1 Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco,
28049 Madrid, Spain
2 HHMI, Department of Molecular and Human Genetics, Baylor College of Medicine,
One Baylor Plaza, Houston, TX 77030, USA
Author for correspondence (e-mail:
jmodol{at}cbm.uam.es)
Accepted 4 January 2005
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SUMMARY |
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Key words: charlatan, Zn-finger transcription factor, achaete/scute, Proneural genes, ASC (AS-C), Bristle development, Drosophila
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Introduction |
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A key step of this process is the expression of the proneural genes
achaete (ac) and scute (sc) in groups of
cells, the proneural clusters, that prefigure the sites of the future
macrochaetae (Cubas et al.,
1991; Romani et al.,
1989
; Skeath and Carroll,
1991
). These genes, members of the achaete-scute complex
(ASC) (reviewed by Campuzano and Modolell,
1992
; Ghysen and
Dambly-Chaudière, 1988
;
Ghysen and Dambly-Chaudière,
1989
), encode transcriptional factors of the basic
helix-loop-helix (bHLH) family. These factors confer to cells the potential to
become SOPs, presumably by implementing neural differentiation programs. From
each proneural cluster, a fixed number of SOPs are born, usually one or two.
The proneural clusters of the wing imaginal disks (the precursors of each
heminotum, wing and mesothoracic pleura) not only appear in constant
positions, but each of them has a characteristic size, shape and time of
appearance and disappearance (Cubas et
al., 1991
; Skeath and Carroll,
1991
). Moreover, a typical cluster that gives rise to one bristle
may consist of 20 to 30 cells, but the SOP is selected from a smaller subgroup
of cells that accumulate higher levels of Ac-Sc proteins than their neighbors,
which constitute the proneural field
(Cubas et al., 1991
;
Cubas and Modolell, 1992
;
Skeath and Carroll, 1991
).
This subgroup and the SOP, which accumulates the highest levels of Ac-Sc,
always occupy the same position within the cluster. Hence, the expression of
ac/sc in proneural clusters is exquisitely regulated.
The regulation of ac/sc is effected by means of two classes of
cis-regulatory sequences, namely, cluster-specific and SOP-specific
enhancers. The first type normally directs expression of both ac and
sc in one specific proneural cluster and defines many of its
characteristics, such as position, size and shape. These cluster-specific
enhancers appear to be controlled by local combinations of transcription
factors that together form a prepattern (reviewed by
Ghysen and Dambly-Chaudière,
1988; Gómez-Skarmeta et
al., 2003
). Expression occurs only at sites with the appropriate
combinations of factors. Although in a few cases some of the prepattern
factors have been identified, most of them remain unknown. Moreover, we still
lack a clear understanding of how the inputs of the prepatterning factors are
integrated into the patterns of proneural gene expression characteristic of
each cluster.
The second type of enhancer mediates the strong expression of proneural
genes in SOPs (Culí and Modolell,
1998) by allowing self-stimulatory loops of expression of ac,
sc and asense (ase), another bHLH member of the ASC
(Brand et al., 1993
;
Domínguez and Campuzano,
1993
; Jarman et al.,
1993a
). The activation of these loops in one of the cells of the
proneural field is an early and essential step of SOP commitment. This loop is
also dependent on the presence of the Senseless (Sens) protein
(Jafar-Nejad et al., 2003
).
The SOP-specific enhancers are also the targets of the inhibitory interactions
that occur within the cells of the proneural cluster mediated by the Notch
signaling pathway via E(spl) proteins
(Culí and Modolell,
1998
; Giagtzoglou et al.,
2003
). By antagonizing these enhancers, N signaling, activated by
Ac/Sc in the cells of the cluster, maintains them in a non-SOP state (mutual
inhibition) (reviewed by Artavanis-Tsakonas
et al., 1995
). However, in a little-understood process, one cell
of the proneural field escapes this inhibition, starts the proneural
self-stimulatory loop and becomes an SOP. The developing SOP then signals via
Notch in order to impede the remaining cells of the field from becoming SOPs
(lateral inhibition) (Heitzler and
Simpson, 1991
; Simpson,
1990
; Simpson,
1997
). These SOP-specific enhancers are also the targets of
positive interactions between the cells of proneural clusters mediated by the
EGFR, which is necessary for the emergence of the SOPs of the notum
macrochaetae (lateral cooperation)
(Culí et al., 2001
). To
prevent the determination of excess SOPs from a proneural cluster, the levels
of EGFR signaling must be regulated. This event seems to be accomplished in
part by a negative effect on EGFR signaling of the N-mediated interactions
that occur among cells of the proneural cluster.
The ac, sc and ase genes are also necessary for the
formation of the external SOs of embryos and larvae
(Dambly-Chaudière and Ghysen,
1987). The process is similar to that in the imaginal disks
(Ruiz-Gómez and Ghysen,
1993
). Other proneural genes are responsible for the development
of the internal chordotonal organs (atonal)
(Jarman et al., 1993b
) and
other neurons of the larval peripheral nervous system (PNS) (amos)
(Huang et al., 2000
;
Villa-Cuesta et al.,
2003
).
Here we report the identification of a novel gene, charlatan (chn), which is involved in the development of the adult pattern of macrochaetae. chn defines a new level of control of ac/sc that is intermediate between the prepattern genes and the ac/sc self-stimulation mediated by the SOP-specific enhancers. Thus, chn, which encodes a zinc finger transcription factor, is activated by ac/sc in the proneural clusters of the wing disk. In turn, chn stimulates the expression of ac/sc in these clusters. This enhanced expression facilitates the formation of SOPs. Our data indicate that the Chn protein reinforces the expression of ac/sc by acting, probably directly, on the proneural cluster-specific enhancers of the ASC. chn is also required for correct development of the embryonic/larval PNS, as its absence removes neurons and causes malformations of chordotonal organs.
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Materials and methods |
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Molecular biology
UAS-chn was prepared by subcloning the cDNA of CG11798
from clone SD05496 (BDGP) into the pUASt vector
(Brand and Perrimon, 1993).
UAS-bda was prepared by subcloning a PCR product containing the
entire ORF of bda that was obtained using genomic Oregon R DNA as
template. The PCR product was sequenced to confirm the fidelity of
amplification. To prepare UAS-chni, a 400 bp fragment of chn
cDNA was amplified by PCR using 5'-GGGATCCCAAGCGGCTGCAGCTGC-3'
upper primer and 5'-TGGAAGCTTCAACTCGTGCACGCC-3' lower primer. The
PCR product was cloned as a BamHI-KpnI fragment in the pHIBS
vector (Nagel et al., 2002
),
to make the pHIBS-chn construct. A BamHI-SacI fragment from
pHIBS-chn was subcloned in the pBluescript vector to generate pBS-chn. A
KpnI-SalI fragment from pHIBS-chn and a
KpnI-SalI fragment from pBS-chn were cloned in opposite
directions in the pUASt vector, thus forming the final RNAi construct. All the
UAS constructs were injected into y w embryos to obtain transgenic
Drosophila lines by standard procedures
(Rubin and Spradling,
1982
).
All overexpression experiments were carried out at 25°C, except for the flies shown in Fig. 1D,E,F, which were raised at 18°C, and those shown in Fig. 1G,H,I, which were cultured at 18°C, shifted to 25°C at 72 hours after egg laying, and returned to 18°C at puparium formation.
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Mosaic analyses
To generate clones of cells mutant for chn, either y w
hs-FLP122, f36a; ck Pf[+] FRT42D/CyO or y w
hs-FLP122; P[ubi-GFP], FRT42D/CyO females (stocks described in FlyBase)
were crossed with w; chnECJ1, FRT42D/CyO males.
Recombination was induced by heat treatment at 37°C for 30 minutes
(Xu and Rubin, 1993). To
generate clones of cells overexpressing UAS-chni, males carrying this
transgene were crossed with y w FLP122; act-FRT
y+ FRT-Gal4 UAS-GFP / SM6a-TM6b Tb females
and recombination was induced by incubation at 37°C for 10 minutes.
Histochemistry
Antibody staining was performed as described
(Cubas et al., 1991). Primary
antibodies were: anti-Sens (Nolo et al.,
2000
), anti-Sc (Skeath and
Carroll, 1991
), anti-Ato
(Jarman et al., 1995
), mAb
22c10 (Zipursky et al., 1984
)
and anti-ß-galactosidase (Cappel). A guinea pig anti-Chn antibody was
prepared against a His-tagged Chn fragment corresponding to amino acids
213-417 cloned into the pET28a vector. However, it only allowed clear
visualization of the Chn protein under overexpression conditions
(Fig. 5N,O). Secondary
antibodies were from Jackson and Amersham. In-situ hybridizations to detect
chn mRNA were performed as described
(González-Crespo and Levine,
1993
) using an antisense DIG-labeled RNA probe.
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Results |
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Overexpression of chn causes supernumerary bristles
When driven by en-Gal4, the EPIL6 insertion induced the
overexpression of both chn and bda transcripts in wing
imaginal disks (not shown). Hence, we created flies carrying either a
UAS-chn or a UAS-bda transgene. Overexpression of
UAS-bda using several drivers (MS1096-Gal4, C765-Gal4,
ap-Gal4 and MS248-Gal4) did not cause noticeable phenotypic
effects (data not shown). By contrast, overexpression of UAS-chn with
these and other drivers gave rise to extra bristles. Ubiquitous expression
with the C765-Gal4 driver
(Gómez-Skarmeta et al.,
1996) caused the appearance of many macrochaetae near wild-type
bristles, and it increased the density of microchaetae [141±10
microchaetae per female heminotum versus 103±7 in Oregon R controls
(averages of 10 heminota) and Fig.
1D]. However, the bristles were always separated by epidermal
cells, suggesting that Notch-mediated lateral inhibition
(Artavanis-Tsakonas et al.,
1999
) was still active. With earlier-expressing drivers such as
MS248-Gal4 (Cavodeassi et al.,
2002
), overexpression gave rise to many bristles (see
Fig. 4D). In addition, this
early expression reduced the size of the heminota and interfered with their
dorsal fusion. With the appropriate drivers many extra bristles appeared on
other regions of the fly body, including wings (C765-Gal4,
MS1096-Gal4 and nub-Gal4), head (MS248-Gal4) and the
metathorax (MS1096-Gal4) (data not shown).
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chn is expressed in the PNS and the CNS
We examined the patterns of expression of chn in embryos and
imaginal disks using in-situ hybridization. In early blastoderm stages, the
expression of chn was ubiquitous, but before stage 5, chn
mRNA disappeared from the poles of the embryo and faint stripes became visible
(data not shown). At stage 5, chn mRNA also accumulated in the dorsal
region, cephalic furrow and in the presumptive mesoderm
(Fig. 2A,B). At stage 11,
chn mRNA was found mostly in the mesoderm
(Fig. 2C), and in ectodermal
patches between the tracheal pits (Fig.
2D,E), where neurons of the PNS appear
(Ruiz-Gómez and Ghysen,
1993). Older embryos (stage 15,
Fig. 2F) showed strong
expression, which was mostly restricted to the central nervous system (CNS)
and PNS. In the latter case, the pattern suggested that expression occurred in
many of the neurons of the ventral, lateral and dorsal clusters of neurons
(Fig. 2F,G; compare with
Fig. 3A).
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Loss of chn causes loss of PNS elements
To examine the effects of the removal of chn, we obtained new LOF
alleles by generating imprecise excisions of the Pl(2)42/18
insertion. One of these, chnECJ1, is probably a null, as
the excision removed at least part of the promoter region of chn
(Fig. 1A), and homozygous
embryos lacked the chn mRNA (as detected by in-situ hybridization,
not shown) and died as embryos. In keeping with the expression of chn
in the cells of the PNS, chnECJ1 embryos displayed
conspicuous anomalies in PNS cells. These included the absence of many
neurons, especially in the dorsal and ventral clusters, and an abnormal
morphology of chordotonal lateral neurons, which appeared bunched and lacked
the typical apical dendrites (Fig.
3A,B, insets). Individually identifiable neurons such as the
v'chn1 and the dbp were generally absent
(Fig. 3B). Some of these
defects were similar to the phenotype described for the original
Pl(2)42/18 insertion (Kania et
al., 1995) but were more severe. Ubiquitous expression of
UAS-chn in the epidermis (69B-Gal4 driver)
(Brand and Perrimon, 1993
)
largely rescued many of the missing neurons
(Fig. 3C) and the morphological
defects of the lateral chordotonal neurons
(Fig. 3C, inset). However,
overexpression of UAS-chn in a wild-type background (using
da-Gal4, 69B-Gal4 and 1407-Gal4 drivers) did not appreciably
affect the larval PNS (data not shown).
In the adult, the effects of chnECJ1 were examined through clonal analysis. Clones of cells that lacked chn often failed to generate macrochaetae (Fig. 4A,B). These were observed to be missing in all notum positions, but scutellar bristles were the most sensitive. However, the penetrance was far from complete: clones including the dorsocentral (n=24) or the posterior postalar (n=8) positions lost approximately 25% of the bristles, while in scutellar clones (n=52) about 45% of the bristles were removed. We also observed, with low frequency, that the bristle shaft was missing, but not the tormogen cell, suggesting a role of chn in formation of the sensory organ. No defects were seen in the pattern of notum microchaetae.
We further examined the effects of the removal of chn in
macrochaetae formation with the help of an RNA-interference construct
(Sharp, 2001),
UAS-chni. A strong (UAS-chniS) and a weak
(UAS-chniW) expressing line were used.
UAS-chniS clearly antagonized chn function,
because it largely rescued the extra bristle and heminota fusion phenotypes of
MS248-Gal4; UAS-chn (Fig.
4D,E). No rescue was observed by replacing
UAS-chniS by UAS-lacZ or UAS-GFP, which
indicated that the effect was not caused by reduced expression of
UAS-chn in the presence of an additional UAS transgene. Moreover,
overexpression of UAS-chniS with MS1096-Gal4
sharply decreased accumulation of the endogenous chn mRNA in the wing
margin (not shown, see below). These experiments indicated that the
UAS-chniS acted as a LOF allele of chn. With the
drivers ap-Gal4 or sca-Gal4 (the latter promotes expression
in proneural clusters), UAS-chniS moderately removed notum
macrochaetae (Table 1), while
microchaetae were not affected. With the MS248-Gal4 driver, the
macrochaetae in the medial part of the notum, dorsocentrals and scutellars,
were often missing (Fig. 4C),
but, similar to the homozygous chnECJ1 clones, in no case
was the phenotype fully penetrant.
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chn promotes sc expression
The strong genetic interaction between the LOF conditions for chn
and ac/sc, together with the presumed activation of chn by
ac/sc (Fig. 2H-J), led
us to examine whether chn might in turn stimulate ac/sc
expression. We first examined whether the overexpression of chn
affected Sc accumulation in third instar wing disks. In these disks,
ac and sc are coexpressed in a stereotyped pattern of
well-resolved proneural clusters from which SOPs emerge
(Fig. 5A)
(Cubas et al., 1991;
Skeath and Carroll, 1991
).
With the MS248-Gal4 driver, UAS-chn promoted strong and
generalized expression of sc in most of the domain of expression of
the driver, namely, the medial and part of the lateral prospective notum
(Fig. 5C). Many SOPs arose from
this enlarged region of Sc accumulation, as detected by the Sens marker
(Nolo et al., 2000
),
consistent with the additional macrochaetae that developed on the notum of
these flies (Fig. 4D). With the
MS1096-Gal4 driver, which is expressed most strongly in the dorsal
part of the wing anlage (Capdevila and
Guerrero, 1994
), there was also ectopic expression of sc
and emergence of extra SOPs in the wing territory
(Fig. 5B). Interestingly,
expression of UAS-chn disrupted the characteristic double row
expression of sc and sens at the wing margin, suggesting
interference with its formation (Fig.
5B). This is also consistent with the presence of small, crumpled
adult wings that carry many bristles and other types of sensilla (data not
shown). Finally, overexpression of UAS-chn with the ubiquitous wing
disk driver C765-Gal4
(Gómez-Skarmeta et al.,
1996
) activated sc but failed to stimulate
atonal (Jarman et al.,
1993b
), a proneural gene which is not a member of the ASC and is
normally expressed in a few cells at the presumptive tegula and ventral radius
(Fig. 5D). Conversely,
overexpression of atonal did not stimulate chn in the wing
disk (not shown).
The expression of ac/sc in proneural clusters is controlled by a
series of separable enhancer elements in the ASC. Each enhancer is responsible
for expression in one or in a few proneural clusters
(Gómez-Skarmeta et al.,
1995). We thus examined whether the ectopic activation of
sc could be mediated by the overexpression of UAS-chn acting
upon these enhancers. As shown in Fig.
5E,F, UAS-chn strongly stimulated the activity of a
construct in which the lacZ gene was under the control of the ASC
L3/TSM enhancer [construct 2.3-lacZ
(Culí and Modolell,
1998
); MS1096-Gal4 driver], which directs expression at
the wing vein L3 and the twin sensilla of the wing margin proneural clusters.
Similar observations were made with the dorsocentral (DC) enhancer [construct
AS1.4DC=DC-lacZ
(García-García et al.,
1999
); C765-Gal4 ubiquitous driver], which promotes
expression in the central part of the notum. It also activated expression
directed by the ANP enhancer
(Gómez-Skarmeta et al.,
1995
) (data not shown). Since the DC-lacZ construct bears
the heterologous hsp70 promoter, these data indicate that the
sc endogenous promoter is dispensable for the stimulation by
UAS-chn. The Ac and Sc proneural proteins were also not essential for
the increased activity of the enhancers, as DC-lacZ expression was
strongly increased by Chn in an In(1)sc10.1 background
(Fig. 5I,J). By contrast, the
sc SOP-dedicated enhancer (SRV-lacZ construct), which is
responsible for the strong accumulation of Sc in SOPs
(Culí and Modolell,
1998
), was only clearly activated by UAS-chn
(C734-Gal4 driver) in the presence of ac/sc, and this
stimulation occurred in individual cells
(Fig. 5N,O). This observation
suggests that the upregulation of this enhancer results from the formation of
ectopic SOPs by the UAS-chn-induced overexpression of sc,
rather than from a direct effect of Chn on the enhancer. Still, the
possibility remains that Chn and Sc cooperate in the activation of this
enhancer.
UAS-chn upregulated the activity of these enhancers, but it did not lead to a generalized expression of lacZ in all the domains of UAS-chn expression. These data indicate that despite the elevated activation, the enhancers are still dependent on the prepattern factors that define their spatial domains of activity. This fact was verified by the observation that the overstimulation of DC-lacZ was strongly dependent on its prepattern activator, the transcription factor Pnr (Fig. 5K-M). Moreover, 2.3-lacZ, which is active only in the wing pouch, was not stimulated by the overexpression of UAS-chn in the prospective notum (MS248-Gal4), which indicates that the sc promoter present in this construct was not responsive to UAS-chn (data not shown).
Loss of function of chn reduces sc and enhancer-lacZ construct expression
Next, we examined in mosaic wing disks whether removal of chn
function affected expression of sc or enhancer-lacZ
constructs in proneural clusters. Homozygous chnECJ1 cells
generally displayed reduced expression of sc
(Fig. 6A) or
ß-galactosidase under the control of proneural enhancers
(Fig. 6B,C,F,G), when compared
with neighboring heterozygous chnECJ1/+ cells. Note,
however, that the expression was not completely abolished. Similar decreased
expression of sc was observed by misexpressing
UAS-chniS in cell clones
(Fig. 6D). These effects appear
to be cell-autonomous. While SOPs could still emerge from homozygous
chnECJ1 cells with reduced levels of Sc (data not shown),
SOPs were often missing (Fig.
6H), in agreement with the partial suppression of macrochaetae
observed within the chnECJ1 clones. When both homozygous
and heterozygous cells were near a position where an SOP emerged, a
heterozygous cell appeared to be preferentially selected
(Fig. 6C,E). These findings
clearly indicate that chn+ was required for proneural
proteins to accumulate in proneural clusters at levels sufficient to ensure
SOP selection. Moreover, the observation that expression of
enhancer-lacZ constructs was reduced in chn-
cells and increased in chn overexpressing cells indicates that the
effect of chn+ was not due to an enhanced perdurance of
the Sc protein, but to the increased transcription of the sc
gene.
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Discussion |
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chn and ac/sc establish a stimulatory loop in proneural clusters
The presence of chn mRNA in the proneural clusters of the wing
disk is dependent on ac/sc. Moreover, ectopic accumulation of Sc
results in ectopic expression of chn. These observations place
chn downstream of ac/sc, and suggest a positive, possibly
direct, regulation of chn by ac/sc. Consistent with this,
two clusters of four and eight E-boxes, putative binding sites for bHLH
proteins of the proneural type (reviewed by
Bertrand et al., 2002), were
found approximately 15 kb upstream of the chn structural sequences
and within the first intron of the gene, respectively.
In turn chn stimulates the accumulation of Sc in proneural
clusters, as loss of function of chn resulted in decreased
accumulation of Sc. However, some Sc still accumulates in the complete absence
of Chn, which probably explains why many SOPs and their corresponding
macrochaetae developed in its absence. The upregulation of sc by
chn is even more manifest by the overexpression of UAS-chn,
which causes a strong accumulation of Sc and leads to the formation of large
numbers of SOPs and extra macrochaetae. ac is also upregulated by
overexpression of chn (L.M.E., unpublished). Although we cannot rule
out that Chn may slow the turnover of Sc/Ac and thereby promote their
accumulation, our data clearly show that Chn stimulates the transcription of
ac/sc. Indeed, the overexpression of chn greatly increases
in vivo the expression of the reporter gene lacZ driven by proneural
group-specific enhancers of the ASC
(Culí and Modolell,
1998;
García-García et al.,
1999
; Gómez-Skarmeta et
al., 1995
) and its removal decreases the expression of these
constructs. The stimulation is also observed with enhancer constructs that do
not have the endogenous sc promoter (rather, they carry an
hsp70 minimal promoter). These data suggest that chn acts
mainly on the ASC enhancers, but we cannot rule out at present that the
endogenous promoter might additionally favor this effect. However, our results
argue against a stimulatory action of Chn directly on the sc and/or
ac promoters, since generalized expression of UAS-chn did
not lead to widespread expression of the constructs carrying the sc
promoter. Moreover, the stimulation was equally observed in the presence or
absence of the endogenous ac/sc genes, which indicates that it is not
mediated by positive feedback loops of ac/sc on the ASC enhancers, in
agreement with previous observations
(Gómez-Skarmeta et al.,
1995
). Considering that the ASC enhancers act in vivo on both the
sc and the ac promoters
(Cubas et al., 1991
;
Gómez-Skarmeta et al.,
1995
; Skeath and Carroll,
1991
), it was to be expected that Chn would also stimulate
ac expression.
Interestingly, Chn not only increased the levels of lacZ
expression within the proneural cluster for which the enhancer was specific,
but in general it also expanded the expression into a larger area surrounding
the proneural cluster, so that more cells were expressing the reporter gene.
Perdurance of ß-galactosidase should not be responsible for this effect,
because when chn was not overexpressed, DC-lacZ directed
ß-galactosidase accumulation only in the cells that also expressed
sc at the DC cluster
(García-García et al.,
1999). Moreover, the stimulation by chn seemed to require
the presence of at least some of the prepattern factors (reviewed by
Ghysen and Dambly-Chaudière,
1988
; Gómez-Skarmeta et
al., 2003
) that normally act on the enhancers and drive the
expression of ac and sc in proneural clusters, as is the
case for Pnr, the prepattern activating factor of the DC cluster
(García-García et al.,
1999
). We propose that excess Chn makes the proneural cluster
enhancers responsive to suboptimal concentrations of the prepattern activators
that are normally too low to permit activity. Hence, the domains of expression
of lacZ are expanded. The dependence of Chn stimulation on different
prepattern factors suggests that Chn acts as a coactivator, increasing the
effective interaction of prepattern activators with the ac and
sc promoters. Moreover, the finding that a fragment of Chn that
contains the five Zn-finger motifs of the protein can bind in vitro to a 316
bp fragment of the DC enhancer DNA further suggests that Chn stimulates
ac/sc expression by directly binding to ASC proneural
cluster-specific enhancers. The possible functional relevance of this binding
is reinforced by the fact that the 316 bp fragment is found within a 508 bp
segment that possesses residual DC enhancer activity and that Chn is capable
of strongly stimulating this activity in vivo.
Specificity of Chn
Chn does not appear to act in vivo as a general stimulator of the enhancer
action of proneural genes. The ASC enhancer(s) responsible for expression of
ac/sc during microchaetae formation did not require Chn, as judged by
the independence of microchaetae density from the activity of chn.
Note that downregulation of ac and/or sc normally leads to a
strong loss of microchaetae
(Ruiz-Gómez and Modolell,
1987). By contrast, overexpression of UAS-chn did
increase their density, suggesting that the microchaetae enhancer(s) can
potentially respond to Chn. chnECJ1 clones and
UAS-chni did not alter the anterior wing margin bristles. However,
overexpression of UAS-chn impaired the expression of sc at
the anterior wing margin (Fig.
5A-C), although we favor the idea that this inhibition results
from an interference of Chn with the general patterning of the wing, as
suggested by the inhibition of sens expression even in the posterior
wing margin (Fig. 5A). The lack
of an identified ASC wing margin enhancer has prevented a more direct test of
these possibilities. We also found that the ASC SOP-specific enhancer
(Culí and Modolell,
1998
) could not be activated in the absence of ac/sc and
that the stimulation that we observed occurred in isolated cells, rather than
in the majority of cells of the domain of UAS-chn expression.
Probably, the stimulation resulted from extra SOPs arising from the
overexpression of the endogenous sc gene. Finally, the proneural gene
atonal, which is not a member of the ASC
(Jarman et al., 1993b
), was
not affected in the wing or in the eye (L. M. E., unpublished) disks by
UAS-chn. We conclude that in the wing disk, Chn is mostly specific
for the ASC enhancers that direct ac/sc expression in the proneural
clusters of the macrochaetae and other landmark sensilla, such as the twin
sensilla of the anterior wing margin (TSM) and the L3 wing vein sensilla
campaniformia.
Genetic levels of control during SOP specification
Taken together, our data indicate that chn and ac/sc form
a mutually stimulatory loop that enhances accumulation of Ac/Sc in the
proneural clusters of the notum macrochaetae
(Fig. 7F). These and other
previous findings suggest the following consecutive levels of genetic control
during SOP selection. The process starts by the deployment of combinations of
prepattern factors that trigger the expression of ac/sc in proneural
clusters (reviewed by Ghysen and
Dambly-Chaudière, 1988;
Gómez-Skarmeta et al.,
2003
) (Fig. 7E).
Then, ac/sc activate chn and their stimulatory loop
reinforces the expression of ac/sc
(Fig. 7F). This allows
increasing levels of Ac/Sc to accumulate in the cells of the proneural cluster
and the formation of the proneural field, which includes the few cells of the
cluster with the highest levels of Ac/Sc
(Cubas and Modolell, 1992
).
The SOP will be selected from one of these cells by the Ac/Sc-mediated
activation of sens, which in turn allows the autostimulatory loops of
the proneural genes mediated by the SOP-specific enhancers
(Culí and Modolell,
1998
; Jafar-Nejad et al.,
2003
; Nolo et al.,
2000
) (Fig. 7G).
These enhancers are the targets of two antagonistic signaling systems, both
triggered by the accumulation of Ac/Sc. The positive one is mediated by the
EGF receptor (Culí et al.,
2001
) and Sens. The EGFR pathway allows the cells of the proneural
cluster to signal positively to each other (lateral cooperation) and helps
activate the SOP-specific enhancers, whereas Sens directly activates proneural
gene expression in a positive feedback loop when the proneurals reach a
certain threshhold in the SOP. Sens and EGFR are in turn antagonized by the
negative loop, which is mediated by the Dl/N pathway and the E(spl) proteins
and prevents more than one cell from turning on the proneural gene
self-stimulation and becoming an SOP (lateral inhibition)
(Artavanis-Tsakonas et al.,
1995
; Culí and
Modolell, 1998
; Giagtzoglou et
al., 2003
; Nolo et al.,
2000
; Heitzler and Simpson,
1991
; Jafar-Nejad et al.,
2003
; Simpson,
1990
; Simpson,
1997
). Thus, three loops of self-stimulation of ac/sc
exist: the first is mediated by chn and targets the proneural cluster
enhancers; the second is mediated by the EGFR pathway and targets the
SOP-specific enhancers; the third is mediated by Sens and also directly
targets the SOP-specific enhancers. It is interesting to note that the first
and third stimulatory loops are mediated by Zn-finger transcription factors of
the C2H2 type with homologs in mammals and other species. The negative loop,
mediated by Dl/N and the E(spl) proteins, maintains most cells of the
proneural cluster in a non-SOP state, allowing them to differentiate as
epidermal cells (Fig. 7E,F). As
previously discussed, it is tempting to speculate that these consecutive
layers of control facilitate the refinement of the position where SOPs arise
within proneural clusters (Culí et
al., 2001
).
Function of chn as a neuronal differentiation gene
In the embryo, chn is expressed in regions where the neurons of
the PNS will arise and later in the developing neural cells. Its removal
causes loss of PNS neurons and defects in the morphology of the chordotonal
organs, suggesting that chn is required for the proper formation of
many or most elements of the PNS. So far, the reported effects of
insufficiency of proneural gene function in the embryonic PNS have mostly been
the removal of neurons and chordotonal organs, rather than defective
morphologies (Dambly-Chaudière and
Ghysen, 1987; Huang et al.,
2000
; Jarman et al.,
1993b
; Villa-Cuesta et al.,
2003
). Hence, we like to suggest that in the embryonic PNS
chn acts more as a neuronal differentiation gene than a proneural
gene activator. In agreement with this suggestion we observed that
overexpression of UAS-chn did not modify the embryonic PNS, as
detected with the 22c10 antibody. By contrast, overexpression of proneural
genes promotes development of extra neurons and chordotonal organs
(Huang et al., 2000
;
Jarman et al., 1993b
;
Villa-Cuesta et al., 2003
).
Moreover, loss of function of cousin of atonal (cato) and
ase, two genes that can act as neuronal differentiation genes, also
causes malformations of the lateral clusters of chordotonal organs
(Goulding et al., 2000
). We do
not know whether the removal of chn may also affect the
differentiation of the adult bristles, but the observation that, with low
frequency, a shaft can be missing, but not the basal cell, also suggests a
role of chn in the differentiation of these SOs. Moreover, the fact
that UAS-chni partially suppressed the extra macrochaetae induced by
UAS-sc (Fig. 4F,G), a
transgene not subjected to chn modulation, may additionally indicate
that chn favors macrochaetae formation. However, it should be kept in
mind that UAS-sc may promote accumulation of Sc not only through its
own expression, but also by the activation of chn, which would in
turn stimulate the endogenous ac/sc genes. This latter stimulation
should be sensitive to UAS-chni and its inhibition might partially
suppress the formation of extra macrochaetae. At present, we cannot decide on
these alternatives.
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ACKNOWLEDGMENTS |
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Footnotes |
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