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Development 129, 25-36 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV2781
Regulation of zebrafish primordial germ cell migration by attraction towards
an intermediate target
Gilbert Weidinger1, Uta Wolke1, Marion Köprunner1, Christine Thisse2, Bernard Thisse2 and Erez Raz1,*
1Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
2Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404
Illkirch cedex, CU de Strasbourg,
France
*Author for correspondence (e-mail: [email protected])
Accepted 3 October 2001
SUMMARY
Migration of primordial germ cells (PGCs) from their site
of specification towards the developing gonad is controlled
by directional cues from somatic tissues. Although in
several animals the PGCs are attracted by signals
emanating from their final target, the gonadal mesoderm,
little is known about the mechanisms that control earlier
steps of migration. We provide evidence that a key step of
zebrafish PGC migration, in which the PGCs become
organized into bilateral clusters in the anterior trunk, is
regulated by attraction of PGCs towards an intermediate
target. Time-lapse observations of wild-type and mutant
embryos reveal that bilateral clusters are formed at early
somitogenesis, owing to migration of PGCs towards the
clustering position from medial, posterior and anterior
regions. Furthermore, PGCs migrate actively relative to
their somatic neighbors and they do so as individual
cells. Using mutants that exhibit defects in mesoderm
development, we show that the ability to form PGC clusters
depends on proper differentiation of the somatic cells
present at the clustering position. Based on these findings,
we propose that these somatic cells produce signals that
attract PGCs. Interestingly, fate-mapping shows that these
cells do not give rise to the somatic tissues of the gonad,
but rather contribute to the formation of the pronephros.
Thus, the putative PGC attraction center serves as an
intermediate target for PGCs, which later actively migrate
towards a more posterior position. This final step of PGC
migration is defective in hands off mutants, where the
intermediate mesoderm of the presumptive gonadal region
is mispatterned. Our results indicate that zebrafish PGCs
are guided by attraction towards two signaling centers, one
of which may represent the somatic tissues of the gonad.
INTRODUCTION
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
In many organisms, the primordial germ cells (PGCs), the
precursors of the gametes, are set aside from the somatic cell
lineages early in development. The somatic cells of the gonad
are specified later and at a different position. Therefore, the
PGCs have to migrate through the embryo to reach the
developing gonad. This process represents an excellent model
in which to study the control of directional cell migration, as
in most organisms the PGCs migrate over long distances and
follow a complex path. PGC migration has been studied most
extensively in Drosophila, Xenopus, chick and mouse (StarzGaiano and Lehmann, 2001). While the timing of migration
and the path taken by the PGCs vary considerably, at least
some of the guidance mechanisms appear to be conserved.
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
Movies available on-line
Key words: Germ cells, Primordial germ cells, Zebrafish, Cell
migration, Chemotaxis
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G. Weidinger and others
(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.
MATERIALS AND METHODS
Zebrafish maintenance and mutant strains
Zebrafish (Danio rerio) were maintained as previously described
(Westerfield, 1995). The mutant strains used are: hands off, hans6
(Yelon et al., 2000); no tail, ntlb160 (Halpern et al., 1993); one-eyedpinhead, oepm134 (Schier et al., 1997); spadetail, sptb104 (Kimmel et
al., 1989). Mutants were identified at the six-somite stage by wholemount RNA in situ hybridization by absence or reduction of
expression of the following markers: spt, myoD in somites; oep,
cathepsin L; spt;ntl, myoD and ntl; spt;oep, myoD and cathepsin L;
oep;ntl, cathepsin L and ntl. hands off mutant embryos were identified
by reduced expression of cmlc2 (Yelon et al., 2000).
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)-5phenyl-tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolylphosphate), 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 (GFPnos1-3′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 antifluorescein-AP antibody and red color reaction according to the twocolor 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.
RESULTS
We have previously described the six discrete steps of zebrafish
PGC migration (Weidinger et al., 1999). During step III,
the PGCs align along the borders of the trunk mesoderm.
Consequently, at the end of gastrulation, most PGCs are found
in two medial-to-lateral lines at the head-trunk border, while
the rest are located in more posterior regions at the lateral
borders of the mesoderm. Between the one- and six-somite
stages, the PGCs located at the head-trunk border migrate
laterally to form two clusters at somite levels 1 to 3 (step IV
Zebrafish germ cell migration
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Fig. 1. Zebrafish PGCs form bilateral clusters in the anterior trunk during early somitogenesis. (A,B) Dorsal views of zebrafish embryos
depicting the movements (arrows) of PGCs that result in bilateral PGC cluster formation. The PGCs (red) are drawn relative to the adaxial
cells, the somites and the lateral edges of the trunk mesoderm. (A) At the end of gastrulation, most PGCs have accumulated in two medial-tolateral lines at the head-trunk border, while the rest aligns along the lateral trunk mesoderm borders in more posterior regions. During early
somitogenesis, both groups of cells migrate towards the lateral mesoderm of the anterior trunk (steps IV and V). (B) At the six-somite stage,
bilateral clusters of PGCs have formed in the anterior trunk, while the posterior trailing PGCs continue to migrate towards the anterior. (C-L)
Fluorescent pictures taken at 16 minute intervals from a time-lapse movie showing migrating PGCs between the bud- and seven-somite stage
on the right side of a wild-type embryo injected with GFP-nos1-3′UTR. Dorsal views, anterior is upwards. Medially located PGCs migrate
laterally (one cell is marked by a green arrow) to join those PGCs that are already located in lateral positions. The forming cluster follows the
general convergence movements medially towards the midline. A single ectopic anterior cell (red arrow) migrates posteriorly and laterally into
the cluster. Note that on this side of the embryo no posterior trailing PGCs can be seen.
of migration, see Fig. 1A,B). At the same time, the posterior
trailing PGCs start to migrate anteriorly and will eventually
join the main clusters (step V, Fig. 1A,B). We have suggested
that both steps IV and V of migration are regulated by
attraction of PGCs towards the lateral mesoderm of the anterior
trunk. If this model is correct, then two predictions can be
made: first, PGCs should actively migrate towards this tissue,
and second, deletion of the putative attraction center should
result in loss of PGC cluster formation.
PGC cluster formation occurs by active migration of
PGCs from medial, posterior and anterior regions
To test whether PGCs actively migrate towards the putative
attraction center, we labeled PGCs with green fluorescent
protein (GFP) in live embryos (see Materials and Methods).
Time-lapse analysis of wild-type embryos showed that
formation of bilateral PGC clusters in the anterior trunk occurs
by migration of PGCs towards this region from medial,
posterior and anterior positions. Medially positioned PGCs
migrate laterally (Fig. 1C-I, green arrow), while PGCs that are
already located at lateral positions follow the general
convergence movement of somatic cells medially towards the
body axis. In addition, some of the PGCs trailing in posterior
regions also migrate anteriorly to join the clusters during early
somitogenesis, while the rest of the posterior trailing cells only
do so at later stages (data not shown).
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 midsomitogenesis. 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.
Migrating PGCs noticeably change their position relative to
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G. Weidinger and others
Fig. 2. Ectopic anterior PGCs migrate posteriorly towards the main clustering position in spt mutant embryos. (A-J) Fluorescent pictures of
embryos injected with GFP-nos1-3′UTR. Dorsal views, anterior is upwards. (A) In wild-type embryos, most PGCs are located in two medialto-lateral lines at the head-trunk border at the one-somite stage. (B-J) Time-lapse cinematography of a spt mutant embryo starting at early
somitogenesis. During the 3.5 hours shown, the embryo was kept at 25°C, thus it developed to approximately the six-somite stage. On each side
of the embryo, a pair of PGCs that were initially located close to each other in ectopic anterior regions is marked by arrowheads and arrows.
Note that two of these cells end up in the ectopic anterior cluster (arrowheads), while the others migrate over a considerable distance posteriorly
towards the main clustering position (arrows).
their somatic neighbors (Fig. 3A-F; Movie 1 on-line) and they
show the morphological features characteristic of motile cells at
all stages analyzed, including extension of numerous cellular
processes (Fig. 3G-I, Movie 2 on-line). The migration patterns
are highly dynamic: individual PGCs frequently change their
speed, direction and position relative to each other during
migration (see Fig. 1, Fig. 2, Fig. 3, and Movies 1 and 3 on-line).
In conclusion, we show that PGCs actively migrate
towards the lateral mesoderm of the anterior trunk
from three different regions and that they do so as
individual cells. These observations could be most
easily explained by assuming that the PGCs are
attracted by signals emanating from the target tissue.
Fig. 3. Zebrafish PGCs migrate actively. (A-F)
Fluorescent pictures taken at the indicated intervals
from a time-lapse movie showing migrating PGCs
during early somitogenesis in a wild-type embryo
injected with GFP-nos1-3′UTR and EGFP-F-globin
marking the outlines of somatic cells. The full movie
can be found at http://dev.biologists.org/supplemental/
(Movie 1). Individual PGCs are marked by colored
asterisks and a single somatic cell by a green arrow.
Note that the PGCs move extensively relative to the
somatic mesodermal cells. The apparent cell shape
changes of somatic cells seen in A-F are largely due to
different focal planes at which the pictures were taken
to keep the PGCs in focus. (G-I) Fluorescent pictures
taken at the indicated intervals from a time-lapse movie
showing migrating PGCs during mid-somitogenesis in a
wild-type embryo injected with EGFP-F-nos1-3′UTR
marking the outlines of PGCs. The full movie can be
found at http://dev.biologists.org/supplemental/ (Movie
2). Note the highly dynamic processes extended by the
PGC on the left side.
PGC cluster formation depends on proper
differentiation of the somatic target tissue
To examine the potential role of the lateral mesoderm of the
anterior trunk as an attraction center for migrating PGCs, we
sought to identify a molecular marker for this tissue. We found
that the zebrafish homolog of Wilm’s tumor suppressor gene 1
(wt1), a transcription factor required for gonad and kidney
Zebrafish germ cell migration
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Fig. 4. wt1 is expressed in the putative
PGC attraction center of the anterior trunk
at early somitogenesis. All pictures shown
are dorsal views of flatmounts of wholemount in situ stained embryos with wt1 in
blue in A-C, nos1 in blue in D and other
markers in red (see Materials and
Methods). (A) At the two-somite stage,
wt1 is exclusively expressed in the lateral
mesoderm of the anterior trunk with a
slight extension into the head, as seen in
comparison with myoD in red, which
stains the adaxial cells and the forming somites. Expression is confined to the mesoderm, as can be seen in side view (data not shown). Prior to
the one-somite stage, no expression can be detected in whole-mount in situ staining. (B) By the six-somite stage, wt1 expression has extended
posteriorly to the fifth somite. (C) Beginning at around the 10-somite-stage, wt1 expression starts to extend into the lateral mesoderm of the
head and into the anterior halves of the first four somites, which do not express myoD. At about this stage, the PGCs start to migrate posteriorly.
(D) The PGC clusters, which form between the one- and six-somite stages, are located within the wt1-expressing region, as seen by comparison
with B and shown here for the eight-somite-stage.
formation in mammals (Kreidberg et al., 1993), is expressed
in the lateral mesoderm of the anterior trunk from the onesomite stage onwards and therefore serves as a suitable marker
for the PGC clustering region (Fig. 4).
We next tested whether proper differentiation of the wt1expressing tissue is required for PGC cluster formation during
early somitogenesis. As demonstrated above, despite the
early PGC migration defect observed in spt mutants during
gastrulation, subsequent clustering of PGCs during early
somitogenesis occurs in these mutants, indicating that the
putative PGC attraction center is active. Consistent with this
notion, we found that wt1 is expressed at almost wild-type
levels in these mutants (Fig. 5C). As no mutation is known to
affect the wt1-expressing lateral mesoderm of the anterior trunk
specifically, we examined double mutants that exhibit more
severe defects in trunk mesoderm development. Such defects
are observed in spadetail;notail (spt;ntl), spadetail;one-eyedpinhead (spt;oep) and one-eyed-pinhead;notail (oep;ntl) double
mutants. Axial, paraxial and ventral mesoderm is severely
reduced or absent in these mutants (G. W., M. K. and E. R.,
unpublished) (Griffin et al., 1998; Schier et al., 1997), while the
lateral mesoderm is differentially affected. Specifically, at the
six-somite stage, wt1 expression is dramatically reduced or
absent in spt;ntl and spt;oep mutant embryos (Fig. 5E,G). In
oep;ntl mutants, however, wt1 is expressed at normal levels
(Fig. 5K). Expression of wt1 is also normal in ntl (data not
shown) and oep single mutants (Fig. 5I). Owing to defects in
paraxial mesoderm development, all three double mutant
combinations show an early PGC migration phenotype during
gastrulation that is very similar to that observed in spt single
mutant embryos. The PGCs fail to align along the head-trunk
border (step III), resulting in random distribution of PGCs along
the anteroposterior axis and absence of medially positioned
PGCs at the end of gastrulation (data not shown). Thus, using
these mutants it is not possible to analyze the ability of the
wt1-expressing target tissue to attract PGCs from medial
positions during step IV of migration, which occurs at early
somitogenesis. However, we could use them to test the
requirement of the putative attraction center for migration of
PGCs from anterior and posterior regions.
Consistent with the suggested role for the wt1-expressing
tissue in attracting PGCs, PGC clusters have not formed in the
anterior trunk in spt;ntl and spt;oep double mutants at the six-
somite stage. Instead, most of the PGCs accumulate at the
ectopic anterior clustering position (Fig. 5F,H, Table 1). spt;ntl
and spt;oep mutants do not recover from their failure to form
PGC clusters during early somitogenesis: even at 24 hours post
fertilization (hpf) the PGCs have not reached their target
position (Fig. 5O, Table 1 and data not shown). As shown in
Fig. 2 and Fig. 5N, in spt mutants, the region between the
ectopic anterior and the main cluster is cleared from PGCs, as
ectopic anterior PGCs migrate back towards the main
clustering position. This process does not occur in spt;ntl and
in most of the spt;oep double mutants and consequently, a
substantial number of PGCs are found in between the two
clusters (Fig. 5O, Table 1 and data not shown). In conclusion,
improper differentiation of the wt1-expressing tissue in spt;ntl
and spt;oep double mutants is correlated with a complete
failure of PGCs to form bilateral clusters.
oep;ntl double mutant embryos express wt1 at normal levels
(Fig. 5K), but otherwise exhibit very similar somatic (Schier
et al., 1997) and early PGC migration defects as spt;ntl and
spt;oep mutants (data not shown). However, even in the most
severely affected oep;ntl mutant embryos, some PGCs are
found in the correct clustering position at the six-somite stage
(Fig. 5L, Table 1). Importantly, the region between the ectopic
anterior and the main clusters is cleared from PGCs between
the one-somite and the 24-hpf stage, while the proportion of
PGCs located in the main clusters rises (Table 1). This
indicates that ectopic PGCs are attracted towards the main
clusters. Compared with oep mutant embryos, which exhibit a
weak and highly variable early PGC migration defect during
gastrulation, oep;ntl double mutants show a smaller PGC
cluster size. We attribute this difference to the fact that the early
migration phenotype is more severe in oep;ntl double mutants,
which leads to accumulation of more PGCs in ectopic anterior
positions (arrowheads in Fig. 5J,L).
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
30
G. Weidinger and others
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
Zebrafish germ cell migration
31
Table 1. PGC cluster formation and distribution of ectopic anterior PGCs in spt;ntl and oep;ntl double mutants
One-somite stage
Embryos
Embryos with
analyzed
PGCs in anterior
(two clutches) clustering position
Wild type†
spt‡
spt;ntl
oep;ntl
10
23
20
13
10%
87%
100%
85%
Proportion of PGCs
in anterior
clustering position
0.3%±1.0%
12.1%±9.9%
13.8%±8.2%
21.1%±20.3%
Embryos with PGCs Proportion of PGCs
in between
in between
anterior and main
anterior and main
clustering regions
clustering regions
0%
100%
95%
100%
0%
11.5%±6.4%
22.0%±16.1%
32.2%±20.1%
Proportion of
Embryos that form PGCs in main
main clusters* clustering region
100%
78%
20%
15%
82.4%±7.6%
41.6%±15.9%
15.3%±11.2%
15.1%±11.9%
Six-somite stage
Embryos
Embryos with
analyzed
PGCs in anterior
(three clutches) clustering position
Wild type†
spt‡
spt;ntl
oep;ntl
32
42
36
9
13%
100%
97%
100%
Proportion of PGCs Embryos with PGCs Proportion of PGCs
in anterior
in between anterior in between anterior
clustering position
and main cluster
and main clusters
0.6%±1.6%
20.1%±11.9%
29.9%±13.2%
45.5%±15.4%
13%
83%
100%
44%
0.5%±1.4%
11.0%±8.0%
25.3%±11.7%
4.4%±7.7%
Proportion of
Embryos that form PGCs in main
main clusters*
clusters
100%
90%
30%
55%
78.2%±13.4%
48.6%±14.8%
18.3%±12.9%
25.6%±16.0%
24 hpf
Embryos
Embryos with
analyzed
PGCs in anterior
(three clutches) clustering position
Wild type†
spt‡
spt;ntl
oep;ntl
28
40
28
20
11%
98%
97%
95%
Proportion of PGCs Embryos with PGCs Proportion of PGCs
in anterior
in between anterior in between anterior
clustering position
and main cluster
and main clusters
0.4%±1.2%
21.2%±12.0%
38.0%±14.5%
38.5%±17.5%
0%
15%
86%
15%
0%
1.0%±3.0%
21.6%±17.6%
0.5%±1.3%
Proportion of
Embryos that form PGCs in main
main clusters*
clusters
100%
98%
11%
100%
98.9%±2.4%
62.3%±18.3%
10.5%±12.5%
50.6%±16.1%
*Embryos with ≥30% of their PGCs in the main clustering region. This region was defined as reaching from the posterior end of the otic vesicle to the first
somite at the one-somite stage, from the first to the fifth somite at the six-somite stage and as the anterior 1/3 of the yolk extension at the 24 hpf stage. At the 24
hpf stage, this region comprises about one-third of the area spanning from the anterior clustering position to and including the main clustering position. If the
PGCs would be randomly distributed in this area, ≤30% of the PGCs should be found in the main clustering area. Therefore, an embryo was counted as forming
main clusters if ≥30% of its PGCs were found in the main clustering region.
†Siblings of spt;ntl embryos that appeared phenotypically wild type.
‡Siblings of spt;ntl embryos that showed just the spt phenotype.
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).
From these experiments, we conclude that cells of the
putative PGC attraction center of the anterior trunk do not
participate in the formation of the gonad. Therefore, the wt1expressing tissue serves as an intermediate target for migrating
PGCs. During later somitogenesis, the PGCs leave this tissue,
possibly attracted by another, definitive target, which would
give rise to the somatic portion of the gonad.
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.
Fig. 5. PGC cluster formation correlates with proper differentiation of the target tissue. (A,C,E,G,I,K) Flat-mounts of embryos at the six- to
eight-somite stage stained for wt1 in blue. (B,D,F,H,J,L) Flat-mounts of embryos at the same stage stained with nos1 in blue to visualize the
PGCs. The position of the main clusters of PGCs in the anterior trunk is marked by an arrow and that of the ectopic anterior clusters by an
arrowhead. Embryos have been stained with other markers in red or blue for identification of mutants and for providing positional landmarks
(see Materials and Methods). Note that wt1 is expressed at normal or only slightly reduced levels in all embryos that show clustering of PGCs
in the anterior trunk (spt, oep and oep;ntl). (M-O) Lateral views of embryos at 24 hpf stained for nos1 in blue. The position of the main PGC
clusters is marked by an arrow and that of the ectopic anterior cluster by an arrowhead. PGC main clusters have formed in the correct region at
the anterior end of the yolk extension in wild-type (M) and spt (N), but not in spt;ntl (O) double mutants. Note the presence of PGCs in
between the ectopic anterior and the main clustering positions in spt;ntl mutants (O).
32
G. Weidinger and others
Fig. 6. The lateral mesoderm of the anterior trunk comprises an
intermediate target of PGCs. (A,B) Fate-mapping of the somatic cells
that surround the main PGC clusters at early somitogenesis. Cells
that contain the uncaged fluorescein lineage tracer are labeled in red
and PGCs in blue using nos1 as probe. (A) Flat-mount of an embryo
at the six-somite stage, fixed immediately after the uncaging
procedure. Note that the uncaged region includes the PGC cluster.
(B) Lateral view of a deyolked embryo at 24 hpf. Note that the cells
containing the lineage tracer (bracket) and the PGCs (arrow) have
separated and that no labeled somatic cells are detectable in the
region of the PGCs.
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.
Second, defects in mesoderm patterning in the presumptive
gonadal region result in failure of PGCs to reach their final
target as observed in hands off (han) mutant embryos.
Mutations in the han locus, which encodes the bHLH
transcription factor hand2, have been shown to affect the
differentiation and morphogenesis of two anterior structures
derived from the lateral plate mesoderm – the heart and the
pectoral fin (Yelon et al., 2000). The hand2 gene is also
expressed in posterior lateral mesoderm, raising the possibility
that it is required for proper differentiation of the gonadal
mesoderm and thus for the migration of PGCs towards their
final target. In han mutant embryos, PGC migration is normal
up to the 16-somite stage; the PGCs correctly form bilateral
clusters in the lateral mesoderm of the anterior trunk, where
wt1 is expressed at normal levels (data not shown). As in wildtype embryos, the PGC clusters leave the wt1-expressing
region around the 10-somite stage and start to migrate
posteriorly (data not shown). However, migration to the
anterior end of the yolk extension is not completed, and at 24
hpf more than 50% of the PGCs are located in between the
wt1-expressing pronephric tissue and the final target (Fig. 8B,
Table 2). In the mutants, the PGCs are located in more anterior
positions relative to other structures too, e.g. relative to the
pancreas, confirming that they indeed fail to migrate
posteriorly (Fig. 8C,D). In addition, 11% of the PGCs are
found in ectopic positions posterior of the final target in han
mutants (Table 2). In wild-type embryos, about the same
number of posterior trailing PGCs has not yet reached the final
target by the 16-somite stage (Weidinger et al., 1999). Thus, it
appears that in han mutants, the PGCs fail to migrate towards
their final target from anterior and posterior regions after the
16-somite stage.
Because the somatic cells of the gonad can only be identified
by histology at later stages and no molecular marker is
available for these cells at the stages analyzed in this study,
we were not able to test directly whether the PGC migration
defect observed in han mutants results from defects in
gonad development. However, we found that the posterior
intermediate mesoderm that gives rise to the pronephric ducts
is mispatterned in the putative gonadal region at day 1 of
development. While in wild-type embryos, the expression level
of pax2.1 in the pronephric mesoderm is much lower in this
area than in more anterior and more posterior regions, such a
gap of expression does not exist in han mutant embryos
(brackets in Fig. 8E,F). In addition, pax2.1 expression along
the whole length of the pronephric ducts is stronger in the
mutants, as more cells express this gene than in wild-type
embryos (Fig. 8E,F and data not shown). Thus, we conclude
that the posterior lateral plate mesoderm, which presumably
gives rise to the somatic tissues of the gonad, is not correctly
patterned in han embryos. It is possible that the observed
increase of pronephric mesodermal cells in these mutants is
accompanied by a reduction in gonadal mesoderm precursors,
resulting in a decreased level of signals required for attraction
of PGCs towards this region.
Fig. 7. PGCs actively migrate towards their final target. (A-D) Fluorescent pictures taken at the indicated intervals from a time-lapse movie (the
full movie can be found at http://dev.biologists.org/supplemental/ (Movie 3) showing the main cluster of PGCs during late somitogenesis on the
right side of an embryo injected with full-vasa-GFP. Dorsal views, anterior is upwards. The Vasa-GFP fusion protein is localized into
perinuclear granules in the PGCs, which migrate posteriorly relative to the somites; one somite boundary is marked with a black line.
Zebrafish germ cell migration
A
33
IV
V
D
2 somites
B
V
6 somites
C
VI
24 hpf
10 somites
Fig. 8. Migration of PGCs towards their final target is defective in
han mutants. (A-D) Dorsal views of the trunk region of embryos at
day 1 of development with the PGCs stained with nos1 in blue. The
PGC clusters are dispersed and located more anterior in han mutants
(B,D) than in wild-type (A,C) relative to the wt1-expressing
glomerulus (arrows in A,B) and relative to the endocrine pancreas
stained in red with preproinsulin (C,D). (E,F) Lateral views of wildtype (E) and han mutant (F) embryos at the 25-somite stage stained
with pax2.1 in blue. Note that a gap of pax2.1 expression in the
pronephric mesoderm is present in wild-type embryos at the anterior
end of the yolk extension (bracket in E), while expression in this
region is stronger in han mutants (bracket in F).
DISCUSSION
An attraction center for zebrafish PGCs in the
anterior trunk
The data presented here suggest that formation of bilateral
PGC clusters in zebrafish is regulated by attraction of PGCs
towards an intermediate somatic target, which does not give
rise to the gonad. We propose that the somatic cells of this
intermediate target, which express the transcription factor wt1
Fig. 9. A model for the regulation of the final steps of zebrafish PGC
migration. Arrows indicate the direction of cell movement.
(A) During early somitogenesis the lateral mesoderm of the anterior
trunk (blue), which is marked by expression of the wt1 gene,
produces signals that attract PGCs. (B) At the six-somite stage PGCs
have formed clusters in the attracting region, while in some embryos
posterior trailing cells are still migrating anteriorly (step V). (C) At
about the 10-somite stage, the attraction center stops to function as
such (light blue) or the PGCs no longer respond. The clusters of
PGCs and remaining trailing PGCs, which have not yet reached the
clusters, start to migrate (downward arrows) towards their final
target, located in the lateral mesoderm around somite levels 8 to 10
(green). It is possible that the final target, which presumably gives
rise to the somatic tissues of the gonad, also attracts PGCs. (D) At 24
hpf, all PGCs have reached the final target (green), while the cells of
the intermediate attraction center (light blue) contribute to formation
of the pronephros.
and contribute to formation of the pronephros, produce signals
that attract PGCs during early somitogenesis (Fig. 9). During
later stages of development, either the attraction center ceases
to produce these signals or the PGCs stop to respond as they
migrate towards their final target located in more posterior
regions.
Table 2. PGC migration phenotype of han mutant embryos at 24 hpf
Wild-type siblings
han
Embryos analyzed
(two clutches)
Embryos with PGCs
between intermediate
and final target*
Proportion of PGCs
between intermediate
and final target*
Embryos with PGCs
posterior of
final target†
Proportion of PGCs
posterior of
final target†
30
29
16%
100%
0.6%±1.4%
57.1%±16.0%
33%
83%
2.5%±4.4%
10.8%±9.4%
*Corresponds to the region between the wt1 expressing nephron primordia and the anterior end of the yolk extension.
†Corresponds to the region posterior of the anterior one-third of the yolk extension.
34
G. Weidinger and others
Our model (Fig. 9) is based on the following observations:
(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 Wilm’s
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
Zebrafish germ cell migration
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)β1. Antibodies directed against TGFβ1 inhibit the ability
of mouse urogenital ridge explants to attract PGCs and mouse
PGCs migrate towards a TGFβ1 source in vitro (Godin and
Wylie, 1991). However, as the expression of TGFβ1 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.
We thank the members of the department for Developmental
Biology at the University of Freiburg, especially Wolfgang Driever,
Zoltan Varga and Gerlinda Wussler, for help and discussions. Tiemo
Klisch, Claudia Hoffmann and Siri Mahler helped with the fish work.
We also thank the zebrafish community for probes and Debbie Yelon
for the han mutant fish. We are grateful to Michal Reichman and Stefan
Rohr for critically reading the manuscript. This work was supported
by grants from the TMR program of the European Commission
(ERBFMBICT983315) and the DFG (RA863). B. T. and C. T. were
supported by funds from the Institut National de la Santé et de la
Recherche Médicale, the Centre National de la Recherche Scientifique,
the Hôpital Universitaire de Strasbourg, the Association pour la
Recherche sur le Cancer and the Ligue Nationale Contre le Cancer.
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