Department of Biochemistry and Cell Biology and The Center for Developmental Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794-5140, USA
* Author for correspondence (e-mail: pgergen{at}life.bio.sunysb.edu)
Accepted 3 February 2003
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SUMMARY |
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Key words: Runx, Hox, Zic, Segmentation, sloppy paired 1, engrailed, wingless, Drosophila
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Introduction |
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This complication makes it difficult to identify the exact roles of these three factors in segment-polarity gene regulation and has obscured our understanding of the combinatorial rules underlying the pair-rule to segment-polarity transition.
One pivotal player in the pair-rule to segment-polarity transition is Runt,
the founding member of the Runx family of transcription factors. Runx proteins
function both as activators and repressors of transcription in multiple
developmental pathways (Coffman,
2003; Komori,
2002
; Shapiro,
1999
; Speck et al.,
1999
; Wheeler et al.,
2000
). Indeed, Runt has separable roles in three developmental
pathways, sex determination, segmentation and neurogenesis, within the first
few hours of Drosophila embryogenesis
(Duffy and Gergen, 1994
).
Ectopic expression experiments indicate a role for Runt in establishing
polarity within each parasegment
(Manoukian and Krause, 1993
).
The four-cell wide run stripes overlap the anterior half of each
ftz stripe and the posterior half of each eve stripe. The
contrasting positive and negative regulatory effects of run on
ftz and eve, respectively, contribute to the graded activity
of these two genes within each parasegment. However, Runt has additional
effects on segment-polarity gene expression beyond modulating ftz and
eve expression. For example, the odd-numbered en stripes are
repressed by Runt, even in cells that express Eve
(Tracey et al., 2000
). The
immediate response of en to transient induction of a heat-inducible
hs-runt transgene strongly suggests this repression is direct.
Additional insights on Runt function have been obtained in other experiments
with hs-runt transgenes (Li and
Gergen, 1999
; Pepling and
Gergen, 1995
; Tsai et al.,
1998
). However, the difficulty in reproducibly controlling the
precise level and timing of expression makes this approach less than ideal for
further dissecting the role of Runt and other pair-rule transcription factors
in segment-polarity gene regulation.
We have recently taken advantage of an alternative strategy to investigate
the segmentation gene network, and in particular the regulatory functions of
Runt. This strategy uses Drosophila lines that maternally express the
yeast transcriptional activator GAL4 to drive expression of GAL4-responsive
UAS transgenes concomitant with the onset of zygotic transcription
during the blastoderm stage of embryogenesis. The transgene construct used to
express GAL4 maternally contains the nanos promoter and the 3'
untranslated region of an -tubulin mRNA and is thus referred to as an
NGT transgene (nanos-GAL4-tubulin). Importantly, the
expression level can be quantitatively and reproducibly manipulated by using
NGT lines that drive different levels of GAL4 expression
(Tracey et al., 2000
).
Experiments with this system have confirmed the potent activity of Runt as a
repressor of the odd-numbered en stripes. Indeed the lethality
associated with NGT-driven Runt expression has provided the basis for
a genetic dissection of en repression
(Wheeler et al., 2002
).
We have used this approach to systematically examine the responses of pair-rule and segment-polarity genes to different levels of Runt. After en, the second most sensitive segmentation gene target of Runt is the slp1 transcription unit of the sloppy paired locus. We find that the combinatorial rules needed to generate two-cell wide slp1 stripes in the posterior half of each parasegment are simpler than the rules needed to generate the single-cell wide stripes of the segment-polarity genes en and wg. Runt is required for slp1 activation in odd-numbered parasegments. This Runt-dependent activation involves cooperation with the zinc-finger transcription factor encoded by the pair-rule gene opa. Indeed, the simple combination of Runt + Opa is sufficient for slp1activation in all somatic blastoderm cells that do not have Ftz. We furthermore find that repression of slp1 in the anterior half of the even-numbered parasegments requires Ftz. Ftz not only blocks Runt-dependent activation in these cells, but the combined action of Runt and Ftz is sufficient for slp1 repression in all blastoderm nuclei. Thus, Runt is switched from an activator to a repressor of slp1 by the Ftz homeodomain transcription factor. Additional experiments indicate that Ftz also modulates the activity of Runt on the segment-polarity genes wg and en. However, in the case of en the combination of Runt + Ftz gives activation rather than repression. These results provide important new insights into the context-dependent activity of Runt in segmentation and also provide a valuable framework for dissecting the mechanisms of transcriptional activation and repression by Runt.
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Materials and methods |
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The GAL4-drivers P{GAL4-nos.NGT}11 (NGT11),
P{GAL4-nos.NGT}40 (NGT40) and P{GAL4-nos.NGT}A
(NGTA) have been described previously
(Tracey et al., 2000;
Wheeler et al., 2002
).
Homozygous NGT40 females produce approximately twice the levels of
maternal GAL4 activity as females homozygous for either NGT11 or
NGTA. Females homozygous for both NGT40 and NGTA
produce
1.5 times more activity than homozygous NGT40 females,
whereas females heterozygous for both NGT40 and NGTA produce
0.75x the activity of homozygous NGT40 females.
The P{UAS-runt.T}14 (UAS-runt[14]),
P{UAS-runt.T}232 (UAS-runt[232]) and
P{UAS-runt.T}15 (UAS-runt[15]) transgenes have been
described previously (Li and Gergen,
1999; Tracey et al.,
2000
). The third chromsome-linked P{UAS-runt.T}13
(UAS-runt[13]) transgene is comparable in activity with
UAS-runt[232]. The P{UAS-opa.VZ}36 (UAS-opa[36])
transgene insertion was created by standard germ line transformation using the
p:
2-3 helper plasmid. This transposon construct was generated by first
digesting pNB40:opa[C] (Benedyk et al.,
1994
) with BstEII and BglII to remove vector
sequences containing the SP6 promoter and 5' untranslated leader of the
Xenopus ß-globin gene as well as 177 nucleotides of the
opa 5' untranslated leader. The digested plasmid was treated
with Klenow polymerase and re-circularized with DNA ligase. A 2.8 kb
EcoRI fragment from this modified opa construct was then
excised and re-cloned into pUAS-T (Brand
and Perrimon, 1993
). The second chromosome-linked
P{UAS-opa.VZ}14 (UAS-opa[14]) insertion, as well as the
third chromosome linked P{UAS-opa.VZ}10 (UAS-opa[10]) and
P{UAS-opa.VZ}12 (UAS-opa[12]) insertions, were obtained by
mobilization of UAS-opa[36]
(Robertson et al., 1988
).
Based on the lethality associated with different levels of NGT-driven
expression we estimate that UAS-opa[12], UAS-opa[14] and
UAS-opa[10] are expressed at 2.5-, 3- and 4-fold higher levels,
respectively, than UAS-opa[36]. The UAS-ftz[261] line was
provided by U. Lohr and L. Pick.
Embryo manipulation and in situ hybridization
Embryos were collected as described
(Tsai and Gergen, 1994). For
experiments with temperature-sensitive mutations, embryos were collected for 1
hour at 25°C, grown for 4.5 hours at the permissive temperature of
18°C, and then shifted to the non-permissive temperature of 30°C for
20 minutes immediately prior to fixation for in situ hybridization.
In situ hybridization with digoxigenin-labeled antisense RNA probes was
carried out as described (Klingler and
Gergen, 1993) with the following modifications: embryos were
digested with Proteinase K (30 µg/ml in PBT=PBS + 0.1% Tween-20) for 3
minutes followed by inactivation with glycine (2 mg/ml) in PBT. To further
reduce non-specific binding, embryos were also pre-treated in a 10% (v/v)
solution of heat inactivated goat serum for 1 hour prior to incubation with
the anti-digoxigenin antibody.
The protocol for double-label in situ hybridization using biotin- and
digoxigenin-labeled RNA probes was adapted from that described by O'Neill and
Bier (O'Neill and Bier, 1994),
with the following modifications: embryos were digested with Proteinase K (50
µg/ml in PBT) for 2.5 minutes; hybridization was carried out at 65°C
overnight; post-hybridization washes were carried out in 1% goat serum, 0.3%
deoxycholate, 0.3% triton-X in PBS in place of BSA/PBT; and the
immunohistochemical detection reaction of HRP-conjugated antibodies with
peroxidase and diaminobenzidine was stopped by washing the embryos four times
in HRP buffer (50 mM citric acid, 50 mM ammonium acetate, pH 5).
The plasmid templates used to generate digoxigenin-labeled riboprobes for
odd-skipped (odd), paired (prd) and
slp1 are described in Wheeler et al.
(Wheeler et al., 2002). The
templates for en and ftz are described elsewhere
(Tracey et al., 2000
;
Tsai and Gergen, 1994
). The
biotinylated ftz riboprobe was synthesized with Biotin-21-UTP
(Clontech) in place of digoxigenin-conjugated UTP. The gooseberry
(gsb) probe was synthesized with T7 RNA polymerase using
SalI-linearized BsH9c2
(Baumgartner et al., 1987
). The
hedgehog (hh) probe was synthesized with T3 RNA polymerase
using NdeI-linearized pB:hh[4/1/8.3] (J. Mohler, personal
communication). The wg riboprobe was synthesized with T3 RNA
polymerase using an EcoRI-linearized pwg-12 template. This
pBluescribe plasmid contains a 1.3 kb HindIII + EcoRI
genomic fragment that encodes much of the 4th and 5th exons (N. Baker,
personal communication).
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Results |
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As described above, the 14-striped slp1 pattern is converted into a seven-stripe pattern in the presence of intermediate as well as high levels of NGT-driven Runt (Fig. 1E,F). These seven stripes are broader than the two-cell wide stripes that normally comprise the posterior half of each parasegment. We used double in situ hybridization to investigate the relationship between these broadened slp1 stripes and the expression of ftz, which identifies cells in even-numbered parasegments. The function of Runt as an activator of ftz is revealed by broadened four-cell wide stripes in gastrula stage embryos that have high levels of NGT-driven Runt (Fig. 2E). In wild-type embryos the ftz mRNA pattern is resolved to 2 cell-wide stripes by this stage. The broadened stripes of slp1 and ftz in these embryos are complementary to each other (Fig. 2E). Thus, uniform expression of Runt in the blastoderm embryo leads to activation of slp1 in all cells within odd parasegments while conversely leading to repression in all cells within even parasegments (Fig. 2F). The changes produced at these high levels of Runt may in part be indirect. Indeed, as will be shown below, the broadening of ftz contributes to the repression of slp1 in even-numbered parasegments. Nevertheless, this result provides compelling evidence that Runt has a dual, parasegment-specific role in slp1 regulation.
Runt and Opa cooperate to activate slp1 transcription
Based on the above results we examined the role of all of the other
pair-rule genes in slp1 regulation. A somewhat surprising result from
these experiments is that slp1 expression in odd-numbered
parasegments is lost in opa mutant embryos
(Fig. 3B). The importance of
Opa is surprising as expression of other segment-polarity genes is reduced,
but not eliminated in opa mutants
(Benedyk et al., 1994;
Cimbora and Sakonju, 1995
).
Moreover, Opa is expressed at uniform levels throughout the pre-segmental
region of the embryo, and thus does not provide positional information that
defines the placement of slp1 stripes relative to other pair-rule
transcription factors. As shown above, the odd-numbered slp1 stripes
require Runt, and are interpreted to expand in response to ectopic Runt. We
tested the requirement for Opa in this Runt-dependent activation by examining
slp1 expression in embryos that have high levels of
NGT-driven Runt and that are also mutant for opa. Expression
of slp1 within the pre-segmental region is lost in these embryos
(Fig. 3E). This result
corroborates our interpretation that the expanded slp1 stripes
produced by NGT-driven Runt correspond to the odd-numbered stripes
and further confirms the importance of Opa for Runt-dependent activation.
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Ftz and Runt cooperate to repress slp1
The four-cell wide ftz stripes identify the even-numbered
parasegments of a mid-blastoderm stage embryo. Expression of slp1
arises in the two posterior-most cells of each of these parasegments, i.e. the
cells that lose expression as the ftz stripes narrow during the
process of cellularization. In ftz mutant embryos, slp1
expression is de-repressed to produce six-cell wide stripes
(Fig. 5A). Double in situ
hybridization experiments with en and slp1 (data not shown)
indicate that this pattern is the result of de-repression in the anterior half
of the even-numbered parasegments (Fig.
5B). As described above, slp1 and ftz are
expressed in complementary patterns in embryos that have high levels of
NGT-driven Runt (Fig.
2E). These complementary patterns are due to repression by Ftz as
slp1 is expressed throughout the presegmental region of a
ftz mutant embryo that has high levels of Runt
(Fig. 5C). This pattern
conforms precisely to the expectation for activation by the combined action of
Runt and Opa (Fig. 5D). Based
on these results, as well as on the close correspondence of the responses of
slp1 and ftz to varying levels of Runt and Opa, we conclude
that Ftz prevents the activation of slp1 by Runt and Opa.
|
Ftz regulates the activity of Runt on the segment-polarity genes wg and en
The above experiments were initiated due to the sensitivity and simplicity
of the slp1 response to NGT-driven Runt. Although
wg is less sensitive than slp1, the parallel responses of
these two genes (Fig. 1F,I)
suggest that Ftz and Runt interact in a similar manner to regulate
wg. The one cell-wide wg stripes identify the posterior-most
cells within each parasegment and correspond to a subset of the
slp1-expressing cells (Fig.
6). As observed for slp1, transient elimination of
run leads to loss of wg expression in odd-parasegments and
expanded expression in a subset of even-numbered parasegments
(Fig. 7A). The odd-numbered
wg stripes are specifically repressed by NGT-driven Ftz
expression (Fig. 7B), whereas
co-expression of Runt and Ftz represses wg in both odd- and
even-numbered parasegments (Fig.
7C). Thus, as found for slp1, Runt and Ftz specifically
cooperate to repress wg. However, the rules for Runt-dependent
activation of wg are more complex than for slp1, as
NGT-driven co-expression of Runt and Opa is not sufficient for
wg activation in the anterior unsegmented region of the embryo
(Fig. 7D). Although the full
set of rules for wg regulation thus remains elusive, these results
demonstrate a pivotal role for Ftz in modulating the regulatory effects of
Runt on wg expression.
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Discussion |
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The expression of slp1 (and slp2) differs from several other segment-polarity genes in that the metameric pattern is comprised of two-cell wide, rather than single-cell wide stripes. These two cell-wide stripes comprise the posterior half of each parasegment (Fig. 6). As shown above, slp1 activation in odd-numbered parasegments requires the cooperative action of Runt and Opa, whereas in even-numbered parasegments Runt works together with Ftz to repress slp1 expression. The simple rules involving these three factors fully account for slp1 regulation in all of the Runt-expressing cells in the blastoderm embryo (Fig. 6) but also raise a question regarding the positional cues that regulate slp1 expression in cells that do not express Runt.
There are four other pair-rule transcription factors that could be involved
in slp1 regulation: Eve, Hairy, Odd and Prd. The phasing of the
pair-rule expression domains of these factors are shown in
Fig. 6. Expression of both Odd
and Prd overlaps the slp1 stripes in a manner that suggests that
neither of these factors provides positional information crucial for
slp1 regulation. Consistent with this, there are no substantial
changes in the early 14-striped slp1 pattern in embryos mutant for
either odd or prd (data not shown). By contrast, elimination
of either Eve or Hairy leads to changes in both the number and spacing of the
slp1 stripes. However, as these are both primary pair-rule genes some
of these changes are certainly indirect and due to alterations in Runt and Ftz
expression (Carroll and Scott,
1986; Ingham and Gergen,
1988
; Klingler and Gergen,
1993
).
Several lines of evidence indicate that Eve has a direct role in slp1 repression. Experiments with the temperature-sensitive eve[ID19] mutation indicate that transient elimination of Eve at the cellular blastoderm stage leads to expanded six cell-wide slp1 stripes because of de-repression in the anterior two cells of each odd-numbered parasegment (data not shown). These two are the cells with the highest level of Eve, indicating that the primary role of Eve at this stage is to repress slp1 expression (Fig. 6). Complementary experiments with an inducible hs-Eve transgene reveal that ectopic Eve blocks slp1 activation in both odd- and even-numbered parasegments (data not shown). This result not only confirms Eve's role as a repressor, but also reveals a crucial difference between Eveand Ftz-dependent repression. As shown above, Ftz-dependent repression is restricted to odd-numbered parasegments unless Runt is also ectopically expressed (Fig. 5). This same restriction is observed in experiments with hs-Ftz transgenes (data not shown), indicating that the difference between Eve and Ftz is not due to the mode of ectopic expression. Taken altogether these results indicate that Eve and Ftz normally have comparable roles in repressing slp1 transcription in the anterior half of the odd- and even-numbered parasegments, respectively, in late blastoderm stage embryos (Fig. 6). The key distinction in the regulation of slp1 by these two homeodomain transcription factors is the critical role that Runt plays in Ftz-dependent repression.
One aspect of slp1 expression not accounted for by the above rules
is the factor (or combination of factors), referred to here as factor X, that
is responsible for slp1 activation in the posterior half of the
even-numbered parasegments (Fig.
6). Activation in these cells is blocked either by the combination
of Runt+Ftz (Fig. 5G) or by
ectopic Eve (data not shown). Runt and Ftz are co-expressed anterior to these
even-numbered stripes and presumably play a role in defining the anterior
margin of these stripes. Conversely, Eve is expressed posterior to these cells
and probably has a role in defining the posterior margins of these stripes.
The sole pair-rule transcription factor that remains as a candidate for Factor
X is Hairy, which is expressed in the posterior half of even-numbered
parasegments (Fig. 6). However,
we do not think that factor X is Hairy for several reasons. All of the
evidence to date indicates that Hairy functions as a repressor
(Barolo and Levine, 1997;
Ish-Horowicz and Pinchin,
1987
; Jimenez et al.,
1997
; Poortinga et al.,
1998
; van Doren et al.,
1994
). Furthermore, NGT-driven expression of Hairy does
not lead to slp1 activation in anterior blastoderm cells similar to
that produced by the co-expression of Runt and Opa (data not shown).
Identification of factor X is clearly important for a complete understanding
of slp1 regulation.
Context-dependent activities of Runt
Previous studies indicated that Runt has roles in both activating and
repressing transcription of different target genes in the Drosophila
embryo (Kramer et al., 1999;
Tracey et al., 2000
;
Tsai and Gergen, 1994
;
Tsai and Gergen, 1995
). The
results presented above provide additional compelling evidence for this dual
activity and also provide insight on factors that contribute to this
context-dependent regulation. The dramatic effects of Ftz on Runt-dependent
slp1 regulation clearly demonstrate that one important component of
context is the specific combination of other transcription factors that are
present in a cell. Indeed, the unique and relatively simple rules for
slp1 regulation make this an especially attractive target for
dissecting the molecular mechanisms whereby Ftz converts Runt from an
activator to a repressor of transcription. It seems likely that the rules
governing the Runt-dependent regulation of slp1 will provide a
foundation for understanding the regulation of wg and gsb,
two segment-polarity genes that are expressed in a subset of
slp-expressing cells and that respond to Runt in a manner similar,
but not identical to slp1.
Our results also point to a second important component of context-dependent
regulation by Runt. The specific combination of Runt + Ftz, which represses
slp1, does not always give repression as these same two factors work
together to activate en in some of these same cells at the same stage
of development. Thus, cellular context alone cannot fully account for the
regulatory differences and there must be a target-gene specific component of
context-dependent regulation. A similar gene-specific example of
context-dependent regulation has recently been described for the Runx protein
Lozenge (Canon and Banerjee,
2003). In this case, the presence of binding sites for the Cut
homeodomain protein helps to stabilize a complex that leads to repression of
deadpan transcription in the same cells in which Lozenge is
responsible for activation of Drosophila Pax2. In a strict parallel
of this model, we would speculate that the slp1 regulatory region
contains binding sites for some factor that helps to stabilize a repressor
complex that includes the Runt and Ftz proteins. In a reciprocal, and not
mutually exclusive model, perhaps there are binding sites for a factor in the
en regulatory region that helps to stabilize a Runt- and
Ftz-dependent transcriptional activation complex. Further studies on the
en and slp1 cis-regulatory regions are needed in order to
address these questions at the molecular level. This future work is crucial
for understanding the context-dependent activity of Runt and thus the
molecular logic of the control system that underlies the pair-rule to
segment-polarity transition in Drosophila segmentation.
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ACKNOWLEDGMENTS |
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