Department of Molecular Biology, Princeton University, Princeton, NJ 0854, USA
* Author for correspondence (e-mail: gdeshpande{at}molbio.princeton.edu)
Accepted 25 November 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Transcriptional quiescence, Germ cells, Drosophila melanogaster
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to their earlier cellularization and slower rate of mitosis,
pole cells differ in their transcriptional activity. Somatic nuclei
substantially upregulate RNA polymerase II transcription after they migrate to
the surface of the embryo. The activation of zygotic gene expression is
essential for these nuclei to respond appropriately to the maternal pathways
that assign positional information along the axes of the embryo. By contrast,
pole cell nuclei shut down RNA polymerase II transcription when they enter the
pole plasm (Zalokar, 1976) and
they then remain transcriptionally quiescent until much later stages of
embryogenesis. Transcriptional quiescence is a hallmark of germline precursor
cells in many organisms (Seydoux and
Strome, 1999
). For example, in C. elegans, RNA polymerase
II transcription is repressed in the germ cell lineage by the product of the
pie-1 gene. Transcriptional inactivity appears to be crucial in
establishing germ cell identity as mutations in pie-1 switch the fate
of these cells to that of a somatic lineage
(Mello et al., 1996
;
Seydoux et al., 1996
).
A number of maternally derived gene products are likely to contribute to
transcriptional quiescence in the pole cells of Drosophila. One of
these is Germ cell less (Gcl), a component of the germ plasm that is necessary
for the formation of pole cells. gcl appears to be involved in the
establishment of transcriptional quiescence and in embryos lacking
gcl activity, newly formed pole buds are unable to silence the
transcription of genes such as sisterless-a and scute.
Conversely, when Gcl protein is ectopically expressed in the anterior of the
embryo it can downregulate the transcription of terminal group genes such as
tailless (tll) and huckebein
(Leatherman et al., 2002). A
second maternally derived gene product involved in transcriptional quiescence
is Nanos. In the soma, Nanos, together with Pumilio, plays a key role in
posterior determination by blocking the translation of maternally derived
hunchback (hb) mRNA
(Irish et al., 1989
;
Lehmann and Nüsslein-Volhard,
1991
). Nanos (Nos) also plays a role in downregulating
transcription in pole cells, and in embryos produced by nos mutant
mothers, genes that are normally active only in somatic nuclei are
inappropriately transcribed in pole cells
(Kobayashi et al., 1996
;
Asaoka et al., 1998
;
Deshpande et al., 1999
). These
include the pair-rule genes fushi tarazu and even
skipped, and the somatic sex determination gene Sex-lethal
(Sxl) (Deshpande et al.,
1999
).
The global effects of nos and gcl mutations on RNA
polymerase II activity in pole cells are analogous to those seen in
pie-1 mutants in C. elegans. In pie-1 mutants,
genes that are normally expressed only in somatic lineages are turned on in
the germ cell lineage. In wild-type C. elegans embryos, the
inhibition of transcription in the germ cell lineage is correlated with a
marked reduction in phosphorylation of the CTD repeats of the large subunit of
RNA polymerase II (Seydoux and Dunn,
1997). The CTD repeats are phosphorylated when polymerase is
transcriptionally engaged (reviewed by
Dahmus, 1996
). PIE-1 protein
may prevent transcription by inhibiting this modification. As in C.
elegans, the RNA polymerase II CTD repeats are underphosphoryted in the
pole cells of wild-type Drosophila embryos. In the pole cells of
gcl and nos mutant embryos, however, the level of CTD
phosphorylation is elevated (Leatherman et
al., 2002
) (G.D., unpublished).
Previous studies have shown that when a heterologous transcriptional
activator, GAL4-VP16 is expressed in pole cells, it is unable to activate
transcription of target gene(s) (Van Doren
et al., 1998). This finding suggested that even if a potent
activator were to be produced in pole cells, it would not be able to overcome
the inhibition of the basal transcriptional machinery by gcl, nos and
other factors. However, as GAL4-VP16 is a chimera of a yeast DNA-binding
domain and a mammalian activation domain, an alternative possibility is that
co-factors essential for its activity may be absent or inactive in
Drosophila pole cells. For these reasons, we decided to test whether
a transcription factor that is normally present and active in the somatic
cells of early Drosophila embryos can promote the transcription of
target genes when inappropriately expressed in pole cells. We chose the
homeodomain protein Bicoid (Bcd), which activates the zygotic transcription of
hb and other genes specifying anterior development. A Bcd protein
gradient is generated in precellular blastoderm embryos from the translation
of maternal mRNA localized at the anterior pole
(Driever and Nüsslein-Volhard,
1988
; Driever and
Nüsslein-Volhard, 1989
;
Struhl et al., 1989
). Although
Bcd is not present in the posterior of wild-type embryos, increasing the
bcd gene dose results in expansion of the gradient toward the
posterior and a concomitant change in the pattern of zygotic gene expression.
This result suggests that co-factors crucial for Bcd function are likely to be
ubiquitous.
We show that ectopic expression of Bcd in pole cells can induce the
transcription of the bcd target gene hb. In addition to
activating hb transcription, Bcd protein perturbs the migration of
the pole cells to the primitive somatic gonad and causes abnormalities in cell
cycle control. These effects on germ cell development resemble those observed
in embryos from nos mutant females. Moreover, as in the case of
nos- pole cells, the Sxl promoter Sxl-Pe
is also turned on in pole cells by Bcd in a sex-nonspecific manner.
Surprisingly, transcriptional activation in pole cells by Bcd requires the
activity of the terminal signaling system. This observation is unexpected, as
previous studies have established that the transcription of a downstream
target gene of the terminal pathway, tailless (tll) is shut
down completely in pole cells (Rudolph, et
al., 1997). Moreover, the doubly phosphorylated active isoform of
MAP kinase ERK, which serves as a sensitive readout of the terminal pathway,
is nearly absent in pole cells (Gabay et
al, 1997
). Taken together, these findings argue that the activity
of terminal signaling pathway in pole cells of wild-type embryos must be
substantially attenuated, but not shut off completely. What mechanisms are
responsible for downregulating terminal signaling in the presumptive germline?
We present evidence indicating that polar granule component
(pgc) functions to attenuate the terminal pathway in newly formed
pole cells. pgc encodes a non-translated RNA that is localized in
specialized germ cell-specific structures called polar granules
(Nakamura et al., 1996
). We
demonstrate that loss of pgc function in newly formed pole cells
results in the ectopic phosphorylation of ERK and the activation of the ERK
dependent target gene tll. We also show that pgc is required
to block the establishment of an active chromatin architecture in pole
cells.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transgene construction and germline transformation
To create the bcd-nos3'UTR hybrid gene, the
3'UTR of the bcd cDNA from the bcd TN3 plasmid
(Driever et al., 1990) was
truncated at the HpaI site and then fused to an
EcoRI-NotI fragment from pHSXgnosbR
(Gavis and Lehmann, 1992
) that
contains the nos 3'UTR and 3' genomic DNA. The Nanos
Response Element within the bcd 3'UTR is removed by the
HpaI truncation. Note that in bcd TN3, the bcd
5'UTR has been replaced with the Xenopus ß-globin
leader (Driever et al., 1990
).
The ß-globin-bcd-nos3'UTR sequences were then
fused to the nos promoter and 5'UTR at an NdeI site
engineered at the nos translation start codon to create
Pnos-ßbcd-nos3'UTR.
Finally, the
Pnos-ßbcd-nos3'UTR
sequences were inserted into the P element vector pDM30
(Mismer and Rubin, 1987
).
Injection of the pDM30
Pnos-ßbcd-nos3'UTR
plasmid into ry506 embryos was carried out according to Spradling
(Spradling, 1986
). Analysis
was carried out using two independent transgenic lines.
Histochemical analysis
Synthesis of digoxigenin-labeled bcd probe and whole-mount in situ
hybridization to bcd RNA was carried out as described previously
(Gavis and Lehmann, 1992).
Whole-mount antibody staining using the Bcd monoclonal antibody 733.3 (gift of
W. Driever) was performed as described previously
(Gavis and Lehmann, 1992
). All
other antibody staining was carried out as described elsewhere
(Deshpande et al., 1995
). Anti
ß-galactosidase antibody (Promega) was used at 1:500. Anti p-H3 antibody
(Upstate Biotechonology) was used at 1:1000. Anti ERK antibody was obtained
from Sigma and used at 1:200. Anti-Vasa antibody (kind gift of P. Lasko) was
used at 1:2000. For confocal analysis, secondary antibodies conjugated with
different fluorophores, anti-rabbit ALEXA-546 (red) and anti rat ALEXA-488
(green) were used (Molecular Probes). All the secondary antibodies were
obtained from Molecular Probes.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine whether Bcd can overcome quiescence, we examined the expression of the bcd target, hunchback (hb) in bcd-nos3'UTR transgenic embryos. By antibody staining, we found that Hb protein could be detected in pole cells of bcd-nos3'UTR embryos but not in pole cells of wild-type embryos (data not shown). As the accumulation of Hb protein in pole cells of transgenic embryos could be due to the inappropriate translation of maternally derived hb mRNA rather than the zygotic transcription of the hb gene, we compared the expression of a paternally derived hb promoter:lacZ reporter transgene (hb:lacZ) in wild-type and bcd-nos3'UTR embryos. In wild-type embryos, hb:lacZ expression is detected in an anterior domain (not shown) and a posterior stripe (Fig. 1A). However, like the endogenous hb gene, no expression of the hb:lacZ reporter is detected in the pole cells (Fig. 1B). By contrast, the hb:lacZ reporter is expressed in pole cells of bcd-nos3'UTR embryos (Fig. 1C,D). These findings indicate that the Bcd transcription factor can overcome the inhibition of the Pol II transcriptional machinery in pole cell nuclei and activate transcription of a known target gene.
|
|
Germ cell migration defects in bcd-nos 3'UTR embryos
In wild-type embryos, pole cells migrate along the dorsal surface of the
embryo during germ band extension. At stage 10, they pass through the
invaginated midgut epithelium to enter the interior of the embryo, where they
migrate along the dorsal side of the endoderm. After reaching the dorsal
mesoderm, the germ cells are segregated into two groups of 10-15 cells,
located on either side of the ventral midline
(Fig. 3A). These two groups of
cells become encapsulated by the somatic gonadal mesoderm, forming the
embryonic gonads (Jaglarz and Howard,
1995; Warrior,
1994
; Moore et al.,
1998
).
|
Interestingly, like nos mutant embryos, bcd-nos3'UTR embryos also exhibit defects in germ cell migration. Germ cell migration in bcd-nos3'UTR embryos is indistinguishable from wild-type until stage 10, after the germ cells migrate to the dorsal mesoderm. Although the germ cells appear to segregate into two clusters on either side of the ventral midline, they either fail to associate with the somatic gonadal mesoderm or sustain contact with the somatic gonad. At stage 13, many of the germ cells in bcd-nos3'UTR embryos are scattered through several segments rather than having coalesced in the somatic gonad (Fig. 3B).
Ectopic Bcd induces Sxl expression
In previous studies (Deshpande et al,
1999), we found that inappropriate expression of Sxl protein in
pole cells induces mitotic and migration defects similar to those observed
here for bcd-nos3'UTR embryos. As Duffy and Gergen
(Duffy and Gergen, 1991
) found
that expression of Sxl protein from the Sxl establishment promoter,
Sxl-Pe, is upregulated in female embryos by Bcd, one possible
explanation for the mitotic and migration defects seen in bcd-nos
3' UTR pole cells is that the Sxl-Pe promoter has
been inappropriately activated in these germ cells by Bcd. Consistent with
this possibility, we found that in marked contrast to the pole cells of
wild-type embryos where Sxl protein is not expressed, Sxl is detected in the
pole cells of blastoderm stage of female and male
bcd-nos3'UTR embryos (data not shown). To show that
the expression of Sxl protein in these cells is due to the transcriptional
activation of Sxl-Pe, we compared the activity of a
Sxl-Pe:lacZ reporter transgene which faithfully mimics the endogenous
Sxl-Pe promoter in wild-type and bcd-nos3'UTR
embryos (Keyes et al., 1992
;
Estes et al., 1995
). In
wild-type embryos, Sxl-Pe:lacZ expression is detected in somatic
cells of female embryos but not in male embryos or in the pole cells of either
sex (Fig. 4A; data not shown).
By contrast, Sxl-Pe:lacZ expression is readily detected in both the
somatic and pole cells of female bcd-nos3'UTR embryos
(Fig. 4B). Sxl-Pe:lacZ
is also expressed in pole cells of male bcd-nos3'UTR
embryos that lack somatic expression (data not shown)
|
The terminal signaling pathway is required for patterning of the anterior-
and posterior-most regions of the embryo
(Janody et al., 2000;
Schaeffer et al., 2000
). This
pathway has been shown to have opposite effects on Bcd activity in different
regions of the embryo. Close to the anterior pole where both terminal
signaling activity and Bcd protein concentration is highest, the terminal
pathway antagonizes transcriptional activation by Bcd
(Ronchi et al., 1993
). By
contrast, near the middle of the embryo where the terminal signaling activity
and Bcd protein concentration are much lower, the terminal pathway potentiates
Bcd function (Janody et al.,
2000
; Schaeffer et al.,
2000
). These observations, together with the differences between
our results and those of Van Doren et al. suggested that the terminal pathway
may be critical for the Bcd-dependent activation of transcription in pole
cells.
To test this possibility, we analyzed the expression of the hb:lacZ reporter in bcd-nos3'UTR embryos mutant for tsl. We found that unlike wild-type bcd-nos3'UTR embryos, hb:lacZ expression is not detected in pole cells from bcd-nos3'UTR embryos mutant for tsl (Fig. 1E,F). As the elimination of tsl function did not alter the amount of Bcd protein at the posterior in bcd-nos3'UTR embryos (not shown), we concluded that Bcd-dependent activation of hb in pole cells requires tsl function. Additionally, this observation suggests that the terminal pathway must be active, at least at some level, in pole cells.
As bcd activation of hb transcription in pole cells is dependent upon the terminal pathway, we wondered whether this was also true for the activation of Sxl-Pe. To test this possibility, the Sxl-Pe:lacZ reporter was introduced into bcd-nos3'UTR embryos mutant for tsl Like hb, the activation of Sxl-Pe:lacZ in pole cells by Bcd requires tsl function (Fig. 4C).
A `gain-of-function' mutation in the terminal signaling pathway gene torso alters gene expression in the soma, but has only minimal effects on transcription in pole cells
The finding that tsl is required for Bcd-dependent activation of
hb and Sxl-Pe transcription in pole cells prompted us to
examine the functioning of the terminal pathway in pole cells in more detail.
The terminal signaling pathway is known to activate the transcription of the
tailless (tll) gene in the soma; however, it does not
normally turn on tll transcription in pole cells
(Rudolph et al, 1997). As the
elevated levels of CTD phosphorylation seen in
bcd-nos3'UTR pole cells suggested that Bcd probably
turns on genes in addition to hb (and Sxl-Pe), we decided to
determine whether one of these is tll. For this purpose we introduced
a tll:lacZ reporter into bcd-nos3'UTR embryos. Although we
could detect ß-galactosidase in bcd-nos3'UTR pole cells, the level
of antibody staining was only marginally above the background staining seen
for the tll:lacZ reporter in wild-type pole cells (data not
shown).
One reason why tll might show little response to ectopic Bcd is that the
terminal signaling pathway has to be fully activated in order to efficiently
induce tll transcription. If the terminal signaling pathway is downregulated
(but not completely off in the pole cells) the tll reporter would remain
repressed. To explore this possibility, we asked whether it was possible to
activate tll expression in pole cells by potentiating terminal signaling. We
used two approaches to upregulate the terminal pathway. In the first, we used
a gain-of-function allele of the terminal pathway receptor gene torso,
torsoRL3, which is active independent of ligand. In the second, we increased
the dose of the torso gene product in pole cells using a nos promoter
transgene that drives the expression of torso protein coding sequences linked
to the nos 3'UTR (Casanova and
Struhl, 1993). As can be seen by comparing ß-galactosidase
expression from the tll:lacZ reporter in wild-type embryos and in
torso-nos3'UTR transgene embryos, potentiating the terminal signaling
pathway weakly activates transcription of the tll reporter in pole cells
(Fig. 5). Similar results were
obtained for the constitutively active torsoRL3 receptor. Although the
expression of the tll reporter was upregulated in the soma of torsoRL3
embryos, there was only a very weak activation of the reporter in the pole
cells (not shown).
|
polar granule component (pgc) downregulates the activity of the terminal group genes in the pole cells
The finding that increasing torso activity induces only a low
level of tll:lacZ expression in pole cells (and has no effect on
Sxl-Pe) suggests that there may be special mechanisms to repress the
terminal pathway in the germline of early embryos. With the aim of identifying
factors that are involved in downregulating the terminal system in pole cells
and blocking tll transcription, we asked if the three genes namely
nos, gcl and polar granule component (pgc) that are
known to function in pole cell differentiation have any effects on the
activity of the tll-lacZ reporter.
Although we found that the tll-lacZ reporter is substantially upregulated in the posterior soma of embryos produced by nos mutant mothers, we failed to detect ß-galactosidase expression in the pole cells (not shown). This observation would suggest that the loss of nos activity in itself is not sufficient to override the inhibition of the terminal signaling pathway in pole cells; however, as tll-lacZ is clearly upregulated in the nearby posterior soma, it would be reasonable to conclude that nos is likely to have a `redundant' role in downregulating the transcription of the terminal pathway gene tll in pole cells. This possibility would be consistent with previous studies that showed that the transcription of Sxl-Pe as well as several segmentation genes is turned on in the pole cells of nos mutant embryos. In the case of gcl, we observed no strong effects on tll-lacZ expression in the soma. Although ß-galactosidase could be detected in the pole cells of gcl mutant embryos, the level was just above background (not shown). Thus, the loss of gcl activity would appear to have only a minor effect on the transcription of this terminal pathway gene. In addition, there were at most only small effects on the activity of Sxl-Pe (not shown).
A different result was obtained in embryos deficient for pgc. For
pgc we used an antisense transgene that is thought to substantially
reduce but not completely eliminate pgc activity
(Nakamura et al., 1996). As
shown in Fig. 6, we found that
the tll-lacZ reporter is activated in pole cells of pgc
embryos, and relatively high levels of ß-galactosidase expression are
observed. Strikingly, the expression of the tll-lacZ reporter in
pgc- pole cells commences between nuclear cycle 9-10 at
the time pole cells first begin to form. This is even prior to the activation
of this reporter in somatic nuclei which normally occurs around cycle 12. As
illustrated in the DAB stained embryos in
Fig. 6, ß-galactosidase
expression is not always restricted to the newly budded pole cells at the very
posterior of the embryo. Instead, some embryos have ß-galactosidase
positive `cells' at positions that can be rather far from the posterior pole.
Double staining with ß-galactosidase and Vasa antibodies indicates that
at least some of these unusual ß-galactosidase positive `cells' are also
Vasa positive (Fig. 6). In
addition in several instances, `cells' that have high levels of
ß-galactosidase often have low levels of Vasa, while `cells' that have
high levels of Vasa have low levels of ß-galactosidase
(Fig. 6). Although this
variability in tll-lacZ expression could reflect the action of other
repressive factors, such as nos, an alternative possibility is that
pgc is not completely inactivated by the antisense transgene. In this
case, even more extreme defects in repressing tll-lacZ and pole cell
formation might be expected in the absence of any pgc activity.
|
Finally, unlike tll-lacZ, the Sxl-Pe:lacZ reporter was
not detectably activated at any point in the pole cells of pgc
embryos (not shown). This finding suggests that pgc may influence the
expression of a different set of genes than either nos or
gcl (Asaoka et al.,
1998; Deshpande et al.,
1999
; Leatherman et al.,
2002
).
The presence of activated MAP kinase correlates with tll transcription in pole cells
The signaling cascade activated by the torso receptor leads to the
phosphorylation of MAP kinase (Gabay et
al., 1997). To determine whether pgc is required to
repress this signaling cascade in pole cells, we used a monoclonal antibody
that recognizes the doubly phosphorylated, active form of MAP kinase, ERK. As
reported previously by Gabay et al. (Gabay
et al., 1997
), we observed a graded activation of ERK in the soma
at anterior and posterior of nuclear cycle 12-13 syncytial embryos
(Fig. 7A). By contrast, the
pole cells of these cycle 12-13 embryos have only a low level of ERK,
indicating that the functioning of the terminal pathway is attenuated in these
cells. We presume that this small amount of ERK is sufficient to cooperate
with ectopically expressed Bcd to activate transcription in pole cells. The
activation of MAP kinase in the soma of early embryos is only transient, and
by cellular blastoderm formation anti-ERK specific staining is greatly
diminished (Fig. 7B).
|
pgc is required to block the formation of an active chromatin structure in pole cells
Although Sxl-Pe was not turned on in pgc pole cells, it
seemed possible that the loss of pgc activity might have more
widespread effects on transcription than just turning on the tll
gene. To investigate this possibility, we determined whether markers for
global transcriptional activity were present in pgc pole cells. One
of these markers is the phosphorylation of ser 2 residues in the Pol II CTD
repeats. As described above, high levels of phosphorylated CTD ser2 are
present in transcriptionally active somatic nuclei of wild-type embryos, while
in the transcriptionally quiescent pole cells, ser2 is largely
unphosphorylated. Consistent with a more general upregulation of transcription
in pgc pole cells, we found that CTD ser2 phosphorylation is elevated
in pgc pole cells of presyncytial blastoderm embryos
(Fig. 2C). Moreover, as was
observed for the tll reporter, the level of CTD ser2 phosphorylation
is greatly reduced in the pole cells of cellularized pgc embryos.
Another marker of global transcription is the methylation of lysine residue
4 in histone H3 (abbreviated as H3MeK4). Schaner et al.
(Schaner et al., 2003) have
shown that there is little if any H3 K4 methylation during the nuclear cycles
preceding the migration of the nuclei to the periphery of the embryo, and even
in nuclear cycle 10/11 embryos only a little H3 K43 methylation is detected in
the somatic nuclei (Fig. 8A).
However, by nuclear cycle 13/14, the level K4 methylation increases
substantially (Fig. 8B), and at
cellular blastoderm formation all somatic nuclei appear to have a high level
of this methylation. By contrast, K4 methylation is not upregulated in
wild-type pole cells during the syncytial blastoderm stage (see
Fig. 8A,B) and there is little
methylated H3 K4 in germ cells until much later in development when they begin
migrating from the midgut towards the somatic gonad.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We show that Bcd protein expressed from a
bcd-nos3'UTR transgene can activate the transcription
of its target gene hb in pole cells, overcoming whatever mechanisms
are responsible for transcriptional quiescence. In addition to activating
transcription of hb, Bcd has other phenotypic effects. It prevents
the pole cells from properly arresting their cell cycle and disrupts their
migration to the somatic gonad. Because similar defects in pole cell
development can be induced by the inappropriate expression of Sxl protein in
these cells (Deshpande et al.,
1999), one plausible hypothesis is that Bcd not only activates the
hb promoter, but also turns on the Sxl establishment
promoter, Sxl-Pe. Consistent with this idea, the Sxl-Pe:lacZ
reporter is turned on in the pole cells of male and female bcd-nos
3' UTR embryos and Sxl protein accumulates in these cells. Although
previous studies by Duffy and Gergen
(Duffy and Gergen, 1991
)
indicate that Sxl-Pe is responsive to Bcd, it is somewhat surprising
that Sxl-Pe is not only inappropriately turned on in pole cells by
Bcd, but that it is activated in both sexes. This suggests that Bcd activation
of Sxl-Pe in pole cells must proceed by a mechanism that bypasses the
X/A chromosome counting system which controls Sxl-Pe activity in the
soma. It is interesting to note that the activation of Sxl-Pe in pole
cells in the absence of nos function also seems to depend upon a
mechanism(s) that circumvents the X/A chromosome counting system.
The behavior of Bcd contrasts with that of a chimeric Gal4-VP16 protein,
which does not activate transcription when expressed in pole cells, although
it does function in the surrounding somatic cells
(Van Doren et al., 1998). The
difference in the activity of Bcd and Gal4-VP16 proteins could reflect a
requirement for different co-factors to activate transcription of their target
genes. For example, the VP16 activation domain has been shown to interact with
the TAFs, TAFII40 and TAFII70 and with the TATA factor
itself (Klemm et al., 1995
;
Nishikawa et al., 1997
). If
one of the TAFs crucial for VP16 function or some as yet unidentified
co-factor is missing or inactive in pole cell nuclei, Gal4-VP16 protein would
not be able to activate transcription in Drosophila germ cells.
Alternatively, as the target enhancers/promoters for Bcd and Gal4-VP16
proteins are different, it is possible that distinct chromatin remodeling
factors are required to access these sequences, and that factors required for
GAL4-VP16 targets are absent in pole cell nuclei.
That Bcd protein depends upon other ancillary factors to turn on
transcription in pole cells is demonstrated by the requirement for
tsl function in the activation of both the hb and
Sxl-Pe promoters. tsl is a component of the maternal
terminal signaling pathway which activates the zygotic genes, tll and
huckebein (hkb), at the poles of the embryo. In addition,
the terminal pathway has opposing effects on the expression of
bcd-dependent gap genes
(Grossniklaus et al., 1994;
Wimmer et al., 1995
;
Gao et al., 1996
). At the
anterior pole, where terminal signaling activity is highest, Bcd targets such
as hb and orthodenticle (otd) are repressed
(Driever and Nüsslein-Volhard,
1989
; Finkelstein and
Perrimon, 1990
; Ronchi et al.,
1993
). At a distance from the anterior pole, where both the
concentration of Bcd protein and the strength of the terminal signaling
cascade is much lower, the terminal pathway has an opposite, positive effect
on hb and otd expression
(Janody et al., 2000
;
Schaeffer et al., 2000
). Two
mechanisms are thought to account for the positive effects of the terminal
pathway on bcd target genes. First, Bcd is a direct target for
phosphorylation by the terminal signaling cascade. Second, regulatory regions
of bcd target genes have sites for other transcription factors whose
activity can be directly modulated by the terminal system.
In our experiments, the concentration of Bcd protein produced by the bcd-nos3'UTR transgene in pole cells is much less than it is at the anterior pole. Similarly, the activity of the terminal signaling cascade in pole cells is much reduced compared with that in the somatic nuclei at the anterior and posterior poles. Thus, in both of these respects, the conditions in the bcd-nos 3' UTR pole cells would appear to most closely approximate those in the region of the embryo where the terminal signaling cascade potentiates rather than inhibits Bcd activity. This would explain why activation of transcription in pole cells by Bcd depends on the terminal signaling pathway and why in this particular instance this pathway does not antagonize the activity of the ectopically expressed Bcd protein.
The fact that the terminal pathway can function in pole cells, yet does not turn on its target gene tll indicates that the activity of this pathway is attenuated in the germline. It seems likely that several different mechanism may be responsible for preventing pole cells from responding to the terminal pathway and turning on tll transcription. One mechanism appears to be an inhibition of the signaling cascade itself. In the posterior and anterior soma of pre-cellular blastoderm embryos the terminal signaling cascade directs the phosphorylation of the MAP kinase ERK. While phosphorylated ERK can also be detected in wild-type pole cells, the amount of activated kinase is much less than in the surrounding soma. Consistent with this observation, potentiating the terminal system using either a gain-of-function torso receptor mutant or by expressing elevated level of the receptor in pole cells using a torso transgene which has the nos 3' UTR had only a small effect on the activity of a tll-lacZ reporter in the germline. By contrast, gain-of-function torso mutation substantially upregulates the tll reporter in the soma.
To identify factors that could be involved in repressing the terminal pathway in pole cells, we examined three genes, nos, gcl and pgc, that are known to play an important role in the early development of the germline and have been implicated in transcriptional quiescence. Of these three, only pgc appeared to have significant effects on the terminal signaling pathway in pole cells. We found that the expression of a tll reporter is turned on in pole cells of embryos deficient in pgc activity. That this is due at least in part to a failure to properly attenuate the terminal signaling pathway in the germline is suggested by the fact that the level of activated ERK is greatly elevated in pgc pole cells compared with wild type. Although these findings implicate pgc in downregulating the terminal pathway, how this is accomplished and whether pgc has a direct rather than an indirect role in this process remains to be determined. In addition, our studies indicate that pgc has functions in addition to attenuating this signaling cascade. First, we found that there are abnormalities in the formation of pole cells in pgc embryos and Vasa-positive `cells' are observed in cycle 9-10 embryos at abnormal locations. Second, the loss of pgc activity may lead to the inappropriate activation of genes in addition to tll. We found that two markers for global transcriptional activity, CTD phosphorylation and histone H3 K4 methylation, are present in pole cells of pgc embryos.
Our results also suggest that multiple and interrelated levels of
regulation are responsible for ensuring transcriptional quiescence in the pole
cells. For example, Sxl-Pe can be upregulated by the
terminal pathway in the soma and requires this pathway to be activated by Bcd
in pole cells. However, this promoter is not activated in pole cells in the
absence of pgc function. Thus, the activation of the terminal
signaling cascade in pole cells is not sufficient in itself to induce
Sxl-Pe. This suggests that mechanisms are in place in pgc
pole cells that would override any effects of activated ERK on Sxl-Pe
activity. Similarly, although loss of nos activity leads to the
activation of Sxl-Pe in pole cells, and the upregulation of
tll in the posterior soma, the tll promoter is not turned on
in nos pole cells. We presume that tll is not activated in
pole cells because it requires the terminal system that still remains
attenuated in nos pole cells. Redundancy is also suggested by the
finding that although the loss of gcl leads to the expression of the
X chromosome counting genes sis-a and scute in pole cells
(Leatherman et al., 2002),
Sxl-Pe is not activated. This suggests nos function is
sufficient to keep Sxl-Pe off in gcl mutant pole cells even
though several X chromosome counting genes are activated. Similarly, we have
not observed an obvious effect of nos mutations on scute
expression in pole cells (G.D, unpublished). This implies that gcl
and nos may be responsible for repressing the transcription of
different sets of genes.
Finally, although transcription is upregulated in pgc pole cells between nuclear cycles 9/10-13, a high level of transcriptional activity is not maintained in the pole cells that are present by the time the cellular blastoderm is formed. The tll reporter is turned off, and both CTD phosphorylation and histone H3 K4 methylation disappear. One possible interpretation of this finding is that pgc has an early function in establishing transcriptional quiescence, but is not required after nuclear cycle 13 because of the activity of other factors such nos or gcl. However, as the number of pole cells at cellularization is reduced compared with the number present earlier, it also possible that the only pole cells that remain are the ones in which the amount of pgc activity is sufficient to establish some degree transcriptional repression. Further studies with bona fide null alleles will be required to resolve this question, and understand how pgc functions during pole cell formation and germ cell determination
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asaoka, M., Sano, H., Obara, Y. and Kobayashi, S. (1998). Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster. Mech. Dev. 78,153 -158.[CrossRef][Medline]
Asaoka, M., Yamada, M., Nakamura, A., Hanyu, K. and Kobayashi, S. (1999). Maternal pumilio acts togother with Nanos in germline development in Drosophila embryos. Nat. Cell Biol. 1,431 -437.[CrossRef][Medline]
Casanova, J. and Struhl, G. (1993). The torso receptor localizes as well as transduces the spatial signal specifying terminal body pattern in Drosophila. Nature 362,152 -155.[CrossRef][Medline]
Dahmus, M. E. (1996). Reversible phosphorylation of the C-terminal domain of RNA polymeraseII. J. Biol. Chem. 265,19185 -19191.
Deshpande, G., Calhoun, G., Yanowitz, J. and Schedl, P. D. (1999). Novel functions of nanos in downregulating mitosis and transcription during the development of the Drosophila germline. Cell 99,271 -281.[Medline]
Deshpande, G., Stukey, J. and Schedl, P. (1995). scute (sis-b). function in Drosophila sex determination. Mol. Cell. Biol. 15,4430 -4440.[Abstract]
Driever, W. and Nüsslein-Volhard, C. (1988). The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95-104.[Medline]
Driever, W. and Nüsslein-Volhard, C. (1989). The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo. Nature 337,138 -143.[CrossRef][Medline]
Driever, W., Siegel, V. and Nüsslein-Volhard, C. (1990). Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen. Development 109,811 -820.[Abstract]
Duffy, J. and Gergen, J. P. (1991). The Drosophila segmentation gene runt acts as a position-specific numerator element necessary for the uniform expression of the sex-determining gene Sex-lethal. Genes Dev. 5,2176 -2187.[Abstract]
Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by Oskar. Nature 358,387 -392.[CrossRef][Medline]
Estes, P. A., Keyes, L. N. and Schedl, P. D. (1995). Multiple response elements in the Sex-lethal early promoter ensure its female-specific expression pattern. Mol. Cell. Biol. 15,904 -917.[Abstract]
Finkelstein, R. and Perrimon, N. (1990). The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development. Nature 346,485 -488.[CrossRef][Medline]
Foe, V. E. and Alberts, B. M. (1983). Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J. Cell Sci. 61,31 -70.[Abstract]
Gabay, L., Seger, R. and Shilo, B. (1997). MAP
kinase in situ activation atlas during Drosophila embryogenesis.
Development 124,3535
-3541.
Gavis, E. R. and Lehmann, R. (1992). Localization of nanos RNA controls embryonic polarity. Cell 71,301 -313.[Medline]
Gavis, E. R. and Lehmann, R. (1994). Translational regulation of nanos by RNA localization. Nature 369,315 -318.[CrossRef][Medline]
Gao, Q., Wang, Y. and Finkelstein, R. (1996). Orthodenticle regulation during embryonic head development in Drosophila. Mech. Dev. 56, 3-15.[CrossRef][Medline]
Grossniklaus, U., Cadigan, K. M. and Gehring, W. J.
(1994). Three maternal coordinate systems cooperate in the
patterning of the Drosophila head.
Development 120,3155
-3171.
Irish, V., Lehmann, R. and Akam, M. (1989). The Drosophila posterior-group gene nanos functions by repressing hunchback activity. Nature 338,646 -648.[CrossRef][Medline]
Jaglarz, M. and Howard, K. (1995). The active
migration of Drosophila primordial germ cells.
Development 121,3495
-3503.
Janody, F., Sturny, R., Catala, F, Desplan, C. and Dostatni,
N. (2000). Phosphorylation of Bicoid on MAP-kinase sites:
contribution to its interaction with the torso pathway.
Development 127,279
-289.
Keyes, L. N., Cline, T. W. and Schedl, P. (1992). The primary sex determination signal of Drosophila acts at the level of transcription. Cell 68,933 -943.[Medline]
Klemm, R. D., Goodrich, J. A., Zhou, S. and Tjian, R.
(1995). Molecular cloning and expression of the 32-kDa subunit of
human TFIID reveals interactions with VP16 and TFIIB that mediate
transcriptional activation. Proc. Natl. Acad. Sci. USA
92,5788
-5792.
Kobayashi, S., Yamada, M., Asaoka, M. and Kitamura, T. (1996). Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature 380,708 -711.[CrossRef][Medline]
Leatherman, J., Levin, L., Boero, J. and Jongens, T. (2002). germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage. Curr. Biol. 12,1681 -1685.[CrossRef][Medline]
Lehmann, R. and Nüsslein-Volhard, C. (1991). The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo. Development 112,679 -691.[Abstract]
Lindsley, D. L. and Zimm, G. G. (1992).The Genome of Drosophila melanogaster . San Diego: Academic Press.
Martin, J., Raibaud, A. and Ollo, R. (1994). Terminal pattern elements in Drosophila embryo induced by the torso-like protein. Nature. 367,741 -745.[CrossRef][Medline]
Mello, C. C., Schubert, C., Draper, B., Zhang, W., Lobel, R. and Priess, J. R. (1996). The PIE-1 protein and germline specification in C. elegans embryos. Nature 382,710 -712.[CrossRef][Medline]
Mismer, D. and Rubin, G. M. (1987). Analysis of
the promoter of the nina E opsin gene in Drosophila
melanogaster. Genetics
116,565
-578.
Moore, L., Broihier, H., van Doren, M., Lunsford, L. and
Lehmann, R. (1998). Identification of genes controlling germ
cell migration and embryonic gonad formation in Drosophila.
Development 125,667
-678.
Nakamura, A., Amikura, R., Mukai, M., Kobayashi, S. and Lasko,
P. (1996). Requirement for a noncoding RNA in Drosophila
polar granules for germ cell establishment. Science
274,2075
-2079.
Nishikawa, J., Kokubo, T., Horikoshi, M., Roeder, R. G. and
Nakatani, Y. (1997). Drosophila TAF(II)230 and the
transcriptional activator VP16 bind competitively to the TATA box-binding
domain of the TATA box-binding protein. Proc. Natl. Acad. Sci.
USA 94,85
-90.
Parisi, M. and Lin, H. (2000). Translational repression: a duet of nanos and pumilio. Curr. Biol. 10,R81 -R83[CrossRef][Medline]
Ronchi, E., Treisman, J., Dostatni, N., Struhl, G. and Desplan, C. (1993). Down-regulation of the Drosophila morphogen bicoid by the torso receptor-mediated signal transduction cascade. Cell 74,347 -355.[Medline]
Rudolph, K. M., Liaw, G. J., Daniel, A., Green, P., Courey, A.
J., Hartenstein, V. and Lengyel, J. A. (1997). Complex
regulatory region mediating tailless expression in early embryonic
patterning and brain development. Development
124,4297
-4308.
Savant-Bhonsale, S. and Montell, D. (1993). torso-like encodes the localized determinant of Drosophila terminal pattern formation. Genes Dev. 7,2548 -2555.[Abstract]
Schaeffer, V., Killian, D., Desplan, C. and Wimmer, E. A.
(2000). High Bicoid levels render the terminal system dispensable
for Drosophila head development. Development
127,3993
-3999.
Schaner, C. E., Deshpande, G., Schedl, P. D. and Kelly, W. G. (2003). A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies. Dev. Cell 5,747 -757.[Medline]
Schultz, C. and Tautz, D. (1995). Zygotic
caudal regulation by hunchback and its role in abdominal
segment formation of the Drosophila embryo.
Development 121,1023
-1028.
Seydoux, G. and Dunn, M. A. (1997).
Transcriptionally repressed germ cells lack a subpopulation of phosphorylated
RNA polymerase II in early embryos of Caenorhabditis elegans and
Drosophila melanogaster. Development
124,2191
-2201.
Seydoux, G. and Strome, S. (1999). Launching
the germline in Caenorhabditis elegans: regulation of gene expression in early
germ cells. Development.
126,3275
-3283.
Seydoux, G., Mello, C. C., Pettitt, J., Wood, W. B., Priess, J. R. and Fire, A. (1996). Repression of gene expression in the embryonic germ lineage of C. elegans. Nature 382,713 -716.[CrossRef][Medline]
Spradling, A. C. (1986). P element-mediated transformation. In Drosophila: A Practical Approach (ed. D. B. Roberts), pp. 175-197. Oxford: IRL Press.
St Johnston, D. (1993). Pole plasm and the posterior group genes. In The Development of Drosophila melanogaster, pp. 325-363. Cold Spring Harbor Laboratory Press.
Struhl, G., Struhl, K. and Macdonald, P. M. (1989). The gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell 57,1259 -1273.[Medline]
Van Doren, M., Williamson, A. L. and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8, 243-246.[Medline]
Wang, C., Dickinson, L. K. and Lehmann, R. (1994). Genetics of nanos localization in Drosophila. Dev. Dyn. 199,103 -115.[Medline]
Warrior, R. (1994). Primordial germ cell migration and the assembly of the Drosophila embryonic gonad. Dev. Biol. 166,180 -194.[CrossRef][Medline]
Williamson, A. and Lehmann, R. (1996). Germ cell development in Drosophila. Annu. Rev. Cell Dev. Biol. 12,365 -391.[CrossRef][Medline]
Wimmer, E. A., Simpson-Brose, M., Cohen, S. M., Desplan, C. and Jäckle, H. (1995). Trans- and cis-acting requirements for blastodermal expression of the head gap gene buttonhead. Mech. of Dev. 53,235 -245.[CrossRef]
Zalokar, M. (1976). Autoradiographic study of protein and RNA formation during early development of Drosophila eggs. Dev. Biol. 49,97 -106.
Zalokar, M. and Erk, I. (1976). Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J. Microsc. Biol. Cell. 25, 97-106.