes,midbrain-hindbrain boundary and eye anlagen).There is one ectopic anterior PGC present inthis wild-type embryo (arrow). (J) A 16-somite-stage embryo stained with myoD. Notethat the PGC clusters have shifted towards theposterior, whereas trailing cells have migratedanteriorly. (K,L) PGCs are located in twolateral lines at the anterior end of the yolkextension at 24 hpf as seen in lateral (K) anddorsal (L) view.


paraxial mesoderm (for the 4-somite stage see Fig. 1G,arrowheads).

Step IIIb, alignment along the lateral border of themesodermPGC clusters located more ventrally also align along the borderof the papc expression domain from the 60% epiboly stage on(shown in Fig. 1E at 90% epiboly). Later these PGCs remainaligned to the outer border of the ventral mesoderm that startsto express pax8 in the anlage of the pronephros (Pfeffer et al.,1998) (Fig. 1F, arrowheads). Cross sections at the 4-somitestage show that these posterior trailing PGCs are in closecontact with the YSL and extend to the ectoderm (Fig. 1H).

At the 2-somite stage, the anterior and the lateral alignmentof PGCs (step IIIa and IIIb) are clearly visible and, dependingon the initial orientation of the clusters, three basicarrangements can be identified (Fig. 1A-C, lower panel).

Step IV, formation of two lateral PGC clustersBetween the 1- and the 5-somite stages, the rows of PGCs atthe level of the 1st somite migrate away from the axis and formtwo clusters lateral to the paraxial mesoderm extending fromthe 1st to the 3rd somite level (compare Fig. 1A-C, lower panelwith Fig. 1I). Interestingly, as the PGCs form these clusters,many cells appear to dissociate from the YSL (Fig. 1G,arrows).

Step V, anterior migration of trailing PGCsFrom the 95% epiboly stage to the 24 hpf stage, the percentageas well as the absolute number of PGCs in regions posterior ofthe main cluster progressively decreases (Fig. 2B and data notshown). Thus, from late gastrulation stages on, PGCs that havealigned along the lateral mesoderm border in posterior regionsmigrate towards the anterior along the pronephros and join themain PGC clusters (for example, compare the position of thesetrailing cells in Fig. 1I and J).

The formal possibility that this phenomenon results fromselective death of posteriorly positioned PGCs is not supportedby our analysis of embryos with abnormal PGC migrationpatterns. PGCs expressing the vasa marker can survive forextended periods of time in various ectopic locations includingposterior positions along their normal route (e.g., in chordinomutant embryos, see below).

Step VI, posterior positioning of the PGC clustersThe two PGC clusters initially form during step IV at the levelof the 1st to 3rd somite. This position shifts towards theposterior so that, at the 16-somite stage, the clusters are locatedat the level of the 5th to the 7th somite and by 24 hpf aroundsomite 8 along the anterior part of the yolk extension (Fig. 1J-L).

A summary of these six steps of early PGC migration thatbring the PGCs from their initial random distribution into thetwo clusters on either side of the axis is presented in Fig. 3.

Migration of PGCs in mutants affected indorsoventral patterningAs a first test for the control of somatic tissue over PGCmigration, we followed the migration pattern in mutantsaffecting the global dorsoventral patterning of the embryo.Genes affecting this process have been identified in zebrafishand, as in Xenopus, a balance between ventralizing bonemorphogenetic proteins (BMPs) and their antagonists secretedby the organizer has been shown to pattern the embryo along

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Fig. 2. In wild-type embryos, the PGCs converge towards the dorsalduring gastrulation and migrate anteriorly during somitogenesisstages. (A) The frequency of embryos showing the symmetrical‘square’ shape arrangement of the four PGC clusters decreases fromthe dome stage to the 60% epiboly stage while more embryos showthe ‘converged’ arrangement where clusters are shifted towards thedorsal. Irregular arrangements (3 or 5 clusters) appear at a constantfrequency of around 10%. n is the number of embryos analyzed.(B) Distribution of PGCs from late gastrula (95% epiboly) to 24 hpf.The number of vasa-positive cells was counted in four regions asindicated in the drawings of embryos at each stage. For the 95%epiboly stage, the regions were sectors along the dorsoventral axis asseen from the vegetal pole. For the 2- to 16-somite stages, anillustration of a flattened embryo is shown with the anterior up; theregions were defined relative to tissues stained by myoD, papc andpax8 for 2 somites (see Fig. 1A-C, lower panel), myoD and pax2.1for 8 somites (see Fig. 1I) and myoD for 16 somites (see Fig. 1J). Forthe 19-somite and 24 hpf stages, the regions were defined relative tostructures like the yolk extension. Note that for all stages the blueregion contains the main cluster of PGCs, whereas the red andyellow regions are of the same absolute size. n is the total number ofPGCs counted; for each stage 26 to 31 embryos were analyzed.

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the dorsoventral axis (reviewed in Mullins, 1999; Schier andTalbot, 1998; Solnica-Krezel, 1999).

Loss of function of the zebrafish chordin homolog chordino,a BMP antagonist, leads to a reduction in dorsal and anteriortissues and concomitant expansion of the ventral-posteriordomain (Hammerschmidt et al., 1996; Schulte-Merker et al.,1997). In 24-hour-old chordino mutant embryos, we find PGCsin ectopic locations (Fig. 4A, B). While many of the cells arriveat the correct position around the level of the 8th somite, on

average 27% of the cells are found posterior of the main cluster(Table 1) and the majority of these ectopic cells is found in thetail around the blood-forming region (not shown). At the 60%and 80% epiboly stages, the distribution of PGCs relative tothe dorsal side of the embryo is normal (Table 1) reflectingmore or less normal execution of migration steps I to III.However, in 2- and 8-somite-stage chordino embryos, we findmore PGCs in the expanded ventral-posterior region of theembryo (Table 1; Fig. 4C,D). Thus, in chordino mutants, stage


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Fig. 3. The six steps of early PGC migration in zebrafish. Schematic drawings of embryos from dome stage to 24 hpf showing the positions andmovements of the four PGC clusters for starting position B (see Fig. 1B). At dome stage, four clusters of PGCs are found close to theblastoderm margin in a symmetrical ‘square’ shape. All possible orientations of the square relative to the dorsal side of the embryo can beobserved. (A) Here, an intermediate arrangement is shown with one cluster close to, but not directly at the dorsal side (see Fig. 1B). Beginningbefore gastrulation, lateral and ventral clusters move towards the dorsal, with ventral clusters migrating more slowly (step I, convergencetowards the dorsal). This movement is shared with somatic cells and can be attributed to early compaction before gastrulation and dorsalconvergence of hypoblast cells during gastrulation. (B) Clusters located very close to the dorsal migrate away from the dorsal midline and aretherefore rarely found on the notochord from the 60% epiboly stage on (step II, exclusion from the dorsal midline). At the 60% epiboly stage,these movements have resulted in loss of the ‘square’ arrangement of PGC clusters; convergence continues, but some PGCs can still be found infar ventral positions until the end of gastrulation. (C) Dorsally located PGCs align along the border between the head and trunk paraxialmesoderm marked by the anterior border of the papc expression domain depicted by a dashed line (step IIIa, alignment along the anteriorborder of the trunk mesoderm). Ventrally located clusters align at the lateral border of the mesoderm that early on is also marked by theborder of papc expression and at late gastrulation starts to express the pronephros marker pax8 (step IIIb, alignment along the lateral borderof the mesoderm). (D) At the 2-somite stage, most PGCs have arrived in two lines at the level of the first somite. These anterior located PGCsmigrate towards the lateral (step IV, formation of two lateral PGC clusters). Cells that were initially located ventrally migrate towards theanterior along the anlage of the pronephros (step V, anterior migration of trailing PGCs). In this illustration, the positions of the PGCs aredrawn relative to the adaxial cells, the somites and the lateral border of the pronephric anlage. (E) At the 8-somite stage, all anterior PGCs arefound lateral to the paraxial mesoderm in a cluster extending from the 1st to the 3rd somite. These clusters start to move towards the posterior(step VI, posterior positioning of the PGC clusters), while the trailing cells tightly align on the lateral border of the pronephros and continueto migrate anteriorly. Here, the PGCs are drawn relative to the expression domains of myoD in the adaxial cells and somites and pax2.1 in thepronephros (see Fig. 1I). (F) At the 19-somite stage, the main clusters have shifted to more posterior positions and in 60% of embryos sometrailing cells are still seen. (G) At 24 hpf, the PGC clusters are located at the anterior end of the yolk extension, which corresponds to the 8th to10th somite level. In most embryos, all PGCs have reached this region, only a few trailing cells are found close to the main clusters. The modeldescribed here holds true for the vast majority of PGCs; in healthy wild-type clutches, about 1% of cells is found in ectopic anterior positionsand, in some cases, we see posterior trailing cells that do not align to the pronephros, but are found in the segmental plate.


V of PGC migration (migration towards the anterior) isspecifically affected. It is possible that the expansion ofventral-posterior fates disrupts the formation of gradedinformation that normally directs trailing PGCs towards theanterior. Additionally, it could be that the deficiency in anteriorstructures leads to a decrease of a signal that normally attractsPGCs. Hence, instead of migrating anteriorly the cells remainin a ventral-posterior position and are finally located in ectopicpositions in the tail.

The swirl mutation, which reveals the function of thezebrafish BMP2b gene, affects dorsoventral patterning of theembryo in an opposite manner (Kishimoto et al., 1997; Mullinset al., 1996). swirl mutant embryos display a strongdorsalization phenotype leading to formation of somitesaround the circumference of the embryo. These mutants showa striking PGC phenotype at the 1- and 5-somite stages withthe cells located in a line along the expanded paraxialmesoderm all around the embryo (Fig. 5A-D). This reductionof convergence of PGCs towards the dorsal midline can betraced back to mid-gastrulation stages; more cells are found inlateral and ventral positions at the 80% epiboly stage and theinitial ‘square’ shape of PGC clusters is maintained in abouthalf of the mutant embryos but is lost in all wild-type siblingsby this stage (Fig. 5E,F). Remarkably, while the PGCs appearto be located in random positions relative to the dorsoventralaxis at the 1- and 5-somite stages, they are found at the correctanteroposterior level of the first somite (Fig. 5A,B). Thus, thePGCs can properly respond to anteroposterior information,which extends around the entire embryo in swirl mutants.

We interpret the swirl phenotype as a manifestation ofdefects in several processes required for normal PGCmigration. First, swirl mutants show reduced convergencemovements (Mullins et al., 1996; Solnica-Krezel, 1999)

leading to a decrease in directional movement of the PGCstowards the dorsal midline of the embryo (Fig. 3A, step I). Theconvergence movement defects shared with somatic cellsposition the PGCs in a random dorsoventral position. Second,ventral mesodermal fates are not specified in swirl mutants(e.g., expression of the pronephric markers pax 8 and pax2.1is not detected (data not shown and Mullins et al., 1996)) sothe lateral edge of the mesoderm along which ventral PGCs arenormally found (e.g., Fig. 1B, lower panel) is not formed,eliminating the option of PGCs to align parallel to theanteroposterior axis. In contrast, in swirl mutants, the anteriorborder of the trunk paraxial mesoderm is formed and extendsventrally all around the embryo. Thus, PGCs at any positionaround the mutant embryo are close to this border as it formsduring gastrulation so that the alignment along the anteriorborder of the trunk mesoderm (step IIIa, Fig. 3C) takes placearound the circumference of the embryo.

Importantly, the lack of ventral fates in swirl mutants affects

Fig. 4. Migration of PGCs inventralized chordino mutant embryos.The PGCs are labeled using the vasaprobe (dark blue) and other structuresare labeled in red with the probesindicated. (A,B) Ectopic PGCs arefound around the expanded bloodforming region in the tail of chordinomutants at 24 hpf; (A) lateral view, (B) dorsal view. (C,D) At the 8-somite stage (stained with myoD and gata2 labeling the IntermediateCell Mass), more PGCs are found in the expanded ventral-posteriorpositions in chordino mutants (D) than in wild-type siblings (C) (seealso Table 1).

Fig. 5. The PGCs are found in random dorsoventral positions indorsalized swirl mutants, but all are located at the sameanteroposterior level. All embryos were stained with vasa in blue anda second staining in red was done with the probes indicated. (A,B) Atthe 1-somite stage, an alignment of PGCs anterior to trunk mesoderm(labeled by papc and myoD) is observed in lateral view of wild-type(A) and swirl mutant (B) embryos (anterior up, dorsal right), butPGCs are found all around the dorsoventral axis in swirl mutantembryos. (C,D) Lateral view of 5-somite-stage embryos stained withpax2.1 and myoD (here only the adaxial cells are labeled by thisprobe). PGCs are still dispersed around the circumference of theembryo in swirl mutants (D); they do not cluster together as they doin wild-type embryos at this stage (C) (see Fig. 1I for a dorsal viewof wild-type). (E,F) PGCs have converged towards the dorsal in wild-type (E), but not swirl mutant (F) embryos at 80% epiboly. Embryosare shown in vegetal view with the dorsal marked by chordinoexpression in the wild-type (E) and circumferential chordinoexpression in the swirl mutant (F).

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another movement normally found in wild-type embryos.Between the 1- and 5-somite stages, anteriorly located PGCsnormally migrate laterally to form a cluster on either side ofthe axis (Fig. 3D, step IV). We do not observe this movementin swirl embryos where the random distribution of PGCsaround the circumference of the embryo does not change fromthe 1- to the 5-somite stage. A likely explanation for this is thata lateral attracting target tissue located at the anteroposteriorlevel of the first somite is not specified in swirl mutants, leavingthe PGCs with no cues required for migration step IV.

Defining intermediate targets for PGCs – migrationalong the lateral border of the mesodermAn intermediate target at the lateral border of the mesodermthat the PGCs could align to during migration step IIIb (Fig.1A-C, bottom panels) is the Intermediate Cell Mass (ICM)which gives rise to blood and blood vessels and expresses thegata2 marker (Detrich et al., 1995). However, at the 7-somitestage, gata2 is expressed at a distance internally to the PGCline and the migration of PGCs in the cloche mutant thataffects hematopoiesis from early stages on (Stainier et al.,1995) is not affected (data not shown). These findings suggestthat the ICM is not required for lateral alignment of PGCs.The possibility that the posterior PGCs align to the lateralborder of the endoderm was ruled out by analyzing theexpression pattern of the endodermal marker fork head-2(Odenthal and Nüsslein-Volhard, 1998) relative to themigrating cells (data not shown).

Another option for an intermediate target along whichposterior PGCs align could be the developing pronephricsystem or simply the lateral edge of the mesoderm. Severalobservations are consistent with a role for these structures inorganizing the posterior PGCs. First, from the 1-somite stageon posterior PGCs are found aligning at the lateral aspect ofthe mesoderm (Fig. 1F,I). When the lateral border of themesoderm and the pronephros are missing, as is the case inswirl mutant embryos, the lateral alignment of trailing PGCsis absent. In addition, alterations in the position of the borderof the mesoderm relative to the dorsal midline can be followedby corresponding alterations in the position of the PGCs. Forexample, in knypek mutant embryos, which are defective inconvergence and extension movements (Solnica-Krezel et al.,1996), the pronephros is found far more lateral than in wild-type embryos, but still the posterior PGCs properly align to it(Fig. 6A).

Currently, no mutation is known that specifically affects thedevelopment of the pronephric anlage as early as the 80%epiboly to 1-somite stages. In the no isthmus mutant, whichshows a late phenotype in the pronephric system (Brand et al.,1996), the early alignment of the ventral PGCs to thepronephric anlage is not affected (data not shown). Therefore,while the developing pronephros is a good candidate for anintermediate PGC target, we could not prove this point usingmutations specifically affecting this structure.

Defining intermediate targets for PGCs – alignmentalong the anterior border of the trunk paraxialmesodermAs described above, PGCs that at the beginning of gastrulationare positioned closer to the dorsal side align at the first somitelevel along the border between the trunk and head paraxial

mesoderm (Fig. 3C, step IIIa, Fig. 6D), but are absent from thedorsal midline (Fig. 3B, step II, Fig. 6D).

The exclusion of PGCs from the midline suggests a role forthe notochord in repelling the PGCs. To test this, we examinedPGC migration in floating head mutants in which thenotochord is replaced by paraxial mesodermal fates (Halpernet al., 1995; Talbot et al., 1995). Clearly evident at 60% epibolyand shown in Fig. 6E for the 90% epiboly stage, PGCs can beobserved in an ectopic position at the dorsoventral level of thenotochord in floating head mutants (3.1±2.3 cells (n=10)compared with 0.4±0.8 in wild-type siblings (n=22)).Therefore, the notochord appears to be required directly orindirectly for exclusion of PGCs from the midline during earlygastrulation (Fig. 3B, step II). This phenotype is however onlytransiently observed; from about the 2-somite stage on whenthe PGCs start to migrate laterally no ectopic cells are foundat the midline of floating head mutants. In addition, both theanterior alignment step (Fig. 3C, step IIIa) and the formationof the lateral clusters around the 5-somite stage (Fig. 3D, stepIV) are normal (data not shown and Fig. 6B,C). We thereforeconclude that differentiated axial mesoderm is not required for


Fig. 6. PGC migration phenotype ofknypek and floating head mutantembryos. All embryos were stainedwith vasa in dark blue and otherprobes in red or light blue asindicated. (A) Trailing PGCs alignalong the abnormally locatedpronephros (stained with pax2.1,adaxial cells and somites with myoD)in knypek mutant embryos at the 7-

somite stage. (B,C) Normal arrangement of PGCs in wild-type (B)and floating head mutant (C) embryos at the 8-somite stagestained with myoD and pax2.1. (D,E) Dorsal view of embryos atthe 90% epiboly stage showing that dorsally located PGCs do notmigrate away from the dorsal midline in floating head mutantembryos. (D) In wild-type embryos, PGCs are not found at theregion of the forming notochord marked by lack of papc staining,but they extend into the dorsal midline that expresses papc infloating head mutant embryos (E).


defining the anteroposterior level of alignment nor for repellingPGCs away from the midline at the 1- to 5-somite stage.

Thus, an attractive possibility is that cues originating in theparaxial mesoderm are responsible for the early alignment ofPGCs at the border of the trunk mesoderm. To test this idea,

we followed the migration of the PGCs in spadetail mutants.The spadetail gene encodes a T-box protein important forproper development of trunk paraxial mesoderm (Griffin et al.,1998). In spadetail mutants, the expression of genes that markthe anterior border of the trunk paraxial mesoderm, like papcand paraxis, is already severely reduced at early stages ofgastrulation (Ho and Kane, 1990; Shanmugalingam andWilson, 1998; Yamamoto et al., 1998). In 24 hpf spadetailmutants, we observe PGCs in several ectopic positions (Fig.7A,B). In 72% of the spadetail mutants, PGCs are located inectopic posterior positions along the yolk extension or in themutant tail (on average 9% of all PGCs show this behavior,Table 2, Fig. 7A). Strikingly, in 96% of the mutant embryos,we find PGCs located in an ectopic anterior position at 24 hpf(on average 16% of all PGCs are located anteriorly; Fig. 7A,B;Table 2). Virtually all of these ectopic anterior PGCs are foundat the same anteroposterior position at the level of the 2nd

branchial arch (Fig. 8A,B). The appearance of ectopic anterior

Fig. 7. Ectopic PGCsare found in spadetailmutant embryos. Allembryos were stainedwith vasa in blue andother probes in red asindicated.(A,B) Ectopic anteriorPGCs are seen betweenthe midbrain-hindbrainboundary and the oticvesicle in 24 hpf stagespadetail mutantembryos and ectopicposterior PGCs arefrequently observed inthe tail; (A) lateralview, (B) dorsal view.(C,D) PGC alignmentat the anterior borderof the papc expresssiondomain at the 80%epiboly stage as seen ina dorsal view of a wild-type embryo (C) is lostin spadetail mutants(D). (E,F) At the 3-somite stage,alignment of PGCs atthe level of the 1st

somite as seen in wild-type embryos (E) islost in spadetailmutants (F) stainedwith myoD, papc andpax8. (G,H) At the 6-somite stage (embryosstained with myoD andpax2.1), the mainclusters of PGCslocated at the 1st to the3rd somite in wild-typeembryos (G) are foundcloser to the oticplacodes in spadetailmutants (H) andectopic anterior PGCsare found mainly inbetween the midbrain-hindbrain boundaryand the otic placodes inthe mutants.

Fig. 8. PGCs maintain their fate at ectopic locations. All embryoswere stained with the vasa probe in blue. (A, B) Double staining ofwild-type (A) and spadetail mutant (B) embryos with vasa andhoxa2 both in blue shows that anterior ectopic PGCs (arrows) arelocated at the anteroposterior level of the 2nd branchial arch(brackets) at 24 hpf. (C, D) Ectopic vasa-expressing cells maintainPGC morphology. Cross-section of a spadetail mutant (C) and awild-type (D) embryo at the indicated levels. Note that both theectopic vasa-positive cells in C and the PGCs located in the correctposition in D are large and show a distinct nuclear shape (see insert).

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PGCs can be traced back to loss of PGC alignment at the head-trunk mesoderm border: at the 80% epiboly stage, dorsal PGCsextend towards the anterior in most of the mutant embryos (Fig.7C,D). Consequently, ectopic anterior PGCs can be seen inspadetail mutant embryos at the 3- and 6-somite stages (Fig.7E-H; Table 2). At the 3-somite stage, the normal alignmentof PGCs at the first somite level is not observed. Instead, allPGCs are found at the lateral border of the mesodermextending into ectopic anterior regions (Fig. 7F). Thus, inspadetail mutant embryos, step IIIa of PGC migration isaffected.

An interesting open question is why all of the ectopicanterior cells in spadetail mutants end up at a defined positionat the anteroposterior level of the 2nd branchial arch. Twopoints relevant to this phenomenon can be made. First, thedefinition of this position of ectopic PGC accumulation is nota result of spadetail loss of function. In wild-type embryos, wealso observed rare cases of ectopic anterior cells (e.g. Fig. 1I,arrow; 1.3% of PGCs in wild type versus 16% in spadetailmutants). The position of these ectopic cells is identical to thatseen in the mutants. Second, tissue sections containing ectopicPGCs in this region in spadetail mutants reveal close cellularinteraction between the PGCs and surrounding branchial archtissue (Fig. 8C). Thus, the PGCs may share cell adhesionproperties with the somatic cells located at this positionresulting in a defined accumulation point for anterior PGCs.

The determination state of early migrating PGCs inzebrafishThe migrating vasa-expressing cells face conflictingrequirements related to their response for cues provided by theenvironment. On one hand, as shown above, the PGCs respondto signals provided by somatic tissues guiding them towardsthe gonad. On the other hand, during their migration, the PGCshave to ignore signals that would lead to their differentiationinto other cell types and to loss of totipotency.

The abnormal migration of PGCs in spadetail mutantembryos allows us to address the question of the determinationstate of the early migrating PGCs in fish. In these mutants,ectopic cells can be observed starting from the 80% epibolystage until at least 2 days of development (data not shown).Since the number of ectopic vasa-positive cells does notdecrease between the 24 hpf stage and the 48 hpf stage (Table2), it appears that vasa-expressing cells can survive in ectopiclocations for at least 2 days.

One possible explanation for this phenomenon is that thePGC fate is lost in the ectopic location and that the observedvasa signal reflects an exceptional stability of this mRNA. Totest this, we injected wild-type embryos with an in vitrotranscribed vasa mRNA. Early overexpression of vasa mRNAdid not lead to a change in the number of vasa-expressing cellsobserved in 24 hpf embryos. Importantly, the injected vasamRNA is not exceptionally stable, so that 24 hourspostinjection no traces of the injected mRNA can be detected(data not shown). The maintenance of the vasa signal by theectopic cells must therefore result from preservation of PGC-specific qualities, which might either preferentially protect thevasa mRNA from degradation (as described in C. elegans forgerm-cell-specific RNAs (Seydoux and Fire, 1994)) or, morelikely, could keep on synthesizing vasa mRNA duringembryogenesis.

To verify that the ectopic cells, in addition to expressingvasa, preserve other PGC characters, we examined theirmorphology. As can be seen in Fig. 8C,D, the morphology ofthe ectopic vasa-expressing cells in spadetail mutants is similarto that of PGCs in normal positions. Both the ectopic PGCsand cells in the normal position are very large and, in mostcases, show a distinct lobular nuclear shape (Fig. 8C, insert).These cellular features are characteristic of PGCs in teleosts(e.g. in B. conchonius (Gevers et al., 1992)). Thus, the ectopiccells maintain vasa gene expression and exhibit morphologicalcharacteristics typical for PGCs, suggesting preservation oftheir fate in the ectopic location at least for the first 48 hoursof development. We believe that at later stages of developmentthe ectopic PGCs die and consider it less likely that at thoselate stages they differentiate and join somatic tissues. However,since we did not follow the final fate of the cells, the possibilityof very late differentiation of ectopic PGC was not ruled out.

DISCUSSION

In this work, we followed the migration of the primordial germcells in early wild-type embryos and characterized six discretesteps of cell movements that bring the PGCs to their positionat 24 hours of development. Some of these movements areobviously shared with somatic cells (e.g., step I, convergencetowards the dorsal). For other steps, for example step IV(formation of two lateral PGC clusters) no correspondingmovements of somatic cells have been described, suggestingthat these movements reflect active PGC migration relative tothe surrounding tissue. Analysis of the behavior of PGCs inmutant embryos allows us to suggest which tissues are requiredfor proper PGC migration during early stages ofembryogenesis and to address questions related to PGC fate.

Migration of PGCs – solutions for fish-specificproblemsWhen compared with other model organisms, the zebrafishshows a unique process of PGC migration. While in mouse,Drosophila, Xenopus and urodeles, the migration starts fromthe same position in each embryo with the PGCs being directedtowards the target in a predictable path, the situation inzebrafish appears more complex. Based on our observations wesuggest a model that helps understanding how PGCs that canoriginate at any position relative to the dorsal tissue are able toarrive at a specific dorsoventral and anteroposterior level withinthe first 12 hours of development.

According to our model, the PGCs, which by the beginningof gastrulation are positioned close to the margin of theblastoderm, initially follow the general behavior described forhypoblast cells, which undergo dorsal convergence. Starting atearly gastrulation, the PGCs align to structures that define theborder of the trunk mesoderm (step IIIa and IIIb). This simplerequirement puts all the PGCs in proximity to an intermediatetarget irrespective of their initial position (Fig. 9A). Throughsuch a mechanism an ordered distribution of PGCs is achievedrelatively early in gastrulation in contrast with the apparentrandom positioning of PGCs a few hours earlier. This earlyarrangement is apparent from the 80% epiboly stage until the1-somite stage when the ventrally located cells are alignedalong the pronephros and cells that originated in the dorsal



align at the level of the first somite (Fig. 9B). The importanceof the early alignment is most strikingly demonstrated inspadetail mutants, which as judged by molecular markers, lacka defined anterior border of the trunk paraxial mesoderm; herethe dorsally located PGCs do not align at the trunk-headmesoderm border and are therefore found in ectopic anteriorlocations. Specific early elimination of the pronephros is notpossible using any existing mutations, so we were not able todirectly test a possible interaction of PGCs with this tissue.However, since PGCs are found in close proximity of thepronephric anlage from early stages onwards, and since otherventral tissues like the intermediate cell mass apparently do notplay a role in directing PGC migration, the pronephros remainsan attractive candidate to serve as an intermediate target forPGCs. Interestingly, in urodeles, positioning of PGCs in thelateral plate mesoderm has been described. While PGCs inurodeles arise in ventral positions (i.e. not random relative tothe dorsal), during somitogenesis these cells are found in aposition reminiscent of that taken by posterior zebrafish PGCswhich align along the pronephros (Nieuwkoop and Sutasurya,1979).

Beginning after the 1-somite stage, we observe directedmigration of cells towards a lateral tissue at the anteroposteriorlevel of the first somite. Both the posteriorly located trailingcells as well as the anteriorly aligned cells can be observed tomigrate towards this position (Fig. 9C). The existence of anattractive signaling center at the lateral anterior part of thetrunk mesoderm is consistent with the normal lateral migrationof PGCs (step IV) in floating head embryos, which arguesagainst repulsion by the notochord at this stage. The swirl andthe spadetail phenotypes also argue for the existence of sucha signaling center. The absence of the laterally directedmovements of the PGCs in swirl mutants could reflect loss ofventral and lateral cell fates including that of the putativesignaling center. In contrast, in spadetail mutants, thisattraction center is not lost, since at 24 hpf the primordial germcells are not randomly distributed anterior to the normal clusteras seen at the 1-somite stage. Instead, in spadetail embryos,most of the PGCs appear to migrate back towards the maincluster while some migrate towards a specific position at thelevel of the 2nd branchial arch.

Specification and maintenance of PGC fateThe identification of the zebrafish vasa homolog and its earlylocalization to the PGCs (Yoon et al., 1997) suggest thatspecification of the PGCs in zebrafish occurs by a mechanismof asymmetric localization of cytoplasmic factors includingmRNAs. Interestingly, when vasa mRNA was injected intoearly embryos, we did not detect a change in the number ofPGCs. We conclude that vasa mRNA per se is not a limitingfactor for PGC specification. It is possible that, in addition tovasa, other cytoplasmic components need to be localized in thefuture PGCs or that an assembly of an active complex ofmolecules takes place during oogenesis. Indications for thetemporal requirement for vasa activity come from Drosophilawhere the Vasa protein is localized to the posterior pole and isfunctionally required during oogenesis for patterning of theabdomen (Hay et al., 1988; Lasko and Ashburner, 1988;Lehmann and Nüsslein-Volhard, 1991).

Once specified, the PGCs should maintain their totipotencyand ignore differentiation signals other than signals important

C 3 somites

B bud

A 60% epiboly

ventral ventraldorsal

Fig. 9. A model for a transition from a random distribution ofPGCs prior to gastrulation to an organized arrangement ofmigrating cells. Schematic drawings show the tissues that providepositional cues for the early migrating PGCs with the notochord ingray, trunk mesoderm in light blue and the PGCs in red. Based onanalysis of multiple embryos, the PGCs are drawn in differentpossible positions; in any single embryo, PGCs are found in onlysome of these locations. Before gastrulation, the PGC clusters arefound in random dorsoventral positions close to the blastodermmargin. (A) At early gastrulation stages, PGCs are found scatteredaround the border of the trunk mesoderm, which they meet ormigrate towards (indicated by black arrows), gradually forming aclear alignment pattern by the end of gastrulation. Cells locateddirectly at or close to the dorsal midline where the notochord isforming are excluded from this region and occupy a more lateralposition. This tendency to align at the borders of the trunkmesoderm leads to an organized arrangement of all PGCs relativeto early developing structures during gastrulation. (B) At the tailbud stage, PGCs are located in a line along the anterior border ofthe trunk mesoderm or align along the pronephros at the lateralborder of the mesoderm. These posteriorly located cells migratetowards the anterior. (C) We propose that, at the 2- to 3-somitestage, a signaling center (dark blue) is established that attractsPGCs located in its vicinity. This leads to migration of PGCslocated at the anterior trunk mesoderm border towards more lateralpositions and may participate in directing posterior trailing cells tothe anterior.

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for their differentiation as gametes. The issue of PGC fate hasbeen investigated extensively in Drosophila. By following thefate of labeled transplanted PGCs, it was found that a largeproportion (up to 50%) of the PGCs end up in regions outsideof the gonad. These cells, however, never differentiated assomatic cells, were never incorporated into adjacent tissuesand eventually died (Kobayashi et al., 1993; Technau andCampos-Ortega, 1986; Underwood et al., 1980). In theexperiments described here, we did not label the PGCs with astable lineage marker, but rather relied on vasa geneexpression. Our ability to follow cells located in severaldifferent ectopic locations for a few days (e.g., in chordino andin spadetail mutants) coupled with the fact that the totalnumber of vasa-positive cells is similar in wild-type andmutant embryos argues for the reliability of vasa as a stablemarker for PGCs. At least during the first 48 hours ofdevelopment, the ectopic cells maintain some PGC charactersas determined by their morphology and by the fact that theyexpress the vasa gene. Our findings are therefore consistentwith those from Drosophila suggesting that PGCs normallydo not contribute to other cell lineages.

These results differ from results in mouse and Xenopus(Stevens, 1970; Wylie et al., 1985). In these systems,transplantation of migrating germ cells into ectopic locationsled to their differentiation into a variety of somatic tissues. Apossible explanation for these conflicting results may be thatthe behavior of the cells was studied at different stages of theirdevelopment. We investigated the determination state of thePGCs at an early stage: the abnormally migrating cells that wefollowed in mutant embryos get into ectopic positions startingas early as mid-gastrulation and thus have not been subjectedto the normal signaling that would be provided by the gonador tissues adjacent to it. The transplanted PGCs in the mouseand in Xenopus have been obtained from stages of migrationthat most likely correspond to later steps in zebrafish. Since thePGCs have to respond to signals from the somatic tissue of thegonad (e.g. Mukai et al., 1999), their resistance todifferentiation cues should be relieved at later stages. Wetherefore propose that these seemingly conflicting resultssimply reflect two different stages in the differentiation of theearly PGCs.

ConclusionsIn this work, we provide a detailed description of germ cellmigration in zebrafish and propose a model to explain theobserved cell behavior in wild-type and mutant embryos.Future studies aimed at testing and refining this first model willutilize newly identified mutations specifically affecting theprocess of germ cell migration and will be enhanced bygeneration of fish lines that allow the migration process to befollowed in live embryos.

We thank colleagues in the zebrafish field for probes and the labsof Christiane Nüsslein-Volhard, Matthias Hammerschmidt andWolfgang Driever for zebrafish strains. We thank Wolfgang Drieverfor help and support, Gerlinda Wussler for taking care of the fishfacility and other members of the department for helpful discussions.We are grateful to Randy Cassada, Iain Drummond and Lila Solnica-Krezel for critical reading of the manuscript. This work was supportedby grants from the European Commission TMR program(ERBFMBICT983315), from the Deutsche Forschungsgemeinschaft(RA863/1-1) and from the Landesschwerpunkt Baden-Württemberg.

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