1 Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
2 Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, CU de Strasbourg, France
*Author for correspondence (e-mail: eraz{at}gwdg.de)
Accepted 3 October 2001
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SUMMARY |
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Key words: Germ cells, Primordial germ cells, Zebrafish, Cell migration, Chemotaxis
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INTRODUCTION |
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First, PGCs reach the gonad primordia by a combination of passive morphogenetic movements and active migration. In Drosophila for example, the PGCs, which are formed at the posterior pole of the embryo, are passively swept into the midgut during gastrulation. From there, they actively migrate through the gut epithelium towards the gonadal mesoderm (Jaglarz and Howard, 1995). Second, the migration path is controlled by cues from the somatic environment and is not autonomous to the PGCs (Cleine, 1986; Jaglarz and Howard, 1994; Wylie et al., 1985). Third, interactions of motile PGCs with the extracellular matrix (ECM) are required for proper migration (Anderson et al., 1999; Bendel-Stenzel et al., 2000; Di Carlo and De Felici, 2000) and contact-mediated interactions have been proposed to play a role also in PGC guidance. For example, Xenopus PGCs appear to be oriented by a polarized cellular or ECM substratum (Heasman et al., 1981; Heasman and Wylie, 1981) and the accumulation of mouse PGCs in the gonad might involve adhesion of pioneer PGCs to the target and subsequent aggregation of interconnected PGCs (Garcia-Castro et al., 1997; Gomperts et al., 1994). Fourth, the gonad primordia appear to produce signals that attract PGCs. This has been shown in mouse, where explants of gonadal tissue can attract PGCs in vitro (Godin et al., 1990), and in chick, where transplanted gonadal tissue can direct accumulation of PGCs in ectopic regions (Kuwana and Rogulska, 1999). Furthermore, in Drosophila, the gene columbus (Hmgcr FlyBase), which is expressed in gonadal mesoderm, is thought to be involved in production of a signal that attracts PGCs (Van Doren et al., 1998). In Drosophila, PGC migration occurs in several distinct steps, some of which do not depend on formation of the somatic gonad (Jaglarz and Howard, 1994; Jaglarz and Howard, 1995; Moore et al., 1998). Apart from the fact that the Drosophila wunen genes appear to be involved in production of a signal that repels PGCs from certain regions of the gut (Starz-Gaiano et al., 2001; Zhang et al., 1997), little is known about the mechanisms that control such intermediate steps in vertebrates and invertebrates. Notably, whether PGCs are attracted towards intermediate targets is not known.
We have previously analyzed the path taken by zebrafish PGCs and the requirement of somatic tissues for controlling PGC migration (Weidinger et al., 1999). As in Drosophila, zebrafish PGC migration can be divided into several discrete steps, some of which are specifically affected by deletion of certain somatic tissues. A key step of migration occurs during early somitogenesis, when the PGCs become organized into two bilateral clusters in the anterior trunk. We show that individual PGCs migrate actively towards the clustering position from several different directions. Furthermore, genetic deletion of the target tissue results in a complete loss of PGC cluster formation. Together, these findings support the notion that the target tissue produces signals that attract PGCs. Interestingly, fate-mapping analysis shows that the somatic gonad, the final target of PGC migration, is not derived from this tissue. Thus, we provide evidence that zebrafish PGC migration is regulated by attraction of PGCs towards an intermediate target.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
Two-color mRNA in situ hybridization was performed as described by Jowett and Lettice (Jowett and Lettice, 1994) with modifications according to Hauptmann and Gerster (Hauptmann and Gerster, 1994) and a combination of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate), both at 175 µg/ml, was used as alkaline phosphatase substrate in the second color reaction producing a red-brown color. The following probes were used: cmlc2 (Yelon and Stainier, 1999), cathepsin L (hgg1 Zebrafish Information Network) (Vogel and Gerster, 1997), krox20 (egr2 Zebrafish Information Network) (Oxtoby and Jowett, 1993), preproinsulin (ins Zebrafish Information Network) (Milewski et al., 1998), myoD (myod Zebrafish Information Network) (Weinberg et al., 1996), nos1 (a zebrafish nanos homolog expressed specifically in PGCs) (Köprunner et al., 2001), ntl (Schulte-Merker et al., 1994), papc (pcdh8 Zebrafish Information Network) (Yamamoto et al., 1998), pax2.1 (pax2a Zebrafish Information Network) (Krauss et al., 1991), pax8 (Pfeffer et al., 1998), wt1 (Serluca and Fishman, 2001).
In vivo observation of PGCs
It is possible to specifically express green fluorescent protein (GFP) in zebrafish PGCs using an RNA coding for mmGFP-5 (Siemering et al., 1996) that contains the nanos1 (nos1) 3' untranslated region (GFP-nos1-3'UTR) (Köprunner et al., 2001) or an RNA encoding a fusion protein of zebrafish Vasa with GFP that also contains the vasa 3'UTR (full-vasa-GFP) (U. W., G. W. and E. R., unpublished). When these RNAs are injected into one-cell stage embryos, most of the PGCs can be detected from mid-gastrula stages onwards. For labeling of somatic cells, a farnesylated EGFP (Clonetech) that is localized to the plasma membrane was ubiquitously expressed using injection of RNA containing the Xenopus globin 3'UTR. For in vivo observation of cytoplasmic processes extended by migrating PGCs, farnesylated EGFP was specifically expressed in PGCs using the nanos1 3'UTR (EGFP-F-nos1-3'UTR). Capped RNA was synthesized from linearized plasmids using the Ambion Message Machine kit. For labeling of PGCs either 160 pg of GFP-nos1-3'UTR RNA, 30 pg of full-vasa-GFP, 140 pg of GFP-nos1-3'UTR plus 10 pg EGFP-F-globin or 80 pg of EGFP-F-nos1-3'UTR were injected according to standard procedures into one-cell stage embryos. Putative spt mutant embryos were selected at late gastrula stages by the presence of ectopic anterior PGCs, observed throughout mid-somitogenesis stages and their phenotype was verified at 24 hpf.
Fate mapping
To map the fate of the wt1-expressing cells at the anterior trunk, about 2 nl of 0.9% DMNB-caged fluorescein dextran (10000 MW, anionic, Molecular Probes, Eugene, Oregon, USA) in 0.2 M KCl were injected into 1- to 2-cell stage embryos that had been dechorionated by pronase treatment. At the 3- to 5-somite stage, embryos were mounted in methylcellulose, viewed under 20x magnification and oriented using DIC optics. A small patch of cells lateral to the most anterior somites on one side of the embryo was labeled by uncaging the fluorescein using illumination with UV light (DAPI filter) for about 1 second. By focusing on the lower cell-layer, uncaging was restricted mainly to mesodermal cells. Some embryos were fixed immediately after the uncaging process, while others were raised up to the 24-hpf stage, fixed and processed for whole-mount in situ hybridization using either wt1 or nos1 digoxigenin-labeled antisense probes. After the first color reaction in blue, the uncaged fluorescein was detected using an anti-fluorescein-AP antibody and red color reaction according to the two-color in situ hybridization protocol. At the 24 hpf stage, labeled somatic cells were detected only anterior of the PGCs in 18 of 19 embryos, while in 1 embryo, a few labeled somatic cells could be detected at the anteroposterior level of the PGCs. We contribute this to an error in labeling the correct region at early somitogenesis, since in 1 of 18 embryos that were fixed immediately after the uncaging procedure cells posterior of the PGC cluster were labeled.
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RESULTS |
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Occasionally, ectopic anterior PGCs, which are rarely present in wild-type embryos, also migrate posteriorly towards the forming clusters (Fig. 1C-L, red arrow). To analyze this phenomenon in more detail, we made use of spadetail (spt) mutant embryos, which exhibit severe defects in early PGC migration (step III), resulting in accumulation of many ectopic PGCs in anterior regions (Weidinger et al., 1999). In these mutants, the PGCs fail to align at the head-trunk border during gastrulation and therefore are randomly distributed along the anteroposterior axis at early somitogenesis stages (Fig. 2B). However, during the next few hours of development (between the one- and six-somite stage) most of the cells accumulate in the normal clustering position in the anterior trunk, while some of the ectopic anterior PGCs form clusters at the anteroposterior level of the second branchial arch (Fig. 2B-J) (Weidinger et al., 1999). Thus, the early dramatic PGC migration defect of spt mutants is largely reversed by mid-somitogenesis. This is achieved by migration of ectopic anterior PGCs posteriorly towards the normal clustering position as observed by time-lapse analysis of live spt mutant embryos (Fig. 2B-J). In addition, PGCs that were located in posterior trunk regions correctly migrate anteriorly towards the forming clusters (step V). PGCs can migrate towards the main clustering position from far anterior regions (arrows in Fig. 2), while cells that were initially adjacent to them can end up in the ectopic anterior cluster (arrowheads in Fig. 2). Because the embryos continue to extend during the observation, and owing to their bent axis, it is difficult to assess the actual distance covered by those PGCs migrating posteriorly, but we estimate that some cells had to migrate at least 10 PGC cell diameters before they reached the main clustering position.
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Analysis of spt;ntl, spt;oep and oep;ntl mutant embryos thus shows that the ability to form PGC clusters correlates with proper differentiation of the lateral mesoderm of the anterior trunk. Hence, this tissue is required to organize clustering of PGCs at early somitogenesis.
The putative attraction center of the anterior trunk is an intermediate target for PGCs
We have provided evidence supporting the notion that bilateral cluster formation of zebrafish PGCs is regulated by attraction of PGCs towards the lateral mesoderm of the anterior trunk. In chick and mouse, the somatic tissues of the gonad have been shown to attract PGCs (Godin et al., 1990; Kuwana and Rogulska, 1999). Therefore, we tested whether the putative zebrafish PGC attraction center gives rise to the gonad, the final target for PGCs.
By day 10 of zebrafish development, PGCs have condensed with somatic cells to form the gonad primordia (Braat et al., 1999; Yoon et al., 1997). The gonads are formed around the anteroposterior level of somite 10, while the putative attraction center that controls bilateral PGC cluster formation at early somitogenesis is located at the level of somites 1 to 3. The PGCs migrate between these two positions during step VI of migration, which starts at about the 10-somite stage and is completed by 24 hpf (Weidinger et al., 1999). To test whether somatic cells of the putative PGC attraction center migrate posteriorly together with the PGCs, we labeled these cells at early somitogenesis and determined their position at 24 hpf. For this purpose, we injected early embryos with a caged fluorescein dextran, uncaged the fluorescein in a small patch of cells lateral of the anterior somites at early somitogenesis and detected the labeled cells using whole-mount in situ hybridization. The region surrounding and including the main PGC cluster could be properly targeted as observed in embryos that were fixed immediately after the uncaging procedure (Fig. 6A). Interestingly, at 24 hpf, labeled mesodermal cells were found only anterior of the PGC clusters (Fig. 6B), indicating that the PGCs separate from the somatic cells of the putative PGC attraction center. These somatic cells stay roughly at the same anteroposterior position, and contribute to formation of the pronephric glomeruli, which continue to express wt1, and to more anterior mesodermal structures (data not shown).
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Defects in mesoderm patterning in the presumptive gonadal region result in failure of PGCs to reach their final target
Two observations support the idea that in zebrafish, too, somatic cells attract the PGCs towards the region of the gonad. First, the bilateral clusters of PGCs actively migrate towards the final target during step VI of migration. Fig. 7 shows four frames from a time-lapse movie (Movie 3 on-line) demonstrating that the PGCs migrate posteriorly relative to the somites. The PGCs do not migrate as an organized cluster, but frequently change their positions relative to each other.
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DISCUSSION |
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(1) The formation of PGC clusters in the lateral mesoderm of the anterior trunk occurs by active migration of PGCs towards this position. In live embryos, GFP-labeled PGCs can be observed to change their position relative to neighboring somatic cells. Notably, those PGCs that migrate from medial to lateral positions actually move in a direction opposite to that of somatic cells, which undergo convergence towards the midline at this time. The PGCs show the characteristic cell shape changes of actively migrating cells as previously reported for migrating PGCs in Drosophila (Jaglarz and Howard, 1995) and mouse embryos (Anderson et al., 2000).
(2) PGCs can migrate towards the clustering position from three different directions. In wild-type embryos, most of the PGCs migrate from medial positions (step IV), while some join the forming clusters from posterior regions (step V). PGCs can, however, migrate towards the clusters from anterior positions as well, which is rarely observed in wild-type, but readily seen in spt mutant embryos.
(3) The proper development of the target tissue is essential for accumulation of PGCs in this region. In double mutants that exhibit severe mesodermal defects, no PGC clusters are formed when wt1, which marks the somatic cells at the clustering region, is not expressed. At the same time, it appears less likely that repulsion by midline tissues plays a role in directing the PGCs towards lateral positions (Weidinger et al., 1999).
(4) PGCs migrate towards the target as individual cells. This is in contrast to Drosophila border cells, for example, whose migration also appears to be regulated by attraction towards the target (Duchek and Rorth, 2001). Proper migration of the border cell cluster appears to depend on guidance cues that are received by leader cells that direct the rest of the cells towards the target (Niewiadomska et al., 1999; Duchek and Rorth, 2001). It has been suggested that leader or pioneer cells are also important for mouse PGC migration from the gut through the dorsal mesentery into the developing genital ridge (Garcia-Castro et al., 1997). Some PGCs enter the site where the genital ridge will develop directly from the gut before the mesentery forms. Later-emerging PGCs are connected with these pioneers via an extensive network of long filopodial processes (Gomperts et al., 1994). PGC aggregation might then cause PGCs to accumulate in the genital ridge (Garcia-Castro et al., 1997). While we do not know to what extent zebrafish PGCs are interconnected, our observations of migrating PGCs in live embryos suggest that they migrate as individual cells. Pairs of ectopic anterior PGCs that are located next to each other can take very different migration paths, e.g., while one migrates into the ectopic anterior cluster the other moves back into the main PGC cluster. In addition, if a group of cells migrates over a certain distance together, the cells frequently change their positions relative to each other. These observations imply that each PGC can read and respond independently to guidance cues.
(5) The fact that neighboring ectopic anterior cells migrate in opposite directions virtually excludes the possibility that their migration is controlled by gradients of adhesive molecules present along the migration path (haptotaxis). Rather, such behavior can be most easily explained by assuming that two attraction centers, the main clustering position in the anterior trunk and another one at the anteroposterior level of the second branchial arch, compete for PGCs (Weidinger et al., 1999).
Taken together, the behavior of migrating PGCs and the fact that genetic deletion of the target tissue results in complete loss of PGC clustering leads us to propose that PGC cluster formation is regulated by chemoattraction of individual cells.
PGC migration is controlled by intermediate targets
In Drosophila, the columbus gene appears to be involved in the production of a signal that attracts PGCs towards their final target: the gonadal mesoderm (Van Doren et al., 1998). Attraction of PGCs by the gonad has also been demonstrated for the mouse and chick (Godin et al., 1990; Kuwana and Rogulska, 1999). Interestingly, the transcription factor Wilms tumor suppressor gene 1 (wt1), which is expressed in the common precursor of kidney and gonad in mouse (Armstrong et al., 1993), is expressed in the putative PGC attraction center of zebrafish. Surprisingly however, our fate-mapping experiments show that the cells forming this center become separated from the PGCs during later development. Thus, they do not comprise the precursors of the somatic gonad. Recently, a fate map of the zebrafish pronephric kidney field was published (Serluca and Fishman, 2001). This study confirms our finding that the wt1-expressing cells remain in the anterior trunk and that they contribute to formation of the glomerulus. By contrast, the clusters of PGCs actively migrate towards more posterior regions, away from the wt1-expressing cells. This final step of migration is defective in han mutants. The PGC clusters initially separate from the wt1-expressing tissue in han mutant embryos, but then fail to complete their posteriorwards migration. At the same time, PGCs that are located in posterior regions appear to stop their migration towards the anterior. Although we were not able to directly test whether these defects in migration towards the final target are associated with defective development of the gonad in han embryos, we could show that the intermediate mesoderm in the target region is mispatterned. Thus, it is possible that the intermediate mesoderm in this region, which is likely to give rise to the somatic tissues of the gonad, fails to attract PGCs. However, a final proof of this model awaits the identification of molecular markers for the gonadal mesoderm.
While it is possible that the final steps of zebrafish PGC migration are controlled by attraction towards the somatic tissues of the gonad, our results underscore the importance of viewing PGC migration as a multistep process that is not solely controlled by attraction of PGCs towards the gonad. This has also been demonstrated in Drosophila, where the whole mesoderm, including the precursors of the somatic gonad, is not required for early steps of migration (Jaglarz and Howard, 1994; Warrior, 1994). The only known mechanism controlling PGC migration before the gonad comes into play is repulsion from specific regions of the gut in Drosophila (Starz-Gaiano et al., 2001; Zhang et al., 1997). We show here that PGC migration can also be regulated by attraction towards an intermediate target. It would be interesting to test whether intermediate attraction centers guide PGCs also in other organisms and whether the developing kidney plays a role in PGC migration in other vertebrates as well.
Little is known about the molecular control of germ cell migration. The WT1 transcription factor can act as a repressor as well as an activator and it has been found to most strongly activate the epidermal growth factor family member amphiregulin in cell culture (Lee et al., 1999). However, PGC migration into the urogenital ridge is normal in Wt1 knockout mice (Kreidberg et al., 1993). In zebrafish, too, wt1 overexpression and knock-down experiments have failed to disturb PGC migration (G. W. and E. R., unpublished). Thus, zebrafish wt1 is probably not directly involved in regulating PGC migration. In mice, the secreted factor steel (Kitl Mouse Genome Informatics) is expressed along the migration path of PGCs and, together with its receptor Kit, which is expressed in PGCs, is required for proper migration and survival of PGCs (Bernex et al., 1996; Matsui et al., 1990). However, instead of acting as a chemoattractant for PGCs, steel is believed to be required for motility of PGCs and the Kit/steel interaction for proper adhesion of PGCs to cellular substrates (Godin et al., 1991; Pesce et al., 1997). In zebrafish, a steel homolog has not been described, and loss-of-function of sparse, a zebrafish Kit ortholog, does not affect PGC migration (Parichy et al., 1999). Another secreted factor that has been suggested to function in attracting PGCs in vertebrates is transforming growth factor (TGF)ß1. Antibodies directed against TGFß1 inhibit the ability of mouse urogenital ridge explants to attract PGCs and mouse PGCs migrate towards a TGFß1 source in vitro (Godin and Wylie, 1991). However, as the expression of TGFß1 has so far not been described in zebrafish, it is unclear whether it represents a candidate for mediating the effects of the proposed PGC attraction centers in this organism.
In view of the fact that the migration paths and the timing of PGC migration are not conserved among different vertebrate groups, it will be interesting to determine whether conservation nonetheless exists at the level of the molecules that control these processes.
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ACKNOWLEDGMENTS |
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