1 Laboratory for Germline Development, RIKEN Center for Developmental Biology,
Kobe, Hyogo 650-0047, Japan
2 Okazaki Institute for Integrative Bioscience, National Institute for Basic
Biology, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787,
Japan
3 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
* Authors for correspondence (e-mail: skob{at}nibb.ac.jp and akiran{at}cdb.riken.jp)
Accepted 22 June 2004
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SUMMARY |
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Key words: Germ cells, Lipid phosphate phosphatase, Cell survival, Cell migration, Drosophila
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Introduction |
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Drosophila PGCs, which are known as pole cells, are formed at the
posterior end of the blastoderm embryo. During gastrulation, pole cells are
carried inside of the embryo with the invaginating posterior midgut
primordium. Pole cells then actively pass through the midgut epithelium to
enter the interior of the embryo, and move dorsally along the midgut surface
towards the mesoderm. Next, pole cells migrate from the midgut to the
mesoderm, where they make contact with the somatic gonadal precursors (SGPs).
Pole cells adhere tightly to the SGPs and finally coalesce into the embryonic
gonad (Starz-Gaiano and Lehmann,
2001).
Nanos and its co-factor Pumilio (Pum) are well-characterized maternal
factors that are essential in a cell-autonomous manner for the proper
migration of pole cells. Pole cells lacking Nanos or Pum are able to pass
through the midgut. After exiting the midgut, however, they remain clustered
on its outer surface, fail to enter the mesoderm and are lost during
subsequent development (Asaoka-Taguchi et
al., 1999; Forbes and Lehmann,
1998
; Kobayashi et al.,
1996
). In addition, Nanos or Pum are essential for transcriptional
regulation in pole cells. Pole cells lacking either of them show premature
activation of several germline-specific enhancers that are normally expressed
at much later stages (Asaoka et al.,
1998
; Asaoka-Taguchi et al.,
1999
; Kobayashi et al.,
1996
), as well as the ectopic expression of genes normally active
only in somatic cells (Deshpande et al.,
1999
). The product of the polar granule component
(pgc) gene also regulates transcriptional repression in pole cells.
In embryos with reduced pgc function, pole cells ectopically express
soma-specific genes such as zerknullt and tailless. In these
pole cells, there is a premature phosphorylation of the Ser2 residue in the
C-terminal domain (CTD) repeats of RNA polymerase II
(Deshpande et al., 2004
;
Martinho et al., 2004
). As
this phosphorylation reflects the active transcription state
(Dahmus, 1996
), it has been
suggested that pgc regulates global transcription in pole cells. As
with nanos and pum, in embryos with reduced pgc
function, pole cells fail to migrate into the gonads and die after exiting the
midgut (Nakamura et al.,
1996
).
In addition to these cell-autonomous mechanisms, intercellular
communication between pole cells and somatic cells is crucial for pole cell
development and migration. For example, trapped in endoderm-1
(tre1) acts in the passage of pole cells through the midgut.
tre1 encodes a G-protein-coupled receptor that is expressed in pole
cells, and is thought to respond to a signal from the midgut to direct
trans-epithelial migration (Kunwar et al.,
2003). Furthermore, pole cells also receive directional cues from
somatic cells that guide their migration to the gonads. Enzymes involved in
isoprenoid biosynthesis are essential for attracting pole cells toward the
gonads (Santos and Lehmann,
2004
; Van Doren et al.,
1998a
). Conversely, zygotic wun and wun2, which
encode a putative ectoenzyme, lipid phosphate phosphatase (LPP), are expressed
in the ventral region of the midgut, and provide a repulsive environment for
pole cell migration (Burnett and Howard,
2003
; Starz-Gaiano et al.,
2001
; Zhang et al.,
1996
; Zhang et al.,
1997
). In addition to repelling pole cells, Wun and Wun2
activities also affect pole cell survival. Overexpression of either of them in
somatic tissues leads to the drastic loss of pole cells during their migration
(Burnett and Howard, 2003
;
Starz-Gaiano et al., 2001
).
However, it remains elusive how zygotic Wun and Wun2 activities in somatic
cells exert their functions on both pole cell migration and survival. We
report that wun2 has a maternal function that is required in pole
cells for their survival. The wun2 function in pole cells was
different from those of nanos, pum or pgc, because
transcriptional regulation in early pole cells was intact in the maternal
wun2 mutant embryo. Furthermore, we provide evidence that pole cell
survival requires a balance of LPP activities in pole cells and somatic cells.
These results indicate that Wun2 in pole cells competes with somatic Wun and
Wun2 for pole cell survival.
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Materials and methods |
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To generate germline clones, w; FRT42B, */T(2;3)apXa females were crossed with y w hs-FLP; FRT42B ovoD1/CyO males. The progeny were heat-shocked twice daily at 37° C for 2 hours during the early pupal stage. Embryos from females with the genotype y w hs-FLP; FRT42B, */FRT42B ovoD1 crossed to wild-type males were collected, fixed and stained for Vasa. Using this strategy, 1156 independent mutagenized lines were screened.
Mapping and identification of the N14 gene
The N14 chromosome was outcrossed to an unmutagenized
FRT42B chromosome to recombine away second mutation(s). A lethal
mutation on the N14 chromosome was placed between FRT42B and
curved by meiotic recombination mapping. N14 was crossed to
the deficiency kit for 2R (Bloomington Stock Center) and tested for
complementations based on lethality and on the maternal-effect mutant
phenotype. During this process, the maternal-effect phenotype of the
N14 mutation was found to be separable from the lethal mutation, and
was uncovered by five overlapping deficiencies in the chromosome region 45C-D:
Df(2R)Np5, Df(2R)w45-30n, Df(2R)w73-1, Df(2R)wun-GL and
Df(2R)w45-19g. The breakpoints of these deficiencies were determined
by semi-quantitative Southern hybridization using PCR-amplified genomic
fragments as the probes.
A series of genomic DNA fragments in the N14 candidate locus was
isolated from a Drosophila genomic DNA library in FIXII (a
gift from B. Suter, McGill University) and subcloned into pCaSpeR3. These
constructs were introduced into flies by a standard method, and were checked
for their ability to rescue the N14 mutant phenotype.
Immunostaining, in situ hybridization and ß-galactosidase staining of embryos
Immunostaining was carried out as described
(Kobayashi et al., 1999),
except that the embryos were devitellinized by hand in the tracer experiment
using the caged fluorescein. The following primary antibodies were used:
rabbit and rat anti-Vasa, rabbit anti-Nanos (K.H.-N. and A.N., unpublished),
rabbit anti-cleaved caspase 3 (Asp175) (Cell Signaling Technology), mouse
anti-RNA polymerase II H5 (Babco) and rabbit anti-ß-galactosidase
(Cappel). Antibody detection was performed using either a biotinylated
secondary antibody followed by the ABC Kit (Vector Laboratory) and DAB
staining, or Alexa Fluor 488-, 568-, 594- and 660-conjugated secondary
antibodies (Molecular Probes). Fluorescence signals were observed under a
laser-scanning confocal microscope (Leica TCS-SP2 AOBS). In situ hybridization
with DIG-labeled RNA probes was carried out as described
(Kobayashi et al., 1999
). For
the nanos, gcl or pgc probes, hybridized signals were
detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche).
For wun2, hybridized signals were detected with a horseradish
peroxidase-conjugated anti-DIG antibody (Roche), amplified with the TSA Biotin
System (Perkin Elmer) and visualized with alkaline phosphatase-conjugated
streptavidin (Vector Laboratory). To detect ß-galactosidase expression,
embryos were stained with X-Gal as described
(Sano et al., 2001
).
Labeling of pole cells using a photoactivatable lineage tracer
A photoactivatable lineage tracer, DMNB-caged fluorescein (Molecular
Probes), was injected into early cleavage stage embryos at a concentration of
1 mg/ml. The injected embryos were allowed to develop until the cellular
blastoderm stage. To photoactivate the tracer, pole cells were exposed to a
two-second pulse of UV irradiation from an epifluorescence microscope fitted
with a DAPI optical filter set. Embryos with pole cells that were successfully
marked with fluorescein were allowed to develop until the indicated stages.
These embryos were then fixed and stained with antibodies.
Generation of a new wun2 allele
EP2650 is a viable insertion that is located 30 bp upstream
of the wun2 locus. Excision lines were generated by crossing virgin
EP2650 females with males possessing
2-3 transposase.
Genomic DNA from the excision lines was prepared and used as PCR templates to
screen lines that carried a deletion within the wun2 locus. The
breakpoints of the deletion were determined by sequencing the PCR
products.
Genetic interactions between maternal wun2 and zygotic wun and wun2
To test the phenotype of wunm+z
wun2mz mutant embryos,
wun2N14/Df(2R)w45-19g females were crossed to
wunCE/CyO, P{en-lacZ} males. Df(2R)w45-19g
uncovers both wun and wun2 loci, and
wunCE allele lacks zygotic expression of both wun
and wun2 (Starz-Gaiano et al.,
2001). Embryos that received a wunCE
chromosome were identified by the loss of ß-galactosidase expression. In
this experiment, half of the lacZ embryos showed a
less severe pole cell death phenotype than that observed in the
wun2mz embryos. Thus, we scored embryos with
weaker pole cell death as well as mis-migrated pole cell phenotypes as
wunm+z wun2mz embryos. For
the overexpression of wun2 both in pole cells and the mesoderm,
twist-Gal4/+; nanos-Gal4-VP16/+ females were crossed to
EP2650 males. Half of the embryos obtained from this cross were
expected to overexpress wun2 in both pole cells and the mesoderm.
These embryos were identified by a strong pole cell migration defect, owing to
the overexpression of wun2 in the mesoderm.
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Results |
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Deficiency mapping revealed that N14 was uncovered by Df(2R)w45-19g. In embryos derived from N14/Df(2R)w45-19g mothers (hereafter referred to as N14m embryos), normal numbers of pole cells were formed (stage 5, Fig. 1A,B; Table 1). These pole cells were carried into the embryo along with the invagination of the posterior midgut, passed through the midgut epithelium, and migrated dorsally along the surface of the midgut (Fig. 1C,D). However, at stage 11, when pole cells normally associate with the mesoderm, the number of Vasa-positive pole cells was dramatically reduced in N14m embryos (Fig. 1E,F). In these embryos, the remaining pole cells, if any, associated with the surface of the midgut (Fig. 1F), and in subsequent development, few or no pole cells were incorporated into the gonads (Fig. 1G,H; Table 1). N14m embryos showed no discernible morphological defects in somatic tissues and developed into adults. However, consistent with the loss of pole cells during embryogenesis, over 80% of the adult females developed from N14m embryos had agametic ovaries (Table 1). This defect in pole cell development was not rescued by a paternally supplied wild-type copy of the N14 gene, and zygotic N14 mutation did not affect the maternal N14 mutant phenotype. These results indicate that maternal N14 function is required for the maintenance of pole cells during their migration to the gonads.
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In N14m embryos, pole cells, which were marked with both fluorescein and anti-Vasa antibody, were observed on the surface of the midgut at stage 10 (Fig. 2A-C). However, at stage 11, the fluorescein-marked cells rapidly disappeared (Fig. 2D-F). We occasionally observed fluorescein-marked pole cells with no or very faint Vasa signals on the surface of the midgut (Fig. 2D-F; arrowheads). At stage 11, the pole cells appeared to have experienced a significant reduction in size accompanied by an increase in the intensity of fluorescein signal (Fig. 2D-F; arrows), and few fluorescein-marked pole cells were detectable by stage 12 (Fig. 2G-I). From these observations, we conclude that pole cells in N14m embryos die after they pass through the midgut epithelium.
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First, we analyzed the distributions of several maternal RNAs and proteins that localize in pole plasm. At the blastoderm stage, distributions of nanos (Fig. 4A,E), germ cell-less (gcl) (Fig. 4B,F) and pgc RNAs (Fig. 4C,G), and the Nanos protein (Fig. 4D,H) in N14m embryos were indistinguishable from those in wild-type embryos, indicating that these pole plasm components are normally incorporated into pole cells in N14m embryos.
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wun2 is the gene responsible for the N14 mutant phenotype
Through complementation tests, we identified five overlapping deficiencies
that uncovered the N14 mutant (see Materials and methods). The
breakpoints of these deficiencies defined the N14 locus within a
100 kb genomic region containing nine identified or predicted genes
(Fig. 6A). To identify the gene
responsible for the N14 mutant phenotype, we generated a series of
transgenes containing genomic DNA fragments in this region
(Fig. 6A). Of these, a
17
kb genomic DNA fragment, which contained two genes, wun2 and
CG13955, completely rescued the N14 mutant. To determine
which gene was responsible for the N14 mutant phenotype, we next made
constructs, in which only one of the genes was intact. Only the 8 kb
HincII fragment (Pwun2-8k), containing the entire
wun2 locus, rescued the N14 mutant. Furthermore, a genomic
fragment that had a partial deletion in the wun2 RNA-coding region
(Pwun2-8k
) failed to rescue the N14 mutant. Finally,
we found that the N14 mutant chromosome had a nonsense mutation in
the wun2 gene at the 111th Trp codon
(Fig. 6B). Thus, we refer to
the N14 mutant as the wun2N14 allele.
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wun2 encodes an LPP that dephosphorylates a number of
phospholipids in vitro (Burnett and Howard,
2003; Starz-Gaiano et al.,
2001
). We next asked whether the phosphatase activity of Wun2 is
responsible for maternal Wun2 function. It has been shown that the putative
catalytic residues of Wun2, His274 or His326, are essential for its activity
(Starz-Gaiano et al., 2001
).
We mutated His274 or His326 into Lys (H274K or H326K) and examined the ability
of such mutant transgenes to rescue the wun2N14 phenotype.
The two mutant Pwun2-8k transgenes with H247K or H326K failed to
rescue the wun2N14 phenotype (data not shown), indicating
that the effect of Wun2 on pole cell survival is dependent on its phosphatase
activity.
Maternal wun2 activity is required in pole cells
We next examined the distribution of wun2 RNA during early
embryogenesis. wun2 RNA was detected ubiquitously in cleavage-stage
embryos (stage 2, Fig. 7A).
Although the signal in the somatic cell region became undetectable by the
cellular blastoderm stage, it remained at high levels in pole cells (stage 4,
Fig. 7B,B'). At stage 5,
wun2 signal was also detected in the somatic region in a posterior
stripe pattern (Fig. 7C). These
observations were consistent with a previous report
(Renault et al., 2002). In
embryos from wun2
/Df(2R)w45-19g females
crossed to wild-type males, wun2 RNA was undetectable in pole cells,
but was expressed in the posterior stripe
(Fig. 7D). These results
indicate that wun2 RNA in pole cells is supplied maternally, while it
is expressed zygotically in the posterior stripe.
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First, we examined the effect of the loss of both zygotic wun and wun2 on pole cell death caused by a maternal wun2 mutation. Embryos lacking both maternal wun2 and zygotic wun and wun2 (referred to as wunm+z wun2mz embryos) showed a partial rescue of the pole cell death phenotype of the maternal wun2 mutation (Fig. 9A,B). In wunm+z wun2mz embryos, an average of 10.5±3.5 pole cells were survived at stage 14-15 (n=28), although they failed to migrate toward the gonads as in the wunm+z wun2m+z embryos. This result revealed that maternal wun2 interacts genetically with zygotic wun and wun2 in regulating pole cell survival.
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Discussion |
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Maternal Wun2 has a novel function in pole cell survival
Several maternal mutations that affect pole cell survival have been
isolated. These include nanos, pum and pgc
(Asaoka-Taguchi et al., 1999;
Forbes and Lehmann, 1998
;
Kobayashi et al., 1996
;
Nakamura et al., 1996
).
However, our results indicate that the phenotypes of maternal wun2
mutant embryos are different from those of nanos, pum or pgc
mutant embryos. For example, although nanos, pum and pgc are
required for transcriptional repression in early pole cells
(Asaoka et al., 1998
;
Asaoka-Taguchi et al., 1999
;
Deshpande et al., 2004
;
Deshpande et al., 1999
;
Martinho et al., 2004
),
maternal wun2 plays no role in either transcriptional repression or
in the onset of zygotic gene expression in pole cells
(Fig. 5). Thus, it is likely
that maternal wun2 is required for pole cell survival at a different
developmental step from nanos, pum and pgc.
Surprisingly, dying pole cells in wun2m embryos
were negative for cleaved caspase 3 (Fig.
3). We also found that the overexpression of the caspase inhibitor
p35 (Hay et al., 1994) in pole
cells did not rescue the pole cell death phenotype in
wun2m embryos (data not shown). Furthermore, we
have never detected TUNEL-positive pole cells in
wun2m embryos (data not shown). Thus, the pole cell
death in wun2m embryos seems to occur via a
mechanism different from typical caspase-dependent apoptosis. It has become
evident that caspase-independent cell death pathways do exist
(Golstein et al., 2003
;
Leist and Jäättela,
2001
; Lockshin and Zakeri,
2002
), and we suppose that such caspase-independent cell death
might be occurring in these pole cells. Further morphological study may reveal
how pole cell death occurs in wun2m embryos.
A balance between LPP activities in pole cells and somatic cells is crucial for pole cell survival and migration
Wun2 belongs to a conserved family of LPPs
(Burnett and Howard, 2003;
Starz-Gaiano et al., 2001
).
LPPs are integral membrane proteins that dephosphorylate a number of bioactive
lipid phosphates in vitro, such as lysophosphatidic acid, phosphatidic acid,
sphingosine-1-phosphate and ceramide-1-phosphate. These lipid phosphates act
as extracellular signaling molecules and/or intracellular second messengers
and affect a variety of cellular processes, including cell survival and
motility (Brindley et al.,
2002
; Sciorra and Morris,
2002
). LPPs can attenuate cell activation by dephosphorylating
bioactive lipid phosphates and/or they can generate alternative signals from
dephosphorylated lipids, such as diacylglycerol, sphingosine and ceramide. The
active sites of LPPs are exposed either on the outer surface of the plasma
membrane or on the luminal surface of intracellular organelles, depending on
their subcellular localization (Brindley et
al., 2002
; Sciorra and Morris,
2002
). It has been proposed that LPPs promote the incorporation of
lipid phosphate substrates into the outer leaflet of the membrane before their
dephosphorylation (Roberts and Morris,
2000
). Although the exact subcellular distribution of the
endogenous Wun2 protein remains elusive, it localizes to the plasma membrane
in Drosophila embryos when overexpressed
(Starz-Gaiano et al., 2001
).
Therefore, Wun2 is likely to be an ecto-enzyme that promotes the uptake and
the dephosphorylation of extracellular lipid phosphate substrates.
Our data indicate that maternally supplied Wun2 acts in a cell-autonomous
manner to promote the survival of pole cells (Figs
7,
8). By contrast, zygotically
expressed Wun and Wun2 act in somatic cells to direct pole cell migration
(Starz-Gaiano et al., 2001;
Zhang et al., 1996
;
Zhang et al., 1997
).
Furthermore, we have shown that pole cell survival requires a balance between
LPP activities in pole cells and somatic cells
(Fig. 9). Considering that Wun
and Wun2 are likely to function as ecto-enzymes
(Burnett and Howard, 2003
;
Starz-Gaiano et al., 2001
),
the same extracellular lipid phosphate could be degraded by both Wun2 in pole
cells, and Wun and Wun2 in somatic cells. Thus, we propose that Wun2 activity
in pole cells competes with somatic Wun and Wun2 activities for the uptake and
the dephosphorylation of a common substrate. When Wun or Wun2 is overexpressed
in somatic cells, the extracellular substrate would be depleted in the
hemocoel surrounding the pole cells, so that Wun2 in pole cells is unable to
produce the survival signal any longer. In embryos overexpressing Wun2 in both
somatic cells and pole cells, increased Wun2 activity in pole cells leads to
the increased incorporation of the substrate, promoting pole cell survival. In
wunm+z wun2mz embryos,
pole cells escaped from cell death (Fig.
9B), suggesting that pole cells are capable of producing the
survival signal even in the absence of pole cell-autonomous Wun2 activity.
There are eight LPP genes, including wun and wun2, in the
Drosophila genome; wun mRNA is also maternally supplied in
cleavage-stage embryos (Renault et al.,
2002
). Although wun mRNA does not become concentrated in
pole cells, it is conceivable that trace amounts of Wun and/or other LPP
become partitioned into pole cells, promoting their survival in the
wunm+z wun2mz embryo.
However, in normal embryos, such activity would have only a subtle effect on
pole cell survival, because it would promote the survival of only a small
number of pole cells.
Somatic LPP activity also functions to repel pole cells. An extracellular
substrate might direct somatic cells to produce a repellant molecule, while
directing pole cells to produce a distinct survival signal. However, based on
our finding that LPP activity in somatic cells competes with that in pole
cells for an extracellular substrate, we favor the idea that LPP activity in
somatic cells provides a repulsive environment that directs pole cell
migration by depleting a substrate that is required by pole cells for their
survival. We expect that similar LPP-mediated mechanisms of cell migration and
survival may be widely used, as LPPs play important roles in various
developmental processes such as axon growth, axis patterning and
extra-embryonic vasculogenesis in mammals
(Bräuer et al., 2003;
Escalante-Alcalde et al.,
2003
). Future work will focus on identifying the endogenous
substrate for Wun2 and resolving the mechanism by which Wun2 exerts its
effects on pole cell survival.
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ACKNOWLEDGMENTS |
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