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First published online 7 March 2007
doi: 10.1242/dev.003426
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Research Report |
Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Author for correspondence (e-mail:
d.stein{at}mail.utexas.edu)
Accepted 10 February 2007
SUMMARY
The establishment of dorsal-ventral (DV) polarity in the Drosophila embryo depends upon a localized signal that is generated in the perivitelline space of the egg through the action of a serine proteolytic cascade. Spatial regulation of this pathway is determined by the expression of the pipe gene in a subpopulation of ventral follicle cells in the developing egg chamber. The Pipe protein exhibits homology to vertebrate glycosaminoglycan sulfotransferases. In a previous study, we demonstrated that embryonic DV polarity depends upon the sulfotransferase activity of Pipe. Surprisingly, however, our results also indicated that formation of the embryonic DV axis does not require the synthesis of the high-energy sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the follicle cells in which Pipe is presumed to function. Here, we resolve this apparent paradox by demonstrating that dorsalized embryos are only produced by egg chambers in which both germline and follicle cells lack PAPS synthetase activity. Thus, PAPS produced either in the germline or in the follicular epithelium can support the requirement for Pipe sulfotransferase activity in embryonic DV patterning. This finding indicates the existence of a conduit for the movement of PAPS between the germline and the follicle cells, which highlights a previously unappreciated mechanism of soma/germline cooperation affecting pattern formation.
Key words: Egg chamber, Sulfation, Sulfonation, Oogenesis, Dorsoventral, PAPS Synthase, papss, pipe, slalom, sugarless, sulfateless
INTRODUCTION
Pattern and polarity along the dorsal-ventral (DV) axis of the
Drosophila embryo depends upon the spatial regulation of a serine
protease cascade in the perivitelline space that surrounds the developing
embryo (Morisato and Anderson,
1995
; Moussian and Roth,
2005; Roth, 2003).
This protease cascade leads ultimately to the ventral activation of the Toll
receptor, which establishes the orientation of DV polarity in the developing
embryo. The spatial parameters of the activated serine protease cascade in the
perivitelline space are determined during oogenesis by the pattern of
expression of the pipe gene (Sen
et al., 1998), which is specifically transcribed in the ventral
follicle cells of the stage-10 egg chamber. The pipe locus encodes up
to 11 distinct protein isoforms (Sergeev
et al., 2001) (see Flybase), produced by alternative splicing,
that all exhibit amino acid similarity to vertebrate heparan sulfate
2-0-sulfotransferase (HSST) and dermatan/chondroitin sulfate
2-0-sulfotransferase (D/CSST) (Kobayashi
et al., 1997; Kobayashi et
al., 1999). These Golgiresident proteins mediate the transfer of
sulfate to the 2-hydroxyl position of uronic acid residues of
glycosaminoglycan (GAG) carbohydrates such as heparin (Hep), heparan sulfate
(HS), chondroitin sulfate (CS) and dermatan sulfate (DS). Like these enzymes,
Pipe is present in the Golgi (Sen et al.,
2000). In previous work we demonstrated that females carrying
follicle cell clones that are mutant for genes required for HS and CS/DS GAG
formation do not produce dorsalized embryos
(Zhu et al., 2005). This, as
well as other observations, indicates that HS and CS/DS GAGs do not serve as
substrates for Pipe sulfotransferase activity, despite the amino acid
similarity between Pipe and vertebrate HSST and D/CSST. However, additional
lines of evidence are consistent with the proposal that Pipe does function as
a sulfotransferase. For example, dorsalized embryos are produced by females
carrying follicle cell clones that are mutant for slalom
(sll), which encodes the Golgi transporter of
3'-phosphoadenosine 5'-phosphosulfate (PAPS)
(Kamiyama et al., 2003;
Luders et al., 2003), the
universal donor in sulfation reactions.
In our previous study, we also tested whether the expression of
papss (also known as Paps - Flybase), the gene encoding PAPS
synthetase (Jullien et al.,
1997), is required in the follicle cell layer to support the
formation of embryonic DV polarity (Zhu et
al., 2005). Surprisingly, we did not detect embryos with DV
defects among the progeny of females carrying follicle cell clones mutant for
papss. This finding was paradoxical, given that the requirement for
sll expression demonstrated a need for PAPS to be transported into
the Golgi of the follicle cells. To explain this result, we proposed that PAPS
is transported between the germline and the follicle cells, perhaps through
gap junctions that are known to exist between follicle cells and the oocyte
(Bohrmann and Haas-Assenbaum,
1993; Giorgi and Postlethwait,
1985; Goldberg et al.,
2004; Waksmonski and Woodruff,
2002). In the work reported here, we have tested this hypothesis
by generating females with egg chambers in which both the germline and clones
of follicle cells were mutant for papss. These females produced
dorsalized embryos, indicating that PAPS is required in the ovary for
embryonic DV patterning, consistent with Pipe's role as a sulfotransferase. In
addition, we show that dorsalized embryos are not generated by females
carrying both germline and follicle cell clones mutant for the GAG
synthesis-related genes sugarless (sgl)
(Binari et al., 1997;
Häcker et al., 1997;
Haerry et al., 1997) or
sulfateless (sfl) (Lin
et al., 1999). These findings definitively rule out HS and CS/DS
GAGs as substrates for Pipe. The results reported here also demonstrate that
although biological sulfation in the follicle cells is crucial for the
production of embryos with normal DV patterning, it is apparently dispensible
for the viability of follicle cell themselves. Our finding that papss
mutant follicle cells can be rescued by a wild-type germline is consistent
with a model in which PAPS can move between the germline and the follicle
cells. This represents a novel mechanism for maintaining homeostasis in an
embryonic patterning pathway.
MATERIALS AND METHODS
Genetics
The wild-type stock was Oregon R. The papss2
(Zhu et al., 2005),
sll7E18 (Luders et
al., 2003), sgl08310
(Häcker et al., 1997) and
sfl03844 (Lin et al.,
1999) mutations were carried on a chromosome bearing an insertion
of P{w[+mW.hs]=FRT(w[hs])}2A at chromosomal interval 79D (FRT79D).
An insertion of the D. melanogaster variant H2A.F/Z-class histone
fused to green fluorescent protein (hGFP)
(Clarkson and Saint, 1999) on
3L of the FRT79D chromosome was used to identify mutant clones in
the ovary. An insertion of P{w[+mC]=ovoD1-18} on 3L
(P[ovoD1]) of the FRT79D chromosome was used to
select for embryos derived from germline clones
(Chou et al., 1993). FLP
expression was induced by heat shock from the hsFLP1 insertion
(Golic and Lindquist, 1989),
and from an insertion of UAS-FLP by the Gal4 enhancer trap insertion
e22c-GAL4 (Duffy et al.,
1998). Larvae carrying hsFLP1 were heat shocked for 1
hour at 37°C on two consecutive days during the second and third larval
instar stages.
Identification and characterization of follicles and embryos from mosaic females
Follicles
Six days after eclosion, females were placed in yeasted vials for one day,
then their ovaries were dissected and fixed in 4% paraformaldehyde in PBS and
heptane for 15 minutes, followed by extensive washing in PBT (PBS+0.1% Tween
20). Ovaries were stained for 5 minutes with 0.2 µg/ml DAPI in PBT followed
by three 5-minute washes in PBT. Stained ovaries were mounted in a 1:1 mix of
glycerol:PBS and photographed using a Zeiss Axioscope II microscope outfitted
with a Zeiss axiocam digital camera. Mutant clones were identified by the
absence of GFP fluorescence in the nuclei.
Embryos
Six days following eclosion, mosaic females were mated to wild-type males
and placed in an egglay collection tube. After an additional 48 hours, embryos
were collected on yeasted apple juice agar plates
(Wieschaus and Nüsslein-Volhard,
1986) that were changed every 24 hours. Cuticle preparations
(van der Meer, 1977) were made
from embryos that were allowed to complete development. Dorsalization of
cuticles was classified according to Roth et al.
(Roth et al., 1991). Cellular
blastoderm-stage embryos were subjected to antibody staining
(Macdonald and Struhl, 1986)
using anti-Twist rabbit polyclonal antibody (1:5000)
(Roth et al., 1989) and a
biotinylated goat anti-rabbit secondary antibody (1:500) that was visualized
with avidin-HRP complex (Vector Laboratories).
RESULTS AND DISCUSSION
To test our hypothesis that papss mutant follicle cells can be
non-autonomously rescued by wild-type germline cells, it was necessary to
generate females carrying mosaic egg chambers in which both germline and
follicle cell clones were mutant for papss. In these follicles, the
germline would be incapable of providing PAPS to the follicle cell layer.
Thus, if PAPS were required for Pipe activity, the embryos resulting from
these follicles would exhibit dorsalized phenotypes. We first determined the
feasibility of generating and detecting egg chambers carrying both follicle
cell and germline clones using FLP/FRT-mediated recombination
(Golic and Lindquist, 1989). To
mark the clones, the FRT79D chromosome was carried in trans to
another FRT79D chromosome with an insertion of the D.
melanogaster variant H2A.F/Z-class histone fused to green fluorescent
protein (hGFP) (Clarkson and Saint,
1999). Following recombination, clones of cells homozygous for the
unmarked FRT79D chromosome were identified by their lack of
fluorescence. To achieve a high frequency of clones in the follicle cell
layer, females carried the e22c-GAL4 enhancer trap insertion and a
UAS-FLP transgene on the second chromosome
(Duffy et al., 1998). To
generate germline clones, FLP expression was induced in response to heat shock
using the hsFLP1 insertion (Golic
and Lindquist, 1989), which places FLP under hsp70
promoter transcriptional control.
In females of the genotype e22c-GAL4, UAS-FLP/+; hGFP, FRT79D/FRT79D, in which FLP was expressed only in the somatic cells, 76% of stage 9-10 egg chambers exhibited at least one mitotic clone in the follicular epithelium (Table 1). When larval heat shock was used to induce FLP expression in hsFLP1/+; hGFP, FRT79D/FRT79D females, 10 out of 760 (1.3%) of the stage 9-10 egg chambers examined carried FRT79D/FRT79D germline cells (Table 1). In the dissected ovaries of hsFLP1/+; e22c-GAL4, UAS-FLP/+; hGFP, FRT79/FRT79D, we identified 8 out of 660 (1.2%) egg chambers in which an oocyte derived from a germline clone was surrounded by an epithelium containing a clone of FRT79D/FRT79D follicle cells (Table 1). Thus, although they are relatively rare, it is possible to generate and identify follicles containing both germline and somatic cell clones.
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) to select for eggs derived from papss
mutant germline clones. In these females, the papss2,
FRT79D chromosome was carried in trans to
P[ovoD1], FRT79D, which carries a transgenic
insertion of the P[ovoD1] allele. Follicles in which the
germline is either heterozygous or homozygous for P[ovoD1]
degenerate and produce no mature eggs
(Oliver et al., 1987). Thus,
the only eggs that are laid by these females must be derived from homozygous
papss2 germline clones. Our previous results demonstrated
that 76% of the follicles carry somatic clones when FLP expression is driven
by e22c-GAL4. Therefore, we expected the hsFLP1/+; e22c-GAL4,
UAS-FLP/+; P[ovoD1],
FRT79D/papss2, FRT79D females, which
were heat shocked as larvae, to produce many eggs that formed in an egg
chamber containing a follicle cell clone. The embryos produced by these
females were collected, allowed to complete embryogenesis, and then cuticle
preparations were made. Of 2736 embryonic cuticles examined, 800 (29%)
exhibited a dorsalized phenotype, including 81 that showed the completely
dorsalized D0 cuticular phenotype (Table
2) (Fig. 2H). To
confirm our assessment of the cuticular phenotype with a molecular marker, we
stained blastoderm-stage embryos with an antibody against Twist
(Thisse et al., 1988), a
marker for mesoderm, which is the most ventral pattern element along the
embryonic DV axis. Many of the embryos produced by these females exhibited
either complete elimination (not shown) or partial disruption of Twist
expression (Fig. 2G). By
contrast, the wild-type pattern of Twist staining was observed in embryos
derived from papss mutant germline clones that presumably developed
in a follicular epithelium in which tissue-specific FRT-mediated recombination
had not been induced (Fig. 2E).
As reported previously, disruption of Twist staining was also seen in the
progeny of females carrying sll mutant follicle cell clones
(Fig. 2C)
(Luders et al., 2003). Thus,
the phenotypic consequence of depleting PAPS is equivalent to eliminating
sll expression in the follicle cells. This is consistent with the
idea that sulfotransferase activity in the follicle cells is crucial for DV
patterning of the embryo.
|
), which encodes Drosophila UDP-glucose dehydrogenase
(Binari et al., 1997;
Häcker et al., 1997;
Haerry et al., 1997). This
finding led us to conclude that HS and CS/DS GAGs could not be the target of
Pipe activity, because the product of Sgl enzymatic activity, UDP-gluronic
acid (UDP-GlcA), is required for their synthesis. Like PAPS, however, UDP-GlcA
is a small molecule (577 Da) that could potentially move between the oocyte
and follicle cells via gap junctions. Our finding that PAPS can be supplied to
papss mutant follicle cells from a wild-type oocyte raised the
possibility that in our previous study, follicle cell clones mutant for
sgl were non-autonomously rescued by the transfer of UDP-GlcA from
the germline. Indeed, observations made in the developing wing disc are
consistent with this possibility. In this tissue, Wingless protein acts as a
morphogen that forms a concentration gradient through a restricted diffusion
mechanism. Wingless diffusion requires the function of the HS proteoglycans
Dally and Dally-like; cells that lack either of these two proteins impede the
movement of Wingless (Han et al.,
2005). Although UDP-glucose dehydrogenase is required for HS
synthesis, cells mutant for sgl do not interfere with Wingless
diffusion (Strigini and Cohen,
2000). This discrepancy could be explained if the UDP-GlcA
required for the formation of the HS chains in Dally and Dally-like were
supplied to the sgl mutant cells by nearby wild-type cells.
Therefore, to rule out a similar mechanism and definitively test the
requirement for HS and CS/DS GAGs in embryonic DV axis formation, we generated
females that simultaneously carried somatic and germline sgl mutant
clones, as described above for papss
(Table 1;
Fig. 1G,H). In contrast to our
finding with papss, none of the 2239 embryos derived from
sgl mosaic females exhibited a dorsalized phenotype, even among the
1485 embryos from females in which P[ovoD1] was used to
select for embryos derived from sgl germline clones
(Table 2). This finding
conclusively demonstrates that the function of Pipe in embryonic DV patterning
does not require the presence of HS or CS/DS GAGs in the follicle cells.
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), an enzyme that mediates the deacetylation and
sulfation of the N-acetyl group on N-acetylglucosamine residues of HS. Sfl is
an integral membrane protein that acts on GAG monosaccharide units in the
lumen of the Golgi. Its product, sulfated HS, is unlikely to pass freely
between cells. In a previous study, we demonstrated that it is possible to
generate females carrying follicle cell clones mutant for sfl and
that they do not produce dorsalized embryos
(Zhu et al., 2005). Here, we
found that it was possible to generate females with coincident germline and
follicle cell clones mutant for sfl
(Table 1)
(Fig. 1I,J). As expected, none
of the 1980 embryos produced by sfl mosaic females, 1310 of which
derived from females with an sfl mutant germline, exhibited a
dorsalized phenotype (Table 2).
Thus, the complete lack of HS GAG synthesis does not impede the establishment
of DV polarity in the embryo, nor does it affect the viability of either the
follicle cells or the germline, or the patterning of the egg chamber.
Despite Pipe's similarity to glycosaminoglycan uronic acid-specific 2-0
sulfotransferases, the findings presented here conclusively demonstrate that
uronic acid-containing GAGS such as HS and CS/DS do not play an essential role
in the establishment of embryonic DV polarity. Our results do support,
however, previous evidence suggesting that Pipe is acting as a
sulfotransferase (Zhu et al.,
2005), as PAPS must be available to the follicle cells for DV
pattern formation to occur normally. Although our data rule out the
possibility that Pipe acts upon HS or CS/DS GAGs, we consider it likely that
Pipe acts on an alternate type of glycoprotein or glycolipid-associated
carbohydrate.
Our ability to generate egg chambers with simultaneous follicle cell and
germline clones mutant for sgl provides evidence for the surprising
conclusion that uronic acid-containing carbohydrates such as HS, CS and DS, as
well modification of other molecules by glucuronidation
(King et al., 2000), are
dispensible for the viability and growth of the oocyte and follicle cells.
Even more strikingly, our results from females carrying both papss
mutant germline and follicle cells indicate that biological sulfation itself
is not essential for follicle cell viability or oocyte maturation. Indeed, the
only requirement for PAPS in the egg chamber that was detected in our
experiments was in embryonic DV patterning, which presumably reflects the
function of PAPS in the sulfation of the Pipe target.
Although PAPS is required for the function of Pipe, our results demonstrate
that PAPS need not be synthesized in the Pipe-expressing ventral follicle
cells as it can be supplied to them from the neighboring germline cells. PAPS
is a highly polar molecule that is unlikely to diffuse freely across lipid
membranes. We consider it likely that in the ovary, PAPS molecules travel
through the gap junctions that are known to exist between the oocyte and
follicle cell layer (Bohrmann and
Haas-Assenbaum, 1993; Giorgi
and Postlethwait, 1985;
Waksmonski and Woodruff,
2002). PAPS (507 Da) is sufficiently small to pass through the gap
junctions, which allow passage of molecules of approximately 1 kDa
(Goldberg et al., 2004). The
gap junctions of insects and other invertebrates are composed of protein
subunits called innexins (Phelan,
2005), for which eight genes have been identified in
Drosophila (Stebbings et al.,
2002). The gonads of flies lacking Innexin 4, encoded by zero
population growth (also known as inx4), contain small numbers of
early germline cells but lack more mature stages
(Tazuke et al., 2002),
indicating that communication via gap junctions is required for gametogenesis.
During the course of oogenesis, the 15 polytene nurse cells provide the
developing oocyte with various metabolites, and the ability of the follicle
cells to receive PAPS from the oocyte provides evidence for a mechanism by
which the follicle cells benefit from the robust synthetic capacity of the
nurse cells. Thus, in addition to a potential influence of gap junctional
communication on embryonic DV patterning, our findings raise the possibility
that communication between germline and soma contributes to the homeostasis of
other metabolites within the egg chamber. Finally, it is worth noting that the
ability of other small metabolites to move from cell to cell should also be
considered as a factor in other developmental contexts.
ACKNOWLEDGMENTS
We are grateful to Drs Joe Duffy, Udo Häcker, James Kennison and Norbert Perrimon, and to the Drosophila Stock Center in Bloomington Indiana for providing Drosophila stocks. This work was supported by grants from the Mizutani Foundation for Glycosciences (040061) and the National Science Foundation (0344888).
Footnotes
* Present address: The Jackson Laboratory, Bar Harbor, ME 04609, USA 
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