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First published online 11 April 2007
doi: 10.1242/dev.003830
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1 Centro de Biología Molecular-C.S.I.C., Facultad de Biología,
Universidad Autónoma-Cantoblanco, 28049 Madrid, Spain.
2 Dpto. Fisiología Animal, Facultad de Biología, Universidad
Autónoma-Cantoblanco, 28049 Madrid, Spain.
* Author for correspondence (e-mail: diazbenjumea{at}cbm.uam.es)
Accepted 11 March 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, nab, squeeze, rotund, Transcriptional co-factors, Proximodistal development, CNS
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). By recruiting co-factors, a DNA-binding
protein can act as co-activator or as co-repressor depending on the context
(Mannervik et al., 1999;
Chinnadurai, 2002). An example
of a co-repressor is the retinoblastoma protein that converts the E2F
transcription factor into a repressor of cell-cycle genes
(Weintraub et al., 1995). The
identification of co-factors and the determination of their precise roles are
crucial for understanding the mechanisms that govern development.
Nab (NGFI-A-binding protein) proteins form an evolutionarily conserved
family of transcriptional regulators. Nab was originally identified in mouse
as a strong co-repressor by virtue of its capacity to interact directly with
the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and
inhibit its activity. Two Nab genes, Nab1 and Nab2, have
been identified in vertebrates. Nab proteins do not bind DNA but they can
repress (Svaren et al., 1998)
or activate (Sevetson et al.,
2000) gene expression by interacting with Egr transcription
factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The
NCD1 domain interacts with the R1 domain of Egr1
(Svaren et al., 1998). The
NCD2 domain is required for transcriptional regulation
(Swirnoff et al., 1998). Mice
harboring targeted deletions of Nab1 and Nab2 have
phenotypes very similar to Egr2 (Krox20)-deficient mice,
suggesting that they act as co-activators of this gene
(Le et al., 2005). In
zebrafish, egr2 controls expression of the Nab gene homologs in the
r3 and r5 rhombomeres of the developing hindbrain
(Mechta-Grigoriou et al.,
2000). Egr2 has been implicated in determining the
segmental identities of r3 and r5 by controlling the expression of several
target genes as well as cell proliferation. Misexpression experiments suggest
that Nab1/Nab2 antagonize Egr2 transcriptional
activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins
might have additional functions as these experiments also led to alterations
of the neural tube not found in Egr2-deficient embryos
(Mechta-Grigoriou et al.,
2000). Conversely, Egr2-deficient mice have a severe
hindbrain segmentation defect that is not found in mice deficient in
Nab1 and Nab2. Nab might also have Egr-independent functions
in mice because, although epidermal hyperplasia has been observed in Nab1
Nab2 double mutant mice, this phenotype has not been observed in mice
lacking any of the Egr proteins (Le et
al., 2005).
In Drosophila, only one Nab gene has been identified; it is highly
homologous to vertebrate Nab genes in the NCD1 and NCD2 domains.
Drosophila nab mutants are early larval lethal. Detection of
nab transcripts by in situ hybridization indicates expression in a
subset of neuroblasts of the embryonic and larval CNS and weak expression in
imaginal discs (Clements et al.,
2003). The role of Nab in Drosophila development is not
known and so far no binding partner has been identified. In this report we
show that nab is a component of the combinatorial code that
determines the number of neurons that express the gene apterous
(ap) in embryonic neural development, and that nab specifies
the Tv neuronal fate in the ap thoracic cluster of neurons.
In early larval development, the wing fate is established in the
distal-most region of the wing disc by a combination of two factors:
activation of the gene vestigial (vg) (Williams et al.,
1991) and repression of the gene teashirt (tsh)
(Ng et al., 1996). Later, in
early third instar larvae, wingless (wg) is activated in a
ring of cells (the inner ring, IR) that borders the vg expression
domain in the presumptive wing region (Fig.
1A). It has been suggested that activation of the IR involves a
signal from the vg-expressing cells to the adjacent cells
(del Álamo Rodríguez et al.,
2002). Interpretation of this signal by the adjacent cells
requires the transcription factors encoded by rotund (rn)
and nubbin (nub) (Ng et
al., 1995) (Fig.
1B,C). Expression of wg in the IR plays a mitogenic role
(Neumann and Cohen, 1996);
hence, as a consequence of wg expression, cells proliferate and the
IR moves away from the vg border
(Fig. 1A'). At a distance
from the source of the signal that drives the initial activation, wg
IR expression is maintained by an autoregulatory loop that involves
homothorax (hth) (Casares
and Mann, 2000). It is thought that an additional mechanism
distally represses wg IR expression and, in so doing, controls cell
proliferation in the wing hinge (del
Álamo Rodríguez et al., 2002;
Liu et al., 2000). In this
report, we show that during imaginal disc development, nab is
strongly expressed in the wing presumptive domain under the control of
vg, and that nab is required in proximodistal axis
development to control the expression of wg in the wing hinge.
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). We
show by pull-down assays that Nab interacts with both proteins via a conserved
C-terminal domain, and present evidence that Nab acts as co-activator of Sqz
in embryo development and as co-repressor of Rn in wing development. Finally,
we propose that there are two mechanisms to repress the activation of
wg expression by Rn in the wing pouch: the first involves Nab as a
co-repressor of Rn; the second involves Sqz as a competitor of Rn for binding
to specific DNA target sites. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fly strains and isolation of the EP#13 line
Fly stocks used were: nabSH143lacZ
(Oh et al., 2003),
nabGal4NP3537 and nabGal4NP1316 (Gal4
Enhancer Trap Insertion Database), sqzlacZ02102, sqzGal4
and UASsqz#7.2 (Allan
et al., 2005), Canton-S, y w1118,
w1118; 
2-3 Sb/TM2, UASGFP, y
w1118 hsFLP122; UbiGFP FRT80/TM2,
UASGFP, (Bloomington Drosophila Stock Center), y
w1118 hsFLP122; Act5C>y+>Gal4
UASGFP (Ito et al.,
1997), DllGal4MD23 and
nubGal4AC62 (Calleja et
al., 1996), dppblkGAL4
(Wilder and Perrimon, 1995),
rn89 (Couso and Bishop,
1998), rn5 and rn20
(Agnel et al., 1989),
UASrn and rnGal4 (St
Pierre et al., 2002), UASvg
(Kim et al., 1996),
vg83b27r (Williams et al., 1993), y
w1118; CyO, EP#720/dppd12
(Rørth et al.,
1998).
EP#720 (inserted in a CyO chromosome) was used as a starting line and nubGal4AC62 was used as a driver in an F1 screen for dominant phenotypes in adult wings. 69,000 flies were scored and two lines with the same phenotype were selected: EP#13 and EP#29. Both EP lines have insertions in the same gene.
Analysis of genetic mosaics
To induce loss-of-function clones, embryos from crosses: (1) y w
hsFLP122; UbiGFP FRT80 females and either nab
FRT80/TM6, Tb or nab FRT80
sqzlacZ02102/TM6, Tb males; and (2) y w
hsFLP122; FRT82 UbiGFP females and FRT82
sqzlacZ02102/TM6 males, were collected over 24 hours
and heat shocked at 37°C for 1 hour in a water bath at 36±12 hours
of development. To induce clones of ectopic expression, y w1118
hsFLP122; Act5C>y+>Gal4 UASGFP
females were crossed either with UASvg, UASnab, UASsqz, UASrn or
UASrn854 males. Embryos were
collected after 24 hours and heat shocked at 34.5°C for 12 minutes in a
water bath at 36±12 hours of development.
In situ hybridization and antibody staining
Standard in situ protocols were used to examine nab and
sqz expression (Tautz and
Pfeifle, 1989). Imaginal discs were fixed and stained for confocal
microscopy following standard protocols. Primary antibodies used were: rat
anti-Ap (1:200)
(Fernández-Fúnez et al.,
1998); mouse anti-ß-galactosidase (1:2000; Promega Z3781);
guinea pig anti-Dimm (1:500) (Allan et al.,
2005); rabbit anti-FMRFa (1:200; Biotrend); rabbit anti-Nab
(1:500; described below); mouse anti-Wg (1:25; Developmental Studies Hybridoma
Bank).
Antibody production
To generate the Nab antibody, two rabbits were immunized with a 6xHis
fusion of the complete Nab protein. After three immunizations, the rabbits
were bled and sera tested on imaginal discs. The two sera gave rise to the
same expression pattern. We confirmed that the antibody recognized Nab by
immunolabeling dppGal4/UASnab wing discs. The expression
patterns revealed by the antibodies were identical to those obtained with the
nabSH143lacZ and nabGal4 lines NP1316
and NP3537.
Mapping of EP insertion lines
The EP element contains 14 Gal4 target sites and is described by
Rørth (Rørth,
1996). A molecular map of the EP#13 insertion site was constructed
by inverse PCR using primers Pry1 and Pry4, as described at the Berkeley
Drosophila Genome Project website
(http://www.fruitfly.org/about/methods/index.html).
Sequencing of the flanking DNA indicated that the P element was inserted at
position 4144528, 86 bp upstream of the nab transcription initiation
site.
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Generation of UASnab and UASrn894
A complete cDNA from the EST LP22227 sequence was cloned into the pUAST
vector and transgenic lines were generated by P-element transformation
(Spradling and Rubin, 1982).
Insertions were tested both by nab RNA in situ hybridization and Nab
antibody staining using dppGal4 as driver. Nab expression was
stronger in the UASnab lines than in the EP#13 insertion.
UASrn894 was generated by
cloning the rn894 fragment into
the pUAST vector.
In vitro GST pull-down assays
For protein interaction assays we used the following procedure:
[35S]-labeled Rn, Sqz and Rn894 were cloned into
pCDNA3 tagged with Flag (Invitrogen) and transcribed/translated with the TNT
Coupled Reticulocyte Lysate System (Promega) and [35S]-L-Met
(Amersham Pharmacia Biotech). nab was cloned into pGEX-4T-2 (GST)
(Amersham Bioscience Research) and purified from bacterial cells that had been
induced by IPTG and incubated with glutathione resin. The
resin-binding/washing buffer contained 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1
mM EDTA, 0.5% NP40, 2 mM DTT. Rn894 was generated by in
vitro site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit,
Stratagene #200518) by the exchange of Asn894 and Lys895 for two stop codons
(TAG). The interaction assay between Nab-GST and a synthetic peptide
containing the 32 amino acids of the C-terminal domain of Rn was run in a
Tricine-SDS-PAGE gel for small proteins as previously described
(Schägger and von Jagow,
1987).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Neumann and Cohen,
1996). By inverse PCR, we identified a unique insertion 86 bp
upstream of the transcription initiation site of nab
(Fig. 1E). We confirmed by RNA
in situ hybridization that this gene (CG33545/LP22227) is misexpressed in
dppGal4>EP#13 wing discs (data not shown). nab was
originally identified in a screen of an embryonic cDNA library using cDNA
fragments from domains NCD1 and NCD2 as probes. Northern analysis revealed a
single transcript encoding a predicted protein of 569 amino acids
(Clements et al., 2003).
Several P-element insertions have been identified in nab, including
the larval lethal insertion P(lacW)l(3)SH143 in the first exon
(Oh et al., 2003), and several
P(GawB) insertions a few nucleotides upstream of the transcription
initiation site (GETDB) (Fig.
1E), one of which, NP3537Gal4, is larval lethal. We were
able to rescue the lethality of the heterozygous combination
nabl(3)SH143/NP3537Gal4 with EP#13, which drives
the gene controlled by the EP#13 insertion in the nab expression
pattern. This result confirms that nab is misexpressed in EP#13. We next analyzed the expression pattern of Nab (Fig. 1F,F''). Antibody against Nab (see Materials and methods) revealed a low level of expression in all imaginal discs. In late third instar wing discs, Nab was strongly expressed in a circular domain that delimits the expression of wg in the IR. Nab expression was first detected in early third instar larvae, in a group of cells of the distal-most wing, and was maintained throughout the remainder of the larval and pupal stages. There was a low level of expression in the rest of the wing disc, except in the hinge where there was no detectable expression. In the eye disc, Nab was detected in a stripe corresponding to the morphogenetic furrow (data not shown).
Vestigial controls nab expression in the wing
We next asked whether, as with other genes involved in proximodistal
patterning, nab expression in the wing was dependent upon
vg. No expression of nab was detected in the distal wing of
vg83b27r wing discs
(Fig. 2A). However,
nab was ectopically expressed in clones of vg-expressing
cells (Fig. 2B,B').
Together, these results indicate that the expression of nab in the
wing depends on vg. In wild-type discs and vg
ectopic-expressing clones, the domain of nab expression is broader
than that of vg, pointing to the nonautonomous control of
nab expression. A similar mechanism has been proposed for other
genes, such as rn and nub, whose expression depends on
vg (del Álamo
Rodríguez et al., 2002). Expression of vg in the
wing starts in second instar larvae, whereas nab expression is first
detected at early third instar. This suggests that some other mechanism
controls the initiation of nab expression.
nab delimits the expression of wg in the wing inner ring
The nabSH143 allele is a P(lacW) insertion in
the first exon. Most larvae homozygous for this allele die in first instar.
Thus, to analyze the role of nab in development of the wing we
generated nabSH143 homozygous mutant clones by mitotic
recombination using the FLP/FRT mitotic recombination system (Xu and Rubin,
1993). In the wing, these clones activated wg ectopically
(Fig. 3A). However, we noted
that not all the clones activated wg (29%; total number of clones
scored=78). It is therefore possible that this allele has some residual
function. In order to isolate new mutant alleles of nab we looked for
imprecise excisions of the EP#13 insertion and identified several new lethal
alleles. We obtained the same results as before with homozygous mutant clones
of the new allele nabR52. As we were not able to detect
any Nab protein in clones of nabSH143 or
nabR52 (data not shown), we conclude that these are very
strong or null alleles. The possibility of functional redundancy between Nab
and other proteins is analyzed below.
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), and the spd enhancer, which drives wg
expression in the IR (Neumann and Cohen,
1996). Previous results suggest that activation by the latter
depends on a nonautonomous signal coming from the vg-expressing cells
(del Álamo Rodríguez et al.,
2002; Liu et al.,
2000). nab co-expresses with wg in the wing
margin and abuts on wg expression in the IR
(Fig. 1F''). We therefore
assumed that Nab should repress activation of the IR enhancer derepressed in
nab clones. To obtain independent evidence that the IR enhancer is
being activated, we tested whether other genes activated in the wing margin
were activated in the nab clones. To this end, we analyzed
cut (ct) and detected no ectopic expression (data not
shown). It has been reported that wg expression can be detected in
the wing after induction of cell death
(del Álamo Rodríguez et al.,
2004; Pérez-Garijo et
al., 2004). To detect cell death in the nab clones we
made use of an antibody that recognizes the activated form of Caspase 3 (Decay
- Flybase) (Thornberry and Lazebnik,
1998), but detected no cell death (data not shown). These results,
together with the pattern of expression
(Fig. 1F',F''),
strongly suggest that the IR enhancer is being activated in the nab
clones and, therefore, that in normal development Nab acts as a repressor of
the wg IR enhancer in the distal wing. To confirm this hypothesis we
expressed nab ectopically in the IR domain using the nubGAL4
driver, which is expressed in a circular domain that includes the IR
(Fig. 3B). In
nubGAL4>UASnab larvae, expression of wg in the
IR was lost, whereas its expression in the wing margin was not affected
(Fig. 3C). We also generated
clones of nab-expressing cells and found that wg expression
was cell-autonomously lost in these clones, whereas wg expression in
the wing margin was not affected (Fig.
3D,D'). In the light of these results, we propose that the
function of nab in wing development is to delimit, distally, the
domain of wg expression in the IR by inhibiting the mechanism of IR
activation.
The Rn zinc-finger transcription factor is a potential partner of Nab in wing development
The mammalian Nab partner Egr1 contains an inhibitory domain called R1.
When this domain is deleted the transcriptional activity of Egr1 increases
15-fold (Gashler et al., 1993;
Russo et al., 1993). It has
been shown that the R1 domain mediates a functional interaction between Nab
and Egr1. Since no R1 domain has been identified in the fly genome and all the
previously identified partners of Nab are Krüppel-type zinc-finger
transcription factors, we examined, as potential Nab partners in the fly,
transcription factors of the Krüppel family expressed in the wing. The
gene rn encodes a Krüppel-like zinc-finger protein
(St Pierre et al., 2002) that
in the wing is expressed in a circular domain slightly broader than the
nab domain (Fig.
1F''). The wg IR enhancer is only active in the
cells that express rn and that do not express nab. Previous
studies have shown that Rn is required for activation of the spd
enhancer (del Álamo
Rodríguez et al., 2002). Our results so far suggest that Rn
could be a partner of Nab in the wing: first, nab is expressed in the
rn-expressing cells that do not express wg; second,
nab loss-of-function clones contain ectopic Wg; and third,
nab misexpression represses the wg IR enhancer.
rn was also expressed in leg discs in a broad ring that
corresponded to three tarsal segments (T2-4)
(Fig. 4A). In rn
mutant legs, the T2-4 tarsal segments were deleted
(Fig. 4B). We would therefore
expect that if Rn were a partner of Nab, ectopic expression of nab in
the leg would generate the same phenotype as the lack of Rn. This proved to be
the case when nab was misexpressed in the rn expression
domain under the control of the rnGal4 driver
(Fig. 4C). The phenotype of
these flies was indistinguishable from the rn mutant phenotype in
both legs and wings (compare Fig. 4B with
C). We examined the specificity of this interaction by rescuing
the phenotype caused by nab misexpression by co-expressing
rn (rnGal4>UASrn+UASnab), as well as by
misexpressing nab in a broader domain using Distal-less Gal4
(DllGal4), which is expressed from mid-tibia to distal leg
(DllGal4>UASrn). In the first experiment, the phenotype
was markedly reduced in both wing and leg (compare
Fig. 4B with D), indicating
that adding more rn antagonizes the inhibitory effect of nab
misexpression. In the second experiment, although nab was
misexpressed in a broader domain of the leg, the phenotype was unaltered and
was restricted to the area where rn was expressed (compare
Fig. 4B with E). Taken
together, these results support a role for Rn as a potential partner of Nab
and that Nab acts as co-repressor of Rn function in the cells where both are
expressed. The rn mutant phenotype in the wing is caused by the loss
of wg expression in the IR (del
Álamo Rodríguez et al., 2002). We wanted to check
whether wg expression was affected in rnGal4 UASnab and
rnGal4 UASnab UASrn wings. In the first case, the IR was found to be
absent (Fig. 4F), whereas in
the second it was partially restored (Fig.
4G). In summary, the results presented here indicate that Nab
functions in wing development by antagonizing the transcriptional activation
function of Rn.
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|
),
C. elegans LIN-28 (Rougvie and
Ambros, 1995) and rat Ciz
(Nakamoto et al., 2000). Sqz
and Rn have two highly homologous domains: the zinc-finger domain (90%
identity) and a 32 amino acid C-terminal domain (over 80% identity).
sqz mutant alleles are larval lethal and have a motility defect.
sqz is first required in embryonic CNS development to define the
number of cells that express the LIM-homeodomain gene ap in the
ap thoracic cluster of interneurons. Later on, it is also involved in
the combinatorial code of transcription factors that specifies the fate of the
Tv neuron in the ap cluster. The Tv neuron is distinguished from the
rest of the neurons in the cluster by the fact that it contains the
neuropeptide FMRFa [FMRFamide-related (Fmrf) - Flybase]. In sqz
mutant embryos, additional ap-expressing neurons are generated and
the Tv neuron is not specified as no FMRFa expression is found
(Allan et al., 2003). To
determine whether Nab is a co-factor of Sqz, we first analyzed the expression
of nab and sqz in stage-17 embryos. We found that a subset
of the CNS neurons that expressed sqz also expressed nab,
whereas other neurons expressed either sqz or nab
(Fig. 5A,B). Two or three
neurons in the ap cluster of stage-17 embryos expressed nab,
one typically at a relatively high level of expression
(Fig. 5C,C'). By the
first instar larval stage only one neuron in the ap cluster expressed
nab. By double staining with anti-FMRFa and anti-Nab we were able to
identify this as the Tv neuron (Fig.
5D,D'). At this stage, sqz was expressed at high
levels in the Tv neuron and at low levels in two other neurons of the
ap cluster. We next analyzed the expression of ap and
FMRFa in nab mutant larvae. In first instar
nabSH143 larvae, we found additional
ap-expressing neurons in the ap cluster
(Fig. 5E,E',H). In
nabSH143 embryos, additional cells expressed the bHLH gene
dimmed (dimm), as shown for sqz mutants
(Hewes et al., 2003)
(Fig. 5E,E',H). We also
examined the expression of FMRFa in the ap clusters of first
instar larvae and found that FMRFa staining was lost or reduced in all the Tv
neurons, mainly in the T1 cluster (Fig.
5G,G',I). We conclude that lack-of-function alleles of
nab and sqz generate the same embryonic phenotypes: the
number of ap-expressing cells in the ap thoracic clusters is
increased, additional dimm-expressing neurons are detected in the
clusters, and Tv neuronal fate is absent. These results strongly suggest that,
unlike the situation in imaginal disc development where Nab acts as a
co-repressor of Rn, in CNS development Nab is required as a co-activator of
Sqz.
|
894). The ability of
Nab-GST to retain the [35S]Rn894 was notably
reduced. We conclude that this conserved domain mediates the direct
interaction of Nab with Rn and Sqz. To further test whether the C-terminal
domain is sufficient to mediate this interaction, we incubated the Nab-GST
with a 32 amino acid peptide containing just the sequence of the C-terminal
domain. Nab-GST did not retain the peptide, indicating that the C-terminal
domain is not sufficient to mediate Nab-Rn interaction (data not shown). As we
have not identified other conserved domains between Rn and Sqz than the
zinc-finger and C-terminal domains, we consider that either secondary
structure or an additional modification of the protein is required for binding
Nab. In order to provide an in vivo functional test of this hypothesis, the
rn894 fragment was cloned into
the pUAST vector and clones of cells misexpressing
UASrn894 were generated
(Act>Gal4>UASrn894).
These clones activated the expression of wg throughout the wing pouch
(Fig. 6D). As a control
experiment, we misexpressed the wild-type version of rn
(Act>Gal4>UASrn). These clones only activated
wg expression in the wing hinge, outside of the nab
expression domain (Fig.
6E).
Sqz competes with Rn in wing disc development
We wished to ascertain whether sqz is expressed in the wing disc.
Because of the high degree of sequence homology between rn and
sqz and to avoid interference with the rn mRNA present in
the wing, we performed an in situ hybridization assay in rn mutant
discs. sqz expression was detected by in situ hybridization in
rn20 wing discs in a circular pattern that faded off
laterally and whose proximal limit coincided with the limit of vg
expression; this corresponded to the distal-most wing fold
(Fig. 7A; compare with
Fig. 1B). To determine whether
sqz plays a role in wing development we analyzed the phenotype of
sqz mutant clones induced by mitotic recombination. These clones had
no adult phenotype, nor did they alter the expression of wg. Since
Sqz and Rn share zinc-finger and the C-terminal domains and differ in their
N-terminal domains, we wondered whether the roles of Sqz and Nab might be
functionally redundant, both repressing Rn activity but by different
mechanisms: Nab would repress Rn activity by direct binding to Rn protein as a
co-repressor, whereas Sqz would compete for binding to the same DNA targets.
To test this hypothesis, we analyzed the effect of misexpressing sqz
in the rn expression domain. rnGal4/UASsqz UASGFP
flies had small deletions of the wing hinge and shortened legs
(Fig. 7B,C), a phenotype that
resembles the nab misexpression and rn mutant phenotypes
(compare with Fig. 4B,C). In
agreement with these results, wg expression in the IR was
downregulated in rnGal4/UASsqz wing discs
(Fig. 7D). An alternative
explanation for these results is that sqz activates nab
expression, but we did not detect nab misexpression in this
experiment. We suggest that there must be some functional redundancy,
irrespective of whether Nab and Sqz play similar roles in the wing by
repressing Rn activity, and this would account for the low penetrance of the
nab mutant clones. Because nab and sqz map on
different chromosome arms it was not possible to generate double-mutant
clones. We therefore generated nabSH143 homozygous clones
in a sqzlacZ/+ background. In this situation, the
frequency of clones misexpressing wg increased (38%; total number of
clones scored=55). We also noted that the clones that showed wg
misexpression were preferentially located in the lateral-most regions of the
wing, which correspond to the regions with the lowest levels of sqz
expression. Taken together, these observations support the hypothesis that Nab
and Sqz play similar roles in wing development: Nab as a co-repressor of Rn
via its conserved C-terminal domain, and Sqz by competing with Rn for binding
to its DNA targets. This function of Sqz would differ from its above-proposed
role as a transcriptional activator in CNS development, and would not require
Nab.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic evidence suggests that Nab is a repressor of Rn function. Rn has
previously been implicated in two aspects of imaginal disc development:
activation of the wg IR enhancer in the wing hinge, and development
of the tarsal segments of the leg. nab misexpression phenocopies
rn loss-of-function in both instances. Moreover, misexpression of
nab outside the normal rn expression domains produces no
phenotype, and the phenotype of nab misexpression is substantially
reduced by simultaneous overexpression of rn. Since nab is
not expressed in the leg, we think that the only function of nab in
imaginal disc development is in the distal wing. In this tissue, rn
and nab are expressed in concentric circles under the control of the
gene vg. Nevertheless, the expression domain of rn is
slightly broader than that of nab. As a result, these two circles
define a ring of cells that express rn but not nab, and it
is precisely in these cells that the wg IR enhancer is activated.
nab misexpression in these cells represses wg, whereas
nab loss-of-function in its expression domain causes misexpression of
wg. These findings, together with the misexpression experiments and
the nab expression pattern, strongly support the proposed role of Nab
as a co-repressor of Rn. Interestingly, wg is not misexpressed in all
the nab loss-of-function clones. There are two possible reasons for
this: first, Sqz might act as a competitor of Rn for DNA binding (see below);
and second, nab loss-of-function might not be sufficient to produce a
complete transformation of the hinge region and full activation of the IR
enhancer. It is important to observe that both rn and nab
are targets of vg, but they are expressed in circular domains of
different sizes. This is probably due to rn being expressed earlier
than nab. This difference in the expression domains permits and
delimits the activation of wg to a narrow ring of cells, which is
crucial for the correct development of the wing. Other genes that play
important roles in wing development, such as nub
(Ng et al., 1995),
dve (Koelzer et al.,
2003; Nakagoshi et al.,
2002) and the vg quadrant enhancer (Williams et al.,
1993), are also expressed at different times in late second and early third
instar larvae. The mechanisms by which the expression of these genes is
temporally controlled are not known.
We have also presented evidence that Nab is a co-activator of Sqz. This
protein has been implicated in two aspects of embryonic ventral nerve cord
development: first, in a Notch-dependent lateral inhibition mechanism that
specifies the number of cells that express ap in the ap
thoracic neuronal cluster; and second, in the specification of the Tv neuronal
fate. nab and sqz are co-expressed in a subset of neurons,
including several of the ap cluster, as well as the Tv neuron.
nab loss-of-function embryos reproduce all the phenotypes of
sqz loss-of-function embryos: additional cells express ap in
the cluster and the Tv neuronal fate is lost. In addition, in both
nab and sqz mutants an increased number of cells in the
clusters express dimm. These findings indicate that Nab is required
for all identified Sqz functions in embryonic development. Although we have
focused our analysis on the ap thoracic cluster of neurons, both
sqz and nab are co-expressed in many cells in the ventral
nerve cord and others expressed either sqz or nab. But no
other functions have been identified for sqz and it is not known how
the expression of sqz is controlled. It has been reported that the
expression of nab in the ventral nerve cord depends on the gene
castor (Clements et al.,
2003). Thus, the results presented here reveal greater complexity
in the mechanisms of neuronal fate specification. The combined expression of
genes, whose expression is individually activated by different mechanisms, is
required to determine specific neuronal fates.
Sqz and Rn share two regions of strong homology: the zinc finger and a
stretch of 32 amino acids in the C-terminal domain. By contrast, only
rn has a long N-terminal domain. Our results indicate that the
C-terminal domain mediates the interaction with Nab. By GST pull-down assays,
we have shown that Nab binds to the full-length Rn protein but not to the
Rn894 version, and clones of cells misexpressing
rn894 activate wg
expression in the nab expression domain. The similarity between
sqz misexpression and rn loss-of-function phenotypes in leg
and wing suggests that Sqz acts like a dominant-negative form of Rn in the
rn domain: both proteins would bind to the same target sites but have
opposite effects, and our results indicate that this role of Sqz would not
require interaction with Nab. It is possible that the long N-terminal region
of Rn is involved in interaction with other partners specifically required for
Rn function.
Thus, our results indicate that Nab has a dual role as co-repressor of Rn
and co-activator of Sqz. Previous studies in vertebrates also suggest that Nab
is involved in both repression and activation of transcription. Co-repressors
are proteins that bridge the interaction of the repressor with its target. Two
main co-repressors have been identified in Drosophila: Groucho and
CtBP. CtBP binds to a specific sequence motif (P-DLS-K) that has been found in
the sequence of three repressors present in the early embryo: Snail, Knirps
and Krüppel. All three are zinc-finger transcription factors, and genetic
evidence suggests that they all require CtBP to repress their targets
(reviewed by Chinnadurai,
2002). Neither Rn nor Sqz have a CtBP-binding motif but we have
identified one in Nab (P-DLS--K). Although the functional significance of this
motif remains to be confirmed, we suggest that Nab is acting as a bridge
between Rn and CtBP.
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ACKNOWLEDGMENTS |
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